Over 30% of human diseases is caused by aberrant mRNA splicing and, among splicing mutations, those at the invariable +1 g nucleotide of donor splice site (5’ss) have the most detrimental effects. A +1g/t mutation (9007 + 1g > t in intron 6) in the gene encoding factor VII (FVII), the protease triggering coagulation whose absence is lethal, represented our model. Homozygotes had undetectable plasma FVII levels and manifested severe bleeding. Expression studies with FVII minigenes in BHK cells indicated that the 9007 + 1 g/t change induces exon 6 skipping, thus leadleading to a frame-shifted mRNA incompatible with proper FVII biosynthesis. Fluorescent labelling of RT-PCR products or realtime RT-PCR, able to detect 1 : 100 000 dilutions of aberrant transcripts, did not reveal normal FVII mRNA. Ectopic FVII splicing patterns in leukocytes from a doubly heterozygous 9007 + 1 g/t-W364X patient, showed transcripts from the W364X allele only. The 9007 + 1 g/t mutation is candidate to impair 5’ss recognition by the spliceosomal snRNP U1, crucial for exon definition. To assess whether compensatory U1-snRNP could rescue FVII expression in vivo, we developed a U1-snRNA+1t complementary to the mutated 5’ss, which however failed to redirect its correct usage. Intriguingly, capillary electrophoresis of transcripts from the mutant minigene identified traces ( 3%) of a 30 bp deleted FVII mRNA resulting from usage of an exon 6 cryptic 5’ss. This predicts a FVII protein lacking 10 residues (Val158-Gln167) in the activation domain but preserving the cleavage site of the serine protease. Expression of the deleted variant results in secretion of low but measurable levels of FVII molecules (0.15% of wt- FVII) with virtually normal specific activity, which in vivo would ensure minimal haemostasis. Our data highlight a poorly recognized feature of aberrant splicing that, by accounting for residual FVII expression levels, might have reverted an otherwise lethal null FVII deficiency caused by a +1 g/t mutation.
Aberrant splicing reverts a potentially lethal coagulation deficiency caused by a +1g/t splicing mutation
CAVALLARI, Nicola;BALESTRA, Dario;RIZZOTTO, Lara;MAESTRI, Iva;BERNARDI, Francesco;PINOTTI, Mirko
2011
Abstract
Over 30% of human diseases is caused by aberrant mRNA splicing and, among splicing mutations, those at the invariable +1 g nucleotide of donor splice site (5’ss) have the most detrimental effects. A +1g/t mutation (9007 + 1g > t in intron 6) in the gene encoding factor VII (FVII), the protease triggering coagulation whose absence is lethal, represented our model. Homozygotes had undetectable plasma FVII levels and manifested severe bleeding. Expression studies with FVII minigenes in BHK cells indicated that the 9007 + 1 g/t change induces exon 6 skipping, thus leadleading to a frame-shifted mRNA incompatible with proper FVII biosynthesis. Fluorescent labelling of RT-PCR products or realtime RT-PCR, able to detect 1 : 100 000 dilutions of aberrant transcripts, did not reveal normal FVII mRNA. Ectopic FVII splicing patterns in leukocytes from a doubly heterozygous 9007 + 1 g/t-W364X patient, showed transcripts from the W364X allele only. The 9007 + 1 g/t mutation is candidate to impair 5’ss recognition by the spliceosomal snRNP U1, crucial for exon definition. To assess whether compensatory U1-snRNP could rescue FVII expression in vivo, we developed a U1-snRNA+1t complementary to the mutated 5’ss, which however failed to redirect its correct usage. Intriguingly, capillary electrophoresis of transcripts from the mutant minigene identified traces ( 3%) of a 30 bp deleted FVII mRNA resulting from usage of an exon 6 cryptic 5’ss. This predicts a FVII protein lacking 10 residues (Val158-Gln167) in the activation domain but preserving the cleavage site of the serine protease. Expression of the deleted variant results in secretion of low but measurable levels of FVII molecules (0.15% of wt- FVII) with virtually normal specific activity, which in vivo would ensure minimal haemostasis. Our data highlight a poorly recognized feature of aberrant splicing that, by accounting for residual FVII expression levels, might have reverted an otherwise lethal null FVII deficiency caused by a +1 g/t mutation.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
