title: CCM3/SERPINI1 bidirectional promoter variants in patients with cerebral cavernous malformations: a molecular and functional study.
abstract: BACKGROUND
  Cerebral cavernous malformations (CCMs) are vascular anomalies of the nervous system mostly located in the brain presenting sporadically or familial. Causes of familial forms are mutations in CCM1 (Krit1), CCM2 (MGC4607) and CCM3 (PDCD10) genes. Sporadic forms with no affected relative most often have only one lesion and no germ line mutations. However, a number of sporadic cases with multiple lesions have been reported and are indeed genetic cases with a de novo mutation or a mutation inherited from an asymptomatic parent.
  
  
  METHODS
  Here, we performed an analysis of regulatory region of CCM genes in 60 sporadic patients, negative for mutations in coding region and intron-exon boundaries and large deletion/duplications in CCM genes by direct sequencing and MLPA. Among 5 variants identified in 851-bp region shared by CCM3 and SERPINI1 genes and acting as asymmetric bidirectional promoter, two polymorphisms c.-639 T > C/rs9853967 and c.-591 T > C/rs11714980 were selected. A case-control study was performed to analyze their possible relationships with sporadic CCMs. Promoter haplotypes activities on CCM3/SERPINI1 genes expression were tested by dual-luciferase assay.
  
  
  RESULTS
  No variants were identified in CCM1 and CCM2 regulatory regions. In CCM3/SERPINI1 asymmetric bidirectional promoter 5 variants, 2 of them unknown and 3 corresponding to polymorphisms c.-639 T > C/rs9853967, c.-591 T > C/rs11714980 and c.-359G > A/rs9834676 were detected. While rs9853967 and rs11714980 polymorphisms fall in a critical regulatory fragment outside the minimal promoter in intergenic region, other variants had no effects on transcription factor binding according to RegRNA tool. Case-control study performed on 60 patients and 350 healthy controls showed frequencies of the mutated alleles significantly higher in the control group than in patients. Furthermore, the functional assay showed a significant reduction of CCM3 expression for C-C haplotype even more than for T-C and C-T haplotypes. In SERPINI1 direction, the reduction was not statistically significant.
  
  
  CONCLUSIONS
  Our data indicated that rs9853967 and rs11714980 polymorphisms could be associated with a protective role in CCM disease.
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy.|Department of Cutting-Edge Medicine and Therapies, Biomolecular Strategies And Neuroscience, Section of Neuroscience-applied, Molecular Genetics and Predictive Medicine, I.E.ME.S.T, Palermo, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Scimone
authors.affiliation: IRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Italy.
authors.forename: Placido
authors.initials: P
authors.lastname: Bramanti
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Ruggeri
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy.|Department of Cutting-Edge Medicine and Therapies, Biomolecular Strategies And Neuroscience, Section of Neuroscience-applied, Molecular Genetics and Predictive Medicine, I.E.ME.S.T, Palermo, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Donato
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Alafaci
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Crisafulli
authors.affiliation: Department of Economy, University of Messina, Messina, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Mucciardi
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy.
authors.forename: Carmela
authors.initials: C
authors.lastname: Rinaldi
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy. asidoti@unime.it.|Department of Cutting-Edge Medicine and Therapies, Biomolecular Strategies And Neuroscience, Section of Neuroscience-applied, Molecular Genetics and Predictive Medicine, I.E.ME.S.T, Palermo, Italy. asidoti@unime.it.
authors.forename: Antonina
authors.initials: A
authors.lastname: Sidoti
authors.affiliation: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, via C. Valeria 1, 98125, Messina, Italy.
authors.forename: Rosalia
authors.initials: R
authors.lastname: D'Angelo
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D016022:Case-Control Studies; D002648:Child; D005260:Female; D020786:Hemangioma, Cavernous, Central Nervous System / Q000235:genetics*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D009479:Neuropeptides / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D015843:Serpins / Q000235:genetics*; D055815:Young Adult; D000097522:Neuroserpin
chemical_list: D051017:Apoptosis Regulatory Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D011518:Proto-Oncogene Proteins; D015843:Serpins; D000097522:Neuroserpin; C495390:PDCD10 protein, human
keywords: Association study; Bidirectional promoter; CCM genes; CCM3/SERPINI1; Dual luciferase-assay; Polymorphism