Objectives: To facilitate genetic screening in complex I deficiency by implementing biochemical and genetic approaches. Patients and Methods: 30 complex I deficient paediatric patients with heterogeneous clinical phenotypes were studied. Complex I deficiency and assembly state were analysed using spectrophotometric assay and Blue Native gel electrophoresis. Candidate gene analysis and homozygosity mapping were used to search for the underlying gene defect. Results: Abnormal complex I subassemblies accumulated in 3 cases: defects in NDUFS4 and a known assembly factor were causative in two cases, whereas the third defect is still unknown. Homozygosity mapping and bioinformatics analysis helped to identify a mutation in the novel complex I chaperone FOXRED1 in a fourth case. Conclusion: By using a combined biochemical and genetic approach we have found the underlying molecular defect in 13/30 [43%] of this complex I deficient patient cohort; further studies are ongoing to identify the genetic defects in the remaining patients. Background: Complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) defect. No therapeutic solutions are available except "neutraceuticals" among which high-fat diet (HFD) has been proposed but never evaluated. Objectives: To evaluate the effect of the long-chain fatty acid palmitate on human CI-deficient fibroblasts viability. Methods: Fibroblasts were obtained from three CI-defective patients genetically identified: two with NDUFS1 mutations and one with NDUFS4 mutations. They were cultured either in glucose-and glutamine-enriched DMEM, or in glucose-free, glutamine-enriched DMEM or in glucose-free, glutamine-, palmitate-enriched DMEM. Cell viability was evaluated daily using an automated cell counter. Succinate oxidation was assessed by polarography. Complex II activity was measured by spectrophotometry. Results: In one of the NDUFS-1 mutated cell line, death occurred within 80 hours after glucose withdrawal with full protection afforded by palmitate. In the two other CI-defective cell lines, death occurred within 170 hours after glucose withdrawal with full palmitate protection. The full protection from death provided by palmitate was associated with a slight but consistent increase (20-30%)in respiratory chain complex II activity. Conclusion: These data show that palmitate protects CI-deficient fibroblasts from death and provide a strong rationale for performing HFD trials in CI-deficiency. Complex I (CI) deficiency is a defect of OXPHOS caused by mutations in the mitochondrial or nuclear genomes. To date disease-causing mutations have been reported in all mitochondrial-encoded subunits and 21 nuclear genes. In about 50% of the patients no mutations are found, suggesting that undiscovered factors are an important cause of disease. In this study we report a consanguineous family from Southern Portugal with three affected children where CI deficiency could not be clarified. The affected children presented similar clinical findings with early onset of the disease having two of the patients a fatal outcome. A reduced activity of CI was detected in two of the patients, which led us to investigate, at the molecular level, some CI associated genes. However, no mutations were detected. Interestingly, all patients presented 3-methylglutaconic acid in the urinary organic acids and it is known that POLG gene is involved in the etiology of these syndromes. In only one of the patients, that also showed mtDNA depletion, the p.G848S/p.Q1236H mutations were found in POLG. It remains unsolved if this family due to the high consanguinity could have two different disorders or if a yet unknown gene, leading to complex I deficiency, could be involved. The role of a secondary respiratory chain deficiency as an additional mechanism to intoxication, leading to development of long-term energydependent complications, has been recently suggested in patients with propionic acidemia (PA). We show for the first time a coenzyme Q10 (CoQ10) functional defect accompanied by a multiple organ oxidative phosphorylation (OXPHOS) deficiency in a child who succumbed to acute heart failure in the absence of metabolic stress. Quinone-dependent activities in the liver (complex I+III, complex II+III) were reduced, suggesting a decrease in electron transfer related to the quinone pool. The restoration of complex II +III activity after addition of exogenous ubiquinone to the assay system suggests CoQ10 deficiency. Nevertheless, we disposed of insufficient material to perform direct measurement of CoQ10 content in the patient's liver. Death occurred before biochemical diagnosis of OXPHOS deficiency could be made. However, this case highlights the usefulness of rapidly identifying CoQ10 defects secondary to PA since this OXPHOS disorder has a good treatment response which could improve heart complications or prevent their appearance. Nevertheless, further studies will be necessary to determine whether CoQ10 treatment can be useful in PA complications linked to CoQ10 deficiency.