Regional heterogeneity of β-thalassemia mutations in the multi ethnic Indian population

Blood Cells, Molecules, and Diseases 42 (2009) 241–246

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Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y b c m d

Regional heterogeneity of β-thalassemia mutations in the multi ethnic Indian population Roshan Colah ⁎, Ajit Gorakshakar, Anita Nadkarni, Supriya Phanasgaonkar, Reema Surve, Pratibha Sawant, Dipika Mohanty, Kanjaksha Ghosh National Institute of Immunohaematology (ICMR), 13th Floor, NMS Building, KEM Hospital Campus, Parel, Mumbai – 4000 12, India

a r t i c l e

i n f o

Article history: Submitted 7 November 2008 Available online 28 February 2009 (Communicated by Sir D. Weatherall, F.R.S., 15 December 2008) Keywords: β-Thalassemia Spectrum of mutations India

a b s t r a c t To determine the frequencies of β-thalassemia mutations in different states of India and to compare this with the available data in Asian Indians for a comprehensive catalogue of molecular defects in the Indian population. β-thalassemia mutations were characterized in 2456 heterozygotes using reverse dot blot hybridization, ARMS and DNA sequencing. 36 β-thalassemia mutations were characterized from 18 different states in India. Seven mutations were common, accounting for 95.8% of mutated alleles. Marked regional diversity was seen in different parts of the country. Among the tribal populations, only 2 mutations (IVS I-5 (G → C) and CD15 (G → A) accounted for over 90% of mutant alleles. A compilation of all the studies in Asian Indians reported so far showed the presence of 63 mutations in the Indian population. This large study adds to the existing data to give a detailed account of the molecular basis of β-thalassemia in India. This information is important for establishing prenatal diagnosis programmes in different states in India as well as other countries in which there is a major influx of Indian immigrants. © 2009 Elsevier Inc. All rights reserved.

Introduction The β-thalassemias are a heterogeneous group of inherited disorders of hemoglobin synthesis and are most common in the Mediterranean region, the Indian subcontinent, South East Asia and the Middle East [1]. The first case of β-thalassemia from India was reported by Mukherji in 1938 [2]. Numerous studies undertaken subsequently have shown that the prevalence of β-thalassemia varies from 1 to 17% in different population groups with an overall frequency of 3–4%. Based on this, it is estimated that there would be 30–40 million carriers and almost 8,000 to 10,000 births each year with severe forms of β-thalassemia. This translates to about 10% of the total thalassemia homozygotes or compound heterozygotes born globally and results in a significant national health burden [3,4]. With gradual control of malnutrition and communicable diseases, children with β-thalassemia major who earlier died young are now surviving and requiring treatment. However, adequate blood transfusions with effective iron chelation are affordable by only about 10% of the cases. This emphasizes the need for a widespread community control programme to reach out to the urban and rural population. Knowing the underlying mutations causing β-thalassemia and their geographic distribution is important for this programme.

⁎ Corresponding author. Fax: +91 22 24138521. E-mail address: [email protected] (R. Colah). 1079-9796/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2008.12.006

Although several studies have been done in the Indian population to analyze the spectrum of mutations, all regions have not been adequately covered. We have undertaken a detailed analysis of the molecular basis of β-thalassemia in India in both caste and tribal populations originating from different regions of the country.

Materials and methods This study includes 2456 β-thalassemia heterozygotes who were identified during screening programmes in schools, colleges, and antenatal clinics as well as couples referred for prenatal diagnosis from various regions of the country over a period of 12 years. 5 ml blood was collected in EDTA after informed consent. Diagnosis of carriers was based on a blood count and hemoglobin analysis with quantitation of HbA2 and HbF on the Variant Hemoglobin Testing system using the “β-thalassemia short programme” (Bio-Rad Laboratories, Hercules, CA, USA). DNA isolation followed the standard phenol chloroform method. The strategy for molecular analysis was to first screen for 6 common Indian β-thalassemia mutations [IVS1–5(G → C), IVS1–1 (G → T), CD 8/9(+G), CD 41/42(−CTTT), CD 15(G → A), CD 30 (G → C)] along with HbS and HbE using reverse dot blot hybridization (RDB) [5]. In the next step we screened for the 7th common mutation, the 619 bp deletion and 28 other known but less common mutations reported among Indians using the amplification refractory mutation system (ARMS) [6]. The majority of the uncharacterized samples were

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R. Colah et al. / Blood Cells, Molecules, and Diseases 42 (2009) 241–246

Table 1 Percentage distribution of β-thalassemia mutations in different states of India States

IVS 1–5 (G → C)

619 IVS 1–1 bp del (G → T)

CD 8/9 CD41/42 (+ G) (− CTTT)

CD15 CD 30 Cap site + 1 CD 5 CD 30 IVS 1–1 (G → A) (G → C) (A → C) (− CT) (G → A) (G → A)

Maharashtra Gujarat Rajasthan Madhya Pradesh Chattisgarh Uttar Pradesh Punjab/Haryana Himachal Pradesh/ Jammu Kashmir West Bengal Bihar Jharkhand Orissa Karnataka Goa Andhra Pradesh Tamil Nadu /Kerala Immigrants (Pakistan) Total

66.5 48.6 72.4 36.9 35.3 64.8 28.8 50.0

1.7 12.6 2.3 19.9 23.5 1.6 7.7 –

1.7 6.5 1.1 10.6 23.5 0.8 9.6 –

0.7 5.0 3.3 11.3 5.9 5.6 17.3 –

2.0 6.9 9.9 5.7 – 9.6 13.5 37.5

17.2 6.1 3.3 5.0 – 4.8 – –

4.2 1.9 1.1 4.3 – 2.4 – –

1.3 2.2 3.3 1.4 5.9 1.6 5.8 12.5

77.2 70.0 75.0 88.6 67.2 15.4 78.8 56.3 15.1

– – – – 0.8 – – – 37.2

1.0 – – 1.2 – – – – 25.1

4.0 – – – 1.6 – – – 15.5

4.0 15.0 – 2.6 0.8 – – 6.31 4.5

2.9 – 8.3 – 11.2 – 6.1 – 0.3

4.0 15.0 16.6 1.2 2.4 23.1 6.1 – 0.3

54.5

9.5

6.2

4.9

4.8

8.2

2.9

IVS II − 837 CD 16 (T → G) (− C)

Othersa UC

Total

0.1 7.3 – – – – 1.9 –

0.8 0.5 – 0.7 – – 5.8 –

0.9 0.5 – 0.7 – 0.8 1.9 –

0.3 – – – – – – –

0.3 0.3 2.2 2.1 5.9 1.6 1.9 –

1.9 0.9 – 0.7 – 4.8 5.8 –

0.4 0.7 1.1 0.7 – 1.6 – –

748 586 90 141 17 125 52 8

2.0 – – – – – 3.0 – 0.3

– – – – – – – – –

– – – 1.2 – 7.7 – – 1.7

– – – – – – 3.0 – –

– – – – 4.0 53.8 – – –

2.0 – – 2.6 – – – – –

2.9 – – – 10.4 – 3.0 37.4 –

– – – 2.6 1.6 – – – –

101 20 12 78 125 13 33 16 291

1.6

1.8

0.8

0.6

0.6

0.7

2.1

0.8 2456

a

Others. Maharashtra: CD – 110 (T → C) − 0.5, CD 111 (− G) − 0.28, IVS 1–130 (G → A) − 0.28, CD 55 (− A)− 0.14, IVS 1 (25 bp del) − 0.14, poly A (T → C) − 0.14, CD 15 (− T) − 0.14, CD 26 (C → T) − 0.14, CD 16 (C → T) − 0.14. Gujarat: Poly A (T → C) − 0.35, IVS 1–130 (G → C) − 0.18, IVS II 654 (C → T) − 0.18, CD47/48 (+ ATCT) − 0.18. Madhya Pradesh: CD126− 131 (− 7 bp) − 0.7. Uttar Pradesh: Poly A (T → C) − 1.6, − 88 (C → T) − 1.6, IVS 1–130 (G → C) − 0.8, CD 22–24 (− 7 bp) − 0.8. Punjab/Haryana/: − 88 (C → T) − 0.19, − 87 (C → T) − 0.19, IVS 1–110 (T → C)− 0.19. West Bengal: CD 15 (− T) − 1.92, − 90 (C → T) − 0.96. Karnataka: CD 15 (− T) − 2.4, poly A (T → C) − 1.6, IVS II − 1 (G → A) − 1.6, CD 121 (G → T) − 1.6, − 88 (C → T) − 0.8, CD 39 (C → T) − 0.8, CD 8 (− AA) − 0.8, IVS II 613 (C → T) − 0.8. Andhra Pradesh: Poly A (T → C) − 3.0. Tamil Nadu: IVS 1 (25 bp del) − 12.5, IVS 1–30 (G → C)− 12.5, IVS II − 613 (C → T) − 6.25, IVS II 745 (C → G) − 6.25. U.C: Uncharacterized.

then directly sequenced on an ABI-310 DNA sequencer (Applied Biosystems, Foster City, CA) using the Big Dye terminator kit. Results The overall distribution of the β-thalassemia mutations in different states of India as well as in the immigrant population from Pakistan

settled in different regions of India is summarized in Table 1. 36 mutations were identified among the 2456 β-thalassemia alleles while 20 alleles (0.8%) remained uncharacterized. Seven mutations were common [IVS1–5(G → C)−54.5%; 619 bp del−9.5%; CD15(G→A)−8.2%; IVS1–1(G→T)−6.2%; CD8/9(+G)–4.9%; CD41/42(−CTTT)−4.8%;CD30(G → C)−2.9%] and accounted for 95.8% of the β-thalassemia alleles. However, their distribution was uneven

Fig. 1. Number of β-thalassemia mutations detected in different states of India.

R. Colah et al. / Blood Cells, Molecules, and Diseases 42 (2009) 241–246

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Fig. 2. Regionwise distribution of β-thalassemia mutations in India.

in different states. Although IVS1–5(G→ C) was the most common mutation, its frequency varied from 15% in the immigrant population and in Goa on the west coast to 88.6% in the eastern state of Orissa. Three mutations, the 619 bp deletion, IVS1–1(G → T) and CD 8/9 (+G), were frequent in the immigrant groups (77.8%): Madhya Pradesh (41.8%) and Chattisgarh (52.9%) in Central India; Gujarat (24.1%) in Western India; and Punjab (34.6%) in the North. They were predominantly seen in the Lohana and Sindhi communities. CD15(G → A) was the second most common mutation in Maharashtra in the West (17.2%) and in the neighbouring southern state of Karnataka (11.2%). In Rajasthan, Uttar Pradesh and Himachal Pradesh/Jammu Kashmir the second most common mutation was CD 41/42(−CTTT) (9.9% 9.6% and 37.5% respectively) while in Bihar in the East both CD 41/42 (−CTTT) and CD 30(G → C) (15.0%) were seen in equal numbers. Goa showed a unique picture with the IVS II-837(T → G) mutation being the most common (53.8%) followed by CD 30(G → C) in 23.1% of alleles, IVS 1–5(G → C) in 15.4% and CD 30 (G → A) in 7.7% of alleles. These were the only mutations seen. IVS II-837(T → G) was also identified in the neighboring coastal areas in Karnataka (4.0%) and occasionally in Maharashtra (0.3%). This mutation was not seen in any other region and the main community having this mutation was Gaud Saraswat Brahmin (GSB).

The capsite mutation often results in a normal or borderline HbA2 level and most of the carriers of this mutation were one of the partners among the couples referred for prenatal diagnosis. This mutation was seen in 1.6% of β-thalassemia alleles and was more common in Himachal Pradesh/Jammu Kashmir (12.5%), Chattisgarh (5.9%) and Punjab (5.8%).

Table 2 Distribution of β-thalassemia mutations among tribal population groups States

CD 15 (G → A)

Others

Maharashtra 28

24

Gujarat Karnataka

16 12

14 –

5

–

CD 41/42 (− CTTT) − 2, Capsite + 1 (A → C) − 1, IVS I-3′ end (25 bp del) − 1, CD 55 (− A) − 1 CD 8/9 (+G) − 1, poly A (T → C)− 1 CD 41/42 (− CTTT) − 1, poly A (T → C)-1, CD 8 (− AA) − 1 IVS 1 − 1 (G → A) − 1

4

–

–

– 38 (32.5%)

– 11 (9.4%)

Andhra Pradesh Madhya Pradesh Bihar Total

IVS 1–5 (G → C)

3 68 (58.1%)

Total 57

32 15 6 4 3 117

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R. Colah et al. / Blood Cells, Molecules, and Diseases 42 (2009) 241–246

Table 3 Spectrum of β-thalassemia mutations in the Indian population

No. of alleles − 90 (C → T) − 88 (C → T) − 87 (C → T) − 80 (C → T) − 29 (A → G) − 28 (A → G) − 25 (A → G) Capsite + 1 (A → C) Int CD (T → C) CD 4 (T → A) + CD 5 (C → T) + CD 6 (G → T) CD 5 (− CT) CD5(−CT)+CD 13(C→T)+CD26 (C→G)+CD27/28(+C) CD7/8 (+G) CD8 (A → G) CD 8 (− AA) CD 8/9 (+G) CD 13 (C → T) CD13(C → T) + CD26 (G → A) + CD27/28 (− C) CD 15 (G → A) CD 15 (− T) CD16 (− C) CD 16 (C → T) CD 17 (A → T) CD22/24 (− 7 bp) CD26 (G → T) CD 27/28 (+ C) CD30 (G → A) CD 30 (G → C) IVS I-1 (G → A) IVS I-1 (G → T) IVS I-5 (G → C) IVSI-110(G → A) IVSI-128(T → G) IVSI-129(A → C) IVSI-130(G → A) IVSI130(G → C) IVS I-25 bp del CD 36/37 (− T) CD36–39 (− 8 bp) CD 39 (C → T) CD 41 (− C) CD41/42(− CTTT) CD 44 (-C) CD47/48(+ ATCT) CD 55 (− A) CD 55 (+A) CD 57/58 (+C) CD 62/64 (− 7 bp) CD 81/87 (− 22 bp) CD 88 (+T) IVS II − 1 (G → A) IVSII 591 (T → C) IVSII 613 (C → T) IVS II 654 (C → T) IVSII 745 (C → G) IVSII 837 (T → G) CD 106/107 (+ G) CD 110 (T → C) CD 111 (− G) CD 121 (G → T) CD126/131(− 17 bp) Poly A (T → C) 619 bp del No mutation UC

Present study

A7

B14

C15

D9,10

E16

F17

G18

H19

I20

J11

K21

L22

M23

N12

O24

P13

Other 25–30

2456 0.04 0.16 0.04 – – 0.04 – 1.6 – –

44 – – – – – – – – – –

102 – 1.9 – – – – – 1.9 – –

110 – 0.9 – – – – – 1.8 – –

702 – 0.2 – – – – – 0.3 – –

146 – – – – – – – – – –

57 – – – – – – – – – –

94 – – – – – – – – – –

80 – – – – – – – 1.3 – –

311 – 1.2 – – – – – 3.5 – –

1050 – – – – – – – 1.1 – –

92 – – – – – – – – – –

356 – 0.3 – – – 0.3 – 2.5 – –

376 – – – – – – – 0.8 – S

1223 – 0.16 – – – – – 0.73 – –

114 – – – – – – – – – –

1545 0.84 0.19 0.06 0.06 0.06 1.55 0.06 0.64 0.13 –

– – – – – – – – – – –

– –

– –

– –

S –

– –

– –

– –

– –

–

–

– – –

– – –

– – –

– – – 16.4 2.7 3.6 13.8 – – – – – – – –

– – – 2.8 – –

– – – 11.8 – –

– – –

2.3 – – 0.6 – – – – – – – – – – – S – 0.9 – – – 10.0 – 38.3 87.7 S – – – – – – – – – – – – – – – – – – – 9.7 8.9 – – – – – – – – – – – – – – – – 0.3 – – – – – – – – – S – – – – – – – – – – – – – 19.2 0.7 – – – –

8.8 – – – – – – – – 3.8 – – 67.9 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 3.8 – 11.6

1.8 –

–

– –

0.04 – – 0.04 – 4.9 2.3 – –

– – –

– – – 19.6 16.5 – – – –

8.1 2.3 4.9 0.12 – – 0.7 2.3 0.9 0.04 – – – – – 0.04 – – 0.04 – – – – – 0.8 – – 2.9 – – 0.6 – – 6.2 2.3 13.6 54.4 34.0 22.3 0.2 – – – – – – – – 0.08 – – 0.24 – – 0.12 2.3 – – – – – – – 0.04 – – – – – 4.8 6.8 11.6 – – – 0.04 – – 0.04 – – – – – – – – – – – – – – – – – 0.08 – – – – – 0.04 – – 0.04 – – 0.04 – – 0.6 – – – – – 0.20 – – 0.08 – – 0.08 – – 0.04 – – 0.32 – – 9.7 29.5 21.4 – – – 0.8 18.2 1.9

S — Sporadic, Figs are in percentages.

2.7 –

–

4.5 – – – – – – – – 5.4 35.5 – – – – – 0.9 – – – – 9.1 – – – – – – – – – – – – – – – – – – – – 22.7 – –

– –

– – 5.5 – – – – – – – – – – – – – – – – – – 19.2 60.1 25.5 – – – – – – – – – – – – – – – – – – – – 7.2 9.6 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 5.5 31.9 – – 18.1 –

0.96

10.1 – –

3.5 –

– 1.9

– – – – – – 0.32 0.64 13.5 32.7 – 0.32 – – – – – – – – – – 0.32 – 0.32 – – – – – – – – – – – – – – – – 17.6 – 0.23

0.4 – –

– – – – –

–

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – 13.0 – –

0.6 – – 0.8 – – – – – – 0.4 – 0.5 – – 15.8 19.6 34.7 22.8 – – – – – – – – – – – – 0.7 – – – – – – – 1.6 – – – – – – – – – – – – 21.0 34.8 1.8 –

–

0.3 S –

–

–

–

– – –

– – –

– – –

– – –

– – –

8.2 – –

5.3 – –

3.4 – – – – – – –

0.81

6.24 – –

2.9 –

2.7 – – – – S 2.5 – 2.0 1.1 0.6 S 6.5 4.5 48.6 58.0 – – – – – – – – – – 0.3 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.6 – – – – – 15.2 10.1 0.8 – 1.4 2.9

0.87

3.5 – –

3.81 – 1.22 – – – – – 0.24 1.38 0.32 11.35 42.5 – – – – – – – – – – – 0.08 0.16 – – – – – – 0.16 – – – – – – – – – – – 21.25 – 2.68

13.1 – – – – – – – – 1.75 – – 71.9 – – – – – – – – – – – – – – – – – – – – – – – – 0.87 – – – – – – 1.75 – –

0.26 – S

1.42 – –

– S S – S S

7.83 – 0.39 – 1.29 – –

–

–

–

– – – –

– – – –

– – –

–

0.06 – 0.06 – 0.06 – – 0.45 – 4.33 – 0.39 – 2.71 – 63.17 – 0.19 – – 0.06 – 0.12 – 0.12 – 0.06 – 0.06 – S 0.06 – 0.26 – 3.49 – 0.19 – – – – S 0.12 – 0.06 – S 0.12 – S 0.12 – – – 0.97 – 0.06 – 0.26 – – – – 3.5 – 3.75 – – 0.19 –

R. Colah et al. / Blood Cells, Molecules, and Diseases 42 (2009) 241–246

The CD 5(− CT) mutation was predominantly seen in Gujarat (7.3%). It was the third most common mutation in this state being particularly frequent among the Prajapati caste group. Fig. 1 shows the total number of mutations seen in each state. The maximum heterogeneity was seen in Maharashtra (22 mutations) followed by Gujarat (16 mutations) and Karnataka (15 mutations). The highest percentage of rare mutations was from Karnataka (10.4%). The data were also analyzed according to 5 regions of the country (Fig. 2). 1424 alleles were from the Western region (Maharashtra, Gujarat and Rajasthan) followed by 211 alleles from the Eastern region (West Bengal, Orissa and Bihar), 187 from the Southern region (Goa, Tamilnadu, Kerala and Andhra Pradesh), 185 from the Northern region (Uttar Pradesh, Punjab, Haryana, Himachal Pradesh and Jammu and Kashmir) and 158 from the Central region (Madhya Pradesh and Chattisgarh), while 291 alleles were from immigrants from the Sindh and Punjab regions of Pakistan. The frequency of IVS 1–5(G → C), the most common mutation varied from 54.05% in the North to 80.57% in the East. The next most common mutation in each region was different i.e. CD 15(G → A) in the West (11.8%) and South (8.56%), 619 bp deletion in Central India (20.25%), CD 30(G → C) in the East (5.69%) and CD 41/42 (−CTTT) in the North (11.89%). 22 different mutations were seen in the South and almost 20% of the β-thalassemia alleles did not have the 7 common mutations. Among the 2456 β-thalassemia heterozygotes, 117 (4.8%) were tribal individuals. The distribution of mutations in the tribal population is shown in Table 2. 76% of those studied were from the tribal belts in Western India (Maharashtra and Gujarat) while the remainder belonged to other States. Only 2 mutations, IVS 1–5(G → C) seen in 58.1% of alleles and CD 15(G → A) seen in 32.5% of alleles accounted for 90.6% of the β-thalassemia alleles among the tribals. The 8 other mutations identified in the remaining 9.4% of alleles were CD 41/42 (−CTTT), CD 8/9(+G), Capsite +1(A→C), IVS 1–1(G→A), polyA (T→C), IVS 1–3′ end (25 bp del), CD 8 (−AA) and CD 55(−A). Table 3 shows the compilation of all the β-thalassemia mutations detected in this study along with the mutations reported among Indians is earlier reports. 63 mutations have been characterized in the Indian population so far. The frequencies of the common Indian mutations have been variable in different studies depending on the region from where the samples were studied. Discussion India is a vast and diverse country with a population of over a billion in which almost 20% of the world's infants, approximately 26 million, are born each year. Over thousands of years of its history there have been several invasions from the Middle East, Central Asia and the West as well as commercial interactions through trade routes with people of Central, Western and South East Asia, the Mediterranean region and Western Europe resulting in a remarkable racial and cultural mix as well as considerable genetic diversity. An intricate caste system and the practice of endogamy have been responsible for regional variations in the distribution of β-thalassemia mutations [7]. Although the presence of β-thalassemia has been reported in most of the population groups studied, with variable frequencies, the profile of mutations has not been fully elucidated. Kazazian et al., 1984 were the first to characterize 7 new mutations in Asian Indians in 44 chromosomes with 18.1% of mutations remaining uncharacterized [8]. The first large study on molecular characterization of β-thalassemia in Indians was done by Varawalla et al. in 1991 [9], in which 702 β-thalassemia carriers from 7 different regions were studied and 11 mutations were detected with 5 common mutations accounting for 93.6% of alleles. 2% of individuals who remained uncharacterized were subsequently shown to have 5 rare mutations, two of which were newly described [IVS II-837 (T → G) and

245

CD 88(+T)]. Three other mutations [CD 5(−CT), IVS 1–110(G → A) and CD 30(G → A)] were also first described in the Indian population [10]. Two other extensive studies undertaken in Northern and Western India identified 13 and 18 mutations in the Indian population, with 1.8 and 2.7% of the alleles respectively remaining uncharacterized [11,12]. Most of the other studies have been much smaller and region specific except for a recent analysis from South India where the majority of the samples studied were from the Southern region as well as WestBengal in the East and where several mutations were characterized [13] (Table 3). As seen in this table the spectrum of mutations in Indians is remarkably wide with 63 mutations reported so far which represents about 30% of mutations seen globally. Individual mutations also had a variable frequency in different studies [14–30]. Our study includes individuals from 18 states in India covering different regions of the country. It also includes β-thalassemia heterozygotes from Madhya Pradesh, Chattisgarh, Orissa, Jharkhand and Goa where much data is not available. This is also the first study to analyze the mutations among tribals from different regions which is important because 8.2% of the population of India (84.3million) is comprised of tribal people (Census of India 2001). IVS 1–5(G → C) and CD 15(G → A) are the two common mutations among the tribals. Only one earlier study of the Gond tribe in Central India described 4 mutations in this group (IVS 1–5(G → C), CD 41/42(−CTTT), CD 30(G → C), and IVS 1–1(G → A) [31]. We describe a much wider spectrum of mutations in different regions. CD 8(− AA) seen in a tribal individual from Maharashtra is common in the Middle East [32]. Three mutations, CD 16(C → T) in a heterozygote from Maharashtra, CD 55(− A) in a tribal individual from Maharashtra and IVS II-613 (C → T) in an individual from Karnataka have been reported by us earlier as novel mutations [33,34]. CD 55 (−A) was also subsequently reported from Sri Lanka [35]. Ten other mutations, though not novel were first reported in the Indian population by us in our earlier studies [−87(C→T), −90(C→T), CD 22/23/24(−7 bp del), CD 26 (G→T), IVS 1−130(G→C), IVS 1–130 (G → A), IVS II 654(C → T), IVS II-745(C → G), CD 121(G → T), CD 126– 131(−17 bp del] [33,34,22,36–38]. In spite of an extensive mutation search, 0.8% of thalassemia alleles remained unidentified. We have seen that not 5 mutations as thought earlier, but 7 mutations, are common covering 95.8% of mutant alleles in Indians. Using reverse dot blot hybridization we were able to identify 86.1% of the mutations in a single step. Our study as well as earlier ones has shown that a large number of rare mutations are encountered in the Southern and Western region of the country, particularly in Karnataka and Maharashtra. The knowledge of the geographic and ethnic distribution of β-thalassemia mutations in the Indian population has far reaching implications. It is not only important for expanding prenatal diagnosis programmes in the country but this information will also be valuable in other countries including the USA, Canada, Europe and Australia in which there is a major influx of Indian migrants who present as ethnic minorities for diagnosis, counseling and management. Acknowledgments This study was supported by the Indian Council of Medical Research (ICMR). We are most thankful to Professor. Sir D. J. Weatherall for his invaluable comments and suggestions. References [1] D.J. Weatherall, J.B. Clegg, The Thalassaemia Syndromes, fourth ed., Blackwell Sci., Oxford, 2001. [2] M. Mukherji, Cooley's anaemia (Erythroblastic or Mediterranean anaemia), Indian J. Pediat. 5 (1938) 1. [3] P.K. Sukumaran, Abnormal hemoglobins in India, In: N.N. Sen, A.K. Basu, J.B. Chatterjea Memorial Committee (Eds.), Trends in Haematology, J.B. Chatterjea Memorial Volume, Calcutta, 1975, pp. 225–261.

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