Rosella Mari

Coexistence of antithrombin deficiency, factor V Leiden and hyperhomocysteinemia in a thrombotic family

We report a thrombotic family with combined type I antithrombin deficiency and factor V Leiden (factor V-R506Q) in which the proposita, affected by recurrent venous and arterial thrombosis, was also characterized by mild hyperhomocysteinemia (28 micromol/l; normal <18.5 micromol/l). Her two thrombotic sisters, with normal antithrombin levels and factor V molecules, showed hyperhomocysteinemia (51 and 30 micromol/l, respectively). Four other members of the family had the combined antithrombin/factor V Leiden defect and two of them had thrombosis. The common A223V mutation in the methylenetetrahydrofolate reductase gene, responsible for the thermolabile variant of the enzyme, was found to be heterozygous in the proposita; the two sisters were homozygous and heterozygous, respectively. The heterozygous sister also had a high titre of antiphospholipid antibodies (85 units of immunoglobulin G antiphospholipid antibody/ml). Furthermore, low plasma folate levels were found in the three hyperhomocysteinemic subjects of the family. This family with several prothrombotic defects is a clear example of the polyfactorial nature of thrombophilia.

Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency.

We report 2 asymptomatic homozygotes for the nonsense p.R462X mutation affecting the carboxy-terminus of coagulation factor VII (FVII, 466 aminoacids). FVII levels of 3–5% and 2.7±0.4% were found in prothrombin time-based and activated factor X (FXa) generation assays with human thromboplastins. Noticeably, FVII antigen levels were barely detectable (0.7±0.2%) which suggested a gain-of-function effect. This effect was more pronounced with bovine thromboplastin (4.8±0.9%) and disappeared with rabbit thromboplastin (0.7±0.2%). This suggests that the mutation influences tissue factor/FVII interactions. Whereas the recombinant rFVII-462X variant confirmed an increase in specific activity (~400%), a panel of nonsense (p.P466X, p.F465X, p.P464X, p.A463X) and missense (p.R462A, p.R462Q, p.R462W) mutations of the FVII carboxy-terminus resulted in reduced secretion but normal specific activity. These data provide evidence for counteracting pleiotropic effects of the p.R462X mutation, which explains the asymptomatic FVII deficiency, and contributes to our understanding of the role of the highly variable carboxy-terminus of coagulation serine proteases.

Replacement of the Y450 (c234) phenyl ring in the carboxyl‐terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity

The interplay between impaired protein biosynthesis and/or function caused by missense mutations, particularly in relation to specific protein regions, has been poorly investigated. As model we chose the severe p.Y450C mutation in the carboxyl‐terminal region of coagulation factor IX (FIX) and, by expression of a panel of recombinant variants, demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl‐terminal mutations produce life‐threatening or mild phenotypes in the presence of similarly reduced protein amounts.

Different Anticoagulant Response to Activated Protein C (APC test) and to Agkistrodon Contortix Venom (ACV test) in a Family with FV-R506Q Substitution

To identify the defect(s) responsible for the thrombotic condition affecting a 55-year-old male and his family, we have utilized a new methodological approach (ProC Global®, Istituto Behring, Milan, Italy) to screen the global anticoagulant activity of the protein C pathway, a defect that accounts for the majority of inherited thrombophilias. The test is based on the activation of endogenous protein C in plasma by Protac®, derived from Agkistrodon contortix snake venom (ACV test). Nineteen members of the family were investigated, 11 showed low responsiveness to ACV (normalized ACV ratios < 0.66; normal > 1. 12); in these individuals specific assays of protein C (PC) and protein S (PS) levels and normalized activated protein C ratios (n-APC-r) were performed. A second test evaluating response to APC, using the classic commercial APC test (n-APC-r 1), detected only 10 subjects with abnormal responses : the propositus and two members of the family with n-APC-r 1 values < 0.54, indicating the homozygous state for the R506Q factor V gene mutation, and seven with values ranging 0.69-0.83, consistent with the heterozygous condition (normal > 0.85). Although only ten subjects presented with low n-APC-r 1 values, DNA analysis, in agreement with the ACV test, detected 11 individual with factor V-R506Q substitution (two homozygotes and nine heterozygotes). Thus the classical APC test failed to identify the APC resistance phenotype in two heterozygous subjects whose values were clearly normal (1.05) in the first case and homozygous (0.53) in the second. The ACV test, however, and the modified APC test with test plasma 1/5 diluted in factor V-deficient plasma (n-APC-r 2) completely matched the DNA analysis. A phenotype/genotype correlation was observed in dilutions higher than 1/3 test plasma factor V-deficient plasma. The presence of unknown mechanisms that influence plasma response to exogenous preformed APC (normal at high factor V-deficient plasma dilutions) but not endogenous ACV activated PC was suspected. The suspected low levels of proteins C and S found in several R506Q members of the family were excluded by reassaying the anticoagulant activities at higher plasma dilution ; this supports the known influence of factor V Leiden on functional PC and PS clotting activity. We conclude that the ACV test is appropriate to evaluate the APC resistance condition, but for a firm diagnosis DNA analysis together with the modified APC test are strongly advised even in the presence of unquestionable APC-r values. Key Words: APC resistance-Factor V Leiden-APC test-ACV test-Diagnosis-Inherited thrombophilia.

Resistance to activated protein C and low levels of protein S activity in nine thrombophilic families: a correct diagnosis.

In order to define the thrombophilic conditions related to activated protein C resistance (APC-R) and protein S (PS) deficiency and to detect the possible combination of these defects, we studied nine unrelated patients selected because of low anticoagulant response to APC and reduced PS activity with at least one first degree relative having the same coagulation feature. The mean APC ratio was 0.64 (normal values > 0.80) and range 0.35-0.80. Three of the patients were heterozygous and two were homozygous for the Leiden mutation (FVR506Q); in the remaining four there was no mutation. The mean PS activity was 41.6% and range 32-54 (normal 65-150%). Five of the patients had low PS activity despite normal total and free antigenic levels, and normal activity when measured at higher plasma dilution; these were carrying at least one gene for the Leiden mutation. In the remaining four patients the crossed-immunoelectrophoresis and the Western-blotting analysis showed a type I PS deficiency confirmed by the familial restriction fragment length polymorphism analysis. Thus, four families were diagnosed with the type I PS defect and five with congenital APC-R. No combined PS/FV Leiden or type II PS defect was found. The only defect found was in the anticoagulant PC pathway. We therefore designed a procedure to diagnose thrombophilic conditions related to this pathway. This study indicates that a specific methodological approach must be used to accurately characterize APC-R and PS deficiency and that care is necessary to avoid the possibility of misdiagnosis.

Coagulation Factor XIIIA (F13A1): Novel Perspectives in Treatment and Pharmacogenetics

Factor XIII (FXIII) is a key molecule in the field of blood coagulation and in the last decades it has weakened attention within the field of angiogenesis and tissue repair. FXIII positively influences wound healing in several tissues by exerting multiple plasma and cellular functions. In the field of haemostasis, FXIII cross-links the neo formed fibrin fibers and supports platelet adhesion to the damaged sub-endothelium warranting a solid architecture. In addition, the pro-angiogenic functions of FXIII are directed by the interaction of vascular endothelial growth factor receptor 2 (VEGFR2) and the integrin αVβ3, on the cell membrane, favouring an important step in the formation of granulation tissue at the wound site for optimal tissue healing. Conversely, the same mechanisms could lead to undesired increased neovascularisation, for example in inflammatory bowel disease or in the retinal degenerative pathologies. The classical symptoms of FXIII deficiency span from intracranial haemorrhage to delay bleeding or the staying of chronic wounds in the skin including impaired mucosal healing. In this view, FXIII bridges primary haemostasis, coagulation and definite tissue healing. Another important recently discovered function ascribed to FXIII is its ability to limit bacterial spreading from the lesion by incorporating specific macromolecules addressed to cellular infiltration, favouring in turn cell migration and survival, as observed also in fibrin-heart cultures for stem cell recruitment. In the field of the novel prognostic biomarkers, the monitoring of the residual circulating FXIII level during acute myocardial infarction has been considered predictive of the post-myocardial infarction healing. Accordingly, adequate FXIII levels can drive and predict the prognosis of complex diseases and the outcome of the associated therapies or interventions. In addition, peculiar pharmacogenetics aspects of the FXIII gene are of extraordinary interest. The present review accounts for the recognized role of FXIII in the healing process and gives some examples on how to use it as prognostic biological/ molecular marker or as potential tailored therapeutic molecule in complex diseases.

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

Differential functional readthrough over homozygous nonsense mutations contributes to the bleeding phenotype in coagulation factor VII deficiency.

Essentials Potentially null homozygous Factor(F)7 nonsense mutations are associated to variable bleeding symptoms. Readthrough of p.Ser112X (life‐threatening) and p.Cys132X (moderate) stop codons was investigated. Readthrough‐mediated insertion of wild‐type or tolerated residues produce functional proteins. Functional readthrough over homozygous F7 nonsense mutations contributes to the bleeding phenotype.

The carboxyl-terminal region is NOT essential for secreted and functional levels of coagulation factor X

The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl‐terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion.

Specific factor IX mRNA and protein features favor drug-induced readthrough over recurrent nonsense mutations

Drug-induced readthrough over premature stop codons (PTCs) is a potentially attractive therapy for genetic disorders, but a wide outcome variability has been observed. Through expression studies, we investigated the responsiveness to the readthrough-inducing drug geneticin of 11 rationally selected factor IX (FIX) nonsense mutations, present in 70% (324/469) of hemophilia B (HB) patients with PTCs. Among the predicted readthrough-permissive TGA variants, only 2 (p.W240X and p.R384X) responded with a remarkable rescue of FIX activity. The amounts of rescued full-length FIX protein for the p.W240X (∼9% of recombinant FIX [rFIX]-wild-type [WT]) slightly exceeded activity (5.2 ± 0.6%). FIX antigen for the p.R384X (1.9 ± 0.3%) was remarkably lower than activity (7.5 ± 0.7%). Data indicate novel specific mechanisms producing functional rescue: (1) prevalent reinsertion of the authentic residue (tryptophan), reverting the nonsense effects for the p.W240X, and (2) gain-of-function for the p.R384X, supported by the fourfold increased activity of the most probable readthrough-mediated missense variant (rFIX-R384W). For most PTCs, impaired secretion/function produced by readthrough-mediated amino acid substitutions prevented a significant functional rescue, which requires combinations of favorable FIX messenger RNA (mRNA) sequence and protein features. This rational approach, applicable to other coagulation disorders, helps with interpreting the poor response reported in the few investigated HB patients, and identifies candidate patients eligible for treatment.