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.

DI LEO, E., Panico, F., Tarugi, P., Battisti, C., Federico, A., & Calandra, S. (2004). 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. HUMAN MUTATION, 24(5), 440-440 [10.1002/humu.9287].

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.

BATTISTI, CARLA;FEDERICO, ANTONIO;
2004

Abstract

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.
File in questo prodotto:
File Dimensione Formato  
di leo.pdf

non disponibili

Tipologia: Post-print
Licenza: NON PUBBLICO - Accesso privato/ristretto
Dimensione 406.36 kB
Formato Adobe PDF
406.36 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11365/27043
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo