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.     

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.     

颅内海绵状血管瘤 CCM1基因第12外显子及其5′端内含子新突变位点

目的　探讨 CCM1基因突变在中国人颅内海绵状血管瘤 ( intracranial cavernous angiomas,ICCA)发病中所起的作用。方法　收集我院神经外科 2 0 0 2年 6月～ 2 0 0 3年 2月收治并经手术病理证实的2 1例 ICCA患者及 15名正常健康对照者 ,从外周静脉血中提取 DNA,PCR法扩增 CCM1基因第 12外显子及其两侧部分内含子序列 ,应用 DNA直接测序技术对扩增产物进行检测。结果　 5例患者中检测出 3处 CCM1基因突变 ,均为首次发现。其中 ,5例患者中均存在 1172 C→ T的错义突变 ,使编码 KRIT1蛋白391位的氨基酸由丝氨酸变成苯丙氨酸。另有 1例患者存在 116 0 A→ C的错义突变 ,使编码 KRIT1蛋白387位氨基酸的谷氨酰胺变成脯氨酸。另一个突变发生在第 12外显子 5′端内含子区域 ,5例患者中有 4例第 4个碱基 C被 T取代。对照组检测结果无异常。结论　中国 ICCA患者存在 CCM1基因第 12外显子的突变 ,并与 ICCA的发病有关     

Mesenteric cysts,lymphatic leak,and cerebral cavernous malformation in a proband with KRIT1-related disease

Cerebral cavernous malformations (CCMs) of the central nervous system arise sporadically or secondary to genomic variation. Established genetic etiologies include deleterious variants in KRIT1 (CCM1), malcavernin (CCM2), and PDCD10 (CCM3). KRIT1-related disease has not been described in conjunction with lymphatic defects, although lymphatic defects with abnormal endothelial cell junctions have been observed in mice deficient in HEG1-KRIT1 signaling. We report a proband with CCMs, multiple chylous mesenteric cysts, and chylous ascites with leaky lymphatic vasculature. Clinical short-read exome sequencing detected a disease-associated KRIT1 variant (NM_194456.1:c.[1927C>T];[=], p.(Gln643*)). We postulate an expansion of KRIT1-related disease to include lymphatic malformations and lymphatic endothelial dysfunction.     

CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis

Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated thin-walled blood vessels that predispose individuals to seizures and stroke. Familial CCM is caused by mutations in KRIT1 (CCM1) or in malcavernin (CCM2), the murine ortholog of which was concurrently characterized as osmosensing scaffold for MEKK3 (OSM). The roles of the CCM proteins in the pathogenesis of the disorder remain largely unknown. Here, we use co-immunoprecipitation, fluorescence resonance energy transfer and subcellular localization strategies to show that the CCM1 gene product, KRIT1, interacts with the CCM2 gene product, malcavernin/OSM. Analogous to the established interactions of CCM1 and beta1 integrin with ICAP1, the CCM1/CCM2 association is dependent upon the phosphotyrosine binding (PTB) domain of CCM2. A familial CCM2 missense mutation abrogates the CCM1/CCM2 interaction, suggesting that loss of this interaction may be critical in CCM pathogenesis. CCM2 and ICAP1 bound to CCM1 via their respective PTB domains differentially influence the subcellular localization of CCM1. Furthermore, we expand upon the established involvement of CCM2 in the p38 mitogen-activated protein kinase signaling module by demonstrating that CCM1 associates with CCM2 and MEKK3 in a ternary complex. These data indicate that the genetic heterogeneity observed in familial CCM may reflect mutation of different molecular members of a coordinated signaling complex.     

Genetic and cellular basis of cerebral cavernous malformations: implications for clinical management

Bacigaluppi S, Retta SF, Pileggi S, Fontanella M, Goitre L, Tassi L, La Camera A, Citterio A, Patrosso MC, Tredici G, Penco S. Genetic and cellular basis of cerebral cavernous malformations: implications for clinical management. Cerebral cavernous malformations (CCMs) are a diffuse cerebrovascular disease affecting approximately 0.5% of the population. A CCM is characterized by abnormally enlarged and leaky capillaries arranged in mulberry‐like structures with no clear flow pattern. The lesion might predispose to seizures, focal neurological deficits or fatal intracerebral hemorrhage. However, a CCM can also remain neurologically silent. It might either occur sporadically or as an inherited disorder with incomplete penetrance and variable expressivity. Due to advances in imaging techniques, the incidence of CCM diagnoses are increasing, and the patient must be managed on a multidisciplinary basis: genetic counselling, treatment if needed, and follow‐up. Advances have been made using radiological and pathological correlates of CCM lesions adding to the accumulated knowledge of this disease, although management of these patients is very variable among centers. This review is aimed at providing an update in genetic and molecular insights of this condition. Included are implications for genetic counselling, and possible approaches to prevention and treatment that derive from the understanding of pathogenetic mechanisms.     

ZPLD1 gene is disrupted in a patient with balanced translocation that exhibits cerebral cavernous malformations

The past few years have seen rapid advances in our understanding of the genetics and molecular biology of cerebral cavernous malformations (CCM) with the identification of the CCM1, CCM2, and CCM3 genes. Recently, we have recruited a patient with an X/3 balanced translocation that exhibits CCM. By fluorescent in situ hybridization analysis, sequence analysis tools and database mining procedures, we refined the critical region to an interval of 200-kb and identified the interrupted ZPLD1 gene. We detected that the mRNA expression level of ZPLD1 gene is consistently decreased 2.5-fold versus control (P=0.0006) with allelic loss of gene expression suggesting that this protein may be part of the complex signaling pathway implicated in CCM formation.     

Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1).

Cerebral cavernous malformations (CCM) are congenital vascular anomalies of the brain that can cause significant neurological disabilities, including intractable seizures and hemorrhagic stroke. One locus for autosomal dominant CCM ( CCM1 ) maps to chromosome 7q21-q22. Recombination events in linked family members define a critical region of approximately 2 Mb and a shared disease haplotype associated with a presumed founder effect in families of Mexican-American descent points to a potentially smaller region of interest. Using a genomic sequence-based positional cloning strategy, we have identified KRIT1, encoding a protein that interacts with the Krev-1/rap1a tumor suppressor, as the CCM1 gene. Seven different KRIT1 mutations have been identified in 23 distinct CCM1 families. The identical mutation is present in 16 of 21 Mexican-American families analyzed, substantiating a founder effect in this population. Other Mexican-American and non-Hispanic Caucasian CCM1 kindreds harbor other KRIT1 mutations. Identification of a common Mexican-American mutation has potential clinical significance for presymptomatic diagnosis of CCM in this population. In addition, these data point to a key role for the Krev-1/rap1a signaling pathway in angiogenesis and cerebrovascular disease.     

Highly variable penetrance in subjects affected with cavernous cerebral angiomas (CCM) carrying novel CCM1 and CCM2 mutations.

Cavernous vascular malformations may affect brain and out-of-brain tissues. In most cases, cerebral cavernous malformations (CCMs) involve the brain alone, and are rarely associated with skin hemangiomas, spinal cord, retinal, hepatic or vertebral lesions. CCMs can cause seizures, intracranial and spinal haemorrhages, focal neurological deficits, and migraine-like headaches. After collecting CCM families of Italian origin and investigating the genetic basis of the disorder we disclosed two novel molecular variations in the KRIT1 and MGC4607 genes. We found a novel CCM1 gene mutation (Q66X) in a family with apparently asymptomatic old-aged mutation carriers and patients who either had skin angiomas alone or the full association of cerebral, spinal, and skin lesions. In this family we report the highest variability in mutation penetrance so far described, including the presence of CCM in one subject since birth (surgery at 19 months of age), a condition to our knowledge so far unreported. In a CCM2 affected family, we also report a novel causative mutation, (54_55delAC) in exon 2 of the MGC4607 gene, that produces a truncated protein containing only 22 amino acids. These data describe novel CCM mutations associated with a particularly high variability of the penetrance causing, in some cases, reduced expression of clinical symptoms and sporadic cases with apparent negative family history. Hence they emphasize the importance of DNA-based diagnostics and genetic counseling to identify unaffected mutation carriers subjects, even at advanced age.     

A novel CCM2 variant in a family with non‐progressive cognitive complaints and cerebral microbleeds

Lobar cerebral microbleeds are most often sporadic and associated with Alzheimer's disease. The aim of our study was to identify the underlying genetic defect in a family with cognitive complaints and multiple lobar microbleeds and a positive family history for early onset Alzheimer's disease. We performed exome sequencing followed by Sanger sequencing for validation purposes on genomic DNA of three siblings with cognitive complaints, reduced amyloid‐beta‐42 in CSF and multiple cerebral lobar microbleeds. We checked for the occurrence of the variant in a cohort of 363 patients with early onset dementia and/or microbleeds. A novel frameshift variant (c.236_237delAC) generating a premature stop codon in the CCM2 gene shared by all three siblings was identified. Pathogenicity of the variant was supported by the presence of cerebral cavernous malformations in two of the siblings and by the absence of the variant exome variant databases. Two siblings were homozygous for APOE‐?4 ; one heterozygous. The cognitive complaints, reduced amyloid‐beta‐42 in CSF and microbleeds suggest preclinical Alzheimer's disease, but the stability of the cognitive complaints does not. We hypothesize that the phenotype in this family may be due to a combination of the CCM2 variant and the APOE status. © 2016 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc.

     

Molecular diagnostic workflow,clinical interpretation of sequence variants,and data repository procedures in 140 individuals with familial cerebral cavernous malformations

Familial cerebral cavernous malformation (FCCM) is an autosomal dominant vascular disorder caused by heterozygous deleterious variants in KRIT1, CCM2 or PDCD10. In a previous study, we presented the clinical and molecular findings in 140 FCCM individuals. In the present work, we report supporting information on (a) applied diagnostic workflow; (b) clinical significance of molecular findings according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology recommendations; (c) standardization of molecular and clinical data according to the Human Phenotype Ontology; (d) preliminary genotype‐phenotype correlations on a subgroup of patients by considering sex, age at diagnosis, neurological symptoms, and number and anatomical site(s) of vascular anomalies; (e) datasets submitted to the Leiden Open Variation Database. An overview of the changes of our diagnostic approach before and after the transition to next‐generation sequencing is also reported. This work presents the full procedure that we apply for molecular testing, data interpretation and storing in public databases in FCCM.     

KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis

Mutations in KRIT1, a protein initially identified based on a yeast two-hybrid interaction with the RAS-family GTPase RAP1A, are responsible for the development of the inherited vascular disorder cerebral cavernous malformations (CCM1). As the function of the KRIT1 protein and its role in CCM pathogenesis remain unknown, we performed yeast two-hybrid screens to identify additional protein binding partners. A fragment containing the N-terminal 272 amino acid residues of KRIT1, a region lacking similarity to any known protein upon database searches, was used as bait. From parallel screens of human fetal brain and HeLa cDNA libraries, we obtained multiple independent isolates of human integrin cytoplasmic domain-associated protein-1 (ICAP-1) as interacting clones. The interaction of KRIT1 and ICAP-1 was confirmed by GST-KRIT1 trapping of endogenous ICAP-1 from 293T cells. The alpha isoform of ICAP-1 is a 200 amino acid serine/threonine-rich phosphoprotein which binds the cytoplasmic tail of beta1 integrins. We show that mutagenesis of the N-terminal KRIT1 NPXY amino acid sequence, a motif critical for ICAP-1 binding to beta1 integrin molecules, completely abrogates the KRIT1/ICAP-1 interaction. The interaction between ICAP-1 and KRIT1, and the presence of a FERM domain in the latter, suggest that KRIT1 might be involved in the bidirectional signaling between integrin molecules and the cytoskeleton. Furthermore, these data suggest that KRIT1 might affect cell adhesion processes via integrin signaling in CCM1 pathogenesis.     

CCM2 gene polymorphisms in Italian sporadic patients with cerebral cavernous malformation: a case-control study

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities that 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 (Krit1), CCM2 (MGC4607) and CCM3 (PDCD10). Among our group of CCM Italian patients, we selected a cohort of sporadic cases negative for mutations in CCM genes. In this cohort, five variants in CCM2 gene were detected, which proved to be the known polymorphisms in intronic regions (IVS2-36A>G and IVS8 +119 C>T) and in coding sequence (c.157 G>A in exon 2, c.358 G>A in exon 4 and c.915 G>A in exon 8). Therefore, we undertook a case-control study to investigate the possible association of these polymorphisms with sporadic CCMs. The five polymorphisms were identified in 91 CCM sporadic patients and in 100 healthy controls by direct sequencing methods using lymphocyte DNA. Polymorphisms IVS2-36A>G and c.915 G>A showed statistically significant differences in frequencies between patients and controls [(χ2, 6.583; P<0.037); (χ2, 14.205; P<0.001)]. The prevalence of the wild-type genotype was significantly lower in the CCM group than in the control sample. Patients with the A/G and G/G genotypes (IVS2-36A>G) had a significant increase for CCM risk (OR, 3.08; 95% CI, 1.5-5.9 and OR, 4.3; 95% CI, 1.4-22.6) and the same was observed for the polymorphism c.915 G> A (genotype G/A OR, 6.1; 95% CI, 3.0-12.6 and genotype A/A OR, 2.79). In addition, the polymorphisms c.358 G>A in exon 4 (χ2, 15.977; P<0.04) and c.915 G>A in exon 8 (χ2, 18.109; P<0.02) were significantly associated with different types of symptoms. Haplotype analysis, performed only on polymorphisms c.358 G>A (p.Val120Ile), c.915 G>A (p.Thr305 Thr) and IVS2-36A>G, shows that haplotype GAG (+--) significantly increased among CCM sporadic patients compared to the control group. Significant differences between patients and controls were observed only for IVS2-36A>G and c.915 G>A polymorphisms indicating their possible association with sporadic CCMs and an increased risk of CCM. On the other hand, polymorphisms c.358 G>A and c.915 G>A were associated with a more benign course of the disease. These data were confirmed by the haplotype GAG (+--) frequencies.     

Phenotype of COL3A1/COL5A2 deletion patients

IntroductionThe diagnosis of Ehlers-Danlos syndrome is usually based on well-defined diagnostic criteria and the result of DNA investigation. Classical (cEDS) and vascular type (vEDS) are the most prevalent subtypes and are caused by heterozygous pathogenic variants in COL5A1, COL5A2, COL1A1 or, respectively, in COL3A1. We describe 3 cases with contiguous deletions resulting in haploinsufficiency of both genes with relative mild features of connective tissue disease.Patients and methodsInformation on medical history, physical information, genetic results (CNV-analysis) and imaging were obtained from the medical file.ResultsThe first patient was a 31 yr old female, diagnosed during pregnancy after the NIPT result showed an interstitial deletion of 2.3 Mb on chromosome 2q32.2, confirmed by XON array. She had normal aortic diameters. She had no signs of cEDS or vEDS except for a relatively thin skin with increased visibility of the veins. Her father died suddenly of a type A/B dissection at the age of 62 years. The second patient was diagnosed at the age of 10 years after she was referred because of her intellectual disability, autism and constipation. She was known with a thin and vulnerable skin and had a bleeding after tooth extraction. Array showed a 14,5 Mb deletion of 2q31.3q32.3 (de novo). Imaging (latest age 17 years) did not show any abnormalities. The third patient, aged 28 years, was diagnosed during pregnancy with an interstitial deletion of circa 6 Mb on chromosome 2q31.1q32.2 3, previously shown in the fetus with bilateral club feet and hydronephrosis. She had no vEDS facial features and the skin was relatively thin. She has thoracolumbar scoliosis and dural ectasia. Imaging did not reveal any vascular abnormalities. Her son, born at 37 weeks 3 days. had club feet but not other clinical signs suggestive of classical or vascular EDS.DiscussionThree patients are described with a contiguous deletion of varying size encompassing the COL3A1 and COL5A2 gene. Due to the mild phenotype a diagnosis of EDS was not suspected and was found coincidental. Since two of the patients were pregnant without major complications these patients may require a less defensive, approach to pregnancy/delivery.     

Novel homozygous LAMB1 in-frame deletion in a pediatric patient with brain anomalies and cerebrovascular event

Biallelic pathogenic variants in LAMB1 have been associated with autosomal recessive lissencephaly 5 (OMIM 615191), which is characterized by brain malformations (cobblestone lissencephaly, hydrocephalus), developmental delay, and epilepsy. Pathogenic variants in LAMB1 are rare, with only 11 pathogenic variants and 11 patients reported to date. Here, we report on a 6-year-old patient from a consanguineous family with profound developmental delay, microcephaly, and a history of a perinatal cerebrovascular event. Brain magnetic resonance imaging (MRI) showed cerebellar cystic defects, signal intensity abnormalities, and a hypoplastic corpus callosum. Trio-exome analysis revealed a homozygous in-frame deletion of Exons 23 and 24 of LAMB1 affecting 104 amino acids including the epidermal growth factor (EGF)-like units 11 and 12 in Domain III. To our knowledge, this is the first reported in-frame deletion in LAMB1. Our findings broaden the clinical and molecular spectrum of LAMB1-associated syndromes.     

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.