Refsum Disease Research Articles

29. Enzyme reduction therapy as adjunct to cord cell transplant in Hurler syndrome

Peroxisomal disorders include a complex spectrum of diseases, characterized by a high heterogeneity from both the clinical and the biochemical points of view. Specific assays are required for the study of peroxisome metabolism. Among these, pipecolic acid evaluation is considered as a supplementary test. We have established the diagnostic role of pipecolic acid in 30 patients affected by a peroxisomal defect (5 Zellweger syndromes, 10 Infantile Refsum diseases, 1 neonatal adrenoleukodystrophy, 6 patients affected by a peroxisomal biogenesis disorder with unclassified phenotype, 1 case of rhizomelic chondrodysplasia punctata (RCDP), 2 acyl-CoA oxidase deficiencies, 2 bifunctional enzyme deficiencies, 2 Refsum diseases, and 1 β-oxidation deficiency). Pipecolic acid was increased in all generalized peroxisomal disorders, while normal pipecolic acid with abnormal very long chain fatty acid concentrations was strong evidence for a single peroxisomal enzyme deficiency. Unexpectedly, hyperpipecolic acidaemia was found also in a child affected by RCDP and in two patients with Refsum disease. In six patients the suggestion of a peroxisomal disorder was raised by the fortuitous finding of a pipecolic acid peak in amino acid chromatography, routinely performed as a general metabolic screening. For all patients, pipecolic acid proved to be a useful parameter in the biochemical classification of peroxisomal disorders.

Ataxia with loss of Purkinje cells in a mouse model for Refsum disease

Refsum disease is caused by a deficiency of phytanoyl-CoA hydroxylase (PHYH), the first enzyme of the peroxisomal alpha-oxidation system, resulting in the accumulation of the branched-chain fatty acid phytanic acid. The main clinical symptoms are polyneuropathy, cerebellar ataxia, and retinitis pigmentosa. To study the pathogenesis of Refsum disease, we generated and characterized a Phyh knockout mouse. We studied the pathological effects of phytanic acid accumulation in Phyh(-/-) mice fed a diet supplemented with phytol, the precursor of phytanic acid. Phytanic acid accumulation caused a reduction in body weight, hepatic steatosis, and testicular atrophy with loss of spermatogonia. Phenotype assessment using the SHIRPA protocol and subsequent automated gait analysis using the CatWalk system revealed unsteady gait with strongly reduced paw print area for both fore- and hindpaws and reduced base of support for the hindpaws. Histochemical analyses in the CNS showed astrocytosis and up-regulation of calcium-binding proteins. In addition, a loss of Purkinje cells in the cerebellum was observed. No demyelination was present in the CNS. Motor nerve conduction velocity measurements revealed a peripheral neuropathy. Our results show that, in the mouse, high phytanic acid levels cause a peripheral neuropathy and ataxia with loss of Purkinje cells. These findings provide important insights in the pathophysiology of Refsum disease.

A novel Refsum-like disorder that maps to chromosome 20

Clinical and genetic characterization of a neurologic disorder resembling Refsum disease in a Norwegian consanguineous family. The affected individuals comprise a brother and sister and their third cousin. The family comes from a small island community and genealogic studies showed that both sets of parents are descendants of a man born in 1585. Based on the hypothesis that this is an autosomal recessive disease and that the patients were homozygous for the same mutation (identical by descent), we used homozygosity mapping to define the genetic locus of this disorder. This slowly progressive disorder starts in childhood with signs of peripheral neuropathy (pes cavus, tendoachilles contracture). Hearing loss and cataract become evident in the third decade. Subsequently, patients develop a disorder of gait due to the combination of ataxia and spasticity, and a pigment retinopathy. While the clinical picture is reminiscent of Refsum disease, affected individuals have normal phytanic and pristanic acid levels in plasma, as well as normal enzymatic activity for alpha-oxidation. We mapped the disease to a 15.96 Mb region on chromosome 20 (20p11.21-q12), containing approximately 200 genes (maximum lod score = 6.3). Sequencing of 23 candidate genes failed to demonstrate detrimental sequence variants. Our findings show that the clinical syndromes that include Refsum disease are more heterogeneous than previously recognized. We have chosen to report the clinical features and mapping of this novel disorder in the hope that this will permit identification of other families and thus proper genetic characterization.

A new disease mimicking Refsum syndrome

In this issue of Neurology ®, Fiskerstrand et al.1 describe a novel family with a “Refsum-like disorder.” In one of those satisfying quirks of timing, it was precisely 25 years ago that another article appeared in Neurology by Refsum and colleagues describing the successful dietary treatment of a patient with Refsum disease.2 Sigvald Refsum (1907–1991), Norway’s best known neurologist, described the disease that bears his name in 1945/1946 and called it “heredopathia atactica polyneuritiformis.”3 He went on to be Chair of the Neurology Department at Oslo from 1954 to 1978. The disease associated with …

Urine acylcarnitine analysis by ESI–MS/MS: A new tool for the diagnosis of peroxisomal biogenesis disorders

Background Patients with peroxisomal biogenesis disorders (PBDs) have an abnormal profile of circulating acylcarnitines (i.e. elevated C16:0-DC-, C18:0-DC-, C24:0-, C26:0-carnitine). We developed an ESI–MS/MS method for quantification of urine acylcarnitines and tested its reliability for the diagnosis of PBDs. Methods Urine from 7 patients with PBDs (5 Zellweger syndrome, 2 infantile Refsum disease), from 2 patients with D-bifunctional protein (D-BP) deficiency, and from 130 healthy controls were analysed by ESI–MS/MS, using a multiple reactions monitoring (MRM) method, and quantified with labelled internal standards. Acylcarnitine levels between groups were analyzed by the STATA™ statistics data analysis and compared by the non parametric Mann–Whitney test. Results In PBDs, the urinary excretion of long-chain dicarboxylylcarnitines (C14:0-DC-, C16:0-DC-, and C18:0-DC-carnitine), and of very long-chain monocarboxylylcarnitines (C22:0-, C24:0-, C26:0-carnitine) were significantly elevated compared to controls ( p < 0.0001). Interestingly, among PBDs the most severe abnormalities of acylcarnitine profile were observed in patients with Zellweger syndrome. One patient with D-BP showed similar abnormalities to PBDs, while in the other only C16:0-DC-carnitine was markedly elevated. Conclusions This study shows that MRM ESI–MS/MS acylcarnitine analysis unequivocally discriminates patients with PBDs and D-BP deficiency from controls, representing a reliable and sensitive method for the diagnosis that requires a short-time analysis with high sample through-put.

Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders

Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 mul) were hydrolyzed, extracted, and derivatized with DAABD-AE in approximately 2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.

End‐stage Liver Disease and Liver Transplant: Current Situation and Key Issues

The aim of this article is to identify the current challenges in pediatric OLT. It is directed to all physicians who care for pediatric patients from the early prospect of liver transplant to posttransplant life. Besides assessing current information on transplant results, the coauthors of this article have identified a number of issues that they consider as the most important that need to be addressed in 2008. Some are only an update of old questions, and others are newly identified problems. The recommendations from the group are summarized at the end of each specific issue and in the consensus guidelines.

Pupil abnormalities in 131 cases of genetically defined inherited peripheral neuropathy

To investigate and correlate the frequency and types of pupil abnormalities that are associated with hereditary peripheral neuropathy in a large cohort of patients prospectively examined. A prospective study between 1998 and 2007. Patients were enrolled and examined after being seen in the neurology clinic. Data were collected on demographics, family and medical history. Patients had eye and pupillography testing carried out as well as being neurologically and genetically investigated. A consecutive series of 131 cases of inherited peripheral neuropathy were seen and categorized into five groups: familial amyloid polyneuropathy (FAP), Charcot Marie Tooth disease (CMT), hereditary neuropathywith liability to pressure palsies (HNPP), Refsum's disease, and hereditary sensory and autonomic neuropathy. A number of unreported mutations were identified in these patient groups. Pupil abnormalities were common in the Refsum's group, with frequent abnormally small pupils. The inherited neuropathies commonly associated with autonomic abnormalities were frequently found to have developed bilateral Horner's syndrome, which was particularly prevalent in our FAP series. Abnormalities were rare in HNPP and CMT type 1, but CMT type 2 showed frequent and varied pupil defects. The results describe the pupil abnormalities that were frequently associated with the particular group of inherited neuropathy patients, but we could not predict the genetic defect or the neuropathy severity. This is the first study of the pupil abnormalities found in the inherited neuropathies and provides an overview of the frequency and type of defects seen in a large number of cases. This series along with the detailed tables will act as an important diagnostic aid in assessing these patients.

Investigational Methods for Peroxisomal Disorders

Peroxisomes play an important role in cellular metabolism. Defects in peroxisome assembly or of a single peroxisomal pathway are associated with a wide variety of inherited disorders, including X-linked adrenoleukodystrophy, Zellweger spectrum disorders, rhizomelic chondrodysplasia punctata, and Refsum disease. A group of peroxisome-specific biomarkers has been shown to be characteristic of specific defects. Patients with defects in peroxisome fatty acid beta-oxidation accumulate very long-chain fatty acids (VLCFA), patients with defects in plasmalogen synthesis are deficient in erythrocyte membrane plasmalogens, and patients with mislocalized pipecolic acid oxidase accumulate pipecolic acid in body fluids. This unit describes three protocols that can be used to measure plasma VLCFA, erythrocyte plasmalogens, and plasma or urine pipecolic acid by capillary gas chromatography (GC) or GC-mass spectrometry. These techniques can be used to identify the majority of patients with known neurogenetic peroxisome disorders.

Cochlear implantation in Refsum's disease

Refsum's disease is a rare disorder characterized by accumulation of phytanic acid in plasma and tissues. The main clinical features are retinitis pigmentosa, cerebellar ataxia and chronic polyneuropathy. Sensorineural hearing loss of varying severity can be an additional feature. This study reports our experience with two patients with Refsum's disease who underwent successful cochlear implantation. Both patients had good audiological outcomes after implantation and were overall very satisfied. Bilateral implantation should be considered with patients with severe dual sensory loss. One patient was implanted bilaterally the other unilaterally, primarily due to prolonged duration of unaided unilateral profound hearing loss. Copyright © 2008 John Wiley & Sons, Ltd.

Cochlear implantation in Refsum's disease

Refsum's disease is a rare disorder characterized by accumulation of phytanic acid in plasma and tissues. The main clinical features are retinitis pigmentosa, cerebellar ataxia and chronic polyneuropathy. Sensorineural hearing loss of varying severity can be an additional feature. This study reports our experience with two patients with Refsum's disease who underwent successful cochlear implantation. Both patients had good audiological outcomes after implantation and were overall very satisfied. Bilateral implantation should be considered with patients with severe dual sensory loss. One patient was implanted bilaterally the other unilaterally, primarily due to prolonged duration of unaided unilateral profound hearing loss. Copyright © 2008 John Wiley & Sons, Ltd.

Non-manifesting Refsum heterozygotes carrying the c.135-2A&gt;G PAHX gene transition

So far, subjects heterozygous for PAHX mutations are regarded as non-symptomatic. In the 24-year-old, HIV-negative daughter and the 26-year-old, HIV-negative son of a patient with Refsum disease due to the homozygous c.135-2A>G transition at the splice site before exon 3 of the PAHX gene, slight abnormalities suggestive of the disease became apparent. The daughter reported a single fever cramp in childhood, recurrent, short-lived amaurotic episodes after getting up from supine, short-sightedness, hypoacusis, and restless legs. The son complained about restless legs, hyperhidrosis, hypoacusis, and bulbar oscillations. Though both children carried the same mutation as their mother in the heterozygous form, clinical neurologic examination, nerve conduction studies and serum phytanic acid concentration were normal in both of them, implying that the described abnormalities were not causally related to the PAHX mutation. In the absence of elevated phytanic acid concentrations, clinical neurologic abnormalities in heterozygous relatives of Refsum patients are not attributable to heterozygosity for PAHX mutations.

The WFN comes of age

The WFN comes of age

A retrospective view on research in neuroscience in Norway

This brief historical review on neuroscience in Norway shows a comparatively high research activity with many important results. The Norwegian zoologist Fridtjof Nansen, who later became a famous Arctic explorer, was the first to formulate the neuron doctrine. 'The Oslo School of Neuroanatomy' contributed enormously to the understanding of the detailed anatomy and chemistry of the central nervous system. Norwegian neurophysiologists made important findings from studies of hippocampus including the inhibitory basket cell, the LTP phenomenon and the 'hippocampal-slice-technique'. In clinical neuroscience the description of Refsum's disease and studies of myasthenia gravis and multiple sclerosis have been of particular importance. Two of 13 centres of excellence in Norway selected in 2003 were from neuroscience, and The Norwegian Research Council has its own programme for neuroscience. The Norwegian Neurological Association arranges annual meetings to promote interest in neurological research.

Thematic review series: Skin Lipids. Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism

Many of the ichthyoses are associated with inherited disorders of lipid metabolism. These disorders have provided unique models to dissect physiologic processes in normal epidermis and the pathophysiology of more common scaling conditions. In most of these disorders, a permeability barrier abnormality "drives" pathophysiology through stimulation of epidermal hyperplasia. Among primary abnormalities of nonpolar lipid metabolism, triglyceride accumulation in neutral lipid storage disease as a result of a lipase mutation provokes a barrier abnormality via lamellar/nonlamellar phase separation within the extracellular matrix of the stratum corneum (SC). Similar mechanisms account for the barrier abnormalities (and subsequent ichthyosis) in inherited disorders of polar lipid metabolism. For example, in recessive X-linked ichthyosis (RXLI), cholesterol sulfate (CSO(4)) accumulation also produces a permeability barrier defect through lamellar/nonlamellar phase separation. However, in RXLI, the desquamation abnormality is in part attributable to the plurifunctional roles of CSO(4) as a regulator of both epidermal differentiation and corneodesmosome degradation. Phase separation also occurs in type II Gaucher disease (GD; from accumulation of glucosylceramides as a result of to beta-glucocerebrosidase deficiency). Finally, failure to assemble both lipids and desquamatory enzymes into nascent epidermal lamellar bodies (LBs) accounts for both the permeability barrier and desquamation abnormalities in Harlequin ichthyosis (HI). The barrier abnormality provokes the clinical phenotype in these disorders not only by stimulating epidermal proliferation, but also by inducing inflammation.

Midlife diagnosis of Refsum Disease in siblings with Retinitis Pigmentosa – the footprint is the clue: a case report

IntroductionRefsum disease is a potentially lethal and disabling condition associated with retinitis pigmentosa in which early treatment can prevent some of the systemic manifestations.Case presentationWe present the cases of two brothers with a diagnosis of retinitis pigmentosa from childhood in whom Refsum disease was subsequently diagnosed midlife, after routine enquiry into hand and feet abnormalities. Subsequent treatment through dietary modification stabilised visual impairment and has prevented development of neurological complications to date.ConclusionIt is therefore important to consider the diagnosis of Refsum disease in any patient with autosomal recessive or simplex retinitis pigmentosa, and to enquire about the presence of "unusual" feet or hands in such patients.

Phytanic acid impairs mitochondrial respiration through protonophoric action.

Refsum disease is a rare, inherited neurodegenerative disorder characterized by accumulation of the dietary branched-chain fatty acid phytanic acid in plasma and tissues caused by a defect in the alphaoxidation pathway. The accumulation of phytanic acid is believed to be the main pathophysiological cause of the disease. However, the exact mechanism(s) by which phytanic acid exerts its toxicity have not been resolved. In this study, the effect of phytanic acid on mitochondrial respiration was investigated. The results show that in digitonin-permeabilized fibroblasts, phytanic acid decreases ATP synthesis, whereas substrate oxidation per se is not affected. Importantly, studies in intact fibroblasts revealed that phytanic acid decreases both the mitochondrial membrane potential and NAD(P)H autofluorescence. Taken together, the results described here show that unesterified phytanic acid exerts its toxic effect mainly through its protonophoric action, at least in human skin fibroblasts.

Structural and mechanistic studies on the peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PhyH)

Phytanic acid (PA) is an epimeric metabolite of the isoprenoid side chain of chlorophyll. Owing to the presence of its epimeric beta-methyl group, PA cannot be metabolized by beta-oxidation. Instead, it is metabolized in peroxisomes via alpha-oxidation to give pristanic acid, which is then oxidized by beta-oxidation. PhyH (phytanoyl-CoA 2-hydroxylase, also known as PAHX), an Fe(II) and 2OG (2-oxoglutarate) oxygenase, catalyses hydroxylation of phytanoyl-CoA. Mutations of PhyH ablate its role in alpha-oxidation, resulting in PA accumulation and ARD (adult Refsum's disease). The structure and function of PhyH is discussed in terms of its clinical importance and unusual selectivity. Most point mutations of PhyH causing ARD cluster in two distinct groups around the Fe(II)- and 2OG-binding sites. Therapaeutic possibilities for the treatment of Refsum's disease involving PhyH are discussed.

Peroxisomal disorders affecting phytanic acid α-oxidation: a review

Peroxisomes are involved in the synthesis and degradation of complex fatty acids. They contain enzymes involved in the alpha-, beta- and omega-oxidation pathways for fatty acids. Investigation of these pathways and the diseases associated with mutations in enzymes involved in the degradation of phytanic acid have led to the clarification of the pathophysiology of Refsum's disease, rhizomelic chondrodysplasia and AMACR (alpha-methylacyl-CoA racemase) deficiency. This has highlighted the role of an Fe(II)- and 2-oxoglutarate-dependent oxygenases [PhyH (phytanoyl-CoA 2-hydroxylase), also known as PAHX], thiamin-dependent lyases (phytanoyl-CoA lyase) and CYP (cytochrome P450) family 4A in fatty acid metabolism. The differential regulation and biology of these pathways is suggesting novel ways to treat the neuro-ophthalmological sequelae of Refsum's disease. More recently, the discovery that AMACR and other peroxisomal beta-oxidation pathway enzymes are highly expressed in prostate and renal cell cancers has prompted active investigation into the role of these oxidation pathways and the peroxisome in the progression of obesity- and insulin resistance-related cancers.

Peroxisomes, Refsum's disease and the α- and ω-oxidation of phytanic acid

In the present paper, we describe the current state of knowledge regarding the enzymology of the phytanic acid alpha-oxidation pathway. The product of phytanic acid alpha-oxidation, i.e. pristanic acid, undergoes three cycles of beta-oxidation in peroxisomes after which the products, including 4,8-dimethylnonanoyl-CoA, propionyl-CoA and acetyl-CoA, are exported from the peroxisome via one of two routes, including (i) the carnitine-dependent route, mediated by CRAT (carnitine acetyltransferase) and CROT (carnitine O-octanoyltransferase), and (ii) the free acid route, mediated by one or more of the peroxisomal ACOTs (acyl-CoA thioesterases). We also describe our recent data on the omega-oxidation of phytanic acid, especially since pharmacological up-regulation of this pathway may form the basis of a new treatment strategy for ARD (adult Refsum's disease). In patients suffering from ARD, phytanic acid accumulates in tissues and body fluids due to a defect in the alpha-oxidation system.