NOVEL PDCD10 PROMOTER VARIANTS IN PATIENTS WITH CEREBRAL CAVERNOUSMALFORMATIONS

Cerebral Cavernous Malformations (CCMs) are vascular anomalies mostly located in the CNS, affecting up to 0.5% of the population and inducing to seizures, recurrent headaches, focal neurological deficits, epileptic attacks and fatal intracerebral hemorrhages. CCMs may occur sporadically or be inherited in an autosomal dominant fashion with a high penetrance. Whereas sporadic cases often present a single lesion, the familial form, usually are characterized by a presence of multiple lesions and have been associated with mutations in Krit1/CCM1, MGC4607/CCM2 and PDCD10/CCM3 genes. Sequencing of all coding exons of the three CCM genes and search for genomic rearrangements using multiplex ligation-dependent probe amplification (MLPA) in Caucasian non-Hispano-American CCM families led to the identification of the causative mutation in 95% of the families. Approximately 72% of families harbored a mutation in Krit1 , 18% in MGC4607 and 10% in PDCD10. The PDCD10 proportion was much lower than expected, based on previous linkage data which suggested that 40% of CCM families were linked to the locus CCM3 (1). In our recent study, in agreement with other data reported in the literature, we have found that the 22-30% of CCM cases with multiple lesions and/or an affected relative, showed no mutations in the coding sequence (2). Therefore, we performed further molecular analysis on promoter regions of three CCM genes and we have detected three novel variants only in promoter region of PDCD10. This region appears to be of particular interest as it is shared by the PDCD10 and SERPINI1, two non homologous genes with a divergent head-to-head orientation, one coding for the PDCD10 programmed cell death and the other for the protease inhibitor SERPINI1 which is down-regulated in brain tumors. Moreover, this promoter region is able to efficiently drive the expressions of the two flanking genes, through a critical regulatory fragment containing a repressive element for SERPINI1 and an enhancer for PDCD10. To determine whether the novel variants had any effect on promoter activity, we cloned the various mutant promoters, into a pGL4.10-Basic luciferase –reporter vector and transfected into human DB-TRG/U373 glioblastoma-astrocytoma cells. The possible effects on promoter activity were investigated using Dual-Luciferase Reporter assay (Promega). 1. Liquori C.L. et al. “Hum Mutat” 2006 2. D’Angelo R. et al. “ Brain Pathology” 2011