Severe factor V deficiency: exon skipping in the factor V gene causing a partial deletion of the C1 domain.

BACKGROUND: Severe factor V (FV) deficiency is a rare coagulation disorder, characterized by very low or unmeasurable plasma levels of functional and immunoreactive FV. Among rare inherited coagulopathies, FV deficiency is the least characterized from a molecular point of view (only 12 mutations have been reported). OBJECTIVES: The aim of this work was to investigate, at the molecular level, the pathogenetic mechanisms responsible for a case of severe FV deficiency. PATIENTS AND METHODS: A 19-year-old Iranian man showing unmeasurable FV activity and severely reduced FV antigen level in plasma was studied. Mutation screening was performed by sequencing. The effect of the identified mutation was investigated both at the mRNA and at the protein level. RESULTS: Molecular analysis of the factor V (FV) gene identified a novel homozygous A-->T transversion at position + 3 of the donor splice site of intron 19 (IVS19 + 3A-->T). Production of mutant mRNA in HeLa cells demonstrated that this mutation causes the entire exon 19 to be skipped from the FV mRNA. The mutant processed transcript codes for a deleted FV, lacking the first 24 amino acids of the C1 domain. Expression of the mutant FV protein in COS-1 cells showed that the deleted protein was synthesized but not secreted; moreover, the intracellular amount of deleted FV was reduced compared to wild type, suggesting intracellular degradation of mutant FV. CONCLUSIONS: This work reports the molecular characterization of the first mutation causing a partial deletion in the FV molecule, resulting in a severe impairment of protein secretion.

Autocrine-acting early secreted mitogenic activity: production and responsiveness in cultures of normal human keratinocytes as a function of in vivo and in vitro age.

Cultured epithelial grafts are used in the clinical treatment of both non-healing and acute partial-thickness wounds, owing to their ability to stimulate endogenous re-epithelialization. We have previously demonstrated that during the first 24 h following plating, human epidermal keratinocytes secrete an autocrine-acting mitogenic activity. Since the biological activity of cultured grafts is believed to decrease with cellular age, the effect of both in vivo and in vitro keratinocyte age on the secretion of this mitogenic activity, as well as on responsiveness to this activity, was studied. Keratinocytes from donors ranging in age from 2 to 81 years were analysed at increasing in vitro population doublings. Secretion into the medium of the mitogenic activity was not affected by either in vivo or in vitro cellular ageing, while responsiveness of keratinocytes to this mitogenic activity was age-related. These results suggest that cultured grafts from elderly donors may be effective in wound treatment.

Coexistence of a novel homozygous nonsense mutation in exon 13 of the factor V gene with the homozygous Leiden mutation in two unrelated patients with severe factor V deficiency.

A novel homozygous 3571C-->T nonsense mutation predicting the synthesis of a truncated factor V (FV) molecule was identified in exon 13 of the human coagulation factor V gene in two unrelated Italian probands with undetectable plasma levels of FV antigen and activity. Both patients were also homozygous for the FV Leiden mutation. Reverse transcription polymerase chain reaction studies showed strongly reduced mRNA levels of the mutant FV allele and FV heavy and light chains were not measurable in the plasma of the probands and reverse transcriptase. Haplotype analysis indicated that the nonsense mutation in both families had a common founder a long time ago.

A novel two base pair deletion in the factor V gene associated with severe factor V deficiency.

We studied a family in which the proband, a 13-year-old boy, had unmeasurable plasma levels of coagulation factor V antigen and activity. Clinical symptoms were severe, with several episodes of haemorrhages in the mucosal tracts (gastrointestinal, nose and urinary) and recurrent haemarthroses that caused permanent arthropathy. Sequence analysis of the factor V gene demonstrated the presence of a novel 2 base pair (bp) homozygous deletion in exon 13 at positions 2833-2834. This mutation, present in the heterozygous state in the asymptomatic mother and absent in the healthy brother, introduced a frameshift and a premature stop at codon 900. This would predict the synthesis of a truncated factor V molecule, lacking part of the B domain and the complete light chain. Because of the existence of a surveillance mechanism that selectively recognizes and degrades mRNA molecules carrying premature termination codons, we analysed the relative abundance of mutant vs. wild-type mRNA molecules in the platelets of the heterozygous proband's mother. The mutant mRNA was significantly reduced in amount (mutant/wild-type ratio 0.35). This is the first reported mutation in the factor V gene causing severe factor V deficiency, the effect of which was quantitatively analysed at mRNA level.