Clinical and functional characterization of a novel homozygous non-canonical splice mutation (c.1910-15_1910-11delinsTTACA) in CEP290 causing Joubert syndrome

Joubert syndrome (JS) is a rare, predominantly autosomal recessive neurodevelopmental disorder characterized by hypotonia, motor delay, intellectual disability, oculomotor apraxia, and the hallmark "molar tooth sign" on axial view of MRI. JS is genetically heterogeneous, with pathogenic variants identified in more than 40 genes involved in primary cilia function. Among these, CEP290 is one of the most frequently mutated genes.

In this study, we investigated two children-an 11-year-old boy (the proband) and his 5-year-old sister-both presenting with a similar phenotype consistent with JS. The parents, who self-identified as Chechen, reported distant consanguinity. The family also included a healthy 13-year-old daughter. The proband had previously been evaluated by a neurologist and underwent whole-genome sequencing (WGS); however, no causative variants were identified initially. After phenotype reassessment by a clinical geneticist, we performed a reanalysis of the raw WGS data and identified a novel homozygous intronic variant of uncertain significance (VUS), c.1910-15_1910-11delinsTTACA in CEP290 (NM_025114.4). Sanger sequencing confirmed that both the proband and his affected sister were homozygous for this variant, which they inherited from their heterozygous parents. Their healthy sister did not carry the variant. mRNA-sequencing and targeted cDNA sequencing (read depth ~ 100,000x) demonstrated that this intronic variant causes completely aberrant splicing of CEP290 pre-mRNA. Predominantly this variant causes the skipping of exon 20 in the main CEP290 transcript. Alternatively, the variant results in partial inclusion of intron 19 into the mRNA, elongation of exon 20 by 58 nucleotides, and a homozygous substitution chr12:88114573 (ACTGTGTA> TTACAGTA). No canonical mRNA isoform was detected when the variant was homozygous. Both the predicted severe truncation and the likely degradation of aberrant transcripts through nonsense-mediated decay (NMD) would correspond to complete loss of CEP290 function. Following the reclassification of this VUS to likely pathogenic, the family was able to pursue in vitro fertilization (IVF) with preimplantation genetic testing for monogenic disorders (PGT-M).

Our study highlights the critical importance of proper phenotyping prior to referral for WES/WGS as well as of combining NGS with functional mRNA studies to achieve a molecular diagnosis for patients with predicted splice-site mutations in JS-associated genes. It also emphasizes the need for functional reassessment of VUS when genomic data are expected to guide reproductive decision-making within affected families.