海胆Runx基因保守序列的克隆与初步分析

Runx(Runt box)是Runt结构域转录因子家族的成员之一。马粪海胆Runx基因第663位和728位碱基发生突变,分别为A→T和C→T。第一个突变为错义突变,使密码子编码的氨基酸由天冬氨酸变为酪氨酸,而第二个突变为同义突变,密码子编码的氨基酸未发生变化。海胆Runx基因保守序列的突变可能会导致其表达产物的变化,进而会影响海胆的生理机能。     

猪H-FABP基因多态片段的序列分析

利用PCR-RFLP方法在猪H-FABP基因内确定了3个变异酶切位点,分别为5′-上游区HinfI-RFLP、内含子2的HaeIII-RFLP和HinfI*-RFLP（HinfI和HinfI*代表不同区域的HinfI酶切反应）。对每个多态片段进行克隆测序分析,结果表明,5′-上游区HinfI-RFLP是由于1324位的碱基T→C的突变引起;在内含子2中,HaeIII-RFLP变异酶切位点在1811位,发生了C到G的突变;HinfI*-RFLP是由于1970位发生T→C的碱基替换。 Abstract:Three variant restriction sites of porcine H-FABP gene,including HinfI-RFLP in 5′-upsream,HaeIII-RFLP and HinfI*-RFLP in intron 2,were confirmed by PCR-RFLP method.The polymorphic fragments were cloned and sequenced.The results revealed a single nucleotide substitution of T→C at position 1324 for HinfI-RFLP,C→G at position 1811 for HaeIII-RFLP and T→C at position 1970 for HinfI*-RFLP,respectively.     

甘肃河西地区西门塔尔杂交类群NGB基因第3外显子的遗传变异研究

旨在对甘肃河西的临泽、甘州、武威、金昌、高台5个地区283头西门塔尔杂交类群NGB基因第3外显子的遗传多态性及变异特征进行系统分析,采用PCR-SSCP方法检测了283头西门塔尔杂交类群NGB基因第3外显子和部分内含子的多态性,且对群体内各等位基因进行了测序。结果显示,5个地区西门塔尔杂交类群共检测出5个等位基因(A、B、C、D、E),表现为5种基因型(AA、AB、AC、AD、AE)。其中甘州、武威、金昌西门塔尔杂交类群NGB基因均只检测到AA、AB 2种基因型,高台西门塔尔杂交类群检测到AA、AE 2种基因型,临泽西门塔尔杂交类群检测到AA、AB、AC、AD 4种基因型。A等位基因和AA基因型的频率在5个群体中最高,为优势基因和优势基因型。对不同SSCP带型的对应片段进行测序分析,共发现6个核苷酸突变位点(75 bp C→T,78 bp C→G,128 bp G→A,214 bp G→A,232 bp C→T,233 bp G→A),其中第75 bp和第78 bp处的突变位点位于内含子区域,其余4处突变位点均位于外显子区域。第214 bp处的核苷酸突变导致甘氨酸(Gly)突变为丝氨酸(Ser),第232 bp处核苷酸突变导致精氨酸(Arg)突变为色氨酸(Trp),第233 bp处核苷酸突变导致精氨酸(Arg)突变为谷氨酰胺(Gln),经χ2检验结果显示,5个地区的西门塔尔杂交类群在此3个突变位点上都处于Hardy-Weinberg平衡状态(P0.05)。群体遗传学分析结果表明,临泽、甘州、武威、金昌、高台西门塔尔杂交类群的多态信息含量(PIC)分别为0.0582、0.0196、0.0196、0.0161、0.0159,均属于低度多态(PIC0.25)。     

鸡MC4R基因的SNPs及其与屠体性状的相关研究

黑素皮质素受体(MC4R, melanocortin-4 receptor)基因的突变与猪、鼠和人等的食欲、肥胖、生长等性状有关, 而鸡MC4R基因的功能却知之甚少. 利用PCR-SSCP(single strand conformation polymorphism)和DNA测序的方法, 对资源家系F₂代鸡群MC4R基因多态性进行了分析, 发现存在4个单核苷酸多态(SNPs, single nucleotide polymorphisms)位点. 其中, 在MC4R基因5′调控区-524 nt发生了碱基的转换突变(C→T), 导致突变型基因比野生型基因多了一个NF-E2和一个cap转录因子结合位点; 在MC4R编码区(61 nt)发生了碱基的错义突变(G→A), 导致此处蛋白质的氨基酸由甘氨酸变为精氨酸; 在MC4R编码区315和336 nt发生了碱基的颠换突变(G→T)和转换突变(C→T), 这两个突变为同义突变. 通过最小二乘分析SNPs与屠体性状的关系, 结果是突变的BB, DD和FF等基因型与鸡的体重、全净膛重(或半净膛重)、腿肉重等存在显著(P<0.05)或极显著(P<0.01)的关系, 但与腹脂重不显著. 结果表明, MC4R基因可以作为影响和控制鸡体重、生长等屠体性状的主要候选基因.     

云南HPV-16型E6、E7基因的突变分析

人乳头瘤病毒(Human papillomavirus,HPV)16型(HPV-16)是引起宫颈癌的一种主要高危型病毒,其2个致癌基因E6和E7的核酸序列变异可能会影响其对宿主细胞的致癌性,已有研究表明其序列突变呈现地域差异性。因此,研究不同地域HPV-16这2个基因的变化情况是宫颈癌流行病学调研的主要内容,也可为研究E6和E7的致癌性积累数据。研究以NCBI登录号为NC_001526.2的HPV-16型病毒的序列为参照,采用Neighbor-joining方法对云南地区74例HPV-16样本的E6、E7的DNA序列构建进化树,结果显示:只有亚洲和欧洲变异亚型,而没有发现非洲1、非洲2、亚-美洲和北美洲这4种变异亚型。DNA序列分析显示:E6的碱基突变以T178G(D25E,59.46%)和T350G(L83V,8.11%)为主,E7的碱基突变主要以A647G(N29S,59.46%)和T846C(同义突变,60.81%)为主。发现E6的新突变有A95G(同义突变,1.35%)和A135G(K11R,1.35%);E7的新突变有C625T(L22F,1.35%)、C627T(同义突变,12.16%)、G689A(G43E,1.35%)、T748G(S63A,1.35%)。此外还发现有一个共突变现象:T178G(D25E,59.46%)-A647G(N29S,59.46%)-T843C(同义突变,21.62%)-T846C(同义突变,60.81%)。     

奶牛β-Lg基因第1外显子PCR-SSCP多态性与泌乳性能的相关性

采用PCR-SSCP技术并结合测序对233头奶牛β乳球蛋白(β-Lg)基因5’端部分序列和外显子1全部序列进行了多态性研究,分析了该基因与奶牛泌乳性状的相关性。结果表明:β-Lg基因5’端和外显子1共存在2个等位基因3种基因型,BB型为优势基因型,B为优势等位基因。该群体在这一位点上偏离Hardy-Weinberg平衡状态,多态信息含量(PIC)为0.3548。测序结果显示,与普通牛该基因序列(X14710)相比,B等位基因在2073 bp、2202 bp和2206 bp处发生了G→C、C→T和A→G的碱基突变,其中2202 bp处的C→T突变导致第11位氨基酸由苏氨酸变为异亮氨酸,而A等位基因在3个位点上与X14710相同。最小二乘法分析表明,BB型305 d乳蛋白量显著高于AA型和AB型(P<0.05);AB型305 d乳脂量显著高于AA型(P<0.05),BB型与AB型之间差异不显著(P>0.05);等位基因B为高乳蛋白量和乳脂量的优势基因,可作为奶牛选育的分子遗传标记。     

中国遗传性胰腺炎患者胰蛋白酶原基因多位点杂合突变及其临床特征

用基因产物直接测序法对2个遗传性胰腺炎家系中胰腺炎患者(共有4例成员)的胰蛋白酶原基因(cationic trypsinogen,PRSS1)5个外显子进行测序,并分析其各自的临床特征.在4例胰腺炎患者中均出现了PRSS1基因杂合突变,但两家系PRSS1基因突变的位点不同,且临床表现差异较大,其中家系1出现6例糖尿病患者且发病年龄较家系2明显延迟,平均发病年龄为29岁,分析其PRSS1基因发现3号外显子336位碱基存在G→A杂合性突变,为中性突变,表达的氨基酸从赖氨酸(Lys)→赖氨酸(Lys),同时在同一外显子的361位碱基还存在另一个G→A杂合性突变,造成121位的丙氨酸(Ala)被苏氨酸(Thr)所取代,胰蛋白酶原的空间结构发生改变,其与抑制因子的结合位点消失,"保护失败"而产生有活性的胰蛋白酶,造成胰腺自身的消化.而家系2未发现糖尿病患者,其胰腺炎患者的血清肿瘤标志物不增高,先证者(Ⅲ8)在胰腺炎发病过程中表现为CD4 T/CD8 Tcell和乙肝表面抗体(anti-HBs)随病程进展逐渐降低,而Ⅲ7不表现出此现象,分析其PRSS1基因发现3号外显子361位碱基同样存在G→A(c.361G→A)突变,而且在415位还存在一个杂合性突变点T→A(c.415T→A),其中c.415T→A不存在于Ⅲ7.胰蛋白酶原基因存在多种形式的突变,而且与临床表型相关.     

一例复合杂合性突变所致多发性内分泌腺瘤病1型的临床表现与基因型

目的分析1例多发性内分泌腺瘤病1型(MEN1)患者的临床特征和基因型,以期提高临床医生对本病的认识与诊断的准确性。方法分析患者病史、临床表现、实验室检查及影像学资料,并对MEN1基因进行扩增与测序。结果发现1例临床症状符合典型的多发性内分泌腺瘤病1型患者,基因测序鉴定本例患者的MEN1基因第9号外显子内存在同义突变,为c.1818位T→C(rs540012),第10号外显子存在两处复合杂合性突变,分别是c.2098位C→T(R527X,rs104894261),造成第527位氨基酸精氨酸(CGA)突变为终止密码子TGA;和c.2140位G→A(A541T,rs2959656),造成第541位丙氨酸(GCA)突变为苏氨酸(ACA)。结论本例患者表现了典型的多发性内分泌腺瘤病1型的临床症状与体征,遗传学分析进一步验证了患者携带MEN1基因的致病突变,提示根据临床表现结合基因学确诊本病的可靠性。     

合作猪SLA-DQA基因第4外显子多态性分析

合作猪的MHC-DQA基因的适应性变异,其抗原识别区域(即外显子4)通过PCR扩增和随后的单链构象多态性(SSCP)和序列分析,结果显示在439个合作猪个体,SLA-DQA第4外显子检出4个等位基因和6个基因型(AA、BB、DD、AB、AC和AD),其中A等位基因和AA基因型的频率最高,为优势基因和优势基因型。对不同型的PCR-SSCP条带测序分析,发现7个突变位点(5 068 bp T→C,5 109 bp和5 149 bp处缺失C,5 131 bp A→G导致丝氨酸变为甘氨酸,5 135 bp C→T,5 234 bp G→A,5 136 bp处插入A)。遗传学分析发现,合作猪多态信息含量(PIC)为0.240 1,属于低度多态,各种基因型的分布不显著。研究结果证实,合作猪SLA—DQA基因第4外显子为低度多态。     

DNA池快速筛查牛STAM1基因SNPs及估算等位基因频率

目的:旨在对不同牛种STAM1基因进行SNPs筛查,为地方牛种选种选育提供一定理论依据.方法:选取生长发育性状明显差异的务川黑牛和贵州荷斯坦奶牛2个牛种构建DNA池,设计1对引物分别扩增2个牛种STAM1基因第14外显子序列总长898bp.切胶回收后对PCR产物进行双向测序.结果:在牛STAM1基因中快速筛查到5个SNPs:A33C、C66G、C356T、T523A、T652C,其中A33C(Tyr→Ser)、C66G(Pro→Arg)、C356T(Glu→Lys)为错义突变,T523A为同义突变,T652C位于内含子区.贵州荷斯坦奶牛在T523A和T652C两个位点基因频率为1.0000,而务川黑牛分别为0.6730和0.8106.生物信息学分析表明:突变前后STAM1的RNA二级结构和蛋白质二级、三级结构均有明显改变.结论:DNA池结合测序技术可快速筛选SNP位点,检测到STAM1基因第14外显子5个SNPs.     

Identification of novel Tapasin polymorphisms and linkage disequilibrium to MHC class I alleles

Tapasin is a Mr 48,000 glycoprotein and has a specialized role in MHC class I-restricted antigen presentation. It is encoded by a gene which maps centromeric to the MHC class II region of human Chromosome 6 within 200 kb of HLA-DP. There is variable dependence upon tapasin for MHC class I expression among different MHC class I alleles. HLA-B*4402 and to a lesser extent HLA-A1 and B8 are tapasin dependent, whereas HLA-B27, A2 and to a lesser extent B7 and A3 are tapasin independent. We investigated whether tapasin is polymorphic and whether these Tapasin alleles are in linkage with any MHC class I alleles. We identified three new mutations within intron 4, which are in a particular linkage with the previously described exon 4 (G16003C) dimorphism. The intronic mutations are G16146T, G16232A, and T16317A (numbering according to cosmid clone F0811; GenBank accession number Z97184). The allele frequency of Tapasin*01 (G16003) was 0.47 and Tapasin*02 (C16003) was 0.53 in this UK population. Four of the eight possible intronic haplotypes were identified and their cis linkage with the tapasin dimorphism ascertained. Tapasin*01 was associated with all the identified haplotypes, while Tapasin*02 was only associated with the wild-type intronic sequence (GGT). There was no significant linkage (P>0.01) of the Tapasin dimorphism or new Tapasin alleles to any of the MHC class I A, B, or C alleles studied or to the extended A1 B8 DR3 haplotype.     

中国人罕见的cisAB变异型分子机制分析

为研究中国人群ABO血型系统中罕见的cisAB变异型的分子遗传背景, 用血型血清学方法鉴定12例ABO血型疑难样本和116例随机无血缘关系样本, 应用聚合酶链反应和DNA序列分析等方法对样本ABO基因酶活性编码区外显子6、7和侧翼内含子进行突变筛选和检测, 并对不同基因型的扩增片段进行单倍体序列分析。血清学检测发现12个样本均为(A强B弱)或(A弱B强), PCR产物直接序列分析表明这些样本均为cisAB变异型, 并存在4种基因型。通过单倍体序列分析, 从中发现2种cisAB等位基因, 其中cisAB01不仅存在外显子7的467C>T和803G>C变异, 还在第6内含子存在未经报道的163T>C和179C>T变异; 另一种等位基因是在B101基础上保留了A101的803G位点, 编码一条176G、235S、266M和268G组合的多肽链。经检索, 未发现该组合的ABO等位基因, 该等位基因已被国际血型抗原基因突变数据库命名为cisAB05等位基因。文章系统研究了中国人群cisAB变异型分子机制, 发现了一种新的cisAB05等位基因, 同时根据内含子序列推测cisAB01等位基因可能是通过A101和B101等位基因间的同源交换而形成的。     

Peptide-presenting similarities among functionally distant HLA-B27 subtypes revealed by alloreactive T lymphocytes of unusual specificity.

Functional dissection of HLA-B27 subtypes using alloreactive or B27-restricted CTL has shown that the structurally related B*2704 and B*2706 are the most distant subtypes relative to the prototype B*2705. In particular, previous studies have failed to find anti-B*2705 CTL cross-reacting with B*2704 or B*2706. Such failure can be accounted for by the drastic effect on T cell recognition of the change at residue 152 in both subtypes relative to B*2705, as established with site-directed mutants. B*2704 and B*2706 are also related in ethnic distribution, as they are restricted to Orientals, jointly being the predominant HLA-B27 subtypes in this population. As far as it is known, there are no differences relative to B*2705 in their linkage to ankylosing spondylitis. In our study, 5 of 13 examined anti-B*2705 limiting dilution CTL lines from a particular HLA-B27- individual were shown to crossreact with B*2704, B*2706 or both. The monoclonal nature of this cross-reaction was established by cold target competition analysis. This result demonstrates that the apparent differences in T cell antigenicity among anti-B27 subtypes are strongly influenced by the responder individual, as the spectrum of clonal specificities in anti-B27 responses may show significant differences among unrelated responders. Fine specificity differences among the cross-reactive CTL allowed unambiguous functional distinction between B*2704 and B*2706. The molecular basis of such cross-reactivity was examined by correlating CTL reaction patterns with the structure of both subtypes, which differ only by two residues located in the beta-pleated sheet bottom of the peptide binding site, and with site-directed mutants mimicking HLA-B27 subtype polymorphism. The results suggest that: 1) distinct peptides are involved in the allospecific epitopes recognized by the various crossreactive CTL, and 2) B*2704, B*2706, and B*2705 differ in their peptide-presenting specificity, but can present some identical or structurally similar peptides.     

Molecular mimicry of an HLA-B27-derived ligand of arthritis-linked subtypes with chlamydial proteins

HLA-B27 is strongly associated with spondyloarthropathies, including ankylosing spondylitis and reactive arthritis. The latter disease is triggered by various Gram-negative bacteria. A dodecamer derived from the intracytoplasmic tail of HLA-B27 was a natural ligand of three disease-associated subtypes (B*2702, B*2704, and B*2705) but not of two (B*2706 and B*2709), weakly or not associated to spondyloarthropathy. This peptide was strikingly homologous to protein sequences from arthritogenic bacteria, particularly to a region of the DNA primase from Chlamydia trachomatis. A synthetic peptide with this bacterial sequence bound in vitro disease-associated subtypes equally as the natural B27-derived ligand. The chlamydial peptide was generated by the 20 S proteasome from a synthetic 28-mer with the sequence of the corresponding region of the bacterial DNA primase. Molecular modeling suggested that the B27-derived and chlamydial peptides adopt very similar conformations in complex with B*2705. The results demonstrate that an HLA-B27-derived peptide mimicking arthritogenic bacterial sequences is a natural ligand of disease-associated HLA-B27 subtypes and suggest that the homologous chlamydial peptide might be presented by HLA-B27 on Chlamydia-infected cells.     

Genetic variation in α1-antichymotrypsin and its association with Alzheimer's disease

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by extracellular neuritic plaques and intracellular neurofibrillary tangles in brain parenchyma. Alpha-1-antichymotrypsin (ACT) is a component of plaque cores, can bind to Abeta, and has been proposed as a possible candidate gene for AD susceptibility. The genetic association between the ACT codon -17*A allele of the signal peptide polymorphism and AD has been shown in some, but not in all studies. One hypothesis is that the ACT codon -17*A allele is in linkage disequilibrium with unknown functional mutation(s) in the ACT gene. This study was undertaken to identify new mutation(s) in the ACT gene by PCR-SSCP-sequencing and, in conjunction with known mutations, to assess their role in affecting the risk of AD. A total of seven new point mutations were observed: 5'UTR(A-->G), Asp128Asn(G-->A), Ser250Ser(C-->T), Leu301Pro(T-->C), Thr324Thr(A-->G), G-->A in intron 4, and 3'UTR C-->A. Of these, mutations at codon 250, codon 324, intron 4 and 3'UTR showed a frequency of 1% or more. Of the known mutations, Thr-17Ala(A-->G), Lys76Lys(A-->G) and Leu241Leu(G-->A) occur at a polymorphic level. The ACT codon -17*A allele was associated with increased risk of AD (OR for AA vs TT: 1.71; 95% CI: 1.16-2.53; P=0.007), especially in the presence of the APOE*4 allele (OR for AA vs TT: 2.35; 95% CI: 1.13-4.85; P=0.02). The codon 241*A allele and the codon 250*T allele were associated with protective effects against AD (OR: 0.36; 95% CI: 0.13-0.86; P=0.02) (OR:0.39; 95% CI: 0.18-0.85; P=0.02). irrespective of the APOE*4 status. The codon 324*G allele was associated with a marginal protective effect (OR:0.57; 95% CI: 0.26-1.26; P=0.17). While the codon 241*A allele was in linkage disequilibrium with the codon -17*A allele, the codon 250*T and codon 324*G alleles were non-randomly associated with the codon -17*T allele. In contrast, the codon 76*G (OR:1.34; 95% CI: 0.92-1.95; P=0.13), codon 227*G (OR:3.96; 95% CI: 0.83-18.8; P=0.08) and intron 4*G (OR:1.47; 95% CI: 0.88-2.29; P=0.15) alleles were associated with a modest risk of AD, and all were in linkage disequilibrium with the codon -17*A allele. EH-based haplotype analysis showed that certain haplotypes are associated with either higher or lower risk of AD. Our data indicate that the ACT gene harbors several potentially important variable sites, which are associated with either an increased or decreased risk of AD. The non-random combination of risk and protective alleles may explain, in part, why the association studies regarding the ACT codon -17*A have been inconsistent, especially if the frequency of other ACT mutations varies between populations.     

Different evolutionary pathway of B*570101 and B*5801 (B17 group) alleles based in intron sequences

Two theories about MHC allele generation have been put forward: (1) point mutation diversification and/or (2) gene conversion events. A model supporting the existence of both of these mechanisms is shown in this paper; the possible evolution of the HLA-B*570101 and HLA-B*5801 alleles (which belong to the HLA-B17 serology group) is studied. The hypothesis favoured is that gene conversion events have originated these alleles, because intron sequences are also analysed. Evolution by point mutation should only be accepted if flanking introns have also been sequenced.The nucleotide sequence data (exons and introns) reported in this paper have been sequenced in our laboratory. They are in the GenBank nucleotide sequence database and have been assigned the accession numbers: B*150101—(a) exon 1, L79939; (b) exon 2 and exon 3, L48400; (c) intron 1, L76249; (d) intron 2, L42468; B*1515—(a) exon 1, exon 2 and exon 3, L49343; (b) intron 1 and intron 2, L76254; B*1539—(a) exon 2, AF033501; (b) exon 3, AF033502; (c) intron 1, AF034961; (d) intron 2 AF034962; B*350101—(a) exon 1, exon 2 and exon 3, L63544; (b) intron 1, L79921; (c) intron 2, L57505; B*510101—(a) exon 1, L77204; (b) exon 2 and exon 3, L47985; (c) intron 1, L76245; (d) intron 2, L42469; B*520102—(a) exon 1, L77205; (b) exon 2 and exon 3, L47984; (c) intron 1, L76244; (d) intron 2, L76251; B*5301—(a) exon 1, intron 1, exon 2, intron 2 and exon 3, U90566; B*1302—(a) intron 1, exon 2, intron 2, exon 3, AF196182; B*400101/02—(a) exon 2 and exon 3, L79937; (b) intron 1, L79919; (c) intron 2, L76629; B*4101—(a) intron 1, exon 2, intron 2 and exon 3, U90560; B*4102 (a) intron 1, exon 2, intron 2 and exon 3, AF 126199; B*4501—(a) intron 1, exon 2, intron 2 and exon 3, U90562; B*570101—(a) intron 1, exon 2, intron 2 and exon 3, AF196183; B*5801—(a) intron 1, exon 2, intron 2 and exon 3, AF196184All exon sequences were officially assigned as confirmatory by the WHO Nomenclature Committee in December 2003: B*1302, B*150101, B*350101, B*400101/02, B*4101, B*510101, B*570101, B*5801, B*5301, B*4501, B*520102, B*1515, B*4102 and B*1539. This follows the agreed policy that, subject to the conditions stated in the Nomenclature Report [Marsh et al. (2002) Tissue Antigens 60:407–464], names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Allelic variation in the intron 2 and 3 of the MICA gene.

We have determined by sequencing the allelic variation in intron 2 and 3 of hte MICA gene for a total of 22 different alleles. Sequencing of introns was performed in two directions, using DNA from homozygous cell lines from families and from unrelated individuals. Intron 2 is 273 bp long and did in the alleles investigated not reveal any length polymorphism. We found a total of eight polymorphic positions which exhibit a strict biallelism, as it is also found in the polymorphisms for exon 2, 3 and 4 of MICA. Intron 3 is 586 bp long an required an additional set of primers placed near the middle of this intron in order to allow a complete bidirectional sequence. In intron 3, a total of 10 polymorphic positions were identified. Interestingly, we found two variants of the allele MICA*002 which are distinguished only by one basepair difference in intron 3. The variant MICA*002A is associated with HLA-B35 and B58, while the allele MICA*002B is associated with B38 and B39.     

Allospecific T cell epitope sharing reveals extensive conservation of the antigenic features of peptide ligands among HLA-B27 subtypes differentially associated with spondyloarthritis

HLA-B*2702, B*2704, and B*2705 are strongly associated with spondyloarthritis, whereas B*2706 is not. Subtypes differ among each other by a few amino acid changes and bind overlapping peptide repertoires. In this study we asked whether differential subtype association with disease is related to differentially bound peptides or to altered antigenicity of shared ligands. Alloreactive CTL raised against B*2704 were analyzed for cross-reaction with B*2705, B*2702, B*2706, and mutants mimicking subtype changes. These CTL are directed against many alloantigen-bound peptides and can be used to analyze the antigenicity of HLA-B27 ligands on different subtypes. Cross-reaction of anti-B*2704 CTL with B*2705 and B*2702 correlated with overlap of their peptidic anchor motifs, suggesting that many shared ligands have similar antigenic features on these three subtypes. Moreover, the percent of anti-B*2704 CTL cross-reacting with B*2706 was only slightly lower than the overlap between the corresponding peptide repertoires, suggesting that most shared ligands have similar antigenic features on these two subtypes. Cross-reaction with B*2705 or mutants mimicking changes between B*2704 and B*2705 was donor-dependent. In contrast, cross-reaction with B*2702 or B*2706 was less variable among individuals. Conservation of antigenic properties among subtypes has implications for allorecognition, as it suggests that shared peptides may determine cross-reaction across exposed amino acid differences in the MHC molecules and that the antigenic distinctness of closely related allotypes may differ among donors. Our results also suggest that differential association of HLA-B27 subtypes with spondyloarthritis is more likely related to differentially bound peptides than to altered antigenicity of shared ligands.     

Identification of novel HLA-B27 ligands derived from polymorphic regions of its own or other class I molecules based on direct generation by 20 S proteasome.

HLA-B27 is strongly associated with ankylosing spondylitis. Natural HLA-B27 ligands derived from polymorphic regions of its own or other class I HLA molecules might be involved in autoimmunity or provide diversity among HLA-B27-bound peptide repertoires from individuals. In particular, an 11-mer spanning HLA-B27 residues 169-179 is a natural HLA-B27 ligand with homology to proteins from Gram-negative bacteria. Proteasomal digestion of synthetic substrates demonstrated direct generation of the B27-(169-179) ligand. Cleavage after residue 181 generated a B27-(169-181) 13-mer that was subsequently found as a natural ligand of B*2705 and B*2704. Its binding to HLA-B27 subtypes in vivo correlated better than B27-(169-179) with association to spondyloarthropathy. Proteasomal cleavage generated also a peptide spanning B*2705 residues 150-158. This region is polymorphic among HLA-B27 subtypes and class I HLA antigens. The peptide was a natural B*2704 ligand. Since this subtype differs from B*2705 at residue 152, it was concluded that the ligand arose from HLA-B*3503, synthesized in the cells used as a source for B*2704-bound peptides. Thus, polymorphic HLA-B27 ligands derived from HLA-B27 or other class I molecules are directly produced by the 20 S proteasome in vitro, and this can be used for identification of such ligands in the constitutive HLA-B27-bound peptide pool.     

HLA-B*2704, an allotype associated with ankylosing spondylitis, is critically dependent on transporter associated with antigen processing and relatively independent of tapasin and immunoproteasome for maturation, surface expression, and T cell recognition: relationship to B*2705 and B*2706

B*2704 is strongly associated to ankylosing spondylitis in Asian populations. It differs from the main HLA-B27 allotype, B*2705, in three amino acid changes. We analyzed the influence of tapasin, TAP, and immunoproteasome induction on maturation, surface expression, and T cell allorecognition of B*2704 and compared some of these features with B*2705 and B*2706, allotypes not associated to disease. In the tapasin-deficient .220 cell line, this chaperone significantly influenced the extent of folding of B*2704 and B*2705, but not their egress from the endoplasmic reticulum. In contrast, B*2706 showed faster folding and no accumulation in the endoplasmic reticulum in the absence of tapasin. Surface expression of B*2704 was more tapasin dependent than B*2705. However, expression of free H chain decreased in the presence of this chaperone for B*2705 but not B*2704, suggesting that more suboptimal ligands were loaded on B*2705 in the absence of tapasin. Despite its influence on surface expression, tapasin had little effect on allorecognition of B*2704. Both surface expression and T cell recognition of B*2704 were critically dependent on TAP, as established with TAP-deficient and TAP-proficient T2 cells. Both immunoproteasome and surface levels of B*2704 were induced by IFN-gamma, but this had little effect on allorecognition. Thus, except for the differential effects of tapasin on surface expression, the tapasin, TAP, and immunoproteasome dependency of B*2704 for maturation, surface expression, and T cell recognition are similar to B*2705, indicating that basic immunological features are shared by the two major HLA-B27 allotypes associated to ankylosing spondylitis in human populations.