Characterization of a novel HLA‐B*40 allele,HLA‐B*40:186:02, by cloning and sequencing

A novel HLA‐B*40 variant, HLA‐B*40:186:02, has been identified by cloning and sequencing in a southern Chinese Han population. Aligned with HLA‐B*40:01:01, HLA‐B*40:186:02 has a nonsynonymous cytosine mutation at nucleotide position 165 in exon 2, leading to amino acid change from glycine to arginine at codon 56. It differs from HLA‐B*40:186:01 by a synonymous change (adenine to cytosine) at position 165 in exon 2.     

A conserved HLA‐A*02:28 associated HLA haplotype,A*02:28‐B*15:11‐DRB1*09:01, restricted to Taiwanese

HLA‐A*02:28, found in a Korean and a Japanese, was reported independently to the IMGT/HLA database in 2003 and 2005, respectively. We report here eight Taiwanese unrelated bone marrow hematopoietic stem cell donors carrying A*02:28 detected during our routine HLA typing exercise. The probable HLA‐A, ‐B and ‐DRB1 haplotype in association with A*02:28 may be deduced from the eight marrow stem cell donor as A*02:28‐B*15:11‐DRB1*09:01. Our result suggests A*02:28‐B*15:11‐DRB1*09:01 is a conserved HLA haplotype restricted to Taiwanese.     

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.     

Genomic sequences of HLA‐A*68:169, HLA‐B*07:298 and HLA‐B*39:129.

Three new HLA class I alleles were described in the Spanish population. HLA‐A*68:169 and ‐B*39:129 show one amino acid replacement at the α1‐domain, compared to A*68:02 (P47 > L47) and ‐B*39:06 (S11 > A11), respectively. HLA‐B*07:298 presents one nucleotide mutation within exon 1, resulting in a new amino acid position ?14, L>Q, which has not been previously described in any HLA protein. Prediction of the B*07:298 signal peptide cleavage did not show significant differences in comparison with that obtained for the rest of HLA‐B genes.     

Identification of the novel HLA‐B*13:02:13 allele in a Taiwanese haematopoietic stem cell donor and the probable HLA haplotype in association with B*13:02:13

Using sequence‐based typing method, we found a new HLA‐B*13:02 variant, B*13:02:13, in a Taiwanese haematopoietic stem cell donor. The DNA sequence of B*13:02:13 is identical to the sequence of B*13:02:01 in exons 2 and 3 except the nucleotide at position 588 where G is replaced by T (codon 172; CTG→CTT). The DNA sequence variation did not alter the amino acid sequence of B*13:02:01. The generation of B*13:02:13 is thought to derive from B*13:02:01 as a result of a silence mutation. The probable HLA‐A, HLA‐B and HLA‐DRB1 haplotype in association with B*13:02:1 may be deduced as HLA‐A*24‐B*13:02:13‐DRB1*07:01 or HLA‐A*02‐B*13:02:13‐DRB1*07:01. The discovery of B*13:02:13 furthers the polymorphism of HLA‐B*13 and HLA‐B*13:02.     

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.     

Characterization of a new HLA‐A allele,HLA‐A*02:07:08, by cloning and sequencing

In this report, we present a novel HLA‐A*02:07 allele, HLA‐A*02:07:08. HLA‐A*02:07:08 was identified in an individual of Han ethnicity in Hunan province, southern China. Following polymerase chain reaction‐sequence‐based typing (PCR‐SBT), this new allele was further confirmed by cloning and sequencing. HLA‐A*02:07:08 differs from HLA‐A*02:07:01 by a single synonymous C to T substitution at nucleotide position 131 in exon 3.     

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.     

Human herpesvirus 6B immediate‐early I protein contains functional HLA‐A*02, HLA‐A*03, and HLA‐B*07 class I restricted CD8+ T‐cell epitopes

Human herpesvirus 6B (HHV‐6B) is a ubiquitous pathogen with frequent reactivation observed in immunocompromised patients such as BM transplant (BMT) recipients. Adoptive immunotherapy is a promising therapeutic avenue for the treatment of opportunistic infections, including herpesviruses. While T‐cell immunotherapy can successfully control CMV and EBV reactivations in BMT recipients, such therapy is not available for HHV‐6 infections, in part due to a lack of identified protective CD8⁺ T‐cell epitopes. Our goal was to identify CD8⁺ T‐cell viral epitopes derived from the HHV‐6B immediate‐early protein I and presented by common human leukocyte Ag (HLA) class I alleles including HLA‐A*02, HLA‐A*03, and HLA‐B*07. These epitopes were functionally tested for their ability to induce CD8⁺ T‐cell expansion and kill HHV‐6‐infected autologous cells. Cross‐reactivity of specific HHV‐6B‐expanded T cells against HHV‐6A‐infected cells was also confirmed for a conserved epitope presented by HLA‐A*02 molecule. Our findings will help push forward the field of adoptive immunotherapy for the treatment and/or the prevention of HHV‐6 reactivation in BMT recipients.     

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.     

Discovery of the novel HLA‐DRB1*09:01:08 allele in a Taiwanese volunteer bone marrow donor and identification of the probable HLA‐A,HLA‐B and HLA‐DRB1 haplotype in association with DRB1*09:01:08

We report here the novel variant of HLA‐DRB1*09:01, DRB1*09:01:08, discovered in a Taiwanese volunteer bone marrow donor by a sequence‐based typing (SBT) method. The DNA sequence of DRB1*09:01:08 is identical to the sequence of DRB1*09:01:02 in exon 2 except a silent mutation at nucleotide position 261(C→T) (GCC→GCT at codon 58). We hypothesize DRB1*09:01:08 was probably derived from DRB1*09:01:02 via a nucleotide point mutation event. The plausible HLA‐A, HLA‐B and HLA‐DRB1 haplotype in association with DRB1*09:01:08 was deduced as A*02:07‐B*46:01‐DRB1*09:01:08.     

Allelic and haplotype diversity of HLA‐A,HLA‐B and HLA‐DRB1 gene at high resolution in the Nanning Han population

The distribution of human leucocyte antigen (HLA) allele and haplotype varied among different ethnic populations. In this study, we investigated the allele and haplotype frequencies of HLA‐A, HLA‐B and HLA‐DRB1 loci in the Nanning Han population who live in Guangxi province of China. We identified 26 HLA‐A, 56 HLA‐B and 31 HLA‐DRB1 alleles in 562 Nanning individuals of Han ethnic group by sequence‐based typing method. Of these, the three most common alleles in HLA‐A, HLA‐B and HLA‐DRB1 loci, respectively, were A*11:01 (32.12%), A*02:07 (12.54%), A*24:02 (12.01%); B*46:01 (14.41%), B*15:02 (13.61%), B*40:01 (11.48%); DRB1*15:01 (14.15%), DRB1*16:02 (11.57%) and DRB1*12:02 (10.14%). With the exception of HLA‐DRB1, the p values of the HLA‐A and HLA‐B loci showed that the HLA allelic distribution in this population was in accordance with Hardy–Weinberg expectation (p > 0.05). A total of 173 HLA~A‐B~DRB1 haplotype with a frequency of >0.1% were presented and the three most common haplotype were HLA‐A*33:03~B*58:01~DRB1*03:01 (6.12%), HLA‐A*11:01~B*15:02~DRB1*12:02 (3.39%) and HLA‐A*11:01~B*15:02~DRB1*15:01 (3.22%). The phylogenetic tree and the principal component analysis suggested that Nanning Han population had a relative close genetic relationship with Chinese Zhuang population and a relative distant genetic relationship with Northern Han Chinese. The information will be useful for anthropological studies, for HLA matching in transplantation and disease association studies in the Chinese population.     

A novel HLA‐B*14 allele – B*14:53 – genetics and serology

HLA‐B*14:53 was found in a UK European normal blood donor prior to registration on the Welsh Bone Marrow Donor Registry. It differs from B*14:13 by one base (103G>T) in exon 2 resulting in a substitution of alanine (A) in B*14:13 to serine (S) in B*14:53. Unique among current HLA‐B*14 alleles, B*14:53 and B*14:13 share a motif of 59 bases between positions 361 and 419 in exon 3. This motif is present in numerous HLA‐B alleles the commonest overall being B*08:01, suggesting that both B*14:53 and B*14:13 arose from intralocus gene conversion events with B*08:01. Thus, B*14:53 probably arose from B*14:01:01 (which has TCC at codon 11 (S), while B*14:13 arose from B*14:02:01:01 which has GCC at codon 11 (A). Additionally, the two likely B*14:53‐bearing and B*14:13‐bearing haplotypes are typical of B*14:01:01‐bearing and B*14:02:01:01‐bearing haplotypes, respectively. Serological testing, using 49 antisera with HLA‐B64, or B64, B65 reactivity, showed that the B*14:53 specificity did not react as a B64 (B*14:01) specificity and may appear as a short/weak HLA‐B14. This implies that residues additional to S at position 11 are involved in HLA‐B64 serological identity; for example, the motif 11S 97W 116F is possessed by B*14:01 and many other B*14 products (and B*39:79 plus some HLA‐C products) but not B65 (B*14:02) or the B*14:53 specificity. B*14:53 was found in a random HLA sequence‐based typed population of 32 530 normal subjects indicating a low precision allele frequency of 0.000015 in subjects resident in Wales.     

HLA‐A,HLA‐B and HLA‐DRB1 allele and haplotype frequencies of 10 918 Koreans from bone marrow donor registry in Korea

The human leucocyte antigen (HLA) system is the most polymorphic genetic system in humans, and HLA matching is crucial in organ transplantation, especially in hematopoietic stem cell transplantation. We investigated HLA‐A, HLA‐B and HLA‐DRB1 allele and haplotype frequencies at allelic level in 10 918 Koreans from bone marrow donor registry in Korea. Intermediate resolution HLA typing was performed using Luminex technology (Wakunaga, Japan), and additional allelic level typing was performed using PCR–single‐strand conformation polymorphism method and/or sequence‐based typing (Abbott Molecular, USA). Allele and haplotype frequencies were calculated by direct counting and maximum likelihood methods, respectively. A total of 39 HLA‐A, 66 HLA‐B and 47 HLA‐DRB1 alleles were identified. High‐frequency alleles found at a frequency of ≥5% were 6 HLA‐A (A*02:01, *02:06, *11:01, *24:02, *31:01 and *33:03), 6 HLA‐B (B*15:01, *35:01, *44:03, *51:01, 54:01 and *58:01) and 8 HLA‐DRB1 (DRB1*01:01, *04:05, *04:06, *07:01, *08:03, *09:01, *13:02 and *15:01) alleles. At each locus, A*02, B*15 and DRB1*14 generic groups were most diverse at allelic level, consisting of 9, 12 and 11 different alleles, respectively. A total of 366, 197 and 21 different HLA‐A‐B‐DRB1 haplotypes were estimated with frequencies of ≥0.05%, ≥0.1% and ≥0.5%, respectively. The five most common haplotypes with frequencies of ≥2.0% were A*33:03‐B*44:03‐DRB1*13:02 (4.97%), A*33:03‐B*58:01‐DRB1*13:02, A*33:03‐B*44:03‐DRB1*07:01, A*24:02‐B*07:02‐DRB1*01:01 and A*24:02‐B*52:01‐DRB1*15:02. Among 34 serologic HLA‐A‐B‐DR haplotypes with frequencies of ≥0.5%, 17 haplotypes revealed allele‐level diversity and majority of the allelic variation was arising from A2, A26, B61, B62, DR4 and DR14 specificities. Haplotype diversity obtained in this study is the most comprehensive data thus far reported in Koreans, and the information will be useful for unrelated stem cell transplantation as well as for disease association studies.     

HLA‐B*15 subtypes in the population of north‐eastern Thailand

The HLA‐B*15 group is the most polymorphic HLA‐B allele and so has several subtypes. These subtypes have not been defined in the population of north‐eastern Thailand (NET). In a previous study, using polymerase chain reaction–sequence‐specific primers (PCR‐SSP), subtypes were categorized into four groups, namely: group I: HLA‐B*15 (01, 04–07, 12, 14, 19, 20, 24, 25, 26N, 27, 32, 33, 34 and 35); group II: HLA‐B*15 (02, 08, 11, 15, 28 and 30); group III: HLA‐B*1503/4802; group IV: HLA‐B*1521. Groups I and II occurred frequently (allele frequency = 8.0 and 2.5%), and thus we optimized the polymerase chain reaction–single‐stranded conformation polymorphism (PCR‐SSCP) method to identify HLA‐B*15 subtypes of groups I and II. Eighty samples of DNA carrying HLA‐B*15 from 300 healthy unrelated individuals were tested. B*1502 (52.5%) and B*1525 (13.8%) were the most common subtypes found in NET. They also showed strong linkage disequilibrium with HLA‐Cw and heterogeneity of HLA‐A, DR, DQ haplotypes. Although limited conclusions can be drawn from this study because of the small number of DNA references used, the baseline data will be useful in the selection of common HLA‐B*15 alleles when subtyping for unrelated donor transplantations.     

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.     

HLA‐B allele dropout in PCR sequence‐specific oligonucleotide probe typing due to intronic polymorphism in the novel B*58:01:01:02 allele

Currently, Luminex technology based on the PCR sequence‐specific oligonucleotide (SSO) probe method has been widely used for HLA genotyping in the immunogenetics laboratories. Here, we reported a case with HLA‐B allele dropout by Luminex technology. The initial HLA‐B result of the Luminex method with a commercial agent kit was inconclusive, and then, the result of PCR‐SBT technology indicated the dropout as a HLA‐B*58 allele. Subsequently, the full‐length sequence of HLA‐B allele was determined by TOPO‐TA cloning, and a novel allele B*58:01:01:02 was identified in the individual. Compared with HLA‐B*58:01:01:01, the novel allele showed some nucleotides difference at 509 C>T, 521 T>G and CCC insertion in position 503 of intron 2. According to the full‐length sequence, the new mutations of intron 2 were contributed to HLA‐B locus allele dropout in the sample. Our results indicated multiplatform should be used to improve the HLA typing accuracy when a conclusive HLA genotype cannot be determined.     

HLA‐A, ‐B and ‐DRB1 allele and haplotype frequencies of 8333 Chinese Han from the Zhejiang province,China

The distribution of human leucocyte antigen (HLA) allele and haplotype is varied among different ethnic populations. In this study, HLA‐A, ‐B and ‐DRB1 allele and haplotype frequencies were determined in 8333 volunteer bone marrow donors of Zhejiang Han population using the polymerase chain reaction sequence‐based typing. A total of 52 HLA‐A, 96 HLA‐B and 61 HLA‐DRB1 alleles were found. Of these, the top three frequent alleles in HLA‐A, HLA‐B and HLA‐DRB1 loci, respectively, were A*11:01 (24.53%), A*24:02 (17.35%), A*02:01 (11.58%); B*40:01 (15.67%), B*46:01 (11.87%), B*58:01 (9.05%); DRB1*09:01 (17.54%),DRB1*12:02 (9.64%) and DRB1*08:03 (8.65%). A total of 171 A‐B‐DRB1 haplotypes with a frequency of >0.1% were presented and the five most common haplotypes were A*33:03‐B*58:01‐ DRB1*03:01, A*02:07‐B*46:01‐DRB1*09:01, A*30:01‐B*13:02‐DRB1*07:01, A*33:03‐B*58:01‐RB1*13:02 and A*11:01‐B*15:02‐DRB1*12:02. The information will be useful for selecting unrelated bone marrow donors and for anthropology studies and pharmacogenomics analysis.     

Epitopes and motifs of the HLA‐B*14 allele family products and related HLA‐B14 cross‐reactive specificities

The split specificities of HLA‐B14 (B64, B65) are assigned to the B*14:01 (B64) and B*14:02 (B65) products only. Of the further 50 B*14 expressed products, only B*14:03 and B*14:06 are officially designated as HLA‐B14. The B*14:08 product differs from B64 by a single amino acid substitution of W97R, while the B*14:53 specificity (which is a “short” B14 and neither B64 nor B65) differs from B64 by three residues (W97S, Y113H and F116Y). Comprehensive testing of B*14:08:01 cells (using 49 alloantisera with B64 or B64, B65 specificities, and five monoclonal antibodies with B65 or B64, B65 activity) showed that the B*14:08 specificity is, like the B*14:53 product, neither B64 nor B65 and appears as a “short” B14 specificity. To help understand the serological reactivity of the B*14:08 and B*14:53 products, and B64 and B65, we identified seven published epitopes (11AV, 97W, 61ICT, 116F, 131S+163T, 170RH and 420) and, by inspection, 29 motifs, that encompass one or more of B64, B65 and various HLA‐B14 cross‐reactive group specificities. We then considered the possession of these epitopes and motifs by the products of B*14:01 to B*14:06, B*14:08 and B*14:53. Seventeen of the 29 motifs fully complied with the one‐/two‐patch functional epitope concept for amino acid proximity, as determined by Cn3D software, the remainder partially complied. The nature and patterns of epitopes and motifs possessed by both B*14:08 and B*14:53 specificities supported their designation as HLA‐B14 but non‐B64/B65. Also that epitope 97W, with 11S or 11A, is critical for serological B64 and B65 reactivity. And conversely, that epitope 116F, and several identified motifs, are probably unimportant for HLA‐B14 antibody reactivity. The previous submission that the B*14:03 specificity is HLA‐B65 was compatible with its epitope/motif pattern. B*14:04 cells would also be expected to react as B65, based on its epitope/motif pattern, and not as B64 as previously implied. Also, from their epitope/motif patterns, and external serological information, it is probable that the B*14:05 and B*14:06 specificities will both appear as “short” HLA‐B14, non‐B64/B65. Several epitopes and motifs encompassed a range of HLA‐B specificities included in the serological HLA‐B14 cross‐reactive group, thus supporting these original serological findings.