Very-Long-Chain Acyl-CoA Dehydrogenase

Straight-chain fatty acids 12–18 carbons in length are metabolized by very-long-chain acyl-CoA dehydrogenase (VLCAD) [6]. This enzyme was originally isolated from rat liver mitochondria and differed from the other human acyl-CoA dehydrogenases because it is a homodimer bound to the inner mitochondrial membrane [11]. The ACADVL gene contains 20 exons, is about 5.4 kb long, and has been mapped to human chromosome 17p11.13-p11 [12].

Long-Chain Acyl-CoA Dehydrogenase

Long-chain branched or unsaturated fatty acids are the likely substrates for long-chain acyl-CoA dehydrogenase (LCAD), an enzyme found within the mitochondrial matrix [13]. This enzyme is a homotetramer of four identical subunits; each subunit binds one molecule of FAD. Lauric acyl-CoA (C₁₂) is the preferred substrate [14, 15]. Expression of ACADL is very low in human liver and cardiac and skeletal muscle, and thus the enzyme likely plays more of a boutique function in cellular metabolism rather than being an important component of energy generation [16]. ACADL encodes LCAD protein and is mapped to chromosome 2q34-p35 [17]. Patients previously described with LCAD deficiency have all been subsequently found to have VLCAD deficiency [17]. To date, no deficiency disorder of LCAD has been identified in humans.

Acyl-CoA Dehydrogenase 9

Long-chain polyunsaturated fatty acids may also be oxidized by acyl-CoA dehydrogenase 9 (ACAD9). This enzyme was originally shown to have a substrate preference for polyunsaturated fatty acids and is the primary long-chain acyl-CoA dehydrogenase expressed in the CNS [18]. However, it is now recognized to also play a role as a respiratory chain complex I assembly factor, and it most frequently manifests with cardiomyopathy and skeletal myopathy [19].Traditionally, the CNS is believed to be glycolytic and to use only glucose and ketones as energy substrates. However, all of the FAO enzymes are synthesized in the CNS [20]. Two additional ACAD proteins were recently described in human and mouse CNS, ACAD10 and ACAD11, whose physiologic substrates remain unknown [21]. However, mice deficient in ACAD10 develop insulin resistant diabetes and obesity. The expression of multiple ACAD enzymes as well as the other FAO enzymes in the CNS suggests an important role in neuronal function but the role FAO plays in energy metabolism or in other essential CNS processes remains to be elucidated.

Medium-Chain Acyl-CoA Dehydrogenase

Chain-shortened acyl-CoAs (C_6–12) become the substrate of medium-chain acyl-CoA dehydrogenase (MCAD). The gene encoding this enzyme, ACADM, contains 12 exons and maps to chromosome 1p31.1 [22]. The enzyme is a homotetramer located in the mitochondrial matrix, similar to LCAD (Figure 34.3). MCAD is an important enzyme in humans, and deficiency of MCAD is the most common FAO defect in Caucasian populations.

Figure 34.3 X-ray crystal structure of MCAD. The top half of the figure shows the monomer. The bottom half of the figure shows the mature homotetramer. Substrate (purple) and FAD (gold) are modeled into the monomer.

Short-chain Acyl-CoA Dehydrogenase

Short-chain acyl-CoA dehydrogenase (SCAD) catalyzes the dehydrogenation of short-chain acyl-CoAs (C_4–6). The gene encoding it, ACADS, consists of 10 exons spanning 13 kb of DNA on chromosome 12q [23]. SCAD is also a homotetramer located in the mitochondrial matrix like LCAD and MCAD, suggesting a common ancestral origin.

Medium-Chain Ketoacyl-CoA Thiolase

The medium-chain ketoacyl-CoA thiolase has also been called mitochondrial 3-oxoacyl-CoA thiolase. The mRNA for this thiolase is highly expressed in human muscle, liver, and fibroblasts. There is some substrate overlap between medium-chain and short-chain 3-ketoacyl-CoA thiolases. The former can cleave C₄ into 2 acetyl-CoA molecules as well as cleave longer fatty ketoacyl-CoA moieties, with highest activity toward C₁₀. It is believed to be the primary thiolase in the medium- and short-chain FAO pathway.

Short-Chain 3-ketoacyl-CoA Thiolase

The short-chain 3-ketoacyl-CoA thiolase is encoded by ACAT1 on chromosome 11q22.3-q.23.1, which contains 12 exons and 11 introns. The cDNA is 1.5 kb and encodes a 427 amino acid residue peptide; the enzyme is a homotetramer of four identical 41 kDa subunits. It cleaves four carbon substrates into two molecules of acetyl-CoA and is thought to be primarily involved in ketone body synthesis.

Carnitine Transport Defects

Carnitine is consumed in the diet from meat, fish, and dairy products but it can also be synthesized from lysine and methionine in the liver. A primary defect in carnitine synthesis has been proposed to be a risk factor in autism in a single study but no follow-up studies have been published, and no other systemic synthetic defects have been reported [35]. Primary/systemic carnitine deficiency more typically refers to a defect in tissue OCTN2 [34]. Tissues such as muscle and heart cannot synthesize carnitine and must take up circulating carnitine via the transporter. Defects in OCTN2 lead to carnitine depletion in muscle, liver, and heart. Muscle depletion of carnitine leads to weakness; cardiac depletion causes cardiomyopathy and arrythmias; and hepatocellular depletion can cause asymptomatic hepatic steatosis or fulminant Reye-like syndrome with hepatic failure, any of which can be precipitated during stress or minor periods of starvation. Sudden unexpected death in infancy or older individuals has been reported in patients with OCTN2 defects. A common variant is present in Faroe Islands and may remain asymptomatic until adulthood.

Carnitine Palmitoyltransferase-IA

Deficiency of CPT-IA causes fasting hypoketotic hypoglycemia and occasionally hepatocellular damage. The heart and muscle are rarely involved because CPT-IA is the liver isoform of the enzyme. One case of CPT-IA deficiency presented with muscle pain and rhabdomyolysis in a previously asymptomatic adult [36]. Hepatic presentation of CPT-I deficiency in one sibling and muscle presentation in another sibling in a family has also been reported. In CPT-IA deficiency, total and free plasma carnitine are characteristically elevated; this observation is unique among all known FAO defects and is otherwise seen only in association with carnitine supplementation or renal insufficiency.

Carnitine Palmitoyltransferase-II

An infantile form of complete CPT-II deficiency presents with severe hypoglycemia, myopathy, and cardiomyopathy and often results in death [37]. Plasma free carnitine is low with elevated acylcarnitines. A milder form is caused by a common mutation that leads to residual enzyme activity and presents with lipid myopathy, recurrent rhabdomyolysis, and myoglobinuria in young adults. The combination of one severe mutation and one mild leads to onset of disease in later infancy or early childhood. Carnitine/acylcarnitine translocase deficiency is clinically similar to infantile CPT-II deficiency. Patients often present in the neonatal or infancy period with severe hypoketotic hypoglycemia; hyperammonemia, probably from liver failure, and elevated acylcarnitines associated with intercurrent illness [26]. Cardiac arrhythmias are common.

Very-Long-Chain Acyl-CoA-Dehydrogenase Deficiency

Deficiency of VLCAD presents with a spectrum of symptoms ranging from a severe infantile presentation with hypoglycemia, hyperammonemia, and liver steatosis and dysfunction similar to MCAD deficiency, to recurrent rhabdomyolysis in adolescents and young adults mimicking CPTII deficiency [38, 39]. Hypoglycemia is rare in late onset patients. Early onset patients typically transition to the muscle phenotype later in childhood (typically age four to six years). Cardiomyopathy may develop at any age and may be dilated or hypertrophic [38, 40]. Cardiac arrhythmias have been reported. Cardiomyopathy may resolve with the initiation of treatment with carbohydrates and MCT supplements but may also progress to demise. VLCAD deficiency is now diagnosed by newborn screening prior to the onset of symptoms in the USA and many other countries [39, 41].

During acute illness, characteristic plasma long-chain acylcarnitines are increased including C14:0, C14:1, C16:0, C16:1, C18:0, C18:1 and C18:2 acylcarnitine esters [42]. Rhabdomyolysis and myoglobinuria are often related to prolonged exercise, an illness with fever or poor oral intake, cold temperatures, or a combination of these factors. Creatine phosphokinase can be extremely elevated and myoglobinuria can cause renal failure requiring hemodialysis.

Adults most often experience exercise intolerance and myoglobinuria without hypoglycemia, but may also have muscle pain and weakness without frank rhabdomyolysis. Symptoms may begin in adolescence and continue intermittently for decades prior to a diagnosis of VLCAD deficiency. Patients may also present with an acute rhabdomyolysis event and no prior history of muscle disease [38–40]. More than 100 different mutations spanning the ACADVL gene have been reported and no common mutation has been identified [43, 44].

Acyl-CoA Dehydrogenase 9

Deficiency of ACAD9 was originally described in three patients with a clinical presentation similar to other FAO disorders including hypoketotic hypoglycemia, fulminant liver failure, and dilated cardiomyopathy [45]. In addition, neurological symptoms including cerebral stroke were present.

Since the initial report, ACAD9 has been shown to be an essential protein for mitochondrial complex 1 assembly and that this function is separate from its enzymatic one [46]. Mutations in ACAD9 are now recognized as a common cause of mitochondrial disease ETC complex 1 deficiency [19]. Two major phenotypes are seen; predominantly cardiomyopathy with milder muscular symptoms, and a more systemic disease as seen in the original patients. The former phenotype is associated with mutations that preserve the ACAD enzymatic functions while the latter are caused by mutations that affect both functions [46]. Many of the isolated respiratory chain deficient patients respond to riboflavin supplementation [19].

Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase

Mutations in the HADHA and HADHB genes can lead to a relative selective loss of the LCHAD activity of TFP or loss of all three TFP activities [47, 48]. All patients share some aspects of clinical course with other fatty acid oxidation disorders (FAODs) including hypoketotic hypoglycemia associated with metabolic acidosis, hepatocellular dysfunction, and sometimes cardiomyopathy. Rhabdomyolysis is common in patients beyond the first few years of life. However, pigmentary retinopathy with vision loss during childhood and progressive peripheral neuropathy are complications specific to LCHAD/TFP deficiency [49]. The former tends to be more severe in isolated LCHAD deficiency, while the latter is usually worse in complete TFP deficiency. While the underlying etiology of the hypoketotic hypoglycemia appears to be a deficiency of energy from depleted carbohydrate stores, the etiology of the other complications is not completely understood [50]. Elevated plasma long-chain hydroxyacylcarnitines, including C16:0-OH, C16:1-OH, C18:0-OH, C18:1-OH, C18:2-OH, are diagnostic of LCHAD or TFP deficiency.

Isolated LCHAD deficiency results from the selective loss of LCHAD activity and relative preservation of hydratase and 3-ketoacyl-CoA thiolase activity of TFP. A common missense mutation, (c.G>C1528) accounts for 87% of the alleles in LCHAD deficiency, affecting the catalytic residue in the active site for this activity [26, 51]. This common mutation decreases LCHAD activity but minimally alters the level of protein of both mitochondrial TFP subunits. Other mutations in both the α- and β-subunits have been shown to result in the loss of all three enzyme activities and TFP deficiency.

There appear to be some clinical differences between LCHAD and TFP deficiencies. Patients who are homozygous for the common LCHAD mutation are more likely to develop progressive chorioretinopathy leading to vision impairment than patients who have β-subunit mutations [50]. The infantile form of TFP deficiency appears to be severe, often resulting in neonatal death [47, 48]. Milder forms of TFP deficiency are associated with severe peripheral neuropathy, impairing mobility, more often than isolated LCHAD deficiency. Patients who are heterozygotes for the common LCHAD mutation and a private mutation may develop some aspects of both the chorioretinopathy with vision loss and peripheral neuropathy with impaired mobility.

Medium-Chain Acyl-CoA Dehydrogenase Deficiency

Deficiency of MCAD is the most common defect in FAO, with an incidence of approximately one in 9,000 live births in western European populations [52]. It is less common in Asians and Africans and the overall incidence in the USA is ~1:26,000 births. The typical acute clinical presentation includes fasting or illness induced hypoketotic hypoglycemia, often associated with metabolic acidosis and hepatocellular dysfunction [53]. Hyperammonemia may be present. Micro- and/or macrovascular steatosis is often observed in symptomatic patients, but typically resolves when patients are well. Prior to the advent of newborn screening for this disorder, as many as half of all patients presented with sudden unexplained death [54]. Even with newborn screening, sudden unexpected death may occur in the first few days after birth (almost exclusively in breast-fed infants) prior to the report of the newborn screen results [55]. Patients become less prone to episodes of decompensation in early childhood (typically by age six to seven years), and symptoms in adults are rare but may be induced by prolonged fasting or severe intercurrent illness. Asymptomatic adults have been described.

In 1990, a common mutation in ACADM, a c.985A>G point mutation, was identified resulting in a lysine to glutamic acid substitution at position 304 of the mature protein [56]. This missense mutation was subsequently shown to account for a large majority of the alleles in identified patients, although with newborn screening identifying affected individuals, the prevalence of this mutation has changed somewhat [52]. Other point mutations have been identified in the first 11 of 12 exons but the c.985A>G point mutation still accounts for the majority of disease-causing alleles [57]. The c.985A>G mutation leads to the normal transcription and translation of a mutant protein that is appropriately imported into mitochondria, but then fails to fold properly to a mature monomer [58].

In countries with newborn screening for MCAD deficiency, most affected infants remain asymptomatic and have an excellent outcome [53]. The vast majority of affected patients who are treated are completely healthy, although they may need to receive intravenous fluids containing glucose when ill or fasting to prevent metabolic crisis. Some neurological impairment has been noted later in life in MCAD-deficient patients, particularly in unscreened populations, but the evidence for neurological impairment in treated patients who have avoided metabolic crisis is scant. Deficits in learning and speech/language communication have been reported in symptomatic individuals prior to newborn screening but not in screened and early effectively treated populations [59].

Short-Chain Acyl-CoA Dehydrogenase Deficiency

Deficiency of SCAD was originally described in patients with a broad spectrum of symptoms including failure to thrive, hypoglycemia, hypotonia, developmental delay, and seizures, but based on patients identified through newborn screening, it is now generally believed to be a biochemical of no clinical relevance [60]. In fact, it is likely that most patients described in the literature with SCAD deficiency had ethylmalonic aciduria due to the presence of one of two common sequence variants in the ACADS gene (c.511>CT and c.625 C>A) [61]. These variants slightly affect enzyme activity leading to increased production of ethylmalonic acid, but are of no clinical consequence [62]. As these variants are present in up to 14% of the general population the associated ethylmalonic aciduria can be coincidentally identified in any clinical scenario.