DNA-pools targeted-sequencing as a robust cost-effective method to detect rare variants: Application to dilated cardiomyopathy genetic diagnosis.

Dilated cardiomyopathy (DCM) is a heart disease characterized by left ventricular dilatation and systolic dysfunction. In 30% of cases, pathogenic variants, essentially private to each patient, are identified in at least one of almost 50 reported genes. Thus, while costly, exons capture-based Next Generation Sequencing (NGS) of a targeted gene panel appears as the best strategy to genetically diagnose DCM. Here, we report a NGS strategy applied to pools of 8 DNAs from DCM patients and validate its robustness for rare variants detection at 4-fold reduced cost. Our pipeline uses Freebayes to detect variants with the expected 1/16 allele frequency. From the whole set of detected rare variants in 96 pools we set the variants quality parameters optimizing true positives calling. When compared to simplex DNA sequencing in a shared subset of 50 DNAs, 96% of SNVs/InsDel were accurately identified in pools. Extended to the 384 DNAs included in the study, we detected 100 variants (ACMG class 4 and 5), mostly in well-known morbid gene causing DCM such as TTN, MYH7, FLNC, and TNNT2. To conclude, we report an original pool-sequencing NGS method accurately detecting rare variants. This innovative approach is cost-effective for genetic diagnostic in rare diseases.

The use of D-dimer in the diagnosis and risk assessment of intracardiac thrombus among patients with dilated cardiomyopathy.

D-dimer is a biomarker of coagulation and fibrinolytic system activation in response to the body's hypercoagulable state. The study aims to investigate the usefulness of D-dimer in diagnosing and assessing the risk of intracardiac thrombus in patients with dilated cardiomyopathy (DCM). Consecutively enrolled in this study were patients with DCM who were admitted to our center for the first time. The diagnostic value was evaluated using the receiver operating characteristic (ROC) curve. Additionally, we used univariate and multivariate logistic regression to investigate the association between D-dimer and intracardiac thrombus. We also performed smooth curve fitting, threshold saturation effect analysis, and subgroup analysis. In total, 534 patients were enrolled in the study, and among them, 65 patients had intracardiac thrombus. Mural thrombus was the predominant type of thrombus, which was mainly located in the left ventricular apex. The optimal cut-off value of D-dimer for the diagnosis of intracardiac thrombus was 484 ng/mL, with a sensitivity and specificity of 0.769 and 0.646, respectively. In both unadjusted and adjusted logistic regression models, a positive association was found between D-dimer and intracardiac thrombus. Curve fitting and threshold effect analysis revealed two inflection points in the relationship between D-dimer and intracardiac thrombus (non-linear test: P = 0.032). When D-dimer was equal to 362 ng/mL, the odds ratio (OR) was 1, and the risk of thrombus gradually increased until it reached 4096 ng/mL, after which the trend no longer increased. Within this range, a twofold increase in D-dimer was associated with a 103.2% increased risk (OR = 2.032; 95% CI 1.293-3.193; P < 0.01). In the subgroup analysis, there was a significant interaction between D-dimer and BMI on intracardiac thrombus (P value for interaction was 0.013), and the risk was higher in patients with a BMI ≥ 25 kg/m2 (OR = 3.44; 95% CI 1.86-6.36; P < 0.01).

Longitudinal Arrhythmic Risk Assessment Based on Ejection Fraction in Patients with Recent-Onset Nonischemic Dilated Cardiomyopathy.

BACKGROUND: Practice guidelines suggest the use of implantable cardioverter-defibrillators in patients with left ventricular ejection fractions (LVEF) ≤ 35% despite 3 to 6 months of guideline-directed medical therapy (GDMT). It remains unclear whether this strategy is appropriate for patients with dilated cardiomyopathy (DCM), who can experience reverse ventricular remodeling for up to 24 months after the initiation of GDMT. The aim of this study was to assess the longitudinal dynamic relationship between LVEF ≤ 35% and arrhythmic risk in patients with recent-onset nonischemic DCM on GDMT. METHODS: A retrospective analysis was conducted among patients with recent-onset DCM (≤6 months) and recent initiation of GDMT (≤3 months) consecutively enrolled in a longitudinal registry. Risk for major ventricular arrhythmic events or sudden cardiac death was assessed in relationship to LVEF ≤ 35% at enrollment and 6 and 24 months after initiation of GDMT. RESULTS: Five hundred forty-four patients met the inclusion criteria. LVEF ≤ 35% identified patients with increased risk for major ventricular arrhythmic events or sudden cardiac death starting from 24 months after initiation of GDMT (hazard ratio, 2.126; 95% CI, 1.065-4.245; P = .03). However, LVEF ≤ 35% at presentation or 6 months after enrollment did not have prognostic significance. Sixty-seven percent of 131 patients with LVEF ≤ 35% at 6 months after initiation of GDMT had improved LVEFs (to >35%) by 24 months. This late LVEF improvement correlated with lower arrhythmic risk (P = .012) and was preceded by a reduction of LV dimensions in the first 6 months of GDMT. CONCLUSIONS: In patients with DCM, the present findings suggest that risk stratification for major ventricular arrhythmic events or sudden cardiac death on the basis of LVEF ≤ 35% is effective after 2 years of GDMT, but not after 6 months. In selected patients with DCM, it would be appropriate to wait 24 months before primary prevention ICD implantation.

Evidence-Based Assessment of Genes in Dilated Cardiomyopathy.

BACKGROUND: Each of the cardiomyopathies, classically categorized as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy, has a signature genetic theme. Hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning >10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. METHODS: An international panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The panel used the Clinical Genome Resource semiquantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories on the basis of the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. RESULTS: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from 8 gene ontologies were classified as having definitive (BAG3, DES, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNT2, TTN) or strong (DSP) evidence. Seven genes (14%; ACTC1, ACTN2, JPH2, NEXN, TNNI3, TPM1, VCL) including 2 additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, 6 were similarly classified for hypertrophic cardiomyopathy and 3 for arrhythmogenic right ventricular cardiomyopathy. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of the 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. CONCLUSIONS: In the curation of 51 genes, 19 had high evidence (12 definitive/strong, 7 moderate). It is notable that these 19 genes explain only a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high-evidence genes; however, genes lacking robust evidence are also commonly included. We recommend that high-evidence DCM genes be used for clinical practice and that caution be exercised in the interpretation of variants in variable-evidence DCM genes.

The combination of carboxy-terminal propeptide of procollagen type I blood levels and late gadolinium enhancement at cardiac magnetic resonance provides additional prognostic information in idiopathic dilated cardiomyopathy - A multilevel assessment of myocardial fibrosis in dilated cardiomyopathy.

AIMS: To determine the prognostic value of multilevel assessment of fibrosis in dilated cardiomyopathy (DCM) patients. METHODS AND RESULTS: We quantified fibrosis in 209 DCM patients at three levels: (i) non-invasive late gadolinium enhancement (LGE) at cardiac magnetic resonance (CMR); (ii) blood biomarkers [amino-terminal propeptide of procollagen type III (PIIINP) and carboxy-terminal propeptide of procollagen type I (PICP)], (iii) invasive endomyocardial biopsy (EMB) (collagen volume fraction, CVF). Both LGE and elevated blood PICP levels, but neither PIIINP nor CVF predicted a worse outcome defined as death, heart transplantation, heart failure hospitalization, or life-threatening arrhythmias, after adjusting for known clinical predictors [adjusted hazard ratios: LGE 3.54, 95% confidence interval (CI) 1.90-6.60; P < 0.001 and PICP 1.02, 95% CI 1.01-1.03; P = 0.001]. The combination of LGE and PICP provided the highest prognostic benefit in prediction (likelihood ratio test P = 0.007) and reclassification (net reclassification index: 0.28, P = 0.02; and integrated discrimination improvement index: 0.139, P = 0.01) when added to the clinical prediction model. Moreover, patients with a combination of LGE and elevated PICP (LGE+/PICP+) had the worst prognosis (log-rank P < 0.001). RNA-sequencing and gene enrichment analysis of EMB showed an increased expression of pro-fibrotic and pro-inflammatory pathways in patients with high levels of fibrosis (LGE+/PICP+) compared to patients with low levels of fibrosis (LGE-/PICP-). This would suggest the validity of myocardial fibrosis detection by LGE and PICP, as the subsequent generated fibrotic risk profiles are associated with distinct cardiac transcriptomic profiles. CONCLUSION: The combination of myocardial fibrosis at CMR and circulating PICP levels provides additive prognostic value accompanied by a pro-fibrotic and pro-inflammatory transcriptomic profile in DCM patients with LGE and elevated PICP.

Case Report: Early Transplant Rejection of a Methanol-Intoxicated Donor Heart in a Young Female Patient. A Diagnostic Approach With CMR, Cardiac Biopsy, and Genetic Risk Assessment.

This case report describes the contributions of multimodality imaging, cardiac biopsy, and genetic sequencing to the diagnosis and management of heart transplant rejection in a 23-year old patient with dilated cardiomyopathy.

Follow-up care by a genetic counsellor for relatives at risk for cardiomyopathies is cost-saving and well-appreciated: a randomised comparison.

Increasing numbers of patient relatives at risk of developing dilated or hypertrophic cardiomyopathy (DCM/HCM) are being identified and followed up by cardiologists according to the ACC/ESC guidelines. However, given limited healthcare resources, good-quality low-cost alternative approaches are needed. Therefore, we have compared conventional follow-up by a cardiologist with that provided at a cardiogenetic clinic (CGC) led by a genetic counsellor. Phenotype-negative first-degree relatives at risk for DCM/HCM were randomly assigned to see either a cardiologist or to attend a CGC. Uptake and resource use were recorded. For 189 participants, we evaluated quality of care experienced, patient satisfaction and perceived personal control (PPC) using validated questionnaires and estimated the average cost difference of these two modes of care. Maximum patient satisfaction scores were achieved more frequently at the CGC (86% vs 45%, P<0.01). In terms of follow-up care provided, the genetic counsellor did not perform worse than the cardiologist (95% vs 59%, P<0.01). The genetic counsellor more often enquired about the relative-at risk's health (100% vs 65%, P<0.01) and family health (97% vs 33%, P<0.01), measured blood pressure (98% vs 29%, P<0.01) and gave disease-specific information (77% vs 52%, P<0.01). Although PPC scores were equal in both groups, the average cost per patient of CGC follow-up was 25% lower. Follow-up of phenotype-negative relatives at risk for DCM/HCM at a CGC led to greater patient satisfaction and is well-appreciated at lower cost. CGC care is a good alternative to conventional cardiological follow-up for this growing group of patients.

Assessment of myocardial deformation with cardiac magnetic resonance strain imaging improves risk stratification in patients with dilated cardiomyopathy.

AIMS: To investigate the prognostic impact of left-ventricular (LV) cardiac magnetic resonance (CMR) deformation imaging in patients with non-ischaemic dilated cardiomyopathy (DCM) compared with late-gadolinium enhancement (LGE) quantification and LV ejection fraction (EF). METHODS AND RESULTS: A total of 210 subjects with DCM were examined prospectively with standard CMR including measurement of LGE for quantification of myocardial fibrosis and feature tracking strain imaging for assessment of LV deformation. The predefined primary endpoint, a combination of cardiac death, heart transplantation, and aborted sudden cardiac death, occurred in 26 subjects during the median follow-up period of 5.3 years. LV radial, circumferential, and longitudinal strains were significantly associated with outcome. Using separate multivariate analysis models, global longitudinal strain (average of peak negative strain values) and mean longitudinal strain (negative peak of the mean curve of all segments) were independent prognostic parameters surpassing the value of global and mean LV radial and circumferential strain, as well as NT-proBNP, EF, and LGE mass. A global longitudinal strain greater than -12.5% predicted outcome even in patients with EF < 35% (P < 0.01) and in those with presence of LGE (P < 0.001). Mean longitudinal strain was further investigated using a clinical model with predefined cut-offs (EF < 35%, presence of LGE, NYHA class, mean longitudinal strain greater than -10%). Mean longitudinal strain exhibited an independent prognostic value surpassing that provided by NYHA, EF, and LGE (HR = 5.4, P < 0.01). CONCLUSION: LV longitudinal strain assessed with CMR is an independent predictor of survival in DCM and offers incremental information for risk stratification beyond clinical parameters, biomarker, and standard CMR.

Clinical use of multimodality imaging in the assessment of dilated cardiomyopathy.

Learning objectives: =To understand the use of cardiovascular imaging for diagnosis, evaluation of prognosis and for supporting treatment decisions and monitoring therapy in patients with dilated cardiomyopathy by providing morphologic, functional and etiologic information, including refined assessment of ventricular function. =To provide to the clinical cardiologist the information on what to expect from each imaging modality and how to work together with the cardiovascular imaging expert to fully explore the potential of complementary imaging techniques. = To provide a look into the future role of new imaging modalities such as molecular imaging.

Usefulness of combined history, physical examination, electrocardiogram, and limited echocardiogram in screening adolescent athletes for risk for sudden cardiac death.

Sudden cardiac death in the young (SCDY) is the leading cause of death in young athletes during sport. Screening young athletes for high-risk cardiac defects is controversial. The purpose of this study was to assess the utility and feasibility of a comprehensive cardiac screening protocol in an adolescent population. Adolescent athletes were recruited from local schools and/or sports teams. Each subject underwent a history and/or physical examination, an electrocardiography (ECG), and a limited echocardiography (ECHO). The primary outcome measure was identification of cardiac abnormalities associated with an elevated risk for sudden death. We secondarily identified cardiac abnormalities not typically associated with a short-term risk of sudden death. A total of 659 adolescent athletes were evaluated; 64% men. Five subjects had cardiac findings associated with an elevated risk for sudden death: prolonged QTc >500 ms (n = 2) and type I Brugada pattern (n = 1), identified with ECG; dilated cardiomyopathy (n = 1) and significant aortic root dilation; and z-score = +5.5 (n = 1). History and physical examination alone identified 76 (11.5%) subjects with any cardiac findings. ECG identified 76 (11.5%) subjects in which a follow-up ECHO or cardiology visit was recommended. Left ventricular mass was normal by ECHO in all but 1 patient with LVH on ECG. ECHO identified 34 (5.1%) subjects in whom a follow-up ECHO or cardiology visit was recommended. In conclusion, physical examination alone was ineffective in identification of subjects at elevated risk for SCDY. Screening ECHO identified patients with underlying cardiac disease not associated with immediate risk for SCDY. Cost of comprehensive cardiac screening is high.

Assessment of myocardial partition coefficient of gadolinium (λ) in dilated cardiomyopathy and its impact on segmental and global systolic function.

PURPOSE: 1) To assess the myocardial partition coefficient (λ) of gadolinium quantified using T1 mapping in dilated cardiomyopathy (DCM); and 2) to assess the impact of increased λ on left ventricular (LV) circumferential strain and ejection fraction in DCM. MATERIALS AND METHODS: Seventeen patients with DCM and 11 controls were prospectively included. All patients and controls underwent a 1.5 T MRI using: 1) cine to quantify LV volumes and function; 2) tagging to quantify circumferential strain in mid-LV; 3) T1 mapping before and 9 minutes after contrast injection to quantify R1, ΔR1, and λ; and 4) inversion recovery 3D Flash was used to assess late gadolinium enhancement (LGE) 10 minutes after Gd DOTA injection (0.2 mmol/kg). We used Student's t-test to compare means, Pearson's test for correlation assessment, and a mixed linear model to integrate the dependency between myocardial segments. RESULTS: No difference in median λ was measured between patients with (0.52 [interquartile range = 0.48-0.56]) and without enhancement on LGE (0.51 [0.47-0.54]; P = 0.07). Circumferential strain value measured in each segment was correlated with the λ measured in the corresponding segment (r = 0.55; P < 0.0001). Multivariate analysis revealed a significant link between the λ in each segment and circumferential strain (0.002 ± 0.001; P = 0.009) and also with ejection fraction (-0.001 ± 0.0008; P = 0.04). CONCLUSION: In DCM, λ correlates independently with circumferential strain and ejection fraction, suggesting that there is a link between λ and systolic function.

Quantitative assessment of cardiac mechanical synchrony using equilibrium radionuclide angiography.

BACKGROUND: Data on normal parameters of cardiac mechanical synchrony is limited, variable and obtained from small cohorts till date. In most studies, software used for such assessment has not been mentioned. The aim of study is to establish normal values of mechanical synchrony with equilibrium radionuclide angiography (ERNA) in a larger population using commercially available software. METHODS: We retrospectively analysed ERNA studies of 108 patients having low pretest likelihood of coronary artery disease, no known history of cardiac disease, normal electrocardiogram and whose ERNA studies were considered normal by experienced observers. In addition, ten patients diagnosed with dilated cardiomyopathy (DCM) and having LVEF ≤ 40% underwent ERNA. Fourier first harmonic analysis of phase images was used to quantify synchrony parameters using commercially available software (XT-ERNA). Intraventricular synchrony for each ventricle was measured as the standard deviation of the LV and RV mean phase angles (SD LVmPA and SD RVmPA, respectively). Interventricular synchrony was measured as LV-RVmPA. Absolute interventricular delay was calculated as absolute difference between LV and RVmPA (without considering ± sign). All variables were expressed in milliseconds (ms) and degree (°). Intra-observer and inter-observer variabilities were assessed. Cut-off values for parameters were calculated from the normal database, and validated against patient group. RESULTS: On phase analysis, LVmPA was observed to be 343 ± 48.5 milliseconds (174.7° ± 18.5°), SD LVmPA was 16.3 ± 5.4 milliseconds (8.2° ± 2.5°), RVmPA was 339 ± 50.4 milliseconds (171.8° ± 18.5°) and SD RVmPA was 37.3 ± 15.7 milliseconds (18.7° ± 7.2°). LV-RVmPA was observed to be 3.9 ± 21.7 milliseconds (2.9° ± 9.6°) and absolute interventricular delay was 16.3 ± 14.8 milliseconds (7.9° ± 6.1°). The cut-off values for the presence of dyssynchrony were estimated as SD LVmPA > 27.1 milliseconds (>13.2°), SD RVmPA > 68.7 milliseconds (>33.1°) and LV-RVmPA > 47.3 milliseconds (>22.1°). There was no statistically significant intra-observer or inter-observer variability. Using these cut offs, 9 patients with DCM showed the presence of left intraventricular dyssynchrony, 5 had right intraventricular dyssynchrony and 2 had interventricular dyssynchrony. CONCLUSIONS: ERNA phase analysis offers an objective and reproducible tool to quantify cardiac mechanical synchrony using commercially available software and can be used in routine clinical practice to assess mechanical dyssynchrony.

Comparison of (99m)Tc-MIBI SPECT/18F-FDG PET imaging and cardiac magnetic resonance imaging in patients with idiopathic dilated cardiomyopathy: assessment of cardiac function and myocardial injury.

OBJECTIVE: The aim of this study is to evaluate the agreement between myocardial F-FDG PET imaging and cardiac magnetic resonance imaging (cMRI) in assessing cardiac function and relationship of cMRI late gadolinium enhancement (cMRI-LGE) and myocardial perfusion/metabolism pattern in patients with idiopathic dilated cardiomyopathy (IDCM). METHODS: Forty-two consecutive patients diagnosed with IDCM were enrolled. All patients underwent Tc-MIBI SPECT, gated F-FDG PET imaging, and cMRI within 3-7 days. Cardiac function parameters were calculated using PET and cMRI. The segments analysis was performed using a 17-segment model. Patterns of perfusion/metabolism were classified as normal, mismatch, mild-to-moderate match, and severe match, and cMRI-LGE was classified into 3 categories (non-LGE, mid-wall LGE, and transmural LGE). RESULTS: The correlation between gated PET and cMRI was excellent for end-diastolic volume (EDV; r = 0.948, P < 0.001), end-systolic volume (ESV; r = 0.939, P < 0.001), and left ventricular ejection fraction (LVEF; r = 0.685, P < 0.001). EDV and ESV were underestimated, whereas LVEF was slightly overestimated by gated PET in comparison to cMRI. Perfusion/metabolism patterns varied in 3 different categories of non-LGE, mid-wall LGE, and transmural LGE (χ = 14.276, P < 0.001). Also, 71.0% (44/62) segments with mid-wall LGE had normal perfusion/metabolism patterns, and 75.9% (63/83) perfusion/metabolism mismatch segments were shown as non-LGE. The incidence of LGE was significantly higher in segments with severe match than the other 3 segment groups (χ = 112.53, P < 0.001). CONCLUSION: There is an excellent agreement between gated PET and cMRI in assessment of cardiac function. LGE-cMRI is much more sensitive in detecting moderate fibrosis, while PET could detect more impaired but viable myocardium. Combining the 2 imaging modalities is useful for providing more comprehensive evaluations of myocardial injury in patients with IDCM.

Copeptin in the assessment of acute lung injury and cardiogenic pulmonary edema.

BACKGROUNDS: Copeptin has been studied as an excellent predictor of outcome in a variety of diseases, its value is even superior to that of B-type natriuretic peptide (BNP) in heart failure, but little is known about its characteristics in acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). We sought to assess the diagnostic and prognostic value of copeptin together with N-terminal pro-BNP (NT-proBNP) in patients with ARDS/ALI or cardiogenic pulmonary edema (CPE). METHODS: Measurement of copeptin and NT-proBNP levels in plasma from 121 consecutive patients with either ARDS/ALI or CPE enrolled in a prospective single center study. RESULTS: In a derivation cohort of 87 patients with ARDS/ALI and 34 patients with CPE, a copeptin threshold of >40.11 pmol/L provided a specificity of 88.2% and a sensitivity of 60.9% for the diagnosis of ARDS/ALI, a NT-proBNP cut point of <2813 pg/ml had a specificity of 79.4% and sensitivity of 65.5% for it. Multivariate Cox regression analysis showed that copeptin was the strongest predictor for mortality in patients with ARDS/ALI [hazard ratio (HR) = 4.72, P < 0.001] and CPE (HR = 3.52, P = 0.019), the association between increasing copeptin and death was statistically significant in patients with ARDS/ALI (HR = 2.64, P = 0.035) and patients with CPE (HR = 1.62, P = 0.029). CONCLUSION: Copeptin of >40.11 pmol/L had a high specificity for the diagnosis of ARDS/ALI in patients presenting with ARDS/ALI or CPE. Compared to NT-proBNP, copeptin was a stronger prognostic marker for short-term mortality.