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Published on: February 25, 2022
An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of
Jun Zou1, Diana Tran1, Mai Baalbaki1
1Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States.
Abstract:
Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.
Insights
Titin truncations cause heart and muscle disease. A newly found internal promoter explains why C-terminal Titin mutations are more severe than N-terminal ones, offering new insights into sarcomere structure and function.
Area of Science:
- Molecular Biology
- Cardiovascular Genetics
- Muscle Physiology
Background:
- Truncating mutations in the giant sarcomeric protein Titin are linked to dilated cardiomyopathy and skeletal myopathy.
- Disease severity in patients with Titin truncations correlates with mutation location, particularly in the C-terminal regions.
Purpose of the Study:
- To investigate the impact of Titin mutation position on disease phenotype using zebrafish models.
- To elucidate the underlying molecular mechanisms responsible for differential disease severity.
Main Methods:
- Generation of six zebrafish lines with CRISPR/Cas9-induced Titin truncations in N-terminal and C-terminal regions.
- Phenotypic analysis of myopathy and cardiac function in mutant zebrafish lines.
- Investigation of Titin gene regulation, including promoter activity and alternative splicing.
Main Results:
- Zebrafish with C-terminal Titin truncations exhibited severe myopathy, while N-terminal truncations resulted in milder phenotypes.
- Neither N-terminal nor C-terminal Titin truncations acted in a dominant-negative manner.
- A conserved internal promoter was identified near the C-terminal mutation site, producing a protein isoform that partially rescued N-terminal truncation phenotypes.
Conclusions:
- Mutation position significantly influences the severity of Titin-related myopathies.
- An internal promoter and alternative protein isoforms play a crucial role in modulating Titin-related muscle diseases.
- These findings provide novel insights into Titin gene regulation, sarcomere architecture, and potential therapeutic strategies for dilated cardiomyopathy and skeletal myopathies.
