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 transfected 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.
Dipartimento di Scienze Biomediche, Università di Modena & Reggio Emilia, Modeno, Italy.
Enza
E
Di Leo
Francesca
F
Panico
Patrizia
P
Tarugi
Carla
C
Battisti
Antonio
A
Federico
Sebastiano
S
Calandra
D000328:Adult; D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005260:Female; D005347:Fibroblasts; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007438:Introns / Q000235:genetics*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D014162:Transfection
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein