In our cohort, 2 (3.4%) patients (Table 1) were found to carry disease-related FLNC variants. Other common variants included MYBP3 (4, 6.9%), GAA (3, 5.2%), and TTN (2, 3.4%) (Supplementary Material 2).

A 3-year-5-month-old female was referred following the detection of a cardiac murmur and recent onset of fever. Initial examination revealed a grade 3/6 systolic murmur audible along the left sternal border at the second intercostal space, with normotensive blood pressure and heart rate. Echocardiography (Fig. 1) demonstrated a reduced ejection fraction (EF), dilated and hypokinetic left ventricle, and bulging of the basal interventricular septum towards the right ventricular side. Based on the left ventricular end-diastolic volume (LVEDV), the Z-score was greater than 2, indicating a significant deviation from the normal range for age. Holter monitoring revealed sinus arrhythmia and B-type pre-excitation. Chest X-ray showed increased pulmonary markings without cardiomegaly. Blood tests suggested an inflammatory response and slightly low thyroid hormone levels. Cardiac enzymes, B-type natriuretic peptide (BNP), high-sensitivity troponin (hsTnI), liver and renal function tests, coagulation profile, immunoglobulins, electrolytes, and ultrasound of the liver and spleen were all within normal limits. She was treated with milrinone, digoxin, diuretics, and anti-infective therapy. After resolving the infection, her EF improved to 51% and she was discharged.

Cardiac evaluation of two patients. A-B: Patient 1’s Holter; C: Left ventricle dilation in Patient 1; D: Septal bulge in Patient 1; E–F: Patient 2’s Holter; G: Right atrium enlargement in Patient 2; H: Thickened septum in Patient 2

Upon discharge, the patient was prescribed oral hydrochlorothiazide, spironolactone, and digoxin. At the 6-month follow-up, her EF had stabilized at over 50%. Subsequently, the treatment regimen was adjusted to include oral propafenone for 3 months. At the 12-month follow-up, a repeat echocardiogram demonstrated further improvement in cardiac function, with the EF ranging between 60 and 70%. While the left ventricular dilation and hypokinesis showed some improvement, the overall clinical status of the patient remained stable, and she continued to show no signs of recurrent infection or worsening cardiac symptoms.

A 3-year-1-month-old girl was referred for cardiac evaluation after presenting with fever, cough, and exertional cyanosis. Chest radiography revealed cardiac silhouette enlargement. She has a history of frequent colds, poor endurance, and reduced physical activity, although she has achieved standard developmental motor milestones. At presentation, her blood pressure was 107/73 mmHg, height was 89 cm (3rd to 10th percentile), and weight was 13 kg (10th to 25th percentile). Coarse breath sounds and scattered crackles were noted bilaterally. Enlargement of the liver was palpable below the costal margin. Cardiac examinations were unremarkable. Echocardiography disclosed a preserved EF with dilated atria, thickened interventricular septum and right ventricular walls, moderate pulmonary hypertension, valvular regurgitations, and a small pericardial effusion. Hepatic ultrasonography suggested congestion with hepatomegaly, altered venous structures, and enlarged portal lymph nodes. Holter monitoring indicated sinus arrhythmia, atrial abnormalities, and signs of myocardial ischemia. Laboratory tests revealed elevated BNP levels, prolonged prothrombin time, increased D-dimer, and evidence of inflammation and infection. CT thorax confirmed pulmonary involvement, pleural effusion, and cardiac silhouette enlargement. No significant deviations were observed in additional biochemical markers or imaging assessments. Upon admission, the patient was treated with hydrochlorothiazide, spironolactone, levocarnitine, and antimicrobial therapy for eight days, resulting in clinical improvement and discharge.

Upon discharge, the patient was prescribed oral spironolactone and hydrochlorothiazide, and she continued to take antibiotics for 5 days as part of her discharge regimen. At the 2-month follow-up post-discharge, the patient exhibited reduced cyanosis, and a repeat echocardiogram demonstrated amelioration of pulmonary hypertension, with other findings consistent with the previous assessment. Since the 2-month follow-up, the patient has not undergone further echocardiographic or electrocardiographic evaluations. However, her clinical symptoms have remained stable, and her activity tolerance is satisfactory. There have been no reports of fever, infection, or cyanosis over the subsequent 5 months. The parents have continued to administer the prescribed medications as directed.

Both patients are the first-born children of their respective parents and have no siblings. Neither patient has a family history of cardiomyopathy or myocarditis extending up to three generations. Both families are of East Asian descent from China.

The de novo heterozygous variations c.3962A > T (p.Glu1321Val) and c.7543C > T (p.Leu2515Phe) in the FLNC gene were respectively identified in the index patients (Fig. 2). The c.3962A > T variant leads to the substitution of glutamic acid with valine at position 1321 of the encoded protein, while the c.7543C > T variant results in the replacement of leucine with phenylalanine at position 2515. According to the ACMG guidelines, both variants were classified as likely pathogenic (PS2 + PM2_Supporting + PP3): 1) the variants were determined to be de novo; 2) The variants were not present in the gnomAD database of normal population; 3) Multiple bioinformatic tools predicted the variants to be deleterious.

Sanger sequencing reveals de novo variants c.3962A > T (p.Glu1321Val) in Patient 1 and c.7543C > T (p.Leu2515Phe) in Patient 2

Conservation analysis with DNAMAN shows strong preservation at FLNC protein positions 1321 (Glu) and 2515 (Leu), with PhyloP scores of 4.854 and 3.35, respectively (Fig. S1). The p.Glu1321Val variant poses a high risk of splicing disruption, supported by an MPA score of 10 and spliceAI predictions suggesting likely alterations in nearby splice donor sites (Fig. S2). Structural modeling, guided by minigene assays and referencing PDB ID Q8BTM8.1.A, suggests that p.Glu1321Val, located within the ROD1 domain, may disrupt this domain, leading to protein truncation. While p.Leu2515Phe, situated in the ROD2 domain, does not significantly affect the overall 3D structure, secondary structure prediction indicates a transition from a random coil to an alpha helix at position 2530 (Fig. S2).

The outcomes of the minigene splicing assay reveal that the c.3962A > T (p.Glu1321Val) variant disrupts canonical splicing, as evidenced by a recurrent 4 base pair deletion at the right flank of Exon 22 across independent cellular analyses. At the molecular level, this deletion is represented as c.3961_3964del (p.Glu1321Alafs*23) in the cDNA sequence, leading to a frameshift variant and the incorporation of a premature stop codon within Exon 23. This de novo variant has not been previously reported and results in a shortened protein isoform of putative 1342 amino acids, diverging significantly from the wild-type protein structure (Fig. 3).

A: Construction strategy diagram of the minigene; B: Agarose gel electrophoresis image from RT-PCR transcript analysis; bands are labeled ‘a’ and ‘b’ in both HeLa and 293 T cells; C: Schematic illustration of the minigene splicing pattern; D: Sequencing results corresponding to the spliced bands depicted

At the molecular level, this deletion is represented as c.3961_3964del in the cDNA sequence, leading to a frameshift variant and the incorporation of a premature stop codon (p.Glu1321Alafs*23) within Exon 23. Consequently, a shortened protein isoform of putative 1342 amino acids may be produced, diverging significantly from the wild-type protein structure (Fig. 3).