PMC8770926_01
Clinical Presentation of the Proband. (A) Chest X ray on admission demonstrating severe cardiomegaly. (B) Three-generation family pedigree: III-1, Index case; II-1, father carries the c.355G>C (p.Val119Leu) variant; II-2, mother carries the c.997C>T (p.Arg333Trp) variant. III-2, older healthy sister that has not been tested; No other family history of heart disease besides [I-1], grandfather with coronary heart disease. (C) Graphs representing summary of serum brain-natriuretic peptide (BNP), cardiac Troponin T, and Lactate levels during the first 10 h after admission. (D) Representative echocardiogram, apical views on admission during systole (a) and diastole (b) demonstrating global left ventricular hypokinesis with estimated EF of 10-15%, significant concentric hypertrophy of the left ventricle, normal coronary arteries take offs, circumferential effusion without chamber compression measuring 14 mm posterior and 8 mm anterior, mild mitral regurgitation. *Please see accompanied Supplementary Videos included in Supplementary Materials.
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F1
The proband is a previously healthy 3-year-old female of non-consanguineous Caucasian parents, who presented with a 10-day history of cough, rhinorrhea, fever, and diarrhea and a 2-day history of emesis, found to have severe cardiomegaly on X-ray (Figure 1A). Three generation-family pedigree is shown in Figure 1B. On admission, a chest X-ray demonstrated profound cardiomegaly (Figure 1A). Her BNP, cardiac troponin, and serum lactate were elevated (Figure 1C). She was found to have severe dilated cardiomyopathy with concentric left ventricular thickening and an ejection fraction (EF) of 10-15% per Echocardiography (Figure 1D and Supplementary Video). Her EKG was profoundly abnormal with huge voltages suggesting a storage disease (Supplementary Figure 1).
Myocardium and Skeletal Muscle Biopsies Reveal Hyperplastic and Polymorphic Mitochondria and Lipid Accumulation. (A) Myocardium: H&E X40 shows diffuse generalized granular vacuolization likely representing mitochondria. (B) Myocardium: Mason Trichrome X40 reveals minimal fibrosis. (C) Myocardium: Periodic Acid Schiff (PAS) X40 Negative staining indicates absence of abnormal glycogen content. (D,E) Myocardium: Electron microscopy- myocardium X11000 (D) and X22000 (E): Mitochondrial hyperplasia, pleoconia, and megaconial forms and loss of internal cristae; Stars indicate mitochondria of variable and abnormal size. (F) Skeletal Muscle: H&E X40 Moderate fiber size variation with occasional atrophic fibers (white arrow). No evidence of degeneration/regeneration/inflammation. (G) Skeletal Muscle: PAS X40 Mild glycogen accumulation. (H) Skeletal Muscle: Oil Red O X10 Increased cytoplasmic lipid. (I) Skeletal Muscle: SDH X40 complex II mitochondrial respiratory chain enzyme-normal appearance. (J,K) Electron microscopy-skeletal muscle direct magnification of X11000 (J) and X8900 (K): Mitochondria are normal in size and shape (yellow arrows); there is slight increase in cytoplasmic Glycogen (orange star) lipid (blue arrowhead).
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F2
Acid Schiff (PAS) staining of the cardiac specimen revealed no glycogen storage abnormalities, and no evidence of inflammation or necrosis was detected (Figures 2A-C). At the ultrastructural level, electron microscopic images revealed inter-myofibrillar mitochondrial hyperplasia with polymorphic features including pelaconia and megaconia forms. Relative paucity of internal cristae was observed, and linear structures representing mitochondrial DNA were noted, together, raising suspicion for a primary mitochondrial disorder (Figures 2D,E). The skeletal muscle showed evidence of lipid and glycogen accumulation suggestive of a combined lipid and glycogen storage myopathy and there was T1 myofiber predominance with preferential type 2 myofiber atrophy (Figures 2F-H). No mitochondrial abnormality was detected by immunohistochemistry (Figure 2I), and electron microscopy analysis of skeletal myotubes was essentially normal (Figures 2J,K).
Compound Heterozygous Variants of TSFM Revealed by Trio (Proband/Parents) Whole Exome Sequencing (WES). (A) Table summary depicts rare compound heterozygous variants in TSFM genes detected by WES. (B) Schematic representation of TSFM gene (upper) and TSFM protein (lower) and known structural motifs. Black arrows indicate exons affected by deleterious mutations. Red arrows indicate the positions of AA substitutions in TSFM protein sequence caused by TSFM variants. (C,D) Integrated genomic viewer windows depict tow heterozygous variants in TSFM in the proband genome, the maternally inherited c.997C>T (C) and the paternally inherited c.355G>C (D). (E) Sanger sequencing confirms that both TSFM variants are in trans configuration in the proband DNA.
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F3
Two novel compound heterozygous variants in the TSFM gene (NM_001172696.1) were detected in trans: c.997C>T [p.(Arg333Trp)] and c.355G>C [p.(Val119Leu)] (Figures 3A-D). Both variants were confirmed in the proband DNA through Sanger sequencing (Figure 3E).
Summary of Clinical Time Course of the Proband. (A) Graph depicts left ventricle ejection fraction percentage overtime. Blue dots represent the time of measurements. Orange dots represent the times of admission and related events (texts). (B) Summary of clinical time course depicts important clinical events over time. HFrEF, heart failure with reduced ejection fraction; ED, emergency department; COXPD3, combined oxidative phosphorylation deficiency 3.
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F4
A summary of clinical time-course and cardiac function, represented by percentage left ventricle ejection fraction (LVEF%), is shown in Figure 4.
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