【作者】 布娟；

【导师】 赵堪兴；

【作者基本信息】 天津医科大学 ， 眼科学， 2006， 博士

【摘要】 目的:应用全基因组扫描、连锁分析的方法对一个常染色体显性遗传的先天性白内障家系进行基因定位;从定位的染色体区域内寻找候选基因,通过基因序列分析对候选基因进行突变筛查;并对家系患者在白内障手术中摘除的晶状体做形态学研究,从而进一步探求致病基因与临床表型之间的关系。 方法:对家系患者进行详细的临床检查,排除其他疾患,确定临床表型。将家系患者在白内障手术中摘除的晶状体做光镜及电镜的形态学检查。提取家系成员外周血DNA,选取分布在GJA8、CRYGS、BFSP2、PTX3、MIP、HSF4、CRYAA、CRYAB、CRYBB1、CRYBB2基因上下2厘摩(centimorgan,cM)范围内的微卫星标记物,通过变性聚丙烯酰胺凝胶电泳和银染法确定单体型;对CRYGC、CRYGD、GJA3及CRYBA1基因直接测序,以确定该家系致病基因是否为已知的常染色体显性遗传先天性白内障(autosomal dominant congenital cataract,ADCC)基因。在全部常染色体范围内选取370对荧光微卫星标记物进行全基因组扫描,相邻微卫星标记物之间的平均距离为10cM。利用聚合酶链反应(Polymerase Chain Reaction,PCR)对全部荧光微卫星标记物进行扩增后,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,并利用Genescan 3.1和Genotyper 2.0软件进行两点法计算LOD值并构建单体型。运用Sim Walk2,Version3.35软件按照多点法分别计算疾病表型与不同染色体上的多个微卫星位点之间的最大优势对数Lod score。根据连更多还原

【Abstract】 ObjectiveTo identify the genetic location of the candidate gene by linkage analysis. To describe the clinical phenotype in a Chinese family with autosomal dominant congenital cataract (ADCC), and to compare the ultrastructure of opaque lenses in the family with transparent normal human lenses. Analyze the sequence of candidate gene by directly sequence to elucidate the genetic background of the phenotype in this ADCC family. MethodsA Chinese family with ADCC (FAN) was collected for this study. A detailed clinical examination including distance visual acuities, slit-lamp examination and funds of ophthalmology was performed for all patients in the family. To observe the morphological changes of the removed lens tissue with light and transmission electron microscopy. The genomic DNA of all family members was extracted from peripheral blood leukocytes, according to the standard methods of protocol. Polymorphic markers were selected from the regions which harbor all known loci linked with ADCC. The markers were amplified by polymerase chain reaction( PCR). Fragments were separated by elecrophoresis through 6% denaturing polyacrylamide gels. Haplotypes were constructed manually according to the pattern of the bands on the gels stained by silver. Two-point lod scores were calculated using the subroutine Mlink of the Linkage package. Mutation analysis of CRYGC, CRYGD, GJA3, CRYBA1 genes in FAN family were performed by screening all coding region of these genes. Linkage analysis and genome-wide linkage screening was conductedusing fluorescent detection of 370 microsatellite markers representing all autosomes at an average resolution of approximately 10 cM. The polymerase chain reactions were carried out to amplify all 370 microsatellite markers. The allele sizes were determined on ABI 3130 —avant genetic analysis according to an internal size standard and the results were analyzed using Genescan 3.1 and Genotyper 2.0 software. Multipoint LOD scores between the disease status and the marker alleles were calculated using the LINKAGE software package of SimWalk2, Version3.35. To further determine the candidate region of disease gene, more microsatellite markers were selected around the location of the microsatellite marker with the largest positive Lod score. Directly sequence was conducted on affected members of FAN family to determine the mutation in all exons of BFSP1 gene. ResultsThe affected members in the family were born with classic phenotype of zonular ADCC. The lens fiber cells showed manifestations of degeneration, including focal degeneration > alterations in the lens fiber cells > reticular change and crystal precipitation. Under transmission electron microscope, the lens fiber cells displayed abnormal inter- and intracellular alterations. There were foci of dense, globular intracellular deposits in the cytoplasm. The cell border’s connection was highly irregular and there were enlarged spaces. Positive Lod score of 370 microsatellite markers in the family occurred on D20S112 and D20S195. Further linkage analysis showed the Markers D20S163 > D20S915 , D20S152 , D20S98 , D20S904 , D20S875 , D20S112> D20S1140> D20S432 co-segregated with the disease locus in all affected members. The maximum Lod Score was 6.02(D20S904), so the candidate region of disease gene in the family was located on 20pl2—20pll.2. Directly sequence showed no mutations in all exons of BFSP1 gene in FAN family. Conclusion All known candidate genes associated with ADCC were excluded from FANfamily. The candidate region of disease gene in the FAN family was located in 20pl2 —20pll.2. No mutations in all exons of BFSPl gene were found in the family. A new disease—causing gene maybe exist in this family.更多还原