Identification of HLA-A*02-B*46 haplotype allele variant in Guangdong Han populations on the basis of PCR-SBT

Background The HLA-A*02-B*46 haplotype is one of most frequent haplotypes among Guangdong Han populations. To explore the characteristics of the HLA-A*02-B*46 haplotype in Guangdong Han populations, the genetic polymorphism of HLA-A*02-B*46 haplotype was analysed by PCR-SBT in our study. Findings Among 88 samples with the homozygotes for HLA-A*02-B*46 in the low resolution, 4 different alleles for A*02 (A*0201, A*0203, A*0206, A*0207) and 1 allele for B*46 (B*4601) were identified by PCR-SBT. Among them, the A*0207 allele was the predominant allele. Inversely, among the samples with HLA-A*2-B*46(-), six alleles were detected for A*02 (A*020101, A*0203, A*0205, A*0206 and A*0207), and the A*0201 allele was predominant. On the other hand, the HLA-A*02-B*46 haplotype presented moderate heterozygosis (32.95%). In addition, the linkage with DRB1 was analysed in HLA-A*2-B*46 haplotype, and there existed 10 alleles with DRB1. With the low resolution for DRB1, the other 10 DRB1 alleles all linked with the HLA-A*02-B*46 haplotype except for DRB1*01, DRB1*10, and DRB1*17. Moreover, we found eight alleles of DRB1 in the HLA-A*0207-B*4601 haplotype. Conclusion The polymorphism distribution of the A*02 allele between the HLA-A*02-B*46 and HLA-A*02-B*46(-) haplotypes among the Guangdong Han populations provides useful information for research on unrelated hematopoietic stem cell transplantation (UHSCT), anthropology, and disease association for populations with the HLA-A*02-B*46 haplotype.


Background
HLA-A*02 is the most common allele in humans [1,2], and is the most heterogeneous HLA-A specificity, with 97 subtypes [3]. HLA-B*46 is the most common allele in East Asian populations and occurs at varying frequencies in different populations, including 14% among the Thais, 13.2% among the Vietnamese, 4.4% among the Japanese, and 10% among the Chinese [4]. The HLA-A*02-B*46 haplotype is one of most common haplotypes in Chinese populations, especially the Guangdong Han population. HLA-A*02-B*46 presents significant linkage disequilibrium, which shows its genetic dominance. Moreover, it is shown that the HLA-A*02-B*46 allele is positively related to certain diseases, such as nasopharyngeal carcinoma, which has a high incidence in Guangdong province [5,6].
The intent of this study was to explore the polymorphism of the HLA-A*02-B*46 haplotype homozygote and its distribution characteristics in the Guangdong Han population, and also to establish a bone marrow bank with HLA-A*02-B*46 haplotype to improve the effect of bone marrow transplantation and decrease the occurrence of GVHD. Therefore, we have analysed the genetic polymorphism of HLA-A*02-B*46 in Guangdong Han population by polymerase chain reaction-sequence-based typing (PCR-SBT) and report our findings in this paper.

Subjects
For all subjects, genomic DNA was isolated from white cells in 5 ml peripheral blood using the DNA Extraction Kit (Qiagen) according to the manufacturer's specifications. Primary genotyping was carried out by PCR-RSSO (Dynal Biotech Ltd, Invitrogen) on 8191 blood samples from unrelated bone marrow donors from the Guangdong Han population. Of the 8191 donors, 88 [mean age (35.5 ± 7.8) years, range 18~55 years] that were typed as HLA-A*02-B*46 positive were used in the present investigation. They were all ethnic Han, and their families have lived in Guangdong Province for at least three generations. As a control, 150 samples with A*02 and without the HLA-B*46 allele were selected in research. These selected samples were homozygotes for HLA-A*02-B*46 in the low resolution (PCR-RSSO). The local ethics committee reviewed the study and informed consent was obtained from all the participants.

HLA-A*02-B*46 genotyping
HLA typing was performed by PCR-SBT analysis as previously described after amplifying the second exon of HLA-A, the third exon of HLA-B, and the second exon of HLA-DRB1 [7]. High resolution typing of the HLA-A*02, B*46, and DRB1 alleles was performed by PCR-SBT using the ABI 3100 with the software Matchtool. The primers used for PCR-amplification and sequencing of HLA-A, B*46, DRB1 were listed in table 1 and 2, respectively.

Statistical analysis
Relative frequencies of the HLA-A*2-B*46 allele were calculated by direct counting. χ 2 analysis was used to assess statistically significant differences between the HLA-A*02-B*46 and HLA-A*02-B*46(-) haplotype, Pc is obtained by P multiplying the number of alleles at each locus. Statistical analyses were conducted with an SPSS software program.

Discussion
The HLA-A*02-B*46 haplotype is one of most frequent haplotypes found in Southern Chinese Han populations, especially in the Guangdong Han population. The HLA-A*02-B*46 haplotype has been reported to possibly be related to the genesis of nasopharyngeal carcinoma, the most common cancer in Guangdong province [5,6]. By means of linkage analysis of the HLA-A*02-B*46 haplotype, it may be possible to detect NPC genetic susceptibility cases in NPC high risk families. In our study, the HLA-A*02, HLA-B*46, HLA-DRB1 alleles and HLA-A*02-B*46 haplotypes in the Guangdong Han population were analysed by high-resolution SBT to study their genetic background. This approach was more efficient than traditional genetic methods, which were based on sequence-specific oligonucleotide probe (SSOP) and sequence-specific primers (SSP).
The HLA-A*02-B*46 haplotype showed high genetic diversity in the Guangdong Han population because of the polymorphism of A*02, although we found only one B*46 allele with B*4601 in HLA-A*02-B*46 haplotypes. HLA-A*02 alleles can be classified into two subgroups, including A*0201 and A*0205, by the diversity of the DNA sequence. Almost all populations have A*0201; however, A*0203, A*0206, A*0207 and A*0210 have only been found in Oriental populations, while A*0205,  Most studies have shown that A*0201 is the prevalent gene in Chinese Han populations and, therefore, is not useful as a genetic marker. However, some investigations have found that at higher resolution, A*0201 does present diversity between North and South Chinese Han populations which could be regarded as a genetic marker [11,12]. In our study, the frequency of A*0201 presents diversity between different haplotypes, so further investigations of the subtypes are important to determine the frequency of A*0201.
High-resolution SBT was applied to analyse the alleles of HLA-A*02-B*46 in our study; this technique has shown that the HLA-A*02-B*46 presents heterozygosis to some extent because of the diversity associated with the A*02 allele. However, the frequency of heterozygosis was lower than the HLA-A*02 haplotype, which has been reported to show 85% heterozygosis [11]. The cause may be the high proportion of A*0207-B*4601. The low heterozygosity of HLA-A*02-B*46 makes it easy to look for unrelated donors with HSCT to transplant into patients with the HLA-A*02-B*46 haplotype.
We have found 10 DRB1 alleles in the HLA-A*02-B*46 haplotype. With the low resolution for DRB1, the other DRB1 alleles all exhibited linked behaviour and linked with the HLA-A*02-B*46 haplotype except for DRB*01, DRB*10, and DRB*17. However, there were only 8 DRB1 alleles in the HLA-A*0207-B*4601 haplotypes, as