Abstract

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 <40 years to 88% in women >60 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 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.

Introduction

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 (≥C22:0) (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 cerebral-adrenoleukodystrophy (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.

Materials and methods

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 compared to those of patients with Parkinson’s disease from the CARPA study (Post et al., 2011).

Neurophysiological testing

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).

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 µM 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 biotin-labelled amplicons) and in lower direction (with upper biotin-labelled 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 ≤10%. 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 ≤4%).

ABCD1 allele-specific expression pattern was defined as ‘severely’ skewed with an expression of the normal allele ≤10% 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. 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.

Figure 1

Age distribution of the cohort.

Figure 1

Age distribution of the cohort.

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.

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.

Figure 3

SF-36 and AMC Linear Disability Scale scores. (A) Graphical representation of the SF-36 domain scores and the physical and 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 < 0.05, **P < 0.01.

Figure 3

SF-36 and AMC Linear Disability Scale scores. (A) Graphical representation of the SF-36 domain scores and the physical and 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 < 0.05, **P < 0.01.

Table 1

Summary of symptoms and signs of all the female participating in the study

Family Age (years) Urinary incontinence Faecal incontinence Gait disorder Sensory complaints Sensory disturbance Spasticity Weakness Pathological reflexes EDSS Mutation ABCD1 protein 
44 No No Yes No No No No Yes 1.0 p.Pro480Thr Absent 
56 Yes Yes No No No No No Yes 1.5 p.Pro480Thr Absent 
AA 45 No No No No No No No No p.Arg660Trp Absent 
AA 59 Yes No Yes No No No Yes Yes 3.5 p.Arg660Trp Absent 
AA 75 Yes No Yes No Yes Yes Yes Yes 6.0 p.Arg660Trp Absent 
42 Yes Yes Yes No Yes Yes Yes Yes 4.0 p.Leu220Pro Reduced 
44 No No No No No No No No p.Leu220Pro Reduced 
44 No No No No No No No No p.Leu220Pro Reduced 
51 No No No Yes Yes No No No 1.0 p.Leu220Pro Reduced 
59 No No No Yes Yes No Yes No 2.0 p.Leu220Pro Reduced 
44 No No No No No No No No p.Gln133* Absent 
38 Yes Yes Yes No Yes Yes Yes Yes 6.0 p.Leu654Pro Absent 
57 Yes No Yes Yes Yes No No Yes 5.5 p.Leu654Pro Absent 
31 No No No No No No No No p.Arg74Trp Absent 
37 No No No No No No No No p.Arg74Trp Absent 
60 No No Yes No Yes Yes Yes Yes 5.5 p.Arg74Trp Absent 
35 No No No No No No No No p.Met1Val Absent 
42 No Yes No No No No No No 1.0 p.Ala245Asp Present 
61 Yes Yes Yes Yes Yes No No Yes 3.5 exon8-10del Absent 
71 No No No No Yes No No Yes 2.0 p.Glu609Lys Absent 
42 No No No No Yes No No Yes 1.5 p.Glu90* Absent 
31 No No No No No No No No p.Pro543Leu Absent 
48 Yes No No No Yes No No Yes 2.5 p.Pro543Leu Absent 
57 No No Yes Yes Yes No Yes Yes 3.5 p.Pro543Leu Absent 
60 Yes No No No Yes No No Yes 3.5 p.Pro543Leu Absent 
51 Yes No Yes No Yes Yes Yes Yes 6.5 p.Ile657del Absent 
22 No No No No No No No No p.Ser149Asn Reduced 
40 No No No No No No No No p.Ser149Asn Reduced 
29 No No No No No No No No p.Arg389His Reduced 
45 Yes No No Yes No No No No 2.0 p.Arg389His Reduced 
57 Yes Yes Yes Yes Yes No No No 3.5 p.Arg389His Reduced 
70 No No Yes No Yes No Yes Yes 3.5 p.Arg389His Reduced 
40 Yes Yes Yes Yes Yes No No Yes 3.5 p.Glu609Lys Absent 
59 Yes Yes Yes Yes Yes Yes Yes Yes 6.0 p.Leu215* Absent 
39 No Yes Yes No Yes No No No 3.0 p.Val208Trpfs Absent 
28 No No No No No No No No p.Pro480Thr Absent 
35 No No No No No No No No p.His283Tyr Reduced 
76 Yes No Yes No Yes No No Yes 2.0 p.His283Tyr Reduced 
51 Yes Yes Yes No Yes No Yes Yes 4.0 p.Gln177* Absent 
47 Yes Yes No No Yes No No No 2.0 p.Arg464* Absent 
56 Yes No Yes No Yes No Yes Yes 2.5 p.Asp442Glyfs Absent 
45 Yes Yes Yes Yes Yes No No Yes 3.5 p.Ala616Thr Absent 
65 No Yes No No No No No No 2.0 p.Ala616Thr Absent 
47 Yes No Yes No No No No Yes 2.5 p.Arg113Alafs Absent 
24 No No No No No No No No p.Glu609Glya Absenta 
50 No No No No Yes No No Yes 2.0 p.Ser633Argfs Absent 
Family Age (years) Urinary incontinence Faecal incontinence Gait disorder Sensory complaints Sensory disturbance Spasticity Weakness Pathological reflexes EDSS Mutation ABCD1 protein 
44 No No Yes No No No No Yes 1.0 p.Pro480Thr Absent 
56 Yes Yes No No No No No Yes 1.5 p.Pro480Thr Absent 
AA 45 No No No No No No No No p.Arg660Trp Absent 
AA 59 Yes No Yes No No No Yes Yes 3.5 p.Arg660Trp Absent 
AA 75 Yes No Yes No Yes Yes Yes Yes 6.0 p.Arg660Trp Absent 
42 Yes Yes Yes No Yes Yes Yes Yes 4.0 p.Leu220Pro Reduced 
44 No No No No No No No No p.Leu220Pro Reduced 
44 No No No No No No No No p.Leu220Pro Reduced 
51 No No No Yes Yes No No No 1.0 p.Leu220Pro Reduced 
59 No No No Yes Yes No Yes No 2.0 p.Leu220Pro Reduced 
44 No No No No No No No No p.Gln133* Absent 
38 Yes Yes Yes No Yes Yes Yes Yes 6.0 p.Leu654Pro Absent 
57 Yes No Yes Yes Yes No No Yes 5.5 p.Leu654Pro Absent 
31 No No No No No No No No p.Arg74Trp Absent 
37 No No No No No No No No p.Arg74Trp Absent 
60 No No Yes No Yes Yes Yes Yes 5.5 p.Arg74Trp Absent 
35 No No No No No No No No p.Met1Val Absent 
42 No Yes No No No No No No 1.0 p.Ala245Asp Present 
61 Yes Yes Yes Yes Yes No No Yes 3.5 exon8-10del Absent 
71 No No No No Yes No No Yes 2.0 p.Glu609Lys Absent 
42 No No No No Yes No No Yes 1.5 p.Glu90* Absent 
31 No No No No No No No No p.Pro543Leu Absent 
48 Yes No No No Yes No No Yes 2.5 p.Pro543Leu Absent 
57 No No Yes Yes Yes No Yes Yes 3.5 p.Pro543Leu Absent 
60 Yes No No No Yes No No Yes 3.5 p.Pro543Leu Absent 
51 Yes No Yes No Yes Yes Yes Yes 6.5 p.Ile657del Absent 
22 No No No No No No No No p.Ser149Asn Reduced 
40 No No No No No No No No p.Ser149Asn Reduced 
29 No No No No No No No No p.Arg389His Reduced 
45 Yes No No Yes No No No No 2.0 p.Arg389His Reduced 
57 Yes Yes Yes Yes Yes No No No 3.5 p.Arg389His Reduced 
70 No No Yes No Yes No Yes Yes 3.5 p.Arg389His Reduced 
40 Yes Yes Yes Yes Yes No No Yes 3.5 p.Glu609Lys Absent 
59 Yes Yes Yes Yes Yes Yes Yes Yes 6.0 p.Leu215* Absent 
39 No Yes Yes No Yes No No No 3.0 p.Val208Trpfs Absent 
28 No No No No No No No No p.Pro480Thr Absent 
35 No No No No No No No No p.His283Tyr Reduced 
76 Yes No Yes No Yes No No Yes 2.0 p.His283Tyr Reduced 
51 Yes Yes Yes No Yes No Yes Yes 4.0 p.Gln177* Absent 
47 Yes Yes No No Yes No No No 2.0 p.Arg464* Absent 
56 Yes No Yes No Yes No Yes Yes 2.5 p.Asp442Glyfs Absent 
45 Yes Yes Yes Yes Yes No No Yes 3.5 p.Ala616Thr Absent 
65 No Yes No No No No No No 2.0 p.Ala616Thr Absent 
47 Yes No Yes No No No No Yes 2.5 p.Arg113Alafs Absent 
24 No No No No No No No No p.Glu609Glya Absenta 
50 No No No No Yes No No Yes 2.0 p.Ser633Argfs Absent 

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.

aMutation inferred from mutation in father and uncle, as this patient did not consent to provide a blood sample and skin biopsy.

Table 2

Symptoms, signs and Expanded Disability Status Scale scores by age group

 18–39 years 40–59 years >60 years All ages 
Incontinence (urine) 1/11 (9) 15/27 (56) 4/8 (50) 20/46 [44% (29–58)] 
Incontinence (faecal) 2/11 (18) 9/27 (33) 2/8 (25) 13/46 [28% (15–42)] 
Gait disorder 2/11 (18) 13/27 (48) 5/8 (63) 20/46 [44% (29–58)] 
Sensory complaints 0/11 (0) 9/27 (33) 1/8 (13) 10/46 [22% (9–34)] 
Sensory disturbance 2/11 (18) 16/27 (59) 7/8 (88) 25/46 [54% (39–69)] 
Spasticity 1/11 (9) 3/27 (11) 2/8 (25) 6/46 [13% (3–23)] 
Weakness 1/11 (9) 8/27 (30) 3/8 (38) 12/46 [26% (13–39)] 
Pathological reflexes 1/11 (9) 16/27 (59) 7/8 (88) 24/46 [52% (37–67)] 
Myelopathy 2/11 (18) 20/27 (74) 7/8 (88) 29/46 [63% (49–78)] 
Neuropathy 2/11 (18) 14/27 (52) 4/8 (50) 20/46 [44% (29–58)] 
Symptomatic 2/11 (18) 22/27 (82) 7/8 (88) 31/46 [67% (53–81)] 
EDSS 0.82 (1.94) 2.41 (1.83) 3.50 (1.56) 2.22 (1.99) 
 18–39 years 40–59 years >60 years All ages 
Incontinence (urine) 1/11 (9) 15/27 (56) 4/8 (50) 20/46 [44% (29–58)] 
Incontinence (faecal) 2/11 (18) 9/27 (33) 2/8 (25) 13/46 [28% (15–42)] 
Gait disorder 2/11 (18) 13/27 (48) 5/8 (63) 20/46 [44% (29–58)] 
Sensory complaints 0/11 (0) 9/27 (33) 1/8 (13) 10/46 [22% (9–34)] 
Sensory disturbance 2/11 (18) 16/27 (59) 7/8 (88) 25/46 [54% (39–69)] 
Spasticity 1/11 (9) 3/27 (11) 2/8 (25) 6/46 [13% (3–23)] 
Weakness 1/11 (9) 8/27 (30) 3/8 (38) 12/46 [26% (13–39)] 
Pathological reflexes 1/11 (9) 16/27 (59) 7/8 (88) 24/46 [52% (37–67)] 
Myelopathy 2/11 (18) 20/27 (74) 7/8 (88) 29/46 [63% (49–78)] 
Neuropathy 2/11 (18) 14/27 (52) 4/8 (50) 20/46 [44% (29–58)] 
Symptomatic 2/11 (18) 22/27 (82) 7/8 (88) 31/46 [67% (53–81)] 
EDSS 0.82 (1.94) 2.41 (1.83) 3.50 (1.56) 2.22 (1.99) 

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.

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 polyneuropathy, as has been reported to occur in males with X-linked adrenoleukodystrophy (van Geel et al., 1996).

Table 3

Summary of nerve conduction studies

 Peripheral neuropathy mean (SD) No peripheral neuropathy mean (SD) Reference interval 
Median nerve 
    Dlt (ms) 3.9 (1.1) 3.3 (0.4) 3.2–3.6 
    Compound muscle action potential (mV) 5.6 (2.2) 6.7 (3.3) 7.1–8.7 
    Motor nerve conduction velocity (m/s) 57.6 (16.7) 59.0 (5.5) 57.1–59.6 
Posterior tibial nerve 
    Dlt (ms) 5.7 (1.1) 4.7 (0.7) 4.4–5.0 
    Compound muscle action potential (mV) 7.1 (3.8) 12.2 (5.6) 8.5–12.2 
    Motor nerve conduction velocity (m/s) 36.2 (3.6) 45.8 (4.6) 44.1–47.2 
Peroneal nerve 
    Dlt (ms) 6.3 (1.7) 4.6 (0.8) 3.9–4.5 
    Compound muscle action potential (mV) 2.5 (1.9) 4.3 (1.3) 4.4–6.1 
    Motor nerve conduction velocity (m/s) 41.8 (12.8) 52.0 (14.4) 46.1–48.6 
Sural nerve    
    Dlt (ms) 4.2 (0.5) 3.7 (0.3) 3.4–3.7 
    Sensory nerve action potential (µV) 5.6 (3.5) 8.4 (5.4) 7.6–10.8 
    Summated compound muscle action potential 15.2 (5.4) 23.3 (8.6) 21.6–26.3 
    Summated sensory nerve action potential 31.0 (14.5) 46.4 (17.9) 39.7–54.5 
 Peripheral neuropathy mean (SD) No peripheral neuropathy mean (SD) Reference interval 
Median nerve 
    Dlt (ms) 3.9 (1.1) 3.3 (0.4) 3.2–3.6 
    Compound muscle action potential (mV) 5.6 (2.2) 6.7 (3.3) 7.1–8.7 
    Motor nerve conduction velocity (m/s) 57.6 (16.7) 59.0 (5.5) 57.1–59.6 
Posterior tibial nerve 
    Dlt (ms) 5.7 (1.1) 4.7 (0.7) 4.4–5.0 
    Compound muscle action potential (mV) 7.1 (3.8) 12.2 (5.6) 8.5–12.2 
    Motor nerve conduction velocity (m/s) 36.2 (3.6) 45.8 (4.6) 44.1–47.2 
Peroneal nerve 
    Dlt (ms) 6.3 (1.7) 4.6 (0.8) 3.9–4.5 
    Compound muscle action potential (mV) 2.5 (1.9) 4.3 (1.3) 4.4–6.1 
    Motor nerve conduction velocity (m/s) 41.8 (12.8) 52.0 (14.4) 46.1–48.6 
Sural nerve    
    Dlt (ms) 4.2 (0.5) 3.7 (0.3) 3.4–3.7 
    Sensory nerve action potential (µV) 5.6 (3.5) 8.4 (5.4) 7.6–10.8 
    Summated compound muscle action potential 15.2 (5.4) 23.3 (8.6) 21.6–26.3 
    Summated sensory nerve action potential 31.0 (14.5) 46.4 (17.9) 39.7–54.5 

Dlt = distal latency time.

Somatosensory evoked potentials

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).

Table 4

Results of somatosensory evoked potentials (median and tibial nerve) and brainstem auditory evoked potentials

 18–39 years 40–59 years >60 years All ages 
Somatosensory evoked potential median nerve 
N9 (ms) 9.50 (0.69) 10.10 (0.93) 10.4 (0.85) 9.99 (0.90) 
N13 (ms) 12.92 (0.82) 13.90 (1.40) 14.07 (0.97) 13.69 (1.29) 
N20 (ms) 19.64 (1.12) 20.74 (1.50) 21.00 (1.22) 20.51 (1.44) 
Abnormal 0/10 (0%) 3/26 (12%) 0/6 (0%) 3/42 (7%) 
Somatosensory evoked potential tibial nerve 
N35 Left (ms) 34.36 (4.44) 36.89 (10.32) 38.83 (5.00) 36.62 (8.56) 
P37 Left (ms) 38.91 (5.25) 44.20 (7.19) 44.94 (5.28) 43.06 (6.78) 
N35 Right (ms) 34.38 (4.59) 37.56 (11.75) 35.90 (3.08) 36.54 (9.52) 
P37 Right (ms) 40.05 (4.19) 45.62 (9.78) 43.95 (4.49) 43.98 (8.29) 
Abnormal 1/10 (10%) 11/27 (41%) 2/7 (29%) 14/44 (30%) 
Brainstem auditory evoked potential 
I–III Left (ms) 2.10 (0.25) 2.31 (0.32) 2.24 (0.24) 2.25 (0.30) 
I–V Left (ms) 4.16 (0.43) 4.38 (0.47) 4.19 (0.23) 4.29 (0.44) 
I–III Right (ms) 2.29 (0.21) 2.38 (0.20) 2.24 (0.14) 2.33 (0.21) 
I–V Right (ms) 4.23 (0.33) 4.46 (0.42) 4.23 (0.32) 4.36 (0.40) 
Abnormal 4/11 (36%) 17/26 (65%) 5/8 (63%) 26/45 (58%) 
 18–39 years 40–59 years >60 years All ages 
Somatosensory evoked potential median nerve 
N9 (ms) 9.50 (0.69) 10.10 (0.93) 10.4 (0.85) 9.99 (0.90) 
N13 (ms) 12.92 (0.82) 13.90 (1.40) 14.07 (0.97) 13.69 (1.29) 
N20 (ms) 19.64 (1.12) 20.74 (1.50) 21.00 (1.22) 20.51 (1.44) 
Abnormal 0/10 (0%) 3/26 (12%) 0/6 (0%) 3/42 (7%) 
Somatosensory evoked potential tibial nerve 
N35 Left (ms) 34.36 (4.44) 36.89 (10.32) 38.83 (5.00) 36.62 (8.56) 
P37 Left (ms) 38.91 (5.25) 44.20 (7.19) 44.94 (5.28) 43.06 (6.78) 
N35 Right (ms) 34.38 (4.59) 37.56 (11.75) 35.90 (3.08) 36.54 (9.52) 
P37 Right (ms) 40.05 (4.19) 45.62 (9.78) 43.95 (4.49) 43.98 (8.29) 
Abnormal 1/10 (10%) 11/27 (41%) 2/7 (29%) 14/44 (30%) 
Brainstem auditory evoked potential 
I–III Left (ms) 2.10 (0.25) 2.31 (0.32) 2.24 (0.24) 2.25 (0.30) 
I–V Left (ms) 4.16 (0.43) 4.38 (0.47) 4.19 (0.23) 4.29 (0.44) 
I–III Right (ms) 2.29 (0.21) 2.38 (0.20) 2.24 (0.14) 2.33 (0.21) 
I–V Right (ms) 4.23 (0.33) 4.46 (0.42) 4.23 (0.32) 4.36 (0.40) 
Abnormal 4/11 (36%) 17/26 (65%) 5/8 (63%) 26/45 (58%) 

Values are mean ± SD.

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 µmol/l; reference values 1.05 ± 0.078 µmol/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).

Table 5

Plasma and fibroblast studies

Plasma C26:0 C26:0/C22:0  
Control 1.05 (0.078) 0.016 (0.0013)  
X-linked adrenoleukodystrophy carriers 
    All 2.26 (0.69) 0.035 (0.012)  
Asymptomatic 2.03 (0.78) 0.031 (0.011)  
    Symptomatic 2.37 (0.63) 0.037 (0.012)  
    20–39 years 2.07 (0.86) 0.034 (0.012)  
    39–59 years 2.32 (0.65) 0.036 (0.012)  
    over 60 years 2.32 (0.64) 0.033 (0.011)  
Plasma C26:0 C26:0/C22:0  
Control 1.05 (0.078) 0.016 (0.0013)  
X-linked adrenoleukodystrophy carriers 
    All 2.26 (0.69) 0.035 (0.012)  
Asymptomatic 2.03 (0.78) 0.031 (0.011)  
    Symptomatic 2.37 (0.63) 0.037 (0.012)  
    20–39 years 2.07 (0.86) 0.034 (0.012)  
    39–59 years 2.32 (0.65) 0.036 (0.012)  
    over 60 years 2.32 (0.64) 0.033 (0.011)  
Fibroblasts C26:0 C26:0/C22:0 β-oxidation 
Control 0.17 (0.077) 0.054 (0.027) 1.70 (0.37) 
X-linked adrenoleukodystrophy carriers 
    All 0.75 (0.32) 0.22 (0.10) 0.73 (0.41) 
    Asymptomatic 0.61 (0.32) 0.19 (0.12) 0.81 (0.42) 
    Symptomatic 0.81 (0.31) 0.24 (0.09) 0.69 (0.41) 
Fibroblasts C26:0 C26:0/C22:0 β-oxidation 
Control 0.17 (0.077) 0.054 (0.027) 1.70 (0.37) 
X-linked adrenoleukodystrophy carriers 
    All 0.75 (0.32) 0.22 (0.10) 0.73 (0.41) 
    Asymptomatic 0.61 (0.32) 0.19 (0.12) 0.81 (0.42) 
    Symptomatic 0.81 (0.31) 0.24 (0.09) 0.69 (0.41) 

C26:0 levels are in μmol/l for plasma and nmol/mg protein for fibroblasts. β-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.

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/D3-C22:0 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).

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.

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.

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.

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.

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.

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.

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.

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’.

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 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 allele-specific 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 (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 >15 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 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 adrenoleukodystrophy should be considered in the differential diagnosis in females with chronic myelopathy. ABCD1 mutation analysis deserves a place in diagnostic protocols for chronic non-compressive 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.

Funding

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.

References

Aalfs
CM
Koelman
JH
Aramideh
M
Bour
LJ
Bruyn
RP
Ongerboer de Visser
BW
Posterior tibial nerve somatosensory evoked potentials in slowly progressive spastic paraplegia: a comparative study with clinical signs
J Neurol
 , 
1993
, vol. 
240
 (pg. 
351
-
6
)
Aaronson
NK
Muller
M
Cohen
PD
Essink-Bot
M
Fekkes
M
Sanderman
R
, et al.  . 
Translation, validation, and norming of the Dutch language version of the SF-36 Health Survey in community and chronic disease populations
J Clin Epidemiol
 , 
1998
, vol. 
51
 (pg. 
1055
-
68
)
Aramideh
M
Hoogendijk
JE
Aalfs
CM
Posthumus Meyjes
FE
de Visser
M
Ongerboer de Visser
BW
Somatosensory evoked potentials, sensory nerve potentials and sensory nerve conduction in hereditary motor and sensory neuropathy type I
J Neurol
 , 
1992
, vol. 
239
 (pg. 
277
-
83
)
Ashworth
B
Preliminary trial of carisoprodol in multiple sclerosis
Practitioner
 , 
1964
, vol. 
192
 (pg. 
540
-
2
)
Bieche
I
Parfait
B
Tozlu
S
Lidereau
R
Vidaud
M
Quantitation of androgen receptor gene expression in sporadic breast tumors by real-time RT-PCR: evidence that MYC is an AR-regulated gene
Carcinogenesis
 , 
2001
, vol. 
22
 (pg. 
1521
-
6
)
Boehm
CD
Cutting
GR
Lachtermacher
MB
Moser
HW
Chong
SS
Accurate DNA-based diagnostic and carrier testing for X-linked adrenoleukodystrophy
Mol Genet Metab
 , 
1999
, vol. 
66
 (pg. 
128
-
36
)
Carrel
L
Willard
HF
X-inactivation profile reveals extensive variability in X-linked gene expression in females
Nature
 , 
2005
, vol. 
434
 (pg. 
400
-
4
)
Dubey
P
Fatemi
A
Huang
H
Nagae-Poetscher
L
Wakana
S
Barker
PB
, et al.  . 
Diffusion tensor-based imaging reveals occult abnormalities in adrenomyeloneuropathy
Ann Neurol
 , 
2005
, vol. 
58
 (pg. 
758
-
66
)
el-Deiry
SS
Naidu
S
Blevins
LS
Ladenson
PW
Assessment of adrenal function in women heterozygous for adrenoleukodystrophy
J Clin Endocrinol Metab
 , 
1997
, vol. 
82
 (pg. 
856
-
60
)
Engelen
M
Kemp
S
de Visser
M
van Geel
BM
Wanders
RJ
Aubourg
P
, et al.  . 
X-linked adrenoleukodystrophy (X-ALD): clinical presentation and guidelines for diagnosis, follow-up and management
Orphanet J Rare Dis
 , 
2012
, vol. 
7
 pg. 
51
 
Engelen
M
Ofman
R
Dijkgraaf
MGW
Hijzen
M
van der Wardt
LA
van Geel
BM
, et al.  . 
Lovastatin in X-Linked Adrenoleukodystrophy
N Engl J Med
 , 
2010
, vol. 
362
 (pg. 
276
-
7
)
Fatemi
A
Barker
PB
Ulug
AM
Nagae-Poetscher
LM
Beauchamp
NJ
Moser
AB
, et al.  . 
MRI and proton MRSI in women heterozygous for X-linked adrenoleukodystrophy
Neurology
 , 
2003
, vol. 
60
 (pg. 
1301
-
7
)
Jangouk
P
Zackowski
KM
Naidu
S
Raymond
GV
Adrenoleukodystrophy in female heterozygotes: underrecognized and undertreated
Mol Genet Metab
 , 
2012
, vol. 
105
 (pg. 
180
-
5
)
Jung
HH
Wimplinger
I
Jung
S
Landau
K
Gal
A
Heppner
FL
Phenotypes of female adrenoleukodystrophy
Neurology
 , 
2007
, vol. 
68
 (pg. 
960
-
1
)
Kemp
S
Mooyer
PA
Bolhuis
PA
van Geel
BM
Mandel
JL
Barth
PG
, et al.  . 
ALDP expression in fibroblasts of patients with X-linked adrenoleukodystrophy
J Inherit Metab Dis
 , 
1996
, vol. 
19
 (pg. 
667
-
74
)
Kemp
S
Theodoulou
FL
Wanders
RJ
Mammalian peroxisomal ABC transporters: from endogenous substrates to pathology and clinical significance
Br J Pharmacol
 , 
2011
, vol. 
164
 (pg. 
1753
-
66
)
Kemp
S
Valianpour
F
Mooyer
PA
Kulik
W
Wanders
RJ
Method for measurement of peroxisomal very-long-chain fatty acid beta-oxidation in human skin fibroblasts using stable-isotope-labeled tetracosanoic acid
Clin Chem
 , 
2004
, vol. 
50
 (pg. 
1824
-
6
)
Kemp
S
Wanders
R
Biochemical aspects of X-linked adrenoleukodystrophy
Brain Pathol
 , 
2010
, vol. 
20
 (pg. 
831
-
7
)
Kurtzke
JF
Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)
Neurology
 , 
1983
, vol. 
33
 (pg. 
1444
-
52
)
Maier
EM
Kammerer
S
Muntau
AC
Wichers
M
Braun
A
Roscher
AA
Symptoms in carriers of adrenoleukodystrophy relate to skewed X inactivation
Ann Neurol
 , 
2002
, vol. 
52
 (pg. 
683
-
8
)
Migeon
BR
Moser
HW
Moser
AB
Axelman
J
Sillence
D
Norum
RA
Adrenoleukodystrophy: evidence for X linkage, inactivation, and selection favoring the mutant allele in heterozygous cells
Proc Natl Acad Sci USA
 , 
1981
, vol. 
78
 (pg. 
5066
-
70
)
Miozzo
M
Selmi
C
Gentilin
B
Grati
FR
Sirchia
S
Oertelt
S
, et al.  . 
Preferential X chromosome loss but random inactivation characterize primary biliary cirrhosis
Hepatology
 , 
2007
, vol. 
46
 (pg. 
456
-
62
)
Moser
AB
Kreiter
N
Bezman
L
Lu
S
Raymond
GV
Naidu
S
, et al.  . 
Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls
Ann Neurol
 , 
1999
, vol. 
45
 (pg. 
100
-
10
)
Moser
HW
Moser
AB
Frayer
KK
Chen
W
Schulman
JD
O'Neill
BP
, et al.  . 
Adrenoleukodystrophy: increased plasma content of saturated very long chain fatty acids
Neurology
 , 
1981
, vol. 
31
 (pg. 
1241
-
9
)
Moser
HW
Moser
AB
Naidu
S
Bergin
A
Clinical aspects of adrenoleukodystrophy and adrenomyeloneuropathy
Dev Neurosci
 , 
1991
, vol. 
13
 (pg. 
254
-
61
)
Moser
HW
Moser
AE
Trojak
JE
Supplee
SW
Identification of female carriers of adrenoleukodystrophy
J Pediatr
 , 
1983
, vol. 
103
 (pg. 
54
-
9
)
Mosser
J
Douar
AM
Sarde
CO
Kioschis
P
Feil
R
Moser
H
, et al.  . 
Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters
Nature
 , 
1993
, vol. 
361
 (pg. 
726
-
30
)
O'Neill
BP
Moser
HW
Saxena
KM
Marmion
LC
Adrenoleukodystrophy: clinical and biochemical manifestations in carriers
Neurology
 , 
1984
, vol. 
34
 (pg. 
798
-
801
)
Ofman
R
Dijkstra
IM
van Roermund
CW
Burger
N
Turkenburg
M
van Cruchten
A
, et al.  . 
The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy
EMBO Mol Med
 , 
2010
, vol. 
2
 (pg. 
90
-
7
)
Penman
RW
Addison's disease in association with spastic paraplegia
Br Med J
 , 
1960
, vol. 
1
 pg. 
402
 
Pilz
P
Schiener
P
Kombination von Morbus Addison und Morbus Schilder bei einer 43 jahrigen Frau
Acta Neuropathol (Berl)
 , 
1973
, vol. 
26
 (pg. 
357
-
60
)
Post
B
Muslimovic
D
van
GN
Speelman
JD
Schmand
B
de Haan
RJ
Progression and prognostic factors of motor impairment, disability and quality of life in newly diagnosed Parkinson's disease
Mov Disord
 , 
2011
, vol. 
26
 (pg. 
449
-
56
)
Restuccia
D
Di Lazarro
V
Valeriani
M
Oliviero
A
Le Pera
D
Colosimo
C
, et al.  . 
Neurophysiological abnormalities in adrenoleukodystrophy carriers. Evidence of different degrees of central nervous system involvement
Brain
 , 
1997
, vol. 
120
 
Pt 7
(pg. 
1139
-
48
)
Salinas
S
Proukakis
C
Crosby
A
Warner
TT
Hereditary spastic paraplegia: clinical features and pathogenetic mechanisms
Lancet Neurol
 , 
2008
, vol. 
7
 (pg. 
1127
-
38
)
Salsano
E
Tabano
S
Sirchia
SM
Colapietro
P
Castellotti
B
Gellera
C
, et al.  . 
Preferential expression of mutant ABCD1 allele is common in adrenoleukodystrophy female carriers but unrelated to clinical symptoms
Orphanet J Rare Dis
 , 
2012
, vol. 
7
 pg. 
10
 
Schmidt
S
Traber
F
Block
W
Keller
E
Pohl
C
von Oertzen
J
, et al.  . 
Phenotype assignment in symptomatic female carriers of X-linked adrenoleukodystrophy
J Neurol
 , 
2001
, vol. 
248
 (pg. 
36
-
44
)
Stradomska
TJ
Tylki-Szymanska
A
Decreasing serum VLCFA levels in ageing X-ALD female carriers
J Inherit Metab Dis
 , 
2001
, vol. 
24
 (pg. 
851
-
7
)
Valianpour
F
Selhorst
JJ
van Lint
LE
van Gennip
AH
Wanders
RJ
Kemp
S
Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry
Mol Genet Metab
 , 
2003
, vol. 
79
 (pg. 
189
-
96
)
Van Asseldonk
JT
Van den Berg
LH
Kalmijn
S
Wokke
JH
Franssen
H
Criteria for demyelination based on the maximum slowing due to axonal degeneration, determined after warming in water at 37 degrees C: diagnostic yield in chronic inflammatory demyelinating polyneuropathy
Brain
 , 
2005
, vol. 
128
 (pg. 
880
-
91
)
van Geel
BM
Draagsterschap van X-gebonden adrenoleukodystrofie
Ned Tijdschr Geneeskd
 , 
2000
, vol. 
144
 (pg. 
1764
-
8
)
van Geel
BM
Assies
J
Wanders
RJ
Barth
PG
X linked adrenoleukodystrophy: clinical presentation, diagnosis, and therapy
J Neurol Neurosurg Psychiatry
 , 
1997
, vol. 
63
 (pg. 
4
-
14
)
van Geel
BM
Assies
J
Weverling
GJ
Barth
PG
Predominance of the adrenomyeloneuropathy phenotype of X-linked adrenoleukodystrophy in The Netherlands: a survey of 30 kindreds
Neurology
 , 
1994
, vol. 
44
 (pg. 
2343
-
6
)
van Geel
BM
Koelman
JH
Barth
PG
Ongerboer de Visser
BW
Peripheral nerve abnormalities in adrenomyeloneuropathy: a clinical and electrodiagnostic study
Neurology
 , 
1996
, vol. 
46
 (pg. 
112
-
8
)
van Roermund
CWT
Visser
WF
Ijlst
L
van Cruchten
A
Boek
M
Kulik
W
, et al.  . 
The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl-CoA esters
FASEB J
 , 
2008
, vol. 
22
 (pg. 
4201
-
8
)
Verhamme
C
van Schaik
IN
Koelman
JH
de Haan
RJ
de
VM
The natural history of Charcot-Marie-Tooth type 1A in adults: a 5-year follow-up study
Brain
 , 
2009
, vol. 
132
 (pg. 
3252
-
62
)
Watkiss
E
Webb
T
Bundey
S
Is skewed X inactivation responsible for symptoms in female carriers for adrenoleucodystrophy?
J Med Genet
 , 
1993
, vol. 
30
 (pg. 
651
-
4
)
Weisscher
N
Post
B
de Haan
RJ
Glas
CA
Speelman
JD
Vermeulen
M
The AMC Linear Disability Score in patients with newly diagnosed Parkinson disease
Neurology
 , 
2007
, vol. 
69
 (pg. 
2155
-
61
)
Zhang
X
De Marcos
LC
Schutte-Lensink
N
Ofman
R
Wanders
RJ
Baldwin
SA
, et al.  . 
Conservation of targeting but divergence in function and quality control of peroxisomal ABC transporters: an analysis using cross-kingdom expression
Biochem J
 , 
2011
, vol. 
436
 (pg. 
547
-
557
)

Author notes

*Present address: INSERM U698, Hôpital Bichat, Paris, France