Genetic Cataract Gene Mutations And Protein Function Change

Objective:Use allele-sharing and linkage analysis strategy, to identify the genetic defects in twoclinical different congenital cataract pedigrees; targeted gene mutations have been found,from bioinformatics and protein structure and function research, analysis the molecularbiology of disease mechanisms.Methods:Part I: Study on the clinical phenotypeInformed consent was obtained from each participant, consistent with the Declaration ofHelsinki. Family history data were recorded. Clinical and ophthalmologic examinationswere performed on affected and unaffected family members at the Beijing Tongren EyeCenter, Capital Medical University, Beijing. Phenotype was documented by slit lampphotography.Part II: Gene maping researchThrough the current available information, analysis the potential relationship betweencongenital cataracts clinical phenotypes and gene mutations, initial identification ofhigh-suspected genetic mutation site; all the members were genotyped with microsatellitemarkers at loci considered to be associated with cataracts. Two-point LOD scores werecalculated using the Linkage software after genotyping, gene sequencing to determine thegenetic mutation site; Denaturing HPLC was used to screen the mutation that wasidentified in the patients, further determine the genetic mutation site.Part III: Bioinformatics ResearchUse vector NTI, DNAMAN, BioEdit and other bioinformatics softwares, as well asNCBI, UCSC databases, analysis the genes and proteins general natures; Swiss-modelsoftware (version 3.5), CLC protein workbench 3 (version 3.0.2), ExPaSy, Radar, Phyresoftware (version 0.2) and other online softwares, analysis the characteristics anddifferences of the mutant and wild-type proteins.PartIV: Proteins structure and function ResearchThe complete gene cDNA sequence was obtained; using site-directed mutagenesisconstructed mutant cDNA sequence; wild and mutant proteins were expressed in E.coliand purified the target proteins; Spectroscopy analysis wild and mutant-type proteins ofhydrophobicity, stability and demonstrate the nature of the characteristics and differencesof wild and mutant type proteins.Results:Affected members of the family gathered from Hebei China had coralliform cataracts. Linkage analysis was obtained at markers, D2S72(LOD score [Z]=3.31, recombination fraction [8]=0.0) and D2S1782 (Z=3.01,θ=0.0). Haplotype analysis indicated that the cataract gene was closely linked to these two markers. Sequencing theγD-crystallin gene (CRYGD) revealed a G>T transversion in exon 2, which caused a substitution of Gly to Cys at codon 61 (p.G61C). This mutation cosegregated with the disease phenotype in all affected individuals and was not observed in any of the unaffected or 100 normal, unrelated individuals. Affected members of the family gathered from Beijing China had lamellar cataracts. Linkage analysis was obtained at markers D3S2322 (LOD score [Z]=7.22, recombination fraction [θ]=0.0) and D3S1541 (Z=5.42,θ=0.0). Haplotype analysis indicated that the cataracts gene was closely linked to these two markers. Sequencing the beaded filament structural protein 2 (BFSP2) gene revealed a G>A transversion in exon 5, which caused a conservative substitution of Arg to His at codon 339 (p.R339H). This mutation cosegregated with the disease phenotype in all affected individuals and was not observed in the unaffected family members or 100 normal, unrelated individuals.Data generated with BioEdit software revealed that the CRYGD protein p.G61C mutation and BFSP2 protein p.R339H mutation located in the evolution conservative region, the mutant protein basic natures and the proportion of amino acid composition changed; In the mutant CRYGD protein, the 3D-structure did not change much but the mutations regional amino acid side chain, protein complexity, hydrophobicity and hydrophobic moment had changed; Mutant BFSP2 protein mutations regional amino acid side chain, secondary structure, hydrophobic moment had changed.Protein structure and function research found: CRYGD protein p.G61C mutant protein shift to an earlier date of 6℃, emergence secondary structure and tertiary structure damaged, early 10℃, emergence a large number of sparse surface exposed; mutant protein than wild-type protein in advance one molar volume, emergence protein structural changes, early 0.6-0.8 molar volume emergence secondary structure destructs, sparse surface more likely to exposed in the water phase; fluorescence phase analysis found: CRYGD protein GuHCl denaturation phase diagram, the point of the whole process more concentrated, mutant protein in GuHCl concentration of 3.2 mol/l, emergence the sharp rise straight line, the point of the entire process more scattered. The largest RRS intensity excitation wavelength of wild-type and mutant BFSP2 protein, in the 350 nm, mutant protein RRS intensity smaller than wild-type protein; wild-type BFSF2 protein RRS intensity growing faster than the mutant-type; at the balance state. Conclude: This is the first found of congenital coralliform cataracts phenotype associated with the mutation of Gly61Cys (p.G61C) in the CRYGD gene; the first found of congenital lamellar cataract phenotype associated with the mutation of Arg339His (p.R339H) in the BFSP2 gene.Large-scale use of a variety of network and stand-alone bio-informatics softwares, found p.G61C CRYGD gene mutation, changes the protein natures in the mutant site, may affect the entire protein hydrophobicity and stability, thereby affecting the function of proteins, which led to the formation of cataracts. BFSP2 gene mutation p.R339H , the pKr (R) values of arginine and histidine large differences, the secondary structure changes, as well as mutant site hydrophobic moment changes, that may affect the function of proteins, which led to the formation of cataract.Successful construct the cDNA sequence of CRYGD gene wild-type, BFSP2 gene wild-type, CRYGD gene p.G61C mutant-type and BFSP2 gene p.R339H mutant-type; Using E. coli expression system, has successful express the CRYGD and BFSP2 wild-type and mutant proteins, found CRYGD protein expressed in the form of soluble and BFSP2 protein expressed in the form of inclusion body; Using electron microscopy, CD, F-2500, to analyze the protein which mutation can lead to cataracts, revealed the special nature of the protein and disease mechanism, found mutant-type CRYGD protein, aggregation increased, stability decreased and environmental resistance reduced, mutant-type BFSP2 protein aggregation decreased and stability increased.