肌萎缩侧索硬化症家系铜锌超氧化物歧化酶基因突变位点的检测

目的：检测一个具有特殊临床表型的肌萎缩侧索硬化症（ALS）家系的铜、锌超氧化物歧化酶（SOD1）基因突变位点,同时比较单链构象多态性（SSCP）和变性高效液相色谱法（DHPLC）的实用价值.方法：采用SOD1基因的5对引物对部分家系成员基因组DNA进行PCR扩增,分别采用SSCP和DHPLC分析各外显子的多态性,异常样本进行DNA直接测序.结果：①两种方法均发现家系成员Ⅱ5、Ⅲ1、Ⅲ3、Ⅲ13、Ⅲ20、Ⅳ1、Ⅳ20的第2外显子和家系成员Ⅲ1、Ⅲ11、Ⅲ20第5外显子存在突变,直接测序证实第2外显子编码区和第5外显子非编码区均插入了一个碱基A.②DHPLC还发现家系成员Ⅲ1的第4外显子有杂合子色谱,直接测序证实其第4外显子存在杂合子,发生了GAA→GGA错义突变.结论：①该家系SOD1基因第2、4、5外显子均存在突变,第2外显子的突变可能是引起该家系发病的原因.②DHPLC技术与PCR-SSCP相比有更高的敏感性,可作为大样本筛查突变位点的一种便捷、可靠的手段.     

CRYGD基因突变与一中国先天性核性白内障家系致病相关

目的对一先天性核性白内障家系进行候选致病基因的筛查,以明确其发病分子基础。方法以家系先证者基因组DNA为模板,聚合酶链反应(PCR)扩增10个白内障候选致病基因的突变热点区域,PCR产物纯化后进行直接DNA测序筛查突变位点。如发现疑似突变位点,则利用直接DNA测序法对该突变位点在家系其他成员的存在情况进行疾病表型与致病突变共分离分析,进一步确认其是否为致病性突变位点。结果该家系三代呈常染色体显性遗传,临床表型大致相同,可确诊为先天性核性白内障。候选致病基因筛查发现在患者的CRYGD基因外显子2发现一个杂合突变c.C70A,正常家系成员无此突变,临床表型与基因型共分离。该突变为错义突变,导致一个CRYGD蛋白第24位的脯氨酸被苏氨酸取代(p.P24T)。结论 CRYGD(p.P24T)突变是导致该先天性核性白内障家系的致病分子基础。     

变性高效液相色谱分析法在人类基因组单核苷酸多态性检测中的应用

目的:检测白种人和汉族人RDH8基因序列中单核苷酸多态性(SNP)位点,探讨变性高效液相色谱分析法(DHPLC)在基因组研究中的效能.方法:建立8个由白种人和汉族人基因组DNA组成的混合样品池,并进行PCR扩增.其产物以DHPLC检测,发现杂合子信号后,对单独DNA样本进行DHPLC检测及直接测序确定其突变类型.以DHPLC结合DNA样本混合技术在150个汉族人和20个欧洲白种人样本中测定SNP的基因频率.结果:在RDH8基因中共检测到17个SNP,其中12个为新发现的,5个为已报道的;11个SNP在筛查阶段发现,6个在基因频率检测阶段发现;2个仅在白种人样本中检测到,3个SNP的MAF在两种人种间有较明显的差异;汉族人基因频率检测确定了7个常见SNP.结论:DHPLC能有效地用于基因组SNP的检测,结合DNA混合技术,更能提高其检测效力,有效地应用于基因频率的测定.     

线粒体DNA13731点突变与脊髓小脑性共济失调相关性研究

目的探索线粒体DNA（mtDNA）突变位点与脊髓小脑性共济失调（SCA）的关系。方法采用聚合酶链反应（PER）对基因确诊的四个SCA家系10例患者及其亲属共34例与40例健康对照的线粒体ND5基因片段进行扩增,扩增产物进行单链构象多态性分析（SSCP）,对SSCP出现异常的样本进行相应mtDNA片段测序。结果在一家系的1名确诊患者及1名症状前患者检测到mtDNA13731（T〉C）点突变。结论脊髓小脑性共济失调的发生、发展可能与mtDNA突变有关。     

用变性高效液相色谱法检测遗传性因子XⅢA链基因突变

目的:观察变性高效液相色谱(DHPLC)法检测凝血因子XIIIA链基因突变的实用价值。方法:用聚合酶链反应(PCR)扩增正常样品和有XIIIA基因603-606delAAGA突变患者的XIIIA链基因外显子5;用单链构象多态性(SSCP)及DHPLC检测分析扩增的外显子5。结果:SSCP显示患者有异常的区带,患者的DHPLC结果出现异常洗脱峰,与SSCP的结果一致。结果表明DHPLC能检测FXIII因子A链基因突变。结论:DHPLC技术是一种筛查基因突变的快速、简单、可靠的方法。     

变性高效液相色谱法筛选鼻咽癌相关基因单核苷酸多态性

目的　利用变性高效液相色谱法（DHPLC）结合测序筛选和鉴定鼻咽癌基因的单核苷酸多态性（SNPs）。方法　PCR扩增30例鼻咽癌患者和28例正常对照者6p21.3区域基因PPP1R11、PP1R10、FLOT1、KIAA0170的4个外显子与1个内含子片断,采用DHPLC技术对扩增片断进行基因变异检测,将不同的类型PCR片断进行全序列测定并与参考序列对照分析。结果　在5个片断中初步鉴定出4个未见报道的新SNP位点,验证了4个已知的SNPs位点和基因型。结论　DHPLC预测结合全序列测序是一种高效、经济、简便、可靠的SNP筛选方法。     

先天性白内障一家系的致病基因研究

目的 对一个来自浙江省杭州地区的三代先天性白内障家系进行常染色体显性遗传基因的突变分析,以寻找其可能的致病基因及突变位点。方法 该家系共10例成员,其中包括4例患者。10 例家系成员在浙江省人民医院眼科中心接受眼科专科检查及全身检查,以排除存在白内障以外的眼部及全身疾患。10例家系成员各抽取外周血5ml,提取基因组DNA。针对国内外文献报道的与常染色体显性遗传先天性白内障相关的18 个基因（CRYAA、CRYAB、CRYBA1、CRYBA2、CRYBA4、CRYBB1、CRYBB2、CRYGC、CRYGD、CRYGS、GJA3、GJA8、MIP、BFSP、HSF4、PITX3、EPHA2、PAX6）设计引物,进行PCR 扩增,对扩产物进行测序和序列分析,了解这10例家系成员的以上基因是否存在相应的序列。结果 临床眼科检查显示该家系先天性白内障类型为粉尘状白内障。候选基因序列测定显示在CRYAA 第1 个外显子中第6 位碱基发生C→T 置换,氨基酸同为天门冬氨酸。该家系中所有患者均有此改变,而所有的正常家系成员均无此改变。结论 CRYAA 第1个外显子中第6位碱基发生C→T的同义突变可能是导致该家系先天性白内障发生的致病原因。     

昆明地区Rh阴性者RhD假基因（RhD ψ）和杂交RhD-CE-D基因的检测和医学应用

目的用PCR基因分型技术分析研究昆明地区常住人口Rh阴性血型的D基因多态性,建立和完善本地区Rh血型的PCRRhD基因检测方法。方法收集46例Rh阴性血型样本,首先采用外显子-SSP进行PCR扩增,筛选出5例D基因部分存在型,再采用内含子-SSP进行PCR扩增,扩增产物用于DNA测序。结果在46例Rh阴性血型样本中,D基因完整缺失者29例（63％）;D基因完整存在者12例（26．1％）,存在全部外显子;D基因部分存在者5例（10．9％）。DNA测序结果显示,2例仅存在D基因的第10外显子的样本均为RhD—CE（3—9）-D杂合基因,1例存在D基因的第4、6外显子为RhD—CE（2—3,5,7—9）-D杂合基因,是新发现的变异等位基因。结论（1）昆明地区常住人群存在高频率的RhD—CE（3—9）-D杂合基因。（2）在昆明人群发现一种新型D变异等位基因即RhD—CE（2—3,5,7—9）-D杂合基因。     

中国人群MEF2A基因突变与冠心病易感性的关系

目的 研究冠心病相关基因MEF2A在中国人群的突变和多态性位点.方法 利用聚合酶链反应-单链构象多态性(polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP)和DNA测序技术检测210例冠心病(CAD)患者及190名健康人MEF2A基因第11外显子.结果 冠心病患者SSCP电泳条带异常样本测序分析发现突变:①密码子451G/T(147191位点G→T)杂合或纯合同义突变;②147108～147131位点之间分别发生1个氨基酸(Q)、2个氨基酸(QQ)、3个氨基酸(QQP)、6个氨基酸(425QQQQQQ430)、7个氨基酸(424QQQQQQQ430)缺失;③密码子435G/A(147143位点)杂合突变.密码子451G/T杂合或纯合同义突变以及氨基酸的缺失是基因多态性,密码子435G/A考虑为新的突变.结论 中国人群在MEF2A基因第11外显子也存在多种基因多态性,其中6或7个氨基酸的缺失和1个147143位点突变可能与冠心病易感性有关.     

原发性肌张力障碍患者DYT1基因突变分析

目的 探讨中国人原发性肌张力障碍患者DYT1基因突变分布特点,评价变性高效液相色谱（DHPLC）技术在DYT1基因突变分析中的应用价值。方法 扩增DYT1基因第5外显子片段,利用DHPLC技术对PCR产物进行检测,并测序确认突变类型,同时用PCR-酶切方法检验DHPLC结果。结果在13例原发性肌张力障碍患者中发现5例存在904-906delGAG（3bp）杂合缺失突变,此突变导致302密码子谷氨酸的丢失;DHPLC与酶切方法结果完全一致。结论 DHPLC方法可用于检测DYT1基因3bp缺失突变;DYT1基因GAG缺失突变可能是导致中国人早发性全身型原发性肌张力障碍的主要致病原因。     

Molecular genetic analysis of autosomal dominant late-onset cataract in a Chinese Family

Congenital cataract is a highly heterogeneous disorder at both the genetic and the clinical-phenotypic levels.A unique cataract was observed in a 4-generation Chinese family,which was characterized by autosomal dominant inheritance and late-onset.Mutations in the 13 known genes (CRYAA,CRYAB,CRYBB1,CRYBB2,CRYGC,CRYBA1/A3,CRYGD,Connexin50,Connexin46,intrinsic membrane protein LIM2,cytoskeletal protein BFSP2,the major intrinsic protein-MIP and the heat shock factor HSF4) have previously been demonstrated to be the frequent reason for isolated congenital cataracts,but the exact molecular basis and underlying mechanisms of congenital cataract still remain unclear.This study was designed to find whether these 13 genes developed any mutation in the family members and to identify the disease-causing gene.Polymerase chain reaction (PCR) and direct DNA sequence analysis were carried out to detect the 13 genes.The results showed that no mutation causing amino acid alternations was found in these potential candidate genes among all patients in the family,and only several single-nucleotide polymorphisms (SNPs) were identified.A transitional mutation in the fourth intron of CRYBB2 and some silent mutations in the first exon of BFSP2 and CRYGD were found in the cataract family,but further study showed that these mutations could also be found in normal controls.It was concluded that some unidentified genes may underlie the occurrence of late-onset cataract in this family.A genome-wide screening will be carried out in the next study.     

应用变性高效液相色谱筛查颅内静脉系统血栓形成AT-Ⅲ基因突变及多态性

目的 应用变性高效液相色谱(DHPLC)方法筛查生育期女性颅内静脉系统血栓形成(CVT)AT-Ⅲ基因的突变及多态性.方法 标本来自于2006年6月～2007年12月南方医院生育期女性CVT患者,及52例严格配伍的健康女性外周静脉血.提取所有受试者全血DNA.PCR扩增后应用变性高效液相色谱(DHPLC)技术分析抗凝血酶-Ⅲ(AT-Ⅲ)基因的启动子区,第1～6外显子及其侧翼序列的基因变异情况.结果 DHPLC技术分析发现病例组出现6种异常峰形,经测序证实1例致病突变为Exon6 G13328A的杂合突变,1例新发现的同义突变Exon4+243 G>A;SNP位点6个,其中4个SNP库已报道的SNP位点.2个新发现的SNP位点,对照组发现一种异常峰型(三峰).结论 DHPLC是一种自动、快速、高通量的基因突变及SNP位点的筛查方法,AT-Ⅲ基因突变可能是导致生育期非妊娠女性CVT的遗传因素之一,功能性SNP位点也可能参与了该人群VTE的发生.

Abstract：

Objective To identify antithrombin Ⅲ(AT- Ⅲ) gene mutation and polymorphisms in pregnant women and parturients with cerebral venous thrombosis (CVT) using denaturing high-performance liquid chromatography (DHPLC). Methods The genomic DNA was extracted from the blood samples of 50 pregnant women and parturients with CVT and 52 matched healthy women for molecular analysis using a PCR/DHPLC assay followed by DNA sequence analysis. Ten primer pairs were designed for amplifying the AT-Ⅲ promoter region and exons 1-6 including the exon/intron boundaries. A rapid screening assay based on DHPLC was established to screen the mutation and polymorphisms of AT- Ⅲ gene. Results Six abnormal peaks were detected in 40 of the patients by DHPLC. Direct DNA sequencing was performed on representative samples detected by DHPLC profiling. One pathogenic heterozygous G13328A missense mutation in exon 6, and a novel silent mutation in exon 4+243 G>A were identified. Six single nucleotide polymorphism (SNP) sites were found, including 4 previously reported ones in the SNP library and two were novel SNP sites. An abnormal peak was detected in the control group by DHPLC. Conclusion DHPLC allows automated and rapid high-throughput detection of AT-Ⅲ gene mutation and polymorphisms in the clinical setting and prenatal diagnosis. Our findings suggested that AT-Ⅲ gene mutation, as well as its polymorphisms, contributes to the occurrence of CVT in pregnant women and parturients.     

家族性阿尔茨海默病早老素-1基因突变位点的检测

目的：探讨早老素－1基因突变在家族性阿尔茨海默病（FAD）发病中的作用。方法：应用聚合酶链反应－单链构象多态性（PCR－SSCP）、变性高效液相（DHPLC）及DNA直接测序技术检测技术,对130人的阿尔茨海默病（AD）家系和50例正常对照组的早老素－1（PS－1）基因第4、5外显子进行了检测。结果：在早老素基因的第5外显子上发现除5例AD患者外,还有4例家系内正常人（Ⅳ－30、37、41、43）PCR－SSCP出现泳动异常。DHPLC检测进一步验证以上9例均表现双峰,提示可能有突变存在。DNA序列分析表明,这9例的早老素－1基因第5外显子的136号密码子发生了GCT→GGT错义突变,使氨基酸由丙氨酸变为甘氨酸（Ala136Gly);第4外显子未发现突变。家系内其他人及正常对照组的PS1基因第4、5外显子经以上检测均未发现异常。结论：PS1基因第5外显子的突变点可能为中国FAD患者早老素基因突变位点之一。     

首次发现肥厚型心肌病肌球蛋白结合蛋白C基因Arg856fs突变

目的： 研究中国人群肥厚型心肌病(HCM)患者的致病基因突变位点，并对基因型与临床表型之间的关系进行分析。方法: 对76例HCM先证者进行聚合酶链反应(PCR)扩增心肌肌球蛋白结合蛋白C基因（MYBPC3）第15 16,18,26,28,34号外显子，产物做单链构象多态性(SSCP)分析，出现异常条带者将其目的片段和正常对照组该片段送检测序。结果: 在1例52岁男性患者的MYBPC3基因第26号外显子上发现了一个新的移码突变位点Arg856fs。由于在100名正常对照组中未见异常，故我们认为此突变位点为该患者的致病基因位点。结论: MYBPC3基因是我国HCM的致病基因之一。     

Mutation analysis of the checkpoint kinase 2 gene in colorectal cancer cell lines

Background Checkpoint kinase 2 （CHK2） is a DNA damage-activated protein kinase which is involved in cell cycle checkpoint control, CHK2 gene could be a candidate gene for colorectal cancer susceptibility, But there are few systematic reports on mutation of CHK2 in colorectal cancer. Methods The mutations of all 14 exons of CHK2 in 56 colorectal cancer cell lines were screened systematically, using denaturing high-performance liquid chromatography （DHPLC） to screen the mismatches of the CHK2 exons amplified products, and then the suspected mutant cell lines were scanned by nucleotide sequence analysis. Results VACO400 in CHK2 exon la was suspected to have mutation by DHPLC and confirmed by sequence, but this was nonsense mutation. C106, CX-1, HT-29, SK01, SW480, SW620 and VACO400 in CHK2 exon lb were confirmed to have the same nonsense mutation in 11609 A〉G. DLD-1 and HCT-15 in CHK2 exon 2 were confirmed to have missense mutation R145W, which was heterozygous C〉T missense mutation at nucleotide 433, leading to an Arg〉Trp substitution within the FHA domain. Conclusions The CHK2 mutation in colorectal cancer is a low frequency event, There are just 10 cell lines to have sequence variations in all the 14 exons in 56 colorectal cancer cell lines and only DLD-1/HCT-15 had heterozygous missense mutation. These findings may give useful information of susceptibility of colorectal cancer as single nucleotide polvmorphvsim.     

DYT1 MUTATIONS AMONGST EARLY ONSET PRIMARY DYSTONIA PATIENTS IN CHINA

Objective To investigate the frequency of GAG deletion in the DYT1 gene among early onset primary dystonia patients in China. Methods Thirteen patients with early onset primary torsion dystonia were screened for mutation in exon 5 of the DYT1 gene using denaturing high-performance liquid chromatography （DHPLC） and DNA sequencing, and the results were confirmed with polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP）. Results The GAG deletion mutation which results in Glu302del in exon 5 of the DYT1 gene was found in 5 patients. The detecting results were consistent between with DHPLC and PCR-RFLP. We did not find any other mutations in the DYT1 gene. Conel~iotm The GAG deletion in the DYT1 gene is common amongst early onset primary torsion dystonia patients in Chin＆ The frequency of DYT1 mutation is not significantly different between European and Asian patients with early onset primary dystonia.     

哮喘家系中的BRS-3编码基因的变异和多态性研究

目的:观察哮喘家系患者外周血白细胞DNA中蛙皮素受体亚型3(BRS-3)的多态性或基因变异,以了解BRS-3与气道高反应疾病遗传性之间的联系,补充和丰富对气道高反应机制的认识。方法:抽取哮喘患者及正常人外周血白细胞DNA,PCR法扩增BRS-3外显子1和外显子3,测序及进行序列分析。结果:在哮喘患者的BRS-3Exon1和Exon3不存在突变或SNP。结论:推测BRS-3的改变有可能不在编码区,但不排除调控区的改变。     

受磷蛋白基因突变与中国成都地区扩张型心肌病的相关性研究

目的研究扩张型心肌病(DCM)患者的肌浆网受磷蛋白(phospholamban,PLN)基因突变情况,探讨该基因与中国DCM患者发病的关系。方法采用聚合酶链反应-单链构象多态性(PCR-SSCP)方法并结合核苷酸序列测定对89例DCM患者及110例对照组的PLN基因进行分析。结果病例组及对照组中均未发现存在异常构象,通过DNA测序未发现存在PLN基因第25核苷酸位点的C→T错义突变及116核苷酸位点的T→G突变。结论本研究未发现中国成都地区DCM患者PLN基因第25和116核苷酸位点存在突变。     

脑血管狭窄患者Nf1全基因的DHPLC分析(附5例报告)

目的 探讨脑血管狭窄患者Nf1基因的突变热点及突变方式.方法 从全血中提取DNA,采用变性高效液相色谱分析(DHPLC)对5例脑血管狭窄病例进行Nf1全基因突变筛查;对DHPLC检测有差异洗脱峰型的区域进行测序分析.结果 5例无危险因素的脑血管狭窄患者Nf1全基因筛查发现:①1例出现5号外显子无义突变位点,c.541C-T;②5例均有14号和15号外显子之间内含子的基因突变;③4例有7号外显子c.702G-A的同义突变位点;④1例出现32号(c.4177G-A,Val-Ile)和46号外显子(c.6918T-G,Asn-Lys)的错义突变.结论 在目前无其他基础疾病的脑血管狭窄患者体内的Nf1基因并非以稳定野生型存在,而是具有不同类型的突变,这些突变集中在第5、7、14、32和46等外显子上.