Clinical and biochemical characterization of four patients with mutations in ECHS1

Abstract

Background: Short-chain enoyl-CoA hydratase (SCEH, encoded by ECHS1) catalyzes hydration of 2-trans-enoyl-CoAs to 3(S)-hydroxy-acyl-CoAs. SCEH has a broad substrate specificity and is believed to play an important role in mitochondrial fatty acid oxidation and in the metabolism of branched-chain amino acids. Recently, the first patients with SCEH deficiency have been reported revealing only a defect in valine catabolism. We investigated the role of SCEH in fatty acid and branched-chain amino acid metabolism in four newly identified patients. In addition, because of the Leigh-like presentation, we studied enzymes involved in bioenergetics.

Methods: Metabolite, enzymatic, protein and genetic analyses were performed in four patients, including two siblings. Palmitate loading studies in fibroblasts were performed to study mitochondrial β-oxidation. In addition, enoyl-CoA hydratase activity was measured with crotonyl-CoA, methacrylyl-CoA, tiglyl-CoA and 3-methylcrotonyl-CoA both in fibroblasts and liver to further study the role of SCEH in different metabolic pathways. Analyses of pyruvate dehydrogenase and respiratory chain complexes were performed in multiple tissues of two patients.

Results: All patients were either homozygous or compound heterozygous for mutations in the ECHS1 gene, had markedly reduced SCEH enzymatic activity and protein level in fibroblasts. All patients presented with lactic acidosis. The first two patients presented with vacuolating leukoencephalopathy and basal ganglia abnormalities. The third patient showed a slow neurodegenerative condition with global brain atrophy and the fourth patient showed Leigh-like lesions with a single episode of metabolic acidosis. Clinical picture and metabolite analysis were not consistent with a mitochondrial fatty acid oxidation disorder, which was supported by the normal palmitate loading test in fibroblasts. Patient fibroblasts displayed deficient hydratase activity with different substrates tested. Pyruvate dehydrogenase activity was markedly reduced in particular in muscle from the most severely affected patients, which was caused by reduced expression of E2 protein, whereas E2 mRNA was increased.

Conclusions: Despite its activity towards substrates from different metabolic pathways, SCEH appears to be only crucial in valine metabolism, but not in isoleucine metabolism, and only of limited importance for mitochondrial fatty acid oxidation. In severely affected patients SCEH deficiency can cause a secondary pyruvate dehydrogenase deficiency contributing to the clinical presentation.

Figures

Fig. 1

Biochemical pathways. The SCEH enzyme encoded by the ECHS1 gene is involved in different metabolic pathways. On the left side the pathways of valine, isoleucine and leucine are depicted. On the right side the pathway of medium/short-chain fatty acid oxidation is depicted. BCAT, branched-chain aminotransferase; BCKD complex, branched-chain alpha-keto acid dehydrogenase complex; HIBCH, 3-hydroxyisobutyryl-CoA hydrolase; HIBADH, 3-hydroxyisobutyrate dehydrogenase; HMG-CoA lyase, 3-hydroxy-3-methylglutaryl-CoA lyase; MHBD, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase; MGH, 3-methylglutaconyl-CoA hydratase; IBD, isobutyryl-CoA dehydrogenase; IVD, isovaleryl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; MCC, 3-methylcrotonyl-CoA carboxylase; MMSDH, methylmalonate semialdehyde dehydrogenase; SBCAD, short branched-chain acyl-CoA dehydrogenase; SCAD, short-chain acyl-CoA dehydrogenase; SCEH, short-chain enoyl-CoA hydratase; SCHAD, short-chain 3-hydroxyacyl-CoA dehydrogenase

Fig. 2

Brain MRI of patient 2. FLAIR images of the brain of patient 2 showing multiple periventricular cystic lesions in addition to attenuated signal in the subcortical white matter

Fig. 3

Brain MRI of patient 4. Brain MRI of patient 4 showing lesions in the basal ganglia (1) and mesencephalon (2). Bilateral hyperintensities are present in the globi pallidi and the left caudate at the age of 12 months (1a) and display progressive degenerative evolution to atrophy after 1 month (1b) and 6 months (1c) of the disease course. The substantia nigra shows acute lesions at 12 months of age (2a) and evolving into atrophy after 6 months (2b)

Fig. 4

SCEH enzyme activity and protein expression in patients fibroblasts (a) SCEH enzyme activity with crotonyl-CoA as substrate measured in fibroblasts of control subjects (n = 10) and fibroblasts of patients 1–4. Whiskers indicate mean ± SD. Patient fibroblasts show a markedly reduced SCEH activity. b Immunoblot analysis with antibodies against SCEH in fibroblasts of two control subjects and fibroblasts of patients 2–4. The lower panel shows the loading control with antibodies against α-tubulin. Patient fibroblasts show reduced SCEH protein expression

Fig. 5

SCEH enzyme activity in patient fibroblasts measured with different substrates. a Formation of tiglyl-CoA by incubation of 2-methyl-butyryl-CoA with recombinantly expressed short branched-chain acyl-CoA dehydrogenase (SBCAD) (left panel) and formation of methacrylyl-CoA by incubation of isobutyryl-CoA with isobutyryl-CoA dehydrogenase (IBD) (right panel). After 2 min all substrate is converted into product. b Enoyl-CoA hydratase enzyme activity with tiglyl-CoA (left graph), methacrylyl-CoA (middle graph) and 3-methylcrotonyl-CoA (right graph) in fibroblasts of two control subjects, SCEH deficient patients and a patient with a complete deficiency of mitochondrial trifunctional protein (MTP) and a patient with 3-methylglutaconyl-CoA hydratase (MGH) deficiency. c Enoyl-CoA hydratase enzyme activity with tiglyl-CoA (left graph), methacrylyl-CoA (middle graph) and crotonyl-CoA (right graph) in liver of two control subjects and two SCEH deficient patients (patient 1 and 2). Residual activity in patients samples is indicated in % of mean activity in two control liver samples

Fig. 6

Pyruvate dehydrogenase subunits in tissue samples. Levels of subunits of pyruvate dehydrogenase are shown in liver (a), muscle (b) and fibroblasts (c). Five control samples are shown followed by samples of patients 1 and 2. Decreased levels of the E2 subunit are shown when probed with a pyruvate dehydrogenase antibody cocktail, as well as with an antibody specific to E2 and E3-binding protein. GAPDH, porin and citrate synthase are shown as loading controls

Fig. 7

Tiglyl-CoA metabolism. The intersecting enzyme activities acting upon tiglyl-CoA in liver tissue