Discovery and Functional Characterization of Novel Non-Coding Variants in a Case of Late-Onset Familial Cerebral Cavernous Malformation [RNA-seq]

ABSTRACT: Background: Cerebral Cavernous Malformation (CCM) is a severe neurovascular disease that shows autosomal dominant inheritance in families. The disease shows wide phenotypic variability, with some patients developing vascular lesions in infancy and others never experiencing symptoms. The majority of familial CCM cases are caused by mutations in KRIT1, CCM2, or PDCD10. However, 5–15% of familial cases and ~40% of sporadic cases with multiple lesions do not exhibit variants in these genes. It remains unknown whether non-coding variants or variants in novel genes contribute to genotype-negative disease or phenotypic variability. Methods: We performed whole genome sequencing (WGS) on a family with two siblings with late-onset CCM and an unaffected third sibling. Next we identified potential causal variants that segregated with disease, were rare (allele frequency <0.001), and were either predicted loss-of-function or in CCM pathway genes. Candidate variants were assessed using in silico splice prediction tools and functional assays to evaluate their effects on protein structure and gene expression. Results: The prioritized causal variants identified with WGS in this family were in the KRIT1 and PIK3CA genes. First we prioritized a single base pair substitution in intron 5 of KRIT1 (c.262+115A>G) predicted to affect splicing. We found the variant leads to inclusion of a novel 129-bp exon containing multiple stop codons in KRIT1 mRNA, likely causing loss of protein function via nonsense-mediated decay or generation of nonfunctional protein. This variant was absent from population databases (ClinVar) and segregated with disease in the family. A second potential disease-relevant variant was in intron 1 of PIK3CA (c.-77+1G>A). Prime editing of this variant in a human aortic endothelial cell line resulted in a 1.5-fold increase in PIK3CA expression. Discussion: This is the first report of WGS identifying causal non-coding single base pair variants in a case of genotype-negative familial CCM. Our findings highlight the diagnostic limitations of exon-focused sequencing and support the application of WGS for variant-negative cases. In this case there is a possible contribution of two non-coding variants that have functional effects on known CCM risk genes. Expanding genetic screening to noncoding regions may improve diagnostic yield, genetic counseling, and CCM risk stratification.

ORGANISM(S): Homo sapiens

PROVIDER: GSE312998 | GEO | 2025/12/12

REPOSITORIES: GEO