Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease

Abstract

The recent discovery of heterozygous human mutations that truncate full-length titin (TTN, an abundant structural, sensory, and signaling filament in muscle) as a common cause of end-stage dilated cardiomyopathy (DCM) promises new prospects for improving heart failure management. However, realization of this opportunity has been hindered by the burden of TTN-truncating variants (TTNtv) in the general population and uncertainty about their consequences in health or disease. To elucidate the effects of TTNtv, we coupled TTN gene sequencing with cardiac phenotyping in 5267 individuals across the spectrum of cardiac physiology and integrated these data with RNA and protein analyses of human heart tissues. We report diversity of TTN isoform expression in the heart, define the relative inclusion of TTN exons in different isoforms (using the TTN transcript annotations available at http://cardiodb.org/titin), and demonstrate that these data, coupled with the position of the TTNtv, provide a robust strategy to discriminate pathogenic from benign TTNtv. We show that TTNtv is the most common genetic cause of DCM in ambulant patients in the community, identify clinically important manifestations of TTNtv-positive DCM, and define the penetrance and outcomes of TTNtv in the general population. By integrating genetic, transcriptome, and protein analyses, we provide evidence for a length-dependent mechanism of disease. These data inform diagnostic criteria and management strategies for TTNtv-positive DCM patients and for TTNtv that are identified as incidental findings.

Conflict of interest statement

Competing Interests: The authors declare that they have no competing interests.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1. Distribution of TTNtv in healthy individuals and DCM patients, and TTN exon usage in the heart

A schematic of the TTN meta-transcript is shown, with sarcomere regions demarcated. The meta-transcript (LRG_391_t1 / ENST00000589042) is a manually curated inferred complete transcript, incorporating all exons of all known TTN isoforms (including fetal and non-cardiac isoforms) with the exception of the large alternative terminal exon 48 (dark green) that is unique to the novex-3 transcript (LRG_391_t2 / ENST00000360870). Exon usage for the two principal adult cardiac isoforms, N2BA and N2B (ENST00000591111, ENST00000460472) is shown, though exon usage in vivo is variable (see below). Novex-1 and novex-2 are rare cardiac isoforms that differ from N2B by the inclusion of a single unique exon each (red and blue respectively within the N2B track). Exon usage in human LV is depicted as the “proportion spliced-in” (PSI, range 0–1; grey bars): the proportion of transcripts that include a given exon. TTNtv are located more distally in cases compared with controls, with A-band and distal I-band enrichment in end-stage (n=155) and unselected DCM patients (n=374) and corresponding depletion in the population (n=3603) and healthy volunteer (n=308) cohorts.

Fig. 2. Factors that discriminate TTNtv in health and disease

A: Usage of TTN exons containing TTNtv across all cohorts. Exon usage is represented as proportion spliced-in (PSI), which is an estimate of the proportion of transcripts that incorporate each exon. Each plotted data point represents the estimated PSI of an exon identified to have a TTNtv, separated by cohort. There was a strong relationship between the PSI of exons containing TTNtv and disease status (P=4.9×10−3, Kruskal-Wallis), with TTNtv in DCM cases found in more highly used exons (P = 4.7×10−4, Mann-Whitney). A similar difference was observed between the replication cohorts (P = 7.5×10−4). B: Relationships between TTNtv location, PSI and disease status. The positions of TTNtv are shown for constitutively expressed exons only (PSI=1).

Fig. 3. TTNtv and survival in DCM

Outcomes in unselected DCM patients with (red) and without (blue) TTNtv. The left panel shows age censored at adverse event (death, cardiac transplant or left-ventricular assist device (LVAD) or at age 70 years. The right panel shows adverse events after enrollment, to control for ascertainment (interval censored from time of enrollment to age 70 years or adverse event). Event free survival is reduced in TTNtv-positive DCM (P = 0.015) due to altered disease progression both before and after presentation. A trend to younger presentation (Table 2), and worse outcomes after enrollment (P = 0.05) combine to give reduced survival overall.

Fig. 4. Allelic dissection of the impact of TTNtv position on cardiac morphology and function

The relationships between TTNtv location and cardiac morphology and function assessed by CMR imaging in an allelic series of DCM cases. Genotype-phenotype relationships are shown for 43 TTNtv in unselected DCM patients. The TTNtv location (X axis) is plotted from the amino- (N) to carboxyl- (C) end of the protein. Distal (C-terminal) TTNtv were associated with worse cardiac contractile performance and associated with diminished indexed stroke volume (SVi) and ejection fraction (EF) of both left and right ventricles as compared to proximal truncations. A regression line is shown for each variable (Tables S14–S15). LV, left ventricle; RV, right ventricle; EDVi, indexed end diastolic stroke volume (ml/m2); ESVi, indexed end systolic volume (ml/m2), SVi, indexed stroke volume (ml/m2), EF ejection fraction (%).

Fig. 5. TTN mRNA and protein expression in LV tissues from DCM patients with and without TTNtv

A. Levels of TTN mRNA in TTNtv-positive (n=18) and negative (n=66) patients (normalised read counts). B. Allelic balance of TTNtv compared to non-truncating TTN single nucleotide polymorphisms (SNPs), a surrogate for the proportion of transcripts with variant alleles (TTNtv or SNPs) among DCM patients with and without TTNtvs. The comparable allelic expression of TTNtv and SNPs does not support substantial nonsense-mediated decay (see also Fig. S9). Bars indicate median and quartiles. C. Protein electrophoresis from a healthy LV (C, lane 1) and LV from DCM patients (lanes 2–12: +, TTNtv-positive; −, TTNtv-negative). Sample IDs are shown for subjects with TTNtv: variant details are shown in Table S4. Truncated protein was not seen in TTNtv-positive samples. The arrows indicate approximate expected sizes of the truncated N2B and N2BA isoforms and T2 denotes TTN degradation product. Semi-quantitative analysis of TTN protein relative to myosin (MHC, myosin heavy chain) showed no reduction of TTN in TTNtv-positive samples.