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Translational studies in X-linked adrenoleukodystrophy

Engelen, M.

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Translational studies in X-linked adrenoleukodystrophy

Translational studies in X-linked adrenoleukodystrophyThesis, University of Amsterdam, Amsterdam

ISBN:

Cover: Joo Yeon Engelen - LeeLay-out: Stephan KempPrinted by Ipskamp Drukkers B.V.

Copyright © 2012 Marc Engelen, The NetherlandsAll rights reserved. No part of this thesis may be reproduced, stored in a retrieval system or transmitted in any way or by any means without prior permission of the author.

Translational studies in X-linked adrenoleukodystrophy

Academisch proefschrift

ter verkrijging van de graad van doctoraan d e Universiteit van Amsterdamop gezag van de Rector Magnificus

prof. dr. D.C. van den Boomten overstaan van een door het

college voor promoties ingestelde commissie,in het openbaar te verdedigen in de Aula der Universiteit

op vrijdag 16 november 2012, te 13:00 uur door

Marc Engelen

geboren te Nijmegen

Promotiecommissie

Promotores: Prof. dr. B.T. Poll – The Prof. dr. M. de Visser

Co-promotores: Dr. S. Kemp Dr. B.M. van Geel

Overige leden: Prof. dr. F. Baas Prof. dr. J.B. van Goudoever Prof. dr. M.S. van der Knaap Prof. dr. F.A. Wijburg Prof. dr. M.A.A.P. Willemsen

Faculteit der Geneeskunde

The work described in this thesis was carried out at the laboratory Genetic Metabolic Diseases and the Departments of Neurology and Pediatric Neurology of the Academic Medical Center, University of Amsterdam, The Netherlands. The research was financially supported by the Departments of Neurology and Pediatric Neurology of the Academic Medical Center and grants from the European Leukodystrophy Association (2005-024I5 & 2006-031I4), the European Union Framework Programme 7 (LeukoTreat 241622), the Stop ALD Foundation and the Netherlands Organization for Scientific Research (91786328).

Gastopponent: Prof. dr. P.A. Aubourg

Printing of this thesis was financially supported by the Academic Medical Center, University of Amsterdam, the Department of Neurology of the Academic Medical Center, Actelion Pharmaceuticals, Genzyme, Shire and the Stop ALD Foundation.

When a thing is done, it’s done.Don’t look back.

Look forward to your next objective.

George C. Marshall

Voor Joo Yeon en Philip

Table of contents

Part I IntroductionChapter 1 X-linked adrenoleukodystrophy (X-ALD) clinical 11 presentation and guidelines for diagnosis, follow-up and management Orphanet Journal of Rare Diseases (2012) 7: 51

Part II In vitro studiesChapter 2 Cholesterol-deprivation increases mono-unsaturated 37 very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy Biochimica et Biophysica Acta (2008) 1781: 105 – 111 Chapter 3 Bezafibrate lowers very long-chain fatty acids in 51 X-linked adrenoleukodystrophy fibroblasts by inhibiting fatty acid elongation Journal of Inherited Metabolic Diseases, in press

Part III Clinical trialsChapter 4 Lovastatin in X-linked adrenoleukodystrophy 65 The New England Journal of Medicine (2010) 362: 276 – 277

Chapter 5 Bezafibrate for X-linked adrenoleukodystrophy 75 PLoS ONE (2012) 7:e41013

Part IV Extension of the phenotypeChapter 6 X-linked adrenomyeloneuropathy due to a novel missense 81 mutation in the ABCD1 start codon presenting as demyelinating neuropathy Journal of the Peripheral Nervous System (2011) 16:353 – 355

Chapter 7 The clinical, biochemical and genetic spectrum of X-linked 85 adrenoleukodystrophy in women: a cross-sectional cohort study manuscript in preparation

Part V Summary and general discussionChapter 8 Summary, general discussion, future research and 105 implications for clinical practice

Part VI Nederlandse samenvattingChapter 9 Van gen naar ziekte; X-gebonden adrenoleukodystrofie 109 Nederlands Tijdschrift voor Geneeskunde (2008) 152: 804 – 808

Chapter 10 Samenvatting in het Nederlands en beschouwing 119

Appendix Acknowledgements / Dankwoord 125 Curriculum vitae 131 List of publications 133

Introduction

1

11

Chapter 1X-linked adrenoleukodystrophy (X-ALD): clinical presentation

and guidelines for diagnosis, follow-up and management

Orphanet Journal of Rare Diseases (2012) 7(1):51

Marc Engelen1,3, Stephan Kemp2,3, Marianne de Visser1, Björn M. van Geel1,4, Ronald J.A. Wanders2, Patrick Aubourg5,6 and Bwee Tien Poll-The1,3

1Department of Neurology; 2Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases;3Department of Pediatric Neurology/Emma Children’s Hospital, Academic Medical Center, University of

Amsterdam, The Netherlands. 4Department of Neurology, Medical Center Alkmaar, Alkmaar, The Netherlands.5Assistance Publique des Hôpitaux de Paris, Department of Pediatric Neurology, Hôpital Kremlin-Bicêtre;

6INSERM UMR745- Université Paris-Descartes, Paris, France.

Chapter 1

1

12

Abstract

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. The disease is caused by mutations in the ABCD1 gene that encodes the peroxisomal membrane protein ALDP which is involved in the transmembrane transport of very long-chain fatty acids (VLCFA; >C22). A defect in ALDP results in elevated levels of VLCFA in plasma and tissues. The clinical spectrum in males with X-ALD ranges from isolated adrenocortical insufficiency and slowly progressive myelopathy to devastating cerebral demyelination. The majority of heterozygous females will develop symptoms by the age of 60 years. In individual patients the disease course remains unpredictable. This review focuses on the diagnosis and management of patients with X-ALD and provides a guideline for clinicians that encounter patients with this highly complex disorder.

Introduction

1

13

Definition

X-ALD is a metabolic disorder characterized by impaired peroxisomal beta-oxidation of very long-chain fatty acids (VLCFA; > C22), which is reduced to about 30% of control levels (Kemp et al 2004; Singh et al 1984). Consequently, there is an accumulation of VLCFA in plasma and all tissues, including the white matter of the brain, the spinal cord and adrenal cortex (Moser et al 1981). It is caused by mutations in the ABCD1 gene located on the X-chromosome (Mosser et al 1993). Mutations in this gene cause the absence or dysfunction of ALDP, a peroxisomal transmembrane protein that transports VLCFacyl-CoA esters from the cytosol into the peroxisome (van Roermund et al 2008; Ofman et al 2010).

Epidemiology

With an estimated birth incidence of 1 in 17,000 newborns (male and female) (Bezman and Moser, 1998), X-ALD is the most common peroxisomal disorder. It occurs in all regions of the world (Kemp et al 2001). Now that newborn screening has become technically feasible and may be implemented in some parts of the world (Hubbard et al 2009) the true prevalence might be even higher.

Clinical features of X-ALD

A short history In retrospect, the first cases of X-ALD were probably described in the late 19th century. In 1897 Heubner described a young boy with rapidly progressive neurologic deterioration consistent with X-ALD, classified as having “diffuse sclerosis” on autopsy (Heubner, 1897). This designation was used at the time for any disease of the white matter causing hardening of the tissue. Cases of “diffuse sclerosis” that resemble X-ALD were also described in 1899 by Ceni (Ceni, 1899) and in 1910 by Haberfield and Spieler (Haberfeld and Spieler, 1910). Shortly thereafter Schilder suggested that “diffuse sclerosis” was too vague and proposed a more accurate pathological classification of the leukodystrophies (Schilder, 1912; Schilder, 1913; Schilder, 1924). He described several cases with lesions in the cerebral white matter and perivascular inflammation that he named “encephalitis periaxialis diffusa”. The syndrome of rapidly progressive cerebral demyelination with inflammatory changes in the white matter on autopsy became known as “Schilder’s disease”. Many cases were described in the following fifty years (Hoefnagel et al 1962). Nevertheless, Siemerling and Creutzfeldt are often credited for having described the first case of X-ALD because they were the first to describe the association of acute cerebral demyelination with clinical and pathological signs of Addison’s disease (Siemerling and Creutzfeldt, 1923).

X-ALD has remained an enigmatic disease for most of the 20th century, although some suspected it might be a metabolic disorder affecting both the central nervous system and adrenal glands (Blaw, 1970; Forsyth et al 1971). Based on the presence of lipid inclusions in adrenal glands, testis, brain and Schwann cells, Schaumburg and Powers suggested the term adrenoleukodystrophy for the disorder and speculated it might be a lipid storage

Chapter 1

1

14

disorder (Schaumburg et al 1972). This hypothesis was confirmed when post-mortem biochemical analysis of brain, adrenal glands and serum of seven patients revealed the presence of cholesterol esters with high amounts of fatty acids longer than C22. These fatty acids were referred to as very long-chain fatty acids (VLCFA) (Igarashi et al 1976). X-linked adrenoleukodystrophy now became a distinct clinical entity with an associated biochemical marker that could be used to confirm the diagnosis in readily accessible materials like blood cells and plasma (Moser et al 1981).

Already in 1910 Addison’s disease associated with spastic paraplegia was described (von Neusser and Wiesel, 1911). Familial Addison disease with or without spastic paraplegia was usually considered to be a variant of hereditary spastic paraplegia (Harris-Jones and Nixon, 1955; Penman, 1960). In 1976, an X-linked adult onset progressive myelopathy that was often associated with Addison’s disease was reported (Budka et al 1976). A year later, five more cases were described and the term adrenomyeloneuropathy (AMN) was proposed because of the involvement of the adrenal cortex, spinal cord and peripheral nerves (Griffin et al 1977). With plasma VLCFA analysis as newly identified biomarker, AMN was soon found to be a form of X-ALD thus extending the phenotypic spectrum of X-ALD (Moser et al 1981). Now, we classify the symptomatology of X-ALD in children and adults in several phenotypes (Table 1). They will be discussed here in more detail.

Phenotypes in male X-ALD patients

Addison-onlyAdrenocortical insufficiency (or even an Addisonian crisis) can be the presenting symptom of X-ALD in boys and men, years or even decades before the onset of neurological symptoms. X-ALD is a frequent cause of Addison’s disease in boys and adult males (Hsieh and White, 2011; Laureti et al 1996a), in particular when circulating adrenocortical autoantibodies are absent (Laureti et al 1996b). A percentage as high as 35 has been reported (Laureti et al 1996a). However, this figure may be overestimated. Indeed, in a study of 40 Dutch men with adrenocortical insufficiency, no patients with X-ALD were identified (B.M. van Geel and J. Assies, unpublished data). Recognizing that Addison’s disease is due to X-ALD has important implications for genetic counseling but also management. It is therefore important to consider X-ALD in any boy or adult male presenting with Addison’s disease. Adrenocortical insufficiency initially affects the glucocorticoid function of the adrenal, but the mineralocorticoid function is ultimately deficient in approximately half of the X-ALD patients (Laureti et al 1996a).

Cerebral ALD (childhood, adolescent and adult)These phenotypes are the most rapidly progressive and devastating phenotypes of X-ALD. They most frequently present in childhood (childhood cerebral ALD; CCALD), however never before the age of 2.5 years (van Geel et al 1997; Moser et al 2001). The onset of CCALD is insidious, with deficits in cognitive abilities that involve visuospatial and visuomotor functions or attention and reasoning. In boys and adolescents it initially results in a decline of school performance. These early clinical symptoms are often misdiagnosed as attention deficit hyperactivity disorder and can delay the diagnosis of CCALD. As the disease progresses, more overt neurologic deficits become apparent, which include withdrawn or hyperactive

Introduction

1

15

Tabl

e 1:

The

X-A

LD p

heno

type

s%

Age

of

onse

tM

yelo

path

yW

hite

matt

er

lesio

ns o

n br

ain

MRI

Beha

vior

al

and

cogn

itive

di

sord

er

Perip

hera

l ne

urop

athy

Endo

crin

e di

sord

erPr

ogre

ssio

n

CCAL

D31

- 35

2.5

- 10

-Ex

tens

ive

+-

often

AD

rapi

dAd

olCA

LD4

- 710

- 21

Poss

ible

at

a pr

eclin

ical

st

age

Exte

nsiv

e+

rare

often

AD

rapi

d

ACAL

D2

- 5>

21+

or -

Exte

nsiv

e+

poss

ible

often

AD

rapi

dAM

N(n

o ce

rebr

al

dise

ase)

40 -

46>

18+

Wal

leria

n de

gene

ratio

n of

th

e co

rtico

spin

al

trac

ts in

bra

in-

stem

, pon

s and

in

tern

al c

apsu

les

-Se

nsor

y-m

otor

, mos

tly

axon

al, r

arel

y de

mye

linati

ng

often

AD

and

testi

cula

r in

suffi

cien

cy

slow

AMN

(c

ereb

ral

dise

ase)

20%

of A

MN

pa

tient

s ove

r a

perio

d of

10

year

s

> 18

+pa

rieto

-occ

ipita

l, fr

onta

l, or

in

volv

ing

the

cent

rum

sem

iova

le

+Se

nsor

y-m

otor

, m

ostly

axo

nal

often

AD

and

testi

cula

r in

suffi

cien

cy

rapi

d

Addi

son

only

Decr

easin

g w

ith a

ge>

2-

--

-AD

-

Wom

en w

ith

X-AL

DU

nkno

wn

how

man

y ar

e sy

mpt

omati

c

high

ly

varia

ble,

m

ostly

>

40

+Ve

ry ra

reW

alle

rian

dege

nera

tion

of

the

corti

cosp

inal

tr

acts

in

brai

nste

m, p

ons

and

inte

rnal

ca

psul

es is

less

co

mm

on th

an in

m

ales

with

AM

N

Very

rare

+/-

AD ra

re

(< 1

%)

slow

- = a

bsen

t; +

= pr

esen

t; CC

ALD

= ch

ildho

od c

ereb

ral A

LD; A

dolC

ALD

= ad

oles

cent

cer

ebra

l ALD

; ACA

LD =

adu

lt ce

rebr

al A

LD; A

MN

=

adre

nom

yelo

neur

opat

hy; A

D =

Addi

son-

dise

ase

Chapter 1

1

16

Figure 1: MRI of the brain in a case of childhood cerebral ALD showing characteristic extensive white matter changes in the parieto-occipital region and internal capsules on FLAIR sequences (A). This area is initially affected in about 80% of cases of cerebral ALD. The rim enhances after administration of gadolinium on T1 sequences (B). In about 20% of cases the site of initial involvement in cerebral ALD is the frontal white matter as shown on this FLAIR image of a different patient with cerebral ALD (C), with prominent rim enhancement after administration of gadolinium on a T1 weighted image (D).

behavior, apraxia, astereognosia, auditory impairment (“word deafness’’ reflecting impairment in acoustic analysis of word sounds), decreased visual acuity, hemiparesis or spastic tetraparesis, cerebellar ataxia and seizures. At this stage progression is extremely rapid and devastating. Affected boys can lose the ability to understand language and walk within a few weeks. Eventually, patients are bedridden, blind, unable to speak or respond,

Introduction

1

17

requiring full-time nursing care and feeding by nasogastric tube or gastrostomy. Usually death occurs two to four years after onset of symptoms, or - if well-cared for - patients may remain in this apparent vegetative state for several years (Moser et al 2001).

The rapid neurologic decline of CCALD is caused by a severe inflammatory demyelination process primarily affecting the cerebral hemispheres. Postmortem histopathological examination of brain tissue reveals extensive demyelination with perivascular infiltration of lymphocytes and macrophages that resembles, to some extent, the demyelinating lesions seen in multiple sclerosis (Powers et al 1992). In 80% of patients the initial demyelinating lesion is localized in the splenium of the corpus callosum and progresses to involve the adjacent parieto-occipital white matter (van der Knaap and Valk, 2005). Alternatively, the initial demyelinating lesions may occur in the genu of corpus callosum and then progress symmetrically or asymmetrically to the white matter of the frontal lobes (van der Knaap and Valk, 2005). The lesions may also initially involve the pyramidal tracts within the pons or the internal capsules and then extend into the white matter of the centrum semiovale. Brain MRI shows abnormal signal intensities (increased signal on T2 and FLAIR sequences, decreased signal on the T1 sequence) in the corpus callosum, parieto-occipital or frontal white matter or pyramidal tracts within the brainstem, pons and internal capsules (Figure 1). Atypical patterns of demyelination, for instance highly asymmetrical disease have been described (Wang et al., 2006). Initially, the demyelinating lesions do not show enhancement on T1 sequences after intravenous gadolinium administration. The presence of gadolinium enhancement of demyelinating lesions occurs usually in a second stage when the disease starts to progress rapidly, reflecting severe inflammation and disruption of the blood brain barrier. This transition to the rapidly progressive neuroinflammatory stage may occur very early, even when the demyelinating lesions are restricted to the corpus callosum or pyramidal tracts or later, once the demyelinating lesions have already significantly extended into the cerebral white matter. In the absence of biomarkers to predict this evolution, brain MRI remains the only tool to detect this evolution in an early stage. A scoring system to grade the demyelinating abnormalities on brain MRI has been developed by Loes (Loes et al., 1994). This score correlates well with neurologic symptoms when the demyelinating lesions involve the parieto-occipital white matter. However, lesions in the frontal white matter can produce severe symptoms (especially behavioral), while the Loes score is still low. Less frequently, cerebral demyelination as the presenting phenotype of X-ALD occurs in adolescence (AdolCALD) or adulthood (ACALD). The symptomatology in these patients strongly resembles CCALD, but the initial progression of symptoms usually is slower. In adults, the early cognitive decline is rarely recognized by their families and friends or at work. As the disease progresses, psychiatric disturbances mimicking schizophrenia or psychosis are not uncommon (Garside et al 1999). In those cases, the diagnosis of X-ALD is often delayed; particularly when no family history of X-ALD is present and when clinical symptoms of Addison’s disease are absent.

Approximately 10% of boys or adolescents with cerebral ALD may not develop the rapidly progressive neuroinflammatory stage of the disease. The same may occur in men with ACALD or in men with AMN who develop secondary cerebral demyelination (see below). This cerebral demyelinating form of X-ALD is often referred to as “chronic or arrested cerebral X-ALD”. The cerebral demyelinating process arrests spontaneously and the patient can remain stable for a decade or even longer. But even after a 10-15 year period of stability,

Chapter 1

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sudden onset of rapid neurologic deterioration may occur, reflecting a full progression to the neuroinflammatory stage of the disease.

Adrenomyeloneuropathy (AMN)Virtually all patients with X-ALD who reach adulthood develop AMN, usually in the 3rd and 4th decade. Initial symptoms are limited to the spinal cord and peripheral nerves. Patients develop gradually progressive spastic paraparesis, sensory ataxia with impaired vibration sense, sphincter dysfunction (mostly urinary), pain in the legs and impotence (Moser et al 2007). The clinical burden of peripheral nerve involvement is usually moderate and difficult to assess because of prominent spinal cord symptoms. If polyneuropathy is investigated electrophysiologically, an axonopathy is found in the majority of the patients (van Geel et al 1996). Rarely, the initial symptomatology may be that of a peripheral neuropathy, either demyelinating or axonal (Kararizou et al 2006; Engelen et al 2011).

Before the availability of MR imaging, AMN was often misdiagnosed as multiple sclerosis or hereditary spastic paraparesis (HSP). The pathological basis of AMN is a noninflammatory distal axonopathy that involves the long tracts of the spinal cord and, to a lesser extent, the peripheral nerves (Powers et al., 2000). This phenotype is in most cases slowly progressive, causing severe motor disability of the lower limbs over one or two decades but mild or no significant deficits in arms and hands. Brain MRI is normal or may show subtle abnormalities such as moderately increased signal intensities of the pyramidal tracts in brainstem, pons and internal capsules on FLAIR and T2 sequences that reflect likely Wallerian degeneration in patients with longstanding symptoms of AMN (Figure 2). These abnormalities are not considered manifestations of cerebral ALD. However, if the increased signal of the pyramidal tracts becomes more intense and extends beyond the internal capsules into the white matter of the centrum semiovale, this is considered as manifestations of cerebral ALD (Figure 3).

Figure 2: MRI of the brain in a patient with AMN showing increased signal in the pyramidal tracts on T2-weighed coronal (A) and axial (B) images indicative of Wallerian degeneration.

Introduction

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19

MRI of the spinal cord eventually shows non-specific atrophy, but no demyelination or gadolinium enhancement as seen in multiple sclerosis. In contrast, magnetization transfer or diffusion tensor-based imaging sequences show significant abnormalities of the spinal cord tracts (Dubey et al., 2005; Fatemi et al., 2005; Smith et al., 2009).

A retrospective study revealed that over a period of 10 years, approximately 20% of AMN patients developed additional cerebral demyelination (van Geel et al., 2001). After an initial progression demyelinating lesions can stabilize spontaneously leading to moderate cognitive deficits. However, once the cerebral demyelinating lesions have entered the active phase of neuroinflammation with gadolinium enhancement, the prognosis is as poor as in CCALD. The neurologic symptoms of AMN patients that develop cerebral ALD are identical to those observed in adults with adult cerebral ALD, with additional symptoms of the pre-existing myelopathy. Approximately 70% of AMN patients have adrenocortical insufficiency (Moser et al., 1991). An equal percentage of affected males have subclinical signs of testicular insufficiency (Assies et al., 1997). Clinical symptoms of testicular insufficiency are rare. Hair of patients with AMN is often thin and sparse; these patients often show balding at an early age (Figure 4). This typical scanty scalp hair was first described in 1955 (Harris-Jones and Nixon, 1955).

Women with X-ALDAs in many X-linked diseases, it was assumed that female carriers remain asymptomatic. However, many women develop AMN-like symptoms (O’Neill et al., 1984). Physical examination of a large group of female carriers attending family conferences in the United States revealed that more than 50% had some kind of abnormality on neurologic examination (Moser et al., 2001). An increasing number of symptomatic heterozygous women are identified as the first member of their family to be affected by X-ALD and the real incidence of AMN in heterozygous women is likely to be close to 65% by the age of 60 years (Engelen et al, manuscript in preparation (Chapter 7)). Onset of neurologic symptoms mainly occurs between the 4th and 5th decade and they are very similar to those observed in adult males with AMN. Sensory ataxia, fecal incontinence and pain in the legs are however often more prominent in symptomatic women with AMN. Cerebral involvement and adrenocortical insufficiency are rare, 2% and 1%, respectively (el-Deiry et al., 1997; Moser et al., 2001). So far, neither have been documented in the Netherlands and France. It is speculated that skewed X-inactivation in neuronal cells may contribute to the manifestation of neurologic symptoms in X-ALD carriers (Maier et al., 2002; Naidu et al., 1997). In a recent study the association of skewed X-inactivation and symptomatic status could not be confirmed (Salsano et al., 2012). However, this may be attributable to differences in mean age between the symptomatic and asymptomatic group in this study.

Asymptomatic and pre-symptomatic patientsA diagnosis of X-ALD must be followed by extended family screening together with a geneticist. This enables the detection of: 1) heterozygous women who can be offered prenatal or in some countries preimplantation diagnosis for future pregnancies, and 2) boys or adult males who are asymptomatic but at risk to develop cerebral demyelination or adrenocortical insufficiency (Kemp et al 2001). It is important to detect these complications as early as possible for treatment, as described in the section on ‘clinical management’.

Chapter 1

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20

Figure 3: MRI of the brain (T2 (A) and FLAIR (C) images; T1 with gadolinium (B, D)) of a patient with AMN who rapidly deteriorated clinically with new symptoms of cognitive decline. On MRI extensive white matter changes were seen in the parieto-occipital white matter and corpus callosum (A), but no enhancement of the lesion after administration of gadolinium (B). A follow-up MRI about 3 months later shows progression of the white matter lesion (C) and there is now faint enhancement of the rim of the lesion after gadolinium administration (D).

Introduction

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21

Evolution over timeX-ALD phenotypes are not static. Presymptomatic males are nearly all at risk to develop neurologic (cerebral ALD, AMN) or endocrinologic (Adisson’s disease) symptoms. Addison-only males can develop AMN or cerebral ALD and AMN males can develop cerebral demyelination. It is estimated that over a period of 10 years about 20% of patients with AMN will progress to a cerebral phenotype (van Geel et al 2001). Presymptomatic women with X-ALD are mostly at risk to develop AMN. Progression of X-ALD in a specific individual can not be predicted.

X-ALD is characterized by the absence of a genotype-phenotype correlation. In spite of identical ABCD1 gene mutations, patients can have markedly divergent neurological and neuropathologic characteristics (Berger et al 1994; Kemp et al 1994). There is ample evidence that other genetic and/or environmental factors may influence the clinical presentation of X-ALD. Segregation analysis suggests that the phenotypic variability is due to at least one autosomal modifier gene (Maestri and Beaty, 1992; Smith et al 1991). Head trauma may be an environmental factor triggering the onset of cerebral ALD (Raymond et al 2010). Other brain lesions, like a stroke, may also trigger cerebral demyelination in patients with X-ALD.

Etiology and Pathophysiology

X-ALD is caused by mutations in the ABCD1 gene located on the X-chromosome. So far, 600 different mutations have been identified (see http://www.x-ald.nl). ABCD1 encodes ALDP,

Figure 4: Thin and scanty scalp hair in a man with X-ALD (AMN phenotype).

Chapter 1

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22

a peroxisomal transmembrane protein involved in the transport of VLCFA-CoA esters from the cytosol into the peroxisome (van Roermund et al 2008; Ofman et al 2010; Kemp et al 2011). ALDP deficiency leads to impaired VLCFA beta-oxidation (about 30% of normal) and the accumulation of VLCFA-CoA esters in cells. The VLCFacyl-CoA esters in the cytosol are incorporated into various lipid fractions and are also substrate for further elongation to even longer fatty acids (Kemp et al 2005; Ofman et al 2010). ELOVL1 (elongation of very-long-chain-fatty acids) is the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X-ALD fibroblasts, but increased synthesis of VLCFA is due to increased substrate availability (Ofman et al 2010).

Various in vitro experiments have demonstrated that VLCFA accumulation is toxic. VLCFA are extremely hydrophobic and their rate of desorption from biological membranes is about 10,000 times slower than that of long-chain fatty acids causing disruptive effects on the structure, stability and function of cell membranes (Ho et al 1995; Knazek et al 1983). Excess of VLCFA in cultured cells decreases the ACTH-stimulated cortisol release by human adrenocortical cells (Whitcomb et al 1988) and causes cell death in astrocytes and oligodendrocytes (Hein et al., 2008). In vivo, VLCFA cause oxidative stress and oxidative damage to proteins (Fourcade et al., 2008; Powers et al., 2005) microglial activation and apoptosis (Eichler et al., 2008). VLCFA-induced oxidative stress may contribute to axonal damage in the spinal cord of Abcd1 knock-out mice that develop an AMN-like phenotype (Fourcade et al., 2008; Pujol et al., 2002). In addition, a dysregulation of oxidative phosphorylation, adipocytokine and insulin signaling pathways, and protein synthesis was recently shown in the spinal cord of Abcd1 knock-out mice (Schluter et al., 2011).

The neuropathological hallmark of AMN is an axonopathy with microgliosis but without significant myelin changes. It is therefore likely that the primary consequence of VLCFA accumulation impairs the capacity of oligodendrocytes and Schwann cells to sustain axonal integrity, resulting in axonal damage.

The VLCFA homeostasis in X-ALD is disturbed (Kemp et al., 2005). This may contribute to the destabilization of the myelin sheath and impair the function of astrocytes and microglia which play an important role in myelin integrity (Eichler et al., 2008; Hein et al., 2008). Not all males with X-ALD develop cerebral ALD corroborating the notion that additional triggers, genetic, epigenetic and/or environmental, modify this process.

In the absence of a relevant X-ALD mouse model that develops cerebral demyelination with neuroinflammation, the pathogenic processes that result in cerebral demyelination and subsequently severe neuroinflammation remains therefore poorly understood.

Biochemical and Molecular Diagnosis

Newborn screening is now technically feasible (Hubbard et al 2009). It is based on the measurement of C26:0 lysophosphatidylcholine (26:0-lyso-PC) in dried blood spots. It will lead to identification of pre-symptomatic patients with X-ALD. Whether this screening is implemented is a matter of national policy and dependent on ethical considerations.

Introduction

1

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If X-ALD is suspected in a male with neurological symptoms (with or without typical brain MRI abnormalities) or Addison’s disease, demonstration that VLCFA are elevated in plasma confirms the diagnosis. For women with X-ALD, the diagnostic test of choice is mutation analysis of the ABCD1 gene, because 15% of women with X-ALD have normal plasma VLCFA levels (Moser et al 2007). Family screening follows the same recommendations.

The ABCD1 gene is the single causative gene for X-ALD (Mosser et al 1993). It is an X-linked inherited disorder. Therefore all daughters of an affected male are obligate carriers whereas his sons can never be affected. When a woman carries the gene for X-ALD, there is a 50% probability for each pregnancy that the gene is transmitted to a son or daughter. The frequency of de novo mutations in the index case is estimated to be around 4% (Wang et al 2011) which indicates that the ABCD1 mutation occured in the germ line. There is evidence of gonadal or gonosomal mosaicism in less than 1% of patients which means an increased risk of an additional affected offspring.

Table 2: Differential diagnosis of chronic myelopathy with normal MRI

Deficiencies: Vitamin B12, folic acid, copperHereditary spastic paraparesis with amyotrophy

Infections (e.g., HTLV-1, HIV)Primary lateral sclerosisRadiation myelopathyCerebrotendinous xanthomatosisMetachromatic leukodystrophyKrabbe disease

Differential diagnosis

SymptomsIn boys and men presenting with Addison’s disease, including those who have only a glucocorticoid deficiency, and in whom there are no detectable steroid-21-hydroxylase or other organ-specific antibodies, X-ALD should be considered and determination of plasma VLCFA levels must be performed (Laureti et al 1998; Mukherjee et al 2006). Young boys and adult males presenting with cognitive and neurological symptoms with (usually enhancing) white matter lesions on brain MRI should be tested for X-ALD.

In adult men, the most common presenting symptom of X-ALD is a chronic myelopathy. In the past, AMN was often misdiagnosed as primary progressive multiple sclerosis or hereditary spastic paraparesis. After ruling out a compressive myelopathy by MRI of the spinal cord and other common causes of chronic myelopathy (some possible diagnoses for chronic myelopathy with a (near) normal MRI are summarized in Table 2) (Wong et al 2008), X-ALD should be considered. A clinical clue to the diagnosis can be the presence of adrenocortical insufficiency and early baldness. However, even in the absence of clinical signs of adrenocortical insufficiency AMN should be considered.

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A category that remains underdiagnosed relatively often are women with AMN. Physicians familiar with X-ALD are aware that it is not uncommon that women with X-ALD develop a myelopathy (Moser et al 1991). However, most neurology textbooks do not list X-ALD in the differential diagnosis for chronic myelopathy in women. The diagnosis can only be excluded by ABCD1 mutation analysis. As mentioned before, there is an increasing number of X-ALD families in which the index case is a woman with clinical symptoms of AMN.

Increased VLCFAIncreased plasma VLCFA are not pathognomonic for X-ALD or might even be false positive, because of hemolysis of the sample or dietary causes, for example a ketogenic diet (Theda et al 1993). Alternatively, some dietary products like rapeseed oil or mustard seed oil that are rich in erucic acid (C22:1) can result in the lowering of C26:0 therefore causing a false negative result (Ann Moser, personal communication). If metabolic screening reveals increased VLCFA, the next step is to confirm the diagnosis by performing ABCD1 mutation analysis. If this is negative, it is important to consider other peroxisomal disorders, such as 1) peroxisomal biogenesis disorders with late onset of symptoms (Regal et al 2010; Sevin et al 2011) (Ebberink et al 2012), and 2) peroxisomal acyl-CoA oxidase 1 (ACOX1) or D-bifunctional enzyme (DBP) deficiency with late onset of symptoms. This requires further testing for bile acid intermediates, phytanic and pristanic acids in plasma, and plasmalogens in erythrocytes (Wanders et al 2010). A further metabolic screening of peroxisomal dysfunction must also be performed on skin fibroblasts, at least by performing catalase immunofluorescence after culturing cells at 37 °C, but also at 40 °C (Wanders and Waterham, 2006).

White matter changes on MRIIn males with confluent white matter changes, X-ALD should be considered, especially when there is increased signal intensity on T2-weighed and FLAIR sequences in the parieto-occipital region and the splenium of the corpus callosum (van der Knaap and Valk, 2005). Rim enhancement of demyelinating lesions is usually observed when boys or adult males present with overt neurological symptoms from cerebral ALD. Therefore, intravenous administration of gadolinium is advised if X-ALD is considered. However, in about 20% of males with X-ALD, the white matter changes occur predominantly in the genu of corpus callosum and frontal lobes, or involve the pyramidal tracts with extension in the white matter of the centrum semiovale (van der Knaap and Valk, 2005). The brain MRI pattern of cerebral X-ALD is usually readily recognized by neuroradiologists. A helpful algorithm for diagnostic work-up of patients with white matter changes on brain MRI is suggested by Schiffmann and Van der Knaap (Schiffmann and van der Knaap, 2009).

There are other peroxisomal disorders that present with MRI abnormalities resembling X-ALD, in particular peroxisomal biogenesis disorders and ACOX1 or DBP deficiency with late onset of symptom (Barth et al 2001). Although the radiologic findings might suggest X-ALD, the clinical presentation is very different and therefore these disorders are easily differentiated.

Genetic counseling and prenatal diagnosis

Genetic counseling must be offered to the parents of affected boys, adult males and women

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with X-ALD and their family to detect: 1) carriers who can be offered prenatal diagnosis, and 2) asymptomatic or pre-symptomatic men or women who can benefit from therapeutic interventions. Regular follow-up in presymptomatic males can prevent serious morbidity and mortality.

ABCD1 mutational analysis can be performed either on a fresh chorionic villus sample at 11-13 weeks of pregnancy or on amniotic cells obtained from amniotic fluid after centrifugation at 15-18 weeks of gestation (Rahil et al 2002). In some countries, pre-implantation genetic diagnosis is available. If the fetus is a female, there is no consensus with respect to prenatal diagnosis and termination of pregnancy, due to the highly variable expression of disease in women with X-ALD. Cases will be evaluated on an individual basis.

Clinical Management

The flowchart in Figure 5 summarizes the recommendations for follow-up of boys and men with X-ALD.

Boys and adult males with X-ALDFollow-up in boys and men with X-ALD is important for two reasons: 1) early detection of adrenocortical insufficiency and 2) early detection of cerebral ALD to propose allogeneic hematopoietic stem cell transplantation (HCT) if a HLA-matched donor or cord blood is available. Despite significant mortality risk, allogeneic HCT remains the only therapeutic intervention that can arrest the progression of cerebral demyelination in X-ALD, provided the procedure is performed very early, i.e., when affected boys or men have no or minor symptoms due to cerebral demyelinating disease (Shapiro et al 2000; Miller et al 2011).

In the future, transplantation of autologous hematopoietic stem cells that have been genetically corrected with a lentiviral vector before re-infusion might become an alternative to autologous HCT, once the very encouraging results obtained in the first two treated patients will have been extended to a larger number of patients with cerebral X-ALD (Cartier et al 2009).

If boys or men do not have Addison’s disease it is recommended that they are evaluated yearly by an endocrinologist for adrenocortical dysfunction by measuring the plasma ACTH levels and performing an ACTH stimulation test (Mukherjee et al 2006). Steroid replacement therapy can then be initiated if necessary.

Boys without neurological deficits should be monitored closely for radiological signs of cerebral ALD. CCALD has not been reported before the age of 2.5 years (Kemp et al 2001). We recommend an MRI of the brain every 6 months in boys aged 3 to 12 years old to screen for early signs of CCALD. If symptoms occur suggestive of cerebral ALD (for instance declining school performance) the MRI should be performed at the earliest available opportunity, but it is our experience that the detection of brain MRI abnormalities precedes any detectable cognitive dysfunction by at least 6 months to 1 year. After the age of 12 years, the incidence of CCALD decreases, but an MRI scan must be performed yearly or earlier if new symptoms occur. It is important to detect cerebral ALD as early as possible, preferably in the

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asymptomatic stage with only moderate radiological abnormalities to discuss the possibility to perform allogeneic HCT. Accordingly, if a brain MRI shows abnormalities, even very limited such as an increased signal intensity on T2 or FLAIR sequences in the splenium or genu of the corpus callosum, brain MRI must be repeated within 3 months to evaluate disease progression and in particular to identify the presence of gadolinium rim enhancement of lesions. Because the disease can be very rapidly progressive, it is strongly advised to discuss the possibility of allogeneic HCT as soon as brain MRI abnormalities typical of cerebral ALD are detected. After a successful transplant, the lesions on MRI stabilize and even regress. Treatment results are better the earlier treatment is started (Miller et al 2011).

For adult men with or without signs of AMN, we advise evaluation by a neurologist yearly or bi-annually to screen for symptoms of AMN and to administer symptomatic treatment if necessary (for instance, medication against spasticity). Referral to a rehabilitation physician and urologist will often become necessary.

Adult men can develop cerebral ALD and in our centers, we offer an MRI of the brain every single year (Moser et al 2001; van Geel et al 2001). There is no proven treatment for cerebral ALD in adults. It seems likely that allogeneic HCT is also effective in adults with early stage cerebral ALD, but there are no published studies or cases describing this treatment. We tend to consider allogeneic HCT in an adult patient with early stage cerebral ALD, after carefully counseling the patient about the lack of evidence for the treatment and the risk of the procedure which is significantly higher than in boys. Whereas the onset of demyelinating lesions involving the corpus callosum and adjacent parieto-occipital or frontal white matter leaves no doubt about the onset of cerebral ALD, the situation is different when there are only slightly increased signal abnormalities in the pyramidal tracts of AMN patients that gradually become more intense and involve the white matter of the centrum semiovale. This can herald the onset of cerebral ALD, but can also reflect Wallerian degeneration in severe AMN.

For AMN there is no effective disease modifying therapy available yet. Although Lorenzo’s oil (LO) had great promise, several open-label trials have shown that the disease progresses even when plasma VLCFA are normalized by LO treatment (Aubourg et al 1993; van Geel et al 1999). A large randomized placebo-controlled clinical trial was designed to provide a definitive answer, but was unfortunately aborted before completion by the safety monitoring board because of presumed side effects of the placebo treatment. There is also a retrospective study suggesting that if presymptomatic boys are started on LO, it may delay the onset of neurological symptoms (Moser et al 2005). We consider the scientific evidence to support the efficacy of LO weak, and do not offer this treatment to our patients. Regular follow-up in AMN remains important, however, mainly to provide symptomatic treatment.

Lovastatin also lowered plasma VLCFA (Singh et al 1998), but a placebo-controlled trial revealed that lovastatin did not have an effect on the C26:0 levels in peripheral blood lymphocytes and erythrocytes nor on the VLCFA content of the low-density lipoprotein fraction (Engelen et al 2010).

More research and new treatments strategies are desperately needed, especially for those affected by AMN, which is relentlessly progressive and causes severe disability. Antioxidants reduce markers for oxidative stress and axonal degeneration in the spinal cord of Abcd1

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knockout mice (Lopez-Erauskin et al 2011). Based on this observation a clinical trial with anti-oxidants in X-ALD is ongoing in Spain.

Women with X-ALDWomen with X-ALD should be evaluated for the development of neurologic symptoms. Since women with X-ALD very rarely develop adrenocortical insufficiency or cerebral involvement, periodic evaluation of adrenocortical function and brain MRI is not mandatory (Moser et al 2007). Greater awareness among physicians that women can develop neurologic symptoms is important for counseling but also to prevent unnecessary diagnostic tests and erroneous diagnosis. We know of cases of women with X-ALD who underwent cervical laminectomy for presumed cervical spondylogenic myelopathy. For symptomatic women with X-ALD, we advise (as for men with AMN) a yearly evaluation by a neurologist to discuss the indication of rehabilitation, the referral to an urologist and treatment of spasticity and neuropathic pain.

Figure 5: Flowchart describing the outpatient management of X-ALD.*If there is no gadolinium enhancement present, consider arrested cerebral ALD and repeat the MRI in 3 months.

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Prognosis

It is debatable whether men with X-ALD can remain really asymptomatic for life. It seems probable that there is only presymptomatic X-ALD in men. Although there is no large prospective cohort study to accurately determine the natural history of the disease, several general observations can be made. It is likely that all patients with X-ALD if they survive into adulthood eventually develop myelopathy, i.e. AMN. Usually, symptoms and signs occur from the 3rd decade of life, but much earlier, or much later, is possible. The severity and progression cannot be predicted for individual patients. There is marked variability ranging from men with X-ALD that are wheelchair bound by the age of 25 and others who are able to walk with a cane while in their seventies. This is important when counseling patients: symptoms will occur, but only time will tell how severely affected an individual will be.

Not every boy or man with X-ALD will develop cerebral ALD. About 35-40% of boys with an ABCD1 mutation will develop CCALD before reaching adulthood. It can still not be predicted who will develop this devastating manifestation of the disease. Previously it was believed that after reaching adulthood this complication was very rare. However, it is now well established that at least 20% of adult males with the AMN phenotype will develop cerebral demyelination later in life (van Geel et al 2001). If this occurs, these patients have the same poor prognosis as boys with inflammatory cerebral ALD.

It is well documented that women develop symptoms that resemble AMN (Moser et al 1991), but there is no prospective study with adequate numbers to really estimate what percentage of women become symptomatic. We recently completed a large prospective cohort study to describe the symptomatology of X-ALD in women (Engelen et al, manuscript in preparation (Chapter 7)).

Unresolved questions and future research

For the majority of patients with X-ALD there is currently no curative or preventive treatment. However, several promising new approaches will hopefully come to fruition in the future. For example, it has been demonstrated in X-ALD cells that small interfering RNA (siRNA)-mediated inhibition of ELOVL1 reduces VLCFA synthesis and levels (Ofman et al., 2010). Compounds that can inhibit ELOVL1 are therefore interesting candidates for new preventive treatments. We demonstrated that bezafibrate which is both an effective, safe and well-tolerated compound lowers the levels of VLCFA in X-ALD cells by inhibiting VLCFA synthesis (Engelen et al 2012). A clinical trial to evaluate its in vivo efficacy in X-ALD patients is ongoing.

As mentioned before, it is likely that several modifier genes play a role in the onset and severity of AMN, the onset of cerebral ALD and the progression to the rapidly progressive neuroinflammatory stage. However, so far no major modifier genes have been identified that could lead to the development of a genetic test to predict phenotype and disease evolution. The eventual phenotype of X-ALD in an individual will most likely be determined by the combination of several epigenetic and environmental modifiers, most of which have not been identified. Much research is currently focused on identifying these modifiers to

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better predict clinical outcome in individual patients.

An important issue is the systematic characterization of disease manifestations in women with X-ALD. Clinical research has always focused on boys and men and women were often considered “carriers” with no or minimal symptoms. For adequate counseling this information is important and should be available.

For clinicians caring for patients with AMN it is well known that cerebral demyelination can occur. It is important to systematically study if an allogeneic HCT can be successful in these cases and even arrest the progression of myelopathy.

Conclusions

Pediatricians, endocrinologists, neurologists and psychiatrists may encounter X-ALD, which is a relatively common metabolic disorder, in their practice. The disorder is associated with severe morbidity and mortality in the majority of affected patients. Recognition of X-ALD is highly important, since in some cases treatment is available, such as allogeneic HCT in the early stage of CCALD and endocrine replacement therapy for adrenocortical insufficiency. Furthermore, prenatal testing to prevent unnecessary new cases of this devastating disease is available.

List of abbreviations

ACALD (Adult cerebral adrenoleukodystrophy), ACOX1 (acyl-CoA oxidase 1), ACTH (adrenocorticotropic hormone), AdolCALD (Adolescent cerebral adrenoleukodystrophy), ALDP (adrenoleukodystrophy protein), AMN (adrenomyeloneuropathy), CCALD (childhood cerebral adrenoleukodystrophy), DBP (D-bifunctional protein), FLAIR (fluid attenuated inversion recovery), HCT (hematopoietic stem cell transplantation), LO (Lorenzo’s oil), MRI (magnetic resonance imaging), VLCFA (very long-chain fatty acids), X-ALD (X-linked adrenoleukodystrophy).

Acknowledgements

This work was supported by a grant from the Netherlands Organization for Scientific Research (91786328 to SK).

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Chapter 2Cholesterol-deprivation increases mono-unsaturated very long-

chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy

Biochimica et Biophysica Acta (2008) 1781(3): 105 – 111

Marc Engelen1,2, Rob Ofman1, Petra A.W. Mooijer1, Bwee Tien Poll – The2, Ronald J.A. Wanders1 and Stephan Kemp1,2

1Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases; 2Department of Pediatric Neurology/Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, The Netherlands

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Abstract

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). X-ALD is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in reduced beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1-14C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.

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Introduction

X-linked adrenoleukodystrophy (X-ALD; MIM300100) is a severe metabolic disorder characterized by impaired peroxisomal beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) and elevated VLCFA levels in plasma and tissues of patients (Moser et al 1981). The clinical presentation of X-ALD is highly variable, even among affected members of the same family (Moser et al 2001). In the most severe phenotype, affected patients develop cerebral demyelination with inflammation which is rapidly progressive and usually fatal within 2 years after onset. This phenotype presents most frequently in early childhood (childhood cerebral ALD; CCALD), but can also occur in adolescence or adulthood. The adrenomyeloneuropathy (AMN) phenotype usually develops between the 3rd and 4th decade of life and is gradually progressive. The main symptoms are spastic paraparesis and incontinence, caused by progressive myelopathy and peripheral neuropathy. Adrenocortical and testicular insufficiency can occur in isolation (Addison-only), but also in combination with any of the other phenotypes.

X-ALD is caused by mutations in the ABCD1 gene (MIM 300371) (Mosser et al 1993), that encodes a peroxisomal transmembrane protein named the adrenoleukodystrophy protein (ALDP), which is classified as a member of the ATP-binding cassette subfamily D of transporters (Mosser et al 1993; Mosser et al 1994). Although the precise function of ALDP is not clear, absence or dysfunction of ALDP causes impaired peroxisomal beta-oxidation of VLCFA in human skin fibroblasts (Singh et al 1981).

Two other peroxisomal transmembrane proteins belonging to the class of ATP-binding cassette transporters have been identified in mammals. These include ALD-related protein (ALDRP) (Lombard-Platet et al 1996) and PMP70 (Kamijo et al 1990). It has been well established that these proteins show functional redundancy, at least partially, and have overlapping substrate specificities. Over-expression of either PMP70 or ALDRP in X-ALD fibroblasts corrects VLCFA beta-oxidation (Kemp et al 1998; Netik et al 1999). Furthermore, in the Abcd1 knockout mouse in which ALDRP was overexpressed the biochemical abnormalities normalized and the neurological AMN-like phenotype was reversed (Pujol et al 2004).

This prompted research with the aim to identify ways to induce the expression of ALDRP, encoded by the ABCD2 gene, which is normally expressed at low levels in most tissues (Berger et al 1999). It has been shown that 4-phenylbutyrate (4-PBA) can induce ALDRP expression in human and mouse primary fibroblasts (Kemp et al 1998). Unfortunately, the dosage needed to obtain a biological effect in humans makes it unpractical for clinical application (Kemp et al 1998; Moser et al 2001).

In 1998 Singh et al (Singh et al 1998a) reported that beta-oxidation of VLCFA was increased markedly by incubating fibroblasts from X-ALD patients with lovastatin, a cholesterol lowering drug that belongs to the class of HMG-CoA-reductase inhibitors. Later it was shown that culturing fibroblasts from X-ALD patients in medium with lipoprotein-deficient fetal calf serum resulted in a reduction in 26:0 levels and increased ABCD2 expression (Weinhofer et al 2002). Since cholesterol depletion leads to activation of the SREBP-pathway (Brown and Goldstein, 1997), a likely mechanism for the observed effects on 26:0 metabolism would be the increased expression of ALDRP. Subsequent studies showed that the promoter of the

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ABCD2 gene indeed contains a functional sterol regulatory element (SRE) (Weinhofer et al 2002). Based on these observations patients with X-ALD have been treated with lovastatin and a low-fat diet in a small open-label study. An approximately 40% reduction in plasma 26:0 levels was observed in 7 out of 12 patients (Singh et al 1998b; Pai et al 2000). In another study in which patients were treated with simvastatin but without dietary fat restriction, no reduction in plasma VLCFA was observed (Verrips et al 2000). Still, based on the aforementioned observations many patients with X-ALD are being treated with lovastatin. Unfortunately, further studies showed that in Abcd1 knockout mice lovastatin (Yamada et al 2000) and simvastatin (Cartier et al 2000) were unable to reduce VLCFA. On the contrary, even higher levels of VLCFA in tissues, notably the brain, were found. With respect to lovastatin treated patients no data is available on the effect on VLCFA levels in tissues other than plasma and erythrocytes. There are, however, indications that interspecies differences are important. Mouse fibroblasts cultured in lipoprotein-deficient culture medium showed a 40% reduction in 26:0 levels compared to a 80% reduction in human fibroblasts (Weinhofer et al 2005). Therefore, the data from mice experiments cannot be extrapolated to humans directly.

The aim of the experiments described in this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD using skin fibroblasts from patients with X-ALD and controls.

Materials and Methods

Fatty acid substratesDeuterium-labeled free fatty acids 16,16,16-D3-16:0 and 17,17,17,18,18-D5-18:0 were purchased from CDN isotopes (Pointe-Claire, Canada) and 12.5 mM stock solutions in DMSO were prepared. Prior to usage stock solutions were vortex mixed and diluted in Ham F-10 tissue culture medium to their final concentration. All chemicals used were of analytical grade.

Cell lines and cell cultureHuman primary skin fibroblasts cell lines were obtained from patients with X-ALD through the neurology outpatient clinic of the Academic Medical Centre. From each patient written informed consent was obtained. Five cell lines from X-ALD patients were used. The clinical phenotype of the patient, the ABCD1 mutation and the effect of the mutation on ALDP expression by means of immunofluorescence were: ALD005 (AMN, p.Ser290X, absent); ALD009 (AMN, p.Glu471fs, absent); ALD011 (Addison-only, p.Arg464X, absent); ALD013 (AMN, p.Met1Val, absent) and ALD014 (AMN, p.Leu220Pro, reduced). Fibroblasts from anonymous controls were obtained from the laboratory cell bank. Cells were grown in HAM F-10 tissue culture medium supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 U/mL) and amphotericin B (250 ng/mL) or in Dulbecco’s Modified Eagle Medium (DMEM), additionally supplemented with HEPES (25 mmol/L). All cell lines were used with passage numbers below 20. For cholesterol deprivation 10% lipoprotein-deficient fetal calf serum, containing less than 3 µmol/L of cholesterol, was used (Bodinco B.V., Alkmaar, The Netherlands). For comparison, regular fetal calf serum contains approximately

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1.5 mmol/L of cholesterol. For cholesterol loading, the lipoprotein-deficient fetal calf serum was supplemented with 10 µg cholesterol (dissolved in EtOH) and 1 µg 25-OH-cholesterol (dissolved in EtOH) per mL serum. Culture media were refreshed every 5 days.

Fatty acid synthesis and fatty acid desaturationCells were seeded in T75 tissue culture flasks at approximately 50% confluency and the medium was refreshed the next day. VLCFA synthesis was measured by incubating the cells for 10 days in the presence of increasing concentrations of D3-16:0. Culture media and D3-16:0 were refreshed after 5 days.For the measurement of long chain fatty acid desaturation activity, cells were incubated for 24 hours in the presence of 100 µM D5-18:0. After incubation, cells were harvested with trypsin, washed twice with phosphate-buffered saline (PBS), once with 0.9% NaCl and taken up in 200 µL deionized water. After sonication protein concentration was determined using BCA (Smith et al 1985). Total cellular fatty acids were analyzed using the electrospray ionization mass spectrometry (ESI-MS) method described previously (Valianpour et al 2003; Kemp et al 2004).

Measurement of VLCFA beta-oxidationCells were cultured in the different media for 7 days and the effect on the peroxisomal beta-oxidation activity was measured using [1-14C]-26:0 as described previously (Wanders et al 1995). Measurements were performed in triplicate for each cell line. As an internal control, mitochondrial beta-oxidation was determined using [1-14C]-16:0 (Wanders et al 1995).

Quantitative RT-PCR analysis of SCD mRNA levelsThe expression levels of SCD mRNA were related to the expression levels of GAPDH (Glyceraldehyde-phosphate-dehydrogenase) mRNA using the LightCycler system (Roche, Mannheim, Germany). Total RNA was isolated from primary skin fibroblasts growing in log phase with TRIzol® reagent (Invitrogen, Carlsbad, USA) and cDNA was synthesized using the first-strand cDNA synthesis kit (Roche, Mannheim, Germany). Quantitative real-time PCR analysis of SCD in skin fibroblasts was performed using the LightCycler FastStart DNA Master SYBR green I kit (Roche, Mannheim, Germany). The following primers for SCD were used: forward 5’-CACCCAGCTGTCAAAGAGAA-3’ and reverse 5’-TCACCCACAGCTCCAAGT-3’. For GAPDH the following primers were used: forward 5’-ACCACCATGGAGAAGGCTGC-3’ and reverse 5’-CTCAGTGCCCAGGATGC-3’. The specificity of amplification was confirmed by agarose gel electrophoresis. Data were analyzed using LightCycler software version 3.5 (Roche) and the LinReg PCR program version 7.5 for analysis of RT-PCR data (Ramakers et al 2003).

Results

Effect of cholesterol deprivation on endogenous VLCFACulturing fibroblasts from X-ALD patients and control subjects in lipoprotein-deficient medium resulted in a 0.29 nmol/mg protein (35%) reduction of 26:0 levels (Fig. 1A and B). Interestingly, these conditions caused a 1.03 nmol/mg protein (70%) increase in the 26:1 levels in fibroblasts from X-ALD patients, but not in control fibroblasts (Fig. 1C and D). This

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experiment was repeated with cells cultured in DMEM instead of HAM F-10 to compare our results with the data from Weinhofer et al who did not investigate the effect on 26:1 levels (Weinhofer et al 2002). The data in Fig. 1B and Fig. 2A show a comparable effect on 26:0 levels: deprivation of cholesterol resulted in a 40% reduction in 26:0 levels in X-ALD cells independent of the type of medium used. Under both culture conditions there was a significant increase in 26:1 (Fig. 1D and 2B). Remarkably, both the 26:0 as well as the 26:1 levels were higher when cells were cultured in DMEM. Compared to Ham F10, 26:0 levels were approximately 1.5-fold higher (compare Fig. 1B with Fig. 2A), and 26:1 levels were about 4-fold higher (compare Fig. 1D with 2B). This could be related to differences in composition between DMEM and Ham F10 culture medium. For example there are clear differences in the concentration of D-glucose (25 mmol/L versus 6 mmol/L) and L-glutamine (1 mmol/L versus 4 mmol/L).

Effect of cholesterol deprivation on VLCFA synthesis from D3-16:0 and 26:0 beta-oxidation To gain more insight into the mechanism by which cholesterol deprivation influences the levels of saturated and mono-unsaturated VLCFA, we measured the effect of cholesterol deprivation on the synthesis of both D3-26:0 and D3-26:1 from D3-16:0. Furthermore, VLCFA beta-oxidation was measured. To this end, X-ALD fibroblasts were cultured in the presence of D3-16:0 for 10 days and the amount of D3-VLCFA was determined. Cholesterol deprivation did not affect net synthesis of saturated VLCFA from D3-16:0 (Fig. 3A). However, the synthesis

Figure 1: Endogenous levels of 26:0 (A and B) and 26:1 (C and D) in fibroblasts from controls (A and C) and X-ALD patients (B and D). Fibroblasts were cultured for 10 days in HAM F10 medium sup-plemented either with 10% fetal calf serum (open bars) or with 10% lipoprotein-deficient fetal calf serum (black bars). Fatty acid levels are expressed as nmol/mg protein. Statistical significance was determined with Student’s unpaired t-test. Error bars represent the standard deviation: (*) p < 0.05; (**) p < 0.01 and (***) p < 0.001.

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of D3-26:1 from D3-16:0 was found to be increased 7-fold under low-cholesterol conditions (Fig. 3B). When X-ALD fibroblasts were grown in lipoprotein-deficient medium, the rate of [1-14C]-26:0 beta-oxidation was not affected (Fig. 4). Furthermore, the beta-oxidation of [1-14C]-16:0 was not different between X-ALD fibroblasts and controls and between the different culture conditions (data not shown).

Effect of cholesterol deprivation on SCD expression and desaturation activity In mammalian cells, the synthesis of mono-unsaturated fatty acids from saturated fatty acids is catalyzed by stearoyl-CoA-desaturase (SCD), an enzyme that introduces a cis -double bond in the ω-9 position (Ntambi and Miyazaki, 2003). The main substrates for this enzyme are palmitoyl-CoA (16:0-CoA) and stearoyl-CoA (18:0-CoA). We investigated whether the increase in mono-unsaturated VLCFA could be explained by an increased expression and activity of SCD. To this end, we performed qPCR analysis using control and X-ALD cell lines cultured on standard HAM F10 medium, lipoprotein-deficient medium, or lipoprotein-deficient medium supplemented with free cholesterol and 25-OH-cholesterol. When

Figure 2: Endogenous 26:0 (A) and 26:1 levels (B) in fibroblasts from X-ALD patients cultured for 10 days in DMEM supplemented either with 10% fetal calf serum (open bars) or with 10% lipoprotein-deficient fetal calf serum (black bars). Fatty acid levels are expressed as nmol/mg protein. Statistical significance was determined with Student’s unpaired t-test. Error bars represent the standard devia-tion: (*) p < 0.05; (**) p < 0.01.

Figure 3: Net synthesis of D3-26:0 (A) and D3-26:1 (B) from D3-16:0 in fibroblasts from X-ALD patients. Fibroblasts were incubated with increasing concentrations D3-16:0 for 10 days in HAM F10 medium supplemented either with 10% fetal calf serum (open squares) or with 10% lipoprotein-deficient fetal calf serum (grey circles). Fatty acid levels are expressed as nmol/mg protein. Error bars represent the standard deviation.

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fibroblasts from both controls and X-ALD patients were cultured in lipoprotein-deficient medium, the SCD mRNA levels increased 4 to 5-fold (Fig. 5). This increase was abolished by the addition of cholesterol and 25-OH-cholesterol to the lipoprotein-deficient culture medium (Fig. 5).

Subsequently, in order to obtain information on the activity of SCD, we incubated X-ALD fibroblasts and controls with labeled D5-18:0 and the amount of D5-18:1 present after 24 hours was measured. Figure 6 shows that the synthesis of D5-18:1 was increased under conditions of cholesterol-deprivation which was reversed by the addition of cholesterol and 25-OH-cholesterol to the lipoprotein-deficient culture medium.

Figure 4: The effect of cholesterol-deprivation on VLCFA (26:0) beta-oxidation in fibroblasts from X-ALD patients. Fibroblasts were cultured for 7 days in HAM F10 medium supplemented either with 10% fetal calf serum (open bars) or with 10% lipoprotein-deficient fetal calf serum (black bars). Control cells cultured under standard conditions were included for comparison. Peroxisomal 26:0 beta-oxidation is expressed as pmol/h/mg protein. Error bars represent the standard deviation. n.d. = not determined.

Figure 5: The effect of cholesterol-deprivation on the expression of SCD was analyzed by quantitative PCR. Control (A) and X-ALD (B) cells were cultured in HAM F10 supplemented either with 10% fetal calf serum (open bars), with 10% lipoprotein-deficient fetal calf serum (black bars), or with 10% lipopro-tein-deficient fetal calf serum with 10 μL cholesterol and 1 μL 25-OH-cholesterol (grey bars). Statistical significance was determined with Student’s unpaired t-test. Error bars represent the standard devia-tion: (*) p < 0.05.

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Figure 6: The effect of cholesterol-deprivation on the activity of SCD was analyzed by measuring the amount of D5-18:1 formed from D5-18:0. Control (A) and X-ALD (B) cells were cultured for 24 hours in HAM F10 with 100 μM D5-18:0 supplemented either with 10% fetal calf serum (open bars), with 10% lipoprotein-deficient fetal calf serum (black bars), or with 10% lipoprotein-deficient fetal calf serum with 10 μL cholesterol and 1 μL 25-OH-cholesterol (grey bars). Fatty acid levels are expressed as nmol/mg protein. Statistical significance was determined with Student’s unpaired t-test. Error bars repre-sent the standard deviation: (*) p < 0.05; (**) p < 0.01.

Discussion

The data presented in this paper confirm the earlier observation that X-ALD fibroblasts grown in lipoprotein-deficient culture medium have a reduced level of 26:0 (Fig. 1B) (Weinhofer et al 2002). However, we also found a large increase in the 26:1 level (Fig. 1D). This has not been reported before.

VLCFA are mostly derived from endogenous synthesis and not from dietary sources (Moser et al 2001), and it has been shown previously that X-ALD fibroblasts have increased 26:1 synthesis from oleate (18:1) (Kemp et al 2005). Saturated VLCFA are synthesized from palmitate (16:0) by chain elongation (Jakobsson et al 2006), whereas elongation of palmitoleate (16:1) yields mono-unsaturated VLCFA. Mono-unsaturated fatty acids in humans are synthesized by SCD, an enzyme that introduces a cis double bond at the ω-9 position. The preferred substrates for SCD are palmitoyl-CoA (16:0-CoA) or stearoyl-CoA (18:0-CoA), and not fatty acyl-CoAs with longer chain length (Ntambi and Miyazaki, 2003). Our results show that cholesterol depletion of the culture medium results in increased 26:1 levels (Fig. 1D) and increased synthesis of D3-26:1 from D3-16:0 in X-ALD fibroblasts (Fig. 3B). Interestingly, this increase in 26:1 was accompanied by an increase in the expression of SCD and increased conversion of D5-18:0 to D5-18:1. SCD is regulated at the transcriptional level and it is a known target of the SREBP class of transcription factors (Ntambi and Miyazaki, 2003). 25-OH-cholesterol is a potent inhibitor of SREBP activation (Brown and Goldstein, 1997), and addition of cholesterol and 25-OH-cholesterol will abolish SREBP activation and can serve as a control condition. The fact that the SREBP signaling pathway is activated by cholesterol depletion (Brown and Goldstein, 1997), may well explain the increase in SCD mRNA levels when X-ALD fibroblasts were cultured in lipoprotein-deficient medium (Fig. 5A). This finding is supported by the increased conversion of D5-18:0 to D5-18:1 by SCD (Fig. 6A). The observation that both SCD mRNA levels and increased conversion of D5-18:0 to D5-18:1 can be reversed by adding free cholesterol and 25-OH-cholesterol to the lipoprotein-deficient medium strongly suggests that the observed effects are indeed attributable to

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cholesterol deprivation and not to other differences between the media.

The findings described in this paper may well be relevant for research aimed towards the generation of new therapeutic approaches for X-ALD currently ongoing and also to clinical practice. It has been shown that increased expression of ALDRP could reverse the deficient VLCFA beta-oxidation in X-ALD fibroblasts (Kemp et al 1998). Furthermore, the compound 4-phenylbutyric acid (4-PBA) reduced VLCFA levels in both human X-ALD fibroblasts as well as in the brain and adrenals of Abcd1 knockout mice. Even though the mode of action has not yet been resolved, this may be caused by peroxisome proliferation, increased ALDRP expression (Kemp et al 1998) or increased mitochondrial beta-oxidation (McGuinness et al 2003). In Abcd1 knockout mice in which ALDRP is overexpressed, there is normalization of the biochemical abnormalities and also reversal of the AMN-like phenotype occurring at the age of 18 months (Pujol et al 2004). This data supports the hypothesis that ALDRP overexpression might be of clinical benefit in X-ALD. Unfortunately, 4-PBA mediated pharmacological overexpression of ALDRP proved unsuitable for application in humans, due to the short half-life of 4-PBA (Moser et al 2001). Therefore, other means to upregulate ALDRP in humans as a treatment for X-ALD are being sought. It has been suggested that cholesterol lowering with lovastatin might accomplish this (Weinhofer et al 2002).

In 1998 the reports that lovastatin reduced the 26:0/22:0 ratio in cultured X-ALD fibroblasts (Singh et al 1998a) and also reduced plasma VLCFA (reported as the sum of 22:0 + 24:0 + 26:0) in X-ALD patients (Singh et al 1998b) offered new hope for a widely applicable and effective treatment for X-ALD. Unfortunately, a follow-up paper from the same group showed less striking effects on 26:0 levels (Pai et al 2000) and a study with simvastatin failed to show any effect on 26:0 (Verrips et al 2000). Later work by Weinhofer et al was aimed to identify the mechanism by which lovastatin reduced the 26:0/22:0 ratio in X-ALD skin fibroblasts (Weinhofer et al 2002). To study this, X-ALD skin fibroblasts were cultured in lipoprotein-deficient medium containing low levels of cholesterol. When grown in this medium, X-ALD fibroblasts showed an increase in ABCD2 expression, as well as a decrease in 26:0 levels (Weinhofer et al 2002). The observed upregulation of ALDRP was taken as a plausible explanation for the observed effect of lipoprotein-deficient culture medium and lovastatin on 26:0 levels in X-ALD fibroblasts.

Our findings, however, shed new light on the feasibility of lowering cholesterol as a therapeutic approach. The decrease in 26:0 induced by culturing X-ALD skin fibroblasts in lipoprotein-deficient medium is offset by a significant increase in 26:1. On the whole, there is no decrease in the total VLCFA pool: the 0.3 nmol/mg protein decrease in 26:0 (Fig. 1A) is smaller than the 1 nmol/mg protein increase in 26:1 (Fig. 2A). The observation that 26:0 beta-oxidation is not increased in lipoprotein-deficient medium is in line with this finding (Fig. 4).

The role of VLCFA accumulation in the pathogenesis of X-ALD is still largely unknown. Since VLCFA accumulation (predominately 26:0) is the biochemical hallmark of X-ALD it seems plausible that this accumulation is somehow related to the development of symptoms. This is supported by experiments by Whitcomb and colleagues showing a direct toxic effect of 26:0 on adrenocortical cells (Whitcomb et al 1988). Adrenocortical cells cultured in the presence of increasing concentrations of 26:0 showed a decreased response to ACTH stimulation. Also, 26:0 has disruptive effects on cell membrane structure and function (Ho

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et al 1995). Whether 26:1 has different effects on cell functioning and membrane properties than 26:0 and whether it is less toxic remains to be resolved, and should be investigated.

The data presented here indicate that it is unclear if 26:0 reduction through cholesterol lowering is a feasible therapeutic approach. Caution should be taken in prescribing lovastatin to X-ALD patients, since data on for example the possible accumulation of mono-unsaturated VLCFA or VLCFA accumulation in tissues is lacking. Currently, we are conducting a clinical trial to evaluate the effect of lovastatin versus placebo on VLCFA in plasma, lymphocytes and erythrocytes of X-ALD patients.

Acknowledgements

The authors thank R. Sanders, I.M.E. Dijkstra, F. Stet and H. van Lenthe for technical assistance. This work was supported by grants from the European Leukodystrophy Association: ELA 2005-024I5 and ELA 2006-031I4.

References

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Brown MS, Goldstein JL. 1997. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331-340.

Cartier N, Guidoux S, Rocchiccioli F, Aubourg P. 2000. Simvastatin does not normalize very long chain fatty acids in adrenoleukodystrophy mice. FEBS Lett 478:205-208.

Ho JK, Moser H, Kishimoto Y, Hamilton JA. 1995. Interactions of a very long chain fatty acid with model membranes and serum albumin. Implications for the pathogenesis of adrenoleukodystrophy. J Clin Invest 96:1455-1463.

Jakobsson A, Westerberg R, Jacobsson A. 2006. Fatty acid elongases in mammals: their regulation and roles in metabolism. Prog Lipid Res 45:237-249.

Kamijo K, Taketani S, Yokota S, Osumi T, Hashimoto T. 1990. The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily. J Biol Chem 265:4534-4540.

Kemp S, Wei HM, Lu JF, Braiterman LT, McGuinness MC, Moser AB, Watkins PA, Smith KD. 1998. Gene redundancy and pharmacological gene therapy: implications for X-linked adrenoleukodystrophy. Nat Med 4:1261-1268.

Kemp S, Valianpour F, Mooyer PA, Kulik W, Wanders RJ. 2004. Method for measurement of peroxisomal very-long-chain fatty acid beta-oxidation in human skin fibroblasts using stable-isotope-labeled tetracosanoic acid. Clin Chem 50:1824-1826.

Kemp S, Valianpour F, Denis S, Ofman R, Sanders RJ, Mooyer P, Barth PG, Wanders RJ. 2005. Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy. Mol Genet Metab 84:144-151.

Lombard-Platet G, Savary S, Sarde CO, Mandel JL, Chimini G. 1996. A close relative of the adrenoleukodystrophy (ALD) gene codes for a peroxisomal protein with a specific expression pattern. Proc Natl Acad Sci U S A 93:1265-1269.

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McGuinness MC, Lu JF, Zhang HP, Dong GX, Heinzer AK, Watkins PA, Powers J, Smith KD. 2003. Role of ALDP (ABCD1) and mitochondria in X-linked adrenoleukodystrophy. Mol Cell Biol 23:744-753.

Moser HW, Moser AB, Frayer KK, Chen W, Schulman JD, O’Neill BP, Kishimoto Y. 1981. Adrenoleukodystrophy: increased plasma content of saturated very long chain fatty acids. Neurology 31:1241-1249.

Moser HW, Smith KD, Watkins PA, Powers J, Moser AB. 2001. X-linked adrenoleukodystrophy. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York: Mc Graw Hill. p 3257-3301.

Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, Poustka AM, Mandel JL, Aubourg P. 1993. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 361:726-730.

Mosser J, Lutz Y, Stoeckel ME, Sarde CO, Kretz C, Douar AM, Lopez J, Aubourg P, Mandel JL. 1994. The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein. Hum Mol Genet 3:265-271.

Netik A, Forss-Petter S, Holzinger A, Molzer B, Unterrainer G, Berger J. 1999. Adrenoleukodystrophy-related protein can compensate functionally for adrenoleukodystrophy protein deficiency (X-ALD): implications for therapy. Hum Mol Genet 8:907-913.

Ntambi JM, Miyazaki M. 2003. Recent insights into stearoyl-CoA desaturase-1. Curr Opin Lipidol 14:255-261.

Pai GS, Khan M, Barbosa E, Key LL, Craver JR, Cure JK, Betros R, Singh I. 2000. Lovastatin therapy for X-linked adrenoleukodystrophy: clinical and biochemical observations on 12 patients. Mol Genet Metab 69:312-322.

Pujol A, Ferrer I, Camps C, Metzger E, Hindelang C, Callizot N, Ruiz M, Pampols T, Giros M, Mandel JL. 2004. Functional overlap between ABCD1 (ALD) and ABCD2 (ALDR) transporters: a therapeutic target for X-adrenoleukodystrophy. Hum Mol Genet 13:2997-3006.

Ramakers C, Ruijter JM, Deprez RH, Moorman AF. 2003. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62-66.

Singh I, Moser HW, Moser AB, Kishimoto Y. 1981. Adrenoleukodystrophy: impaired oxidation of long chain fatty acids in cultured skin fibroblasts an adrenal cortex. Biochem Biophys Res Commun 102:1223-1229.

Singh I, Pahan K, Khan M. 1998a. Lovastatin and sodium phenylacetate normalize the levels of very long chain fatty acids in skin fibroblasts of X- adrenoleukodystrophy. FEBS Lett 426:342-346.

Singh I, Khan M, Key L, Pai S. 1998b. Lovastatin for X-linked adrenoleukodystrophy. N Engl J Med 339:702-703.

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85.

Valianpour F, Selhorst JJ, van Lint LE, van Gennip AH, Wanders RJ, Kemp S. 2003. Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol Genet Metab 79:189-196.

Verrips A, Willemsen MA, Rubio-Gozalbo E, De JJ, Smeitink JA. 2000. Simvastatin and plasma very-long-chain fatty acids in X-linked adrenoleukodystrophy. Ann Neurol 47:552-553.

Wanders RJ, Denis S, Ruiter JP, Schutgens RB, van Roermund CW, Jacobs BS. 1995. Measurement of peroxisomal fatty acid beta-oxidation in cultured human skin fibroblasts. J Inherit Metab Dis 18 Suppl 1:113-124.

Weinhofer I, Forss-Petter S, Zigman M, Berger J. 2002. Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy. Hum Mol Genet 11:2701-2708.

Weinhofer I, Forss-Petter S, Kunze M, Zigman M, Berger J. 2005. X-linked adrenoleukodystrophy mice demonstrate abnormalities in cholesterol metabolism. FEBS Lett 579:5512-5516.

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Whitcomb RW, Linehan WM, Knazek RA. 1988. Effects of long-chain, saturated fatty acids on membrane microviscosity and adrenocorticotropin responsiveness of human adrenocortical cells in vitro. J Clin Invest 81:185-188.

Yamada T, Shinnoh N, Taniwaki T, Ohyagi Y, Asahara H, Horiuchi, Kira J. 2000. Lovastatin does not correct the accumulation of very long-chain fatty acids in tissues of adrenoleukodystrophy protein-deficient mice. J Inherit Metab Dis 23:607-614.

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Chapter 3Bezafibrate lowers very long-chain fatty acids in

X-linked adrenoleukodystrophy fibroblasts byinhibiting fatty acid elongation

Journal of Inherited Metabolic Diseases (2012) in press

Marc Engelen1,3, Martin J.A. Schackmann2, Rob Ofman2, Robert-Jan Sanders2, Inge M.E. Dijkstra2, Sander M. Houten2, Stéphane Fourcade4, Aurora Pujol4,5, Bwee Tien

Poll – The1,3, Ronald J.A. Wanders2 and Stephan Kemp2,3

1Department of Neurology; 2Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases; 3Depart-ment of Pediatric Neurology/Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, The Netherlands. 4Neurometabolic Diseases Laboratory, The Bellvitge Institute of Biomedical Research (IDIBELL), Cen-ter for Biomedical Research on Rare Diseases (CIBERER), Barcelona, Spain. 5ICREA (Institució Catalana de Recerca i

Estudis Avançats), Barcelona, Spain.

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Abstract

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding ALDP, an ATP-binding-cassette (ABC) transporter located in the peroxisomal membrane. ALDP deficiency results in impaired peroxisomal beta-oxidation and the subsequent accu-mulation of very long-chain fatty acids (VLCFA; > C22:0) in plasma and tissues. VLCFA are primarily derived from endogenous synthesis by ELOVL1. Therefore inhibiting this enzyme might constitute a feasible therapeutic approach. In this paper we demonstrate that bezafi-brate, a PPAR pan agonist used for the treatment of patients with hyperlipidaemia reduces VLCFA levels in X-ALD fibroblasts. Surprisingly, the VLCFA-lowering effect was independent of PPAR activation and not caused by the increase in either mitochondrial or peroxisomal fatty acid beta-oxidation capacity. In fact, our results show that bezafibrate reduces VLCFA synthesis by decreasing the synthesis of C26:0 through a direct inhibition of fatty acid elon-gation activity. Taken together, our data indicate bezafibrate as a potential pharmacothe-rapeutic treatment for X-ALD. A clinical trial is currently ongoing to evaluate the effect in patients with X-ALD.

Take-home message: Pharmacological inhibition of ELOVL1 with bezafibrate lowers very long-chain fatty acids in X-linked adrenoleukodystrophy fibroblasts and might constitute a new approach to treatment.

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Introduction

X-linked adrenoleukodystrophy (X-ALD: OMIM 300100) is an inherited metabolic disorder characterized by impaired peroxisomal β-oxidation of very long-chain fatty acids (VLCFA; ≥C22) and accumulation of VLCFA (mainly ≥C26:0) in plasma and tissues of patients (Moser et al 2001). It is caused by mutations in the ABCD1 gene (http://www.x-ald.nl), encoding a peroxisomal transmembrane protein named ALD protein (ALDP: OMIM 300371) (Mosser et al 1993). Clinically, X-ALD is characterized by a striking and unpredictable variation in phe-notypic expression, ranging from the rapidly progressive childhood cerebral form (CCALD) to the more slowly progressive adult form adrenomyeloneuropathy (AMN) and variants wit-hout neurological involvement (“Addison-only” phenotype) (Moser et al 2001). Experimental pharmacotherapy in X-ALD was aimed at normalizing VLCFA beta-oxidation and VLCFA levels. Over the years several compounds have been investigated, such as Loren-zo’s oil (Aubourg et al 1993; van Geel et al 1999), 4-phenylbutyrate (Kemp et al 1998), and lovastatin (Singh et al 1998; Engelen et al 2010). These treatments were shown to be either unpractical or ineffective in clinical trials and therefore other drugs are needed.Fenofibrate (a PPAR-α agonist) was shown to induce expression of ALDR (Abcd2) in the liver of Abcd1-/- mice (Netik et al 1999). ALDRP is a functional homolog of ALDP (Kemp et al 1998). However, fenofibrate has no effect on Abcd2 expression in the brain of Abcd1-/- mice, possibly because it is a substrate for the Mdr1 transporter at the blood brain barrier and therefore does not penetrate into the brain very effectively (Berger et al 1999). For this reason, we investigated the effect of several other drugs known to activate PPAR on VLCFA metabolism in cultured skin fibroblasts from patients with X-ALD. The results described in this paper show that bezafibrate (BF), but not fenofibrate, clofibrate or other PPAR agonists, could reduce VLCFA in cultured fibroblasts from patients with X-ALD. The VLCFA which accumulate in X-ALD, are partly absorbed from the diet (Kishimoto et al 1980), but mostly result from endogenous synthesis through elongation of long-chain fatty acids (Tsuji et al 1981). Recently, we identified ELOVL1 as the key enzyme responsible for the synthesis of VLCFA and demonstrated that knock-down of ELOVL1 resulted in lower VLCFA synthesis and reduced levels of VLCFA in cultured X-ALD fibroblasts (Ofman et al 2010). Hence, inhibiting fatty acid elongation (for example by inhibition of ELOVL1) by pharmaco-logical means could be a potential treatment for X-ALD. Here, we show that BF lowers VLCFA in X-ALD fibroblasts by direct inhibition of fatty acid elongation.

Materials and Methods

ChemicalsDeuterium-labeled palmitate-16,16,16-D3 acid (D3C16:0) was purchased from CDN isotopes (Pointe-Claire, Canada). A 12.5 mM stock solution in dimethyl sulfoxide (DMSO) was pre-pared. BF, fenofibrate and clofibrate, WY14643, GW501516, and rosiglitazone were pur-chased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions in DMSO were made of 400 mM (BF and clofibrate), 50 mM (fenofibrate), 10 mM (WY14643 and rosiglitazone), 500 µM (GW501516). MK-886 was purchased from Cayman Chemical (Ann Arbor, MI, USA) and a stock solution of 50 mM in DMSO was used. Prior to usage the stock solutions were vortex mixed and diluted in HAMF10 tissue culture medium to the final concentration. All chemi-cals used were of analytical grade.

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Cell lines and cell culturePrimary human skin fibroblasts were obtained from X-ALD patients through the Neurology Outpatient Clinic of the Academic Medical Center. From each patient written informed con-sent was obtained. X-ALD diagnosis was confirmed by VLCFA and ABCD1 mutation analysis. Control fibroblasts were from male anonymous volunteers. Cells from patients with a per-oxisomal biogenesis disorder were obtained from the laboratory cell bank. Cells were grown in HAMF10 supplemented with 10% fetal calf serum, 2.5 mM HEPES, 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM glutamine. Cells were used between passage numbers 6 and 20. Culture media were refreshed every 5 days.

Fatty acid synthesis Synthesis of D3-VLCFA in intact cells was measured using D3-C16:0. Assays were performed in triplicate. Cells were seeded at 40% confluency in T75 flasks. The next day, medium was replaced by fresh medium supplemented with D3-C16:0 (dissolved in DMSO) at a final con-centration of 50 µM. After 72 hr, cells were harvested and VLCFA analyzed as described (Valianpour et al 2003).

Measurement of fatty acid beta-oxidationMitochondrial beta-oxidation activity of intact fibroblasts was measured by quantifying the production of 3H2O from [9,10-3H(N)] oleic acid as described previously (Moon and Rhead 1987). Peroxisomal beta-oxidation activity of intact fibroblasts was measured using [1-14C]-26:0 as described previously (Wanders et al 1995). All measurements were performed in triplicate for each cell line.

Immunofluorescence and counting of peroxisomesFibroblasts where grown on glass microscopy slides in 6-well plates with or without 400 µM BF. Cells were fixed with paraformaldehyde and permeabilized with Triton X-100. Peroxiso-mes were visualized by catalase immunofluorescence microscopy as described previously (Kemp et al 1996). To count peroxisomes, we made images of immunofluorescence-stained cells after focusing on the cell nucleus, and determined the peroxisome number per cell with the aid of a colony counter. For each cell line and condition, 10 cells were counted at random.

Quantitative RT-PCR analysisELOVL1, ELOVL4, ELOVL6, ACOX1, ABCD3 and CPT1a mRNA levels in control and X-ALD fibro-blasts growing in log phase were determined as described (Engelen et al 2008), with primer sets presented in Table S1 (Supporting Information Table S1, online only).

Purification of mouse liver microsomesMicrosomes were isolated from livers from wild type and transgenic ELOVL1 over-expres-sing mice (Kemp et al, manuscript in preparation) by differential centrifugation as described by Baudhuin et al (Baudhuin et al 1964), with minor modifications. Livers were washed with ice-cold homogenization buffer containing 250 mM sucrose, 2 mM EDTA, 2 mM DTT and 5 mM MOPS (pH 7.4), minced and homogenized using a potter tissue grinder with Teflon pestle with 5 strokes at 500 rpm. A post-nuclear supernatant was produced by centrifuga-tion at 600 g for 10 min. The supernatant was centrifuged at 22,500 g for 10 min and the pellet was discarded. Next, the supernatant was centrifuged for 1 h at 100,000 g to obtain

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a microsomal fraction. The pellet was resuspended in homogenization buffer containing 10 mg/mL methyl-beta-cyclodextrin and sonicated for 4 times 5 seconds at 8 W with a 1 minute interval. The microsomal membranes were collected by centrifugation at 100,000 g for 1 h. Finally, the microsomes were resuspended in homogenization buffer and stored at -80 °C until further use. All steps were carried out at 4 °C. Protein concentrations were determined using BCA as described (Smith et al 1985).

Fatty acid elongation assayThe fatty acid elongation assay was carried out using a method adapted from Nagi et al (Nagi et al 1989). The reaction mixture contained 50 mM potassium phosphate buffer (pH 6.5), 10 mg/mL α-cyclodextrin, 1 mM NADPH, 5 µM rotenone, 60 µM [2-14C] malonyl-CoA (6.5 dpm/pmol) (American Radiolabeled Chemicals, St Louis, MO) and 20 µM C16:0-CoA or 20 µM C22:0-CoA (Avanti Polar Lipids, Alabaster, AL), in a total volume of 200 μL. The reaction was started by adding 100 µg protein of the microsomal fraction and allowed to proceed for 30 min at 37°C. NADPH dependency was tested by performing the reaction without NADPH. Reactions were carried out with or without BF or BF-CoA (100 – 400 µM). The reaction was stopped by adding 200 μL 5 M KOH in 10% methanol, saponified at 65°C for 1 h and acidified by adding 200 μL 5 N HCl and 200 μL 96% ethanol. Fatty acids were extracted 3 times with 1 mL hexane and the hexane phases were collected in a scintillation vial to which 10 mL scintil-lation cocktail (Ultima-Gold, Perkin Elmer) was added and radioactivity counted.

Synthesis of bezafibroyl-CoA (BF-CoA)BF-CoA was synthesized by a method adapted from Rasmussen et al (Rasmussen et al 1990). Dichloromethane (DCM) and tetrahydrofuran (THF) (Merck) were dried with molecular sie-ve deperox (Fluka). Triethylamine and ethylchloroformate (Merck) were diluted to 1 M with dry DCM. The reaction contained 36 μmol BF dissolved in 1.4 mL DCM/THF (5:2) and 40 μL 1 M triethylamine. The reaction mixture was incubated at room temperature for 10 min under constant stirring and under an atmosphere of nitrogen. After 10 min, 40 μL 1 M ethylchlo-roformate was added and the incubation was continued for 45 min. After the incubation, the mixture was dried under nitrogen and dissolved in 0.5 mL tert-butanol. Next, 40 μmol CoA trilithium salt (Sigma-Aldrich) dissolved in 0.5 mL 0.4 M potassium bicarbonate was added and the sample was incubated for 30 min at room temperature. The reaction was stopped by adding 100 μL 0.1 N HCl. BF-CoA was purified using a C18 solid phase extraction column (JTBaker). The column was eluted by a gradient of acetonitrile and 40 mM ammo-nium acetate, starting with 10% acetonitrile and 90% 40 mM ammonium acetate increasing to 50% acetonitrile and 50% 40 mM ammonium acetate. Acetonitrile was evaporated and purity was checked by HPLC. BF-CoA was quantified using 5,5’-dithiobis-(2-nitrobenzoic) acid (DTNB) (Sigma-Aldrich). The method used was an adaptation of that of Ellman (Ellman 1959). BF-CoA was diluted in 20 μM MES buffer pH 6.0, an equal volume of 2 M NaOH was added and the sample was incubated for 30 min at 50°C. The reaction was neutralized with 2 M HCl. Absorbance at 412 nm was measured and the concentration was determined using a calibration curve of CoA trilithium salt (Sigma-Aldrich).

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Results

Effect of BF on endogenous VLCFA levels and de novo C26:0 synthesis in fibroblasts from patients with X-ALDWe tested the effect of several drugs from the fibrate class, but found that only BF reduces C26:0 levels, both fenofibrate and clofibrate being ineffective (Fig. 1A). BF is a PPAR pan-agonist activating all three PPARs. To determine whether the effect of BF on C26:0 levels is mediated by activation of either PPARα, PPARβ/δ or PPARγ, X-ALD fibroblasts were in-cubated with the PPARα ligand WY14643, PPARβ/δ ligand GW501516, or the PPARγ ligand rosiglitazone either alone or in all possible combinations. Only treatment with BF reduced C26:0 levels by about 30% after 7 days (Fig. 1B). To examine if longer incubations with BF would result in a further decrease of C26:0 levels, we cultured X-ALD fibroblasts for up to 21 days. This resulted in a small additional decrease of C26:0 of 10% after 14 days and 15% after 21 days, respectively (Fig. 1C). No signs of cyto-

Figure 1: Only BF, and not other drugs from the fibrate class, reduces C26:0 in X-ALD fibroblasts. (A) C26:0 levels in 3 X-ALD cell lines cultured for 7 days without drugs (black bar), in the presence of BF (grey bar), fenofibrate (white bar), or clofibrate (hatched bar). (B) C26:0 levels in 3 X-ALD cell lines cultured for 7 days without drugs (black bar), in the presence of BF (grey bar), agonists of PPAR-α (WY 14643), PPAR-β/δ (GW 501516) and PPAR-γ (rosiglitazon), or with different combinations of PPAR ago-nists (white bars). (C) C26:0 levels in 8 control (white bar) and 8 X-ALD cell lines cultured without (black bar) or with 400 µM BF (grey bars) for up to 3 weeks. Fatty acid levels are in nmol/mg protein. Data are mean ± SD. * = p < 0.05, ** = P < 0.01, *** = P < 0.001 by ANOVA followed by Dunnett’s multiple comparison test compared with untreated X-ALD cells.

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toxicity or impaired growth as determined by the MTS cell proliferation assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay) were observed (data not shown). Previously, we validated the use of stable-isotope labeled fatty acids to study VLCFA de novo synthesis in whole cells and demonstrated that the synthesis of D3-C26:0 from D3-C16:0 is elevated in X-ALD fibroblasts (Ofman et al 2010). Earlier work has shown that BF is a potent inhibitor of long-chain fatty acid elongation, while clofibrate is not (Sanchez et al 1993; Vazquez et al 1995). Therefore, we tested the effect of BF, clofibrate and fenofibrate on C26:0 de novo synthesis. Of all fibrates tested, only BF affected the de novo D3-C26:0 synthesis (Fig 2A). BF reduced D3-C26:0 synthesis in a concentration-dependent manner. At 400 µM BF, the syn-thesis of D3-C26:0 was reduced by 75% compared with untreated X-ALD fibroblasts (Fig. 2B). At this concentration, there was no difference in the amount of newly synthesized D3-C26:0 between X-ALD and control cells. We performed further experiments with BF, and not the other fibrates or PPAR agonists, to identify by which mechanism BF reduces C26:0 levels in fibroblasts from patients with X-ALD.

Figure 2: BF, but not other fibrates, inhibits D3-C26:0 synthesis. (A) D3-C26:0 synthesis from D3-C16:0 in 5 X-ALD cell lines cultured for 3 days without drugs (black bar) and in the presence of BF (grey bar), fenofibrate (FF, white bar), or clofibrate (CF, hatched bar). (B) D3-C26:0 analysis from D3-C16:0 in 4 control (white bar) and 4 X-ALD cell lines cultured for 3 days without (black bar) or with increasing concentrations of BF (grey bars). Fatty acid levels are in nmol/mg protein. Data are mean ± SD. ** = P < 0.01, *** = P < 0.001 by ANOVA followed by Dunnett’s multiple comparison test compared with untreated X-ALD cells.

The effect of BF on C26:0 levels is independent of induction of mitochondrial or peroxisomal beta-oxidationBF and PPAR ligands in general are known to induce mitochondrial and peroxisomal beta-oxidation (Cabrero et al 2001; Islinger et al 2007; Bonnefont et al 2009; Pyper et al 2010). The effect of BF treatment on the rate of mitochondrial and peroxisomal fatty acid beta-oxidation was determined in X-ALD fibroblasts. Exposure of X-ALD fibroblasts to BF caused a 50% increase in C16:0 beta-oxidation (Fig 3A), and a 35% increase in C26:0 beta-oxidation (Fig. 3B). Since it is known that fibrates induce peroxisome proliferation in rodents (Islinger et al 2007), we determined the amount of peroxisomes in X-ALD fibroblasts incubated for 10 days with BF. We did not find any evidence for an effect of BF treatment on peroxisome abundance (Fig. 3C).

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Figure 3: Effect of BF on mitochondrial and peroxisomal beta-oxidation and peroxisome number. Measurement of (A) C16:0 beta-oxidation and (B) C26:0 beta-oxidation activity in 5 control cell lines (white bar) and 5 X-ALD cell lines cultured without (black bar) or with 400 µM BF (grey bar) for 7 days. Activities are in pmol/mg/hour. Data are mean ± SD. * = P < 0.05 and *** = P < 0.001 by ANOVA fol-lowed by Dunnett’s multiple comparison test compared with untreated X-ALD cells. (C) Peroxisome number in 10 X-ALD cell lines cultured without (white bar) or with 400 µM BF (grey bar) for 3 days. Mean and quartiles are indicated, the error bars represent the range.

Figure 4: C26:0 reduction by BF is not mediated by increased peroxisomal beta-oxidation. Analy-sis of de novo D3-C26:0 synthesis in 3 X-ALD (black bar) cell lines and 3 cell lines from patients with a peroxisomal biogenesis disorder (PBD, white bar). Cells were incubated with 50 µM D3-C16:0 for 3 days without or with 400 µM BF (cross-hatched bars). Fatty acid levels are in nmol/mg protein. Data are mean ± SD. *** = p < 0.001 by student’s unpaired t-test.

BF reduced C26:0 levels in cultured X-ALD fibroblasts, while other drugs of the fibrate class or other synthetic PPAR agonists could not. This strongly suggested that the effect of BF on VLCFA levels is not dependent solely on induction of mitochondrial or peroxisomal beta-oxidation, since the other compounds should have been effective as well then (Blaauboer et al 1990; Kemp et al 2011). BF treatment resulted in a 35% increase in the peroxisomal C26:0 beta-oxidation capacity (Fig 3B). To test if the induction of peroxisomal beta-oxidation by BF caused the reduction in C26:0 levels, we incubated fibroblasts from patients with a peroxisomal biogenesis disorder (PEX1, PEX6 and PEX26) with BF and measured the effect on D3-C26:0 de novo synthesis. The synthesis of D3-C26:0 from D3-C16:0 was reduced rou-ghly 50% in peroxisome-deficient fibroblasts incubated with BF (Fig 4). This clearly indicated that the effect of BF on D3-C26:0 synthesis and C26:0 levels is only partially mediated by an induction of the peroxisomal beta-oxidation capacity.

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To investigate whether the induction of mitochondrial beta-oxidation by BF is responsible for the reduction in C26:0, we used the PPARα inhibitor MK-886 (Kehrer et al 2001). The induction of mitochondrial beta-oxidation by BF in X-ALD fibroblasts could be reversed with 50 µM MK-886, suggesting that PPARα activation is completely blocked at this concentration (Fig 5A). Next, we measured D3-C26:0 de novo synthesis in X-ALD fibroblasts incubated with BF in the presence of MK-886. Addition of MK-886 did not affect the reduction of D3-C26:0 synthesis (Fig 5B). Combined, these data are highly suggestive that the reduction of C26:0 levels by BF is PPAR independent and can not be explained by the increase in mitochondrial beta-oxidation capacity and only partially by the increase in peroxisomal beta-oxidation ca-pacity. This suggests that BF inhibits the formation of D3-C26:0.

BF directly inhibits fatty acid elongationThe amount of D3-C26:0 present is the net result of the elongation of D3-C16:0 to D3-C26:0 by ELOVL6 and ELOVL1, respectively (Ofman et al 2010) and the degradation of D3-C26:0 by peroxisomal beta-oxidation. The inhibitory effect of BF on the formation of D3-C26:0 could either be indirect by affecting gene expression or direct by inhibition of key enzymes in-volved in VLCFA de novo synthesis (Ofman et al 2010). To investigate the effect of BF on gene expression levels of several key enzymes involved in VLCFA synthesis, peroxisomal beta-oxidation and mitochondrial beta-oxidation we performed quantitative RT-PCR. As shown in Fig 6, there is only a small reduction in the mRNA levels of ELOVL6, which is not statistically significant. There was no increase in the expression of ACOX1. This is in line with previously published data (Blaauboer et al 1990) and our own data (Fig 3C) showing that there is no peroxisome proliferation, at least in cultured human cells, treated with fibrates, in contrast to rodents (Blaauboer et al 1990). BF did not induce the expression of ABCD3. Induction of this gene was detected in mice treated with fenofibrate, and considered to be the mecha-

Figure 5: C26:0 reduction by BF is not mediated by increased mitochondrial beta-oxidation. (A) C18:1 beta-oxidation in 3 X-ALD cell lines cultured without (black bar), or for 48 hours with 400 µM BF (grey bar), or with BF and increasing concentrations of MK-886 (white bars). Activities are in pmol/mg/hour. Data are mean ± SD. (B) De novo D3-C26:0 syn-thesis in 4 X-ALD cell lines incubated with 50 µM D3-C16:0 without (black bar) or with 400 µM BF (grey bar), 50 µM MK-886 (white bar), or 400 µM BF and 50 µM MK-886 (cross-hatched bar). Fatty acid levels are in nmol/mg protein. Data are mean ± SD. ** = p < 0.01; *** = p < 0.001 by ANOVA followed by Dun-nett’s multiple comparison test compared with un-treated X-ALD cells.

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nism by which fibrates might be useful in correcting the metabolic defect in X-ALD (Berger et al 1999; Netik et al 1999). Our data show that induction of ABCD3 did not occur in human X-ALD fibroblasts upon exposure to BF. In line with previous data (Djouadi et al 2005), BF treatment resulted in increased expression of CPT1a.The previous experiments suggested that the effect of BF on C26:0 levels and C26:0 synthe-sis could be mediated by a direct inhibiting effect on fatty acid elongation. VLCFA are synthe-sized by the concerted action of ELOVL6 and ELOVL1 (Ofman et al 2010). ELOVL6 elongates C16:0 to C22:0 and ELOVL1 elongates C22:0 to C26:0. We measured the effect of free BF and BF esterified to coenzyme CoA (BF-CoA) on C16:0-CoA and C22:0-CoA elongation. In the elongation assay BF had no effect. However, BF-CoA inhibited the chain elongation activity of both C16:0 and C22:0 in a concentration dependent manner (Fig. 7). Both fenofibrate and clofibrate did not inhibit C22:0-CoA elongation (Fig. 7B). These data demonstrate that BF-CoA lowers C26:0 levels by direct inhibition of fatty acid chain elongation.

Discussion

Synthetic PPAR alpha ligands, like fibrates, are potentially interesting compounds to investi-gate as therapeutic agents in X-ALD because they are known to activate mitochondrial and peroxisomal fatty acid beta-oxidation (Vazquez et al 2001). They might therefore reduce VLCFA accumulation by increasing VLCFA degradation. Indeed, Brown and colleagues de-monstrated that treatment of two CCALD patients with clofibrate resulted in a reduction in VLCFA (Brown et al 1982). However, this reduction was not sustained. More recent expe-riments showed that fenofibrate induced expression of both ALDRP (ABCD2) and PMP70 (ABCD3) in the liver of Abcd1-deficient mice, but not in brain (Berger et al 1999). In Mdr1-/- knockout mice induction of ALDRP and PMP70 in brain did occur, suggesting that fenofibrate is indeed cleared from the brain by Mdr1 (Berger et al 1999). These studies, however, did not report the effect on VLCFA levels in tissues. In this paper, we studied the effect of se-veral classical fibrates and other synthetic PPAR ligands in a cell model for X-ALD, and show that BF but not the other fibrates reduced endogenous C26:0 levels. This C26:0 reducing effect could not be mimicked by other PPAR-ligands which means that the effect is PPAR independent. BF has been demonstrated to induce mitochondrial beta-oxidation (Djouadi et al 2005; Bonnefont et al 2009). Blocking the induction of mitochondrial beta-oxidation

Figure 6: Effect of BF on gene expression of ge-nes involved in fatty acid metabolism. Expression levels of genes involved in fatty acid metabolism in 3 X-ALD cell lines cultu-red without (black bars) or with 400 µM BF (grey bars) for 48 hours were analyzed by quantitative PCR. Data are mean ± SD. * = p < 0.05 by student’s unpaired t-test.

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Figure 7: BF inhibits fatty acid elongation. (A) Fatty acid elongation activity of C16:0-CoA (a substrate of ELOVL6) in the presence of an increasing concentration of BF (o) and BF-CoA (●). (B) Fatty acid elon-gation activity of C22:0-CoA (a substrate of ELOVL1) in the presence of an increasing concentration of BF (o) and BF-CoA (●) or with an increasing concentration of fenofibrate (FF, □) or clofibrate (CF, ∆). Fatty acid elongation activity was measured using 20 µM C16:0-CoA or C22:0-CoA as substrate with concentrations of the inhibitor up to 400 µM. Error bars represent the standard deviation.

with MK-886 did not prevent the reduction of D3-C26:0 de novo synthesis in X-ALD fibroblast by BF. We also showed that the peroxisomal C26:0 beta-oxidation capacity in X-ALD skin fibroblasts increased with 35% upon treatment with BF. However this does not seem to be the only mechanism of reduction of VLCFA in fibroblasts incubated with BF, because in cells from patients with a peroxisome biogenesis disorder in which peroxisomal beta-oxidation is completely deficient, BF lowered D3-C26:0 levels as well. This strongly suggested that BF might reduce C26:0 levels primarily by inhibiting C26:0 synthesis. To test this we measured de novo synthesis of D3-C26:0 from D3-C16:0 in X-ALD fibroblasts. BF indeed decreased de novo synthesis of D3-C26:0 in a concentration-dependent manner. At a concentration of 400 µM BF, D3-C26:0 levels in X-ALD fibroblasts were at the level of control fibroblasts. The rate limiting enzymes involved in synthesis of C26:0 from C16:0 are ELOVL6 (elongation of C16:0 to C22:0) and ELOVL1 (elongation of C22:0 to C26:0) (Kemp and Wanders 2010; Ofman et al 2010). By qPCR we showed that expression levels of these enzymes are not affected in fibroblasts incubated with BF suggesting a direct inhibition of VLCFA synthesis. Previously using rat liver microsomes other investigators showed that BF inhibits palmitoyl-CoA (C16:0-CoA) elongation in an in vitro assay (Sanchez et al 1993; Vazquez et al 1995). We used pu-rified microsomes from wild type and ELOVL1 over-expressing mice to test the effect of BF on elongation of both long-chain fatty acids (LCFA) and VLCFA. Our results demonstrate that BF-CoA, but not free BF, is a potent inhibitor of both LCFA and VLCFA elongation. It should be noted, however, that these results do not allow us to demonstrate at which level BF inhibits VLCFA synthesis. Fatty acid elongation requires four sequential reaction steps: (i) condensation between the fatty acyl-CoA and malonyl-CoA to form 3-ketoacyl-CoA; (ii) reduction using NADPH to form 3-hydroxyacyl-CoA; (iii) dehydration to trans-2-enoyl-CoA;

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and (iv) reduction to fully elongated fatty acyl-CoA. The initial condensation reaction is cata-lyzed by the enzyme referred to as “elongation of very long-chain fatty acids” (ELOVL) and is considered to be rate limiting (Cinti et al 1992). While seven elongases have been identified in mammals (designated ELOVL1-ELOVL7)(Jakobsson et al 2006), only a single enzyme has been identified yet for the subsequent reaction step (Jakobsson et al 2006). Identification of the specific enzyme(s) affected by BF is not a trivial thing and requires detailed analysis of all enzymes involved, including: 3-ketoacyl-CoA reductase (HSD17B12), 3-hydroxyacyl de-hydratase (HACD3) and the trans-2,3-enoyl-CoA reductase (TECR). This will be the subject of future studies.

Concluding remarks

The work described in the paper shows that inhibition of VLCFA synthesis by pharmacolo-gical means could be a feasible treatment option for X-ALD. BF is a good candidate for this approach. BF lowers the levels of C26:0 by a direct inhibition of the synthesis. Mouse studies to evaluate the in vivo effect of BF treatment on VLCFA in X-ALD mice would be interesting; however, the effect of fibrates is quite different in rodents and humans. Watanabe et al de-monstrated that rats and mice are unusable as a model system (for primates) to the study the effect of BF (Watanabe et al 1989). BF has a proven safety profile for (long-term) use in humans. With a daily dose of 200 mg of BF peak plasma levels of 50 µM can be reached, with a maximum daily dose of 800 mg of BF therapeutic levels might be reached in plasma (Miller and Spence 1998). A small scale proof of principle clinical trial is currently ongoing to evaluate the effect in X-ALD patients.

Acknowledgements

We thank Femke Stet, Petra Mooyer, and Henk van Lenthe for expert technical assistance. This research was supported by the Netherlands Organization for Scientific Research (VIDI-grant number 91786328), the European Union Framework Programme 7 (grant number LeukoTreat 241622). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Chapter 4Lovastatin in X-linked adrenoleukodystrophy

The New England Journal of Medicine (2010) 362(3): 276 – 277(published in abbreviated form)

Marc Engelen1,2, Rob Ofman3, Marcel G.W. Dijkgraaf4, Michiel Hijzen3, Lucinda A. van der Wardt5, Björn M. van Geel1,6, Marianne de Visser1, Ronald J.A. Wanders3,

Bwee Tien Poll-The1,2 and Stephan Kemp2,3

Departments of Neurology1, Pediatric Neurology2, Laboratory Genetic Metabolic Diseases3, Clinical Epidemiology, Biostatistics & Bio-informatics4 and Nutrition5, Emma Children’s Hospital/Academic Medical Center, University of

Amsterdam, The Netherlands. Department of Neurology6, Medical Center Alkmaar, Alkmaar, The Netherlands.

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Abstract

BackgroundX-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder and is characterized by rapidly progressive cerebral demyelination or a gradually progressive myelopathy with or without adrenocortical insufficiency. The disease is caused by mutations in the ABCD1 gene resulting in impaired peroxisomal beta-oxidation and the subsequent accumulation of very long-chain fatty acids (VLCFA; >C22) in plasma and tissues. In 1998 it was reported in this Journal that lovastatin reduces total plasma VLCFA in X-ALD patients. This finding led to tre-atment with lovastatin in many patients with X-ALD despite contradicting data on the effect of lovastatin on plasma VLCFA. The aim of this clinical trial was to confirm that in patients with X-ALD lovastatin treatment can reduce C26:0 levels in plasma, but also in lymphocytes and erythrocytes. MethodsThe study was designed as a randomized double-blind placebo controlled cross-over trial (ISRCTN31565393). With a total of 14 patients, a 50% reduction of C26:0 levels can be detec-ted with a power of 80%. The primary outcome measures were reduction of LDL-cholesterol in plasma, C26:0 levels in plasma and LDL-lipoprotein particles, lymphocytes and erythro-cytes.

ResultsFourteen male patients with X-ALD were enrolled in the trial. LDL-cholesterol in plasma was reduced by 40%. Plasma C26:0 levels were reduced by almost 20% (but this effect was also observed for non-VLCFA). There was no effect on C26:0 levels in lymphocytes, erythrocytes and LDL-lipoprotein particles.

ConclusionThe reduction of plasma C26:0 is non-specific and linked to a reduction in LDL-lipoprotein particles. Lovastatin is not a treatment option for X-ALD.

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Introduction

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder with a birth incidence of 1:17.000 (Bezman et al 2001). It is characterized by impaired peroxisomal beta-oxidation of very long-chain fatty acids (VLCFA; > C22:0), which accumulate in all tis-sues (Moser et al 2001). X-ALD is caused by mutations in the ABCD1 gene (see also the X-ALD database at http://www.x-ald.nl) resulting in the absence or dysfunction of a peroxisomal transmembrane protein, the ALD protein (ALDP) (Mosser et al 1993). The disease is charac-terized by a highly variable clinical expression, even within families (Kemp et al 2001). Often, the first manifestation of the disease in childhood is isolated adrenocortical insufficiency (“Addison-only” phenotype) without neurological dysfunction. About 40% of affected male patients develop rapidly progressive cerebral demyelination causing severe disability and death usually within 2 years after symptom onset (Moser et al 2001). This usually occurs between the age of 3 and 10 years (childhood cerebral ALD; CCALD), but is more rarely also seen in adolescence (adolescent cerebral ALD; AdolCALD), or adulthood (adult cerebral ALD; ACALD) (Moser et al 2001). In adulthood the most common phenotype is adrenomyelo-neuropathy (AMN), a gradually progressive myelopathy and peripheral neuropathy, causing severe disability. Symptoms usually appear in the 3rd or 4th decade. Patients with AMN can also develop secondary cerebral demyelination (“AMN cerebral”)(Van Geel et al 2001). Currently, treatment options are very limited and are mostly symptomatic. Only in a very early stage of CCALD progression can be halted or reversed by hematopoietic stem cell transplantation (Peters et al 2004). Treatment with Lorenzo’s oil, a 4:1 mixture of C18:1 and C22:1 in combination with a low-fat diet rapidly normalizes plasma VLCFA, but seems to have no effect on disease progression in several open-label clinical trials (Aubourg et al 1993; van Geel et al 1999).Previously, it was reported that lovastatin lowers VLCFA in patients with X-ALD (Singh et al 1998b; Pai et al 2000). This finding, however, could not be reproduced with simvastatin (Ver-rips et al 2000). Subsequently performed animal experiments showed that lovastatin has no effect on brain and adrenal VLCFA levels in Abcd1 knockout mice (Yamada et al 2000), and simvastatin even caused accumulation of VLCFA in these tissues (Cartier et al 2000). In vitro experiments with cultured skin fibroblasts grown in cholesterol-depleted culture me-dium showed a reduction in VLCFA levels (Weinhofer et al 2002). We recently demonstrated that this reflects a shift to increased synthesis of mono-unsaturated VLCFA (Engelen et al 2008). This trial was designed to investigate if lovastatin indeed has a biochemical effect in vivo in patients with X-ALD, and to help us decide whether a large scale trial with clinical outcome parameters is warranted.

Methods

Eligible patientsPatients were recruited from the AMC neurology outpatient clinic which is a referral center for peroxisomal disorders and through the patient support organization. Men with the AMN phenotype of X-ALD, confirmed by plasma VLCFA analysis and/or ABCD1 mutation analysis, were eligible. Exclusion criteria were the use of Lorenzo’s oil and/or cholesterol lowering therapy, contraindications for lovastatin use (liver- or kidney failure), or impairment so se-

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vere that the patient was unable to visit the outpatient clinic.The study protocol was approved by the local institutional review board. The trial was regis-tered with the Dutch Trial Register (#852) and Current Controlled Trials (ISRCTN31565393).

TreatmentThe study was designed as a randomized double-blind cross-over placebo controlled trial (Figure 1). Eligible patients were randomized and started on a run-in phase with a low fat diet. A randomization list was created by the Biostatistics department. Prepackaged medica-tion sets with identical appearance were created by the Stichting Haarlemse Ziekenhuisapo-theken (GCP certified foundation of hospital pharmacies). Patients entering the trial were given consecutive trial numbers, matching a set of prepackaged medication (twelve bottles numbered consecutively). After a one month run-in phase in which a standard low fat diet was administered by the dietician (as recommended by the American Heart Association) (Lichtenstein et al., 2006), medication or placebo was started, followed by washout and switch to the other treatment arm. Adverse effects were recorded. Treatment assignments were concealed from all investigators. Blood samples were taken at 0, 4, 12, 26, 30, 38 and 52 weeks. The code was broken after all patients had completed the follow-up visits and the data analyzed.

Laboratory studiesAt each visit four standard Vacutainer tubes (2 with heparin, 2 with EDTA) were collec-ted. Routine measurements (creatine kina-se and transaminase activities in serum, and a plasma lipid spectrum) were performed at the Department of Clinical Chemistry. The samples for VLCFA measurements were processed the same day at the Laboratory for Genetic Metabolic Diseases. Blood was centrifuged in Leukoprep tubes for 20 mi-nutes at 2000 rpm. The plasma fraction was stored in separate tubes at -80 °C after snap freezing in liquid nitrogen. The lymphocyte pellet was resuspended in lysisbuffer twice to remove erythrocytes, and then washed with cold saline and stored at -80 °C. The erythrocytes were washed twice with cold saline and stored at -80 °C. VLCFA were measured as described previously (Kemp et al 2005). Lipoprotein fractions were iso-lated from plasma as described previously (Innis-Whitehouse et al 1998).

Outcome measuresEndpoints were plasma total cholesterol, LDL-cholesterol, C18:1, C24:0 and C26:0 levels, lymphocyte and erythrocyte C26:0 levels, and C18:1, C24:0 and C26:0 content of the LDL-

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lipoprotein fraction at 22 weeks after start of treatment. For plasma total cholesterol, LDL-cholesterol, C18:1, C24:0 and C26:0 levels, and lymphocyte C26:0 levels intermediate end-points at 8 weeks after start of treatment were assessed as well.

Sample sizeIt was assumed that the mean baseline C26:0 level prior to treatment would be about 2.94 µmol/L with a standard deviation of 0.87 µmol/L (Valianpour et al 2003). It was further assumed that lovastatin treatment might result in a 50% drop of this level. Based on the crossover design, all patients were to be analyzed within their randomization groups by comparing the change scores over the treatment periods (see below). The change scores in the groups would then be plus 1.47 (placebo minus lovastatin) and minus 1.47 (lovasta-tin minus placebo) respectively. Assuming a conservatively estimated zero correlation bet-ween the outcome measurements for the first and the second treatment period within each group, the standard deviation of the change scores in each group would be 1.23. Using a two groups t-test with a 0.05 two-sided significance level to detect a difference in these chan-ge scores of 2.94 assuming a common standard deviation of 1.739, 80% power would be achieved with a sample size of 7 in each group of the 2x2 cross-over design (or 14 in total).

Statistical analysisAvailable pre-diet score distributions for plasma total cholesterol, LDL-cholesterol, C18:1, C24:0, C26:0 and lymphocyte C26:0 were assessed for normality by one sample Kolmo-gorov-Smirnov tests. Given the small number of patients in both trial arms, the pre-diet scores were tested for inequality with two-sample two-sided Student’s t-tests.Based on the cross-over design, all patients were analyzed within their randomization groups. The analysis was performed with correction for non-significant pre-treatment base-line differences following the run-in phase with standard low fat diet, that were in the same direction as the expected differences between placebo and lovastatin under the alterna-tive hypothesis that there is a significant difference between lovastatin and placebo treat-ment. The correction was performed by using the changes from baseline in each treatment period. In the group receiving placebo followed by lovastatin, we first subtracted baseline from outcome scores during placebo treatment as well as during treatment with lovasta-tin. Subsequently, change scores over treatment periods were determined by subtracting the observed changes under the lovastatin regimen from the observed changes under the placebo regimen. In the group receiving lovastatin followed by placebo, we first subtracted baseline from outcome scores during treatment with lovastatin as well as during placebo. Subsequently, change scores over treatment periods were determined by subtracting the observed changes under the placebo regimen from the observed changes under the lovas-tatin regimen. Data was analyzed using the SPSS software for Windows, version 16 (SPSS, Chicago, IL). P-values below 0.05 indicated statistical significance. Graphs were made in GraphPad Prism (GraphPad Software, La Jolla, CA).

Results

PatientsTwenty-two patients were considered for inclusion in the study. Five were excluded from

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participation (one was on Lorenzo’s oil, 2 were already on statin therapy, 2 were too severely impaired to complete the trial). Three patients declined to participate. Fourteen patients were randomized. No significant differences were observed for the randomized groups. There were no serious adverse events. All patients completed the study.

Biochemical resultsIn Table 1 data and change scores from baseline for 8 weeks and 22 weeks following start of treatment are presented for the all outcome parameters. The data is further summari-zed in scatter plots (Figure 2). At the first (8 weeks of treatment) and second (22 weeks of treatment) time point, there is a significant decrease in total and LDL-cholesterol levels in plasma of 38%. The plasma C24:0 and C26:0 levels are decreased initially by 20% and 18% respectively, although the difference is no longer statistically significant at the second time point for C26:0. There is a reduction of 17% in C18:1 levels at the first and second time point. There is no change in C26:0 levels in erythrocytes or lymphocytes at either time point. The C18:1, C24:0 and C26:0 content of LDL lipoprotein particles are unchanged.

Discussion

This study shows that lovastatin initially reduces C26:0 levels by about 20% in the plasma of patients with X-ALD, but fails to do so in lymphocytes or erythrocytes. It has been reported previously that lovastatin lowers plasma VLCFA in patients with X-ALD (Singh et al 1998b; Pai et al 2000). This prompted many patients worldwide to take lovastatin, even though no clinical trial with clinical endpoints showing a beneficial effect has been performed and both in vitro and in vivo data on results with lovastatin and cholesterol-lowering are conflicting (Singh et al 1998a; Cartier et al 2000; Yamada et al 2000; Weinhofer et al 2002; Engelen et al 2008). A trial with clinical endpoints is difficult to perform, considering the low incidence of X-ALD and the unpredictable disease course, requiring large groups and a long follow-up. It seemed premature to perform this trial for lovastatin since the “biological plausibility” is low, considering the conflicting data in the literature mentioned above. Therefore, our aim was to first perform a “proof of principle” trial with biochemical endpoints. Compliance in the trial was excellent, since there was a clear decrease in LDL-cholesterol levels of almost 40% during the lovastatin treatment period. There was also no drop-out in this small trial with highly motivated participants. There were no adverse effects. We demonstrated a de-crease in C26:0 levels in plasma of roughly 20%. However, even with this decrease, levels remain between 2 and 3 times above the control level of 0.67 ± 0.13 µmol/L (Valianpour et al 2003) and the effect seems to diminish over time (the decrease is no longer statistically significant after 22 weeks of treatment). The decrease is not specific for VLCFA since C18:1 (a long chain mono-unsaturated fatty acid) is also reduced in plasma by lovastatin. It is of note that treatment with lovastatin does not affect VLCFA at the cellular level, since C26:0 levels in erythrocytes and lymphocytes were unchanged. Since VLCFA are virtually water insoluble and only a small fraction binds to albumin (Ho et al 1995), most of the plasma VLCFA is trans-ported as cholesterol-esters in lipoprotein particles like LDL. To establish whether plasma VLCFA reduction is linked to a reduction in LDL cholesterol we measured the C18:1, C24:0 and C26:0 content of the LDL lipoprotein fraction. Lovastatin treatment did not decrease the C18:1, C24:0 or C26:0 content of LDL-cholesterol (Table 1). Based on these observations we conclude that lovastatin leads to a small decrease in C24:0 and C26:0 levels in plasma which

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71

has to be considered a non-specific result of the LDL-cholesterol decrease. This is corrobo-rated by the finding that C18:1 is also reduced and further supported by the lack of effect on C26:0 levels in peripheral blood lymphocytes and erythrocytes, and in LDL-lipoprotein fraction VLCFA content.

Tabl

e 1:

Maj

or o

utco

me

mea

sure

s afte

r 8 w

eeks

(if a

pplic

able

) and

22

wee

ks o

f tre

atm

ent w

ith lo

vast

atin

at a

dos

e of

40

mg

once

dai

ly*

8 w

k of

trea

tmen

t22

wk

of tr

eatm

ent

Base

line

Mea

n ch

ange

95%

CI

P va

lue

Mea

n ch

ange

95%

CI

P va

lue

Plas

ma

Tota

l cho

lest

erol

5.52

1.48

± 0

.25

0.94

to 2

.02

<0.0

011.

45 ±

0.2

40.

92 to

1.9

8<0

.01

LDL

3.54

1.44

± 0

.20

1.02

to 1

.87

<0.0

011.

35 ±

0.2

10.

89 to

1.8

2<0

.01

C18:

12.

370.

38 ±

0.1

70.

0 to

0.7

60.

050.

44 ±

0.1

70.

06 to

0.8

20.

03

C24:

077

.114

.2 ±

2.0

9.8

to 1

8.5

<0.0

0110

.7 ±

3.8

02.

4 to

19.

00.

02

C26:

02.

560.

39 ±

0.1

10.

15 to

0.6

30.

004

0.23

± 0

.16

-0.1

2 to

0.5

80.

18

Lipo

prot

ein

Frac

tion

C18:

1 in

LDL

1540

ND

NA

NA

-84

± 69

-240

to 8

00.

26†

C24:

0 in

LDL

64N

DN

AN

A3.

4 ±

2.5

-2.0

to 8

.90.

19

C26:

0 in

LDL

9.7

ND

NA

NA

-1.9

± 1

.1-4

.3 to

0.6

0.12

Cells

C26:

0 (ly

mph

ocyt

es)

0.35

-0.0

1 ±

0.02

-0.0

4 to

0.0

30.

640.

03-0

.02

to 0

.08

0.22

C26:

0 (e

ryth

rocy

tes)

0.18

ND

NA

NA

0.00

2 ±

0.00

3-0

.01

to 0

.01

0.53

* Pl

us–m

inus

val

ues

are

mea

ns ±

SE. T

he m

ean

redu

ction

indi

cate

s th

e ab

solu

te c

hang

e fr

om b

asel

ine

leve

ls aft

er tr

eatm

ent.

P va

lues

wer

e ca

lcul

ated

with

the

use

of a

two-

sided

, unp

aire

d St

uden

t’s t-

test

. Apo

B de

note

s ap

olip

opro

tein

B, C

I con

fiden

ce in

terv

al, C

18:1

ole

ic a

cid,

C2

4:0

tetr

acos

anoi

c ac

id, C

26:0

hex

acos

anoi

c ac

id, N

A no

t app

licab

le, a

nd N

D no

t det

erm

ined

.†

Equa

l var

ianc

es w

ere

not a

ssum

ed.

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Figure 2: Scatter plots of the effect of lovastatin on levels of plasma total cholesterol (A), LDL-cholesterol (B), plasma C24:0 (C) and C26:0 (D), C24:0 (E) and C26:0 (F) in LDL lipoprotein parti-cles, C26:0 in lymphocytes (G) and C26:0 in erythrocytes (H). TP1 and TP2 are 8 or 22 weeks. Units are mmol/L (A and B), µmol/L (C and D), pmol/mmol ApoB (E and F) and nmol/mg protein (G) and % of total fatty acids (H). Error bars indicate the mean and standard deviation. *** p < 0.001.

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Conclusion

It seems unnecessary to invest a great deal of resources and time in a trial with clinical end-points. Physicians should not prescribe lovastatin as a VLCFA lowering therapy to patients with X-ALD, since evidence does not support it.

Acknowledgements

The authors gratefully acknowledge the patients who were willing to participate in this trial. The work described here was supported by grants from the European Leukodystrophy As-sociation (ELA: 2005-024I5 to BPT and 2006-031I4 to SK) and the Netherlands Organization for Scientific Research (NWO-VIDI-grant 91786328 to SK).

References

Aubourg P, Adamsbaum C, Lavallard-Rousseau MC, Rocchiccioli F, Cartier N, Jambaque I, Jakobezak C, Lemaitre A, Boureau F, Wolf C, . 1993. A two-year trial of oleic and erucic acids (“Lorenzo’s oil”) as treatment for adrenomyelo-neuropathy. N Engl J Med 329:745-752.

Bezman L, Moser AB, Raymond GV, Rinaldo P, Watkins PA, Smith KD, Kass NE, Moser HW. 2001. Adrenoleukodys-trophy: incidence, new mutation rate, and results of extended family screening. Ann Neurol 49:512-517.

Cartier N, Guidoux S, Rocchiccioli F, Aubourg P. 2000. Simvastatin does not normalize very long chain fatty acids in adrenoleukodystrophy mice. FEBS Lett 478:205-208.

Engelen M, Ofman R, Mooijer PA, Poll-The BT, Wanders RJ, Kemp S. 2008. Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy. Bio-chim Biophys Acta 1781:105-111.

Ho JK, Moser H, Kishimoto Y, Hamilton JA. 1995. Interactions of a very long chain fatty acid with model membranes and serum albumin. Implications for the pathogenesis of adrenoleukodystrophy. J Clin Invest 96:1455-1463.

Innis-Whitehouse W, Li X, Brown WV, Le NA. 1998. An efficient chromatographic system for lipoprotein fractiona-tion using whole plasma. J Lipid Res 39:679-690.

Kemp S, Pujol A, Waterham HR, van Geel BM, Boehm CD, Raymond GV, Cutting GR, Wanders RJ, Moser HW. 2001. ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical cor-relations. Hum Mutat 18:499-515.

Kemp S, Valianpour F, Denis S, Ofman R, Sanders RJ, Mooyer P, Barth PG, Wanders RJ. 2005. Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy. Mol Genet Metab 84:144-151.

Lichtenstein AH, Appel LJ, Brands M, Carnethon M, Daniels S, Franch HA, Franklin B, Kris-Etherton P, Harris WS, Howard B, Karanja N, Lefevre M, Rudel L, Sacks F, Van HL, Winston M, Wylie-Rosett J. 2006. Diet and lifestyle re-commendations revision 2006: a scientific statement from the American Heart Association Nutrition Committee. Circulation 114:82-96.

Moser HW, Smith KD, Watkins PA, Powers J, Moser AB. 2001. X-linked adrenoleukodystrophy. In: Scriver CR, Beau-det AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York: Mc Graw Hill. p 3257-3301.

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Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, Poustka AM, Mandel JL, Aubourg P. 1993. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 361:726-730.Pai GS, Khan M, Barbosa E, Key LL, Craver JR, Cure JK, Betros R, Singh I. 2000. Lovastatin therapy for X-linked adreno-leukodystrophy: clinical and biochemical observations on 12 patients. Mol Genet Metab 69:312-322.

Peters C, Charnas LR, Tan Y, Ziegler RS, Shapiro EG, DeFor T, Grewal SS, Orchard PJ, Abel SL, Goldman AI, Ramsay NK, Dusenbery KE, Loes DJ, Lockman LA, Kato S, Aubourg PR, Moser HW, Krivit W. 2004. Cerebral X-linked adrenoleuko-dystrophy: the international hematopoietic cell transplantation experience from 1982 to 1999. Blood 104:881-888.

Singh I, Pahan K, Khan M. 1998a. Lovastatin and sodium phenylacetate normalize the levels of very long chain fatty acids in skin fibroblasts of X- adrenoleukodystrophy. FEBS Lett 426:342-346.

Singh I, Khan M, Key L, Pai S. 1998b. Lovastatin for X-linked adrenoleukodystrophy. N Engl J Med 339:702-703.

Valianpour F, Selhorst JJ, van Lint LE, van Gennip AH, Wanders RJ, Kemp S. 2003. Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol Genet Metab 79:189-196.

van Geel BM, Assies J, Haverkort EB, Koelman JH, Verbeeten B, Jr., Wanders RJ, Barth PG. 1999. Progression of ab-normalities in adrenomyeloneuropathy and neurologically asymptomatic X-linked adrenoleukodystrophy despite treatment with “Lorenzo’s oil”. J Neurol Neurosurg Psychiatry 67:290-299.

Van Geel BM, Bezman L, Loes DJ, Moser HW, Raymond GV. 2001. Evolution of phenotypes in adult male patients with X-linked adrenoleukodystrophy. Ann Neurol 49:186-194.

Verrips A, Willemsen MA, Rubio-Gozalbo E, De JJ, Smeitink JA. 2000. Simvastatin and plasma very-long-chain fatty acids in X-linked adrenoleukodystrophy. Ann Neurol 47:552-553.

Weinhofer I, Forss-Petter S, Zigman M, Berger J. 2002. Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy. Hum Mol Genet 11:2701-2708.

Yamada T, Shinnoh N, Taniwaki T, Ohyagi Y, Asahara H, Horiuchi, Kira J. 2000. Lovastatin does not correct the accu-mulation of very long-chain fatty acids in tissues of adrenoleukodystrophy protein-deficient mice. J Inherit Metab Dis 23:607-614.

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Chapter 5Bezafibrate for X-linked adrenoleukodystrophy

PLoS ONE (2012) 7(7): e41013

Marc Engelen1,3, Luc Tran2, Rob Ofman2, Josephine Brennecke2, Ann B. Moser4, Inge M.E. Dijkstra2, Ronald J.A. Wanders2, Bwee Tien Poll-The1,3, Stephan Kemp2,3

1Department of Neurology, 2Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, and 3De-partment of Pediatric Neurology/Emma Children’s Hospital, Academic Medical Center, University of Amsterdam,

Amsterdam, The Netherlands. 4Department of Neurogenetics, Kennedy Krieger Institute, Baltimore, MD, USA.

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Abstract

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene and is characterized by impaired beta-oxidation of very-long-chain fatty acids (VLCFA) and sub-sequent VLCFA accumulation in tissues. In adulthood X-ALD most commonly manifests as a gradually progressive myelopathy, (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate (BF), a drug used for the treatment of hyperlipidaemia, reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1, an enzyme involved in the VLCFA synthesis. We therefore designed a proof-of-principal clinical trial to determine whether BF reduces VLCFA levels in plasma and lymphocytes of X-ALD patients. Ten males with AMN were treated with BF for 12 weeks at a dose of 400 mg daily, followed by 12 weeks of 800 mg daily. Every 4 weeks patients were evaluated for side effects and blood samples were taken for analysis. Adherence was good as indicated by a clear reduction in triglycerides. There was no reduction in VLCFA in either plasma or lymphocytes. Plasma levels of BF did not exceed 25 µmol/L. We concluded that BF, at least in the dose given, is unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients. It is unclear whether this is due to the low levels of BF reached in plasma. Our future work is aimed at the identification of highly-specific inhibitors of ELOVL1 that act at much lower concentrations than BF and are well tolerated. BF appears to have no thera-peutic utility in X-ALD.

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Introduction

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder characterized by impaired beta-oxidation of very long-chain fatty acids (VLCFA) and accumulation of these VLCFA in tis-sues (Moser et al 2007). It is caused by mutations in the ABCD1 gene (www.x-ald.nl) (Mos-ser et al 1993). The disease is highly variable in clinical expression, however, in adulthood it most frequently manifests as a gradually progressive myelopathy and peripheral neuropa-thy (adrenomyeloneuropathy phenotype or AMN) (Moser et al 2007). Treatment for AMN is purely symptomatic and currently there is no proven intervention that can halt progression of the disease (Moser et al 2007). We identified ELOVL1 as the enzyme responsible for the synthesis of VLCFA (Ofman et al 2010), and demonstrated that siRNA-mediated knockdown of ELOVL1 lowers VLCFA levels in X-ALD fibroblasts (Ofman et al 2010). Next, we showed that bezafibrate (BF) reduces VLCFA levels in X-ALD fibroblasts by directly inhibiting ELOVL1 (Engelen et al 2012). BF is a drug of the fibrate class for the treatment of dyslipidaemia and has a proven safety profile for (long-term) use in humans (Miller and Spence, 1998). We therefore designed a proof of principal clinical trial to test whether BF can reduce VLCFA levels in the plasma and lymphocytes of patients with X-ALD.

Figure 1: Schematic representation of the BEZA trial design.

Methods

The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1 (online only). The BEZA trial study protocol was approved by the Institutional Review Board (Medisch Ethische Toetsings Commissie) of the Academic Medical Center. The trial is registered at clinicaltrials.gov (NCT01165060). Adult men with biochemically and genetically proven X-ALD without contra-indications for the use of BF were eligible for inclusion. All participating patients were evaluated at baseline for eligibility and received trial medication after written informed consent was obtained. They were evaluated at intervals of 4 weeks until the end of the trial at 24 weeks. The initial dose of BF was 400 mg per day, which was subsequently increased to 800 mg per day at week 12 (Figure 1). At each visit side effects were monitored, a general physical examination including weight was performed and blood samples taken. Blood samples were taken in the morning after an overnight fast before the first medication dose. Blood samples were analyzed at the laboratory for clinical chemistry for routine laboratory tests. VLCFA and BF levels were analyzed as previously described (Masnatta et al 1996; Valianpour et al 2003). Lysophosphatidylcholine-C26:0 (C26:0 lysoPC) was analyzed in bloodspots (Hubbard et al 2009). Data were analyzed with PASW statistics, version 18 (IBM). Statistical significance was evaluated with Student’s paired t-test.

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Results

Ten males with AMN participated in the trial. No side effects that necessitated discontinu-ation of the trial medication occurred. Body weight was unchanged (Table 1). There was a clear reduction in plasma triglycerides (1.34 mmol/L to 0.70 mmol/L at BF 400 mg and 0.71 mmol/L at BF 800 mg), and to a lesser extent a decrease in total cholesterol and LDL-cho-lesterol. There was also an increase in HDL-cholesterol (Table 1). These are known effects of BF and confirm patient adherence. There was no consistent reduction in C26:0 in plasma or lymphocytes, neither at 400 nor at 800 mg BF per day (Table 1). We observed an increase in plasma C22:0 and C24:0 at a dose of 800 mg BF per day. The amount of C26:0 lysoPC was unchanged in blood spots after 24 weeks of treatment with BF. The plasma level of BF did not exceed 25 µmol/L at the highest dose of 800 mg BF per day.

Table 1: Summary of the different parameters measured at the indicated time point in the trial.

Plasma Baseline BF 400 mg BF 800 mg

Total cholesterol (mmol/L)

5.57 ± 1.42 4.80 ± 0.88** 4.85 ± 0.84*

LDL (mmol/L) 3.67 ± 1.17 2.93 ± 0.80** 2.90 ± 0.73*

HDL (mmol/L) 1.39 ± 0.25 1.57 ± 0.25*** 1.64 ± 0.26***

TG (mmol/L) 1.34 ± 0.79 0.70 ± 0.31** 0.71 ± 0.22*

C22:0 (µmol/L) 43.32 ± 9.25 45.37 ± 8.12 55.02 ± 9.74**

C24:0 (µmol/L) 62.56 ± 11.68 64.08 ± 11.78 82.62 ± 12.50***

C26:0 (µmol/L) 3.26 ± 0.96 2.56 ± 0.50** 2.99 ± 0.49

C26:0/C22:0 ratio 0.075 ± 0.015 0.058 ± 0.012 0.056 ± 0.012**

Bezafibrate (µmol/L) n.d. n.d. 10.1 ± 6.7

Lymphocytes Baseline BF 400 mg BF 800 mg

C22:0 (nmol/mg) 5.89 ± 1.03 5.85 ± 1.65 5.10 ± 1.47

C24:0 (nmol/mg) 6.03 ± 0.78 6.45 ± 1.85 6.42 ± 1.44

C26:0 (nmol/mg) 0.35 ± 0.040 0.37 ± 0.084 0.40 ± 0.11

C26:0/C22:0 ratio 0.06 ± 0.01 0.06 ± 0.01 0.08 ± 0.03

bloodspots Baseline BF 400 mg BF 800 mg

C26:0 lysoPC 2.84 ± 1.40 2.50 ± 0.89 2.63 ± 1.18

Weight (kg) 86.7 ± 9.3 n.d. 87.9 ± 10.5

Summary of the different parameters measured at the indicated time point in the trial. Values are mean ± the standard deviation. Statistically significant differences from the baseline value are indica-ted. * p < 0.05, ** p < 0.01, *** p < 0.001. n.d. = not determined.

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Discussion

The pathophysiology of X-ALD is not well understood, although it seems likely that accu-mulation of VLCFA is toxic and related to neurodegeneration (Kemp et al 2012). Therefore drugs that reduce the level of VLCFA might be effective in halting or slowing progression of the disease.Recently, we showed that it is possible to reduce VLCFA in fibroblasts from X-ALD patients by inhibiting the synthesis of VLCFA by the enzyme ELOVL1 (Ofman et al 2010). We later showed that this can also be accomplished by incubating fibroblasts from X-ALD patients with BF (Engelen et al 2012).BF is a drug that has been in use for decades for the treatment of hypertriglyceridaemia and has an excellent safety profile (Miller and Spence, 1998). Therefore we decided to initiate this small scale proof of principle clinical trial to investigate whether BF reduces VLCFA in plasma and lymphocytes of X-ALD patients. In a previous clinical trial with lovastatin we demonstrated that reduction of plasma VLCFA can be an artifact of LDL reduction and does not reflect a reduction in blood cells (Engelen et al 2010). Unfortunately, we could not show a reduction of plasma or lymphocyte VLCFA levels. Con-versely, there was an unexpected increase in C22:0 and C24:0 levels in plasma. We did not observe this in blood cells or bloodspots.Our results show that there is no rationale for a large follow-up trial with clinical endpoints utilizing this compound. The concept of treating X-ALD patients with an inhibitor of VLCFA synthesis remains a feasi-ble option. It seems that BF is simply not efficacious enough. Our previous work suggests that BF is a competitive inhibitor of ELOVL1 (Engelen et al 2012). In our cell culture experi-ments a high concentration of BF of 400 µmol/L was required to achieve a maximal effect on the level of VLCFA. At this concentration the de novo VLCFA synthesis was reduced to the level in control cells. It is likely that even with the high dose of 800 mg BF per day, the intra-cellular levels of BF remained inadequate. Indeed, at the highest BF dosage plasma levels did not exceed 25 µmol/L with an average of 10 µmol/L (Table 1). These levels are not peak levels, but rather residual plasma levels. It is unlikely that concentrations even approaching 400 μmol/L were reached. This may explain the lack of in vivo efficacy of BF on our outcome parameters. To achieve the effect of VLCFA reduction, significantly higher BF concentrations are necessary as compared to concentrations indicated for reduction of TG.Future research will be focused on the identification of specific inhibitors of ELOVL1 that act at much lower concentrations than BF and are well-tolerated. In conclusion, BF appears to have no therapeutic utility in X-ALD.

Acknowledgements

This research was supported by the Netherlands Organization for Scientific Research (VIDI-91786328), the European Union Framework Programme 7 (LeukoTreat 241622) and The Stop ALD Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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References

Engelen M, Schackmann MJ, Ofman R, Sanders RJ, Dijkstra IM, Houten SM, Fourcade S, Pujol A, Poll-The BT, Wan-ders RJ, Kemp S. 2012. Bezafibrate lowers very long-chain fatty acids in X-linked adrenoleukodystrophy fibroblasts by inhibiting fatty acid elongation. J Inherit Metab Dis.

Engelen M, Ofman R, Dijkgraaf MGW, Hijzen M, van der Wardt LA, van Geel BM, de Visser M, Wanders RJA, Poll-The BT, Kemp S. 2010. Lovastatin in X-Linked Adrenoleukodystrophy. N Engl J Med 362:276-277.

Hubbard WC, Moser AB, Liu AC, Jones RO, Steinberg SJ, Lorey F, Panny SR, Vogt RF, Jr., Macaya D, Turgeon CT, Torto-relli S, Raymond GV. 2009. Newborn screening for X-linked adrenoleukodystrophy (X-ALD): validation of a combined liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Mol Genet Metab 97:212-220.

Kemp S, Berger J, Aubourg P. 2012. X-linked adrenoleukodystrophy: Clinical, metabolic, genetic and pathophysiolo-gical aspects. Biochim Biophys Acta 1822:1465-74.

Masnatta LD, Cuniberti LA, Rey RH, Werba JP. 1996. Determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma by high-performance liquid chromatography. J Chromatogr B Biomed Appl 687:437-442.

Miller DB, Spence JD. 1998. Clinical pharmacokinetics of fibric acid derivatives (fibrates). Clin Pharmacokinet 34:155-162.

Moser HW, Mahmood A, Raymond GV. 2007. X-linked adrenoleukodystrophy. Nat Clin Pract Neurol 3:140-151.

Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, Poustka AM, Mandel JL, Aubourg P. 1993. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 361:726-730.

Ofman R, Dijkstra IM, van Roermund CW, Burger N, Turkenburg M, van Cruchten A, van Engen CE, Wanders RJ, Kemp S. 2010. The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy. EMBO Mol Med 2:90-97.

Valianpour F, Selhorst JJ, van Lint LE, van Gennip AH, Wanders RJ, Kemp S. 2003. Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol Genet Metab 79:189-196.

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Chapter 6X-linked adrenomyeloneuropathy due to a novel missense

mutation in the ABCD1 start codon presentingas demyelinating neuropathy

Journal of the Peripheral Nervous System (2011) 16: 353 – 355

Marc Engelen1,2, Anneke J. van der Kooi1, Stephan Kemp2, Ronald J.A. Wanders2, Erik A. Sistermans3, Johannes T.M. Koelman1, Hans R. Waterham2, Björn M. van

Geel1, 4, Marianne de Visser1

1Department of Neurology and 2Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam; 3Department of Clinical Genetics, VU Medical Center, Amsterdam; and 4Department of Neurology, Medical Center

Alkmaar, The Netherlands

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X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene which encodes the adrenoleukodystrophy protein (ALDP), a peroxisomal transmembrane pro-tein (Mosser et al 1993; Moser et al 2001). Several often overlapping phenotypes can be distinguished. Approximately, half of all patients develop adrenomyeloneuropathy (AMN), characterized by slowly progressive spastic paraparesis, peripheral neuropathy, sphincter dysfunction, and adrenocortical insufficiency (Moser et al 2001). We present a patient with an unusual neuropathy who was shown to have X-ALD caused by a mutation in the ABCD1 gene. A 27-year-old man presented with exercise-related weakness and fatigue in the legs which progressed slowly over the last 2 years. Three years prior to presentation he shortly used testosterone replacement therapy for decreased libido. Family history was negative for neuromuscular and endocrinological disorders. Physical examination revealed slight ge-neralized wasting and weakness of the legs and decreased sensation for light touch at the dorsum of both feet with decreased position sense of the toes. Pes cavus was not present. Deep tendon reflexes were normal, and plantar reflexes were flexor. Electrophysiologic fin-dings were compatible with a mild, symmetric demyelinating neuropathy confined to the lower limbs (Table 1). Serum creatine kinase activity was elevated (270 U/L, normal <193 U/L). Normal or negative results were obtained for thyroid stimulating hormone, sodium and potassium, and vasculitis parameters (anti-neutrophil cytoplasmic antibody, antinuclear antibody and complement levels). Cerebrospinal fluid examination showed elevated protein (0.66 g/L, upper limit of normal 0.49). DNA analysis revealed no mutations in the PMP22 gene. Refsum’s disease was considered, but the concentration of phytanic acid in plasma was normal. When phytanic acid levels are determined in our laboratory, we routinely in-clude very long-chain fatty acids (VLCFA) as well. The VLCFA plasma C26:0 concentration was 4.32 μmol/L (normal 0.45–1.32 μmol/L), and C26:0/C22:0 ratio was 0.09 (normal ≤0.02). These results prompted mutation analysis of the ABCD1 gene, which revealed a missense mutation (c.1A>G) in the start codon of the ABCD1 gene, resulting in the substitution of the initiator methionine to a valine residue (p.Met1Val). The effect of the mutation on the stability of ALDP was investigated in cultured skin fibroblasts by means of immunofluores-cence (IF) microscopy analysis and western blot analysis (Ligtenberg et al 1995). Both IF and western blot analysis showed no detectable ALDP (Figure 1). These data indicate that most likely translation is not initiated. Endocrinological investigations revealed mild adreno-cortical insufficiency with a dehydroepiandrosteron sulphate level of 5.9 µmol/L (normal 8–17) that did not increase after administration of tetracosactide. The basal morning cor-tisol level was 250 μmol/L (normal 220–650) with a normal increase after administration of tetracosactide. The adrenocorticotropic hormone level was 14 ng/L (normal <55). There was also testicular insufficiency with a testosterone level of 7.9 nmol/L (normal 12.3–20), sex hormone binding globulin 18 nmol/L (normal 16–38), free androgen index 59.5 (normal 49–89), luteinizing hormone (LH) 4.0 U/L (<15), and follicle stimulating hormone (FSH) 3.1 U/L (normal 1.0–10.0). MRI of the cerebrum (Loes X-ALD severity score 0) (Loes et al., 1994) and spinal cord was normal. Additional DNA analysis revealed that the patient’s mother was an asymptomatic carrier for X-ALD. Recently, the patient’s tendon reflexes have become more brisk, suggestive of spinal cord involvement. X-ALD is a clinically heterogeneous disorder. Most patients either have childhood cerebral adrenoleukodystrophy or the AMN phenotype (Moser et al 2001). Our patient’s presen-tation is unusual in that he presented with exercise-related weakness and fatigue in the legs and was found to have a demyelinating neuropathy, only in the legs. We found one similar case in a series of teenagers with polyneuropathy (Kararizou et al 2006). Detailed

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genetic analysis and nerve conduction findings were not reported. Recently a patient with pes cavus, spasticity and a primarily demyelinating neuropathy with variable, focal slowing of motor nerve conduction velocities (NCVs) but relative preservation of motor and sensory

Tabl

e 1:

Ele

ctro

neur

ogra

phic

al fe

atur

es.

Mot

or

nerv

eDi

stal

late

ncy

(ms)

Dist

al

com

poun

d m

uscl

e ac

tion

pote

ntial

(m

V)

Prox

imal

co

mpo

und

mus

cle

actio

n po

tenti

al

(mV)

Mot

or n

erve

co

nduc

tion

velo

city

(m

/s)

Sens

ory

nerv

eDi

stal

la

tenc

y (m

s)

Sen-

sory

ner

ve

actio

n po

tenti

al

(µV)

Sens

ory

nerv

e co

n-du

ction

ve

loci

ty

(m/s

)M

edia

n L

3.5

20.5

18.6

52M

edia

n L

4.0

18.3

51U

lnar

R3.

219

.519

.852

Uln

ar R

3.3

18.4

53Pe

rone

al L

6.7

6.0

4.7

28Su

ral R

6.6

5.0

34Ti

bial

R10

.33.

21.

931

H-w

aves

F-w

aves

Ner

veM

-wav

e (m

s)H-

wav

e (m

s)N

erve

Mus

cle

Min

imal

late

ncy

(ms)

Tibi

al L

6.3

43.4

Med

ian

LAb

duct

or p

ollic

is br

evis

29.6

Tibi

al R

5.3

44.0

Uln

ar R

Abdu

ctor

dig

iti q

uinti

31.5

Pero

neal

LEx

tens

or d

igito

rum

br

evis

76.4

Skin

tem

pera

ture

was

kep

t at 3

2 °C

. Abn

orm

al fi

gure

s are

show

n in

bol

d. L

, left

; R, r

ight

.

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84

amplitudes was described who was found to have both a PMP22 gene deletion consistent with a hereditary neuropathy with liability to pressure palsies and a c.1166G>A mutation in the ABCD1 gene, causing an amino acid substitution at codon 389 (p.Arg389His) (Hodapp et al 2006). Whether polyneuropathy in AMN patients is primarily demyelinating or axonal by nerve conduction studies is controversial. We have reported previously that polyneuropathy in this condition is predominantly axonal on electrophysiological examination (van Geel et al 1996), whereas Chaudhry et al. have reported that demyelination occurs more frequently (Chaudhry et al 1996). This is corroborated by histological studies of nerve biopsies of pa-tients with AMN in whom also either a predominantly demyelinating or axonal neuropathy can be observed even within the same family (Schröder et al 1996). It is important to realize that X-ALD should be considered in the differential diagnosis of polyneuropathy at presenta-tion, whether it is predominantly axonal or demyelinating.

ReferencesChaudhry V, Moser HW, Cornblath DR. 1996. Nerve conduction studies in adrenomyeloneuropathy. J Neurol Neu-rosurg Psychiatr 61:181–185.

Hodapp JA, Carter GT, Lipe HP, Michelson SJ, Kraft GH, Bird TD. 2006. Double trouble in hereditary neuropathy: con-comitant mutations in the PMP-22 gene and another gene produce novel phenotypes. Arch Neurol 63:112–117.

Kararizou E, Karandreas N, Davaki P, Davou R, Vassilopoulos D. 2006. Polyneuropathies in teenagers: a clinicopatho-logical study of 45 cases. Neuromuscul Disord 16:304–307.

Ligtenberg MJ, Kemp S, Sarde CO, van Geel BM, Kleijer WJ, Barth PG, Mandel JL, van Oost BA, Bolhuis PA. 1995. Spectrum of mutations in the gene encoding the adrenoleukodystrophy protein. Am J Hum Genet 56:44–287.

Loes DJ, Hite S, Moser H, Stillman AE, Shapiro E, Lockman L, Latchaw RE, Krivit W. 1994. Adrenoleukodystrophy: a scoring method for brain MR observations. AJNR Am J Neuroradiol 15:1761–1766.

Moser H, Smith K, Watkins P, Powers J. 2001. X-linked adrenoleukodystrophy. In: The Metabolic & Molecular Bases of Inherited Disease. Scriver R, Beaudet AL, Sly WS, and Valle D (Eds). McGraw-Hill Inc., New York, pp 3257–3301.

Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, Poustka AM, Mandel JL, Aubourg P. 1993. Putative X-linked adrenoleukodystrophy gene shares unexpected Homology with ABC transporters. Nature 361:726–730.

Schröder JM, Mayer M, Weis J. 1996. Mitochondrial abnormalities and intrafamilial variability of sural nerve biopsy findings in adrenomyeloneuropathy. Acta Neuropathol 92:64–69.

van Geel BM, Koelman JH, Barth PG, Ongerboer de Visser BW. 1996. Peripheral nerve abnormalities in adrenomy-eloneuropathy: a clinical and electrodiagnostic study. Neurology 46:112–118.

Wanders RJ, Dekker C, Ofman R, Schutgens RB, Mooijer P. 1995. Immunoblot analysis of peroxisomal proteins in liver and fibroblasts from patients. J Inherit Metab Dis 18 (Suppl1):101–112.

Figure 1: The p.Met1Val mutation affects ALDP stability. The level of ALDP in fibroblasts from controls and X-ALD patients was determined by western blot analysis as des-cribed previously (Wanders et al., 1995). A monoclonal antibody, directed against an epitope between the amino acids 279 and 482 of the human ALDP was used. Lanes 1 and 4, protein extracts from two different control sub-jects; lane 2, the index patient; lane 3, protein extract from an X-ALD patient with a p.Arg554His mutation that results in no detectable ALDP (www.x-ald.nl).

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Chapter 7The clinical, biochemical and genetic spectrum of X-linked

adrenoleukodystrophy in women:a cross-sectional cohort study

manuscript in preparation

Marc Engelen1,2, Mathieu Barbier3,4, Inge M.E. Dijkstra5, Remmelt Schür2, Rob M.A. de Bie1, Camiel Verhamme1, Marcel G.W. Dijkgraaf6, Patrick A. Aubourg3,4,

Ronald J.A. Wanders5, Björn M. van Geel7, Marianne de Visser1,Bwee Tien Poll – The1,2 and Stephan Kemp2,5

Departments of 1Neurology and 2Pediatric Neurology/Emma Children’s Hospital, Academic Medical Center,University of Amsterdam, Amsterdam, The Netherlands. 3Assistance Publique des Hôpitaux de Paris, Department of Pediatric Neurology, Hospital Kremlin-Bicêtre, Paris, France. 4INSERM UMR745- University Paris-Descartes, Paris,

France. 5Laboratory Genetic Metabolic Diseases and 6Clinical Epidemiology, Biostatistics & Bio-informatics,Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 5Department of Neurology,

Medical Center Alkmaar, Alkmaar, The Netherlands.

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Abstract

X-linked adrenoleukodystrophy is a peroxisomal disorder caused by mutations in the ABCD1 gene. It is characterized by impaired very long-chain fatty acid (VLCFA) beta-oxidation and VLCFA accumulation in plasma and tissues. Clinically, the disease has been well characterized in boys and men. However, women can develop symptoms and are not merely carriers. In this cross-sectional cohort study we investigated symptoms and prevalence of these symptoms in women with X-ALD. A secondary goal was to determine if the X-inactivation pattern of the ABCD1 gene was associated with symptomatic status. We found that women with X-ALD develop signs and symptoms of myelopathy (38/46, 70%) and/or peripheral neuropathy (17/46, 37%). Especially striking was the occurrence of fecal incontinence (13/48, 28%). The prevalence of symptomatic women increased sharply with age (from 36% in women under 40 to 88% in women over 60 years of age). Most X-ALD carriers had increased VLCFA in plasma and/or fibroblasts, and/or decreased VLCFA beta-oxidation in fibroblasts. We did not find an association between the X-inactivation pattern and symptomatic status. X-ALD carriers developed an adrenomyeloneuropathy-like phenotype and there is a strong association between symptomatic status and age. Clinicians should consider X-ALD in women with a myelopathy (especially with early fecal incontinence).

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Introduction

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder characterized by deficient beta-oxidation of very long-chain fatty acids (VLCFA; ≥C22:0) and accumulation of VLCFA in plasma and tissues (Moser et al 2007). It is caused by mutations in the ABCD1 gene (Mosser et al 1993). In men with X-ALD, disease course and symptomatology have been studied extensively, as reviewed by Moser et al (Moser et al 2007). In adult men with X-ALD, the most frequent phenotype is adrenomyeloneuropathy (AMN), with or without adrenocortical insufficiency (Moser et al 2007). It is characterized by a slowly progressive myelopathy and peripheral neuropathy, manifesting as a spastic paraparesis, sensory disturbance in the lower extremities and incontinence. Onset is typically in the 3rd decade of life, but can be sooner or much later (Moser et al 2007).

However, the phenotype of X-ALD in women has not been systematically investigated in a large prospective study. There have been several studies and observations concerning women with X-ALD, although these are rare compared to the many studies on X-ALD in men. In 1984, 21 obligate carriers of X-ALD were described and several were shown to have neurological symptoms, mostly signs and symptoms of myelopathy (O’Neill et al 1984). It was estimated that that roughly 50% of women with X-ALD develop symptoms at some point in their life (Moser et al 1991). In a small study 8 women with X-ALD were clinically characterized and were found to have a myelopathy (Schmidt et al 2001). In a retrospective study from the Netherlands many women were found to show neurological symptoms and the prevalence of symptoms increased with age (van Geel, 2001). It is known that adrenal insufficiency is rare in women with X-ALD (el-Deiry et al 1997), and there are only a few reports of cerebral X-ALD in women (Pilz et al 1973; Jung et al 2007). These studies provide no information on the prevalence of neurologic symptoms in the entire population of women with X-ALD, since data was acquired in small groups and may be biased towards women with symptoms. The spectrum in women with X-ALD may be wider than in affected men, ranging from asymptomatic to severely affected. Some studies suggest this could be related to the pattern of X-inactivation, while others can not confirm this correlation (Migeon et al 1981; Watkiss et al 1993; Maier et al 2002; Salsano et al 2012; Naidu et al 1997).

To summarize, the studies done so far are prone to selection bias since they favor inclusion of those women with symptoms and might overestimate the true prevalence of neurologic symptoms in women with X-ALD.

The primary goal of our study was to determine the proportion of women with X-ALD that have symptoms and to characterize these symptoms both clinically and with ancillary investigations. The secondary goal was to investigate whether symptomatic women differ biochemically from women without a clinical phenotype.

Methods

Research populationThe study protocol was approved by the Institutional Review Board of the Academic Medical Center. This study was designed as a prospective cross-sectional cohort study. All female

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88

carriers over the age of 18 years were eligible to participate. Women with X-ALD were recruited from the outpatient clinics of the Academic Medical Center (a reference center for peroxisomal disorders in the Netherlands) and Medical Center Alkmaar. To prevent bias for symptomatic women, we tried to examine all female relatives with X-ALD of the women participating. Through the Dutch X-ALD patient organization letters to invite women to participate were sent to all members. After informed consent was obtained, women visited the outpatient clinic for assessment, including questionnaires, neurological examination, neurophysiological tests, blood samples and skin biopsy.

Clinical assessmentA careful history with emphasis on neurologic complaints and neurological examination was performed. In particular, symptoms of incontinence, gait disorder and maximum walking distance, and sensory disturbance were recorded. Urinary incontinence was defined as urge incontinence. Stress incontinence was not considered to be a symptom of myelopathy in this study. 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 or pinprick, vibration- or position sense in the lower extremities. Muscle strength was rated using the MRC scale, and spasticity was rated using the Ashworth scale. An EDSS value (Kurtzke, 1983) was scored based on the information in the medical records. Patients with a peripheral neuropathy or myelopathy were considered symptomatic. A myelopathy was considered to be present if 1) there were symptoms of myelopathy (for instance sphincter disturbances) and 2) signs of myelopathy on neurological examination (pyramidal tract or dorsal column signs) were present. A peripheral neuropathy was considered present if the NCV and/or myography were abnormal (as described below).All participants completed the SF36 (quality of life assessment) and the AMC Linear Disability Scale (ALDS) (Weisscher et al 2007). SF36 values can be compared to reference values for the Dutch population, matched for gender and age. The ALDS values are compared to those of patients with Parkinson’s disease from the CARPA study (Post et al 2011).

Neurophysiological testingNerve conduction studies and myography were performed according to a fixed protocol, allowing comparison with reference values and possibly follow-up in the future (Verhamme et al 2009). An axonal peripheral neuropathy was diagnosed if in two separate nerves at least 1 parameter was outside the 95% confidence interval and the criteria for demyelinating peripheral neuropathy were not fulfilled (van Asseldonk et al 2005). Somatosensory evoked potentials (SSEP) of left median and left and right posterior tibial nerve (Aramideh et al 1992; Aalfs et al 1993) and brainstem auditory evoked potentials (BAEP) on the left and right side were registered, conforming to local protocols.

Blood samplesVenous blood samples were taken and plasma and lymphocytes were isolated and stored as described previously (Engelen et al 2010). VLCFA were determined in these samples as described previously (Valianpour et al 2003).

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Cell culture and biochemical analysisFrom skin biopsy material primary fibroblast cell lines were isolated. Cell lines were cultured in HAMF10 culture medium supplemented with 10% fetal calf serum. For all subjects for whom material was available VLCFA were determined in plasma and fibroblasts as described previously (Valianpour et al 2003). Peroxisomal beta-oxidation activity was measured as described previously by incubating cells with deuterium labeled C22:0 (Kemp et al 2004). ALDP levels were determined by immunofluorescence (IF) as well as by quantitative immunoblot (Kemp et al 1996; Zhang et al 2011). Genetic analysis and ABCD1 allelic specific expression in fibroblastsMutation analysis of the ABCD1 gene was performed as described previously (Boehm et al 1999). The ABCD1 allelic specific expression (ABCD1 ASE) of 38 carriers was measured by pyrosequencing. The RNA from primary fibroblasts was extracted. The cDNA synthesis has been described elsewhere (Bieche at al 2001). The ratio of wild-type versus mutant allele expressed was measured by pyrosequencing. We PCR-amplified the cDNA using biotin-labeled primers and performed quantitative pyrosequencing using a PyroMark Q96 ID instrument (Qiagen, Venlo, The Netherlands). Biotin-labeled single stranded amplicons were isolated according to protocol using the Qiagen Pyromark Q96 Work Station and underwent pyrosequencing with sequencing primer. The list of PCR- and sequencing-primers is indicated in supplemental table S1 (online only). Each sample was pyrosequenced in upper direction (with lower biotin-labeled amplicons) and in lower direction (with upper biotin-labeled amplicons). The ratio of wild-type and mutant allele was measured by the Pyro Mark ID software (Qiagen). For each carrier, the ABCD1 ASE value represents the mean of upper and lower reads (two independent experiments). For included samples, variations between upper and lower reads were ≤ 10%. For a subset of samples (n=6), we repeated the experiments using different PCR-primers or different cDNAs. The ABCD1 ASE values were similar (variations ≤ 4%).For five patients it was not technically possible to determine the ABCD1 ASE with this technique due to complex mutational events. For these five cases, values were calculated using ALDP immunofluorescence and determining the ratio of ALDP positive and negative cells with IF-microscopy. We defined the ratio of ABCD1 allele expressed as: “severely” skewed when the ratio of normal or mutated allele was between 0% and 10%; “moderately” skewed when the ratio of normal or mutated allele was between 11% and 25%; “randomly” when the ratio of normal or mutated allele was between 26% and 74%, similar to a previous report (Miozzo et al 2007). In the manuscript ABCD1 ASE will be referred to as X-inactivation.

Data entry and statistical analysisData were entered in and analyzed with IBM SPSS statistics version 19 (IBM Inc, New York, U.S.A.) and Prism version 5 (GraphPad Software, La Jolla, U.S.A.). The relationship between symptomatic status, age and X-inactivation pattern (ABCD1 ASE) was analyzed by logistic regression.

Results

Demographic and clinical characteristicsA total of 46 women from 26 kindreds were enrolled in the study. Of these participants

Chapter 7

7

90

Fam

ilyAg

eU

Inc

F In

cG

ait

Sens

CSe

ns D

SpPa

rP

Ref

EDSS

Mut

ation

ALDP

A56

Yes

Yes

No

No

No

No

No

Yes

1.5

p.Pr

o480

Thr

abse

nt

A44

No

No

Yes

No

No

No

No

Yes

1.0

p.Pr

o480

Thr

abse

nt

AA45

No

No

No

No

No

No

No

Yes

0p.

Arg6

60Tr

pab

sent

AA59

Yes

No

Yes

No

No

No

Yes

Yes

3.5

p.Ar

g660

Trp

abse

nt

AA75

Yes

No

Yes

No

Yes

Yes

Yes

Yes

6.0

p.Ar

g660

Trp

abse

nt

B42

Yes

Yes

Yes

No

Yes

Yes

Yes

Yes

4.0

p.Le

u220

Pro

redu

ced

B44

No

No

No

No

No

No

No

No

0p.

Leu2

20Pr

ore

duce

d

B44

No

No

No

No

No

No

No

No

0p.

Leu2

20Pr

ore

duce

d

B51

No

No

No

Yes

Yes

No

No

Yes

1.0

p.Le

u220

Pro

redu

ced

B59

No

No

No

Yes

Yes

No

Yes

No

2.0

p.Le

u220

Pro

redu

ced

C44

No

No

No

No

No

No

No

No

0p.

Gln1

33*

abse

nt

D38

Yes

Yes

Yes

No

Yes

Yes

Yes

Yes

6.0

p.Le

u654

Pro

abse

nt

D57

Yes

No

Yes

Yes

Yes

No

No

Yes

5.5

p.Le

u654

Pro

abse

nt

E31

No

No

No

No

No

No

No

No

0p.

Arg7

4Trp

abse

nt

E37

No

No

No

No

No

No

No

No

0p.

Arg7

4Trp

abse

nt

E60

No

No

Yes

No

Yes

Yes

Yes

Yes

5.5

p.Ar

g74T

rpab

sent

F35

No

No

No

No

No

No

No

No

0p.

Met

1Val

abse

nt

G42

No

Yes

No

No

No

No

No

No

1.0

p.Al

a245

Asp

pres

ent

H61

Yes

Yes

Yes

Yes

Yes

No

No

Yes

3.5

exon

8-10

del

abse

nt

I71

No

No

No

No

Yes

No

No

Yes

2.0

p.Gl

u609

Lys

abse

nt

J42

No

No

No

No

Yes

No

No

Yes

1.5

p.Gl

u90*

abse

nt

K31

No

No

No

No

No

No

No

No

0p.

Pro5

43Le

uab

sent

K48

Yes

No

No

No

Yes

No

No

Yes

2.5

p.Pr

o543

Leu

abse

nt

Table 1: Summary of demographic, clinical and genetic data of all the women participating in the study.

Age: age at examination, U Inc: urinary incontinence, F Inc: fecal incontinence, Gait: gait disorder, Sens C: sensory complaints, Sens D: sensory symptoms on neurological examinations, Sp: spasticity, Par: paresis of the lower extremities, P Ref: pathological reflexes (lower extremities), EDSS: expanded disability status scale, Mutation: mutation in ABCD1, ALDP: effect of mutation in ABCD1 on ALDP as determined by Western blot. & mutation inferred from mutation identified in father and uncle.

Extension of the phenotype

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Fam

ilyAg

eU

Inc

F In

cG

ait

Sens

CSe

ns D

SpPa

rP

Ref

EDSS

Mut

ation

ALDP

K57

No

No

Yes

Yes

Yes

No

Yes

Yes

3.5

p.Pr

o543

Leu

abse

nt

K60

Yes

No

No

No

Yes

No

No

Yes

3.5

p.Pr

o543

Leu

abse

nt

L51

Yes

No

Yes

No

Yes

Yes

Yes

Yes

6.5

p.Ile

657d

elab

sent

M22

No

No

No

No

No

No

No

No

0p.

Ser1

49As

nre

duce

d

M40

No

No

No

No

No

No

No

No

0p.

Ser1

49As

nre

duce

d

N29

No

No

No

No

No

No

No

No

0p.

Arg3

89Hi

sre

duce

d

N45

Yes

No

Yes

Yes

No

No

No

No

2.0

p.Ar

g389

His

redu

ced

N57

Yes

Yes

Yes

Yes

Yes

No

No

No

3.5

p.Ar

g389

His

redu

ced

N70

No

No

Yes

No

Yes

No

Yes

Yes

3.5

p.Ar

g389

His

redu

ced

O40

Yes

Yes

Yes

Yes

Yes

No

No

Yes

3.5

p.Gl

u609

Lys

abse

nt

P59

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

6.0

p.Le

u215

*ab

sent

Q39

No

Yes

Yes

No

Yes

No

No

No

3.0

p.Va

l208

Trpf

sab

sent

R28

No

No

No

No

No

No

No

No

0p.

Pro4

80Th

rab

sent

S35

No

No

No

No

No

No

No

No

0p.

His2

83Ty

rre

duce

d

S76

Yes

No

Yes

No

Yes

No

No

Yes

2.0

p.Hi

s283

Tyr

redu

ced

T51

Yes

Yes

Yes

No

Yes

No

Yes

Yes

4.0

p.Gl

n177

*ab

sent

U47

Yes

Yes

No

No

Yes

No

No

No

2.0

p.Ar

g464

*ab

sent

V56

Yes

No

Yes

No

Yes

No

Yes

Yes

2.5

p.As

p442

Glyf

sab

sent

W45

Yes

Yes

Yes

Yes

Yes

No

No

Yes

3.5

p.Al

a616

Thr

abse

nt

W65

No

Yes

No

No

No

No

No

No

2.0

p.Al

a616

Thr

abse

nt

X47

Yes

No

Yes

No

No

No

No

Yes

2.5

p.Ar

g113

Alaf

sab

sent

Y24

No

No

No

No

No

No

No

No

0p.

Glu6

09Gl

y&ab

sent

&

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12 were recruited from the outpatient clinic, 34 were female relatives of these women or responded to the letter sent to members of the patient organization.Complete clinical and electrophysiological data are available from 46 women, blood samples are available from 45 women and skin biopsies were obtained from 43 women (one obligate carrier refused venapuncture, two refused skin biopsy, one skin biopsy failed due to a yeast infection). The age of the women included in the study ranged from 22 to 76 years (average 48 ± 13 years). The age distribution is shown in Figure 1. Mutation analysis was done in 45 women (as for one obligate carrier no leukocytes were available), the mutation in that subject was considered to be the mutation found in her father and paternal uncle. Complaints of incontinence (both urinary and fecal), abnormal gait, and sensory symptoms were common in the group studied, as listed in Table 1. Data is summarized in Table 2. There was a myelopathy (as defined in the methods section) in 32/46 (70%) of the women. Of the women in the cohort 34/46 (74%) were considered symptomatic because of either a myelopathy and/or a peripheral neuropathy. Demographic and clinical data is shown in Figure 1 and Table 1. Women who have signs and symptoms suggestive of myelopathy increase in frequency with age (Table 2). The percentage of women that are symptomatic increases from 4/11 (36%) in the youngest age group, and increases to 30/35 (86%) in the older age groups. Logistic regression shows a strong relationship between age and symptomatic status, with the probability of being symptomatic increasing strongly with age (Wald 9.620, p = 0.002). The SF-36 physical and mental compound scores and the ALDS score are reported for the entire cohort by age group (Table 2).

Electrophysiological testing

Nerve conduction studies and myographyOf all participants NCV, EMG and evoked potentials (BAEP and SSEP) were available. Of the women 17/46 (37%) had a sensorimotor axonal neuropathy according to local electrophysiological criteria (Table 3).

Figure 1: Age distribution of the cohort.

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Somatosensory evoked potentials (median nerve and posterior tibial nerve)The SSEP from the median nerve was abnormal in 3/42 (6.5%) of cases (4 registrations failed because of technical reasons), of the tibial nerve 14/44 (30.4%) was abnormal (2 registrations failed). Of the female carriers who were asymptomatic on neurological examination (11/46), the median nerve SSEP was normal in all cases and the SSEP of the posterior tibial nerve was abnormal in only 1/11 (9%). Of the women who were symptomatic on neurological examination (35/46), 3/38 (8.6%) had an abnormal median nerve SSEP and 13/35 (37.1%) had an abnormal tibial nerve SSEP. The average latency of the cortical peak increases with age for median nerve and tibial nerve SSEP. The percentage of abnormal SSEP increases with age. The findings from the SSEP from median and tibial nerve for the complete cohort are reported in Table 4.

Brainstem auditory evoked potentialsIn the entire cohort BAEP measurements were done in 45 women. The measurement failed for technical reasons in one participant. BAEP results are shown in Table 4. For the entire group 26/46 (41.3%) women had an abnormal BAEP, mostly consisting of an increase I – V and I – III interval. There were no significant left – right differences. Of the women who were symptomatic, 23/35 (65.7%) had an abnormal BAEP result. In the asymptomatic group this proportion is 3/7 (27.3%).

18 - 39 yrs 40 - 59 yrs > 60 yrs all ages

Incontinence (urine) 1/11 (9) 15/27 (56) 5/8 (63) 21/46 (46)

Incontinence (fecal) 2/11 (18) 9/27 (33) 2/8 (25) 13/46 (28)

Gait disorder 2/11 (18) 15/27 (56) 5/8 (63) 22/46 (48)

Sensory complaints 1/11 (9) 12/27 (44) 2/8 (25) 15/46 (33)

Sensory disturbance 2/11 (18) 12/27 (44) 6/8 (75) 23/26 (50)

Spasticity 1/11 (9) 3/27 (11) 1/8 (13) 5/46 (11)

Weakness 1/11 (9) 7/27 (26) 2/8 (25) 10/46 (22)

Pathological reflexes 2/11 (18) 19/27 (70) 6/8 (75) 27/46 (59)

Abnormal history 3/11 (27) 21/27 (78) 6/8 (75) 30/46 (65)

Abnormal examination 4/11 (36) 22/27 (82) 6/8 (75) 32/46 (70)

Symptomatic 4/11 (36) 23/27 (85) 7/8 (88) 34/46 (74)

EDSS 0.82 (1.94) 2.41 (1.83) 3.50 (1.56) 2.22 (1.99)

SF-36 physical 50.0 (10.6) 44.0 (15.1) 44.4 (13.4) 45.3 (13.7)

SF-36 mental 51.4 (4.3) 51.5 (8.1) 51.1 (5.8) 51.4 (6.87)

ALDS 89.1 (0.7) 87.4 (4.2) 84.4 (7.0) 87.4 (4.4)

Table 2: Symptoms and signs by age group.

Symptoms and signs (reported as absolute number (%)), EDSS, SF-36 and ALDS scores (reported as mean ± SD) for the entire cohort, and stratified by age group. SF-36 physical: SF-36 physical com-pound score, SF-36 mental: SF-36 mental compound score.

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Table 3: Summary of nerve conduction studies.

Mean (SD) Reference interval

Median nerve

dlt (ms) 3.6 (0.8) 3.2 – 3.6

CMAP (mV) 6.2 (2.9) 7.1 – 8.8

MNCV (m/s) 58.4 (11.6) 56.9 – 59.5

Posterior tibial nerve

dlt (ms) 5.1 (1.0) 4.4 – 5.0

CMAP (mV) 10.0 (5.5) 8.5 – 12.2

MNCV (m/s) 41.6 (6.4) 44.1 – 47.2

Sural nerve

dlt (ms) 3.9 (0.5) 2.9 – 3.2

SNAP (uV) 7.2 (4.9) 7.5 – 10.8

Summated CMAP 19.8 (8.4) 21.6 – 26.3

Summated SNAP 39.8 (18.1) 39.7 – 54.5

18 - 39 yrs 40 – 59 yrs > 60 yrs all ages

SSEP arm

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 (11.5) 0/6 (0) 3/42 (6.5)

SSEP leg

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 (10.0) 11/27 (40.7) 2/7 (28.6) 14/44 (30.4)

BAEP

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 (36.4) 17/26 (65.4) 5/8 (62.5) 26/45 (57.8)

Table 4: Results of somatosensory evoked potentials (median and tibial nerve) and brainstem auditory evoked potentials.

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Biochemical and genetic analysis

Plasma and leukocytesFor 45/46 participant blood samples were obtained and VLCFA levels in plasma were determined. The data are summarized in Table 5. VLCFA were clearly increased with an average of 2.26 µmol/L ± 0.69 µmol/L (reference values 0.88 – 1.21 µmol/L). 31/46 women (68.9%) had abnormal plasma VLCFA levels.

VLCFA levels, ALDP levels and beta-oxidation activity in fibroblastsVLCFA levels, VLCFA beta-oxidation in skin fibroblasts, ABCD1 mutation analysis, fibroblast ALDP levels (as determined by immunoblot) and fibroblast ALDP immunofluorescence were determined if material was available. In 37/43 (86%) C26:0 levels in fibroblasts were increased, and 36/43 the C26:0/C22:0 (84%) ratio was abnormal. In 26/43 (60%) the D3-C16:0/D3-C22:0 was decreased, indicating reduced peroxisomal VLCFA beta-oxidation. Residual levels of ALDP were determined by immunoblot and correlate with residual VLCFA beta-oxidation and C26:0 levels, i.e. the more residual ALDP the higher the VLCFA beta-oxidation and the lower fibroblast C26:0 levels. Results are summarized in Table 5 and Figure 2.

ABCD1 allelic specific expression (ABCD1 ASE) studies in fibroblasts and correlation of X-inactivation with symptomatic or asymptomatic status.Data for ABCD1 ASE is shown in Figure 3. It is randomly distributed and correlates well with beta-oxidation activity and ALDP expression in cultured fibroblasts (Figure 4). This suggests the methodology used to determine ABCD1 ASE is valid. To correlate ABCD1 ASE with symptomatic status it is important to correct for age, since there is a strong correlation between age and symptomatic status (Table 2). There seems to be no correlation between symptomatic status and the ABCD1 ASE pattern.

Discussion

Women with X-ALD can develop neurological symptoms. In fact, one of the earliest descriptions of an AMN-like phenotype in X-ALD concerns a female patient (Penman, 1960).

Mean (SD) Reference interval

Plasma

C26:0 (µmol/L) 2.26 (0.69) 0.88 – 1.21

C26:0/C22:0 ratio 0.035 (0.012) 0.013 – 0.019

Fibroblasts

C26:0 (nmol/mg protein) 0.75 (0.32) 0.12 – 0.22

C26:0/C22:0 ratio 0.22 (0.10) 0.035 – 0.072

D3-C16:0/D3-C22:0 ratio 0.73 (0.41) 1.58 – 1.82

Table 5: Results of plasma and fibroblast studies.

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This is, however, not so obvious for most physicians (Jangouk et al 2011). We know of several cases in which women with X-ALD underwent cervical laminectomy for suspected cervical spondylogenic myelopathy (van Geel et al 2007).

We found in the largest prospective cohort of female carriers to date that neurological abnormalities are found in approximately 70% of female carriers and that the frequency increases with age. The main symptoms are consistent with a myelopathy, as in men with X-ALD and the AMN phenotype. It is striking how often fecal incontinence is reported, often as an early symptom. One should bear in mind that it is often not voluntarily reported because it is felt to be embarrassing. To our knowledge, this is not reported often in men

Figure 2: ALDP levels as determined by Western blot correlate with the residual peroxisomal beta-oxi-dation activity (A), C26:0 synthesis (B), C26:0 levels (C) and the C26:0/C22:0 ratio (D) in skin fibroblasts from female carriers. Fatty acids are in nmol/mg protein.

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with AMN or in other degenerative myelopathies, for instance hereditary spastic paraplegia (Salinas et al 2008). In fact, in our cohort sphincter disturbance is a frequent and early symptom, and even occurs in a few symptomatic carriers without other signs of myelopathy.

Signs of peripheral neuropathy were mostly absent or minor clinically, and do not seem to contribute much to the overall disability. However, on electrophysiological testing, though, about 37% of the women in this cohort fulfilled the criteria for an axonal sensorimotor neuropathy.

Signs and symptoms present in our cohort caused reduced quality of life. Comparing the SF36 scores to the Dutch norm population, symptomatic women show a reduced quality of life in all domains. The compound physical score was lower than that of age matched controls, corresponding to the signs of myelopathy frequently present in our cohort. The compound psychological score was reduced even more strikingly. Possibly this is related not only to the distress caused by the physical disability also by the fact that many women with X-ALD suffered the loss of children to X-ALD in their families. Furthermore, not only was quality of life reduced, analysis of the ALDS scale revealed that women with X-ALD in the symptomatic category had the same levels of disability as patients with Parkinson’s disease three years after the diagnosis. The group of patients with Parkinson’s disease is significantly older (average age 67 versus 48 years) than our cohort but still levels of disability were similar, suggesting that the burden of disease in women with X-ALD is considerable.

SSEP was reported to be very sensitive in detecting abnormalities in women with X-ALD in a presymptomatic stage (Restuccia et al 1997). In our cohort SSEP of the arms (median nerve) were normal in all asymptomatic women, and were abnormal in only 9% of the symptomatic women. This is not surprising, since in none of the symptomatic women were the arms affected clinically. SSEP of the legs (posterior tibial nerve) were only abnormal in 27% of the asymptomatic women and in 67% of the symptomatic women. Therefore, we conclude that SSEP is not superior to clinical examination in detecting myelopathy in our cohort.

BAEP were abnormal in a large proportion of all women in our cohort, as described previously (Restuccia et al 1997). A larger proportion of the symptomatic women has an abnormal BAEP test result (66% versus 27%). The findings were aspecific, mostly consisting of an increased

Figure 3: Distribution of ABCD1 allele spe-cific expression in skin fibroblasts from fe-male carriers.

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I – V or III – V interval. This indicates a slowing of conduction in the brainstem. For clinical practice BAEP has no value as a diagnostic tool.

As has been known for several decades, 15 – 20% of heterozygotes have normal plasma VLCFA (Moser et al 1999). In our cohort we found that a slightly higher proportion (30%) had normal VLCFA. Furthermore, even in cultured fibroblasts the VLCFA levels can be normal (Moser et al 1983). Combining VLCFA test result in plasma and fibroblasts marks a higher proportion of the women in our cohort as biochemically abnormal. In this cohort, by combining VLCFA levels in plasma and fibroblasts 41 of 43 women (95.3%) for whom plasma and fibroblasts were available could be identified. This corresponds to an earlier observation that by combining VLCFA measurements in plasma and fibroblasts 93% of carriers can be identified. We also performed a beta-oxidation assay in cultured fibroblasts. For the VLCFA beta-oxidation results in fibroblasts vary from completely normal to levels as low as those in men with X-ALD. By adding this parameter, 44 of 45 women with X-ALD (97.8%) for whom plasma and/or fibroblasts were available could be identified. However, this still does not reliably identify all women with X-ALD. In women the diagnostic test of choice is ABCD1 mutation analysis.

We show that the biochemical abnormality in fibroblasts correlates with the X-inactivation pattern, i.e. the more skewed to the mutant allele, the more abnormal the fibroblasts are biochemically. Our study shows that in fibroblasts, ABCD1 allele specific expression (ABCD1 ASE) predicts the biochemical phenotype. This validates our methodology.

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 (Figure 4). In fact, the median skewing is 49.3, suggesting a random ABCD1 ASE pattern. For over 2 decades there has been controversy over whether X-inactivation predicts the symptomatic status of women with X-ALD. In 1993 it was reported that symptomatic status and X-inactivation in fibroblasts do not correlate in a small sample of 12 women (Watkiss et al 1993). A more recent report suggests that skewed X-inactivation patterns in leukocytes do correlate with symptoms in women with X-ALD (Maier et al 2002). However, the assay used in that study (HUMARA) has limitations. It is now known that X-inactivation is not uniform through the entire X-chromosome, but can differ from locus to locus (Carrel et al 2005). The most recent study compares a group of symptomatic carriers and asymptomatic carriers and cannot find an association between X-inactivation in fibroblasts and symptomatic status (Salsano et al 2012). It should be noted that there is an average age difference of more than 15 years between the groups of symptomatic and asymptomatic women with X-ALD, making a comparison difficult considering that our data show that age is a strong predictor for symptomatic status in women with X-ALD. In our study we can not establish a link between X-inactivation as determined by ABCD1 ASE) and symptomatic status when taking age into account. This still does not exclude X-inactivation as an important modifier. A major weakness, as in the other studies, is that non-neural tissue was used to obtain material for X-inactivation studies. Also, even though this is the largest cohort of women with X-ALD studied systematically so far,

Figure 4: ABCD1 ASE correlates with ALDP levels as determined with IF (A) or Western blot (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 female carriers.

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the number of participants might 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.

In this cohort imaging of the brain was not performed routinely since brain involvement is rare and this has been studied by others (Fatemi et al 2003). MRI of the spinal cord of men with AMN and symptomatic women with X-ALD shows abnormalities on magnetization transfer imaging (Dubey et al 2005). This is compatible with the clinical findings in our cohort. An MRI of the brain and spinal cord was only performed in three women to exclude other disorders, and was normal in each case.

A limitation of our study is that not all women with X-ALD in the Netherlands were examined. Even though we tried to minimize bias by including not only women from the outpatient clinic (around 75% of the participants were recruited through the X-ALD patient organization), it is still possible that the participants are not a totally random sample of women with X-ALD. Based on the prevalence it can be estimated there are at least 200 women with X-ALD in the Netherlands. Our population therefore probably contains no more than 25 to 30% of all women with X-ALD in the Netherlands. It is therefore possible that those without symptoms were less likely to participate. Although there was no cohort of age matched controls, it is unlikely that the symptoms described are normal aging. Myelopathy and peripheral neuropathy can not be considered a part of healthy aging. Co-morbidity, like diabetes, was rare in the cohort.

In summary, women with X-ALD are highly likely to develop symptoms during their lifetime. The most important predictor is age, with virtually all women with X-ALD having some clinical manifestation if older than 60. Clinical manifestations are mostly related to myelopathy. Especially striking is the high incidence of fecal incontinence. Peripheral neuropathy is not prominent clinically, although based on nerve conduction studies 37% has a sensorimotor axonal peripheral neuropathy. Biochemical abnormalities are common, but cannot exclude X-ALD with certainty in women. The biochemical abnormalities in fibroblasts in women with X-ALD 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. Women with X-ALD should not be considered merely carriers, but they constitute a distinct AMN-like phenotype of X-ALD. Physicians should consider the diagnosis in women with a chronic myelopathy and ABCD1 mutation analysis should have a place in diagnostic protocols for chronic non-compressive myelopathy.

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Summary and General Discussion

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Chapter 8Summary, general discussion, future research and

implications for clinical practice

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Summary of the thesisX-linked adrenoleukodystrophy (X-ALD) is a relatively common metabolic disorder (Moser et al 2007). It causes severe disability in the majority of affected men and women. For most patients there is currently no curative treatment. We review current recommendations for management in Chapter 1.

The biochemistry of X-ALD has been studied extensively. Although there are still unresolved issues, there is a reasonable understanding of the molecular mechanisms of this disease at the cellular level, as reviewed by Kemp and Wanders (Kemp and Wanders 2010). This, and an excellent in vitro model system available for research (i.e. cultured primary skin fibro-blasts) offers possibilities to screen for compounds that might be of therapeutic use in this disorder. In Chapters 2 and 3 we describe the use of this in vitro model to test potential very long-chain fatty acid (VLCFA) lowering compounds.

It is important to stress that the hypotheses generated by in vitro experiments always need to be validated in clinical trials, maybe first with biochemical endpoints, but eventually with relevant clinical endpoints. Patients with X-ALD are often desperate for a cure and willing to take medication based on pathophysiological theories or inconclusive and poorly designed clinical trials. If one claims a compound might be effective based on in vitro data there is a moral obligation to validate these claims in a clinical trial. We show that even if compounds or strategies seem promising in the laboratory (and even in laboratory animals), they can be totally ineffective in a clinical trial. We performed two clinical trials to test if lovastatin (Chapter 4) or bezafibrate (Chapter 5) can lower VLCFA in X-ALD. Lovastatin was widely be-lieved to lower VLCFA, but we show that the VLCFA lowering in plasma by lovastatin is an ar-tefact. Bezafibrate is also ineffective, probably because plasma concentrations required for a VLCFA lowering effect can not be reached even with the maximum tolerable dose. Because a biochemical effect was absent further studies with clinical endpoints seem unnecessary.

As with all disorders, the clinical spectrum often expands with time as more “atypical ca-ses” are diagnosed and described. In Chapter 6 we describe a man with X-ALD presenting with signs and symptoms of a demyelinating peripheral neuropathy. Usually, the peripheral neuropathy in X-ALD is not prominent and classified as an axonal sensomotor peripheral neuropathy (van Geel et al 1997). We conclude that in demyelinating peripheral neuropathy without a specific cause X-ALD can be considered. In Chapter 7 we describe a large cohort of women with X-ALD. It is clear that women with X-ALD are not merely carriers but develop signs and symptoms of myelopathy and peripheral neuropathy. Symptomatic status corre-lates with age, after 60 years of age most women can be considered symptomatic. There is a striking phenotypic heterogeneity; we could not find an association between the X-inactiva-tion pattern in fibroblasts and symptomatic status. As we discuss in the paper, however, it is possible that expression in other (neural) tissues does correlate. These tissues are not easily accessible and so have not been studied by us.

General discussion and future researchResearch to find the cause and possibly a cure for rare diseases is sometimes considered less relevant on the basis of the relatively small amount of people that would benefit. Howe-ver, studying these disorders can sometimes lead to new insights in human physiology that

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might be applicable to other disorders. Also, even though the number of affected individuals is small, the burden of disease is not for those affected and their families. We will continue with our efforts to improve the outcome in this devastating disease.

Unfortunately, the compound we identified in vitro as a VLCFA lowering drug, is not effective in a clinical trial. The concept, however, of decreasing VLCFA synthesis pharmacologically remains a plausible strategy for future studies. Currently, we are trying to identify inhibitors of ELOVL1 (the rate limiting enzyme involved in VLCFA synthesis) in vitro. We hope this will yield new drugs to stabilize the disease in the future.

Clinical trials in X-ALD to test clinical efficacy are difficult. The disease course is highly varia-ble, age of onset and progression differ enormously between patients. To determine if a new drug offers clinical benefit trials with large groups of patients are necessary. This is a consi-derable challenge in a rare disease. Furthermore, long follow-up of at least 3 years is neces-sary because the disease is usually gradually progressive. Effects on surrogate endpoints (like reduction of plasma VLCFA) should not be considered proof of clinical efficacy. Loren-zo’s oil normalizes VLCFA, but does not prevent progression of disease in X-ALD (Aubourg et al 1993; van Geel et al 1999). Still, clinical trials with biochemical endpoints can be useful as pilot studies. Because trials with clinical endpoint take enormous effort potential new drugs can first be evaluated in small clinical trials with biochemical endpoint. If there is an effect on the surrogate endpoints a follow-up trial with relevant clinical endpoints can be perfor-med. It is important to stress that plasma VLCFA can be misleading. In Chapter 4 we show that if plasma LDL is reduced, plasma VLCFA are lower because of reduced VLCFA transport capacity of the blood. It is vital to also determine VLCFA in leukocytes and/or erythrocytes.

In a university hospital patient care and clinical research are sometimes intertwined. We are planning to create a large cohort of men and women with X-ALD and follow them prospec-tively according to a fixed protocol. Our new collaboration with the VUmc will allow us to implement new MRI techniques in the follow-up, that could lead to earlier detection of the onset of cerebral ALD. This will provide well documented information on the natural history of X-ALD and might also be useful in future therapeutic research.

Research into the pathophysiology of cerebral ALD has been seriously hampered by the lack of a suitable mouse model. The current “X-ALD mouse” develops an AMN-like phenotype. We hope that the new mouse currently developed by Dr. S. Kemp will develop full blown cerebral ALD. This would provide many new research opportunities to unravel the pathop-hysiology of cerebral ALD.

The clinical course in X-ALD is unpredictable, there is no genotype-phenotype correlation (Kemp et al 2012). The identification of genetic or environmental modifiers could help in identifying those patients at risk for cerebral ALD. This would improve management, be-cause a “tailor made” follow-up program can then be offered. New genetic modifiers have been discovered in collaboration with Professor Aubourg that bring this goal a step closer. In the women with X-ALD we hoped to be able to predict symptomatic status by analyzing the X-inactivation pattern in fibroblasts. We were unable to show an association, as described in Chapter 7. We are planning to analyze the X-inactivation pattern in different tissues and to increase the number of patients in our analysis by collaborating with other research groups.

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Implications for clinical practiceX-linked adrenoleukodystrophy is a complex disorder. Patients with X-ALD benefit from care-ful follow-up, as described in Chapter 1. Timely identification of adrenocortical insufficiency and hormonal substitution therapy reduces morbidity and mortality. There is a narrow win-dow of opportunity for bone marrow transplantation after the onset of cerebral ALD and follow-up with routine MRI scans is recommended. Furthermore, virtually all patients will gradually develop neurological deficits and symptomatic treatment of bladder- or bowel dysfunction and spasticity should be started. This follow-up should be offered in a large center by physicians with experience in this field. Ideally, this follow-up should be done according to a fixed protocol. This will most likely result in better patient care and allows us to systematically study the natural history of the disorder. We are currently working on improving the X-ALD outpatient clinic.

References

Aubourg P, Adamsbaum C, Lavallard-Rousseau MC, Rocchiccioli F, Cartier N, Jambaqué I, Jakobezak C, Lemaitre A, Boureau F, Wolf C, et al. A two-year trial of oleic and erucic acids (“Lorenzo’s oil”) as treatment for adrenomyelo-neuropathy. N Engl J Med. 9;329(11):745-52.

Moser HW, Mahmood A, Raymond GV. 2007. X-linked adrenoleukodystrophy. Nat Clin Pract Neurol 3(3):140-51.

Kemp S, Wanders R. 2010. Biochemical aspects of X-linked adrenoleukodystrophy. Brain Pathol 20(4):831-7.

Kemp, S., Berger, J. & Aubourg, P. 2012. X-linked adrenoleukodystrophy: Clinical, metabolic, genetic and pathophy-siological aspects. Biochim Biophys Acta 1822(9):1465–1474.

van Geel BM, Koelman JH, Barth PG, Ongerboer de Visser BW. 1996. Peripheral nerve abnormalities in adrenomy-eloneuropathy: a clinical and electrodiagnostic study. Neurology 46(1):112-8.

van Geel BM, Assies J, Haverkort EB, Koelman JH, Verbeeten B Jr, Wanders RJ, Barth PG. 1999. Progression of ab-normalities in adrenomyeloneuropathy and neurologically asymptomatic X-linked adrenoleukodystrophy despite treatment with “Lorenzo’s oil”. J Neurol Neurosurg Psychiatry. 67(3):290-9.

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Chapter 9Van gen naar ziekte; X-gebonden adrenoleukodystrofie

Nederlands Tijdschrift voor Geneeskunde (2008) 152(14): 804 – 808

Marc Engelen, Stephan Kemp en Björn M. van Geel

Afdeling Neurologie en het Laboratorium voor Genetische Metabole Ziekten, Academisch Medisch Centrum/Universiteit van Amsterdam, Amsterdam. Afdeling Neurologie, Medisch Centrum Alkmaar, Alkmaar.

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De ziekte

X-gebonden adrenoleukodystrofie (X-ALD) is de frequentst voorkomende peroxisomale stofwisselingsziekte met als biochemisch kenmerk een stapeling in plasma en weefsels van zeerlangeketenvetzuren (ZLKV), dit zijn vetzuren met meer dan 22 koolstofatomen (Moser et al 2001). De symptomen zijn zeer variabel, ook bij familieleden met dezelfde mutatie (Kemp et al 2001). Vooralsnog is het niet mogelijk de klinische manifestaties bij een individuele patiënt te voorspellen. Er wordt een aantal fenotypen onderscheiden (Tabel).

Cerebrale X-ALD. De cerebrale vormen van X-ALD zijn het ernstigst. Bij ongeveer 30% van alle mannelijke patiënten ontwikkelt dit fenotype zich op de kinderleeftijd, meestal tussen het 3e en het 10e levensjaar. Deze vorm wordt ook wel ‘childhood-onset’ cerebrale ALD (CCALD) genoemd. Soms manifesteert de ziekte zich op hogere leeftijd en heet dan ‘adolescent cerebral’ ALD of ‘adult cerebral’ ALD (Moser et al 2001). Deze vormen hebben een sluipend begin met vaak als eerste verschijnselen gedragsverandering en afname van schoolprestaties, gevolgd door visusproblemen, doofheid, spastische tetraparese en epilepsie. De verschijnselen zijn snel progressief; na twee jaar verkeren de meeste patiënten in een vegetatieve toestand of zijn overleden. De klachten worden veroorzaakt door een inflammatoire reactie met demyelinisatie in de hersenen (Powers et al 1992). Bij MRI-onderzoek van de hersenen worden kenmerkende verschijnselen gevonden (Figuur 1) (Loes et al 1994). Een groot deel van de jongens heeft bijnierschorsinsufficiëntie.

Figuur 1: MRI-afbeeldingen van het cerebrum van een jongen met ‘childhood-onset’ cerebrale adrenoleukodystrofie: (a) T2-gewogen opname met uitgebreide hyperintense wittestofafwijkingen pariëto-occipitaal; (b) T1-gewogen opname na intraveneus contrast met aankleuring van de rand van de laesie.

Adrenomyeloneuropathie (AMN). De meeste patiënten met X-ALD die de adolescentie gepasseerd zijn, krijgen adrenomyeloneuropathie, doorgaans tussen het 20e en het 40e levensjaar (Moser et al 2001). AMN wordt gekenmerkt door langzaam progressieve spastische paraparese, gestoorde sensibiliteit in de benen en mictie- en defecatieproblemen. Daarnaast heeft 80% van de mannen bijnierschorsinsufficiëntie en 75% tekenen van

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gonadale insufficiëntie (Assies et al 1997; Moser et al 2001). Kenmerkend is de spaarzame beharing van het hoofd (Figuur 2). De diagnose wordt vaak pas laat gesteld (van Geel et al 1993).

Pathologisch is er axonale degeneratie in het ruggenmerg en in de perifere zenuwen, zonder ontstekingscomponent (van Geel et al 1996; Powers et al 2000). Patiënten met AMN hebben een grote kans (20% in 10 jaar) op additionele cerebrale demyelinisatie (van Geel et al 2001). Hoewel de verdeling van de cerebrale laesies bij AMN er iets anders uitziet, is de prognose net zo slecht als die van CCALD (van Geel et al 2001).

Figuur 2: Typische spaarzame beharing van het hoofd van een patiënt met adrenomyeloneuropathie.

‘Addison-only’-fenotype. Sommige patiënten hebben aanvankelijk bijnierschorsinsufficiëntie zonder neurologische verschijnselen. De symptomen zijn hetzelfde als die van de ziekte van Addison (Moser et al 2001). Hyperpigmentatie van de handlijnen is bijvoorbeeld een uiting van deze bijnierschorsinsufficiëntie (Figuur 3). Bij jongens en mannen met een primaire bijnierschorsinsufficiëntie dient onderzoek te worden ingezet naar X-ALD, zeker bij afwezigheid van antilichamen tegen bijnierschorscellen (Mukherjee et al 2006).

Patiënten bij wie afwijkingen aan het zenuwstelsel aanvankelijk ontbreken, kunnen deze gedurende het leven alsnog krijgen, zodat het ene fenotype in het andere kan overgaan. Uiteindelijk ontstaan bij de meeste patiënten met dit fenotype toch neurologische uitvalsverschijnselen.

Asymptomatische en atypische vormen. Bij screenen van de familie van indexpatiënten kunnen mannen gevonden worden die tot op hoge leeftijd geen of nauwelijks klachten krijgen. Ook zijn er patiënten beschreven met een atypisch klachtenpatroon, zoals de in Azië beschreven spinocerebellaire varianten (Ohno et al 1984).

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Draagsters. Zoals vaker bij X-chromosomaal overervende ziekten is waargenomen, kunnen ook draagsters symptomen krijgen. Op patiëntenbijeenkomsten in de Verenigde Staten zijn draagsters onderzocht en van hen bleek 53% neurologische afwijkingen te hebben (Moser et al 1991). In Nederland bleek 48% van de draagsters verschijnselen van AMN te hebben. Dit percentage neemt toe met de leeftijd en in de leeftijdscategorie 60-69 jaar heeft zelfs 79% klachten die samenhangen met X-ALD (van Geel, 2000). Bijnierschorsinsufficiëntie en cerebrale afwijkingen zijn bij draagsters zeer zeldzaam (Moser et al 2001).

Figuur 3. Gehyperpigmenteerde hand-lijnen bij bijnierschorsinsufficiëntie als gevolg van adrenoleukodystrofie.

Het gen en het eiwit

X-ALD wordt gekenmerkt door een verhoging van ZLKV in plasma en weefsels (Moser et al 1981). De afbraak van ZLKV via beta-oxidatie is een peroxisomale functie. Al sinds 1984 is bekend dat de peroxisomale beta-oxidatie in cellen van patiënten met X-ALD sterk verminderd is, tot 25-30% van normaal (Singh et al 1984). In 1993 is het gen geïdentificeerd dat verantwoordelijk is voor het defect bij X-ALD (Mosser et al 1993). Dit ABCD1-gen codeert voor een peroxisomaal transmembraaneiwit dat geclassificeerd is als een ATP-‘binding cassette’(ABC)-‘half transporter’. De functie is nog niet onomstotelijk vastgesteld, maar verondersteld wordt dat het eiwit een rol speelt bij het transport van ZLKV over de peroxisomale membraan (Figuur 4) (Kemp and Wanders, 2007).

De cel

ZLKV accumuleren in alle cellen en weefsels, maar uiteindelijk ontstaat bij X-ALD schade aan het zenuwweefsel en de steroïdhormoonproducerende weefsels (Moser et al 2001). Specifieke celpopulaties die te gronde gaan, zijn de hormoonproducerende cellen in de zona fasciculata en reticularis van de bijnierschors (en minder in de zona glomerulosa), de leydigcellen in de testes en oligodendrocyten in het centrale zenuwstelsel (Moser et al 2001).

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Pathologisch zijn er 2 duidelijk verschillende patronen. Enerzijds is er de niet-inflammatoire axonopathie in ruggenmerg en perifere zenuwen (Powers et al 2000), die gevonden wordt bij AMN, en de atrofie van de bijnieren (Powers et al 1980). Anderzijds ontwikkelt zich bij een deel van de patiënten een hevige inflammatoire reactie in het centrale zenuwstelsel met perivasculaire lymfocytaire infiltraten en snel progressieve cerebrale demyelinisatie (Powers et al 1992). De rol van ZLKV in de pathofysiologie hiervan is onduidelijk.

Het is onbekend hoe het komt dat bij sommige patiënten cerebrale demyelinisatie ontstaat en bij anderen niet. Relaties tussen fenotype en ABCD1-mutatie (Kemp et al 2001) of plasma-ZLKV-spiegels zijn tot op heden niet gevonden (Moser et al 1999). Waarschijnlijk zijn er modificerende genen die het uiteindelijke fenotype mede bepalen (Asheuer et al 2005).

figuur 4. Schematisch overzicht van het biochemisch defect in X-gebonden adrenoleukodystrofie (X-ALD). Mutaties in het ABCD1-gen veroorzaken een afwezig of defect ALD-proteïne (ALDP). Zeerlangeketenvetzuren (ZLKV) worden gemaakt door enzymen in de membraan van het endoplasmatisch reticulum uit langeketenvetzuren. Als door een defect ALDP de afbraak van ZLKV verminderd is, ontstaat stapeling ervan in de cel.

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De populatie

X-ALD heeft een geboorte-incidentie van rond de 1:17.000 (Bezman et al 2001). Dit betekent dat in Nederland elk jaar 12 kinderen (jongens en meisjes) met een afwijking in het gen worden geboren. In Nederland zijn enige honderden patiënten en draagsters bekend.

Diagnostiek en behandeling

Mutatieanalyse van het ABCD1-gen heeft inmiddels meer dan 600 verschillende mutaties opgeleverd (www.x-ald.nl). Ook het bepalen van de concentratie ZLKV in plasma is zeer betrouwbaar voor het stellen van de diagnose bij mannen. Hierbij worden de concentratie van de ZLKV C26:0 en de C26:0/C22:0- en de C24:0/C22:0-ratio’s bepaald. Echter, 20% van de draagsters kan normale ZLKV-waarden in plasma hebben (Moser et al 2001). Voor hen is mutatieanalyse van het ABCD1-gen dan ook de enige betrouwbare test. Ook prenatale diagnostiek is mogelijk en zeer betrouwbaar.

Vooralsnog is er geen curatieve behandeling beschikbaar, maar allogene beenmerg-transplantatie (BMT) of hematopoëtische stamceltransplantatie (HSCT) leidt tot stabilisatie en zelfs regressie van symptomen bij CCALD, mits deze vroeg in het beloop wordt toegepast (Peters et al 2004). Voor zover bekend is BMT niet effectief voor de fenotypen waarbij cerebrale afwijkingen ontbreken. Uit de follow-up van patiënten die op jonge leeftijd een transplantatie hebben ondergaan, zal moeten blijken of deze ingreep de ontwikkeling van AMN vertraagt of voorkomt.

Dieettherapie bestaande uit vetbeperking in combinatie met gebruik van Lorenzo’s olie, een mengsel van glyceroltrioleaat en glyceroltrierucaat in de verhouding 4:1, normaliseert de ZLKV-concentratie in plasma binnen 1 maand (Moser et al 2001). Dit gegeven sprak zo tot de verbeelding dat er in 1992 zelfs een speelfilm over is gemaakt: Lorenzo’s oil (Barth, 1993). Echter, in openlabelstudies werd geen effect van Lorenzo’s olie op het beloop van CCALD en AMN gevonden (Uziel et al 1991; van Geel et al 1999). In Nederland wordt deze behandeling daarom nauwelijks meer toegepast. Momenteel loopt in de VS een groot gerandomiseerd dubbelblind onderzoek naar het effect van Lorenzo’s olie bij AMN. Jongens jonger dan 10 jaar zonder neurologische symptomen die met Lorenzo’s olie worden behandeld, krijgen volgens een in de VS uitgevoerd retrospectief onderzoek bij grote therapietrouw minder vaak cerebrale afwijkingen dan jongens die de olie minder goed gebruikten. Daarom wordt het gebruik van Lorenzo’s olie in de VS wel aangeprezen voor jongens zonder neurologische symptomen (Moser et al 2005). Thans wordt ook onderzoek verricht naar gentherapie voor X-ALD, dit is echter nog niet breed toepasbaar (Benhamida et al 2003).

Hoewel curatieve behandeling vooralsnog ontbreekt, is het toch zeer belangrijk X-ALD tijdig te herkennen. Er zijn namelijk levensreddende behandelingen mogelijk, zoals hormoonsuppletie voor bijnierschorsinsufficiëntie. BMT of HSCT kan bij vroege cerebrale verschijnselen een gunstig effect hebben. De behandeling van spasticiteit en daarmee samenhangende symptomen is niet anders dan bij andere neurologische aandoeningen, zoals multiple sclerose.

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Literatuur

Asheuer M, Bieche I, Laurendeau I, Moser A, Hainque B, Vidaud M, Aubourg P. 2005. Decreased expression of ABCD4 and BG1 genes early in the pathogenesis of X-linked adrenoleukodystrophy. Hum Mol Genet 14:1293-1303.

Assies J, Gooren LJ, Van GB, Barth PG. 1997. Signs of testicular insufficiency in adrenomyeloneuropathy and neurologically asymptomatic X-linked adrenoleukodystrophy: a retrospective study. Int J Androl 20:315-321.

Barth PG. 1993. [‘Lorenzo’s oil’]. Ned Tijdschr Geneeskd 137:640-641.

Benhamida S, Pflumio F, Dubart-Kupperschmitt A, Zhao-Emonet JC, Cavazzana-Calvo M, Rocchiccioli F, Fichelson S, Aubourg P, Charneau P, Cartier N. 2003. Transduced CD34(+) cells from adrenoleukodystrophy patients with HIV-derived vector mediate long-term engraftment of NOD/SCID mice. Mol Ther 7:317-324.

Bezman L, Moser AB, Raymond GV, Rinaldo P, Watkins PA, Smith KD, Kass NE, Moser HW. 2001. Adrenoleukodystrophy: incidence, new mutation rate, and results of extended family screening. Ann Neurol 49:512-517.

Ho JK, Moser H, Kishimoto Y, Hamilton JA. 1995. Interactions of a very long chain fatty acid with model membranes and serum albumin. Implications for the pathogenesis of adrenoleukodystrophy. J Clin Invest 96:1455-1463.

Kemp S, Pujol A, Waterham HR, van Geel BM, Boehm CD, Raymond GV, Cutting GR, Wanders RJ, Moser HW. 2001. ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations. Hum Mutat 18:499-515.

Kemp S, Wanders RJ. 2007. X-linked adrenoleukodystrophy: Very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment. Molecular Genetics and Metabolism 90:268-276.

Loes DJ, Hite S, Moser H, Stillman AE, Shapiro E, Lockman L, Latchaw RE, Krivit W. 1994. Adrenoleukodystrophy: a scoring method for brain MR observations. AJNR Am J Neuroradiol 15:1761-1766.

Moser HW, Moser AB, Frayer KK, Chen W, Schulman JD, O’Neill BP, Kishimoto Y. 1981. Adrenoleukodystrophy: increased plasma content of saturated very long chain fatty acids. Neurology 31:1241-1249.

Moser HW, Bergin A, Naidu S, Ladenson PW. 1991. Adrenoleukodystrophy. Endocrinol Metab Clin North Am 20:297-318.

Moser AB, Kreiter N, Bezman L, Lu S, Raymond GV, Naidu S, Moser HW. 1999. Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls. Ann Neurol 45:100-110.

Moser HW, Smith KD, Watkins PA, Powers J, Moser AB. 2001. X-linked adrenoleukodystrophy. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease: McGraw Hill, New York. p 3257-3301.

Moser HW, Raymond GV, Lu SE, Muenz LR, Moser AB, Xu J, Jones RO, Loes DJ, Melhem ER, Dubey P, Bezman L, Brereton NH, Odone A. 2005. Follow-up of 89 Asymptomatic Patients With Adrenoleukodystrophy Treated With Lorenzo’s Oil. Arch Neurol 62:1073-1080.

Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, Poustka AM, Mandel JL, Aubourg P. 1993. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 361:726-730.

Mukherjee S, Newby E, Harvey JN. 2006. Adrenomyeloneuropathy in patients with ‘Addison’s disease’: genetic case analysis. J R Soc Med 99:245-249.

Ohno T, Tsuchida H, Fukuhara N, Yuasa T, Harayama H, Tsuji S, Miyatake T. 1984. Adrenoleukodystrophy: a clinical variant presenting as olivopontocerebellar atrophy. J Neurol 231:167-169.

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Peters C, Charnas LR, Tan Y, Ziegler RS, Shapiro EG, DeFor T, Grewal SS, Orchard PJ, Abel SL, Goldman AI, Ramsay NKC, Dusenbery KE, Loes DJ, Lockman LA, Kato S, Aubourg PR, Moser HW, Krivit W. 2004. Cerebral X-linked adrenoleukodystrophy: the international hematopoietic cell transplantation experience from 1982 to 1999. Blood 104:881-888.

Powers JM, Schaumburg HH, Johnson AB, Raine CS. 1980. A correlative study of the adrenal cortex in adreno-leukodystrophy--evidence for a fatal intoxication with very long chain saturated fatty acids. Invest Cell Pathol 3:353-376.

Powers JM, Liu Y, Moser AB, Moser HW. 1992. The inflammatory myelinopathy of adreno-leukodystrophy: cells, effector molecules, and pathogenetic implications. J Neuropathol Exp Neurol 51:630-643.

Powers JM, DeCiero DP, Ito M, Moser AB, Moser HW. 2000. Adrenomyeloneuropathy: a neuropathologic review featuring its noninflammatory myelopathy. J Neuropathol Exp Neurol 59:89-102.

Singh I, Moser AE, Moser HW, Kishimoto Y. 1984. Adrenoleukodystrophy: impaired oxidation of very long chain fatty acids in white blood cells, cultured skin fibroblasts, and amniocytes. Pediatr Res 18:286-290.

Uziel G, Bertini E, Bardelli P, Rimoldi M, Gambetti M. 1991. Experience on therapy of adrenoleukodystrophy and adrenomyeloneuropathy. Dev Neurosci 13:274-279.

van Geel BM, Assies J, Haverkort EB, Barth PG, Wanders RJ, Schutgens RB, Keyser A, Zwetsloot CP. 1993. Delay in diagnosis of X-linked adrenoleukodystrophy. Clin Neurol Neurosurg 95:115-120.

van Geel BM, Koelman JH, Barth PG, Ongerboer de Visser BW. 1996. Peripheral nerve abnormalities in adrenomyeloneuropathy: a clinical and electrodiagnostic study. Neurology 46:112-118.

van Geel BM, Assies J, Haverkort EB, Koelman JH, Verbeeten B, Wanders RJ, Barth PG. 1999. Progression of abnormalities in adrenomyeloneuropathy and neurologically asymptomatic X-linked adrenoleukodystrophy despite treatment with “Lorenzo’s oil”. J Neurol Neurosurg Psychiatry 67:290-299.

van Geel BM. 2000. Draagsterschap van X-gebonden adrenoleukodystrofie. Ned Tijdschr Geneeskd 144:1764-1768.

van Geel BM, Bezman L, Loes DJ, Moser HW, Raymond GV. 2001. Evolution of phenotypes in adult male patients with X-linked adrenoleukodystrophy. Ann Neurol 49:186-194.

Whitcomb RW, Linehan WM, Knazek RA. 1988. Effects of long-chain, saturated fatty acids on membrane microviscosity and adrenocorticotropin responsiveness of human adrenocortical cells in vitro. J Clin Invest 81:185-188.

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Chapter 0Samenvatting in het Nederlands en beschouwing

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Samenvatting van dit proefschrift

Voor een beschrijving van de ziekteverschijnselen, diagnostiek naar en behandeling van X-gebonden adrenoleukodystrofie (X-ALD) verwijs ik naar Hoofdstuk 1 en 9. De biochemie van X-ALD is door de jaren heen in veel detail bestudeerd en voor een groot deel opgehelderd (Kemp et al., 2010). Kort samengevat is er sprake van een verminderde afbraak van zeer lang-keten vetzuren (ZLKV; vetzuren met 22 of meer koolstofatomen) in een celorganel dat het peroxisoom heet. Deze gestoorde afbraak wordt veroorzaakt door mutaties in het ABCD1 gen gelegen op het X-chromosoom dat codeert voor het eiwit ALDP. Dit eiwit is belangrijk voor het transport van ZLKV het peroxisoom in waar de afbraak plaatsvindt. Het gevolg van de gestoorde afbraak is stapeling van ZLKV, en dan vooral van C26:0 (een ZLKV met 26 koolstofatomen en geen dubbele bindingen). Deze ZLKV stapelen in het bloedplasma en alle weefsels en leiden uiteindelijk tot weefselschade. Het is aannemelijk dat het verlagen van ZLKV concentraties een gunstig effect zal hebben op de beloop van de ziekte. Stoffen die ZLKV kunnen verlagen zijn dan ook potentiële geneesmiddelen voor deze aandoening.

Er is een uitstekend modelsysteem beschikbaar voor het bestuderen van het effect van stoffen op ZLKV metabolisme in het laboratorium, namelijk gekweekte huidcellen (fibroblasten) van patiënten met X-ALD. Eerder onderzoek suggereerde dat het verlagen van cholesterol met een cholesterol verlagend medicijn (lovastatine) bij patiënten met X-ALD ook een verlaging van ZLKV tot gevolg heeft (Singh et al, 1998). In het eerder genoemde celmodel bleek dat huidcellen van patiënten met X-ALD die gekweekt werden in kweekmedium zonder cholesterol inderdaad een lagere concentratie ZLKV hadden dan cellen gekweekt in standaard medium (Weinhofer et al., 2002). Wij verrichtten experimenten om het mechanisme van deze daling in ZLKV in cellen van patiënten met X-ALD in medium zonder cholesterol te achterhalen. Onze experimenten toonden aan dat er weliswaar een daling is van C26:0, maar daar staat tegenover een sterke toename van C26:1 (een ZLKV met een dubbele binding, een zogenaamd onverzadigd vetzuur). Er is geen sprake van een verlaging van ZLKV, maar van een verschuiving van verzadigde naar onverzadigde ZLKV (Hoofdstuk 2). Dit maakt cholesterolverlaging als therapeutische optie voor X-ALD minder aantrekkelijk, want er blijft sprake van stapeling. In hetzelfde celmodel tonen we aan dat bezafibraat de concentratie van ZLKV kan verlagen in fibroblasten van patiënten met X-ALD (Hoofdstuk 3). Het mechanisme van deze verlaging is niet het herstellen van de afbraak van ZLKV, maar het remmen van de aanmaak van nieuwe ZLKV door de cel. Middelen die de aanmaak van ZLKV door de cel kunnen remmen lijken dus potentiële geneesmiddelen voor X-ALD.

Hypotheses gegenereerd met een in vitro model zoals hierboven beschreven zijn slechts het beginpunt bij het ontwikkelen van nieuwe medicijnen. Validatie van de bevindingen in een goede klinische trial is nodig voordat potentiële behandelingen aan patiënten worden aangeboden. Idealiter hebben die trials relevante klinische uitkomstmaten. Dit is echter vooral bij X-ALD lastig. De klinische verschijnselen zijn hoogst onvoorspelbaar en langzaam progressief. Om een definitieve uitspraak te doen over de werkzaamheid van een therapie bij X-ALD zijn dus grote groepen patiënten nodig en een lange follow-up. Dat is tijdrovend onderzoek en lijkt alleen geïndiceerd voor zeer kansrijke nieuwe therapieën. Wij hebben er daarom voor gekozen voor de middelen die eerder zijn genoemd eerst een biochemisch effect aan te tonen. Alleen stoffen waar een biochemisch effect overtuigend aangetoond is komen dan in aanmerking voor een trial met klinische uitkomstmaten. Patiënten met zeldzame ziektes waarvoor geen goede behandeling beschikbaar is zijn vaak wanhopig

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en bereid om medicatie te gebruiken zonder dat de werkzaamheid (of zelfs veiligheid) afdoende bewezen is. In het geval van X-ALD zijn hier meerdere voorbeelden van te noemen. Lorenzo’s olie (LO), een mengsel van C18:1 en C22:1, werd op basis van proeven met fibroblasten snel ook geprobeerd bij patiënten met X-ALD. Biochemisch was er een spectaculair effect. In het bloed normaliseerde C26:0 bij patiënten die een vetbeperkt dieet gebruikten in combinatie met de olie. Al snel werd er enorme druk op artsen uitgeoefend om Lorenzo’s olie voor te schrijven. Het verrichten van een klinische trial met een placebo groep was niet meer mogelijk, dit werd onethisch geacht. Helaas bleek later dat patiënten die Lorenzo’s olie gebruikten nog steeds klinisch achteruit te gaan (zie bijvoorbeeld Aubourg et al., 1993; van Geel et al., 1999). Aangezien er echter nooit een goede klinische trial is verricht met een controlegroep blijven er “believers” die menen dat LO het ziektebeloop vertraagd. Een ander voorbeeld van een “therapie” die te snel is toegepast is lovastatine. In 1998 werden resultaten gepubliceerd van experimenten waarbij fibroblasten van patienten met X-ALD aan lovastatine (een cholesterol verlagend medicijn) werden blootgesteld (Singh et al., 1998). Dit zou een spectaculaire daling van ZLKV laten zien. Kort daarna volgde een kleine klinische trial met een twijfelachtige biochemische uitkomstmaat (Singh et al., 1998). Dit leidde tot grote opwinding en vele patienten gingen op basis van deze gegevens al lovastatine gebruiken (soms zonder recept). In onze eigen trial (Hoofdstuk 4) laten wij zien dat de kleine daling van C26:0 in het bloed van patiënten met X-ALD behandeld met lovastatine slechts een artefact is. De “lovastatine mythe” is echter hardnekkig. Nog steeds komen er regelmatig vragen van collega’s uit het buitenland of van patiënten zelf of een behandeling met lovastatine niet te overwegen valt. Ons eigen onderzoek naar het effect van bezafibraat in vitro heeft ook geleid tot een klinische trial (Hoofdstuk 5). Helaas is er bij de mens geen effect op de ZLKV in bloed of bloedcellen omdat de benodigde concentratie niet bereikt wordt met de maximaal toegestane dosis bezafibraat. Voor zowel lovastatine als bezafibraat lijkt het niet zinvol een grote en langdurige klinische trial te verrichten met klinische uitkomstparameters, aangezien er helemaal geen biochemisch effect is. Het gebruik van deze medicamenten is dan ook niet geïndiceerd voor de behandeling van patiënten met X-ALD.

Voor veel stofwisselingsziekten geldt dat naarmate de diagnostische mogelijkheden toenemen en er meer gevallen ontdekt worden, het klinisch spectrum steeds breder wordt. Dit geldt zeker ook voor X-ALD. In het begin van de 20e eeuw werden enkele gevallen van childhood cerebrale ALD (CCALD) beschreven (toen nog de ziekte van Schilder genoemd), pas in 1975 toen stapeling van ZLKV herkend werd bleek dat adrenomyeloneuropathie (AMN) een verschijningsvorm van dezelfde ziekte was. In dit proefschrift beschrijven wij in Hoofdstuk 6 een man met X-ALD die zich presenteerde met een demyeliniserende polyneuropathie, wat ongebruikelijk is. Verder is de laatste jaren meer en meer aandacht gekomen voor vrouwen met X-ALD. Het is nu duidelijk dat het niet alleen maar “draagsters” betreft die de ziekte aan hun zoons kunnen doorgegeven, maar dat deze vrouwen zelf niet zelden klachten ontwikkelen. Het percentage vrouwen dat klachten en symptomen heeft die te wijten zijn aan X-ALD neemt sterk toe met de leeftijd. Het betreft klachten die lijken op adrenomyeloneuropathie bij mannen. De klinische, neurofysiologische en biochemische kenmerken van 46 vrouwen met X-ALD in een prospectief verzameld cohort worden beschreven in Hoofdstuk 7.

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Algemene beschouwing en plannen voor de toekomst

Onderzoek naar zeldzame ziektes wordt vaak beschouwd als minder relevant omdat er zo weinig mensen door getroffen worden. Toch is het belangrijk dat ook voor deze ziektes aandacht is. Enerzijds omdat het leed veroorzaakt door een zeldzame ziekte voor aangedane patiënten en families niet minder is dan voor “gewone” ziekten, maar anderzijds leidt het bestuderen van deze zeldzame ziektes vaak tot nieuwe pathofysiologische inzichten. Het bestuderen van X-ALD bijvoorbeeld heeft bijgedragen aan kennis over de functie van peroxisomen in menselijke cellen, de functie van het eiwit ALDP, heeft geleid tot het identificeren van het enzym dat verantwoordelijk is voor de aanmaak van ZLKV (Ofman et al., 2010), etcetera. In ieder geval hebben wij nog genoeg plannen om onderzoek te blijven doen naar peroxisomale aandoeningen met als doel verbetering van diagnostiek, ophelderen van de pathofysiologie en uiteindelijk het ontwikkelen van betere behandelingen. Hieronder een opsomming van de aandachtspunten van het onderzoek naar X-ALD in het AMC de komende jaren.

Het concept van het verlagen van ZLKV bij X-ALD met een stof die de aanmaak van ZLKV remt (Hoofdstuk 3) is een interessante strategie voor het ontwikkelen van nieuwe behandelingen voor X-ALD. Hopelijk lukt het om in de toekomst een stof te identificeren die net als bezafibraat niet toxisch is en de aanmaak van ZLKV kan remmen door inhibitie van ELOVL1, maar bij veel lagere concentraties dan bezafibraat.

Zoals eerder kort besproken zijn klinische trials bij X-ALD om een effect op het ziektebeloop aan te tonen moeilijk. Er zijn enorme verschillen in leeftijd waarop klachten ontstaan en de mate van progressie tussen patiënten. Hierdoor zijn grote groepen patiënten nodig, wat een grote uitdaging is bij een zeldzame aandoening. Bovendien is lange follow-up nodig wegens de over het algemeen geleidelijke progressie van symptomen. Zoals eerder beschreven zijn “surrogaat uitkomstmaten” (zoals ZLKV) waarschijnlijk nuttig bij het selecteren van medicijnen die mogelijk ook een effect hebben op het ziektebeloop. De medicijnen die een effect hebben op een surrogaat uitkomstmaat kunnen dan vervolgens in een grote klinische trial verder worden onderzocht. Het is wel belangrijk dat dit ook vervolgens ook gedaan wordt, aangezien de geschiedenis met LO aantoont dat effect op een surrogaat uitkomstmaat (in dit geval ZLKV in bloedplasma) niet betekent dat er ook daadwerkelijk een klinisch effect is. Daarnaast is uit onderzoek, opgenomen in dit proefschrift (Hoofdstuk 4), duidelijk geworden dat plasma ZLKV een bijzonder slechte uitkomstmaat zijn voor X-ALD; in ieder geval moet ook gekeken worden naar ZLKV in bijvoorbeeld leukocyten.

In een universiteitsziekenhuis zijn patiëntenzorg en onderzoek soms nauw verweven. De zorg voor patiënten met X-ALD biedt unieke kansen. Hoewel er door de jaren heen veel kennis is opgedaan over de verschijnselen van X-ALD is er nooit een groot cohort patiënten met X-ALD over langere tijd (bijvoorbeeld 10 jaar lang) prospectief gevolgd. Wij willen graag een grote natuurlijk beloop studie doen (met mannen, vrouwen en kinderen) waaraan hopelijk alle patiënten die nu gevolgd worden op onze polikliniek aan deel zullen nemen. De samenwerking met de collega’s van het VUmc maakt het ook mogelijk aan ons follow-up protocol nieuwe MRI technieken toe te voegen waarmee het optreden van cerebrale ALD mogelijk al in een vroeger stadium ontdekt kan worden. Dit zal informatie opleveren over het natuurlijk beloop van de ziekte die enerzijds betere counseling mogelijk maakt, maar misschien ook nuttig kan zijn bij onderzoek naar het evalueren van het effect van nieuwe

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interventies.

Het onderzoek naar de pathofysiologie van cerebrale X-ALD mist een geschikt muismodel. Er is weliswaar een “X-ALD muis” met alle biochemische kenmerken van X-ALD, maar die ontwikkelt een AMN-achtig fenotype op hoge leeftijd en geen cerebrale afwijkingen. Hopelijk krijgt de nieuwe muis ontwikkeld door dr. S. Kemp een fenotype dat lijkt op cerebrale ALD. Dit zou vele nieuwe interessante mogelijkheden bieden om de “triggers” voor cerebrale ALD te ontrafelen en nieuwe behandelingen te testen in de muis.

Het klinisch beloop van X-ALD is zeer onvoorspelbaar ook binnen een en dezelfde familie, er is geen enkele genotype-fenotype correlatie (Kemp et al., 2001). Het identificeren van genetische en/of omgevingsfactoren die het fenotype bepalen (zogenaamde “modifiers”) zouden ons in staat stellen patiënten met een hoog risico op het ontwikkelen van cerebrale ALD “tailor made” follow-up of zelfs preventieve behandeling aan te bieden. In samenwerking met Prof. Aubourg uit Parijs zijn nieuwe genetische modifiers ontdekt die dit doel een stap dichterbij brengen. Wij hoopten op basis van het X-inactivatiepatroon in fibroblasten van vrouwen met X-ALD te kunnen voorspellen of zij symptomatisch worden of niet. Zoals beschreven in Hoofdstuk 7 konden wij echter geen associatie aantonen tussen X-inactivatie patroon in fibroblasten en symptomatische toestand na correctie voor leeftijd. Er zijn plannen om samen te werken met andere onderzoeksgroepen om een nog grotere groep vrouwen te kunnen analyseren. Daarnaast willen we dit onderzoek herhalen in andere weefsels (in eerste instantie met lymfocyten).

Implicaties voor de klinische praktijk

X-gebonden adrenoleukodystrofie is een ingewikkelde aandoening. Regelmatige follow-up is nodig door artsen met ervaring met deze aandoening, zoals beschreven in Hoofdstuk 1. Tijdige onderkenning van bijnierschorsinsufficiëntie en zo nodig suppleren van hydrocortison voorkomt morbiditeit en zelfs mortaliteit. Verder is er maar klein therapeutisch interval voor het laten verrichten van een beenmergtransplantatie als er afwijkingen passend bij cerebrale ALD ontstaan. Ook ontwikkelen vrijwel alle patiënten met X-ALD uiteindelijk een myelopathie met incontinentie en spasticiteit. Hiervoor is symptomatische behandeling beschikbaar. Wij werken aan verbetering van de X-ALD polikliniek en het ontwikkelen van een vast protocol voor follow-up. Dit zal de kwaliteit van de patiëntenzorg zeker ten goede komen en biedt ook mogelijkheden voor het bestuderen van het natuurlijk beloop van de ziekte.

Referenties

Aubourg P, Adamsbaum C, Lavallard-Rousseau MC, Rocchiccioli F, Cartier N, Jambaqué I, Jakobezak C, Lemaitre A, Boureau F, Wolf C, et al. A two-year trial of oleic and erucic acids (“Lorenzo’s oil”) as treatment for adrenomyeloneuropathy. N Engl J Med. 329(11):745-52.

Engelen M, Ofman R, Dijkgraaf MG, Hijzen M, van der Wardt LA, van Geel BM, de Visser M, Wanders RJ, Poll-The BT, Kemp S. 2010. Lovastatin in X-linked adrenoleukodystrophy. N Engl J Med. 362(3): 276 – 7.

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Kemp S, Wanders R. 2010. Biochemical aspects of X-linked adrenoleukodystrophy. Brain Pathol. 20(4):831-7.

Ofman R, Dijkstra IM, van Roermund CW, Burger N, Turkenburg M, van Cruchten A, van Engen CE, Wanders RJ, Kemp S. 2010. The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy. EMBO Mol Med. 2(3): 90 – 97.

van Geel BM, Assies J, Haverkort EB, Koelman JH, Verbeeten B Jr, Wanders RJ, Barth PG. 1999. Progression of abnormalities in adrenomyeloneuropathy and neurologically asymptomatic X-linked adrenoleukodystrophy despite treatment with “Lorenzo’s oil”. J Neurol Neurosurg Psychiatry. 67(3):290-9.

Weinhofer I, Forss-Petter S, Zigman M, Berger J. 2002. Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy. Hum Mol Gen. 11(22): 2701 – 8.

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Appendix

Dit proefschrift is het resultaat van onderzoek dat vele jaren in beslag heeft genomen en begon in 2005. Vooral het begin was moeilijk, iets wat de meeste promovendi zullen herkennen; de mislukte experimenten en de onzekerheid of het ooit nog zou gaan lukken. Wat het ingewikkelder maakte is de combinatie van onderzoek in een laboratorium (Laboratorium Genetische Metabole Ziekten) met ook patiëntgebonden onderzoek (via de kinderneurologie en neurologie). Het aanleren van nieuwe vaardigheden (zoals het netjes kweken van fibroblasten) kost tijd en ging niet altijd naar wens de eerste maanden. Dit, en andere factoren, maakte dat ik mijzelf het eerste jaar enige malen vervloekt heb dat ik gekozen had voor juist dit project. Uiteindelijk, zoals dat vaak gaat, kan ik alleen maar zeggen dat ik geluk heb gehad. Ik heb de kans gekregen om mijzelf de verdiepen in een fascinerende groep ziektes op het grensvlak van (kinder)neurologie, stofwisselingsziekten en fundamenteel onderzoek. De groep van peroxisomale aandoeningen leent zich uitermate goed voor het verrichten van echt translationeel onderzoek. Daarom denk en hoop ik dat dit proefschrift niet het einde is, maar het begin van verder translationeel onderzoek. Vele mensen hebben bijgedragen aan het tot stand komen van dit proefschrift en wil ik graag hieronder speciaal bedanken.

Allereerst, alle patiënten die bereid waren om medicijnen te slikken, naar het ziekenhuis te komen, en huidbiopten af te staan voor onderzoek.

Prof. dr. B.T. Poll – The, beste Bwee Tien, toen ik in 2004 werd ingepland voor mijn stage kinderneurologie was ik er stellig van overtuigd dat ik er in ieder geval nooit mijn aandachtsgebied van zou willen maken. Die mening heb ik na het doorlopen van de stage kinderneurologie herzien. Je bent voor mij als clinicus een groot voorbeeld en ik ben er trots op door jou opgeleid te zijn tot kinderneuroloog. Ik hoop nog vele jaren te kunnen profiteren van al je kennis, met name van de zeldzame (en niet zo zeldzame) stofwisselingsziekten. Veel dank voor de mogelijkheid om mij te verdiepen in het onderzoeksveld van de peroxisomale ziekten en voor het in mij gestelde vertrouwen. Beste Prof. dr. M. de Visser, beste Marianne, met je heldere blik en onderzoekservaring heb je op de juiste momenten geholpen met het onderzoek vlot trekken en mij bijgestuurd toen dat nodig was. Een manuscript was altijd snel (soms binnen enige uren!) weer retour, met nauwkeurige en zinnige suggesties. Zonder jouw inzet was Hoofdstuk 6 nooit gepubliceerd, een goede les in doorzettingsvermogen. Mijn co-promotor, Dr. S. Kemp, beste Stef, inmiddels kennen wij elkaar alweer meer dan 7 jaar. Het begin bij “lab GMZ” was niet makkelijk voor mij en ik moet eerlijk toegeven dat je het eerste jaar niet tot mijn favoriete personen behoorde (en waarschijnlijk vice versa ook niet). Dat is behoorlijk veranderd de afgelopen jaren: ik beschouw je inmiddels niet alleen als co-promotor maar als vriend. Zonder jouw hulp en ideeën was dit proefschrift er niet geweest. Je bent een zeer integere wetenschapper. Ik heb veel van je geleerd: met name goed naar data kijken en niet meteen het experiment naar de vuilnisbak verwijzen. Ik hoop dat we de komende jaren samen kunnen blijven werken. Je hebt als principal investigator een geweldig functionerende X-ALD onderzoeksgroep gecreëerd, en daar mag je met recht trots op zijn. En natuurlijk ook duizendmaal dank voor de prachtige opmaak van dit proefschrift! Dr. B.M. van Geel, beste Björn, de “godfather van X-ALD” in Nederland, ik ken jou inmiddels al sinds je als neuroloog ging werken in Medisch Centrum Alkmaar. Het was erg leuk om je weer tegen te komen bij dit onderzoeksproject. Dank dat ik gebruik kon maken van je uitgebreide ervaring met X-ALD en dank voor het uitermate nauwgezet bestuderen van alle manuscripten door de jaren heen. Hopelijk kunnen we nog

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lang samenwerken op een “X-ALD polikliniek nieuwe stijl”. Prof. dr. R.J.A. Wanders, beste Ronald, veel dank voor de gelegenheid die jij mij geboden hebt om te werken aan X-ALD. Je enthousiaste bijdragen (vaak kracht bijgezet met termen die ik hier beter niet kan herhalen) en ideeën tijdens de werkbesprekingen waren waardevol. Het is jammer dat je in Japan bent tijdens de verdediging van dit proefschrift. Prof. dr. M. Vermeulen, beste Rien, zelf weet je het misschien niet meer, maar jij hebt mij destijds (in 2004) naar Bwee Tien gestuurd toen ik aangaf dat ik ook graag een promotieplek wilde. Bovendien ben jij degene geweest die de opzet voor de trial met lovastatine hebt bedacht (Hoofdstuk 4). Dank je wel voor je hulp. Dr. M. Dijkgraaf, beste Marcel, je hulp bij het uitwerken van de lovastatine trial was onmisbaar.

De leden van de promotiecommissie Prof. dr. F. Baas, Prof. dr. J.B. van Goudoever, Prof. dr. M.S. van der Knaap, Prof. dr. F.A. Wijburg, Prof. dr. M.A.A.P. Willemsen voor het beoordelen van het manuscript. Prof. dr. P.A. Aubourg, dear Patrick, thank you for taking the time and effort to participate in the defense of my thesis.

Alle mensen die werken op het laboratorium voor Genetische Metabole ziekten hebben door de jaren heen bijgedragen aan het proefschrift. Dank! Een aantal mensen wil ik graag in het bijzonder noemen. Rob Ofman, beste Rob, jouw vaardigheid en praktische ideeën waren onmisbaar. Het isoleren van lipoproteïne fracties en het meten van ZLKV daarin was zonder jou zeker niet gelukt. Inge Dijkstra, beste Inge, dank voor het leren van de qPCR techniek en dank voor al het werk dat je hebt gestoken in het kweken, blotten, en meten in de fibroblasten van de vrouwen met X-ALD. Alle mede-(ex)-AIOs: Jasper Koomen, Robert-Jan Sanders, Riekelt Houtkooper, Michel van Weeghel, Saskia Mandey, Naomi van Vlies, Marit Schneiders, Linda Henneman, Malika Chegari, Ference Loupatti, Nellie Lensink, Wouter Visser, Roos Cuperus, Catherine van Engen, Martin Schackmann en Maxim Boek: dank voor alle praktische hulp, maar natuurlijk ook de gezellige gesprekken (was buitengewoon goed voor mijn algemene ontwikkeling). Petra Mooijer, je zult in het begin wel gek van mij geworden zijn, maar dank dat je mij cellen hebt leren kweken en ook natuurlijk bedankt voor alle beta-oxidatie assays. Annelies Tromp, beste Annelies, zonder jou waren alle samples absoluut nooit meer boven water gekomen.

De afdeling klinische neurofysiologie van het AMC: Dr. Hans Koelman, Dr. Fleur van Rootselaar, Dr. Camiel Verhamme, Marijke Dekker-v.d. Sloot, Jose Dilai, Dwar Sewgobind, Erik Mans, Stephanie Golsteijn, Janny Ree, Edwin Blok, Marianne de Wrede-Spitteler, Claudia jonkhout en Thijs Boeree. Bedankt voor alle EMGs, BAEPs en SSEPs die er altijd bij konden, ook als het druk was. Ik mis de KNF nog steeds zelfs nu er geen koffie machine meer staat!

De studenten die hun wetenschappelijk stage project hebben besteed aan X-ALD: Michiel Hijzen, in no time heb je ongelofelijk veel plasma’s verwerkt, met een geweldig resultaat.Luc Tran, dank voor je ondersteuning bij de trial met bezafibraat. Remmelt Schür en Farnaz Namazi al jullie werk aan het opschonen en invullen van de database met gegevens van de vrouwen met X-ALD heeft mijn leven een stuk makkelijker gemaakt.

Prof.Dr. J.J. Kastelein, ik weet niet of we zonder uw bemiddeling ooit lovastatine hadden bemachtigd voor onze trial.

Dit promotietraject staat niet los van mijn opleiding tot neuroloog en kinderneuroloog. Als

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ik niet in het AMC terecht was gekomen, had ik nooit de kans gekregen om te werken aan dit proefschrift. Dat ik in het AMC terecht ben gekomen heb ik zeker te danken aan wat ik heb geleerd tijdens een fantastische tijd als AGNIO in Medisch Centrum Alkmaar. Dr. R. ten Houten, Dr. M. Aramideh, Dr. J. Brans, Dr. B.M. van Geel, Dr. M.M. Veering: ik heb veel van jullie geleerd. Het AMC is een geweldige en stimulerende plek om opgeleid te worden tot neuroloog, met volop aandacht en mogelijkheden voor patiëntgebonden onderzoek. Tijdens mijn opleiding heb ik een geweldige tijd gehad. Prof.dr J. Stam, beste Jan, ik ben blij dat ik je heb mogen meemaken zowel bij hoorcolleges neurologie tijdens de studie en later ook als opleider neurologie. Naast een “principal investigator” ben je ook beslist een “principal educator”. Dank ook aan alle neurologen, mede-assistenten, en natuurlijk de verpleegkundigen en andere medewerkers van de verpleegafdeling neurologie. Door de jaren heen heb ik van iedereen wel iets geleerd. Veel van het promotieleed heb ik kunnen delen met mijn voormalige kamergenoten op H2: Edo Richard (je hebt mij als student gered van dakloosheid en dankzij jou ben ik Gummbah gaan waarderen), Vincent Odekerken (mede-volgeling van Flying Spaghetti Monster), Daan Velseboer en Constant Verschuur. Dank voor af en toe aanhoren van mijn gezeur over de METC (of DEC) en dergelijke.

Alle medewerkers van de polikliniek neurologie en het secretariaat: Pieta Mooijweer, Harriet Kuil (dank je voor je hulp met allerlei proefschrift gerelateerde zaken), Farida Romeo-Melehi, Aziza Ain Drou, Monique Idzinga-Zijp, Ellisenka Asselman, Maggy van Gils, Carla Erkelens-van der Bosch, Carrie Steevens, Jacqueline Jansen, Yvonne Molenaar, Tineke van Nieuwkoop, Marcia Breuren, Loes de Rink en Petra Stigter. Jullie waren altijd weer flexibel als om mij een kamer toe te kennen als er plots weer iemand met X-ALD voor een van de trials voor de balie stond. Farida en Aziza, bedankt voor het nemen van al die huidbiopten (Hoofdstuk 7) door de jaren heen.

Nadine Fleitour van het onderzoeksbureau: dank voor je hulp bij de LOVA trial.

Mijn collega’s bij de kinderneurologie, Dr. W.C.G. Overweg – Plandsoen en Dr. J.F.M. Niermeijer. Beste Truus en Jikke-Mien, dank voor het extra werk dat jullie op je genomen hebben om mij de kans te geven dit proefschrift af te maken tijdens de zomermaanden.

Mijn goede vrienden en paranimfen: Thomas Cherpanath, beste Thomas, wij zijn al vrienden sinds ons eerste studiejaar in 1995. Ik ben vereerd dat jij mijn paranimf wilt zijn. Ik hoop over een aantal jaren paranimf te zijn op jouw promotie. Ewout Schut, beste Ewout, in januari 2003 begonnen we samen als AGNIO neurologie in het AMC en al snel werden we goede vrienden. Je enorme kennis was onmiddellijk opvallend. Ik ben blij dat er twee opleidingsplaatsen beschikbaar waren, anders ben ik bang dat ik mijn carrière elders had moeten voortzetten. Robert-Jan Sanders, beste Robert-Jan, ik veel aan je gehad als lab maatje, passend dat je nu ook paranimf bent.

Roel en Benne, lieve broertjes, het is dan weliswaar een proefschrift zonder differentiaalvergelijkingen geworden (inderdaad een beetje een alpha proefschrift), maar ik hoop dat jullie het toch eens doorbladeren. Roel, nu je eindelijk weer in Nederland woont (misschien zelfs in Amsterdam binnenkort?) kunnen we weer eens wat bier gaan drinken?

Jos en Marlein, lieve pap en mam, dank voor alles: jullie liefde en steun, aanmoediging, en natuurlijk de bereidheid om altijd op Philip te passen als er weer eens een au pair uitviel. Pap,

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ik weet dat je soms twijfelde of ik ooit nog wel zou promoveren (want welke wetenschapper is nou niet gepromoveerd voor de 30!?) maar kijk, het is toch gelukt!

친애하는 장모님, 장인 어르신, 제 박사학위식에 참석차 먼길 네덜란드까지 와주셔서 저는 정말 행복합니다.

Lieve Joo Yeon, 사랑하는 주연, 당신을 사랑 해요. 당신이있어 행복합니다.

Lieve Philip, je bent het leukste en liefste kind dat ik mij kon wensen. Altijd ondernemend en vrolijk, en je neemt het mij nooit kwalijk als ik toch eerst nog even moest werken. Ik ben trots op je.

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Curriculum vitae

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Curriculum vitae

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Ik ben geboren op 10 oktober 1977. Ik studeerde geneeskunde aan de Universiteit van Amsterdam en behaalde mijn artsexamen in 2002. Ik werkte als AGNIO neurologie in het Medisch Centrum Alkmaar (MCA), en volgde vervolgens de opleiding tot neuroloog in het Academisch Medisch Centrum in Amsterdam (opleider: Prof. dr. J. Stam, opleider KNF: Dr. J.H.T.M. Koelman, opleider perifere stage MCA: Dr. R. ten Houten). De opleiding werd twee jaar onderbroken waarin ik ben begonnen met het onderzoeksproject beschreven in dit proefschrift. In 2009 werkte ik een jaar als arts-assistent kindergeneeskunde in het Universitair Medisch Centrum Utrecht in het kader van de subspecialisatie tot kinderneuroloog (opleider: Dr. J. Frenkel). Registratie als neuroloog volgde in 2011. Het fellowship kinderneurologie werd afgerond in 2012.

List of Publications

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List of Publications

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Docherty CC, Kalmar-Nagy J, Engelen M, Nathanielsz PW. 2001. Development of fetal vascular reponses to endothelin-1 and acetylcholine in the sheep. American Journal of Physiology 280 (2): R554 – 562.

Docherty CC, Kalmar-Nagy J, Engelen M, Koenen SV, Nijland M, Kuc RE, Davenport AP, Nathanielsz PW. 2001. Effect of in vivo fetal infusion of dexamethasone at 0.75 GA on fetal ovine resistance artery responses to ET-1. American Journal of Physiology 281 (1): R261 – 268.

Boekholdt SM, Trip MD, Peters RJG, Engelen M, Boer JMA, Feskens EJM, Zwinderman AH, Kastelein JJP, Reitsma PH. 2002. Thrombospondin-2 polymorphism is associated with a reduced risk of premature myocardial infarction. Arteriosclerosis Thrombosis and Vascular Biology 22 (12): e24 – 27.

Engelen M, Tijssen MAJ. 2005. Paroxysmal non-kinesiogenic dyskinesia in antiphospholipid syndrome. Movement Disorders 20 (1): 111 – 113.

Jong de BA, Engelen M, Schaik van IN, Vermeulen M. 2005. Confusing Cochrane reviews on treatment in multiple sclerosis. Lancet Neurology 4 (6): 330 – 331.

Engelen M, Ofman R, Mooijer PAW, Poll – The BT, Wanders RJA, Kemp S. 2008. Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy. Biochimica Biophysica Acta 1781 (3): 105 – 111.

Engelen M, Kemp S, Geel van BM. 2008. Van Gen tot Ziekte: X-gebonden adrenoleukodystrofie. Nederlands Tijdschrift voor Geneeskunde 152 (14): 804 – 808.

Engelen M, Nederkoorn PJ, Smits M, Beek van de D. 2009. Delayed life-threathening subdural hematoma after minor head injury in a patient with severe coagulopathy: a case report. Cases Journal 2: 7587.

Engelen M, Ofman R, Dijkgraaf MGW, Hijzen M, Wardt van der LA, Geel van BM, Visser de M, Wanders RJA, Poll – The BT, Kemp S. 2010. Lovastatin in X-linked adrenoleukodystrophy. New England Journal of Medicine 362 (3): 276 – 277.

Engelen M, Westhoff D, Gans de J, Nederkoorn PJ. 2011. A 64-year old man presenting with carotid artery occlusion and corticobasal syndrome: a case report. Journal of Medical Case Reports 5: 357.

Engelen M, Kooi van der AJ, Kemp S, Wanders RJA, Sistermans EA, Waterham HR, Koelman JTM, Geel van BM, Visser de M. 2011. X-linked adrenomyeloneuropathy due to a novel missense mutation in the ABCD1 start codon presenting as demyelination neuropathy. Journal of the Peripheral Nervous System 16 (4): 353 – 355.

Gevers S, Nederveen AJ, Fijnvandraat K, Berg van den SM, Ooij van P, Heijtel DF, Heijboer H, Nederkoorn PJ, Engelen M, Osch van MJ, Majoie CB. 2012. Arterial spin labeling measurement of cerebral perfusion in children with sickle cell disease. Journal of Magnetic Resonance Imaging 35 (4): 779 – 787.

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Engelen M, Tran L, Ofman R, Brennecke J, Moser AB, Dijkstra IME, Wanders RJA, Poll – The BT, Kemp S. 2012. Bezafibrate for X-linked adrenoleukodystrophy. PLoS One 7(7): e41013.

Engelen M, Kemp S, Visser de M, Geel van BM, Wanders RJA, Aubourg PA, Poll – The BT. 2012. Orphanet Journal of Rare Diseases 7(1): 51.

Engelen M, Poll – The BT. 2012. Peroxisomal Leukoencephalopathy. Seminars in Neurology 32 (1): 42 – 50.

Engelen M, Schackmann MJA, Ofman R, Sanders RJ, Dijkstra IME, Houten SM, Fourcade S, Pujol A, Poll – The BT, Wanders RJA, Kemp S. 2012. Bezafibrate lowers very long-chain fatty acids in X-linked adrenoleukodystrophy fibroblasts by inhibiting fatty acid elongation. Journal of Inherited Metabolic Diseases DOI: 10.1007/s10545-012-9471-4