Mitochondrion 15 (2014) 59–64
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Leukoencephalopathy with cysts and hyperglycinemia may result from NFU1 deﬁciency Mathilde Nizon a, Audrey Boutron b, Nathalie Boddaert c, Abdelhamid Slama b, Hélène Delpech d, Claude Sardet d, Anaïs Brassier a, Florence Habarou a,e, Agnès Delahodde f, Isabelle Correia b, Chris Ottolenghi a,e, Pascale de Lonlay a,⁎ a
Reference Center of Inherited Metabolic Diseases, University Paris Descartes, Hospital Necker Enfants Malades, APHP, Paris, France Department of Biochemistry, Hospital Bicêtre, Le Kremlin Bicêtre, France c Department of Pediatric Radiology, University Paris Descartes, Hospital Necker Enfants Malades, Paris, France d Department of Molecular Genetics, CNRS UMR 5535, Montpellier, France e Department of Biochemistry, University Paris Descartes, Hospital Necker Enfants Malades, Paris, France f Paris-Sud University, CNRS-UMR8621, Genetics and Microbiology Institute, Orsay, France b
a r t i c l e
i n f o
Article history: Received 20 May 2013 Received in revised form 29 December 2013 Accepted 15 January 2014 Available online 22 January 2014 Keywords: Leucoencephalopathy with cysts Pyruvate dehydrogenase deﬁciency Hyperglycinemia NFU1 Lipoic acid
a b s t r a c t Lipoic acid metabolism defects are new metabolic disorders that cause neurological, cardiomuscular or pulmonary impairment. We report on a patient that presented with progressive neurological regression suggestive of an energetic disease, involving leukoencephalopathy with cysts. Elevated levels of glycine in plasma, urine and CSF associated with intermittent increases of lactate were consistent with a defect in lipoic acid metabolism. Support for the diagnosis was provided by pyruvate dehydrogenase deﬁciency and multiple mitochondrial respiratory chain deﬁciency in skin ﬁbroblasts, as well as no lipoylated protein by western blot. Two mutations in the NFU1 gene conﬁrmed the diagnosis. The p.Gly208Cys mutation has previously been reported suggesting a founder effect in Europe. © 2014 Elsevier B.V. and Mitochondria Research Society.
1. Introduction Lipoic acid is a sulfur-containing cofactor covalently attached to and essential for the function of several key enzymatic complexes such as pyruvate dehydrogenase complex (PDHc), α-ketoglutarate dehydrogenase (α-KGDH), branched chain keto acid dehydrogenase (BCKDH) activity and H protein activity from the glycine cleavage system (GCS) (Cameron et al., 2011; Navarro-Sastre et al., 2011). Lipoic acid is formed in mitochondria by a series of reactions involving the transfer of an octanoyl-ACP derived from fatty acid biosynthesis onto an apoprotein by LIPT2 and the addition of the sulfur component in a reaction catalyzed by lipoic acid synthase (LIAS), an enzyme possessing two [4Fe–4S] clusters (Hiltunen et al., 2010). The [4Fe–4S] cluster, which is a cofactor of LIAS as well as other mitochondrial proteins (see below), is assembled by a complex metabolic pathway involving proteins such as NFU1 (NFU Iron–Sulfur cluster scaffold homolog (S. cerevisiae), ISCU (Iron–Sulfur cluster scaffold homolog), BOLA3 (bolA family member 3 (Escherichia coli)) and IBA57 (IBA57, iron-sulfur ⁎ Corresponding author at: Reference Center of Metabolic Disease Unit, Université Paris Descartes, Hôpital Necker-Enfants Malades, INSERM-U781, 149 rue de Sèvres, 75015, Paris, France. Tel.: +33 1 44 49 48 52; fax: +33 1 44 49 48 50. E-mail address: [email protected]
(P. de Lonlay). 1567-7249/$ – see front matter © 2014 Elsevier B.V. and Mitochondria Research Society. http://dx.doi.org/10.1016/j.mito.2014.01.003
cluster assembly homolog (Saccharomyces. cerevisiae)) (Ajit Bolar et al., 2013; Cameron et al., 2011). In this pathway NFU1 probably acts as a scaffold downstream of ISCU during Fe–S cluster biogenesis (Navarro-Sastre et al., 2011), whereas BOLA3 is related to proteins that bind glutaredoxins, which play an unknown role in Fe–S cluster biogenesis. Mutations in either NFU1 or BOLA3 disrupt the function of LIAS which adds lipoate moiety to key subunits E2 of PDHc and α-KGDH, and to the related enzymes involved in branched chain amino acid metabolism and glycine degradation. IBA57 is part of the iron–sulfur cluster assembly machinery (Ajit Bolar et al., 2013). Defects in iron–sulfur cluster biosynthesis pathway ([Fe–S] clusters) lead to abnormal function of the enzyme-bound cofactor lipoate but also of many proteins involved in intermediary metabolism and oxidative phosphorylation, as they participate in electron transfer reactions and in complex I, II and III functions (Rouault and Tong, 2008). This explains the multiple mitochondrial dysfunction syndrome associated with NFU1, BOLA3, LIAS, ISCU and IBA57 mutations (Ajit Bolar et al., 2013; Cameron et al., 2011; Haack et al., 2013; Kollberg et al., 2009; Navarro-Sastre et al., 2011). Up to now, two reports describe patients with NFU1 mutations, all presenting with infantile encephalopathy and neurological regression leading to death before the age of 15 months. Most patients had
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pulmonary hypertension, and hyperglycinemia and lactic acidosis were common ﬁndings. Interestingly, one mutation, p.Gly208Cys, was frequent in European patients. Some patients apparently presented with leukoencephalopathy but no brain MRI were available (Navarro-Sastre et al., 2011). Here, we report on a new patient of French origin with two NFU1 mutations, one shared with the Spanish patients and the other one not reported as yet. The ﬁndings at presentation included neurological regression, leukoencephalopathy with cysts and hyperglycinemia. The association of neurological regression mimicking an energetic disease, even with intermittent hyperlactatemia in the basal state, along with hyperglycinemia suggested the diagnosis. Administration of oral lipoic acid was without effect. 2. Materials and methods 2.1. Patient The patient, a girl, was the ﬁrst child of non-consanguineous French parents. She was born after an uneventful pregnancy and spontaneous delivery at term with normal birth parameters. Up to 14 months old, psychomotor development was normal. She could support her head at age one month, sit unaided at age 7 months and had an excellent follow with eyes. The course of the disease was marked by two episodes of metabolic acidosis at 5 and 18 months of age. The ﬁrst episode occurred in a context of vomiting and dehydration and was associated with mild hyperlactatemia (2.7 mmol/L), liver cytolysis and reversible pulmonary hypertension under symptomatic treatment. The second episode occurred in a context of motor regression without fever, with transient motor deﬁciency of the right arm, hyperlactatemia (6.9 mmol/L), hypoglycemia (2.6 mmol/L) and acute painful syndrome. Starting from 14 months old, the patient progressively lost standing and sitting abilities, and failure to thrive with poor feeding was noticed. Neurological examination revealed axial hypotonia, severe spastic tetraparesis and extrapyramidal syndrome with numerous opisthotonos episodes daily. A new episode of motor deﬁciency of the right arm with right facial paralysis occurred at 18 months old, requiring hospitalization in our unit. Progressive aggravation led to artiﬁcial feeding. At this time, electroencephalogram (EEG) was pathologic without any physiological rhythms but no burst suppression pattern. At 2 1/2 years, she had abnormal movements and erratic myoclonies. Focal seizures were observed and the EEG showed frontal and temporal spikes and focal discharges suggesting secondary lesional epilepsy. 2.2. Metabolic investigation The ﬁrst metabolic workup was performed when the patient was referred to our unit at age 17 months. Lactate and pyruvate levels were determined in plasma and CSF by enzymatic methods. Plasma, urinary and CSF amino acids were assayed by nihydrin colorimetry (Jeol Aminotac Analyzers) and urinary organic acids by gas chromatography– mass spectrometry (300MS triple quadrupole, Brüker). 2.3. Enzymatic analysis and respiratory chain investigation Pyruvate dehydrogenase activity was measured by the release of CO2 from 0.2 mM [1-14C]pyruvate, using suspensions of leukocytes or DCA-activated ﬁbroblasts disrupted by sonication (Sperl et al., 1993). Polarographic and spectrophotometric assay of mitochondrial respiratory chain complex activities were measured in leukocytes and skin ﬁbroblasts according to standard procedures (Rustin et al., 1994). 14
2.4. Molecular analysis DNA was extracted from white blood cells, collected from the patient and her parents after informed consent. The LIAS (GenBank
NG_032111.1), BOLA3 (GenBank NG_031910) and NFU1 (GenBank NG_031931.1) genes were sequenced using intronic primers (purchased from Applied Biosystems, Forster City, CA). 2.5. Immunoblot analysis in NFU1 and PDHA1 mutant ﬁbroblasts Total protein extracts were prepared by lysing cultured ﬁbroblasts generated from either control individual or from patients with either NFU1 mutation, or true PDH E1-α defect. Cell pellets were lysed in Triton X-100 lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM β-glycerophosphate, 1 mM Na orthovanadate, 10− 4 M PMSF, 10− 6 M leupeptin, 10− 6 M pepstatin A and 1% Triton X-100). 40 μg of total protein extracts was separated by SDSpolyacrylamide-gel electrophoresis and transferred to nitrocellulose membranes, blocked in TBS containing 5% nonfat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. The following antibodies were used: anti-Lipoic Acid (Abcam), antiPyruvate Dehydrogenase E1-α subunit (Abcam), anti-DLD (Santa Cruz Biotechnology, Inc.) and anti-α Tubulin (Sigma-Aldrich). Quantitative analyses of immunoblots were performed with the Odyssey infrared image system (LiCor) using DyLight™ 800 conjugated secondary antibodies from LiCor. 2.6. In vivo and in vitro supplementation A trial of lipoic acid (100 mg/kg/day) then ketogenic diet (lipids 60% of calories), was performed during six months and 24 h respectively. This work has been approved by our institutional ethical committee. Fibroblasts from skin biopsies from a control individual and the patient were cultured in monolayer ﬂasks with HamF10 medium containing 12% fetal calf serum and 100 UI/mL penicillin G and 100 μg/mL streptomycine. The ﬂasks were incubated at 37 °C with 5% carbon dioxide. Culture medium was supplemented with lipoic acid (Sigma) to a ﬁnal concentration of 10 (+2 mg) and 100 (+20 mg) μM during three weeks. PDH and MRC activities were analyzed on cultured skin ﬁbroblasts before and after three weeks of supplementation. 3. Results 3.1. Brain MRI Brain magnetic resonance imaging (MRI) revealed progressive leukoencephalopathy with extensive signal abnormalities in the periventricular cerebral white matter and in the corpus callosum (Fig. 1). The abnormal white matter and corpus callosum were partially cystic or with cavitation. Basal ganglia, cerebellum and brain stem were normal. The MRS spectroscopy with long TE (144) shows no peak of lactate. There was no argument for a stroke-like episode. 3.2. Metabolic investigation Biochemical analysis revealed a mild metabolic acidosis (bicarbonates 15 mmol/L, N N 20 mmol/L) but normal level of lactate and pyruvate in blood (lactate 1.5 mmol/L, N b 2.2 mmol/L; pyruvate 0.07 mmol/L, N b 0.20 mmol/L), in urine (lactate 75 μmol/mmol of creatinine, N b 75 μmol/mmol of creatinine) and CSF (lactate 1.8 mmol/L, N b 1.95 mmol/L; pyruvate 0.11 mmol/L, N b 0.20 mmol/L). These parameters remained normal in the follow-up while lactate was reported as elevated during the two episodes of regression at ages 5 and 18 months (2.7 mmol/L and 6.9 mmol/L respectively; pyruvate not available). Glycine was elevated in blood (1178 μmol/L, N b 264 μmol/L), in urine (3395 μmol/mmol of creatinine, N b 356 μmol/mmol of creatinine) and slightly elevated in CSF (20 μmol/L, N b 16 μmol/L). All other amino acids were normal or low-normal in CSF, plasma and urine. An isolated increase of glycine was observed in all subsequent tests (plasma and urine). Urinary organic acid analysis
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Fig. 1. A 2.3-year-old girl with NFU1 mutation and leukoencephalopathy with cysts or cavitations. The MRI reveals extensive signal abnormalities in the periventricular cerebral white matter and in the corpus callosum. Axial T1 (A), axial diffusion (B), axial Flair (D) and axial T2 (E) images show conﬂuent white matter lesions that also affect the corpus callosum. The axial FLAIR images show that the abnormal white matter is also partially cystic (or with cavitation) (D). The corpus callosum is severely involved on sagittal T1 (C) with cysts or cavitations of the genu and splenium of the corpus callosum. After contrast, no enhancement within the lesion is seen. The MRS spectroscopy with long TE (144) shows no peak of lactate (F). The posterior fossa, the internal capsule, the basal ganglia, the cortex and the U ﬁbers are spared.
showed mildly increased excretion of tiglylglycine, glutaric acid, 2-hydroxyglutaric acid, 3-hydroxyglutaric acid and Krebs cycle intermediates, and α-ketoglutaric acid was strongly increased (approx. 1500 μmol/mmol of creatinine, N b 79 μmol/mmol of creatinine). On subsequent tests, all the organic acids were normalized except for multiple acylglycines that increased to various extents (2-methylbutyl, isovaleryl-, tiglyl- and hexanoylglycine showed 2–10 fold ratios above upper normal levels). Methyltetrahydrofolate was slightly reduced in CSF (30 nmol/L, N N 63 nmol/L). Plasma acylcarnitine analysis showed slight elevation of all acylcarnitines (data not shown). 3.3. NFU1 disease Pyruvate dehydrogenase complex activity measured in ﬁbroblast homogenates was found at 25% of the control values (i.e.,
0.36 nmol/min/mg proteins, control 1.2 nmol/min/mg proteins) whereas normal activity was found in leukocytes (Table 1). A generalized mitochondrial complex deﬁciency predominant on complex II was revealed in patient's cultured skin ﬁbroblasts (i.e., CII 4 nmol/min/mg of protein, normal control range from 10.8 to 17 nmol/min/mg of protein). In leukocytes the only anomaly was weak oxygen consumption in the polarographic assay using succinate as substrate (6.8 nmol O2/min/mg of protein, reference range from 9 to 15.2 nmol O2/min/mg of protein). The LIAS, BOLA3 and NFU1 genes were sequenced, leading to the identiﬁcation in NFU1 gene of a previously reported glycine to cysteine substitution at codon 208 (p.Gly208Cys, c.622G N T) and a novel p.Gly189Arg mutation (c.565G NA), both heterozygous and mapping in the iron–sulfur cluster assembly domain encoded by exon 7. Pathogenicity of the p.Gly208Cys mutation has already been demonstrated in
Table 1 Pyruvate dehydrogenase activity and mitochondrial respiratory chain polarographic measurement on patient's and control's leukocytes and skin ﬁbroblasts. Skin ﬁbroblasts were supplemented or not with lipoic acid during three weeks. CII–CIV mitochondrial respiratory chain complexes II–IV; CS: citrate synthase; PDHc: pyruvate dehydrogenase complex; enzyme activities are expressed in nmol/min/mg protein. Enzyme activities
PDHc CS CII CII + III CIII CIV CS a
In leukocytes Reference range
LIPOIC Ac 0.001 mmol/La
LIPOIC Ac 0.010 mmol/L
LIPOIC Ac 0.100 mmol/L
1.82–3.68 62–142 14–32.6 27–76 75–237 85–269 36–85
2.69 136 17 36 100 168 86
1.37–3.10 36–90 10.8–17 21–42 98–180 72–143 32–72
0.36 67 4 6 37 35 35
0.21 51 6 11 111 85 41
0.33 62 6 11.6 124 125 55
Standard concentration in Ham F10 medium.
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yeast models suggesting a role in the delivery of Fe–S clusters (NavarroSastre et al., 2011). The affected glycine residue at codon 189 is conserved amongst NFU1 orthologs. In silico analysis of the missense mutation was achieved using the integrated ALAMUT V.2.0 software (Interactive Biosoftware: http://www.interactive-biosoftware.com). The mutation was predicted to be deleterious. Sequence analysis of NFU1 from 60 control chromosomes did not reveal this mutation. Furthermore this affected residue was not revealed in the exome sequencing project including at least 13 000 alleles [Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (URL: http:// evs.gs.washington.edu/EVS/)]. The parents were heterozygous for one or the other of the two mutations. No mutation was identiﬁed in LIAS and BOLA3 gene. We used an antibody to lipoate to identify the lipoate containing subunits of oxo acid dehydrogenases (PDHc, α-KGDHc and BCKDHc) in ﬁbroblasts. Anti lipoate failed to detect the expected lipoylated proteins of PDHc, α-KGDHc and BCKDHc in NFU1 mutated patient ﬁbroblasts whereas normal bands were seen for PDH E1-α and PDH E3 subunits (Fig. 2). 3.4. Therapeutic trial and in vitro experiments A six-month trial of oral lipoic acid did not prevent neurological regression and appeared to be without any effect. Lipoic acid treatment has been stopped after six months. By contrast a trial of ketogenic diet immediately worsened the patient who presented an acute episode of dystonia and metabolic attack within 24 h following this test. Thus, ketogenic diet was immediately stopped. No modiﬁcation was observed in patient's and control's ﬁbroblasts after lipoic acid supplementation, neither for PDH nor for MRC complex activity (Table 1). 4. Discussion Here, we report on a child with neurological regression, leukoencephalopathy with cysts and hyperglycinemia due to NFU1 mutations. Our report raises the possibility that this disease may be underdiagnosed, as biochemical clues of energetic disease such as hyperlactatemia were only intermittently present. The atypical leukoencephalopathy was associated with elevated levels of glycine,
and the combined features are suggestive of the diagnosis. Finally one of the detected mutations, p.Gly208Cys, in the NFU1 gene, was previously reported in several patients of a large cohort in Spain, suggesting that this mutation is common in Europe possibly due to a founder effect. Despite intermittently increased levels of lactate, the neurological episode of regression associated with hyperglycinemia was consistent with a defect of lipoic acid metabolism. The transient episodes of hyperlactatemia at ages 5 and 18 months reinforced this hypothesis. Pyruvate dehydrogenase and mitochondrial respiratory chain activities were subsequently measured in patient's ﬁbroblasts and leukocytes (with predominant complex II defect) whereas skeletal muscle biopsy was canceled, as the diagnosis had been possible in ﬁbroblasts. Classical nonketotic hyperglycinemia is characterized by hypotonia, myoclonic jerks, seizures and intellectual deﬁciency and results from impaired glycine cleavage system. Classically, seizures are difﬁcult to treat and occur early. The initial electroencephalogram shows burstsuppression pattern that evolves into hypsarrhythmia and/or multifocal spikes. Known leukodystrophies with cavitation such as megalencephalic leukoencephalopathy with subcortical cysts are not associated with blood glycine elevation and the pathophysiological mechanism is very different (van der Knaap et al., 2012). In our patient, nonketotic hyperglycinemia was unusual biochemically as the raise of CSF glycine was very moderate. In particular, the ratio of CSF to plasma glycine (1%) was much lower than the 4% cut off that is recommended for the diagnosis of classical nonketotic hyperglycinemia (Korman et al., 2006). Nevertheless, no other amino acid was above normal levels in CSF, and late onset cases of protein P or T deﬁciency show CSF/plasma ratios b 4% (unpublished data). Acylglycines were presumably increased secondarily to increased glycine. Clinically, our patient presented with partial seizures since the age of 2 1/2 years and after the development of many cerebral cysts. The initial electroencephalogram at 18 months did not show burst suppression pattern. This chronology suggests that seizures were secondary to cerebral lesions rather than hyperglycininemia. Furthermore, αketoglutarate elevation in urine might have resulted from αcetoglutarate dehydrogenase deﬁciency and tiglyglycine excretion in urine from branched chain keto acid dehydrogenase deﬁciency, according to the lipoate synthesis metabolism impairment including complex dehydrogenase E2 subunit and [Fe–S] cluster proteins. Fe–S proteins are required for the function of succinate dehydrogenase and aconitase, two proteins from the tricarboxylic acid cycle and respiratory chain
Fig. 2. A. Immunobloting analyses of ﬁbroblast extracts generated from control individual and from patients with either PDHA1 or NFU1 mutations. Immunoblotted proteins were probed with the indicated antibodies directed against lipoylated E2 proteins (PDH, BCKDH and α-KGDH), the PDH E1-α and DLD subunits (E3 subunit of PDH, BCKDH and α-KGDH), respectively. Immunoblotting with anti-α Tubulin is used to assess loading of total proteins. B. Quantiﬁcation of immunoblots by the Odyssey infrared image system (LiCor) showing Lipoic Acid/α-Tubulin, PDH E1-α subunit/α-Tubulin and DLD/α-Tubulin proteins ratios in these ﬁbroblasts.
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complexes I–III (Rouault, 2012). Finally, the H-protein participates in 5,10-methylenetetrahydrofolate formation, presumably accounting for the mild CSF folate deﬁciency observed in this case (Kikuchi et al., 2008). Some metabolites that were found increased only once and moderately in urine can be rationalized with the mechanisms of the disorder (2-hydroxyglutarate and glutarate) and others may represent nonspeciﬁc features of unclear signiﬁcance (3-hydroxyglutarate). The normalization of most organic acids in the follow-up of our patient suggests a variability in the biochemical results as already described in other Krebs' cycle defects (Brassier et al., 2013). Disruption of Fe–S cluster biogenesis in the different parts of the pathway causes very different disease phenotypes suggesting that the transfer of Fe–S clusters to speciﬁc target proteins downstream of the ISCU complex depends on a dedicated pathway involving speciﬁc recipient proteins (Ye and Rouault, 2010). Indeed, mutations in NFU1 or BOLA3 interfere quite speciﬁcally with activity of a Fe–S enzyme, lipoic acid synthase (LIAS) (Liu et al., 2009). Clinical data suggest an overlap between the different syndromes described for this pathway (Cameron et al., 2011; Navarro-Sastre et al., 2011; Seyda et al., 2001). The three patients reported with BOLA3 mutations presented with epileptic encephalopathy, dilated or hypertrophic cardiomyopathy, muscular hypotonia and elevated blood and CSF glycine levels with an early death at the age of three to eleven months (Cameron et al., 2011; Haack et al., 2013; Seyda et al., 2001). ISCU mutations were associated with Swedish myopathy and exercise intolerance with aconitase and succinate dehydrogenase deﬁciency (Kollberg et al., 2009). In two sibs, homozygous mutation was identiﬁed in the IBA57 gene. The patients died in the neonatal period and had severe hypotonia, respiratory insufﬁciency, brain malformations, increased level of lactate and glycine in blood and CSF associated with multiple respiratory chain defect (Ajit Bolar et al., 2013). Otherwise, a homozygous missense mutation in LIAS has been described in a boy who died at age 4 years, presenting with neonatal-onset epilepsy, muscular hypotonia, spastic tetraparesis, multicystic encephalopathy with hydrocephalus ex vacuo on brain ultrasonography, and mild hypertrophic cardiomyopathy. Biochemical analyses revealed intermittent lactic acidosis and elevated glycine concentration in plasma and urine. Investigations showed decreased pyruvate dehydrogenase complex activity while respiratory chain was normal (Mayr et al., 2011). Both neurological and cardiopulmonary systems are involved in patients with mutations in BOLA3, NFU1 and LIAS genes. Notably 7/13 patients with NFU1 mutations had pulmonary hypertension which occurred though only transiently in our case as well. We also emphasize the tissue speciﬁcity of the NFU1 mutation effect on E2 subunit of PDHc and respiratory chain complex activities, as no alteration was observed in lymphocytes. A decrease in protein lipoylation has already been reported in cultured skin ﬁbroblasts, skeletal muscle and liver but not in brain, kidney and lung from some NFU1 patients (Ferrer-Cortès et al., 2013). Tissue speciﬁc effects were also noticed for the ISCU myopathy (Nordin et al., 2012). It is worth to notice that currently, the child is still alive at age two years and a half whereas the other reported patients died by 15 months. Nevertheless, we have been unable to devise an effective treatment. The ketogenic diet administrated in order to compensate for the pyruvate dehydrogenase deﬁciency worsened the condition of the patient, possibly because of the partial block of the Krebs cycle at the α-cetoglutarate dehydrogenase step. Folate supplementation was administered but also without effect (data not shown). Presumably because eukaryotes are strictly dependent on the de novo synthesis of the lipoyl group within mitochondria and because of dysfunctional iron–sulfur cluster, lipoic acid supplementation did not have any effect, neither clinically nor in vitro on PDH activity, despite a mild effect in vitro on respiratory chain activities as previously described (Arivazhagan et al., 2001). However, consistently with our negative results, lipoic acid added in growth medium of a yeast lip5 lipoic acid synthase mutant did not rescue the respiratory chain deﬁciency (Sulo and Martin, 1993) and lipoic acid administration to the mouse Lias knockout mutant did not rescue the embryonic lethality (Yi and Maeda, 2005).
In conclusion, lipoic acid biosynthesis is at the crossroad of critical metabolic pathways and is associated with clinical and biological features that include progressive leukoencephalopathy with cysts and hyperglycinemia without ketosis. Both radiological and biochemical symptoms suggest the diagnosis of lipoic acid metabolism dysfunction, i.e. NFU1 or LIAS mutations. Screening for the p.Gly208Cys mutation in NFU1 gene could provide a useful diagnostic test in European patients. Further investigations are needed on the underlying pathophysiology, particularly because lipoic acid is not effective as a supportive therapy. Acknowledgments We are grateful to the patient and her family. This work was supported by Association Française contre les Myopathies [grant number 15947], Fondation Bettencourt, Association Noa Luu, Association Nos Anges and Fondation Lejeune [grant 2011 – 2012]. 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