FDXR Mutations Cause Sensorial Neuropathies and Expand the Spectrum of Mitochondrial Fe-S-Synthesis Diseases
2017; Elsevier BV; Volume: 101; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2017.09.007
ISSN1537-6605
AutoresAntoine Paul, Anthony Drecourt, Floriane Petit, D. Dupin Deguine, Christelle Vasnier, Myriam Oufadem, Cécile Masson, Crystel Bonnet, Saber Masmoudi, Isabelle Mosnier, L. Mahieu, D. Bouccara, Josseline Kaplan, Georges Challe, C. Domange, Fanny Mochel, Olivier Sterkers, S. Gerber, Patrick Nitschké, Christine Bôle‐Feysot, Laurence Jonard, Souad Gherbi, Oriane Mercati, Ines Aïssa, Stanislas Lyonnet, Agnès Rötig, Agnès Delahodde, Sandrine Marlin,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoHearing loss and visual impairment in childhood have mostly genetic origins, some of them being related to sensorial neuronal defects. Here, we report on eight subjects from four independent families affected by auditory neuropathy and optic atrophy. Whole-exome sequencing revealed biallelic mutations in FDXR in affected subjects of each family. FDXR encodes the mitochondrial ferredoxin reductase, the sole human ferredoxin reductase implicated in the biosynthesis of iron-sulfur clusters (ISCs) and in heme formation. ISC proteins are involved in enzymatic catalysis, gene expression, and DNA replication and repair. We observed deregulated iron homeostasis in FDXR mutant fibroblasts and indirect evidence of mitochondrial iron overload. Functional complementation in a yeast strain in which ARH1, the human FDXR ortholog, was deleted established the pathogenicity of these mutations. These data highlight the wide clinical heterogeneity of mitochondrial disorders related to ISC synthesis. Hearing loss and visual impairment in childhood have mostly genetic origins, some of them being related to sensorial neuronal defects. Here, we report on eight subjects from four independent families affected by auditory neuropathy and optic atrophy. Whole-exome sequencing revealed biallelic mutations in FDXR in affected subjects of each family. FDXR encodes the mitochondrial ferredoxin reductase, the sole human ferredoxin reductase implicated in the biosynthesis of iron-sulfur clusters (ISCs) and in heme formation. ISC proteins are involved in enzymatic catalysis, gene expression, and DNA replication and repair. We observed deregulated iron homeostasis in FDXR mutant fibroblasts and indirect evidence of mitochondrial iron overload. Functional complementation in a yeast strain in which ARH1, the human FDXR ortholog, was deleted established the pathogenicity of these mutations. These data highlight the wide clinical heterogeneity of mitochondrial disorders related to ISC synthesis. Hearing loss and visual defects have mostly genetic origins. Several syndromes, including Usher (OMIM: 276903) and Wolfram syndromes (OMIM: 606201), are associated with hearing and visual impairments. Sensorial neuropathies are less frequent than cochlear and retinal defects. Auditory neuropathies (ANs) represent about 8% of congenital deafness,1Nikolopoulos T.P. Auditory dyssynchrony or auditory neuropathy: understanding the pathophysiology and exploring methods of treatment.Int. J. Pediatr. Otorhinolaryngol. 2014; 78: 171-173Crossref PubMed Scopus (19) Google Scholar, 2Moser T. Starr A. Auditory neuropathy--neural and synaptic mechanisms.Nat. Rev. Neurol. 2016; 12: 135-149Crossref PubMed Scopus (179) Google Scholar and the prevalence of optic atrophy (OA) is 1/40,000.3Bocquet B. Lacroux A. Surget M.O. Baudoin C. Marquette V. Manes G. Hebrard M. Sénéchal A. Delettre C. Roux A.F. et al.Relative frequencies of inherited retinal dystrophies and optic neuropathies in Southern France: assessment of 21-year data management.Ophthalmic Epidemiol. 2013; 20: 13-25Crossref PubMed Scopus (40) Google Scholar The association between ANs and OA can be part of a multiorgan syndrome, usually implicating other neurologic systems. Examples include Friedreich ataxia (OMIM: 606829), Charcot-Marie-Tooth disease (OMIM: 159440), Brown-Vialetto-Van-Laere syndrome (OMIM: 614707), and Mohr-Tranebjaerg syndrome (OMIM: 304700). The AN-OA association is due to specific rare OPA1 (OMIM: 605290) and TMEM126A (OMIM: 612988) mutations.4Namba K. Mutai H. Takiguchi Y. Yagi H. Okuyama T. Oba S. Yamagishi R. Kaneko H. Shintani T. Kaga K. Matsunaga T. Molecular impairment mechanisms of novel OPA1 mutations predicted by molecular modeling in patients with autosomal dominant optic atrophy and auditory neuropathy spectrum disorder.Otol. Neurotol. 2016; 37: 394-402PubMed Google Scholar, 5Meyer E. Michaelides M. Tee L.J. Robson A.G. Rahman F. Pasha S. Luxon L.M. Moore A.T. Maher E.R. Nonsense mutation in TMEM126A causing autosomal recessive optic atrophy and auditory neuropathy.Mol. Vis. 2010; 16: 650-664PubMed Google Scholar Interestingly, several of the implicated genes encode mitochondrial proteins. Here, we report on eight subjects, two boys and six girls, from four different families (F1–F4) affected by AN and OA. The first clinical signs were observed during childhood or adolescence. The four families are from different origins (F1—Tunisia; F2—Algeria; F3—France; and F4—Azerbaijan and Russia). All parents were healthy, and there were no cases of sensorial defect in any relatives. Recurrence of the AN-OA was observed in two families, F1 and F4, with 2/5 and 2/4 of the children being affected, respectively (Figure 1, Table 1, and Supplemental Note). Consanguinity was noted only in family 1. All subjects were born at term, after normal pregnancy and delivery, with normal parameters. They all had normal psychomotor development except that subject 7 had a slight language delay; she spoke her first words at 2 years of age (Table 1 and Supplemental Note). For all subjects the diagnosis of AN was made on the basis of severe impaired auditory brainstem responses (ABRs), discrepancies between tonal and vocal audiometry, and the presence of otoacoustic emissions on both ears (Figure S1A). All subjects underwent ophthalmological fundus, electroretinogram (ERG), visual evoked potential (VEP) tests, and optic coherence tomography (OCT) and showed a bilateral defect of the optic nerves (Figure S1B). Magnetic resonance imaging (MRI) of cerebral and temporal bones did not reveal any abnormalities in supra-tentorial regions, posterior fossa, or inner ears (data not shown) in any index cases. Subjects 1–5 and 7–8 had isolated sensorial neurologic defects. Bilateral retinitis pigmentosa was diagnosed in subject 6 at 2 years of age because of low vision and nystagmus. ERG waves were indiscernible, and the fundus was altered. At the age of 20, his neurologic examination showed only hypopallesthesia of the lower limbs. His cerebral MRI spectroscopy study and electromyogram were normal at this age.Table 1Clinical Data of Affected Individuals and FDXR GenotypesFamiliesF1F2F3F4Familial originTunisiaAlgeriaFranceRussia and AzerbaijanIndividuals12345678GendersFFFFMMGGOnset of hearing defect13 yr13 yr15 yr11 yrteenage20 yr17y5yHearing phenotypeBANBANBANBANBANBANBANBANOnset of visual defect13 y13y36y31y31y2 and 20yChildhood17yVisual phenotypeBOABOAinfraclinical BOAinfraclinical BOAinfraclinical BOABRP and BOABOABOAFDXR variationsc.916 C>T Hoc.916 C>T Hoc.916 C>T Hoc.916 C>T Hoc.916 C>T c.1255 C>Tc.724C>T, c.979C>Ac.643C>G, c.1429 G>Ac.643C>G, c.1429 G>AProtein changep.Arg306Cysp.Arg306Cysp.Arg306Cysp.Arg306Cysp.Arg306Cys, p.Gln419∗p.Arg242Trp, p.Arg327Serp.Leu215Val, p.Glu477Lysp.Leu215Val, p.Glu477LysExAC browser allele frequency8.85 × 10−68.85 × 10−68.85 × 10−68.85 × 10−68.85 × 10−60000Score prediction, SIFT0.5850.5850.5850.5850.04NA00.300.0300.03Score prediction, Mutation Taster11111NA111111F1–4: families 1–4. M: male. F: female. BAN: bilateral auditory neuropathy. BOA: bilateral optic atrophy. BRP: bilateral retinitis pigmentosa. Ho: homozygous. NA: not available. Open table in a new tab F1–4: families 1–4. M: male. F: female. BAN: bilateral auditory neuropathy. BOA: bilateral optic atrophy. BRP: bilateral retinitis pigmentosa. Ho: homozygous. NA: not available. None of the index cases (subjects 1 and 5–7) harbored mutations in OPA1, TMEM126A, or mitochondrial DNA. Molecular analysis of 35 different genes involved in severe congenital retinitis pigmentosa did not identify any causative variation. Because of a high degree of consanguinity in family 1 and the absence of disease in any parent, autosomal-recessive inheritance was hypothesized, and whole-exome-sequencing (WES) for subjects 1 and 3 was performed. Informed consent for diagnostic and research studies was obtained for all subjects in accordance with the Declaration of Helsinki protocols, and the local institutional review boards in Paris (Comité de Protection des Personnes, Ile de France II) approved the study. DNA was extracted from leucocytes. Exome capture was performed with the Sure Select Human All Exon kit (Agilent Technologies). Agilent Sure Select Human All Exon (58 Mb, V6) libraries were prepared from 3 μg of genomic DNA sheared with an Ultrasonicator (Covaris) as recommended by the manufacturer. Barcoded exome libraries were pooled and sequenced with a HiSeq2500 system (Illumina), generating paired-end reads. After demultiplexing, sequences were mapped on the human genome reference (NCBI build 37, hg19 version) with BWA. Variant calling was carried out with the Genome Analysis Toolkit (GATK), SAMtools, and Picard tools. Single-nucleotide variants were called with GATK Unified Genotyper, whereas indel calls were made with the GATK IndelGenotyper_v2. All variants with a read coverage ≤2× and a Phred-scaled quality ≤20 were filtered out. All the variants were annotated and filtered with PolyWeb, an in-house-developed annotation software. For subject 1's library, a 86× mean depth of coverage was obtained, and 86% of the targeted exonic bases were covered by at least 15 independent sequencing reads (86% at 15×). The mean depth of coverage obtained for the exome library of subject 3 was 106×, and >97% of the targeted exonic bases were covered by at least 15 independent sequencing reads (>97% at 15×). Variant-filtering strategies and familial segregation led to the identification of only one homozygous variant shared by subjects 1 and 3 in FDXR (NM_024417.4): c.916C>T (p.Arg306Cys). This allele has been reported with a frequency of 8.85 × 10−6, 1/112999 alleles (ExAc Browser Beta). We detected the FDXR c.916C>T homozygous variation in subjects 1–4 by Sanger sequencing (Figure S2). This variation co-segregated with the disease as expected for autosomal-recessive inheritance (Figure 1). We subsequently performed FDXR Sanger sequencing in subjects 5–8. Subject 5 was found to be compound heterozygous for the same c.916C>T FDXR variation and for c.1255C>T (p.Gln419∗). Unfortunately, we did not have access to DNA of other members of this family. Subject 6 had two compound heterozygous missense variations: c.724C>T (p.Arg242Trp) and c.979C>A (p.Arg327Ser) (Figure 1 and Figure S2). Subjects 7 and 8 were found to be compound heterozygous for two other missense mutations, c.643C>G (p.Leu215Val) and c.1429G>A (p.Glu477Lys) (Figure 1 and Figure S2). DNA of other family members was not available. The pathogenicity clues for these FDXR variations are presented in Table 1. No FDXR mutation could be found in 12 independent subjects who demonstrated hearing loss due to a cochlear defect and optic atrophy, 12 subjects with isolated auditory neuropathy, 58 subjects with isolated optic atrophy, or four subjects with auditory neuropathy associated with peripheral neuropathy. FDXR (OMIM 103270) encodes a mitochondrial NADPH: adrenodoxin oxidoreductase or ferredoxin reductase, the sole human ferredoxin reductase involved in the biosynthesis of iron-sulfur (Fe-S) clusters and heme formation.6Ewen K.M. Kleser M. Bernhardt R. Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins.Biochim. Biophys. Acta. 2011; 1814: 111-125Crossref PubMed Scopus (74) Google Scholar, 7Jung Y.S. Gao-Sheridan H.S. Christiansen J. Dean D.R. Burgess B.K. Purification and biophysical characterization of a new [2Fe-2S] ferredoxin from Azotobacter vinelandii, a putative [Fe-S] cluster assembly/repair protein.J. Biol. Chem. 1999; 274: 32402-32410Crossref PubMed Scopus (56) Google Scholar, 8Stehling O. Wilbrecht C. Lill R. Mitochondrial iron-sulfur protein biogenesis and human disease.Biochimie. 2014; 100: 61-77Crossref PubMed Scopus (199) Google Scholar We built a structural-homology model of ferredoxin reductase on the basis of the crystal structure of Bos taurus ferredoxin reductase (Figure 2A). This in silico modeling showed that the amino acids modified by the mutations are scattered in various parts of the protein but are not located in FAD or Fe-S binding sites. Three amino acid changes are predicted to create new H bonds: p.Leu215Val with Ala211 and Leu212, p.Arg242Trp with Leu279, and p.Arg327Ser with Gln313 (Figures 2B, 2C, and 2E). On the contrary, p.Arg306Cys is predicted to abolish the H bond with Glu349 (Figure 2D). These modifications are thus expected to destabilize ferredoxin reductase. Indeed, immunoblot analyses showed a major decrease of ferredoxin reductase in fibroblasts of subjects 1 and 5 (Figure 3A). To assess the consequences of FDXR mutations on ISC synthesis, we measured in-gel activity of mitochondrial and cytosolic aconitase, both containing ISC, that were found to be decreased (Figure 3B). Moreover, the amounts of heme oxygenase (HO-1) (Figure 3B) and SDHB, an iron-sulfur cluster (ISC)-containing subunit of respiratory chain complex II (Figure 3D), were severely decreased in subjects' fibroblasts. Finally, we also observed decreased activities of complexes I and III in these cells (Table 2). No fibroblasts were available for other affected subjects.Table 2Respiratory-Chain Enzyme Activities in Cultured Skin FibroblastsS1S5CAbsolute Activities (nmol/min/mg protein)CI7614 ± 3CII292622 ± 2CIII82125190 ± 23CIV61144107 ± 14CV101421 ± 3Activity RatiosCI/CII0,230,220,57 ± 0,11CIII/CII2,94,98,8 ± 0,8CIV/CII2,15,74,7 ± 0,5CV/CII0,340,560,87 ± 0,09CI–V: complexes I–V. Abnormal values are shown in bold. Open table in a new tab CI–V: complexes I–V. Abnormal values are shown in bold. Several mitochondrial proteins are involved in ISC assembly; these include frataxin, encoded by FXN (MIM: 606829), the mutations of which result in Friedreich ataxia.11Campuzano V. Montermini L. Moltò M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. et al.Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2302) Google Scholar In yeast, Arh1 and Yfh1, the orthologs of human ferredoxin reductase and frataxin (FXN), respectively, are two factors involved in the first step of ISC assembly. Considering that (1) human FXN mutations induce mitochondrial iron accumulation and decreased activity of ISC enzymes,8Stehling O. Wilbrecht C. Lill R. Mitochondrial iron-sulfur protein biogenesis and human disease.Biochimie. 2014; 100: 61-77Crossref PubMed Scopus (199) Google Scholar, 12Schmucker S. Argentini M. Carelle-Calmels N. Martelli A. Puccio H. The in vivo mitochondrial two-step maturation of human frataxin.Hum. Mol. Genet. 2008; 17: 3521-3531Crossref PubMed Scopus (110) Google Scholar (2) yeast Yfh1 defects result in mitochondrial iron overload,13Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia.Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar and (3) yeast Arh1 defects induce a cellular iron uptake and disturbed iron trafficking within the cell,14Li J. Saxena S. Pain D. Dancis A. Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis.J. Biol. Chem. 2001; 276: 1503-1509Crossref PubMed Scopus (109) Google Scholar we examined iron homeostasis in fibroblasts of subjects with FDXR mutations. Iron enters cells either via internalization of transferrin (Tf)-bound iron by transferrin receptor 1 (TfR1)-mediated endocytosis or by non-Tf-bound iron (NTBI) uptake.15Lane D.J.R. Merlot A.M. Huang M.L. Bae D.H. Jansson P.J. Sahni S. Kalinowski D.S. Richardson D.R. Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease.Biochim. Biophys. Acta. 2015; 1853: 1130-1144Crossref PubMed Scopus (224) Google Scholar, 16Sohn Y.-S. Ghoti H. Breuer W. Rachmilewitz E. Attar S. Weiss G. Cabantchik Z.I. The role of endocytic pathways in cellular uptake of plasma non-transferrin iron.Haematologica. 2012; 97: 670-678Crossref PubMed Scopus (37) Google Scholar An increase of the TfR1 steady-state level was observed in the fibroblasts of subjects 1 and 5 compared to control (187% and 184% of the control amount, respectively), and highly elevated amounts of mitochondrial superoxide dismutase 2 (SOD2) were also observed (Figure 3A and Figure S3). In keeping with this, increased mitochondrial superoxide production in S1 and S5 fibroblasts was detected by flow cytometry with MitoSOX (Figure 3C). These results suggest that mutations in the gene encoding ferredoxin reductase induce abnormal iron uptake by TfR1 and that they result in a major oxidative stress in mitochondria. Homeostasis of cellular iron is regulated by a post-transcriptional mechanism that involves iron-regulatory proteins (IRPs) and that modulates TfR1 levels, so that iron uptake does not take place in high-iron conditions. When grown for 3 days in low-iron conditions (fetal beef serum [FBS]-free DMEM, i.e., devoid of Tf-bound iron), a major increase in the ferritin steady-state level was observed in cultured fibroblasts of subject 1 and to a lesser extent those of subject 5 (Figure 3D and Figure S3), reflecting iron overload. Ferric ammonium citrate (FAC) is a soluble form of iron known to enhance NTBI uptake in fibroblasts17Buys S.S. Martin C.B. Eldridge M. Kushner J.P. Kaplan J. Iron absorption in hypotransferrinemic mice.Blood. 1991; 78: 3288-3290PubMed Google Scholar by a poorly understood mechanism that might involve the endocytic pathway.16Sohn Y.-S. Ghoti H. Breuer W. Rachmilewitz E. Attar S. Weiss G. Cabantchik Z.I. The role of endocytic pathways in cellular uptake of plasma non-transferrin iron.Haematologica. 2012; 97: 670-678Crossref PubMed Scopus (37) Google Scholar After 3 days of incubation with FAC, control cells increased amounts of ferritin as expected, but S1 and S5 fibroblasts displayed a much higher ferritin steady-state level, showing that both fibroblast cultures abnormally increased their iron content (Figure 3D and Figure S3). As expected, in high-iron conditions, amounts of ACO1 (also called IRP1) decreased in control and subjects' fibroblasts, and amounts of TfR1 decreased in control cells. Cultured fibroblasts of subject 5 also had decreased amounts of TfR1 in high-iron conditions, but not only did TfR1 amounts fail to decrease in S1 fibroblasts, they actually increased, allowing abnormal iron uptake. We therefore quantified iron content in fibroblasts. When grown in low-iron conditions (no FAC and no FBS), control and affected individual fibroblasts showed a similar amount of iron. After a 3 day incubation with FAC, FDXR mutant fibroblasts exhibited a major increase in cellular iron content (a 50- and 100-fold change for S5 and S1, respectively), whereas control fibroblasts displayed a 2- to 3-fold upregulation (Figure 3E and Figure S3). Accordingly, SOD1 and SOD2 increased in high-iron conditions, reflecting the oxidative stress induced by iron overload. Nevertheless, mitochondrial ROS production was not modified in high- versus low-iron conditions (Figure 3C), suggesting that SOD2 induction efficiently protects against ROS overproduction. The mitochondrial aconitase (ACO2) and SDHB amounts were decreased in subjects' fibroblasts in low- and high-iron conditions because of the ISC defect induced by FDXR mutations. Altogether, these results suggest that the defect in ferredoxin reductase induced a failure to repress iron uptake, as previously demonstrated in FDXR knock-down human cells18Shi Y. Ghosh M. Kovtunovych G. Crooks D.R. Rouault T.A. Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis.Biochim. Biophys. Acta. 2012; 1823: 484-492Crossref PubMed Scopus (120) Google Scholar and in ARH1-deficient yeast cells.14Li J. Saxena S. Pain D. Dancis A. Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis.J. Biol. Chem. 2001; 276: 1503-1509Crossref PubMed Scopus (109) Google Scholar To definitively demonstrate the pathogenic nature of the identified FDXR mutations, we studied their effect on the growth of a Saccharomyces cerevisiae strain in which ARH1 was deleted. Yeast mitochondrial Arh1 shares 35% identity with ferredoxin reductase (Figure 2F) and is essential for cell viability. Consistent with previous studies,19Manzella L. Barros M.H. Nobrega F.G. ARH1 of Saccharomyces cerevisiae: a new essential gene that codes for a protein homologous to the human adrenodoxin reductase.Yeast. 1998; 14: 839-846Crossref PubMed Scopus (49) Google Scholar the ARH1-null mutation (arh1Δ) led to lethality that was rescued by overexpression of ARH1 (Figures 4A and 4B ). Using a well-known plasmid-shuffling system,20Boeke J.D. Trueheart J. Natsoulis G. Fink G.R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics.Methods Enzymol. 1987; 154: 164-175Crossref PubMed Scopus (1075) Google Scholar we found, contrary to findings of the Manzella study,19Manzella L. Barros M.H. Nobrega F.G. ARH1 of Saccharomyces cerevisiae: a new essential gene that codes for a protein homologous to the human adrenodoxin reductase.Yeast. 1998; 14: 839-846Crossref PubMed Scopus (49) Google Scholar that overexpression of wild-type (wt) human FDXR cDNA was able to rescue arh1Δ lethality (Figures 4A and 4B), demonstrating that FDXR is the true functional ortholog of ARH1, as suggested by Webert et al.,22Webert H. Freibert S.A. Gallo A. Heidenreich T. Linne U. Amlacher S. Hurt E. Mühlenhoff U. Banci L. Lill R. Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin.Nat. Commun. 2014; 5: 5013Crossref PubMed Scopus (113) Google Scholar who used purified human ferredoxin reductase instead of Arh1 in an in vitro reconstitution of yeast Fe/S cluster synthesis on Isu1. Subsequently, we introduced amino acid changes into the ferredoxin reductase sequence for 3/5 missense mutations. p.Arg242Trp was completely unable to complement the arh1Δ growth defect (Figure 4A), indicating that this mutation disrupts the ferredoxin reductase function. p.Arg306Cys and p.Leu215Val changes did not modify cell growth in this condition but induced a growth defect on non-fermentable (respiratory) substrate (YPG, Figure 4B). Because Arh1 is required for ISC assembly, we further measured the enzyme activity of mitochondrial aconitase, which contains an ISC. Spectrophotometric analyses revealed that aconitase activity in arh1Δ cells expressing p.Arg306Cys and p.Leu215Val was decreased in comparison to that in cells expressing WT Fdxr (Figure 4C), thus confirming the deleterious nature of the FDXR variants. By using exome sequencing, we found mutations in the gene encoding the mitochondrial ferredoxin reductase in eight subjects from four independent families, and functional studies in yeast clearly established that the variations tested cause disease in these subjects. Mutations in several genes involved in ISC assembly have been reported in human diseases (Friedreich ataxia due to FXN mutations is the most frequent).11Campuzano V. Montermini L. Moltò M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. et al.Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2302) Google Scholar These mutations result in a wide panel of variably severe clinical presentations, ranging from fatal infantile leukodystrophy to mitochondrial myopathy.23Beilschmidt L.K. Puccio H.M. Mammalian Fe-S cluster biogenesis and its implication in disease.Biochimie. 2014; 100: 48-60Crossref PubMed Scopus (80) Google Scholar Interestingly, whereas mutations in FDX1L, encoding ferredoxin-2 (FDX2), result in mitochondrial myopathy and myoglobinuria,24Spiegel R. Saada A. Halvardson J. Soiferman D. Shaag A. Edvardson S. Horovitz Y. Khayat M. Shalev S.A. Feuk L. Elpeleg O. Deleterious mutation in FDX1L gene is associated with a novel mitochondrial muscle myopathy.Eur. J. Hum. Genet. 2014; 22: 902-906Crossref PubMed Scopus (57) Google Scholar we showed here that mutations in the gene encoding its reductase (FDXR) result in sensorial neuropathies. Those points highlight the wide clinical heterogeneity of mitochondrial disorders even when the disease-causing mutations arise in a same pathway. Ferredoxin reductase is also able to reduce ferredoxin-1 (FDX1), which is not involved in ISC assembly but rather in steroid metabolism. Nevertheless, none of the subjects with FDXR mutations show any defect in steroid metabolism, suggesting that these specific mutations do not modify FDX1 function or steroid metabolism. Why FDXR mutations result in sensorial neuropathies is intriguing, and further studies will be needed to help us understand it. Nevertheless, several examples of isolated deafness or optic atrophy have been described in mitochondrial disorders and result from mitochondrial DNA mutations (m.1555A>G)25Ruiz-Pesini E. Lott M.T. Procaccio V. Poole J.C. Brandon M.C. Mishmar D. Yi C. Kreuziger J. Baldi P. Wallace D.C. An enhanced MITOMAP with a global mtDNA mutational phylogeny.Nucleic Acids Res. 2007; 35: D823-D828Crossref PubMed Scopus (468) Google Scholar or nuclear gene mutations, such as OPA121Delettre C. Lenaers G. Griffoin J.M. Gigarel N. Lorenzo C. Belenguer P. Pelloquin L. Grosgeorge J. Turc-Carel C. Perret E. et al.Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.Nat. Genet. 2000; 26: 207-210Crossref PubMed Scopus (1155) Google Scholar and PNPT1.26von Ameln S. Wang G. Boulouiz R. Rutherford M.A. Smith G.M. Li Y. Pogoda H.M. Nürnberg G. Stiller B. Volk A.E. et al.A mutation in PNPT1, encoding mitochondrial-RNA-import protein PNPase, causes hereditary hearing loss.Am. J. Hum. Genet. 2012; 91: 919-927Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Indirect evidence of mitochondrial iron overload in arh1Δ yeast cells expressing human mutant proteins is provided by increased SOD2 levels and defective mitochondrial aconitase activity. Moreover, we detected a major iron overload and increased mitochondrial ROS production in fibroblasts of affected individuals, which is in accordance with previous studies performed in human FDXR-knockdown cells18Shi Y. Ghosh M. Kovtunovych G. Crooks D.R. Rouault T.A. Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis.Biochim. Biophys. Acta. 2012; 1823: 484-492Crossref PubMed Scopus (120) Google Scholar as well as FDX2-knockdown cells.27Sheftel A.D. Stehling O. Pierik A.J. Elsässer H.P. Mühlenhoff U. Webert H. Hobler A. Hannemann F. Bernhardt R. Lill R. Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 11775-11780Crossref PubMed Scopus (214) Google Scholar We also observed increased TfR1 amounts that could be ascribed to the increased binding of IRP1 to the iron-responsive element; this increased binding activity was induced by an ISC defect, as previously demonstrated.18Shi Y. Ghosh M. Kovtunovych G. Crooks D.R. Rouault T.A. Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis.Biochim. Biophys. Acta. 2012; 1823: 484-492Crossref PubMed Scopus (120) Google Scholar, 27Sheftel A.D. Stehling O. Pierik A.J. Elsässer H.P. Mühlenhoff U. Webert H. Hobler A. Hannemann F. Bernhardt R. Lill R. Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 11775-11780Crossref PubMed Scopus (214) Google Scholar Finally, we detected a considerable amount of iron in FDXR mutant cells when we grew these in high-iron conditions, suggesting that these cells are unable to regulate iron entry. Nevertheless, ferritin is not repressed in FDXR mutant fibroblasts, suggesting that an additional defect of iron homeostasis could occur in cultured skin fibroblasts. In conclusion, we have shown that biallelic mutations in FDXR lead to sensorial neuropathies, confirming the critical role of the Fe-S biogenesis in the function of optic and auditory neurons. We acknowledge the parents, the patients, and the physicians for their participation; Institut IMAGINE and the association S'entendre for their support; and Dr. Patrizia Amati Bonneau for OPA1 and TMEM126A studies. This work was partially funded through the E-Rare project GENOMIT (01GM1207) and the Agence Nationale de la Recherche (ANR-10-IAHU-01). A.D. was supported by a fellowship from ApoPharma. Download .pdf (1.78 MB) Help with pdf files Document S1. Supplemental Note and Figures S1–S3 UCSC genome browser, https://genome.ucsc.edu/ExAC Browser, http://exac.broadinstitute.org/Online Mendelian Inheritance in Man (OMIM), https://www.ncbi.nlm.nih.gov/omimEnsembl database, http://www.ensembl.org/index.htmlSaccharomyces genome database, http://www.yeastgenome.org/Basic local alignment search tool (Blast), https://blast.ncbi.nlm.nih.gov/Blast.cgiExpression atlas, http://www.ebi.ac.uk/gxa/
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