Congenital Cataract Spontaneous Mutation Gene Identified In Mice By Positoinal Cloning

Congenital cataract is lens opacity at birth or after birth occurred within the first year. There are 0.4% of newborns are congenital cataracts and congenital cataract is a common blind causing disease in children. Approximately 26%～51% of congenital cataract is caused by genetic factors. The fundamental way of prevention for the disease is dissecting the congenital cataracts by molecular mechanisms.In 2006 years, two spontaneous mutant cataract mice were found in the F1 hybrids of ICR an outbred mouse strain crossed to inbred strain BALB/cJ. The disease allele maintained a stable autosomal dominant inheritance pattern at further backcross to BALB/cJ mice.In this study, homozygotes and heterozygotes of the disease mice were identified. Heterozygous mutants expressed a nuclear and radial cataract, which was observed during split lamp examination. The homozygotes have already developed the cataract at day 13 after birth, without observable pathology of corneal, behavior, reproductive performance and development.For mapping of the mutation, heterozygous carriers were mated to wildtypeC3H/HeJ mice. Offsprings (F1 generation) with cataracts were backcrossed to thewildtype C3H/HeJ mice. In the F2 generation, a total of 83 samples were selected forgene identification. A genome-wide linkage analysis was performed with, 44microsatellite markers evenly spaced in autosomal chromosomes. The mutation wasmapped to chromosome 1 between the markers D1MU410 (17cM) and D1Mit102(73cM). Next, sevsral markers were further selected between the markers DlMit410(17cM) and D1Mit102 (73cM) for fine mapping. The mutation was mapped tochromosome 1 between D1Mit236 (25.7cM) and D1Mit46 (43.1cM) by haplotypeanalysis. 13 samples of different haplotype were used for mapping. Two additional markers D1Mit19 (36.9cM) and D1Mit7 (41cM))were genotyped and fine mapping culminated in 11cM interval on chromosome 1 between D1Mit236 and D1Mit19. This position of the further mapped interval leads to suspicion that theγ-crystallin encoding gene Cryg (32cM) is the candidate gene in terms of the gene expression and known function of the gene family. All six members of the Cryg gene cluster were amplified using gene-specific PCR methods from cDNA. cDNA Sequence analysis revealed a deletion of 1 -bp at the 209 base of exon 3 of Crygc gene, leading to a stop codon at the 76 of exon 3 of Crygc gene which produces a truncated protein. To confirm the deletion mutations as cause of cataracts reasons, the result of large samples for sequencing and single nucleotide identification were performed and the results show that the deletion genotype and phenotype of cataract is linked.Although the RT-PCR results show that mRNA transcription of Crygc can be normal, however, as the mutation is located in conserved fourth Greek-key motif which is shorted in the mutant mice, when mRNA is translated into comprised protein, the function of gene is changed. Bioinformatic analysis of the mutantγC-crystallin reveals that the HMM of second domain is from 1 to 66, and 15 amino acids are lost in the mutant mice which cause conserved region of second structural domain to be damaged. Based on the amino acid sequence information, the conservative segment is short, whereas the conserved sequences of is critical for the maintenance of protein structure and function. Shortened conservative segment affects the function of the lens proteins and leads to the formation of cataracts. In the three-dimensional structure analysis of protein, the C-terminal of mutant protein deletes aβ-sheet. And in the detection of health degrees of three structural models, because mutant protein are truncated at 76 of exon 3 of Crygc gene, resulting in an increase of Anolea mean force potential for quality assessment of the final models, the structural of protein is instable.On the basis of experimental results from genetic studies and structural analysis, the thesis paper concludes that single-base deletion of Crygc is the molecular cause of cataract mice which were spontaneously mutated in natural breedings.