The Genetics of Uveal Coloboma

National Eye Institute

Investigators

Linked publications & trials

Abstract

1. Clinical and Genomic Studies Recruitment of coloboma patients in the Genetics of Uveal Coloboma protocol (13-EI-0049) and the Whole Exome and Whole Genome Sequencing for Genotyping of Inherited and Congenital Eye Conditions protocol (14-EI-0064) continues after the slow down imposed by the Covid-19 pandemic. We have recruited and examined 185 families and a total of 673 patients and family members. All 185 probands and their first-degree relatives (when available) have completed ophthalmic exams. General physical examinations and targeted systemic testing were performed on probands, as needed. Lymphoblastoid cell lines were established for all participants. We received IRB approval for a new protocol Potential Environmental Causes of Uveal Coloboma (000366-EI). The aim is to explore maternal factors and exposures during the first trimester of pregnancy as potential causes of uveal coloboma and to correlate exposure data to clinical data from affected children. This study focuses on the mothers of children with coloboma. They will be administered a questionnaire over the phone about their health, lifestyle and habits before and during pregnancy. The questionnaire is adapted from the National Birth Defects Prevention Study (NBDPS) Mother Questionnaire. Furthermore, the study will use existing data from NBDPS and NIH studies, including family data such as eye exam, genetic test results, and family history of coloboma. 2. Laboratory Studies Work in the laboratory has suffered a major slowing down because of Covid-19 and the limitations in personnel allowed to the physical workplace. A. The RICO mouse model of coloboma The RICO (Retinal & Iris COloboma) mouse arose from the random insertion of a transgene (NSE-VEGF) on chromosome 13 in the C57BL/6 background. Both homozygous and heterozygous mutants developed coloboma and the homozygous transgene insertion was lethal. The genomic organization at the insertion site included approximately 30 copies of the transgene, an inversion, three duplications and a deletion in a gene desert. We detected transient VEGF mRNA and protein expression during eye and brain development. Upon outcrossing of the RICO mouse from the C57BL/6 to the 129/SvJ background, the homozygous mutant survived to adulthood and the severity of the coloboma phenotype persisted, albeit with reduced penetrance in the heterozygous mice. Since the phenotype was maintained across a different background, it remained to be tested whether it was a direct consequence of VEGF overexpression or of genomic rearrangements at the site of transgene insertion. To this end, we generated additional transgenic mouse lines (NSE-VEGF) using the same construct as in the RICO mouse. Three lines showed integration at different chromosomal sites with various degrees of VEGF transcript expression. Mice backcrossed to the C57BL/6 background are just becoming available and will be analyzed for VEGF expression in the developing eye and for coloboma phenotype. B. Coloboma candidate gene studies (Zfp703/Nlz1 and Zfp503/Nlz2) We identified two zinc-finger motif-containing genes, Zfp703/Nlz1 and Zfp503/Nlz2, that are important in regulating optic fissure closure in zebrafish (Brown et al., Proc Natl Acad Sci U S A. 2009). Both Nlz1 knockout (KO) and Nlz2 KO mice were perinatally lethal and exhibited coloboma. Protein expression studies in mouse eye indicated that Nlz2 is expressed transiently during development in the retinal pigment epithelium (RPE) at the time around optic fissure closure, and in developing and adult amacrine and ganglion cells. Nlz2 ko mice displayed altered patterns of expression of transcription factors markers of eye development and abnormal retinal pigment epithelium (RPE) and retina morphogenesis. Systemic abnormalities were also observed (manuscript under review). C. CRISPR screening for genes associated with optic fissure closure Using CRISPR/Cas9-mediated genome editing as a screening method, we generated KO zebrafish lines to investigate the roles of candidate genes from Brown et al., Proc Natl Acad Sci U S A. 2009, in the ontogenesis of coloboma. We generated 72 CRISPR F0 lines, mostly containing insertions/deletions (indels) in the exonic regions of targeted genes. Of those, 19 lines displayed coloboma in F1 compound heterozygous zebrafish; 11 of the 19 lines also presented other anatomical defects. In situ hybridization at 24 hours post-fertilization (hpf), 32 hpf, and 48 hpf for most tested genes showed localization to the eye. Of the 19 genes, 14 encoded signaling pathway proteins and 5 encoded transcription factors. Some of the genes are currently being further investigated for their role in optic fissure closure. D. Live imaging of optic fissure closure in zebrafish Another aspect to understanding the onset of coloboma is to visually record in real time how different cells/tissues behave when the two opposing epithelial sheets come together during optic fissure closure. We have injected zebrafish embryos at single cell stage with CAAX-GFP RNA which marks cell membranes green and H2A-m-Cherry RNA which marks the nuclei red. We imaged zebrafish embryos under the confocal microscope from 32hpf, the time at which the fissure margins are in close opposition, until fissure closure. Since relying on RNA may be problematic due to photo bleaching, we generated zebrafish lines transgenic for CAAX-GFP and H2A-m-Cherry. These lines have also the advantage that they can be crossed to the mutant lines from the CRISPR/Cas9 screen to visualize altered cell/tissue behavior by live imaging. Because of the long acquisition times, this project could not be pursued during the past year. We hope to be able to restart soon.