Novel mechanisms of Microphthalmia, Anophthalmia and Coloboma

Microphthalmia, anophthalmia and coloboma (MAC) form a spectrum of potentially blinding ocular anomalies with a combined prevalence of approximately 1.2 per 10,000 live births. Defined as a reduction in the size of the globe (microphthalmia), absence of the globe (anophthalimia) or failure of the optic fissure to close (coloboma), the MAC spectrum is a highly heterogeneous congenital ocular malformation with over 80 currently known genes and 40-60% of cases still unexplained. In this study, we aimed to use whole exome sequencing (WES) to identify novel factors involved in the MAC spectrum and further analyze their pathogenicity and developmental role using zebrafish as a model.;The second aim was to discover novel factors involved in the MAC spectrum and further evaluate them using zebrafish; this work resulted in two additional publications. Using WES data from the 27 probands negative for variants in known genes, we identified two novel factors associated with MAC. Trio analysis identified a de novo frameshift EFTUD2 variant in a patient with microphthalmia, coloboma, retinal dystrophy, microcephaly and craniofacial defects. Dominant mutations in EFTUD2 cause Mandibulofacial Dysostosis with variable mild ocular features, however MAC has not previously been reported. TALENs were utilized to knockout eftud2. Homozygous mutants displayed reduced head size, smaller eyes, curved body, massive cell death and early embryonic lethality. This is the first patient with a variant in EFTUD2 to be associated with severe ocular phenotypes.;WES of two first cousins affected with coloboma, microcornea, cataracts and skeletal dysplasia revealed a shared heterozygous MAB21L2 variant, p.(Arg51Gly). The variant completely co-segregated with disease phenotype in affected family. TALEN mediated genome editing created a frameshift loss-of-function allele, mab21l2Q48Sfs*5, and an inframe two amino acid deletion, mab21l2R51_F52del, encompassing the orthologous Arg51. Homozygous embryos for mab21l2Q48Sfs*5 presented with severe lens and retinal defects while homozygous mab21l2R51_F52del mutants displayed a milder lens phenotype and more severe coloboma. Both lines demonstrated optic vesicle invagination defects, increased cell death/abnormal proliferation and altered expression of several ocular markers. Importantly, mRNA rescue assays demonstrated that wild type MAB21L2 mRNA was able to rescue the mab21l2Q48Sfs*5 phenotype while mRNA encoding for the p.(Arg51Gly) variant was not able to do the same. These findings support the identification of MAB21L2 as a novel factor involved in human coloboma.;The third aim of this study was to further explore the role of the MAB21L/mab21l family during development. Screening of MAB21L1 in patients with MAC revealed a heterozygous variant of uncertain significance, p.(Arg62Cys). A frameshift loss-offunction allele, mab21l1K36Rfs*7, was created using TALENs to further explore MAB21L1/mab21l1's role in development. Homozygous mab21l1K36Rfs*7 embryos lacked an obvious embryonic phenotype, but homozygous adults displayed ocular coloboma. These findings suggest an independent and important function for MAB21L1/mab21l1 in ocular development.;The first aim of this study was to screen MAC patients for pathogenic variants in known ocular genes. We examined the WES data of 32 MAC probands, as well as available family members, for variants in known MAC and other ocular genes. We identified likely pathogenic variants in COL4A1, OTX2, PAX6 and NDP. In the case of COL4A1, we were the first group to demonstrate a clear association of this gene with MAC phenotypes. For PAX6, the novel missense variant we identified is the first variant associated with aphakia. These findings were reported in two separate first-author publications.;To further explore previous reports of genetic interactions between Pax6 and Mab21l2, we created double heterozygous zebrafish with the loss-of-function pax6bsa86 allele and either mab21l2Q48Sfs*5 or mab21l2R51_F52del alleles. The double homozygous embryos (mab21l2/pax6b), as well as embryos with both a homozygous mutation (mab21l2 or pax6b) and a heterozygous mutation (pax6b or mab21l2), demonstrated more severe phenotypes than single homozygous embryos for either gene with novel ocular features present in some cases. This indicates that mab21l2 and pax6b are likely involved in overlapping as well as parallel pathways.;Finally, to gain a better understanding of mab21l2 function, we performed microarray analysis to identify transcriptome wide changes in gene expression due to mab21l2 deficiency. Analysis of differentially expressed transcripts identified enrichment of genes associated with cataracts, microphthalmia and other ocular phenotypes. Genes belonging to tbx and vsx families, as well as several genes involved in the retinoic acid signaling pathway, were the most significantly affected.;In conclusion, this study expanded the genetic and phenotypic spectrum of MAC disorders. Most importantly, this project identified a novel human disease gene, MAB21L2, and obtained an initial insight into molecular mechanisms associated with its deficiency. This project additionally generated several genetic models of mab21l1 and mab21l2 deficiency in zebrafish that can now be utilized for further studies of this debilitating visual disorder and development of new therapeutic strategies.