Ocular coloboma results from abnormal embryonic development and is often associated with additional ocular and systemic features. Coloboma is a highly heterogeneous disorder with many cases remaining unexplained. Whole exome sequencing from two cousins affected with dominant coloboma with microcornea, cataracts, and skeletal dysplasia identified a novel heterozygous allele in MAB21L2, c.151 C>G, p.(Arg51Gly); the mutation was present in all five family members with the disease and appeared de novo in the first affected generation of the three-generational pedigree. MAB21L2 encodes a protein similar to C. elegans mab-21 cell fate-determining factor; the molecular function of MAB21L2 is largely unknown. To further evaluate the role of MAB21L2, zebrafish mutants carrying a p.(Gln48Serfs*5) frameshift truncation (mab21l2Q48Sfs*5) and a p.(Arg51_Phe52del) in-frame deletion (mab21l2R51_F52del) were developed with TALEN technology. Homozygous zebrafish embryos from both lines developed variable lens and coloboma phenotypes: mab21l2Q48Sfs*5 embryos demonstrated severe lens and retinal defects with complete lethality while mab21l2R51_F52del mutants displayed a milder lens phenotype and severe coloboma with a small number of fish surviving to adulthood. Protein studies showed decreased stability for the human p.(Arg51Gly) and zebrafish p.(Arg51_Phe52del) mutant proteins and predicted a complete loss-of-function for the zebrafish p.(Gln48Serfs*5) frameshift truncation. Additionally, in contrast to wild-type human MAB21L2 transcript, mutant p.(Arg51Gly) mRNA failed to efficiently rescue the ocular phenotype when injected into mab21l2Q48Sfs*5 embryos, suggesting this allele is functionally deficient. Histology, immunohistochemistry, and in situ hybridization experiments identified retinal invagination defects, an increase in cell death, abnormal proliferation patterns, and altered expression of several ocular markers in the mab21l2 mutants. These findings support the identification of MAB21L2 as a novel factor involved in human coloboma and highlight the power of genome editing manipulation in model organisms for analysis of the effects of whole exome variation in humans.

The PITX2 (paired-like homeodomain 2) gene encodes a bicoid-like homeodomain transcription factor linked with several human disorders. The main associated congenital phenotype is Axenfeld-Rieger syndrome, type 1, an autosomal dominant condition characterized by variable defects in the anterior segment of the eye, an increased risk of glaucoma, craniofacial dysmorphism and dental and umbilical anomalies; in addition to this, one report implicated PITX2 in ring dermoid of the cornea and a few others described cardiac phenotypes. We report three novel PITX2 mutations—c.271C > T, p.(Arg91Trp); c.259T > C, p.(Phe87Leu); and c.356delA, p.(Gln119Argfs*36)—identified in independent families with typical Axenfeld-Rieger syndrome characteristics and some unusual features such as corneal guttata, Wolf-Parkinson-White syndrome, and hyperextensibility. To gain further insight into the diverse roles of PITX2/pitx2 in vertebrate development, we generated various genetic lesions in the pitx2 gene via TALEN-mediated genome editing. Affected homozygous zebrafish demonstrated congenital defects consistent with the range of PITX2-associated human phenotypes: abnormal development of the cornea, iris and iridocorneal angle; corneal dermoids; and craniofacial dysmorphism. In addition, via comparison of pitx2M64* and wild-type embryonic ocular transcriptomes we defined molecular changes associated with pitx2 deficiency, thereby implicating processes potentially underlying disease pathology. This analysis identified numerous affected factors including several members of the Wnt pathway and collagen types I and V gene families. These data further support the link between PITX2 and the WNT pathway and suggest a new role in regulation of collagen gene expression during development.

Pediatric cataracts are observed in 1–15 per 10,000 births with 10–25% of cases attributed to genetic causes; autosomal dominant inheritance is the most commonly observed pattern. Since the specific cataract phenotype is not sufficient to predict which gene is mutated, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 23 pedigrees affected with familial dominant cataract. Review of WES data for 36 known cataract genes identified causative mutations in nine pedigrees (39%) in CRYAA, CRYBB1, CRYBB3, CRYGC (2), CRYGD, GJA8 (2), and MIP and an additional likely causative mutation in EYA1; the CRYBB3 mutation represents the first dominant allele in this gene and demonstrates incomplete penetrance. Examination of crystallin genes not yet linked to human disease identified a novel cataract gene, CRYBA2, a member of the βγ-crystallin superfamily. The p.(Val50Met) mutation in CRYBA2 cosegregated with disease phenotype in a four-generation pedigree with autosomal dominant congenital cataracts with incomplete penetrance. Expression studies detected cryba2 transcripts during early lens development in zebrafish, supporting its role in congenital disease. Our data highlight the extreme genetic heterogeneity of dominant cataract as the eleven causative/likely causative mutations affected nine different genes and the majority of mutant alleles were novel. Furthermore, these data suggest that less than half of dominant cataract can be explained by mutations in currently known genes.