Role of Genetic Thrombophilia Markers in Thrombosis Events in Elderly Patients with COVID-19.

Thrombosis is an extremely dangerous complication in elderly patients with COVID-19. Since the first months of the pandemic, anticoagulants have been mandatory in treatment protocols for patients with COVID-19, unless there are serious contraindications. We set out to discover if genetic thrombophilia factors continue to play a triggering role in the occurrence of thrombosis in patients with COVID-19 with prophylactic or therapeutic anticoagulants. We considered the following genetic markers as risk factors for thrombophilia: G1691A in the FV gene, C677T and A1298C in the MTHFR gene, G20210A and C494T in the FII gene, and (-675) 4G/5G in the PAI-I gene. In a cohort of 176 patients, we did not obtain a reliable result indicating a higher risk of thrombotic complications when taking therapeutic doses of anticoagulants in carriers of genetic markers for thrombophilia except the C494T mutation in the FII gene. However, there was still a pronounced tendency to a higher incidence of thrombosis in patients with markers of hereditary thrombophilia, such as FV G1691A and FII G20210A mutations. The presence of the C494T (Thr165Met) allele in the FII gene in this group of patients showed a statistically significant effect of the mutation on the risk of thrombotic complications despite anticoagulant therapy.

Contribution of genotypes in Prothrombin and Factor V Leiden to COVID-19 and disease severity in patients at high risk for hereditary thrombophilia.

Thrombotic and microangiopathic effects have been reported in COVID-19 patients. This study examined the contribution of the hereditary thrombophilia factors Prothrombin (FII) and Factor V Leiden (FVL) genotypes to the severity of COVID-19 disease and the development of thrombosis. This study investigated FII and FVL alleles in a cohort of 9508 patients (2606 male and 6902 female) with thrombophilia. It was observed that 930 of these patients had been infected by SARS-CoV-2 causing COVID-19. The demographic characteristics of the patients and their COVID-19 medical history were recorded. Detailed clinical manifestations were analyzed in a group of cases (n = 4092). This subgroup was age and gender-matched. FII and FVL frequency data of healthy populations without thrombophilia risk were obtained from Bursa Uludag University Medical Genetic Department's Exome Databank. The ratio of males (31.08%; 27.01%) and the mean age (36.85 ± 15.20; 33.89 ± 14.14) were higher among COVID-19 patients compared to non-COVID-19 patients. The prevalence of FVL and computerized tomography (CT) positivity in COVID-19 patients was statistically significant in the thrombotic subgroup (p < 0.05). FVL prevalence, CT positivity rate, history of thrombosis, and pulmonary thromboembolism complication were found to be higher in deceased COVID-19 patients (p < 0.05). Disease severity was mainly affected by FVL and not related to genotypes at the Prothrombin mutations. Overall, disease severity and development of thrombosis in COVID-19 are mainly affected by the variation within the FVL gene. Possible FVL mutation should be investigated in COVID-19 patients and appropriate treatment should be started earlier in FVL-positive patients.

Integrative analyses of genes about venous thromboembolism: An umbrella review of systematic reviews and meta-analyses.

BACKGROUND: In recent years, many studies have found possible links between gene polymorphisms and venous thromboembolism (VTE). By identifying genetic risk factors before facing environmental risk factors such as surgical interventions and COVID-19 vaccination, we could rapidly respond to the risk of VTE. The aim of this study was to perform an umbrella review of genetic variants related to VTE. Integrative gene analysis of VTE was performed to identify critical genetic variations. METHODS: This study conducted an umbrella review of systematic reviews and meta-analyses. All included studies were selected from the PubMed/MEDLINE database. To select eligible studies, the following variables were extracted: first author name; effect size of each study genetic variant; year of publication; the number of studies included in each article; ethnicity, sample size, P values, and heterogeneity estimates. To assess cumulative evidence in genetic epidemiology about effects of gene polymorphisms on VTE, Human Genome Epidemiology Network's Venice criteria were used. Methodological quality assessment was conducted with JBI Critical Appraisal Checklist for Systematic Reviews and Research Syntheses. RESULTS: Genes provided in the present study with genetic variants associated with VTE were FVL (G1691A), Prothrombin (G20210A), MTHFR (C677T, A1298C), PAI-1 (4G/5G), factor VII activating protease (1601G > A), and endothelial protein C receptor (g.6936A_G, c.4600A_G). Among them, variants in FVL, Prothrombin, MTHFR, and PAI-1 showed high significance. Particularly, variants in Prothrombin (G20210A), MTHFR (C677T), and PAI-1 (4G/5G) had more than 2 types of model significance. CONCLUSION: The present study performed a systematic review of genetic variants associated with VTE. Our results could lead to a more comprehensive understanding of VTE etiology. These results could give a strategy of prediagnosis about evaluating individual risks of VTE who might be exposed to environmental risk factors.

Thromboembolic events in deceased patients with proven SARS-CoV-2 infection: Frequency, characteristics and risk factors.

BACKGROUND: Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) results in respiratory syndromes but also in vascular complications such as thromboembolism (TE). In this regard, immunothrombosis, resulting from inflammation in SARS-CoV-2 infected tissues, has been described. Data on TE in COVID-19 are mainly based on clinical observational and/or incomplete autopsy studies. The true burden of TE and the relevance of genetic predisposition, however, have not been resolved. OBJECTIVES: Here, we report on a consecutive cohort of 100 fully autopsied patients deceased by SARS-CoV-2 infections during the first wave of the pandemic (March to April 2020). We investigated the localization of TE, potential clinical risk factors, and the prothrombotic gene mutations, factor V Leiden and prothrombin G20210A, in postmortem blood or tissue samples. RESULTS: TE was found in 43/100 autopsies. 93 % of TE events were venous occlusions, with 23 patients having pulmonary thromboembolism (PT) with or without lower-extremity deep vein thrombosis. Of these, 70 % showed PT restricted to (sub)segmental arteries, consistent with in situ immunothrombosis. Patients with TE had a significantly higher BMI and died more frequently at an intensive care unit. Hereditary thrombophilia factors were not associated with TE. CONCLUSIONS: Our autopsy results show that a significant proportion of SARS-CoV-2 infected patients suffer from TE, affecting predominantly the venous system. Orthotopic peripheral PT was the most frequent finding. Hereditary thrombophilia appears not to be a determinant for TE in COVID-19. However, obesity and the need for intensive care increase the risk of TE in these patients.

Whole blood samples for faster real-time PCR analysis of thrombophilic mutations in SARS-CoV-2 virus positive patients.

High incidence of thrombosis and venous thromboembolism was reported in patients with COVID-19. In this study, we focused on analysis of thrombophilic mutations performed without a standard DNA extraction step. In one hundred of COVID-19 positive outpatients, real-time PCR for Leiden mutation in the FV gene and G20210A mutation in the FII gene was carried out from DNA extracts and modified whole blood samples, and their cycle threshold (Ct) values were evaluated. In the extracts, healthy homozygotes (wt/wt), heterozygotes (M/wt), and homozygous carriers of Leiden mutation (M/M) provided median Ct values of 18.5, 19.4/22.0, and 20.9. In the whole blood, Ct values were 25.3 (wt/wt), 24.8/27.2 (M/wt), and 26.9 (M/M). Median Ct values for G20210A in the extracts were 19.6 for homozygotes (wt/wt), and 19.7/20.4 for heterozygous carriers. The whole blood samples provided Ct values of 23.9 in healthy homozygotes and 26.3/27.2 in heterozygotes for G20210A mutation. No homozygous subjects for G20210A and no double heterozygotes (for Leiden and G20210A mutations) were found. Despite significant differences in the Ct values, genotyping showed complete result concordance of the DNA extracts and the whole blood samples. The integrity and amplificability of DNA molecules in the whole blood samples during 28 days of deep freezing, interrupted by four cycles of thawing, did not significantly change. In conclusion, we demonstrated a new protocol for the detection of the thrombophilic mutations via real time PCR on the modified whole blood of COVID-19 positive patients. The blood modification was reliable, easy, cheap, and saving costs and turnaround time of the whole laboratory process.

A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis.

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.