Rare truncating variants in the sarcomeric protein titin associate with familial and early-onset atrial fibrillation

Abstract

A family history of atrial fibrillation constitutes a substantial risk of developing the disease, however, the pathogenesis of this complex disease is poorly understood. We perform whole-exome sequencing on 24 families with at least three family members diagnosed with atrial fibrillation (AF) and find that titin-truncating variants (TTNtv) are significantly enriched in these patients (P = 1.76 × 10-6). This finding is replicated in an independent cohort of early-onset lone AF patients (n = 399; odds ratio = 36.8; P = 4.13 × 10-6). A CRISPR/Cas9 modified zebrafish carrying a truncating variant of titin is used to investigate TTNtv effect in atrial development. We observe compromised assembly of the sarcomere in both atria and ventricle, longer PR interval, and heterozygous adult zebrafish have a higher degree of fibrosis in the atria, indicating that TTNtv are important risk factors for AF. This aligns with the early onset of the disease and adds an important dimension to the understanding of the molecular predisposition for AF.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Pedigrees of the families with TTNtv and a loss-of-function variant in DSC2. Square: male. Circle: female; Black filled: AF affected individual; White filled: unaffected individual; Gray filled: individual with symptoms of heart disease; Diagonal line: diseased individual. Presence of mutation is indicated with + for presence and − for absence (persons with available exomes). Patient II_6 also had thyroid disease and aortic regurgitation. Tachy.Ind.: tachycardia induced, HF: heart failure, AMI: acute myocardial infarction, DM2: diabetes type 2, HCL: hypercholesterolemia, DCM: dilated cardiomyopathy

Fig. 2

Compromised sarcomere structure in adult heterozygous zebrafish mutants. TEM images (13,500 × ) from adult atria (a, b) and ventricles (c, d). a WT atria show well-defined sarcomeres, with distinguishable Z-discs (red arrows) and I-bands (yellow arrows) throughout the tissue. Scale bar 2 µm. b In heterozygous mutant atria the sarcomere structure is less organized. The Z-discs appear blurred (red arrows), and the I-bands are absent (yellow arrows). Scale bar 2 µm. c Ventricle sarcomeres appear well defined in WT siblings, with clear Z-discs (red arrows), I-bands (yellow arrows), and M-lines (blue arrow). Scale bar; 2 µm. d In heterozygous mutants, there is a distinct lack of I-bands and M-lines, and the Z-discs appear blurry and increased in thickness (Scale bar; 2 µm). The length of the sarcomeres, as measured from Z-disc to Z-disc, are significant different between WT and heterozygous in atria

Fig. 3

Truncating mutation in ttn.2 cause increase fibrosis and electrophysiological defects in adult heterozygous mutants. Sirius staining of isolated whole hearts from adult WT (a) and heterozygous mutant siblings (b) show increased fibrotic lesions in the heterozygous heart (b) compared with the WT (a). This increase appears to be more pronounced in the atria of the heterozygous hearts (d) (n = 4) compared with that of the WT siblings (c) (n = 4). Scale bars: a, b 200 µm; c, d; 50 µm. ECG surface recordings of adult WT fish (e) revealed a regular ECG pattern with well-defined P-waves and QRS complexes, with regular PR intervals (g) and RR intervals, with low beat-to-beat variability, as shown by the Poincaré plot (h), indicative of a regular heart rhythm (n = 8). In the heterozygous siblings, the ECG pattern was equally well defined (f), but with a larger PR interval, compared with the WT siblings. Furthermore, the RR interval and beat-to-beat variability was irregular, as demonstrated by the Poincaré plot (i), indicating an irregular heart rhythm in the heterozygous siblings (n = 8)