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Sperm motility is directly related to the ability of sperm to move through the female reproductive tract to reach the ovum. Sperm motility is a complex trait that is influenced by environmental and genetic factors and is associated with male fertility, oocyte penetration rate, and reproductive success of cattle. In this study we carried out a GWAS in Italian Holstein bulls to identify candidate regions and genes associated with variations in progressive and total motility (PM and TM, respectively). After quality control, the final data set consisted of 5,960 records from 949 bulls having semen collected in 10 artificial insemination stations and genotyped at 412,737 SNPs (call rate95%; minor allele frequency5%). (Co)variance components were estimated using single trait mixed models, and associations between SNPs and phenotypes were assessed using a genomic BLUP approach. Ten windows that explained the greatest percentage of genetic variance were located on Bos taurus autosomes 1, 2, 4, 6, 7, 23, and 26 for TM and Bos taurus autosomes 1, 2, 4, 6, 8, 16, 23, and 26 for PM. A total of 150 genes for TM and 72 genes for PM were identified within these genomic regions. Gene Ontology enrichment analyses identified significant Gene Ontology terms involved with energy homeostasis, membrane functions, sperm-egg interactions, protection against oxidative stress, olfactory receptors, and immune system. There was significant enrichment of quantitative trait loci for fertility, calving ease, immune response, feed intake, and carcass weight within the candidate windows. These results contribute to understanding the architecture of the genetic control of sperm motility and may aid in the development of strategies to identify subfertile bulls and improve reproductive success.

Background: Cerebral venous thrombosis (CVT) is an uncommon cause of stroke in young adults. We aimed to determine the impact of age, gender and risk factors (including sex-specific) on CVT onset. Methods: We used data from the BEAST (Biorepository to Establish the Aetiology of Sinovenous Thrombosis), a multicentre multinational prospective observational study on CVT. Composite factors analysis (CFA) was performed to determine the impact on the age of CVT onset in males and females. Results: A total of 1309 CVT patients (75.3% females) aged ⩾18 years were recruited. The overall median (IQR-interquartile range) age for males and females was 46 (35–58) years and 37 (28–47) years ( p Conclusions: Women suffer CVT 9 years earlier in comparison to men. Female patients with multiple (⩾1) risk factors suffer CVT ~12 years earlier compared to those with no identifiable risk factors.

Donations after circulatory death (DCD) are still challenging in Italy because of prolonged ischemia time (tWIT) due to the law and logistical issues. This cohort study was primarily aimed at assessing the association between successful transplantation and DCD types in the North Italy Transplant program. Adjusted risk ratios (RR) and 95% confidence intervals (CIs) for type III versus type II DCD were estimated using a Poisson regression model with a robust error variance. All consecutive DCD between 2008 and 2020 were included. Among 142 DCD, 102 were eligible for liver donation, and 96 were proposed: 68/69 (99%) and 28/33 (85%) type III and II DCD, respectively. Sixty-nine livers were recovered, 51/68 (75%) from type III and 18/28 (64%) from type II DCD, respectively (RR: 1.18; 95% CI: 0.87-1.60). After ex-vivo perfusion, 50/68 (74%) and 14/28 (50%) livers from type III and type II DCD were transplanted (RR: 1.49; 95% CI: 1.01-2.19). The estimate decreased after further controlling for tWIT (RR: 1.11; 95% CI: 0.55-2.24). Five patients (7.8%) experienced a PNF, 3/50 and 2/14 from type III and type II DCD, respectively. Type III DCD livers were more likely to be transplanted than type II. Warm ischemia time might explain this difference.

Primary brain tumors are associated with an increased risk of pulmonary embolism (PE), particularly in the early post-operative period. The pathophysiological mechanisms of PE are poorly understood. This study aims to describe prospectively extracellular vesicles (EVs) levels and investigate whether or not their variations allow to identify patients at increased risk of post-operative PE. Consecutive meningioma or glioma patients candidate to tumor resection were included in the study if a pulmonary perfusion scan (Q-scan) performed before surgery ruled out PE. EVs derived from platelets (CD41+) or endothelial cells (CD144+), tissue factor-bearing EVs (CD142+) and their procoagulant subtype (annexin V+) were analyzed by flow cytometry before surgery (T0), within 24 h (T1), two (T2) and seven days (T7) after surgery. Q-scan was repeated at T2. Ninety-three patients with meningioma, 59 with glioma and 76 healthy controls were included in the study. CD142+ and annexin V+/CD142+ EVs were increased at T0 in meningioma and glioma patients compared to healthy controls. Twenty-nine meningioma (32%) and 16 glioma patients (27%) developed PE at T2. EVs levels were similar in meningioma patients with or without PE, whereas annexin V+ and annexin V+/CD142+ EVs were significantly higher at T1 and T2 in glioma patients with PE than in those without. Procoagulant EVs, particularly annexin V+/CD142+, increase after surgery and are more prevalent in glioma patients who developed PE after surgery than in those who did not.

Limited data are available on breakthrough COVID- 19 in patients with hematologic malignancy (HM) after anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Adult patients with HM, ≥1 dose of anti-SARS-CoV-2 vaccine, and breakthrough COVID-19 between January 2021 and March 2022 were analyzed. A total of 1548 cases were included, mainly lymphoid malignancies (1181 cases, 76%). After viral sequencing in 753 cases (49%), the Omicron variant was prevalent (517, 68.7%). Most of the patients received ≤2 vaccine doses before COVID-19 (1419, 91%), mostly mRNA-based (1377, 89%). Overall, 906 patients (59%) received COVID-19-specific treatment. After 30-day follow-up from COVID-19 diagnosis, 143 patients (9%) died. The mortality rate in patients with the Omicron variant was 7.9%, comparable to other variants, with a significantly lower 30-day mortality rate than in the prevaccine era (31%). In the univariable analysis, older age (P < .001), active HM (P < .001), and severe and critical COVID-19 (P = .007 and P < .001, respectively) were associated with mortality. Conversely, patients receiving monoclonal antibodies, even for severe or critical COVID-19, had a lower mortality rate (P < .001). In the multivariable model, older age, active disease, critical COVID-19, and 2-3 comorbidities were correlated with a higher mortality, whereas monoclonal antibody administration, alone (P < .001) or combined with antivirals (P = .009), was protective. Although mortality is significantly lower than in the prevaccination era, breakthrough COVID-19 in HM is still associated with considerable mortality. Death rate was lower in patients who received monoclonal antibodies, alone or in combination with antivirals.

Objective Cerebral venous thrombosis (CVT) is an uncommon form of stroke affecting mostly young individuals. Although genetic factors are thought to play a role in this cerebrovascular condition, its genetic etiology is not well understood. Methods A genome-wide association study was performed to identify genetic variants influencing susceptibility to CVT. A 2-stage genome-wide study was undertaken in 882 Europeans diagnosed with CVT and 1,205 ethnicity-matched control subjects divided into discovery and independent replication datasets. Results In the overall case-control cohort, we identified highly significant associations with 37 single nucleotide polymorphisms (SNPs) within the 9q34.2 region. The strongest association was with rs8176645 (combined p = 9.15 x 10(-24); odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.76-2.31). The discovery set findings were validated across an independent European cohort. Genetic risk score for this 9q34.2 region increases CVT risk by a pooled estimate OR = 2.65 (95% CI = 2.21-3.20, p = 2.00 x 10(-16)). SNPs within this region were in strong linkage disequilibrium (LD) with coding regions of the ABO gene. The ABO blood group was determined using allele combination of SNPs rs8176746 and rs8176645. Blood groups A, B, or AB, were at 2.85 times (95% CI = 2.32-3.52, p = 2.00 x 10(-16)) increased risk of CVT compared with individuals with blood group O. Interpretation We present the first chromosomal region to robustly associate with a genetic susceptibility to CVT. This region more than doubles the likelihood of CVT, a risk greater than any previously identified thrombophilia genetic risk marker. That the identified variant is in strong LD with the coding region of the ABO gene with differences in blood group prevalence provides important new insights into the pathophysiology of CVT. ANN NEUROL 2021

Surgery for breast cancer has changed in the last few decades in favor of more conservative approaches, without compromising loco‐regional control and survival. Common immediate complications fol ‐lowing breast surgery are hematoma, seroma, and wound infection.1Traditionally, surgeons have implemented the use of closed‐suction drains in this setting with the aim of preventing these complications, which can cause discomfort, morbidity, increased follow‐up visits,and possible delay in the beginning of adjuvant therapies.2 On the other hand, potential benefits of performing breast procedures without using a drain have been increasingly considered.3 There is lack of modern and official guidelines on use of drains in breast sur ‐gery, with no consensus regarding their management and the appro‐priate use of antibiotics in this setting. Almost all reports agree withthe use of drains after breast procedures, especially if they are asso‐ciated with reconstruction or axillar lymph node dissection (ALND), but an extreme variability is reported regarding practice patterns.4A national survey regarding the management of drains afterbreast cancer surgery was sent by email on January 2018 to all Breast Centers (BC) registered with Senonetwork Italia. Data col ‐lected by the administrative office of Senonetwork were de‐iden‐tified and exported for statistical analysis. A Steering Committeecomposed of general surgeons and plastic surgeons was appointedto coordinate the survey, which consisted of 22 multiple choicequestions, designed to inquire about surgeons' demographics, use of drains after breast procedures with and without reconstructionand/or ALND, antibiotic use and to characterize drain managementin their clinical practice.

Single-antigen bead (SAB) platform permits the identification of antibodies not detectable by complement-dependent lymphocytotoxicity test, but their clinical significance is not completely understood. The aim of this study was to evaluate whether the presence of pretransplant SAB-detected antibodies is associated with the development of allograft failure. This is a single-center cohort study with 10-year follow-up in which 573 kidney recipients with negative pretransplant complement-dependent lymphocytotoxicity crossmatch who received transplants at the Kidney Transplant Center of Policlinico, Milan, from deceased donors between 1996 and 2005 were evaluated. Pretransplant plasma samples were retrospectively analyzed by SAB assay. Survival analyses were performed to assess the risk of allograft failures by SAB-detected antibodies. Pretransplant antibodies were found in 160 (28.0%) recipients, of whom 42 subsequently developed an allograft failure for a survival rate of 70.9% (95% confidence interval [CI), 63.5–78.4). Among those without antibodies, 58 (14.0%) returned to dialysis with a survival rate of 84.7% (95% CI, 81.0–88.4). In Cox regression analyses, patients with SAB-positivity had 2-fold higher risk of allograft failure than those who were SAB-negative (hazard ratio, 2.07; 95% CI, 1.39–2.79). Results did not change after adjustment for putative confounders. In conclusion, in this single-center cohort, 10-year allograft survival rate was significantly influenced by the presence of SAB-detected antibodies.

Background Lung transplantation is an established therapeutic option for patients with end-stage pulmonary disease. In May 2005, the lung allocation score (LAS) was introduced in the United States to maximize the benefit to the recipient population and reduce waiting list mortality. The LAS has been applied in a region of Italy since March 2016 on a provisional basis. The aims of the study were describing waiting list characteristics and short-term outcomes after lung transplantation before and after LAS introduction. Methods All the patients who received transplants between January 1, 2011, and March 15, 2017, were included in our retrospective study. The study population was divided into 2 cohorts (historical cohort and post-LAS cohort) and a comparison among the main perioperative data was performed. Results The historical cohort consisted of 415 patients on the waiting list with 91 deaths and 199 lung transplants; the post-LAS cohort consisted of 134 patients with 10 deaths on the waiting list and 51 transplants. Median waiting time and mortality on the list decreased from 223 to 106 days (P = .03) and from 11.2% to 7.5% (P > .05), respectively. The transplantation rate increased from 25% to 38% (P = .001) and the probability to receive a transplant in the first year in the post-LAS era increased significantly (P = .004). Conclusions The results of the introduction of the LAS system in our region are encouraging and have not shown any adverse short-term effects. The regional coordination decided to prolong the experimental application of LAS in order to accumulate more data and to evaluate medium-term outcomes.

Primary brain tumors are associated with an increased risk of pulmonary embolism (PE), particularly in the early post-operative period. The pathophysiological mechanisms of PE are poorly understood. This study aims to describe prospectively extracellular vesicles (EVs) levels and investigate whether or not their variations allow to identify patients at increased risk of post-operative PE. Consecutive meningioma or glioma patients candidate to tumor resection were included in the study if a pulmonary perfusion scan (Q-scan) performed before surgery ruled out PE. EVs derived from platelets (CD41

INTRODUCTION solid organ transplantation may be considered challenging for waitlist patients during Coronavirus disease 2019 (COVID-19) pandemic. AIM the aim of this study was to investigate COVID-19 incidence and mortality in patients transplanted in the North Italy Transplant program (NITp) area during the outbreak. MATERIALS AND METHODS all consecutive patients transplanted from February 20 to April 3, 2020 (6 weeks) were included in our cohort and followed for at least 4 weeks. Survival analyses were performed. RESULTS one-hundred and twenty-four patients were transplanted with 12 (9.7%) hearts, 4 (3.2%) lungs, 39 (31.4%) livers, 67 (54%) kidneys, 2 (1.6%) combined kidney-pancreas. Recipients’ mean age was 51 years (SD±16.6) and 76/124 (61%) were male. Five (4%) developed COVID-19 after a mean of 13 days (SD±6.7) with a cumulative incidence of 4.0% (CI95%, 0.5-7.5). During the follow-up, 5/124 (4%) recipients died for an overall mortality of 4.3% (CI 95%, 0.6-8.0) of whom only one for COVID-19, for a COVID-19-related mortality of 0.8% (CI95%, 0-6.0). CONCLUSIONS this study showed a low COVID-19 incidence and related-mortality in patients transplanted during the COVID-19 era. Further studies with a longer follow-up are mandatory to confirm the safety of transplant procedures., Highlights • Coronavirus disease 2019 outbreak has unfavourably influenced the overall donation activity • Five (4%) transplanted recipients developed COVID-19 during the outbreak period with a cumulative incidence of 4.0% (CI95%, 0.5-7.5). • COVID-19-related mortality was of 0.8% (CI95%, 0-6.0).

Introduction coronavirus disease 2019 (COVID-19) outbreak has unfavourably influenced the overall donation activity. Aim the aim of this study was to investigate effect of COVID-19 on transplantation in the North Italy Transplant program (NITp). Material and methods this was a cross-sectional study in which all consecutive potential deceased donors proposed in the NITp during 6 weeks from February 21, 2020 (period A) were included and compared to all potential donors during the same time frame of the previous years (period B) and all potential donors during six weeks before February 20, 2020 (period C). Results fifty-eight deceased donors were proposed during period A, 95 during Period B and 128 Period C. After evaluation process 32/58 (55.2%), 60/95 (63.2%) and 79/128 (61.7%) donors were used for organ donation in period A, B and C, respectively (p-value 0.595). Results: this study showed an important reduction of donations and transplants during the COVID-19 pandemic. A strict monitoring should be mandatory to ensure a safety procedure., Highlights • Coronavirus disease 2019 outbreak has unfavourably influenced the overall donation activity • We observed an almost 47-60% of donation reduction during the outbreak period • Donors' decrease led to a 44%-59% reduction in transplant activity ABSTRACT

Essentials The role of cerebral venous thrombosis (CVT) recanalization on neurologic outcome is still debated. We studied a large cohort of 508 CVT patients with 419 patient years of radiological follow-up. Recanalization rate is high during the first months after CVT and neurologic outcome is favorable. High recanalization grade of CVT independently predicts good neurological outcome. Summary: Background Studies with limited sample size and with discordant results described the recanalization time-course of cerebral venous thrombosis (CVT). The neurological outcome after a first episode of CVT is good, but the role of recanalization on neurological dependence is still debated. Objectives The aim of the study is to assess the recanalization rate after cerebral venous thrombosis (CVT) and its prognostic role in long-term neurological outcome. Patients/Methods In a retrospective observational multicenter cohort study, patients with an acute first episode of CVT with at least one available imaging test during follow-up were enrolled. Patency status of the vessels was categorized as complete, partial or not recanalized. Neurological outcome was defined using the modified Rankin scale (mRS) as good (mRS = 0–1) or poor (mRS = 2–6). Results Five-hundred and eight patients (median [IQR] age, 39 [28.5–49] years; 26% male) were included. Complete or partial recanalization was not differently represented in patients undergoing scans at different periods of time (from 28-day to 3 month-period up to a 1–3 year-period). mRS at the time of follow-up imaging was available in 483 patients; 92.8% of them had a mRS of 0-1. CVT recanalization (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.59–4.13) was positively associated, whereas cancer (OR, 0.29; 95% CI, 0.09–0.88), and personal history of venous thromboembolism (VTE) (OR, 0.36; 95% CI, 0.14–0.92) were negatively associated as independent predictors of favorable (mRS = 0–1) outcome at follow-up. Conclusions Most patients with a first CVT had complete or partial recanalization at follow-up. Recanalization was independently associated with a favorable neurological outcome.

Essentials Isolated distal deep vein thrombosis (IDDVT) is frequently associated with cancer. No study has specifically evaluated the long-term clinical course of cancer-associated IDDVT. Patients with cancer-associated IDDVT are at very high risk of symptomatic recurrence and death. We observed low rates of major bleeding during anticoagulation. SummaryBackground Although isolated distal deep vein thrombosis (IDDVT) is frequently associated with cancer, no study has specifically evaluated the long-term clinical course of IDDVT in this setting. Aim To provide data on the rate of recurrent venous thromboembolism (VTE), major bleeding events and death in IDDVT patients with active cancer. Patients and Methods Consecutive patients with active cancer and an objective IDDVT diagnosis (January 2011 to September 2014) were included from our files. We collected information on baseline characteristics, IDDVT location and extension, VTE risk factors, and type and duration of anticoagulant treatment. Results A total of 308 patients (mean age 66.2 [standard deviation (SD), 13.2 years]; 57.1% female) with symptomatic IDDVT and a solid (n = 261) or hematologic (n = 47) cancer were included at 13 centers. Cancer was metastatic in 148 (48.1%) patients. All but three (99.0%) patients received anticoagulant therapy, which consisted of low-molecular-weight heparin in 288 (93.5%) patients. Vitamin K antagonists were used for the long-term treatment in 46 (14.9%) patients, whereas all others continued the initial parenteral agent for a mean treatment duration of 4.2 months (SD, 4.6 months). During a total follow-up of 355.8 patient-years (mean, 13.9 months), there were 47 recurrent objectively diagnosed VTEs for an incidence rate of 13.2 events per 100 patient-years. During anticoagulant treatment, the annual incidence of major bleeding was 2.0 per 100 patient-years. Conclusions Cancer patients with IDDVT have a high risk of VTE recurrence. Additional studies are warranted to investigate the optimal intensity and duration of anticoagulant treatment for these patients.

Background Red cell distribution width (RDW) is a marker of cardiovascular diseases and venous thromboembolism, but its role in cerebral vein thrombosis (CVT) is unknown. Aims To investigate whether high values of RDW are associated with an increased risk of CVT. Methods A case–control study of CVT patients (≥ 18 years-old) referred to our center contrasted with healthy individuals. Odds ratios (ORs) were calculated for RDW values > 90th percentile by multivariable logistic regression and adjusted for demographic characteristics, hemorheological parameters, renal function, fibrinogen and CRP. Quartiles based on the distribution of RDW values were used in an additional model to assess a dose–response relationship. The risk of CVT associated with the combined presence of high RDW and either thrombophilia abnormalities or oral contraceptive use was also estimated. Results 143 cases (median age 36 years, 18–79) and 352 controls (42 years, 18–80) were investigated. RDW values > 90th percentile (> 14.6%) were associated with an increased risk of CVT (OR 2.44, 95% CI 1.39–4.28). The association remained after further adjustment for hemorheological parameters (OR 3.73, 95% CI 1.72–8.09), inflammatory markers (OR 3.77, 95% CI 1.72–8.25) and renal function (OR 3.62, 95% CI 1.53–8.55). The risk appeared restricted to these extreme levels (> 14.6%), as there was no graded association between values of RDW and CVT risk over quartiles. There was a synergistic effect on the risk of CVT for the combination of high RDW and thrombophilia abnormalities (OR 33.20, 95% CI 6.95–158.55) or oral contraceptive use (OR 37.99, 95% CI 8.78–164.45). Conclusions Values of RDW > 90th percentile are associated with CVT.

Spontaneous pregnancy loss and implantation failure after assisted reproductive technologies (ART) are very common occurrences. Although 50–60% of all cases remains unexplained, various predisposing factors, including thrombophilias, have been identified. Thus, the potential benefit of a prophylaxis with low-molecular-weight heparins in improving outcomes has been often investigated over the years. However, the majority of studies are observational and results from randomized clinical trials (RCTs) are inconclusive, probably due to heterogeneity and limited sample size. To cover these unmet needs and to have further data mainly based on the real-life clinical management, we designed these multicenter registries. OTTILIA (Observational sTudy on antiThrombotic prevention in thrombophILIA and pregnancy loss) and FIRST (recurrent Failures in assIsted Reproductive Techniques) registries are two prospective, multicenter, observational studies to evaluate pregnancy or ART outcomes in consecutive women with previous reproductive failures after spontaneous or assisted conception, respectively. All enrolled women are observed from their first visit after positive pregnancy test (OTTILIA) or before commencing a new ART cycle (FIRST) until the end of pregnancy or ART procedure (negative pregnancy test/end of pregnancy, if successful cycle), respectively. Data are collected by means of questionnaires and recorded in a central database. Follow-up investigations are performed during hospital stay, routine clinical follow-up visits or telephone interviews. Primary outcome is live birth rate in the OTTILIA register and clinical pregnancy rate in the FIRST. Although RCTs are the ‘gold standard’ for evaluating treatment outcomes, we believe that our registries represent a valid alternative in improving knowledge on mechanisms involved in reproductive failures and supporting future clinical decisions. NCT 02385461 , retrospectively registered 5 March 2015 (OTTILIA); NCT 02685800 , registered 10 February 2016 (FIRST).

Introduction Oral contraceptive (OC) use increases the risk of venous thromboembolism (VTE), but the effect of duration of use remains to be elucidated. Patients and methods This case-control study was aimed to investigate the duration of OC use on the risk of VTE according to women age, periods of use, prevalence of other risk factors and the role of thrombophilia abnormalities. Seven-hundred patients and 209 controls who used OC were stratified into short users (≤ 1 year), long users (1 to 5 years), and very long users (> 5 years). Results and conclusions Compared to non-users, the odds ratio (OR) for VTE was 9.0 (95% CI 6.9–12.2) in short, 6.5 (95% CI 4.8–83.7) in long and 5.9 (95% CI 4.4–8.1) in very long users. The risk of VTE in short users was highest in women ≤ 30 years and in the first year of use (OR 13.1, 95% CI 7.7–22.4) and decreased afterward (OR 7.7, 95% CI 5.0–11.9). This trend was not observed in women > 30 years. Compared to non-carriers and non-users, a joint effect of thrombophilia abnormalities and OC use on VTE risk was observed particularly in short users (OR 62.2, 95% CI 29.8–129.6), but also afterward (OR 25.4, 95% CI 16.5–39.2). Other transient risk factors for VTE were present in 25% of very long and 16% of short users. In conclusion, the risk of VTE in OC users decreases over time only before 30 years and in first users. Thrombophilia abnormalities strongly interact with the duration of OC use in determining VTE.

Introduction The interest in health-related quality of life (HR-QoL) has increased in the past few years. Aim The aim of this study was to evaluate the HR-QoL before and after transplantation in solid organ recipients referred to the North Italy Transplant program. Material and Methods This cross-sectional study was performed between 2010 and 2011. All consecutive recipients on the waiting list for liver, heart, or kidney transplantation were included and compared to all consecutive transplanted patients at 6 and 24 months of follow-up after transplantation. The HR-QoL was evaluated with the 36-item Short Form Health Survey (SF–36) and the Profile of Mood States (POMS). Questionnaires were self-reported anonymously. Descriptive statistical analyses were performed. Results Four hundred eleven patients were interviewed: 146 patients (35.5%) were on the waiting list, 137 (33.3%) were transplanted 6 months before the interview, and 128 (31.1%) were transplanted 24 months before the interview. Patients on the waiting list had a lower SF–36 score for all items than did transplanted patients after both 6 and 24 months. According to POMS results, patients on the waiting list had a higher prevalence of depression, tension, anger, fatigue, and confusion than did transplanted patients. Conclusions Patients on the waiting list showed a worse quality of life compared to patients after transplantation as demonstrated by SF–36 and POMS results. These findings should be confirmed in a cohort study.

Background Cerebral vein thrombosis (CVT) is a rare, life-threatening disease affecting one adult per 100,000 per year. Genetic risk factors are deficiencies of the natural anticoagulant proteins antithrombin, protein C, protein S or single nucleotide polymorphisms such as factor V Leiden and prothrombin 20210A. In 20% of patients, the cause of CVT remains unknown. Aim To identify novel genetic risk factors for CVT using targeted next-generation DNA sequencing (NGS). Methods We investigated 171 CVT patients and 298 healthy controls. Patients were selected using the following criteria: objective diagnosis of CVT, no active cancer. We performed targeted NGS analysis of the protein-coding regions of 734 candidate genes related to hemostasis and inflammation, 150 ancestry informative markers and 28 thrombosis-associated variants. Results We identified 3723 common and low frequency variants with minor allele frequency (MAF) >1% in 590 genes. Single variant association testing using logistic regression analysis identified rs8176719 insertion/deletion (indel) variant in the ABO gene associated with CVT (age and sex adjusted OR 2.03; 95% CI 1.52–2.73; P = 2.07 × 10−6; Bonferroni P = 0.008). In addition, we identified 8839 rare variants (MAF ≤ 1%) in 723 genes. Gene-based association analysis of these rare variants using a burden test revealed only a tentative association of non-coding variants located in the F8 locus with CVT. Conclusion Targeted NGS identified a common indel variant rs8176719 in the ABO gene. Gene-based tests of association failed to reveal genomic loci with a cumulative burden of rare variants associated with CVT.

After acute proximal deep vein thrombosis (DVT) the thrombotic mass decreases, especially during the first months of anticoagulation. The persistence of residual vein obstruction (RVO) may predict future recurrence in patients with cancer-associated DVT. We aimed to evaluate the proportion of patients with RVO after an episode of cancer associated isolated distal DVT (IDDVT), to identify variables associated with RVO, and to provide initial evidence of its association with recurrent VTE. We performed a post-hoc analysis of a multicenter cohort study of patients with isolated cancer-associated acute IDDVT. We included patients who underwent a control ultrasonography at the end of the anticoagulant treatment between day 30 and day 365 after index IDDVT, given that no recurrent VTE had already occurred on anticoagulant treatment. A total of 153 patients had ultrasonographic follow-up after a median of 92 days from index IDDVT: 45.8% had RVO and 54.2% exhibited complete recanalization. Female sex, Body Mass Index > 30Kg/m2 and involvement of axial calf veins showed the strongest association with RVO. The risk of recurrence was twofold higher in patients with (versus without) RVO. RVO persisted in approximately half of patients with an episode of cancer-associated IDDVT at anticoagulant discontinuation. Patients with RVO appeared to be at a higher risk for recurrent events.

Rivaroxaban, which targets factor Xa and does not reduce proteins C/S, was chosen to treat a 6-year-old girl with homozygous protein S (PS) deficiency who developed skin necrosis while on warfarin. Owing to the lack of experience with rivaroxaban in children, the girl was started with 5 mg once-daily, which was gradually increased to 40 mg daily. The increasing dosage was driven by the need to avoid recurrence of skin necrosis. During dose-escalation, four pharmacokinetics assays were carried out measuring drug plasma concentrations and their effect on hemostatic parameters. We report the laboratory work-up, with special reference to parameters of thrombin-generation. Rivaroxaban concentrations by HPLC were correlated with those by the anti-factor Xa assay (r 2 = 0.92, p < 0.01), but there was an overestimation by HPLC. Thrombin-generation parameters, such as the area under the curve (referred to as ETP), peak-thrombin, and velocity-index, when measured after addition of thrombomodulin, showed unexpected changes: ETP decreased, but peak-thrombin and velocity-index increased. Similar patterns were obtained in a PS-depleted plasma and in plasma from patients with heterozygous PS deficiency, but not in plasma from controls. In conclusion, these preliminary results suggest that PS may be a determinant of velocity and peak-thrombin, but not of the total amount of thrombin generated.

Background An association between high red cell distribution width (RDW) and venous thromboembolism (VTE) has been observed. However, it is not known whether this association differs within various manifestations of VTE, nor if there is an interaction between RDW and thrombophilia abnormalities on the risk of VTE. Aims To investigate whether RDW is a marker of the risk of VTE; to identify subgroups of patients in which the association between RDW and VTE is stronger; to investigate a possible interaction between RDW and thrombophilia abnormalities. Methods Case–control study on 730 patients with a first objectively-confirmed VTE episode (300 unprovoked and 430 provoked) consecutively referred to our Center between 2007 and 2013, and 352 healthy controls. Blood was taken for a thrombophilia work-up and a complete blood count, including RDW, at least three months after VTE. Results Individuals with RDW above the 90 th percentile (> 14.6%) had a 2.5-fold increased risk of VTE compared to those with RDW ≤ 90 th percentile, independently of age, sex, body mass index, other hematological variables and renal function (adjusted odds ratio: 2.52 [95%CI:1.42-4.47]). The risk was similar for unprovoked and provoked VTE, and slightly higher in patients with pulmonary embolism (adjusted odds ratio 3.19 [95%CI:1.68-6.09]) than in those with deep vein thrombosis alone (2.29 [95%CI:1.22-4.30]). No interaction between high RDW and thrombophilia abnormalities on the risk of VTE was observed. Conclusion Our findings confirm RDW as an independent and easily available marker for stratification of the risk of VTE.

Background Sudden sensorineural hearing loss (ISSHL) is idiopathic in 85% of cases and cochlear micro-thrombosis has been hypothesized as pathogenic mechanism. The role of thrombophilia and cardiovascular risk factors in ISSHL is controversial and whether these risk factors influence the clinical outcome of ISSHL is unknown. Methods and patients To investigate the role of thrombophilia and cardiovascular risk factors in ISSHL and to evaluate their influence on clinical outcome of the disease, 118 patients with a first episode of ISSHL and 415 healthy controls were investigated. Thrombophilia screening included measurements of antithrombin, protein C, protein S, factor V Leiden, prothrombin G20210A, antiphospholipid antibodies, fibrinogen, factor VIII and homocysteine. Results Deficiencies of antithrombin, protein C or S taken together, high factor VIII and hyperhomocysteinemia were significantly associated with ISSHL (OR [95%CI]: 7.55 [1.05-54.47], 2.91 [1.31-6.44] and 2.69 [1.09-6.62], respectively), whereas no association was found with the remaining thrombophilia markers. A 2-fold increased risk of poor clinical outcome was observed for every 5 μmol/L increase of fasting homocysteine levels (adjusted OR [95%CI]) 2.13 [1.02-4.44]) until levels of approximately 15 μmol/L, then the risk increased slowly. Cardiovascular risk factors (arterial hypertension, hyperlipidemia, diabetes and smoking) were associated with an increased risk of ISSHL (OR [95%CI] 1.88 [1.17-3.03]) and with a poor clinical outcome (OR [95%CI] 2.22 [0.93-5.26]). Conclusions Hyperhomocysteinemia, high factor VIII and, with more uncertainty, deficiencies of antithrombin, protein C or S and cardiovascular risk factors increase the risk of ISSHL. Hyperhomocysteinemia and cardiovascular risk factors are associated with a poor clinical outcome of ISSHL.

Background The role of thrombophilic and cardiovascular risk factors in different manifestations of retinal vein occlusion (RVO), i.e., central or branch RVO, and at different ages is still debated. Aims To evaluate the association between thrombophilic and cardiovascular risk factors and the risk of RVO (overall, separately for central and branch RVO, and at different ages). Methods Case-control study on 313 patients with a first objectively-confirmed RVO (216 central and 97 branch RVO) and 415 healthy individuals. Results Antithrombin, protein C or protein S deficiency (adjusted odds ratio [95%CI]: 15.60 [2.01–121]; p = 0.009), hyperhomocysteinemia (HHCy; 3.22 [1.38–7.49]; p = 0.007), high factor VIII (FVIII) levels (3.08 [1.20–7.89]; p = 0.019), factor V Leiden (2.93 [0.97–8.86]; p = 0.058) and the presence of at least one cardiovascular risk factor (1.79 [1.00–3.23]; p = 0.050) were associated with an increased risk of branch RVO. The association was weaker for central RVO, and limited to HHCy (2.15 [1.09–4.24]; p = 0.027) and high FVIII (1.99 [0.90–4.42]; p = 0.091). For HHCy, high FVIII and cardiovascular risk factors the association with the risk of RVO was stronger at an age > 50 years (3.41[1.29–8.99], p = 0.013; 2.57[1.00–6.68], p = 0.050; and 2.03[1.16–3.56], p = 0.013, respectively) than ≤ 50 years (1.93[0.85–4.36], p = 0.114; 1.67[0.54–5.12], p = 0.371; and 1.22[0.73–2.03], p = 0.454, respectively), whereas classic inherited thrombophilia (antithrombin, protein C or protein S deficiencies, factor V Leiden and prothrombin G20210A mutation) was slightly more prevalent at an age ≤ 50 years (1.62 [0.76–3.45], p = 0.210) than > 50 years (1.11[0.44–2.79], p = 0.833). Conclusions Thrombophilic and cardiovascular risk factors are associated with RVO, particularly branch RVO. The risk of RVO associated with HHCy, high FVIII and cardiovascular risk factors is higher at an older age.

Venous thromboembolism (VTE), i.e., deep vein thrombosis and pulmonary embolism, are among the main causes of morbidity and mortality in pregnancy with an estimated incidence of 1 per thousand women years. Recommendations on antithrombotic prophylaxis are weak. The aim of the "Pregnancy Health-care Program" (PHP) was to evaluate the individual risk of VTE in a cohort of pregnant women, and manage them with the aim of reducing the rate of VTE. The study was conducted from Jan 2008 to Dec 2010 in the hospital and obstetrical outclinics in Cremona, Italy, and included 1,787 pregnant women who received clinical observation (n = 1,197), above-knee compression stockings (n = 437) or compression stockings and low-molecular-weight heparin (LMWH) (n = 85) depending on their individual VTE risk, evaluated with a risk score (0.5-3.0) derived from the relative risk estimates of a number of risk factors. Visits were scheduled at the end of each trimester and at hospital discharge after delivery. No VTE occurred, but one superficial vein thrombosis was observed 3 days after vaginal delivery in a woman who received clinical observation (0.6 per thousand). No bleeding was observed in LMWH users. The PHP based on stratification of individual VTE risk resulted in a low incidence of VTE events.

BACKGROUND: A bleeding diathesis is a common feature of Noonan syndrome, and various coagulation abnormalities have been reported. Platelet function has never been carefully investigated. METHODS: The degree of bleeding diathesis in a cohort of patients with Noonan syndrome was evaluated by a validated bleeding score and investigated with coagulation and platelet function tests. If ratios of prothrombin time and/or activated partial thromboplastin time were prolonged, the activity of clotting factors was measured. Individuals with no history of bleeding formed the control group. RESULTS: The study population included 39 patients and 28 controls. Bleeding score was ≥2 (ie, suggestive of a moderate bleeding diathesis) in 15 patients (38.5%) and ≥4 (ie, suggestive of a severe bleeding diathesis) in 7 (17.9%). Abnormal coagulation and/or platelet function tests were found in 14 patients with bleeding score ≥2 (93.3%) but also in 21 (87.5%) of those with bleeding score CONCLUSIONS: Nearly 40% of patients with the Noonan syndrome had a bleeding diathesis and >90% of them had platelet function and/or coagulation abnormalities. Results of these tests should be taken into account in the management of bleeding or invasive procedures in these patients.

Purpose The development of donor specific human leukocyte antigen (HLA) antibodies (DSA) has already been identified as a risk factor for graft dysfunction and poor survival in the context of lung transplantation (LuTx), but available evidence is not conclusive. The aim of our study was to evaluate the possibly existing correlation between pre-existing and/or post transplant developing DSA and recipients’ outcome. Methods This was a prospective, cohort study on consecutive LuTx recipients from January 2013 to December 2017. Every individual underwent DSA surveillance with blood specimens collection before and after LuTx. Three groups were defined based on pre-formed antibody setting at time of LuTx: silent, if no antibody was found on their blood; reactive, patients with non-DSA antibodies; and activated, patient with DSA antibodies. Correlation between antibodies panel (before and after LuTx) and outcomes (lung allograft dysfunction and survival) was then investigated. Results 132 patients were considered; 70 (53%) males, median age at LuTx 36 (26; 51) years. Among the “silent” patients: 63% remained negative after LuTx, 20% developed non-DSA and 17% DSA. Among “reactive” recipients: 15% became negative after LuTx, 39% maintained non-DSA and 46.2% developed de-novo DSA. Finally, considering “activated” individuals: the vast majority (80%) maintained pre-transplant DSA and developed new ones, while the remaining 20% lost pre-transplant DSA but showed de-novo non DSA. DSA tended to appear much earlier in pre-transplant allosensitized patients (1 vs. 3 months). Image 1 depicts these results. Finally, both pre and post LuTx DSA showed correlation with the development of chronic lung allograft dysfunction (CLAD) (HR 3.43; 1.19-9.89 and HR 3.35; 1.46-7.68). Conclusion These preliminary findings support the role of both pre-existing and post LuTx developed DSA as a potentially dangerous risk factor for CLAD development, while they did not correlate with acute cellular rejection and/or survival.

We report a case of a 6-year-old girl with severe protein S deficiency due to a homozygous mutation and recurrent episodes of skin necrosis. She developed purpura fulminans at birth and a catheter-related venous thrombosis complicated by massive pulmonary embolism at the sixth day of life. Long-term oral anticoagulant therapy with a vitamin K-antagonist was started with a therapeutic range of the international normalized ratio of prothrombin time between 2.0 and 3.0. Unfortunately, this common range was not sufficient because recurrent episodes of warfarin-induced skin necrosis developed if the international normalized ratio was

Summary. Background: Inherited deficiencies of antithrombin (AT), protein C (PC) and protein S (PS) are risk factors for venous thromboembolism (VTE). They are usually defined by laboratory cut-offs (in our setting 81, 70 and 63 IU dL−1, respectively), which give only a rough idea of the VTE risk associated with plasma levels of these proteins. Objectives: We investigated whether the risk of VTE associated with the plasma deficiencies of AT, PC or PS has a dose–response effect, and whether low borderline levels of these proteins are associated with an increased risk of VTE, both in the whole study population and separately in carriers of either factor V Leiden or G20210A prothrombin gene mutation. Patients/Methods: A case–control study of 1401 patients with a first objectively-documented VTE and 1847 healthy controls has been carried out. Results: A dose–response effect on the VTE risk was observed for all the three anticoagulant proteins. Compared with individuals with AT, PC or PS levels > 100 IU/dL, the adjusted odds ratio (95%CI) of VTE was 2.00 (1.44–2.78) for AT levels between 76 and 85 IUdL−1, 2.21 (1.54–3.18) and 1.84 (1.31–2.59) for PC and PS levels between 61 and 75 IUdL−1. The risk of unprovoked VTE in factor V Leiden or prothrombin G20210A carriers appears 2 to 3-fold increased when levels of AT or PS are low borderline. Conclusions: Low borderline plasma levels of AT, PC and PS are associated with a 2-fold increased risk of VTE and should be considered in the assessment of the individual VTE risk.

Summary. Background: It is currently unclear whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm. Objective: To determine the prevalence of the JAK2 V617F mutation in patients with a first episode of cerebral venous thrombosis. Patients: In this retrospective cohort study, patients with cerebral venous thrombosis were tested for the JAK2 V617F mutation and were followed until the development of a myeloproliferative neoplasm or censored at the end of follow-up. Results: Ten of 152 patients (6.6%) carried the JAK2 V617F mutation. Three of them had known acquired risk factors for thrombosis, and five had thrombophilia. Six patients met the diagnostic criteria for myeloproliferative neoplasm at the time of cerebral venous thrombosis, and three additional patients developed the disease during the follow-up (median duration 7.8 years, range 6 months to 21.3 years), giving an annual incidence of 0.26% patient-years (95% confidence interval 0.05–0.64). The last patient has no evidence of disease after 3 years of follow-up. Patients without the JAK2 V617F mutation at the time of cerebral venous thrombosis were retested at the end of the follow-up and remained negative, with normal blood counts (log-rank test χ2: 159 [P

Background— The clinical course of cerebral sinus-venous thrombosis (CSVT) is largely unknown because prospective studies with a long follow-up and with the goal to assess thrombosis recurrence rate and predisposing factors for recurrence are lacking. Methods and Results— One hundred forty-five patients with a first CSVT were followed up for a median of 6 years after discontinuation of anticoagulant treatment. End points were recurrent CSVT or other clinical manifestations of venous thromboembolism. CSVT recurred in 5 patients (3%) and other manifestations of venous thromboembolism (deep vein thrombosis of the lower limbs or pulmonary embolism) were seen in 10 additional patients (7%), for a recurrence rate of 2.03 per 100 person-years (95% confidence interval, 1.16 to 3.14) for all manifestations of venous thromboembolism and 0.53 per 100 person-years (95% confidence interval, 0.16 to 1.10) for CSVT. Nearly half of the recurrences occurred within the first year after discontinuation of anticoagulant therapy. Risk factors for recurrent venous thrombosis were male sex (adjusted hazard ratio, 9.66; 95% confidence interval, 2.86 to 32.7) and, for thromboses other than CSVT, severe thrombophilia resulting from antithrombin, protein C, protein S deficiency, anti-phospholipid antibodies, or combined abnormalities (adjusted hazard ratio, 4.71; 95% confidence interval, 1.34 to 16.5). Conclusions— The risk of recurrent CSVT is low and is higher in the first year after discontinuation of anticoagulant treatment and among men. Mild thrombophilia abnormalities are not associated with recurrent CSVT, but severe thrombophilia entails an increased risk of deep vein thrombosis of the lower limbs or pulmonary embolism.

Rare mutations in PROC, PROS1 or SERPINC1 as well as common variants in F5, F2, F11 and SERPINC1 have been identified as risk factors for deep vein thrombosis (DVT). To identify novel genetic risk factors for DVT, we have developed and applied next-generation DNA sequencing (NGS) of the coding area of hemostatic and proinflammatory genes. Using this strategy, we previously identified a single nucleotide variant (SNV) rs6050 in the FGA gene and novel, rare SNVs in the ADAMTS13 gene associated with DVT. To identify novel coding variants in the genetic predisposition to DVT, we applied NGS analysis of the coding area of 186 hemostatic and proinflammatory genes in 94 DVT cases and 98 controls and we identified 18 variants with putative role in DVT. A group of 585 Italian idiopathic DVT patients and 550 healthy controls was used to genotype all the 18 risk-associated variants identified by NGS. Replication study in the Italian population identified the rs2232710 variant in the protein Z-dependent protease inhibitor (ZPI) gene to be associated with an increased risk of DVT (OR 2.74; 95% CI 1.33–5.65; P = 0.0045; Bonferroni P = 0.081). However, the rs2232710 SNV showed no association with DVT in two Dutch replication cohorts the LETS study (454 patients and 451 controls) and the MEGA study (3799 patients and 4399 controls), indicating that the rs2232710 variant is not a risk factor for DVT.

In family studies, the risk for venous thromboembolism (VTE) in relatives with factor V Leiden (FVL) or G20210A prothrombin (PT20210A) gene polymorphisms may differ according to genotype and clinical presentation of the proband. To address this hypothesis, a retrospective cohort family study was carried out on 192 kindreds with at least one member with homozygous FVL or PT20210A, for a total of 886 relatives. The proband of the family was heterozygous in 68 and homozygous or with both polymorphisms in 124 kindreds. Twenty-three probands were asymptomatic, 11 had had arterial thrombosis, 7 obstetrical complications, and 151 venous thrombosis (122 VTE and 29 superficial vein thrombosis). The incidence of VTE (per 1000 patient-years) in relatives was higher when the proband had heterozygous rather than homozygous polymorphism (1.25 [95\% confidence interval (CI), 0.73-1.91] vs 0.44 [0.19-0.78]) and when the proband had had VTE instead of other or no clinical manifestations (0.95 [0.57-1.42] vs 0.50 [0.19-0.96]). Compared with relatives belonging to kindreds with homozygous probands without VTE, the adjusted hazard ratio of VTE for relatives selected from kindreds with heterozygous probands with VTE was 4.14 (95\% CI, 1.17-14.71). The genotype and clinical presentation of the proband influence the risk for VTE in relatives with FVL or PT20210A.

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Among the so called antiphospholipid (aPL) antibodies Lupus Anticoagulant (LAC) is considered the strongest risk factor for thromboembolic events. In individuals without a previous thromboembolic event (carriers), LAC is a risk factor when associated with the presence of anticardiolipin (aCL) and aβ2-Glycoprotein I (aβ2GPI) antibodies. On the other hand, data on carriers of isolated LAC positivity are sparse and inconclusive. The aim of this study was to prospectively determine the incidence of thrombosis in a cohort of carriers of isolated LAC positivity. One-hundred seventy-nine carriers of LAC confirmed twelve weeks apart and in a reference laboratory were studied. During a total follow up of 552 person-years, there were seven thromboembolic events (1.3% person-y). All the seven patients had at least one adjunctive major risk factor for thrombosis. The cumulative incidence of thromboembolic events was 3.1% (95% CI 0.6-5.6) after 2years, and 5.9% (95% CI 1.2-10.6) after 5 and 10years. On a multivariate regression analysis considering age, sex, autoimmune disease, risk factors for arterial and venous thrombosis, use of aspirin, only age was found to be an independent predictor of thromboembolic events (HR=1.1, 95% CI 1.0-1.2, p=0.02). These data might be relevant in clinical practice and underline the importance of differentiating LAC carriers in terms of isolated positivity or positivity associated with the presence of antibodies to aCL and β2-glycoprotein I.

Cerebral venous sinus thrombosis (CVST) is a rare life-threatening disease with an estimated annual incidence of three to four cases per million in adults and seven cases per million in neonates. Brain tumors, cerebral infections or traumas, oral contraceptive use, pregnancy, and puerperium are established risk factors for CVST but in 15–20% of patients the disease is apparently unprovoked, i.e., occurring in the absence of predisposing factors. In the last decade there has been increasing evidence that early diagnosis and anticoagulant treatment reduce morbidity of CVST and improve survival. However, the optimal duration of anticoagulant treatment is not well established, because limited information is available on the rate of CVST recurrence after anticoagulant discontinuation. Thrombophilia is a hypercoagulable state leading to a thrombotic tendency, and young patients with CVST, either provoked or unprovoked, deserve investigation. Thrombophilic abnormalities can be inherited (deficiency of the natural anticoagulant proteins antithrombin, protein C, or protein S, mutations in the factor V gene (factor V Leiden) or prothrombin gene (prothrombin G20210A)), acquired (antiphospholipid antibodies), or “mixed,” i.e., either congenital or acquired (hyperhomocysteinemia). The risk of thrombosis is different according to each abnormality. The aim of this chapter is to review the literature about the role of thrombophilia in CVST and to give suggestions for management of the disease.

Five thousand eight hundred and thirty-seven healthy young men, aged 18-19, from two distinct regions of Central Italy, Abruzzo and Marche, have been investigated during the military medical examination to evaluate pigmentary traits and skin phototypes. They were examined over the entire body except the scalp and ano-genital area for pigmented lesions, which included common acquired melanocytic nevi (MN), clinically atypical nevi and solar lentigines. Eye and hair color were also recorded. Information was gathered by self-questionnaire, personal interview and clinical skin examination performed by two independent dermatologists. For sunlight susceptibility and sun exposure, each subject was classified from I to IV skin phototype according to the classification of sun-reactive skin types as proposed by Fitzpatrick. Almost half of the entire population (44.5%) had affirmed that they tanned easily and rarely burned (phototype IV), 10.3% had skin phototype I or II, 11.2% had more than 40 common MN, and 37.2% had atypical nevi with a diameter > or = 5 mm. The common MN and atypical nevi count had a significant association with skin phototype. Subjects with phototype I or II, and solar lentigo had significantly higher nevus counts than individuals without these characteristics.

A retrospective clinical study was carried out to compare results obtained with just endoscopic resection and with adjuvant use of the Bacillus Calmette-Guérin (BCG), epirubicin and mitomycin C after TURB in patients with superficial bladder tumour (stage pTa/1 grade 1-3). At an average follow-up of 44 months, a decrease in the percentage of recurrences was noted in all the adjuvant therapy groups compared to that in the group subjected to TURB only. The risk of recurrence for the BCG group was 0.427, for the epirubicin group 0.609 and for the mitomycin C group 0.668. There was progression of the disease in: 5.6% of the TURB, 9.1% of the BCG, 5% epirubicin and 3.6% mitomycin C, for a total of 26 patients (7.3%). Twenty-four cystectomies were performed and 2 patients died due to progression of the disease. At 44 months average follow-up, 353 (98.6%) patients are alive and 329 (93.2%) still have their own bladder.

Introduction Cerebral venous thrombosis (CVT) is a rare cerebrovascular condition accounting for Methods and analysis To better understand the genetic basis of CVT, we have established an international biobank of CVT cases, Biorepository to Establish the Aetiology of Sinovenous Thrombosis (BEAST) which aims to recruit highly phenotyped cases initially of European descent and later from other populations. To date we have recruited 745 CVT cases from 12 research centres. As an initial step, the consortium plans to undertake a genome-wide association analysis of CVT using the Illumina Infinium HumanCoreExome BeadChip to assess the association and impact of common and low-frequency genetic variants on CVT risk by using a case–control study design. Replication will be performed to confirm putative findings. Furthermore, we aim to identify interactions of genetic variants with several environmental and comorbidity factors which will likely contribute to improve the understanding of the biological mechanisms underlying this complex disease. Ethics and dissemination BEAST meets all ethical standards set by local institutional review boards for each of the participating sites. The research outcomes will be published in international peer-reviewed open-access journals with high impact and visibility. The results will be presented at national and international meetings to highlight the contributions into improving the understanding of the mechanisms underlying this uncommon but important disease. This international DNA repository will become an important resource for investigators in the field of haematological and vascular disorders.

Background Deep vein thrombosis (DVT) genetic predisposition is partially known. Objectives This study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies. Methods Wild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity of recombinant ADAMTS13 were measured by ELISA and FRETS-VWF73 assays, respectively. The replication studies were performed in an Italian case-control study (Milan study; 298/298 patients/controls) using a next-generation sequencing approach and in a Dutch case-control study (MEGA study; 4306/4887 patients/controls) by TaqMan assays. Results In vitro results showed reduced ADAMTS13 activity for three SNVs (p.Val154Ile [15%; 95% confidence interval [CI] 14–16], p.Asp187His [19%; 95%[CI] 17–21], p.Arg421Cys [24%; 95%[CI] 22–26]) similar to reduced plasma ADAMTS13 levels of patients carriers for these SNVs. Therefore these three SNVs were interrogated for risk association. The first replication study identified 3 heterozygous carriers (2 cases, 1 control) of p.Arg421Cys (odds ratio [OR] 2, 95%[CI] 0.18–22.25). The second replication study identified 2 heterozygous carriers (1 case, 1 control) of p.Asp187His ([OR] 1.14, 95%[CI] 0.07–18.15) and 10 heterozygous carriers (4 cases, 6 controls) of p.Arg421Cys ([OR] 0.76, 95%[CI] 0.21–2.68). Conclusions Three SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced ex vivo and in vitro ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT.

Introduction Isolated distal deep vein thrombosis (IDDVT) accounts for one-fourth to one-half of all deep vein thrombosis (DVT) of the leg. Patients with IDDVT are frequently treated for a shorter period of time compared to patients with proximal DVT and/or pulmonary embolism (PE) due to a perceived lower risk of recurrence. About 10-20% of patients with venous thromboembolic events (VTEs) have concomitant cancer. Guidelines recommend long-term anticoagulant treatment in this group of patients due to their high risk of VTE recurrence. Unfortunately, information on the clinical history of IDDVT patients is limited and, to date, no study has evaluated the long-term risk of VTE recurrence in IDDVT patients with cancer. Aim To provide information on the clinical history of IDDVT patients with active cancer. Materials and Methods A multicenter, cohort study including active-cancer patients with an objective diagnosis of IDDVT (between January 2011 and September 2014) was conducted. Information on baseline characteristics, thrombosis location and extension, concomitant risk factors, type and duration of treatment was collected. All patients were followed for a minimum of 12 months and up to 24 months. During follow-up, VTE recurrence, major bleeding episodes and death were registered. Potential risk factors for VTE recurrence were evaluated. Results 308 patients (mean age 66.2 ± 13.2 years, female 57.1%) in 13 centers were included, Table 1; 261 patients had solid cancer and 47 patients hematologic cancer. At the time of IDDVT diagnosis, the disease was metastatic in 148 patients (48.1%); 99.0% of patients received anticoagulant treatment: 288 patients (93.5%) were initially treated with low molecular weight heparin, 15 with fondaparinux (5.2%) and 1 with unfractionated heparin; vitamin K antagonists were used in 46 patients (14.9%) only. Total follow-up was 389 patient-years, mean follow-up 15.2 months. Mean duration of treatment was 4.2 months. During the study period there were 47 episodes of VTE recurrence (36 proximal DVT or PE) for a incidence rate of 13.2 events per 100 patient-years; 7 patients had major bleeding (2.3%) and 137 died (44.5%). At multivariate analysis, previous VTE was associated with an increased risk of recurrence (OR 2.10; 95% 1.06, 4.14), whereas patients with gastrointestinal cancer had a lower risk of recurrence (OR 0.26; 95% CI 0.08, 0.86). Table 1 . Baseline characteristics of the population Number of patients 308 Age (years), mean ± SD 66.2 ± 13.2 Women 176 (57.1%) Men 132 (42.9%) Body-mass index > 30 Kg/m 2 25 (8.1%) Concomitant risk factors for VTE Recent surgery or trauma 79 (25.6%) In-patients/immobilization 45 (14.6%) Prolonged bed rest 47 (15.3%) Local or systemic infections 19 (6.2%) Qualifying distal venous thrombosis Axial calf veins 135 (43.8%) Muscolar calf veins 149 (48.4%) Medial gastrocnemius veins 113 (36.7%) Lateral gastrocnemius veins 45 (14.6%) Soleal veins 73 (23.7%) Bilateral venous thrombosis 22 (7.1%) More than one vein involved 127 (41.2%) Previous history of VTE 45 (14.6%) Family history of VTE 16 (5.2%) Primary cancer site Breast 54 (17.5%) Gastrointestinal 51 (16.6%) Pancreas 18 (5.8%) Hepatic 9 (2.9%) Lung 44 (14.3%) Hematologic 47 (15.3%) Prostate 17 (5.5%) Brain 15 (4.9%) Other 53 (17.2%) Metastases 148 (48.1%) Cancer therapy Systemic chemotherapy 174 (56.5%) Radiotherapy 20 (6.5%) Hormonal therapy 36 (11.7%) Anticoagulant therapy 305 (99%) Low-molecular-weight heparin 288 (93.5%) Fondaparinux 16 (5.2%) Unfractionated heparin 1 (0.3%) Heparin-Vitamin K antagonists 46 (14.9%) VTE = venous thromboembolism. Conclusions Cancer patients with IDDVT have a high risk of VTE recurrence. Other studies are warranted to address the adequate management of these patients.

Summary Background The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Rare single-nucleotide variants (SNVs) of the coding areas of hemostatic genes may explain part of this missing heritability. The ADAMTS13 and VWF genes encode two interconnected proteins with fundamental hemostatic functions, the disruption of which may result in thrombosis. Objectives To study the distribution and burden of rare coding SNVs of ADAMTS13 and VWF found by sequencing in cases and controls of DVT. Patients/Methods The protein-coding areas of 186 hemostatic/proinflammatory genes were sequenced by next-generation technology in 94 thrombophilia-negative patients with DVT and 98 controls. Gene-specific information on ADAMTS13 and VWF was used to study the association between DVT and rare coding SNVs of the two genes. Results More than 70 billion base pairs of raw sequence data were produced to sequence the 700-kb target area with a median redundancy of × 45 in 192 individuals. Most of the 4366 SNVs identified were rare and non-synonymous, indicating pathogenetic potential. Rare (frequency of

Cerebral sinus-venous thrombosis (CSVT) is a rare life-threatening disease with an estimated annual incidence of 3–4 cases per million in adults and 7 cases per million in neonates. Brain tumors, cerebral infections and traumas are local risk factors for CSVT, but the commonest encountered risk factors are oral contraceptive use, pregnancy and puerperium that make the disease predominant in female sex. In 15–20 % of patients, the disease remains unprovoked, i.e., occurring in the absence of predisposing factors. Thrombophilic abnormalities either inherited [deficiency of the natural anticoagulant proteins antithrombin, protein C or protein S, mutations in the factor V gene (factor V Leiden) or prothrombin gene (prothrombin G20210A)] or acquired (antiphospholipid antibodies) are worthy to be investigated in patients with CSVT, as well as hyperhomocysteinemia. In a small proportion of patients, CSVT is the first manifestation of a myeloproliferative neoplasm. The proportion of patients with recurrent CSVT is low, but venous thromboembolism (deep vein thrombosis in the lower limbs or pulmonary embolism) can develop particularly in patients with a first idiopathic CSVT. In the past decade, there has been increasing evidence that early diagnosis and anticoagulant treatment reduce morbidity of CSVT and improve survival. However, the optimal duration of anticoagulant treatment is not well established, because limited information is available on the rate of CSVT recurrence after anticoagulant discontinuation.

Background Next-generation DNA sequencing is opening new avenues for genetic association studies in common diseases that, like deep vein thrombosis (DVT), have a strong genetic predisposition still largely unexplained by currently identified risk variants. In order to develop sequencing and analytical pipelines for the application of next-generation sequencing to complex diseases, we conducted a pilot study sequencing the coding area of 186 hemostatic/proinflammatory genes in 10 Italian cases of idiopathic DVT and 12 healthy controls. Results A molecular-barcoding strategy was used to multiplex DNA target capture and sequencing, while retaining individual sequence information. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples) and enriched for target DNA sequences. Sequencing was performed on ABI SOLiD-4 platforms. We produced > 12 gigabases of raw sequence data to sequence at high coverage (average: 42X) the 700-kilobase target area in 22 individuals. A total of 1876 high-quality genetic variants were identified (1778 single nucleotide substitutions and 98 insertions/deletions). Annotation on databases of genetic variation and human disease mutations revealed several novel, potentially deleterious mutations. We tested 576 common variants in a case-control association analysis, carrying the top-5 associations over to replication in up to 719 DVT cases and 719 controls. We also conducted an analysis of the burden of nonsynonymous variants in coagulation factor and anticoagulant genes. We found an excess of rare missense mutations in anticoagulant genes in DVT cases compared to controls and an association for a missense polymorphism of FGA (rs6050; p = 1.9 × 10-5, OR 1.45; 95% CI, 1.22-1.72; after replication in > 1400 individuals). Conclusions We implemented a barcode-based strategy to efficiently multiplex sequencing of hundreds of candidate genes in several individuals. In the relatively small dataset of our pilot study we were able to identify bona fide associations with DVT. Our study illustrates the potential of next-generation sequencing for the discovery of genetic variation predisposing to complex diseases.