| 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. |
PDF Full Text Request