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DIAGNOSIS OF HAEMOGLOBINOPATHIES: FROM SCREENING TO CONFIRMATION OF GENETIC DEFECT

Written by: Assoc. Prof. Dr. Raja Zahratul Azma Raja Sabudin Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia

Haemoglobinopathies 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

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].

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

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 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-Polymerase 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.

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