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Robyn V Jamieson, Alan Ma, John R Grigg, Rebecca Greenlees; Genomic approaches in disease gene identification in congenital cataract: molecular diagnoses and new insights to Rap1 signalling in the lens. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1346.
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© ARVO (1962-2015); The Authors (2016-present)
There is marked genetic heterogeneity in diseases affecting the lens that lead to congenital cataracts, anterior segment abnormalities and microphthalmia. Our purpose is to use genomic approaches to rapidly identify known disease genes in patients, followed by strategies for novel disease gene identification in those where no known disease gene is found. Our work provides genetic diagnoses for patients as well as new understanding in ocular and cellular biology.
We applied TruSeq Custom Amplicon or Trusight One Clinical Exome capture (Illumina Inc CA USA) followed by next-generation sequencing (NGS), for disease gene identification in 47 apparently non-syndromic patients with congenital cataracts. Known cataract-causing genes were examined for mutations. Genomic investigation in a patient with a chromosomal translocation was undertaken for novel disease gene identification. Functional studies were performed in the Caco2 epithelial cell line, zebrafish and mouse, using shRNA, morpholinos and a genetrap knockout respectively.
We identified pathogenic variants in over 70% of our congenital cataract patient cohort, including variants in crystallin and gap junction genes, VIM, MIP, BFSP1, PAX6 and MAF. Mutations were also found in the X-linked genes, NHS and BCOR, in four patients with previously unrecognised syndromic features providing new diagnoses for these families. We identified a novel lens human disease gene implicated in the Rap1 signalling pathway. Loss of function of this gene led to lens abnormality, cataract formation and microphthalmia in our zebrafish and mouse models. In our mouse model there was abnormal E-cadherin, F-actin and ZO1 staining in anterior epithelial cells of the lens, indicating abnormalities of adhesion and polarity. There was also aberrant αSMA expression indicating a critical role for proper regulation of Rap1 signalling for prevention of epithelial to mesenchymal transition in the lens.
Our NGS strategy provides a major advance in clinical molecular diagnosis for patients with congenital cataracts. Our genomic work has also led to new knowledge with implications for regulation of Rap1 signalling and insights to the role of this pathway in cell adhesion, cytoskeletal integrity, polarity and epithelial to mesenchymal transition.
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