Disorders of Lipid Metabolism

C H A P T E R

50

Disorders of Lipid Metabolism Stefano Di Donato and Franco Taroni Fondazione IRCCS Istituto Neurologico “Carlo Besta,” Milan, Italy

INTRODUCTION Abnormalities of lipid catabolism as possible causes of human disease were first suggested in the late 1960s by morphological observations of excessive accumulation of lipid droplets within muscle fibers of a young woman who had attacks of muscle weakness lasting from a few weeks to several years.1 Lipid myopathy associated with muscle pain and cramps and occasional myoglobinuria was later described by Engel (1970) in two twin sisters who, when fed with long-chain fatty acids (LCFAs), showed low ketone production but generated a normal amount of ketones after a medium-chain fatty acid meal.2 These findings suggested that the patients might suffer from a specific defect in the oxidation of LCFAs. Following these seminal observations, primary lipid myopathy associated with pathogenic carnitine deficiency in muscle was first described by Angelini and Engel (1973),3 and carnitine palmitoyltransferase (CPT) deficiency was discovered as the first enzyme defect of mitochondrial fatty-acid (FA) oxidation by DiMauro and Melis-DiMauro (1973),4 in a 29-year-old man who suffered from recurrent episodes of muscle pain and pigmenturia triggered by prolonged exercise. Since this latter description, 18 autosomal recessive defects have been identified, involving almost all enzyme steps in the pathway5–9 (see Tables 50.1 and 50.2, pages 560 and 562, respectively). With the exception of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, which has a high frequency (1 in 10,000–30,000 births) among Northern European Caucasians,10,11 these disorders are uncommon and the prevalence rate is unknown for most of them.

PATHOPHYSIOLOGY The immediate source of chemical energy for muscle contraction is the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). ATP can be regenerated from ADP and the high-energy compound phosphocreatine, but during long-term exercise the rephosphorylation of ADP to ATP requires the utilization of other fuels, such as carbohydrate, FA, and ketones. Although anaerobic glycogenolysis in the cytosol can generate ATP up to 100 times faster than aerobic oxidation of glucose, it yields only 2 moles of ATP per mole of glucose as compared to 38 moles of ATP per mole of glucose yielded by mitochondrial oxidative phosphorylation (OXPHOS). Furthermore, it rapidly leads to the accumulation of toxic fatigue-promoting metabolic end products (mainly lactic acid). Therefore, OXPHOS is the primary energy source for the regeneration of ATP during muscle work. Although both carbohydrate and fatty-acid catabolic pathways converge into acetyl-coenzyme A (acetyl-CoA) for final intramitochondrial oxidation through the tricarboxylic acid cycle (TCA) and the respiratory chain (OXPHOS), the pattern of muscle fuel utilization is determined primarily by the intensity and duration of exercise. At rest, most muscle energy is provided by mitochondrial oxidation of LCFA (C14–C20) and the respiratory quotient (respiratory exchange ratio; RER) of resting muscle is close to 0.8, indicating an almost total dependence on the oxidation of FA.6 During the early phase of exercise (up to ≈ 45 minutes), energy is derived mainly from catabolism of muscle glycogen stores and blood glucose. After approximately 90 minutes of exercise at an intensity of ≈ 70% of maximum oxygen uptake (VO2 max), muscle glycogen stores are depleted and there is a gradual shift from glucose to fatty-acid utilization. After a few hours, about 70% of the skeletal muscle energy requirement is met by the oxidation of fatty acids. Although the mobilization and rate of energy production from fatty acids are slow as compared with those of glycogen, complete oxidation of a fatty-acid molecule is highly exergonic. For example, the oxidation of one molecule of palmitate (C16:0) has a net yield of 129 ATPs.6 Heart is also largely dependent on LCFA oxidation for its functional activity.6 Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease http://dx.doi.org/10.1016/10.1016/B978-0-12-410529-4.00050-4

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© 2015 Elsevier Inc. All rights reserved.

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50.  Disorders of Lipid Metabolism

TABLE 50.1  Main Clinical Features of Fatty-Acid β-Oxidation Disorders Myopathic symptoms

Disorder

Acutea

Chronic

Hepatic symptoms

Cardiomyopathy

Metabolic Hypoketotic encephalo­ hypoglycemia pathyb

Abnormal organic acids

Other features

MIM No.c

LONG-CHAIN FATTY-ACID OXIDATION Fatty-acid transport CT

−

++

+++

+

+

−

Endocardial fibroelastosis

212140

CPT1

−

−

−

+++

+++

−

Renal tubular acidosis

255120

CACT

?

++

+++d

+++

+++

+/−

212138

CPT2, type 1 (muscular)

+++

−

−

−

−

−

255110 600650

++

++

++

++

+/−

Recurrent pancreatitis

600649 600650

++

+++

+++

+++

+/−

Brain and kidney dysplasia

600649

CPT2, type 2 +/− (hepatocardiomuscular) CPT2, type 3 (lethal neonatal)

−

β-Oxidation spiral VLCADe

+

++

++

++

++

+++

201475

MTP, type 1 (LCHAD)

++

++

++

+++

+++

+++

Retinitis pigmentosa, 600890 AFLP, HELLP, lactic acidemia

MTP, type 2 (LCEH/ LCHAD/ LCKT)

++

++

++

+++

+++

+++

Retinitis pigmentosa, 143450 peripheral neuropathy, hypoparathyroidism

+++

+++

201450

+/−

++

Hypotonia, hypertonia, mental retardation

201470

MEDIUM- AND SHORT-CHAIN FATTY-ACID OXIDATION MCAD

+/−

+/−

−

+++

SCAD

−

?

f

−

+/−

HAD

−

−

−

++

+/−

+++

Congenital hyperinsulinism

231530

MCKAT

+++

−

−

−

++

+++

Vomiting, hyperammonemia

602199

g

UNSATURATED FATTY-ACID OXIDATION 2,4-Dienoyl− CoA reductase ACAD9j

+/−

++h

−

−

−

−i

Microcephaly, dysmorphism

222745

+/−

+

++

++

++k

Brain atrophy, cerebellar infarct, chronic thrombocytopenia

611126

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561

Pathophysiology

TABLE 50.1  Main Clinical Features of Fatty-Acid β-Oxidation Disorders—cont’d Myopathic symptoms

Disorder

Acutea

Chronic

Hepatic symptoms

Cardiomyopathy

Metabolic Hypoketotic encephalo­ hypoglycemia pathyb

Abnormal organic acids

Other features

MIM No.c

Congenital anomalies, renal dysplasia, dysmorphism

231680 130410 231675

MULTIPLE ACYL-COA DEHYDROGENATION DEFECTS ETF or − ETF:QO, severe

−

−

+++

+++

+++l

ETF or − ETF:QO, mild

+

+/−

+++

+++

+++l,m

+++

−

+++

+

+++

Riboflavinresponsive MADDn

−

231680 130410 231675 Leukodystrophy, coenzyme Q10 deficiency

231680

Abbreviations: CT, carnitine transporter; CPT, carnitine palmitoyltransferase; CACT, carnitine/acylcarnitine translocase; VLCAD, very long-chain acyl-CoA dehydrogenase; MTP, mitochondrial trifunctional protein; LCHAD, long-chain 3-hydroxyacyl-CoA dehydrogenase; LCEH, long-chain 2-enoyl-CoA hydratase; LCKT, long-chain 3-ketoacyl-CoA thiolase; MCAD and SCAD, medium- and short-chain acyl-CoA dehydrogenase, respectively; MCKAT, medium-chain 3-ketoacyl-CoA thiolase; HAD, l-3-hydroxyacyl-CoA dehydrogenase; ACAD9, acyl-CoA dehydrogenase 9; ETF, electron transfer flavoprotein; ETF:QO, ETF:coenzyme Q oxidoreductase; MADD, multiple acyl-CoA dehydrogenation deficiency; AFLP, acute fatty liver of pregnancy; HELLP, hypertension or hemolysis, elevated liver enzymes, and low platelets. a Myoglobinuria; bReye-like episodes; cMendelian Inheritance in Man (MIM; McKusick VA. Mendelian Inheritance in Man: A Catalog of Human Genes and Genetic Disorders, 12th ed. Baltimore: Johns Hopkins University Press; 1998); Online MIM database (OMIM™): http://www.ncbi.nlm.nih.gov/omim; dventricular arrhythmias in most cases; e includes cases previously reported as defects of the long-chain acyl-CoA dehydrogenase; fhypotonia; gketotic hypoglycaemia; hthe only patient reported had persistent hypotonia in the neonatal period; iurinary excretion of the unusual carnitine ester 2-trans,4-cis-decadienoylcarnitine; jmostly active against unsaturated long-chain acyl-CoA substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA); kabnormal unsaturated long-chain acylcarnitines (C18:1 and C18:2) in postmortem liver extract; lglutaric aciduria type II (GAII); methylmalonic-adipic aciduria; n some patients have mutations in the ETFDH (ETF:QO) gene (see text for details); other patients have been reported to have Coenzyme Q deficiency and mutations in the ETFDH gene (see text for details).

Mitochondrial oxidation of lipids is a complex process that requires a series of enzymatic reactions8,10,12,13 (Figure 50.1). Schematically, plasma free FAs delivered into the cytosol are first activated to their corresponding acyl-coenzyme A (CoA) thioesters at the outer mitochondrial membrane by acyl-CoA synthetase(s). Unlike shortchain (C4–C6) and medium-chain (C8–C12) acyl-CoAs, long-chain (C14–C20) acyl-CoAs cannot enter mitochondria directly. The mitochondrial CPT enzyme system, in conjunction with a carnitine/acylcarnitine translocase (CACT), provides the active carnitine-dependent mechanism whereby long-chain acyl-CoAs are transported from the cytosolic compartment into the mitochondrion, where β-oxidation occurs. l-carnitine is supplied for this reaction by a plasma-membrane sodium-dependent carnitine transporter (CT). Once in the mitochondria, FAs are oxidized by repeated cycles of four sequential reactions, acyl-CoA dehydrogenation, 2-enoyl-CoA hydration, l-3-hydroxy-acylCoA dehydrogenation, and 3-ketoacyl-CoA thiolysis. The final step of each cycle in the β-oxidation spiral is the release of one molecule of acetyl-CoA and a fatty acyl-CoA, which is two carbon atoms shorter. Each reaction is catalyzed by multiple enzymes that exhibit partially overlapping chain-length specificity.12 Complete catabolism of long-chain acyl-CoAs in mitochondria is accomplished by the action of two distinct, albeit coordinated, β-oxidation systems.14 One is located on the mitochondrial inner membrane and is specifically involved in the oxidation of LCFA. The other system, composed of soluble enzymes located in the mitochondrial matrix, is responsible for the β-oxidation of medium- and short-chain acyl-CoAs (Figure 50.1). Finally, mitochondrial FA β-oxidation is tightly coupled to both the tricarboxylic acid (TCA) and the respiratory chain. Thus, while acetyl-CoA released can enter the TCA cycle, the electrons of the flavin adenine dinucleotide (FAD)-dependent acyl-CoA dehydrogenases and the nicotinamide adenine dinucleotide (NAD+)-dependent l-3-hydroxy-acyl-CoA dehydrogenases are transferred to the respiratory chain.

Control of FA β-Oxidation and Synthesis The regulatory control of FA oxidation and synthesis occurs at multiple levels, and with diverse mechanisms in different organs: it is a highly complex interactive system, not yet fully understood, which involves transcriptional and nontranscriptional components.15 Essential information in the control of lipid metabolism relevant to this chapter is given below.

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50.  Disorders of Lipid Metabolism

TABLE 50.2  Molecular Genetics of Fatty-Acid β-Oxidation Disorders Deficiency

MIM no.a

Gene name

Chromosomal localization

Gene structure

cDNA, coding region

Mutations identified

Prevalent mutations

LONG-CHAIN FATTY-ACID OXIDATION Fatty-acid transport CT

212140

SLC22A5

5q33.1

10 exons

1674 bp

++

None

CPT1

255120

CPT1A

11q13.1-q13.5

20 exons

2322 bp

+

None

CACT

212138

SLC25A2

3p21.31

9 exons

903 bp

+

None

CPT2

255110 600650 600649

CPT2

1p32

5 exons

1974 bp

++

c.439C > T p.Ser113Leu

VLCAD

201475

ACADVL

17p11.2-11.13

20 exons

1968 bp

+++

None

MTP, type 1 (LCHAD)

600890

HADHA

2p23

20 exons

2289 bp

+

c.1528G > C p.Glu474Gln

MTP, type 2 143450 (LCEH/LCHAD/ LCKT)

HADHB

2p23

16 exons

1422 bp

+

None

β-Oxidation spiral

MEDIUM- AND SHORT-CHAIN FATTY-ACID OXIDATION MCAD

201450

ACADM

1p31

12 exons

1263 bp

+++

c.985A > G p.Lys304Glu

SCAD

201470

ACADS

12q22

10 exons

1239 bp

++

c.625G > A p.Gly185Ser c.511C > T p.Arg147Trp

HAD

231530

HADH

4q22-q26

8 exons

945 bp

+

None

MCKAT

602199

−

n.d.

n.d.

n.d.

−

n.d.

UNSATURATED FATTY-ACID OXIDATION 2,4-Dienoyl-CoA reductase

222745

DECR1

8q21.3

10 exons

1008 bp

−

n.d.

ACAD9

611126

ADAD9

3q26

22 exons

1866 bp

+

none

MULTIPLE ACYL-COA DEHYDROGENATION DEFECTS ETF α subunit

231680

ETFA

15q23-25

12 exons

1002 bp

+

c.797C > T p.Thr266Met

ETF β subunit

130410

ETFB

19q13.3

6 exons

768 bp

+

None

ETF:QO

231675/231680

ETFDH

4q32-q35

13 exons

1854 bp

+

None

Riboflavinresponsive MADDb

231680

ETFDH

4q32-q35

13 exons

1854 bp

++

None

Abbreviations: CT, carnitine transporter; CPT, carnitine palmitoyltransferase; CACT, carnitine/acylcarnitine translocase; VLCAD, very long-chain acyl-CoA dehydrogenase; MTP, mitochondrial trifunctional protein; LCHAD, long-chain 3-hydroxyacyl-CoA dehydrogenase; LCEH, long-chain 2-enoyl-CoA hydratase; LCKT, longchain 3-ketoacyl-CoA thiolase; MCAD and SCAD, medium- and short-chain acyl-CoA dehydrogenase, respectively; HAD, l-3-hydroxyacyl-CoA dehydrogenase; MCKAT, medium-chain 3-ketoacyl-CoA thiolase; ETF, electron transfer flavoprotein; ETF:QO, ETF:coenzyme Q oxidoreductase; MADD, multiple acyl-CoA dehydrogenation disorders; n.d., not determined. a Mendelian Inheritance in Man (MIM; McKusick VA. Mendelian Inheritance in Man: A Catalog of Human Genes and Genetic Disorders, 12th ed. Baltimore: Johns Hopkins University Press; 1998); Online MIM database (OMIM™): http://www.ncbi.nlm.nih.gov/omim; bsome patients have mutations in the ETFDH gene (see text for details), other patients have been reported to have coenzyme Q deficiency and mutations in the ETFDH gene (see text for details).

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Pathophysiology

563

FIGURE 50.1  Schematic representation of the functional and physical organization of fatty-acid β-oxidation enzymes in mitochondria. Abbreviations: CT, plasma membrane high-affinity sodium-dependent carnitine transporter (OCTN2); CPT1, carnitine palmitoyltransferase 1; CACT, carnitine/acylcarnitine translocase; CPT2, carnitine palmitoyltransferase 2; VLCAD, LCAD, MCAD, SCAD, very-long-, long-, medium-, and short-chain acyl-CoA dehydrogenase, respectively; ACAD9, acyl-CoA dehydrogenase 9; MTP, mitochondrial trifunctional protein; Hydratase, 2-enoyl-CoA hydratase; HAD, l-3-hydroxyacyl-CoA dehydrogenase; KT, 3-ketoacyl-CoA thiolase; ETF, electron transfer flavoprotein (ox, oxidized; red, reduced); ETF:QO, ETF:coenzyme Q oxidoreductase; I, respiratory chain complex I (NDH, NADH:coenzyme Q reductase); II, respiratory chain complex II (SDH, succinate dehydrogenase); CoQ, coenzyme Q; III, respiratory chain complex III (b, cytochrome b; c1, cytochrome c1); Cyt c, cytochrome c; IV, respiratory chain complex IV (cytochrome c oxidase) (a, cytochrome a; a3, cytochrome a3); V, respiratory chain complex V (ATP synthase). Enzymes which use FAD as a coenzyme are indicated in red.

Hormonal and Allosteric Control of FA Oxidation in Liver In the fed state, under low glucagon/insulin ratio, the liver avidly takes up glucose from blood. Glucose is partly degraded and oxidized and partly stored as glycogen. FA and triglyceride liver synthesis is high, whereas FA oxidation and ketone body production are shut off because of the high cellular levels of malonyl-CoA, the most potent allosteric suppressor of CPT1 activity. Malonyl-CoA concentration is in turn increased due to the abundance of citric acid and TCA substrates and to the activation of acetyl-CoA carboxylase. 16 In physiological fasting conditions, such as night fasting, or forced nutrient deprivation, glucagon/insulin ratio dramatically increases, and glucagon signaling induces activation of the 5′-AMP-activated protein kinase (AMPK). Activated AMPK phosphorylates ­acetyl-CoA carboxylase enzymes, promptly turning off their activity. These metabolic events cause a dramatic drop in malonyl-CoA concentration, hence relieving the inhibition on CPT1 and turning on ketone body (acetoacetate and β-hydroxybutyrate) production. Ketone bodies represent the vital energy substrate

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50.  Disorders of Lipid Metabolism

for all peripheral organs in the fasting state, except for the brain, which primarily requires oxygen and glucose for survival.16 Therefore, the essential mechanisms created by nature to prevent episodes of life-threatening ­hypoglycemia during fasting are embodied in the metabolic-allosteric mechanisms determined by malonyl-CoA concentration in liver cells and by the hormonal balance governed by pancreatic β-cells.

Transcriptional Control of Mitochondrial β-Oxidation At the transcriptional level, several hormone nuclear receptors (NRs), including the peroxisome proliferatoractivated receptor alpha (PPARα) and the estrogen-related receptor alpha (ERRα), govern FA β-oxidation in mitochondria, although the full set of known NRs coordinately regulates in a more general sense metabolic activation, including mitochondrial mass and respiratory function.17,18 As regards lipid metabolism, PPARα and ERRα target a series of genes encoding FA oxidation enzymes.15 However, induction of an active transcriptional program implies the assembly of large multiprotein complexes in order to turn on properly the transcription– translation of genes encoding β-oxidation enzymes.17 Among the proteins of the transcription complex, a crucial role is played by the transcriptional coactivators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and beta (PGC1β).18 PGC1α is the most potent activator of the transcriptional activity driven by PPARα and ERRα.17,18 PGC1α activity is in turn controlled by the reversible side-chain acetylation of its lysine residues, a process that depends upon two additional enzymes: the GCN5 acetyltransferase and the SIRT1 deacetylase.15,17 Notably, SIRT1 is induced by glucose/nutrient deprivation, AMPK activation, and activation of cAMP-dependent protein kinase A (cAMP/PKA).15 Therefore, the critical steps for FA enzymes synthesis governed by SIRT1 activation/PGC1α deacetylation in muscle and heart is reminiscent of the process ruled by ­malonyl-CoA and glucagon/insulin ratio in the liver. Notably, a recent report describes the power of oleic acid, but not of saturated long-chain fatty acids, to stimulate FA mitochondrial β-oxidation in skeletal muscle through cAMP/PKA-mediated SIRT1 activation.15 The oleic ­acid-dependent activation of FA mitochondrial oxidation in skeletal muscle is a neat example of an additional signaling–transcriptional control of this crucial mitochondrial function.15

CLINICAL FEATURES Defects of FA mitochondrial β-oxidation are autosomal recessive disorders of infancy and childhood, though some patients present later in life. Their classification and main clinical and genetic features are illustrated in Tables 50.1 and 50.2. Overall, the clinical syndromes associated with FA oxidation disorders result from the failure of FA-oxidizing tissues to respond to increased energy demands. Clinical manifestations range from a predominantly myopathic disease, either acute or chronic, to life-threatening systemic metabolic dysfunction (Table 50.3). Symptomatic hypoglycemia, characteristically associated with impaired ketogenesis, is often the earliest clinical manifestation and can be observed in nearly all these disorders.14 Recurrent episodes of hypoketotic hypoglycemia, with or without concomitant brain involvement, are the most common presentation in the newborn or infant. Nausea and vomiting, hypotonia, drowsiness, and coma are also frequent. Sometimes, attacks are triggered by fasting or minor viral infections. The acute and frequently life-threatening presentation in early infancy requires differential diagnosis from other encephalopathies of infancy because: 1) a few defects of β-oxidation can be effectively cured, such as carnitine deficiency and riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency; and 2) early diagnosis may help to prevent acute metabolic attacks, mental retardation, epilepsy, severe brain damage, and death.19–21 Some infants survive the acute metabolic attacks but show poor growth, impaired psychomotor development, dystonia, spastic tetraplegia, and intractable seizures. Nervous system involvement is usually secondary to severe acidotic and hypoglycemic attacks, though patients with trifunctional protein deficiency may have retinitis pigmentosa and peripheral neuropathy.14,22,23 Infants with severe defects of CPT2, ETF, or ETF:QO, however, can present congenital malformations of the brain (microgyria, neuronal heterotopia) and, sometimes, facial dysmorphism reminiscent of Zellweger syndrome, suggesting that LCFA may play a role during human development.24 In addition to metabolic symptoms, patients often have cardiomyopathy; primary carnitine deficiency, carnitine/acylcarnitine translocase deficiency, CPT2 deficiency, VLCAD deficiency, and MTP deficiency are all associated with various forms of heart disease.14,21 Patients with onset in late infancy, childhood, or adulthood tend to have more chronic disorders characterized by progressive myopathy or cardiomyopathy, sometimes associated with mild metabolic symptoms, such as nausea

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Clinical features

565

TABLE 50.3  C  linical Features Associated with Mitochondrial Fatty-Acid β-Oxidation Disorders HEPATIC SIGNS Hypoglycemia associated with low ketones (hypoketotic hypoglycemia) Reye-like syndrome Steatosis Acute hepatic failure Sudden infant death syndrome (SIDS) MUSCLE SIGNS Hypotonia Weakness and wasting Proximal myopathy with lipid storage Exercise intolerance and muscle pain with increased levels of creatine kinase Episodic rhabdomyolysis (with occasional paroxysmal myoglobinuria) CARDIAC SIGNS Hypertrophic and dilated cardiomyopathy Progressive heart failure Arrhythmias Cardiac arrest Sudden infant death syndrome (SIDS) NERVOUS SYSTEM SIGNS Permanent brain damage due to hypoglycemia, arrhythmias, or cardiac arrest Microgyria, cortical atrophy, and neuronal heterotopia Pigmentary retinopathy Peripheral sensorimotor neuropathy MALFORMATIONS Renal dysplasia and nephromegaly* Polycystic kidney Facial dysmorphism Brain malformations *

Proximal and distal tubulopathy is observed in CPT1 deficiency.

and drowsiness, or with altered laboratory tests, such as hypoglycemia or poor rise of blood ketone concentrations in provocative tests. Disorders of lipid mitochondrial metabolism may cause two main clinical syndromes in muscle, namely 1) progressive weakness with hypotonia (e.g., carnitine transporter and carnitine/acylcarnitine translocase defects) or 2) acute, recurrent, reversible muscle dysfunction with exercise intolerance and acute muscle breakdown (rhabdomyolysis) with myoglobinuria (e.g., deficiencies of CPT2, very-long-chain acyl-CoA dehydrogenase, or trifunctional protein).6,7 Approximately 40% of patients affected with all kinds of FA oxidation disorders, except CPT1 and MCAD deficiencies, present with significant muscular involvement.19 Because of the dependence of heart and skeletal muscle upon LCFA oxidation, cardiomyopathy, typically hypertrophic but sometimes dilated, and skeletal muscle myopathy, either chronic (lipid storage myopathy) or acute (paroxysmal myoglobinuria), are commonly observed in LCFA oxidation defects while they are extremely rare in disorders of medium- and shortchain FA oxidation.21 Because clinical presentation is of limited help in differential diagnosis, the only way to reach a definitive diagnosis is to analyze body fluids for accumulating metabolites and to study tissues for specific enzymes of fatty acid metabolism. Because most of the organic acids accumulating in β-oxidation defects are effectively cleared from the blood by the kidneys, gas chromatography–mass spectrometry (GC–MS) analysis of 24-hour urine specimens usually reveals a pattern of metabolites characteristic of a specific disease and is therefore the test of choice.5 When available, analysis of plasma acylcarnitine profile by tandem (MS/MS) or electrospray mass spectrometry is the most specific and direct approach for the specific diagnosis of most of the FA oxidation disorders.5 It is very sensitive and

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can be easily performed on Guthrie cards for newborn screening.11 Finally, genetic testing is now available for almost all these disorders, and prevalent mutations have been identified in some of them which makes molecular screening feasible and cost-effective.20,25

DEFECTS OF MITOCHONDRIAL FATTY-ACID OXIDATION Carnitine Transporter Deficiency (Primary Carnitine Deficiency) l-carnitine (β-hydroxy-γ-N-trimethylamino-butyrate) is required for the active transport of LCFA into mitochondria. Primary carnitine deficiency (PCD) is characterized by increased urinary carnitine loss and severely decreased carnitine concentration in plasma, heart, and skeletal muscle. The disease is autosomal recessive and has a frequency of 1 : 37,000–1 : 100,000 newborns, as determined by neonatal screening of carnitine levels.26 CLINICAL FEATURES

Two major clinical presentations are associated with PCD.10,26 The most common phenotype is characterized by slowly progressive hypertrophic or dilated cardiomyopathy with lipid storage myopathy (Figure 50.2A and B), occurring between 1 and 7 years of age. A second phenotype, more frequent before 2 years of age, is characterized by acute recurrent episodes of nonketotic hypoglycemic encephalopathy. These two phenotypes are not mutually exclusive, as both metabolic and cardiomuscular presentations have been described in some families.6 LABORATORY FINDINGS

PCD has to be distinguished from secondary carnitine deficiency that can be associated with a number of acquired or inherited diseases, including other FA oxidation defects.6,27 In PCD, carnitine content is very low (< 5% of normal) both in tissues (muscle, heart, liver) and in plasma, and analysis of plasma and urine does not show an abnormal

FIGURE 50.2  Muscle biopsies from patients with fatty-acid oxidation defects. (A) and (B) Lipid storage myopathy in a patient with primary carnitine deficiency (PCD) caused by a defect of the high-affinity plasma carnitine transporter (CT); (A) modified Gomori trichrome staining showing numerous vacuoles mostly in type 1 fibers. ×160. (B) Oil Red O stain showing numerous large lipid droplets within fibers. ×250. (C) Recurrent paroxysmal myoglobinuria in a young adult with CPT2 deficiency, harboring the common p.Ser113Leu mutation in the CPT2 gene. Muscle biopsy performed 10 days after an acute episode shows mild nonspecific morphological alterations. There is evidence of fiber loss and modest variability of fiber diameter. Some fibers show central nuclei. Hematoxylin & eosin, ×160. (D) and (E) Recurrent paroxysmal myoglobinuria and interictal chronic proximal myopathy in a young woman with VLCAD deficiency. (D) Hematoxylin & eosin stain shows mild nonspecific morphological alterations. There is fine vacuolization in some fibers and fiber diameter variability. ×160; (E) Oil Red O stain shows signs of mild lipid accumulation with numerous fine droplets within most fibers. Lipid droplets exhibit a subsarcolemmal distribution. ×250.

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Defects of mitochondrial fatty-acid oxidation

567

acylcarnitine profile nor dicarboxylic aciduria which are usually seen in patients with other FA oxidation defects.6 Once suspected, the transporter defect should be ultimately confirmed by carnitine uptake assay in cultured skin fibroblasts28 or by molecular analysis.29 MOLECULAR GENETICS

PCD is caused by mutations in the SLC22A5 gene encoding the high-affinity plasma membrane carnitine transporter OCTN2.30 Most of the mutations are nonsense mutations associated with no residual carnitine transport activity.26,31 In a few cases, “leaky” missense mutations associated with residual carnitine transport activity have been identified.29 THERAPY

If therapy is started before irreversible organ damage occurs, PCD patients respond very well to high-dose oral l-carnitine supplementation (usually 100–600 mg per kg per day),26,32 which may avoid cardiac transplant. Hypoglycemic episodes also tend to disappear.26

Carnitine Palmitoyltransferase Deficiencies The carnitine palmitoyltransferase (CPT) system is composed of two distinct acyltransferases, CPT1 on the outer mitochondrial membrane, and CPT2 on the inner mitochondrial membrane.33 CPT1 is expressed in at least three tissue-specific isoforms encoded by distinct genes,33 whereas CPT2 is present in all tissues in a single form encoded by a gene on chromosome 1.33,34 CPT1 Deficiency This disorder is commonly referred to as the “hepatic” form of CPT deficiency. The disease manifests before the second year of life with encephalopathy, fasting hypoglycemia, hypoketonemia, low plasma insulin concentrations, and elevated plasma carnitine levels.19,33,35 Since the disease is caused by mutations in the CPT1A gene on chromosome 11q13.1 encoding the liver isoform of CPT1,35 there is no cardiomuscular involvement, which makes this defect unique among the disorders of LCFA oxidation19 (Table 50.1). CPT2 Deficiency CLINICAL FEATURES

Three different clinical phenotypes are associated with CPT2 deficiency (Table 50.1): 1) a myopathic form with juvenile–adult onset; 2) an infantile form with hepatic, muscular, and cardiac involvement; and 3) a lethal neonatal form with developmental abnormalities. In all cases, the enzyme defect can be demonstrated in every tissue examined (e.g., skeletal muscle, liver, fibroblasts, platelets, leukocytes).6,33,36 The “muscular” form of CPT deficiency is the most common disorder of lipid metabolism in muscle, one of the most common inherited disorders of mitochondrial FA oxidation,19 and a major cause of hereditary recurrent myoglobinuria in both children and young adults.9,33,37 The clinical hallmark of the disease is paroxysmal myoglobinuria. Attacks of myoglobinuria are most often precipitated by prolonged exercise (exertional myoglobinuria).37 Prolonged fasting, infections, usually of viral etiology, and/or fever are the primary precipitating factors in the younger patients.9,33,37 In approximately 20% of cases, attacks may occur without any apparent cause. True cramps are not a feature, but patients describe instead a feeling of “tightness” and pain in exercising muscles before the appearance of myoglobinuria and weakness. Persistent weakness is very uncommon.6 The classic “muscular” form of CPT2 deficiency is usually a benign disease with a favorable evolution, provided that acute renal insufficiency, a potential complication of massive myoglobinuria, is adequately managed.38 There are usually no clinical signs of liver dysfunction. Cardiac involvement is very unusual.6 In rare cases, CPT2 deficiency can manifest as a severe life-threatening infantile hepatocardiomuscular form (CPT2 deficiency type 2), characterized by nonketotic hypoglycemia, liver failure, cardiomyopathy, and mild signs of muscle involvement, or a fatal neonatal-onset form (CPT2 deficiency type 3) with acute metabolic decompensation and features of brain and kidney dysgenesis.2,5,6,13,14,36,39–42 LABORATORY FINDINGS

Outside episodes of myoglobinuria and at rest, serum creatine kinase (CK) levels are normal. During acute episodes of rhabdomyolysis, there is a massively elevated urinary excretion of myoglobin (≥ 200 ng per mL) and greatly

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increased levels of serum CK (20–400-fold) of muscle origin (CK-MM). Prolonged fasting or mild exercise may also provoke an increase in serum CK (2- to 20-fold above normal). Glycemia, ketonemia, ketonuria, urinary organic acid profile and serum and muscle carnitine levels are usually normal. Acute tubular necrosis, a life-threatening condition, may develop in patients excreting more than 1000 ng per mL of myoglobin. Following attacks, serum CK levels usually return to normal by 8–10 weeks. Between attacks, routine laboratory tests are not contributive to the diagnosis. In most cases, muscle biopsies in interictal periods are normal or may show mild signs of muscle involvement with regenerating fibers (Figure 50.2C). Diagnosis is ultimately made by demonstrating the enzyme defect in muscle or, more conveniently, in peripheral blood leukocytes.6 However, since only one gene is associated with CPT2 deficiency, molecular genetic testing currently provides the most convenient means for noninvasive, rapid, and specific diagnosis. MOLECULAR GENETICS

More than 70 mutations in the human CPT2 gene have been identified.33,36,42,43 Although most of the mutations are “private,” a “common” mutation (p.Ser113Leu) can be identified in approximately 80% of patients with muscular CPT2 deficiency, being present in ≥ 50% of mutant alleles in patients of different ethnic origins.6,37,43,44 There is some genotype–phenotype correlation. The muscular form of the disease is always associated with residual CPT2 activity, whereas mutations that abolish enzyme activity are invariably found in patients with the lethal early-onset form.33,39,44,45 THERAPY

Effective prevention of attacks may be accomplished by instituting a high-carbohydrate diet with a low amount of long-chain fats and with frequent and regularly scheduled meals, by avoiding the known precipitating factors (fasting, cold, prolonged exercise) and by increasing slow-release carbohydrate intake during intercurrent illness or sustained exercise.19,33,41,46 More recently, agonists of PPARα such as bezafibrate have been shown to restore CPT2 activity and LCFA oxidation in fibroblasts from patients with the muscular form of CPT2 deficiency and to provide long-term subjective improvement of clinical conditions.47 These results have not been confirmed by a randomized clinical trial.48

Carnitine/Acylcarnitine Translocase Deficiency Along with infantile and neonatal CPT2 deficiency, CACT deficiency is one of the most severe mitochondrial FA oxidation defects. More than 30 patients have been reported since the first description in 1992.10,49,50 CLINICAL FEATURES

Patients exhibit life-threatening episodes in the neonatal period, characterized by neonatal distress with hyperammonemia, variable hypoglycemia, heart beat disorders, and muscle involvement with weakness and high serum CK.26 The disease is often fatal within the first 2 years of life because of the deleterious combination of energy impairment and the toxic consequences of long-chain acylcarnitine accumulation, which may cause untreatable episodes of arrhythmia.26,49,50 LABORATORY FINDINGS

Diagnosis is suspected from the abnormal plasma acylcarnitine profile with low free carnitine and elevated C16–C18.6,26,51 MOLECULAR GENETICS

More than 35 mutations in the SLC25A20 (solute carrier family 25 [carnitine/acylcarnitine translocase], member 20) gene have been reported thus far,50 most of which are private. Two-thirds of mutations are nonsense, frameshift, or splice-site mutations resulting in premature stop codons (null mutations).49 Functional analysis of missense mutations has been performed in few cases.51 Null mutations are associated with rapidly progressive disease whereas hypomorphic mutations cause a milder phenotype with a near normal development with appropriate therapy.26,51 THERAPY

Therapy is based on low-LCFA and high-carbohydrate diet in an intensive protocol characterized by frequent or continuous feeding.6,26 Whether supplementation with carnitine is advisable or potentially hazardous is still to be established, as it could induce an increase in toxic long-chain acylcarnitine production.50,52 III.  NEUROMETABOLIC DISORDERS



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Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency CLINICAL FEATURES

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency53 has been reported in more than 400 cases.54 It appears to be the most common long-chain fatty-acid oxidation defect, with a disease prevalence of up to 1 : 30,000.55 The defect is clinically heterogeneous and can cause three major phenotypes: 1) an acute presentation with exercise-induced rhabdomyolysis and myoglobinuria—myalgia is more severe and episodes more numerous than in CPT2 deficiency;56 2) a severe childhood form, with early onset of dilated or hypertrophic cardiomyopathy, recurrent episodes of hypoketotic hypoglycemia, and high mortality rate (50–75%); and 3) a milder childhood form, with later onset of hypoketotic hypoglycemia and dicarboxylic aciduria, low mortality, and rare cardiomyopathy. Overall, acute metabolic decompensation is the most frequent form of presentation in VLCAD-deficient patients and most patients suffer from the severe cardiomyopathic form with early onset and poor outcome.25 LABORATORY FINDINGS

In the muscle form, serum CK markedly increases during attacks (20- to > 200-fold). However, patients do not exhibit hypoketotic hypoglycemia nor dicarboxylic aciduria and increase of plasma long-chain acylcarnitines is rarely observed.6 Plasma LCFA profile by GC-MS can be helpful for diagnosis because it may reveal an increase of tetradecenoic (C14:1) acid, which persists even after the patient has fully recovered.57,58 As in CPT2 deficiency, muscle biopsy may not provide any clue to the diagnosis. It may show mild nonspecific morphological alterations with no evidence of lipid accumulation (Figure 50.2D) or may demonstrate a diffuse increase of fat droplets mostly in type 1 fibers57,58 (Figure 50.2E). MOLECULAR GENETICS

More than 80 disease-causing mutations have been identified in the ACADVL gene, none of which seemed to predominate.25,53,59,60 There is some genotype–phenotype correlation and mutations that result in some residual enzyme activity are usually found in patients with the milder phenotypes.25 THERAPY

VLCAD-deficient patients should be treated with a dietary regimen consisting of avoidance of fasting and a high-carbohydrate, low-LCFA diet. The beneficial effect of medium-chain triglycerides (MCT) is controversial and available evidence indicates that MCT ingestion does not ameliorate exercise performance in VLCAD-deficient myopathic patients.61 As for CPT2 deficiency, the use of bezafibrate was found to ameliorate the biochemical and cellular phenotype,62 but a recent randomized clinical trial did not demonstrate its clinical efficacy.48

Mitochondrial Trifunctional Protein Deficiency The mitochondrial trifunctional protein (MTP) is a complex enzyme composed of four α subunits, harboring longchain 2-enoyl-CoA hydratase (LCEH) and long-chain l-3-hydroxyacyl-CoA dehydrogenase (LCHAD) activities, and four β subunits harboring long-chain 3-ketoacyl-CoA thiolase (LCKT) activity. MTP deficiency is relatively frequent, with more than 80 patients reported thus far.63,64 CLINICAL FEATURES

The clinical manifestations of the disease are characteristically associated with urinary excretion of C6–C14 3-hydroxydicarboxylic acids. Patients can be classified into two groups:65 LCHAD DEFICIENCY  The vast majority (≥ 85%) of MTP-deficient patients have an isolated deficiency of LCHAD activity.65 LCHAD deficiency appears to be a relatively common β-oxidation defect (1 in 50,000 births in Northern Europe).65 The disease is clinically heterogeneous. In infancy and early childhood, hypoglycemic encephalopathy with or without severe hepatic involvement and cardiomyopathy is the most common presentation. Mortality is high (≈ 50%). However, cardiomyopathy in patients who survive acute episodes tends to resolve with dietary therapeutic measures.65,66 Later in childhood, the predominant manifestation is paroxysmal rhabdomyolysis and myoglobinuria. Among the distinctive features of LCHAD deficiency are progressive pigmentary retinopathy67 and peripheral neuropathy,68 which are not observed in patients with any other β-oxidation defect. Also characteristic of this disorder is the occurrence of acute fatty liver disease in pregnant women with an affected fetus.21,65,69

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MTP DEFICIENCY (COMBINED ENZYME DEFICIENCY)  In a smaller group of patients, all the three activities harbored by MTP are deficient, albeit to different extents. Clinical manifestations are similar to those observed in patients with isolated LCHAD deficiency, although, in general, the clinical presentation is more severe with a higher mortality rate.69 MOLECULAR GENETICS

A prevalent missense mutation (c.1528G > C, p.E510Q) in the LCHAD domain of the α subunit gene (HADHA) can be detected in approximately 90% of LCHAD-deficient alleles,65 thus making molecular screening for the disease quite feasible. No apparent genotype–phenotype correlation has been observed, as patients homozygous for this mutation show widely different phenotypes.65 Unlike LCHAD deficiency, the molecular basis of MTP deficiency is heterogeneous and different mutations have been identified in both HADHA and HADHB genes with poor ­genotype–phenotype correlation.63,65 THERAPY

The mainstay of therapy is avoidance of fasting and a high-carbohydrate, low-LCFA diet associated with MCT oil supplementation.66,70 Deficiency of docosahexaenoic acid (DHA), an essential n-3 polyunsaturated FA necessary for nerve myelination, has been documented in MTP-deficient patients, and encouraging response to cod liver oil extract, high in DHA content, has been observed.66,71,72

Medium-Chain Acyl-CoA Dehydrogenase Deficiency Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common FA oxidation disorder, with a frequency in the United States of 1 : 10,000, as determined by newborn screening.25,73 CLINICAL FEATURES

Typical symptoms include fasting intolerance, nausea, vomiting, hypoketotic hypoglycemia, lethargy, and coma beginning within the first 2 years of life. Approximately 20% of patients die suddenly at first presentation of the disease because of acute metabolic decompensation in response to either prolonged fasting or intercurrent and common infections.74 Clinical manifestations, however, are variable, and some patients may be asymptomatic, being recognized through family screening. Skeletal muscle and heart involvement is extremely rare.6,14 LABORATORY FINDINGS

Patients have medium-chain dicarboxylic aciduria and secondary carnitine deficiency. The disease is characterized by urinary excretion of C6–C10 dicarboxylic acids (with a characteristic pattern C6 > C8 > C10), acylglycine and acylcarnitine conjugates (hexanoylglycine, phenylpropionylglycine, suberylglycine and octanoylcarnitine).75 C12–C14 dicarboxylic acids, the hallmarks of VLCAD deficiency, are absent. Ketones tend to be inappropriately low in plasma.75 Deficiency of MCAD can be documented in most tissues, including cultured fibroblasts and peripheral blood lymphocytes.75 MOLECULAR GENETICS

A prevalent mutation (c.985A > G/p.K329E) in the ACADM gene (chromosome 1p31) is found in 90% of patients of Northern European descent.25 MCAD deficiency is prevalently observed in this Caucasian population, in which the carrier frequency for the common c.985A > G mutation is approximately 1 : 40.5 The p.K329E mutation causes impairment of tetramer assembly and instability of the protein.76 More than 30 mutations account for the remaining alleles73 and are usually (> 90%) present only in compound heterozygous form.75 THERAPY

Early diagnosis and treatment of MCAD deficiency can result in good long-term prognosis. Avoidance of fasting and maintenance of adequate caloric intake may prevent life-threatening metabolic attacks.

Multiple Acyl-CoA Dehydrogenase Deficiency (Glutaric Aciduria Type II) Multiple acyl-CoA dehydrogenation deficiency (MADD) or glutaric aciduria type II (GAII) is an autosomal recessive disorder of FA, amino acid, and choline oxidation, resulting from a generalized defect in intramitochondrial acyl-CoA dehydrogenation due to defective electron transport from the acyl-CoAs to ubiquinone (coenzyme Q; CoQ) in the mitochondrial respiratory chain (Figure 50.1). The function of some 14 FAD-containing dehydrogenases is affected.21 III.  NEUROMETABOLIC DISORDERS



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CLINICAL FEATURES

Three different phenotypes have been described:6,77,78 1) two lethal neonatal forms—with or without multiple congenital anomalies—characterized by hypotonia, hepatomegaly, severe hypoglycemia, and metabolic acidosis; and 2) a milder, late-onset form characterized by potentially life-threatening episodes of metabolic decompensation, ethylmalonic–adipic aciduria, and progressive lipid storage myopathy. LABORATORY FINDINGS

The disease is characterized by urinary excretion of numerous organic acids (not only glutaric acid, as in glutaric aciduria type I, but also lactic, ethylmalonic, butyric, isobutyric, 2-methyl-butyric, and isovaleric acids) and by multiple elevation of plasma acylcarnitines of different lengths (C4–C16).77 MOLECULAR GENETICS

In most cases, regardless of the phenotype, the disease is due to a defect in the genes encoding the α (ETFA) or β subunits (ETFB) of electron transfer flavoprotein (ETF) or the electron transfer flavoprotein ubiquinone oxido­ reductase (ETF:QO) (ETFDH)6,77–79 (Tables 50.1 and 50.2). Most patients do not respond to riboflavin (vitamin B2) supplementation.21

Riboflavin-Responsive Multiple Acyl-CoA Dehydrogenase Deficiency Riboflavin is the precursor of the coenzyme flavin adenine dinucleotide (FAD) which is the redox prosthetic group of several flavoproteins including the acyl-CoA dehydrogenases of the β-oxidation system and the electron transfer flavoproteins ETF and ETF:QO6,80 (Figure 50.1). A subset of MADD patients have been recently characterized who respond to pharmacological doses of riboflavin both clinically and biochemically. CLINICAL FEATURES

Riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD) is mostly characterized by impaired oxidation of fatty acids due to multiple deficiencies of short-chain acyl-CoA dehydrogenase (SCAD), MCAD, long-chain acyl-CoA dehydrogenase (LCAD) and VLCAD. There are two major clinical phenotypes: 1) an “infantile form” with nonketotic hypoglycemia, hypotonia, failure to thrive, and acute metabolic episodes reminiscent of Reye syndrome; and 2) a “juvenile form” characterized by progressive proximal lipid storage myopathy.80 LABORATORY FINDINGS

There is usually a complex abnormal pattern of urinary excretion of organic acids (glutaric aciduria type II [GAII] or ethylmalonic–adipic aciduria), which indicates a multiple acyl-CoA dehydrogenation defect.6,21 Activities and protein levels of SCAD, MCAD, and VLCAD are reduced in isolated muscle mitochondria.80 MOLECULAR GENETICS

Recessive mutations in the ETFDH gene encoding ETF:QO have been identified in some RR-MADD patients presenting with encephalopathy or muscle weakness or a combination of both.81 Whether ETFDH mutations represent a common cause of RR-MADD still remains to be elucidated. Molecular analysis of 23 of our familial patients with RR-MADD has shown a robust prevalence of subjects with a variety of ETFDH mutations. Notably, among these patients, one family presented a dominant pattern of transmission (F. Taroni, S. Di Donato, and C. Gellera, unpublished data). ETFDH mutatons have also been reported in some patients with Coenzyme Q (CoQ) deficiency presenting with lipid storage myopathy and late-onset GAII.82,83 In these cases, however, response to therapy was not uniform, with some patients improving following riboflavin or CoQ10 (150–500 mg/day) monotherapy, and others requiring the combined therapy. Interestingly, most ETFDH mutations in RR-MADD patients are located around the ubiquinone binding pocket.81,82 Since riboflavin is the precursor of FAD, it has been proposed that riboflavin responsiveness may result from the ability of FAD to act as a chemical chaperone that promotes folding of certain misfolded ETF:QO proteins, thereby ameliorating or normalizing disease symptoms.84 THERAPY

The clinical, morphological, and biochemical responses to oral riboflavin supplementation (100–400 mg per day oral riboflavin) are usually dramatic,6,80 with rapid improvement of muscle weakness and wasting and disappearance of signs of lipid accumulation at muscle biopsy. A prompt response to riboflavin treatment is also observed in encephalopathic patients.81 Riboflavin supplementation also normalizes the activities of SCAD and MCAD, and III.  NEUROMETABOLIC DISORDERS

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restores to normal the amount of protein mass.6,80 Since riboflavin is the precursor of FAD, and riboflavin responsiveness results from the ability of FAD to act as a chemical chaperone that promotes folding of certain misfolded ETF:QO proteins, the effect is to ameliorate or normalize disease symptoms.84

OTHER DISORDERS OF FATTY-ACID β-OXIDATION Short-Chain Acyl-CoA Dehydrogenase Deficiency This is a rare disorder which was first reported in 1987.85 Since then, approximately 25 patients have been reported worldwide, based upon reduced or absent short-chain acyl-coA dehydrogenase (SCAD) activity in vitro and the presence of ethylmalonic aciduria (EMA).86,87 Clinical manifestations range from hypoglycemia and vomiting to hypotonia and seizures accompanied with developmental delay and dysmorphic features.25,85,87–89 Notably, fasting ketogenesis is not impaired.89 Laboratory diagnosis is based on ethylmalonic aciduria and elevated plasma C4-acylcarnitine concentration and is confirmed by enzyme assay in muscle tissue and the detection of disease mutations in the ACADS gene.25,88 In addition to the small number of patients with SCAD inactivating mutations, a vast number of other patients with predominantly neuromuscular symptoms and EMA were found to carry two common SCAD gene variants (c.511C > T/p.G209S and c.625G > A/p.R171W).25,89,90 These alleles are not regarded as true disease-causing mutations nor are they polymorphisms, but rather mutations that confer disease susceptibility.88,90

Medium-Chain 3-Ketoacyl-CoA Thiolase Deficiency So far, this defect has been described in only one case, a neonate presenting with vomiting, metabolic acidosis, liver dysfunction, and terminal rhabdomyolysis and myoglobinuria.91 No information is yet available on the molecular bases of the disorder. l-3-Hydroxyacyl-CoA

Dehydrogenase Deficiency

In earlier reports and literature reviews, the disorder now known as l-3-hydroxyacyl-CoA dehydrogenase (HAD) deficiency was described as short-chain HAD (SCHAD) deficiency.5 It has now become clear that HAD, rather than SCHAD, provides the majority of 3-hydroxyacyl-CoA dehydrogenase activity for mitochondria.92 Furthermore, HAD, encoded by the HADH gene on chromosome 4q22, has a preference for medium-chain straight ­3-hydroxyacyl-CoAs, whereas SCHAD, also known as type 10 17β-hydroxysteroid dehydrogenase, encoded by the HSD17B10 gene on chromosome Xp11.2, acts on a wide spectrum of substrates, including steroids, cholic acids, and fatty acids, with a preference for short-chain methyl-branched acyl-CoAs.92 To date, approximately 10 patients have been reported with missense mutations in the HADH gene.93,94 Presentation of these patients was heterogeneous with either mild late-onset nonketotic hypoglycemia or severe neonatal nonketotic hypoglycemia associated with hyperinsulinism.93,94 Urine organic acids showed increased dicarboxylic and 3-hydroxydicarboxylic acids with 6–14 carbons. Elevated C4-hydroxyacylcarnitine was present in plasma.93,94 Deficient HAD activity was seen in fibroblasts or other tissues.93,94 HAD deficiency is the only FA oxidation disorder associated with congenital hyperinsulinism. Interestingly, HAD mRNA and activity are particularly high in the pancreas and especially in the islets of Langerhans, which suggests an important role for HAD in insulin secretion, possibly through a novel glucose–fatty acid cycle.93,94 The hyperinsulinism associated with HAD deficiency is responsive to diazoxide.93,94

Defects of Unsaturated-Fatty-Acid Oxidation 2,4-Dienoyl-CoA Reductase Deficiency 2,4-Dienoyl-CoA reductase is an enzyme required in the degradation of unsaturated fatty acids with an even number of double bonds, such as linoleic acid (9-cis,12-cis-C18:2). It converts 2,4-dienoyl-CoA to 3-trans-enoyl-CoA. 2,4-Dienoyl-CoA reductase deficiency is a very rare disorder, as it has been described in one female infant only, presenting with hypotonia and fatal respiratory acidosis.95 She also had microcephaly with a short trunk, arms, and fingers, small feet, and a large face. Organic acid profile was normal but an unusual acylcarnitine species (2-trans,4-cis-decadienoylcarnitine) was detected in plasma and urine. Enzyme activity was reduced in liver and muscle. No information is available on the molecular basis of the disorder. III.  NEUROMETABOLIC DISORDERS



REFERENCES

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Acyl-CoA Dehydrogenase 9 Deficiency Several acyl-CoA dehydrogenases (ACAD9, ACAD10, and ACAD11) putatively involved in fatty-acid oxidation have been recently described, but their role in the pathophysiology of human disease is not fully elucidated.21,96 ACAD9, which closely resembles VLCAD, has been shown to have maximum activity with unsaturated long-chain acyl-CoAs.97 Enzyme defect and mutations in the ACAD9 gene have been described in three patients presenting with recurrent episodes of acute liver dysfunction and hypoglycemia, cardiomyopathy, and chronic neurologic dysfunction.97 More recently, however, ACAD9 mutations have also been found in patients with a defect of complex I of the respiratory chain98 while evidence indicates a role for ACAD9 in the biogenesis of complex I.99

ACKNOWLEDGEMENTS Part of the original work cited here was made possible by the valuable contribution of Drs. Silvia Baratta, Barbara Castellotti, Patrizia Cavadini, Barbara Garavaglia, Cinzia Gellera, Federica Invernizzi, Eleonora Lamantea, Marco Rimoldi, and Elisabetta Verderio, and the generous support of Telethon-Italia to F.T.

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