中国天疱疮患者的免疫遗传易感性研究

目的探讨中国天疱疮患者的免疫易感基因位点.方法本实验应用序列特异性引物聚合酶链式反应(SSP-PCR)技术对HLA-II类等位基因进行特异性体外扩增,分析了天疱疮患者HLA基因的DR位点和DRB1、DQB1多态性,并将天疱疮患者的基因频率与正常人结果进行χ2检验,以探讨中国天疱疮易感性基因位点.结果天疱疮患者中DR4的频率为93.3%(28/30),DR14的频率为63.3%(19/30),天疱疮患者中的DRB1*04和DRB1*14的频率分别为40%(12/30)和53.3%(16/30),经χ2分析,与正常对照组相比具有显著的差异性;在28个DR4+的天疱疮患者中有13个DRB1*0402,在19个DR14+天疱疮患者中DRB1*1401频率显著增高(P<0.01).所有DRB1*04+患者均为DQB1*0302,所有DRB1*14患者均为DQB1*0503.结论中国人天疱疮易感性与HLA的DR4、DR14抗原以及DRB1*04、DRB1*14、DRB1*0402、DRB1*1401基因位点与高度相关.     

HLA-DRB1、DQB1基因与汉族人群寻常型天疱疮的相关性研究

目的　探讨 HL A- DRB1、DQB1位点基因在汉族人群寻常型天疱疮易感性中的作用。方法用序列特异性引物 -聚合酶链反应方法 ,对 6 1例寻常型天疱疮 (pemphigus vulgaris,PV)患者和 5 7名正常对照进行了 HL A- DRB1、DQB1等位基因的分型 ,并分析了 DRB1、DQB1基因在两组中的分布。结果　与正常对照组比较 ,PV组 DR4、DRB1* 14 (* 14 0 1、* 14 0 4、* 14 0 5 )基因频率明显增高 (Pc分别 <0 .0 5及P<0 .0 1) ,差异有显著性 ;PV组 DQB1* 0 5 0 3、DQB1* 0 30 2基因频率明显增高 (Pc均 <0 .0 5 ) ,差异有显著性。对 DR4阳性样本的组内基因亚型分型结果发现 ,PV组中 DRB1* 0 4 0 3、DRB1* 0 4 0 6频率显著增高(Pc<0 .0 5 ) ,差异有显著性。 PV患者组单倍型 HL A- DRB1* 0 4 ,DQB1* 0 30 2和 HL A- DRB1* 14 ,DQB1* 0 5 0 3频率明显增高 (P<0 .0 5 )。结论　HL A- DRB1* 0 4 ,DQB1* 0 30 2和 HL A- DRB1* 14 ,DQB1* 0 5 0 3可能是汉族人 PV推测的易感单倍型。     

寻常型天疱疮自身抗原Dsg3片段的重组表达和特异性细胞反应

目的 :探讨寻常型天疱疮自身抗原Dsg3在特异性T细胞反应中的作用 ,为自身免疫性疾病机制的研究提供依据。方法 :根据Genbank中的Dsg3序列分析 ,采用RT PCR法克隆自身抗原Dsg3E1,E2 ,E3,E4,E5多肽片段的cDNA ,定向插入表达载体PGEX 2T ,导入大肠杆菌JM10 9中表达重组融合蛋白并经GST层析柱纯化 ;进一步与PV患者及疾病对照组、正常对照组T细胞混合培养 ,观察T细胞增殖反应。结果 :Dsg3E1,E2和E4,E5可刺激PV患者T细胞反应 ,而不与疾病对照组、正常对照组反应。结论 :Dsg3E1,E2和E4,E5中包含T B细胞作用相关的抗原表位 ,在PV发病中起重要作用。     

导常型天疱疮自身抗原Dsg3片段的重组表达和特异性细胞反应

目的：探讨寻常型天疱疮自身抗原Dsg3在特异性T细胞反应中的作用，为自身免疫性疾病机制的研究提供依据。方法：根据Genbank中的Dsg3序列分析，采用RT-PCR法克隆自身抗原Dsg3E1,E2,E3,E4,E5多肽片段的cDNA,定向插入表达载体PGEX-2T，导入大肠杆菌JM109中表达重组融合蛋白并经GST层析柱纯化；进一步与PV患者及疾病对照组、正常对照组T细胞混合培养，观察T细胞增殖反应。结果：DsgE1，E2和E4，E5可刺激PV患者T细胞反应，而不与疾病对照组、正常对照组反应。结论：Dsg3E1,E2和E4，E5中包含T-B细胞作用相关的抗原表位，在PV发病中起重要作用。     

中国东北地区汉族人寻常型天疱疮与HLA相关的研究

目的 探讨中国人寻常型天疱疮（pemphigus vulgaris,PV)相关的HLA抗原及等位基因。方法 应用标准微量淋巴细胞毒试验方法及聚合酶链反应－序列特异性引物（PCR－SSP）技术对27例PV患者HLAI类分子及Ⅱ类分子DR、DQ等位基因检测，并和健康对照组比较。结果 PV组中HLA－A3、A26（10）、B13和B60（40）抗原频率明显增高，校正P值后，HLA－A3、A26（10）、B60（40）与对照组差异仍有显著意义。HLA－DRB1＊140x(1401、1404、1405、1407、1408）、DRB1＊120x和DQB1＊0503等位基因出现频率明显高于对照组，校正P值后两组差异仍有显著意义。结论 中国汉族PV患者中，PV的发生与HLA显著相关。     

应用PCR/SSO方法进行西安汉族人群HLA-DR,DQ位点的DNA分型

对80例西安汉族人HLA第Ⅱ类抗原的DRB、DQA1和DQB1的等位基因多态性进行分析,发现的特异性包括HLA-DRB1 27种,DRB3 3种,DRB5 3种,DQA1 8种,DQB1 14种,共55种。按血清学所定的特异性将DRB1位点的等位基因对应分类,出现频率由高到低排列依次为DR5,DR2,DR6,DR9,DR4,DR7,DR8,DR3,DR1,DR10。在DQA1和DQB1等位基因中,DQA1*0301和DQB1*0301、DQB1*0303的频率是最高的。和以前所报道的北方人群中DRB1*1301与DQB1*0604相连锁的结论不同的是,该人群中DRB1*1301与DQB1*0603相连锁,且未发现北方人群中所报道的DRB1*1403。这一结论与地处南方     

中国北方汉族寻常型天疱疮与HLA-Ⅱ类基因单倍型的相关性研究

目的：探讨HLA—DR、DQ基因单倍型与中国东北地区汉族人寻常型天疱疮的相关性。方法：应用序列特异性引物-聚合酶链反应（PCR—SSP）技术对27例中国东北汉族PV患者的HLA—DRB1、DQB1等位基因测定，进行单倍型分析，并与99例健康对照者进行比较。结果：与对照组比较，寻常型天疱疮患者组中单倍型DRB1＊140x-DQB1＊0503、DRB1＊140x-DQB1＊0201、DRB1＊120x-DQB1＊0503和DRB1＊140x—DQB1＊0302的频率明显增高，经统计学检验差异有显著意义（P〈0．05）。结论：特异单倍型可能是决定寻常型天疱疮发生的重要因素。     

云南汉族系统性红斑狼疮的易感及抵抗单体型研究

目的：探讨云南汉族系统性红斑狼疮（SLE）在HLA－DRB1、DQA1、DQB1等座位的易感抵抗单体型,方法：采用多聚酶链反应－序列特异性引物（PCR－SSP）技术对63例动态汉族SLE患者及54名同民族健康对照进行DRB1、DQA1、DQB1基因分型。结果：与正常对照组比较,SLE病人中有5个单体型频率显著升高;11个单体型频率在病例组中明显降低。结论：云南汉族SLE的易感单体型为DQA1^*0102-DQB1^*0601,DR15-DQA1^*0102-DQB1^*0601,DR15-DQA1^*0102-DQB1^*0602,DR15-DQA1^*0101-DQB1^*0601,DR15-DQA1^*0103-DQB1^*0601;其余均为低抗单体型。     

乙型肝炎疫苗免疫失败成人T细胞亚群、HLA-DR区基因表型分析

目的 研究成人乙肝疫苗免疫失败与HLA-DR、T细胞亚群相关性,探讨与成人乙肝疫苗免疫失败相关因素.方法 随机选取接种10μg重组酵母乙型肝炎疫苗免疫失败者及成功者各20名,检测T细胞亚群及HLA-DR、HLA-B27、HLA DRB1 * 07、DRB1 * 04、DRB1 * 1001、DQB1 * 0401等DR区基因表型水平.结果 免疫失败组CD4-/CD8-含量低于对照组,免疫失败组HLA-DR及HLA-B27平均水平高于对照组,两组间HLA DRB1 * 07基因频率差异有统计学意义(P<0.05),而两组间CD3、CD4、CD8、CD5、CD7、CD4+/CD8+、CD4-/CD8+、CD4+/CD8-含量及HLA DRB1 * 04、DRB1 * 1001、DQB1*0401水平差异均无统计学意义.结论 成人乙肝免疫失败可能与HLA-DR、HLA-B27、HLA DRB1 *07、CD4-/CD8-等相关.     

寻常型银屑病血清瘦素水平与HLA等位基因相关性研究

目的:研究中国北方汉族人寻常型银屑病(PV)患者血清瘦素(leptin)水平与HLA等位基因的关联性。方法:采用序列特异性引物-聚合酶链反应(PCR-SSP)方法对91例PV患者和102例健康人进行HLA-A、B、Cw、DRB1及DQB1分型,放射免疫分析(RIA)技术测定其血清瘦素水平。结果:(1)PV患者血清瘦素水平(7.63±4.44 ng/ml)较正常对照(5.03±2.72 ng/ml)显著增高(P<0.001);男性及女性PV患者血清瘦素水平分别较男性及女性正常对照显著增高(P=0.004,P<0.001)。(2)北方汉族人PV与HLA-A*010x(Pc=0.039 4)、A*300x(Pc=0.012 9)、B*570x(Pc=0.013 7)、Cw*0602(Pc=0.000 9)、Cw*060x(除外Cw*0602)(Pc<1×10-4)、DRB1*0701/0702(Pc=0.001 2)及DQB1*0201(Pc=0.005 6)等位基因呈正相关,与Cw*040x(Pc=0.018 8)呈负相关。(3)携带HLA-DRB1*15及DRB1*0701/0702等位基因的男性PV患者和携带HLA-A*010x、A*24、Cw*040x、DRB1*15、DQB1*0301等位基因的女性患者的血清瘦素水平均显著增高(P<0.05)。结论:(1)HLA-A*010x、A*300x、B*570x、Cw*0602、Cw*060x(除外Cw*0602)、DRB1*0701/0702及DQB1*0201等位基因可能是北方汉族人PV的易感基因或与易感基因相连锁,HLA-Cw*040x可能是预防PV发病的“保护因子”。(2)PV患者血清瘦素水平显著增高,并与HLA等位基因相关联,提示HLA等位基因和瘦素参与PV发病过程。     

Presence of the DRB4*0103102N null allele in different DRB1*04-positive individuals

The DRB4 gene encoding the DR53 antigen is present in DRB1*04-, DRB1*07- and DRB1*09-positive individuals. Eight allelic variants of DRB4 have been recognized, 5 resulting in an expressed DR53 antigen and 3 belonging to the null alleles. So far the DRB4*0103102N null allele had been found exclusively in individuals carrying the haplotype DR7,-DQ9. High-resolution typing of HLA class II by polymerase chain reaction using sequence-specific primers (PCR-SSP) and/or sequence-based typing of kidney patients and their families revealed the presence of the DRB4*0103102N null allele segregating with DRB1*04 and DQB1*03 in 4 different families. Three different haplotypes on which the null allele was located, were recognized by family studies: DRB1*0401, DQB1*0301; DRB1*0402, DQB1*0302 and DRB1*0404, DQB1*0302. Determination of the DR53 specificity of antisera reacting with DR53-positive individuals has always been difficult due to the simultaneous presence of DR4, 7 or 9. Identification of DR4-positive DR53-negative individuals as described here, provided the serological reactions with DR53-antisera and revealed the antibody specificities in the antisera used.     

HLA-DRB1 polymorphisms and autoimmune responses to desmogleins in Japanese patients with pemphigus.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are caused by autoantibodies against keratinocyte adhesion molecules desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1), respectively. To determine possible major histocompatibility complex (MHC) class II associations with autoantibody responses to desmogleins, haplotype and allele distributions, along with molecular polymorphisms of HLA-DR and -DQ genes were analyzed based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) results in 85 Japanese patients with pemphigus. Each of 55 PV patients carried at least one allele of HLA-DRB1*04 and DRB1*14 subtypes, with significant increases of HLA-DRB1*0406/DQA1*0301/DQB1*0302, DRB1*14/DQA1*0104/DQB1*05 and DRB1*1406/DQA1*0503/ DQB1*0301 haplotypes compared to normal controls. The HLA-DRB1*04 and DRB*14 alleles carried by PV patients shared hydrophobic amino acid residues Phe26, Leu67 and Val86, as well as hydrophilic amino acid residues at positions 70 and 71 on the DRB1 beta chain. HLA-DR/DQ distributions did not differ among PV patients according to the presence or absence of anti-Dsg1 co-existing with anti-Dsg3. Thirty PF patients, all producing autoantibodies only to Dsg1, showed more diverse HLA-DR/DQ distributions, sharing hydrophobic amino acid residues at positions 26 and 67, as well as hydrophilic amino acid residues at positions 70 and 71, of the DRB1 chain. These findings suggest that autoantibody responses to desmogleins might be regulated by amino acid residues at positions 26, 67, 70, 71 and 86 at peptide binding sites of HLA-DRB1 molecules, and that autoimmune responses to Dsg3 might be more strictly regulated by specific amino acid residues at these positions on the HLA-DRB1 chain than responses to Dsg1.     

Pemphigus vulgaris in Jewish patients is associated with HLA-A region genes: mapping by microsatellite markers

Pemphigus vulgaris (PV) is the most severe autoimmune blistering disorder of the skin that is mediated by circulating autoantibodies against desmoglein 3 (Dsg3). It has been reported that in Jews the associated haplotype in PV is human leukocyte antigen (HLA) B38, DRB1*0402, DQB1*0302. Significant associations with HLA were observed also in non-Jews. Dsg3-specific T-cell responses were detected in PV patients but also in healthy individuals who were either carriers of the PV-associated DRB1*0402 allele or alleles that share similar or identical peptide binding motifs to DRB1*0402. This suggests that genes other than the classical major histocompatibility complex (MHC) genes are associated with the development of the autoimmune response. We used 16 microsatellite probes that span the entire MHC region to screen DNA samples from 38 PV patients and 76 healthy controls. Results demonstrated that some markers were associated with class II region including a TAP associated marker. However, four probes, D6S265, C_527, D6S510, and MOGC, which are all mapped to the region of HLA-A, were highly associated with PV. These results suggest that a gene, or genes in the class I region are important in the initiation of the autoimmune cascade. Activation/suppression of these genes might act as the trigger mechanism that starts the autoimmune destructive process.     

Allelic distribution and the effect of haplotype combination for HLA type II loci in the celiac disease population of the Valencian community (Spain)

The association between human leukocyte antigen (HLA) class II antigens and celiac disease (CD) was analyzed in a Spanish population. No association with DRB1*04 and DQB1*0302 was noted. The main associated haplotype (70.8%) was DRB1*03–DQB1*0201–DQA1*0501(DR3–DQ2), followed by DRB1*07–DQB1*0202–DQA1*0201 (DR7–DQ2) haplotype, which is associated with DRB1*11–DQB1*0301–DQA1*0505 (DR11–DQ7). The combinations of DR3–DQ2 with DR7–DQ2, and DR7–DQ2 with DR11–DQ7, present a twofold risk compared with each haplotype in homozygosis. An independence test in DR3-DQ2 haplotype found that association with CD was attributable to the whole haplotype, but for DR7-DQ2 was secondary to DQB1/DQA1. There is no need of a double gene dosage to increase the risk. CD-associated alleles typing demonstrates a very high negative predictive value to exclude CD in risk groups.     

HLA haplotypes and class II molecular alleles in Sardinian and Italian patients with pemphigus vulgaris

HLA class II antigens and DRB1, DQA1, DQB1 alleles were studied in 16 Italian and in 16 Sardinian patients with pemphigus vulgaris (PV). In the last group the complete HLA A-DQ haplotypes, including the complotypes, were defined by family studies. As in other populations, two PV susceptibility haplotypes were found: HLA-DRB 1*0402, DQA1*0301, DQB1*0302 and HLA-DRB 1*1401, DQA1*0104, DQB 1*0503. The first haplotype was largely prevalent in the Sardinian patients and was a part of the extended haplotype HLA-A2, Cw4, B35, S31, DR4, DQ8. The strength of the allele associations to PV is in agreement with the view that the main PV susceptibility genes are the DRB 1*0402 and DQB 1*0503 alleles. A genetic resistance to PV seems to be conferred by the HLA-DR3, DQ2 haplotype in the Sardinian population.     

Combinations of HLA DR and DQ molecules determine the susceptibility to insulin-dependent diabetes mellitus in Koreans

The association of HLA-DRB1 and DQB1 genes with IDDM in Koreans was assessed using 115 IDDM patients and 140 nondiabetic controls. DQB1*0201 is the only DQB1 allele positively associated with IDDM while DQB1*0602, *0601 and *0301 are negatively associated. Three DRB1 alleles (DRB1*0301, DRB1*0407 and DRB1*0901) are positively associated while four DR allele groups (DRB1*15, DRB1*12, DRB1*10 and DRB1*14) are negatively associated. However, Haplotype analyses indicated that DQB1*0302, DRB1*0405 and DRB1*0401 may confer susceptibility because the DRB1*0405-DQB1*0302 and DRB1*0401-DQB1*0302 haplotypes are positively associated with the disease. The lack of association in Koreans with the DQB1*0302 allele, which appears predisposing in studies of non-Orientals, is due to its strong linkage disequilibrium (LD) with the protective DRB1*0403 and *0406 alleles, while the lack of association with DRB1*0405 is because of its strong LD with the protective DQB1*0401 allele. Nine DR/DQ genotypes confer significantly increased risk to IDDM. Seven of the nine genotypes (DR3/4s, DR1/4s, DR4s/13, DR4s/8, DR4s/7, DR9/13 and DR3/9) were also found to be at high risk to IDDM in other populations, while the two others (DR1/9 and DR9/9) are only found in Koreans. Surprisingly, DR4/4 homozygotes are not associated with high risk to IDDM in Koreans. This observation can be explained by the high frequency of protective DR4 subtypes and the protective DQ alleles (0301 and 0401) associated with the susceptible DR4 alleles. Our analyses indicate that the counterbalancing act between susceptible DRB1 and protective DQB1, and vice versa, that has already been observed in Chinese and Japanese, is the major factor responsible for the low incidence of diabetes in Koreans.     

HLA class II polymorphism contributes to specify desmoglein derived peptides in pemphigus vulgaris and pemphigus foliaceus

Susceptibility to Pemphigus, an autoimmune disease of the skin, has been previously linked to DRB1*0402, 1401/04 and DQB1*0503 in pemphigus vulgaris (PV), to DRB1*0102, 0404, 1402/06 in endemic pemphigus foliaceus in Brazil and to DRB1*04 in Italian patients suffering from pemphigus foliaceus (PF). The disease is caused by autoantibodies against desmoglein (Dsg1 in PF, Dsg3 in PV).Molecular typing of 57 French patients suffering from PV (37) and from PF (20) confirmed previous results concerning PV and showed that DRB1*0102 and 0404 are susceptible molecules to PF in France.We have analysed the characteristics of the 'pockets' of the susceptibility-associated molecules to PV and PF and we showed that (i) in PV, two kinds of Dsg3 derived peptides may be presented by HLA-DR according to HLA polymorphism (DRB1*0402 or DRB1*14/0406), (ii) the same Dsg1 peptides may be presented by DRB1*0102, DQB1*0404 or DRB1*14 in PF, (iii) the DRB1*14/0406 PV-related molecules may be able to present Dsg1 and Dsg3 peptides thereby providing an explanation for the cases of PV with combined responses to Dsg1 and to Dsg3 which are typified by a muco-cutaneous clinical phenotype.     

HLA-DRB1*0402 haplotypes without DQB1*0302 in Venezuelan patients with pemphigus vulgaris

The two basic forms of autoimmune intraepidermal blistering diseases, pemphigus vulgaris (PV) and pemphigus foliaceus (PF), affect different layers of the skin, have different symptoms and target different antigens. We have defined human leukocyte antigen (HLA)-DRB1-DQB1 alleles and haplotypes in a case-control study of 66 non-Jewish patients attending a public reference Hospital over the past 10 years. The control group consisted of 101 matched individuals tested also by medium to high-resolution polymerase chain reaction-sequence-specific oligonucleotide with primers and probes from the 12th and 13th International Histocompatibility Workshop. Patients and controls were descendants of three-generation individuals born in the country. Among the patients, 49 had PV, 50% showed predominantly mucosal involvement, 50% showed predominantly the cutaneous clinical phenotype and 17 had PF. Statistically significant HLA-DR frequency differences between patients with PV and controls were found only for DRB1*0402 and DRB1*1401 [odds ratio (OR) = 27.22, confidence interval (CI) 94.7-7.82, P= 1.1 x 10(-14) and OR = 46.56, CI 801.4-2.70 P= 7.5 x 10(-6), respectively]. Both alleles were also increased in the patients with PF compared with the controls (OR = 7.0, P= 0.038 and OR = 21.64, P= 0.009, respectively), but the significance of the difference did not resist Bonferroni correction. Haplotype analysis showed that DRB1*0402 was always present with DQB1*0302 and DRB1*1401 with DQB1*0503, but no independent effect of the DQB1*0302 in the former haplotype was evident. Our results support the hypothesis that the DRB1*0402 without DQB1*0302 is the most relevant HLA-DRB1 allele responsible for the pathogenesis of pemphigus in Venezuelan patients with PV and discard the DQB1*0302 influence observed in other populations.     

Relative predispositional effects of HLA class II DRB1-DQB1 haplotypes and genotypes on type 1 diabetes: a meta-analysis

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.