Within the sarcomere, titin spans from the Z-disc to the M-band,² making it the third most abundant myofilament in striated muscle, with a size of approximately 33,000 amino acids.³ The properties of titin vary along its length, depending on its location within the sarcomere.² Several isoforms of titin exist, with N2B and N2BA being predominant in the heart. The TTN gene also encodes a distinct cardiac isoform, novex-3 titin, which is 5,600 amino acids long and lacks the A-band and M-band segments of titin.³ The functional characteristics of titin are significantly influenced by alternative splicing of the TTN gene. More than a third of TTN's 363 exons, particularly those in the I-band, can undergo alternative splicing, generating a variety of isoforms. These isoforms result in titin proteins with different lengths and stiffness, affecting both passive tension and force production within the sarcomere. During fetal development, longer and more flexible isoforms are predominant, while shorter and stiffer isoforms, such as N2BA and N2B, replace them after birth. In adult hearts, these isoforms exist in a typical ratio of 70:30 to 60:40, respectively. Isoform switching, regulated by the master splicing regulator RNA binding motif 20 (RBM20), modulates sarcomere tension in response to changes in loading conditions and pathological processes. Variants affecting RBM20 are also linked to the DCM phenotype. Additionally, post-translational modifications, like phosphorylation, rapidly alter titin's stiffness in response to physiological and pathological stress.⁴

Truncating variants in the TTN gene (TTNtv), including nonsense variants that result in premature termination codons, insertions and deletions (indels) causing frameshifts, and variants affecting canonical splice sites, are now recognized as a major genetic cause of DCM. These variants are predominantly found in the A-band of titin. Cohort studies have analyzed the distribution of truncating variants in TTN among DCM patients and controls. The titin protein is segmented into different bands: Z-disc (red), I-band (blue), A-band (green), and M-band (purple) (Figure 2).⁵

Interpreting TTN variants is challenging due to the presence of multiple transcripts with variable expression across different tissues (table 1). Alternative splicing means not all exons are included in every transcript, which can affect the impact of a variant depending on the exon involved. The recommended approach for variant reporting involves the inferred full meta-transcript, as annotated by the Havana Group. Truncating variants in the A-band region of cardiac isoforms N2B and N2BA, which are more highly expressed in cardiac tissue, are particularly associated with DCM. TTN contains various domains, including Ig-like, Z-repeat, PEVK, Fibronectin type III, and Protein kinase domains. For more details on TTN exons, transcripts, and domains, resources like https://www.cardiodb.org/ are valuable. Variant interpretation requires careful consideration of phenotype, inheritance pattern, and variant classification, following the American College of Medical Genetics and Genomics (ACMG) guidelines.⁶ Variants are classified as benign, likely benign, variants of uncertain significance, likely pathogenic, and pathogenic, based on criteria including variant type (nonsense, missense, frameshift) and frequency in control populations. Although missense variants in TTN are frequently found during genomic analysis, they are rarely reported as the primary pathogenic mechanism in TTN-related DCM is truncating variants. Variants of uncertain significance should be periodically re-evaluated as new data emerge, and family segregation studies can aid in reclassification. Determining pathogenicity for TTN truncating variants requires considering the variant's position in the gene and correlating with clinical or functional data, such as previous descriptions in symptomatic individuals and the associated clinical phenotype (DCM, myopathy). Thus, caution is advised when interpreting TTN variants.

The expressivity of TTN gene variants refers to the spectrum of phenotypes observed in individuals carrying the same variant. TTN variants have a well-established link with DCM, typically showing an autosomal dominant inheritance pattern, and TTN-related myopathy, which generally follows an autosomal recessive pattern (https://clinicalgenome.org/). The association of TTN variants with hypertrophic cardiomyopathy is limited.³

DCM can be caused by variants in several genes, but TTN truncating variants are responsible for most genetic cases. TTNtv variants in the A-band are present in up to 25% of familial DCM cases and 18% of sporadic cases.⁷ Family studies suggest that the penetrance of truncating TTN variants in individuals over 40 years old is close to 95%.³ However, TTN truncating variants are also found in about 1% of healthy individuals, indicating incomplete penetrance and variable expressivity, which may involve other yet-to-be-understood mechanisms. When evaluating TTN variants, it is important to consider the phenomena of incomplete penetrance and variable expressivity. Not everyone with a pathogenic TTN variant will exhibit related signs or symptoms, and clinical presentations may vary even within the same family. This variability complicates the prediction of cardiomyopathy onset. Additional genetic and environmental factors, such as hemodynamic stress,⁸ alcohol consumption,⁹ chemotherapy,¹⁰ and pregnancy,¹¹ may contribute to this phenotypic variability, although the precise mechanisms remain unclear.

Identifying a causal TTN variant has important prognostic implications. Compared to DCM patients without identified variants, those with TTN variants exhibit similar clinical manifestations, morbidity, and mortality.³ However, DCM patients with TTN variants show a higher rate of reverse remodeling in response to heart failure treatment, with 53.2% responding favorably compared to only 11.1% of patients with desmosomal gene variants.¹²

Genetic evaluation offers benefits beyond the index patient, extending to family members, even during subclinical phases. Family screening enables early diagnosis, allowing for early treatment and potentially better outcomes.⁷ Genetic counseling and evaluation of first-degree relatives of DCM patients are recommended with a Class I recommendation and Level of Evidence B.⁷

Although established heart failure treatments, such as renin-angiotensin-aldosterone system blockade and beta-adrenergic antagonism, effectively reduce mortality and improve quality of life, they do not directly address the underlying mechanisms caused by TTN variants. Understanding the pathophysiology and molecular pathways involved in titin modification opens new avenues for promising therapeutic strategies and targets.¹³ Some truncated TTN variants lead to overexpression of the mTOR (mammalian target of rapamycin) complex, suggesting that mTOR inhibitors like rapamycin could be evaluated in randomized clinical trials.¹⁴ In animal models, antisense oligonucleotides have been shown to mediate and alter splicing processes, excluding the reading of the mutated exon and preventing premature termination of titin translation. This strategy has proven effective in other conditions, such as Duchenne muscular dystrophy, and is already FDA-approved for that indication.¹⁵ A recent study demonstrated that using CRISPR technology could increase TTN gene expression and titin production in a cell model, improving cellular contractile capacity in patients with haploinsufficiency.¹⁶