Dataset Information

Identification of pathogenic mechanisms of COCH mutations, abolished cochlin secretion, and intracellular aggregate formation: genotype-phenotype correlations in DFNA9 deafness and vestibular disorder.

ABSTRACT: Mutations in COCH (coagulation factor C homology) cause autosomal-dominant nonsyndromic hearing loss with variable degrees of clinical onset and vestibular malfunction. We selected eight uncharacterized mutations and performed immunocytochemical and Western blot analyses to track cochlin through the secretory pathway. We then performed a comprehensive analysis of clinical information from DFNA9 patients with all 21 known COCH mutations in conjunction with cellular and molecular findings to identify genotype-phenotype correlations. Our studies revealed that five mutants were not secreted into the media: two von Willebrand factor A (vWFA) domain mutants, which were not transported from the endoplasmic reticulum to Golgi complex and formed high-molecular-weight aggregates in cell lysates, and three LCCL domain mutants, which were detected as intracellular dimeric cochlins. Mutant cochlins that were not secreted and accumulated in cells result in earlier age of onset of hearing defects. In addition, individuals with LCCL domain mutations show accompanying vestibular dysfunction, whereas those with vWFA domain mutations exhibit predominantly hearing loss. This is the first report showing failure of mutant cochlin transport through the secretory pathway, abolishment of cochlin secretion, and formation and retention of dimers and large multimeric intracellular aggregates, and high correlation with earlier onset and progression of hearing loss in individuals with these DFNA9-causing mutations.

SUBMITTER: Bae SH

PROVIDER: S-EPMC4373469 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Identification of pathogenic mechanisms of COCH mutations, abolished cochlin secretion, and intracellular aggregate formation: genotype-phenotype correlations in DFNA9 deafness and vestibular disorder.

Bae Seung-Hyun SH Robertson Nahid G NG Cho Hyun-Ju HJ Morton Cynthia C CC Jung Da Jung DJ Baek Jeong-In JI Choi Soo-Young SY Lee Jaetae J Lee Kyu-Yup KY Kim Un-Kyung UK

Human mutation 20141201 12

Mutations in COCH (coagulation factor C homology) cause autosomal-dominant nonsyndromic hearing loss with variable degrees of clinical onset and vestibular malfunction. We selected eight uncharacterized mutations and performed immunocytochemical and Western blot analyses to track cochlin through the secretory pathway. We then performed a comprehensive analysis of clinical information from DFNA9 patients with all 21 known COCH mutations in conjunction with cellular and molecular findings to ident ...[more]

PMID: 25230692

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Project description:Five missense mutations in the FCH/LCCL domain of the COCH gene, encoding the protein cochlin, are pathogenic for the autosomal dominant hearing loss and vestibular dysfunction disorder, DFNA9. To date, the function of cochlin and the mechanism of pathogenesis of the mutations are unknown. We have used the biological system of transient transfections of the entire protein coding region of COCH into several mammalian cell lines, to investigate various functional properties of cochlin. By western blot analysis of lysates prepared from transfected cells, we show that cochlin is a secreted protein. Immunocytochemistry shows concentrated localisation of cochlin in perinuclear structures consistent with the Golgi apparatus and endoplasmic reticulum, showing intracellular passage through these secretory compartments. We detected that cochlin is proteolytically cleaved between the FCH/LCCL domain and the downstream vWFA domains, resulting in a smaller cochlin isoform of approximately 50 kDa. Interestingly, this isoform lacks the entire mutation bearing FCH/LCCL domain. We have also shown that cochlin is N-glycosylated in its mature secreted form. Previous studies of the FCH/LCCL domain alone, expressed in bacteria, have demonstrated that three of four DFNA9 mutations cause misfolding of this domain. Characteristic eosinophilic deposits in DFNA9 affected inner ear structures could be the result of aberrant folding, secretion, or solubility of mutated cochlins, as in certain other pathological states in which misfolded proteins accumulate and aggregate causing toxicity. To examine the biological consequences of cochlin misfolding, we made separate constructs with three of the DFNA9 mutations and performed parallel studies of the mutated and wild type cochlins. We detected that mutated cochlins are not retained intracellularly, and are able to be secreted adequately by the cells, through the Golgi/ER secretory pathway, and also undergo proteolytic cleavage and glycosylation. These results suggest that DFNA9 mutations may manifest deleterious effects beyond the point of secretion, in the unique environment of the extracellular matrix of the inner ear by disrupting cochlin function or interfering with protein-protein interactions involving the FCH/LCCL domain. It is also possible that the mutations may result in aggregation of cochlin in vivo over a longer time course, as supported by the late onset and progressive nature of this disorder.

Dataset Information

Identification of pathogenic mechanisms of COCH mutations, abolished cochlin secretion, and intracellular aggregate formation: genotype-phenotype correlations in DFNA9 deafness and vestibular disorder.

Publications

Identification of pathogenic mechanisms of COCH mutations, abolished cochlin secretion, and intracellular aggregate formation: genotype-phenotype correlations in DFNA9 deafness and vestibular disorder.

Similar Datasets

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