Indian Journal of Human Genetics Medknow Publications on behalf of Indian Society of Human Genetics ISSN: 0971-6866 EISSN: 1998-362x Vol. 16, Num. 3, 2010, pp. 149-153

Indian Journal of Human Genetics, Vol. 16, No. 3, September-December, 2010, pp. 149-153

Original Article

Molecular analysis of human leukocyte antigen class I and class II allele frequencies and haplotype distribution in Pakistani population

T Moatter, M Aban, S Tabassum, U Shaikh, S Pervez

Department of Pathology & Microbiology, Aga Khan University Hospital, Karachi, Pakistan

Correspondence Address:S Pervez, Department of Pathology & Microbiology, Aga Khan University Medical Centre, Stadium Road, Karachi - 74800, Pakistan shahid.pervez@aku.edu

Code Number: hg10031

DOI: 10.4103/0971-6866.73408

Abstract

Keywords: DQB1, DRB1, human leukocyte antigen polymorphism, human leukocyte antigen, HLA-A, HLA-B, HLA-C, Pakistan, population genetics, sequence-specific oligonucleotide analysis

Introduction

The human leukocyte antigen (HLA) system spans a 4-Mb region on the short arm of chromosome 6, band p21.3. It consists of class I, II, and III non-overlapping segments which encode for cell-surface heterodimeric glycoproteins. ^[1] The genes for these antigens are highly polymorphic and play a central role in the immune response. ^[2] Allele and haplotype frequencies of the HLA loci differ among various human populations and the studies of polymorphism in HLA system are useful for tracing the evolution of different populations and identification of conserved combination of different alleles. ^[3],[4] Moreover, HLA alleles have been implicated to both disease susceptibility and progression and, in some instances, even resistance to certain diseases because of their crucial role in immune system. ^[5] Several disease association studies have shown association of different HLA alleles with the same disease in different populations. ^[6] HLA system also plays a major role in allograft rejection. HLA molecules expressed by transplanted organs are strongly immunogenic, and if not matched with donor HLA, are recognized as non-self and initiate T-cell proliferation and destruction of the transplanted organ, and in association with other cellular and antibody responses, may lead to graft rejection. ^[7] Therefore, for predicting the probability of finding compatible donors in unrelated bone marrow transplantation, it is valuable to have reliable estimates of HLA allele and haplotype frequencies.

In this study, we carried out molecular analysis of class I HLA-A, HLA-B, HLA-C and class II HLA-DRB1, HLA-DQB1 genes at medium resolution level in a population of Pakistani nationals, using polymerase chain reaction and sequence specific primers (PCR-SSP) technique. The distribution of various allele frequencies and the putative haplotypes are compared with those of different populations.

Materials and Methods

Subjects

In this study, we investigated a sample of 1000 individuals representative of the Pakistani population for the distribution of HLA class I and II alleles. The sample population of 1000 unrelated subjects (men, women) had a mean age 26.7 ± 9.5 years.

DNA extraction and HLA-A, B, C, DRB1 and DQB1 typing

Blood samples (5 ml each) of individuals who participated in this study were obtained in tubes containing ethylenediamine tetraacetic acid (EDTA) as anticoagulant. White blood cells were separated on Ficoll/Hypaque and DNA was extracted according to the method of Miller et al.[8] HLA alleles were identified using PCR-SSP technique as described by the manufacturer (One Lambda, Canoga Park, CA, USA). The HLA typing method used in this study could discriminate all of the HLA class I and class II alleles officially recognized by the WHO nomenclature committee for Factors of the HLA system. ^[9] Allele frequencies and haplotype frequencies were calculated using the software Pypop Win32-0.7.0. ^[10]

Results

The frequency distribution of HLA-A, -B, -C alleles was obtained by DNA typing of 1000 unrelated healthy Pakistani nationals comprising 340 females and 660 males, representing all the major regions of Pakistan. Sixteen distinct alleles of HLA-A, 28 alleles of HLA-B and 13 different alleles of HLA-Cw were identified [Table - 1]. The most frequent class I alleles observed were AFNx0102, BFNx0135 and CwFNx0107, showing a frequency of 19.2, 13.7 and 20%, respectively. Other most common HLA-A alleles were AFNx0101, FNx0124, FNx0111 and FNx0133, whereas HLA-A FNx0166, FNx0169 and FNx0174 were rare and present in less than 1.5% of the sample population. HLA-B FNx018, FNx0115, FNx0140, FNx0151 and FNx0152 were detected in a large number of individuals. For HLA-C, six alleles were the most common and were identified in 83% of the subjects. These were Cw FNx0103, FNx0104, FNx0106 FNx0107, FNx0112 and FNx0115. The genotypes of HLA-A, AFNx0102:AFNx0102 and AFNx0102:AFNx0168, showed a significant difference between the observed and expected frequencies (P < 0.5). In addition, genotypes of HLA-B, FNx0135:FNx0158, showed statistically significant differences between the observed and expected frequencies (P < 0.5).

The allele frequencies and distribution of HLA-DRB1 and HLA-DQB1 loci are shown in [Table - 2]. Thirteen distinct HLA-DRB1 alleles and five HLA-DQB1 alleles were recognized. Frequently observed DRB1 alleles were FNx0103, FNx0107, FNx0111 and FNx0115, representing greater than 60% of the samples. The commonest HLA-DRB1 type was FNx0115; its frequency was 21%. DRB1FNx0115 alleles of DR2 family were most frequently found in this study. DRB1FNx0115 constituted 91% of the total DR2 subtypes. Out of 914 HLA-DRB1 genotypes observed, genotype FNx0103:FNx0115 was observed in a smaller proportion of the population than was expected and this difference was statistically significant (P < 0.001).

[Table - 3] shows the estimated frequencies of two-locus haplotypes extended from HLA-DRB1 to HLA-DQB1. Sixty-three different haplotypes and 285 unique genotypes for DRB1-DQB1 were recognized. The most common haplotype was HLA-DRB1FNx0115-DQB1FNx0106; its frequency and relative linkage disequilibrium values were 16.9 and 1.00, respectively. Besides DQB1FNx0106, DRB1FNx0115 was found in association with DQB1FNx0103 and FNx0105. However, for all the different haplotypes for DRB1FNx0115 DQB1 alleles identified, HLA-DRB1FNx0115 was found in strong association with DQB1FNx0106 (82%; P < 0.001). HLA-DQB1FNx0102 was recognized to be in association with HLA-DRB1FNx0103 as the second most common haplotype (15.8%) in Pakistani population. No single HLA-DRB1 allele that showed strong association with multiple HLA-DQB1 alleles was identified.

Out of 2608 haplotypes estimated for HLA-A, -B and C, the frequencies of 10 most common haplotypes which constituted 26% of the total haplotypes are listed in [Table - 3]. The haplotypes AFNx0102, BFNx0140 and CwFNx0115 were the most frequent in Pakistanis. As shown in [Table - 3], HLA-AFNx0102 was involved in 9% of the class I haplotypes primarily in association with BFNx0140, BFNx0150, BFNx0152, CwFNx0115, CwFNx0106 and CwFNx0112. Analysis of HLA-B and C associations showed that BFNx0135 was most tightly associated with CwFNx0104, whereas with HLA-A, strong association of BFNx0140 was identified with HLA-AFNx0102. A total of 2747 unique genotypes and 1757 haplotypes of HLA-A, B and DRB1 were identified in this study [Table - 4]. Further analysis of HLA-A, B and DRB1 haplotypes have demonstrated associations and haplotype frequencies of 3.3% for AFNx0126, BFNx0108 and DRB1FNx0103. Other commonly found A:B:DRB1 haplotypes included AFNx0102:BFNx0140:DRB1FNx0115 showing 3% frequency. Estimation of association between HLA-B and HLA-DRB1 alleles shows that the most frequent DRB1 allele FNx0115 was in association with HLA-BFNx0140 and BFNx0152, both of which contributed to more than 10% of the total haplotypes.

Discussion

HLAs are a key player in shaping the ability of the immune system to distinguish between self and non-self. PCR-SSCP based typing has been used to identify many novel alleles at HLA class I and class II loci, making it a necessary tool for studying new alleles with variations in regions previously not known to be polymorphic. HLA system is a useful tool for distinguishing and relating populations. Moreover, the study of HLA frequencies and genetic distances can be used to assess the existence or absence of gene flow among neighboring populations. ^[11] In this study, multi-locus haplotype analysis was carried out for HLA class I and class II, and the data were compared with those of other populations of the world to evaluate the genetic relations with other populations. Our analysis has shown that for HLA-A, the most frequent allele was AFNx0102. Higher frequency of HLAFNx0102 has been reported in other populations including Caucasians (28%), Russians (26%), South Indians, Kerala Hindu population (25%), and Turks (29%). ^{[12],[13],[14],[15]} In Pakistani population, HLA-A genotype HLA AFNx0102: AFNx0102 was significantly more common (P < 0.05) when compared with other HLA-A genotypes.

The most common HLA B types identified in Pakistan were BFNx0135, FNx0140, FNx0151 and FNx0152. The distribution of HLA-B alleles in our population was comparable to Iranian and Oriental populations, except for HLA BFNx0152 which was proportionally higher in Pakistan (2.6% vs. 8%). ^[16],[17] In Caucasian and Negroid populations, the frequency of BFNx0135, BFNx0140, BFNx0151 and BFNx0152 was lower than that of Pakistani population. ^[12],[16] For HLA-C locus, CwFNx0107 was found in 20% Pakistani individuals when compared with other populations. CwFNx0107 was most frequently seen in Russian and Caucasian populations (36% and 32.6%, respectively). ^[12],[13] It was noted that at HLA-C locus, the most common genotype was CwFNx0107: CwFNx0112 and rare genotype was CwFNx0103: CwFNx0104 (P < 0.001).

The analysis of our data shows that the most frequent allele from HLA-DRB1 locus is DRB1FNx0115, with a frequency of 20.5% The frequency of this allele in other populations examined including Iranian (12%), Caucasian (9.3%) and North Africans (10.7%) was relatively lower. ^{[12],[18],[19]} However, similar frequency of HLA-DRB1FNx0115 was noted in North Indian population. In the Far East including Japan and China, the frequencies for DRB1FNx0115 were 2.9 and 4.4%, respectively, compared to 20.5% in the Pakistani population. ^{[20],[21],[22]} The most frequent DQB1 allele in the Pakistani population was found to be DQB1FNx0102 with a frequency of 39%. This allele existed at a higher frequency in North Indian (32%), Tunisian (32.6%) and African (30.5%) populations. ^{[21],[22],[23],[24]} In contrast, in the Bulgarian and Chinese populations, its frequency was reported to be 13.5 and 17.8%, respectively. ^[25],[26]

The common two locus haplotype in Pakistani population was DRB1FNx0115-DQB1FNx0106 (17%). This haplotype with a frequency of about 15% was reported in Caucasoids, Africans (26.6%). ^[23],[27] This haplotype is also very common in the Indian population (13.2%), whereas in Iran and Yemeni populations DRB1FNx0115-DQB1FNx0106 was present at a lower level with a frequency of 2.7 and 2.0%, respectively. ^[28],[29] The haplotype DRB1FNx0107-DQB1FNx0102 was identified in the Pakistani population in 12.2% of the normal individuals, this haoplotype was also found at a very high frequency in the Tunisian (19.5%) and Yemeni (22.5%) and Iranian (6.8%) populations, but it was not reported in the Caucasians of the Western Europe. ^[29],[30] The class I haplotype AFNx0102-BFNx0140 identified in the Pakistani population was also reported in the Oriental populations of Hong Kong (1.8%) and Philippines (3.2%). ^[31] The distribution of HLA haplotypes in the Pakistani population indicates that it has an influence of Caucasians and Oriental populations.

In conclusion, our study has revealed the frequency of HLA class 1 and II alleles and their haplotypes in the Pakistani population. The data presented here may be effectively utilized for analysis of disease association and anthropological studies in the local population, besides its importance in bone marrow transplantation.

References

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