Automated assessment of cardiac dynamics in aging and dilated cardiomyopathy Drosophila models using machine learning.

The Drosophila model is pivotal in deciphering the pathophysiological underpinnings of various human ailments, notably aging and cardiovascular diseases. Cutting-edge imaging techniques and physiology yield vast high-resolution videos, demanding advanced analysis methods. Our platform leverages deep learning to segment optical microscopy images of Drosophila hearts, enabling the quantification of cardiac parameters in aging and dilated cardiomyopathy (DCM). Validation using experimental datasets confirms the efficacy of our aging model. We employ two innovative approaches deep-learning video classification and machine-learning based on cardiac parameters to predict fly aging, achieving accuracies of 83.3% (AUC 0.90) and 79.1%, (AUC 0.87) respectively. Moreover, we extend our deep-learning methodology to assess cardiac dysfunction associated with the knock-down of oxoglutarate dehydrogenase (OGDH), revealing its potential in studying DCM. This versatile approach promises accelerated cardiac assays for modeling various human diseases in Drosophila and holds promise for application in animal and human cardiac physiology under diverse conditions.

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.

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.

Systematic large-scale assessment of the genetic architecture of left ventricular noncompaction reveals diverse etiologies.

PURPOSE: To characterize the genetic architecture of left ventricular noncompaction (LVNC) and investigate the extent to which it may represent a distinct pathology or a secondary phenotype associated with other cardiac diseases. METHODS: We performed rare variant association analysis with 840 LVNC cases and 125,748 gnomAD population controls, and compared results to similar analyses on dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). RESULTS: We observed substantial genetic overlap indicating that LVNC often represents a phenotypic variation of DCM or HCM. In contrast, truncating variants in MYH7, ACTN2, and PRDM16 were uniquely associated with LVNC and may reflect a distinct LVNC etiology. In particular, MYH7 truncating variants (MYH7tv), generally considered nonpathogenic for cardiomyopathies, were 20-fold enriched in LVNC cases over controls. MYH7tv heterozygotes identified in the UK Biobank and healthy volunteer cohorts also displayed significantly greater noncompaction compared with matched controls. RYR2 exon deletions and HCN4 transmembrane variants were also enriched in LVNC, supporting prior reports of association with arrhythmogenic LVNC phenotypes. CONCLUSION: LVNC is characterized by substantial genetic overlap with DCM/HCM but is also associated with distinct noncompaction and arrhythmia etiologies. These results will enable enhanced application of LVNC genetic testing and help to distinguish pathological from physiological noncompaction.

Assessment of PDK4 and TTN gene variants in 48 Doberman Pinschers with dilated cardiomyopathy.

OBJECTIVE: To evaluate the frequency of variants in the pyruvate kinase dehydrogenase 4 (PDK4) and titin (TTN) genes in a group of Doberman Pinschers with dilated cardiomyopathy (DCM) and to determine whether there were unique clinical attributes to each variant. ANIMALS: 48 Doberman Pinschers with DCM. PROCEDURES: Doberman Pinschers with recently diagnosed DCM were identified, and genomic DNA from each was genotyped with a PCR assay for detection of PDK4 and TTN genetic variants. Dogs were grouped on the basis of whether they had the TTN variant alone, PDK4 variant alone, both variants, or neither variant. Descriptive statistics were compiled for dog age, body weight, and left ventricular dimensions and fractional shortening and for the presence of ventricular and supraventricular arrhythmias and heart failure. Results were compared across groups. RESULTS: Of the 48 dogs, 28 had the TTN variant alone, 10 had both variants, 6 had neither variant, and 4 had the PDK4 variant alone. The mean age was younger for dogs with the PDK4 variant alone, compared with other dogs. However, the number of dogs with the PDK4 variant alone was very small, and there was an overlap in age across groups. No other meaningful differences were detected across groups, and independent genotype-phenotype relationships were not identified. CONCLUSIONS AND CLINICAL RELEVANCE: Although findings indicated that the TTN variant was most common, 6 dogs had neither variant, and this fact supported the concept of ≥ 1 other genetic contributor to DCM in Doberman Pinschers. Future studies are warranted to evaluate genotype-phenotype relationships in Doberman Pinschers with DCM.

An assessment of the role of vinculin loss of function variants in inherited cardiomyopathy.

The ACMG/AMP variant classification framework was intended for highly penetrant Mendelian conditions. While it is appreciated that clinically relevant variants exhibit a wide spectrum of penetrance, accurately assessing and expressing the pathogenicity of variants with lower penetrance can be challenging. The vinculin (VCL) gene illustrates these challenges. Model organism data provide evidence that loss of function of VCL may play a role in cardiomyopathy and aggregate case-control studies suggest low penetrance. VCL loss of function variants, however, are rarely identified in affected probands and therefore there is a paucity of family studies clarifying the clinical significance of individual variants. This study, which aggregated data from >18,000 individuals who underwent gene panel or exome testing for inherited cardiomyopathies, identified 32 probands with VCL loss-of-function variants and confirmed enrichment in probands with dilated cardiomyopathy (odds ratio [OR] = 9.01; confidence interval [CI] = 4.93-16.45). Our data revealed that the majority of these individuals (89.5%) had pediatric onset of disease. Family studies demonstrated that heterozygous loss of function of VCL alone is insufficient to cause cardiomyopathy but that these variants do contribute to disease risk. In conclusion, VCL loss-of-function variants should be reported in a diagnostic setting but need to be clearly distinguished as having lower penetrance.

A cost-effectiveness model of genetic testing and periodical clinical screening for the evaluation of families with dilated cardiomyopathy.

PURPOSE: To assess the relative cost-effectiveness of cascade genetic testing in asymptomatic relatives of patients with dilated cardiomyopathy (DCM) compared with periodical clinical surveillance. METHODS: A decision-analytic model, combining a decision tree and a Markov model, was used to determine the lifetime costs and quality-adjusted life years (QALYs) for the two strategies. Deterministic and probabilistic sensitivity analyses were undertaken to assess the robustness of findings and to explore decision uncertainty. RESULTS: The incremental cost per additional QALY of cascade genetic testing prior to periodical clinical surveillance of first-degree relatives compared with periodical clinical surveillance alone was estimated at approximately AUD $6100. At established thresholds of cost-effectiveness, there is a 90% probability that cascade genetic testing is cost-effective. Extensive sensitivity analyses, including the addition of second-degree relatives, did not alter the conclusions drawn from the main analysis. CONCLUSION: Using cascade genetic testing to guide clinical surveillance of asymptomatic relatives of patients with DCM is very likely to be cost-effective. As the DCM pathogenic variant detection rate rises and new evidence for personalized treatment of at-risk individuals becomes available, the cost-effectiveness of cascade testing will further increase.

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.

Health Status and Psychological Distress in Patients with Non-compaction Cardiomyopathy: The Role of Burden Related to Symptoms and Genetic Vulnerability.

BACKGROUND: Non-compaction cardiomyopathy (NCCM) is a cardiomyopathy characterized by left ventricular tribeculae and deep intertrabecular recesses. Because of its genetic underpinnings and physical disease burden, noncompaction cardiomyopathy is expected to be associated with a lower health status and increase in pscyhological distress. PURPOSE: This study determined the health status and psychological distress in NCCM patients. We also examined the potential contribution of genetic predisposition and cardiac symptoms to health status and distress in NCCM, by comparing NCCM patients with (1) patients with familial hypercholesterolemia (FH) and (2) patients with acquired dilated cardiomyopathy (DCM). METHODS: Patients were recruited from the Erasmus Medical Center, Rotterdam, The Netherlands. Using a case-control design, NCCM patients (N = 45, mean age 46.7 ± 15.1 years, 38 % male) were compared with 43 FH patients and 42 DCM patients. Outcome measures were health status (Short Form Health Survey-12), anxiety (Generalized Anxiety Disorder 7-item scale) and depression (Patient Health Questionnaire 9-item scale). RESULTS: NCCM patients showed significantly worse health status (Physical Component Score F(1,84) = 9.58, P = .003; Mental Component Score F(1,84) = 16.65, P < .001), anxiety (F(1,85) = 9.63, P = .003) and depression scores (F(1,82) = 5.4, P = .023) compared to FH patients, also after adjusting age, sex, comorbidity, educational level and time since diagnosis. However, NCCM patients did not differ from DCM patients (Physical Component Score F(1,82) = 2,61, P = .11; Mental Component Score F(1,82) = .55, P = .46), anxiety (F(1,82) = 1.16, P = .28) and depression scores (F(1,82) = 1,95, P = .17). CONCLUSION: Cardiac symptoms are likely to play a role in the observed poor health status and elevated levels of anxiety and depressive symptoms in NCCM, whereas the burden of having a genetic condition may contribute less to these health status and psychological measures.

A group approach to genetic counselling of cardiomyopathy patients: satisfaction and psychological outcomes sufficient for further implementation.

The introduction of next-generation sequencing in everyday clinical genetics practise is increasing the number of genetic disorders that can be confirmed at DNA-level, and consequently increases the possibilities for cascade screening. This leads to a greater need for genetic counselling, whereas the number of professionals available to provide this is limited. We therefore piloted group genetic counselling for symptomatic cardiomyopathy patients at regional hospitals, to assess whether this could be an acceptable alternative to individual counselling. We performed a cohort study with pre- and post-counselling patient measurements using questionnaires, supplemented with evaluations of the group counselling format by the professionals involved. Patients from eight regional hospitals in the northern part of the Netherlands were included. Questionnaires comprised patient characteristics, psychological measures (personal perceived control (PPC), state and trait anxiety inventory (STAI)), and satisfaction with counsellors, counselling content and design. In total, 82 patients (mean age 57.5 year) attended one of 13 group sessions. Median PPC and STAI scores showed significantly higher control and lower anxiety after the counselling. Patients reported they were satisfied with the counsellors, and almost 75% of patients were satisfied with the group counselling. Regional professionals were also, overall, satisfied with the group sessions. The genetics professionals were less satisfied, mainly because of their perceived large time investment and less-than-expected group interaction. Hence, a group approach to cardiogenetic counselling is feasible, accessible, and psychologically effective, and could be one possible approach to counselling the increasing patient numbers in cardiogenetics.

Assessment of DNA damage using cytokinesis-block micronucleus cytome assay in lymphocytes of dilated cardiomyopathy patients.

Studies on the extent of DNA damage are undertaken to elucidate the nature and causes of genomic instability in any syndrome or disease progression in human. In this study, cytokinesis-block micronucleus cytome (CBMN Cyt) assay was employed to evaluate the extent of chromosomal instability or DNA damage in lymphocytes of patients suffering from dilated cardiomyopathy (DCM), a serious cardiac muscle disorder. Effect of DNA damage on the disease was also assessed by analysis of mutations in cardiac Troponin C type I (TNNC1) gene. Blood samples were collected from 48 DCM patients and 48 age- and sex-matched controls from Vellore region of South India. Significantly high frequencies of micronuclei (MNi) and genomic damage such as nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs) were observed in the patient group as compared with the control group (P < 0·001). Molecular analysis revealed that no mutations were found in the TNNC1 gene. It was observed that although there was a high frequency of DNA damage in the lymphocytes of the patients, no correlation between severity of the phenotype and the frequencies of MNi, NPBs and NBUDS could be established. Our study appears to be the first one in which chromosomal instability was estimated using CBMN Cyt assay for DCM patients. Studies with a larger population size may help in validating the use of genetic markers for establishing frequencies and type of DNA damage in DCM. It will also help in understanding the effect of DNA damage on this disease.

Sex and gender differences in myocarditis and dilated cardiomyopathy.

Heart failure due to nonischemic dilated cardiomyopathy (DCM) contributes significantly to the global burden of cardiovascular disease. Myocarditis is, in turn, a major cause of acute DCM in both men and women. However, recent clinical and experimental evidence suggests that the pathogenesis and prognosis of DCM differ between the sexes. This seminar provides a contemporary perspective on the immune mediators of myocarditis, including interdependent elements of the innate and adaptive immune response. The heart's acute response to injury is influenced by sex hormones that appear to determine the subsequent risk of chronic DCM. Preliminary data suggest additional genetic variations may account for some of the differences in epidemiology, left ventricular recovery, and survival between men and women. We highlight the gaps in our knowledge regarding the management of women with acute DCM and discuss emerging therapies, including bromocriptine for the treatment of peripartum cardiomyopathy.

Assessment of cardiac function in three mouse dystrophinopathies by magnetic resonance imaging.

Lack of dystrophin results in skeletal muscle dystrophy and dilated cardiomyopathy in humans and animal models. To achieve a basic understanding of the natural development of cardiomyopathy in different dystrophinopathy mouse models, left and right ventricular heart function was assessed at different ages in three dystrophinopathy mouse models (mdx, mdx/utrn(+/-) model and mdx/utrn(-/-)) using magnetic resonance imaging. Left ventricular function was significantly decreased, already at 2months in the most severely affected mdx/utrn(-/-) mice. Furthermore, whereas heart function was stable in wild-type mice over time, both mdx and mdx/utrn(+/-) showed a clear decrease at 10months of age, most prominently in the right ventricle. Therefore magnetic resonance imaging is an adequate technique to determine heart function in dystrophinopathy mouse models and can be used to assess the effect of potential therapies.

Assessment of LMNA copy number variation in 58 probands with dilated cardiomyopathy.

The contribution of copy number variation (CNV) to dilated cardiomyopathy (DCM) is unknown. However, estimates have suggested that CNVs could constitute 15% of mutations underlying Mendelian disease. This is of particular relevance to DCM, where only approximately 35% of genetic cause has been identified. We have previously reported 19 point mutations in LMNA, the gene encoding Lamin A/C, in a cohort of 324 unrelated DCM probands (5.9%), making it the most common genetic cause of DCM. Recently a large deletion was reported in LMNA in 1 of 25 DCM probands. To further assess the contribution of CNVs in LMNA cardiomyopathy, we used Multiplex Ligation Probe Amplification (MLPA) to screen for large deletions and duplications in 58 DCM probands negative for point mutations in LMNA. Despite excellent quality control and robust MLPA results, our study failed to identify any deletions or duplications. We conclude that at least for LMNA, point mutations are the major source of DCM causation.

Evaluation of second-generation sequencing of 19 dilated cardiomyopathy genes for clinical applications.

Medical sequencing for diseases with locus and allelic heterogeneities has been limited by the high cost and low throughput of traditional sequencing technologies. "Second-generation" sequencing (SGS) technologies allow the parallel processing of a large number of genes and, therefore, offer great promise for medical sequencing; however, their use in clinical laboratories is still in its infancy. Our laboratory offers clinical resequencing for dilated cardiomyopathy (DCM) using an array-based platform that interrogates 19 of more than 30 genes known to cause DCM. We explored both the feasibility and cost effectiveness of using PCR amplification followed by SGS technology for sequencing these 19 genes in a set of five samples enriched for known sequence alterations (109 unique substitutions and 27 insertions and deletions). While the analytical sensitivity for substitutions was comparable to that of the DCM array (98%), SGS technology performed better than the DCM array for insertions and deletions (90.6% versus 58%). Overall, SGS performed substantially better than did the current array-based testing platform; however, the operational cost and projected turnaround time do not meet our current standards. Therefore, efficient capture methods and/or sample pooling strategies that shorten the turnaround time and decrease reagent and labor costs are needed before implementing this platform into routine clinical applications.

Restarting the clock...again: ethical considerations in retransplantation.

Cardiac and pulmonary transplantation has revolutionized end-stage heart and lung therapy. With the advent of cyclosporine and other immunosuppressive therapies, many patients lead productive lives. Unfortunately, other patients who have undergone cardiac and/or pulmonary transplantation do not have favorable results. In fact, some require retransplantation to live. Because of organ scarcity, healthcare professionals and patients must examine not only retransplantation survival rates but also the ethical considerations when dealing with resource-limited organs. Given that retransplantation survival rates are not as favorable as those for primary transplantation and that no studies involving quality of life and morbidity could be located, considerable thought should be given to this controversial practice.