云南汉族2型糖尿病与HLA-DQA1基因的关联研究

目的　探讨云南汉族 2型糖尿病及 2型糖尿病肾病与 HL A- DQA1基因的关联性。方法　采用聚合酶链反应 -序列特异性引物技术对云南汉族 10 8例 2型糖尿病患者及 5 6名同地区同民族健康对照人群进行 DQA1基因分型。结果　云南汉族 2型糖尿病患者与正常对照组比较 ,DQA1* 0 30 1( RR=3.0 92 ,P<0 .0 1) ,DQA1* 0 5 0 1( RR=3.2 5 7,P<0 .0 5 )等位基因频率明显增高 ,DQA1* 0 4 0 1( RR=0 .371,P<0 .0 1)等位基因频率显著下降。糖尿病合并肾病组与正常对照组及不合并肾病的 2型糖尿病组比较 ,糖尿病合并肾病患者 HL A- DQA1* 0 30 2等位基因频率显著升高 ( RR=3.35 6 ,P<0 .0 1) ,各期糖尿病肾病比较中 DQA1* 0 30 2频率差异无显著性 ( P>0 .0 5 )。结论　 HL A- DQA1* 0 30 1,DQA1* 0 5 0 1是云南汉族 2型糖尿病的易感基因 ,HL A- DQA1* 0 4 0 1是云南汉族 2型糖尿病的抵抗基因 ;HL A- DQA1*0 30 2是 2型糖尿病合并肾病的易感基因     

HLA-DQ多态性基因与系统性红斑狼疮易感性

采用PCR-SSO方法对江苏籍汉族SLE患者和正常对照组HLA-DQ作基因分型.结果显示,患者组中DQA1*0102频率(RR=3.43.Pc=0.03164)及HLA-DQA1*0102,DQB1*0601和HLA-DQA1*0102,DQB1*0602单倍型频率(RR=9.4,P=0.027和RR=12.4.P=0.007)均明显高于正常对照组.相反,DQA1*0601频率则显著低于正常对照组(RR=0.29,Pc=0.0461).但没发现任何DQB1等位基因与SLE有关.这提示在汉族SLE与HLA-DQ基因的相关性方面,DQA1*0102起主导作用.DQA1*0102或某个与其紧密连锁的其它基因可能是汉族SLE的易感基因,而DQA1*0601则可能对SLE的发病有一定的保护性.     

人类白细胞Ⅱ类抗原DR、DQ基因型与妊娠期糖尿病的相关性研究

目的探讨人类白细胞II类抗原DR、DQ基因型与妊娠期糖尿病的相关性。方法对26例GDM孕妇及同期入院的42例正常健康孕妇,采用序列特异性引物聚合酶链反应技术(PCR-SSP)检测HLA-II类抗原DR和DQ的等位基因。结果研究中发现,DQA1*0101、DQA1*0201、DQB1*0609、DRB l*07-DQA1*0201-DQB1*0201基因频率在GDM中显著高于正常对照组,两组比较,统计学有差异(Ρ<0.05)。DQB1*0301基因频率在GDM中显著低于正常对照组,两组比较,有统计学差异(Ρ<0.05)。结论人类白细胞II类抗原DR、DQ基因型与GDM的易感性和保护性存在关联。DQA1*0101、DQA1*0201、DQB1*0609、DRB l*07-DQA1*0201-DQB1*0201基因是GDM的易感基因。DQB1*0301基因是GDM的保护基因。     

中国汉族人HLA-DQAl~*0601基因转录启动子区显示新的核苷酸序列

本文测定了中国汉族人HLA纯合细胞SMY-43A的DQA1~*0601基因启动子区(QAP)核苷酸序列,发现顺式作用元件W box中-215和-216位核苷酸组成与已报道的白种人DQA1~*0601的QAP序列不同,由于SMY-43A的Ⅱ类基因单倍型组成为中国人特有,提示相应的QAP多态性可能具有人种差异.     

HLA-DQA1*0301及*0103与腔隙性脑梗死和原发性高血压的遗传易感性研究

目的：针对腔隙性脑梗死（lacunar stroke,LS）和原发性高血压（essential hypertension,EH)及多基因致病特点,进行HLA－DQA1位点的基因分型,分析其遗传易感性。方法：采用PCR－SSP方法对62例LS、52例EH和64例正常对照进行HLA－DQA1位点的基因分型。结果：①HLA－DQA1*0301等位基因与LS及EH呈显著正相关。②HLA－DQA1＊0103等位基因与LS及EH呈显著负相关。结论：HLA－DQA1*0301等位基因与LS及EH的发病有一定的关联,而HLA－DQA1＊0103等位基因可能是LS及EH的保护性基因。     

HLA-DQA1基因多态性与HBV感染结局相关

目的探讨中国汉族人群人类白细胞抗原(HLA)-DQA1基因多态性是否与乙型肝炎病毒(HBV)感染结局相关联。方法以213例HBV自限性感染者和420例慢性乙肝患者为研究对象,应用聚合酶链反应-序列特异性引物(PCR-SSP)技术进行HLA-DQA1基因分型,用EPI和SPSS软件分析DQA1多态性的分布频率及其组间差异。结果DQA1*0102在慢性乙肝组的分布频率显著低于HBV自限性感染组(15.47%比较20.42%,P<0.05),而DQA1*0201在慢性乙肝组的分布频率显著高于HBV自限性感染组(10.48%比较6.10%,P<0.05)。调整性别、年龄等混杂因素影响的非条件logistic回归分析结果显示,与HLA-DQA1其他等位基因相比,携带DQA1*0102者降低慢性乙肝发生的风险(P<0.05,OR=0.69,95%C I:0.49-0.96),而携带DQA1*0201者增加慢性乙肝发生的风险(P<0.05,OR=1.77,95%C I:1.09-2.87)。结论HLA-DQA1基因多态性可能是影响HBV感染结局的重要宿主遗传因素。     

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。     

HLA-DQA1、-DQB1基因多态性与高原红细胞增多症的相关性研究

目的:探讨移居高原汉族男性人群HLA-DQA1、-DQB1基因多态性是否与其发生高原红细胞增多症(HAPC)的易感性相关联.方法:以60例移居高海拔地区男性HAPC患者和印例移居同地区健康男性为研究对象,应用聚合酶链反应-序列特异性引物(PCR-SSP)技术进行HLA-DQA1、-DQB1基因分型,并使用SPSS软件分析HLA-DQA1、-DQB1多态性的分布频率及其组问差异.结果:移居高海拔地区的HAPC患者组的HIA-DQA1*0401和*050 1分布频率分别为0.125 0和0.258 3;而同地区健康对照组HLA-DOA1*0401和*0501分布频率分别为0.041 7和0.158 3.与对照组相比,HAPC组HLA-DQA1*0401相对危险性为3.67,校正P值<0.05;*050l危险性为2.31,校正P值<0.05.而HAPC组和对照组在DQA1*0101/0104、DQA1*0103、DQB1*0201、DQB1*0601等位基因未显示显著性差异.结论:HLA-DQA1*0401、*0501位点町能与HAPC的易感性关联.     

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     

原发性肥厚型心肌病与HLA-DQ的相关性研究

本文用PCR/SSO方法对41例原发性肥厚型心肌病患者和52例正常人HLA-DQA1和DQB1基因的多态性进行分析.发现,在肥厚型心肌病患者中,DQA1＊0201,DQB1＊0504,0502等位基因频率明显较低,其相对风险值分别为9.51、5.87和11.60;而DQA1*0501,DQB1＊03031的频率明显地高,相对风险值分别为2.93和6.65.初步认为,原发性肥厚型心肌病与某些HLA-DQ基因相关联.     

采用PCR-RFLP技术分析中国人HLA-DR5亚型

采用等位特异的限制性内切酶降解经PCR扩增的中国人HLA纯合细胞的DRB基因片段,根据相应的限制性片段长度多态性(PCR-RFLP),可十分快速而准确地从DNA水平区分DR5的两个亚型,并显示属于DRW12亚型的中国人HLA纯合细胞与参考细胞间电泳格局的差异,提示新的DRW12变异体的存在。该技术不必采用等位或顺序特异的寡核苷酸探针进行杂交,因而无需使用放射性同位素,展示了应用于HLA基因分型的良好前景。     

PCR—RFLP技术在HLA—DQA1基因分型中的应用

采用ＰＣＲ－ＲＦＬＰ方法对５４例上海人随机样本作ＨＬＡ－ＤＱＡ１基因分型，其中２４例已有ＰＣＲ－ＳＳＯＰＨ分型结果。通过分析比较，发现这两种方法得出的结论基本一致。     

HLA-DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers

We previously introduced HLA-DQA1, -DPB1 and DQB1 genotyping with the modified PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or alternatively no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. In this study, 43 HLA-DRB1 alleles, excluding DRB1*1103 and *1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR-RFLP method combined with 7 pairs of group-specific primers. It is impossible to distinguish DRB1*0701 and DRB1*0702 as they are identical for the second exon of DRB1. For DR1-DRB1, DR2-DRB1, DR4-DRB1, DR7-DR1, DR9-DRB1, DRw10-DRB1 or DRw52 associated antigens (DR3, w11, w12, w13, w14, and DRw8)-DRB1 gene amplification, the second exon of the DRB1 gene was selectively amplified using each group-specific primer from genomic DNAs of 70 HLA-homozygous B-cell lines and healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR-RFLP method can be successfully applied to all possible DRB1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR-SSO method, because the PCR-RFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR-RFLP method is technically simple, practical and inexpensive for determination of the HLA-DRB1 alleles for routine HLA typing work.     

A nested PCR-RFLP method for high-resolution typing of HLA-A alleles

We developed a nested polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method for high-resolution typing of HLA-A alleles. HLA-A alleles can be identified by this method without the need for other information such as serological type. The first PCR was performed using outer primers, ASP5 and ASP3, specific for the HLA-A gene, and a 991-bp DNA fragment extending from exon 1 through exon 3 was amplified. In the second PCRs, exon 2 and exon 3 of the HLA-A gene were amplified separately from the diluted first PCR product using nested primers. Computer analysis of cleavage patterns for 78 HLA-A alleles showed that 31 RFLP patterns could be obtained by digestion of the exon 2 PCR product using eight restriction endonucleases and 42 RFLP patterns by digestion of the exon 3 PCR product using 11 restriction endonucleases, and all alleles could be discriminated based on combinations of these RFLP patterns except for nine allele groups or pairs: A*0201/ 0207/ 0215N/0220/0222, A*0205/0208/0214, A*0206/ 0221, A*0212/ 0213, A*2402/2405, A*2406/2413, A*2601 / 2605, A*2603/2606 and A*7401/7402. Thus, 65 PCR-RFLP patterns were predicted from the results of analysis of digestion patterns of 78 HLA-A alleles. Among 2145 possible homozygous and heterozygous combinations of the 65 distinguishable PCR-RFLP patterns, 82 combinations were predicted to have the same PCR-RFLP patterns. In PCR-RFLP analysis, although the nested primers were not specific for the HLA-A gene, clear RFLP banding patterns were obtained because specificity was guaranteed by the use of the outer primers, ASP5 and ASP3 in the first PCR. A*0201 and A*0207 occur relatively frequently in the Asian populations among indistinguishable allele groups or pairs using the present PCR-RFLP method. We also developed a PCR sequence-specific primers (PCR-SSP) method for distinguishing between A*0201/0220/0222 and A*0207/0215N. We could identify 39 alleles (groups) upon HLA-A typing of 50 Japanese individuals, 40 cell lines of the Fourth Asia-Oceania Histocompatibility Workshop, and 80 cell lines of the UCLA International Cell Exchange Program using the present PCR-RFLP and PCR-SSP methods.     

HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers.

We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplified by DQw1 group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQw1-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB1*0602 and DQB1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.     

NUCLEOTIDE SEQUENCING OF HLA-DQ GENE SECOND EXONS IN CHINESE HOMOZYGOUS CELLS

Six HLA class I and class II-homozygous Chinese cell lines with unique HLA-Dw types were studied. Since the majority of HLA class II nucleotide sequence polymorphism is localized within the second exons of the genes, we used the polymerase chain reaction (PCR) to amplify these regions in HLA-DQA and DQB genes and subsequently determined the nucleotide sequences. No unique DQA1 or DQB1 alleles were found. However, a new haplotype of DQA1*601-DQB1*0301-DRB1*1202 was found in two cells; and DQA1*03011 was found in association with DR9 in another two cells. This indicates that new DR-DQ associations may explain the observed new HLA-Dw types. The DQB2 sequences were identical in all six cells and were identical to a sequence previously reported in a DR6 haplotype. The DQA2 sequences from two clones obtained from two cells differed from each other and from previously reported sequences. The results show that the DQA1 and DQB1 alleles in the Chinese individuals studied are as previously reported in Caucasian populations and as such may be typed by restriction fragment-length polymorphism (RFLP) or PCR-sequence-specific oligonucleotide typing (PCR-SSO) or PCR-RFLP using conventional probe or restriction enzyme sets.     

HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers

Abstract: We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB 1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplifed by DQwl group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQwl-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB 1*0602 and DQB 1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB 1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping.

We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping

Abstract: We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacryl-amide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, Fokl, MboII and Mnll can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, Fokl, Ddel, BsaJI, BssHII, Cfr13I, Rsal, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

A simple and rapid method for HLA-DP genotyping by digestion of PCR-amplified DNA with allele-specific restriction endonucleases

We previously reported a simple and rapid method for HLA-DQA genotyping by digestion of polymerase chain reaction-amplified DQA genes with allele-specific restriction endonucleases. Here we report the application of this method to DP genotyping. The second exon of the HLA-DPB genes was selectively amplified from genomic DNAs of 72 HLA-D homozygous B-cell lines by the polymerase chain reaction method. Amplified DNAs were digested with ApaI, SacI, BstUI, FokI, and RsaI, which can recognize allelic sequence variations in the polymorphic segments of the DPB second exon and then subjected to electrophoresis in polyacrylamide gels. Sixteen different polymorphic patterns of the restriction fragments were found, and twelve were identical to patterns predicted from the known DNA sequences correlating with each HLA-DPw specificity defined by cellular typing. The other four patterns were distinct from those of the known DPw specificities, suggesting the presence of novel DP alleles. This polymerase chain reaction-restriction fragment length polymorphism method provides a simple and rapid technique for accurate definition of HLA-DP types at the nucleotide level, replacing the technically demanding method of primed lymphocyte typing.