High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR–SSOP–Luminex method in the Japanese population

Abstract

We have developed a new high-throughput, high-resolution genotyping method for the detection of alleles at the human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 loci by combining polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes (SSOPs) protocols with the Luminex 100 xMAP flow cytometry dual-laser system to quantitate fluorescently labeled oligonucleotides attached to color-coded microbeads. In order to detect the HLA alleles with a frequency of more than 0.1% in the Japanese population, we created 48 oligonucleotide probes for the HLA-A locus, 61 for HLA-B, 34 for HLA-C, and 51 for HLA-DRB1. The accuracy of the PCR-SSOP-Luminex method was determined by comparing it to the nucleotide sequencing method after subcloning into the plasmid vector using 150 multinational control samples obtained from the International HLA DNA Exchange University of California Los Angeles. In addition, we performed the PCR-SSOP-Luminex method for HLA allele typing on DNA samples collected from 1,018 Japanese volunteers. Overall, the genotyping method exhibited an accuracy of 85.91% for HLA-A, 85.03% for HLA-B, 97.32% for HLA-C, and 90.67% for HLA-DRB1 using 150 control samples, and 100% for HLA-A and -C, 99.90% for HLA-B, and 99.95% for HLA-DRB1 in 1,018 Japanese samples. The PCR-SSOP-Luminex method provides a simple, accurate, and rapid approach toward multiplex genotyping of HLA alleles to the four-digit or higher level of resolution in the Japanese population. It takes only approximately 5 h from DNA extraction to the definition of HLA four-digit alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci for 96 samples when handled by a single typist.

Host-Specific Adaptation of HIV-1 Subtype B in the Japanese Population

“…Also consistent with previous observations in Caucasian, African, Chinese, and Mexican populations (1,6,11,15,18), the number of HLA-B-associated polymorphisms in our cohort was higher than that of HLA-A-or HLA-C-associated polymorphisms, further supporting a dominant role of HLA-B in HIV evolution (32). An interesting feature of the Japanese population is that approximately 70% of individuals carry HLA-A*24:02 (23). Despite sufficient statistical power to detect HLA-A*24:02-associated polymorphisms in our cohort, we identified only 9 of them, 6 of which were located in epitopes identified by our group (33)(34)(35).…”

“…A total of 78 unique HLA class I alleles, defined at subtype level (four-digit) resolution, were observed in our cohort (see Fig. S1 in the supplemental material) at frequencies consistent with those in the published literature (23). Of these, 37 (including 9 HLA-A, 17 HLA-B, and 11 HLA-C alleles) were observed in at least 10 individuals and thus were included in the statistical analysis of HLA-APs (see Materials and Methods).…”

“…HLA-B*57, -B*58, and -B*27 are not present at appreciable frequencies in Japan (23). It is therefore perhaps unsurprising that no correlations between HLA-associated substitutions in Gag and HIV clinical parameters were observed in our cohort.…”

No Contribution of the ABCB11 p.444A Polymorphism in Japanese Patients with Drug-Induced Cholestasis

“…The following primers were used: V444A sense, 59-CTTTGGGGGCATACATAAAC-39; V444A antisense, 59-CTGACT-GAAATGTTGCTATG-39; rs3815676 sense, 59-ATGGGTGAAGAGCAGGAGAG-39; rs3815676 antisense, 59-TGCCTATGTGATAACATGCTGA-39; rs7577650 sense, 59-CTTTCAGTGCCAGTAGGATG-39; and rs7577650 antisense, 59-GGAAA-TAATTAGCAAGTATCACTACC-39. HLA genotypes, including HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DPB1, were analyzed using the Luminex method (Itoh et al, 2005), and the results were compared with those of pooled Japanese controls (n = 3002).…”

Reduced Frequency, Diversity, and Function of Human T Cell Leukemia Virus Type 1-Specific CD8+ T Cell in Adult T Cell Leukemia Patients

“…HLA allele types were determined by the PCR-sequence-specific oligonucleotide probes protocols with the Luminex 100 xMAP flow cytometry dual-laser system method using high m.w. DNA isolated from the cryopreserved PBMC as described (G&G Science) (36). Briefly, amplification was as follows: target DNA was PCR-amplified using 5Ј-biotin-labeled primers that are highly specific to certain sequences of HLA genes.…”