人白细胞抗原DQB1基因与1型糖尿病相关性研究

目的　研究四川地区汉族人群人类白细胞抗原 (humanleucocyteantigen ,HLA)DQB1基因与 1型糖尿病 (type 1diabetesmellitus ,T1DM )发病年龄及糖尿病自身抗体的相关性。 方法　应用聚合酶链反应 序列特异性引物方法对 46例T1DM患者和 5 2名正常人进行HLA DQB1基因分型 ,并对T1DM患者以酶联免疫吸附法定性检测谷氨酸脱羧酶抗体 (glutamicaciddecarboxylaseantibody ,GADA)及抗胰岛细胞抗体 (isletcellantibody ,ICA)。结果　DQB1 0 2 0 1基因阳性率T1DM组高于对照组 (OR =18,P <0 .0 0 5 ) ;而DQB1 0 60 1、 0 60 2基因阳性率对照组高于T1DM组 (OR分别为 0 .0 7、0 3 1,P <0 .0 5 )。DQB1 0 60 2基因阳性率在发病年龄≥ 2 0岁T1DM组高于 <2 0岁组 (P <0 .0 5 )。携带DQB1 0 2 0 1基因的患者中 ,GADA阳性率明显高于此基因阴性的患者 (P <0 .0 2 5 )。结论　四川地区汉族人群中DQB1 0 2 0 1可能是 1型糖尿病的易感基因 ,而DQB1 0 60 2、 0 60 1是保护性基因 ,且DQB1 0 60 2基因的存在有可能推迟 1型糖尿病的发病 ;DQB1 0 2 0 1的阳性率与GADA阳性率正相关。     

山东地区汉族人 HLA-DPB1基因单核苷酸多态性

使用第十一届国际组织相容性会议提供的HLA－DPB1引物序列，由PCR技术扩增人HLA－DPB1基因第二外显子。产物纯化后，直接核酸序列分析确定个体的HLA－DPB1外显子核酸序列。使用基因定型软件与国际上公布的HLA－DPB1核酸序列构建的基因型核酸序列数据库进行比较，确定个体的基因型别、确定等位基因类型。研究结果提示中国人HLA－DPB1第二外显子基因序列与国际上公布的序列基本一致，但有不同之处，多处存在单核苷酸多态性（SNP），提示存在有新的等位基因。对51例个体研究显示，出现频率最高的等位基因是DPB1＊02012。     

人白细胞抗原DQB15′—调控区序列与Ⅰ型糖尿病的关联

应用聚合酶链反应（ＰＣＲ）、ＰＣＲ／ＳＳＣＰ和克隆测序等方法比较分析汉族１－型糖尿病患者与正常对照者ＨＬＡ－ＤＱＢ１５′－调控区多态性。结果显示携带不同等位基因的患者与对照者ＤＱＢ１５′－调控区ｙ、ｓｂｏｘ核苷酸序列相同,且与白种人基因结构一致;ｙｂｏｘ核苷酸序列存在二各结构,ＣＣＴＡＧＡＧＡＣＡＧＡＴＴ序列常常与ＤＱ１＊０３２等位基因在同一单倍型;转录起始位点至ｙｂｏｘ间－４４至－６１位存在多态     

用聚合酶链反应-直接测序分型法研究江苏汉族人群HLA-DQA1和DQB1基因多态性

目的检测江苏地区汉族人群HLA-DQA1和DQB1等位基因及单倍型的频率，分析该人群DQA1、DQB1基因多态性和DQA1-DQB1单倍型特点。方法应用聚合酶链反应-直接测序分型法（PCR-sequence-based typing ，PCR-SBT）方法对100名健康、无血缘关系的江苏汉族人群的HLA-DQA1和DQB1进行基因分型。结果共检出7个DQA1等位基因和13个DQB1等位基因。DQA1等位基因中，DQA1＊0301／02／03的基因频率最高（29．5％），其次为DQA1＊0501（18．5％）、DQA1＊0102（17．0％）、DQA1＊0201（12．5％）；DQB1等位基因中，DQB1＊0201／02（21．5％）、DQB1＊0301／09（14．5％）、DQB1＊0303（13．5％）和DQB1＊0603（11．5％）最为常见。分析得出30种DQA1-DQB1单倍型，DQA1＊0301／02／03-DQB1＊0303（12．5％）、DQA1＊0201．DOB1＊0201／02（10．5％）、DQA1＊0501-DQB1＊0201／02（9．5％）、DQA1＊0501-DQB1＊0301／09（7．0％）为常见的单倍型。结论江苏汉族人群HLA-DQA1和DQB1基因具有较为丰富的多态性，基因频率分布具有中国北方群体的特征且具有一定的独特性。     

浙江汉族人群HLA-DPA1和-DPB1基因多态性分析

目的 检测浙江地区汉族人群HLA-DPA1和HLA-DPB1等位基因及单倍型频率.方法 应用PCR-直接测序分型法对100名健康、无血缘关系的浙江汉族人外周血标本进行HLA-DPA1和HLADPB1等位基因分析.结果 在100份标本中检出8个HLA-DPA1等位基因和19个HLA-DPB1等位基因.HLA-DPA1等位基因频率较高的依次为DPA1*020202(47.0%)、DPA1*010301(38.5%)和DPA1*020101(10.5%).HLA-DPB1等位基因中,频率较高的依次为DPB1*0501(39.5%)、DPB1*020102(13.5%)和DPB1*040101(13.0%).连锁分析发现共有44个HLA-DPA1-DPB1单倍型,单倍型频率最高为DPA1*020202-DPB1*0501(29.5%).结论 浙江地区汉族人群HLA-DPA1和DPB1基因座等位基因具有丰富的多态性,2个基因座呈现连锁不平衡.     

浙江汉族人群HLA-DPA1和-DPB1基因多态性分析

目的 检测浙江地区汉族人群HLA-DPA1和HLA-DPB1等位基因及单倍型频率.方法 应用PCR-直接测序分型法对100名健康、无血缘关系的浙江汉族人外周血标本进行HLA-DPA1和HLADPB1等位基因分析.结果 在100份标本中检出8个HLA-DPA1等位基因和19个HLA-DPB1等位基因.HLA-DPA1等位基因频率较高的依次为DPA1*020202(47.0%)、DPA1*010301(38.5%)和DPA1*020101(10.5%).HLA-DPB1等位基因中,频率较高的依次为DPB1*0501(39.5%)、DPB1*020102(13.5%)和DPB1*040101(13.0%).连锁分析发现共有44个HLA-DPA1-DPB1单倍型,单倍型频率最高为DPA1*020202-DPB1*0501(29.5%).结论 浙江地区汉族人群HLA-DPA1和DPB1基因座等位基因具有丰富的多态性,2个基因座呈现连锁不平衡.     

浙江汉族人群HLA-DPA1和-DPB1基因多态性分析

目的 检测浙江地区汉族人群HLA-DPA1和HLA-DPB1等位基因及单倍型频率.方法 应用PCR-直接测序分型法对100名健康、无血缘关系的浙江汉族人外周血标本进行HLA-DPA1和HLADPB1等位基因分析.结果 在100份标本中检出8个HLA-DPA1等位基因和19个HLA-DPB1等位基因.HLA-DPA1等位基因频率较高的依次为DPA1*020202(47.0%)、DPA1*010301(38.5%)和DPA1*020101(10.5%).HLA-DPB1等位基因中,频率较高的依次为DPB1*0501(39.5%)、DPB1*020102(13.5%)和DPB1*040101(13.0%).连锁分析发现共有44个HLA-DPA1-DPB1单倍型,单倍型频率最高为DPA1*020202-DPB1*0501(29.5%).结论 浙江地区汉族人群HLA-DPA1和DPB1基因座等位基因具有丰富的多态性,2个基因座呈现连锁不平衡.     

昆明白族儿童HLA-DRB1、DQB1位点基因多态性分析

主要组织相容性复合体（MHC）是染色体上的一群紧密连锁的基因群，可作为一个遗传单彰单倍型而遗传，属共显性等位基因。人类的MHC称人类白细胞抗原（HLA）系统，位于人的第6对染色体短臂上。HLA-DR、DQ抗原是异基因移植免疫反应中最重要的抗原之一，决定其特异性的主要编码基因DRB1、DQB1具有高度多态性，其对HLA与疾病相关性及人类遗传学等研究均具有重要意义。     

人类白细胞抗原HLA-DPA1和HLA-DPB1基因高通量测序分型的研究

目的 建立可靠的人类白细胞抗原(human leukocyte antigen,HLA)基因HLA-DPA1和HLA-DPB1同步测序分型方法,研究南方汉族人群HLA-DPA1和HLA-DPB1基因的多态性.方法 根据HLA-DPA1和HLA-DPBI等位基因的全长序列,分别采用位点特异性引物扩增HLA-DPA1和HLA-DPBI目的基因片段,涵盖完整的第2外显子.PCR产物经磁珠纯化,用自行设计的测序引物及优化的测序反应体系对HLA-DPA1和HLA-DPB1第2外显子进行双向测序.纯化的测序反应产物经ABI 3730测序仪测序,用Assign 3.5 SBT软件分析结果.结果 PCR扩增获得了清晰的HLA-DPA1和HLA-DPB1基因目的片段,对HLA-DPA1和HLA-DPB1基因的第2外显子进行的双向测序结果中,序列无背景信号和杂峰.在176名南方汉族健康无关个体中,共检出了4种HLA-DPA1等位基因,其频率分布依次为:DPAI* 02:02(0.589)＞DPAl* 01:03(0.284)＞ DPAI* 02:01(0.096)＞ DPAl* 04:01(0.031).HLA-DPB1检出了14种等位基因,其中频率大于5％的4种等位基因为:DPBl* 05:01、DPBl* 02:01、DPBl* 04:01和DPBl* 02:02,频率介于1％～5％的7种,其余3种等位基因的频率均为1％以下.HLA-DPB1测序分型的结果与用AtriaAlleleSEQR商品化测序分型试剂盒检测的结果一致.结论 建立了HLA-DPA1及HLA-DPB1测序分型的方法,在群体遗传学及疾病关联研究等领域具有广泛的实用价值.     

慢性鼻窦炎伴鼻息肉与HLA-DPB1、DQB1基因相关性研究

目的:探讨慢性鼻窦炎伴鼻息肉患者在HLA-DPB1、DQB1两个基因位点的易感因素及保护性因素,初步探讨HLA-DPB1、DQB1两个基因与慢性鼻窦炎伴鼻息肉的相关性,寻找易感基因类型.方法:通过对HLA-DPB1、DQB1两个基因位点进行PCR扩增,用ABI 3700自动测序仪对扩增产物进行测序,从而对46例广东籍慢...     

Sequenase sequence profiles used for HLA-DPB1 sequencing-based typing

Abstract: Sequencing-based HLA typing (SBT) is a PCR based high resolution HLA typing method in which polymorphic regions of the gene are sequenced and directly used for typing. Currently, for class II SBT, alleles are identified by comparison of the exon 2 sequence with their corresponding allele sequence library. Routine SBT requires reliable identification of heterozygosity, and automated assignment of the alleles. In sequencing strategies different enzymes can be used for primer extension. The most characteristic difference between sequences obtained by two protocols using Sequenase®, or Taq-cycle sequencing, respectively, is a difference in incorporation of nucleotides in the primer extension leading to different sequence profiles. In Taq-cycling sequencing variable nucleotide incorporation results in irregular, but reproducible peak patterns, whereas Sequenase® incorporates nucleotides in nearly equal amounts, resulting in more even peak patterns. In a previously published multi-center study we evaluated HLA-DPB1 SBT using Taq-cycle sequencing, and showed that typing can reliably be performed, considering the specific sequence profiles. In this study the applicability of Sequenase® for HLA-DPB1 SBT was tested. A panel of samples were typed by SBT at five test sites which participate in the Sequencing Based Typing component of the 12^th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. The assignment of homozygosity and heterozygosity was validated by Multi-Sequence Analysis, performing cluster analysis of chromatographic data of all sequences at each position. Sequence characteristics were examined and considered for appropriate assignment. Data reveals that Sequenase® sequencing can also reliably be used for HLA-DPB1 typing.     

Sequencing-based typing reveals new insight in HLA-DPA1 polymorphism

Abstract: An HLA-DPA1 sequencing-based typing (SBT) system has been developed to identify DPA1 alleles. Up to now eight DPA1 alleles have been defined. Six can be discriminated based upon exon 2 polymorphism. The three subtypes of DPA1*01: DPA1*0101, DPA1*0102 and DPA1*0103, have identical exon 2 sequences but show differences in exon 4. Exon 4 sequences were known for only the three DPA1*01 subtypes and for DPA1*0201. We now present additional sequence information for exon 4 and the unknown segments at the 3' end of exon 2. Additionally with the use of this sequencing technique it is also possible to identify previously unidentified polymorphism. We have studied the exon 2 and exon 4 polymorphism of DPA1 in 40 samples which include all known DPA1 alleles. A new allele, DPA1*01 new, was identified which differs by one nucleotide in exon 2 from DPA1*0103, resulting in an aspartic acid at codon 28. The DPA1*01 subtypes DPA1*0101 and DPA1*0102 could not be confirmed in samples which previously were used to define these subtypes, and consequently they do not exist. The exon 4 sequence of DPA1*0201 is corrected based on sequence data of DAUDI, the cell line in which DPA1*0202 was originally defined. The exon 4 regions of the remaining four alleles were resolved: the exon 4 regions of the alleles DPA1*02021 and DPA1*02022 were found to be identical to the — corrected — DPA1*0201 whereas the exon 4 region of DPA1*0301 differs by one nucleotide compared to DPA1*0103. The DPA1*0401 exon 4 region differs by one nucleotide compared to the corrected DPA1*0201. As is found in other class II genes, all exons of DPA1 show some polymorphism, but the polymorphism in exon 2 is sufficient to identify the different alleles.     

An evaluation of a multicenter study on HLA-DPB1 typing using solid-phase Taq-cycle sequencing chemistry

Abstract: In HLA Class II genes, polymorphism is mainly located in the second exon. Most DNA based typing methods are confined to the identification of specific sequence motifs in the second exon. In contrast, Sequencing Based Typing (SBT) elucidates the entire exon 2 sequence for typing. Comparison of the obtained exon 2 sequence with an allele sequence library results in allele assignment. We tested the applicability of SBT using a protocol for amplification followed by solid phase Taq-cycle sequencing for HLA-DPB1 typing. A panel of 32 samples were typed by SBT at five test sites which are participating in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. In this multicenter study we focused on the reliability of analyzing heterozygous sequences for HLA typing. A multi-sequence analysis approach, Polall, was developed to evaluate sequences obtained. The assignment of homozygosity and heterozygosity was validated by cluster analysis of chromatographic data of all sequences. Sequence characteristics were examined and considered for appropriate assignment. Differences in sequence characteristics that occurred between the test sites are considered in detail. The evaluation of data of 5 test sites reveals that Taq-cycle sequencing can reliably be performed for HLA-DPB1 SBT.     

人白细胞抗原-DPB1基因直接测序分型技术

研究建立准确的基于人白细胞抗原一ＤＰＢ１（ＨＬＡ－ＤＰＢ１）核酸序列的基因分型技术,为疾病与ＨＬＡ基因相关性研究及器官、组织移植提供准确的ＨＬＡ基因分型方法。使用第十一届国际组织相容性会议提供的引物,经ＰＣＲ技术扩增、分离相应的基因片段,纯化后用ＰＥ公司四色荧光染料终止剂标记循环测序技术直接测定核酸序列,获得个体基因型序列资料,通过与基同型资料数据库比较确定基因型别。这一技术可以准确地确定个体的 ＨＬＡ－ＤＰＢ１的基因型别并得到核酸序列。为进一步研究奠定了基础。本技术可用于其它类似的研究工作。     

Sequence-based typing of HLA class II DQB1

Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.     

Crystallization of oral fluid components in patients with type 1 diabetes mellitus

We systematized and described morphological criteria characterizing crystalline aggregates in mixed saliva from patients with type 1 diabetes mellitus.__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 22–24, January, 2005     

HLA-DPB1 typing by polymerase chain reaction amplification with sequence-specific primers

DPB1 is the second most polymorphic class II locus with currently 84 recognized alleles, i.e. DPB1*0101 to DPB1*8101. Most of the alleles have been described during the last few years using oligonucleotide and sequencing techniques and relatively little is known about the role and importance of the polymorphic residues as regards to the function of DP molecules. In the present study, polymerase chain reaction (PCR) primers were designed for identification of all the phenotypically different DPB1 alleles by PCR amplification with sequence-specific primers. Forty-eight standard genomic PCR reactions per sample were performed in order to achieve this resolution. Unique amplification patterns were obtained in 2983 of 3160 (94.4%) possible genotypes. The primers were combined so that only very rare genotypes gave rise to ambiguous patterns. Sixty-four Histocompatibility Workshop cell lines and 150 DNAs provided by the UCLA DNA exchange were investigated by the DPB1 primer set. All typing results were conclusive. Analysis of the distribution of DPB1 alleles was performed in 200 Caucasian samples, 100 African samples and 40 Oriental samples. The population study by the DPB1 PCR-SSP method showed a characteristic distribution of HLA-DPB1 alleles. Each ethnic group had one, or two, frequent DPB1 allele(s) and the frequency of homozygotes was high, suggesting that balancing selection does not appear to be affecting the evolution of the DPB1 locus.     

Two new HLA-DQB alleles routinely identified by sequence-based typing: DQB1*030103 and DQB1*0505

Here, we report the identification of two novel human leukocyte antigen-DQB1 alleles, DQB1*030103 and DQB1*0505, found by routine typing using commercial kits.     

DNA methylation and mRNA expression of HLA‐DQA1 alleles in type 1 diabetes mellitus

Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA‐DQA1 gene. Our aim was to investigate DNA methylation of HLA‐DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA‐DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA‐DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA‐DQA1 promoter alleles was analysed. Individual mRNA HLA‐DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA‐DQA1 allele 02:01 expression (PPIA normalization, P_corr = 0·041; DRA normalization, P_corr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA‐DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA‐DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA‐DQA1 allele in patients with T1D have been described for the first time.