The Genetics of Uveal Coloboma

National Eye Institute

Investigators

Linked publications, trials & patents

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. As of February 2022, we had enrolled 245 participants (85 affected) from 79 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 followed by reflex full exome 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. 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 are now using long-read sequencing to better evaluate this genomic architecture. 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 have been examined and do not have coloboma. However, the total number of copies of the NSE-VEGF transgene incorporated are low compared to parent RICO line, making interpretation of a negative result difficult. 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. 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. Since the time of the last BSC, 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)37, 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, 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. Out of 84 genes targeted with CRISPR, 17 displayed coloboma in the F1 progenies with underlying compound heterozygous/homozygous variants, although numbers were small in many cases and will need further confirmation. Of these, we have studied netrin1 (ntn1)encoding a protein associated with axon guidance regulation of cell migration and epithelial plasticitymost extensively, showing that mutation/knockdown consistently produced a coloboma phenotype; the gene coding for a Ntn1 receptor, Dcc, was also identified in ourscreen. Notably, ntn1 was similarly identified in an RNA-seq-based screen in chick OF closure and our data were incorporated into a collaborative manuscript. The CRISPR screen has been hampered by several factors: 1) systemic pathology caused by some induced gene mutations and 2) loss of mutant alleles with outcrossing of mutation-carrying F0 fish to wild-type (WT) fish; leading us to readdress this project using a CRISPRi strategy. Because the numbers of fish with coloboma in the F1 generation were low, we did not pursue transcriptomics or proteomics at present, but feel that such techniques will be useful in our future studies. In light of recent progress by others with live imaging of OF closure, we have temporarily de-emphasized this line of research*. We have engaged Dr. Kristen Kwan, who has developed an elegant live imaging system to look at early eye morphogenesis and OF closure, and we intend to forge collaborations around the investigation of specific mutants.