FV Leiden Mutation Research Articles

P-432 The association of early pregnancy loss and thrombophilia

Abstract Study question Is there a correlation between thrombophilia and the occurrence of pregnancy loss? Summary answer No statistically significant association is observed between thrombophilia and the live births. What is known already The understanding of recurrent pregnancy loss is a subject of controversy, encompassing both its causes and potential treatments and there is currently no consensus regarding the criteria for examining patients experiencing pregnancy loss, the specific tests to be conducted, or the appropriate treatment approaches. The connection between hereditary thrombophilia and pregnancy loss remains unclear. Study design, size, duration Within the timeframe of 01/12/2020 to 01/06/2022, pregnancy loss clinic of a tertiary university hospital received a total of 296 patients who had experienced at least one pregnancy loss. Following examination, these patients were encouraged to return to clinic if they became pregnant again. Among them, 192 patients did become pregnant and sought further care. These patients received treatment based on pregnancy loss clinic’s management algorithm, and their subsequent pregnancies were closely monitored. Participants/materials, setting, methods This prospective study involved 192 patients who, after undergoing examination, experienced subsequent pregnancies. These patients were categorized into two groups based on their obstetric outcomes: live birth and pregnancy loss. The primary focus of the study was to compare hereditary and acquired thrombophilia between these two groups. Additionally, secondary outcomes included a comparison of obstetric history, genetics, uterine anomalies, and results from biochemical and hormonal examinations between the two groups. Main results and the role of chance 150 of 192 patients achieved live birth (78.13%). The prevalence of hereditary thrombophilia mutations showed no significant differences between the two groups. Specifically, in the pregnancy loss group, the FV Leiden mutation rate was 12.5%, compared to 13.7% in the live birth group (p = 0.836). The Prothrombin gene mutation was observed in 7.5% of the pregnancy loss group and 7.25% of the live birth group (p = 1.000). Additionally, the PAI 4G/5G polymorphism was present in 60% of the pregnancy loss group and 52% of the live birth group (p = 0.347), the MTHFR/C677T polymorphism was detected in 62.5% of the pregnancy loss group and 56.5% of the live birth group (p = 0.500), and the MTHFR/1298C polymorphism was identified in 62.5% of the pregnancy loss group and 60.1% of the live birth group (p = 0.788). While antithrombin III levels were comparable between the two groups (p = 0.53), a noteworthy decline in antithrombin III levels was observed with an increasing history of losses: 110.2 for one loss, 106.0 for two losses, and 100.2 for three or more losses (p = 0.033). The homocysteine level was found to be 10.85 μM (range: 6.60-24.80 μM) in the pregnancy loss group and 9.40 μM (range: 4.90-37.00 μM) in the live birth group (p = 0.023). Limitations, reasons for caution A limitation of our study is its single-center nature. Additionally, we did not include a healthy control group with no history of pregnancy loss, preventing a direct comparison between the pregnancy loss group and a group unaffected by such experiences. Wider implications of the findings Our study provides valuable insights into questioning the live birth expectations of women who have experienced pregnancy loss and underscores the significance of thrombophilia in the context of pregnancy loss. Trial registration number not applicable

Survival Advantage Associated with Heterozygous Factor V Leiden Mutation in Sars-Cov-2-Infected K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involves virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associated coagulopathy (CAC) which is driven by cellular and molecular mechanisms that are still unclear. We developed a mouse model based on the K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Plasma analysis revealed virus-induced upregulation of endothelial activation markers and alterations of all the measured coagulation factors' activities, resulting first in increased vascular leakage in the lung on day 5 post-infection, followed by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events on day 7. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection (p.i.). Prothrombin time showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state was also reflected in the alteration of Factor(F) VIII and FX activity increasing to ~150% compared to uninfected controls. FIX showed the most significant alteration and was ~200% of the normal activity on day 3 p.i. and gradually increased over time, reaching ~350%. We also observed an increase in the activity of FV starting 3 days p.i. at ~210% of the normal level and persisting unchanged up to day 7 p.i. As reported during human infections, FV activity increased without significant changes in protein concentration or FVa levels. Natural anticoagulant and fibrinolytic mechanisms were dysregulated during virus infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of plasma protein C concentration which was not associated with any alteration of liver function, while plasma protein S concentration remained unaltered. Given the procoagulant environment generated after SARS-CoV-2 infection, reducing activated protein C (APC) anticoagulant and cytoprotective activities by blocking APC's active site with the SPC-54 monoclonal antibody worsened disease outcome and brought the mortality rate from 50% to 90%. In this observed procoagulant pathological context, where we observe increased activity of FV and where a deficiency of APC functions resulted in increased mortality, it wouldn't be surprising to observe an even more severe phenotype with the addition of APC resistance. To test this idea, we generated K18-hACE2 mice harboring the homologous human FV Leiden (FV L) mutation (R504Q). The Leiden mutation in FV eliminates one of several sites in FVa that are substrates for proteolysis by APC, resulting in diminished inactivation of FVa by APC and preventing the formation of the anticoagulant form of FV that serves as a cofactor for protein S-dependent inactivation of FVIIIa by APC. Upon SARS-CoV-2 infection, the presence of the homozygous FV L mutation in K18-hACE2 mice didn't affect the severity of the disease as measured by body weight loss during the course of the infection, and homozygous mutated mice had the same mortality rate as FV WT mice. Surprisingly, K18-hACE2 male mice carrying a heterozygous FV L mutation were extremely protected from SARS-CoV-2 infection, as their mortality dropped from 50% to only 15%. Histological analysis reveals how heterozygous FV L mice displayed a great reduction in immune infiltrate-associated fibrin deposition, suggesting an unexpected protective role of this prothrombotic mutation in the development of CAC. The protective effect of FV L heterozygosity may be attributable to enhanced thrombin formation which boosts inflammation and enhanced APC generation controlling proinflammatory and prothrombotic signaling, thus showing a delicate balance achieved by FV L heterozygosity that was lost for FV L homozygosity. As seen also in mouse models of bacterial sepsis, such favorable effects associated with FV L support the somewhat counterintuitive speculation that a partial increase in thrombin production in the early phase of the infection may support the resolution of COVID-19 complications.

Congenital thrombophilia in East-Asian venous thromboembolism population: a systematic review and meta-analysis

BackgroundVarious inherited traits contribute to the overall risk of venous thromboembolism (VTE). In addition, the epidemiology of thrombophilia in the East-Asian VTE population remains unclear; thus, we aimed to assess the proportion of hereditary thrombophilia via a meta-analysis. MethodsPublications from PubMed, EMBASE, web of science, and Cochrane before December 30, 2022, were searched. Studies from Japan, Korea, China, Hong Kong, Taiwan, Singapore, Thailand, Vietnam, Myanmar, and Cambodia were included. Congenital thrombophilia was described as diseases including protein C (PC) deficiency, protein S (PS) deficiency, antithrombin (AT) deficiency, factor (F)V Leiden (FVL), and prothrombin G20210A mutations. Studies were selected by 2 reviewers for methodological quality analysis. A random-effects model was used for the meta-analysis, assuming that estimated effects in the different studies are not identical. ResultsForty-four studies involving 6453 patients from 7 counties/regions were included in the meta-analysis. The prevalence of PC, PS, and AT deficiencies were 7.1%, 8.3%, and 3.8%, respectively. Among 2924 patients from 22 studies, 5 patients were carriers of FVL mutation. Among 2196 patients from 10 studies, 2 patients were carriers of prothrombin G20210A mutation in a Thailand study. ConclusionThe prevalence of PC, PS, and AT deficiencies was relatively high, while a much lower prevalence of FVL and prothrombin G20210A mutations were identified in East-Asian patients with VTE. Our data stress the relative higher prevalence of PC, PS, and AT deficiencies for thrombophilia in the East-Asian VTE population.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

The impact of hereditary thrombophilias in recurrent pregnancy loss

Introduction: Recurrent pregnancy loss (RPL) is defined as two or more consecutive pregnancy loss which occurs before the 20th weeks of pregnancies for the last menstrual period. Hereditary cause of thrombophilic gene mutations and polymorphism may play an essential role in RPLs. Material and Method: 291 women with a history of two or more consecutive abortions as a study group and 61 women without the history of miscarriages as a control group were included in a study. In this study we analysed the effects of Factor II Prothrombin mutation ,FV Leiden mutation, MTHFR C677T, MTHFT A1298C, PAI-1, ?-fibrinogen, Factor XIIIA (V34L) and Glycoprotein IIIa (L33P) polymorphisms on RPL by using pyrosequencing. Chi-square and multiple regression analysis were used for statistical analysis. Results: FII prothrombin mutation, FV Leiden mutation, MTHFR C677T, MTHFR A1298C, PAI1 and Beta fibrinogen were found statistically significant in the chi-square test. Heterozygous FV G1691A (OR:8.092, CI: 1.280-51.165), homozygous MTHFR A1298C (OR:17.621, CI: 3.644 - 85.203), Heterozygous MTHFR C677T (OR: 2.921 CI: 0.811-10.515), Homozygous MTHFR C677T (OR: 3.619 CI: 1.647-7.954), heterozygous MTHFR A1298C (OR: 5.989, CI: 2.574-13.934), homozygous PAI1 (OR: 8.756, CI: 2.805 -27.334), heterozygous PAI1 ( OR: 7.114, CI: 3.145- 16.096) homozygous FibrinogenG455A (4.085, CI: 1.438-11.610) were found statistically significant in logistic regression analysis for RPL(p<0.05). Discussion: This study indicated that there is a significant association between thrombophilias and RPL. Therefore, it is important to detect thrombophilic mutations in RPL.

DOUBLE HETEROZYGOTIC FV DEFECT WITH HETEROZYGOTIC FV LEIDEN MUTATION AND FV DEFICIENCY IN THROMBOSIS

FV Leiden mutation causes activated protein C (APC) resistance and causes an increase in thrombin level. Although moderate bleeding is seen in severe factor V deficiency, less than 1% of patients experience bleeding. Cases in which thrombosis is prominent in the presence FV Leiden mutation and FV deficiency have been reported. Here, we present a patient with FV deficiency with FV Leiden heterozygous mutation in the etiology of recurrent abortion. A 41-year-old female patient who applied to her primary care physician with bilateral lumbar pain upon finding INR: 1.43 (0.8-1.2) and APTT: 37.6 seconds (25-36.5), the patient was recommended to apply to our out patient clinic. The patient who described two spontaneous abortions (at the age of 25, the first in the 2nd trimester and the other in the 3rd trimester), also had a history of ecchymosis in the extremities caused by minor trauma at intervals. PT, INR and APTT returned to normal with the mixing test performed on the patient (12.1 sEC, 1.03 and 28.6 sec, respectively).Afterwards, FV, which is one of the factors in the common pathway of coagulation, was found low in the examination repeated twice (12.3% and 10.2%) (N: 62-139%). The APCR studied twice in screening for thrombophilia was 1.4 and 2.4 (N: 2.61-3.32) Protein C, protein S, antithrombin III levels were within normal limits, LAC and APA were negative. According to this result, FV Leiden heterozygous mutation was detected in the genetic thrombophilia panel. Also the patient had FV deficiency . Authors termed the coexistence of heterozygous FV Leiden mutation and type1 FV deficiency as pseudohomozygous FV Leiden mutation. In our and other studies, we concluded that thrombosis was clinically significant, where as bleeding was rare and mild. We think that prolonged PT and APTT results in patients with a history of thrombosis with FV Leidenmutation are also stimulating in evaluating FV activity.

Factor V H1299r (Hr2) Heterozygosity: A Risk Factor For Recurrent Implantation Failure Particularly In Non-Carriers For Factor V Leiden Mutation-A Case-Control Study

Objective: The association between recurrent implantation failure and thrombophilia is still controversial depending on the published reports with conflicting results. In this study, we aimed to assess the clinical relevance of screening women with recurrent implantation failure for some thrombophilic variants including factor V H1299R (FV HR2) haplotype. Study Design: A total of 279 women were recruited in this case-control study. 229 women with a history of recurrent implantation failure and 50 fertile control with no history of pregnancy losses were screened for eight specific gene mutations, regarding factor V G1691A gene (FV Leiden), FV HR2, factor II prothrombin G20210A, factor XIII V34L, PAI-1 4G/5G, MTHFR C677T, MTHFR A1298C and A3 haplotype of the endothelial cell protein C receptor gene. Results: Recurrent implantation failure group displayed a significantly higher prevalence of FV HR2 heterozygosity than fertile controls while the frequency of FV Leiden mutation was comparable between groups (p=0.011; p=0.619). Additionally, the difference in the prevalence of other specific or total gene mutations among women with recurrent implantation failure was also insignificant. Discussion: The primer outcome of this study was the co-existence of the higher prevalence of FV HR2 haplotype and the insignificant percentage of FV Leiden mutation in women with recurrent implantation failure. Thus, we emphasize that the HR2 haplotype may be associated with recurrent implantation failure particularly in non-carriers for FV Leiden mutation. In the necessity of screening for thrombophilia in recurrent implantation failure, HR2 haplotype should be involved in the searched gene panel particularly in the absence of FV Leiden mutation. Further large-scale prospective studies are needed to investigate whether screening or treatment for HR2 haplotype has any detrimental impact on implantation success in cases of recurrent implantation failure.

Ovarian hyperstimulation syndrome and the risk of internal jugular vein thrombosis

Introduction: Thromboembolic events are a rare complication of severe ovarian hyperstimulation syndrome (OHSS). In contrast to the classical left ilio-femoral deep vein thrombosis casesduring pregnancy,atypical localization in upper extremity, including internal jugular vein (IJV)is also reported. Review of the literature: We reviewed 39 cases of IJV thrombosis associated with OHSS mostly during assisted reproductive treatment. The diagnosis was made during first trimester, mainly with Doppler ultrasound, and treatment with heparin was initiated in all cases. In 14 patients another risk factor for thromboembolic event was reported (50% had FV-Leiden mutation). The IJV was extended in upper extremityincluding axillary, subclavian or superior vena cava in 46%, whereas pulmonary embolism was reported in 10% of cases. Discussion: Obstetricians should be aware of the rare complication of deep vein thrombosis in upper extremity including thrombosis of IJV, even weeks after OHSS symptoms have resolved.Patients with OHSS after assisted reproductive treatmentshould be prescribed heparin in prophylactic dose during the first trimester.

Evaluation of Clinical, Laboratory Findings and Prognosis of Paediatric Thrombosis

The number of cases suffering from thrombotic events during childhood is gradually increasing. Despite being quite uncommon among children compared to adults, symptomatic thrombosis may lead to mortality and life-long disabling sequela when occurs at pediatric ages as well.The aim of our study is determining the risk groups for childhood thrombotic events along with their prognosis by examining clinical and laboratory features of cases. Files belonging to all patients aged 0 to 18 years with a diagnosis of thrombotic event confirmed between the dates of 1 January 2009 and 30 June 2016 at Hacettepe University, Department of Paediatric Hematology were studied retrospectively.A total of 227 patients are included. 95 (41.9%) of them are girls and 132 (%58.1) are boys. Median age of diagnosis is 41 months (1month-240 months) where 69 patients are aged under one year (≤1 year), 103 patents are aged between 1 and 12 years (˃1-≤12 years ) and 55 patients are adolescents (˃12 years) at the age of diagnosis. When classified due to thrombosis origin, it is noticed that 50 patients (22%) had arterial thrombosis while 177 patients (88%) had venous. 90% of patients (n=205) presented with acute thrombosis while 10% of patients (n=22) were suffering from chronic thrombosis. An underlying disease is detected at 195 patients (86%), cardiac diseases and malignities occupying first and second ranks when listed. The frequency of sufferring from an underlying disease or an acute infection is found to be significantly higher at children aged under one year compared to other age groups. Besides, the frequency of congenital diseases of heart as the underlying cause of thrombosis is found to be significantly higher at children aged under one year and aged between 1 and 12 years, compared to adolescents. It is revealed that the incidence for implementation of a catheter into the circulatory system is significantly higher for patients suffering from venous thrombosis compared with those from arterial thrombosis. However, significantly higher numbers of complications are observed at patients with arterial thrombosis in comparison to those with venous thrombosis. It is detected that venous thrombotic cases are occasionally treated with LMWH. No statistically significant difference is found between arterial and venous thrombotic cases in terms of FV Leiden or Protrombin 20210A mutation prevalences.There are a total of 36 patients applied with recurrence, 14 (39%) of them being girls and 22 (61%) being boys. 22 of patients has suffered from recurrence for once while 14 of them has referred with a recurrence more than once. For both group of patients suffering from recurrence for once and more than once, the most frequent site of the first thrombotic episode is observed to be deep venous thrombosis. Adolescents are demonstrated to be more prone to recurrence for more than once wih a statistically significant higher rate compared with other age groups. Coexistence of MTHFR C677T mutation heterozygosity and MTHFR A1298C mutation heterozygosity (compound heterozygotic pattern for MTHFR) is discovered to be a risk factor for recurrence given the statistically significant relative higher incidence in this specific group.Death of 47 (23%) patients are recorded within the median follow-up duration of patients which is 12 months. 77% of those patients passed away due to their underlying primary disease. Venous thrombotic cases are found to have a significantly higher mortality rate compared to arterial thrombotic cases. It is also found that mortality rate is significantly higher among children under age of one year in comparison with other age groups regardless of thrombosis origin.206 of 227 patients are evaluated for overall survival. Mean survival duration is came out to be 72.5 ±3.1 months with probability of overall survival for one years duration calculated to be 76.6% for all patients. Factors affecting mortality probability are evaluated via Cox regression (proportional hazard) analysis. When age, sex, recurrence and origin of thrombosis are used for estimation altogether as factors which could affect mortality, probability of death under age of one year is increased by 3.46 times than adolescence. Using same method, death risk due to venous thrombosis is calculated to be 3.8 times higher than arterial thrombosis DisclosuresNo relevant conflicts of interest to declare.

Impedimetric Sensing of Factor V Leiden Mutation by Zip Nucleic Acid Probe and Electrochemical Array.

A carbon nanofiber enriched 8-channel screen-printed electrochemical array was used for the impedimetric detection of SNP related to Factor V Leiden (FV Leiden) mutation, which is the most common inherited form of thrombophilia. FV Leiden mutation sensing was carried out in three steps: solution-phase nucleic acid hybridization between zip nucleic acid probe (Z-probe) and mutant type DNA target, followed by the immobilization of the hybrid on the working electrode area of array, and measurement by electrochemical impedance spectroscopy (EIS). The selectivity of the assay was tested against mutation-free DNA sequences and synthetic polymerase chain reaction (PCR) samples. The developed biosensor was a trustful assay for FV Leiden mutation diagnosis, which can effectively discriminate wild type and mutant type even in PCR samples.

ARMS TaqMan real-time PCR for genotyping factor V Leiden mutation in Han Chinese.

Factor V Leiden (FVLeiden ) is a missense mutation of 1691 position (G1691A) in exon 10 of FV gene, and being a genetic risk for venous thrombosis. Currently, there are several PCR-based methods for detecting FVLeiden mutation; however, these methods have disadvantages such as time-consuming, cumbersome steps and potentially hazardous gels. The aims of present study were to develop a simple, time-saving, accurate, and gel-free method, called amplification refractory mutation system (ARMS) TaqMan real-time PCR, for detecting FVLeiden mutation. We severally designed two specific reverse primers for mutant and wild-type through intentional introduction of mismatched nucleotide at the penultimate 3' position. Although target amplicon amplification efficiency is reduced, but another corresponding amplicon is almost completely inhibited. Then, specific TaqMan-probe was designed to detect target amplicon. Established method was used to detect 500 unselected samples in Han Chinese, the results showed 499 cases of wild-type and one heterozygote. Afterward, 50 randomly picked wild-type cases and one heterozygote were reexamined by bidirectional DNA sequencing, which is considered as "Gold standard method." Exhilaratingly, the results detected by the two methods were completely consistent. At last, allelic frequency of FVLeiden was calculated the in Han Chinese. Given the above results, A FVLeiden heterozygote has been found in 500 random samples in Han Chinese, and the allelic frequency was 0.1%. In conclusion, the ARMS TaqMan real-time PCR is an ideal detecting system for genotyping FVLeiden mutation in clinical application, and FVLeiden mutation exists in Han Chinese despite extremely low prevalence.

Feature of distribution of polymorphic options of genes associated with thrombophilia and arterial hypertension in children with hemolytic-uremic syndrome

INTRODUCTION. The basis of hemolytic-uremic syndrome (HUS) is the pathology of hemostasis with the development of a condition that threatens the patient's life. Violations of the hemostatic system are probably largely due to a hereditary predisposition to thrombophilia.THE AIM. To study the characteristics of allelic polymorphism of genes associated with hereditary thrombophilia in 15 children with hemolytic-uremic syndrome.PATIENTS AND METHODS. A study was conducted of 15 children with HUS from the age of 1 to 4 years who were treated in the nephrology department of the clinic of St. Petersburg State Medical University. A typical HUS was diagnosed in 14 children. One child was diagnosed with atypical HUS confirmed by laboratory tests (increased levels of antibodies to protein H). At the Research Institute of Hematology and Transfusiology of the FMBA of Russia, PCR for all 15 patients and two parents analyzed the polymorphism of genes associated with thrombophilia.RESULTS. In all examined children, polymorphism of genes associated with various links of hemostasis was found. Two patients showed a mutation in the factor V gene (FV Leiden). In 7 patients out of 15 subjects, the homozygous state of the plasminogen activator inhibitor (PAI-1 - 675 4G / 5G) was revealed.CONCLUSION. The FV Leiden mutation, as the main reason for the resistance of activation of protein C (an inhibitor of the formation of thrombin), was found in two children, one with a typical HUS, and one with an atypical HUS. In 7 children out of 15 subjects, a homozygous state of plasminogen activator inhibitor (PAI-1 - 675 4G / 5G) was revealed, which may indicate insufficient fibrinolytic activity in these children, as an important factor contributing to the development of TMA.

Breast reconstruction using Deep Inferior Epigastric Perforator (DIEP) flap in a patient with heterozygous Factor V Leiden: a case report

Factor V Leiden (FVLeiden) mutation is the commonest type of inherited thrombophilia. Free tissue transfer is very rarely used in such patients due to concerns about microvascular thrombosis and flap failure. Even when rarely offered, the peri-operative antithrombotic regimen for such cases is widely varied and based of personal experiences and preference. These regimens usually include multiple pharmacologic agents but their real benefit is based merely on anecdotal evidence. On the other hand, the potential complications of such drugs are well established in literature. Deep Inferior Epigastric Perforator (DIEP) flap is the most widely used autologous breast reconstruction technique. Here, we report a case of successful free DIEP flap breast reconstruction in a lady with heterozygous FVLeiden mutation. We have described our approach to this patient, including the simple single drug anticoagulation regimen used as advised by the hematologists for venous thromboembolism (VTE) prophylaxis. We have compared our approach to that of others who have reported similar cases before. Our report shows that it is feasible to offer successful free tissue breast reconstruction to these patients by following meticulous surgical technique without the need of additional systemic antithrombotic agents over and above the simple regimen indicated for general VTE prophylaxis.

Thrombotic Outcomes Following Hormonal Treatment of Heavy Menstrual Bleeding in Pediatric Patients Taking Anticoagulation

Introduction: Anticoagulant-associated heavy menstrual bleeding (HMB) is a common and under recognized complication in women who are being treated for venous thromboembolism (VTE), leading to complications such as anemia and decreased quality of life. The appropriate management of HMB in this population is complex and often requires hormonal therapy which may significantly increase the risk for VTE. Additionally, combined oral contraceptives may need to be used to control heavy menstrual bleeding despite contributing to the overall risk for VTE. Due to limited adult and pediatric data, there are currently no expert consensus guidelines for the treatment of anticoagulant-associated HMB. We describe a single institutional experience and thrombotic outcomes following hormonal therapy for anticoagulation-associated HMB in adolescents. Methods: A retrospective cohort of pediatric females ages 9-21 years treated at Children's Healthcare of Atlanta were identified between January 2016 and July 2019. Patients were identified using common ICD codes for both HMB and anticoagulation/thrombosis. Patient demographic information, clinical characteristics, and thrombotic outcomes were abstracted from review of the electronic medical record. Patients receiving anti-platelet therapy with clopidogrel were included due to the reported similarity of bleeding complications compared to anticoagulants. Summary statistics were performed and Student's t test, Fisher's exact test, and Chi square test were used for statistical analysis (SAS v9.4, 2012, Cary, NC, USA). A p value <0.05 was considered statistically significant. Results: The cohort was comprised of 17 adolescent females with confirmed anticoagulation-associated HMB. Among the cohort, 59% identified as White, 35% Black, and 6% Asian. Body Mass Index was normal in 41%, while 24% were obese and 35% were underweight. Indications for anticoagulation included VTE (n=11), pulmonary embolism (n=6), May-Thurner Syndrome with placement of iliac vein stent (n=3), and other vascular anomalies with confirmed stenosis/thrombosis (n=2). The most common risk factor for thrombosis was initiation of combined hormone oral contraceptives within 3 months of the event that led to initiation of anticoagulation (35%). Anticoagulant medications used included lovenox (n=13), clopidogrel (n=3), and lovenox/aspirin (n=1). The median age at initiation of hormonal therapy for HMB was 17.3 years (min-max, 12.6-18.3 years), with a median time between starting anticoagulation and initiation of hormonal therapy of 11 days (min-max, 0-529 days). Medications used to treat HMB included medroxyprogesterone (n=9), norethindrone (n=6), norethindrone-ethinyl estradiol (n=1), and etonogestrel implant (n=1). After initiating hormonal therapy, amenorrhea or menstrual suppression was reported in all but one individual (n=16/17). Following initiation of hormonal therapy with medroxyprogesterone, only one adolescent experienced a new VTE event. This adolescent had the additional risk factors of May-Thurner anatomy, heterozygous FV Leiden and prothrombin gene mutations, and antiphospholipid antibody syndrome. The median length of follow-up from initiation of hormonal therapy was 325 days (min-max, 43-932 days). Statistical differences between groups were not feasible due to small sample size and few outcome events. Conclusions: The proper treatment of anticoagulation-associated HMB using hormonal therapy in the pediatric population is unclear, but our data suggest that treatment with hormonal therapy reduces anticoagulation-associated HMB while adding minimal risk for VTE. The only VTE event that occurred in our cohort was in the setting of multiple strong risk factors for VTE. Larger pediatric studies are required in order to confirm these findings. Disclosures White: National Hemophilia Foundation: Other: Shire Clinical Fellowship Program. Sidonio:Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Kedrion: Research Funding; Uniqure: Membership on an entity's Board of Directors or advisory committees; Biomarin: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Electrochemical Detection of Solution Phase Hybridization Related to Single Nucleotide Mutation by Carbon Nanofibers Enriched Electrodes.

In the present study, a sensitive and selective impedimetric detection of solution-phase nucleic acid hybridization related to Factor V Leiden (FV Leiden) mutation was performed by carbon nanofibers (CNF) modified screen printed electrodes (SPE). The microscopic and electrochemical characterization of CNF-SPEs was explored in comparison to the unmodified electrodes. Since the FV Leiden mutation is a widespread inherited risk factor predisposing to venous thromboembolism, this study herein aimed to perform the impedimetric detection of FV Leiden mutation by a zip nucleic acid (ZNA) probe-based assay in combination with CNF-SPEs. The selectivity of the assay was then examined against the mutation-free DNA sequences as well as the synthetic PCR samples.

PREVALENCE OF FACTOR V LEIDEN AND PROTHROMBIN G20210A IN WOMEN WITH VON WILLEBRAND DISEASE TYPE 1

Background.Von Willebrand disease is a hereditary malfunction of the blood coagulation system caused by waveform quantitative and/or qualitative deficiency of von Willebrand factor (vWF).Aim.To evaluate the frequency of occurrence of FVLeiden and FII G20210A mutations in female patients with von Willebrand type 1 disease.Materials and methods.136 women aged from 18 to 45 years (mean 31.7 ± 0.5 years) were enrolled in a study conducted during the January 2011 — December 2017 period. Questionnaire was used to reveal hemorrhagic diathesis. Inclusion criteria were as follows: no less than 3 positive responses to questions 1–7, or 2 positive responses to questions 1–7 plus no less than 100 points of the evaluated menstrual blood loss. An independent inclusion criterion was 180 points or more in the question concerning menstrual blood loss. A mandatory inclusion criterion was the confirmation of absence of thromboembolic events in a proband and first line relatives. The study included assessment of such parameters as ristocetin-cofactor activity of von Willebrand factor (vWF:RCo), von Willebrand factor antigen (vWF:Ag), factor VIII (FVIII:C), platelet aggregation induced with ADP, ristomycin, collagen, as well as molecular-genetic assay of factor V (FVLeiden) and gene (FII G20210A) polymorphism using allele-specific polymerase chain reaction.Results.No mutations of FVLeiden and FII G20210A were revealed in 102 women with von Willebrand disease type 1. Heterozygous mutation of FVLeiden was found in 12 (8.8 %) subjects with von Willebrand disease type 1 (vWF:RCo from 27 to 47 % (mean 37.3 ± 0.8 %), vWF:Ag from 25 to 46 % (mean 37.5 ± 0.8 %), FVIII:C from 29 to 49 % (mean 44.1 ± 0.5 %). Homozygous mutation of FVLeiden was identified in 3 (2.2%) women with von Willebrand disease type 1, with vWF:RCo being 40, 43 and 45 %, vWF:Ag — 39, 44 and 42 %, FVIII:C — 47, 45 and 48 %, respectively. Heterozygous mutation FII G20210A was detected in 19 (13.9 %) subjects with von Willebrand disease type 1 (vWF:RCo from 36 to 49 % (mean 43.0 ± 0.4 %), vWF:Ag from 32 to 46 % (mean 42.2 ± 0.6 %), FVIII:C from 30 to 49 % (mean 45.1 ± 0.4 %).Conclusion.By means of diminishing the coagulation potential of the blood coagulation system, a decrease in the activity of VIII and von Willebrand factors may compensate possible negative effects associated with FVLeiden and FII G20210A gene mutations in female patients with von Willebrand type 1 disease.

ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation

The development of a low-cost and disposable biosensing technologies has received a great interest of healthcare for the sensitive and reliable detection of single nucleotide mutation related to single nucleotide polymorphisms (SNPs). In the present study, an impedimetric biosensing platform based on zip nucleic acids (ZNA) was developed for the sensitive detection of Factor V Leiden (FV Leiden) mutation. After optimization of experimental parameters, the sequence selective hybridization between ZNA probe and target related to FV Leiden mutation was evaluated via electrochemical impedance spectroscopy technique (EIS) by measuring changes at the charge transfer resistance, Rct. Sensitive and selective impedimetric analysis was performed using carbon nanofiber (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (MULTIx8 CNF-SPE) resulting in a relatively shorter time in comparison to conventional methods. The selectivity of ZNA probe to mutation-free DNA sequences was also investigated. The applicability of single-use ZNA biosensor was also tested in synthetic PCR samples containing a single base mutation.

Effect of multiple thrombophilic gene mutations on uterine artery blood flow in nonpregnant recurrent pregnancy loss patients: are we searching enough?

Objective: To investigate whether multiple thrombophilic mutations had a significant effect on uterine artery blood flow of nonpregnant recurrent pregnancy loss (RPL) patients.Materials and methods: Among 71 RPL patients, FV Leiden (FV), prothrombin G20210A (PTGM) and MTHFR mutations, deficiency of protein S, protein C and antithrombin III (AT3), association of Val34Leu polymorphism of the FXIII (FXIII), 4G/5G polymorphism of plasminogen activator inhibitor (PAI), −455-G/A polymorphism of β-fibrinogen (fibrinogen), and HPA-1 a/b L33P polymorphism of GPIIIa (GPIIIa) genes were investigated. Doppler flow measurements of RPL patients and healthy controls were performed at mid-luteal phase.Results: Twenty-two patients who had no thrombophilic gene mutations were grouped as unexplained RPL. Also, while 25 patients had multiple mutations, 24 patients had single mutation. In the multiple mutation RPL group, the most frequent mutations were PAI (22%), MTHFR C677T (20%), MTHFR A1298C (19%), and fibrinogen (11%). Only in the multiple mutation RPL group, mean PI and dominant PI values were significantly higher than the control group.Conclusions: Our data showed negative effects of multiple thrombophilic gene mutations on uterine artery blood flow and clarified the different effects of single and multiple thrombophilic factors on uterine artery vasculature. It is concluded that investigating more thrombophilic mutations could ameliorate prognostic factors of RPL and interactions to improve uterine artery blood flow could bring benefit to obstetric outcome.