Loss of heterozygosity in CCM2 cDNA revealing a structural variant causing multiple cerebral cavernous malformations.

Loss-of-function variants in CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes are identified in the vast majority of familial cases with multiple cerebral cavernous malformations. However, genomic DNA sequencing combined with large rearrangement screening fails to detect a pathogenic variant in 5% of the patients. We report a family with two affected members harboring multiple CCM lesions, one with severe hemorrhages and one asymptomatic. No causative variant was detected using DNA sequencing of the three CCM genes, CNV detection analysis, and RNA sequencing. However, a loss of heterozygosity in CCM2 was observed on cDNA sequences in one of the two affected members, which strongly suggested that this locus might be involved. Whole genome sequencing (WGS) identified a balanced structural variant on chromosome 7 with a breakpoint interrupting the CCM2 gene, preventing normal mRNA synthesis. These data underline the importance of WGS in undiagnosed patients with typical multiple CCM.

The dural angioleiomyoma harbors frequent GJA4 mutation and a distinct DNA methylation profile.

The International Society for the Study of Vascular Anomalies (ISSVA) has defined four vascular lesions in the central nervous system (CNS): arteriovenous malformations, cavernous angiomas (also known as cerebral cavernous malformations), venous malformations, and telangiectasias. From a retrospective central radiological and histopathological review of 202 CNS vascular lesions, we identified three cases of unclassified vascular lesions. Interestingly, they shared the same radiological and histopathological features evoking the cavernous subtype of angioleiomyomas described in the soft tissue. We grouped them together with four additional similar cases from our clinicopathological network and performed combined molecular analyses. In addition, cases were compared with a cohort of 5 soft tissue angioleiomyomas. Three out 6 CNS lesions presented the same p.Gly41Cys GJA4 mutation recently reported in hepatic hemangiomas and cutaneous venous malformations and found in 4/5 soft tissue angioleiomyomas of our cohort with available data. Most DNA methylation profiles were not classifiable using the CNS brain tumor (version 12.5), and sarcoma (version 12.2) classifiers. However, using unsupervised t-SNE analysis and hierarchical clustering analysis, 5 of the 6 lesions grouped together and formed a distinct epigenetic group, separated from the clusters of soft tissue angioleiomyomas, other vascular tumors, inflammatory myofibroblastic tumors and meningiomas. Our extensive literature review identified several cases similar to these lesions, with a wide variety of denominations. Based on radiological and histomolecular findings, we suggest the new terminology of "dural angioleiomyomas" (DALM) to designate these lesions characterized by a distinct DNA methylation pattern and frequent GJA4 mutations.

Movement disorders in patients with alternating hemiplegia: "Soft" and "stiff" at the same time.

OBJECTIVE: To assess nonparoxysmal movement disorders in ATP1A3 mutation-positive patients with alternating hemiplegia of childhood (AHC). METHODS: Twenty-eight patients underwent neurologic examination with particular focus on movement phenomenology by a specialist in movement disorders. Video recordings were reviewed by another movement disorders specialist and data were correlated with patients' characteristics. RESULTS: Ten patients were diagnosed with chorea, 16 with dystonia (nonparoxysmal), 4 with myoclonus, and 2 with ataxia. Nine patients had more than one movement disorder and 8 patients had none. The degree of movement disorder was moderate to severe in 12/28 patients. At inclusion, dystonic patients (n = 16) were older (p = 0.007) than nondystonic patients. Moreover, patients (n = 18) with dystonia or chorea, or both, had earlier disease onset (p = 0.042) and more severe neurologic impairment (p = 0.012), but this did not correlate with genotype. All patients presented with hypotonia, which was characterized as moderate or severe in 16/28. Patients with dystonia or chorea (n = 18) had more pronounced hypotonia (p = 0.011). Bradykinesia (n = 16) was associated with an early age at assessment (p < 0.01). Significant dysarthria was diagnosed in 11/25 cases. A history of acute neurologic deterioration and further regression of motor function, typically after a stressful event, was reported in 7 patients. CONCLUSIONS: Despite the relatively limited number of patients and the cross-sectional nature of the study, this detailed categorization of movement disorders in patients with AHC offers valuable insight into their precise characterization. Further longitudinal studies on this topic are needed.

Novel CCM2 missense variants abrogating the CCM1-CCM2 interaction cause cerebral cavernous malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations mostly located within the central nervous system. Most deleterious variants are loss of function mutations in one of the three CCM genes. These genes code for proteins that form a ternary cytosolic complex with CCM2 as a hub. Very few CCM2 missense variants have been shown to be deleterious by modifying the ternary CCM complex stability. OBJECTIVES: To investigate the causality of novel missense CCM2 variants detected in patients with CCM. METHODS: The three CCM genes were screened in 984 patients referred for CCM molecular screening. Interaction between CCM1 and CCM2 proteins was tested using co-immunoprecipitation experiments for the CCM2 missense variants located in the phosphotyrosine binding (PTB) domain. RESULTS: 11 distinct CCM2 rare missense variants were found. Six variants predicted to be damaging were located in the PTB domain, four of them were novel. When co-transfected with CCM1 in HEK293T cells, a loss of interaction between CCM1 and CCM2 was observed for all six variants. CONCLUSION: We showed, using co-immunoprecipitation experiments, that CCM2 missense variants located in the PTB domain were actually damaging by preventing the normal interaction between CCM1 and CCM2. These data are important for diagnosis and genetic counselling, which are challenging in patients harbouring such variants.

Clinical and Molecular Features of 5 European Multigenerational Families With Moyamoya Angiopathy.

Background and Purpose Moyamoya angiopathy (MMA) is a rare cerebral vasculopathy outside of Asia. In Japanese patients, a vast majority of patients carry the founder p.R4810K variant in the RNF213 gene, and familial cases are around 10%. In European patients, data about familial occurrence are limited. The aim of this study was to characterize the clinical and molecular features of several European families with a parent-to-child transmission of MMA. Methods Out of 126 MMA probands referred, we identified 113 sporadic probands and 13 familial probands. Segregation analysis showed a vertical parent-to-child pattern of inheritance in the families of 5 of these probands. All 5 families were of German or Dutch ancestry. We investigated the clinical features of affected members and used whole-exome sequencing to screen RNF213 and 13 genes involved in Mendelian MMA and to identify genes recurrently mutated in these families. Results Twelve affected MMA patients were identified, including 9 females and 3 males. Age at clinical onset ranged from 11 to 65 years. In 3 of 5 families, associated livedo racemosa was found. We did not detect any deleterious variants in the 13 known MMA genes. RNF213 rare missense variants predicted to be pathogenic were detected in all affected members of 2 of these families, as well as 2 candidate variants of the PALD1 gene. Conclusions Nonsyndromic MMA was identified in 5 European families, including 2 to 3 clinically affected cases segregating with a parent-to-child pattern of inheritance in each family. Molecular screening detected rare deleterious variants within RNF213 and PALD1 in all affected members of 2 of these 5 families, as well as in some clinically unaffected members. Altogether these data raise the difficult and, to date unanswered, question of the medical indication of presymptomatic screening.

Mutations in the netrin-1 gene cause congenital mirror movements.

Netrin-1 is a secreted protein that was first identified 20 years ago as an axon guidance molecule that regulates midline crossing in the CNS. It plays critical roles in various tissues throughout development and is implicated in tumorigenesis and inflammation in adulthood. Despite extensive studies, no inherited human disease has been directly associated with mutations in NTN1, the gene coding for netrin-1. Here, we have identified 3 mutations in exon 7 of NTN1 in 2 unrelated families and 1 sporadic case with isolated congenital mirror movements (CMM), a disorder characterized by involuntary movements of one hand that mirror intentional movements of the opposite hand. Given the diverse roles of netrin-1, the absence of manifestations other than CMM in NTN1 mutation carriers was unexpected. Using multimodal approaches, we discovered that the anatomy of the corticospinal tract (CST) is abnormal in patients with NTN1-mutant CMM. When expressed in HEK293 or stable HeLa cells, the 3 mutated netrin-1 proteins were almost exclusively detected in the intracellular compartment, contrary to WT netrin-1, which is detected in both intracellular and extracellular compartments. Since netrin-1 is a diffusible extracellular cue, the pathophysiology likely involves its loss of function and subsequent disruption of axon guidance, resulting in abnormal decussation of the CST.

Mutations in DCC cause isolated agenesis of the corpus callosum with incomplete penetrance.

Brain malformations involving the corpus callosum are common in children with developmental disabilities. We identified DCC mutations in four families and five sporadic individuals with isolated agenesis of the corpus callosum (ACC) without intellectual disability. DCC mutations result in variable dominant phenotypes with decreased penetrance, including mirror movements and ACC associated with a favorable developmental prognosis. Possible phenotypic modifiers include the type and location of mutation and the sex of the individual.

APP Mutations in Cerebral Amyloid Angiopathy with or without Cortical Calcifications: Report of Three Families and a Literature Review.

BACKGROUND: Specific APP mutations cause cerebral amyloid angiopathy (CAA) with or without Alzheimer's disease (AD). OBJECTIVE: We aimed at reporting APP mutations associated with CAA, describe the clinical, cerebrospinal fluid AD biomarkers, and neuroimaging features, and compare them with the data from the literature. METHODS: We performed a retrospective study in two French genetics laboratories by gathering all clinical and neuroimaging data from patients referred for a genetic diagnosis of CAA with an age of onset before 66 years and fulfilling the other Boston revised criteria. We studied the segregation of mutations in families and performed a comprehensive literature review of all cases reported with the same APP mutation. RESULTS: We screened APP in 61 unrelated French patients. Three mutations, located in the Aß coding region, were detected in five patients from three families: p.Ala692Gly (Flemish), p.Glu693Lys (Italian), and p.Asp694Asn (Iowa). Patients exhibited CAA and progressive cognitive impairment associated with cortical calcifications in the Iowa and Italian mutation carriers, but not the patient carrying the Flemish mutation. CONCLUSIONS: This is the first evidence of cortical calcification in patients with an APP mutation other than the Iowa mutation. We discuss the radiological, cerebrospinal fluid, and clinical phenotype of patients carrying these mutations in the literature.

Cerebral cavernous malformations associated to meningioma: High penetrance in a novel family mutated in the PDCD10 gene.

Multiple familial meningiomas occur in rare genetic syndromes, particularly neurofibromatosis type 2. The association of meningiomas and cerebral cavernous malformations (CCMs) has been reported in few patients in the medical literature. The purpose of our study is to corroborate a preferential association of CCMs and multiple meningiomas in subjects harbouring mutations in the PDCD10 gene (also known as CCM3). Three members of an Italian family affected by seizures underwent conventional brain Magnetic Resonance Imaging (MRI) with gadolinium contrast agent including gradient echo (GRE) imaging. The three CCM-causative genes were sequenced by Sanger method. Literature data reporting patients with coexistence of CCMs and meningiomas were reviewed. MRI demonstrated dural-based meningioma-like lesions associated to multiple parenchymal CCMs in all affected individuals. A disease-causative mutation in the PDCD10 gene (p.Gln112PhefsX13) was identified. Based on neuroradiological and molecular data as well as on literature review, we outline a consistent association between PDCD10 mutations and a syndrome of CCMs with multiple meningiomas. This condition should be considered in the differential diagnosis of multiple/familial meningioma syndromes. In case of multiple/familial meningioma the use of appropriate MRI technique may include GRE and/or susceptibility-weighted imaging (SWI) to rule out CCM. By contrast, proper post-gadolinium scans may aid defining dural lesions in CCM patients and are indicated in PDCD10-mutated individuals.

Congenital mirror movements: mutational analysis of RAD51 and DCC in 26 cases.

OBJECTIVE: We screened a large series of individuals with congenital mirror movements (CMM) for mutations in the 2 identified causative genes, DCC and RAD51. METHODS: We studied 6 familial and 20 simplex CMM cases. Each patient had a standardized neurologic assessment. Analysis of DCC and RAD51 coding regions included Sanger sequencing and a quantitative method allowing detection of micro rearrangements. We then compared the frequency of rare variants predicted to be pathogenic by either the PolyPhen-2 or the SIFT algorithm in our population and in the 4,300 controls of European origin on the Exome Variant Server. RESULTS: We found 3 novel truncating mutations of DCC that segregate with CMM in 4 of the 6 families. Among the 20 simplex cases, we found one exonic deletion of DCC, one DCC mutation leading to a frameshift, 5 missense variants in DCC, and 2 missense variants in RAD51. All 7 missense variants were predicted to be pathogenic by one or both algorithms. Statistical analysis showed that the frequency of variants predicted to be deleterious was significantly different between patients and controls (p < 0.001 for both RAD51 and DCC). CONCLUSION: Mutations and variants in DCC and RAD51 are strongly associated with CMM, but additional genes causing CMM remain to be discovered.

Cerebral cavernous malformations arise independent of the heart of glass receptor.

BACKGROUND AND PURPOSE: The Heart of Glass (HEG) receptor binds KRIT1 and functions with KRIT1, CCM2, and PDCD10 in a common signaling pathway required for heart and vascular development. Mutations in KRIT1, CCM2, and PDCD10 also underlie human cerebral cavernous malformation (CCM) and postnatal loss of these genes in the mouse endothelium results in rapid CCM formation. Here, we test the role of HEG in CCM formation in mice and in humans. METHODS: We constitutively or conditionally deleted Heg and Ccm2 genes in genetically modified mice. Mouse embryos, brain, and retina tissues were analyzed to assess CCM lesion formation. RESULTS: In postnatal mice, CCMs form with Ccm2-/- but not with Heg-/- or Heg-/-;Ccm2+/- endothelial cells. Consistent with these findings, human patients with CCM who lack exonic mutations in KRIT1, CCM2, or PDCD10 do not have mutations in HEG. CONCLUSIONS: These findings suggest that the HEG-CCM signaling functions during cardiovascular development and growth, whereas CCMs arise because of loss of HEG-independent CCM signaling in the endothelium of the central nervous system after birth.

CCM molecular screening in a diagnosis context: novel unclassified variants leading to abnormal splicing and importance of large deletions.

Loss of function mutations in CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 gene are identified in about 95 % of familial cases of cerebral cavernous malformations and 2/3 of sporadic cases with multiple lesions. In this study, 279 consecutive index patients referred for either genetic counseling or for diagnosis of cerebral hemorrhage of unknown etiology were analyzed for the three cerebral cavernous malformations (CCM) genes by direct sequencing and quantitative studies, to characterize in more detail the mutation spectrum associated with cerebral cavernous malformations and to optimize CCM gene screening. Analysis of the cDNA was performed when possible to detect the consequences of the genomic variations. A pathogenic mutation was identified in 122 patients. CCM1 was mutated in 80 patients (65 %), CCM2 in 23 (19 %), and CCM3 in 19 (16 %). One hundred patients harbored a loss of function point mutation (82 %) and 22 had a large deletion (18 %). Novel unclassified variants were detected in the patients among whom six led to a splicing defect. The causality of three missense variants that did not modify the splicing could not be established. These findings expand the CCM mutation spectrum and highlight the importance of screening the three CCM genes with both direct sequencing and a quantitative method. In addition, six new unclassified variants were shown to be deleterious because they led to a splicing defect. This underlines the necessity of the cDNA analysis when an unknown variant is detected.

Antithrombotic therapy and bleeding risk in a prospective cohort study of patients with cerebral cavernous malformations.

BACKGROUND AND PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Activating NOTCH3 mutation in a patient with small-vessel-disease of the brain.

The most common causative diagnosis of hereditary small-vessel-disease of the brain, CADASIL, is due to highly stereotyped mutations in the NOTCH3 receptor. NOTCH3 has 33 exons but all CADASIL mutations occur within the Epidermal Growth Factor-like Repeats encoded by exons 2-24, lead to an odd number of cysteine residues and are associated with GOM deposits and abnormal NOTCH3 protein accumulation. The majority of CADASIL mutations appear to retain normal level of signaling activity, while very few mutations show reduced activity. Herein we identified a novel heterozygous missense mutation (c.4544T>C) in exon 25 of NOTCH3 in a patient with cerebral small-vessel-disease but lacking GOM deposits and NOTCH3 accumulation. The mutation should result in a p.L1515P substitution in the evolutionarily highly conserved juxtamembranous region of NOTCH3, which constitutes the heterodimerization domain. The p.L1515P mutant exhibits increased canonical NOTCH3 signaling, although in a ligand-independent fashion. Biochemical analysis suggests that the mutation renders NOTCH3 hyperactive through destabilization of the heterodimer. Therefore, our study suggests that the p.L1515P mutation falls in a novel mechanistic class of NOTCH3 mutations and that NOTCH3 activating mutations should be further considered for molecular analysis of patients with cerebral small-vessel-disease.

Genotype-phenotype correlations in cerebral cavernous malformations patients.

OBJECTIVE: To compare clinical features of CCM1, CCM2, and CCM3 mutation carriers. METHODS: A detailed clinical and molecular analysis of 163 consecutive cerebral cavernous malformation (CCM) families was performed. RESULTS: 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). INTERPRETATION: 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.