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BRAIN
A JOURNAL OF NEUROLOGY
X-linked adrenoleukodystrophy in women: a
cross-sectional cohort study
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–The
1,2
and Stephan Kemp
2,4
1 Department of Neurology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
2 Department of Paediatric Neurology/Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
3 Assistance Publique des Ho
ˆpitaux de Paris, Department of Paediatric Neurology, Hospital Kremlin-Bice
ˆtre, Paris, France
4 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-Bice
ˆtre, Paris, France
7 Department of Neurology, Medical Centre Alkmaar, Alkmaar, The Netherlands
*Present address: INSERM U698, Ho
ˆpital 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: m.engelen@amc.uva.nl
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 symp-
toms 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
doi:10.1093/brain/awt361 Brain 2014: Page 1 of 14 |1
Received June 26, 2013. Revised October 17, 2013. Accepted November 10, 2013.
ßThe Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Brain Advance Access published January 29, 2014
at Universiteit van Amsterdam on February 5, 2014http://brain.oxfordjournals.org/Downloaded from
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
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 (5C22:0) (van Roermund et al., 2008). ABCD1 defi-
ciency 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 esti-
mated 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 car-
riers (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 ancil-
lary 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 car-
riers 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 neuro-
logical examination was performed. In particular, symptoms of incon-
tinence, 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 examin-
ation 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 exam-
ination, 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 com-
pared to reference values for the Dutch population, matched for
gender and age. The AMC Linear Disability Scale values were
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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 ful-
filled (Van Asseldonk et al., 2005). Somatosensory evoked potentials
of left median and the posterior tibial nerves on both sides and brain-
stem auditory evoked potentials on both sides were registered, con-
forming 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 mM deuterium-labelled C22:0
(D
3
-C22:0) (Kemp et al., 2004). ABCD1 protein levels were deter-
mined 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 pyrosequen-
cing. 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 pyrosequen-
cing. 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
410%. For validation, we repeated the experiment for a subset of
samples (n=6) using different PCR primers or independent comple-
mentary 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 ‘moder-
ately’ 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 meas-
ures 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 venepunc-
ture) 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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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 myel-
opathy, 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 regres-
sion showed a significant relationship between age and symptom-
atic 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 car-
riers. However, on measures that are related to physical disability
(Fig. 3A), there was a trend towards a statistically significant dif-
ference. 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
polyneuropathy, as has been reported to occur in males with
X-linked adrenoleukodystrophy (van Geel et al., 1996).
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 registra-
tions 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 somato-
sensory evoked potential. The average latency of the cortical peak
increased with age for median nerve and tibial nerve somatosen-
sory evoked potential. The percentage of abnormal somatosensory
evoked potentials increased with age (Table 4).
Brainstem auditory evoked potentials
Brainstem auditory evoked potential measurement failed for tech-
nical reasons in one participant (Table 4). For the entire group,
26/45 (58%) had an abnormal brainstem auditory evoked poten-
tial, mostly consisting of an increased I–V and I–III interval. In the
asymptomatic group, 3/13 (23%) had a normal brainstem audi-
tory 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 D
3
-C16:0/D
3
-
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 correl-
ation 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 1 Age distribution of the cohort.
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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
A 44 No No Yes No No No No Yes 1.0 p.Pro480Thr Absent
A 56 Yes Yes No No No No No Yes 1.5 p.Pro480Thr Absent
AA 45 No No No No No No No No 0 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
B 42 Yes Yes Yes No Yes Yes Yes Yes 4.0 p.Leu220Pro Reduced
B 44 No No No No No No No No 0 p.Leu220Pro Reduced
B 44 No No No No No No No No 0 p.Leu220Pro Reduced
B 51 No No No Yes Yes No No No 1.0 p.Leu220Pro Reduced
B 59 No No No Yes Yes No Yes No 2.0 p.Leu220Pro Reduced
C 44 No No No No No No No No 0 p.Gln133* Absent
D 38 Yes Yes Yes No Yes Yes Yes Yes 6.0 p.Leu654Pro Absent
D 57 Yes No Yes Yes Yes No No Yes 5.5 p.Leu654Pro Absent
E 31 No No No No No No No No 0 p.Arg74Trp Absent
E 37 No No No No No No No No 0 p.Arg74Trp Absent
E 60 No No Yes No Yes Yes Yes Yes 5.5 p.Arg74Trp Absent
F 35 No No No No No No No No 0 p.Met1Val Absent
G 42 No Yes No No No No No No 1.0 p.Ala245Asp Present
H 61 Yes Yes Yes Yes Yes No No Yes 3.5 exon8-10del Absent
I 71 No No No No Yes No No Yes 2.0 p.Glu609Lys Absent
J 42 No No No No Yes No No Yes 1.5 p.Glu90* Absent
K 31 No No No No No No No No 0 p.Pro543Leu Absent
K 48 Yes No No No Yes No No Yes 2.5 p.Pro543Leu Absent
K 57 No No Yes Yes Yes No Yes Yes 3.5 p.Pro543Leu Absent
K 60 Yes No No No Yes No No Yes 3.5 p.Pro543Leu Absent
L 51 Yes No Yes No Yes Yes Yes Yes 6.5 p.Ile657del Absent
M 22 No No No No No No No No 0 p.Ser149Asn Reduced
M 40 No No No No No No No No 0 p.Ser149Asn Reduced
N 29 No No No No No No No No 0 p.Arg389His Reduced
N 45 Yes No No Yes No No No No 2.0 p.Arg389His Reduced
N 57 Yes Yes Yes Yes Yes No No No 3.5 p.Arg389His Reduced
N 70 No No Yes No Yes No Yes Yes 3.5 p.Arg389His Reduced
O 40 Yes Yes Yes Yes Yes No No Yes 3.5 p.Glu609Lys Absent
P 59 Yes Yes Yes Yes Yes Yes Yes Yes 6.0 p.Leu215* Absent
Q 39 No Yes Yes No Yes No No No 3.0 p.Val208Trpfs Absent
R 28 No No No No No No No No 0 p.Pro480Thr Absent
S 35 No No No No No No No No 0 p.His283Tyr Reduced
(continued)
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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 fibro-
blasts. 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 adreno-
myeloneuropathy-like phenotype in X-linked adrenoleukodystro-
phy 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 myelop-
athy (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 incontin-
ence 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’.
Table 1 Continued
Family Age
(years)
Urinary
incontinence
Faecal
incontinence
Gait
disorder
Sensory
complaints
Sensory
disturbance
Spasticity Weakness Pathological
reflexes
EDSS Mutation ABCD1
protein
S 76 Yes No Yes No Yes No No Yes 2.0 p.His283Tyr Reduced
T 51 Yes Yes Yes No Yes No Yes Yes 4.0 p.Gln177* Absent
U 47 Yes Yes No No Yes No No No 2.0 p.Arg464* Absent
V 56 Yes No Yes No Yes No Yes Yes 2.5 p.Asp442Glyfs Absent
W 45 Yes Yes Yes Yes Yes No No Yes 3.5 p.Ala616Thr Absent
W 65 No Yes No No No No No No 2.0 p.Ala616Thr Absent
X 47 Yes No Yes No No No No Yes 2.5 p.Arg113Alafs Absent
Y 24 No No No No No No No No 0 p.Glu609Gly
a
Absent
a
Z 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.
a
Mutation inferred from mutation in father and uncle, as this patient did not consent to provide a blood sample and skin biopsy.
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Signs of peripheral neuropathy were mostly absent or minor
clinically, and did not seem to contribute much to the overall dis-
ability. It is possible that signs and symptoms were masked by the
more prominent signs of myelopathy. Notwithstanding, on elec-
trophysiological 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 meas-
ures related to physical symptoms (Fig. 3A), but the standard de-
viation 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), correspond-
ing to the signs of myelopathy frequently present in our cohort.
Analysis of the AMC Linear Disability Scale revealed that mani-
festing X-linked adrenoleukodystrophy carriers had similar disabil-
ity 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), suggest-
ing that the burden of disease in X-linked adrenoleukodystrophy
carriers is considerable.
Somatosensory evoked potential was reported to be very sensi-
tive 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 clin-
ically. 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 car-
riers (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 brain-
stem 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 fibro-
blasts 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 fibro-
blasts 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
Table 2 Symptoms, signs and Expanded Disability Status Scale scores by age group
18–39 years 40–59 years 460 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.
Figure 2 Symptomatic status and age. The bars indicate the
percentage of X-linked adrenoleukodystrophy carriers con-
sidered 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.
X-linked adrenoleukodystrophy in women Brain 2014: Page 7 of 14 |7
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beta-oxidation activity in cultured fibroblasts varied from com-
pletely normal to values found in males with X-linked adrenoleu-
kodystrophy. By combining very long-chain fatty acids
measurements and beta-oxidation activity, 44/45 X-linked adre-
noleukodystrophy carriers (98%) could be identified. However,
this still does not reliably identify all X-linked adrenoleukodystro-
phy 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 bio-
chemically. 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 repro-
duced 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 correl-
ate in a small sample of 12 females. A more recent report sug-
gested 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 be-
tween the human androgen receptor (Xq12) and ABCD1 gene
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. *P50.05, **P50.01.
8|Brain 2014: Page 8 of 14 M. Engelen et al.
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(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 symp-
tomatic 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 dif-
ficult 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 be-
tween 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 adrenomyelo-
neuropathy and symptomatic X-linked adrenoleukodystrophy
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 (mV) 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.
Table 4 Results of somatosensory evoked potentials (median and tibial nerve) and brainstem auditory evoked potentials
18–39 years 40–59 years 460 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.
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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 adre-
noleukodystrophy carriers.
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)
Fibroblasts C26:0 C26:0/C22:0 b-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 mmol/l for plasma and nmol/mg protein for fibroblasts. b-oxidation activity in fibroblasts is expressed as the ratio between D
3
-C16:0/D
3
-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.
10 |Brain 2014: Page 10 of 14 M. Engelen et al.
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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 adrenoleukody-
strophy 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 dia-
betes, 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
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.
X-linked adrenoleukodystrophy in women Brain 2014: Page 11 of 14 |11
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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.
12 |Brain 2014: Page 12 of 14 M. Engelen et al.
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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.
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