Impact of h globin gene mutations on the clinical phenotype of h thalassemia in India

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1 Blood Cells, Molecules, and Diseases 33 (2004) Impact of h globin gene mutations on the clinical phenotype of h thalassemia in India Roshan Colah*, Anita Nadkarni, Ajit Gorakshakar, Supriya Phanasgaonkar, Reema Surve, P.G. Subramaniam, Nagnath Bondge, Kamala Pujari, Kanjaksha Ghosh, Dipika Mohanty Institute of Immunohaematology (ICMR), K.E.M. Hospital Campus, Parel, Mumbai , India Submitted 17 May 2004 (Communicated by E. Beutler, M.D., 20 May 2004) Abstract The h thalassemias are one of the commonest group of autosomal recessive disorders in India. Although majority of patients are severe and transfusion-dependent, about 10 15% of cases have a milder phenotype. We evaluated the role of h gene mutations in modulating the clinical presentation of 342 h thalassemia patients which included 278 severe thalassemia major (TM) and 64 thalassemia intermedia (TI) cases (severe TI: 27; mild TI: 37) from this region. Thirteen h thalassemia mutations were characterized by reverse dot blot hybridization or amplification refractory mutation system (ARMS); denaturing gradient gel electrophoresis (DGGE) analysis and DNA sequencing helped to characterize the remaining nine mutations. Majority of the patients in the thalassemia major and thalassemia intermedia groups had severe h + or h 0 mutations. IVS 1-5 (GYC) was the commonest mutation in the three groups. The six severe and common Indian mutations [(IVS 1-5 (GYC), 619 bp deletion, IVS 1-1 (GYT), codons 8/9 (+G), codon 15 (GYA), codons 41/42 (-CTTT)] accounted for 92.0% of molecular lesions in the thalassemia major group, 86.8% in the severe TI group, and 72.9% in the mild TI group. IVS 1-1 (GYT) and codon 30 (GYC) were significantly more common in thalassemia intermedia cases. The mild capsite +1 (AYC) mutation was present in both severe and mild cases. Three other mild h + mutations, poly A (TYC), -28 (AYG), and -88 (CYT), were seen only in the thalassemia intermedia cases. These four mild mutations in combination with other severe h + or h 0 mutations resulted in a very variable clinical presentation. This study reveals that, in majority of Indian patients, the h genotype cannot predict the phenotype. D 2004 Elsevier Inc. All rights reserved. Keywords: h thalassemia mutations; Phenotypes; India Introduction * Corresponding author. Institute of Immunohaematology (ICMR), 13th Floor K.E.M. Hospital Campus, Parel, Mumbai , India. Fax: address: colah@vsnl.com (R. Colah). The h thalassemias are an extremely heterogeneous group of single gene disorders due to an absence or reduction of h globin synthesis. Homozygotes and compound heterozygotes exhibit a varied clinical expression ranging from the severe transfusion-dependent disorder from early infancy or thalassemia major to the relatively milder thalassemia intermedia phenotype [1]. This diversity is primarily related to the degree of imbalance of globin chain synthesis which could be due to the nature of the h gene mutations or due to interaction with a or g loci [1]. In this study, we have characterized the h thalassemia mutations in severe and mild cases to evaluate their contribution in clinical diversity in Indian patients /$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi: /j.bcmd

2 154 R. Colah et al. / Blood Cells, Molecules, and Diseases 33 (2004) Material and methods The study included 342 patients who were referred from different hospitals. Our patients were classified into severe thalassemia major (TM) and the milder thalassemia intermedia (TI) based on (1) the age of onset of symptoms, (2) degree of anemia and age when first transfused, (3) transfusion requirements per year, and (4) hepatosplenomegaly, growth, and development. The TM group included patients who presented within 2 years of age and required regular blood transfusions for survival. The TI group included patients who presented between 2 and 30 years and were able to maintain their hemoglobin levels between 7 and 9 g/dl without transfusion or required infrequent transfusions. There were 278 severe cases (TM) and 64 milder ones (TI). In the thalassemia major (TM) group, majority of the patients (91.7%) presented within 6 months to 1 year of age, while the remaining became symptomatic between 1 and 2 years, and all of them required 8 to 12 blood transfusions per year. The age at presentation and transfusion requirements of the thalassemia intermedia (TI) cases was very variable. Twenty-seven cases (42.1%) presented between 2 and 4 years (severe thalassemia intermedia). Of these, 22.2% maintained their hemoglobin level between 7 and 9 g/dl without transfusions; 44.5% of cases required intermittent transfusions (1 4 per year), and 33.3% of cases became transfusion-dependent after 3 years of age (10 12 per year). Thirty-seven cases (57.9%) became symptomatic between 4 and 30 years (mild thalassemia intermedia). In this group, 64.9% of cases had a transfusion-independent survival, 21.6% of cases required intermittent transfusions, while 13.3% became transfusion-dependent between 4 and 10 years of age. Parents and siblings were also studied in most of the cases. Blood was collected in EDTA after informed consent. Hematological analysis was done including the complete blood count and measurement of HbA 2 and HbF by automated HPLC on the Variant Hemoglobin Testing System using the h Thal Short program (Biorad Laboratories, Hercules, CA, USA). DNA was extracted from peripheral blood leukocytes using a standard protocol [2]. The h thalassemia mutations were characterized using a combination of reverse dot blot hybridization for six common Indian mutations [IVS1-5 (GYC), IVS1-1 (GYT), codons 8/9 (+G), codon 15 (GYA), codon 30 (GYC), codons 41/42 (-CTTT)] [3], amplification refractory mutation system (ARMS) for the 619 bp deletion and six rarer mutations [capsite +1 (AYC), codon 16 (-C), codon 5 (-CT), codon 30 (GYA), IVS1-1 (G-A), IVSII-1 (G-A)] [4,5]. Denaturing gradient gel electrophoresis (DGGE) analysis was done in the remaining uncharacterized samples to localize the mutation [6], followed by DNA Table 1 h thalassemic alleles in thalassemia major and thalassemia intermedia cases Mutation Phenotype No. of chromosomes(%) Thalassemia major Thalassemia intermedia Severe IVS 1-5 (GYC) Severe h (52.5%) 22 (40.7%) 28 (37.8%) 619 bp Deletion h 0 79 (14.1%) 8 (14.8%) 4 (5.4%) IVS 1-1 (GYT) h 0 37 (6.7%) 9 (16.5%) 9 (12.2%) Codons 8/9 (+G) h 0 50 (9.0%) 5 (9.2%) 7 (9.4%) Codons 41/42 (-CTTT) h 0 21 (3.8%) 1 (1.9%) 2 (2.7%) Codon 15 (GYA) h 0 30 (5.4%) 2 (3.7%) 4 (5.4%) Codon 30 (GYC) h 0 9 (1.6%) 2 (3.7%) 4 (5.4%) Capsite (+1) (AYC) Mild h + 7 (1.3%) 1 (1.9%) 4 (5.4%) IVS 1-1 (GYA) h 0 5 (0.9%) 0 0 Codon 30 (GYA) h 0 3 (0.5%) 0 0 IVS II-837 (TYG) h + or h 0 5 (0.9%) 1 (1.9%) 0 Codon 16 (-C) h 0 3 (0.5%) 1 (1.9%) 0 Codon 121 (GYT) h 0 2 (0.4%) 0 0 Codon 5 (-CT) h 0 6 (1.1%) 0 0 IVS II-1 (GYA) h 0 2 (0.4%) (CYT) Mild h (2.7%) Poly A (AATAAAYAACAAA) Mild h (1.9%) 1 (1.4%) -28 (AYG) Mild h (1.4%) IVS (GYC) h (1.4%) Codon 55 (-A) h 0 1 (0.2%) bp Deletion codons h 0 2 (0.4%) 0 0 IVS II-654 (CYT) h 0 1 (0.2%) 0 0 yh Thal 0 1 (1.9%) 2 (2.7%) No mutation in the h gene (4.0%) Mutation uncharacterized (uc) 1 (0.21%) 2 (2.7%) Total Mild

3 R. Colah et al. / Blood Cells, Molecules, and Diseases 33 (2004) sequencing by Sanger dideoxy chain termination method or on the ABI Prism 310 DNA sequencer (Applied Biosystems, Foster City, CA, USA) [7]. Results Twenty-two h thalassemia mutations could be characterized (Table 1) The six common Indian mutations, IVS1-5(GYC), 619 bp deletion, IVS 1-1 (GYT), codons 8/9 (+G), codons 41/42 (-CTTT), and codon 15(GYA), accounted for 92.0% of the molecular lesions in the TM group, 86.8% in the severe TI group, and 72.9% in the mild TI group. The severe h + mutation, IVS 1-5 (GYC) was more common in the thalassemia major cases. The h 0 mutation, 619 bp deletion, was equally prevalent in thalassemia major and severe TI cases but not in the milder TI group. The h 0 mutations, IVS1-1 (GYT) and codon 30 (GYC), were more common in thalassemia intermedia cases. The milder capsite mutation was present in a compound heterozygous state in thalassemia major and thalassemia intermedia cases, although it was more prevalent in the milder TI group. The other milder mutations, -88 (CYT), poly A (AATAAA- YAACAAA), and -28 (AYG), were seen only in thalassemia intermedia cases. The rare mutations IVS1-1 (GYA), codon 30 (GYA), codon 121 (GYT), codon 5 (-CT), IVS II- 1(GYA), codon 55 (-A), 17 bp deletion, and IVS II 654 (CYT) were seen only in the TM group. Three cases of thalassemia intermedia were compound heterozygotes of yh thalassemia and h thalassemia. In another three cases in this group, only one mutation was present after scanning the entire h globin gene and its flanking regions by DGGE and they were able to maintain their Hb levels between 7 and 8 g/dl. In three chromosomes (TM-1, TI-2), the mutation was localized by DGGE but remained uncharacterized. Table 2 shows the h genotypes seen in the three groups. The severe h + /severe h + genotype was more common in the TM group (42.1%) compared to both the severe TI group (25.9%) and the mild TI group (18.9%). However, the prevalence of the severe h + /h 0 genotype was lower in the Table 2 h Globin genotypes in thalassemia major and thalassemia intermedia cases Genotype Thalassemia major Thalassemia intermedia Severe Mild Severe h + /Severe h (42.1%) 7 (25.9%) 7 (18.9%) Severe h + /h 0 60 (21.6%) 8 (29.7%) 4 (10.9%) h 0 /h 0 94 (33.8%) 10 (37.0%) 11 (29.7%) Severe h + /mild h + 2 (0.7%) 1 (3.7%) 4 (10.8%) Mild h + /h 0 4 (1.4%) 0 4 (10.8%) h 0 /yh (5.4%) Mild h + /yh 0 1 (3.7%) 0 Severe h+/uc (13.5%) Mild h+/uc 1 (0.4%) 0 0 Total Table 3 Clinical phenotypes in compound heterozygotes having one mild h + thalassemia mutation Mutations mild TI group, while the prevalence of the h 0 /h 0 genotype was not significantly different in the severe and milder patients. A mild h + mutation was more common in the milder thalassemia intermedia cases. We analyzed the clinical phenotypes arising from compound heterozygosity of four of the milder mutations along with a second mutation (Table 3). The mild h + capsite +1 (AYC) mutation was found to be present along with seven other mutations with a variable clinical presentation. Majority of patients (8 of 12) inheriting this mild mutation along with the severe h + mutation IVS 1-5 (GYC) or other h 0 mutations had a severe transfusion-dependent disorder from 6 to 11 months of age. The other mild mutations [-88 (CYT), -28 (AYG), and poly A (TYC)] in combination with severe h +, h 0,oryh thalassemia mutations resulted in a thalassemia intermedia phenotype. None of our patients had inherited two mild alleles. Discussion Capsite+1 (AYC) -88 (CYT) -28 (AYG) PolyA (AATAAA YAACAAA) IVS 1-5 TM a -2 TI b -1 TI c -1 (GYC) TI d bp Deletion TM a -1 Codons 8/9 (+G) TI d -1 Codons 41/41 (-CTTT) TM a -1 IVS 1-1 (GYT) TM a -1 TI e -1 TI d -1 Codon 16 (-C) TM a -1 IVS (GYC) TI f -1 Gg(Agyh) 0 Thal TI g -1 uc TM a -1 a Severe transfusions /year from 6 11 months onwards. b 6 years, untransfused. c 4 years, transfused at 3 years. d 4.5 to 16 years, untransfused or 1 2 transfusions per year, Hb 7 9 g/dl. e 4 years, asymptomatic, Hb 10.8 g/dl. f 11 years, intermittent transfusion after 7 years, Hb 8 9 g/dl. g 2 years, untransfused Hb 8 9 g/dl. In many population groups worldwide, the most important contributing factor for a mild clinical presentation in h thalassemia homozygotes and compound heterozygotes is a mild h + thalassemia mutation. There are several mutations at different positions upstream of the h globin gene which reduce the severity of the disease. These include the -88 (CYT) mutation in American Blacks and Asian Indians, -101 (CYT) in Italians and Mediterranean groups, -28 (AYG) in Chinese, -29 (AYG) in the Black population, and the capsite +1 ( AYC) mutation in Asian Indians. The

4 156 R. Colah et al. / Blood Cells, Molecules, and Diseases 33 (2004) other mild h + mutations are those in the consensus sequence required for polyadenylation [8]. In Israeli patients with TI, a compound heterozygosity with the -101 (CYT) mutation led to a consistently mild phenotype [9]. In Lebanon, 68% of mild cases had one of the mild mutations, IVS 1-6, codons 29, -88, or -87 [10]. In Italian patients from eastern Sicily, two mutations IVS 1-6 (TYC) and codon 6 (-A) were statistically prevalent in TI cases [11]. In Southern Iran, IVSII-1 (GYA) was the most prevalent mutation in milder cases (24%); however, a large number of alleles could not be characterized [12]. In our study, the severe h + mutation IVS1-5 (GYC) was the most common defect in the TI group, although its prevalence in the mild TI group was significantly lower than in the severe thalassemia major group ( P b 0.01). The prevalence of IVS1-1 (GYT) and codon 30 (GYC) were significantly higher ( P b and P b 0.025) in the mild thalassemia intermedia group compared to the TM group. In an earlier study, Ho et al. [13] had reported that IVS1-1 (GYT) was the most common mutation encountered in 50 Asian Indians with TI [13]. Twenty-two h thalassemia mutations were characterized among the 342 patients studied by us, and majority of the cases in both the TM and TI groups had severe h + or h 0 mutations. Codon 121 (GYT) has been reported as a dominant mutation in some populations [8]. In our series, the two alleles with this mutation were from a homozygote with a transfusion-dependent disorder. The parents were both heterozygous for codon 121 (GYT) and were asymptomatic. Codon 55 (-A) was first reported by us as a novel mutation [14]. This was present along with the IVS1-5 (GYC) mutation leading to a severe phenotype. Codon 55 (-A) has also been recently reported in four patients from Sri Lanka; three of whom were compound heterozygotes for the IVS1-5 (GYC) or codon 15 (-T) mutations and one was homozygous for this mutation [15]. The capsite +1 (AYC) mutation was first reported in Asian Indians and was associated with a mild form of h thalassemia [16]. In the present study, 12 cases were compound heterozygotes with the capsite mutation along with another mutation (Table 3). Seven of these cases had a severe TM phenotype and required regular blood transfusions from 6 to 11 months of age. This is contrary to an earlier report where all compound heterozygotes of the capsite mutation with a second mutation in Asian Indians had a thalassemia intermedia phenotype, although other ameliorating factors like a-thalassemia and the -158 (CYT) Gg mutation were absent in some of these cases [13]. One of our cases of thalassemia intermedia had the poly A(TYC) mutation along with the Gg (Agyh) 0 thalassemia Indian inversion (Table 3). This rare combination of mutations has not been reported earlier. This child was diagnosed at the age of 2 years and was maintaining a hemoglobin level of 8 9 g/dl without transfusions. However, he was later on transfused after 4 years of age. The poly A (TYC) mutation was first reported by Orkin et al. [17] in a Black family. Subsequently, it was found along with the IVS II-1 (GYA) mutation in a Turkish family where three children had moderately severe anemia [18]. This mutation has been recently seen in seven alleles in Sri Lanka [15]. The two other milder alleles that we found in the TI group were the -88 (CYT) mutation in compound heterozygosity with IVS1-130 (GYC) another very rare mutation in Indians and the -28 (AYG) mutation which was reported by us for the first time in an Indian along with the common IVS 1-5 (GYC) mutation [19]. These mild alleles have been reported in other population groups [20]. In three TI chromosomes, the entire h globin gene and its flanking regions were normal by DGGE analysis, and the mutation may lie elsewhere, possibly in the locus control region. Such uncharacterized alleles are found in many populations [8]. In India, majority of the molecular diagnostic centers undertake h thalassemia genotyping, but a genotyping is not routinely done. Our aim was to evaluate whether knowing the h globin genotype would help the clinician in predicting the severity of the disease in a large series of patients. In our study, only 7.4% of severe thalassemia intermedia cases and 21.6% of mild thalassemia intermedia cases had a mild h thalassemia mutation in combination with another severe mutation, and mutations per se are not the major contributor to a milder clinical presentation in Indian patients. Thus, the knowledge of the h globin genotype alone may not be sufficient in clinical practice for prediction of the phenotype during genetic counseling in Indian patients. Acknowledgments We thank all the hematologists and pediatricians from different hospitals for referring the patients. References [1] D.J. Weatherall, J.B. Clegg (Eds.), The Thalassemia Syndromes, Blackwell Science, Oxford, 2001, pp [2] J.M. Old, D.R. Higgs, The thalassemias, in: D.J. Weatherall (Ed.), Methods Hematol., vol. 6, Churchill Livingstone, Edinburgh, 1983, pp [3] R.B. Colah, A.C. Gorakshakar, C.Y. Lu, A.H. Nadkarni, S.N. Desai, A.R. Pawar, C.P. Lulla, R. Krishnamurthy, D. Mohanty, Application of covalent reverse dot blot hybridization for rapid prenatal diagnosis of the common Indian thalassemia syndromes, Indian J. Hematol Transfus. Med. 15 (1997) [4] N.Y. Varawalla, J.M. Old, R. Sarkar, R. Venkatesan, D.J. 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