Abstract
BackgroundCerebral 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.MethodsHere, 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.ResultsNo 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.ConclusionsOur data indicated that rs9853967 and rs11714980 polymorphisms could be associated with a protective role in CCM disease.
Highlights
Cerebral cavernous malformations (CCMs) are vascular anomalies of the nervous system mostly located in the brain presenting sporadically or familial
Linkage analyses predicts that 40 % of patients with familial forms is linked to CCM1 locus, 40 % to CCM3 and, only 20 % to CCM2 [10,11,12,13]; according with reported experimental data, these frequencies are not confirmed by an our recent screening of a cohort of Italian patients with CCMs [9]
Our data showed that 54 % of patients leads CCM1 mutations, a very lower percent was observed for CCM3 (6 %) while 18 % leads mutation at CCM2 locus; the absence of any mutation in 22 % of patients, leads to consider other possible elements involving in the development of disease like a somatic mosaicism of a de novo mutation that occurred during gestation and is not detectable in DNA extracted from peripheral blood [5, 6, 14], the possibility of large deletions or duplications not detected by direct sequencing or mutations located in regulatory regions of CCM genes, and the existence of other as yet unidentified genes
Summary
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. CCMs are associated with loss-of-function mutations in any one of the three CCM genes CCM1/KRIT1, CCM2/ MGC4607 or CCM3/PDCD10 and occur in both sporadic and familial forms [5], inherited in an autosomal dominant fashion with a high penetrance. Our data showed that 54 % of patients leads CCM1 mutations, a very lower percent was observed for CCM3 (6 %) while 18 % leads mutation at CCM2 locus; the absence of any mutation in 22 % of patients, leads to consider other possible elements involving in the development of disease like a somatic mosaicism of a de novo mutation that occurred during gestation and is not detectable in DNA extracted from peripheral blood [5, 6, 14], the possibility of large deletions or duplications not detected by direct sequencing or mutations located in regulatory regions of CCM genes, and the existence of other as yet unidentified genes
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