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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
Author(s) -
Di Leo Enza,
Panico Francesca,
Tarugi Patrizia,
Battisti Carla,
Federico Antonio,
Calandra Sebastiano
Publication year - 2004
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.9287
Subject(s) - minigene , exon , biology , intron , frameshift mutation , point mutation , rna splicing , microbiology and biotechnology , splice site mutation , exon trapping , mutant , mutation , polypyrimidine tract , stop codon , genetics , messenger rna , gene , alternative splicing , rna
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.