Case Reports

.2019 Apr 30;27(5):1376-1386.e6.

doi: 10.1016/j.celrep.2019.04.005.

Functional Assessment of Lipoyltransferase-1 Deficiency in Cells, Mice, and Humans

Min Ni¹, Ashley Solmonson², Chunxiao Pan², Chendong Yang², Dan Li², Ashley Notzon², Ling Cai³, Gerardo Guevara², Lauren G Zacharias², Brandon Faubert², Hieu S Vu², Lei Jiang⁴, Bookyung Ko², Noriko Merida Morales², Jimin Pei⁵, Gonçalo Vale⁶, Dinesh Rakheja⁷, Nick V Grishin⁸, Jeffrey G McDonald⁶, Garrett K Gotway⁹, Markey C McNutt⁹, Juan M Pascual¹⁰, Ralph J DeBerardinis¹¹

Affiliations

¹ Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address: min.ni@utsouthwestern.edu.
² Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
³ Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Quantitative Biomedical Research Center, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁴ Department of Molecular and Cellular Endocrinology, City of Hope, Duarte, CA 91010, USA.
⁵ Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁶ Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁷ Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁸ Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁹ Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁰ Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Neurology and Neurotherapeutics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹¹ Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address: ralph.deberardinis@utsouthwestern.edu.

PMID:31042466
PMCID: PMC7351313
DOI: 10.1016/j.celrep.2019.04.005

Case Reports

Functional Assessment of Lipoyltransferase-1 Deficiency in Cells, Mice, and Humans

Min Ni et al. Cell Rep.2019.

.2019 Apr 30;27(5):1376-1386.e6.

doi: 10.1016/j.celrep.2019.04.005.

Authors

Affiliations

¹ Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address: min.ni@utsouthwestern.edu.
² Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
³ Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Quantitative Biomedical Research Center, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁴ Department of Molecular and Cellular Endocrinology, City of Hope, Duarte, CA 91010, USA.
⁵ Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁶ Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁷ Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁸ Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁹ Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁰ Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Neurology and Neurotherapeutics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹¹ Children's Medical Center Research Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address: ralph.deberardinis@utsouthwestern.edu.

PMID:31042466
PMCID: PMC7351313
DOI: 10.1016/j.celrep.2019.04.005

Abstract

Inborn errors of metabolism (IEMs) link metabolic defects to human phenotypes. Modern genomics has accelerated IEM discovery, but assessing the impact of genomic variants is still challenging. Here, we integrate genomics and metabolomics to identify a cause of lactic acidosis and epilepsy. The proband is a compound heterozygote for variants in LIPT1, which encodes the lipoyltransferase required for 2-ketoacid dehydrogenase (2KDH) function. Metabolomics reveals abnormalities in lipids, amino acids, and 2-hydroxyglutarate consistent with loss of multiple 2KDHs. Homozygous knockin of a LIPT1 mutation reduces 2KDH lipoylation in utero and results in embryonic demise. In patient fibroblasts, defective 2KDH lipoylation and function are corrected by wild-type, but not mutant, LIPT1 alleles. Isotope tracing reveals that LIPT1 supports lipogenesis and balances oxidative and reductive glutamine metabolism. Altogether, the data extend the role of LIPT1 in metabolic regulation and demonstrate how integrating genomics and metabolomics can uncover broader aspects of IEM pathophysiology.

Keywords: 2-ketoacid dehydrogenase; epilepsy,developmental delay; fatty acid oxidation; genomics; inborn errors of metabolism; lactic acidosis; lipogenesis; lipoylation; metabolomics.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

**Figure 1.. Metabolic Defects in a Patient with Compound Heterozygous Mutations ofLIPT1**
(A) Patient (P3–002) and affected siblings are indicated in black. TheLIPT1 genotype is indicated under the individuals subjected to molecular analysis. (B) Chromatogram ofLIPT1 sequences from the patient (II.5, P3–002) and healthy sibling (II.3). (C) Schematic of LIPT1 proteins encoded from paternal and maternal alleles. The N44S and S292X mutations in the lipoyltransferase domain are indicated in red. (D) Conservation of N44 (red frame) and three interacting residues (blue frames) in 16 species. (E) Three-dimensional structure of bovine Liptl (left panel) and an enlargement (right panel) showing hydrogen bonding among Asn44, Ser36, Ser38, and Asn68. The locations of cofactors, AMP-conjugated lipoic acid (lipoyl-AMP) and magnesium ion, are also indicated. (F) Plasma lactate concentrations from P3–002 over 12 years. The upper limit of the reference range (0.7–2.5 mmol/L) is indicated in red. (G) Plasma alanine, proline, and glutamate in P3–002. The dashed lines indicate the upper limit of the normal range for each amino acid. (H) Principal-component analysis (PCA) of plasma metabolomics from patient P3–002, the healthy sibling (II.3), and healthy controls (n = 60}. (I) Quantitative plasma abundance of total 2-hydroxyglutarate (2-HG), L-2-hydroxyglutarate (L-2-HG), and D-2-hydroxyglutarate (D-2-HG) in P3–002, the healthy sibling (II.3), and controls (n = 6). Data are the mean and SD from two (sick) or six (control) replicates. (J) Heatmap of plasma lipidomics showing species with variable importance in the projection (VIP) scores ≥1.0 between patient P3–002 and healthy controls (n = 16).

**Figure 2.. Functional Analysis of LIPT1 Variants in Patient-Derived Fibroblasts**
(A) Immunobiot analysis of primary human fibroblasts from five healthy subjects and patient P3–002. DLAT, DLST, and DBT are the E2 subunit of PDH, AKGDH, and BCKDH complexes, respectively. (B) Metabolite set enrichment analysis of metabolites decreased in the P3–002 fibroblasts compared to the control primary fibroblasts. The metabolomic profiling was performed on primary human fibroblasts from five healthy subjects and patient P3–002. (C) Comparison of selected metabolites determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the fibroblasts from five controls and patient P3–002. Data are the mean and SD from 3 replicates for P3–002 and 15 replicates for controls. An unpaired, two-tailed t test was used to assess statistical significance. ****p< 0.0001, ***p< 0.001. (D) PDH activity determined by the amount of CO₂ released from¹⁴C-pyruvate in control primary fibroblasts and P3–002 fibroblasts. Data are the mean and SD from four replicates. ****p < 0.0001, ***p < 0.001, calculated by unpaired two-tailed t test. (E) PDH activity measured by the amount of CO₂ released from¹⁴C-pyruvate in P3–002 fibroblasts with or without ectopic expression of WT LIPT1. Data are the mean and SD from four replicates. ****p < 0.0001, calculated by unpaired two-tailed t test. (F) Seahorse assay to measure the maximal palmitate-stimulated respiration in P3–002 fibroblasts with or without ectopic expression of LIPT1. Data are the mean and SEM from six replicates. **p< 0.01, *p< 0.05, calculated by unpaired two-tailed t test. (G) Lipogenesis rate measured with³H₂O in H460 cells containing or lacking LIPT1. Data are the mean and SD from nine replicates. ****p < 0.0001, calculated by unpaired two-tailed t test. The immunobiot analysis of LIPT1 is shown in the insert panel. (H) Immunobiot analysis of human neonatal fibroblast cell (HNFC), P3–002 fibroblasts, and P3–002 fibroblasts with ectopic expression of WTLIPT1 or either of the twoLIPT1 variants (N44S and S292X) observed in the patient. (I) Relative PDH activity in P-3002 fibroblasts and in P3–002 fibroblasts with ectopic expression of the WTLIPT1 or theLIPT1 mutants (N44S and S292X). Data are the mean and SD from five replicates. ****p < 0.0001, ***p < 0.001, **p < 0.01, calculated by unpaired two-tailed t test.

**Figure 3.. Homozygosity of theLipt1 N44S Variant Causes Mid-embryonic Demise in Mice**
(A) Genotype frequencies of embryos derived fromLipt1^N44S/+ intercrosses. (B) Lateral view of mouse embryos at embryonic day (E) 10.5 with the denotedLiptl genotypes. Scale bars: 1 mm. (C) Immunoblot analysis of embryos with WT Liptl(Liptl^+/+) or with heterozygosity {*LiptT^N44S/+*) or homozygosity (*Lipt1^N44S/N44S*) for the N44S variant. All embryos on the blot are from the same litter; these results are representative of two independent litters. (D) PDH activity in mouse embryos with the denoted genotypes. The data are the mean and SD ofLipt1^+/+ (n = 3) orLipt1^N44S/+ (n = 8) embryos. The data from two individualLipt1^N44S/N44S embryos are also shown. Unpaired two-tailed t tests were used to assess statistical significance. *p < 0.05; n.s., not significant.

**Figure 4.. Oxidative and Reductive Glutamine Metabolism Support TCA Cycle Replenishment, Fatty Acid Synthesis, and Growth of LIPT1Deficient Fibroblasts**
(A) Representative phase contrast images showing the morphology of P3–002 fibroblasts in culture when LIPT1 was ectopically expressed or when the indicated metabolites were supplemented in conditional medium (2 mM glucose, 0 mM glutamine, 8mM galactose, and 2.5% dialyzed fetal bovine serum [FBS]). Scale bar: 100 (B) P3–002 fibroblast viability and bioenergetics were evaluated in conditional medium supplemented with the indicated metabolites for 48 h. The results are shown as mean and SD from three replicates, and p values are calculated by unpaired two-tail t test. ***p< 0.001, **p< 0.01, *p< 0.05. (C) Immunoblot analysis of P3–002 fibroblasts cultured with the indicated metabolites in the conditional medium for 48 h. (D) Heatmap of metabolic pathways enriched after LIPT1 expression or metabolite supplementation in P3–002 fibroblasts. The color scale represents the impact score on pathways using metabolomic pathway analysis (MetPA). Gray indicates no enrichment of the indicated pathway. (E) Cellular respiration rate measured in P3–002 fibroblasts in the presence or absence of glutamine (2 mM). Data are the mean and SEM from eight replicates. ****p< 0.0001, calculated by unpaired two-tail t test. {F) Fractional enrichment of the citrate isotopologues in P3–002 fibroblasts with or without ectopic LIPT1 expression (P3–002+LIPT1). The cells were cultured in [U-¹³C]glutamine for 24 h. Data are the mean and SD from three replicates. **p < 0.01, calculated by unpaired two-tail t test. (G) Fractional enrichment of the malate isotopologues in P3–002 or P3–002+LIPT1 fibroblasts after culture with [U-¹³C]glutarmine for 24 h. Data are the mean and SD from three replicates. **p < 0.01, calculated by unpaired two-tail t test. (H) Fractional enrichment of the citrate isotopologues in WT or L1PT1-knockout (KO) H460 cell clones after culture with [U-¹³C]glutamine for 6 h. (I) Fractional enrichment of the malate isotopologues in WT or LIPT1-KO H460 cell clones after cultured with [U-¹³C]glutamine for 6 h. (J) Mass isotopologue distribution in palmitate from WT or LIPT1-KO H460 cells after culture with [U-¹³C]glutamine for 24 h. Data are the mean and SD from three replicates. (K) Mass isotopologue distribution in palmitate from WT or LIPT1-KO H460 cells after culture with [U-¹³C]glucose for 24 h. Data are the mean and SD from three replicates. (L) Major metabolic alterations associated with LIPT1 deficiency in humans. Green arrows indicate elevation, and red arrows indicate reduction.

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Functional Assessment of Lipoyltransferase-1 Deficiency in Cells, Mice, and Humans

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Functional Assessment of Lipoyltransferase-1 Deficiency in Cells, Mice, and Humans

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