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Author's Personal Copy J Mol Neurosci DOI 10.1007/s12031-015-0606-6

Detection of Novel Mutation in Ccm3 Causes Familial Cerebral Cavernous Malformations Concetta Scimone 1 & Placido Bramanti 2 & Alessia Ruggeri 1 & Zoe Katsarou 3 & Luigi Donato 1 & Antonina Sidoti 1 & Rosalia D’Angelo 1

Received: 15 May 2015 / Accepted: 19 June 2015 # Springer Science+Business Media New York 2015

Abstract Cerebral cavernous malformations are vascular lesions that usually involve brain micro-vessels. They can occur both in a sporadic form and familial one. Causes of familial forms are mutations at three loci: CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10. Here, we describe a novel CCM3 missense mutation (c.422T>G) detected in two Greek brothers showing multiple lesions at magnetic resonance imaging; to date, only the youngest is symptomatic. Bioinformatics tools showed this novel variant causes a loss of function in Pdcd10 protein due to its localization in the eighth helix and, particularly, affects Leu141, a highly conserved amino acid. Roles of Pdcd10 in angiogenesis regulation and its association with early development of cerebral cavernous malformations were also considered.

Keywords Familial cerebral cavernous malformations . CCM3 mutation . Incomplete penetrance . Impaired angiogenesis

* Antonina Sidoti [email protected]

Introduction Cerebral cavernous malformations (CCMs) represent approximately 20 % of all cerebrovascular malformations (Bacigaluppi et al. 2013). They consist of enlarged capillaries characterized by deficits at intercellular junctions and cell extracellular matrix (ECM) adhesions and by the absence of pericytes (Lee and Spetzler 1989). CCMs are mostly located at the central nervous system (CNS) and do not involve brain parenchyma. They usually exhibit focal neurological deficits, seizures, headaches, and intracerebral hemorrhages (ICH). However, only about 70 % of patients are clinically manifest (Haasdijk et al. 2012). CCMs can occur as sporadic forms or be inherited with an autosomal dominant pattern with incomplete penetrance and variable expressivity (Labauge et al. 2007). Germ line mutations at three loci—CCM1/KRIT1 (7q21.2), CCM2/MGC4607 (7p13), and CCM3/PDCD10 (3q26.1)—are genetic causes of familial forms (Felbor et al. 2006). While genotype–phenotype correlations for CCM1 and CCM2 mutations are still unclear, a great aggressiveness and an early onset were observed in CCM3 mutations carriers (Shenkar et al. 2014). The three CCM genes encode for proteins involved in regulation of angiogenesis (Fischer et al. 2013) and stress response (Guazzi et al. 2012; Uhlik et al. 2003). Here, we describe a case of two brothers with multiple lesions caused by a novel missense mutation in CCM3. Case Presentation and Molecular Analysis

1

Department of Biomedical Sciences and Morpho-functional Images, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, Via C. Valeria 1, 98125 Messina, Italy

2

IRCCS Centro Neurolesi BBonino-Pulejo^, Contrada Casazza, SS113, 98124 Messina, Italy

3

Department of Neurology, Hippokration General Hospital, 49 Konstantinoupoleos Street, Thessaloniki, Greece

The proband is a Greek 50-year-old man (Fig. 1, III-2) who manifested sudden onset of blurred vision and unsteadiness of gait; he was fully ambulatory but needed mild support to walk at open spaces. Neurological examination revealed mild paresis of the left cranial nerve VI as well as mild pyramidal and cerebellar signs on his right side. MRI scan of his brain

Author's Personal Copy J Mol Neurosci

Fig. 1 Pedigree of the family with cerebral cavernous malformations. Filled circles and squares indicate affected individuals. Symbols with a diagonal indicate deceased individuals. For the brothers are also reported the genotypes

revealed multiple lesions ranging from a few millimeters to 1 cm (Fig. 1, MRI a). His mother died at the age of 81 due to ICH and had similar findings in MRI (Fig. 1, II-1, MRI b). A brain MRI scan was also performed for his elder (54 years old) asymptomatic brother (Fig. 1, III-1, MRI c), and exam revealed multiple brain lesions similar to those of his brother indicative of the same disease. Diagnosis of cavernomatosis was established.

DNA was isolated from peripheral blood; coding exons, intron–exon boundaries, and promoter regions of the three genes were screened using primers (available upon request) designed according to the CCM1, CCM2, and CCM3 published nucleotide sequence of GenBank (accession nos. NG_012964.1, NG_016295.1, and NG_008158.1, respectively). PCR products were sequenced on 3500 Genetic Analyzer (Applied Biosystems, CA, USA), using the BigDye


Terminator v3.1 chemistry, following the manufacturer’s procedures. Analysis was performed on both brothers, a maternal uncle (Fig. 1, II-3), and a maternal aunt (Fig. 1, II-4). Multiplex ligation-dependent probe amplification (MLPA) assay was performed using SALSA MLPA Kits P130 and P131 CCM (MRC Holland) to exclude the presence of large genomic rearrangements. Statistical analysis was performed by Coffalyser software. PCR-RFLP (restriction fragment length polymorphism) was conducted on 300 control samples by HpyCH4IVenzyme (5′A/CGT3′) to estimate the variant’s frequency. To assess potential dangerous effects of this novel variant on protein function, an in silico analysis was performed using two different tools: PolyPhen-2 and MutationTaster. For cDNA analysis, RNA was purified from lymphocytes isolated from peripheral blood and retro-transcribed by GeneAmp RNA PCR Core Kit (Applied Biosystems). CCM3 whole coding sequence was amplified and then sequenced. All patients were fully informed and written consent was obtained.

Results and Discussions Sequencing analysis of all coding exons of the three CCM genes was performed, and a novel missense mutation c.422T>G in CCM3 was found only in the two brothers. Moreover, in the elder asymptomatic one, two SNPs (single nucleotide polymorphisms), rs11552377 and rs575724801, in CCM2 were detected. SNP rs2027950 at CCM1, instead, was detected only in the symptomatic one (Fig. 1). No promoters’ variants were identified. Missense mutation c.422T>G affecting exon 7 of CCM3 causes the amino acid change p.L141R. No large insertions/deletions were detected by MLPA analysis for the three genes. The new substitution is not reported in the following databases: CCM mutation database (http://www. angiomaalliance.org/pages.aspx?content=348#.VImlG9KGAg), HGMD (http://www.hgmd.cf.ac.uk/ac/index.php), NCBI SNP (http://www.ncbi.nlm.nih.gov/snp/), and Ensembl (http://www.ensembl.org/Homo_sapiens/Gene/ Sequence?db=core;g=ENSG00000114209;r=3:167683298167734939), and results obtained from PCR-RFLP analysis (not shown) confirm that the c.422T>G variant is a mutation and due to its segregation with affected family members, it is linked to CCM development. Deleterious effects of this missense mutation were confirmed by both the two prediction tools considered. Particularly, for PolyPhen-2, this mutation is predicted to be Bprobably damaging^ (http://genetics.bwh. harvard.edu/ggi/pph2/c32a522f25da470f3c0e50e4a72796 7052c1c225/2981176.html) and Bdisease causing^ for MutationTaster (http://www.mutationtaster.org/cgi-bin/ MutationTaster/MutationTaster69.cgi). No splicing

alterations were detected by direct sequencing of full-length CCM3 cDNA. Moreover, the presence of the c.422T>G variant also in cDNA’s electropherogram (not shown) allows to confirm that defective mRNA is not broken down by a nonsense-mediated decay mechanism. The novel CCM3 missense mutation c.422T>G affects BαG-helix^ of the focal adhesion targeting (FAT)-homology domain. This is involved in Pdcd10 interaction with Ccm2 and with paxillin (Li et al. 2010). Multiple sequence alignment (http://genetics.bwh.harvard.edu/cgi-bin/pph2/display_ alignment.cgi?sid=c32a522f25da470f3c 0e50e4a727967052c1c225&id=Q9BUL8&mut= L141R&web=0) shows that p.L141 is a highly conserved amino acid as well as the entire domain’s structure is maintained during evolution and mutations at the FAThomology domain are deleterious for protein’s activity. Pdcd10 can bind a large number of proteins via its C-terminal FAT domain. The three CCM proteins co-localize at the cell periphery and form a ternary complex that is involved in cell– cell adhesion and cell adhesion to the ECM (Zawistowski et al. 2005; Voss et al. 2007). Pdcd10 is implicated in the regulation of focal adhesions by binding to paxillin (Chen et al. 2013). Interaction with VEGFR2 during early embryogenesis, instead, underlines Pdcd10’s role in angiogenesis regulation and involves Pdcd10’s FAT-homology domain (He et al. 2010). Moreover, Pdcd10 is also involved in the regulation of DLL4–Notch signaling, a negative regulator of angiogenesis (You et al. 2013). Pdcd10 also takes part in the formation of the STRIPAK complex (Goudreault et al. 2009) that contributes to the control of neurovascular development. Involvement of Pdcd10 in the regulation of many cellular processes is the result of its multiple interactions; these interactions can be mutually exclusive, and they are tightly linked to cellular physiological or pathological conditions, to the cell context, and to the developmental stage considered. In order to elucidate CCM pathogenesis, early activity of Pdcd10 on VEGFR2 could explain why CCM3 germ line mutations’ carriers develop prematurely lesions that occur with a more serious symptomatology than patients with CCM1 and CCM2 mutations, genes that are involved in tardive vascular differentiation. An important aspect of this case regards why only one of the two brothers is symptomatic despite that both are carriers of the same mutation and affected by multiple lesions. Incomplete penetrance of familial CCMs was already described; however, a hypothesis is that the SNPs detected in the asymptomatic brother are associated with symptomatology’s absence, balancing deleterious effects of the c.422T>G CCM3 mutation; in fact, rs11552377 (p.Val120Ile) was associated with a less severe symptomatology (D’Angelo et al. 2012). Acknowledgments We are thankful to Dr. Eugenia Avdellidou and Dr. Eleni Fourkala of the Department of Neurology, Hippokration General Hospital, Thessaloniki, Greece, for their contribution to this study.


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