William B. Rizzo

Mitochondrial Fatty-Acid Oxidation Disorders

Inherited defects in mitochondrial fatty-acid beta-oxidation comprise a group of at least 12 diseases characterized by distinct enzyme or transporter deficiencies. Most of these diseases have a variable age of onset and clinical severity. Symptoms are often episodic and associated with mild viral illness, physiologic stress, or prolonged exercise that overwhelms the ability of mitochondria to oxidize fatty acids. Depending on the specific genetic defect, patients develop fasting hypoketotic hypoglycemia, cardiomyopathy, rhabdomyolysis, liver dysfunction, or sudden death. Neuropathy and pigmentary retinopathy are seen in some of the diseases. The diagnosis is based on finding an accumulation of specific biochemical markers such as acylcarnitine metabolites in blood and urinary dicarboxylic acids and acylglycines. Confirmatory testing requires enzymatic studies and DNA analysis. Therapeutic approaches are generally effective in preventing severe symptomatic episodes, including sudden death. Newborn screening for fatty-acid oxidation disorders promises to identify many affected patients before the onset of symptoms.

Dietary erucic acid therapy for X‐linked adrenoleukodystrophy

We investigated the biochemical and clinical efficacy of dietary erucic acid (C22:1) therapy for X-linked adrenoleukodystrophy (ALD). In a double-blind crossover study of patients who were on chronic oleic acid (C18:1) therapy, addition of erucic acid to the diet led to a further reduction in plasma hexacosanoic acid (C26:0) concentration. We treated 12 newly diagnosed ALD patients with a diet enriched with erucic acid and oleic acid for 2 to 19 months. Mean plasma C26:0 concentration decreased to normal by 4 weeks, and the C26:0 composition of plasma sphingomyelin and phosphatidylcholine became normal by 4 months on therapy. Fatty acid analysis of postmortem tissues from 1 boy treated for 10 months suggested that dietary erucic acid entered the heart, liver, adrenal gland, and brain. Eight patients remained on treatment long enough (mean, 12 ± 3 months) to evaluate their clinical response; 6 of these patients with moderate to advanced disease deteriorated neurologically or showed progression of white matter disease on brain magnetic resonance imaging whereas 2 mildly affected patients remained clinically stable after 10 and 19 months. No adverse effects of the diet occurred. We conclude that dietary erucic acid therapy is effective in lowering plasma C26:0 to normal in ALD patients, and may prevent further demyelination in some mildly affected boys.

Adrenoleukodystrophy Oleic acid lowers fibroblast saturated C22‐26 fatty acids

Monounsaturated fatty acids, especially oleic acid (C18:1), decreased the content of saturated very-long-chain fatty acids (VLFA) in cultured skin fibroblasts from patients with adrenoleukodystrophy (ALD) and controls. When confluent ALD fibroblasts were incubated with oleic acid for 5 days in lipid-free medium, which eliminates uptake of exogenous VLFA, the mean cell content of C26:0 was decreased by 33.7 ± 10.1%. Oleic acid inhibited C26:0 synthesis in ALD fibroblasts by 58% and total fatty acid synthesis by 68 to 78%. Therefore, the elevated C26:0 levels in ALD cells may be lowered by inhibiting fatty acid biosynthesis, and inhibition of saturated VLFA synthesis by oleate may be useful in treating ALD.

Sjögren-Larsson syndrome. Deficient activity of the fatty aldehyde dehydrogenase component of fatty alcohol:NAD+ oxidoreductase in cultured fibroblasts.

Sjögren-Larsson syndrome (SLS) is an inherited disorder associated with impaired fatty alcohol oxidation due to deficient activity of fatty alcohol:NAD+ oxidoreductase (FAO). FAO is a complex enzyme which consists of two separate proteins that sequentially catalyze the oxidation of fatty alcohol to fatty aldehyde and fatty acid. To determine which enzymatic component of FAO was deficient in SLS, we assayed fatty aldehyde dehydrogenase (FALDH) and fatty alcohol dehydrogenase in cultured fibroblasts from seven unrelated SLS patients. All SLS cells were selectively deficient in the FALDH component of FAO, and had normal activity of fatty alcohol dehydrogenase. The extent of FALDH deficiency in SLS cells depended on the aliphatic aldehyde used as substrate, ranging from 62% of mean normal activity using propionaldehyde as substrate to 8% of mean normal activity with octadecanal. FALDH activity in obligate SLS heterozygotes was partially decreased to 49 +/- 7% of mean normal activity using octadecanal as substrate. Differential centrifugation studies in fibroblasts indicated that this FALDH enzyme was largely particulate; soluble FALDH activity was normal in SLS cells. Intact SLS fibroblasts oxidized octadecanol to fatty acid at less than 10% of the normal rate, but oxidized free octadecanal normally, suggesting that the FALDH affected in SLS is chiefly involved in the oxidation of fatty alcohol to fatty acid. These results show that the primary enzymatic defect in SLS is the FALDH component of the FAO complex, which leads to deficient oxidation of fatty aldehyde derived from fatty alcohol.

The Molecular Basis of Sjögren-Larsson Syndrome: Mutation Analysis of the Fatty Aldehyde Dehydrogenase Gene

Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). To define the molecular defects causing SLS, we performed mutation analysis of the FALDH gene in probands from 63 kindreds with SLS. Among these patients, 49 different mutations-including 10 deletions, 2 insertions, 22 amino acid substitutions, 3 nonsense mutations, 9 splice-site defects, and 3 complex mutations-were found. All of the patients with SLS were found to carry mutations. Nineteen of the missense mutations resulted in a severe reduction of FALDH enzyme catalytic activity when expressed in mammalian cells, but one mutation (798G-->C [K266N]) seemed to have a greater effect on mRNA stability. The splice-site mutations led to exon skipping or utilization of cryptic acceptor-splice sites. Thirty-seven mutations were private, and 12 mutations were seen in two or more probands of European or Middle Eastern descent. Four single-nucleotide polymorphisms (SNPs) were found in the FALDH gene. At least four of the common mutations (551C-->T, 682C-->T, 733G-->A, and 798+1delG) were associated with multiple SNP haplotypes, suggesting that these mutations originated independently on more than one occasion or were ancient SLS genes that had undergone intragenic recombination. Our results demonstrate that SLS is caused by a strikingly heterogeneous group of mutations in the FALDH gene and provide a framework for understanding the genetic basis of SLS and the development of DNA-based diagnostic tests.

Sjögren-Larsson syndrome. Impaired fatty alcohol oxidation in cultured fibroblasts due to deficient fatty alcohol:nicotinamide adenine dinucleotide oxidoreductase activity.

Lipid metabolism was studied in cultured skin fibroblasts from patients with the inherited disorder, Sjögren-Larsson syndrome (SLS). Intact SLS fibroblasts incubated in the presence of [1-14C]palmitate accumulated more radioactive hexadecanol than did normal cells, whereas incorporation of radioactivity into other cellular lipids was unaltered. The hexadecanol content of SLS fibroblasts was abnormally elevated. Hexadecanol accumulation was not due to increased fatty alcohol synthesis nor its deficient utilization for glycerol ether synthesis. The half-life of intracellular hexadecanol loaded into SLS fibroblasts was increased (70 min) compared with normal (15 min), and intact SLS fibroblasts showed impaired oxidation of [14C]-hexadecanol to fatty acid. Fatty alcohol:NAD+ oxidoreductase, the enzyme catalyzing this reaction, was deficient in SLS fibroblasts. Mean total activity in SLS fibroblasts (n = 5) was 13% of that in normal fibroblasts, and palmitoyl CoA-inhibitable activity was 1% of normal. Fibroblasts from two obligate SLS heterozygotes had enzyme activities intermediate between that in normal fibroblasts and individuals with SLS. These results suggest that the primary defect in SLS is deficiency of fatty alcohol:NAD+ oxidoreductase. SLS represents the first inherited disorder in man associated with an isolated abnormality in fatty alcohol metabolism.

Recessive Mutations in ELOVL4 Cause Ichthyosis, Intellectual Disability, and Spastic Quadriplegia

Very-long-chain fatty acids (VLCFAs) play important roles in membrane structure and cellular signaling, and their contribution to human health is increasingly recognized. Fatty acid elongases catalyze the first and rate-limiting step in VLCFA synthesis. Heterozygous mutations in ELOVL4, the gene encoding one of the elongases, are known to cause macular degeneration in humans and retinal abnormalities in mice. However, biallelic ELOVL4 mutations have not been observed in humans, and murine models with homozygous mutations die within hours of birth as a result of a defective epidermal water barrier. Here, we report on two human individuals with recessive ELOVL4 mutations revealed by a combination of autozygome analysis and exome sequencing. These individuals exhibit clinical features of ichthyosis, seizures, mental retardation, and spasticity-a constellation that resembles Sjögren-Larsson syndrome (SLS) but presents a more severe neurologic phenotype. Our findings identify recessive mutations in ELOVL4 as the cause of a neuro-ichthyotic disease and emphasize the importance of VLCFA synthesis in brain and cutaneous development.

A clinical approach to the diagnosis of patients with leukodystrophies and genetic leukoencephelopathies.

Leukodystrophies (LD) and genetic leukoencephalopathies (gLE) are disorders that result in white matter abnormalities in the central nervous system (CNS). Magnetic resonance (MR) imaging (MRI) has dramatically improved and systematized the diagnosis of LDs and gLEs, and in combination with specific clinical features, such as Addisons disease in Adrenoleukodystrophy or hypodontia in Pol-III related or 4H leukodystrophy, can often resolve a case with a minimum of testing. The diagnostic odyssey for the majority LD and gLE patients, however, remains extensive--many patients will wait nearly a decade for a definitive diagnosis and at least half will remain unresolved. The combination of MRI, careful clinical evaluation and next generation genetic sequencing holds promise for both expediting the diagnostic process and dramatically reducing the number of unresolved cases. Here we present a workflow detailing the Global Leukodystrophy Initiative (GLIA) consensus recommendations for an approach to clinical diagnosis, including salient clinical features suggesting a specific diagnosis, neuroimaging features and molecular genetic testing. We also discuss recommendations on the use of broad-spectrum next-generation sequencing in instances of ambiguous MRI or clinical findings. We conclude with a proposal for systematic trials of genome-wide agnostic testing as a first line diagnostic in LDs and gLEs given the increasing number of genes associated with these disorders.

Sjögren-Larsson syndrome: Inherited defect in the fatty alcohol cycle*

We investigated fatty alcohol metabolism in eight patients with Sjögren-Larsson syndrome, and in nine obligate heterozygotes. Fatty alcohol: nicotinamide-adenine dinucleotide oxidoreductase (FAO) activity was deficient in cultured skin fibroblasts (mean 18% of normal, n = 8) and peripheral blood leukocytes (mean 22% of normal, n = 3) from patients with Sjögren-Larsson syndrome. The palmitoyl coenzyme A-inhibitable component of FAO activity was decreased to 10% and 15% of normal in fibroblasts and leukocytes, respectively, of patients with Sjögren-Larsson syndrome. Most affected patients accumulated long-chain fatty alcohol in plasma, with a greater relative accumulation of octadecanol (mean threefold greater than normal) than hexadecanol (mean twofold greater than normal). Erythrocyte lipid alkyl ether linkages derived from hexadecanol were slightly increased in three of four patients. Fibroblasts and leukocytes from heterozygotes with Sjögren-Larsson syndrome showed mean FAO activities that were intermediate between those seen in homozygotes and in normal control subjects. The heterozygotes had normal fatty alcohol concentrations in plasma. These studies demonstrate FAO deficiency in patients with Sjögren-Larsson syndrome, and suggest that accumulation of fatty alcohol or its metabolic products may be important in the pathogenesis of this disorder.

Peroxisome biogenesis disorders in the Zellweger spectrum: An overview of current diagnosis, clinical manifestations, and treatment guidelines

Peroxisome biogenesis disorders in the Zellweger spectrum (PBD-ZSD) are a heterogeneous group of genetic disorders caused by mutations in PEX genes responsible for normal peroxisome assembly and functions. As a result of impaired peroxisomal activities, individuals with PBD-ZSD can manifest a complex spectrum of clinical phenotypes that typically result in shortened life spans. The extreme variability in disease manifestation ranging from onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults presents practical challenges in disease diagnosis and medical management. Recent advances in biochemical methods for newborn screening and genetic testing have provided unprecedented opportunities for identifying patients at the earliest possible time and defining the molecular bases for their diseases. Here, we provide an overview of current clinical approaches for the diagnosis of PBD-ZSD and provide broad guidelines for the treatment of disease in its wide variety of forms. Although we anticipate future progress in the development of more effective targeted interventions, the current guidelines are meant to provide a starting point for the management of these complex conditions in the context of personalized health care.