Nonsense-mediated mRNA decay process in nine alleles of Niemann-Pick type C patients from Spain.

Mutations in NPC1 or NPC2 genes are responsible of Niemann-Pick type C disease (OMIM #257220), an autosomal recessive neurodegenerative lysosomal storage disorder caused by a non-regulation of intracellular lipid trafficking. Alterations such as nonsense or frame shift mutations generate a premature termination-codon (PTC). Nonsense-mediated mRNA decay (NMD) is a natural cellular process that degrades mRNAs that encode a prematurely truncated protein. In this study we have analyzed 9 NPC1 mutations which generate a PTC (p.R116X, p.Q119VfsX8, p.W260X, p.S425X, p.A558GfsX12, p.Q775X, p.G993EfsX4, p.R1059X and p.I1061NfsX4), in order to determine if their mRNAs suffer NMD process. To achieve this objective we compared fibroblasts of patients carrying these alleles with and without cycloheximide (CHX) treatment using conventional PCR and real-time PCR. The results of conventional PCR of untreated fibroblasts showed a reduction of the amount of NPC1 mRNA compared to control in all patients. After CHX-treatment, a recovery of mRNA was detected but not in all the alleles. However, when real-time PCR was used, the recovery was observed including those alleles that qualitatively showed no apparent increase in mRNA level. In conclusion, we confirmed that NMD process is responsible for the mRNA decay for all the analyzed NPC1 PTC-encoding mutations.