A point mutation in the lariat branch point of intron 6 of NPC1 as the cause of abnormal pre‐mRNA splicing in Niemann‐Pick type C disease

The lariat branch point sequence (BPS) is crucial for splicing pre‐mRNA even if BPS mutations have infrequently been reported in human disease. In two siblings with Niemann‐Pick type C (NPC) disease we identified two mutations of the NPC1 gene: i) one in exon 20 (c.2932C>T) (p.R978C) previously reported in NPC patients; ii) the other (c.882‐28A>G) unreported, in the highly conserved adenosine of a putative lariat BPS of intron 6. Using RT‐PCR we found that, besides the normally spliced mRNA, patients' fibroblasts contained minute amounts of an mRNA devoid of exon 7. The exon 6 ‐ exon 8 junction in this mRNA causes a frameshift and a premature stop codon, predicted to result in a truncated protein. To assess the effect of c.882‐28A>G mutation we constructed two minigenes (wild type and mutant), spanning from intron 5 to intron 8, which were inserted into a pTarget vector and transfectd in COS1 cells. The wild type minigene generated an mRNA of the expected size and sequence; the mutant minigene generated only an mRNA devoid of exon 7. This is the first example of a splicing defect due to a mutation in the lariat BPS in an intron of NPC1 found in NPC patients. © 2004 Wiley‐Liss, Inc.