Phenotypic spectrum of cardiac conduction disturbance and cardiomyopathy linked to titin canonical splice-site variants

Aims Truncating variations in the titin gene (TTNtv) are the most common genetic cause of dilated cardiomyopathy (DCM) and have been implicated in various arrhythmic and heart failure phenotypes. Nonetheless, predicting the pathogenicity of a distinct subtype of TTNtv, canonical splice-site variations (TTNcsv), remains challenging. Furthermore, the precise transcriptional and phenotypic consequences associated with TTNcsv remain unclear. We evaluated the transcriptional profiles of TTN in vitro, focusing on TTNcsv found in patients with cardiac dysfunction, and compared them with their phenotypic manifestations. Methods and Results Genome-wide linkage analysis, whole-exome sequencing and whole-genome sequencing were performed on a five-generation family with cardiac conduction disturbance (CCD). In addition, whole-genome sequencing was performed on 402 Japanese biobank patients with cardiomyopathy or unidentified cardiac dysfunction. Transcriptional profiles of TTNcsv were evaluated by RNA-Seq of induced pluripotent stem cell-derived cardiomyocytes and endomyocardial biopsy specimens, and by minigene assays. A rare segregating TTNcsv (c.49049-2A>C) was identified in the five-generation family with CCD. RNA-Seq and minigene assays revealed complex aberrant TTN splicing transcripts: predominant non-truncating transcripts caused by an 18-bp in-frame deletion (83–90%) and minor truncating transcripts (10–17%). Two additional TTNcsv were identified in biobank participants by whole-genome sequencing. A minigene assay of TTNcsv c.67348+1G>A, identified in a CCD patient with mild cardiac dysfunction, revealed predominant non-truncating transcripts (95%), caused by retention of a 90-bp in-frame intron, along with 5% truncating TTN transcripts, caused by a 50-bp frameshift deletion, mirroring the TTNcsv observed in the familial CCD. In contrast, the DCM-associated TTNcsv c.67637-2A>G generated 87% truncating TTN transcripts, attributed to frameshift intron retention and cryptic splicing. Conclusions The clinical data from this family suggest a close association between TTNcsv and CCD, which may involve an increase in non-truncating transcripts. Further studies are required to determine their precise relationship and the underlying mechanisms.