PMC:3905285 / 9659-21381 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3905285","sourcedb":"PMC","sourceid":"3905285","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3905285","text":"Results\n\nClinical phenotype\nThe patient, a boy, was the first child of non-consanguineous French parents. He was born at term after an uneventful pregnancy and spontaneous delivery with normal birth parameters. Psychomotor development was delayed and associated with hypotonia. At age 15 months, when the patient was referred to our unit, metabolic investigations were regarded as normal, including brain magnetic resonance imaging (MRI), muscle biopsy, plasma amino acids and urinary organic acids (data not shown). The course of the disease was marked by an episode of metabolic acidosis at 18 months of age that occurred in the context of vomiting and dehydration associated with gastro-enteritis. The patient had hyperlactatemia (8 mmol/L), liver cytolysis (AST 305 u/l, ALT 220 u/l, N \u003c 40) and psychomotor regression that occurred suddenly and included severe trunk hypotonia, coma with dystonia and no head control. After a few days, he was bed- and wheel-chair bound, could not stand or sit unaided. Severe spastic tetraparesis and extrapyramidal syndrome were observed. He could not speak but understood simple orders. He was otherwise fully conscious, alert, and he could smile, laugh and follow with eyes. Major swallowing difficulties led to gastrostomy. Brain MRI revealed cerebellar atrophy, an important sus-tentorial cortical atrophy with ventricular dilatation, bilateral thalamic anomalies, bi-frontal white matter anomalies and delayed myelinisation (Additional file 1: Figure S1). During the decompensation, the MRS spectroscopy with long TE (144) showed a peak of lactate (Additional file 1: Figure S1).\n\nBiochemical parameters\nThe most striking biochemical features involved the plasma amino acid profile that associated increased levels of glutamine and proline at presentation as well as on repeated sampling, and low levels of lysine at presentation (Table 1). This profile was consistent with multiple α-keto acid dehydrogenase deficiency, particularly involving PDH and α-KGDH, thus suggestive of E3 subunit/DLD deficiency. However, no or only slight elevations were noted for glutamate, α-amino, α-hydroxy or α-ketoadipic acids, thus contrasting with the large increases and sometimes massive amounts observed in typical forms of NFU1 deficiency ([2] and data not shown). In addition, in sharp contrast to all previously reported patients with deficiency in the de novo lipoic acid synthesis pathway, in our patient glycine was not increased in CSF, plasma or urine (Table 1). This was not consistent with deficiency of the glycine cleavage system. Furthermore, the branched chain amino acids unexpectedly showed from very low to low-normal levels, thus not suggestive of BCKDH deficiency (Table 1).\nTable 1 Relevant plasma, urinary and CSF amino acids and urinary organic acid levels Reference intervals are provided as the range of the age-matched reference population. Median and range of plasma amino acid values are also provided for the patient samples (N = 8) collected during follow-up. Mild metabolic acidosis (bicarbonates 16 mmol/L, N \u003e 20 mmol/L) associated with increased level of lactate and pyruvate in blood (lactate 8 mmol/L, N \u003c 2, pyruvate 0.44 mmol/L, N \u003c 0.2), in urine (lactate 252 μmol/mmol of creatinine) and CSF (lactate 6.60 mmol/L, pyruvate 0.36 mmol/L). Urinary α-ketoglutarate was highly increased (Table 1). On subsequent tests, lactate and pyruvate levels as well as urinary α-ketoglutarate were normalized.\nA severe decrease of PDH and α-KGDH activities was found in patient fibroblasts (Table 2), and BCKDH activity was also strongly reduced, as inferred by a 13C6 labeled leucine loading test that showed levels comparable to fibroblasts from a maple syrup urine disease patient. In contrast, mitochondrial respiratory chain activities and E3 subunit (DLD) activity measured in fibroblast homogenates were normal (data not shown). Polarographic assay in fibroblasts showed reduced oxygen production using pyruvate as substrate (2.2 nmol O2/min/mg of protein, reference range from 3.3 to 6.8 nmol O2/min/mg of protein).\nTable 2 Biochemical investigations PDHc, α-KGDHc activities and polarographic studies on patient and control skin fibroblasts, CO2 production after administration of 14C-3OHbutyrate, 14C-glucose or 14C- butyrate in fibroblasts of the patient and a control.\nFigure 1 Western blots of patient fibroblasts with antibodies against LIPT1 (A), lipoic acid (B), and tubulin (Sigma-Aldrich) or actine in cultures in basal conditions (A, B) and after LIPT1 transfection (C). A and B: Control, patients (P) with PDHA1, NFU1 and LIPT1 mutations were as indicated. A: the LIPT1 antibody failed to detect the LIPT1 protein in patient fibroblasts. B: Anti-lipoate antibody failed to detect the expected lipoylated proteins of ketoacid dehydrogenases (PDHc, α-KGDHc and BCKDHc) in the patient fibroblasts as well as in fibroblasts of another patient with NFU1 mutations, whereas normal bands were seen in patient fibroblasts with PDHA1 mutations and in control. C: Control (C), Patient with LIPT1 mutations (P), patient fibroblasts transfected with LIPT1 (P + LIPT1). Anti-lipoate antibody revealed normalized and very moderately increased amounts of BCKDH (x8) and α-KGDH (x1.8) proteins, respectively, in patient fibroblasts after LIPT1 transfection whereas no band was detected for PDH protein. Oxidation rates of butyrate (fatty acid), 3-hydroxybutyrate (ketone body) and glucose were measured by incubating patient-derived fibroblasts with 1 and 10 mmol/l 1-14C labeled substrates. We found that CO2 production by the Krebs cycle and mitochondrial respiratory chain activity in fibroblast’s patient were very low compared to controls after administration of each of these substrates (Table 2), suggesting a defect in both the Krebs’ cycle and PDH.\n\nMolecular investigations\nTo identify the causative gene mutation, we first excluded the genes PDHA1, PDHB, PDHX, DLAT, DLD[1], LIAS (GenBank NG_032111.1), BOLA3 (GenBank NG_031910) and NFU1 (GenBank NG_031931.1) by direct sequencing using DNA extracted from white blood cells. Subsequently, exome capture was performed and resulted in a list of 25 candidate genes, including a mitochondrial protein, LIPT1 (MIM 610284). The compound heterozygous c.875C \u003e G and c.535A \u003e G transitions in LIPT1 gene (NP_660200.1) on chromosome 2 resulted in a stop mutation (p.Ser292X) and the substitution of a threonine by an alanine (p.Thr179Ala) in the protein, respectively. Sanger sequencing confirmed these mutations in the affected individual and showed that the parents were heterozygous for either mutation. These changes were predicted to be “damaging” and “deleterious” by Polyphen and SIFT softwares, respectively, and were located in a highly conserved domain of the protein. In addition, these mutations were not found in 100 controls of French origin or in exome sequencing projects including at least 13 000 alleles [http://evs.gs.washington.edu/EVS/].\n\nImmunoblotting\nA LIPT1 antibody failed to detect the LIPT1 protein in patient fibroblasts (Figure 1A). Anti-lipoate antibody failed to detect the expected lipoylated proteins of ketoacid dehydrogenases (PDHc, α-KGDHc and BCKDHc) in the patient fibroblasts as well as in fibroblasts of another patient with NFU1 mutations, whereas normal bands were seen in patient fibroblasts with PDHA1 mutations and in control (Figure 1B). By contrast anti PDH E1-α (Abcam) antibody detected normal bands in the LIPT1-mutated patient fibroblasts (data not shown).\n\nLIPT1 transfection\nControl and patient fibroblasts were transfected using the pLenti-GIII-CMV-hLIPT1-HA vector and analyzed after 36 h. As shown in Figure 1C after LIPT1 transfection, anti-lipoate antibody revealed normalized (×8) and moderately (×1.8) increased amounts of BCKDH and α-KGDH proteins, respectively, in patient fibroblasts (quantification was performed with image J software) whereas no band was detected for PDH protein. PDH and α-KGDH activities increased moderately after LIPT1 transfection (PDH: 90 and 93 to 210 and 215 pmoles/min/mg protein; α-KGDH: 0.9 to 3 nmoles/min/mg protein). Lactate (L) and pyruvate (P) levels were significantly increased in fibroblast supernatants compared to controls and they were dramatically decreased after LIPT1 transfection (Figure 2A-B). Interestingly, the level of 3OHbutyrate also decreased with a concomitant increase of glucose suggesting that the cells metabolized 3OHbutyrate upon partial rescue of α-KGDH activity (Figure 2C-D).\nFigure 2 Levels of lactate (A), pyruvate (B), glucose (C) and 3OHbutyrate (D) in supernatants from patient fibroblast’s cultured in basal condition and after LIPT1 transfection. P: patient, C: control. A-B: Lactate (L) and pyruvate (P) levels were significantly increased in fibroblast supernatants compared to controls and they were dramatically decreased after LIPT1 transfection. C-D: the level of 3OHbutyrate also decreased with a concomitant increase of glucose suggesting that the cells metabolized 3OHbutyrate upon partial rescue of α-KGDH activity. Data are represented as mean ± SEM.\n\nLipoic acid administration on yeast model and human fibroblasts\nWild-type and Δlip3 strains, deleted for lip3, the gene orthologous to human LIPT1[15], were grown on glucose and ethanol medium for 3 and 7 days at 28°C and 36°C. A growth defect of Δlip3 cells on ethanol was observed at 28°C and was exacerbated at 36°C (Figure 3A).\nFigure 3 Effects of lipoic acid administration on Δlip3-deleted yeast strain and patient fibroblasts. A: Growth of Δlip3 on glucose and ethanol at different temperature and after lipoic acid administration. Δlip3 yeasts failed to growth on ethanol at 28°C and the effect was stronger at 36°C. Lipoic acid was added in growth medium of Δlip3 strains and strikingly improved Δlip3 growth (2 ng/ml). B: PDH, α-KGDH and citrate synthase (CS) activities measured on cultured fibroblasts in basal condition and after administration of lipoic acid. In patient fibroblasts, this led to absent, or only moderate increase of α-KGDH and PDH activity. C: Lactate levels in cultured fibroblast supernatants in basal condition and after administration of lipoic acid. P: patient, C: control. Lactate level decreased significantly and this effect contrasted with the increase observed in control fibroblasts. Data are represented as mean ± SEM. D: Labeled to natural isotopic ratios for 3-hydroxyisovaleric acid (an isovaleryl-CoA derivative) in the LIPT1-deficient patient compared to a MSUD and control patient. The panel shows that LIPT1 deficiency leads to a severe loss of metabolic flux involving BCKDH that is similar to BCDKH deficiency. The (+) and (-) signs stand for whether lipoate was added. Y-axis: ratio of stable isotope labeled vs natural 3-hydroxyvaleric acid. Error bars indicate standard deviations from triplicate experiments. Lipoic acid (2 ng/mL) was added in growth medium of Δlip3 cells to test its potential therapeutic effect and it markedly improved Δlip3 growth (Figure 3A).\nBecause of this result, patient and control fibroblast cultures were supplemented with lipoic acid to a final concentration of 10 or 100 μM during three weeks. In the patient fibroblasts, this led to absent, or only moderate increase of αKGDH and PDH activities respectively (Figure 3B), yet lactate level decreased significantly and this effect contrasted with the lactate increase observed in control fibroblasts (Figure 3C). Nevertheless, the 13C6-labeled leucine loading test showed no effect of lipoic acid on BCKDH activity (Figure 3D). Thus, lipoic acid supplementation might have beneficial, though only partial effects, through mechanism(s) that remain to be determined.\n","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":1624}},{"label":"Title","span":{"begin":9,"end":27}},{"label":"Section","span":{"begin":1626,"end":5826}},{"label":"Title","span":{"begin":1626,"end":1648}},{"label":"Table caption","span":{"begin":2728,"end":3025}},{"label":"Table caption","span":{"begin":4084,"end":4343}},{"label":"Figure caption","span":{"begin":4343,"end":5371}},{"label":"Section","span":{"begin":5828,"end":6979}},{"label":"Title","span":{"begin":5828,"end":5852}},{"label":"Section","span":{"begin":6981,"end":7529}},{"label":"Title","span":{"begin":6981,"end":6995}},{"label":"Section","span":{"begin":7531,"end":9119}},{"label":"Title","span":{"begin":7531,"end":7549}},{"label":"Figure 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