Diagnosis of pediatric mitochondrial diseases via targeted next-generation sequencing (NGS): real-world data with the Blueprint Genetics® platform

Background: The diagnosis of mitochondrial disorders (MDs) in pediatric patients is complex and often delayed due to heterogeneous clinical presentations and limited access to invasive confirmation tests such as muscle biopsy. Objective: To evaluate the diagnostic sensitivity and specificity of a targeted next-generation sequencing (NGS) panel (Blueprint Genetics® “FLEX Comprehensive Epilepsy Panel Plus”) for detecting MDs in a cohort of pediatric patients with neurological symptoms compared with gold standard tissue biopsy and clinical follow-up. The primary endpoint was the diagnostic accuracy of targeted NGS panel testing for pediatric MDs. The secondary endpoints were the identification of clinical predictors and biochemical correlations associated with increased diagnostic yield and the assessment of age-dependent diagnostic patterns. Methods: We enrolled 36 pediatric patients between May 2022 and July 2023 on the basis of predefined clinical criteria suggestive of MDs. All patients underwent buccal swab-based targeted NGS testing. Confirmatory tissue biopsies and functional studies were performed in selected cases as the gold standard reference. True positives were defined as pathogenic/likely pathogenic variants confirmed by tissue biopsy and/or strong clinical correlation. Statistical analyses included Pearson correlation, Fisher’s exact test, and univariate logistic regression, stratifying data by age of onset and clinical features. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines to ensure methodological rigor and transparency. This was a convenience sample without a priori power calculation. Results: Five patients (5/34 out of 36 patients who completed the study, 14.7%) harbored variants in MD-associated genes. Pathogenic or likely pathogenic variants were confirmed in three cases through tissue biopsy, acting as true positives. The sensitivity was 100% (95% CI: 29.2–100%), the specificity was 94% (95% CI: 79.6–99.3%), the accuracy was 94% (95% CI: 80.3–99.3%), and the positive predictive value was 60% (95% CI: 14.7–94.7%). The positive predictive value reflects the presence of variants of uncertain significance (VUS) requiring further validation. Statistically significant correlations were observed between the presence of MD variants and elevated blood lactate (R = 0.51, p < 0.05) and GGT levels (R = 0.46, p < 0.05) and reduced hemoglobin (R=-0.42, p < 0.05). Eight specific clinical signs, occurring at < 12 months of age, strongly predicted MD-related variants with statistical significance (p < 0.05). Conclusion: Targeted NGS via buccal swabs demonstrates high accuracy and specificity in diagnosing pediatric MDs when guided by a structured clinical phenotype and confirmed by tissue analysis. Early onset of specific clinical signs should prompt NGS testing and may reduce unnecessary invasive procedures. Early diagnosis enables improved patient management and family counseling.