Localization And Cloning The Disease-associated Gene Of Progressive Polymorphic Congenital Cataract

Although surgical techniques and visual prognosis have been improved recently, congenital cataracts remain the leading cause of visual disability in children worldwide. The incidence of congenital cataracts has been estimated to be 0.6-6 per 10,000 births , and genetic mutation is the most common cause. In fact, about one third of all congenital cataracts are inherited, with the most common being the nonsyndromic autosomal dominant form . The frequrent autosomal inheritance provides a tool to identify the genes involved in lens development and cataract formation. To date, more than 19 candidate loci have been identified and 14cataract-related genes characterized. These genes can be considered in four groups: (1) Crystallin genes encoding greater than 90% of the structural proteins in the lens. Mutations in 7 crystallin genes have been identified as the cause of autosomal dominant congenital cataract (ADCC) including: CRYAA , CRYAB, CRYBA1 , CRYBB1 , CRYBB2 , CRYGC , CRYGD and CRYGS . (2) Genes encoding membrane transport proteins including: MIP , GJA3 and GJA8 (3) Genes encoding cytoskeletal proteins such as BFSP2 (4) Genes encoding transcription factors such as PITX3 and HSF4 .Identical mutations can result in different phenotypes and the identical cataract phenotypes have also been mapped to different loci . At present, there are two major methods used in cataract candidate disease-associated gene research: one is basing on major functional protein associated with cataractgenesis, mapping the candidate gene location by hybridization; the other is mapping the locus for congenital cataract in typical family using genomic genescan, candidate gene linkage analysis and candidate gene screening to identifying the disease-associated gene.We studied a large, four-generation Chinese family containing members affected by congenital progressive polymorphic cataract. Opacification of the lens was bilateral in all affected cases, but the appearance of white opacities distributed in the nucleus and cortex were highly variable, which included pulverulent, dot, strip, star-like and sheet shapes. Autosomal dominant inheritance of the congenital cataract was demonstrated by Singles Method of segregation analysis. We studied thecandidate disease-associated gene of this special fasciculiform congenital cataract, clarifying the congenital cataract mechanisms in human. Informed consent in accordance with the Declaration of Helsinki and Zhejiang Institutional Review Board approval was obtained from all participants. Informed consent was also obtained from the subjects after explanation of the nature and possible consequences of the study.Part I The progressive polymorphic congenital cataract familyForty-one individuals participated in the study: 18 affected individuals and 23 unaffected individuals of whom 11 were spouses. Affected status was determined by a history of cataract extraction or ophthalmologic examination, which including slit-lamp examination under dilated pupils, visual acuity testing and fundus examination. The cataract phenotype of the patients who had had cataract extraction was learned from the history records. Opacification of the lens was bilateral in all affected cases, but the appearance of white opacities distributed in the nucleus and cortex were highly variable, which included pulverulent, dot, strip, star-like and sheet shapes. In addition, the opacities became denser as age increased. Visual acuity in the unoperated eyes of those affected individuals ranged from 1.0 to 0.1. Most affected individuals noticed their visual impairments before the age of ten, and then their visual acuity decreased gradually until surgery was required to improve their visual function after the age of 40. Also, there was no family history of other ocular orsystemic abnormalities aside from age-related disorders.Part II Estimation of the family inherited modelBased on the presence of affected individuals in each of the four generations, approximate numbers of affected males and females and male-to-male transmission, autosomal dominant inheritance of the cataract was supported. To identify the family inherited model, we performed the Single Method of segregation analysis: the segregated ratio was 46.9% and the 95% credible interval was between 29.65% and 64.15%, suggestting the autosomal dominant inheritance.Part III Candidate disease-associated gene mappingBlood specimens (5ml) were collected in EDTA and leukocyte genomic DNA was extracted. The initial strategy consisted of screening 18 known loci related to ADCC formation and 41 fluorescent short tandem repeat polymorphic markers (ABI PRISM? Linkage Mapping Set, Version 2.0) were used. Mutiplex PCR products were resolved using a ABI Prism^TM 377XL DNA sequencer and the data were collected and analyzed using these computer programs: (ABI Prism^TM Genescan 3.0 and Genescan analysis(?) 2.1). Two-point lod scores between the cataract locus and markers were calculated by the MLINK program of LINKAGE package (version 5.1) for a full range of θ values. A significant two point LOD score was generated at marker D22S420, D22S539 and D22S315 for 22q 11 -12.1. The highest observedLOD score was 6.26 (θ=0.00) with marker D22S315.Part IV Mutational analysis of CRYBB2 geneWe screened all 6 exons of the candidate gene, CRYBB2, by DNA sequencing and Restrict Fragment Length Polymorphm (RFLP) Analysis for mutations. The genomic sequence of CRYBB2 gene was obtained from http://www.ncbi.nlm.nih.gov and http://genome.ucsc.edu (CRYBB2; NM₀00496). Six gene-specific PCR primer pairs were designed to amplify the 6 exons and flanking intron sequences of CRYBB2. PCR products were TA cloned and sequenced commercially. Two affected and two unaffected individuals were compared. By sequencing of exon 6 of the CRYBB2 gene, we found a base change (C→T) at position 475 of the CRYBB2 cDNA. This mutation creates a premature stop codon, and this nonsense mutation creates a novel SpeI restriction site that segregated with all affected members in this Chinese family, but was not detected in the 100 unrelated normal controls and unaffected pedigree members.Part V Computer prediction and analysis of the mutatant β B2- crystallin proteinβ B2- crystallin plays an important factor in human lens development and cataract formation and it's tertiary structure is highly conserved. Both wild type and mutant β B2-crystallin structure predictions were done by SWISS-MODEL usingbovine β B2-crystallin (1BLB, 2BB2) as templates and by PROSPECT 2.0 using bovine β B2-crystallin (2BB2) as a template. Models were viewed in Swiss-Pdb Viewer. The mutant β B2-crystallin protein predicted by SWISS-MODEL and PROSPECT software showed that 51 amino acid in c-terminal were truncated and the fourth "Greek Key" motif in the second domain couldn't be formation. The mutant β B2-crystallin also demonstrated having prevalent negative surface electrostatic potential and decreased hydrogen bonds.Conclusion:1. This is the first report about the prgressive polymorphic congenital cataract family in China.2. Autosomal dominant inheritance was demonstrated in this family.3. Mutation analysis of the candidate gene, CRYBB2, identified a C→T heterozygous transversion at nucleotide position 475 in the exon6 of CRYBB2, which co-segregated with the presence of the prgressive polymorphic congenital cataract in this family and was not observed in 100 unrelated controls.4. The mutant β B2-crystallin protein, which was predicted by SWISS-MODEL and PROSPECT 2.0, had the changed tertiary structure, surface electrostatic potential and decreased hydrogen bonds.5. This study confirmed that the congenital cataracts were phenotypically and genetially heterogeneous and the relative relationship between the genetype and the phenotype, further supporting the notion that β B2-crystallin play an important role in human lens development and cataract formation.