Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network

Abstract

Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted highthroughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2.

Aggregation of population‐based genetic variation over protein domain homologues and its potential use in genetic diagnostics

“…This specific variant in NOTCH4 (p.Cys815Gly, rs150079294) has an allele frequency 0.1632% in ExAC. dbSNP suggests that this variant is benign based on a single study (Chassaing et al., ; Sherry et al., ), whereas our results further support the notion that this variant is problematic for this domain because of almost complete absence of common variation across the homologues. Even more interesting are the positions that are not evolutionary conserved (>0.6 relative entropy), but nevertheless depleted of population‐based missense variation.…”

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome

“…For zebrafish studies, sample size was based on prior experiments to evaluate similar phenotypes 25,82 . For mouse studies, given that we were looking for a dichotomous effect, we set up the CRISPR experiment with both wild-type and mutant repair template, which is our standard practice for modeling heterozygous variants.…”

Ectopic expression of Hoxb1 induces cardiac and craniofacial malformations

“…As mesenchyme, they surround the optic vesicle as it grows towards the lateral ectoderm, contributing such periocular and anterior chamber structures as the matrix of the cornea, the melanocytes of the iris and ciliary body, the pericytes of the choroid, hyaloid and retinal blood vessels, and the sclera. In mice, the choroid is also heavily invested by NCC‐derived pericytes, in juxtaposition to the retinal pigmented epithelium (RPE; Chassaing et al, ). Mildly affected Wnt1‐Cre;CAG‐Hoxb1 fetuses consistently have ocular malformations such as coloboma (Figure f), while severely affected animals undergo secondary microphthalmia, showing only rudiments of remaining RPE (Figure b).…”