用PCR指纹图作HLA－DR快速配型的研究

ＰＣＲ指纹图技术选用通用型引物扩增ＨＬＡ－ＤＲＢ基因第二外显子，其产物在ＰＡＧＥ电泳中显示特定的“卫星”条带。可用于个体间作ＨＬＡ－ＤＲ的基因水平交叉配型，确定供受体间ＤＲＢ基因的相容性。采用ＨＬＡ纯合的Ｂ淋巴细胞系，分析单倍型上ＤＲＢＩ、ＤＲＢ３等基因异同时指纹图格局的变化，并通过１５对细胞间组合和比较，证实经ＤＮＡ交叉配合后的指纹图格局能十分敏感地反映个体间ＤＲ基因间的差异。在此基础上进一步对１７对随机个体作ＰＣＲ指纹图及交叉配型分析，表明这一技术在判别供受体间ＤＲ基因匹配性上十分有效，并有简单、快速、准确等优点。     

用PCR单链构象多态性快速判别个体间HLA－DP相容性并检出新等位基因

先确定和比较ＨＬＡ纯合细胞ＤＰＡＩ和ＤＰＢＩ基因ＰＣＲ扩增产物的单链构象多态性（ＰＣＲ－ＳＳＣＰ），然后分析了２０对无血缘关系个体单链ＤＮＡ及其两两混合状态下的构象多态性变化，在分子水平测定异体间ＤＰ匹配性，并和已有的ＤＰ基因分型（ＰＣＲ－ＲＦＬＰ）格局相比较，结果表明，ＲＦＬＰ不同的个体其ＳＳＣＰ必定不同；而ＲＦＬＰ相同的个体，ＳＳＣＰ一般相同，但有例外。从例外个体异常的ＳＳＣＰ格局中发现了新ＤＰ等位基因。本研究证实ＰＣＲ－ＳＳＣＰ在检测等位基因之间微小差异方面十分敏感，并具有简单、快速、经济等优点，对移植中的ＤＰ交叉配型有实际应用价值。     

应用微量SSP法进行器官移植供受者HLA-Ⅱ类基因分型

目的：探讨应用于临床器官移植的组织配型新技术。方法：采用快速ＤＮＡ抽提技术，建立微量ＰＣＲ－ＳＳＰ－ＨＬＡ－ＤＲＢ／ＤＱＢ基因分型方法，并与单克隆抗体一步法分型结果进行对比研究。结果：应用２种方法对７３份标本进行ＨＬＡ－Ⅱ类抗原／基因分型，结果完全相符，但微量ＰＣＲ－ＳＳＰ法不但快速，需血量少，而且分辨率高。结论：微量ＰＣＲ－ＳＳＰ法具有快速，准确，省血等优点，适合在临床器官移植配型中推广应用。     

一种新的HLA—II类基因分型方法——PCR／SSP技术的建立

ＰＣＲ／ＳＳＰ是一种新的基因多态性分析技术，可用于ＨＬＡ复合体及其他具有多态性特点２的基因分型。我室于１９９３年建立这种方法，并对正常人、重症肌无力患者、全身性红斑狼疮患者、骨髓移植供／受者进行了ＨＬＡ－ＤＲＢ１、－ＤＱＢ１基因型别分析。与其它ＨＬＡ基因分型方法相比，ＰＣＲ／ＳＳＰ具有简便、快速、准确及重量性好等特点。本文介绍ＰＣＲ／ＳＳＰ技术的原理、方法、特点以及常见问题的处理。     

PCR指纹法及PCR／SSO技术用于骨髓移植供者选择的比较研究

在１１个家系内进行了骨髓移植的配型工作。用ＰＣＲ分别扩增了供受才是最具有多态性的ＤＲＢ和ＤＱＢ基因的第二外显子，这些ＰＣＲ产物在非变性的聚丙烯酰胺凝胶电泳上显示具有单倍型特异性的，各不相同的带型格局，称为ＰＣＲ指纹。ＰＣＲ指纹和交叉配型具有简单、经济、灵敏等优点，对骨髓移植供才是的选择，有突出的优越性     

一种新的HLA─Ⅱ类基因分型方法——PCR／SSP技术的建立

ＰＣＲ／ＳＳＰ（Ｓｅｑｕｅｎｃｅｓｐｅｃｉｆｉｃｐｒｉｍｅｒ，序列特异性引物）是一种新的基因多态性分析技术，可用于ＨＬＡ复合体及其他具有多态性特点的基因分型。我室于１９９３年建立这种方法，并对正常人、重症肌无力患者、全身性红斑狼疮患者、骨髓移植供／受者进行了ＨＬＡ－ＤＲＢ１、－ＤＱＢ１基因型别分析。与其它ＨＬＡ基因分型方法相比，ＰＣＲ／ＳＳＰ具有简便、快速、准确及重复性好等特点。本文介绍ＰＣＲ／ＳＳＰ技术的原理、方法、特点以及常见问题的处理。     

广东汉族人类风湿关节炎易感性与HLA—DRB1基因相 …

探讨ＨＬＡ－ＤＲＢ１基因与类风湿关节炎相关性。方法 采用ＰＣＲ－ＳＳＰ方法对４７例广东汉族人ＲＡ患者进行ＨＬＡ－ＤＲＢ１基因分型，并与相应人群健康者１０２例结果比较。结果 ＨＬＡ－ＤＲ１基因在ＲＡ组显著增高，ＤＲ１６在ＲＡ组也高于正常；而ＥＲ９基因在ＲＡ组显著减少。     

HLA－DRB1等位基因与中国湖北汉族人SLE关联的研究

在国内首次借助ＰＣＲ／ＳＳＰ技术对５２例湖北汉族人ＳＬＥ患者进行了ＨＬＡ－ＤＲＢ１基因型别分析。结果发现，实验组ＨＬＡ－ＤＲＢ１＊０３０１基因频率为２４％，表型频率为４４．２％，ＲＲ＝４．７６，χ２＝２１．２，Ｐ＜０．０１；其它等位基因频率在实验组与对照组间差异无显著性。该结果提示ＨＬＡ－ＤＲＢ１＊０３０１基因与ＳＬＥ有关联     

中国胰岛素依赖性糖尿病病人（HLA－DR9）的HLA－DQB1基因顺序分析

用血清学方法研究显示中国人胰岛素依赖性糖尿病（ＩＤＤＭ）与ＨＬＡ－ＤＲ９相关。鉴于白种人中的研究显示ＩＤＤＭ与ＨＬＡ－ＤＱβ链第５７位氨基酸相关，Ａｓｐ－５７对ＩＤＤＭ呈抗性，ｎｏｎ－Ａｓｐ与ＩＤＤＭ易感性相关。我们用ＰＣＲ技术扩增了中国人中血清学ＤＲ９纯合的ＩＤＤＭ患者和正常对照的ＨＬＡ－ＤＱＢ１基因第二外显子并测定了核苷酸顺序，结果未发现ＩＤＤＭ特异ＨＬＡ－ＤＱＢ１等位基因，但发现ＩＤＤＭ病人ＨＬＡ－ＤＱＢ１５７位均为天冬氨酸。表明中国ＩＤＤＭ患者中的ＨＬＡ－ＤＱＢ１５７位天冬氨酸不一定具有保护个体抵抗ＩＤＤＭ的足够能力。ＩＤＤＭ易感性可能涉及多个基因位点的变化，另外还可能与其它遗传因素及环境因素有关。     

中国胰岛素依赖性糖尿病病人（HLA－DR9）的HLA－DQB1基因顺序分析

用血清学方法研究显示中国人胰岛素依赖性糖尿病（ＩＤＤＭ）与ＨＬＡ－ＤＲ９相关。鉴于白种人中的研究显示ＩＤＤＭ与ＨＬＡ－ＤＱβ链第５７位氨基酸相关，Ａｓｐ－５７对ＩＤＤＭ呈抗性，ｎｏｎ－Ａｓｐ与ＩＤＤＭ易感性相关。我们用ＰＣＲ技术扩增了中国人中血清学ＤＲ９纯合的ＩＤＤＭ患者和正常对照的ＨＬＡ－ＤＱＢ１基因第二外显子并测定了核苷酸顺序，结果未发现ＩＤＤＭ特异ＨＬＡ－ＤＱＢ１等位基因，但发现ＩＤＤＭ病人ＨＬＡ－ＤＱＢ１５７位均为天冬氨酸。表明中国ＩＤＤＭ患者中的ＨＬＡ－ＤＱＢ１５７位天冬氨酸不一定具有保护个体抵抗ＩＤＤＭ的足够能力。ＩＤＤＭ易感性可能涉及多个基因位点的变化，另外还可能与其它遗传因素及环境因素有关。     

HLA-DRB3 typing by restriction digestion of locus-specific amplified DNA

Locus HLA-DRB3 codes for the serologically defined supertypic specificity DRw52 in HLA-DR3, -5 and -w6 haplotypes. Three specificities of DRw52 (DRw52a, -b and -c) can further be distinguished by cellular techniques or by DNA typing with allele-specific oligonucleotide probes. These specificities were recently reported to have significant importance in antigen presentation. To avoid a time-consuming hybridization procedure, we have developed a simple typing system using PCR and subsequent digestion by allele-specific restriction endonucleases. A system was established with locus-specific amplification of HLA-DRB3 and digestion by the enzymes KpnI, ScaI and HinfI which recognize unique restriction sites within the amplified region. This allowed HLA-DRB3 typing on agarose gel by determining whether the amplification product has been digested or not. This typing system was compared to conventional oligotyping by analyzing 145 RFLP-typed individuals for their DRw52 specificity using both methods. Agarose typing correlated well with oligotyping and was shown to be more simple and practical even in heterozygous individuals.     

HLA-DR and DQB1 gene polymorphism in the North-western Colombian population

HLA-DRB1, DRB3, DRB4, DRB5 and DQB1 polymorphisms were studied using molecular methods in a population of 100 unrelated healthy individuals from an area in north-west Colombia (Medellin) inhabited by the "Paisa", a community with features of a genetically isolated group. The most frequently observed specificities at the DRB1 locus were *07 (16.4%) and *15 (12%), and at the DQB1 locus *02 (18.8%) and *03 (33.6%), of which *0302 was the most prevalent allele (14.3%). The most polymorphic specificities were DRB1*04, 13 and 11, and DQB1*06. Both the HLA-DRB1 and DQB1 loci were in linkage disequilibrium. Haplotypes were estimated using maximum likelihood methods. The most frequent two locus haplotype was DRB1*07-DQB1*02 (6.6%) and these specificities were in linkage disequilibrium. Several unusual possible haplotypes were observed. Both the HLA-DRB1 and DQB1 locus were in Hardy-Weinberg equilibrium.     

The nature of polymorphism of the HLA-DRB intron sequences is lineage specific

Abstract: The sequence database of HLA-DRB genes is mainly derived from mRNA analysis or has focused exclusively on the polymorphism of the 2nd exon. Little is known about the non-coding sequences of the different DRB alleles which represent about 94% of the genes. In this study we have determined the sequence of the 3' 500 bp intron 1 fragment adjacent to exon 2 in all serologically defined HLA-DRB genes and their most frequent allelic subtypes. The intron sequences turned out to be highly polymorphic. Similar to the class I introns, this variability was not characterized by random point mutations but by a highly systematic diversity reflecting the lineage-specific relationship of the HLA-DR alleles. With a few exceptions in DRB1*15, 13 and 08 as well as DRB 4 and 5, the variability mirrors the serological diversity. As well as delivering insight into the genetic relationship between the different DRB alleles, these sequences will provide an extremely valuable basis for developing advanced DRB sequencing strategies for clinical purposes.     

HLA-DRB1基因与子痫前期相关性研究

目的探讨HLA-DRB1基因与子痫前期相关性。方法采用序列特异性引物技术（PCR—SSP）对119例子痫前期患者、117例正常孕妇及其新生儿进行HLA-DRB1等位基因分型，比较其基因频率。结果共检出13种等位基因，两组孕母或两组新生儿间，其HLA-DRB1等位基因频率差异无统计学意义（Pc〉0．05）；子痫前期组和正常晚孕组母婴所携带HLA-DRB1＊014、-DRB1＊10、-DRB1＊14等位基因配伍差异有统计学意义（P〈0．05）。结论母婴所携带某些HLA-DRB1基因配伍与子痫前期易感性或抗性相关；父源性HLA-DRB1＊014基因与子痫前期易感性相关。     

Shared class II MHC polymorphisms between humans and chimpanzees

To gain an insight into the evolution of the major histocompatibility complex alleles, three DRB and one DRA genes were isolated from chimpanzee cDNA libraries. The nucleotide sequences of the chimpanzee DRB (ChLA-DRB) genes were then compared with those of the available HLA-DRB alleles by constructing unrooted phylogenetic trees. All three ChLA-DRB genes were found to be more closely related to certain HLA-DRB alleles than unrelated HLA-DRB alleles are to each other. Since available evidence does not support the convergent evolution of MHC alleles, this result is consistent with the idea that closely related ChLA-DRB and HLA-DRB alleles are derived from common ancestral alleles, the existence of which predates the divergence of human and chimpanzee lineages. The predicted amino acid sequences of mature ChLA-DRA and HLA-DRA molecules differ by only one amino acid.     

HLA class II DRB high resolution genotyping by pyrosequencing: comparison of group specific PCR and pyrosequencing primers

Sequencing of alleles of the highly polymorphic, multiple loci HLA-DRB gene family was performed by pyrosequencing using purified DNA from the 11(th) International Histocompatibility Workshop human lymphoblastiod cell lines as well as genomic DNA isolated from blood samples obtained from healthy adult volunteers. Genomic DNA was prepared from donors whose blood had been stored either frozen or as dried blood spots. Pyrosequence-based typing was optimized for identifying alleles of the HLA-DRB1, -3, -4, and -5 genes. The procedure should be applicable to other HLA loci including the class I genes HLA-A and -B that, along with HLA-DRB, are crucial for histocompatibility matching of tissue antigens during transplantation. Computer simulation of pyrosequencing data suggest that alleles of HLA-DRB1, -3, -4, and -5 were readily identifiable by pyrosequencing as were their heterozygous allelic combinations. Pyrosequencing primers were designed to specifically sequence HLA loci of interest even in a background of other amplified, closely related sequences such as alleles of the pseudogene HLA-DRB6, -7, -8, and -9. Polymorphic residues of HLA-DRB genes were identified within each pyrosequencing reaction, obtained by 50 to 70 nucleotide read lengths. Heterozygous allelic combinations of HLA genes were analyzed and compared successfully to genotyping of alleles by sequence-specific oligonucleotide probe hybridization as well as allele specific polymerase chain reaction protocols. Pyrosequence-based typing is compatible with genotyping of allelic combinations expected from heterozygous individuals, resulting in nucleotide resolution of the highly polymorphic HLA system. Using a single pyrosequence instrument, complete typing of HLA-DRB genes can be performed daily on hundreds of individuals for high resolution histocompatibility genotyping studies.     

HLA-DRB3 gene alleles in Caucasian patients with Graves' disease

Summary Graves' disease (GD) is a human leukocyte antigen (HLA) linked organ-specific autoimmune disease. In German GD patients the disease is associated with HLA specificities of the HLA-DRw52 family (HLA-DR3, -DR5, and DR6; HLA-DRB3 positive HLA haplotypes). Recently, a strong association with a HLA-DRB3 restriction fragment length polymorphism gene has been described. To study HLA-DRB3 alleles and their association with the disease, a large cohort of controls (n = 3724) and GD patients (n = 304) was analyzed. HLA-DR allelic combinations revealed an increase in HLA-DR3/DR5 heterozygous patients (relative risk 2.9; P<0.001). HLA-DRB3 alleles, as defined by DNA typing in HLA-DR matched groups revealed a significant increase in DRB3^*0101 homozygosity (relative risk 17.5; P< 0.001) in HLA-DR3 homozygous patients. In GD patients with ophthalmopathy (grade II or higher, according to Werner) DRB3^*0101/^*0202 heterozygosity revealed an increased relative risk of 5.5 (P<0.001). Non-HLA-DR3 homozygous, DRB3^*0101/^*0202 heterozygous patients were at the highest risk for endocrine ophthalmopathy (relative risk 10; P<0.001). Our data, based on DNA typing methods of HLA-D genes, provide evidence that the susceptibility is strongly associated with HLA-DRB3 genes.Abbreviations EF etiological fraction - GD Graves' disease - HLA human leukocyte antigen - RR relative risk     

Generic HLA-DRB1 gene oligotyping by a nonradioactive reverse dot-blot methodology.

HLA-DRB1 allelic specificities can be determined using SSOs annealing to their complementary PCR-amplified target DNA. To perform HLA-DR oligotyping routinely for donors and recipients of bone marrow transplantation, a "reverse" dot-blot technique has been developed that consists in the hybridization of labeled PCR-amplified target DNA to SSOs that have been first attached to nitrocellulose membranes. The 15 oligonucleotides chosen enabled the following HLA-DRB1 "generic" specificities to be defined: DR1, BON, 2, 3, 4, 11, 11 JVM, 12, 13, 13 HAG, 14, 7, 8, 9, 10. The genomic DNA was amplified by asymetric PCR with incorporation of biotinylated deoxynucleotides predominantly to generate labeled single-stranded DNA. Hybridization between specific immobilized oligoprobes and target DNA was nonradioactively detected by a colorimetric reaction using alkaline phosphatase. The reverse dot-blot methodology was successfully tested, first, for the determination of HLA-DR4 subspecificities, and then the procedure was routinely applied to the generic HLA-DR oligotyping of bone-marrow donors and recipients.     

Description of a polymorphism in the regulatory region of the HLA-DRA gene.

It was previously reported that the cell membrane expression of HLA class II molecules is tissue specific and variable among individuals. This variation could be explained at the level of gene regulation. Due to the fact that a coordinate regulation of the HLA-DR genes seems to exist, we focused on the HLA-DRA monomorphic gene. In order to study the polymorphism of the HLA-DRA gene regulatory region, we used restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified genomic DNA. The DdeI and PvuII digestions of the amplified DNA permitted the definition of the two alleles according to the absence (allele A) or the presence (allele B) of the two polymorphic restriction sites. The presence of these sites was confirmed by direct sequencing after PCR. Using homozygous typing cells, a close relationship between the HLA-DRB coding region and the HLA-DRA regulatory region polymorphisms was shown. Furthermore, a linkage between the HLA-DRA regulatory region and DRB3 gene coding region polymorphisms could be established. These results suggested a structural argument for different levels of HLA class II genes and, consequently, of cell-surface expression of class II antigens according to the allelic specificities of DRA, DRB1, and DRB3.