Haemoglobinopathy

Review Article Indian J Med Res 134, October 2011, pp 552-560 encroaching(a) & non- invasive approaches for antepartum diagnosing of haemoglobinopathies Experiences from India R. B. Colah, A. C. Gorakshakar & A. H. Nadkarni subject area Institute of Immunohaematology (ICMR), Mumbai, India Received October 29, 2010 The thalassaemias and sickle cell disease argon the commonest monogenic disorders in India. There are an estimated 7500 12,000 babies with ? -thalassaemia major natural every year in the country. While the overall prevalence of carriers in divers(prenominal) States varies from 1. to 4 per centime, recent work has shown considerable variations in frequencies even inside States. Thus, micromapping would help to determine the true burden of the disease. Although screening in antenatal clinics is being through with(p) at many centres, only 15-20 per cent of pregnant women render in antenatal clinics in public hospitals in the first trimester of pregnancy. There are on ly a handful of centres in major cities in this immense country where antepartum diagnosing is done. There is considerable molecular heterogeneity with 64 variances identified, of which 6 to 7 common fluctuations account for 80-90 per cent of mutant alleles. offshoot trimester foetal diagnosis is done by chorionic villus sampling (CVS) and deoxyribonucleic acid analysis using reverse dot blot hybridization, amplification refractory chromosomal mutation system (ARMS) and desoxyribonucleic acid sequencing. Second trimester diagnosis is done by cordocentesis and foetal blood analysis on HPLC at a few centres. Our experience on prenatal diagnosis of haemoglobinopathies in 2221 pregnancies has shown that 90 per cent of couples were referred for prenatal diagnosis of ? -thalassaemia after having one or more affected children while about 35 per cent of couples were referred for prenatal diagnosis of sickle cell disorders prospectively.There is a clear need for more data from India on non-invasive approaches for prenatal diagnosis. Key words Haemoglobinopathies India invasive and non-invasive approaches prenatal diagnosis Introduction The inherited disorders of haemoglobin are the almost common monogenic disorders globally. Around 7 per cent of the population worldwide are carriers with more than 3,00,000 severely affected babies born every year1. Prenatal diagnosis is an integral component of a community control programme for haemoglobinopathies. Estimating the disease burden, generating consciousness in the population, screening 552 o identify carriers and couples at happen and heritable counselling are prerequisites for a successful prevention programme. The remarkable success of such programmes in the 1970s in Cyprus, Italy, Greece and the UK led to the development of control programmes in many other countries2-6. The extent of the problem in India ? -thalassaemia has been reported in most of the communities that invite been screened so far in India . While the overall prevalence varies from 1. 5 to 4 per COLAH et al PRENATAL DIAGNOSIS OF HAEMOGLOBINOPATHIES IN INDIA 553 ent in different States, communities like Sindhis, Punjabis, Lohanas, Kutchi Bhanushalis, Jains and Bohris have a higher prevalence (4-17%)7-12. Different reportshaveestimatedthat7500-12,000? -thalassaemia major babies would be born in India each year12 -14. It has also been shown recently by micromapping at the district level in two States, Maharashtra and Gujarat in westernIndiathattheprevalenceof? -thalassaemiatrait in different districts within these States is variable (0 9. 5%). Based on these estimates there would be more or less 1000birthsof? thalassaemiamajorbabieseachyear in these two States alone15. Thus, such data should be obtained from different States to know the true burden of the disease and for planning and executing control programmes. Haemoglobin S (Hb S) is prevalent in central India and among the tribal belts in western, eastern and southe rn India, the carrier rates varying from 1-40 per cent16-18. It has been estimated that over 5000 babies with sickle cell disease would be born each year19. Haemoglobin E is widespread in the labor union eastern States in Assam, Mizoram, Manipur, ArunachalPradesh and Tripura, the prevalence of Hb E trait being highest (64%) among the Bodo-Kacharis in Assam and going up to 30-40 per cent in some other populations in this region20-22. In eastern India the prevalence of Hb E trait varies from 3-10 per cent in West Bengal8,23. Both Hb E andHbSwhenco-inheritedwith? -thalassaemiaresult in a disorder of variable clinical severity24-26. These inherited haemoglobin disorders cause considerable pain and poor to the patients and their families and are a major drain on health resources in the country. The need for accurate identification of carries and couples at peril Classical ? thalassaemia carriers have typically reduced red cell indices mean corpuscular volume (MCV)T) ? + 3. -87 (CT) ? + 4. -80 (CT) ? + 5. -29 (AG) ? + 6. -28 (AG) ? + 7. -25 (AG) ? + B. Cap site 1. +1 (AC) ? + C. Initiation codon 1. ATG ACG ? 0 D. RNA processing mutations i) Splice continuative site 1. Codon 30 (GC) ? 0 2. Codon 30 (GA) ? 0 3. IVS 1-1 (GT) ? 0 4. IVS 1-1 (GA) ? 0 5. IVS 1-129 (AC) ? 0 6. IVS 1-130 (GC) ? 0 7. IVS 1-130 (GA) ? 0 8. IVS II-1 (GA) ? 0 (ii) Consensus site 1. IVS 1-5 (GC) ? + 2. IVS 1-128 (TAG GAG) ? + 3. IVS II-837 (TG) ? (iii) IVS changes 1.IVS I-110 (GA) ? + 2. IVS II-591 (TC) ? + 3. IVS II-613 (CT) ? + 4. IVS II-654 (CT) ? + 5. IVS II-745 (CG) ? + iv) Coding region changes 1. Codon 26 (GA) Hb E ? + E. RNA translational mutations i) Nonsense 1. Codons 4,5,6 (ACT CCT GAG ACA TCT ? 0 TAG) 2. Codon 5 (-CT), Codon 13 (CT), Codon 26 ? (GC), Codons 27/28 (+C) in cis 3. Codon 6 (GAG TAG) and on the aforementioned(prenominal) ? 0 chromosome Codon 4 (ACT ACA) , Codon 5 (CCTTCT) 4. Codon 8 (AG) ? 5. Codon 13 (CT), Codon 26 (GA), Codons ? 27/28 (-C) in cis 6. Codon 15 (TG G TAG) ? 0 7. Codons 62-64 (7 bp del) ? 0 8. Codons 81-87 (22 bp del) ? 9. Codon 121 (GT) ? 0 Contd. themselves, today their relatives and all-embracing families are coming forward to get screened38. There is only one centre in Lucknow in north India which offers a formal course for genetic counsellors and there is a need for more such courses throughout the country. Counsellors should be aware that couples at risk of havingachildwith? -thalassaemiamajor,sicklecell disease, Hb S ? -thalassaemia, Hb E ? -thalassaemia, ? -thalassaemia, Hb Lepore ? -thalassaemia and Hb SD disease should be given the option of prenatal diagnosis to avoid the birth of a child with a severe disorder.However, couples at risk of having a child with Hb D disease, Hb D ? -thalassaemia and Hb E disease do not require prenatal diagnosis as these disorders are mild. InSardinia,identificationofthemaximumnumber of carriers followed by effective genetic counselling helpedtoreducethebirthrateof? -thalassaemiamaj or babies from 1250 to 1400039. Prenatal diagnosis The first initiatives in India Facilities for prenatal diagnosis became available in India in the mid 1980s40. Until then, although prenatal diagnosis was offered by a few centres, foetal samples were sent to the UK and other countries for analysis.Foetal blood sampling by fetoscopy done between 18 and 22 wk gestation and diagnosis by globin chain synthesis were done for the next 4 to 5 geezerhood at 2 centres in Mumbai40,41. Chorionic villus sampling and DNA analysis in the first trimester In the 1990s first trimester foetal diagnosis by chorionic villus sampling (CVS) and DNA analysis was established at 4-5 centres in the north in Delhi42, in the west in Mumbai41,43,44 and in the south in Vellore45. These services then expanded to other cities like Lucknow and Chandigarh in the north46,47, and Kolkata in the east48.However, these services are still limited to major cities where couples are referred to or CVS samples are sent fro m surrounding areas. Molecular analysis ? -thalassaemia is extremely heterogeneous with more than 200 mutations described worldwide49. In India, about 64 mutations have been characterized by studies done at different centres30,31,49-51 (Table I). Six to seven mutations IVS 1-5 (G? C), 619 bp deletion, IVS 1-1 (G? T), Codon 8/9 (+G), Codons 41/42 (-CTTT), COLAH et al PRENATAL DIAGNOSIS OF HAEMOGLOBINOPATHIES IN INDIA (ii) Frameshift 1. Codon 5 (-CT) 2. Codons 7/8 (+G) 3. Codon 8 (-AA) 4. Codons 8/9(+G) 5.Codon 13 (CT) 6. Codon 15 (-T) 7. Codon 16 (-C) 8. Codon 16 (CT) 9. Codon 17 (AT) 10. Codons 22-24 (7 bp del) 11. Codon 26 (GT) 12. Codon 35 (AG) 13. Codons 36/37 (-T) 14. Codons 36-39 (8 bp del) 15. Codon 39 (CT) 16. Codon 44 (-C) 17. Codons 47/48 (+ATCT) 18. Codon 55 (+A) 19. Codon 55 (-A) 20. Codons 57/58 (+A) 21. Codon 88 (+T) 22. Codons 106/107 (+G) 23. Codon 110 (TC) 24. Codon 111 (-G) 25. Codon 135 (CT) F. RNA cleavage and polyadenylation mutation 1. AATAAAAACAAA G. Deletional mutations 1. 619 bp deletion 3end 2. 10. 3 kb deletion 3. Codons 126-131 (17 bp deletion) Source Refs 30, 31, 49-51 55 ?0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? 0 ? + ? 0 ? + ? 0 ? 0 ? 0 Fig. 1. Regionaldistri butionof? -thalassaemiamutationinIndia. molecular techniques like covalent reverse dot blot hybridization (CRDB), amplification refractory mutation system (ARMS), denaturing gradient gel electrophoresis (DGGE), and DNA sequencing43,44,52. Foetal blood analysis in the arcminute trimester Most of the prenatal diagnosis programmes in the Mediterranean countries started with second trimester foetal blood analysis but they were able to switch over tofirsttrimesterdiagnosisinashortspan5,39.In India, second trimester diagnosis is still done as manycouplesatriskareidentifiedlateduringpregnancy. Foetal blood sampling is done by cordocentesis at 18 to20wkgestationandafterconfirmingthatthereisno motherly contamination in the foetal sample by foetal cell staining using the Kleihauer-Betke method, it is analysed by HPLC on the Variant haemoglobin Testing System (Bio Rad Laboratories, Hercules, USA). The HbA levels in foetuses affected with ? -thalassaemia major have ranged from 0 to 0. 5 per cent and these were distinguishable from heterozygous babies where the Hb A levels were 1. per cent in different studies. However, there was some overlap in Hb A levels between heterozygotes and normals53-55. Sickle cell disease and Hb E thalassaemia have also been diagnosed in this way. On the other hand, experience in Thailand showed that while ? 0 thalassaemia homozygotes and HbE-? 0 thalassaemia compound heterozygotes could be diagnosed by HPLC analysis of foetal blood, ? ++ thalassaemia homozygotes may be misdiagnosed as heterozygotes56. Amniotic fluid cells have not been used extensively in India for prenatal diagnosis of haemoglobinopathies. Codon 15 (G? A), Codon 30 (G?C) are common accounting for 85-95 per cent of mutant alleles. However, regional differences in their frequencies have been noted30,31,50,51 (Fig. 1). The prevalence of IVS 1 -5 (G? C), the most common mutation in India varies from 15-88 per cent in different States. Codon 15 (G? A) is the second most frequent mutation in Maharashtra and Karnataka and Codon 5 (-CT) is the third most common mutation in Gujarat. The -88 (C? T) and the Cap site +1 (A? C) mutations are more common in the northern region30,31,50. The 619 bp deletion is the most common mutation among the immigrant population from Pakistan.This cognition on the distribution of mutations in different regions and in people of different ethnic backgrounds has facilitated prenatal diagnosis using 556 INDIAN J MED RES, OCTOBER 2011 Experience at National Institute Immunohaematology (NIIH), Mumbai of Bothfirstandsecondtrimesterprenataldiagnosis for the ? -thalassaemias and sickle cell disorders are done at National Institute of Immunohaematology, Mumbai, and over the last 25 years 2,221 pregnancies at risk have been investigated (Table II). While majority of the couples were at risk of having children with ? thalassaemia major, a significant number of couples at risk of having children with sickle cell disorders have been referred for prenatal diagnosis in the last 4 to 5 years. Our experience in western India has shown that there are still very few couples (G or codon 35 ? (A? G) at alpha -beta chain interfaces. 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