MyHVP Newsletter | Vol. 5 | Issue: 1 | June 2020

MyHVP Newsletter Volume 05 | Issue 01 | Jan-June 2020

GLOBAL GLOBIN 2020 CHALLENGE MEETING AND CONFERENCE 2019

“Enhancing Partnership in Genomic Capacity and Equitable Healthcare” 28th – 30th October 2019 The Headquarters of UNESCO, Paris, France

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he Global Globin 2020 Challenge (GG2020) is a project under the Human Variome Project (HVP) initiative aimed at a global collection, consolidation and compilation of genomic data, clinical differences and individual phenotypes of diseases from all ethnic groups around the world and allow this information for open access to all (www.humanvariomeproject.org).

For the year 2019, the GG2020 conference held at the UNESCO headquarters in Paris was co-organised with Professor Dr. Jacques Elion from the Paris Diderot University Medical School.

The conference was hosted by Malaysian Ministry of Education comprising committee members from Universiti Sains Malaysia, Malaysian Node of the Human Variome Project (MyHVP), Universiti Kebangsaan Malaysia, International Islamic University Malaysia, Ministry of Health Malaysia, and The Cyprus Institute of Neurology and Genetics (CING).

plenary lectures and eight symposiums. There were also 19 posters and nine free oral papers were registered for presentations. In addition, three pre-conference meetings held namely HVP Board of Director Meeting, ClinGen Panel Expert Meeting and GG2020 Closed-door Meeting. A key outcome of the closed-door meeting was the rebranding of GG2020 as Global Globin Network.

The next meeting and conference for 2020 will be held in Cairo, Egypt to be chaired by Professor Dr Ghada el-Kamah from Human Genetics and Genome Research Division, National Research Centre, Egypt. Professor Dr Zilfalil Alwi from Malaysian Node Human Variome Project, was appointed as the co-chairman. However, due to Covid-19 outbreak that has affected worldwide in this year, the Global Globin Network (GGN) 2020 is postponed into 2021.

The conference was officiated by GG2020 advisor, Dr Hamidah Mat. The conference attracted 80 participants. The scientific programme presented one workshop, one keynote lecture, four Contact us: | Secretariat Office: Human Variome Project Malaysian Node & South-east Asian Node School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kota Bharu, Kelantan, Malaysia | Phone :(60) 097676543 / 6531 | Fax: (60) 097676543 | Email : myhvp@usm.my | Website: hvpmalaysia.kk.usm.my

MyHVP Newsletter

Head’s Address

Board of Editors 2020

ISSN: 2550-1747 | Volume 05 | Issue 01| Year 2020

Editor in Chief Professor Dr. Zilfalil Alwi

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Managing Editor Prof. Dr. Wan Zaidah Abdullah

Editorial Board Members

Prof. Ida Madieha binti Azmi Assoc. Prof. Dr. Muhammad Farid Johan Assoc. Prof. Dr. Endom Ismail Assoc. Prof. Dr Rosnah Bahar Dr. Nik Norliza Bt Nik Hassan Dr. Azlina bt Ahmad Annuar Mr. Abdul Halim Fikri Bin Hashim

English Editor Amyzar Alwi

Contents

2 Head’s Address 6-7 Postgraduate 3 Report 8 Photo Diary

4-5 When a Geneticist Writes

© 2020. All rights reserved. The information in this newsletter is provided by the Malaysian Node of the Human Variome Project (MyHVP) members including South-east Asian Node (HVPSEA Node) for educational / information purpose only. It is not a substitute for professional medical care and medical advice.

The contents express the opinions of the authors who alone are responsible for their view expressed. MyHVP does not accept any legal responsibility for their contents.

Writers, Invited!

lobal Globin Challenge 2020 (GG2020) is one of two global initiatives of the Human Variome Project (HVP). The other initiative is the BRCA challenge. This issue highlights the Global Globin 2020 Conference that was held at the UNESCO headquarters on 28-30 October 2019. A total of 80 medical practitioners, researchers, scientists, haematologists, and experts in thalassaemia and sickle cell disease participated in the 3-day meeting. The theme of the conference is “Enhancing Partnership in Genomic Capacity and Equitable Healthcare”.

A total of 46 lectures were delivered, and 9 presentation of papers including 19 posters. Six awards were given for the best oral and poster presenters. The keynote lecture entitled “Gene Therapy of the Beta-haemoglobinopathies: Success and Challenges” was given by Dr. Emmanuel Payen on behalf of Prof. Philippe Leboulch, a world-renowned expert on gene therapy for thalassaemia.

GG2020 also successfully published its conference proceedings in the Hemoglobin Journal. This publication marks an important milestone for HVP and Global Globin. All the lectures and papers presented at the conference can be accessed in the supplementary issue of Hemoglobin, Volume 43, 2019 (https://www.tandfonline.com/toc/ihem20/43/6).

The board of editors of MyHVP newsletter would like to acknowledge all the contributions made by its managing editor, Prof. Dr. Wan Zaidah Wan Abdullah who officially retired from Universiti Sains Malaysia (USM) on 1 May 2020, after an illustrious 19 years of service to the university. We wish her the best in her future undertaking and hope that she will continue to contribute to the progress of this newsletter and MyHVP. As usual, we welcome articles or news from members of MyHVP. Do email us your articles or news related to genetics or genomic at myhvp@usm.my for publication in this newsletter.

Thank you.

Prof. Dr. Zilfalil Alwi Head, Malaysian Node of the Human Variome Project (MyHVP)

MyHVP Newsletter is issued biannually. For the coming issue, the public from various fields and specialties are invited to their lived experience in dealing with the issues of medicine or biomedicine. Writers can contribute their writing based on these following criteria: 1. Length (Max. 1page A4 size, and it may be edited for our use) 2. The committee has the right to share your writing for further issues.

3. To suit the needs of the publication, and your writing won’t be returned 4. Emailed the article to myhvp@usm.my

Published by

Malaysian Node of the Human Variome Project (MyHVP) School of Medical Sciences Universiti Sains Malaysia Health Campus 16150, Kubang Kerian, Kelantan, Malaysia Tel: +6097676531 /6543 | Email: myhvp@usm.my

MyHVP Newsletter | Jan-June 2020| page 2

Report WORLD DOWN SYNDROME DAY 2020 Reported by: Prof Zilfalil Bin Alwi, Dr Mohd Zulkifli Abdul Rahim, Pn Wan Noriah Wan Ramli, Dr Nur Suhaila Idris, Pn Rosnani Zakaria For Persatuan Sindrom Down Negeri Kelantan (PSDNK)

W

orld Down Syndrome Day is observed on the 21st of March every year to signify the underlying abnormality in Down syndrome in which there is the presence of a third (an extra) chromosome on chromosome 21.

World Down Syndrome Day is a global awareness day, observed to raise public awareness of Down syndrome. Various organizations, as well as civil society, including non-governmental organizations (NGO) and private sectors all over the world, observe World Down Syndrome Day through continuous life skills programs.

Activities and events are organised to help raise aware- ness of what it means to have Down syndrome, and how people with Down syndrome play a vital role in our lives and communities.

Individuals with Down syndrome have equal rights to adequate access to health care, early intervention programmes, and to inclusive education, as well as appropriate research vital to the growth and development of the individual. Therefore it is important that awareness and initiatives are put in place to help people with Down syndrome live life to their fullest potential.

The theme for this year World Down Syndrome Day is “We De- cide” based on the principle that people with Down syndrome should have full participation in decision-making about matters affecting their lives. This is in line with the United Nation Convention on the Rights of Persons with Disabilities (CRPD), which state that full, effective and meaningful participation is a core human rights principle.

Events are organised to show how effective and meaningful participation of persons with Down syndrome can be achieved by providing good support, effective communication, acces- sible information and inclusive consultation. Empowerment given to persons with Down syndrome and to those sup- porting them, including their representative associations will lead them to a more effective and meaningful participation.

More effort should be made to reach out to key stakeholders, including educators, employers, healthcare professionals, media, NGOs and government agencies so that the message can be effectively disseminated and bring about meaningful change.

tails and what it means for their child. Without support, they would struggle to accept their child and may not be able to provide the loving and secure environment that the child needs.

As PSDNK grew in size and strength, more activities are organ- ised for parents and their children at different locations in Kelan- tan. Besides the usual family support group activities, social and independent living skills and vocational skills activities are also organised. These weekly activities provide parents a chance to listen to the lived experiences of people with Down syndrome. Through these social skills activities, the children are provided opportunities to develop their communication skills, self-confidence and self-regulation (such as queuing and taking turns). These social skills may also involve the ability and skill to use public transport, using currency as well as shopping.

For vocational training, PSDNK is working closely with Universiti Sains Malaysia (USM) Disability Transformation Unit (DTU) in the supported employment training programme where children with Down syndrome will get the opportunity to be trained with vocational skills that will prepare them for future employment.

PSDNK is also working closely with Kelantan state government and its agencies as well as several NGOs in the state, to plan for a vocational training center in agriculture aimed at providing future opportunity for employment in the field of agriculture.

One of the highlights of the social and living skill activities was a trip to Singapore organised last year by PSDNK for parents and their Down syndrome children. In Singapore, they were hosted by a representative of a local NGO who took them to various tourist spots and treated them to meals. PSDNK also met their counterpart, Singapore Down Syndrome As- sociation and exchanged ideas and shared their experience in managing their associations.

While the children thorough- ly enjoyed this valuable experience of visiting another coun- try and the social skills that was picked up during the trip, it was the return trip to Kelantan that became the highlight. The 29-hour train ride from Singapore to Wakaf Bahru, Kelan- tan offered the children the chance to interact with people of different backgrounds through use of appropriate social skills and behaviour, as well as learning the safety aspect of life within the confine of a moving train over a long period of time.

While there has been a lot of progress made for children with Down syndrome in our country, more can be done to reach out to and engage key stake- holders, increase awareness among the public and policy makers in the country and bring about positive change and provide empowerment to persons with Down syndrome.

Yes, together we can make the change and give them a better life.

“We Decide”!

In Malaysia, the Kelantan Down Syndrome Association or Persatuan Sindrom Down Negeri Kelantan (PSDNK) as it is more popularly known, is a parent support group that is active in organizing activities for children with Down Syndrome in the state of Kelantan.

Established in 2016 by a group of parents of children with Down syndrome, PSDNK currently has 73 members from various districts in Kelantan. It was established initially to provide parents with emotional support and accurate information about Down syndrome so that they are better prepared to support and care for their child. Often parents whose child is diagnosed with Down syndrome will initially feel anger, fear and sadness and often do not understand what Down syndrome enMyHVP Newsletter | Jan-June 2020 | page 3

When a Geneticist Writes DIAGNOSIS OF HAEMOGLOBINOPATHIES: FROM SCREENING TO CONFIRMATION OF GENETIC DEFECT Written by: Prof. Dr. Raja Zahratul Azma Raja Sabudin Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia

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aemoglobinopathies is one of the most common genetic disorders affecting worldwide. Haemoglobinopathies are broadly classified into thalassaemias (α, β, δβ) and abnormal structural variants. Few structural variants such as Haemoglobin (Hb) Lepore and HbE clinically resemble thalassaemic phenotype. Due to global migration, haemoglobinopathies has emerged as a health problem in developing countries including Malaysia. The prevalence of carriers of haemoglobin disorders in Malaysia is 4.5–11% [1-4]. As such, knowledge of the prevalence and heterogeneity of haemoglobinopathies in a target population is key to the selection of the most suitable laboratory methods to be utilised at screening centres.

In Malaysia, thalassaemia screening programmes were implemented in 2005 [4] through the initial approach of antenatal and cascade screening. More recently, a nationwide compulsory screening programme involving form four students in secondary schools in Malaysia was launched by the Ministry of Health [5].

The first line of laboratory screening approach for haemoglobinopathy is the full blood count measurement using fully automated haematology analyser which is highly sensitive for carriers. This screening approach provides measurements of red cell indices such as haemoglobin (Hb), mean corpuscular haemoglobin (MCH) value, mean corpuscular volume (MCV) value, red cell distribution width (RDW) and red cell count (RCC) are helpful in differentiating carriers from patients with underlying iron deficiency anaemia. Patients with haemoglobinopathies showed hypochromia (MCH<27pg) red cells associated with eryhtrocytosis (RBC>4.75x10^12/dl) [6], while iron deficiency anaemia patients tend to have hypochromia with lower RBC [7]. Morphology of red cells in carriers was more homogenous compared to those with iron deficiency [8].

Detection of Haemoglobin H (HbH) inclusion bodies by peripheral blood smear stain with methylene blue has been used to diagnose α-thalassaemia. However, the sensitivity of this test was often unsatisfactory and a study has shown that none of single or two α-gene deletions thalassaemia patients showed positive results in this test [9]. Only three and four gene deletion forms of α-thalassaemia will have positive results with HbH inclusion.

Hb analysis using High Performance Liquid Chromatography (HPLC) or Capillary Electrophoresis is the next method in line.

They are relatively expensive but have been successfully in detecting majority of β-thalassaemia, three and four gene deletion forms of α-thalassaemia; and some of structural variants such as Hb Constant Spring (HbCS). However, the test is not useful in the detection of α-thalassaemia carriers and most of these cases have been missed for years. Even though HbCS ‘peak’ may appear in Hb analysis, quite a number of heterozygotes cases have been missed using HPLC [10].

The accurate determination of gene abnormalities in haemoglobinopathies is very important. Both α and β-thalassaemias which show heterogenous genetic abnormalities and increasing number of cases now require advanced methods of molecular analysis for confirmation. The molecular defects of α and β- thalassaemia in the major ethnics in Malaysia have been established. The commonest causes of α-thalassaemia are the α-gene deletions (αα/--SEA, αα/-α3.7, α α /- α 4.2) and a non-deletional abnormality i.e a HbCS [HBA2: TAA>CAA] [3,11].

Multiplex polymerase chain reaction (PCR) (GAP and amplification refractory mutation system (ARMS)) allows rapid detection of deletion and non-deletion α-gene abnormalities respectively. However, the applicability requires the definition of the breakpoints limited to known and well defined genetic deletions and mutations. Thus less common but not less important deletional and non-deletional α-gene abnormalities, as well as undiscovered genetic abnormalities could be missed and these could be novel to the Malaysian population. Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a simple technique that is suitable for rapid and mass screening of gene deletions. MLPA has been applied succesfully in a number of genes in which deletions and duplications are common [12]. From our recent study, MLPA was able to detect all deletional gene abnormalities with 95% concordance rate with conventional multiplex ARMS [13]. However, even though MLPA showed 100% sensitivity and specificity in detecting HbCS, the only non-deletional α-thalassaemia available in MLPA method, it was unable to differentiate homozygous from heterozygous states of HbCS [13]. Multiplex ARMS is still the best method for deferentiating the zygosity of HbCS even thouh it requires an additional run for wild type. Another good molecular method available is real time PCR (Taqman@ SNP genotyping assays) where it easily differentiates homozygous from heterozygous states of HbCS, but this method is expensive and laborious [10,11]. Hb Adana [HBA2: c.179G>A] is another non-deletional alpha thalassaemia which is more frequently detected since the introduction of multiplex ARMS for non-deletional α-thalassaemia in molecular laboratories in Malaysia [11,14,15]. Of recent, a case of 1VS-I-1G>A [HBA2: c.95+1G>A] [16], a rare α2-gene mutation has been discovered with the advent of better molecular skills and techniques.

The estimated carrier rate for β-thalassaemia in Malaysia is 4.5% predominated by Malays and Chinese [17,18]. The common β-globin gene mutations affecting the Malays are Cd26 (G>A) HbE, IVS1-5 (G>C), IVS 1-1 (G>T), Cd 19 (A>G) Hb Malay and Cd 17 (A>T) while Chinese have Cd41/42 (-TCTT), IVS 2-654 (C>T), -28 (A>G), Cd17 (A>T) and Cd71/72 (+A) mutations [18].

Filipino β0-deletion (45kb deletion) is seen more commonly in indigenous population of Sabah and Sarawak in East Malaysia [19]. β-thalassaemia shows considerable phenotypic variations posing diagnostic difficulties with HPLC or CE. A considerable MyHVP Newsletter | Jan-June 2020| page 4

When a Geneticist Writes number of cases showed unequivocal Hb A2 levels and some cases of thalasaemia intermedia expressed Hb F levels a typical beta thalassaemia. These differences in phenotypic expressions may be accounted for by genetic modifiers such as the coexistence of alpha thalassaemia, a silent beta gene variant e.g mutation at -28 ATC, Hb Malay and a possible inter- action with delta beta thalasaemia gene e.g Hb Lepore [20]. It is important to characterize all beta thalassaemia cases at molecular level. Our recent experience in analysing molecular genetic of β-thalassaemia cases detected by Hb analysis, using multiplexes-PCR and flow-through hybridization (FTH) techniques showed both methods were able to detect gene abnormalities in around 95% of cases. FTH was designed using 25 probes while multiplex-PCR (ARMS and GAP) with 28 probes were able to detect most mutations and deletion types of beta gene abnormalities in our Malaysia population [21]. However, FTH was less laborious, rapid and useful in diagnostic centres with high workload. It is also the best method for determining the zygosity of beta-thalassaemia cases, in which it needs only one run for analysis. DNA sequencing is another good method for the diagnosis of thalassaemia but it is a laborious method and deletional type of thalassaemia might be missed [20].

and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-depen ent probe amplification. Journal of medical genetics. 2005; 42(12), 922–31.

13. Farah-Azima AM, Maizatul-Husna, Azma RZ, Hafiza A, Azlin I, Zarina AL, Hamidah A, Noor-Farisah AR, Shuhaila A, Ainoon O. The use of multiplex ligation-dependent probe amplification (MLPA) assay in detecting alpha thalassaemia gene abnormalities: Comparison with multiplex PCR. Abstract of the 14th Annual Scientific Meeting, Malaysian Society of Haematology, 2017.

14. Ezalia Esa, Tan Jen Ern, Rahimah Ahmad, Nur Aisyah Aziz, Zubaidah Zakaria and Azlinda Abu Bakar (2014). A rare case of compound heterezygous haemaglobin Q-Thailand and haemoglobin Adana. International Journal of Health Sciences and Research 4(10): 327-332.

15. Hafiza A, Noor Adilah J, Azma RZ, Azlin I, Farisah AR et al. A case series of H-inclusion negative α-thalassaemia intermedia due to compound heterozygosity of Haemoglobin Adana (HBA2: c179G>A p.Gly60Asp) with other α-thalassaemias in Malay families. Hemoglobin. 2014. 2014; 38(4): 277 – 281.

16. Hafiza A, Tang YL, Azma RZ, Azlin I, Loh CK et al. A severe α-thalassaemia due to compound heterozygosity for rare Codon 59 (GGC>GAC) with IVS I nt I (G/A) mutations in α2-gene. Abstract of the International Conference on Medical and Health Sciences (ICMHS), 2013.

17. Hassan S, Ahmad R, Zakaria Z, Zulkafli Z & Abdullah WZ. Detection of β-Globin Gene Mutations among β-Thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System-Polymer-

In conclusion, definitive molecular diagnosis of inherited genetic disorders is crucial for optimum management, genetic counseling, and prevention.

ase Chain Reaction. Malaysian Journal of Medical Sciences. 2013; 20(1), 13–20.

18. George E, & Tan JA MA. Genotype-Phenotype Diversity of β-Thalassaemia in Malaysia: Treatment Options and Emerging Therapies. Med J Malaysia. 2010; 65(4), 256-260.

19. Teh LK, George E, Lai MI, Tan JAMA, Wong L et al. Molecular Basis of Transfusion Dependent Beta-Thalassaemia Major Patients in Sabah. Journal of Human Genetics, 2014; 59(3), 119–23.

20. Hafiza A, Azma RZ, Madzlifah A, Suziana MN, Azlin I et al. Co-inheritance of compound heterozygous Hb Lepore and -thalassaemia with single gene deletion -thalassaemia (–α3.7 type): A case report. Malaysian J Pathol. 2015; 37(3): 275-292 21. Norunaluwar J, Azma RZ, Hafiza A, Azlin I, Khairiliah AK et al. Detection of beta thalassaemia alleles – multiplex amplification refractory mutation system versus flow-through hybridization kit. Abstract of the XIIIth Malaysian National Haematology Scientific Meeting, 2016.

References 1. Wee YC, Tan KL, Chow TWP, Yap SF, and Tan MAJA. Heterogeneity in α-thalassaemia interactions in Malay, Chinese and Indian in Malaysia. Journal of Obstetrics and Gynaecology Research. 2005; 31(6): 540–546.

2. Azma RZ, Ainoon O, Azlin I, Hamenuddin H, Hadi NA et al. Prevalence of iron deficiency anaemia and thalassaemia trait among undergraduate medical students. Clin Ter. 2012; 163(4): 287- 291.

3. Rahimah AN, Nisha S, Safiah B, Roshida H, Punithawathy Y et al. Distribution of alpha thalassaemia in 16-year-old Malaysian students in Penang, Melaka and Sabah. Med J Malaysia, 2012; 67(6): 565-570.

4. Ezalia E, Irmi Elfina R, Elizabeth G, Wan Hayati MY, Norhanim A et al. Thalassaemia Screening among Healthy Blood Donors in Hospital Tengku Ampuan Rahimah, Klang Med & Health 2014; 9(1): 44-52.

5. Daily Express, Independent National Newspaper of East Malaysia. Thumbs up for free thalassaemia screening. 2016.

6. Tripathi, N., Soni, J.P , Sharma, P.K , Verma, M. Role of Haemogram Parameters and RBC Indices in Screening and Diagnosis of Beta-Thalassaemia Trait in Microcytic, Hypochromic Indian Children. 2015; International Journal of Hematological Disorders 2(2): 43-46.

7. Soliman AR, Kamal G, Elsalakawy A, Mohamed TH. Blood indices to differentiate between Beta-Thalassaemia trait and iron deficiency anaemia in adult healthy Egyptian blood donors. 2014; Egypt J Haematol 39: 91-92.

8. Bain Bain BJ. 2006. The , ,  and  thalassaemias and related conditions. Haemoglobinopathy diagnosis, Second Edition: 63 – 127.

9. Wang C, Beganyl L, Fernandes BJ. Measurements of red cell parameters in alpha thalassaemia trait: Correlation with the genotype. Lab Hematol. 2000; 6: 163-166.

10. Azma RZ, Khamisah, MG, Suria AA, Hafiza A, Azlin, I et al. Detection of homozygous Haemoglobin Constant Spring by capillary electrophoresis method.

ARC Journal of Haematology. 2016; 1(1): 28-32.

11. Azma RZ, Ainoon O, Hafiza A, Azlin I, Noor Farisah AR et al.

Molecular characteristic of alpha thalassaemia among patients diagnosed in UKM Medical Centre. Malays J Pathol 2014;37(1): 27-32.

12. Harteveld, C. L., Voskamp, a, Phylipsen, M., Akkermans, N., den Dunnen, J. T., White, S. J. & Giordano, P. C. 2005. Nine unknown rearrangements in 16p13.3

Continued from references page 6 9. Gatell, J. M. (2011). Antiretroviral therapy for HIV: do subtypes matter?

Clin Infect Dis, 53(11), 1153-1155. doi: 10.1093/cid/cir686 10. Hu, W., Kaminski, R., Yang, F., Zhang, Y., Cosentino, L., Li, F., . . . Khalili, K. (2014). RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc Natl Acad Sci U S A, 111(31), 11461-11466. doi: 10.1073/pnas.1405186111 11. Lu, D. Y., Yarla, N. S., Xu, B., Ding, J., Lu, T. R., & Wu, H. Y. (2017). HAART in HIV/AIDS Treatments, Future Trends. Infect Disord Drug Targets. doi: 10.217 4/1871526517666170505122800 12. Ng, K. T., Ong, L. Y., Lim, S. H., Takebe, Y., Kamarulzaman, A., & Tee, K. K. (2013). Evolutionary history of HIV-1 subtype B and CRF01_AE transmission clusters among men who have sex with men (MSM) in Kuala Lumpur, Malaysia. PLoS One, 8(6), e67286. doi: 10.1371/journal.pone.0067286 13. Saraswathy, T. S., Ng, K. P., & Sinniah, M. (2000). Human immunodeficiency virus type 1 subtypes among Malaysian intravenous drug users.

Southeast Asian J Trop Med Public Health, 31(2), 283-286.

14. Taylor, B. S., Sobieszczyk, M. E., McCutchan, F. E., & Hammer, S. M. (2008).

The challenge of HIV-1 subtype diversity. N Engl J Med, 358(15), 1590-1602. doi: 10.1056/NEJMra0706737 15. Zhu, W., Lei, R., Le Duff, Y., Li, J., Guo, F., Wainberg, M. A., & Liang, C. (2015).

The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology, 12, 22. doi: 10.1186/s12977-015-0150-z

MyHVP Newsletter | Jan-June 2020 | page 5

Postgraduate CRISPR/CAS9: GENOME EDITING FOR THE POTENTIAL ELIMINATION OF GENETICALLY DIVERSE HIV SUBTYPES IN MALAYSIA Ravichantar Nithya and Theva Das Kumitaa *Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Kepala Batas, Penang

H

IV/AIDS has claimed more than 35 million lives globally since its discovery 30 years ago (WHO 2018). HIV-1 can be divided into four groups (M, N, O and P) and among them, group M is responsible for most of the HIV-1 infections worldwide.

Group M has nine distinct subtypes: A, B, C, D, F, G, H, J and K and nearly 100 Circulating Recombinant Forms (CRF). There is approximately a 25%-30% genetic difference between clades which explains the dissimilarities in transmission rate, natural history, rate of CD4 T cells decline, progression to AIDS, and recovery rate in response to combination antiretroviral therapy (cART) (Taylor, Sobieszczyk, McCutchan, & Hammer, 2008).

Although 50% of global HIV infections are caused by subtype C (AVERT 2018), subtype B is the most researched subtype as it is the predominant strain in US and Western Europe. Currently, cART is the only treatment available to control mortality and morbidity of the infection. However, cART does not cure HIV, has side effects and leads to drug resistance when patients are not compliant. cART also does not eradicate latent reservoirs, and may not work effectively against non-B HIV subtypes as it was developed against subtype B (Desai, Iyer, & Dikshit, 2012; Gatell, 2011; Lu et al., 2017).

In Malaysia, an analysis of the early 15,100 cases showed that patients were mostly infected with HIV subtypes B, C and E (now known as CRF01_AE) (Brown et al., 1996; Saraswathy, Ng, & Sinniah, 2000). At present, we have 93,089 individuals living with HIV, 3,391 new infections and 911 cases of AIDS-related deaths in Malaysia (MAC 2016), with CRF01_AE (40.9%) being the predominant subtype followed by CRF33_01B (20.5%), subtype B (10.1%) and new CRFs and URFs (Chow et al., 2016). The shift in prevalent subtypes is mostly due to the change in transmission, going from intravenous drug users (IVDU) to men engaging in same gender sex (MSM). The change of subtypes in such a short duration emphasizes the need to have a treatment that would combat genetically diverse HIV subtypes, and not just HIV subtype B. The latest genome editing tool, Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein, Cas9 (CRISPR/Cas9) is a promising tool in HIV cure efforts. CRISPR/ Cas9 is a bacterial adaptive immune system consisting of guide RNA that binds complementarily to our target sequence in the genome, and Cas9, which creates a double stranded break at the binding site of the guide RNA. The double stranded break caused by CRISPR/Cas9 leads to insertions and deletions at the target site, which eventually disrupts the targeted gene and causes it to be non-functional. Hence, in the event of a dis- ease such as HIV, disrupting the HIV genome would be an effi- cient method of eradicating the virus.

In our study, we designed a CRISPR/Cas9 that recognizes all three subtypes of HIV prevalent in Malaysia, B, C and CRF01_AE. We also targeted six different genes that are important for viral replication, infectivity and pathogenesis, specifically LTR, Pol, Gag, Rev, Tat and Vif (Figure 1). Simultaneously knocking down

the conserved regions of multiple HIV genes would facilitate large deletions that prevent viral escape variants while rendering the genome non-functional. CRISPR/Cas9 also targets both transcriptionally active and inactive provirus (Zhu et al., 2015) by cleaving the nascent HIV-1 proviral DNA intermediate prior to integration into host cell genome, which is not possible with cART. Our in-vitro functional testing shows that our CRISPR/Cas9 reduces HIV viral load by 4-fold while remaining safe to humans.

Figure 1: LTR, Pol, Gag, Rev, Tat and Vif, HIV genes targeted by our CRISPR/Cas9 In summary, CRISPR/Cas9 promises complete HIV genome inactivation (Ebina, Misawa, Kanemura, & Koyanagi, 2013; Hu et al., 2014) and serves as a promising gene therapy that works across different subtypes to prevent transmission and is a potential cure against HIV. Acknowledgement This study is supported by Fundamental Research Grant Scheme (203/CIPPT/6711440) and Short-Term Grant (304/ CIPPT/6313090) provided by Malaysian Ministry of Higher Education (MOHE) and Universiti Sains Malaysia respectively.

References 1. WHO, HIV/AIDS, 2017. http://www.who.int/mediacentre/factsheets/ fs360/en/. Accessed 20 August 2018.

2. Avert, Global HIV and AIDS statistics, 2017.https://www.avert.org/professionals/hiv-science/types-strains. Accessed 20 August 2018 3. Malaysian Aids Council, HIV statistics, 2016. https://www.mac.org.my/v3/ resources/hiv-statistics/. Accesed 24 August 2018 4. Brown, T. M., Robbins, K. E., Sinniah, M., Saraswathy, T. S., Lee, V., Hooi, L. S., . . . Kalish, M. L. (1996). HIV type 1 subtypes in Malaysia include B, C, and E. AIDS Res Hum Retroviruses, 12(17), 1655-1657. doi: 10.1089/aid.1996.12.1655 5. Chow, W. Z., Bon, A. H., Keating, S., Anderios, F., Halim, H. A., Takebe, Y., . . . Tee, K. K. (2016). Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries. PLoS One, 11(8), e0161853. doi: 10.1371/journal.pone.0161853 6. Chow, W. Z., Ong, L. Y., Razak, S. H., Lee, Y. M., Ng, K. T., Yong, Y. K., . . . Tee, K. K. (2013). Molecular diversity of HIV-1 among people who inject drugs in Kuala Lumpur, Malaysia: massive expansion of circulating recombinant form (CRF) 33_01B and emergence of multiple unique recombinant clusters. PLoS One, 8(5), e62560. doi: 10.1371/journal.pone.0062560 7. Desai, M., Iyer, G., & Dikshit, R. K. (2012). Antiretroviral drugs: critical issues and recent advances. Indian J Pharmacol, 44(3), 288-298. doi: 10.4103/0253-7613.96296 8. Ebina, H., Misawa, N., Kanemura, Y., & Koyanagi, Y. (2013). Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep, 3, 2510. doi: 10.1038/srep02510

MyHVP Newsletter | Jan-June 2020| page 6

Postgraduate CRISPR GENE EDITING IN G6PD DEFICIENT THP-1 CELL LINE Lelamekala Vengidasan1, Ida Shazrina Ismail1, Muhammad Amir Yunus2, Kumitaa Theva Das2, Badrul Hisham Yahaya1, Narazah Mohd Yusoff1* 1 Regenerative Medicine Cluster, Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, Pulau Pinang, Malaysia 2 Infectomics Cluster, AMDI, Universiti Sains Malaysia, Pulau Pinang, Malaysia * Email: narazah@usm.my *Contact number: (+6) 04-5622395

C

RISPR gene editing based technology can be applied in numerous approaches including correction or inactivation of deleterious mutations, addition of therapeutic transgene and disruption of the viral DNA. Progress in genome editing is expeditious and few works have been successfully translated to pre-clinical and phase 1 clinical trial such as on Duchenne muscular dystrophy (DMD) (Aiuti, Roncarolo, & Naldini, 2017; Long et al., 2014; Ousterout et al., 2013), Hepatitis B virus (HBV) (Bloom, Ely, Mussolino, Cathomen, & Arbuthnot, 2013; Lin et al., 2014), cataracts (Wu et al., 2013), cystic fibrosis (Schwank et al., 2013) and hereditary tyrosinaemia (Yin et al., 2014). Our study was aimed at correcting G6PD deficiency in THP-1 cell line using ribonucleic CRISPR/Cas9 system. This correction utilized a homology directed repair employing plasmid donor template by fixing the mutation at single site.

G6PD deficiency is the most common genetic enzyme deficiencies with approximately 400 million people living with it (Cappellini and Fiorelli, 2008). G6PD deficiency is an X-linked genetic disorder affecting individuals. Our work has successfully shown that this deficiency can be corrected by using genome editing system, CRISPR-Cas9. Although the percentage of the correction was low, techniques to improve its efficiency can be enhanced.

Among the methods that can be used to improve the efficiency of the system is the utilisation of a unique and optimised guide RNA to reduce the off-target effects (Cho et al., 2014). Instead of transferring Cas9-encoded gene into a human cell line, the delivery of purified recombinant Cas9 protein can achieve an editing efficiency as high as 79 % (Kim, Kim, Cho, Kim, & Kim, 2014). Besides that, the Cas9 protein might serve a better choice compared to the one used in the work, Cas9 plasmid DNA. Op- timisations, as discussed, are needed to improve the system for a higher percentage of gene correction in CRISPR-Cas9 system.

In addition, to increase the percentage of the HDR for single site mutation, the utilisation of ssODN is preferred instead of plasmid circular DNA. It has been shown that donor DNA by utilising ssODN results a far better percentage of gene correction in monogenic related disease.

Figure 1: Illustrated guide RNA which is mapped to WT G6PD mRNA full sequences. The green box shows a gRNA (I) while the blue box shows gRNA (II).

(Legends: Green box : gRNA I, Blue box : gRNA II, Red box : Mutation site at 871 aa.)

References 1. Aiuti, A., Roncarolo, M. G., & Naldini, L. (2017). Gene therapy for ADA‐ SCID, the first marketing approval of an ex vivo gene therapy in Europe: paving the road for the next generation of advanced therapy medicinal products. EMBO Molecular Medicine, e201707573.

2. Bloom, K., Ely, A., Mussolino, C., Cathomen, T., & Arbuthnot, P. (2013).

Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Molecular Therapy, 21(10), 1889-1897.

3. Cho, S. W., Kim, S., Kim, Y., Kweon, J., Kim, H. S., Bae, S., & Kim, J.-S. (2014). Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome research, 24(1), 132-141.

4. Kim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J.-S. (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome research, 24(6), 1012-1019.

5. Lin, S.-R., Yang, H.-C., Kuo, Y.-T., Liu, C.-J., Yang, T.-Y., Sung, K.-C., . . .

Shen, Y.-C. (2014). The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Molecular Therapy—Nucleic Acids, 3(8), e186.

6. Long, C., McAnally, J. R., Shelton, J. M., Mireault, A. A., Bassel-Duby, R., & Olson, E. N. (2014). Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA. Science, 345(6201), 1184-1188.

7. Ousterout, D. G., Perez-Pinera, P., Thakore, P. I., Kabadi, A. M., Brown, M. T., Qin, X., . . . Gersbach, C. A. (2013). Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Molecular Therapy, 21(9), 1718-1726.

8. Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature biotechnology, 32(4), 347355.

9. Schwank, G., Koo, B.-K., Sasselli, V., Dekkers, J. F., Heo, I., Demircan, T., . . . van der Ent, C. K. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell, 13(6), 653-658.

10. Wu, Y., Liang, D., Wang, Y., Bai, M., Tang, W., Bao, S., . . . Li, J. (2013).

Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell, 13(6), 659-662.

11. Yin, H., Xue, W., Chen, S., Bogorad, R. L., Benedetti, E., Grompe, M., . . .

Anderson, D. G. (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature biotechnology, 32(6), 551.

MyHVP Newsletter | Jan-June 2020 | page 7

Photo Diary Key events in the year 2019 and first half of 2020 (Jan - June 2020)

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Note: Programmes scheduled in the half year of 2020 have been postponed due to the worldwide COVID-19 pandemic .

Photo 1 - 7 EduVariome Programme at SMK Batu Lintang, Sarawak (Photo 1-2) 9 April 2019

MRSM Pengkalan Chepa, Kelantan (Photo 3-5) - 7 July 2019

SMK Tengku Mahmud, Besut, Terengganu (Photo 6-7) - 9 February 2020

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Photo 9-12

Meeting with the Mufti of Kelantan. A discussion on abortion of fetal thalassemia diagnosis from an islamic perspective.

Details on this report will be published in the next issue 2020.

Global Globin 2020 Challenge Conference 2019

July 9, 2019

October 28-30, 2019 The Headquarters of UNESCO, Paris, France

Kota Bharu, Kelantan, MyHVP Newsletter | Jan-July 2019| page 8