Truncations of titin causing dilated cardiomyopathy

Abstract

Background: Dilated cardiomyopathy and hypertrophic cardiomyopathy arise from mutations in many genes. TTN, the gene encoding the sarcomere protein titin, has been insufficiently analyzed for cardiomyopathy mutations because of its enormous size.

Methods: We analyzed TTN in 312 subjects with dilated cardiomyopathy, 231 subjects with hypertrophic cardiomyopathy, and 249 controls by using next-generation or dideoxy sequencing. We evaluated deleterious variants for cosegregation in families and assessed clinical characteristics.

Results: We identified 72 unique mutations (25 nonsense, 23 frameshift, 23 splicing, and 1 large tandem insertion) that altered full-length titin. Among subjects studied by means of next-generation sequencing, the frequency of TTN mutations was significantly higher among subjects with dilated cardiomyopathy (54 of 203 [27%]) than among subjects with hypertrophic cardiomyopathy (3 of 231 [1%], P=3×10(-16)) or controls (7 of 249 [3%], P=9×10(-14)). TTN mutations cosegregated with dilated cardiomyopathy in families (combined lod score, 11.1) with high (>95%) observed penetrance after the age of 40 years. Mutations associated with dilated cardiomyopathy were overrepresented in the titin A-band but were absent from the Z-disk and M-band regions of titin (P≤0.01 for all comparisons). Overall, the rates of cardiac outcomes were similar in subjects with and those without TTN mutations, but adverse events occurred earlier in male mutation carriers than in female carriers (P=4×10(-5)).

Conclusions: TTN truncating mutations are a common cause of dilated cardiomyopathy, occurring in approximately 25% of familial cases of idiopathic dilated cardiomyopathy and in 18% of sporadic cases. Incorporation of sequencing approaches that detect TTN truncations into genetic testing for dilated cardiomyopathy should substantially increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy. Defining the functional effects of TTN truncating mutations should improve our understanding of the pathophysiology of dilated cardiomyopathy. (Funded by the Howard Hughes Medical Institute and others.).

Figure 1. Spatial Distribution of Truncating Mutations in Titin

The cardiac sarcomere (top) consists of titin (orange), the third major filament, in addition to the thick filaments (green rods with globular heads) and thin filaments (green coiled ovals). Regions of the sarcomere are demarcated as follows: Z-disk (red), I-band (blue), A-band (green), and M-band (purple). The sarcomere depiction is adapted from Granzier and Labeit. TTN mutations are shown as thin vertical bars (middle), with overlapping mutations appearing as thicker bars. Splicing and copy-number mutations (blue) and nonsense and frameshift mutations (red) in subjects with dilated cardiomyopathy are shown, including two frameshift mutations previously reported to be linked to dilated cardiomyopathy (Table 14 in the Supplementary Appendix). All types of truncating mutations in controls and subjects with hypertrophic cardiomyopathy (black) are also indicated, as are truncating mutations previously identified in patients with congenital myopathy (light purple) or limb-girdle muscular dystrophy (dark purple). Titin isoforms and sequence variants are shown relative to the titin sequence (UniProt sequence Q8WZ42, www.uniprot.org) (bottom). Analysis of the N2BA class of cardiac titin isoforms excluded exons (black carets) with little evidence for cardiac expression (see Table 15 in the Supplementary Appendix). In cardiac tissue, TTN isoforms N2BA and N2B span the sarcomere, whereas novex-3 titin is shorter and less abundant (the exon specific to novex-3 titin is shown as a black bar).

Figure 2. Kaplan–Meier Estimates of Age at Diagnosis and Clinical Progression in Subjects with Dilated Cardiomyopathy Caused by TTN Mutations

Panel A shows data for the time of diagnosis, and Panels B, C, and D show freedom from cardiac transplantation, implantation of left ventricular assist device (LVAD), and death from cardiac causes. Panels A and B show data for the 67 subjects who had mutations and the 228 who did not. Panels C and D show data for the 94 subjects (from 19 families) who had truncating mutations (Fig. 2 in the Supplementary Appendix; also see the Supplementary Appendix for a description of the effect of sex). Hatch marks indicate subjects with censored data.

Figure 3. Histopathological Abnormalities in Cardiac Specimens from Two Subjects with TTN Truncating Mutations

Specimens from the cardiac interventricular septum in two subjects (Panel A and Panel B, hematoxylin and eosin) show myocyte nuclei with abnormal morphologic characteristics (arrowheads).