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Cree leukoencephalopathy and CACHVWM disease are allelic at the EIF2B5 locus.

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Cree Leukoencephalopathy
and CACH/VWM Disease
Are Allelic at the
EIF2B5 Locus
Anne Fogli, PhD,1 Kondi Wong, MD,2
Eleonore Eymard-Pierre, PhD,1 Jack Wenger, BSc,2
John-Paul Bouffard, MD,2 Ehud Goldin, PhD,3
Deborah N. Black, MD,4
Odile Boespflug-Tanguy, MD, PhD,1
and Raphael Schiffmann, MD3
Cree leukoencephalopathy is a rapidly fatal infantile autosomal recessive leukodystrophy of unknown cause observed in the native North American Cree and Chippewayan indigenous population. We found in the brain of
affected individuals the typical foamy cells with the oligodendroglial phenotype described in central hypomyelination syndrome/vanishing white matter, a syndrome related to mutations in the genes encoding the five
subunits of the eucaryotic translation initiation factor
eIF2B. In three patients of two Cree families, we found a
homozygous missense mutation resulting in a histidine
substitution at arginine 195 of ␧-eIF2B.
Ann Neurol 2002;52:506 –510
An infantile leukoencephalopathy among the native
Cree and Chippewayan indigenous population in
Northern Quebec and Manitoba was first described in
1988.1 The onset of Cree leukoencephalopathy (CLE)
is between 3 and 9 months of age with death in 100%
by 21 months of age. Hypotonia often is noted in early
infancy followed by relatively sudden onset of seizures,
spasticity, hyperventilation, vomiting, and diarrhea, often in the setting of a febrile illness. Onset is followed
by developmental regression, lethargy, blindness, and
cessation of head growth seen as flattening of the head
circumference curve.1 Computerized tomography of
From the 1Institut National de la Santé et de la Recherche Médicale
UMR 384, Facultéde Médecine, Clermont-Ferrand, France; 2Department of Neuropathology at the Armed Forces Institute of Pathology, Washington, DC; 3Developmental and Metabolic Neurology Branch, National Institutes of Health, Bethesda, MD; and
Department of Psychiatry, Université de Montréal, Montréal, Quebec, Canada.
Received Apr 24, 2002, and in revised form Jun 13. Accepted for
publication Jun 14, 2002.
Published online Aug 28, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10339
Address correspondence to Dr Boespflug-Tanguy, INSERM UMR
384, Faculté de Médecine, 28, place Henri Dunant BP 38, 63001
Clermont-Ferrand Cedex, France. E-mail: odile.boespflug@inserm.
© 2002 Wiley-Liss, Inc.
the head shows symmetrically hypodense white matter.
A similar image was seen on T1-weighted head magnetic resonance imaging that showed symmetrical diffuse attenuation of hemispherical and often cerebellar
white matter.2 T2-weighted magnetic resonance images
showed hyperintense white matter that included the
subcortical fibers, basal ganglia, and thalamus. Previous
gross neuropathological examination showed that white
matter was grayish white with translucent zones and
subcortical cavitation. Microscopic examination showed
diffuse white matter vacuolation in some cases and astrogliosis with presence of oligodendrocytes and cells described as lipid-laden macrophages.1,2 Parents of affected
children are normal, and because there is a strong history of consanguinity in this population, CLE is considered autosomal recessive.
We microscopically investigated three brains of CLE
patients and found the typical foamy oligodendrocytes
we earlier reported in childhood ataxia with diffuse
central hypomyelination syndrome (CACH)3,4 also
called myelinopathia centralis diffusa5 or vanishing
white matter disease (VWM).6 Mutations in each of
the five subunits of the translation initiation factor 2B
(eIF2B) have been reported recently in this syndrome.7,8 We first investigated the EIF2B5 gene,
which encodes the ε subunit, and is localized in the
3q27 region,9 a region involved in CACH/VWM including in a severe variant we recently reported.10,11
Subjects and Methods
Light Microscopy
Paraffin blocks of tissue were histologically sectioned to a 9␮m
thickness and stained with standard hematoxylin and eosin,
periodic acid–Schiff (PAS)/Luxol fast blue, Bielschowsky, and
modified Alcian blue (8GX)/PAS. The modifications to the
standard AB8GX/PAS staining procedure consisted of placing
the histological sections in 3% acetic acid at pH 2.5 for 5
minutes before the Alcian blue 8GX reagent in 3% acetic acid
solution for 30 minutes. After a 5-minute wash, the sections
were placed in periodic acid solution for 10 minutes followed
by Schiff’s reagent (Colmann’s Feulgen reagent) for 20 minutes. The sodium metabisulfite rinse was purposefully omitted.
The slides were washed in warm tap water until the water was
pink, and the slides were left to stand in the warm pink tap
water for 5 minutes. (The rational being that the pink dye
neutralizes chlorine; otherwise, chlorine bleaches the Schiff’s
reagent resulting in loss of coloration.) The sections then were
counterstained with Richard Allen Scientific (Kalamazoo, MI)
hematoxylin I counterstain.
EIF2B5 Analysis
SUBJECTS. We analyzed two CLE affected families (894
and 866; Fig 1). Patients II1 and II2 of Family 894 had mild
motor developmental delay during early infancy followed by
acute neurological deterioration during a febrile illness at
ages 7 months and 5 months, respectively. Both patients
died within 10 days of onset of acute neurological symptoms. The patient of Family 866 presented at age 4 months
with developmental delay followed by progressive deteriora-
Fig 1. EIF2B5 mutations in Cree leukoencephalopathy (CLE) families. A G584A mutation was found in an homozygous state in
the three CLE affected individuals (Family 866-II1, Family 894-II1 and II2) and in an heterozygous state in both parents (Family
866-I1 and I2, Family 894-I1 and I2) and in one unaffected individual (Family 894-II4). Two individuals were homozygous for
the wild-type G nucleotide (Family 894-II3 and Family 894-II5).
Fogli et al: CLE and EIF2B
tion to a decerebrate, nonresponsive state by age 13 months
and death 1 month later. The appropriate institutional review boards approved the study, and written informed consent was obtained from all participating subjects or from
their legal guardian.
DNA AND SEQUENCE ANALYSES. Genomic DNA was extracted from blood lymphocytes using the Nucleon kit (Amersham, Buckinghamshire, UK). We used 10 sets of oligonucleotide primers to amplify by polymerase chain reaction and
sequence the 16 coding regions of the EIF2B5 gene including 39 to 173 nucleotides of the intronic regions (primer
sequences and conditions are available upon request).
“Foamy” Oligodendrogial Cells Are Observed in Cree
Leukoencephalopathy Brains
Cerebral cortical and cerebellar white matter was rarefied, with profound myelin loss, but with relative
sparing of the axons. The cerebellar cortex was atrophic
with thinning of the foliae and loss of cells in the granular layer. Marked neuronal loss also was observed in
the hippocampus. Numerous cells with round to oval
nuclei and abundant foamy-vacuolated cytoplasm were
scattered throughout the cerebral and cerebellar deep
white matter on hematoxylin and eosin staining (Fig
2A and B). The foamy cells had light blue cytoplasm
and a bright red Golgi region on modified PAS/Alcian
blue 8GX staining (see Fig 2C). The foamy cells were
strongly immunoreactive to myelin oligodendrocyte
glycoprotein antibody (see Fig 2D), as were the foamy
oligodendroglial cells seen in CACH/VWM (see Fig
2F). The abundance of abnormal foamy oligodendroglial cells was in direct proportion to the amount of
white matter left in the brain. There were no parenchymal macrophages identifiable with the CD68
marker (see Fig 2E). Glial fibrillary acidic protein
staining showed atypical astrogliosis with blunted processes (see Fig 2G) and many nests of atypical gliofibrillar cells scattered throughout the white matter (see
Fig 2H and I).
The Gene Causing Cree Leukoencephalopathy
Syndrome Is EIF2B5
Similarities in pathological features between CLE and
CACH/VWM led us to look for possible mutations in
the EIF2B genes. Because a severe variant of CACH is
linked to chromosome 3q27, we first investigated the
EIF2B5 gene encoding the ε-subunit of the eIF2B factor. We found a homozygous G584A missense mutation in exon 4 of EIF2B5 in all three affected individuals resulting in a histidine substitution at arginine 195
of ε-eIF2B (see Fig 1). Unaffected parents in both families were heterozygous for the mutation as well as one
unaffected sibling, whereas two nonaffected children
were homozygous for the wild-type allele (see Fig 1).
Annals of Neurology
Vol 52
No 4
October 2002
This homozygous G584A mutation was not present in
a control group of 110 individuals including 50 nonaffected adults from the Cree population. In this Cree
control group, five individuals have this mutation in a
heterozygous state yielding a mutated allele frequency
of 1 of 20.
We recently noticed similarities and differences between CLE and a severe variant of CACH/VWM. Investigating microscopically the brain of three CLE
patients, we found neuropathological hallmarks of
CACH/VWM: cavitating orthochromatic leukodystrophy with rarity of myelin breakdown and relative sparing of axons.12 In all three Cree brains examined, we
observed abnormal cells identical to the foamy oligodendroglial cells that we described in CACH/VWM.4
Atypical astrogliosis with blunted processes and many
nests of atypical gliofibrillar cells scattered throughout
the white matter were observed in CLE and absent in
CACH/VWM. Subsequently, using molecular analysis,
we demonstrated that CLE is allelic to CACH/VWM
at the 3q27 locus: all affected Cree infants analyzed
showed a founder homozygous missense mutation
R195H in the ε-eIF2B.
The eucaryotic translation initiation factor 2B
(eIF2B) is composed of five different subunits (␣, ␤,
␥, ␦, and ε) and converts the protein synthesis initiation factor 2 (eIF2) from an inactive guanine diphosphate (GDP)-bound form to an active eIF2–guanine tri-phosphate (GTP) complex. In CACH/VWM
patients, mutations has been found in each of these
subunits without correlation between severity of the
phenotype and the mutations detected in the eIF2B
genes; both childhood and juvenile forms were observed in siblings.7 In contrast, all CLE patients express a very severe phenotype. Homozygous R195H
mutation that affects the guanine exchange factor domain of the ε-eIF2B has never been reported in
CACH/VWM7,8 to our knowledge, or in our large
patient population, including in a recently reported
severe form of CACH/VWM. We recently identified
a heterozygous R195H ε-eIF2B mutation in only one
CACH/VWM family from Highlands in Scotland (A.
Fogli, D. Rodriguez, E. Bertini, R. Shiffmann, D.
Boespflug-Tanguy, unpublished results). The northern Quebec Cree first encountered Europeans in the
early 1700s; these were Scottish fur traders from the
Hudson Bay Trading Company.14 Probands from the
two Cree families in this report can trace their paternal ancestors to three English Hudson Bay Company
employees around 1770 (J. Pachano, personal communication). A founder effect arising from a common
ancestor may explain the presence of the ε-eIF2B mutation in this population and perhaps also in Scotland. The indigenous North American population of
Fig 2. Foamy oligodendroglial cells in the white matter of Cree leukoencephalopathy (CLE) brains. Light microscopy of CLE white
matter brain sections (A, B, hematoxylin and eosin stain) shows numerous cells having small, eccentrically placed, round to oval
nuclei (A, arrows) with fine chromatin and abundant foamy cytoplasm. When the nucleus is out of the plane of section, only a
membrane-bound agglomeration of foamy cytoplasm is visible (A, arrowheads). Cytoplasmic vacuolations (B, arrows) occasionally
are identified in the foamy cells that range in size from 12␮m to over 35␮m in diameter (B, arrowhead). Modified periodic acid–
Schiff/Alcian blue 8GX staining (C) shows light blue–stained cytoplasm with a bright red Golgi region (C, arrow). The foamy cells
react strongly to antibodies against myelin oligodendrocyte glycoprotein (MOG; D) but are nonreactive to CD68 (E) indicating an
oligodendroglial rather than a monocytic phenotype. Compare the MOG immunoreactivity of foamy oligodendroglial cells in CLE
(D, arrowheads) with those in central hypomyelination syndrome/vanishing white matter (F, arrowhead). Glial fibrillary acidic
protein immunostaining (G–I) highlights atypical astrogliosis with coarse, blunted glial processes (G, arrowheads) and unusual gliofibrillary structures (H, I; arrowheads). A to D and F are ⫻400 original magnification; E and G to I are ⫻200 original
northern Quebec may have evolved an adaptation to
an extremely cold environment, rendering them particularly susceptible to dysregulation of protective
mechanisms that respond to temperature elevation.
Regulation of eIF2B represents a major protective
mechanism of cells in response to heat stress.15 This
mutation and other EIF2B5 mutations may increase
the binding affinity of GTP/GDP preventing the cell
from sensing a decrease in nucleotide availability during a traumatic event, thereby causing the protein to
stay constitutively active. As a consequence during
stress (eg, fever), eIF2B may be unable to decrease
protein synthesis causing accumulation of denatured
proteins. However, a reduction of eIF2B activity cannot be ruled out currently.
A long presymptomatic phase, despite the presence
Fogli et al: CLE and EIF2B
of severe white matter abnormalities on magnetic resonance imaging, has been observed in CACH/VWM,
suggesting a pre-existing dysfunction in myelinogenesis.3 The basal ganglia and thalamic abnormalities described in CLE2 have not been observed in CACH/
VWM but are classically observed in other severe
leukoencephalopathies with vacuolation of the white
matter such as Alexander’s disease that is caused by
astrocytic dysfunction caused by glial fibrillary acidic
protein mutations (OMIM 137780).16 CLE may represent the most severe observed form of eIF2B-related
disorders, possibly because of an exaggerated response
to heat stress induced by a common infectious illness.
In this population with a very high rate of consanguinity, the ability to detect heterozygous carriers and
affected fetuses provides the possibility for genetic
counseling and prevention of this very devastating
This work was supported by grants from the European Leukodystrophy Association (ELA, G. Alba, president), the Institut National
de la Santé et de la Recherche Médicale (INSERM projet PROGRES), the Jean Pierre and Nancy Boespflug myopathic research
foundation, and the National Institute of Neurological Disorders
and Stroke (protocol 97-N-0170).
We gratefully acknowledge the participation of the patients’ families
and the support of the Eeyou Awaash Foundation (A. Bearskin,
President; W. Neacappo, Vice-President). We also thank L. Dauche,
P. Combes, F. Gauthier, and G. Giraud for technical help in processing blood samples and in sequencing, and D. G. Schoenberg,
MS, for editorial assistance.
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