This study was approved by the ethics committee of West China Second Hospital of Sichuan University (approval number: 2021–069). Written informed consent was obtained from the parents of the patients for whole-exome sequencing (WES) as well as inclusion of any clinical and imaging details in the publication.

Peripheral blood samples were collected from the proband and his parents in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes and stored at 4°C for less than 6 h. DNA extraction was then performed using the Blood Genome Column Medium Extraction Kit (Tiangen Biotech, Beijing, China) according to manufacturer instructions.

WES was conducted using the NovaSeq 6000 platform (Illumina, San Diego, CA, United States), and the raw data were processed using FastP to remove any adapters and filter low-quality reads. The paired-end reads were aligned to the Ensembl GRCh38/hg38 reference genome using the Burrows–Wheeler aligner. Variant annotations were performed in accordance with database-sourced minor allele frequencies (MAFs) and practical guidelines on pathogenicity issued by the American College of Medical Genetics. The MAF annotations were based on the 1000 Genomes (1000G), dbSNP, ESP, ExAC, and Chigene inhouse MAF databases, while Provean, Sift, Polypen2_hdiv, and Polypen2_hvar databases were used for further annotations using R software (R Foundation for Statistical Computing, Vienna, Austria).

The AlphaFold3 tool (https://golgi.sandbox.google.com/) was employed to establish the protein crystal structures and analyze the mutant sites of DMD and TNNI3K. Then, Pymol software was used to illustrate the molecular structures of the wild-type and mutant forms of the target genes. Additionally, AlphaFold3 was used to predict the potential interactions between dystrophin and TNNI3K proteins.

A 15-month-old male patient presented to our emergency department with respiratory failure characterized by dyspnea, dysphonia, and non-productive cough. The clinical manifestations included perioral cyanosis upon exertion, lethargy, hyperhidrosis, reduced lacrimation, and peripheral coldness. The patient exhibited no pyrexia but developed hemoptysis with a pink frothy sputum. The proband, born at 38 weeks of gestation via caesarean section due to oligohydramnios, was the first offspring of non-consanguineous parents and had a birth weight of 2,850 g. The prenatal ultrasonography had revealed renal cystic changes, and the neonatal period was complicated by pneumonia that required a week-long period of hospitalization, during which ultrasonographic examination confirmed autosomal dominant polycystic kidney disease with concomitant left hydronephrosis. The patient’s medical history included one episode of urinary tract infection. The developmental history revealed feeding difficulties since birth, dental eruption at 10 months, and current inability to achieve independent standing. Language development was limited to maternal recognition without further vocabulary acquisition. Physical examination revealed distinctive craniofacial features, including a high-arched palate, hypertelorism, and low-set ears with thickened pinnae. Marked perioral cyanosis was evident during crying, which was accompanied by hoarse vocalization. Respiratory assessment demonstrated soft cervical tissues without tracheal tug, absent tripod positioning, and decreased left-lung breath sounds with prominent coarse crackles predominantly in the left field. Cardiac examination revealed irregular rhythm with diminished heart sounds and significant murmurs across all valve areas. The abdomen was soft with mild distention and hepatomegaly extending 3 cm below the costal margin; the spleen was non-palpable. Peripheral examinations showed no significant pitting edema or digital clubbing, along with preserved peripheral perfusion and normal digital temperature.

Laboratory investigations revealed electrolyte disturbances characterized by hyponatremia (Na⁺: 130.7 mmol/L), elevated creatine kinase-MB (7.43 μg/L), increased β-hydroxybutyrate (0.94 mmol/L), and markedly elevated B-type natriuretic peptides (>5,000.00 pg/mL), with the cTnI assessment demonstrating normal levels. Metabolomic analysis of the blood demonstrated elevated medium- and long-chain acylcarnitines (C14, C16:1-OH, C18, C18:1, and C18:2), suggesting underlying fatty-acid oxidation dysfunction. Urinary organic acid analysis revealed decreased excretion of 2-ketoglutarate-OX-2 (1) and homovanillic acid-2. Notably, comprehensive hematological, hepatic, renal, and thyroid function tests showed that the results were within the reference ranges. Additional parameters, including pyruvate, lactate, and ammonia concentrations, along with urinalysis and fecal examination showed no significant abnormalities. Autoantibody screening yielded negative results. Thoracic, cervical, and cerebral computed tomography revealed bilateral pulmonary inflammation with interstitial changes, marked cardiomegaly with consequent left main bronchus compression, minimal right-sided pleural effusion, and mild bilateral ventricular dilatation. Some additional findings included right submandibular gland enlargement, pharyngeal wall thickening, maxillary sinus mucosal hyperplasia, and bilateral cervical lymphadenopathy. Renal imaging demonstrated superior pole enlargement with irregular morphology. Renal ultrasonography confirmed bilateral nephromegaly with parenchymal echogenic changes, multiple cystic formations consistent with infantile polycystic kidney disease, left hydronephrosis, and partial right-sided collecting system dilatation. The abdominal ultrasonography was unremarkable.

Echocardiography revealed severe left-ventricular dysfunction, left-ventricular enlargement with altered echogenicity, severe mitral regurgitation, minimal pericardial effusion, and bilateral ventricular systolic dysfunction (Figure 1). Electrocardiography revealed sinus arrhythmia with junctional escape rhythm, axis deviation, left-ventricular hypertrophy, and T-wave abnormalities in leads II, III, aVF, V3, and V5. Twenty-four-hour Holter monitoring showed sinus arrhythmia, isolated atrial ectopy (8 events in 24 h), ventricular ectopy (28 events in 24 h, <0.1% of total beats) with paired morphologies, T-wave changes, and moderately reduced heart-rate variability (SDNN: 80 m).

Interestingly, cardiac magnetic resonance imaging demonstrated a different presentation compared to that observed in general DMD patients (Figure 2). The cardiac cine sequence showed that the left ventricle was enlarged, with significantly reduced left-ventricular motion and systolic function (ejection fraction (EF) = 21%). Hypertrabecularization was observed in the free wall and apex of the left ventricle. Late gadolinium enhancement and T1 mapping showed diffuse myocardial fibrosis, especially in the muscle layer of the interventricular septal wall and subendocardial layer of the left ventricle.

As unexplained severe heart failure was identified and a rare cardiomyopathy was suspected, genetic testing was conducted to explore any associated variants. WES was performed for the proband and their parents. According to the results of WES, a hemizygous variant was identified as c.1540G>T (p.V514L) in the DMD gene (Figure 3A); this allele variant was inherited from the maternal side, and the DMD c.1540G>T variant had never been reported in the ExAC and 1000G databases. Further potential compound variants were searched based on the WES results. In detail, we identified all potential cardiomyopathy-associated variants and validated the pathogenic or likely pathogenic variants. Then, careful screening was completed based on the MAFs, CADD scores, and potential correlations with the clinical manifestations of the proband. Next, a heterozygous variant was also identified as c.1633G>T (p.G545C) in the TNNI3K gene, which was absent in both the paternal and maternal sides and was treated as a de novo variant; this allele variant has also never been reported, which makes it a novel genotype (Figure 3B). Moreover, we excluded other potential variants involved in cardiovascular, metabolic, and muscular disorders. Then, we excluded all variants that were reported as pathogenic or likely pathogenic, and none of these were confirmed to be associated with the phenotype of the proband as heart failure and cardiomyopathy. Hence, we suspected that the compound variants of DMD c.1540G>T and TNNI3K c.1633G>T contributed to the pathogenic phenotype of this proband as a complicated manifestation of clinical cardiac dysfunction. To elucidate the molecular structures of the human DMD and TNNI3K genes, MutationTaster was used with R software to predict the pathogenicities of DMD c.1540G>T and TNNI3K c.1633G>T as well as assess the impacts of these two mutations on specific protein structures. Then, AlphaFold3 tool was used to establish the protein crystal structure and analyze the mutant sites of DMD and TNNI3K (Figure 3C) (Varadi et al., 2022; Jumper et al., 2021). Modeling analysis with proteins was performed using AlphaFold3 (https://golgi.sandbox.google.com/) for the mutant sites in the wild-type genes using Pymol software. We illustrate the molecular structural differences between the wild-type and mutant varieties of DMD and TNNI3K. The amino acid residues around site 514 had changed in DMD p.V514L, with alterations in the surrounding residues without labeling of the corresponding hydrogen bonds (Figure 3C), which coincided with the pathogenic predictions (Figure 3C). The relationship between the residues around the mutant site indicated that structural change owing to the variant of DMD p.V514L would cause protein dysfunction. The amino acid residues around site 545 had changed in TNNI3K p.G545C. According to the analysis results using ChimeraX software, after G545C substitution, the hydrogen bond network of the TNNI3K protein (green dashed line) did not change but the contact points with the water molecules (yellow dashed line) increased significantly; at the same time, the hydrophobic distribution of the protein showed that the hydrophobic region (orange yellow) increased and hydrophilic region (blue) decreased. In addition, free-energy difference analysis predicted that this substitution could cause decreased protein stability (ΔΔG<0). G545 is located in the serine/threonine kinase domain of TNNI3K and is a critical region for its function. The G545C substitution introduces the sulfhydryl group of cysteine, which increases the hydrophobicity of this site and could enhance its interactions with the surrounding hydrophobic groups, thereby affecting the overall conformation and stability of the protein. Such conformational changes may lead to alterations in the kinase activity or substrate binding capacity, which can in turn interfere with the normal function of TNNI3K (Figure 3C). Furthermore, we used AlphaFold3 to predict the potential interactions between DMD and TNNI3k (Figure 3D), and the results demonstrated that these genes could interact with each other at specific residues, where the C terminals of TNNI3K could come into contact with the N terminals of DMD (Figure 3E). However, the compound variants of DMD p.V514L and TNNI3K p.G545C can lead to significant changes in the structural predictions between these two proteins, implying that the original interactions between DMD and TNNI3K can collapse and the space folds of DMD itself may also be disrupted. Thus, the illustrations demonstrate that these single mutations definitely impair protein formation. Additionally, the combined mutations can aggravate molecular function collapse as these two proteins serve as a complex in maintaining myofilament hemostasis and biological functions to regulate cardiomyocyte contractions.

Upon analyzing the clinical manifestations as well as conducting imaging assessments and genetic tests, the patient was diagnosed with DMD accompanied by early-onset heart failure. Then, a long-term follow-up and therapy plan was suggested, including Sacubitril Valsartan Digoxin (0.15 mg daily), sodium (15 mg twice a day), Empagliflozin (2.5 mg daily), metoprolol (25 mg daily), spironolactone, and antiepileptic medication administration. Following therapeutic intervention, the patient demonstrated partial clinical improvement characterized by enhanced exercise tolerance and mild tachypnea within the compensatory physiological limits, accompanied by strengthened myocardial contractility with decreased arrhythmic events. Objective measurements revealed progressive reduction of hepatomegaly to 1 cm subcostal, consistent with diminished hepatic congestion. Although these parameters indicate positive therapeutic response, the residual symptomatology persists, necessitating ongoing cardiovascular monitoring and serial hepatic assessments to evaluate disease progression and treatment efficacy. Thus, the patient’s heart failure has been evaluated as irreversible, and a suggestion of heart transplantation has been provided to the patient, who is currently on the wait list.