Refsum disease due to the splice-site mutation c.135-2A>G before exon 3 of the PHYH gene, diagnosed eight years after detection of retinitis pigmentosa

Journal of the Neurological Sciences 266 (2008) 182 – 186 www.elsevier.com/locate/jns

Short communication

Refsum disease due to the splice-site mutation c.135-2ANG before exon 3 of the PHYH gene, diagnosed eight years after detection of retinitis pigmentosa Josef Finsterer a,⁎, Günther Regelsberger b , Till Voigtländer b a

b

Krankenanstalt Rudolfstiftung, Vienna, Austria Institute of Neurology, Medical University of Vienna, Währinger Gürtel 18-20, A-1097 Vienna, Austria Received 20 June 2007; received in revised form 30 August 2007; accepted 6 September 2007 Available online 1 October 2007

Abstract Objectives: If Refsum disease (RD) is not considered as a differential at onset of the initial manifestations the diagnosis of RD remains unrecognized for a long time as in the following case. Case report: A 55-y old Caucasian female with hyperextensible joints developed progressive visual impairment due to retinitis pigmentosa and sensorimotor polyneuropathy of the lower limbs since age 32 y. Screening for causes of polyneuropathy at age 40 y revealed markedly elevated serum phytanic acid (PA) with a maximum value of 293.6 μg/ml (n:b 6 μg/ml) why RD was diagnosed. Since age 48 y slowly progressive hypacusis was noted. RD was caused by the known transition A135G in exon 3 of the PHYH gene. Additionally, the polymorphism T153C in exon 3 of the PHYH gene was detected. Upon strict adherence to the Chelsea diet PA levels slightly decreased since onset of this therapy. Conclusion: This case confirms that RD remains unrecognized for a long time if RD is not considered as a differential of retinitis pigmentosa as the initial manifestation of the disease. Early recognition of RD is important since there is the therapeutic option of starting a diet. © 2007 Elsevier B.V. All rights reserved. Keywords: Phytanic acid; Alpha-oxidation; Peroxisomes; Lipid metabolism; Fatty acids; Multi-system disease; Polyneuropathy; Retinitis pigmentosa; Deafness

1. Introduction Adult Refsum disease (RD), also known as heredopathia atactica polyneuritiformis (OMIM 266500), is a genetically heterogeneous peroxisomal disorder, characterized by retinitis pigmentosa, anosmia, sensorimotor polyneuropathy, impaired hearing, cerebellar ataxia, ichthyosis, and, rarely, cardiac abnormalities or skeletal malformations [1,2]. In a few cases psychiatric disturbances have been observed. RD results from accumulation of phytanic acid (PA, 3,7,11,15tetramethylhexadecanoic acid, a 3-methyl-branched acid [3]) due to a defect in one of the enzymes involved in α-oxidation

⁎ Corresponding author. Postfach 20, 1180 Vienna, Austria. Tel.: +43 1 71165; fax: +43 1 4781711. E-mail address: [email protected] (J. Finsterer). 0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2007.09.005

(most frequently phytanoyl-CoA α-hydroxylase, PHYH) or due to a general peroxisomal dysfunction [4]. If RD is not considered as a differential at onset of the initial manifestations the diagnosis remains unrecognized for a long time, as in the following case. 2. Case report The patient is a 55-y old Caucasian female with a history of slowly progressive visual impairment due to retinitis pigmentosa diagnosed at age 32 y, leading to blindness since age 38 y, and slight numbness of the distal lower limbs since age 32 y. At age 40 y polyneuropathy was diagnosed upon the clinical presentation and nerve conduction studies. When screening for causes of polyneuropathy RD was detected at age 40 y upon a markedly elevated PA in the serum, 8 y after onset of the first clinical manifestations (Table 1). Hyperextensible joints were first noted at age 41 y. Also at age 41 y

J. Finsterer et al. / Journal of the Neurological Sciences 266 (2008) 182–186 Table 1 Results of serological investigations for diphytanyl triglycerides, monophytanyl triglycerides, nonphytanyl triglycerides, and phytanic acid over 14 years Age

DPT a

MPT a

NPT a

PA

GC-FID b

(y)

(%)

(%)

(%)

(%)

RL 40 40 40 41 42 42 42 43 43 44 44 45 45 46 46 47 47 48 48 49 49 51 51 52 53 54 54 55

≤2 27 nd 16 31 6 6 6 4 3 12 7 11 18 19 18 11 17 10 6 18 0 23 0 20 1 25 27 9

≤20 44 34 46 45 33 23 29 31 22 29 28 39 37 31 39 33 40 27 26 40 33 37 26 53 22 22 30 31

29 52 38 24 61 71 65 65 75 49 65 50 45 50 43 56 43 63 68 42 67 40 74 27 77 53 43 60

nd 16 17 30 11 11.2 12.3 8.3 9 17 7 12 14 14 14 12 10 12 10.6 14 10 13 12 10 13 nd nd nd

(μg/ml) ≤1000 nd nd nd nd nd nd nd nd nd nd nd nd nd 390 407 710 230 521 329 276 414 372 231 318 323 nd nd nd

c

GC-MS (μg/ml) d ≤600 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 216 220 223 222 294 209

DPT: diphytanyl triglycerides; MPT: monophytanyl triglycerides; NPT: nonphytanyl triglycerides (relative DPT, MPT, and NPT values in percent, determined by thin-layer chromatography [38], show the degree of PA integration into fats); RL: reference limit; nd: not determined. a Normal value of 100% nonphytanyl triglycerides. b PA in percent of total fatty acids as determined by GC-FID. c PA concentration determined by GC-FID. d PA concentration determined by GC-MS. Since PA concentrations were determined by different methods (GC-FID and GC-MS) and various extraction protocols and internal standards were applied during the disease course, the absolute values are not comparable but have to be related to the reference values determined for each method.

a colon carcinoma without local or distal dissemination was diagnosed, requiring partial colon resection but no radiation or chemotherapy. Nine years later colonoscopy revealed a relapse of the carcinoma, requiring abdominal surgery again, without a further relapse since then. Since age 43 y slowly progressive hypacusis was noted, supplied with a hearing device on from age 52 y. During several years she repeatedly had noted cramps of the thigh muscles bilaterally, particularly in the morning. Clinical neurologic examination at age 53 y showed bilateral blindness with atrophy of the bulbs, symmetrical, diffuse wasting of the upper and lower limbs, a springing finger, weakness for foot extension with right-sided predominance (Medical Research Council 5-), hyperextensible joints, and absent deep tendon reflexes on the lower limbs. Routine blood

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chemical investigations at age 54 y revealed slightly elevated cholesterol of 225 mg/dl (n: b 200 mg/dl) but was otherwise normal. Serum concentrations of diphytanyl triglycerides and monophytanyl triglycerides and of PA were elevated (Table 1). Ultrasonography of the liver, pancreas, and kidneys was normal. Nerve conduction studies showed prolonged distal latency (left median nerve), reduced motor conduction velocity (left median, left ulnar nerves), reduced sensory nerve conduction velocity (left ulnar nerve), absent response to adequate motor (right peroneal nerve) or sensory stimulation (left median, right sural nerves), or reduced amplitude of the compound muscle action potentials (left ulnar nerve) in the absence of partial or complete conduction blocks. Since age 41 y until age 54 y (14 y) the index patient was on a low PA diet. Two months before detection of the carcinoma she was eating salads and corn exclusively. She was regularly taking folic acid and vitamin B12. Since age 54 y she had switched to a diet proposed by the Chelsea & Westminster Refsum clinic [personal communication], which resulted not only in a subjective improvement of the complaints but also in a decrease of the biochemical abnormalities (Table 1). She had never undergone plasmapheresis, lipapheresis, or cascade filtration. 3. Methods Originally, determination of PA concentration was performed by gas chromatography equipped with flame ionization detection (GC-FID) as described by Molzer et al., 1989 [5]. Since the age of 51 y gas chromatography combined with mass spectrometry (GS-MS), using a modified procedure of Vreken et al. [6], was applied. One hundred μl plasma were added to 2 ml 0.5 M hydrochloric acid in acetonitrile and 100 μl internal standard (consisting of stable-isotope-labeled 2 μg/ml (3-methyl-2H3)-phytanic acid (obtained from Dr. ten Brink (Free University Hospital, Amsterdam, the Netherlands) in toluene) and incubated for 45 min at 110°C. After cooling, 2 ml 1.0 M NaOH in methanol were added or incubated for 45 min at 110°C. After cooling to room temperature, 0.5 ml of 10 M hydrochloric acid and 4 ml hexane were added. Samples were shaken and centrifuged at 1120 ×g for 10 min. The upper phase was dried under a stream of nitrogen in a waterbath at 45°C. After addition of 50 μl pyridine and 50 μl N-Methyl-N(tert-butyldimethyl-silyl)trifluoroacetimide (MTBSTFA; Sigma-Aldrich Co.), samples were incubated at 80°C for 30 min, then allowed to cool for 10 min at room temperature and diluted in 1 ml heptane. GC-MS analyses were performed on a TRACE MS Plus gas chromatograph single quadrupol mass spectrometer from Thermo Finnigan (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a J&W Scientific DB-1 ms capillary column (30 m × 0.25 mm I.D., film thickness 0.25 μm; Agilent Technologies Inc., Santa Clara, CA, USA). The initial oven temperature was 60°C, maintained for 1 min. The temperature was then increased by 30°C/min to 160°C, thereafter by 5°C/ min to 230°C, and finally by 20°C/min to 320°C, held for

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10 min. Injection, interface, and source temperature were adjusted at 300°C. A sample volume of 1 μl was injected at splitless mode with a splitless time of 1.5 min. Helium was used as carrier gas at constant pressure of 50 kPa. Electron impact ionization was utilized at 70 eV. Single ion monitoring was used to detect the (M-57)+ ions of the fatty acid silyl esters. Data were analyzed with the Finnigan XcaliburTM software package (Thermo Fisher Scientific Inc.). For DNA analysis genomic DNA was extracted from leukocytes using the QIAamp DNA mini kit (Qiagen, Hilden, Germany). Sequencing included the complete coding regions of the PHYH gene, including exons 1 to 9 and the exon–intron boundaries, which were amplified by PCR using appropriate primers. The PCR products were purified by agarose gel electrophoresis. Relevant DNA fragments were excised, extracted from the gel and sequenced in forward and reverse direction using the dideoxy Cy5/Cy5.5 Dye Primer sequencing kit (Visible Genetics, Toronto, Canada) and the OpenGene DNA sequencing system (Visible Genetics). DNA sequence analysis for mutations in the PHYH gene at age 53y revealed a previously described homozygous c.135– 2ANG transition [1], resulting in the elimination of a splice site. Additionally, the homozygous polymorphism c.153CNT in exon 3 of the PHYH gene was detected. The mother of the patient had been blind and had suffered from deafness and a springing finger before decease. The patient's son complained about restless legs, hyperhidrosis, slight hypacusis, and bulbar oscillations after forced concentration, but clinical neurologic examination was normal. The patient's daughter reported a single fever cramp in childhood, recurrent, short-lived amaurotic episodes since childhood after getting up from supine, and restless legs after exercise, but clinical neurologic examination was normal. Nerve conduction studies in the daughter were normal. Both children carried the splice-site mutation and polymorphism found in their mother, the first in a heterozygous form and the second in a homozygous form. PA levels were normal in both children. 4. Discussion RD is a rare (estimated prevalence 1:500,000–1:1,000,000 [7,8]), heterogeneous, peroxisomal disorder, biochemically characterized by the accumulation of the dietary, branched chain fatty acid PA (a saturated fatty acid of 20 carbon atoms with isoprenoic structure [9]), in the serum and various tissues, such as brain, retina, myocardium, kidney, liver, myelin sheaths, or fat [10,11]. Clinically, RD is characterized by the presence of cerebellar ataxia (sailor's walk), anosmia, retinitis pigementosa, deafness, sensorimotor polyneuropathy, bony changes, and increased protein in the cerebro–spinal fluid [11,12]. Rarely, there may be psychomotor retardation [13] or ichthyosis [14]. Hypacusis may be due to inner ear affection or, rarely, auditory nerve neuropathy [15]. Though clinical manifestations may start already at infancy or adulthood RD remains often unrecognized for a long time and is often diagnosed too late. Early diagnosis is important since the

course of the disease can be modified by diet treatment. Single manifestations of RD respond to high caloric, high docosahexaenoic acid, and low PA diet [2]. All food products originating from ruminants (meat, diary products) as well as fasting (to avoid mobilization of PA stores in fat) should be avoided. Though questionable, some patients may respond to black cumin oil (nigella sativa) [16]. Helpful for acute manifestations may be plasmapheresis, membrane differential filtration [16], lipapheresis [17], cascade filtration [17], or liver transplantation [18]. RD is genetically heterogeneous and caused by various mutations in at least two genes, encoding for phytanoyl-CoA α-hydroxylase (PHYH) on chromosome 10pter-p11.2 [19] and the PTS2 receptor (PEX7) on chromosome 6q21-q22.2 [20]. The PHYH gene extends over 21 kb and contains 9 exons and 8 introns [21]. Mutations in the PHYH gene account for most of the RD cases [2,22–24]. The remainder is caused by deficient PEX7 [20] and very rarely, by deficient racemase [25]. Over-expression of the PHYH-associated protein PHYHAP1 is made responsible for the cardiac manifestations of RD [26]. Mutations in the PHYH gene lead to impaired PHYH function and thus impaired breakdown of PA to pristanic acid at the initial α-oxidation step in peroxisomes and accordingly accumulation of PA [12,21,24]. The cytotoxic effect of PA is not fully elucidated yet but seems to be due to a combined effect on calcium regulation, mitochondrial depolarization due to facilitated opening of the mitochondrial permeability transition pore, decreased ATP production, or increased generation of reactive oxygen species [27,28]. Peroxisomes are single-membrane-bound cell organelles, in which various metabolic processes take its course, in particular α- and βoxidation of fatty acids [29,30]. α-Oxidation of PA is necessary before β-oxidation due to the presence of a blocking methyl group at the 3-position [23]. The presented case of an adult RD is interesting for the delayed diagnosis of RD, the hyperextensive joints, the markedly elevated PA serum levels, and the apparent response of the increased PA levels to the Chelsea diet. Whether hyperextensible joints were a manifestation of RD or due to other causes remains speculative. RD is frequently associated with short metacarpals or metatarsals [2]. If this was the cause of hyperextensible joints in the presented patient remains speculative, since dilative bands or flabby capsules have not been reported in association with RD. Whether the colon carcinoma was due to RD, the diet or other causes remains questionable. Previously, however, increased PA levels were associated with an increased risk of prostate cancer [31,32]. Though PA levels were high in the presented patient, PA levels up to 6400 μmol/l have been reported [33]. Sudden elevation of PA at age 41y (Table 1) was attributed to weight loss from the strict diet or the malignoma. The effect of the Chelsea diet on serum levels of PA is not unusual but future determinations in the described patient may show if this effect is truly attributable to the diet or just a pathophysiologic fluctuation. The Chelsea diet did not have an effect on any of the clinical manifestations so far. Expected

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clinical improvement from the Chelsea diet may concern hearing, or polyneuropathy but not impaired vision [34]. The conspicuous elevation of the PA serum levels throughout the observational period has only been rarely reported. The considerable elevation of serum PA in the presented patient may be due to a faulty diet and thus ingestion of too high amounts of PA or due to markedly impaired metabolization of PA. Low adherence is rather unlikely since compliance of the patient was high. More likely, the patient's low PA diet was not highly effective. Most likely, however, high PA levels are attributable to impaired metabolization of PA. The c.135–2ANG PHYH gene mutation leads to skipping of exon 3, consisting of 111 nucleotides. The mutation causes an inframe deletion of 37 amino acids and altered protein (p.Y46–R82del), which is clearly detectable by Western blot analysis when heterozygously expressed in S. cerevisiae but completely lacks enzymatic activity [35]. That the PHYH gene polymorphism c.153CNT contributed to the increase of the PA levels is rather unlikely given the fact that this polymorphism is a silent mutation and the most frequent variant according to the sequence tag database (estDB). An argument in favor of a causative role is that in other disorders, such as Leber's hereditary optic neuropathy, polymorphisms significantly influence the degree of the phenotypic expression [36]. The high levels of PA in the presented patient could be also due to an additional mutation in one of the enzymes involved in the omega-hydroxylation, an alternative pathway of PA breakdown [37]. In conclusion, this case shows that the diagnosis of RD may be delayed if not considered as a differential at the initial manifestations. RD should be considered if there is retinitis pigmentosa, impaired smelling, impaired hearing, or polyneuropathy alone or in combination and confirmed by the demonstration of an isolated plasma PA accumulation N 200 μmol/l vs. 10–30 μmol/l in non-affected controls. Early recognition of RD is important since there is the therapeutic option of starting a diet. References [1] Jansen GA, Waterham HR, Wanders RJ. Molecular basis of Refsum disease: sequence variations in phytanoyl-CoA hydroxylase (PHYH) and the PTS2 receptor (PEX7). Hum Mutat 2004;23:209–18. [2] Wierzbicki AS, Lloyd MD, Schofield CJ, Feher MD, Gibberd FB. Refsum's disease: a peroxisomal disorder affecting phytanic acid alpha-oxidation. J Neurochem 2002;80:727–35. [3] Sonneveld W, Haverkamp Begemann P, van Beers GJ, Keuning R, Schogt JCM. J Lipid Res 1962;3:351–5. [4] van den Brink DM, Wanders RJ. Phytanic acid: production from phytol, its breakdown and role in human disease. Cell Mol Life Sci 2006;63:1752–65. [5] Molzer B, Kainz-Korschinsky M, Sundt-Heller R, Bernheimer H. Phytanic acid and very long chain fatty acids in genetic peroxisomal disorders. J Clin Chem Clin Biochem 1989;27:309–14. [6] Vreken P, van Lint AEM, Bootsma AH, Overmas H, Wanders RJA, van Gennip AH. Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatographyelectron impact mass spectrometry. J Chromatogr B 1998;713:281–7.

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[7] Chapman E. Adult Refsum's disease; 2006. www.refsumdisease.org/ clinicians/nomenclature.shtml. [8] Gibberd FB, Feher MD, Sidey MC, Wierzbicki AS. Smell testing: an additional tool for identification of adult Refsum's disease. J Neurol Neurosurg Psychiatry 2004;75:1334–6. [9] Schonfeld P, Kahlert S, Reiser G. A study of the cytotoxicity of branched-chain phytanic acid with mitochondria and rat brain astrocytes. Exp Gerontol 2006;41:688–96. [10] Young SP, Johnson AW, Muller DP. Effects of phytanic acid on the vitamin E status, lipid composition and physical properties of retinal cell membranes: implications for adult Refsum disease. Clin Sci (Lond) 2001;101:697–705. [11] Cakirer S, Savas MR. Infantile Refsum disease: serial evaluation with MRI. Pediatr Radiol 2005;35:212–5. [12] Mukherji M, Chien W, Kershaw NJ, Clifton IJ, Schofield CJ, Wierzbicki AS, et al. Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease. Hum Mol Genet 2001;10:1971–82. [13] Baumgartner MR, Jansen GA, Verhoeven NM, Mooyer PA, Jakobs C, Roels F, et al. Atypical Refsum disease with pipecolic acidemia and abnormal catalase distribution. Ann Neurol 2000;47:109–13. [14] Ruther K. Adult Refsum disease. A retinal dystrophy with therapeutic options. Ophthalmologe 2005;102:772–7. [15] Oysu C, Aslan I, Basaran B, Baserer N. The site of the hearing loss in Refsum's disease. Int J Pediatr Otorhinolaryngol 2001;61:129–34. [16] Straube R, Gackler D, Thiele A, Muselmann L, Kingreen H, Klingel R. Membrane differential filtration is safe and effective for the long-term treatment of Refsum syndrome — an update of treatment modalities and pathophysiological cognition. Transfus Apher Sci 2003;29:85–91. [17] Gutsche HU, Siegmund JB, Hoppmann I. Lipapheresis: an immunoglobulin-sparing treatment for Refsum's disease. Acta Neurol Scand 1996;94:190–3. [18] Van Maldergem L, Moser AB, Vincent MF, Roland D, Reding R, Otte JB, et al. Orthotopic liver transplantation from a livingrelated donor in an infant with a peroxisome biogenesis defect of the infantile Refsum disease type. J Inherit Metab Dis 2005;28: 593–600. [19] Jansen GA, Ofman R, Ferdinandusse S, Ijlst L, Muijsers AO, Skjeldal OH, et al. Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene. Nat Genet 1997;17:190–3. [20] van den Brink DM, Brites P, Haasjes J, Wierzbicki AS, Mitchell J, Lambert-Hamill M, et al. Identification of PEX7 as the second gene involved in Refsum disease. Am J Hum Genet 2003;72:471–7. [21] Jansen GA, Hogenhout EM, Ferdinandusse S, Waterham HR, Ofman R, Jakobs C, et al. Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis of Refsum's disease. Hum Mol Genet 2000;9:1195–200. [22] Foulon V, Asselberghs S, Geens W, Mannaerts GP, Casteels M, Van Veldhoven PP. Further studies on the substrate spectrum of phytanoylCoA hydroxylase: implications for Refsum disease? J Lipid Res 2003;44:2349–55. [23] Wanders RJ, Jansen GA, Lloyd MD. Phytanic acid alpha-oxidation, new insights into an old problem: a review. Biochim Biophys Acta 2003;1631:119–35. [24] Wierzbicki AS, Mitchell J, Lambert-Hammill M, Hancock M, Greenwood J, Sidey MC, et al. Identification of genetic heterogeneity in Refsum's disease. Eur J Hum Genet 2000;8:649–51. [25] Ferdinandusse S, Denis S, Clayton PT, Graham A, Rees JE, Allen JT, et al. Mutations in the gene encoding peroxisomal alpha-methylacylCoA racemase cause adult-onset sensory motor neuropathy. Nat Genet 2000;24:188–1891. [26] Koh JT, Jeong BC, Kim JH, Ahn YK, Lee HS, Baik YH, et al. Changes underlying arrhythmia in the transgenic heart overexpressing Refsum disease gene-associated protein. Biochem Biophys Res Commun 2004;313:156–62. [27] Kahlert S, Schonfeld P, Reiser G. The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and

186

[28]

[29] [30]

[31]

[32]

[33]

J. Finsterer et al. / Journal of the Neurological Sciences 266 (2008) 182–186 mitochondria, and reduces cell viability in rat hippocampal astrocytes. Neurobiol Dis 2005;18:110–8. Schonfeld P, Kahlert S, Reiser G. In brain mitochondria the branchedchain fatty acid phytanic acid impairs energy transduction and sensitizes for permeability transition. Biochem J 2004;383:121–8. Depreter M, Espeel M, Roels F. Human peroxisomal disorders. Microsc Res Tech 2003;61:203–23. Wanders RJ, Waterham HR. Peroxisomal disorders: the single peroxisomal enzyme deficiencies. Biochim Biophys Acta 2006;1763: 1707–20. Ferdinandusse S, Rusch H, van Lint AE, Dacremont G, Wanders RJ, Vreken P. Stereochemistry of the peroxisomal branched-chain fatty acid alpha- and beta-oxidation systems in patients suffering from different peroxisomal disorders. J Lipid Res 2002;43:438–44. Xu J, Thornburg T, Turner AR, Vitolins M, Case D, Shadle J, et al. Serum levels of phytanic acid are associated with prostate cancer risk. Prostate 2005;63:209–14. Britton TC, Gibberd FB, Clemens ME, Billimoria JD, Sidey MC. The significance of plasma phytanic acid levels in adults. J Neurol Neurosurg Psychiatry 1989;52:891–4.

[34] Marcaud V, Defontaines B, Jung P, Degos CF. Refsum's disease: evolution 35 years after diagnosis. Rev Neurol (Paris) 2002;158: 225–9. [35] Jansen GA, Hogenhout EM, Ferdinandusse S, Waterham HR, Ofman R, Jakobs C, et al. Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis of Refsum's disease. Hum Mol Genet 2000;9:1195–200. [36] Feuk L, Marshall CR, Wintle RF, Scherer SW. Structural variants: changing the landscape of chromosomes and design of disease studies. Hum Mol Genet 2006;15(Spec No 1):R57–66. [37] Xu F, Ng VY, Kroetz DL, de Montellano PR. CYP4 isoform specificity in the omega-hydroxylation of phytanic acid, a potential route to elimination of the causative agent of Refsum's disease. J Pharmacol Exp Ther 2006;318:835–9. [38] Molzer B, Bernheimer H, Barolin GS, Höfinger E, Lenz H. Di-, monoand nonphytanyltriglycerides in the serum: a sensitive parameter of the phytanic acid accumulation in Refsum's disaease. Clin Chim Acta 1979;91:133–40.