Two novel tau mutations associated to a Frontotemporal Dementia Sporadic case: a molecular study

Objective: To investigate the molecular effect of a novel heterozygous mutation, a T to C transition at position -15 in intron 9 identified in the MAPT gene of a FTD neuropathologically ascertained patient in which we previously identified another novel MAPT mutation (+4 intron 10). Methods: Minigenes were designed to contain MAPT exons 9-11 and intronic minimal flanking sequences. Fragment with the t(-15)c mutation was generated from patient genomic DNA, control fragment was generated from a FTD-Ub+ DNA. Each fragment was inserted into pcDNA3 vector and costructs were transfected into HeLa and MCF7 cells. The relative amount of exon 10 (E10) splicing was quantified by RT-PCR analyses of transcripts derived from minigene constructs. Results: The intron 9 transition shows, in both cell lines analyzed, a decrease of transcripts excluding E10 when compared with FTD-Ub+ control. Transcripts derived from minigene construct carried the intron 10 +4 mutation have shown a high expression of transcripts lacking E10. Studies performed on RNA extracted from post-mortem brain tissue have shown an altered alternative splicing due to a strong increase of tau3R mRNAs. Clinical and neuropathological data are reported by Colao et al. in this meeting. Conclusions: The molecular effect of the -15 intron 9 transition decreases E10 exclusion and seems to be in contrast with that showed by the +4 mutation. We have to underline that the transition occurs in a regulatory region for E10 alternative splicing and the +4 mutation in the 5’ splice site of intron 10. Considering the altered 4R:3R ratio observed in brain tissue, with the 3R increase, we cannot exclude a trans-acting effect of the two mutations or a binding inhibition for splicing factors, that may have contributed to the neurodegeneration process.