上海市汉族多囊肾病基因2单核苷酸多态性的检测

在多囊肾病基因2（PKD2）中存在若干单核苷酸多态性（SNP）。PKD2突变可导致常染色体显性多囊肾病（ADPKD）。目前在PKD2中已检出数个SNP，但筛选的样本均来自高加索人。有学者认为，基因组平均每1001bp就有1个SNP，PKD2全长68000bp，理论上应有60多个SNP，这些遗传标记对于ADPKD发病机制的阐明具有重要意义。本研究检测PKD2中的SNP及其在上海市汉族人群中的频率分布。现报告如下。     

肌萎缩侧索硬化一家系的临床特征及SOD1基因分析

目的探讨家族性肌萎缩侧索硬化（FALS）一家系的临床特点及SOD1基因突变规律。方法分析一个FALS大家系的临床特征、肌电图改变和遗传方式,用PCR—SSCP法检测SOD1基因第1～5号外显子的突变。结果该家系有4代46人,其中6人患病,5人死于肌萎缩侧索硬化（ALS）,具典型ALS症状。PCR—SSCP法检测SOD1基因第1～5号外显子未发现突变。结论该家系为非SODI基因突变的FALS家系,临床特征与SOD1基因突变家系有明显差异。     

中国汉族人群PKD2基因多态性检测

目的：检测中国汉族人群PKD2基因多态性。方法：选取50名健康志愿者，提取外周血白细囊DNA，应用聚合酶链反应-单链构象多态性分析技术(PCR-SSCP)进行多态性检测，取异常条带标本进行核苷酸序列测定，判别PKD2外显子基因多态性位点及类型。结果：从50名健康人中成功检测出2种多态性。第1种为PKD2外显子7的1716位碱基由鸟嘌呤置换为腺嘌呤，编码氨基酸仍为赣氨酸。第2种为PKD2外显子1的第420位碱基由鸟嘌呤置换为腺嘌呤，编码氨基酸仍为甘氨酸。结论：建立了PCR-SSCP直接检测我国汉族人PKD2基因多态性的方法，并成功检测出2种PKD2基因多态性，为开展常染色体显性遗传性多囊肾病基因诊断奠定了基础。     

检测结核分枝杆菌rpoB基因突变的研究

目的：建立聚合酶链反应－单链构象多态性（PCR－SSCP）检测结核分枝杆菌rpoB基因突变的方法，并评价共临床应用的价值。方法：依据结核分枝杆菌rpoB基因的耐利福平决定区设计引物，用PCR从临床分离株和直接从痰标本中扩增rpoB基因片段；对扩得的rpoB基因片段做DNA SSCP分析，并随机测定rpoB片段的序列。结果：PCR从所有212株结核分枝杆菌中均扩得230bp片段135份抗酸染色阳性的痰标本中，有113份扩得阳性片段（83．7％），在SSCP分析中，140份经L－J药敏试验检测为利福平耐受的结核分枝杆菌，有130份有rpoB基因突变（符合率为92．9％），72份经L－J药敏试验检测为利福平敏感的菌，有67份的rpoB基因无突变（符合率为93．1％），测序分析发现57份经SSCP检测为突变的rpoB片段均有序列改变，10份经SSCP分析为无突变的有8份无序列改变，SSCP与测序结果的符合率为94．0％，在本研究的菌株中，耐多药结核病（MDR－TB）占76．4％（107／140），有92．5％（99／107）耐多药菌株经SSCP检测有rpoB突变。结论：建立的PCR－SSCP分析方法，是一种较准确和稳定的检测结核分枝杆菌rpoB基因突变的方法，可用于临床快速分析患者结核分枝杆菌对利福平的耐药性，并可作为MDR－TB判断的一个重要指标。     

多重PCR在瘢痕疙瘩Fas基因突变分析中的应用

目的：建立瘢痕疙瘩Fas基因常见突变外显子的多重PCR扩增反应体系，以利进行批量瘢痕疙瘩基因突变的筛选和检测。方法：取正常皮肤、增生瘢痕和瘢痕疙瘩组织DNA样本，根据瘢痕疙瘩Fas基因易发突变的外显子6，8，9之序列，按照多重PCR引物设计原则，建立3对相应引物，先分别找出各外显子的最佳扩增条件，再进行综合分析，最终找到一个理想的多重PCR扩增条件。结果：各外显子单一扩增片段和多重PCR扩增条带均清晰，产量较高，无非特异性扩增，产物长度与理论值一致；DNA测序证实，各扩增产物即各外显子基因片段。结论：成功建立了瘢痕疙瘩Fas基因常发突变外显子的多重PCR扩增体系，与以往单基因分次扩增相比，实现了一次同时扩增3个基因片段，为瘢痕疙瘩基因诊断和进一步相关的分子生物学研究提供了一个经济、快捷的方法，对于瘢痕疙瘩Fas基因的突变研究具有较高的应用价值。     

遗传性蛋白C缺陷症家系的一个基因突变

目的 研究一个遗传性蛋白C（PC）缺陷症家系的遗传表型及基因特征。方法 PC活性用凝固法测定，PC抗原用ELISA方法测定。用PCR扩增2代家系12个成员中4个PC活性及抗原减低的PCⅡ-Ⅸ号外显子片段，用单链构象多态性（SSCP）分析cDNA变性后的差异，用测序法检测突变点。用限制性酶切验证突变点，同时分析家系的基因型。结果 该家系2代4名成员PC抗原水平在34．3％－67．8％（参考值80％－120％）。PC活性在22％－49％（参考值70％－130％），较正常参考范围明显减低。限制性酶切分析该家系12名成员时发现9名成员存在基因的突变。基因突变位点在Ⅶ号外显子第6219位核苷酸G→A突变，使正常编码的CGG精氨酸突变为CAG谷氨酰胺。结论 该家系为I型PC缺陷症，基因分析证明先证为杂合子型。在PCⅦ号外显子上第6219位核苷酸G→A突变，在蛋白质合成过程中第169位精氨酸被谷氨酰胺替代（R→Q），为目前国内献中尚未报道的一个基因突变点。     

ALK-1基因无义突变Arg 479 Stop导致的遗传性出血性毛细血管扩张症

目的：对一个遗传性出血性毛细血管扩张症家系进行Endoglin基因和ALK1基因突变检测，以探讨其病因。方法：用PCR法对先证者的Endoglin基因14个外显子和ALK-1基因10个外显子及其侧翼序列和5′端非翻译区(5′UTR)序列进行扩增，PCR产物纯化后直接测序，检测其基因突变。针对家系成员3代23人基因突变区域进行扩增后测序。结果：发现先证者及家系其他患者ALK-1基因第10外显子1437C→T，导致479位氨基酸Arg(CGA)变为Stop(UGA)，均为杂合突变。结论：杂合无义突变C1437T引起的Arg 479 stop是导致本家系遗传性出血性毛细血管扩张症的原因。     

Glu29→Gly纯合子突变导致的遗传性凝血酶原缺乏症

目的 对1个遗传性凝血酶原缺乏症家系进行凝血酶原(FⅡ)基因突变的检测。方法 用活化的部分凝血活酶时间（APTT)，凝血酶原时间(PT)及FⅡ促凝活性(FⅡ：C)、FⅡ抗原(FⅡ：Ag)测定进行表型诊断；用PCR法对先证的FⅡ基因14个外显子及其侧翼序列和5′端非翻译区(5′UTR)、3′端非翻译区(3′UTR)序列进行扩增，PCR产物纯化后直接测序，检测其基因突变。家系成员DNA在先证FⅡ基因突变区域扩增后测序。突变位点经限制性内切酶分析证实。103名健康献血作对照。结果 先证表型诊断为凝血酶原缺乏症(Ⅰ型)；FⅡ外显子区共发现3个与献报道的FⅡ基因序列不同的位点，其中位于第2外显子区的为纯合突变A601G。家系分析表明先证父亲、母亲和外祖母均为A601G杂合子。结论 纯合错义突变A601G引起的Glu29→Gly是导致该例遗传性凝血酶原缺乏的原因。     

七例多囊肾患者及其家属PKD基因突变分析

目的 研究太原市中心医院肾内科收集的7 例多囊肾患者及其家属外周血PKD 基因的突变情况.方法 采用聚合酶链式反应(PCR)技术扩增PKD1 和PKD2 部分外显子,PCR 产物直接测序,据测序图谱进行突变分析.结果 1 例患者及其三个女儿PKD2 的4 号外显子35689 位C(T)杂合突变,致使氨基酸由精氨酸突变为终止密码子,发生无义突变;7 例患者及其家属均在PKD1 的11、12 号外显子出现杂合突变,其中27752 位点位于12 内含子14 位,不会影响氨基酸的变化,目前还不确定是否为致病突变.11 号外显子共计发现5 个突变位点,其中26310、26547 为同义突变,26411、26438、26495 为错义突变.结论 外周血PKD 基因的突变检测可作为多囊肾病症前及产前筛查有效的分子生物学诊断技术.     

抗凝血酶基因13389G缺失导致的I型抗凝血酶缺乏症

目的：对一先天性I型抗凝血酶缺乏症家系进行基因突变的检测。方法：用PCR法对先证者抗凝血酶基因的7个外显子及其侧翼内含子序列进行 扩增，PCR产物纯化后直接测序，检测其基因突变。结果：先证者表现为抗凝血酶基因外显子6区13389G缺失，引起移码突变。结论：该突变是先天性抗凝血酶缺乏症的一个新的突变位点，可以导致血栓形成。     

Mutation analysis of autosomal dominant polycystic kidney disease genes in Han Chinese

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2. The complexity of these genes, particularly PKD1, has complicated genetic screening, though recent advances have provided new opportunities for amplifying these genes. In the Han Chinese population, no complete mutational analysis has previously been conducted across the entire span of PKD1 and PKD2. Here, we used single-strand conformation polymorphism (SSCP) analysis to screen the entire coding sequence of PKD1 and PKD2 in 85 healthy controls and 72 Han Chinese from 24 ADPKD pedigrees. In addition to 11 normal variants, we identified 17 mutations (12 in PKD1 and 5 in PKD2), 15 of which were novel ones (11 for PKD1 and 4 for PKD2). We did not identify any seeming mutational hot spots in PKD1 and PKD2. Notably, we found several disease-associated C-T or G-A mutations that led to charge or hydrophobicity changes in the corresponding amino acids. This suggests that the mutations cause conformational alterations in the PKD1 and PKD2 protein products that may impact the normal protein functions. Our study is the first report of screenable mutations in the full-length PKD1 and PKD2 genes of the Han Chinese, and also offers a benchmark for comparisons between Caucasian and Han ADPKD pedigrees and patients.     

A single strand conformation polymorphism study of CD40 ligand. Efficient mutation analysis and carrier detection for X-linked hyper IgM syndrome.

Mutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed. 11 different mutations were found in DNA from all 13 patients whose activated T cells failed to bind a recombinant CD40 construct. The exact nature of four of these mutations, a deletion and three splice defects, could not be determined by cDNA sequencing. In addition, SSCP analysis permitted rapid carrier detection in two families in whom the source of the mutation was most likely a male with gonadal chimerism who passed the disorder on to some but not all of his daughters. These studies document the utility of SSCP analysis for both mutation detection and carrier detection in X-linked hyper IgM syndrome.     

Molecular taxonomy using single-strand conformation polymorphism (SSCP) analysis of mitochondrial ribosomal DNA genes

Single-strand conformation polymorphism (SSCP) analysis detects single point mutations in DNA molecules. We demonstrate that SSCP analysis of mitochondrial ribosomal DNA (rDNA) genes is a sensitive taxonomic tool because these genes often differ at numerous sites among closely related species. Using conserved primers, portions of the 12S or 16S rDNA genes were amplified using the polymerase chain reaction (PCR) in congeneric species of ticks, leaf hoppers, mosquitoes, and closely related endoparasitic wasps. SSCP was performed and products were visualized with silver staining. Species-specific patterns were observed in all taxa. Intraspecific variation at the level of single nucleotide substitutions was detected. SSCP diagnostics are less expensive and time consuming to develop than PCR with species-specific primers, and, unlike PCR with arbitrary primers, there is minimal concern with DNA contamination from non-target organisms.     

Simultaneous use of two retroviral vectors in human gene marking trials: feasibility and potential applications.

Two Moloney murine leukemia virus (Mo-MLV)-based neoR retroviral vectors--LNL6 and G1Na--were used to transduce various human tumor-infiltrating lymphocytes (TIL) populations. These groups included bulk CD(8+)- and CD(4+)-enriched TIL from human renal cell carcinomas and melanomas. Transduction efficiencies averaged 5% for single 4-hr supernatant infections. Integrated provirus could be detected for up to 4 weeks of in vitro culture. LNL6 provirus could be distinguished from G1Na provirus using specific polymerase chain reaction (PCR) primers. A single neomycin phosphotransferase (neoR) gene copy could be detected in 10(5) TIL. Using quantitative PCR, the relative ratio of LNL6 to G1Na copies in the same sample could be determined even at low copy numbers. These preclinical studies demonstrate the feasibility of using two retroviral marking vectors in human gene therapy efforts.     

Characterization of microsatellite markers to diagnose ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) maps to chromosome 16p13.3 (PKD1) and to chromosome 4q21-23 (PKD2), with the likelihood of a third unmapped locus. The size and genomic complexity of the PKD1 gene make it impractical to detect mutations for prenatal diagnosis. Therefore, pedigree-based linkage analysis remains useful for diagnosis of ADPKD. Since, the complete genome sequences of chromosome 16p13.3 and 4q21-23 including PKD1 and PKD2, respectively, were reported very recently, in order to do more precise diagnosis of ADPKD, we tried to find microsatellite markers. We performed database searches of 2000 kb of genome sequence across the 16p13.3 and the 4q21-23. To determine the distribution of alleles and the degree of polymorphism of the microsatellites, genotyping experiments were performed on 48 Korean individuals. We found novel 14 microsatellite markers around ADPKD that are more polymorphic and closer to PKD1 or PKD2 than the known markers. The novel microsatellite markers were applied to diagnose ADPKD families. These novel microsatellite markers are not only useful for presymptomatic and prenatal diagnosis of ADPKD, but also applicable in the study of positional cloning, human evolution and tumor biology.     

The molecular genetic analysis of polycystic kidney disease]

Autosomal dominant polycystic kidney disease (ADPKD) is one of common single gene disorders. The development of molecular genetic techniques has shown that mutant PKD1 gene assigned to ADPKD was closely linked to alpha-globin on the short arm of chromosome 16. This location was established when genetic linkage was found between ADPKD and a highly polymorphic region at the 3' end of the alpha-globin cluster (3' HVR). The discover of genetic linkage markers such as 3' HVR probe has provided a diagnostic test in presymptomatic stage. We performed this diagnostic test using DNA probes in 3 patients with ADPKD of one Japanese family. They also showed PKD1 gene linkage as previously described by Reeders et al. Linkage analysis of the PKD1 gene might be available to diagnostic test of ADPKD. DNA diagnosis of ADPKD however has to be performed carefully because of an ethical standpoint.     

逆转录套式聚合酶链反应检测庚型肝炎病毒

目的建立敏感、特异的逆转录套式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄ－ＰＣＲ）方法，以检测中国人庚型肝炎病毒（ＨＧＶ）感染。方法根据中国人感染ＨＧＶ者ＮＳ５区部分核苷酸序列分析结果，设计套式ＰＣＲ引物，用于ＨＧＶＲＮＡ的检测，并与用国外报道引物所作的一次ＰＣＲ和套式ＰＣＲ检测结果进行比较。结果共检测标本１３３份。以国外报道引物作一次ＰＣＲ和套式ＰＣＲ检出率分别为８．３％和１１．３％，作者建立的套式ＰＣＲ检出率为１８．０％，对部分ＰＣＲ产物进行序列分析证实为ＨＧＶ特异性基因。结论建立的方法可在中国人群中显著提高ＨＧＶＲＮＡ检出率。