A novel homozygous TTN splice-site variant causing aberrant pre-mRNA splicing and an in-frame insertion/deletion (p.Arg32498_Glu32504delinsGln) was identified in a hypertrophic cardiomyopathy proband, with functional validation via minigene assay supporting reclassification from VUS to tepid VUS (scoring 3 points).
Key Findings
Results
A novel homozygous TTN splice-site variant was identified in a 42-year-old male proband with nonobstructive HCM and paroxysmal atrial arrhythmias via whole-exome sequencing.
The proband was a 42-year-old Chinese male diagnosed with nonobstructive hypertrophic cardiomyopathy.
The variant was predicted to cause a 14-base pair deletion at a splice acceptor site.
Two asymptomatic offspring were found to carry the heterozygous variant.
Initial classification under variant interpretation guidelines placed the variant as a variant of uncertain significance (VUS), meeting only the PM2_supporting criterion.
Results
Minigene splicing assay confirmed aberrant pre-mRNA splicing caused by the TTN variant, resulting in an in-frame insertion/deletion at the protein level.
The minigene assay demonstrated altered pre-mRNA splicing leading to an in-frame insertion/deletion designated p.Arg32498_Glu32504delinsGln.
In silico splicing prediction tools were used prior to the minigene assay to predict the splicing impact.
The functional verification confirmed that the mutation conforms to the PM4 criterion.
Meeting PM4 supported reclassification of the variant as a 'tepid VUS' scoring 3 points.
Results
Among 28 previously reported TTN splicing variants associated with HCM, the majority localized to the I-band region of titin.
57.1% (16/28) of previously reported HCM-associated TTN splicing variants localized to the I-band region.
21.4% (6/28) were situated in the A-band domain of titin.
21.4% (6/28) co-occurred with pathogenic variants in other sarcomeric genes (MYH7 or MYBPC3), correlating with more severe clinical phenotypes.
Reclassification and reinterpretation of all 28 variants revealed that none met the level of likely pathogenic or higher.
Results
Co-occurrence of TTN splicing variants with pathogenic variants in other sarcomeric genes was associated with more severe clinical phenotypes.
6 of 28 (21.4%) previously reported TTN splicing variants in HCM co-occurred with pathogenic variants in MYH7 or MYBPC3.
This co-occurrence correlated with more severe clinical phenotypes.
The literature review covered 28 previously reported splicing variants in TTN associated with HCM.
Background
The TTN gene encodes a crucial structural protein within cardiac sarcomeres and its variants may contribute to both hypertrophic cardiomyopathy and dilated cardiomyopathy, though phenotype and genotype differ between these conditions.
TTN variants have been associated with both HCM and dilated cardiomyopathy.
Phenotype and genotype relationships differ between the two cardiomyopathy subtypes.
The present case focuses on the Mendelian genetic basis of the proband's cardiomyopathy phenotype.
What This Means
This research describes a genetic investigation of a 42-year-old Chinese man with hypertrophic cardiomyopathy (HCM), a condition where the heart muscle becomes abnormally thick. Scientists used whole-exome sequencing—a technique that reads the protein-coding portions of a person's DNA—and discovered a rare mutation in both copies of his TTN gene, which provides instructions for making titin, a giant protein that acts like a molecular spring in heart muscle cells. The mutation was located at a 'splice site,' meaning it affects how the genetic instructions are processed before being used to make the protein. The man's two children each inherited one copy of the mutated gene but showed no symptoms.
To confirm that this mutation actually disrupts the normal processing of the TTN gene's instructions, the researchers used a laboratory test called a minigene assay. This test showed that the mutation does indeed cause abnormal splicing of the genetic message, resulting in a slightly altered titin protein with a small segment of amino acids changed (p.Arg32498_Glu32504delinsGln). Because the change is 'in-frame,' the overall protein structure is maintained rather than being completely disrupted. This functional evidence allowed the researchers to upgrade the variant's classification from a standard 'variant of uncertain significance' to a 'tepid VUS' with a slightly higher evidence score, though it still did not reach the threshold of 'likely pathogenic.'
The researchers also reviewed 28 previously reported TTN splice-site variants linked to HCM. They found that most (57.1%) were located in a specific structural region of titin called the I-band, and that about one in five patients also had mutations in other heart muscle genes (MYH7 or MYBPC3), which was associated with more severe disease. This research suggests that TTN splicing variants can play a role in HCM, and that laboratory testing—rather than genetic prediction alone—is important for understanding the true impact of these mutations. The findings also highlight how complex the genetics of HCM can be, particularly when multiple gene variants are present.
Sun X, Wang D, Wang S, Wan R, Jiang Z, Zhang W, et al.. (2026). Functional Validation of a Novel Homozygous TTN Splice-Site Variant Reveals Aberrant Splicing in Hypertrophic Cardiomyopathy.. Human mutation. https://doi.org/10.1155/humu/4229370