HLA-B40交叉反应组高分辨度DNA分型

目的　采用顺序特异引物聚合酶链反应技术（ＰＣＲＳＳＰ） ， 建立汉族人群ＨＬＡＢ４０ 交叉反应组高分辨度ＤＮＡ 分型方法。方法　Ｂ４０ 组标准ＤＮＡ１０ 份，血清学Ｂ４０ 阳性临床样本１６４ 份，快速盐析法提取模板ＤＮＡ。设计合成１３ 个特异引物和１ 对阳性对照引物，组成８ 个ＰＣＲ 反应，建立一步法ＰＣＲＳＳＰ 快速ＨＬＡＢ４０ 组高分辨度ＤＮＡ 分型方法。结果　所有样本和标准ＤＮＡ 采用ＰＣＲＳＳＰ 分型均获得成功，可准确分辨出Ｂ４０ 组所有等位基因。无假阳性和假阴性出现，重复率１００ ％ ，总耗时４ ～５ 小时。分型结果经双盲验证符合。临床应用结果显示： 三分之一的汉族人群存在Ｂ４０组抗原，Ｂ６０ 占绝大多数（６７ ．３ ％ ） ，血清学对Ｂ４０ 组的误差率达１０ ％ 。本方法比血清学更加准确， 对血清学分型存在困难、亚型分辨不清的纯合子或杂合子均能作出准确的分型。结论　本研究建立的ＨＬＡＢ４０ 组ＰＣＲＳＳＰ 高分辨度ＤＮＡ 分型方法， 分辨度高、特异性强、结果精确可靠、明显优于血清学方法，适合于临床应用。     

用顺序特异引物聚合酶链反应技术对HLA-B基因分型

目的采用顺序特异引物聚合酶链反应技术（ＰＣＲ－ＳＳＰ）建立汉族人群ＨＬＡ－Ｂ基因分型方法。方法肾移植供受者临床样本ＤＮＡ３１９份，Ｂ基因标准系列ＤＮＡ６２份。设计合成Ｂ座位５２个特异性引物和１对阳性对照引物，组成４１个ＰＣＲ反应，建立ＰＣＲ－ＳＳＰ方法。一步法完成对Ｂ座位的基因分型。结果经双盲验证，并与血清学方法比较。结果所有临床样本和标准ＤＮＡ，ＰＣＲ－ＳＳＰ基因分型均获得成功。可准确分辨ＨＬＡ－Ｂ座位等位基因４１个，实际检出汉族人群Ｂ抗原特异性３２个。无假阳性和假阴性，４０份样本的重复率１００％，总耗时５小时。分型结果经双盲验证完全符合。与血清学分型结果比较显示：７８份血清学空白中１７例存在第二个基因，２６份血清学分型结果错误。总误差率１３．５％。结论用ＰＣＲ－ＳＳＰ技术行ＨＬＡ－Ｂ基因分型，分辨率高、特异性强、重复性好、相对简捷快速，分型结果较血清学方法更加精确可靠，适合于临床应用。     

采用 PCR-SSP法检测HLA-B27基因

目的：建立顺序特异引物聚合酶链反应（ＰＣＲ－ＳＳＰ）方法检测ＨＩＡ－Ｂ２７基因并与血清学方法比较。方法：设计合成Ｂ２７特异物３个和内源性阳性对照２个,建立ＰＣＲ－ＳＳＰ方法,用于Ｂ２７基因分型。血清学分型为一步法单克隆抗估方法。临床样本１００份,不源于可疑强直性脊柱炎患者。快速酚氯仿法提取模板ＤＮＡ。结果：１００例临床样本ＰＣＲ－ＳＳＰ行Ｂ２７基因分型均获成功。总耗时４ｈ。其中Ｂ２７阴性５８例,阳     

用PCR—SSP作HAL—B位点分型及B22亚型分析

目的：用ＤＮＡ分型弥补血清学ＨＬＡ－Ｂ位点分型的欠缺并了解南方汉族Ｂ２２亚型分布情况。方法；采用标准细胞作参照，建立Ｂ位点的ＰＣＲ－ＳＳＰ分型方法地血清学Ｂ２２阳性标本进行Ｂ２２亚型分析。结果：１０４株标准细胞ＰＣＲ－ＳＳＰ分型结果与已知结果完全一致，１７例白血病人及同胞分型与血清学一致，１例不符；Ｂ２２亚型以３＾＊５４０１最多，Ｂ＾＊５６０１较少，１例分型格局异常，可能为新Ｂ２２等位基因或错配的     

HLA-A位点PCR-SSP基因分型和血清学分型的比较

目的：建立优化ＨＬＡ－Ａ位点ＰＣＲ－ＳＰ分型方法。方法：采用普通扩增仪和Ｅｐｐｅｎｄｏｒｆ管对细胞系ＤＮＡ和３７个健康正常人进行基因分型,血清学检测采用标准淋巴细胞毒试验方法。结果：发现引物浓度和浓度比、ＤＮＡ的纯度及酶的选用,是影响ＨＬＡ－Ａ位点ＰＣＲ－ＳＳＰ分型准确性的重要因素。该方法对３７个标本的基因分型结果与血清学结果相符合。结论：ＰＣＲ－ＳＳＰ方法具有简便准确的优点,可以作为ＨＬＡ－Ａ位     

用PCR—RFLP方法调查上海汉族人群中HLA—DP多态性

用聚合酶链反应－限制性内切酶片段长度多态性（ＰＣＲ－ＲＦＬＰ）技术对１５０名上海地区健康汉族人所作的ＨＬＡ－ＤＰ分型，结果在本群体中共检出２种ＤＰＡ１和１２种ＤＰＢ１等位基因。ＤＰＡ１０１和ＤＰＡ１０２频率分别为３１％和６９％。在ＤＰＢ１等位基因中，以ＤＰＢ１０５０１（３８．６７％），０２０１（１７．３３％），０４０２（１１．６７％）和０４０１（１０．６７％）最为常见。对不同人种中ＨＬＡ－ＤＰ分布特点加以比较，并对ＰＣＲ－ＲＦＬＰ技术在ＨＬＡ－ＤＰ分型应用中的优点进行了讨论。     

北方汉族过敏性紫癜与HLA相关性研究

为了研究过敏性紫癜（ＡＰ）的发病机理中是否有免疫遗传因素参与，采用国际通用的ＮＩＨ标准微量淋巴细胞毒试验方法检测４０例ＡＰ患者的ＨＬＡ－Ⅰ类抗原，并与１００例北方汉族正常人ＨＬＡ－Ⅰ类抗原频率进行比较。利用聚合酶链反应－序列特异性引物技术（ＰＣＲ－ＳＳＰ）对其中３０例ＡＰ患者进行ＨＬＡ－Ⅱ类基因分型，并与１０４例北方汉族正常人的ＨＬＡ－Ⅱ类基因频率进行了比较。发现ＡＰ患者ＨＬＡ－Ａ３０＋３１、Ｂ１３、Ｂ３５、Ｂ４０抗原频率较对照组明显增高（Ａ３０＋３１：Ｐｃ＜０．０１，ＲＲ＝７．９７；Ｂ１３∶ＰＣ＜０．０１，ＲＲ＝６．００，Ｂ３５∶Ｐｃ＜１０－５，ＲＲ＝１０．４０；Ｂ４０∶Ｐｃ＜０．０５，ＲＲ＝３．８５）。ＨＬＡ－ＤＲ１０基因频率在ＡＰ患者较正常对照组明显增高（ＤＲ１０∶Ｐｃ＜１０－５，ＲＲ＝２１．８８），而ＨＬＡ－ＤＱ３、ＤＱ６基因频率较正常对照组明显降低（ＤＱ３∶Ｐｃ＜１０－５，ＲＲ＝０．１３；ＤＱ６∶Ｐｃ＜０．０５，ＲＲ＝０．２３）。提示ＡＰ与ＨＬＡ－Ａ３０＋３１、Ｂ１３、Ｂ３５、Ｂ４０、ＤＲ１０正相关，与ＨＬＡ－ＤＱ３ＤＱ６负相关。     

中国人HLA—B27、B7、B13和B40的检测及其相互杂？…

目的 检测１１９４例初诊怀疑患强直性脊柱炎（ＡＳ）和其它关节性疾病患者的ＨＬＡ－Ｂ２７、Ｂ７、Ｂ１３和Ｂ４０位点抗原的分布，并分析这些位点的相互杂合和其抗原的交叉反应性。方法 微量细胞毒反应法，在同一块反应盘上同时检测。结果 １．在１１９４例患者中，ＨＬＡ－Ｂ２７抗原阳性率达３８．６％；Ｂ７的抗原阳性率为７．４％；Ｂ１３为１４．６％；Ｂ４０为１３．４％。２．在ＨＬＡ－Ｂ２７阳性人群中，Ｂ７抗原的阳     

Ⅰ型糖尿病患者的HLA-DR-DQ基因单体型分析

应用ＨＬＡ－ＤＲＢ，ＤＱＢ１序列特异性引物ＰＣＲ扩增方法，鉴定８１例ＩＤＤＭ患者，７个家系和８４例正常对照汉族人群的ＤＲＢ基因多态性及ＩＤＤＭ的ＨＬＡ－ＤＲ－ＤＱ基因单体型。结果表明：（１）ＩＤＤＭ患者ＤＲＢ１＊０３，ＤＲＢ１＊０９等位基因频率明显高于对照组，其频率分别为８．６４％ｖ．ｓ３．０％和２８．４％ｖ．ｓ１６．１（Ｐ＜０．０５）。（２）患者中ＤＲＢ１－ＤＲＢ３／ＤＲＢ１－ＤＲＢ４基因型频率明显高于对照组，即１９．８％ｖ．ｓ６．７％（Ｐ＜０．０５）。（３）首次证明ＤＲＢ１＊１２等位基因与中国人ＩＤＤＭ正相关。上述结果提示不同种族ＨＬＡ－ＤＲ型别分布的差异，可能是不同种族人群ＩＤＤＭ发病率变化的分子基础。     

胰岛素依赖型糖尿病患者的HLA—DQA1等位基因分析

应用聚合酶链反应与序列特异寡核苷酸探针杂交技术（ＰＣＲ／ＳＳＯＰ），分析了北京地区６１例汉族胰岛素依赖型糖尿病（ＩＤＤＭ）患者和５０例非糖尿病对照者的ＨＬＡ－ＤＱα链第５２位氨基酸的编码基因。结果表明：１．８９％的患者ＨＬＡ－ＤＱα链５２位是精氨酸，其编码基因为ＨＬＡ０ＤＱＡ１０３０１和（或）０５０１，其中１０．８％为０３０１纯合子，７０．３％为０３０１杂合子，１８．９％为０５０１纯合子；２．患者     

应用顺序特异性引物聚合酶链反应检测华南人群HLA-B座位第4外显子变异

目的：为探明中国华南人群中因HLA-B座位发生基因突变而引起表达变异存在的可能性。方法：用顺序特异性引物聚合酶链反应（PCR-SSP）对75例经血清血分型为纯合型样本再次分型，对有差异的结果使用PCR-SSP方法检测第四外显子突变情况。结果：75例血清学指定为纯合型标本经DNA分型证实有12例为杂合型。12例结果有差异的样本经PCR-SSP法表达变异的筛查发现有1例存在第四外显子额外胞嘧啶插入，经DNA分型发现的第二基因为沉默基因。结论：应用PCR-SSP方法可检测HLA-B等位基因突变，该突变导致HLA-B基因表达无效，进行HLA基因表达变异的检测可提高HLA分型的准确性，更加有效的指导临床进行器官和骨髓的选配。     

HLA-B27基因检定技术

用聚合酶链反应和血清学方法检测ＨＬＡ－Ｂ２７，并进行比较。方法：采用ＰＣＲ技术７８例可疑为强直性脊椎炎患者样本的ＨＬＡ－Ｂ２７基因，并与血清学比较。结果：３６例具有Ｂ２７基因者中２４例同时作血清学试验，５例血清学难以判定结果，并与ＰＣＲ结果不一致。     

血小板活化因子乙酰水解酶基因994(G→T)突变与脑梗塞的关联性

目的 研究血浆型血小板活化因子乙酰水解酶（platelet-activating factor-acetylhydrdase，PAF-AH）基因994（G→T）点突变与脑梗塞常见类型的相关性。方法 应用聚合酶链反应技术，检测108例脑梗塞患者（分为动脉粥样硬化性脑梗塞组、腔隙性脑梗塞组和心源性脑梗塞组）和215名正常对照中，血浆型PAF-AH基因该突变的基因型频率和等位基因频率。结果 对照组994（G→T）突变的基因型频率为20．46％（杂合子：18．60％；纯合子：1．86％）。全部脑梗塞患者人群中，突变基因型频率为35．19％（杂合子：32．41％；纯合子：2．78％），明显高于对照组（P〈0．01）；其中动脉粥样硬化性脑梗塞组该突变基因型频率为38．10％（杂合子：34．92％；纯合子：3．18％），与对照组相比差异有统计学意义（P〈0．01）。腔隙性脑梗塞组和心源性脑梗塞组的该突变基因型频率分别为t32．35％（杂合子：29．41％；纯合子：2．94％）和27．27％（杂合子：27．27％；纯合子：0）；与对照组比较差异无统计学意义（P〉0．05）。结论 血浆型PAF-AH基因994（G→T）T点突变与中国人脑梗塞的发生显著相关，其相关性主要来源于动脉粥样硬化性脑梗塞。而与腔隙性脑梗塞可能无明显关联。     

MICB polymorphisms and haplotypes with MICA and HLA alleles in Koreans

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) is located within the human MHC class I region. The location of MICB in the MHC region may imply the presence of linkage disequilibrium with polymorphic MICA and human leukocyte antigen (HLA) loci. MICB is also polymorphic; however, MICB polymorphisms have not been investigated in Koreans. Using sequence-based typing (SBT), we estimated the allelic frequencies of MICB and haplotypes with MICA, HLA-B, and HLA-DRB1 at high resolution in a population of 139 unrelated Korean individuals. Eight MICB alleles were identified. The most frequent allele was MICB*005:02/*010 (57.2%), followed by *002 (11.5%), *004 (8.3%), *005:03 (8.3%), and *008 (6.8%). The most common two-locus haplotypes were MICB*005:02/*010-MICA*010 (19.4%), MICB*005:02/*010-DRB1*15:01 (6.5%), and MICB*005:02/*010-B*15:01 (10.4%); the most common three-locus haplotypes were B*15:01-MICA*010-MICB*005:02/*010 (5.8%) and MICA*010-MICB*005:02/*010-DRB1*04:06 (10.4%); and the most common four-locus haplotype was B*15:01-MICA*010-MICB*005:02/*010-DRB1*04:06 (5.8%). This is the first study to provide information about MICB allele frequencies and haplotypes with HLA in Koreans. These study results should help understand mechanisms of disease association between the MICB locus and neighboring loci in Koreans.     

HLA antigen and gene frequencies in Eskimos of East Greenland

A population of 78 Ammassallik Eskimos was tested for HLA‐A, B, Cw, DR and DQ specificities using serological techniques and HLA‐Cw, DRB, DQB1 and DPB1 using three different genomic techniques. The application of two new genomic techniques for HLA‐Cw (PCR‐SSP phototyping) and HLA‐DPB1 (PCR‐RLFP) typing confirmed serological results and gave a higher resolution, with more heterozygotes than previously found. High gene frequencies of HLA‐A24 (0.80), B51 (0.21), B61 (0.30), B62 (0.21), Cw*0303 (0.27), Cw*0304 (0.51), DRB1*0401 (0.45), DRB1*1402 (0.24), DQ7 (0.88), DPB1*0201 (0.18), DPB1*0401 (0.40) and DPB1*0402 (0.30) were found. A limited number of alleles were found at all HLA loci. The Ammassallik Eskimos of East Greenland are genetically homogenous with little, if any, admixture with European populations. This contrasts with other Greenlandic populations formerly tested. The pattern of alleles indicates a close genetic relationship with other Eskimo populations.     

血清学指定HLA-B座位纯合型误差分析及HLA-B座位表达变异检测的意义

目的 :用DNA分型法对HLA B座位抗原纯合型进行分型 ,探明是否存在HLA B基因发生变异引起结果差异。方法 :经血清学鉴定HLA B座位纯合型样本 75例 ,用PCR SSP法进行DNA分型和表达变异筛查。结果 :75例血清学鉴定为纯合型后经DNA分型证实有 12例存在第二基因 ,经PCR SSP法进行变异筛查发现有 1例存在第四外显子额外胞嘧啶插入 ,经DNA分型发现的第二基因实为沉默基因。结论 :用PCR SSP法进行HLA B基因分型可弥补血清学方法的不足 ,同时筛查表达变异使分型结果更加准确 ,能有效地指导临床应用。     

Impact of High-Frequency HLA Haplotypes on Clinical Cytomegalovirus Reactivation in Allogeneic Hematopoietic Stem Cell Transplantation

Some studies support the hypothesis that HLA genes and haplotypes evolved by natural selection through their protective abilities against specific infectious pathogens. However, very little is known regarding the impact of high-frequency HLA haplotypes on the risk of relevant infectious diseases among a given ethnic group. We evaluated the impact of high-frequency HLA haplotypes on cytomegalovirus (CMV) reactivation and infection in allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a Japanese population as a model of infectious disease that has coexisted with humans. A total of 21,127 donor-patient pairs were analyzed. HLA-A-B-DRB1 haplotypes were estimated using the maximum probability algorithm. Seven haplotypes with >1% frequency were defined as high-frequency haplotypes (HfHPs). Homozygotes of HfHP and heterozygotes had significantly lower risk of CMV reactivation and infection (hazard ratio [HR] = 0.88, P = .009 and HR = 0.93, P = .003, respectively) than homozygotes of low-frequency HLA haplotypes (LfHPs). In subgroup analyses of a different donor source, these associations were statistically significant in unrelated donor transplants. Finally, CMV risk for homozygotes and heterozygotes of each HfHP was compared with that of homozygotes of LfHPs. The 2 most predominant HfHP groups (A*24:02-B*52:01-DRB1*15:02 group and A*24:02-B*07:02-DRB1*01:01 group) had a significantly lower risk of CMV reactivation and infection (HR = 0.86, P < .001 and HR = 0.91, P = .033, respectively). Our findings suggest that HfHPs may be protective against CMV reactivation and infection and that increased care regarding CMV reactivation and infection may be necessary for patients with LfHP after allo-HSCT.     

Allelic frequencies of the HLA-B17 antigen group: comparative analysis by serology, IEF and PCR-SSOP typing

Abstract: Current typing technology for class I HLA antigens uses serological and/or isoelectric focusing gel electrophoresis. DNA typing for the HLA class I antigens can accurately identify the class I genotype of individuals and cell lines. Here, we report correlation of DNA typing results with serological and IEF results for the B17 group. The B17 antigens are relatively common, being carried by almost 9% of Caucasians and 28% of blacks. In this study, five 10th International Histocompatibility Workshop cell lines carrying B17 and 106 individuals in 61 families carrying B17 were DNA typed for B17 using B17-allele-specific amplification and sequence specific oligonucleotide probe hybridization pattern analysis. 38 (55.07%) out of 69 unrelated haplotypes had B*5701, 23 (33.33%) had B*5801, 6 (8.70%) had B*5702, and 2 (2.90%) had B*5802. DNA typing results correlated well with serological and isoelectric focusing results. In general, there was high degree of agreement between all three methods, although heterozygosity for B17 poses a particular problem for serological and IEF methodology. Both B*5701 and B*5801 have the same electrophoretic mobility on IEF gel, corresponding to B17.2, B*5702 corresponds to B17.1, while B*5802 corresponds to B17.3.     

HLA antigen and gene frequencies in Eskimos of East Greenland.

A population of 78 Ammassallik Eskimos was tested for HLA-A, B, Cw, DR and DQ specificities using serological techniques and HLA-Cw, DRB, DQB1 and DPB1 using three different genomic techniques. The application of two new genomic techniques for HLA-Cw (PCR-SSP phototyping) and HLA-DPB1 (PCR-RLFP) typing confirmed serological results and gave a higher resolution, with more heterozygotes than previously found. High gene frequencies of HLA-A24 (0.80), B51 (0. 21), B61 (0.30), B62 (0.21), Cw*0303 (0.27), Cw*0304 (0.51), DRB1*0401 (0.45), DRB1*1402 (0.24), DQ7 (0.88), DPB1*0201 (0.18), DPB1*0401 (0.40) and DPB1*0402 (0.30) were found. A limited number of alleles were found at all HLA loci. The Ammassallik Eskimos of East Greenland are genetically homogenous with little, if any, admixture with European populations. This contrasts with other Greenlandic populations formerly tested. The pattern of alleles indicates a close genetic relationship with other Eskimo populations.     

Clarification of HLA-B serologically ambiguous types by automated DNA sequencing

Abstract: Assignment of HLA-B types can be hampered by ambiguous reactivity of the typing sera resulting in inaccurate HLA-B assignments. In this study, 19 Korean samples exhibiting ambiguous serologic reactivities were characterized by DNA sequencing. Alleles identified from 7 samples were previously undetected in this population (B*1517, B*4101, B*4701, B*5001, and B*5106) and from 9 samples were common alleles in this population (B*4002, B*4003, B*4006, B*1501, B*1401, B*67012, and B*5401). Three samples were putative HLA-B homozygotes. Three major factors causing serologic ambiguity were identified: weak or false negative reactivity of typing sera (52.4%); cross or false positive reactivity of the sera (38.1%); and absence of information on the reaction patterns due to the lack of appropriate sera in the typing kit (e.g. B*4101 encoded molecule) or to the presence of recently characterized molecules (e.g. B*5106 encoded molecule) (9.5%). Overall, sequencing was helpful in clarifying ambiguous serologic reaction patterns improving the HLA typing for the Korean population.