Tsa Conference Book by Tuberous Sclerosis Association

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Hosted at Castle Hotel Windsor

#TSCResearch

2015 International Tuberous Sclerosis Complex Research Conference: From Treatment to Prevention

This conference is supported by a grant from Novartis and presented by

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2015 International Tuberous Sclerosis Complex Research Conference: From Treatment to Prevention Conference at a glance: Thursday 10 September

12:00 - 13:30

Registration and lunch

13:30 - 14:00

Opening address: • Aims • Objectives • Structure for the event • NIH update

14:00 - 15:30 16:00 - 17:30 19:00 - 19:30 19:30 - 21:30 Friday 11 September

07:30 - 08:30 08:30 - 10:15 10:45 - 12:30 12:30 - 13:40 13:40 - 15:35

Windsor and Georgian

Session 1 Early diagnosis and the impact of genetics on diagnosis

Windsor and Georgian

Reception (Cash bar available before dinner)

Windsor and Georgian

Early morning tea and coﬀee

Georgian and Conference Foyer

Session 3 The potential for preventing epileptogenesis in TSC

Windsor and Georgian

Lunch

Eighteen Restaurant and Georgian

Session 2 Impact and gaps of mTOR inhibitor treatment of TSC

Windsor and Georgian

Conference dinner with keynote speaker, Michael Hall and Gomez Award presentation

Windsor and Georgian

Session 4 Breakthroughs in the understanding of lung and renal manifestations of TSC

Windsor and Georgian

Session 5 Breakthroughs in understanding the pathogenesis of TSC at the cellular level

Windsor and Georgian

17:00 - 18:30

Session 6 Late breaking research

Canapé reception and poster viewing

Windsor and Georgian

07:30 - 08:30

Early morning tea and coﬀee

Georgian and Conference Foyer

08:30 - 10:15

Session 7 Burden of TSC and engaging those aﬀected to impact research

16:00 - 17:00

Saturday 12 September

Eighteen Restaurant and Georgian

10:45 - 12:30

12:30 - 13:30

Sandringham, Balmoral and Conference Foyer

Windsor and Georgian

Session 8 Translating research into improvements in care and outcomes

Windsor and Georgian

Main conference close and lunch.

Eighteen Restaurant and Georgian

Summing up: Where next?

Dear Conference Participants, Welcome to Windsor, Royal Berkshire, UK. On behalf of the Tuberous Sclerosis Association UK and the Tuberous Sclerosis Alliance USA, we welcome all our global delegates joining us for what we anticipate will be an informative and impactful three days in conference. The main conference goal is to provide a forum for exchanging results of research and developing collaborations for future tuberous sclerosis complex (TSC) research. The overarching conference theme is the search for a cure for TSC. To this end our conference will also serve as a collaborative gathering for developing an early intervention trial to address the most serious effects of TSC. The planning committee created a rich conference programme that explores the latest developments in our understanding of TSC at the cellular level; diagnosis and the impact of genetic analysis; the manifestation of TSC in terms of epilepsy, lung and kidney; progress and gaps in treatment; understanding the burden of the disease; and the translation of research into improvements in care outcomes. During our conference dinner on Thursday evening, we are privileged to hear from our keynote speaker, Professor Michael Hall of the University of Basel, who will discuss his pioneering work in the discovery of TOR (Target of Rapamycin). On Friday evening, a poster reception will feature more than 50 posters on display. Please do join us for drinks and canapés 17:00-18:30 when researchers will be available with their posters. We would like to thank the sponsors who are generously supporting this event. We are very grateful this conference is supported by a grant from Novartis, while The Rothberg Institute for Childhood Diseases is a presenting sponsor. Support is also provided by The Brian O’Brien Family, GW Pharmaceuticals, Bcureful and the Castle Hotel Windsor. We would also like to recognise the National Institutes of Health (USA), the National Institute of Neurological Disorders and Stroke and the National Center for Advancing Translational Sciences which provided grant support for this year's research conference. Finally we wish to thank our invited speakers and poster presenters for contributing to this important conference as well as everyone’s ongoing support in advancing our global efforts within your community, laboratories and practice. Sincerely,

Jayne Spink CEO Tuberous Sclerosis Association

Kari Luther Rosbeck President and CEO Tuberous Sclerosis Alliance

2015 International Conference Organising Committee Co-Chair: J. Chris Kingswood, MSc, FRCP, TSA, Head of Research Strategy Co-Chair: Steven L. Roberds, PhD, TS Alliance, Chief Scientific Officer Alison Cooper, MPH, TSA, Head of Research Annie Jones, MSc, MBA, TSA, Chair Research Committee Jo Anne Nakagawa, TS Alliance, Director of Clinical Projects & TSC Clinic Liaison Kari Luther Rosbeck, TS Alliance, President and Chief Executive Officer Katie Smith, TS Alliance, Manager, Research and Global Affairs Jayne Spink, PhD, TSA, Chief Executive Officer TSCure Steering Group and Conference Scientific Advisory Committee John Bissler, MD John Blenis, PhD Paolo Curatolo, MD David N. Franz, MD Michael Hall, PhD Elizabeth Henske, MD Sergiusz Józwiak, MD, PhD Darcy Krueger, MD, PhD David Mowat, MBBS, MRCGP, DRACOG, FRACP Finbar O'Callaghan, MA, MB, MSc, PhD Julian Sampson, DM, FRCP, FMedSci Elizabeth Thiele, MD, PhD Petrus de Vries, MBChB, MRCPsych, PhD Michael Wong, MD, PhD

Table of contents

Page

Conference agenda

Keynote speaker, Michael Hall

Speaker biographies: speakers, community speakers and session chairs

Index of speaker abstracts

Session 1 - Early diagnosis and the impact of genetics on diagnosis

Session 2 - Impact and gaps of mTOR inhibitor treatment of TSC

Session 3 - The potential for preventing epileptogenesis in TSC

Session 4 - Breakthroughs in the understanding of lung and renal manifestations of TSC

Session 5 - Breakthroughs in understanding the pathogenesis of TSC at the cellular level

Session 6 - Late breaking research

Session 7 - Burden of TSC and engaging those affected to impact research

Session 8 - Translating research into improvements in care and outcomes

Poster listing

Poster abstracts

Vicky H. Whittemore Travel Awards

Exhibitors

Tuberous Sclerosis Association, UK About the Tuberous Sclerosis Association Research strategy List of grants Committees

Tuberous Sclerosis Alliance, USA About the Tuberous Sclerosis Alliance Research strategy List of grants Board of Directors International Scientific Advisory Board

Tuberous Sclerosis Complex International (TSCi)

Conference Support Acknowledgements

Conference Agenda Day One: Thursday 10 September 2015 12:00 - 13:30 13:30 - 14:00

Registration and Lunch Opening address, aims, objectives and structure for event, NIH update – Jayne Spink and Steven L. Roberds

14:00 - 15:30

15:15 - 15:30

Session 1: Early diagnosis and the impact of genetics on diagnosis, Chaired by Hope Northrup Community Speaker – Philippa Ward (UK) Julian Sampson – Genetics and early diagnosis of TSC Darcy Krueger – Early treatment in TSC with vigabatrin and mTOR inhibitors: Enthusiasm, caution, or both? Rebecca Ihrie – Neural stem cell patterning and the origin of SEGAs

15:30 - 16:00

Break

16:00 - 17:30 16:00 - 16:30 16:30 - 16:45 16:45 - 17:15 17:15 - 17:30

Session 2: Impact and gaps of mTOR inhibitor treatment of TSC, Chaired by Steve Roberds Chris Kingswood – mTOR inhibitors, where are we now Sophie Thompson – Beyond mTOR David Franz – mTOR inhibitors, where next Jadwiga Schreiber – Toward a better understanding of epilepsy

19:00 - 19:30

Reception (Cash bar will be available)

19:30 - 21:30

Conference Dinner Keynote speaker – Professor Michael Hall, mTOR signaling in growth and metabolism Gomez Award presentation, Kari Luther Rosbeck

14:00 - 14:15 14:15 - 14:45 14:45 - 15:15

Day Two: Friday 11 September 2015 07:30 - 08:30

Early morning tea and coffee

08:30 - 10:15

Session 3: The potential for preventing epileptogenesis in TSC, Chaired by Elizabeth Thiele Community Speaker – Ron Heffron (USA) Sergiusz Jóźwiak – EPISTOP and other new data on early intervention for epilepsy in TSC Martina Bebin – The potential for early intervention to prevent the onset of epilepsy in TSC

8:30 - 8:45 8:45 - 9:15 9:15 - 9:45

9:45 - 10:15

Michael Wong – New concepts on the genesis of epilepsy in TSC: looking beyond mTOR

10:15 - 10:45

Break

10:45 - 12.30 10:45 - 11:00 11:00 - 11:30 11:30 - 12:00 12:00 - 12:30

Session 4: Breakthroughs in the understanding of lung and renal manifestations of TSC, Chaired by Chris Kingswood Carmen Priolo – Aberrant lipid metabolism as a TSC biomarker Wei Shi – A novel mouse model of LAM Frank McCormack – mTOR inhibitors and beyond in the lung John Bissler – TSC renal failure and cystic disease

12:30 - 13.40

Lunch and networking

13:40 - 15:35

15:05 - 15:35

Session 5: Breakthroughs in understanding the pathogenesis of TSC at the cellular level, Chaired by Julian Sampson Community Speaker – Wesley Gomes (Brazil) Brendan Manning – Alternative therapeutic targets for TSC uncovered downstream of mTORC1 Gina Lee – mTORC1-mediated regulation of mRNA biogenesis in TSC and LAM Andy Tee – Combination therapy with nelfinavir: mechanism of drug action and selective targeting of Tuberous Sclerosis Complex 2 deficient cells John Blenis – mTOR, metabolism and therapeutic vulnerabilities

15:35 - 16:00

Break

16:00 - 17:00 16:00 - 16:20

Session 6: Late breaking research, Chaired by Chris Kingswood Iris Overwater – Sirolimus treatment in children with Tuberous Sclerosis Complex and intractable epilepsy: a randomized controlled trial Kandice Varcin – Social visual perception in tuberous sclerosis complex Juxiang Cao – The TSC protein complex regulates melanogenesis through mTOR and GSK3ß, explaining the pathogenesis of TSC ‘white spots’

13:40 - 13:50 13:50 - 14:20 14:20 - 14:35 14:35 - 15:05

16:20 - 16:40 16:40 - 17:00

17:00 - 18:30

Canapé reception and poster viewing

Day Three: Saturday 12 September 2015 07:30 - 8:30

Early morning tea and coffee

08:30 - 10:15

Session 7: Burden of TSC and engaging those affected to impact research, Chaired by Paolo Curatolo Community Speaker – Marie James (UK) Petrus de Vries – The neuropsychiatric burden and methods that work to lessen that burden Anna Jansen – Burden of TSC and health economics

8:30 - 8:45 8:45 - 9:15 9:15 - 9:45

9:45 - 10:15

Patrick Bolton – TS2000: the impact of TSC in early years

10:15 - 10:45

Break

10:45 - 12:30

Session 8: Translating research into improvements in care and outcomes, Chaired by David Mowat David Kwiatkowski – Genetics and potential for personalized medicine Lisa Henske – Treatments on the horizon for TSC and LAM Mustafa Sahin – The potential for early intervention to prevent development of autism

10:45 - 11:15 11:15 - 11:45 11:45 - 12:15

12:15 - 12:30

Summing up: Where next? Chaired by Chris Kingswood

12:30 - 13:30

Main Conference Close, lunch and networking

Conference Dinner Thursday 10 September Keynote Speaker Professor Michael Hall, BS, PhD, Biozentrum, University of Basel, Switzerland Michael N. Hall was born (1953) in Puerto Rico and grew up in South America (Venezuela and Peru). He received his PhD from Harvard University and was a postdoctoral fellow at the Pasteur Institute (Paris, France) and the University of California, San Francisco. He joined the Biozentrum of the University of Basel (Switzerland) in 1987 where he is currently Professor and former Chair of Biochemistry. Hall is a pioneer in the fields of TOR signaling and cell growth control. In 1991, Hall and colleagues discovered TOR (Target of Rapamycin) and subsequently elucidated its role as a central controller of cell growth and metabolism. TOR is a highly conserved, nutrient- and insulin-activated protein kinase. The discovery of TOR led to a fundamental change in how one thinks of cell growth. It is not a spontaneous process that just happens when building blocks (nutrients) are available, but rather a highly regulated, plastic process controlled by TOR-dependent signaling pathways. As a central controller of cell growth and metabolism, TOR plays a key role in development and aging, and is implicated in disorders such as TSC, cancer, cardiovascular disease, diabetes, and obesity. Hall is a member of the US National Academy of Sciences, has received numerous awards, including the Louis-Jeantet Prize for Medicine (2009), the Marcel Benoist Prize for Sciences or Humanities (2012), the Breakthrough Prize in Life Sciences (2014), and the Canada Gairdner International Award (2015), and has served on several editorial and scientific advisory boards. He and his wife Sabine (née Carrère) live in Basel with their daughters Zoé and Léa.

Presenting: mTOR signaling in growth and metabolism TOR (target of rapamycin) is a highly conserved serine/threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress. TOR was originally discovered in yeast but is conserved in all eukaryotes including plants, worms, flies, and mammals. TOR is found in two structurally and functionally distinct multiprotein complexes termed TORC1 and TORC2. The two TOR complexes, like TOR itself, are highly conserved. Thus, the two TOR complexes constitute an ancestral signaling network conserved throughout eukaryotic evolution to control the fundamental process of cell growth. As a central controller of cell growth, TOR plays a key role in development and aging, and is implicated in disorders such as TSC, cancer, cardiovascular disease, obesity, and diabetes. While the role of TOR in controlling growth of single cells is relatively well understood, the challenge now is to understand the role of TOR signaling in disease and in coordinating and integrating overall body growth and metabolism in multicellular organisms. This will require elucidating the role of TOR signaling in individual tissues. Data on the role of mammalian TORC1 (mTORC1) and/or mTORC2 in controlling cellular processes and in specific tissues will be presented.

Biographies: Speakers, community speakers and session chairs Martina Bebin, MD, MPA, Pediatric Neurologist/ Epileptologist, Co-Director, TSC Clinic, University of Alabama at Birmingham, Birmingham, AL. Dr Martina Bebin is a Professor of Neurology and Pediatrics at the University of Alabama at Birmingham. Her primary research interest is in tuberous sclerosis complex (TSC), and she serves as co-director of the UAB TSC Clinic with Dr Bruce Korf. She has served as principal investigator (PI) on numerous pediatric antiepileptic clinical trials over the last 20 years. Since 2009, she has served on the DOD-CDMRP grant review program for tuberous sclerosis complex. She is currently the administrative PI for the NIH grant entitled Potential EEG biomarkers and antiepileptogenic strategies for epilepsy in TSC and the site PI for the ACE autism grant investigating early biomarkers for ASD in infants with TSC. Dr Bebin has been involved with the Tuberous Sclerosis Alliance for more than 10 years and currently serves on the Tuberous Sclerosis Alliance Board of Directors.

John Blenis, PhD, Professor of Pharmacology, Weill Cornell Medical College, New York, NY. Dr John Blenis is the Anna-Maria and Stephen Kellen Professor of Cancer Research and Professor of Pharmacology at Weill Cornell Medical College. He completed his BA degree (1977) at the University of California, Berkeley, USA, his PhD (1983) at Michigan State University, Michigan, USA, and his postdoctoral research at Harvard University, Cambridge, Massachusetts, USA (1983-1987). Dr Blenis was an Assistant Professor at Northwestern University (1987-1989), and then Assistant, Associate and Tenured Professor of Cell Biology from 1989-2014 at Harvard Medical School before moving his laboratory to the Meyer Cancer Center at Weill Cornell Medical College. The major objectives of his research are to define and characterize the molecular basis of cellular signaling with a focus on the Ras-ERK-RSK and PI3K-mTOR-S6K1 pathways, and how these signaling systems contribute to the normal and disease-associated physiology.

John J. Bissler, MD, Professor of Pediatrics and FedEx Chair of Excellence, University of Tennessee Health Science Center, and Director, Tuberous Sclerosis Center of Excellence and Division of Nephrology, St. Jude Children’s Research Hospital and Le Bonheur Children’s Hospital. Dr Bissler is a Nephrologist at Le Bonheur Children’s Hospital and St. Jude Children’s Research Hospital, at the University of Tennessee in Memphis. Dr Bissler’s experience and interest in tuberous sclerosis renal disease led him to be the principle investigator for the critical first studies demonstrating the effectiveness of mTORC1 inhibition for renal angiomyolipomata associated with TSC that were published in the New England Journal of Medicine and The Lancet. He is Principle Investigator for the Exist-2. He also has helped to start up the multidisciplinary Tuberous Sclerosis Clinic at Cincinnati Children’s and is the Director of the TSC Centre of Excellence in Memphis. Dr Bissler conducts basic, translational, and clinical studies to find ways to help his patients. He currently serves on the Tuberous Sclerosis Alliance Board of Directors.

Patrick Bolton, BSc, MA, PhD, MBBS, FRCPsych, Professor of Child & Adolescent Psychiatry, King's College London, Institute of Psychiatry, London. Patrick Bolton studied psychology and medicine at London University and Guy’s Hospital. He trained in child psychiatry at the MRC Child Psychiatry Unit at the Institute of Psychiatry and Maudsley Hospital. After completing his training, he was appointed Lecturer and Honorary Consultant at the Institute of Psychiatry and, subsequently, Senior Lecturer at Cambridge University and Trinity College Cambridge. In 2003, he was appointed Professor of Child & Adolescent Psychiatry at the Institute of Psychiatry, Kings College London and Honorary Consultant Child and Adolescent Psychiatrist at the Maudsley Hospital, where he leads the National Specialist Clinical Service for children with autism spectrum and related disorders. His research has focussed on identifying the causes of autism and related disorders of social function and communication. His research is supported by awards from the MRC, Wellcome Trust, Autism Speaks, the UK Tuberous Sclerosis Association, Action Medical Research and other research charities.

Juxiang Cao, PhD, Postdoctoral Research Associate Brigham and Women's Hospital, Boston, MA. Juxiang Cao is currently a Postdoctoral fellow at Brigham and Women’s Hospital, Harvard Medical School. She earned her PhD from Auburn University in Molecular Biology and Biochemistry Program in the department of Biological Sciences in 2008. She pursued her postdoctoral training in cancer biology. Dr Cao’s research interest includes skin biology and cancers, and has broad interests in all types of cancers, including lung cancer, breast cancer, and kidney cancer. Her current research focuses on uncovering the molecular mechanisms associated with tumors and hypomelanotic macules in TSC patients, exploring biochemical and signaling pathways and therapeutic approaches. She is particular interested in the pathogenesis of the tumors and skin lesions that occur in TSC, including lymphangioleiomyomatosis (LAM), angiomyolipomas and hypomelanotic macules.

Paolo Curatalo, MD, Professor, Head of Section of Pediatric Neurology and Psychiatry, the Department of Neuroscience, and Professor of Pediatric Neurology and Psychiatry, the University of Rome Tor Vergata. Professor Curatolo’s main areas of research are tuberous sclerosis complex, epilepsy, developmental disabilities, autism spectrum disorders, and ADHD. He has published more than 300 articles on child neurology in peer-reviewed international journals and more than 65 on tuberous sclerosis. He serves on numerous editorial boards, including the Journal of Child Neurology, the European Journal of Pediatric Neurology, Brain & Development, and Pediatric Neurology. He served as President of the International Child Neurology Association (2002-2006) and is a 20-year member of both the Executive Board of the European Pediatric Neurology Society and the International Child Neurology Association.

He is the author of three monographs: • Tuberous Sclerosis Complex: from basic science to clinical phenotypes, Cambridge University Press 2003 • Neurocutaneous syndromes, John Libbey Eurotext, 2006 • Malformations of the nervous system, Elsevier 2008

Petrus de Vries, MBChB, MRCPsych, PhD, Sue Struengmann Professor of Child & Adolescent Psychiatry, Division of Child and Adolescent Psychiatry, University of Cape Town, Cape Town. Petrus de Vries is the Sue Struengmann Professor of Child & Adolescent Psychiatry and Director of the Centre for Autism Research in Africa and the Adolescent Health Research Unit at the University of Cape Town. He trained in Medicine at Stellenbosch University in South Africa before moving to the UK where he completed his clinical training in Psychiatry and Child & Adolescent Psychiatry, and earned a PhD in Developmental Neuropsychiatry at the University of Cambridge. Professor de Vries is chairman of the Society for the Study of Behavioural Phenotypes (SSBP), an international, interdisciplinary research organization, is on the WHO ICF-CY steering group for autism spectrum disorders and ADHD, and is a member of the Executive Committee of IACAPAP (the International Association for Child & Adolescent Psychiatry and Allied Professions). Apart from his work in autism spectrum disorders and adolescent health, he also has a programme of research in the neuropsychiatry of tuberous sclerosis complex (TSC) with an interest both in the molecular mechanisms underlying neurocognitive and neurodevelopmental deficits associated with the TSC1/2-mTOR signalling pathway and in community-based participatory research. He is a Medical Advisor to the Tuberous Sclerosis Association (UK), a member of the Professional Advisory Board and International Scientific Advisory Board of the Tuberous Sclerosis Alliance (USA), a Specialist Advisor to TSDeutschland and scientific advisor to Stichting Michelle (Netherlands). He was chairman of the Neuropsychiatry Panel of the international consensus group that revised the diagnostic criteria and treatment guidelines for TSC in 2012 and has been on the study steering committee of three phase III clinical trials for mTOR inhibitors in TSC. He is also a member of the working committee of the TOSCA international natural history database project of TSC. David N. Franz, MD, Pediatric Neurologist, Founding Director, TSC Clinic, Children's Hospital Medical Center, Cincinnati, OH. David Neal Franz, MD, was born and raised in Dayton, Ohio. He received his undergraduate degree in History and Literature from Earlham College in Richmond, IN. After completing his training he served as Assistant Professor of Neurology and Pediatrics at Wright State University before returning to Cincinnati Children’s Hospital Medical Center. He established the Cincinnati Tuberous Sclerosis Clinic in 1992 to assist in the medical care of patients who have or are suspected of having tuberous sclerosis complex (TSC). His clinic was the first to initiate clinical trials of the mTOR inhibitors rapamycin (2003) and later everolimus (2006) for TSC-related brain tumors, epilepsy, angiomyolipomas, and pulmonary disease (lymphangioleiomyomatosis). This research has resulted in FDA approval of everolimus to treat brain and kidney tumors associated with TSC. Dr Franz’s specialty clinic for TSC is currently the largest in the world and follows more than 850 adults and children with the disease.

Wesley Gomes, Community speaker, Brazil. Wesley Gomes is an Industrial Engineer and Vice President of the Associação Brasileira de Esclerose Tuberosa (ABET). In addition, he is partner and CEO of a German company subsidiary in Brazil, which is also responsible for the Latin American market. Wesley’s sister Flávia was diagnosed with TSC when she was two years old and, by that time, the doctor gave the family the terrifying prognosis she would not live more than 15 years. Since then, Flávia and her family have been struggling and fighting against the perverse consequences of the disease, especially the seizures. When Flávia was 12 years old, she went in a coma for almost two months, but managed to recover without major sequels. When she was 15, her parents (Márcia and Wesley Assis) founded ABET. ABET is a non-profit association dedicated not only to attend patients with TS and their families from all over Brazil, but also children with major brain damage. On a daily basis, the association provides therapeutic treatment – such as psychology, physiotherapy, occupational therapy, speech therapy, music therapy and social assistance – to around 20 children. The major challenges of the association at the moment are to raise funds for its subsistence and to set up a fixed clinical staff. Nevertheless, this year, the association together with the families was able to organize the 2nd Brazilian TS Congress, which featured the participation of leading experts, including Dr. John Bissler. Last August, Flávia celebrated her 30th birthday and started treatment with everolimus.

Ron Heffron, PE, Community speaker, USA. Ron Heffron is an engineer specializing in the planning and design of ports and shipping terminals globally. He and his wife Ann have an 11 year old son with TSC, Bao Heffron, who was diagnosed at 5 months with infantile spasms. Bao has endured four resection surgeries to control his seizures, conducted at NYU and UCLA. He is high-functioning and loves to travel, but he still cannot chew and all his food must be pureed. He also has learning disabilities, behavioral issues, and anxieties that are disruptive at times. On the positive side, he was mainstreamed in his school last year for the first time in 5th grade and he was very proud and motivated to stay in the mainstream class. With intensive tutoring and hours of homework every night, he brought home a report card at the end of the year with straight As! Ron is a past board member of the TS Alliance and a current member of the organization’s Science & Medical Committee. He has participated in the TSCRP research proposal review process for 7 years, and is a familiar face at TSC research conferences.

Elizabeth P. Henske, MD, Oncologist, Brigham and Women's Hospital, Harvard Medical School, Boston, MA. Dr Elizabeth (Lisa) Petri Henske is Director of the Center for LAM Research and Clinical Care at the Brigham and Women’s Hospital/Harvard Medical School, a medical oncologist at the Dana-Farber Cancer Institute and an Associate Member of the Broad Institute of MIT and Harvard. She earned her undergraduate degree summa cum laude from Yale, where she majored in Molecular Biophysics and Biochemistry and was elected to Phi Beta Kappa, and her MD degree from Harvard Medical School. She completed her Residency in Internal Medicine and Fellowship in Hematology/Oncology at the Massachusetts General Hospital. Dr Henske’s laboratory discovered that LAM is caused by mutations in the tuberous sclerosis complex (TSC) genes. Currently, her research is focused on the cellular and metabolic mechanisms underlying the pathogenesis of lymphangioleiomyomatosis (LAM). She is the Principal Investigator of the Sirolimus and Autophagy Inhibition in LAM (SAIL) clinical trial. Dr Henske has received awards for her research from the Tuberous Sclerosis Alliance (the Gomez Award), the LAM Foundation (the Scientific Advancement Award), and the Society for Women's Health Research (the Medtronic Prize). Dr Henske’s laboratory research is focused on the cellular and metabolic mechanisms through which mutations in the TSC genes lead to tumorigenesis and lymphangioleiomyomatosis (LAM). She chairs the International Scientific Advisory Board of the Tuberous Sclerosis Alliance and has been a member of the LAM Foundation Scientific Advisory Board since 1999.

Rebecca Ihire, PhD, Assistant Professor, Cancer Biology and Neurological Surgery, Vanderbilt University, Nashville, TN. Rebecca Ihire earned a PhD in Cancer Biology at Stanford University, followed by a postdoctoral fellowship in the laboratory of Arturo Alvarez-Buylla at UC San Francisco. She is now an assistant professor in Cancer Biology and (by courtesy) Neurological Surgery at Vanderbilt University. Her laboratory is focused on understanding how cells in the healthy and diseased brain control their proliferation and their ability to make the different cell types of the brain. The lab works with basic researchers, medical researchers, and neurosurgeons to study stem cells in the normal brain and stem-like cells in multiple brain tumor types. It uses specialized stereotaxic targeting and single-cell assays (including confocal microscopy, mass cytometry, and advanced data visualization methods) to identify populations of stem cells and test their contribution to brain tumor development. Her research is particularly interested in dissecting the biological mechanisms underlying subependymal giant cell astrocytoma development, and leveraging this knowledge to find new candidate treatments for these tumors.

Marie James, Community speaker, UK. Marie is married to Perry and their youngest son, Trystan, was diagnosed with TSC in 1987. She has gained a wealth of hands on, diverse experiences through raising Trystan. Marie has been involved on various steering groups and campaigns with Learning Disability Wales, Mencap, Life Options Project, SNAP Cymru, Contact a Family Wales, RhAGPHA (Welsh Medium SEN Organisation), NAS Cymru, Wales Neurological Alliance and the Genetic Alliance UK. Marie also

volunteers for other user organisations with an interest in disabled children, young people and their families within Wales. She is a member of the Carmarthenshire ASD Steering and the Carer’s Training Groups. She was instrumental in forming Carmarthenshire’s National Autistic Society Branch 14 years ago and has since been responsible for its management, supporting more than 300 families. Marie has contributed numerous TSC awareness articles for press, TV and radio, in both Welsh and English. The family has also taken part in TV documentaries relating to TSC linked with education, health, autism and carer’s issues. Marie remains committed to making a difference in the lives of people affected by TSC – from lending support, fundraising and raising the profile of the TSA in all communities, to liaising with Local Authorities and Welsh Assembly Government and campaigning on the barriers to accessing treatment and services.

Anna Jansen, MD, PhD, Pediatric Neurologist UZ Brussel Hospital, Brussels. Dr Anna Jansen is a pediatric neurologist at the UZ Brussel Hospital in Belgium. She runs a multidisciplinary TSC clinic for both children and adults, together with Dr Peter Janssens, nephrologist. Dr Jansen is co-investigator in the EPISTOP trial (FP7/2007-2013 GA 602391) and is responsible for the dissemination of the project results (www.epistop.eu). She is the Vice-Chair of the TOSCA Scientific Advisory Board and coordinates the TOSCA SEGA research project. She was part of the TSC-associated neuropsychiatric disorders committee of the 2012 TSC Consensus Conference and participated in the development of the TAND checklist which she continues to promote.

Annie Jones, MBA, MSc, Chair of TSA Research Committee. Annie has more than 30 years’ experience in the pharmaceutical industry. After graduating from the University of York, she began her career as a Sales Representative with Reckitt & Coleman UK. Joining Bayer UK as a Sales Representative she was promoted to a Teaching Hospital Sales Specialist, Regional Trainer and Teaching Hospital Manager. During this time she also completed her MBA at Leeds University. She assumed the role of Scientific Affairs Manager in the UK after a secondment working in the USA and was then promoted to International Scientific Affairs Manager with Bayer AG, and relocated to Germany. In 2000, Annie returned to the UK and established herself as a consultant. She has also worked with the UK government’s Detection & Identification of Infectious Agents (DIIA) project. Annie’s main areas of professional expertise are antibiotic resistance; strategic publication planning; development of primary abstracts, posters, clinical papers and scientific supplements; advisory boards; opinion leader development; and management of scientific symposium. Since 2011 she has been the patient representative for St. Catherine’s Hospice and a member of the Supportive Care, Early Diagnosis and Advanced disease (SEDA) Research Group. In February 2012, the newest addition to the family was diagnosed with Tuberous Sclerosis. She became a Trustee and a member of the research committee for the charity in 2014. Leisure interests include “the grandchildren,” Celtic and classical harp, gardening and scuba diving.

Sergiusz Jóźwiak, MD, PhD, Pediatric Neurologist, Chair, Department of Pediatric Neurology, Medical University in Warsaw. Until May 2015 Professor Jóźwiak was head of the Department of Neurology and Epileptology in the largest Polish pediatric hospital: the Children’s Memorial Health Institute in Warsaw. Since June 2015 he chairs the position of Head of newly established Pediatric Neurology Department at Medical University in Warsaw. In years 2009-2014 he was also Poland’s national consultant for child neurology. Professor Jóźwiak’s research focuses mainly on neurocutaneous disorders and epilepsy, infantile spasms in particular. For more than 20 years, he has directed a special program for tuberous sclerosis complex (TSC) patients and determined practical guidelines for TSC management. He also conducts clinical research and directs basic studies on TSC pathogenesis. Other interests include pediatric multiple sclerosis, neuromuscular disorders, and metabolic diseases. In 2009, Professor Jóźwiak received the prestigious Manuel Gomez Award, which was established by the Tuberous Sclerosis Alliance for "creative or pioneering efforts that have appreciably improved either the understanding of the disease or the clinical care available for individuals with tuberous sclerosis complex." In addition, he was the recipient of the Polish Academy of Science award for scientific merit. Professor Jóźwiak’s publications include more than 240 scientific papers, 107 book chapters, and editorships of 32 textbooks.

Chris Kingswood, MSc, FRCP, Nephrologist, Sussex Kidney Unit, Royal Sussex County Hospital, Brighton. Dr Chris Kingswood is a UK-based physician in nephrology at the Sussex Kidney Unit, Brighton & Sussex University Hospitals Trust, and a visiting consultant at St. George’s Hospital. Dr Kingswood’s experience and interest in the tuberous sclerosis complex (TSC) led him to research the natural history and treatment of TSC. He is chief UK investigator for the Exist-2 and TOSCA studies. He also helped set up the multidisciplinary Tuberous Sclerosis Clinic at St. George's Hospital.

Darcy Krueger, MD, PhD, Associate Professor of Clinical Pediatrics and Neurology, Director, Tuberous Sclerosis Clinic, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Dr Krueger is Director of the Tuberous Sclerosis Clinic, Associate Professor of Clinical Pediatrics and Neurology, and Associate Director of Research in Neurology at Cincinnati Children’s Hospital Medical Center. He also was a founding member of the Tuberous Sclerosis Complex Clinical Research Consortium and served as its first director from 2011 to 2013 and continues as a member of its Senior Advisory Board. Dr Krueger received PhD and MD degrees from Saint Louis University in 2000 and 2002, respectively, and completed a combined residency in pediatrics, neurology, and child neurology at Cincinnati Children's Hospital Medical Center (CCHMC) and the University of Cincinnati College of Medicine in 2007. Active preclinical, translational, and clinical research projects with both CCHMC and external collaborators are aimed at better understanding the underlying molecular mechanisms involved in TSC disease pathogenesis and treatment outcome. Dr Krueger was a leading investigator for the landmark clinical trial using mTOR inhibitors to treat subependymal giant cell astrocytomas that led to the first ever FDA-approved treatment for TSC in 2010. Currently, Dr

Krueger is principal investigator, project leader, or major co-investigator for multiple TSC-focused studies of the TSC Clinical Research Consortium funded by the National Institutes of Health, including early identification of TSC infants at-risk for autism and epilepsy (TSC Autism Center of Excellence Network and TSC Epilepsy Biomarkers Study), and evaluating common mechanisms of autism in rare genetic syndromes linked to the mTOR pathway (Rare Diseases Consortium Research Network for Developmental Synaptopathies).

David Kwiatkowski, MD, PhD, Harvard Medical School, Brigham and Women’s Hospital, an Dana Farber Cancer Institute. David J. Kwiatkowski is a Professor of Medicine and Senior Physician at Harvard Medical School, Brigham and Women’s Hospital, and Dana Farber Cancer Institute. He has worked on the genetics, basic and translational research on TSC for more than 25 years. He was the lead editor for the worldwide reference book on this disorder, Tuberous Sclerosis Complex: Genes, Clinical Features, and Therapeutics (Wiley, 2010). He is a past recipient of the TS Alliance’s Gomez Award and Program Leader for the Dana Farber Harvard Cancer Center Cancer Genetics Program.

Gina Lee, PhD, Postdoctoral Fellow, Department of Pharmacology, Weill Cornell Medical College. Gina Lee is a postdoctoral fellow in John Blenis lab at Weill Cornell Medical College (WCMC). During her graduate study at KAIST and Seoul National University, she characterized novel cell growth regulating genes using a Drosophila genetic model system. The results of which were published in several peer-reviewed journals. She also received travel and poster awards from international cancer research conferences including FASEB and Gordon Research Conferences. During her postdoctoral training, she aimed to conduct in-depth biochemical and cell biological mechanistic studies to identify potential targets for drug development and therapy. For more than 25 years, the Blenis lab has focused on investigating Ras/ERK/RSK and PI3K/mTORC1/S6K signaling-associated questions, especially in the context of tumor biology. Their lab is uniquely positioned to identify novel mTORC1 and ERK-MAPK signaling processes from recent large-scale phosphoproteomic screen efforts. She found that mRNA splicing machinery is controlled by mTORC1 signaling and identified cell growth and metabolism regulating genes as downstream targets. She has published a paper studying the regulation of tumor metabolism by mTORC1-mediated mRNA translation. Finally, the Blenis lab collaborates with the world’s experts in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), which will help her apply her tools to patient-derived cells and translate basic research results to more preclinical and clinical studies.

Brendan Manning, PhD, Professor, Harvard University’s School of Public Health (HSPH), Faculty Member of the Dana-Farber/Harvard Cancer Center, Boston, MA. Following receipt of his PhD from Yale in 2000, Dr Manning joined the laboratory of Lewis Cantley at Harvard Medical School for his postdoctoral research. While there, he discovered that the tuberous sclerosis complex (TSC) tumor suppressors were the molecular link between the PI3K and mTOR pathways, thereby connecting a signaling pathway activated in the majority of human cancers to a nutrient-sensing pathway that controls cell growth and metabolism. In 2004, he joined the faculty of the then newly established Department of Genetics and Complex Diseases at HSPH to continue

research at the interface of tumors and metabolism. Ongoing studies in the Manning laboratory are delineating how the mTOR signaling network coordinates nutrient availability with cellular metabolism and how dysregulation of this network contributes to cancer and other diseases, including TSC and lymphangioleiomyomatosis (LAM). Dr Manning is also Director of the PhD Program in Biological Sciences in Public Health at Harvardâ&#x20AC;&#x2122;s Graduate School of Arts and Sciences and serves on the Board of Directors of the Tuberous Sclerosis Alliance and the Scientific Advisory Board of the LAM Foundation.

Frank McCormack, MD, Professor of Medicine, University of Cincinnati, OH. Dr McCormack is Taylor Professor and Director of the Division of Pulmonary and Critical Care Medicine at the University of Cincinnati. He received his training in Internal Medicine at the University of Michigan and completed his Pulmonary and Critical Care Medicine Fellowship at the University of Colorado. He has an active NIH and VA Merit funded research program focused on the role of the alveolar epithelium in innate immunity, rare disease pathogenesis and heritable pulmonary fibrosis. His clinical interest is interstitial lung disease, especially genetic lung disorders such as lymphangioleiomyomatosis.

David Mowat, MBBS, MRCGP, DRACOG, FRACP, Senior Staff Specialist, Clinical Geneticist, Sydney Children's Hospital, Sydney. David has a specific interest in the diagnosis and management of different causes of intellectual disability and autism. He has been the co-director of the multidisciplinary Tuberous Sclerosis clinic based at Sydney Childrenâ&#x20AC;&#x2122;s Hospital since its commencement in 2005. The clinic follows about 200 individuals with TSC and their families.

Hope Northup, MD, Geneticist and Pediatrician, Director, TSC Clinic, The University of Texas Medical School at Houston, Houston, TX. Hope Northrup, MD, is a Medical Geneticist and Pediatrician who began working on tuberous sclerosis complex (TSC) during her Medical Genetics training in 1987. She has cared for numerous patients with TSC and their families as well as contributing to research and education about TSC. Her research spans the spectrum in discoveries about TSC beginning with gene linkage, progressing on to gene cloning and studies understanding the molecular mechanisms of the TSC-causing genes now leading to clinical drug trials for treatment of the disease. Dr. Northrup has published many scientific/medical articles about TSC. She has been actively involved with the Tuberous Sclerosis Alliance (TS Alliance) throughout her career providing presentations at national and regional educational meetings to further aid families in understanding TSC. Dr. Northrup was delighted to be chosen as the Co-Chair of the 2012 TSC Consensus Conference that resulted in updated diagnostic criteria for TSC as well as defining new surveillance and management guidelines for TSC that set the standard of care for TSC patients. Dr. Northrup is the founder and co-director of the TSC Center of Excellence at the University of Texas and Memorial Hermann Hospital in Houston, Texas.

Iris Overwater, MSc, PhD Researcher, Department of Pediatric Neurology, ENCORE-TSC Expertise Center, Erasmus Medical Centre. Iris Overwater’s PhD project is aimed at studying the underlying causes and possible treatment of epilepsy and intellectual disability in children with TSC. In addition to recently finishing a trial on mTOR inhibitors for epilepsy treatment, she is currently coordinating a trial to investigate the effect of mTOR inhibitors on cognitive development and behavior in children with TSC.

Carmen Priolo, MD, PhD, Instructor in Medicine/Junior Faculty member, Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA. Carmen is a physician-scientist who completed her specialty training in Medical Oncology at the University of Messina and the Regina Elena Cancer Institute (Rome, Italy) and her post-doctoral training at the Dana-Farber Cancer Institute (Boston, USA). Dr Priolo’s research interests include patient-derived pre-clinical models of cancer (primary cell cultures and human tumors implanted in mice) and the links between genetics and metabolism in cancer. Dr Priolo’s current research goal is to uncover cellular metabolic abnormalities that can be targeted therapeutically and used to improve diagnostics in TSC and LAM.

Steven L. Roberds, PhD, Chief Scientific Officer, Tuberous Sclerosis Alliance, Silver Spring, MD. Steve Roberds has led the scientific and clinical efforts of the TS Alliance since June 2011. Under his leadership, the organization’s research funding strategy emphasizes career development of young investigators while strengthening translational science — biomarkers, drug screening, and clinical research — to bring forward innovative treatments for TSC. During his first year with the organization, the TS Alliance played a critical role in launching a TSC Clinical Research Consortium that initiated two clinical studies in early 2013. Also in his first year, the TS Alliance developed and implemented the first TSC Clinical Consensus Conference since 1998 to update recommendations for the diagnosis, surveillance and management of TSC. During his 16-year career in the pharmaceutical industry prior to joining the TS Alliance, Steve successfully led global, multi-disciplinary research teams to rapidly advance projects along the continuum from idea conception to designing Phase 2 proof-of-concept studies in humans. He received his PhD in Pharmacology from Vanderbilt University and his Bachelor of Science degree in Pharmacy from Butler University.

Kari Luther Rosbeck, President and CEO, Tuberous Sclerosis Alliance, Silver Spring, MD. Kari Luther Rosbeck, President and CEO, has been with the TS Alliance since June 2001 and is responsible for the overall management and administration of the organization. During her tenure as President and CEO since November 2007, the TS Alliance established a Drug Screening Program to foster collaboration with industry and academia in order to move treatments for tuberous sclerosis complex (TSC) forward in a more expedited way and played an active role in the development of the first clinical trial for neurocognition in TSC. Because of Kari’s leadership, the organization has taken

an active role in educating the TSC community about clinical trials to diminish the time for recruitment. Kari previously developed the TS Alliance’s national volunteer outreach program and was responsible for fund development. She developed and implemented a vast national network of more than 30 volunteer branches called Community Alliances; increased volunteer participation from 90 individuals to more than 2,000; and through her involvement in special events and major gift fundraising, helped increase the organization’s annual revenue by more than 50 percent. Kari serves on the Board of Directors for the National Organization for Rare Diseases (NORD), the American Thoracic Society Public Advisory Roundtable and has been involved in nonprofit fundraising and volunteer management for 20-plus years.

Mustafa Sahin, MD, PhD, Pediatric Neurologist, Director, TSC Program and Translational Neuroscience Center, Children's Hospital, Boston, MA. Mustafa Sahin, MD, PhD is Associate Professor of Neurology at Harvard Medical School. After receiving his BS degree from Brown University, Dr Sahin earned an MD and a PhD from Yale University School of Medicine. He completed a pediatrics residency at Children's Hospital of Philadelphia and a child neurology residency at Boston Children's Hospital. Dr Sahin directs the Multidisciplinary Tuberous Sclerosis Program and the Translational Neuroscience Center at Boston Children’s Hospital. His lab has developed evidence that the TSC/mTOR pathway plays crucial roles in axon specification, guidance, myelination, regeneration and more recently in cerebellar circuitry and circadian rhythms. These experiments support the notion that neurological defects in TSC-deficient mice can be blocked by postnatal mTORC1 inhibition. This work underpins the design of a clinical trial investigating the effect of an mTORC1 inhibitor on neurocognition in individuals with TSC.

Julian Sampson, DM, FRCP, FMedSci, Geneticist, Professor and Head of Department, Medical Genetics, Institute of Medical Genetics, Wales College of Medicine, Cardiff University, Cardiff, Wales. Professor Sampson is a clinical geneticist working at the School of Medicine, Cardiff University, UK where he is Head of the Institute of Medical Genetics and Director of the Division of Cancer and Genetics and with the NHS All Wales Medical Genetics Service. His interests are in the clinical and molecular genetics of disease and tuberous sclerosis is a major focus for his research. He led the international consortium that identified TSC2 and worked as a member of the consortium that identified TSC1. He made the first reports of contiguous deletion of TSC2 and PKD1 in patients with tuberous sclerosis and polycystic kidney disease and undertook genotype-phenotype studies including the first report of differential severity of TSC1 and TSC2-associated disease. He subsequently developed a team of researchers in Cardiff working on the cell biology of TSC, preclinical trials in transgenic mouse models and clinical trials including the TESSTAL and TRON trials of mTOR inhibitors in angiomyolipoma and neurocognitive function in TSC. His group provided DNA diagnostics for TSC to more than 20 countries and made a major contribution to the catalogue of genetic variation on the TSC1 and TSC2 mutation databases.

Jadwiga Schreiber, PhD, MSc, Postdoc, Erasmus Medical Center. Jadwiga Schreiber obtained her PhD in Developmental Neurobiology from the Max Delbrück Center (MDC) for Molecular Medicine/Free University in Berlin, Germany. Since 2010 she is a postdoc in Professor Ype Elgersma’s lab at the Neuroscience Department of the Erasmus Medical Center in Rotterdam, the Netherlands. In Elgersma’s lab she leads the Tuberous Sclerosis Complex (TSC) research projects. She and her team are in particular interested in cellular and network changes occurring after TSC-loss, which lead to onset of epilepsy and in understanding which therapeutic strategies could be efficient in interfering with these epileptogenic processes. By making use of unique mouse models for TSC and by applying highly sensitive techniques including electrophysiology and EEG-recordings they investigate physiological processes of epileptogenesis and modulatory effects of drugs on these processes. Elgersma’s TSC-team collaborates closely with Dr Marie Claire de Wit, neurologist and the head of the TSC clinic at the ENCORE expertise center for neurodevelopmental disorders in the Sophia Children’s Hospital in Rotterdam, and with Dr Mark Nellist from the Department of Clinical Genetics, Erasmus Medical Center, The Netherlands.

Wei Shi, PhD, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California. Wei Shi, PhD, is a tenured Associate Professor in the Developmental Biology and Regenerative Medicine Program at the Saban Research Institute of Children’s Hospital Los Angeles and in the Department of Surgery at Keck School of Medicine, University of Southern California. Dr Shi obtained his PhD degree at University of Tennessee, Memphis. He had his postdoctoral training on lung development and cell/molecular biology at Children’s Hospital Los Angeles before he was promoted to be on the faculty at USC. He has been studying growth factor signaling in regulating lung development, injury repair and regeneration with the support of NIH and other funding agencies for over a decade. In the past a few years, Dr Shi’s research focuses on lung mesenchymal stem/progenitor cells in lung development and diseases using several transgenic mouse models. One of these projects is developing lung mesenchyme-specific Tsc2 conditional knockout mouse model in collaboration with Dr. Lisa Henske.

Jayne Spink, PhD, CEO, Tuberous Sclerosis Association. Jayne Spink (Chief Executive) joined the TSA in January 2012. Jayne has a BSc and PhD in Genetics and completed 6 years of post-doctoral research (biomedical – Reading University, Royal Postgraduate Medical School & Institute of Psychiatry) prior to joining the Department of Health in 1998. Jayne attained Principal Scientist grade at DH and was Head of Genetic Science Policy, Safety and Regulation until 2005 when she left to join the National Institute of Clinical Excellence (NICE) as an Associate Director. From 2007-2011 Jayne was Director of Policy & Research with the Multiple Sclerosis Society. Jayne has a long-standing and active interest in medicines regulation, patient involvement in regulatory decision making and access to healthcare.

Andrew Tee, PhD, BSc, Senior Lecturer, Institute of Cancer and Genetics, Cardiff University. Andrew Tee, PhD, BSc, trained at Dundee and Harvard University, and his main research strengths

include dissection of novel cell signalling mechanisms relating to TSC and mTORC1. He first became interested in TSC at Harvard (in Professor John Blenis’s lab as an EMBO travelling fellow) and made several landmark discoveries regarding delineation of the TSC1/2->Rheb->mTORC1 signalling pathway. Within the United Kingdom (UK), Dr. Tee is a leading expert on Rheb and has an established lab that works on TSC and mTORC1. He later uncovered that TSC1/2-mTORC1 was upstream of STAT3, and STAT3 is necessary for gene expression of HIF1α. This is an important contribution that uncovers a novel signalling mechanism that is directly linked to angiogenic signalling and metabolic transformation. Dr. Tee recently discovered that nelfinavir and chloroquine selectively kills TSC2-deficient cell lines, indicating that targeting endoplasmic reticulum stress in combination with lysosomal inhibition could be a viable strategy to treat TS patients. He has key research interests in autophagy regulation and a TSC-related genetic disorder, called Birt-HoggDubé.

Elizabeth Thiele, MD, PhD, Pediatric Neurologist/ Epileptologist, Director, The Carol and James Herscot Center for Children and Adults with TSC, Massachusetts General Hospital, Department of Neurology, Boston, MA. Elizabeth A. Thiele is Director of the Pediatric Epilepsy Program and Director of the Herscot Center for Tuberous Sclerosis Complex at Massachusetts General Hospital. Dr Thiele is also Professor of Neurology at Harvard Medical School. Dr Thiele serves on the Board of Directors of the Tuberous Sclerosis Alliance and is also a member of the American Epilepsy Society, the Child Neurology Society, and on the Scientific Advisory Boards of the Charlie Foundation and the Angelman Syndrome Foundation. Dr Thiele directs clinical research efforts in the neurologic aspects of Tuberous Sclerosis Complex including epilepsy, autism and mental health issues. She also works on improved dietary therapies for epilepsy, including Low Glycemic Index Treatment. Dr Thiele has received awards for teaching in residency programs at Massachusetts General Hospital and the Longwood Neurology Program and for research mentoring from the Harvard MIT Health Sciences and Technology program. She also received the Gomez Award from the Tuberous Sclerosis Alliance in 2008, the inaugural John M. Freeman award in 2014 and the Infantile Spasms Hero Award from the Child Neurology Foundation in 2014. Dr Thiele received her AB from Washington University, St. Louis in 1983 and her MD and PhD from The Johns Hopkins University School of Medicine in 1990. She completed a residency in pediatrics at The Johns Hopkins Hospital in Baltimore and a residency in child neurology with the Longwood Program and Children’s Hospital in Boston. In addition, she completed a neuroscience fellowship at Children’s Hospital in Boston. Dr Thiele is board certified in neurology with a special qualification in child neurology, and in clinical neurophysiology.

Sophie Thomson, PhD, Post-doctoral Researcher, Centre for Integrative Physiology, Hugh Robson Building, University of Edinburgh. Dr Sophie Thomson is a post-doctoral researcher at the University of Edinburgh. Sophie was awarded her PhD from the University of Edinburgh in 2014, for work on Spinal Muscular Atrophy. In 2014, Sophie joined the lab of Dr Emily Osterweil, where she researches protein synthesis disregulation in autism spectrum disorders, such as Tuberous Sclerosis (TSC) and Fragile X Syndrome (FXS).

Kandice Varcin, PhD, Postdoctoral Fellow, Boston Children’s Hospital, Harvard Medical School, Boston, MA. Kandice Varcin, PhD, is a postdoctoral research fellow in the Laboratories of Cognitive Neuroscience at Boston Children’s Hospital, Harvard Medical School. Kandice also completed a Bachelor of Psychology (Hons) and a Masters of Psychology (Clinical) at the University of New South Wales in Sydney, Australia.

Philippa Ward, Community Speaker, UK. Philippa Ward lives in York with her husband, David, and two young sons, Benjamin (5) and Thomas (4). A modern languages graduate, she works full time as a Communications Specialist for a pharmaceutical company, while David is a sports journalist at the local BBC radio station. Philippa and David first became aware of tuberous sclerosis complex in February 2012 when their youngest son, Thomas, was diagnosed with the condition. He was just five months old at the time. Thomas has tubers in his brain, which have caused epilepsy and significant developmental delay. He is non-verbal, has autistic tendencies and sensory processing disorder, and is currently on three different medicines to help control his seizures. He is on the Exist-3 clinical trial and is responding well to treatment with everolimus. He attends the local special school where they have access to all the kinds of people Thomas needs to help him fulfill his potential (speech therapists, physiotherapists, occupational therapists to name but a few). Since Thomas’s diagnosis, life has changed immeasurably for Philippa, David and Ben, but they are grateful to have found the Tuberous Sclerosis Association. The support the TSA offers to people at all stages of their TS journey and the TSA network the family has been able to tap into have been invaluable. Philippa is an advocate of the TSA and supports the charity however she can, including helping run the Yorkshire support group. She is currently training for the 13-mile Great North Run, which takes place this Sunday (13 September). At the time of going to print, she had raised more than £1,300 in donations for the charity. In her spare time, Philippa enjoys knitting, reading and watching the world go by with a good cup of coffee.

Michael Wong, MD, PhD, Pediatric Neurologist/ Epileptologist, Director, TSC Clinic, Washington University School of Medicine, St. Louis, MO. Dr Wong is the Allen P. and Josephine B. Professor of Pediatric Neurology at Washington University School of Medicine in St. Louis and Director of the Washington University Tuberous Sclerosis Clinic at St. Louis Childrenâ&#x20AC;&#x2122;s Hospital. He is active in basic science research in epilepsy and tuberous sclerosis complex, funded by the National Institutes of Health, Department of Defense, and the Tuberous Sclerosis Alliance. He has published more than 100 research papers, reviews, and commentaries.

Index of speaker presentations: Martina Bebin John Blenis John J. Bissler Patrick Bolton Juxiang Cao

Petrus de Vries David N. Franz Elizabeth P. Henske Rebecca Ihire Anna Jansen Sergiusz Jóźwiak Chris Kingswood Darcy Krueger David Kwiatkowski Gina Lee Brendan Manning Frank McCormack Iris Overwater

Carmen Priolo Mustafa Sahin Julian Sampson Jadwiga Schreiber Wei Shi Andrew Tee

Sophie Thomson Kandice Varcin Michael Wong

The potential for early intervention to prevent the onset of epilepsy in TSC TSC renal failure and cystic disease mTOR, metabolism and therapeutic vulnerabilities TS2000: the impact of TSC in early years The TSC protein complex regulates melanogenesis through mTOR and GSK3ß, explaining the pathogenesis of TSC ‘white spots’ The neuropsychiatric burden and methods that work to lessen that burden mTOR inhibitors, where next Treatments on the horizon for TSC and LAM Neural stem cell patterning and the origin of SEGAs Burden of TSC & health economics EPISTOP and other new data on early intervention for epilepsy in TSC mTOR inhibitors, where are we now Early treatment in TSC with vigabatrin and mTOR inhibitors: Enthusiasm, caution, or both? Genetics and potential for personalized medicine mTORC1-mediated regulation of mRNA biogenesis in TSC Alternative therapeutic targets for TSC uncovered downstream of mTORC1 mTOR inhibitors and beyond in the lung Sirolimus treatment in children with Tuberous Sclerosis Complex and intractable epilepsy: a randomized controlled trial Aberrant lipid metabolism as a TSC biomarker The potential for early intervention to prevent development of autism Genetics and early diagnosis of TSC Toward a better understanding of epilepsy A novel mouse model of LAM Combination therapy with nelfinavir: mechanism of drug action and selective targeting of Tuberous Sclerosis Complex 2 deficient cells Beyond mTOR Social visual perception in tuberous sclerosis complex New concepts on the genesis of epilepsy in TSC: looking beyond mTOR

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Session 1 â&#x20AC;&#x201C; Early diagnosis and the impact of genetics on diagnosis Thursday 10 September 2015 14:00 - 15:30 Windsor and Georgian Session Chair: Hope Northup, MD, Geneticist and Paediatrician, Director, TSC Clinic, The University of Texas Medical School at Houston, Houston, TX. 14:00 - 14:15 Community Speaker: Philippa Ward, UK. Session presentations: 14:15 - 14:45 Genetics and early diagnosis of TSC Julian Sampson, DM, FRCP, FMedSci, Geneticist, Professor and Head of Department, Medical Genetics, Institute of Medical Genetics, Wales College of Medicine, Cardiff University, Cardiff, Wales. 14:45 - 15:15 Early treatment in TSC with vigabatrin and mTOR inhibitors: Enthusiasm, caution, or both? Darcy Krueger, MD PhD, Associate Professor of Clinical Pediatrics and Neurology, Director, Tuberous Sclerosis Clinic, Cincinnati Childrenâ&#x20AC;&#x2122;s Hospital Medical Center, Cincinnati, OH. 15:15 - 15:30 Neural stem cell patterning and the origin of SEGAs Rebecca Ihire, PhD, Assistant Professor, Cancer Biology and Neurological Surgery, Vanderbilt University, Nashville, TN.

Genetics and early diagnosis of TSC Julian Sampson, DM, FRCP, FMedSci, Geneticist, Professor and Head of Department, Medical Genetics, Institute of Medical Genetics, Wales College of Medicine, Cardiff University, Cardiff, Wales. Successes in the targeted medical treatment of TSC-associated growth manifestations including SEGA, angiomyolipoma, lymphangioleiomyomatosis and facial angiofibromas using mTOR inhibitors represent a stunning advance and a paradigm for the treatment of Mendelian disorders. Efficacy in relation to seizures, neurodevelopmental and neurocognitive manifestations of TSC is under investigation with the hope that mTOR inhibitors will also have benefits in these top priority areas. The responses seen to mTOR inhibitors in patients with TSC have been durable, but partial and reversible. Of great interest, therefore, is the potential for earlier and preventive approaches using these agents. Because of variable severity and the time course of emergence of the TSC phenotype, concerns have been raised about the balance of risks and benefits that might be associated with very early intervention with mTOR inhibitors. Patients with TSC2 mutations are at higher risk of poor outcomes than those with TSC1 mutations, although some specific TSC2 missense mutations are associated with mild phenotypes. Genotype-based selection may be one way of optimising the balance of risks and benefits involved for very young patients in prevention trials.

Early treatment in TSC with vigabatrin and mTOR inhibitors: Enthusiasm, caution, or both? Darcy Krueger, MD PhD, Associate Professor of Clinical Pediatrics and Neurology, Director, Tuberous Sclerosis Clinic, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA Numerous studies have demonstrated the efficacy of vigabatrin for the treatment of infantile spasms and partial-onset seizures in TSC. Also there is now clear evidence that establishes the effectiveness of sirolimus and everolimus to treat many additional clinical manifestations of TSC. Accordingly and appropriately, vigabatrin and mTOR inhibitors are included in the published surveillance and treatment recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Group. As increasingly numbers of TSC patients are being diagnosed prenatally or first years of life, symptomatic treatment not unexpectedly has extended down to earlier ages and stages of the disease. Early symptomatic treatment and even a potential shift from symptomatic treatment to presymptomatic, disease-modifying or disease-prevention uses raise additional need to assess the safety of early and potential long-term exposure of vigabatrin and mTOR inhibitors in infants with TSC. The focus of this presentation is to summarize and discuss published and unpublished experience with use of these agents in infants and children with TSC, with particular emphasis on safety.

Neural stem cell patterning and the origin of SEGAs Rebecca Ihire, PhD, Assistant Professor, Cancer Biology and Neurological Surgery, Vanderbilt University, Nashville, TN. Most TSC patients develop subependymal nodules – small, benign tumors surrounding the ventricles – but only a smaller subset go on to develop SEGAs, which are much larger and require clinical intervention. Both small and large tumors are thought to arise from the same cells - the neural stem and progenitor cells which exist in a specialized niche around the lateral ventricles. However, unlike subependymal nodules, SEGAs are well-known to present in a specific, ventral location within this niche. During the past decade, research has revealed that neural stem cells, rather than being a

single homogenous mass, are arranged in a tightly patterned mosaic. The position of a stem cell predicts the types of neurons it will make, and this â&#x20AC;&#x153;positional identityâ&#x20AC;? is retained even upon culture or transplantation. We hypothesized that the location-specific patterning of healthy neural stem cells might drive the location-specific presentation of SEGAs â&#x20AC;&#x201C; that is, that ventral neural progenitors might be uniquely susceptible to the effects of Tsc1 or Tsc2 mutation. We are now directly testing this model by disrupting the Tsc2 gene in localized populations of neural stem cells in the mouse. I will present preliminary findings which suggest that neural stem cells in a specific subregion of the niche are a likely cell of origin for SEGAs. If ventral cells are uniquely capable of generating these tumors, this would suggest new avenues for targeting or preventing these tumors without impacting the stem cell niche as a whole.

Session 2 â&#x20AC;&#x201C; Impact and gaps of mTOR inhibitor treatment of TSC Thursday 10 September 2015 16:00 - 17:30 Windsor and Georgian Session Chair: Steven L. Roberds, PhD, Chief Scientific Officer, TS Alliance, Silver Spring, MD. Session presentations: 16:00 - 16:30 mTOR inhibitors, where are we now Chris Kingswood, MSc FRCP, Nephrologist, Sussex Kidney Unit, Royal Sussex County Hospital, Brighton. 16:30 - 16:45 Beyond mTOR: differential mRNA translation in autism spectrum disorders Sophie R. Thomson, PhD, Post-doctoral Researcher, Centre for Integrative Physiology, Hugh Robson Building, University of Edinburgh. 16:45 - 17:15 mTOR inhibitors, where next David N. Franz, MD, Pediatric Neurologist, Founding Director, TSC Clinic, Children's Hospital Medical Center, Cincinnati, OH. 17:15 - 17:30 Toward a better understanding of epilepsy Jadwiga Schreiber, PhD, MSc, Postdoc, Erasmus Medical Center.

mTOR inhibitors, where are we now Chris Kingswood, MSc FRCP, Nephrologist, Sussex Kidney Unit, Royal Sussex County Hospital, Brighton. The lifetime risk of a serious health problem over in TSC is high; SEGA in 10-15%, renal bleed or CKD in 40-50%, symptomatic LAM in 5-40%, refractory epilepsy in 40%, disfiguring rash in 75% and TAND in 90%. A series of pilot and phase two studies have shown that mTOR inhibitors are efficacious in ameliorating many of these problems. They are now licensed for treatment of SEGA, renal AML and LAM following phase 3 studies. Other studies to determine their benefits in refractory epilepsy, neurocognition and ASD are underway. The pioneering studies in Cincinnati, TESSTAL and the USA multicentre study have demonstrated mTORIs halt growth and shrink most SEGAs and AMLs. One Cincinnati study has also suggested remarkable efficacy in refractory epilepsy. Exist-1 and Exist-2 have confirmed the effects on SEGA and AML and that this effects is durable over 3-5 years. They have also shown significant improvement in angiofibromas, as have studies of topical treatment. The Exist-1 and 2 studies have revealed which adverse events are true side effects. Serious side effects are rare and the prevalence of all side effects diminishes over the first 3 years of therapy. Side effects of special interest that require long term monitoring to assess their significance include; amenorrhoea, issues of fertility and sexual maturation, hypophosphataemia and proteinuria. Further studies are needed to determine the optimal dosing regime including; ideal starting dose, whether induction / maintenance doses and daily or intermittent doses result in the best balance of efficacy versus risk. Optimal duration of treatment and whether adjunctive treatment (Vigabatrin, ketogenic diet, or agents to promote TS cell apoptosis) enhances efficacy have yet to be studies. We also need to establish if long-term efficacy is improved by starting at a younger age or earlier in the evolution of any individual organ involvement. TSC is a systemic disease â&#x20AC;&#x201C; the benefits of treatment should be assessed holistically not just by single organ protection. The results of on-going and future studies will allow us to re-calculate the balance between benefit and risk of mTORIs and determine if we can progress from the era of rescue therapy to prevention with early intervention. Beyond mTOR: differential mRNA translation in autism spectrum disorders Sophie R. Thomson, PhD, Post-doctoral Researcher, Centre for Integrative Physiology, Hugh Robson Building, University of Edinburgh.

Tuberous sclerosis (TSC) is a devastating neurodevelopmental disorder, where a large proportion of patients suffer from epilepsy, intellectual disability and autism. The genes that are mutated that cause TSC (TSC1 or TSC2) play a major role in regulating protein synthesis downstream of the translational regulator mammalian target of rapamycin (mTOR). Previous work with a mouse model of TSC (Tsc2+/) shows that it exhibits dysregulated protein synthesis in the hippocampus, which is believed to contribute to deficiencies in long-term synaptic depression (LTD) downstream of metabotropic glutamate receptor 5 (mGluR5), and in behavioural learning paradigms (Auerbach et al, 2011). Importantly, correcting the protein synthesis, using either an mTOR inhibitor or an mGluR5 positive allosteric modulator (PAM), ameliorates the observed deficits in both mGluR-LTD and in hippocampal learning (Auerbach et al, 2011). Our hypothesis is that dysregulation of protein synthesis in TSC is responsible for synaptic plasticity deficits in TSC, and this can be corrected with either application of an mTOR inhibitor or an mGluR5 PAM. Our goal is to isolate and identify proteins that are aberrantly translated in order to gain insight into the pathological mechanism of TSC, and develop novel treatment strategies for TSC. In order to do this, we have developed a translating ribosome affinity purification (TRAP) assay in CA1 pyramidal neurons to isolate the abnormally translated mRNAs. Using this system, we can define the proteins which are aberrantly translated, and corrected with either an mTOR inhibitor or an mGluR5 PAM. This information will be vital in determining dysregulated signalling pathways that function downstream of mTOR, and could represent new therapeutic targets in TSC.

mTOR inhibitors, where next David N. Franz, MD, Pediatric Neurologist, Founding Director, TSC Clinic, Children's Hospital Medical Center, Cincinnati, OH. mTOR inhibition has provided a new therapeutic option for individuals with TSC with many different types of involvement. The current and possible future uses of these drugs in TSC will be discussed, with an emphasis on practical patient management.

Toward a better understanding of epilepsy Jadwiga Schreiber, PhD, MSc, Postdoc, Erasmus Medical Center. Understanding the molecular and cellular mechanisms underlying epilepsy in TSC is profoundly important for the improvement of existing treatments for TSC-dependent epilepsy and the identification of novel drug candidates, which reduce mTOR hyperactivation. In TSC patients, the precise primary molecular cause of epilepsy is known (a TSC mutation, which results in mTOR activation), however epilepsy represents a complex interplay of primary and secondary effects, which cannot easily be distinguished from each other. To gain insights into the mechanisms underlying TSC induced epileptogenesis, we generated an inducible TSC mouse model. This model has proven to be a unique and very useful tool to identify and distinguish between changes that cause the epilepsy and changes that are consequences of the epilepsy. We are in particular interested in processes occurring prior to epilepsy onset and whether they can be reversed by treatment with mTOR inhibitor, rapamycin, and/or commonly used anti-epileptic drugs (AEDs). Specifically, we want to know if early (preventive) treatment with mTOR inhibitor or AEDs can efficiently lower mTOR activity in the brain and thereby prevent development of epilepsy. In

addition, we aim to determine whether there is an added value of preventive (prior to seizure onset) versus symptomatic (after seizure onset) treatment on seizure outcome. In ongoing experiments we are testing anti-epileptogenic effect of rapamycin, and three commonly used GABA-ergic AEDs including vigabatrin, tigabine and clobazam. With our approach we aim to determine the most critical step in the development of epilepsy and to assess its mTOR-dependency. Our principal goal is to identify effective and safe treatments which reduce mTOR activity in the brain and significantly improve epileptic outcome.

Session 3 – The potential for preventing epileptogenesis in TSC Friday 11 September 2015 08:30 - 10:15 Windsor and Georgian Session Chair: Elizabeth Thiele, MD, PhD, Pediatric Neurologist/Epileptologist, Director, The Carol and James Herscot Center for Children and Adults with TSC, Massachusetts General Hospital, Department of Neurology, Boston, MA. 08:30 - 08:45 Community Speaker: Ron Heffron, PE, Community speaker, USA. Session presentations: 08:45 - 09:15 EPISTOP and other new data on early intervention for epilepsy in TSC Sergiusz Jóźwaik, MD, PhD, Pediatric Neurologist, Chair, Department of Pediatric Neurology, Medical University in Warsaw. 09:15 - 09:45 The potential for early intervention to prevent the onset of epilepsy in TSC E. Martina Bebin, MD, MPA, Pediatric Neurologist/ Epileptologist, Co-Director, TSC Clinic, University of Alabama at Birmingham, Birmingham, AL. 09:45 - 10:15 New concepts on the genesis of epilepsy in TSC: looking beyond mTOR Michael Wong, PhD, Pediatric Neurologist/ Epileptologist, Director, TSC Clinic, Washington University School of Medicine, St. Louis, MO.

EPISTOP and other new data on early intervention for epilepsy in TSC Sergiusz J贸藕waik, MD, PhD, Pediatric Neurologist, Chair, Department of Pediatric Neurology, Medical University in Warsaw. J贸藕wiak S, and EPISTOP Consortium members According to the existing definition, epilepsy requires the occurrence of at least one epileptic seizure. However, it is now widely accepted that clinical seizures are preceded by a latent period of epileptogenesis. This cascade of cellular and molecular events may be triggered by diverse brain insults, including trauma, infection or genetic predisposition, and leads to the formation of hyperexcitable neural networks ultimately resulting in spontaneous epileptiform activity. This process continues with onset of clinical seizures, leading to the development of established, drugresistant epilepsy, and secondary comorbidities. Thus far, studies aimed at understanding the molecular and cellular mechanisms of epileptogenesis have been possible only in animal models. In humans, epileptogenesis studies are difficult because the patients usually present after seizures and little is known about the earlier stages of the disease. There is an urgent need for better understanding of epileptogenesis in humans. Currently, there are no recognized biomarkers enabling the diagnosis of epilepsy before the clinical seizure appear. There is also no established antiepileptogenic strategy or medication to prevent the development of epilepsy and its comorbidities. Introduction of novel antiepileptogenic drugs is not possible due to the poor understanding of epileptogenesis itself and a shortage of specific targets for such drugs. EPISTOP is the first prospective study of epileptogenesis in humans, beginning BEFORE seizures and continuing through age 2+ years, permitting detailed analysis of epilepsy onset, drug-resistant epilepsy, and its comorbidities. To maximize information derived from the study we have chosen homogenous group of patients with prenatal or early infantile diagnosis of Tuberous Sclerosis Complex (TSC). A clinical randomized study of pre-seizure treatment in TSC infants is an important part of the project. The aim of EPISTOP is to examine the risk factors and biomarkers of epilepsy and to identify possible new therapeutic targets to block or otherwise modify epileptogenesis in humans. Biomarker analysis will be performed by a multidisciplinary, systematic approach in three clinical settings: 1/ prospective study of epilepsy development in infants with TSC, including analysis of clinical, neuroimaging, and molecular, blood-derived biomarkers at predefined time points: before the onset of seizures, after the onset of epileptiform discharges on EEG, at seizure onset and at the age of 24 months; 2/ prospective study of blood-based biomarkers in young children with TSC treated with antiepileptic drugs prior to seizure onset in comparison to children treated only after clinical seizures appearance; 3/ analysis of biomarkers of epileptogenesis and drug-resistant epilepsy in brain specimens obtained from patients with TSC who have had surgery for refractory epilepsy and TSC autopsy cases. Clinical data supporting the concept of EPISTOP will be presented. Potential impact of early prevention of epileptogenesis in other epileptic encephalopathies will be discussed.

Acknowledgements: The work reported in this paper was partially supported by the 7th Framework Programme of European Commission within the Large-scale Integrating Project EPISTOP (Proposal No: 602391-2)

The potential for early intervention to prevent the onset of epilepsy in TSC E. Martina Bebin, MD, MPA, Pediatric Neurologist/ Epileptologist, Co-Director, TSC Clinic, University of Alabama at Birmingham, Birmingham, AL. Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease that affects ~1 in 6000 people and the neurological manifestations in TSC are common and in children represent the most disabling problems of the disease, including epilepsy, intellectual disabilities, psychiatric problems and autism. Epilepsy is particularly prevalent, affecting about 80% of individuals with TSC with over 60% having seizures that are severe and refractory. Almost half of TSC infants develop epileptic spasms, which is associated with poor neurological prognosis. Increasingly, patients are diagnosed with TSC at a young age before the onset of epilepsy due to non-neurological findings, such as cardiac rhabdomyomas. The earlier diagnosis of TSC provides a unique opportunity to identify and validate a biomarker for epilepsy. A predictive biomarker would allow earlier intervention that may alter or curtail epileptogenesis and its adverse effects. A recent open-label study suggests treating TSC patients with an abnormal EEG prior to onset of epileptic spasms with vigabatrin may improve neurological outcome (Jozwiak 2011). An earlier retrospective study reported similar benefit with early treatment (Bombardieri 2010). Nonetheless, the use of clinical EEG as a reliable biomarker of epilepsy has not been rigorously validated and has been limited to retrospective analyses which are subject to referral, recording, and recall biases. The presentation will discuss the interim results of the NIH funded study entitled Potential EEG biomarker and antiepileptogenic strategies for epilepsy and TSC. This study provides a unique opportunity to document the evolution of epileptogenesis, development of clinical seizures, and the utility of EEG as an early biomarker for epilepsy in TSC. It is a multicenter prospective observational study which recruited TSC infants who were seizure-free and on no antiepileptic drugs at the time of enrollment. All subjects underwent serial physical examinations and video-EEG throughout the study. Parental education on seizure recognition was completed at initial enrollment. Once seizure onset occurred, standard of care was applied, and subjects were followed until 24 months. Developmental assessments were completed at 6, 12 and 24months of age. Forty patients were enrolled, 28 over age 12 months with completed EEG evaluation at the time of the interim analysis. Of those, 19 (67.8%) developed seizures. Epileptic spasms occurred in 10 (52.6%), focal seizures occurred in 5 (26.3%), generalized tonic-clonic seizure in 1 (5.3%), and a combination of epileptic spasms and focal seizures in 3 (15.7%). Fourteen infants (73.6%) had the first emergence of epileptiform abnormalities on EEG at mean age of 4.3 months, preceding seizure onset by an average of 2.8 months. Hypsarrhythmia was not evident on any of the subjects EEGs prior to onset of epileptic spasms. All children with epileptiform discharges subsequently developed epilepsy (100% positive predictive value), and the negative predictive value for not developing epilepsy after a normal EEG was 64%. Based on the interim analysis we concluded that serial routine EEGs in TSC infants is a feasible strategy to identify those at high risk for epilepsy.

New concepts on the genesis of epilepsy in TSC: looking beyond mTOR Michael Wong, PhD, Pediatric Neurologist/ Epileptologist, Director, TSC Clinic, Washington University School of Medicine, St. Louis, MO. Epilepsy is a common, disabling neurological manifestation of Tuberous Sclerosis Complex (TSC), occurring in up to 90% of patients and typically involving multiple seizure types, including infantile spasms. TSC patients with epilepsy are often refractory to currently available antiseizure medications and are frequently not appropriate candidates for epilepsy surgery due to the multifocal nature of the seizures, leaving many patients with life-long intractable epilepsy. Furthermore, most current antiseizure medications are symptomatic therapies, but do not have antiepileptogenic or disease-modifying effects to prevent the development or progression of epilepsy. mTOR inhibitors and vigabatrin are being investigated as potential disease-modifying agents for epilepsy in TSC, but have significant side effects. So, novel treatments are needed to alleviate intractable epilepsy in TSC and prevent the emergence of epilepsy in the first place. Development of more effective therapies for epilepsy depends on identifying and targeting specific mechanisms that cause epileptogenesis in TSC on the pathological, cellular, and molecular levels. On the pathological level, cortical tubers may be central to generating seizures in many patients; however, there is accumulating evidence that non-tuber cellular and molecular abnormalities may also promote epileptogenesis. On the cellular level, neuronal abnormalities not surprisingly may promote seizure generation in TSC, but astrocytes and microglia have also been implicated in the pathophysiology of epilepsy in TSC. On the molecular level, animal models have demonstrated that the mTOR pathway is critical for epileptogenesis in TSC and that mTOR inhibitors have antiepileptogenic effects in these models. Beyond mTOR, a variety of downstream or independent molecular mechanisms have also been implicated in epileptogenesis, related to such processes as inflammation, metabolism, and synaptic plasticity. Rational targeting of these mechanisms involved in different aspects of epileptogenesis in TSC has the potential to develop novel, more effective antiseizure and antiepileptogenic therapies.

Session 4 – Breakthroughs in the understanding of lung and renal manifestations of TSC Friday 11 September 2015 10:45 - 12:30 Windsor and Georgian Session Chair: Chris Kingswood, MSc, FRCP, Nephrologist, Sussex Kidney Unit, Royal Sussex County Hospital, Brighton. Session presentations: 10:45 - 11:00 Aberrant lipid metabolism as a TSC biomarker Carmen Priolo, MD, PhD, Instructor in Medicine/Junior Faculty member, Division of Pulmonary and Critical Care Medicine , Brigham and Women’s Hospital and Harvard Medical School, Boston, MA. 11:00 - 11:30 A novel mouse model of LAM Wei Shi, PhD, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California. 11:30 - 12:00 mTOR inhibitors and beyond in the lung Frank McCormack, MD, Professor of Medicine, University of Cincinnati, OH. 12:00 - 12:30 TSC renal failure and cystic disease John Bissler, MD, Professor of Pediatrics and FedEx Chair of Excellence, University of Tennessee Health Science Center, and Director, Tuberous Sclerosis Center of Excellence and Division of Nephrology, St. Jude Children’s Research Hospital and Le Bonheur Children’s Hospital.

Aberrant lipid metabolism as a TSC biomarker Carmen Priolo, MD, PhD, Instructor in Medicine/Junior Faculty member, Division of Pulmonary and Critical Care Medicine , Brigham and Womenâ&#x20AC;&#x2122;s Hospital and Harvard Medical School, Boston, MA. Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease characterized by benign tumors of the brain, skin, heart, and kidneys. About 30-80% of women with TSC develop lymphangioleiomyomatosis (LAM), a cystic destruction of the lung associated with proliferation of smooth muscle actin-positive cells. LAM can also occur as a sporadic disorder, characterized by somatic mutations of the TSC genes.TSC2 deficiency leads to activation of mTORC1, which stimulates cell growth and metabolism. Clinical trials of TSC and LAM with the mTORC1 inhibitor rapamycin showed partial response of tumor lesions and stabilization of pulmonary function; however, both tumor growth and pulmonary function decline resumed when treatment was stopped. These studies highlight more efficient therapies as an unmet need in TSC and LAM. In this context, the availability of sensitive biomarkers of disease progression and therapeutic response would streamline the design of clinical trials in TSC. We found enhanced choline phospholipid metabolism in TSC2-deficient cells relative to controls, with diagnostic and therapeutic implications. Through an unbiased plasma metabolite profile, we also found that four choline lysophospholipids (LPC) were elevated in LAM (or TSC-LAM) patients versus healthy subjects. The same LPC were elevated in conditioned media from TSC2-deficient cells, supporting the hypothesis that metabolic defects in TSC2-deficient cells may be associated with distinctive plasma metabolite profiles in patients. Surprisingly, accumulation of these LPC in TSC2deficient cells was not suppressed by either rapamycin or the mTOR catalytic inhibitor torin1. Finally, we show that TSC2-dependent metabolic abnormalities can be tackled to identify novel biomarkers for imaging TSC lesions and measure tumor burden. These studies may ultimately have clinical impact revealing ways to monitor disease progression and response to the mTORC1 inhibitor rapamycin or other targeted therapies in TSC and LAM.

A novel mouse model of LAM Wei Shi, PhD, Childrenâ&#x20AC;&#x2122;s Hospital Los Angeles, Keck School of Medicine, University of Southern California. Tuberous sclerosis complex (TSC) is caused by Tsc1 or Tsc2 germline mutation. Lymphangioleiomyomatosis (LAM) is a major clinical presentation of TSC lung disease, which is a significant cause of morbidity in women with TSC. LAM is characterized by proliferation of abnormal smooth muscle-like cells (LAM cells) and pulmonary cystic injury that result in destruction of lung structure and failure of respiratory function. The cellular origin of LAM cells and the mechanisms underlying the progressive cystic lung destruction in LAM are incompletely understood. One major challenge is the development of TSC-LAM disease animal models, as conventional homozygous Tsc1 or Tsc2 knockout mice die embryonically, and heterozygous Tsc1 or Tsc2 knockout mice do not develop LAM-like lesions. We have recently developed a mouse line, in which lung mesenchymal cells can be genetically targeted at different stages by a Tbx4-lung enhancer-driven Tet-On inducible system. By crossing with floxed-Tsc2 mouse line, we have successfully knocked out Tsc2 specifically

in lung mesenchymal cells during or after lung development, which was verified by western blot and immunohistochemistry. Interestingly, deletion of Tsc2 in lung mesenchyme during lung development resulted in reduced alveolar formation at early postnatal age, and subsequent alveolar cyst formation in adulthood. Moreover, spontaneous focal proliferative lesion was also detected in lungs of adult Tsc2 conditional knockout mice, predominantly in females. These phenotypic changes resemble the pulmonary pathology of TSC-LAM. Therefore, our study suggests that Tsc2 in lung mesenchymal cells plays critical roles in promoting alveolar development in early life and maintaining alveolar homeostasis in adulthood. Loss of Tsc2 function in lung mesenchymal progenitor cells may be associated with LAM pathogenesis.

mTOR inhibitors and beyond in the lung Frank McCormack, MD, Professor of Medicine, University of Cincinnati, OH. Lymphangioleiomyomatosis (LAM) is a metastasizing neoplasm that is driven by mTOR activating mutations in tuberous sclerosis genes. LAM arises from an unknown source, spreads via the blood and lymphatics and specifically targets the lung, resulting in extensive cystic remodelling. Symptomatic LAM is restricted almost entirely to women, suggesting that hormonal influences are important in disease pathogenesis. Lung function declines at the rate of about 7-15% per year, and most patients are short of breath with daily activities within 10 years of symptom onset. Therapy with mTOR inhibitors results in stabilization of lung function and improvement in functional performance, but the beneficial effects of treatment require continuous drug exposure. Trials to define the lowest effective dose and the safety and durability of long term suppressive therapy with mTOR inhibitors are under development. Future trials aimed at inducing remission will be based on strategies targeting autophagy, metabolism or other cellular mechanism that is uniquely altered in cells harboring mTOR mutations.

TSC renal failure and cystic disease John J. Bissler, MD, FedEx Chair of Excellence, Director, Tuberous Sclerosis Center of Excellence, Director, Division of Nephrology at St. Jude Childrenâ&#x20AC;&#x2122;s Research Hospital and LeBonheur Childrenâ&#x20AC;&#x2122;s Hospital. Dave Bridges, Ying Yang, Asim F. Choudri, Ryan Sullivan, Kenneth W. Gross, John J. Bissler. Tuberous sclerosis renal disease involves the loss of normal blood filtering because of solid and cystic lesions. These can arise as part of development or later in life. Recent advances have demonstrated that angiomyolipoma appear to be derived from a vascular mural cell. Once thought to be a structural cell, the vascular pericyte is endowed with stem cell capacities. Like pericytes, angiomyolipoma cells are responsive to certain vascular signalling pathways. TSC renal macrocystic disease can range from early onset and severe polycystic kidney disease phenotype or as simple cyst, and these can occur in patients with disease linked to either TSC1 or TSC2. There are multicystic and cortical cystic phenotypes. Microcystic cortical renal cystic disease is rare but associated with a rapid decay in renal function. This later disease process can even appear to have normal kidneys on sonogram or can appear to have increase cortical echogenicity.

To begin to better study preventative and curative therapies, we used a recombination strategy that developmentally expresses Cre recombinase, as well as expresses disease on a much-reduced scale later in life. Cellular targets included renal pericytes and distal tubular cells. The resulting animal develops variable renal cystic disease that include microcystic and macrocystic diseases. Early investigation reveals adipose-like lesions that is HMB-45, SMA, pS6 positive lesions and is associated with hemorrhage. Early intervention with an mTORC inhibitor appears to prolong survival and reduce renal cystic phenotype progression.

Session 5 â&#x20AC;&#x201C; Breakthroughs in understanding the pathogenesis of TSC at the cellular level Friday 11 September 2015 13:40 - 15:35 Windsor and Georgian Session Chair Julian Sampson, DM, FRCP, FMedSci, geneticist, Professor and Head of Department, Medical Genetics, Institute of Medical Genetics, Wales College of Medicine, Cardiff University, Cardiff, Wales. 13:40 - 13:50 Community Speaker: Wesley Gomes, Brazil. Session presentations: 13:50 - 14:20 Alternative therapeutic targets for TSC uncovered downstream of mTORC1 Brendan Manning, PhD, Professor, Harvard Universityâ&#x20AC;&#x2122;s School of Public Health (HSPH), Faculty Member of the Dana-Farber/Harvard Cancer Center, Boston, MA. 14:20 - 14:35 mTORC1-mediated regulation of mRNA biogenesis in TSC and LAM Gina Lee, PhD, Postdoctoral Fellow, Department of Pharmacology, Weill Cornell Medical College. 14:35 - 15:05 Combination therapy with nelfinavir: mechanism of drug action and selective targeting of Tuberous Sclerosis Complex 2 deficient cells Andrew R. Tee, PhD, Senior Lecturer, Institute of Cancer and Genetics, Cardiff University. 15:05 - 15:35 mTOR, metabolism and therapeutic vulnerabilities John Blenis, PhD, Professor of Pharmacology, Weill Cornell Medical College, New York, NY.

Alternative therapeutic targets for TSC uncovered downstream of mTORC1 Brendan Manning, PhD, Professor, Harvard Universityâ&#x20AC;&#x2122;s School of Public Health (HSPH), Faculty Member of the Dana-Farber/Harvard Cancer Center, Boston, MA. The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) has the ability to sense and integrate growth signals in various forms, including intracellular nutrients and energy, growth factors, and cellular stresses. mTORC1 senses many of these signals through a small G protein switch involving the tuberous sclerosis complex (TSC) tumor suppressors, TSC1 and TSC2, and the Ras-related small G protein Rheb. Importantly, genetic lesions disrupting the function of TSC1 and TSC2 underlie the development and pathology of both TSC and LAM, and aberrant activation of mTORC1 is a major driver of disease development and progression caused by these genetic events. However, we are just starting to understand the downstream consequences of mTORC1 activation in both normal and disease states. Through unbiased genomic and metabolomic approaches, we have found that, in addition to its established role in promoting protein synthesis, mTORC1 stimulates changes in specific metabolic pathways to promote anabolic cell growth and proliferation. Our latest unpublished findings on the metabolic functions downstream of mTORC1 and their promise as novel therapeutic targets to selectively kill TSC tumor cells will be presented.

mTORC1-mediated regulation of mRNA biogenesis in TSC and LAM Gina Lee, PhD, Postdoctoral Fellow, Department of Pharmacology, Weill Cornell Medical College. In recent years mRNA biogenesis (expression and processing) has become appreciated as a frequent source of gene-product variation that alters stability, cellular localization, activity and even confers antagonistic biological functions for many oncogenic or tumor suppressive proteins. The serine/arginine-rich (SR) protein kinases (SRPKs) regulate mRNA splicing through the phosphorylation of SR proteins that bind pre-mRNA splice sites. Although these enzymes are not frequently altered in cancer, altered splicing may be due to changes in signaling that control SRPKs. However, a major gap exists in our knowledge of which signals control mRNA processing. This knowledge would drastically alter our understanding of how oncogenic signaling reshapes molecular networks and adaptively redirects the flow of biological information. Dysregulated mTORC1 signaling is a common feature of genetic tumor syndromes including Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM). Through SILAC-based phosphoproteomics and S6K1/mTORC1 interaction screening efforts, we identified SRPKs as S6K1 and mTORC1 substrates. We have defined the molecular basis of SRPK regulation and provide evidence that these protein kinases links nutrient and growth factor signalling to the regulation of mRNA processing and translation, protein diversity, metabolism and carcinogenesis.

Combination therapy with nelfinavir: mechanism of drug action and selective targeting of Tuberous Sclerosis Complex 2 deficient cells Andrew R. Tee, PhD, Senior Lecturer, Institute of Cancer and Genetics, Cardiff University. Loss of Tuberous Sclerosis Complex 2 and aberrant signalling through mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is known to enhance the basal levels of endoplasmic reticulum (ER) stress. Tsc2 null cells likely exhibit enhanced endoplasmic reticulum (ER) stress, due to

a combination of inappropriately activated protein synthesis, impaired autophagy, and oxidative stress that leads to the accumulation of misfolded protein. The unfolded protein response (UPR) pathway restores ER homeostasis by three main mechanisms: by slowing the rates of global protein translation, by targeting unfolded protein to proteolytic degradation pathways, and finally through enhancement of protein folding that is orchestrated by molecular protein chaperones. Nelfinavir was originally approved for the treatment of human immunodeficiency virus infection as a dual HIV1 and HIV-2 protease inhibitor. However, nelfinavir has a broader drug action where one molecular target is through binding and inhibition of heat shock protein 90 (HSP90). Nelfinavir possesses anticancer activity as a single drug agent which is likely attributed to its ability to inhibit HSP90, which prevents efficient protein folding within the ER and induces cell death. This study utilises druginduced ER stress through nelfinavir in order to examine ER stress tolerance in cell lines with loss of Tuberous Sclerosis Complex 2 (TSC2) and hyper-active mTORC1 signalling. Our initial findings in wild type cells showed nelfinavir inhibited mTORC1 signalling and upregulated autophagy, as determined by decreased rpS6 and S6K1 phosphorylation, and SQTSM1 protein expression, respectively. In contrast, cells with hyper-active mTORC1 displayed basally elevated levels of ER stress which was greatly exaggerated following nelfinavir treatment, seen through increased CHOP mRNA and XBP1 splicing. To further enhance the effects of nelfinavir, we impaired proteolytic degradation pathways with either chloroquine (as an autophagy inhibitor) or bortezomib (as a proteasome inhibitor). Combination of nelfinavir with either chloroquine or bortezomib significantly increased ER stress and caused selective cell death in multiple cell line models that are deficient of Tsc2, whilst control cells tolerated treatment. Our research demonstrates that combination therapies with nelfinavir may have therapeutic potential for the treatment of Tuberous Sclerosis.

mTOR, metabolism and therapeutic vulnerabilities John Blenis, PhD, Professor of Pharmacology, Weill Cornell Medical College, New York, NY. The mTOR Complex 1 (mTORC1) signaling pathway has evolved to sense and respond to cellular energy status, nutrient availability and surrounding oxygen concentrations. In addition, mTORC1 can be further activated by mitogen- and hormone-stimulated kinases including Akt, ERK and RSK, and suppressed by mTORC1-regulated S6K1 via a variety of negative feedback loops. The integration of these multiple inputs control the strength and duration of downstream signaling, which is important in differentially regulating mTORC1-dependent processes such as protein synthesis and cellular metabolism. I will discuss how mTORC1 and S6K1 regulate aspects of nutrient metabolism, mRNA metabolism and protein production; biological processes critical to the control of cell growth while at the same time creating vulnerabilities that may provide important therapeutic opportunities in cancers with activated mTORC1 signaling.

Session 6 - Late breaking research Friday 11 September 2015 16:00 - 17:00 Windsor and Georgian Session Chair: Chris Kingswood, MSc , FRCP, Nephrologist, Sussex Kidney Unit, Royal Sussex County Hospital, Brighton, UK. Session Presentations: 16:00 - 16:20 Sirolimus treatment in children with Tuberous Sclerosis Complex and intractable epilepsy: a randomized controlled trial Iris Overwater, MSc, PhD, Researcher, Department of Pediatric Neurology , ENCORE-TSC Expertise Center, Erasmus Medical Center. 16:20 - 16:40 Social visual perception in Tuberous Sclerosis Complex Kandice Varcin, PhD, Postdoctoral Fellow, Boston Children’s Hospital, Harvard Medical School, Boston, MA. 16:40 - 17:00 The TSC protein complex regulates melanogenesis through mTOR and GSK3ß, explaining the pathogenesis of TSC ‘white spots’ Juxiang Cao, PhD, Postdoctoral Research Associate, Brigham and Women's Hospital, Boston, MA.

Sirolimus treatment in children with Tuberous Sclerosis Complex and intractable epilepsy: a randomized controlled trial Iris Overwater, MSc, PhD, Researcher, Department of Pediatric Neurology , ENCORE-TSC Expertise Center, Erasmus Medical Center. Iris E. Overwater, AndrĂŠ B. Rietman, Karen Bindels-de Heus, Caspar W. N. Looman, Floor E. Jansen, Henriette A. Moll, Ype Elgersma, Marie-Claire Y. de Wit. Background Tuberous Sclerosis Complex (TSC) is a genetic disorder caused by inactivating mutations in the TSC1 or TSC2 genes. Mutations in these genes cause disinhibition of the mTOR pathway, which has been shown to be involved in epilepsy in patients with TSC. Up to 90% of TSC patients suffer from epilepsy, and as many as 63% are intractable to anti-epileptic drugs (AEDs). Animal studies and an uncontrolled study in children suggest that mTOR inhibitors may be potent drugs for reducing seizure frequency. Methods We performed the first randomized clinical trial into the effect of the mTOR inhibitor sirolimus on intractable epilepsy in TSC patients. Eligible children were aged 3 months-12 years, had a definite clinical diagnosis of TSC and had at least one seizure per week at baseline. Participants were randomized in a cross-over design to receive add-on sirolimus treatment immediately or after six months. Total duration of the trial was twelve months. Primary outcome of the trial was frequency of epileptic seizures. Secondary outcomes were cognitive and motor development, and EEG abnormalities. Results Twenty-three children aged 1.8-10.9 years were randomised between September 2011 and December 2013. At baseline epilepsy was severe, with seizure onset at =6 months of age in 18 children (78%), and 14 children (61%) suffering from infantile spasms. After six months of sirolimus treatment, nine children (39%) had a seizure frequency decrease of 50% or more compared to baseline seizure frequency. Six children (26%) had such a response after six months of the control period. Cognition and behaviour did not show a significant change. Adverse events occurred in all children. The most common adverse event was upper respiratory tract infection, which occurred equally often in the intervention (20) and the nonintervention (19) period. Five children (22%) discontinued sirolimus prematurely due to adverse events. Conclusions This is the first randomised controlled trial showing the effect of mTOR inhibition on epileptogenesis in intractable epilepsy due to TSC. Funding These investigations were supported by the Dutch Epilepsy Foundation, project number 11-15.

Social visual perception in Tuberous Sclerosis Complex Kandice Varcin, PhD, Postdoctoral Fellow, Boston Childrenâ&#x20AC;&#x2122;s Hospital, Harvard Medical School, Boston, MA. Kandice J. Varcin, Shafali S. Jeste, Charles A. Nelson. TSC confers a high risk of neurodevelopmental disorders, most commonly, autism spectrum disorder (ASD) and intellectual disability. Up to 80% of children with TSC will experience cognitive delay and up to 60% will meet criteria for ASD. We conducted the first prospective, longitudinal study of development in infants with TSC with a goal of moving beyond diagnostic categorization to 1) a deeper characterization of the social communication and cognitive profiles of infants with TSC and 2) better defining the timing and emergence of developmental delays in TSC. Recently, we identified delays in non-verbal skills development within the first two years of life in TSC that were most

prominent in infants who developed ASD. The current study extends this work by using eye-tracking and electroencephalographic (EEG) methodologies to investigate the processing of socially relevant information (e.g., faces) across early development in TSC. Methods: High-density EEG recordings were conducted with infants with TSC and typically developing (TD) controls at 12 (n=8 TSC; n=8 TD) and 24 months (n=13 TSC; n=7 TD) while infants viewed images of faces and objects. Eye-gaze fixations to familiar vs. unfamiliar faces were recorded at 12, 18, 24 and 36 months in infants with TSC and TD controls and compared to infants with ASD without a known genetic disorder. Results: At 12 months, there were no differences in neural responses to faces between infants with TSC and TD controls. However, by 24 months, infants’ with TSC demonstrated delayed latencies in their neural processing of faces compared to TD infants. Similarly, while at 12 months there were no group differences in patterns of visual fixations to faces, at 18 and 24 months of age, infants with TSC showed reduced rates of visual fixations to the eye region of faces compared to TD infants and infants with ASD without a known genetic disorder. Conclusions: Infants with TSC show differences in their processing of socially relevant information. Specifically, infants with TSC show latency delays in their neural responses to faces relative to TD infants, and they show reduced visual fixations to the eye region of faces relative to TD infants and infants with ASD without a genetic disorder. Importantly, we found that these alterations in social visual processing emerge between 1–2 years of age, pointing to the second year of life as an important developmental window for interventions targeting non-verbal communication in TSC.

The TSC protein complex regulates melanogenesis through mTOR and GSK3ß, explaining the pathogenesis of TSC ‘white spots’ Juxiang Cao, PhD, Postdoctoral Research Associate Brigham and Women's Hospital, Boston, MA. Juxiang Cao, Magdalena E. Tyburczy, Thomas N. Darling, Hans R. Widlund and David J. Kwiatkowski Tuberous sclerosis complex (TSC) is due to inactivating mutations in either TSC1 or TSC2, which cause aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1). A classic clinical manifestation of TSC is the hypomelanotic macules (white spots), which are seen in the majority of TSC patients, and have previously been unexplained. Using two highly pigmented human cell lines, one is the SK-MEL-30 melanoma cell line and the other an immortalized primary human melanocyte cell line, we examined the molecular basis of pigment loss in TSC. Knockdown of either TSC1 or TSC2 led to similar findings in the two lines, with mTORC1 activation and loss of pigmentation. Marked reduction in expression of MITF, PGC-1 alpha, tyrosinase and other pigment genes was seen. This reduction in pigmentation was completely reversed by treatment with rapamycin, or forced expression of MITF. MITF expression did not affect mTORC1 activation including autophagy inhibition seen in these cells. AKT inactivation, reduced pS9-GSK-3 beta, increased GSK-3 beta, increased pS33,37/T41-beta-catenin, and markedly reduced total and nuclear beta-catenin were all seen in the TSC1/TSC2 knockdown cells. Inhibition of GSK3 beta with any of three inhibitors, or reconstitution with wildtype or dominant-active beta-catenin fully restored pigmentation in the TSC1/TSC2 knockdown cells, also without effect on mTORC1 activation or autophagy inhibition. Analysis of cultured primary melanocytes isolated from a white spot from each of two TSC patients demonstrated similar pigmentation loss, mTORC1 activation, and loss of TSC2 protein, while genetic studies showed biallelic mutation in TSC2 in one of these two cultures, not seen in DNA from a fibroblast culture from the same white spot. Hence our studies indicate that

white spots in TSC form due to two hit loss of TSC1 or TSC2 in clonal melanocytic patches, with subsequent activation of mTORC1, inactivation of AKT, activation of GSK-3 beta and reduced free beta-catenin, leading to reduced MITF and pigment formation. These effects are reversed both by rapamyin and GSK-3 beta inhibitor treatments.

Session 7 - Burden of TSC and engaging those affected to impact research Saturday 12 September 2015 08:30 - 10:15 Windsor and Georgian Session Chair: Paolo Curatalo, MD, Professor of Pediatric Neurology and Child Psychiatry, Director, Pediatric Neuroscience Unit, Tor Vergata University Hospital, Rome. 08:30 - 08:45 Community Speaker: Marie James, UK. Session presentations: 08:45 - 09:15 The neuropsychiatric burden and methods that work to lessen that burden Petrus J. de Vries, MBChB, MRCPsych, PhD, Sue Struengmann Professor of Child & Adolescent Psychiatry, Division of Child and Adolescent Psychiatry, University of Cape Town, Cape Town. 09:15 - 09:45 Burden of TSC and health economics Anna Jansen, MD, PhD, Pediatric Neurologist, UZ Brussel Hospital, Brussels. 09:45 - 10:15 TS2000: The impact of TSC in early years Patrick Bolton, BSc, MA, PhD, MBBS, FRCPsych, Professor in Child and Adolescent Psychiatry, Institute of Psychiatry, King's College London.

The neuropsychiatric burden and methods that work to lessen that burden Petrus J. de Vries, MBChB, MRCPsych, PhD, Sue Struengmann Professor of Child & Adolescent Psychiatry, Division of Child and Adolescent Psychiatry, University of Cape Town, Cape Town. Tuberous Sclerosis Complex (TSC) is associated with wide-ranging multi-system involvement. However, it is the TSC-Associated Neuropsychiatric Disorders (TAND) that cause some of the greatest burden to individuals who live with TSC, their families and their support networks. Even though almost 90% of people with TSC will have elements of TAND at some stage in their lives, very few people receive appropriate evaluation and intervention for these disorders. In this presentation we will draw on data from three different user/carer surveys performed over the last few years to describe what people with TSC perceive as their priorities relating to the neuropsychiatric burden of TSC. We will then discuss some current and future strategies to reduce the burden of TSC-associated neuropsychiatric disorders.

Burden of TSC and health economics Anna Jansen, MD, PhD, Pediatric Neurologist, UZ Brussel Hospital, Brussels. Aim: The lecture will review what is known about the physical, psychological and financial burden of TSC for the patient and his family, the healthcare system and society. Rationale: To give a context to the potential benefits and risks of early preventative treatment, balancing the relief of that burden against the risks and costs of treatment.

TS2000: The impact of TSC in early years Patrick Bolton, BSc, MA, PhD, MBBS, FRCPsych, Professor in Child and Adolescent Psychiatry, Institute of Psychiatry, King's College London. The TS 2000 study is a prospective longitudinal study of the natural history of tuberous sclerosis complex (TSC). It was initiated in 2001 with support and funding from the UK Tuberous Sclerosis Association. All newly diagnosed cases of tuberous sclerosis in the UK born between 2001-2005 identified through a network of clinical centres (n=125). In phase 1 of the TS 2000 study cases were systematically evaluated to determine the nature of the mutation and to document the physical manifestations at diagnosis (including brain cortical tuber count and location), as well as the type and severity of epilepsy and the profile of intellectual abilities. In phase 2 of the study, all cases have been re-evaluated with in depth assessment of the cognitive and behavioural features, focusing on symptoms of the autism spectrum and attention deficit hyperactivity. The subset of the TS 2000 cohort who were presented in infancy prior to the onset or establishment of seizures were the focus of a special study that aimed to chart cognitive development before and after seizure onset. The findings from the study have demonstrated the value of genetic diagnosis in cases of possible tuberous sclerosis and the fact that cases are increasingly being identified prenatally or in the early post-natal period. They have also illustrated how the developmental trajectories of infants with TSC are associated with the onset, type and severity of seizure disorder, in addition to the nature of

genetic mutation and extent of brain involvement. The data also show the range of physical, cognitive and behavioural problems in a population representative cohort. The TS 2000 cohort study is the first prospective longitudinal study of the natural history of TS. Its success would not have been possible without the support of the UK Tuberous Sclerosis Association or the commitment of the families and individual with TSC. Engagement and participation in the study have been promoted through a study web site, regular newsletters, Facebook and reports in the UK TSA publications. The UK TSA support has been fundamental to ensuring he long term viability of the study and its success in attracting additional funding and support from a range of charities. The TS 2000 cohort study represents a model of research and family participation that can be adopted in other countries and other rare disorder study groups.

Session 8 - Translating research into improvements in care and outcomes Saturday 12 September 2015 10:45 - 12:30 Windsor and Georgian Session Chair: David Mowat, MBBS, MRCGP, DRACOG, FRACP, Senior Staff Specialist, Clinical Geneticist, Sydney Children's Hospital, Sydney. Session presentations: 10:45 - 11:15 Genetics and potential for personalized medicine David Kwiatkowski, MD, PhD, Harvard Medical School, Brigham and Womenâ&#x20AC;&#x2122;s Hospital, and Dana Farber Cancer Institute. 11:15 - 11:45 Treatments on the horizon for TSC and LAM Elizabeth P. Henske, MD, Oncologist, Brigham and Women's Hospital, Harvard Medical School, Boston, MA. 11:45 - 12:15 The potential for early intervention to prevent development of autism Mustafa Sahin, MD, PhD, Pediatric Neurologist, Director, TSC Program and Translational Neuroscience Center, Children's Hospital, Boston, MA. 12:15 - 12:30 Summing up: Where next? Chris Kingswood, MSc, FRCP

Genetics and potential for personalized medicine David Kwiatkowski, MD, PhD, Harvard Medical School, Brigham and Womenâ&#x20AC;&#x2122;s Hospital, Dana Farber Cancer Institute. Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing. Magdalena E. Tyburczy, Kira A. Dies, Jennifer Glass, Susana Camposano, Yvonne Chekaluk, Aaron R. Thorner, Ling Lin, Darcy Krueger, David N. Franz, Elizabeth A. Thiele, Mustafa Sahin, David J. Kwiatkowski. Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene syndrome due to mutations in either TSC1 or TSC2. 10-20% of TSC individuals have no mutation identified (NMI) after thorough conventional molecular diagnostic assessment. 53 TSC subjects who were NMI were studied using next generation sequencing to search for mutations in these genes. Blood/saliva DNA including parental samples were available from all subjects, and skin tumor biopsy DNA was available from six subjects. We identified mutations in 45 of 53 subjects (85%). Mosaicism was observed in the majority (26 of 45, 58%), and intronic splice site mutations were also unusually common, seen in 18 of 45 subjects (40%). Seventeen (38%) mutations were seen at an allele frequency < 5%, five at an allele frequency < 1%, and two were identified in skin tumor biopsies only, and were not seen at appreciable frequency in blood or saliva DNA. These findings illuminate the extent of mosaicism in TSC, indicate the importance of full gene coverage and next generation sequencing for mutation detection, show that analysis of TSC-related tumors can increase the mutation detection rate, indicate that it is not likely that a third TSC gene exists, and enable provision of genetic counselling to the substantial population of TSC individuals who are currently NMI.

Treatments on the horizon for TSC and LAM Elizabeth P. Henske, MD, Oncologist, Brigham and Women's Hospital, Harvard Medical School, Boston, MA. Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene disorder characterized by seizures, cognitive deficits, and tumors of the brain, heart, skin, and kidney. The majority of women with TSC develop lymphangioleiomyomatosis (LAM), a destructive lung disease caused by the diffuse proliferation of smooth muscle-like LAM cells. LAM also occurs in a sporadic setting, in women who do not have germline TSC mutations. Both sporadic and TSC-LAM are caused by bi-allelic TSC gene inactivation leading to activation of mTORC1 (Carsillo et al. PNAS 2000). LAM can lead to oxygen-dependency and premature death, and can recur after lung transplantation. Multiple clinical studies indicate that Rapamycin and its analogs induce cytostatic, rather than cytocidal, effects on TSC2-deficient cells, including LAM cells. Autophagy is a critical metabolic node in TSC2-deficient cells. TSC2-null cells have decreased basal and stress-induced autophagy levels. A marked increase in the autophagy substrate p62/SQSTM1 (sequestosome 1) is observed in TSC2-deficient cells and in human TSC and LAM tumors. Downregulation of p62 is sufficient to block tumorigenesis of TSC2-null cells in mice, and loss of one allele of Beclin1 inhibits tumor formation in Tsc2+/- mice, supporting the hypothesis that TSC tumorigenesis is autophagy-dependent. In preclinical studies, combining mTORC1 inhibition (with Rapamycin) with autophagy inhibition (with Chloroquine) is more effective than either treatment alone in inhibiting the growth of TSC2-null xenograft tumors and the development of spontaneous

renal tumors in Tsc2+/- mice (Parkhitko et al. PNAS 2011). We are currently testing dual targeting of mTORC1 and autophagy in LAM in a Phase I clinical trial, the Sirolimus and Autophagy Inhibition in LAM (SAIL) trial. Metabolic reprogramming induced by autophagy inhibition leads to pentose phosphate pathway "addiction" selectively in Tsc2-deficient cells; combined inhibition of the PPP and autophagy induces cell death (Parkhitko et al. Mol Cancer Res 2014). A high throughput screen identified chelerythrine chloride as a selective inducer of death in TSC2-null cells, via a reactive oxygen species-dependent mechanism (Medvetz et al. Mol Cancer Res 2015). Taken together, these data provide critical proofof-concept that the metabolic consequences of mTORC1 hyperactivation can be therapeutically targeted without inhibiting mTORC1 itself. By averting metabolic dormancy and inducing cytocidal rather than cytostatic responses, we predict that Rapalog-independent therapies will lead to more complete and durable clinical responses for tumors with mTORC1 hyperactivation, including TSC and LAM.

The potential for early intervention to prevent development of autism Mustafa Sahin, MD, PhD, Pediatric Neurologist, Director, TSC Program and Translational Neuroscience Center, Children's Hospital, Boston, MA. Accumulating evidence suggests that TSC patients have non-tuber abnormalities that contribute to the development of the neurological and behavioral phenotype. Using mouse models of TSC, work from our lab and from our collaborators has started to shed light on the cellular and neural circuit abnormalities underlying the neurobehavioral problems. We have shown that TSC1/2 proteins regulate axon specification, guidance, myelination and regeneration. We can detect aberrant wiring in both mouse models and on diffusion tensor imaging of TSC patients. Recently, we generated a conditional knockout of Tsc1 only in cerebellar Purkinje cells. Mutant mice displayed autistic-like behaviors – social impairment, restrictive/repetitive behaviors, and abnormal vocalizations. Importantly, treatment of mutants with the mTOR inhibitor, rapamycin, prevented both pathological and behavioral deficits. Taken together, these preclinical studies have led to collaborative biomarker and treatment trials that are currently ongoing. We have formed a clinical consortium of five children’s hospitals geographically distributed throughout the USA (known as TSC Autism Centers of Excellence Network – TACERN). This network and the biomarker studies will accelerate the progress toward clinical trials in TSC associated neuropsychiatric disorders. Our ultimate goal is to detect autism symptoms as early as possible in infants with TSC and provide an effective treatment

Poster Listing The poster session is supported by GW Pharmaceuticals. 11. Disease management in a rare disease: Comparisons of Tuberous Sclerosis Complex (TSC) patients treated in TSC Centers of Excellence (COE) versus other settings Nicole M. Engel-Nitz

1. Arrhythmias and functional disorders in 43 patients with Tuberous Sclerosis Fatou Aw 2. Everolimus for renal angiomyolipoma associated with Tuberous Sclerosis Complex: Efficacy and safety over 3.5 years of treatment in the EXIST-2 study John J. Bissler

12. Everolimus For Fast Expanded aCcess in TSC SEGA (EFFECTS) Andras Fogarasi

3. Treatment patterns for renal angiomyolipoma in patients with Tuberous Sclerosis Complex Katherine Cappell

13. Everolimus for subependymal giant cell astrocytoma associated with tuberous sclerosis complex: final long-term results from EXIST-1 David N. Franz

4. The Swedish TSC population â&#x20AC;&#x201C; A retrospective registry analysis Peter Carlqvist

14. Effect of everolimus on skin lesions in patients being treated for subependymal giant cell astrocytoma: results from the final 4-year analysis of EXIST-1 David N. Franz

5. Characterising STAT3 as a therapeutic target for TSC & LAM Kayleigh M. Dodd

15. Examination of patients with progression while treated with everolimus for subependymal giant cell astrocytoma: results from the final 4-year analysis of EXIST-1 David N. Franz

6. Clinical report: a rare co-occurrence of tuberous sclerosis complex and Rett syndrome in a girl with mental retardation, epilepsy and autism.ve proposal Marina Y. Dorofeeva

7. New Pathways: Improved molecular characterization of the Burt Hogg DubĂŠ tumour syndrome Peter F. Doubleday 8. Loss of TSC2 sensitises cells to nelfinavirbortezomib dual therapy Elaine A. Dunlop

17. Effect of everolimus on subependymal giant cell astrocytoma in patients being treated for renal angiomyolipoma: results from the 3.5-year update of EXIST-2 Michael Frost

9. Lessons from the TSC1 and TSC2 Variation Databases Rosemary Ekong, 10. The clinical and genetic outcome of an antenatal diagnosis of cardiac rhabdomyoma Frances Elmslie

18. Monitoring testing patterns among patients with tuberous sclerosis complexrelated angiomyolipomas Michael Frost

19. DTI as a biomarker of self-injurious behavior in children with tuberous sclerosis complex Tanjala T. Gipson

29. Identification of Pro-survival Rapamycindependent microRNA (Rapa-miRs) in Tuberous Sclerosis Complex Hilaire C. Lam

20. Ganglioside D3 overexpression in TSC deficient tissues: relevance to immunotherapy Steven W. Henning

30. Malignancy in Tuberous Sclerosis Complex Anna M. Larson 31. Can perinatal risk factors influence neuropsychiatric outcome in children with Tuberous Sclerosis? Holan Liang

21. The combination of rapamycin and resveratrol for treatment of TSC/LAM Marina K. Holz 22. A new model of cortical tuber associated epilepsy Lawrence S. Hsieh

32. A case of neonatal subependymal giant cell astrocytoma (SEGA) treated with everolimus Philippe Major

23. Autism symptoms in toddlers with TSC: Is there a distinctive phenotype that informs treatment targets? Shafali Spurling Jeste

33. Reduction in retinal nerve fiber layer thickness in Tuberous Sclerosis Complex Sara Marciano

24. Topical Everolimus for facial angiofibromas in the Tuberous Sclerosis Complex. A pilot study Nebojsa J. Jovic

34. A genetic and clinical overview of 18 years molecular diagnostics of Tuberous sclerosis complex in Germany Karin Mayer

25. Epilepsy management in Tuberous Sclerosis: The role of thermal ablation as a substitute for resective surgery Karen Keough

35. Psychiatric outcomes in the TS 2000 Cohort Study (TS2000): Autistic traits and autism spectrum disorder (ASD) Fiona S. McEwen

26. Real-world assessment of renal involvement in tuberous sclerosis complex patients in the United Kingdom: a retrospective cohort study in the Clinical Practice Research Datalink Chris Kingswood

36. Diffusion tensor imaging reveals white matter abnormalities in tuberous sclerosis patients with refractory seizures Romina Moavero 37. Symptoms of Tuberous Sclerosis Complex: Comparison of TSC1 versus TSC2 gene mutation Jo Anne Nakagawa

27. Effect of everolimus on renal angiomyolipoma in pediatric patients from the final analysis of EXIST-1 Chris Kingswood

38. Functional analysis of TSC1 and TSC2 variants Mark Nellist

28. TuberOus SClerosis registry to increase disease Awareness (TOSCA) â&#x20AC;&#x201C; baseline data analysis for 2093 patients Chris Kingswood on behalf of TOSCA Consortium and TOSCA investigators

39. Demographic and clinical characteristics of patients with tuberous sclerosis complex in a TSC patient database Hope Northrup

40. Tuberous Sclerosis Complex and malignant Tumors: A retrospective study in an Italian cohort Angela Peron

associated with tuberous sclerosis complex: results from the 4-year final analysis of EXIST-1 Steven P. Sparagana

41. Diminished rates of protein synthesis in a mouse model of Tuberous Sclerosis Complex: an mTORC1-dependent phenomenon R. Michelle Reith

50. Understanding families living with TSC in Australia Clare P. Stuart 51. Calcium signaling induces TSC2 phosphorylation independently of Akt Tsukasa Suzuki

42. A single cell biology platform to investigate the effects of stem cell positional identity on SEGA development Gabrielle V. Rushing

52. Sleep problems in Tuberous Sclerosis Complex: A questionnaire based study and a systematic literature review. Panagiota Sykioti

43. GSK2126458, a dual PI3K/mTOR inhibitor, is less effective than rapamycin for treatment of renal lesions in a genetically engineered Tsc2+/- mouse model Ming Hong Shen

53. Genetic mechanisms associated with tuberous sclerosis and tuberous sclerosis-like phenotypes Laura Thomas

44. Evaluation of combined treatment of rapamycin with atorvastatin for therapy of renal lesions in a genetically engineered Tsc1+/- mouse model Ming Hong Shen

54. A risk pathway to intellectual disability in Tuberous Sclerosis Complex Charlotte Tye

45. Heterogeneous response of renal tumours to sunitinib and sorafenib in genetically engineered TSC mouse models Ming Hong Shen

55. Attention deficit hyperactivity disorder and executive dysfunction in Tuberous Sclerosis Complex Charlotte Tye

46. mTORC1 inhibition rescues primary cilia expression and affects Wnt/PCP signalling in TSC2-deficient renal cystic epithelial cells Brian J. Siroky

56. Autistic-like phenotype in Tuberous Sclerosis animal model: remission by mTORinhibitor Robert Waltereit

47. A simplified management approach for general practitioners for skin lesions In patients with Tuberous Sclerosis Complex (TSC) Keyoumars Soltani

57. Attention to social and non-social stimuli in Tuberous Sclerosis Complex Lucy Wilde 58. Tuberous sclerosis complex 1(TSC1) regulates the Caveolae mediated endocytosis via controlling the Rab5 activity. Yuji Yamamoto

48. Pancreatic neuroendocrine tumors in pediatric patients with Tuberous Sclerosis Complex: A case study Steven P. Sparagana 49. Everolimus exposure and overall tolerability in patients treated for subependymal giant cell astrocytoma

rapamycin (mTOR) inhibitor, for the treatment of renal angiomyolipoma associated with either tuberous sclerosis complex (TSC) or sporadic lymphangioleiomyomatosis (sLAM). Here we present longer-term safety and efficacy data for everolimus including the open-label extension phase (cutoff date April 1, 2014). Methods: 118 patients with renal angiomyolipoma associated with TSC (n=113) or sLAM (n=5) were randomly assigned 2:1 to receive 10 mg/day everolimus (n=79) or placebo (n=39). Angiomyolipoma response rate, the primary endpoint, was defined as the proportion of patients with confirmed =50% reduction in sum of volumes of all target angiomyolipoma relative to baseline in the absence of an increase in kidney volume (>20% from nadir), no new angiomyolipoma lesions, and no angiomyolipoma-related bleeding of = grade 2. Results: At the initial cutoff (June 30, 2011), everolimus was superior to placebo for angiomyolipoma response rate (42% vs 0%; P?0.0001). Following these positive results, all patients still in the study were offered open-label everolimus in an extension phase. As of April 1, 2014, 112 patients (median age, 32 years [range 18-62]) received =1 dose of everolimus. Median duration of everolimus exposure was 39.8 months (range, 0.5-57.3). Angiomyolipoma response rate was 56.3% (95% confidence interval [CI], 46.6%-65.6%). At week 96 (n=98), 80.6% and 63.3% of patients had reductions =30% and =50%, respectively, in angiomyolipoma volume. Progression-free rate at 48 months was 78.3% (95% CI, 63.6%87.6%). AEs were similar to previous reports and remained mostly grade 1 or 2. AEs commonly reported in =20% of patients were nasopharyngitis (44.6%), stomatitis (42.9%), hypercholesterolemia (35.7%), acne and headache (31.3% each), urinary tract infection (30.4%), aphthous stomatitis (25.9%), diarrhea (24.1%), hypertension (23.2%), cough (21.4%), and nausea (20.5%). Incidence of emerging AEs decreased over the study period. The most frequent grade 3 AEs (=3%) were blood phosphorus decreased (4.5%) and amenorrhea (5.6%; n=4 of 71 females aged 10-55 years). The only grade 4 event occurring in =2 patients was blood uric acid increased (2.7%). No patient had a renal bleed while taking everolimus. Conclusions: A sustained, increasing reduction in angiomyolipoma volume was seen with continued everolimus treatment. No new safety concerns.

Poster Abstracts 1. Arrhythmias and functional disorders in 43 patients with Tuberous Sclerosis Aw F, Leye M, Major P, Gagnon H, Brière J, Dahdah N CHU Ste-Justine, Université de Montréal, Montréal, Qc, Canada Background: Tuberous Sclerosis Complex is an autosomal dominant disease. It leads to the formation of benign tumors in various organs, including the heart. Commonly called cardiac rhabdomyomas (RHM) it is most frequently found during childhood. RHM regresses in most cases spontaneously but can be complicated by serious arrhythmias or obstruction that may compromise cardiac output. Methods: We conducted a retrospective analysis of 30 patients and then classified patients according to the initial volume of RHM in early childhood. We subsequently conducted a prospective cardiac evaluation of mass. We compared the prevalence of arrhythmia based on the initial volume of the RHM comparatively between the case and the family control subjects with TSC but no RHM. Results: The median follow-up time was 4.5 years. The Prevalence of RHM was 46.8% (37 RHM) between 0-5 years, 39.2% (31 RHM) between 12-33 years and 13.9% (11 RHM) between 6-11 years. RHM causes in 20% an obstacle to inflow or outflow tract. Patients with RHM were classified according to the total volume of initial RHM among whom 16 cases (64%) with history of small RHM (?25% of left ventricular mass) at birth versus 11 (36%) with history of large RHM. The encountered benign arrhythmias founded were isolated ventricular premature beats (55%) and supraventricular premature beats (50%), which were 2 times fold and 4 times fold more compared to reported prevalence in the general pediatric population, respectively (p<0.05). Significant arrhythmia was noted in 3 (30%) patients with large RHM: one patient with Wolff Parkinson White syndrome had supraventricular tachycardia treated with antiarrhythmic agent, one case had atrial fibrillation equally treated with anti-arrhythmic agent, with resolved arrhythmia concomitantly with the regression of the tumor, a third patient had symptomatic paroxysmal supraventricular tachycardia requiring percutaneous cryoablation. There was a trend which did not reach statistical significance difference in the prevalence of cardiac arrhythmias between cases with small versus large RHM (p=0.07). Conclusion: In this series, arrhythmia was more prevalent in TSC compared to general pediatric population, with a trend towards higher prevalence in cases with larger RHM. This is the first series to report RHM size correlation with the prevalence of arrhythmia included cases with absent RHM. It is possible that such arrhythmias are caused by subanatomical lesions.

3. Treatment Patterns for Renal Angiomyolipoma in Patients with Tuberous Sclerosis Complex Katherine Cappell, Xue Song, Zhimei Liu, Judith Prestifilippo, John Bissler, Hearns Charles, John Hulbert Truven Health Analytics Inc., Ann Arbor, MI (KC, XS); Novartis Pharmaceuticals Corporation, East Hanover, NJ (ZL, JP); LeBonheur Children’s Hospital and St. Jude Children’s Research Hospital, Memphis, TN (JB); NYU Langone Medical Center, New York, NY (HC); Urologic Physicians, Edina, MN (JH)

2. Everolimus for Renal Angiomyolipoma Associated With Tuberous Sclerosis Complex: Efficacy and Safety Over 3.5 Years of Treatment in the EXIST-2 Study John J. Bissler, J Christopher Kingswood, Elzbieta Radzikowska, Bernard A. Zonnenberg, Michael Frost, Elena Belousova, Matthias Sauter, Norio Nonomura, Susanne Brakemeier, Petrus J. de Vries, Noah Berkowitz, Severine Peyrard, Klemens Budde

Background: Renal pathology is common in patients with tuberous sclerosis complex (TSC). Surgical treatment for renal angiomyolipoma includes renal artery embolization (EMB), partial nephrectomy (PN), and total nephrectomy (TN). Pharmacological treatments include everolimus and sirolimus. Objectives: This study used large national claims databases to examine treatment with everolimus and sirolimus, EMB, PN, and TN, in a TSC population. Methods: Patients with =1 diagnosis of TSC (ICD-9-CM 759.5) and renal angiomyolipoma (ICD-9-CM 233.0) were extracted using Treatment Pathways based on the MarketScan® Commercial (1/1/2010-10/31/2014) and Medicaid (10/1/2010-9/30/2013) Databases. Index date was 1st TSC or renal angiomyolipoma diagnosis date. Patients were followed until the end of data. The proportion of patients using medications or procedures was estimated during the variable follow-up. Results: Patients were grouped into 4 cohorts: Commercial age <18 (N=204, mean age 10.0, 46.6% male), Commercial age =18 (N=279, mean age 36.0, 30.5%

St Jude Children’s Research Hospital and Le Bonheur Children’s Hospital, Memphis, TN, USA (JJB); Royal Sussex County Hospital, Brighton, UK (JCK); National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland (ER); Universitair Medisch Centrum, Utrecht, Netherlands (BAZ); Minnesota Epilepsy Group, St. Paul, MN, USA (MF); Moscow Research Institute of Pediatrics and Pediatric Surgery, Moscow, Russia (EB); Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Objective: EXIST-2 (NCT00790400), a randomized, double-blind, phase 3 trial, assessed everolimus, an oral mammalian target of

male), Medicaid age <18 (N=71, mean age 9.1, 45.1% male), and Medicaid age =18 (N=43, mean age 29.3, 34.9% male). TSC diagnosis occurred prior to renal angiomyolipoma diagnosis in most patients (59.1% - 88.7% across all cohorts). In the Commercial cohort, up to 37.3% received medications or procedures, including dialysis and renal transplant. 13.9% of Commercial patients had everolimus, higher than the 9.5% rate reported in the literature. 11.6% of Commercial patients had sirolimus, 9.5% were embolized, 3.5% had PN, and 6.2% had TN. Medicaid patients had lower treatment rates; the corresponding proportions for everolimus, sirolimus, EMB, PN, and TN were 4.5%, 6.5%, 3.7%, 2.0%, and 2.0% in Medicaid. Further analysis is being conducted to understand treatment pattern changes over time and duration of drug therapy using the most complete and updated claims data. Conclusions: The rate of drug use captured in the most recent data appeared to be higher while the rate of procedures appeared to be lower than those previously reported in the literature, suggesting that the treatment pattern has changed over time. A higher proportion of Commercial patients had everolimus and procedures than did their Medicaid counterparts.

everolimus. Only 2% were treated with takrolimus. A total of 27 patients died during the study period, making any quantitative analysis of mortality difficult. 5. Characterising STAT3 as a therapeutic target for TSC & LAM Kayleigh M. Dodd & Andrew R. Tee Cardiff University Hypoxia inducible factor-1a (HIF-1a) is a key mediator of the hypoxic response and controls a number of cellular processes involved in tumour growth, including angiogenesis, glucose metabolism and cell survival. We previously demonstrated that mTORC1 drives HIF-1a protein accumulation translationally via 4E-BP1 and S6K1, and on a transcriptional level via Signal Transducer and Activator of Transcription-3 (STAT3). We demonstrate that STAT3 is crucial for induction of the HIF-1a protein, whereby knockdown of STAT3 abrogates mTORC1 mediated HIF-1a activity. mTORC1 promotes the activity of STAT3 via direct phosphorylation at Ser727. Of interest, we also observe that the JAK2 regulated Tyr705 phosphorylation site of STAT3 is elevated in TSC deficient cell lines, which suggests that the JAK2/STAT3 signalling pathway may also contribute to TSC pathology. STAT3 is not only a key mediator of angiogenesis via HIF-1a, but also promotes other cellular processes including cellular survival and cell migration. We demonstrate that inhibition of STAT3 can block the migration of angiomyolipomaderived cell lines, where this inhibition is further enhanced by co-treatment with the mTOR kinase inhibitor KU-0063794, indicating that STAT3 drives migration of these cells via mTORdependent and -independent mechanisms. STAT3 inhibition also promotes cell survival via activation of anti-apoptotic genes bcl-x, bcl-2, mcl-1, cyclin D1, and c-myc, in concordance with this, 24 h treatment with the JAK2/STAT3 inhibitor, FLLL31, induced cell death in Tsc2-deficient cell lines. Furthermore, knockdown of STAT3 in these AML-derived cells also significantly reduced average tumour spheroid volume in soft agar. Our data indicates that targeting JAK2/STAT3 in combination with mTORC1 may enhance the anti-tumour efficacy of rapalogues currently undergoing clinical trials in TS patients.

4. The Swedish TSC population – A retrospective registry analysis Carlqvist P. Welin K-O. Lawenius E., Althin R. Wilén-Koort A. Rask, O. Novartis Sverige AB, Täby, Sweden (PC, EL, AWK) The Swedish Institute for Health Economics, Lund, Sweden (KOW, RA) Department of Clinical Sciences, Lund University, Malmö University Hospital, Malmö, Sweden (OR) Background Tuberous sclerosis complex (TSC) is a rare genetic disease that causes non-malignant tumors to grow in vital organs. Symptoms and severity vary depending on the location of tumors, from hardly noticeable to severe. The treatment of TSC includes surgery and treatments aimed at controlling symptoms, e.g. antiepileptics. Recent studies have shown that pharmaceutical treatment with everolimus may reduce subependymalt giant cell astrocytoma (SEGA) and renal angiomyolipoma (AML). Due to the rarity of the condition, previous research about the epidemiology and treatment of patients with TSC is limited. Objective The objectives of this retrospective registry study were to estimate the prevalence, comorbidities, treatment pathway and mortality of patients with TSC in Sweden. Methods Three National registries were used for this study: the National Patient Registry (NPR) including all inpatient (since 1987) and all outpatient (since 2001) hospital visits, the Swedish Prescribed Drug Registry (PDR) including all prescribed pharmacy drug dispenses, and the Swedish Cause of Death Registry (CDR). All three registries are mandatory to report to, ensuring good coverage. Data from July 2005 to December 2013 was used in the analyses. Prevalence, common manifestations of TSC, and comorbidities were estimated. In addition to manifestations and comorbidities the PDR was used to estimate the use of drugs in the treatment of TSC. Results A total of 545 unique patients with TSC were identified in the NPR during the study period. The estimated prevalence of TSC by the end of 2013 was found to be 5.37 per 100 000 individuals. The mean [SD] (median) age at the first observation of a TSC diagnosis was 20.91 (17) years. Of the 545 patients 9% had a diagnosis of SEGA, which was the most common manifestation of TSC (the diagnoses associated with the largest proportion of patients). Of the defined comorbidities, epilepsy was the most prevalent with 63% having at least one diagnosis associated with epilepsy. Of the 545 identified patients with TSC, a majority (77%) had at least one dispensation of one of the defined drugs (antiepileptics, antipsycotics, anxiolytics, sirolimus, takrolimus, and everolimus). The most common was antiepileptics followed by anxiolytics, 12% were treated with either sirolimus or

6. Clinical report: a rare co-occurrence of tuberous sclerosis complex and Rett syndrome in a girl with mental retardation, epilepsy and autism.ve proposal Elena D. Belousova, Marina Y. Dorofeeva, Dmitrii V. Vlodavetz Department of Psychoneurology and Epileptology, Research and Clinical Institute for Pediatrics, Pirogov Russian National Research Medical University, Moscow, Russia; 2, Taldomskaya str, Moscow, 127412, Russia Introduction. There are some genetic disorders with combination of mental retardation, epilepsy and autism in which the abnormal m-TOR signaling is implicated. The most important of them is tuberous sclerosis complex (TSC), but the disturbances of m -TOR pathway can also be detected in Rett syndrome (RTT), Fragile X syndrome and Dawn syndrome. We are describing the rare case of co-occurrence of TSC and RTT. Case study. The female child was born at term by normal delivery after non-complicated pregnancy. Family history was negative for epilepsy and mental retardation. Neonatal period was uneventful and psychomotor development normal before the child became 1,5 years old. At the age of 18 months the girl developed hand-wringing stereotypes, facial hypotonia, ataxia and gait apraxia. She loosed verbal contact with relatives, eyeto-eye contact and became indifferent to the surrounding environment. When she was 2 years old focal adversive seizures started, they were easily controlled with carbamazepine. Cerebral cortical and subcortical tubers, cerebral white-matter radial migration lines and subependymal

nodules on brain MRI together with hypomelanotic macules suggested the presence of TSC. Diagnosis was confirmed at age of 3 years by a heterozygous mutation c.5161-2A>G in TSC2 gene on chromosome 16p13. But the rude regression of psychomotor development and speech, autistic features alongside with characteristic hand-wringing stereotypes were unexplained until at age of 4,5 years RTT was diagnosed by finding a heterozygous missense mutation in exon 4 of the MECP2 gene c.455C>T, resulting in a P152R substitution in the methyl-binding domain of the protein. At age of 5 the patient is not able to walk independently and has no expressive speech, she is autistic, has ataxia, limb rigidity, hyperreflexia, lack of purposeful hand movements, verbal and motor stereotypies. Discussion. Presence of two mutations (one characteristic for TSC2 and one – characteristic for RTT) significantly worsened the developmental and motor delay and autistic features in our patient. This may be due to well established m-TOR activation in TSC and recently described in animal models of RTT reduced m-TOR signaling (S. Ricardi et al, 2011).

models of TSC2-deficiency. We reveal that multiple TSC2-null cell lines are sensitive to nelfinavir and bortezomib cytotoxicity through ER stress-mediated cell death, while normal cells are able to tolerate this drug combination through intact cell survival mechanisms. We show that the two drugs synergise and can significantly block the growth of TSC2-deficient tumour spheroids. Repositioning these two clinically relevant drugs could prove beneficial for the treatment of tuberous sclerosis complex. 9. Lessons from the TSC1 and TSC2 Variation Databases Rosemary Ekong, Nicola Migone, Ivo Fokkema, Mark Nellist, Steve Sparagana, Johan den Dunnen, Sue Povey Department of Genetics, Evolution and Environment, University College London, UK (RE, SP) Department of Genetics, Biology and Biochemistry, University of Turin, Italy (NM) Human and Clinical Genetics, Leiden University Medical Center, The Netherlands (IF, JdD) Functional Unit, Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands (MN) Texas Scottish Rite Hospital for Children, 2222 Welborn Street, Dallas, TX 75219 (SS)

7. New Pathways: Improved molecular characterization of the Burt Hogg Dubé tumour syndrome Peter F Doubleday1,3, Dr Elaine A Dunlop1, Dr Bryan A Ballif2, Dr Andrew R Tee1

The publicly available Tuberous Sclerosis Variation Databases (www.lovd.nl/TSC1 and www.lovd.nl/TSC2) have contributed to progress in the clinical care of patients and to advances in the understanding of the function of the gene products. The ability to collect information on TSC1 and TSC2 variants from various sources, and from different parts of the world into one resource, as well as integrate data from large population databases, has made it possible to reach conclusions that would otherwise have been difficult to make if such information were held separately. To date, we have 807 different TSC1 and 2363 different TSC2 variants, including >442 not yet displayed but available on querying. These include large rearrangements and somatic variants found in other conditions (bladder cancer, renal cell carcinoma, AML and LAM). Germline variants have also been seen in Autism spectrum disorder (ASD). Apart from published variants, a large proportion of the variants in the TSC databases now come from diagnostic labs. Some findings that have been possible through the TSC databases are that we have been able to clearly ascertain two TSC2 exons (25 and 31 as currently described by the TSC community) in which no pathogenic variants have been confirmed in clinically diagnosed TSC patients. Various sources of data indicate that many human adult tissues lack these exons in the majority of TSC2 transcripts and we suggest that variants within these exons are unlikely to cause clinically diagnosable TSC. Other findings include pathogenic intronic variants in regions previously given little attention; a number of exonic predicted missense variants that cause aberrant splicing and are probably pathogenic; and the classification of variants previously designated as ‘unclassified’ by integrating data from large multi-population databases. An overview of the TSC databases, our criteria for pathogenicity assessment and lessons learnt will be presented. The TSC variation databases are funded by the TSA and the TS Alliance.

1. Institute of Cancer and Genetics, University of Cardiff, Wales; 2. Department of Biology, University of Vermont, Burlington; 3. Department of Molecular Biosciences, Northwestern University, Chicago. Highly related to Tuberous Sclerosis Complex, the Burt Hogg Dube tumour syndrome is caused by germline loss-of-function mutations in the FLCN gene, leading to fibrofolliculomas and an increased predisposition to renal neoplasm development. The FLCN gene product has been shown to participate in mTORC1, AMPK and ULK1 mediated signaling events; however, further functional characterization is required to elucidate disease progression and improve treatment options. Therefore, here, we use proteomic approaches to identify targets of FLCN and FLCN post-translational modifications which have not been previously described. We also use a cell culture-based model of disease progression to better understand how loss of functional FLCN may promote tumourigenesis at the molecular level. 8. Loss of TSC2 sensitises cells to nelfinavir-bortezomib dual therapy Elaine A. Dunlop*, Charlotte E. Johnson*, Julian R. Sampson, Rachel J. Errington, D. Mark Davies, Andrew R. Tee Institute of Cancer and Genetics, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK. Rapalogs have proved to be a successful treatment for tuberous sclerosis complex (TSC) tumours, resulting in a significant reduction in the size of renal angiomyolipomas in TSC and sporadic LAM patients. However, tumours often regrow following cessation of treatment, so current research is focussing on more cytotoxic drug treatments. TSC2-deficient cells are known to have elevated levels of endoplasmic reticulum (ER) stress due to hyperactivity of mammalian/mechanistic target of rapamycin complex 1 (mTORC1). We hypothesised that a further enhancement of ER stress using nelfinavir, in combination with simultaneous blockade of the proteasome survival pathway using bortezomib, would selectively target TSC2-deficient cells. This drug combination has already proved cytotoxic to breast cancer, acute myeloid leukaemia, non-small cell lung cancer and myeloma cell models (Shim et al., 2012, Kraus et al., 2014, Kawabata et al., 2012) and has entered clinical trials as a cancer therapy. This study evaluates the efficacy of combined nelfinavir and bortezomib drug cytotoxicity within genetic cell

10. The clinical and genetic outcome of an antenatal diagnosis of cardiac rhabdomyoma Frances Elmslie (1), Baskaran Thilaganathan (2), Aris Papageorghiou (2) Tessa Homfray (1) 1.

South West Thames Regional Genetics Service, St George’s University Hospitals NHS Foundation Trust 2. Fetal Medicine Unit, St George’s University Hospitals NHS Foundation Trust A number of studies have suggested that an antenatal diagnosis of cardiac rhabdomyoma is associated with a diagnosis of

Tuberous Sclerosis in 50 to 88% of cases (1,2). We followed up a group of 17 women with an antenatal diagnosis of cardiac rhabdomyoma who presented to our fetal medicine unit over a period of 10 years. None had a prior family history of tuberous sclerosis. We found that 16 of the 17 pregnancies were definitely affected by tuberous sclerosis (94%), following further investigation, in 1 of the 17 the diagnosis remains uncertain. We will discuss the antenatal and genetic findings, the natural history and the pregnancy-related decisions made by this group of patients, and suggest guidelines for management.

other settings, and also appeared to be more likely to receive imaging studies suggested by disease treatment guidelines. 12. Everolimus For Fast Expanded aCcess in TSC SEGA (EFFECTS) Andras Fogarasi, Liesbeth De Waele, Gabriella Bartalini, Sergiusz Jozwiak, Nicola Laforgia, Helene Verhelst, Borivoj Petrak, Jean-Michel Pedespan, Olaf Witt, Ramon Castellana, Stefania Crippa, Gabriella Gislimberti, Zsuzsanna Gyorsok Bethesda Children’s Hospital, Budapest, Hungary (AF); National Institute of Neurosciences, Budapest, Hungary (ZG) Background: EXamining everolimus In a Study of Tuberous sclerosis complex (EXIST-1), a randomized, double-blind, phase 3 study demonstrated the efficacy and safety of everolimus (a mammalian target of rapamycin inhibitor) in the treatment of subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex (TSC). Here, we present the results of the Everolimus For Fast Expanded aCcess in TSC SEGA (EFFECTS) study, which was designed to provide everolimus access to patients with SEGA associated with TSC and to assess the safety and efficacy of everolimus in a real-world setting. Methods: Patients (3 years of age or older) with a definite diagnosis of TSC, and with at least 1 SEGA lesion were eligible to be included in this phase 3b, multicenter, expanded access study. Eligible patients received once-daily everolimus, the dose of which was adjusted to attain a trough level of 5 to 15 ng/mL. The primary objective was evaluation of safety, which included collection of adverse events (AEs) and serious AEs with their severity and relationship to everolimus. The secondary objective was evaluation of efficacy and was based on the best overall response as per medical judgment at the end of the study. Results: One hundred of the 120 enrolled patients (83.3%) completed the study. Median age of patients was 11 years (range, 1-47). Median daily dose of everolimus was 5.82 mg (range, 2.0-11.8) and median duration of exposure was 56.5 weeks (range, 0.3-130). Overall, 89 (74.2%) patients had at least 1 AE. The most common AEs reported were aphthous stomatitis (n = 18, 15.0%), pyrexia (n = 18, 15.0%), bronchitis (n = 11, 9.2%), and stomatitis (n = 10, 8.3%). Grade 3 and grade 4 AEs were reported in 25 (20.8%) and 3 (2.5%) patients, respectively. Suspected drug-related AEs were reported in 62 (51.7%) patients, of which 15 (12.5%) were of grade 3 or 4. AEs lead to drug discontinuation in 8 (6.7%) patients. With regard to the best overall response, a partial response was seen in 81 (67.5%) patients, a stable disease in 35 (29.2%) patients, and disease progression in 1 (0.8%) patient. In 3 (2.5%) patients, the response was unknown. Conclusion: The results of EFFECTS confirm the acceptable safety profile of everolimus in patients with SEGA associated with TSC in a real-world setting. In addition, this study further supports the efficacy of everolimus in the treatment of SEGA associated with TSC. (EudraCT: 2010022583-13)

References 1. 2.

Harding and Pagon, Am J Med Genet 1990; 37(4):443-6 Chao et al, Ultrasound Obstet Gynecol 2008; 31(3):289-95

11. Disease Management in a Rare Disease: Comparisons of Tuberous Sclerosis Complex (TSC) Patients Treated in TSC Centers of Excellence (COE) versus Other Settings Nicole M. Engel-Nitz PhD, Cori Blauer-Peterson, MPH, Jonathan C. Johnson, MS, Aylin Altan, PhD, Zhimei Liu, PhD, Tanjala T. Gipson, MD, E. Steve Roach, MD, James Wheless, MD Optum, Eden Prairie, MN (NEN, CBP, JJ, AA); Novartis Pharmaceuticals Corporation, East Hanover, NJ (ZL); Nationwide Children’s Hospital, Columbus, OH (SR); Kennedy Krieger Institute, Baltimore, MD (TG), Le Bonheur Children’s Hospital, Memphis, TN (JW) Background: TSC Centers of Excellence (COE) provide multidisciplinary teams of providers with experience treating the multi-system conditions associated with TSC. Understanding which patients are treated at TSC COE versus in other settings may illuminate the benefits associated with an integrated disease management program. Objective: To compare the characteristics and treatment patterns for patients with TSC treated in TSC COE versus other settings. Methods: This retrospective study used medical and pharmacy claims and enrollment data from 2 large US national health plan databases. Included TSC subjects (ICD-9-CM 759.5x) were identified between Jan 2000- Dec 2011 and had 6 months preand 12 months post-index health plan enrollment. Index dates were first TSC COE visit or a date of a visit for TSC-related conditions for patients not treated in COE. Patients were stratified into three cohorts: patients who had =1 visit to a TSCCOE; patients who were treated by providers associated with TSC COE, but not at the COE sites; other included all other TSC patients. Results: Among TSC patients, 20% (n=324) were treated in a TSC COE, and remainder by associated providers (14%, n=225) or other providers (67%, n=1,108). COE-treated patients were younger (mean 18 years) than the associated provider group (mean 35 years) or the other cohort (mean 37 years; p<0.001 for each vs. COE). COE-treated patients were more than twice as likely to have a seizure disorder, with 31% vs. 14% in the associated provider and 12% in the other cohort (p<0.001 each vs. COE). More COE-treated patients had TSCrelated cardiac conditions (6%, vs. 5% associated provider [NS] and 4%, other, p=0.039), as well as ocular disorders (4% COE vs. <1% associated provider and other, p<0.05). Dermatologic and renal disorders were frequent but not statistically different across the cohorts. COE-treated patients were twice as likely to receive renal (19%) and brain scans (19%) as patients in the associated provider and other cohort (p<0.01 for each vs. COE). Conclusions: Patients treated in TSC-COE appeared to be younger and had more severe disease than patients treated in

13. Everolimus for subependymal giant cell astrocytoma associated with tuberous sclerosis complex: final long-term results from EXIST-1 David N. Franz, Elena Belousova, Steven Sparagana, E. Martina Bebin, Michael Frost, Rachel Kuperman, Olaf Witt, Michael H. Kohrman, J. Robert Flamini, Joyce Y. Wu, Paolo Curatolo, Petrus J. de Vries, Noah Berkowitz, Julie Niolat, Sergiusz Jozwiak Cincinnati Children’s Hospital (DNF); Moscow Research Institute of Pediatrics (EB); Texas Scottish Rite Hospital (SS); UAB School of Medicine (EMB); Minnesota Epilepsy Group (MF); Children’s Hospital and Research Center (RK); University of Heidelberg Medical Center (OW); University of Chicago (MHK); Children’s Healthcare of Atlanta (JRF); Mattel Children’s Hospital (JYW); University of Rome Tor Vergata (PC); University of Cape Town (PJVD); Novartis (NB, JN); Medical University of Warsaw (SJ)

improvement; Grade 3-1=moderate improvement to almost clear; Grade 0=completely clear). Assessments were conducted every 12 weeks. Skin lesions included hypomelanotic macules, shagreen patches, periungual or subungual fibromas, facial angiofibromas, and forehead plaques. Skin lesion response rate was defined as the proportion of patients achieving either complete clinical response (Grade 0) or partial response (Grade 1-3) and confirmed by a second assessment. Results: In total, 111 patients received =1 dose of everolimus; 105 (57% male, median age 9.48 years) had presence of =1 skin lesion at baseline. Median duration of exposure in this group was 47 months (range, 1.9-58.3 months) at study completion. Best overall skin lesion response rate was 58.1% (n=61/105; 95% confidence interval: 48.1-67.7). Complete clinical response was noted in 8.6% of patients (n=9) and partial response was noted in 49.5% (n=52). Single assessments of partial and complete clinical responses were observed as early as week 12 and 24 of everolimus treatment, respectively. After week 48, the proportion of responders remained stable at around 50%. Overall, the most commonly reported AEs in this group (>30%) were stomatitis (45.7%), convulsion (36.2%), nasopharyngitis (35.2%), and mouth ulceration (34.3%). Conclusion: Systemic everolimus treatment improved the appearance of TSC-related skin lesions with responses sustained over 4 years of treatment.

Objective: Oral mTOR inhibitors are indicated for treatment in pediatric and adult patients with tuberous sclerosis complex (TSC) who have subependymal giant cell astrocytoma (SEGA) that requires therapeutic intervention but cannot be curatively resected. Everolimus, an mTOR inhibitor, has demonstrated efficacy in reducing SEGA volume. However, long-term use of mTOR inhibitors may be necessary. This analysis explored the long-term efficacy and safety of everolimus in treating SEGA associated with TSC from the conclusion of the EXIST-1 study (NCT00789828). Methods: Patients with new or growing TSCassociated SEGA were randomly assigned to receive everolimus 4.5 mg/m2/day (titrated to a target blood trough of 5–15 ng/mL) or placebo. Upon achieving positive results for the primary endpoint (SEGA response rate) during the core phase (cutoff March 2, 2011), all remaining patients were offered open-label everolimus in an extension phase. SEGA response rate was defined as the proportion of patients achieving a confirmed response of =50% reduction in the sum volume of target SEGA lesions from baseline in the absence of worsening non-target SEGA lesions, new target SEGA lesions, and new or worsening hydrocephalus. Results: In total, 111 patients (median age 9.5 years) received =1 dose of everolimus. Median duration of everolimus exposure was 47.1 months (last patient last treatment, October 2, 2014). SEGA response rate increased from 34.6% in the core phase to 57.7% (95% confidence interval [CI], 47.9-67.0) at study conclusion. SEGA progression was observed in 13 patients (11.7%). Estimated progressionfree survival rate at 3 years was 88.8% (95% CI, 80.6%-93.6%). Incidence of adverse events (AEs) was comparable to previous reports and generally decreased over time. Most common AEs (=20%) were stomatitis (44.1%), convulsion (36.9%), nasopharyngitis (35.1%), mouth ulceration (34.2%), pyrexia (28.8%), vomiting and cough (26.1% each), pneumonia and upper respiratory tract infection (25.2% each), and diarrhea (24.3%). Eleven patients (9.9%) experienced an AE that led to study discontinuation. Conclusions: Everolimus showed increased tumor response over the course of prolonged treatment, with a stable and expected safety profile during more than 4 years of treatment for TSC-associated SEGA.

15. Examination of patients with progression while treated with everolimus for subependymal giant cell astrocytoma: results from the final 4-year analysis of EXIST-1 David N. Franz, Elena Belousova, Steven Sparagana, Paolo Curatolo, Noah Berkowitz, Julie Niolat, Sergiusz Jozwiak Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA (DNF); Moscow Research Institute of Pediatrics & Pediatric Surgery, Moscow, Russia (EB); Texas Scottish Rite Hospital for Children, Dallas, TX, USA (SS); University of Rome Tor Vergata, Rom Objective: To examine patients who experienced progression of subependymal giant cell astrocytoma (SEGA) during treatment with everolimus for SEGA associated with tuberous sclerosis complex (TSC) from the final 4-year analysis of EXIST-1. Methods: Patients of any age with new or growing TSCassociated SEGA were randomly assigned to receive everolimus 4.5 mg/m2/day (titrated to trough blood concentration of 5-15 ng/mL) or placebo. After the results from the double-blind core phase favored everolimus (cutoff date March 2, 2011), all remaining patients were offered open-label everolimus in an extension phase. SEGA progression was defined as an increase in SEGA volume =25% from the lowest value to a value greater than baseline, and/or the appearance of new SEGA lesions =1 cm in longest diameter, worsening of non-target lesions, or new or worsening hydrocephalus. Results: In total, 111 patients received =1 dose of everolimus. Over the ~4 year treatment (study completion date October 2, 2014), 64 patients (57.7%) achieved a best overall SEGA response of “response.” SEGA progression was noted in 13 patients (11.7%, 7 males and 6 females, median age 4.85 [range, 1.1-9.6] years, median duration of everolimus exposure 44 months), and occurred approximately 250 to 1700 days after everolimus initiation. Only one patient discontinued everolimus treatment due to SEGA progression. For 6 patients, progression was detected at the end of treatment visit after everolimus had already been discontinued for other reasons (completed treatment [n=2], adverse event [n=2] noncompliance [n=1], withdrew consent [n=1]). AEs leading to discontinuation were sinus infection (grade 1) in 1 patient, and viral infection (grade 2), blood alkaline phosphatase increased (grade 2), and acinetobacter bacteremia (grade 3) in 1 patient. In another 6 patients, although progression was noted, everolimus was continued at the discretion of the investigator. Five of the 13 patients who progressed had achieved a prior SEGA response. Conclusions:

14. Effect of everolimus on skin lesions in patients being treated for subependymal giant cell astrocytoma: results from the final 4-year analysis of EXIST-1 David N. Franz, Elena Belousova, Steven Sparagana, Paolo Curatolo, Noah Berkowitz, Julie Niolat, Sergiusz Jozwiak Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA (DNF); Moscow Research Institute of Pediatrics & Pediatric Surgery, Moscow, Russia (EB); Texas Scottish Rite Hospital for Children, Dallas, TX, USA (SS); University of Rome Tor Vergata, Rome Background: Skin lesions are a common manifestation of tuberous sclerosis complex (TSC). The randomized, doubleblind, placebo-controlled, phase III EXIST-1 study (NCT00789828) examined the effect of everolimus on TSCassociated subependymal giant cell astrocytoma (SEGA) with its effect on TSC-associated skin lesions as a secondary objective. We present long-term skin lesion data as of the study conclusion (October 2, 2014). Methods: Patients of any age with new or growing TSC-associated SEGA were randomly assigned to receive oral everolimus 4.5 mg/m2/day (titrated to target trough 5-15 ng/mL) or matching placebo. Upon achieving positive results for the primary endpoint (SEGA response rate) during the core phase (cutoff March 2, 2011), all remaining patients were offered open-label oral everolimus in an extension phase. Using the Physician’s Global Assessment of Clinical Condition, investigators evaluated severity of skin disease compared to baseline on a 7-point grading scale (Grade 6=worse than baseline; Grade 5-4=no change or slight

During this long-term study of everolimus in a population with new or growing SEGA, only a small percentage of patients had SEGA progression. Only one patient discontinued treatment due to SEGA progression, whereas half of the others continued at the discretion of the investigator, suggesting that everolimus was still providing benefit despite progression.

everolimus is a systemic therapy and patients with TSC often have multiple organs affected, an exploratory objective was to examine changes in subependymal giant cell astrocytomas (SEGAs) in this population. Methods: 118 adult patients with renal angiomyolipoma associated with TSC (n=113) or sLAM (n=5) were randomly assigned 2:1 to 10 mg/day everolimus (n=79) or placebo (n=39). After results from the core phase (cutoff June 30, 2011) favored everolimus for renal angiomyolipoma response rate (primary endpoint), all remaining patients were offered open-label everolimus in an extension phase. In patients with SEGA at baseline, the SEGA response rate was defined as the proportion of patients with best overall SEGA response of “response” (=50% reduction in the sum volume of target SEGA lesions from baseline in the absence of worsening non-target SEGA lesions, new target SEGA lesions, and new or worsening hydrocephalus, confirmed by a second scan). Adverse events (AEs) were monitored at each visit. Results: In total, 112 patients with renal angiomyolipoma received =1 dose of everolimus; 50 had target SEGA lesions (longest diameter =1 cm) at baseline. Median duration of everolimus exposure in this group was 40.6 months (range, 0.5-57.3). As of the longer-term cutoff date (April 1, 2014), 40% of patients achieved SEGA response (n=20/50; 95% confidence interval, 26.4-54.8) and 50% of patients achieved stable disease (n=25/50), an increase from 10.3% (n=4/39) in the core phase. The percentage of patients with =50% reduction in SEGA volume increased from 13.5% at week 12 (n=5/37) to 55.9% at week 144 (n=19/34). By week 192 (n=13), 53.8% exhibited =50% reduction in SEGA volume. The most commonly reported AEs (=30%) were stomatitis (46%), nasopharyngitis and urinary tract infection (36% each), acne and hypercholesterolemia (34% each), and headache (30%). Conclusions: Treatment of TSC- or sLAM-associated renal angiomyolipoma with everolimus provided an added benefit for patients also diagnosed with SEGA. In addition to reducing angiomyolipoma volume, everolimus treatment reduced SEGA volume, and the proportion of patients achieving SEGAresponse increased over approximately 3.5 years of treatment.

16. The effect of everolimus on growth and sexual maturation in patients treated for subependymal giant cell astrocytoma associated with tuberous sclerosis complex: results from the 4-year final analysis of EXIST-1 David N. Franz, Rachel Kuperman, Joyce Y. Wu, Noah Berkowitz, Julie Niolat, Steven Sparagana Cincinnati Children’s Hospital Medical Center, Cincinnati, OH (DNF); Children’s Hospital and Research Center, Oakland, CA (RK); Mattel Children’s Hospital at UCLA, Los Angeles, CA (JYW); Novartis Pharmaceuticals Corporation, East Hanover, NJ (NB); Novartis Pharmaceuticals S.A.S., Rueil-Malmaison, France (JN); Texas Scottish Rite Hospital for Children, Dallas, TX (SS) Objective: To assess the effect of everolimus on growth and sexual maturation in patients with subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex in the EXIST-1 study (NCT00789828). Methods: Patients with =1 SEGA lesion (longest diameter =1 cm) were randomized (2:1) to 4.5 mg/m2/day everolimus (target trough 5-15 ng/mL) or placebo. After achieving positive results for the primary endpoint (SEGA response rate, cutoff March 2, 2011), all patients were offered open-label everolimus in an extension phase. Tanner stage assessments for sexual development were completed annually. Patient growth (in patients aged <18 years) was assessed through standard deviation scores (SDS) for height and weight. Results: In total, 111 patients (median age 9.5 [range, 1.1-27.4] years) received =1 dose of everolimus. At study end (October 2, 2014), the median duration on everolimus was 47.1 months (range, 1.9-58.3). More than half were at Tanner stage 1 at baseline (34/64 male patients, 27/47 female patients for breast development, and 25/47 female patients for pubic hair). Normal progression in Tanner stage was usually seen; median age to attain Tanner stage 2 was 12.1 years for genitalia and 12.0 years for pubic hair in males (range, 9.5-14.9), and 10.4 years for breast development and 11.0 years for pubic hair in females (range, 6.6-13.0). SDS for height and weight in patients aged <18 years were comparable prior to and after starting everolimus. Conclusion: Final clinical data from more than 4 years of treatment in EXIST-1 did not show an impact of everolimus on growth and development or sexual maturation.

18. Monitoring testing patterns among patients with tuberous sclerosis complex-related angiomyolipomas Elyse Swallow, MPP; Sarah King, BA; Jinlin Song, PhD; Miranda Peeples, BA; James Signorovitch, PhD; Zhimei Liu, PhD; Judith Prestifilippo, MD; Michael Frost, MD; Michael Kohrman, MD; Bruce Korf, MD, PhD; Darcy Krueger, MD, PhD; Hope Northrup, MD; Steven Sparagana, MD, Analysis Group, Inc. (ES, SK, JS, MP, JS); Novartis Pharmaceuticals Corporation (ZL, JP); Minnesota Epilepsy Group (MF); University of Chicago (MK); University of Alabama at Birmingham (BK); Cincinnati Children's Hospital Medical Center (DK); University of Texas Medical School at Houston (HN); Texas Scottish Rite Hospital for Children (SS)

17. Effect of everolimus on subependymal giant cell astrocytoma in patients being treated for renal angiomyolipoma: results from the 3.5-year update of EXIST-2 Michael Frost, John J. Bissler, Klemens Budde, Elena Belousova, Noah Berkowitz, Severine Peyrard, J. Christopher Kingswood, E. Martina Bebin

Introduction: The 2012 International Tuberous Sclerosis Complex Consensus Group updated surveillance and management guidelines for tuberous sclerosis complex (TSC)related angiomyolipomas (TSC-AML) to recommend monitoring with magnetic resonance imaging (MRI). Previous guidelines from 1999 had recommended monitoring TSC-AML with renal ultrasonography every 1-3 years. This study used the TSC Natural History Database to assess trends in TSC-AML monitoring over time. Methods: This study analyzed TSC-AML patients in the TSC Natural History Database, which contains information on demographics, clinical features, diagnostic and follow-up tests, and treatments for more than 1,300 TSC patients enrolled in 16 participating clinics since 2006. The analysis included imaging tests conducted following angiomyolipoma diagnosis. We assessed the frequency of tests by type and calendar year (2000-2012). Additionally, we assessed monitoring sequences over the first 3 years after TSC-

Minnesota Epilepsy Group, St. Paul, MN, USA (MF); St Jude Children’s Research Hospital and Le Bonheur Children’s Hospital, Memphis, TN, USA (JJB); Charité Universitätsmedizin, Berlin, Germany (KB); Moscow Research Institute of Pediatrics and Pediatric Surgery, Moscow, Russia (EB); Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA (NB); Novartis Pharmaceuticals S.A.S., Rueil-Malmaison, France (SP); Royal Sussex County Hospital, Brighton, UK (JCK); University of Alabama School of Medicine Objective: EXIST-2 (NCT00790400), a double-blind, placebocontrolled, phase III trial, evaluated everolimus in treating renal angiomyolipoma associated with tuberous sclerosis complex (TSC) or sporadic lymphangioleiomyomatosis (sLAM). Because

AML diagnosis for the subset of patients diagnosed with TSCAML in 2006 or later with at least 3 years of data following that diagnosis. Results: Among the 621 patients with TSC-AML, median age at TSC diagnosis was 0.7 years, and median age at AML diagnosis was 9.8 years. Nearly half (46%) were male and a majority (77%) were white. Most patients (n=521, 84%) had at least 1 monitoring test. The most commonly used tests were MRI (65% of patients), ultrasound (62%), and CT (41%). Between 2000 and 2012, MRI made up an increasingly large proportion of the total number of monitoring tests, while ultrasound declined as a proportion of all tests. Of the 521 patients with at least 1 test, 236 (45%) were diagnosed with angiomyolipoma in 2006 or later; of these patients, 179 (76%) had at least 3 complete years of observable data following diagnosis. Among these 179 patients, 92% received an imaging test during the first 3 years of follow-up; 51% received an MRI during the first 3 years. Conclusions: In this analysis of TSC-AML patients, a majority of patients received at least 1 imaging test in the 3 years following angiomyolipoma diagnosis, consistent with 1999 guidelines. MRI was increasingly used over the study period, though only half of the patients received an MRI in the first 3 years of follow-up. Given that the 2012 guidelines recommend MRI surveillance of TSC-related renal disease, it is possible that MRI use will increase over time.

20. Ganglioside D3 overexpression in TSC deficient tissues: relevance to immunotherapy Steven W. Henning, Adam Hammer, Levi Barse, Jonathan M. Eby, Edward Kessler, Emily Gilbert, Daniel F. Dilling, Maria Picken, I. Caroline Le Poole Departments of Pathology and Medicine/ Oncology Research Institute, Loyola University Chicago, Chicago (IL), USA (SWH, AH, LB, JME, EK, EG, DFD, MPI, ICLP) Hyperactivity of mTORC1 induces cholesterol synthesis and formation of membrane lipid rafts that incorporate ganglioside D3. We hypothesized that loss of TSC-1 or TSC-2 function may be associated with consistent overexpression of this glycosphingolipid on the surface of affected cells. Overexpression of GD3 has likewise been reported for neuroendocrine tumor cells, and its expression has been exploited to develop immunotherapy. Specifically, antibodies to GD3 have been engaged in stage I-III clinical trials to treat melanoma or small cell lung carcinoma. We tested our hypothesis by immunostaining of TSC affected tissues obtained from the NDRI tissue procurement core. We compared GD3 expression, T cell infiltration, NK cell and NKT cell infiltration in 3 tissue samples of affected cutaneous, pulmonary, renal and brain samples compared to 3 normal tissue samples. The abundance of anti-GD3 antibodies in serum samples from 10 affected TSC donors and 5 controls were measured by ELISA. Indirect staining revealed a 5.5-fold increase in GD3 expression in affected pulmonary tissues, whereas 13.2-fold overexpression was observed in angiomyolipomas compared to unaffected kidney tissue. In either case, the presence of TSC affected cells was confirmed by gp100 expression. In brain and skin, overexpression of GD3 was 5.4-fold and 1.4-fold, respectively. In the latter tissues, GD3 overexpression did not co-localize with detection of gp100-expressing cells. Consistent GD3 overexpression was not accompanied by increased T cell, NK cell or NKT cell infiltration to affected kidney or lung tissue. A trend towards increased macrophage infiltration, as reported for TSC affected skin tissue, did not reach significance in our studies. Meanwhile, GD3 overexpression was accompanied by markedly reduced anti-GD3 titers in serum (P<0.001). These studies support the notion that loss of TSC-1 or -2 is consistently associated with overexpression of GD3, but such overexpression is not accompanied by enhanced immune responses to the ganglioside. This finding is of importance as GD3 overexpression is a predictable consequence of loss of TSC function. This lends credibility to the notion that prophylactic vaccination to elicit anti-GD3 immune responses can restrict the outgrowth of tubers and hamartomas in TSC. In this respect, our newly developed DNA vaccine encoding GD3 synthase can serve to activate DCs and elicit NKT cell responses towards GD3 to keep TSC-associated lesions in check.

19. DTI as a biomarker of self-injurious behavior in children with tuberous sclerosis complex Tanjala T. Gipson, Andrea Poretti, Jeffrey Baum, Sarah A. Kelley, Gwendolyn J. Gerner, Kathryn A. Carson, Michael V. Johnston, Thierry A. G. M. Huisman Section of Pediatric Neuroradiology, Division of Pediatric Radiology, Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, USA (AP, JB, TAGMH) Tuberous Sclerosis Center of Excellence, Department of Neurology and Developmental Medicine, Kennedy Krieger Institute, Baltimore, MD, USA (TTG, MVJ) Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA (TTG, MVJ) Department of N Purpose: Self-injurious behavior (SIB) refers to behavior resulting in physical injury to one’s own body and has been observed in tuberous sclerosis complex (TSC). We investigated children with TSC and SIB for volumetric and microstructural changes in selected anatomical brain regions compared to children with TSC without SIB. Materials and Methods: We performed an atlas-based analysis of diffusion tensor imaging data and calculated number of voxels, fractional anisotropy (FA), mean, axial, and radial diffusivity following regions: globus pallidus, putamen, caudate nucleus, amygdala, nucleus accumbens, hippocampus, hypothalamus, postcentral gyrus, and superior parietal gyrus. Abnormalities in these regions have been reported in other diseases with SIB. The diagnosis of SIB was made based on clinical criteria. Regression analysis adjusting for age was performed. Results: We included 6 children with TSC and SIB (mean age 8.9±4.0 years) and 10 children with TSC without SIB (mean age 8.8±4.9 years). In children with TSC and SIB we found a reduced number of voxels for the bilateral globus pallidus (right, 216.17±23.73 vs. 285.20±48.88, p<0.001; left, 219.83±18.62 vs. 276.00±34.34, p=0.001) and caudate nucleus (right, 719.33±112.49 vs. 930.90±198.86, p=0.003; left, 748.33±116.84 vs. 904.90±116.83, p<0.001) as well as reduced FA for the bilateral globus pallidus (right, 0.236±0.014 vs. 0.272±0.016, p<0.001; left, 0.219±0.016 vs. 0.249±0.014, p<0.001) and left caudate nucleus (0.162±0.019 vs. 0.189±0.020, p<0.001) compared to children with TSC without SIB. Conclusion: These findings suggest a specific role for the globus pallidus and caudate nucleus in the pathogenesis of SIB in children with TSC.

21. The combination of rapamycin and resveratrol for treatment of TSC/LAM Anya Alayev, Yang Sun, Peter F. Doubleday, Rachel S. Salamon, Rose B. Snyder, Chenggang Li, Naomi, S. Schwartz, Sara Malka Berger, Bryan A. Ballif, Jane J. Yu, Marina K. Holz Albert Einstein College of Medicine, Yeshiva University, New York, NY (AA, RSS, RBS, NSS, SMB, MKH) Brigham and Women’s Hospital and Harvard Medical School, Boston, MA (YS, CL, JJY) University of Vermont, Burlington, VT (PFD, BAB) Introduction: The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is hyperactivated in TSC and LAM, providing rationale for therapeutic use of mTOR inhibitors (rapamycin or rapalogs). The concern with the use of rapalogs is that they induce autophagy and suppress the negative feedback loop to Akt, which promotes cell survival, causing the therapy to be only partially effective, and disease

progression occurs upon cessation of treatment. Results: We investigated the use of rapamycin in combination with resveratrol, a naturally-derived polyphenol, in TSC2-deficient cells. We tested whether such combination would prevent rapamycin-induced upregulation of autophagy and shift the cell fate towards apoptosis. We found that the treatment blocked rapamycin-induced upregulation of autophagy and restored inhibition of Akt. Interestingly, the combination of rapamycin and resveratrol selectively promoted apoptosis of TSC2deficient cells. The combination of rapamycin and resveratrol produced promising results in in vivo models. First, the addition of resveratrol to rapamycin treatment reduced lung homing of TSC2-deficient cells when tested in mouse tail-vein injection model. Second, resveratrol synergized with rapamycin in reducing TSC2-null xenograft tumor size. Analysis of tumors revealed that rapamycin and resveratrol caused attenuation of PI3K/mTOR signaling, and upregulation of apoptosis, as seen by caspase-3 cleavage and TUNEL staining. Finally, mass spectrometry-based identification of cellular targets of resveratrol revealed specific modulation of the PI3K and mTOR pathway components, converging onto mTORC1 inhibitory protein PRAS40 and S6 kinase 1, providing mechanistic insight into the mode of action of resveratrol. Conclusions: The addition of resveratrol to rapamycin treatment may be a promising option for selective and targeted therapy for diseases with TSC2 loss and mTORC1 hyperactivation.

neurons still lead to seizures. Together these data nicely demonstrate the utility of our new model of epilepsy associated CTs that fully recapitulates the etiology, symptomology, and pathology of the human disease. With this model we have also determined that rapamycin treatment while effective must be life-long. 23. Autism symptoms in toddlers with TSC: Is there a distinctive phenotype that informs treatment targets? Shafali Spurling Jeste, MD Kandice J Varcin, PhD, Scott Huberty Charles Nelson, PhD Amanda Gulsrud, PhD (SSJ, SH, AG): UCLA David Geffen School of Medicine, Center for Autism Research and Treatment (KV, CAN): Harvard Medical School, Boston Childrens Hospital Background: Tuberous Sclerosis Complex (TSC) is strongly associated with cognitive impairment, behavioral disturbances, and autism spectrum disorders (ASD), with rates of ASD ranging from 30-60% (Jeste, 2008). By 12 months of age, delays in both verbal and non-verbal cognition are associated with the development of ASD, and infants later diagnosed with ASD exhibit a decline in non-verbal cognition from 12-36 months (Jeste, 2014). Aims: This study draws from a well-characterized cohort of toddlers with non-syndromic ASD and directly compares their behaviors to a cohort of age-matched toddlers with TSC. This comparison facilitates the identification of a behavioral profile that may be specific to toddlers with TSC regardless of autism diagnostic categorization. We hypothesize that nonverbal communication and motor skills will be more impaired in the TSC group. Methods: 81 toddlers with ASD and 25 toddlers with TSC participated in the study. Participants were matched on chronological age (ASD=31.42 months, TSC= 30.56; F(1,104)=.88, p=.35) and IQ (ASD=68.60, TSC=62.94; F(1,104)=1.35, p=.25). The Autism Diagnostic Observation Schedule (ADOS)- Module 1 and the Mullen Scales of Early Learning were administered. To compare the developmental profile of the TSC and ASD participants we conducted a profile analysis of the ADOS, using a repeated measures ANOVA. Results: There was a significant item * diagnosis interaction (F(28,2828)=1.7, p=.01) indicating a difference in the structure of the profiles between the two groups. There was a main effect of diagnosis (F(28,2828)=67.1, p<.01). On average, the TSC group showed less impairments on the ADOS. Post hoc tests show that profiles diverged most in the domain of social interaction, with the TSC group showing less impairment in eyecontact, social smiling, gesturing, integration of gaze and social overtures, and overall social quality. TSC scored similarly to the ASD group on items related to communication and repetitive behaviors. Conclusions: Future analyses will include dividing toddlers with TSC based on clinical diagnosis of ASD. However, these findings demonstrate that toddlers with TSC exhibit a distinctive behavioral profile characterized by less impairment in eye-contact, social smiling, communicative gesturing and the overall quality of the social interaction. These specific areas of strength help to inform the treatment targets for a new behavioral intervention study for infants and toddlers with TSC.

22. A New Model of Cortical Tuber Associated Epilepsy Lawrence S. Hsieh, John Wen, Kumiko Claycomb, Yuegao Huang, Felicia Harrsch, Janice R. Naegele, Fahmeed Hyder, Gordon F. Buchanan, and Angelique Bordey Departments of Neurosurgery (LSH, JW, AB), Neurology (KC, GFB), and Magnetic Resonance Research Center (YH, FH), Yale University School of Medicine, New Haven, CT; Department of Biology, Wesleyan University, Middletown, CT (FH, JRN) Cortical tubers (CTs) consist of misplaced neurons with abnormal morphology and connectivity. It is commonly associated with seizures in Tuberous Sclerosis Complex (TSC). Despite tremendous advances in our understanding of the etiology behind TSC, mechanisms underlying the development of seizures in TSC patients are still unclear. One of the reasons is because current efforts in modeling seizures associated with TSC do not recapitulate the right context in which CTs give rise to seizures. Often times, models of spontaneous seizures exhibit tubers so severe and global that animals die prematurely. Hence, we set out to develop a novel mouse model of epilepsy associated focal CTs, in which we can identify epileptogenic brain regions and determine components of CTs that are necessary for the development of seizures. Since hyperactivation of mechanistic target of Rapamycin (mTOR) in the brain is likely the underlying cause of neurological syndromes in TSC, we hypothesized that focal upregulation of mTOR signaling in the medial prefrontal cortex (mPFC), a brain region commonly involved in epileptogenesis, should sufficiently mimic seizure development. Using in utero electroporation to deliver DNA encoding a constitutively active mutant of the canonical activator of mTOR â&#x20AC;&#x201C; Ras homolog enriched in brain (Rheb) â&#x20AC;&#x201C; we have successfully generated mPFC tubers associated with spontaneous seizures in mice. Like in humans, these tubers are detectable by magnetic resonance imaging and seizures are detectable by electroencephalography (EEG), which last about 40 seconds on average and show uncanny similarities to human epileptiform activity with postictal slow wave activity. Widespread astrogliosis is also observed only in the epileptic brains. Interestingly, CTs in the somatosensory cortex alone do not cause seizures. Although rapamycin is effective in suppressing seizures and neural dysmorphogenesis in our model, seizures return when treatment stops. Surprisingly, tubers without heterotopic

24. Topical Everolimus for Facial Angiofibromas in the Tuberous Sclerosis Complex. A pilot study Nebojsa J.Jovic, Ana Kosac, Jela Milic, Danina Krajisnik Neurology and Psychiatry Children and Adolescents Clinic, Faculty of Pharmacy, Dept. Of Pharmaceutical Technology and Cosmetology, Belgrade, Serbia Multiple facial angiofibromas are a major feature of tuberous sclerosis complex (TSC). These progressive, disfiguring lesions are present in up to 80% of patients. In the last 5 years, case reports and small case series have reported a beneficial effect of topical use of mammalian target of rapamycin (mTOR) inhibitors on facial angiofibromas.

seizure-free for 3 weeks after ablation but then recurred with daily seizures on 3 medications. All 3 patients who underwent ablations had intracranial monitoring with depth electrodes to confirm localization of onset prior to ablation. All 3 were discharged home the day after the ablation procedure, and none had any perceptible functional deficit on examination. Based on these early data, thermal ablation can result in excellent seizure control with a substantial decrease in hospitalization and recovery time. Longer follow-up is necessary to ensure that the outcomes remain favorable in comparison to resective surgery.

PATIENTS and METHODS: Eight patients, one male and seven females, aged 14– 46 years, diagnosed with definitive TSC and who did not qualify for a systemic mTOR therapy, were enrolled in this study. Two patients had history of ineffective laser therapy. Topical everolimus was prescribed as a compassionate use. Everolimus 0.2% was extracted from tablets and mixed with the ointment and gel. Petrolatum was used in the vehicle. Lesions were treated with the ointment (3 patients) or gel (5) twice daily for 6 months. Subjects were monitored monthly and photographs were taken. Efficacy was evaluated by scoring improvements in redness, papule size and flatness. Study was approved by the local Ethics committee. Informed consent was obtained from all patients or parents. The Quality of Life in Epilepsy Inventory (QOLIE-31) and SF-36® Health Survey, 2002 (SerbianVersion 1.0) were used before and after treatment.

26. Real-world assessment of renal involvement in tuberous sclerosis complex patients in the United Kingdom: a retrospective cohort study in the Clinical Practice Research Datalink Chris Kingswood, Paola Nasuti, Dirk Demuth, Keyur Patel, Clement Erhard, Elizabeth Gray, Alun Pinnegar, Matthew Magestro

RESULTS: Patients tolerated the regimen well. All symptoms, except papule size, began to improve during the 4th week after treatment started. Flatness improved the most. After 3 months, the angiofibromas had improved markedly, including reduction in both number and size of lesions and better facial texture. Blood levels of everolimus were undetectable. Favourable effects were maintained at the 6 month therapy visit. One month after stopping the treatment, redness recurred to 60– 80% of baseline, but the reduction of papule size and flatness continued in 6/8.Two patients experienced one short episode of skin burning and irritation. Facial lesions severity significantly influenced QOLIE-31 domains: overall quality of life, emotional well-being and social function. Data positively correlated with SF-36 (rho = 0.74)

The Royal Sussex County Hospital, Brighton, England (CK); IMS Health, London, UK (PN, DD, KP); CE Analytics, Cambridge, UK (CE); Novartis Pharmaceuticals UK Ltd, Frimley, UK (EG, AP); Novartis Pharmaceuticals Corporation, East Hanover, NJ (MM) Objectives: Renal manifestations are among the most common clinical features of tuberous sclerosis complex (TSC), and these include chronic kidney disease (CKD), polycystic kidney disease (PKD), renal cysts, and angiomyolipomas. This study sought to examine renal involvement in TSC and associated health care resource utilization (HCRU) and costs in the United Kingdom. Methods: TSC patients in the Clinical Practice Research Datalink and linked Hospital Episodes Statistics databases between January 1987 and June 2013 were retrospectively identified. Renal involvement was determined via diagnostic and procedure codes. HCRU and costs were analyzed in a subgroup of TSC patients from April 1997 to March 2012. An agematched cohort was used for comparison purposes. Results: Of 334 patients with TSC, 25.4% (N=85; 54.1% male) had renal involvement (suspected angiomyolipoma or kidney neoplasm [12.0%], CKD [9.9%], hematuria [6.3%], renal cyst [4.8%], or PKD [3.9%]). Anemia developed in 22.4% of these 85 patients. One third (33.8%) of TSC patients aged =18 years had renal involvement vs 4.3% of patients aged <18 years. Of all TSC patients with renal involvement, 29.4% underwent a kidneyrelated surgical intervention (nephrectomy [14.1%], renal embolization [12.9%], kidney transplantation [3.5%], or excision of kidney lesion [7.0%]) and 14.1% had a blood transfusion. Each year, the TSC patients with renal involvement averaged 16.9 general practitioner visits (33.6 visits, including administrative encounters), 1.1 hospitalizations (average length of stay: 4.3 days), and 5.1 specialist consultations. Almost half (45.8%) of these hospitalizations presented via the accident and emergency rooms. Specifically, visits to a nephrologist or urologist occurred on average 1.5 times per patient per year. Conclusion: Compared with other studies, the proportion of patients with TSC who had renal involvement in this retrospective database study was low and may reflect an opportunity to improve patient monitoring and early detection. Nevertheless, the high rate of surgical interventions and CKD is suggestive of substantial morbidity and considerable HCRU and costs. A treatment approach that could modify disease progression would likely reduce morbidity in TSC patients with renal involvement.

CONCLUSIONS: Topical 1% everolimus seems to be a favourable and safe therapeutic option for patients with facial angiofibromas who do not require systemic treatment. Fading of redness occurred and relapsed rapidly, while decreases of size and thickness appeared to be slow but continued. Improvement of the facial lesions positively influenced quality of life of treated patients. 25. Epilepsy management in Tuberous Sclerosis: The role of thermal ablation as a substitute for resective surgery Keough K, Clarke D, Briggs D, Lee M, Berhane R, Sharp K and Perkins F. Dell Children’s Medical Center and University of Texas at Austin Dell Medical School, Austin, TX Thermal ablation is a less invasive alternative for definitive treatment of a medically-refractory seizure focus compared to surgical resection. Tuberous sclerosis (TS) patients often have multiple epileptogenic cortical lesions. Many patients undergo epilepsy surgery and some require multiple simultaneous or sequential procedures. We aimed to define the frequency of epilepsy surgical intervention in our patient population, and to compare the morbidity, duration of hospitalization and seizure outcome of ablation versus resection. We reviewed records of all 27 TS patients evaluated in our comprehensive TS clinic. Four were excluded due to inadequate information. Of 23 patients ranging in age from 9 months to 31 years, four patients (17%) had never experienced clinical seizures. Eight patients (35%) were medically controlled on one or two seizure medications. Eight (35%) have undergone resective surgery, and 3 (13%) have had thermal ablation. Among patients who had undergone resection, 50% were seizure-free on 1-2 seizure medications (follow-up 7 mo-8 years), and 3/8 improved but still had seizures on 1-3 seizure medications (6-31 months follow-up). One has just completed resection and is too early to report outcome. Of the 3 patients who have undergone ablation, two are fully controlled on 2 medications with 7-8 months of follow-up. One of the 3 ablation patients was

27. Effect of everolimus on renal angiomyolipoma in pediatric patients from the final analysis of EXIST-1 J. Christopher Kingswood, David N. Franz, Elena Belousova, Noah Berkowitz, Thomas Brechenmacher, Sergiusz Jozwiak, John J. Bissler

or were newly diagnosed with TSC. TOSCA includes a “core” section to record general information on patients’ backgrounds and subsections to collect additional data on specific disease manifestations. A follow-up observation period of up to 5 years is planned. Baseline data from the entire cohort are presented here. Results: As of September 30, 2014, baseline core data from 2093 patients were available. Median age at consent was 13 years (0-71); median age at diagnosis of TSC was 1 year (069). Molecular testing was done in 902 (43.1%) patients. Subependymal giant cell astrocytomas (SEGAs) were reported in 510 (24.4%) patients; median age at diagnosis was 8 years (051). Of the 422 (82.7%) patients with ongoing SEGAs, 81 (19.2%) had SEGAs growing since previous scans. Subependymal nodules were reported in 1636 (78.2%) patients and cortical tubers in 1721 (82.2%). Epilepsy was reported in 1748 (83.5%) patients; 15.9% of patients with infantile spasms and 35.9% with focal seizures were refractory to treatment. Of the 822 patients for whom intelligence quotient data were available, 451 (54.9%) had mild to profound degree of intellectual disability. Renal angiomyolipomas were reported in 987 (47.2%) patients; median age at diagnosis was 13 years (067). Of the 946 (95.8%) patients with ongoing renal angiomyolipomas, 396 (41.9%) had multiple and bilateral lesions, 329 (34.8%) had lesions > 3 cm, and 204 (21.6%) had growing lesions. Lymphangioleiomyomatosis (LAM) was reported in 144 (6.9%; 8 male and 136 female) patients; median age at diagnosis was 35 years (9-61). Cardiac rhabdomyomas were reported in 717 (34.3%) patients; median age at diagnosis was < 1 year (0-60). Facial angiofibromas were reported in 1199 (57.3%) patients; median age at onset was 6 years (0-67). Conclusions: For a TSC study, 2093 patients represent a large sample. This is not a population based cohort; therefore the prevalence of different features of the disease will reflect the referral patterns of the clinics contributing patients to the cohort. However, future detailed description of their problems and natural history will add valuable insights into the necessity for monitoring, the timing, and indications for treatment in this disease.

Royal Sussex County Hospital, Brighton, UK (JCK); Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA (DNF); Moscow Research Institute of Pediatrics & Pediatric Surgery, Moscow, Russia (EB); Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA (NB); Novartis Pharmaceuticals S.A.S., Rueil-Malmaison, France (TB); Medical University of Warsaw, Warsaw, Poland (SJ); St Jude Children’s Research Hospital and Le Bonheur Children’s Hospital, Memphis, TN, USA (JJB) Objectives: To assess the long-term effect of everolimus on renal angiomyolipoma in pediatric patients being treated for tuberous sclerosis complex (TSC)–associated subependymal giant cell astrocytoma (SEGA). Data from the final analysis of the EXIST-1 study (NCT00789828) are presented. Methods: Patients (any age) with TSC and new or worsening SEGA were randomly assigned (2:1) to receive everolimus 4.5 mg/m2 (target blood trough 5-15 ng/mL) or placebo. After achieving positive results for SEGA response rate (primary endpoint) during the study’s core phase (cutoff March 2, 2011), all remaining patients could receive open-label everolimus in an extension phase. This post hoc analysis focuses on the subset of patients <18 years of age with =1 target renal angiomyolipoma lesion (longest diameter =1.0 cm). Renal angiomyolipoma response rate was defined as the proportion of patients with =50% reduction in renal angiomyolipoma volume relative to baseline, with no new lesions =1 cm in longest diameter, no increase in kidney volume =20% from nadir, and no angiomyolipoma-related bleeding grade =2 (CTCAE, version 3.0). Adverse events (AEs) were assessed every visit and graded using CTCAE, version 3.0. Results: In total, 33 patients aged <18 years with target renal angiomyolipoma received =1 dose of everolimus and were included in this analysis (cutoff October 2, 2014). Median (range) age was 11.5 (5.4-17.5) years. Median duration of everolimus exposure was 44.8 months. Renal angiomyolipoma response rate was 75.8% (95% confidence interval, 57.7%-88.9%). The mean decrease from baseline in the sum of volumes of target angiomyolipoma lesions increased from 47% at week 12 to 71% at week 96, and to 72% at week 240. The most common AEs experienced in =20% of these patients included convulsion and mouth ulceration (45.5% each), stomatitis (42.4%), cough (27.3%), nasopharyngitis (24.2%), and headache, sinusitis, and upper respiratory tract infection (21.2% each). Four patients (12.1%) experienced a drug-related serious AE. Three patients (9.1%) discontinued due to an adverse event. Conclusions: Treatment of TSC-associated SEGA with everolimus provided an added benefit for patients also diagnosed with renal angiomyolipoma. In addition to reducing SEGA volume, everolimus treatment reduced angiomyolipoma volume, and the proportion of patients achieving renal angiomyolipoma response increased over almost 4 years of treatment.

29. Identification of Pro-survival Rapamycin-dependent microRNA (Rapa-miRs) in Tuberous Sclerosis Complex Hilaire C. Lam, Harilaos Filippakis, Katherine A. Cottrill, Alicia Llorente Lope, Ana Pereira1, Douglas A. Medvetz, Damir Khabibullin, Carmen Priolo, Stephen Y. Chan, Elizabeth P. Henske Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Boston, MA, USA (HCL, HF, ALL, AP, DAM, DK, CP, EPH) Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA, US (KAC, SYC) Background: Tuberous sclerosis complex (TSC) is a multisystem, hamartomatous disease associated with tumors of the brain, skin and kidney, as well as progressive cystic lung destruction characteristic of lymphangioleiomyomatosis (LAM). TSC is caused by mutations in TSC1 or TSC2, resulting in hyperactive mechanistic Target of Rapamycin (mTOR) 1 signaling. Rapamycin and related rapalogs inhibit mTORC1 activity and stabilize tumors in TSC patients, but tumor progression proceeds at pretreatment rates following cessation of therapy. MicroRNA repress target gene expression and are commonly dysregulated in cancers. In previously published work, we identified a group of rapamycin responsive miRNA, rapa-miRs. Surprisingly the most upregulated rapa-miRs, miR-21 in particular, are known pro-survival oncogenic miRNA (oncomiRs). We hypothesize that inhibiting Rapa-miRs and downstream Rapa-miR targets may lead to more complete and durable responses to mTORC1 inhibitors. Methods: We utilized qRT-PCR to determine the kinetics of miRNA induction with rapamycin in various cell types and in mouse xenografts. Mimics and inhibitors were used to determine the functional

28. TuberOus SClerosis registry to increase disease Awareness (TOSCA) – baseline data analysis for 2093 patients Anna Jansen, Guillaume Beaure d’Augères, Ramon Castellana, Stefania Crippa, Petrus J. de Vries, Carla Fladrowski, Gabriella Gislimberti, Christoph Hertzberg, Sergiusz Jozwiak, John Lawson, Rima Nabbout, Finbar O’Callaghan, Matthias Sauter, Renaud Touraine, J. Chris Kingswood on behalf of TOSCA Consortium and TOSCA investigators

Pediatric Neurology Unit, UZ Brussel, VUB, Brussels, Belgium (AJ); Sussex Kidney Unit, Royal Sussex County Hospital, Brighton, United Kingdom (JCK) Introduction and Methods: TOSCA is an international disease registry designed to address knowledge gaps in the natural history and management of TSC. Eligible patients had a documented visit for TSC within 12 months before participation

TSC2 cases being more impaired. The genes code for proteins involved in activation of the mTOR pathway. Co-morbid neurodevelopmental disorders such as intellectual disability (ID) and autistic spectrum disorders (ASD) are common, yet little is known about risk factors influencing outcome other than gene mutation. One factor consistently associated with ASD is obstetric risk. The only study of obstetric complications in TS found no difference between TS children and their nonaffected siblings, and no association with ASD outcome. These findings warrant replication controlling for genotype.

consequences of miRNA. Expression profiling has been used to identify miRNA target genes. Bioinformatics and network analysis approaches have identified biological relationships between these miRNA targets. Identification of potential biomarkers has begun by profiling miRNA in LAM patient serum. Results: Our initial screens identified miR-29b, -21, -24, 221, -106a and -199a as candidate rapa-miRs. Rapamycin induced miR-21 greater than 1.5 fold in diverse cell lines and in mouse xenografts. Network analysis using the Consolidated Interactome (CI) revealed that rapa-miRs may inhibit multiple key pro-apoptotic pathways. We have also identified pro-cell death targets, PPIF, TP53BP2 and PDCD4, which are negatively regulated by miR-21 upon rapamycin treatment. These data suggest that rapa-miRs regulate proteins critical for cell fate determination and survival in response to rapamycin treatment. Finally, hierarchical clustering distinguished 4/5 LAM patients from controls in pilot LAM serum miRNA profiles. Conclusions: Rapamycin induces pro-survival oncomiRs, which may be valuable targets for therapeutic intervention in TSC. miRNA may serve as biomarkers in LAM.

Method: This study uses data from the TS2000 Study, a population based, longitudinal study of TS. 125 children diagnosed with TS between 2001 and 2005 were ascertained and genetic testing undertaken. In 2013, consenting participants were assessed for ASD symptoms using the ADI-R, IQ using the WISC and obstetric histories of cases and siblings were assessed using the Obstetric Enquiry schedule (OES) and coded using a Modified Gillberg Optimality Scale (MGOS). Preliminary Results: Data was available for 51 children with a diagnosis of TS and 57 siblings (47% male, mean age: 14 years). Of cases, the average IQ score was 68.9 and mean ADI score was 30.5. 38 cases had known TSC1 or 2 gene mutation (5 TSC1, 33 TSC2). Mean MGOS scores in TS cases was 4.0, and in siblings 3.0. Although TS cases had significantly higher (MGOS) scores compared to siblings (t=2.01, p = .047) the significance was lost in paired sample t-tests. Birth order was not associated with birth optimality, but children with TSC1 gene mutation had significantly poorer birth optimality scores (t=-2.53, p = .016), although this finding is limited by very small number of TSC1 cases. Suboptimal birth had no association with IQ or ADI score.

30. Malignancy in Tuberous Sclerosis Complex Alexandra L. Geffrey (1), Anna M. Larson (1), Magdelena E. Tyburczy, Jan L. Paolini, Elias A. Shaaya, Patrick Li, Sarah F. Pollack, Anat Stemmer-Rachamimov, Norbert J. Liebsch, Elahna Paul, David J. Kwiatkowski, Elizabeth A. Thiele Massachusetts General Hospital, Boston, MA (ALG, AML, JLP, EAS, PL, SFP, ASR, NJL, EP, EAT) Brigham and Women’s Hospital, Boston, MA (MET, DJK) Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that exhibits numerous benign hamartomas in multiple organ systems, including the skin, brain, heart, kidneys, lungs, and eyes. However, malignant pathologies in otherwise typical TSC patients have also been described in numerous case reports and small case series over the past half century. This study reports on the comprehensive examination of a cohort of 448 TSC patients for malignancies. Renal cell carcinoma, pancreatic neuroendocrine tumors, clival chordoma, and breast cancer were the cancer types observed with the greatest frequency. Four TSC patients had multiple malignancies in different tissues over time. The proportion of TSC patients who were found to have a history of malignancy was 7% (31/448), and the median age at diagnosis was 37 years. This population shows both a lower age at diagnosis and a higher 5-year cancer survival rate than the general population. Although the mechanism of the relatively favorable outcome for TSC patients with these malignancies is uncertain, it may relate to early detection due to more intensive medical care in this population, or to the benign nature of these malignancies, which may be similar to the classic hamartomas seen in TSC. Further studies are necessary to explore the genetic basis of malignant development in TSC and the potential benefit of mTOR inhibitors as treatment for these diverse malignancies.

Conclusion: Our data replicates previous non-significant findings and suggest that TSC1 gene mutations may be associated with more suboptimal birth but this finding is limited by small sample size. It may also be the case that as TSC1 cases are generally milder, only those that have additional suboptimal births present to clinicians. Other environmental factors need exploration in differentiating outcome. 32. A case of neonatal subependymal giant cell astrocytoma (SEGA) treated with everolimus Philippe Major, Lucie Côté, Isabelle Goyer, Nagib Dahdah Department of Neurosciences, CHU Sainte-Justine, University of Montreal, Montreal, Quebec, Canada (PM, LC) Department of Pharmacy, CHU Sainte-Justine, University of Montreal, Montreal, Quebec, Canada (IG) Division of Pediatric Cardiology, CHU Sainte-Justine, University of Montreal, Montreal, Quebec, Canada (ND) Subependymal giant cell astrocytomas (SEGAs) affect 10-20% of patients with TSC and they are associated with significant morbidity and mortality. Surgical resection was the only therapeutic option available until successful SEGA shrinkage was reported using mTOR inhibitors. Neonatal SEGAs are very rare and their natural history is poorly understood. We report the effects of off-label everolimus treatment in a neonate with SEGA. This male patient is presently 16 months old and was born at 34 weeks’ gestation. TSC was suspected before birth because of the identification of cardiac rhabdomyomas on prenatal ultrasounds. A brain MRI done five days after birth showed a voluminous SEGA (11x6.2x7mm) near the right foramen of Monro associated with ventriculomegaly. Enteral everolimus 0.1 mg was started nine days after birth. Follow-up MRI at nine weeks showed a reduction in SEGA size (10x4.8x6mm). At three months, the rhabdomyomas were undetectable. At four months, everolimus was stopped for one

31. Can Perinatal Risk Factors influence neuropsychiatric outcome in children with Tuberous Sclerosis? Holan Liang, Fiona S. McEwen, Charlotte Tye, Emma Woodhouse, Lisa Underwood, Henry Rogers, Funmilola Oluwo, Ciara Halpin, Sri Wulandari, Patrick F. Bolton TS2000 Research Team, King’s College London Aims: To assess whether obstetric risk influences neuropsychiatric outcome in children with TSC gene mutation. Background: Tuberous sclerosis (TS) is a genetic disorder caused by a mutation in either the TSC1 or TSC2 gene, with

week due to mouth ulcers. At seven months, despite being on everolimus, the patient started having infantile spasms. These were refractory to vigabatrin and topiramate, but they were stopped by adrenocorticotropic hormone (ACTH). At ten months, he was admitted to the intensive care unit because he required ventilatory support due to a severe pneumonia. Both everolimus and ACTH were stopped. Clobazam, valproic acid and levetiracetam were subsequently added for the treatment of focal seizures. Oral prednisone was started at 14 months because of recurrence of infantile spasms. Brain MRI done at 16 months showed a stable lesion (9x5x6mm) despite having been off everolimus for six months. No TSC-causing mutation was identified. This is a rare case of neonatal SEGA successfully treated with everolimus. Contrary to what is observed with SEGA in older patients, there was no observed regrowth, even six months after being off everolimus. Cardiac rhabdomyomas also rapidly shrunk, but infantile spasms were not prevented by everolimus.

Germany. We performed mutation analyses in a total of 797 individuals who had a definite (492), probable or possible (336) clinical diagnosis of TSC based on the 1998 diagnostic criteria. According to the availability and improvement of mutation detection techniques over the years, different combinations of SSCP, HDA, PTT, Southern blotting, FISH, long range PCR, MLPA, Sanger sequencing and NGS were applied. A probable pathogenic mutation was found in 57% (452/797) of the index patients. The mutation detection rate achieved 68% (336/492) in patients with a definite diagnosis and declined to 38% (116/305) regarding those with a probable or possible diagnosis. Comprehensive sequencing combined with MLPA reached the highest sensitivity with 95%. The spectrum of mutations in each gene was similar to that previously reported with 73% (332/452) occurring in TSC2 and 27% (120/452) occurring in TSC1. The mutations included 59 (13%) variants of unknown significance, seven (1,5%) in TSC1 and 52 (11,5%) in TSC2, representing mostly missense and intronic variants. In 43% (345/797) of patients no mutation was identified (NMI), in 31% (153/492) with a definite clinical diagnosis and in 57% (192/336) with a probable or possible diagnosis. In 12 out of 20 prenatal analyses based on conspicuous ultrasound findings, TSC could be genetically confirmed. Genotype-phenotype correlations were studied for 398 index patients with sufficient clinical information. In accordance with previous studies, renal angiomyolipoma, renal cysts, retinal hamartomas and cardiac rhabdomyomas were all significantly more common in individuals with TSC2 mutations. Similarly, subependymal nodules, SEGAs, facial angiofibroma, hypomelanotic macules and ungual fibromas were predominant in the TSC2 group, however not reaching statistical significance. Although the response to therapies with mTOR inhibitors seems to be independent of mutation type or location the inclusion of genetic testing results in the updated TSC diagnostic criteria emphasises the significance of highly efficient mutation analyses, preferentially NGS in the future, especially in patients with a possible diagnosis allowing early implementation of surveillance and treatment schemes.

33. Reduction in Retinal Nerve Fiber Layer Thickness in Tuberous Sclerosis Complex Sara Marciano(2), Leonardo Emberti Gialloreti(1,6), Romina Moavero(2,3), , Matteo Pardini(4,5), Francesca Benassi(6), Maria Giulia Mutolo(7), Paolo Curatolo(2) Tor Vergata University Hospital,Rome(SM,LEG,RM,PC) Bambino Gesù Children’s Hospital(RM) University of Genoa, Genoa, Italy(MP) CNAPP, Rome, Italy(LEG,FB) University of Rome “Sapienza”(MGM) Abstract Purpose. Diffuse microstructural white matter abnormalities reflecting axonal disorganization, reduced/altered myelination or gliosis have been described in individuals with TSC. Aim of our study was to non-invasively investigate central nervous system axonal integrity in patients with tuberous sclerosis complex (TSC). Optical Coherence Tomography (OCT) is a fast, easy-to-perform, non-invasive and cost-efficient method to assess retinal morphology in vivo and to measure the thickness of the retinal nerve fiber layer (RNFL). Methods. In order to assess central nervous system axonal integrity, 8 subjects with TSC have been investigated by OCT to evaluate RNFL and have been compared with matched healthy controls. Results. When comparing mean overall RNFL thicknesses of the TSC group with the control group, the TSC group presented with significantly lower RNFL values, compared to the control group, in the temporal quadrant (62.5±6.9 vs. 76.9±5.4; t = 14.438; p<0.0001). Conclusions. Since a reduced RNFL thickness might be seen as an indicator of chronic axonal degeneration or lack of appropriate neuronal development, our results support the presence of axonal alterations in TSC, and also that WM disorganization could be much more diffuse than originally thought. Since axonal alterations directly derive from mTOR overactivation, which occurs early during fetus development, the RNFL thinning we observed could represent one of the facets of such early neurodevelopmental abnormalities. Keywords: Tuberous Sclerosis Complex; Retinal nerve fiber layer thickness; Optical coherence tomography (OCT); White matter; axons; mTOR

35. Psychiatric outcomes in the TS 2000 Cohort Study (TS2000): Autistic traits and autism spectrum disorder (ASD) Fiona S. McEwen, Charlotte Tye, Holan Liang, Emma Woodhouse, Lisa Underwood, Henry Rogers, Funmilola Oluwo, Ciara Halpin, Sri Wulandari, Patrick F. Bolton King's College London, Institute of Psychiatry, Psychology & Neuroscience, London, UK (FSM, CT, HL, EW, LU, HR, FO, CH, SW, PFB) South London & Maudsley NHS Foundation Trust, London, UK (PFB) Background: TSC increases the risk for autism spectrum disorder (ASD), although the mechanism is not fully understood. A number of risk factors may be important, such as mutation type, number and location of brain cortical tubers, and epilepsy. Furthermore, there is marked variability in outcome such that some individuals meet full criteria for ASD while others are unaffected. It is not known to what extent the risk for ASD is dichotomous (diagnosis of ASD versus unaffected) or whether there is a more general increase in risk for autistic traits, even in those that do not meet diagnostic criteria for clinical disorder. Aims: (1) To establish the prevalence of ASD in a population-representative sample of children and young adults with TSC; (2) To explore whether the distribution of autistic traits in individuals with TSC shows a bimodal distribution (ASD vs. unaffected) or whether autistic traits are continuously distributed; (3) To investigate which risk factors predict ASD and autistic traits, including mutation (TSC1 vs. TSC2), cortical tuber load, and type and severity of epilepsy. Methods: TS2000 is the first UK population-representative, prospective longitudinal study to chart the development of TSC throughout childhood. All cases identified as newly diagnosed

34. A genetic and clinical overview of 18 years molecular diagnostics of Tuberous sclerosis complex in Germany Karin Mayer, Manuela Scholz, Maria A.K. Schmid, Imma Rost Center for Human Genetics and Laboratory Diagnostics, Martinsried, Germany (KM, MS, IR) Childrens Hospital, LudwigMaximilians-University of Munich, Munich, Germany (MAKS) Tuberous sclerosis complex (TSC) is caused by inactivating mutations in one of both genes, TSC1 or TSC2. This report describes the genetic and clinical findings achieved over a period of 18 years of TSC molecular genetic diagnostics in

during 2001-2006 were recruited (N=125) and have been followed for up to 14 years, with a focus on psychiatric outcomes. In-depth assessments for ASD have been carried out, and medical and genetic data have been collected. Results: Fifty-two percent of participants in TS2000 met criteria for ASD (on parent-report Autism Diagnostic Interview-Revised; AGRE criteria), and a further 23% showed subthreshold autistic traits. A minority (24%) had relatively low levels of autistic traits. Autistic traits were continuously distributed throughout the sample. Mutation was not significantly associated with ASD (31% of TSC1 vs. 53% of TSC2 with ASD; p>.20). Higher levels of autistic traits were associated with higher cortical tuber load (rho=.30; p=.024); epilepsy severity in the 1st (rho=.33; p=.003), 2nd (rho=.44; p<.001), and subsequent (rho=.46, p<.001) years of life, and a history of infantile spasms (d=0.91, p<.001). Conclusions: Children with TSC are at very high risk for ASD, as well as social/communication difficulties that fall short of diagnosis of ASD but might nonetheless be clinically important. More severe epilepsy – particularly infantile spasms – carries a poorer prognosis.

TSC including demographics, genotypes, conditions affecting various organs in the human body, diagnostic and follow-up tests, and treatments. A total of 1,328 individuals were registered in the first iteration of the database. Of these, 40% (n=534) of individuals’ records contained genetic data specifying mutations in the TSC1 or TSC2 gene. Categorical variables were compared between groups using Fisher’s Exact test. Results: Two-thirds of the reported mutations (n=363) were in the TSC2 gene, whereas one-third (n=171) were in the TSC1 gene. Individuals with a TSC1 mutation were significantly older compared to patients with the TSC2 mutation (mean age [SD] = 7.6 [12.0] versus 2.0 [5.9], respectively; p<0.001). No significant difference in gender or race was observed between the two groups. Consistent with other studies and as would be expected from the relatively young age of those with genetic data, neurologic conditions were the most prevalent. Data will be presented comparing various manifestations between genotypes, but generally the prevalence of most manifestations is similar in both groups. Conclusion: The difference in ages of individuals with TSC1 vs. TSC2 mutations may reflect a younger age at diagnosis of individuals with TSC2 mutations, which would be consistent with historical observations that TSC1 mutations may result in a “less severe” phenotype. However, other genetic factors impact progression of TSC including the type of mutation (e.g., missense vs. deletion), somatic mosaicism, and modifier genes. Knowledge of which gene is mutated in a given individual is currently insufficient to predict the manifestations that will arise in a given individual, but research to measure the impact of other genetic factors will undoubtedly lead to a better understanding of individualized risk and, therefore, treatment options.

36. Diffusion tensor imaging reveals white matter abnormalities in tuberous sclerosis patients with refractory seizures Romina Moavero, Antonio Napolitano, Raffaella Cusmai, Federico Vigevano, Lorenzo Figà-Talamanca, Giuseppe Calbi, Paolo Curatolo, Bruno Bernardi Systems Medicine Department, Child Neurology and Psychiatry Unit, Tor Vergata University Hospital of Rome, Italy 2. Neuroscience Department, Neurology Unit, “Bambino Gesù” Children’s Hospital, IRCCS, Rome, Italy 3. Enterprise Risk Management, Medical P

38. Functional analysis of TSC1 and TSC2 variants Marianne Hoogeveen-Westerveld, Rosemary Ekong, Sue Povey, Mark Nellist1.

Background and aims. Tuberous sclerosis complex (TSC) is associated with early onset epilepsy, a high rate of drug resistant seizures, and mental retardation. Aims of this study were to evaluate the effects of early onset and refractory seizures on white matter by using diffusion tensor imaging (DTI). Methods. We enrolled 20 children with TSC and epilepsy onset in the first 3 years of life, and grouped them according to seizure persistence or freedom. All patients underwent brain MRI with DTI. Specific ROIs have been placed to generate tracks to calculate fractional anisotropy (FA) and apparent diffusion coefficient (ADC). Statistical analysis has been performed by ANOVA. Results. Children with persistent seizures presented an overall reduced FA, with statistically significant differences on the temporal trunk, cingulum bundle, external capsula and superior cerebellar peduncles. Conclusions. Our study demonstrates that children with persistent seizures present more significant alterations of brain connectivity in areas crucial for executive functions and verbal abilities, implying a higher risk for deficits in these areas as well as for attention deficit hyperactivity disorder and autism.

Functional Unit, Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands. (MH-W, MN) Department of Genetics, Evolution and Environment, University College London, London, U.K. (RE, SP) In most individuals with tuberous sclerosis complex (TSC) a pathogenic TSC1 or TSC2 mutation can be detected. However, in some cases, variants of uncertain clinical significance are identified. These changes are often non-terminating, causing either amino acid substitutions or in-frame insertion/deletions that are unlikely to prevent TSC1 or TSC2 expression, but could affect the function of the TSC1-TSC2 complex. Investigating the effects of unclassified variants on TSC1-TSC2 activity is a useful adjunct to standard genetic testing and has been implemented as a diagnostic test in our laboratory. To date, we have studied the effects of >80 TSC1 and >200 TSC2 variants on TSC1-TSC2 complex formation and stability and on TORC1 signaling. Our work has provided insight into the genetic risks in the families segregating the tested variants, into possible genotypephenotype correlations, and into the structure and function of the TSC1-TSC2 complex. The results of our functional assessments have been submitted to the TSC1 and TSC2 Leiden Open Variation Databases (LOVD) (see www.lovd.nl/TSC1 and www.lovd.nl/TSC2). We will present an overview of our work.

37. Symptoms of Tuberous Sclerosis Complex: Comparison of TSC1 versus TSC2 Gene Mutation Jo Anne Nakagawa, William Irish, Michael Ryan, Joe Womeldorff, Steven Roberds Tuberous Sclerosis Alliance, Silver Spring, MD (JN, SR); CTI HECOR, Cincinnati, OH (WI, MR, JW)

39. Demographic and clinical characteristics of patients with tuberous sclerosis complex in a TSC patient database Elyse Swallow, MPP; Sarah King, BA; Jinlin Song, PhD; Miranda Peeples, BA; James Signorovitch, PhD; Zhimei Liu, PhD; Judith Prestifilippo, MD; Michael Frost, MD; Michael Kohrman, MD; Bruce Korf, MD, PhD; Darcy Krueger, MD, PhD; Hope Northrup, MD; Steven Sparagana, MD,

Introduction: Tuberous sclerosis complex (TSC) is a genetic disorder characterized by the abnormal cell growth and numerous tumors. TSC is caused by mutations in the TSC1 or TSC2 gene. TSC symptoms vary greatly from person-to-person, and some of this variability is likely attributable to the type of TSC gene mutation. Methods: Data was obtained from the Tuberous Sclerosis Alliance TSC natural history database, which was launched in 2006 to collect information on individuals with

Analysis Group, Inc. (ES, SK, JS, MP, JS); Novartis Pharmaceuticals Corporation (ZL, JP); Minnesota Epilepsy

Group (MF); University of Chicago (MK); University of Alabama at Birmingham (BK); Cincinnati Children's Hospital Medical Center (DK); University of Texas Medical School at Houston (HN); Texas Scottish Rite Hospital for Children (SS)

lo studio e la cura della Sclerosi Tuberosaâ&#x20AC;? located at the San Paolo Hospital of Milan have been recorded in a database since 2001. 230 patients (1-63 years of age, 85 under 18 yrs) were included in the study. We retrospectively evaluated the presence of malignant tumors in these patients. The subtypes of malignancies were analyzed, and a preliminary genotypephenotype correlation was done. A deeper analysis of renal tumors with clinical data was performed and compared to the current data in the literature. Results: Fourteen out of 230 patients developed malignant tumors (6.1%). Four tumors were diagnosed in children, while the other 10 developed in adults (median age at diagnosis 35 years; range 20-58 years). We found 7 renal tumors, of which 5 were renal cell carcinomas (2.2%), a Wilms tumor, and an oncocytoma. Among non-renal cancers, the following types of malignant tumors were found: 2 uterine carcinomas, 2 colon adenocarcinomas, an abdominal sarcoma, an osteosarcoma, and a glioblastoma multiforme. Children showed exclusively renal and brain malignancies. Six patients had a germline mutation in TSC1, and 5 had a germline mutation in TSC2. In one patient no mutations were identified, and genetic analysis was not performed in 2 other patients. Among the ones with renal carcinomas, 3 had a mutation in TSC1 and 2 a mutation in TSC2. Conclusions: The prevalence of renal cell carcinomas in our cohort was concordant with the data in the scientific literature. However, we found that the prevalence of the other malignancies overlaps with the prevalence in the general population (3% vs 4% in Italy), and it is only slightly increased when all malignant tumors are included (6.1% vs 4% in Italy). In conclusion, TSC patients do not seem to have an increased risk of developing malignant tumors besides renal cancer. Further studies are needed to confirm these data.

Introduction: The TSC Natural History Database is the first large database capturing clinical and genetic information for patients with tuberous sclerosis complex (TSC), providing a unique opportunity to assess variation in patient characteristics by gene mutation. This analysis examined characteristics of the overall TSC population and the subsets of patients with mutations in the TSC1 and TSC2 genes. Methods: This study used the TSC Natural History Database, which contains information regarding demographics, diagnosed clinical features, treatments, and monitoring for patients with TSC in the United States, to assess demographic and clinical characteristics of TSC patients overall and stratified by TSC1 vs. TSC2 mutation. Results: Among all patients (N=1,328), median age at diagnosis was 0.8 years (interquartile range [IQR]: 0.34.3 years) and half (49%) were male. Patients were white (74%), Hispanic (15%), African American (9%), Asian (4%), and/or another race (4%). The most commonly diagnosed clinical features were brain tubers (86%), epilepsy (84%), subependymal nodules (82%), hypomelanotic macules (73%), angiofibroma (60%), angiomyolipoma (49%), and rhabdomyoma (38%). Except for angiomyolipoma, these were also the most common first presenting clinical features. Gene mutation information was available for approximately half of the patients (53%). Of those, 52% had a TSC2 mutation, 24% had a TSC1 mutation, <1% had a PKD1 mutation, and 24% had no identified gene mutation. Relative to TSC1 patients (n=171), TSC2 patients (n=363) were diagnosed earlier (median age of 0.5 vs. 2.3 years); otherwise, demographic characteristics were similar between groups. Rates of most clinical features were higher among TSC2 patients, particularly angiomyolipoma (62% vs. 20%), renal cystic lesions (52% vs. 30%), rhabdomyoma (50% vs. 32%), angiofibroma (66% vs. 48%), ophthalmological hamartoma (22% vs. 8%), delayed cognitive development (30% vs. 19%), brain tubers (94% vs. 85%), and epilepsy (89% vs. 80%). Rates of first presenting clinical features were similar across groups, except for rhabdomyoma (20% in TSC2 vs. 11% in TSC1). Conclusions: Neurological and dermatological features were present in most patients. Rates of clinical features were higher among TSC2 patients than among TSC1 patients, consistent with literature reporting a more severe disease burden among TSC2 patients.

41. Diminished rates of protein synthesis in a mouse model of Tuberous Sclerosis Complex: an mTORC1-dependent phenomenon R. Michelle Reith, Tianjian Huang, Tom Burlin, Carolyn Beebe Smith National Institute of Mental Health Tuberous Sclerosis Complex (TSC) is an autosomal dominant neurogenetic disorder affecting about 1 in 6,000 people, leading to benign growths throughout the brain and other vital organs. TSC usually has effects on the central nervous system manifested by a high incidence of seizures, intellectual disability, and autism. TSC is caused by mutations in either TSC1 or TSC2, which encode for proteins that form a complex and interact with a small GTP-binding protein, RHEB, to inhibit mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is a central regulator of ribosomal biogenesis and translation initiation, and loss of TSC1/2 function results in increased activity of mTORC1. Nevertheless, heterozygous loss of Tsc2 leads to diminished protein synthesis both in vitro and in vivo. Specifically, with the in vivo quantitative autoradiographic L-[114C]leucine method, we found reduced rates of cerebral protein synthesis (rCPS) in Tsc2+/- mice at 3 months of age, particularly in the parietal cortex, dorsal and ventral hippocampus, median raphe, and visual cortex. We treated mice with rapamycin to determine if we could reverse this phenotype. We performed both acute and chronic rapamycin studies. For acute studies, we injected 3mg/kg rapamycin or vehicle (about 5% ethanol) i.v., 30 min prior to tracer infusion. For chronic studies, we treated mice from P21 until P90-P105 with rapamycin-enriched chow to achieve a daily dose of approximately 2.2mg/kg. Preliminary results indicate that acute rapamycin treatment of Tsc2+/- mice (n=5) increased rCPS in many brain regions, including thalamus, hippocampus, and parietal cortex compared to vehicle-treated Tsc2+/- mice (n=4). Rapamycin treatment did not significantly alter rCPS in controls. In vehicle-treated animals, we confirm decreased protein

40. Tuberous Sclerosis Complex and Malignant Tumors: a Retrospective Study in an Italian Cohort Angela Peron, Aglaia Vignoli, Francesca La Briola, Graziella Cefalo, Giulia Scornavacca, Massimo Corona, Enrica Riva, Maria Paola Canevini, and the Tuberous Sclerosis Group of the San Paolo Hospital of Milan Child Neurology Unit, Epilepsy Center, San Paolo Hospital, DISS, University of Milan, Milan, Italy (AP, AV, FLB, GS, MPC) Pediatrics Department, San Paolo Hospital, DISS, University of Milan, Milan, Italy (GC, ER) DAMA (Disabled Advanced Medical Assistance) Unit, San Paolo Hospital, DISS, University of Milan, Milan, Italy (MC) Aim: TSC is characterized by the presence of benign tumors (hamartomas). The known causes are mutations of the TSC1 or TSC2 tumor suppressor genes. TSC patients have a higher risk of developing renal cell carcinomas (prevalence 2-4%). However, there are no data regarding the prevalence of other malignant tumors in these patients. The aim of our study was to assess the prevalence of malignancies in a single center in an Italian population of children and adults affected by TSC. Materials and methods: Clinical and genetic data of all the patients referred to the â&#x20AC;&#x153;Centro di riferimento regionale/nazionale per

synthesis in Tsc2+/- mice (n=4) compared to controls (n=3). Our preliminary results suggest a complex role of mTORC1 in the regulation of cerebral protein synthesis, a role that is sensitive to rapamycin treatment.

from a phase 1 trial for advanced solid tumours suggested 2.5 mg as daily maximum tolerated dose (MTD) while several other phase 1 trials are still under way. In this study, we have assessed the therapeutic efficacy of GSK2126458 on renal lesions in a genetically engineered Tsc2+/ mouse model. We first determined 2 mg/kg of GSK2126458 for males and 1 mg/kg for females as the daily MTD. We treated Tsc2+/- mice from the age of 13 months with vehicle, rapamycin (4 mg/kg) and GSK2126458 (MTD) (n=9 per group) 5 times a week via intraperitoneal injection for two months. Tumour burden was estimated from total whole areas and cellular areas respectively of all lesions (cystic, papillary and solid) observed in 6 coronal sections at a150 µm interval prepared from each kidney of a mouse. Compared to vehicle, both rapamycin (P=0.0004 for whole areas; P=0.0006 for cellular areas) and GSK2126458 (P=0.002 for whole areas; P=0.010 for cellular areas) treated mice showed significant reduction in overall tumour burden but efficacy for GSK2126458 was significantly less than that for rapamycin (P=0.0643 for whole areas; P=0.0135 for cellular areas).

42. A Single Cell Biology Platform to Investigate the Effects of Stem Cell Positional Identity on SEGA Development Gabrielle V. Rushing, Nalin Leelatian, Asa A. Brockman, Cary Fu, Kevin C. Ess, Jonathan M. Irish, Rebecca A. Ihrie Vanderbilt University Management of subependymal giant cell astrocytoma (SEGA) growth is a significant concern for TSC patients. Both subependymal nodules and SEGAs present in the ventricularsubventricular zone (V-SVZ) and are thought to originate from the stem cells that reside there. If left untreated, SEGAs can grow and become lethal due to obstruction of cerebrospinal fluid flow through the ventricles. However, the factors that cause SEGAs to present in a specific position within the ventral V-SVZ are not well understood. Recent work has demonstrated that stem cells in the V-SVZ have a “positional identity”: the position of the stem cell predicts the types of progeny that will be generated, suggesting intrinsic differences between stem cell microdomains. This introduces the intriguing possibility that SEGAs may derive from a specific, ventral population of stem cells that is more susceptible to the effects of TSC1/2 mutation. However, it is not known whether the intrinsic patterning of VSVZ stem cells corresponds to measurable proliferative signaling differences that may influence tumor development. We are currently using a mouse model of TSC to test the effects of positional identity on SEGA development. We have developed assays that apply phospho-specific flow cytometry to cultured neural stem/progenitor cells (NPCs), and can simultaneously measure the per-cell levels of multiple relevant phospho-proteins both upstream and downstream of mTORC1. Preliminary analyses in wild type and Tsc2 heterozygous dorsal and ventral NPCs suggests that ventral cells have elevated basal mTOR pathway activity compared to dorsal NPCs and therefore may have increased sensitivity to TSC-related mutations. In the future, this platform will be used for identification of agents that specifically target the ventral NPCs, with the ultimate goal of eliminating SEGAs without harming the remaining germinal niche. This finding would provide an avenue for identifying novel therapeutic strategies to treat SEGAs by targeting properties specific to ventral NPCs. Critically, the phospho-flow platform can be readily adapted to future drug screening efforts. Ultimately, our research will elucidate whether SEGAs develop from a ventral cell of origin and which mTOR pathway components differ in ventral NPCs as compared to other microdomains of the V-SVZ. Determining the molecular basis for SEGA development and identifying candidate therapies would be a major advance for TSC patients.

44. Evaluation of combined treatment of rapamycin with atorvastatin for therapy of renal lesions in a genetically engineered Tsc1+/- mouse model Paulina Samsel, Kalin Narov, Jian Yang, Julian Sampson and Ming Hong Shen Institute of Medical Genetics, Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK Atorvastatin is a statin used to lower blood cholesterol and to reduce risks of cardiovascular disease-associated complications through inhibition of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase. Statins including atorvastatin have demonstrated anti-tumour activity in in vitro and in vivo preclinical studies. Atorvastatin inhibits Akt/mTOR signalling in tumour cells and Tsc2 null cells. However, atorvastatin alone does not suppress tumour growth in the kidneys and the liver of 129Sv/Jae Tsc2+/- mice. In this study, we have tested whether combined treatment of rapamycin with atorvastatin could be more effective than rapamycin or atorvastatin alone in treating renal lesions in a genetically engineered Tsc1+/ mouse model. We treated Tsc1+/- mice from the age of 12 months with vehicle (i.p.), rapamycin (5 mg/kg, i.p.), atorvastatin (100 mg/kg; gavage) and a combination of rapamycin with atorvastatin (n=10 per group) 5 times a week for two months. Tumour burden was estimated from total whole areas and cellular areas of all lesions (cystic, papillary and solid) respectively observed in both kidneys in each mouse. Compared to vehicle, significant reductions in tumour burden were observed with rapamycin (P=0.0058 for whole areas; P=0.0003 for cellular areas) or rapamycin plus atorvastatin (P=0.0006 for whole areas; P=0.0001for cellular areas) but not atorvastatin alone (P=0.4295 for whole areas; P=0.3077 for cellular areas). A slightly greater reduction in tumour burden was observed in mice treated with rapamycin and atorvastatin together than rapamycin alone but the difference was not statistically significant (P=0.3077 for whole areas; P=0.0751 for cellular areas). We conclude that combined treatment of rapamycin with atorvastatin is unlikely to confer significant advantages over treatment with rapamycin alone.

43. GSK2126458, a dual PI3K/mTOR inhibitor, is less effective than rapamycin for treatment of renal lesions in a genetically engineered Tsc2+/- mouse model Kalin Narov*, Paulina Samsel*, Jian Yang*, Ashley Jones, Julian Sampson and Ming Hong Shen (*Equal contributors) Institute of Medical Genetics, Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK

45. Heterogeneous response of renal tumours to sunitinib and sorafenib in genetically engineered TSC mouse models Jian Yang, Paulina Samsel, Anna Hellmann, Kalin Narov, Julian Sampson and Ming Hong Shen

Potential rapamycin induced activation of Akt necessitates the development of new strategies and agents for TSC therapy. GSK2126458 is a highly potent and orally bioavailable dual ATPcompetitive inhibitor of PI3K and mTOR, and may help to overcome problems caused by inhibitors that target mTORC1 only. Preclinical studies have demonstrated broad anti-tumour activity of GSK2126458 in vitro and in vivo. Preliminary data

Institute of Medical Genetics, Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK

localization of gene products identified in the PCR screen were observed by western blotting and confocal microscopy.

Tumours from patients with TSC are highly vascular. Renal angiomyolipoma (AML) cells express vascular endothelial growth factor (VEGF) and circulating VEGF-D levels are elevated in patients with AML and/or LAM. Sunitinib and sorafenib are small-molecule multiple kinase inhibitors whose targets include VEGF receptors. Both drugs have demonstrated clinical efficacy in some types of cancer including renal cell carcinoma. In this study, we investigated potential molecular targets and antitumour efficacy of sunitinib and sorafenib in renal lesions of genetically engineered Tsc1+/- or Tsc2+/- mice. We found that renal lesions usually had increased expression of VEGF and VEGF receptor 1 (VEGFR1) but decreased expression of VEGFR2 and platelet-derived growth factor receptor ß (PDGFRß). We treated Tsc1+/- mice from the age of 12 months with vehicle, sunitinib (45 mg/kg), rapamycin (5 mg/kg) or sunitinib (45 mg/kg) plus rapamycin (5 mg/kg) (n=8 per group). We also treatedTsc2+/- mice from the age of 11 months with vehicle, sorafenib (42 mg/kg), RAD001 (everolimus) (10 mg/kg) or sorafenib (30 mg/kg) plus RAD001 (6 mg/kg) (n=10 per group). All mice were treated 5 times a week via gavage for two months and then sacrificed for assessment of tumour burden in the kidneys. By comparison, treatment with rapamycin, RAD001, rapamycin plus sunitinib or RAD001 plus sorafenib significantly reduced tumour burden (P<0.01). Compared to vehicle, sunitinib or sorafenib treated mice showed increased apoptosis/necrosis in a small proportion of solid tumours but no significant reduction in overall tumour burden (P>0.05). We conclude that renal lesions show a heterogeneous response to sunitinib and sorafenib in Tsc1+/- or Tsc2+/- mice.

RAD001 significantly increased cilia length and expression in TSC2-deficient cells, as well as protein levels of acet. α-tubulin and kinesin-2. Expression of Invs, a Wnt signaling component, was increased at 3 hours of RAD001 treatment, and Axin2 and Vangl2, components of Wnt and PCP signaling, were upregulated at 72 hours. Cilia localization of inversin was observed in RAD001-treated EKT2 cells, and lateral membrane localization was increased. β-catenin, a Wnt signaling mediator, levels were elevated in RAD001-treated cells, and lateral membrane localization was increased. These findings suggest that cellular abnormalities such as defective cilia structure and altered Wnt/PCP signalling are correctable by mTORC1 inhibition in renal cystic cells, and highlight its potential as preventative therapy for TSC renal cystic disease. 47. A Simplified Management Approach For General Practitioners For Skin Lesions In Patients With Tuberous Sclerosis Complex (TSC) Duri Yun, MD, Keyoumars Soltani, MD University of Chicago, Section of Dermatology Often the skin lesions of TSC become physically, functionally, aesthetically and emotionally disturbing. Except for major medical centers where TSC cases are managed by a team of experts, most patients are cared for by individual providers e.g. family physicians, skilled nurses, and physician assistants. We hereby suggest practical guidelines for skin lesions that can treat in their private offices, and cases to be referred to specialists in major centers. The diagnostic and therapeutic guidelines have been updated by the TSC International Panel in 2012 Facial angiofibromas: Topical sirolimus applied BID is very effective in the treatment and prevention of facial lesions and erythema. More severe cases may be referred for: dermabrasion, laser abrasion, and pulse dye laser therapy. Fibrous cephalic plaques: Small, relatively flat and stable lesions may simply be observed and followed. Enlarging plaques, especially on the scalp may grow significantly and are best referred for excision before needing extensive procedures and wound repairs. Ungual fibromas: Small ungual fibromas can be managed with topical sirolimus and cryosurgery (Liq. N2). Larger lesions can deform or destroy the affected nails and should be referred for excision and/or laser treatment. Hypomelanotic macules: Hypomelanotic macules are usually on the covered areas and do not bother patients. When on exposed skin, they can be covered by make up foundation in women, or sunless tanning lotions in men and women. Commonly associated skin lesions: Skin tags (fibroepithelial polyps) occur often and in large numbers on the neck, axillae and groin, and can be snipped off with scissors. Seborrheic dermatitis is common especially in TSC patients with epilepsy. Similarly acne vulgaris often coexist with facial angiofibromas, and will require treatment.

46. mTORC1 inhibition rescues primary cilia expression and affects Wnt/PCP signalling in TSC2-deficient renal cystic epithelial cells Brian J. Siroky, Alyssa Wilson, Nolan Pachciarz, John J. Bissler Division of Nephrology and Hypertension, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio (BJS, AW, NP); Tuberous Sclerosis Complex Center of Excellence, Le Bonheur Children’s Hospital, University of Tennessee College of Medicine, Memphis, Tennessee (JJB) Rapamycin is a potent mTORC1 inhibitor, and is used for treatment of renal tumors in TSC patients, but a therapeutic option for TSC renal cysts has not been identified. Primary cilia are antenna-like structures present at the apical cell surface, defects of which contribute to most forms of inherited renal cystic disease. TSC protein deficiency and resulting increased mTORC1 activity has been found to affect renal cilia. However, the mechanism(s) by which cilia are affected is not known, nor whether TSC renal cystic disease is related to a ciliary defect. Furthermore, alterations in Wnt and Planar Cell Polarity (PCP) signaling have been observed in models of TSC, are related to primary cilia abnormalities, and are thought to contribute to cystogenesis. We examined cilia length/expression in TSC2-deficient renal cystic epithelial cells, and quantitatively and qualitatively evaluated effects of the mTORC1 inhibitor RAD001. We also measured changes in expression/localization of “ciliome” genes/proteins in response to RAD001 treatment of EKT2 cells.

48. Pancreatic Neuroendocrine Tumors in Pediatric Patients with Tuberous Sclerosis Complex: A Case Study Erin K. Fanning, Steven P. Sparagana MD

Fluorescence microscopy was used to determine the percentage of ciliated cells. Cilia length of live cells labelled with the membrane dye Di-8-ANNEPS was measured from 3D images obtained by confocal microscopy. Western blotting for acet. α-tubulin and kinesin-2 was performed to complement imaging findings. Change in expression of 84 cilia-related genes was quantified in RAD001-treated EKT2 cells at 3, 24 and 72 hours by PCR arrays. Changes in amount and subcellular

Texas Scottish Rite Hospital for Children- Neurology Division, Dallas, TX, USA Objective: To analyze three cases of pancreatic neuroendocrine tumors (PanNET) associated with Tuberous Sclerosis Complex (TSC) in pediatric patients. Case Report 1: 8-year-old male with a TSC1 mutation presented with a cystic mass (1.1 x 1.2 x 1.2cm) within the abdominal left upper quadrant between the

pancreas and small bowel on surveillance MRI of the abdomen. Follow-up MRI 2 months later identified the mass as an exophytic lesion projecting from the inferior margin of the pancreatic tail now measuring 1.5 cm by 1.2 cm. The patient was followed with serial surveillance scans which showed a gradual increase in size of the mass. After 6 years, MRI showed the mass to be 1.5 x 2.1 x 2.3cm. Growth of the mass and elevated insulin levels (147.7 microIU/mL) led to the decision to resect the mass in June 2014. Pathology indicated a neuroendocrine tumor. Case Report 2: 13-year-old male with a TSC2 mutation presented with a lesion within the tail of the pancreas measuring 1.7 x 1.5 x 1.6cm on abdominal MRI. On abdominal CT 2 years later, the mass had grown to 3.3 x 2.5 x 2.5cm. The mass was presumed to be a neuroendocrine tumor and was resected in June 2014. Distal pancreas, spleen, an aortic venacava lymph node, and a mesenteric lymph node were removed. The pancreas specimen showed a welldifferentiated, low grade neuroendocrine tumor. The other tissues were negative for tumor. Case Report 3: 10-year-old female with a TSC2 mutation had an abdominal MRI February 2014 with a pancreatic lesion noted measuring 1.6 x 1.4cm. Patient was started on everolimus for a subependymal giant cell astrocytoma and for enlarged angiomyolipomas. Follow-up MRI 4 months later showed the pancreatic lesion has shrunk to 1.3cm X 1.0cm. Her other tumors had also become smaller. She will continue to have follow-up MRIs to monitor her TSC associated lesions. Discussion: PanNETs occur rarely in TSC but can grow, and some may be endocrinologically active. There is little literature on management of these tumors. Dedicated imaging of the abdomen, not just the kidneys, could lead to more accurate and timely diagnosis. While PanNETs of concern may be resected, we did see a decrease in size of the PanNET of our patient on everolimus. Everolimus has an indication for advanced PanNETs in adults without TSC that cannot be treated with surgery, and everolimus may have a role in management of PanNETs in TSC.

course were generally between 5-7 ng/mL. Eighty patients (72%) required a dose reduction or interruption due to an AE; 8 patients (7.2%) discontinued the study due to a drug-related AE. One accidental death occurred (not related to study drug). The most common AEs suspected to be treatment-related were stomatitis (43.2%) and mouth ulceration (32.4%). The incidence of newly emergent AEs generally decreased over time. Clinically significant laboratory abnormalities were mainly grade 1 or 2 and resolved spontaneously. Conclusion: Exposure to everolimus over 4 years showed no new safety concerns in patients with TSC-associated SEGA. 50. Understanding families living with TSC in Australia Clare P. Stuart Tuberous Sclerosis Australia Rationale There has been limited research into the experience of individuals and families living with tuberous sclerosis complex (TSC) in Australia. Tuberous Sclerosis Australia (TSA) wanted to understand their experiences of TSC and the services provided by TSA. Methods Survey with a web-based and paper option distributed to families through TSAs magazine, email and social media. Survey was adapted from the family survey conducted by Tuberous Sclerosis Association (UK). Results The survey was completed by 88 individuals. 92% of respondents were parents or carers of a person with Tuberous Sclerosis. Seven individuals with TSC responded to the survey. Respondents lived in all parts of Australia and in metro, rural and remote areas roughly reflecting the Australian population. 82% of respondents were Australian born and 95% speak English at home. 99% reported that they speak English very well. There were no responses from people of Aboriginal or Torres Strait Islander descent. The respondents reported skin problems (88%), epilepsy (80%), learning difficulties (75%) and kidney tumours (72%) as the most common symptoms of TSC. The conditions that were reported most as having severe impact were learning difficulties, intellectual disability, epilepsy, autism, and challenging behaviour. Open text responses demonstrated the major impact of TSC-associated neuropsychiatric disorders (TAND), as well as epilepsy, socialising, fear, and access to medical specialists knowledgeable in TSC. Most (88%) reported having a single general practitioner (GP) but few (15%) reported their GP taking a care co-ordination role, which was instead done by neurologists or the parent. 51% were aware of the guidelines for TSC diagnosis, surveillance and management. Respondents prioritised TSA providing information, connecting them with health professionals and funding research. There was a desire for shorter events and webinars. Conclusions Australians with TSC have a similar experience of TSC as reported elsewhere. This includes difficulties with TAND, epilepsy and psycho-social factors. They have a positive opinion of TSA and more could be done to meet their diverse needs. Further research could be done to identify unmet needs and reach more adults with TSC.

49. Everolimus exposure and overall tolerability in patients treated for subependymal giant cell astrocytoma associated with tuberous sclerosis complex: results from the 4-year final analysis of EXIST-1 Steven Sparagana, Joyce Y. Wu, Rachel Kuperman, Noah Berkowitz, Julie Niolat, David N. Franz Texas Scottish Rite Hospital for Children, Dallas, TX (SS); Mattel Children’s Hospital at UCLA, Los Angeles, CA (JYW); Children’s Hospital and Research Center, Oakland, CA (RK); Novartis Pharmaceuticals Corporation, East Hanover, NJ (NB); Novartis Pharmaceuticals S.A.S., Rueil-Malmaison, France (JN); Cincinnati Children’s Hospital Medical Center, Cincinnati, OH (DNF) Objective: To assess everolimus exposure and overall tolerability during the long-term EXIST-1 study (NCT00789828) in patients with subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex (TSC). Methods: Patients were initially randomly assigned (2:1) to everolimus or placebo. After positive results for the primary endpoint (SEGA response rate; data cutoff March 2, 2011), all patients could continue on open-label everolimus for up to 4 years after the last patient was randomized. Everolimus starting dose was 4.5 mg/m2/day, which was titrated to a target trough level of 5-15 ng/mL based on tolerability. Everolimus blood trough concentrations were measured throughout the study. Safety assessments included adverse event (AE) reporting and laboratory monitoring. Results: In total, 111 patients (median age 9.5 [range, 1.1-27.4] years) received =1 dose of everolimus. Median duration of everolimus exposure was 47.1 (range, 1.958.3) months at study conclusion (October 2, 2014), and median daily dose intensity was 5.89 (range, 1.0-13.8) mg/m2. Median everolimus trough concentrations over the study

51. Calcium signaling induces TSC2 phosphorylation independently of Akt Tsukasa Suzuki, Kanako Sato, Jun-ichi Iwata, Machiko Kazami, Ken-ichi Kobayashi, Yuji Yamamoto Department of Applied Biology and Chemistry, Tokyo University of Agriculture TSC2, a mediator of insulin signaling, forms a complex with TSC1 and controls protein synthesis by inhibiting mTORC1 activity. TSC2 has been also found to interact with calmodulin (CaM). However, it is not clear whether this interaction has a physiological function. Here, we focused how calcium signaling, especially intracellular Ca2+ affects insulin signaling via TSC2. To increase intracellular Ca2+ concentration in vitro, HeLa cells

were exposed to ionomycin (iono) and intracellular Ca2+ level was monitored. The intracellular Ca2+ level was maximum in 2 min and stayed at a relatively high concentration for a total of 10 min. Based on this result, insulin signaling mediators, such as TSC2, Akt, mTOR, and S6 phosphorylation, were analyzed at the 2 min point and 10 min point after iono treatment. We found that iono treatment enhanced the phosphorylation levels of TSC2, mTOR, and S6. However, phospho-Akt was not affected, suggesting that calcium signaling induced TSC2 phosphorylation to regulate mTORC1 signaling. To clarify whether calcium signaling mediator including Ca2+, CaM, and CaMKII involved in these phosphorylation change, cells were also treated with BAPTA-AM (Ca2+ chelator), W-7 (CaM antagonist), or STO-609 (CaMKII inhibitor), respectively. These inhibitors reduced ionoinduced TSC2 and S6 phosphorylation. Interestingly inhibition of AKT by LY294002, TSC2 was still phosphorylated and the level of S6 phosphorylation was induced by iono treatment. It is important for the lysosomal localization of mTORC1 to be activated. Recent study has also showed that TSC2 localization on the lysosomal membrane is required for inhibition of mTORC1 activity. In contrast, AKT-induced TSC2 phosphorylation promotes removal of TSC2 from lysosome, indicating that TSC2 phosphorylation is important for its own localization. We found that Ca2+ stimulation altered the localization of TSC2 from lysosomal membrane to the cytoplasm, suggesting that calcium signaling-induced TSC2 phosphorylation also regulates TSC2 localization. These findings provide a novel crosstalk between insulin signaling and calcium signaling, via TSC2. This crosstalk may play an important role for homeostasis of the mTORC1 signal.

included in the analysis. The rate of sleep problems in the TSC group varied between 31% to 58% with the main sleep problems being increased sleep latency and awakenings and reduced sleep time. The polysomnography study reported disturbances in sleep architecture such as reduced sleep time and decreased REM. Conclusion Children with TSC have difficulties with reduced sleep hours and increased sleep latency. TSC children with comorbid ASD are more likely to have sleep difficulties. Full population data is expected by 09/2015. 53. Genetic mechanisms associated with tuberous sclerosis and tuberous sclerosis-like phenotypes Laura Thomas, Angharad Walters, Peter Giles, Kevin Ashelford, Matthew Mort, James Colley, Julian Sampson Institute of Medical Genetics, Cardiff University, UK Mutations in the TSC1 or TSC2 genes are causative in at least 80% of cases of tuberous sclerosis (TSC). Their identification is important for genetic counselling, diagnosis, and as a resource to better understand the pathophysiology of TSC. The remaining patients are classified as no mutation identified (NMI). It is expected that a number of different genetic mechanisms may account for their phenotypes. They may have mutations in TSC1 or TSC2 that have escaped detection (e.g. mutations in un-screened promoters or untranslated regions, or low level mosaicism for conventional mutations), and/or there may be mutations in other genes that confer similar phenotypes to TSC. This study employs targeted ultra deep sequencing (UDS) and whole exome sequencing (WES) to identify the genetic causes of TSC and TSC-like phenotypes in NMI patients. Trios of samples (an affected proband and their unaffected parents) have been analysed to aid in novel, de novo variant detection. To date, 36 samples (12 trios) have been analysed by targeted UDS of the 104,500bp region containing TSC1 and TSC2, and 15 of these samples (5 trios) have been analysed by WES. We have evaluated two different sequence capture techniques; Long Range PCR (LR-PCR) and Haloplex (Agilent) on a HiSeq (Illumina). Following sequence alignment (hg19), two SNP calling pipelines: GATK, and VarScan were employed to identify putative SNVs and indels, which were subsequently verified using a combination of standard PCR, COLD-PCR and Direct Sequencing. For 8 of the patients, RNA was also available and a TSC1 and TSC2 allelic imbalance assay was completed using direct sequencing. UDS identified putative de novo SNVs and indels in all 12 NMI patients, however, re-sequencing failed to verify the variants in all but one, in whom a likely pathogenic de novo TSC2 mutation was confirmed (c.2590C>T; p.Q864X). This mutation was mosaic; present in only 1% of alleles. No imbalance of allelic expression was detected in the 8 patients investigated. Further investigation of non-TSC1/2 de novo mutations identified by WES is being undertaken.

52. Sleep Problems in Tuberous Sclerosis Complex: A questionnaire based study and a systematic literature review. Panagiota Sykioti, Holan Liang, Fiona McEwen, Charlotte Tye & Patrick Bolton TS2000 Research Team, King’s College London Background Sleep problems are common in children with neuropsychiatric symptoms and are a major source of morbidity for children and their families. Tuberous sclerosis complex (TSC) is a multisystem genetic disorder with prominent central nervous system involvement and patients suffering from TSC are at high risk for sleep problems. The aim of our study was to describe the sleep problems in children with TSC and to systematically review epidemiological and clinical features of sleep problems in TSC, in order to understand the interplay between common comorbidities and sleep disturbance in TSC. Methods This study uses data from the TS2000 Study, a population based, longitudinal study of TSC. Between 2001-05, 125 children diagnosed with TSC were ascertained and genetic testing was undertaken. In 2014, consenting participants were assessed using a parent-report questionnaire enquiring about Autistic Spectrum Disorder (ASD), epilepsy and sleep, assessed via the Composite Sleep Diagnostic Interview (CSDI). A systematic literature review was conducted in 07/2014 in all major databases. Preliminary Results Until now, data of 31 participants (41.9% females, age 13 ±3.5 years) have been analyzed. 41.9% reported co-morbid ASD. Sleep problems were subjectively reported by 55.0% of parents. The mean total CSDI score was 5.0±3.8. 45.0% had problems with sleep latency and 52% reported total sleep hours <7. The univariate analysis of the characteristics of the ASD versus the non ASD group showed that the groups did not differ significantly regarding gender or age. All children with ASD had comorbid epilepsy, compared to 75% of the children without ASD. Patients with ASD showed significantly higher mean CSDI scores compared to children without (6.8 vs 3.7, p=0.025) From the systematic literature review we identified 314 papers. Following removal of duplicates and eligibility assessment 307 articles were excluded and 7 papers were

54. A risk pathway to intellectual disability in Tuberous Sclerosis Complex Charlotte Tye, Fiona S. McEwen, Holan Liang, Emma Woodhouse, Lisa Underwood, Henry Rogers, Funmilola Oluwo, Ciara Halpin, Sri Wulandari, Tuberous Sclerosis 2000 Study Group, Patrick F. Bolton Institute of Psychiatry, Psychology & Neuroscience, King's College London Background: Individuals with mutations in the TSC genes develop a variable and diverse array of physical, cognitive and behavioural manifestations. Epilepsy is present in 60-90% of cases and is typically seen during the first year of life. A large proportion go on to develop intellectual disability (ID). The causal mechanisms that underlie variable intellectual outcomes

are not fully understood. This study aimed to investigate the risk factors associated with intellectual development in TSC within a longitudinal design. Method: The TS 2000 Study comprises a nationally ascertained sample of all newly diagnosed cases of TSC identified in the period 2001-2006. In Phase 1 of the study, baseline data were collected on intellectual ability and longitudinal prospective data on epilepsy, tuber count and other clinical correlates were gathered. A follow-up phase was conducted in 2013-2014 to update information on developmental outcome. Intellectual ability (here on IQ) was measured using the Mullen Scales of Early Learning, the Wechsler Abbreviated Scale of Intelligence and/or the Vineland Adaptive Behaviour Scale, depending on age and ability of the individual (phase 1 n=; 121 phase 2 n= 85). Results: There was a moderate correlation between IQ at Phase 1 and Phase 2. Individuals who showed a significant reduction in IQ between Phase 1 and Phase 2 (more than 15 points) had greater epilepsy severity in the first year of life compared to those with an increase in IQ. There were significant correlations between lower IQ at Phase 2 and earlier age of seizure onset, greater severity of epilepsy in the first year of life and cortical tuber count. Structural equation modelling supported a causal pathway from genetic abnormality to cortical tuber count to epilepsy severity in the first year of life to intellectual outcome at 8+ years. Conclusion: The strong support for a likely causal association between seizure severity during early development and intellectual impairment in later childhood and adolescence, supports the clinical importance of early, prompt and effective treatment or prevention of epilepsy in TSC.

mutation in TSC1. Increased variability was associated a higher number of cortical tubers in the parietal lobe, while increased error rates were associated with cortical tubers in the frontal lobe. Structural equation modelling indicated a risk pathway from genetic mutation, through brain, to cognitive deficits. The findings indicate fluctuations in attention are a feature of ADHD in TSC, and demonstrate a homology between idiopathic and syndromic cases. Poor task performance was associated with the level of brain involvement in specific regions. These findings hold key implications for cognitive neuroscience and for the understanding of ADHD. Identification of risk factors for poor developmental outcome in TSC will aid in targeting more specific treatment and intervention strategies. 56. Autistic-like phenotype in Tuberous Sclerosis animal model: remission by mTOR-inhibitor Miriam Schneider (1), Kai Schönig (1), Petrus de Vries (2), Robert Waltereit (1,3) (1) Central Institute of Mental Health, Mannheim, Germany (2) University of Cape Town, South Africa (3) University Hospital Carl Gustav Carus, Dresden, Germany The monogenetic disease Tuberous Sclerosis Complex (TSC) has high rates of autism spectrum disorder (ASD), intellectual disability and epilepsy. In TSC, up to 50% of all patients have ASD and, in turn, TSC mutations account for up to 4% of all patients with ASD. Epilepsy is an independent risk factor for ASD in TSC. In rats with Tsc2 haploinsufficiency we had found deficient social interaction that we interpreted as a correlate of the clinical manifestation in TSC patients with ASD. This social deficit was accelerated by developmental epilepsy induced during childhood in these Tsc2+/- (Eker)-rats, resembling the human situation. A key molecular mechanism of TSC pathology is increased activity of the mammalian target of rapamycin (mTOR). Autistic-like behaviour was described in Purkinje cellspecific Tsc1-mutant mice. The mTOR-inhibitor Rapamycin rescued the social deficit behaviour. Here we studied the effect of mTOR-inhibitor Everolimus on social interaction and cognition in Tsc2+/- (Eker)-rats with previously induced developmental epilepsy. The experimental group that modelled TSC pathology carried the Tsc2+/- (Eker)-mutation and was challenged with pharmacologically induced status epilepticus at P7 and P14 (n=12). The control group was made up of wild-type carriers from the Eker rat line and did not receive developmental epilepsy (n=11). At the age of our month, all animals were investigated in the pre-treatment behavioural analysis (T1). They were then during one week treated three times with 1 mg/kg Everolimus by intraperitoneal injection. Finally, they were retested in the post-treatment behavioural analysis (T2). Locomotor activity was reduced in the Tsc2+/(Eker) plus developmental epilepsy group, but not affected by Everolimus. Both anogential and non-anogenital social exploration were impaired in Tsc2+/- (Eker) plus developmental epilepsy rats. This effect was completely abrogated by Everolimus treatment. Finally, we investigated social cognition. Similarly, social cognition was impaired in Tsc2+/- (Eker) plus developmental epilepsy rats. This effect was fully reversed by the mTOR-inhibitor. Everolimus successfully treated the social interaction and cognition deficits in Tsc2+/- (Eker)-rats with developmental epilepsy induced during childhood age.

55. Attention deficit hyperactivity disorder and executive dysfunction in Tuberous Sclerosis Complex Charlotte Tye, Katherine Johnson, Simon Kelly, Lisa Underwood, Emma Woodhouse, Fiona S. McEwen, Holan Liang, Patrick F. Bolton Institute of Psychiatry, Psychology & Neuroscience, King's College London (CT, LU, FSM, HL, EW, PFB) Melbourne School of Psychological Sciences, University of Melbourne (KJ) School Of Electric, Electronic & Communications Engineering, University College Dublin (SK) Tuberous Sclerosis Complex (TSC) is one of the best-established causes of attention deficit hyperactivity disorder (ADHD), with between 30-60% of individuals meeting criteria for a diagnosis. Individuals with idiopathic (no known cause) ADHD demonstrate poor performance on a range of cognitive tasks, with a consistent finding being increased response variability. This study sought to test whether individuals with TSC and ADHD demonstrate similar cognitive deficits, and which risk factors these deficits are related to. Participants (n=54) were recruited from the TS 2000 Study Cohort, a nationally ascertained longitudinal study of TSC diagnosed between 20012006. In Phase 1 of the study, data on epilepsy, tuber count and other clinical correlates were gathered. A follow-up phase was conducted in 2013-2014 to collect new data on developmental outcome and cognitive functioning. The Development and Wellbeing Assessment (DAWBA) was used to assess ADHD. A Go/No-Go task was administered, whereby a motor response must be executed or inhibited depending on whether a ‘go’ or no-go’ stimulus appeared on a computer screen. Reaction times (RTs) were modelled using an ex-Gaussian curve and the sequence of reaction times was analysed using a fast Fourier transform (FFT) to examine fast moment-to-moment variability and gradual variability occurring over the course of the task. Increased response variability (RT standard deviation, tau, fast variability and slow variability) was associated with ADHD symptoms and was demonstrated in individuals meeting diagnostic criteria for ADHD. Individuals with a mutation in the TSC2 gene had higher RT variability compared to those with a

57. Attention to social and non-social stimuli in Tuberous Sclerosis Complex Lucy Wilde, Hayley Crawford, Joe McCleery & Chris Oliver Cerebra Centre for Neurodevelopmental Disorders, School of Psychology, University of Birmingham (LW, HC, CO) Centre for Research in Psychology, Behaviour &

Achievement Faculty of Health and Life Sciences Coventry University (HC) School of Psychology, Uni

58. Tuberous sclerosis complex 1(TSC1) regulates the Caveolae mediated endocytosis via controlling the Rab5 activity. Yuji Yamamoto, Shintaro Sato, Kazuyuki Tsuchiya, Ikue Tsuchiya, Kenichi Kobayasi, Yoshimitsu Matsui-Ito and Tsukasa Suzuki

Technical Summary: Background: Tuberous Sclerosis Complex (TSC) is associated with increased rates of Autism Spectrum Disorder (ASD). Processing of social stimuli has been shown to be atypical in ASD, with reduced attention to social, compared to non-social, stimuli reported. Because of increased prevalence of ASD in TSC it may be expected that social processing is also atypical in this disorder. To further explore whether attention to social stimuli is atypical in TSC, the current study used eye tracking methodology to examine spontaneous attention allocation to social versus non-social stimuli in children with TSC. Methods: Eye tracking methodology was used with seventeen children with TSC (10 male, 7 female, mean chronological age 7.64 years) and a contrast group of 25 typically developing (TD) children (15 male, 10 female, mean chronological age 5 years). No significant difference was found between estimates of developmental age derived for the samples. Children viewed paired video clips presented side by side, with one social video (showing a person) and one non-social video (showing objects). Half of these paired videos were directed (subject facing towards the camera) and half were non-directed (subject not facing towards the camera). Results: Both dwell time (percentage time looking at social and non-social videos) and time to first fixation to social and non-social videos were analysed, comparing â&#x20AC;&#x2DC;preferenceâ&#x20AC;&#x2122; for social versus non-social videos. No differences were found between groups in dwell time. However, for time to first fixation an interaction suggests that when videos were directed preference for social videos (over non-social videos) did not differ between groups but when they were non-directed preference for social videos became significantly weaker in children with TSC in comparison to typically developing children. Discussion: Findings suggest potential atypicalities in the allocation of attention to social and non-social stimuli when stimuli are less salient (i.e. not directed at the individual), with children with TSC showing reduced social preference for less salient stimuli. Given the importance of attending to social stimuli for social development, this reduced attention to social stimuli which are less salient may provide insight into a potential pathway to the social impairments observed in TSC.

Department of Applied Biological Chemistry, Tokyo University of Agriculture TSC1 and TSC2 form a hetero-complex and negatively regulate the mTOR activity via serving as the Rheb GTPase activating protein (GAP). The activity of TSC1/TSC2 complex is controlled through the phosphorylation by AKT/PKB and the suppressive effect towards mTOR activity is removed. We have previously reported that the TSC2 is a component of the caveolae, the small invaginations of the plasma membrane that is composed by caveolin-1 protein and in rich of cholesterol and sphingolipids. The role of caveolae still remains not fully understood. However, caveolae seem to go through endocytic pathway, and we have found that this endocytosis is controlled through small G-protein family member, Rab5 activity. Here we report that TSC1 is controlling the caveolae mediate endocytosis via regulating the Rab5 activity. The interaction of TSC1 and caveolin-1 was first conformed in HeLa cells by IP using anti-TSC1 antibody. This interaction was observed in the early endosomes analyzed by the confocal laser scanning microscopy. We then knocked down the TSC1 expression by siRNA and studied whether the caveolae mediate endocytosis was affected. Cholera toxin B subunit (CTxB), one of the ligand known to be taken in the cell through caveolae was reduced and remained on the plasma membrane. We also examined whether the rab5 activity was affected by the TSC1. Cell extracts from HeLa cells transiently expressing either wild, constitutively activated or inactivated Rab5 were subjected to IP analysis using anti-TSC1 antibody. We found that TSC1 strongly interacted with inactivated Rab5. We further measured whether the TSC1 its self regulates the Rab5 activity by reducing the expression of TSC1by siRNA. Rab5 activity measured by GST-R5BD pull down assay, showed a reduction of Rab5 activity. Finally we examine the insulin stimulation can enhance the caveolin-1-TSC1-Rab5 interaction. These results suggest that TSC1 may have a role to control the caveolae mediate endocytosis by regulating the Rab5 activity.

Vicky H. Whittemore Travel Awards Poster Presentations

Platform Presentations

Fatou Aw, CHU Ste-Justine, Université de Montréal. Arrhythmias and functional disorders in 43 patients with Tuberous Sclerosis

Juxiang Cao, Brigham and Women's Hospital, Inc. The TSC protein complex regulates melanogenesis through mTOR and GSK3ß, explaining the pathogenesis of TSC ‘white spots’

Elaine Dunlop, Cardiff University. Loss of TSC2 sensitises cells to nelfinavirbortezomib dual therapy

Iris Overwater, Erasmus University Medical Center. Sirolimus treatment in children with Tuberous Sclerosis Complex and intractable epilepsy: a randomized controlled trial

Lawrence Hseih, Yale University. A New Model of Cortical Tuber Associated Epilepsy Hilaire Lam, Brigham and Women's Hospital, Inc. Identification of Pro-survival Rapamycindependent microRNA (Rapa-miRs) in Tuberous Sclerosis Complex

Kandice Varcin Boston, Children's Hospital. Social Visual Perception in Tuberous Sclerosis Complex

Gabrielle Rushing, Vanderbilt University. A Single Cell Biology Platform to Investigate the Effects of Stem Cell Positional Identity on SEGA Development Charlotte Tye, Kings College London. Attention deficit hyperactivity disorder and executive dysfunction in Tuberous Sclerosis Complex

Exhibitors

About the Tuberous Sclerosis Association, UK The Tuberous Sclerosis Association (TSA) was established in 1977. The TSA supports individuals and families throughout the United Kingdom and Northern Ireland affected by tuberous sclerosis complex (TSC). The TSA’s mission is “to provide care for today and hope for tomorrow” and its three main objectives are to: • Support individuals affected by TSC, together with their families or caregivers. • Encourage and support research into the causes and management of TSC. • Provide education and information. The TSA has committed more than £500,000 over the next three years to help fund research into the causes and management of TSC. Raising awareness of TSC and its care and management are pivotal to our work. In the last two years we have: • Delivered TSC Awareness Training to 50 health, social care and education professionals, which has resulted in a greater understanding of meeting the needs of people affected by TSC. • Partnered with British Medical Journal Learning to produce an accredited online training module. This has given us an effective way of reaching and educating substantial numbers of GPs and other non-specialists. The module has also been accredited outside the UK and accessed by healthcare professionals worldwide. • Influenced policy and provision by lobbying parliamentarians and ministers and promoting awareness of TSC through engagement with national, regional and local media. Each year the TSA provides in excess of 3,000 hours of support direct to families and individuals. This includes working with people affected by TSC to help them to navigate the health, social care and welfare system, providing emotional and practical support throughout. Our aim is to ensure people affected by TSC achieve the best possible outcomes and maximize their quality of life. TSA Research Strategy The TSA began funding research into TSC in 1980. Since then we have given financial support to a wide range of projects spanning the research spectrum from the laboratory to the clinic. Funding by the TSA has contributed to the following medical advances: • Identification of the TSC genes • DNA testing for TSC • Discovering the natural history of TSC • Understanding what goes wrong in TSC cells • Clinical trials of mTOR inhibitors There is very little government funded TSC research in the UK. For this reason, it is absolutely vital that the TSA targets its research spend effectively. Our strategy focuses on understanding TSC and its impacts and treating and preventing manifestations of TSC. Our objectives are to: 1. Increase our level of investment in TSC research. We will target our investment with a view to encouraging young or new researchers into the field of TSC research and through awarding ‘seed grants’ to innovative projects. We will continue to support successful active research centres.

2. Raise the profile of TSC research, actively seeking, fostering and promoting collaborations. 3. To maximise the involvement of people affected by TSC in research. We will provide support and information that will enable people affected by TSC to understand and participate in TSC research at all levels; helping to set the research agenda, shape and review our investments, raise funding to support research and participate in research projects. Our strategic themes are: Theme 1: Understanding TSC and its impacts • Natural history of TSC, outcome or prognosis– supporting a UK register / database of natural history • Social & service Research – stimulating social research to reveal the unmet needs of people affected by TSC such that resources can be targeted to relieve them • Molecular genetics research • Understanding individual manifestations of TSC, for example is epilepsy alone involved in SUDEP or are there other contributory factors? Theme 2: Treating and preventing the manifestations of TSC • Epilepsy: anticonvulsant drugs trials in epilepsy • Neuropsychological/psychiatric problems: including the understanding of and alleviation of autistic behaviours • Improving outcomes in neurocognition • Treatment of tumours: including the topical treatment for skin tumours • Early intervention studies to ameliorate the long-term effects of TSC. • Treatment of kidney and lung problems Our vision: To facilitate the development of a major early invention study. This is likely to be based around the use of an mTOR inhibitor to in early childhood and likely to focus outcomes in the following areas: • Prevention of the progression of kidney disease in children with known kidney problems • Prevention of the worse aspects of TSC amongst neonates including: Epilepsy, ASD, Intellectual impairment, SUDEP, skin rash, kidney and lung disease etc. This is a major driving focus for TSA research. It has become our major research programme known as TSCure. It requires large scale preparation and collaboration. This will include: • Commissioning of specific reviews and primary research • Development of a task force to determine the focus and parameters of early intervention studies • Lobbying governmental bodies, funding councils, international TSC organisations and pharmaceutical companies to secure appropriate funding for this major undertaking. • The use of the 2015 International conference as a platform for developing methodological aspects of the studies and building collaboration. • Promoting or assisting with other studies that help build the body of knowledge important to the development of TSCure.

TSA Funded Grants: 2015-F01 Dr Charlotte Tye, Kings College London, UK. Designing a protocol to measure the developmental trajectory of infants with Tuberous Sclerosis.

Problem behaviours in Tuberous Sclerosis Complex (PhD student: Stacey Bissell). 2013-F02 Dr Kayleigh Dodd, Cardiff University, UK. Investigating mTORC1 independent functions of TSC2.

2015-S01 Dr Andrew Tee, Cardiff University, UK. New drug therapies to kill TSC2-deficient cell lines (PhD Student: Henry McCann).

2013-P01 Professor Sue Povey, University College London, UK. The TSC1 and TSC2 Variation Databases.

2015-F02 Professor. Ype Elgersma, Erasmus Medical Center, The Netherlands. Towards a better understanding and better treatment of TSC-related epilepsy (Fellow: Dr Jadwiga Schreiber).

2013-P02 Dr Ming Hong Shen, Cardiff University, UK. Prevention of renal lesions by fine-tuning mTOR signalling in a mouse model of Tuberous Sclerosis.

2014-F04 Dr Sophie Thomson, University of Edinburgh, UK. Targeting the mGluR5-FMRP signaling pathway for the treatment of TSC.

2013-P05 Dr Andrew Tee, Cardiff University, UK. Restoring TSC pathology through autophagy induction and mTORC1 inhibition. 20121 Dr Lucy Wilde, University of Birmingham, UK. Challenging behaviour and autism spectrum disorder in tuberous sclerosis complex: from research to practice.

2014-F06 Dr Lucy Wilde, Cerebra Centre, University of Birmingham, UK. Delineating impulsive behaviour in Tuberous Sclerosis Complex.

20122 Professor Patrick Bolton, Kings College, London, UK. Autism Spectrum and Attention Deficit Hyperactivity Disorders in the Tuberous Sclerosis 2000 Cohort Study (Fellow: Holan Liang).

2014-S01 Professor Petrus de Vries, University of Cape Town, South Africa. Understanding and Treating TSC-Associated Neuropsychiatric Disorders (TAND) (PhD Student: Loren Leclezio). 2014-S02 Dr Ming Hong Shen, Cardiff University, UK. Dual inhibition of glycolysis and glutaminolysis for anti-tumour therapy in a Tsc2+/- mouse model (PhD Student: Ashley Jones).

Professor Julian Sampson, Cardiff University, UK. Contribution toward patient expenses for: TRON: A randomised, double blind, placebocontrolled study of RAD001 (Everolimus) in the treatment of neurocognitive problems in Tuberous Sclerosis.

2014-S03 Professor Chris Oliver, University of Birmingham, UK.

TSA Committees Board of Trustees: President Christine Naylor Honorary Life Vice Presidents Anne Carter Tom Carter Esther Galbraith Trustee Officers Martin Short, Chairman Philip Goldenberg, Deputy Chair and Company Secretary Rob Vaughan, Honorary Treasurer Other Trustees Martin Balfour-Allen Janet Bower Nick Dale Marie James Annie Jones Dr Chris Kingswood Sophie Lording Christine Naylor Lisa Suchet Jean Wilson Research Committee (RESCOM) Prof Harold Baum John Blay Jeannie Blay Paula Davis Nick Davis Catherine Falconer Dr Alan Fryer Carole Hagan Perry James Annie Jones (Chair) Dr Chris Kingswood

About the Tuberous Sclerosis Alliance, USA As a registered 501(c)(3) organization, the Tuberous Sclerosis Alliance (TS Alliance) is the only US-based voluntary health organization dedicated to finding a cure for TSC while improving the lives of those affected. In 1974, four mothers from Southern California with a dream formed the TS Alliance to provide fellowship, generate awareness, pursue knowledge and provide hope to those who shared the unfortunate common bond of tuberous sclerosis complex (TSC). These goals remain the driving force of the organization today. For more than 40 years, the TS Alliance has been the leading resource for individuals, families and healthcare providers, helping optimize care and offering a wide range of support services, information and resources for those affected by TSC. Over the years, we have expanded our mission to improve quality of life for individuals and families affected by TSC through: • The stimulation and sponsorship of research; • The development of programs, support services and resource information; and • The development and implementation of public and professional education programs designed to heighten awareness of TSC. Since 1984, the TS Alliance has funded almost $18 million in research grants. Because of our advocacy for research and promoting young investigators, we funded and cultivated a new generation of senior scientists who today receive generous funding from sources such as the National Institutes of Health and the Department of Defense Congressionally Directed Medical Research program. Because of the strength of our grassroots volunteers, more than $267 million has been spent on TSC research over the past 12 years from these important sources. Visit www.tsalliance.org for a wide array of resources, including medical and support information, research grants and clinical trial listings, materials for parents/caregivers as well as adults with TSC, school-related issues, outreach and awareness campaigns, online support and discussion groups, government advocacy program details, events calendar, Community and Global Alliance listings and contact information, resources for adults with TSC, online donations, videos, and much more. Research Strategy The TS Alliance’s Research Program stimulates and supports basic, translational, and clinical research on the various manifestations of tuberous sclerosis complex to further the development of clinical therapies and, ultimately, a cure for TSC. The TS Alliance research grants program funds projects focused on TSC through postdoctoral fellowships, research grants, and Rothberg Courage Awards. Also, collaborations between basic and clinical researchers are encouraged and fostered by actively participating in and providing financial support for larger research initiatives and consortia. Implemented in 2006, the TSC Natural History Database captures clinical data to document the impact of the disease on a person’s health over a lifetime. As of June 2015, 1674 people with tuberous sclerosis complex were enrolled in the project from among 17 U.S.-based sites. The TS Alliance provides funding to participating clinics to perform data entry, monitors the integrity of the

database, and makes data available to investigators to answer specific research questions and identify potential participants for clinical trials and studies. The TSC Biosample Repository is a new TS Alliance directed project that will impact research over the next ten years or more. High-quality biosamples such as blood, DNA, and tissues linked to detailed clinical data are required for researchers to understand why TSC is so different from person to person. Such samples are currently unavailable, and the TS Allianceâ&#x20AC;&#x2122;s Science and Medical Committee identified this as a gap that can be filled effectively with leadership of the TS Alliance, guided by a steering committee of clinicians and researchers. The TS Alliance contracted with the Van Andel Research Institute in Grand Rapids, Michigan, to host and distribute biosamples. The TS Alliance is soon launching a TSC Preclinical Research Consortium. The research community recognizes that prioritization of ideas for clinical trials of potential new treatments will be best achieved by comparing head-to-head data using consistent animal models and testing procedures. Selection of the best candidate drugs from among drug screening hits is best achieved in a collaborative way, bringing together the best minds in TSC biology and experts in the process of drug discovery and development. The TS Alliance is ideally positioned to drive this collaborationâ&#x20AC;&#x201D;with input from academic, regulatory, and industry stakeholdersâ&#x20AC;&#x201D;by maintaining constant focus on the needs of those affected by the disorder. The organization also continues to be a key partner in the TSC Clinical Research Consortium. The five clinics comprising this consortium received National Institutes of Health grants to conduct two clinical studies initiated in 2013 to determine what early signs or tests can identify infants with TSC at highest risk of developing autism by age three, as well as measure the ability of EEG and brain imaging to assess the risk of newly diagnosed infants with TSC to develop infantile spasms. The biomarkers resulting from studies will have a major impact on our ability to intervene very early to prevent some of the most devastating manifestations of TSC. Additionally, the coordinated work to execute both of these studies has developed infrastructure and processes to form the basis of an ongoing and growing TSC Clinical Research Network.

TS Alliance Funded Grants Michael Higley, MD, PhD Yale University, New Haven, CT. Analysis and treatment of altered cortical network dynamics in TSC.

FY2014 Postdoctoral Fellowship Award Gina Lee, PhD Sponsor: John Blenis, PhD Weill Cornell Medical College, New York, NY. mTORC1-mediated mRNA alternative splicing in Tuberous Sclerosis and LAM Co-funded with The LAM Foundation.

Wei Shi, PhD Children’s Hospital, Los Angeles, CA. Lung mesenchymal TSC2 deletion in TSC lung disease.

FY2013

Gerta Hoxhaj, PhD Sponsor: Brendan Manning, PhD Harvard University School of Public Health, Boston, MA. Role of the TSC complex component TBC1D7 in cell migration and invasion.

Research Grant Award Rebecca Ihrie, PhD Vanderbilt University, Nashville, TN. Dissecting the effects of stem cell patterning on subependymal tumors.

Research Grant Award David Kwiatkowski, MD, PhD Brigham and Women’s Hospital, Boston, MA EPISTOP: clinical and molecular biomarkers of epilepsy in TSC. Co-funded with the European Commission.

Sue Povey, MD, MA University College London, London, UK. The TSC1 and TSC2 variation database Co-funded with the Tuberous Sclerosis Association (UK).

Carmen Priolo, MD, PhD Brigham and Women’s Hospital, Boston, MA. Aberrant lipid metabolism in TSC diagnostics and therapeutic

Gabriella D’Arcangelo, PhD Rutgers, The State University of New Jersey, Piscataway, NJ. Identifying TSC cellular phenotypes using patient-derived iPSCs.

Board of Directors The Tuberous Sclerosis Alliance Board of Directors have general oversight, fiduciary, policy development and ambassadorial responsibilities. The board develops and approves strategic plans that define the framework that guides the TS Alliance staff in daily activities, which move the organization closer to achieving its mission. Laura Lubbers, PhD, Chair Darren Miles, Treasurer Perkasie, PA Minneapolis, MN David Fitzmaurice, Vice Chair Wyckoff, NJ

Keith Hall, Past Chair East Lyme, CT

Rebecca Anhang Price, PhD, Secretary Chevy Chase, MD

Martina Bebin, MD, MPA Athens, AL

John Bissler, MD Memphis, TN

Ted Mastroianni Washington, DC

Cassandra Carroll Oakland, CA

David Michaels Scarsdale, NY

April Cooper Irvine, CA

Debora Moritz Scottsdale, AZ

Beth Dean Chicago, IL

Courtney O'Malley New York, NY

Rita DiDomenico Staten Island, NY

David Parkes Fairfield, CT

Steven Goldstein Westport, CT

Henry Shapiro Santa Monica, CA

Colleen Johns Narberth, PA

Elizabeth Thiele, MD, PhD Newton, MA

Brendan Manning, PhD Boston, MA International Scientific Advisory Board The TS Alliance International Scientific Advisory Board (ISAB) is comprised of both U.S. and international leaders in all areas of science relating to TSC research. This board assists the TS Alliance in defining the role of the TS Alliance in both current and future TSC scientific endeavors and will work with the TS Alliance Professional Advisory Board to identify and prioritize translational research opportunities. ISAB members meet biennially through a TS Alliance-sponsored conference, which also provides a forum for promoting collaborative efforts between the international and U.S. TSC research communities. The mission of the ISAB is to help guide the TS Alliance’s future research strategies by: • • • •

Providing direction for immediate and long term TSC research initiatives; Identifying and prioritizing translational TSC research opportunities; Enhancing awareness of TSC and the TS Alliance in the member’s respective research communities; and Aligning with the TS Alliance Professional Advisory Board to position the TS Alliance as the leader in privately-funded TSC research.

Chair Elizabeth Petri Henske, MD Brigham and Women's Hospital Harvard Medical School Boston, MA.

John Blenis, PhD Department of Pharmacology, Weill Cornell Medical College, New York, NY

Peter Crino, MD, PhD Department of Neurology Temple University School of Medicine Philadelphia, PA.

Brendan Manning, PhD Department of Genetics and Complex Disease Harvard School of Public Health Boston, MA.

Petrus J. de Vries, MBChB, MRCPsych, PhD Sue Struengmann Professor of Child & Adolescent Psychiatry University of Cape Town Rondebosch, Cape Town, South Africa.

David Mowat MBBS, MRCGP, DRACOG, FRACP Senior Staff Specialist Clinical Geneticist Sydney Children's Hospital Sydney, Australia.

Howard Goodkin, MD, PhD Department of Neurology University of Virginia Charlottesville, VA.

Hope Northrup, MD Professor of Pediatrics Director, Division of Medical Genetics University of Texas–Houston Houston, TX.

Luciana Amaral Haddad, MD, PhD Departamento de Genética e Biologia Evolutiva, Universidade de São Paulo São Paulo, Brazil.

Mustafa Sahin, MD, PhD Department of Neurology Children’s Hospital/Harvard Boston, MA.

Gregory Holmes, MD Chair, Department of Neurological Sciences University of Vermont School of Medicine Burlington, VT

Julian Sampson, DM, FRCP, FMedSci Professor and Head of Department, Medical Genetics Institute of Medical Genetics Wales College of Medicine Cardiff University Cardiff, Wales, UK.

Chris Kingswood, MSc, FRCP Brighton & Sussex University Hospitals Trust Renal Unit Brighton, UK.

Andrew Tee, PhD Medical Genetics Institute of Medical Genetics Wales College of Medicine Cardiff University Cardiff, Wales, UK.

David Kwiatkowski, MD, PhD Professor of Medicine Brigham & Women’s Hospital & Harvard Medical School Boston, MA. Richard Lamb, PhD Calgary University Calgary, Canada.

Michael Wong, MD, PhD Department of Neurology Washington University School of Medicine St. Louis, MO.

Raymond Yeung, MD Department of Surgery University of Washington Seattle, WA.

Hongbing Zhang, MD, PhD Institute of Basic Medical Sciences Chinese Academy of Medical Sciences Peking Union Medical College Beijing, China.

Tuberous Sclerosis Complex International (TSCi) is a consortium of organizations that support individuals with TSC around the world. Initiated in the mid 1980s, TSCi serves as a forum for the organizations to share information, exchange ideas and methods, co-fund research projects, and support individuals with tuberous sclerosis complex (TSC) and their families, caregivers, educators and health care providers. Additional information can be found on the TSCi website (www.tscinternational.org). ARGENTINA Asociación Argentina de Esclerosis Tuberosa (ARGET) Telephone: 011 1565474229 Email: asociacionarget@gmail.com Website: www.esclerosis-tuberosa.org; www.asociacionarget.blogspot.com.ar Representatives: Constanza Mavroyani Cynthia Huenuman

BRAZIL Brazilian Association of Tuberous Sclerosis (ABET) Address: Rua Joaquim Linhares, 30 Anchieta, Belo Horizonte, MG, Brazil Telephone: (31) 3221 12 44, (31) 3223 78 75 Email: abet@abet.org.br Website: abet.org.br/jla2 Representatives: Marcia da Silva Wesley de Assis Gomes, Email: wesleyag@uol.com.br Dr. Luciana A. Haddad, University of São Paulo, Email: grupotsc@usp.br

AUSTRALIA Tuberous Sclerosis Australia Address: 17 Linksview Rd, Springwood NSW 2777, Australia Telephone: 1300 733 435 (Australia only) Email: info@atss.org.au Website: www.atss.org.au Representatives: Debbie Crosby Clare Stuart

CANADA Tuberous Sclerosis Canada Sclérose Tubéreuse Address: 132 Commerce Park Drive, Unit K, Suite 125, Barrie, ONT, L4N-0Z7, Canada Telephone:1-888-223-2410 (English and French Toll-Free) Website: www.tscanada.ca Representatives: Jennifer Flinn, Email: jflinn@outlook.com

AUSTRIA Tuberöse Sklerose Complex Mitanand Address: Berg 44 b, A-6840 Götzis, Austria Website: www.tuberoesesklerose.at Email: info@tuberoesesklerose.at Representative: Jeanette Bobos, Email: Bobos@tsdev.org Andrea Schmidt

CHINA TSC China Alliance Telephone: 010-56230920 13391565385 Email: scchina@126.com; help@tscchina.org Website: www.tscchina.org Representatives: June Lu, Email: lumj@tscchina.org Jane Yu, Email: yum@tscchina.org Dr. Hongbing Zhang

BELGIUM Tubereuse Sclerosis in Vlaanderen Telephone: 014 43 36 40 Email: info@tsiv.be Website: www.tsiv.be Representative: May Stoops, Email: maystoops@scarlet.be

COLUMBIA FUNCOLAMEST, Columbian Foundation for Patients with LAM & TSC Telephone: 8675151

Cell Phone: 011-57- 314 344 1730 Website: http://destulamoca.wix.com/funcolamest Representative: Deicy Ester Mojica Casallas, Email: destulamoca@gmail.com

Address: Israeli T.S.C., 4/1 Irit Street, Tel Aviv 68047, Israel Telephone:+972 3 6595593 +972-(0)50-627-8882 (cell) Website: www.tsc.org.il Representative: Dan Bloom, E-mail: dan@tsc.org.il Yosef Savion, E-mail: saviony@netvision.net

DENMARK Dansk Forening for Tuberøs Sclerose (DFTS) Address: Hortensiavej 21, 8270 Højbjerg, Denmark Telephone: 1-888-223-2410 (English Toll-Free) 1 866 558 7278 (Téléphone en Francais) Website: www.tsdanmark.dk Representative: Liselotte W. Andersen, Tel: 8627 7714, Email: brandersen@webspeed.dk

ITALY Associazione Sclerosi Tuberosa- AST Onlus Address: Via Anagnina Nuova,13 -, c/o Villaggio "Eugenio Litta"-00046, GROTTAFERRATA (RM) Italy Telephone/Fax: (+39) 06 9415001 - 335-8282000 Telephone: +39 338 7177673 Email: info@sclerosituberosa.org Website: www.sclerosituberosa.org Representative: Carla Fladrowski Ferrara, Email: LaCarloski@gmail.com

FRANCE Association sclérose tubéreuse de Bourneville (ASTB) Address: 3 rue des Coquelicots, 67150 Hindisheim, France Telephone: +33 9 70 44 06 01 Email: astb.siege@yahoo.fr Website: www.astb.asso.fr Representatives: Guillaume Beaure d'Augères, Email: gba.astb@laposte.net Nathalie Rudelle, Email: astb.rudelle@orange.fr

JAPAN The Japanese Society of Tuberous Sclerosis Complex Address: 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-8421 Telephone: 03-5802-1038 Email: tscres@juntendo.ac.jp Website: jstsc.kenkyuukai.jp Representatives: Dr. Okio Hino, Juntendo University, ohino@juntendo.ac.jp Megumi Takei, mtakei.intermedd@sky.bbexcite.jp

GERMANY Tuberoese Sklerose Deutschland e.V. (TSD) Address: Im Brückfeld 15, 65207 Wiesbaden, Germany Telephone:+49-611-469-2707 +49-611-469-2708 Website: www.tsdev.org Representatives: Angelika Kodat, Email: Angelika@Kodat.net Dr. Karin Mayer, Email: mayer@tsdev.org

MACEDONIA Dajte ni krilja Address: Institute of Radiotherapy and Oncology fSkopje, Vodnjanska 17 1000, Skopje, Macedonia Telephone: +38975410444 Fax: 0030 210 7660989 Website: www.dajtenikrilja.mk/ Representative: Dr. Milan Risteski, Email: milanristeski@yahoo.com

GREECE Tuberous Sclerosis Association of Greece Address: Timotheou 99-101, 162 32 Athens, Greece Telephone: 0030 210 7600289 Fax: 0030 210 7660989 Email: tsahellas@ath.forthnet.gr Website: www.pespa.gr Representatives: Marianna Lambrou, Email: tsahellas@ath.forthnet.gr Dr. Sotiris Giouroukos

MEXICO Asociación Mexicana de Esclerosis Tuberosa, A.C. (AMET) Address: MexCalle 40 N°405B, por 29 y 27 Colonia Jesús Carranza, CP 97109, Mérida Yucatán - México Telephone: 9262097 Website: www.amet.org.mx Email: amet@amet.org.mx; esclerosistuberosa@hotmail.com Representatives:

ISRAEL Tuberous Sclerosis Alliance of Israel

Ary Agami Arrieta Maria Luisa Pardo, Email: esclerosistuberosa@hotmail.com

RUSSIA TSA Russia Address: Lazo str. 10/1-51, Moscow 111398, Russia Telephone: 7-095-309-2452 Website: www.epileptologhelp.ru Representatives: Marina Dorofeeva, Email: mdorofeeva@inbox.ru Leonid Satanin, Email: lsatanin@nsi.ru

NETHERLANDS Stichting Tubereuze Sclerosis Nederland (STSN) Address: Secretariaat STSN, Couwenhoven 53-09, 3703 ES Zeist, The Netherlands Telephone: +31 (0)6 52 82 46 60 Email: info@stsn.nl Website: www.stsn.nl Representative: Hans Ploegmakers, Email: algemeen@stsn.nl

SLOVAKIA ASTUS – Asociácia tuberóznej sklerózy Address: ASTUS, n.o. Záhorácka 5272/21A, 90101 Malacky, Slovakia Telephone: +421 (0) 907 228 284 (mobile); +421 (0) 948 384 886 Website: www.diagnozatsc.sk Email: astus@astus.sk or info@diagnozatsc.sk Representative: Barbora ZAJAČEK (Hlavicová), Email: barbora@astus.sk Andrej Melicher

NEW ZEALAND Tuberous Sclerosis Complex New Zealand (TSCNZ) Address: 11 Riverlea Drive, Katikati 3129, New Zealand Telephone: 0064211547017 Email: info@tsc.org.nz Website: www.atss.org.au/nz/ Representatives: Helen Willacy NORWAY Norsk Forening for Tuberøs Sklerose (NFTS) Address: 2130 KNAPPER, Norway Telephone: 45 67 27 51 Website: www.nfts.no Representative: Torbjørn Hodne Wenche Røknes

SOUTH AFRICA TSC South Africa Representatives: Professor Petrus de Vries Email: rehana.effendi@uct.ac.za, +27 21 6854103 Loren Leclezio Email: loren.leclezio@uct.ac.za, +27 21 6854103 SPAIN Asociación Nacional de Esclerosis Tuberosa Address: Calle Camarena nº 119, Bajoº Local, 28047 - Madrid Telephone: Madrid: 917 19 36 85, Barcelona: 619310914 Email: Madrid: escletuber10@gmail.com, Barcelona: infobcn@esclerosistuberosa.org Representatives: Yolanda Palomo Castano, Email: escletuber10@gmail.com Katja Kramer

POLAND Stowarzyszenie Chorych na Stwardnienie Guzowate Address: ul. Morska 53A, 81-332 Gdynia, Poland Telephone: 914-227-1740 (English) Website: www.stwardnienie-guzowate.eu Email:stowarzyszenie.stw.guz@wp.pl Representatives: Andrzej Chaberka, Email: andrzej.chaberka@wp.pl Joanna Hensel, Email: henseljoanna@gmail.com (all English correspondence) Aneta Jurko, Email: ajurko@o2.pl PORTUGAL Associação de Esclerose Tuberosa Portugal Address: Rua das Rosas N-34, 2750-260 Cascais, Portugal Telephone: Mobile: +351 916 521 322 Website: www.esclerosetuberosa.org.pt Email: esclerosetuberosa@gmail.com Representatives: Micaela Rozenberg Catarina Paulino

SWEDEN TSC Sverige Telephone: 0046 8 649 18 61 or cell phone number: 0046 70 680 89 54 Website: www.tsc-sverige.se Representatives: Berit Oberg, Email: lennart.berit@telia.com Bo Karlberg, Email: bo.karlberg@anchem.su.se

SWITZERLAND STB Suisse Address: Case postale 323, 3065 Bolligen Telephone: 031 921 01 06 Email: stbsuisse@postmail.ch Website: www.stbsuisse.ch Representative: Doris Friedrich, Email: Doris@bluewin.ch (German Speaking Only)

Tuberous Sclerosis Association Address: CAN Mezzanine, 32026 Loman Street, London, E1 0EH, United Kingdom Telephone: 0208 690 2288 Email: jayne.spink@tuberous-sclerosis.org Website: www.tuberous-sclerosis.org Representatives: Dr. Chris Kingswood Dr Jayne Spink

TAIWAN Taiwan Tuberous Sclerosis Complex (TTSC) Association Address: Hengyang Road, Taipei 8F-7 No. 6, Taiwan Telephone: 0223318852 Email: TSCare@gmail.com Website: www.ttsc.org.tw Representatives: Robert Luu Jerry Yu

USA Tuberous Sclerosis Alliance Address: 801 Roeder Road, Suite 750, Silver Spring MD 20910 Telephone: 1-301-562-9890 1-800-225-6872 (Toll-Free) Fax: 1-301-562-9870 Email: info@tsalliance.org Website: www.tsalliance.org Representatives: Katie Smith, E-mail: ksmith@tsalliance.org Kari Luther Rosbeck, E-mail: krosbeck@tsalliance.org

UNITED KINGDOM

Conference Support Acknowledgements

The Tuberous Sclerosis Association and the Tuberous Sclerosis Alliance are very grateful for the support received from the following groups in helping make the conference successful.

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“it is one of the most exciting timies in medicine and especially in TSC research... The goals of the NYU Langone Medical Center and Bcureful are to help organize collaborative efforts to focus research on projects that can truly advance both our understanding of TSC, and most importantly, to improve the care and treatment in the near future.”

-Dr. Orrin Devinsky, NYU Langone Medical Center

Join us as we go forward in the direction of a cure for Tuberous Sclerosis Complex

www.bcureful.org

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Holyrood

Blenhelm Boardroom

1st Floor

Hampton

Chatsworth

Ground Floor

Warwick

Lodge

Sandringham Suite

OďŹ&#x192;ce

Entrance Foyer

Stage

Lobby

Bar

Windsor Suite

Balmoral

Georgian Room

Castle Hotel Windsor

Conference & Banqueting Suite Floor Plan