12.9 Disorders of peroxisomal metabolism in adults

12.9 Disorders of peroxisomal metabolism in adults 2157

ESSENTIALS The peroxisome is a specialized organelle which employs molecular oxygen in the oxidation of complex organic molecules including lipids. Enzymatic pathways for the metabolism of fatty acids, including very long-​chain fatty acids (VLCFA), enable this organelle to carry out
β-​oxidation in partnership with mitochondria. A  peroxisomal pathway for isoprenoid lipids derived from chlorophyll, such as phytanic acid, utilizes α-​oxidation, but a default mechanism involving ω-​oxidation may also metabolize phytanic acid and its derivatives. The biochemical manifestations, molecular pathology, and di- verse clinical features of many peroxisomal disorders have now been clarified, offering the promise of prompt diagnosis, better manage- ment, and useful means to provide appropriate genetic counselling for affected families. At the same time, specific treatments including rigorous dietary interventions and plasmapheresis to remove undegraded toxic metabolites offer credible hope of improvement and prevention of disease in affected individuals. Inborn errors of peroxisomal metabolism usually present in in- fancy and childhood, but some disorders typically become manifest later in life and in adults, in whom the progress is often slow. Particular adult peroxisomal disorders X-​linked adrenoleukodystrophy (X-​ALD)—​due to mutation in the gene for an ATP-​binding cassette (ABC) protein of unknown function and characterized by accumulation of unbranched saturated VLCFAs, particularly hexacosanoate (C26), in the cholesterol esters of brain white matter, adrenal cortex, and certain sphingolipids of the brain. The disease has multiple phenotypes: it may present in adolescence with slowly progressive stiffness, clumsiness, weakness, weight loss, and skin pigmentation typical of Addison’s disease; it may present in adults with primarily psychiatric manifestations. Most cases de- velop increasing handicap; management is palliative and supportive in most instances. Adult Refsum’s disease—​due in most cases to mutations in the gene for phytanoyl-​CoA hydroxylase (PHYH) such that patients are un- able to detoxify phytanic acid by α-​oxidation and have greatly ele- vated levels of this in their plasma. Usually presents in late childhood with progressive deterioration of night vision, the occurrence of progressive retinitis pigmentosa, and anosmia; late features include deafness, ataxia, polyneuropathy, ichthyosis, and cardiac arrhyth- mias. Treatment is by restriction of dietary phytanic acid, with or without its elimination by plasmapheresis or apheresis. Neuropsychiatric adult peroxisomal disorders Historical perspective The likely first description of X-​linked adrenoleukodystrophy (X-​ ALD; OMIM 300100) was in 1910 when a 6-​year-​old child devel- oped abnormal eye movements, apathy, and mental deterioration. His gait then deteriorated and skin darkening was noted prior to his death a few months later. Examination of the brain by Schilder showed central demyelination, perivascular lymphocytes, foam cells, and gliosis which he termed encephalitis periaxalis diffusa. Other cases he later described are likely due to other leukodystrophies. Adrenoleukodystrophy was defined in 1970, with its characteristic adrenal changes of excess very long-​chain fatty acids (VLCFAs) and cholesterol esters present in cell inclusion bodies. These VLCFAs were later recognized as pathognomonic and identifiable in plasma samples and the primary defect was identified as an inability to me- tabolize them. The gene was mapped to Xq18 and identified as a member of the ATP-​binding cassette (ABC) transporter family. X-​ ALD was localized to the peroxisome. Subsequently, mouse models have been developed which show some clinical features of human disease such as adrenomyeloneuropathy but typically lack the cere- bral changes seen in man. Aetiology Adrenoleukodystrophy is characterized by the accumulation of un- branched saturated VLCFAs with a chain length of 24 to 30 carbons, particularly hexacosanoate (C26), in the cholesterol esters of brain white matter, in the adrenal cortex, and in certain sphingolipids of the brain. The disorder shows X-​linked inheritance with expression in female heterozygotes. The disruptive effects of the accumulation of VLCFAs, especially hexacosanoic acid (C26:0), on cell membrane structure and function may explain the neurological manifestations seen in adrenoleukodystrophy patients. VLCFAs cause alterations in membrane fluidity and affect cortisol secretion from cultured cells 12.9 Disorders of peroxisomal metabolism in adults Anthony S. Wierzbicki

section 12  Metabolic disorders 2158 of adrenal cortical origin. In addition, albumin has only one C26 binding site compared with more than six for shorter fatty acids, so limiting its efficacy as a reverse transport protein for excess VLCFAs. Clinical features X-​ALD is heterogeneous: seven phenotypes occur in males and five are recognized in females. Childhood cerebral adrenoleukodystrophy presents between the ages of 5 and 10 years with emotional lability, hyperactivity/​withdrawal, and mental deterioration, mimicking at- tention deficit disorder which evolves to parietal lobe dysfunction with apraxia, astereognosis, and later dementia. MRI shows a char- acteristic pattern of symmetric involvement of the posterior parieto-​ occipital white matter in 85% of patients, frontal involvement in 10%, and an asymmetric pattern in the rest. The clinical phenotype of X-​ALD shows a variable progression which may be interrupted by periods of arrest on MRI sometimes lasting 5 to 10 years. The adolescent form is adrenomyeloneuropathy which presents with slowly progressive stiffness, clumsiness, weakness, weight loss, and skin pigmentation typical of Addison’s disease. Autonomic function including micturition and erectile function are affected later. Somatosensory, auditory, and brainstem evoked potential are abnormal with some cases of abnormal visual and peripheral nerve conduction abnormalities. Brain MRI scans are abnormal in 50% of men and 80% of women, usually affecting corticospinal tracts with later parenchymal changes. Depression and emotional lability are common. Adult cerebral adrenoleukodystrophy is a variant of adrenomyeloneuropathy occurring after age 20 without spinal cord symptoms. The primary signs are psychiatric with a presentation of psychotic mania and may include schizophrenia or dementia. Some cases show a pure initial Addisonian picture with no neurological involvement; all are autoantibody negative. The onset of Addison’s disease is usually in childhood but the neurological changes follow in 20 to 30 years. Subtle hyper-​reflexia or impaired vibration sense and subtle MRI or neurophysiological signs may be detected earlier in these cases. It had been considered that neurological changes were mild or absent in carriers of X-​ALD, but up to 20% are symptom- atic. Women who are X-​ALD heterozygotes usually present with adrenomyeloneuropathy at age 30 to 40. Subtle signs are often de- tected prior to presentation but eventually the full picture occurs, with late-​onset dementia. A  recent prospective study of 46 fe- male carriers found an age-​dependent phenotype of myelopathy occurring in 63% and faecal incontinence in 28% independent of X-​inactivation status. These were associated with abnormalities in plasma VLCFAs and decreased fibroblast β-​oxidation of VLCFAs. In female adrenoleukodystrophy heterozygotes, adrenal cortical insufficiency rarely develops, although isolated min- eralocorticoid insufficiency may occur but may be difficult to diagnose. Furthermore, adrenoleukodystrophy heterozygotes are predisposed to hypoaldosteronism related to the use of nonsteroidal anti-​inflammatory drugs (NSAIDs). A subclinical decrease in gluco- corticoid reserve, as measured by synthetic ovine corticotropin-​ releasing hormone testing, may be present in most of these women. Aldosterone levels should be included in ACTH stimulation testing done to detect adrenal insufficiency in affected women. NSAIDs should be considered a risk factor for the develop- ment of hypoaldosteronism in women who are heterozygous for adrenoleukodystrophy. Rare presentations include olivopontocerebellar atrophy which has been described as X-​ALD ataxia in Japanese. Other uncommon presentations include unilateral masses which can mimic brain tumours and cases of spontaneous remission of neurological symptoms. A clinical syndrome that mimics features of ALD is acyl-​coenzyme A (CoA) oxidase deficiency but most cases present with severe neo- natal disease. Neuropathology There are two distinct forms of neuropathology associated with X-​ ALD. Pure adrenomyeloneuropathy is a distal axonic neuropathy while the cerebral forms are associated with inflammation. In cere- bral X-​ALD, brain pathology is often grossly normal though with signs of cerebral atherosclerosis. Grey matter is unaffected but white matter disease occurs in a rostrocaudal direction with demyelination prominent in the parieto-​occipital cortex and the cerebellum. The detailed pathology shows oligodendroglial cell loss, astrocytosis, and a perivascular inflammatory infiltrate. In the noncerebral form, de- myelination is seen in the corticospinal tracts with no obvious in- flammation and only mild gliosis and occasional macrophages. In the adrenal cortex, cells are filled with lamellar deposits of cholesterol es- ters with primary cortical atrophy and no evidence of inflammation or antibodies, with milder changes in the adrenomyeloneuropathy form. In men with X-​ALD, the testes show Leydig cell alterations, again with lamellar deposits. It has been estimated that at least 10% of males with Addison’s disease (adrenocortical failure) have X-​linked adrenomyeloneuropathy or unrecognized X-​ALD. Metabolism of VLCFAs VLCFAs are derived from the diet and endogenous synthesis, with between 20 and 80% derived from synthesis depending on the study. The synthetic pathway occurs in brain microsomes with re- peated additions of malonyl-​CoA units to palmitic (C16:0) or stearic (C18:0) acid precursors. There are probably separate pathways for C20:0 and C22:0 (behenic) fatty acids with the C22:0 pathway also elongating C22:1 (erucic) acid. Synthesis of VLCFAs starts with use of a specific activator pro- tein SLC27A4—​the fatty acid transport protein 4 (FATP4). The synthesis of saturated VLCFA, monounsaturated VLCFA (MUFA), and polyunsaturated fatty acids (PUFAs) occurs in the endoplasmic reticulum by four distinct enzymes; elongation of very long-​chain fatty acids ligase (ELOVL), 3-​ketoacyl-​CoA reductase (HSD17B12), 3-​hydroxyacyl dehydratase (HACD3), and trans-​2,3,-​enoyl-​CoA reductase (TECR). The initial condensation reaction catalysed by ELOVL is usually rate limiting. Mammals have seven different ELOVL enzymes (ELOVL1–​7) but only a single enzyme has been identified so far for each synthetic pathway. ELOVL1, ELOVL3, ELOVL4, and ELOVL6 are involved in the synthesis of saturated and monounsaturated fatty acids and ELOVL2, ELOVL4, ELOVL5, and ELOVL7 are essential for PUFA metabolism. The synthesis of C24:0 and C26:0 VLCFAs is carried out by the concerted action of ELOVL6 (C18:0–​C22:0) and ELOVL1 (C24:0–​C26:0) of which the latter is the key rate-​limiting step. Degradation of VLCFAs occurs by β-​oxidation within peroxi- somes after activation by specific acyl-​CoA ligases which are chain-​ length specific. Again, FATP4 plays a key role in activating the fatty acid.

12.9  Disorders of peroxisomal metabolism in adults 2159 Molecular genetics: the X-​ALD protein and its homologues The X-​ALD gene was mapped to a region of the X-​chromosome close to the glucose-​6-​phosphate dehydrogenase gene. The gene was established to code for an ABC protein of still unknown function but likely to involve the translocation of a variety of substrates across extra-​ and intracellular membranes, including lipids, sterols, and drugs. The ABCD1 protein (adrenoleukodystrophy protein) maps to Xq28 and is mutated in X-​ALD. ABCD1 is a member of the ABC transporter superfamily. It ex- presses a half transporter which is located in the peroxisome. The gene has an open reading frame of 2235 bases which encodes a 745-​ amino acid protein with 38.5% amino acid identity and 78.9% simi- larity to another peroxisomal protein (ABCD3). Mutations in ABCD1 result in X-​ALD in animal models, with elevated VLCFAs. ABCD1 is one of four related peroxisomal trans- porters that are found in the human genome, the others being ABCD2 (adrenoleukodystrophy related protein) (OMIM 601081), ABCD3 (peroxisomal membrane protein 70) (OMIM 170995), and ABCD4 (P70R/​PMP69) (OMIM 603214). The adrenoleukodystrophy pro- tein and the adrenoleukodystrophy-​related protein are expressed on oligodendroglia, while the adrenoleukodystrophy-​related protein and peroxisomal membrane protein 70 are found in neurons of the central nervous system. These genes are highly conserved in evolu- tion, and two homologous genes are present in the yeast genome, PXA1 and PXA2, which also transport long-​chain fatty acids. The 80-​kDa protein encoded by this gene is absent in patients with X-​ ALD, in whom X-​ALD mRNA was undetectable. Most of the ABCD1 mutations (>450) in X-​ALD are point mutations, but large deletions have been described. There is no correlation between genotype and phenotype. In 15 to 20% of obligate female heterozygotes, false-​ negative results occur for plasma VLCFAs. Mutation analysis is the only reliable method for the identification of heterozygotes. Overexpression of the adrenoleukodystrophy protein and its homologue, the adrenoleukodystrophy-​related protein (ABCD2), can restore the impaired peroxisomal β-​oxidation in the fibro- blasts of adrenoleukodystrophy patients. However, it seems that functional replacement of the adrenoleukodystrophy protein by adrenoleukodystrophy-​related protein is not due to stabiliza- tion of the mutated adrenoleukodystrophy protein. Similarly, the adrenoleukodystrophy-​related protein and peroxisomal membrane protein 70 could restore the peroxisomal β-​oxidation defect in the liver of adrenoleukodystrophy protein-​deficient mice by stimulating Aldr and Pmp70 gene expression through a dietary treatment with the peroxisome proliferator fenofibrate. These results suggested that a correction of the biochemical defect in adrenoleukodystrophy might be possible by drug-​induced overexpression or ectopic expression of the adrenoleukodystrophy-​related gene. The adrenoleukodystrophy protein transporter may facilitate the interaction between per- oxisomes and mitochondria, the two sites within the cells where β-​oxidation of VLCFAs occurs. The phenotype of X-​ALD was thought to be based on microglial activation for cerebral effects, while inflammation is less involved in adrenomyeloneuropathy but transcriptome studies show that a combination of effects of the defi- ciency on oxidative phosphorylation and adipocytokine and insulin signalling are responsible for the phenotypes. Many papers have described mutations in the ABCD1 gene in X-​ ALD patients, indeed more than 600 different mutations have now been described (http://​www.x-​ald.nl) of which 51% are missense, 28% frame-​shift, and 12% nonsense mutations; 6% are small in- sertions/​deletions and 13% are exon deletions. About 75% of all nonrecurrent ABCD1 mutations result in the absence of ABCD1 protein (http://​www.x-​ald.nl). Nonsense and frame-​shift mutations as well as large deletions lead to a truncated protein. Many missense mutations result in unstable protein whose detection is likely to be dependent on the specificity and sensitivity of the method used. The lack of anti-​ABCD1 immunofluorescence (IF) using microscopy in cultured fibroblasts is commonly used to assess ABCD1 pro- tein expression. However, fibroblasts express relatively low levels of ABCD1 and so this method may miss ABCD1 expression detectable by the more sensitive western blot methods. Re-​investigation of ‘IF negative’ cell lines using improved techniques has confirmed this supposition, hence some previously suspected cases may require re-​analysis. Disease-​associated missense mutations are not equally distributed over the ABCD1 protein. Analysis of 300 missense mu- tations showed clustering in two major regions (Fig. 12.9.1). Epidemiology Screening and diagnostic records suggest that the prevalence is a minimum of 1 in 22 500 to 1 in 62 000. In contrast, the use of the Hardy–​Weinberg approach and genetic frequency data suggests a combined male to female frequency of 1 in 18 000 similar to phenyl- ketonuria (1 in 12 000). Differential diagnosis The differential diagnosis of neuropsychiatric abnormalities is shown in Table 12.9.1. X-​ALD can mimic attention deficit disorder, multiple sclerosis, organic dementias, and psychoses among neuro- logical diseases, and Addison’s disease and hypogonadism among endocrine disorders (Table 12.9.2). The critical clinical differential element is the finding of abnormal ACTH concentrations and skin pigmentation with neurological signs, however subtle. Clinical investigation Clinical biochemistry The primary abnormality in X-​ALD is an accumulation of VLCFAs (>C22) which occur in myelin. C26:0 can account for up to 5% of brain cerebrosides and sulphatides. In X-​ALD, both saturated and unsaturated forms of C26:0 (cerotic) and C24:0 (lignoceric) acids ac- cumulate with reductions in C24:1(n-​9) (nervonic) acid. Normally, shorter fatty acids accumulate in brain cholesterol esters, but in X-​ALD, by contrast, these are mostly C26:0 and are enriched in myelin and in areas of demyelination. Similarly, C26:0 accumu- lates in white matter phosphatidylcholine phospholipids, C24:0 and C24:1 in gangliosides. Erythrocytes, plasma, and cultured fibroblasts all contain a 2-​ to 10-​fold excess of VLCFAs. The diagnostic test re- lies on measurement of C26:0 levels and the ratios of C26 to C22:0 (docosahexaenoic acid) and C26:0 to C24:0 (tetracosanoic acid). Some neonatal paediatric screening programmes have begun to implement screening for C26:0 phosphatidylcholine as a marker of X-​ALD in their dried blood spot analysis programmes. Results can be confirmed by fibroblast studies or by the use of sequencing techniques. Highly elevated VLCFA levels are also found in peroxisomal biogenesis disorders but these show a different clinical presentation to X-​ALD or transiently with ketogenic diets for seizures. False-​negative results may occur in patients consuming excess C22:1;

section 12  Metabolic disorders 2160 Table 12.9.1  Psychiatric signs and inborn errors of metabolism in adolescents and adults Disorder Confusion Mental retardation Behavioural disturbance Catatonia Visual hallucination Psychosis Depression Urea cycle defect + +/​–​ + + + + + Homocysteine disorders + + + + + +/​–​ + Porphyria + + + +/​–​ +/​–​ Wilson’s disease +/​–​ + +/​–​ + CTX + + + + MLD + + GM2 gangliosidosis + + + + + Mannosidoses + + + + + X-​ALD + + + Acyl-​CoA oxidase (pseudoneonatal adrenoleukodystrophy) + + + Nonketotic hyperglycinaemia + + Monoamine oxidase A deficiency + + Creatine transporter deficiency + + Succinic semi-​aldehyde dehydrogenase deficiency + + Niemann–​Pick C + + + + + CTX, cerebrotendinous xanthomatosis; MLD, metachromatic leukodystrophy; X-​ALD, X-​linked adrenoleukodystrophy. Reproduced from Sedel F et al. (2007a). Psychiatric manifestations revealing inborn errors of metabolism in adolescents and adults. J Inherit Metab Dis, 30, 631–​41, with permission. 10 8 6 4 2 0 50 100 N mPTS Transmembrane domain Number of disease-causing mutations per block of 10 amino acids Evolutionary conservation (% AA identity per block of 10 amino acids) Walker A Walker B C 0 20 40 60 80 100 150 200 250 300 350 400 450 500 550 600 650 700 750 Fig. 12.9.1  The degree of interspecies conservation and location of human mutations in the ABCD1 gene. The first region of conservation/​disease-​causing mutations is located in the transmembrane domain region (amino acids 83–​344) and the second is located in the ATP-​ binding domain (amino acids 500 and 668). The N-​terminal 73 amino acids and the C-​terminal 50 amino acids are mostly spared, hence caution is warranted when interpreting sequencing data suggesting missense mutations outside these key regions. Reproduced with permission from Wiersinger C, Eichler FS, Berger J. The genetic landscape of X-​linked adrenoleukodystrophy: inheritance, mutations, modifier genes, and diagnosis. Appl Clin Genet. 2015; 8: 109–​ 121. Copyright © 2015 Wiesinger et al.

12.9  Disorders of peroxisomal metabolism in adults 2161 ω-​9 (erucic acid; Lorenzo’s oil) which is found in mustard and rapeseed oils. A few affected males (0.1%) have borderline normal C26:0 levels and 15% of obligate female carriers have normal results. Effective mu- tation detection in these families is therefore fundamental to the un- ambiguous determination of genetic status. Of particular concern are female members of kinships with segregating X-​ALD mutations, be- cause normal levels of VLCFA do not guarantee a lack of carrier status. Prenatal diagnosis is possible from cultured amniocytes or chorionic villus cells. Abnormal liver function tests are a common finding in adrenoluekodystrophies and occur secondary to disturbances in di-​ and trihydroxycholestanoic acid (DHCA and THCA) metabolism. Radiology A MRI scan often reveals biochemical changes before the develop- ment of clinical symptoms. Eighty per cent of childhood cerebral adrenoleukodystrophy patients have symmetric periventricular white matter changes in the posterior parietal and occipital lobes with a dorsocaudal progression with time (Fig. 12.9.2a). Patients with adrenomyeloneuropathy typically have abnormalities in the pyramidal tracts (Fig 12.9.2b). Contrast studies show up areas of active demye- lination, inflammation with breakdown of the blood–​brain barrier, and gliosis. The Loes score (34-​point X-​ALD severity score) based on the five patterns of disease visible on MRI is used to determine severity and prognosis and is used as a decision aid prior to bone marrow transplantation. The presence of demyelination and gadolinium en- hancement are used to differentiate stable from likely progressive in- flammatory changes on MRI scanning (Fig. 12.9.2c). Proton magnetic resonance spectroscopy shows only mild reduction in N-​acetyl aspar- tate, normal choline and myo-​inositol, and normal lactate in patients Table 12.9.2  Differential diagnosis of X-​ALD Presentation Differential diagnosis Childhood neurological with normal endocrinology Hyperactivity, attention deficit disorder Epilepsy/​seizures Brain tumour Metachromatic/​globoid leukodystrophy Postencephalitic syndromes, e.g. subacute sclerosing panencephalitis Myelinoclastic diffuse sclerosis Childhood neurological with hypoadrenalism Addison’s disease with post-​hypoglycaemic damage X-​linked glycerol kinase deficiency Central pontine myelinolysis Glucocorticoid deficiency with achalasia Hypoadrenalism Secondary causes of hypoadrenalism Adrenomyeloneuropathy Multiple sclerosis Familial or other spastic parapareses Spinocerebellar/​olivopontocerebellar degeneration Cervical spondylosis Spinal cord tumour, e.g. ependymoma Adult cerebral Schizophrenia Depression Epilepsy/​organic psychosis Alzheimer’s disease or other dementias Brain tumour Heterozygote with symptoms Multiple sclerosis Chronic spinal disease Spinal cord tumour Cervical spondylosis (A) (a) (B) (C) (D) Fig. 12.9.2  (a) MRI of the brain in a case of childhood cerebral adrenoleukodystrophy (ALD) showing characteristic extensive white matter changes in the parieto-​occipital region and internal capsules on FLAIR sequences (A). This area is initially affected in about 80% of cases of cerebral ALD. The rim enhances after administration of gadolinium on T1 sequences (B). In about 20% of cases the site of initial involvement in cerebral ALD is the frontal white matter as shown on this FLAIR image of a different patient with cerebral ALD (C), with prominent rim enhancement after administration of gadolinium on a T1-​weighted image (D). (b) MRI of the brain in a patient with adrenomyeloneuropathy showing increased signal in the pyramidal tracts on T2-​weighed coronal (A) and axial (B) images indicative of Wallerian degeneration. (c) MRI of the brain (T2 (A) and FLAIR (C) images; T1 with gadolinium (B, D)) of a patient with adrenomyeloneuropathy who rapidly deteriorated clinically with new symptoms of cognitive decline. On MRI, extensive white matter changes were seen in the parieto-​occipital white matter and corpus callosum (A), but no enhancement of the lesion after administration of gadolinium (B). A follow-​up MRI about 3 months later shows progression of the white matter lesion (C) and there is now faint enhancement of the rim of the lesion after gadolinium administration (D). Reproduced with permission from Engelen M, Kemp S, de Visser M, van Geel BM, Wanders RJ, Aubourg P, Poll-​The BT. X-​linked adrenoleukodystrophy (X-​ALD): clinical presentation and guidelines for diagnosis, follow-​up and management. Orphanet J Rare Dis. 2012 Aug 13;7:51. doi: 10.1186/​1750-​1172-​7-​51.
Copyright © 2012 Engelen et al.; licensee BioMed Central Ltd.

section 12  Metabolic disorders 2162 with arrested as compared with progressing disease where N-​acetyl aspartate levels are significantly reduced while choline compounds, myo-​inositol, and lactate are raised. 18Fluorodeoxyglucose positron emission tomography shows increased glucose uptake in the frontal lobes with decreased activity in the temporal lobes and cerebellum in patients with X-​ALD. The increase in frontal activity correlated with scores from psychological evaluations. Proton spectroscopy using N-​acetyl aspartate shows up neuronal loss, while choline compound studies assaying phosphocholine and glycerophosphocholine indicate membrane turnover and demyelin- ation, and myo-​inositol compounds seem to be indices of gliosis. The presence of lactate indicates the anaerobic metabolism of the inflammatory cell infiltrate. In the adrenomyeloneuropathy brain, MRIs may be normal in 50% of men and 80% of women but diffuse spinal cord atrophy is present. Endocrinology Overt hypoadrenalism occurs in 40% of patients with child- hood cerebral adrenoleukodystrophy and 80% have a deficient cortisol response on Synacthen testing. In childhood disease, 80% show abnormal adrenal stimulation test results, while in adrenomyeloneuropathy, between 30 and 50% show normal re- sponses. Clinical Addison’s disease is found in 1% of female hetero- zygotes. In adrenoleukodystrophy heterozygotes, adrenal cortical insufficiency rarely develops, although hypoaldosteronism may occur, especially if NSAIDs are being used. ACTH levels are in- creased in male patients. Levels of follicle-​stimulating hormone or luteinizing hormone are increased in 50 to 70% of patients with adrenomyeloneuropathy, while testosterone levels are reduced in 20% with low normal levels of dehydroepiandrosterone sulphate. Neurophysiology Hearing is normal but brainstem auditory evoked potentials are abnormal in 95% of adrenomyeloneuropathy patients and 42% of heterozygote patients. Abnormalities in visual evoked potentials are also found as latencies and are increased in 20% of men with adrenomyeloneuropathy but in more than 70% with childhood cere- bral disease. Electroretinograms are normal. Subtle demyelination and axonal loss patterns of nerve conduction are found in 90% of men and 67% of women with adrenomyeloneuropathy, usually af- fecting the legs more than the arms. Neuropsychological tests can show up deficits in parieto-​occipital function affecting visuospatial parameters and auditory processing, while frontal lobe lesions affect executive functions, emotions, problem solving, and anticipatory processing. Treatment The progressive nature of X-​ALD means that comprehensive family and professional management support services are required. Leukodystrophies are associated with progressive learning diffi- culties, psychiatric disturbance, and increasing disability. Painful muscle spasms are common and should be managed with diazepam, baclofen, or gabapentin. Bulbar muscle function may be lost with disease progression, thus requiring special attention to feeding to reduce the risk of aspiration pneumonia. The routine management of patients with X-​ALD includes regular clinical reviews allied with MRI scanning at approximately 3–​6-​month intervals depending on the rate of progression. Endocrine assessment is performed at baseline and repeated if the clinical syndrome includes features of hypoadrenalism. Dietary therapy was based on the restriction of the intake of C26:0 to less than 15% of normal intake, but early trials showed no effect of this on levels of VLCFA levels. Addition of oleic acid normalized VLCFA levels in fibroblasts and oral glyceryl trioleate reduced VLCFA levels by 50% with an improvement in nerve con- duction measures. A  4:1 combination of glyceryl trioleate and trierucate (Lorenzo’s oil) normalized VLCFA levels within 1 month and prompted mass use of this intervention. No evidence of a clin- ically relevant benefit from dietary treatment with Lorenzo’s oil has been seen in many studies of patients with neurological involvement and X-​ALD, and asymptomatic thrombocytopenia was noted in 30% of patients. The fatty acid composition of the plasma and liver, but not that of the brain, improves with this therapy, suggesting that (A) (B) (b) (A) (c) (B) (C) (D) Fig. 12.9.2  Continued

12.9  Disorders of peroxisomal metabolism in adults 2163 little erucic acid crossed the blood–​brain barrier. Thus, dietary sup- plementation with Lorenzo’s oil is of limited value in correcting the accumulation of saturated VLCFAs in the brain of patients with es- tablished neurological adrenoleukodystrophy. In a study of 89 asymptomatic boys with X-​ALD who had normal MRI scans, Lorenzo’s oil and moderate fat restriction were pre- scribed for 6.9 ± 2.7 years. Plasma fatty acids and clinical status were followed as measures of outcome. Twenty-​four per cent devel- oped MRI abnormalities and 11% developed neurological and MRI abnormalities. The trial concluded that the reduction of C26:0 by Lorenzo’s oil was associated with a reduced risk of developing MRI abnormalities. Lorenzo’s oil therapy is indicated in asymptomatic boys with X-​ALD who have normal brain MRI scans. Experience with other adrenoleukodystrophy patients indicated that total fat in- take in excess of 30 to 35% of total calories may counteract or nullify the C26:0-​reducing effect of Lorenzo’s oil. Patients who develop progressive MRI abnormalities should be considered for haematopoietic stem cell transplantation, but the 5-​year mortality is 38% and survival is increased by 8 months on average. Results in 283 boys with X-​ALD who received haematopoietic cell bone marrow transplantation showed that the estimated 5-​ year survival was 66%. The leading cause of death was disease pro- gression. Donor-​derived engraftment occurred in 86% of patients. Demyelination involved parietal–​occipital lobes in 90%, leading to visual and auditory processing deficits in many boys. Bone marrow transplantation must be considered very early, even in a child without symptoms but with signs of demyelination on MRI, if a suit- able donor is available. There are few data on the usefulness of bone marrow transplantation in adrenomyeloneuropathy. Adrenal function must be monitored since 80% of asymptomatic patients with adrenoleukodystrophy develop evidence of adrenal insufficiency and adrenal hormone replacement therapy should be provided when indicated by laboratory findings. Given the inflammation associated with X-​ALD, a number of immunosuppressive regimens have been investigated. Studies of cyclophosphamide, immunoglobulin, and interferon-​β have been unsuccessful. Prognosis The prognosis in X-​ALD depends on the presentation. As yet, there are no methods of determining which type of disease will result from a given mutation as genotype–​phenotype correlation is poor. Once leukodystrophy begins, the prognosis is poor as progression is inevitable. Data from inherited error bone marrow transplant regis- tries shows prolongations in life with transplantation in X-​ALD but do not record improvements in quality of life. Future developments Other potential therapeutic approaches to X-​ALD include the use of lipid-​lowering drugs. Lowering cholesterol activates human ABCD2 in cultured cells. In mice, a sterol regulatory element exists in the Abcd2 promoter and overlaps sites for liver X receptor/​retinoid X receptor heterodimers. Adipose Abcd2 is induced by SREBP1c, whereas hepatic Abcd2 expression is down-​regulated by concur- rent activation of liver X receptor-​α and SREBP1c. Hepatic Abcd2 expression in liver X receptor-​α/​β mice is inducible to levels vastly exceeding wild type. Statins (3-​HMG-​CoA reductase inhibitors) are capable of nor- malizing VLCFA levels in primary skin fibroblasts derived from X-​ALD patients. They block the induction of proinflammatory cyto- kines through effects on rho kinase. Twelve patients with X-​ALD were treated with lovastatin for up to 12 months. Levels of C26:0 declined from pretreatment values and stabilized at various levels during a period of observation of up to 12 months, which does not correlate with the type of adrenoleukodystrophy gene mutation. In six patients, erythrocyte C26:0 levels fell by 50%. All patients with adrenomyeloneuropathy remained neurologically stable. However, follow-​up trials have been unsuccessful. The PPAR-​α agonist-​mediated induction of ABCD2 expression seems to be indirect and possibly mediated by the sterol-​responsive element-​binding protein 2 in mice. In addition PPAR-​α is involved in the regulation of ELOVL1, a key step in VLCFA synthesis. In vitro CoA esters of both bezafibrate and gemfibrozil inhibit ELOVL1 and could form starting points for novel drug development. However, a study of the pan-​PPAR agonist bezafibrate in 10 male patients failed to show any effects of plasma or erythrocyte VCLFA concentrations despite reducing plasma triglyceride levels as predicted. Studies in animal models have suggested that the PPAR-​γ (with some PPAR-​α activity) agonist pioglitazone may reduce axonal degeneration but there have been no studies in humans. Sodium 4-​phenylbutyrate reduces VLCFA levels through its ef- fects on peroxisomal function and increases adrenoleukodystrophy-​ related protein levels. However, human studies have failed to show consistent beneficial effects. ω-​Oxidation is an alternative oxidation route for VLCFAs. These fatty acids are substrates for the ω-​oxidation system in human liver microsomes and are converted into ω-​hydroxy fatty acids and further oxidized to dicarboxylic acids via cytochrome P450 (CYP)-​mediated reactions. The high sensitivity towards the specific CYP inhibitor 17-​octadecynoic acid suggested that ω-​hydroxylation of VLCFAs is catalysed by the CYP4A/​F subfamilies, particularly CYP4F2 and CYP4F3B, and that therapies capable of increasing ω-​oxidation may have the potential to reduce the progression of the disease. Previously gene therapy has been attempted for X-​ALD using lenti- virus transformation of white cells and 9 to 14% of cells showed re- constitution of ABCD1 expression over 24 months. A more modern gene therapy approach using an adeno-​associated virus construct AAV-​9/​ABCD1 shows appropriate neurological tropism following intracerebroventricular or intravenous injection and reduces plasma and brain VLCFA levels in Abcd1-​deficient mice. Given the extensive oxidative stress associated with demyelin- ation in X-​ALD there has been interest in antioxidant therapies in the treatment of X-​ALD. A combination of the antioxidants α-​ tocopherol, N-​acetyl-​cysteine, and α-​lipoic acid reduced demyelin- ation in Abcd1-​deficient mice. There are no studies of this approach in humans. There has been an explosion of interest in novel therapeutic strat- egies for inherited errors of metabolism. Classical enzyme replace- ment therapy for X-​ALD is impossible given the need to replace a peroxisomal transporter molecule, but other strategies utilizing technologies such as stabilized mRNA technology combined with liposome or other shielded delivery technologies allied with various methods of delivering tissue specificity may be more successful. These show promise in cell culture models but no studies have yet been performed in human X-​ALD. None of these technologies

section 12  Metabolic disorders 2164 have reached animal models let alone human trials in peroxisomal diseases. Neuro-​ophthalmic adult peroxisomal disorders Introduction Though survival is improving for peroxisomal biogenesis disorders and more subtle defects are now diagnosed, most still present in the neonatal period or in infancy. This is also true for most single en- zyme peroxisomal deficiencies. Only one group of disorders presents later, with the onset of symptoms often in early teenage years but, due to delays in diagnosis, many are not identified until they reach adulthood. In contrast to the neuropsychiatric or endocrine pres- entation associated with adrenoleukodystrophy, these peroxisomal disorders present as central and peripheral neuropathies—​a neuro-​ ophthalmic picture. They are often termed Refsum’s disease though, given the multiple underlying genetic defects, it would be better to refer to them as Refsum’s syndrome. The syndrome comprises three genetic disorders:  phytanoyl-​CoA hydroxylase deficiency (clas- sical adult Refsum’s disease), atypical rhizomelic chondrodysplasia punctata type 1, and the newly described α-​methylacyl-​CoA racemase deficiency. Historical perspective Adult Refsum’s disease (OMIM 266510), also called heredopathia atactica polyneuritiformis, is a hereditary sensory motor neur- opathy type IV. It was first described in 1947, but only recognized as a syndrome by Refsum in 1962. He described a constellation of signs comprised of retinitis pigmentosa, anosmia, deafness, ataxia, and polyneuropathy allied with raised levels of protein in the cere- brospinal fluid. The biochemical defect was identified in 1963 when phytanic acid was noted in the plasma of affected patients and de- fective α-​oxidation was later suggested as the cause of adult Refsum’s disease. This disease was thought to be unifactorial with admittedly some rare aberrant complementation studies until 1995 when, after the localization of the gene for phytanoyl-​CoA hydroxylase, up to 50% of cases in one series were shown not to be linked to chromo- some 10 but to chromosome 6. Eventually, the novel defect was iden- tified as a variant of rhizomelic chondrodysplasia punctata type 1 and caused by mutations in peroxin 7. In parallel with this discovery, three patients were described in 1997 with a phenotype of sensory neuropathy and a subtle bile acid disorder but whose families in- cluded siblings with a Refsum’s-​like syndrome which was identified as due to a deficiency in α-​methylacyl-​CoA racemase. A clinical phenocopy associated with polyneuropathy, hearing loss, ataxia, ret- initis pigmentosa, and cataract (PHARC) (OMIM 612674) has re- cently been described. In contrast to other Refsum-​like syndromes, phytanic acid levels are normal in this condition. Clinical features In contrast to Zellweger’s syndrome (OMIM 214100), neonatal adrenoleukodystrophy (OMIM 202370), infantile Refsum’s dis- ease (OMIM 266500), and rhizomelic chondrodysplasia (OMIM 601757), adult Refsum’s disease usually presents in late childhood with progressive deterioration of night vision, the occurrence of progressive retinitis pigmentosa, and anosmia (Table 12.9.3). Anosmia, contrary to early reports, is a constant feature of adult Refsum’s disease. After 10 to 15 years, deafness, ataxia, polyneur- opathy, ichthyosis, and cardiac arrhythmias can occur. Short meta- carpals or metatarsals are found in about one-​third of patients. Rare findings include psychiatric disturbance and proteinuria. Premature death may result from cardiac arrhythmias. α-​Methylacyl-​CoA racemase (OMIM 604489)  presents with adult-​onset sensorimotor neuropathy (Table 12.9.3). It may be ac- companied by retinitis pigmentosa, visual field restriction and loss of acuity, axonal sensorimotor neuropathy, and myopathy-​like adult Refsum’s disease. Other features described have included primary hypogonadism, hypothyroidism, spastic paraparesis, epileptic seiz- ures, and mild developmental delay. More severe childhood-​onset cases have shown a phenotype of defects in bile acid synthesis al- lied with fat-​soluble vitamin deficiencies, coagulopathy, and cholestatic liver disease and a resemblance to a Niemann–​Pick type C phenotype. Table 12.9.3  Comparison of clinical features of underlying metabolic defects associated with adult Refsum’s disease Adult Refsum’s disease
(n c. 300) Rhizomelic chondrodysplasia (n c.5) α-​Methylacyl-​CoA racemase (n c.6) PHARC (n c.30) Retinitis pigmentosa Age >12 Age >12 Age >20 Age >30 Cataract Age >30 Age >30 ? Age >5 Anosmia All All ? Absent Sensorineural deafness Age >40 Age >40 ? Age >5 Sensory neuropathy Age >20 PA dependent Age >20 PA dependent Age >30 Axonal/​ demyelinating Age >30 Variable progressive Ataxia Age >20 PA dependent Age >20 PA dependent Variable Progressive Cholestasis No No Yes No Biochemistry: Phytanic acid Pristanic acid (μmol/​L)

300 <0.2 100 <0.2 <200 10 Normal (<10) Normal (0.5–​3) PA, phytanic acid; PHARC, polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract.

12.9  Disorders of peroxisomal metabolism in adults 2165 PHARC shares many clinical features of Refsum’s disease but lacks the anosmia and possibly the osteological changes (Table 12.9.3). Some mitochondrial disorders in the Leigh’s syndrome spectrum, including neurogenic muscle weakness, ataxia, and retinitis pig- mentosa (NARP), caused in many cases by mutations in mitochon- drial MT-​ATP6, may also share some clinical features with adult Refsum’s disease. Aetiology Phytanic acid (3R,S,7R,11R,15-​tetramethylhexadecanoic acid) is an isoprenoid lipid derived from the phytol side chain of chlorophylls by bacterial degradation in ruminants, invertebrates, or pelagic fish (see Fig. 12.9.3). Phytol can be oxidized to an unsaturated fatty acid, phytenic acid, and this is saturated to phytanic acid by a pathway involving fatty aldehyde dehydrogenase 10 (FALDH-​10) in micro- somes. The significance of this pathway in humans is unclear though high phytanic acid levels have been described in some patients defi- cient in FALDH-​10 with Sjögren–​Larsson syndrome. Most phytanic acid is ingested from the adipose tissue and muscle of herbivores or pelagic fish. The average human daily dietary intake of phytanic acid in Western societies is between 50 and 100 mg, of which about 50% is absorbed and metabolized. Phytanic acid is transported in plasma bound to very low-​density lipoprotein and later low-​density lipoprotein, with its elimination allied to reverse cholesterol transport (high-​density lipoprotein). Phytanic acid is preferentially taken up by the liver and may account for up to 50% of the free fatty acid pool in hepatocytes. This pool is labile and can be acutely mobilized by stress, infection, or starva- tion, resulting in rapid phytanic acid release. Plasma phytanic acid concentrations are less than 10% of the levels found in adipose tissue and neurons, which accumulate phytanic acid because of its hydro- phobicity. The elimination half-​life of total body phytanic acid is usually between 1 and 2 years. Most fatty acids are metabolized by the β-​oxidation path- ways in peroxisomes and mitochondria. Phytanic acid cannot be metabolized by this route due to the presence of a β-​methyl group. Instead, phytanic acid is metabolized either by α-​oxidation to pristanic acid, or by ω-​oxidation from the other end of the molecule. Using radiolabelled [14C]-​phytanic acid as a substrate, an enzyme activity responsible for the α-​oxidation of phytanic acid in cell lys- ates was described in 1967. This activity was eventually localized within peroxisomes and, after 30 years, the pathway responsible for α-​oxidation has been clarified. α-​Oxidation of phytanic acid Most phytanic acid metabolism occurs in the liver and kidney by α-​oxidation, though skin fibroblasts are used for clinical diagnostic purposes. Phytanic acid from plasma enters the peroxisome in asso- ciation with the sterol carrier protein-​2 (SCP-​2) and is metabolized by a four-​step initial α-​oxidation pathway (Fig. 12.9.4). Unusually, it appears this pathway can metabolize two stereoiso- mers of its substrate equally well. One carbon atom is then removed from the latter in a lyase reaction to give pristanal and formyl-​CoA. Pristanal is then oxidized to pristanic acid which is thio-​esterified using CoA to give a racemic mixture. The action of α-​methylacyl-​ CoA racemase converts the (2R)-​epimer to the (2S)-​epimer. Further degradation of (2S)-​pristanic acid by the stereospecific β-​oxidation pathway then occurs, with the release of propionyl and acetyl-​CoA units. Further β-​oxidation reactions (including epimerization) are required to generate the dimethylundecanoic and dimethylnonanoic and methyl-​heptanoic acid derivatives, which are finally exported for mitochondrial β-​oxidation. Disordered ω-​oxidation of phytanic acid Patients with adult Refsum’s disease are unable to detoxify phytanic acid by α-​oxidation, and so the ω-​oxidation pathway is the only metabolic pathway available for its degradation (Fig. 12.9.5). This pathway produces 3-​methyladipic acid as the final metab- olite, which is excreted in the urine. Thus, 3-​methyladipic acid concentrations can be used as an index of the molar activity of the 0 0 10 20 30 Age (years) 40 50 60 RP Ichthyosis Ataxia Deafness Neuropathy Anosmia 2 4 6 8 10 Number of patients 12 14 16 Fig. 12.9.3  Cumulative incidence of clinical features on presentation of 15 patients with Refsum’s disease. RP, retinitis pigmentosa. Reproduced from Wierzbicki AS, et al. (2002). Refsum’s disease: a peroxisomal disorder affecting phytanic acid alpha-​ oxidation. J Neurochem, 80, 727–​35, with permission.

section 12  Metabolic disorders 2166 ω-​oxidation pathway. After ingestion of a test load of phytanic acid, 3-​methyladipic acid is detected in the urine of healthy controls and adult Refsum’s disease heterozygotes showing that ω-​oxidation plays a significant role in postprandial metabolism of phytanic acid in humans. The activity of the ω-​oxidation pathway is approximately doubled in patients with adult Refsum’s disease, but this microsomal pathway has considerable reserve capacity. The balance of intake of phytanic acid and its ω-​oxidation is likely to determine long-​term concentra- tions of the lipid. Patients with adult Refsum’s disease often clinically relapse during episodes of illness or drastic weight loss. Fasting in- duces ketosis and lipolysis and acute mobilization of phytanic acid in hepatocyte and adipocyte fatty acid pools. This process can in- duce a release of 5000 mg (c.15 mmol) per day of phytanic acid (50 times normal). In experimental ketosis, following acute starvation, phytanic acid doubled in 29 h in patients with adult Refsum’s dis- ease and an 80% rise was seen in urinary 3-​methyladipic acid levels, indicating that ω-​oxidation was buffering part of this rise. Phytanic acid concentrations can exceed the capacity of the residual α-​ and ω-​oxidation pathways. Excess phytanic acid is excreted by low-​ affinity pathways. Phytanic acid can be glucuronidated and it can also be lost nonspecifically in the urine as nephropathy is a feature of adult Refsum’s disease. The enzymology of the ω-​oxidation pathway in adult Refsum’s disease has been clarified and occurs through the microsomal CYP4A system as well as the peroxisome. The capacity of the ω-​ oxidation pathway has been measured by the excretion of 2,6-​ dimethyloctanedioic acid (the C10 ω-​2-​methyl thioester derivative of phytanic acid) at 30 mg phytanic acid (89 µmol) per day. However, other studies measuring 3-​methyladipic acid excretion showed a far lower capacity of 6.9 mg (20.4 µmol) per day. These differences in activity may reflect the metabolic fates of the respective markers. Both 2,6-​dimethyloctanedioic acid and 3-​hexanedioic acid are products of the initial steps of ω-​oxidation and may be dependent on carnitine ester formation for activation and further metabolism. The initial steps of ω-​oxidation appear to have a greater capacity than that of the whole pathway when measured by the final product 3-​methyladipic acid. Molecular toxicology of Refsum’s syndrome The exact mechanism of the toxicity of phytanic acid to neuronal, cardiac, and bone tissue is gradually being clarified. Structural Fig. 12.9.4  Metabolic pathway for α-​oxidation of phytanic acid and β-​oxidation of pristanic acid producing 4,8 dimethynonanoyl-​ CoA, propionyl CoA, and acetyl-​CoA as end-​products. This pathway is integrated with PXMP-​2 porin responsible for transport of small molecules, PMP34 for ATP, and a transporter for phytanoyl-​CoA. Reproduced with permission from Wanders RJA, Komen J, Ferdinandusse S. Phytanic acid metabolism in health and disease. Biochimica et Biophysica Acta (BBA) -​ Molecular and Cell Biology of Lipids. Copyright © 2011 Elsevier B.V. Fig. 12.9.5  ω-​Oxidation mediated by a microsomal CYP4A enzyme yields phytane 1,16 dioic acid which can be metabolized to a wide spectrum of metabolites by β-​oxidation. Reproduced with permission from Wanders RJA, Komen J, Ferdinandusse S. Phytanic acid metabolism in health and disease. Biochimica et Biophysica Acta (BBA) -​ Molecular and Cell Biology of Lipids. Copyright © 2011 Elsevier B.V.

12.9  Disorders of peroxisomal metabolism in adults 2167 homology between phytanic acid and vitamin A, vitamin E, geranyl pyrophosphate, and farnesyl pyrophosphate has been noted and it has been suggested that phytanic acid may have a role in the regu- lation of isoprenoid metabolism and protein prenylation. Recent studies have identified that phytanic acid and also pristanic acid are direct toxins to mitochondria and it has been found that phytanic acid has a rotenone-​like action in uncoupling complex I in the oxida- tive phosphorylation chain in the mitochondrial inner membrane, with subsequent likely production of reactive oxygen species, causes secondary calcium-​driven changes through GPR40, and induces apoptosis in neuronal cells through a histone deacetylase-​mediated mechanism. This metabolic toxicity may explain why neuronal or allied retinal pigment tissues rich in mitochondria are the prime tis- sues affected in adult Refsum’s disease. The molecular toxicology of pristanic acid is unknown, although it is likely that the mild ophthalmic features seen in some cases may relate to phytanic acid toxicity as for phytanoyl-​CoA hydroxylase deficiency. Although both di-​ and trihydroxycholestanoic acids levels are elevated in α-​methylacyl-​CoA racemase, there is no phenotype of itching associated with this disorder. The cause of the sensory neuropathy in α-​methylacyl-​CoA racemase still remains to be determined. Molecular genetics The defect in adult Refsum’s disease was soon identified as being due to the lack of an α-​oxidase. It took 30 years for the enzyme responsible, phytanoyl-​CoA hydroxylase, to be identified. Two groups identified the gene for phytanoyl-​CoA hydroxylase simul- taneously in 1997. The phytanoyl-​CoA hydroxylase gene includes nine exons and codes for a 338-​amino acid protein including the 30-​amino acid signal domain, which is cleaved on entry into the peroxisome. Like all the peroxisomal targeting sequence type 2 proteins, phytanoyl-​CoA hydroxylase is transported into the peroxisomes by the protein transporter peroxin 7. Deficiency in this transporter is responsible for rhizomelic chondrodysplasia punctata (RCDP) type 1. Phytanoyl-​CoA hydroxylase is an iron (II) and 2-​oxoglutarate-​dependent oxygenase, with little overall sequence similarity to other human oxygenases. Numerous point and splice mutations in phytanoyl-​CoA hydroxylase have now been described in adult Refsum’s disease patients, many of which affect 2-​oxoglutarate conversion. Significantly, all cause complete inactivation of the protein; no partial function mutations have yet been identified. Genetic mapping studies have shown that in most cases, but not all, classical adult Refsum’s disease maps to chromosome 10. The locus for the second form of adult Refsum’s disease, comprising about 10% of cases, was localized to chromosome 6q22–​q24 and biochemical studies of fibroblasts from patients with adult Refsum’s disease established that these patients have subtle deficiencies of per- oxisomal targeting sequence type 2-​dependent enzyme functions (plasmalogen synthesis) consistent with mild variants of RCDP, though they lack any clinical signs specific to childhood-​onset form of RCDP where significant deficiencies in plasmalogen syn- thesis result in intellectual impairment and other neurological signs. Ironically, one of the original patients described with adult Refsum’s disease turned out to have the RCDP variant. A limited number of mutations have been described that cause Refsum’s–​RCDP and it is unclear why these mutations should preferentially lead to specific mislocalization of phytanoyl-​CoA hydroxylase in contrast to the other peroxin 7 imported proteins. Epidemiology Neuropathic adult peroxisomal disorders are rare, with a prevalence of 1 in 106 in Europe and, for unexplained reasons, 10-​fold less in the United States of America. As with all recessive conditions, they are more common in cultures or localities with strong founder ef- fects where consanguineous marriages are frequent. The classical Refsum’s phenotype is usually found in genetic ophthalmic services where it may represent 1% of retinitis pigmentosa cases. No sur- veys have been performed on the incidence of α-​methylacyl-​CoA racemase among patients with neuropathy. Differential diagnosis The differential diagnoses of the neuropathic disorders and relevant signs and investigations are shown in Tables 12.9.4 and 12.9.5. With classical adult Refsum’s disease, the differential diagnosis includes the various genetic retinitis pigmentosa syndromes if neurological signs are subtle and other rare neurological disorders (Table 12.9.6). Clinical investigation The key investigations in the case of suspected neuropathic adult Refsum’s disease are the measurement of phytanic acid (for adult Refsum’s disease) and pristanic acid (for suspected α-​methylacyl-​ CoA racemase). These are diagnostic. For clinical staging purposes, electroretinograms are often performed but often show flat responses characteristic of well-​ established retinitis pigmentosa. Visual fields should be assessed regularly as functional diplopia is a long-​term complication of adult Refsum’s disease. Slit-​lamp examination for cataracts is also indi- cated, as these can be treated. Ideally, retinal photography should be performed so that the extent of retinitis pigmentosa and its pro- gression can be monitored on a long-​term basis. Anosmia can be detected by screening using the standard four-​bottle smell test, but is best quantified by more extensive profiles (e.g. the University of Pennsylvania smell identification test). Auditory function should be assessed by auditory evoked potentials and hearing tests and moni- tored every 5 years. Peripheral neuropathy should be investigated by peripheral nerve conduction studies for somatosensory poten- tials and electromyography. A  nonspecific demyelination pattern is typical of adult Refsum’s disease. Osteo-​ or chondrodysplasia is best identified by a radiological survey of hands and feet for short metatarsals and knee radiology for signs of current or previous chondrodysplasia. Subtler signs that may accompany these definitive tests include an electrolyte profile showing mild hypokalaemia and a Fanconi-​ like aminoaciduria which can occur in adult Refsum’s disease. Liver function tests should be performed. If bilirubin is raised or α-​methylacyl-​CoA racemase is suspected, a detailed bile acid profile should be performed by mass spectrometric methods. As the differ- ential diagnoses include vitamin deficiencies, vitamin A and E levels should be measured to exclude retinol-​deficiency retinopathy and tocopherol-​deficient ataxia. Vitamin B12 and folate determinates are used to exclude cobalamin/​folate deficient neuropathy. To differentiate phytanoyl-​CoA hydroxylase from peroxin 7 adult Refsum’s disease, it is necessary to measure plasma VLCFAs and plasmalogens. However, often the deficiencies are subtle and these

section 12  Metabolic disorders 2168 investigations may appear normal. For a definitive diagnosis, a skin biopsy should be taken, fibroblasts grown, and detailed enzyme and immunofluorescence profiles examined in a specialist peroxisomal laboratory. Criteria for diagnosis The pathognomonic finding in adult Refsum’s disease is greatly ele- vated phytanic acid concentrations in the plasma (>200 µmol/​litre; normal <30 µmol/​litre), in contrast to other peroxisomal disorders where levels are usually lower and other metabolic abnormalities are also present. Unlike in rhizomelic chondrodysplasia punctata or the peroxisomal biogenesis disorders, no intellectual defects are seen, bone abnormalities are mild (if present at all), and there is no de- fect in plasmalogen synthesis. In infantile Refsum’s disease, which is a mild clinical variant of the peroxisomal biogenesis disorder encompassing Zellweger’s disease as its most severe form, numerous Table 12.9.4  Differential diagnosis of treatable adult neuropathies caused by inborn errors of metabolism Disease Onset Neurology Signs Chemistry Treatment Screening Fabry’s disease 10–​20 Small fibre, sensory Stroke; cardiomyopathy; renal Low
α-​galactocerebrosidase ERT WBC
α-​galactocerebrosidase Serine deficiency 10–​20 Axonal Growth delay; ichthyosis Low CSF/​plasma serine Serine Plasma amino acids Cerebrotendinous xanthomatosis 10–​40 Axonal, demyelination, sensorimotor Mental retardation; ataxia, spastic paraparesis. Tendon xanthomata Cholestanol Chenodeoxycholate Cholestanol Adult Refsum’s disease/​syndrome 10–​50 Demyelination, sensorimotor Retinitis pigmentosa, ataxia, anosmia Phytanic acid Low phytanic acid diet Phytanic acid Porphyrias 10–​50 All Neuropsychiatric Dermatological PBG and δALA Various PBG and δALA Wilson’s disease 15–​50 Axonal, demyelination, sensorimotor Movement disorder Copper/​caeruloplasmin Chelation Copper/​ caeruloplasmin CSF, cerebrospinal fluid; δALA, δ-​aminolaevulinic acid; PA, phytanic acid; PBG, porphobilinogen. Reproduced from Sedel F et al (2007) Peripheral neuropathy and inborn errors of metabolism in adults. J Inherit Metab Dis, 30, 642–​53, with permission. Table 12.9.5  Differential diagnosis of other adult neuropathies caused by inborn errors of metabolism Disease Age of onset Neuropathy Signs Chemistry Treatment Screening Mitochondrial myopathy 15–​50 All Retinitis pigmentosa, epilepsy, ataxia CSF/​plasma lactate None Lactate, muscle biopsy Metachromatic leukodystrophy 15–​50 Demyelination, sensorimotor Psychiatry, ataxia Aryl-​sulphatase A None/​bone marrow transplant Aryl-​sulphatase A Krabbe’s disease 15–​50 Demyelination, sensorimotor Spastic paraparesis WBC galactocerebrosidase None/​Bone marrow transplant WBC galactocerebrosidase GM2 gangliosidosis 15–​50 All Psychiatry; ataxia, Hexosaminidase None WBC hexosaminidase AMACR 10–​50 Demyelination, sensorimotor Retinitis pigmentosa, ataxia, anosmia, IQ Pristanic acid, D/​ THCA Low PA diet Pristanic acid Abetalipoproteinaemia 5–​20 Axonal, sensory, sensorimotor Ataxia, movement disorder, retinitis pigmentosa, acanthocytes Low cholesterol, low apolipoprotein B, vitamins A and E Vitamins A and E Apolipoprotein B Vitamin E deficiency 10–​20 Axonal, demyelination, sensorimotor Ataxia, movement disorder, retinitis pigmentosa, acanthocytes Vitamin E Vitamin E Vitamin E Homocysteine metabolism (CblC) 15–​50 Axonal, motor neuron disease, sensorimotor Psychiatric, stroke, leukoencephalopathy; Macrocytosis Homocysteine; methylmalonic acid Folate, vitamins B12 and B6, betaine Homocysteine X-​ALD 15–​50 Axonal, demyelination, sensorimotor Neuropsychiatric leukoencephalopathy; adrenal failure Very long-​chain fatty acids ?Lorenzo’s oil Very long-​chain fatty acids NARP 5–​20 Demyelination, sensorimotor Retinitis pigmentosa ataxia Mitochondrial—​MT-​ ATP6 None; supportive Mitochondrial DNA PHARC 10–​30 Demyelination, sensorimotor Retinitis pigmentosa ataxia, cataract Endocannabinoid(?) None None AMACR, α-​methylacyl-​CoA racemase; CSF, cerebrospinal fluid; D/​THCA, di-​/​trihydroxycholestanoic acid; PA, phytanic acid; WBC, white blood cell. Adapted from Sedel F, et al. (2007). Peripheral neuropathy and inborn errors of metabolism in adults. J Inherit Metab Dis, 30, 642–​53, with permission.

12.9  Disorders of peroxisomal metabolism in adults 2169 subtle peroxisomal defects are present and the condition presents from birth. In α-​methylacyl-​CoA racemase neuropathy, the pathognomonic findings are raised levels of pristanic acid (>100 µmol/​litre) allied with increases in di-​ and trihydroxycholestanoic acids. A secondary elevation of phytanic acid may be seen, but levels are usually be- tween 50 and 100 µmol/​litre. Treatment Long-​term prospects for the treatment of adult Refsum’s disease (or at least for some forms) are good as it is one of the few inherited disorders of metabolism with an exogenous precipitating cause. The disease is treated symptomatically by restriction of phytanic acid in- take in the diet or its elimination by plasmapheresis or apheresis. These regimens reduce plasma phytanic acid levels by between 50 and 70%, to values typically around 100 to 300 µmol/​litre, and can eliminate phytanic acid completely from fat stores in some patients. There is long-​term efficacy and safety data for a phytanic acid re- strictive diet and to a lesser extent for plasmapheresis/​apheresis, which are used in a few centres. Treatment is generally most effective in resolving symptoms of ichthyosis, less so sensory neuropathy and least so ataxia, and it has uncertain effects on the progression of ret- initis pigmentosa, anosmia or deafness, although it seems to sta- bilize these signs. Prognosis The prognosis in adult Refsum’s disease depends on the degree to which phytanic acid concentrations are decreased. In untreated disease, presentation is with progressive weakness and neuropathy usually following an acute infective illness which leads to anorexia and acute hepatic phytanic acid release exacerbating the condition. Concentrations of phytanic acid in the plasma usually exceed 1000 µmol/​litre. Left untreated, cardiomyopathy and sudden death can occur. If phytanic acid levels are reduced by plasmapheresis and by adequate parenteral nutrition, and then a low phytanic acid diet is followed, prognosis is good. Any myopathy usually resolves within 2 to 3 weeks, though acute visual and auditory deterioration may be irrecoverable. In long-​term cases, patients are blind, deaf, and anosmic, have extensive peripheral myopathy, and are often wheelchair bound. In acute adult Refsum’s disease, once phytanic acid levels fall to less than 500 µmol/​litre, ichthyosis resolves followed by improvement in Table 12.9.6  Differential diagnosis of retinitis with neurological signs (apart from adult Refsum’s disease) Presentation OMIM Neurological and other signs Abetalipoproteinaemia 200100 Ataxia, movement disorder, retinitis pigmentosa, acanthocytes Vitamin E deficiency 600415 Ornithine aminotransferase deficiency 258870 Gyeate atrophy Myopathy Usher’s syndrome Ia 276900 Congenital deafness, ataxia Usher’s syndromes II 276901 Moderate progressive deafness Bardet–​Biedl–​Moon syndrome 209900 Polydactyly Truncal obesity Hypogonadism Short stature Mental retardation Kearns–​Sayre syndrome 530000 Ophthalmoplegia Cardiomyopathy Ceroid lipofuscinosis (Batten’s disease) 204300 Seizures Dyskinesia Dementia X-​linked macular degeneration 304020 Ataxia Myoclonic encephalopathy NARP 551500 Neurogenic proximal muscle weakness Ataxia Dementia Seizures PHARC 612674 Polyneuropathy Hearing loss Ataxia Cataract OMIM, Online Mendelian Inheritance in Man.

section 12  Metabolic disorders 2170 myopathy and neuropathy. If phytanic acid levels can be restored to normal values, then it is likely that ophthalmological changes will be minimal or slow, but sudden step-​like deteriorations can occur. The principal long-​term disability is increasing loss of visual field with subsequent diplopia and progressive cataract formation. Auditory function generally remains good unless phytanic acid levels are sub- stantially raised, in which case audiological deterioration occurs with the need for cochlear implants. Although acute myopathy re- solves, patients may have muscle spasms or contractures which may be either related to the adult Refsum’s disease or secondary to the osteodystrophy. Splints and the surgical correction of osteopathy may be required. Other issues Adult Refsum’s disease is a potentially treatable cause of retinitis pig- mentosa or neuropathy. The average delay to diagnosis is 12 years and a simple biochemical screening test exists for these disorders. Given that earlier implementation of dietary restriction of phytanic acid would likely arrest the disease process before retinitis is estab- lished, screening for phytanic and pristanic acidaemias should be considered as an important investigation in retinitis pigmentosa or peripheral neuropathy. Future developments The causes of neuropathic adult peroxisomal disorders are in- completely delineated. Cases of pristanic acidaemia with an adult Refsum’s disease phenotype exist for which no cause has yet been found. Similarly, all cases of adult Refsum’s disease currently de- scribed are null-​function variants, so the phenotype associated with low partial function has not been identified. It may be entirely normal, but it is possible that some cases of retinal dystrophy or peripheral neuropathy may actually be caused by mild phytanoyl-​ CoA hydroxylase mutations. No cases of deficiency of phytanic acid lyase have been described, although the condition should show signs of an adult Refsum’s disease phenotype with some overlap into the α-​oxidation pathway-​associated features of α-​methylacyl-​ CoA racemase and possibly non-​neurological effects of 2-​hydroxy-​ fatty acid deficiency. A number of lyase enzymes with peroxisomal targeting signal motifs remain to be placed on the α-​oxidation pathway and these may be associated with neuropathy or retinitis pigmentosa syndromes. Reduction of dietary phytanic acid is already successful in ameli- orating most nonophthalmic symptoms in long-​term studies with diet and to a lesser extent apheresis. Orlistat has been shown to re- duce phytanic acid in some patients. Newer, more efficacious, ther- apies are still required to fully reverse the progression of this disease. The signalling pathways which regulate α-​oxidation in humans are unclear. In rodents, the retinoid X receptor β and peroxisomal proliferator-​activating receptor α pathways control α-​oxidation and thus fibrate (PPAR-​α agonist) therapy increases activity, but this does not seem to be true in humans. As ω-​oxidation is capable of large increases in activity and is principally mediated through CYP enzymes, it forms a good candidate for therapeutic interventions to induce enzyme activity and reduce phytanic acid levels in Refsum’s disease. However, at the present time, no drug therapy trials of com- pounds capable of modulating either the α-​ or the ω-​oxidation path- ways have been conducted in humans. PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract) syndrome A phenocopy sharing many features of adult Refsum’s disease has recently been described (Table 12.9.3). This disorder forms part of a newly designated group of phospholipid, sphingolipid, and fatty acid synthesis disorders. The synthesis of phospholipids involves multiple steps from dihydroxyacetone phosphate (DHAP) and gly- cerol (Fig. 12.9.6). Clinical features PHARC is a progressive syndrome and has the key clinical features of polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cata- ract (MIM 612674). It is marked by early-​onset cataract and hearing loss, retinitis pigmentosa, and involvement of both the central and peripheral nervous systems, including demyelinating sensorimotor polyneuropathy and cerebellar ataxia. Approximately 30 cases have been described. Most seem to have an adult Refsum’s disease-​type phenotype but lack the key feature of anosmia which is universally present in adult Refsum’s disease. PHARC seems to be a progressive disorder, unlike adult Refsum’s disease, and seems not to show tem- poral or environmental variability in symptoms. Clinically, the onset of retinitis pigmentosa seems to be later in PHARC than in adult Refsum’s disease (approximately age 35–​40 vs 15–​20 years), cataracts occur earlier (age 20–​30 vs 30–​50 years), and early-​onset deafness is common (age <15 years vs 50 years in adult Refsum’s disease). Patients have a demyelinating neuropathy and develop chronic ataxia at varying ages. Cerebellar atrophy is more common on MRI (25%) than in adult Refsum’s disease, but milder phenotypes have retinitis pigmentosa associated with only mild sensorineural hearing loss and cataract and no other signs or symptoms. Diagnosis The biochemical profile differs from adult Refsum’s disease or other α-​oxidation disorders in showing no abnormality in phytanic or pristanic acid concentrations or in any other peroxisomal metabolites. PHARC was originally mapped to chromosome 20 and is caused by mutations in α/​β hydrolase-​12 (ABHD12), an enzyme involved in hydrolysing the endocannabinoid 2-​arachidonyl-​glycerol and possibly other structurally related substrates including very long chain fatty acids. ABDH12 can be inhibited by triterpenoids (e.g. betulonic acid). Phytol and phytanic acid are diterpenes. The me- tabolism of 2-​arachidonyl-​glycerol in the brain is complex as 85% is metabolized by mono-​acylglycerol lipase (MAGL) or ABDH6, while ABDH12 has a far greater role in microglial 2-​arachidonyl-​glycerol metabolism suggesting that microglial dysfunction is the patho- genic mechanism behind PHARC. Both ABDH12 and ABDH16 are involved in cellular lyso-​phosphatidylserine metabolism and modu- late cytokine production. How these biochemical findings relate to causing the full clinical phenotype is unclear. Treatment No specific treatment has been defined for PHARC. Symptomatic treatment is recommended for individual deficits. The effective- ness of a dietary restriction of phytanic acid in this condition is

12.9  Disorders of peroxisomal metabolism in adults 2171 Dihydroxyacetone 3-phosphate Glycerol-3-phosphate Glycerol Monoacylglycerol ADP ATP CTP PPi 14 18 15 16 17 O P O O O CO CH2 CH2 CH2 CH2 CO CH O CH2 CH2 CO Cardiolipin CO O O O C O H HO P CH O 19 4 3 5 7 8 6 1 1 2 4 Lysophosphatidic Acid Phosphatidic Acid ADP ATP CDP Diacylglycerol Diacylglycerol Diacylglycerol Triacylglycerol Phosphatidyl Serine Phosphatidyl Inositol Phosphatidyl Choline Phospho- choline choline ADP ATP CMP CDP- choline Phosphatidyl Ethanolamine Myoinositol CMP Phosphoglycero- phosphate Phosphatidyl- Glycerol Phospho- glycerol Lyso- phospholipids COOH Prostaglandins Leukotriens Arachidonic Acid 2-Arachidonoyl Glycerol Glycerol H2O H2O H2O Fatty acid Fatty acid 10 9 9 13 12 11 + Acyl CoA CoA-SH Acyl CoA CoA-SH Fig. 12.9.6  Major reactions involved in phospholipids biosynthesis: CDP, cytidyldiphosphate; CMP, cytidylmonophosphate; CoA, coenzyme A; CTP, cytidyltriphosphate. Phospholipids are synthesized de novo from glycerol or from glyceraldehyde 3-​phosphate and dihydroxyacetone phosphate (DHAP). Glycerol-​ 3-​phosphate is esterified twice with acyl-​CoA by acyl-​CoA transferase (1) to form lysophosphatidic (LPA) acid and then phosphatidic acid (PA). In adipose, muscular tissues, and skin, LPA is converted into PA by α/​β-​hydrolase-​5 (ABHD5) (2). In mitochondria, PA and LPA are obtained from monoacylglycerol (MAG) and diacylglycerol (DAG), respectively, a reaction catalysed by acylglycerol kinase (4). PA can be converted either into DAG by phosphatidic acid phosphatase (3), or into CDP-​DAG by phosphatidic acid cytidyltransferase (14). DAG is an essential inter-​mediate for the synthesis of triglycerides and of various phosphoglycerides: phosphatidyl serine (PS) by PS-​synthase (5), which is transformed to phosphatidyl ethanolamine (PE) by PS-​carboxylase (6), phosphatidyl inositol (PI) by PI-​synthase (7) and phosphatidyl choline (PC) by PC-​synthase (8). On the other side, CDP diacylgly-​cerol is converted, by the sequential action of phosphatidic acid glycerol phosphate synthase (15) and phosphatase (16), into phosphatidylglycerophosphate—​a precursor of cardiolipin, an important phospholipid of the mitochondrial membrane. This is catalysed by cardiolipin synthase (17) and enriched by linoleic acid by the remodelling enzyme, monolysocardiolipin acyl transferase (also called tafazzin). The alternative pathway for DAG and fatty acids synthesis is their release from membrane phospholipids by specific phospholipases (PLA2) (9) and lysophospholipases (Neuropathy target esterase) (10). Phospholipase C (11) leads to the membrane release of DAG and its further conversion by diacylglycerol lipase (12) into 2-​arachidonoyl glycerol, which can be hydrolysed into arachidonic acid by α/​β-​hydrolase-​12 (ABDH12) (13). Arachidonic acid is the starting molecule of many complex fatty acids like prostaglandins and leukotrienes. Reproduced with permission from Lamari, F., Mochel, F., Sedel, F. et al. J Inherit Metab Dis (2013) 36: 411. https://​doi.org/​ 10.1007/​s10545-​012-​9509-​7. Copyright © 2012 SSIEM and Springer.

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12.9  Disorders of peroxisomal metabolism in adults 2173 Wierzbicki AS, et al. (2002). Refsum’s disease: a peroxisomal disorder affecting phytanic acid alpha-​oxidation. J Neurochem, 80, 727–​35. Wierzbicki AS, et  al. (2003). Metabolism of phytanic acid and
3-​methyl-​adipic acid excretion in patients with adult Refsum’s dis- ease. J Lipid Res, 44, 1481–​88. Websites Adult Refsum’s Disease website (information for patients, carers and clinicians): http://​refsumdisease.org. United Leukodystrophy Foundation website: http://​www.ulf.org. X-​linked Adrenoleukodystrophy database: http://​www.X-​ald.nl.