[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.

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.

[Analysis of genetic polymorphisms and a novel tri-allelic cloning sequence for the D13S317 locus among an ethnic Han Chinese population].

OBJECTIVE: To study the genetic polymorphisms of short-tandem repeats (STR) for the D13S317 locus among an ethnic Han Chinese population and verify a novel tri-allelic pattern identified for the locus. METHODS: A total of 378 paternity test cases from Guangdong Forensic Authentication Institute from October 17, 2017 to December 28, 2017 were selected as the study subjects. A GlobalFilerTM Express kit was used for the STR genotyping. Samples suspected for having a novel tri-allelic pattern were verified with a PowerPlex 21 kit. Potential variant of the primer-binding region and flanking sequences underlying the tri-allelic pattern was excluded by molecular cloning and sequencing. RESULTS: Six alleles were detected for the D13S317 locus, with the characteristic distribution frequencies being 8 (29.1%), 9 (13.1%), 10 (15.21%), 11 (24.21%), 12 (13.89%) and 13 (3.44%), respectively. In one of the families, the D13S317 locus of the proband was suspected to harbor a triband allele (8, 9, 10). A re-test has confirmed the result of initial test. Molecular cloning and sequencing analysis of the D13S317 locus in the proband and his daughter has failed to find allelic variants in the primer-binding region and flanking sequence, which has confirmed the novel tri-allelic pattern for the locus. CONCLUSION: A novel type 2 tri-allelic pattern (8, 9, 10) at the D13S317 locus has been identified among the ethnic Han Chinese population. The pattern has not been transmitted to the female offspring, and has been included in the international STRBase database for the first time.

[Genetic analysis of a Chinese pedigree with an allele dropout at the HLA-B locus].

OBJECTIVE: To delineate a deletional mutation of the HLA-B gene in a Chinese pedigree. METHODS: A female patient with acute myeloid leukemia who had visited Liuzhou People's Hospital in April 2022 was selected as the study subject. Routine human leukocyte antigen (HLA) was determined by using PCR-sequence specific oligonucleotide polymorphism (PCR-SSOP) and PCR-sequence-based typing (PCR-SBT) methods. Next generation sequencing (NGS) was used to validate the candidate variant in the HLA-B gene. RESULTS: The PCR-SBT and SSOP results for the HLA-B locus were inconsistent for the patient and her daughter. The SSOP results of the two individuals were HLA-B*35:01, 40:02 and HLA-B*35:01, 40:01, respectively. However, the PCR-SBT results has indicated a mismatch with the nearest HLA-B*35:01 at exon 4. NGS results showed that the HLA-B*35:01 had a 9 bp deletion in the intron 5. The patient's husband was HLA-B*40:01, 58:01, which was normal. CONCLUSION: The variant in intron 5 of the HLA-B gene in this pedigree has mapped to a primer-binding region for the SBT reagent, which has affected the accuracy of PCR-SBT results.

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.

Characterization of a novel variant allele, HLA-B*56:04:05, identified in a Chinese Han individual.

The novel HLA-B *56:04:05 allele differs from its most closely related allele B*56:04:01:01 in exon 4.

[Establishment of sequence-based typing assay for KIR2DS4 gene and identification of a new allele KIR2DS4*016].

OBJECTIVE: To establish a reliable sequence-based typing method for KIR2DS4 and study its allele polymorphism in Chinese Han population. METHODS: Using PCR-SSP method to detect the positive or negative of KIR2DS4 gene in 222 random Chinese Han individuals, and then using the method of high fidelity and long-fragment PCR-SBT to amplify, sequence and genotype the exons 4 and 5 of KIR2DS4 positive individuals. RESULTS: We successfully amplified the fragment with 3.2 kb length contains exons 4 and 5 of KIR2DS4 and detected the KIR2DS4 allele frequency in Chinese Han population. 209 KIR2DS4 positive individuals were detected, and the positive rate is 94.1%. By sequence-based typing, we identified 12 genotypes and 7 alleles of KIR2DS4. The 6 known alleles and their detection frequency is as follows: KIR2DS4* 00101/011 (180, 81.1%), KIR2DS4* 010 (53, 23.9%), KIR2DS4* 004 (34, 15.3%), KIR2DS4* 003 (15 and 6.8%), KIR2DS4* 006 (2, 0.9%) and KIR2DS4* 015 (1, 0.5%). In this study, we found a new allele, KIR2DS4* 016, with the difference in exon 5 comparing its most similar allele KIR2DS4* 010. In the exon 5 of KIR2DS4* 010, there is a 22bp-deletion, while the exon 5 of KIR2DS4* 016 is normal. This is not a rare allele because it was detected 3 times in studied population and with the frequency of 1.4%. The sequence of the new allele sequence has been submitted to GenBank (accession no.: KC414890) and the IPD -KIR database (submission no.: IWS40001804), and was nominated by WHO nomenclature committee for HLA system. CONCLUSION: In this study, a sequence-based typing method for KIR2DS4 was established, and the polymorphism data of KIR2DS4 in Chinese Han population was enriched by studying the allele polymorphism and new allele.

Establishment of Rapid Detection Methods for rs76971248 Related to Leukemia.

Background: The HLA-E gene is a member of the HLA-I gene family. Its genetic polymorphism is regarded as associated with numerous diseases. Establishing a rapid and accurate detection method of disease-related SNP sites in HLA-E is particularly important. Methods: Blood samples from 226 healthy blood donors and 228 leukemia patients were collected, and DNA was extracted. Three typing methods based on PCR-sequence-based typing, TaqMan genotyping, and high-resolution melting curve were established to identify rs76971248 (G>T). The Chi-square test was used for statistical analysis by SPSS. Results: Three methods based on PCR-SBT, TaqMan genotyping, and HRM were all able to identify rs76971248. The software for analyzing the results of HLA-E sequencing was easy to use, and the results were accurate. The frequency of rs76971248 in different types of leukemia patients was significantly lower than that in healthy blood donors (p < 0.05). And the frequency of the G/G genotype in leukemia patients was significantly higher than that in healthy blood donors (p < 0.05). Conclusions: For the screening of known SNP sites in large-scale populations, among the three methods, the TaqMan genotyping method had the advantage of shortest time consumption, simplest operation, and greatest specificity, which was the most appropriate method for this experiment. The analysis software for HLA-E gene sequencing needed to be further optimized. rs76971248 had a protective effect against leukemia. And the G/G genotype was a risk factor for leukemia.

Characterization of a novel variant allele, HLA-C*03:587, identified in a Chinese Han individual.

The novel HLA-C*03:587 allele differs from the closest allele C*03:03:01:01 in exon 5.

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).

Identification of HLA-DPB1*1104:01 by next-generation sequencing in a Chinese individual.

HLA-DPB1*1104:01 differs from HLA-DPB1*540:01 by a single nucleotide change in exon 2.

Adaptive Admixture of HLA Class I Allotypes Enhanced Genetically Determined Strength of Natural Killer Cells in East Asians.

Human natural killer (NK) cells are essential for controlling infection, cancer, and fetal development. NK cell functions are modulated by interactions between polymorphic inhibitory killer cell immunoglobulin-like receptors (KIR) and polymorphic HLA-A, -B, and -C ligands expressed on tissue cells. All HLA-C alleles encode a KIR ligand and contribute to reproduction and immunity. In contrast, only some HLA-A and -B alleles encode KIR ligands and they focus on immunity. By high-resolution analysis of KIR and HLA-A, -B, and -C genes, we show that the Chinese Southern Han (CHS) are significantly enriched for interactions between inhibitory KIR and HLA-A and -B. This enrichment has had substantial input through population admixture with neighboring populations, who contributed HLA class I haplotypes expressing the KIR ligands B*46:01 and B*58:01, which subsequently rose to high frequency by natural selection. Consequently, over 80% of Southern Han HLA haplotypes encode more than one KIR ligand. Complementing the high number of KIR ligands, the CHS KIR locus combines a high frequency of genes expressing potent inhibitory KIR, with a low frequency of those expressing activating KIR. The Southern Han centromeric KIR region encodes strong, conserved, inhibitory HLA-C-specific receptors, and the telomeric region provides a high number and diversity of inhibitory HLA-A and -B-specific receptors. In all these characteristics, the CHS represent other East Asians, whose NK cell repertoires are thus enhanced in quantity, diversity, and effector strength, likely augmenting resistance to endemic viral infections.

Characterization of the novel variant allele, HLA-B*13:82, identified in a Chinese Han individual.

The novel HLA-B*13:82 allele differs from the closest allele B*13:02:01:01 in exon 3.

Characterization of a novel variant allele, HLA-A*11:155, identified in a Chinese Han individual.

The novel HLA-A*11:155 allele differs from the closest allele A*11:01:01:01 in exon 3.

Natural Killer Cells Offer Differential Protection From Leukemia in Chinese Southern Han.

Interactions of human natural killer (NK) cell inhibitory receptors with polymorphic HLA-A, -B and -C molecules educate NK cells for immune surveillance against tumor cells. The KIR A haplotype encodes a distinctive set of HLA-specific NK cell inhibiting receptors having strong influence on immunity. We observed higher frequency of KIR A homozygosity among 745 healthy Chinese Southern Han than 836 adult patients representing three types of leukemia: ALL (OR = 0.68, 95% CI = 0.52-0.89, p = 0.004), AML (OR = 0.76, 95% CI = 0.59-0.98, p = 0.034), and CML (OR = 0.72 95% CI = 0.51-1.0, ns). We observed the same trend for NHL (OR = 0.47 95% CI = 0.26-0.88 p = 0.017). For ALL, the protective effect of the KIR AA genotype was greater in the presence of KIR ligands C1 (Pc = 0.01) and Bw4 (Pc = 0.001), which are tightly linked in East Asians. By contrast, the C2 ligand strengthened protection from CML (Pc = 0.004). NK cells isolated from KIR AA individuals were significantly more cytotoxic toward leukemic cells than those from other KIR genotypes (p < 0.0001). These data suggest KIR allotypes encoded by East Asian KIR A haplotypes are strongly inhibitory, arming NK cells to respond to leukemogenic cells having altered HLA expression. Thus, the study of populations with distinct KIR and HLA distributions enlightens understanding of immune mechanisms that significantly impact leukemia pathogenesis.

Characterization of a novel variant allele, HLA-C*08:125, identified in a Chinese Han individual.

The novel HLA-C*08:125 allele differs from the closest allele C*08:02:01:01 in exon 3.

Clinical significance of HLA-E genotype and surface/soluble expression levels between healthy individuals and patients with acute leukemia.

Human leukocyte antigen (HLA)-E is a nonclassical HLA molecule with limited polymorphisms. Genotype frequency and expression of HLA-E were examined here for the first time in acute leukemia patients and healthy controls. The frequency of HLA-E*01:03/*01:03 individuals was significantly higher (p = .008, OR = 1.845), while the frequency of HLA-E*01:01/*01:01 individuals was much lower in the patient group (p = .002, OR = .363) than in control group. The surface expression on HLA-E*01:03/*01:03 individuals was found to be significantly higher than on HLA-E*01:01/*01:01 individuals in both of acute leukemia and control groups, but no significant difference was observed between the corresponding genotypes in two groups. However, the level of expression of soluble HLA-E is significantly higher in patients than in the control group, but there was no genotype-specific expression in either group. These findings indicate that soluble HLA-E secretion and HLA-E*01:03/*01:03 genotype that brings higher surface expression might play important roles in the mechanisms underlying tumor escape in acute leukemia.

The genetic contribution of HLA-E*01:03 and HLA-E*01:03-G*01:01 to Posner-Schlossman syndrome in southern Chinese.

BACKGROUND: The polymorphisms of classical HLA-Ia and HLA-II loci have been associated with Posner-Schlossman syndrome (PSS) in the southern Chinese population. However, the associations of non-classical HLA-Ib (e.g., HLA-E and HLA-G) loci with PSS have not been reported for in the southern Chinese population. This study aimed to evaluate the associations of the HLA-E and HLA-G loci with PSS in a southern Chinese Han population group. METHODS: Ninety-seven unrelated patients with PSS and 90 ethnically matched control subjects were recruited from the Shenzhen Eye Hospital in China. The full-length sequences of HLA-E and HLA-G genes were amplified by long-range high-fidelity PCR, and the third exon of the HLA-E gene and the coding region of the HLA-G gene were sequenced. RESULTS: The allele frequency of HLA-E*01:03 in patients with PSS was significantly higher than that in the control group (P=0.017, corrected P=0.034, OR =1.66). The genotype frequencies of HLA-E*01:01/01:03 and HLA-E*01:03/01:03 in the PSS group were significantly higher than that in the control group (P=0.027, OR =2.62; P=0.011, OR =3.05; respectively). There were no significant differences in the frequency of HLA-G alleles and genotypes between the two groups (all P>0.05). The haplotype frequency of HLA-E*01:03-G*01:01 in the PSS group was significantly higher than that in the control group (P=0.019, OR =1.63), although this association did not survive the Bonferroni correction (corrected P=0.13). CONCLUSIONS: This study proved for the first time that HLA-E*01:03 and HLA-E*01:03-G*01:01 might be risk factors for PSS.

[Polymorphisms of MICA gene and their linkage disequilibrium with HLA-B among ethnic Han Chinese from Shenzhen].

OBJECTIVE: To study the distribution of MICA alleles among ethnic Han Chinese blood donors from Shenzhen and their linkage disequilibrium with HLA-B gene. METHODS: For 143 randomly selected blood donors, the MICA and HLA-B alleles were determined with a PCR-sequence based typing (SBT) method. Allelic frequency, haplotypic diversity and linkage disequilibrium were analyzed with a Pypop software. RESULTS: Thirteen MICA and 35 HLA-B alleles were identified among the 143 blood donors, among which MICA*008:01 had the highest frequency (76/286), whilst MICA*008:01-HLA-B*40:01 and MICA*010-HLA-B*46:01 were the most common haplotypes. No novel allele was identified. CONCLUSION: The allele frequencies, haplotype diversities and linkage disequilibrium parameters under a high resolution can facilitate further studies and applications of the MICA and HLA-B genes.

[Genetic polymorphisms of KIR2DS4 gene among ethnic Hans from southern China].

OBJECTIVE: To study genetic polymorphisms of the KIR2DS4 gene among ethnic Hans from southern China. METHODS: Genomic DNA was isolated from 306 unrelated individuals and amplified with KIR2DS4-specific PCR primers. KIR2DS4-positive samples were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic genotypes was accomplished by using Assign 3.5 software. For samples with inconclusive SBT results, RT-PCR products covering the entire coding sequence of the KIR2DS4 gene were subjected to cloning and haplotype sequencing. RESULTS: Among all tested samples, 297 were demonstrated to have carried the KIR2DS4 framework gene. For KIR2DS4-positive samples subjected to SBT for the entire coding sequences, no background was observed with the obtained sequences. Three of the seven identified alleles were of novel types, which were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for Factors of HLA System. The observed frequencies for the 7 alleles were KIR2DS4*00101 (78.8%), *003 (10.5%), *004 (16.0%), *010 (23.2%), *017 (0.3%), *00105 (0.3%) and *018 (0.7%), respectively. Allele KIR2DS4*007 was not found. The overall frequency for normal cell-surface expression KIR2DS4 alleles including 2DS4*00101, *017 and *00105 was 79.4%, and that for non cell-surface expression alleles including 2DS4*003, *004, *010 and *018 was 50.4%. The ratio between the two was 1.6:1. CONCLUSION: The present study has elucidated the allelic diversity of KIR2DS4 among ethnic Hans from southern China, which may provide valuable data for transplantation as well as studies on KIR-associated disease and evolution.