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.     

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。     

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.     

Gene mapping of autosomal dominant retinitis pigmentosa in a Chinese family

Background The autosomal dominant form of retinitis pigmentosa （ADRP） can be caused by mutations in 14 genes and further loci remains to be identified. This study was intended to identify mutations in a Chinese pedigree with ADRP.
Methods A large Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite （STR） markers tightly linked to genes known to be responsible for ADRP were selected for linkage analysis. Exons along with adjacent splice junctions of PRPF31 were amplified by polymerase chain reaction （PCR） and screened by direct sequencing.
Results The caused gene of ADRP was mapped to 19q13.4 between markers D19S572 and D19S877, with a maximum LOD score of 3.01 at marker D19S418 （recombination fraction=0）.
Conclusion The affected gene linked to the 19q13.4 in a Chinese family with ADRP, which is different from other mutations at the same loci in other Chinese families.     

视紫红质和Peripherin/RDS基因在视网膜色素变性家系中的突变检测

目的 对一常染色体显性视网膜色素变性(autosomal dominant retinitis pigmentosa,adRP) 家系进行视紫红质基因(rhodopsin,RHO)、盘膜边缘蛋白/ 视网膜变性慢基因(Peripherin/retinal degeneration slow,Peripherin/RDS)、视杆外节盘膜蛋白1 基因(retinal outer segment membrane protein 1,ROM1)、神经视网膜亮氨酸拉链基因(neural retinal leucine zipper,NRL) 和视锥杆细胞同源盒基(cone-rod homeobox-containing gene,CRX) 基因的突变检测.方法 采集一连续3 代发病的adRP 家系28 名成员外周血,提取基因组DNA,采用聚合酶链反应(polymerase chain reaction,PCR) 和直接测序技术,对RHO、Peripherin/RDS、ROM1、NRL 和CRX 基因进行检测,结果与标准核酸序列进行比对和分析.结果 该家系成员在RHO、Peripherin/RDS、ROM1、NRL 和CRX 基因中未发现致病突变,但是在Peripherin/RDS 基因第1 外显子和第3 外显子编码区发现4 处单核苷酸改变.结论 该家系在RHO、Peripherin/RDS、ROM1、NRL 和CRX 基因中未检测到致病突变,Peripherin/RDS 基因外显子中4 处单核苷酸改变属于单核苷酸多态性(single nucleotide polymorphisms,SNPs).     

两个家系中X连锁视网膜色素变性的RP2 基因无义突变

目的 检测引起2个家系产生X连锁视网膜色素变性的RP2基因突变。方法 根据RP2基因外显子的内含子DNA序列合成8对引物，以人基因组DNA为模板，PCR扩增出包含RP2基因所有外显子的8个片段。扩增产物化后直接测序。通过比较病人和正常人相应的DNA序列，检测基因突变位点。结果 在2个家系中首次检测到RP2基因的同一个无义突变38C→T。突变位于RP2基因的第2外显子。它使该基因编码精氨酸的遗传密码CGA变为终止密码TGA，引起发病。结论 该突变的检出有助于RP2蛋白的功能分析和X连锁视网膜色素变性的基因诊断。     

SCREENING FOR MUTATIONS IN A NOVEL RETIAL—SPECIFIC GENE AMONG CHINESE PATIENTS WITH RETINTITS PIGMENTOSA

Objective.To identify and evaluate mutations in the RP1 gene among Chinese patients with retinitis pigmentosa(RP).Methods.Leukocyte DNA of 92 RP patients were collected in Hong Kong.Sequence changes of the entire coding region of the RP1 gene were examined using PCR,conformation sensitive gel electrophoresis and DNA sequencing.Results.In total,1 nonsense mutation and 1 nonsense variant as well as 10 missense alterations were identified in the RP1 gene,among which,Arg 677 Ter was found in 1 RP patient and another nonsense variant,Arg 1933Ter ,was identified in 3 normal individuals and 1 patient with Stargardt‘s disease,suggesting its nonpatogenicity,Arg667 Ter is expected to lead to large disruptions of the encoded protein.Couclusions.The nonpathogenicity of Arg 1933 Ter indicates that the C-terminal 224 residues of RP1 protein may be not critical for RP1.The most C-terminal truncation previously reported was due to Tyr1053(1-bp del) and occurred in RP patients.Thus RP can be caused by reduction in the level of the region of RP1 protein after condon 1052 but before 1933.T o ascertain such a proposition,genotypes of more RP patients may reveal more RP cousative mutations and more sequence alterations different than those of other ethnic groups.     

SCREENING FOR MUTATIONS IN A NOVEL RETINA-SPECIFIC GENE AMONG CHINESE PATIENTS WITH RETINITIS PIGMENTOSA

To identify and evaluate mutations in the RPl gene among Chinese patients with retinitis pigmen-tosa (RP).Methods. Leukocyte DNA of 92 RP patients were collected in Hong Kong. Sequence changes of the entire coding region of the RP1 gene were examined using PCR, conformation sensitive gel electrophoresis and DNA sequencing.Results. In total, 1 nonsense mutation and 1 nonsense variant as well as 10 missense alterations were identified in the RPl gene, among which, Arg677Ter was found in 1 RP patient and another nonsense variant, Argl933Ter, was identified in 3 normal individuals and 1 patient with Stargardt' s disease, suggesting its nonpathogenicity. Arg677Ter is expected to lead to large disruptions of the encoded protein.Conclusions. The nonpathogenicity of Argl933Ter indicates that the C - terminal 224 residues of RPl protein may be not critical for RPl. The most C - terminal truncation previously reported was due to Tyr1053 (1-bp del) and occurred in RP patients. Thus RP can be caused by reduction in     

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。     

应用微卫星多态位点对X连锁型视网膜色素变性疾病进行遗传连锁分析的研究

目的应用遗传连锁分析方法对X连锁型视网膜色素变性家系进行分析,确定其致病基因的所在位点。方法在2个目前常见的X-连锁遗传位点——RP2和RP3处分别选取具有高信息量的微卫星位标,对2例疑似X连锁型视网膜色素变性家系进行遗传连锁分析。通过对家系成员的单倍型分析,并使用两点法计算其最大优势时数（LOD Score）值,确定致病基因所在的染色体位置。结果微卫星标记DXS993与2例遗传家系表型间最大LOD值分别为：〈-2和0．8;DXS 1068在2例遗传家系LOD值分别为0．4和0．8。结论RP2基因可能不是XY家系的致病性基因;在ZLK家系,怀疑疾病位点与RP2或RP3基因连锁。用遗传连锁分析方法确定致病基因所在染色体的范围起到重要的作用。     

两例疑为X连锁型视网膜色素变性家系的单倍型分析研究

目的应用遗传连锁分析方法对X连锁型视网膜色素变性家系进行分析,定位其致病基因的所在位点.方法选取已知X连锁视网膜色素变性候选基因附近的短串联重复序列多态性标记(short tandem repeat polymorphism, STRP),即在RP2、RP3、RP6、RP23和RP24处分别选取具有高信息量的微卫星位标,对2例疑似为X连锁型视网膜色素变性家系进行遗传连锁分析;通过对家系成员的单倍型分析,并进行两点法连锁分析,确定其致病基因所在染色体的大致位置.结果 2例家系在DXS 993处得到的最大LOD值分别为:1.18和1.03;在DXS 1068处得到的最大 LOD值分别为:0.58和-2.69;在DXS 1214处得到的最大LOD值分别为:-2.33和-2.45;在DXS 8051处得到的最大LOD值分别为:-2.34和-2.51;在DXS 8043处得到的最大LOD值分别为:-2.23和-2.62.结论 ZCF家系的致病基因,可能不在RP3、RP6、RP23或RP24位点;而对于FYJ家系,我们则怀疑致病基因位于RP2位点,但也不排除与RP3连锁;用遗传连锁分析方法对确定致病基因所在染色体的范围起到重要的作用.     

Clinical evidence in concurrence of retinitis pigmentosa and glaucoma

Glaucoma is rarely complicated by retinitis pigmentosa (RP). To provide clinical evidences for this rare situation, we report the concurrence of these two diseases in two children of a Chinese family. In the present two-child Chinese family without positive history, the older sister presented with bilateral sector RP and coexisting chronic angle-closure glaucoma, and the brother with bilateral whole RP but without coexisting glaucoma. Clinical evidences in concurrence of variants of RP and glaucoma because of possible different gene mutations from the same genetic background represent a rare situation, which may provide clues for future researches in molecular pathogenesis of these rare diseases.

     

Two novel mutations of the retinitis pigmentosa GTPase regulator gene in two Chinese families with X-linked retinitis pigmentosa

目的：检测引起两个中国家系产生X连锁视网膜色素变性的RPGR基因突变。方法：以人类基因组DNA为模板,用位于内含子的引物扩增出RPGR基因外显子1-19的PCR片段。PCR产物经SSCP分析后直接进行测序。通过比较病人和正常人相应的DNA顺序,检出基因突变位点。结果：在两个家系检测到两个新突变c1536delC和E332X,它们分别位于RPGR基因的第12和第9外显子（这是两个外显子首次发现的突变）。两突变均可导致翻译提前终止,产生不具备正常功能的截短蛋白。结论：两个突变是相应空系产生视网膜色素变性的遗传学基础,所得结果有助于分析RPGR蛋白功能。     

视网膜色素变性家系应用助视器的效果随访分析

目的探讨助视器在视网膜色素变性(RP)患者中的应用价值。方法分析一例RP家系的患病家谱、患者的生活视力以及验配助视器后的效果。结果该家系4代人中患病12例,其中3人日常生活视力均≤0.12,应用2.8×2.8倍眼镜式远用助视器后,其康复视力分别达到了0.4、0.4、0.6,摆脱了低视力困扰。结论助视器可提高RP患者的生活视力和生活质量,其临床价值应得到重视。     

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…     

Clinical evidences in concurrence of retinitis pigmentosa and glaucoma

Objective To provide clinical evidences for concurrence of retinitis pigmentosa and glaucoma in two children of a Chinese family. Design Case series. Participants 2 cases with varying degrees of retinitis pigmentosa and glaucoma in a two-child Chinese family. Methods Complete ophthalmic examinations of the two present cases are conducted. The interesting concurrence of retinitis pigmentosa and glaucoma is reviewed and discussed. Main Outcome Measures The clinical findings including general ocular examinations, visual fields, ultrasound biomicroscopy, B ultrasonography, flourescein angiography, optical coherence tomography and electrophysiologic tests. Results In the present two-child Chinese family without positive history, the older sister presented with bilateral sector retinitis pigmentosa and coexisting chronic angle-closure glaucoma, and the brother with bilateral whole retinitis pigmentosa but without coexisting glaucoma. Conclusions Clinical evidences in concurrence of variants of retinitis pigmentosa and glaucoma because of possible different gene mutations from the same genetic background represent a rare situation, which may provide clues for future researches in molecular pathogenesis of these rare diseases.     

一个疑似X连锁型视网膜色素变性家系的遗传连锁分析的研究

目的对一个疑似X连锁型视网膜色素变性(X-linked retinitis pigmentosa,XLRP)家系进行分析,确定其致病基因的所在位点。方法选取已知XLRP候选基因附近的微卫星位标,用多点参数分析方法计算其最大优势对数值(LOD score),并通过对家系成员单倍型分析,确定该家系致病基因所在的染色体位置。结果位于X染色体长臂的微卫星标记DXS8043与该例遗传家系间最大LOD值小于-2,其他位于X染色体短臂的11个微卫星标记与该例遗传家系LOD值保持在0.5上下,无明显的高值。单倍型结果表明该家系中2例患者兄弟(Ⅲ1、Ⅲ6)和他们的携带者母亲(Ⅱ2)在DXS8051与DXS1214之间拥有在正常成员中不存在的基因型,表明该基因型与疾病共分离,进一步确定了他们X性连锁遗传模式,并且可将致病基因初步定位于DXS8051与DXS1214之间的RP23和RP6基因。结论可以排除该例家系的致病位点与X染色体长臂上的RP24基因的连锁,高度怀疑连锁位点存在于X染色体的短臂的RP23和RP6基因,需另增加位标的信息量,以进一步缩小致病基因所在的具体范围。     

Genetic diagnosis of Liddle’s syndrome by mutation analysis of SCNN1B and SCNN1G in a Chinese family

Background  Liddle’s syndrome is a rare autosomal-dominant monogenic form of salt-sensitive hypertension. This study aimed to screen the gene mutation in β and γ subunits of the epithelial sodium channel (ENaC) of a Chinese family with Liddle’s syndrome, an autosomal dominant form of hypertension.

Methods  DNA samples from the proband with early-onset, treatment-resistant hypertension and suppressed plasma renin activity were initially screened for mutations in the C-terminal exons of the ENaC β or γ subunit genes, using amplification by polymerase chain reaction and direct DNA sequencing. We also screened the C-terminus of SCNN1B and SCNN1G in family members, and screened for the mutation in 150 controls.

Results  Genetic analysis of the β ENaC gene revealed a missense mutation of CCC to TCC at codon 616 in the proband, her mother and her grandmother. One hundred and fifty randomly selected controls had not the mutation, indicating that this is not a common genetic polymorphism. There was no mutation of the γ ENaC gene in any of the individuals examined.

Conclusions  Through direct DNA sequencing analysis, we established the diagnosis of Liddle’s syndrome for the proband and her families, and provided tailored therapies to this abnormality. These results provide further evidence that Pro616Ser is a critical amino acid that has a key role in the inhibition of sodium channel activity.

     

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     

载脂蛋白E与视网膜疾病关系的研究

目的 :通过检测中国汉族人视网膜色素变性患者的载脂蛋白E表型及孔源性视网膜脱离病人视网膜下液(SRF)及血液中载脂蛋白E(apoE)的含量 ,探讨载脂蛋白E与视网膜疾病的关系。方法 :采用等电聚焦及免疫印迹方法检测正常人和视网膜色素变性患者的aopE表型及ELISA方法测定孔源性视网膜脱离病人视网膜下液(SRF)及血液中载脂蛋白E(apoE)的含量。结果 :aopE的ε4 等位基因与视网膜色素变性的发病有关联 (RR =2 5 0 71P <0 0 1)。SRF中apoE的含量与视网膜脱离的范围及时间相关 (P <0 0 1)。结论 :aopE的ε4 基因可能是视网膜色素变性发病的又一遗传因素 ;apoE与孔源性视网膜脱离的损伤修复有关