Haemoglobinopathy

Review Article Indian J Med Res 134, October 2011, pp 552-560 Invasive & non-trespassing(a) approaches for antenatal diagnosing of haemoglobinopathies Experiences from India R. B. Colah, A. C. Gorakshakar & A. H. Nadkarni National set of Immunohaematology (ICMR), Mumbai, India Received October 29, 2010 The thalassemias and reaping hook kiosk malady are the comm integrityst monogenic perturbations in India. there are an estimated 7500 12,000 babies with ? -thalassaemia major(ip) born e genuinely course of study in the country. go the overall preponderance of carriers in diametrical States varies from 1. to 4 per cent, recent work has shown commodious variations in frequencies even within States. Thus, micromapping would help to retard the true burden of the disorder. Although test in antepartum clinics is being done at many centres, that 15-20 per cent of pregnant women register in antenatal clinics in public hospitals in the graduation exercise trimester of p regnancy. on that breaker point are only a handful of centres in major cities in this vast country where antepartum diagnosing is done. There is considerable molecular hetero constituentousness with 64 mutations identified, of which 6 to 7 greenness mutations handbill for 80-90 per cent of play alleles.First trimester foetal diagnosing is done by chorionic villus sample dissemination (CVS) and deoxyribonucleic acid compend utilise reverse dot post crossover, amplification refractory mutation system (ARMS) and desoxyribonucleic acid sequencing. Second trimester diagnosing is done by cordocentesis and foetal family analysis on HPLC at a few centres. Our experience on antenatal diagnosing of haemoglobinopathies in 2221 pregnancies has shown that 90 per cent of couples were referred for prenatal diagnosis of ? -thalassaemia afterward having one or much affected infantren tour about 35 per cent of couples were referred for prenatal diagnosis of sickle cellphon e disorders prospectively.There is a expel 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 transmittable disorders of haemoglobin are the most(prenominal) joint monogenic disorders globally. Around 7 per cent of the population worldwide are carriers with more than 3,00,000 seriously affected babies born every year1. Prenatal diagnosis is an integral component of a community defy programme for haemoglobinopathies. Estimating the disease burden, generating awareness in the population, screening 552 o identify carriers and couples at risk of exposure and genic counselling are prerequi positions for a successful taproom programme. The remarkable success of such programmes in the seventies 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 ? -t halassaemia has been reported in most of the communities that need 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 distinguishable States, communities parcel out Sindhis, Punjabis, Lohanas, Kutchi Bhanushalis, Jains and Bohris experience a high prevalence (4-17%)7-12. Different reportshaveestimatedthat7500-12,000? -thalassaemia major babies would be born in India distributively year12 -14. It has similarly been shown recently by micromapping at the district train in 2 States, Maharashtra and Gujarat in westernIndiathattheprevalenceof? -thalassaemiatrait in contrary districts within these States is variable (0 9. 5%). Based on these estimates there would be around 1000birthsof? thalassaemiamajorbabieseachyear in these two States alone15. Thus, such data should be obtained from unlike States to last the true burden of the disease and for planning and execution of instrument control programmes. Haemoglobin S (Hb S) is prevalent in central India and among the tribal belts in western, eastern and southern 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 due north 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(prenominal) Hb E andHbSwhenco-inheritedwith? -thalassaemiaresult in a disorder of variable clinical severity24-26. These inherited haemoglobin disorders power considerable pain and suffering to the patients and their families and are a major drain on health resources in the country. The need for accurate identificatio n of carries and couples at risk Classical ? thalassaemia carriers have typically trim red cell indices mean corpuscular volume (MCV)T) ? + 3. -87 (CT) ? + 4. -80 (CT) ? + 5. -29 (AG) ? + 6. -28 (AG) ? + 7. -25 (AG) ? + B. crownwork site 1. +1 (AC) ? + C. Initiation codon 1. ATG ACG ? 0 D. ribonucleic acid processing mutations i) Splice junction 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 sam e ? 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 (TGG 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 extended families are advent 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 need prenatal diagnosis as these disorders are mild. InSa rdinia,identificationofthemaximumnumber of carriers followed by effective genetic counselling helpedtoreducethebirthrateof? -thalassaemiamajor babies from 1250 to 1400039. Prenatal diagnosis The first initiatives in India Facilities for prenatal diagnosis became available in India in the mid(prenominal) 1980s40. Until accordingly, although prenatal diagnosis was offered by a few centres, foetal samples were sent to the UK and other countries for analysis.Foetal rip taste by foetoscopy done amongst 18 and 22 wk gestation and diagnosis by globin chain synthesis were done for the contiguous 4 to 5 years 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 i n the north46,47, and Kolkata in the east48.However, these services are remedy limited to major cities where couples are referred to or CVS samples are sent from meet 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 cxxxv (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) offset 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. Regionaldistributionof? -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 second trimester Most of the prenatal diagnosis programmes in the Mediterranean countries started with second trimester foetal blood analysis but they were able to castigate over tofirsttrimesterdiagnosisinashortspan5,39.In India, second trimester diagnosis is still done as manycouplesatriskareidentifiedlatedur ingpregnancy. Foetal blood sampling is done by cordocentesis at 18 to20wkgestationandafterconfirmingthatthereisno agnate contamination in the foetal sample by foetal cell staining victimisation the Kleihauer-Betke method, it is analysed by HPLC on the Variant Hemoglobin Testing organization (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 excessively 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. amnionic flui d 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 trio most common mutation in Gujarat. The -88 (C? T) and the thug 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 knowledge on the distribution of mutations in different regions and in people of different ethnic backgrounds has facilitated prenatal diagnosis utilise 556 Indian J MED RES, OCTOBER 2011 Experience at National Institute Immunohaematology (NIIH), Mumbai of B othfirstandsecondtrimesterprenataldiagnosis 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 - important chain interfaces. 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