Study On Localization And Function Of The Disease-associated Gene Of A Progressive Congenital Cortical Punctate Cataract Family

BackgroundCataract is the leading cause of blindness worldwide. Especially congenital cataract has become the primary cause of childhood blindness and visual impairment. It has been reported that the incidence of this disease is about1to6every ten thousand and it is the second cause of blindness in children. The etiology of congenital cataract is numerous and complicated. Approximate1/3of congenital cataracts are inherited. Therefore, current researches of inherited cataract mainly focus on the location of disease-associated genes and mutations. To date, at least26genes and40genotypes have been identified to be linked with different forms of inherited cataract. However, the genetic heterogeneity of inherited cataract, which ranges widely in phenotype and severity, poses huge difficulty on the identification of disease-causing genes. Moreover, a new post-genomic era will come after the accomplishment of human genome project (HGP). It means the functional study of disease-related genes and its pathogenic mechanism will become a hotspot, especially for inherited diseases. On the one hand, the study of genes and molecular mechanism underling hereditary cataract will enrich current genebank of disease and provide a means of presymptomatic and genetic diagnosis. On the other hand, it will shed light on the pathogenesis of lens opacity and make it possible to understand of the mechanism of this disease. This improvement will be helpful for the future gene therapy of inherited cataract.In this paper, we studied on a Chinese family affected with progressive congenital cortical punctate cataract by locating and functional detecting of the disease-associated gene.MethodsWe collected a detailed family history and clinical data from affected and unaffected individuals in the family, through a number of ophthalmologic examinations (including visual acuity, slit-lamp examination and fundus examination). Inherited mode was analyzed through pedigree. Genomic DNA samples were extracted from peripheral blood of the family members. All the exons and flanking intronic sequences of candidate gene were amplified by polymerase chain reaction (PCR) and screened for mutation by direct bidirectional DNA sequencing. Computational algorithms, including Polyphen-2and SIFT, are used to predict whether a specific amino acid substitution is deleterious or neutral to the structure and function of the protein. Wild type cDNA was PCR amplified from the cDNA library of normal lens, then the product was cloned into pcDNA3.1(-). Site directed mutagenesis was used to construct the mutant plasmid. Cellular distribution of AQPO was examined by expression in HeLa cells:protein distribution was detected by immunofluorescene and observed by confocal fluorescence microscopy. Results1. The phenotype of the affected family members is progressive cortical punctate cataract. The family’s inherited mode is autosomal dominant. By direct sequencing of the reported candidate genes, including CRYAA. CRYBA1、CRYGD、CRYGS、MIP、 GJA3, we detected a heterozygous c.448G>C change in the coding region of the MIP gene, resulting in the substitution of conserved asparagic acid to histidine (D150H), this change was not observed in all unaffected family menbers and200unrelated individuals. The results of Polyphen-2and SIFT indicated D150H substitution was likely deleterious and possibly contributed to the disease.2. We successfully constructed plasmid of wildtype AQPO and AQP0D150H. After transfection into HeLa cells, we detected the mutant protein mainly aggregating in cytoplasm and it was rarely observed locating at plasma membrane. This indicated that the D150H substitution impaired the normal trafficking mechanism of AQPO.Conclusions1. Here we reported a four generation Chinese family with autosomal dominant congenital cataract, and identified a novel MIP gene mutation c.448G>C (p.D150H), which co-segregated with the presence of the progressive cortical punctate cataract in this family.2. This mutation may impair the trafficking mechanism of AQPO and reduce the protein at plasma membrane significantly.3. Our study widened the mutation spectrum of hereditary cataract and revealed the pathogenic mechanism of cataract caused by MIP mutation.