常染色体显性先天性白内障一家系致病基因的连锁分析

摘要

Background About one third of congenital cataract is associated with inheriting factor.The inherited heterogeneity has been found in congenital cataract.To seek the pathogenic gene is essential for the gene therapy. Objective Present study was to map and identify the causal gene for autosomal dominant congenital cataract (ADCC) in a Chinese family. Methods The clinical features of all affected members in this family were examined.Blood samples were collected from eleven family members for genetic linkage analysis.Polymorphic microsatellite markers were selected from the regions which harbor all known loci linked with ADCC.Universal fluorescent-labeled M13 primer was used in linkage analysis.Direct genomic sequencing was used to evaluate the candidate gene for example CRYBB2 gene.This study followed Helsinki Declaration and was proved by Tianjin City Ethic Committee.Written informed consent was obtained from each SUbject before any medical procees. Results The maximum two-point LOD score of 1.20 was obtained for marker D22S315 (θ=0).The LOD score of 0.6 was obtained for marker D16S3068.No mutation in all exons of CRYBB2 gene was found in the family. Conclusion CRYBB2 gene associated with ADCC was excluded from the family.A genome-wide linkage screening should be conducted.Genotyping with microsatellite markers using Universal fluorescent-labeled M13 primer can decrease the cost and obtain the same result.%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.