Gene Dosage of F5 c.3481C>T Stop-Codon (p.R1161Ter) Switches the Clinical Phenotype from Severe Thrombosis to Recurrent Haemorrhage: Novel Hypotheses for Readthrough Strategy

Abstract

Inherited defects in the genes of blood coagulation essentially express the severity of the clinical phenotype that is directly correlated to the number of mutated alleles of the candidate leader gene (e.g., heterozygote vs. homozygote) and of possible additional coinherited traits. The F5 gene, which codes for coagulation factor V (FV), plays a two-faced role in the coagulation cascade, exhibiting both procoagulant and anticoagulant functions. Thus, defects in this gene can be predisposed to either bleeding or thrombosis. A Sanger sequence analysis detected a premature stop-codon in exon 13 of the F5 gene (c.3481C>T; p.R1161Ter) in several members of a family characterised by low circulating FV levels and contrasting clinical phenotypes. The propositus, a 29 y.o. male affected by recurrent haemorrhages, was homozygous for the F5 stop-codon and for the F5 c.1691G>A (p.R506Q; FV-Leiden) inherited from the heterozygous parents, which is suggestive of combined cis-segregation. The homozygous condition of the stop-codon completely abolished the F5 gene expression in the propositus (FV:Ag < 1%; FV:C < 1%; assessed by ELISA and PT-based one-stage clotting assay respectively), removing, in turn, any chance for FV-Leiden to act as a prothrombotic molecule. His father (57 y.o.), characterised by severe recurrent venous thromboses, underwent a complete molecular thrombophilic screening, revealing a heterozygous F2 G20210A defect, while his mother (56 y.o.), who was negative for further common coagulation defects, reported fully asymptomatic anamnesis. To dissect these conflicting phenotypes, we performed the ProC^®Global (Siemens Helthineers) coagulation test aimed at assessing the global pro- and anticoagulant balance of each family member, investigating the responses to the activated protein C (APC) by means of an APC-sensitivity ratio (APC-sr). The propositus had an unexpectedly poor response to APC (APC-sr: 1.09; n.v. > 2.25), and his father and mother had an APC-sr of 1.5 and 2.0, respectively. Although ProC^®Global prevalently detects the anticoagulant side of FV, the exceptionally low APC-sr of the propositus and his discordant severe-moderate haemorrhagic phenotype could suggest a residual expression of mutated FV p.506QQ through a natural readthrough or possible alternative splicing mechanisms. The coagulation pathway may be physiologically rebalanced through natural and induced strategies, and the described insights might be able to track the design of novel treatment approaches and rebalancing molecules.

Keywords: FV Leiden; blood coagulation; cis-segregation; inherited thrombophilia; premature stop-codon (PTC); readthrough.

Figure 1

Full pedigree of the original family. Black symbols refer to the combined F5 cis-defect (i.e., p.R506Q; p.R1161Ter); grey symbols refer to F2 G20210A substitution. The asterisk indicates the haemorrhagic phenotype (III1), the black circle the thrombotic phenotype (II1). Strike-through symbol indicates a dead individual (I1). Laboratory analyses in subject (I1) have not been determined due to his death at the time of investigation; nevertheless, the F5 cis-heterozygosity (i.e., p.R506Q; p.R1161Ter) has been shown after excluding this haplotype in his wife (I2). The arrow indicates the index patient (i.e., propositus). (I3), (I4) and (II2) did not report clinical manifestations ascribable to a positive history of coagulopathy, nevertheless subjects I3 and I2 carry the F5 cis-defect (i.e., p.R506Q; p.R1161Ter) and the F2 G20210A respectively.

Figure 2

Sanger sequencing in F5 exon 13 showing the c.3481C>T stop-codon. (a–d) normal control, heterozygous pattern in the father (II1) and in the mother (II2), and homozygous status in the propositus (III1) respectively. In the nucleotide sequence, Y indicates that both C and T have been detected.

Figure 3

Anticoagulant response of PC, PS and FV assessed as ratio of sensitivity to APC (APC-sr) of different scalar dilutions (0–100%) in comparison to that of FV-Leiden and FVIII. Black, pink and light-blue diamonds (on the left), show the undiluted APC sensitivity ratios of the three family members (II1, II2, and III1, respectively). “FV Leiden” indicates FV 506QQ sample dilution with normal pool plasma, and “FV Leiden 2” indicates FV 506QQ sample dilution in FV-immune-depleted plasma as detailed in the Methods section. Note that, to obtain measurable APTT seconds, the lowest proportion of “FV Leiden 2” in FV-deficient plasma starts at 5% of factor defect (%), similarly the highest dilution of “FV” ends at 95% of factor defect (%), this because both samples cannot yield measurable APTT seconds at FV concentration below 5%. The remaining samples are in the full range (0–100%).