Estimation of the 6‐digit level allele and haplotype frequencies of HLA‐A, ‐B, and ‐C in Koreans using ambiguity‐solving DNA typing

Abstract

Because Korean society is fast becoming multi-ethnic, the determination of ambiguous human leukocyte antigen (HLA) types using HLA allele frequencies is becoming less applicable. In this study, we focused on the development of new technical methods to directly resolve the ambiguities arising from HLA genotyping. One hundred and fifty unrelated healthy Korean adults were included in this study. All alleles from each HLA locus were first divided into 2-4 groups, with each group amplified in a single PCR tube (multi-group-specific amplification, MGSA). To resolve phase ambiguities, some allele groups were also amplified separately in small group-specific amplification (SGSA) tubes. In order to then resolve incomplete sequence ambiguities, primers for MGSA and SGSA were initially designed to cover additional exons. If needed, a heterozygous ambiguity resolving primer (HARP) or sequence specific primer (SSP) was also used. When MGSA and SGSA methods were applied, the rate of phase ambiguity was greatly reduced to an average of 6% (1.3% in HLA-A, 15.7% in -B, and 2.0% in -C). Additional HARP and SSP methods could resolve all the phase ambiguities. Using our proposed method, we also detected three alleles that have not been previously reported in Korea, C*04:82, C*07:18, and C*08:22, and report 6-digit level HLA allele and haplotype frequencies among Koreans. In conclusion, the use of MGSA/SGSA for the initial amplification step is a cost-effective method facilitating timely and accurate reporting, given the continuing increase in the ethnic diversity of the Korean population. The MGSA described here can be applicable to various populations and thus could be shared by the majority of HLA typing laboratories. However, efforts to solve HLA ambiguity should continue, because SGSA, HARPs and SSPs would be specific to a particular population.

Identification of 8-Digit HLA-A, -B, -C, and -DRB1 Allele and Haplotype Frequencies in Koreans Using the One Lambda AllType Next-Generation Sequencing Kit

“…The base position of samples showing discrepant results between NGS and previous Sanger sequencing HLA genotypes obtained by Jun, et al [12] was confirmed using the BIOWI-THUS SBT Analyzer (Biowithus Inc., Seoul, Korea). Next, we performed PCR using primer sets (Avita Plus Kit, Biowithus Inc., Seoul, Korea) harboring the necessary additional exon and Sanger sequencing according to previous methods to confirm the sequence difference in the base position of the additional exon [12].…”

“…Samples were obtained by collecting 10-20 mL of venous blood from each subject, and the concentration of the extracted DNA was adjusted to 40-100 ng/µL. These samples were previously analyzed for 6-digit HLA types using Sanger sequencing with Big Dye Terminator 3.1 (ABI Inc., Foster City, CA, USA) and an ABI 3730 DNA analyzer (ABI Inc.) [12]. Samples were stored in a -70°C deep freezer until our analysis.…”

Identification of a novel HLA‐B allele, HLA‐B*55:81

“…HLA‐B*55 is relatively common in Orientals compared with other ethnic groups with an average frequency of 3.67% (0.0%‐22.6%) . High‐resolution genotyping of HLA‐B*55 has shown mostly B*55:02 (2.00%) in Koreans . Here, we report a novel HLA‐B*55 allele, officially named as B*55:81 , which was identified by cloning and sequencing in a Korean individual.…”

Identification of the novel HLA‐C*03 allele, HLA‐C*03:03:35

“…HLA-C*03 is the most frequent allele with average frequency of 27.32% in the Korean population: C*03:02 (7.66%), C*03:03 (9.33%), and C*03:04 (10.33%). 1 Serological equivalence of C*03:03 is assigned as Cw9 which is different from Cw10 encoded by the C*03:02 and C*03:04 alleles. 2 Therefore, 4-digit level of HLA typing is necessary to assign split antigens for HLA-C*03 alleles.…”