Cerebral Cavernous Malformations Patients Research Articles

Endoport-assisted microsurgical resection of cerebral cavernous malformations

The aim of this case series is to describe the surgical technique and postoperative outcomes for endoport-assisted microsurgical resection (EAMR) of cerebral cavernous malformations (CCM). Significant manipulation of subcortical white matter tracts may be necessary for the successful resection of CCM located in deep brain regions. Minimally invasive neurosurgical devices such as endoport systems can decrease disruption of the cortex and white matter tracts overlying deep-seated CCM through small cranial and dural openings. The role of endoport technology in modern CCM surgery is incompletely understood. Three patients with symptomatic CCM underwent EAMR at our institution using the BrainPath endoport system (NICO Corporation, Indianapolis, IN, USA). Complete resection was achieved in two patients. One patient with a large 4.5cm callosal CCM was left with a small residual lesion. There were no postoperative complications and all patients were functionally independent (modified Rankin Scale score 2 or less) at follow-up. Based on our initial experience with EAMR for CCM we believe the endoport can be an effective alternative to traditional retractor systems. Due to the nature of the small craniotomy and durotomy performed for endoport placement EAMR has the potential to improve surgical outcomes by reducing postoperative pain, analgesic requirements and hospital stays. Therefore, EAMR may be considered for appropriately selected CCM patients, although additional experience is necessary to improve our understanding of its role in CCM management.

Abstract W P419: Correlation of Quantitative Susceptibility Mapping in Cerebral Cavernous Malformations with Clinical Features

Quantitative susceptibility mapping (QSM) is a novel noninvasive magnetic resonance imaging (MRI) technique that was shown to accurately quantify the iron content in cerebral cavernous malformation (CCM) lesions. In this study, we aimed to correlate the baseline characteristics of CCM patients and their clinical activities with QSM measurements. Methods: a total of 65 patients (mean age: 39, age range: 3 - 76, 16 male, 49 female, 27 sporadic, 38 familial) were included. Imaging was performed on 3T systems. Lesions from all sporadic cases were included in the final analysis. In familial cases, only lesions identified on T2 weighted images and have maximum cross-sectional diameter greater than 5mm were included. A total of 241 lesions were analyzed. Results: Pearson’s correlation revealed that patient age at scan was positively correlated with the mean lesional susceptibility per patient (p<0.05, Figure 1a). Mean susceptibility for lesions with prior bleeding history was significantly higher than lesions without (p<0.05, Figure 1b). Longitudinal changes in lesional susceptibility were small in a short term (<14 months) in patients who were clinically stable (Figure 1c). In addition, a mixture of susceptibility levels was observed in different lesional blood byproduct depositions, as determined by contrast on the conventional MRIs (Figure 1d). Conclusions: Our prelimiary data suggested that: 1) lesions with longer duration harbor more iron; 2) lesions with prior bleeds harbor more iron deposition; 3) there are little changes in lesional iron deposition in patients who are clinically stable in a short time frame. In sum, QSM offered additional insights into the natural progression of the CCM disease in terms of the accumulation of lesional iron deposition.

Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis

Cerebral cavernous malformations (CCMs) are vascular lesions affecting the central nervous system. CCM occurs either sporadically or in an inherited, autosomal dominant manner. Constitutional (germline) mutations in any of three genes, KRIT1, CCM2 and PDCD10, can cause the inherited form. Analysis of CCM lesions from inherited cases revealed biallelic somatic mutations, indicating that CCM follows a Knudsonian two-hit mutation mechanism. It is still unknown, however, if the sporadic cases of CCM also follow this genetic mechanism. We extracted DNA from 11 surgically excised lesions from sporadic CCM patients, and sequenced the three CCM genes in each specimen using a next-generation sequencing approach. Four sporadic CCM lesion samples (36%) were found to contain novel somatic mutations. Three of the lesions contained a single somatic mutation, and one lesion contained two biallelic somatic mutations. Herein, we also describe evidence of somatic mosaicism in a patient presenting with over 130 CCM lesions localized to one hemisphere of the brain. Finally, in a lesion regrowth sample, we found that the regrown CCM lesion contained the same somatic mutation as the original lesion. Together, these data bolster the idea that all forms of CCM have a genetic underpinning of the two-hit mutation mechanism in the known CCM genes. Recent studies have found aberrant Rho kinase activation in inherited CCM pathogenesis, and we present evidence that this pathway is activated in sporadic CCM patients. These results suggest that all CCM patients, including those with the more common sporadic form, are potentially amenable to the same therapy.

Mutation Prevalence of Cerebral Cavernous Malformation Genes in Spanish Patients

ObjectiveTo study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients.MethodsWe analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing.ResultsA total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported.ConclusionsAnalysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

Deep intronic KRIT1 mutation in a family with clinically silent multiple cerebral cavernous malformations

Loss-of-function mutations in CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10 genes are identified in the vast majority of familial cases with multiple cerebral cavernous malformations (CCMs). However, genomic DNA sequencing combined to large rearrangement screening fails to detect a mutation in 5% of those cases. We report a family in which CCM lesions were discovered fortuitously because of the investigation of a developmental delay in a boy. Three members of the family on three generations had typical multiple CCM lesions and no clinical signs related to CCM. No mutation was detected using genomic DNA sequencing and quantitative multiplex PCR of short fluorescent fragments (QMPSF). cDNA sequencing showed a 99-nucleotide insertion between exons 5 and 6 of CCM1, resulting from a mutation located deep into intron 5 (c.262+132_262+133del) that activates a cryptic splice site. This pseudoexon leads to a premature stop codon. These data highly suggest that deep intronic mutations explain part of the incomplete mutation detection rate in CCM patients and underline the importance of analyzing the cDNA to provide comprehensive CCM diagnostic tests. This kind of mutation may be responsible for apparent sporadic presentations due to a reduced penetrance.

Sporadic Cerebral Cavernous Malformations: Report of Further Mutations of CCM Genes in 40 Italian Patients

Cerebral cavernous malformations (CCMs) are vascular lesions characterized by abnormally enlarged capillary cavities, affecting the central nervous system. CCMs can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1 (K-Rev interaction trapped 1 (KRIT1)), CCM2 (MGC4607), and CCM3 (PDCD10). CCMs occur as a single or multiple malformations that can lead to seizures, focal neurological deficits, hemorrhagic stroke, and headache. However, patients are frequently asymptomatic. In our previous mutation screening, performed in a cohort of 95 Italian patients, both sporadic and familial, we have identified several mutations in CCM genes, three of which in three distinct sporadic patients. In this study, representing further molecular screening of the three CCM genes, in a south Italian cohort of CCM patients enrolled by us in the last three years, we report the identification of other four new mutations in 40 sporadic patients with either single or multiple CCM.

Antithrombotic Therapy and Bleeding Risk in a Prospective Cohort Study of Patients With Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are one of the most frequently diagnosed vascular malformations of the brain and constitute a potential source of intracranial hemorrhage. In CCM patients suffering ischemic stroke or heart disease, the use of anticoagulants or antiplatelet therapy is generally avoided by fear of hemorrhagic complications, but no systematic studies exist to support this hypothesis. We prospectively followed-up consecutive patients with a diagnosis of one or more CCMs in a prospective database since 2008. Retrospective data collection was used for patients with a diagnostic event or imaging studies done before first assessment. Symptomatic hemorrhage and other focal neurological events during prospective follow-up were defined according to the current guidelines of the Angioma Alliance Scientific Advisory board. A total of 87 patients were prospectively enrolled in our cohort [50 women (57%), mean age 44.8 years (SD±17.6), mean follow-up 3.9 years], harboring a total of 738 CCMs. Fifty-five patients (63%) had a single CCM, and 32 patients (37%) had multiple CCMs. Longitudinal follow-up included 16 (18%) patients receiving long-term antithrombotic therapy by antiplatelet treatment (n=11) or oral anticoagulants (n=5). During 5536 lesion-years of observation, none of the patients under antithrombotic therapy experienced CCM hemorrhage on follow-up. Our observational data suggest that long-term antithrombotic treatment by antiplatelet drugs or warfarin does not increase the frequency of CCM-related hemorrhage. Patients harboring single or multiple CCMs suffering ischemic stroke or heart disease should not be withheld antithrombotic therapy.

Conditional deletion of Ccm2 causes hemorrhage in the adult brain: a mouse model of human cerebral cavernous malformations

Cerebral cavernous malformations (CCM) are irregularly shaped and enlarged capillaries in the brain that are prone to hemorrhage, resulting in headaches, seizures, strokes and even death in patients. The disease affects up to 0.5% of the population and the inherited form has been linked to mutations in one of three genetic loci, CCM1, CCM2 and CCM3. To understand the pathophysiology underlying the vascular lesions in CCM, it is critical to develop a reproducible mouse genetic model of this disease. Here, we report that limited conditional ablation of Ccm2 in young adult mice induces observable neurological dysfunction and reproducibly results in brain hemorrhages whose appearance is highly reminiscent of the lesions observed in human CCM patients. We first demonstrate that conventional or endothelial-specific deletion of Ccm2 leads to fatal cardiovascular defects during embryogenesis, including insufficient vascular lumen formation as well as defective arteriogenesis and heart malformation. These findings confirm and extend prior studies. We then demonstrate that the inducible deletion of Ccm2 in adult mice recapitulates the CCM-like brain lesions in humans; the lesions display disrupted vascular lumens, enlarged capillary cavities, loss of proper neuro-vascular associations and an inflammatory reaction. The CCM lesions also exhibit damaged neuronal architecture, the likely cause of neurologic defects, such as ataxia and seizure. These mice represent the first CCM2 animal model for CCM and should provide the means to elucidate disease mechanisms and evaluate therapeutic strategies for human CCM.

Mutation Analysis of CCM1, CCM2 and CCM3 Genes in a Cohort of Italian Patients with Cerebral Cavernous Malformation

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K-Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three-gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at-risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified,nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255-4delGTA; c.1682-1683 delTA in CCM1; c.48A > G; c.82-83dupAG in CCM2; and c.395 + 1G > A in CCM3 genes [corrected].The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at-risk relatives.

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions.

Recent insights into cerebral cavernous malformations: the molecular genetics of CCM

Cerebral cavernous malformations (CCM) are vascular lesions which can occur as a sporadic (80% of the cases) or familial autosomal dominant form (20%). Three CCM genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Almost 80% of CCM patients affected with a genetic form of the disease harbor a heterozygous germline mutation in one of these three genes. Recent work has shown that a two-hit mechanism is involved in CCM pathogenesis which is caused by a complete loss of any of the three CCM proteins within endothelial cells lining the cavernous capillary cavities. These data were an important step towards the elucidation of the mechanisms of this condition.

The association between high VEGF levels and multiple probable punctuate cavernous malformations

Cerebral cavernous malformations (CCMs) are congenital abnormalities of the cerebral vessels. The de novo development of new lesions in this disease has been reported. However, the underlying mechanism of progressive CCMs in such patients remains unclear. This report documents two cases of multiple probable CCMs that showed a progressive behaviour. The plasma levels of vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1) were measured using an enzyme-linked immunosorbent assay (ELISA). The concentration of both VEGF and TGF-beta1 in plasma was increased in these patients. A relationship was observed between high concentrations of growth factors and progressive CCMs. Even though a causal linkage between these conditions cannot be confirmed, a continuous high VEGF level in plasma could be a possible clinical indicator for subsequent intracerebral haemorrhages in the CCM patients.

C329X in KRIT1 is a founder mutation among CCM patients in Sardinia

Cerebral cavernous malformations (CCMs) are CNS vascular anomalies associated with seizures, headaches and hemorrhagic strokes and represent 10–20% of cerebral lesions. CCM is present in 0.1–0.5 of the population. This disorder most often occurs sporadically but may also be familial. Familial cases are inherited as a dominant trait with incomplete penetrance and are estimated to account for KRIT1 10–40% of the patients. The identification of the genes involved in such disorders allows to characterize carriers of the mutations without clear symptoms. The first gene involved in CCM1 is KRIT1. In addition to two other genes have been described: MGC4607 (CCM2) and PDCD10 (CCM3). We selected 13 patients belonging to seven Sardinian families on the basis of clinical symptoms and Magnetic Resonance results. In MGC4607 gene an undescribed exon five deletion likely producing a truncated protein was identified in one family. In two patients with clear phenotype and in three asymptomatic relatives a 4 bp deletion in exon 9 of KRIT1 gene, leading to a premature stop codon, was detected. A unique nonsense mutation (C329X) has been found in seven patients and two asymptomatic subjects belonging to four unrelated families. Haplotype analysis revealed a common origin of this mutation. These data suggest a “founder effect” in Sardinia for the C329X mutation, similar to other mutations described in different populations.

Structural and functional differences between KRIT1A and KRIT1B isoforms: A framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein–protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein–protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal β-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure–function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

Study of cerebral cavernous malformation in Spain and Portugal

We aimed to study clinical, radiological and molecular genetic features of patients with cerebral cavernous malformations (CCMs) from the Iberian Peninsula. We screened Krit1(CCM1), MGC4607(CCM2), and PDCD10(CCM3) by systematic SSCP and direct sequencing of coding exons in 48 nuclear families and 30 sporadic cases of CCM from Spain and Portugal. Screening of CCM patients detected nine different mutations in 19 families. We found four new mutations in Krit1. Three of them were caused by either a small insertion or deletion, which lead to frameshift and premature termination codons. We also found a missense L308H mutation located in a highly conserved sequence within the ankyrin domain of Krit1. In CCM2, we found a redundant 14 bp deletion in exon 5 of MGC4607 which predicts a truncated protein at residue 230. We did not find mutations in CCM3. Finding that the 14 bp deletion was present in eleven families from the Iberian Peninsula indicates a high prevalence of this mutation. This redundant CCM2 mutation is worth considering in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

Genotype-phenotype correlations in cerebral cavernous malformations patients.

To compare clinical features of CCM1, CCM2, and CCM3 mutation carriers. A detailed clinical and molecular analysis of 163 consecutive cerebral cavernous malformation (CCM) families was performed. A deleterious mutation was detected in 128 probands. Three hundred thirty-three mutation carriers were identified (238 CCM1, 67 CCM2, and 28 CCM3). Ninety-four percent of the probands with an affected relative had a mutation compared with 57% of the probands with multiple lesions but no affected relative (p < 0.001). The number of affected individuals per family was lower in CCM3 families (p < 0.05). The proportion of patients with onset of symptoms before 15 years of age was higher in the CCM3 group (p < 0.0025). Cerebral hemorrhage was the most common initial presentation in CCM3 patients. The average number of T2-weighted imaging lesions was similar in the three groups, in contrast with a significantly lower number of gradient-echo sequence lesions in CCM2 patients (p < 0.05). The number of gradient-echo sequence lesions increased more rapidly with age in CCM1 than in CCM2 patients (p < 0.05). Despite similarities among the three groups, there is a significantly lower number of affected individuals in CCM3 pedigrees, CCM3 mutations may confer a higher risk for cerebral hemorrhage, particularly during childhood, and the increment of gradient-echo sequence lesions with age differs between CCM1 and CCM2 patients.

De novo lesions in familial form of cerebral cavernous malformations: clinical and MR features in 29 non-Hispanic families

BACKGROUND To evaluate clinical and MR features of de novo lesions (DNL) in the familial form of cerebral cavernous malformation (CCM) in 40 patients belonging to 29 unrelated non-Hispanic families. METHODS Forty patients followed up by serial cerebral MR examinations were included in this retrospective study. First and last available MR examinations were retrospectively analyzed and compared for each patient to diagnose DNL. Gradient-echo (GRE) sequences were performed in only 11 of the 40 patients and were not considered for this study. Incidence of DNL was evaluated in terms of lesions/patient-year. All DNL were characterized by their clinical and MR features (location, size, type). Type of CCM was determined according to the classification of Zabramski (1994). Patient groups with and without DNL were compared for sex, age, number of pre-existing CCMs, and follow-up. RESULTS Twenty-three DNL were recorded in 11 patients (27.5%) and the incidence was 0.2 lesions/patient-year (mean follow-up = 3.2 years). All but one DNL were asymptomatic. Twenty DNL were supratentorial and three were infratentorial. Mean diameter was 8 mm (2–35 mm). Six DNL were classified as type 1 (subacute hemorrhage), six as type 2 (hemorrhages and thromboses of varying ages) and 11 as type 3 (chronic hemorrhage with hemosiderin staining). No statistical difference between groups was found in terms of sex, age, or number of pre-existing CCMs. On the other hand, duration of follow-up was significantly longer in the group with DNL. CONCLUSION The occurrence of DNL seems to be a hallmark of the familial form of CCM in non-Hispanic families as well as in Hispanic families. Such DNL are usually asymptomatic and are mainly classified as type 3 (chronic hemorrhage with hemosiderin staining). Within the limits of the retrospective study design and potential selection bias introduced by the varying indications for MR scanning, it does seem that DNL may occur at any time in the lifespan of CCM patients, and occurrence does not seem to depend on age, sex, or the total number of pre-existing lesions.