Family-based whole-genome and mitochondrial DNA analysis in fatal infantile cardiomyopathy: findings from blood and myocardium

Background Fatal cardiomyopathy in infancy, particularly when accompanied by metabolic abnormalities and multiorgan failure, may result from pathogenic variation in either the nuclear or mitochondrial genome. Establishing causality remains challenging when candidate variants are of uncertain significance, show incomplete segregation, or are also present in clinically unaffected relatives. Methods Family-based whole-genome sequencing was performed on blood-derived DNA from the affected infant and both parents and on myocardial DNA from the infant. Primary variant analysis used a GATK-based pipeline with HaplotypeCaller and VEP annotation, followed by clinical interpretation and prioritization with VarSome Clinical and Franklin (Genoox) according to ACMG/AMP criteria, population frequency, predicted effect, biological relevance, inheritance, and family segregation. Structural variants were detected and analysed with DELLY v0.9.1. For computational confirmation of the mitochondrial findings, all four WGS datasets were reanalysed in Galaxy Europe using GATK 4.6.2.0 Mutect2 in mitochondrial mode , followed by HaploGrep3 haplogroup assignment. Results The infant developed rapidly progressive cardiomyopathy with severe systolic dysfunction and multiorgan failure and died at 8 months of age. Fractional shortening decreased from 26% to 10%, and a large circumferential pericardial effusion required drainage of approximately 150 mL. Urinary lactate reached 2,192 mmol/mol creatinine, followed by hepatic and renal failure. Myocardial histology demonstrated diffuse interstitial fibrosis, extensive loss of myofibrillar structures, cytoplasmic vacuolization, and inclusion bodies, without evidence of myocarditis. Nuclear WGS identified candidate variants in FOXO6 , VWF , and TSC1 , but segregation and phenotype assessment did not support any as the primary disease cause. DELLY identified 27,214 structural-variant records; after stringent quality, segregation, genotype, and read-support filtering, four constitutional de novo and six myocardium-associated candidates remained, but none was considered causal without further validation. Dedicated mitochondrial-mode reanalysis reproduced near-homoplasmic m.4401A>G in proband blood (99.8%; 2,056×), myocardium (99.9%; 2,052×), and maternal blood (99.9%; 2,030×), with no raw Mutect2 call in paternal blood. The maternal-lineage variants m.772A>G and m.16288T>C showed a similar segregation pattern. HaploGrep3 assigned proband blood, myocardium, and maternal blood to haplogroup J1d3a, whereas paternal blood was assigned to N1b1a. The m.4401A>G variant was classified as a global private mutation relative to J1d3a rather than a haplogroup-defining variant and showed no enrichment in affected myocardial tissue. Conclusion Neither nuclear-genome nor mitochondrial-genome analyses identified a variant that could be confidently established as the cause of the observed phenotype. The mitochondrial variant m.4401A>G remains of biological interest but currently lacks sufficient genetic evidence for pathogenicity in this family.