Refsum Disease Research Articles

Diagnosis of hereditary neuropathies in adult patients.

This paper reviews the clinical diagnostic approach to hereditary neuropathies in adults by analysing: elements that point to a neuropathy of inherited origin, different modalities of presentation, laboratory and instrumental diagnostic tests, including molecular tests, symptoms and signs of involvement of other organs. Different phenotypes may be identified according to: disease course; involvement of motor, sensory, autonomic fibres; site of lesion (neuropathy versus neuronopathy); calibre of involved fibres (small-fibre versus large-fibre neuropathy); presence of distinctive symptoms (neuropathic pain); involvement of other organs or apparatus. Charcot-Marie-Tooth disease, Familial Amyloid Polyneuropathy, Hereditary Sensory and Autonomic Neuropathy, Fabry disease, Tangier disease, Porphyric Neuropathies, Refsum disease, Hereditary Neuropathy with liability to Pressure Palsies, Hereditary Neuralgic Amyotrophy, and other rare disorders involving the peripheral nervous system are reviewed.

Identification of PEX7 as the second gene involved in Refsum disease.

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid α-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.

Ichthyosis: etiology, diagnosis, and management.

The ichthyoses are a heterogeneous group of disorders with both inherited and acquired forms. Clinical presentation, pattern of inheritance, and laboratory evaluation may establish a precise diagnosis, which can assist in prognosis and genetic counseling. Congenital autosomal recessive ichthyosis (CARI) usually presents at birth, often as a collodion baby. CARI can progress into any one of a spectrum of disorders. Lamellar ichthyosis is characterized by dark, plate (armor)-like scale. This disease is often caused by mutations in the gene encoding the enzyme transglutaminase 1. Congenital ichthyosiform erythroderma is another phenotype within CARI, marked by generalized redness and fine white scale. Epidermolytic hyperkeratosis is an autosomal dominant disorder characterized by hyperkeratosis and blistering, and at least six clinical phenotypes have been described. It may be due to mutations in the gene encoding the intermediate filament proteins keratin 1 and 10. Ichthyosis vulgaris is the most common ichthyosis, and is inherited in an autosomal dominant pattern. Involvement is generally mild and may vary greatly with climate and humidity. X-linked ichthyosis, due to a defect in the enzyme steroid sulfatase, affects males with generalized scaling that usually begins soon after birth. There may be associated corneal opacities that do not affect vision. Sjögren-Larsson syndrome is an autosomal recessive ichthyosis associated with progressive spastic paralysis and mental retardation. This condition is caused by mutations in the gene for fatty aldehyde dehydrogenase. Refsum's disease, due to accumulation of phytanic acid, results in ichthyosis and progressive neurologic dysfunction. The erythrokeratodermas are characterized by hyperkeratosis and localized erythema. Erythrokeratodermia variabilis is autosomal dominant and characterized by generalized or localized hyperkeratosis and migratory red patches. Mutations in the genes encoding the gap junction proteins, connexins, underlie this disorder. Netherton's syndrome is an autosomal recessive disorder characterized by ichthyosis, a hair shaft abnormality and atopy. The ichthyosis may present at birth with erythroderma or in some cases a collodion presentation. However, a frequent characteristic skin manifestation is ichthyosis linearis circumflexa. Netherton's syndrome has been found to be due to an abnormality in a serum protease inhibitor. Acquired ichthyosis can have a variety of underlying causes including neoplastic, infectious, drugs, endocrine, metabolic, autoimmune, malabsorptive states, and hereditary. Topical, and in more severe cases, systemic, therapy are useful in managing this array of disorders of cornification.

Novel organelle-targeting signals in viral proteins.

Peroxisomes are vital cellular organelles controllingimportant metabolic functions like hydrogen peroxidedegradation, fatty acid metabolism, gluconeogenesis,toxin degradation, etc. (Flatmark et al., 1988; van denBosch et al., 1992; Masters, 1997; Lopez-Huertas etal., 2000). Certain human metabolic disorders such asZellweger syndrome, neonatal adrenoleukodystrophy,infantile Refsum disease and rhizomelic chondrodysplasiapunctata are associated with the peroxisomal defect inprotein import and/or biogenesis (Gould and Valle, 2000).Intracellular transport of proteins into peroxisomes de-pends on either type 1 peroxisomal targeting signal (PTS1)or, type 2 (PTS 2). Type 1 signal sequence is a tripeptidemotif present in the carboxy terminal of the protein(consensus sequence [STAGQCN]-[KRH]-[LIVMAFY];Gould et al., 1987, 1989; Swinkels et al., 1991; Olivieret al., 2000). Whereas, PTS2 constitutes a combina-tion of nine amino acid bipartite sequences (consensus[RKS]-[ILVH]-x(5)-[QH]-[LAE]) in the N-terminal halfof the protein (Gould et al., 1989; Swinkels et al., 1991;Terlecky et al., 1996). Receptors for PTS1 and PTS2 havebeen identified as PEX5 and PEX7 respectively (Terleckyet al., 1996; Lametschwandtner et al., 1998; Subramani,1998). The first report of PTS was in the firefly luciferasegene more than a decade ago (Gould et al., 1987, 1989).Following its discovery, except for viruses, virtually alleukaryotes, parasites and fungi have been identified tohave PTS-containing proteins and demonstrated to targetsuch proteins to the organelle (Fung and Clayton, 1991;Aitchison et al., 1992; Subramani, 1998). Recently weidentified the presence of a functional and conserved PTS1in one of the rotaviral proteins (VP4) which prompted usto look into other viral protein sequences available in thedatabase for potential PTS1 and PTS2 motifs.

Genetic heterogeneity in Japanese patients with peroxisome biogenesis disorders and evidence for a founder haplotype for the most common mutation in PEX10 gene.

We have identified 31 Japanese patients with peroxisome biogenesis disorders (PBDs) for 20 years as the only PBDs diagnostic center in Japan: 27 patients with Zellweger syndrome (ZS), including 2 siblings cases, 3 with neonatal adrenoleukodystrophy (NALD) and 1 with rhizomelic type chondrodysplasia punctata (RCDP). No patient with infantile Refsum disease has been detected. In genetical, those were subdivided into complementation group A (5 ZS and 1 NALD), B (11 ZS), C (4 ZS), E (5 ZS and 2 NALD), F (2 ZS) and R (1 RCDP) and mutation analysis of PEX 1, 2, 6, 7 and 10 has been done in these patients. We demonstrated all 11 ZS patients from group B had a common mutation, 2 base pair deletion in PEX 10 gene, homozygously. To determine whether the high frequency of the PEX 10 mutation is due to a founder effect, we analyzed three single nucleotide polymorphisms in the PEX 10 gene among the patients and Japanese controls, which suggested that this mutation arose once on an ancestral chromosome in the Japanese population. We detected 24 PBDs patients in the last 10 years, which means that the incidence of the PBDs in Japan was estimated to be approximately 1 in 500,000 births.KeywordsJapanese PatientFounder EffectCommon MutationComplementation GroupZellweger SyndromeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Biochemical markers predicting survival in peroxisome biogenesis disorders.

To identify prognostic markers reflecting the extent of peroxisome dysfunction in primary skin fibroblasts from patients with peroxisome biogenesis disorders (PBD). PBD are a genetically heterogeneous group of disorders due to defects in at least 11 distinct genes. Zellweger syndrome is the prototype of this group of disorders, with neonatal adrenoleukodystrophy and infantile Refsum disease as milder variants. Common to these three disorders are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness. Because genotype-phenotype studies are complicated by the genetic heterogeneity among patients with PBD, the authors evaluated a series of biochemical markers as a measure of peroxisome dysfunction in skin fibroblasts. Multiple peroxisomal functions including de novo plasmalogen synthesis, dihydroxyacetonephosphate acyltransferase (DHAPAT) activity, C26:0/C22:0 ratio, C26:0 and pristanic acid beta-oxidation, and phytanic acid alpha-oxidation were analyzed in fibroblasts from a series of patients with defined clinical phenotypes. A poor correlation with age at death was found for de novo plasmalogen synthesis, C26:0/C22:0 ratio, and phytanic acid alpha-oxidation. A fairly good correlation was found for pristanic acid beta-oxidation, but the best correlation was found for DHAPAT activity and C26:0 beta-oxidation. A mathematic combination of DHAPAT activity and C26:0 beta-oxidation showed an even better correlation. DHAPAT activity and C26:0 beta-oxidation are the best markers in predicting life expectancy of patients with PBD. Combination of both markers gives an even better prediction. These results contribute to the management of patients with PBD.

Formation of (R)-2,3-dihydrogeranylgeranoic acid from geranylgeraniol in rat thymocytes.

In a metabolic study of [1-(14)C]geranylgeranial involving rat thymocytes, the radioactivity was mainly incorporated into two metabolites, Z1 and Z2, the latter moving slower than the former on a silica-gel thin-layer plate. The time course of Z1 and Z2 formation superficially suggested a precursor-product relationship between Z1 and Z2. The two metabolites were chemically converted to their methyl esters on treatment with trimethylsilyl diazomethane. Z1 was cochromatographed with E,E,E-geranylgeranoic acid (GGA). Z2 was prepared in a large quantity from geranylgeranial using thymocytes, and purified by TLC followed by ESI (negative ion mode) or EI mass-spectrometry. The observation of a negative ion at m/z 305 on ESI and a molecular ion at m/z 306 (C(20)H(34)O(2)) with fragments similar to GGA on EI implied that Z2 was dihydroGGA, which has been detected in the urine and serum of patients with Refsum disease. The EI mass spectrum of (R)-2,3-dihydroGGA was identical to that of Z2. The diastereomeric amide synthesized from metabolite Z2 with (R)-1-(1-naphtyl)ethylamine was cochromatographed with (R acid, R) amide, not with (S acid, R) amide, which were similarly synthesized from (R)- and (S)-2,3-dihydroGGAs, respectively. In another metabolic study on [1-(14)C]geranylgeraniol (GGOH), the radioactivity was similarly incorporated into a metabolite corresponding to (R)-2,3-dihydroGGA. (R)-2,3-DihydroGGA induced DNA ladder formation with a maximum at 15 mciroM in thymocytes. However, 2,3-dihydrofarnesoic acid did not induce it at all.

Utilization of Sterol Carrier Protein-2 by Phytanoyl-CoA 2-Hydroxylase in the Peroxisomal α Oxidation of Phytanic Acid

Since it possesses a 3-methyl group, phytanic acid is degraded by a peroxisomal α-oxidation pathway, the first step of which is catalyzed by phytanoyl-CoA 2-hydroxylase (PAHX). Mutations in human PAHX cause phytanic acid accumulations leading to Adult Refsum's Disease (ARD), which is also observed in a sterol carrier protein 2 (SCP-2)-deficient mouse model. Phytanoyl-CoA is efficiently 2-hydroxylated by PAHX in vitro in the presence of mature SCP-2. Other straight-chain fatty acyl-CoA esters were also 2-hydroxylated and the products isolated and characterized. Use of SCP-2 increases discrimination between straight-chain (e.g., hexadecanoyl-CoA) and branched-chain (e.g., phytanoyl-CoA) substrates by PAHX. The results explain the phytanic acid accumulation in the SCP-2-deficient mouse model and suggest that some of the common symptoms of ARD and other peroxisomal diseases may arise in part due to defects in SCP-2 function caused by increased phytanic acid levels.

Identification of the peroxisomal β-oxidation enzymes involved in the degradation of leukotrienes

Leukotrienes (LTs) are metabolically inactivated via ω-oxidation and subsequent β-oxidation from the ω-end. This β-oxidation process takes place in peroxisomes. In this study we investigated the role of different enzymes involved in peroxisomal β-oxidation in the degradation of LTs. We analyzed LTB 4, LTE 4, and their oxidation products in urine of patients with Infantile Refsum's disease (IRD), d-bifunctional protein (DBP) deficiency, Rhizomelic Chondrodysplasia Punctata (RCDP) type 1, and X-linked adrenoleukodystrophy (XALD). We found that patients with IRD and DBP deficiencies excrete increased amounts of LTB 4, LTE 4, ω-carboxy-LTB 4, and ω-carboxy-LTE 4 in their urine, whereas the β-oxidation products were not detectable. These results show that DBP plays an essential role in the degradation of LTs. In urine of patients with XALD and RCDP type 1 we found normal levels of LTB 4, LTE 4, and their oxidation products, indicating that the adrenoleukodystrophy protein and peroxisomal 3-ketoacyl-CoA thiolase are not involved in the metabolic inactivation of LTs.

Postnatal expression and distribution of Refsum disease gene associated protein in the rat retina and visual cortex: effect of binocular visual deprivation

Previously, phytanoyl-CoA alpha-hydroxylase-associated protein 1 (PAHX-AP1) was isolated as a novel neuron-specific protein to interact with Refsum disease (RfD) gene PAHX. Its expression in the brain increased after eyelid opening, and the elevated level was maintained through adulthood. In this report, to verify the hypothesis that light could trigger this increase, we have examined the developmental distribution pattern of PAHX-AP1 in rat retina and visual cortex, and changes of its expression by binocular deprivation. Northern blot analyses demonstrated PAHX-AP1 expression reached its highest level in the visual cortex and eyeball at 4 weeks after birth, and these levels were maintained through adult life. Two weeks after visual deprivation, its expression in the eyeball and visual cortex decreased compared with the control. In situ hybridization analyses of the retina showed that PAHX-AP1 expression was limited to the ganglionic cell layer at 10 days after birth, but expressed in the inner nuclear cell layer and extended to the outer nuclear cell layer at 2 and 3 weeks after birth, respectively. Two weeks after visual deprivation, however, it decreased in the ganglionic and inner nuclear cell layer, and disappeared in the rod and cone cell layers. In the visual cortex, strong signals of PAHX-AP1 were detected in layers IV and VI, and II–VI at 10 days and 2 weeks after birth, respectively. Its expression decreased after 2 weeks of visual deprivation. These results indicate that visual stimulation is essential for the maintenance of PAHX-AP1 expressions in the retina, especially in the rod and cone cell layers, and visual cortex, and suggest that PAHX-AP1 may be involved in the developmental regulation of the photoreceptor’s function.

Refsum's disease: a peroxisomal disorder affecting phytanic acid alpha-oxidation.

Refsum's disease (hereditary motor sensory neuropathy type IV, heredopathia atactica polyneuritiformis) is an autosomal recessive disorder the clinical features of which include retinitis pigmentosa, blindness, anosmia, deafness, sensory neuropathy, ataxia and accumulation of phytanic acid in plasma- and lipid-containing tissues. The transport and biochemical pathways of phytanic acid metabolism have recently been defined with the cloning of two key enzymes, phytanoyl-CoA 2-hydroxylase (PAHX) and 2-hydroxyphytanoyl-CoA lyase, together with the confirmation of their localization in peroxisomes. PAHX, an iron(II) and 2-oxoglutarate-dependent oxygenase is located on chromosome 10p13. Mutant forms of PAHX have been shown to be responsible for some, but not all, cases of Refsum's disease. Certain cases have been shown to be atypical mild variants of rhizomelic chondrodysplasia punctata type 1a. Other atypical cases with low-plasma phytanic acid may be caused by alpha-methylacyl-CoA racemase deficiency. A sterol-carrier protein-2 (SCP-2) knockout mouse model shares a similar clinical phenotype to Refsum's disease, but no mutations in SCP-2 have been described to-date in man. This review describes the clinical, biochemical and metabolic features of Refsum's disease and shows how the biochemistry of the alpha-oxidation pathway may be linked to the regulation of metabolic pathways controlled by isoprenoid lipids, involving calcineurin or the peroxisomal proliferator activating alpha-receptor.

Tackling rare syndromes

In their letter about the anaesthetic management of a child with Soto's syndrome (Varvinski et al. Anaesthesia 2001; 56: 809), the authors make a plea for an anaesthetic website listing these rare syndromes and their management. They need a copy of Victor C. Baum and Jennifer E. O'Flaherty's Anaesthesia for Genetic, Metabolic and Dysmorphic Syndrome of Childhood (Lippincott Williams and Wilkins, 1999). This book gives concise details of the clinical features and anaesthetic implications of just about every syndrome you have ever heard of and many that you haven't. I anaesthetise regularly for Special Needs patients requiring dental surgery and faced at short notice with a day case who has Johanson–Blizzard Syndrome or Sanfilippo Syndrome or CHARGE Association or Angelman Syndrome (all of which have come my way fairly recently) this book has been invaluable. It is also a useful teaching aid and enables one to impress colleagues with one's detailed knowledge of Rett Syndrome, Refsum Disease and similar exotica! Get a copy. It's the best £70 I've spent in ages.

Effects of phytanic acid on the vitamin E status, lipid composition and physical properties of retinal cell membranes: implications for adult Refsum disease

Effects of phytanic acid on the vitamin E status, lipid composition and physical properties of retinal cell membranes: implications for adult Refsum disease

Late onset white matter disease in peroxisome biogenesis disorder.

To report late onset cerebral white matter disease as a distinctive phenotype in peroxisome biogenesis disorder (PBD). There is phenotypic and genetic overlap among the PBD known as Zellweger syndrome (ZS), infantile Refsum disease (IRD), and neonatal adrenoleukodystrophy (NALD). Distinctive external features are variable among these three disorders, and neurologic deficit has its onset at birth or in infancy. In a structured follow-up cohort of 25 patients with PBD, not including ZS, three patients had an unusual pattern of cerebral white matter disease with onset past the age of 1, not conforming to any of the classic PBD phenotypes. Clinical phenotyping and follow-up, peroxisomal biochemical determinations in body fluids and fibroblasts, identification of affected PEX gene by genetic complementation in fibroblasts, and MRI studies. Two unrelated patients with PBD without distinctive external features had normal neurodevelopmental milestones during their first year, followed by rapid deterioration including severe hypotonic pareses, seizures, retinopathy, and deafness. A third patient initially diagnosed with IRD developed cerebral white matter degeneration in the third year of life, complicating the original diagnosis. MRI in all three patients showed cerebral demyelination with sparing of subcortical fibers and pronounced central cerebellar demyelination. Late-onset cerebral white matter disease may occur in PBD, either following IRD or following normal early development and in the absence of distinctive external features. Peroxisome biogenesis disorder should be included in the differential diagnosis of post-infantile onset of cerebral white matter disease

Effects of phytanic acid on the vitamin E status, lipid composition and physical properties of retinal cell membranes: implications for adult Refsum disease

Adult Refsum disease is an inherited disorder in which phytanic acid accumulates in tissues and serum. Two hypotheses have been proposed to explain the pathogenesis of this condition. The molecular distortion hypothesis suggests that phytanic acid may alter membrane composition and structure, thereby affecting membrane function(s). The anti-metabolite hypothesis suggests that an accumulation of phytanic acid in membranes may interfere with vitamin E function. These two hypotheses were investigated by studying the effects of modulating phytanic acid and alpha-tocopherol concentrations on the fatty acid composition and certain physical parameters of cultured retinal cells. Results showed that (a) the phospholipid fraction of retinal cells readily incorporated phytanic acid, (b) the incorporation of phytanic acid increased membrane fluidity, (c) there was no competition for uptake between phytanic acid and alpha-tocopherol, and (d) the incorporation of phytanic acid did not increase the susceptibility of membranes to lipid peroxidation in vitro. These results obtained with cultured retinal cells suggest that the molecular distortion hypothesis, but not the anti-metabolite hypothesis, could explain the pathogenesis of adult Refsum disease. In vitro tissue culture models can, however, only approximate to the much more complex situation that occurs in vivo.

Refsum disease, peroxisomes and phytanic acid oxidation: a review.

Refsum disease was first recognized as a distinct disease entity by Sigvald Refsum in the 1940s. The discovery of markedly elevated levels of the branched-chain fatty acid phytanic acid in certain patients marked Refsum disease as a disorder of lipid metabolism. Although it was immediately recognized that the accumulation of phytanic acid is due to its deficient breakdown in Refsum disease patients, the true enzymatic defect remained mysterious until recently. A major breakthrough in this respect was the resolution of the mechanism of phytanic acid alpha-oxidation in humans. In this review we describe the many aspects of Refsum disease from the clinical signs and symptoms to the enzyme and molecular defect plus the recent identification of genetic heterogeneity in Refsum disease.

Degenerative Disorders of the Central Nervous System

Degenerative Disorders of the Central Nervous System

Peroxisomal Straight-chain Acyl-CoA Oxidase and D-bifunctional Protein Are Essential for the Retroconversion Step in Docosahexaenoic Acid Synthesis

Docosahexaenoic acid (DHA, C22:6n-3) is essential for normal brain and retinal development. The nature and subcellular location of the terminal steps in DHA biosynthesis have been controversial. Rather than direct Delta4-desaturation of C22:5n-3, it has been proposed that this intermediate is elongated to C24:5n-3, desaturated to C24:6n-3, and "retroconverted" to DHA via peroxisomal beta-oxidation. However, this hypothesis has recently been challenged. The goal of this study was to determine the mechanism and specific enzymes required for the retroconversion step in human skin fibroblasts. Cells from patients with deficiencies of either acyl-CoA oxidase or D-bifunctional protein, the first two enzymes of the peroxisomal straight-chain fatty acid beta-oxidation pathway, exhibited impaired (5-20% of control) conversion of either [1-14C]18:3n-3 or [1-14C]22:5n-3 to DHA as did cells from peroxisome biogenesis disorder patients comprising eight distinct genotypes. In contrast, normal DHA synthesis was observed in cells from patients with rhizomelic chondrodysplasia punctata, Refsum disease, X-linked adrenoleukodystrophy, and deficiency of mitochondrial medium- or very long-chain acyl-CoA dehydrogenase. Acyl-CoA oxidase-deficient cells accumulated 2-5 times more radiolabeled C24:6n-3 than did controls. Our data are consistent with the retroconversion hypothesis and demonstrate that peroxisomal beta-oxidation enzymes acyl-CoA oxidase and D-bifunctional protein are essential for this process in human skin fibroblasts.

The site of the hearing loss in Refsum's disease

Refsum's disease is a disorder of lipid metabolism with pigmentary retinopathy, demyelinating neuropathy, ataxia, and hearing loss. Previous histological studies have located the site of hearing impairment in the inner ear, but it has never been confirmed audiologicaly in the literature. In this reported case of Refsum's disease, despite hearing loss and absence of response in ABR, robust otoacoustic emissions were measured. Together with these and other audiological findings, we conclude that our case might be the first reported case of Refsum's disease with auditory neuropathy. The site of the hearing abnormality in Refsum's disease may be ‘post-outer hair cells’ in some cases as in the current case. Because of their limited benefits and risk of noise-induced damage to outer hair cells, the use of hearing aids before otoacoustic emission measurements should be considered cautiously in Refsum's disease.

Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease.

Refsum's disease is a neurological syndrome characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaemia. Many cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PAHX) which catalyses the initial alpha-oxidation step in the degradation of phytanic acid. Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a requirement for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate. Sequence analysis in the light of crystallographic data for other members of the 2-oxoglutarate-dependent oxygenase super-family led to secondary structural predictions for PAHX, which were tested by site-directed mutagenesis. The H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands. The clinically observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assayed for both 2-oxoglutarate and phytanoyl-CoA oxidation. The P29S mutant was fully active, implying that the mutation resulted in defective targeting of the protein to peroxisomes. Mutation of Arg-275 resulted in impaired 2-oxoglutarate binding. The Q176K, G204S and N269H mutations caused partial uncoupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation. The results demonstrate that the diagnosis of Refsum's disease should not solely rely upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation.