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Ricci et al. Vessel Plus 2021;5:31 https://dx.doi.org/10.20517/2574-1209.2021.28 Page 9 of 14

The genomic DNA sequencing followed by copy number analysis allows identifying pathogenic variants in
[13]
87%-98% of all familial CCM cases . In a minority of patients with a positive family history of CCM or
with multiple CCM lesions, however, this approach does not allow detecting any mutations. For these cases,
pathogenic variants outside of the standard diagnostic target regions may be considered. In addition, in that
event, cDNA analysis is advisable. It can reveal splicing anomalies produced by deep intronic variants, not
revealed by standard sequencing, or a loss of heterozygosity causing the lack of expression of one of the
CCM alleles. Some CCM deep intronic variants have been identified by using this approach. An example is a
deep intronic KRIT1 gene deletion (c.262+132_262+133delAA), which leads to the insertion of a 99-bp
pseudo-exon causing a premature stop codon in the open reading frame .
[75]

Finally, rare structural anomalies, such as inversions, can be detected using whole genome sequencing
(WGS) and bioinformatics analysis. An example is the recent identification of a 24-kb inversion involving
exon 1 of CCM2 gene . However, this kind of approach is currently performed in a research context and is
[76]
not part of the standard diagnostic process.

A flow-chart summarizing the approach to CCM genetic screening is shown in Figure 7.

GENETIC COUNSELING
Genetic testing for KRIT1, CCM2, and PDCD10 genes can confirm the clinical diagnosis in patients and
guide the genetic counseling. However, to correctly evaluate the genetic risk of CCM, it is necessary to
assess whether the patient has a family history of disease and presents with single or multiple CCM lesions,
in the absence of developmental venous anomaly or history of brain radiation. Thus, a detailed three-
generation pedigree providing information about probable CCM symptoms, such as seizures, recurrent
headaches, cerebral hemorrhages, focal neurological deficits, and a proband’s brain MRI, including gradient
echo (GRE) or susceptibility-weighted imaging (SWI) sequences, should be obtained.

It is necessary to take into account that the family history can be negative because the disease is not
recognized in other family members. This could be due to parents’ death before symptom onset, reduced
penetrance, or phenotypic variability. Thus, in the presence of an apparently negative family history, it is
still advisable to perform the appropriate evaluations in the proband’s parents.

In the case of familial forms of CCM, the genetic screening sensitivity for the three CCM genes in a proband
with an affected relative is more than 90%. When the proband carries a mutation, the sensitivity of
screening in his/her relatives reaches 100% . If a mutation is identified in a proband, genetic testing of at-
[77]
risk family members can be offered. Genetic counseling is fundamental to give patients and relatives all
details necessary to make an informed choice. In particular, asymptomatic individuals should be carefully
informed about the possible psychological implications of a positive test before they take a decision.
Moreover, proband’s family members should be informed that about 40% of the CCM mutation carriers
remain asymptomatic and intrafamilial phenotypic variability may be high. Predictive testing in minors
[54]
[24]
raises ethical issues and should not be carried out .
In the case of sporadic forms of CCM, in individuals with a single lesion, the presence of CCM gene
mutations is exceedingly rare. A study on KRIT1 gene mutations in sporadic cases showed the presence of
mutations in 29% of sporadic cases with multiple lesions, while no mutation was identified in patients with
[5]
single lesions . For this reason, genetic testing is usually not indicated in sporadic forms with a single
lesion.