Genotype–phenotype correlation in inherited brain
myelination defects due to proteolipid protein gene
Fabrice Cailloux, Fernande Gauthier-Barichard, Corinne Mimault, Valerie Isabelle,
Virginie Courtois, Genevi` eve Giraud, Bernard Dastugue, Odile Boespflug-Tanguy and
Clinical European Network on Brain Dysmyelinating Disease†
INSERMU384, Facult´ e de M´ edecine, Clermont-Ferrand, France
Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type2 (SPG2) are X-linked developmental
defects of myelin formation affecting the central nervous system (CNS). They differ clinically in the onset
and severity of the motor disability but both are allelic to the proteolipid protein gene (PLP), which
encodes the principal protein components of CNS myelin, PLP and its spliced isoform, DM20. We
investigated 52PMD and 28SPG families without large PLP duplications or deletions by genomic PCR
amplification and sequencing of the PLP gene. We identified 29 and 4abnormalities respectively. Patients
with PLP mutations presented a large range of disease severity, with a continuum between severe forms of
PMD, without motor development, to pure forms of SPG. Clinical severity was found to be correlated with
the nature of the mutation, suggesting a distinct strategy for detection of PLP point mutations between
severe PMD, mild PMD and SPG. Single amino-acid changes in highly conserved regions of the DM20
protein caused the most severe forms of PMD. Substitutions of less conserved amino acids, truncations,
absence of the protein and PLP-specific mutations caused the milder forms of PMD and SPG. Therefore,
the interactions and stability of the mutated proteins has a major effect on the severity of PLP-related
diseases. European Journal of Human Genetics (2000) 8, 837–845.
Keywords: proteolipid protein gene (PLP); Pelizaeus Merzbacher disease (PMD); spastic paraplegia (SPG); myelin
disorder; X chromosome; molecular diagnosis
The distinction between inherited defects in central nervous
system (CNS) myelination (dysmyelinating diseases) and
genetic diseases leading to myelin destruction (demyelinat-
ing diseases) was first made at the beginning of the twentieth
century by Pelizaeus and Merzbacher, who described a
dysmyelinating disease that became the prototype of this
class of disorders. They defined this disease as involving (i)
early-onset motor disability inherited via an X -linked reces-
sive trait and (ii) a neuropathological pattern of myelin
sheath disappearance strictly limited to the CNS, with no
nerve cell or axon involvement and no inflammation.
Introduction of electrophysiological and magnetic reso-
nance imaging (MRI) techniques to demonstrate the abnor-
mal formation of CNS myelin, defined new diagnostic
criteria for PMD.1The proteolipid protein gene (PLP) was
identified as the causative locus for PMD.2This gene, which
maps to X q22, encodes two proteolipid proteins in oligoden-
drocytes, PLP and its spliced isoform, DM20, which account
for more than 50% of the total protein mass of CNS myelin.
Correspondence: Odile Boespflug-Tanguy, INSERM U384, Facult´ e de
M´ edecine, 28, place Henri Dunant, BP38, 63001Clermont-Ferrand,
Cedex, France. Tel: + 33473608024; Fax: + 33473276132; E-mail:
†ENBDD: Clinical European Network on Brain Dysmyelinating Disease:
M Baethmann, E Bertini, B Chabrol, JM Cuisset, J Gaertner, F Hanefeld,
A Kohlschutter, P Landrieu, JM Lopes-Terradas, M Mayer, S Peudenier,
JM Prats-Vinas, D Rodriguez, D Rating, R Surtees, G Uziel, L Vallee and
T Voit, with the collaboration of J Aicardi, P Amarenco, I Bernard, J
Boulloche, ML Chauvet, G Cioni, I Desguerre, C DeSouza, D Fontan, A
Gal, C H¨ ubner, I Kr¨ ageloh-Mann, G Kurlemann, D Lacombe, C LeBerre,
B Lemarec, J Lopez-Pison, C Moraine, K Muller, A Nivelon-Chevalier, JP
Nuyts, K Pohl, E Scalais, W Schrank, G Sebire, M Spada, H Steinbock, S
St¨ ocker, M Tardieu, A Toutain and L Zelante
Received 23 July 1999; revised 5 June 2000; accepted 20 June 2000
European Journal of Human Genetics (2000) 8, 837–845
© 2000 Macmillan Publishers LtdAll rights reserved 1018–4813/00 $15.00 y
PLP is a major structural component of CNS myelin,3whereas
DM20 which is produced earlier in CNS development may be
involved in oligodendrocyte differentiation and survival.4,5A
wide spectrum of PLP mutations have been found in animal
models6and PMD patients: duplication of the entire PLP
gene is the most frequent causative mutation,7,8with other
mutations in the coding and non-coding regions of PLP
accounting for 10 to 25% of cases.9PLP gene mutations have
also been associated with X -linked spastic paraplegia2
(SPG2).10–12This disease involves early-onset progressive gait
difficulties associated in most patients with neurological
signs classically found in PMD patients (nystagmus, ataxia,
mild mental retardation), defining complicated forms of
However, SPG2 differs from PMD in that motor milestone
achievement is almost normal in the first year of life for
SPG2. We investigated 52PMD and 28SPG families with no
large PLP duplications or deletions by PCR amplification and
sequencing of the seven coding regions and the splice sites of
PLP. Abnormalities were identified in 29PMD and 4SPG2
families, missense mutations being the most common.
The 52PLP-mutated patients presented a large range of
disease severity, which we graded according to the best motor
function achieved between 1 and 10years of age. Clinical
severity was found to be correlated with the nature of the
mutation, suggesting a distinct strategy for detection of PLP
point mutations between severe PMD, mild PMD and SPG.
The interactions and stability of the mutated proteins was
found to have a major effect on the severity of PLP-related
Selection of patients
Patients with PMD or SPG were referred to us by paediatric
neurologists from various European countries.
104PMD patients were selected for PLP analysis based on
clinical, electrophysiological and magnetic resonance imag-
ing criteria described elsewhere.2The 52patients with no
large PLP duplications or deletions were eligible for PLP
analysis by PCR amplification and sequencing of the coding
and splicing regions of the gene.
Twenty-nine SPG patients were selected according to the
following clinical criteria:
(ii) early-onset5PG (before 10years of age), with almost
normal milestone achievement during the first year of
(iii) slow progression,
(iv) low CNS conduction velocities on somatosensory or
brainstem evoked potential evaluation, and/or ‘hypo-
myelinated’ areas of the brain on MRI (iso or hyper T1
associated with hyper T2 signals of the white matter
relative to the grey matter),
(v) no other known cause (including normal results for:
spinal cord MRI, amino acid and organic acid gas
chromatography, lactate/pyruvate ratio, lysosomal
enzymes, very long chain fatty acids, HTLV1
The 28patients with no large PLP duplications or deletions
were eligible for subsequent PLP analysis.
Classification of PLP-mutated families
In the 33families with identified PLP mutations, detailed
clinical information was collected from 52 of the 56patients
reported to be affected. These patients had a mean age of
18.5 + /–17.5years at the time of clinical evaluation.
The PMD group (42patients) presented early impairment
(before 3months) of normal motor development associated
with neurological signs, which were gradually modified by
the maturing nervous system: early nystagmus was observed
in all patients, except family 35, at a mean age of
1.2 + /–1.5months; bobbing movements of the head and
trunk, choreoathetotic movements of the limbs and cer-
ebellar ataxia were observed between 6 and 18months of age;
progressive spastic paraplegia with pyramidal tract signs were
clearly present after 2–4years. In all cases, motor handicap
was greater than impairment of psycho-intellectual develop-
ment. No rapid degradation was observed. Few patients had
no motor acquisition but the performance of most improved
slowly until age 10–12years. Performance differed between
patients. After reaching a plateau, a slow deterioration was
observed after 10–20years, accompanied by the development
of cortical atrophy on MRI. Five patients died (12%) at a
mean age of 30 + /–11years (range: 3–60years).
We classified patients into five forms according to the best
motor function they were able to achieve between the ages of
1 and 10years:
• form 0 included 8patients (19%), with a mean age of
5.6 + /–2.3years at clinical evaluation, with no motor
achievement; one presented stridor (family 49), one
(family 26) had seizures well controlled by anti-epi-
leptic drugs and one died at age3 (family 107);
• form 1 included 12patients (28%), with a mean age of
8.3 + /–5years at clinical evaluation, who achieved
head control between 2 and 4years; one died at
10years (family 29);
• form 2 included 6patients (14%), with a mean age of
22.1 + /–20 at clinical evaluation, able to achieve a
sitting position between 2 and 5years; half achieved a
dysarthric but comprehensible speech;
• form 3 included 13patients (31%), with a mean age of
19.3 + /–15 at clinical evaluation, able to achieve a
sitting position between 1 and 2years, to walk with
Genotype–phenotype correlation in PLP disease
F Cailloux et al
European Journal of Human Genetics
depending on changes in the interactions between mutated
proteolipid protein monomers. Substitutions of very con-
served aa and truncated proteins with large changes in
C-terminal structure have the largest effect on the intra-
cellular interactions of mutated proteins. However, the
cellular mechanisms responsible for oligodendrocyte death
This work was supported by grants from the Institut National de la
Sant´ e et de la Recherche M´ edicale (R´ eseau Recherche Clinique,
PROGRES), European Leukodystrophies Association (ELA France), the
EU Biomed2 (BMH4–961405) project and the Jean Pierre and Nancy
Boespflug Foundation for Myopathic Research.
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European Journal of Human Genetics