Characterization of a Novel Splicing Variant in Acylglycerol Kinase (AGK) Associated with Fatal Sengers Syndrome.

Mitochondrial functional integrity depends on protein and lipid homeostasis in the mitochondrial membranes and disturbances in their accumulation can cause disease. AGK, a mitochondrial acylglycerol kinase, is not only involved in lipid signaling but is also a component of the TIM22 complex in the inner mitochondrial membrane, which mediates the import of a subset of membrane proteins. AGK mutations can alter both phospholipid metabolism and mitochondrial protein biogenesis, contributing to the pathogenesis of Sengers syndrome. We describe the case of an infant carrying a novel homozygous AGK variant, c.518+1G>A, who was born with congenital cataracts, pielic ectasia, critical congenital dilated myocardiopathy, and hyperlactacidemia and died 20 h after birth. Using the patient's DNA, we performed targeted sequencing of 314 nuclear genes encoding respiratory chain complex subunits and proteins implicated in mitochondrial oxidative phosphorylation (OXPHOS). A decrease of 96-bp in the length of the AGK cDNA sequence was detected. Decreases in the oxygen consumption rate (OCR) and the OCR:ECAR (extracellular acidification rate) ratio in the patient's fibroblasts indicated reduced electron flow through the respiratory chain, and spectrophotometry revealed decreased activity of OXPHOS complexes I and V. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts and describe the possible molecular mechanism underlying the pathogenicity of this novel AGK variant. Experimental validation using in vitro analysis allowed an accurate characterization of the disease-causing variant.

Identification of a Novel Variant in EARS2 Associated with a Severe Clinical Phenotype Expands the Clinical Spectrum of LTBL.

The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient's clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this disease.

Identification and Characterization of New Variants in FOXRED1 Gene Expands the Clinical Spectrum Associated with Mitochondrial Complex I Deficiency.

Complex I (nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase) is the largest complex of the mitochondrial oxidative phosphorylation system (OXPHOS) system. Forty-four subunits encoded in nuclear and mitochondrial genomes compose this multiprotein complex, its assembly being a highly complex process involving at least 15 additional nuclear encoded assembly factors. Complex I deficiency is a mitochondrial disorder usually associated with early-onset severe multisystem disorders characterized by highly variable clinical manifestations. Flavin adenine dinucleotide (FAD)-dependent oxidoreductase domain-containing protein 1 (FOXRED1) is a complex I assembly factor. To date, only five patients with mitochondrial complex I deficiency due to mutations in FOXRED1 have been characterized. Here, we describe a child with ataxia, epilepsy and psychomotor developmental delay carrying two heterozygous FOXRED1 variants, c.920G>A (p.Gly307Glu) and c.733+1G>A. We demonstrate the molecular mechanism supporting the pathogenicity of the FOXRED1 variants, showing a clear deficiency of complex I activity. The reduction in the steady-state level of complex I holoenzyme in patient fibroblasts, confirmed the pathogenicity of the variants and showed the molecular mechanism behind their pathogenicity. A comparison of the clinical presentation of the index case with the previously described cases allowed deepening our knowledge about the clinical variability associated with FOXRED1 defects.

High-throughput assay to measure oxygen consumption in digitonin-permeabilized cells of patients with mitochondrial disorders.

BACKGROUND: Muscle biopsy analysis is regarded as the gold standard in diagnostic workups of patients with suspected mitochondrial disorders. Analysis of cultured fibroblasts can provide important additional diagnostic information. The measurement of individual OXPHOS complexes does not always provide sufficient information about the functional state of the complete mitochondrial energy-generating system. Thus, we optimized a high-throughput fluorescence-based methodology for oxygen consumption analysis in patient-derived cells. METHODS: We analyzed mitochondrial respiration in digitonin-permeabilized cells in the presence of a substrate mix containing pyruvate and malate, using a phosphorescent probe, 96-well plates, and a fluorescence plate reader. RESULTS: In control fibroblasts, we observed clear stimulation by ADP of the pyruvate + malate-driven respiration. Known inhibitors of the OXPHOS system and the Krebs cycle significantly reduced respiration. In patient fibroblasts with different OXPHOS deficiencies, ADP-stimulated respiratory activity was decreased in comparison to control cells. In several patients with reduced ATP production rate in muscle tissue but with normal OXPHOS enzyme activities, the fibroblasts displayed reduced respiratory activity. Finally, we observed a clear difference between control and complex I-deficient transmitochondrial cybrid cells. CONCLUSIONS: These results confirm the validity of the assay as a high-throughput screening method for mitochondrial function in digitonin-permeabilized cells. The assay allows primary and secondary mitochondrial abnormalities in muscle to be differentiated, which is of great importance with respect to counseling, and also will facilitate the search for new genetic defects that lead to mitochondrial disease.