Molecular Analysis and Identification of Rare HbE/Beta Thalassemia Mutations in Peninsular Malaysia

Nur-Aisyah, Aziz

Following the Policy of the National Regulation 3.3 , page 17, on CME disclosures, dated 5 November 2009, and on behalf of the Provider , - Collage S.p.A.- n. 309

I, NUR AISYAH AZIZ HERE DECLARE

DISCLOSURE OF RELEVANT FINANCIAL RELATIONSHIPS

NO, have no relevant personal financial relationship in the medical/health field.

DISCLOSURE OF PROMOTIONAL TALKS

NO, I have not presented any promotional talks for any pharmaceutical companies within the past 12 months

I understand that continuing education accreditation guidelines prohibit me from accepting any reimbursement (financial, gifts or in-kind exchange) for this presentation from any source other than the accredited CME provider ( Collage S.p.A.)

15-16 September, 2017 Nur Aisyah Aziz

Outline

Statement of the Problem

The purpose of this study

Methodology

Conclusion & Significance

Epidemiology of HbE/Beta-thalassemia

Hemoglobin E (HbE) is one of the most common structural beta-globin variant in the world.

WHO – High estimates for India, Sri Lanka, Malaysia, Southern China and Maldives.

Important health problem in the Indian subcontinent and Southeast Asia.

The heterogeneity of the mutations makes it difficult to identify the mutation in some beta-thalassemia patients.

Clinical presentation: Asymptomatic; NTDT Transfusion dependent, TDT.

In Malaysia - substantial variation in incidence of HbE attributable to ancestry

than their geographic locations.

The highest prevalence was documented in aboriginal groups; Semai &

Temiar, with a gene frequency of 26% and 45% respectively.

The frequency of HbE occurred 10x in Malaysian Malay > Malaysian Chinese.

Incidence of 4x HbE/-thal > Homozygous -thal.

In Malaysia, HbE is one of the most common beta-globin mutations with 5%

carrier rate among the Malays.

Genotype-Phenotype correlation has not been completely defined.

HbE/-thalassemia: Malaysia Scenario

Statement of the Problem:

The clinical management of HbE/beta thalassemia has been challenging due to its marked heterogeneity at the molecular level.

Previous reports have recommended DNA-based analysis to distinguish the HbE-related disorders.

In Malaysia, DNA analysis for thalassemia syndromes had only been established in the last five years.

Discovery and understanding of molecular heterogeneity of HbE-related disorders in Malaysia are still limited.

The purpose of this study:

To characterize beta-globin gene mutations using DNA-based techniques in the patients with the clinical

impression of HbE/beta-thalassemia in Peninsular Malaysia.

Methodology1. Medical Research and Ethics Committee (MREC); NMRR-12-1119-13926.

2. A total of 300 HbE/beta-thal cases from nine hospitals in seven states of Peninsular Malaysia were recruited between Sept-2013 and Jan-2016.

3. Informed consent from each subject.

4. Isolation of genomic DNA

5. Molecular analysis (HBB: NG_000007.3)

• Multiplex ARMS-PCR

• Multiplex Gap-PCR

• Sequencing

• Multiplex ligation-dependent probe amplification (MLPA)

300 subjects, ~2.5mL of blood & Data collection; 9hospitals

http://www.nationsonline.org/oneworld/map/malaysia_map.htm

Methodology (cont.)

HbE/Beta-Thal Genotypes

Phase 2: To detect the uncommon β-thal mutations

SequencingMultiplex ligation probe-dependent

amplification (MLPA)

Phase 1: To detect β-thal mutations

Multiplex ARMS-PCR for 21 common beta thalassemia mutations

Multiplex gap-PCR for 10 beta-globin gene cluster deletions

Methodology (cont.)

Conclusion & Significance:

1. DNA-based techniques implemented in this study have effectively facilitated the confirmation of HbE/beta thalassemia mutations.

2. 22 types (17 βE/β° and 5 βE/β+) of comp. HbE/beta-thal mutations were detected in 91% (n=274/300)

3. 8% (n=24/300) Non-HbE/beta-thal &

4. 1% (n=2/300) HbE/beta-variant

5. Five most common comp. het-HbE:IVS1-5(G>C), Codon 41/42(-TTCT), IVS1-1(G>T), Codon 17(AAG>TAG) & IVS2-654(C>T).

Conclusion & Significance (cont.):3. Ten rare beta-globin mutations detected in this study were nine of β°

[Codon 35 (TAC→TA-), IVS 1-2 (T>C), Codon 123/124/125 (-8bp), IVS 1 25bp del, Codon 41 (TTC>TT-), Codon 35 (TAC>TAA), 619bp del, Codon 6 (GAG>G-G), Codon 30 (AGG>ACG)] and one de novo βvariant [Codon 98 (GTG>GGG)].

4. This study provides updates on molecular heterogeneity of HbE/beta thalassemia in Malaysia.

5. More variability of βE/β°-allele was seen in this study compared to βE/β+-allele.

6. Thus, in view of marked clinical heterogeneity and presence of modifier gene, this study highlights the importance of definitive diagnosis at molecular level for HbE/beta thalassemia in Malaysia.

Main Bibliography1. Cao A. and Galanello R. Beta-thalassemia. Genetics IN Medicine. 2010. Vol. 12 (2)

2. Fuchareon S & Weatherall D.J. The Hemoglobin E Thalassemias. Cold Spring Harb Perspect Med 2012. doi:10.1101/cshperspect.a011734.

3. George E. HbE β-thalassemia in Malaysia: Revisited. J Hematol Thromb Dis. 2013. 1:1. http://dx.doi.org/10.4172/2329-8790.1000101.

Acknowledgement:

We would like to thank all of the participating hospitals (HKL, HAmpang, HTAR, HPenang, HSB, HSAS, HSI, HAS & HRPB, HSNZ) for full cooperation

in this study.

Also, would like to thank the Director General of Health, Ministry of Health Malaysia for permission to present this poster.

This study was funded by National Health Institute (NIH), Ministry of Health Malaysia (JPP-12-037).

Thank you