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.

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.

Cis-Segregation of c.1171C>T Stop Codon (p.R391*) in SERPINC1 Gene and c.1691G>A Transition (p.R506Q) in F5 Gene and Selected GWAS Multilocus Approach in Inherited Thrombophilia.

Inherited thrombophilia (e.g., venous thromboembolism, VTE) is due to rare loss-of-function mutations in anticoagulant factors genes (i.e., SERPINC1, PROC, PROS1), common gain-of-function mutations in procoagulant factors genes (i.e., F5, F2), and acquired risk conditions. Genome Wide Association Studies (GWAS) recently recognized several genes associated with VTE though gene defects may unpredictably remain asymptomatic, so calculating the individual genetic predisposition is a challenging task. We investigated a large family with severe, recurrent, early-onset VTE in which two sisters experienced VTE during pregnancies characterized by a perinatal in-utero thrombosis in the newborn and a life-saving pregnancy-interruption because of massive VTE, respectively. A nonsense mutation (CGA > TGA) generating a premature stop-codon (c.1171C>T; p.R391*) in the exon 6 of SERPINC1 gene (1q25.1) causing Antithrombin (AT) deficiency and the common missense mutation (c.1691G>A; p.R506Q) in the exon 10 of F5 gene (1q24.2) (i.e., FV Leiden; rs6025) were coinherited in all the symptomatic members investigated suspecting a cis-segregation further confirmed by STR-linkage-analyses [i.e., SERPINC1 IVS5 (ATT)5-18, F5 IVS2 (AT)6-33 and F5 IVS11 (GT)12-16] and SERPINC1 intragenic variants (i.e., rs5878 and rs677). A multilocus investigation of blood-coagulation balance genes detected the coexistence of FV Leiden (rs6025) in trans with FV HR2-haplotype (p.H1299R; rs1800595) in the aborted fetus, and F11 rs2289252, F12 rs1801020, F13A1 rs5985, and KNG1 rs710446 in the newborn and other members. Common selected gene variants may strongly synergize with less common mutations tuning potential life-threatening conditions when combined with rare severest mutations. Merging classic and newly GWAS-identified gene markers in at risk families is mandatory for VTE risk estimation in the clinical practice, avoiding partial risk score evaluation in unrecognized at risk patients.

The Active Metabolite of Warfarin (3'-Hydroxywarfarin) and Correlation with INR, Warfarin and Drug Weekly Dosage in Patients under Oral Anticoagulant Therapy: A Pharmacogenetics Study.

OBJECTIVES: Warfarin oral anticoagulant therapy (OAT) requires regular and frequent drug adjustment monitored by INR. Interindividual variability, drug and diet interferences, and genetics (VKORC1 and CYP2C9) make the maintenance/reaching of stable INR a not so easy task. HPLC assessment of warfarin/enantiomers was suggested as a valid monitoring-tool along with INR, but definite results are still lacking. We evaluated possible correlations between INR, warfarin/3'-hydroxywarfarin, and drug weekly dosage aimed at searching novel alternatives to OAT monitoring. VKORC1/CYP2C9 pharmacogenetics investigation was performed to account for the known influence on warfarin homeostasis. METHODS: 133 OAT patients were recruited and assessed for warfarin/3'-hydroxywarfarin serum levels (HPLC), INR, and VKORC1 and CYP2C9 genotypes. A subgroup of 52 patients were monitored in detail (5 consecutive controls; c0-c4) till the target INR was reached. Correlation analyses were performed in both groups. RESULTS: In the whole OAT group both warfarin and 3'-hydroxywarfarin correlate with INR at comparable degree (r2 = 0.0388 and 0.0362 respectively). Conversely, warfarin weekly dosage better correlates with warfarin than with 3'-hydroxywarfarin (r2 = 0.0975 and r2 = 0.0381 respectively), but considering together warfarin plus 3'-hydroxywarfarin the correlation strongly increased (r2 = 0.1114; p<0.0001). Interestingly, 3'-hydroxywarfarin reached a strong correlation at c4 respect to warfarin (r2 = 0.2157 and r2 = 0.0549; p = 0.0005 and p = 0.0944 respectively) seeming less affected by drug adjustments in the subgroup of 52 patients who started OAT. The multivariate analyses aimed at estimating the true contribution of 3'-hydroxywarfarin on INR value ascribed it the unique significant value (p = 0.0021) in spite of warfarin who lost association. The pharmacogenetics studies confirmed that patients carrying the VKORC1 variant-allele required lower warfarin maintenance dosage and that the combination of VKORC1 and CYP2C9 yielded a warfarin responsive index (WRI) inversely related to the number variant alleles. CONCLUSION: Our results overall suggest that 3'-hydroxywarfarin monitoring could be of great advantage in INR monitoring respect to classical warfarin assessment showing significant contribution also in multivariate analysis. Therefore, additional active metabolites should be recognized and investigated as novel useful indicators.

Factor XIII-A dynamics in acute myocardial infarction: a novel prognostic biomarker?

After acute myocardial infarction (MI) the damaged heart has to be repaired. Factor XIII (FXIII) is considered a key molecule in promoting heart healing. FXIII deficiency was associated to cardiac rupture and anomalous remodelling in MI. During MI, FXIII contributes firstly to the intracoronary thrombus formation and shortly after to heal the myocardial lesion. To quantify the real contribution of FXIII in this process, and to explore its possible prognostic role, we monitored the FXIII-A subunit levels in 350 acute MI patients during the first six days (d0-d5) plus a control at 30-60 days (d30). A one-year follow-up was performed for all the patients. A transient drop in the FXIII-A mean level was noted in the whole cohort of patients (FXIII-Ad0 99.48 ± 30.5 vs FXIII-Ad5 76.51 ± 27.02; p< 0.0001). Interestingly, those who developed post-MI heart failure showed the highest drop (FXIII-Ad5 52.1 ± 25.2) and they already presented with low levels at recruitment. Similarly, those who died showed the same FXIII-A dynamic (FXIII-Ad5 54.0 ± 22.5). Conversely, patients who remained free of major adverse cardiac events, had lower consuming (FXIII-Ad0 103.6 ± 29.1 vs FXIII-Ad5 84.4 ± 24.5; p< 0.0001). Interestingly, the FXIII-A drop was independent from the amount of injury assessed by TnT and CKMB levels. The survival analysis ascribed an increased probability of early death or heart failure inversely related to FXIII-A quartiles (FXIII-A25th< 59.5 %; hazard ratio 4.25; 2.2-5.1; p< 0.0001). Different FXIII-A dynamics and levels could be utilised as early prognostic indicators during acute MI, revealing the individual potential to heal and suggesting tailored treatments to avoid heart failure or its extreme consequence.