Objective Respiratory chain complex (RCC) I is the second most common biochemical abnormality causing Leigh syndrome (LS), an early-onset neurodegenerative disorder. NDUFS4 gene presents a hotspot of mutations leading to an inappropriate assembly of complex I. Our aim was to describe a 67-days-old female presenting with severe hypotonia, abnormal eye movements, lethargy, seizures and respiratory failure leading to death on day 5 of evolution. Plasma lactate was 5.83 mmol/L and amino acids and organic acids were normal. MRI showed swelling lesions in the basal ganglia and brainstem with restricted diffusion and a lactate peak on MRS. Methods Pyruvate dehydrogenase (PDH) and oxidation-reduction activity assays were performed in fibroblasts and muscle. Spectrophotometric assays of RCC were performed in muscle. Genetic analysis of nuclear genes involved in mitochondrial disorders was assessed by targeted exome sequencing using the TruSight One Sequencing Panel (Illumina). Results The patient showed a significative reduction in PDH activity in fibroblasts and muscle, with values of 0.22 mmol/min*mg prot (normal values 0.34–2.6) and 0.5 mmol/min*mg prot, (normal values 0.8–3.4) respectively, and reduced oxidation of pyruvate in fibroblasts. Muscle analysis of RCC showed a 50% reduction in the residual activity of CI and CIII compared to control. Muscle Q10 was low when related to cytrate synthase (2.18 nmol/U CS, reference values 2.7–8.4). Massive sequencing revealed a previously described homozygous mutation c.291delG (p. Trp97Ter) in NDUFS4 gene, which was further confirmed by Sanger sequencing. No disease-causing mutations were detected in the PDHc genes. Conclusion NDUFS4 defect in our patient led to a rapidly progressive and fatal Leigh encephalopathy in the second month of life. A combined defect in pyruvate metabolism and respiratory chain complexes was misleading; probably, mild Q10 deficiency was secondary to inappropriate assembly of complex I. Finally, NGS techniques allowed genetic diagnosis in our patient.