Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes

Human Molecular Genetics, 2014, Vol. 23, No. 7 doi:10.1093/hmg/ddt585 Advance Access published on November 20, 2013 1907–1915 Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes Frederic Tort1,2, Xènia Ferrer-Cortès1,2, Marta Thió3, Aleix Navarro-Sastre1,2, Leslie Matalonga1,2, Ester Quintana1, Núria Bujan1, Angela Arias1,2, Judit Garcı́a-Villoria1,2, Cecile Acquaviva4, Christine Vianey-Saban4, Rafael Artuch2,5, Àngels Garcı́a-Cazorla2,3, Paz Briones1,2,6 and Antonia Ribes1,2,∗ 1 Received September 27, 2013; Revised November 12, 2013; Accepted November 13, 2013 Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient’s fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans. INTRODUCTION Cofactor disorders of mitochondrial energy metabolism constitute a heterogeneous and emerging group of diseases with a wide variety of clinical symptoms associated with particular metabolic profiles and variable enzymatic defects. Although most of the genes involved in mitochondrial cofactor biosynthesis still remain to be elucidated, an increasing number of them have been recently identified and described to be involved in human diseases (1 – 9). Lipoic acid is an essential cofactor necessary for the activity of four mitochondrial enzyme complexes: pyruvate dehydrogenase (PDHC), 2-ketoglutarate dehydrogenase (2-KGDH), branchedchain 2-keto acid dehydrogenase (BCKDH) and the glycine cleavage system (GCS) (10). Lipoic acid is covalently attached ∗ To whom correspondence should be addressed. Tel: +34 932275600; Fax: +34 932275668; Email: # The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: Secció d’Errors Congènits del Metabolisme, Servei de Bioquı́mica i Genètica Molecular, Hospital Clinic, IDIBAPS, C/Mejı́a Lequerica s/n, Barcelona 08028, Spain, 2CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain, 3Servei de Neurologı́a, Hospital Sant Joan de Déu, Carretera d’Esplugues, Esplugues de Llobregat 08950, Spain, 4Service Maladies Héréditaires du Métabolisme, Centre de Biologie Est, CHU de Lyon, Bron 69500, France, 5Department de Bioquı́mica, Hospital Sant Joan de Déu, Carretera d’Esplugues, Esplugues de Llobregat 08950, Spain and 6Consejo Superior de Investigaciones Cientı́ficas (CSIC), Barcelona, Spain 1908 Human Molecular Genetics, 2014, Vol. 23, No. 7 RESULTS Clinical and biochemical findings suggestive for a combined defect of 2-ketoacid dehydrogenase complexes The severe clinical presentation associated with lactic acidosis, increase of a-alanine and of 2-ketoglutarate led us to suspect a mitochondrial disease, but the enzyme activities and assembly of the respiratory chain complexes in muscle biopsy and skin fibroblasts were normal (Table 1, Supplementary Material, Fig. S1). However, PDHC activity was clearly reduced (Table 1), whereas the partial PDHC reactions catalyzed by pyruvate decarboxylase (PDH-E1) and dihydrolipoamide dehydrogenase (PDH-E3) subunits were normal. Dihydrolipoamide acetyl transferase (PDH-E2) activity was not measured, but analysis of the cDNA sequence of DLAT (MIM 608770), encoding for PDH-E2, did not reveal any mutation. Moreover, sequence analysis of the genes encoding for the remaining PDHC subunits [PDHA1 (MIM 300502), PDHB (MIM 179060), DLD (MIM 238331) and PDHX (MIM 608769)] did not reveal sequence variations, excluding a primary PDHC deficiency. The rates of oxidation of 14-C-substrates (pyruvate and leucine) in fibroblasts were low, whereas the rates of oxidation of glutamate and succinate were slightly below the reference range (Table 1). Altogether these findings led us to speculate that a deficiency of a common cofactor of the 2-ketoacid dehydrogenase complexes might be the cause of the disease. The two candidates were lipoic acid and thiamin as they are known to be necessary for the activity of these enzymatic complexes. However, the low PDHC activity, measured in the presence of thiamin in the incubation mixture, made the diagnosis of thiamin deficiency unlikely (3). Defective lipoylation of the E2 subunits of the PDH and 2-KGDH complexes was deficient whereas it was normal for the H protein of the GCS To determine whether lipoic acid metabolism was impaired in this individual, we analyzed fibroblasts from this patient by immunostaining using an antibody that specifically recognizes lipoic acid bound to proteins. Immunohistochemical analysis showed complete absence of protein-bound lipoic acid (Fig. 1A). Immunoblot staining in control cell lines detected two predominant lipoylated proteins of 65 and 50 kDa, corresponding to lipoic acid-bound PDH-E2 and 2-KGDH-E2, respectively (Fig. 1B) (17). In contrast, a complete absence of lipoylated E2 subunits of both PDHC and 2-KGDH was observed in the patient’s fibroblasts, whereas PDH-E2 protein content was normal (Fig. 1B). Patients carrying mutations in genes causing disruption of lipoic acid biosynthesis (NFU1, BOLA3, LIAS and IBA57) also showed high levels of glycine in plasma, urine and cerebrospinal fluid (CSF) owing to a defect in the lipoylation of the H protein of the CGS (1,2,4,7,8). Interestingly, the patient reported here showed no evidence of glycine elevation. Accordingly, the lipoylation of the H pr (...truncated)