title: Identification of two novel mutations and of a novel critical region in the KRIT1 gene.
abstract: Cerebral cavernous malformations (CCMs) represent a common autosomal dominant disorder that predisposes patients to hemorrhagic strokes and focal neurological signs. Mutations in three genes (KRIT1, MGC4607, and PDCD10) have been associated with CCMs. We investigated the role of two new mutations in the KRIT1 gene in two Italian families affected by CCMs. Whole blood DNA was extracted and the mutations were detected after polymerase chain reaction (PCR), denaturing high-performance liquid chromatography screening, and sequencing of the coding regions of the three CCMs-associated genes. Total RNA was extracted, and the KRIT1 cDNA was sequenced and subsequently subjected to real-time quantitative PCR in order to examine the translational outcome of each genomic mutation. A novel splicing acceptor site deletion of the exon 14 in one family and an intronic nucleotide change close to the exon 19 in the other one were identified, both in the KRIT1 gene. These mutations were proven to alter the correct splicing mechanism, resulting, respectively, in a truncated protein of 432 amino acids and in a protein lacking an internal segment. We report two novel cases of splicing affecting genomic variants, suggesting a careful reanalysis of previously identified splice site variations in KRIT1 to look for their possible causative roles of similar missplicing events and their consequent involvement in the pathogenesis of CCMs. Moreover, our genotype-phenotype functional correlation suggests that the C-terminal portion of the KRIT1 protein is likely to contain a short, previously unrecognized segment necessary for its activity.
authors.affiliation: Medical Genetics Service, Poliambulatorio Giovanni Paolo II, IRCCS Casa Sollievo della Sofferenza Hospital, Viale Cappuccini, 71013, San Giovanni Rotondo (FG), Italy. v.guarnieri@operapadrepio.it
authors.forename: Vito
authors.initials: V
authors.lastname: Guarnieri
authors.forename: Lucia A
authors.initials: LA
authors.lastname: Muscarella
authors.forename: Rosina
authors.initials: R
authors.lastname: Amoroso
authors.forename: Alessandro
authors.initials: A
authors.lastname: Quattrone
authors.forename: Massimo E
authors.initials: ME
authors.lastname: Abate
authors.forename: Michelina
authors.initials: M
authors.lastname: Coco
authors.forename: Domenico
authors.initials: D
authors.lastname: Catapano
authors.forename: Vincenzo A
authors.initials: VA
authors.lastname: D'Angelo
authors.forename: Leopoldo
authors.initials: L
authors.lastname: Zelante
authors.forename: Leonardo
authors.initials: L
authors.lastname: D'Agruma
mesh_terms: D000293:Adolescent; D001932:Brain Neoplasms / Q000235:genetics / Q000473:pathology; D005091:Exons; D005190:Family; D005260:Female; D006392:Hemangioma, Cavernous / Q000235:genetics* / Q000473:pathology; D006801:Humans; D007438:Introns; D000075927:KRIT1 Protein; D008279:Magnetic Resonance Imaging; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D009154:Mutation; D010375:Pedigree; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*
chemical_list: D000075927:KRIT1 Protein; C107928:KRIT1 protein, human; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins