Haemoglobinopathy

Review Article Indian J Med Res 134, October 2011, pp 552-560 Invasive & non-invasive approaches for prenatal diagnosis of haemoglobinopathies: Experiences from India R. B. Colah, A. C. Gorakshakar & A. H. Nadkarni National Institute of Immunohaematology (ICMR), Mumbai, India Received October 29, 2010 The thalassaemias and sickle cell disease are the commonest monogenic disorders in India. There are an estimated 7500 – 12,000 babies with ? -thalassaemia major born every year in the country. While the overall prevalence of carriers in different States varies from 1. to 4 per cent, recent work has shown considerable variations in frequencies even within States. Thus, micromapping would help to determine the true burden of the disease. Although screening in antenatal clinics is being done at many centres, only 15-20 per cent of pregnant women register in antenatal clinics in public hospitals in the first trimester of pregnancy. There are only a handful of centres in major cities in this vast country where prenatal diagnosis is done. There is considerable molecular heterogeneity with 64 mutations identified, of which 6 to 7 common mutations account for 80-90 per cent of mutant alleles. First trimester foetal diagnosis is done by chorionic villus sampling (CVS) and DNA analysis using reverse dot blot hybridization, amplification refractory mutation system (ARMS) and DNA 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 most 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 awareness in the population, screening 552 o identify carriers and couples at – risk and genetic 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 have 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 around 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 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 north eastern States in Assam, Mizoram, Manipur, Arunachal Pradesh 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 suffering 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 risk Classical ? thalassaemia carriers have typically reduced red cell indices [mean corpuscular volume (MCV)T) ? + 3. -87 (C>T) ? + 4. -80 (C>T) ? + 5. -29 (A>G) ? + 6. -28 (A>G) ? + 7. -25 (A>G) ? + B. Cap site 1. +1 (A>C) ? + C. Initiation codon 1. ATG > ACG ? 0 D. RNA processing mutations i) Splice junction site 1. Codon 30 (G>C) ? 0 2. Codon 30 (G>A) ? 0 3. IVS 1-1 (G>T) ? 0 4. IVS 1-1 (G>A) ? 0 5. IVS 1-129 (A>C) ? 0 6. IVS 1-130 (G>C) ? 0 7. IVS 1-130 (G>A) ? 0 8. IVS II-1 (G>A) ? 0 (ii) Consensus site 1. IVS 1-5 (G>C) ? + 2. IVS 1-128 (TAG > GAG) ? + 3. IVS II-837 (T>G) ? (iii) IVS changes 1. IVS I-110 (G>A) ? + 2. IVS II-591 (T>C) ? + 3. IVS II-613 (C>T) ? + 4. IVS II-654 (C>T) ? + 5. IVS II-745 (C>G) ? + iv) Coding region changes 1. Codon 26 (G>A) 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 (C>T), Codon 26 ? (G>C), Codons 27/28 (+C) in cis 3. Codon 6 (GAG > TAG) and on the same ? 0 chromosome Codon 4 (ACT> ACA) , Codon 5 (CCT>TCT) 4. Codon 8 (A>G) ? 5. Codon 13 (C>T), Codon 26 (G>A), 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 (G>T) ? 0 Contd†¦. themselves, today their relatives and extended 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,sicklecel l 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? -thalassaemiamajor babies from 1:250 to 1:400039. 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 foetoscopy done between 18 and 22 wk gestation and diagnosis by globin chain synthesis were done for the next 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 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 from 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 (C>T) 6. Codon 15 (-T) 7. Codon 16 (-C) 8. Codon 16 (C>T) 9. Codon 17 (A>T) 10. Codons 22-24 (7 bp del) 11. Codon 26 (G>T) 12. Codon 35 (A>G) 13. Codons 36/37 (-T) 14. Codons 36-39 (8 bp del) 15. Codon 39 (C>T) 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 (T>C) 24. Codon 111 (-G) 25. Codon 135 (C>T) F. RNA cleavage and polyadenylation mutation 1. AATAAA>AACAAA G. Deletional mutations 1. 619 bp deletion; 3’end 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. 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 switch over tofirsttrimesterdiagnosisinashortspan5,39. In India, second trimester diagnosis is still done as manycouplesatriskareidentifiedlateduringpregnancy. Foetal blood sampling is done by cordocentesis at 18 to20wkgestationandafterconfirmingthatthereisno maternal contamination in the foetal sample by foetal cell staining using the Kleihauer-Betke method, it is analysed by HPLC on the Variant Hemoglobin 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, ? ++ thalassae mia 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 knowledge 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. Ann Hematol 2009; 88 : 1269-71. 52. Old JM, Varawalla NY, Weatherall DJ. The rapid detection and prenatal diagno sis of ? -thalassemia in theAsian Indian and Cyproit populations in the UK. Lancet 1990; 336 : 834-7. 53. Rao VB, Natrajan PG, Lulla CP, Bandodkar SB. Rapid midtrimester prenatal iagnosis of beta-thalassaemia and other haemoglobinopathies using a non- radioactive anion exchange HPLC technique – an Indian experience. Prenat Diagn 1997; 17 : 725-31. 54. Wadia MR, Phanasgaokar SP, Nadkarni AH, Surve RR, Gorakshakar AC, Colah RB, et al. Usefulness of automated chromatography for rapid fetal blood analysis for second trimester prenatal diagnosis of beta-thalassemia. Prenat Diagn 2002; 22 : 153-7. 559 55. Rao S, Saxena R, Deka D, Kabra M. Use of HbA estimation by CE-HPLC for prenatal diagnosis of beta-thalassemia; experience from a tertiary care centre in north India: a brief report. Hematology 2009; 14 : 122-4. 56. Winichagoon P, Sriphanich R, Sae-Mgo WB, Chowthaworm J, Tantisirin P, Kanokpongsakdi S, et al. Application of automated HPLC in prenatal diagnosis of thalassemia. Lab Hematol 2002; 8 : 29-35. 57. Holzgreve W. Will ultrasound screening and ultrasound guided procedures be replaced by non-invasive techniques for the diagnosis of fetal chromosome anomalies? Ultrasound Obstet Gynecol 1997; 9 : 217-9. 58. Steele CD, Wapner RJ, Smith JB, Haynes MK, Jackson LG. Prenatal diagnosis using fetal cells isolated from maternal peripheral blood. Clin. Obstet Gynecol 1996; 39 : 801-13. 59. Mesker WE, Ouwerkerk-vn Velzen MC, Oosterwijk JC, Bernini LF, Golbus MS, Kanhai HH, et al. Two colour immunocytochemical staining of gamma and epsilon type hemoglobin in fetal red cells. Prenat Diagn 1998; 18 : 1131-7. 60. Takabayashi H, Kuwabara S, Ukita T, Ikawa K, Yamafuji K, Igarashi T. Development of non-invasive fetal DNA diagnosis from maternal blood. Prenat Diagn 1995; 15 : 74-7. 61. Cheung MC, Goldberg JD, Kan YW. Prenatal diagnosis of sickle cell anemia and thalassemia by analysis of fetal cells in maternal blood. Nat Genet 1996; 14 : 264-8. 62. Di Naro E, Ghezzi F, Vitucci A, Tannoia N, Campanale D, D’ Addario V, et al. Prenataldiagnosisof? -thalassemiausing fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod 2000; 6 : 571-4. 63. Kolialexi A, Vrettou C, Traeger-Synodinos J, Burgemeister R, Papantoniou N, Kanavakis E, et al. Non invasive prenatal diagnosisof? -thalassemiausingindividualfetalerythroblasts isolated from maternal blood after enrichment. Prenat Diagn 2007; 27 : 1228-32. 64. D’Souza E, Sawant PM, Nadkarni AH, Gorakshakar A, Mohanty D, Ghosh K, et al. Evaluation of the use of monoclonal antibodies and nested PCR for non-invasive prenatal diagnosis of hemoglobinopathies in India. Am J Clin Pathol 2008; 130 : 202-9. 65. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350 : 485-7. 66. Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitaive analysis of fetal DNA in maternal plasma and serum: implications for non invasive prenatal diagnosis. Am J Hum Genet 1998; 62 : 768-75. 67. Lun FMF, Chiu RWK, Allen Chan KC, Lau TK, Leung TY, Dennis Lo YM. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008; 54 : 1664-72. 68. Li Y, Zimmermann B, Rusterholz C, Kang A, Holzgrave W, Hahn S. Size separation of circulating DNA in maternal plasma permits ready detection of fetal DNA polymorphisms. Clin Chem 2004; 50 : 1002-11. 69. Chiu RW, Lau TK, Leung TK, Chow KC, Chui DH, Lo YM. Prenatal exclusion of beta thalassemia major by examination of maternal plasma. Lancet 2002; 360 : 998-1000. 560 INDIAN J MED RES, OCTOBER 2011 beta thalassemia point mutation by MALDI – TOF mass spectrometry. Fetal Diagn Ther 2009; 25 : 246-9. Papasavva T, Kalikas I, Kyrri A, Kleanthous M. Arrayed primer extension for the noninvasive prenatal diagnosis of beta thalassemia based on detection of single nucleotide polymorphism. Ann N Y Acad Sci USA 2008; 1137 : 302-8. Li Y, Di Naro E, Vitucci A, Zimmermann B, Holzgreve W, Hahn S. Detection of paternally inherited fetal point mutations for beta thalassemia using size fractionated cell free DNA in maternal plasma. J Am Med Assoc 2005; 293 : 843-9. Chan K, Yam I, Leung KY, Tang M, Chan TK, Chan V. Detection of paternal alleles in maternal plasma for noninvasive prenatal diagnosis in beta thalassemia: a feasibility study in southern China. Eur J Obstet Gynecol Repord Biol 2010; 150 : 28-33. Lo YMD. Non invasive prenatal diagnosis in 2020. Prenat Diagn 2010; 30 : 702-3. 70. Papasavva T, Kalakoutis G, Kalikas I, Neokli E, Papacharalambous S, Kyrri A, et al. Non-invasive prenatal diagnostic assay for the detection of beta thalassemia. Ann NY Acad Sci USA 2006; 1075 : 148-53. 71. Tungwiwat W, Fucharoen G, Fucharoen S, Ratanasiri T, Sanchaisuriya K, Sae- Ung N. Application of maternal plasma DNA analysis for noninvasive prenatal diagnosis of Hb E beta thalassemia. Transl Res 2007; 150 : 319-25. 72. Lazaros L, Hatzi E, Bouba I, Makrydimas G, Dalkalitsis N, Stefos T, et al. Noninvasivefirsttrimesterdetectionofpaternal beta globin gene mutations and polymorphisms as predictors of thalassemia risk at chorionic villus sampling. Eur J Obstet Gynecol Repord Biol 2008; 140 : 17-20. 73. Li Y, Di Naro E, Vitucci A, Grill S, Ahong XY, Holzgreve W, et al. Size fractionation of cell free DNA in maternal plasma improves the detection of a paternally inherited 74. 75. 76. 77. Reprint requests: Dr Roshan Colah, Scientist F, National Institute of Immunohaematology (ICMR), 13th Floor, NMS Bldg, KEM Hospital Campus, Parel, Mumbai 400 012, India e-mail: [email  protected] com