Identification of HLA-A*0224: implications for PCR-SSP HLA typing

We describe a novel HLA-A*02 allele, A*0224, that was identified after a comparison of DNA and serological typing revealed a discrepancy in the HLA-A types: HLA-A2 was defined by serology but was not detected by the polymerase chain reaction using sequence-specific primers (PCR-SSP). DNA sequencing indicated the presence of a variant HLA-A*02 allele that differed from A*0201 by a single base (C/A) at position 453. This base substitution corresponded to the annealing site of a primer common to the two A*02-amplifying PCR-SSP mixtures used in the method. This provides an explanation for the results and highlights a limitation of PCR-SSP methods even where two PCR mixtures are used to detect alleles. Serological titration studies suggested that A*0201, A*0205 and A*0224 are unlikely to be differentiated during routine serological typing.     

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.     

sHLA-A*0201-抗原肽四聚体的构建与功能检测

目的：构建有功能的sHLA-A＊0201-抗原肽四聚体，建立一套HLA Ⅰ类分子／抗原肽的四聚体制备技术。方法：利用基因工程及体外折叠技术构建sHLA-A＊0201-LMP2A426-434复合物分子，并在BirA酶的作用下生物素化。与荧光标记的亲和素衍生物以分子数4：1结合而形成HLA-A＊0201-LMP2A426-434四聚体。获得的四聚体对长期混合淋巴细胞培养中增殖的抗原特异性CTL进行检测，同时用细胞毒实验验证。结果：荧光标记的亲和素与生物素化的HLA-A＊0201-抗原肽复合物分子正确结合，制备的四聚体能够与长期混合淋巴细胞培养出的特异性T细胞结合。结论：从结构与功能上证实成功地获得有功能的HLA-A＊0201-抗原肽复合物四聚体。为进一步研究T细胞识别机制与功能奠定了基础，也为过程复杂的HLA-抗原肽四聚体制备提供了可行的免疫学监控方法。     

整合法预测HBV相关抗原的HLA-A* 0201限制性结合肽

目的 预测HBV的相关抗原HBsAg、HBcAg、DNA polymerase新的HLA-A*0201限制性结合肽.方法 选取SYFPEITHI、BIMAS、SVMHC、IEDB、EpiJen预测工具以及整合法预测抗原对应蛋白序列P03146,P03138,P03156的HLA-A*0201限制性结合肽,并与现有表位数据库中的结合肽比较.结果 整合法与单个预测算法相比提高了预测的灵敏度和特异性,对3种抗原的预测准确性均有提高,MR(misclassification rate)值分别降为1.14%、3.41%、1.46%;利用此方法发现3种抗原尚未得到实验验证的HLA-A*0201结合肽,分别为,HBcAg:ELMTLATWV(64-72)、LMTLATWVGV(65-74);HBsAg:LMT-LATWVGV(246-254)、IIFLFILLL(244-252)、FLFILLLCU(246-255)、SLYSILSPFL(367-375)以及LLCLIFLLvL(251-260);DNA polymerase:SLFTAVTNFL(513-522)、GLLGFAAPFT(554-563);在HBsAg蛋白序列中发现了31个氨基酸的免疫热点区域FIIFLFILLLCLIFLLVLLDYQGMLPVCPLI(243-273).结论 整合法比单个预测算法性能更好,能够更准确地预测抗原的HLA限制性结合肽,采用该方法所发现的9条可能表位和1个免疫热点区域为后续HBV新表位的鉴定及其功能研究提供了有价值的线索.     

A new HLA-A*02 null allele, HLA-A*9213N

A novel HLA-A*02 null allele, differing from HLA-A*02010101 at codon 60 (TGG tryptophan-->TAG stop), is described.     

Identification and nucleotide sequence of HLA-A*03013

We have identified a further HLA-A*03 allele, A*03013, in three unrelated Caucasoid individuals. The allele was initially detected by the polymerase chain reaction using sequence-specific primers (PCR-SSP) and was shown to differ from HLA-A*03011 by a single non-coding substitution of G to T at position 167 in exon 2 by DNA sequencing. The A*03013 bearing haplotype in one of the three individuals, was HLA-A*03013, B*07, Cw*0702, DRB1*1101, DRB3*0202, DQA1*05, DQB1*0301. The estimated phenotype and gene frequencies for A*03013 was <0.02% and <0.00007, respectively.     

Identification and nucleotide sequence of HLA-A*02015

A novel HLA-A*02 allele, A*02015, is described that possess a unique non-coding substitution of G to A at position 113 in exon 3.     

PCR-SSP结合测序分析快速筛选HLA-A*0201亚等位基因

建立相对简单经济的检测HLA A 0 2 0 1等位基因的方法 ,以便于从特定人群中快速筛选HLA A 0 2 0 1阳性的个体 ,满足科研的需要。根据第十二届HLA国际联合会提供的标准操作规程和一对HLA A位点特异性的引物 ,从外周血中提取基因组DNA ,扩增HLA A基因 ;再设计两对HLA A2组特异性引物 ,采用套式PCR分别扩增HLA A基因的 5’端和 3’端 ,如出现特异条带 ,则将后两对引物的PCR产物进行测序分析 ,对照HLA A2组等位基因的第 2和第 3外显子序列图 ,确定是否为HLA A 0 2 0 1 1～ 0 2 0 1 6的 6个等位基因。结果 :以HLA A 0 2 0 1阳性的T2细胞株为阳性对照进行检测 ,结果完全正确 ,并设立非HLA A 0 2 0 1的A2细胞株RML (HLA A 0 2 0 4 )及Raii细胞株 (非A2组 )为对照 ,同时随机检测 30份门诊病人血样 ,其中 7份为HLA A2阳性 ,经测序 ,2例为HLA A 0 2 0 1 1 ,1例为HLA A 0 2 1 1或 0 2 35 ,1例是HLA A 0 2 0 6或0 2 2 1或 0 2 4 1或 0 2 4 4或 0 2 5 1或 0 2 5 4 ,1例是HLA A 0 2 0 5或 0 2 1 4 ,1例是HLA A 0 2 34或 0 2 35 ,1例是 0 2 5 0。本方法利用位点特异性引物进行 2～ 3次PCR扩增 ,结合PCR产物测序便可相对简便经济地检测出HLA A 0 2 0 1的全部 6个亚等位基因 ,为快速检测其他特定HLA等位基因?     

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.     

基于SCORE函数估算HLA-A*0201限制性CTL表位亲和性

目的 建立HLA-A*0201限制性CTL表位亲和性的定量预测方法。方法基于SCORE打分函数,运用定量构效关系的理论和方法研究了HLA-A*0201限制性CTL表位九肽结构与亲和性间的定量关系,并建立了SCORE得分与亲和性的定量关系模型,并用外部样本(5个HLA-A*0201限制性CTL表位九肽)作为预测集用于检验模型的预测能力。结果基于SCORE打分函数建立的定量模型具有较好的相关性(r=0．9165,RMS=0．38)和对外部样本的预测能力(rpred=0.9847,RMS=0.135)。结论基于SCORE打分函数,运用定量构效关系研究的理论和方法建立了HLA-A*0201限制性CTL表位亲和性的定量预测方法,为实验鉴定高亲和性HLA-A*0201限制性CTL表位提供了理论依据。     

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.     

The quantity of naturally processed peptides stably bound by HLA-A*0201 is significantly reduced in the absence of tapasin

Tapasin plays a critical role in promoting peptide binding by major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum. In its absence, cell surface expression of most allotypes is significantly reduced. Two exceptions are HLA-A*0201 and HLA-B*2705. In this study, the repertoire of peptides bound endogenously by these allotypes in the absence of tapasin was examined and stability of the HLA class I/peptide complexes assessed. Similar quantities of peptides were recovered from B*2705 complexes expressed in the absence and presence of tapasin and the composition of the peptide pools were not radically different. However, the stability of B*2705 molecules expressed at the surface of tapasin-deficient cells was found to be reduced which suggests there are subtle changes to the peptide repertoire. The impact of the absence of tapasin was more dramatic for A*0201. Although equivalent levels of cell surface A*0201 are expressed in the presence and absence of tapasin, very little A*0201 glycoprotein was recovered from tapasin-deficient cells suggesting the complexes readily dissociate. Consistent with reduced stability, A*0201 complexes were found to be rapidly lost from the surface of tapasin-deficient cells. Analysis of the small quantity of endogenously bound peptides recovered from A*0201 expressed in the absence of tapasin revealed a complex mixture typical of A*0201 molecules expressed in normal cells. Therefore these molecules are unable to exploit the alternative supply of TAP-independent A*0201-binding peptides present in the endoplasmic reticulum. Loading of A*0201 with peptides from both TAP-dependent and TAP-independent sources is significantly compromised without tapasin.     

Five novel HLA-A alleles, HLA-A*030108, A*2491, A*2498, A*330303, A*3317 were identified by polymerase chain reaction sequence based typing

Five HLA-A variant alleles HLA-A*030108, A*2491, A*2498, A*330303, A*3317 were identified in Chinese individuals.     

Genetic polymorphism within HLA-A*02: significant allelic variation revealed in different populations

Abstract: HLA-A2 is present at high frequency in most populations, as identified by serological and biochemical means. The value of these methods is limited by their failure to discriminate between the products of the 14 known allelic HLA-A*02 variants. The great majority of genetic polymorphism which defines the allelic variants is found in exons 2 and 3 of the A*02 genes. These exons encode the α-1 and α-2 domains of the HLA Class I molecules, and variation within the genes may influence the peptide binding specificity of the gene products of each allele. Failure to accurately assign the allelic types has implications in transplantation, in interpretation of cellular assays and in the understanding of HLA disease associations. We have developed a method for determining the 14 Known alleles of HLA-A*02 by use of ARMS–PCR to determine the degree of variation of HLA-A*02 alleles in 3 different population groups. Considerable variation was found in the relative frequencies of particular A*02 alleles between Caucasian, oriental and black individuals. Our results indicate the importance of ethnic origin in terms of the expected HLA-A*02 allelic profile, and emphasize the functional significance of allele supecific subtyping of HLA-A*02.     

CT抗原KM-HN-1 HLA-A * 0201限制性表位预测及其与HLA-A * 0201结合强度分析

目的通过对CT抗原（cancer-testis antigen）KM-HN-1进行HLA-A＊0201限制性表位预测,并对候选表位肽与HLA-A＊0201分子结合亲和力及复合物稳定性进行分析,为探索基于KM-HN-1的免疫治疗奠定基础。方法利用基于蛋白酶体剪切位点特异性的算法PAProc及基于肽MHC-I结合的算法BIMAS和SYFPEITHI对KM-HN-1进行HLA-A＊0201限制性表位预测．合成KM-HN-1相关候选表位肽KM-HN-I321-329（KLLPFRETV）,KM-HN-I303-211,（FLPTAPPNV）,KM-HN-I629-637。（TLLQIIETV）,KM-HN-I87-95（ILNKSIIEV）,KM-HN-I538-596。（QMMEALDQL）及阳性对照肽HBVcAg18-27（FLPSDFFPSV）;对这些合成肽与HIA-A＊0201分子结合亲和力及其复合物稳定性根据文献报道的方法进行分析。结果KM-HN-I321-329（KLLPERETV）结合亲和力最低,KM-HN—I203-211（FLPTAPPNV）结合亲和力最高,其余3条肽结合亲和力介于2者之间;稳定性实验（DC50）结果显示：KM-HN-I538—546（QMMEALDQL）DC50小于2h,KM—HN-I321-329（KLLPERETV）的DC50介于2～4h之间,KM-HN-I87-95。（ILNKSIIEV）的DC50介于6～8h之间,KM-HN-I233-211（HLPTAPPNV）及KM-HN-I629—633（TLLQIIETV）的DC50均大于8h。结论基于蛋白酶体剪切位点特异性的算法及基于肽MHC-I结合的算法对KM-HN-1进行HLA-A＊0201限制性表位预测,结合候选表位肽与HLA-A＊0201分子结合的亲和力与复合物稳定性实验分析,为该抗原HLA-A＊0201限制性表位的鉴定奠定了基础。