Cerebral Cavernous Malformations: An Update on Prevalence, Molecular Genetic Analyses, and Genetic Counselling

Based on the latest gnomAD dataset, the prevalence of symptomatic hereditary cerebral cavernous malformations (CCMs) prone to cause epileptic seizures and stroke-like symptoms was re-evaluated in this review and calculated to be 1:5,400-1:6,200. Furthermore, state-of-the-art molecular genetic analyses of the known CCM loci are described which reach an almost 100% mutation detection rate for familial CCMs if whole genome sequencing is performed for seemingly mutation-negative families. An update on the spectrum of CCM1, CCM2, and CCM3 mutations demonstrates that deep-intronic mutations and submicroscopic copy-number neutral genomic rearrangements are rare. Finally, this review points to current guidelines on genetic counselling, neuroimaging, medical as well as neurosurgical treatment and highlights the formation of active patient organizations in various countries

Inactivation of Cerebral Cavernous Malformation Genes Results in Accumulation of von Willebrand Factor and Redistribution of Weibel-Palade Bodies in Endothelial Cells

Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1−/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1−/− BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1−/− BOECs which was observed in CCM1+/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells

Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations

Autosomal dominant cerebral cavernous malformation (CCM) represents a genetic disorder with a high mutation detection rate given that stringent inclusion criteria are used and copy number variation analyses are part of the diagnostic workflow. Pathogenic variants in either CCM1 (KRIT1), CCM2 or CCM3 (PDCD10) can be identified in 87–98% of CCM families with at least two affected individuals. However, the interpretation of novel sequence variants in the 5′-region of CCM2 remains challenging as there are various alternatively spliced transcripts and different transcription start sites. Comprehensive genetic and clinical data of CCM2 patients with variants in cassette exons that are either skipped or included into alternative CCM2 transcripts in the splicing process can significantly facilitate clinical variant interpretation. We here report novel pathogenic CCM2 variants in exon 3 and the adjacent donor splice site, describe the natural history of CCM disease in mutation carriers and provide further evidence for the classification of the amino acids encoded by the nucleotides of this cassette exon as a critical region within CCM2. Finally, we illustrate the advantage of a combined single nucleotide and copy number variation detection approach in NGS-based CCM1/CCM2/CCM3 gene panel analyses which can significantly reduce diagnostic turnaround time

FAM222B Is Not a Likely Novel Candidate Gene for Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are prevalent slow-flow vascular lesions which harbour the risk to develop intracranial haemorrhages, focal neurological deficits, and epileptic seizures. Autosomal dominantly inherited CCMs were found to be associated with heterozygous inactivating mutations in 3 genes, CCM1(KRIT1), CCM2(MGC4607), and CCM3(PDCD10) in 1999, 2003 and 2005, respectively. Despite the availability of high-throughput sequencing techniques, no further CCM gene has been published since. Here, we report on the identification of an autosomal dominantly inherited frameshift mutation in a gene of thus far unknown function, FAM222B(C17orf63), through exome sequencing of CCM patients mutation-negative for CCM1-3. A yeast 2-hybrid screen revealed interactions of FAM222B with the tubulin cytoskeleton and STAMBP which is known to be associated with microcephaly-capillary malformation syndrome. However, a phenotype similar to existing models was not found, neither in fam222bb/fam222ba double mutant zebrafish generated by transcription activator-like effector nucleases nor in an in vitro sprouting assay using human umbilical vein endothelial cells transfected with siRNA against FAM222B. These observations led to the assumption that aberrant FAM222B is not involved in the formation of CCMs

Identifizierung und Charakterisierung eines neuen Kandidatengens für kavernöse Gefäßmalformationen des zentralen Nervensystems

Zerebrale kavernöse Malformationen sind Gefäßfehlbildungen des menschlichen zentralen Nervensystems, die mit einer Prävalenz von etwa 1:650 in der Bevölkerung auftreten und zu rezidivierenden Kopfschmerzen, Krampfanfällen und Gehirnblutungen führen können. Diese Läsionen treten sowohl sporadisch als auch als Konsequenz von erblichen Mutationen (familiäre Kavernomatose) mit unvollständiger Penetranz und variabler Expressivität auf. Kausale Mutationen sind für die Gene CCM1 ( KRIT1), CCM2 (Malcavernin) und CCM3 (PDCD10) beschrieben. Die vorliegende Dissertationsarbeit mit dem Titel „Identifizierung und Charakterisierung eines neuen Kandidatengens für kavernöse Gefäßmalformationen des zentralen Nervensystems“ hatte die Suche neuer CCM-assoziierter Gene und deren Beschreibung zur Aufgabe. Als Ausgangspunkt dienten fünf Indexpatienten aus der Kohorte Stahl et al. 2008, bei denen keine ursächliche Mutation in den bekannten CCM-Genen identifiziert werden konnte. Die Exomsequenzierung mittels SOLiD™ 5500XL ergab für die vier isolierten und den familiären Fall mehr als 210.000 Varianten. Nach Filterung und Priorisierung dieser Veränderungen wurden acht Kandidatengene definiert, von denen fünf mittels klassischer Sanger-Sequenzierung validiert werden konnten. Das vielversprechendste Kandidatengen, FAM222B (C17orf63), in dem 2012 keine Loss-of-function Mutationen bekannt waren und das für ein Protein unbekannter Struktur und Funktion kodiert, wurde für die weitere Charakterisierung ausgewählt. Zunächst konnten durch einen Yeast Two-Hybrid Screen Interaktionspartner identifiziert werden, die sich in die bekannten CCM-Signalwege integrieren ließen. Funktionelle Studien mittels Morpholino- und TALEN-Technik im Zebrafischmodell und mit humanen Nabelschnurvenenendothelzellen zeigten jedoch keinen signifikanten Effekt von FAM222B auf die Angiogenese. Auch eine detaillierte Bewertung der Informationen, die erst Ende 2014 in der ExAC-Datenbank veröffentlicht wurden, spricht in Zusammenschau mit den experimentellen Daten eher dagegen, FAM222B als neues Kandidatengen für die Entstehung von CCMs einzustufen. Parallel zu den funktionellen Studien wurde die Kohorte von Stahl et al. 2008 kontinuierlich erweitert. Bei den molekulargenetischen Analysen fanden sich mehr kausale Mutationen im CCM3-Gen als bisher angenommen. Ferner konnte gezeigt werden, dass rund ein Drittel der Probanden vor Erreichen des Erwachsenenalters und ein Fünftel der Mutationsträger bereits vor dem 10. Lebensjahr erkranken.Cerebral cavernous malformations are vascular anomalies of the central nervous system that occur with a prevalence of around 1:650 in the general population and can lead to recurrent headaches, epileptic seizures, and cerebral haemorrhage. These lesions appear sporadically or as a consequence of hereditable mutations (familial form) with incomplete penetrance and variable expressivity. Causal mutations are described in one of three genes, CCM1 (KRIT1), CCM2 (Malcavernin), and CCM3 (PDCD10). The current thesis entitled "Identification and Characterization of a New Candidate Gene for Cavernous Vascular Malformations of the Central Nervous System" dealt with the search for new CCM-associated genes and their description. Using the mutation-negative cohort from Stahl et al. 2008 as starting point, exome sequencing of four isolated and one familial case was performed on a SOLiD 5500XL platform. Thereby, more than 210.000 variants could be identified, from which eight were left after filtering and priorization. Five of them could be validated by Sanger sequencing and one promising candidate gene, FAM222B (C17orf63) was selected for further testing. A yeast 2-hybrid screen with the FAM222B protein as bait revealed interactions fitting well into the known CCM-associated signaling pathways. However, functional studies using Morpholino and TALEN techniques in zebrafish as well as siRNA interference in spheroid-based sprouting assays with human umbilical vein endothelial cell cultures showed no significant effect for FAM222B on angiogenesis. A detailed evaluation of the information that was published in the end of 2014 within the ExAC database in synopsis with the experimental data argues against the classification of FAM222B as a new CCM candidate gene. In parallel to performing functional studies, the cohort from Stahl et al. 2008 was steadily extended. Diagnostic genetic testing revealed more causal variants in CCM3 than previously thought. Furthermore, it could be shown that nearly one third of probands become symptomatic before adulthood and one fifth of mutation carriers before the age of 10