Gene Mapping And Analysis Of Candidate Genes In Three Chinese Families With Congenital Cataract

ObjectiveTo determine the clinical phenotypes of three Chinese family(CA-001,CA-002,CA-003)with autosomal dominant congenital cataract(ADCC).Identify the genetic location of the candidate gene by linkage analysis.Analyze the sequence of candidate gene by directly sequence to elucidate the genetic basis of the three ADCC family and to investigate the relationship between gene mutation and clinical phenotype in ADCC.Methods1.Three Chinese family with ADCC(CA-001,CA-002,CA-003)was collected for this study.A detailed clinical examination including disease history,distance visual acuities,slit-lamp examination and direct funduscopy was performed for all patients in the three family.2.Identify the genetic location of the candidate gene by linkage analysis.The genomic DNA of all family members was extracted from peripheral blood leukocytes,using DNA Isolation kits for Mammalian Blood(Robe Biochimical,Inc).Polymorphic microsatellite markers were selected from the regions which harbor all known loci linked with ADCC.The markers were amplified by polymerase chain reaction(PCR).The allele sizes were determined on ABI3130-avant genetic analyses according to an internal size standard and the results were analyzed using Genescan analysis v3.7 software and Genetyper v3.7software(Applied Biosystems).Two point analyses for these markers were calculated using the MILINK program of LINKAGE package(Version 5.1).3.Gene sequence analysis To identify the genetic location of the candidate gene according the linkage analysis result.Mutation analysis of CRYAB,CRYGC,CRYGD genes in CA-001 family were performed by screening all coding region of these genes.Mutation analysis of CRYBB2 genes in CA-002 family were performed by screening all coding region of these genes.Mutation analysis of CRYAB genes in CA-003 family were performed by screening all coding region of these genes.Results1.Kindred CA-001The affected members in the family were born with classic phenotype of coralliform ADCC.The maximum lod score of 1.51 was acquired between microsatellite marker D2S325 and ADCC in this family.The lod score of 1.20 was acquired between microsatellite marker D11S925 and ADCC in this family.The CRYGC/CRYGD gene located on 2q33-q35,the CRYAB gene located on 11q22-q22.3.So the CRYGC/CRYGD and CRYAB gene may be the candidate gene in the family.Sequencing of the coding regions of the CRYGD genes showed the presence of a heterozygous C→A transversion at nucleotide 70 in exon 2 of CRYGD that is associated with cataracts in this family.This mutation resulted in a proline to threonine substitution at amino acid 23 of the protein in the first of the four Greek key motifs that characterise this protein.No mutations in all exons of CRYGCand CRYABgene were found in the family.2.Kindred CA-002The affected members in the family were born with phenotype of nuclear or cortical catarct.Two patients suffered from nystagmus.The maximum lod score of 1.20 was acquired between microsatellite marker D22S315and ADCC in this family.The CRYBB2 gene located on 22q11.2.So the CRYBB2 gene may be the candidate gene in the family.But no mutations in all exons of CRYBB2 gene were found in the family.3.Kindred CA-003The affected members in the family were born with phenotype of nuclear or cortical catarct.Some patients suffered from microcornea and esotropia.The maximum lod score of 0.96 was acquired between microsatellite marker D3S1569 and ADCC in this family.The lod score of 0.60 was acquired between microsatellite marker D1S2842,D11S898,D21S266,22S539 and ADCC in this family.No mutations in all exons of CRYAB gene were found in the family.Conclusion1.Pedigree analysis demonstrated that the congenital cataract conditions in three families are inherited as an autosomal dominant(ADCC)trait.2.Direct DNA sequence analysis revealed that a C-to-A transition at nucleotide 70 of the CRYGD gene,resulting in an Pro58-to-Thr substitution,segregated with the phenotype of aculeiform cataract in CA-001 families.3.CRYBB2 genes associated with ADCC were excluded from CA-002 family.A genome-wide linkage screening should be conducted.4.CRYAB genes associated with ADCC were excluded from CA-003 family.A genome-wide linkage screening should be conducted.