Loss-Of-Function CCM Gene Variants in Japanese Patients With Cerebral Cavernous Malformations

Hereditary cerebral cavernous malformations (CCMs) are characterized by lesion multiplicity and the formation of de novo lesions, in contrast to sporadic forms. We performed sequencing analyses covering the known autosomal dominant loci CCM1/KRIT1, CCM2/CCM2 (Malcavernin), and CCM3/PDCD10 in 11 patients from eight families with CCMs. All patients had multiple lesions, and de novo lesions were observed in four patients (36.4%). Heterozygous loss-of-function variants were identified in seven families, including four variants of KRIT1, two of CCM2, and one of PDCD10. Among them, c.1730 + 4_1730 + 7delAGTA in KRIT1 and c.609G>A (p.Lys203=) in CCM2 were putative splicing variants located outside the invariant GT/AG dinucleotides at the splice junction because AI-based prediction tools indicated that both variants may disrupt normal splicing. In vitro splicing assays confirmed that the AI-based predictions were correct, and the same aberrant splicing pattern was reproduced by reverse transcription PCR using tissue-derived RNA for the c.609G>A variant in CCM2. In one patient with numerous CCM lesions, whole-genome sequencing was performed, which showed no abnormalities in the three known CCM loci, including deep intronic and structural variants. This may indicate the technological limitations of current bioinformatics analyses or the presence of a novel CCM gene.