A deep intronic TCTN2 variant activating a cryptic exon predicted by SpliceRover in a patient with Joubert syndrome.

The recent introduction of genome sequencing in genetic analysis has led to the identification of pathogenic variants located in deep introns. Recently, several new tools have emerged to predict the impact of variants on splicing. Here, we present a Japanese boy of Joubert syndrome with biallelic TCTN2 variants. Exome sequencing identified only a heterozygous maternal nonsense TCTN2 variant (NM_024809.5:c.916C >T, p.(Gln306Ter)). Subsequent genome sequencing identified a deep intronic variant (c.1033+423G>A) inherited from his father. The machine learning algorithms SpliceAI, Squirls, and Pangolin were unable to predict alterations in splicing by the c.1033+423G>A variant. SpliceRover, a tool for splice site prediction using FASTA sequence, was able to detect a cryptic exon which was 85-bp away from the variant and within the inverted Alu sequence while SpliceRover scores for these splice sites showed slight increase (donor) or decrease (acceptor) between the reference and mutant sequences. RNA sequencing and RT-PCR using urinary cells confirmed inclusion of the cryptic exon. The patient showed major symptoms of TCTN2-related disorders such as developmental delay, dysmorphic facial features and polydactyly. He also showed uncommon features such as retinal dystrophy, exotropia, abnormal pattern of respiration, and periventricular heterotopia, confirming these as one of features of TCTN2-related disorders. Our study highlights usefulness of genome sequencing and RNA sequencing using urinary cells for molecular diagnosis of genetic disorders and suggests that database of cryptic splice sites predicted in introns by SpliceRover using the reference sequences can be helpful in extracting candidate variants from large numbers of intronic variants in genome sequencing.