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.     

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.     

*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）     

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。     

Avellino角膜营养不良家系致病基因的定位研究

目的：对收集到的一个常染色体显性遗传性Avellino角膜营养不良家系的致病基因进行初步定位。
　　方法：采集家系中所有成员的外周静脉血,从中提取基因组DNA样本。在热点区域内选取微卫星标记进行基因扫描,分别利用LINKAGE软件和CYRILLIC软件进行连锁分析及单体型分析,以确定候选基因所在的染色体区域。
　　结果：该Avellino角膜营养不良家系的连锁分析结果在D5 S396和D5 S393这两个微卫星标记处获得最大优势对数计分(LOD)值,Zmax=3.01(θ=0.00)。单体型分析将致病基因定位于微卫星标记D5 S808和D5 S638之间。
　　结论：该Avellino角膜营养不良家系的致病基因初步定位于染色体5q上的遗传距离约为11.2厘摩( cM)的一段区域内。     

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。     

常染色体显性遗传性白内障一家系致病基因的研究

目的:对一个4代常染色体显性遗传先天性白内障家系进行致病基因研究。方法:对15例家系成员(8例患者,7例非患者)进行眼部检查,采集静脉血,提取基因组DNA,选取已报道的与常染色体显性遗传性白内障相关的19个位点附近的微卫星标记,PCR扩增后进行基因型分析,用连锁分析进行定位;对提示连锁的标记计算Lod值,并构建单体型;对定位区域内已知候选基因测序。结果:该家系患者表型为绕核性白内障;患者在17q11-12有共享基因型,该位点微卫星标记与致病基因间的两点连锁最大Lod值为2.71,证实该位点与该家系的致病基因连锁;测序未发现CRYBA1/BA3突变。结论:该家系的致病突变不是由于CRYBA1/A3外显子和调控区突变,可能是未被发现基因突变或机制参与该家系的发病。     

先天性膜性白内障一家系致病基因的遗传分析

目的 分析一个先天性白内障家系的遗传规律,对其突变基因进行初步研究.方法 选取一先天性膜性白内障家系,对家系成员进行临床检查并采集静脉血.标准饱和酚/氯仿抽提法提取DNA,选取多态性微卫星遗传标记,合成引物,聚合酶链反应,聚丙烯酰胺凝胶电泳,基因分型,等位基因共享分析法对已知候选基因进行排除性定位.结果 该家系为常染色体显性遗传性先天性白内障家系.其致病基因与D22S315联系紧密,重组发生在以D22S303和D22S1167为上下边界的范围内.对该范围内已知的先天性白内障致病基因CRYBB1、CRYBB2、CRYBB3、CRYBA4进行DNA直接测序,未发现突变.结论 该家系致病基因定位于22q11.2～q12.1的2.4 Mbp范围内,其致病基因与已知基因座不同.该范围内可能存在导致先天性膜性白内障的新的致病基因.     

HSF4基因突变导致常染色体显性遗传核性白内障

目的 鉴定一个延续5代常染色体显性遗传核性白内障家系的致病基因。方法 根据已知先天性白内障致病基因在染色体上的定位，选择了D16S539分子标记，对该家系进行连锁分析，通过基因测序鉴定致病基因。结果 该69名家系成员中有16例患有先天性核性白内障，致病基因定位于16q21-q22，并在候选基因HSF4外显子3检测到一新的突变杂合子134456G-A，该突变导致112E的同义突变，而在家系正常成员中则未检测到该突变。结论 该家系核性白内障表型很可能系由HSF4基因134456G-A突变所致，且此突变尚未见报道。     

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）     

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。     

A novel locus for Leber congenital amaurosis (LCA4) with anterior keratoconus mapping to chromosome 17p13

PURPOSE: A two-generation consanguineous Pakistani family with autosomal recessive Leber congenital amaurosis (LCA, MIM 204,000) and keratoconus was identified. All affected individuals have bilateral keratoconus and congenital pigmentary retinopathy. The goal of this study was to link the disease phenotype in this family. METHODS: Genomic DNA was amplified across the polymorphic microsatellite poly-CA regions identified by markers. Polymerase chain reaction (PCR) products were separated by nondenaturing polyacrylamide gel electrophoresis. Alleles were assigned to individuals, which allowed calculation of LOD scores using the Cyrillic and MLINK software program. The retinal guanylate cyclase (RETGC-1, GDB symbol GUC2D) and pigment epithelium-derived factor (PEDF) genes were analyzed by heteroduplex analysis and direct sequencing for mutations in diseased individuals. RESULTS: Based on a whole genome linkage analysis the first locus for this combined phenotype has been mapped to chromosome 17p13. Linkage analysis gave a two point LOD score of 3.21 for marker D17S829. Surrounding this marker is a region of homozygosity of 15.77 cM, between the markers D17S1866 and D17S960; however, the crossover for the marker D17S1529 refines the region to 10.77 cM within which the disease gene is predicted to lie. Mutation screening of the nearby RETGC-1 gene, which has been shown to be associated with LCA1, revealed no mutations in the affected individuals of this family. Similarly, another prime candidate in the region PEDF was also screened for mutations. The factor has been shown to be involved in the photoreceptor differentiation and neuronal survival. No mutations were found in this gene either. Furthermore, RETGC-1 was physically excluded from the critical disease region based on the existing physical map. CONCLUSIONS: It is therefore suggested that this combined phenotype maps to a new locus and is due to an as yet uncharacterized gene within the 17p13 chromosomal region.     

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.     

A novel locus for autosomal dominant nuclear cataract mapped to chromosome 2p12 in a Pakistani family

PURPOSE: To map the disease locus in a four-generation, consanguineous Pakistani family affected by autosomal dominant congenital nuclear cataract (adNCat). All affected individuals had early onset of bilateral nuclear cataract. METHODS: Genomic DNA from family members was typed for alleles at more than 300 known polymorphic genetic markers by polymerase chain reaction. The lod scores were calculated by using two-point linkage analysis of the genotyping data. RESULTS: The maximum lod score, 4.05, was obtained for the marker D2S2333. Proximal and distal crossovers were observed with markers D2S286 and D2S1790, respectively. These crossovers define the critical disease locus to an interval of approximately 9 centimorgans (cM). CONCLUSIONS: Linkage analysis identified a novel locus for adNCat on chromosome 2p12 in a Pakistani family. A genome database analysis of the target interval is being undertaken to identify candidate gene(s) for the disease.     

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.