JAGT - Issue 2 2016

The Journal

of the Association of Genetic Technologists

Volume 42 • Number 2 • Second Quarter 2016

Brain Tickler

Column Editor: Helen Lawce

Brain Tickler

Submitted by:

A peripheral blood sample was received from a 3-year-old boy presenting with global developmental delay, cognitive impairment, failure to thrive and convulsions.

Rikki Harrison Cytogenetics Laboratory Oregon Health & Sciences University Portland, Oregon

The answer to this Brain Tickler appears on page 18.

The Journal of the Association of Genetic Technologists Second Quarter 2016 Volume 42, Number 2

Table of Contents

The official journal of the AGT

Brain Tickler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inside Front Cover Column Editors and Review Board . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 A Note from the Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Editorial Information Editor Mark Terry, BSc Associate Editors Turid Knutsen, MT(ASCP), CLSp(CG) Helen Lawce, BSc, CLSp(CG) Heather E. Williams, MS, CG(ASCP)CM Su Yang, BSc, CLSp(CG) Book Review Editor Helen Lawce, BSc, CLSp(CG) Copyright © 2016 by the AGT. All rights reserved. Contents are not to be reproduced or reprinted without permission of the AGT Editor.

The Journal of the Association of Genetic Technologists is published four times a year and is available to individuals and libraries at a subscription rate of $105 per year. The subscription rate for members of the AGT is included in the annual membership dues.

Back issues can be purchased for members at $5 per issue and for non-members at $25 per issue as long as supplies are available.

Material intended for publication or correspondence concerning editorial matters should be sent to the editor.

JAGT Editor Mark Terry 1264 Keble Lane Oxford, MI 48371 586-805-9407 (cell) 248-628-3025 (phone/fax) Email: markterry@charter.net

Case Study CML in Chronic Phase with Novel Secondary Cytogenetic Abnormalities: A Case Report.

Hiral S. Patel, Manisha M. Brahmbhatt, Pina J. Trivedi, Dharmesh M. Patel, Ankita A. Sugandhi, Aanal U. Mehta, Binita V. Patel, Prabhudas S. Patel. . . . . . . . . . . . . . . . . . . . 57 Case Study An Adult Male Presenting with Concurrent Plasma Cell Myeloma Involving a CCND1-IGH Translocation and Chronic Myelogenous Leukemia with a Variant (9;22) Translocation Peter M. Lee, Ken Siangchin, Sophie Song, David Shabsovich, Yalda Naeini and Carlos A. Tirado. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Profiles and Perspectives Dr. Rachel Burnside Interviewed by Hon Fong L. Mark. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Government Regulations 2016 ASCLS Legislative Symposium Overview Jennifer Crawford-Alvares, CG(ASCP)CM.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Brain Tickler Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Molecular Diagnostics A Journey of Continuous Learning Through Teaching Michelle Mah. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Genetics in the News The Roaring Molecular Revolution of Editing the Genomes of Many Living Organisms, Including Humans, Using Programmable Nucleases Jaime Garcia-Heras. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Continuing Education Opportunities Test Yourself #2, 2016 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 AGT Journal Clubs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Placement service items of less than 150 words and advertisements, requests for back issues, reprint orders, and questions about subscriptions and advertising costs should be sent to the AGT Executive Office at agt-info@kellencompany.com. Acceptance of advertisements is dependent on approval of the editor-in-chief.

Association Bu siness Association of Genetic Technologists BOD Contacts. . . . . . . . . . . . . . . . . . . . . . . . . . 89 Letter from the President . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 New Membership Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Product Order Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

ISSN 1523-7834

The Journal of the Association of Genetic Technologists is indexed in the life sciences database BIOSIS and in the National Library of Medicine’s PubMed. The Journal of the Association of Genetic Technologists 42 (2) 2016

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The Journal of the Association of Genetic Technologists Staff

Column Editors Abstract Reviews/Genetics in the News Jaime Garcia-Heras, MD, PhD Director of Cytogenetics The Center for Medical Genetics 7400 Fannin, Suite 700 Houston, TX 77054 713-432-1991 713-432-1661 FAX jgarcia@geneticstesting.com Brain Tickler/Book Review Editor Helen Lawce, BSc, CLSp(CG) Clinical Cytogenetics Laboratory Oregon Health Sciences University 3181 SW Sam Jackson Parkway MP-350 Portland, OR 97201 503-494-2790 503-494-6104 FAX lawceh@ohsu.edu

Genetics, Government & Regulation Helen Bixenman, MBA, CLSup, CLSp(CG) San Diego Blood Bank 3636 Gateway Center Avenue, Suite 100 San Diego, CA 92102 619-400-8254 hbixenman@sandiegobloodbank.org Jennifer Crawford-Alvares Cytogenetic Technologist II Section of Hematology/Oncology The University of Chicago Medicine 5841 S. Maryland Ave.,Rm. I-304 Chicago, IL jen.crawford34@gmail.com Office: 773-702-9153

Meeting Notices Jun Gu, MD, PhD, CG(ASCP)CM University of Texas MD Anderson Cancer Center School of Health Professions Cytogenetic Technology Program 1515 Holcombe Blvd., Unit 2 Houston, TX 77030 713-563-3094 jungu@mdanderson.org Molecular Diagnostics Michelle Mah, MLT, MB(ASCP)CM Advanced Molecular Diagnostics Laboratory Princess Margaret Cancer Centre University Health Network Toronto, Ontario, Canada 416-946-4501 ext.5036 michelle.mah@uhn.ca

Letters to the Editor Mark Terry, JAGT Editor 1264 Keble Lane Oxford, MI 48371 586-805-9407 (cell) 248-628-3025 (phone/FAX) markterry@charter.net

Special Interests Turid Knutsen, MT(ASCP), CLSp(CG) 17836 Shotley Bridge Place Olney, MD 20832 301-570-4965 knutsent@earthlink.net Test Yourself Sally J. Kochmar, MS, CG(ASCP)CM Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu

Profiles & Perspectives Hon Fong Louie Mark, PhD, FACMG President KRAM Corporation 2 Pine Top Road Barrington, RI 02806 401-246-0487 HonFong_Mark@Brown.edu

Review Board Linda Ashworth, BSc, CLSp(CG) (Cytogenetics, Molecular genetics)

Jaime Garcia-Heras, MD, PhD (Clinical cytogenetics)

Jennifer L. McGonigle, BA, CLSp(CG) (Cytogenetics)

Debra Saxe, PhD (Prenatal diagnosis, Cytogenetics)

Helen Bixenman, BSc, CLSp(CG), CLSup (Prenatal diagnosis)

Robert Gasparini, MS, CLSp(CG) (Prenatal diagnosis, Cytogenetics)

Judith Brown, MS, CLSp(CG), CLSp(MB) (Cytogenetics)

Barbara K. Goodman, PhD, MSc, CLSp(CG) (Molecular cytogenetics)

Karen Dyer Montgomery, PhD, FACMG (Cancer cytogenetics, Cytogenetics, Molecular cytogenetics)

Jack L. Spurbeck, BSc, CLSp(CG) (Cancer cytogenetics, Molecular genetics)

Kim Bussey, PhD (Cancer genetics, Molecular genetics, Microdissection/PCR/DNA) Mona CantĂş, BSc, CLSp(CG) (Cytogenetics) Anthony Ciminski, CG(ASCP)CM Molecular Genetics, Molecular Cytogenetics Adam Coovadia, CLSpP(CG, MG) (Traditional, Molecular, Regulatory) Philip D. Cotter, PhD, FACMG (Prenatal diagnosis, Chromosome rearrangements, Molecular genetics) Jennifer Costanzo, MS, CLSp(CG) (Cytogenetics, Molecular genetics) Janet Cowan, PhD (Cytogenetics, Cancer genetics, FISH, Solid tumors) Lezlie Densmore, BSc, CLSp(CG) (Cytogenetics, Molecular genetics) Janet Finan, BSc, CLSp(CG) (Hemic neoplasms, Somatic cell hybridization) Sue Fox, BSc, CLSp(CG) (Bone marrow cytogenetics, Prenatal diagnosis, Supervisory/Management)

Stephen R. Moore, PhD, ABMG Peggy Stupca, MSc, CLSp(CG) (Clinical cytogenetics, radiation biology, (Cytogenetics, Prenatal diagnosis, toxicology; clinical molecular genetics) Breakage syndromes, FISH, Regulations/ Michelle M. Hess, MS, CLSp(CG) QA) (Cytogenetics, Cancer cytogenetics) Rodman Morgan, MS, CLSp(CG) (Cancer cytogenetics) Nancy Taylor, BSc, CLSp(CG), MT(ASCP) Lynn Hoyt, BSc, CLSp(CG), CLSup (Cytogenetics, Cancer cytogenetics) (Classical cytogenetics) Susan B. Olson, PhD (Cancer cytogenetics, Molecular Thomas Wan, PhD Peter C. Hu, PhD, MS, MLS(ASCP), CG, MB (Cytogenetics, Molecular genetics, genetics, Prenatal diagnosis, OB/GYN, (Cytogenetics, Molecular cytogenetics, Cancer genetics) Counseling, Cytogenetics) Education) James Waurin, MSc, CLSp(CG) Jonathan P. Park, PhD Denise M. Juroske, MSFS, MB(ASCP)CM (Cytogenetics, Molecular genetics, (Prenatal diagnosis, Counseling) (Cytogenetics, Molecular, Education) Cell biology) Sara Wechter, BSc Julia Kawecki, BSc, CLSp(CG) (Cytogenetics, Cancer) David Peakman, AIMLT, CLSp(CG) (Cytogenetics, Molecular genetics) (Prenatal diagnosis) Heather E. Williams, MS, CG(ASCP)CM Turid Knutsen, MT(ASCP), CLSp(CG) (Cytogenetics, Molecular Genetics) Carol Reifsteck, BA (Cancer cytogenetics, CGH, SKY) (Breakage syndromes, Fanconi’s Su Yang, BSc, CLSP(CG) anemia, Prenatal diagnosis) Brandon Kubala, BSc, CLSp(CG) (Education, Traditional Cytogenetics) (Traditional Cytogenetics) Gavin P. Robertson, PhD Jason A. Yuhas, BS, CG(ASCP)CM (Cytogenetics, Molecular genetics, Anita Kulharya, PhD (Cytogenetics, Molecular cytogenetics) Somatic cell genetics, Tumor suppressor (Molecular genetics, Clinical genes, Cancer genes) cytogenetics) James Zabawski, MS, CLSp(CG) (Education, Traditional Cytogenetics) Laurel Sakaluk-Moody, MSc, MLT(CG) Helen Lawce, BSc, CLSp(CG) (Cytogenetics, Developmental biology, (Prenatal diagnosis, Solid tumors, FISH, Prenatal cytogenetics) Chromosome structure, Evolution) Hon Fong Louie Mark, PhD, FACMG (Molecular genetics, Somatic cell genetics, Cancer cytogenetics, Breast cancer, Trisomies, Laboratory practices, Regulatory practices, FISH)

The Journal of the Association of Genetic Technologists 42 (2) 2016

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A Note from the Editor

THIS, THAT & THE OTHER As I write this, in late April, spring has finally made its way to Michigan (for the most part), the New York primary was last night, with the winners being Donald Trump and Hillary Clinton, and I personally am heading toward two major landmarks — my youngest son, Sean, will graduate from high school in May and my oldest son, Ian, will also graduate from college in May. By the time you’re reading this, which should be in early June, summer should be here for pretty much everyone, the presidential runoff will still be ongoing, and hopefully you’re looking forward to AGT’s annual meeting in Anaheim.

Time passes, in other words, while things happen and/or get done.

Inside the Covers “Inside the covers” is kind of an interesting phrase in terms of digital versions of anything, but I digress.

This issue has some very interesting things in it, including a couple case studies, Michelle Mah’s column that places next generation sequencing into a real-world context, and Jaime Garcia-Heras’s fascinating overview of the technological, ethical and moral challenges of genomic editing with programmable nucleases.

And, of course, the “usual suspects” are here as well — the Test Yourself, Pat Dowling’s column, association news and others.

I hope you find value in the pages “between the covers.”

The Association I also hope that you find value in The Association of Genetic Technologists. The organization has been challenged — as have many professional associations — by general long and short-term economic trends, and what some view as demographic changes, i.e., younger people don’t necessarily seem to join professional organizations. Or so claim the media.

For my own entertainment I typed “millennials” into Google and the first story to come up was a news story from today in a site called “Quartz” titled, “Traumatized by millennials, employers are already desperate to make Generation Z happy.”

The article cites research out of a business administration group called ADP, which surveyed 2,400 full-time and part-time employees of different age groups to evaluate how they think the global workplace had changed. That seems like a pretty huge extrapolation, 2,400 people in 13 countries used to show workplace trends globally, but there you have it.

One of the things they say is, “’The need for meaning has certianly evolved over the years,’” the ADP researchers wrote.

“’Today, the younger generation of Millennials places more of an emphasis on a search for meaning within their jobs than previous generations, who tended to look for meaning outside of work.’”

Hmmm. Well, I suspect there’s a lot of ways to interpret that, but for this column for this audience today, let’s just ask: What do you want out of your job?

And, related to this association, What do you want out of The Association of Genetic Technologists?

They’re not necessarily the same thing. Money is always one answer, presumably, from your job. Otherwise it’s a hobby. And I’m crass and money-fixated enough to think that sometimes when an employer starts talking about how meaningful and important your work is, there may be an undercurrent of, “Yeah, we can’t pay you better, and we’re about to cut your benefits again, but what you do is important!”

Hey, I’ve been there. I sat through the meetings where administrators told us what great work we were doing and how the patients benefit from our professionalism, skill and hard work, while at the same time telling us why there was a pay freeze and we had to pay more out-of-pocket for health insurance.

The point isn’t to complain about the realities of being in the workplace.

What is the Point, Mark?

The point is, basically, what do you want AGT to do for you? Advocate for higher pay? Provide input on state and federal licensure and certification issues that directly affect you? Conduct salary surveys so your employer can advocate for better pay on your behalf? Provide opportunities for continuing education?

Because AGT does that and more. But if it’s not being communicated clearly to the membership, we need to know that.

Or is is possible that these issues aren’t that important to you?

I suspect they really are, but they seem rather nebulous out in the working world. And if AGT disappeared, many of you would eventually find that you had little voice in the professional world concerning your profession … or if profession is too ambitious a word or concept, your job.

So the point, ultimately is: If you want that voice, support AGT and consider becoming involved with the organization.

Cheers, Mark Terry, Editor

AGT Website: www.AGT-info.org Member’s Only area – User Name: First initial, Last initial and AGT ID# (Ex. CR12345) Password: genetics

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Case Study

CML in Chronic Phase with Novel Secondary Cytogenetic Abnormalities: A Case Report.

Hiral S. Patel1, Manisha M. Brahmbhatt1, Pina J. Trivedi1, Dharmesh M. Patel1, Ankita A. Sugandhi1, Aanal U. Mehta1, Binita V. Patel1, Prabhudas S. Patel1* 1.

The Gujarat cancer & Research Institute, Asarwa, Ahmedabad, India

Abstract The clonal evolution in t(9;22)-positive Chronic Myelocytic Leukemia (CML) patients is well established. Four major changes occur in more than 70% of patients: +8, i(17q), +19, and an extra Philadelphia chromosome. Here, we present a case with CML-Chronic phase (CML-CP) and novel t(9;13)(q34;q12~13) in addition to t(9;22)(q34;q11.2). Fluorescence in situ hybridization (FISH) using dual color dual fusion probe analysis on interphase and metaphase cells confirmed the t(9;13)(q34;q12~13) as clonal evolution and secondary event to Philadelphia chromosome. This suggests minor route additional chromosomal aberrations which might affect prognosis. Further studies are required to ascertain the expression of genes that play a role in CML-blast crisis in order to to explore its therapeutic significance and prognostic value.

Introduction

count 499Ă—109/L, and white blood cell count of 142.7Ă—109/L with 127.4 absolute neutrophil count, 6% lymphocytes, 4.8% monocytes, 4% eosinophiles and 0.1% basophiles. The bone marrow aspirate revealed hypercellularity with 3% blasts, 1% promyelocytes, 37% myelocytes, 12% metamyelocytes, 13% band cells, 23% polymorphs, 2% eosinophils, 3% basophils, 2% lymphocytes, 4% late normoblasts and 0% early normoblasts. The marrow showed increased myeloid precursors with an increased myeloid to erythroid ratio, and a myelo-poly peak thus suggesting CML-CP. The patient was treated with Imatinib Mesylate (IM) at 400 mg/day soon after the diagnosis.

Chronic Myelocytic Leukemia (CML) is a clonal hematological disorder, which progresses from a chronic phase to a more aggressive accelerated stage called blast phase (Faderl et al., 1999). During the chronic phase, nearly all CML patients show the chromosomal t(9;22)(q34;q11) or a variant translocation rendering a fusion between the BCR and ABL1 genes (Nowell and Hungerford, 1960; Rowley JD, 1973; Kurzrock et al., 1988). The appearance of secondary chromosomal abnormalities in CML patients usually is considered the hallmark of the blast phase (Muehleck et al., 1984).

Such secondary chromosomal abnormalities are nonrandom, the most frequent being: additional Philadelphia (Ph)- chromosome, trisomy 8, 9, 19, 20 or 21; isochromosome 17; monosomy 7; and deletion of the Y chromosome. These chromosomal aberrations are designated as major-route additional chromosomal aberrations which include frequently observed abnormalities and minor-route additional chromosomal aberrations, which include rarely observed aberrations such as t(3;12), t(4;6), t(2;16), and t(1;21) (Mitelman et al., 1976; Babicka et al., 2006; Tarkan-Arguden et al., 2009; Fabarius et al., 2011). However, a minor percentage of patients show different secondary chromosomal abnormalities (Mitelman, 1993).

Occurrence of additional chromosomal abnormalities besides the Ph- chromosome is defined as Clonal Evolution (CE) and is considered to be a marker of disease progression. It reflects the genetic instability of the highly proliferative CML progenitors (Shah et al., 2015).

In the present study, we present a cytogenetically unique case of CML-CP with novel secondary chromosome change, t(9;13) (q34;q12~13) in addition to standard t(9;22)(q34;q11.2). To the best of our knowledge, such a case has not been reported to date as a secondary chromosomal change in the literature.

Conventional Cytogenetics

Chromosome-banding analysis was performed on bone marrow cells in unstimulated short-term (16 hours) cultures. The cells were treated with a colcemid and hypotonic solution, the pellet was fixed and washed in methanol-acetic acid (3:1), and the cells were resuspended in fixative and dropped onto slides. Chromosomes were examined after GTG-banding techniques. Twenty metaphases were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2013) (Verma et al., 1995; Shaffer et al., 2013).

Fluorescence in situ hybridization (FISH):

FISH was performed using Locus Specific BCR/ABL dual color dual fusion (DCDF) translocation probe and Whole Chromosome Painting (WCP) probe for chromosome 13 and 9 (Abbott Molecular, USA). Twenty metaphase spreads were analyzed, using aepi-fluorescence microscope (AxioImager. Z1 mot, Zeiss, USA) equipped with appropriate filter sets. Image capturing and processing were carried out using an ISIS FISH imaging system (MetaSystems, Germany). The WCP FISH was carried out to determine the nature of the translocation.

Material and Methods

Results

Case details:

Conventional Cytogenetics

A 65-year-old female was referred to The Gujarat Cancer and Research Institute, Ahmedabad, India, in June 2015 with chief complaints of generalized weakness and headache. The hematologic parameters were as follows: hemoglobin 9 g/L, platelet

Karyotyping was done before initiation of treatment, and the results were described as, 46,XX,t(9;13)(q34;q12~13),t(9;22) (q34;q11.2)[20] (Fig. 1). The results were further characterized by

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Case Study CML in Chronic Phase with Novel Secondary Cytogenetic Abnormalities: A Case Report.

Figure 1. A G-banded karyotype result showing 46,XX,der(9)t(9;13)(q34;q12~13),t(9;22)(q34;q11.2).

Arrow indicates abnormal chromosomes.

molecular cytogenetic studies.

severe block in differentiation and apoptosis. BCR-ABL is directly or indirectly responsible for progressive genomic instability or epigenetic changes, which occur at the CML stem cell level and/or in later CML progenitor cells. The degree of genomic instability is proportional to the level of BCR-ABL kinase activity. CE can occur in any phase of CML. Its frequency increases with the advancing stage, rising from 30% in accelerated phase to 80% in blast crisis (Mitelman et al., 2015). Here, we present a novel secondary change with t(9;13)(q34;q12~13) in addition to the Ph chromosome, which is observed in the chronic phase of CML patients. We confirmed t(9;13)(q34;q12~13) as secondary chromosomal change because: 1) FISH for BCR/ABL showed a typical positive signal pattern and no variant signal pattern, and 2) The WCP FISH results for chromosome 9 and 13 showed a gap between chromosome 9 and chromosome 13 signals, which indicated that t(9;13)(q34;q12~13) took place as a second event to t(9;22)(q34;q11). These secondary events to Ph chromosome are referred to as CE and advanced disease state.

The chromosome 13 rearrangements in blast crisis comprise both translocations and deletions. A common region of deletion is identified at 13q12-14. Deletion of 13q is sometimes found in the accelerated phase of CML disease. The t(12;13) as the sole additional abnormality in CML cases with myeloid blast crisis is

Fluorescence in situ hybridization (FISH) BCR/ABL-DCDF FISH:

The FISH results with DCDF-BCR/ABL revealed a typical positive signal pattern, i.e. 1G1O2F and confirmed BCR/ABL fusion gene (Fig. 2). The results were as follows: nucish (ABL1, BCRx3) (ABL1 con BCRx2)[200]. The FISH results did not reveal any variant translocation; this was a typical positive case for positive BCR/ABL translocation.

Whole Chromosome Paint (WCP) FISH

The WCP FISH for chromosome 13 Spectrum Orange (SO) and chromosome 9 Spectrum Green (SG) confirmed the translocation between chromosome 9 and chromosome 13 (Fig. 3). The results were as follows: 46,XX,der(9)t(9;13)(q34;q12~13),t(9;22)(q34;q11.2), ish der(9)t(9;13)(q34;q12~13)(wcp9+,wcp13+).

The final karyotype after WCP FISH was as follows: 46,XX,der(9) t(9;13)(q34;q12~13),t(9;22)(q34;q11.2)[20].

Discussion: In CML, progression to advanced phase is accompanied by a

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Case Study CML in Chronic Phase with Novel Secondary Cytogenetic Abnormalities: A Case Report.

Figure 3. The metaphase image is depicting WCP FISH results. The derivative chromosome 9 (spectrum green) and 13 (spectrum orange) are indicated with arrows. The green arrow shows the der(9q34), orange arrow shows the part of der(13q12~13) on der(9)(q34) and yellow arrow shows the gap between der(9q34) and der(13q12~13). The WCP FISH confirmed t(9;13)(q34;q12~13).

Figure 2. Result of BCR/ABL-DCDF FISH with typical positive signal pattern, 1G1O2F; a metaphase showing typical positive signal pattern for BCR/ABL translocation. Green arrow shows BCR gene on chromosome 22, orange arrow shows ABL gene on chromosome 9 and yellow arrows show fusion genes on derivative 9 and 22.

reported to be directly related to progression of the disease (Boyer et al., 2002). Deletions of genetic material from a specific area of chromosome 13, particularly q14, which is a site for miR 15a/16-1, has been strongly linked to the development of haematological malignancies such as chronic lymphocytic leukaemia, acute myeloid leukemia, myeloproliferative disorders and myelodysplastic syndromes (Mitelman et al., 2015). Gao et al. (2011) hypothesized miR 15A/16-1 might function as a tumor suppressor gene by downregulating the WT1 oncogene. Shah et al. (2015) reported involvement of 13q in novel translocation t(5;13)(q12;p13) categorized under minor-route additional chromosomal aberrations associated with disease progression and Imatinib resistance. This suggests minor-route additional chromosomal aberrations might affect prognosis. Prognostic significance of CE is heterogeneous and dependent on the time of occurrence as well as time of initiation of therapy. However, it is a poor prognostic factor and represents multistep progression of CML. This case study suggests that minor-route additional chromosomal aberrations might affect prognosis. Further studies are required to ascertain the expression of genes and the role of CML in blast crisis, and to explore its therapeutic significance and prognostic value. Such cases indicate possible concern for advanced disease states and further alternative treatment strategies. Furthermore, this case study emphasizes the significance of the combination of conventional and molecular cytogenetic studies.

2011;118(26): 6760-68.

Faderl S, Talpaz M, Estrov Z, O’Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med. 1999;341: 164–72.

Gao S, Xing C, Chen C, Lin S, Dong P, Yu F. miR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level. J Exp Clin Cancer Res CR. 2011;30(1): 110.

Kurzrock R, Gutterman JU, Talpaz M. The molecular genetics of Philadelphia chromosome-positive leukemias. N Engl J Med. 1988;319: 990–8.

Mitelman F, Levan G, Nilsson PG, Brandt L. Non-random karyotypic evolution in chronic myeloid leukemia. Int J Cancer. 1976;18(1): 24-30.

Mitelman F. The cytogenetic scenario of chronic myeloid leukemia. Leuk Lymphoma. 1993;11: 11–5.

Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2015). Mitelman F, Johansson B and Mertens F (eds.), http:// cgap.nci.nih.gov/Chromosomes/Mitelman [As Accessed online on September 30, 2015].

Muehleck SD, McKenna RW, Arthur DC, Parkin JL, Brunning RD. Tra n sfor mation of ch ronic myelogenou s leu kemia: clinical, morphological and cytogenetic features. Am J Clin Pathol. 1984;82: 1–14.

Nowell PC, Hungerford DA. A minute chromosome in human chronic myelogenous leukemia. Science. 1960;132: 1497–9.

Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243: 290–1.

Shah B, Gajendra S, Gupta R, Sharma A. Novel cytogenetic aberrations in a patient of chronic myeloid leukemia with blast crisis. J Clin Diagn Res. 2015 May;9(5): XD05-XD06.

Shaffer LG, McGowan-Jordan J, Schmid M (eds.). ISCN 2013: An International System for Human Cytogenetic Nomenclature. Basel, Switzerland: Karger; 2013.

Tarkan-Arguden Y, Ar MC, Yılmaz S, Ongoren S, Kuru D, Ure U, et al.

Cytogenetic clonal evolution in patients with chronic myeloid leukemia.

BiotechnolBiotechnol Equip. 2009;23(4): 1515–20.

Verma RS, Babu Arvind. Human Chromosomes, Manual of Basic Techniques. Oxford, UK: Pergamon Press; 1995.

References: Babicka L, Zemanova Z, Pavlistova L, Brezinova J, Ransdorfova S, Houskova L, Moravcova J, Klamova H, Michalova K. Complex chromosomal rearrangements in patients with chronic myeloid leukemia. Cancer Genet Cytogenet. 2006;168(1): 22-29.

Boyer J. del(13q) in myeloid malignancies. Atlas Genet Cytogenet Oncol Haematol. 2002;6(1): 31-33.

Fabarius A, Leitner A, Hochhaus A, Müller MC, Hanfstein B, Haferlach C, et al. Schweizerische Arbeitsgemeinschaft fur Klinische Krebsforschung (SAKK) and the German CML Study Group. Impact of additional cytogenetic aberrations at diagnosis on prognosis of CML: long-term observation of 1151 patients from the randomized CML Study IV. Blood.

*Corresponding author Dr. Prabhudas S. Patel Professor & Head, Department of Cancer Biology The Gujarat Cancer & Research Institute prabhudas_p@hotmail.com

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Case Study

An Adult Male Presenting with Concurrent Plasma Cell Myeloma Involving a CCND1-IGH Translocation and Chronic Myelogenous Leukemia with a Variant (9;22) Translocation Peter M. Lee1, Ken Siangchin1, Sophie Song1, David Shabsovich1, Yalda Naeini1 and Carlos A. Tirado1 1. Department of Pathology & Laboratory Medicine, UCLA, Los Angeles, CA 90024

Abstract

treatments such as chemotherapy, radiation, lenalidomide or bortezomib (Sawyer, 2011). CML is cytogenetically characterized by the Philadelphia chromosome bearing a constitutively active tyrosine kinase, which deregulates cell signaling pathways that control cell growth, resulting in an uncontrolled proliferation of granulocytes (Ragupathi et al., 2013). The coexistence of PCM and CML is rarely reported and raises questions regarding the prognosis and the mechanism by which these two malignancies occur and interact with one another.

The t(11;14)(q13;q32) involving IGH and CCND1 and t(9;22) (q34;q11.2) involving BCR and ABL1 are common abnormalities in plasma cell myeloma (PCM) and chronic myelogenous leukemia (CML), respectively. However, the concurrence of the two malignancies is extremely rare. Herein, we present a case of an 87-year-old male who presented with anemia and monocytosis.

FISH studies on a bone marrow sample enriched for plasma cells detected a t(11;14) positive for IGH and CCND1 fusion in 92% of nuclei. However, cytogenetic analysis of the bone marrow revealed a t(9;22)(q34;q11.2) in 40% of the metaphases. Interphase and metaphase FISH studies on the sample confirmed the presence of the BCR-ABL1 fusion in 88% of nuclei but did not show any signals corresponding to the derivative 9, suggesting a variant t(9;22) with a deletion or additional material of unknown origin at the 9q34 band of the derivative 9 and a derivative 22 bearing the BCR-ABL1 fusion gene. The concurrence of plasma cell myeloma and chronic myelogenous leukemia is extremely rare with less than 20 cases reported. The molecular pathway in which the multiple malignancies arise is still poorly understood, and this case provides insight into the concurrence of PCM and CML.

CLINICAL PRESENTATION Patients and Samples An 87-year-old male presented with anemia and monocytosis.

CBC data included a hemoglobin of 12.0 g/dL and white blood cell count of 34.89 K/uL. A bone marrow biopsy showed a mildly hypercellular marrow (50% cellularity) with myeloid hyperplasia. Plasma cells were mildly increased in number, with 8-10% monotypic kappa-restricted plasma cells highlighted by immunohistochemical studies, consistent with a plasma cell myeloma. Flow cytometry performed on the bone marrow revealed an abnormal plasma cell population (2.8% of total cells) and left shifted granulocytes. This population showed kappa light-chain restriction, and was positive for CD38, CD138, partial CD45, CD20, dim CD27, and negative for CD19, CD56, CD117, CD28, and CD81, consistent with plasma cell dyscrasia.

INTRODUCTION Plasma cell neoplasms (PCN) include plasma cell myeloma (PCM), plasmacytoma, and the syndromes defined by the consequence of tissue immunoglobulin deposition, primary amyloidosis, and light- and heavy-chain deposition diseases (Swerdlow et al., 2008). In particular, PCM is characterized by an uncontrolled proliferation of a single plasma cell clone leading to overproduction of a monoclonal immunoglobulin (Swerdlow et al., 2008). Chronic myelogenous leukemia (CML) is a clonal myeloproliferative neoplasm characterized by uncontrolled granulopoiesis. CML is divided into chronic, accelerated, and blastic phases, based on the blast count. Chronic phase generally lasts up to 3-5 years and tends to be associated with a favorable prognosis, but when it progresses to accelerated and subsequently blastic phases, it can become lethal and does not respond as well to treatments such as tyrosine kinase inhibitors (TKI) (Cortez et al., 1996). The t(11;14)(q13;q32) involving IGH and CCND1 occurs in 15% of the PCM patients, while t(9;22)(q34;q11.2) involving BCR and ABL1 occurs in 95% of the CML patients (Nowell and Hungerford 2004; Sawyer, 2011). A promoter exchange between the CCND1 gene, which encodes for a cyclin D1 protein that binds to a cyclin dependent kinase 4 or 6 to form a complex, and the highly expressed IGH promoter region causes an overexpression of the CCND1 protein that results in the deregulation of the cell cycle, giving rise to plasma cell dyscrasia (Mateos et al., 2002). Patients with this translocation tend to respond well to

MATERIAL AND METHODS A) Conventional Cytogenetics

Chromosome analysis was performed on the bone marrow using standard cytogenetic techniques. The karyotypes were described according to the ISCN 2013 nomenclature (Shaffer et al., 2013).

B) FISH -

B.1 Unenriched Sample FISH on cultured BM interphase cells (n=200) was performed using the following probes: D13S319/13q34; IGH/FGFR3 DC, DF Probes, EGR1/D5S23, D5S721 and BCR/ABL1/ASS1 Tri-Color Probe Kit (Abbott Molecular, Des Plaines, Il).

Additional FISH studies using CKS1B/CDKN2C(P18) and IGH/MAF Dual Fusion probes obtained from Cytocell were performed.

FISH was also performed on previously G-banded metaphases using a BCR/ABL1/ASS1 Tri-Color DF FISH Probe Kit (Abbott Molecular).

B.2 Enriched CD 138+ Sample

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Case Study An Adult Male Presenting with Concurrent Plasma Cell Myeloma Involving a CCND1-IGH Translocation and Chronic Myelogenous Leukemia with a Variant (9;22) Translocation FISH was performed on 100 interphase nuclei from a sample enriched for CD138 plasma cells, obtained using the RoboSep-S Human CD138 Positive Selection Protocol (Yao-Shan, 2002), using the CCND1-IGH Dual Color Dual Fusion and TP53/CEP 17 FISH Probes (Abbott Molecular).

These findings were described as: • nuc ish(ASS1x1,ABL1X2,BCRX2)(ABL1 con BCRX1) [176/200] No other abnormal signal patterns were observed using other probes.

• nuc ish(CKS1B,CDKN2C)x2[200] • nuc ish(FGFR3,IGH)x2[200] • nuc ish(EGR1,D5S23/D5S721)x2[200] • nuc ish(D13S319,D13S1020)x2[200] • nuc ish(IGH,MAF)x2[200]

RESULTS Cytogenetics (Unenriched Sample)

Chromosome analysis revealed an abnormal male karyotype with an apparent reciprocal translocation between chromosomes 9 and 22 (Philadelphia positive) in 8/20 metaphases analyzed. This karyotype was described as 46,XY,t(9;22)(q34;q11.2)[8]/46,XY[12].

Metaphase FISH studies using the same probe on the previously G-banded metaphases bearing the t(9;22) revealed a fusion signal on the derivative 22 corresponding to a BCR-ABL1 fusion and no signals on the der(9) corresponding to a deletion of the 9q34

Figure 1. Karyotype showing a t(9;22)(134;q11.2)

B) FISH Figure 3. Metaphase FISH showing BCR-ABL1 fusions [The Ph and the der(9) chromosomes]

B.1 Unenriched Sample Interphase FISH studies revealed one BCR-ABL1 fusion and loss of 9q specific signals in 88% of nuclei analyzed, consistent with a variant t(9;22) with deletion of the 9q34 region on the resulting derivative 9 (Figure 2).

region, consistent with the interphase FISH findings (Figure 3).

In light of the conventional cytogenetic and FISH findings, the karyotype was described as follows: 46,XY,add(9)(q34),ins(22;9)(q11.2;q34q34)[8]/46,XY[12]. ish add(9)(q34)(ABL1-,BCR-),ins(22;9)(q11.2;q34q34) (BCR+,ABL1+;ABL1+) B.2 Enriched CD 138+ Sample FISH studies revealed the presence of CCND1-IGH signals confirming the presence of a t(11;14)(q13;q32) in 92/100 nuclei examined. However, FISH did not reveal any abnormal signal pattern using the Vysis TP53/CEP 17 FISH Probes. The results were summarized as: • nuc ish (CCND1,IGH)x3(CCND1 con IGHx2)[92/100] • nuc ish (TP53,D17Z1)x2[100] (Figure 4).

Discussion The concurrence of plasma cell myeloma (PCM) and chronic myelogenous leukemia is extremely rare with less than 20 cases present in literature, and the mechanism behind their concurrence is not yet fully understood (Alsidawi et al., 2014). Plasma cell myeloma is characterized by complex chromosomal aberrations, but the most

Figure 2. Interphase nuclei showing BCR-ABL1 fusions [The Ph and the der(9) chromosomes]

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Case Study An Adult Male Presenting with Concurrent Plasma Cell Myeloma Involving a CCND1-IGH Translocation and Chronic Myelogenous Leukemia with a Variant (9;22) Translocation patients with cervical cancer and patients with ankylosing spondylitis who received radiation therapy who showed a significant dose-response relationship for the development of CML (Little et al., 1999; Ragupathi et al., 2013).

A more plausible explanation for the concurrence of CML and PCM is the common ancestry stem cell relationship between the cells that constitute the two neoplasms. One hypothesized mechanism is the occurrence of neoplastic transformation and proliferation along myeloid-granulocytic cell lines as well as lymphoplasmacytic cell lines leading to the development of both CML and PCM (Klenn et al., 1993).

CML is known to give rise to clonal populations of cells that give them a proliferative advantage utilizing the constitutively active tyrosine kinase. Consistent with these observations, the BCR-ABL1 fusion was identified in macrophages, immunoglobin synthesizing B lymphocytes, megakaryocytes, and erythropoietic cells (Ragupathi et al., 2013). To further strengthen the pluripotent stem cell theory, there is evidence to suggest that samples of blood from healthy adults, children, and umbilical cord blood (UCB) show clear but low expression of the BCRABL1 mRNA transcript (Biernaux et al., 1995). Results showed no expression of the transcript in UCB, one case in children’s WBC, and 22/73 cases in adults suggesting a linear relationship with increasing age (Biernaux et al., 1995). With advancements in technology, these BCR-ABL1 transcripts have been detected more readily with RT-PCR, and healthy individuals have been found to carry one or more of these leukemia and lymphoma associated chromosomal translocations (Song et al., 2011). Given that patients without hematological malignancies are able to bear a t(9;22) in their cells, it is hypothesized that the secondary abnormality is what triggers the quiescent tyrosine kinase of the Ph chromosome making it active and leading to concurrence of CML and another malignancy. Furthermore, even though the simultaneous occurrence is rare, it is not a surprise to see CML patients exhibiting Ph+ B-lymphoblastoid cells during the chronic phase (Klenn et al., 1993).

There is also a possible association between the blast phase of CML and the concurrence of PCM and CML. CML blast phase is usually characterized by a blast count of 30% in peripheral blood or bone marrow (Sawyers et al., 2002). However, in the current case, the patient has mild myeloid hyperplasia despite an overwhelming population of Ph-positive cells. A possible theory would be that the concurrence of PCM and CML affects the expression of the BCR-ABL1 gene. There is another case in which H929 plasma cell myeloma cells exhibited BCR-ABL1 fusion protein using immunoprecipitation and tandem mass spectrometry suggesting another molecular pathway from the common ancestry stem cell. However, other tests suggested a crosscontamination between K-562 CML cell lines refuting the claim that H929 plasma cell myeloma cells exhibited the BCR-ABL1 fusion. These processes support the theory in which the disease originates early in the hematopoietic stem cell disorder, manifesting the malignancies in the mature stages (Naparstek et al., 1980; Lewis et al., 1986; Klenn et al., 1993). Additional literature showed how dual markers for plasma cell and myeloid antigens were identified in myeloid cells and the transformation of lymphoid cells in the blastic phase of CML indicates the presence of a progenitor cell that can differentiate into myeloid and lymphoid cell lineages (Schwarzmeier et al., 2003).

In the present study, we report a case of concurrent plasma cell myeloma involving a CCND1-IGH translocation and chronic myelogenous leukemia with a variant t(9;22). This constellation of

Figure 4. Interphase nuclei showing CCND1-IGH fusions

frequently observed translocation is t(11;14)(q13;q32), which is found in 15% of the PCM patients and is associated with a standard risk prognosis (Chesi et al., 1998; Fonseca et al., 2002; Bergsagel et al., 2005; Sawyer 2011). Translocations common in plasma cell myeloma are mediated by errors in B-cell specific DNA modifications such as the IGH switch recombination (Bergsagel et al., 2001). In many cases, abnormal clones have low proliferative activity in culture, leading to the observation of normal karyotypes by conventional cytogenetic analysis, despite the chromosomal complexity of PCM involving numerous numerical and structural abnormalities (Sawyer, 2011). In comparison to plasma cell myeloma, chronic myeloid leukemia is driven by the constitutively active BCR-ABL1 tyrosine kinase which promotes cell proliferation and survival leading to malignant transformation of the disease. Chronic myelogenous leukemia is consistently associated with a Philadelphia chromosome resulting in a fusion gene consisting of BCR at chromosome 22q11 and ABL1 at 9q34 (Malvestiti, 2014). The Philadelphia chromosome is detected in 90% of the patients, and the variant deletions on der(9) present in 9-15% of the cases do not appear to have a poor prognostic factor when treated with imatinib and second generation TKIs (Castagnetti et al., 2010; QuintĂĄs-Cardama et al., 2011). On the other hand, there is contradicting evidence to suggest that the deletions on the derivative 9 result in disease progression.

There are patients from the onset of diagnosis who progressed to blast or accelerated phase in a mean duration of 32.8 months, while patients without deletions progressed in 62.4 months (Lee et al., 2003).

Contradicting evidence suggests an inconclusive prognostic indication of deletions on the derivative 9 in CML patients.

There are several possible theories that explain the coexistence of plasma cell myeloma and CML. Firstly, there is a suggested link between PCM and CML with patients diagnosed with PCM following a long term treatment course of CML with tyrosine kinase inhibitor, imatinib (Derghazarian et al., 1974; Ide et al., 2010; Alsidawi et al., 2014); however, almost half of the reported cases describe patients who either exhibited PCM followed by the development of CML, or simultaneously developed both malignancies, undermining the link between imatinib treatment and development of PCM (Schwarzmeier et al., 2003; Offiah et al., 2012; Alsidawi et al., 2014). Alternatively, there is no evidence in the literature of patients treated with lenalidomide or bortezomib leading to the development of CML, undermining the link between PCM treatment and the development of CML (Alsidawi et al., 2014). Another possible mechanism, even though extremely rare, is presented in specific instances in which radiation can induce CML. Ragupathi et al. noted in an earlier study that there have been

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Case Study An Adult Male Presenting with Concurrent Plasma Cell Myeloma Involving a CCND1-IGH Translocation and Chronic Myelogenous Leukemia with a Variant (9;22) Translocation abnormalities is not typically observed in other cases with concurrent plasma cell myeloma and chronic myelogenous leukemia because plasma cell myeloma is typically diagnosed by the analysis of the bone marrow aspirate, blood and urine tests, and plasmacytoma on tissue biopsy.

In the case presented, the plasma cell myeloma and chronic myelogenous leukemia were discovered simultaneously. There was no medication administered nor radiation therapy offered between diagnoses. As a result, the relationship between the treatment and the development of the diseases remains inconclusive. Neoplastic transformation and proliferation of a common stem cell that leads to PCM and CML remains a plausible theory, but unfortunately, due to limited sample material, we could not further test the t(9;22) on the enriched samples nor the t(11;14) on the sample not enriched, providing no definitive evidence to support this claim.

This study further emphasizes the importance of enriching plasma cells when detecting plasma cell myeloma. Without enriching the sample for plasma cells, the karyotype of the samples typically only shows signs of chronic myelogenous leukemia without signs of plasma cell myeloma in this context. As a result, only CML-related karyotypic abnormalities will be observed regardless, since conventional cytogenetic analysis cannot be done on enriched samples because those cells cannot be cultured. This particular case demonstrates the importance of coupling cytogenetic findings with FISH when working with plasma cells in particular because FISH is the cytogenetic methodology that can provide the most information in this context.

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Castagnetti F, Testoni N, Luatti S, Marzocchi G, Manchini M, Kerim S, Giugliano E, Albano F, Cuneo A, Abruzzese E, Martino B, Panandri F, Amabile M, Iacobucci I, Alimena G, Pane F, Martinelli G, Saglio G, Baccarani M, Rosti G. Deletions of the derivative chromosome 9 do not influence the response and the outcome of chronic myeloid leukemia in early chronic phase treated with imatinib mesylate: GIMEMA CML Working Party analysis. J Clin Oncol. 2010;28(16): 2748-2754.

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Corresponding author Carlos A. Tirado, Ph.D. ctirado@mednet.ucla.edu, carlostirado@hotmail.com

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Profiles and Perspectives

Column Editor: Hon Fong L. Mark, PhD, MBA, FACMG

Dr. Rachel Burnside Interviewed by Dr. Hon Fong L. Mark Dr. Rachel Burnside received her education from the University of Houston at Clear Lake (UHCL) in 1998 (bachelor’s) and the University of Kentucky (UK) in 2006 (PhD). Following a clinical cytogenetics fellowship at the University of Alabama at Birmingham, she joined LabCorp as a director, where she has been since 2008. Dr. Burnside has been involved in the American College of Medical Genetics and Genomics as past-Chair of the Membership Committee, and in that position, helped establish the Student Session at the annual ACMG meeting. In addition, she has participated in a number of trainee-mentor events. She has been invited to present the work from her lab at several meetings, including ACMG, CAGdb, and a regional FGT conference in Durham, NC. This column editor first visited LabCorp in North Carolina as a CAP inspector.

Dr. Rachel Burnside

their families. I knew going in that I wanted to further my education with a graduate or professional degree, but I never had a plan to return to cytogenetics as a director.

HFLM: As a bright young woman, obviously there were many options open to you when you decided to go to college (Mark, 1999; Mark, 2000). What was the main reason that you chose those academic institutions?

DRB: I took seven years to get my undergraduate degree, and I did not do well academically for the first several years. I initially began my undergraduate studies at Texas A&M, but I graduated from the University of Houston at Clear Lake. There were a number of factors going into the time it took to get my degree and the various schools attended along the way, but I was not a very serious student until later in my undergrad studies. It IS possible to achieve high academic goals, such as pursuing a graduate or professional degree, with a less-than-stellar academic record. Don’t be afraid to go for it!

HFLM: What was the field of molecular genetics like when you started? Were there many clinical laboratories? Please describe your training.

DRB: Cytogenetics was already a mature lab specialty in the late 1990s, but there were not so many molecular genetics labs. FISH had been offered clinically for a few years, and array technology was really still only at Baylor. I recall a conversation I had in the lunch room when I was a tech with one of the PhD employees from a molecular identity lab next door, who was telling me she was looking to take a job at Baylor because they had this new thing called a DNA chip that could examine the whole genome. I had no idea what she was talking about, but it sounded very cool.

Turns out, this was the BAC array comparative genomic hybridization technique that was developed at Baylor and eventually spun out the vendor, Spectral Genomics. That little DNA chip ultimately began the era of cytogenomics and microarrays as we now know them.

I ultimately decided to go to the University of Kentucky (UK) because my husband is from Kentucky, and we decided to move closer to his family for a while. UK does not have a genetics department, so I decided to work in Dr. Martha Peterson’s lab in the department of Microbiology, Immunology, and Molecular Genetics because her research used molecular genetic techniques, which was as close as I could come to genetics in a human model system. There were other labs in the Biology department that studied genetics, but the idea of dealing with fruit flies all day was not appealing to me.

HFLM: Why did you choose to enter the specialized field of genetics?

DRB:

Both Drs. Cheung and Smith have PhDs in Clinical Genetics, which is no longer offered as a graduate degree, so the career path to a director position was not clear to me.

Also, students entering basic science graduate programs are trained to do research, not clinical laboratory science, so I actually lost sight of cytogenetics as a career during the years of my PhD program. It wasn’t until I began to look for postdoctoral positions that I came across the clinical cytogenetics fellowship program. I knew immediately that this was going to be my career, and I was fortunate enough to be accepted for the fellowship at UAB in Birmingham, Alabama. My first day in the lab was like getting back on a bike. Having the experience as a tech helped me get the fellowship position, and I was able to start on day one with scope work and to contribute to the production of the lab.

I think the decision came over time as a result of working as a technologist in a cytogenetics lab after graduating from UHCL. I was hired by Dr. Sau Wai Cheung (now at Baylor College of Medicine) and Dr. Jan Smith (also at Baylor) in what was then a privately owned cytogenetics lab in Houston—a lab that ironically is now owned by LabCorp.

I had no idea what cytogenetics was when I started; I was just happy to have a paycheck with a comma in it. During my two years as a tech, I really came to love the field and its impact on the healthcare decisions for patients and

When I began to research clinical cytogenetics fellowships, the program at UAB was particularly appealing as it is a strictly clinical program; there was no research component required, and no grant funding required. My training probably looked very similar to many others’. Training programs typically involve learning all aspects of the wet lab work for all sample types, attending clinics with the clinical geneticists and residents to learn about different disorders, presenting abnormal cases to the clinical genetics department in case conference, taking coursework in all the lab specialties, as well as genetic counseling, writing

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Profiles and Perspectives

Dr. Rachel Burnside

abstracts for conferences, and learning about how to interpret results, troubleshoot, and write reports with the directors. I learned a great deal from the directors of the lab, Drs. Drew Carroll, Paula Cosper (retired), and Fady Mikhail. I was also able to publish a couple of case reports and work on side projects associated with Children’s Oncology Group (COG) cases and some osteosarcomas with one of the pathologists, Dr. Gene Siegal. UAB is one of two institutions in the U.S. that is a central review site for COG B-cell ALL, and what I learned there has helped in my current position.

Please elaborate.

DRB:

HFLM: Who do you consider your most important mentors in genetics at the undergraduate and graduate levels?

DRB: I had a really great advisor at UHCL, Carroll Lassiter (retired), who made a big impact on me getting myself together, academically speaking. He called me at home one Saturday afternoon and challenged me to do better.

He knew that I had the intellectual capacity to make good grades, but that I wasn’t applying myself. My genetics professor at UHCL, Dr. Alma Moon Novotny (now at Rice University) was a really difficult instructor, but she was a great teacher and I really enjoyed her classes—and did well in them. Drs. Cheung and Smith both made a big impact on my choice of career, even if I wasn’t aware of it until much later. When I decided to do the clinical postdoctoral fellowship, no one in my department at UK could offer any advice because they had no idea what I was getting myself into. I reached back out to Dr. Cheung for advice and talking with her reassured me that cytogenetics was indeed the career choice I wanted. I also still have regular interactions with Drs. Carroll and Mikhail.

I have been active in the American College of Medical Genetics and Genomics since I was a trainee. I am a past Chair of the Membership Committee and have been a member of workgroups within the Lab QA Committee.

While on the Membership Committee, we began a session at the annual meeting targeted towards students (undergrad, grad, genetic counseling, medical, etc.) to provide information about careers in all the specialties of medical genetics and get students interested in medical genetics as a career. This session has been a great success and it continues to grow and evolve every year based on the feedback of the participants.

HFLM: What do you think is the most urgent scientific question to be answered in our field in the coming years?

DRB: The new hot topic at most of the medical genetics conferences these days is genomic sequencing. New mutations and previously unknown disorders are being described at a pace not seen before in science. The biggest issue in the laboratory specialties continues to be how to interpret variants of unknown clinical significance.

HFLM: What do you think is the most pressing nonsciencerelated problem facing molecular geneticists today?

DRB: Since the time of Copernicus and probably before then, science has been subject to ridicule and skepticism by those who do not understand it. Simply explaining scientific and medical principles is often not sufficient to avoid the skepticism. National Geographic published a great article last year in the March (2015) issue talking about the reasons non-scientists often don’t believe in science, even after explanation. This will continue to be a part of reality for those of us in scientific and medical fields.

HFLM: What do you consider to be the most interesting or most important project that you have ever done? Please elaborate.

DRB: A recent project that I completed was a study of all of our microarray patients and a review of all the primary literature for 22q11.2 deletions (Burnside, 2015). As you know, proximal 22q has a cluster of segmental duplications, called LCR22A-LCR22H (A-H) that mediate deletions and duplications of different but recurrent regions. The most commonly deleted region is the A-D interval that results in DiGeorge syndrome (also known as 22q11.2 Microdeletion syndrome). The literature regarding the different possible deletions was all over the map, with various names such as proximal, distal, atypical, nested, and central. It became very confusing trying to sort out the regions and which patients had which deletion. I brought everything together in one article, clearly defining which regions were proximal, central, and distal, the key features reported for each region, frequency of inheritance, and which papers reported which deletions. It has greatly helped the directors and genetic counselors in our lab with respect to report writing and citing relevant literature, and I hope it is helpful to other directors and counselors, as well.

HFLM: In what direction do you see molecular genetics going in the next century?

DRB

Genomic sequencing and understanding the consequences of mutations will become more clinically useful as the technologies develop. The demise of cytogenetics has been predicted for as long as I have been in the field. However, I think what we are seeing instead is the convergence of cytogenetic, cytogenomic, and molecular techniques for whole genome studies. There are useful applications for all methodologies, and as yet there is no one test that can detect all abnormalities. For example, I recently published a report about a patient for whom the microarray appeared relatively straightforward with an unbalanced translocation derivative chromosome, but the chromosomes were more complex that the array showed (Burnside, 2014). Alternatively, seemingly simple rearrangements by chromosome analysis can actually be more complex with microarray. These sorts of findings have impacts on recurrence risks for families.

HFLM: What advice would you give a young person today who has an interest in going into the field of molecular genetics?

HFLM: I understand that you are an active participant in the American College of Medical Genetics and Genomics.

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Profiles and Perspectives

Dr. Rachel Burnside

DRB: I agree. Science is all around us and involves every aspect of life, so it needs the participation of all people.

But it’s not just science in which women have not been sufficiently represented. Of the Fortune 500 companies, fewer than 20% of executive positions are held by women.

There are myriad reasons why women do not always achieve leadership positions (I highly recommend Lean In by Sheryl Sandberg—to both men and women), and external support from society and mentors certainly has an impact. For women with families, partners that fully share responsibilities at home are vital. Having women to emulate is important for career choice, and I think that women in positions of leadership also need to advocate for the advancement of more women coming up behind them.

DRB: Yes, do it! This is such a great field, and it’s still small enough that you develop a great network of colleagues from all over. I can’t speak to medical school programs, but for PhD programs, look for genetics programs specifically.

Studying human genetics is not necessary, but it does help.

Once you identify the few schools which you are interested in attending, look on their department websites at what the faculty researchers are doing. Remember, you will spend your time working in one lab throughout a PhD program, so it’s important that the research is interesting to you.

Once you find some labs that sound interesting, contact those PIs directly. Ask them questions about the program and whether they are accepting new students in their lab, and tell them you are interested to rotate through their lab.

Doing rotations in the first and/or second year of graduate school is a great way to find your lab home. You may find out that the lab you were first interested in is not a great fit. Once you are nearing completion of your PhD, start contacting the program directors or training directors of programs to which you intend to apply for fellowships.

Having a technician/technologist background on top of a PhD will make you a very strong candidate. Many fellows are now getting dual boarded, especially if cytogenetics is one of the specialties. Cyto/molecular dual fellowships are very popular but also very competitive. I have known of fellows that have had to wait two or three years to get into programs after they got their PhD, so start looking early and ask directly what will make you a top candidate for any fellowship program you are considering.

HFLM: This column often ends with a saying or an important piece of advice. Before ending this very interesting interview, is there something you would like to leave with our readers?

DRB: I can speak for directors everywhere when I say that we so appreciate all the work of our techs and supervisors.

Without you, we could not do what we do, and it’s your work by which the quality of labs is measured. If you aspire to achieve more with your career, be bold and go for it. It isn’t easy, and it takes time, but it is well worth the effort.

References Cited Achenbach J. The Age of Disbelief. National Geographic. Mar. 2015.

Burnside RD, Spudich L, Rush B, Kubendran S,· Schaefer GB. Secondary Complex Chromosome Rearrangement Identified by Chromosome Analysis and FISH Subsequent to Detection of an Unbalanced Derivative Chromosome 12 by SNP Array Analysis. Cytogenet Genome Res. 2014;142(2): 92-100.

Burnside RD. 22q11.21 Deletion Syndromes: A Review of Proximal, Central, and Distal Deletions and Their Associated Features. Cytogenet Genome Res. 2015;146(2): 89-99.

Mark HFL. Cytogenetics in the 1960s. J Assoc Genet Technol. 1999;26: 72-73.

Mark HFL. Medical Cytogenetics. New York: Marcel Dekker; 2000.

Sandberg S and Scovell N. Lean In: women, work, and the will to lead. New York: Alfred A. Knopf; 2013.

HFLM: If there is one thing that you can change about the genetics marketplace today, what would it be?

DRB: It would probably be the consistent interpretation of results by different labs. Several organizations are working very hard to promote consistent interpretation of variants, including ClinGen, ACMG, ClinVar, and others. I participate in a workgroup of ClinGen that evaluates genes for dosage sensitivity, and what compelled me to join this group was a lack of consistency in reporting microarray results from various labs around the country. Part of this inconsistency is the experience (or lack of experience) with respect to variants that have been encountered by that lab, and part of it is whether there is information in public databases or in the medical literature describing such variants. The effort of these groups is and will be vital to having consistent interpretation of results, regardless of what lab does the testing.

Hon Fong L. Mark, Ph.D.,M.B.A.,F.A.C.M.G., Editor of the cytogenetics textbook, “Medical Cytogenetics,” is President of KRAM Corporation, a small consulting firm specializing in medical genetics, grant review and scientific review administration. Dr. Mark is a Clinical Cytogeneticist boardcertified by the ABMG (1993), and was formerly Director of Cytogenetics and Clinical Professor at the Lifespan Academic Medical Center/Brown University in Providence, RI, Director of Human Genetics, RIDOH, also in Providence, RI, and Director of the Cytogenetics Department at Presbyterian Laboratory Services/Novant Health in Charlotte, N.C. She was recruited to the Boston University School of Medicine as Director of the Cytogenetics Laboratories (and Clinical Professor of Pathology and Laboratory Medicine) in 2004, a position from which she resigned in 2007.

This column is dedicated to the technologists and laboratory directors in all the cytogenetics laboratories in the U.S. and throughout the world whom she had the good fortune of meeting through the years.

HFLM: As a former Chairperson of the Faculty Committee on the Status of Women at Brown University and a former member of the Executive Committee and Commissioner of the Rhode Island Commission on Women appointed by the Governor, my views may be somewhat biased, but there are many others who believe that women in science have not been given sufficient support by society to fully realize their full potential. Of course, there are others out there who disagree with this point of view. What do you have to say to that?

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Government & Regulation

Column Editor: Jennifer Crawford-Alvares, CG(ASCP)CM

2016 ASCLS LEGISLATIVE SYMPOSIUM OVERVIEW Every year the Association of Clinical Laboratory Science (ASCLS) hosts a legislative symposium in Washington, D.C. This year, although I was unable to attend, I have written a summary of the items discussed based on meeting minutes and conference planning calls. The symposium was held at the Hilton Old Town in Alexandria, VA on March 14th and 15th and included speakers from ASCLS, Clinical Laboratory Management Association (CLMA), American Society for Clinical Pathology (ASCP) and the Association for Molecular Pathology (AMP). Four main topics were discussed: harmonization of laboratory testing, Patient Access to Medicare Act (PAMA) and laboratories, laboratory developed tests (LDTs), and the clinical laboratory workforce initiative.

Number (TIN), which is the current method. This is an extremely important issue to genetics and molecular diagnostics as many of our laboratory tests are still utilizing old, nominal fee codes to bill for new, high-tech testing methods.

Everyone in the laboratory community is in agreement that Laboratory Developed Tests (LDT) should be regulated, but we are requesting Congress that we, the laboratory community, be provided sufficient time to review and report on what we know to be the most effective and efficient way of providing oversight of LDTs. In light of the volume that the Federal Drug Administration (FDA) will be required to review, an estimated 60,000 LDTs, we ask that the FDA partner with CMS for advanced oversight of LDTs in order to ensure a timely and coordinated effort in the approval process.

The harmonization of laboratory testing is one of the newest topics to be discussed at the legislative symposium and has come to the forefront based on a 2014 decision by Congress to request that The Centers for Disease Control and Prevention (CDC) “partner with the private sector in harmonizing clinical laboratory test results.” Although clinical laboratories are working hard to provide high quality results, they are often only accurate based on the devices or methods each individual lab uses. For clinical genetics this may be the difference in cut-off values for reporting abnormal FISH results or establishing breakpoints for chromosomes based on visible band length. Results such as these are non-harmonized and could mean inaccurate clinical decisions are being made, which could negatively impact patient outcomes.

The issue of a Clinical Laboratory Workforce Initiative is not being listed as last because it is of least importance, but mostly because this is an issue that for many years we have been strongly advocating on Capitol Hill for critically needed funding. We are continually urging Congress to address clinical laboratory workforce shortages with a specific need to provide well-trained laboratory personnel to the underserved urban and rural communities.

Based on a 2014 workforce survey by ASCP, the employment outlook for clinical laboratory personnel shows a high percentage of retiring workers and a small percentage of highly-skilled graduates currently entering the workforce. This alarming difference is thought to be a product of clinical laboratory educational program cutbacks. We believe this is an issue that requires the federal government to step in and reissue vital allied health funding in order to ensure the future progress and adequate staffing of our laboratories.

The laboratory community and medical device manufacturers have created an international consortium and are working with the CDC to urge Congress to appropriate the necessary funding to harmonize clinical laboratory testing. AGT has recently joined many other laboratory organizations to cosign a request to Congress for Fiscal Year 2017 appropriations to be appointed for the CDC to continue harmonizing many more laboratory tests.

As you can see by reading this review of the 2016 ASCLS Legislative Symposium, there are many governmental issues that are facing our clinical laboratories today. There are two easy ways to get involved in being part of the change: respond quickly to email requests by AGT’s governmental affairs committee when asked to contact your local government officials for an upcoming vote, or by becoming a part of the AGT governmental affairs committee. You can learn more by contacting me, Jen CrawfordAlvares, at jen.crawford34@gmail.com

One topic which was reviewed at the symposium and has been addressed before is the Patient Access to Medicare Act (PAMA).

Unfortunately, CMS has misssed congressionally mandated deadlines for a recalculation of the Clinical Laboratory Fee Schedule (CLFS). We are advocating for a new fee schedule date of January 1, 2018, which is one year further out than CMS has currently mandated.

In addition, the use of a facility CLIA number would reflect true market-based reimbursement rates rather than those being calculated by our parent organization’s Taxpayer Identification

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Brain Tickler

Brain Tickler Summary (see inside front cover)

Karyotype: 46,XY,del(21)(q22.3) Microarray was performed and found a 3.32Mb deletion of the distal end of chromosome 21q.

Confirmation using a FISH probe mixture, Abbott ToTelVysion Mix #4 (4ptel (SG)/4qtel (SO)/21tel (SG+SO)/AML1 (SA) and LSI 21 (aqua). One 21 was missing the signal for the telomere specific region.

The 4p/4q telomere signals are not shown.

Deletion of 21q is a unique syndrome and the phenotype can vary greatly. See Genomic analysis of partial 21q monosomies with variable phenotypes.

Elisha DO Roberson, Elizabeth Squibb Wohler, Julie E HooverFong, Emily Lisi, Eric L Stevens, George H Thomas, Jay Leonard, Ada Hamosh, Jonathan Pevsner. Eur J Hum Genet.

2011 Feb;19(2): 235–238.

Also see: http://www.rarechromo.org/ information/Chromosome%2021/21q%20 deletions%20FTNW.pdf.

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Molecular Diagnostics

Column Editor: Michelle Mah, MLT, MB(ASCP)CM

A Journey of Continuous Learning Through Teaching For the first quarter of 2016 I wrote briefly about the increasing access to next-generation sequencing (NGS) technologies. As manufacturers make sequencers smaller and cheaper, we can expect clinical utility to expand in all areas of genetic testing.

I think it is incredibly exciting to implement NGS, but I fear I may have created a daunting undertone about the challenges of increased workflow complexity and data handling because I have written articles about numerous platforms and talked about the looming possibility of genomical amounts of sequencing data.

Even my first column from the fall of 2014 discussed the more complicated workflow and results that may take close to one hour of pure computational analysis followed by hours of manual interpretation. While I did not exaggerate those facts, experience has allowed me to reflect on how well our lab has improved workflow and handled data analysis over the past two years. We offer several NGS assays on the testing menu, and as the technologies have matured, so has the technical support and intra-lab expertise.

Not too long ago, in February, I delivered an NGS data processing and analysis workshop to the current class of Genetics Technology students at The Michener Institute in Toronto. The curriculum at the Institute has evolved over the last few years, and in addition to providing a formal lecture on NGS technologies and applications, the instructors desired some practical experience for student technologists. The goal was to distill information and focus on relevance. Relevance was key. We were not going to dive deep into computational analysis or spend hours on variant annotation. Those skills can certainly be mastered and maybe even required in some labs, but as technologists those are likely not our areas of expertise.

In the workshop, I gave an overview of common sequencing data formats, the processing workflow from raw sequences to variant reports that a technologist would likely review, and we even managed to go over several curated cases of variant filtering and genotyping. We discussed the upfront analysis, which primarily consists of data transfer and format conversion to input files containing base calls directly into commercially purchased software for genome alignment and variant calling. We shied away from saying mutations at this point because not all nucleotide changes are clinically meaningful given the breadth of sequence variations. At this point, I acknowledged that this is an account of the work the technologists in my lab performs and that different labs will have different analysis pipelines and expectations.

Setting up the files for analysis was quite straightforward to the class, and I was told by the instructors afterwards that the variant reports were not what they had imagined, but were instead a manageable list. Contrary to the thousands of sequence variants produced by each NGS test, labs are commonly only interested in certain genes and genomic regions pertaining to the disease of interest and treatment. The final list of variants after extensive filtering can range from five to less than 20 (sometimes more).

From this list of 20 variants, for example, it is certainly possible to highlight and record only the most clinically significant variants.

The last half of the workshop was spent on evaluating the

clinical significance of variants. Aside from a handful of known clinically significant variants or mutations, the evaluation process relies on predetermined quality criteria and the abundance of database information, clinical studies and published literature.

The predetermined quality criteria is analogous to the quality of an electropherogram (i.e., strong signal intensities and clean discernable peaks). For NGS variants we evaluate sequencing depth, which is the number of times a nucleotide was sequenced and correctly aligned to the reference base. Sequencing depth can be used interchangeably with the term coverage. The phenomenon of NGS technology is the massively parallel sequencing that generates millions of sequencing reads. For example, if a single nucleotide variant has a coverage of 500x, this means the base at which the change occurred was sequenced 500 times, or one may think of it as 500 sequenced reads that contain the position of interest.

Coverage is crucial because it reflects the degree of confidence that the base is called correctly, the minimum coverage required to determine acceptability will differ for different applications and all thresholds are determined during assay validation.

If we can accept the coverage for a given variant, the next questions to ask is: What is the frequency or percentage of this sequence variation?, and, Is the variant called correctly? Unlike Sanger sequencing, which has a limit of detection at approximately 15%, low-level frequencies at 2% to 5% are easily detected by NGS assays. Therefore, it is important to set a minimum percentage that meets both the analytical sensitivity and specificity to determine true positives and negatives in a given assay.

Once variant percentage is assessed, time is spent verifying the nomenclature provided by the software. In many instances, insertions and deletions need to be doubled-checked by examining how the sequence is aligned to the reference. This can be done directly on the software’s visualization interface and is similar to checking if the peaks on the electropherogram are called correctly where the nucleotide changes occur. There is a lot of excellent software and open sourced tools for labs to exploit but that does not mean we can simply press the “start sequencing� button and assume all the resulting variants are accepted.

Finally, if the coverage and percentage meets criteria, the results are forwarded to annotation specialists or senior technologists for variant interpretation. This means multiple databases and annotation software are employed to determine benign polymorphisms, functional consequences, or in some cases, variants with limited information may be classified as a variant of unknown significance at this time.

I think it was somewhat reassuring to the class that the current skill sets we use to analyze conventional applications still apply to NGS results, albeit on a relatively larger scale. The technologies may have changed but the fundamentals have not. I would like to thank the class of 2017 for allowing me to share the work that we do.

References Kulkarni S and Pfeifer J (eds). Clinical Genomics. Massachusetts: Academic Press; 2014.

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AGT 41 AGT 41 ANNUAL ANNUAL MEETING MEETING STST

Registration Brochure

June 23-25, 2016 Hyatt Regency Orange County Garden Grove, Calif.

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JOIN US! JOIN US! The education program and registration for the AGT 41st Annual Meeting is now available at www.AGT-info.org!

You will not want to miss out on this year’s outstanding scientific program, you can still register on-site at the conference!

We hope to see you in Orange County!

Connect With Us!

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Genetics in the News

Column Editor: Jaime Garcia-Heras

The Roaring Molecular Revolution of Editing the Genomes of Many Living Organisms, Including Humans, Using Programmable Nucleases Programmable nucleases allow the editing of the genome in a broad range of organisms. It is now possible to correct mutations associated with diseases, modify genes of interest, delete sequences or genes from the DNA, and introduce genes or sequences to the DNA. These changes can be done without alterations in the genetic background of the host.

This technology has opened unprecedented possibilities to treat human genetic disorders, improve crops and farm animals for food supply, create novel animal models to study diseases, and replicate “in vitro” or “in vivo” genomic rearrangements (including chromosome abnormalities) that trigger disease, including cancer. This technological revolution has brought considerable advances in many fields and will continue to do so in the foreseeable future.

which are made of a zinc-finger DNA binding domain and the nuclease domain of FokI. The transcription activator-like effector nucleases (TALENs) were the second type of programmable nucleases available.

TALENs also have the nuclease domain of FokI but they have as a DNA binding domain, repeats derived from transcription activator-like effectors, which are proteins secreted by the Xanthomonas spp bacteria.

RNA-guided nucleases (CRISPR-Cas9 system) adapted from a microbial adaptive immune defense system from bacteria became available in 2013. The CRISPR-Cas9 system consists of a nuclease Cas9 protein and a single guide RNA (sgRNA) derived from the CRISPR RNA (crRNA) and a transacting CRISPR RNA. The sgRNA pairs to distinctive DNA bases and guides the Cas9 protein to generate site-specific double-strand breaks (DSBs).

Introduction

Genomic editing to study human diseases due to gene mutations and genomic rearrangements

The modification of genes has been an effective research tool to study gene function, often through the observation of effects on the phenotype of cells or whole organisms. Later on came the applications to improve crops or livestock with the incorporation of desirable traits (e.g. resistance to diseases, higher content of nutrients), and the development of gene therapy for human diseases with the goals of correcting pathogenic genes and restoring normal function.

The power to edit DNA with programmable endonucleases has expanded progress in studying human diseases with the development of other models of transgenic animals and novel “in vitro” cell systems.

Genetically modified transgenic mice have been the standard for studying human diseases for many years (Doyle et al., 2012). This animal model has allowed the characterization of many gene disorders but it is, however, very labor intensive and requires a considerable investment in capital, time and resources. By contrast, genomic editing with programmable endonucleases is now a quicker and more efficient way to target a disease-gene (or any gene of interest) and create transgenic mice. Moreover, many transgenic models have been developed using the same technology in other species. These include the fruit fly Drosophila melanogaster, the worm Caenorhabditis elegans, the yeast Sacharomyces cerevisiae, and non-traditional animals such as rats and goats (Riordan et al., 2015).

Conventional gene targeting has been a widely used and powerful method to inactivate genes via homologous recombination and evaluate gene function. This methodology to modify genes has had limitations, however, because homologous recombination is very efficient in yeast, but is a very rare event in higher eukaryotes. Nevertheless, in spite of the drawbacks in eukaryotes, gene targeting applied to mouse embryonic stem cells was instrumental in developing transgenic mice (Bouabe and Okkenhaug, 2013) and has been the gold standard to study gene function in mammals (Capecchi, 2005).

The most recent tools of gene editing via programmable nucleases (also called targeted genome engineering) that surfaced in this decade are a turning point in the quest to modify genes (Chandrasegaran and Carroll, 2016). This technology allows changes in almost any DNA sequence through double-strand breaks (DSBs) at precisely chosen sites that are induced by programmable nucleases. Thereafter, these DBSs are subject to either homologous recombination repair (HRR) if normal homologous sequences are available as a template, or are processed by an error-prone non-homologous end joining (NHEJ) that generates mutations. This revolutionary editing of the genome mediated by programmable nucleases has allowed studies that were previously difficult or impossible to accomplish.

It is important to mention that transgenic mice have provided a lot of useful information about human genetic diseases but they do not always fully model the clinical features of humans (e.g. cystic fibrosis and Duchene muscular dystrophy). Such discrepancies prompted the search for alternative models in large animals, looking for phenotypes more compatible with human genetic disorders. The domestic pig has been seen as a good candidate since it has many common anatomical, physiological and genetic attributes as humans and there are models of human diseases already available in transgenic pigs (Flisikowska et al., 2014). Likewise, non-human primates have been considered since they currently provide suitable models to study development, diseases, and therapy in humans. In recent years the technology of programmable nucleases has allowed substantial progress in developing models in both species. Of particular interest are a Rhesus monkey model with Duchene muscular dystrophy due to a knockout of the dystrophin gene that recapitulates key features of the disease in humans (Chen et al., 2015) and a model for Rett syndrome in Rhesus and Cynomolgus monkeys (Liu H et al., 2014). In the pig there is a model for familial hypercholesterolemia caused by inactivation of the LDLR gene that

Programmable nucleases are chimeric proteins that were designed in the laboratory for the first time in 1994. They consist of a DNA binding domain fused to the nuclease domain of the restriction enzyme FokI (Kim and Chandrasegaran, 1994). The DNA binding domain can be customized to recognize and bind to a specific DNA sequence while the nuclease domain generates DSBs in the DNA. The first programmable nucleases used for genome editing were the zinc-finger nucleases (ZFNs)

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Genetics in the News The Roaring Molecular Revolution of Editing the Genomes of Many Living Organisms, Including Humans, Using Programmable Nucleases encodes the low-density lipoprotein (LDL) receptor (Carlson et al., 2012).

incorporate a “healthy” gene copy that replaces the mutated gene.

There are compelling examples of genome editing restoring normal gene function in human genetic diseases that give hope for more effective treatments in the future. Hematologic and immune disorders were the early focus of intense research due to their frequency, severity of the clinical picture, and the insufficient treatments. The first groundbreaking success was the correction of the IL2R gamma receptor gene that is mutated in X-linked severe combined immunodeficiency (Urnov et al., 2005). Based on protocols established for gene editing of CD34+ hematopoietic stem cells (HSCs) and human induced pluripotent stem cells (hIPSCs) it is now possible to offer alternatives for the treatment of sickle cell disease and beta-thalassemia. There has been steady progress in other diseases as well. In cystic fibrosis (CF) the CFTR locus has been corrected in cultured intestinal stem cells from patients (Schwank et al., 2013). TALENs were used to correct fibroblasts carrying COL7A1 gene mutations associated with recessive dystrophic epidermolysis bullosa (RDEB) (Osborn et al., 2013). These results highlight the potential to correct “ex-vivo” genetic abnormalities in cells from patients and use the “healthy” cells in a subsequent transplant.

With regard to novel in vitro systems, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be used to study human genetic diseases. hESCs and iPSCs have the conspicuous advantages that they can grow in culture for extended periods of time, retain a normal karyotype and pluripotency, and allow a molecular manipulation of their DNA in the laboratory. It is now feasible to treat these cells with programmable endonucleases and generate gene mutations or rearrangements linked to the etiology of various genetic diseases (Chiba and Hockemeyer, 2015). In this way we can model “in vitro” genetic diseases with a recognized genetic abnormality and study key aspects that include the cell biology, gene regulation, metabolism, development, and potentially evaluate therapies.

The induction “in vitro” of specific genomic rearrangements that replicate in cultured eukaryotic cells events that would have occurred “in vivo” is another application of programmable nucleases. These include large deletions (Lee et al., 2010), translocations due to DBSs on different chromosomes (Piganeau et al., 2013), and inversions or duplications (Lee et al., 2012; Li et al., 2015). There have been impressive advances in human cancer, first generating cell lines with chromosome abnormalities resembling those reported in patients with AML and the t(8;21)(22;q22), Ewing’s sarcoma with the t(11;22) (q24;q22), lung adenocarcinoma with the t(5;6)(q33;q22) and the inv(2) (p21p23), and anaplastic large cell lymphoma with the t(2;5)(p23;q35) (Piganeau et al., 2013; Choi et al., 2014; Torres et al., 2014). This progress was followed by the development of “in vivo” mouse models carrying the inv(2)(p21;23) that faithfully recapitulated the features of non-small cell lung cancer in humans (Blasco et al., 2014; Maddalo et al., 2014).

Some studies proposed to correct somatic cells carrying mutations first and then generate induced pluripotent stem cells (iPSCs) from them (e.g. Osborn et al., 2013 for RDEB). Others proposed to start correcting directly iPSCs derived from patients (Li et al., 2015 for Duchene muscular dystrophy). The iPSCs obtained with either protocol could be programmed to generate adult cells or tissues adequate for autologous transplantation without the risk of rejection by the recipient. The hope is that in the case of Duchene muscular dystrophy (DMD) patient-derived iPSCs with a normal dystrophin gene become an effective therapy to rebuild the affected muscles, a goal that has been elusive so far. In addition, there has been strong interest in gene editing “in vivo,” especially for diseases in which the organs involved are not easy to transplant like the liver. Promising headway was made on this subject with mouse models to deliver factor IX cDNA for hemophilia B (Li et al., 2011) and correct the FAH gene that is implicated in hereditary tyrosinemia I (Yin et al., 2014). The prevention of genetic disease has also been reported in the laboratory mouse, most conspicuously with DMD. Long et al. showed a halt in the onset of disease by a correction of the Dmd mutation in the germline of an mdx mouse model (Long et al., 2014).

Treatment of human genetic diseases based on genomic editing There are numerous hereditary monogenic diseases in humans caused by gene mutations. Unfortunately, there is no cure in most cases, and quite often the therapy is only palliative. There have been successes with standard gene therapy for recessive disorders of the hematopoietic system such as SCID and Wiskott-Aldrich syndrome.

In other instances RNA interference has been effective in clinical trials. The recent advent of genomic editing with programmable nucleases now offers chances to supersede the limitations of standard gene therapy and proceed to inactivate deleterious mutations, correct or delete defective genes, or add therapeutic genes into specific sites of the genome (Maeder and Gersbach, 2016). Genomic editing has been recently tested on a research basis with impressive success in the laboratory and is steadily making its way into the clinic. Programmable nucleases targeting a faulty gene can generate double-strand breaks (DBSs) that are repaired by either the error-prone nonhomologous end-joining (NHEJ) or the error-free homology-directed repair (HDR).

When NHEJ takes place it operates without a template and deletions or insertions often occur at the sites of DSBs. As a result of NHEJ a faulty gene (mutation) can be inactivated through a deletion that disrupts the reading frame. Meanwhile the normal gene copy on the homologous chromosome remains intact and can take over the normal function. In the case of homology directed repair (HDR) it works with the aid of an exogenous DNA source as a normal template to

The most remarkable success with therapeutic genome editing has been achieved in the treatment of HIV/AIDS in humans. The HIV/ AIDS virus requires the CCR5 co-receptor in the outer membrane of macrophages and CD4+ T-cells in order to attach to and infect these cells, start its own replication, and ultimately trigger the onset of disease. This critical role of CCR5 is consistent with observations that individuals homozygous for a loss-of-function mutation of this receptor are resistant to the infection by the HIV/AIDS virus while heterozygous individuals usually have a slower course of the disease.

In a recent Phase 1 clinical trial, CD4+ T cells were isolated from patients infected with HIV, edited with Zinc Finger Nucleases (ZFNs) to inactivate the CCR5 gene encoding the CCR5 receptor, and then transplanted to the same patients as an autologous transplant. The early results showed that this procedure is in general safe, it provides a stable population of normal CD4+ T cells, and most importantly patients acquired a partial genetic resistance to the HIV infection

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Genetics in the News

The Roaring Molecular Revolution of Editing the Genomes of Many Living Organisms, Including Humans, Using Programmable Nucleases goals were accomplished by disabling with TALENs the myostatin (MSTN) gene, a negative regulator that restricts muscle growth (Luo et al., 2014; Proudfoot et al., 2015).

(Tebas et al., 2014).

The controversy about editing of genes in human embryos

It has been possible to generate a breed of cattle that gives milk without the allergens linked to cow’s milk allergy. This is a common disease mostly seen during infancy and early childhood that is caused by allergens from the casein fraction of proteins and the whey proteins of milk (Lifschitz and Szajewska, 2015). This new breed was generated by a knock-out with ZFNs of beta-lactoglobulin (BLG), a gene that encodes a major allergen present in the whey protein fraction of milk in cows (Yu et al., 2011).

While the pace of genomic editing with programmable nucleases gained momentum, the scientific community anticipated controversies about its use if it was applied to human embryos or germ cells. The time came in May 2015 when Liang et al. reported for the first time genome editing in human embryos (Liang et al., 2015). The experiments were done in tripronuclear (3PN) human zygotes that were discarded by fertility clinics because they could not progress into a pregnancy and a viable liveborn. Liang et al. demonstrated that it was possible to cleave the beta globin gene (HBB) that is mutated in beta-thalassemia.

However, the efficiency of HDR repair was low, many of the embryos tested after the genome editing were mosaic and would not be screened accurately by preimplantation diagnosis, off-target cleavage events in sites other than the beta globin were apparent, and there were also unwanted mutations. Taking all the findings together Liang et al. cautioned against the premature use of genome editing for clinical applications and urged for a thorough investigation about the use of genome editing technology.

Diseases that cause significant economic impact in livestock have also stimulated the use of targeted genome editing to develop breeds resistant to them. One instructive case is the resistance to mastitis acquired by transgenic cows with transgenes for the human lysozyme and a bacterial lysostaphin inserted in the beta-casein locus (Liu X et al., 2013; Liu X et al., 2014). This is a quite significant accomplishment considering that mastitis is a recurrent disease that causes enormous financial losses to the dairy industry. Another informative example is the increased resistance to tuberculosis caused by Mycobacterium bovis in cattle provided by the knockin of the mouse SP110 gene (Wu et al., 2015). The expectations are that this genetic change will help to control bovine tuberculosis, which is a serious public health threat in less developed areas of the world.

The results of Liang et al. sparked attention from the public, generated a heated debate, raised significant ethical and scientific concerns, prompted the immediate recommendations to scrutinize embryo editing (Doudna, 2015), and ultimately called for a broad discussion about guidelines for a proper use of genome editing in humans (Baltimore et al., 2015). A meeting held in Washington, D.C. on December 2015 recommended against a cessation in research on human gene editing, and at the same time to stop the research and applications of modified human embryos to establish a pregnancy.

There was also a consensus that genome editing to correct genetic defects in nonreproductive cells after birth should continue (Reardon, 2015). A few months later another report about gene editing in human embryos continued to fuel controversies (Kang et al., in press).

This study tried to inactivate the CCR5 gene in nonviable embryos introducing the CCR5 delta32 allele and make them resistant to the HIV infection. Of all the 26 embryos that were targeted, only four incorporated the CCR5 delta32 allele while the other alleles kept the wild type CCR5 or carried indel mutations. The authors concluded that many technical hurdles remained to be solved before the procedure could be implemented in the clinic. For the time being Kang et al. advocated for a moratorium on the genome editing of the germline until a rigorous scientific evaluation and ethical discussion were undertaken.

Regarding xenotransplantation, there are hopes that large animals become donors that alleviate the shortages of organs for human transplantation. Given the similarities in anatomy, physiology and size with humans, the domestic pig is perceived as an attractive source.

But, the porcine genome carries the alpha-1,3 galactosyl-transferase (GGTA1) gene of an enzyme that synthesizes Gal epitopes present in the cell surface of all tissues. As a result, transplants from pigs to primates are rejected due to a hyperacute rejection (HAR) mediated by preformed antibodies that recognize the Gal epitopes. In a breakthrough to overcome HAR, biallelic knock-outs of GGTA1 were generated in pigs with the aid of zinc-finger nucleases (Hauschild et al., 2011; Li et al., 2013). To eventually have organs from pigs suitable for xenotransplantation, however, it will be necessary to address other major immunological reactions triggered by xenotransplantation on the host and the existence of porcine endogenous retrovirus (Luo et al., 2012).

The contributions of genome editing for the improvement of crops Conventional breeding has been used for a long time to improve crops and incorporate into them beneficial traits that are carried on by subsequent generations. This is, however, a long procedure restricted by the genetic variation available in nature that requires extensive back-crossing between species to introduce the selected traits in the genome. For these reasons conventional breeding is very laborintensive, time-consuming, and expensive. Nowadays the modern tools of genome editing have become an alternative to traditional plant breeding because they allow biologists to make heritable and unique modifications at distinct sites of the genome in a shorter time frame and very efficiently.

Genome editing of farm animals Programmed nucleases have been successfully exploited to create farm animals that carry traits of high commercial value, are more resistant to certain diseases, especially those that impair productivity or the wellbeing, or allow diverse applications in biotechnology or medicine (Tan et al., in press).

At this time there are breeds of cattle and sheep with increased muscle mass available to simultaneously boost economic profits and maximize the output in the production of marketable lean meat. These

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Genetics in the News The Roaring Molecular Revolution of Editing the Genomes of Many Living Organisms, Including Humans, Using Programmable Nucleases (Buermans and den Dunnen, 2014; van Dijk et al., 2014). In just a few years, particularly after 2012 which delivered the first report of the CRISPR/Cas9 systems (Jinek et al., 20132), the clinical and research innovations with genome editing came continuously at an astonishing pace. We should acknowledge that the limits of what we can accomplish with genome editing using programmable nucleases are only posed by the imagination of the experimenter.

There are multiple reports of genome editing in plants for commercial applications, and to enhance resistance to diseases or pathogens, and pesticides. In general, these developments were mostly focused on crops with high economic interest that are critical to feed large populations.

On the subject of resistance to disease for example, the rice bacterial blight susceptibility gene OsSWEET14 was targeted with TALENs to generate disease-resistant rice (Li et al., 2012). Wang et al. modified homeo-alleles that encode the mildew-resistance locus (MLO) in bread wheat to create heritable mutants with resistance to powdery mildew disease, a trait that is not found in natural populations (Wang et al., 2014).

Given the vulnerable status of the food chain in many regions of the world, often magnified by climate changes, decreases in arable land due to urbanization, scarce water for irrigation and the continuous growth of populations, genome editing of livestock and crops may provides opportunities to alleviate the shortages of both. There is still a pressing need to expand the food supply despite that the Green Revolution in agriculture has been a success and plant biotechnology has played a major role over the last two decades. Fortunately, there has been promising progress with genome editing that led to enhancements in resistance to disease, and in output of beneficial dietary traits in livestock and crops. At this point, though, it seems that the feasibility of implementing genome editing to maximize the supply of food will be determined by the approval or ban of these novel crops and livestock by regulatory frameworks, and by the acceptance or rejection of them by societies or consumers. We must keep in mind the negative perception or plain opposition to genetically modified organisms (GMOs) in many countries, which may spill over products from genome editing (PFGE), and sometimes regulatory bodies put restrictions for the consumption of GMOs that eventually may include PFGE.

Editing of genes related to resistance to pesticides that could allow farmers a more liberal use of them has been fulfilled too. Mutations in the acetolactate synthase (ALS) genes SuRA and SuRB were designed to acquire resistance to imidazolinone and sulphonylurea herbicides in tobacco (Townsend et al., 2009). The protoporphyrinogen oxidase (PPOX) gene was edited to generate mutations that result in resistance to the herbicide butafenil in Abrabidopsis (de Pater et al., 2013).

The capability to stack in the same locus multiple useful traits is another innovative commercial application of genome engineering with programmable nucleases (Sprink et al., 2015). These stacked traits can then segregate together at a single locus in a normal Mendelian fashion. This approach is much more efficient than trying the conventional breeding, which is very tricky and often results in the segregation of these traits in the progeny. The thought is that stacking could be used to develop multiresistant traits and produce different pharmaceutical proteins (Sprink et al., 2015).

There seem to be enormous opportunities for successful genome editing in the clinic, particularly to cure or alleviate diseases without a current satisfactory therapy and for the prevention of genetic disease.

In the case of severe debilitating diseases ascertained postnatally, like Duchene muscular dystrophy and sickle cell anemia, for example, these may turn out to be curable or at least alleviated if we take into account the progress already made on a research basis. It is remarkable that in the future induced pluripotent stem cells (iPSCs) may be part of the new therapeutic arsenal against genetic diseases. Used concurrently with genome editing, iPSCs could generate adult tissues adequate for transplants once the underlying genetic abnormality has been corrected in a sample from a patient. Without any doubt the most noteworthy therapeutic success with genome editing has been its application to patients affected with AIDS. This is a milestone because a vaccine against this disease has been elusive and the antiretroviral therapies available are not a cure, are very expensive, and resistance to them may arise.

The potential control of vectors responsible of infectious diseases with high public health significance Recent research proposed an intriguing idea, the use of genome editing to control vectors of serious infectious diseases that affect humans. These vectors include the mosquito Aedes aegypti that transmits the chikungunya, yellow fever and dengue viruses (Kistler et al., 2015), and mosquitos from the genus Anopheles that transmit malaria (Smidler et al., 2013). This vector control is based on modifications in the genome of the mosquitos to render them resistant to infection by these viruses or parasites, or disable the adequate physiological environment that allows mosquitos to become a suitable reservoir. Prospects of releasing engineered male mosquitos in the wild to propagate these features of vector control have also been considered.

But as a whole, there are concerns about the ecological impact and the odds of unleashing strong selective pressures in these viruses and parasites (Ainsworth, 2015).

Given the current consensus to refrain from genome editing embryos to establish a pregnancy or germ cells, it is too early to know if it may be offered to patients in the future. It is fair to say, though, that it would be very helpful in certain clinical situations, for example, combined with preimplantation genetic screening for couples who carry deleterious gene traits and only generate embryos with a genetic disease through assisted reproductive technology. Furthermore, voices of concern have pointed out that banning genome editing of germ cells would jeopardize the best possible prevention for the transmission of genetic diseases, would discourage scientific innovations, and drive its practice underground (Church, 2015). There was a recent development in this topic in the UK: The approval to edit the genome of embryos

Concluding remarks The dawn of genomic editing with programmable nucleases has been a landmark innovation in molecular genetics. It is comparable to the first accounts of restriction fragment length polymorphisms (RFLPs) (Botstein et al., 1980), and the coming of age of the techniques of polymerase reaction (PCR) and next generation sequencing (NGS)

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The Roaring Molecular Revolution of Editing the Genomes of Many Living Organisms, Including Humans, Using Programmable Nucleases for just research purposes by the Human Fertilization and Embryology Authority, a move that may set a precedent to do the same research in other countries (Callaway, 2016). This project intends to modify genes that are active after fertilization and will be stopped after seven days when the embryos will be discarded. The findings may give clues about gene regulation and function during the early stages of human embryonic development, may explain some of the failures with assisted reproductive technology, and in time, lead to improvements in treatments for infertility.

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Chandrasegaran S, Carroll D. Origins of programmable nucleases for genome engineering. J Mol Biol. 2016;428: 963-989.

Chen Y, Zheng Y, Kang Y, Yang W, Niu Y, Guo X, Tu Z, Si C,Wang H, Xing R, Pu X, Yang S-H, Li S, Ji W, Li X-J. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Hum Mol Genet. 2015;24: 3764-3774.

Chiba K, Hockemeyer D. Genome editing in human pluripotent stem cells using site-specific nucleases. Meth Mol Biol. 2015;1239: 267-280.

Choi PS, Meyerson M. Targeted genomic rearrangements using CRISPR/Cas technology. Nat Commun. 2014;5: 4728.

Church G. Encourage the innovators. Nature. 2015:528: S7. de Pater S, Pinas JE, Hooykaas PJJ, van der Zaal BJ. ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation. Plant Biotechnol J. 2013;11: 510-515.

Doudna J. Embryo editing needs scrutiny. Nature. 2015;528: S6.

Doyle A, McGarry MP, Lee NA, Lee JJ. The construction of transgenic and gene knockout/knock in mouse models of human disease. Transgenic Res. 2012;21: 327-349.

Flisikowska T, Kind A, Schnieke A. Genetically modified pigs to model human diseases. J Appl Genet. 2014;55: 53-64.

Hauschild J, Petersen B, Santiago Y, Queisser A-L, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, Niemann H. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Si USA. 2011;108: 12013-12017.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337: 816-821.

Kang X, He W, Huang Y, Yu Q, Chen Y, Gao X, Sun X, Fan Y. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Casmediated genome editing. J Assist Reprod Genet. 2016;33: 581-588.

Kim Y-G, Chandrasegaran S. Chimeric restriction endonuclease. Proc Natl Acad Sci USA. 1994;91: 883-887.

Kistler KE, Vosshall LB, Matthews BJ. Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. Cell Rep. 2015;11: 51-60.

Lee HJ, Kim E, Kim J-S. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 2010;20: 81-89.

Lee HJ, Kweon J, Kim E, Kim S, Kim J-S. Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res. 2012;22: 539-548.

Li H, Haurigot V, Doyon Y, Li T, Wong SY, Bhagwat AS, Malani N, Anguela XM, Sharma R, Ivanciu L, Murphy SL, Finn JD, Khazi FR, Zhou S, Paschon DE, Rebar EJ, Bushman FD, Gegory PD, Holmes MC, High KA. In vitro genome editing restores haemostasis in a mouse model of haemophilia. Nature. 2011;475: 217-221.

Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, Tanaka M, Amano N, Watanabe A, Sakurai H, Yamamoto T, Yamanaka S, Hotta A. Precise correction of the dystrophin gene in Duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Rep. 2015;4: 143-154.

Li J, Shaou J, Guo Y, Tang Y, Wu Y, Jia Z, Zhai Y, Chen Z, Xu Q, Wu Q. Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9. J Mol Biol. 2015;7: 284-298.

Li P, Estrada JL, Burlak C, Tector AJ. Biallelic knockout of the α-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases. J Surg Res. 2013;181; E39-E45.

Li T, Liu B, Spalding MH, Weeks DP, Yang B. High-efficiency TALEN-based

The mouse models recently developed with programmable nucleases that recapitulate typical rearrangements associated with human cancer, especially those that create fusion of genes with oncogenic properties, have been a defining event because they may supersede if not replace completely the traditional transgenic mice. This new tool is much more efficient than the protocols to create transgenic mice and gives unparalleled opportunities to study molecular drivers of cancer, the response to therapy especially with new agents, and discover the onset of drug resistance. In the case of Ewing’s sarcoma, for example, it would be valuable to evaluate new therapies considering that there hasn’t been progress for a long time. Furthermore, given the continuous reporting of unique rearrangements in tumors, it is foreseeable that compared to traditional transgenic mice, these latest genetically engineered mice will speed up and facilitate the study of these malignancies.

Overall we ought to be optimistic about all the feats of genome editing achieved so far, but at the same time we should not put aside some significant concerns. One of them has already emerged and was broadly discussed, the potential of unethical or frivolous uses, including applications in eugenics and the need to stop (for now) genomic editing in human embryos and germ cells. Another concern is how to ensure fair access to this technology at a reasonable cost to all who need it, whether farmers in need of better crops or livestock, patients who require highly specialized treatment, or others. Needless to say, the latter concern is prominent in the third world as well as in highly developed countries with marked economic or social inequalities. Finally, individuals and societies will have to reconsider thoroughly their standards of what is considered a genetic disease, and the acceptable goals of prevention and treatment related to genome editing.

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Continuing Education Opportunities

Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

Test Yourself #2, 2016 Readers of The Journal of the Association of Genetic Technologists are invited to participate in this “open book test” as an opportunity to earn Contact Hours. AGT offers 3 Contact Hours for this Test Yourself based on articles in Volume 42, Number 1, First Quarter 2016 of the Journal.

Test Yourself is free to AGT members and $30 for non-members. To take this exam, send a copy of your completed Answer Sheet along with the completed Contact Hours Reporting Form to the AGT Education Committee Representative in your region. The list of representatives is on page 201 of this issue. Non-members should submit a check payable to AGT for $30 with their answer sheet. Entry material must be post-marked on or before September 12, 2016.

Passing score is 87% or 21 out of 24 questions answered correctly. Compiled by Doina Ciobanu and Sally Kochmar.

The following questions are from Chow R. et al. A t(17;19) (q22;p13.3) involving TCF3, a t(1;9)(p13;p13), and a 5’ IGH deletion in a case of adult B-cell acute lymphoblastic leukemia.

J Assoc Genet Technol. 2016;42(1): 6-14.

The following questions are from Conant J et al. Fetal Tissue Procurement for Karyotype Analysis: Clinician or PathologistWhich is Better? J Assoc Technol. 2016;42(1): 15-18.

5. Stillbirth occurs in 1 out of every ______ pregnancies in the United States. a. 100 b. 200 c. 180 d. 160

1. The t(17;19)(q22;p13) which results in the TCF3-HLF fusion gene is found in less than ______of cases. a. 3% b. 5% c. 1% d. 10%

6. All of the following are true, except: a. Chromosomal abnormalities are detected in up to 13% of stillbirths. b. More than 20% of stillbirths exhibit developmental anomalies. c. Up to 30% of attempts to culture cells fail. d. Lack of growth may be due to bacterial contamination, insufficient cells or non viable tissue.

2. According to this article: I. TCF3 encodes transcription factors which function as transcription activators for cell growth .

II. The t(17;19) involving TCF3 and HLF generates a chimeric transcription factor which prevents apoptosis.

III. the TCF3-HLF fusion gene can result from two types of rearrangements.

IV. B-cell involvement of TCF3 is mostly seen in conjunction with PBX1. a. I, II and III b. I, III and IV c. I and II only d. all of the above

7. On average, tissue for chromosome analysis was collected by the clinician _____ hours after delivery. a. 10 b. 15 c. 36.5 d. 2

3. Monosomy 7 has been found in about 5% of ALL cases. a. true b. false

8. This study shows that collection of stillborn tissue samples by a pathologist has a lower rate of microbial contamination than tissue collected by a clinician at the time of delivery. a. true b. false

4. All of the following are correct, except: a. At second relapse, cytogenetic studies showed a t(1;19) (p13;p13), a t(17;19) and complex rearrangements involving chromosomes 5,7 and 14. b. FISH at second relapse detected three copies of 19p13.3 in 38.3% of analyzed nuclei. c. FISH studies at first relapse detected a 5’ IGH deletion in 80.3% of the nuclei analyzed. d. Cytogenetic results at initial diagnosis showed a t(17;19) (q22;p13.3) in 12 of the 20 cells analyzed.

The following questions are from Mark HFL. Dr. Jean Amos Wilson. J Assoc Genet Technol. 2016;42(1): 20-21.

9. All of the following statements are correct except: a. Dr. Amos Wilson is an ABMG-certified Clinical Molecular Geneticist. b. She was the 2012-2013 President of the California Clinical Laboratory Association. c. She was VP of Operations for Berkeley Heart Lab. d. She holds a NYSDOH Certificates of Qualification in Genetic Testing and Molecular Tumor Markers.

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Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

10. Where did Dr. Amos Wilson finish her post-doctoral fellowship? a. b. c. d.

16. The t(16;21)(p11;q22) results in the replacement of the RNA binding motifs of TLS/FUS with the ETS DNA binding motifs downstream of ERG in the C-terminal, and fusion of the resulting protein to the N-terminal of TLS/FUS. a. true b. false

Cleveland, Ohio Ohio State University Massachusetts General Hospital Alameda, CA

17. How many total cases were compiled in this article? a. 68 b. 60 c. 50 d. 78

11. According to Dr. Amos Wilson, today’s biggest challenge for clinical molecular genetics is the decreased reimbursement and the impact on test availability and innovation. a. true b. false

18. Choose the correct answer: a. 45% of the cases had secondary abnormalities b. 7% of cases had trisomy 10 as a secondary abnormality c. Recurrent secondary abnormalities were rare d. del (13)(q12q14) was observed in 9 cases

The following questions are from Mah M. Making NGS Technologies Increasingly Accessible. J Assoc Genet Technol.

2016;42(1): 23.

12. Choose the correct statement: a. Qiagen launched a complete NGS workflow in August 2015. b. The Actionable Insights Tumor Panel is testing for 10 genes. c. The Gene Reader’s NGS System scope of testing is more limited than most NGS panels. d. Labs can use Qiagen products only for NGS automated sample preparation.

The following questions are from Lawce H. Book Review: The Deeper Genome: Why there is more to the human genome that meets the eye. J Assoc Genet Technol 2016; (42)1:34.

19. All of the following are true about author John Parrington except: a. He is a lecturer in Molecular and Cellular Pharmacology at the University of Cambridge. b. He published in journals such as Nature, Current Biology and Journal of Cell Biology. c. He writes popular science articles. d. He worked as a science journalist for the Times.

13. If labs move in the direction of whole exome and whole genome sequencing, new IT infrastructure will be required. a. true b. false

20. Parrington analyzes the many roles of RNA and discusses at length the role of epigenetics. a. true b. false

The following questions are from Buchanan J. et al. A t(16;21) (p11;q22) in Acute Myeloid Leukemia (AML) resulting in Fusion of the FUS(TLS) and ERG Genes: A review of the literature. J Assoc Genet Technol 2016;42(1): 24-33.

The following questions are from Garcia-Heras J. The 2015 Nobel Prize in Chemistry. The discovery of essential mechanisms that repair DNA damage. J Assoc Genet Technol.

2016;(42)1: 35-38.

14. The t(16;21)(p11;q22): I. appears in about 1% of AML cases.

II. is associated with a good prognosis.

III. median survival rate of patients carrying this translocation is 16 months.

IV. results in expression of the FUS/TLS-ERG chimeric protein. a. I, II and IV b. I, III and IV c. II, III and IV d. I, II and III

21. Xeroderma pigmentosum is a rare autosomal recessive disease with an incidence of _____ in Western Europe. a. 1 per 250,000 b. 1 per 4,500,000 c. 1 per 4,500 d. 1 per 450,000

15. TLS/FUS is located on ______, while ERG is located on_____. a. 16p11, 22q11 b. 16q11, 22q22 c. 16p11, 21q11 d. 16p11, 21q22

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Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

24. All of the following statements are true, except: a. The new IOM report was issued in September 2015. b. The recommendations were grouped into eight goals. c. The Measure and Reduce goal aims to identify, reduce and learn from diagnostic errors. d. Only five goals have recommendations for opportunities in future research.

22. Patients with xeroderma pigmentosum: I. have atrophic dry skin.

II. have a high incidence of early tumors of the skin.

III. 20% of them develop more severe phenotypes IV. Some present neurodegeneration and premature aging. a. b. c. d.

I, II and III I, II and IV I, III and IV all of the above

The following questions are from Crawford-Alvares J. A brief review of IOM’s latest report. J Assoc Genet Technol.

2016;(42)1: 40-41.

23. According to the author, from the eight goals IOM recommended, our profession can create its biggest impact by improving cross-divisional teamwork and communication, highlighting the importance of professional training opportunities and developing a revised system for reporting and learning from errors. a. true b. false

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Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

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Continuing Education Opportunities

The AGT Education Committee's Journal Club Journal Clubs are a great way to earn Contact Hours without leaving your home or lab! Journal Clubs can be completed as a group or individually. Each Journal Club includes a reading list, several discussion questions and a post-test. The discussion questions provide a starting point for group discussion and give individuals taking a Journal Club questions to consider while reading the articles. The post-test is taken after reading the articles and is returned to the regional representatives of the Education Committee to be graded.

Each successfully completed Journal Club is worth 4.0 Contact Hours. Journal Clubs can be ordered through the AGT Executive Office.

READING LIST 48 – General Content Area: Hematologic Malignancies–2004 1. Molecular Cytogenetic Aspects of Hematological Malignancies: Clinical Implications

Congenital Malformations: Delineation of 7q21.11 Breakpoints 4. Use of Targeted Array-Based CGH for the Clinical Diagnosis of Chromosomal Imbalances: Is Less More?

READING LIST 49 – General Content Area: Miscellaneous Topics–2004

READING LIST 53 – General Content Area: Genetic Syndromes–2005

1. The Burden of Genetic Disease on Inpatient Care in a Children’s Hospital 2. Contribution of Malformations and Genetic Disorders to Mortality in a Children’s Hospital 3. A Study of Reciprocal Translocations and Inversions Detected by Light Microscopy With Special Reference to Origin, Segregation, and Recurrent Abnormalities

READING LIST 50 – General Content Area: Cancer Genetics–2005 1. Cytogenetics and Molecular Genetics of Lung Cancer 2. Chromosome Abnormalities May Correlate With Prognosis in Burkitt/ Burkitt-Like Lymphomas of Children and Adolescents: A Report from Children’s Cancer Group Study CCG-E08 3. Clinical Applications of BCR-ABL Molecular Testing in Acute Leukemia

READING LIST 51 – General Content Area: Cancer Genetics–2005 1. Cytogenetic Profile of Myelodysplastic Syndromes with Complex Karyotypes: An Analysis Using Spectral Karyotyping 2. Classical Hodgkin Lymphoma is Associated with Frequent Gains of 17q 3. Specific Chromosome Aberrations in Peripheral Blood Lymphocytes are Associated with Risk of Bladder Cancer

READING LIST 52 – General Content Area: New Technologies–2005 1. Colour-Changing Karyotyping: An Alternative to M-FISH/SKY 2. A New Chromosome Banding Technique, Spectral Color Banding (SCAN), for Full Characterization of Chromosomal Abnormalities 3. Molecular Cytogenetic Analysis of Complex Chromosomal Rearrangements in Patients with Mental Retardation and

1. Five Years of Molecular Diagnosis of Fragile X Syndrome (1997-2001): A Collaborative Study Reporting 95% of the Activity in France 2. Changing Demographics of Advanced Maternal Age (AMA) and the Impact on the predicted incidence of Down Syndrome in the United States: Implications for Prenatal Screening and Genetic Counseling 3. “Everybody in the World is My Friend” Hypersociability in Young Children with Williams Syndrome

READING LIST 54 – General Content Area: Chromosome Breakage Syndromes–2006 1. Chromosome Breakage Syndromes and Cancer 2. DEB Test for Fanconi Anemia Detection in Patients with Atypical Phenotype 3. Nijmegen Breakage Syndrome: Clinical Manifestation of Defective Response to DNA Doublestrand Breaks

READING LIST 55 – General Content Area: Array Based Prenatal Genetics–2006 1. Array-based Comparative Genomic Hybridization Facilitates Identification of Breakpoints of a Novel der(1)t(1;18) (p36.3;q23)dn in a Child Presenting with Mental Retardation 2. Detection of Cryptic Chromosome Aberrations in a Patient with a Balanced t(1;9)(p34.2;p24) by Array-based Comparative Genomic Hybridization 3. Jumping Translocations in Multiple Myeloma

READING LIST 56 – General Content Area: Leukemia–2007 1. Fluorescence in situ Hybridization Analysis of Minimal Residual Disease and the Relevance of the der(9) Deletion in Imatinib-treated Patients with Chronic Myeloid Leukemia

2. Characterization of the t(17;19) Translocation by Gene-specific Fluorescent in situ Hybridizationbased Cytogenetics and Detection of the E2A-HLF Fusion Transcript and Protein in Patient’s Cells 3. Combination of Broad Molecular Screening and Cytogenetic Analysis for Genetic Risk Assignment and Diagnosis in Patients with Acute Leukemia

READING LIST 57 – General Content Area: Premature Chromosome Condensation–2007 1. Premature Chromosome Condensation in Humans Associated with Microcephaly and Mental Retardation: A Novel Autosomal Recessive Condition 2. Chromosome Condensation: DNA Compaction in Real Time 3. Phosphatase Inhibitors and Premature Chromosome Condensation in Human Peripheral Lymphocytes at Different CellCycle Phases

READING LIST 58 – General Content Area: Solid Tumor and FISH–2007 1. Methylthioadenosine Phosphorylase Gene Deletions are Frequently Detected

by Fluorescence in situ Hybridization in Conventional Chondrosarcoma 2. Solid Pseudopapillary Neoplasms of the Pancreas are Associated with FLI-1 Expression, but Not with EWS/FLI-1 Translocation 3. High Incidence of Chromosome 1 Abnormalities in a Series of 27 Renal Oncocytomas: Cytogenetic and Fluorescent In Situ Hybridization Studies

READING LIST 59 – General Content Area: Treatment of Prader-Willi Syndrome with Growth Hormone–2008 1. Two Years of Growth Hormone Therapy in Young Children with Prader-Willi Syndrome: Physical and Neurodevelopmental Benefits - American Journal of Medical Genetics Part A, Volume 143A, Issue 5, pages 443-448, 1 March 2007 2. Growth Hormone Therapy and Scoliosis in Patients with Prader-Willi Syndrome 3. Cause of Sudden, Unexpected Death

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Continuing Education Opportunities of Prader-Willi Syndrome Patients with or without Growth Hormone Treatment

READING LIST 60 – General Content Area: Generics of Autism–2008 1. 15q11-13 GABAa Receptor Genes are Normally Biallelically Expressed in Brain yet are Subject to Epigenetic Dysregulation in Autism-Spectrum Disorders 2. Characterization of an Autism-Associated Segmental Maternal Heterodisomy of the Chromosome 15q11-13 Region 3. 15q Duplication Associated with Autism in a Multiplex Family with a Familial Cryptic Translocation t(14;15)(q11.2;q13.3) Detected Using Array-CGH

READING LIST 61 – General Content Area: Genetics of Nicotine Addiction–2008 1. Fine Mapping of a Linkage Region on Chromosome 17p13 Reveals that GABARAP and DLG4 are Associated with Vulnerability to Nicotine Dependence in European-Americans 2. Genomewide Linkage Scan for Nicotine Dependence: Identification of a Chromosome 5 Risk Locus 3. Genetic Linkage to Chromosome 22q12 for a Heavy-Smoking Quantitative Trait in Two Independent Samples

READING LIST 62 – General Content Area: Somatic Mutation Detection–2007 1. Inferring Somatic Mutation Rates Using the Stop-Enhanced Green Fluorescent Protein Mouse 2. Paternal Age at Birth is an Important Determinant of Offspring Telomere Length 3. Genome-Wide SNP Assay Reveals Structural Genomic Variation, Extended Homozygosity and Cellline Induced Alterations in Normal Individuals

READING LIST 63 – General Content Area: Polyglutamine Neurodegenerative Disorders–2007 1. CAG- Encoded Polyglutamine Length Polymorphism in the Human Genome 2. Polyglutamine Neurodegenerative Diseases and Regulation of Transcription: Assembling the Puzzle 3. Pathogenesis and Molecular Targeted Therapy of Spinal and Bulbar Muscular Atrophy

READING LIST 64 – General Content Area: Clinical Applications of Noninvasive Diagnostic Testing–2008 1. Digital PCR for the Molecular Detection of Fetal Chromosomal Aneuploidy 2. Noninvasive Testing for Colorectal Cancer: A Review 3. Novel Blood Biomarkers of Human Urinary Bladder Cancer

READING LIST 65 – General Content Area: Diabetes–2010

READING LIST 70 – General Content Area: Molecular Cardiology–2010

1. The Development of c-MET Mutation Detection Assay 2. Molecular Mechanisms of Insulin Resistance in Chronic Hepatitis C 3. A Genetic Diagnosis of HNF1A Diabetes Alters Treatment and Improves Glycaemic Control in the Majority of Insulin-Treated Patients

1. Identification of a Pleiotropic Locus on Chromosome 7q for a Composite Left Ventricular Wall Thickness Factor and Body Mass Index: The HyperGEN Study 2. Novel Quantitative Trait Locus is Mapped to Chromosome 12p11 for Left Ventricular Mass in Dominican Families: The Family Study of Stroke Risk and Carotid Atherosclerosis 3. Genome-Wide Association Study Identifies Single-Nucleotide Polymorphism in KCNB1 Associated with Left Ventricular Mass in Humans: The HyperGEN Study

READING LIST 66 – General Content Area: Diabetes–2010 1. Distribution of Human Papillomavirus Genotypes in Invasive Squamous Carcinoma of the Vulva 2. Distribution of HPV Genotypes in 282 Women with Cervical Lesions: Evidence for Three Categories of Intraepithelial Lesions Based on Morphology and HPV Type 3. Evaluation of Linear Array Human Papillomavirus Genotyping Using Automatic Optical Imaging Software

READING LIST 67 – General Content Area: Pancreatic Cancer and its Biomarkers–2010 1. Molecular Profiling of Pancreatic Adenocarcinoma and Chronic Pancreatitis Identifies Multiple Genes Differentially Regulated in Pancreatic Cancer 2. Effect of Recombinant Adenovirus Vector Mediated Human Interleukin-24 Gene Transfection on Pancreatic Carcinoma Growth 3. Highly Expressed Genes in Pancreatic Ductal Adenocarcinomas: A Comprehensive Characterization and Comparison of the Transcription Profiles Obtained from Three Major Technologies

READING LIST 68 – General Content Area: Influenza A(H1N1) Virus–2010 1. Detection of Influenza A(H1N1)v Virus by Real-Time RT-PCR 2. Economic Consequences to Society of Pandemic H1N1 Influenza 2009 – Preliminary Results for Sweden 3. Response after One Dose of a Monovalent Influenza A (H1N1) 2009 Vaccine — Preliminary Report

READING LIST 69 – General Content Area: The Development of c-MET Mutation Detection Assay–2010 1. Somatic Mutations in the Tyrosine Kinase Domain of the MET Proto-Oncogene in Papillary Renal Carcinomas 2. Expression and Mutational Analysis of MET in Human Solid Cancers 3. Role of cMET Expression in Non-Small-Cell Lung Cancer Patients Treated with EGFR Tyrosine Kinase Inhibitors

READING LIST 71 – General Content Area: Detection of Clarithromycin Resistance in H. Pylori–2010 1. Rapid Detection of Clarithromycin Resistance in Helicobacter Pylori Using a PCR-based Denaturing HPLC Assay 2. Rapid Screening of Clarithromycin Resistance in Helicobacter Pylori by Pyrosequencing 3. Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

READING LIST 72 – General Content Area: Werner Syndrome Gene–2010 1. Telomeric protein TRF2 protects Holliday junctions with telomeric arms from displacement by the Werner syndrome helicase 2. WRN controls formation of extrachromosomal telomeric circles and is required for TRF2DeltaBmediated telomere shortening 3. Sequence-specific processing of telomeric 3' overhangs by the Werner syndrome protein exonuclease activity

READING LIST 73 – General Content Area: Diagnosis of Melanoma Using Fluorescence in Situ Hybridization–2011 1. Using Fluorescence in situ Hybridization (FISH) as an Ancillary Diagnostic Tool in the Diagnosis of Melanocytic Neoplasms 2. Transcriptomic versus Chromosomal Prognostic Markers and Clinical Outcome in Uveal Melanoma 3. Detection of Copy Number Alterations in Metastatic Melanoma by a DNA Fluorescence In situ Hybridization Probe Panel and Array Comparative Genomic Hybridization: A Southwest Oncology Group Study (S9431)

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Continuing Education Opportunities READING LIST 74 – General Content Area: Role of Short Interfering RNA in Gene Silencing–2011 1. Highly Specific Gene Silencing by Artificial miRNAs in Rice.

2. Gene silencing by RNAi in mouse Sertoli cells.

3. Retrovirus-delivered siRNA.

READING LIST 75 – General Content Area: Multiple Myeloma: Molecular Markers and Tests–2010 1. Multiple Myeloma: Lusting for NF-B 2. Functional Interaction of Plasmacytoid Dendritic Cells with Multiple Myeloma Cells: A Therapeutic Target 3. High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients

READING LIST 76 – General Content Area: Colorectal Cancer and Loss of Imprinting of IGF2–2010 1. Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer 2. Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene.

3. Epigenetics at the Epicenter of Modern Medicine

READING LIST 77 – General Content Area: Health Effects Associated with Disruption of Circadian Rhythms–2011 1. Circadian Polymorphisms associated with Affective Disorders 2. A new approach to understanding the impact of Circadian Disruption on Human Health 3. Circadian Rhythm and its Role in Malignancy

READING LIST 78 – General Content Area: Role of Hedgehog Signaling Pathway in Diffuse Large BCell Lymphoma–2010 1. Sonic hedgehog signaling proteins and ATP-bindig cassette G2 are aberrantly expressed in diffuse large B-cell lymphoma 2. Sonic Hedgehog Signaling Pathway is Activated in ALK-Positive Anaplastic Large Cell Lymphoma 3. Sonic Hedgehog is Produced by Follicular Dendritic Cells and Protects Germinal Center B Cells from Apoptosis

READING LIST 79 – General Content Area: Whole Genome Amplification & 1986 Chernobyl, Ukraine Nuclear Power Plant Accident–2010 1. BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer.

2. Whole Genome Amplification

Technologies - Eliminating the Concern Over Running Out of DNA Samples Mid Experiment.

3. A break-apart fluorescence in situ hybridization assay for detecting RET translocation in papillary thyroid carcinoma.

READING LIST 80 – General Content Area: Expression of miRNA in Diffuse Large B-Cell Lymphoma–2010 1. Differentiation stage specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas.

2. Coordinated Expression of MicroRNA-155 and Predicted Target Genes in Diffuse Large B-cell Lymphoma.

3. Specific expression of miR-17-5p and miR-127 in testicular and central nervous system diffuse large B-cell lymphoma.

READING LIST 81 – General Content Area: The Genetics of Bipolar Disorder–2010 1. Gene-wide analyses of genomewide association data sets: evidence for multiple common risk alleles for schizophrenia and bipolar disorder and for overlap in genetic risk 2. Subcortical Gray Matter Volume Abnormalities in Healthy Bipolar Offspring: Potential Neuroanatomical Risk Marker for Bipolar Disorder?

3. Genetic and Environmental Influences on Pro-Inflammatory Monocytes in Bipolar Disorder

READING LIST 82 – General Content Area: Role and Detection of Human Endogenous Retroviruses in Rheumatoid Arthritis–2011 1. Increase in Human Endogenous Retrovirus HERV-K(HML-2) Viral Load in Active Rheumatoid Arthritis.

2. A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis 3. Lack of Detection of Human Retrovirus-5 Proviral DNA in Synovial Tissue and Blood Specimens From Individuals With Rheumatoid Arthritis or Osteoarthritis.

READING LIST 83 – General Content Area: Roles of Oncogenes in Breast Cancer–2010 1. The Nuclear Receptor Coactivator Amplified in Breast Cancer-1 Is Required for Neu (ErbB2/HER2) Activation, Signaling, and Mammary Tumorigenesis in Mice.

2. Dysregulated miR-183 inhibits migration in breast cancer cells.

3. Current and emerging biomarkers in breast cancer: prognosis and prediction

READING LIST 84 – General Content Area: Elevated Levels of Human Endogenous Retrovirus-W in Patients With First Episode of Schizophrenia–2010 1. Elevated Levels of Human Endogenous Retrovirus-W Transcripts in Blood Cells From Patients With First Episode Schizophrenia.

2. Endogenous Retrovirus Type W GAG and Envelope Protein Antigenemia in Serum of Schizophrenic Patients.

3. Reduced Expression of Human Endogenous Retrovirus (HERV)– W GAG Protein in the Cingulate Gyrus and Hippocampus in Schizophrenia, Bipolar Disorder, and Depression.

READING LIST 85 – General Content Area: Esophageal Cancer–2010 1. The Changing Face of Esophageal Cancer 2. Epidermal Growth FactorInduced Esophageal Cancer Cell Proliferation Requires Transactivation of-Adrenoceptors 3. Esophageal cancer risk by type of alcohol drinking and smoking: a casecontrol study in Spain

READING LIST 86 – General Content Area: p53 Family and Its Role In Cancer–2010 1. Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence.

2. Shaping genetic alterations in human cancer: the p53 mutation paradigm.

3. p53 polymorphisms: cancer implications.

READING LIST 87 – General Content Area: Proteins Involved with Chronic Myleloid Leukemia and Other Myleoprolifertive Disorders–2011 1. Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders.

2. Kinase domain mutants of Bcr enhance Bcr-Abl oncogenic effects.

3. Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML).

READING LIST 88 – General Content Area: DNA Topology–2010 1. The why and how of DNA unlinking.

2. Bacterial DNA topology and infectious disease.

3. DNA topoisomerase II and its growing repertoire of biological functions.

READING LIST 89 – General Content Area: LPL Waldenstrom Macroglobulinemia–2010 1. Spontaneous splenic rupture in Waldenstrom's macroglobulinemia.

2. How I Treat Waldenstrom's Macroglobulinemia.

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Continuing Education Opportunities 3. International prognostic scoring system for Waldenström Macroglobulinemia.

READING LIST 90 – General Content Area: Next Generation Sequencing Platforms–2010

of highthroughput sequence data.

3. Next-Generation Sequencing: From Basic Research to Diagnostics.

READING LIST 100 – General Content Area: Early onset of autosomal dominant Alzheimer disease–2011

READING LIST 95 – General Content Area: Cell Death–2011

1. Genetics of Alzheimer Disease.

2. New mutation in the PSEN1 (E120G) gene associated with early onset Alzheimer’s disease.

3. Evidence for Three Loci Modifying Ageat-Onset of Alzheimer’s Disease in EarlyOnset PSEN2 Families.

1. Rapid whole-genome mutational profiling using next-generation sequencing technologies.

2. Combining Next-Generation Sequencing Strategies for Rapid Molecular Resource Development from an Invasive Aphid Species.

3. Evaluation of next generation sequencing platforms for population targeted sequencing studies.

1. Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3.

2. Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxiainduced cell death.

3. Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains.

READING LIST 91 – General Content Area: Hutchinson-Gilford Progeria Syndrome–2011

READING LIST 96 – General Content Area: Genetic Associations of Cerebral Palsy– 2011

1. Epidermal expression of the truncated prelamin a causing Hutchinson– Gilford progeria syndrome: effects on keratinocytes, hair and skin 2. Defective Lamin A-Rb Signaling in Hutchinson-Gilford Progeria Syndrome and Reversal by Farnesyltransferase Inhibition 3. Increased expression of the Hutchinson– Gilford progeria syndrome truncated lamin a transcript during cell aging.

1. Mannose-binding lectin haplotypes may be associated with cerebral palsy only after perinatal viral exposure.

2. Methylenetetrahydrofolate Reductase Gene Polymorphisms and Cerebral Palsy in Chinese Infants.

3. Apolipoprotein E genotype and cerebral palsy.

READING LIST 92 – General Content Area: Severe Combined Immunodeficiency Screening and Patient Studies–2011 1. Long-term Outcome after Hematopoietic Stem Cell Transplantation of a Singlecenter Cohort of 90 Patients with Severe Combined Immunodeficiency.

2. Why Newborn Screening for Severe Combined Immunodeficiency Is Essential: A Case Report.

3. Development of a Routine Newborn Screening Protocol for Severe Combined Immunodeficiency.

READING LIST 93 – General Content Area: Biological and Physical Hazards Encountered in the Laboratory–2011 1. Lab Safety Matters.

2. Virus Transfer from Personal Protective Equipment to Healthcare Employees’ Skin and Clothing. Emerging Infectious Diseases.

3. Prevalence of Hepatitis C Virus Infection Among Health-Care Workers: A 10-Year Survey.

READING LIST 94 – General Content Area: Rapid whole-genome mutational profiling using nextgeneration sequencing technologies–2011 1. Comparison of next generation sequencing technologies for transcriptome characterization.

2. ShortRead: a bioconductor package for input, quality assessment and exploration

READING LIST 97 – General Content Area: Treatments for HIV/AIDs–2011 1. Early Antiretroviral Therapy Reduces AIDS Progression/Death in Individuals with Acute Opportunistic Infections: A Multicenter Randomized Strategy Trial.

2. Asia can afford universal access for aids prevention and treatment.

3. Trends in reported aids defining illnesses (adis) among participants in a universal antiretroviral therapy program: an observational study.

READING LIST 98 – General Content Area: Myosin Light Chain Kinase (MYLK) Gene Mutation Affect in Smooth Muscle Cells– 2012 1. Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice.

2. Mutation in myosin light chain kinase cause familial aortic dissections.

3. Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

READING LIST 99 – General Content Area: Chromosome 6 and Its Associated Diseases–2011 1. Novel Cleft Susceptibility Genes in Chromosome 6q.

2. A susceptibility locus on chromosome 6q greatly increases risk lung cancer risk among light and never smokers.

3. The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer.

READING LIST 101 – General Content Area: Multiplex PCR and Emerging Technologies for the Detection of Respiratory Pathogens–2011 1. A multiplex one-step real-time RT-PCR assay for influenza surveillance.

2. Taking New Tack, PrimeraDx Offers MDx Tech as Open Platform for Test Developers.

3. Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children.

READING LIST 102 – General Content Area: Single Nucleotide Polymorphism (SNP) Array Analysis–2011 1. A fast and accurate method to detect allelic genomic imbalances underlying mosaic rearrangements using SNP array data.

2. SAQC: SNP array quality control.

3. Calibrating the performance of SNP arrays for whole-genome association studies.

READING LIST 103 – General Content Area: Research of BRAF Gene Related to Cancer–2011 1. Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF. 2. Distinct patterns of DNA copy number alterations associate with BRAF mutations in melanomas and melanoma derived cell lines.

3. Pharmacodynamic Characterization of the Efficacy Signals Due to Selective BRAF Inhibition with PLX4032 in Malignant Melanoma.

READING LIST 104 – General Content Area: Microarray Single Nucleotide Polymorphism (SNP) Troubleshooting–2011 1. Model-based clustering of array CGH data.

2. Application of a target array comparative genomic hybridization to prenatal diagnosis.

3. A model-based circular binary segmentation algorithm for the analysis of array CGH data.

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Continuing Education Opportunities READING LIST 105 – General Content Area: Inflammasome Activation by Proteins–2011 1. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1 2 in type 2 diabetes.

2. ER stress in Alzheimer’s disease: A novel neuronal trigger for inflammation and Alzheimer’s pathology.

3. The inflammasome: a caspase-1activation platform that regulates immune responses and disease pathogenesis.

READING LIST 106 – General Content Area: DNA Barcoding–2011 1. Commercial Teas Highlight Plant DNA Barcode Identification Successes and Obstacles.

2. Mutational Patterns and DNA Barcode for Diagnosing Chikungunya Virus.

3. The Barcode of Life Data Portal: Bridging the Biodiversity Informatics Divide for DNA Barcoding.

READING LIST 107 – General Content Area: HERV-K and Its Correlation With Melanoma Cells–2011 1. Expression of human endogenous retrovirus K in melanomas and melanoma cell lines Cancer.

2. Expression of HERV-K correlates with status of MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells cancer.

3. An endogenous retrovirus derived from human melanoma cells.

READING LIST 108 – General Content Area: Refractory Myeloma–2011 1. Pomalidomide plus low-dose dexamethasone in myeloma refractory to both bortezomib and lenalidomide: comparison of 2 dosing strategies in dual-refractory disease.

2. Relapse/Refractory Myeloma Patient: Potential Treatment Guidelines.

3. Emerging role of carfilzomib in treatment of relapsed and refractory lymphoid neoplasms and multiple myeloma.

READING LIST 109 – General Content Area: Short Tandem Repeat (STR) Technology in Forensic Community–2011 1. An integrated microdevice for highperformance short tandem repeat genotyping.

2. A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

3. Generating STR profile from "Touch DNA".

READING LIST 110 – General Content Area: Methods of Screening and Evaluation of Hepatitis C Virus–2011

3. CD5-negative Blastoid Variant Mantle Cell Lymphoma with Complex CCND1/ IGH and MYC Aberrations.

1. Hepatitis c virus: prevention, screening, and interpretation of assays.

2. Serial follow-up of repeat voluntary blood donors reactive for anti-hcv elisa.

3. Comparison of fibrotest-actitest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis b and c.

READING LIST 115 – General Content Area: Cystic Fibrosis - 2014

READING LIST 111 – General Content Area: Pharmacogenomics–2011 1. Pharmacogenomic testing: Relevance in medical practice: Why drugs work in some patients but not in others.

2. Clinical assessment incorporating a personal genome.

3. Genomics and drug response.

READING LIST 112 – General Content Area: Adrenoleukodystrophy–2011 1. Novel exon nucleotide deletion causes adrenoleukodystrophy in a Brazilian family.

2. X-linked adrenoleukodystrophy: ABCD1 de novo mutations and mosaicism.

3. Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with Xlinked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes.

1. Rapid Detection of the ACMG/ACOGRecommended 23 CFTR DiseaseCausing Mutations Using Ion Torrent Semiconductor Sequencing 2. Long-Term Evaluation of Genetic Counseling Following False-Positive Newborn Screen for Cystic Fibrosis 3. Rapid Transport of Muco-Inert Nanoparticles in Cystic Fibrosis Sputum Treated with N-acetyl cysteine

READING LIST 116 – General Content Area: Autism - 2015 1. Intellectual disability and autism spectrum disorders: Causal genes and molecular mechanisms.

2. Aberrant tryptophan metabolism: the unifying biochemical basis for autism spectrum disorders?

3. Decreased tryptophan metabolism in patients with autism spectrum disorders

READING LIST 113 – General Content Area: Quality Assurance and Quality Control of Microarray Comparative Genomic Hybridization–2011 1. Customized oligonucleotide array-based comparative genomic hybridization as a clinical assay for genomic profiling of chronic lymphocytic leukemia.

2. Comparison of familial and sporadic chronic lymphocytic leukaemia using high resolution array comparative genomic hybridization.

3. Microarray-based comparative genomic hybridization.

READING LIST 114 – General Content Area: mFISH–2012 1. Human interphase chromosomes: a review of available molecular cytogenetic technologies.

2. Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization.

Copyright law prohibits AGT from supplying readers with the actual journal articles (electronically or otherwise). Availability of articles online does not imply the service is free.

Some journals require a subscription or impose a fee. The web addresses are included for the convenience of those wishing to obtain the articles in this way.

The Journal of the Association of Genetic Technologists 42 (2) 2016

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Continuing Education Opportunities

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The Journal of the Association of Genetic Technologists 41 (4) 2015

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Association Business

AGT, The Organization for Cytogenetic & Molecular Professionals AGT, originally founded in 1975 as the Association of Cytogenetic Technologists, serves to: • promote the scientific and professional development of all areas of genetics; • foster the exchange of information between those interested in genetics; • encourage cooperation between those persons actively or formerly engaged in genetics; and • stimulate interest in genetics as a career.

AGT has approximately 1,000 members. Membership is open to all who are employed or interested in genetics. All regular members are entitled to hold office, vote in elections, attend all AGT meetings, and receive The Journal of the Association of Genetic Technologists and access the AGT International Online Membership Directory.

Board of Directors Officers President Patricia K. Dowling, PhD Pathline Labs 535 E. Crescent Ave. Ramsey, NJ 07446 PDowling@pathlinelabs.com President-Elect Jason A. Yuhas, BS, CG(ASCP)CM Mayo Clinic Division of Laboratory Genetics Cytogenetics Lab 200 First St. SW Rochester, MN 55905 507-538-7634 yuhas.jason@mayo.edu Secretary-Treasurer Denise Juroske Short, MSFS, MB(ASCP)CM 219 Timberland Trail Lane.

Lake City, TN 37769 dmj4565@gmail.com Public Relations Director Ephrem Chin MBA, MB(ASCP)CM, QLC Oxford Gene Technology 520 White Plains Road, Suite 500 Tarrytown, NY 10591 404-579-9995 nzelman@gmail.com Education Director Sally J. Kochmar, MS, CG(ASCP)CM Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu

Annual Meeting Director Adam H. Sbeiti, MT(ASCP)CMCGCMD LMCM Quest Diagnostics-Nichols Institute 33608 Ortega Highway San Juan Capistrano, CA 92690 949-728-4376 adam.h.sbeiti@questdiagnostics.com Annual Meeting Co-Director Jennifer N. Sanmann, PhD UNMC Human Genetics Laboratory 985440 NE Med. Center Omaha, NE 68198-5440 402-559-3145 jsanmann@unmc.edu

Representative to NAACLS Term: 9/12 – 9/16 Peter C. Hu, PhD, MS, MLS(ASCP)CM CGCM, MBCM University of Texas M.D. Anderson Cancer Center School of Health Sciences 1515 Holcomb Blvd., Box 2 Houston, TX 77030 713-563-3095 pchu@mdanderson.org Representative to Foundation for Genetic Technology Term: 7/10 – 6/16 Patricia LeMay, MT(ASCP), CG(ASCP)CM Monmouth Medical Center Department of Pathology 300 Second Ave. Long Branch, NJ 07740 732-923-7369 plemay1945@aol.com

Council of Representatives Representative to CCCLW Term: 7/14 – 6/20 Hilary E. Blair, BS, MS, CG(ASCP)CM Mayo Clinic 200 First St. SW Rochester, MN 55905 507-255-4385 blair.hilary@mayo.edu

Representative to CAP/ACMG Term: 1/16 – 12/120 Jun Gu, MD, PhD, CG(ASCP)CM University of Texas MD Anderson Cancer Center School of Health Professions Cytogenetic Technology Program 1515 Holcombe Boulevard, Unit 2 Houston, TX 77030 (713) 563-3094 jungu@mdanderson.org

Representatives to BOC Term: 10/12 – 9/15 Helen Bixenman, MBA/HCM, CHC, CG(ASCP)CMDLMCM, QLC 4095 W. Shannon St. Chandler, AZ 85226-2195 helen.bixenman@gmail.com

Publications

Term: 10/11 – 9/16 Amy R. Groszbach, MEd, MLT(ASCP)CM MBCM Mayo Clinic Molecular Genetics Laboratory – Hilton 920 200 First St. SW Rochester, MN 55905 507-284-1229 groszbach.amy@mayo.edu

AGT Journal Editor Mark D. Terry 1264 Keble Lane Oxford, MI 48371 248-628-3025 markterry@charter.net

Visit AGT’s Website at www.AGT-info.org The Journal of the Association of Genetic Technologists 42 (2) 2016

89

Other Contacts Liaison to ASCLS Governmental Affairs Committee Kathryn Sudduth, BA, CG(ASCP)CMDLMCM 2713 Brookmere Road Charlottesville, VA 22901 434-973-0690 kas3m2@embarqmail.com FGT Board of Trustees President Robin A. Vandergon, CG(ASCP)CMDLMCM 8767 E. Los Altos Ave. Clovis, CA 93619 559-392-0512 rrink@quixnet.net

Executive Office P.O. Box 19193 Lenexa, KS 66285 Overnight Mail Address: 18000 W. 105th St. Olathe, KS 66061 913-895-4605 913-895-4652 FAX AGT-info@goAMP.com www.AGT-info.org

Staff Contacts: Monica Evans-Lombe, Executive Director 913-895-4781 mevanslombe@kellencompany.com Christie Ross, Education Program Director 913-895-4776 cbross@kellencompany.com Diane Conner, Administrative Assistant 913-895-4801 dnorthup@kellencompany.com

Association Business

Letter from the President Greetings fellow genetics professionals!!

I just got back from a vacation in Paradise—Puerto Plata, Dominican Republic. I go there at least twice a year, but this time was special. The group I traveled with made a visit (one we’ve made before) to Casa Nazaret, Nazareth House, an orphanage for unadoptable children and adolescents. They are considered unadoptable because of the severe disabilities that most of them have, ranging from cerebral palsy and other defects of the birth process to intellectual disabilities to obvious genetic abnormalities, like Down syndrome, and to other severe dysmorphisms that, as a geneticist, aroused my suspicion of some sort of chromosome disorder.

This year we brought a new wheelchair for a seven-year-old girl with cerebral palsy, as well as lots of other stuff. As we touched and played and fed and communicated as best we could with these beautiful children I was reminded of how fragile life is, but how even with disabilities it is still possible to feel joyful and loved. We saw that and more. I was also struck by how lucky I was to have the knowledge of genetics, the essence of our being, that I have, and how important it is to keep up with my “lifetime of learning.”

The field of genetics is expanding very quickly, but there is one thing that is very obvious, input from all techniques is frequently necessary to paint the complete picture of a genetic malady. A DNA-based test can determine that a mutation of some kind exists, but cytogenetics can perhaps define the mechanism of how the mutation occurred.

I am lucky to have knowledge of both cytogenetics and molecular genetics. I encourage my cytogenetics colleagues to learn more molecular, and my molecular colleagues to learn more cytogenetics. It’s fun!! I also encourage everyone to learn more biochemical genetics—what a trip!! AGT is the only association dedicated to the professional and technical needs of technologists in molecular genetics, biochemical genetics and cytogenetics.

In my opinion, the technologists are the ones who know more about how to do genetics than any director (unless you are a director, like me, who started as a technologist and whose heart is still at the bench…). So, fellow genetics professionals, what is keeping you from joining or renewing your membership in AGT? We did a survey (thanks to those who participated!) and some folks just forgot, imagine that! It’s never too late, dear colleagues!

Some folks have left the field due to retirement or loss of position. I guess I understand this, but, then, there is the “lifetime of learning.” Some folks think that $95 per year is too expensive since most institutions don’t support their employees’ professional organization dues. I say whose career is it? Yours or your institution’s?

Some folks say WIFM. I say if you have to ask this, then perhaps you are not a professional. Never mind. Go back to what you were doing. If anything I have said here strikes a chord with you, please consider joining or renewing your membership to AGT. We have listened to your concerns and are working hard to improve your AGT experience. See you in Anaheim (or nice to have seen you in Anaheim!!).

Pat Dowling, AGT President

The Journal of the Association of Genetic Technologists 42 (2) 2016

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Student Members. Student membership shall be available to persons who are pursuing a full or part-time course of study at an educational institution or school and who are interested in pursuing a career in the field of genetics.

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Mail application form and appropriate fee for membership in correct U.S currency. Money order or check in U.S. funds drawn on a U.S. bank only. CHECKS DRAWN ON INTERNATIONAL BANKS WILL NOT BE ACCEPTED. Make checks payable to Association of Genetic Technologists. For your convenience, you may pay by credit card. Applications received after September 15 are applied toward the next membership year. NOTE: Membership expires on December 31 of each year.

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ISCN (2009): A Reference Guide Select method of delivery:

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Lab Manual is only available on a flash drive. The 4th Edition is scheduled to be released in 2016.]

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How does AGT membership benefit your organization?

GROW YOUR ORGANIZATION As a member, you’ll access the resources and tools you need to help innovate and inspire your organization. AGT also promotes the interests of geneticists and genetic technologists on a National scale, with representation on organizations such as NAACLS, BOC, CCCLW, CAP/ACMG and others.

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BE PREPARED All organizations look different, but the challenges they face are often the same.

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THREE EASY WAYS TO JOIN: Online: http://www.agtinfo.org/Pages/joinagt.aspx Mail: Return the application to the address listed With an Event: Sign up for membership at the same time you register for one of our events. It’s that easy!

ISSN 1523-7834