Intragenic TTN Deletions in a Single Family with Dilated Cardiomyopathy

Titin (TTN) is known as the largest sarcomeric protein that resides within the heart muscle. Due to alternative splicing of TTN, the heart expresses two major isoforms (N2B and N2BA) that incorporate four distinct regions termed the Z-line, I-band, A-band, and M-line. Next-generation sequencing allows a large number of genes to be sequenced simultaneously and provides the opportunity to easily analyze giant genes such as TTN. Mutations in the TTN gene can cause cardiomyopathies, in particular dilated cardiomyopathy (DCM). DCM is the most common form of cardiomyopathy, and it is characterized by systolic dysfunction and dilation of the left ventricle. TTN truncating variants have been described as the most common cause of DCM, while the real impact of TTN missense variants in the pathogenesis of DCM is still unclear. In a recent population screening study, rare missense variants potentially pathogenic based on bioinformatic filtering represented only 12.6% of the several hundred rare TTN missense variants found, suggesting that missense variants are very common in TTN and are frequently benign. The aim of this review is to understand the clinical role of TTN mutations in DCM and in other cardiomyopathies. Whereas TTN truncations are common in DCM, there is evidence that TTN truncations are rare in the hypertrophic cardiomyopathy (HCM) phenotype. Furthermore, TTN mutations can also cause arrhythmogenic right ventricular cardiomyopathy (ARVC) with distinct clinical features and outcomes. Finally, the identification of a rare TTN missense variant cosegregating with the restrictive cardiomyopathy (RCM) phenotype suggests that TTN is a novel disease-causing gene in this disease. Clinical diagnostic testing is currently able to analyze over 100 cardiomyopathy genes, including TTN; however, the size and presence of extensive genetic variation in TTN presents clinical challenges in determining significant disease-causing mutations. This review discusses the current knowledge of TTN genetic variations in cardiomyopathies and the impact of the diagnosis of TTN pathogenic mutations in the clinical setting.

Background: Mutations in the TTN gene are the most common causes of dilated cardiomyopathy (DCM). The clinical significance of TTN gene variants remains inadequately understood.Methods: Whole-exome sequencing and phenotypic characterisation were performed, and patients were followed up for a median of 44 months.Results: We analyzed the association of the TTN variants with the clinical outcomes in a prospective study of 1,041 patients with sporadic DCM. TTN truncating variants (tTTN) were detected in 120 (11.5%) patients as compared with 2.4/10,000 East Asian populations in the Genome Aggregation Database (GnomAD; p < 0.0001). Pathogenic TTN missense variants were also enriched in DCM as compared with the GnomAD populations (27.6 vs. 5.9%, p < 0.0001). DCM patients with tTTN had a lower left ventricular ejection fraction (28.89 ± 8.72 vs. 31.81 ± 9.97, p = 0.002) and a lower frequency of the left bundle branch block (3.3 vs. 11.3%, p = 0.011) than those without or with mutations in other known causal genes (OCG). However, tTTN were not associated with the composite primary endpoint of cardiac death and heart transplantation during the follow-up period [adjusted hazard ratio (HR): 0.912; 95% confidence interval: 0.464–1.793; p = 0.790]. There was also no sex-dependent effect. Concomitant tTTN and pathogenic variants in OCG were present in only eight DCM patients and did not affect the outcome.Conclusion: The phenotype of DCM caused by tTTN, major causes of sporadic DCM, is not distinctly different from those caused by other causal genes for DCM.

We have previously described 19 pedigrees with apparent lamin (LMNA)-related dilated cardiomyopathy (DCM) manifesting in affected family members across multiple generations. In 6 of 19 families, at least 1 individual with idiopathic DCM did not carry the family's LMNA variant. We hypothesized that additional genetic cause may underlie DCM in these families. Affected family members underwent exome sequencing to identify additional genetic cause of DCM in the 6 families with nonsegregating LMNA variants. In 5 of 6 pedigrees, we identified at least 1 additional rare variant in a known DCM gene that could plausibly contribute to disease in the LMNA variant-negative individuals. Bilineal inheritance was clear or presumed to be present in 3 of 5 families and was possible in the remaining 2. At least 1 individual with a LMNA variant also carried a variant in an additional identified DCM gene in each family. Using a multivariate linear mixed model for quantitative traits, we demonstrated that the presence of these additional variants was associated with a more severe phenotype after adjusting for sex, age, and the presence/absence of the family's nonsegregating LMNA variant. Our data support DCM as a genetically heterogeneous disease with, at times, multigene causation. Although the frequency of DCM resulting from multigenic cause is uncertain, our data suggest it may be higher than previously anticipated.

Dilated cardiomyopathy (DCM), which include genetic and nongenetic forms, is the most common form of cardiomyopathy. DCM is characterized by left ventricular or biventricular dilation with impaired contraction. In the United States, DCM is a burden to healthcare that accounts for approximately 10,000 deaths and 46,000 hospitalizations annually. In this review, we will focus on the genetic forms of DCM and on recent advances in the understanding of cytoskeletal, sarcomeric, desmosomal, nuclear membrane, and RNA binding genes that contribute to the complexity and genetic heterogeneity of DCM. Although mutations in TTN remain the most common identifiable cause of genetic DCM, there is a growing appreciation for arrhythmogenic-prone DCM due to mutations in LMNA, desmosomal genes, and the recently described FLNC gene encoding the structural filamin C protein. Mutations in RBM20 highlight the relevance of RNA splicing regulation in the pathogenesis of DCM. Although expanded genetic testing has improved access to genetic diagnostic studies for many patients, the molecular mechanisms in the pathogenesis of the disease remained largely unknown. : The identification of the molecular causes and subsequent insight into the molecular mechanisms of DCM is expanding our understanding of DCM pathogenesis and highlights the complexity of DCM and the need to develop multifaceted strategies to treat the various causes of DCM.

Dilated cardiomyopathy (DCM) is an autosomal dominant disease characterised by dilation of the left ventricle and reduced systolic function. Although hundreds of mutations in more than 40 genes have been attributed to DCM, affecting a wide variety of structural proteins, conduction pathways and contraction mechanisms, this accounts for approximately 40% of familial genetic causes. Additionally, due to low prevalence of any particular single gene mutation in the population, as well as genetic overlap with other cardiomyopathies and diagnostic technology limitations, the diagnosis of DCM remains difficult. The introduction of high‐throughput deoxyribonucleic acid (DNA) sequencing through multigene testing panels has improved diagnostic sensitivity. Furthermore, functional studies have provided innovative approaches to advance collective understanding of DCM aetiology, pathogenesis and diagnosis. Key Concepts: DCM is an autosomal dominant disease affecting 1 in 2500 individuals with up to 50% of cases attributed to genetic causes. Approximately 40 genes have been discovered as part of DCM aetiology, but this accounts for only 40% of genetic cases. Mutations causing DCM have been discovered in multiple cardiomyocyte proteins and pathways, with most mutations representing a rare cause of DCM and each contributing a low frequency of DCM cases. Mutated genes involved in DCM include those encoding sarcomeric, cytoskeleton, nuclear membrane and ion channel proteins; most mutations leads to a similar DCM phenotype. DCM‐associated rare gene variants found both in research and clinical settings require confirmatory and follow‐up studies, because some of them may eventually result in benign common variations. Functional studies model systems and animal models, such as in zebrafish, are useful for clearly observing the heart, and these studies have been successful in demonstrating distinct cardiac phenotypes for DCM‐related gene mutations.

Heart failure is a major health burden, affecting 40 million people globally. One of the main causes of systolic heart failure is dilated cardiomyopathy (DCM), the leading global indication for heart transplantation. Our understanding of the genetic basis of both DCM and systolic heart failure has improved in recent years with the application of next-generation sequencing and genome-wide association studies (GWAS). This has enabled rapid sequencing at scale, leading to the discovery of many novel rare variants in DCM and of common variants in both systolic heart failure and DCM. Identifying rare and common genetic variants contributing to systolic heart failure has been challenging given its diverse and multiple etiologies. DCM, however, although rarer, is a reasonably specific and well-defined condition, leading to the identification of many rare genetic variants. Truncating variants in titin represent the single largest genetic cause of DCM. Here, we review the progress and challenges in the detection of rare and common variants in DCM and systolic heart failure, and the particular challenges in accurate and informed variant interpretation, and in understanding the effects of these variants. We also discuss how our increasing genetic knowledge is changing clinical management. Harnessing genetic data and translating it to improve risk stratification and the development of novel therapeutics represents a major challenge and unmet critical need for patients with heart failure and their families.

Titin truncating mutations: A rare cause of dilated cardiomyopathy in the young

Truncating variants in the TTN gene (TTNtv) are the most common genetic cause of dilated cardiomyopathy (DCM) but also occur as incidental findings in the general population. This study investigated factors associated with the clinical manifestation of TTNtv. An international multicentre retrospective observational study was performed in families with TTNtv-related DCM. Shared frailty models were used to estimate associations of variant characteristics with lifetime risk of DCM, and logistic regression to estimate odds ratios (ORs) for individual-level clinical risk factor profiles (cardiac conditions, cardiovascular comorbidities, lifestyle) and DCM. A total of 3158 subjects in 1043 families with TTNtv-related DCM were studied. TTNtv-positive subjects were 21-fold more likely to develop DCM [OR, 21.21; 95% confidence interval (CI), 14.80-30.39]. Disease onset was earlier in males, but was similar for TTNtv of different types and locations. The presence of clinical risk factors was associated with earlier DCM onset (OR, 3.41; 95% CI, 2.06-5.64), with a prior history of atrial fibrillation having a two-fold increased odds of DCM (OR, 2.05; 95% CI, 1.27-3.32). The prevalence of clinical risk factors increased with age; however, the strength of the DCM association was greatest for young-onset (<30 years) disease (OR, 4.75; 95% CI, 2.35-9.60). Administration of beta-adrenergic receptor or renin-angiotensin system-blocking drugs prior to overt DCM was associated with 87% reduced odds of DCM (OR, .13; 95% CI, .08-.23). Disease onset in TTNtv-associated familial DCM is dependent on individual patient context and is potentially modifiable by risk factor management and prophylactic therapeutic intervention.

Dilated cardiomyopathy (DCM) is the leading indication for heart transplantation. TTN gene truncating mutations account for about 25% of familial DCM cases and for 18% of sporadic DCM cases. The clinical relevance of specific variants in TTN has been difficult to determine because of the sheer size of the protein for which TTN encodes, as well as existing extensive genetic variation. Clinicians should communicate novel clinically-relevant variants and genotype–phenotype associations, so that animal studies evaluating the molecular mechanisms are always conducted with a focus on clinical significance. In the present study, we report for the first time the novel truncating heterozygous variant NM_001256850.1:c.72777_72783del (p.Phe24259Leufs*51) in the TTN gene and its association with DCM in a family with sudden death. This variant occurs in the A-band region of the sarcomere, in a known mutational hotspot of the gene. Truncating titin variants that occur in this region are the most common cause of DCM and have been rarely reported in asymptomatic individuals, differently from other pathogenic TTN gene variants. Further studies are warranted to better understand this particular clinically-relevant variant.

Idiopathic dilated cardiomyopathy (DCM) is a common cardiomyopathy with the prevalence of 1:250, and at least one-third of all the cases are inherited. Mutations in the TTN gene are considered as the most frequent cause of inherited DCM and cover 10–30% of the cases. The studies were mainly focused on the adult or mixed age group of patients with DCM. The mutation rate in the TTN gene, the characteristics of manifestations and their prognostic significance in childhood have not been studied. To determine TTN mutation rate in children with DCM and the relevance of including this gene in the DNA diagnostic protocol for paediatric DCM, complete clinical and instrumental examination of 36 DCM patients (up to 18 years) with the manifestation of the disease was conducted in specialised cardiology centres. Molecular genetic testing included sequencing of coding and adjacent regulatory regions of the major cardiac TTN isoform N2BA using IonTorrent ™ semiconductor sequencing (for 25 isolated cases) and trio whole exome sequencing (trio WES)on the Illumina platform (for 11 family cases). Our pilot group included 36 probands with DCM diagnosis first established on the basis of the generally accepted criteria at the age of 5 days to 18 years(average age: 6.5 years). The sex ratio (M:F) was 23: 8. There were 25 sporadic DCM cases and 11 cases of familial DCM (at least one of the parents and/or siblings were also diagnosed with DCM). The only likely pathogenic truncating variant p.Arg33703*in the TTN gene (TTNtv) was found in a 16-year-oldmale proband out of 36 (3%). Apparently, TTN-dependent forms of DCMs manifest later at a young (but older than 18 years) or more mature age, and TTN gene cannot be considered as the first-line genetic testing for DCM in the paediatric group, despite several studies have reported a generally high mutation rate in this gene with DCM. Further research is needed to compare the representation of mutations in the TTN gene in different age groups of DCM patients.

Protein-truncating variants in the TTN gene are a well-established cause of dilated cardiomyopathy (DCM). We report a novel case of DCM caused by a mobile element insertion (MEI) in TTN, through which we highlight the key features of MEIs in next-generation sequencing data. Because of the rarity of MEIs, the next-generation sequencing data features associated with these events may be mistaken as noise, potentially leading to missed diagnoses. Next-generation sequencing gene panel testing for DCM was performed on a 17-year-old male patient presenting with severe left ventricular dilatation and systolic dysfunction. Manta was used for structural variant detection, followed by manual review of NGS data for potential structural variants. Manta detected a potential insertion in TTN. Manual review identified hallmark features consistent with a LINE-1 MEI. This finding was orthogonally confirmed by long-range polymerase chain reaction and gel electrophoresis, which indicated an insertion of approximately 4 to 5 kilobase pairs. The insertion disrupted the reading frame of TTN within an A-band exon, resulting in protein truncation that was classified as likely pathogenic. This case expands the mutational spectrum of TTN protein-truncating variants. It also underscores the importance of recognizing rarer types of pathogenic variants (eg, MEIs) to produce accurate genetic diagnostics.

Dilated cardiomyopathy (DCM) is a heart muscle disease that represents an important cause of morbidity and mortality, yet causal mechanisms remain largely elusive. Here, we perform a large-scale genome-wide association study and multitrait analysis for DCM using 9,365 cases and 946,368 controls. We identify 70 genome-wide significant loci, which show broad replication in independent samples and map to 63 prioritized genes. Tissue, cell type and pathway enrichment analyses highlight the central role of the cardiomyocyte and contractile apparatus in DCM pathogenesis. Polygenic risk scores constructed from our genome-wide association study predict DCM across different ancestry groups, show differing contributions to DCM depending on rare pathogenic variant status and associate with systolic heart failure across various clinical settings. Mendelian randomization analyses reveal actionable potential causes of DCM, including higher bodyweight and higher systolic blood pressure. Our findings provide insights into the genetic architecture and mechanisms underlying DCM and myocardial function more broadly.

Introduction. The causes of dilated cardiomyopathy can be divided into ischemic and nonischemic. Although the importance of thyroid hormones for proper functioning of the cardiovascular system is well known, dilated cardiomyopathy is a rare presentation of hypothyroidism. Case Report. This is a case report of a 38-year-old man admitted to the Intensive Cardiac Care Unit with signs and symptoms of advanced heart failure. Dilated cardiomyopathy was established by echocardiography, while blood test showed highly elevated levels of thyroid stimulating hormone and decreased levels of free triiodothyronine and free thyroxine. The ejection fraction was significantly improved with levothyroxine replacement therapy. Conclusion. Hypothyroidism should always be considered as the cause of dilated cardiomyopathy. Thyroid hormone tests should be performed in all patients with dilated cardiomyopathy.

To determine whether Borrelia burgdorferi is implicated in the pathogenesis of dilated cardiomyopathy in the United Kingdom. A controlled prospective study. Patients' notes were reviewed for evidence of Lyme disease and serum samples were tested by enzyme linked immunoadsorbent assay (ELISA) for antibodies to B burgdorferi. Samples with raised antibody concentrations were subsequently analysed by immunoblotting to determine their antibody binding specificity. Tertiary referral centre. 97 consecutive patients with dilated cardiomyopathy diagnosed according to World Health Organisation criteria were studied. Serum samples were taken from two matched control groups. The first group (n = 38) was age, sex, and geographically matched. The second control group (n = 39) was environmentally matched and consisted of members of the patients' own households. Clinical evidence of Lyme disease. Presence of raised antibody concentrations to B burgdorferi. No patients had a previous illness compatible with Lyme disease. Analysis of the ELISA data showed eight of 97 patients with dilated cardiomyopathy (8.2%) and two of 77 controls (3.9%) had raised antibody concentrations. Immunoblot analysis, however, did not show binding patterns consistent with the presence of IgG specific for B burgdorferi in any of these samples. There was no clinical or serological evidence to implicate B burgdorferi in the pathogenesis of idiopathic dilated cardiomyopathy in the United Kingdom. In the absence of specific symptoms or likely exposure to B burgdorferi routine serological testing for Lyme disease in this group of patients is not recommended. Furthermore, raised antibodies to B burgdorferi are not diagnostic of active infection and ELISA results should be interpreted with caution unless specific B burgdorferi antibody bands have been found by immunoblot analysis.

Dilated cardiomyopathy (DCM) is one of the most frequent causes of heart failure of unknown origin. One possible cause of DCM is considered to be a sequel to myocarditis. However, the mechanism of progression from viral myocarditis to DCM is still not clear. The expression of the immunoregulatory cytokines interferon (IFN)-gamma and interleukin (IL)-2 and the proinflammatory cytokines IL-1 beta and tumor necrosis factor (TNF)-alpha in the heart tissue was studied in a murine model of postmyocarditis DCM induced by encephalomyocarditis virus. IFN-gamma, IL-1 beta, and TNF-alpha mRNA increased 3 days after virus inoculation. IL-2 mRNA was detectable 7 days after inoculation. The peak expression of all cytokine genes examined was seen 7 days after inoculation. The expression of these cytokine genes decreased thereafter but persisted 80 days after inoculation. IL-1 beta gene expression in the chronic stage was relatively high compared with other cytokines and was correlated with the ratio of heart weight to body weight and the extent of fibrotic lesions. Immunohistochemical analysis revealed that some of the mononuclear cells, endothelial cells, and interstitial macrophages were positive for IL-1 beta or TNF-alpha and fibroblasts were positive for IL-1 beta in the heart tissue of mice 80 days after inoculation. Persistent expression of cytokines was seen in a murine model of postmyocarditis DCM. These cytokines may have important implications in the pathogenesis of DCM.