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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.

Spontaneous abortion is a pregnancy complication characterized by complex and multifactorial etiology. About 5% of childbearing women are globally affected by early pregnancy loss (EPL) and most of them experience recurrence (RPL). Epigenetic mechanisms and controlled inflammation are crucial for pregnancy maintenance and genetic predispositions may increase the risk affecting the maternal-fetal crosstalk. Combined analyses of global methylation, inflammation and inherited predispositions may contribute to define pregnancy loss etiopathogenesis. LINE-1 epigenetic regulation plays crucial roles during embryo implantation, and its hypomethylation has been associated with senescence and several complex diseases. By analysing a group of 230 women who have gone through pregnancy interruption and comparing those experiencing spontaneous EPL (n = 123; RPL, 54.5%) with a group of normal pregnant who underwent to voluntary interruption (VPI, n = 107), the single statistical analysis revealed significant lower (P < 0.00001) LINE-1 methylation and higher (P < 0.0001) mean cytokine levels (CKs: IL6, IL10, IL17A, IL23) in EPL. Genotyping of the following SNPs accounted for different EPL/RPL risk odds ratio: F13A1 rs5985 (OR = 0.24; 0.06-0.90); F13B rs6003 (OR = 0.23; 0.047-1.1); FGA rs6050 (OR = 0.58; 0.33-1.0); CRP rs2808635/rs876538 (OR = 0.15; 0.014-0.81); ABO rs657152 (OR = 0.48; 0.22-1.08); TP53 rs1042522 (OR = 0.54; 0.32-0.92); MTHFR rs1801133/rs1801131 (OR = 2.03; 1.2-3.47) and FGB rs1800790 (OR = 1.97; 1.01-3.87), although Bonferroni correction did not reach significant outputs. Principal Component Analysis (PCA) and logistic regression disclosed further SNPs positive/negative associations (e.g. APOE rs7412/rs429358; FGB rs1800790; CFH rs1061170) differently arranged and sorted in four significant PCs: PC1 (F13A, methylation, CKs); PC3 (CRP, MTHFR, age, methylation); PC4 (F13B, FGA, FGB, APOE, TP53, age, methylation); PC6 (F13A, CFH, ABO, MTHFR, TP53, age), yielding further statistical power to the association models. In detail, positive EPL risk association was with PC1 (OR = 1.81; 1.33-2.45; P < 0.0001) and negative associations with PC3 (OR = 0.489; 0.37-0.66; P < 0.0001); PC4 (OR = 0.72; 0.55-0.94; P = 0.018) and PC6 (OR = 0.61; 0.46-0.81; P = 0.001). Moreover, significant inverse associations were detected between methylation and CKs levels in the whole group (r IL10  = - 0.22; r IL17A  = - 0.25; r IL23  = - 0.19; r IL6  = - 0.22), and methylation with age in the whole group, EPL and RPL subgroups (r 2 TOT  = 0.147; r 2 EPL  = 0.136; r 2 RPL  = 0.248), while VPI controls lost significance (r 2 VPI  = 0.011). This study provides a valuable multilayer approach for investigating epigenetic abnormalities in pregnancy loss suggesting genetic-driven dysregulations and anomalous epigenetic mechanisms potentially mediated by LINE-1 hypomethylation. Women with unexplained EPL might benefit of such investigations, providing new insights for predicting the pregnancy outcome and for treating at risk women with novel targeted epidrugs., (© 2024. The Author(s).)

Background: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents., Objectives: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy., Methods: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation., Results: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 μM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo., Conclusion: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation., Competing Interests: Declaration of competing interests T.M.H. is cofounder and shareholder of Coagulation Profile BV as well as coinventor on the thermostable inhibitor of contact activation patent WO2013028069A1. The other authors declare no conflicts of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Myocardial infarction (MI) is one of the leading causes of death in Western countries. An early diagnosis decreases subsequent severe complications such as wall remodeling or heart failure and improves treatments and interventions. Novel therapeutic targets have been recognized and, together with the development of direct and indirect epidrugs, the role of non-coding RNAs (ncRNAs) yields great expectancy. ncRNAs are a group of RNAs not translated into a product and, among them, microRNAs (miRNAs) are the most investigated subgroup since they are involved in several pathological processes related to MI and post-MI phases such as inflammation, apoptosis, angiogenesis, and fibrosis. These processes and pathways are finely tuned by miRNAs via complex mechanisms. We are at the beginning of the investigation and the main paths are still underexplored. In this review, we provide a comprehensive discussion of the recent findings on epigenetic changes involved in the first phases after MI as well as on the role of the several miRNAs. We focused on miRNAs function and on their relationship with key molecules and cells involved in healing processes after an ischemic accident, while also giving insight into the discrepancy between males and females in the prognosis of cardiovascular diseases.

Background: Development and worldwide availability of safe and effective vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to fight severe symptoms of coronavirus disease 2019 (COVID-19) and block the pandemic have been a great achievement and stimulated researchers on understanding the efficacy and duration of different vaccine types. Methods: We investigated the levels of anti-SARS-CoV-2 antibodies (IgG) and neutralizing antibodies (NAbs) in 195 healthy adult subjects belonging to the staff of the University-Hospital of Ferrara (Italy) starting from 15 days up to 190 days (about 6 months) after the second dose of the BNT162b2 (Pfizer-BioNTech) mRNA-based vaccine (n = 128) or ChAdOx1 (AstraZeneca) adenovirus-based vaccine (n = 67) using a combined approach of serological and genomics investigations. Results: A strong correlation between IgG and NAb levels was detected during the 190 days of follow-up ( r 2 = 0.807; p < 0.0001) and was confirmed during the first 90 days (T1) after vaccination ( r 2 = 0.789; p = 0.0001) and 91-190 days (T2) after vaccination ( r 2 = 0.764; p = 0.0001) for both vaccine types ( r 2 = 0.842; p = 0.0001 and r 2 = 0.780; p = 0.0001 for mRNA- and adenovirus-based vaccine, respectively). In addition to age ( p < 0.01), sex ( p = 0.03), and type of vaccine ( p < 0.0001), which partially accounted for the remarkable individual differences observed in the antibody levels and dynamics, interesting genetic determinants appeared as significant modifiers of both IgG and NAb responses among the selected genes investigated ( TP53 , rs1042522; APOE , rs7412/rs429358; ABO , rs657152; ACE2 , rs2285666; HLA-A rs2571381/rs2499; CRP , rs2808635/rs876538; LZTFL1 , rs35044562; OAS3 , rs10735079; SLC6A20 , rs11385942; CFH , rs1061170; and ACE1 , ins/del, rs4646994). In detail, regression analysis and mean antibody level comparison yielded appreciable differences after genotype stratification (P 1 and P 2 , respectively, for IgG and NAb distribution) in the whole cohort and/or in the mRNA-based vaccine in the following genes: TP53 , rs1042522 (P 1 = 0.03; P 2 = 0.04); ABO , rs657152 (P 1 = 0.01; P 2 = 0.03); APOE , rs7412/rs429358 (P 1 = 0.0018; P 2 = 0.0002); ACE2 , rs2285666 (P 1 = 0.014; P 2 = 0.009); HLA-A , rs2571381/rs2499 (P 1 = 0.02; P 2 = 0.03); and CRP , rs2808635/rs876538 (P 1 = 0.01 and P 2 = 0.09). Conclusion: High- or low-responsive subjects can be identified among healthy adult vaccinated subjects after targeted genetic screening. This suggests that favorable genetic backgrounds may support the progression of an effective vaccine-induced immune response, though no definite conclusions can be drawn on the real effectiveness ascribed to a specific vaccine or to the different extent of a genotype-driven humoral response. The interplay between data from the polygenic predictive markers and serological screening stratified by demogeographic information can help to recognize the individual humoral response, accounting for ethnic and geographical differences, in both COVID-19 and anti-SARS-CoV-2 vaccinations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gemmati, Longo, Gallo, Silva, Secchiero, Zauli, Hanau, Passaro, Pellegatti, Pizzicotti, Serino, Singh and Tisato.)

Background: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels., Objectives: To investigate the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK)., Methods: We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an analysis of infused FIX PK parameters in patients (n = 30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42)., Results: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants results in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. Pharmacokinetics analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 h, 95% confidence interval 44.3-114.5) in patients (n = 7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, P = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX., Conclusions: FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

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.

In December 2019, a novel severe acute respiratory syndrome (SARS) from a new coronavirus (SARS-CoV-2) was recognized in the city of Wuhan, China. Rapidly, it became an epidemic in China and has now spread throughout the world reaching pandemic proportions. High mortality rates characterize SARS-CoV-2 disease (COVID-19), which mainly affects the elderly, causing unrestrained cytokines-storm and subsequent pulmonary shutdown, also suspected micro thromboembolism events. At the present time, no specific and dedicated treatments, nor approved vaccines, are available, though very promising data come from the use of anti-inflammatory, anti-malaria, and anti-coagulant drugs. In addition, it seems that males are more susceptible to SARS-CoV-2 than females, with males 65% more likely to die from the infection than females. Data from the World Health Organization (WHO) and Chinese scientists show that of all cases about 1.7% of women who contract the virus will die compared with 2.8% of men, and data from Hong Kong hospitals state that 32% of male and 15% of female COVID-19 patients required intensive care or died. On the other hand, the long-term fallout of coronavirus may be worse for women than for men due to social and psychosocial reasons. Regardless of sex- or gender-biased data obtained from WHO and those gathered from sometimes controversial scientific journals, some central points should be considered. Firstly, SARS-CoV-2 has a strong interaction with the human ACE2 receptor, which plays an essential role in cell entry together with transmembrane serine protease 2 (TMPRSS2); it is interesting to note that the ACE2 gene lays on the X-chromosome, thus allowing females to be potentially heterozygous and differently assorted compared to men who are definitely hemizygous. Secondly, the higher ACE2 expression rate in females, though controversial, might ascribe them the worst prognosis, in contrast with worldwide epidemiological data. Finally, several genes involved in inflammation are located on the X-chromosome, which also contains high number of immune-related genes responsible for innate and adaptive immune responses to infection. Other genes, out from the RAS-pathway, might directly or indirectly impact on the ACE1/ACE2 balance by influencing its main actors (e.g., ABO locus, SRY , SOX3, ADAM17 ). Unexpectedly, the higher levels of ACE2 or ACE1/ACE2 rebalancing might improve the outcome of COVID-19 in both sexes by reducing inflammation, thrombosis, and death. Moreover, X-heterozygous females might also activate a mosaic advantage and show more pronounced sex-related differences resulting in a sex dimorphism, further favoring them in counteracting the progression of the SARS-CoV-2 infection., Competing Interests: The authors declare no conflicts of interest.

Emicizumab has been approved in several countries for regular prophylaxis in patients with congenital haemophilia A and FVIII inhibitors because it substantially reduces their bleeding risk and improves quality of life. However, although significantly less frequent, some breakthrough bleeds may still occur while on emicizumab, requiring treatment with bypassing or other haemostatic agents. Thrombotic complications have been reported with the associated use of activated prothrombin complex concentrates. In addition, when surgery/invasive procedures are needed while on emicizumab, their management requires multidisciplinary competences and direct supervision by experts in the use of this agent. Given this, and in order to expand the current knowledge on the use of emicizumab and concomitant haemostatic agents, and reduce the risk of complications in this setting, the Italian Association of Haemophilia Centres (AICE) here provides guidance on the management of breakthrough bleeds and surgery in emergency situations in patients with haemophilia A and inhibitors on emicizumab prophylaxis. This paper has been shared with other National Scientific Societies involved in the field.

Childhood acute lymphoblastic leukemia (ALL) peaks around age 2-4, and in utero genetic epigenetic mother-fetus crosstalk might tune ALL onset during childhood life. Folate genes variably interact with vitamin status on ALL risk and prognosis. We investigated DHFR and MTHFR gene variants in 235 ALL children and their mothers to disclose their role in determining ALL onset age and survival. Pyrosequence of DHFR 19bp ins/del (rs70991108; W/D), MTHFR C677T (rs1801133; C>T), and MTHFR A1298C (rs1801131; A>C) was assessed in children and in 72% of mothers for dyad-analysis comparison. DHFR DD-children had delayed ALL onset compared to WW-children (7.5 ± 4.8 vs. 5.2 ± 3.7 years; P = 0.002) as well as MTHFR 1298 CC-children compared to AA-children (8.03 ± 4.8 vs. 5.78 ± 4.1 years; P = 0.006), and according to the strong linkage disequilibrium between MTHFR 677 T-allele and 1298C-allele, MTHFR TT-children showed early mean age of onset though not significant. Offspring of MTHFR 677 TT-mothers had earlier ALL onset compared to offspring of 677 CC-mothers (5.4 ± 3.3 vs. 7 ± 5.3 years; P = 0.017). DHFR / MTHFR 677 polymorphism combination influenced onset age by comparing DD/CC vs. WW/TT children (8.1 ± 5.7 vs. 4.7 ± 2.1 years; P = 0.017). Moreover, mother-child genotype combination gave 5.5-years delayed onset age in favor of DD-offspring of 677 CC-mothers vs. WW-offspring of 677 TT-mothers, and it was further confirmed including any D-carrier children and any 677 T-carrier mothers ( P = 0.00052). Correction for multiple comparisons maintained statistical significance for DHFR ins/del and MTHFR A1298C polymorphisms. Unexpectedly, among the very-early onset group (<2.89 years; 25th), DD-genotype inversely clustered in children and mothers (4.8% vs. 23.8% respectively), and accordingly ALL offspring of homozygous DD-mothers had increased risk to have early-onset (adjusted OR (odds ratio) = 3.08; 1.1-8.6; P = 0.03). The opposite effect DHFR promoter variant has in tuning ALL onset-time depending on who is the carrier (i.e., mother or child) might suggest a parent-origin-effect of the D-allele or a two-faced epigenetic role driven by unbalanced folate isoform availability during the in-utero leukemogenesis responsible for the wide postnatal childhood ALL latency.

Abdominal adhesions (AA) account for the most common complication of peritoneal surgery with bowel obstruction being the severest problem in the absence of effective predicting biomarkers. Anti-AA-barriers or adhesiolysis did not completely prevent bowel obstruction, although there is evidence they might reduce related complications requiring reoperation. In addition, gender-related predispositions have not been adequately investigated. We explored the role of coagulation Factor XIII (F13A1 and F13B subunit-genes) in patients following laparotomy, mostly median/lower median incision line. Globally, 426 patients (54%,♀), were PCR-SNP-genotyped for FXIIIA V34L (rs5985), FXIIIA P564L (rs5982), FXIIIA Y204F (rs3024477) and FXIIIB H95R (rs6003). Patients' clinical phenotypes were: Group-A (n = 212), those who developed AA, and 55.2% of them developed bowel obstruction (subgroup-A1), the remaining were subgroup-A2; Group B (n = 214) were those who did not develop AA (subgroup-B1; 53.3%) or symptoms/complications (subgroup-B2). Among different laparotomy, colon surgery associated with AA at a major extent (OR = 5.1; 3.24-7.8; P < 0.0001) with different gender scores (♀OR = 5.33; 2.32-12.23; P < 0.0001 and ♂OR = 3.44; 1.58-7.49; P < 0.0001). Among SNPs, P564L (OR = 4.42; 1.45-13.4; P = 0.008) and Y204F (OR = 7.78; 1.62-37.3; P = 0.01) significantly predicted bowel obstruction and survival-analyses yielded interesting gender distinctions (♀HR = 5.28; 2.36-11.8; P = 0.00005; ♂HR = 2.22; 1.31-3.85; P = 0.0034). Active compounds preventing AA belong to the anticoagulant/fibrinolysis areas, suggesting them candidate investigation targets. We identified novel prognostic markers to predict AA/bowel obstruction giving insights to design novel therapeutic and gender prevention programs.

Factor XIIIA (FXIIIA) levels are independent predictors of early prognosis after acute myocardial infarction (AMI) and the Valine-to-Leucine (V34L) single nucleotide polymorphism (SNP) seems associated with lower AMI risk. Since the long-term AMI prognosis merits deeper investigation, we performed an observational study evaluating relationships between FXIIIA residual levels, cardiovascular risk-factors, and inherited genetic predispositions. FXIIIA V34L was genotyped in 333 AMI patients and a five-year follow-up was performed. FXIIIA levels assessed at day-zero (d0) and four days after AMI (d4), and conventional risk factors were analyzed, focusing on the development of major adverse cardiovascular events (MACE). FXIIIA assessed at d0 and d4 was also an independent MACE predictor in the long-term follow-up (FXIIIA d0 , Odds Ratio (OR) = 3.02, 1.79⁻5.1, p = 0.013; FXIIIA d4 , OR = 4.46, 2.33⁻8.55, p = 0.0001). FXIIIA d4 showed the strongest MACE association, suggesting that the FXIIIA protective role is maximized when high levels are maintained for longer time. Conversely, FXIIIA levels stratified by V34L predicted MACE at a lesser extent among L34-carriers (Hazard Risk (HR) VV34 = 3.89, 2.19⁻6.87, p = 0.000003; HR L34-carriers = 2.78, 1.39⁻5.57, p = 0.0039), and V34L did not predict all MACE, only multiple-MACE occurrence ( p = 0.0087). Finally, in survival analysis, heart failure and death differed significantly from stroke and recurrent ischemia ( p = 0.0013), with FXIIIA levels appreciably lower in the former ( p = 0.05). Overall, genetically-determined FXIIIA levels have a significant long-term prognostic role, suggesting that a pharmacogenetics approach might help to select those AMI patients at risk of poor prognosis in the need of dedicated treatments.

Cognitive impairments of different aetiology share alterations in iron and lipid homeostasis with mutual relationships. Since iron and cholesterol accumulation impact on neurodegenerative disease, the associated gene variants are appealing candidate targets for risk and disease progression assessment. In this light, we explored the role of common single nucleotide polymorphisms (SNPs) in the main iron homeostasis genes and in the main lipoprotein transporter gene (APOE) in a cohort of 765 patients with dementia of different origin: Alzheimer's disease (AD) n = 276; vascular dementia (VaD), n = 255; mild cognitive impairment (MCI), n = 234; and in normal controls (n = 1086). In details, four genes of iron homeostasis (Hemochromatosis (HFE: C282Y, H63D), Ferroportin (FPN1: -8CG), Hepcidin (HAMP: -582AG), Transferrin (TF: P570S)), and the three major alleles of APOE (APOE2, APOE3, APOE4) were analyzed to explore causative interactions and synergies. In single analysis, HFE 282Y allele yielded a 3-fold risk reduction in the whole cohort of patients (P<0.0001), confirmed in AD and VaD, reaching a 5-fold risk reduction in MCI (P = 0.0019). The other iron SNPs slightly associated with risk reduction whereas APOE4 allele resulted in increased risk, reaching more than 7-fold increased risk in AD homozygotes (P = 0.001), confirmed to a lower extent in VaD and MCI (P = 0.038 and P = 0.013 respectively) as well as in the whole group (P<0.0001). Comparisons of Mini Mental State Examination (MMSE) among AD showed appreciable lowering in APOE4 carriers (P = 0.038), confirmed in the whole cohort of patients (P = 0.018). In interaction analysis, the HFE 282Y allele completely extinguished the APOE4 allele associated risk. Conversely, the coexistence in patients of a substantial number of iron SNPs accrued the APOE4 detrimental effect on MMSE. Overall, the analysis highlighted how a specific iron-allele burden, defined as different combinations of iron gene variants, might have different effects on cognitive impairment and might modulate the effects of established genetic risk factors such as APOE4. Our results suggest that established genetic risk factors might be affected by specific genetic backgrounds, making patients differently suited to manage iron accumulation adding new genetic insights in neurodegeneration. The recently recognized interconnections between iron and lipids, suggest that these pathways might share more than expected. We therefore extended to additional iron gene variants the newly proposed influencing mechanisms that HFE gene has on cholesterol metabolism. Our results have a strong translational potential promoting new pharmacogenetics studies on therapeutic target identification aimed at optimally tuning brain iron levels.

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

Macular degenerations represent leading causes of central blindness or low vision in developed countries. Most of these severe visual disabilities are due to age-related macular degeneration (AMD) and pathologic myopia (PM), both of which are frequently complicated by subfoveal choroidal neovascularization (CNV). Photodynamic therapy with verteporfin (PDT-V) is still employed for CNV treatment in selected cases or in combined regimen. In Caucasian patients, the common polymorphism G185T of factor XIII-A gene (FXIII-A-G185T; rs5985) has been described as predictor of poor angiographic CNV responsiveness to PDT-V. Nevertheless, the prognostic implications of this pharmacogenetic determinant on long-term visual outcome after a PDT-V regimen have not been evaluated. We retrospectively selected Caucasian patients presenting with treatment-naive CNV and receiving standardized PDT-V protocol for two years. The study population included patients affected by subfoveal CNV secondary to AMD or PM. We assessed the correlations between the polymorphic allele T of FXIII-A-G185T and: (1) total number of photodynamic treatments; and (2) change in visual acuity from baseline to the end of the follow-up period. Considering a total study population of 412 patients with neovascular AMD or PM, the carriers of 185 T-allele of FXIII-A (GT or TT genotype) received a higher number of photodynamic treatments than patients without it (GG wild-type genotype) (p < 0.01; mean number of PDT-V: 5.51 vs. 3.76, respectively). Moreover, patients with 185 T-allele of FXIII-A had a more marked worsening of visual acuity at 24 months than those with the GG-185 wild genotype (p < 0.01; mean difference in logMAR visual acuity: 0.22 vs. 0.08, respectively). The present findings show that the G185T polymorphism of the FXIII-A gene is associated with significant differences in the long-term therapeutic outcomes of patients treated with standardized PDT-V protocol. The comprehensive appraisal of both anti-thrombophilic effects due to FXIII-A G185T variant and photo-thrombotic action of PDT-V toward CNV provides several clues about the rationale of this intriguing pharmacogenetic correlation. Further investigations are warranted to outline the appropriate paradigm for guiding PDT-V utilization in the course of the combined therapeutic protocol for neovascular macular degeneration.

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.

Despite great advances in haemophilia care in the last 20 years, a number of questions on haemophilia therapy remain unanswered. These debated issues primarily involve the choice of the product type (plasma-derived vs. recombinant) for patients with different characteristics: specifically, if they were infected by blood-borne virus infections, and if they bear high or low risk of inhibitor development. In addition, the most appropriate treatment regimen in non-inhibitor and inhibitor patients compel physicians operating at the haemophilia treatment centres (HTCs) to take important therapeutic decisions, which are often based on their personal clinical experience rather than on evidence-based recommendations from published literature data. To know the opinion on the most controversial aspects in haemophilia care of Italian expert physicians, who are responsible for common clinical practice and therapeutic decisions, we have conducted a survey among the Directors of HTCs affiliated to the Italian Association of Haemophilia Centres (AICE). A questionnaire, consisting of 19 questions covering the most important topics related to haemophilia treatment, was sent to the Directors of all 52 Italian HTCs. Forty Directors out of 52 (76.9%) responded, accounting for the large majority of HTCs affiliated to the AICE throughout Italy. The results of this survey provide for the first time a picture of the attitudes towards clotting factor concentrate use and product selection of clinicians working at Italian HTCs., (© 2014 John Wiley & Sons Ltd.)

Summary: Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by a lack or decrease of coagulation factor VIII activity. The molecular diagnosis of HA is challenging and a variety of different mutations have been identified throughout the F8 gene. Our aim was to detect the causative mutation in 266 HA patients from Emilia-Romagna region (Italy) and in all suspected carriers. Molecular analysis of F8 in 201 HA patients (152 index cases) was performed with a combination of several indirect and direct molecular approaches, such as long distance polymerase chain reaction, multiplex ligation-dependent probe amplification, denaturing high performance liquid chromatography and direct sequencing. The analysis revealed 78 different mutations, 23 of which were novel, not having been reported in national or international databases. The detection rate was 100%, 86% and 89% in patients with severe, moderate and mild HA, respectively. The information provided by this registry will be helpful for monitoring the treatment of HA patients in Emilia-Romagna and also for reliable genetic counselling of affected families in the future.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

Thrombosis of the cerebral veins or sinuses is a rare cerebrovascular disorder, which seldom represents a complication of acute promyelocytic leukemia. To the best of our knowledge, it never occurred during treatment with all-trans retinoic acid. We report a case of a 35-year-old woman affected by acute promyelocytic leukemia, who developed massive thrombosis of the cerebral sinuses and veins when she was in complete morphological and molecular remission after all-trans retinoic acid and idarubicin treatment. Anticoagulant therapy contributed to progressive dissolution of the thrombosis as documented by magnetic resonance imaging with complete disappearance of neurological signs without sequelae.

It has been demonstrated recently that coagulation factor XIII (FXIII) plays an extraordinary role in myocardial healing after infarction, improving survival in a mouse model. Common FXIII gene variants (i.e. FXIIIA-V34L and FXIIIB-H95R) significantly influence the molecular activity. To evaluate whether there is a relationship between the two FXIII gene variants and survival in patients after myocardial infarction (MI), V34L and H95R were PCR-genotyped in a cohort of 560 MI cases and follow-up was monitored. Cases with ST-segment elevation MI (STEMI) were 416 (74.3%) and 374 of these were treated with primary percutaneous coronary intervention (PCI) (89.9%). The remaining 144 patients showed non-ST-segment elevation MI (NSTEMI) at enrollment. The combined endpoint was the occurrence of death, re-infarction, and heart failure. Kaplan-Meier analysis at one year yielded an overall rate for adverse events of 24.5% with a lower incidence in the L34-carriers (28.8% vs 17.1%; log-rank, P = 0.00025), similar to that of the 416 STEMI (23.8%) being (28.0% and 16.9%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Primary PCI-group had a slight lower incidence (22.9%) of adverse events (26.8% and 17.1%; VV34- and L34-carriers respectively; log-rank, P = 0.009). During hospitalization, 506 patients received PCI (374 primary PCI and 132 elective PCI). Significance was conserved also in the overall PCI-group (28.6% and 17.8%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Similar findings were observed at 30 days follow-up. Cases carrying both FXIII variants had improved survival rate (log-rank, P = 0.019). On the other hand, minor bleeding complications were found increased in L34-carriers (P = 0.0001) whereas major bleeding complications were not. Finally, more direct evidence on the role of FXIII molecule on survival might come from the fact that despite significant FXIII antigen reductions observed in cases after MI, regardless the FXIII genotype considered, L34-carriers kept almost normal FXIII activity (VV34- vs L34-carriers; P < 0.001). We conclude that FXIII L34-allele improves survival after MI in all the groups analyzed, possibly through its higher activity associated with assumable positive effects on myocardial healing and recovered functions. Genetically determined higher FXIII activity might influence post-MI outcome. This paves the way for using FXIII molecules to improve myocardial healing, recovery of functions, and survival after infarction.

Matrix metalloproteinases (MMPs) are overexpressed in venous leg ulcers, determining a breakdown of the main extracellular matrix (ECM) components owing mainly to collagenase activities, and so playing a crucial role in ulcer pathogenesis. The authors studied the effects of coagulation factor XIII (FXIII), which cross-links collagen and other ECM components, in human fibroblast cultured cells in the presence and in the absence of matrix metalloproteinases from Clostridium histolyticum collagenase. Clostridium collagenase at concentrations of 2.0, 1.0, and 0.5 mg/mL was added to normal human dermal fibroblasts cultured in the presence of 0.0, 1.0, and 5.0 U/mL of FXIII concentrate (Fibrogammin P, Aventis Behring). Cell counting and metabolically active fibroblast evaluation in the cultures were monitored for 72 hours, by means of trypan-blue dye and MTT test, respectively. The MTT test showed that at the highest collagenase concentration (2.0 mg/mL), the cell number decreased more than 95% in 72 hours of treatment and no significant differences were observed regardless of the FXIII concentrations utilized. At lower collagenase concentration (1.0 mg/mL), in absence or in presence of FXIII (1.0 U/mL), the cell number decreased by about 80% in 72 hours. In contrast, in the presence of higher FXIII levels (5.0 U/mL), cells suffered globally significantly less collagenase effects (p = 0.011) and the gain was appreciable at each time tested. Finally, at 0.5 mg/mL of collagenase concentration, in the absence of FXIII, the cell number decreased by about 60% in 72 hours, whereas in presence of FXIII 1.0 U/mL and 5.0 U/mL, cells decreased significantly less, by about 35% and 20%, respectively (p < 0.025 and p < 0.01, respectively). These data were also confirmed by direct cell counting utilizing the trypan-blue test. Factor XIII contrasts effectively the detrimental action of Clostridium collagenases in human fibroblast cultured cells. These results support several in vivo reports about the effectiveness of its topical application in order to enhance the venous ulcer healing processes.

Low Factor XIII (FXIII) activity has been reported in the blood of patients with chronic venous leg ulcer (CVU). In vivo studies have described increased wound healing in CVU patients treated with FXIII concentrate, and in vitro studies have shown increased regenerative capacity in FXIII-treated fibroblasts. In addition, a common G-to-T polymorphism in the FXIIIA-subunit gene (V34L) significantly increases the activity and modifies the cross-linking properties of the FXIII molecule and this variant has been investigated as a protective factor against thrombosis, a recognized risk factor for CVU establishment. Therefore, the role of FXIII levels, FXIII V34L, FVR506Q, and FIIG20210A, common gene polymorphisms in the pathogenesis of CVU was investigated. Ninety-one patients with CVU and 195 healthy controls (91 of them sex- and age-matched) were PCR-genotyped for the FXIIIV34L, FVR506Q, and FIIG20210A substitutions and FXIIIA-subunit levels were determined by immuno-electrophoresis. The extent of the venous ulcer surface in patients was measured by computer software. The allele frequency and the genotype distribution of the FXIII polymorphism did not show significant differences between the whole group of cases and controls as well as prothrombin variants did. On the contrary, the FVR506Q variant (FV Leiden) allele was more frequent in patients, yielding a significant OR value of 5.93 (95 percent CI, 1.83-19.17; p= 0.003). Considering only CVU cases secondary to a post-thrombotic syndrome (n= 24), FV Leiden yielded a greater OR value of 16.08 (95 percent CI, 4.33-59.6; p < 0.0001). When the CVU cases were stratified by the three possible FXIII genotypes, a significant trend toward a lower mean value of the ulcerated area was clearly evident as the number of the polymorphic alleles (L34) increased in the genotype of patients (VV = 11.9 cm(2,)+/- 23.6; VL = 6.1 cm(2,)+/- 6.9; LL = 4.1 cm(2,)+/- 2.8; p= 0.01). On the other hand, FXIIIA antigen levels were similar between CVU cases and matched controls, but 11 percent of cases had FXIII deficiency (FXIIIA

Folate and methionine metabolism is involved in DNA synthesis and methylation processes. Polymorphisms in the genes of folate metabolism enzymes have been associated with some forms of cancer. In a case-control study, we evaluated whether four common polymorphisms in methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MS A2756G), and methionine synthase reductase (MTRR A66G) genes may have a role in altering susceptibility to adult acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). We analyzed DNA of 120 adult ALL, 200 NHL, and 257 healthy control subjects. Individual carrying the MTHFR 677TT genotype showed a 3.6-fold decreased ALL risk [odds ratio (OR) 0.28, 95% confidence interval (95% CI) 0.12-0.72] than wild-types. Similarly, MS 2756GG individuals showed a 5.0-fold decreased ALL risk (OR 0.20, 95% CI 0.02-1.45) than wild-types. In combined results, subjects with the MTHFR 677CT/TT and MS 2756AG/GG genotypes revealed a 3.6-fold ALL risk reduction (OR 0.28, 95% CI 0.14-0.58) and those with the MTHFR 677TT and MTRR 66AG genotypes revealed a 4.2-fold ALL risk reduction (OR 0.24, 95% CI 0.06-0.81). Finally, those with the MS 2756AG/GG and MTRR 66AG/GG genotypes revealed a 2.2-fold ALL risk reduction (OR 0.45, 95% CI 0.10-0.85). Single analysis for NHL did not show any significant difference for all the polymorphisms investigated, but in the low-grade NHL subgroup, we found a 2.0-fold risk reduction for the MTRR 66GG homozygous genotype (OR 0.50, 95% CI 0.25-0.99), which was higher (OR 0.37, 95% CI 0.14-0.85) when analyzed in combination with MS 2756AA genotype. These data are in accordance with the hypothesis that polymorphisms in the genes for folate and methionine metabolism might play a greater role in the occurrence of ALL than NHL by influencing DNA synthesis and/or DNA methylation.

To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC(R) Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85-100 and 30-90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.

The role of a common polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recently investigated as a protective genetic factor against arterial and venous thrombosis. In addition, the less frequent Leu34 allele has been described as a risk factor for intracerebral hemorrhage. We evaluated the prevalence of this polymorphism by PCR in three case-control studies of patients diagnosed as having primary intracerebral hemorrhage (PCH, n = 130), coronary heart diseases (CHD, n = 240; myocardial infarction/no myocardial infarction, 120/120), and cerebrovascular diseases (CVD, n = 240; cerebral infarction/transient ischaemic attack, 120/120). The matched control groups consisted of patients admitted to the hospital without history of vascular disease. In addition, 200 healthy subjects were investigated. The frequency of the mutated allele (Leu34) was higher in patients with PCH than in controls (33.8% vs. 23.1%, P = 0.009) and lower in CHD and CVD patients compared to controls (18.1% vs. 25.2%, P = 0.010 and 17.3% vs. 24.2%, P = 0.011, respectively). Moreover, among the patients with CHD, the Leu34 allele was underrepresented in cases with myocardial infarction than without (12.9% vs. 23.3%, P = 0.004) and than in controls (12.9% vs. 25.2%, P < 0.001). Similar findings were obtained in patients with CVD comparing the cases with cerebral infarction versus cases with transient ischaemic attack (12.5% vs. 22.1%, P = 0.008) and versus controls (12.5% vs. 24.2%, P < 0.001). Finally, considering altogether the groups of ischaemic patients (CHD and CVD, n = 480), it was noted a trend towards a higher mean age of the clinical onset in homozygotes for the Leu allele than in the wild types (P = 0.078). This study indicates that in our population possession of the FXIII Val34Leu mutation predisposes to the occurrence of primary intracerebral hemorrhage and protects against cerebral and myocardial infarction. A wider modulatory role in the progression and onset of atherothrombotic diseases could be ascribed to FXIII Val34Leu., (Copyright 2001 Wiley-Liss, Inc.)

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject--the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations--showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus' uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus' two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents., (Copyright 2001 S. Karger AG, Basel)

Background and Objective: Hyperhomocysteinemia, due to a combination of genetic and environmental factors, is considered to be a risk factor for vascular disease. Individuals with the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR), due to homozygous C677T MTHFR gene mutation, have significantly raised plasma levels of homocysteine and may be at increased risk of vascular disease. However, it is still controversial a direct association between C677T homozygosity and the occurrence of vascular disease is still controversial., Design and Methods: To clarify the contribution of C677T MTHFR mutation in arterial occlusive disease (AOD) or venous thromboembolism (VTE), we performed a case-controlled study including 160 cases with AOD and 180 cases with VTE attending our referral center and compared them with 200 matched healthy controls. MTHFR gene mutation was evaluated by PCR and odds ratios (OR) and the 95% confidence intervals (CI) were used to estimate the risk for venous or arterial thrombosis., Results: There was a high prevalence of homozygotes for the mutated MTHFR allele among the whole group of cases with arterial disease (OR = 2.35, p = 0.001). Considering the AOD cases with and those without associated risk factors for arterial disease separately the difference remained significant only in the latter group (p = 0.168 and P<0.001 respectively). In contrast, the prevalence of mutated homozygotes among the whole group of cases with VTE was not significantly different from that in the control group (OR = 1.67; p = 0.070). Excluding VTE cases with inherited thrombophilia or with circumstantial risk situations the value increased in both subgroups (OR = 2.26; p = 0.006 and OR = 2.03; p = 0.033 respectively). Considering only VTE cases with neither inherited thrombophilia nor circumstantial risk situations the risk increased further (OR = 2.57; p = 0.017)., Interpretation and Conclusions: These data suggest that in selected patients homozygosity for the MTHFR mutation increases the risk of both arterial and venous thromboses and that differences in selection criteria for the patient group may be responsible in part for the controversial association of the MTHFR mutation and vascular disease.

Several studies have indicated that mild to moderate hyperhomocystinemia is a common cause of arterial occlusive disease. Whether hyperhomocystinemia per se is an independent risk factor for vein thromboembolism (VTE) is still somewhat controversial. Both genetic and nutritional factors influence plasma homocysteine levels. Therefore, we evaluated plasma total homocysteine (tHcy), folate, and vitamin B12 levels and established, by polymerase chain reaction, the presence of the C677T mutation (A223V) in the methylenetetrahydrofolate reductase (MTHFR) gene in 220 cases with VTE without well-established prothrombotic defects. As a control group, 220 healthy subjects from the same geographic area as the cases were investigated. Hyperhomocystinemia was defined as a plasma tHcy level above the 95th percentile in the controls (18.05 micromol/L). Hyperhomocystinemia was found in 16% of cases (odds ratio=3.59; P<0.001); deficiencies of folate (<2.47 ng/mL) or vitamin B12 (<165 pg/mL), defined as values below the 5th percentile in controls, were found in 17.7% (P<0.001) and 12.3% (P=0.015) of cases, respectively. The homozygous condition for the MTHFR mutation (VV) was present in 28.2% of cases and 17.7% of controls (odds ratio=1.82; P=0.013). Comparing only the idiopathic forms of VTE (n=80/220; 36.3%) with normal controls, individuals with hyperhomocystinemia, or individuals homozygous for MTHFR mutation increased the odds ratios to 4.03 (P=0.005) and 2.11 (P=0.018), respectively. No statistically significant difference was observed in the MTHFR genotype distribution of cases and controls with hyperhomocystinemia (P=0.386); however, the normal MTHFR genotype (AA) appeared in control subjects only when tHcy levels were below the 80th percentile (10.57 micromol/L) of the distribution, whereas in case patients, it was present at the highest tHcy levels. A strong association between mutated homozygosity (VV), low folate levels, and hyperhomocystinemia was found in both groups. We conclude that in patients with VTE who do not have coexisting prothrombotic defects, hyperhomocystinemia increases the risk of developing idiopathic and venous thrombosis; the homozygous condition for the MTHFR mutation confers a moderate risk but, together with low folate levels, it is the main determinant of mild hyperhomocystinemia in normal and thromboembolic populations.

Identifying a defect affecting the protein C/protein S (PC/PS) anticoagulant system, using a single global test, has recently become possible thanks to a new methodological approach based on the activation of endogenous plasma PC by Protac, derived from Agkistrodon Contortix snake venom (ACV). The introduction of a commercial test (ProC Global), ACV-based, provides a useful tool for the screening of thrombotic patients since the most frequent causes of inherited thrombophilia are found in the PC/PS system. The test provides information only on the global activity of the anticoagulant pathway but not on PC and PS activity or on the factor V related conditions (e.g., FV Leiden). The present study shows that by carrying out the test alternating the presence of PC-, PS-, or FV-deficient plasma and using appropriate amounts of ACV, it is possible to increase the specificity of the test to correctly evaluate respectively the PC or PS activities or the activated protein C resistance condition (APC-R). These simple modifications applied to the original commercial test allow to detect exactly, using a single, basic methodology, the principal defects affecting the PC/PS anticoagulant pathway. Furthermore, carrying out the tests on an automated coagulometer, in combination or not with the classic ProC Global assay, it is possible to use a unique reagent profile to simultaneously investigate in the same or different samples, the PC, PS, and APC-R defect.

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.

Hemostatic parameters of 495 beta-thalassemic patients (421 with thalassemia major and 74 with thalassemia intermedia) were analyzed, to assess their association with the described thrombophilic condition and to verify the role of additional risk factors (e.g. persistent postsplenectomy thrombocytosis, insulin dependent diabetes mellitus, estrogen-progestin treatment and atrial fibrillation). The prevalence of thromboembolic accidents was 5.2% and in four patients (15.3%) inherited or acquired predisposing defects were recognized. The incidence of thromboembolic events and the associated relative risk due to hemocoagulative abnormalities in these patients are discussed.

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.

We studied a patient affected by von Willebrand disease type 2A who experienced several mild bleeding episodes and was characterized by markedly reduced haemostatic parameters. In the exon 28 of von Willebrand factor (vWF) gene a T to C transition at nucleotide 8680, resulting in the missense mutation Leu817Pro, was found in the heterozygous form in the patient and in two affected relatives. As suggested by the presence in platelets of a complete spectrum of vWF multimers as well as by the increased vWF antigen levels and improved haemostasis after DDAVP treatment, the mutation is compatible with normal multimerization, and could be responsible for a reduced stability or an impaired physiological secretion of vWF.

The photometric method of Fickenscher et al. for the determination of factor XIII (FXIII) activity has been used in the study of 35 patients with severe chronic hepatopathy, in comparison with 25 normal subjects. The FXIII proteic fractions a and b were determined by quantitative immuno-electrophoresis after Laurell. The plasmatic FXIII activity, as well as the proteic fractions a and b, were significantly reduced in hepatopatic patients, in comparison to controls, and proportional to the prolongation of prothrombin times. Ratios between functional and immunological levels of FXIII in hepatopatics were similar to those observed in controls. These results confirm the involvement of fibrin stabilization deficiency in the coagulation defect of severe chronic hepatopathies. The correlations between functional and antigenic values are in agreement with the hepatic origin of FXIII. The method of Fickenscher has been proved to be rapid and simple, and it may be useful in the routine study of hepatopathies, for a better knowledge of the role of FXIII deficiency in the complex coagulopathy of liver diseases, as well as of other acquired FXIII deficiencies.

The lupus anticoagulant (LAC) and anticardiolipin antibody (ACA) syndromes require particular therapeutic approaches: thrombotic accidents are an indication for oral anticoagulant therapy (OAT), whereas severe thrombocytopenia may require the special treatments used for immunologic thrombocytopenic purpura (ITP). We describe the case of a 21-year-old male who presented with axillary vein thrombosis associated with LAC and ACA at high titers in December 1990. OAT was begun and, due to repeated episodes of thrombocytopenia, high-dose steroid therapy was later added with success. The daily steroid dose was reduced because of patent hypercortisolism, but the platelet count fell to 4 x 10(9)/L. A bone marrow biopsy was characteristic for ITP. Splenectomy was performed in June 1993, and the platelet count rapidly normalized. Platelet antibodies were always detectable before and after splenectomy. The patient is currently asymptomatic, with platelet counts above 300 x 10(9)/L at one and a half years after splenectomy. This case indicates that ACA-associated thrombocytopenia, like ITP and HIV-related thrombocytopenias, can be successfully treated with steroids and splenectomy, even though different pathogenetic mechanisms are responsible for the antibody-induced platelet consumption.

The authors emphasize the clinical importance of US in evaluating the extent and the topographic relationships of haematomas. Seven haemorrhagic episodes occurred in this group of patients have been studied using US. For retrobulbar haematomas we have utilized a single water bath transducer operating at 5 and 7.5 MHz, while for soft tissue (muscle) we have utilized linear array transducer operating at 3.5 MHz. The haemorrhagic complications consisted of two deep haematomas involving the ileo-psoas muscle, with different evolution; two relapsing haematomas of the soleus muscle followed by different clinical course; two post-traumatic relapsing retrobulbar haematomas which gave rise to severe complications; two haematomas which occurred simultaneously in the right iliac fossa. Our experience suggests that US can give both accurate diagnostic information and useful clues concerning the solution and therapeutic management of haematomas. This leads to a better prognosis, mostly when the instrumental investigation is carried out early.