Fatal infantile leukodystrophy
A severe variant of CACH/VWM syndrome, allelic to
chromosome 3q27
P. Francalanci, PhD; E. Eymard–Pierre, PhD; C. Dionisi–Vici, MD; R. Boldrini, MD; F. Piemonte, PhD;
R. Virgili, BS; G. Fariello, MD; C. Bosman, MD; F.M. Santorelli, MD; O. Boespflug–Tanguy, MD;
and E. Bertini, MD

Article abstract—Objective: To describe clinical and neuropathologic studies and linkage analysis on two sisters with a
severe form of leukodystrophy. Methods: A detailed study was performed on the second sister. Genotyping markers for
chromosome 3, including eight additional markers surrounding the vanishing white matter (VWM) locus, were used.
Results: During the first year of life, two sisters developed a severe neurologic condition after an intercurrent infection. It
was accompanied by irritability and stupor with rapid loss of their motor abilities. Results of extensive metabolic studies
were negative. Brain MRI showed severe and diffuse abnormalities of the encephalic white matter. Neuropathologic
examination showed a severe lack of myelin with diffuse vacuolating white matter lesions in the brain, associated with an
increased density of oligodendrocytes and a reduced number of astrocytes on morphometric analysis. In sharp contrast,
the spinal cord white matter was preserved. The affected sibpairs shared a common haplotype for a broad region in
chromosome 3. They were homozygous between markers D3S1565 and D3S3669, including the VWM locus. Conclusions:
This condition is an unusual variant of childhood ataxia with diffuse central hypomyelination (CACH)/VWM, with
characteristic shrinking and perivascular clustering of astrocytes. Haplotype analysis suggests that this variant is allelic
to the VWM locus located on chromosome 3q27.
NEUROLOGY 2001;57:265–270

Childhood ataxia with diffuse CNS hypomyelination
(CACH),1 myelinosclerosis centralis diffusa (MCD),2
and the leukodystrophy named vanishing white matter (VWM) by van der Knaap et al.3 (OMIM #603896)
all identify a form of genetically determined progressive leukodystrophy with a relapsing-remitting
course and an unknown molecular basis.4
This condition is clinically characterized by a progressive ataxic diplegia starting between the ages of
1.5 and 5 years after normal early neurologic development. Seizures, dysarthria, and optic atrophy are
frequent. In some patients, the disease may start
acutely and may progress with recurrent episodes of
neurologic deterioration and lethargy, exacerbated
by febrile illness and minor head trauma.5,6 We describe an unusual familial leukodystrophy with early
onset, rapidly progressive course, and fatal outcome.
Brain MRI and postmortem examination were compatible with a severe variant of CACH/VWM leukodystrophy, allelic to the VWM locus recently located
on chromosome 3q27.4
Case report. Patient 1. A girl born to healthy and unrelated parents had a normal perinatal and neonatal development until the age of 11 months when she suddenly

lost all motor abilities and became irritable after an upper
respiratory tract infection. At admission, physical examination showed normal body weight and length and occipital–frontal circumference. A brain CT showed complete
hypodensity of the cerebral white matter. Brain MRI showed
generalized hypointensity of the white matter in T1-weighted
images, which turned hyperintense in T2-weighted sections.
There was diffuse hypointensity of the encephalic white matter in intermediate-weighted images. Extensive metabolic
workup, including serum creatine kinase, ammonia, orotate,
lactate and pyruvate, very long chain fatty acids, and phytanic acid, was normal. Urinary organic acids GC/MS, urinary sulfites, urinary xanthine, hypoxanthine, uridine,
pseudouridine, and amino acids, as well as urinary oligosaccharides, ␤-galactocerebrosidase, and arylsulfatase enzyme
activities in leukocytes, were normal. CSF analysis showed a
normal immunoglobulin profile without oligoclonal bands or
pleocytosis. Motor and sensory nerve conduction velocities,
morphologic examination of a muscle biopsy specimen, biochemical assay for respiratory chain enzyme complexes in
muscle, and examination for the most frequently encountered mtDNA mutations were all normal.
The patient did not improve during follow-up. The clinical picture was worsened by the presence of nystagmus,
tetraplegia, intermittent dystonia, opisthotonus, seizures,
and feeding difficulties. The girl died at age 18 months.

From the Division of Pathology (Drs. Francalanci, Boldrini, and Bosman, and R. Virgili), Unit of Molecular Medicine, Department of Neurosciences (Drs.
Dionisi–Vici, Piemonte, Fariello, Santorelli, and Bertini), Bambino Gesu’ Research Hospital, IRCCS, Rome, Italy; and INSERM U 384 (Drs. Eymard–Pierre
and Boespflug–Tanguy), Faculté de Médecine, Clermont–Ferrand Cedex, France.
Supported by Ricerca Finalizzata Strategica 2000.
Received December 15, 2000. Accepted in final form March 9, 2001.
Address correspondence and reprint requests to Dr. Enrico Bertini, Dept. of Neurosciences, Unit of Molecular Medicine, Bambino Gesu’ Children’s Hospital,
P.za S. Onofrio, 4, 00165 Rome, Italy; e-mail: ebertini@tin.it
Copyright © 2001 by AAN Enterprises, Inc.

265

Figure 1. (A through F) MRI of Patient
2 performed at 12 months of age.
Fluid-attenuated inversion recovery
(FLAIR) (6000/150) (A) and
intermediate-weighted (3000/40) (B)
images show a diffuse hypointense signal, particularly in FLAIR, suggesting
a cavitation process. (D and E) T1weighted images (587/15) show hypointensity of the ventral mesencephalic
corticospinal tracts and of the cerebellar white matter. (C and F) Diffuse and
symmetric hyperintensity of the cerebral
white matter, internal capsule, and
pontine corticospinal tracts in T2weighted (3000/100) images, with moderate swelling of the telencephalic white
matter.

Patient 2. Patient 2, the youngest sister of Patient 1,
experienced a similar clinical condition at the age of 10
months, after an infective episode. Brain MRI showed generalized hyperintensity in the white matter of the brain,
including the U-fibers, cerebellum, mesencephalon, and
pons in T2-weighted sections (figure 1, C and F) with moderate swelling. Hypointense areas were evident in
intermediate-weighted but particularly in fluid-attenuated
inversion recovery (FLAIR)–weighted areas of the brain
(figure 1, A and B), and in T1-weighted images showing
diffuse and marked hypointensity of the white matter of
the brain, cerebellum, mesencephalic ventral feet, and
pontine corticospinal tracts (figure 1, D through E). This
girl died at age 13 months. Autopsy was performed and
authorized 2 hours after death.
Materials and methods. Neuropathologic examination.
Samples from frontal–temporal– occipital lobes, cerebellum, and brainstem were snap frozen and others were
fixed in 4% formalin and embedded in paraffin and cut into
4-␮m sections. Samples for histology were stained with
hematoxylin-eosin and Luxol fast blue (LFB). In Patient 2
and in a 12-month-old control, who had died of septic
shock, we used a mouse monoclonal antibody for human
2'-3' cyclic nucleotide 3'phosphodiesterase (CNPase,
Chemicon, Temecula, CA) to stain for oligodendrocytes;
amouse anti-human glial fibrillar acid protein (GFAP,
Dako ChemMate, Carpinteria, CA) for astrocytes; and a
mouse monoclonal anti-human CD68 (Dako ChemMate)
for microglia–macrophages. Mitotic activity was assessed
using a mouse monoclonal antibody against nuclear antigen 2KI67,MIB1 (Immunotech, MA). Apoptotic processes
were looked for using terminal deoxynucleotide transferase
266 NEUROLOGY 57

July (2 of 2) 2001

(TdT)-mediated-biotin nick-end labeling (TUNEL) and a
mouse monoclonal antibody against nuclear antigen P53.
Fragments from telencephalic white matter, cerebellar
white matter, pons, medulla oblongata, and cervical spinal
cord were fixed in Karnowski’s fluid, post-fixed in buffered
1% osmium tetroxide, and embedded in Epon 812. Onemicrometer–thick sections were stained with toluidine
blue. Thin sections (400 Å) were collected on uncoated
copper-rhodium grids, stained with uranyl acetate and
lead citrate, and observed at the electron microscope Zeiss
10C (Jena) operating at 70 kV.
Morphometric analysis. Cell counts were performed on
matched white matter specimens (left parietal cortex) from
Patient 2 and a control, using a semiautomatic image analysis system (Quantimet 570, Leica, Cambridge, UK). Total
cells were counted in an area of 3 mm2 that included 35
fields at the magnification of ⫻40. The same area was
analyzed in serial sections immunostained with GFAP,
CNPase, and CD68 to count astrocytes, oligodendrocytes,
and macrophages, respectively.
Statistical analysis employed the Statview 4.1 software
package (Abacus Concepts, SAS Institute, Cary, NC).
Protein studies. Equal amounts of proteins were extracted from postmortem frozen cerebral white specimens
of the patient and of the control and loaded onto a
12%polyacrylamide gel with 5% stacking gel. Electrophoresis was carried out at 100 V and the wet gel was transferred at 200 V for 1 hour in standard buffer. Filters were
immunostained for proteolipid protein (PLP), myelin basic
protein (MBP), and myelin oligodendrocyte glycoprotein
(MOG) as described.7
Linkage analysis. Genomic DNA was extracted from
fibroblasts of the two affected patients and from blood

samples of both parents. Genotyping data corresponding to
chromosome 3 were performed using the markers of the
ABI Linkage Mapping Set version 2 (LMS2) according to
conditions mentioned by the manufacturer (Perkin-Elmer,
Foster City, CA). In addition, 12 markers surrounding the
VWM locus were analyzed in the same conditions, according
to the primer sequence of the Genethon microsatellite linkage map (ftp://ftp.genethon.fr/pub/Gmap/Nature-1995/data/
data_chrom3). PCR products were sized using an ABI 377
sequencer using Genescan 2.1 and Genotyper 2.0 programs
(Perkin Elmer, Foster City, CA).
Results. Neuropathologic examination. Brain weight
was 980 g, in the high range values compared to normal

age-matched controls (886 ⫾ 23). Gross examination of the
brain showed no cerebral or cerebellar atrophy. On sectioning, all gray matter structures appeared normal. The ventricles were not enlarged. There was diffuse softening and
swelling of the white matter that appeared grayish and
gelatinous with multiple cavitated areas, particularly in
the telencephalic regions. Cerebellar white matter was
also swelled and there was a grayish discoloration of the
dentate nucleus. The brainstem appeared normal.
Histologic examination showed that hypomyelination
was associated with cavitation and absence of inflammation without prominent axonal and neuronal changes. Reactive astrogliosis and increased microglial response did
not accompany the severity of white matter changes. My-

Figure 2. (A through E) Synopsis of the
optic microscopic observations in Patient
2. A semi-schematic design of the different encephalic structures is represented
on the left side and the essential optic
microscopic abnormalities are summarized on the right side. Images on the
left have fields covered by oblique lines
that correspond to abnormal areas detailed at the optic microscopic level on
the right. (A) (Luxol fast blue [LFB], parietal cortex; the molecular layer is on
the right side of the photograph) shows
absence of myelin stain and multiple
cavitations. (B) (LFB) shows discoloration and vacuolization of the cerebellar
white matter with a relative preservation of the medullary layer. C (LFB)
shows that vacuolization of the pontine
white matter was particularly prominent in the corticospinal tract and tractus tegmentalis centralis, and (D) (LFB)
shows that the same abnormality was
present in the inferior olivary hilus. E
(LFB, spinal cervical level) does not
show any prominent abnormality.

July (2 of 2) 2001

NEUROLOGY 57

267

Figure 3. (A through F) Histochemical
examination of the telencephalic white
matter (left parietal region) of Patient 2
(A, C, and E) and of a control (B, D, F).
A (closed arrowhead) and B (halfopened arrowhead) are immunostained
for CNPase (oligodendrocytes) and C
through F are immunostained for GFAP
(astrocytes). Notice that CNPase immunostaining is limited to the cytoplasm of
oligodendrocytes in A (closed arrowhead), with a normal aspect. (C) Astrocytes frequently had a perivascular
clustering in the patient but not in the
control. C and E show marked astrocytic shrinkage (closed arrowhead) with
reduced astrocytic network (neuropile)
whereas D and F show a normal pattern, in which astrocytes are swollen
(half-open arrowheads) for postmortem
events. Bar ⫽ 100 ␮m.

elinated fibers were completely absent with LFB stain in
the telencephalic white matter (figure 2A) and mesencephalic cortical–spinal tract. The white matter was severely
involved in these regions with spongiform network and
multiple cavitations (figure 2A). Prominent discoloration
and vacuolization was also evident in the cerebellar white
matter (figure 2B), the pontine cortical–spinal tract (figure

2C), and the tractus tegmentalis centralis. In the medulla
oblongata we found vacuolating white matter lesions restricted to the inferior olivary hilus (figure 2D). No alterations were found in the spinal cord (figure 2E). Myelin
sheets were also absent in the telencephalic regions with
CNPase immunostaining (figure 3A). Most of the axons
were preserved and loosely mixed with phagocytic macro-

Figure 4. (A through D) Ultrastructural
examination of Patient 2. (A and B) Telencephalic white matter. A (bar ⫽ 8 ␮m)
shows complete absence of myelination,
three normal oligodendrocytes, and axonal swelling. In B (bar ⫽ 1.6 ␮m) mitochondria are well-preserved (arrow). The
extracellular space is not increased and
there is axonal swelling. C (bar ⫽ 2.5
␮m) and D (bar ⫽ 1.2 ␮m) (spinal cord)
show two myelinated axons with intramyelin vacuolization (half-opened arrowheads). Myelin has a normal structure
and compaction (closed arrowhead).

268 NEUROLOGY 57

July (2 of 2) 2001

Morphometric analysis showed that the mean value of
total cell count was 63 ⫾ 24.5 in the patient and 61.9 ⫾
7.99 in the control. We found an increased number of oligodendrocytes (30.1 ⫾ 5.97 versus 20.2 ⫾ 5.49, p ⬍ 0.0001)
and macrophages (10.2 ⫾ 3.8 versus 3.94 ⫾ 1.5, p ⬍
0.0001) in the patient. Conversely, astrocytes were reduced
in number (16.1 ⫾ 5.49 versus 38.5 ⫾ 6.34, p ⬍ 0.0001).
There were rare KI67.MIB1-positive cells in the cerebral
white matter ruling out an active mitotic activity. TUNEL
assay and P53 antibody were negative excluding apoptotic
cellular events.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of myelin proteins and Western blot analysis for
PLP, MBP, MOG, and CNPase did not show abnormal
bands (courtesy of Dr. J.M. Matthieu).
Ultrastructural findings. Ultrastructural examination
of the brain confirmed the complete absence of myelin in
the telencephalic and cerebellar white matter. At the periphery of the cavitating areas, all the axons were completely devoid of myelin sheets, and most of them were
moderately swelled. Intermingled with these “naked” axons we found numerous oligodendrocytes without any abnormality (figure 4A). The extracellular space was not
increased (figure 4B). A few macrophages contained myelin debris and lipid droplets. Astrocytes had no cytoplasmic
inclusions. In the spinal cord white matter, the axons retained their myelin sheets with preservation of myelin
structure and compaction (figure 4, C and D). However,
many axons showed very thin myelin layer and some
showed frequent intra-myelinic vacuolization in the inner
and outer surface (figure 4, C and D). Peripheral nerves
and roots did not show any myelin abnormalities.
Haplotypes analysis. The affected sibpairs shared a
common haplotype for a broad chromosome 3 region (figure
5). In addition, patients were homozygous between markers D3S1565 and D3S3669, including the VWM locus.

Figure 5. Haplotype analysis using 27 microsatellite
markers along the long arm of chromosome 3. Boxed
markers delimit the region in which the two affected sibs
are identical by descent showing the same homozygote
haplotype. All markers of the vanishing white matter locus
(in bold) are included in this region of homozygosity.
phages and oligodendrocytes. CD68-positive macrophages
were relatively increased in the white matter with a preferential perivascular displacement. With GFAP stain astrocytes were shrinking and the astrocytic network was
markedly reduced when compared to control (figure 3, C
through F). It was frequent to find astrocytic perivascular
aggregates (figure 3C) that were not prominent in subpial
regions.

Discussion. Infantile onset and a rapid outcome
characterize the clinical course of this familial fatal
leukodystrophy. Neuroradiologic and neuropathologic findings closely resemble what has been described as VWM leukodystrophy or CACH,8 despite
the lowest age at onset and a more rapid and severe
clinical course. In both patients the acute neurologic
symptoms started after a febrile illness, mimicking a
metabolic or an acquired postinfectious disorder.
These etiologies were ruled out after an extensive
clinical and biochemical workup.
Brain MRI showed marked and diffuse T1hypointense images affecting all the white matter of
the brain, including U-fibers, the cerebellum, and
the cortical–spinal tracts of the mesencephalon and
pons. In addition, intermediate-weighted but particularly FLAIR-weighted images correlated with the
widespread vacuolization observed at the neuropathologic examination. We found no ventricular dilatation and the white matter appeared to be
moderately swelled although the patients had no
macrocephaly. Altogether, these neuroimaging findings have the characteristics of a severe variant of
CACH/VWM syndrome, as reported.9-11 Neuropathologically, we found a profound hypomyelination of
July (2 of 2) 2001

NEUROLOGY 57

269

the cerebral and cerebellar white matter, and of the
brainstem descending cortical–spinal pathways. In
addition, we observed characteristic demyelinating
lesions in the ileum of inferior olivary nuclei as recently described in CACH/VWM.8 The degree of hypomyelination had a cranio-caudal gradient ranging
from absence of myelin in the brain to a normal
amount in the spinal cord, following backward the
sequence of CNS myelination during development.12
These findings may be due to a demyelinating process that is more pronounced in the cerebral white
matter than in the spinal cord. Alternatively, hypomyelination can affect preferentially white matter
areas that have not yet completed myelination. In
this case, it can be hypothesized that the disease
process does not interfere with myelin development
until myelin breakdown and disturbed myelination
is triggered by trauma or infectious episodes.
Increased density of oligodendrocytes without evidence of increased mitotic activity has been proposed
as a hallmark of CACH/VWM.8 Likewise, we have
observed these same neuropathologic characteristics
in our patient. Moreover, we found shrinking of astrocytes with reduced GFAP immunostaining of the
neuropil. Astrocytes were reduced in number and
frequently in a perivascular position. This feature
has never been reported in CACH/VWM. At the
gross examination of the brain we observed that
weight was increased to the high range weight values, and that there was swelling of the white matter,
giving the impression of an increase of water or solutes in the white matter. Ultrastructurally, white
matter axons were swelled and the extracellular
space was preserved. Overall, the distinguishing features of this disease are shrinking of astrocytes associated with axonal swelling.
Astrocytes invest nearly all the extrasynaptic neuronal surfaces and of the various functions, they are
implicated in transport mechanisms and in buffering
mechanisms.13-14 Astrocytes also promote outgrowth
of oligodendrocytes, an early event of myelinogenesis,15 and absence or overexpression of the astrocytespecific GFAP in mouse model produces severe
leukoencephalopathies.16,17 More recently, GFAP mutations have been reported in patients with Alexander
disease, a severe leukoencephalopathy characterized
by progressive macrocephaly with white matter cavitation and presence of Rosenthal fibers within astrocytes.18 Thus, a dysfunction of astrocytes may be
responsible for hypomyelination in this condition. Family data have indicated that CACH/VWM syndrome
has an autosomal recessive inheritance with age dependent penetrance.6 A genome linkage screening of 19
VWM families suggested single gene localization on
chromosome 3q274 without genetic heterogeneity between childhood and juvenile onset forms. We demonstrate that the sibpairs affected by this severe variant

270 NEUROLOGY 57

July (2 of 2) 2001

of CACH/VWM are homozygous for the critical region
containing the gene for the CACH/VWM leukodystrophy, and do not exclude linkage to this locus. Our combined neuropathologic, neuroradiologic, and genetic
results suggest that this variant is allelic to the VWM
locus, broadening the phenotypic variability of this
disorder.
References
1. Schiffmann R, Moller JR, Trapp BD, et al. Childhood ataxia
with diffuse central nervous system hypomyelination. Ann
Neurol 1994;35:331–340.
2. 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 magnetic
resonance spectroscopy. Neuropediatrics 1993;24:244 –248.
3. van der Knaap MS, Barth PG, Gabreels FJ, et al. A new
leukoencephalopathy with vanishing white matter. Neurology
1997;48:845– 855.
4. Leegwater PA, Konst AA, Kuyt B, et al. The gene for leukoencephalopathy with vanishing white matter is located on chromosome 3q27. Am J Hum Genet 1999;65:728 –734.
5. Tedeschi G, Schiffmann R, Barton NW, et al. Proton magnetic
resonance spectroscopic imaging in childhood ataxia with diffuse central nervous system hypomyelination. Neurology
1995;45:1526 –1532.
6. van der Knaap MS, Kamphorst W, Barth PG, et al. Phenotypic variation in leukoencephalopathy with vanishing white
matter. Neurology 1998;51:540 –547.
7. Gardinier MV, Amiguet P, Linington C, et al. Myelin/oligodendrocyte glycoprotein is a unique member of the immunoglobulin superfamily. J Neurosci Res 1992;33:177–187.
8. Rodriguez D, Gelot A, della Gaspera B, et al. Increased density of oligodendrocytes in childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological
and biochemical study of two cases. Acta Neuropathol 1999;
97:469 – 480.
9. Van der Knaap MS, Valk J. Magnetic resonance of myelin,
myelination and myelin disorders, 2nd edition. Berlin:
Springer, 1995.
10. van der Knaap MS, Valk J, Barth PG, et al. Leukoencephalopathy with swelling in children and adolescents: MRI patterns
and differential diagnosis. Neuroradiology 1995;37:679 – 686.
11. van der Knaap MS, Barth PG, Stroink H, et al. Leukoencephalopathy with swelling and a discrepantly mild clinical course
in eight children. Ann Neurol 1995c;37:324 –334.
12. Brody BA, Kinney HC, Kloman AS, et al. Sequence of central
nervous system myelination in human infancy. I. An autopsy
study of myelination. J Neuropathol Exp Neurol 1987;46:283–
301.
13. Albers RW, Siegel GJ. Membrane transport in basic neurochemistry. In: Siegel GJ, Agranoff BW, Albers RW, Fisher SK,
Uhler MD, eds. Philadelphia: Lippincott-Raven, 1999.
14. Baslow MH. Molecular water pumps and the aetiology of
Canavan disease: a case of the sorcerer’s apprentice. J Inherit
Metab Dis 1999;22:99 –101.
15. Oh LY, Yong VW. Astrocytes promote process outgrowth by
adult human oligodendrocytes in vitro through interaction between bFGF and astrocyte extracellular matrix. Glia 1996;17:
237–253.
16. Liedtke W, Edelmann W, Bieri PL, et al. GFAP is necessary
for the integrity of CNS white matter architecture and longterm maintenance of myelination. Neuron 1996;17:607– 615.
17. Messing A, Head MW, Galles K, et al. Fatal encephalopathy
with astrocyte inclusions in GFAP transgenic mice. Am J
Pathol 1998;152:391–398.
18. Brenner M, Johnson AB, Boespflug-Tanguy O, et al. Mutations in glial fibrillary acidic protein (GFAP) associated with
Alexander disease. Nat Genet 2001;27:117–120.