Anti-HLA-DPB1 monoclonal antibody, clone 7D7

Next generation sequencing (NGS) methods have been established as an efficient approach for HLA typing because unlike traditional Sanger sequencing, they provide unambiguous results at a reasonable cost. We previously developed a multi-locus index method to genotype four HLA loci (A, B, C, and DRB1) on the Illumina MiSeq platform. We have now expanded this method to include two additional loci, HLA-DPB1 and DQB1. Contiguous full-length amplicons from 5'UTR through 3'UTR regions were generated using one long-range PCR reaction per locus for each of the six loci from 96 individuals of different ethnicities. The six amplicons from each donor were pooled, enzymatically fragmented and given a donor-specific index. This approach enabled sequencing of 576 loci from 96 individuals in a single MiSeq run. Donor-specific sequence reads were demultiplexed, and allele calls were generated from FASTQ files using commercially available software. Comparison to HLA genotypes generated from Sanger sequence-based typing (SBT) identified no discordances among any of the alleles analyzed in this study. Importantly, this method was able to resolve 22 DPB1 and 20 DQB1 alleles that were ambiguous with the SBT method. Furthermore, a novel allele in each of these two loci was identified, with the DQB1*05:01:24 allele having a frequency of greater than five percent. This method was subsequently validated against a blinded panel of 22 samples from the 17th International HLA and Immunogenetics Workshop. The flexibility of the method is further highlighted by successful genotyping of eight loci comprising all classical HLA loci for a subset of the samples. We now present a high-throughput, high-resolution, scalable NGS HLA typing method to accurately and efficiently genotype all classical HLA class I and II loci.

Recent genome-wide association studies have revealed the HLA region on chromosome 6p21 as a susceptibility locus for hepatitis B virus (HBV) infection, a finding subsequently replicated in independent samples. However, only limited single nucleotide polymorphisms (SNPs) were analyzed in most of these studies, and it remains to be determined which SNPs contribute to the detected association. After genotyping 140 SNPs within this genomic region in a total of 1657 HBV-positive patients and 1456 HBV-negative controls, we conducted a series of genetic epidemiological and bioinformatics analysis, including individual SNP-based association analysis, haplotype-based association analysis, and conditional analysis. We identified 76 SNPs and 5 LD blocks in HLA-DP/DQ clusters that are significantly associated with HBV infection, with the smallest P value being 3.88 x 10(-18) for rs9277535 in HLA-DPB1. With conditional analysis, we further revealed that the genes contributing to the effects of variants in HLA-DP/DQ on infection are independent of each other, and the LD block 5 in the 3'-UTR region of HLA-DPB1 had a predominant effect in the association of HLA-DP with HBV infection. We also found that the SNPs in the 3'-UTR region of HLA-DPB1 were significant between the subgroups of inactive HBV carrier, chronic hepatitis B, or hepatic cirrhosis from the case group and the spontaneous HBV-clearance subgroup from the control group. Finally, we did further association analysis of SNPs in this region with different subgroups from the case group, which revealed no association of these SNPs with the progression of HBV-related diseases. In sum, we showed, for the first time, that the HLA-DP/DQ clusters contribute independently to HBV infection, and the 3'-UTR region of HLA-DPB1 represents an important functional region involved in HBV infection.