[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.

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.

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.

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.

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.

[A study of HLA-DPA1 and DPB1 matching status for unrelated donor-recipient pairs matched at allele level for HLA-A, -B, -C, -DRB1 and -DQB1 loci].

OBJECTIVE: To analyze the status of HLA-DPA1 and DPB1 matching for unrelated donor-recipient pairs matched at high-resolution allele level for HLA-A, B, C, DRB1 and DQB1 loci. METHODS: A total of 76 unrelated donor-recipient pairs matching at allele level for HLA-A, B, C, DRB1 and DQB1 loci were subjected to HLA-DPA1 and DPB1 sequence-based typing (SBT). HLA-DPA1and DPB1 matching status at high-resolution allelic level was also analyzed. RESULTS: The allelic identity ratio for single HLA-DPA1 and DPB1 were 17.1% and 9.2%, respectively. HLA-DPA1 and DPB1 allelic identity ratio were both very low. The majority of unrelated donor-recipient pairs (73.7%) had an incompatibility at 1 HLA-DPA1 allele, 9.2% of pairs had an incompatibility at 2 DPA1 alleles. As for the high-polymorphic HLA-DPB1 gene, 57.9% of studied donor-recipient pairs had an incompatibility at 1 HLA-DPB1 allele, almost 1/3 (32.9%) of them were completely incompatible. When HLA-DPA1 and DPB1 genes were analyzed together, the donor-recipient pairs matched at 2/4 was the most common (51.4%), 4/4 allelic complete matched pairs accounted for 5.6%, and 0/4 matched pairs accounted for 8.3%. CONCLUSION: Our results indicated that the ratio of HLA-DPA1 and DPB1 complete match in the unrelated donor-recipient pairs matching at allelic level for HLA-A, B, C, DRB1 and DQB1 loci were very low. The effect of HLA-DPA1 and DPB1 matching status on clinical unrelated stem cell transplantation still needs to be elucidated.

[Establishment of a large-scale bi-directional sequencing and genotyping platform for MICA gene exons 2 to 4].

OBJECTIVE: To establish a stable and large-scale bi-directional sequencing platform for genotyping MICA gene exons 2 to 4, and to analyze single nucleotide polymorphisms(SNP) of the region. METHODS: Primers for particular alleles of MICA gene exons 2 to 5 were designed. Optimal conditions for PCR amplification and sequencing reaction were explored. A commercialized one-way sequencing kit for MICA allele was used as a parallel control. Four samples carrying a MICA *010 allele were subjected to cloning and haplotype sequencing. RESULTS: Results of MICA allele typing of 100 samples for a parallel control group were confirmed by the establish method. Twenty-two SNP in MICA gene exons 2 to 4 were detected in Chinese population. Two novel allelic sequences were accepted by GenBank and IMGT/HLA database and officially named as MICA*065 and MICA*066 by the WHO Nomenclature Committee. A novel SNP in MICA gene intron 3 was discovered, with allelic sequence submitted to GenBank and IMGT/HLA database. CONCLUSION: The bi-directional sequencing genotyping platform may be applied for large-scale study of MICA allelic polymorphisms, tissue typing, organ transplantation and disease research.

[Study on HLA nucleotide sequence matching in epitope positions among recipient-donor pairs for allogenic hematopoietic stem-cell transplantation].

OBJECTIVE: To analyze the human leukocyte antigens(HLA)-A, -B, -Cw, -DRB1 and DQB1 nucleotide sequences between patients waiting for allogenic hematopoietic stem-cell transplantation (HSCT) and donors in Chinese population, and to establish strategy for maximizing optimal donor selection. METHODS: HLA high-resolution typing in a total of 537 recipient-donor pairs was determined by sequence based typing (SBT) method. The nucleotide BLAST tool was used to compare the nucleotide sequences among recipient-donor pairs. RESULTS: Only 16.20% (88/537) of recipient-donor pairs were found to fully match for nucleotide sequences of all HLA-A,-B,-Cw, -DRB1 and -DQB1 loci. Mismatch rate in single locus were 8.38% in HLA-A, 0.74% in HLA-B, 12.29% in HLA-C, 2.42% in HLA-DRB1, and 2.79% in HLA-DQB1, respectively. Mismatch rate in two or multiple HLA loci was 42.65%. Nonpermissive allele mismatch combinations (A 02:01-A 02:06, A 02:06-A 02:07, Cw 03:04-Cw 15:02, Cw 03:03-Cw 04:01, Cw 03:04-Cw 14:02, Cw 03:03-Cw 08:01, DRB1 04:03:01-DRB1 04:05) were detected in single mismatch HLA locus of recipient-donor pairs, mismatches of B 07:05:01-B 07:06, Cw 07:01:01-Cw 07:06 combinations outside of epitope positions were detected in two recipient-donor pairs. CONCLUSION: Our data suggested that attention should be paid in comparing nucleotide sequences between recipient and donor, and in distinguishing nucleotide sequence mismatches within and outside of the epitope positions. These results could serve as guidelines for donor selection.

[Recombination of HLA haplotypes in Guangdong Han nationality].

OBJECTIVE: To analyze the human leukocyte antigen complex class I (-A, -B & -C) and class II (-DRB1 & -DQB1) linked haplotypes of Guangdong Han nationality and to study the recombination events of five classical loci in the inheritance of HLA haplotypes. METHODS: A total of 939 peripheral blood samples were collected from 198 families in Guangdong Han nationality who came to our center for HLA typing from 2000 August to 2009 December. HLA-(A, B & DRB1) and HLA-(C & DQB1) alleles were typed by low-resolution polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSO) and PCR-sequence specific primers (PCR-SSP) methods respectively. Then the recombination sites were analyzed by familial study. The samples of 52 individuals from the families with exchange recombination were analyzed by the sequence-based typing (SBT) to judge whether the recombination was interallelic or interlocus exchange. RESULTS: Among 543 offspring individuals of 198 families in Guangdong Han nationality, 9 individuals with HLA-A-C-B-DRB1-DQB1 linked haplotypes had a recombination rate of 1.657%. Among 9 HLA haplotypes recombined families, 3 of them were found to have a crossover between HLA-A and -Cw loci and 6 of them a crossover between HLA-B and -DRB1 loci. Four of these recombination events occurred in the most common haplotypes A*3303-Cw*0302-B*5801-DRB1*0301-DQB1*0201 of Guangdong Han nationality. Among 9 cases of recombination, 5 of them were formed by a crossover between maternal chromosomes and 4 cases a crossover between paternal chromosomes. Three individuals with an exchange between A/Cw loci were all females. Among 6 cases with an exchange between B/DRB1 loci, 5 of them were males and 1 case was female. CONCLUSION: During the inheritance, recombination of HLA linked haplotype mainly occurred between A/Cw loci and B/DRB1 loci, the recombination is related to the haplotype-specificity and sex-specificity.

[Cloning and sequencing HLA-A and -B genomic DNA and analyzing polymorphism in regulatory regions in Chinese Han individuals].

In the present study, a high-resolute method for cloning and sequencing genomic full-length HLA-A and -B using 20 Chinese Han individuals was established. We detected 10 HLA-A allele sequences 4.2 kb in length and 6 HLA-B allele sequences 3.7 kb in length, and the sequences included all exons, all introns, 5'promoter, and 3'UTR of the two genes. All sixteen sequences have been submitted to GenBank and IMGT/HLA database. A*1153 is a novel allele, and the introns of B*151101 are firstly reported here. The 5'promoter and 3'UTR sequences of 5 HLA-A alleles and 2 HLA-B alleles are also firstly disclosed, and all other alleles have extended the genomic full length sequences released in IMGT/HLA database. The polymorphic structures of upper 5'promoter and downstream 3'UTR, which were uncovered in IMGT/HLA database, are firstly depicted in Chinese Han individuals. Twenty-six single nucleotide polymorphisms (SNPs) and one 3 bp-insertion/deletion (Indel) were located in the upper 5'promoter and 14 SNPs were located in the 3'UTR of HLA-A. In addition, five SNPs and one 1 bp-indel were located in the upper 5'promoter and 5 SNPs were located in the 3'UTR of HLA-B. Through analyzing the phylogenetic trees of 5'promoter, exons and 3'UTR of the two genes, we found that the evolution history of regulatory regions and exons is different between the two genes. The regulatory regions are tightly linked with exons in most of HLA-A alleles excluding A*24020101. On the contrary, recombinant events may occur frequently between regulatory regions and exons in most HLA-B alleles.