Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy (DCM) and skeletal myopathy. The most severely affected DCM 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 (Z-disk and I-band) and C-terminal (A-band) 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.
Animal experimentation: All zebrafish and mouse experimental work conformed to the 'Guide for the Care and Use of Laboratory Animals' published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animal work was performed according to institutional guidelines with the full approval of the University of California Institutional Animal Care and Use Committee (protocols AN090013-03 and AN107039-01)
Human subjects: Human genetic studies were performed according to institutional guidelines and with the full approval of the University of California San Francisco Committee on Human Research (CHR#10-00207) and all studies performed were in keeping with the original informed consent forms. Informed consent and consent to publish was obtained from all participants.
- Harry C Dietz, Howard Hughes Medical Institute and Institute of Genetic Medicine, Johns Hopkins University School of Medicine, United States
© 2015, Zou et al.
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