Prevalence of Titin Truncating Variants in General Population

Abstract

Background: Truncating titin (TTN) mutations, especially in A-band region, represent the most common cause of dilated cardiomyopathy (DCM). Clinical interpretation of these variants can be challenging, as these variants are also present in reference populations. We carried out systematic analyses of TTN truncating variants (TTNtv) in publicly available reference populations, including, for the first time, data from Exome Aggregation Consortium (ExAC). The goal was to establish more accurate estimate of prevalence of different TTNtv to allow better clinical interpretation of these findings.

Methods and results: Using data from 1000 Genomes Project, Exome Sequencing Project (ESP) and ExAC, we estimated the prevalence of TTNtv in the population. In the three population datasets, 52-54% of TTNtv were not affecting all TTN transcripts. The frequency of truncations affecting all transcripts in ExAC was 0.36% (0.32% - 0.41%, 95% CI) and 0.19% (0.16% - 0.23%, 95% CI) for those affecting the A-band. In the A-band region, the prevalences of frameshift, nonsense and essential splice site variants were 0.057%, 0.090%, and 0.047% respectively. Cga/Tga (arginine/nonsense-R/*) transitional change at CpG mutation hotspots was the most frequent type of TTN nonsense mutation accounting for 91.3% (21/23) of arginine residue nonsense mutation (R/*) at TTN A-band region. Non-essential splice-site variants had significantly lower proportion of private variants and higher proportion of low-frequency variants compared to essential splice-site variants (P = 0.01; P = 5.1 X 10-4, respectively).

Conclusion: A-band TTNtv are more rare in the general population than previously reported. Based on this analysis, one in 500 carries a truncation in TTN A-band suggesting the penetrance of these potentially harmful variants is still poorly understood, and some of these variants do not manifest as autosomal dominant DCM. This calls for caution when interpreting TTNtv in individuals and families with no history of DCM. Considering the size of TTN, expertise in DNA library preparation, high coverage NGS strategies, validated bioinformatics approach, accurate variant assessment strategy, and confirmatory sequencing are prerequisites for reliable evaluation of TTN in clinical settings, and ideally with the inclusion of mRNA and/or protein level assessment for a definite diagnosis.

Fig 1. Influence of 1000 genomes project evolution on titin truncation prevalence.

Abbreviations: P1V1 –phase 1 version 1; P1V2 –phase 1 version 2; P1V3 –phase 1 version 3; P3V5 –phase 3 version 5.

Fig 2. Flowchart for analysis of TTN truncations in publicly available reference populations.

Abbreviations: 1KG– 1000 Genomes project; ESP–Exome Sequencing Project; ExAC—Exome Aggregation Consortium; LV–Left ventricle.

Fig 3. Spatial distribution of titin frameshift, nonsense and splice-site mutations in reference populations.

Titin is linearly depicted with its 152 Ig-like domains in green and 132 fibronectin type III domains in purple. TTNtv are shown as lollipops and bars. Depicted are nonsense (red) and frameshift (green) mutations in various phases of the 1000 Genomes project. Dark grey bars represent the complete map of various TTN mutations from ExAC. Variants are shown relative to the titin Uniprot Sequence identifier Q8WZ42. The dashed lines below the protein schematic indicate the location of variants within the sarcomere. Abbreviations: 1KG– 1000 Genomes project; ESP–Exome Sequencing project; ExAC—Exome Aggregation Consortium; V–version.

Fig 4. Titin population splice site/region allele frequency spectrum.

TTN population non-essential splice-site variants (>2bp) have significantly lower proportion of private variants and higher proportion of low-frequency variants compared to essential splice-site variants (P = 0.01; P = 5.1 × 10⁻⁴, respectively). The P-values are shown for comparison between essential splice-site (1-2bp) and non-essential splice-site variants: 3–4bp (red), 5–6bp (green), and (blue) for combined comparison.