一特殊表型的常染色体显性遗传先天性白内障家系致病基因的筛选与鉴定

背景遗传因素是先天性白内障的主要致病因素之一,致病基因的筛查是研究先天性白内障发病分子机制的重要步骤。目的明确一结晶样晶状体混浊的先天性常染色体显性遗传白内障（ADCC）家系的致病基因。方法收集山西省榆社县一个四代先天性结晶样混浊白内障家系22名成员,其中患者10例。在获得知情同意后,该家系成员进行家系调查以确定遗传方式。经裂隙灯显微镜检查和常规眼科临床检查确定表型。采集其中17例家系成员的外周静脉血5ml并提取DNA,ADCC的17个已知致病基因周围选取22个荧光标记的微卫星,通过对微卫星标志物的扩增和基因型分析对该家系进行基因两点连锁分析,并计算对数优势评分（LOD）值。对筛选的候选基因进行直接测序分析。结果该家系患者晶状体混浊表型非常类似,家系分析表明为四代垂直遗传,符合单基因ADCC的特点。基因连锁分析提示,该家系与微卫星D2S325位点和D2S2358位点连锁,最大LOD值分别为1．20（0=0）和0．22（0=0）,位于此区域内的CRYGD基因测序后发现一个已经报道的错义突变c．C70A（p．P23T）。结论CRYGD基因P23T突变是该家系结晶样晶状体混浊的致病原因。     

两个先天性珊瑚状白内障家系CRYGD基因P23T突变的检测

背景先天性白内障的致病基因及临床表型具有明显的异质性,通过连锁分析进行基因定位,直接测序法筛选致病基因是目前常用的分析方法。目的对2个先天性珊瑚状白内障家系（CCl和CC2）进行致病基因研究。方法收集确诊为常染色体显性遗传的2个先天性珊瑚状白内障家系17名成员的外周静脉血各5ml,包括ll例患者、4名正常成员和2名配偶,提取基因组DNA。选取与已知常染色体显性遗传先天性白内障相关的位点进行基因组扫描,利用微卫星标记物进行PCR扩增并进行序列分析。采用两点法计算LOD值对致病基因进行连锁分析。采用直接测序法对候选致病基因以及3个单核苷酸多态性（sNP）位点（rs2305429,rs2305430,rs2242074）进行序列分析。利用SNP对2个家系的先证者进行单体型分析。结果CCl和CC2家系中的先证者裂隙灯下均可见双眼晶状体核中央混浊区呈珊瑚状,经两点法计算LOD值,家系CCl在微卫星位点D2S325获得的最大LOD值为3．28,而CC2家系在D2S325获得的最大LOD值为1．50,连锁分析结果支持2个家系均与位于2q的候选致病基因CRYGC和CRYGD连锁。基因序列分析发现2个珊瑚状白内障家系均携带CRYGD基因c．C70A．（P．P23T）突变体,而2个家系中的正常人及100名正常对照则无此基因突变。2个家系先证者携带不同类型的单体型结构。结论CRYGD基因C．C70A．（P．P23T）突变是导致2个不同祖先来源的先天性珊瑚状白内障家系CCl和CC2致病的丰要原因。     

先天性白内障一家系致病基因的排除性定位

目的 明确一个国人常染色体显性先天性白内障（ADCC）家系致病基因的染色体位点是否位于已知的22个非综合征型ADCC致病位点内，从而初步定位该ADCC家系致病基因的染色体位点。设计 家系遗传研究。研究对象 一个先天性白内障家系。方法 对26例家系成员中的16例进行临床检查、采集静脉血样、提取基因组DNA；在已知的22个非综合征型ADCC致病位点内，分别选取3-6个多态性微卫星标记，对该ADCC家系进行遗传连锁分析。主要指标 先天性白内障临床表型、Lod值。结果 该家系患者为晶状体前囊膜及前囊膜下混浊；所有多态性微卫星标记与致病基因两点间的Lod值均≤-2，证实微卫星标记所在的染色体区域与该ADCC家系的致病基因不连锁。结论 该ADCC家系致病基因的染色体位点不在已知的22个非综合征型ADCC致病位点内，可能是一个新的致病基因导致了该家系的临床表型。     

常染色体显性遗传性先天性白内障一家系致病基因的初步定位

目的 初步定位常染色体显性遗传性先天性白内障(ADCC)一家系的致病基因。方法 收集ADCC一家系资料,在已知先天性白内障致病基因和位点附近,选择合适的短串联重复序列多态性标记(STRP),对ADCC一家系进行连锁分析,使用Mlink软件采用对数优势记分法(LOD)计算LOD值。结果 在STRP中,D17S805、D17S1294及D17S1293与致病基因位点连锁的最大LOD值分别为2．03、2．49及2．22(重组率0=0)。结论 该ADCC家系的致病基因初步定位在第17对染色体上;CRYBA1基因为候选基因。（中华眼科杂志,2004,40：824-827)     

花冠状常染色体显性遗传性先天性自内障致病基因初步定位

目的 初步定位具有花冠状表型的常染色体显性遗传性先天性白内障一家系的致病基因.方法 收集家系成员的资料,提取基因组DNA,据文献报道在已知先天性白内障致病基因和位点附近,选择合适的短串联重复序列多态性标记,使用LINKAGE 5.1软件计算标准LOD值,对此家系进行连锁分析.结果 此表型先天性白内障的致病基因定位在3q22.3-q25.2,即D3S3612至D3S1594之间15.2 cM范围内.在D3S1569和D3S3599处,得到与致病基因住点连锁的最大LOD值均为3.01(重组率=0.00).结论 该花冠状常染色体显性遗传性先天性白内障致病基因初步定位在第3对染色体上3q22.3-q25.2.     

先天性核性白内障家系致病基因的定位与候选基因突变检测

目的 对中国一常染色体显性遗传性先天性核性白内障家系进行致病基因的定位与候选基因突变检测.方法 实验研究.采集家系成员的外周静脉血,提取基因组DNA.用约400个中密度微卫星标记进行基因扫描,平均遗传距离10厘摩(cM).利用LINKAGE软件包进行连锁分析.在阳性定位区域内选取更为精细的微卫星标记进行精细定位.利用CYRILLIC软件进行单体型分析,确定候选基因所在染色体区域.候选基因直接测序检测基因突变.结果两点间连锁分析在微卫星标记D2S325处获得最大对数优势计分(LOD)值Zmax=2.29(θmax=0.00).精细定位和单体型分析将致病基因定位于微卫星标记D2S117和D2S2382之间,遗传距离约19.04 cM,染色体位置为2q32.3-q35.候选基因直接测序发现CRYGC基因第3外显子第470碱基一个G→A的点突变.结论本研究将我国一个先天性核性白内障家系的致病基因定位于2号染色体2q32.3-q35约19.04cM区域内,并在CRYGC基因发现一个新的点突变与此家系共分离.(中华眼科杂志,2009,45:234-238)     

东北地区一遗传性白内障家系的临床遗传学及基因定位研究

目的:分析一先天性核型白内障家系的遗传方式及致病基因所在位置。方法:收集一个3代遗传性白内障家系成员的临床资料;提取家系成员外周血DNA,选取62个态性微卫星标记进行连锁分析。应用LINKAGE软件(version 5.2)中的MLINK程序计算两点连锁LOD值,并人工构建家系成员的单体型。结果:确定该家系为一常染色体显性遗传性白内障大家系,在微卫星标记D22S689可获得最大LOD值2.71(θ=0时),单体型提示该家系表型可能与染色体22q11.2-12.1区域连锁。该区域含有CRYBB1,CRYBB2,CRYBB3,CRYBA44个候选基因。结论:本研究先天性核型白内障家系符合常染色体显性遗传规律,其致病基因定位于22q11.2-12.1区域。     

常染色体显性先天性白内障一家系致病基因的连锁分析

背景先天性白内障约1／3的病例是由遗传所致,已发现遗传性白内障有着极为明显的遗传异质性,了解先天性白内障的致病基因对其基因治疗极为重要。目的分析一个具有常染色体显性遗传特点的先天性白内障家系的临床表型特征,进行已知致病基因的筛查定位。方法对遗传性先天性白内障一家系共16名成员眼部进行详细的临床检查,包括6例患者,确定为本家系白内障患者的临床表型。收集其中11名家系成员的血液样本提取DNA,包括3名正常家系成员及其配偶、5例患者。利用连锁分析进行排除定位,并采用Schuelke报道的新方法,只合成普通引物及一种荧光标记的通用引物M13,进行聚合酶链反应（PCR）,对连锁区域内的候选基因进行基因序列分析。结果本家系的白内障遗传方式符合常染色体显性遗传特征。基因连锁分析表明,在D22S315得到最高LOD值为1．20,在D16S3068得到LOD值为0．6。CRYBB2基因所有编码区及外显子与内含子交界处未发现基因序列突变。结论本家系初步排除了CRYBB2基因与此家系先天性白内障的相关性。对这个家系的基因定位需要更进一步的全基因组扫描,以发现致病基因在染色体上的可疑区间。连锁分析中进行微卫星位点的PCR扩增时,利用合成荧光标记的通用引物M13,可以显著降低成本,并取得同样的实验结果。     

一常染色体显性遗传性白内障家系致病基因的排除性定位

目的对常染色体显性遗传先天性白内障家系进行致病基因的定位研究。方法对4代11例家系成员（6例患者）进行眼部和全身检查,采集静脉血,提取基因组DNA,选取已报道的与遗传性白内障相关位点附近的微卫星标记,PCR扩增后进行基因型分析,用连锁分析进行排除;没有排除的位点,基因外显子测序。结果 35例家系成员中,追溯调查共有10例患者,其中第1代1例,第2代2例,第3代5例,第4代2例。该家系患者表型为完全性白内障;绝大多数位点,患者没有共享基因型;微卫星标记与致病基因间的2点连锁Lod值〈-2,证实这些位点与该家系的致病基因不连锁;有3个多态性标记（D10S1239、D22S286、D22S926）0〈Lod值≤0.6,Lod值虽然不是〈-2,但在家系患者中没有共享等位基因;测序未发现外显子有突变。结论此家系的致病基因不是已报道位点的致病基因,其致病基因有待进一步研究。     

特殊表型遗传性先天性白内障的超微结构和基因定位

目的 探讨一表型特殊、晶状体呈簇状混浊的常染色体显性遗传性先天性白内障(ADCC)的超微结构,并初步定位该疾病的相关候选基因。方法 收集特殊表型ADCC一家系资料,对家系成员行眼部检查;在光学显微镜和透射电镜下观察晶状体细胞超微结构的改变;选择γ-晶状体蛋白基因附近多个微卫星位点,对该家系ADCC疾病相关候选基因进行连锁分析。结果 光学显微镜下特殊表型ADCC患者晶状体纤维细胞失去正常排列规则,产生不规则折光,并可见网格样改变、黏液样变性及结晶样物质析出等局灶性退行性变;透射电镜下可见细胞皱缩、变形,失去正常长六边形形态,细胞间隙增宽,细胞内可见异常高密度球形颗粒沉着。连锁分析结果显示,该家系ADCC疾病相关候选基因与微卫星位点D2S2208、D2S2382及D2S164连锁,最大LOD值为3．34。结论 特殊表型ADCC的特异性病理学改变集中在晶状体纤维细胞,其疾病相关候选基因极可能为γ-晶状体蛋白基因。（中华眼科杂志,2004,40：306-310)     

先天性前极白内障家系致病基因的定位与候选基因突变检测

目的 对中国一常染色体显性遗传先天性前极白内障家系进行致病基因的定位与候选基因突变检测.方法 采集家系成员外周静脉血,提取基因组DNA.选用ABI公司提供的约400个遗传标记物进行基因扫描.基因扫描分初步扫描和精细扫描两步进行.首先对已报道的先天性白内障候选区域进行初步扫描,之后在阳性区域内进行精细扫描.数据经连锁分析,初步确定致病基因所在染色体区域.在阳性区域内选取更高密度的荧光标记物进行精细扫描,并进行单体型分析.候选基因直接测序检测基因突变.结果 两点间连锁分析在微卫星标记D21S1252处获得最大对数优势计分(LOD)值Zmax=3.23(θmax=0.00).精细定位和单体型分析将致病基因定位于微卫星标记D21S263和D21S266之间,遗传距离约18.47厘摩(cM),染色体位置为21q22.11-q22.3.候选基因直接测序发现CRYAA基因第3外显子第347碱基一个G→A的点突变.结论 本研究将一中国先天性前极白内障家系的致病基因定位于21号染色体21q22.11-q22.3区域内,并在CRYAA基因发现一个点突变与此家系共分离.

Abstract：

Objective To map the gene mutation responsible for autosomal dominant inherited congenital anterior polar cataract in a Chinese family. Methods Peripheral blood samples were collected from the members in this congenital cataract family. DNA was extracted from the blood samples. A genescan was performed using approximately 400 microsatellite markers (ABI). Linkage analysis was processed to define the region of mutated gene. High density primers labeled with fluorescent stain for the positive region were adopted for fine targeting and haplotype analysis was performed. Mutation detection was carried out by sequencing candidate genes. Results The maximum two-point LOD score was obtained at D21S1252,Zmax = 3. 23 ( θmax = 0. 00). After fine targeting and haplotype analysis,the mutated gene was located within a 18. 47 cM region between D21S263 and D21S266 on chromosome 21q22.11 -q22.3. Direct sequencing of the candidate gene revealed a G→ A transition in exon 3 of CRYAA. Conclusion The present study has identified a missense mutation in CRYAA associated with congenital anterior polar cataract in a Chinese family.     

*常染色体显性先天性缝合状白内障家系致病基因的定位

目的对我国一常染色体显性先天性缝合状白内障家系进行致病基因的定位。方法采集家系成员外周静脉血提取基因组DNA。选用美国Applied Biosystems公司提供的约400个遗传标记物进行基因扫描。数据经Linkage软件包进行连锁分析,初步确定致病基因所在染色体区域。在阳性区域内选取更高密度的荧光标记物进行精细扫描,用Cyrillic软件进行单体型分析。结果两点间连锁分析在D3S1279处获得最大对数优势记分（LOD）值Zmax=2．32（θmax=0．00）。通过精细扫描和单体型分析将致病基因定位于D3S1267和D3S1614之间约38．6厘摩（cM）区域内。结论先天性缝合状白内障家系的致病基因位于3号染色体3q21．1-q26．2约38．6cM区域内。（中华腥科杂志,2006,42：908—912）     

先天性白内障一家系全基因组扫描基因定位及候选基因序列分析

目的应用全基因组扫描、连锁分析的方法对常染色体显性遗传性先天性白内障（ADCC）一家系进行基因定位、寻找候选基因并进行突变筛查。方法提取该家系成员外周血DNA,进行全基因组扫描。在ABI 3130-avant全自动遗传分析仪上读取370对微卫星标记物的等位基因片段大小,并采用Genescan 3.1和Genotyper 2.0软件进行两点法计算LOD值并构建单体型。根据连锁分析的结果,对该区域内在晶状体中呈高表达,且对维持晶状体纤维细胞的分化状态起重要作用的基因-BFSP1进行直接的序列分析。结果该家系的致病基因位于20p12-20p11.2的13.96 cm区域内。在该区域内的基因-BFSP1全部外显子及外显子与内含子交界处均未发现任何突变。结论首次将一常染色体显性遗传绕核型先天性白内障家系的致病基因定位于20p12-20p11.2的13.96 cm区域内。     

Genetic linkage analyses and Cx50 mutation detection in a large multiplex Chinese family with hereditary nuclear cataract

Purpose: The aim of the study was to characterize the underlying mutation in a large multiplex Chinese family with hereditary nuclear cataract.

Methods: A 6-generation Chinese family having hereditary nuclear cataract was recruited and clinically verified. Blood DNA samples were obtained from 53 available family members. Linkage analyses were performed on the known candidate regions for hereditary cataract with 36 polymorphic microsatellite markers. To identify mutations related to cataract, a direct sequencing approach was applied to a candidate gene residing in our linkage locus.

Results: A linkage locus was identified with a maximum 2-point LOD score of 4.31 (recombination fraction?=?0) at marker D1S498 and a maximum multipoint LOD score of 5.7 between markers D1S2344 and D1S498 on chromosome 1q21.1, where the candidate gene Cx50 is located. Direct sequencing of Cx50 showed a 139 G to A transition occurred in all affected family members. This transitional mutation resulted in a replacement of aspartic acid by asparagine at residue 47 (D47N) and led to a loss-of-function of the protein.

Conclusions: The D47N mutation of Cx50 causes the hereditary nuclear cataract in this family in an autosomal dominant mode of inheritance with incomplete penetrance.     

The telomere of human chromosome 1p contains at least two independent autosomal dominant congenital cataract genes

AIMS: Multiple genetic causes of congenital cataract have been identified, both as a component of syndromes and in families that present with isolated congenital cataract. Linkage analysis was used to map the genetic locus in a six generation Australian family presenting with total congenital cataract. METHODS: Microsatellite markers located across all known autosomal dominant congenital cataract loci were genotyped in all recruited family members of the Tasmanian family. Both two point and multipoint linkage analysis were used to assess each locus under an autosomal dominant model. RESULTS: Significant linkage was detected at the telomere of the p arm of chromosome 1, with a maximum two point LOD of 4.21 at marker D1S507, a maximum multipoint exact LOD of 5.44, and an estimated location score of 5.61 at marker D1S507. Haplotype analysis places the gene inside a critical region between D1S228 and D1S199, a distance of approximately 6 megabases. The candidate gene PAX7 residing within the critical interval was excluded by direct sequencing in affected individuals. CONCLUSION: This is the third report of congenital cataract linkage to 1ptel. The critical region as defined by the shared haplotype in this family is clearly centromeric from the Volkmann cataract locus identified through study of a Danish family, indicating that two genes causing autosomal dominant congenital cataract map to the telomeric region of chromosome 1p.     

遗传性核性金黄色结晶样白内障患者γ晶状体蛋白基因错义突变

目的确定中国北方常染色体显性遗传性白内障（ADCC）-家系的致病基因。方法收集ADCC-家系资料,提取血液白细胞DNA,运用微卫星位点多态性连锁分析,对提示连锁的染色体区域内的候选基因测序,寻找突变。结果该家系致病基因定位在2q33．3-34区域内,对其候选基因γ晶体蛋白基因簇各基因进行测序,发现γD晶体蛋白基因第二外显子有一个杂合子的错义突变（109C→A）与家系患者共分离,此突变可导致其编码的第36位精氨酸被丝氨酸取代。结论此γ晶体蛋白基因突变引起该家系核性结晶样先天性白内障,是由1D晶体蛋白基因109C→A（R36S）突变引起的。（中华腰群杂志,2006,42：913—917）     

Posterior Polar Cataract: Genetic Analysis of a Large Family

Congenital cataracts are clinically and genetically heterogeneous. Loci for autosomal dominant posterior polar cataracts have been mapped to chromosomes 1p36, 11q22-q22.3, 16q22, and 20p12-q12. We investigated a large four-generation family with 20 individuals affected with congenital posterior polar cataracts. After exclusion of known loci for posterior polar cataracts, a genome-wide screen was conducted. In this family, we mapped dominant congenital posterior polar cataracts to chromosome 10q24. On haplotype analysis, we identified an 11-cM interval between loci D10S1680 and D10S467, which included the PITX3 gene. On sequencing the coding region of PITX3, we found a 17-base-pair duplication in exon 4. Although the same genotype was described in a family with ASMD and cataracts, the common phenotype of this mutation is probably posterior polar cataract; a modifier gene is presumed to cause anterior segment abnormalities in the previously described patients. The same mutation was recently identified in four families with congenital cataracts. This study provides further evidence of genetic heterogeneity of autosomal dominant posterior polar cataract.     

Posterior polar cataract: genetic analysis of a large family

Congenital cataracts are clinically and genetically heterogeneous. Loci for autosomal dominant posterior polar cataracts have been mapped to chromosomes 1p36, 11q22-q22.3, 16q22, and 20p12-q12. We investigated a large four-generation family with 20 individuals affected with congenital posterior polar cataracts. After exclusion of known loci for posterior polar cataracts, a genome-wide screen was conducted. In this family, we mapped dominant congenital posterior polar cataracts to chromosome 10q24. On haplotype analysis, we identified an 11-cM interval between loci D10S1680 and D10S467, which included the PITX3 gene. On sequencing the coding region of PITX3, we found a 17-base-pair duplication in exon 4. Although the same genotype was described in a family with ASMD and cataracts, the common phenotype of this mutation is probably posterior polar cataract; a modifier gene is presumed to cause anterior segment abnormalities in the previously described patients. The same mutation was recently identified in four families with congenital cataracts. This study provides further evidence of genetic heterogeneity of autosomal dominant posterior polar cataract.