S.-Y. Ko

Analysis of hepatitis B virus drug-resistant mutant haplotypes by ultra-deep pyrosequencing

Direct sequencing and reverse hybridization are currently the main methods for detecting drug-resistance mutations of hepatitis B virus (HBV). However, these methods do not enable haplotype analysis so they cannot be used to determine whether the mutations are co-located on the same viral genome. This limits the accurate identification of viral mutants that are resistant to drugs with a high genetic barrier. In our current study, ultra-deep pyrosequencing (UDPS) was used to detect HBV drug-resistance mutations in 25 entecavir-treated and five treatment-naive patients. Of the 25 entecavir-treated patients, 18 had experienced virological breakthrough and two exhibited reduced susceptibility to entecavir. The results obtained by UDPS were compared with those of direct sequencing, and the haplotypes of the drug-resistant HBV mutants were analysed. The average number of reads per patient covering the region in which drug-resistance mutations are located was 1735 (range 451-4526). UDPS detected additional drug-resistance mutations not detected by direct sequencing in 19 patients (mutation frequency range 1.1-23.8%). Entecavir-resistance mutations were found to be co-located on the same viral genome in all 20 patients displaying virological breakthrough or reduced susceptibility to entecavir. In conclusion, UDPS was not only sensitive and accurate in identifying drug-resistance mutations of HBV but also enabled haplotype analysis of the mutants. This method may offer significant advantages in explaining and predicting the responses of patients with HBV to antiviral therapy.

MICB polymorphisms and haplotypes with MICA and HLA alleles in Koreans

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) is located within the human MHC class I region. The location of MICB in the MHC region may imply the presence of linkage disequilibrium with polymorphic MICA and human leukocyte antigen (HLA) loci. MICB is also polymorphic; however, MICB polymorphisms have not been investigated in Koreans. Using sequence-based typing (SBT), we estimated the allelic frequencies of MICB and haplotypes with MICA, HLA-B, and HLA-DRB1 at high resolution in a population of 139 unrelated Korean individuals. Eight MICB alleles were identified. The most frequent allele was MICB*005:02/*010 (57.2%), followed by *002 (11.5%), *004 (8.3%), *005:03 (8.3%), and *008 (6.8%). The most common two-locus haplotypes were MICB*005:02/*010-MICA*010 (19.4%), MICB*005:02/*010-DRB1*15:01 (6.5%), and MICB*005:02/*010-B*15:01 (10.4%); the most common three-locus haplotypes were B*15:01-MICA*010-MICB*005:02/*010 (5.8%) and MICA*010-MICB*005:02/*010-DRB1*04:06 (10.4%); and the most common four-locus haplotype was B*15:01-MICA*010-MICB*005:02/*010-DRB1*04:06 (5.8%). This is the first study to provide information about MICB allele frequencies and haplotypes with HLA in Koreans. These study results should help understand mechanisms of disease association between the MICB locus and neighboring loci in Koreans.

B*39: 60, a novel HLA-B*39 allele identified by sequence-based typing

The new allele B*39:60 showed one nucleotide difference with B*39:01:01 at codon 152 (GTG/GCG).

Development and clinical evaluation of a microarray for HLA-A and -DRB1 genotyping

Microarray technology makes high-throughput genotyping possible by permitting the simultaneous analysis of large sets of genes on a small reaction slide. Human leukocyte antigen (HLA) loci showing high polymorphisms are suitable targets for microarray. In this study, we developed a microarray kit with newly designed oligonucleotide probes for the genotyping of HLA-A and -DRB1. In total, 42 probes were designed to hybridize to polymorphic sites for HLA-A and 36 for HLA-DRB1. Asymmetric polymerase chain reaction (PCR) using four primers was performed to amplify exon 2 of HLA-DRB1, whereas symmetric PCR was performed to amplify both exons 2 and 3 of HLA-A. Evaluation of performance using samples from 138 Koreans disclosed consistent microarray results with all sequence-based typing at the low-resolution level. Despite the occurrence of ambiguities in 35 HLA-A (25.4%) and 5 HLA-DRB1 (3.6%) cases, correct genotypes were assigned with high certainty by referring to allele distribution in Koreans. These data clearly indicate that our newly developed microarray kit is optimal in determining correct genotypes at the low-resolution level in Koreans.

Anthropological analysis of Koreans using HLA class II diversity among East Asians.

Human leukocyte antigens (HLAs) are useful markers for anthropological investigations because the allele and haplotype distributions at these loci vary widely among ethnic groups. HLA frequencies in Koreans, however, have not previously been analyzed on a phylogenetic basis. We determined the allele frequencies of four HLA class II (HLA-DRB1, -DQA1, -DQB1, and -DPB1) loci in 149 unrelated Korean individuals using a sequence-based typing method. A total of 29 HLA-DRB1, 17 HLA-DQA1, 16 HLA-DQB1, and 15 HLA-DPB1 alleles were identified. The most common allele at each locus was DRB1*0901, DQA1*0102, DQB1*0301, and DPB1*0501, respectively. Four-locus allelic association analysis showed the existence of 25 DRB1-DQA1-DQB1-DPB1 haplotypes with a frequency greater than 0.010. A dataset comprising ethnicity-specific information from published literature and the dbMHC database, as well as the allele frequencies determined in this study, was subjected to phylogenetic analysis. The analysis showed that Koreans are most closely related to Japanese and Han Chinese from Shandong province. Correspondence analyses showed that the current Korean population is located in a position intermediate between the northern and southern East Asian groups, supporting the theory of a bi- and/or multidirectional route of migration of early Korean settlers. This report can be used for anthropological studies, and may also be of use in the International Hematopoietic Stem Cell Sharing program.

Identification of a novel HLA-B*40 null allele, HLAB*40:155N

The new allele B*40:155N showed five nucleotide insertion between nucleotide 594 and 595 (codon 174 and 175) compared to B*40:01:01.

HLA-DRB1*13:99, a novel HLA-DRB1*13 allele identified by sequence-based typing

The new allele DRB1*13:99 showed one nucleotide difference with DRB1*13:02:01 at codon 51 (ACG/AAG).

HLA-DQB1*05:06, a novel HLA-DQB1*05 allele identified by sequence-based typing

The new allele DQB1*05:06 showed one nucleotide difference with DQB1*05:03:01 at codon 40 (TTC/TTG).

HLA‐C*03:93, a novel HLA‐C*03 allele identified by sequence‐based typing

The new allele C*03:93 showed one nucleotide difference with C*03:04:01 at codon 140 (GCT/ACT).

Sequencing of a new HLA-A allele:HLA-A*11:01:54: NEW ALLELE Alerts

The new allele A*11:01:54 shows one nucleotide difference from A*11:01:01 at codon 35 (CGG/AGG).