VKORC1 and CYP2C9 polymorphisms related to adverse events in case-control cohort of anticoagulated patients

Vitamin K antagonists (VKAs) are highly effective but have a narrow therapeutic index and require routine monitoring of the INR. The primary aim of pharmacogenetics (PGx) is to optimize patient care, achieving drug treatments that are personalized according to the genetic profile of each patient. The best-characterized genes involved in VKA PGx involve pharmacokinetics (VKORC1) and pharmacodynamics (CYP2C9) of VKA metabolism. The role of these genes in clinical outcomes (bleeding and thrombosis) during oral anticoagulant (OAC) therapy is controversial. The aim of the present study was to evaluate any potential association between genotype VKORC1 and CYP2C9 and adverse events (hemorrhagic and/or thrombotic), during initiation and long-term VKA treatment, in Caucasian patients. Furthermore, we aimed to determine if the concomitant prescription of other selected drugs affected the association between genotype and adverse events.We performed a retrospective, matched case-control study to determine associations between multiple gene variants, drug intake, and any major adverse effects in anticoagulated patients, monitored in 2 Italian anticoagulation clinics.Our results show that anticoagulated patients have a high risk of adverse events if they are carriers of 1 or more genetic polymorphisms in the VKORC1 (rs9923231) and CYP2C9 (rs1799853 and rs1057910) genes.Information on CYP2C9 and VKORC1 variants may be useful to identify individualized oral anticoagulant treatment for each patient, improve management and quality of VKA anticoagulation control, and monitor drug surveillance in pharmacovigilance programs

Relationship between gender differences and clinical outcome in patients with the antiphospholipid syndrome

Antiphospholipid syndrome (APS), characterized by artherial and/or venous thrombosis, pregnancy morbidity and "antiphospholipid" antibodies (aPLs), is more common in women than in men, with a female to male ratio of about 3.5:1. Only few studies have investigated the clinical differences between male and female patients with APS. Therefore, this study was aimed to analyze the differences of clinical manifestations and laboratory tests, at diagnosis, between female and male APS patients and the clinical outcome. We enrolled 191 consecutive APS patients (125 with primary APS, PAPS, and 66 with secondary APS, SAPS) with a female predominant ratio of approximately 3:1 (142 vs 49). The prevalence of PAPS was higher in males than females (p<0.001). The analysis of aPL profile revealed that high IgM anti-cardiolipin (aCL) and high-medium IgG aCL titers were more frequent in males. In thrombotic APS peripheral arterial thrombosis was more common in male than female patients (p=0.049), as well as myocardial infarction (p=0.031). Multivariate analysis to correct for cardiovascular risk factors, high titer of aPLs and triple positivity for aPLs, revealed that the odds ratio for myocardial infarction in male was 3.77. Thus, APS may be considered as a disease in which serological (IgM titer) and clinical profiles are influenced by gender

Alarmin HMGB1 and Soluble RAGE as New Tools to Evaluate the Risk Stratification in Patients With the Antiphospholipid Syndrome

Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by arterial and/or venous thrombosis, pregnancy morbidity in the presence of circulating “anti-phospholipid antibodies” (aPL). One of the main target antigens of aPL is β2-glycoprotein I (β2-GPI). APS may occur as a primary syndrome or associated with Systemic Lupus Erythematosus (SLE). High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different type of cells during activation and/or cell death and may act as a proinflammatory mediator through ligation to its receptors, including RAGE. There is accumulating evidence that HMGB1 contributes to the pathogenesis of inflammatory and autoimmune diseases, especially SLE. In a previous study we demonstrated increased serum levels of HMGB1 in both primary and secondary APS patients. In this work we analyzed: (i) in vitro whether anti-β2-GPI antibodies from APS patients may induce both a HMGB1 cellular relocation by activation of its putative receptor RAGE in platelets and monocytes and, (ii) ex vivo, serum levels of HMGB1/soluble RAGE (sRAGE) in APS patients and their possible correlation with clinical manifestations. Platelets and monocytes from healthy donors were incubated with affinity purified anti-β2-GPI antibodies. HMGB1 and RAGE expression were analyzed by Western Blot. Sera from 60 consecutive APS patients (primary or secondary), diagnosed according to the Sydney Classification Criteria, were enrolled. As a control, 30 matched healthy subjects were studied. Serum levels of HMGB1 and sRAGE were analyzed by Western Blot. In vitro results showed that anti-β2-GPI antibodies were able to induce RAGE activation and HMGB1 cellular relocation in both monocytes and platelets. HMGB1 and sRAGE serum levels were significantly increased in APS patients in comparison with healthy subjects (p<0.0001). Interestingly, APS patients with spontaneous recurrent abortion showed significantly higher levels of sRAGE; moreover, in APS patients a direct correlation between serum levels of HMGB1 and disease duration was detected. Our observations suggest that anti-β2-GPI antibodies may trigger RAGE activation and HMGB1 cellular relocation during APS. Monitoring these molecules serum levels may represent an useful tool to evaluate the pathogenesis and risk stratification of clinical manifestations in APS

Protein Aggregation Landscape in Neurodegenerative Diseases: Clinical Relevance and Future Applications

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable β-sheet enriched intermediates, which are stabilized by intermolecular interactions with other β-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders

Diagnosis of catastrophic anti-phospholipid syndrome in a patient tested negative for conventional tests

Catastrophic antiphospholipid syndrome (CAPS) is a severe variant of APS, characterised by clinical evidence of multiple organ involvement developing over a very short period of time, histopathological evidence of multiple small vessel occlusions and laboratory confirmation of the presence of aPL (lupus anticoagulant and/or anticardiolipin antibodies and/or anti-Beta2-glcyoprotein I antibodies). Here we report a case of a 39-year-old woman patient who developed a CAPS which was negative to the conventional aPL but positive for aPL in thin layer chromatography immunostaining and vimentin/cardiolipin antibodies by ELISA test. The patient was treated with high doses of glucocorticoids, intravenous immunoglobulins plasma exchange and immunoadsorbent apheresis with a significant improvement of the ischaemic lesions of the hands even though the necrosis of the feet progressively worsened. As a result, the patient underwent partial surgical amputation of the feet. To our knowledge, this is the first ever reported case of CAPS diagnosed by means of thin layer chromatography immunostaining and vimentin/cardiolipin antibody ELISA test

Anti-vimentin/cardiolipin IgA in the antiphospholipid syndrome: a new tool for “seronegative” diagnosis

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder defined by the simultaneous presence of vascular clinical events, pregnancy morbidity and antiphospholipid antibodies (aPL). In clinical practice it is possible to find patients with APS who are persistently negative for the routine aPL tests (“seronegative APS”, SN-APS). Recently, the identification of aPL IgA and/or anti-β2-GPI IgA was shown to represent a further test in SN-APS patients. In this study we analyzed the presence of anti-vimentin/cardiolipin (aVim/CL) IgA in a large cohort of patients with SN-APS, evaluating their possible association with clinical manifestations of the syndrome. This study includes 60 consecutive SN-APS patients, 30 patients with APS and 40 healthy donors. aVim/CL IgA were detected by ELISA. Results show that twelve out of 30 APS patients (40%) and 16/60 SN-APS patients (26.7%) resulted positive for aVim/CL IgA. Interestingly, SN-APS patients tested positive for aVim/CL IgA showed a higher prevalence of arterial thrombosis (p=0.017, likelihood positive ratio of 5.7). This study demonstrates for the first time the presence of anti-Vim/CL IgA in sera of patients with APS. In particular, they revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients tested positive for aVim/CL IgA reported a high likelihood ratio to have the clinical features of APS, this test may be considered a suitable approach in the clinical evaluation of SN-APS

Carbamylation of β2-GPI generates new autoantigens for Antiphospholipid Syndrome. a new tool for diagnosis of "seronegative" patients

Objectives: Antiphospholipid syndrome (APS) is a prothrombotic condition defined by recurrent thrombosis, pregnancy complications and circulating antiphospholipid antibodies (aPL), including anti-β2-Glycoprotein I (β2-GPI). In clinical practice it is possible to find patients with APS persistently negative for the aPL tests according to Sydney criteria ("seronegative APS", SN-APS). Recently, several autoimmune responses have been described as a consequence of post-translational modifications of their target autoantigens. This study was undertaken to test carbamylated-β2-GPI (Carb-β2-GPI) as a new autoantigen of APS. Methods: β2-GPI was carbamylated by potassium cyanate and used to investigate its effect on monocyte-derived DC (moDC) phenotype and function. Sera from 114 SN-APS patients, 60 APS, 20 patients with Rheumatoid Arthritis, 20 NON-APS thrombosis and 50 healthy donors were analyzed for anti-Carb-β2-GPI by ELISA. Results: Carb-β2-GPI is able to activate moDCs, inducing up-regulation of CD80, CD86, and CD40, activation of ERK, p38 MAPK and NF-κB and IL-12p70 release.Serological results showed that both 37/114 SN-APS (32.45%) and 23/60 APS patients (38.33%) resulted positive for anti-Carb-β2-GPI. Interestingly, SN-APS patients tested positive for anti-Carb-β2-GPI showed a higher prevalence of thrombocytopenia (p= 0.04, likelihood positive ratio of 3.9). Conclusion: Data obtained from both functional tests on moDCs and from immunologic approaches prompted identification of Carb-β2-GPI as a "new" antigenic target in APS. In particular, anti-Carb-β2-GPI revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients tested positive for anti-Carb-β2-GPI reported a high risk of thrombocytopenia, this test may be considered a suitable approach in the clinical evaluation of SN-APS

“Non-criteria antiphospholipid antibodies”: bridging the gap between seropositive and seronegative Antiphospholipid Syndrome

Objective: We aimed to analyze the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL-anti-cardiolipin antibodies (aCL), anti-β2-glycoprotein I antibodies (aβ2-GPI), and lupus anticoagulant (LA) - named seronegative APS (SN-APS). Methods: Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. Results: We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, 3 cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. Conclusions: This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitoring and providing adequate targeted therapy, which improve SN-APS prognosis

A monocentric cohort of obstetric seronegative anti-phospholipid syndrome

The present study was conducted to diagnose obstetric anti-phospholipid syndrome (OAPS) in patients with clinical signs suggestive of anti-phospholipid syndrome (APS), but persistently negative for conventional anti-phospholipid antibodies (aPL). Sera from 61 obstetrical seronegative APS (SN-APS) patients were analyzed for anti-cardiolipin antibodies (aCL) using thin-layer chromatography (TLC)-immunostaining, for anti-cardiolipin/vimentin antibodies (aCL/Vim), anti-phosphatidylserine/prothrombin antibodies, IgA anti-β2glycoprotein I antibodies (aβ2GPI), and IgA aCL antibodies by enzyme-linked immunosorbent assay. Taken together, our findings show that in 50 out of 61 SN-APS (81.9%) at least one aPL/cofactor antibody was detected using the assays under test. Results revealed that 76% of SN-APS patients resulted positive for aCL by TLC-immunostaining, 54% for aCL/Vim, 12% for aPS/PT, 4% for IgA aβ2GPI, and 2% for IgA aCL. Thirty-five out of 61 patients were followed up and the tests were repeated on two occasions, at least 12 weeks apart. Twenty-six out of 35 SN-APS (74.3%) were positive at least one non-conventional test; only 2 patients (5.7%) did not confirm the positivity to the second test. These findings suggest that non-conventional tests, mainly aCL/Vim and aCL detected by TLC-immunostaining, seem to be the most sensitive approaches for finding out aPL in patients with obstetric SN-APS. The use of these tests can be useful for accurate and timely diagnosis of patients with obstetrical APS who are negative for conventional laboratory criteria markers