[The Polymorphism Analysis of HLA Class II Alleles Based on Next-Generation Sequencing and Prevention Strategy for Allele Dropout].

OBJECTIVE: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system. METHODS: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods. RESULTS: A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System. CONCLUSION: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

Characterization of the novel HLA-A*31:01:53 allele by next generation sequencing in a Chinese individual.

HLA-A*31:01:53 differs from HLA-A*31:01:02:01 by one nucleotide change at nucleotide 900 in exon 5 from G to A.

Two novel HLA class II alleles, HLA-DRB1*09:57 and HLA-DRB1*09:58 in Chinese individuals.

Compared with HLA-DRB1*09:01:02:05, the alleles HLA-DRB1*09:57 and HLA-DRB1*09:58 each show one nucleotide change, respectively.

Next-generation HLA sequencing reveals the novel HLA-A*33:244 allele.

The novel HLA-A*33:244 allele contains a c.553G>A substitution in exon 3 compared with A*33:03:01:01.

Characterization of the novel HLA-A*30:211 allele by next generation sequencing in a Chinese individual.

HLA-A*30:211 differs from HLA-A*30:01:01:01 by one nucleotide change at nucleotide 344 in exon 3 from G to C.

The novel HLA-B*13:179 variant detected by next generation sequencing in a Chinese individual.

HLA-B*13:179 differs from HLA-B*13:99 by one nucleotide substitution at position 829(A>G) in exon 4.

The novel HLA-B*15:664 allele, identified by next-generation sequencing in a Chinese individual.

B*15:664 differs from B*15:02:01:01 by one nucleotide change at nucleotide 755 in exon 4 from C to G.

A single nucleotide substitution in exon 5 produced the novel allele, HLA-B*40:01:83.

HLA-B*40:01:83, carrying a single nucleotide substitution in exon 5 is described.

Full-length sequence of the novel HLA-B*40:537 allele by next-generation sequencing in a Chinese individual.

One nucleotide substitution in codon 116 of HLA-B*40:06:01:12 results in a novel allele, HLA-B*40:537.

[Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP].

OBJECTIVE: To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method. METHODS: All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology. RESULTS: A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database. CONCLUSION: The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.

A novel HLA-DQB1*04 variant, HLA-DQB1*04:90, identified in a Chinese Han individual.

Identification of the novel HLA-DQB1*04:90 allele that differs from DQB1*04:01:01:01 at nucleotide 183 in exon 2 from A to T.

Full-length sequence of the novel HLA-C*03:566 allele by next-generation sequencing in a Chinese individual.

HLA-C*03:566 differs from HLA-C*03:04:01:02 by one nucleotide substitution at nucleotide 92 in exon 2 from A to G.

HLA-DRB1*14:239, a novel HLA-DRB1 allele with one exonic mutation.

HLA-DRB1*14:239 differs from HLA-DRB1*14:03:01 by one nucleotide substitution in codon 82 in exon 2.

A novel HLA-C*15 allele, HLA-C*15:192, identified by next generation sequencing in a Chinese individual.

Identification of the novel HLA-C*15:192 allele that differs from HLA-C*15:02:01:01 at one position in exon 2.

Identification of the novel HLA-DRB3*02:02:19 allele.

The HLA-DRB3*02:02:19 allele differs from DRB3*02:02:01:02 by a single nucleotide change in exon 2.

Identification of a novel HLA-A*11 allele, HLA-A*11:399, in a Chinese Individual.

One nucleotide substitution in codon 90 of HLA-A*11:01:01:01 results in a novel allele, HLA-A*11:399.

Discovery of the novel HLA-C*01:179 allele in a southern Chinese patient.

One nucleotide substitution in codon 189 of HLA-C*01:02:01:01 results in a novel allele, HLA-C*01:179.

A substitution in exon 2 resulted in the novel HLA-A*30:140 variant identified in a Chinese individual.

HLA-A*30:140 differs from HLA-A*30:01:01 by one nucleotide change in exon 2 at position 341 (C > A).

Discovery of the novel HLA-A*11:396 allele in a Chinese Han individual.

One nucleotide substitution in codon 73 of HLA-A*11:01:01:01 results in a novel allele, HLA-A*11:396.

Identification of the HLA-DPA1*02:33 allele by next-generation sequencing in a Chinese individual.

The HLA-DPA1*02:33 allele differs from DPA1*02:02:02:04 by two nucleotide change in exon 4.