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HLA-B*37:107 differs from HLA-B*37:01:01:01 in codon 25 in exon 2., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The range of HLA typing for successful hematopoietic stem cell transplantation (HSCT) is gradually expanding with the next-generation sequencing (NGS)-based improvement in its quality. However, it is influenced by the allocation of finances and laboratory conditions. HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 alleles were genotyped at the 3-field level by amplicon-based NGS using MiSeqDx system and compared to our previous study employing long-range PCR and NGS using TruSight HLA v2 kit, in healthy donors from South Korea. Exon 2, exons 2/3, exons 2/3/4 or 5 of 11-loci were amplified by multiplex PCR. The sequence reads of over 53 depth counts were consistently obtained in each sample exon, depending on the target exon determined to match the reference sequence contained in the IPD-IMGT/HLA Database. HLA alleles were investigated by combinations of the determined exons. A total of 18 alleles with a frequency over 10% were found at the 11 HLA loci. Three ambiguities of HLA-A, -C, and -DRB1 were resolved. We observed a total of 26 HLA-A ~ C ~ B and 6 HLA-DRB1 ~ DQA1 ~ DQB1 ~ DPA1 ~ DPB1 haplotypes having significant linkage disequilibrium between alleles at all neighboring HLA loci. This result was compatible with the previous one, using TruSight HLA v2 kit. Advantages are simple and short progress time because one plate is used for each PCR step in one PCR machine and 11-loci HLA typing is possible even if only eight samples. These data suggested that expanded 11-loci HLA typing data by amplicon-based NGS might help perform HSCT., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Allele frequencies and haplotype frequencies of HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 have been rarely reported in South Koreans using unambiguous, phase-resolved next generation DNA sequencing. In this study, HLA typing of 11 loci in 173 healthy South Koreans were performed using next generation DNA sequencing with long-range PCR, TruSight® HLA v2 kit, Illumina MiSeqDx platform system, and Assign™ for TruSight™ HLA software. Haplotype frequencies were calculated using the PyPop software. Direct counting methods were used to investigate the association with DRB1 for samples with only one copy of a particular secondary DRB locus. We compared these allele types with the ambiguous allele combinations of the IPD-IMGT/HLA database. We identified 20, 40, 26, 31, 19, 16, 4, and 16 alleles of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1, respectively. The number of HLA-DRB3/4/5 alleles was 4, 5, and 3, respectively. The haplotype frequencies of most common haplotypes were as follows: A*33:03:01-B*44:03:01-C*14:03-DRB1*13:02:01-DQB1*06:04:01-DPB1*04:01:01 (2.89%), A*33:03:01-B*44:03:01-C*14:03 (4.91%), DRB1*08:03:02-DQA1*01:03:01-DQB1*06:01:01-DPA1*02:02:02-DPB1*05:01:01 (5.41%), DRB1*04:05:01-DRB4*01:03:01 (12.72%), DQA1*01:03:01-DQB1*06:01:01 (13.01%), and DPA1*02:02:02-DPB1*05:01:01 (30.83%). In samples with only one copy of a specific secondary DRB locus, we examined its association with DRB1. We, thus, resolved 10 allele ambiguities in HLA-B, -C (each exon 2+3), -DRB1, -DQB1, -DQA1, and -DPB1 (each exon 2) of the IPD-IMGT/HLA database. Korean population was geographically close to Japanese and Han Chinese populations in the genetic distances by multidimensional scaling (MDS) plots. The information obtained by HLA typing of the 11 extended loci by next generation sequencing may be useful for more exact diagnostic tests on various transplantations and the genetic population relationship studies in South Koreans., Competing Interests: The authors have declared that no competing interests exist.

HLA genes play a pivotal role for successful hematopoietic stem cell transplantation (HSCT). There is an increasing need for sophisticated screening of donor HLA genotypes for unrelated HSCT. Next generation sequencing (NGS) has emerged as an alternative for classical Sanger sequence for HLA typing. In this study, HLA-A, -B, and -DRB1 alleles were genotyped at the allelic (6-digit) level using MiSeqDx in 26,202 volunteers from the Korean Network for Organ Sharing. Exon 2 and 3 of HLA-A and -B and exon 2 of HLA-DRB1 were amplified by polymerase chain reaction (PCR) and each allele was determined by matching the targeted exons and the reference sequence consisting of the IPD-IMGT/HLA Database. Seventy alleles of HLA-A, 102 alleles of HLA-B, and 69 alleles of HLA-DRB1 were identified. According to common and well-documented catalogs, 34 alleles in HLA-A, 61 in HLA-B, and 45 in HLA-DRB1 locus were common alleles, and 12, 14, and 11 kinds, were well-documented alleles, respectively. Thirteen novel alleles including 3 alleles in HLA-A, 8 alleles in HLA-B, and 2 alleles in HLA-DRB1 loci were found. Ten haplotypes with a frequency of more than 1.0% accounted for 22.4% of the total haplotype frequencies. Cis/trans ambiguities of HLA-A and -B loci by combination of exons 2 and 3 were analyzed to be 0.17% of 3 and 3.95% of 22 genotypes, respectively. This information on rare and novel alleles found by accurate HLA typing with NGS may be helpful for unrelated HSCT among Koreans., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*35:01:64 differs from HLA-B*35:01:01:01 in codon 58 in exon 2., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*15:529 differs from HLA-B*15:01:01:01 in codon 138 in exon 3., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*51:284 differs from HLA-B*51:01:01:01 in codon 44 in exon 2., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*54:41 differs from HLA-B*54:01:01:01 in codon 121 in exon 3., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*46:01:26 differs from HLA-B*46:01:01:01 in codon 66 in exon 2., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*44:454 differs from HLA-B*44:03:02:01 in codon 81 in exon 2., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*46:67 differs from HLA-B*46:01:01 in codons 94, 95, and 103 in exon 3., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*40:330 differs from HLA-B*40:06:01:01 in codons 113 and 116 in exon 3., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*54:35 and -B*54:38 differ from HLA-B*54:01:01 in codons in exons 2 and 3., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)