EP18.48: A novel splicing variant of CC2D2A gene causes Joubert syndrome

Joubert syndrome is distinguished by hypotonia, ataxia, delayed development, abnormal ocular movements, and deregulated respiratory function. Until now, mutations in 34 genes have been identified as causative agents for Joubert syndrome. Notably, the CC2D2A gene stands out as a frequent pathogenic contributor to this syndrome, transmitted in an autosomal recessive pattern. Herein, we present a case study of a 4-year-old girl exhibiting developmental delays, mild mental retardation, strabismus, and cerebellar vermis hypoplasia with the molar tooth sign on brain MRI, leading to a clinical diagnosis of Joubert syndrome. Trio whole-exome sequencing (Trio-WES) revealed the presence of biallelic CC2D2A variants in the child c.439-5T>G and c.4732 G>A. Initially, these variants were classified as variants of uncertain significance (VUS) according to ACMG guidelines. However, further analysis using splicing prediction software suggested that the c.439-5T>G variant might potentially affect mRNA splicing. To delve deeper into the pathogenic potential of these variants, reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to investigate whether the c.439-5T>G variant indeed interfered with pre-mRNA splicing. Additionally, a minigene vector was constructed to facilitate in vitro expression studies. These experiments confirmed that the c.439-5T>G variant indeed caused abnormal mRNA splicing, leading to mRNA degradation via the nonsense-mediated decay (NMD) pathway. Collectively, bioinformatics predictions and functional assays (RT-PCR, minigene expression) provided compelling evidence for the pathogenicity of the c.439-5T>G variant. Based on these findings, this variant can now be classified as pathogenic according to ACMG guidelines. Furthermore, given that the c.4732 G>A mutation is in trans with the pathogenic mutation site, this adds further weight to its potential pathogenicity, elevating it to a possible pathogenic variant.