Titin-truncating Variants Research Articles

Prognostic implications of genotype findings in non-ischemic dilated cardiomyopathy: a network meta-analysis

Abstract Background Evidence on the relative impact of diverse genetic backgrounds associated with non-ischemic dilated cardiomyopathy (DCM) remains contradictory. Aim This study sought to synthesize the available data regarding long-term outcomes of different gene groups in DCM. Methods Electronic databases were systematically screened to identify studies reporting prognostic data on pre-specified gene groups. Those included pathogenic/likely pathogenic (P/LP) variants, truncating titin variants (TTNtv), lamin A/C mutations (LMNA), and desmosomal proteins. Outcomes were divided into composite adverse events (CAEs), major ventricular arrhythmic events (MVAEs) and heart failure events (HFEs). Random effects meta-analyses were conducted to quantify the pooled odds ratio (ORs) for each gene group. A frequentist network meta-analysis was executed to allow indirect within-gene group comparisons. Results Twenty-six studies comprising 7,255 genotyped DCM individuals were included. Patients with P/PL variants had a higher risk for CAEs [OR, 2.10 (95% CI, 1.67-2.65)], MVAEs [OR, 1.86 (95% CI, 1.52-2.26)], and HFEs [OR, 2.01 (95% CI, 1.08-3.73)] than genotype negative patients. The presence of TTNtv was linked to a higher risk for CAEs [OR, 1.78 (95% CI, 1.20-2.63)], but not MVAEs or HFEs. LMNA and desmosomal groups suffered a higher risk for CAEs, MVAEs, and HFEs compared to non-LMNA and non-desmosomal groups respectively (Picture 1). When genes were indirectly compared, the presence of LMNA resulted in a more detrimental effect that TTNtv, with respect to all composite outcomes but no significant difference was found between LMNA and desmosomal genes. Desmosomal genes harbored a higher risk for MVAEs compared to TTNtv (Picture 2). Conclusion Different genetic substrates associated with DCM result in divergent natural histories. Routine utilization of genetic testing should be employed to refine risk stratification and inform therapeutic strategies in DCM.Natural history of DCM based on genotypeNetwork meta-analysis of gene variants

Exercise training improves cardiovascular fitness in dilated cardiomyopathy caused by truncating titin variants

BackgroundParticipation in regular exercise activities is recommended for patients with chronic heart failure. However, less is known about the effect of exercise in patients with genetic dilated cardiomyopathy (DCM). We...

Inferring disease course from differential exon usage in the wide titinopathy spectrum.

Biallelic titin truncating variants (TTNtv) have been associated with a wide phenotypic spectrum, ranging from complex prenatal muscle diseases with dysmorphic features to adult-onset limb-girdle muscular dystrophy, with or without cardiac involvement. Given the size and complexity of TTN, reaching an unequivocal molecular diagnosis and precise disease prognosis remains challenging. In this case series, 12 unpublished cases and one already published case with biallelic TTNtv were collected from multiple international medical centers between November 2022 and September 2023. TTN mutations were detected through exome or genome sequencing. Information about familial and personal clinical history was collected in a standardized form. RNA-sequencing and analysis of TTN exon usage were performed on an internal sample cohort including postnatal skeletal muscles, fetal skeletal muscles, postnatal heart muscles, and fetal heart muscles. In addition, publicly available RNA-sequencing data was retrieved from ENCODE. We generated new RNA-seq data on TTN exons and identified genotype-phenotype correlations with prognostic implications for each titinopathy patient (whether worsening or improving in prenatal and postnatal life) using percentage spliced in (PSI) data for the involved exons. Interestingly, thanks to exon usage, we were also able to rule out a titinopathy diagnosis in one prenatal case. This study demonstrates that exon usage provides valuable insights for a more exhaustive clinical interpretation of TTNtv; additionally, it may serve as a model for implementing personalized medicine in many other genetic diseases, since most genes undergo alternative splicing.

Abstract We103: Integration of deep learning assisted high-content screening and deep tissue-phenotyping to identify cardioprotective compounds in dilated cardiomyopathy

With a prevalence of 4:10,000, dilated cardiomyopathy (DCM) is the most common hereditary heart disease, characterized by dilatation of one or both ventricles and impaired systolic and diastolic function. Numerous mutations have been identified to cause DCM. TITIN-truncating variants (TTNtv) are commonly observed in 20% of the genetic DCM cases. Progress in drug discovery for DCM has been hampered by the lack of robust high-throughput/high-content analysis pipelines of disease-specific cell and tissue platforms for deep structural and functional phenotyping. To address this challenge, we have developed a two-tiered human induced pluripotent stem cell (iPSC)-based screening approach, comprised of high-content cardiomyocyte imaging with artificial intelligence (AI)-driven image and data analyses as well as deep tissue phenotyping in engineered heart muscle (EHM) models. As a DCM use case, we have developed several isogenic human iPSC-lines with and without a previously reported and pathologically relevant A-band-TTNtv mutation (c.70692_70693insAT; p.T23565SfsX5; Gerull et al. 11788824, Gramlich et al. 25759365, Fomin et al. 34731013) Using fixed weights from a convolutional deep neural network trained on ImageNet, we generated unbiased deep embeddings from each 2D cell model and applied these to train machine learning (ML) models to detect morphological disease phenotypes. Our ML model is able to confidently separate healthy controls from TTNtv cells with high accuracy (>93%), specificity (WT >79%) across different batches/plate layouts and generate concentration-response curves, demonstrating platform robustness and sensitivity. Building upon this work, we will present our latest efforts applying the platform technology for a chemogenomic screening campaign to identify novel cardioprotective drugs using a library of 7,100 bioactive compounds and validation assays, including measuring functional changes in EHMs. The phenotypic profiling platform, as presented here, can be readily adapted to other disease-relevant cell types and pathologies. Our results demonstrate the power of combining iPSC-CMs with cytological profiling and deep learning as well as tissue-level phenotyping to accelerate drug discovery for patients with DCM.

Titin truncating variants, cardiovascular risk factors and the risk of atrial fibrillation and heart failure.

High-proportion spliced-in (hiPSI) titin truncating variant (TTNtv) carriers have a higher risk of atrial fibrillation and heart failure1. However, the role of cardiovascular risk factors in modifying the risk of atrial fibrillation and heart failure attributed to hiPSI TTNtv carriers is unknown. Here, we investigate the role of cardiovascular risk, quantified using the pooled cohort equations (PCEs), in influencing the hazard of outcomes attributed to hiPSI TTNtvs among UK Biobank participants without baseline cardiovascular disease. The cohort was stratified based on hiPSI TTNtv carrier status and cardiovascular risk (low: <5%, intermediate: 5.0-7.5% and high: >7.5%). The primary outcome was a composite of atrial fibrillation, heart failure or death. TTNtv noncarriers with low cardiovascular risk were used as the reference group for all analyses. Among 179,752 participants (median age: 56 (49, 62) years; 57.5% female), the risk of the primary outcome was lower in hiPSI TTNtv carriers with low cardiovascular risk (adjusted hazard ratio: 2.23 (95% confidence interval: 1.62-3.07)) than those with high cardiovascular risk (adjusted hazard ratio: 8.21 (95% confidence interval: 6.63-10.18)). A favorable cardiovascular risk factor profile may partially offset the risk of clinical outcomes among hiPSI TTNtv carriers.

Prognostic implications of genotype findings in non-ischaemic dilated cardiomyopathy: A network meta-analysis.

Evidence on the relative impact of diverse genetic backgrounds associated with non-ischaemic dilated cardiomyopathy (DCM) remains contradictory. This study sought to synthesize the available data regarding long-term outcomes of different gene groups in DCM. Electronic databases were systematically screened to identify studies reporting prognostic data on pre-specified gene groups. Those included pathogenic/likely pathogenic (P/LP) variants, truncating titin variants (TTNtv), lamin A/C variants (LMNA), and desmosomal proteins. Outcomes were divided into composite adverse events (CAEs), malignant ventricular arrhythmic events (MVAEs) and heart failure events (HFEs). A total of 26 studies (n = 7255) were included in the meta-analysis and 6791 patients with genotyped DCM were analysed. Patients with P/LP variants had a higher risk for CAEs (odds ratio [OR] 2.10, 95% confidence interval [CI] 1.67-2.65), MVAEs (OR 1.86, 95% CI 1.52-2.26), and HFEs (OR 2.01, 95% CI 1.08-3.73) than genotype-negative patients. The presence of TTNtv was linked to a higher risk for CAEs (OR 1.78, 95% CI 1.20-2.63), but not MVAEs or HFEs. LMNA and desmosomal groups suffered a higher risk for CAEs, MVAEs, and HFEs compared to non-LMNA and non-desmosomal groups, respectively. When genes were indirectly compared, the presence of LMNA resulted in a more detrimental effect that TTNtv, with respect to all composite outcomes but no significant difference was found between LMNA and desmosomal genes. Desmosomal genes harboured a higher risk for MVAEs compared to TTNtv. Different genetic substrates associated with DCM result in divergent natural histories. Routine utilization of genetic testing should be employed to refine risk stratification and inform therapeutic strategies in DCM.

Alcoholic cardiomyopathy: an update.

Alcohol-induced cardiomyopathy (AC) is an acquired form of dilated cardiomyopathy (DCM) caused by prolonged and heavy alcohol intake in the absence of other causes. The amount of alcohol required to produce AC is generally considered as >80 g/day over 5 years, but there is still some controversy regarding this definition. This review on AC focuses on pathogenesis, which involves different mechanisms. Firstly, the direct toxic effect of ethanol promotes oxidative stress in the myocardium and activation of the renin-angiotensin system. Moreover, acetaldehyde, the best-studied metabolite of alcohol, can contribute to myocardial damage impairing actin-myosin interaction and producing mitochondrial dysfunction. Genetic factors are also involved in the pathogenesis of AC, with DCM-causing genetic variants in patients with AC, especially titin-truncating variants. These findings support a double-hit hypothesis in AC, combining genetics and environmental factors. The synergistic effect of alcohol with concomitant conditions such as hypertension or liver cirrhosis can be another contributing factor leading to AC. There are no specific cardiac signs and symptoms in AC as compared with other forms of DCM. However, natural history of AC differs from DCM and relies directly on alcohol withdrawal, as left ventricular ejection fraction recovery in abstainers is associated with an excellent prognosis. Thus, abstinence from alcohol is the most crucial step in treating AC, and specific therapies are available for this purpose. Otherwise, AC should be treated according to current guidelines of heart failure with reduced ejection fraction. Targeted therapies based on AC pathogenesis are currently being developed and could potentially improve AC treatment in the future.

High-proportion spliced-in titin truncating variants in African and European ancestry in the All of Us Research Program.

High-proportion spliced-in titin truncating variants (hiPSI TTNtvs) have been associated with an increased risk of atrial fibrillation, dilated cardiomyopathy (DCM) and heart failure in individuals of European ancestry1. However, similar data in individuals of African ancestry are lacking. Here we examined the association of hiPSI TTNtvs with atrial fibrillation, DCM and heart failure in individuals of African ancestry using data from the All of Us Research Program. Among 38,154 individuals of African ancestry, 169 (0.4%) individuals carried a hiPSI TTNtv. hiPSI TTNtv carriers were at a higher risk of developing atrial fibrillation (adjusted hazard ratio (HRadj) 2.42, 95% confidence interval (CI) 1.52-3.85), DCM (HRadj 2.82, 95% CI 1.81-4.39) and heart failure (HRadj 2.07, 95% CI 1.43-3.00) compared with noncarriers. The association of hiPSI TTNtvs with atrial fibrillation, DCM and heart failure was similar in individuals of African ancestry and those of European ancestry. Therefore, genetic testing for hiPSI TTNtvs may permit early identification of carriers and support preventive measures to reduce the likelihood of heart failure development both in individuals of European ancestry and in individuals of African ancestry.

Relation between non-sustained ventricular tachycardia and major arrhythmic events in patients with dilated cardiomyopathy caused by titin truncating variants

Abstract Background Truncating titin variants (TTNtv) are the most frequent monogenetic cause of dilated cardiomyopathy (DCM). Current data suggest that nonsustained ventricular arrhythmia is common in patients with TTVtv but its relation to the risk of major arrhythmic events (MAE) is uncertain. Purpose To investigated the relationship between NSVT recorded on Holter ECG or implanted device and MAE in patients with DCM. Methods We retrospectively studied consecutively evaluated patients with DCM (left ventricle ejection fraction (LVEF) below 50%) on echocardiography and a pathogenic TTNtv. Patients with obstructive coronary artery disease, valvular disease, ventricular tachycardia ablation during follow-up time and follow-up less than one month were excluded. Survival was modelled from the initial Holter or device implantation. NSVT was defined as ≥3 consecutive ventricular beats at a rate &amp;gt;120 beats per minute, lasting for &amp;lt;30 s. MAE was defined as sustained ventricular tachycardia (VT), ventricular fibrillation (VF), sudden cardiac death, implantable cardioverter defibrillator (ICD) treated ventricular arrhythmias (appropriate shock and ATP). The clinical endpoint was MAE (sustained VT, VF, sudden cardiac death, ICD shocks and ATP) and multivariable analysis was performed using Cox proportional hazards models. Event-free survival probabilities were estimated using the Kaplan-Meier method, and comparisons of Kaplan-Meier curves were made using log-rank tests. Results The cohort comprised 58 men and 30 women [mean age 49 ± 15 years; mean follow-up 3.5 ± 1.8 years]. Symptoms were NYHA class I in 56 patients, class II in 25 patients, class III in 7 patients and class IV in none. Holter ECGs were performed in 77 patients, 41 of whom had NSVT. Twenty five patients had pacemaker/ICD in situ; 14 had NSVT. Six patients had MAE; 4 with NSVT. Univariate analysis association between MAE and LVEF ≤35% (hazard ratio (HR), 9.03; 95% confidence interval (CI), 1.49-54.71; P=0.017), prolonged (&amp;gt;8 beats) NSVT (HR, 6.71; 95% CI, 1.22-36.98; P=0.029) and fast (&amp;gt;200 bpm) NSVT (HR, 5.09; 95% CI, 1.02-25.47; P=0.048). Kaplan-Meier showed similar differences in freedom from MAE (LVEF ≤35%; P=0.004, long NSVT; P=0.025, fast NSVT; P=0.031). Conclusion Fast and prolonged NSVT is associated with a higher risk of MAE in patients with TTNtv.Characteristics at the beginningSurvival probability for freedom

Influence of sarcomeric protein mutations on myocardial function in a 3D-disease model of human iPSC-derived bioartificial cardiac tissue

Abstract Background Peripartum cardiomyopathy (PPCM) is a rare type of cardiomyopathy that occurs in women without prior history of heart disease at later stages of pregnancy or several months after childbirth (1). The disease is characterized by lowered left ventricular ejection fraction (LVEF&amp;lt;45%) and can lead to heart failure if left untreated (1). Approximately 20% of PPCM patients harbor sarcomeric mutations, from which titin truncating variants (TTNtv-s) have the highest pathogenic potential (2). However, the role of such genetic defects of the contractile apparatus on PPCM pathogenesis is not well understood. Purpose The study aims to establish an in vitro 3D disease model of PPCM to elucidate the role of sarcomeric mutations, including titin mutations, in its etiology. We hypothesize, that the disease phenotype can be uncovered in vitro via the stimulation of PPCM patient-derived Bioartificial Cardiac Tissues (BCTs), utilizing pregnancy-related factors in our custom-made bioreactors (3). Methods We selected three patients carrying previously unstudied mutations in the TTN gene for the study. The iPSC reprogramming was performed using a commercial, Sendai virus-based kit. Pluripotency was confirmed via flow cytometry and immunofluorescence stainings. The presence of mutations was confirmed by exome sequencing. Cardiomyocyte differentiation (4) and BCT generation (3) were carried out via established protocols. Following maturation, BCTs were stimulated with the pregnancy-related factors, and tissue forces were measured in the bioreactors. These forces include intrinsically generated (spontaneous) and electrically induced (paced) contraction forces (mN), as well as passive forces, which correlate with tissue stiffness. Results We successfully generated two clonal induced pluripotent stem cell (iPSC) lines from each patient. We confirmed that all of the cell lines were &amp;gt;90% positive for pluripotency markers. After differentiation, CMs contracted spontaneously from day 7 onwards and were &amp;gt;95% positive for cardiac markers (SA, MF20, cTnT). The BCTs remodeled and started contracting on days 1-2 after generation, similarly to wild-type tissues in the baseline conditions. Prolactin treatment of Patient A, harboring a missense mutation in titin, did not show significant functional effects. We confirmed, that patients B and C harbor TTNtvs, and tissues from these patients show a trend of altered stiffness after stimulation with repeated mechanical stretch cycles or β-adrenergic agonist isoprenaline. Further analysis of the underlying mechanisms investigating metabolic changes, signaling pathways, and transcriptomics are ongoing. Conclusion The established iPSC and tissue engineering technologies offer a valuable tool for the development of an iPS-CM-based 3D in vitro disease model for PPCM, which can be used for risk stratification of individual and combined sarcomeric mutations and the development of tailored therapeutic strategies.

Abstract 13979: Titin-Truncating Variants Predispose to Dilated Cardiomyopathy in Patients Genetically Similar to African Reference Populations

Introduction: Heterozygous mutations within the TTN gene can cause the translation of shortened forms of the titin protein known as titin-truncating variants (TTNtvs) leading to dilated cardiomyopathy (DCM) among certain individuals. We previously reported an odds ratio (OR) of 18.7 for DCM among individuals genetically similar to the European reference population (EUR) with high percentage spliced in (hiPSI) TTNtvs, however we were unable to show a similar relationship among individuals genetically similar to the African reference population (AFR). Methods: Penn Medicine Biobank volunteers with whole exome sequencing were screened for TTNtvs with minor allele frequency &lt;0.001 and percent spliced in &gt;0.9. Participants were also screened for a DCM diagnosis defined using International Classification of Diseases, ninth and tenth revision diagnoses codes. Left ventricular ejection fraction (LVEF) values were extracted from structured transthoracic echocardiography reports. Logistic and linear regression were employed to evaluate the association between hiPSI TTNtvs and DCM or LVEF adjusting for age, sex, and first five genetic principal components. Results: Of the 43,731 participants with sequencing data, 11,136 (27.1%) were genetically similar to the AFR reference population, 50.3% were male, and the median age was 57.8 years (IQR: 44.9-69.5 years). One percent (418 individuals) had a hiPSI TTNtv. The age- and sex-adjusted OR of having DCM was similar among AFR and EUR participants carrying hiPSI TTNtvs (AFR OR: 4.37, 95% CI 2.68 to 7.13, P &lt; 0.001; EUR OR: 5.86, 95% CI 4.58 to 7.49, P &lt; 0.001; meta OR: 5.48, 95% CI 4.31 to 6.97, P &lt; 0.001). Among those individuals with echocardiography data available, hiPSI TTNtvs were associated with significantly decreased minimum LVEF independent of genetic reference population similarity (EUR β: -10.08, 95% CI -12.71 to -7.44, P &lt; 0.001; AFR β: -12.83, 95% CI -18.39 to -7.27, P &lt; 0.001; Meta β: -10.58, 95% CI -12.96 to -8.20, P &lt; 0.001). Conclusions: Our analysis demonstrates a strong association between hiPSI TTNtv and both DCM and minimum LVEF in diverse populations suggesting that genetic testing and counseling recommendations should not differ between these populations.

Enrichment of titin-truncating variants in exon 327 in dilated cardiomyopathy and its relevance to reduced nonsense-mediated mRNA decay efficiency.

Titin truncating variants (TTNtvs) are the most common genetic cause of dilated cardiomyopathy (DCM). Among four regions of titin, A-band enrichment of DCM-causing TTNtvs is widely accepted but the underlying mechanism is still unknown. Meanwhile, few reports have identified exon 327 as a highly mutated A-band exon but the degree of exon 327 enrichment has not been quantitatively investigated. To find the real hotspot of DCM-causing TTNtvs, we aimed to reassess the degree of TTNtv enrichment in known titin regions and in exon 327, separately. In addition, we tried to explain exon 327 clustering in terms of nonsense-mediated mRNA decay (NMD) efficiency and a dominant negative mechanism recently proposed. Research papers focusing on TTNtvs found in patients with DCM were collected. A total of 612 patients with TTNtv-realated DCM were obtained from 10 studies. In the four regions of TTN and exon 327, the degree of TTNtvs enrichment was calculated in a way that the effect of distribution of highly expressed exons was normalized. As a result, exon 327 was the only region that showed significant enrichment for DCM-related TTNtv (p < .001). On the other hand, other A-band exons had almost the same number of TTNtv of random distribution. A review of RNAseq data revealed that the median allelic imbalance deviation of exon 327 TTNtvs was .04, indicating almost zero NMD. From these findings, we propose that the widely accepted A-band enrichment of DCM-related TTNtv is mostly attributable to exon 327 enrichment. In addition, based on the recently demonstrated dominant negative mechanism, the extremely low NMD efficiency seems to contribute to exon 327 enrichment.

Titin-truncating variants in hiPSC cardiomyocytes induce pathogenic proteinopathy and sarcomere defects with preserved core contractile machinery.

Titin-truncating variants (TTNtv) are the single largest genetic cause of dilated cardiomyopathy (DCM). In this study we modeled disease phenotypes of A-band TTNtv-induced DCM in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using genome editing and tissue engineering technologies. Transcriptomic, cellular, and micro-tissue studies revealed that A-band TTNtv hiPSC-CMs exhibit pathogenic proteinopathy, sarcomere defects, aberrant Na+ channel activities, and contractile dysfunction. These phenotypes establish a dual mechanism of poison peptide effect and haploinsufficiency that collectively contribute to DCM pathogenesis. However, TTNtv cellular defects did not interfere with the function of the core contractile machinery, the actin-myosin-troponin-Ca2+ complex, and preserved the therapeutic mechanism of sarcomere modulators. Treatment of TTNtv cardiac micro-tissues with investigational sarcomere modulators augmented contractility and resulted in sustained transcriptomic changes that promote reversal of DCM disease signatures. Together, our findings elucidate the underlying pathogenic mechanisms of A-band TTNtv-induced DCM and demonstrate the validity of sarcomere modulators as potential therapeutics.

Abstract 10026: Augmentation of Slow Repolarizing Potassium Channel (I Ks ) Links Truncated Titin Mutations to Atrial Fibrillation

Introduction: Rare titin-truncating variants ( TTN tv) are associated with early-onset atrial fibrillation (AF) but the pathogenic mechanisms and therapeutic implications remain unclear. To determine the function of a highly conserved immunoglobulin-like domain of the A-band of TTN , a region enriched in AF-associated variants, we employed CRISPR-Cas9-mediated deletion of 9 amino acids (Δ9) in human induced pluripotent stem cells and then derived atrial cardiomyocytes (hiPSC-aCMs) for electrophysiological (EP) analysis. Hypothesis: We hypothesized that the TTN -Δ9 mutation results in augmentation of the slow delayed rectifier potassium current (I Ks ) resulting in shortened action potential duration (APD). Methods: TTN -WT hiPSC-aCMs (healthy control) and TTN -Δ9 hiPSC-aCMs (isogenic mutant) were matured with metabolic conditioning, electrical stimulation, co-culture with atrial fibroblasts, and micropatterning. We used transmission electron microscopy (TEM) to assess sarcomeric defects, RNA-sequencing and RT-qPCR to assess molecular ion channel remodeling, and high throughput automated patch clamp and optical voltage mapping to assess the EP properties of TTN -Δ9 hiPSC-aCMs. We also performed targeted pharmacological inhibition of I Ks . Results: We showed that TTN -Δ9 hiPSC-aCMs displayed disrupted organization and myofibril assembly ( Fig. 1A-B ), upregulated voltage-gated potassium channel complexes and increased KCNQ1 expression ( Fig 1C-D ), and shortened APD ( Fig. 1E-F ). I Ks was significantly augmented in TTN -Δ9 hiPSC-aCMs ( Fig. 1G-H ). Infusion of the I Ks -specific blocker HMR-1556, and not I Kr blocker dofetilide, fully restored the shortened APD 90 and partially recovered defects in contractility in TTN -Δ9 hiPSC-aCMs compared to TTN -WT hiPSC-aCMs ( Fig. 1I-J ). Conclusion: Our findings suggest that augmentation of I Ks contributes to the pathogenic mechanism of TTN -mediated AF, pointing to I Ks as a potential therapeutic target.

Titin-Related Dilated Cardiomyopathy: The Clinical Trajectory and the Role of Circulating Biomarkers in the Clinical Assessment

Titin truncating variants (TTNtv) are known as the leading cause of inherited dilated cardiomyopathy (DCM). Nevertheless, it is unclear whether circulating cardiac biomarkers are helpful in detection and risk assessment. We sought to assess 1) early indicators of cardiotitinopathy including the serum biomarkers high-sensitivity cardiac troponin T (hs-cTnT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in clinically stable patients, and 2) predictors of outcome among TTNtv carriers. Our single-center cohort consisted of 108 TTNtv carriers (including 70 DCM patients) from 43 families. Clinical, laboratory and follow-up data were analyzed. The earliest abnormality was left ventricular dysfunction, present in 8, 26 and 47% of patients in the second, third and fourth decade of life, respectively. It was followed by symptoms of heart failure, linked to NT-proBNP elevation and severe left ventricular systolic dysfunction, and later by arrhythmias. Hs-cTnT serum levels were increased in the late stage of the disease only. During the median follow-up of 5.2 years, both malignant ventricular arrhythmia (MVA) and end-stage heart failure (esHF) occurred in 12% of TTNtv carriers. In multivariable analysis, NT-proBNP level ≥650 pg/mL was the best predictor of both composite endpoints (MVA and esHF) and of MVA alone. In conclusion, echocardiographic abnormalities are the first detectable anomalies in the course of cardiotitinopathies. The assessment of circulating cardiac biomarkers is not useful in the detection of the disease onset but may be helpful in risk assessment.

418 Titin mutations and female sex characterize dilated cardiomyopathy in the elderly

Abstract Aims Dilated cardiomyopathy (DCM) is frequently caused by genetic factors. Studies identifying deleterious rare variants have predominantly focused on early-onset cases, and little is known about the genetic underpinnings of the growing numbers of patients with DCM who are diagnosed after 60 years of age (i.e. late-onset DCM). The aim is to investigate the prevalence, type, and prognostic impact of disease-associated rare variants in late-onset DCM patients. Methods and results We analysed a population of late-onset DCM patients who had undergone genetic testing in seven international tertiary referral centres worldwide. A positive genotype was defined as the presence of ‘pathogenic’ or ‘likely pathogenic’ (P/LP) variants. The study outcome was all-cause mortality. 184 patients over age 60 years (56% females, mean age 67 ± 6 years, mean left ventricular ejection fraction 32 ± 10%) were studied. Sixty-six patients (36%) were carriers of a P/LP variant. Titin truncating variants (TTNtv) were the most prevalent (present in 25% of the total population and accounting for 69% of all genotype-positive patients). During a median follow-up of 42 months (interquartile range: 10–115), 23 patients (13%) died; 17 of these (25%) were carriers of P/LP variants while six patients (5.1%) were genotype-negative (P &amp;lt; 0.001). Conclusions In the largest series worldwide, to date, of patients with late-onset DCM, we found a high prevalence of female sex and a high genetic mutation burden, largely due to TTNtv. Patients with a positive genetic test had higher mortality than genotype-negative patients. These findings support the extended use of genetic testing also in the elderly.

Genetic risk factors for dilated cardiomyopathy

Aim. To study the diagnostic significance of genetic testing in patients with dilated cardiomyopathy (DCM), identify predictors of life-threatening ventricular tachyarrhythmias (VTAs) and assess adverse clinical outcomes in different genetic groups.Material and methods. The study included 126 unrelated patients with verified DCM as follows: 70 (55,6%) probands with criteria for familial DCM and 56 (44,4%) individuals with a probable hereditary component. All patients (age, 43,1±11,3 years; men, 92 (73%); left ventricular ejection fraction, 30,6±8,43%; left ventricular enddiastolic diameter, 68,3±8,36 mm; follow-up period — median, 49 months) receive a complex of diagnostic investigations, including genetic screening using nextgeneration sequencing, followed by verification of variants by the Sanger method.Results. Pathogenic and likely pathogenic genetic variants were found in 61 (48,4%) of 126 patients with DCM. The dominant mutations were titin-truncating variants (TTNtvs), identified in 16 individuals (12,7%), and variants of lamin A/C (LMNA), identified in 13 probands (10,3%). Mutations in the other 19 genes were found in 32 (25,4%) patients. The following primary endpoints were assessed: sudden cardiac death (SCD), episodes of VTA (sustained ventricular tachycardia/ventricular fibrillation) and appropriate shocks of implanted cardiac resynchronization therapy (CRT)/cardioverter defibrillators (CVD) devices. As a result of ROC analysis, the following independent risk factors for SCD were identified: mutations in the LMNA gene (AUC, 0,760; p=0,0001) and non-sustained ventricular tachycardia (cut-off heart rate ≥161 bpm: AUC, 0,788; p=0,0001). When comparing the phenotypes and genotypes of DCM, TTNtv genotype was associated with a lower prevalence of complete left bundle branch block (χ2=7,46; p=0,024), a lower need for CRT/CVD implantation (χ2=5,70; p=0,017) and more rare episodes of sustained ventricular tachycardia/ventricular fibrillation (χ2=30,1; p=0,0001) compared with LMNA carriers. Kaplan-Meier analysis showed the worst prognosis in carriers of LMNA mutations both in relation to life-threatening VTA (log rang χ2=88,5; p=0,0001) and in achieving all unfavorable outcomes (χ2=27,8; p=0,0001) compared with groups of genenegative individuals, carriers of TTNtv and other genotypes.Conclusion. The phenotypes of DCM with TTNtv did not significantly differ in the incidence of VTAs and adverse outcomes compared with the gene-negative group and other genotypes (with the exception of LMNA). The contribution of the associations of LMNA mutations with VTAs on prognosis was confirmed, which shows the important role of LMNA genotype diagnosis for SCD risk stratification in patients with DCM.

NEW GENES, NEW TECHNIQUES IN NEUROMUSCULAR DISORDERS: O.12 Muscle biopsy and RNAseq in the diagnosis of titin related diseases

The ACMG/AMP guidelines for variant interpretation underline that splicing defects are an important mechanism of pathogenicity. In a standard diagnostic pipeline, in silico tools are needed to assess the possible effect on the splice of any identified genetic change (including synonymous variants) and a direct analysis of RNA, cDNA and protein is crucial to support variant classification. Due the sheer size of the TTN transcripts, the interpretation of titin variants is challenging and many unsolved myopathy patients (up to 6%) carry a single heterozygous TTN causative variant. We re-evaluated by SpliceAI all the possible single nucleotide changes in the titin coding sequence, identifying 9,732 variants predicted to alter the splicing (threshold 0.5). Of these, 2,078 are synonymous or missense variants and 317 are nonsense variants. Few previously reported causative ‘missense’ variants are predicted to impact the splicing. At the same time, we analysed by RNA sequencing 14 samples from patients with a confirmed recessive titinopathy, 24 samples from patients with a single heterozygous disease-causing variant in TTN and six from patients with a clinical suspicion of titinopathy. Variants causing a premature stop codon, located in exons out of the M-band, result in a nonsense mediated decay of titin transcripts. Vice versa, the in-depth characterization of 15 splice variants demonstrates that most of them cause in-frame losses or gains, still resulting in a near–full length protein. Using current bioinformatic approaches, RNAseq fails to identify elusive variants in most unsolved patients with heterozygous TTNtv and/or TTN-compatible phenotype, suggesting three possible scenarios: a causative role of the identified missense changes, more complex mechanisms involving heterozygous titin truncating variants, or alternative molecular diagnoses. At the same time, our study demonstrates the clinical importance of TTN transcript and protein studies in the diagnostic pipeline of neuromuscular disorders.

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.