HLA-A*02 subtype distribution in Caucasians from northern Italy: identification of A*0220

This study describes a comprehensive, easy to perform PCR-SSOP typing approach suitable for complete genomic subtyping of HLA-A*02. A single 1.6 kb PCR–amplificate spanning exons 2,3 and 4 of the HLA-A*02 gene was used for hybridization with a panel of twenty-four SSOPs. This allowed unequivocal assignment of all so far known HLA-A2 subtypes, including A*0209 and A*0215N which differ for nucleotide substitutions in exon 4, without the need for two separate amplifications. Using this approach, HLA-A*02 subtype distribution was analyzed in 218 samples from unrelated, healthy individuals from northern Italy enrolled in the Italian Bone Marrow Registry and typed as HLA-A2 by serology or generic molecular analysis. As expected, A*0201 was found in the majority (92.6%) of samples. However, a significant number (6.8%) of individuals carried A*0205. Furthermore, A*0202, A*0208, A*0209 and A*0217, so far not described in Caucasians, were detected in a low number of samples (frequency ranging from 0.45% to 1.8%). Finally, a novel HLA-A*02 subtype, A*0220, was detected in 0.9% of the samples. As confirmed by DNA sequencing of exons 2 and 3, this allele is identical to A*0201 except for a single nucleotide substitution in codon 66 which changes the predicted amino acid sequence form Lys to Asn. The findings of this study have implications for the selection of HLA-A*02+ donors in unrelated bone marrow transplantation and of patients for specific immunotherapy with HLA-A*02 restricted peptide vaccines.     

HLA-A02 subtyping in a Portuguese population

Allelic polymorphisms identified between HLA-A^*02 variants allow differentiation between potentially functionally different HLA molecules.

Given the differences in population distribution described in previous studies we aimed to evaluate the relative frequency of A^*02 alleles in Portuguese Caucasoids. The allele frequency of HLA-A^*02 has been defined by serology in this population at 26.6%.

Genomic DNA from 45 individuals from the central region of Portugal, previously assigned as HLA-A2 by serology, were studied using the HLA-A^*02 SSP ARMS-PCR subtyping kit provided within HLA Class I DNA typing component of the 12th IHW, which discriminates 17 different alleles.

HLA-A^*0201 was found the most frequent, present in 88.9% of this population although three other alleles were identified: A^*0205 (8.9%), A^*0207 (2.2%) and A^*0217 (2.2%). One A^*0205 sample was found in heterozygous combination with A^*0201. From 22 HLA-A^*02 related haplotypes analyzed, no significant association between HLA-A^*02 and other HLA alleles was seen. The haplotype HLA-A^*0201-B44-DRB1^*-DQA1^*0300-DQB1^*0301 was present in three samples and HLA-A^*0201-B14-DRB1^*01-DQA1^*0101-DQB1^*0501 and HLA-A^*0201-B51-DRB1^*11-DQA1^*0501-DQB1^*0301 were found in two individuals each. Similarly, HLA-A^*0205-B51-DRB1^*07-DQA1^*0201-DQB1^*0201 was seen in two out of four samples with HLA-A^*0205.

Even though the limited number of samples studied, A^*0201 frequency was similar to those reported for Caucasoid populations. In addition, A^*0207 allele previously found exclusively in Chinese populations and A^*0205, with a moderate significant frequency in African and North American Blacks, were described in this investigation.     

HLA-A*02 allele frequencies and haplotypic associations in Koreans

We have investigated the frequencies of HLA-A*02 alleles and their haplotypic associations with HLA-B and -DRB1 loci in 439 healthy unrelated Koreans, including 214 parents from 107 families. All of the 227 samples (51.7%) typed as A2 by serology were analyzed for A*02 alleles using polymerase chain reaction (PCR)-low ionic strength-single-strand conformation polymorphism (LIS-SSCP) method. A total of six different A*02 alleles were detected (A*02 allele frequency 29.6%): A*0201/9 (16.6%), *0203 (0.5%), *0206 (9.3%), *0207 (3.0%), and one each case of *0210 and *02 undetermined type. Two characteristic haplotypes showing the strongest linkage disequilibrium were A*0203-B38-DRB]*1502 and A*0207-B46-DRB1*0803. Besides these strong associations, significant two-locus associations (P<0.001) were observed for A*0201 with B61, DRB1*0901 and DRB1*1401, and for A*0206 with B48 and B61. HLA haplotypes carrying HLA-A2 showed a variable distribution of A*02 alleles, and all of the eight most common A2-B-DR haplotypes occurring at frequencies of > or =1% were variably associated with two different A*02 alleles. These results demonstrate that substantial heterogeneity is present in the distribution of HLA-A*02 alleles and related haplotypes in Koreans.     

Development of PCR-SSOP for the identification of HLA-A*02 subtypes and determination of HLA-A*02 frequencies within different ethnic populations

A PCR-SSOP typing method, involving a single PCR amplification in conjunction with 19 digoxigenin labelled oligonucleotide probes, has been developed for the identification of 17 known HLA-A*02 alleles. The method has been applied to four populations (Northern Ireland, Singapore Chinese, Shetland Island and Mexican) and percentages of HLA-A*02 alleles determined within each population.     

HLA-A, -B and -DRB1 allele frequencies in the Bangladeshi population

Population genetic studies have become an invaluable tool because of the extreme polymorphism found at some of the loci of the human leukocyte antigen (HLA) system. In this study, we are reporting for the first time the genetic polymorphism of 141 healthy unrelated Bangladeshi Bangalees living in central region of Dhaka. We studied the HLA-A, -B and -DRB1 loci using polymerase chain reaction with sequence-specific primers. The allelic frequencies, two and three locus haplotype frequencies were statistically analyzed. A total of 16 HLA-A alleles, 26 HLA-B alleles and 14 HLA-DRB1 alleles were detected. A*33-B*44 (8.15%) was the most common two loci class 1 haplotype, whereas A*33-B*44-DRB1*07 (6.38%) was the most frequent three loci haplotype. The most common HLA-A, HLA-B and HLA-DRB1 alleles were A*33 (17.02%), B*15 (19.5%) and DRB1*15 (29.07%), respectively. Construction of phylogenetic tree using average linkage between groups and correspondence analysis showed close associations with Indian non-tribal random Dravidians, north Indian Hindus and some relations with Mongolian and Pakistani populations. We believe this data will provide useful information for bone marrow registry, legal medicine, disease association and anthropological studies.     

中华骨髓库湖北分库志愿者HLA-A*02亚型基因多态性分析

目的:检测中华骨髓库湖北分库志愿者HLA-A*02基因亚型,了解其亚型基因多态性分布特点。方法:采用序列特异性寡核苷酸探针聚合酶链反应技术(PCR-SSOP)及聚合酶链反应-直接测序技术(PCR-SBT)对分库1 364名志愿者进行HLA-A高分辨率分型。结果:1 364例HLA-A基因分型结果中,共691例A*02阳性,A*02低分辨基因型频率为0.301,检出A*0201、A*0203、A*0205、A*0206、A*0207、A*0210、A*0211、A*0290,8个等位基因。其中A*0201是优势基因,A*0207、A*0206、A*0203为常见型基因,本研究人群中还检测出了东亚人群罕见型基因:A*0205、A*0211、A*0290。其基因频率分布与台湾慈济脐血库、日本、北爱尔兰、亚裔美国人比较,存在统计学差异。此人群A*02低、高分辨杂合度分别为83.4%、96.7%,其高分辨率纯合子呈现规律性的分布。结论:中华骨髓库湖北分库志愿者HLA-A*02亚型基因频率呈高度多态性,其基因多态性与台湾慈济脐血库、日本、北爱尔兰、亚裔美国人存在显著性差异。     

HLA-A,B,C gene and haplotype frequencies in the Finnish population

The HLA-A,B,C gene and haplotype frequencies calculated by the direct gene counting method from the genotypes of 550 unrelated Finns are reported. The typings were done during the years 1978-1980. The delta values are given for the HLA-A,B and B,C haplotypes. Some comparisons between Finns and other European populations are stated.     

Characterization of three new alleles HLA-A*02:241, HLA-A*02:242 and HLA-A*30:04:02

We describe the identification of the novel HLA-A*24:135 allele [nucleotide 397 (T→C) in exon 3, Phe→Leu at codon 109 in α2 domain, compared with A*24:02:01] by sequence-based typing (SBT).     

Molecular diversity of HLA-A*02 in Asian Indians: predominance of A*0211

The North Indians are considered predominantly Caucasoid with an admixture of genes from the Mongoloid and Aryan races. The present study was undertaken to investigate the genetic diversity of HLA-A*02 in the North Indian population and determine the frequency distribution of its molecular subtypes at the population level. The study revealed a high occurrence of A*0211 (33.8%) in this population along with increased frequencies of the common Oriental alleles, A*0206 (7.5%) and A*0207 (32.5%) and also of HLA-A*0205 (15%) commonly observed in negroid populations. HLA-A*0211 has only been reported with very low frequencies among the Ticuna Jews, Thai population, and Colombian Blacks in the malaria endemic areas of Africa. Significantly, we observed an unexpectedly low frequency of A*0201 (3.8%) in contrast to its distribution in Western Caucasians in whom it constitutes 95% of the HLA-A2 repertoire. Prevalence of HLA-A*0211 at very high frequencies among North Indians may be a consequence of the founder effect, racial admixture or selection pressure due to environmental factors in this population.     

A simplified PCR-SSP method for HLA-A2 subtype in a population of Wuhan, China

HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allellc distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers （PCR-SSP） to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles （〉 99% allele frequency） reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A＊0209 and HLA-A＊0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis （FCM） with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A＊0201 ranks the first （allele frequency = 15.5%）, followed by A＊0207 （5.8%）, A＊0206 （4.7%）, A＊0203 （2.6%）, A＊0210 （0.7%）, and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study indicates that the developed typing method is simple and reliable for HLA-A2 subtyping in Chinese, and the profile of allelic distribution of HLA-A2 subtypes is revealed in the population of Wuhan, China.     

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.     

Molecular diversity of HLA-A*19 group of alleles in south Indian population

To determine the genetic diversity of the human leucocyte antigen (HLA)-A*19 group of alleles in the south Indian Tamil population, we studied 100 random healthy unrelated individuals. The frequency of HLA-A*19 was 37% with A*33 (45.9%), A*32 (29.7%), A*31 (16.2%), A*30 (5.4%), A*29 (2.7%) and A*74 (0%). The frequency distribution of the HLA-A*19 alleles was distinct and revealed marked similarities and variations with other populations.     

Identification of a new HLA-A*02 allele, HLA-A*02:305 N, which differs from the HLA-A*02:01:01 allele by a single nucleotide deletion in exon 4

The HLA-A*02:305N new allele lacks a nucleotide in exon 4 compared to HLA-A*02:01:01 allele.     

Sequence analysis of the novel allele HLA-A*110106 in the Chinese population

A novel HLA-A*110106 allele was identified in the Chinese individual by polymerase chain reaction sequence-based typing.