A gene encoding a putative FAD-dependent L-2-hydroxyglutarate dehydrogenase is mutated in L-2-hydroxyglutaric aciduria

Abstract

The purpose of this study was to identify the biochemical and genetic defect in L-2-hydroxyglutaric aciduria, a neurometabolic disorder characterized by the presence of elevated concentrations of L-2-hydroxyglutaric acid in urine, plasma, and cerebrospinal fluid. Evidence is provided for the existence in rat tissues of a FAD-dependent enzyme catalyzing specifically the oxidation of L-2-hydroxyglutarate to alpha-ketoglutarate. This enzyme is mainly expressed in liver and kidney but also at lower levels in heart, brain, and other tissues. Subcellular fractionation indicates that the liver enzyme is present in mitochondria, where it is bound to membranes. Based on this information, a database search led to the identification of a gene encoding a human hypothetical protein homologous to bacterial FAD-dependent malate dehydrogenases and targeted to mitochondria. The gene encoding this protein, present on chromosome 14q22.1, was found to be in a region homozygous in patients with L-2-hydroxyglutaric aciduria from two consanguineous families. Three mutations that replaced a highly conserved residue (Lys-71-Glu and Glu-176-Asp) or removed exon 9 were identified in homozygous state in patients from three distinct families and were found to cosegregate with the disease. It is concluded that L-2-hydroxyglutarate is normally metabolized to alpha-ketoglutarate in mammalian tissues and that L-2-hydroxyglutaric aciduria is caused by mutations in the gene that most likely encodes L-2-hydroxyglutarate dehydrogenase. The pathological findings observed in this metabolic disorder must therefore be due to a toxic effect of L-2-hydroxyglutarate on the central nervous system.

Fig. 1.

Elution profile of l-2-hydroxyglutarate dehydrogenase from a DEAE-Sepharose column (A) and substrate saturation curve (B). (A) A rat liver extract prepared from 10 g of liver was chromatographed on DEAE Sepharose. The release of tritium from dl-2-hydroxy[2-³H]glutarate (○) (8), l-2-hydroxyglutarate dehydrogenase activity (•), and A₂₈₀ (▵) were determined in the indicated fractions. (B) The activity was determined with the spectrophotometric assay on fraction 21 of the DEAE-Sepharose column in the presence of the indicated substrates. Protein was measured according to the Bradford method (15).

Fig. 2.

Effect of FAD and FMN on the activity of l-2-hydroxyglutarate dehydrogenase. The rat liver enzyme was partially purified by chromatography on a Q-Sepharose column in the presence of detergents. It was assayed with the radiochemical assay in the presence of 100 μM unlabeled l-2-hydroxyglutarate, 1.5 mM INT, and the indicated concentrations of FAD or FMN.

Fig. 3.

Tissue distribution of l-2-hydroxyglutarate dehydrogenase. The activity was determined in homogenates from the indicated rat tissues with the spectrophotometric (spectr.) assay (Upper) or the radiochemical assay (Lower).

Fig. 4.

Alignment of the putative human l-2-hydroxyglutarate dehydrogenase with homologous proteins from different species. The alignment shows sequences from the following species: Homo sapiens (Hs; GenBank accession no. NP-079160.1); Mus musculus (Mm; GenBank accession no. NP-663418.1); Synechocystis sp. (Sy; GenBank accession no. NP-442886.1), and E. coli (Ec; GenBank accession no. NP-289209.1). The coding region of the human cDNA was amplified from liver RNA by RT-PCR, and its sequence was confirmed (GenBank accession no. AY757363). The putative cleavage site (><) for the mitochondrial presequence in the human sequence as determined withtarget p (18), and the mutations found in the three different families (E, D, and x) and a conserved stretch of hydrophobic residues (h) are indicated above the alignment.

Fig. 5.

Structure of the putative l-2-hydroxyglutarate dehydrogenase gene (A) and cosegregation of mutations in this gene with l-2-hydroxyglutaric aciduria in three consanguineous families (B–D). (A) The polymorphic markers used to study the L2HGDH locus in families 1 and 2. (B–D) Alleles of these polymorphic markers and of the L2HGDH gene are indicated below each investigated individual in the families. In family 1 (B), the Δ allele corresponds to the removal of exon 9. In families 2 (C) and 3 (D), the one-letter code is used to designate either Lys (K) or Glu (E) at position 81 and Glu or Asp (D) at position 176. In the large, 11-child-kindred family, the first and last affected children had healthy children (data not shown).