Frontotemporal Dementia Sporadic Case associating two novel Tau mutations. Part two: molecular effect on Exon 10 alternative splicing

Objective: To investigate the molecular effect of two novel heterozygous intronic mutations localized in the regions surrounding exon 10, identified in the MAPT gene of a FTD neuropathologically ascertained patient. The T to C transition [t(-15)c] is localized at position -15 of intron 9 and the A to C transversion at position +4 of intron 10 (E10 +4). Methods: Minigenes were designed to contain MAPT intact exons 9-11 and intronic minimal flanking sequences. Fragments with the two mutations were 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 cells. The relative amount of exon 10 (E10) splicing was quantified by RT-PCR analysis of transcripts derived from minigene constructs. Results: Transcripts derived from minigene constructs carrying the two mutations cause an increase in E10 splicing that gives rise to a higher increase of mRNAs transcripts excluding E10. Our data on the index patient's parents demonstrate that if only one mutation is present in heterozygous condition the ratio of E10 including/ E10 excluding transcripts is quite normal. In the index patient both mutations are important for the dramatic altered ratio, that give rise to an increase on Tau3R mRNA transcripts as shown on studies performed on RNA extracted from post-mortem brain tissue. Conclusions: We hypothesize that a trans-acting effect of both mutations, localized in the regulatory region for E10 alternative splicing, may have contributed to the neurodegenerative process, increasing E10 splicing.