Plasma apolipoprotein M predicts overall survival in metastatic breast cancer patients.

PURPOSE: Apolipoprotein M (APOM) is a plasma apolipoprotein closely involved with lipid metabolism and inflammation. In vitro studies suggest that APOM may also have a tumor-suppressive role in breast cancer. In the present study, we aimed to evaluate the impact of plasma APOM levels on the prognosis of breast cancer patients. METHODS: We measured APOM levels using an enzyme-linked immunosorbent assay in 75 patients with ER-positive/HER2-negative metastatic breast cancer. The endpoint was overall survival (OS) at 24 months. RESULTS: During the 24-month follow-up period, 34.7% of the patients died. Baseline APOM levels were significantly reduced in patients who deceased during follow-up compared to survivors (42.7 ± 14.5 µg/mL versus 52.2 ± 13.8 µg/mL; P = 0.003). Cox regression analysis showed a hazard ratio of 0.30 [95% confidence interval 0.15-0.61]; P < 0.001 per doubling of APOM levels. Correction for age, C-reactive protein, menopausal state, histology of the primary tumor, metastatic site, number of metastases, endocrine resistance, scheduled therapy line, and kind of scheduled therapy indicated that circulating APOM predicted OS independently of these parameters (HRper doubling = 0.23 [0.09-0.56; P = 0.001). CONCLUSIONS: Our study suggests that circulating APOM is significantly linked with reduced mortality in metastatic breast cancer patients.

Myricetin attenuates hypoxia-induced inflammation in human adipocytes.

BACKGROUND: Adipose tissue hypoxia plays a crucial role in the development of chronic low-grade systemic inflammation which has been associated with the pathogenesis of obesity-related diseases. Myricetin is a natural compound present in numerous plant-based foods with presumed anti-inflammatory and beneficial health effects. The impact of this flavonoid on hypoxia-induced expression of inflammatory adipokines and hypoxia-regulated pathways is unknown so far and has been addressed in the present study. METHODS: Differentiated human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were cultured with or without myricetin under normoxic and hypoxic conditions for varying time periods. The effect of hypoxia and myricetin on the expression of the investigated adipokines was measured by real-time RT-PCR. Western blot analysis was used for the detection of transcription factors involved in hypoxia-regulated pathways. RESULTS: Myricetin interfered in the hypoxia-induced regulation of adipokines and the underlying pathways, which are involved in transmitting the inflammatory response. It strongly repressed hypoxia-induced expression of apelin, leptin, chemerin, asprosin, and DPP-4 and HIF-1α accumulation in the nucleus was diminished. Furthermore, the activation of the key regulators in the inflammatory response NF-κB, Akt, and CREB was suppressed by myricetin under hypoxic conditions. Myricetin also decreased hypoxia-induced accumulation of the pro-tumorigenic transcription factors Snail and Slug in the nucleus. CONCLUSION: Taken together, our results indicated that myricetin regulated hypoxia-induced expression of adipokines and hypoxia-regulated pathways in human adipocytes. Our study therefore provided evidence of the anti-inflammatory effects of myricetin in hypoxia-treated human adipocytes.

Lipid profiles of patients with manifest coronary versus peripheral atherosclerosis - Is there a difference?

AIM: Peripheral arterial disease (PAD) and coronary artery disease (CAD) are both caused by atherosclerosis. Serum lipids and lipoproteins are predictive of the development of atherosclerosis but it is not clear if they differ in the two manifestations, PAD and CAD. We tested whether a more detailed characterization of the lipid and lipoprotein patterns of PAD and CAD allows a clear differentiation between the two atherosclerotic phenotypes. METHODS: A cohort of 274 statin-naïve patients with either newly diagnosed imaging proven PAD (n = 89) or stable CAD (n = 185) was characterized using nuclear magnetic resonance- and liquid chromatography-tandem mass spectrometry-based advanced lipid and lipoprotein analysis. An independent cohort of 1239 patients with PAD and CAD was used for validation. RESULTS: We found a significant difference in markers of inflammation as well as ceramide and phosphatidylcholine levels between patients with PAD and CAD. In contrast, basic lipid markers including total cholesterol, LDL cholesterol, HDL cholesterol, lipoprotein(a) or detailed lipoprotein profiles did not differ significantly between patients with PAD and CAD. Applying ratios and scores derived from ceramides and phosphatidylcholines further improved the discrimination between PAD and CAD. These significant differences were independent of body composition, from the status of smoking or type 2 diabetes mellitus, and also from apolipoprotein C-III and other inflammatory parameters which were different between CAD and PAD. CONCLUSION: The present study clearly suggests that PAD and CAD differ in terms of their ceramide- and phosphatidylcholine-based lipid patterns but not in lipoprotein characteristics.

Efficient protection of microorganisms for delivery to the intestinal tract by cellulose sulphate encapsulation.

BACKGROUND: Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough. RESULTS: We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS), that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for at least 4 h without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase at concentrations found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract. CONCLUSION: Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.

Real-time PCR based detection of the lactase non-persistence associated genetic variant LCT-13910C>T directly from whole blood.

Primary hypolactasia is the main cause of lactose intolerance in adults. It is strongly associated with the single genetic variant LCT-13910C>T, located upstream of the lactase encoding gene. Consequently, analysis of LCT-13910C>T has been recommended as a direct genetic test for the trait. The aim of our study was to develop a TaqMan probe based real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood, circumventing DNA isolation. The LCT-13910C>T variant was determined using the DirectBlood Genotyping PCR Kit (myPOLS Biotec, Konstanz, Germany) together with an in-house TaqMan primer-probe assay. Validity and specificity of the assay was evaluated using EDTA anti-coagulated whole blood samples and corresponding DNA samples. Results from real-time PCR were compared with results obtained by Sanger sequencing from 105 blinded whole blood samples. Validity and specificity of the assay using whole blood were comparable to those using purified genomic DNA as substrate in PCR. Genetic analysis of blood samples were in complete agreement with results obtained by Sanger sequencing. In conclusion, we present a reliable real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood further facilitating diagnosis of primary hypolactasia in symptomatic patients.

Circulating Glypican-4 Is a Predictor of 24-Month Overall Survival in Metastatic Breast Cancer.

INTRODUCTION: Endocrine treatment combined with CDK4/6 inhibitors is the preferred treatment strategy in patients presenting with ER-positive/HER2-negative breast cancer, but the clinical course remains highly variable among individual patients. There is an unmet need for prognostic or predictive biomarkers in this important group of patients. Recently, we have identified circulating glypican-4 (GPC4) as a new biomarker of inferior outcomes in patients with metastatic colorectal cancer. The impact of plasma GPC4 levels on the survival of breast cancer patients is unknown and has been addressed in the present study. METHODS: Our study included 47 patients with ER-positive/HER2-negative metastatic breast cancer prior to treatment with CDK4/6 inhibitors combined with endocrine therapy. The endpoint was overall survival (OS) at 24 months. GPC4 levels were measured in plasma using an enzyme-linked immunosorbent assay. RESULTS: Increased circulating GPC4 levels were significantly linked to advanced age, postmenopausal state, visceral metastases, and invasive lobular carcinoma. During the 2-year observational follow-up period, 25.5% of patients died. The area under the receiver operating characteristic curve (ROC-AUC) analysis revealed an AUC of 0.713 [0.555-0.871]; p = 0.029 for OS; and an optimal cutoff value of GPC4 for predicting OS of 4.77 ng/mL. No patient showing GPC4 values below this cutoff died during the observational period. Cox regression analysis showed a hazard ratio of 2.14 [95% confidence interval: 1.24-3.67]; p = 0.006 for one standard deviation change of plasma GPC4. CONCLUSIONS: Our study suggests circulating GPC4 as a significant predictor of poor survival in metastatic breast cancer patients.

Coronary Event Risk Test (CERT) as a Risk Predictor for the 10-Year Clinical Outcome of Patients with Peripheral Artery Disease.

(1) Background: Ceramides are a new kind of lipid biomarker and have already been demonstrated to be valuable risk predictors in coronary patients. Patients with peripheral artery disease (PAD) are a population with a worse prognosis and higher mortality risk compared to coronary artery disease (CAD) patients. However, the value of ceramides for risk prediction in PAD patients is still vague, as addressed in the present study. (2)Methods: This observational study included 379 PAD patients. The primary endpoint was all-cause mortality at 10 years of follow-up. A set of ceramides was measured by LC-MS/MS and combined according to the Coronary Event Risk Test (CERT) score, which categorizes patients into one of four risk groups (low risk, moderate risk, high risk, very high risk). (3) Results: Kaplan-Meier survival curves revealed that the overall survival of patients decreased with the increasing risk predicted by the four CERT categories, advancing from low risk to very high risk. Cox regression analysis demonstrated that each one-category increase resulted in a 35% rise in overall mortality risk (HR = 1.35 [1.16-1.58]). Multivariable adjustment, including, among others, age, LDL-cholesterol, type 2 diabetes, and statin treatment before the baseline, did not abrogate this significant association (HR = 1.22 [1.04-1.43]). Moreover, we found that the beneficial effect of statin treatment is significantly stronger in patients with a higher risk, according to CERT. (4) Conclusions: We conclude that the ceramide-based risk score CERT is a strong predictor of the 10-year mortality risk in patients with PAD.

Circulating glypican-4 is a new predictor of all-cause mortality in patients with heart failure.

BACKGROUND: Heart failure confers a high burden of morbidity and mortality. However, risk prediction in heart failure patients still is limited. Blood-based biomarkers hold promise to improve clinical risk assessment. Recently we have identified circulating glypican-4 (GPC4) as a significant predictor of mortality in coronary angiography patients and patients with peripheral artery disease. The impact of serum GPC4 on mortality in patients with heart failure is unknown and is addressed in this prospective cohort study. METHODS: We prospectively recorded all-cause mortality in 288 patients with heart failure. GPC4 levels were measured using an enzyme-linked immunosorbent assay at baseline. RESULTS: During the 24-month follow-up period, 28.1% (n = 81) of the patients died. Serum GPC4 significantly predicted all-cause mortality (hazard ratio (HR) per doublingof GPC4 = 3.57 [2.31-5.53]; P < 0.001). Subgroup analysis showed that GPC4 was significantly associated with all-cause mortality in patients with reduced ejection fraction (HR per doubling = 3.25 [1.75-6.04]; P < 0.001) as well as in those with preserved ejection fraction (HR per doubling = 3.07 [1.22-7.70]; P = 0.017). The association between serum GPC4 and all-cause mortality remained significant in multivariable Cox regression analysis correcting for traditional risk factors (P = 0.035). Results from C-statistics indicated an additional prognostic value of GPC4 relative to NT-proBNP for the prediction of two-year all-cause mortality (P = 0.030). CONCLUSION: Circulating GPC4 independently predicts all-cause mortality in patients with heart failure.

Cell-free circulating RAS mutation concentrations significantly impact the survival of metastatic colorectal cancer patients.

PURPOSE: RAS mutations are predictors of an adverse outcome in EGFR-targeted therapies and have been proposed as prognostic biomarkers of survival in metastatic colorectal cancer (mCRC). The analysis of circulating tumor DNA from plasma samples, known as liquid biopsies, has indicated that the RAS mutation status may change over time, potentially affecting patients' prognosis. To further evaluate the clinical validity of RAS mutation retesting using liquid biopsies, we prospectively investigated the impact of the circulating quantitative RAS mutation status on the course of mCRC. METHODS: The present study included 81 consecutively recruited patients with mCRC. We used targeted next-generation sequencing of circulating cell-free DNA to determine and quantify plasma RAS mutation status. RESULTS: Patients with a RAS mutation detected by liquid biopsy (37%; n = 30) were at increased risk of death during the follow-up period compared to RAS wild-type patients. Patients with evidence of a RAS mutation in the primary tumor but a putative RAS mutation loss in plasma (28%; n = 11) showed a prolonged survival compared to patients with a preserved RAS mutation status. Also, circulating RAS mutation concentrations significantly affected the outcome: The mortality risk of patients with a high RAS mutation concentration increased fivefold compared to subjects with a putative RAS mutation loss or low RAS mutation concentration. CONCLUSION: Our results emphasize the clinical value of circulating RAS mutations in managing mCRC.

Data on the association of serum glypican-4 with future major adverse cardiovascular events and mortality in patients undergoing coronary angiography.

This data article is associated to the research article titled 'Serum glypican-4 is a marker of future vascular risk and mortality in coronary angiography patients' (Muendlein et al., 2022). The present article provides additional prospective data on the association of serum glypican-4 (GPC4) with the incidence of future major adverse cardiovascular events (MACE), vascular mortality, and overall mortality in a cohort of 760 coronary angiography patients. Serum GPC4 levels significantly differed between patients with or without an event during follow up. The results were confirmed in subgroup analyses with respect to age, sex, type 2 diabetes mellitus, obesity, the presence of significant coronary stenoses, and renal function, as well as medical treatment. That said, an interaction term between GPC4 and impaired renal function and between GPC4 and the use of beta blockers on the incidence of future fatal events reached statistical significance. In addition, C-statistics were performed showing an additional predictive value of categorized GPC4 to a basic risk model including traditional risk factors for overall mortality.

Evaluation of the association of serum glypican-4 with prevalent and future kidney function.

Serum glypican-4 (GPC4) has been identified as an insulin-sensitizing adipokine serving as a marker for body mass index and insulin resistance in humans. The association of circulating GPC4 with kidney function is to date largely unexplored. Therefore, we aimed to evaluate the association between serum GPC4 and prevalent as well future kidney function in a prospective cohort study. The study included 456 Caucasian coronary angiography patients. After a median follow up period of 3.4 years, data on kidney function was reassessed in all patients. Chronic kidney disease (CKD) was defined by decreased estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 or albuminuria. At baseline, serum GPC4 was significantly associated with decreased eGFR (adjusted odds ratio (OR) per standard deviation = 4.75 [2.66-8.48]; P < 0.001), albuminuria (OR = 1.49 [1.15-1.92]; P = 0.002), and, accordingly, with CKD (OR = 1.75 [1.35-2.26]; P < 0.001). GPC4 levels also significantly and independently predicted the incidence of newly diagnosed decreased eGFR (OR = 2.74 [1.82-4.14]; P < 0.001, albuminuria (OR = 1.58 [1.01-2.46]; P = 0.043, and CKD (OR = 2.16 [1.45-3.23]; P < 0.001). ROC analysis indicated an additional predictive value of GPC4 to a basic prediction model for newly diagnosed CKD and eGFR < 60 mL/min/1.73 m2. Our study, therefore, indicates that high serum GPC4 is associated with decreased prevalent and future kidney function.

Serum glypican-4 is a marker of future vascular risk and mortality in coronary angiography patients.

BACKGROUND AND AIMS: Glypican-4 (GPC4) is a cell surface protein, but can be released into circulation under various clinical conditions. The association of circulating GPC4 with the risk of future cardiovascular events or death is unclear. In the present study, we aimed to investigate the association between serum GPC4 and major adverse cardiovascular events (MACE), vascular mortality, and all-cause mortality in a prospective cohort study. METHODS: Our study included 760 patients undergoing coronary angiography. During a mean follow up period of 6.3 years, the incidence of MACE, vascular mortality, and all-cause mortality was recorded. Serum GPC4 levels were determined using an enzyme-linked immunosorbent assay. RESULTS: Serum GPC4 was highly significantly associated with increased age, body mass index, brain natriuretic peptide, and oxidized low density lipoprotein, as well as with decreased estimated glomerular filtration rate. During the follow-up period, 145 patients died, including 67 vascular deaths. MACE occurred in 137 patients. Serum GPC4 was significantly associated with MACE, vascular mortality, and all-cause mortality independently of traditional cardiovascular risk factors, with adjusted hazard ratios (HR) and 95% confidence intervals for one standard deviation change of serum GPC4 of 1.32 [1.10-1.58], 1.38 [1.06-1.78], and 1.53 [1.29-1.82], respectively. The best cut-off value for serum GPC4 for predicting MACE, vascular mortality, and all-cause mortality was 7.24 ng/ml for all three endpoints. Respective adjusted HRs were 1.61 [1.07-2.43], 2.85 [1.62-5.01], and 2.92 [2.00-4.27]. CONCLUSIONS: Our study indicates that elevated serum GPC4 levels are significantly associated with an increased risk of MACE, vascular mortality, and all-cause mortality.

Serum glypican-4 is associated with the 10-year clinical outcome of patients with peripheral artery disease.

BACKGROUND: Patients with peripheral artery disease (PAD) are at increased risk of cardiovascular events and mortality compared with non-PAD populations. Blood based biomarkers may improve clinical risk assessment. Recently, we found significant associations of serum glypican-4 (GPC4) with cardiovascular events and mortality in coronary angiography patients. The impact of serum GPC4 on the clinical outcome in PAD patients is unknown and has been addressed in a prospective cohort study. METHODS: We measured GPC4 levels using an enzyme-linked immunosorbent assay in 295 PAD patients. The primary endpoint was major adverse cardiovascular events (MACE); we further investigated vascular mortality and all-cause mortality over 10 years of follow-up. RESULTS: Serum GPC4 levels were positively linked with age, low kidney function, C-reactive protein (CRP), and the use of cardiovascular medications. During the 10-year follow-up period, MACE, vascular mortality, and all-cause mortality occurred in 43.1%, 33.4%, and 45.4%, respectively, of the patients. High serum GPC4 was significantly associated with all three endpoints (each log-rank P-value <0.001). In Cox regression analysis serum GPC4 significantly predicted MACE, vascular mortality, and all-cause mortality independently from traditional risk factors including age, sex, T2DM, hypertension, low kidney function, severity of PAD, smoking, and CRP, with adjusted hazard ratios [95% confidence interval] for one standard deviation change of serum GPC4 of 1.38 [1.06-1.80], 1.84 [1.28-2.64], and 1.94 [1.51-2.51], respectively. CONCLUSION: We conclude that serum GPC4 is a predictor of the 10-year clinical outcome in patients with PAD.

SARS-CoV-2 RBD-specific and NP-specific antibody response of healthcare workers in the westernmost Austrian state Vorarlberg: a prospective cohort study.

OBJECTIVES: Austria, and particularly its westernmost federal state Vorarlberg, developed an extremely high incidence rate during the COVID-19 pandemic. Healthcare workers (HCWs) worldwide are known to have an increased risk of contracting the disease within the working environment and, therefore, the seroprevalence in this population is of particular interest. We thus aimed to analyse SARS-CoV-2-specific antibody dynamics in Vorarlberg HCWs. DESIGN: Prospective cohort study of HCWs including testing at three different time points for the prevalence of anti-SARS-CoV-2 IgG antibodies specific for nucleocapsid protein (NP) and receptor-binding domain (RBD). SETTING: All five state hospitals of Vorarlberg. PARTICIPANTS: A total of 395 HCWs, enrolled in June 2020 (time point 1 (t1)), 2 months after the end of the first wave, retested between October and November at the beginning of the second wave (time point 2 (t2)) and again at the downturn of the second wave in January 2021 (time point 3 (t3)). MAIN OUTCOMES: We assessed weak and strong seropositivity and associated factors, including demographic and clinical characteristics, symptoms consistent with COVID-19 infection, infections verified by reverse transcription PCR (RT-PCR) and vaccinations. RESULTS: At t1, 3% of HCWs showed strong IgG-specific responses to either NP or RBD. At t2, the rate had increased to 4%, and at t3 to 14%. A strong response was found to be stable for up to 10 months. Overall, only 55% of seropositive specimen had antibodies against both antigens RBD and NP; 29% had only RBD-specific and 16% only NP-specific antibodies. Compared with the number of infections found by RT-PCR, the number of HCWs being seropositive was 38% higher. CONCLUSION AND RELEVANCE: Serological testing based on only one antigen implicates the risk of missing infections; thus, the set of antigens should be broadened in the future. The seroprevalence among participating HCWs was comparable to the general population in Austria. Nevertheless, in view of undetected infections, monitoring and surveillance should be reconsidered.

Value of total cholesterol readings earlier versus later in life to predict cardiovascular risk.

BACKGROUND: Prognostic implications of blood cholesterol may differ at different stages of life. This cohort study compares the value of total cholesterol (TC) readings earlier versus later in life for the prediction of coronary atherosclerosis, cardiovascular events, and cardiovascular death. METHODS: In a cardiovascular observation study (CVOS) we performed coronary angiography and prospectively recorded cardiovascular events in 1090 patients over up to 19 years. These patients had participated in a health survey (HS) 15 years prior to the CVOS baseline. TC was measured twice, first at the earlier HS and then later at CVOS recruiting. FINDINGS: Patients in the highest versus the lowest TC-category of the HS had an OR of 4.30 [2.41-7.65] for significant CAD at angiography, a HR of 1.74 [1.10-2.76] for cardiovascular events, and a HR of 7.55 [1.05-54.49] for cardiovascular death after multivariate adjustment. In contrast, TC as measured at the baseline of the CVOS was neither significantly associated with significant CAD (OR= 0.75 [0.49-1.13]) nor with cardiovascular events or death during follow-up (HR= 0.86 [0.62-1.18] and 0.79 [0.41-1.53], respectively). Moreover, the ESC/EAS-SCORE was found to be more powerful in predicting cardiovascular mortality when using earlier instead of later TC, with a continuous net reclassification improvement of 0.301 (p<0.001). INTERPRETATION: Early measurement not only enables early intervention in keeping with the concept of lifelong exposure to atherogenic lipoproteins. These data also suggest that cardiovascular risk prediction is more accurate if using earlier in life TC readings. FUNDING: The present study did not receive any particular funding.

Serum Parathyroid Hormone Predicts Mortality in Coronary Angiography Patients with Type 2 Diabetes.

BACKGROUND: Elevated serum levels of parathyroid hormone (PTH), one of the main regulators of calcium homeostasis and vitamin D metabolism, have been proposed as predictors of mortality. The impact of type 2 diabetes mellitus (T2DM) on the putative association between PTH and mortality has not been investigated thus far. AIM: The aim of our study was to investigate the impact of T2DM on the power of PTH to predict mortality risk. METHODS: Serum PTH levels were determined in 904 consecutive Caucasian patients referred to coronary angiography for the evaluation of established or suspected stable coronary artery disease (CAD), including 235 patients with T2DM. Prospectively, deaths were recorded over a mean follow-up period of 6.3 years. RESULTS: PTH at baseline did not differ significantly between patients with and without T2DM (P = .307). Cox regression analysis revealed that the serum PTH level strongly predicted all-cause mortality in patients with T2DM (hazard ratio [HR] = 2.35 [1.37-4.03]; P = .002), whereas PTH did not predict all-cause mortality in patients without T2DM (HR = 1.04 [0.81-1.32]; P = .766). The interaction term PTH × T2DM was significant (P = .006), indicating a significantly stronger impact of PTH on mortality risk in patients with T2DM than in individuals without diabetes. The impact of PTH on mortality risk in patients with T2DM remained significant after adjustment for glycated hemoglobin A1c, diabetes duration, classical cardiovascular risk factors, serum levels of vitamin D, and kidney function (HR = 2.10 [1.10-4.10]; P = .030). CONCLUSION: We conclude that PTH is a significantly stronger predictor of all-cause mortality in patients with T2DM than in those without T2DM.

Direct blood PCR: TaqMan-probe based detection of the venous thromboembolism associated mutations factor V Leiden and prothrombin c.20210G>A without DNA extraction.

BACKGROUND: Practically, the initial step of genetic analysis is the extraction of DNA from blood or other cells, which is often time consuming and cost-intensive. We aimed at establishing a real-time PCR protocol for the detection of the venous thromboembolism associated mutations factor V Leiden (F5 c.1691G>A; p.R506Q) and prothrombin (F2) c.20210G>A from whole blood, without DNA extraction. METHODS: F5 c.1691G>A (p.R506Q) and F2 c.20210G>A mutations were determined in 205 EDTA anti-coagulated whole blood samples from patients who underwent routine clinical genotyping using the DirectBlood Genotyping PCR Kit (myPOLS Biotec, Konstanz, Germany) together with in-house developed TaqMan primer-probe assays. RESULTS: Validity score values of genotype calls using whole blood were similar and did not significantly differ compared to those using genomic DNA as substrate in PCR. Mutation analysis of 205 whole blood samples showed a negligible PCR dropout rate (one in 410 reactions) and were in 100% concordance with results obtained by conventional genotyping. CONCLUSION: We successfully established a robust and valid real-time PCR protocol for the detection of the venous thromboembolism associated mutations F5 c.1691G>A (p.R506Q) and F2 c.20210G>A directly from whole blood.

Are SGLT2 polymorphisms linked to diabetes mellitus and cardiovascular disease? Prospective study and meta-analysis.

Inhibition of the sodium glucose co-transporter 2 (SGLT2) reduces cardiovascular morbidity, and mortality in patients with type 2 diabetes mellitus (T2DM) with atherosclerotic, cardiovascular disease. So far, a link between common genetic variations of the SGLT2 encoding gene SLC5A2 and glucose homeostasis as well as cardiovascular disease has not been established. The present study, therefore, aimed to investigate SLC5A2 single nucleotide polymorphisms (SNPs) in relation to type 2 diabetes and coronary artery disease (CAD) and prospectively the incidence of cardiovascular events. We genotyped the SLC5A2 tagging SNPs rs9934336, rs3813008, and rs3116150 in a total of 1684 high risk cardiovascular patients undergoing coronary angiography, including 400 patients with T2DM. Additionally, we performed a meta-analysis combining results from the present study and the literature. Variant rs9934336 was significantly associated with decreased HbA1c (P = 0.023). Further, rs9934336 was significantly inversely associated with the presence of T2DM in univariate (OR = 0.82 [0.68-0.99]; P = 0.037) as well as in multivariate analysis (OR = 0.79 [0.65-0.97]; P = 0.023). The association between rs9934336 and T2DM was confirmed in a meta-analysis including results from two previous observations which by themselves had failed to show a significant association of the polymorphism with T2DM (OR = 0.86 [0.78-0.95]; P = 0.004). Polymorphisms rs3813008 and rs3116150 were associated neither with glycemic parameters nor with T2DM. None of the SNPs tested was significantly associated with the baseline presence of CAD or the incidence of cardiovascular events. We conclude that genetic variation within the SLC5A2 gene locus is significantly related to the manifestation of T2DM.

The novel adipokine CTRP1 is significantly associated with the incidence of major adverse cardiovascular events.

BACKGROUND AND AIMS: The recently identified adiponectin paralogue C1q and tumor necrosis factor-related protein 1 (CTRP1) has been associated with obesity-linked disorders and coronary atherosclerosis. So far, the impact of circulating CTRP1 on the incidence of future cardiovascular events is unclear. Therefore, we aimed at investigating the association between CTRP1 and future cardiovascular risk. METHODS: We measured CTRP1 serum levels in 539 patients undergoing coronary angiography for the evaluation of established or suspected stable coronary artery disease (CAD). Prospectively, we recorded major adverse cardiovascular events (MACE), defined as the incidence of cardiovascular death, non-fatal myocardial infarction and non-fatal stroke over a follow-up period of 8 years. RESULTS: At baseline, obesity, the metabolic syndrome, type 2 diabetes, and non-alcoholic fatty liver disease were significantly associated with increased CTRP1 (all p-values ≤0.001). Prospectively, MACE rates were lowest in the first quartile (15.3%) and increased over the second (23.7%) to the third and fourth quartile (each 29.0%; ptrend = 0.008). Moreover, after multivariable adjustment, CTRP1 was significantly associated with future MACE, with adjusted HRs of 1.83 [1.04-3.23]; p=0.037, 2.16 [1.25-3.75]; p=0.006, and 1.80 [1.03-3.15]; p=0.038, for CTRP1 quartiles two, three and four, respectively, when compared to quartile one. CONCLUSIONS: We conclude that high serum levels of CTRP1 are significantly associated with future MACE.

Data on the association between CTRP1 and future major adverse cardiovascular events in patients undergoing coronary angiography.

This article provides additional data on the association of the new adipokine CTRP1 with the incidence of future major adverse cardiovascular events in a prospective cohort of patients undergoing coronary angiography. In this regard, multivariable Cox proportional hazards models taking into account cardiac risk markers are presented. Additionally, data on the impact of baseline variables including metabolic traits and co-morbidities on the incidence of future major adverse cardiovascular events are shown. This data article is associated to the research article titled 'The Novel Adipokine CTRP1 is Significantly Associated with the Incidence of Major Adverse Cardiovascular Events' Muendlein et al., 2019.