常染色体显性视网膜色素变性家系基因定位

目的探讨一常染色体显性视网膜色素变性（autosomal dominant retinitis pigmentosa,adRP）家系致病基因与盘膜边缘蛋白/视网膜变性慢基因（eripherin/retinal degeneration slow,RDS）、视杆外节盘膜蛋白1基因（retinal outer segment membraneprotein 1,ROM1）、视锥杆细胞同源盒基因（cone-rod homeobox gene,CRX）、神经视网膜亮氨酸拉链基因（neural retinalleucine zipper,NRL）、鸟甘酸环化酶激活1B基因（guanylate cyclase activator 1B,GUCA1B）、肌动蛋白同源2基因（fascinhomolog 2,FSCN2）和拓扑异构酶结合1基因（topoisomerase I binding,TOPORS）多发突变位点的关系。方法采集一个连续4代发病的RP家系14个成员外周血4~5 ml,提取基因组DNA,采用聚合酶链反应（plymerase chain reaction,PCR）对常见的7个adRP候选基因的17个外显子多发突变位点进行扩增,PCR产物纯化后直接测序,测序结果与美国国立生物技术信息中心（National Center for Biotechnology Information,NCBI）数据库中公布的核酸标准序列进行比对分析。结果该家系视网膜色素变性为常染色体显性遗传,家系成员在RDS、ROM1、CRX、NRL、GUCA1B、FSCN2和TOPORS基因中未发现致病突变,仅在RDS基因第1外显子和第3外显子编码区发现5处单核苷酸改变。结论 RDS、ROM1、CRX、NRL、GUCA1B、FSCN2和TOPORS不是本研究家系的致病基因,RDS基因外显子中的5处单核苷酸的改变为单核苷酸多态性（single nucleotide polymorphism,SNP）。     

一个常染色体显性视网膜色素变性家系表型分析和RHO基因突变检测

目的分析一常染色体显性视网膜色素变性(autosomal dominant retinitis pigmentosa,adRP)家系的临床表型,确定该家系与视紫红质(rhodopsin,RHO)基因的关系。方法依据RP诊断标准及患者临床表型,确定一连续4代发病的adRP家系遗传的特点;采集家系中13位成员外周血8-10ml,提取基因组DNA;聚合酶链反应(polymerase chain reaction,PCR)扩增RHO基因的第1-5外显子基因片段,产物纯化后直接测序;测序结果与美国国立生物技术信息中心(National Center forBiotechnology Information,NCBI)数据库上公布的核酸标准序列进行比对分析。结果该家系临床特点为所有患者均于10岁左右出现夜盲,1例22岁出现视野损害,2例40岁左右出现视野损害,并且于50岁左右双眼相继发生急性闭角型青光眼和并发性白内障。该家系5例患者在RHO基因外显子、上下游非编码序列以及内含子及外显子拼接部中均未发现碱基改变。结论本研究家系患者存在遗传异质性和表型异质性,RHO基因不是该家系的致病基因。     

一汉族先天性视网膜劈裂家系RS1基因检测分析

背景先天性视网膜劈裂症是青少年黄斑变性的主要原因,主要表现为黄斑区轮辐状视网膜神经感觉层劈裂,RS1基因是其最常见的致病基因。目的检测一个汉族先天性视网膜劈裂家系的致病突变基因,探讨其遗传学基础。方法对参与研究的所有家系成员进行详细的眼科检查。利用直接测序技术对该家系进行RS1基因全部外显子及剪接位点测序,数据经分析比对,候选突变进行致病性评估。结果该汉族家系共4代,遗传模式符合X-性连锁遗传,家系中患病者眼底特征为视网膜劈裂。直接测序获得致病突变RS1 c.697GA(p.C219Y),该纯合错义突变在该家系中呈共分离。结论 RS1 c.697GA(p.C219Y)是该家系的致病突变。     

华人遗传性混合息肉病综合征BMPR1A基因种系突变检测

[目的]探讨遗传性混合息肉病综合征华人家系中BMPR1A基因是否存在种系突变.[方法]34个家系成员外周血提取基因组DNA,利用聚合酶链式反应分别扩增BMPR1A基因全部外显子,进行DNA测序与突变分析,对突变外显子PCR扩增产物作变性聚丙烯酰胺凝胶电泳鉴定.[结果]家系12和2所有发病成员BMPR1A基因2号外显子位于codon23处缺失11个碱基(AAAGTCAGAAA),同时2号外显子PCR扩增产物变性聚丙烯酰胺凝胶电泳也发现异常迁移条带,而家系中正常成员的检测未发现这一突变.[结论]两华人HMPS家系中BMPR1A基因缺失突变与疾病共遗传,该基因极可能是HMPS华人家系的致病基因.     

华人遗传性混合息肉病综合征BMPR1A基因种系突变检测

【目的】探讨遗传性混合息肉病综合征华人家系中BMPR1A基因是否存在种系突变。【方法】34个家系成员外周血提取基因组DNA，利用聚合酶链式反应分别扩增BMPR1A基因全部外显子，进行DNA测序与突变分析，对突变外显子PCR扩增产物作变性聚丙烯酰胺凝胶电泳鉴定。【结果】家系12和2所有发病成员BMPR1A基因2号外显子位于codon23处缺失11个碱基(AAAGTCAGAAA)，同时2号外显子PCR扩增产物变性聚丙烯酰胺凝胶电泳也发现异常迁移条带，而家系中正常成员的检测未发现这一突变。【结论】两华人HMPS家系中BMPR1A基因缺失突变与疾病共遗传，该基因极可能是HMPS华人家系的致病基因。     

广东地区视网膜色素变性两大家系RHO基因突变分析

目的 分析广东地区两个常染色体显性遗传视网膜色素变性(autosomal dominant retinitis pigmentosa，ADRP)家系视紫红质（rhodopsin, RHO）基因突变，探讨基因突变与临床表型的关系。 方法 收集广东地区两个常染色体显性遗传视网膜色素变性家系的临床资料，对家系成员进行视力、视野、眼底镜检查；应用聚合酶链反应（PCR）和直接测序技术，对两个家系所有现存成员进行RHO基因检测。 结果 发现一家系患者为P347S杂合性突变，另一家系患者存在P171L杂合性突变，两家系的临床表现为发病早，病情进展快，表型较严重，该两家系正常成员均未发现RHO基因突变。 结论 RHO基因的P347S与P171L突变分别为该两家系视网膜色素变性的病因。该两种突变均导致较严重的临床表型，与分子基础一致。     

常染色体显性遗传玻璃体视网膜脉络膜病变一家系的致病基因筛查及临床分析

目的 使用外显子结合目标区域捕获测序检测常染色体显性遗传玻璃体视网膜脉络膜病变(autosomal dominant vitreoretinochoroidopathy,ADVIRC)家系的致病基因,并分析其临床表型.方法 收集重庆地区常染色体显性遗传玻璃体视网膜脉络膜病变先证者及4代成员的临床资料,完善眼科检查.采集该家系4例患者及8例健康成员的外周静脉血,提取基因组DNA,运用外显子结合目标区域捕获测序芯片进行测序,并对测序结果进行分析后得到候选致病基因性突变位点.运用PCR和直接测序进行验证,确定致病基因.根据致病基因的临床表型对该家系进行临床分析.结果 基因检测发现第六外显子的杂合突变BEST1(c.704TR＞C),家系患者中出现的小角膜、先天性白内障、周边视网膜脉络膜发育不良、晚期易发生青光眼等符合该突变基因的临床表型.结论 BEST1基因的c.704TR＞C杂合突变为该ADVIRC家系的致病基因之一.     

全外显子组测序发现一家族性扩张型心肌病致病基因

 目的 对一扩张型心肌病(dilated cardiomyopathy, DCM)家系行全基因组外显子测序以寻找该家系的致病基因。方法 收集在复旦大学附属中山医院就诊的1位DCM患者及其家系成员的临床资料,采集相关家系成员外周血并抽提DNA,对该家系5名成员行全基因组外显子测序,寻找致病基因,用Sanger测序对家系其他成员进行验证。结果 通过对家系患者与正常人测序结果比对分析,同时经过多个生物数据库数据过滤,发现LMNA基因6号外显子上存在的杂合突变 c.961 C>T (p.Arg321Ter)为该家系的可能致病基因突变。LMNA c.961 C>T无义突变导致LMNA编码蛋白质过程提前终止,相应蛋白质功能异常,进而导致该家系中此突变基因的携带者出现心功能异常。结论 本研究应用全基因组外显子测序从一DCM家系中发现其致病基因及突变位点:LMNA c.961 C>T (p.Arg321Ter),突变导致该家系相关成员心功能异常。此位点在汉族人群中尚属首次报道。     

一个Paget骨病家系SQSTM1及TNFRSF11A基因突变分析

目的 研究一Paget骨病家系SQSTM1基因及TNFRSF11A基因突变情况。方法 收集一Paget骨病家系,家系成员外周血中提取基因组DNA,应用聚合酶链式反应、直接测序对该家系成员SQSTM1及TNFRSF11A基因外显子进行测序。测序结果与GenBank公布的SQSTM1和TNFRSF11A基因正常序列对比,寻找有无突变。结果 在该家系中未发现与Paget骨病共分离的致病基因突变,检测到14个已知的单核苷酸多态性。结论 该家系成员的发病情况与SQSTM1、TNFRSF11A基因中发现的SNP无相关性,排除其为该Paget骨病家系致病基因的可能性。     

利用变性高效液相色谱方法进行HCM家系MYH7基因突变筛查研究

目的:研究利用变性高效液相色谱技术(DHPLC)检测家族性肥厚型心肌病(hypertrophic cardiomyopathy,HCM)的主要致病基因β-肌球蛋白重链(beta-myosin heavy chain gene,MYH7)突变情况.方法:采用DHPLC结合DNA测序方法对3个新疆地区HCM家系成员的MYH7基因8、14外显子及附近上下游序列进行检测分析.结果:在其中一个家系中发现MYH7基因14外显子中存在Thr441Met突变,该突变在中国人中是首次发现.另外两个家系也发现有不同位点的突变.结论:MYH7基因在HCM家系中具有较高的突变率,不同突变基因型以及基因突变携带个体在临床表型上有所差异.运用变性高效液相色谱技术,能够快速有效地进行基因突变筛查,有利于对HCM家族成员的诊断、患病风险预测及疾病早期预防和治疗.     

Mass genetics study of rhodopsin point mutations in retinitis pigmentosa

Objective: To evaluate the incidence and pattern of rhodopsin (RHO) mutations in Chinese patients with retinitis pigmentosa (RP). Methods: Conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing were apphed to detect point mutations that occurred in the five coding exons and splice sites of RHO gene in 98 index patients with RP. Results: Four patients of one ADRP family were found to have a missense mutation at codon 347, Pro347Leu. One late-onset RP patient and her daughter, without clinical expression at present, were discovered to have a novel frameshift mutation at codon 327, Pm327 (1-bp del). Neither of the two mutations was found in 100 normal controls. Ma299Ser was found in one RP patient. Two control subjects also had Ma299Ser, suggesting its nonpathogenicity and just single nucleotide polymorphism (SNP). Conclusion: Two RP patients had rhodopsin mutations, thus the expected frequency of RHO mutations in RP is about 2.0% (95% confidence interval: 0.3%-4.4%). A highly conserved C-terminal sequence QVS（A) PA was altered due to Pro347Leu and thereby misdirecting rhodopsin to incorrect subcellular location. Loss of all phosphorylation sites at the C-tenninns and a highly conserved sequence QVS(A) PA may occur because of Pm327(1-bp del). To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying Pm327(1-bp del) would be of great value.     

Peripherin/RDS gene in Indonesian patients with retinitis pigmentosa: geographic comparison of polymorphic variations

To analyze peripherin/RDS (retinal degeneration slow) gene alterations in Indonesian patients with retinitis pigmentosa. We examined the gene in 13 unrelated Indonesian patients with retinitis pigmentosa and in 24 normal individuals. Peripheral venous blood was extracted, and genomic DNAs were amplified by polymerase chain reaction (PCR). The PCR products were directly sequenced. Each subject underwent ocular examination. The prevalence of the gene alteration was compared to that reported in Japanese and Caucasian populations. Among 13 patients, 3 concurrently had Glu304Gln and Gly338 Asp alterations at exon 3 of the peripherin/RDS gene. Two patients had heterozygous alterations and one had a homozygous variation. The prevalence of the alterations (23%) in Indonesian patients was similar to that in Japanese patients (26%) and was lower than that in Caucasian patients (30-70%). The alterations were also observed in 7 of 24 (29%) normal healthy Indonesian individuals. Peripherin/RDS gene polymorphisms (Glu304Gln and Gly338Asp) were found in Indonesian patients with retinitis pigmentosa. The prevalence of alterations in Indonesian patients was similar to that in Japanese patients and lower than in Caucasian patients.     

A COMPLETE SCREEN FOR MUTATIONS OF THE RHODOPSIN GENE IN A PANEL OF CHINESE PATIENTS WITH AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA

Objective To evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. Methods We have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families. Two RHO mutations, Pro347Leu and Pro327 (l-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years.The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (l-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been fiat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger daughter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls. Conclusions The frequency of RHO mutations in Chinese patients was lower than that in Europe and North America.The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (l-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.     

视网膜色素变性患者视紫红质基因突变分析

目的 研究中国人视网膜色素变性(RP)患者视紫红质(RHO)基因的突变频率及特征,探讨其在RP发病机制中的作用.方法 运用DNA直接测序法,对55例中国内地汉族RP先证者及55例对照者进行RHO全基因突变检测分析.结果 共检出7种碱基变异,其中2种为非致病错义突变,其余5种为非编码区单核苷酸多态性,RP组和对照组各单核苷酸多态性位点突变频率比较,差异均无统计学意义(P＞0.05).结论 RHO基因在中国华南地区RP 患者中的突变率低于国外报道.检出的已报道的单核苷酸多态性位点与RP无显著相关性.     

与缓慢型视网膜变性基因突变相关的弥漫型视网膜色素变性伴黄斑部病变的研究

目的 研究有缓慢型视网膜变性（ＲＤＳ）基因突变的临床表现,以探讨视网膜色素变性（ＲＰ）的分型方法,方法 对查出ＲＤＳ基因突变的ＲＰ病例进行家系分析,以及视力,裂隙灯显微镜,直接检眼镜,视网膜电图,动态和／或静态视野,Ｄ－１５色相配列试验,暗适应,眼底荧光血管造影或彩色眼底照相等眼科临床检查,结果 本研究４例ＲＰ患者在中青年时发病,夜盲及视力下降几乎同时出现视力下降明显,视网膜视维,视杆细胞功能明显     

Mutation Identification in A 5-Generation Pedigree with Autosomal Dominant Retinitis Pigmentosa

RetinitisPigmentosa (RP)referstoagroupofinheritedretinaldystrophiesthatarecharacterizedbyprogressivephotoreceptordegeneration InWesterncountriesRPhasareportedprevalenceof 19- 2 7per10 0 0 0 0 [1] Thesimilarprevalenceof 2 5 per 10 0 0 0 0wasalsoobservedinChina[2 ] GenescausingRPhavebeenidentifiedbyacombinationoflinkagemapping ,cloningandcandidatetesting Geneticheterogeneity ,allelicheterogeneityandclinicalheterogeneityhavebeendemonstratedamongpatientswithautosomaldominantRP (adRP) ,auto…     

香港地区汉族人视紫红质基因和视网膜色素变性1基因与视网膜色素变性的双基因关联分析

目的研究视紫红质基因(RHO)和视网膜色素变性1(RP1)基因在香港地区汉族视网膜色素变性(RP)患者中的突变频率及特征,并进一步探讨它们在RP发病机理中潜在的相互作用。方法运用高通量构象敏感凝胶电泳和直接测序方法,对151例香港地区汉族RP先证者和150名对照者进行了RHO和RP1基因全编码区和邻近剪切位点的内含子区域序列突变的检测。对携带基因突变的12例RP先证者的46名亲属进一步作分离分析。运用单因素分析、多因素分析和基因型家系不平衡分析研究RHO和RP1基因对RP的作用。结果RHO基因和RP1基因的突变检出率各为1.3%。RHO基因中-26G>A可增高RP危险性,而RP1基因中R872H则可降低RP危险性。多因素Logistic回归分析揭示了RHO基因IVS423G>A分别与RP1基因N985Y和C2033Y之间存在相互作用。结论在香港地区汉族RP患者中,RHO和RP1基因的突变率比其他人群中RP患者的突变率低。除了致病性基因突变,非编码区的序列改变也可改变RP的易感性。部分RP患者由双基因突变引起,当然多基因共同作用也完全可能存在。     

X-连锁隐性视网膜色素变性的分子遗传学分析

目的探讨1个X-连锁隐性视网膜色素变性(X-linkedrecessiveretinitispigmentosa,XLRRP)家系先证者分子遗传学基础。方法应用聚合酶链反应-直接测序,检测与XLRP相关基因视网膜GTP酶调节因子(retinitispigmentosaGTPaseregulator,RPGR)基因的所有外显子和突变热区15号外显子开放阅读框(exonopenreadingframe15,ORF15)及其与内含子交界处序列。结果检测到2种新的同义突变,c.2166A>G(Glu722)和c.3396C>T(Asp1132),都位于ORF15;以及4种已知多态c.29-15G>A,c.469 63C>T,c.1227 67A>G和c.1675-101A>T。结论尚不能确定此XLRP家系的疾病相关基因。