一原发性开角型青光眼家系OPTN基因4号和14号外显子基因突变研究

目的 研究中国大陆地区一原发性开角型青光眼(POAG)家系OPTN基因4号和14号外显子的突变情况.方法 采用聚合酶链反应(PCR)扩增POAG家系39例成员的OVTN基因4号和14号外显子,并对PCR产物进行单链构象多态性(SSCP)分析,对14号外显子同时利用内切酶Pag Ⅰ进行限制件片段长度多态性(RFLP)分析.同时用100例与该家系无亲缘关系的健康体检者进行对照试验.结果 该POAG家系成员OPTN基因4号和14号外显子通过SSCP分析均未发现突变,酶切14号外显子发现有2例可疑情况.正常对照组中SSCP分析和酶切分析均未发现突变及其可疑情况.结论 该家系开角型青光眼的发病可能并非由于OPTN基因4号和14号外显子的基因突变引起,寻找致病原因仍需作进一步研究.同时,SSCP分析和酶切方法的联合应用,可以提高检测的敏感性.     

遗传性对称性色素异常症一家系ADAR基因突变研究

目的研究遗传性对称性色素异常症家系的RNA特异性的腺苷脱氨酶(RNA-specific adeno-sine deaminase,ADAR)基因的突变。方法收集一个遗传性对称性色素异常症家系的临床资料,采用聚合酶链反应及直接测序法对家系内成员进行ADAR基因突变位点检测,同时对50名无血缘关系健康对照者的该位点进行直接测序,并对国内外报道的ADAR基因突变进行总结。结果该家系中所有患者均存在ADAR基因c.2447G>A突变,导致p.R816K改变,而在家系内非患者及正常对照者中均未发现该突变。目前国内外已报道ADAR基因突变为64种。结论ADAR基因突变是中国人群中遗传性对称性色素异常的致病原因之一,ADAR基因各功能区域均可发生突变,但ADEAMc可能为其热点突变区域。     

一个眼牙指发育不良家系的基因突变分析

目的 旨在报道一个罕见的中国汉族眼牙指发育不良家系,并对该家系进行基因突变分析.方法 采用PCR及测序方法检测该家系先证者GJA1基因编码区及其侧翼序列的突变,然后在家系成员中验证,并通过Weblogo软件对可疑变异位点进行保守性分析;同时利用PCR、电泳及测序分析方法,检测HOXD13基因突变,排除患者由HOXD13基因突变致病的可能.结果 该家系内患者均携带GJA1基因的杂合突变c.605 C>T,正常个体均无此突变;该突变导致Cx43蛋白第202位氨基酸由精氨酸变成组氨酸(p.R202H);HOXD13基因未见异常.结论GJA1基因c.605 C>T(p.R202H)突变是该中国汉族眼牙指发育不良家系的致病突变,该突变为中国人群中首次报道.     

一个视网膜色素变性家系的视紫红质基因突变分析

目的　确定常染色体显性遗传视网膜色素变性家系的致病基因及其突变位点,并研究其临床表型。方法　对一个常染色体显性遗传视网膜色素变性(autosomal dominat retinitis pigmentosa,ADRP)家系成员进行了视力、视野及眼底镜检查,并对该家系中先证者进行了视网膜电流图分析。应用聚合酶链反应和直接测序技术,对该家系的所有现存人员的视紫红质基因的外显子进行测序分析。结果　该家系的2 5名成员中12例患者有视紫红质基因(rhodopsin,RH O)的5 12 C>T(P171L)突变,均呈杂合子,该错义突变使密码子171由CCA变成CTA。而未受累者的视紫红质基因表现为野生型。该家系患者的临床表现为5～6岁时出现夜盲,在2 0～30岁逐渐出现视力和视野损害,并先后在4 0～5 0岁前后失明,其中2例患者并发青光眼,先证者的闪烁视网膜电图呈熄灭型。结论　视紫红质基因RH O的一种已知突变5 12 C>T(P171L)是该家系的病因。与国外相同的基因突变类型相比较,该家系发病早、病情进展快、视功能损害较重。     

一个中国近端指(趾)骨间关节黏连家系NOG基因新突变鉴定

目的 检测一个中国近端指(趾)骨间关节黏连家系的致病基因突变.方法 收集该家系患者和家系成员的临床资料,采集外周血提取基因组DNA.运用聚合酶链式反应和Sanger测序法筛查先证者的NOG和GDF5基因.确定突变位点后,在家系成员中进行共分离分析并在200名正常对照中筛查该突变.结果 该家系患者中存在NOG基因c.502 T＞C错义突变,该突变导致其编码蛋白Noggin第168位氨基酸由苯丙氨酸变为亮氨酸(p.F168L).在家系正常成员和200名正常对照中未检测到相同突变.该突变在dbSNP、ExAC等数据库中未见报道.在GDF5基因上未发现可疑突变.结论 NOG基因c.502 T＞C错义突变是该家系发病的原因.     

10个中国汉族寻常型鱼鳞病家系的FLG基因突变分析

目的 对10个寻常型鱼鳞病(ichthyosis vulgaris,Ⅳ)家系的FLG基因进行突变检测,并分析中国汉族人Ⅳ患者FLG基因的突变热点.方法 用聚合酶链反应扩增10个Ⅳ家系的所有患者及正常成员FLG基因的全部外显子,对扩增产物直接进行DNA测序检测突变,同时选取100名无亲缘关系的正常人作为对照.结果 在7个家系中发现了3种FLG基因的致病突变,包括c.3321delA、c.5757delCCAG和c.8138C＞T(p.S2706X),其中有两例患者的突变类型为3321delA的纯合突变,在另外3个家系中未发现FLG基因的突变.所有家系的正常成员和100名正常对照均未发现c.5757delCCAG和c.8138C＞T(p.S2706X),而在2名正常对照中发现其携带c.3321delA杂合突变.结论 在中国汉族Ⅳ家系检测到3种FLG基因的突变类型,其中c.3321delA是最常见的突变类型(46.9％).首次发现c.5757delCCAG和c.8138C＞T(p.S2706X)突变与Ⅳ相关.部分家系未检测到FLG基因突变,推测可能由其他致病基因导致.     

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.     

一个遗传性脊髓小脑型共济失调大家系的致病基因分析

目的探讨一个遗传性脊髓小脑型共济失调（SCA）大家系的遗传特点和基因突变分析。方法对一个遗传性脊髓小脑型共济失调（SCA）大家系进行家系调查,绘制系谱图,抽取家系成员外周血,采用聚合酶链反应和毛细管电泳对致病基因进行分析检测。结果该家系的遗传性脊髓小脑型共济失调（SCA）为常染色体显性遗传,6代45人中有15人为SCA患者,4人为携带致病基因的无症状患者。患者ATX3基因的CAG三核苷酸重复65-73次。结论该家系为常染色体显性遗传的SCA3型（SCA/MJD）,患者基因突变检测分析显示异常扩增的CAG突变数与发病年龄呈明显的负相关。基因突变检测在疾病诊断和早期发现无症状患者方面有重要作用,从遗传生殖角度阻断该病的遗传有重要意义。     

中国汉族1型Marie Unna遗传性少毛症家系U2HR的1个新突变

目的 对重庆地区一个常染色体显性遗传的遗传性少毛症家系进行基因诊断,并探讨其临床特点.方法 对先证者进行病史和家族史询问以及详细的临床检查.提取所有家系成员的基因组DNA,扩增HR基因及其上游开放阅读框基因的所有编码序列直接进行序列测定,与数据库中的参考序列进行比对分析.结果 包括先证者在内的所有患者自幼头发眉毛稀少,随年龄增长头发生长缓慢、易断,自青春期开始出现头发脱落.查体见弥漫性头发稀疏,发干粗糙质硬,呈现不规则扭曲样外观,睫毛、腋毛以及阴毛稀少.对先证者U2 HR基因测序发现编码区第103位碱基由T变为C(c.103T＞C),导致第35位终止密码子TAG丢失而编码谷氨酰胺(p.X35Q),家系内的正常成员以及家系外同地区的50名健康对照均未发现该突变位点.结论 该家系的临床表现和遗传方式符合1型MUHH的诊断,基因诊断发现的U2HR突变位点103T＞C为中国汉族遗传性少毛症患者人群中首次报道.     

中国人腓骨肌萎缩症小热休克蛋白27基因突变分析

目的 探讨中国人腓骨肌萎缩症（Clmrcot-Marie-Tooth disease，CMT）小热休克蛋白27基因（small heat-shock protein 27，HSP27）的突变特点。方法 应用聚合酶链反应结合DNA序列分析方法，对114个CMT家系的先证者进行HSP27基因突变研究，并进一步对基因突变家系进行单体型分析。结果 在4个常染色体显性遗传CM32家系中发现一个HSP27基因错义突变C379T，单体型分析提示这4个家系很可能具有共同祖先。结论 中国人CMT患者存在HSP27基因突变，但突变率较低（0．90％）。HSP27基因C397T突变除引起远端型遗传性运动神经病外尚可导致CMT2，进一步证实同一CMT疾病基因的同一突变可引起不同的表现型。     

Analysis of myocilin gene mutations in Japanese patients with normal tension glaucoma and primary open-angle glaucoma

The myocilin gene was identified as a gene (MYOC) that caused primary open-angle glaucoma (POAG). Although a normal tension glaucoma (NTG) patient with the myocilin gene mutation was previously reported, no study using large numbers of patients with NTG has been reported. Single-strand conformation polymorphism analysis and subsequent sequence analysis were performed for genotyping the myocilin gene in 114 unrelated Japanese patients with NTG. One hundred and nineteen patients with POAG and 100 control subjects without glaucoma were studied as reference subjects. Five amino acid sequence changes of the myocilin were identified: Arg46Stop (one NTG), Arg76Lys (four NTG, 10 POAG, seven control), Arg158Gln (one NTG, one POAG, one control) found in only Japanese, Asp208Glu (four NTG, three POAG, one control), Pro481Ser (one control). Pro481Ser was novel. Arg76Lys always occurred with 1-83 from G to A in the promoter as it was reported in Chinese. Although some Japanese patients with NTG had sequence changes of the myocilin gene, there were no apparent specific mutations in patients with NTG.     

Low frequency of myocilin mutations in Indian primary open-angle glaucoma patients

Glaucoma is one of the major causes of blindness in the Indian population. Mutations in the myocilin (MYOC) gene have been reported in different populations. However, reports on MYOC mutations in Indian primary open-angle glaucoma (POAG) patients and juvenile open-angle glaucoma (JOAG) patients are sparse. We therefore screened 100 unrelated POAG/JOAG patients for MYOC mutations. Patients with POAG/JOAG were clinically diagnosed. Genomic DNA from such patients was collected and studied for MYOC mutations by direct sequencing. Nucleotide variations were compared with unrelated healthy controls by restriction enzyme digestion. Secondary structure prediction for the sequence variants was performed by Chou-Fasman method. A novel mutation in exon 1 (144 G-->Alpha) resulting in Gln48His substitution was observed in 2% of the patients. Four other polymorphisms were also observed. The novel mutation was seen in four other affected family members of a JOAG patient. The novel mutation was found to alter the secondary structure in the glycosaminoglycan initiation site of the protein. MYOC mutations were found in 2% of the population studied. MYOC gene may not be playing a significant role in causing POAG in the Indian population.     

Myocilin allele-specific glaucoma phenotype database

Glaucoma, a complex heterogenous disease, is the leading cause for optic nerve-related blindness worldwide. Since 1997, when mutations in the myocilin (MYOC) gene were identified as causing juvenile onset as well as a proportion of primary open-angle glaucoma (POAG), more than 180 variants have been documented. Approximately one in 30 unselected patients with POAG have a disease-causing myocilin mutation and it has been shown that firm genotype-phenotype correlations exist. We have compiled an online catalog of myocilin variants and their associated phenotypes. This locus-specific resource, to which future submissions can be made, is available online (www.myocilin.com; last accessed 28 August 2007). The database, constructed using MySQL, contains three related sheets that contain data pertaining to the information source, variant identified, and relevant study data, respectively. The website contains a list of all identified variants and summary statistics as well as background genomic information, such as the annotated sequence and cross-protein/species homology. Phenotypic data such as the mean+/-standard deviation (SD) age at POAG diagnosis, mean+/-SD maximum recorded intraocular pressure, proportion of patients requiring surgical intervention, and age-related penetrance can be viewed by selecting a particular mutation. Approximately 40% of the identified sequence variants have been characterized as disease causing, with the majority ( approximately 85%) of these being missense mutations. Preliminary data generated from this online resource highlight the strong genotype-phenotype correlations associated with specific myocilin mutations. The large-scale assimilation of relevant data allows for accurate comprehensive genetic counseling and the translation of genomic information into the clinic.     

原发性先天性淋巴水肿一家系VEGFR3基因突变分析

目的 对1个患原发性先天性淋巴水肿(prjmary congenital lymphoedema,PCL)汉族大家系进行遗传学研究,了解先天性淋巴水肿患病的分子遗传学基础.方法 对该家系12名成员(10名直系亲属、2名配偶)采样并提取DNA,选择已知的3个PCL相关致病基因位点,用荧光微卫星标记进行基因连锁定位.确定VEGFR3为致病基因后,对家系中的患者进行VEGFR3基因的突变检测,并与100名正常人进行对照分析.结果 该家系疾病与5q35.3区的微卫星标记D5S408连锁.对该区域的VEGFR3进行DNA测序,发现患者含有1个c.C3341T转换,该突变导致VEGFR3蛋白发生p.Pro1114Leu;该家系中所检测患者均发现携带该杂合突变.100名正常对照该位点的测序分析未能检测到该突变.结论 VEGFR3基因是最重要的PCL致病基因,该家系成员VEGFR-3的p.Pro1114Leu突变是患者患淋巴水肿的遗传基础.     

Glaucoma-causing myocilin mutants require the Peroxisomal targeting signal-1 receptor (PTS1R) to elevate intraocular pressure

Glaucoma is a leading cause of worldwide irreversible visual impairment and blindness and is a clinically and genetically heterogenous group of optic neuropathies. Specific mutations in the myocilin (MYOC) gene cause primary open angle glaucoma (POAG) with varying age-of-onset and degree of severity. We show a mutation-dependent, gain-of-function association between human myocilin and the peroxisomal targeting signal type 1 receptor (PTS1R). There was correlation between the glaucoma phenotype and the specific MYOC mutations, with the more severe early-onset POAG mutations having a higher degree of association with PTS1R. Expression of human myocilin glaucomatous mutations in mouse eyes causes elevated intraocular pressure, which is a major phenotype of MYOC glaucoma. This is the first demonstration of a disease resulting from mutation-induced exposure of a cryptic signaling site that causes mislocalization of mutant protein to peroxisomes and the first disease-gene-based animal model of human POAG.     

Novel mutations in the myocilin gene in Japanese glaucoma patients

Myocilin is a gene responsible for juvenile onset primary open angle glaucoma (POAG) mapped as the GLC1A locus and, many mutations have been reported worldwide. Some mutations were found not only in patients with juvenile onset POAG, but also in patients with late onset POAG and in patients with normal tension glaucoma. To investigate the mutation prevalence in Japan, we performed a mutation analysis in 140 unrelated Japanese patients. We have identified the 10 sequence variants, of which four were highly probable for disease-causing mutations (Arg46ter, Arg158Gln, Ile360Asn, and Ala363Thr), and six polymorphisms (Gln19His, Arg76Lys, Asp208Glu, Val439Val, Arg470His, and Ala488Ala). Thus, myocilin mutations were found at the rate of 4/140 (2.9%) probands, similar to previous reports with other ethnic populations.     

一个先天性无虹膜合并白内障家系PAX6基因突变研究

目的 鉴定一个先天性无虹膜合并白内障家系的致病是否与PAX6基因突变有关.方法 提取该家系全部12名存活成员和96名正常人外周血白细胞DNA,PCR扩增PAX6基因的第4～13编码外显子及侧翼内含子剪切区域,通过直接测序比较家系患者与正常人序列差异以确定致病突变.结果 对PAX6基因序列分析结果显示,该家系3例患者中均存在一个无义突变,而在正常人和家系中非受累成员中则未发现.无义突变位于PAX6基因第10外显子1143位核苷酸c.1143C>T,突变导致精氨酸替换为终止密码(R261X).结论 PAX6基因突变R261X是中国人先天性无虹膜合并白内障的致病突变.     

先天性厚甲症Ⅰ型一家系角蛋白6A基因突变研究

目的 研究1个先天性厚甲症Ⅰ型(pachyonychia congenital type Ⅰ,PC-Ⅰ)家系中的角蛋白6A(keratin 6A,K6A)基因突变情况.方法 收集1个先天性厚甲症Ⅰ型家系,并提取该家系中2例患者及3名表型正常者和100名无亲缘关系健康个体的外周血标本,通过聚合酶链反应结合其产物DNA直接测序的方法 ,检测K6A基因的突变情况.结果 该家系中患者存在K6A基因上第521位碱基胸腺嘧啶(T)转化成胞嘧啶(C),使得K6A基因的第1外显子174位密码子由TTT突变成TCT,导致所编码的苯丙氨酸被丝氨酸取代,而该家系的正常人及无关健康个体对照不存在此突变.结论 该PC-Ⅰ家系中患者的表现型是K6A基因的错义突变(521 T→C)引起.     

中国闭角型青光眼病一家系MYOC和CYP1B1基因两突变分析

目的 对一个中国闭角型青光眼病家系进行分子遗传学研究.方法 对家系进行连锁分析,通过测序和单链构象多态性方法鉴定致病基因突变.结果 在家系患者中均发现MYOC基因的一个无义突变(Arg46Stop)以及CYP1B1基因的一个氨基酸改变(Leu432Val).而在无亲缘关系的健康中国汉族人群中没有检测到同时存在上述突变.结论 在一个闭角型青光眼病家系内鉴定了MYOC基因突变以及CYP1B1基因多态.该研究结果提示闭角型青光眼病的发病机制可能是二者协同作用的结果,并支持开角和闭角型青光眼病可能存在相同的发病机理的假说.     

Identification of a new GLC1A mutation in a sporadic,primary open-angle glaucoma in Japan

Glaucoma is a major cause of blindness characterized by progressive degeneration of the optic nerve and elevated intraocular pressure. Recent studies have revealed a genetic basis for a substantial proportion of cases of familial primary open-angle glaucoma (POAG) and the gene causing the abnormality has been identified. Sequence variations that meet the criteria for a probable disease-causing mutation have been found in the American and European populations. In this study, we examined 58 cases of sporadic glaucoma from Japan to clarify the relationship between the mutations of the GLC1A gene and sporadic glaucoma in Japan. We have examined 33 POAG, 17 primary closed-angle glaucomas, 6 normal-tension glaucomas and 2 steroid-induced glaucomas for mutation of the GLC1A gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing studies. We identified a previously unreported GGT right curved arrow GAT transition at codon 451 in exon 3, resulting in a glycine to asparagine substitution in one POAG patient. No other mutations of the GLC1A gene were found in other types of glaucoma. These findings further emphasize the importance of GLC1A mutation in the development of POAG.