Pathogenic cryptic variants detectable through exome data reanalysis significantly increase the diagnostic yield in Joubert syndrome

Abstract

Joubert syndrome (JS) is a genetically heterogeneous neurodevelopmental ciliopathy. Despite exome sequencing (ES), several patients remain undiagnosed. This study aims to increase the diagnostic yield by uncovering cryptic variants through targeted ES reanalysis. We first focused on 26 patients in whom ES only disclosed heterozygous pathogenic coding variants in a JS gene. We reanalyzed raw ES data searching for copy number variants (CNVs) and intronic variants affecting splicing. We validated CNVs through real-time PCR or chromosomal microarray, and splicing variants through RT-PCR or minigenes. Cryptic variants were then searched in additional 44 ES-negative JS individuals. We identified cryptic "second hits" in 14 of 26 children (54%) and biallelic cryptic variants in 3 of 44 (7%), reaching a definite diagnosis in 17 of 70 (overall diagnostic gain 24%). We show that CNVs and intronic splicing variants are a common mutational mechanism in JS; more importantly, we demonstrate that a significant proportion of such variants can be disclosed simply through a focused reanalysis of available ES data, with a significantly increase of the diagnostic yield especially among patients previously found to carry heterozygous coding variants in the KIAA0586, CC2D2A and CPLANE1 genes.

Fig. 1. Comparison of frequencies of LOF alleles in five JS major genes showing a strong enrichment in the JS cohort (blue) compared to gnomAD population database (orange), NIG-ExIT database (gray) and non-JS internal ES database (yellow).

The y-axis shows the frequencies expressed on a logarithmic scale.

Fig. 2. For each experiment, schematic splicing alterations (left), agarose gel electrophoresis on cDNA (middle) and electropherograms (right) are reported.

A Direct RNA assay, the variant causes skipping of exon 13; (B) Direct RNA assay, a 63 bp pseudo-exon is inserted between exons 15 and 16; (C) Minigene, the variant disrupts the acceptor splicing site of exon 24, causing the loss of 15 bp; (D) Minigene, the variant introduces a new acceptor splicing site upstream exon 17, causing a 16 bp intron retention; (E, F) Minigenes, the variants cause the skipping of exons 34 and 44, respectively; (G) Minigene, a 57 bp pseudo-exon is inserted between exons 9 and 10. CTR control, PT patient, WT wild-type, MUT mutated, b1, b2: pSPL3 synthetic exons; black pins: wild-type bands; red pins: mutated bands; white asterisks: unexpected bands probably due to an internal splice site present in both the wild-type and mutated constructs in minigenes experiments.