Role of Titin Missense Variants in Dilated Cardiomyopathy

Abstract

Background: The titin gene (TTN) encodes the largest human protein, which plays a central role in sarcomere organization and passive myocyte stiffness. TTN truncating mutations cause dilated cardiomyopathy (DCM); however, the role of TTN missense variants in DCM has been difficult to elucidate because of the presence of background TTN variation.

Methods and results: A cohort of 147 DCM index subjects underwent DNA sequencing for 313 TTN exons covering the N2B and N2BA cardiac isoforms of TTN. Of the 348 missense variants, we identified 44 "severe" rare variants by using a bioinformatic filtering process in 37 probands. Of these, 5 probands were double heterozygotes (additional variant in another DCM gene) and 7 were compound heterozygotes (2 TTN "severe" variants). Segregation analysis allowed the classification of the "severe" variants into 5 "likely" (cosegregating), 5 "unlikely" (noncosegregating), and 34 "possibly" (where family structure precluded segregation analysis) disease-causing variants. Patients with DCM carrying "likely" or "possibly" pathogenic TTN "severe" variants did not show a different outcome compared with "unlikely" and noncarriers of a "severe" TTN variant. However, the "likely" and "possibly" disease-causing variants were overrepresented in the C-zone of the A-band region of the sarcomere.

Conclusions: TTN missense variants are common and present a challenge for bioinformatic classification, especially when informative families are not available. Although DCM patients carrying bioinformatically "severe" TTN variants do not appear to have a worse clinical course than noncarriers, the nonrandom distribution of "likely" and "possibly" disease-causing variants suggests a potential biological role for some TTN missense variants.

Keywords: cardiomyopathy; cardiovascular genetics; dilated cardiomyopathy; heart failure; missense variants.

Figure 1

Bioinformatic and segregation analysis in 134 DCM Probands. TTN variants were filtered with bioinformatic algorithms as described and classified into “severe” or “nonsevere” categories. Mutations initially identified as “severe” were studied by segregation analysis when samples were available and further classified as “likely,” “possibly,” or “unlikely” to be disease causing. DCM indicates dilated cardiomyopathy; TTN, titin gene.

Figure 2

Pedigrees for “likely” TTN families harboring disease‐causing pathogenic variants. Squares, circles, plus signs, and arrows indicate males, females, positive TTN variant status, and probands, respectively. An obligate carrier is indicated by a dotted circle. Black shading indicates individuals affected with DCM. Double plus symbols mean double heterozygous variants. DNFDC014 has variant rs72648231, TSFDC044 has variants rs72648236 and rs72650088, DNFDC131 has variant rs72648220, and DNFDC074 has variant rs72650078. DCM indicates dilated cardiomyopathy; TTN, titin gene.

Figure 3

Exon structure of human TTN gene with identified DCM missense mutations. P indicates “possible” disease‐causing mutation; L, “likely” disease‐causing mutation. Indicated are also titin domain numbers (top) and exon numbers (bottom). Red rectangle represents immunoglobulin‐like domain; white, fibronectin type 3 domain; blue, unique sequence; green, z‐repeat domain; yellow, PEVK domain; black, titin kinase domain (gene structure based on Bang et al17). DCM indicates dilated cardiomyopathy; TTN, titin gene.

Figure 4

Missense variants mapped on TTN gene. A, Protein domain structure of largest full‐length titin isoform (UniProt entry Q8WZ42‐12, which includes all exons except for Novex‐3; this is the same as Ensembl transcript Ttn‐018Ensembl) with indicated locations of “possibly” (blue arrowheads) and “likely” (red arrowheads) SNPs. Top: Z‐disk and I‐band region of titin with tandem immunoglobulin segments (proximal [prox.], middle and distal subsegments), N2B and N2BA elements, and PEVK segment of I‐band indicated separately and I/A junction. Bottom: A‐band and M‐band regions of titin with D‐zone (6 super‐repeats each containing 7 domains) and C‐zone (11 super‐repeats with 11 domains each), C‐M junction, and M‐band regions indicated. I‐band region of titin expressed in N2B cardiac titin (the dominant isoform in the LV) is shown by the gray underline (the A‐band region is constitutively expressed in all full‐length isoforms). B, Pearson's χ² goodness‐of‐fit tests were used to test if the distribution of the “likely” and “possibly” categorized nonsynonymous missense SNPs were random or not, incorporating the size in amino acids of each protein region tested as one group and the remainder of titin protein as a second group (df=1). Mutations in the C‐zone repeats are overrepresented (indicated by direction of arrow). C, The SNPs in the C‐zone are not randomly distributed over the 11 domains of the super‐repeats but appear to be biased toward the last 3 Fn‐III domains (the 11 super‐repeats were aligned and the number of SNPs in each of the 11 domains summed). LV indicates left ventricle; SNPs, single nucleotide polymorphisms; TTN, titin gene.

Figure 5

Long‐term survival curves in TTN variant carriers. Kaplan–Meier event‐free survival for CVD or HTx based on TTN variant categories: TRUNC, “likely” and “possibly”; NC, “unlikely.” CVD indicates cardiovascular death; HTx, heart transplant; NC, noncarrier; TRUNC, truncations; TTN indicates titin gene.