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. As of August 2023, we had enrolled approximately ___ participants (___ affected) from ___ families. Whenever possible, all affected individuals and their first degree family members have undergone a complete ophthalmic examination and blood draw for genetics. Affected individuals have undergone a battery of systemic testing looking for potential phenotypic associations. They have CLIA-certified sequencing of known developmental eye disease genes on an exome background followed by reflex full genome sequencing for negative reports. The protocol has been modified recently to include individuals with microphthalmia and anophthalmia--two phenotypes on the same continuum of developmental abnormalities. This change leverages our recently-funded U01 research effort with Drs. Philip Lupo and Laura Mitchell to perform population-based genetic epidemiology for microphthalmia/anophthalmia/coloboma phenotypes (MAC) as part of the Texas Birth Defects Registry. Recruitment numbers have included patients that have been ascertained through the MAGIC protocol. Our protocol "Potential Environmental Causes of Uveal Coloboma" (000366-EI) explores maternal factors and exposures during the first trimester of pregnancy as potential causes of uveal coloboma to correlate exposure data to clinical data from affected children. We have begun administering a questionnaire over the phone about parents' 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. Enrollment has commenced. 2. Laboratory Studies 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. . Long-read sequencing detected approximately fifteen copies of the transgene at the insertion site, an inversion, one duplications and a deletion in a gene desert on chromosome 13. We detect hVEGF in the developing eye. An open question is whether coloboma is caused by this abnormal expression of VEGF and/or disruption of gene expression caused by the genomic rearrangement. Creation of two additional transgenic lines with NSE-VEGF did not result in coloboma, although copy numbers were considerably lower; as such, a dosage-dependent effect cannot be ruled out. We are pursuing RNA-Seq experiments to identify changes in gene expression, particularly those surrounding the integration site. B. Coloboma candidate gene studies (Zfp703/Nlz1 and Zfp503/Nlz2) Using developmental gene profiling, we previously identified two zinc-finger motif-containing genes, Zfp703/Nlz1 and Zfp503/Nlz2, that are important in regulating optic fissure closure in zebrafish. 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. Zfp503 knockout embryos display coloboma and hypopigmentation of the presumptive RPE (pRPE) at 100% penetrance. Around the time of OF closure, the pRPE becomes hyperplastic in a ventral-to-dorsal fashion and expresses VSX2 immunostaining, indication of a more neural-retina-like phenotype. We have characterized viable Zfp503+/- mice, showing congenital optic nerve excavation by OCT and histology. We completed the assessment of developmentally regulated ocular transcription factors, revealing down-regulation of MITF and OTX2 and up-regulation and/or anatomically expanded expression of PAX6, PAX2, VSX2 proteins, and Vax1 and Vax2 transcripts, particularly in the ventral, proximal pRPE, accompanied by an expansion of cell number. Zfp503-/- mice were never observed in live born litters, likely because of hypoplastic lung, and/or rib cage abnormalities. Gene expression profiling by RNA-Seq revealed significant downregulation of melanin pigment-related genes(e.g., Tyr, Dct, Slc45A2, Slc24a5, Gpnmb, Pmel, Gpr132, Mlana, Mlph) and RPE signature genes (<9.5x10-15, hypergeometric testing), consistent with a defect in RPE differentiation. A number of differentially expressed genes overlapped with those we previously identified by molecular profiling of OF closure (p<3.2x10-9, hypergeometric testing), including Strmn4, Insm1, Itgb8, Dcc, Tox3, Atoh7, Ascl1, Dkk3, Myb, Hes5, Fgf15, and Onecut1. Sequencing of a cohort of patients with uveal coloboma did not reveal convincing loss-of-function alleles. These data were published during the reporting period in IOVS. 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 developmental gene profiling--combining data from our experiments as well as those from similar studies published since. We are pursuing validating several candidates that have coloboma using standard markers and rescue experiments. D. Downstream effectors of FAT1 Our collaborators and we have reported that biallelic mutations in FAT1 result in a syndromic form of uveal coloboma. We have since sought to identify potential downstream effectors of FAT1 in ocular development. Recent work has focused on the role of the Hippo signaling pathway and the RERE pathway. We have shown that the zebrafish rerea mutant (babyface) robustly recapitulates optic fissure closure defects resulting from loss of RERE function, as observed in humans with NEDBEH syndrome. Mutants exhibit expansion of proximal retinal optic stalk and reduced expression of some ventral retinal fate genes due to deregulated protein signaling. Using zebrafish and cell-based assays, we determined that NEDBEH-associated human RERE variants function as hypomorphs in their ability to repress shh signaling; some exhibit abnormal nuclear localization. Inhibiting shh signaling by the protein inhibitor HPI-1 rescues coloboma, confirming our observation that coloboma in rerea mutants is indeed due to deregulation of shh signaling. We have also confirmed that