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Association of a Null Mutation in the CNTF Gene With Early Onset of Multiple Sclerosis
Ralf Giess, MD;
Mathias Mäurer, MD;
Ralf Linker, MD;
Ralf Gold, MD;
Monika Warmuth-Metz, MD;
Klaus V. Toyka, MD;
Michael Sendtner, MD;
Peter Rieckmann, MD
Arch Neurol. 2002;59:407-409.
ABSTRACT
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Background Immune-mediated demyelination and axonal damage lead to early functional
impairment in multiple sclerosis (MS). Ciliary neurotrophic factor (CNTF)
is a potent survival factor for neurons and oligodendrocytes and may be relevant
in reducing tissue destruction during inflammatory attacks.
Subjects and Methods We screened 288 unselected patients with multiple sclerosis (MS) (mean
age, 40.2 ± 10.2 years; range, 18-71 years) for a previously described
homozygous null mutation within the CNTF gene leading
to a truncated, biologically inactive protein. The G-to-A CNTF null mutation at position -6 of the second exon was identified
by a HaeIII polymorphism of the polymerase chain
reaction–amplified genomic DNA.
Results The homozygous CNTF null mutation (CNTF-/-) was found in 7 (2.4%) of the 288 randomly selected
patients with MS. Patients with the CNTF-/-
genotype had a significantly earlier onset of disease (17 vs 27 years; Mann-Whitney
test, P = .007) with predominant motor symptoms.
Conclusions These results suggest that CNTF contributes to time and site of early
clinical manifestation. The frequency of patients with MS with a homozygous CNTF null mutation in this population was not higher than
in control groups, indicating that the CNTF null
mutation is not a risk factor for development of MS.
INTRODUCTION
MULTIPLE sclerosis (MS) is a chronic inflammatory demyelinating disease
of the central nervous system with presumed autoimmune origin. In addition
to exogenous factors, genetically determined dispositions that cause deviations
in the regulatory balance of proinflammatory and anti-inflammatory cytokines1 are regarded as relevant for disease susceptibility
and expression. As has been demonstrated for the HLA system,2
functional relevant polymorphisms in candidate genes could modify both the
onset and course of the disease.
However, in light of the ongoing discussion about mechanisms leading
to axonal damage in MS, not only genes encoding for immunoregulatory molecules
are of particular interest, but also genes that control oligodendrocyte and
neuronal survival. In this context, the gene-encoding ciliary neurotrophic
factor (CNTF) is an interesting candidate to reduce immune-mediated structural
damage in MS. The CNTF was identified as a survival and differentiation factor
for a variety of neuronal cell types, including motoneurons and sensory and
sympathetic neurons.3 In the central nervous
system, CNTF is expressed mainly by astrocytes and promotes mitosis and maturation
of oligodendrocyte precursor cells in vitro and in vivo.4
Moreover, CNTF protects oligodendrocytes from tumor necrosis factor–induced
apoptotic cell death.5 Therefore, CNTF may
be involved in repair mechanisms in MS lesions in the course of the disease.
In this study, we screened patients with MS for a previously described
homozygous null mutation within the CNTF gene leading
to a truncated, biologically inactive protein6
and correlated the CNTF-/- genotype with
disease activity.
PATIENTS AND METHODS
The study was approved by the local ethics committees. After written
informed consent was obtained, blood was collected from 288 unselected adult
German patients (mean [SD] age, 40.2 ± 10.2 years; range, 18-71 years)
with clinically definite MS. The patients were tracked in our MS outpatient
clinic under highly standardized follow-up conditions.
DNA was isolated from 1 mL of EDTA-treated blood. For extraction of
chromosomal DNA, a commercially available kit (QIAamp Blood Midi Kit; QIAGEN
GmbH, Hilden, Germany) was used according to the manufacturer's instructions.
The G-to-A CNTF null mutation at position -6
of the second exon of the CNTF gene, which leads
to a frameshift in the derived CNTF messenger RNA
coding for a truncated, biologically inactive protein, was identified by a HaeIII polymorphism of the polymerase chain reaction (PCR)–amplified
genomic DNA as recently described.6 The mutation
eliminates a HaeIII restriction site. Therefore,
the PCR product with a length of 134 base pairs (bp) including the intron
1–exon 2 boundary remains undigested, whereas the PCR product of the
wild-type allele is cleaved by HaeIII, resulting
in 2 fragments of 94 and 40 bp (Figure 1).
For confirmation, PCR products that showed the digestion pattern for the homozygous CNTF null mutation were sequenced on an automated DNA sequencer
(Perkin-Elmer 373 A; Applied Biosystems, Foster City, Calif). All PCRs were
performed at least twice with patient DNA to avoid PCR errors.
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Figure 1. Identification of the CNTF null mutation by a HaeIII restriction
polymorphism of polymerase chain reaction–amplified genomic DNA in a
2% agarose gel stained with ethidium bromide. The 3 different genotypes were
identified by characteristic digestion patterns: CNTF+/+,
2 bands of 94 and 40 base pairs (bp); CNTF+/-,
3 bands of 134, 94, and 40 bp; and CNTF-/-,
1 band of 134 bp.
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Magnetic resonance (MR) imaging and MR spectroscopy were performed in
patients with the CNTF-/- genotype and
age- and sex-matched patients with the CNTF+ allele
with a 1.5-T scanner (Magnetom Vision, standard head coil; Siemens, Erlangen,
Germany). The T1-weighted (repetition time, 532 milliseconds; echo time, 17
milliseconds) and T2-weighted (repetition time, 2000 milliseconds; echo time,
20/80 milliseconds) images were obtained by the nonangulated conventional
double spin-echo technique (thickness, 6 mm). The MR spectroscopy was performed
according to standard protocols (SVS [single-voxel] PRESS [point resolved
spectroscopy in steady state] technique, 2-dimensional–CSI [chemical
shift imaging]–spectroscopic imaging).7
Clinical data for patients with the CNTF-/-
genotype and those with the CNTF+ allele were compared
by  2 test, and statistical analysis of age at disease onset
in relation to the homozygous CNTF null mutation
was performed with the Mann-Whitney test (GraphPad Prism; GraphPad Software,
San Diego, Calif).
RESULTS
The homozygous CNTF null mutation (CNTF-/-) was found in 7 (2.4%) of the 288 randomly selected
patients with MS. This frequency corresponds to numbers previously reported
for a Japanese (2.3%) and a German (2%) population.6, 8
Four of the 7 patients with MS and the CNTF-/-
genotype (mean age, 36.3 ± 15.5 years; Expanded Disability Status Scale
score, 4.1 ± 2.4; disease duration, 16.0 ± 15.5 years) had relapsing-remitting
MS, and 3 had a secondary progressive disease course at the time of sample
collection. Six (85%) of 7 patients carrying the CNTF-/-
genotype had disease onset with marked motor or brainstem symptoms and incomplete
remission, which was detected in only 21% of our population with MS (P = .01) and at least 1 functional CNTF allele. To our surprise, patients with the CNTF-/- genotype had a significantly earlier onset of clinical
disease (median, 17 years; 25th percentile, 16.3 years; 75th percentile, 24
years), compared with patients carrying at least 1 functional CNTF gene (median, 27.0 years; 25th percentile, 22 years; 75th percentile,
33 years; Mann-Whitney test, P = .007) (Figure 2).
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Figure 2. Patients with multiple sclerosis
carrying CNTF wild-type alleles (CNTF+/+, CNTF+/-; n = 288) vs patients
with multiple sclerosis carrying a homozygous null mutation of the CNTF gene (CNTF-/-; n = 7) leading
to a truncated, biologically inactive CNTF protein. Boxes indicate median
and 25% to 75% intervals; bars, 10th and 90th percentiles; and asterisk, P = .007, Mann-Whitney test.
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With particular focus on structural damage, we performed MR spectroscopy
of the normal-appearing white matter of the 7 patients and compared the values
for the N-acetylaspartate–creatine ratio with
values obtained from 7 age- and sex-matched control patients with MS. With
this technique, no difference between the 2 groups could be detected. Nevertheless,
the diameters of the third and lateral ventricles were greater in patients
with the CNTF-/- genotype than in control
patients (third ventricle: 5.2 ± 1.8 mm vs 3.5 ± 2.6 mm, P = .1; lateral ventricle, 17.4 ± 3.7 mm vs 13.9
± 2.3 mm, P = .09), demonstrating a trend
toward a more pronounced brain atrophy in patients carrying the CNTF-/- genotype.
COMMENT
In this study, the CNTF-/- mutation
was associated with a significantly earlier onset of disease and motor disability
in patients with MS. The frequency of patients with MS with a homozygous CNTF null mutation in this population was not higher than
in control groups, indicating that the CNTF null
mutation is not a risk factor for development of MS, but that this genotype
may rather predispose to earlier disease onset and early motor involvement.
Our data suggest that trophic support of neurons and oligodendrocytes as provided
by CNTF may be critical to reduce early damage in the inflammatory lesions
typical of MS. The lack of significant data from MR spectroscopic studies
might be due to our heterogeneous study group, with patients having varying
disease length up to 25 years. On the other hand, measurements that represent
the net effect of tissue destruction, such as atrophy measurements, show greater
values in patients with the CNTF-/- genotype.
The homozygous CNTF null mutation is very rare.
Therefore, the observation that patients carrying the CNTF-/- genotype have an earlier disease onset is based on
findings in 7 patients. However, our results are in complete accordance with
the observations made in experimental allergic encephalomyelitis in CNTF knock-out mice. After induction of myelin oligodendrocyte
glycoprotein, CNTF-/- mice with experimental
autoimmune encephalomyelitis showed a significantly earlier disease onset
and a delayed recovery from relapses.9
A number of recent studies established that patients with a first attack
may already have signs of previous central nervous system lesions by MR imaging.
Moreover, numerous lesions remain latent without overt clinical bouts, and
the brain may even show mild atrophy early in the disease.10-11
Against this background, failure to produce neurotrophic factors like CNTF
could be responsible for a more extended structural damage during an inflammatory
attack (eg, due to proinflammatory cytokines) and may lead to a shorter preclinical
interval between silent lesion development and first clinical symptoms of
MS. This hypothesis is also supported by findings in experimental autoimmune
encephalomyelitis in CNTF-/-, since these
mice histopathologically showed a pronounced vesicular demyelination, resembling
tumor necrosis factor  –mediated oligodendrogliopathy.9
Our study clearly demonstrates that, in addition to evaluation of the
immunoregulatory mechanism, it is necessary to evaluate the role of neurotrophic
factors in MS. Moreover, this study supports the view of MS as an inflammatory
neurodegenerative disease. This change of paradigm could also widen the therapeutic
approaches in MS. Administration of CNTF or other trophic factors, by adequate
pharmacologic approaches, could be useful to enhance regeneration, particularly
in patients lacking endogenous CNTF.
AUTHOR INFORMATION
Accepted for publication September 28, 2001.
Author contributions: Study
concept and design (Drs Giess, Mäurer, Gold, Toyka, Sendtner,
and Rieckmann); acquisition of data (Drs Giess, Mäurer,
Linker, and Warmuth-Metz); analysis and interpretation of
data (Drs Giess, Mäurer, Linker, Warmuth-Metz, and Rieckmann); drafting of the manuscript (Drs Giess, Mäurer, and
Linker); critical revision of the manuscript for important
intellectual content (Drs Gold, Warmuth-Metz, Toyka, Sendtner, and
Rieckmann); statistical expertise (Drs Giess, Mäurer,
Linker, and Toyka); obtained funding (Dr Rieckmann); administrative, technical, and material support (Drs Giess,
Mäurer, Gold, Toyka, Sendtner, and Rieckmann); study
supervision (Dr Rieckmann).
This study was supported by the Gemeinnützige Hertie-Stiftung,
Frankfurt, Germany, and the German Multiple Sclerosis Society, Hannover.
We thank Daniela Waffler for excellent technical assistance.
Corresponding author and reprints: Peter Rieckmann, MD, Department
of Neurology, University of Würzburg, Josef Schneider Straße 11,
D-97080 Würzburg, Germany (e-mail: p.rieckmann{at}mail.uni-wuerzburg.de).
From the Division of Neuroradiology (Dr Warmuth-Metz), Department of
Neurology (Drs Giess, Mäurer, Linker, Gold, Toyka, and Rieckmann), and
Institute of Clinical Neurobiology (Dr Sendtner), Bayerische Julius-Maximilians
Universität, Würzburg, Germany.
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