Short Report

VOLUME 16 NUMBER 6 November/December 2003

Distribution of C282Y and H63D mutations in the HFE gene in healthy Asian Indians and patients with thalassaemia major: [PDF]
Gurvinder Kaur, C. Chen Rapthap, M. Xavier,
R. Saxena, V. P. Choudhary, S. K. Reuben, N. K. Mehra

Background. Mutations in the HFE gene have been shown to be strongly associated with hereditary haemochromatosis, an autosomal recessive disease of iron overloading. The majority of patients with hereditary haemochromatosis possess a homo-zygous mutation C282Y that disrupts the binding of the HFE gene with b2 microglobulin and prevents its surface expression. Another HFE mutation H63D is known to increase the relative risk of developing hereditary haemochromatosis. This disease is rare in India although secondary haemochromatosis is commonly seen among children suffering from thalassaemia major. The status of HFE mutations has not been explored among Indians, particularly in patients with thalassaemia major. It is also possible that in India clinical haemochromatosis could be masked by iron deficiency.

Methods. We examined a cohort of 59 unrelated, healthy individuals from north India, 57 from south India and 75 thalassaemia major patients from north India for HFE mutations (C282Y and H63D) in cis/trans by the polymerase chain reaction sequence-specific primer method.

Results. The C282Y and H63D mutations in the HFE gene were rare among Indians. Although the HFE mutations were increased among patients of thalassaemia their effect on iron burden or disease pathogenesis remains unclear.

Conclusions. Hereditary haemochromatosis is rarely observed among Indians and so are the C282Y and H63D mutations in the HFE gene. Long term follow up studies would be required to determine whether the relatively higher frequency of these mutations among patients of thalassaemia has any influence on iron accumulation.
Natl Med J India 2003;16:309–10


Hereditary haemochromatosis is an autosomal recessive disorder of iron overload affecting approximately 5 per 1000 Caucasians while 1 in every 12 Caucasians in northwestern Europe is a carrier.1 It is a multigenic disorder with the possible involvement of several genetic loci that include HFE-1 (C282Y, H63D and S65C mutations in HFE at 6p21),2 HFE-2 (1q in juvenile haemochromatosis), HFE-3 (Y250X and other mutations in TFR-2 at 7q22),3 NRAMP-2 (duodenal metal transporter, DMT-1),4 SFT (stimulator of iron transport)5 and other unknown factors.

The HFE gene, originally called human histocompatibility leucocyte-H (HLA-H), is a non-peptide binding major histocompatibility complex (MHC) class I homologue that binds with b2 microglobulin and modulates iron transport by binding the transferrin receptor (TfR)6 in a pH-dependent manner. Two mutations in the HFE gene, namely (i) C282Y that results from a change in cysteine (C) at position 282 to tyrosine (Y), and (ii) H63D that results from a change in histidine (H) at position 63 to aspartate (D) disrupt cell surface expression of the mutant HFE gene, and alter binding with TfR, respectively. These mutations are strongly associated with predisposition to hereditary haemochromatosis and are also implicated in other disorders such as rheumatoid arthritis,7 type 2 diabetes mellitus,8 porphyria cutanea tarda9 and coronary heart disease.10

Considerable ethnic variation is observed in the frequency distribution of HFE mutations. Hereditary haemochromatosis is rare in India11 and the status of HFE mutations is virtually unexplored. On the other hand, iron overload is frequently observed among patients with thalassaemia major, a widely prevalent haemoglobinopathy.12 We investigated the prevalence of HFE mutations in two healthy populations from north and south India and in patients with thalassaemia major from north India.

This study was done on 116 healthy, unrelated subjects (59 from north India [from the states of Punjab, Delhi, Haryana and Uttar Pradesh] and 57 from south India [from the states of Kerala, Andhra Pradesh, Karnataka and Tamil Nadu]) and 75 patients with thalassaemia major from north India referred to the All India Institute of Medical Sciences, New Delhi. All the patients with thalassaemia major had received multiple blood transfusions and iron chelation therapy. Blood samples were collected after informed consent and processed for HFE genotyping using the polymerase chain reaction-sequence specific primer method and primer sequences as previously published.13 Four possible HFE genotypes, namely 63H-282C, 63H-282Y, 63D-282C, 63D-282Y, were analysed and their frequencies scored.

The relative frequencies of HFE mutations in the healthy subjects and the thalassaemia major patients were compared and the significance of association was tested by 2×2 contingency c2 analysis and Fischer exact text. Serum ferritin levels were also measured in 27 of the 75 thalassaemia patients using the standard biochemical procedure.14

Genotyping of C282Y and H63D mutations
The C282Y mutation was seen in only 1 of the 59 healthy subjects from north India (1.7%), but in none of those from south India. On the other hand, the allele frequency of H63D was 4 of 59 (6.8%) in subjects from north India, and 6 of 57 (10.5%) in those from south India. The C282Y and H63D mutations were observed in 3 (4%) and 9 (12%) patients with thalassaemia major, respectively.
An analysis of the genotypes of these mutations revealed that the frequency of genotype CY was higher among patients with thalassaemia major (6 of 75) than healthy controls (1 of 116; p=0.03). There was no significant difference in the prevalence of HH, HD or DD genotypes between thalassaemia patients and healthy controls. We did not encounter any compound heterozygotes among the subjects tested (Table I).

Analysis of ferritin levels
High ferritin levels (2050–11 000 mg/L) were observed in all but 1 (96.3%) of these patients. However, there was no correlation between C282Y and H63D mutations and serum ferritin levels.

Table I. HFE genotypes in healthy Indians and patients with thalassaemia major
Subjects from
North India
South India
Genotypes HD and DD represent the presence of mutation (H to D) at amino acid residue 63 in heterozygous and homozygous conditions, respectively. The genotypes CY and YY represent presence of mutation (C to Y) at amino acid residue 282 in the heterozygous and homozygous conditions, respectively.

We describe the occurrence of HFE gene mutations, C282Y and H63D, in two healthy populations from north and south India as well as thalassaemia major patients from north India. Generally these mutations occur at lower frequencies in Indians as compared with healthy Europeans.1 For example, the C282Y mutation occurs with a frequency of 6.4% in the British population,1 5.6% in the Hungarian,15 3.1% in the Polish16 and 3.9% in the Germans, but is rare in Greeks (1.4%) and is absent in the Finnish, African, Chinese and Turkish populations. Similarly, the frequency of H63D mutation is 12.8% in the British, 10.4% in Russians, 13.6% in Europeans, but only 2.8% in Chinese, and is absent in Papua New Guineans and Australian Aboriginals.

Homozygosity of the C282Y mutation has been reported in over 90% of patients with hereditary haemochromatosis in the UK by the UK Haemochromatosis Consortium, 82.3% in the USA,17 92.4% in Britain18 and in 100% of Australian patients with hereditary haemochromatosis and a positive family history.19 Thus, the C282Y mutation is a very good marker for hereditary haemochromatosis, given the fact that HFE is not the only gene responsible for this condition since the role of other factors (genetic, dietary and environmental) cannot be ruled out. Hereditary haemochromatosis is rarely seen in the Asia–Pacific region20 including India, Japan,21 China22 and Korea.23 The observed low frequencies of the C282Y and H63D mutations in these populations correlates with this observation.
Our results suggest that the relatively infrequent occurrence of the C282Y and H63D mutations among Indians could explain the absence or rare occurrence of hereditary haemochromatosis. These HFE mutations might be involved in the process of secondary iron overload observed in some patients with thalassaemia major.

We thank the Department of Biotechnology, Ministry of Science and Technology and the Indian Council of Medical Research for granting financial support for the study.

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All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India
Gurvinder Kaur, C. Chen Rapthap, M. Xavier,
N. K. Mehra Department of Transplant Immunology and Immunogenetics
R. Saxena, V. P. Choudhary Department of Haematology
Lady Doak College, Madurai, Tamil Nadu, India
S. K. Reuben Department of Biology and Research Centre
Correspondence to N. K. MEHRA;


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