Systematic permutation testing in GWAS pathway analyses: identification of genetic networks in dilated cardiomyopathy and ulcerative colitis

Background: Genome wide association studies (GWAS) are applied to identify genetic loci, which are associated with complex traits and human diseases. Analogous to the evolution of gene expression analyses, pathway analyses have emerged as important tools to uncover functional networks of genome-wide association data. Usually, pathway analyses combine statistical methods with a priori available biological knowledge. To determine significance thresholds for associated pathways, correction for multiple testing and over-representation permutation testing is applied. Results: We systematically investigated the impact of three different permutation test approaches for over-representation analysis to detect false positive pathway candidates and evaluate them on genome-wide association data of Dilated Cardiomyopathy (DCM) and Ulcerative Colitis (UC). Our results provide evidence that the gold standard - permuting the case–control status – effectively improves specificity of GWAS pathway analysis. Although permutation of SNPs does not maintain linkage disequilibrium (LD), these permutations represent an alternative for GWAS data when case–control permutations are not possible. Gene permutations, however, did not add significantly to the specificity. Finally, we provide estimates on the required number of permutations for the investigated approaches. Conclusions: To discover potential false positive functional pathway candidates and to support the results from standard statistical tests such as the Hypergeometric test, permutation tests of case control data should be carried out. The most reasonable alternative was case–control permutation, if this is not possible, SNP permutations may be carried out. Our study also demonstrates that significance values converge rapidly with an increasing number of permutations. By applying the described statistical framework we were able to discover axon guidance, focal adhesion and calcium signaling as important DCM-related pathways and Intestinal immune network for IgA production as most significant UC pathway

Prohormones in the early diagnosis of cardiac syncope

Background--The early detection of cardiac syncope is challenging. We aimed to evaluate the diagnostic value of 4 novel prohormones, quantifying different neurohumoral pathways, possibly involved in the pathophysiological features of cardiac syncope: midregional-pro-A-type natriuretic peptide (MRproANP), C-terminal proendothelin 1, copeptin, and midregionalproadrenomedullin. Methods and Results--We prospectively enrolled unselected patients presenting with syncope to the emergency department (ED) in a diagnostic multicenter study. ED probability of cardiac syncope was quantified by the treating ED physician using a visual analogue scale. Prohormones were measured in a blinded manner. Two independent cardiologists adjudicated the final diagnosis on the basis of all clinical information, including 1-year follow-up. Among 689 patients, cardiac syncope was the adjudicated final diagnosis in 125 (18%). Plasma concentrations of MRproANP, C-terminal proendothelin 1, copeptin, and midregional-proadrenomedullin were all significantly higher in patients with cardiac syncope compared with patients with other causes (P < 0.001). The diagnostic accuracies for cardiac syncope, as quantified by the area under the curve, were 0.80 (95% confidence interval [CI], 0.76-0.84), 0.69 (95% CI, 0.64-0.74), 0.58 (95% CI, 0.52-0.63), and 0.68 (95% CI, 0.63-0.73), respectively. In conjunction with the ED probability (0.86; 95% CI, 0.82-0.90), MRproANP, but not the other prohormone, improved the area under the curve to 0.90 (95% CI, 0.87-0.93), which was significantly higher than for the ED probability alone (P=0.003). An algorithm to rule out cardiac syncope combining an MRproANP level of < 77 pmol/L and an ED probability of < 20% had a sensitivity and a negative predictive value of 99%. Conclusions--The use of MRproANP significantly improves the early detection of cardiac syncope among unselected patients presenting to the ED with syncope

Atlas of the clinical genetics of human dilated cardiomyopathy

[Abstract] Aim. Numerous genes are known to cause dilated cardiomyopathy (DCM). However, until now technological limitations have hindered elucidation of the contribution of all clinically relevant disease genes to DCM phenotypes in larger cohorts. We now utilized next-generation sequencing to overcome these limitations and screened all DCM disease genes in a large cohort. Methods and results. In this multi-centre, multi-national study, we have enrolled 639 patients with sporadic or familial DCM. To all samples, we applied a standardized protocol for ultra-high coverage next-generation sequencing of 84 genes, leading to 99.1% coverage of the target region with at least 50-fold and a mean read depth of 2415. In this well characterized cohort, we find the highest number of known cardiomyopathy mutations in plakophilin-2, myosin-binding protein C-3, and desmoplakin. When we include yet unknown but predicted disease variants, we find titin, plakophilin-2, myosin-binding protein-C 3, desmoplakin, ryanodine receptor 2, desmocollin-2, desmoglein-2, and SCN5A variants among the most commonly mutated genes. The overlap between DCM, hypertrophic cardiomyopathy (HCM), and channelopathy causing mutations is considerably high. Of note, we find that >38% of patients have compound or combined mutations and 12.8% have three or even more mutations. When comparing patients recruited in the eight participating European countries we find remarkably little differences in mutation frequencies and affected genes. Conclusion. This is to our knowledge, the first study that comprehensively investigated the genetics of DCM in a large-scale cohort and across a broad gene panel of the known DCM genes. Our results underline the high analytical quality and feasibility of Next-Generation Sequencing in clinical genetic diagnostics and provide a sound database of the genetic causes of DCM.Hôpitaux de Paris; PHRC AOM0414

Direct Comparison of Cardiac Myosin-Binding Protein C with Cardiac Troponins for the Early Diagnosis of Acute Myocardial Infarction

Cardiac myosin-binding protein C (cMyC) is a cardiac-restricted protein that is more abundant than cardiac troponins (cTn) and is released more rapidly after acute myocardial infarction (AMI). We evaluated cMyC as an adjunct or alternative to cTn in the early diagnosis of AMI.Unselected patients (N=1954) presenting to the emergency department with symptoms suggestive of AMI, concentrations of cMyC, and high-sensitivity (hs) and standard-sensitivity cTn were measured at presentation. The final diagnosis of AMI was independently adjudicated using all available clinical and biochemical information without knowledge of cMyC. The prognostic end point was long-term mortality.Final diagnosis was AMI in 340 patients (17%). Concentrations of cMyC at presentation were significantly higher in those with versus without AMI (median, 237 ng/L versus 13 ng/L

Impact of age on the performance of the ESC 0/1h-algorithms for early diagnosis of myocardial infarction.

Aims We aimed to evaluate the impact of age on the performance of the European Society of Cardiology (ESC) 0/1h-algorithms and to derive and externally validate alternative cut-offs specific to older patients. Methods and results We prospectively enrolled patients presenting to the emergency department (ED) with symptoms suggestive of acute myocardial infarction in three large diagnostic studies. Final diagnoses were adjudicated by two independent cardiologists. High-sensitivity cardiac troponin (hs-cTn) T and I concentrations were measured at presentation and after 1 h. Patients were stratified according to age [<55 years (young), ≥55 to <70 years (middle-age), ≥70 years (old)]. Rule-out safety of the ESC hs-cTnT 0/1h-algorithm was very high in all age-strata: sensitivity 100% [95% confidence interval (95% CI) 94.9-100] in young, 99.3% (95% CI 96.0-99.9) in middle-age, and 99.3% (95% CI 97.5-99.8) in old patients. Accuracy of rule-in decreased with age: specificity 97.0% (95% CI 95.8-97.9) in young, 96.1% (95% CI 94.5-97.2) in middle-age, and 92.7% (95% CI 90.7-94.3) in older patients. Triage efficacy decreased with increasing age (young 93%, middle-age 80%, old 55%, P < 0.001). Similar results were found for the ESC hs-cTnT 0/1h-algorithm. Alternative, slightly higher cut-off concentrations optimized for older patients maintained very high safety of rule-out, increased specificity of rule-in (P < 0.01), reduced overall efficacy for hs-cTnT (P < 0.01), while maintaining efficacy for hs-cTnI. Findings were confirmed in two validation cohorts (n = 2767). Conclusion While safety of the ESC 0/1h-algorithms remained very high, increasing age significantly reduced overall efficacy and the accuracy of rule-in. Alternative slightly higher cut-off concentrations may be considered for older patients, particularly if using hs-cTnI. Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT00470587, number NCT00470587 and NCT02355457 (BACC)

Direct comparison of 2 rule-out strategies for acute myocardial infarction: 2-h accelerated diagnostic protocol vs 2-h algorithm

We compared 2 high-sensitivity cardiac troponin (hs-cTn)-based 2-h strategies in patients presenting with suspected acute myocardial infarction (AMI) to the emergency department (ED): the 2-h accelerated diagnostic protocol (2h-ADP) combining hs-cTn, electrocardiogram, and a risk score, and the 2-h algorithm exclusively based on hs-cTn concentrations and their absolute changes.Analyses were performed in 2 independent diagnostic cohorts [European Advantageous Predictors of Acute Coronary Syndrome Evaluation (APACE) study, Australian-New Zealand 2-h Accelerated Diagnostic Protocol to Assess patients with chest Pain symptoms using contemporary Troponins as the only biomarker (ADAPT) study] employing hs-cTnT (Elecsys) and hs-cTnI (Architect). The final diagnosis was adjudicated by 2 independent cardiologists.AMI was the final diagnosis in 16.5% (95% CI, 14.6%-18.6%) of the 1372 patients in APACE, and 12.6% (95% CI, 10.7%-14.7%) of 1153 patients in ADAPT. The negative predictive value (NPV) and sensitivity for AMI were very high and comparable with both strategies using either hs-cTnT or hs-cTnI in both cohorts (all statistical comparisons nonsignificant). The percentage of patients triaged toward rule-out was significantly lower with the 2h-ADP (36%-43%) vs the 2-h algorithm (55%-68%) with both assays and in both cohorts (P < 0.001). The sensitivity of the 2h-ADP was higher for 30-day major adverse cardiovascular events.Both algorithms provided very high and comparable safety as quantified by the NPV and sensitivity for AMI and major adverse cardiac events (MACE) at 30 days in patients triaged toward rule-out, although sensitivity for MACE at 30 days was lower with both algorithms in cohort 2. Although the 2-h algorithm was more efficacious, not all patients ruled out for AMI by this algorithm were appropriate candidates for early discharge. The 2h-ADP seems superior in the selection of patients for early discharge from the ED.APACE: http://clinicaltrials.gov/show/NCT00470587ADAPT: Australia-New Zealand Clinical Trials Registry ACTRN12611001069943

Early kinetics of cardiac troponin in suspected acute myocardial infarction.

INTRODUCTION AND OBJECTIVES Release kinetics of high-sensitivity cardiac troponin (hs-cTn) T and I in patients with acute myocardial infarction (AMI) are incompletely understood. We aimed to assess whether hs-cTnT/I release in early AMI is near linear. METHODS In a prospective diagnostic multicenter study the acute release of hs-cTnT and hs-cTnI within 1 and 2hours from presentation to the emergency department was quantified using 3 hs-cTnT/I assays in patients with suspected AMI. The primary endpoint was correlation between hs-cTn changes from presentation to 1 hour vs changes from presentation to 2hours, among all AMI patients and different prespecified subgroups. The final diagnosis was adjudicated by 2 independent cardiologists, based on serial hs-cTnT from the serial study blood samples and additional locally measured hs-cTn values. RESULTS Among 2437 patients with complete hs-cTnT data, AMI was the adjudicated diagnosis in 376 patients (15%). For hs-cTnT, the correlation coefficient between 0- to 1-hour change and 0- to 2 hour change was 0.931 (95%CI, 0.916-0.944), P <.001. Similar findings were obtained with hs-cTnI (Architect) with correlation coefficients between 0- to 1-hour change and 0- to 2 hour change of 0.969 and hs-cTnI (Centaur) of 0.934 (P <.001 for both). Findings were consistent among type 1 and type 2 AMI and in the subgroup of patients presenting very early after chest pain onset. CONCLUSIONS Patients presenting with early AMI showed a near linear release of hs-cTnT and hs-cTnI. This near linearity provides the pathophysiological basis for rapid diagnostic algorithms using 0- to 1-hour changes as surrogates for 0- to 2 hour or 0- to 3 hour changes. Registered at ClinicalTrials.gov (Identifier: NCT00470587)

Early kinetics of cardiac troponin in suspected acute myocardial infarction Cinética temprana de troponina en pacientes con sospecha de infarto agudo de miocardio

Introduction and objectives: Release kinetics of high-sensitivity cardiac troponin (hs-cTn) T and I in patients with acute myocardial infarction (AMI) are incompletely understood. We aimed to assess whether hs-cTnT/I release in early AMI is near linear. Methods: In a prospective diagnostic multicenter study the acute release of hs-cTnT and hs-cTnI within 1 and 2 hours from presentation to the emergency department was quantified using 3 hs-cTnT/I assays in patients with suspected AMI. The primary endpoint was correlation between hs-cTn changes from presentation to 1 hour vs changes from presentation to 2 hours, among all AMI patients and different prespecified subgroups. The final diagnosis was adjudicated by 2 independent cardiologists, based on serial hs-cTnT from the serial study blood samples and additional locally measured hs-cTn values. Results: Among 2437 patients with complete hs-cTnT data, AMI was the adjudicated diagnosis in 376 patients (15%). For hs-cTnT, the correlation coefficient between 0- to 1-hour change and 0- to 2 hour change was 0.931 (95%CI, 0.916-0.944),