Genetic Mapping And Mutation Analysis In Chinese Families With Congenitial Cataract

ObjectiveHuman congenital cataract has a diverse aetiology. In the proportion of cases where the cause is genetic, the disease shows wide phenotypic and genetic heterogeneity. Over the past few years, much research has been devoted to map the genes that underlie the disorder. Most progress to date has been in the identification of genetic mutations causing autosomal domimant congenital cataracts. Twenty - three loci for congenital cataracts have been localized to specific chromosome regions and 11 disease causing genes have been identified. Overall there is good correlation between the genetic mutations so far identified and the resulting lens phenotype but it is clear that mutations at more then one locus may give rise to similar forms of cataract. The identification of genes causing inherited forms of cataract will improve our understanding of the mechanisms underlying cataractogenesis in childhood and will also provide further insights into development and physiology of normal lens. Perhaps more importantly, it is likely that some of the genes causing early onset congenital cataracts will be implicated in age related cataracts which remain the commonest cause of blindness in the world.Materials and MethodsProbands and family members from 6 Chinese families with congenital cataracts were investigated. For phenotypic classification, McKusick's OMIM (Online Mendelian Inheritance in Man) was referred.After informd consent was obtained, peripheral blood samples (5 ml per individual) were collected from family members (affected and unaffected). Ge-nomic DNA was extracted from whole blood using standard phenol/chloroform -proteinase K method. A total of 23 microsatellite polymorphic markers at chromosome 1q21 -q25, 1p22.3, 2q33 -q36, 11q22. 1 - q23.21, 17q11 -q12, 21q22. 3 and 22q11. 2 - ql2. 1 were selected and genotyped for linkage analysis. Sequence information for PCR primers were obtained from the NCBI website. Primers were commercially synthesized. All available individuals from the 6 families were subjected to PCR genotyping. The PCR fragments were separated through 8% denaturing polyacrylamide gel electrophoresis. Two - point linkage analysis between the cataract phenotype and the genetic markers were performed using the MLINK component of the LINKAGE package. Haplotype was constructed using allele information in each family.To detect pathogenic mutations of the genes in the CRYG cluster, primers were designed from the regions which allowed amplification of all coding exons and their flanking intronic sequences of each individual gene. PCR products were examined on 2% agarose gels to confirm the amplification and purified u-sing the QIAquick Gel Exiraction Kit. The purified PCR fragments were submitted for direct automatic sequencing. To confirm the R139X mutation of the CRYGD gene in family 2, a mismatch primer was designed to introduce a Msp I restriction site in the normal allele by semi - nested PCR, and the resulted PCR fragments of 223bp were analyzed by Msp I digestion and polyacrylamide gel e-lectrophoresis.A large Chinese family with congenital cataract - aniridia syndrome was also clinically and genetically investigated. Blood samples were collected and ge-nomic DNA was extracted as described above. All coding exons and their flanking intronic sequences of the PAX6 gene were amplified using specific primers, PCR products were purified and then subjected to direct automatic sequencing. To confirm the R254X mutation of the PAX6 gene, a mismatch primer was designed to introduce a Msp I restriction site in the normal allele by semi - nested PCR, and the resulted PCR fragments of 141bp were analyzed by Msp I digestion and polyacrylamide gel electrophoresis.ResultsA total of 203 individuals (109 males and 94 females) , including 62 patients (26 mals and 36 females) , from the 6 families with congenital cataract were retrospectively investigated. And 158 individuals were available for examination. Blood samples were collected from 72 individuals, including 43 patients. The pattern of phenotype transmission in all 6 families suggested a mode of autosomal dominant inheritance. Of the 6 families, 4 were diagnosed as nuclear cataract, 1 as lamellar cataract, and 1 as disc - shaped cataract.Microsatellite polymorphic markers, mainly of tetranucleotide repeats, were successfully genotyped by polyacrylamide gel electrophoresis and silver staining. Two - point linkage analysis did not provide evidence for linkage to markers from chromosome regions 1q21 - q25 , 1p22. 3 , 11q22. 1 - q23. 21, 17q11 - q12, 21q22.3 and 22q11.2-q12. 1 in any of the 6 families. However, markers at chromosome 2q33 - q36 (D2S1385, D2S1369, D2S1782, D2S2944 and D2S1371) showed potential linkage to the desease phenotype in families 2 and 5. A maximum LOD score of 1. 51 and 2.44 were obtained for marker D2S2499 (6 =0.00) in family 2 and 5, respectively, suggesting possible involvement of the CRYG gene cluster in the cataractogenesis in the families. Haplolype in all the 6 families was also constructed based on the allele information.PCR direct sequencing of 4 individual genes in the CRYG gene cluster revealed a nonsense mutation of the CRYGD gene, R139X (418C >T) , in family 2 with nuclear cataract, and a single nucleotide deletion within the CRYGD gene, 475delG, in family 5 with lamellar cataract. Msp I restriction analysis showed cosegregation of the R139X mutation with all affected individuals, but not with normal individuals, in family 2. Since these two mutations will result in protein truncation, they are therefore pathogenic mutations. These mutations were not reported in the literature and are novel CRYGD mutations which are associated with congenital cataract.A total of 58 individuals (38 males and 20 females) , including 21 patients (13 mals and 8 females) , from the large family with congenital cataract -...