fig7

Figure 7. Stepwise protocol of CCM gene screening in familial CCM cases (probands) and sporadic CCM cases with multiple lesions. After genomic DNA extraction from white blood cells, sequencing of all exons and intron-exon boundaries of the three CCM genes is performed, using serial single gene testing by Sanger sequencing or multigene panels by Next Generation Sequencing (NGS). If no mutation is detected, the analysis is extended to exon copy number variations (CNV) using quantitative MLPA or QMPSF. If negative, RNA extracted from blood leukocytes is reverse transcribed and sequenced by Sanger sequencing to evaluate the presence of aberrant splicing. This approach is also used in the case of variants of uncertain significance identified in the first step of the analysis. These approaches are routinely performed in diagnostic labs. These steps are part of the standard diagnostic process. If no alteration is detected, whole genome sequencing (WGS) may be performed to assess the presence of rare structural anomalies (usually in a research context). CCM: Cerebral cavernous malformations.