Immobility due to COVID-19 confinement, use of contraceptives and presence of the G20210A mutation. Validity of the Virchow triad for the presentation of Deep Venous Thrombosis and Pulmonary Thromboembolism.

Venous thromboembolic disease is a complex and multifactorial pathology, the result of the interaction of both genetic and environmental factors. The Virchow triad, first described in 1859, is still valid to explain the pathogenesis of thrombosis, where three main factors are reflected: a) hypercoagulable state; b) impaired blood flow; c) endothelial injury. The outbreak of a new COVID-19 pandemic has led to drastic confinement measures, with the consequent syndrome of immobility, as occurred in Spain between the months of March to June 2020. Pregnancy and hormonal contraception have proven to be an environmental factor predisposing to venous thrombosis. The presence of genetic factors, such as the mutation of the prothrombin gene G20210A, has been shown to be a risk factor for the presentation of venous thrombosis. We present the case of a 23-year-old non-smoking woman, a heterozygous carrier of a prothrombin gene mutation G20210A (hypercoagulability), who after confinement due to COVID-19 (impaired blood flow due to immobility) and use of hormonal patch contraceptives (endothelial abnormality), triggered deep vein thrombosis (DVT) / pulmonary thromboembolism (PE) that required hospital admission; and who, after rapid withdrawal of anticoagulant treatment, presented a second and a third episode of DVT. We highlight the usefulness of evaluating risk factors in G20210A heterozygous patients and the proper management of anticoagulation to avoid recurrences in patients susceptible to DVT / PE.

Immobility due to COVID-19 confinement, use of contraceptives and presence of the G20210A mutation. Validity of the Virchow triad for the presentation of Deep Venous Thrombosis and Pulmonary Thromboembolism.

Venous thromboembolic disease is a complex and multifactorial pathology, the result of the interaction of both genetic and environmental factors. The Virchow triad, first described in 1859, is still valid to explain the pathogenesis of thrombosis, where three main factors are reflected: a) hypercoagulable state; b) impaired blood flow; c) endothelial injury. The outbreak of a new COVID-19 pandemic has led to drastic confinement measures, with the consequent syndrome of immobility, as occurred in Spain between the months of March to June 2020. Pregnancy and hormonal contraception have proven to be an environmental factor predisposing to venous thrombosis. The presence of genetic factors, such as the mutation of the prothrombin gene G20210A, has been shown to be a risk factor for the presentation of venous thrombosis. We present the case of a 23-year-old non-smoking woman, a heterozygous carrier of a prothrombin gene mutation G20210A (hypercoagulability), who after confinement due to COVID-19 (impaired blood flow due to immobility) and use of hormonal patch contraceptives (endothelial abnormality), triggered deep vein thrombosis (DVT) / pulmonary thromboembolism (PE) that required hospital admission; and who, after rapid withdrawal of anticoagulant treatment, presented a second and a third episode of DVT. We highlight the usefulness of evaluating risk factors in G20210A heterozygous patients and the proper management of anticoagulation to avoid recurrences in patients susceptible to DVT / PE.

The Association Between Parameters of Erythrocytes Morphology and Thrombophilia-Related Mutations

Background: Alterations in erythrocyte morphology parameters have been identified and associated with hematological disorders and other chronic and cardiovascular diseases. Erythrocytes are abundant in thrombus content. Their hemoglobin density and differences in the ratio of macrocytic and microcytic cells may be associated with hypercoagulopathy in those with a history of thrombosis. Objective: This cross-sectional study aimed to investigate the relationship between hemogram parameters and thrombophilia genetic parameters. Method: A total of 55 patients whose thrombophilia panel was reviewed due to the diagnosis of thrombosis were included in the study. %MIC, %MAC, %HPO, %HPR and all hemogram parameters were measured using Abbott Alinity HQ. Prothrombin G20210A, MTHFR C677T, MTHFR A1298C, Factor V Leiden G169A and PAI-1 4G/5G mutations were studied using Real Time-PCR. Results: The MTHFR C677T mutation was detected in 58.2% of the patients. The Factor V Leiden mutation was detected in 5.5% of the patients. The MTHFR A1298C mutation was detected in 58.2%, The PAI mutation was detected in 74.5%, and the Factor 13 mutation was detected in 29% of the patients. Prothrombin G20210A mutation was not detected in any of the patients. Red blood cell (RBC) and Hct values were higher in Factor 13 mutant group; the Hgb and Htc values were higher in the MTHFR C677T mutant group. Conclusion: The MTHFR C677T and Factor 13 mutations may be associated with high Hct and RBC, Hgb, and Htc values, respectively and coagulation tendency in patients with a history of thrombosis.

The Factor II (FII) Expression Quantitative Trait Locus (eQTL) Prothrombin G20210A Is Pleiotropically Associated with Plasma Fibrinogen Levels and Has a Profound Effect on Obesity in Mexican Americans of South Texas

The literature on risk factors for venous thromboembolism (VTE) is replete with complex combinations of genetic and environmental determinants. Here we apply statistical genetic models to data from Mexican Americans of South Texas participating in the San Antonio Family Study (SAFS) to help disentangle some of this complexity. The SAFS has data for nearly 50 large extended pedigrees (Figure A) that are extensively phenotyped, especially for traits related to the pathophysiology of cardiovascular disease. Using a linear mixed model approach, while accounting for age, sex, and their interactions (including age-squared, age-by-sex, and age-squared-by-sex) as confounders, we found significant heritability for plasma FII (46%; N=640; p=6.9E-12) and fibrinogen (28%; N=759; p=1.6E-06) coagulant activity levels and that the Prothrombin G20210A mutation was significantly associated with both traits (FII: p=0.002; fibrinogen: p=0.037; Figure B). We also examined a dichotomous obesity variable based on the Adult Treatment Panel III criterion of sex-specific waist circumferences (>102 cm in males; >88 cm females) denoted as OBWC. Under a threshold and liability model, we found a significant heritability of the liability of OBWC (71%; N=654; p=4.4E-08) and that Prothrombin G20210A was a significant predictor (p=0.031), while still adjusting for age, sex, and their interactions. As can be seen in Figure C, the G20210A mutation profoundly impacts the liability of OBWC such that obesity prevalence, where the prevalence parameter is denoted by Kp in the figure, increases by 27% from individuals homozygous for the major G-allele (G/G) with a prevalence of 34% to heterozygous (G/A) individuals with a prevalence of 61%. To the best of our knowledge, this appears to be the largest single-allele-dose effect for obesity reported in the literature. We next performed bivariate trait analysis (each time accounting for age, sex, and their interactions as confounders) to discover potentially meaningful correlations between the three traits of interest and to see if these would influence their association with G20210A. Under a bivariate model for any two traits, denoted as trait A and B say, the parameters of the following equation are estimated: r_p(A,B) = r_g(A,B)*sqrt(h2_A)*sqrt(h2_B) + r_e(A,B)* sqrt(1-h2_A)*sqrt(1-h2_B), where r_p(A,B), r_g(A,B), and r_e(A,B) respectively denote the phenotypic, genetic, and environmental correlation coefficients of A and B, and where h2_A and h2_B denote the trait heritabilities. Moreover, we can test their significance using likelihood-based inferential statistics. For the FII and OBWC bivariate analysis, none of the correlation coefficients were significant. For the fibrinogen and OBWC bivariate analysis, we found significant phenotypic (r_p=0.24; p=2.0E-05) and genetic (r_g=0.41; p=0.024) correlations. For the FII and fibrinogen bivariate analysis, we found significant phenotypic (r_p=0.26; p=3.1E-10) and environmental (r_e=0.28; p=0.003) correlations. For all bivariate analyses, the Prothrombin G20210A mutation was always a significant predictor for both traits of any given pair. In conclusion, Prothrombin G20210A is pleiotropically associated with plasma FII and fibrinogen coagulant activity levels, and OBWC, and profoundly impacts the prevalence of obesity in our sample. The associations of the prothrombin G20210A mutation are not affected by the consideration of inter-correlations between the three traits examined and thus appears to be fairly robust. Figure 1 Figure 1. Disclosures DeFronzo: AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Intarcia: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding.

Cerebral venous sinus thrombosis: a complication of nitrous oxide abuse

Nitrous oxide (NO) is an inhalant that has become increasingly popular as a recreational drug. While it is presumed to be harmless, a number of adverse effects of NO have been described. We discuss the case of a 24-year-old man with no medical history, who initially presented to the emergency department with progressive polyneuropathy caused by vitamin B12 deficiency after NO abuse. Two days after being discharged with hydroxocobalamin supplementation, the patient returned with a severe headache, blurry vision and slurred speech. Imaging revealed cerebral venous sinus thrombosis. Hypercoagulability workup showed slightly elevated homocysteine and normalised vitamin B12 after supplementation. Genetic testing showed a heterozygous prothrombin G20210A mutation. He was treated with low-molecular-weight heparin followed by dabigatran. We hypothesise that NO use may increase the risk of developing cerebral venous thrombosis, especially in patients with multiple risk factors and elevated homocysteine levels.

Pregnancy complications in G20210A mutation carriers associated with high prothrombin activity

Objective To study the association between high activity of Factor II (prothrombin) in blood plasma with G20210A mutation and the development of great obstetrical syndromes. Material and methods A prospective clinical cohort study was conducted on 290 pregnant women (average age 31.7 ± 4.7 years old). The main group was made up of 140 G20210A patients, while the control group comprised 150 women with the wild G20210G type. The aim was to evaluate the activity of Factor II in the venous blood plasma during the stages of pregnancy with regard to trophoblast invasion waves. As per results, association analysis of Factor II activity value and gestational complications was carried out. Results In the control group, the median (Me) of Factor II activity ranged from 108% (preconception period) to 144% (pregnancy) [95% CI 130–150]. In patients with the GA type, the value was significantly higher in related periods, ranging from 149 to 181% [95% CI 142–195], p < 0.0001. With Factor II activity ranging from 148.5 to 180.6%, pregnancies in the main group had no complications. Higher levels of Factor II activity were associated with the development of early and/or severe preeclampsia (PE) and fetal growth retardation (FGR). Conclusion The data obtained regarding Factor II activity in blood plasma, juxtaposed with the development of great obstetrical syndromes, allow to assume that manifestation of G20210A in early and/or severe PE and FGR is associated with this coagulation factor’s level of activity. Threshold value of the Factor II activity with G20210A mutation, allowing to predict the development of PE, comprised 171.0% at the preconception stage (AUC – 0.86; p < 0.0001) and within 7–8 weeks of gestation it was 181.3% (AUC – 0.84; p < 0.0001).

Carrier frequencies for thrombophilia-related genetic variants in a Romanian cohort

Genetic testing for hereditary thrombophilia, an inherited predisposition to thrombotic events, is increasingly available. To evaluate the rate of positive thrombophilia tests in our laboratory we analyzed the carrier status for common thrombophilia-related gene variants in a consecutive unselected cohort of 360 Romanian patients. Genetic tests were performed on a Real-Time PCR platform. Majority of patients (98.6%) carried at least one thrombophilic variant. The carrier frequencies for classical prothrombotic mutations in F5 (Factor V Leiden) and F2 genes (prothrombin G20210A mutation) were 11.67% (10.27% heterozygous, 1.4% homozygous) and 6.95% (6.39% heterozygous, 0.56% homozygous), respectively. Concurrently, high carrier frequencies for MTHFR c.677C>T, MTHFR c.1298A>C, and PAI-1 4G/5G variants, that are controversially associated with thrombophilia, were observed: 65.28% (52.5% heterozygous, 12.78% homozygous), 53.61% (45% heterozygous, 8.61% homozygous), and 78.61% (49.44% heterozygous, 29.17% homozygous), respectively. The impact of MTHFR genotypes on plasma homocysteine levels was also determined. Male carriers of TT homozygous genotype and CT heterozygous genotype of MTHFR C677T polymorphism had significantly higher levels of plasma homocysteine unrelated to age, compared to those harboring CC homozygous genotype (P=0.028). In unselected patients a high rate of positive thrombophilia tests was observed and the clinical implications of such results need to be carefully examined.

Pregnancy Complications In G20210A Mutation Carriers Associated With High Prothrombin Activity

Objective to study the association between high activity of Factor II (prothrombin) in blood plasma with G20210A mutation and the development of great obstetrical syndromes. Material and methods A prospective clinical cohort study was conducted on 290 pregnant women (average age 31.7±4.7 years old). The main group was made up of 140 G20210A patients, while the control group comprised 150 women with the wild G20210G type. The aim was to evaluate the activity of Factor II in the venous blood plasma during the stages of pregnancy with regard to trophoblast invasion waves. As per results, association analysis of Factor II activity value and gestational complications was carried out. Results In the control group, the median (Me) of Factor II activity ranged from 108% (preconception period) to 144% (pregnancy) [95% CI 130-150]. In patients with the GA type, the value was significantly higher in related periods, ranging from 149% to 181% [95% CI 142-195], p<0.0001. With Factor II activity ranging from 148.5% to 180.6%, pregnancies in the main group had no complications. Higher levels of Factor II activity were associated with the development of early and/or severe preeclampsia (PE) and fetal growth retardation (FGR). Conclusion The data obtained regarding Factor II activity in blood plasma, juxtaposed with the development of great obstetrical syndromes, allow to assume that manifestation of G20210A in early and/or severe PE and FGR is associated with this coagulation factor's level of activity. Threshold value of the Factor II activity with G20210A mutation, allowing to predict the development of PE, comprised 171.0% at the preconception stage (AUC – 0.86; p<0.0001) and within 7-8 weeks of gestation it was 181.3% (AUC – 0.84; p<0.0001).

Thrombophilia Testing Practices: The Mayo Clinic Experience

Introduction: The 2013 ASH Choosing Wisely campaign recommends against thrombophilia testing in patients with major transient risk factors for venous thromboembolism (VTE). Our Special Coagulation Laboratory (SCL) offers an algorithmic approach to thrombophilia testing which includes assays for lupus anticoagulant, dysfibrinogenemia, anticoagulant proteins (protein C, protein S, antithrombin), activated protein C resistance with reflex to factor v Leiden (if indicated), and prothrombin G20210A mutation. Samples are received through Mayo Clinic Laboratories (MCL), national and international reference laboratory (often with limited or no clinical information) and from internal Mayo Clinic practice. We hypothesized that thrombophilia testing would decline in cases where it was recommended against following the publication of testing guidelines. Methods: We audited the external thrombophilia testing samples between2013-2019 and internal samples between 2014-2019 (periods during which they were available). For the internal samples, complete test volumes were only available 2014-2019. Because external clients may either adopt internal testing or contract with a different reference laboratory, many clients may not have been retained over the entire observed period. To better understand the ordering practices of consistent clients, external clients which did not have thrombophilia testing sent to MCL each year of the observed period were excluded. We separated internal ordering practices by hematology and oncology or thrombophilia clinic staff and trainees contrasted with those of other specialties. Results: MCL received 18,529 external thrombophilia testing samples from 322 external healthcare systems during the observed period. From 37 clients, 5,878 (38.2%) samples met inclusion criteria. Annual volume of samples ranged from 890 in 2013 to 861 in 2019 (861-1046). Special coagulation lab processed 11,639 internal thrombophilia tests during the observed periods. There was a consistent small annual increase in testing with 1,398 performed in 2014 and 2,430 in 2019. Of 11651 tests ordered, only 18.6% (2167) were ordered by people most likely to be familiar with ASH choosing wisely campaign. Annual thrombophilia testing ordered by hematology and oncology or thrombophilia clinic staff increased from 307 in 2014 to 387 in 2019 (307-432). However, the ordering practices of these providers as a proportion of overall practices declined from 22.0% (307/1398) in 2014 to 15.9 % (387/2430) in 2019. Table Discussion: Our preliminary data showed no significant trend in thrombophilia ordering practices among included external clients since publication of the ASH Choosing Wisely guidelines on thrombophilia testing. Internally we found a consistent small rise in numbers of thrombophilia tests ordered since 2014 but this may reflect changes in patient volume. We observed that the majority of internal thrombophilia testing was ordered by non-hematology/oncology or thrombophilia providers who are perhaps less likely to be familiar with ASH Choosing Wisely guidelines. The proportion of testing ordered by hematology & oncology or thrombophilia providers declined during the observed period. Our findings are limited by lacking information on the indication and appropriateness for testing as well as possibility of change in patient population. However the overall trend in test volumes and specialty of ordering providers deserves attention and highlight the value in educating other medical societies on ASH Choosing Wisely recommendations for thrombophilia testing. Future work will focus on appropriateness for thrombophilia testing including the indication and time (remote from anticoagulation and acute thrombotic episode), location of testing (inpatient vs. outpatient), as well as investigating if testing has changed patient management, which may help in creating new Choosing Wisely recommendations for thrombophilia testing. Figure 1. Disclosures Pruthi: HEMA Biologics: Honoraria; Bayer Healthcare: Honoraria; Merck: Honoraria; Instrumentation Laboratory: Honoraria; Genentech Inc.: Honoraria; CSL Behring: Honoraria.