Whole-Exome Sequencing Identifies Three Novel TTN Variants in Chinese Families with Dilated Cardiomyopathy

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Dilated cardiomyopathy (DCM), a severe heart disease, is the leading cause of heart failure and sudden cardiac death worldwide. DCM is defined by a dilated and deficient systolic left ventricle (LV), and is a major risk factor for morbidity and mortality worldwide. DCM progression can be ascribed to genetic and non-genetic factors, including hypertension, infectious agents, toxins, and drugs. Sarcomere genes play crucial roles in myocardial cells’ physical structure and physiological function. Various cardiomyopathies can be attributed to variations in sarcomere genes [1]. TTN, the largest protein in the human body, is important in sarcomere structure and function; variations in the TTN gene are associated with many hereditary cardiomyopathies, including DCM, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy/dysplasia, catecholamine-induced polymorphic ventricular tachycardia, and left ventricular non-compaction [2].

The review board of the Second Xiangya Hospital of Central South University approved this study. The three patients involved in the research and their families provided written informed consent to participate in the study. Blood was obtained from the patients and sent to the Berrygenomics Bioinformatics Institute (Beijing, China) for exome capture, high-throughput sequencing, and filtering. The novel TTN mutations were verified by Sanger sequencing.

We identified three Chinese patients with characterized DCM (Figure 1A). Patient 1 was a 51-year-old man from Hunan province with a sudden DCM attack, who showed polypnea, palpitation, arrhythmia, and cardiac dilation. B- and M-mode ultrasound suggested an LV diameter of 55 mm, a left atrium diameter of 33 mm, and an ejection fraction of 48%. Patient 2 was a 29-year-old man from Hunan province with an almost 5 year history of DCM. B- and M-mode ultrasound suggested an LV diameter of 77 mm, a left atrium diameter of 52 mm, and an ejection fraction of 30%. Patient 3 was a 42-year-old man from Hunan province, suffered sudden cardiac death due to dilated cardiomyopathy. Forensic autopsy indicated a clearly dilated heart and enlarged heart cavity; the heart weight was 602 g, the left ventricular wall thickness was 13 mm, and the right ventricular wall thickness was 3 mm. No methamphetamine, MDMA, ketamine, cocaine, methadone, codeine, dichlorvos, parathion, or dimethoate were detected in the blood.

Figure 1

(A) Pedigrees associated with clinical phenotypes. Ultrasound testing results for patient 1 and patient 2. Forensic autopsy of patient 3, showing a dilated heart. (B) Sanger sequencing results of the TTN variants. (C) Distribution of TTN variants found in this study.

Data filtering excluded shared common variants from the 1000 Genomes Project, ESP, GnomAD, and ExAC databases. After filtering of cardiomyopathy-associated genes, only a novel splicing TTN variant (c.35485+1G>A) was identified in patient 1, a novel frameshift deletion TTN variant (c.82137del: p.A27380Lfs*3) was identified in patient 2, and a novel frameshift insertion variant (c.80415insA: p.V26806Sfs*3) was identified in patient 3. Sanger sequencing was used to verify the variants (Figure 1B). Three bioinformatics programs (Polyphen-2, Mutationtaster, and SIFT) predicted that these variants had high pathogenicity and could cause disease.

We used whole-exome sequencing combined with cardiomyopathy-associated gene filtering to explore the possible causative genes in three patients with dilated cardiomyopathy. Through this approach, a novel splicing variant (c.35485+1G>A), a novel frameshift deletion variant (c.82137del: p.A27380Lfs*3), and a novel frameshift insertion variant (c.80415insA: p. V26806Sfs*3) in TTN were identified in these three patients. The splicing variant (c.35485+1G>A) affected the I-band region of TTN. Two frameshift variants were located in the domain of A-band TTN (Figure 1C). Our findings highlight the diverse spectrum of genetic variants associated with DCM and underscore the importance of comprehensive genetic analysis in elucidating disease mechanisms.

 

What is the scientific question being addressed?

DCM is a severe heart disease with high morbidity and mortality. Our study implies the vital role of TTN in DCM.

What is the main novel finding?

Here, we employed WES and identified three novel TTN variants in Chinese DCM patients.

In a large sequenced DCM cohort, robust disease associations with 12 genes have been observed, thus underscoring their critical roles in DCM pathogenesis [3]. TTN was included among these genes and therefore might have diagnostic utility in clinical settings. Titin plays critical roles in the contraction and relaxation of striated muscle, and acts as an architectural protein attached to many essential proteins, including actin, obscurin, myosin, and protein kinases G and A. Structurally, TTN acts as a bidirectional molecular spring connecting the Z-disk to the M-line. TTN consists of four subunits: the N-terminal Z-line, the I-band, the A-band, and the C-terminal M-line [4]. In our study, the splicing variant (c.35485+1G>A) affected the I-band region of TTN. This variant may affect the PEVK domain in the I-band, which is largely responsible for TTN’s elastic properties, thereby affecting the connection between the Z-disk and M-line, and decreasing stability and elasticity. The frameshift deletion variant (c.82137del: p.A27380Lfs*3) and the frameshift insertion variant (c.80415insA: p. V26806Sfs*3) were both located in the A-band domain of TTN. The A-band is considered a stabilizer for assembly of the thick filament via multiple interactions with myosin. These frameshift variants may hinder the stable anchor in myosin binding during muscle contraction. The splicing and frameshift variants caused loss function of the Iband and A-band to different degrees.

In summary, through a combination of whole-exome sequencing and filtering of cardiomyopathy-associated genes, we identified a novel splicing variant (c.35485+1G>A), a novel frameshift deletion variant (c.82137del: p.A27380Lfs*3), and a novel frameshift insertion variant (c.80415insA: p. V26806Sfs*3) in TTN as possible causative genes of DCM in three Chinese patients. Our study broadens the spectrum of TTN variants and may help with genetic screening for related diseases.

In conclusion, this study supplements the TTN gene mutation library and improves genetic diagnosis strategies for DCM. Additional functional studies of the TTN protein with these variants are recommended, and may contribute to ascertaining the molecular and pathological mechanisms of DCM.

Data Availability Statement

The data supporting the conclusions are included in the article. Raw data are available upon request.

Ethics Statement

The study was approved by the Second Xiangya Hospital of Central South University (ethics number Z0146-02).

Acknowledgments

We thank the patients and their families for participating in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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References

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