Revealing hidden genetic diagnoses in the ocular anterior segment disorders

Abstract

Purpose: Ocular anterior segment disorders (ASDs) are clinically and genetically heterogeneous, and genetic diagnosis often remains elusive. In this study, we demonstrate the value of a combined analysis protocol using phenotypic, genomic, and pedigree structure data to achieve a genetic conclusion.

Methods: We utilized a combination of chromosome microarray, exome sequencing, and genome sequencing with structural variant and trio analysis to investigate a cohort of 41 predominantly sporadic cases.

Results: We identified likely causative variants in 54% (22/41) of cases, including 51% (19/37) of sporadic cases and 75% (3/4) of cases initially referred as familial ASD. Two-thirds of sporadic cases were found to have heterozygous variants, which in most cases were de novo. Approximately one-third (7/22) of genetic diagnoses were found in rarely reported or recently identified ASD genes including PXDN, GJA8, COL4A1, ITPR1, CPAMD8, as well as the new phenotypic association of Axenfeld-Rieger anomaly with a homozygous ADAMTS17 variant. The remainder of the variants were in key ASD genes including FOXC1, PITX2, CYP1B1, FOXE3, and PAX6.

Conclusions: We demonstrate the benefit of detailed phenotypic, genomic, variant, and segregation analysis to uncover some of the previously "hidden" heritable answers in several rarely reported and newly identified ocular ASD-related disease genes.

Keywords: exome and genome sequencing; eye; genomic medicine; ocular anterior segment dysgenesis; ophthalmology.

Fig. 1. Yield of study and mode of inheritance.

a Yield of study, by gene, for 22 patients with a genetic diagnosis. The proportion of genetic diagnoses found in the relevant genes is shown in this chart. The group of rarely reported genes includes six genes: COL4A1, PXDN, CPAMD8, ADAMTS17, ITPR1, and GJA8 (two variants). b Mode of inheritance, before and after testing, for the 22 patients with a genetic diagnosis. This figure demonstrates the breakdown of inheritance among the 22 solved cases. On referral, 19/22 were thought to be sporadic, and 3 familial with autosomal dominant (AD) inheritance. After testing, of the sporadic cases, 11 were found to be due to de novo autosomal dominant variants, 6 were due to autosomal recessive (AR) inheritance, and 2 were familial autosomal dominant cases with subtle clinical features in parents. Of the 3 familial cases, 2 were confirmed as familial autosomal dominant and one was found to be due to autosomal recessive inheritance in an inbred population group leading to pseudodominance. Hence overall after testing, there were 11 de novo autosomal dominant cases, 4 familial autosomal dominant cases, and 7 autosomal recessive cases.

Fig. 2. Representative clinical images of this cohort demonstrate broad range of severity across multiple genotypes.

a Patient 26 with COL4A1 heterozygous variant. Photograph of right eye with Peters anomaly and failed corneal graft. b Previously undiagnosed affected mother of patient 26, with right eye showing mild features of anterior segment disorder (ASD) including Rieger anomaly, with strands of iris adhesions to the overlying cornea and mild iris hypoplasia (white asterisk). c Patient 21 with PXDN homozygous variant. Right eye (i) has previously undergone penetrating keratoplasty at age 4 years. Now failed corneal graft with central corneal opacity (black asterisk). Scleromalacia surrounding this with choroidal tissue visible through the residual sclera. Left eye (ii) shows sclerocornea with a residual small oval opaque central corneal tissue (black asterisk) with injected and dilated superficial corneoscleral vessels. No clear view of iris structures through cornea. d Patient 9 with homozygous variant in ADAMTS17. Left eye slit lamp view of anterior segment demonstrating features of Axenfeld–Rieger anomaly: corectopia, polycoria (arrows), peripheral iridocorneal adhesions, anterior iris stroma hypoplasia (white asterisk). ( e) Patient 30 with CYP1B1 variants: left eye shows generalized corneal opacification. In addition, surgical scarring is visible superiorly from previous glaucoma filtration surgery. f Patient 28 with CPAMD8 variants: right (i) and left (ii) eyes of proband showing corectopia of pupils (white arrows) and iris hypoplasia with the iris sphincter muscle visible (white asterisk). g Patient 36 with PITX2 heterozygous variant: image shows the clinical features of primary congenital corneal opacification, commonly termed sclerocornea. This case has central area of clear cornea, which on corneal topography has low (flat) keratometry in the range meeting definition of cornea plana. The peripheral cornea is scleralized (arrow) making identification of the peripheral iris difficult as well. h Patient 14 with PITX2 heterozygous variant: image shows features of Axenfeld–Rieger anomaly with iris hypoplasia (asterisk), corectopia, polycoria, and posterior embryotoxon (black arrow).

Fig. 3. Variants in extracellular matrix-associated genes COL4A1, PXDN, ADAMTS17, and recently reported anterior segment disorder (ASD) genes CPAMD8 and ITPR1.

Variants reported in the rarely reported ASD genes a COL4A1, b PXDN, c ADAMTS17, d CPAMD8, e ITPR1. Variants above the gene were found in this study, and previously reported variants are listed underneath the gene diagrams. Note: in COL4A1 over 50 missense variants, mostly involving glycine residues in the triple helical domain, have been reported in the literature. Several well reported variants are displayed. Also, the ADAMTS17 variant we report is the first associated with an Axenfeld–Rieger anomaly (ARA) phenotype.

Fig. 4. Novel variants in FOXC1 Wing 2 domain, PITX2, and a PAX6 deletion identified on genome sequencing (GS).

(a) FOXC1 gene, with key domains and regions of the major forkhead domain, with wing 2 (W2) region highlighted. b Alignment demonstrates the highly conserved residues of this domain across the FOXC1 paralog FOXC2, and the phylogenetic tree. Previously reported pathogenic variants in the wing 2 domain are labeled, and the Arg173 highlighted in pink. c PITX2 gene and three novel variants found in this study. d PAX6 deletion is also demonstrated, with exon numbering in the diagram.