三个Fabry病家系GLA基因突变与临床表型

目的 分析3个Fabry病家系GLA基因突变及其与临床表型的关系.方法 应用PCR结合DNA测序技术,检测先证者及相关成员GLA基因编码序列与剪切位点DNA序列变异,分析致病性突变与临床表型关系.结果 在家系1先证者GLA基因第5外显子中发现1个未经报道的错义突变c.797A＞C(D266A),家系2先证者GLA基因第5外显子中发现1个错义突变c.644A＞G(N215S),家系3先证者GLA基因第2外显子中发现1个无义突变c.355C＞T(Ql19X).家系1与家系3先证者主要表现为皮肤损害和慢性肾功能不全,家系2先证者临床则以肥厚性心肌病为特点.结论 首次发现的GLA基因c.797A＞C(D266A)突变是第266位密码子第6个被证实的错义突变,已报道的另5种突变均有致病性,在正常非相关对照中未发现该突变,提示GLA基因c.797A＞C突变很可能是该家系的致病原因.N215S和Q119X系首次发现于中国Fabry病家系的突变.GLA基因不同位点的突变具有较为显著的表型差异.     

复合杂合性蛋白C基因突变导致的遗传性蛋白C缺陷症家系分析

目的 对1个遗传性蛋白C(proteinC,PC)缺陷症家系进行蛋白 C基因(protein C gene,PROC)突变检测,探讨其分子发病机制.方法 通过检测先证者及其家系成员(共4代15人)血浆蛋白C活性(protein C activity,PC∶A)、蛋白C抗原含量(protein C antigen,PC∶Ag)及其他凝血指标进行表型分析;用PCR法扩增先证者PROC的9个外显子及其侧翼序列,PCR产物纯化后测序,发现突变位点则反向测序予以证实;针对先证者的突变位点,对其家系成员进行相应基因突变检测.结果 先证者PC∶ A和PC∶Ag明显降低,分别为26％和18.60％,家系中其他成员中有7人PC∶A降低,6人PC∶Ag降低.先证者的PROC第7外显子存在g.6128T＞G杂合错义突变导致p.Phe139Val,第9外显子存在g.8478G＞C杂合错义突变导致p.Asp255His;其祖母、父亲、三姑和四姑均存在g.6128T＞G杂合突变,母亲、二舅、妹妹和儿子均存在g.8478G＞C杂合突变.存在g.8478G＞C突变的成员PC∶A下降明显.结论 先证者PROCg.6128T＞G和g.8478G＞C突变分别来自其父系家族和母系家族,该复合杂合错义突变是导致遗传性PC缺陷症的分子基础.     

线粒体糖尿病家系基因突变的遗传学筛查

目的 探索与糖尿病发病相关的线粒体基因突变位点。方法 采用PCR、DNA直接测序技术对28个临床疑似线粒体基因突变糖尿病家系(mitochondrial diabetes mellitus，MDM)进行线粒体基因突变高发区域(tRNA^Leu(UUR)基因及NADH脱氢酶1基因)的筛查。结果 两个家系发现与糖尿病发病有关的突变位点。其中1个家系(16号)同时携带nt3243A→G突变和16S rRNA3205C→T突变。该家系两例患者均有消瘦、耳聋、β细胞功能低下、发病年龄低的特点，先证者血乳酸水平在正常高限。在另1先证者患耳聋的家系(28号)发现NDl基因3434A→G突变，导致氨基酸改变，与家系中糖尿病呈共同分离。上述突变均未在正常人中检测到。结论 3243位点突变在本组家系中的发现率为3．08％。16S rRNA3205C→T突变可能与糖尿病发病有关或与nt3243A→G突变协同作用。NDl基因3434A→G突变与28号家系糖尿病的发生有关。     

一个中国G6PD缺乏症家系致病基因的突变分析

目的对一个葡萄糖-6-磷酸脱氢酶(G6PD)缺乏症家系成员G6PD基因13个外显子全面测序,识别致病的突变位点及其遗传模式。方法在G6PD缺乏症高发区广东惠州地区收集到一个G6PD缺乏症核心家系,包括先证者(儿子)、患病母亲和正常父亲。取家系成员的外周血样,并提取基因组DNA,用PCR和DNA测序法对G6PD基因全部外显子进行序列分析。结果先证者及其母亲在G6PD基因第2号外显子出现同一点突变(c.95AG,p.His32Arg),该突变引起组氨酸被精氨酸替换(CACCGC)。然而先证者父亲在G6PD基因的13个外显子均未出现突变。3种生物信息学软件均预测该突变对蛋白质功能具有较强的危害性。结论该家系中c.95AG呈X连锁显性遗传,是G6PD缺乏症的致病突变位点之一。     

长QT综合征 KCNQ1基因突变筛查方法

目的 研究中国人长QT综合征(long QT syndrome，LQTS)与编码缓慢激活延迟整流钾通道基因(postassium voltage—gated channel，KQT—like subfamily member 1，KCNQl)突变的关系。方法 根据心电图T波的特征对31个家系进行基因分型的初步预测。对10个预测为LQTl家系的家庭成员，用聚合酶链反应-单链构像多态性(polymerase chain reaction—single strand conformation polymorphism,PCR—SSCP)方法进行KCNQ1基因16个外显子及剪接位点的筛查，SSCP异常者进行DNA测序。为避免遗漏心电图表现不典型的LQT1，同时也为了进行方法学比较，对其它21个非LQT1家系只对先证者进行16个外显子的PCR和DNA直接测序。对测序有异常者，分析其家系成员相应外显子的疾病分离情况。若异常只存在于患者，则检查该异常在50个正常对照者中的情况。结果 (1)在心电图预测分型为LQT1的家系中发现了位于第5外显子的S277L(1个家系)和G306V(1个家系)2个错义突变。另外发现了3个多态性，分别为435C→T(1145I)(7个家系)、1632C→A(S546S)(1个家系)、IVS1 9C→G(3个家系)。(2)在心电图分型预测为非LQT1的先证者中只发现了1个剪接突变IVS1 5G→A(2个家系)和1个多态性IVS1 18C→T(1个家系)。3个突变位点均位于KCNQ1基因功能区域，各突变家系内患者存在同样的异常条带或序列，而正常对照无此异常。结论 在中国人LQTS患者中发现TKCNQ1基因上的2个新错义突变、1个剪接突变和4个多态性。结合心电图分型预测，PCR-SSCP法可发现绝大部分突变，是筛查LQTS突变的简便而经济的方法。     

眼皮肤白化病Ⅰ型产前基因诊断

目的对已生育过眼皮肤白化病Ⅰ型（oculocutaneous albinism type Ⅰ，OCA1）患儿的两个家系进行酪氨酸酶（tyrosinase，TYR）基因TYR的突变研究和产前基因诊断。方法应用PCR技术扩增TYR基因各外显子、外显子．内含子交界区及启动子区，直接以DNA序列测定技术分析先证者或其父母的基因突变，明确致病性突变后，检测胎儿TYR基因相应位点的DNA序列，获知胎儿的基因型。结果家系1的先证者为R278X和929insC突变复合杂合子；胎儿未获得这2种突变等位基因，基因型和表型均正常。家系2先证者的父母分别为IVS4＋3A→T和G253E突变的杂合子，胎儿只获得了父源性的IVS4＋3A→T突变等位基因，未获得母源性G253E突变等位基因，胎儿为表型正常的致病基因携带者。结论此为中国大陆首次真正意义上的OCA1产前基因诊断；应用上述基因分析方法进行OCA1产前基因诊断是可行的。     

一例ABO新等位基因B112的分子生物学研究及其家系分析

目的 研究1例新的ABO亚型B112的分子机制,并对其家系进行分析.方法 应用单克隆抗体检测先证者红细胞ABO血型抗原,标准A、B、O红细胞检测先证者血清中的ABO抗体.采用聚合酶链反应(po1ymerase chain reaction,PCR)技术扩增先证者ABO基因的第5至7外显子序列,PCR产物经双酶切后直接测序分析第6和7外显子.同时采用基因组单链抽提技术分离先证者的两条单倍型,对分离的单倍型扩增后进行ABO基因测序分析.家系调查采集先证者父母的标本进行ABO血清学实验和ABO基因第6和7外显子测序分析.结果 先证者血清学表型符合B表型特性,直接测序分析显示第6和7外显子有261G/缺失、297A/G、526C/G、559C/T、657C/T、703G/A、796C/A、803G/C、930G/A杂合,推断基因型为BO.基因组单链抽提技术将先证者B和O基因分离后,测序得到两个等位基因为O01和B112.与B101相比,B112第559位C→T导致第187位精氨酸变成半胱氨酸.家系调查显示先证者B112基因从母亲遗传所得,母亲标本ABO血型血清学特性和测序分析结果与先证者完全一致.结论 发现1例559C＞T突变的ABO亚型新等位基因B112,其B抗原表达正常,提示α-1,3-半乳糖基转移酶第187位精氨酸变成半胱氨酸并不影响B转移酶的活性.     

一个遗传性凝血因子Ⅴ缺乏症家系中凝血因子Ⅴ基因两种新突变的鉴定

目的对一个遗传性凝血因子Ⅴ缺乏症家系进行凝血因子Ⅴ（factorⅤ，FⅤ）基因突变的检测，探讨先天性凝血因子Ⅴ缺乏症的分子发病机理。方法PCR结合直接测序方法对先证者的FⅤ基因全部25个外显子及其旁侧序列进行分析，鉴别其中可能存在的基因变异。应用PCR产物反向测序法或PCR限制性内切酶分析技术对突变位点进行确证。随机选择100名健康体检者作为正常对照。结果先证者FⅤ基因存在两种突变，分别是第11外显子区的A1763C错义突变和位于第16内含子3′端剪接位点的G-T突变；家系分析表明这两个突变是双杂合子型，前者遗传自父亲，后者遗传自母亲。100名健康对照者均未发现A1763C错义突变。结论第11外显子A1763C错义突变和第16内含子3′端的剪接位点突变使先证者呈现双重杂合子型，可能是先证者FⅤ先天性缺乏的原因。     

眼皮肤白化病Ⅰ型产前基因诊断二例

目的对生育过眼皮肤白化病（oculocutaneous albinism,OCA）患儿的2个家系进行基因诊断分型,并在此基础上提供产前基因诊断。方法采用PCR扩增先证者OCA 1型疾病相关基因TYR的所有5个编码外显子,PCR产物直接测序,在确定致病突变的基础上对及家系成员进行综合分析。结果 2个OCA先证者均携带TYR基因复合杂合突变,确定2例先证者均为OCA1型患者。TYR基因共检测到3种突变：c.71G〉A,c.896G〉A和c.929ins C。产前诊断：第1个家系提示胎儿基因型与先证者一致,家属选择终止妊娠;第2个家系中胎儿为TYR基因野生型携带者,继续妊娠至足月分娩,新生儿随访正常。结论利用基因检测可为眼皮肤白化病患者提供确切的临床分型,并在此基础上提供有效的产前基因诊断。     

纯合子Cys329Gly导致的遗传性凝血因子Ⅶ缺陷症家系基因突变分析

目的对1个姨表近亲婚配的遗传性凝血因子Ⅶ(FⅦ)缺陷症家系进行表型和F7基因突变分析,探讨其分子发病机制。方法检测先证者及其家系成员(共4代9人)凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、血浆FⅦ活性(FⅦ:C)等来明确诊断。PCR扩增先证者全部外显子及其侧翼序列、5′和3′非翻译区及家系成员相应的突变位点区域,PCR产物纯化后直接测序,寻找突变位点,以反向测序验证所发生的突变;使用生物信息学软件(Poly Phen-2和Mutation Taster)预测突变位点对蛋白质功能的影响,利用Py MOL软件构建正常FⅦ蛋白空间模型,进行定点突变观察构型改变。结果先证者PT(36.1s)和FⅦ:C(2%)明显异常,家系中其余8位成员PT均略高于正常对照组和FⅦ:C均略低于正常对照组;基因分析显示先证者F7基因第8外显子存在c.1165 TG纯合型突变,即TGT→GGT(Cys329Gly),8位家系成员的F7基因分析均显示c.1165有杂合型突变;两种生物信息学软件都提示此突变会引起蛋白功能变化,有致病性;F7基因蛋白构型显示突变后329位点与310位点之间二硫键消失,周边静电磁场发生改变。结论该家系F7基因存在Cys329Gly突变,是引起FⅦ缺陷症的主要分子机制,推测先证者纯合Cys329Gly突变基因分别遗传自近亲结婚的杂合子父母。     

中国人遗传性非息肉病性结直肠癌MSH6基因胚系突变的测序研究

目的探讨中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系中MSH6基因胚系突变。方法采用PCR-直接测序的方法检测39个无胚系MSH2及MLH1基因突变、符合不同临床标准的中国人HNPCC家系先证者MSH6基因各外显子胚系突变;对137名正常人胚系基因组DNA进行错义突变相应外显子的测序分析。应用Envision二步法检测有突变的先证者肿瘤组织MSH6蛋白表达。结果在39个HNPCC先证者中共发现6个MSH6基因的胚系突变,分别位于第4、6、9和第10外显子;突变类型为4个错义突变、1个无义突变、1个剪接区的插入突变;对4个错义突变的相应外显子的测序分析显示:137名正常人胚系基因组DNA5例具有第6外显子1163密码子处的c.3488A>T的错义突变,约占3.65%(5/137),为单核苷酸多态性(single nucleotide polymorphism,SNP);其余错义突变在正常人群中均未发现。在6例有MSH6基因胚系突变家系的肿瘤组织中免疫组化染色除1例为SNP的肿瘤组织MSH6蛋白阳性表达外,其余均为阴性表达。经过查询国际HNPCC突变数据库及SNP数据库证实上述突变中5个为国际上尚未报道的病理性突变,1个为新发现的SNP。结论MSH6基因胚系突变在符合不同临床标准的中国人HNPCC中均起一定作用,对无MSH2及MLH1基因胚系突变的先证者行MSH6基因胚系突变的测序分析对确诊HNPCC家系是必要的。     

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene. Fabry disease (FD) is an X-linked lysosomal storage disorder with a heterogeneous spectrum of clinical manifestations that are caused by the deficiency of α-galactosidase A (α-Gal-A) activity. Although useful for diagnosis in males, enzyme activity is not a reliable biochemical marker in heterozygous females due to random X-chromosome inactivation, thus rendering DNA sequencing of the α-Gal-A gene, alpha-galactosidase gene (GLA), the most reliable test for the confirmation of diagnosis in females. The spectrum of GLA mutations is highly heterogeneous. Many polymorphic GLA variants have been described, but it is unclear if haplotypes formed by combinations of such variants correlate with FD, thus complicating molecular diagnosis in females with normal α-Gal-A activity. We tested 67 female probands with clinical manifestations that may be associated with FD and 110 control males with normal α-Gal-A activity. Five different combinations of GLA polymorphic variants were identified in 14 of the 67 females, whereas clearcut pathogenetic alterations, p.Met51Ile and p.Met290Leu, were identified in two cases. The latter has not been reported so far, and both mutant forms were found to be responsive to the pharmacological chaperone deoxygalactonojirimycin (DGJ; migalastat hydrochloride). Analysis of the male control population, as well as male relatives of a suspected FD female proband, permitted the identification of seven different GLA gene haplotypes in strong linkage disequilibrium. The identification of haplotypes in control males provides evidence against their involvement in the development of FD phenotypic manifestations.     

Identification of four novel mutations in five unrelated Korean families with Fabry disease

Fabry disease is a X-linked recessively inherited metabolic disorder, which results from the deficient activity of the lysosomal hydrolase alpha-galactosidase A leading to the systemic deposition of glycosphingolipids with terminal alpha-galactosyl moieties. Single-strand conformation polymorphism (SSCP) analysis was performed, followed by DNA sequencing of PCR amplified exons of the human alpha-galactosidase A gene in 5 unrelated Korean patients with classic Fabry disease. Five different mutations were identified; two nonsense mutations (Y86X and R342X), one missense mutation (D266N), and two small deletions (296del2 and 802del4). Except for R342X mutation, four were novel mutations (Y86X, D266N, 296del2, 802del4). A T to G transversion at nucleotide position 5157 in exon 2 caused a tyrosine-to-stop substitution at codon 86. A G to A transition at position 10287 in exon 5 substituted an asparagine for an aspartate at codon 266. Mutation 296del2 in exon 2 resulted in a frame shift with a stop signal at the 22th codon downstream from the mutation, whereas mutation 802del4 resulted in a stop codon at the site of 4 bp deletion. In addition, the 802del4 was found to be a de novo mutation. This is the first report on mutation analysis of the human alpha-galactosidase A gene in Korean patients with Fabry disease.     

一个遗传性出血性毛细血管扩张症家系的临床和遗传学分析

目的 :对一个中国遗传性出血性毛细血管扩张症 (HereditaryHemorrhagicTelangiectasia ,HHT)家系的临床表现和遗传缺陷进行分析。方法 :对该HHT家系进行家系调查 ,并对所有成员进行临床检测。用PCR方法扩增ALK 1(Ac tivinReceptor LikeKinase 1)和Endoglin(ENG)基因的所有外显子、外显子 -内含子交界区和 5’、3’非翻译区 ,采用直接测序的方法查找基因突变。结果 :该家系的先证者有反复发作的鼻出血和皮肤粘膜的毛细血管扩张 ,并均有阳性家族史。各器官均未发现动静脉血管畸形。在家系的先证者及其他 4个成员中发现一个位于ALK 1基因第 8外显子G12 32A的错义突变 ,导致Arg 4 11Gln。结论 :HHT的临床表现多样。新错义突变G12 32A(Arg4 11Gln)可能导致HHT2的分子缺陷。该突变为国内首次报道。     

Germline mutations of E-cadherin gene in Korean familial gastric cancer patients

Gastric cancer is the most common cancer in Korea. Germline mutations of the E-cadherin gene have recently been identified in familial gastric cancer patients. We screened five Korean familial gastric cancer patients to investigate germline mutations of the E-cadherin gene. These patients fulfilled the following criteria: presence of at least two gastric cancer patients within first-degree relatives and one patient diagnosed before the age of 50 years. Abnormal band patterns were found in exons 6 and 10 in two familial gastric cancer patients by polymerase chain reaction-single strand conformation polymorphism analysis (probands from the SNU-G2 and SNU-G1001 families, respectively). DNA sequencing analysis of the E-cadherin gene of these two patients revealed missense mutations in each exon. The SNU-G2 proband harbored a missense mutation from aspartic acid (GAT) to glycine (GGT) at codon 244 in exon 6 of the E-cadherin gene, and the SNU-G1001 proband had a missense mutation from valine (GTG) to alanine (GCG) at codon 487 in exon 10. The SNU-G2 proband was diagnosed with gastric cancer at the age of 38; three brothers and two sisters had died of gastric cancer under the age of 50, and their mother had died of gastric cancer at the age of 63. The SNU-G1001 proband was diagnosed with gastric cancer at the age of 42 and one brother had died of gastric cancer at the age of 49. In summary, we found germline mutations of the E-cadherin gene in two of five Korean familial gastric cancer patients screened. Received: January 4, 1999 / Accepted: February 4, 1999     

中国人遗传性非腺瘤病性结直肠癌家系hMSH2和hMLH1基因突变分析

目的 分析符合不同临床标准的中国遗传性非腺瘤病性结直肠癌(HNPCC)家系hMSH2和hMLH1基因种系突变状况,评价不同临床标准预示突变检测的敏感性。方法应用DNA直接测序对24个符合Amsterdam标准、15个符合日本标准家系先证者和19个符合Bethesda指导纲要患者(字系中仅1例患者)进行hMSH2和hMLH1基因种系突变检测。对检出突变的家系进行家庭成员的突变筛选。并对检出突变患者进行肿瘤组织突变的检测。结果在16例家系先证者中检测到6个hMSH2突变和11个hMLHl种系突变,其中12个突变是国际上尚未报道过的新突变。突变位于不同外显子中,其中6个突变位于hMLHl第14-16外显子。Amsterdam标准家系突变阳性率为50％(12／24),以日本标准所筛家系突变阳性率为3／15,以上两组家系以外的Bethesda指导纲要患者突变阳性率为1／19。突变类型包括移码突变、无义突变、剪接异常、框架内插入或缺失以及错义突变。基因突变与疾病共分离,检出突变家系先证者的肿瘤组织错配修复基因表现出3种不同基因型：(1)野生型等位基因丢失;(2)肿瘤组织基因型与生殖细胞一致;(3)突变型等位基因丢失。结论中国人HNPCC家系hMSH2和hMLHl突变谱广泛,突变类型多样,hMLHl突变较hMSH2突变多见,突变较为集中于hMLHl外显子14-16。不同临床标准预示突变的敏感性不同。突变基因型与疾病表现型共分离。家系成员中尚未发病的突变携带者应予密切监测。     

Molecular basis of fabry disease: Mutations and polymorphisms in the human α-galactosidase A gene

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the α-galactosidase A gene at Xq22.1. Studies of the mutations in unrelated Fabry families have identified a variety of lesions indicating the molecular genetic heterogeneity underlying the disease. Forty-nine different mutations have been described including five partial gene deletions, one partial gene duplication, nine small deletions and insertions, three splice junction consensus site alterations, and 31 coding region single base substitutions. Most mutations resulted in the classical disease phenotype; however, five missense mutations were detected in atypical hemizygotes who were asymptomatic or had symptoms confined to the heart, including N215S, which was described in three unrelated atypical males. Most mutations were confined to a single pedigree with the exception of N215S, R227Q, R227X, R342Q, and R342X, which were each found in several unrelated families. Five of the 14 coding region CpG dinucleotides were sites of point mutations including the CpGs in codons 227 and 342, which were each mutated in both orientations. The identification of the mutation in a given Fabry family permits precise prenatal diagnosis and heterozygote detection of other family members with this X-linked recessive disease. Studies of additional Fabry families will provide information on the nature and frequency of the mutations causing this disease as well as potential insights into the structure/ function relationships of this lysosomal hydrolase. © 1994 Wiley-Liss, Inc.