Mitochondrial Fatty Acid Oxidation Disorders Associated with Short-Chain Enoyl-CoA Hydratase (ECHS1) Deficiency

Mitochondrial fatty acid β-oxidation (FAO) is the primary pathway for fatty acid metabolism in humans, performing a key role in liver, heart and skeletal muscle energy homeostasis. FAO is particularly important during times of fasting when glucose supply is limited, providing energy for many organs and tissues, including the heart, liver and brain. Deficiencies in FAO can cause life-threatening metabolic disorders in early childhood that present with liver dysfunction, hypoglycemia, dilated hypertrophic cardiomyopathy and Reye-like Syndrome. Alternatively, FAO defects can also cause ‘milder’ adult-onset disease with exercise-induced myopathy and rhabdomyolysis. Short-chain enoyl-CoA hydratase (ECHS1) is a key FAO enzyme involved in the metabolism of fatty acyl-CoA esters. ECHS1 deficiency (ECHS1D) also causes human disease; however, the clinical manifestation is unlike most other FAO disorders. ECHS1D patients commonly present with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy traditionally associated with defects in oxidative phosphorylation (OXPHOS). In this article, we review the clinical, biochemical and genetic features of the ESHS1D patients described to date, and discuss the significance of the secondary OXPHOS defects associated with ECHS1D and their contribution to overall disease pathogenesis.


Mitochondrial Metabolism
Mitochondria are the 'powerhouses' of the cell, producing 95% of all cellular energy in the form of adenosine triphosphate (ATP) [1]. ATP is expended for essential processes such as growth, reproduction, cell signaling and differentiation [2]. Additionally, mitochondria are involved in other important cellular functions including apoptosis, calcium homeostasis, biosynthesis of heme and iron-sulfur clusters, as well as innate immune responses [2,3].
Through a series of four enzymatic reactions, dehydrogenation, hydration, a second dehydrogenation and thiolysis, fatty acyl-CoA chains within the mitochondria are processed to yield one acetyl-CoA molecule, two electrons and a fatty acyl-CoA shortened by two carbons. This series of reactions is then performed repeatedly until only two acetyl-CoA molecules remain (commonly termed the β-oxidation spiral) ( Figure 1). Both dehydrogenation steps are cofactor-dependent, resulting in the reduction of NAD + and FAD to NADH and FADH 2 , which are subsequently oxidized by OXPHOS complexes I and II respectively [7].

ECHS1 is a Multifunctional Enzyme
Short-chain enoyl-CoA hydratase (ECHS1; EC 4.2.1.17) is responsible for the second step of FAO. ECHS1 activity was first observed by Stern and Del Campillo [10] in ox heart and liver, with human ECHS1 cDNA clones first isolated in 1993 [10]. The 11 kb ECHS1 gene locus was subsequently mapped to chromosome 10q26.2-q26.3 by fluorescence in situ hybridization, encoding eight exons with the 5 and 3 untranslated regions contained within exons I and VIII respectively [11].
ECHS1 is transcribed as a single 1.4 kb mRNA, with expression observed in hepatocytes, fibroblasts and myocytes [10]. The translated 290 amino acid precursor protein contains a 27-amino-acid N-terminal mitochondrial targeting signal that is cleaved upon entry into the mitochondrial matrix [12], where the resulting 28.3-kDa mature protein forms an active 188 kDa homohexamer composed of a 'dimer of trimers' [13,14] (Figure 2A). ECHS1 catalyzes the conversion of trans-∆ 2 -enoyl-CoA thioesters to 3-L-hydroxyacyl-CoA thioesters by stereospecific hydration of the trans double bond between carbons two and three [15] ( Figure 2B). ECHS1 has strongest substrate affinity for the 4-carbon crotonyl-CoA, but can bind enoyl-CoA chains up to 10 carbon atoms in length [14]. While ECHS1 has considerably higher specificity for straight-chain enoyl-CoA thioesters as part of the FAO pathway, it also exhibits moderate activity for degrading methacrylyl-CoA (valine pathway), 3-methylcronytyl-CoA (leucine pathway) and tiglyl-CoA (isoleucine pathway) [16,17]. Interestingly, only metabolites of the valine pathway have been detected in plasma and urine of patients with ECHS1 deficiency (ECHS1D), suggesting that ECHS1 is vital for valine metabolism, but not leucine or isoleucine metabolism [16,17].

FAO Disease
Defects in FAO were first described in the 1970 s in patients with carnitine O-palmitoyltransferase deficiencies [18][19][20], with the first pathogenic mutations identified in ACADM (which encodes the medium-chain acyl-CoA dehydrogenase) [21][22][23]. Pathogenic mutations have now been identified in at least 22 different FAO genes, and can affect up to 1 in 10,000 individuals in certain populations [1]. Patients can present in early childhood with severe (often lethal) liver dysfunction, hypoglycemia and Reye-like syndrome (a combination of encephalopathy due to acute brain swelling and liver dysfunction caused by fat accumulation) [24]. Cardiac symptoms, such as dilated hypertrophic cardiomyopathy and arrhythmias, are also common [25,26]. Alternatively, 'milder' adult-onset disease, presenting with exercise-induced myopathy and rhabdomyolysis (breakdown of muscle fibers), has also been reported [27].
Disease presentation may not be persistent, with patients showing no symptoms or biochemical deficiencies until an episode of metabolic crisis. These episodes can be triggered by various circumstances, including prolonged fasting, exercise, infection, exposure to cold, or a fat-rich diet [7,28,29]. Treatment options are limited, focusing on restricting dietary long-chain fatty acids that cannot be metabolized (and which become toxic), as well as maintaining blood glucose levels.
FAO deficiencies are also believed to cause about 1-3% of unexplained sudden infant deaths [30], with deficiencies in MCAD and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) reported [31,32]. In addition, mutations in the genes encoding MCAD, SCAD, LCHAD and CPT2 are associated with acute fatty liver of pregnancy (AFLP) and hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome, both of which carry significant neonatal and maternal morbidity and mortality during pregnancy [33,34].

ECHS1 Deficiency (ECHS1D)
ECHS1D onset is usually at birth or in early childhood, with death occurring within the first two days of life in some cases [35]. The clinical presentation of ECHS1D is typified by Leigh syndrome (subacute necrotizing encephalomyelopathy) or Leigh-like syndrome, with symptoms including (but not limited to) developmental delay, dystonia, metabolic acidosis, cardiomyopathy and apnea. Leigh syndrome is a progressive neurodegenerative disease characterized by bilateral symmetric brain lesions and psychomotor regression [36], and is not typically observed in other FAO disorders [37]. Leigh syndrome has been associated with more than 75 genes, mostly involved in OXPHOS complex I structure and assembly [38]. Brain magnetic resonance imaging (MRI) findings have revealed T 2 bilateral hyperintensities, a hallmark of Leigh syndrome, in almost all reported cases of ECHS1D. Despite this, there are disparities between the clinical presentation in ECHS1D and the classical features of Leigh syndrome (as defined in Rahman, et al. [39]). Indeed, Haack, et al. [40] proposed that ECHS1D is a distinct form of Leigh-like syndrome associated with severe progressive encephalopathy, accompanied by bilateral brain lesions and mitochondrial dysfunction.
More recently, three patients were identified with clinical symptoms that expand the phenotypic spectrum of ECHS1D [41][42][43]. One patient harboring ECHS1 variants previously associated with Leigh-like syndrome also presented with cutis laxa, a connective tissue disorder characterized by loose, inelastic skin [43]. Another two patients displayed symptoms of paroxysmal exercise-induced dyskinesia (PED) [41,42]. PED is a much milder form of ECHS1D that offers a more optimistic prognosis, being characterized by recurrent attacks of abnormal dystonic movement that is triggered by prolonged exercise [44]. While these PED patients were unrelated, they shared a common ECHS1 mutation (c.518C > T; p.Ala173Val), suggesting an association between this variant and PED. Interestingly, T 2 hyperintensities were still observed in these patients, but Leigh-like symptoms were absent. Conversely, a sibling of one of these patients did suffer from Leigh-like syndrome with severe generalized dystonia, further exemplifying the substantial clinical heterogeneity of ECHS1D [42].

Pathogenic Mutations in ECHS1
Forty-two patients (from 33 families) with ECHS1D have been described to date ( Table 1). As with almost all primary disorders of FAO, ECHS1D follows an autosomal recessive pattern of inheritance. Human mutations in ECHS1 were first described in two infant siblings [45], with all subsequent cases exhibiting compound heterozygosity or homozygosity due to consanguinity. Twenty-seven of the 30 different ECHS1 mutations identified are missense, suggesting that null mutations may be incompatible with life [40,46]. Other noteworthy variants include nonsense mutations, frameshift mutations, and duplications resulting in protein truncation [35,40,42]. The only incidence of homozygosity for a truncating mutation was reported in two siblings with a very severe phenotype and death within 48 h, establishing some evidence for a genotype-phenotype relationship [35].
While most ECHS1 mutations are novel, the c.476A > G; p.Gln159Arg variant has been identified in seven unrelated ECHS1D patients with diverse racial backgrounds [17,40,43,47,48]. Furthermore, this variant may also represent a common founder mutation, as it has been independently reported in two families of Pakistani origin [40,48]. A second possible founder mutation in ECHS1 (c.538A > G; p.Thr180Ala) has also been identified in four French-Canadian patients [47]. Interestingly, this mutation has also been identified in an Irish Traveler family with a haplotype shared with the French-Canadian patients, suggesting an Irish ancestral origin with subsequent migration to Canada [48].

Biochemical and Metabolic Characterization of ECHS1D
ECHS1 may play a redundant role in FAO, as exemplified by the unremarkable acylcarnitine profiles in most patient cells [16,17,35,40,[47][48][49][50]52]. However, extremely high acylcarnitine levels (of C6 and C4 chain lengths) were reported in one patient who died within one day of birth [46]. As such, elevated acylcarnitines may act as an indicator of disease in only the most severe cases of ECHS1D.
Loss of ECHS1 activity for valine metabolism also appears to play a role in disease pathology. ECHS1 acts on both methacrylyl-CoA from the valine pathway and acryloyl-CoA from an alternate pathway of odd-chain FAO that feeds into the valine pathway [54]. In ECHS1D, these two intermediates accumulate, becoming toxic by spontaneous reaction with thiol groups and other mitochondrial cysteine residues, resulting in impaired ATP production and metabolic acidosis [55]. Specifically, methacrylyl-CoA and acryloyl-CoA react with lipoyl domains of the E2 subunit of the pyruvate dehydrogenase complex (PDC), inhibiting its function [17]. Indeed, reduced PDC activity has been observed in many ECHS1D patients, explaining the commonly observed symptom of lactic acidosis [17,35,40,43,45,47,48,51,52]. Notably, an inhibitory effect on two other lipoyl domaincontaining mitochondrial enzymes was not detected [17], suggesting inhibition is specific to the PDC (via an unknown mechanism).
These findings have led to tentative correlations between ECHS1D severity, degree of lactic acidosis and reduced PDC activity. In cases where ECHS1D caused early death, lactate levels were high and PDC activity low [35]. Conversely, in mild cases of ECHS1D, lactic acidosis was absent [16,41,42].

Secondary OXPHOS Defects in ECHS1D
FAO and OXPHOS are tightly linked biochemically, with the oxidation of fatty acids generating NADH and FADH 2 for oxidation by OXPHOS complexes I and II. In addition, studies have also reported physical interactions between FAO and OXPHOS proteins: OXPHOS complex I can bind the FAO protein LCHAD [56], while OXPHOS complex III can be purified in complexes with the FAO electron transfer flavoprotein (ETF) [57]. More recently, the FAO proteins VLCAD, MCAD, LCHAD, and ETF were shown to co-migrate with the OXPHOS supercomplex in a metabolically active super-structure that can oxidize fatty acids [58].
Interestingly, patients with primary deficiencies in LCHAD can exhibit significant OXPHOS enzyme defects [26,59]. These secondary OXPHOS defects were historically attributed to the accumulation of inhibitory fatty-acyl CoA intermediates. However, it now appears that more complex mechanisms are involved. Indeed, we recently showed that loss of the FAO protein MCAD can disrupt OXPHOS complex assembly and stability, resulting in reduced mitochondrial respiration and increased ROS generation in the presence of OXPHOS inhibitors [60].
Secondary OXPHOS defects have also been identified in ECHS1D. These range from isolated defects in complex I, complex III or complex IV to multiple complex I/III/IV or complex I/IV/V defects [40,[47][48][49]53]. Furthermore, blue native polyacrylamide gel electrophoresis (BN-PAGE) has identified reduced steady-state levels of mature complex IV in one patient [47]. Conversely, no differences in complex IV levels were detected in three other ECHS1D patients [17,49].
While it is not clear what causes these secondary OXPHOS defects in ECHS1D, disruption of complex I activity may be explained via its interaction with the pyruvate dehydrogenase complex (PDC). As discussed above, toxic metabolites of the valine pathway can accumulate in ECHS1D and inhibit PDC activity [17]. This inhibition may disrupt the binding of complex I to the PDC [56], with a potential loss of both complex I stability and NADH dehydrogenase activity.
Overall, it is difficult to predict the effect of ECHS1D on OXPHOS. However, it is apparent that patients with secondary OXPHOS defects generally exhibit more severe Leigh-like symptoms (Table 1).

Concluding Remarks
Forty patients have been described with pathogenic mutations in ECHS1 since the identification of the first two patients with ECHS1D in 2014. Notably, most ECHS1D patients present with Leigh syndrome or Leigh-like syndrome, a severe disorder traditionally associated with defects in OXPHOS complex I activity. While loss of ECHS1 disrupts both FAO and valine metabolism, secondary OXPHOS defects have also been identified in some patients with ECHS1D. Furthermore, these secondary OXPHOS defects are associated with a more severe clinical phenotype, suggesting that they also contribute to disease pathology.
Like other FAO deficiencies, secondary OXPHOS defects in ECHS1D may be due to the accumulation of toxic fatty acid and/or valine metabolites, which can directly inhibit OXPHOS complex activity. Alternatively, the effects of these metabolites may be indirect by disrupting the interaction between the pyruvate dehydrogenase complex (PDC) and OXPHOS complex I. Whichever mechanism is involved, further research is required to clarify the relationship between ECHS1D and OXPHOS dysfunction, and to determine if the stability and/or biogenesis of the OXPHOS complexes is also disrupted by the loss of ECHS1. This knowledge will be invaluable for our understanding of the complex interactions between the FAO and OXPHOS pathways, and will help to advance the diagnosis and treatment of mitochondrial disorders such as ECHS1D.