Investigating a possible role of R3HCC1L in embryonic development and ocular disease
Peters anomaly (PA) is an anterior segment ocular disorder with wide phenotypic variability and genetic heterogeneity. Here we report a family consisting of a male with a diagnosis of syndromic PA and his unaffected parents, with no causative variants identified in known developmental ocular genes. Exome sequencing analysis identified compound heterozygous missense variants, c. 1022A>T p.(Asp341Val) and c.1457T>A p.(Phe486Tyr), in R3HCC1L. Both variants are ultra-rare in control populations and have CADD scores of 21.9 and 23.3, respectively, suggesting possible deleterious effects. The R3HCC1L transcript variants encode three different protein isoforms all sharing two conserved C-terminal domains, an RNA recognition motif (RRM) and a coiled-coil domain (CCD), and likely represent an RNA-binding protein involved in post-transcriptional gene regulation; the identified patient variants are located within the N-terminal part of the protein shared by two of the three protein isoforms, upstream of the RRM and CCD domains. To investigate the possible role of R3HCC1L in embryonic development, the single zebrafish ortholog of R3HCC1L, r3hcc1l, was examined for its expression and function. In situ expression studies showed that zebrafish r3hcc1l is expressed in the developing lens, cornea, retina and hyaloid vasculature, supporting its possible role in ocular development in vertebrates. CRISPR-Cas9 gene editing was used to generate a zebrafish line with a 4-bp deletion in r3hcc1l, c.623-626del, that is predicted to result in a nonsense-mediated decay and, if expressed, a nonfunctional truncated protein (p.Thr208fs*39) lacking 80% of the RRM and the entire CCD. The resultant r3hcc1lc.623-626del heterozygous and homozygous animals did not show any visible structural abnormalities in the eye or any other systems, with normal survival of all genotypes to adulthood, providing no support for its possible role in the congenital phenotype of interest. However, the function of r3hcc1l may not be completely conserved with human R3HCC1L, and/or zebrafish may have compensatory mechanisms that are not present in humans. In addition, the engineered zebrafish variant disrupts the most conserved C-terminal region of R3HCC1L/r3hcc1l shared by all protein isoforms and likely leads to a complete loss-of-function of this gene, which may be different from the disease mechanism associated with the specific missense alleles identified in the patient. Finally, while it is important to consider the possible limitations of animal models, it is also necessary to highlight that the identified R3HCC1L variants may not have any role in the phenotype observed in this single patient. Identification of new R3HCC1L variants of interest in families affected with PA or other congenital phenotypes, if successful, will provide further support for the possible developmental function of this gene.