FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2740414557> ?p ?o ?g. }

• W2740414557 endingPage "290" @default.
• W2740414557 startingPage "283" @default.
• W2740414557 abstract "Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals’ fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2. Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals’ fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2. Lipoic acid (LA) is an essential cofactor of major mitochondrial enzyme complexes (Figure S1), including the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases, namely pyruvate dehydrogenase (PDHc; pyruvate oxidation), α-oxoglutarate dehydrogenase (α-KGDHc; Krebs cycle), branched chain α-oxoacid dehydrogenase (BCKDHc; leucine, isoleucine, and valine catabolism), and 2-oxoadipate dehydrogenase (2-OADH, lysine catabolism). This cofactor is covalently bound to a conserved lysine residue of the E2 subunits of PDHc, BCKDHc, 2-OADH, and α-KGDHc as well as the H protein of GCS. In addition, lipoylation of the E3-binding protein (E3BP) of PDHc has been observed. Based on studies in yeast, the LA biosynthesis pathway involves mitochondrial fatty acid synthesis up to eight carbon length (octanoyl moiety bound to an acyl carrier protein, ACP) and three lipoate-specific sequential enzymes, LIPT2, LIAS, and LIPT1.1Fujiwara K. Okamura-Ikeda K. Motokawa Y. Purification and characterization of lipoyl-AMP:N epsilon-lysine lipoyltransferase from bovine liver mitochondria.J. Biol. Chem. 1994; 269: 16605-16609PubMed Google Scholar, 2Fujiwara K. Suzuki M. Okumachi Y. Okamura-Ikeda K. Fujiwara T. Takahashi E. Motokawa Y. Molecular cloning, structural characterization and chromosomal localization of human lipoyltransferase gene.Eur. J. Biochem. 1999; 260: 761-767Crossref PubMed Scopus (34) Google Scholar, 3Booker S.J. Unraveling the pathway of lipoic acid biosynthesis.Chem. Biol. 2004; 11: 10-12Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Schonauer M.S. Kastaniotis A.J. Kursu V.A. Hiltunen J.K. Dieckmann C.L. Lipoic acid synthesis and attachment in yeast mitochondria.J. Biol. Chem. 2009; 284: 23234-23242Crossref PubMed Scopus (100) Google Scholar, 5Hiltunen J.K. Autio K.J. Schonauer M.S. Kursu V.A. Dieckmann C.L. Kastaniotis A.J. Mitochondrial fatty acid synthesis and respiration.Biochim. Biophys. Acta. 2010; 1797: 1195-1202Crossref PubMed Scopus (101) Google Scholar In brief, LIPT2 transfers an octanoyl moiety from octanoyl-ACP on the GCS H protein, LIAS adds two sulfur atoms to produce a lipoyl residue, and LIPT1 transfers lipoyl residues from the GCS H protein to the E2 subunits of PDHc, BCKDHc, 2-OADH, and α-KGDHc (Figure S1). We and others have recently shown that mutations involving LIAS (MIM: 614462) and LIPT1 (MIM: 616299) lead to severe clinical conditions.6Mayr J.A. Zimmermann F.A. Fauth C. Bergheim C. Meierhofer D. Radmayr D. Zschocke J. Koch J. Sperl W. Lipoic acid synthetase deficiency causes neonatal-onset epilepsy, defective mitochondrial energy metabolism, and glycine elevation.Am. J. Hum. Genet. 2011; 89: 792-797Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar, 8Tort F. Ferrer-Cortès X. Thió M. Navarro-Sastre A. Matalonga L. Quintana E. Bujan N. Arias A. García-Villoria J. Acquaviva C. et al.Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes.Hum. Mol. Genet. 2014; 23: 1907-1915Crossref PubMed Scopus (42) Google Scholar Because LIAS is an iron-sulfur cluster (ISC) protein, lipoylation deficiency is a prominent feature of defective iron-sulfur [4Fe-4S] cluster cofactor biosynthesis (e.g., NFU1 [MIM: 605711], BOLA3 [MIM: 614299], IBA57 [MIM: 615330], LYRM4 [MIM: 615595]9Navarro-Sastre A. Tort F. Stehling O. Uzarska M.A. Arranz J.A. Del Toro M. Labayru M.T. Landa J. Font A. Garcia-Villoria J. et al.A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins.Am. J. Hum. Genet. 2011; 89: 656-667Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 10Haack T.B. Rolinski B. Haberberger B. Zimmermann F. Schum J. Strecker V. Graf E. Athing U. Hoppen T. Wittig I. et al.Homozygous missense mutation in BOLA3 causes multiple mitochondrial dysfunctions syndrome in two siblings.J. Inherit. Metab. Dis. 2013; 36: 55-62Crossref PubMed Scopus (74) Google Scholar, 11Ajit Bolar N. Vanlander A.V. Wilbrecht C. Van der Aa N. Smet J. De Paepe B. Vandeweyer G. Kooy F. Eyskens F. De Latter E. et al.Mutation of the iron-sulfur cluster assembly gene IBA57 causes severe myopathy and encephalopathy.Hum. Mol. Genet. 2013; 22: 2590-2602Crossref PubMed Scopus (90) Google Scholar, 12Lim S.C. Friemel M. Marum J.E. Tucker E.J. Bruno D.L. Riley L.G. Christodoulou J. Kirk E.P. Boneh A. DeGennaro C.M. et al.Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes.Hum. Mol. Genet. 2013; 22: 4460-4473Crossref PubMed Scopus (84) Google Scholar, 13Nizon M. Boutron A. Boddaert N. Slama A. Delpech H. Sardet C. Brassier A. Habarou F. Delahodde A. Correia I. et al.Leukoencephalopathy with cysts and hyperglycinemia may result from NFU1 deficiency.Mitochondrion. 2014; 15: 59-64Crossref PubMed Scopus (41) Google Scholar, 14Ferrer-Cortès X. Narbona J. Bujan N. Matalonga L. Del Toro M. Arranz J.A. Riudor E. Garcia-Cazorla A. Jou C. O’Callaghan M. et al.A leaky splicing mutation in NFU1 is associated with a particular biochemical phenotype. Consequences for the diagnosis.Mitochondrion. 2016; 26: 72-80Crossref PubMed Scopus (17) Google Scholar, 15Tonduti D. Dorboz I. Imbard A. Slama A. Boutron A. Pichard S. Elmaleh M. Vallée L. Benoist J.F. Ogier H. Boespflug-Tanguy O. New spastic paraplegia phenotype associated to mutation of NFU1.Orphanet J. Rare Dis. 2015; 10: 13Crossref PubMed Scopus (25) Google Scholar). ISC cofactors also participate in electron transfer reactions and are required for respiratory chain complexes I, II, and III,16Rouault T.A. Tong W.H. Iron-sulfur cluster biogenesis and human disease.Trends Genet. 2008; 24: 398-407Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 17Booker S.J. Cicchillo R.M. Grove T.L. Self-sacrifice in radical S-adenosylmethionine proteins.Curr. Opin. Chem. Biol. 2007; 11: 543-552Crossref PubMed Scopus (94) Google Scholar thereby accounting for combined lipoic acid and OXPHOS deficiency.11Ajit Bolar N. Vanlander A.V. Wilbrecht C. Van der Aa N. Smet J. De Paepe B. Vandeweyer G. Kooy F. Eyskens F. De Latter E. et al.Mutation of the iron-sulfur cluster assembly gene IBA57 causes severe myopathy and encephalopathy.Hum. Mol. Genet. 2013; 22: 2590-2602Crossref PubMed Scopus (90) Google Scholar, 18Cameron J.M. Janer A. Levandovskiy V. Mackay N. Rouault T.A. Tong W.H. Ogilvie I. Shoubridge E.A. Robinson B.H. Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes.Am. J. Hum. Genet. 2011; 89: 486-495Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Furthermore, defects in mitochondrial transport of S-adenosylmethionine (SAM), mediated by SLC25A26, result in defective LA synthesis, since SAM is a substrate for the lipoic acid synthetase (MIM: 616794).19Kishita Y. Pajak A. Bolar N.A. Marobbio C.M. Maffezzini C. Miniero D.V. Monné M. Kohda M. Stranneheim H. Murayama K. et al.Intra-mitochondrial methylation deficiency due to mutations in SLC25A26.Am. J. Hum. Genet. 2015; 97: 761-768Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Here we report on three individuals from two unrelated families (Figure S2) with a lipoate-related disease involving α-oxoacid dehydrogenase dysfunction due to mutations in LIPT2, encoding the enzyme that catalyzes the first dedicated step of the LA biosynthesis pathway. This study was approved by the local ethics committees. Informed consent was obtained from the parents. Individuals presented with severe neonatal-onset encephalopathy and abnormal EEG summarized in the Supplemental Note (see also Figure S3 and Table S1). Both clinical and radiological findings were reminiscent of LIPT1 deficiency,7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar, 8Tort F. Ferrer-Cortès X. Thió M. Navarro-Sastre A. Matalonga L. Quintana E. Bujan N. Arias A. García-Villoria J. Acquaviva C. et al.Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes.Hum. Mol. Genet. 2014; 23: 1907-1915Crossref PubMed Scopus (42) Google Scholar whereas first-line biochemical findings were abnormal yet little specific. Individual P1 is alive with no episodes of metabolic decompensation. Brain MRI showed marked supra-tentorial cortical atrophy with ventricular dilatation, bi-frontal white matter abnormalities, and delayed myelination (Figure 1A, middle), similar to those observed in a previously described LIPT1-deficient individual7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar (Figure 1A, top). MRS spectroscopy with a long TE (144) showed a lactate peak (Figure 1B). Laboratory testing showed hyperlactatemia with a high lactate/pyruvate ratio at 16 months of age, but CSF lactate was normal as well as bicarbonates and urinary organic acid analysis except for a slight increase in lactate (Table S2). Moderate biochemical abnormalities in plasma amino acids included moderate hyperglycinemia contrary to LIPT1 deficiencies previously described,7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar, 8Tort F. Ferrer-Cortès X. Thió M. Navarro-Sastre A. Matalonga L. Quintana E. Bujan N. Arias A. García-Villoria J. Acquaviva C. et al.Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes.Hum. Mol. Genet. 2014; 23: 1907-1915Crossref PubMed Scopus (42) Google Scholar increased alanine and decreased branched-chain amino acids (Table S2). At that time, these results were not suggestive of combined α-oxoacid dehydrogenase deficiency or E3 deficiency (MIM: 246900) but rather suggested denutrition. During follow-up, the lactate levels normalized (age 5 years). As in LIPT1 deficiency,7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar, 8Tort F. Ferrer-Cortès X. Thió M. Navarro-Sastre A. Matalonga L. Quintana E. Bujan N. Arias A. García-Villoria J. Acquaviva C. et al.Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes.Hum. Mol. Genet. 2014; 23: 1907-1915Crossref PubMed Scopus (42) Google Scholar no elevations were noted for α-hydroxy or α-oxoadipic acids, thus contrasting with the large increases and sometimes massive amounts observed in typical forms of NFU1 deficiency.9Navarro-Sastre A. Tort F. Stehling O. Uzarska M.A. Arranz J.A. Del Toro M. Labayru M.T. Landa J. Font A. Garcia-Villoria J. et al.A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins.Am. J. Hum. Genet. 2011; 89: 656-667Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar Mitochondrial respiratory chain activities in skeletal muscle and liver and E3 subunit activity measured in fibroblast homogenates were normal (data not shown).20Rustin P. Chrétien D. Bourgeron T. Gérard B. Rötig A. Saudubray J.M. Munnich A. Biochemical and molecular investigations in respiratory chain deficiencies.Clin. Chim. Acta. 1994; 228: 35-51Crossref PubMed Scopus (1092) Google Scholar Using blood DNA, Sanger sequencing did not identify pathogenic gene variations in PDHA1 (MIM: 312170), PDHB (MIM: 614111), PDHX (MIM: 245349), DLAT (MIM: 245348), DLD,21Imbard A. Boutron A. Vequaud C. Zater M. de Lonlay P. de Baulny H.O. Barnerias C. Miné M. Marsac C. Saudubray J.M. Brivet M. Molecular characterization of 82 patients with pyruvate dehydrogenase complex deficiency. Structural implications of novel amino acid substitutions in E1 protein.Mol. Genet. Metab. 2011; 104: 507-516Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar LIAS, BOLA3, NFU1, and LIPT1. Subsequently, exome sequencing was performed as previously described7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar and resulted in a list of 35 candidate genes, including only one encoding a mitochondrial protein, LIPT2. In P1, the two heterozygous c.89T>C transition and c.377T>G transversion were found in LIPT2 (GenBank: NM_001144869.2; see below). To ensure that LIPT2 mutations led to a decrease in lipoic-acid-dependent enzymatic activities, we measured oxygen consumption using pyruvate as a substrate and PDHc and α-KGDHc activities in fibroblasts.22Brivet M. Garcia-Cazorla A. Lyonnet S. Dumez Y. Nassogne M.C. Slama A. Boutron A. Touati G. Legrand A. Saudubray J.M. Impaired mitochondrial pyruvate importation in a patient and a fetus at risk.Mol. Genet. Metab. 2003; 78: 186-192Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Both metabolic investigations were severely reduced (Tables S3 and S4) as in LIPT1-deficient individual.7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar In P1 fibroblasts, consistent with a defect in both the Krebs’ cycle and PDHc activity, 1 mmol/L 13C5-labeled glutamine loading test revealed decreased fumarate versus normal proline labeling (Figure 2A), and 1 mmol/L 13C6-labeled leucine loading test (a modification of a previously described radioactive test24Yoshida I. Sweetman L. Nyhan W.L. Metabolism of branched-chain amino acids in fibroblasts from patients with maple syrup urine disease and other abnormalities of branched-chain ketoacid dehydrogenase activity.Pediatr. Res. 1986; 20: 169-174Crossref PubMed Scopus (11) Google Scholar) revealed decreased BCKDHc activity, but to a lesser extent than in LIPT1-deficient individual (Figure 2A). Lipoic acid administration was orally tested over 3 months at a daily dosage of 25 mg/kg/day in two doses. No clinical modification was observed by the parents, and the neurological examination remained unchanged.Figure 2Labeled Glutamine and Leucine Loading Tests and Oxygen Consumption Rates in Fibroblasts from Control Subjects and Individuals with LIPT1 and LIPT2 DeficienciesShow full caption(A) Labeled to natural ratios for 3-hydroxyisovaleric acid after a 13C6 leucine loading test in fibroblasts of an individual with LIPT1 deficiency and LIPT2-P1 and for fumaric acid and proline after a 13C5 glutamine loading test in LIPT2-P1 and control fibroblasts. Labeled leucine and glutamine loading tests are consistent with decreased BCKDHc activity and Krebs cycle activity defects. Labeled amino acids were acquired from Eurisotop. Organic acids derived from labeled substrates were measured by gas chromatography-mass spectrometry (GC-436 Scion-TQD, Brüker Daltonics). Results are presented as means ± SD of triplicates.(B) Oxygen consumption rates measured in fibroblasts from healthy subjects and individuals with LIPT1 and LIPT2 deficiencies. Basal OCR levels did not differ significantly in fibroblasts from healthy control subjects and individuals with LIPT17Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar and LIPT2 deficiencies. By contrast, when challenged with a mitochondrial uncoupler (FCCP), fibroblasts from healthy individuals responded with the expected increase in oxygen consumption, whereas the response in cells from individuals with LIPT1 and LIPT2 deficiency was significantly lower. OCR was measured using the XF Cell Mito Stress Test Kit and XFe96 analyzer (Seahorse Bioscience, Agilent Technologies) following the manufacturer’s protocols.23Vergnes L. Chin R. Young S.G. Reue K. Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance.J. Biol. Chem. 2011; 286: 380-390Crossref PubMed Scopus (67) Google Scholar Cells were seeded at the density 30,000 cells/well in an XFe96cell culture microplate and allowed to attach for 3 hr before the measurement. Basal OCR was measured, followed by sequential treatment with Oligomycin A (1 μM), FCCP (1 μM), and Antimycin A (1 μM). Each treatment was measured every 3 min (3 min measurement) three times and a minimum of six replicates were utilized per condition. All compounds and materials were obtained from Seahorse Bioscience. Protein concentrations in each well were determined with the BCA method (Pierce) in cell lysates after the measurement. Data are presented as mean ± SEM normalized to protein content in each well. Statistical test was performed using ANOVA test; ∗p ≤ 0.05 control versus affected individuals’ cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Labeled to natural ratios for 3-hydroxyisovaleric acid after a 13C6 leucine loading test in fibroblasts of an individual with LIPT1 deficiency and LIPT2-P1 and for fumaric acid and proline after a 13C5 glutamine loading test in LIPT2-P1 and control fibroblasts. Labeled leucine and glutamine loading tests are consistent with decreased BCKDHc activity and Krebs cycle activity defects. Labeled amino acids were acquired from Eurisotop. Organic acids derived from labeled substrates were measured by gas chromatography-mass spectrometry (GC-436 Scion-TQD, Brüker Daltonics). Results are presented as means ± SD of triplicates. (B) Oxygen consumption rates measured in fibroblasts from healthy subjects and individuals with LIPT1 and LIPT2 deficiencies. Basal OCR levels did not differ significantly in fibroblasts from healthy control subjects and individuals with LIPT17Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar and LIPT2 deficiencies. By contrast, when challenged with a mitochondrial uncoupler (FCCP), fibroblasts from healthy individuals responded with the expected increase in oxygen consumption, whereas the response in cells from individuals with LIPT1 and LIPT2 deficiency was significantly lower. OCR was measured using the XF Cell Mito Stress Test Kit and XFe96 analyzer (Seahorse Bioscience, Agilent Technologies) following the manufacturer’s protocols.23Vergnes L. Chin R. Young S.G. Reue K. Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance.J. Biol. Chem. 2011; 286: 380-390Crossref PubMed Scopus (67) Google Scholar Cells were seeded at the density 30,000 cells/well in an XFe96cell culture microplate and allowed to attach for 3 hr before the measurement. Basal OCR was measured, followed by sequential treatment with Oligomycin A (1 μM), FCCP (1 μM), and Antimycin A (1 μM). Each treatment was measured every 3 min (3 min measurement) three times and a minimum of six replicates were utilized per condition. All compounds and materials were obtained from Seahorse Bioscience. Protein concentrations in each well were determined with the BCA method (Pierce) in cell lysates after the measurement. Data are presented as mean ± SEM normalized to protein content in each well. Statistical test was performed using ANOVA test; ∗p ≤ 0.05 control versus affected individuals’ cells. Individuals P2 and P3, two siblings whose healthy parents are of German origin, died in the first year of life without any psychomotor acquisition (see Supplemental Note). Lactate concentration of individual P2 was 3.7 mmol/L at birth and increased to values between 8 and 10, in a single measurement up to 111 mmol/L (N < 1.8 mmol/L). Brain MRI showed periventricular cystic changes. A ketogenic diet was started, which resulted in a decrease of lactate concentrations but also led to a weight loss and worsening of his condition. Organic acids in urine were elevated (Table S2). Biochemical investigations in fibroblasts found decreased PDHc activity (Table S3) while respiratory chain enzyme activities were normal (data not shown). Individual P3 presented with severe lactic acidosis up to 17 mmol/L at birth. Brain MRI showed enlarged lateral ventricles and formation of cysts in the cortex and white matter of the whole cerebral structures. Furthermore, gyration of the cerebral hemispheres was decreased (Figure 1A, bottom). Metabolite investigations showed elevated lactate concentrations in blood and cerebrospinal fluid and elevated pyruvate in blood, with a lactate/pyruvate ratio of 30 (N < 20) (Table S2). Amino acid analysis in plasma showed elevation of alanine and proline, moderate increase of glycine, and decrease of branched-chain amino acids (Table S2). Investigation of organic acids in urine revealed elevation of lactate, pyruvate, and 2-oxoglutarate. A muscle biopsy performed at the age of 2 weeks revealed some variability in fiber diameters (7–19 μm) but no ragged-red or cytochrome c oxidase negative fibers. Electron microscopy showed some subsarcolemnal glycogen accumulation but normal structure of mitochondria. The activity of respiratory chain enzymes complexes I, II, III, and IV was normal (data not shown). Investigation of pyruvate dehydrogenase in fibroblasts was decreased (Table S3). In P2, panel diagnostics for gene mutations associated with mitochondrial diseases was performed but did not reveal any abnormal result. Exome sequencing, performed as previously described,25Kremer L.S. Danhauser K. Herebian D. Petkovic Ramadža D. Piekutowska-Abramczuk D. Seibt A. Müller-Felber W. Haack T.B. Płoski R. Lohmeier K. et al.NAXE mutations disrupt the cellular NAD(P)HX repair system and cause a lethal neurometabolic disorder of early childhood.Am. J. Hum. Genet. 2016; 99: 894-902Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 26Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar, 27Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31559) Google Scholar revealed compound heterozygous mutations in LIPT2 c.314T>G (p.Leu105Arg; not reported in gnomAD) and c.377T>G (p.Leu126Arg; frequency reported in gnomAD: 0.0001878), the latter being shared with P1. Sanger sequencing confirmed mutations in P1, P2, and P3 and showed that the parents were heterozygous for one of these mutations. The mutations c.314T>G and c.377T>G were predicted to be “probably damaging” by PolyPhen-2 (Table S5) and were located in a conserved domain of the protein (Figure S2). The c.89T>C variation is a SNP (rs539962457, minor allele frequency [MAF] < 0.0002/1; frequency reported in gnomAD: 0.00003238). It changes a leucine into a proline (p.Leu30Pro) at the second last position of a conserved alpha-helix, which is the predicted mitochondrial targeting sequence of LIPT2 protein and was considered as “possibly damaging” by PolyPhen. These variations were not found in more than 1,000 exome-sequencing projects performed in the Imagine Institute. To determine whether lipoic acid metabolism was impaired in individuals with LIPT2 mutations, we used an antibody that specifically recognizes lipoic acid bound to proteins as previously described7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.Orphanet J. Rare Dis. 2013; 8: 192Crossref PubMed Scopus (41) Google Scholar and anti-porin antibody. Anti-lipoate antibody detected strongly decreased levels of the expected lipoylated E2 subunits of α-oxoacid dehydrogenases in fibroblasts of individuals P1 and P2, whereas normal bands were seen in the control (Figure 3A). In fibroblasts of an individual with LIPT1 deficiency,7Soreze Y. Boutron A. Habarou F. Barnerias C. Nonnenmacher L. Delpech H. Mamoune A. Chrétien D. Hubert L. Bole-Feysot C. et al.Mutations in human lipoyltransferase gene" @default.
• W2740414557 created "2017-08-08" @default.
• W2740414557 creator A5003048807 @default.
• W2740414557 creator A5006648785 @default.
• W2740414557 creator A5009947949 @default.
• W2740414557 creator A5011228927 @default.
• W2740414557 creator A5012173616 @default.
• W2740414557 creator A5017552542 @default.
• W2740414557 creator A5019467915 @default.
• W2740414557 creator A5020430798 @default.
• W2740414557 creator A5022734186 @default.
• W2740414557 creator A5023844947 @default.
• W2740414557 creator A5028326580 @default.
• W2740414557 creator A5033772913 @default.
• W2740414557 creator A5034526107 @default.
• W2740414557 creator A5034813741 @default.
• W2740414557 creator A5035939606 @default.
• W2740414557 creator A5038805113 @default.
• W2740414557 creator A5044517259 @default.
• W2740414557 creator A5045218892 @default.
• W2740414557 creator A5047685808 @default.
• W2740414557 creator A5049343245 @default.
• W2740414557 creator A5050007993 @default.
• W2740414557 creator A5052438294 @default.
• W2740414557 creator A5054481926 @default.
• W2740414557 creator A5054486997 @default.
• W2740414557 creator A5056001305 @default.
• W2740414557 creator A5065678737 @default.
• W2740414557 creator A5070618342 @default.
• W2740414557 creator A5070875825 @default.
• W2740414557 creator A5071796847 @default.
• W2740414557 creator A5072329970 @default.
• W2740414557 creator A5081327741 @default.
• W2740414557 creator A5088052212 @default.
• W2740414557 date "2017-08-01" @default.
• W2740414557 modified "2023-10-18" @default.
• W2740414557 title "Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy" @default.
• W2740414557 cites W1527284807 @default.
• W2740414557 cites W1556287700 @default.
• W2740414557 cites W1607569982 @default.
• W2740414557 cites W1967185435 @default.
• W2740414557 cites W1991290396 @default.
• W2740414557 cites W1993538003 @default.
• W2740414557 cites W1995630908 @default.
• W2740414557 cites W2001986473 @default.
• W2740414557 cites W2005870809 @default.
• W2740414557 cites W2015761412 @default.
• W2740414557 cites W2019042812 @default.
• W2740414557 cites W2029446405 @default.
• W2740414557 cites W2029595673 @default.
• W2740414557 cites W2031212222 @default.
• W2740414557 cites W2042687493 @default.
• W2740414557 cites W2048522862 @default.
• W2740414557 cites W2062700139 @default.
• W2740414557 cites W2074576093 @default.
• W2740414557 cites W2075253085 @default.
• W2740414557 cites W2091614510 @default.
• W2740414557 cites W2092152103 @default.
• W2740414557 cites W2103441770 @default.
• W2740414557 cites W2108234281 @default.
• W2740414557 cites W2113439382 @default.
• W2740414557 cites W2119506420 @default.
• W2740414557 cites W2120339601 @default.
• W2740414557 cites W2124733101 @default.
• W2740414557 cites W2130262043 @default.
• W2740414557 cites W2145845104 @default.
• W2740414557 cites W2163132490 @default.
• W2740414557 cites W2170465531 @default.
• W2740414557 cites W2186620924 @default.
• W2740414557 cites W2190635170 @default.
• W2740414557 cites W2520649374 @default.
• W2740414557 doi "https://doi.org/10.1016/j.ajhg.2017.07.001" @default.
• W2740414557 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5544388" @default.
• W2740414557 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28757203" @default.
• W2740414557 hasPublicationYear "2017" @default.
• W2740414557 type Work @default.
• W2740414557 sameAs 2740414557 @default.
• W2740414557 citedByCount "49" @default.
• W2740414557 countsByYear W27404145572017 @default.
• W2740414557 countsByYear W27404145572018 @default.
• W2740414557 countsByYear W27404145572019 @default.
• W2740414557 countsByYear W27404145572020 @default.
• W2740414557 countsByYear W27404145572021 @default.
• W2740414557 countsByYear W27404145572022 @default.
• W2740414557 countsByYear W27404145572023 @default.
• W2740414557 crossrefType "journal-article" @default.
• W2740414557 hasAuthorship W2740414557A5003048807 @default.
• W2740414557 hasAuthorship W2740414557A5006648785 @default.
• W2740414557 hasAuthorship W2740414557A5009947949 @default.
• W2740414557 hasAuthorship W2740414557A5011228927 @default.
• W2740414557 hasAuthorship W2740414557A5012173616 @default.
• W2740414557 hasAuthorship W2740414557A5017552542 @default.
• W2740414557 hasAuthorship W2740414557A5019467915 @default.
• W2740414557 hasAuthorship W2740414557A5020430798 @default.
• W2740414557 hasAuthorship W2740414557A5022734186 @default.
• W2740414557 hasAuthorship W2740414557A5023844947 @default.
• W2740414557 hasAuthorship W2740414557A5028326580 @default.
• W2740414557 hasAuthorship W2740414557A5033772913 @default.

• next