5. 3
VRIJE UNIVERSITEIT
Vanishing white matter
A study of phenotypic variation and the relationship
between genotype and phenotype
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. V. Subramaniam,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de Faculteit der Geneeskunde
op dinsdag 1 december 2015 om 13.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Hanna Ditta Willemina van der Lei
geboren te Bussum
6. 4
promotor: prof.dr. M.S. van der Knaap
copromotoren: dr. G.C. Scheper
dr. T.E.M. Abbink
9. CONTENT
Chapter 1 General Introduction
Chapter 2 Phenotypic variation in vanishing white matter
disease
Chapter 3 Characteristics of early MRI in children and
adolescents with vanishing white matter
Chapter 4 Restricted diffusion in vanishing white matter
Chapter 5 Genotype - phenotype correlation in vanishing
white matter disease
Chapter 6 Severity of vanishing white matter disease does
not correlate with deficits in eIF2B activity or the
integrity of eIF2B complexes
Chapter 7 Summary, discussion and future perspectives
Chapter 8 Samenvatting, discussie en toekomstperspectieven
List of publications
Curriculum vitae
Dankwoord
9
31
65
77
93
111
135
147
156
157
158
12. 10
Chapter 1
GENERAL INTRODUCTION
Vanishing white matter (VWM; OMIM number 603896)1
is a genetic leukoencephalopathy linked to
mutations in either of the five genes encoding eukaryotic translation initiation factor 2B (eIF2B).2,3
It is a disease of all ages. Patients experience slowly progressive neurologic deterioration with
additional episodes of rapid clinical decline triggered by physical stress like febrile infections and
minor head trauma. The disease is fatal. VWM is one of the most prevalent inherited childhood white
matter disorders4
, although its exact incidence has not been determined. The diagnosis of VWM
can be made with confidence in individuals presenting with typical clinical findings, characteristic
abnormalities on cranial MRI, and identifiable mutations in one of five genes, encoding the subunits
of eIF2B.4,5
There is no specific treatment for VWM. Management is at present supportive, based on
treatment of symptoms, avoidance of stress situations known to provoke deterioration, prevention
of secondary complications and genetic counselling of individuals and families.6
HISTORY
The history of VWM is longer than usually assumed.5-7
Probably one of the first descriptions of
the disease that can be found dates back to 1962 when Eicke8
described clinical features and
autopsy findings characteristic for VWM in a 36-year-old woman who presented at age 31 years
with gait difficulties and secondary amenorrhoea. She experienced chronic progressive disease
with episodes of rapid deterioration after minor physical trauma. At autopsy a diffuse, cystic
destruction of the cerebral white matter was seen with around the cystic areas high numbers
of oligodendrocytes. Only mild fibrillary astrocytosis and scant sudanophilic lipids were present.
The diagnosis was “atypical diffuse sclerosis”. Similar neuropathological case descriptions by
Watanabe9
, Girard10
, Anzil11
, Deisenhammer12
, Gautier13
, and Graveleau14
and their co-workers
were published. Cavitatory degeneration of the cerebral white matter and the presence of
increased numbers of oligodendrocytes were central findings.8-14
Some mentioned febrile
infections and minor trauma as provoking factors.8,9,10
The disease was not recognised as one
disease entity until 1993, when Hanefeld15
and Schiffmann16
and colleagues described series of
patients with a disease characterised by a childhood-onset, progressive leukoencephalopathy
with an autosomal recessive mode of inheritance. Minor head trauma as a provoking factor
was recognized15
and the typical proton magnetic resonance spectroscopy (MRS) findings were
described: a decrease of all MRS signals in the affected white matter.15-17
Brain biopsy findings in
two patients were interpreted as indicative of hypomyelination and the name “childhood ataxia
with central nervous system hypomyelination” was proposed.16
Van der Knaap and colleagues
described another series of patients with a larger clinical variation in age of onset and rate of
progression and recognised both febrile infections and minor head trauma as provoking factors
for the disease.1,18
MRI and MRS findings were interpreted as indicative of progressive cystic
degeneration of the cerebral white matter rather than hypomyelination, which was confirmed
by autopsy findings.1,4
In line with these observations the name “vanishing white matter” was
proposed.1,4
Brück and co-workers used the name “myelinopathia centralis diffusa”.19
13. 11
General introduction
In 2001 and 2002 it became known that the disease is caused by mutations in any of the five
genes, encoding the subunits of eukaryotic translation initiation factor 2B (eIF2B), which has an
important role in protein synthesis and in the regulation of protein synthesis rates under dif-
ferent conditions, including cellular stress.20,21
The known clinical variation has been expanding
ever since. The term “eIF2B-related disorders” was proposed to include all clinical phenotypes
related to mutations in eIF2B subunit genes.6,22,23
CLINICAL MANIFESTATIONS
VWM is in its so-called classical form characterized by chronic progressive neurological deterioration
with cerebellar ataxia, less prominent spasticity and relatively mild mental decline.1,15,16
In addition,
rapid deterioration may occur during febrile illness or following minor head trauma or fright.24-26
The disease shows an extremely wide phenotypic variation ranging from severe congenital or
early infantile forms up to patients with an onset in adulthood with slowly progressive neuro-
logical decline.6,18,22-24,27
The brain is the most severely affected organ in all variants.24
The age
of onset is predictive of disease severity.18,22,23
An overview of all reported patients world-wide
showed that approximately 20% of the patients have an onset before the age of 2 years, 45%
between ages 2 and 5, 20% between ages 6 and 16, and 15% after the age of 16 years.28
The
time course of disease progression varies from individual to individual even within the same
family18,20,29-31
ranging from rapid progression with death occurring within a few months up to
very slow progression with death occurring many years after onset.1,5,18,31
In the literature different clinical phenotypes have been described based on age of onset.6,22-24
Severe phenotype: antenatal – infantile onset
The antenatal/congenital onset form is characterized by a severe encephalopathy. The most se-
vere variants of VWM known, present in the third trimester of pregnancy with decreased fetal
movements, contractures, oligohydramnios, growth failure and microcephaly. A rapid decline
soon after birth occurs with feeding difficulties, failure to thrive, vomiting, axial hypotonia, limb
hypertonia or hypotonia, cataract and microcephaly. Apathy, irritability, intractable seizures,
and finally apneic episodes and coma follow. In addition to signs of a serious encephalopathy
and ovarian dysgenesis in females, only the antenatal onset patients may display growth failure,
microcephaly, cataracts, hepatosplenomegaly, pancreatic abnormalities, and kidney hypoplasia.
Death follows within a few months.22,32
A slightly milder, but also severe and rapidly fatal form of VWM is characterized by an onset in
the first year of life with death before the age of two. 33-35
Francalanci et al.33
describe two sisters
with irritability, stupor, and rapid loss of motor abilities following an intercurrent infection at
age 10 to 11 months and death at age of 21 months. “Cree leukoencephalopathy”, described
among the native North American Cree and Chippewayan indigenous population, has its onset
between 3 and 9 months and death occurs before the age of 2 years.35,36
14. 12
Chapter 1
Classical phenotype: early childhood onset
The most frequent, ‘classical’ variant of VWM has its onset in early childhood, between the
ages of 2 and 6 years.1,15,16,18
Initially motor and intellectual development is normal or mildly
delayed, followed by chronic progressive neurological deterioration, although patients may also
be stable for a long period at any stage of the disease. Cerebellar ataxia usually dominates the
clinical picture, whereas spasticity is less prominent and intellectual abilities are relatively pre-
served.1,15,16,18
Epilepsy, often mild and well treatable, may occur. 1,15,16,18
Exceptional cases with
more serious epilepsy have been reported.37
Optic atrophy may develop with loss of vision at
later stages, but not in all patients.16
In a few cases peripheral neuropathy has been reported,
although in most patients there is no clinical and neurophysiologic evidence of involvement of
peripheral nerves.38,39
The head circumference is normal in most patients but especially in more
severe patients progressive macrocephaly may occur in the context of rapidly progressive cystic
degeneration of the cerebral white matter.40,41
Additionally episodes of rapid deterioration may occur, during which patients rapidly lose mo-
tor skills and become hypotonic. Irritability, vomiting, and seizures are followed by somnolence
and lowering of consciousness.1,18
The decline may end in coma and death. If recovery occurs, it
is usually incomplete. The episodes are provoked by febrile infections, minor head trauma and,
rarely, fright. With head trauma and fright, the deterioration occurs instantaneously, whereas
the deterioration occurs in the days after the beginning of febrile infections, independent of
the course of the infection and recovery from it. Strikingly, not every provoking incident is fol-
lowed by deterioration. Most patients die a few years after disease onset, but some do so after
only a few months while other patients remain relatively stable for decades.1,15,16,18
Mild phenotype: late-childhood – adult onset
Over time milder variants with an adolescent or adult onset of VWM were recognized.6,18,28,42-44
The latest onset of disease that has been reported is 62 years.28
The clinical presentation be-
comes more variable with an onset at later age. Later onset disease generally has a more in-
sidious onset, a slower course and the stress-provoked episodes of rapid deterioration are less
common.28
In some adults, the disease starts with motor deterioration, similar to the classical
phenotype.45
However, alteration in intellectual abilities and behavioral changes can be the ini-
tial sign in adult onset forms.29,31,43,44,46
, Occasional seizures29
, complicated migraines, psychiatric
symptoms28,29,46
and presenile dementia28,47
have been described as first signs of the disease. Un-
expectedly rapid progression and death within a few months has also been published.18
In females with VWM primary or secondary amenorrhea related to ovarian failure is frequently
observed.32,48
The signs of ovarian failure may precede or follow the neurological deterioration.28
Asymptomatic cases
A- or presymptomatic patients have been described, also with a typically affected sibling.2,29,46,49
15. 13
General introduction
Ovarian failure
The juvenile and adult forms are often associated with primary or secondary ovarian failure in
females, a syndrome referred to as “ovarioleukodystrophy”. 48,50
Ovarian dysgenesis, however,
may occur in all different disease severities.1,8,22,32,48,50
At autopsy in infantile and childhood cases
ovarian dysgenesis has been found. The affected individuals were prepubertal and the ovarian
dysgenesis was clinically not manifest.1,22,32
Premature ovarian failure in the absence of leukoen-
cephalopathy is not associated with mutations in EIF2B1-5.51
Phenotypic spectrum
It is becoming clear that VWM may occur at all ages.5,6,28
Whereas VWM was initially regarded a
disease of children, an increasing number of adults has been diagnosed. At present limited in-
formation is available on the relative occurrence and phenotypic presentation over all ages.
MAGNETIC RESONANCE
The second step in the diagnosis of VWM is the cranial magnetic resonance imaging (MRI). Vali-
dated MRI criteria allow an MRI-based diagnosis of VWM in patients with a typical MRI.5 MRI is
an effective tool for the diagnosis; the correlation between in MRI findings typical of VWM and
detection of mutations in the EIF2B1-5 genes is very high.4,5,52,53
Figure 1 | Normal axial T2-weighted (a) and FLAIR (b), and sagittal T1-weighted (c) images of a 3-year-old child.
On T2-weighted (a) and FLAIR (b) images, cortex, basal ganglia and thalami are gray; myelinated white matter
structures are dark-gray. CSF is white on T2-weighted images and black on FLAIR images. On T1-weighted
images (c), cortex is gray, myelinated white matter is white and CSF is black.
In healthy persons normal, myelinated white matter has a low signal on T2-weighted, proton
density and FLAIR images. The signal is high on T1-weighted images (figure 1). CSF has a high
signal on T2-weighted images and a low signal on proton density, fluid-attenuated inversion
recovery (FLAIR) and T1-weighted images (figure 1).6
b ca
16. 14
Chapter 1
Figure 2 | MR images of a 2-year-old patient with VWM. The axial T2-weighted images (a, b) show the diffuse
signal abnormality of the cerebral white matter (a). The globus pallidus (a), cerebellar white matter (b), mid-
dle cerebellar peduncles (b), central tegmental tracts in the pontine tegmentum (b) and pyramidal tracts in
the basis of the pons (b) also have an abnormal signal. Axial FLAIR images (c, d) show that all cerebral white
matter is abnormal, in part having a high signal and in part a low signal, similar to CSF, indicative of cystic
degeneration. Within the rarefied and cystic white matter, dots and stripes are seen, indicative of remaining
tissue strands (c, d). The sagittal T1-weighted image (e) shows a pattern of radiating stripes within the abnor-
mal white matter, representing the remaining tissue strands. Axial diffusion-weighted images (f) show a high
signal, suggestive of restricted diffusion, in the directly subcortical white matter, corpus callosum and internal
capsule. The remainder of the white matter has a low signal, suggesting increased diffusion (f). The ADC
map (g) confirms the decreased diffusion in the areas mentioned with low ADC values (40-60), and increased
diffusion in the remainder of the white matter with high ADC values (160-220). NB Normal myelinated white
matter has ADC values of approximately 70–90 × 10−5 mm2/sec.6
In VWM MRI typically shows symmetrically diffuse abnormality of all or almost all the cerebral
hemispheric white matter with evidence of progressive white matter rarefaction in a “melt-
ing-away” pattern. Well-delineated cysts are rare. The U-fibres may be relatively spared.1,18,54
This change is best shown by proton density and FLAIR images. In contrast to MRI in healthy
individuals the abnormal white matter has a high signal on proton density, T2-weighted and
FLAIR images and a low signal on T1-weighted images (figure 2). Cystic white matter has the
signal behaviour of CSF, different from abnormal white matter on proton density and FLAIR
images (figure 2). A fine meshwork of remaining tissue strands is usually visible within the areas
of CSF-like white matter, with a typical radiating appearance on sagittal and coronal images and
a b c d
e f g
17. 15
General introduction
a dot-like pattern in the centrum semiovale on the transverse images (figure 2). Over time, MRI
shows evidence of progressive rarefaction and cystic degeneration of the affected white matter,
which is replaced by fluid.1,3,5,18,54
In the end-stage, all white matter has disappeared between the ependymal lining and the cor-
tex. A fluid-filled space remains, although the cerebral cortex does not collapse (figure 2).6
Using genetic analysis as the ‘golden standard’, the proposed MRI criteria have 95% sensitivity
and 94% specificity.1,5,18
MRI CRITERIA FOR THE DIAGNOSIS OF VWM5
Obligatory criteria
1. The cerebral white matter exhibits either diffuse or extensive signal abnormalities; only the
immediately subcortical white matter may be spared.
2. Part or all of the abnormal white matter has a signal intensity close to or the same as CSF on
proton density or FLAIR images, suggestive of white matter rarefaction or cystic destruction.
3. If proton density and FLAIR images suggest that all cerebral white matter has disappeared,
there is a fluid-filled distance between ependymal lining and the cortex, and not a total col-
lapse of the white matter.
4. The disappearance of the cerebral white matter occurs in a diffuse “melting away” pattern.
5. The temporal lobes are relatively spared, in the extent of the abnormal signal, degree of
cystic destruction, or both.
6. The cerebellar white matter may be abnormal, but does not contain cysts. 7. There is no con-
trast enhancement.
Suggestive criteria
1. Within the abnormal white matter there is a pattern of radiating stripes on sagittal and
coronal T1-weighted or FLAIR images; on axial images, dots and stripes are seen within the
abnormal white matter as cross-sections of the stripes.
2. Lesions within the central tegmental tracts in the pontine tegmentum.
3. Involvement of the inner blade of the corpus callosum, whereas the outer blade is spared.
18. 16
Chapter 1
Figure 3 | Axial T2- images of a VWM patient, obtained at 6 days (a) and 5 months (b). The initial MRI (a) shows
broadening of gyri and a mildly swollen aspect of the cerebral white matter. Its signal intensity is normal for
unmyelinated white matter. The follow-up MRI (b) shows an impressive atrophy of the cerebral white matter
with highly dilated lateral ventricles. What remains of the white matter has too high a signal intensity, even for
unmyelinated white matter.6
a b
a
a b
c d
19. 17
General introduction
Figure 4 | The axial FLAIR image of a 15-year-old boy with recent onset disease (a) shows extensive cerebral
white matter abnormalities, sparing the subcortical white matter. The inner blade of the corpus callosum is
affected whereas the outer blade is better preserved. There is no evidence of white matter rarefaction. The
axial FLAIR image of a 46-year-old woman (b), who has been symptomatic for approximately 10 years, shows
the same with additional white matter atrophy. The axial FLAIR image of a 42-year-old man (c), who has been
symptomatic for 18 years, shows the same picture as the previous patient, with additional cystic degeneration
of the cerebral white matter. The cerebral white matter atrophy is more severe. In contrast, the axial FLAIR
image of a 37-year-old woman (d), who has been symptomatic for 2 years, shows the classical MRI picture,
comparable to figures 2c and 2d.6
In the most severe, and also in de mildest cases or earliest stages of the disease at any age, MRI
findings may be atypical and the MRI criteria may not apply.1,6,29,55
In early infantile VWM the
gyral pattern may look immature and the white matter may look swollen preceding the stage of
rarefaction. The cerebral white matter may become highly atrophic over time, with the ependy-
mal lining touching the depth of the gyri (figure 3).6,22,32,54
In late onset cases, teenagers and adults, the rarefaction or cystic degeneration in the white mat-
ter is usually less prominent or even absent (figure 4). Atrophy is often present (figure 4).28,29,48
Several presymptomatic and mildly symptomatic patients underwent MRI with initially not nec-
essarily evidence of white matter rarefaction. For example, in an asymptomatic child at the age
of 2 a diffuse leukoencephalopathy was seen without cavitation. One year later cystic degener-
ation was found.1
In addition, absence of any evidence of white matter rarefaction on MRI was
found in an 18-year-old woman who only experienced a tonic-clonic seizure.29
On diffusion-weighted images, the rarefied and cystic white matter demonstrates an increased
diffusivity.56,57
Areas of restricted diffusion can be found within the non-rarefied white mat-
ter.56,57
The histopathologic correlate of the diffusion restriction is unclear.
Proton magnetic resonance spectroscopy
In VWM the findings with proton MRS depend on the stage of white matter rarefaction. The
white matter spectrum is relatively preserved when there is little white matter degeneration.
Follow-up investigations reveal progressive reduction of all the white matter metabolites. In
the end stage, the spectrum is similar to that of CSF with some lactate and glucose and no or
minor “normal” signals. This may be seen in any brain disease with cystic degeneration and is
not diagnostic for VWM. The cortical, gray matter spectrum stays well preserved throughout the
disease course.1,6,15-18,55, 58
20. 18
Chapter 1
GENETICS
The diagnosis VWM is completed by demonstrating that both alleles of one of the genes encod-
ing the subunits of eukaryotic translation initiation factor eIF2B contain a pathogenic mutation.
History
The step-wise search for the genetic cause of VWM started in the late nineteen nineties when
a genetic linkage study was initiated using exclusively MRI criteria to select patients for this
study.1,6,18
The focus on Dutch patients lowered the risk of genetic heterogeneity and two found-
er effects in The Netherlands were each key to finding disease-causing mutations in a gene. The
two genes, EIF2B5 and EIF2B2, are both encoding a subunit of eIF2B. 2,3,53,59,60
Subsequently, it was
shown that VWM could be related to mutations in any of the five genes (EIF2B1-5), encoding the
five subunits of eIF2B (eIF2Bα, β, γ, δ and ε). 2,3,53,59,60
Mutations
Several reports of the VWM-causing mutations have been published.6,61,62
Almost 170 different
mutations have been published.6,63
(94, 24, 17, 19 and 8 in EIF2B5, EIF2B4, EIF2B3, EIF2B2, and
EIF2B1, respectively), of which approximately 80% are missense mutations. If patients are com-
pound heterozygous for two mutations, the mutations always affect the same gene.5,21,22
,34,35,48,61
Mutations in EIF2B5 are most frequent; two-thirds of the patients with VWM have mutations
in EIF2B5. It is the largest subunit, but it also contains a disproportionately high number of
mutations.6,21,53,62
Frameshifts and nonsense mutations are rare and have been reported only in the compound-het-
erozygous state. Patients never have two null-mutations. Patients have at most one null-muta-
tion, invariably in combination with a missense mutation.6
The pathogenic mutation leading to the amino acid change p.Arg113His in the eIF2Bε subunit is
by far the most frequently observed mutation. This mutation is found in approximately 40% of
the patients.6,21,64,61
Other more frequent amino acid changes affect Thr91, Arg315 and Arg339 in
eIF2Bε and Glu213 in eIF2Bβ. The eIF2B complex is highly conserved in all eukaryotes.6,21,64,61
The
low number of non-synonymous single nucleotide polymorphisms (SNPs) occurring in the EIF2B1-5
genes reflect the importance of sequence conservation.6
Genotype-phenotype correlation
A wide variability in severity has been observed among VWM patients, even among patients
with the same mutations, and among patients within families 2,18,29-31
That is why the existence
of a genotype-phenotype correlation was questioned and why it was concluded that
environmental and/or genetic factors other than the eIF2B mutations determine at least part
of the phenotype.5,6,7
However, it is clear that some mutations are consistently associated with
a relatively benign phenotype, such as p.Arg113His in eIF2Bε and p.Glu213Gly in eIF2Bβ.21,28,29
A high percentage of patients with adult onset VWM with slow disease progression have
21. 19
General introduction
the p.Arg113His mutation in eIF2Bε in the homozygous state.28,29
This mutation is also most
frequently found in women with ovarioleukodystrophy.48,65,31
Arg113 is not conserved even
among mammals; histidine is the normal amino acid at the equivalent position in mouse and
rat, which could explain why p.Arg113His is responsible for a milder phenotype in humans.7,48
In the other end of the spectrum of VWM, specific mutations, including p.Arg195His in eIF2Bε
(the Cree founder mutation), p.Val309Leu in eIF2Bε, p.Pro247Leu in eIF2Bδ and p.Gly200Ala in
eIF2Bβ are consistently associated with a severe phenotype.6,7,22,23,34,35,52
All in all, there is evidence for a genotype-phenotype correlation, but a confirmatory study on
the subject is lacking.
MALE-FEMALE RATIO
Males and females are equally affected among the patients with infantile and childhood onset
of the disease.6
Surprisingly, among adult onset VWM patients, a predominance of females has
been observed.28
The reason for the predominance of females among the older patients is not
understood. It has been suggested that with mild mutations, females are more prone to disease
presentation, while more males remain asymptomatic.28
PATHOPHYSIOLOGY OF VWM
The genes mutated in VWM, EIF2B1-5, encode the subunits of a pentameric complex that is
involved in protein synthesis, the eukaryotic initiation factor 2B (eIF2B).2,21
Physiology of eIF2B
eIF2B is an enzyme that is crucial for the initiation step of the translation of all mRNAs. It ac-
tivates its substrate eIF2 through the exchange of GDP for GTP (figure 5). Only eIF2-GTP and
not eIF2-GDP can form a ternary complex with initiator methionyl-tRNA. This complex binds to
the 40S ribosomal subunit, which only then binds the 5’ cap structure of an mRNA and starts
scanning for an AUG start codon in the 5’ untranslated region (5’UTR) of a gene. Upon AUG
start codon recognition by the tRNA anti-codon loop, the 60S ribosomal subunit joins the com-
plex and forms a translation-competent 80S ribosome. Simultaneously, eIF2-GTP is hydrolyzed to
eIF2-GDP, which subsequently leaves the translation complex. The guanine nucleotide exchange
(GEF) activity of eIF2B is indispensable to regenerate active eIF2-GTP to allow new rounds of
initiation to occur.66,67
The best-studied pathway of regulation of the activity of eIF2B occurs through the phosphoryla-
tion of the α-subunit of eIF2. When phosphorylated on its α-subunit, eIF2 binds eIF2B so tightly
that it inhibits its activity, leading to a reduction or shut-down of overall protein synthesis.68
This
makes eIF2B a key regulator of general protein synthesis.
22. 20
Chapter 1
Figure 5 | The purpose of the initiation of translation is to position a translation competent ribosome
on the start codon of the messenger RNA. This process starts by binding of a ternary complex consist-
ing of eIF2, GTP and charged initiator methionyl-tRNA to the small ribosomal subunit (40S), which leads
to formation of the 43S pre-initiation complex. Subsequent binding of the mRNA results in 48S forma-
tion. The ribosome will scan the 5ʹuntranslated region for an AUG start codon. Upon recognition of the
start codon the large ribosomal subunit (60S) binds to form an 80S ribosomal complex. Concomitant-
ly, the GTP on eIF2 is hydrolysed to GDP and eIF2 is released from the ribosome. The 80S ribosome will
enter the elongation phase of translation. The inactive eIF2⋅GDP is reactivated by exchanging GDP for
GTP. eIF2B is essential in this step by dissociating GDP from eIF2. The main mechanism to regulate the ac-
tivity of eIF2B is through phosphorylation of eIF2 on the α-subunit. Phosphorylated eIF2 binds tightly to
eIF2B and acts as a competitive inhibitor of the GDP-GTP exchange reaction. Several other translation
initiation factors that are involved in the initiation process were omitted from this drawing for clarity.6
Down-regulation of eIF2B activity is part of the cellular stress response. Protein synthesis is
downregulated under different stress condition, for example heme deficiency, amino acid star-
vation, misfolded proteins in the endoplasmic reticulum, and during viral infections as part
of the interferon response. This response is important to guarantee cell survival under harm-
ful conditions and could link to the clinical observation that VWM patients rapidly deteriorate
during systemic infections and head trauma.6,69-73
Altered eIF2B activity
The functional effects of mutations in eIF2B can affect the eIF2B activity in diverse ways: by loss
of function of the affected subunit, altering the stability of individual subunits, failure to form
complexes with the other subunits, altering its catalytic activity, affecting the interaction with
the substrate eIF2, or a combination of these.74-77
23. 21
General introduction
At first mutations in eIF2B were reported to decrease eIF2B activity by 20 to 70% as measured
in patient-derived lymphoblasts or fibroblasts.52
The severity of the decrease was reported to
correlate with the clinical severity, although later data showed inconsistencies in this correla-
tion.52,78
In patients’ lymphoblasts and fibroblasts, the decreased eIF2B activity was not found
to affect the rate of global protein synthesis, before, during or after stress (e.g. heat shock or
recovery after), or the ability of these cells to proliferate and survive.76,79,80
These observations
suggest that basal eIF2B activity by itself may not or not straightforwardly explain the disease.6,7
This conclusion warrants further investigations. One reason for this is that assessment of eIF2B
activity in patient-derived lymphoblasts or fibroblasts has been proposed as a tool in the diagno-
sis of VWM78
and lack of correlation with disease mechanisms raises the question what is actually
assessed when eIF2B activity is measured.
Pathology findings
VWM is a cavitating orthochromatic leukoencephalopathy. Characteristic neuropathological
findings include tissue rarefaction and cystic degeneration of the white matter with surprisingly
meagre reactive gliosis, dysmorphic astrocytes, and paucity of myelin despite a striking increase
in oligodendrocytic cellular density.1,6,7,18,19,81,82
On macroscopic examination the cerebral white matter varies from appearing grayish and ge-
latinous to more cystic and cavitary (figure 6). The frontoparietal
white matter, particularly deep and periventricular, is more commonly involved with relative
sparing of the temporal lobe, optic tracts, corpus callosum, anterior commissure, and internal
capsule. The cortex and other gray structures are normal.1,18,19,81,82
In contrast with children, neo-
nates and infants show brain swelling with flattening of the gyri, while adults display a variable
degree of atrophy.1,6,81
24. 22
Chapter 1
Figure 6. | Gross morphology of VWM, Luxol fast blue staining. A coronal section of the left hemisphere
demonstrates myelin loss of the centrum semiovale extending to the gyral white matter but sparing the
U-fibers. Note the relative preservation of the striatal and pallidal white matter and of the internal capsule.
Cortical and subcortical gray matter appears to be uninvolved.6
Microscopic examination of VWM brain tissue shows that white matter oligodendrocytes and
astrocytes bear the brunt of the disease in this disease (figure 7).1,19,83
Increased numbers of
oligodendrocytes are present around cystic areas and in less affected white matter.18,81,82
Part of
the oligodendrocytes display an abundant foamy cytoplasm and are in that way a distinguishing
pathological feature of VWM.6,82
The paradoxical coexistence of increased numbers of oligoden-
drocytes and paucity of myelin in relatively preserved areas prompted a question regarding the
functional maturity of oligodendrocytes in VWM.
25. 23
General introduction
Figure 7. | Macroglial cells in the white matter of a VWM patient, hematoxylin-eosin staining, magnification ×
400. Astrocytes (a) have blunt, coarse processes instead of the fine arborisations seen in normal reactive cells
(insert). Oligodendrocytes (b) have abundant and finely granular cytoplasma; a normal cell (insert) is given
for comparison. 6
Astrocytes are dysmorphic with short blunt processes instead of the fine arborisations seen in
activated normal astrocytes.6,81,82
The abnormal appearance of astrocytes may be explained by
abnormality in the cytoskeletal composition, with an abnormal increase in the cytoskeletal pro-
tein GFAP-delta.84
Recent studies on maturation of macroglia in VWM brains confirmed that
the maturation status of astrocytes and oligodendrocytes is affected. Astrocytes proliferate but
remain immature, which probably explains the lack of astrogiosis in damaged white matter.84
Oligodendrocyte precursor cells are highly increased in numbers. A block in their maturation
may explain the striking concurrence of oligodendrocytosis and myelin paucity.84
Additionally,
high molecular weight hyaluronan, a known inhibitor of oligodendrocyte maturation, and its
receptor CD44 were found to be elevated in VWM white matter.83,84
Hyaluronan is produced by
astrocytes. A correlation was shown between the level of high molecular weight hyaluronan
and the degree of white matter damage in VWM.
eIF2B and involvement of specific tissues
The reason why the white matter of the central nervous system and, less consistently, the ovaries
are selectively vulnerable to mutations in genes coding for eIF2B is as yet not understood.
Aims/Scope and outline of this thesis
In the nineties VWM was recognizes as disease entity. In 2001 and 2002, before the start of this
study, the genetic defect underlying VWM was found. This discovery made it possible to study
different aspects of this currently untreatable disorder. This thesis describes the research that
has been done to increase our understanding of the phenotypic variation and correlation be-
tween genotype and phenotype in VWM.
a b
26. 24
Chapter 1
Large studies on phenotypic variation in VWM are scarce. In chapter 2 a cross-sectional observa-
tional study is presented. We investigated the disease course in a cohort of 228 patients. We col-
lected data on prevalence and characteristics of subgroups of patients defined by age of onset
and explored male versus female differences. One aim of this study is to increase our knowledge
of the clinical phenotype of VWM and in that way increase insight into the disease. A second
aim is to collect historical control information, which may be needed for trials on therapies that
do not allow blinding, such as cell-based therapies.
In VWM MRI typically shows diffuse and symmetrical abnormalities of the cerebral white matter.
Over time the cerebral white matter becomes progressively rarefied and cystic. Before DNA test-
ing was available, the diagnosis of VWM was made by clinical and MRI criteria. Some patients,
however, underwent MRI in the presymptomatic or early symptomatic stage and their MRIs may
not fulfill the criteria. Insight in early MRI characteristics is lacking. We therefore performed a
study on early MRI characteristics in VWM. In chapter 3 the results are presented.
In chapter 4 we focus on diffusion-weighted imaging (DWI). DWI reveals increased diffusion of
the rarefied and cystic regions in VWM, but we also observed areas with restricted diffusion in
some patients. It is unclear what the underlying histology is in the areas with restricted diffu-
sion. We investigated the occurrence of restricted diffusion in VWM, the affected structures, the
time of occurrence in the disease course and the histopathologic correlate.
The disease onset, clinical severity and disease course of VWM patients vary greatly and the
influence of genotype and gender on the phenotype is unclear. A study on the genotype-phe-
notype correlation is hampered by the great number of private mutations, but careful selection
of patient groups sharing mutations allowed the study presented in chapter 5.
VWM is caused by mutations of the genes encoding eIF2B, the enzyme that catalyses the ex-
change of GDP for GTP on eIF2 (GEF activity). It is at present unclear what the correlation be-
tween decreased GEF activity measured in patient-derived lymphoblasts and the disease is. In
chapter 6 we focus on the functional effects of selected VWM mutations in eIF2B-β, -γ, -δ and
-ε by co-expressing mutated and wild-type subunits in human cells and on measurement of the
GEF activity in patient derived cells.
The implications/results of these chapters are summarized and discussed in chapter 7.
27. 25
General introduction
1. Van der Knaap MS, Barth PG, Gabreëls FJM, et al. A new leukoencephalopathy with
vanishing white matter. Neurology 1997;48:845.
2. Leegwater PA, Vermeulen G, Könst AA, et al. Subunits of the translation initiation fac-
tor eIF2B are mutant in leukoencephalopathy with vanishing white matter. Nat Genet
2001;29:383.
3. Van der Knaap MS, Leegwater PA, Könst AA, et al. Mutations in each of the five subu-
nits of translation initiation factor eIF2B can cause leukoencephalopathy with vanishing
white matter. Ann Neurol 2002;51:264.
4. Van der Knaap MS, Breiter SN, Naidu S, et al. Defining and categorizing leukoencepha-
lopathies of unknown origin: MR imaging approach. Radiology 1999;213:121.
5. Van der Knaap MS, Pronk JC, Scheper GC : Vanishing white matter disease. Lancet Neurol
2006;5:413.
6. Van der Knaap MS, Bugiani M, Boor I, et al. Vanishing White Matter. In: Scriver C, et al.,
editors. OMMBID. Chap 235.1. New York: McGraw-Hill; 2010. Available online.
7. Bugiani M, Boor I, Powers JM, et al. Leukoencephalopathy with vanishing white matter.
A review. J Neuropathol Exp Neurol 2010;69:987.
8. Eicke WJ. Polycystische umwandlung des marklagers mit progredientem verlauf. Atypis-
che diffuse sklerose? Arch Psychiat Nervenkr 1962;203:599.
9. Watanabe I, Muller J. Cavitating “diffuse sclerosis” J Neuropathol Exp Neurol
1967;26:437.
10. Girard PF, Tommassi M, Rochet M, et al. Leuco- encéphalopathie avec cavitations mas-
sives, bilatérales et symétriques: syndrome de décortication post-traumatique. Presse
Med 1968;76:163.
11. Anzil AP, Gessaga E. Late-life cavitating dystrophy of the cerebral and cerebellar white
matter. Eur Neurol 1972;7:79.
12. Deisenhammer E, Jellinger K. Höhlenbindendeneurtralfett-leukodystrophie mit schub-
verlauf. Neuropediatrics 1976;7:111.
13. Gautier JC, Gray F, Awada A, et al. Leucodystrophie orthochromatique cavitaire de
l’adulte. Prolifération et inclusion oligodendrogliales. Rev Neurol 1984;140:493.
14. Graveleau P, Gray F, Plas J, et al. Leucodystrophie orthochromatique cavitaire avec modi-
fications oligodendrogliales. Un cas sporadique adulte. Rev Neurol 1985;141:713.
15. Hanefeld F, Holzbach U, Kruse B, et al. Diffuse white matter disease in three children: an
encephalopathy with unique features on magnetic resonance imaging and proton mag-
netic resonance spectroscopy. Neuropediatrics 1993;24:244.
16. Schiffmann R, Moller JR, Trapp BD, et al. Childhood ataxia with diffuse central nervous
system hypomyelination. Ann Neurol 1994;35:331.
17. Tedeschi G, Bonavita S, Barton NW, et al. Proton magnetic resonance spectroscopic im-
aging in the clinical evaluation of patients with Niemann-Pick type C disease. J Neurol
Neurosurg Psychiatry 1998;65:72.
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55. Mascalchi M, De Grandis D, Ginestroni A, et al. Early MR imaging and spectroscopy ap-
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General introduction
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32.
33. CHAPTER 2
Phenotypic variation in vanishing
white matter disease
H. D. W. van der Lei*
E. M. Hamilton*
J. A. M. Gerver,
G. E. M. Abbink
C. G. M. van Berkel
M. S. van der Knaap
* these two individuals should be considered as joint first authors
who made equal contributions to this study
34. 32
Chapter 2
ABSTRACT
Objective
Vanishing white matter (VWM) is a chronic leukoencephalopathy with additional stress-pro-
voked episodes of rapid deterioration. VWM is caused by recessive mutations in the genes en-
coding eukaryotic initiation factor 2B. Phenotypic variation is wide; large studies on the subject
are scarce. The aim of the present study is to better describe the phenotypic variation.
Methods
We performed a large cross-sectional observational study in all 228 genetically confirmed VWM
patients (200 families) from the Amsterdam VWM database up to August 2011. We used clinical
questionnaires to collect information on disease course and reviewed the mutations.
Results
The clinical inventory involved 223 patients, of which 120 were female; 5 patients were excluded
because of co-morbidity. Mean age of onset was 8 years (median 3 years, range antenatal peri-
od - 54 years). Fifty-six patients were deceased; mean age of death was 9 years (median 5 years,
range 3 months - 46 years). There wa=s a clear correlation between age at disease onset and
disease severity. Patients with onset < 2 years had the most severe disease course with delayed
motor development, early loss of unsupported walking, sometimes involvement of extracere-
bral organs, more episodes of rapid deterioration, more comas and earlier fatality than patients
with later onset. Female patients outnumbered male patients in the teenage and adult onset
categories and tended to have milder disease.
Conclusions
The VWM disease spectrum consists of a continuum of phenotypes with extremely wide vari-
ability. The younger the first neurological signs appear, the more severe the disease course is.
35. 33
Phenotypic variation in vanishing white matter disease
INTRODUCTION
Vanishing white matter (VWM),1,2
also called childhood ataxia with central hypomyelination
(CACH)3
or eIF2B-related disorder4
, is one of the most prevalent inherited childhood leukoen-
cephalopathies. The disease course is characterized by chronic progressive neurological de-
terioration mainly due to cerebellar ataxia and to a lesser degree spasticity, with additional
stress-provoked episodes of rapid deterioration after febrile infections, minor head trauma,
and, less often, acute fright.1-3,7,8
Rapid loss of motor skills, hypotonia, irritability, seizures, vom-
iting and somnolence characterize the episodes, which may lead to coma and death.
VWM is caused by recessive mutations in the genes EIF2B1-5 encoding the five subunits of eu-
karyotic initiation factor 2B (eIF2B).5,6
eIF2B is essential in all cells for initiation of translation of
mRNAs into proteins and for regulation of the rate of protein synthesis under different condi-
tions, including stress.9,10
About 160 different mutations have been described in VWM and most
patients are compound-heterozygous for two different mutations in one of the five genes.20
Initially VWM was recognized as a disorder of young children, most often with an onset be-
tween 2 and 6 years of age1,3,7
, but it has become apparent that disease onset and severity vary
widely. Patients with antenatal onset die within the first months of life.4
Early infantile forms,
like the Cree encephalopathy, lead to demise before 2 years of age.4,11,12
Much milder variants
start in adolescence or adulthood and are mostly characterized by slow disease progression,
although some patients die within a few months or years.2,13-17
Subdivisions in groups based on
age of onset, have been published.16, 26
The wide phenotypic variation has a complex explanation. There is evidence that the genotype
influences the phenotype 25
, as some mutations are specifically associated with a mild or severe
clinical course. 12,14-16, 25
On the other hand, striking phenotypic heterogeneity within families has
been reported2,15,16,18
, indicating that environmental or other genetic factors also influence the
phenotype. An effect of gender has been suggested as well.19, 25
Large studies on phenotypic variation in VWM are scarce. In this cross-sectional observational
study we investigated the disease course in a cohort of 228 VWM patients in order to obtain
insight into the clinical variation of VWM. We collected data on prevalence and characteristics
of subgroups of patients defined by age of onset and explored male versus female differences.
PATIENTS AND METHODS
Study design
We performed a cross-sectional observational study and included all genetically proven patients
in our VWM patient database until August 2011. The database contains all patients referred to
VU University Medical Center for mutational analysis for VWM.
Standard protocol approvals, registrations, and patient consents
Written informed consent for research was obtained from all patients, or guardians of the pa-
36. 34
Chapter 2
tients, participating in the study. Approval of the ethical standards committee was received for
retrospective analysis of clinical information, collected by questionnaires.
Phenotype
Clinical questionnaires were completed by the patient’s physician (38%) or the patient and fam-
ily members (15%). For the remaining patients, clinical information was derived from medical
records by the authors of the paper (JAMG, HDWvdL and EMH). The inventory involved items on
demographic details, pregnancy and delivery, early motor development, early cognitive develop-
ment, disease onset and signs, provoking factors, disease course and survival. Patients with anoth-
er disease affecting neurological function in addition to VWM were excluded.
We used age of onset to categorize the patients into the following five groups: antenatal-infantile
(<2 years), early juvenile (2 - <6 years), late-juvenile (6 - <12 years), teenage (12 - <18 years) and
adult (≥ 18 years) onset. The disease onset was considered the age at which the first neurological
sign was noted. The disease duration was defined as the time between the disease onset and the
latest clinical observation or death. Patients were scored as having lost walking without support
when they could walk with support only and they were scored as fully wheelchair dependent when
they were not able to walk both without and with support. Patients who never achieved walking
without or with support were scored as having lost ambulation at the age of 18 months. Patients
who died before the age of 18 months were not included in the analyses of achieving and losing
of ambulation. Involvement of ovaries was assessed in females who were older than 16 years at
the last clinical observation. Regarding disease course, three different aspects were considered: the
phase of disease onset, the steadily progressive component and the episodes of rapid deterioration.
Statistical analysis
Summary statistics were used to describe the clinical phenotype. The skewness statistic test and
non-parametric Kolmogorov-Smirnov test for uniform distribution were used to test the distribu-
tion of age of onset. The five age of onset groups were compared with respect to age and dura-
tion of disease at loss of ambulation and at death using the Kruskal-Wallis test. The same items
were analysed for differences between male and female patients using the Mann-Whitney U test.
Nominal and ordinal data were analysed by Chi-square testing or Fisher’s exact test. The probabili-
ties of individuals to lose the ability to walk without support, become fully wheelchair dependent
or die relative to the disease duration were estimated through Kaplan-Meier curves. Individuals
in whom the event of loss of walking without support, becoming wheelchair dependent or death
had not occurred within the study period were indicated as censored for the respective analysis.
Subgroups were formed by age of onset category and gender and compared by log-rank statistics.
All statistical analyses were performed using SPSS 20.
Genotype inventory
Mutation analysis was performed in our laboratory in 224 patients and in an outside lab in four. Ge-
nomic DNA was extracted from whole blood, lymphoblasts or fibroblasts. The exons and flanking
intron DNA of the genes EIF2B1, -2, -3, -4, and -5 were amplified by PCR as previously described.6
37. 35
Phenotypic variation in vanishing white matter disease
RESULTS
Patients
The total number of patients included was 228 from 200 families; 121 patients were female. Five
patients were excluded from the inventory on clinical characteristics because of co-morbidity (i.e.,
Down syndrome, biliary atresia, galactosemia, glutaric aciduria type 1, and a brain developmental
anomaly). In case of limited clinical information, patients were only excluded from analysis for the
subjects of the missing data. For each item, the number of patients available for analysis is shown
in brackets or in tables 1 or 2.
Fifty six patients were deceased; mean age at death was 9 years (median age 5 years, range 3
months - 46 years). The duration of the disease at time of death ranged from 1 month to 27 years
(mean 5 years, median 3 years).
The mean age of the living patients at the latest clinical evaluation was 17 years, (median 13 years,
range 0 – 59 years, n=167). The mean duration of follow up was 8 years, median 5 years, range 0 -
31 years. The residence of the patients was Europe (n=130), North America (n=46), South America
(n=24), Africa (n=4), Asia (n=14) and Australia and New Zealand (n=5).
Table 1 | Overview of presenting signs in 201 patients. y, years; m, months
Presenting sign Frequency Range age of onset
Gait problems 61 2-48y
Loss of motor skills following head trauma 33 3-18y
Loss of motor skills following infection 24 12m-16y
Loss of motor skills 18 2-42y
Seizures 18 2m-22y
Ataxia 16 18m-25y
Weakness / hypotonia 12 6m-20y
Cognitive/memory/behavior problems 10 6-54y
Developmental delay 7 14m-2y
Sleepiness / coma following infection 6 20m-5y
Antenatal signs 6 antenatal
Sleepiness / coma following minor head trauma 6 13m-20y
Depression 4 27-48y
So far asymptomatic 3 12m-9y
Severe headache/migraine 2 7-13y
Amenorrhea 2 19-27y
Delayed mental development 1 18m
Loss of activeness 1 4y
Vision loss 1 31y
41. 39
Phenotypic variation in vanishing white matter disease
Age of onset
The mean age at which the first neurological signs were noted was 8 years (median 3 years,
range 0 - 54 years, n=210); 87 % of the patients had an onset before the age of 18 years and
69% before the age of 6 (figure 1). The most frequent age of onset was 2 years (45 patients),
followed by 3 years (31 patients) and 1 year (28 patients).
Sixteen patients were symptomatic before the age of 1 year, six of whom most likely had an
antenatal onset because of intrauterine growth retardation, reduced fetal movements, contrac-
tures at birth, oligohydramnios or a combination of these features. They showed neurological
signs very early in life. Three patients were still asymptomatic at the latest clinical observation
(at ages of 1, 6 and 10 years). They had been diagnosed because of an affected sibling, an inci-
dental finding on CT scan, which was made because of head trauma without neurological signs,
and because of an incidental finding on MRI scan, which was made because of an episode of
dizziness, respectively.
Figure 1 | Age of onset: *6 patients had an onset before birth.
There was a significant positively skewed distribution of age of onset (skewness statistic = 2.3,
p<0.001, figure 2). For the interval disease onset 18 – 54 years, the disease followed a rather uni-
form distribution (one sample Kolmogorov-Smirnov test of uniform distribution p= 0.38 (figure 2).
The nature of the first signs was different for different ages of onset (table 1). At all ages,
patients mainly presented with motor problems; a minority of later childhood or adult onset
patients however, presented with cognitive or psychiatric problems.
42. 40
Chapter 2
Figure 2 | Distribution of age of onset.
Early motor development
Twenty-five patients had a delayed early motor development (see table 2). This concerned 54%
of the patients with an onset before 2 years; 19 % of the patients with an onset between 2- <6
years and 6% in the 6- <12 years at onset group. Patients with teenage or adult onset all had a
normal early motor development.
Loss of ambulation
Five percent of the patients who reached the age of 18 months never achieved walking without
support; 4% never achieved walking without or with support (see table 2). Seventy-two percent
of the patients lost walking without support at a mean age of 10 years (median 4 years, range
12 months - 53 years). Fifty-five percent became fully wheelchair dependent at a mean age of
11 years (median 6 years, range 18 months – 47 years).
Provoking factors
Episodes of deterioration were provoked by head trauma (reported in 57% of patients), febrile
infections (71%) and acute psychological stress or fright (24%). The younger the patients were,
the more sensitive they were to infections; deterioration was provoked by fever in 92% of pa-
tients with onset < 2 years, while that was the case in 53% of patients with onset ≥ 18 years (see
table 2). Head trauma as provoking factor was reported in 57% of patients, with the highest
rate in juvenile onset male patients (2-<12 years; 81%). Other provoking factors mentioned less
often were heat (n=5) and anesthesia (n=5).
Involvement of ovaries
Information on ovarian function was available for 44 of 55 women older than 16 years at the
latest clinical evaluation. In 64% of these 44 women signs of ovarian failure were reported. In
10 patients there was secondary amenorrhoea; seven patients had primary amenorrhoea; three
patients had amenorrhoea without further specification reported; seven patients had irregular
menses and one patient was infertile. Additionally, ovarian dysgenesis was found at autopsy in
two patients who died at the ages of 10 months27
and 6 years.1
43. 41
Phenotypic variation in vanishing white matter disease
Involvement of other organs than the brain and ovaries
The following clinical abnormalities in other organs were found in 13 patients: congenital cat-
aract (n=4, all antenatal onset), retinopathy (n=1, age of disease onset 10 years), renal failure
(n=2, age of onset 2-3 years), renal hypo-dysplasia (n=2, antenatal onset), liver dysfunction with
episodic icterus (n=1, age of onset 5 years), hepatosplenomegaly with non-specific abnormal-
ities at biopsy (n=1, antenatal onset), cholelithiasis (n=1, age of onset 17 years), leukopenia
(n=1, age of onset 7 months), and adrenal insufficiency (n=1, age of onset 35 years; see table
2). Furthermore, the autopsy of a girl with antenatal disease onset revealed mild pancreatitis.
Disease course
The disease course is depicted in table 2, describing the course 1) just after disease onset, 2) re-
garding episodes of deterioration and 3) regarding the chronic phase. After disease onset, the
majority of patients (77%) showed increasing problems. In these patients, the course was char-
acterized by slow progression, episodes of rapid deterioration or a combination of these (tables
2 and 3). Patients rarely showed complete recovery after an episode of rapid deterioration; they
more often recovered partially or remained seriously handicapped. Some patients, especially
young children, died in the course of an episode. The chronic phase consisted of stable or slowly
progressive disease in most patients, but rapid disease progression was seen frequently in early
onset patients, and occasionally in older onset patients.
Correlation between age of onset and disease progression
When studying the relation between age of onset and disease course, earlier onset was related
to a more severe disease. There was a significant difference in survival between the five age of
onset categories as defined (table 2), especially between patients with onset <2 years versus
later onset patients. Life span was particularly reduced in the six antenatal onset patients, while
most patients with onset >6 years are still alive (table 2, figure 3; log rank analysis p<0.001).
The earlier the disease onset, the more often patients had disturbed early motor development
(table 2). In the <2 years at onset group, only 52% percent achieved walking without support. In
the group with onset at 2- <6 years, 98% achieved walking without support and patients with a
later disease onset all achieved walking without support.
Cognitive development was disturbed in 36% of patients with an onset <2 years and in 4% of
patients with an onset at 2- <6 years. In older onset patients the initial cognitive function was
reported as normal.
44. 42
Chapter 2
Table 3 | Clinical characteristics male and female patients
number
All patients
223
Female
120
Male
103
p-value
Survival
Age of death
(median, range); number
5y (3m-46y)
56
4y (3m-46y)
29
6y (3m-27y)
27
0.95
Disease duration at death
(median, range); number
3y (1m-27y)
55
2y (1m-27y)
28
3y (1m-21y)
27
0.51
Disease duration living patients
(median, range); number
5y (0-31y)
167
5y (0-31y)
91
5y (0-25y)
76
Neurological development and symptomatology
Delayed motor development
(percentage, number)
19%
135
18%
78
21%
57
0.65
Delayed cognitive development
(percentage, number)
8%
165
6%
95
10%
70
0.39
Achieved walking without support (per-
centage, number)
95%
139
94%
77
97%
62
0.46
Age at loss of walking without
support (median, range); number
4y
(16m-53y)
100
4y
(16m-44y)
53
3y
(18m-53y)
47
0.10
Duration at loss of walking without
support (median, range); number
6m (0-19y )
100
6m (0-19y)
53
6m (0-13y)
47
0.63
Age at full wheelchair dependency
(median, range); number
6y
(18m-47y)
77
9y (2-47y)
42
4y
(18m-26y)
35
0.03
Duration at full wheelchair dependency
(median, range); number
2y (0-22y)
77
2y (0-22y)
42
12m (0-13y)
35
0.08
Epilepsy
(percentage, number)
50%
67/134
50%
34/68
50%
33/66
1.00
Episode(s) of coma
(percentage, number)
29%
122
28%
64
29%
58
1.00
Involvement of extracerebral
organs
9%
141
9%
58
10%
83
0.54
Disease course just after start
(number)
n=187 n=103 n=84
No further problems (percentage) 6% 6% 6% 0.97
Stable problems (percentage) 17% 21% 12% 0.09
Increasing problems (percentage) 77% 73% 82% 0.13
Disease course if deterioration occurred
(number)
n=151 n=85 n=66
45. 43
Phenotypic variation in vanishing white matter disease
Slowly progressive (percentage) 44% 47% 39% 0.35
Episodes of rapid deterioration
(percentage)
24% 25% 23% 0.77
Combination (percentage) 32% 28% 38% 0.21
Recovery after episodes of deterioration
(number)
n=108 n=59 n=49
Complete recovery (percentage) 9% 14% 4% 0.11
Partial recovery (percentage) 44% 41% 49% 0.92
Remained seriously handicapped
(percentage)
33% 30% 35% 0.68
Combination (percentage) 6% 5% 6% 1.00
Death (percentage) 8% 10% 6% 0.51
Chronic phase (number) n=151 n=85 n=66 0.87
Stable (percentage) 32% 32% 32% 0.99
Slowly progressive (percentage) 52% 52% 52% 0.98
Rapid progression in months
(percentage)
14% 13% 15% 0.70
Combination (percentage) 3% 4% 1% 0.45
Factors provoking deterioration
Head trauma (percentage, number)
57%
116
47%
61
67%
55
0.03
Infections with fever
(percentage, number)
70%
123
64%
62
75%
61
0.19
Acute psychological stress or acute fright
(percentage, number)
24%
82
19%
42
30%
40
0.25
Affected gene (number) n=120 n=103
EIF2B1 (percentage) 0% 4%
EIF2B2 (percentage) 20% 10%
EIF2B3 (percentage) 7% 7%
EIF2B4 (percentage) 7% 7%
EIF2B5 (percentage) 66% 72%
In italic the number of patients in which an event has occurred is shown relative to the total number of
patients in whom information on the clinical manifestation was available. P-values concern the comparison
between male and female patients.y; year, m; months, n.a.; not applicable
46. 44
Chapter 2
Figure 3 | Disease duration at death
The earlier the disease onset, the earlier patients lost walking without support and the earlier
they became wheelchair dependent (table 2). When evaluating the duration of disease at loss of
walking without support, the loss of ambulation occurred considerably earlier in patients with
an onset < 6 years than in patients with onset at or after 6 years (figure 4). Adult onset patients
became wheelchair dependent sooner after disease onset than late juvenile or teenage onset
patients.
47. 45
Phenotypic variation in vanishing white matter disease
Figure 4 | Disease duration at loss of ambulation per age of onset category
Comas were most prevalent in the <2 years at onset group (42% versus an overall occurrence
of 29%). Most patients had 1 episode of coma, four patients with an onset before the age of 6
years had multiple (2 - 6) episodes.
Epilepsy was not significantly related to age of onset, although most prevalent in the <2 years
onset category (67%). The prevalence was lowest in adult onset patients (31%).
Patients with onset <2 years more often had episodes of rapid deterioration (40%) and had
the highest occurrence of death after an episode of deterioration (25%). Teenage and adult
onset patients most often showed complete (25%) or partial (56%) recovery after an episode
of deterioration. In the category with the earliest onset, the chronic phase of the disease was
less often slowly progressive (19%) than in patients with later onset (50-82%) and more often
characterized by rapid progression (46% versus 0-10%).
Involvement of organs outside the brain occurred more frequently in patients with an onset <2
years (table 2).
Genotype
The EIF2B1-5 mutations of all 228 patients are listed in supplementary table 1; in table 2 the
frequency of occurrence of mutations are shown for each gene. The majority of patients were
compound heterozygous (n=136) and the total number of different genotypes was 124. A
unique genotype was found in 67 individuals and in 13 sibling pairs. The genetic heterogeneity
hampered the study of a genotype-phenotype correlation. Seven groups of at least five patients
48. 46
Chapter 2
with the same genotype could be formed; A) EIF2B2, c.599G>T / p.Gly200Val with c.871C>T / p.
Pro291Ser (n=5), B) EIF2B2, c.638A>G / p.Glu213Gly with c.599G>T / p.Gly200Val (n=5), C) EIF2B2,
c.638A>G / p.Glu213Gly homozygous (n=12), D) EIF2B5, c.271A>G / p.Thr91Ala homozygous
(n=8), E) EIF2B5, c.271A>G / p.Thr91Ala with c.1015C>T / p.Arg339Trp (n=5) F) EIF2B5, c.338G>A
/ p.Arg113His with c.1016G>A / p.Arg339Gln (n=6), and G) EIF2B5, c.338G>A / p.Arg113His ho-
mozygous (n=29). In five groups (A, B, C, E and F), there was consistency regarding age of onset
and mortality; all patients in these groups were categorized in only two successive age of onset
categories and mortality rates and ages at death were in the same range. In groups D and G on
the other hand, there was quite a large variability in the ages of onset, mortality rate and ages at
death. There were, however, no cases in which patients with an onset at <2 years and ≥18 years
had the same genotype. The homozygous c.338G>A, p.Arg113His genotype was most frequent
(n=29). This genotype has previously been associated with a mild phenotype.25
Median age of
onset in patients with this genotype in the current cohort was 17 years (range 2 - 54 years of age).
There were no patients with onset <2 years and onset at 2- <6 years was rare (n=3). The most fre-
quent onset category was ≥18 years; n=13). Mortality rate was low (n=3, age 30 - 36 years).
On the subject of involvement of ovaries, no relation with genotype was found. In the group pa-
tients with homozygous c.338G>A / p.Arg113His mutations, 67% suffered from ovarian failure,
as compared to 61 % of the total studied female population older than 16 years.
Influence of gender
In total, 103 male and 120 female patients were clinically phenotyped. In the infantile and ju-
venile onset groups (<12 years), there were no substantial differences in male: female ratio. In
the teenage and adult onset group (≥12 years), female patients outnumbered male patients (30
females versus 12 male patients; figure 1).
Summary statistics on clinical characteristics for males and females are shown in table 3. There
were no significant differences in survival between males and females. The mean age at death
was higher in females (11 years versus 8 years in males), while the median age of death was high-
er in males (6 years versus 4 years in females), but for this subject the larger representation and
therefore higher number of deaths of females at older ages should be taken into account (figure
5). Up to the age of 7 years there are no differences in male and female survival (figure 5).
49. 47
Phenotypic variation in vanishing white matter disease
Figure 5 | Disease duration at death
When comparing the ages at loss of ambulation, there was a trend for earlier loss of walking
without support in males and a significant difference in age at full wheelchair dependency.
When looking at the duration of the disease at loss of ambulation, there were, however, no
significant differences, although there was still a trend of sooner loss of ambulation in males
(figure 6, p=0.19 and figure 7, p=0.16). Regarding epilepsy, coma and disease course no signifi-
cant gender differences were found.
50. 48
Chapter 2
Figure 6 | Disease duration at loss of walking without support
Figure 7 | Disease duration at full wheelchair dependency
51. 49
Phenotypic variation in vanishing white matter disease
Intrafamilial difference
Within the studied cohort, there were 23 families with two affected siblings and two families
with three affected siblings (supplementary table 1, page 64). Regarding age of onset, the ma-
jority was categorized in the same age of onset group. In six families, patients were categorized
in two subsequent age of onset categories. There were no substantial differences between fam-
ilies regarding mortality; the most striking observation was a difference in survival of 10 years
between two affected siblings.
DISCUSSION
We investigated the phenotypic variation among VWM patients in the largest cohort so far.
VWM was initially defined as an early juvenile onset disorder, but the spectrum was soon found
to be much broader, with on the one extreme very severely affected patients with antenatal
onset4
, and on the other extreme mildly affected patients with onset in late adulthood, up
to the age of 62 years.19
We suspect that until now especially adult onset, mild variants of
VWM have largely been underdiagnosed, because of the less typical presentation and the lack
of awareness of adult neurologists. The same has been described in X-linked adrenoleukodys-
trophy, which was originally described as a rapidly progressive childhood onset disorder.28
The
initial phenotype was later named ‘Childhood cerebral ALD’. Later on, the milder, adult onset
variant adrenomyeloneuropathy was recognized more and more, and is now recognized to be
the most common form of X-ALD.29, 30
The finding that the VWM spectrum continues to expand on both extremes suggests that it is
a continuum and that even more extreme phenotypes are currently missed. It is, for instance,
unknown how many miscarriages and stillbirths are caused by severely pathogenic mutations in
one of the eIF2B genes. On the other hand, we suspect that there are adults with very subtle,
perhaps subclinical neurological symptomatology due to mild eIF2B mutations in whom the
diagnosis is never established.
We observed a clear relation between age of onset and disease severity. Particularly patients
with an onset before 2 years of age were very fragile, with rapid loss of function and higher and
earlier mortality. The later the onset, the more likely that the disease course is stable, while early
onset patients more often experience episodes of deterioration. Such episodes are especially
provoked by febrile infections, especially in children with disease onset < 2 years. In older boys
head trauma is a frequent provoking factor.
Interestingly, there was not a linear relation between age of onset and duration of the disease at
which loss of walking without support or full wheelchair dependency occurred. Patients with an
onset before 6 years lost ambulation soonest, but patients with an adult onset lost walking soon-
er than late juvenile and teenage onset patients. The rapid loss of ambulation in the early onset
patients is in line with the more severe disease in these patients. Perhaps adult onset patients are
52. 50
Chapter 2
more susceptible to loss of ambulation due to a lower adaptive capacity than adolescents.
Intriguing findings are the higher occurrence of VWM among teenage and adult females in this
cohort, as well as the trend for higher survival rates and less rapid loss of ambulation among
females. Larger numbers of patients are required to find out whether this male:female imbal-
ance is consistent in VWM and whether the male disadvantage is more prominent in VWM than
explained by the general ‘life expectancy gap’. In 2012, the global adult mortality rate (proba-
bility of dying between 15 - 60 years of age per 1000 population) was 187 in males and 124 in
females.31
Also during childhood and adolescence males are more likely to die than their female
peers, regardless of the underlying condition (relative risk from birth to age 20 years 1.44, 95%
confidence interval 1.44-1.45).32
In infancy the gender differences are less pronounced (relative
risk 1.12 95% confidence interval 1.11-1.12).32
Balsara et al., suggest the existence of a male vul-
nerability factor, attributed to a complex interplay of factors including acquired risks, heath-re-
porting behavior, illness behavior, health care utilization as well as an underlying biological
difference. In the current cohort of VWM patients, the longer survival in females can partially
be explained by the overrepresentation of females in the later onset categories in which the
phenotype is milder, a phenomenon that has been described before by Labauge et al (2009).19
Another aspect that may contribute to the male:female imbalance is the frequent occurrence of
ovarian failure in affected females. It is possible that in the category of mildly affected individu-
als who exhibit only subtle neurological signs, this feature advances diagnosis in woman, while
the diagnosis in equally mildly affected males is missed.
A formal genotype-phenotype correlation analysis was hampered by the wide genetic hetero-
geneity of patients, but certain genotypes are undoubtedly related to particular phenotypes,
as described before.25
Also the relative homogeneity in the phenotypes of affected individuals
from the same family suggests a certain correlation between genotype and phenotype. In the
current cohort compound heterozygosity for c.599G>T and c.871C>T in EIF2B2 in three families
appeared to be associated with a strikingly severe phenotype. The study of still larger numbers
of patients, as well as the acquisition of more complete clinical inventories will be helpful to
further characterize phenotypic aspects in relation to genotype.
It has previously been suggested that for individuals with severe forms of VWM, the genotype
would supersede the effect of environmental or other genetic factors on the symptomatology,
while in milder forms, environmental or other genetic factors would play a greater role.16
This
concept could explain the clinical differences observed in groups of later onset patients affected
by the same mutations, as well as in individuals within the same family.
We are aware of shortcomings of our clinical variation study. Retrospectively collected data are
of lower quality than prospective data. The involvement of numerous different physicians who
examined the patients and interpreted the findings will have added to the variations described
for patients. Different aspects, such as the influence of medical resources on diagnostic pro-
cedures and treatment, religious or cultural perceptions, differences between physicians and
families filling in the questionnaires, selection bias and information bias all may have hampered
a truly objective evaluation of the clinical course in VWM patients. It is possible that a selection
bias has been present, at least in the beginning of the study, as VWM was initially recognized as
53. 51
Phenotypic variation in vanishing white matter disease
a disease in childhood with a possible underestimation in the older age of onset groups.
Although our study involves the largest described VWM cohort, still the numbers are in the
subgroups are small. Especially the subgroups of patients with an onset > 6 years consisted of
small numbers of patients, which may not be representative of the entire patient population
of these categories. Rare features, such as the possibility of involvement of organs outside the
brain, require further study. At present, the idea that severe, antenatal onset patients are at
risk for dysfunction of extracerebral organs is generally accepted. In milder, late onset disease,
it remains to be elucidated whether pathology outside the brain, for instance choleliathias, is a
consequence of VWM or a coincidental comorbidity.
Finally, it should be appreciated that for certain clinical items, a rather substantial bias of miss-
ing data must be taken into account. Missing data are often not random, for example missing
data on early childhood in adult patients.
More and larger follow up studies will further contribute to a more representative description
of the clinical spectrum in VWM patients.
54. 52
Chapter 2
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