A bird's eye view on the use of whole exome sequencing in rare congenital ophthalmic diseases

Abstract

Phenotypic and genotypic heterogeneity in congenital ocular diseases, especially in anterior segment dysgenesis (ASD), have created challenges for proper diagnosis and classification of diseases. Over the last decade, genomic research has indeed boosted our understanding in the molecular basis of ASD and genes associated with both autosomal dominant and recessive patterns of inheritance have been described with a wide range of expressivity. Here we describe the molecular characterization of a cohort of 162 patients displaying isolated or syndromic congenital ocular dysgenesis. Samples were analyzed with diverse techniques, such as direct sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing (WES), over 20 years. Our data reiterate the notion that PAX6 alterations are primarily associated with ASD, mostly aniridia, since the majority of the cohort (66.7%) has a pathogenic or likely pathogenic variant in the PAX6 locus. Unexpectedly, a high fraction of positive samples (20.3%) displayed deletions involving the 11p13 locus, either partially/totally involving PAX6 coding region or abolishing its critical regulatory region, underlying its significance. Most importantly, the use of WES has allowed us to both assess variants in known ASD genes (i.e., CYP1B1, ITPR1, MAB21L1, PXDN, and PITX2) and to identify rarer phenotypes (i.e., MIDAS, oculogastrointestinal-neurodevelopmental syndrome and Jacobsen syndrome). Our data clearly suggest that WES allows expanding the analytical portfolio of ocular dysgenesis, both isolated and syndromic, and that is pivotal for the differential diagnosis of those conditions in which there may be phenotypic overlaps and in general in ASD.

Fig. 1

Summary of patients’ phenotypes. The figure represents the percentage of patients with a diagnosis of aniridia, coloboma, Axenfeld–Rieger syndrome, Peters anomaly, WAGR, and morning glory anomaly, together with the percentage distribution between familial and sporadic cases

Fig. 2

Distribution of PAX6 alterations in our cohort. A Percentage of alterations affecting the 11p13 locus considering both SNVs and SVs. B Percentage of SNVs affecting the 11p13 locus found in our cohort

Fig. 3

Spectrum of novel coding PAX6 mutations assessed in this study. The figure represents novel variants, i.e., alterations not included in publicly available databases, within the PAX6 coding region identified in our cohort. Boxes represent the 13 PAX6 exons and their colors represent respective protein domains. Moreover, canonical PAX6 protein is represented with its functional domains. PAI-RED paired domain; HD homeodomain; PST proline–serine–threonine domain

Fig. 4

Schematic representation of 11p13 deletions assessed in our cohort. 3′ of PAX6. Genes are represented by light gray and gray boxes. Arrows indicate the direction of transcription. Light green eclipses represent enhancers. Dark green eclipses represent SIMO and E180B. Black lines represent pathogenic and likely pathogenic deletions found in our cohort, while the orange line represents the VUS. Vertical dashed lines represent the 244 kb and the 18 kb PAX6 critical regulatory regions identified by [20] and [21], respectively. Some of these deletions are also described in [13]. Genomic coordinates are based on human genome assembly hg19