Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement

Abstract

Objective: Short-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal-recessive ECHS1 deficiency.

Methods: Using exome sequencing, we identified ten unrelated individuals carrying compound heterozygous or homozygous mutations in ECHS1. Functional investigations in patient-derived fibroblast cell lines included immunoblotting, enzyme activity measurement, and a palmitate loading assay.

Results: Patients showed a heterogeneous phenotype with disease onset in the first year of life and course ranging from neonatal death to survival into adulthood. The most prominent clinical features were encephalopathy (10/10), deafness (9/9), epilepsy (6/9), optic atrophy (6/10), and cardiomyopathy (4/10). Serum lactate was elevated and brain magnetic resonance imaging showed white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism. Analysis of patients' fibroblast cell lines (6/10) provided further evidence for the pathogenicity of the respective mutations by showing reduced ECHS1 protein levels and reduced 2-enoyl-CoA hydratase activity. While serum acylcarnitine profiles were largely normal, in vitro palmitate loading of patient fibroblasts revealed increased butyrylcarnitine, unmasking the functional defect in mitochondrial β-oxidation of short-chain fatty acids. Urinary excretion of 2-methyl-2,3-dihydroxybutyrate - a potential derivative of acryloyl-CoA in the valine catabolic pathway - was significantly increased, indicating impaired valine oxidation.

Interpretation: In conclusion, we define the phenotypic spectrum of a new syndrome caused by ECHS1 deficiency. We speculate that both the β-oxidation defect and the block in l-valine metabolism, with accumulation of toxic methacrylyl-CoA and acryloyl-CoA, contribute to the disorder that may be amenable to metabolic treatment approaches.

Figure 1

Short-chain enoyl-CoA hydratase (ECHS1) functions. Proposed functions of ECHS1 in the mitochondrial amino acid and fatty acid metabolism with illustration of the level of HIBCH (3-hydroxyisobutyryl-CoA hydrolase) deficiency.

Figure 2

Pedigrees of investigated families and short-chain enoyl-CoA hydratase (ECHS1) structure and conservation of identified mutations. (A) Pedigrees of 10 families with mutations in ECHS1. Mutation status of affected (closed symbols) and unaffected (open symbols) family members. (B) Gene structure of ECHS1 with known protein domains of the gene product and localization and conservation of amino acid residues affected by mutations. Intronic regions are not drawn to scale.

Figure 3

Spectrum of brain MRI and autopsy changes in ECHS1 patients. (A) MRI (T2) at age 8 days in individual F1, II:2 showing widespread diffuse white matter changes and brain atrophy. (B) MRI (T2) at age 8 months in individual F6, II:1 (#376) showing brain atrophy and bilateral symmetric signal hyperintensity in caput nucleus caudatus and putamen. (C) MRI (FLAIR) at age 2.2 years in individual F9, II:2 (#57277) showing increased signal in putamen, globus pallidus and caput nucleus caudatus. (D) MRI (FLAIR) at age 15 years in individual F10, II:1 (#52236) showing bilateral symmetric signal hyperintensity in caput nucleus caudatus and putamen. (E) Autopsy at age 11 months in individual F2, II:1 (#42031) showing necrotizing encephalopathy of the caudate and lenticular nuclei. MRI, magnetic resonance imaging; ECHS1, short-chain enoyl-CoA hydratase.

Figure 4

Analysis of ECHS1 levels and enzymatic activity. (A) Analysis of ECHS1 steady state levels by immunoblotting show a decrease in the amount of ECHS1 in patient-derived cell lines compared to controls. (B) Analysis of residual ECHS1 enzymatic activity indicates reduced 2-enoyl-CoA hydratase activity in cell lysates from patients’ fibroblasts compared to controls. Results shown are from at least three experiments performed in triplicates and controls (n = 4). Boxplot whiskers indicate range from 5th to 95th percentile. *P < 0.001; two-tailed unpaired t-test. (C) Analysis of palmitate-dependent OCR in fibroblast cell lines revealed impaired respiration in patients’ cells in comparison to controls. The experiment was performed several times with very similar results. The data are shown from one experiment performed with more than 10 replicates for each cell line grown and treated in parallel. Error bars indicate 1 SD. *P < 0.001; two-tailed unpaired t-test. ECHS1, short-chain enoyl-CoA hydratase; OCR, oxygen consumption rate.