A novel missense mutation in tropomyosin 1 gene associated with hypertrophic cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder that can lead to heart failure or sudden death. Family-based identification of rare sarcomeric variants can support molecular diagnosis and cascade screening in inherited HCM. This study aimed to identify and evaluate a novel TPM1 variant found in a Vietnamese family with HCM. The proband, a 3-year-old boy diagnosed with HCM, and eight relatives from three generations underwent clinical and genetic evaluation. A candidate variant initially identified by targeted next-generation sequencing was validated by PCR and Sanger sequencing. Familial segregation analysis was performed, and variant pathogenicity was assessed according to ACMG guidelines with support from in silico prediction and structural modeling. Sanger sequencing confirmed a heterozygous missense variant in exon 6 of TPM1 NM_001018005.2:c.576G > C, p.(Glu192Asp), in the proband, his father, and paternal grandfather, all of whom exhibited clinical signs of HCM. The variant was absent in unaffected relatives and in public population databases. Based on ACMG criteria (PM1, PM2, PM5, and PP3), the variant was classified as likely pathogenic. This novel TPM1 variant segregated with HCM in a Vietnamese family, expands the known mutational spectrum of TPM1 in hypertrophic cardiomyopathy, and warrants further functional investigation and familial genetic evaluation.

Highlights

• •A new TPM1 mutation found in a Vietnamese family with hypertrophic cardiomyopathy.
• •The variant tracks with the disease in three affected generations.
• •ACMG evaluation and in silico data support a likely pathogenic role.
• •The altered site lies in a key actin-binding region of tropomyosin.
• •Adds new information on TPM1 variants linked to inherited heart disease.

1. Introduction

Hypertrophic cardiomyopathy (HCM) is one of the most common inherited cardiovascular disorders, characterized by unexplained left ventricular hypertrophy, myocyte disarray, and myocardial fibrosis.¹ The disease is primarily associated with pathogenic variants in sarcomeric protein genes, and usually follows an autosomal dominant pattern, conferring a 50% risk of transmission to offspring.¹ Clinical manifestations are highly heterogeneous, ranging from asymptomatic disease to exertional dyspnea, chest pain, syncope, heart failure, arrhythmias, and sudden cardiac death.¹ HCM affects approximately 1 in 500 individuals in the population, including young and competitive athletes, as first estimated using echocardiography by the CARDIA Study in 1995.² A more recent global analysis suggests the true prevalence may be substantially higher because many affected individuals remain undiagnosed.³

Diagnosis of HCM is primarily based on the presence of unexplained left ventricular hypertrophy identified by echocardiography or cardiac magnetic resonance imaging in the absence of abnormal loading conditions or secondary causes of hypertrophy.⁴ Additional diagnostic evaluation includes electrocardiography, family history assessment, and genetic testing, which may aid in distinguishing HCM from phenocopy conditions or physiological hypertrophy in athletes. Clinical management is guided by symptoms, left ventricular outflow tract obstruction, and the estimated risk of sudden cardiac death. Long-term management also includes periodic clinical follow-up, lifestyle counseling, family screening, and genetic counseling.⁴

The etiology of HCM is predominantly genetic, with more than 1800 variants across at least 50 genes implicated in disease pathogenesis.⁵ Variants in genes encoding thick filament sarcomeric proteins, such as MYBPC3 and MYH7, account for the majority of cases; however, variants in thin-filament genes, including TPM1, have also been associated with HCM.¹ The TPM1 gene encodes the tropomyosin alpha-1 chain, a critical component regulating calcium-dependent actin-myosin interaction during contraction.⁶ Although TPM1 variants represent only 2% of genotype-positive HCM cases globally,⁷ their prevalence has been reported at over 20% in Vietnamese patients, suggesting that TPM1 should be carefully evaluated in Vietnamese families with inherited cardiomyopathy.⁸

The human TPM1 gene is located on chromosome 15q22.2, spans approximately 29 kb,⁹ and contains nine exons that undergo alternative splicing to generate tissue-specific isoforms.¹⁰ The canonical cardiac transcript encodes tropomyosin α-1 chain (TPM1), a highly conserved coiled-coil dimeric protein consisting of 284 amino acids that polymerizes along the length of actin filaments within the sarcomere. TPM1 is predominantly expressed in cardiac and skeletal muscle tissues, and plays a central regulatory role in calcium-dependent myocardial contraction. Together with the troponin complex, tropomyosin modulates the interaction between actin and myosin by controlling access of myosin heads to actin-binding sites. During diastole, tropomyosin sterically blocks actin-myosin interaction, whereas calcium binding to troponin C induces conformational changes that shift tropomyosin away from the myosin-binding groove, thereby permitting cross-bridge formation and force generation.¹¹

Consequently, pathogenic TPM1 variants may alter thin filament activation, calcium sensitivity, contractility, and myocardial relaxation, contributing to sarcomeric dysfunction and cardiac hypertrophy.¹² Therefore, this study aimed to validate the TPM1 NM_001018005.2:c.576G > C, p.(Glu192Asp) variant initially detected by targeted next-generation sequencing, assess its segregation with HCM in a Vietnamese family, and reclassify its pathogenicity according to ACMG/AMP criteria. The overall workflow of genetic analysis and variant interpretation is summarized in Fig. 1.

Fig. 1.

Workflow for identification and pathogenicity assessment of the novel TPM1 variant.

2. Materials and methods

2.1. Study subjects

This study protocol was approved by the Ethics Committee of the University of Health Sciences-VNUHCM (Approval Code: 11/OD-IRB-VN01.017). A Vietnamese family with a 3-year-old male proband diagnosed with hypertrophic cardiomyopathy was recruited for this study. Eight family members, including the paternal grandparents, father, mother, paternal uncle, paternal uncle's wife, and two paternal cousins, also consented to participate in this study. All participants provided informed consent and were thoroughly counseled about the study.

2.2. Targeted next-generation sequencing

Genomic DNA was extracted from buccal swabs of the proband and his father and subjected to targeted next-generation sequencing using the G4500 gene panel at Gene Solutions Lab, Ho Chi Minh City, Vietnam. Sequencing libraries were prepared using New England BioLabs reagents, and target regions were enriched with IDT DNA capture probes. Sequencing was performed on an Illumina NextSeq platform, with an average depth of approximately 100× and at least 95% of target regions covered at >10×. Next, sequence reads were aligned to the GRCh38 human reference genome. Rare variants with a population frequency < 1% in databases (1000 Genomes Project and ESP), were retained for interpretation. Variant classification was performed using ClinVar and ACMG guidelines. The assay covered single-nucleotide variants and short insertions/deletions within coding regions and nearby splice regions of genes included in the panel.

2.3. Sanger validation and segregation analysis

Saliva samples were collected using sterile collection tubes with standard procedures. Genomic DNA was extracted using the ammonium sulfate method. DNA concentration and purity were assessed using a spectrophotometer (A260/A280 and A260/A230 ratios) and by 1% agarose gel electrophoresis. The identified variant in TPM1 exon 6 (c.576G > C) was validated by PCR and Sanger sequencing using the following primers: TPM1-F: 5′-CCTTCTGCTGTTGCGAGGTTG-3′ and TPM1-R: 5′-ACTGTAAGTGTTGCTTTCTGGCAG-3′. Primers were designed to amplify a 353-bp fragment. PCR products were confirmed by gel electrophoresis and sequenced using an ABI 3130 Genetic Analyzer. Sequences were aligned to the TPM1 reference (NG_007557.1, NM_001018005.2).

2.4. Variant pathogenicity assessment

Variant pathogenicity was classified according to ACMG guidelines. Variant frequency was evaluated using the Genome Aggregation Database (gnomAD v4.1.0). Segregation analysis was performed across family members. The location and conservation of the altered residue were analyzed using InterPro, UniProt and multiple sequence alignment. Published reports and ClinVar were searched for other variants affecting the same residue. MutationTaster2025, PhyloP, and phastCon were used to predict the functional impact and evolutionary conservation of the variant. Similar workflows combining NGS-based variant detection, Sanger confirmation, and segregation analysis have been widely used for the characterization of rare pathogenic variants in various disorders.13, 14, 15

2.5. TPM1 protein structure analysis

The potential effect of the p.(Glu192Asp) variant on tropomyosin structure was explored with SWISS-MODEL and comparative visualization of the wild-type and mutant proteins.

3. Results

3.1. Case report

The proband was a 3-year-old boy diagnosed with HCM at Children's Hospital 1, Ho Chi Minh City, Vietnam, after detection of a cardiac murmur and a family history of cardiomyopathy. Clinically, he presented with easy fatigability, excessive sweating on exertion, and intermittent mild palpitations, without syncope, chest pain, cyanosis, or significant dyspnea. Transthoracic echocardiography revealed asymmetric interventricular septal hypertrophy with systolic anterior motion of the mitral valve, resulting in left ventricular outflow tract obstruction (LVOTO) with peak pressure gradients ranging from 41 to 110 mmHg on serial examinations (Fig. 2A). Left ventricular systolic function was hyperdynamic, with an estimated ejection fraction of approximately 87%, and mild-to-moderate mitral regurgitation was observed. The overall findings were consistent with obstructive HCM. The patient was managed conservatively with beta-blocker therapy and regular cardiology follow-up.

Fig. 2.

Echocardiographic and genetic assessment of the proband and family. (A) Doppler echocardiographic assessment of mitral inflow in the proband.

(B) Family pedigree across three generations, with proband indicated by an arrow. Males are shown as squares and females as circles; shaded symbols represent individuals clinically affected by HCM, while unshaded symbols indicate unaffected individuals. (C) Sanger sequencing results. The red rectangle highlights position c.576 of exon 6 of TPM1. The proband, his father, and paternal grandfather show a heterozygous G > C substitution, c.576G > C, p.(Glu192Asp), whereas the other examined family members are homozygous for the reference G nucleotide. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The family history was notable for cardiomyopathy in his father and paternal grandfather, while other examined relatives were clinically unaffected (Fig. 2B). The familial segregation pattern was consistent with autosomal dominant inheritance. Given the early disease onset, obstructive phenotype, and familial history, molecular genetic analysis was subsequently performed to investigate a potential pathogenic variant associated with familial HCM.

3.2. Genetic analysis

Targeted sequencing identified the same heterozygous TPM1 variant, NM_001018005.2:c.576G > C, p.(Glu192Asp), in both the proband and his father. The variant had not been reported in ClinVar at the time of testing and was initially classified as a variant of uncertain significance. Because the variant was found in two affected family members and involved an HCM-associated gene, it was selected for Sanger validation and segregation analysis.

PCR amplification of the TPM1 exon 6 region produced a single, specific band of 353 bp, confirming successful amplification and suitability for Sanger sequencing (Fig. S1). Sanger sequencing confirmed a heterozygous missense mutation in exon 6 of the TPM1 NM_001018005.2:c.576G > C, p.(Glu192Asp) in the proband, his father, and paternal grandfather. All other family members were wild-type, consistent with their unaffected phenotype (Fig. 2C). According to ACMG/AMP guidelines, the variant fulfilled lines of evidence supporting pathogenicity: PM1 (location within a critical and highly conserved tropomyosin functional region), PM2 (absent from population databases including gnomAD), PM5 (a different pathogenic missense change previously reported at the same residue), and PP3 (supportive computational tools and evolutionary conservation evidence). Together, these lines of evidence support classification of the c.576G > C variant as “likely pathogenic”.

3.3. TPM1 protein structure analysis

The p.(Glu192Asp) variant results in substitution of glutamic acid with aspartic acid at position 192, both of which are acidic but differ in side-chain length. This conservative change may alter local interactions critical for actin-tropomyosin binding. Structural modeling using SWISS-MODEL showed a shorter acidic side chain in the mutant model, suggesting possible local alterations in intermolecular interactions within the actin–tropomyosin complex (Fig. 3).

Fig. 3.

Comparative structural modeling of the TPM1 protein and schematic representation of the amino acid at position 192 in wild-type and mutant forms. (A) Predicted wild-type TPM1 structure generated by SWISS-MODEL, with the arrow indicating the schematic structure of the wild-type amino acid (glutamic acid) generated by HOPE. (B) Predicted mutant TPM1 structure carrying p.(Glu192Asp), generated by SWISS-MODEL, with the arrow indicating the schematic structure of the mutant amino acid (aspartic acid) generated by HOPE. The shared backbone is highlighted in red, while the unique side chains are shown in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

Mutations in TPM1 are known to cause HCM, albeit at a lower frequency compared to MYH7 and MYBPC3.¹ However, a previous Vietnamese cohort reported a comparatively high frequency of TPM1 variants, some of which were associated with severe clinical manifestations.⁸ Our study identified a novel TPM1 missense variant, p.(Glu192Asp), in a Vietnamese HCM family, which was reclassified as likely pathogenic according to ACMG/AMP criteria. Another pathogenic variant affecting the same residue, p.(Glu192Lys), has previously been reported in patients with cardiomyopathy, supporting the functional importance of this amino acid position. The p.(Glu192Lys) mutation, at the same codon, has been described in a proband with LVNC who experienced severe symptoms and had a family history of sudden cardiac death. The same report also noted a previous case of HCM with atrial septal defect carrying p.(Glu192Lys).¹⁶ These findings suggest that even subtle alterations at residue Glu192 may disrupt sarcomeric regulation and contribute to disease development.

This variant represents a conservative amino acid substitution located within a critical functional region of tropomyosin involved in actin–myosin interaction. This finding is consistent with previous reports of pathogenic TPM1 variants, including p.(Asp175Asn) and p.(Glu180Gly),¹⁷ which affect the stability of the thin filament and influence actin-myosin interaction, leading to abnormal muscle contraction. Together with segregation and rarity, these observations support p.(Glu192Asp) as a candidate disease-associated variant.

The TPM1 p.(Glu192Asp) variant identified in this study provides a potential molecular marker for evaluating at-risk relatives and supports cascade genetic screening, particularly among first-degree members, to identify asymptomatic carriers. However, this study is limited by the small sample size of a single family, which precludes conclusions regarding the broader prevalence, penetrance, or pathogenic significance of this variant. In addition, functional studies (in vitro or in vivo) were not performed to directly assess the biochemical and physiological impact of the p.(Glu192Asp) mutation on TPM1 protein structure, stability, sarcomeric interactions, or calcium sensitivity. Future studies involving larger cohorts and functional validation using cell lines or animal models are therefore required to clarify the biological and clinical significance of this variant, establish genotype-phenotype correlations, and improve diagnostic accuracy, prognostic assessment, and personalized treatment strategies for patients with HCM.

In conclusion, a novel TPM1 variant, NM_001018005.2:c.576G > C, p.(Glu192Asp), was identified in a Vietnamese family with HCM. The variant segregated with the disease phenotype among available relatives and was reclassified in this study as likely pathogenic according to ACMG/AMP criteria. The findings expand the known mutational spectrum of TPM1 and support familial genetic evaluation and further functional investigation of this variant.

Declaration of generative AI usage

During the preparation of this work the authors used ChatGPT in order to correct grammatical mistakes. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

CRediT authorship contribution statement

Kim Nhuong Thi Nguyen: Writing – original draft, Methodology, Formal analysis. Ngoc Thuy Nguyen: Writing – original draft, Methodology, Formal analysis. Minh Thu Thi Nguyen: Methodology, Formal analysis, Data curation. Thanh Liem Nguyen: Methodology, Formal analysis, Data curation. Minh Thong Le: Methodology, Formal analysis, Data curation. Thu Suong Thi Nguyen: Writing – review & editing, Supervision, Resources, Project administration, Conceptualization. Minh Nam Nguyen: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Informed consent

This study protocol received approval from the Ethics Committee of the University of Health Sciences-VNUHCM (Approval Code: 11/OD-IRB-VN01.017). Informed consent for participation and publication of anonymized data was obtained from all subjects.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material