PP_Hannemieke van der Lei_FINALpdf

Vanishing white matter

A study of phenotypic variation and the relationship between genotype and

phenotype

Hannemieke van der Lei

Vanishing white matterA study of phenotypic variation and the relationship between genotype

and phenotype

Hannemieke van der Lei

2

ISBN: 978-94-6259-876-8

Printed by: Ipskamp Drukkers

Lay-out: Persoonlijk Proefschrift, by Lyanne Tonk

Cover design: Painting by Jennifer Konings, design by Lyanne Tonk

Study funding: Supported by the Optimix Foundation for Scientific Research, the Dutch

Organisation for Scientific Research (ZonMw TOP 9120.6002 and ZonMw AGIKO 920-

03-308), and the Dr WM Phelps Foundation (2008029 WO). The funding agencies had no

direct involvement with the contents of the study. Financial support for printing this thesis

was kindly provided by Stichting Researchfonds Kindergeneeskunde, VU University Medical

Center, Amsterdam, The Netherlands

© H. van der Lei 2015.

All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by

any means, without prior permission of the author.

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

4

promotor: prof.dr. M.S. van der Knaap

copromotoren: dr. G.C. Scheper

dr. T.E.M. Abbink

5

If life was easy

it would be boring

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

CHAPTER 1General introduction

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

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

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

13


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

9

b c a

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

15


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.

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 ba

a b

c d

17


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

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

19


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.

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

21


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

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.

23


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

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.

25


1. Van der Knaap MS, Barth PG, Gabreels 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.

REFERENCES

26

Chapter 1

18. van der Knaap MS, Kamphorst W, Barth PG, Kraaijeveld CL, Gut E, Valk J : Phenotypic

variation in leukoencephalopathy with vanishing white matter. Neurology 1998;51:540.

19. Brück W, Herms J, Brockmann K, et al. Myelinopathia centralis diffusa (vanishing white

matter disease): Evidence of apoptotic oligodendrocyte degeneration in early lesion

development. Ann Neurol 2001;50:532.



2001;29:383.

21. Van der Knaap MS, Leegwater PA, Könst AA, et al. Mutations in each of the five subu-


white matter. Ann Neurol 2002;51:264.

22. Van der Knaap MS, van Berkel CG, Herms J, et al. eIF2B-related disorders: antenatal on-

set and involvement of multiple organs. Am J Hum Genet 2003;73:1199.

23. Fogli A, Schiffmann R, Bertini E, et al. The effect of genotype on the natural history of

eIF2B-related leukodystrophies. Neurology 2004;62:1509.

24. Van der Knaap MS, Pronk JC, Scheper GC. Vanishing white matter disease. Lancet Neurol

2006;5:413.

25. Vermeulen G, Seidl R, Mercimek-Mahmutoglu S, Rotteveel JJ, Scheper GC, van der

Knaap MS. Fright is a provoking factor in vanishing white matter disease. Ann Neurol

2005;57:560.

26. Kaczorowska M, Kuczynski D, Jurkiewicz E, et al. Acute fright induces onset of symptoms

in vanishing white matter disease-case report. Eur J Paediatr Neurol 2006;10:192.

27. Fogli A, Boespflug-Tanguy O. The large spectrum of eIF2B-related diseases. Biochem Soc

Trans. 2006;34:22.

28. Labauge P, Horzinski L, Ayrignac X, et al. Natural history of adult-onset eIF2B-related

disorders: a multi-centric survey of 16 cases. Brain 2009;132:2161.

29. van der Knaap MS, Leegwater PAJ, van Berkel CGM, et al. Arg113His mutation in eIF2ep-

silon as a cause of leukoencephalopathy in adults. Neurology 2004;62:1598.

30. Mierzewska H, van der Knaap MS, Scheper GC, et al. Leukoencephalopathy with van-

ishing white matter due to homozygous EIF2B2 gene mutation. First Polish cases. Folia

Neuropathol 2006;44:144.

31. Damon-Perriere N, Menegon P, Olivier A, et al. Intra-familial phenotypic heterogeneity

in adult onset vanishing white matter disease. Clin Neurol Neurosurg 2008;110:1068.

32. Boltshauser E, Barth PG, Troost D, et al. “Vanishing white matter” and ovarian dysgene-

sis in an infant with cerebro-oculo-facio-skeletal phenotype. Neuropediatrics 2002;33:57.

33. Francalanci P, Eymard-Pierre E, Dionisi-Vici C, et al. Fatal infantile leukodystrophy:

a severe variant of CACH/VWM syndrome, allelic to chromosome 3q27. Neurology

2001;57:265.

34. Fogli A, Dionisi-Vici C, Deodato F, et al. A severe variant of childhood ataxia with central

hypomyelination/vanishing white matter leukoencephalopathy related to EIF2B5 muta-

tion. Neurology 2002;59:1966.

27


35. Fogli A, Wong K, Eymard-Pierre E, et al. Cree leukoencephalopathy and CACH/VWM

disease are allelic at the EIF2B5 locus. Ann Neurol 2002;52:506.

36. Black DN, Booth F, Watters GV, et al. Leukoencephalopathy among native Indian infants

in northern Quebec and Manitoba. Ann Neurol 1988;24:490.

37. Jansen AC, Andermann E, Niel F, et al. Leucoencephalopathy with vanishing white mat-

ter may cause progressive myoclonus epilepsy. Epilepsia 2008;49:910.

38. Federico A, Scali O, Stromillo ML, et la. Peripheral neuropathy in vanishing white matter

disease with a novel EIF2B5 mutation. Neurology 2006;67:353.

39. Huntsman RJ, Seshia S, Lowry N, et al. Peripheral neuropathy in a child with Cree leukod-

ystrophy. J Child Neurol 2007;22:766.

40. Passemard S, Gelot A, Fogli A, et al. Progressive megalencephaly due to specific eIF2Be

mutations in two unrelated families. Neurology 2007;69:400.

41. Pineda M, R-Palmero A, Baquero M, et al. : Vanishing white matter disease associated

with progressive macrocephaly. Neuropediatrics 2008;39:29.

42. Carra-Dalliere C, Horzinski L, Avrignac X, et al. Natural history of adult-onset eIF2B-relat-

ed disorders: a multicentric survey of 24 cases. Rev Neurtol 2010;167:802.

43. Damasio J, van der lei HD, van der Knaap MS, et al. Late onset vanishing white matter

disease presenting with learning difficulties. J Neurol Sci 2012;314:169.

44. Ghezzi L, Scarpini E, Rango M, et al. A 66-year-old patient with vanishing white matter

disease due to the p.Ala87Val EIF2B3 mutation. Neurology 2012;79:2077.

45. Matsui M, Mizutani K, Miki Y, et al. Adult-onset leukoencephalopathy with vanishing

white matter. Eur J Radiol Extra 2003;46:90.

46. Ohtake H, Shimohata T, Terajima K, et al. Adult-onset leukoencephalopathy with vanish-

ing white matter with a missense mutation in EIF2B5. Neurology 2004;62:1601.

47. Prass K, Bruck W, Schroder NW, et al. Adult-onset leukoencephalopathy with vanishing

white matter presenting with dementia. Ann Neurol 2001;50:665.

48. Fogli A, Rodriguez D, Eymard-Pierre E, et al. Ovarian failure related to eukaryotic initia-

tion factor 2B mutations. Am J Hum Genet 2003;72:1544.

49. Biancheri R, Rossi A, Di Rocco M, et al. Leukoencephalopathy with vanishing white mat-

ter: an adult onset case. Neurology 2003;61:1818.

50. Schiffmann R, Tedeschi G, Kinkel P, et al. Leukodystrophy in patients with ovarian dys-

genesis. Ann Neurol 1997;41:654.

51. Fogli A, Gauthier-Barichard F, Schiffmann R, et al. Screening for known mutations in

EIF2B genes in a large panel of patients with premature ovarian failure. BMC Womens

Health 2004;4:8.

52. Fogli A, Schiffmann R, Hugendubler L, et al. Decreased guanine nucleotide exchange

factor activity in eIF2B-mutated patients. Eur J Hum Genet 2004;12:561.

53. Pronk JC, van Kollenburg B, Scheper GC, et al. Vanishing white matter disease: a review

with focus on its genetics. Ment Retard Dev Disabil Res Rev 2006;12:123.

54. Van der Knaap MS, Valk J. Magnetic resonance of myelination and myelin disorders. 3rd

edition. Springer, Heidelberg, 2005.

28

Chapter 1

55. Mascalchi M, De Grandis D, Ginestroni A, et al. Early MR imaging and spectroscopy ap-

pearance of eIF2B-related leukoencephalopathy. Neurology 2006;67:537.

56. Patay Z. Diffusion-weighted MR imaging in leukodystrophies. Eur Radiol 2005;15:2284.

57. Ding XQ, Bley A, Ohlenbusch A, et al. Imaging evidence of early brain tissue degener-

ation in patients with vanishing white matter disease; a multimodal MR study. J Magn

Reson Imaging 2012;35:926.

58. Dreha-Kulaczewski SF, Dechent P, Finsterbusch J, et al. Early reduction of total

N-acetyl-aspartate-compounds in patients with classical vanishing white matter disease.

A long-term follow-up MRS study. Pediatr Res 2008;63:444.

59. Leegwater PA, Könst AA, Kuyt B, et al. The gene for leukoencephalopathy with vanish-

ing white matter is located on chromosome 3q27. Am J Hum Genet 1999;65:728.

60. Leegwater PA, Pronk JC, van der Knaap MS. Leukoencephalopathy with vanishing

white matter: from magnetic resonance imaging pattern to five genes. J Child Neurol

2003;18:639.

61. Maletkovic J, Schiffmann R, Gorospe JR, et al. Genetic and clinical heterogeneity in

eIF2B-related disorders. J Child Neurol 2008;23:205.

62. Scali O, Di Perri C, Federico A. The spectrum of mutations for the diagnosis of vanishing

white matter disease. Neurol Sci 2006;27:271.

63. Shimada S, Miva K, Oda N, et al. An unmasked mutation of EIF2B2 due to submicroscopic

deletion of 14q24.3 in a patient with vanishing white matter disease. Am J Med Genet A

2012;158A:1771.

64. Ohlenbusch A, Hanneke M, Brockmann K, et al. Identification of ten novel mutations in

patients with eIF2B-related disorders. Hum Mutat 2005;25:411.

65. Mathis S, Scheper GC, Baumann N, et al. The ovarioleukodystrophy. Clin Neurol Neuro-

surg 2008;110:1035.

66. Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translation initiation

and principles of its regulation. Nature reviews 2010;10:113.

67. Hinnebusch AG. Molecular Mechanism of Scanning and Start Codon Selection in Eukary-

otes. Microbiology 2011;75:434.

68. Kimball SR : Eukaryotic initiation factor eIF2. Int J Biochem Cell Biol 1999;31:25.

69. Chen JJ. Regulation of protein synthesis by the heme-regulated eIF2alpha kinase: rele-

vance to anemias. Blood 2007;109:2693.

70. Hinnebusch AG. The eIF-2 alpha kinases: regulators of protein synthesis in starvation and

stress. Semin Cell Biol 1994;5:41.

71. Clemens MJ. KR--a protein kinase regulated by double-stranded RNA. Int J Biochem Cell

Biol 1997;29:945.

72. Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplas-

mic-reticulum-resident kinase. Nature 1999;397:271.

73. Harding HP, Novoa I, Zhang Y, et al. Regulated translation initiation controls stress-in-

duced gene expression in mammalian cells. Mol Cell 2000;6:1099.

74. Boesen T, Mohammad SS, Pavitt GD, et al. Structure of the catalytic fragment of trans-

29


lation initiation factor 2B and identification of a critically important catalytic residue. J

Biol Chem 2004;279:10584.

75. Richardson JP, Mohammad SS, Pavitt GD. Mutations causing childhood ataxia with cen-

tral nervous system hypomyelination reduce eukaryotic initiation factor 2B complex

formation and activity. Mol Cell Biol 2004;24:2352.

76. Li W, Wang X, Van Der Knaap MS, Proud CG. Mutations linked to leukoencephalopathy

with vanishing white matter impair the function of the eukaryotic initiation factor 2B

complex in diverse ways. Mol Cell Biol 2004;24:3295.

77. Hiyama TB, Ito T, Imataka H, et al. Crystal structure of the alpha subunit of human trans-

lation initiation factor 2B. J Mol Biol 2009;392:937.

78. Horzinsky L, Huyghe A, Cordoso MC, et al. Eukaryotic initiation factor 2B (eIF2B) GEF

activity as a diagnostic tool for EIF2B-related disorders. PLoS One 2009;15:e8318.

79. Kantor L, Harding HP, Ron D, et al. Heightened stress response in primary fibroblasts

expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients. Hum Genet

2005;118:99.

80. Van Kollenburg B, Thomas AA, Vermeulen G, et al. Regulation of protein synthesis in

lymphoblasts from vanishing white matter patients. Neurobiol Dis 2006;21:496.

81. Rodriguez D, Gelot A, della Gaspera B, et al. Increased density of oligodendrocytes in

childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropatho-

logical and biochemical study of two cases. Acta Neuropathol 1999;97:469.

82. Wong K, Armstrong RC, Gyure KA, et al. Foamy cells with oligodendroglial phenotype in

childhood ataxia with diffuse central nervous system hypomyelination syndrome. Acta

Neuropathol 2000;100:635.

83. Bugiani M, Boor I, van Kollenburg B, et al. Defective glial maturation in vanishing white

matter disease. J Neuropathol Exp Neurol 2011;70:69.

84. Bugiani M, Postma N, Polder E, et al. Hyaluronan accumulation and arrested oligoden-

drocyte progenitor maturation in vanishing white matter disease. Brain 2013;136:209.

33

CHAPTER 2Phenotypic variation in vanishing white matter disease

H. D. W. van der Lei*E. M. Hamilton*J. A. M. Gerver, G. E. M. AbbinkC. G. M. van BerkelM. S. van der Knaap

* these two individuals should be considered as joint first authors

who made equal contributions to this study

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.

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-

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

35


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

36

Chapter 2

Tabl

e 2

| Clin

ical

cha

ract

eris

tics

per

age

of

onse

t ca

tego

ry. I

n it

alic

the

num

ber

of p

atie

nts

in w

hich

an

even

t ha

s oc

curr

ed is

sho

wn

rela

tive

to

the

tota

l num

ber

of p

atie

nts

in

who

m in

form

atio

n on

the

clin

ical

man

ifes

tati

on w

as a

vaila

ble.

P-v

alue

s co

ncer

n th

e co

mpa

riso

n of

the

five

age

of

onse

t gr

oups

. y; y

ear,

m; m

onth

s, n

.a.;

not

appl

icab

le, F

; fem

ale

nu

mb

erA

ll p

atie

nts

223

0-<

2yrs

462-

<6

yrs

102

6-<

12 y

rs24

12 -

<18

yrs

14≥1

8 yr

s28

p-v

alu

e

Surv

ival

Ag

e o

f d

eath

(m

edia

n, r

ang

e); n

um

ber

5y (

3m-4

6y)

5612

m (

3m-1

2y)

278y

( 2

-29y

)21

26y

(17-

36y)

225

y (1

6-33

y)2

34y

(27-

46y)

4<

0.00

1

Dis

ease

du

rati

on

at

dea

th

(med

ian

, ran

ge)

; nu

mb

er

3y (

1m-2

7y)

557m

(1m

-10y

)26

6y (

2m-2

4y )

2117

y (8

-27y

)2

9y (

3m -

18 y

)2

6y (

3-12

y)

40.

001

Dis

ease

du

rati

on

livi

ng

pat

ien

ts

(med

ian

, ran

ge)

; nu

mb

er5y

(0-

31y)

167

2y (

1m-1

1y)

196y

(0-

31y)

817y

(1m

-28y

)22

8y (

2y-2

6y)

125y

(0-

30y)

24

Neu

rolo

gic

al d

evel

op

men

t an

d s

ymp

tom

ato

log

y

Del

ayed

mo

tor

dev

elo

pm

ent

(p

erce

nta

ge,

nu

mb

er)

19%

135

54%

2416

%69

6% 180% 8

0% 15<

0.00

1

Del

ayed

co

gn

itiv

e d

evel

op

men

t(p

erce

nta

ge,

nu

mb

er)

8% 165

36%

284% 86

0% 220% 12

0% 16<

0.00

1

Ach

ieve

d w

alki

ng

wit

ho

ut

sup

po

rt

(per

cen

tag

e, n

um

ber

)95

%13

962

%21

98%

7410

0% 1710

0% 1010

0% 20<

0.00

1

Ag

e at

loss

of

wal

kin

g w

ith

ou

t su

pp

ort

(m

edia

n, r

ang

e); n

um

ber

4y

(1.

4-53

y)10

018

m (

16m

-3y)

153y

(18

m-1

7y)

57

15y

(9-2

9y)

916

y (1

2-32

y)6

35y

(19-

53 y

)13

<0.

001

Du

rati

on

at

loss

of

wal

kin

g w

ith

ou

t su

pp

ort

m

edia

n, r

ang

e); n

um

ber

6m (

0-19

y)10

03m

(0-

18m

)15

6m (

0-12

y)57

6y (

6m-1

9y)

96m

(0-

16y)

64y

(0-

13y)

13<

0.00

1

Ag

e at

fu

ll w

hee

lch

air

dep

end

ency

(m

edia

n, r

ang

e); n

um

ber

6y (

18m

-47y

)77

2.5y

(18

m-7

y)9

4y (

2-18

y)47

20y

(10-

30y)

518

y (1

2-33

y) 7

29y

(24-

47y)

9<

0.00

1

Du

rati

on

at

full

wh

eelc

hai

r d

epen

den

cy

(med

ian

, ran

ge)

; nu

mb

er2y

(0-

22y)

7710

m (

1m-5

y)9

18m

(0-

15y)

4711

y (1

2m-2

0y)

52y

(0-

17 y

)7

5y (

2 -2

2y)

90.

01

Epile

psy

(p

erce

nta

ge,

nu

mb

er)

50% 13

467

%27

44%

7061

%13

50% 8

31%

130.

16

Epis

od

e(s)

of

com

a

(per

cen

tag

e, n

um

ber

)29

%12

242

%31

27%

5525

%12

14% 7

15%

130.

39

37


Invo

lvem

ent

extr

acer

ebra

l org

ans

(p

erce

nta

ge,

nu

mb

er)

9% 141

27%

264% 68

7% 1510

%10

5% 210.

02

Dis

ease

co

urs

e ju

st a

fter

sta

rt (

nu

mb

er)

n=

187

n=

37n

=87

n=

22n

=14

n=

26

No

fu

rth

er p

rob

lem

s (p

erce

nta

ge)

6%3

%7%

4%7%

4%0.

93

Stab

le p

rob

lem

s (p

erce

nta

ge)

17%

5%21

%23

%22

%15

%0.

21

Incr

easi

ng

pro

ble

ms

(per

cen

tag

e)77

%92

%72

%73

%71

%81

%0.

13

Dis

ease

co

urs

e if

det

erio

rati

on

occ

urr

ed (

nu

mb

er)

n=

151

n=

33n

=71

n=

17n

=10

n=

20

Slo

wly

pro

gre

ssiv

e (p

erce

nta

ge)

44%

30%

41%

59%

60%

55%

0.42

Epis

od

es o

f ra

pid

det

erio

rati

on

(p

erce

nta

ge)

24%

40%

23%

6%20

%20

%0.

11

Co

mb

inat

ion

(p

erce

nta

ge)

32%

30%

36%

35%

20%

25%

0.80

Rec

ove

ry a

fter

ep

iso

des

of

det

erio

rati

on

(n

um

ber

)n

=10

8n

=24

n=

58n

=10

n=

5n

=11

Co

mp

lete

rec

ove

ry (

per

cen

tag

e)9%

8.5%

7%0%

40%

18%

0.10

Part

ial r

eco

very

(p

erce

nta

ge)

44%

29%

44%

60%

40%

64%

0.16

Rem

ain

ed s

erio

usl

y h

and

icap

ped

(per

cen

tag

e)33

%29

%40

%30

%20

%9%

0.35

Co

mb

inat

ion

(p

erce

nta

ge)

6%8.

5%7%

0%0%

0%1.

00

Dea

th (

per

cen

tag

e)8%

25%

2%10

%0%

9%0.

01

Ch

ron

ic p

has

e (n

um

ber

)n

=15

1n

=26

n=

77n

=17

n=

8n

=22

Stab

le (

per

cen

tag

e)32

%31

%30

%18

%50

%41

%0.

19

Slo

wly

pro

gre

ssiv

e (p

erce

nta

ge)

52%

19%

56%

82%

50%

54%

<0.

001

Rap

id p

rog

ress

ion

in m

on

ths

(per

cen

tag

e)14

%46

%10

%0%

0%5%

<0.

001

Co

mb

inat

ion

(p

erce

nta

ge)

3%4%

4%0%

0%0%

0.30

Fact

ors

pro

voki

ng

det

erio

rati

on

Hea

d t

rau

ma

(p

erce

nta

ge,

nu

mb

er)

57%

11

629

%

2169

%64

85%

13

17%

6

40%

10

<0.

001

Infe

ctio

ns

wit

h f

ever

(p

erce

nta

ge,

nu

mb

er)

70%

12

392

%

2874

%

6533

%

943

%

750

%

120.

001

38

Chapter 2

Acu

te p

sych

olo

gic

al s

tres

s o

r ac

ute

fri

gh

t (p

erce

nt-

age,

nu

mb

er)

24%

82

0%

16

28%

46

33%

6

20%

5

50%

8

0.03

Aff

ecte

d g

ene

(nu

mb

er)

n=

223

n=

46n

=10

2n

=24

n=

14n

=28

EIF2

B1

(per

cen

tag

e)2%

0%3%

0%0%

0%

EIF2

B2

(per

cen

tag

e)15

%15

%17

%17

%7%

3.5%

EIF2

B3

(per

cen

tag

e)7%

11%

4%4%

7%11

%

EIF2

B4

(per

cen

tag

e)7%

13%

6%12

%0%

3.5%

EIF2

B5

(per

cen

tag

e)69

%61

%70

%67

%86

%82

%

39


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.

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

41


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.

42

Chapter 2

Table 3 | Clinical characteristics male and female patients

number

All patients

223

Female

120

Male

103p-value

Survival

Age of death

(median, range); number

5y (3m-46y)

56

4y (3m-46y)

29

6y (3m-27y)

270.95

Disease duration at death


3y (1m-27y)

55

2y (1m-27y)

28

3y (1m-21y)

270.51

Disease duration living patients


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%

570.65

Delayed cognitive development


8%

165

6%

95

10%

700.39

Achieved walking without support (per-

centage, number)

95%

139

94%

77

97%

620.46

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)

470.63

Age at full wheelchair dependency


6y

(18m-47y)

77

9y (2-47y)

42

4y

(18m-26y)

35

0.03

Duration at full wheelchair dependency


2y (0-22y)

77

2y (0-22y)

42

12m (0-13y)

350.08

Epilepsy


50%

67/134

50%

34/68

50%

33/661.00

Episode(s) of coma


29%

122

28%

64

29%

581.00

Involvement of extracerebral

organs

9%

141

9%

58

10%

830.54

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

43


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


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


Factors provoking deterioration

Head trauma (percentage, number)57%

116

47%

61

67%

550.03

Infections with fever


70%

123

64%

62

75%

610.19

Acute psychological stress or acute fright


24%

82

19%

42

30%

400.25

Affected gene (number) n=120 n=103

EIF2B1 (percentage) 0% 4%





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

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.

45


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

53

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).

47


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.

48

Chapter 2

Figure 6 | Disease duration at loss of walking without support

Figure 7 | Disease duration at full wheelchair dependency

49


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

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

51


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.

52

Chapter 2

1. Van der Knaap MS, Barth PG, Gabreels FJ, et al. A new leukoencephalopathy with van-

ishing white matter. Neurology 1997;48:845-855.

2. Van der Knaap MS, Kamphorst W, Barth PG, et al. Phenotypic variation in leukoencepha-

lopathy with vanishing white matter. Neurology 1998;51:540-547.


hypomyelination. Ann Neurol 1994;35:331-340.

4. Van der Knaap MS. Van Berkel GM, Herms J, et al. eIF2B-related disorders: antenatal

onset and involvement of multiple organs. Am J Hum Genet 2003;73:1199-1207.



2001;29:383-388.

6. Van de Knaap MS, Leegwater PA, Könst AA, et al. Mutations in each of the fine subunits

of translation initiation factor eIF2B can cause leukoencephalopathy with vanishing

white matter. Ann Neurol 2002;51:264-270.

7. 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;24:244-248.

8. Vermeulen G, Seidl R, Mercimek-Mahmutoglu S, et al. Fright is a provoking factor in

vanishing white matter disease. Ann Neurol 2005;57:560-563.

9. Sonenberg N, Hershey JW, Merrick WC. Translational control of gene expression. 1st ed.

New York; CSHL Press, 2000.

10. Pavitt GD. eIF2B, a mediator of general and gene-specific translational control. Biochem

Soc Trans 2005;33:1487-1492.

11. Black DB, Harris R, Schiffmann R, Wong K. Fatal infantile leukodystrophy: a severe vari-

ant of CACH/VWM syndrome, allelic to chromosome 3q27. Neurology 2002;58:161-162.


disease are allelic at EIF2B5 locus. Ann Neurol 2002;52:506-510.

13. Prass K, Brück W, Schröder NW, et al. Adult-onset leukoencephalopathy with vanishing

white matter presenting with dementia. Ann Neurol 2001;50:665-668.


ter: an adult onset case. Neurology 2003;61:1818-1819.

15. Van der Knaap MS, Leegwater PAJ, van Berkel CGM, et al. p.Arg113His mutation in eIF-

2Bε as cause of leukoencephalopathy in adults. Neurology 2004;62:1598-1600.


eIF2B-related leukodystrophies. Neurology 2004;62:1509-1517.


ing white matter with a missense mutation in EIF2B5. Neurology 2004;62:1601-1603.

18. Damon-Perriere N, Menegon P, Olivier A, et al. Intra-familial heterogeneity in adult on-

REFERENCES

53


set vanishing white matter disease. Clin Neurol Neurosurg 2008;110:1068-1071.


disorders: a multi-centric survey of 16 cases. Brain 2009;132:2161-2169.

20. Pronk JC, van Kollenburg B, Scheper GC, van der Knaap MS. Vanishing white matter dis-

ease: a review with focus on its genetics. Ment Retard Dev Disabil Res Rev 2006;12:123-

128.

21. Ohlenbusch A, Henneke M, Brockmann K, et al. Identification of ten novel mutations in


22. Fogli A, Boespflug-Tanguy O. The large spectrum of eIF2B-related disorders. Biochem Soc

Trans 2006;34:22-29.


white matter disease. Neurol Sci 2006;27:271-277.


eIF2B-related disorder. J Child Neurol 2008;23:205-215.

25. van der Lei HD, van Berkel CG, van Wieringen WN, et al. Genotype-phenotype correla-

tion in vanishing white matter disease. Neurology 2010;75:1555-1559.

26. Schiffmann R, Fogli A, van der Knaap MS, et al. Childhood ataxia with central nervouw

system hypomyelination/vanishing white matter. 2003 Feb 20 [Updated 2012 Aug 9]. In:

Pagon RA, Adam MP, Birg TD, et al., editors. GeneReviews

[Internet]. Seattle (WA): Washington, Seattle; 1993-2013. Available from: http://www.

ncbi.nlm.nih.gov/books/NKB1258/

27. Boltshauser E, Barth PG, Troost D, et al. “Vanishing white matter” and ovarian dys-

genesis in an infant with cerebro-oculo-facio-skeletal phenotype. Neuropediatrics.

2002;33:57-62.

28. Schaumburg HH, Powers JM, Raine CS, et al. Adrenoleukodystrophy. A clinical and

pathological study of 17 cases. Arch Neurol. 1975;32:577-91.

29. Bezman, L, Moser, HW Incidence of X-linked adrenoleukodystrophy and the relative

frequency of its phenotypes. (Editorial) Am. J. Med. Genet. 1998;76: 415-419.

30. Kemp S, Pujol A, Waterham HR, et al. ABCD1 mutations and the X-linked adrenoleu-

kodystrophy mutation database: role in diagnosis and clinical correlations. Hum Mutat.

2001;18:499-515.

31. World Health Organization. World health statistics 2014, http://www.who.int/gho/publi-

cations/world_health_statistics/2014/en/ (accessed 03 June 2014).

32. Balsara SL, Faerber JA, Spinner NB, Feudtner C. Pediatric mortality in males versus fe-

males in the United States, 1999-2008. Pediatrics. 2013;132:631-638.

65

54

Chapter 2

Sup

ple

men

tary

tab

le 1

| O

verv

iew

of

gen

oty

pe,

ag

e o

f o

nse

t an

d s

urv

ival

VW

M p

atie

nts

PtFa

mily

Sexe

Gen

eM

uta

tio

n 1

Am

ino

aci

d

chan

ge

1M

uta

tio

n 2

*A

min

o a

cid

ch

ang

e 2

Dis

ease

o

nse

tC

urr

ent

age

1F1

mEI

F2B

1c.

115+

1G>

A

p.G

lu5_

Lys3

8del

a,b

c.62

2A>

Tp

.Asn

208T

yr2-

<6

yrs

11 y

rs

2F2

mEI

F2B

1c.

253-

23T>

Cp

.Asp

85Ph

e.fs

11c.

911A

>G

p.T

yr30

4Cys

19 y

rs

3F3

mEI

F2B

1c.

833C

>G

p.P

ro27

8Arg

2-<

6 yr

s14

yrs

4F4

mEI

F2B

1c.

878C

>T

p.P

ro29

3Leu

2-<

6 yr

s11

yrs

5F5

fEI

F2B

2c.

512C

>T

p.S

er17

1Ph

ec.

947T

>A

p.V

al31

6Asp

≥18

yrs

† 46

yrs

6F6

mEI

F2B

2c.

548d

elG

p.A

rg18

3fs

c.81

8A>

Gp

.Lys

273A

rg2-

<6

yrs

6 yr

s

7F7

fEI

F2B

2c.

599G

>T

p.G

ly20

0Val

c.87

1C>

Tp

.Pro

291S

eran

ten

atal

† 0

yrs

8F7

fEI

F2B

2c.

599G

>T

p.G

ly20

0Val

c.87

1C>

Tp

.Pro

291S

eran

ten

atal

† 0

yrs

9F7

fEI

F2B

2c.

599G

>T

p.G

ly20

0Val

c.87

1C>

Tp

.Pro

291S

eran

ten

atal

† 0

yrs

10F8

mEI

F2B

2c.

599G

>T

p.G

ly20

0Val

c.87

1C>

Tp

.Pro

291S

er0-

<2

yrs

† 0

yrs

11F9

fEI

F2B

2c.

599G

>T

p.G

ly20

0Val

c.87

1C>

Tp

.Pro

291S

er0-

<2

yrs

† 2

yrs

12F1

0m

EIF2

B2

c.59

9G>

Tp

.Gly

200V

alc.

880G

>T

p.V

al29

4Ph

e12

-<18

yrs

18 y

rs

13F1

1m

EIF2

B2

c.de

l607

-612

,insT

Gp

.Glu

202f

sc.

986G

>T

p.A

rg32

9Val

2-<

6 yr

s21

yrs

14F1

2f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

del

529-

543

p.d

el17

7-18

12-

<6

yrs

11 y

rs

15F1

3f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

547C

->T

p.A

rg18

3X2-

<6

yrs

25 y

rs

16F1

4f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

551i

nsA

p.L

ys18

4fs

2-<

6 yr

s12

yrs

17F1

5f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

599G

>T

p.G

ly20

0Val

0-<

2 yr

s4

yrs

18F1

6m

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

599G

>T

p.G

ly20

0Val

0-<

2 yr

s8

yrs

19F1

7f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

599G

>T

p.G

ly20

0Val

2-<

6 yr

s7

yrs

20F1

8f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

599G

>T

p.G

ly20

0Val

-34

yrs

21F1

9f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

599G

>T

p.G

ly20

0Val

2-<

6 yr

s17

yrs

55


22F2

0f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

599G

>T

p.G

ly20

0Val

2-<

6 yr

s3

yrs

23F2

1m

EIF2

B2

c.63

8A>

Gp

.Glu

213G

lyc.

del6

07-6

12,in

sTG

p.G

lu20

2fs

2-<

6 yr

s4

yrs

24F2

2m

EIF2

B2

c.63

8A>

Gp

.Glu

213G

ly2-

<6

yrs

36 y

rs

25F2

3f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

ly2-

<6

yrs

14 y

rs

26F2

4f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

ly2-

<6

yrs

6 yr

s

27F2

5f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

ly6-

<12

yrs

23 y

rs

28F2

6f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

ly6-

<12

yrs

17 y

rs

29F2

7f

EIF2

B2

c.63

8A>

Gp

.Glu

213G

ly2-

<6

yrs

23 y

rs

30F2

7f

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

2-<6

yrs

16 y

rs

31F2

7f

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

6-<1

2 yr

s14

yrs

32F2

8m

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

6-<1

2 yr

s7

yrs

33F2

9m

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

-25

yrs

34F2

9m

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

-22

yrs

35F3

0f

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

2-<6

yrs

5 yr

s

36F3

1f

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

c.94

7T>

Ap.

Val3

16A

sp2-

<6 y

rs26

yrs

37F3

2f

EIF2

B2

c.63

8A>G

p.G

lu21

3Gly

c.94

7T>

Ap.

Val3

16A

sp2-

<6 y

rs8

yrs

38F3

3f

EIF2

B2

c.65

3C>T

p.Th

r218

Ile2-

<6 y

rs12

yrs

39F3

4m

EIF2

B3

c.32

G>T

p.G

ly11

Val

c.65

7-97

5del

p.Se

r220

Cys

fsX5

60-

<2 y

rs†

1 yr

40*

F35

mEI

F2B

3c.

144T

>Ap.

Phe

48Le

u-

-

41F3

6f

EIF2

B3

c.26

0C>T

p.A

la87

Val

c.74

A>G

p.Ly

s25A

rg2-

<6 y

rs11

yrs

42F3

7f

EIF2

B3

c.26

0C>T

p.A

la87

Val

c.14

0G>A

p.G

ly47

Glu

2-<6

yrs

5 yr

s

43F3

8f

EIF2

B3

c.26

0C>T

p.A

la87

Val

12-<

18 y

rs28

yrs

44F3

9f

EIF2

B3

c.26

0C>T

p.A

la87

Val

≥18

yrs

23 y

rs

45F4

0f

EIF2

B3

c.26

0C>T

p.A

la87

Val

c.34

4A>C

p.H

is11

5Pro

≥18

yrs

44 y

rs

56

Chapter 2

46F4

1m

EIF2

B3

c.27

2G>A

p.A

rg91

His

c.10

04C

>Tp.

Pro

335L

eupr

esym

p.6

yrs

47F4

2m

EIF2

B3

c.31

9G>A

p.A

sp10

7Asn

c.52

1C>A

p.A

la17

4Glu

2-<6

yrs

5 yr

s

48F4

3m

EIF2

B3

c.60

2A>G

p.A

sp20

1Gly

0-<2

yrs

† 1

yr

49F4

3m

EIF2

B3

c.60

2A>G

p.A

sp20

1Gly

0-<2

yrs

† 4

yrs

50F4

4f

EIF2

B3

c.60

2A>G

p.A

sp20

1Gly

0-<2

yrs

† 2

yrs

51F4

5f

EIF2

B3

c.67

4G>A

p.A

rg22

5Gln

6-<1

2 yr

s10

yrs

52F4

6m

EIF2

B3

c.67

4G>A

p.A

rg22

5Gln

c.11

93de

lTG

p.Va

l398

fs2-

<6 y

rs†

12 y

rs

53F4

7m

EIF2

B3

c.68

7T>G

p.Ile

229M

et≥1

8 yr

s50

yrs

54F4

8f

EIF2

B3

c.11

24T>

Gp.

Ile37

5Ser

0-<2

yrs

† 0

yrs

55F4

9f

EIF2

B4

c.13

4A>G

p.G

ln45

Arg

2-<6

yrs

8 yr

s

56F5

0f

EIF2

B4

c.62

6A>G

p.A

rg20

9Gln

c.49

9-1G

>Cp.

Val1

67H

isfs

X47

6-<1

2 yr

s9

yrs

57F5

0f

EIF2

B4

c.62

6A>G

p.A

rg20

9Gln

c.49

9-1G

>Cp.

Val1

67H

isfs

X47

6-<1

2 yr

s8

yrs

58F5

1f

EIF2

B4

c.68

3C>T

p.A

rg22

8Val

c.11

91+1

G>A

p.C

ys33

8Trp

fsX1

7a, b

2-<6

yrs

14 y

rs

59F5

2m

EIF2

B4

c.72

5C>T

p.P

ro24

2Ser

c.11

20C

>Tp.

Arg

374C

ys2-

<6 y

rs15

yrs

60F5

3m

EIF2

B4

c.87

7_87

9del

-G

AG

p.G

lu29

3del

2-<6

yrs

5 yr

s

61*

F54

mEI

F2B

4c.

935T

>Cp.

Ile31

2Thr

c.13

99C

>Tp.

Arg

467T

rp-

-

62F5

5m

EIF2

B4

c.11

21G

>Tp.

Arg

374L

euc.

1370

+1in

sT

p.Va

l398

Met

-fs

X24

p.Va

l398

Met

fsX

24

a, c

0-<2

yrs

† 0

yrs

63F5

6m

EIF2

B4

c.11

20C

>Tp.

Arg

374C

ysc.

1070

G>A

p.A

rg35

7Gln

6-<1

2 yr

s35

yrs

64F5

7f

EIF2

B4

c.11

20C

>Tp.

Arg

374C

ysc.

1090

C>T

p.

Arg

364T

rp0-

<2 y

rs†

2 yr

s

65F5

8f

EIF2

B4

c.11

20C

>Tp.

Arg

374C

ys2-

<6 y

rs10

yrs

66F5

9f

EIF2

B4

c.11

72C

>Ap.

Arg

391A

span

tena

tal

† 0

yrs

67*

F60

fEI

F2B

4c.

1399

C>T

p.A

rg46

7Trp

--

68F6

1f

EIF2

B4

c.14

00G

>Tp.

Arg

467L

eu≥1

8 yr

s26

yrs

57


69F6

2m

EIF2

B4

c.14

47C

>T

p.A

rg48

3Trp

ante

nata

l†

0 yr

s

70F6

2f

EIF2

B4

c.14

47C

>Tp.

Arg

483T

rpan

tena

tal

† 0

yrs

71F6

3m

EIF2

B4

c.14

62T>

Cp.

Tyr4

88H

is2-

<6 y

rs14

yrs

72F6

3m

EIF2

B4

c.14

62T>

Cp.

Tyr4

88H

is0-

<2 y

rs13

yrs

73F6

4f

EIF2

B5

c.5C

>Tp.

Ala

2Val

0-<2

yrs

3 yr

s

74F6

5m

EIF2

B5

c.5C

>Tp.

Ala

2Val

2-<6

yrs

7 yr

s

75F6

6m

EIF2

B5

c.5C

>Tp.

Ala

2Val

c.63

1A>G

p.A

rg21

1Gly

2-<6

yrs

7 y

rs

76F6

7f

EIF2

B5

c.16

1G>C

p.A

rg54

Pro

c.94

3C>T

p.A

rg31

5Cys

2-<6

yrs

24 y

rs

77F6

7m

EIF2

B5

c.16

1G>C

p.A

rg54

Pro

c.94

3C>T

p.A

rg31

5Cys

2-<6

yrs

21 y

rs

78F6

8m

EIF2

B5

c.16

7T>C

p.P

he56

Ser

c.13

60C

>Tp.

Pro

454S

er2-

<6 y

rs17

yrs

79F6

9f

EIF2

B5

c.20

3T>C

p.Le

u68S

erc.

685_

768d

elp.

Ser2

29_V

al25

6del

c2-

<6 y

rs†

2 yr

s

80F7

0f

EIF2

B5

c.21

7G>A

p.Va

l73M

et0-

<2 y

rs5

yrs

81F7

1m

EIF2

B5

c.21

8T>G

p.Va

l73G

lyc.

338G

>Ap.

p.A

rg11

3His

6-<1

2 yr

s18

yrs

82F7

1m

EIF2

B5

c.21

8T>G

p.Va

l73G

lyc.

338G

>Ap.

p.A

rg11

3His

6-<1

2 yr

s14

yrs

83F7

2f

EIF2

B5

c.23

0A>G

p.A

sp77

Gly

c.40

7G>A

p.A

rg13

6His

0-<2

yrs

1 yr

84F7

3m

EIF2

B5

c.23

6C>T

p.Th

r79I

lec.

338G

>Ap.

p.A

rg11

3His

2-<6

yrs

† 4

yrs

85F7

4m

EIF2

B5

c.24

7del

Cp.

Leu8

3Xc.

475A

>Gp.

Ile15

9Val

2-<6

yrs

4 yr

s

86F7

4f

EIF2

B5

c.24

7del

Cp.

Leu8

3Xc.

475A

>Gp.

Ile15

9Val

pres

ymp.

1 yr

87F7

5f

EIF2

B5

c.25

1C>T

p.Th

r84I

lec.

274T

>Ap.

Phe

92Ile

2-<6

yrs

8 yr

s

88F7

6f

EIF2

B5

c.27

1A>G

p.Th

r91A

la6-

<12

yrs

38 y

rs

89F7

7m

EIF2

B5

c.27

1A>G

p.Th

r91A

la2-

<6 y

rs†

24 y

rs

90F7

8m

EIF2

B5

c.27

1A>G

p.Th

r91A

la≥1

8 yr

s34

yrs

91F7

8m

EIF2

B5

c.27

1A>G

p.Th

r91A

la6-

<12

yrs

32 y

rs

92F7

9f

EIF2

B5

c.27

1A>G

p.Th

r91A

la2-

<6 y

rs28

yrs

93F8

0f

EIF2

B5

c.27

1A>G

p.Th

r91A

la2-

<6 y

rs34

yrs

58

Chapter 2

94F8

0m

EIF2

B5

c.27

1A>G

p.Th

r91A

la2-

<6 y

rs†

29 y

rs

95F8

1f

EIF2

B5

c.27

1A>

Gp

.Th

r91A

la12

-<18

yrs

† 16

yrs

96F8

2m

EIF2

B5

c.27

1A>

Gp

.Th

r91A

lac.

1016

G>

Ap

.Arg

339G

ln0-

<2

yrs

2 yr

s

97F8

3m

EIF2

B5

c.27

1A>

Gp

.Th

r91A

lac.

1016

G>

Cp

.Arg

339P

ro0-

<2

yrs

† 2

yrs

98F8

4f

EIF2

B5

c.27

1A>

Gp

.Th

r91A

lac.

1015

C>

Tp

.Arg

339T

rp0-

<2

yrs

† 4

yrs

99F8

5m

EIF2

B5

c.27

1A>

Gp

.Th

r91A

lac.

1015

C>

Tp

.Arg

339T

rp0-

<2

yrs

† 10

yrs

100

F85

mEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.10

15C

>T

p.A

rg33

9Trp

2-<

6 yr

s†

14 y

rs

101

F86

fEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.10

15C

>T

p.A

rg33

9Trp

2-<

6 yr

s†

6 yr

s

102

F86

fEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.10

15C

>T

p.A

rg33

9Trp

2-<

6 yr

s†

5 yr

s

103*

F87

mEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.12

08C

>T

p.A

la40

3Val

--

104

F88

mEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.12

08C

>T

p.A

la40

3val

0-<

2 yr

s6

yrs

105

F89

fEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.12

08C

>T

p.A

la40

3val

2-<

6 yr

s†

3 yr

s

106

F90

fEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.17

45+

5G>

Ap

.Tyr

583X

≥18

yrs

47 y

rs

107

F91

mEI

F2B

5c.

271A

>G

p.T

hr9

1Ala

c.18

82T>

Cp

.Trp

628A

rg0-

<2

yrs

† 12

yrs

108

F92

mEI

F2B

5c.

318C

>T

p.L

eu10

6Ph

e2-

<6

yrs

3 yr

s

109

F92

mEI

F2B

5c.

318C

>T

p.L

eu10

6Ph

e0-

<2

yrs

4 yr

s

110

F93

fEI

F2B

5c.

318C

>T

p.L

eu10

6Ph

e2-

<6

yrs

12 y

rs

111

F94

mEI

F2B

5c.

318C

>T

p.L

eu10

6Ph

e0-

<2

yrs

6 yr

s

112

F95

fEI

F2B

5c.

318C

>T

p.L

eu10

6Ph

ec.

406C

>T

p.A

rg13

6Cys

2-<

6 yr

s4

yrs

113

F96

fEI

F2B

5c.

318C

>T

p.L

eu10

6Ph

ec.

944G

>A

p.A

rg31

5His

0-<

2 yr

s0

yrs

114

F97

mEI

F2B

5c.

318C

>T

p.L

eu10

6Ph

ec.

1946

T>C

p.Il

e649

Thr

2-<

6 yr

s5

yrs

115

F98

mEI

F2B

5c.

331T

>C

p.L

eu10

6Ph

ec.

1360

C>

Tp

.Pro

454S

er2-

<6

yrs

5 yr

s

59


116

F99

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.24

1G>

Ap

.Glu

81Ly

s2-

<6

yrs

† 11

yrs

117

F99

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.24

1G>

Ap

.Glu

81Ly

s2-

<6

yrs

16 y

rs

118

F100

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.27

1A>

Gp

.Th

r91A

la6-

<12

yrs

13 y

rs

119

F101

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.27

1A>

Gp

.Th

r91A

la2-

<6

yrs

4 yr

s

120

F102

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.31

4A>

Gp

.His

105A

rg2-

<6

yrs

4 yr

s

121

F103

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.31

8A>

Tp

.Leu

106P

he

6-<

12 y

rs10

yrs

122

F104

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.33

7C>

Tp

.Arg

113C

ys≥1

8 yr

s†

27 y

rs

123

F105

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

55 y

rs

124

F106

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

2-<

6 yr

s31

yrs

125

F107

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

6-<

12 y

rs17

yrs

126

F108

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

12-<

18 y

rs24

yrs

127

F109

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

33 y

rs

128

F110

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

59 y

rs

129

F111

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

12-<

18 y

rs19

yrs

130

F112

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

2-<

6 yr

s9

yrs

131

F113

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

50 y

rs

132

F114

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

43 y

rs

133

F115

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

6-<

12 y

rs8

yrs

134

F116

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

12-<

18 y

rs26

yrs

135

F117

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

55 y

rs

136

F118

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

6-<

12 y

rs32

yrs

137

F119

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

6-<

12 y

rs15

yrs

138

F119

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

12-<

18 y

rs25

yrs

60

Chapter 2

139

F120

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

12-<

18 y

rs24

yrs

140

F121

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

37 y

rs

141

F122

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

31 y

rs

142

F123

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

6-<

12 y

rs†

36 y

rs

143

F124

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

† 30

yrs

144

F125

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

2-<

6 yr

s8

yrs

145

F126

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

30 y

rs

146

F127

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

-19

yrs

147

F128

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

38 y

rs

148

F129

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

6-<

12 y

rs17

yrs

149

F130

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

12-<

18 y

rs†

33 y

rs

150

F131

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

48 y

rs

151

F132

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

≥18

yrs

43 y

rs

152

F133

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

p.A

rg13

6His

-5

yrs

153

F134

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

p.A

rg13

6His

2-<

6 yr

s8

yrs

154

F135

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.46

8C>

Gp

.Ile1

56M

et2-

<6

yrs

27 y

rs

155

F136

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.51

1A>

Gp

.Arg

171G

ly2-

<6

yrs

18 y

rs

156

F137

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.59

2G>

Ap

.Glu

198L

ys≥1

8 yr

s54

yrs

157

F138

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.71

3T>

Ap

.Val

238G

lu2-

<6

yrs

12 y

rs

158

F139

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.75

8C>

Ap

.Ser

253T

yr12

-<18

yrs

17 y

rs

159

F139

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.75

8C>

Ap

.Ser

253T

yr6-

<12

yrs

14 y

rs

160

F140

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.79

2del

Tin

sAC

Ap

.Ph

e264

fs2-

<6

yrs

† 8

yrs

161

F141

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.79

2del

Tin

sAC

Ap

.Ph

e264

fs2-

<6

yrs

15 y

rs

162

F142

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.80

5C>

Gp

.Arg

269G

ly2-

<6

yrs

7 yr

s

163

F143

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.89

6G>

Ap

.Arg

299H

is2-

<6

yrs

27 y

rs

61


164

F144

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.89

6G>

Ap

.Arg

299H

is12

-<18

yrs

22 y

rs

165

F145

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.89

6G>

Ap

.Arg

299H

is2-

<6

yrs

† 3

yrs

166

F145

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.89

6G>

Ap

.Arg

299H

is2-

<6

yrs

† 13

yrs

167

F146

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.89

6G>

Ap

.Arg

299H

is6-

<12

yrs

† 17

yrs

168

F147

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.94

3C>

Tp

.Arg

315C

ys≥1

8 yr

s30

yrs

169

F148

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.94

4G>

Ap

.Arg

315H

is2-

<6

yrs

7 yr

s

170

F149

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.94

4G>

Ap

.Arg

315H

is6-

<12

yrs

15 y

rs

171

F150

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.94

4G>

Ap

.Arg

315H

is2-

<6

yrs

6 yr

s

172

F151

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.94

7G>

Ap

.Arg

316G

ln2-

<6

yrs

4 yr

s

173

F152

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.99

7del

Cp

.Gln

333A

rg-

fsX

230-

<2

yrs

5 yr

s

174

F153

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

15C

>T

p.A

rg33

9Trp

2-<

6 yr

s25

yrs

175

F154

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

15C

>T

p.A

rg33

9Trp

2-<

6 yr

s7

yrs

176

F155

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

15C

>T

p.A

rg33

9Trp

2-<

6 yr

s3

yrs

177

F156

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

16G

>A

p.A

rg33

9Gln

2-<

6 yr

s9

yrs

178

F157

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

16G

>A

p.A

rg33

9Gln

2-<

6 yr

s5

yrs

179

F158

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

16G

>A

p.A

rg33

9Gln

2-<

6 yr

s6

yrs

180

F159

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

16G

>A

p.A

rg33

9Gln

2-<

6 yr

s10

yrs

181

F160

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

16G

>A

p.A

rg33

9Gln

0-<

2 yr

s†

±8

yrs

182

F160

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

16G

>A

p.A

rg33

9Gln

2-<

6 yr

s†

3 yr

s

183

F161

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

21d

el A

CA

p.d

elA

sn34

12-

<6

yrs

10 y

rs

184

F162

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

49T>

Cp

.Leu

350P

ro0-

<2

yrs

4 yr

s

185

F163

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.10

51G

>A

p.G

ly35

1Ser

12-<

18 y

rs41

yrs

186

F164

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.11

57G

>T

p.G

ly38

6Val

2-<

6 yr

s†

7 yr

s

187

F164

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.11

57G

>T

p.G

ly38

6Val

2-<

6 yr

s†

3 yr

s

188

F165

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.12

08C

>T

p.A

la40

3Val

0-<

2 yr

s2

yrs

62

Chapter 2

189

F166

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.12

08C

>T

p.A

la40

3Val

2-<

6 yr

s†

10 y

rs

190

F167

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.12

64C

>T

p.A

rg42

2X2-

<6

yrs

†16

yrs

191

F168

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.12

89T>

Cp

.Val

430A

la2-

<6

yrs

16 y

rs

192

F169

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.12

89T>

Cp

.Val

430A

la2-

<6

yrs

2 yr

s

193

F170

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.12

95C

>T

p.T

hr4

32Ile

2-<

6 yr

s5

yrs

194

F171

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.13

60C

>T

p.P

ro45

4Ser

≥18

yrs

† 39

yrs

195

F172

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.18

10C

>T

p.P

ro60

4Ser

2-<

6 yr

s18

yrs

196

F173

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.18

13d

elC

p.L

eu60

5fs

2-<

6 yr

s†

10 y

rs

197

F174

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.18

24G

>A

p.M

et60

8Ile

pre

sym

p.

10 y

rs

198

F175

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.19

46T>

Cp

.Ile6

49Th

r12

-<18

yrs

20 y

rs

199

F176

fEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.19

48G

>A

p.G

lu65

0Lys

2-<

6 yr

s†

4 yr

s

200

F176

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.19

48G

>A

p.G

lu65

0Lys

2-<

6 yr

s†

12 y

rs

201

F177

mEI

F2B

5c.

338G

>A

p.A

rg11

3His

c.20

51G

>C

p.T

rp68

4Ser

0-<

2 yr

s3

yrs

202

F178

mEI

F2B

5c.

380T

>C

p.L

eu12

7Pro

c.10

15C

>T

p.A

rg33

9Trp

2-<

6 yr

s13

yrs

203

F179

fEI

F2B

5c.

406C

>T

p.A

rg13

6Cys

0-<

2 yr

s2

yrs

204

F180

fEI

F2B

5c.

406C

>T

p.A

rg13

6Cys

0-<

2 yr

s4

yrs

205

F181

mEI

F2B

5c.

406C

>T

p.A

rg13

6Cys

2-<

6 yr

s5

yrs

206

F182

mEI

F2B

5c.

449T

>G

p.L

eu15

0Arg

c.13

55A

>G

p.H

is45

2Arg

2-<

6 yr

s5

yrs

207

F183

fEI

F2B

5c.

562T

>A

p.S

er18

8Th

r2-

<6

yrs

4 yr

s

208

F184

fEI

F2B

5c.

578A

>T

p.P

ro19

3Leu

c.14

44+

19G

>A

p.T

yr48

3Val

fsX

48≥1

8 yr

s37

yrs

209

F184

fEI

F2B

5c.

578A

>T

p.P

ro19

3Leu

c.14

44+

19G

>A

p.T

yr48

3Val

fsX

48≥1

8 yr

s31

yrs

210

F185

mEI

F2B

5c.

631A

>G

p.A

rg21

1Gly

c.12

01C

>T

p.A

rg40

1X2-

<6

yrs

3 yr

s

211

F186

fEI

F2B

5c.

685-

14A

>G

p.Se

r229

_Gln

255d

elc.

1543

T>G

p.T

rp51

5Gly

≥18

yrs

23 y

rs

212

F187

mEI

F2B

5c.

806G

>A

p.A

rg26

9Gln

0-<

2 yr

s†

1 yr

213

F187

mEI

F2B

5c.

806G

>A

p.A

rg26

9Gln

0-<

2 yr

s10

yrs

63


214

F188

fEI

F2B

5c.

943C

>T

p.A

rg31

5His

c.39

5G>

Cp

.Gly

132A

la12

-<18

yrs

24 y

rs

215

F189

fEI

F2B

5c.

943C

>G

p.A

rg31

5Gly

2-<

6 yr

s12

yrs

216

F189

mEI

F2B

5c.

943C

>G

p.A

rg31

5Gly

2-<

6 yr

s2

yrs

217

F190

mEI

F2B

5c.

943C

>G

p.A

rg31

5Gly

0-<

2 yr

s†4

yrs

218

F191

fEI

F2B

5c.

943C

>T

p.A

rg31

5Cys

c.12

08C

>T

p.A

la40

3Val

0-<

2 yr

s2

yrs

219

F192

mEI

F2B

5c.

1015

C>

Tp

.Arg

339T

rpc.

1360

C>

Tp

.Pro

454S

er6-

<12

yrs

10 y

rs

220*

F193

mEI

F2B

5c.

1016

G>

Ap

.Arg

339G

lnc.

1254

G>

Cp

.Glu

418A

sp-

-

221

F194

mEI

F2B

5c.

1016

G>

Cp

.Arg

339P

ro0-

<2

yrs

1 yr

222

F195

mEI

F2B

5c.

1244

A>

Gp

.Asp

415G

lyc.

1280

C>

Tp

.Pro

427L

eu2-

<6

yrs

12 y

rs

223

F196

fEI

F2B

5c.

1289

T>C

p.V

al43

0Ala

c.13

40C

>T

p.S

er44

7Leu

0-<

2 yr

s†0

yrs

224

F197

fEI

F2B

5c.

1309

G>

Ap

.Val

437M

etc.

271A

>G

p.T

hr9

1Ala

0-<

2 yr

s†5

yrs

225

F198

fEI

F2B

5c.

1360

C>

Tp

.Pro

454S

erc.

1208

C>

Tp

.Ala

403V

al2-

<6

yrs

7 yr

s

226

F199

fEI

F2B

5c.

1459

G>

Ap

.Glu

487L

ys≥1

8 yr

s49

yrs

227

F200

mEI

F2B

5c.

1484

A>

Gp

.Tyr

495C

ys0-

<2

yrs

†0 y

rs

228

F200

fEI

F2B

5c.

1484

A>

Gp

.Tyr

495C

ys0-

<2

yrs

†0 y

rs

The

tab

le is

org

aniz

ed b

y as

cen

din

g s

equ

ence

of

mu

tati

on

1 a

nd

, in

cas

e o

f h

eter

ozy

go

sity

, th

e se

qu

ence

of

mu

tati

on

2. P

atie

nts

wit

h s

imila

r g

eno

typ

es a

re m

arke

d in

the

sam

e co

lou

r (w

hit

e o

r g

ray)

.

*Pat

ien

t w

as e

xclu

ded

fo

r cl

inic

al p

hen

oty

pin

g b

ecau

se c

om

orb

idit

y

m, m

ale;

f, f

emal

e; h

et, h

eter

ozy

ou

s; h

om

, ho

mo

zyg

ou

s; y

rs: y

ears

; pre

sym

p.,

pre

sym

pto

mat

ic; †

: pat

ien

t d

ecea

sed

a as

sum

ing

exo

n s

kip

pin

g (

Tale

rico

an

d B

erg

et, M

ol C

ell B

iol 1

990;

10:

629

9–64

05)

b c

han

ge

at D

NA

an

d p

rote

in le

vel a

men

ded

fro

m o

rig

inal

pu

blic

atio

n (

van

der

Kn

aap

et

al, A

nn

Neu

rol 2

002;

51:2

64-2

70)

c ch

ang

e at

DN

A a

nd

pro

tein

leve

l am

end

ed f

rom

ori

gin

al p

ub

licat

ion

(Pr

on

k JC

et

al, M

ent

Ret

ard

Dev

Dis

abil

Res

Rev

200

6;12

:123

-

CHAPTER 3Characteristics of early MRI in children and adolescents with vanishing white matter

H. D. W. van der LeiM. E. SteenwegF. BarkhofT. J. de GrauwM. D’HoogheR. E. MortonS. ShahN. I. WolfM. S. van der Knaap

Neuropediatrics. 2012;43:22-6

66

Chapter 3

ABSTRACT

Objective

MRI in vanishing white matter typically shows diffuse abnormality of the cerebral white matter,

which becomes increasingly rarefied and cystic. We investigated the MRI characteristics preced-

ing this stage.

Design

In a retrospective observational study we evaluated all available MRIs in our database of

DNA-confirmed VWM patients and selected MRIs without diffuse cerebral white matter abnor-

malities and without signs of rarefaction or cystic degeneration in patients below 20 years of

age. A previously established scoring list was used to evaluate the MRIs.

Results

An MRI of seven patients fulfilled the criteria. All had confluent and symmetrical abnormalities

in the periventricular and bordering deep white matter. In young patients, myelination was

delayed. The inner rim of the corpus callosum was affected in all patients.

Conclusions

In early stages of VWM, MRI does not necessarily display diffuse cerebral white matter involve-

ment and rarefaction or cystic degeneration. If the MRI abnormalities do not meet the criteria

for VWM, it helps to look at the corpus callosum. If the inner rim (the callosal-septal interface)

is affected, VWM should be considered.

67

Characteristics of early MRI in children and adolescents with vanishing white matter

INTRODUCTION

Vanishing white matter (VWM; MIM #603896) is a leukoencephalopathy with autosomal re-

cessive inheritance, characterized by slowly progressive ataxia and spasticity with additional

stress-provoked episodes of rapid deterioration after febrile infections, mild head trauma or

even acute fright.4,8,19 Although VWM most frequently occurs in children, it affects people of all

ages.1,2,4,8,9,13 The disease is caused by mutations in the genes EIF2B1-5 encoding the five subunits

of eukaryotic initiation factor eIF2B. This protein complex is essential in all cells of the body

given its pivotal role in protein synthesis and its regulation in stress conditions.12

MRI typically shows diffuse and symmetrical abnormalities of the cerebral white matter. Over

time the cerebral white matter becomes progressively rarefied and cystic (Fig 1C).8,9 Before DNA

testing was available, the diagnosis of VWM was made by clinical and MRI criteria.8,9

Some patients, however, undergo MRI in the presymptomatic or early symptomatic stage and

their MRIs may not fulfill the criteria.2,8,9,16 We therefore performed a study on early MRI char-

acteristics in VWM.


Study design

Approval of the institutional review board was received for retrospective analysis of clinical and

MRI information with waiver of informed consent.

In this retrospective observational study we looked at all available MRIs in our database up to

February 1, 2011. The database contains MRIs of VWM patients referred for DNA analysis.

68

Chapter 3

Figure 1 | Early MRI with delayed myelination. T2-weighted images (A, D) in patient 3 at age 1.7 years show

abnormal periventricular and bordering deep white matter, while the subcortical white matter is not myeli-

nated, indicative of significantly delayed myelination. On FLAIR (B, E), abnormal but non-rarefied white mat-

ter is hyperintense. At age 3.2 (C, F), both FLAIR images show extensive cerebral white matter abnormalities

and with rarefaction of the white matter (arrows).

The inclusion criteria for the present study were:

1. Genetic confirmation of the diagnosis VWM.

2. Age at MRI below 20 years.

3. No diffuse cerebral white matter abnormalities on MRI.

4. No MRI signs of rarefaction or cystic degeneration of the cerebral white matter

If a patient had more than one MRI fulfilling criteria 3 and 4, the first MRI was included. We

also looked at follow-up MRIs to document the evolution of the abnormalities. We noted age

of onset, age at MRI, disease duration and clinical signs at time of MRI. Disease duration was

defined as time between disease onset and first available MRI.

69


Evaluation of MRIs

All available MRIs of VWM patients were assessed by consensus of three investigators (HDWvdL,

MES and MSvdK). A previously established scoring list was used to evaluate the MRI studies.10

Items were scored only as absent or present to minimize the effects of subjective rating.

White matter abnormalities were defined as areas of T2-hyperintensity. White matter rarefac-

tion was defined as T2-hyperintense white matter with low signal on FLAIR images, but not as

low as cerebrospinal fluid. Cystic degeneration was defined as T2-hyperintense areas with on

FLAIR images a signal as low as that of cerebrospinal fluid.

RESULTS

The database contains the MRIs of 224 DNA-confirmed VWM patients. An MRI of seven patients

fulfilled the inclusion criteria. Age, age of onset, disease duration, EIF2B1-5 mutations and MRI

abnormalities are summarized in Table 1.

All MRIs showed abnormalities in the periventricular and deep cerebral white matter and the

inner rim of the corpus callosum (the callosal-septal interface) (figure 1,2). All white matter

abnormalities were confluent and symmetrical. There was no predominance of white matter ab-

normalities in frontal, parietal, occipital or temporal regions. The five youngest patients showed

deficient myelination. In four the myelin deficiency was minor, only apparent in mild T2 hyper-

intensity in directly subcortical and basotemporal areas. In patient 3 myelin deficiency was more

prominent and diffuse (figure 1). The central tegmental tracts in the pons were involved in four

young patients. No gray matter abnormalities were found.

We evaluated the 12 available follow-up MRIs of five patients (for numbers and ages see table 1,

page 83). Over time, there was a shift from predominant involvement of the periventricular and

bordering deep white matter towards diffuse white matter abnormalities with more extensive

involvement of deep and later subcortical white matter. With time, the classical MRI pattern of

VWM with signs of rarefaction of the cerebral white matter was found in all patients. In patient

3 progress of myelination was noted on follow-up. In patients 2 and 4 follow-up MRIs showed

diffuse white matter disease, making assessment of progress of myelination impossible.

With respect to clinical findings at time of the first MRI, patient 1 underwent MRI in the pre-

symptomatic stage after her brother was diagnosed with VWM. Patients 2 and 5 had one epi-

sode with transient deterioration following a febrile infection or fall, in patient 2 followed by

slowly progressive spasticity and ataxia. Patient 3 presented with mild developmental delay,

hypotonia and growth retardation. Patients 4, 6 and 7 had headaches; two had migraines with

aura.

70

Chapter 3

Figure 2 | Early MRI in adolescent. Axial T2-weighted (A) and sagittal FLAIR images (B,C) of patient 7 at age

15.8 years show signal abnormalities in the periventricular and bordering deep white matter and neither de-

layed myelination nor atrophy. The inner rim of the corpus callosum is affected (C, arrow). At age 19.3, sagittal

FLAIR image (D) shows more extensive abnormalities and rarefaction of the white matter (arrow).

DISCUSSION

Central MRI criteria to diagnose VWM are (1) extensive or diffuse cerebral white matter abnor-

malities and (2) evidence of rarefaction or cystic degeneration of part of or all cerebral white

matter.8,9,16 We were aware of the fact that these MRI criteria are not suitable to diagnose VWM

in the earliest stages of the disease.8,16 In this study, we focused on the MRI pattern in early stag-

es of VWM in patients younger than 20 years.

Young patients with a more severe disease variant (patients 1-5) had signal abnormalities in the

periventricular and deep white matter and additionally signs of variably deficient myelination

(figure 1). On follow-up, the classical MRI picture of VWM with diffuse cerebral white matter

abnormalities and white matter rarefaction followed soon. Patients with teenage onset (6 and

7) showed signal abnormalities in the periventricular and bordering deep white matter without

signs of deficient myelination. The MRIs of these patients also evolved into the classical VWM

MRI picture (figure 2).

Independent of age of onset, all patients displayed a gradient in the cerebral white matter

signal abnormalities. The periventricular and bordering deep white matter was affected from

the beginning. Over time, the rest of the deep and then the subcortical cerebral white matter

became affected. In all patients the inner rim of the corpus callosum was involved, which is a

known finding suggestive of VWM (figure 2).16 Most young patients showed lesions in the cen-

tral tegmental tracts. Such lesions are known to occur in VWM, but have also been observed in

other conditions and are, in fact, nonspecific.5

Consistent with earlier observations8,9, we did not find normal or almost normal MRIs in the be-

ginning. Even in the presymptomatic stage, the cerebral white matter already shows extensive

abnormalities (patient 1). But in contrast to what was previously thought8,9,16, the cerebral white

matter abnormalities are not diffuse or almost diffuse from the presymptomatic stage onwards.

Initial white matter abnormalities are present in the periventricular and bordering deep white

matter and spread out to the directly subcortical white matter.

The differential diagnosis is difficult in the early MRI stages.16 In patients presenting with rapid

71


neurological deterioration after a febrile infection, disorders to consider are encephalitis, acute

demyelinating encephalomyelitis (ADEM) and mitochondrial defects. In contrast to VWM, MRI

typically shows asymmetrical multifocal white matter lesions in ADEM 6 and variable lesions in

white as well as grey matter in encephalitis.7 In both conditions one may find contrast enhance-

ment and prominent diffusion restriction of the affected areas, unlike in VWM.6,7 In VWM the

diffusion restriction is seen in the relatively spared areas.17 In mitochondrial leukoencephalop-

athies with rapid deterioration following an infection, MRI may show a picture similar to that

of VWM on T2-weighted and FLAIR images, but contrast enhancement and diffusion restriction

within the lesions again help in the differentiation from VWM.3 Additionally, mitochondrial dis-

orders are usually associated with lactate elevations in body fluids and MR spectroscopy, which

is not the case in VWM.3

In patients with subacute or chronic neurological deterioration mitochondrial leukoenceph-

alopathies, lysosomal storage disorders (especially metachromatic leukodystrophy or Krabbe

disease) and peroxisomal disorders are important disorders in the differential diagnosis. MRI

features allow distinction from VWM in most cases.15 A hint towards the diagnosis VWM is the

selective involvement of the inner rim of the corpus callosum.

In most VWM patients with an early inconclusive MRI, evidence of white matter rarefaction and

cystic degeneration follows soon, allowing an MRI-based diagnosis of VWM. Exceptions are the

adult onset variants of VWM. In those patients, the cerebral white matter abnormalities may be

slow to become diffuse, may mainly show atrophy and no signs of rarefaction or cystic degen-

eration for many years after onset, making an MRI-based diagnosis difficult.2,14 For all ages it is

true that if the MRI abnormalities do not meet the criteria for VWM, it helps to look at the cor-

pus callosum. If the inner rim is affected, VWM should be considered. If the MRI findings remain

inconclusive, it may be worthwhile to assess the known biochemical markers for VWM, such as

cerebrospinal fluid glycine and asialotransferrin.11,18

72

Chapter 3


eIF2B-related leukodystrophies. Neurology 2004; 62: 1509-1517.


disorders: a multi-centric survey of 16 cases. Brain 2009; 132: 2161-2169.

3. Saneto RP, Friedman SD, Shaw DW. Neuroimaging of mitochondrial disease.

Mitochondrion 2008; 8: 396-413.


hypomyelination. Ann Neurol 1994; 35: 331-340.

5. Shioda M, Hayashi M, Takanashi J, Osawa M. Lesions in the central tegmental tract in

autopsy cases of developmental brain disorders. Brain Dev 2011; 33: 541-547.

6. Tenenbaum S, Chitnis T, Ness J, Hahn JS; International Pediatrics MS Study Group.

Acute disseminated encephalitis. Neurology 2007; 68: S23-S36.

7. Tunkel AR, Glaser CA, Bloch KC, et al. The management of encephalitis: clinical practice

guidelines by the Infectious Diseases Society of America. Clin Infect Dis 2008; 47: 303-

327.

8. Van der Knaap MS, Barth PG, Gabreels FJ, et al. A new leukoencephalopathy with

vanishing white matter. Neurology 1997; 48: 845-855.

9. Van der Knaap MS, Kamphorst W, Barth PG, et al. Phenotypic variation in

leukoencephalopathy with vanishing white matter. Neurology 1998; 51: 540-547.

10. Van der Knaap MS, Breiter SN, Naidu S, et al. Defining and categorizing

leukoencephalopathies of unknown origin: MR imaging approach. Radiology 1999;

213: 121-133.

11. Van der Knaap MS, Wevers RA, Kure S, et al. Increased cerebrospinal fluid glycine: a

biochemical marker for a leukoencephalopathy with vanishing white matter. J Child

Neurol 1999; 14: 728-731.

12. Van der Knaap MS, Leegwater PA, Könst AA, et al. Mutations in each of the fine

subunits of translation initiation factor eIF2B can cause leukoencephalopathy with

vanishing white matter. Ann Neurol 2002; 51: 264-270.

13. Van der Knaap MS, van Berkel CG, Herms J, et al. eIF2B-related disorders: antenatal

onset and involvement of multiple organs. Am J Hum Genet 2003; 73: 1199-1207.

14. van der Knaap MS, Leegwater PA, van Berkel CG, et al. Arg113His mutation in

eIF2Bepsilon as cause of leukoencephalopathy in adults. Neurology 2004; 62: 1598-

1600.

15. van der Knaap MS, Valk J. Magnetic resonance of myelination and myelin disorders.

3rd edition. Springer, Heidelberg, 2005.

16. van der Knaap MS, Pronk JC, Scheper GC. Vanishing white matter disease. Lancet

Neurol 2006; 5: 413-423.

17. van der Lei HD, Steenweg ME, Bugiani M, et al. Restricted diffusion in vanishing white

matter. Arch Neurol. 2012;69:723-727.

REFERENCES

73


18. Vanderver A, Schiffmann R, Timmons M, et al. Decreased asialotransferrin in

cerebrospinal fluid of patients with childhood ataxia and central nervous system

hypomyelination or vanishing white matter disease. Clin Chem 2005; 51: 2031-2042.

19. Vermeulen G, Seidl R, Mercimek-Mahmutoglu S, et al. Fright is a provoking factor in

vanishing white matter disease. Ann Neurol 2005; 57: 560-563.

74

Chapter 3

Tab

le 1

| Pa

tien

t an

d M

RI c

har

acte

rist

ics

pat

ien

t1

23

45

67

gen

der

F

FF

FM

FM

age

at fi

rst

MR

I (y)

1.0

1.5

1.7

3.5

4.4

13.2

15.8

nu

mb

er/a

ge

at f

ollo

w-u

p M

RI (

y)0/

-1/

2.3

2/2.

3-3.

21/

8.1

0/-

6/13

.4-1

9.9

2/16

.5-1

9.3

age

at o

nse

t (y

)n

o o

nse

t1.

51.

71.

04.

413

.015

.0

dis

ease

du

rati

on

(y)

no

on

set

00

2.5

00.

20.

8

gen

e m

uta

ted

EIF2

B5

EIF2

B5

EIF2

B2

EIF2

B4

EIF2

B5

EIF2

B5

EIF2

B2

mu

tati

on

1c.

247d

elC

,p

.Leu

83X

c.33

8G>

A,

p.A

rg11

3His

c.59

9G>

T,p

.Gly

200V

alc.

499-

1G>C

,p.

Val

167H

isfs

X47

c.5C

>T,

p.A

la2V

alc.

338G

>A

,p

.Arg

113H

isc.

599G

>T,

p.G

ly20

0Val

mu

tati

on

2c.

475A

>G

,p

.Ile1

59V

alc.

1208

C>

T,p

.Ala

403V

alc.

638A

>G

,p

.Glu

213G

lyc.

626A

>G

,p

.Arg

209G

lnc.

631A

>G

,p

.Arg

211G

lyc.

1946

T>C

,p

.Ile6

49Th

rc.

880G

>T,

p.V

al29

4Ph

e

Ab

no

rmal

itie

s o

n t

he

firs

t M

RI

per

iven

tric

ula

r W

Mye

sye

sye

sye

sye

sye

sye

s

dee

p W

Mye

sye

sye

sye

sye

sye

sye

s

sub

cort

ical

WM

no

no

an

oa

no

an

oa

no

an

oa

cere

bel

lar

WM

yes

yes

yes

no

no

no

no

po

ns-

CTT

no

yes

yes

yes

yes

no

no

inte

rnal

cap

sule

-po

ster

ior

limb

no

no

no

no

no

no

no

exte

rnal

/ext

rem

e ca

psu

len

on

on

on

on

on

on

o

corp

us

callo

sum

-in

ner

rim

yes

yes

yes

yes

yes

yes

yes

corp

us

callo

sum

-ou

ter

rim

no

no

no

no

no

no

no

del

ayed

mye

linat

ion

yes

yes

yes

yes

yes

no

no

y, y

ear(

s); W

M, w

hit

e m

atte

r; C

TT, c

entr

al t

egm

enta

l tra

cts;

aal

mo

st c

om

ple

tely

sp

ared

exc

ept

for

smal

l are

as

75


CHAPTER 4Restricted diffusion in vanishing white matter

H. D. W. van der LeiM. E. SteenwegM. BugianiP. J. W. PouwelsI. M. VentF. BarkhofW. N. van WieringenM. S. van der Knaap

Arch Neurol. 2012;69:723-727

78

Chapter 4

ABSTRACT

Objective

Vanishing white matter (VWM) is a leukoencephalopathy characterized by slowly progressive

ataxia, spasticity and stress-provoked episodes of rapid deterioration. MRI shows diffuse in-

volvement of the cerebral white matter, which becomes rarefied and is eventually replaced by

fluid. Diffusion-weighted imaging (DWI) reveals increased diffusion of the rarefied and cystic

regions. We also observed areas with restricted diffusion in some patients. We investigated the

occurrence of restricted diffusion in VWM, the affected structures, the time of occurrence in the

disease course and the histopathologic correlate.

Design

In a retrospective observational study we evaluated all available DWI studies in our database

and recorded the areas that displayed restricted diffusion in one or more patients. We measured

the mean ADC of these areas in all patients and used the putamen for internal quality control.

We recorded age and disease duration at MRI. We obtained an MRI of a postmortem VWM brain

slice and subsequently performed histopathologic stainings.

Results

Forty-six patients were included. Areas with decreased ADC values were found in the U-fibers

(21 patients), cerebellar white matter (18), middle cerebellar peduncle (8), pyramids (8), genu

(8) or splenium of the corpus callosum (9) and posterior limb of the internal capsule (10). Pa-

tients showing restricted diffusion (32) were overall younger and had shorter disease duration.

Histopathology of the brain slice revealed that regions with restricted diffusion had a higher

cell density.

Conclusion

In VWM, restricted diffusion can be found in relatively spared regions with high cellularity, par-

ticularly in young patients with short disease duration.

79

Restricted diffusion in vanishing white matter

INTRODUCTION

Leukoencephalopathy with vanishing white matter (VWM; MIM #603896)1, also called child-

hood ataxia with diffuse central nervous system hypomyelination (CACH)2, is a white matter

disorder characterized by ataxia and spasticity with a variable rate of progression1-4 and ad-

ditional episodes of major deterioration provoked by stress.1,3-6 It is one of the most prevalent

inherited childhood white matter disorders7, but may affect people of all ages.4,8,9 The disease is

caused by mutations in the genes encoding the eukaryotic translation initiation factor eIF2B.10,11

MRI typically shows a diffuse and symmetrical involvement of the cerebral white matter, which

becomes progressively rarefied and is eventually replaced by fluid (figure 1).1,4 Relatively spared

regions are the U-fibers, corpus callosum, internal capsule, anterior commissure, brain stem and

cerebellar white matter.1,4

Only few studies mention the results of diffusion-weighted images (DWI) in VWM.12-16 In gen-

eral, DWI reveals increased diffusion of the rarefied and cystic white matter related to highly

expanded extracellular spaces.12,13 However, diffusion restriction has recently been reported in

two DNA-confirmed VWM patients in the corpus callosum and U-fibers.16

We decided to perform a systematic study on the subject. We investigated the occurrence of

restricted diffusion in a large series of VWM patients, the affected structures and the time of oc-

currence during the disease course. We obtained an MRI of a postmortem brain slice of a VWM

patient and investigated its histopathology to correlate the DWI findings with histopathology.


Study design

We performed a retrospective observational study and included all available digital diffu-

sion-weighted MR studies in our database up to January 1, 2010. The database contains all VWM

patients referred to our center for DNA analysis, and their MRIs. If a patient had more than one

DWI study, the first was used for primary analysis.

89

90

80

Chapter 4

Figure 1 | MR imaging in VWM disease. 1-year-old VWM patient. Axial T2-weighted image (A) shows diffusely

abnormal white matter. On FLAIR (B), abnormal but intact white matter is hyperintense, rarefied white matter

is hypointense. Central white matter is rarefied, corpus callosum, internal capsule and U-fibers are not (B).

DWI (C) shows low signal in rarefied white matter and high signal in abnormal, non-cystic regions. Low ADC

values (D) indicate restricted diffusion in non-rarefied regions.


Approval of the ethical standards committee was received for retrospective analysis of clinical

and MRI information with waiver of informed consent.

Patients and controls

All patients were diagnosed with VWM on the basis of two mutations in one of the genes en-

coding eIF2B (EIF2B1-5). We excluded those lacking clinical information and those affected by an

additional neurological disease. We used age and disease duration at MRI as clinical parameters.

A dataset of DWI studies of control persons (n=37; male/female=18/19; mean age 5.3 years;

81


median 2.6; range 0.1-24.1) was used to establish reference values (supplementary figure e-1,

figure e-2, see page 101-102). The control group comprised 31 diagnostic MRIs, obtained at a 1.5

T scanner, without structural abnormalities and 6 MRIs of healthy volunteers.

MR images evaluation

All available MRIs of VWM patients and controls were scored by consensus of two investigators

(HDWvdL and MES).

For the identification of studies with restricted diffusion we reviewed both DWI and ADC maps.

For the definitive assessment of diffusion, we only used apparent diffusion coefficient (ADC)

maps in order to avoid the problem of T2-shine-through. Regions of interest (ROIs) were drawn

manually to measure the mean ADC per structure. Special care was taken to minimize partial

volume effects caused by adjacent structures, ventricles and cystic areas. The size of each ROI

was adapted to the size of the structure. Only structures clearly visible and large enough to draw

a ROI within the structure boundaries on axial images were analyzed. ROI sizes varied between

6 mm2 (pyramids) and 70 mm2 (putamen).

For each structure investigated, a scatter plot of the ADC values of the controls was created and

a fitted 5% prediction line was determined to use as the lower level of normal per age (supple-

mentary figure e-1, figure e-2, page 103-105). A mean ADC of a structure below the reference

ADC for that age, scored by both investigators, was used as criterion for restricted diffusion.

The MRIs were collected from many different centers and, consequently, different MRI scanners

and DWI pulse sequences had been used, resulting in potentially different ADC values. All MRI

scanners were 1.5 T machines. We used the mean ADC of a structure that is not affected in VWM

for internal quality control. We chose the putamen because of its size.14 If the mean ADC of the

putamen in a patient was below the reference ADC for that age, the DWI study was excluded

from the analysis. We also excluded all poor quality DWI studies.

We evaluated all available ADC maps of VWM patients for areas of restricted diffusion. All re-

gions that displayed restricted diffusion in at least one VWM patient were then systematically

analyzed in all VWM patients and controls. We noted the signal behavior of the selected areas

on FLAIR images.

Postmortem brain tissue: MRI and histopathology

An MRI of a formalin-fixed brain slice of one of the deceased VWM patients was performed

to correlate restricted diffusion to histopathology. The study was conducted on a 1.5 T whole

body MR scanner (Siemens, Sonata, Erlangen, Germany). The 1.7 cm thick brain slice was placed

in a slice holder, which fits into an 8-channel phased-array head coil. The MR imaging protocol

included a dual-echo proton density (PD)/T2-weighted fast spin echo sequence (TR 2500 ms; TE

24/85 ms; 4 measurements; slice thickness 4 mm, in-plane resolution 1 x 1 mm, interpolated to

0.5 x 0.5 mm) and a single-slab 3D-FLAIR sequence17 (TR 6500 ms; TE 355 ms; TI 2200 ms; 1 meas-

urement; slice thickness 1.25 mm, in plane resolution 1.1 x 1.1 mm). DWI was performed with a

single shot STEAM sequence18,19 (TR 5200 ms; TE 48 ms; 80 averages, each consisting of a refer-

ence image with b=0 s/mm2 and a 3-scan trace-weighted diffusion image with b=750 s/mm2; slice

82

Chapter 4

thickness 5 mm, in-plane resolution 1.17 x 1.17 mm). The PD/T2 and DWI images were located

at the center of the brain slice, which was covered by several thin FLAIR images. After imaging

the brain slice was cut at the level of the MRI study and embedded in paraffin. Eight micron thick

sections were obtained and stained with Hematoxylin and Eosin using standard techniques.


Summary statistics (mean and standard deviation, the latter in brackets) of clinical variables are

given in years. Clinical variables of patient subgroups were compared using either the two-sam-

ple Student t-test or one way ANOVA. For all brain structures investigated, scatter plots were

created from the mean ADC values of the controls by age. By robust regression analysis (to

accommodate possible outliers) of the log-transformed variables, the 5% prediction line per

structure was determined, and after back transformation to the original scale, used as the lower

level of normal.

Analyses were performed using SPSS for Windows version 15.

RESULTS

Restricted diffusion in VWM patients

The database contained 72 DWI studies of 56 patients. One patient (1 DWI study) was excluded

because of co-morbidity (encephalocele, abnormal gyration and neuronal heterotopias) and 4

patients (4 DWI studies) because of a lack of any clinical information. Five DWI studies (excluding

1 patient) were excluded because of poor image quality and 6 studies (excluding 4 patients) be-

cause the ADC value of the putamen was below 5% of the reference. Of the remaining 56 DWI

studies obtained in 46 patients, we used the first 46 MRIs for our primary study. We evaluated

the 10 follow-up MRIs (4 patients had 1 follow-up MRI; 3 patients had 2 follow-up MRIs) to see

what happened with restricted diffusion over time.

The 46 patients included in the study had a male/female ratio of 16/30; mean age of 13.2 years,

age range of 0.3-47.6 years; age at onset of 7.7 (range 0.2-37.0) and a disease duration of 5.5 years

(range 0-28.8).

Decreased ADC values were found on the first available MRI in 32 of the 46 patients, and included

the U-fibers (21 patients), cerebellar white matter (18), middle cerebellar peduncles (8), pyramids

(8), genu (8) or splenium of the corpus callosum (9) and posterior limb of the internal capsule (10).

All regions with restricted diffusion were hyperintense rather than hypointense on FLAIR images

(figure 1), indicative of tissue abnormality without cystic degeneration.

Age and disease duration at the time of MRI of patients with and without restricted diffusion

are given in Table 1. ADC values of patients and control persons for each structure can be found

in supplementary figure e-1, figure e-2 and table e-1 (see page 89-91). Patients with restricted

diffusion had a lower age and shorter disease duration. This effect was most marked for patients

with restricted diffusion in the U-fibers, cerebellar white matter, pyramids, or genu of the corpus

callosum. To a lesser degree the trend of lower age and shorter disease duration was visible for

83


Tab

le 1

| R

estr

icte

d p

roto

n d

iffu

sio

n p

er s

tru

ctu

re: n

um

ber

of

MR

Is, a

ge

and

dis

ease

du

rati

on

at

firs

t M

RI

all p

atie

nts

pat

ien

ts w

ith

re

stri

cted

dif

fusi

on

pat

ien

ts w

ith

ou

t re

stri

cted

dif

fusi

on

p-v

alu

e***

stru

ctu

ren

*ag

e at

MR

I, y*

*d

isea

se

du

rati

on

, yn

age

at

MR

I, y

dis

ease

d

ura

tio

n, y

nag

e at

M

RI,

yd

isea

se

du

rati

on

, yag

e at

M

RI

dis

ease

d

ura

tio

n

fro

nta

l U fi

ber

s46

13.2

(±

13.5

)5.

5 (±

8.4)

123.

3 (±

2.7)

0.4

(±0.

9)34

16.7

(±

14.0

)7.

3 (±

9.1)

<0.

001

<0.

001

par

ieta

l U fi

ber

s46

13.2

(±

13.5

)5.

5 (±

8.4)

143.

7 (±

3.4)

1.1

(±2.

3)32

17.4

(±

14.2

)7.

3 (±

9.4)

<0.

001

0.00

1

occ

ipit

al U

fib

ers

4613

.2 (

±13

.5)

5.5

(±8.

4)18

4.5

(±3.

9)0.

9 (±

1.2)

2818

.8 (

±14

.5)

8.4

(±9.

7)<

0.00

1<

0.00

1

tem

po

ral U

fib

ers

4613

.2 (

±13

.5)

5.5

(±8.

4)14

4.3

(±4.

3)0.

7 (±

0.9)

3217

.1 (

±14

.3)

7.6

(±9.

3)<

0.00

1<

0.00

1

cere

bel

lar

wh

ite

mat

ter

4513

.4 (

±13

.6)

5.6

(±8.

5)18

w7.

8 (±

10.8

)1.

9 (±

6.1)

2717

.2 (

±14

.1)

8.0

(±9.

0)0.

016

0.00

9

mid

dle

cer

ebel

lar

ped

un

cle

4413

.6 (

±13

.7)

5.7

(±8.

6)8

7.3

(±14

.2)

3.3

(±9.

2)36

15.1

(±

13.3

)6.

2 (±

8.4)

0.14

60.

397

pyr

amid

al t

ract

s44

13.6

(±

13.7

)5.

7 (±

8.6)

83.

9 (±

5.2)

0.7

(±0.

7)36

15.8

(±

14.0

)6.

7 (±

9.1)

<0.

001

<0.

001

gen

u o

f co

rpu

s ca

llosu

m39

13.0

(±

13.7

)4.

7 (±

7.8)

82.

2 (±

1.1)

0.3

(±0.

5)

3115

.8 (

±14

.1)

5.9

(±8.

4)<

0.00

10.

001

sple

niu

m o

f co

rpu

s ca

llosu

m42

13.0

(±

13.7

)4.

9 (±

8.0)

95.

6 (±

9.9)

3.3

(±9.

6)

3315

.1 (

±13

.9)

5.4

(±7.

7)0.

032

0.50

3

po

ster

ior

limb

of

inte

rnal

ca

psu

le45

13.5

(±

13.6

)5.

6 (±

8.5)

109.

4 (±

15.4

)3.

1 (±

8.1)

35

14.6

(±

13.0

)6.

3 (±

8.6)

0.28

80.

310

all s

tru

ctu

res*

***

4613

.2 (

±13

.5)

5.5

(±8.

4)32

8.2

(±10

.6)

2.7

(±6.

7)14

24.8

(±

12.5

)11

.7 (

±8.

8)<

0.00

10.

003

*to

tal n

um

ber

of

scan

s in

wh

ich

th

e st

ruct

ure

co

uld

be

eval

uat

ed *

*val

ues

are

mea

n (

± S

D)

in y

ears

***

p-v

alu

e o

f co

mp

aris

on

of

pat

ien

ts w

ith

an

d w

ith

ou

t re

stri

cted

dif

-

fusi

on

***

*co

mp

aris

on

of

all p

atie

nts

wit

h o

ne

or

mo

re s

tru

ctu

res

wit

h r

estr

icte

d d

iffu

sio

n v

ersu

s p

atie

nts

wit

ho

ut

any

rest

rict

ed d

iffu

sio

n.

84

Chapter 4

patients with restricted diffusion in the middle cerebellar peduncles, splenium of the corpus callo-

sum or posterior limb of the internal capsule.

Of the patients with multiple DWI studies, two showed no restricted diffusion at all; in one re-

stricted diffusion arose on the second MRI; in one it was initially present and disappeared; in two

it partially disappeared; in one it was initially present, disappeared and arose again.

DWI of postmortem brain tissue and histopathologic correlation

The scanned post mortem coronal brain slice was of a girl who died at 5.6 years. MRI at age 1.6 years

had shown restricted diffusion in the U-fibers, cerebellar white matter, middle cerebellar peduncles,

pyramids, genu and splenium of the corpus callosum, and posterior limb of the internal capsule on

both sides. At age 2.1 diffusion restriction was limited to the U-fibers and posterior limb of inter-

nal capsule. The postmortem ADC map of the brain slice showed restricted proton diffusion in the

U-fibers (figure 2).

Macroscopically, the white matter appeared diffusely grayish and gelatinous to frankly cystic in

the periventricular and deep hemispheric regions. Microscopic examination revealed that the re-

gions showing restricted diffusion had a highly increased cellular density with relative myelin

preservation. No signs of acute tissue degeneration with cytotoxic edema were detected (figure

3). The areas had the typical characteristics of the relatively spared regions in vanishing white mat-

ter disease with a high cell density of oligodendrocytes and oligodendrocyt precursor cells.1,2,4,20-24

Figure 2 | Postmortem diffusion-weighted imaging in a brain slice of a VWM patient. Coronal brain slice of

a VWM patient, who died at 5.6 years. FLAIR (A) shows a large cystic area and abnormal, high signal in the

non-cystic white matter. DWI (B) shows that part of the subcortical white matter has a relatively high signal

(1); the remainder of the white matter has a lower signal (2). The ADC map (C) displays low signal in area 1

and a high signal in the rest of the white matter.

97

85


patients with restricted diffusion in the middle cerebellar peduncles, splenium of the corpus callo-

sum or posterior limb of the internal capsule.

Of the patients with multiple DWI studies, two showed no restricted diffusion at all; in one re-

stricted diffusion arose on the second MRI; in one it was initially present and disappeared; in two

it partially disappeared; in one it was initially present, disappeared and arose again.

DWI of postmortem brain tissue and histopathologic correlation

The scanned post mortem coronal brain slice was of a girl who died at 5.6 years. MRI at age 1.6 years

had shown restricted diffusion in the U-fibers, cerebellar white matter, middle cerebellar peduncles,

pyramids, genu and splenium of the corpus callosum, and posterior limb of the internal capsule on

both sides. At age 2.1 diffusion restriction was limited to the U-fibers and posterior limb of inter-

nal capsule. The postmortem ADC map of the brain slice showed restricted proton diffusion in the

U-fibers (figure 2).

Macroscopically, the white matter appeared diffusely grayish and gelatinous to frankly cystic in

the periventricular and deep hemispheric regions. Microscopic examination revealed that the re-

gions showing restricted diffusion had a highly increased cellular density with relative myelin

preservation. No signs of acute tissue degeneration with cytotoxic edema were detected (figure

3). The areas had the typical characteristics of the relatively spared regions in vanishing white mat-

ter disease with a high cell density of oligodendrocytes and oligodendrocyt precursor cells.1,2,4,20-24

Figure 2 | Postmortem diffusion-weighted imaging in a brain slice of a VWM patient. Coronal brain slice of

a VWM patient, who died at 5.6 years. FLAIR (A) shows a large cystic area and abnormal, high signal in the

non-cystic white matter. DWI (B) shows that part of the subcortical white matter has a relatively high signal

(1); the remainder of the white matter has a lower signal (2). The ADC map (C) displays low signal in area 1

and a high signal in the rest of the white matter.

97

Figure 3 | High cell density in spared regions. A section of the scanned brain slice (A), stained with Hematoxilin

and Eosin, shows rarefaction (2) and cystic degeneration of most white matter, while the U-fibers are partially

spared (1). At higher magnification a remarkably increased number of cells is observed in a relatively spared

region (B, area 1 in A). A much lower density of cells is present in a rarefied area (C, area 2 in A). The cells have

the morphology of oligodendrocytes and oligodendrocyte precursor cells. Original magnifications: B,C, X100.

DISCUSSION

We focused on restricted diffusion in VWM. Increased diffusion generally reflects increased ex-

tracellular spaces, whereas decreased diffusion is seen in conditions of decreased extracellular

spaces. In conditions characterized by acute tissue degeneration, decreased diffusion is gener-

ally caused by cytotoxic edema.25,26 Cytotoxic edema is associated with cell swelling and com-

pression of the extracellular spaces. Decreased diffusion is, however, also seen in conditions of

storage of substances, myelin vacuolation and intramyelinic edema, and high cellularity, such as

in tumors with a high cell density and abscesses.25-29

We observed decreased ADC values in specific white matter structures in VWM: U-fibers, corpus

callosum, internal capsule, cerebellar white matter, middle cerebellar peduncles and pyramids.

These are regions known to be relatively spared in VWM.1,2,4,20-24 In all patients with areas of

restricted diffusion, the FLAIR images confirmed that these areas were affected but not rarefied

or cystic. In VWM, less affected regions may have a high cellular density with much higher cell

86

Chapter 4

numbers than in control brain tissue.4,21,23,24. Especially high numbers of oligodendrocytes21-24 and

oligodendrocyte precursor cells30 have been observed in better preserved regions. Our DWI-his-

topathology correlation confirms that areas of restricted diffusion are relatively spared regions

with high cellularity. The morphology of the cells in those areas is compatible with oligodendro-

cytes and precursor cells.

We found restricted diffusion mainly in younger patients with short disease duration, suggest-

ing it is an early feature of the disease. The two VWM patients in whom restricted diffusion has

been mentioned before, have the Cree encephalopathy variant of VWM, which occurs in infants

and young children.16 Not all patients with short disease duration, however, show areas with

restricted diffusion and we also found restricted diffusion in some older patients. At present, we

have no explanation for these observations.

In conclusion, restricted diffusion in metabolic disorders is often easily ascribed to tissue necrosis

and cytotoxic edema. Strikingly, however, in VWM restricted diffusion is seen in relatively spared

regions with a high cell density.

87





system hypomyelination. Ann Neurol 1994;35:331-340.

3. Hanefeld F, Holzbach U, Kruse B, Wilichowski E, Christen HJ, Frahm J. Diffuse white

matter disease in three children: an encephalopathy with unique features on magnet-

ic resonance imaging and proton magnetic resonance spectroscopy. Neuropediatrics

1993;24:244-248.

4. Van der Knaap MS, Kamphorst W, Barth PG, Kraaijeveld CL, Gut E, Valk J. Phenotypic varia-

tion in leukoencephalopathy with vanishing white matter. Neurology 1998;51:540-547.

5. Vermeulen G, Seidl R, Mercimek-Mahmutoglu S, Rotteveel JJ, Scheper GC, van der

Knaap MS. Fright is a provoking factor in vanishing white matter disease. Ann Neurol

2005;57:560-563.

6. Kaczorowska M, kuczynski D, Jurkiewicz E, et al. Acute fright induces onset of symptoms

in vanishing white matter disease-case report. Eur J Paediatr Neurol 2006;10:192-193.

7. Van der Knaap MS, Breiter SN, Naidu S, Hart AA, Valk J. Defining and categorizing leuko-

encephalopathies of unknown origin: MR imaging approach. Radiology 1999;213:121-133.

8. Van der Knaap MS, van Berkel CG, Herms J, et al. eIF2B-related disorders: antenatal onset

and involvement of multiple organs. Am J Hum Genet 2003;73:1199-1207.





2001;29:383-388.

11. Van der Knaap MS, Leegwater PA, Könst AA, et al. Mutations in each of the fine subunits

of translation initiation factor eIF2B can cause leukoencephalopathy with vanishing white

matter. Ann Neurol 2002;51:264-270.

12. Sijens PE, Boon M, Meiners LC, Brouwer OF, Oudkerk M. 1H chemical shift imaging,

MRI, and diffusion-weighted imaging in vanishing white matter disease. Eur Radiol

2005;15:2377-2379.

13. Patay Z. Diffusion-weighted MR imaging in leukodystrophies. Eur Radiol 2005;15:2284-

2303.

14. Van der Knaap MS, Pronk JC, Scheper GC. Vanishing white matter disease. Lancet Neurol

2006;5:413-423.

15. Van der Knaap MS, Schiffmann R, Scheper GC. Conversion of a normal MRI into an MRI

showing a cystic leukoencephalopathy is not a known feature of vanishing white matter.

Neuropediatrics 2007;38:264.

16. Harder S, Gourgaris A, Frangou E, et al. Clinical and neuroimaging findings of Cree leu-

kodystrophy: a retrospective case series. Am J Neuroradiol 2010;31:1418-1423.

REFERENCES

88

Chapter 4

17. Moraal B, Roosendaal SD, Pouwels PJ, et al. Multi-contrast, isotropic, single-slab 3D MR

imaging in multiple sclerosis. Eur Radiol 2008;18:2311-2320.

18. Koch MA, Glauche V, Finsterbusch J, et al. Distortion-free diffusion tensor imaging of

cranial nerves and of inferior temporal and orbitofrontal white matter. Neuroimage

2002;17:497-506.

19. Van der Voorn JP, Pouwels PJ, Powers JM, et al. Correlating quantitative MR imaging with

histopathology in X-linked adrenoleukodystrophy. Am J Neuroradiol 2011;32:481-489.

20. Brück W, Herms J, Brockmann K, Schultz-Schaeffer W, Hanefeld F. Myelinopathia centralis

diffusa (vanishing white matter disease): evidence of apoptotic oligodendrocyte degenera-

tion in early lesion development. Ann Neurol 2001;50:532-536.

21. Rodriquez D, Gelot A, della Gaspera B, et al. Increased density of oligodendrocytes in

childhood ataxia with diffuse central nervous system hypomyelination (CACH) syndrome:

neuropathological and biochemical study of two cases. Acta Neuropathol 1999;97:469-480.


childhood ataxia with diffuse central nervous system hypomyelination syndrome. Acta

Neuropathol 2000;100:635-646.

23. Francalanci P, Eymard-Pierre E, Dionisi-Vici C, et al. Fatal infantile leukodystrophy: a severe

variant of CACH/VWM syndrome, allelic to chromosome 3q27. Neurology 2001;57:265-270.

24. Van Haren K, van der Voorn JP, Peterson DR, van der Knaap MS, Powers JM. The life and

death of oligodendrocytes in vanishing white matter disease. J Neuropathol Exp Neurol

2004;63:618-630.

25. Schaefer PW, Grant PE, Gonzalez RG. Diffusion-weighted MR imaging of the brain. Radiol-

ogy 2000;217:331-345.

26. Stadnik TW, Demaerel P, Luypaert RR, et al. Imaging turorial: differential diagnosis of

bright lesions on diffusion-weighted MR images. RadioGraphics 2003;23:e7.

27. Sugahara T, Korogi Y, Kochi M, et al. Usefulness of diffusion-weighted MRI with echo-pla-

nar technique in the evaluation of cellularity in gliomas. J Magn Reson Imaging 1999;9:53-

60.

28. Vermathen P, Robert-Tissot L, Pietz J, Lutz T, Boesch C, Kreis R. Characterization of white

matter alterations in phenylketonuria by magnetic resonance relaxometry and diffusion

tensor imaging. Magn Reson Med 2007;58:1145-1156.

29. Oguz KK, Anlar B, Senbil N, Cila A. Diffusion-weighted imaging findings in juvenile me-

tachromatic leukodystrophy. Neuropediatrics 2004;35:279-282.

30. Bugiani M, Boor I, van Kollenburg B, et al. Defective glial maturation in vanishing white

matter disease. J Neuropath Exp Neurol 2011;70:69-82.

89


Table e-1 | Number of patients with restricted diffusion per hemisphere

right hemisphere left hemisphere patients

structure n* n n**

frontal U fibers 7 11 12

parietal U fibers 12 11 14

occipital U fibers 13 13 18

temporal U fibers 12 11 14

cerebellar white matter 6 18 18

middle cerebellar peduncle 5 7 8

pyramidal tracts 5 7 8

genu of corpus callosum - - 8

splenium of corpus callosum - - 9

posterior limb of internal capsule 6 9 10

*total number of patients showing restricted diffusion in the structure in this hemisphere **total number of patients showing restriction in the right and/or the left hemisphere

90

Chapter 4

91


Figure e-2. ADC values of controls and patients

Legend. Marks reflect ADC values (average of both investigators) of patient (red triagles) at first MRI and control persons (black circles) by age. Fifty (black line) and five percent prediction line (black striped line) of control values. See table e-1 for total numbers of patients with restricted diffusion per structure.

40

8010

012

0Pyramidal tract R

age (years)

mea

n A

DC

(x 1

0-5

mm

2 /s)

0 10 20 30

020

4060

40

8010

012

0

Pyramidal tract L

age (years)m

ean

AD

C (x

10-

5 m

m2 /

s)

0 10 20 30

020

4060

40

8010

012

0

Genu of corpus callosum

age (years)

mea

n A

DC

(x 1

0-5

mm

2 /s)

020

4060

0 302010

40

8010

012

0

Splenium of corpus callosum

age (years)

mea

n A

DC

(x 1

0-5

mm

2 /s)

0 10 20 30

020

4060

40

8010

012

0Posterior limb of internal capsule R

age (years)

mea

n A

DC

(x 1

0-5

mm

2 /s)

0 302010

020

4060

40

8010

012

0

Posterior limb of internal capsule L

age (years)m

ean

AD

C (x

10-

5 m

m2 /

s)0 302010

020

4060

40

8010

012

0

Putamen R

age (years)

mea

n A

DC

(x 1

0-5

mm

2 /s)

0 302010

020

4060

40

8010

012

0

Putamen L

age (years)

mea

n A

DC

(x 1

0-5

mm

2 /s)

0 302010

060

4020

CHAPTER 5Genotype - phenotype correlation in vanishing white matter disease

H. D. W. van der LeiC. G. M. van BerkelW. N. van WieringenC. BrennerA. FeigenbaumS. Mercimek-MahmutogluM. PhilippartB. TatliE. WassmerG. C. ScheperM. S. van der Knaap

Neurology 2010;75:1555-1559

94

Chapter 5

ABSTRACT

Objective

Vanishing white matter (VWM) is an autosomal-recessive leukoencephalopathy characterized

by slowly progressive ataxia and spasticity with additional stress-provoked episodes of rapid

and major deterioration. The disease is caused by mutations in the genes encoding the subunits

of eukaryotic initiation factor 2B, which is pivotal in translation of mRNAs into proteins. The

disease onset, clinical severity and disease course of VWM patients vary greatly. The influence of

genotype and gender on the phenotype is unclear.

Methods

From our database of 184 patients with VWM, we selected those with the following muta-

tions in the gene EIF2B5: p.Arg113His in the homozygous state (n=23), p.Arg113His in the com-

pound-heterozygous state (n=49), p.Thr91Ala in the homozygous state (n=8), p.Arg113His/p.

Arg339any (n=9), and p.Thr91Ala/p.Arg339any (n=7). We performed a cross-sectional observa-

tional study. Evaluated clinical characteristics were gender, age of onset, age at loss of walking

without support and age at death. Means, male/female ratios and Kaplan-Meier curves were

compared.

Results

Patients homozygous for p.Arg113His had a milder disease than patients compound-heterozy-

gous for p.Arg113His and patients homozygous for p.Thr91Ala. Patients with p.Arg113His/p.

Arg339any had a milder phenotype than patients with p.Thr91Ala/p.Arg339any. Overall females

tended to have a milder disease than males.

Conclusions

The clinical phenotype in VWM is influenced by the combination of both mutations. Females

tend to do better than males.

95

Genotype - phenotype correlation 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 an autosomal recessive5,6 leukoencephalopathy. The course

is chronic progressive with additional stress-provoked episodes of rapid deterioration.1-3,7,8

VWM is caused by mutations in the genes EIF2B1-5 encoding the subunits of eukaryotic initia-

tion factor 2B (eIF2B).5,6 eIF2B is indispensable for translation initiation and regulation of pro-

tein synthesis under different conditions, including cell stress.9,10

Although VWM was initially recognized as a disorder of young children1,3,7, it has become clear

that the variation in disease severity is extremely wide. Severe forms start in the antenatal or

early infantile period and lead to early demise.4,11,12 Much milder variants start in adolescence or

adulthood and are characterized by slow disease progression.2,13-17

The explanation for this wide phenotypic variation is complex. There is evidence that the gen-

otype influences the phenotype. Certain mutations are consistently associated with a mild or

severe phenotype.12,14-16 In addition, striking intra-familial phenotypic heterogeneity has been

reported2,15,16,18, suggesting that environmental or other genetic factors also influence the phe-

notype. Recently, an effect of gender was suggested.19

Many different mutations have been described in VWM and most patients are compound-het-

erozygous for two different mutations in one of the five disease genes.20 We addressed the

question whether the clinical phenotype of compound-heterozygous patients is determined

by the mildest mutation, the most severe mutation or by both. Our hypothesis was that both

mutations determine the phenotype. In addition, we hypothesized that the disease severity is

different in man and women.


Study design

We performed a cross-sectional observational study and included all available patients with

specific mutations from our VWM patient database until February 2009. The database contains

all patients referred to VU University Medical Center for mutational analysis for VWM.


Written informed consent for research was obtained from all patients, or guardians of the pa-

tients, participating in the study. Approval of the ethical standards committee was received for

retrospective analysis of clinical information, collected by questionnaires.

Mutation-based selection of patients

Approximately 70% of the VWM patients in our database have mutations in EIF2B5 and 30%

have mutations in EIF2B1-4. We focused on the patients with EIF2B5 mutations. P.Arg113His

in EIF2B5 is the most frequent mutation and the mildest known mutation. In the homozygous

state it is almost invariably associated with adulthood onset and protracted disease course.14-16

107

108

96

Chapter 5

To answer the question whether the mildest mutation, the most severe mutation or the combi-

nation of both mutations determines the phenotype, we compared the phenotypes of patients

homozygous for p.Arg113His, compound-heterozygous for p.Arg113His and another mutation

(p.Arg113His/p.any) and patients in whom p.Arg113His was not involved (p.any/p.any).

Another relatively large group in our database has the so-called Dutch founder mutation

(p.Thr91Ala in EIF2B5).5 The next largest group has a mutation involving arginine (Arg) at posi-

tion 339 of EIF2B5. At this position we have observed amino acid substitutions to Gln, Trp and

Pro (collectively referred to as p.Arg339any). To investigate the effect of two different EIF2B5

mutations on the phenotype we selected patients homozygous for p.Arg113His, patients homo-

zygous for p.Thr91Ala, and patients with one of these mutations in the compound-heterozy-

gous state with p.Arg339any. None of our patients is homozygous for p.Arg339any.

Phenotype

We used clinical questionnaires to be filled in by either the family or the physician following

the patient. In order to avoid the problem of differences in rating the disability, we chose ro-

bust clinical outcomes for the present study to characterize the phenotype: age of onset, age

at loss of walking without support and age at death. The disease onset was considered the age

at which the first neurological symptom appeared. Delayed early development was not includ-

ed. Patients were scored as having lost independent walking when they could no longer walk

without support and not when they started to use a wheelchair for part of the time. If the pa-

tients never walked without support, they were scored as having lost the ability of unsupported

walking at the age of 1½ years. Hence, if patients died before the age of 1½, they where not

included in the analysis of loss of walking without support. The disease duration was defined

as the time between the disease onset and the latest clinical observation. Both disease duration

and age were used for analysis of Kaplan-Meier curves.

To test whether the disease severity differs between man and women, we analyzed male/female

differences in the entire group of EIF2B5-mutated patients and in the groups of patients with

the EIF2B5 mutations specified above.

Patients with another disease affecting neurological function in addition to VWM were exclud-

ed. Patients, for whom no follow-up data were known, were also excluded from the analysis.


Summary statistics (mean and standard deviation, the latter in brackets) of clinical variables are

given in years. Non-censored clinical variables of patient subgroups were compared using either

the two-sample Student t-test or one way ANOVA. The cumulative probabilities of individuals

to lose the ability to walk without support or to die, in relation to the disease duration or

the age of the patient, were estimated through Kaplan-Meier curves. Log-rank statistics were

performed to compare subgroups with respect to censored clinical variables. Individuals not

reaching an event (i.e., age at loss of independent walking or death) within the study period

were considered to be censored. Analyses were performed using SPSS for Windows version 15.

97


RESULTS

Patients

Of the 184 patients in our database, 126 (68%) had mutations in EIF2B5. The number of patients,

age of onset, age at loss of independent walking and age at death for the different groups are

given in Tables 1 and 2. Sixty-two patients were male and 64 female. The gender and genotype

of all patients included in the study can be found in the supplementary Table e-1 (page 102).

Three patients of the 126 patients were excluded from further analysis: two because of co-mor-

bidity (i.e., Down syndrome and a developmental anomaly in the form of encephalocele, ab-

normal gyration and neuronal heterotopias) and one because of a lack of clinical information.

Table 1 | Disease severity for patients with EIF2B5 mutations: p.Arg113His versus the other mutations

*Mean and standard deviation (in brackets) are indicated in years.

**Number of patients of whom data were available and an event had occurred.

Comparison of p.Arg113His/p.Arg113His, p.Arg113His/p.any and p.any/p.any

Comparing the patients homozygous for p.Arg113His with the patients compound-heterozygous

for this mutation, differences were found concerning average age of onset (p<0.001), average age

at loss of unsupported walking (p<0.001), and average age at death (p=0.012) in favor of patients

homozygous for p.Arg113His (table 1). Comparison of the Kaplan-Meier plots demonstrated that

patients homozygous for p.Arg113His retained the ability to walk independently longer, both in

age (p<0.001) and in disease duration (p<0.001) and had a longer survival in age (p<0.001) and

disease duration (p=0.009) than patients compound-heterozygous for p.Arg113His (figure 1).

Comparing the patients homozygous for p.Arg113His with the patients with two other EIF2B5

mutations than p.Arg113His (p.any/p.any), we found differences in average age of onset

(p=0.001), age at loss of unsupported walking (p<0.001) and age at death (p<0.001) in favor

of patients homozygous for p.Arg113His. Log rank tests confirmed that patients homozygous

for p.Arg113His retained the ability to walk independently longer, both in age (p<0.001) and

in disease duration (p=0.001) and had a longer survival in age (p=0.002) and disease duration

(p=0.013) than the p.any/p.any group (figure 1).

There were no differences between patients who were compound heterozygous with one p.Ar-

g113His mutation and the patients with two other mutations (figure 1).

Patient group

Age of onset of the disease

Age at loss of unsupported walking

Age at death

p.Arg113His/ p.Arg113His 19.2 (±14.1)* 21.9 (±10.7) 33.0 (±4.2)

(n=23) 19** 13 2

p.Arg113His/p.any 6.2 (±8.0) 7.4 (±10.6) 12.4 (±9.8)

(n=49) 47 38 15

p.any/p.any 6.1 (±9.9) 8.0 (±11.8) 7.2 (±7.4)

(n=51) 50 42 7

98

Chapter 5

Table 2 | Disease severity for patients with EIF2B5 mutations: the effect of p.Arg113His, p.Thr91Ala and p.Arg339any


**Number of patients of whom data were available and an event had occurred.

Figure 1 | Survival. Patients homozygous for the p.Arg113His mutation survive longer than patients with p.Ar-

g113His/p.any and p.any/p.any. There is no difference between patients with p.Arg113His/p.any and p.any/p.

any. The curves are showing the cumulative probability of patients to survive estimated from the age of onset.

Patient group



Age at death

p.Arg113His/ p.Arg113His 19.2 (±14.1)* 21.9 (±10.7) 33.0 (±4.2)

(n=23) 19** 13 2

p.Thr91Ala/ p.Thr91Ala 6.9 (±4.8) 13.6 (±8.4) 22.8 (±8.9)

(n=8) 7 7 2

p.Arg113His/ p.Arg339any 2.4 (±0.9) 3.7 (±1.1) 3.0 (-)

(n=9) 9 7 1

p.Thr91Ala/ p.Arg339any 1.7 (±0.5) 2.1 (±0.5) 7.3 (±4.3)

(n=7) 7 6 6

0 5 10 15 20 25 30

0.0

0.2

0.4

0.6

0.8

1.0

duration of the disease from onset (years)

cum

ulat

ive

prob

abili

ty to

sur

vive

p.Arg113His/p.Arg113His

p.Arg113His/p.any

p.any/p.any

99


Figure 2 | Ability to walk. Patients homozygous for the p.Arg113His mutation retain the ability to walk in-

dependently longer than patients with p.Thr91Ala/p.Thr91Ala, p.Arg113His/p.Arg339any and p.Thr91Ala/p.

Arg339any. The latter do worse in the order mentioned. The curves are showing the cumulative probability of

the ability to walk independently estimated from birth.

Comparison of p.Arg113His, p.Thr91Ala, and p.Arg339any

We found a difference in age of onset between patients homozygous for p.Arg113His and

patients homozygous for p.Thr91Ala (p=0.003), the first group doing better than the second

group (table 2). We found a trend for age at loss of unsupported walking (p=0.092) and age at

death (p=0.323), the first group doing better. Comparing Kaplan-Meier plots demonstrated that

patients homozygous for p.Arg113His retained the ability to walk independently longer in age

(p=0.05), but not in disease duration (p=0.451) than patients homozygous for p.Thr91Ala (figure

2). There was no difference with respect to overall survival.

Patients with p.Arg113His/p.Arg339any and patients with p.Thr91Ala/p.Arg339any differed in

average age at loss of unsupported walking (p=0.008), the first group doing better. The same

trend was demonstrated for age of onset (p=0.077). The number of patients with p.Arg113His/p.

Arg339any who died was too small for a valid comparison. Comparing Kaplan Meier plots con-

firmed that patients with p.Arg113His/p.Arg339any retained the ability to walk longer in age

(p<0.001) and tended to have a longer disease duration (p=0.092). There was a difference in age

at death (p=0.039). Also the disease duration until death tended to be longer for patients with

p.Arg113His/p.Arg339any than for patients with p.Thr91Ala/p.Arg339any (p=0.089).

0 10 20 30 40 50 60

0.0

0.2

0.4

0.6

0.8

1.0

age of patients (years)

cum

ulat

ive

prob

abili

ty to

wal

k in

depe

nden

tly

p.Arg113His/p.Arg113His

p.Arg113His/p.Arg339any

p.Thr91Ala/p.Thr91Ala

p.Thr91Ala/p.Arg339any

100

Chapter 5

Influence of gender in EIF2B5-mutated patients

Age of onset, age at loss of independent walking and age at death for females and males in the

different groups are given in supplementary Table e-2 (page 106).

An imbalance between males and females was only noticed in the subgroup of patients homo-

zygous for p.Arg113His. The ratio was 8 males / 15 females in this group, versus 26 / 23 in the

p.Arg113His/p.any group, 4 / 4 in the p.Thr91Ala/p.Thr91Ala group, 4 / 5 in the p.Arg113His/p.

Arg339any group, and 4 / 3 in the p.Thr91Ala /p.Arg339any group.

Within the entire group of EIF2B5-mutated patients (males / females = 60 / 63) differences were

found between males and females concerning average age of onset (p=0.01) and average age

at loss of unsupported walking

(p=0.01) in the sense that females did better. There was no difference in average age at death

(p=0.256). Comparison of the Kaplan-Meier plots revealed that females tended to retain the

ability to walk longer in age (p=0.090). There were no differences between males and females

in survival.

We also analyzed the subgroups of patients with p.Arg113His/p.Arg113His, p.Arg113His/p.any,

p.Thr91Ala/p.Thr91Ala, p.Arg113His/p.Arg339any or p.Thr91Ala /p.Arg339any separately. In the

group of patients homozygous for p.Arg113His the age at loss of unsupported walking was

higher in females than in males (p=0.072). Females with p.Arg113His/p.Arg339any tended to

have a later onset (p=0.109) and to lose the ability to walk later (p=0.107). The latter was also

found in the log rank test (p=0.098). No other gender-related trends were found in the different

subgroups.

DISCUSSION

In our study of the influence of genotype on phenotype in VWM, we focused on patients with

EIF2B5 mutations, being the largest group. We selected the three most frequent mutations:

p.Arg113His, p.Thr91Ala and p.Arg339any, associated with a mild, mild to intermediate, and se-

vere phenotype, respectively. We treated patients with p.Arg339any as a single group, although

p.Arg339Trp, p.Arg339Pro and p.Arg339Gln may not have exactly the same effect on the phe-

notype. Mutations at this position are, however, consistently associated with a severe disease.

No patients homozygous for p.Arg339any are known and homozygosity for the mutation may

be incompatible with life.

Our findings demonstrate that patients homozygous for p.Arg113His have a milder disease than

patients compound-heterozygous for p.Arg113His. This indicates that the phenotype is not de-

termined by the mildest mutation alone, but either by the most severe mutation or by a combi-

nation of both mutations. Patients homozygous for p.Arg113His tend to have a milder disease

than patients homozygous for p.Thr91Ala. Patients homozygous for p.Arg113His and homozy-

gous for p.Thr91Ala have a milder disease than patients with p.Arg113His/p.Arg339any and

patients with p.Thr91Ala/p.Arg339any. Patients with p.Arg113His/p.Arg339any have a milder

phenotype than patients with p.Thr91Ala/p.Arg339any. These findings indicate that the pheno-

115

101


Influence of gender in EIF2B5-mutated patients

Age of onset, age at loss of independent walking and age at death for females and males in the

different groups are given in supplementary Table e-2 (page 106).

An imbalance between males and females was only noticed in the subgroup of patients homo-

zygous for p.Arg113His. The ratio was 8 males / 15 females in this group, versus 26 / 23 in the

p.Arg113His/p.any group, 4 / 4 in the p.Thr91Ala/p.Thr91Ala group, 4 / 5 in the p.Arg113His/p.

Arg339any group, and 4 / 3 in the p.Thr91Ala /p.Arg339any group.

Within the entire group of EIF2B5-mutated patients (males / females = 60 / 63) differences were

found between males and females concerning average age of onset (p=0.01) and average age

at loss of unsupported walking

(p=0.01) in the sense that females did better. There was no difference in average age at death

(p=0.256). Comparison of the Kaplan-Meier plots revealed that females tended to retain the

ability to walk longer in age (p=0.090). There were no differences between males and females

in survival.

We also analyzed the subgroups of patients with p.Arg113His/p.Arg113His, p.Arg113His/p.any,

p.Thr91Ala/p.Thr91Ala, p.Arg113His/p.Arg339any or p.Thr91Ala /p.Arg339any separately. In the

group of patients homozygous for p.Arg113His the age at loss of unsupported walking was

higher in females than in males (p=0.072). Females with p.Arg113His/p.Arg339any tended to

have a later onset (p=0.109) and to lose the ability to walk later (p=0.107). The latter was also

found in the log rank test (p=0.098). No other gender-related trends were found in the different

subgroups.

DISCUSSION

In our study of the influence of genotype on phenotype in VWM, we focused on patients with

EIF2B5 mutations, being the largest group. We selected the three most frequent mutations:

p.Arg113His, p.Thr91Ala and p.Arg339any, associated with a mild, mild to intermediate, and se-

vere phenotype, respectively. We treated patients with p.Arg339any as a single group, although

p.Arg339Trp, p.Arg339Pro and p.Arg339Gln may not have exactly the same effect on the phe-

notype. Mutations at this position are, however, consistently associated with a severe disease.

No patients homozygous for p.Arg339any are known and homozygosity for the mutation may

be incompatible with life.


patients compound-heterozygous for p.Arg113His. This indicates that the phenotype is not de-

termined by the mildest mutation alone, but either by the most severe mutation or by a combi-

nation of both mutations. Patients homozygous for p.Arg113His tend to have a milder disease

than patients homozygous for p.Thr91Ala. Patients homozygous for p.Arg113His and homozy-

gous for p.Thr91Ala have a milder disease than patients with p.Arg113His/p.Arg339any and

patients with p.Thr91Ala/p.Arg339any. Patients with p.Arg113His/p.Arg339any have a milder

phenotype than patients with p.Thr91Ala/p.Arg339any. These findings indicate that the pheno-

115

type is not determined by the most severe mutation alone, but by the effect of both mutations.

This conclusion is important for clinicians and genetic counsellors providing information to pa-

tients and families. One should be careful, however, with definitive predictions for new pa-

tients. Further studies on larger groups of patients would make conclusion more certain.

Recently, an overrepresentation of females was found among adult VWM patients, mainly pa-

tients with p.Arg113His.19 A higher susceptibility of females to disease expression was suggest-

ed, assuming that affected males remain asymptomatic for a longer period of time. The present

study, however, demonstrates that females with mutations in EIF2B5 tend to do better than

males. All differences found were in favor of the females. Larger subgroups of patients must be

studied before it can be concluded that females do better for all mutations. Strikingly, and in

agreement with this recent study19, we also found an overrepresentation of females among the

p.Arg113His-mutated patients. It is at present unexplained why there are more female patients

with this mutation.

116

102

Chapter 5

Table e-1 | EIF2B5-mutated patients in the study

Patient Family Gender Amino acid change Amino acid change

1 F1 M p.Arg113His p.Arg113His

2 F2 F p.Arg113His p.Arg113His

3 F3 F p.Arg113His p.Glu81Lys


5 F4 M p.Arg113His p.Glu650Lys


7 F5 M p.Tyr495Cys p.Tyr495Cys

8 F5 F p.Tyr495Cys p.Tyr495Cys

9 F6 F p.Ile156Met p.Arg113His


11 F8 F p.Ser171Phe p.Val316Asp

12 F9 F p.Ser229_Gln253del p.Leu68Ser


14 F11 M p.Arg113His p.Arg339Gln

15 F12 M p.Arg171Gly p.Arg113His

16 F13 M p.Thr91Ala p.Ala403Val


18 F15 F p.Val437Met p.Thr91Ala

19 F16 M p.Asp415Gly p.Pro427Leu

20 F17 M p.Arg113His p.His105Arg



23 F19 F p.Arg339Trp p.Thr91Ala

24 F19 F p.Thr91Ala p.Arg339Trp


26 F21 M p.Leu106Phe p.Leu106Phe

27 F22 F p.Arg422X p.Arg113His


29 F24 M p.Thr91Ala p.Thr91Ala

30 F24 F p.Thr91Ala p.Thr91Ala

31 F25 M p.Arg113His p.Arg339Trp

32 F26 F p.Thr91Ala p.Ala403Val

33 F27 F p.Arg113His p.Arg339Trp

34 F28 M p.Arg113Cys p.Arg113His

35 F29 F p.Pro454Ser p.Arg113His

117

103


Table e-1 | EIF2B5-mutated patients in the study

Patient Family Gender Amino acid change Amino acid change







7 F5 M p.Tyr495Cys p.Tyr495Cys

8 F5 F p.Tyr495Cys p.Tyr495Cys

9 F6 F p.Ile156Met p.Arg113His


11 F8 F p.Ser171Phe p.Val316Asp

12 F9 F p.Ser229_Gln253del p.Leu68Ser


14 F11 M p.Arg113His p.Arg339Gln

15 F12 M p.Arg171Gly p.Arg113His



18 F15 F p.Val437Met p.Thr91Ala

19 F16 M p.Asp415Gly p.Pro427Leu

20 F17 M p.Arg113His p.His105Arg



23 F19 F p.Arg339Trp p.Thr91Ala




27 F22 F p.Arg422X p.Arg113His




31 F25 M p.Arg113His p.Arg339Trp

32 F26 F p.Thr91Ala p.Ala403Val


34 F28 M p.Arg113Cys p.Arg113His

35 F29 F p.Pro454Ser p.Arg113His

117



38 F32 M p.Ala2Val p.Ala2Val



411 F35 F p.Arg315His p.Leu106Phe

42 F36 M p.Arg269Gln p.Arg269Gln

43 F36 M p.Arg269Gln p.Arg269Gln

44 F37 M p.Val73Gly p.Arg113His

45 F37 M p.Val73Gly p.Arg113His

46 F38 F p.Arg113His p.Ser253Tyr

47 F38 F p.Arg113His p.Ser253Tyr


49 F40 M p.Arg339Trp p.Pro454Ser

50 F41 M p.Arg339Gln p.Arg113His

51 F41 M p.Arg339Gln p.Arg113His


53 F43 F p.Arg113His p.Arg629Gly

54 F44 F p.Thr91Ala p.Tyr583X



57 F47 M p.Phe264fs p.Arg113His

58 F48 F p.Ala403val p.Ser447Leu


60 F50 M p.Arg339Trp p.Thr91Ala

61 F50 M p.Arg339Trp p.Thr91Ala

623 F51 M p.Glu418Asp p.Arg339Gln

63 F52 F p.Arg113His p.Ile649Thr

64 F53 F p.Leu605fs p.Arg113His

65 F54 M p.Thr91Ala p.Arg339Pro


67 F56 M p.Trp111Arg p.Pro454Ser

68 F57 M p.Arg136Cys p.Arg136Cys

69 F58 M p.Arg339Trp p.Leu127Pro

70 F59 M p.Asn341del p.Arg113His


72 F61 F p.Arg339Gln p.Arg113His

73 F62 F p.Pro454Ser p.Ala403Val

104

Chapter 5

74 F63 F p.Arg315Gly p.Arg315Gly

75 F64 M p.Val238Glu p.Arg113His

76 F65 M p.Arg113His p.Phe264LeufsX14

77 F66 F p.Arg113His p.Pro604Ser

78 F67 M p.Arg113His p.Gly386Val

79 F67 M p.Arg113His p.Gly386Val

80 F68 F p.Val73Met p.Val73Met


82 F70 M p.Thr79Ile p.Arg113His


84 F72 M p.Trp628Arg p.Thr91Ala


86 F74 F p.Ala403Val p.Arg113His






92 F78 M p.Arg315Gly p.Arg315Gly

93 F79 F p.Cys545Thr p.Thr182Met

94 F80 F p.Thr84Ile p.Phe92Ile

95 F81 M p.Phe56Ser p.Pro454Ser


97 F83 F p.Arg113His p.Val430Ala



100 F86 M p.Ile649Thr p.Leu106Phe

101 F87 F p.Arg113His p.Arg339Gln

102 F88 M p.Arg113His p.Met608Ile

103 F89 F p.Arg315Cys p.Ala403Val


105 F91 M p.Leu350Pro p.Arg113His


107 F93 F p.Arg113His p.Arg315Cys



110 F96 F p.Thr91Ala p.Arg113His

111 F97 F p.Tyr483ValfsX48 p.Pro193Leu

105


112 F97 F p.Tyr483ValfsX48 p.Pro193Leu



115 F100 M p.Thr91Ala p.Arg339Gln



118 F102 F p.Arg113His p.Arg339Gln

119 F103 M p.Ala2Val p.Arg211Gly

120 F104 M p.Arg113His p.Thr432Ile

121 F105 M p.Arg113His p.Gln333ArgfsX23

122 F106 F p.Arg113His p.Leu106Phe



125 F108 F p.Leu106Phe p.Arg136His


1 Patient was excluded because of lack of clinical information.

2 Patient was excluded because of a developmental anomaly (encephalocele with abnormal gyration and

neuronal heterotopias).

3 Patient was excluded because of Down syndrome.

120

106

Chapter 5

Table e-2 | Disease severity for patients with EIF2B5 mutations: the effect of gender


**The number of patients of whom data were available and an event had occurred.

Patient group



Age at death

Overall

male 5.6 (±8.2)* 6.5 (±9.1) 9.0 (±6.8)

n=60 55** 45 17

female 10.7 (±12.7) 12.8 (±13.7) 13.0 (±12.8)

n=63 61 48 17

p.Arg113His/p.Arg113His 9 7 1

male 16.5 (±14.7) 14.0 (±9.0) -

n=8 6 4 0

female 20.5 (±14.2) 25.4 (±9.8) 33.0 (±4.2)

n=15 13 9 2

p.Arg113His/p.any

male 5.6 (±14.1) 7.6 (±11.9) 10.4 (±4.2)

n=26 24 21 10

female 6.8 (±4.8) 7.2 (±9.1) 16.4 (±8.9)

n=23 23 17 5

p.Thr91Ala/p.Thr91Ala

male 5.3 (±3.2) 9.0 (±5.0) -

n=4 3 3 0

female 8.1 (±5.8) 17.1 (±9.3) 22.8 (±8.9)

p.Arg113His/p.Arg339any

male 1.9 (±1.1) 2.9 (±0.2) 3.0

n=4 4 3 1

female 2.9 (±0.5) 4.3 (±1.2) -

n=5 5 4 0

p.Thr91Ala/p.Arg339any

male 1.6 (±0.6) 1.9 (±0.5) 9.1 (±6.0)

n=4 4 3 3

female 1.7 (±0.5) 2.3 (±0.4) 5.5 (±1.3)

n=3 3 3 3

107


122

REFERENCES



2. Van der Knaap MS, Kamphorst W, Barth PG, Kraaijeveld CL, Gut E, Valk J. Phenotypic var-

iation in leukoencephalopathy with vanishing white matter. Neurology 1998;51:540-547.


hypomyelination. Ann Neurol 1994;35:331-340.

4. Van der Knaap MS. Van Berkel GM, Herms J, et al. eIF2B-related disorders: antenatal

onset and involvement of multiple organs. Am J Hum Genet 2003;73:1199-1207.



2001;29:383-388.


of translation initiation factor eIF2B can cause leukoencephalopathy with vanishing


7. Hanefeld F, Holzbach U, Kruse B, Wilichowski E, Christen HJ, Frahm J. Diffuse white

matter disease in three children: an encephalopathy with unique features on magnet-

ic resonance imaging and proton magnetic resonance spectroscopy. Neuropediatrics

1993;24:244-248.

8. Vermeulen G, Seidl R, Mercimek-Mahmutoglu S, Rotteveel JJ, Scheper GC, van der Knaap

MS. Fright is a provoking factor in vanishing white matter disease. Ann Neurol

2005;57:560-563.

9. Sonenberg N, Hershey JW, Merrick WC. Translational control of gene expression. 1st ed.

New York; CSHL Press, 2000.


Soc Trans 2005;33:1487-1492.

11. Black DB, Harris R, Schiffmann R, Wong K. Fatal infantile leukodystrophy: a severe vari-

ant of CACH/VWM syndrome, allelic to chromosome 3q27. Neurology 2002;58:161-162.


disease are allelic at EIF2B5 locus. Ann Neurol 2002;52:506-510.

13. Prass K, Brück W, Schröder NW, et al. Adult-onset leukoencephalopathy with vanishing

white matter presenting with dementia. Ann Neurol 2001;50:665-668.


ter: an adult onset case. Neurology 2003;61:1818-1819.

15. Van der Knaap MS, Leegwater PAJ, van Berkel CGM, et al. Arg113His mutation in eIF2Bε as cause of leukoencephalopathy in adults. Neurology 2004;62:1598-1600.




ing white matter with a missense mutation in EIF2B5. Neurology 2004;62:1601-1603.

123

108

Chapter 5

18. Damon-Perriere N, Menegon P, Olivier A, et al. Intra-familial heterogeneity in adult on-

set vanishing white matter disease. Clin Neurol Neurosurg 2008;110:1068-1071.


disorders: a multi-centric survey of 16 cases. Brain 2009;132:2161-2169.

20. Pronk JC, van Kollenburg B, Scheper GC, van der Knaap MS. Vanishing white matter dis-

ease: a review with focus on its genetics. Ment Retard Dev Disabil Res Rev 2006;12:123-

128.



22. Fogli A, Boespflug-Tanguy O. The large spectrum of eIF2B-related disorders. Biochem Soc

Trans 2006;34:22-29.


white matter disease. Neurol Sci 2006;27:271-277.


eIF2B-related disorder. J Child Neurol 2008;23:205-215.

109


CHAPTER 6Severity of vanishing white matter disease does not correlate with deficits in eIF2B activity or the integrity of eIF2B complexes

H. D. W. van der Lei*R. Liu*X. Wang*N. C. Wortham H. Tang, C. G. M. van BerkelT. A. MufundeW. Huang M. S. van der KnaapG. C. ScheperC. G. Proud

* these three individuals should be considered as joint first authors who made equal contributions to this study

Hum Mutat 2011;32:1036-1045

112

Chapter 6

ABSTRACT

Autosomal recessive mutations in eukaryotic initiation factor 2B (eIF2B) cause leukoenceph-

alopathy vanishing white matter (VWM; OMIM 603896) with a wide clinical spectrum. eIF2B

comprises five subunits (α-ε; genes EIF2B1, 2, 3, 4 and 5) and is the guanine nucleotide-exchange

factor (GEF) for eIF2. It plays a key role in protein synthesis. Here, we have studied the functional

effects of selected VWM mutations in EIF2B2-5 by co-expressing mutated and wildtype subunits

in human cells. The observed functional effects are very diverse, including defects in eIF2B com-

plex integrity; binding to the regulatory a-subunit; substrate binding; and GEF activity. Activity

data for recombinant eIF2B complexes agree closely with those for patient-derived cells with the

same mutations. Some mutations do not affect these parameters even though they cause severe

disease. These findings are important for three reasons; they demonstrate that measuring eIF2B

activity in patients’ cells has limited value as a diagnostic test; they imply that severe disease

can result from alterations in eIF2B function other than defects in complex integrity, substrate

binding or GEF activity and, lastly, the diversity of functional effects of VWM mutations implies

that seeking agents to manage or treat VWM should focus on downstream effectors of eIF2B,

not restoring eIF2B activity.

113

Severity of vanishing white matter disease does not correlate with deficits in eIF2B

INTRODUCTION

eIF2B (eukaryotic initiation factor 2B) plays a key role in protein synthesis (mRNA translation)

and in its regulation.1 It acts as a GDP-dissociation stimulator protein (GDS)2 to mediate guanine

nucleotide exchange on eIF2, the factor that (as eIF2.GTP) brings the initiator methionyl-tRNA

(Met-tRNAiMet) to the ribosome to recognize the start codon during each ‘round’ of translation

initiation. eIF2B is thus often referred to as a guanine nucleotide-exchange factor (GEF). The

observations that overexpressing eIF2B increases protein synthesis rates in cardiomyocytes and

HEK293 cells3,4, and induces growth of the former, indicates that eIF2B is a critical rate-deter-

mining component of the protein synthesis machinery. Consistent with this, eIF2B activity can be

regulated in several different ways.1

eIF2B comprises five different subunits (α-ε), encoded, respectively, by the genes EIF2B1-5

(OMIM accession numbers 606686, 606454, 606273, 606687 and 603945). The largest one, eIF-

2Bε, contains the catalytic domain (approximately residues 527-726 in human eIF2Bε) within

its C-terminal region which mediates GDP/GTP exchange on eIF2.5,6 The extreme C-terminus of

eIF2B ε interacts with eIF2.7 Large sections of the sequences of eIF2B γ and eIF2Bε show mutual

sequence similarity.8 These two subunits form a binary complex termed the ‘catalytic subcom-

plex’.9 The other three subunits, α, β and δ, also show mutual sequence similarity10,11 and form a

‘regulatory subcomplex’9, so named because it confers on the holocomplex sensitivity to inhibi-

tion by the substrate eIF2 when the latter protein is phosphorylated at Ser51 in its α-subunit.12

This provides one important means of controlling eIF2B activity.

The importance of the integrity and/or activity of eIF2B complexes for normal cell function is

underlined by the fact that mutations in eIF2B lead to a neurodegenerative disorder, which is

often severe and is termed ‘vanishing white matter’ (VWM; OMIM 603896)) or ‘childhood ataxia

with central nervous system hypomyelination’ (CACH).13-15 Mutations in any one of the five sub-

units of eIF2B can cause this disease, although the majority of VWM patients have mutations in

eIF2Bε.13,16 A striking feature of this condition is its phenotypic variation, some mutations giving

rise to very severe congenital disease while others are associated with mild adult and late-child-

hood-onset forms. Furthermore, while neurological features are always present, additional or-

gans and tissues are affected in some patients, mainly infants with severe disease.

Few VWM mutations have so far been studied in terms of their effects on human eIF2B function,

all of which proved to be partial loss-of-function mutations17, affecting, e.g., the integrity of

eIF2B complexes and/or eIF2B activity. Since our earlier study, a large number of additional VWM

mutations in eIF2B subunit genes have been described (about 160). Of these, 95 mutations are

in EIF2B5; 24 in EIF2B4, 17 in EIF2B3, 17 in EIF2B2, and 8 in EIF2B1.16

Here, we characterize a range of these additional VWM mutations within several eIF2B subu-

nits, including for the first time some that cause the most severe known variants of VWM. Our

data extend earlier work indicating that VWM mutations affect eIF2B complexes in a variety of

114

Chapter 6

ways. In particular, we find no evidence for a relationship between the degree of impairment

of eIF2B function and the severity of the disease associated with the mutations tested here. An

important implication of this finding is that measuring eIF2B activity in patient derived samples

is of very limited diagnostic value. The data also suggest that some VWM mutations may affect

novel functions of eIF2B distinct from its guanine nucleotide exchange activity.

MATERIALS AND METHODS

Vectors and site-directed mutagenesis

The vectors for myc- or his/myc-tagged eIF2Bβ and e used here are based on those described

earlier.4,17 The vectors for his/myc-tagged eIF2Bγ and eIF2Bδ were created in a similar way.

All vectors were fully resequenced after mutagenesis. The amounts of the vectors encoding

different subunits of eIF2B were adjusted to achieve similar levels of expression of each

subunit. Throughout this report, the nucleotide numbering reflects cDNA numbering with +1

corresponding to the A of the ATG translation initiation codon in the reference sequence. The

initiation codon is numbered codon 1.

Cell culture, transfection and lysis

The approach involves co-transfecting human cells with, usually, five different vectors for the

α-ε subunits of eIF2B. We employ human embryonic kidney (HEK) 293 cells and the calcium

phosphate-based transfection method18 as modified by Hall-Jackson et al.19 as they provide

very high efficiency (typically >95% of cells are transfected as judged from routine parallel

transfections using a vector encoding the green fluorescent protein, GFP). The amounts of the

vectors encoding different subunits of eIF2B used for transfections were carefully adjusted to

achieve similar levels of expression of each subunit. Experiments showing lower transfection

efficiencies were rejected. All experiments were conducted at least three times and data from

all experiments compared to enable us to accurately assess the effects of each eIF2B mutation

tested. HEK293 cells were propagated, transfected and lysed as described earlier.4,19 The lysis

buffer used contains 25mM HEPES-KOH (pH 7.6); 25mM b-glycerol phosphate, 10% (v/v) glyc-

erol, 50mM KCl, 15mM imidazole, 14mM b-mercaptoethanol, and 0.5% Triton X-100, plus the

following proteinase and protein phosphatase inhibitors: 0.5mM NaVO3; 1mM, benzamidine

hydrochloride; 0.1mM phenylmethylsulfonylfluoride; 1 mg/ml each of pepstatin, antipain and

leupeptin.

Transformed lymphoblastoid cell lines were obtained by immortalizing peripheral blood lym-

phocytes with Epstein-Barr virus as described.20 Lymphoblasts and primary fibroblasts were cul-

tured and prepared as described earlier21,22 with minor modifications for the fibroblasts, which

were harvested after 5 days and assayed immediately. Beta-glycerophosphate was used as pro-

tein phosphatase inhibitor in the lysis buffer for lymphoblasts and fibroblasts.

115


Analysis of eIF2B complexes

In all cases, crude lysates from transfected HEK293 cells were analyzed by SDS-PAGE/western

blot (using anti-myc) in order to monitor the levels of expression of the eIF2B subunits.

(Meantime, lysates were stored at -80C in small aliquots, which were subsequently thawed

only once and then discarded). Based on these data, appropriate quantities of lysate were

used for the purification of the eIF2B complexes. This was performed by purifying the

hexahistidine/myc-tagged subunit under study together with any associated myc-tagged

subunits on Ni-NTA (nickel-chelate nitriloacetic acid) beads. The bound material was then

analysed by SDS-PAGE followed by western blot with anti-myc, as described earlier.17 Typically,

50-100 mg of cell lysate protein was applied to 10 ml of the resin (topped up to a total of 0.5

ml with lysis buffer containing 20 mM imidazole). After mixing for 1h at 4oC, the beads were

washed twice with lysis buffer containing 0.15% Triton X-100, and then once with the eIF2B

assay buffer (20mM Tris-HCl, pH 7.5, 50mM KCl, 1mM dithiothreitol). Typically five parallel

pull-downs were performed of equal volumes of each lysate; three were used for the eIF2B

activity assays; two were combined and analysed by SDS-PAGE/western blot with anti-myc or

antibodies for eIF2α or eIF2α phosphorylated at Ser51 (eIF2(αP)). Antibodies to human eIF2Bβ

(sc-9979), eIF2Bδ (sc-9981) and eIF2α (sc-11386) were obtained from Santa Cruz; anti-eIF2Bα

(ab40744) was purchased from Abcam. Anti-myc (M4439-100UL) was from Sigma-Aldrich.

Antibodies to phosphorylated eIF2α (#9721S) were from Cell Signaling Technology.

Measurement of eIF2B nucleotide exchange activity

Care was taken to ensure that all assays were performed within the linear range of the assay,

which corresponds to release of 25-30% of the bound [3H]GDP, in order to ensure that our as-

says accurately reflect the activity of the eIF2B tested.23 Any assays which fell outside this range

were repeated with a reduced quantity of eIF2B complexes. All assays were performed at 30°C,

in triplicate and on three independent preparations of each type of complex. Any discrepancies

were resolved by performing additional experiments in triplicate. Data are expressed as mean ±

SEM (n ³ 3), with the activity of complexes containing only wildtype (WT) subunits being set at 1.

Patient-derived cell lines

The eIF2B activity in lysates of patient-derived cells was performed as described earlier.21,22 Pro-

tein concentrations were equalized at 0.5 µg/µl before measuring activity. Briefly, to a tube con-

taining eIF2.[3H]GDP complex and a 100-fold excess of non-radiolabelled GDP, cell supernatant

(30mg total protein) was added. The reaction mixture was incubated at 30oC. Aliquots were

removed from the reaction mix at 0, 2 ,4 and 6 minutes. The linear decrease in binding of eIF2.

[3H]GDP to nitrocellulose filter disks was quantified by measuring radiation by liquid scintilla-

tion spectrometry. Experiments were carried out twice and activities per lysate were measured

in duplicate.

116

Chapter 6

RESULTS

Validation of the assays for eIF2B activity

To study the effects of VWM mutations on the properties of the eIF2B complex, we employed an

approach that we have reported previously.17 This involves expressing the subunit under study

(either WT or mutant) equipped with a hexahistidine tag and a myc tag, along with myc-tagged

WT versions of all four other subunits, in HEK293 cells. Subsequently, the cells are lysed and

lysates are applied to nickel beads on which the histidine-tagged subunit and any associated

proteins (other eIF2B subunits, eIF2, etc) will be retained. After purification, samples are im-

mediately analysed for guanine nucleotide-exchange activity using a standard assay employing

eIF2.[3H]GDP complexes as substrate. Data are normalized after SDS-PAGE/western blot analysis

using anti-myc of an aliquot of each sample to ascertain the levels of eIF2B subunits, especially

the catalytic ε-subunit, and to examine the association of other eIF2B subunits with the his6/

myc-tagged eIF2B polypeptide. This analysis will reveal changes in the ability of the mutated

subunit to form complexes with other eIF2B polypeptides, but cannot report more subtle chang-

es in conformation which may affect GEF activity. Careful comparison of western blot data from

lysates from multiple experiments confirmed that none of the missense mutations tested here

affected the expression levels of the mutated polypeptides, indicating that these mutations do

not affect protein stability (data not shown). This contrasts with the decreased expression levels

observed for the nonsense (premature stop) mutants we tested previously17 and which clearly do

result in decreased stability of the (truncated) polypeptides.

Figure 1 | validation of the preparation and analysis of recombinant eIF2B complexes. (A)

GEF assays were performed using purified wildtype eIF2B complexes using two different concentrations of

eIF2.[3H]GDP complexes. (B) HEK293 cells were transfected with vectors encoding the indicated eIF2B sub-

units, all with myc tags; eIF2B ε also had a his-tag. After purification on Ni-NTA beads, complexes were

analysed by SDS-PAGE and western blot using either anti-myc (top section, detects all five subunits [where

present]; ‘ns’ indicates non-specific cross-reaction) or antibodies that detect eIF2Bα β or δ, as indicated.

117


To confirm that our measurements of eIF2B GEF activity accurately reflect effects of mutations

on the properties of eIF2B, we conducted some further validation. Thus, in addition to ensuring

that data, lay within the linear range for this assay, we also assessed the dependency of the rate

of the GEF reaction on the amount of substrate, eIF2.[3H]GDP. We tested the rate of the reac-

tion at the usual level of eIF2.[3H]GDP complexes (9nM) and at three times that concentration

(27nM). The rate of the reaction (release of [3H]GDP) tripled when three times as much substrate

was used (figure 1A). This demonstrates that the substrate concentrations used are well below

the Km and thus that any effects of mutations on either the Km or Vmax will be manifested as

changes in the observed activity. As described earlier, we employ a substantial excess of eIF2

as substrate relative to the eIF2 introduced into the assay with the eIF2B.23 This will overcome

effects due to the competitive inhibition of eIF2B by any phosphorylated eIF2 introduced into

the assays due to its association with the eIF2B complexes.24

Secondly, we assessed the extent to which the host cells’ own endogenous eIF2B subunits as-

sociated with the recombinant complexes studied here. To do this, cells were transfected with

vectors for all five subunits, as usual, or with vectors for eIF2Bβ-ε or eIF2Bγ and eIF2Bε only.

We have previously shown that these combinations of subunits form stable complexes that can

be isolated.17 In principle, endogenous subunits might associate with the ectopically-expressed

eIF2B polypeptides. This would be assessed most easily by looking for the presence of endog-

enous eIF2Bα in the complexes obtained when cells are transfected with vectors for eIF2Bβ-ε

and for endogenous eIF2Bα, β and δ, when only the eIF2Bγ and e vectors are used. The purified

complexes were then purified and analysed by western blot using both anti-myc, to detect the

recombinant subunits, and antisera for eIF2Bα, β or δ, to detect copurifying endogenous poly-

peptides.

As shown in Figure 1B, no endogenous subunits were detected in the complexes from cells trans-

fected with eIF2Bβ-ε or eIF2Bγ/ε, although the signals for the recombinant subunits detected us-

ing the same antibodies were strong. This implies that any association of endogenous subunits

with the recombinant ones is, at most, very low. Thus, the data reported here are not influenced

by the association of endogenous subunits with the recombinant complexes. In most cases (with

the sole exception of Figure 5), this would not affect the data in any case, since, apart from the

myc, tag the human subunits expressed from the vectors are identical to the endogenous human

eIF2B polypeptides.

118

Chapter 6

Figure 2 | Analysis of the effects of VWM in eIF2Bε. HEK293 cells were transfected with vectors encoding his/

myc-tagged WT eIF2Bε or the indicated mutants plus myc-tagged versions of the other four eIF2B subunits.

After analysis of the resulting lysates by SDS-PAGE western blot using anti-myc, samples containing similar

amounts of his/myc-eIF2Bε were subjected to purification on Ni-NTA beads to isolate recombinant eIF2Bε

and associated subunits. Samples of the purified material were either analysed by SDS-PAGE and western

blot using anti-myc (A) or assayed for eIF2B (nucleotide exchange) activity (B). In (A), the positions of the

myc-tagged eIF2B subunits are shown. Membranes were also probed for eIF2α or eIF2α phosphorylated on

Ser51 (eIF2(αP)), as indicated. * indicates the his-tagged subunit (i.e., here it is eIF2Bε). *, p < 0.05 vs. wildtype

(Student’s t-test).

119


Effects of VWM mutations in the NT-homology region of eIF2BεOne of the most N-terminal VWM mutations in eIF2Bε is A16D. This residue is widely conserved

(table 1). The A16D mutation had no effect on either the formation of eIF2B complexes (figure

2A) or eIF2B GEF activity (figure 2B). The A16D mutation is only known to occur in a compound

heterozygous state with the mild R113H mutation, and is associated with ‘classical’ VWM.22,25,26

We realize one should be careful making statements on disease severity in patients with com-

pound heterozygous mutations. The clinical phenotype appears to be determined by the com-

bined effect of both mutations and not by either the mildest or the most severe mutation.27

The N-terminal part of eIF2Bε contains a region with homology to nucleotidyl-transferases (NTs;

residues 44-1658). Within this region are several VWM mutations, some of which occur at very

highly conserved residues (table 1). All of these mutants formed holocomplexes similarly to the

WT subunit, with no defect in association of any subunit or of the eIF2B complex with its sub-

strate, eIF2 (figure 2A and data not shown; summarized in table 2). It should be noted that (i) no

eIF2 is observed in Ni-NTA pulldowns from cells transfected with empty vector or with the five

vectors for myc- tagged eIF2B subunits (i.e., without a hexahistidine tag; data not shown) and

(ii) the amount of eIF2 phosphorylated at Ser51 (eIF2(αP)) also did not vary.

The F56V21 and L68S28 mutants, which have been identified in compound heterozygous patients

and are associated with classical or severe infantile VWM, showed slightly reduced exchange ac-

tivity while complexes containing eIF2Bε[V73G]14 actually displayed enhanced activity (increased

by about 60%; figure 2B).

R136C is a further example of a VWM mutation in the NT domain involving a highly conserved

residue (table 1). We have identified two patients that are homozygous for this mutation and

exhibit a classical disease phenotype with an onset at about two years of age. This mutation did

not affect complex formation (figure 2A, right hand section) but did cause a 40% drop in eIF2B

activity (figure 2B), indicating that mutations in the NT domain can influence activity. Mutation

of the corresponding arginine residue in mouse eIF2Bε to a histidine, which is also a clinically

reported mutation16, results in a loss of eIF2B activity of approximately 25%.29

Interestingly, the R269G mutation (which lies between the NT and I-patch domains;28 showed a

slight increase in eIF2B activity (figure 2B), although this did not reach statistical significance (p

= 0.08). This mutation did not affect complex formation (figure 2A).

120

Chapter 6

Table 1 | Genes and mutants studied in this report

HGNC Gene name (eIF2B subunit)

OMIM and MIM

numbers

Accession number

DNA mutation

Amino acid

change

Conservation

Pt Mm Rn Oc Bt Dr Sc

EIF2B2

(eIF2B β)

eif2b2606454

NM_014239.2 c.512C>T S171F S S S S S S S

c.599G>T G200V G G G G G G A

c.638A>G E213G E E E E E E E

c.818A>G K273R K K K K K K K

c.871C>T P291S P P P P P P S

c.986G>T G329V G G G G G G G

EIF2B3

(eIF2Bγ)

eif2b3606273

NM_020365.2 c.407A>C Q136P Q Q Q Q Q Q K

c.674G>A R225Q R R R R R R A

c.1023T>G H341Q H H H H H H I

EIF2B4

(eIF2B δ)

eif2b4606687

NM_001034116.1 c.1069C>T R357W R R R R R R K

c.1172C>A A391D A A A A A L L

c.1447C>T R483W R R R R R G N

EIF2B5

(eIF2Bε)

eif2b5603845

NM_003907.2 c.47C>A A16D A A A A V A A

c.166T>G F56V F F F F F F F

c.203T>C L68S L L L L L L L

c.217G>A V73G V V V V V V V

c.236C>T T79I T T T T T T T

c.271A>G T91A T T T T T T V

c.338G>A R113H R H H R R R -

c.406C>T R136C R R R R R R R

c.805C>G R269G R R R R R R R

c.806G>A R269Q R R R R R R R

c.896G>A R299H R R R R R R R

c.1208C>T A403V A A A A A A -

c.1459G>A E487K E E E E E E I

c.1484A>G Y495C Y Y Y Y Y Y Y

c.1745+5G>A Y583X -- -- -- -- -- -- --

c. 1882T>C W628R W W W W W W W

c.1946T>C I649T I I I I I M I

c.1948G>A E650K E E E E E E K

Conservation: the species listed are: Pt, Pan troglodytes; Mm, Mus musculus; Rn, Rattus norvegicus; Oc, Oryc-

tolagus cuniculus; Bt, Bos taurus, Dr, Danio rerio; Sc, Saccharomyces cerevisiae.

‘-‘ indicates an amino acid change in a region with little conservation. ‘- -‘ nonsense mutation.

121


As shown in Figure 2A, and consistent with our earlier data17, some eIF2 co-purifies with the

eIF2B complexes. In the case of the mutants described above, the amount of eIF2 (assessed by

blotting for its α-subunit) and the amount of eIF2α which is phosphorylated on Ser51 (eIF2(αP))

was similar in all cases. This associated eIF2 will not therefore affect the measured GEF activity

in comparison with WT complexes.

Analysis of the effects of VWM mutations in the C-terminal regions of eIF2BεThe Y495C mutation in eIF2Bε is associated with very severe disease (infantile form30) in patients

that are homozygous for this variant. This residue is highly conserved (table 1) suggesting it may

have an important function. It is therefore quite surprising that this mutation did not affect

complex integrity, activity or binding to eIF2 (figure 2A,B; see table 2 for summary).Although

almost 100 VWM mutations have now been identified in eIF2Bε, only six missense mutations

lie within its catalytic domain, P604S31, M608I16, W628R14, I649T32, E650K14 and W684S16. If the

mutations were randomly distributed, then about sixteen would be expected to occur in this

domain. We previously studied the W628R mutation.17 Neither I649T nor E650K had a discern-

ible effect on complex formation but each caused a marked decrease in activity (figure 2A,B).

The amount of eIF2 and eIF2(αP) associated with the Y495C mutant was similar to that seen for

complexes containing WT eIF2Bε (figure 2A) but was decreased in the cases of the two catalytic

domain mutants I649T and E650K (figure 2A). The reduced binding of eIF2 to these two mutants

is similar to the observations we made earlier for the only other catalytic domain mutant so far

tested in this way, W628R.17 Their decreased ability to bind eIF2 may, at least in part, explain

their reduced GEF activity against eIF2.GDP.

122

Chapter 6

Table 2 | Summary of data from studies on recombinant eIF2B complexes.

Subunit (gene /OMIM accession Number)

Mutation Disease severity Effect on

GEF

activity

Effect on

holocomplex

formation

Comments

eIF2Bβ;(EIF2B2/606454)

S171F (heterozygous) ± No effect

G200V (heterozygous) Nd Complete loss

K273R (heterozygous) ↓↓ No effect

P291S (heterozygous) Nd ↓↓↓

G329V (heterozygous) ↓↓↓ eIF2Ba lost

eIF2Bγ(EIF2B3/606273)

Q136P intermediate

/mild***

↓ No effect

R225Q classical* ± No effect Decreased eIF2 binding

H341Q classical*** ↓ No effect

eIF2Bδ(EIF2B4/606687)

R357W classical *** ↓↓↓ ↓↓↓

A391D severe** ± No effect

R483W severe** ↓↓↓ ↓↓↓

eIF2Bε(EIF2B5/603945)

A16D (heterozygous) ± No effect

F56V (heterozygous) ↓ No effect

L68S (heterozygous) ± No effect

V73G (heterozygous) ↑↑ No effect

R136C classical* ↓↓ No effect

R269G severe**** ↑ No effect

Y495C severe** ± No effect

I649T (heterozygous) ↓↓↓ No effect Decreased eIF2 binding

E650K (heterozygous) ↓↓↓ No effect Decreased eIF2 binding

* unpublished, own data ** (van der Knaap et al., 2003); *** (Fogli et al., 2004a); **** (Ohlenbusch et al., 2005).

# GEF activity (relative to control) ↑↑ >150%; ↑ >130%; ↓ 70-90%; ↓↓ 50-70%; ↓↓↓ <50%; ± indicates no

significant difference from controls.

Analysis of the effects of selected additional VWM mutations in eIF2BγTogether with eIF2Bγ, eIF2Bε forms the so-called ‘catalytic subcomplex’.9 Several mutations in

human eIF2Bγ are associated with VWM but their functional consequences have not previously

been studied. Therefore, we characterized the effects of three mutations in EIF2B3, the gene en-

coding eIF2Bγ. These mutations, Q136P, R225Q and H341Q, are associated with mild or ‘classical’

infantile forms of VWM.14,33 Each had little, if any, effect on the integrity of eIF2B complexes (fig-

ure 3A) and only slightly decreased the nucleotide exchange activity of the complex (figure 3B).

123


Figure 3 | Analysis of the effects of VWM mutations in eIF2Bγ (A,B) and eIF2Bδ (C,D). Please see legend to Fig.

1 for details. (A,C) Analysis of purified recombinant eIF2B complexes by SDS-PAGE and western blot. The posi-

tions of the myc-tagged eIF2B subunits are shown. Membranes were also probed for eIF2α or eIF2α phospho-

rylated on Ser51 (eIF2(αP)), as indicated; (B,D) activity data for complexes containing the indicated mutants

of eIF2B subunits. * indicates the his-tagged subunit in each case. *, p < 0.05 vs. wildtype (Student’s t-test).

The amounts of copurifying eIF2 and eIF2(αP) were similar for WT, Q136P and H341Q, but a de-

crease in both was seen for the R225Q mutant (Fig. 3A). Given that bound eIF2 impairs the GEF

activity of eIF2B (figure 1A), our assays may overestimate the activity of complexes containing

eIF2Bγ [R225Q]. Thus, this mutation may cause an appreciably larger decrease in GEF activity

than is evident from the data in Fig. 3B.

Characterisation of effects of mutations in eIF2Bb and eIF2Bd

Several VWM mutations occur at highly conserved residues in EIF2B4, the gene encoding eIF2Bδ (ta-

ble 1). Interestingly, some of them are associated with the most severe forms of VWM. To date, none

of these mutations in human eIF2Bδ had yet been functionally characterized. We analysed three

mutants: A391D and R483W, which both result in congenital disease30; and R357W, which causes

severe infantile VWM.25 The R357W mutant showed a greatly decreased ability to form complexes

with other eIF2B subunits and a corresponding decrease in apparent activity (likely because eIF2Bε,

the catalytic subunit, is largely absent from the complexes containing this mutation; figure 3C,D).

124

Chapter 6

The R483W mutant of eIF2Bδ also showed a marked deficit in complex formation and a similar ef-

fect on eIF2B activity (figure 3C,D; see table 2 for a summary). The A391D mutation had only a mod-

est effect at most on complex formation and, surprisingly, had no significant effect on GEF activity.

The eIF2Bδ(A391D) mutation did not affect the amount of bound eIF2 or eIF2(αP), but reduced

binding was seen for the R357W and R483W mutants (figure 3C). This decrease likely reflects the

fact that these two mutations compromise formation of eIF2B holocomplexes, although we can-

not rule out that these mutations affect eIF2 binding in additional ways.

We also tested five further mutations in eIF2Bβ: S171F, G200V, K273R, P291S and G329V, all at

highly conserved positions (table 1; figure 4) 14,30,33-35, in addition to those we previously stud-

ied.17 All of the mutations tested here are found in a compound heterozygous manner in pa-

tients.13,16 Three of these mutations affected the integrity of eIF2B complexes: the P291S mutant

showed severely reduced ability to bind the other subunits, and the G200V mutant was incapa-

ble of binding any other subunits (figure 4A). The G329V mutant was able to form a complex

containing eIF2Bγ, δ and ε, but the complexes lacked eIF2Bα (figure 4A). The S171F, K273R and

G329V mutations did not affect the ability to bind eIF2 (figure 4A). The degree of association

with phosphorylated eIF2 mirrored the binding of eIF2.

Figure 4 | Analysis of the effects of VWM mutations in eIF2Bβ. Please see legend to Fig. 1 for details. (A)

Analysis of purified recombinant eIF2B complexes by SDS-PAGE and western blot. The positions of the myc-

tagged eIF2B subunits are shown. Membranes were also probed for eIF2α or eIF2α phosphorylated on Ser51

(eIF2(αP)), as indicated; (B) activity data for complexes containing the indicated mutants of eIF2B subunits. *

indicates the his-tagged subunit. *, p < 0.05 vs. wildtype (Student’s t-test).

Since the G200V mutant of eIF2Bβ cannot bind the catalytic subunit, and the P291S mutant

showed greatly decreased binding, it was not appropriate to perform activity analyses for them.

Therefore, we assessed the activities of complexes containing the eIF2Bβ mutants which can in-

teract with other eIF2B subunits (S171F, K273R and G329V). All three mutants showed decreased

125


activity, with the greatest decrease being seen for G329V, the mutant that cannot bind eIF2Bα

(figure 4B). We have previously observed36,37 that complexes lacking eIF2Bα show decreased

activity (see also below), although there are also reports to the contrary.38

Although mutants such as eIF2Bβ[G200V] and [P291S] and eIF2Bδ[R357W] and [R483W] can-

not form eIF2B holocomplexes, binary eIF2Bγ/ε ‘catalytic’ subcomplexes may still form in cells

expressing these mutants and may provide GEF activity for eIF2. To test this possibility, we ex-

pressed eIF2Bε together with all four other subunits, with all except eIF2Bα or with eIF2Bγ

only. Complexes were isolated and activity assays performed as described. These data (figure

5A) demonstrate that, mammalian eIF2Bγ and e can form stable complexes in the absence of

the other subunits (Fig. 5A), in broad agreement with our previous data.17 However, this binary

complex displays only about 20% of the activity of the holocomplex (figure 5B). Similarly, while

complexes lacking eIF2Bα are able to form, they show a 50% reduction in activity compared to

the complete eIF2B complex (figure 5). Given that the recombinant complexes do not contain

significant amounts of endogenous eIF2B subunits (figure 1B), the activities measured here do

accurately affect the properties of two- or four-subunit eIF2B complexes.

Figure 5 | eIF2B complexes. (A) HEK293 cells were transfected with vectors encoding the indicated eIF2B sub-

units, all with myc tags; eIF2Bε also had a his-tag. Samples of cell lysate (input, left) or the material pulled

down on Ni-NTA beads (right) were analysed by SDS-PAGE and western blot with anti-myc. Positions of eIF2B

subunits are indicated. (B) activity data for the complexes shown in (A) normalized to that of the eIF2B holo-

complex. Data are mean ± SEM, n = 3. *, p < 0.01 vs. complete complex (Student’s t-test).

126

Chapter 6

Analysis of eIF2B activity in lysates from patient-derived cells

We considered it important to compare the activity data for mutated eIF2B complexes

expressed in HEK293 cells with the eIF2B activity in lysates of cells derived from patients with

the same mutations. Lysates of fibroblast cell lines derived from patients (table 3) with selected

homozygous mutations were therefore analyzed for eIF2B activity (figure 6A). Although

the suitability of patient-derived fibroblasts for this purpose has been questioned22, in our

hands the assays gave consistent and reproducible results. The data show that the cells from

a patient with the eIF2Bδ[R483W] mutation showed a 60% loss of activity while samples from

patients with the eIF2Bδ mutation A391D or the eIF2Bε mutations R269Q or Y495C showed

little or no alteration in eIF2B activity.

Figure 6 | Analysis of eIF2B (nucleotide exchange) activity in lysates from patient-derived cells. Lysates from

primary fibroblast cell lines (A) or from immortalized lymphoblast cell lines derived from VWM patients (B)

were assayed for eIF2B GEF activity. Data are expressed pmol [3H]GDP released/minute as mean ± SEM. *, p <

0.01 vs. control 1 (Student’s t-test).

127


Table 3 | Patient derived cell lines: patient characteristics and eIF2B activity data

Patient Cell type Gene Mutation 1

Mutation 2

Onset (yrs)*

Death or present age

(yrs)

Disease severity

Effect on GEF activity#

patient 1 Fibroblast EIF2B4 A391D A391D 0 0.8 severe ↓

patient 2 Fibroblast EIF2B4 R483W R483W 0 0.3 severe ↓↓↓

patient 3 Fibroblast EIF2B5 R269Q R269Q 1.5 10.6; still alive classical/

intermedi-ate

±

patient 4 Fibroblast EIF2B5 Y495C Y495C 0.7 0.8 severe ↓

patient 5 lymphoblast EIF2B2 E213G E213G 5 37.3; still alive mild ↓↓↓

patient 6 lymphoblast EIF2B5 R113H R113H 54 59.5; still alive mild ↓↓↓

patient 7 lymphoblast EIF2B5 T91A Y583X 37 49.5; still alive mild ↓↓↓

patient 8 lymphoblast EIF2B5 E487K E487K 42 49; still alive mild ↓↓

patient 9 lymphoblast EIF2B5 T79I R113H 2.4 4.9 severe ↓↓

patient 10 lymphoblast EIF2B5 T91A A403V 1.5 3.8 severe ↓↓

patient 11 lymphoblast EIF2B5 T91A W628R 1.5 12.1 classical ↓↓↓

patient 12 lymphoblast EIF2B5 R113H R299H 1.5 8.2 classical ↓↓

* age at first reported neurological symptom

# for explanation of symbols, see Table 1

These data are in close agreement with those from our studies using recombinant eIF2B mu-

tants. In particular, even mutations that cause severe disease (Y495C, A391D) do not lead to a

change in eIF2B activity in lysates from patient-derived cells, in accordance with their lack of

effect on the activity of recombinant eIF2B complexes.

We extended our study to measure eIF2B activity in lysates of lymphoblastoid cells derived from

patients with different disease severities (table 3; figure 6B). These samples showed decreased

eIF2B activity relative to controls. However, there was no correlation between the extent of the

defect in eIF2B activity and the reported severity of the disease associated with each combina-

tion of mutations. Indeed, two of the mild mutations (the homozygous eIF2Bβ[E213G/E213G]

and eIF2Bε[R113H/R113H]) showed the largest reduction in activity, whereas combinations re-

sulting in more severe disease (T79I/R113H and T91A/A403V in eIF2Bε) showed much smaller

defects in activity (Fig. 6B).

145

128

Chapter 6

DISCUSSION

In this study, we have extended our earlier investigations on the functional effects of disease-

causing mutations in eIF2B to examine numerous mutations in four of the five subunits of this

protein complex. We also examined the effects of mutations on the activity of eIF2B in samples

from patient-derived material. This has allowed us to compare the functional consequences of

such mutations using two independent approaches, i.e., using recombinant eIF2B complexes

and cells from VWM patients. The major findings of this study are that:

(i) in contrast to our earlier conclusions from a more limited set of mutations17, and other studies21,39,

several VWM mutations have little or no effect on the nucleotide exchange activity of eIF2B;

(ii) some mutations that cause severe disease affect neither complex formation nor activity;

(iii) data obtained using recombinant eIF2B complexes in human cells correspond closely with activi-

ty data from cells of patients with the corresponding mutation, thus validating the former approach

(which we reported earlier17); and

(iv) taking into account the above points, measurement of eIF2B activity in samples from patients is

of limited diagnostic value: while decreased eIF2B GEF activity in patient-derived samples is indica-

tive of VWM, normal GEF activity clearly cannot be taken to indicate the absence of VWM.

Furthermore, our present data suggest that some mutations linked to VWM disease may actual-

ly enhance, rather than impair, the nucleotide exchange activity of eIF2B.

The biochemical phenotypes of the mutations studied here fall broadly into four categories,

which we shall discuss separately.

(I) Mutations that cause an obvious defect in complex formation or substrate binding also have

decreased activity (summarized in Table 2). These mutations include two in the catalytic domain

of eIF2Bε (I649T and E650K), which do not affect complex formation but do display a markedly

decreased ability to bind the substrate, eIF2, and, likely as a consequence of this (given that our

assays use substrate [eIF2] concentrations below the Km), lower activity. This is very similar to the

behaviour of the only other catalytic domain mutation we have tested, W628R.17

Also in this category are the R357W and R483W mutations in eIF2Bδ, which show impaired

complex formation and decreased activity. This decreased activity presumably reflects the defect

in complex formation since such complexes largely lack the catalytic ε-subunit (and its partner,

eIF2Bγ). This would leave those subunits free to form binary complexes, which we have shown can

form in human cells but possess markedly decreased activity. Thus, in cells from patients with these

mutations, eIF2B may occur mainly as the partially active eIF2Bγε complex which has reduced ac-

tivity. Since such complexes lack the regulatory subunit, they will be insensitive to inhibition by

phosphorylated eIF2. Thus, the lesion in such cells likely reflects decreased and uncontrolled eIF2B

function. Incidentally, the lower activity of mammalian eIF2Bγε complexes differs from the situation

for the corresponding yeast binary complex, which is more active than the eIF2B holocomplex.9

The G329V mutation in eIF2Bβ leads to a defect in binding to eIF2Bα, although the other four

subunits do form a complex. Since mammalian eIF2Bα is required for sensitivity to phosphoryl-

129


ated eIF2α40, such complexes would be resistant to the inhibition of eIF2B function caused by ac-

tivation of, e.g., PERK during the unfolded protein response. This could be especially important

in cells that make and secrete large amounts of proteins such as oligodendrocytes, effects on

which are likely of major importance in VWM.16 The resulting complexes show roughly 50% of

WT activity, in line with the activity of eIF2Bβ-ε complexes containing WT eIF2Bβ. The observa-

tion that loss of eIF2Bα impairs eIF2B function agrees with our earlier observations for purified

mammalian eIF2B2,36, but conflicts with data for mammalian complexes expressed in insect cells,

where eIF2Bα appeared to be dispensable for function.38 Two other mutations in eIF2Bβ (G200V

and P291S) caused complete loss of complex formation. Again, in cells expressing these mutants,

partially active, eIF2α(P)-insensitive binary complexes are likely to provide GEF activity for eIF2.

(II) Mutations without a clear effect on complex formation or eIF2 binding but with reduced

activity (see table 2). Several mutants showed no defect in complex formation but gave rise to

decreased activity, although the decreases were often modest. This group includes the eIF2Bε

mutations F56V and R136C, which can still bind to eIF2. The K273R mutation in eIF2Bβ and the

Q136P and H341Q mutations in eIF2Bg show similar effects. The reasons for the reduced activi-

ties of these mutated complexes remain to be established.

(III) A particularly interesting finding is that certain mutations elicit no biochemical defect, al-

though some of them even cause severe disease (table 2). These include Y495C in eIF2Bε and

A391D in eIF2Bδ. In both cases, the data for the activity and integrity of the recombinant com-

plexes accord closely with the activity data from patient-derived cells (summarized in table 3),

leading to the clear conclusion that severe disease can be caused by mutations that affect nei-

ther the integrity of eIF2B complexes nor exchange activity. Interestingly, A391 in eIF2Bδ is a

highly conserved residue not just in this subunit, but is also conserved in the α and β subunits as

well in an equivalent position. Furthermore, it is conserved in a number of proteins from differ-

ent species containing a similar Rossmann fold, which is commonly found in nucleotide-binding

proteins, but of different functions, including ribose-1,5-bisphosphatase from T. kodakaraensis

(pdb 3A9C), which is highly homologous to eIF2Bδ. Thus, this site may have further, as yet un-

identified functions, possibly associated with nucleotide binding. Equally interesting are the

effects of the two clinically observed mutations at R269 of eIF2Bε (R269G and R269Q), which

are associated with severe or classical disease. The R269G mutation slightly increased the activity

of the recombinant complexes. The R269Q mutation was present in one of the patient derived

samples, and showed no significant effect on eIF2B activity. The ability of such mutations to

cause the disease might be due to effects on other functions of eIF2B. However, despite indica-

tions that eIF2B may play an additional role(s) in translation initiation (discussed in (Hinnebusch,

2000)41), it is not clear what these may be and we cannot therefore assess them.

The A16D and L68S mutations in eIF2Bε; and the S171F mutation in eIF2Bβ also had little or no

effect on eIF2B activity, but, as they are only known to occur in compound heterozygous pa-

tients, we cannot comment on their association with disease severity (see table 2).

130

Chapter 6

(IV) Mutations that increase activity. Two mutations in eIF2Bε appear to increase activity (V73G

(considerable increase) and R269G (slight increase)) and are associated, respectively, with mild

or severe disease (table 2).13,25 It is not clear how enhanced activity is connected to VWM dis-

ease. These findings reinforce the conclusions that VWM disease is not always associated with

decreased activity of eIF2B and that measuring this parameter in patient-derived samples is not

a useful or reliable diagnostic tool.

The data from the patient-derived samples are valuable for two main reasons: firstly, the altera-

tions in eIF2B activity observed in lysates from cells with homozygous mutations are very similar

to the values obtained from studies on isolated recombinant complexes containing the same

mutations. This applies both to the set of mutations studied here and to those we reportedly

earlier (e.g., R113H in eIF2Bε and E213G in eIF2Bβ).17 The data therefore validate the approach

that we have used here and previously17 of characterizing the properties of wildtype or mutant

subunits/complexes expressed in human cells. Secondly, data for samples from patients with

compound heterozygous mutations corroborate our conclusion that there is no simple rela-

tionship between the extent of the defect in eIF2B activity and the severity of the associated

disease (see figure 6B and table 3). These findings indicate that measurements of eIF2B GEF

activity in samples from patients are of limited diagnostic value. In particular, reliance on such

assays would lead to instances of VWM being overlooked. Nevertheless, a decrease in eIF2B GEF

activity measured in samples from patient-derived cells can be taken as an indication of VWM,

although further (DNA sequence) data are clearly essential to confirm this.

Further work is needed to improve our understanding of how diverse defects in both defined

and perhaps as yet undefined functions of eIF2B caused by VWM-associated mutations lead to

neurological disease: such diversity is probably not surprising given that VWM mutations occur

in many regions of any subunit of eIF2B. The creation of transgenic knock-in mice, recently

reported for a mutant in eIF2Bε29, will undoubtedly help to achieve this. Finally, our data show

that VWM mutations have very diverse effects on eIF2B activity – e.g., decreased, increased or

unaltered GEF activity. Importantly, this implies that the search for therapeutic agents to help

manage or treat VWM should focus on understanding the downstream effectors of eIF2B that

are affected in VWM, rather than on restoring eIF2B activity.

131


REFERENCES


Soc Trans 2005;33:1487-1492.

2. Williams DD, Loughlin AJ, Proud CG. Characterisation of the initiation factor eIF2B

complex from mammalian cells as a GDP-dissociation stimulator protein. J Biol Chem

2001;276:24697-24703.

3. Hardt SE, Tomita H, Katus HA, Sadoshima J. Phosphorylation of eukaryotic translation

initiation factor 2Bepsilon by glycogen synthase kinase-3beta regulates beta-adrenergic

cardiac myocyte hypertrophy. Circ Res 2004;94:926-935.

4. Wang X, Proud CG. A Novel Mechanism for the Control of Translation Initiation by Ami-

no Acids, Mediated by Phosphorylation of Eukaryotic Initiation Factor 2B. Mol Cell Biol

2008;28:1429-1442.

5. Gomez E, Mohammad SS, Pavitt GD. Characterization of the minimal catalytic domain

within eIF2B: the guanine-nucleotide exchange factor for translation initiation. EMBO J

2002;21:5292-5301.

6. Gomez E, Pavitt GD. Identification of domains and residues within translation initiation

factor eIF2Bepsilon required for guanine nucleotide-exchange reveals a novel activation

function promoted by eIF2B complex formation. Mol Cell Biol 2000;20:3965-3976.

7. Asano K, Krishnamoorthy T, Phan L, et al. Conserved bipartite motifs in yeast eIF5 and

eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation,

mediate binding to their common substrate eIF2. EMBO J 1999;18:1673-1688.

8. Koonin EV. Multidomain organization of eukaryotic guanine nucleotide exchange factor

eIF-2B revealed by analysis of conserved sequence motifs. Protein Science 1995;4:1608-

1617.

9. Pavitt GD, Ramaiah KVA, Kimball SR, Hinnebusch AG. eIF2 independently binds two

distinct eIF2B subcomplexes that catalyse and regulate guanine-nucleotide exchange.

Genes Dev 1998;12:514-526.

10. Bushman JL, Asuru AI, Matts RL, Hinnebusch AG. Evidence that GCD6 and GCD7, trans-

lational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for

eIF-2 in Saccharomyces cerevisiae. Mol Cell Biol 1993;13:1920-1932.

11. Paddon CJ, Hannig EM, Hinnebusch AG. Amino acid sequence similarity between GCN3

and GCD2, positive and negative translational regulators of GCN4: evidence for antago-

nism by competition. Genetics 1989;122:551-559.

12. Pavitt GD, Yang W, Hinnebusch AG. Homologous segments in three subunits of the gua-

nine nucleotide exchange factor eIF2B mediate translational regulation by phosphoryla-

tion of eIF2. Mol Cell Biol 1997;17:1298-1313.


Trans 2006;34:22-29.

14. Leegwater PAJ, Vermeulen G, Koenst AAM, et al. Subunits of the translation initiation

factor eIF2B are mutated in leukoencephaly with vanishing white matter. Nat Genetics

132

Chapter 6

2001;29:383-388.

15. van der Knaap M, Leegwater PAJ, Könst AAM, et al. Mutations of each of the five subu-



16. Van der Knaap MS, Bugiani M, Boor I, et al. Vanishing White Matter. In: 2010 Scriver C,

et al., editors. OMMBID. New York: McGraw-Hill.

17. Li W, Wang X, van der Knaap M, Proud CG. Mutations linked to leukoencephalopathy

with vanishing white matter impair the function of the eukaryotic initiation factor 2B

complex in diverse ways. Mol Cell Biol 2004;24:3295-3306.

18. Chen CA, Okayama H. Calcium phosphate-mediated gene transfer: a highly efficient

transfection system for stably transforming cells with plasmid DNA. Biotechniques

1988;6:632-638.

19. Hall-Jackson CA, Cross DA, Morrice N, Smythe C. ATR is a caffeine-sensitive, DNA-ac-

tivated protein kinase with a substrate specificity distinct from DNA-PK. Oncogene

1999;18:6707-6713.

20. van Kollenburg B., Thomas AA, Vermeulen G, et al. Regulation of protein synthesis in

lymphoblasts from vanishing white matter patients. Neurobiol Dis 2006;21:496-504.

21. Fogli A, Schiffmann R, Hugendubler L, et al. Decreased guanine nucleotide exchange

factor activity in eIF2B-mutated patients. Eur J Hum Genet 2004;12:561-566.

22. Kantor L, Harding HP, Ron D, et al. Heightened stress response in primary fibroblasts

expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients. Hum Genet

2005;118:99-106.

23. Welsh GI, Proud CG. Regulation of protein synthesis in Swiss 3T3 fibroblasts. Rapid ac-

tivation of the guanine-nucleotide-exchange factor by insulin and growth factors. Bio-

chem J 1992;284:19-23.

24. Rowlands AG, Panniers R, Henshaw EC. The catalytic mechanism of guanine nucleotide

exchange factor action and competitive inhibition by phosphorylated eukaryotic initia-

tion factor 2. J Biol Chem 1988;263:5526-5533.



26. van der Knaap M, Leegwater PA, Van Berkel CG, et al. Arg113His mutation in eIF2Bepsi-

lon as cause of leukoencephalopathy in adults. Neurology 2004;62:1598-1600.

27. van der Lei HDW, Van Berkel CG, van Wieringen WN, et al. Genotype-Phenotype correla-

tion in vanishing white matter disease. Neurology 2010;75:1555-1559.



29. Geva M, Cabilly Y, Assaf Y, et al. A mouse model for eukaryotic translation initiation

factor 2B-leucodystrophy reveals abnormal development of brain white matter. Brain

2010;133:2448-2461.

30. van der Knaap M, Van Berkel CG, Herms J, et al. eIF2B-Related Disorders: Antenatal On-

set and Involvement of Multiple Organs. Am J Hum Genet 2003;73:1199-1207.

133


31. Kaczorowska M, Kuczynski D, Jurkiewicz E, et al. Acute fright induces onset of symptoms

in vanishing white matter disease-case report. Eur J Paediatr Neurol 2006;10:192-193.

32. Pronk JC, van Kollenburg B, Scheper GC, Van der Knaap MS. Vanishing white matter disease:

a review with focus on its genetics. Ment Retard Dev Disabil Res Rev 2006;12:123-128.

33. Fogli A, Dionisi-Vici C, Deodato F, et al. A severe variant of childhood ataxia with central

hypomyelination/vanishing white matter leukoencephalopathy related to EIF2B5 muta-

tion. Neurology 2002;59:1966-1968.

34. Fogli A, Rodriguez D, Eymard-Pierre E, et al. Ovarian failure related to eukaryotic initia-

tion factor 2B mutations. Am J Hum Genet 2003;72:1544-1550.


ing white matter with a missense mutation in EIF2B5. Neurology 2004;62:1601-1603

36. Craddock BL, Proud CG. The a-Subunit of Mammalian Initiation Factor eIF-2B is Essential

for Catalytic Activity. Biochem Biophys Res Commun 1996;220:843-847.

37. Williams DD, Pavitt GD, Proud CG. Characterisation of the initiation factor eIF2B and its

regulation in Drosophila melanogaster. J Biol Chem 2001;276:3733-3742.

38. Fabian JR, Kimball SR, Heinzinger NK, Jefferson LS. Subunit assembly and guanine nucle-

otide exchange factor activity of eukaryotic initiation factor eIF2B subunits expressed in

Sf9 cells. J Biol Chem 1997;272:12359-12365.

39. Richardson JP, Mohammad SS, Pavitt GD. Mutations causing childhood ataxia with cen-

tral nervous system hypomyelination reduce eukaryotic initiation factor 2B complex

formation and activity. Mol Cell Biol 2004;24:2352-2363.

40. Kimball SR, Fabian JR, Pavitt GD, et al. Regulation of guanine nucleotide exchange

through phosphorylation of eukaryotic initiation factor eIF2α: role of the α- and δ-subu-

nits of eIF2B. J Biol Chem 1998;273:12841-12845.

41. Hinnebusch AG. Mechanism and regulation of methionyl-tRNA binding to ribosomes. In:

Sonenberg N, Hershey JWB, Mathews MB, editors. Translational Control of Gene Expres-

sion. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 2000. p 185-243.

CHAPTER 7Summary, discussion and future perspectives

136

Chapter 7

VANISHING WHITE MATTER DISEASE

Vanishing white matter disease1 (VWM) is a puzzling leukoencephalopathy caused by mutations

in any of the five genes encoding eukaryotic translation initiation factor 2B (eIF2B), an ubiqui-

tously expressed protein complex with a crucial role in initiation of mRNA translation for virtually

every protein in the human body.2-5

VWM is one of the most prevalent inherited childhood white matter disorders6, but the disease may

occur at all ages.7,8 Most patients with VWM show signs of progressive neurologic deterioration with

predominantly signs of motor dysfunction. In the past years it has become known that the clinical

spectrum is extremely wide with, for example, migraines, psychiatric symptoms and presenile demen-

tia as start of the disease at older age. In some patients the disease also affects other organs, especially

the ovaries. Only in patients with the most severe variant of the disease, the antenatal onset variant,

other organs can be affected as well.7-12 The list of known VWM causing mutations is still expanding.8,13

The disease is fatal. There is no specific treatment for VWM. Management is at present supportive.8

Typical findings on MRI are symmetrical diffuse abnormality of the cerebral hemispheric white

matter with rarefaction and cystic degeneration in a “melting-away” pattern.1,7 Neuropathologi-

cal findings show a cavitating white matter disease with meagre reactive gliosis and involvement

of the glial lineage with a maturation defect of astrocytes and oligodendrocytes.1,8,9,14-17

The link between the selective vulnerability of the white matter of the brain and the mutated

eIF2B protein complex is not understood.

The main goal of this thesis was to increase our understanding of the phenotypic variation and

the correlation between genotype and phenotype in VWM.

CLINICAL VARIATION IN VWM

The disease onset, clinical severity and disease course of VWM patients vary greatly.7-12 Large

studies on phenotypic variation in VWM are scarce. Chapter 2 provides data collected and analyzed

from our VWM patient database, the largest cohort so far, to gain more systematic knowledge on

the clinical variation in VWM. Data were collected on prevalence and characteristics of subgroups

of patients defined by age of onset.

The large clinical spectrum of VWM was confirmed in the dataset. The VWM disease spectrum

consists of a continuum of phenotypes with extremely wide variability. The spectrum continues to

expand on both extremes suggesting that even more extreme phenotypes are currently missed.

The division in certain age of onset groups is arbitrary, though clinically useful. Most patients pres-

ent in childhood. Disease severity clearly correlates with age at onset of symptoms; the younger

the first neurological signs appeared, the more severe the disease course is with a higher sensi-

tivity to stress. We suspect that especially adult onset, mild variants of VWM have largely been

underdiagnosed, because of the less typical presentation and the lack of awareness under adult

neurologists. More and larger follow-up studies will further contribute to a more representative

description of the clinical spectrum in VWM patients.

137

Summary, discussion and future perspectives

MALE-FEMALE DIFFERENCES

In VWM the male-female ratio is not equal for patients of all ages. Among adult onset VWM

patients, a predominance of females has been observed.18 This is unexpected for an autosomal

recessive disorder. Labauge and colleagues hypothesized that the reason for the imbalance in

ratio between males and females is that with mild mutations, females are more prone to disease

presentation, while more males remain asymptomatic.18

In chapter 2 and 5 we studied male-female differences. We also found a higher number of VWM

teenage and adult females, with a trend for higher survival rates and less rapid loss of ambu-

lation among females; so a milder disease than males. However, if males would tend to have a

more severe disease, one would expect more males in the younger age at onset groups, which

we did not find. It is important to note that in general, male have a shorter life expectancy

than females and at all ages more men than women die. Balsara et al.19 suggest the existence

of a male vulnerability factor, attributed to a complex interplay of factors including acquired

risks, health-reporting behavior, illness behavior, health care utilization as well as an underlying

biological difference explaining the general male disadvantage in life expectancy.19 However, in

infantile and early childhood onset VWM, these gender differences are less pronounced. Larg-

er numbers of patients are required to find out whether the general male:female imbalance

explains the imbalance in VWM or whether the male disadvantage is more prominent in VWM

than explained by the general ‘life expectancy gap’.19,20 A factor that could contribute to the

observed male-female imbalance is that females with mild variants of VWM are more readily

diagnosed because of the ovarian failure.18 It is possible that in the category of mildly affected

individuals who exhibit only subtle neurological signs, this feature advances diagnosis in wom-

an, while the diagnosis in equally mildly affected males is missed.

We are aware of shortcomings of our clinical variation studies. Retrospectively collected data

are of lower quality than prospective data. Our study on clinical variation is the largest described

VWM cohort, but still the numbers are often small in the subgroups. The characteristics of our

phenotypic variation study with its international and multi-institutional nature with physicians

and families filling in the questionnaires, both having a different background, may all hamper a

truly objective evaluation of the clinical course in VWM patients. Missing data are often not ran-

dom, for example missing data on early childhood in adult patients. It is possible that over time

a selection bias occurred as VWM was initially recognized as a disease in childhood with a possi-

ble underestimation in the older age of onset groups. For future studies on clinical variation, a

study at a larger scale and with longer follow-up of VWM patients could give more insight into

the true clinical variation. If treatment would become available and case-control studies would

be unethical, a well-documented, large database of historical controls is mandatory. So, the Am-

sterdam VWM database is a long-term project with new information being entered.

138

Chapter 7

MRI CHARACTERISTICS IN VWM

Before DNA testing was available, the diagnosis of VWM was made by clinical and MRI criteria.

Nowadays, using genetic analysis as the ‘gold standard’, the proposed MRI criteria have 95%

sensitivity and 94% specificity.1,7,9

MRI in early stages of VWM disease

The MRI criteria are suitable to diagnose cases with typical presentation, but are not suitable for

identifying unusual MRI variants like the most severe and the mildest variants.7,8 Additionally,

MRI criteria may not be met in the earliest stages of the disease. In chapter 3 we studied the MRI

characteristics of the early stages of the disease, preceding the stage of rarefaction and cystic

white matter degeneration. The study showed that in early stages of VWM, MRI does not nec-

essarily display diffuse cerebral white matter abnormalities and also rarefaction or cystic degen-

eration is not necessarily present. All patients included in the study had confluent and symmet-

rical abnormalities in the periventricular and bordering deep white matter. In young patients,

myelination was delayed. The inner blade of the corpus callosum was affected in all patients.

A conclusion of this study is that if the MRI abnormalities do not meet the criteria for VWM, it

helps to look at the corpus callosum: if the inner blade is affected, VWM should be considered.

Restricted diffusion

Only a few studies mention the results of diffusion-weighted imaging (DWI) in VWM.7,21-23 On

diffusion-weighted images, the rarefied and cystic white matter demonstrates an increased

diffusivity related to highly expanded extracellular spaces.7,8,21-24 However, areas of restricted

diffusion are found in some patients.8,23,24 It has been suggested that the restricted diffusion in

VWM reflects acute brain tissue degeneration or acute demyelination23,24, but the exact histo-

pathological characteristics underlying restricted diffusion remain unknown.

In chapter 4 the occurrence of restricted diffusion in vanishing white matter was investigated.

Areas with decreased apparent diffusion coefficient values were found in the U fibers, cerebel-

lar white matter, middle cerebellar peduncle, pyramids, genu or splenium of the corpus callo-

sum, and posterior limb of the internal capsule. All are relatively spared regions in VWM.9,15,25,26

Patients showing restricted diffusion were younger and had shorter disease duration. Histo-

pathologic analysis of a brain slice revealed that the regions with restricted diffusion had a

higher cell density and did not show tissue degeneration. These findings are in agreement with

the observation that relatively spared regions, which show diffusion restriction, have the high-

est cell density in VWM.26

Our findings added to the knowledge on MRI in early stage of the disease and DWI findings in

VWM. A still difficult problem is posed by MRIs of (young) adults that show diffuse white-matter

abnormalities without rarefaction or cystic degeneration.7-9,27,28 A valid question is whether in

such cases mutational analysis of the EIF2B1-5 genes should be performed. The contribution of

the known biochemical markers glycine (ratio of CSF to plasma)29 and asiolotransferin30,31 has

139


not been evaluated in such cases.7,8,28 Our findings suggest that involvement of the inner blade

of the corpus callosum helps in the decision. Meoded and colleagues32 propose adding spinal

imaging, as they found involvement of the cervical posterior spinal tracts and mild global spinal

cord atrophy in a single patient. It is at present unclear if these findings are sufficiently specific

for VWM to be of help. Histopathology findings show that in the spinal cord is only partially

affected.1,9,15,16,25

GENOTYPE-PHENOTYPE CORRELATIONS

The explanation for the wide phenotypic variation in VWM is complex. Certain mutations are

consistently associated with a mild or severe phenotype.11,27,33,34 On the other hand, environmen-

tal and/or other genetic factors than the eIF2B mutations appear to determine at least part of

the phenotype, as within families phenotypic heterogeneity has been reported.7-9,11,17,27,35

In chapter 5 we looked at the correlation between genotype and phenotype and addressed the

question whether the clinical phenotype of compound-heterozygous patients is determined

by the mildest mutation, the most severe mutation or by both. We selected the three most fre-

quent mutations in EIF2B5: p.Arg113His, p.Thr91Ala and p.Arg339any, associated with a mild,

mild to intermediate, and severe phenotype, respectively.


patients compound-heterozygous for p.Arg113His and patients homozygous for p.Thr91Ala. Pa-

tients with p.Arg113His/p.Arg339any have a milder phenotype than patients with p.Thr91Ala/p.

Arg339any.

These findings indicate that the phenotype is not determined by the most severe or mildest

mutation alone, but by the effect of both mutations. This conclusion is important for clinicians

and genetic counsellors providing information to patients and families.

One should be careful, however, with definitive predictions for new patients. Further studies on

larger groups of patients would make conclusions more definitive. In view of the many different

mutations increasing the study scale is conditional for better insight into genotype-phenotype

correlation.

eIF2B DYSFUNCTION

Mutations in eIF2B affect eIF2B function in diverse ways.36-39 When tested in patient-derived

lymphoblasts and fibroblasts mutations were reported to decrease eIF2B activity as guanosine

exchange factor (GEF).40 The severity of the decrease was suggested to correlate with the clinical

severity, although later data showed inconsistencies in this correlation.40,41

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 or cell proliferation and sur-

vival.38,47,48 These observations suggest that basal eIF2B activity by itself may not or not straight-

forwardly explain the disease.8,17 Assessment of eIF2B activity in patient-derived cell lines has

140

Chapter 7

been proposed as a tool in the diagnosis of VWM41, but considering the above observations, it is

questionable whether eIF2B activity as measured in cells reflects anything significant regarding

the disease and whether it can be used as a marker of the disease or of disease severity.

In chapter 6 we focused on the functional effects of selected VWM mutations by co-express-

ing mutated and wild-type subunits in human cells and combined these studies with measure-

ment of the GEF activity of eIF2B in patient derived cells with the same mutations. Our findings

showed that the observed functional effects are diverse, including defects in eIF2B complex

integrity, binding to the regulatory alpha-subunit, substrate binding and GEF activity. Strikingly,

some of the studied mutations causing severe disease did not alter eIF2B function in the tested

parameters. Some even resulted in elevated GEF activity.

Measurement of eIF2B GEF activity in patient-derived lymphoblasts and fibroblasts as published

has therefore limited value as a diagnostic test. It is at present unclear if the protocol used for

measurement of GEF activity reflects physiological conditions, if the used patient-derived cell

types are the right system for such measurements, or if the disease is altering eIF2B function in

another way than measured.

An experimental set-up with cultured brain cells derived from VWM patients would be ideal to

study effects of VWM mutations on cells from the most affected organ, the brain, to study for ex-

ample GEF activity. This could also be done with cells from the ovaries and other affected organs

in de severe antenatal forms.

A mutant mouse model provides an alternative for this option. A mutant mouse model for VWM

has been developed42-44, but this mouse lacks a clear phenotype. Although interpretation of results

obtained from mouse models for human diseases is hampered by species differences, a represen-

tative mutant VWM mouse would allow studies on all types of cells at all stages of the disease.

Another option would be to use VWM patient derived induced pluripotent stem cells that can

be differentiated into neural cells, especially oligodendrocytes and astrocytes. Such cells could be

more suitable for the study of effects of mutant eIF2B on cell functions, including GEF activity.

The total amount of eIF2B per cell and eIF2/eIF2B ratio have been shown to vary between dif-

ferent tissues.45 The eIF2B concentration is the rate-limiting step in mRNA translation velocity.

Different expression levels of eIF2B and differences in eIF2/eIF2B ratio in brain cells or other cell

types may influence the sensitivity of the translation initiation process to the control exerted by

phosphorylation of eIF2α in such cells. The total amount of eIF2B and the

eIF2/eIF2B ratio have not been determined for different human brain cell types and for cells in

different regions of the brain, while such differences may contribute to the selective vulnera-

bility of the white matter and white matter regions in VWM. Studies in a mutant mouse model

may contribute to the insights on this subject.

Several authors have investigated the hypothesis that the decreased eIF2B activity might impair

the cellular stress response and improperly activate the unfolded protein response (UPR).46-48

These suggestions can be confirmed in mutant mice with VWM.

141


Aberrant control of translation of specific mRNAs has been put forward as possible effect of

eIF2B dysfunction.49-51 Decreased eIF2B activity leads to reduced general rates of protein synthe-

sis, but to increased synthesis of some proteins, depending on the structures of the 5’untranslat-

ed region of the mRNA. It could be that such proteins are central in the disease mechanisms of

VWM. It is also possible that eIF2B serves additional functions, apart from those in translation

initiation8,52 and it could be that a defect in those functions in fact cause the disease VWM.

Until now, research on VWM has been most of all hypothesis-driven. With this approach small

pieces of the VWM puzzle have been laid, as discussed above. However, we are far away from

understanding the big VWM picture. Further research should be more open, not based on an a

priori hypothesis. With a more open mind we may start to begin to gain better insight into the

pathophysiology of VWM. Such insight is essential for developing treatment.

142

Chapter 7

1. Van der Knaap MS, Barth PG, Gabreels FJM, et al. A new leukoencephalopathy with vanish-

ing white matter. Neurology 1997;48:845.

2. Leegwater PA, Pronk JC, van der Knaap MS. Leukoencephalopathy with vanishing white

matter: from magnetic resonance imaging pattern to five genes. J Child Neurol 2003;18:639.


of translation initiation factor eIF2B can cause leukoencephalopathy with vanishing white

matter. Ann Neurol 2002;51:264-270.

4. Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translation initiation and

principles of its regulation. Nature reviews 2010;10:113.

5. Hinnebusch AG. Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes.

Microbiology 2011;75:434.

6. Van der Knaap MS, Breiter SN, Naidu S, et al. Defining and categorizing leukoencephalopa-

thies of unknown origin: MR imaging approach. Radiology 1999;213:121.

7. Van der Knaap MS, Pronk JC, Scheper GC. Vanishing white matter disease. Lancet Neurol.

2006;5:413.

8. 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.

9. Van der Knaap MS, Kamphorst W, Barth PG, et al. Phenotypic variation in leukoencephalop-

athy with vanishing white matter. Neurology 1998;51:540

10. Van der Knaap MS, van Berkel CG, Herms J, et al. eIF2B-related disorders: antenatal onset

and involvement of multiple organs. Am J Hum Genet 2003;73:1199.


eIF2B-related leukodystrophies. Neurology 2004;62:1509.


Trans. 2006;34:22.

13. Shimada S, Miva K, Oda N, et al. An unmasked mutation of EIF2B2 due to submicroscopic

deletion of 14q24.3 in a patient with vanishing white matter disease. Am J Med Genet A.

2012;158A:1771.

14. Brück W, Herms J, Brockmann K, et al. Myelinopathia centralis diffusa (vanishing white mat-

ter disease): Evidence of apoptotic oligodendrocyte degeneration in early lesion develop-

ment. Ann Neurol 2001;50:532.

15. Rodriguez D, Gelot A, della Gaspera B, et al. Increased density of oligodendrocytes in child-

hood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological and

biochemical study of two cases. Acta Neuropathol. 1999;97:469.


childhood ataxia with diffuse central nervous system hypomyelination syndrome. Acta Neu-

ropathol 2000;100:635.

17. Bugiani M, Boor I, Powers JM, et al. Leukoencephalopathy with vanishing white matter. A

review. J Neuropathol Exp Neurol 2010;69:987.

REFERENCES

143


18. Labauge P, Horzinski L, Ayrignac X, et al. Natural history of adult-onset eIF2B-related disor-

ders: a multi-centric survey of 16 cases. Brain 2009;132:2161.

19. Balsara SL, Faerber JA, Spinner NB, Feudtner C. Pediatric mortality in males versus females in

the United States, 1999-2008. Pediatrics. 2013;132:631-8.

20. World Health Organization. World health statistics 2014, http://www.who.int/gho/publica-

tions/world_health_statistics/2014/en/ (accessed 03 June 2014).

21. Sijens PE, Boon M, Meiners LC, et al. 1H chemical shift imaging, MRI and diffusion-weighted

imaging in vanishing white matter disease. Eur Radiol. 2005;15:2377.

22. Patay Z. Diffusion-weighted MR imaging in leukodystrophies. Eur Radiol 2005;15:2284.

23. Harder S, Gourgaris A, Frangou E, et al. Clinical and neuroimaging findings of Cree leukod-

ystrophy: a retrospective case series. Am J Neuroradiol. 2010;31:1418.

24. Ding XQ, Bley A, Ohlenbusch A, et al. Imaging evidence of early brain tissue degeneration in

patients with vanishing white matter disease; a multimodal MR study. J Magn Reson Imag-

ing 2012;35:926.

25. Francalanci P, Eymard-Pierre E, Dionisi-Vici C, et al. Fatal infantile leukodystrophy: a severe

variant of CACH/VWM syndrome, allelic to chromosome 3q27. Neurology 2001;57:265.

26. Van Haren K, van der Voorn JP, Peterson DR, et al. The life and death of oligodendrocytes in

vanishing white matter disease. J Neuropathol Exp Neurol 2004;63:618.

27. Van der Knaap MS, Leegwater PAJ, van Berkel CG, et al. Arg113His mutation in eIF2Bε as

cause of leukoencephalopathy in adults. Neurology 2004;62:1598.

28. Mascalchi M, De Grandis D, Ginestroni A, et al. Early MR imaging and spectroscopy appear-

ance of eIF2B-related leukoencephalopathy. Neurology 2006;67:537.

29. Van der Knaap MS, Wevers RA, Kure S, et al. Increased cerebrospinal fluid glycine: a bio-

chemical marker for a leukoencephalopathy with vanishing white matter. J Child Neurol

1999;14:728.

30. Vanderver A, Schiffmann R, Timmons M, et al. Decreased asialotransferrin in cerebrospinal

fluid of patients with childhood-onset ataxia and central nervous system hypomyelination/

vanishing white matter disease. Clin Chem 2005;51:2031.

31. Vanderver A, Hathout Y, Maletkovic J, et al. Sensitivity and specificity of decreased CSF asia-

lotransferrin for eIF2B-related disorder. Neurology 2008;70:2226.

32. Meoded A, Poretti A, Yoshida S, et al. Leukoencephalopathy with vanishing white matter:

serial MRI of the brain and spinal corg including diffusion tensor imaging. Neuropediatrics

2011;42:82.

33. Fogli A, Wong K, Eymard-Pierre E, et al. Cree leukoencephalopathy and CACH/VWM disease

are allelic at EIF2B5 locus. Ann Neurol 2002;52:506.

34. Biancheri R, Rossi A, Di Rocco M, et al. Leukoencephalopathy with vanishing white matter:

an adult onset case. Neurology 2003;61:1818.

35. Damon-Perriere N, Menegon P, Olivier A, et al. Intra-familial heterogeneity in adult onset

vanishing white matter disease. Clin Neurol Neurosurg 2008;110:1068.

36. Boesen T, Mohammad SS, Pavitt GD, Andersen GR. Structure of the catalytic fragment of

translation initiation factor 2B and identification of a critically important catalytic residue. J

144

Chapter 7

Biol Chem 2004;279:10584.

37. Richardson JP, Mohammad SS, Pavitt GD. Mutations causing childhood ataxia with central

nervous system hypomyelination reduce eukaryotic initiation factor 2b complex formation

and activity. Mol Cel Biol 2004; 24: 2352.

38. Li W, Wang X, van der Knaap MS, Proud CG. Mutations linked to leukoencephalopathy with

vanishing white matter impair the function of the eukaryotic initiation factor 2B complex in

diverse ways. Mol Cell Biol 2004; 24: 3295.

39. Hiyama TB, Ito T, Imataka H, Yokoyama S. Crystal structure of the alpha subunit of human

translation initiation factor 2B. J Mol Biol 2009;392:937.

40. Fogli A, Schiffmann R, Hugendubler L, et al. Decreased guanine nucleotide exchange factor

activity in eIF2B-mutated patients. Eur J Hum Genet 2004;12:561.

41. Horzinsky L, Huyghe A, Cordoso MC, et al. Eukaryotic initiation factor 2B (eIF2B) GEF activity

as a diagnostic tool for EIF2B-related disorders. PLoS One 2009;15:e8318.

42. Geva M, Cabilly Y, Assaf Y, et al. A mouse model for eukaryotic translation initiation factor

2B-leucodystrophy reveals abnormal development of brain white matter. Brain 2010:133;

2448.

43. Marom L, Ulitsky I, Cabilly Y, et al. A point mutation in translation initiation factor

eIF2B leads to function- and time-specific changes in brain gene expression. PLoS One

2011;6:e26992.

44. Cabilly Y, Barbi M, Geva M, et al. Poor cerebral Inflammatory response in eIF2B knock-

in mice: implications for the aetiology of vanishing white matter disease. PLoS ONE

2012;7:e46715.

45. Oldfield S, Jones BL, Tanton D, et al. Use of monoclonal antibodies to study the structure and

function of eukaryotic protein synthesis initiation factor eIF2B. Eur J Biochem 1994;221:399.

46. Schiffmann R, Elroy-Stein O. Childhood ataxia with CNS hypomyelination/vanishing white

matter disease--a common leukodystrophy caused by abnormal control of protein synthesis.

Mol Genet Metab 2006;88:7.

47. Kantor L, Harding HP, Ron D, et al. Heightened stress response in primary fibroblasts express-

ing mutant eIF2B genes from CACH/VWM leukodystrophy patients. Hum Genet 2005;23:1

48. Van Kollenburg B, van Dijk J, Garbern J, et al. Glia-specific activation of all pathways of the

unfolded protein response in vanishing white matter disease. J Neuropathol Exp Neurol

2006;65:707.

49. Komar AA, Hatzoglou M. Internal ribosome entry sites in cellular mRNAs: mystery of their

existence. J Biol Chem 2005;280:23425.

50. Kozak M. A second look at cellular mRNA sequences said to function as internal ribosome

entry sites. Nucleic Acids Res 2005;33:6593.

51. Resch AM, Ogurtsov AY, Rogozin IB, et al. Evolution of alternative and constitutive regions

of mammalian 5ʹ′UTRs. BMC Genomics 2009;10:162.

52. Klein U, Raminez MT, Kobilka BK, von Zastrow M. A novel interaction between adr-

energic receptors and the alpha-subunit of eukaryotic initiation factor 2B. J Biol Chem

1997;272:19099.

145


CHAPTER 8Samenvatting, discussie en toekomstperspectieven

148

Chapter 8

VANISHING WHITE MATTER

Vanishing white matter (VWM) is een intrigerende wittestofziekte, die veroorzaakt kan worden

door fouten (mutaties) in elk van de vijf genen die coderen voor eukaryotische translatie ini-

tiatie factor 2B (eIF2B), een eiwit complex dat in elke cel van het lichaam aanwezig is en een

cruciale rol speelt bij het begin (de initiatie) van de vertaling (translatie) van vrijwel elk mRNA

naar eiwit in het menselijk lichaam.

VWM is één van de vaakst voorkomende erfelijke ziekten van de witte stof van de hersenen op

de kinderleeftijd, maar de ziekte kan op elke leeftijd voorkomen. De meeste VWM patienten

vertonen tekenen van toenemende neurologische achteruitgang met overwegend motorische

problemen. In de afgelopen jaren is het duidelijk geworden dat de klinisch variatie zeer groot

is met bijvoorbeeld migraine, psychiatrische symptomen of preseniele dementie als eerste teken

van de ziekte op oudere leeftijd. Bij sommige patienten tast de ziekte ook andere organen aan,

met name de eierstokken. Alleen bij patienten met de ernstigste vorm van de ziekte, die al voor

de geboorte begint, kunnen ook andere organen zijn aangedaan. De lijst van bekende VWM

mutaties wordt nog steeds langer. De ziekte is fataal. Er is geen specifieke behandeling voor

VWM. Op dit moment is het enige dat gedaan kan worden klachten bestrijden.

Kenmerkende bevindingen op hersenscans (MRI) zijn symmetrische diffuse afwijkingen in de

witte stof van de grote hersenen, die verdwijnt en degenereert in een soort “wegsmeltend”

patroon. Er ontstaan gaten in de witte stof. Neuropathologische bevindingen laten een

caviterende wittestofziekte zien, waarbij slechts weinig littekenweefsel (reactieve gliose)

wordt gezien. Vooral zogenaamde astrocyten en oligodendrocyten (samen de macroglia

of kortweg glia genoemd) zijn afwijkend, onrijp en falen in hun functie.Hoe het verband is

tussen de kwetsbaarheid van specifiek de witte stof van de hersenen en het gemuteerde eIF2B

eiwitcomplex, is niet bekend.

Het belangrijkste doel van dit proefschrift was om onze kennis van de klinische variatie en

de correlatie tussen specifieke mutaties (het genotype) en de klinische verschijnselen (het

fenotype) bij VWM te vergroten.

KLINISCHE VARIATIE BIJ VWM

De leeftijd bij begin van de ziekte, de ernst van de verschijnselen en het ziekteverloop

varieren enorm onder VWM patienten. Grote studies over fenotypische variatie bij VWM

zijn schaars. Hoofdstuk 2 bevat de verzamelde gegevens van onze VWM patient-database,

het grootste cohort tot nu toe. Wij verzamelden systematische gegevens over de klinische

variatie bij VWM. In dit hoofdstuk worden gegevens gepresenteerd met betrekking tot

subgroepen van patienten, die gedefinieerd zijn op basis van leeftijd bij begin van de ziekte.

De grote klinische variatie van VWM werd bevestigd in de dataset. Het VWM ziekte spectrum

bestaat uit een continuüm van fenotypen met extreem grote variabiliteit. Het spectrum dijt aan

149

Samenvatting, discussie en toekomstperspectieven

beide uiterste kanten nog steeds uit, hetgeen suggereert dat de nog extremere fenotypen op dit

moment nog worden gemist. De verdeling in groepen op basis van de leeftijd bij het begin van de

ziekte is willekeurig, maar wel klinisch bruikbaar. Bij de meeste patienten openbaart de ziekte zich

op de kinderleeftijd. Er is een duidelijke correlatie tussen de ernst van de ziekte en de leeftijd bij

het begin van de eerste ziekteverschijnselen: hoe jonger de patient is als de eerste neurologische

symptomen zichtbaar worden, hoe ernstiger het ziekteverloop is en hoe groter de gevoeligheid

voor stress. We vermoeden dat vooral milde vormen van VWM met een begin van de ziekte op

volwassen leeftijd worden gemist vanwege de weinig kenmerkende presentatie en het gebrek

aan herkenning door neurologen. Meer en grotere vervolgonderzoeken kunnen bijdragen aan

een steeds representatievere beschrijving van het klinische spectrum van VWM patienten.

MAN-VROUW VERSCHILLEN

Bij VWM is de man-vrouw verhouding niet gelijk voor patienten van alle leeftijden. Bij volwas-

sen VWM patienten zijn er verhoudingsgewijs meer vrouwen. Dit is niet wat verwacht wordt

bij een autosomaal recessief overervende aandoening. Labauge en collega’s opperden dat de

reden voor de onevenwichtige verhouding tussen mannen en vrouwen is, dat bij milde mutaties

vrouwen vatbaarder voor de ziekte zijn, terwijl mannen vaker zonder verschijnselen blijven.

In hoofdstuk 2 en 5 bestudeerden we de man-vrouw verschillen. We vonden ook een groter

aantal vrouwelijke tiener- en volwassen patienten dan mannelijke. Wij vonden tevens een trend

voor hogere overlevingskansen en later verliezen van de loopvaardigheid van vrouwen vergele-

ken met mannen, hetgeen suggereert dat vrouwen een mildere variant van de ziekte dan man-

nen. Echter, als de mannen een ernstiger ziektebeloop zouden hebben, zou men meer mannen

in de jongere leeftijdsgroepen verwachten, hetgeen we niet vonden. Het is hierbij belangrijk op

te merken dat mannen in het algemeen een kortere levensverwachting hebben dan vrouwen en

dat op alle leeftijden meer mannen sterven dan vrouwen. Balsara et al. suggereerden het bestaan

van een mannelijke kwetsbaarheidsfactor samenhangend met een complex samenspel van fac-

toren, waaronder risicogedrag, ander ziektegedrag, minder gezondheidszorggebruik en een

onderliggend biologische verschil, die de achterstand van mannen in levensverwachting zouden

verklaren. Echter, onder VWM patienten met een begin op de zuigelingen-, peuter-, kleuter- en

kinderleeftijd ontbreken sekseverschillen en zijn mannen niet duidelijk oververtegenwoordigd.

Grotere studies met meer patienten zijn nodig om te onderzoeken of dit algemene man-vrouw

verschil de oververtegenwoordiging van volwassen vrouwelijke VWM patienten verklaart of

dat het mannelijke nadeel bij VWM meer uitgesproken is dan verklaard kan worden door dit al-

gemene gat in levensverwachting.19,20 Een factor die kan bijdragen aan de waargenomen overver-

tegenwoordiging van vrouwen onder volwassen VWM patienten is dat vrouwen met een mild-

ere variant van VWM sneller worden gediagnosticeerd door verschijnselen van dysfunctie van

de eierstokken. Het is mogelijk dat in de categorie mild aangedane individuen met slechts subti-

ele neurologische verschijnselen de betrokkenheid van de eierstokken een snellere diagnose bij

vrouwen in de hand werkt, terwijl de diagnose bij even licht getroffen mannen wordt gemist.

150

Chapter 8

We zijn ons bewust van de tekortkomingen van onze studie betreffende de klinische variatie.

Retrospectief verzamelde gegevens zijn van lagere kwaliteit dan prospectieve data. Ons VWM

cohort is het grootst beschreven cohort tot nu toe, maar de aantallen in de subgroepen zijn

toch vaak nog klein. Onze fenotypische variatie studie heeft een aantal kenmerken, die in haar

nadeel werken: het internationale en multi-institutionele karakter van de studie; het feit dat

zowel artsen als families de vragenlijsten invulden, beide met een andere achtergrond. Deze

factororen kunnen de resultaten van het onderzoek nadelig beïnvloeden. Ontbrekende ge-

gevens zijn vaak niet willekeurig, bijvoorbeeld ontbrekende gegevens over de vroege jeugd

bij volwassen patienten. Het is ook mogelijk dat er een selectiebias is, aangezien VWM aan-

vankelijk werd gezien als een ziekte van de kindertijd met een mogelijke onderschatting van

het voorkomen van VWM op oudere leeftijd. Voor toekomstige studies over klinische variatie,

zou een studie op een grotere schaal en met een langere follow-up van VWM patienten beter

inzicht in de werkelijke klinische variatie geven. Als behandeling beschikbaar zou worden en

case-control studies zouden onethisch zijn, is een goed gedocumenteerde, grote database van

historische controles noodzakelijk. Derhalve is de Amsterdamse VWM database een langdurig

project waaraan nog steeds nieuwe informatie wordt toegevoegd.

MRI KENMERKEN VWM

Voordat DNA diagnostiek beschikbaar was, werd de diagnose VWM gesteld aan de hand van

klinische en MRI criteria. Tegenwoordig, met behulp van genetische analyse als de ‘gouden

standaard’, blijken de beschreven MRI criteria een sensitiviteit van 95% en een specificiteit van

94% te hebben.

MRI in vroege stadia van VWM

De MRI criteria zijn goed toepasbaar voor diagnostiek van de ziektegevallen met een typische

presentatie, maar zijn niet geschikt voor het identificeren van ongewone MRI varianten, zoals

gezien worden bij de ernstigst en de mildst aangedane patienten. Bovendien voldoen de

MRI criteria niet altijd in hele vroege stadia van de ziekte. In hoofdstuk 3 hebben we de MRI

kenmerken van de vroege stadia van de ziekte, nog voorafgaand aan de fase

van cysteuze degeneratie van de witte stof, onderzocht. De studie toonde aan dat in de vroege

stadia van VWM, MRI niet noodzakelijkerwijs diffuus afwijkende witte stof laat zien en ook pre-

cysteuze of cysteuze degeneratie niet perse aanwezig hoeft te zijn. Alle patienten in de studie

had confluerende en symmetrische afwijkingen in de periventriculaire en aangrenzende diepe

witte stof. Bij jonge patienten was de myelinisatie vertraagd. De binnenrand van het corpus

callosum was aangedaan bij alle patienten. Een van de conclusies van deze studie is dat als de

MRI niet aan de criteria voor VWM voldoet, het helpt om te kijken naar het corpus callosum: als

het binnenrand is aangedaan, moet VWM worden overwogen.

176

151


We zijn ons bewust van de tekortkomingen van onze studie betreffende de klinische variatie.

Retrospectief verzamelde gegevens zijn van lagere kwaliteit dan prospectieve data. Ons VWM

cohort is het grootst beschreven cohort tot nu toe, maar de aantallen in de subgroepen zijn

toch vaak nog klein. Onze fenotypische variatie studie heeft een aantal kenmerken, die in haar

nadeel werken: het internationale en multi-institutionele karakter van de studie; het feit dat

zowel artsen als families de vragenlijsten invulden, beide met een andere achtergrond. Deze

factororen kunnen de resultaten van het onderzoek nadelig beïnvloeden. Ontbrekende ge-

gevens zijn vaak niet willekeurig, bijvoorbeeld ontbrekende gegevens over de vroege jeugd

bij volwassen patienten. Het is ook mogelijk dat er een selectiebias is, aangezien VWM aan-

vankelijk werd gezien als een ziekte van de kindertijd met een mogelijke onderschatting van

het voorkomen van VWM op oudere leeftijd. Voor toekomstige studies over klinische variatie,

zou een studie op een grotere schaal en met een langere follow-up van VWM patienten beter

inzicht in de werkelijke klinische variatie geven. Als behandeling beschikbaar zou worden en

case-control studies zouden onethisch zijn, is een goed gedocumenteerde, grote database van

historische controles noodzakelijk. Derhalve is de Amsterdamse VWM database een langdurig

project waaraan nog steeds nieuwe informatie wordt toegevoegd.

MRI KENMERKEN VWM

Voordat DNA diagnostiek beschikbaar was, werd de diagnose VWM gesteld aan de hand van

klinische en MRI criteria. Tegenwoordig, met behulp van genetische analyse als de ‘gouden

standaard’, blijken de beschreven MRI criteria een sensitiviteit van 95% en een specificiteit van

94% te hebben.

MRI in vroege stadia van VWM

De MRI criteria zijn goed toepasbaar voor diagnostiek van de ziektegevallen met een typische

presentatie, maar zijn niet geschikt voor het identificeren van ongewone MRI varianten, zoals

gezien worden bij de ernstigst en de mildst aangedane patienten. Bovendien voldoen de

MRI criteria niet altijd in hele vroege stadia van de ziekte. In hoofdstuk 3 hebben we de MRI

kenmerken van de vroege stadia van de ziekte, nog voorafgaand aan de fase

van cysteuze degeneratie van de witte stof, onderzocht. De studie toonde aan dat in de vroege

stadia van VWM, MRI niet noodzakelijkerwijs diffuus afwijkende witte stof laat zien en ook pre-

cysteuze of cysteuze degeneratie niet perse aanwezig hoeft te zijn. Alle patienten in de studie

had confluerende en symmetrische afwijkingen in de periventriculaire en aangrenzende diepe

witte stof. Bij jonge patienten was de myelinisatie vertraagd. De binnenrand van het corpus

callosum was aangedaan bij alle patienten. Een van de conclusies van deze studie is dat als de

MRI niet aan de criteria voor VWM voldoet, het helpt om te kijken naar het corpus callosum: als

het binnenrand is aangedaan, moet VWM worden overwogen.

176

Restrictie van de diffusie

Slechts enkele studies presenteren resultaten van diffusie-gewogen beelden bij VWM. Op dif-

fusie-gewogen beelden toont de verdwijnende en cysteuze witte stof een verhoogde diffu-

siviteit, die gerelateerd is aan de vergrote extracellulaire ruimte. Echter bij sommige patient-

en met VWM worden gebieden met verminderde diffusie gevonden. Er is gesuggereerd dat

de beperkte diffusie bij VWM acute hersenweefseldegeneratie of acute demyelinisatie weer-

spiegelt, maar de exacte histopathologische kenmerken van de beperkte diffusie zijn onbekend.

In hoofdstuk 4 werd het voorkomen van diffusiebeperking bij VWM onderzocht. Gebieden

met een lagere diffusiecoefficient werden gevonden in de U-vezels, cerebellaire witte stof,

middelste cerebellaire pedunkels, piramidebanen, genu of splenium van het corpus callo-

sum, en crus posterius van de capsula interna, allemaal relatief gespaarde gebieden bij VWM.

Patienten met restrictie van de diffusie waren jonger en hadden een kortere duur van de

ziekte. Histopathologische analyse van een hersenplak toonde aan dat de gebieden met re-

strictie van de diffusie een hogere celdichtheid lieten zien en geen weefsel degeneratie ver-

toonden. Deze bevindingen zijn in overeenstemming met de observatie dat relatief gespaarde

gebieden, die restrictie van de diffusie laten zien, de hoogste celdichtheid hebben bij VWM.

Onze bevindingen voegen kennis toe over MRI in een vroeg stadium van de ziekte en over dif-

fusie-gewogen beelden bij VWM. De MRI’s van (jong) volwassenen met diffuus afwijkende witte

stof zonder (pre-)cysteuze degeneratie blijven wel een diagnostisch probleem. Een terechte vraag

is of in dergelijke gevallen mutatie-analyse van de genen EIF2B1-5 zou moeten worden uitgevo-

erd. De toegevoegde waarde van de bekende biochemische markers glycine (verhouding van

liquor ten opzichte van plasma) en asiolotransferine is niet geevalueerd voor dergelijke gevallen.

Onze bevindingen suggereren dat de betrokkenheid van de binnenrand van het corpus callosum

helpt bij de evaluatie. Meoded en colleagues stellen het toevoegen van beeldvorming van het

ruggenmerg voor, omdat zij afwijkingen van de cervicale achterstrengen en milde algehele

atrofie van het ruggenmerg vonden bij een VWM patient. Het is op dit moment onduidelijk of

deze bevindingen voldoende specifiek zijn voor VWM om van waarde te zijn. Histopathologische

bevindingen tonen aan dat het ruggenmerg slechts gedeeltelijk aangedaan is.

GENOTYPE-FENOTYPE CORRELATIE

De verklaring voor de grote fenotypische variatie in VWM is complex. Bepaalde mutaties

zijn consequent geassocieerd met een mild of juist ernstig fenotype. Anderzijds kunnen

omgevingsfactoren en/of andere genetische factoren dan de eIF2B mutaties ten minste een deel van

het fenotype verklaren, gezien de gerapporteerde fenotypische heterogeniteit binnen families.

In hoofdstuk 5 hebben wij gekeken naar de correlatie tussen genotype en fenotype en naar

de vraag of het klinisch fenotype van samengesteld heterozygote patienten wordt bepaald

door de mildste mutatie, de meest ernstige mutatie of door beide. We selecteerden de drie

meest voorkomende mutaties in EIF2B5: p.Arg113His, p.Thr91Ala en p.Arg339any, mutaties

152

Chapter 8

die respectievelijk geassocieerd zijn met een mild, licht tot intermediair en ernstig fenotype.

Onze bevindingen tonen dat patienten die homozygoot zijn voor de p.Arg113His mutatie een

mildere ziekte hebben dan patienten die samengesteld heterozygoot zijn voor p.Arg113His

en een andere mutatie en dan patienten die homozygoot zijn voor p.Thr91Ala. Patienten met

p.Arg113His / p.Arg339ieder hebben een milder fenotype dan patienten met p.Thr91Ala /

p.Arg339ieder.

Deze bevindingen geven aan dat het fenotype niet wordt bepaald door de ernstigste of mildste

mutatie alleen, maar wordt bepaald door het samenspel van beide mutaties. Deze conclusie is

belangrijk voor artsen en genetici die informatie verstrekken aan patienten en families.

Men moet voorzichtig zijn met precieze voorspellingen voor nieuwe patienten. Verder

onderzoek bij grotere groepen patienten kunnen bovenstaande conclusies meer definitief

maken. Gezien de vele verschillende mutaties is een studie op grotere schaal een voorwaarde

voor beter inzicht in genotype-fenotype correlaties.

eIF2B DYSFUNCTIE

Mutaties in eIF2B beïnvloeden de eIF2B functie op verschillende manieren. Bij testen van

gekweekte lymfoblasten en fibroblasten van patienten werd gerapporteerd dat mutaties

de eIF2B activiteit verlagen wanneer deze werd getest op guanosine exchange factor (GEF)

activiteit. De grootte van de daling correleerde met de ernst van de ziekte, hoewel latere studies

inconsistenties in deze correlatie lieten zien.

Bij onderzoek van lymfoblasten en fibroblasten van patienten werd niet gevonden dat de ver-

minderde eIF2B activiteit de snelheid van globale eiwitsynthese, vóór, tijdens of na stress beïn-

vloedt dan wel de celproliferatie en celoverleving verandert. Deze waarnemingen suggereren

dat verminderde GEF activiteit van eIF2B de ziekte niet of niet volledig verklaart. Het testen van de

eIF2B activiteit in cellen van patienten is eerder geopperd als een hulpmiddel bij het diagnostic-

eren van VWM, maar gezien bovenstaande opmerkingen valt te betwijfelen of eIF2B activiteit,

getest zoals hierboven beschreven, wel kan worden gebruikt als een marker van de ziekte of

ernst van de ziekte.

In hoofdstuk 6 richtten we ons op functionele effecten van geselecteerde mutaties door

co-expressie van gemuteerde en wild-type subunits in humane cellen. We combineerden deze

waarnemingen met metingen van de GEF activiteit van eIF2B in cellen van patienten met

dezelfde mutaties. Onze bevindingen lieten zien dat de waargenomen functionele effecten

van de onderzochte mutaties divers zijn, met defecten in integriteit van het eIF2B complex,

verminderde binding aan de regulerende alfa-subunit, verstoorde binding van substraat en

verminderde GEF activiteit. Opvallend was dat sommige van de onderzochte mutaties er-

nstige ziekte veroorzaken maar geen impact hebben op één van de geteste eIF2B parame-

ters. Sommige mutaties resulteerden zelfs in een verhoogde GEF activiteit.Meting van eIF2B

153


GEF-activiteit in gekweekte lymfoblasten en fibroblasten van patienten, zoals eerder gepub-

liceerd, heeft dus geen of een zeer beperkte waarde als diagnostische test. Het is onduidelijk

of het protocol voor het meten van GEF activiteit fysiologische omstandigheden weerspiege-

lt, of de gebruikte gekweekte celtypen van patienten het juiste systeem zijn voor dergelijke

metingen, of de ziekte eIF2B functie wijzigt op een andere vlak dan gemeten.

Een proefopstelling met gekweekte hersencellen afkomstig van VWM patienten zou ideaal zijn

om effecten van VWM mutaties te bestuderen in cellen van het meest aangetaste orgaan, de

hersenen, en daarin bijvoorbeeld de GEF activiteit te testen. Dit zou ook kunnen met cellen

van de eierstokken en andere organen die bij de ernstigste vorm van de ziekte aangedaan zijn.

Een mutant muismodel biedt een alternatief. Een mutant muismodel voor VWM is reeds ontwik-

keld, maar deze muis heeft geen duidelijk fenotype. Hoewel de interpretatie van de resultaten

verkregen uit muismodellen voor humane ziekten wordt belemmerd door verschillen tussen muis

en mens, zou een representatieve VWM muis experimenten mogelijk maken in alle soorten cellen

in alle stadia van de ziekte. Een andere optie zou zijn om gebruik te maken van geïnduceerde

pluripotente stamcellen van VWM patienten die kunnen worden gedifferentieerd richting neu-

rale cellen, vooral oligodendrocyten en astrocyten. Dergelijke cellen kunnen geschikt zijn voor

bestuderen van effecten van gemuteerd eIF2B op cel functies, waaronder GEF activiteit.

De totale hoeveelheid eIF2B per cel en de eIF2/eIF2B ratio zijn verschillend in verschillende

weefsels. De eIF2B concentratie is de snelheid beperkende

stap bij mRNA translatie. Verschillende expressieniveaus van eIF2B en verschillen in eIF2/eIF2B

ratio in hersencellen of andere celtypen kunnen de gevoeligheid van de translatie initiatie

beïnvloeden voor regulatie door fosforylatie van eIF2α in dergelijke cellen. De totale hoeveelheid

van eIF2B en de eIF2/eIF2B verhouding zijn niet vastgesteld voor de verschillende menselijke

celtypes in de hersenen en voor cellen in verschillende regio’s van de hersenen, terwijl dergelijke

verschillen kunnen bijdragen aan de selectieve kwetsbaarheid van de witte stof en regio’s van

de witte stof bij VWM. Experimenten in een mutant VWM muismodel kunnen bijdragen aan de

inzichten over dit onderwerp.

Verschillende auteurs hebben de hypothese dat de verminderde eIF2B activiteit de cellulaire reactie

op stress zou kunnen aantasten en abnormale activatie van de ‘unfolded protein response’ (UPR)

geeft, onderzocht. Deze hypotheses kunnen in mutante muizen met VWM worden onderzocht.

Afwijkende regulatie van translatie van specifieke mRNA’s werd gesuggereerd als mogelijk

effect van eIF2B dysfunctie. Verminderde eIF2B activiteit leidt tot verminderde snelheid van

eiwitsynthese, maar verhoogde synthese van sommige eiwitten, afhankelijk van de structuur van

het ‘5’ onvertaalde gebied van het mRNA. Het kan zijn dat dergelijke eiwitten centraal staan in het

ziektemechanisme van VWM. Het is ook mogelijk dat eIF2B andere functies heeft, naast de bekende

in translatie initiatie en het kan zijn dat een defect in die functies in feite de ziekte veroorzaken.

154

Chapter 8

Tot nu toe is het onderzoek naar VWM vooral hypothese gestuurd geweest. Met deze

benadering zijn kleine stukjes van de VWM puzzel gelegd, zoals hierboven besproken. Maar

we zijn nog ver weg van het volledig begrijpen van VWM. Verder onderzoek moet opener

zijn, niet gedreven door een a priori hypothese. Op die manier kunnen we een beter inzicht te

krijgen in de pathofysiologie van VWM. Deze inzichten zijn essentieel voor de ontwikkeling van

behandelingen.

156

LIST OF PUBLICATIONS

THIS THESIS

H.D.W. van der Lei, M.E. Steenweg, F. Barkhof en M.S. van der Knaap. Early MRI characteristics in

children and adolescents with vanishing white matter. Neuropediatrics 2012;43:22-26.

H.D.W. van der Lei, M.E. Steenweg, M. Bugiani, P.J.W. Pouwels, I.M. Vent, F. Barkhof, W.N. van

Wieringen en M.S. van der Knaap. Restricted diffusion in vanishing white matter. Archives of

Neurology 2012;69:723-727

R. Liu*, H.D.W. van der Lei*, X. Wang*, N.C. Wortham, C.G.M. van Berkel, W. Huang en M.S.

van der Knaap, G.C. Scheper en C.G. Proud. Severity of vanishing white matter disease does not

correlate with biochemical deficits in eIF2B function. Human Mutation 2011;32:1036-1045

* these three individuals should be considered as joint first authors who made equal contribu-

tions to this study

H.D.W. van der Lei, C.G.M. van Berkel, W.N. van Wieringen, C. Brenner, A. Feigenbaum, S. Merci-

mek-Mahmutoglu, M. Philippart, B. Tatli, E. Wassmer, G.C. Scheper en M. S. van der Knaap. Gen-

otype-phenotype correlation in vanishing white matter disease. Neurology 2010;75:1555-1559.

OTHERS

J. Damásio, H.D.W. van der Lei, M.S. van der Knaap, E. Santos. Late onset vanishing white matter

disease presenting with learning difficulties. Journal of the Neurological Sciences 2012;314:169-170.

M. van Vliet, M. Diamant, H.D.W. van der Lei, H. Budde en I.A. von Rosenstiel. Het metabool

syndroom bij kinderen met overgewicht in Amsterdam-West. Welke definitie definieert kinder-

en met een hoog risico? Tijdschrift voor Kindergeneeskunde 2009;77:1-10.

I. Koomen, D.E. Grobbee, A. Jennekens-Schinkel, J.J. Roord, H.D.W. van der Lei, M.A.C. Kraak

en A.M. van Furth. Prediction of learning and behavioral problems in school-age survivors of

bacterial meningitis. Acta Paediatrica 2004;93:1378-85.

157

CURRICULUM VITAE

The author of this thesis, Hanna (Hannemieke) Ditta Willemina van der Lei, was born on the

7th of June 1977 in Bussum, the Netherlands. She moved to Epe at age one and went to pri-

mary school there. In 1995, she graduated from secondary school at the Heertganck in Heerde.

For one year she studied medicine at the Catholic University of Leuven, whereafter she contin-

ued to study medicine at the VU University Medical Center. Her research internship concerned

sequelae after bacterial meningitis in children. The curiosity for science was born and she pro-

longed her commitment to the Meningitis Project working as a student assistent of dr. Irene

Koomen en prof.dr. Marceline van Furth. She was involved in a project on metabolic syn-

drome in overweight children at the Slotervaart Hospital in Amsterdam during her internships.

After finishing medical school at the end of 2004 she worked at the department of pediatrics

of the Rijnstate Ziekenhuis, Arnhem and the VU University Medical Center in Amsterdam.

In 2007 started her PhD project at the Center for Childhood White Matter Disorders super-

vised by prof.dr. Marjo van der Knaap, dr. Gert Scheper and dr. Truus Abbink which resulted

in the thesis you are holding. She received the best poster award at the EPNS 2011 congress

in Dubrovnik, Croatia for the work described in chapter 6. In 2012 she started her residency

training in rehabilitation medicine. She is currently working as a resident at the rehabilitation

department of the Spaarne Gasthuis in Hoofddorp. Hannemieke is married to Michiel Hopman

and they live in Diemen with their three daughters Willemijn, Laura and Suze.

158

DANKWOORD

Het zit erop! Het boekje is gedrukt.

Het zijn jaren geweest waarin ik veel heb geleerd op wetenschappelijk vlak maar ook op per-

soonlijk vlak. Promoveren en onderzoek doen kun je niet alleen, dus ik wil iedereen die op wel-

ke manier dan ook aan dit proefschrift heeft bijgedragen heel hartelijk bedanken. Het is helaas

onmogelijk iedereen persoonlijk te bedanken, enkele mensen wil ik in het bijzonder noemen.

Allereerst wil ik alle patienten, hun families en dokters die aan de onderzoeken hebben mee-

gewerkt bedanken, zonder u was onderzoek als dit niet mogelijk.

Mijn promotor, Prof.dr. M.S. van der Knaap, beste Marjo, dank dat je me aannam ondanks dat

ik geen laboratoriumervaring had en dat je me hebt opgeleid als wetenschapper. Bedankt voor

alle kansen en mogelijkheden die je me hebt geboden. Dank ook voor je geduld en aansporing

toen het lastig werd met afronden. Ik heb veel bewondering voor je werk, je ideeen, je visie.

Het is een voorrecht bij je te werken!

En dan natuurlijk mijn copromotoren. Vanaf het eerste uur was dat Dr. G.C. Scheper. Beste Gert,

je hebt me meegenomen en wegwijs gemaakt in de wereld van het laboratoriumonderzoek. Je

had altijd tijd en niet te vergeten humor. Dank je wel!

Dr. T.E.M. Abbink, beste Truus, tijdens de afronding van mijn proefschrift nam je het stokje over

van Gert, je vertrouwen, je enthousiasme, het sparren, ik waardeer het enorm. Dank je wel.

Leden van de leescommissie, Prof. dr. B Uitdehaag, Prof. dr. O.F. Brouwer, Dr. P. Zwijnenburg, Dr.

A. Thomas, hartelijk dank dat u de tijd nam het manuscript te lezen en te beoordelen. Prof. dr.

B. Uitdehaag, Prof. dr. O.F. Brouwer, Dr. P. Zwijnenburg, Dr. A. Thomas, Dr. M. Engelen en Prof.

dr. J.G. Becher, veel dank dat u plaatsneemt in de promotiecommissie.

I would like to thank all the co-authors for their input.

Lieve Marjan, dank voor je vriendschap en wat fijn dat je me bijstaat als paranimf! Dank ook

voor het samenwerken bij verschillende onderzoeken in dit proefschrift. Lieve Irene, het begon

allemaal in Utrecht bij het Julius Centrum tijdens je eigen promotieonderzoek. Nu al jaren een

zeer waardevolle vriendschap, dank je wel. Super dat ook jij me wilt bijstaan vandaag.

Jan Gerver, je was mijn ‘student’, maar in de zin van wijze levenslessen was dat zeker andersom.

Dank je wel daarvoor en natuurlijk voor al het werk dat je deed voor de klinische variatie data-

verzameling.

Jennifer Konings, dank je wel voor je kunstwerk dat dit proefschrift siert.

159

Ik ben natuurlijk ook veel dank verschuldigd aan iedereen van de wittestofgroep (Margreet, Ilja,

Marianna, Emiel, Nienke, Barbara, Machiel, Koen, Nicole, Carola, Laura, Eline, Stephanie, Lody,

Mohit, Vivi, Annet, Anne) dank voor de vele vruchtbare dinsdagochtendbesprekingen. Dank jul-

lie wel voor alle hulp op het lab, alle discussies en de gezelligheid ook. Speciale dank aan Carola

voor alle hulp bij het kweken. Eline, dank voor het samenwerken bij de studie naar de klinische

variatie en het schrijven van hoofdstuk twee. Veel succes met het vervolg!

Ook wil ik iedereen van de afdeling Medical Genomics van Prof. dr. Heutink bedanken voor de

hulp op het lab en de gezelligheid.

Scot Kimball and Lydia Kutzler thank you very much for all the help with the eIF2B assay. It was

a pleasure to stay in Hershey and perform and learn about the assay in your lab.

Ik heb op verschillende plekjes een bureau gehad, en daarmee allerlei kamergenoten die ik

dankbaar ben voor het meedenken, meeleven en de gezelligheid. Onder andere Femke en

Sebastiaan “de la”. Later de collega’s van de kindergeneeskunde op PK-4X (Suzanne, Marieke,

Gerrit, Jolice, Ilse, Raphaele, Marc, Hester, Katja, Eline, Marleen, Annelies, Sandra, Stefanie, Wil-

lemijn, Miret, Femke, Ineke, Anneke en Annemieke) wat was het vaak gezellig met de lunch en

de bijbehorende vaak hilarische, soms serieuze discussieonderwerpen.

Opleiders, revalidatieartsen en arts-assistenten van het OOR-VUmc veel dank voor de ruimte die

ik kreeg om mijn promotie af te ronden. Speciaal dank aan Prof. dr. Becher, beste Jules, heel veel

dank voor je stimulans en ondersteuning.

In het bijzonder wil ik Hanneke, Margriet, Olga, Rachel, Iris, Rimke, Aileen, Tracy en Evelien

noemen, dank voor jullie aanhoudende interesse en meeleven.

Optimix Foundation for Scientific research, ZonMw en de Dr. Phelps Stichting hartelijk dank voor

de financiele ondersteuning van mijn promotietraject.

Afdeling kindergeneeskunde VUmc en Hoppert B.V. hartelijk dank voor de financiele bijdrage

aan het drukken van dit proefschrift.

Lieve familie en vrienden, bedankt voor al jullie steun, interesse en alle mooie momenten van

het leven die we samen beleven en vieren.

Lieve pa en ma, dank voor alle kansen en mogelijkheden die jullie me hebben geboden. En

niet in de laatste plaats voor veelvuldig oppassen zodat ik naast de opleiding tijd vrij had voor

afronden van mijn promotie. Mijn dank is groot.

Lieve Michiel, ik hou van jou! Dank je wel voor alles.

Willemijn, Laura en Suze, wat voel ik me rijk als jullie moeder.

PP_Hannemieke van der Lei_FINALpdf

Documents

Transcript of PP_Hannemieke van der Lei_FINALpdf