doi:10.1093/brain/awt361
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BRAIN A JOURNAL OF NEUROLOGY
X-linked adrenoleukodystrophy in women: a cross-sectional cohort study
1 2 3 4
Department of Neurology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Department of Paediatric Neurology/Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Assistance Publique des Hoˆpitaux de Paris, Department of Paediatric Neurology, Hospital Kremlin-Biceˆtre, Paris, France Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands 5 Department of Clinical Epidemiology, Biostatistics and Bio-informatics, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands 6 INSERM U986, Le Kremlin-Biceˆtre, Paris, France 7 Department of Neurology, Medical Centre Alkmaar, Alkmaar, The Netherlands *Present address: INSERM U698, Hoˆpital Bichat, Paris, France Correspondence to: M. Engelen, MD, PhD Department of Paediatric Neurology/Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1100DD Amsterdam, The Netherlands E-mail: [email protected]
X-linked adrenoleukodystrophy is the most common peroxisomal disorder. The disease is caused by mutations in the ABCD1 gene that encodes the peroxisomal transporter of very long-chain fatty acids. A defect in the ABCD1 protein results in elevated levels of very long-chain fatty acids in plasma and tissues. The clinical spectrum in males with X-linked adrenoleukodystrophy has been well described and ranges from isolated adrenocortical insufficiency and slowly progressive myelopathy to devastating cerebral demyelination. As in many X-linked diseases, it was assumed that female carriers remain asymptomatic and only a few studies addressed the phenotype of X-linked adrenoleukodystrophy carriers. These studies, however, provided no information on the prevalence of neurological symptoms in the entire population of X-linked adrenoleukodystrophy carriers, since data were acquired in small groups and may be biased towards women with symptoms. Our primary goal was to investigate the symptoms and their frequency in X-linked adrenoleukodystrophy carriers. The secondary goal was to determine if the X-inactivation pattern of the ABCD1 gene was associated with symptomatic status. We included 46 X-linked adrenoleukodystrophy carriers in a prospective cross-sectional cohort study. Our data show that X-linked adrenoleukodystrophy carriers develop signs and symptoms of myelopathy (29/46, 63%) and/or peripheral neuropathy (26/46, 57%). Especially striking was the occurrence of faecal incontinence (13/46, 28%). The frequency of symptomatic women increased sharply with age (from 18% in women 540 years to 88% in women 460 years of age). Virtually all (44/45, 98%) X-linked adrenoleukodystrophy carriers had increased very long-chain fatty acids in plasma and/or fibroblasts, and/or decreased very long-chain fatty acids beta-oxidation in fibroblasts. We did not find an association between the X-inactivation pattern and symptomatic status. We conclude that X-linked
Received June 26, 2013. Revised October 17, 2013. Accepted November 10, 2013. Advance Access publication January 29, 2014 ß The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
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Marc Engelen,1,2 Mathieu Barbier,3,* Inge M. E. Dijkstra,4 Remmelt Schu¨r,2 Rob M. A. de Bie,1 Camiel Verhamme,1 Marcel G. W. Dijkgraaf,5 Patrick A. Aubourg,3,6 Ronald J. A. Wanders,4 Bjorn M. van Geel,7 Marianne de Visser,1 Bwee T. Poll–The1,2 and Stephan Kemp2,4
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adrenoleukodystrophy carriers develop an adrenomyeloneuropathy-like phenotype and there is a strong association between symptomatic status and age. X-linked adrenoleukodystrophy should be considered in the differential diagnosis in women with chronic myelopathy and/or peripheral neuropathy (especially with early faecal incontinence). ABCD1 mutation analysis deserves a place in diagnostic protocols for chronic non-compressive myelopathy.
Keywords: adrenoleukodystrophy; X-inactivation; myelopathy; faecal incontinence; carriers
Materials and methods
X-linked adrenoleukodystrophy is a peroxisomal disorder caused by mutations in the ABCD1 gene (http://www.x-ald.nl) (Mosser et al., 1993), which codes for the transporter of very long-chain fatty acids (5C22:0) (van Roermund et al., 2008). ABCD1 deficiency impairs peroxisomal very long-chain fatty acid degradation resulting in increased cytosolic very long-chain fatty acid-CoA levels (Kemp et al., 2011), which are further elongated by the very long-chain fatty acid-specific elongase, ELOVL1 (Ofman et al., 2010), thus causing very long-chain fatty acid accumulation (Moser et al., 1981; Kemp and Wanders, 2010). In males, disease course and symptomatology have been studied extensively (Engelen et al., 2012). The clinical spectrum ranges from isolated adrenocortical insufficiency, slowly progressive myelopathy and peripheral neuropathy in adulthood (adrenomyeloneuropathy) to a rapidly progressive and fatal cerebral demyelinating disease in boys or adult men (cerebral-adrenoleukodystrophy). The most frequent phenotype in males is adrenomyeloneuropathy (van Geel et al., 1994; Engelen et al., 2012). There have been several studies and observations concerning the phenotype of X-linked adrenoleukodystrophy carriers. O’Neill et al. (1984) described 21 obligate carriers and several were shown to have signs and symptoms of myelopathy. It was estimated that 50% develop symptoms at some point in their life (Moser et al., 1991). In a small study, eight females were found to have myelopathy and in a retrospective study the frequency of symptoms increased with age (van Geel, 2000; Schmidt et al., 2001). These studies provide no information on the prevalence of neurological symptoms in the entire population of X-linked adrenoleukodystrophy carriers, as data were acquired in small groups and may be biased towards females with symptoms. Adrenal insufficiency is rare in X-linked adrenoleukodystrophy carriers (el-Deiry et al., 1997), and there are only a few reports of X-linked adrenoleukodystrophy carriers that developed cerebraladrenoleukodystrophy (Pilz and Schiener, 1973; Jung et al., 2007). Some studies suggested that disease severity could be related to the pattern of X-inactivation, whereas others refuted this observation (Migeon et al., 1981; Watkiss et al., 1993; Maier et al., 2002; Salsano et al., 2012). The primary goal of our study was to estimate the proportion of X-linked adrenoleukodystrophy carriers that develop symptoms and to characterize these symptoms both clinically and with ancillary investigations. A secondary goal was to determine if the X-inactivation pattern of the ABCD1 gene was associated with symptomatic status.
Research population We designed a prospective cross-sectional cohort study. Female carriers over the age of 18 years were eligible to participate. They were recruited from the outpatient clinics of the Academic Medical Centre and the Medical Centre Alkmaar from 2008–10. To prevent bias for symptomatic women, we tried to examine all female relatives with X-linked adrenoleukodystrophy of the women participating. Through the Dutch X-linked adrenoleukodystrophy patient organization, letters to invite X-linked adrenoleukodystrophy carriers to participate were sent to all members. The study protocol was approved by the local Institutional Review Board. After informed consent was obtained, women visited the outpatient clinic for assessment, which included questionnaires, neurological examination, neurophysiological tests, blood samples and skin biopsy.
Clinical assessment A careful history with emphasis on neurological complaints and neurological examination was performed. In particular, symptoms of incontinence, gait disorder, maximum walking distance, and sensory disturbance were recorded. Urinary incontinence was defined as urge incontinence. Stress incontinence was not considered a symptom of myelopathy. A gait disorder was considered present if the walking distance was significantly reduced or running was not possible. Sensory complaints were recorded if there were paraesthesias or numbness in the lower extremities. Sensory disturbances on examination were considered present if there was reduced sensation to touch, pinprick, vibration or position sense in the lower extremities. Muscle strength was rated using the Medical Research Council Scale and spasticity was rated using the Ashworth scale (Ashworth, 1964). An Expanded Disability Status Scale value was scored based on the history and physical examination performed during the visit and recorded in the medical chart (Kurtzke, 1983). Carriers with peripheral neuropathy or myelopathy were considered symptomatic. Myelopathy was considered present if: (i) there were symptoms of myelopathy (for instance sphincter disturbance); and (ii) signs of myelopathy on neurological examination (pyramidal tract or dorsal column signs) were present. If only symptoms were present (for instance faecal incontinence) but no signs on neurological examination, myelopathy was not scored as present. Peripheral neuropathy was considered present if nerve conduction studies and/or EMG were abnormal (as described below). All participants completed the Dutch version of the SF-36 (Quality of Life Assessment) (Aaronson et al., 1998), and the AMC Linear Disability Scale (Weisscher et al., 2007). SF-36 values can be compared to reference values for the Dutch population, matched for gender and age. The AMC Linear Disability Scale values were
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Introduction
X-linked adrenoleukodystrophy in women compared to those of patients with Parkinson’s disease from the CARPA study (Post et al., 2011).
Neurophysiological testing
Blood samples Venous blood samples were taken and plasma and lymphocytes stored as described previously (Engelen et al., 2010).
Cell culture and biochemical analysis From skin biopsy material primary fibroblast cell lines were generated and cultured as described (Valianpour et al., 2003). Very long-chain fatty acids (C26:0 and the C26:0/C22:0 ratio) were determined in plasma and fibroblasts as described (Valianpour et al., 2003). Peroxisomal beta-oxidation activity was measured essentially as described by incubating cells with 30 mM deuterium-labelled C22:0 (D3-C22:0) (Kemp et al., 2004). ABCD1 protein levels were determined by immunofluorescence and quantitative immunoblot (Kemp et al., 1996; Zhang et al., 2011).
Genetic analysis and ABCD1 allele-specific expression in fibroblasts Mutation analysis of the ABCD1 gene was performed as described (Boehm et al., 1999). ABCD1 allele-specific expression (ABCD1 allele-specific expression) of 38 carriers was measured by pyrosequencing. RNA was extracted from primary fibroblasts and complementary DNA synthesized as described (Bieche et al., 2001). The ratio of normal versus mutant allele expressed was measured by pyrosequencing. Complementary DNA was PCR-amplified using biotin-labelled primers followed by quantitative pyrosequencing using a PyroMark Q96 ID instrument (Qiagen). Biotin-labelled single-stranded amplicons were isolated following the protocol using the Qiagen PyroMark Q96 Workstation pyrosequenced with a sequencing primer (PCR- and sequencing-primers are provided in Supplementary Table 1). Each sample was pyrosequenced in the upper direction (with lower biotinlabelled amplicons) and in lower direction (with upper biotin-labelled
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amplicons). The ratio of normal versus mutant allele was measured using the PyroMark ID software (Qiagen). For each carrier, the ABCD1 allele-specific expression value represents the mean of upper and lower reads (determined in two independent experiments). For included samples, variations between upper and lower reads were 410%. For validation, we repeated the experiment for a subset of samples (n = 6) using different PCR primers or independent complementary DNA samples. The ABCD1 allele-specific expression values were similar (variations 44%). ABCD1 allele-specific expression pattern was defined as ‘severely’ skewed with an expression of the normal allele 410% and ‘moderately’ skewed with an expression of the normal allele between 11–25%, similar to a previous report (Miozzo et al., 2007). In the manuscript ABCD1 allele-specific expression will be referred to as X-inactivation. For five patients the ABCD1 allele-specific expression analysis was not possible because of the complexity of the mutation. Instead, ABCD1 allele-specific expression values were calculated using ABCD1 protein immunofluorescence and determination of the ratio of ABCD1 protein positive and negative cells.
Data entry and statistical analysis Data were analysed with IBM SPSS statistics version 19 (IBM Inc.), Microsoft Excel from Office 2010 and Prism version 5 (GraphPad Software). Depending on the distributional properties, outcome measures were expressed as means standard deviation (SD) or as medians with ranges. Statistical significance was assessed by independent sample Student’s t-test for normally distributed continuous data and the Wilcoxon test for non-normally distributed continuous data. All reported P-values are two-sided and were not adjusted for multiple testing. The SF-36 scores were converted to z-scores based on the Dutch norm population, using the appropriate age groups. AMC Linear Disability Scale scores were analysed as described previously (Weisscher et al., 2007). The relationship between symptomatic status, age and the pattern of X-inactivation in skin fibroblasts were analysed by logistic regression.
Results Demographic and clinical characteristics Forty-six females from 26 kindreds were enrolled in the study (one kindred with five females, two with four females, two with three, five with two, and 17 with one). Twelve were recruited through the outpatient clinic, 34 were relatives of these females or responded to the letter sent to members of the Dutch X-linked adrenoleukodystrophy patient organization. Complete clinical and electrophysiological data were available from 46 females, blood samples were available from 45 females (one refused venepuncture) and skin biopsies were obtained from 43 women (two refused skin biopsy, one fibroblast culture failed because of a yeast infection). The age of the included females ranged from 22 to 76 years (average 48 13 years) (Fig. 1). Mutation analysis was done in 45 females. For one obligate carrier no leucocytes were available, the mutation in that subject was considered to be the mutation found in her father and paternal uncle (Table 1). Symptoms, signs and Expanded Disability Status Scale scores are presented in Table 1 and summarized by age group in Table 2.
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Nerve conduction studies and EMG were performed according to a fixed protocol, allowing comparison with reference values and possibly follow-up in the future (Verhamme et al., 2009). Compound muscle action potential amplitudes, baseline to negative peak, were recorded from the abductor pollicis brevis, abductor hallucis brevis, and extensor digitorum brevis on one side. A summated compound muscle action potential amplitude was calculated. Motor nerve conduction velocities were calculated after distal and proximal stimulation of the right median, tibial, and peroneal nerves. Sensory nerve conduction velocity, calculated based on negative peak markers, and sensory nerve action potentials amplitudes, baseline to negative peak, were investigated for the left median nerve and right sural nerve. A summated sensory nerve action potential amplitude was calculated. Electromyography was done of the left anterior tibial, and interosseus I/II muscles. An axonal peripheral neuropathy was diagnosed if in two separate nerves at least one parameter was outside the 95% confidence interval (CI) and the criteria for demyelinating peripheral neuropathy were not fulfilled (Van Asseldonk et al., 2005). Somatosensory evoked potentials of left median and the posterior tibial nerves on both sides and brainstem auditory evoked potentials on both sides were registered, conforming to local protocols (Aramideh et al., 1992; Aalfs et al., 1993).
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M. Engelen et al. polyneuropathy, as has been reported to occur in males with X-linked adrenoleukodystrophy (van Geel et al., 1996).
Somatosensory evoked potentials
Complaints of incontinence (both urinary and faecal), abnormal gait, and sensory symptoms were common. Myelopathy was found in 29/46 (63%) and peripheral neuropathy in 26/46 (57%). Two of 46 (4%) had peripheral neuropathy without myelopathy, and 11/46 (24%) had myelopathy without peripheral neuropathy. Thirty-one of 46 (67%) were considered symptomatic because of either myelopathy and/or peripheral neuropathy. The percentage of women with signs and symptoms suggestive of myelopathy increased with age (Table 2). The percentage of symptomatic females increased from 2/11 (18% in the youngest age group) to 7/8 (88% in the oldest age group). Logistic regression showed a significant relationship between age and symptomatic status, with the probability of being symptomatic increasing clearly with age [P = 0.002; odds ratio (OR) 1.2 (1.07–1.34); Fig. 2]. Quality of life, as measured by the SF-36, was not statistically significant different between asymptomatic and symptomatic carriers. However, on measures that are related to physical disability (Fig. 3A), there was a trend towards a statistically significant difference. There were no differences in emotional or mental health between asymptomatic and symptomatic carriers. The levels of disability were measured by the AMC Linear Disability Scale scores (Fig. 3B) and these were compared to data from the CARPA study, a prospective cohort study in Parkinson’s disease (Post et al., 2011). The AMC Linear Disability Scale scores were lower in the group of symptomatic X-linked adrenoleukodystrophy carriers compared with the asymptomatic carriers. Symptomatic carriers had AMC Linear Disability Scale scores comparable to patients with Parkinson’s disease at baseline and 1 year after diagnosis.
Electrophysiological testing Nerve conduction studies and electromyography Of the X-linked adrenoleukodystrophy carriers 26/46 (57%) had a peripheral neuropathy. The abnormalities mostly consisted of a reduction in compound muscle and sensory nerve action potential amplitudes, with a marginal slowing in nerve conduction velocity (Table 3). This is consistent with a sensorimotor axonal peripheral
Brainstem auditory evoked potentials Brainstem auditory evoked potential measurement failed for technical reasons in one participant (Table 4). For the entire group, 26/45 (58%) had an abnormal brainstem auditory evoked potential, mostly consisting of an increased I–V and I–III interval. In the asymptomatic group, 3/13 (23%) had a normal brainstem auditory evoked potential. In the symptomatic group this proportion was 23/32 (72%) had an abnormal brainstem auditory evoked potential. There were no significant left–right differences.
Biochemical and ABCD1 allele-specific expression analysis Plasma Plasma C26:0 were increased (2.26 0.69 mmol/l; reference values 1.05 0.078 mmol/l; Table 5). Thirty-one of 45 (69%) had abnormal plasma very long-chain fatty acids levels. There were no significant differences in plasma C26:0 levels between asymptomatic and symptomatic carriers (P = 0.16). In contrast to what we reported before, C26:0 levels did not increase with age (Stradomska and Tylki-Szymanska, 2001).
Fibroblasts Fibroblast C26:0 levels were increased in 37/43 (86%), and the C26:0/C22:0 ratio in 36/43 (84%) (Table 5). The D3-C16:0/D3C22:0 flux-ratio was decreased in 26/43 (60%), indicating reduced peroxisomal very long-chain fatty acids beta-oxidation capacity. Two of 43 (5%) carriers had normal C26:0 levels in plasma and fibroblasts, and 1/45 (2%) had normal C26:0 levels and normal peroxisomal beta-oxidation. There was a clear correlation between the ABCD1 protein levels in fibroblasts and the residual peroxisomal beta-oxidation capacity, C26:0 synthesis and very long-chain fatty acids levels (Fig. 4).
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Figure 1 Age distribution of the cohort.
The somatosensory evoked potential from the median (arm) nerve was abnormal in 3/42 (7%) of X-linked adrenoleukodystrophy carriers (four registrations failed because of technical reasons), of the tibial (leg) nerve 14/44 (30%) were abnormal (two registrations failed) (Table 4). The median nerve somatosensory evoked potential was normal in all 15 asymptomatic carriers. The posterior tibial nerve somatosensory evoked potential was abnormal in one (7%) asymptomatic carrier. Three of 27 (11%) symptomatic carriers had an abnormal median nerve somatosensory evoked potential and 13/29 (45%) had an abnormal tibial nerve somatosensory evoked potential. The average latency of the cortical peak increased with age for median nerve and tibial nerve somatosensory evoked potential. The percentage of abnormal somatosensory evoked potentials increased with age (Table 4).
Age (years)
44 56 45 59 75 42 44 44 51 59 44 38 57 31 37 60 35 42 61 71 42 31 48 57 60 51 22 40 29 45 57 70 40 59 39 28 35
A A AA AA AA B B B B B C D D E E E F G H I J K K K K L M M N N N N O P Q R S
No Yes No Yes Yes Yes No No No No No Yes Yes No No No No No Yes No No No Yes No Yes Yes No No No Yes Yes No Yes Yes No No No
Urinary incontinence No Yes No No No Yes No No No No No Yes No No No No No Yes Yes No No No No No No No No No No No Yes No Yes Yes Yes No No
Faecal incontinence Yes No No Yes Yes Yes No No No No No Yes Yes No No Yes No No Yes No No No No Yes No Yes No No No No Yes Yes Yes Yes Yes No No
Gait disorder No No No No No No No No Yes Yes No No Yes No No No No No Yes No No No No Yes No No No No No Yes Yes No Yes Yes No No No
Sensory complaints No No No No Yes Yes No No Yes Yes No Yes Yes No No Yes No No Yes Yes Yes No Yes Yes Yes Yes No No No No Yes Yes Yes Yes Yes No No
Sensory disturbance No No No No Yes Yes No No No No No Yes No No No Yes No No No No No No No No No Yes No No No No No No No Yes No No No
Spasticity No No No Yes Yes Yes No No No Yes No Yes No No No Yes No No No No No No No Yes No Yes No No No No No Yes No Yes No No No
Weakness Yes Yes No Yes Yes Yes No No No No No Yes Yes No No Yes No No Yes Yes Yes No Yes Yes Yes Yes No No No No No Yes Yes Yes No No No
Pathological reflexes
Mutation p.Pro480Thr p.Pro480Thr p.Arg660Trp p.Arg660Trp p.Arg660Trp p.Leu220Pro p.Leu220Pro p.Leu220Pro p.Leu220Pro p.Leu220Pro p.Gln133* p.Leu654Pro p.Leu654Pro p.Arg74Trp p.Arg74Trp p.Arg74Trp p.Met1Val p.Ala245Asp exon8-10del p.Glu609Lys p.Glu90* p.Pro543Leu p.Pro543Leu p.Pro543Leu p.Pro543Leu p.Ile657del p.Ser149Asn p.Ser149Asn p.Arg389His p.Arg389His p.Arg389His p.Arg389His p.Glu609Lys p.Leu215* p.Val208Trpfs p.Pro480Thr p.His283Tyr
EDSS 1.0 1.5 0 3.5 6.0 4.0 0 0 1.0 2.0 0 6.0 5.5 0 0 5.5 0 1.0 3.5 2.0 1.5 0 2.5 3.5 3.5 6.5 0 0 0 2.0 3.5 3.5 3.5 6.0 3.0 0 0
(continued)
Absent Absent Absent Absent Absent Reduced Reduced Reduced Reduced Reduced Absent Absent Absent Absent Absent Absent Absent Present Absent Absent Absent Absent Absent Absent Absent Absent Reduced Reduced Reduced Reduced Reduced Reduced Absent Absent Absent Absent Reduced
ABCD1 protein
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Family
Table 1 Summary of symptoms and signs of all the female participating in the study
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Age = age at examination; urinary incontinence = urge incontinence; faecal incontinence = soiling and urge incontinence; gait = gait disorder, for instance unable to run; sensory complaints = numbness or tingling; sensory disturbance = sensory symptoms on neurological examinations; spasticity = spasticity of the lower extremities; weakness = paresis of the lower extremities, Pathological reflexes: pathological reflexes in the lower extremities, EDSS = expanded disability status scale; mutation = mutation in ABCD1; ABCD1 protein = effect of mutation in ABCD1 on ABCD1 protein as determined by immunoblot. Asterisk indicates the introductions of a stop codon. a Mutation inferred from mutation in father and uncle, as this patient did not consent to provide a blood sample and skin biopsy.
Reduced Absent Absent Absent Absent Absent Absent Absenta Absent p.His283Tyr p.Gln177* p.Arg464* p.Asp442Glyfs p.Ala616Thr p.Ala616Thr p.Arg113Alafs p.Glu609Glya p.Ser633Argfs 2.0 4.0 2.0 2.5 3.5 2.0 2.5 0 2.0 Yes Yes No Yes Yes No Yes No Yes No Yes No Yes No No No No No No No No No No No No No No 76 51 47 56 45 65 47 24 50 S T U V W W X Y Z
Yes Yes Yes Yes Yes No Yes No No
No Yes Yes No Yes Yes No No No
Yes Yes No Yes Yes No Yes No No
No No No No Yes No No No No
Yes Yes Yes Yes Yes No No No Yes
Weakness Spasticity Sensory disturbance Sensory complaints Gait disorder Faecal incontinence Urinary incontinence Age (years) Family
M. Engelen et al.
Correlation studies of X-inactivation with asymptomatic or symptomatic status The distribution of ABCD1 allele-specific expression (which will be referred to as the pattern of X-inactivation) is shown in Fig. 5A. There was no evidence for skewing to either the mutated or normal ABCD1 allele. The pattern appeared random and the median value was exactly 0.5. The pattern of X-inactivation was similar between age groups (Fig. 5B). This contrasts with previous reports that documented a highly skewed pattern of X-inactivation in X-linked adrenoleukodystrophy carriers (Migeon et al., 1981). We subsequently correlated the pattern of X-inactivation with biochemical parameters in fibroblasts, i.e. ABCD1 protein levels, residual beta-oxidation activity and very long-chain fatty acids levels (Fig. 6). A clear correlation was found between the pattern of X-inactivation and the biochemical parameters in the fibroblasts. Indeed, if there was preferential expression of the normal allele, the level of ABCD1 protein was higher, residual rates of beta-oxidation were higher, very long-chain fatty acids synthesis was lower, and very long-chain fatty acids levels were lower, and vice versa. To determine if the pattern of X-inactivation correlates with symptomatic status it is important to consider age as there is a strong correlation between age and symptomatic status (Fig. 2). Logistic regression showed no correlation between symptomatic status and the ABCD1 allele-specific expression pattern, also after adjustment for age [P = 0.74; OR 1.00 (0.97–1.02)]. In Fig. 5C and D the distribution of X-inactivation in asymptomatic and symptomatic carriers is plotted.
Discussion X-linked adrenoleukodystrophy carriers develop neurological symptoms. In fact, one of the earliest descriptions of an adrenomyeloneuropathy-like phenotype in X-linked adrenoleukodystrophy was a female patient (Penman, 1960). This is not common knowledge among physicians (Jangouk et al., 2012). Several X-linked adrenoleukodystrophy carriers were reported to undergo cervical laminectomy for suspected cervical spondylogenic myelopathy (van Geel et al., 1997). In this largest prospective cross-sectional cohort of female carriers to date, we found that neurological abnormalities are common and that the frequency increases steeply with age. The main symptoms were consistent with myelopathy, as in males with adrenomyeloneuropathy. It is striking how often faecal incontinence is reported as an early symptom. One should bear in mind, however, that it is often not voluntarily reported because it is felt to be embarrassing. To our knowledge, this is not reported often in males with adrenomyeloneuropathy or in other degenerative myelopathies, for instance, hereditary spastic paraplegia, albeit that this feature may not have been specifically asked for (Salinas et al., 2008). In our cohort sphincter disturbance is a frequent (13/46) and early symptom, and even occurs in a few (2/17) symptomatic carriers without other signs of myelopathy. Interestingly, most females with X-linked adrenoleukodystrophy were actually relieved to find out that their symptoms were related to X-linked adrenoleukodystrophy, as often they had been told that an X-linked disease does not cause symptoms in ‘carriers’.
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Pathological reflexes
EDSS
Mutation
ABCD1 protein
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Table 1 Continued
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Table 2 Symptoms, signs and Expanded Disability Status Scale scores by age group
Incontinence (urine) Incontinence (faecal) Gait disorder Sensory complaints Sensory disturbance Spasticity Weakness Pathological reflexes Myelopathy Neuropathy Symptomatic EDSS
18–39 years
40–59 years
460 years
All ages
1/11 (9) 2/11 (18) 2/11 (18) 0/11 (0) 2/11 (18) 1/11 (9) 1/11 (9) 1/11 (9) 2/11 (18) 2/11 (18) 2/11 (18) 0.82 (1.94)
15/27 (56) 9/27 (33) 13/27 (48) 9/27 (33) 16/27 (59) 3/27 (11) 8/27 (30) 16/27 (59) 20/27 (74) 14/27 (52) 22/27 (82) 2.41 (1.83)
4/8 (50) 2/8 (25) 5/8 (63) 1/8 (13) 7/8 (88) 2/8 (25) 3/8 (38) 7/8 (88) 7/8 (88) 4/8 (50) 7/8 (88) 3.50 (1.56)
20/46 [44% 13/46 [28% 20/46 [44% 10/46 [22% 25/46 [54% 6/46 [13% 12/46 [26% 24/46 [52% 29/46 [63% 20/46 [44% 31/46 [67% 2.22 (1.99)
(29–58)] (15–42)] (29–58)] (9–34)] (39–69)] (3–23)] (13–39)] (37–67)] (49–78)] (29–58)] (53–81)]
Figure 2 Symptomatic status and age. The bars indicate the percentage of X-linked adrenoleukodystrophy carriers considered symptomatic within each age group (i.e. diagnosed with a myelopathy and/or a peripheral neuropathy). The dots show each individual X-linked adrenoleukodystrophy carrier in the cohort, classified as either symptomatic or asymptomatic.
Signs of peripheral neuropathy were mostly absent or minor clinically, and did not seem to contribute much to the overall disability. It is possible that signs and symptoms were masked by the more prominent signs of myelopathy. Notwithstanding, on electrophysiological testing about 57% of the females in this cohort fulfilled the criteria for an axonal sensorimotor neuropathy. There was no statistically significant difference in SF-36 scores between asymptomatic and symptomatic carriers (Fig. 3A). Although any symptom may affect quality of life, it seems that the majority of symptomatic carriers have symptoms that do not impact the SF-36 enough to be visible at group level. The z-scores are usually below zero for the symptomatic carriers on the measures related to physical symptoms (Fig. 3A), but the standard deviation is large, supporting the previous assumption. However, symptomatic females show a trend towards reduced quality of life in domains related to physical symptoms (Fig. 3A), corresponding to the signs of myelopathy frequently present in our cohort.
Analysis of the AMC Linear Disability Scale revealed that manifesting X-linked adrenoleukodystrophy carriers had similar disability as patients with Parkinson’s disease 1 year after the diagnosis. However, compared to our cohort, the Parkinson’s disease cohort was significantly older (average age 67 versus 48 years), suggesting that the burden of disease in X-linked adrenoleukodystrophy carriers is considerable. Somatosensory evoked potential was reported to be very sensitive in detecting abnormalities in X-linked adrenoleukodystrophy carriers in a presymptomatic stage (Restuccia et al., 1997). In our cohort, however, somatosensory evoked potentials of the median nerve (arm) were normal in all asymptomatic, and abnormal in only 11% of the symptomatic females. This is not surprising, as in none of the symptomatic females the arms were affected clinically. Somatosensory evoked potentials of the posterior tibial nerve (legs) were only abnormal in 7% of the asymptomatic and 45% of the symptomatic females. Therefore, we conclude that somatosensory evoked potential is not superior to clinical examination in detecting myelopathy in our cohort. In agreement with an earlier report, brainstem auditory evoked potential was abnormal in 58% (Restuccia et al., 1997). A larger proportion of the symptomatic carriers had an abnormal brainstem auditory evoked potential compared with the asymptomatic carriers (72% versus 23%). The findings were non-specific, mostly consisting of an increased I–V or III–V interval. This indicates a slowing of conduction in the brainstem. For clinical practice brainstem auditory evoked potential has no value as a diagnostic tool. As has been known for several decades, 15–20% of X-linked adrenoleukodystrophy carriers have normal plasma C26:0 (Moser et al., 1999). We found that a slightly higher proportion (31%) had normal plasma C26:0. Furthermore, even in cultured fibroblasts the C26:0 can be normal (Moser et al., 1983), as was the case in 6/43 (14%). Combining C26:0 levels in plasma and fibroblasts marks 41/43 (95%) of the females in our cohort as biochemically abnormal. This corresponds to an earlier observation that by combining very long-chain fatty acids measurements in plasma and fibroblasts 93% of carriers can be identified (Moser et al., 1983). Peroxisomal very long-chain fatty acids
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Symptoms and signs [reported as absolute number (% with 95% CI)] and Expanded Disability Status Scale (EDSS) (mean SD) scores for the entire cohort and stratified by age group.
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mental compound scores. For each of the SF-36 scores a z-score was calculated based on data from the Dutch normative population. (B) AMC Linear Disability Scale scores for asymptomatic and symptomatic X-linked adrenoleukodystrophy carriers and patients with Parkinson’s disease (PD) from the CARPA cohort at baseline and up to 5 years after diagnosis (PD1–PD5). There is a significant difference between symptomatic and asymptomatic carriers. Symptomatic carriers have levels of disability comparable to patients with Parkinson’s disease 1 year after diagnosis. Statistical significant differences are indicated. *P 5 0.05, **P 5 0.01.
beta-oxidation activity in cultured fibroblasts varied from completely normal to values found in males with X-linked adrenoleukodystrophy. By combining very long-chain fatty acids measurements and beta-oxidation activity, 44/45 X-linked adrenoleukodystrophy carriers (98%) could be identified. However, this still does not reliably identify all X-linked adrenoleukodystrophy carriers. Therefore, in females, only ABCD1 mutation analysis identifies all carriers. We showed that the biochemical abnormalities in fibroblasts correlated with the X-inactivation pattern, i.e. the more skewed to the mutant allele, the more abnormal the fibroblasts were biochemically. Our study showed that in fibroblasts, ABCD1 allelespecific expression (ABCD1 allele-specific expression) predicts the biochemical phenotype.
X-inactivation was considered to be skewed preferentially to the mutant allele (Migeon et al., 1981). That finding cannot be reproduced in our data set (Fig. 5). In fact, the median skewing was 49:51, suggesting a random ABCD1 allele-specific expression pattern. For over two decades there has been controversy over whether X-inactivation predicts the symptomatic status of X-linked adrenoleukodystrophy carriers. Watkiss et al. (1993) reported that symptomatic status and X-inactivation in fibroblasts do not correlate in a small sample of 12 females. A more recent report suggested that skewed X-inactivation patterns in leukocytes did correlate with symptoms in X-linked adrenoleukodystrophy carriers (Maier et al., 2002). However, the assay used in that study (HUMARA) has limitations because of the genetic distance between the human androgen receptor (Xq12) and ABCD1 gene
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Figure 3 SF-36 and AMC Linear Disability Scale scores. (A) Graphical representation of the SF-36 domain scores and the physical and
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Table 3 Summary of nerve conduction studies No peripheral neuropathy mean (SD)
Reference interval
3.9 (1.1) 5.6 (2.2) 57.6 (16.7)
3.3 (0.4) 6.7 (3.3) 59.0 (5.5)
3.2–3.6 7.1–8.7 57.1–59.6
5.7 (1.1) 7.1 (3.8) 36.2 (3.6)
4.7 (0.7) 12.2 (5.6) 45.8 (4.6)
4.4–5.0 8.5–12.2 44.1–47.2
6.3 (1.7) 2.5 (1.9) 41.8 (12.8)
4.6 (0.8) 4.3 (1.3) 52.0 (14.4)
3.9–4.5 4.4–6.1 46.1–48.6
4.2 5.6 15.2 31.0
3.7 8.4 23.3 46.4
3.4–3.7 7.6–10.8 21.6–26.3 39.7–54.5
(0.5) (3.5) (5.4) (14.5)
(0.3) (5.4) (8.6) (17.9)
Dlt = distal latency time.
Table 4 Results of somatosensory evoked potentials (median and tibial nerve) and brainstem auditory evoked potentials 18–39 years Somatosensory evoked potential median nerve N9 (ms) 9.50 (0.69) N13 (ms) 12.92 (0.82) N20 (ms) 19.64 (1.12) Abnormal 0/10 (0%) Somatosensory evoked potential tibial nerve N35 Left (ms) 34.36 (4.44) P37 Left (ms) 38.91 (5.25) N35 Right (ms) 34.38 (4.59) P37 Right (ms) 40.05 (4.19) Abnormal 1/10 (10%) Brainstem auditory evoked potential I–III Left (ms) 2.10 (0.25) I–V Left (ms) 4.16 (0.43) I–III Right (ms) 2.29 (0.21) I–V Right (ms) 4.23 (0.33) Abnormal 4/11 (36%)
40–59 years
460 years
All ages
10.10 (0.93) 13.90 (1.40) 20.74 (1.50) 3/26 (12%)
10.4 (0.85) 14.07 (0.97) 21.00 (1.22) 0/6 (0%)
9.99 (0.90) 13.69 (1.29) 20.51 (1.44) 3/42 (7%)
36.89 (10.32) 44.20 (7.19) 37.56 (11.75) 45.62 (9.78) 11/27 (41%)
38.83 (5.00) 44.94 (5.28) 35.90 (3.08) 43.95 (4.49) 2/7 (29%)
36.62 (8.56) 43.06 (6.78) 36.54 (9.52) 43.98 (8.29) 14/44 (30%)
2.31 (0.32) 4.38 (0.47) 2.38 (0.20) 4.46 (0.42) 17/26 (65%)
2.24 (0.24) 4.19 (0.23) 2.24 (0.14) 4.23 (0.32) 5/8 (63%)
2.25 (0.30) 4.29 (0.44) 2.33 (0.21) 4.36 (0.40) 26/45 (58%)
Values are mean SD.
(Xq28). It is now known that X-inactivation is not uniform through the entire X-chromosome, but can differ from locus to locus (Carrel and Willard, 2005). Another recent study compared a group of asymptomatic and symptomatic carriers and did not find an association between X-inactivation in fibroblasts and symptomatic status (Salsano et al., 2012). Importantly, there was an average age difference of 415 years between the groups of asymptomatic and symptomatic carriers, making a comparison difficult considering that our data show that age is a strong predictor for symptomatic status in X-linked adrenoleukodystrophy carriers. When taking age into account, we could not establish a link between ABCD1 allele-specific expression and symptomatic status.
This still does not exclude X-inactivation as an important modifier. A limitation, in all studies, is that non-neural tissue was used to obtain material for X-inactivation studies. Even though this is the largest cohort of X-linked adrenoleukodystrophy carriers studied systematically so far, the sample size might still be too small to detect the relationship. In future studies, we will keep expanding the cohort and possibly extend X-inactivation studies to different tissues. Imaging of the brain was not performed routinely as brain involvement is rare and this has been studied by others (Fatemi et al., 2003). MRI of the spinal cord of men with adrenomyeloneuropathy and symptomatic X-linked adrenoleukodystrophy
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Median nerve Dlt (ms) Compound muscle action potential (mV) Motor nerve conduction velocity (m/s) Posterior tibial nerve Dlt (ms) Compound muscle action potential (mV) Motor nerve conduction velocity (m/s) Peroneal nerve Dlt (ms) Compound muscle action potential (mV) Motor nerve conduction velocity (m/s) Sural nerve Dlt (ms) Sensory nerve action potential (mV) Summated compound muscle action potential Summated sensory nerve action potential
Peripheral neuropathy mean (SD)
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Table 5 Plasma and fibroblast studies Plasma
C26:0
C26:0/C22:0
Control X-linked adrenoleukodystrophy carriers All Asymptomatic Symptomatic 20–39 years 39–59 years over 60 years
1.05 (0.078)
0.016 (0.0013)
2.26 2.03 2.37 2.07 2.32 2.32
0.035 0.031 0.037 0.034 0.036 0.033
Fibroblasts
C26:0
C26:0/C22:0
Control X-linked adrenoleukodystrophy carriers All Asymptomatic Symptomatic
0.17 (0.077)
0.054 (0.027)
1.70 (0.37)
0.75 (0.32) 0.61 (0.32) 0.81 (0.31)
0.22 (0.10) 0.19 (0.12) 0.24 (0.09)
0.73 (0.41) 0.81 (0.42) 0.69 (0.41)
(0.69) (0.78) (0.63) (0.86) (0.65) (0.64)
(0.012) (0.011) (0.012) (0.012) (0.012) (0.011) b-oxidation
Figure 4 ALDP expression and functional correlate. ALDP levels as determined by immunoblot correlate with the residual peroxisomal beta-oxidation activity (A), C26:0 synthesis (B), C26:0 levels (C) and the C26:0/C22:0 ratio (D) in skin fibroblasts from X-linked adrenoleukodystrophy carriers.
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C26:0 levels are in mmol/l for plasma and nmol/mg protein for fibroblasts. b-oxidation activity in fibroblasts is expressed as the ratio between D3-C16:0/D3-C22:0, meaning that higher values correspond to a higher C26:0 beta-oxidation (details in the ‘Materials and methods’ section). Values are mean SD.
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carriers shows abnormalities on magnetization transfer imaging (Dubey et al., 2005). This is compatible with the clinical findings in our cohort. MRI of the brain and spinal cord was only performed in three females to exclude other disorders, and was normal in each case. A limitation of our study is that not all X-linked adrenoleukodystrophy carriers in The Netherlands were examined. Even though we tried to minimize selection bias by including not only females from the outpatient clinic, and 75% of the participants were recruited through the X-linked adrenoleukodystrophy patient organization (with 52 female members) and relatives, it is still possible that the participants are not a totally random sample of X-linked adrenoleukodystrophy carriers. Based on the frequency in our cohort it can be estimated there are at least 200 X-linked adrenoleukodystrophy carriers in the Netherlands. Our cohort therefore probably contains no more than 25–30% of all X-linked adrenoleukodystrophy carriers in The Netherlands. It cannot be excluded that those without symptoms were less
likely to participate. Although there was no age-matched control cohort, it is unlikely that the symptoms described are attributable to normal ageing. Myelopathy and peripheral neuropathy cannot be considered a part of healthy ageing. Co-morbidity, like diabetes, was rare in the cohort. In summary, X-linked adrenoleukodystrophy carriers are highly likely to develop symptoms. The most important predictor is age, with most carriers having some clinical manifestation beyond age 60. Clinical manifestations are mostly related to myelopathy. Especially striking is the high incidence of faecal incontinence. Peripheral neuropathy is not prominent clinically, although based on nerve conduction studies 57% have a sensorimotor axonal peripheral neuropathy. Biochemical abnormalities are common, but cannot exclude X-linked adrenoleukodystrophy with certainty in females. The biochemical abnormalities in fibroblasts are clearly related to the X-inactivation pattern. We were not able to show a link between X-inactivation and symptomatic status, but this may be related to the limitations stated above. X-linked
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Figure 5 ABCD1 allele-specific expression in skin fibroblasts. (A) Distribution of ABCD1 allele specific expression (ABCD1 ALE) in skin fibroblasts from X-linked adrenoleukodystrophy carriers (B) the distribution of ABCD1 allele-specific expression is similar in all age groups (C) between asymptomatic and (D) symptomatic carriers patterns of ABCD1 X-inactivation in skin fibroblasts seem similar.
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Figure 6 Correlation between ABCD1 allele-specific expression and biochemical parameters in skin fibroblasts. ABCD1 allele-specific expression correlates with ABCD1 protein levels as determined with immunofluorescence (A) or immunoblot (B), the residual peroxisomal beta-oxidation activity (C), C26:0 synthesis (D), C26:0 levels (E) and the C26:0/C22:0 (F) ratio in skin fibroblasts from X-linked adrenoleukodystrophy carriers.
X-linked adrenoleukodystrophy in women adrenoleukodystrophy should be considered in the differential diagnosis in females with chronic myelopathy. ABCD1 mutation analysis deserves a place in diagnostic protocols for chronic noncompressive myelopathy.
Acknowledgements We thank patients and their families for their participation in this study. We greatly appreciate the help of Ms. Mercan Akyuz for technical assistance.
This work was supported by a grant from the Netherlands Organization for Scientific Research (NWO grant 91786328).
Supplementary material Supplementary material is available at Brain online.
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BRAIN A JOURNAL OF NEUROLOGY
X-linked adrenoleukodystrophy in women: a cross-sectional cohort study
1 2 3 4
Department of Neurology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Department of Paediatric Neurology/Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Assistance Publique des Hoˆpitaux de Paris, Department of Paediatric Neurology, Hospital Kremlin-Biceˆtre, Paris, France Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands 5 Department of Clinical Epidemiology, Biostatistics and Bio-informatics, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands 6 INSERM U986, Le Kremlin-Biceˆtre, Paris, France 7 Department of Neurology, Medical Centre Alkmaar, Alkmaar, The Netherlands *Present address: INSERM U698, Hoˆpital Bichat, Paris, France Correspondence to: M. Engelen, MD, PhD Department of Paediatric Neurology/Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1100DD Amsterdam, The Netherlands E-mail: [email protected]
X-linked adrenoleukodystrophy is the most common peroxisomal disorder. The disease is caused by mutations in the ABCD1 gene that encodes the peroxisomal transporter of very long-chain fatty acids. A defect in the ABCD1 protein results in elevated levels of very long-chain fatty acids in plasma and tissues. The clinical spectrum in males with X-linked adrenoleukodystrophy has been well described and ranges from isolated adrenocortical insufficiency and slowly progressive myelopathy to devastating cerebral demyelination. As in many X-linked diseases, it was assumed that female carriers remain asymptomatic and only a few studies addressed the phenotype of X-linked adrenoleukodystrophy carriers. These studies, however, provided no information on the prevalence of neurological symptoms in the entire population of X-linked adrenoleukodystrophy carriers, since data were acquired in small groups and may be biased towards women with symptoms. Our primary goal was to investigate the symptoms and their frequency in X-linked adrenoleukodystrophy carriers. The secondary goal was to determine if the X-inactivation pattern of the ABCD1 gene was associated with symptomatic status. We included 46 X-linked adrenoleukodystrophy carriers in a prospective cross-sectional cohort study. Our data show that X-linked adrenoleukodystrophy carriers develop signs and symptoms of myelopathy (29/46, 63%) and/or peripheral neuropathy (26/46, 57%). Especially striking was the occurrence of faecal incontinence (13/46, 28%). The frequency of symptomatic women increased sharply with age (from 18% in women 540 years to 88% in women 460 years of age). Virtually all (44/45, 98%) X-linked adrenoleukodystrophy carriers had increased very long-chain fatty acids in plasma and/or fibroblasts, and/or decreased very long-chain fatty acids beta-oxidation in fibroblasts. We did not find an association between the X-inactivation pattern and symptomatic status. We conclude that X-linked
Received June 26, 2013. Revised October 17, 2013. Accepted November 10, 2013. Advance Access publication January 29, 2014 ß The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
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Marc Engelen,1,2 Mathieu Barbier,3,* Inge M. E. Dijkstra,4 Remmelt Schu¨r,2 Rob M. A. de Bie,1 Camiel Verhamme,1 Marcel G. W. Dijkgraaf,5 Patrick A. Aubourg,3,6 Ronald J. A. Wanders,4 Bjorn M. van Geel,7 Marianne de Visser,1 Bwee T. Poll–The1,2 and Stephan Kemp2,4
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adrenoleukodystrophy carriers develop an adrenomyeloneuropathy-like phenotype and there is a strong association between symptomatic status and age. X-linked adrenoleukodystrophy should be considered in the differential diagnosis in women with chronic myelopathy and/or peripheral neuropathy (especially with early faecal incontinence). ABCD1 mutation analysis deserves a place in diagnostic protocols for chronic non-compressive myelopathy.
Keywords: adrenoleukodystrophy; X-inactivation; myelopathy; faecal incontinence; carriers
Materials and methods
X-linked adrenoleukodystrophy is a peroxisomal disorder caused by mutations in the ABCD1 gene (http://www.x-ald.nl) (Mosser et al., 1993), which codes for the transporter of very long-chain fatty acids (5C22:0) (van Roermund et al., 2008). ABCD1 deficiency impairs peroxisomal very long-chain fatty acid degradation resulting in increased cytosolic very long-chain fatty acid-CoA levels (Kemp et al., 2011), which are further elongated by the very long-chain fatty acid-specific elongase, ELOVL1 (Ofman et al., 2010), thus causing very long-chain fatty acid accumulation (Moser et al., 1981; Kemp and Wanders, 2010). In males, disease course and symptomatology have been studied extensively (Engelen et al., 2012). The clinical spectrum ranges from isolated adrenocortical insufficiency, slowly progressive myelopathy and peripheral neuropathy in adulthood (adrenomyeloneuropathy) to a rapidly progressive and fatal cerebral demyelinating disease in boys or adult men (cerebral-adrenoleukodystrophy). The most frequent phenotype in males is adrenomyeloneuropathy (van Geel et al., 1994; Engelen et al., 2012). There have been several studies and observations concerning the phenotype of X-linked adrenoleukodystrophy carriers. O’Neill et al. (1984) described 21 obligate carriers and several were shown to have signs and symptoms of myelopathy. It was estimated that 50% develop symptoms at some point in their life (Moser et al., 1991). In a small study, eight females were found to have myelopathy and in a retrospective study the frequency of symptoms increased with age (van Geel, 2000; Schmidt et al., 2001). These studies provide no information on the prevalence of neurological symptoms in the entire population of X-linked adrenoleukodystrophy carriers, as data were acquired in small groups and may be biased towards females with symptoms. Adrenal insufficiency is rare in X-linked adrenoleukodystrophy carriers (el-Deiry et al., 1997), and there are only a few reports of X-linked adrenoleukodystrophy carriers that developed cerebraladrenoleukodystrophy (Pilz and Schiener, 1973; Jung et al., 2007). Some studies suggested that disease severity could be related to the pattern of X-inactivation, whereas others refuted this observation (Migeon et al., 1981; Watkiss et al., 1993; Maier et al., 2002; Salsano et al., 2012). The primary goal of our study was to estimate the proportion of X-linked adrenoleukodystrophy carriers that develop symptoms and to characterize these symptoms both clinically and with ancillary investigations. A secondary goal was to determine if the X-inactivation pattern of the ABCD1 gene was associated with symptomatic status.
Research population We designed a prospective cross-sectional cohort study. Female carriers over the age of 18 years were eligible to participate. They were recruited from the outpatient clinics of the Academic Medical Centre and the Medical Centre Alkmaar from 2008–10. To prevent bias for symptomatic women, we tried to examine all female relatives with X-linked adrenoleukodystrophy of the women participating. Through the Dutch X-linked adrenoleukodystrophy patient organization, letters to invite X-linked adrenoleukodystrophy carriers to participate were sent to all members. The study protocol was approved by the local Institutional Review Board. After informed consent was obtained, women visited the outpatient clinic for assessment, which included questionnaires, neurological examination, neurophysiological tests, blood samples and skin biopsy.
Clinical assessment A careful history with emphasis on neurological complaints and neurological examination was performed. In particular, symptoms of incontinence, gait disorder, maximum walking distance, and sensory disturbance were recorded. Urinary incontinence was defined as urge incontinence. Stress incontinence was not considered a symptom of myelopathy. A gait disorder was considered present if the walking distance was significantly reduced or running was not possible. Sensory complaints were recorded if there were paraesthesias or numbness in the lower extremities. Sensory disturbances on examination were considered present if there was reduced sensation to touch, pinprick, vibration or position sense in the lower extremities. Muscle strength was rated using the Medical Research Council Scale and spasticity was rated using the Ashworth scale (Ashworth, 1964). An Expanded Disability Status Scale value was scored based on the history and physical examination performed during the visit and recorded in the medical chart (Kurtzke, 1983). Carriers with peripheral neuropathy or myelopathy were considered symptomatic. Myelopathy was considered present if: (i) there were symptoms of myelopathy (for instance sphincter disturbance); and (ii) signs of myelopathy on neurological examination (pyramidal tract or dorsal column signs) were present. If only symptoms were present (for instance faecal incontinence) but no signs on neurological examination, myelopathy was not scored as present. Peripheral neuropathy was considered present if nerve conduction studies and/or EMG were abnormal (as described below). All participants completed the Dutch version of the SF-36 (Quality of Life Assessment) (Aaronson et al., 1998), and the AMC Linear Disability Scale (Weisscher et al., 2007). SF-36 values can be compared to reference values for the Dutch population, matched for gender and age. The AMC Linear Disability Scale values were
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Introduction
X-linked adrenoleukodystrophy in women compared to those of patients with Parkinson’s disease from the CARPA study (Post et al., 2011).
Neurophysiological testing
Blood samples Venous blood samples were taken and plasma and lymphocytes stored as described previously (Engelen et al., 2010).
Cell culture and biochemical analysis From skin biopsy material primary fibroblast cell lines were generated and cultured as described (Valianpour et al., 2003). Very long-chain fatty acids (C26:0 and the C26:0/C22:0 ratio) were determined in plasma and fibroblasts as described (Valianpour et al., 2003). Peroxisomal beta-oxidation activity was measured essentially as described by incubating cells with 30 mM deuterium-labelled C22:0 (D3-C22:0) (Kemp et al., 2004). ABCD1 protein levels were determined by immunofluorescence and quantitative immunoblot (Kemp et al., 1996; Zhang et al., 2011).
Genetic analysis and ABCD1 allele-specific expression in fibroblasts Mutation analysis of the ABCD1 gene was performed as described (Boehm et al., 1999). ABCD1 allele-specific expression (ABCD1 allele-specific expression) of 38 carriers was measured by pyrosequencing. RNA was extracted from primary fibroblasts and complementary DNA synthesized as described (Bieche et al., 2001). The ratio of normal versus mutant allele expressed was measured by pyrosequencing. Complementary DNA was PCR-amplified using biotin-labelled primers followed by quantitative pyrosequencing using a PyroMark Q96 ID instrument (Qiagen). Biotin-labelled single-stranded amplicons were isolated following the protocol using the Qiagen PyroMark Q96 Workstation pyrosequenced with a sequencing primer (PCR- and sequencing-primers are provided in Supplementary Table 1). Each sample was pyrosequenced in the upper direction (with lower biotinlabelled amplicons) and in lower direction (with upper biotin-labelled
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amplicons). The ratio of normal versus mutant allele was measured using the PyroMark ID software (Qiagen). For each carrier, the ABCD1 allele-specific expression value represents the mean of upper and lower reads (determined in two independent experiments). For included samples, variations between upper and lower reads were 410%. For validation, we repeated the experiment for a subset of samples (n = 6) using different PCR primers or independent complementary DNA samples. The ABCD1 allele-specific expression values were similar (variations 44%). ABCD1 allele-specific expression pattern was defined as ‘severely’ skewed with an expression of the normal allele 410% and ‘moderately’ skewed with an expression of the normal allele between 11–25%, similar to a previous report (Miozzo et al., 2007). In the manuscript ABCD1 allele-specific expression will be referred to as X-inactivation. For five patients the ABCD1 allele-specific expression analysis was not possible because of the complexity of the mutation. Instead, ABCD1 allele-specific expression values were calculated using ABCD1 protein immunofluorescence and determination of the ratio of ABCD1 protein positive and negative cells.
Data entry and statistical analysis Data were analysed with IBM SPSS statistics version 19 (IBM Inc.), Microsoft Excel from Office 2010 and Prism version 5 (GraphPad Software). Depending on the distributional properties, outcome measures were expressed as means standard deviation (SD) or as medians with ranges. Statistical significance was assessed by independent sample Student’s t-test for normally distributed continuous data and the Wilcoxon test for non-normally distributed continuous data. All reported P-values are two-sided and were not adjusted for multiple testing. The SF-36 scores were converted to z-scores based on the Dutch norm population, using the appropriate age groups. AMC Linear Disability Scale scores were analysed as described previously (Weisscher et al., 2007). The relationship between symptomatic status, age and the pattern of X-inactivation in skin fibroblasts were analysed by logistic regression.
Results Demographic and clinical characteristics Forty-six females from 26 kindreds were enrolled in the study (one kindred with five females, two with four females, two with three, five with two, and 17 with one). Twelve were recruited through the outpatient clinic, 34 were relatives of these females or responded to the letter sent to members of the Dutch X-linked adrenoleukodystrophy patient organization. Complete clinical and electrophysiological data were available from 46 females, blood samples were available from 45 females (one refused venepuncture) and skin biopsies were obtained from 43 women (two refused skin biopsy, one fibroblast culture failed because of a yeast infection). The age of the included females ranged from 22 to 76 years (average 48 13 years) (Fig. 1). Mutation analysis was done in 45 females. For one obligate carrier no leucocytes were available, the mutation in that subject was considered to be the mutation found in her father and paternal uncle (Table 1). Symptoms, signs and Expanded Disability Status Scale scores are presented in Table 1 and summarized by age group in Table 2.
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Nerve conduction studies and EMG were performed according to a fixed protocol, allowing comparison with reference values and possibly follow-up in the future (Verhamme et al., 2009). Compound muscle action potential amplitudes, baseline to negative peak, were recorded from the abductor pollicis brevis, abductor hallucis brevis, and extensor digitorum brevis on one side. A summated compound muscle action potential amplitude was calculated. Motor nerve conduction velocities were calculated after distal and proximal stimulation of the right median, tibial, and peroneal nerves. Sensory nerve conduction velocity, calculated based on negative peak markers, and sensory nerve action potentials amplitudes, baseline to negative peak, were investigated for the left median nerve and right sural nerve. A summated sensory nerve action potential amplitude was calculated. Electromyography was done of the left anterior tibial, and interosseus I/II muscles. An axonal peripheral neuropathy was diagnosed if in two separate nerves at least one parameter was outside the 95% confidence interval (CI) and the criteria for demyelinating peripheral neuropathy were not fulfilled (Van Asseldonk et al., 2005). Somatosensory evoked potentials of left median and the posterior tibial nerves on both sides and brainstem auditory evoked potentials on both sides were registered, conforming to local protocols (Aramideh et al., 1992; Aalfs et al., 1993).
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M. Engelen et al. polyneuropathy, as has been reported to occur in males with X-linked adrenoleukodystrophy (van Geel et al., 1996).
Somatosensory evoked potentials
Complaints of incontinence (both urinary and faecal), abnormal gait, and sensory symptoms were common. Myelopathy was found in 29/46 (63%) and peripheral neuropathy in 26/46 (57%). Two of 46 (4%) had peripheral neuropathy without myelopathy, and 11/46 (24%) had myelopathy without peripheral neuropathy. Thirty-one of 46 (67%) were considered symptomatic because of either myelopathy and/or peripheral neuropathy. The percentage of women with signs and symptoms suggestive of myelopathy increased with age (Table 2). The percentage of symptomatic females increased from 2/11 (18% in the youngest age group) to 7/8 (88% in the oldest age group). Logistic regression showed a significant relationship between age and symptomatic status, with the probability of being symptomatic increasing clearly with age [P = 0.002; odds ratio (OR) 1.2 (1.07–1.34); Fig. 2]. Quality of life, as measured by the SF-36, was not statistically significant different between asymptomatic and symptomatic carriers. However, on measures that are related to physical disability (Fig. 3A), there was a trend towards a statistically significant difference. There were no differences in emotional or mental health between asymptomatic and symptomatic carriers. The levels of disability were measured by the AMC Linear Disability Scale scores (Fig. 3B) and these were compared to data from the CARPA study, a prospective cohort study in Parkinson’s disease (Post et al., 2011). The AMC Linear Disability Scale scores were lower in the group of symptomatic X-linked adrenoleukodystrophy carriers compared with the asymptomatic carriers. Symptomatic carriers had AMC Linear Disability Scale scores comparable to patients with Parkinson’s disease at baseline and 1 year after diagnosis.
Electrophysiological testing Nerve conduction studies and electromyography Of the X-linked adrenoleukodystrophy carriers 26/46 (57%) had a peripheral neuropathy. The abnormalities mostly consisted of a reduction in compound muscle and sensory nerve action potential amplitudes, with a marginal slowing in nerve conduction velocity (Table 3). This is consistent with a sensorimotor axonal peripheral
Brainstem auditory evoked potentials Brainstem auditory evoked potential measurement failed for technical reasons in one participant (Table 4). For the entire group, 26/45 (58%) had an abnormal brainstem auditory evoked potential, mostly consisting of an increased I–V and I–III interval. In the asymptomatic group, 3/13 (23%) had a normal brainstem auditory evoked potential. In the symptomatic group this proportion was 23/32 (72%) had an abnormal brainstem auditory evoked potential. There were no significant left–right differences.
Biochemical and ABCD1 allele-specific expression analysis Plasma Plasma C26:0 were increased (2.26 0.69 mmol/l; reference values 1.05 0.078 mmol/l; Table 5). Thirty-one of 45 (69%) had abnormal plasma very long-chain fatty acids levels. There were no significant differences in plasma C26:0 levels between asymptomatic and symptomatic carriers (P = 0.16). In contrast to what we reported before, C26:0 levels did not increase with age (Stradomska and Tylki-Szymanska, 2001).
Fibroblasts Fibroblast C26:0 levels were increased in 37/43 (86%), and the C26:0/C22:0 ratio in 36/43 (84%) (Table 5). The D3-C16:0/D3C22:0 flux-ratio was decreased in 26/43 (60%), indicating reduced peroxisomal very long-chain fatty acids beta-oxidation capacity. Two of 43 (5%) carriers had normal C26:0 levels in plasma and fibroblasts, and 1/45 (2%) had normal C26:0 levels and normal peroxisomal beta-oxidation. There was a clear correlation between the ABCD1 protein levels in fibroblasts and the residual peroxisomal beta-oxidation capacity, C26:0 synthesis and very long-chain fatty acids levels (Fig. 4).
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Figure 1 Age distribution of the cohort.
The somatosensory evoked potential from the median (arm) nerve was abnormal in 3/42 (7%) of X-linked adrenoleukodystrophy carriers (four registrations failed because of technical reasons), of the tibial (leg) nerve 14/44 (30%) were abnormal (two registrations failed) (Table 4). The median nerve somatosensory evoked potential was normal in all 15 asymptomatic carriers. The posterior tibial nerve somatosensory evoked potential was abnormal in one (7%) asymptomatic carrier. Three of 27 (11%) symptomatic carriers had an abnormal median nerve somatosensory evoked potential and 13/29 (45%) had an abnormal tibial nerve somatosensory evoked potential. The average latency of the cortical peak increased with age for median nerve and tibial nerve somatosensory evoked potential. The percentage of abnormal somatosensory evoked potentials increased with age (Table 4).
Age (years)
44 56 45 59 75 42 44 44 51 59 44 38 57 31 37 60 35 42 61 71 42 31 48 57 60 51 22 40 29 45 57 70 40 59 39 28 35
A A AA AA AA B B B B B C D D E E E F G H I J K K K K L M M N N N N O P Q R S
No Yes No Yes Yes Yes No No No No No Yes Yes No No No No No Yes No No No Yes No Yes Yes No No No Yes Yes No Yes Yes No No No
Urinary incontinence No Yes No No No Yes No No No No No Yes No No No No No Yes Yes No No No No No No No No No No No Yes No Yes Yes Yes No No
Faecal incontinence Yes No No Yes Yes Yes No No No No No Yes Yes No No Yes No No Yes No No No No Yes No Yes No No No No Yes Yes Yes Yes Yes No No
Gait disorder No No No No No No No No Yes Yes No No Yes No No No No No Yes No No No No Yes No No No No No Yes Yes No Yes Yes No No No
Sensory complaints No No No No Yes Yes No No Yes Yes No Yes Yes No No Yes No No Yes Yes Yes No Yes Yes Yes Yes No No No No Yes Yes Yes Yes Yes No No
Sensory disturbance No No No No Yes Yes No No No No No Yes No No No Yes No No No No No No No No No Yes No No No No No No No Yes No No No
Spasticity No No No Yes Yes Yes No No No Yes No Yes No No No Yes No No No No No No No Yes No Yes No No No No No Yes No Yes No No No
Weakness Yes Yes No Yes Yes Yes No No No No No Yes Yes No No Yes No No Yes Yes Yes No Yes Yes Yes Yes No No No No No Yes Yes Yes No No No
Pathological reflexes
Mutation p.Pro480Thr p.Pro480Thr p.Arg660Trp p.Arg660Trp p.Arg660Trp p.Leu220Pro p.Leu220Pro p.Leu220Pro p.Leu220Pro p.Leu220Pro p.Gln133* p.Leu654Pro p.Leu654Pro p.Arg74Trp p.Arg74Trp p.Arg74Trp p.Met1Val p.Ala245Asp exon8-10del p.Glu609Lys p.Glu90* p.Pro543Leu p.Pro543Leu p.Pro543Leu p.Pro543Leu p.Ile657del p.Ser149Asn p.Ser149Asn p.Arg389His p.Arg389His p.Arg389His p.Arg389His p.Glu609Lys p.Leu215* p.Val208Trpfs p.Pro480Thr p.His283Tyr
EDSS 1.0 1.5 0 3.5 6.0 4.0 0 0 1.0 2.0 0 6.0 5.5 0 0 5.5 0 1.0 3.5 2.0 1.5 0 2.5 3.5 3.5 6.5 0 0 0 2.0 3.5 3.5 3.5 6.0 3.0 0 0
(continued)
Absent Absent Absent Absent Absent Reduced Reduced Reduced Reduced Reduced Absent Absent Absent Absent Absent Absent Absent Present Absent Absent Absent Absent Absent Absent Absent Absent Reduced Reduced Reduced Reduced Reduced Reduced Absent Absent Absent Absent Reduced
ABCD1 protein
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Family
Table 1 Summary of symptoms and signs of all the female participating in the study
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Age = age at examination; urinary incontinence = urge incontinence; faecal incontinence = soiling and urge incontinence; gait = gait disorder, for instance unable to run; sensory complaints = numbness or tingling; sensory disturbance = sensory symptoms on neurological examinations; spasticity = spasticity of the lower extremities; weakness = paresis of the lower extremities, Pathological reflexes: pathological reflexes in the lower extremities, EDSS = expanded disability status scale; mutation = mutation in ABCD1; ABCD1 protein = effect of mutation in ABCD1 on ABCD1 protein as determined by immunoblot. Asterisk indicates the introductions of a stop codon. a Mutation inferred from mutation in father and uncle, as this patient did not consent to provide a blood sample and skin biopsy.
Reduced Absent Absent Absent Absent Absent Absent Absenta Absent p.His283Tyr p.Gln177* p.Arg464* p.Asp442Glyfs p.Ala616Thr p.Ala616Thr p.Arg113Alafs p.Glu609Glya p.Ser633Argfs 2.0 4.0 2.0 2.5 3.5 2.0 2.5 0 2.0 Yes Yes No Yes Yes No Yes No Yes No Yes No Yes No No No No No No No No No No No No No No 76 51 47 56 45 65 47 24 50 S T U V W W X Y Z
Yes Yes Yes Yes Yes No Yes No No
No Yes Yes No Yes Yes No No No
Yes Yes No Yes Yes No Yes No No
No No No No Yes No No No No
Yes Yes Yes Yes Yes No No No Yes
Weakness Spasticity Sensory disturbance Sensory complaints Gait disorder Faecal incontinence Urinary incontinence Age (years) Family
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Correlation studies of X-inactivation with asymptomatic or symptomatic status The distribution of ABCD1 allele-specific expression (which will be referred to as the pattern of X-inactivation) is shown in Fig. 5A. There was no evidence for skewing to either the mutated or normal ABCD1 allele. The pattern appeared random and the median value was exactly 0.5. The pattern of X-inactivation was similar between age groups (Fig. 5B). This contrasts with previous reports that documented a highly skewed pattern of X-inactivation in X-linked adrenoleukodystrophy carriers (Migeon et al., 1981). We subsequently correlated the pattern of X-inactivation with biochemical parameters in fibroblasts, i.e. ABCD1 protein levels, residual beta-oxidation activity and very long-chain fatty acids levels (Fig. 6). A clear correlation was found between the pattern of X-inactivation and the biochemical parameters in the fibroblasts. Indeed, if there was preferential expression of the normal allele, the level of ABCD1 protein was higher, residual rates of beta-oxidation were higher, very long-chain fatty acids synthesis was lower, and very long-chain fatty acids levels were lower, and vice versa. To determine if the pattern of X-inactivation correlates with symptomatic status it is important to consider age as there is a strong correlation between age and symptomatic status (Fig. 2). Logistic regression showed no correlation between symptomatic status and the ABCD1 allele-specific expression pattern, also after adjustment for age [P = 0.74; OR 1.00 (0.97–1.02)]. In Fig. 5C and D the distribution of X-inactivation in asymptomatic and symptomatic carriers is plotted.
Discussion X-linked adrenoleukodystrophy carriers develop neurological symptoms. In fact, one of the earliest descriptions of an adrenomyeloneuropathy-like phenotype in X-linked adrenoleukodystrophy was a female patient (Penman, 1960). This is not common knowledge among physicians (Jangouk et al., 2012). Several X-linked adrenoleukodystrophy carriers were reported to undergo cervical laminectomy for suspected cervical spondylogenic myelopathy (van Geel et al., 1997). In this largest prospective cross-sectional cohort of female carriers to date, we found that neurological abnormalities are common and that the frequency increases steeply with age. The main symptoms were consistent with myelopathy, as in males with adrenomyeloneuropathy. It is striking how often faecal incontinence is reported as an early symptom. One should bear in mind, however, that it is often not voluntarily reported because it is felt to be embarrassing. To our knowledge, this is not reported often in males with adrenomyeloneuropathy or in other degenerative myelopathies, for instance, hereditary spastic paraplegia, albeit that this feature may not have been specifically asked for (Salinas et al., 2008). In our cohort sphincter disturbance is a frequent (13/46) and early symptom, and even occurs in a few (2/17) symptomatic carriers without other signs of myelopathy. Interestingly, most females with X-linked adrenoleukodystrophy were actually relieved to find out that their symptoms were related to X-linked adrenoleukodystrophy, as often they had been told that an X-linked disease does not cause symptoms in ‘carriers’.
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Pathological reflexes
EDSS
Mutation
ABCD1 protein
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Table 1 Continued
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Table 2 Symptoms, signs and Expanded Disability Status Scale scores by age group
Incontinence (urine) Incontinence (faecal) Gait disorder Sensory complaints Sensory disturbance Spasticity Weakness Pathological reflexes Myelopathy Neuropathy Symptomatic EDSS
18–39 years
40–59 years
460 years
All ages
1/11 (9) 2/11 (18) 2/11 (18) 0/11 (0) 2/11 (18) 1/11 (9) 1/11 (9) 1/11 (9) 2/11 (18) 2/11 (18) 2/11 (18) 0.82 (1.94)
15/27 (56) 9/27 (33) 13/27 (48) 9/27 (33) 16/27 (59) 3/27 (11) 8/27 (30) 16/27 (59) 20/27 (74) 14/27 (52) 22/27 (82) 2.41 (1.83)
4/8 (50) 2/8 (25) 5/8 (63) 1/8 (13) 7/8 (88) 2/8 (25) 3/8 (38) 7/8 (88) 7/8 (88) 4/8 (50) 7/8 (88) 3.50 (1.56)
20/46 [44% 13/46 [28% 20/46 [44% 10/46 [22% 25/46 [54% 6/46 [13% 12/46 [26% 24/46 [52% 29/46 [63% 20/46 [44% 31/46 [67% 2.22 (1.99)
(29–58)] (15–42)] (29–58)] (9–34)] (39–69)] (3–23)] (13–39)] (37–67)] (49–78)] (29–58)] (53–81)]
Figure 2 Symptomatic status and age. The bars indicate the percentage of X-linked adrenoleukodystrophy carriers considered symptomatic within each age group (i.e. diagnosed with a myelopathy and/or a peripheral neuropathy). The dots show each individual X-linked adrenoleukodystrophy carrier in the cohort, classified as either symptomatic or asymptomatic.
Signs of peripheral neuropathy were mostly absent or minor clinically, and did not seem to contribute much to the overall disability. It is possible that signs and symptoms were masked by the more prominent signs of myelopathy. Notwithstanding, on electrophysiological testing about 57% of the females in this cohort fulfilled the criteria for an axonal sensorimotor neuropathy. There was no statistically significant difference in SF-36 scores between asymptomatic and symptomatic carriers (Fig. 3A). Although any symptom may affect quality of life, it seems that the majority of symptomatic carriers have symptoms that do not impact the SF-36 enough to be visible at group level. The z-scores are usually below zero for the symptomatic carriers on the measures related to physical symptoms (Fig. 3A), but the standard deviation is large, supporting the previous assumption. However, symptomatic females show a trend towards reduced quality of life in domains related to physical symptoms (Fig. 3A), corresponding to the signs of myelopathy frequently present in our cohort.
Analysis of the AMC Linear Disability Scale revealed that manifesting X-linked adrenoleukodystrophy carriers had similar disability as patients with Parkinson’s disease 1 year after the diagnosis. However, compared to our cohort, the Parkinson’s disease cohort was significantly older (average age 67 versus 48 years), suggesting that the burden of disease in X-linked adrenoleukodystrophy carriers is considerable. Somatosensory evoked potential was reported to be very sensitive in detecting abnormalities in X-linked adrenoleukodystrophy carriers in a presymptomatic stage (Restuccia et al., 1997). In our cohort, however, somatosensory evoked potentials of the median nerve (arm) were normal in all asymptomatic, and abnormal in only 11% of the symptomatic females. This is not surprising, as in none of the symptomatic females the arms were affected clinically. Somatosensory evoked potentials of the posterior tibial nerve (legs) were only abnormal in 7% of the asymptomatic and 45% of the symptomatic females. Therefore, we conclude that somatosensory evoked potential is not superior to clinical examination in detecting myelopathy in our cohort. In agreement with an earlier report, brainstem auditory evoked potential was abnormal in 58% (Restuccia et al., 1997). A larger proportion of the symptomatic carriers had an abnormal brainstem auditory evoked potential compared with the asymptomatic carriers (72% versus 23%). The findings were non-specific, mostly consisting of an increased I–V or III–V interval. This indicates a slowing of conduction in the brainstem. For clinical practice brainstem auditory evoked potential has no value as a diagnostic tool. As has been known for several decades, 15–20% of X-linked adrenoleukodystrophy carriers have normal plasma C26:0 (Moser et al., 1999). We found that a slightly higher proportion (31%) had normal plasma C26:0. Furthermore, even in cultured fibroblasts the C26:0 can be normal (Moser et al., 1983), as was the case in 6/43 (14%). Combining C26:0 levels in plasma and fibroblasts marks 41/43 (95%) of the females in our cohort as biochemically abnormal. This corresponds to an earlier observation that by combining very long-chain fatty acids measurements in plasma and fibroblasts 93% of carriers can be identified (Moser et al., 1983). Peroxisomal very long-chain fatty acids
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Symptoms and signs [reported as absolute number (% with 95% CI)] and Expanded Disability Status Scale (EDSS) (mean SD) scores for the entire cohort and stratified by age group.
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mental compound scores. For each of the SF-36 scores a z-score was calculated based on data from the Dutch normative population. (B) AMC Linear Disability Scale scores for asymptomatic and symptomatic X-linked adrenoleukodystrophy carriers and patients with Parkinson’s disease (PD) from the CARPA cohort at baseline and up to 5 years after diagnosis (PD1–PD5). There is a significant difference between symptomatic and asymptomatic carriers. Symptomatic carriers have levels of disability comparable to patients with Parkinson’s disease 1 year after diagnosis. Statistical significant differences are indicated. *P 5 0.05, **P 5 0.01.
beta-oxidation activity in cultured fibroblasts varied from completely normal to values found in males with X-linked adrenoleukodystrophy. By combining very long-chain fatty acids measurements and beta-oxidation activity, 44/45 X-linked adrenoleukodystrophy carriers (98%) could be identified. However, this still does not reliably identify all X-linked adrenoleukodystrophy carriers. Therefore, in females, only ABCD1 mutation analysis identifies all carriers. We showed that the biochemical abnormalities in fibroblasts correlated with the X-inactivation pattern, i.e. the more skewed to the mutant allele, the more abnormal the fibroblasts were biochemically. Our study showed that in fibroblasts, ABCD1 allelespecific expression (ABCD1 allele-specific expression) predicts the biochemical phenotype.
X-inactivation was considered to be skewed preferentially to the mutant allele (Migeon et al., 1981). That finding cannot be reproduced in our data set (Fig. 5). In fact, the median skewing was 49:51, suggesting a random ABCD1 allele-specific expression pattern. For over two decades there has been controversy over whether X-inactivation predicts the symptomatic status of X-linked adrenoleukodystrophy carriers. Watkiss et al. (1993) reported that symptomatic status and X-inactivation in fibroblasts do not correlate in a small sample of 12 females. A more recent report suggested that skewed X-inactivation patterns in leukocytes did correlate with symptoms in X-linked adrenoleukodystrophy carriers (Maier et al., 2002). However, the assay used in that study (HUMARA) has limitations because of the genetic distance between the human androgen receptor (Xq12) and ABCD1 gene
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Figure 3 SF-36 and AMC Linear Disability Scale scores. (A) Graphical representation of the SF-36 domain scores and the physical and
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Table 3 Summary of nerve conduction studies No peripheral neuropathy mean (SD)
Reference interval
3.9 (1.1) 5.6 (2.2) 57.6 (16.7)
3.3 (0.4) 6.7 (3.3) 59.0 (5.5)
3.2–3.6 7.1–8.7 57.1–59.6
5.7 (1.1) 7.1 (3.8) 36.2 (3.6)
4.7 (0.7) 12.2 (5.6) 45.8 (4.6)
4.4–5.0 8.5–12.2 44.1–47.2
6.3 (1.7) 2.5 (1.9) 41.8 (12.8)
4.6 (0.8) 4.3 (1.3) 52.0 (14.4)
3.9–4.5 4.4–6.1 46.1–48.6
4.2 5.6 15.2 31.0
3.7 8.4 23.3 46.4
3.4–3.7 7.6–10.8 21.6–26.3 39.7–54.5
(0.5) (3.5) (5.4) (14.5)
(0.3) (5.4) (8.6) (17.9)
Dlt = distal latency time.
Table 4 Results of somatosensory evoked potentials (median and tibial nerve) and brainstem auditory evoked potentials 18–39 years Somatosensory evoked potential median nerve N9 (ms) 9.50 (0.69) N13 (ms) 12.92 (0.82) N20 (ms) 19.64 (1.12) Abnormal 0/10 (0%) Somatosensory evoked potential tibial nerve N35 Left (ms) 34.36 (4.44) P37 Left (ms) 38.91 (5.25) N35 Right (ms) 34.38 (4.59) P37 Right (ms) 40.05 (4.19) Abnormal 1/10 (10%) Brainstem auditory evoked potential I–III Left (ms) 2.10 (0.25) I–V Left (ms) 4.16 (0.43) I–III Right (ms) 2.29 (0.21) I–V Right (ms) 4.23 (0.33) Abnormal 4/11 (36%)
40–59 years
460 years
All ages
10.10 (0.93) 13.90 (1.40) 20.74 (1.50) 3/26 (12%)
10.4 (0.85) 14.07 (0.97) 21.00 (1.22) 0/6 (0%)
9.99 (0.90) 13.69 (1.29) 20.51 (1.44) 3/42 (7%)
36.89 (10.32) 44.20 (7.19) 37.56 (11.75) 45.62 (9.78) 11/27 (41%)
38.83 (5.00) 44.94 (5.28) 35.90 (3.08) 43.95 (4.49) 2/7 (29%)
36.62 (8.56) 43.06 (6.78) 36.54 (9.52) 43.98 (8.29) 14/44 (30%)
2.31 (0.32) 4.38 (0.47) 2.38 (0.20) 4.46 (0.42) 17/26 (65%)
2.24 (0.24) 4.19 (0.23) 2.24 (0.14) 4.23 (0.32) 5/8 (63%)
2.25 (0.30) 4.29 (0.44) 2.33 (0.21) 4.36 (0.40) 26/45 (58%)
Values are mean SD.
(Xq28). It is now known that X-inactivation is not uniform through the entire X-chromosome, but can differ from locus to locus (Carrel and Willard, 2005). Another recent study compared a group of asymptomatic and symptomatic carriers and did not find an association between X-inactivation in fibroblasts and symptomatic status (Salsano et al., 2012). Importantly, there was an average age difference of 415 years between the groups of asymptomatic and symptomatic carriers, making a comparison difficult considering that our data show that age is a strong predictor for symptomatic status in X-linked adrenoleukodystrophy carriers. When taking age into account, we could not establish a link between ABCD1 allele-specific expression and symptomatic status.
This still does not exclude X-inactivation as an important modifier. A limitation, in all studies, is that non-neural tissue was used to obtain material for X-inactivation studies. Even though this is the largest cohort of X-linked adrenoleukodystrophy carriers studied systematically so far, the sample size might still be too small to detect the relationship. In future studies, we will keep expanding the cohort and possibly extend X-inactivation studies to different tissues. Imaging of the brain was not performed routinely as brain involvement is rare and this has been studied by others (Fatemi et al., 2003). MRI of the spinal cord of men with adrenomyeloneuropathy and symptomatic X-linked adrenoleukodystrophy
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Median nerve Dlt (ms) Compound muscle action potential (mV) Motor nerve conduction velocity (m/s) Posterior tibial nerve Dlt (ms) Compound muscle action potential (mV) Motor nerve conduction velocity (m/s) Peroneal nerve Dlt (ms) Compound muscle action potential (mV) Motor nerve conduction velocity (m/s) Sural nerve Dlt (ms) Sensory nerve action potential (mV) Summated compound muscle action potential Summated sensory nerve action potential
Peripheral neuropathy mean (SD)
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Table 5 Plasma and fibroblast studies Plasma
C26:0
C26:0/C22:0
Control X-linked adrenoleukodystrophy carriers All Asymptomatic Symptomatic 20–39 years 39–59 years over 60 years
1.05 (0.078)
0.016 (0.0013)
2.26 2.03 2.37 2.07 2.32 2.32
0.035 0.031 0.037 0.034 0.036 0.033
Fibroblasts
C26:0
C26:0/C22:0
Control X-linked adrenoleukodystrophy carriers All Asymptomatic Symptomatic
0.17 (0.077)
0.054 (0.027)
1.70 (0.37)
0.75 (0.32) 0.61 (0.32) 0.81 (0.31)
0.22 (0.10) 0.19 (0.12) 0.24 (0.09)
0.73 (0.41) 0.81 (0.42) 0.69 (0.41)
(0.69) (0.78) (0.63) (0.86) (0.65) (0.64)
(0.012) (0.011) (0.012) (0.012) (0.012) (0.011) b-oxidation
Figure 4 ALDP expression and functional correlate. ALDP levels as determined by immunoblot correlate with the residual peroxisomal beta-oxidation activity (A), C26:0 synthesis (B), C26:0 levels (C) and the C26:0/C22:0 ratio (D) in skin fibroblasts from X-linked adrenoleukodystrophy carriers.
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C26:0 levels are in mmol/l for plasma and nmol/mg protein for fibroblasts. b-oxidation activity in fibroblasts is expressed as the ratio between D3-C16:0/D3-C22:0, meaning that higher values correspond to a higher C26:0 beta-oxidation (details in the ‘Materials and methods’ section). Values are mean SD.
X-linked adrenoleukodystrophy in women
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carriers shows abnormalities on magnetization transfer imaging (Dubey et al., 2005). This is compatible with the clinical findings in our cohort. MRI of the brain and spinal cord was only performed in three females to exclude other disorders, and was normal in each case. A limitation of our study is that not all X-linked adrenoleukodystrophy carriers in The Netherlands were examined. Even though we tried to minimize selection bias by including not only females from the outpatient clinic, and 75% of the participants were recruited through the X-linked adrenoleukodystrophy patient organization (with 52 female members) and relatives, it is still possible that the participants are not a totally random sample of X-linked adrenoleukodystrophy carriers. Based on the frequency in our cohort it can be estimated there are at least 200 X-linked adrenoleukodystrophy carriers in the Netherlands. Our cohort therefore probably contains no more than 25–30% of all X-linked adrenoleukodystrophy carriers in The Netherlands. It cannot be excluded that those without symptoms were less
likely to participate. Although there was no age-matched control cohort, it is unlikely that the symptoms described are attributable to normal ageing. Myelopathy and peripheral neuropathy cannot be considered a part of healthy ageing. Co-morbidity, like diabetes, was rare in the cohort. In summary, X-linked adrenoleukodystrophy carriers are highly likely to develop symptoms. The most important predictor is age, with most carriers having some clinical manifestation beyond age 60. Clinical manifestations are mostly related to myelopathy. Especially striking is the high incidence of faecal incontinence. Peripheral neuropathy is not prominent clinically, although based on nerve conduction studies 57% have a sensorimotor axonal peripheral neuropathy. Biochemical abnormalities are common, but cannot exclude X-linked adrenoleukodystrophy with certainty in females. The biochemical abnormalities in fibroblasts are clearly related to the X-inactivation pattern. We were not able to show a link between X-inactivation and symptomatic status, but this may be related to the limitations stated above. X-linked
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Figure 5 ABCD1 allele-specific expression in skin fibroblasts. (A) Distribution of ABCD1 allele specific expression (ABCD1 ALE) in skin fibroblasts from X-linked adrenoleukodystrophy carriers (B) the distribution of ABCD1 allele-specific expression is similar in all age groups (C) between asymptomatic and (D) symptomatic carriers patterns of ABCD1 X-inactivation in skin fibroblasts seem similar.
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Figure 6 Correlation between ABCD1 allele-specific expression and biochemical parameters in skin fibroblasts. ABCD1 allele-specific expression correlates with ABCD1 protein levels as determined with immunofluorescence (A) or immunoblot (B), the residual peroxisomal beta-oxidation activity (C), C26:0 synthesis (D), C26:0 levels (E) and the C26:0/C22:0 (F) ratio in skin fibroblasts from X-linked adrenoleukodystrophy carriers.
X-linked adrenoleukodystrophy in women adrenoleukodystrophy should be considered in the differential diagnosis in females with chronic myelopathy. ABCD1 mutation analysis deserves a place in diagnostic protocols for chronic noncompressive myelopathy.
Acknowledgements We thank patients and their families for their participation in this study. We greatly appreciate the help of Ms. Mercan Akyuz for technical assistance.
This work was supported by a grant from the Netherlands Organization for Scientific Research (NWO grant 91786328).
Supplementary material Supplementary material is available at Brain online.
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