Recognition of HLA‐A*24:137 allele in a Taiwanese unrelated bone marrow stem cell donor and the plausible HLA haplotype associated with A*24:137

We detected a rare HLA‐A*24:137 allele in an unrelated Taiwanese haematopoietic stem cell donor during a routine SBT (sequence‐based typing) HLA typing exercise. The DNA sequence of A*24:137 is identical to the sequence of A*24:02:01:01 in exons 2 and 3 except at codon 21 where CGC was replaced with CAA. The DNA variation caused an amino acid alteration at amino acid residue 21 (R‐>Q). The HLA haplotype in association with A*24:137 may be deduced as A*24:137‐B*15‐DRB1*14. The formation of A*24:137 was probably the result of a nucleotide point mutation involving A*24:02:01:01. It remains to be determined whether A*24:137 is restricted to Taiwanese/Chinese ethnicity.     

Frequency of HLA‐A alleles in the Syrian population genotyped by sequence‐based typing

HLA‐A molecules are highly polymorphic. Their accurate typing at a high‐resolution level is crucial for successful organ, bone marrow and cord blood transplantation. Furthermore, several HLA alleles have been involved in susceptibility to autoimmune diseases, allergies, cancers and inflammations. In order to determine common HLA‐A alleles in Syria and their frequencies, sequence‐based typing (SBT) was used to genotype HLA‐A alleles at high resolution (four digit level) among one hundred and thirty randomly selected Syrian individuals. Exons 2, 3 and 4 of the HLA‐A gene were amplified by PCR and sequenced. The sbt ‐engine software was used for allele assignment. Ambiguities were solved using group‐specific sequencing primers (GSSPs). We could identify 32 different HLA‐A alleles which were divided into 3 groups: high frequency (approximately 10%, A*01:01; A*24:02; A*03:01; A*02:01), moderate frequency (approximately 3%, such as A*02:05, A*31:01 and A*33:01), and low frequency (approximately 1%, such as A*02:11, A*29:01, A*02:02 and A*36:01). Homozygosity rate was higher than expected (11.5% vs. 7.15%). For high frequency alleles, our results show similarity to neighbouring countries. However, 15 alleles (such as A*02:04, A*02:06, A*02:11 and A*02:17) found in our cohort in low frequencies were never reported in some or all neighbouring countries. This is the first report on HLA‐A allele frequencies in Syria. In spite of the relatively low number of tested subjects, our results revealed a high degree of diversity, with 32 different alleles, reflecting the high ethnic heterogeneity of the Syrian population. The identification of alleles rarely or never reported in neighbouring countries indicates a higher genetic diversity in Syria.     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

Characterization of a novel MICA allele,MICA*012:05, by cloning and sequencing

A new MICA allelic variant, MICA*012:05, has been identified in a Chinese Mongolian population. Following polymerase chain reaction–sequence‐based typing (PCR‐SBT), this new allele was further confirmed by cloning and sequencing. MICA*012:05 was linked to an HLA‐A*24‐C*01‐B*55:02‐DRB1*09 haplotype. MICA*012:05 differs from MICA*012:01 by a single synonymous C to T substitution at nucleotide position 269 in exon 3.     

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‐A locus allelic dropout in Sanger sequence‐based typing due to the single nucleotide polymorphism of exon 1

The DNA‐based method is used widely for HLA genotyping in routine work, but some allele may be dropout in the genotyping procedure. Here, we reported a case with HLA‐A allele dropout in the Sanger PCR‐SBT test. The initial PCR‐SBT method with a commercial agent kit was not characterized, and the result of Luminex technology indicated the dropout as a HLA‐A*02 allele. Subsequently, the sequences of exons 2–4 were fully matched with the A*02:07 and A*11:01:01 by allele group‐specific primer amplification PCR‐SBT. On further analysis, a novel allele A*02:07:07 was identified, which has one nucleotide difference from A*02:07:01 at position 6 C>G of exon 1. According to the sequencing for 5′‐UTR to 3′‐UTR, the novel single nucleotide polymorphism of exon 1 was contributed to HLA‐A locus allele dropout in the sample. Our results indicated multiplatform analysis is necessary when a conclusive HLA type cannot be determined by a single methodology.     

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 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.     

Discovery of the rare HLA‐B*39:77 allele in an unrelated Taiwanese bone marrow stem cell donor using the sequence‐based typing method

We detected a rare HLA‐B locus allele, B*39:77, in a Taiwanese unrelated marrow stem cell donor in our routine HLA sequence‐based typing (SBT) exercise for a possible haematopoietic stem cell donation. In exons 2, 3 and 4, the DNA sequence of B*39:77 is identical to the sequence of B*39:01:01:01 except one nucleotide at nucleotide position 733 (G‐>A) in exon 4. The nucleotide variation caused one amino acid alteration at residue 221 (Gly‐>Ser). B*39:77 was probably derived from a nucleotide substitution event involving B*39:01:01:01. The probable HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 haplotype in association with B*39:77 may be deduced as A*02:01‐B*39:77‐C*07:02‐DRB1*08:03‐DQB1*06:01. Our discovery of B*39:77 in Taiwanese adds further polymorphism of B*39 variants in Taiwanese population.     

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.     

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.