Two Novel KRIT1 and CCM2 Mutations in Patients Affected by Cerebral Cavernous Malformations: New Information on CCM2 Penetrance

Abstract

Wide comprehension of genetic features of cerebral cavernous malformations (CCM) represents the starting point to better manage patients and risk rating in relatives. The causative mutations spectrum is constantly growing. KRIT1, CCM2, and PDCD10 are the three loci to date linked to familial CCM development, although germline mutations have also been detected in patients affected by sporadic forms. In this context, the main challenge is to draw up criteria to formulate genotype-phenotype correlations. Clearly, genetic factors determining incomplete penetrance of CCM need to be identified. Here, we report two novel intronic variants probably affecting splicing. Molecular screening of CCM genes was performed on DNA purified by peripheral blood. Coding exons and intron-exon boundaries were sequenced by the Sanger method. The first was detected in a sporadic patient and involves KRIT1. The second affects CCM2 and it is harbored by a woman with familial CCM. Interestingly, molecular analysis extended to both healthy and ill relatives allowed to estimate, for the first time, a penetrance for CCM2 lower than 100%, as to date reported. Moreover, heterogeneity of clinical manifestations among those affected carrying the same genotype further confirms involvement of modifier factors in CCM development.

Keywords: CCM; CCM genotype-phenotype correlation; CCM2 penetrance; pathogenesis; splicing variants.

Figure 1

MRI scan of patients. MRI images obtained by T2- weighted gradient echo sequences. Each panel refers to a single patient. (a) Single lesion detected in the sporadic patient. (b–e) Lesions of affected family members. In reference to Figure 2: (b) II-1; (c) II-3; (d) II-4; (e) III-13.

Figure 2

Familial pedigree of case report 2. The arrow indicates the proband. Healthy members are indicated by empty symbols. Empty circles vertically crossed refer to healthy carrier mutation. Affected ones are represented by black filled. By question mark are indicated consanguineous of which no genotype or phenotype data are available.

Figure 3

Novel IVS15-66A>T mutation detected in KRIT1 gene. The figure shows both wild-type (A) and mutated (B) electropherograms. The heterozygous substitution is indicated by the arrow.

Figure 4

Novel IVS10-1G>A mutation detected in CCM2 gene. The figure shows both wild-type (A) and mutated (B) electropherograms. The heterozygous substitution is indicated by the arrow. The novel heterozygous variant causes splicing alteration that occurs including also the first nucleotide of exon 10, as shown by frameshift observed in electropherogram of the coding sequence (C). The arrow indicates the point of the mutation.

Figure 5

Secondary structure prediction of mutated malcavernin. The image compares native (A) and mutated (B) malcavernin secondary structures. As indicated by the arrows, the main structural modifications affect harmonin-homology domain (HHD) at C-terminus with consequent loss of two helices. Images were obtained by “Structure Prediction” module of RaptorX tool.