Genetic Mapping And Mutation Analysis In Chinese Family With Congenital Cataract

ObjectiveCongenital cataract is a common blindness threatening disease among children. About 40 million children suffer from the disorder worldwide,among which 1/3 are hereditary cataract.Hereditary cataract includes autosomal dominant congenital cataract (ADCC),autosomal recessive cataract and X-linked ones.ADCC is the most common type.It spreads generation by generation in the family,and brings about a poor living condition for the descendants.The elimination of hereditary diseases is the targeted proposition of the national health care.This is a populational country which is rich of hereditary resources.To find out novel causative genes and to better understand their mechanism is our goal to benefits human being and prenatal investigation and inquiry. ADCC is gene defect,which is being workout in molecular level.The outcomes of the research work in this field will fully support the hereditary interference and genomic therapy theoretically.The present study introduces a Northeasten resident family which covers 4-generation and 10 members with hereditary cataract.A series of research work had been done in screening and identifying the novel gene mutation.Materials and MethodsWe investigated 4 hereditary cataract families and gave them physical examination to exclude their general diseases and other ocular abnormalities.Pupil had been dilatated and anterior segment photographies had been documented.Phenotypes of each family had been identified clinically and pedigrees had been drawn carefully.After signed the written consent,Peripheral blood samples(4 ml per individual)were collected from the family members including non-affected members.Genomic DNA was extracted from their whole blood using standard phenol/chloroform-proteinase K method.A total 26 microsatellite polymorphic markers at chromosome 1q21-25,2q33-36,3q21-25,3q27.3,10q5,11q22.1-23.21,12q13-14,13q12.11,16q23.1,16q22.1,17q11-12,19q13.33,20p12.1,21q2.3,22q11.2-12.1 were selected and genotyped for linkage analysis.Sequence information for PCR primers were obtained from the NCBI website.Primers were commercially synthesized.All family members were subject to PCR genotyping.The PCR fragments were separated though 8% denaturing polyacrylamide gel electrophoresis.Two-point linkage analysis between the cataract phenotype and genetic markers were performed using the MLINK component of the LINKAGE package.Haplotype was constructed using allele information of family3.To calculate LOD score:LOD≥3,the two-point linkage exists.LOD≤-2,fail to linkage.LOD score between- 2 and 3,indicates linkage relation.Acording to the allele and the relation between family member,artificially construct the family haplotype.From NCBI download web site complete exon sequence of human GJAB.To amplify the DNA sequence by elongate the either side of DNA to 100bp.Design and synthesize primers.Amplify the exon of GJA8 by using primer GJASF/GJASR and using DNA of proband and an affected family member.Purify and collect PCR product.To purified PCR fragments were submitted for direct automatic sequencing.Reaction cycle according to fragment length.A less-than-500bp tested a reaction,a greater-than-500bp took sequence reaction bidirectionally.For the suspected locus, further restriction analysis is needed to identify the mutation.ResultsA total of 71 individuals,including 26 patients(10 males and 16 females),from the 4 families with congenital cataract,were investigated retrospectively.And 66 individuals were available for examination.Blood samples were collected from 49 individuals,including 25 patients.The pattern of phenotype transmission in all families suggested a mode of autosomal dominant inheritance.Of the 4 families,2 were zonular and another 2 were nuclear 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 1q21-25,2q33-36,3q21-25,3q27.3,10q25,11q22.1-23.21,12q13-14,13q12.11,16q23.1,16q22.1,17q11-12,19q13.33,20p12.1,21q22.3 and 22q11.2-12.1.However,a maximum LOD score of 2.36 were obtained at 1q21-25 for marker D1S1156(θ=0)in family 3,suggesting possible involvement of the GJA8 gene cluster in the cataractogenesis in this family.PCR direct sequencing of the proband genes in the GJA8 gene cluster revealed a missenss mutation of the GJA8 gene,S258F(773C＞T),leading to the substitution of serine for phenylalamine,which suggested that the novel mutation is pathogenic mutation.Mismatch primer GJA8MF and GJA8R amplified DNA of 20 family members from family 3 and 100 nomal individuals,which produced 169bp DNA fragments.PCR products were digested by Hinf I and analysed by 8%PAGE.The result showed that all the affected individuals contained the mutation which was exactly the same as the proband,but all nomal individuals did not.Conclusion1,Four families with congenital cataract were found and autosomal dominance was indicated as the mode of inheritance.2,The phenotype of ADCC in all families was different.Two families expressed zonular cataract and the other two were nuclear cataract.3,Two-point linkage analysis using 26 microsatellite polymorphic markers from 12 candidate chromosome regions excluded chromosome 2,3,10,11,12,13,16,17, 19,20,21 and 22 in family 3.4,Two-point linkage analyses showed the linkage relation with 1q21-25region in family 3.5,A novel GJA8 mutation was found in family3[S258F(773C＞T)].6,The phenotype of Chinese ADCC families exist hereditary hetrogenicity.