The complete deficiency of Factor VII (FVII), the protease triggering blood coagulation, is potentially lethal, as suggested by the absence of homozygous large gene deletions in humans and knock-out experiments in mice. We characterized the IVS6+1G>T mutation at the IVS6 donor splice site (5'ss) of FVII gene. The mutation was identified in homozygous condition in two severe FVII deficient patients from Thailand who experienced gastrointestinal bleeding in the first months of life and were subsequently kept under prophylaxis regimen. Plasma FVII activity levels, measured by PT-based and fluorogenic functional assays (FXa and thrombin generation) were strikingly below the commercial FVII deficient/depleted plasma (negative controls). The FVII mRNA was investigated in leukocyte mRNA from a heterozygous subject, which did not reveal any trace of normal transcripts deriving from the mutant IVS6+1T allele. Through the expression of extended FVII minigenes in a human liver cell line (Hep3B), we demonstrated that the mutation primarily induced exon 6 skipping and, to a lower extent, total and partial IVS6 retention, thus producing frameshifts incompatible with FVII biosynthesis and function. The IVS6+1G>T is predicted to impair recognition of the donor splice site by the spliceosomal small nuclear ribonucleoprotein U1 (U1-snRNP), a crucial step in exon definition. To assess whether compensatory U1-snRNP could rescue FVII expression, we developed a modified U1- snRNA+1T complementary to the mutated 5'ss. However, cotransfection with this U1snRNA did not increase, to any appreciable extent, usage of the correct 5'ss. Altogether these findings do not provide experimental evidence even for minimal expression of this FVII gene mutation, affecting the invariable GT dinucleotide of the 5ss and abolishing correct FVII mRNA processing. Intriguingly, our findings also suggest that very low FVII levels, below the detection threshold of recombinant methodology, cause a lifethreatening disorder but can be compatible with life.
The complete impairment of factor VII gene expression by the IVS6+1g/t mutation is compatible with a severe but not lethal bleeding disorder
CAVALLARI, Nicola;BALESTRA, Dario;RIZZOTTO, Lara;MARIANI, Guglielmo;PAGANI, Franco;BERNARDI, Francesco;PINOTTI, Mirko
2010
Abstract
The complete deficiency of Factor VII (FVII), the protease triggering blood coagulation, is potentially lethal, as suggested by the absence of homozygous large gene deletions in humans and knock-out experiments in mice. We characterized the IVS6+1G>T mutation at the IVS6 donor splice site (5'ss) of FVII gene. The mutation was identified in homozygous condition in two severe FVII deficient patients from Thailand who experienced gastrointestinal bleeding in the first months of life and were subsequently kept under prophylaxis regimen. Plasma FVII activity levels, measured by PT-based and fluorogenic functional assays (FXa and thrombin generation) were strikingly below the commercial FVII deficient/depleted plasma (negative controls). The FVII mRNA was investigated in leukocyte mRNA from a heterozygous subject, which did not reveal any trace of normal transcripts deriving from the mutant IVS6+1T allele. Through the expression of extended FVII minigenes in a human liver cell line (Hep3B), we demonstrated that the mutation primarily induced exon 6 skipping and, to a lower extent, total and partial IVS6 retention, thus producing frameshifts incompatible with FVII biosynthesis and function. The IVS6+1G>T is predicted to impair recognition of the donor splice site by the spliceosomal small nuclear ribonucleoprotein U1 (U1-snRNP), a crucial step in exon definition. To assess whether compensatory U1-snRNP could rescue FVII expression, we developed a modified U1- snRNA+1T complementary to the mutated 5'ss. However, cotransfection with this U1snRNA did not increase, to any appreciable extent, usage of the correct 5'ss. Altogether these findings do not provide experimental evidence even for minimal expression of this FVII gene mutation, affecting the invariable GT dinucleotide of the 5ss and abolishing correct FVII mRNA processing. Intriguingly, our findings also suggest that very low FVII levels, below the detection threshold of recombinant methodology, cause a lifethreatening disorder but can be compatible with life.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
