Identification of a new HLA-B*40 allele, HLA-B*4081, in a Chinese individual

A novel human leukocyte antigen (HLA)-B*40 allele, officially named B*4081, was identified during routine high-resolution sequence-based typing in a Chinese potential hematopoietic stem cell transplantation donor. The HLA-B*4081 allele shows one nucleotide difference from B*400101 in exon 2 at nucleotide position 124 where G→C (codon 18 GGG→CGG) resulting in a coding change, 18Gly is changed to Arg, this is a unique nucleotide change among the HLA class I alleles, suggesting a point mutation mechanism.     

A novel HLA-B*40 sequence--B*40:92

A new allele, HLA‐B*40:92, was identified in a north‐western European subject during polymerase chain reaction using sequence‐specific priming (PCR‐SSP)‐based typing of haematopoietic stem cell (HSC) donors. B*40:92 differs from B*40:01:01 by six nucleotides at positions 559, 560, 603, 605, 610 and 618 in exon 3 which represents a substitution motif of at least 60 nucleotides. This motif, which occurs in numerous HLA alleles including the relatively high frequency B*15 and B*35 allele families, encode four amino acid changes at positions 163 (glutamic acid > leucine), 177 (aspartic acid > glutamic acid), 178 (lysine > threonine), 180 (glutamic acid > glutamine) and a silent substitution, conserved alanine, at codon 182. Thus, it is likely that HLA‐B*40:92 occurred following a gene conversion‐like or interallelic recombination event involving B*40:01:01 and probably a B*15 or more likely a B*35 family allele. HLA‐B*40:92 was found on a haplotype with HLA‐A*02:01, B*40:92, C*03:04, DRB1*13:02, DRB3*03:01, DQA1*01:02, DQB1*06:04, DPA1*02:02, DPB1*05:01. Tests on 69 selected B40 and B35 antisera and Lambda Monoclonal Trays? show that B*40:92 encodes a ‘short’ B40/B60 serological specificity which displays some HLA‐B35 reactivity. The HLA‐B40 and HLA‐B35 motifs (possible epitopes) responsible for this serological reactivity were identified. This single example of HLA‐ B*40:92 was found in 56,823 consecutive HLA PCR‐SSP typed HSC donors indicating a carriage frequency of 0.00176% (allele frequency 0.00001) in blood donors resident in Wales. An Epstein‐Barr virus transformed B‐cell line from the HLA‐B*40:92 donor is available.     

HLA-A*02:251新等位基因克隆测序鉴定及序列分析

目的 鉴定中国人群人类白细胞抗原(human leukocyte antigen,HLA)A*02:251新等位基因,分析新等位基因遗传特征.方法 采用聚合酶链反应-测序分型法(polymerase chain reaction-sequence based typing,PCR-SBT)对组织配型健康供、患者进行HLA基因分型,发现先证者核苷酸杂合序列与已知序列不匹配,不能指定先证者HLA等位基因型,对先证者DNA扩增HLA-A位点第2～4外显子,PCR产物经克隆到PMD18-T质粒载体中以获得单链核苷酸序列,对克隆所得产物进行HLA-A基因的第2～4外显子双向测序分析.结果 发现先证者的一个HLA-A*02:06:01基因被确认,而另一个HLA-A基因为新等位基因,其序列被GenBank接受(编号为HM245348).新等位基因序列通过IMGT/HLA 数据库BLAST,与最相近的A*02:01:01:01相比,在第3外显子上有1个核苷酸的不同,即第383位 G>C,密码子 128 GAG→GAC,氨基酸由谷氨酸(Glu)→天门冬氨酸(Asp).供、患者HLA-A、B、C、DQB1位点等位基因不匹配.结论 该等位基因为新的HLA-A*02:251等位基因.中国人群HLA-A 位点第3外显子核苷酸序列存在多态性.

Abstract：

Objective To identify a novel human leukocyte antigen (HLA) allele A*02:251 and analyze the sequences in Chinese population. Methods Routine HLA-A, -B, -DRB1 high resolution genotyping for healthy Chinese donors and patients was performed with polymerase chain reaction-sequence based typing. An unknown HLA-A allele was initially detected by HLA typing in the healthy donor. Genomic DNA of the HLA-A locus in the proband was amplified, the amplified product was cloned by PMD18-T to split the two alleles, and selected clones were sequenced. Results The sequencing results showed that a normal A*02:06:01 and a novel A*02:251 variant allele were identified. The sequence of the novel allele has been submitted to GenBank (HM245348). Nucleotide sequence alignments with HLA-A allele from the IMGT/HLA Sequence Database showed that the novel A*02 variant allele differed from the closest allele A*02:01:01:01 by nt 383 G>C (codon 128 GAG>GAC) in exon 3, which resulted in one amino acid substitution of Glu>Asp. The HLA-A, B, C and DQB1 alleles of the healthy donor did not match with that of the patient. Conclusion This novel allele is officially designated as HLA-A*02:251 by World Health Organization(WHO) Nomenclature Committee (Submission ID HWS10010755). The sequence of HLA-A locus in exon 3 is confirmed to be polymorphic in Chinese population.     

Three new HLA‐C alleles (HLA‐C*14:02:13, HLA‐C*15:72 and HLA‐C*15:74) in Saudi bone marrow donors

Three new HLA‐C alleles were identified by sequence‐based typing method (SBT) in donors for the Saudi Bone Marrow Donor Registry (SBMDR). HLA‐C*14:02:13 differs from HLA‐C*14:02:01 by a silent G to A substitution at nucleotide position 400 in exon 2, where lysine at position 66 remains unchanged. HLA‐C*15:72 differs from HLA‐C*15:22 by a nonsynonymous C to A substitution at nucleotide position 796 in exon 3, resulting in an amino acid change from phenylalanine to leucine at position 116. HLA‐C*15:74 differs from HLA‐C*15:08 by a nonsynonymous C to T substitution at nucleotide position 914 in exon 3, resulting in an amino acid change from arginine to tryptophan at position 156.     

Detection of the rare HLA‐B*40:97 allele in an unrelated Taiwanese bone marrow donor

We detected a rare HLA‐B locus allele, B*40:97, in a Taiwanese unrelated donor in our routine HLA SBT (sequence‐based typing) exercise for a possible hematopoietic stem cell donation. In exons 2, 3 and 4, the sequence of B*40:97 is identical to the sequence of B*40:02:01 except one nucleotide at nucleotide position 760 (C‐>T) in exon 4. The nucleotide variation caused one amino acid alteration at residue 230 (L‐>F). B*40:97 was probably derived from a nucleotide substitution event where C was replaced by T at nucleotide 760 involving B*40:02:01. The HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1 haplotype in association with B*40:97 may be deduced as A*26:01‐B*40:97‐C*03:03‐DRB1*11:01‐DQB1*03:03. Our recognition of B*40:97 in Taiwanese helps to fill the void of ethnic information for the allele B*40:97 reported to the IMGT/HLA Database.     

The HLA‐C*05 family allele – C*05:142

The sequencing of exons 2–7 of a likely new HLA‐C*05 allele identified the second example of HLA‐C*05:142, in a male UK European, within a few months of the first example being found in Germany. C*05:142 differs from C*05:01:01:01 by a single base (395G>C) in exon 3 resulting in an amino acid substitution of R108P. Comprehensive serological HLA‐Cw5 typing, using 19 antisera, indicated that C*05:142 encodes a “normal” Cw5 specificity. Failure to identify the involvement of position 108 in published HLA‐C epitopes supported this assertion. The likely HLA class I C*05:142‐bearing haplotype is A*02:01~C*05:142~B*44:02. This new allele has a maximum frequency of 0.00001, in 34,743 sequenced‐based typed subjects, contrasting with that of C*05:01 (allele frequency 0.10441), in our local, largely UK European, blood donors.     

Characterization of a novel allelic variant in HLA‐B*40 lineage,HLA‐B*40:298:02, by cloning and sequencing

A novel allelic variant in HLA‐B*40 lineage, HLA‐B*40:298:02, has been identified in an individual of Han ethnicity afflicted with nasopharyngeal carcinoma in Hunan province, southern China. Following polymerase chain reaction–Sanger sequence‐based typing (PCR–SBT), this new variant was further confirmed by two distinct strategies of cloning and sequencing. HLA‐B*40:298:02 differs from HLA‐B*40:298:01 by a single synonymous cytosine substitution at nucleotide position 26 (T→C) in exon 3, which corresponds to codon 99 of the mature HLA‐B mRNA molecule. This new allele has an estimated frequency of 0.0002, in about 2,500 sequence‐based typed subjects from the same population.     

HLA class I null alleles and new alleles affect unrelated bone marrow donor searches

Unrecognized HLA null alleles or new alleles may affect the outcome of bone marrow transplants using unrelated donors. Some reports suggest that null alleles occur in the range of 0.003-0.07% (1, 2), which has led some transplant programs to stop performing serologic typing. We describe nine cases involving expression variants or new alleles. Three cases involved expression variants, including two null alleles and A*24020102L. One of the null alleles was a new variant of A*02. Seven cases involved new alleles. In five cases, there where discrepancies between HLA typing by serology and PCR-SSP. These included the three expression variants, one new B40 allele that typed serologically as B41 and one new B*07 allele that typed serologically as B42. Eight of these cases were found in the course of typing bone marrow transplant patients or potential unrelated donors since May of 2001 (total tested, 710 patients, 1914 donors). Thus, the incidence of null alleles was two in 2,624 (0.08%). Sequence-based typing (SBT) was performed on 676 of these samples. The decision to perform SBT was influenced by finding a serologic typing discrepancy in two cases. In one of those cases, SBT would probably have been performed at a later time, prior to final selection of a donor. Thus, the incidence of new alleles was between 4 and 6 of 676 (0.59-0.89%). We conclude that new HLA alleles and null alleles are uncommon but not extremely rare, and they continue to affect a significant number of unrelated donor searches.     

Identification of a novel HLA-Cw*08 variant allele, Cw*0824

A novel HLA-C allele, Cw*0824, which was identified from an individual of the Han Chinese, differs from Cw*080101 at codon 222 (GAG > AAG ) in exon 4, which results in an amino acid change Glu222Lys.
In recent years, many human leukocyte antigen (HLA)-C alleles have been identified. Up to date, 23 different Cw*08 alleles have been identified according to the IMGT/HLA Database release 2.25.2 May 2009 (1) . Here, we describe the identification of the novel allele HLA-Cw*0824 that was found during routine high resolution sequence-based typing (SBT) of a Chinese stem cell voluntary donor. The HLA alleles of the donor were typed as A*11, 24; B*15, 46; and DRB1*09, 12.     

Two novel HLA‐C alleles,HLA‐C*02:02:34 and HLA‐C*03:369, were identified in the same individual

Two new HLA class I alleles, HLA‐C*02:02:34 and HLA‐C*03:369, were characterized in a single Polish bone marrow donor.     

The distributions of HLA‐A,HLA‐B,HLA‐C,HLA‐DRB1 and HLA‐DQB1 allele and haplotype at high‐resolution level in Zhejiang Han population of China

The distributions of HLA allele and haplotype are variable in different ethnic populations and the data for some populations have been published. However, the data on HLA‐C and HLA‐DQB1 loci and the haplotype of HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1 loci at a high‐resolution level are limited in Zhejiang Han population, China. In this study, the frequencies of the HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1 loci and haplotypes were analysed among 3,548 volunteers from the Zhejiang Han population using polymerase chain reaction sequencing‐based typing method. Totals of 51 HLA‐A, 97 HLA‐B, 45 HLA‐C, 53 HLA‐DRB1 and 27 HLA‐DQB1 alleles were observed. The top three frequent alleles of HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1 loci were A*11:01 (23.83%), A*24:02 (17.16%), A*02:01 (11.36%); B*40:01 (14.08%), B*46:01 (12.20%), B*58:01 (8.50%); C*07:02 (18.25%), C*01:02:01G (18.15%), C*03:04 (9.88%); DRB1*09:01 (17.52%), DRB1*12:02 (10.57%), DRB1*15:01 (9.70%); DQB1*03:01 (22.63%), DQB1*03:03 (18.26%) and DQB1*06:01 (10.88%), respectively. A total of 141 HLA‐A‐C‐B‐DRB1‐DQB1 haplotypes with a frequency of ≥0.1% were found and the haplotypes with frequency greater than 3% were A*02:07‐C*01:02:01G‐B*46:01‐DRB1*09:01‐DQB1*03:03 (4.20%), A*33:03‐C*03:02‐B*58:01‐DRB1*03:01‐DQB1*02:01 (4.15%), A*30:01‐C*06:02‐B*13:02‐DRB1*07:01‐DQB1*02:02 (3.20%). The likelihood ratios test for the linkage disequilibrium of two loci haplotypes was revealed that the majority of the pairwise associations were statistically significant. The data presented in this study will be useful for searching unrelated HLA‐matched donor, planning donor registry and for anthropology studies in China.     

Analysis for complete genomic sequence of HLA-B and HLA-C alleles in the Chinese Han population

In the present study, we have determined the complete genomic sequence and analysed the intron polymorphism of partial HLA‐B and HLA‐C alleles in the Chinese Han population. Over 3.0 kb DNA fragments of HLA‐B and HLA‐C loci were amplified by polymerase chain reaction from partial 5′ untranslated region to 3′ noncoding region respectively, and then the amplified products were sequenced. Full‐length nucleotide sequences of 14 HLA‐B alleles and 10 HLA‐C alleles were obtained and have been submitted to GenBank and IMGT/HLA database. Two novel alleles of HLA‐B*52:01:01:02 and HLA‐B*59:01:01:02 were identified, and the complete genomic sequence of HLA‐B*52:01:01:01 was firstly reported. Totally 157 and 167 polymorphism positions were found in the full‐length genomic sequence of HLA‐B and HLA‐C loci respectively. Our results suggested that many single nucleotide polymorphisms existed in the exon and intron regions, and the data can provide useful information for understanding the evolution of HLA‐B and HLA‐C alleles.     

Study on the polymorphisms of HLA‐ABCDQB1DRB1 alleles and haplotypes in Hubei Han population of China

The present study aimed to analyse the frequencies of human leukocyte antigen HLA‐ABCDQB1 and HLA‐DRB1 alleles and haplotypes in a subset of 3,732 Han population from Hubei of China. All samples were typed in the HLA‐ABCDQB1 and HLA‐DRB1 loci using the sequence‐based typing method; subsequently, the HLA polymorphisms were analysed. A total of 47 HLA‐A, 89 HLA‐B, 43 HLA‐C, 49 HLA‐DRB1 and 24 HLA‐DQB1 alleles were identified in the Hubei Han population. The top three most frequent alleles in the HLA‐ABCDQB1 and HLA‐DRB1 were A*11:01 (0.2617), A*24:02 (0.1590), A*02:07 (0.1281); B*46:01 (0.1502), B*40:01 (0.1409) and B*58:01 (0.0616); C*01:02 (0.2023), C*07:02 (0.1691) and C*03:04 (0.1175); and DQB1*03:01 (0.2000), DQB1*03:03 (0.1900), DQB1*06:01 (0.1187); DRB1*09:01 (0.1790), DRB1*15:01 (0.1062) and DRB1*12:02 (0.0841), respectively. Meanwhile, the three most frequent two‐loci haplotypes were A*02:07‐C*01:02 (0.0929), B*46:01‐C*01:02 (0.1366) and DQB1*03:03‐DRB1*09:01 (0.1766). The three most frequent three‐loci haplotypes were A*02:07‐B*46:01‐C*01:02 (0.0883), B*46:01‐DQB1*03:03‐DRB1*09:01 (0.0808) and C*01:02‐DQB1*03:03‐DRB1*09:01 (0.0837). The three most frequent four‐loci haplotypes were A*02:07‐B*46:01‐C*01:02‐DQB1*03:03 (0.0494), B*46:01‐DRB1*09:01‐C*01:02‐DQB1*03:03 (0.0729) and A*02:07‐B*46:01‐DQB1*03:03‐DRB1*09:01 (0.0501). The most frequent five‐loci haplotype was A*02:07‐B*46:01‐C*01:02‐DQB1*03:03‐DRB1*09:01 (0.0487). Heat maps and multiple correspondence analysis based on the frequencies of HLA specificity indicated that the Hubei Han population might be described into Southern Chinese populations. Our results lay a certain foundation for future population studies, disease association studies and donor recruitment strategies.     

A nonsense mutation in exon 3 results in the HLA-B null allele B*5127N

A new HLA-B null allele has been identified within the B*51 group by combined serological and molecular typing of an Italian Caucasoid family. Serological data indicated that the proband typed homozygous for A2 and B60. Confirmatory typing using sequence specific oligonucleotide hybridization (SSPOH) detected a second B allele within the B*51 group. Allele specific typing (SSP) for B*51 subtypes, including the known B*5111N allele, was performed, and typing results were consistent with B*5101, suggesting the presence of a new null variant. Cloning and sequencing of this allele identified a B*5101 variant with a nonsense mutation in exon 3. This new null allele has been designated B*5127N. The combined use of serologic and DNA-based typing methods facilitates the identification of null and low-expression alleles. An overview of null alleles of class I HLA is presented.     

Complete nucleotide sequence characterization of DRB5 alleles reveals a homogeneous allele group that is distinct from other DRB genes

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A better and comprehensive sequence assessment can be achieved by the inclusion of full gene sequences of all the common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of two alleles available. In the present study the DRB5 genes from 18 subjects alleles were cloned and sequenced; haplotype analysis showed that 17 of them had a single copy of DRB5 and one consanguineous subject was homozygous at all HLA loci. Methodological approaches including robust and efficient long-range PCR amplification, molecular cloning, nucleotide sequencing and de novo sequence assembly were combined to characterize DRB5 alleles. DRB5 sequences covering from 5′UTR to the end of intron 5 were obtained for DRB5*01:01, 01:02 and 02:02; partial coverage including a segment spanning exon 2 to exon 6 was obtained for DRB5*01:03, 01:08N and 02:03. Phylogenetic analysis of the generated sequences showed that the DRB5 alleles group together and have distinctive differences with other DRB loci. Novel intron variants of DRB5*01:01:01, 01:02 and 02:02 were identified. The newly characterized DRB5 intron variants of each DRB5 allele were found in subjects harboring distinct associations with alleles of DRB1, B and/or ethnicity. The new information provided by this study provides reference sequences for HLA typing methodologies. Extending sequence coverage may lead to identify the disease susceptibility factors of DRB5 containing haplotypes while the unexpected intron variations may shed light on understanding of the evolution of the DRB region.     

HLA-B*4012: a new allele with unique serological features

We have defined the new allele HLA-B*4012, which had been isolated from a black individual. It was initially recognized as a serologically unique allele when typing her father for renal transplantation. The HLA class I phenotype was A*0201,*6602; B*4001,*4012; Bw6; Cw*0304,*1505. Sequencing from exon 1 through intron 3 of B*4012 was performed. B*4012 is identical to B*4001 and B*4010 in exon 3, and in the 3' part of exon 2, but it is unique in that exon 1 and the 5' part of exon 2 are identical to B*1503, B*1509, B*1510, B*1518, B*1523, and B*1529. The generation of this allele is best explained by a recombination event in exon 2 (break point between nucleotides 205 and 222 from the beginning of the coding region) of B*4001 or B*4010 with one of these B*15 variants as a donor allele. Its unique serological feature (B48, B60, B70, and B72 reactivity) is consistent with the sequence data of its donor alleles.     

Identification of an HLA-B7 serological variant and its characterization by sequencing based typing

We have identified an HLA-B*07 variant allele, B*0716, in a Caucasoid cadaver kidney donor. The HLA class I type by polymerase chain reaction using sequence-specific primers (PCR-SSP) was A*01, 32; B*07, 08; Cw*07. Serological typing, using monoclonal and polyclonal anti-HLA antisera, gave disparate results for the B antigens. Monoclonal antibodies identified B7 and B8 antigens but polyclonal antisera recognised only the B8 antigen. PCR using sequencing based typing (PCR-SBT) confirmed the presence of both B*0703 and B*0801 alleles but with a mutation in one of the alleles. The HLA-B*07 allele was isolated by allele-specific PCR and was shown to have a mutation, G-->T, at 292 in exon 2. This mutation changes codon 74, which encodes aspartic acid (GAC) present in all previously identified B*07 alleles, to tyrosine (TAC) in the variant. The serological results suggest that codon 74 is a crucial part of a B7 antigen-specific epitope recognised by tissue typing polyclonal antisera.     

Two novel alleles HLA‐A*02:433 and HLA‐A*02:434 identified in Saudi bone marrow donors using sequence‐based typing

In this report, we present two novel HLA‐A alleles: HLA‐A*02:433 and HLA‐A*02:434. These alleles were identified by sequence‐based typing method (SBT), in two donors for the Saudi Bone Marrow Donor Registry (SBMDR). Allele A*02:433 is identical to A*02:05:01G except for a G to A substitution at nucleotide position 449 in exon 2. This substitution results in glycine to serine substitution at position 83. Whereas, allele A*02:434 is identical to A*02:01:01G except for a C to A substitution at nucleotide position 245 in exon 2, which results in phenylalanine to threonine substitution at position 15. The generation of both alleles appears to be the result of nucleotide point mutation involving 02:01:01 and 02:05:01.     

HLA-B*8202 identified in a Caucasoid potential bone marrow donor

Sequence analysis of HLA class I alleles has continued to reveal the true extent of polymorphism, particularly for B-locus alleles. This diversity can arise through reshuffling of polymorphic sequences generated by point mutation, resulting in interallelic recombination or intergenic recombination (1). Here we describe a new B-locus allele, B*8202, which is structurally most similar to B*8201, having only one nucleotide difference in exon 3 at nucleotide 557, resulting in an amino acid change of aspartic acid to glycine at residue 162. Glycine is the consensus amino acid for B-locus alleles, which suggests that B*8202 is older than B*8201 in evolutionary terms. B*8201 was found to be a hybrid of B*4501 and B*5602 that may have arisen through recombination events, explaining the serological patterns observed with these allotypes. The importance of high-resolution typing is emphasised here as routine typing suggested the presence of B*8201 and the new variant allele may have been missed had it not been typed further by sequence-based typing.     

序列分析及确认HLA新等位基因B*55:46

背景：近几年来,随着中华骨髓库的建立和人类白细胞抗原分型技术的不断发展和提高,中国人类白细胞抗原新等位基因不断被发现。 目的：探索中国人的人类白细胞抗原新等位基因。 方法：应用PCR-序列特异性寡核苷酸探针基因分型技术,对1名27岁男性汉族造血干细胞志愿捐献者进行HLA基因分型,并应用基于测序的方法分析该基因序列及与最相近等位基因序列的差异。 结果与结论：PCR-序列特异性寡核苷酸探针结果显示该样本HLA-B基因座反应格局出现异常提示;基因测序结果表明其B基因座第3外显子序列与所有已知HLA-B等位基因序列均不一致,在所检测的第2、3外显子中,与序列最相近的等位基因B*55:02:01的差异只是在第3外显子发生了nt 412 A→G一个核苷酸替代,导致第138位密码子由AAC→GAC,相应的编码的天冬酰胺改变为天冬氨酸。将其序列提交国际基因数据库及IMGT/HLA 数据库,证实该HLA等位基因为国际上首次发现,被世界卫生组织织人类白细胞抗原因子命名委员会正式命名为HLA-B*55:46 (HM989018)。