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

Abstract

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.

Keywords: FOXRED1; complex I deficiency; epilepsy; mitochondrial disorders.

Figure 1

Reverse sequence chromatograms showing Sanger sequencing results. The missense variant c.920G>A (p.Gly307Glu), located in exon 8, was inherited from the mother and the splicing variant, c.733+1G>A, located in intron 6, was inherited from the father. The patient and affected brother harbored both variants in FOXRED1 in a compound heterozygous condition.

Figure 2

(A) Domain structure of FOXRED1 with all the variants identified in the described cases. In light blue, the cleavable mitochondrial targeting sequence is shown or displayed. DAO—FAD-dependent oxidoreductase. (B) FOXRED1 protein modeling wild-type and FOXRED1 protein modeling with the mutation p.G307E, reflecting the predicted consequences in the spatial protein structure due to the change of the non-polar amino acid Glycine to the negatively charged Glutamic acid.

Figure 3

(A) Oxygen Consumption Rate (OCR) is measured before and after the addition of inhibitors. The Seahorse XF Cell Mito Stress Test uses compounds of respiration that target components of the Electron Transport Chain (ETC) in the mitochondria to reveal key parameters of metabolic function. These modulators are ETC inhibitors (oligomycin, FCCP, and a mix of rotenone and antimycin A) which were serially injected to measure ATP (adenosine triphosphate) production, maximal respiration (Max Resp), non-mitochondrial respiration (Non-Mito Resp), proton leak, spare respiratory capacity (SRC) and basal respiration (Basal Resp). (B) The basal energy metabolism of each cell line was assessed by analyzing OCR/ECAR ratios through sequential injections of the inhibitors. (C) Measurement of enzyme activities for the different oxidative phosphorylation system (OXPHOS) complexes in patient and control fibroblasts. FOXRED1 migrated at its predicted size of 54 kDa. CS—Citrate synthase. (D) SDS-PAGE immunodetections of FOXRED1 in control and patient fibroblasts. (M) Marker, (C) Control, and (P) Patient. Blot was cropped from different parts of the same gel to show results from samples of interest. A full-length gel is included in Supplementary Material. (E) One-dimensional Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) analysis of complex I in gel activity and western blot immunodetection showing differences in complex I amount between control (C) and patient (P) fibroblasts. Blot was cropped from different parts of the same gel to show results from samples of interest. A full-length gel is included in Supplementary Material.