一家族性高胆固醇血症家系低密度脂蛋白受体基因突变分析

为分析一家族性高胆固醇血症家系低密度脂蛋白受体的基因突变，提取患儿及其父母外周血基因组DNA，用聚合酶链反应扩增低密度脂蛋白受体基因的18个外显子。用单链构象多态性分析检测聚合酶链反应产物，对单链构象多态性分析电泳结果异常者进行DNA序列分析。结果发现，单链构象多态性分析发现患者及其母亲第10外显子存在一异常条带。DNA测序结果证实患者第10外显子的471位密码子由AGA同义突变为AGG，483住密码子由TGG突变为TAG，导致在483住提前出现终止密码子。本研究利用聚合酶链反应一单链构象多态性分析方法报道了一个新的低密度脂蛋白受体突变位点。     

家族性高胆固醇血症患者低密度脂蛋白受体基因新突变一例

目的 分析中国家族性高胆固醇血症（FH）患儿低密度脂蛋白受体（LDL－R）基因突变的情况，并为在婴幼儿时期此病的症前筛查提供确诊方法。方法 以患儿及其父母的基因组DNA为模板，首先用聚合酶链反应（PCR）扩增该基因的启动子和全部18个外显子，然后用单链构象多态性（SSCP）方法分析PCR产物，最后对电泳结果异常进行DNA测序。结果 在1个家系中检测出患儿和其父亲LDL－R基因的一种新突变，即在第4外显子的444位碱基发生杂合突变（T→A），相应的氨基酸由半胱氨酸变成终止密码子，其母亲LDL－R基因正常。结论 LDL－R基因此位点的突变可引起FH，PCR－SSCP方法可用于筛查出的高危人群的确诊。     

中国特发性扩张型心肌病患者与δ-肌聚糖基因的相关性研究

目的探讨中国特发性扩张型心肌病是否与δ-肌聚糖基因（SGCD）的突变相关。方法用人工合成的寡核苷酸引物，通过聚合酶链反应（PCR）-单链构象多态性分析（SSCP）-DNA测序法对70名特发性心肌病患者和70例健康对照SGCD基因的全部9个外显子进行分析。结果在病例组和对照组外显子2，3，6，9m，9p的单链构象多态性图谱均呈相同形态，DNA测序发现两组的核苷酸序列没有差异；经反复改变电泳条件仍未能分离出外显子4，5，7，8的SSCP电泳条带。结论未证实δ-肌聚糖基因（SGCD）是特发性心肌病患者的致病基因。     

低密度脂蛋白受体基因突变类型的筛查

目的调查我国家族性高胆固醇血症(FH)患者低密度脂蛋白受体基因突变类型。方法以患者的基因组DNA为模板,用聚合酶链反应扩增该基因的18个外显子。用单链构象多态性方法分析检测PCR产物,对电泳结果异常者进行DNA测序;错配引物PCR引入Msp Ⅰ酶切位点检测最常见的载脂蛋白B基因突变类型R3500Q。结果经单链构象多态性分析发现,7个先证者有异常电泳条带出现。DNA测序证实,先证者存在9个突变位点。经文献检索发现除C255R外,其余均系尚未报道的新突变类型。在所有FH患者中未检测出载脂蛋白B基因突变。结论中国人FH患者可能存在独特的低密度脂蛋白受体基因突变谱。     

特发性扩张型心肌病与δ-肌聚糖基因相关性研究

目的　探讨特发性扩张型心肌病患者中是否有δ-肌聚糖基因的突变。方法　用人工合成的寡核苷酸引物 ,通过聚合酶链反应 ( PCR) -单链构象多态性分析 ( SSCP) ,结合 DNA测序对δ-肌聚糖基因的可疑突变部位外显子 6与外显子 9p进行分析。结果　分析了 5 0例扩张性心肌病患者和 5 0例对照组该基因的外显子 6和外显子 9p,在非变性聚丙烯酰氨凝胶电泳中 ,未发现异常构象 ,通过 DNA测序证实两组的核苷酸序列没有差异。结论　小样本研究未能证实δ-肌聚糖基因是扩张性心肌病患者的致病基因     

X-连锁隐性遗传的腓骨肌萎缩症与Cx32基因突变

目的：探讨X－连锁隐性遗传的腌骨肌萎缩症（CMTXR）与Cx32基因突变的关系。方法：应用聚合酶链反应－单链构象多态性（PCR－SSCP）结合DNA序列方法检测了一个X－连锁隐性遗传的腌骨肌萎缩症家系中4名患者,9名有血缘关系的正常人及家系外50名无血缘关系的正常人。结果：发现该家系中4例患者及3名有血缘关系的正常人均出现异常SSCP条带,经测序证实为Arg15Gln突变。结论：Cx32基因突变可以导致X－连锁隐性遗传的腓骨肌萎缩症。应用PCR－SSCP结合DNA序列分析方法可对由Cx32基因突变所致的X－连锁隐性遗传的腓骨肌萎缩症进行基因诊断。     

Peutz-Jeghers综合征STK13基因突变分析

目的 研究STK13基因在部分Peutz-Jeghers综合征(PJS)家系中的突变情况，判断它是否为中国人PJS新的致病基因，为基因诊断奠定基础。方法 采用聚合酶链反应-单链构象多态性分析(PCR—SSCP)和DNA测序的方法，选择10个无STK11基因突变的PJS家系，对STK13基因进行突变分析。结果 所有7个外显子均未检测到致病突变。结论 STK13基因不是中国人PJS患者新的致病基因。     

姐妹2人同患家族性高胆固醇血症家系LDL-R基因突变分析

目的：对临床确诊的家族性高胆固醇血症（FH）两姐妹及其家系成员进行低密度脂蛋白受体（LDL-R）基因突变分析,探讨其发病机制。方法：提取患者外周血基因组DNA,聚合酶链反应分别扩增启动子和18个外显子片断,采用单链构象多态性（PCR-SSCP）结合银染技术,对异常电泳条带进行核苷酸序列分析。结果：姐妹2人及其父亲,叔叔,祖母均发现LDL-R基因第13外显子存在一个错义突变,与GeneBank对照证实第1879位G→A碱基置换,氨基酸的改变为丙氨酸→苏氨酸（A606T突变）,其母亲和女儿经测序并未发现此突变位点。结论：姐妹2人均为LDL-R基因存在A606T杂合错义突变,并均来自其父系亲属；可能是该家系发病的分子基础。     

中国大陆人散发性先天性巨结肠症患者内皮素受体B基因的研究

目的 探讨中国大陆人散发性先天性巨结肠症（sHD）易患基因内皮素受体B（EDNRB）的突变与多态性特征。方法 以92例sHD及其中32例患儿双亲为研究对象,并以60例正常儿为对照。提取受检者外周静脉血DNA,采用聚合酶链反应-单链构象多态性技术（PCR-SSCP）对EDNRB基因外显子-2（exon-2）进行分析,并通过DNA测序检测阳性标本的核苷酸改变方式,与文献报道的其他种族sHD同一基因特征做比较。结果 全部标本EDNRB基因exon-2均未发现突变与多态性位点的存在。结论 中国大陆人sHD患者EDNRB基因的exon-2不存在突变与多态性位点。     

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(PCR)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.     

低密度脂蛋白受体功能与基因突变的关系

目的：分析家族性高胆固醇血症低密度脂蛋白受体（LDLR）功能变化,寻找基因突变位点;阐明该基因突变类型对LDLR功能的影响。方法：患儿及其你系、母系三代共30人检查血脂和临床表现,作系谱分析,确定该患儿符合家族性高胆固醇血症纯合子的诊断;培养患儿皮肤成纤维细胞,用受体的放射性配体结合技术,定量测定细胞的LDLR的功能;提取外周血基因组DNA对LDLR基因的相应外显子进行PCR－SSCP及DNA序列分析。结果：系谱分析发现11个杂合子及1个纯合子;纯合子患儿LDLR结合功能基本正常,但其内移和降解功能只有正常人的3．6％及1．7％;DNA测序结果证实患儿第17外显子的第599和600密码子间插入一个碱基G,导致框移突变;第842密码子发生CCA→CCG的无义突变。结论：首次报道了一个新的LDLR突变位点;初步明确该突变位点对患者的影响较为显著。     

Spectrum of LDL receptor gene mutations in Denmark: implications for molecular diagnostic strategy in heterozygous familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (FH) is one of the most common potentially fatal single-gene diseases leading to premature coronary artery disease, but the majority of heterozygous FH patients have not been diagnosed. FH is due to mutations in the gene coding for the low-density lipoprotein (LDL) receptor, and molecular genetic diagnosis may facilitate identification of more FH subjects. The Danish spectrum of 29 different mutations, five of which account for almost half of heterozygous FH, is intermediate between that of countries such as South Africa, where three mutations cause 95% of heterozygous FH in the Afrikaners, and Germany or England, where there are many more mutations. In clinical practice, a strategy for the genetic diagnosis of heterozygous FH, tailored to the mutational spectrum of patients likely to be seen at the particular hospital/region of the country, will be more efficient than screening of the whole LDL receptor gene by techniques such as single-strand conformation polymorphism (SSCP) analysis in every heterozygous FH candidate. In Aarhus, Denmark, we have chosen to examine all heterozygous FH candidates for the five most common LDL receptor gene mutations (W23X, W66G, W556S, 313 + 1G --> A, 1846 - 1G --> A) and the apoB-3500 mutation by rapid restriction fragment analysis. Negative samples are examined for other mutations by SSCP analysis followed by DNA sequencing of the exon indicated by SSCP to contain a mutation. If no point mutation or small insertion/deletion is detected, Southern blot or Long PCR analysis is performed to look for the presence of large gene rearrangements. In conclusion, our data suggest that an efficient molecular diagnostic strategy depends on the composition of common and rare mutations in a population.     

家族性高胆固醇血症家系低密度脂蛋白受体活性及其基因突变分析

目的:对1例临床诊断为家族性高胆固醇血症(FH)纯合子患者家系进行低密度脂蛋白受体(LDL-R)活性研究及其基因突变分析,旨在探讨其分子病理机制。方法:对先证者及其父母进行血脂、颈动脉超声等检查;采用流式细胞仪检测先证者及其父母外周血淋巴细胞LDL-R表达量和功能;以先证者基因组DNA为模板,扩增载脂蛋白B100(ApoB100)基因3500区域片断,PCR产物进行核苷酸序列分析,排除由该突变导致家族性ApoB100缺陷症;用降落式PCR方法,在同一程序中分别扩增LDL-R基因的启动子和全部18个外显子片段,扩增产物进行核苷酸序列分析;发现突变后验证其父母是否存在相同基因突变。结果:先证者血清TC和LDL水平明显升高、颈动脉内中膜明显增厚;LDL-R的表达量和结合功能显著下降(分别为正常人的13.6%和21.1%);ApoB100基因3500区域未见突变,LDL-R基因第1864位碱基T取代了G发生纯合错义突变,导致第601位氨基酸天冬氨酸变成脯氨酸(D601Y)。其父母LDL-R基因发现了相同的杂合突变,经检索该突变在我国未见报道。结论:证实家系先证者存在LDL-R基因突变,分别来源于父系和母系的遗传,该突变可能是家系发病的分子基础;D601Y也可能是我国FH患者LDL-R基因一种新的突变类型。     

家族性高胆固醇血症患者低密度脂蛋白受体基因复合纯合突变一例

目的 检测家族性高胆固醇血症(FH)患者低密度脂蛋白受体(LDL—R)基因点突变，分析基因型与临床表型间的关系，探讨其分子病理机制。方法以1例临床诊断为FH纯合子患儿及其父母的基因组DNA为模板，用降落PCR方法，在同一程序中分别对LDL—R基因的启动子和全部18个外显子片段进行扩增，琼脂糖凝胶电泳检测，扩增产物直接进行核苷酸序列分析，并检索FH突变数据库(www．ucl．ac．uk／fh)。此外采用PCR-限制性内切酶技术，检测载脂蛋白(Apo)B100基因Q3500R突变，以排除家族性Apo B100缺陷症。结果该患儿及其父亲第4外显子发生Cys^122→Tyr(C122Y)杂合错义突变，同时患儿及其母第9外显子发生Thr^273→Ile(17383I)杂合错义突变，因此该患儿LDLR基因第4、9外显子各存在一个杂合突变，上述突变尚未在FH突变数据库见到。未检测出患儿及其父母Apo B100Q3500R突变。结论此患儿为LDL—R基因存在C122Y、T3831合纯合突变并分别来自其父母；以上两位点突变可引起FH；是FH患者LDL-R基因的一种新突变类型。     

Familial hypercholesterolemia in Utah kindred with novel R103W mutations in exon 4 of the LDL receptor gene

Heterozygous familial hypercholesterolemia (FH) is a serious disorder causing twice normal low-density lipoprotein cholesterol levels early in childhood and very early coronary disease in both men and women. Previously published blood cholesterol criteria greatly under-diagnosed new cases of FH among members of known families with FH and over-diagnosed FH among participants of general population screening. Thus, there is a need for accurate and genetically validated criteria for the early diagnosis of heterozygous FH. In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To carry out molecular genetic diagnosis of the disease, we screened DNA samples for mutations in all 18 exons and the exon- intron boundaries of the low-density lipoprotein (LDL) receptor gene. Novel point mutations were identified in the proband: a C-to-T transversion at nucleotide position 369, causing substitution of Tryptophan for Arginine at codon 103 in exon 4 of the LDL receptor gene. The SSCP method was used to examine seven members of the family recruited for the diagnosis. This method helped to unequivocally diagnose only the proband as heterozygous for this particular LDL receptor mutation while excluding the remaining six individuals from carrier status with FH.     

Familial hypercholesterolemia in Utah kindred with novel 2412-6 Ins G mutations in exon 17 of the LDL receptor gene

Familial hypercholesterolemia (FH) is a monogenic disorder associated with primary hypercholesterolemia. FH is characterized by autosomal co-dominant inheritance with strikingly elevated LDL-cholesterol, the presence of xanthoma and premature atherosclerosis. In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To determine the genetic etiology of the lipoprotein abnormalities, we screened DNA samples from the family for mutations in all 18 exons and the exon- intron boundaries of the low-density lipoprotein receptor (LDLR) gene. Novel point mutations were identified in the proband: a one-base insertion of G to a five-G stretch at nucleotides 2412-6 (codons 783-785), causing a frameshift in exon 17 of the LDL receptor gene. The direct sequencing method was used to examine six members of the family recruited for the diagnosis. This method helped to unequivocally diagnose the five individuals as heterozygous for this particular LDL receptor mutation. This method also helped us to diagnose with FH, or to exclude from carrier status, three children between ages 6 and 11.     

姐妹2人同患家族性高胆固醇血症家系LDL-R基因突变分析

目的:对临床确诊的家族性高胆固醇血症(FH)两姐妹及其家系成员进行低密度脂蛋白受体(LDL-R)基因突变分析,探讨其发病机制。方法:提取患者外周血基因组DNA,聚合酶链反应分别扩增启动子和18个外显子片断,采用单链构象多态性(PCR-SSCP)结合银染技术,对异常电泳条带进行核苷酸序列分析。结果:姐妹2人及其父亲,叔叔,祖母均发现LDL-R基因第13外显子存在一个错义突变,与GeneBank对照证实第1879位G→A碱基置换,氨基酸的改变为丙氨酸→苏氨酸(A606T突变),其母亲和女儿经测序并未发现此突变位点。结论:姐妹2人均为LDL-R基因存在A606T杂合错义突变,并均来自其父系亲属;可能是该家系发病的分子基础。     

c-kit gene mutation at exon 17 or 13 is very rare in sporadic gastrointestinal stromal tumors

BACKGROUND AND AIM: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the human gut. They frequently have gain-of-function mutations of the c-kit gene, which encodes a receptor, tyrosine kinase. The mutations were found at exon 11 in most cases, and either at exon 9 or at exon 13 in rare cases. Recently, we found a family with multiple GIST and a gain-of-function mutation at exon 17. The family was the first reported GIST case with c-kit gene mutation at exon 17 including sporadic GIST. Although we previously reported that the c-kit gene mutation at exon 17 was not detected in 124 sporadic GIST by single-strand conformation polymorphism (SSCP) analysis, the mutation at exon 17 observed in the familial GIST was detectable by the use of direct sequencing but not by our SSCP method. In the present study, we examined the mutations at exon 17 and exon 13 by using direct sequencing. METHODS: Genomic DNA was extracted from formalin-fixed, paraffin-embedded GIST tissues. We could obtain 143 sporadic GIST cases appropriate for DNA analysis at exon 17 and 141 at exon 13. Exons 17 and 13 were amplified by using polymerase chain reaction and direct sequencing was conducted. RESULTS: No mutation was found at exon 17, and only one case with the mutation at exon 13 was observed. The GIST with the mutation at exon 13 was large and showed frequent mitosis, and the patient died of the recurrent GIST 3 years after the first operation. CONCLUSION: The mutation at exons 17 or 13 was considered to be very rare in sporadic GIST.     

Rearrangements in the LDL receptor gene in Dutch familial hypercholesterolemic patients and the presence of a common 4 kb deletion

DNA samples from 53 unrelated Dutch patients with familial hypercholesterolemia (FH) were screened for rearrangements in the gene for the LDL receptor (LDLR) by Southern analysis. Four different mutations have been detected by hybridisation of BglII digested genomic DNA with an exon 10-14 containing cDNA probe. The mutations are defined by a 7 kb insertion near exon 11, a partial gene duplication encompassing exons 9-12, a 4 kb deletion of exons 7 and 8 and an 0.4 kb deletion comprising the 5'-part of exon 16. These four different rearrangements in the LDLR gene account for 17% of the mutations in the Dutch FH population sample. Interestingly, the 4 kb deletion was detected in 5 unrelated FH patients (9.5%) and appeared to be identical to the deletion previously described (Russell, D.W. et al., Arteriosclerosis, 9 (Suppl. I) (1989) I-8; Russell, D.W. et al., Cold Spring Harbor Symp. Quant. Biol., 51 (1987) 401). in an FH patient of Dutch origin. This suggests that the 4 kb deletion is a common mutation in the Dutch FH population.