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Identification and functional analysis of a novel missense mutation in GJA8, p.Ala69Thr.

ABSTRACT: BACKGROUND:To explore the molecular genetic cause of a four-generation autosomal dominant congenital cataract family in China. METHODS:Targeted region sequencing was performed to screen for the potential mutation, and Sanger sequencing was used to confirm the mutation. The homology model was constructed to identify the protein structural change, PolyPhen-2 and Provean were used to predict the mutation impact. Functional and cellular analysis of the wild and mutant GJA8 were performed in DF-1 cells by western blotting, dye uptake assay, immunofluorescence, Annexin V-FITC staining. RESULTS:A novel heterozygous mutation (c.205G?>?A; p.Ala69Thr) was identified within GJA8, which cosegregated with congenital cataract phenotype in this family. Bioinformatics analysis showed the mutation was located in a highly conserved region, and the mutation was predicted to be pathogenic. Function analysis indicated that the mutation inhibited GJA8 hemichannel activity, reduced cell tolerance to oxidative stress, changed the protein distribution pattern and inhibited the cell growth. CONCLUSIONS:We have identified a novel missense mutation in GJA8 (c.205G?>?A, p.Ala69Thr) in a four-generation Chinese family and our results will further broaden the gene mutation spectrum of GJA8.

SUBMITTER: Li D

PROVIDER: S-EPMC7678044 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Identification and functional analysis of a novel missense mutation in GJA8, p.Ala69Thr.

Li Dandan D Xu Chenjia C Huang Dandan D Guo Ruru R Ji Jian J Liu Wei W

BMC ophthalmology 20201120 1

<h4>Background</h4>To explore the molecular genetic cause of a four-generation autosomal dominant congenital cataract family in China.<h4>Methods</h4>Targeted region sequencing was performed to screen for the potential mutation, and Sanger sequencing was used to confirm the mutation. The homology model was constructed to identify the protein structural change, PolyPhen-2 and Provean were used to predict the mutation impact. Functional and cellular analysis of the wild and mutant GJA8 were perfor ...[more]

PMID: 33218330

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Project description:Purpose: To broaden the mutation and phenotype spectrum of the GJA8 and CHMP4B genes and to reveal genotype-phenotype correlations in a cohort of Chinese patients with congenital cataracts (CCs). Methods: Six Chinese Han families with CCs inherited in an autosomal dominant (AD) pattern were recruited for this study. All patients underwent full ocular examinations. Genomic DNA was extracted from the leukocytes of peripheral blood collected from all available patients and their unaffected family members. Whole-exome sequencing (WES) was performed on all probands and at least one of their parents. Candidate variants were further confirmed by Sanger sequencing. Bioinformatic analysis with several computational predictive programs was performed to assess the impacts of the candidate variants on the structure and function of the proteins. Results: Four heterozygous candidate variants in three different genes (CRYBB2, GJA8, and CHMP4B) were identified in affected individuals from the six families, including two novel missense variants (GJA8: c.64G > C/p. G22R, and CHMP4B: c.587C > G/p. S196C), one missense mutation (CRYBB2: c.562C > T/p. R188C), and one small deletion (GJA8: c.426_440delGCTGGAGGGGACCCT/p.143_147delLEGTL). The three missense mutations were predicted as deleterious in all four computational prediction programs. In the homologous model, the GJA8: p.143_147delLEGTL mutation showed a sequence deletion of five amino acids at the cytoplasmic loop of the Cx50 protein, close to the third transmembrane domain. Patients carrying mutations in the same gene showed similar cataract phenotypes at a young age, including total cataracts, Y-sutural with fetal nuclear cataracts, and subcapsular cataracts. Conclusion: This study further expands the mutation spectrum and genotype-phenotype correlation of CRYBB2, GJA8, and CHMP4B underlying CCs. This study sheds light on the importance of comparing congenital cataract phenotypes in patients at the same age stage. It offers clues for the pathogenesis of CCs and allows for an early prenatal diagnosis for families carrying these genetic variants.

Dataset Information

Identification and functional analysis of a novel missense mutation in GJA8, p.Ala69Thr.

Publications

Identification and functional analysis of a novel missense mutation in GJA8, p.Ala69Thr.

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