Abstract
Uveal coloboma is a potentially blinding congenital ocular malformation caused by the failure of optic fissure closure during the fifth week of human gestation. We performed custom capture high‐throughput screening of 38 known coloboma‐associated genes in 66 families. Suspected causative novel variants were identified in TFAP2A and CHD7, as well as two previously reported variants of uncertain significance in RARB and BMP7. The variant in RARB, unlike previously reported disease mutations in the ligand‐binding domain, was a missense change in the highly conserved DNA‐binding domain predicted to affect the protein's DNA‐binding ability. In vitro studies revealed lower steady‐state protein levels, reduced transcriptional activity, and incomplete nuclear localization of the mutant RARB protein compared with wild‐type. Zebrafish studies showed that human RARB messenger RNA partially reduced the ocular phenotype caused by morpholino knockdown of rarga gene, a zebrafish homolog of human RARB. Our study indicates that sequence alterations in known coloboma genes account for a small percentage of coloboma cases and that mutations in the RARB DNA‐binding domain could result in human disease.
Highlights
Custom capture or targeted gene sequencing has been a cost‐effective approach to detect mutations in genes known to be associated with a specific disease condition
We identified novel variants in genes such as TFAP2A and CHD7, and previously reported variants in RARB and BMP7, indicating that mutations in known coloboma‐associated genes account for only 4.5%
Congenital disorders such as coloboma and other conditions were shown to exhibit high genetic heterogeneity and targeted sequencing of disease‐associated genes may yield the limited outcome of potential variants (DaRe, Vasta, Penn, Tran, & Hahn, 2013; Perez Millan et al, 2018; Redin et al, 2014)
Summary
The first stage custom capture (CC‐1) sequencing included 42 families and analysis was performed on 34 families which had at least trios with 110 study subjects. Quality scores computed by MPG (“Most Probable Genotype” caller) algorithm (https://www.ncbi.nlm.nih.gov/pubmed/20810667). Immunofluorescence staining was performed following a standard protocol as previously described (George et al, 2016). Cultured HEK 293 cells were harvested for protein in radioimmunoprecipitation assay (RIPA) buffer (Cat # 89901; Invitrogen) containing protease inhibitors cocktail (REF # 04693159001; Roche). Renilla luciferase reporter plasmid DNA (pRL‐TK) and CYP26A1 promoter region cloned firefly luciferase reporter plasmid DNA (pGL4.10) were cotransfected with pcDNA3.1+‐DYK‐tagged RARB or RARB‐mutant plasmids into HEK 293 cells in culture. The cultured cells were lysed in passive lysis buffer and samples prepared as per manufacturer protocol using the Dual‐Luciferase Reporter assay system (Cat # E1960; Promega).
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