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Ciliary neurotrophic factor genotype does not influence clinical phenotype in amyotrophic lateral sclerosis.

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Ciliary Neurotrophic
Factor Genotype Does
Not Influence Clinical
Phenotype in Amyotrophic
Lateral Sclerosis
Ammar Al-Chalabi, PhD, MRCP,1,2
Margaret D. Scheffler, BA,2 Bradley N. Smith, BSc,1
Matthew J. Parton, PhD, MRCP,1
Merit E. Cudkowicz, MD,3 Peter M. Andersen, DMSc,4
Douglas L. Hayden, MA,5 Valerie K. Hansen, BSc,1
Martin R. Turner, MRCP,1
Christopher E. Shaw, MD, FRCP,6
P. Nigel Leigh, PhD, FRCP,1
and Robert H. Brown, Jr, MD, DPhil2
Ciliary neurotrophic factor (CNTF) maintains survival of
adult motor neurons. Mice lacking the CNTF gene develop mild, progressive motor neuron loss. In the normal
human population, 1 to 2.3% are homozygous for a null
allele, and reports suggest this mutant is associated with a
younger onset of amyotrophic lateral sclerosis (ALS). We
have tested this hypothesis in a study of 400 subjects
with ALS and 236 controls. There was no difference in
age of onset, clinical presentation, rate of progression, or
disease duration for those with one or two copies of the
null allele, excluding CNTF as a major disease modifier
in ALS.
Ann Neurol 2003;54:130 –134
Amyotrophic lateral sclerosis (ALS) is a disease in
which there is progressive degeneration of motor neurons resulting in paralysis and death, usually over approximately 3 years. Approximately 10% of cases show
autosomal dominant inheritance. Mutations in the
gene for Cu/Zn superoxide dismutase (SOD1) account
From the 1Department of Neurology, Academic Neuroscience Centre, Institute of Psychiatry, King’s College London, London, United
Kingdom; 2Day Neuromuscular Research Laboratory, MGH East,
Charlestown, MA; 3Neurology Clinical Trials Unit, Massachusetts
General Hospital, Boston, MA; 4Department of Neurology, Umeå
University, Umeå, Sweden; 5General Clinical Research Center, Massachusetts General Hospital, Boston, MA; and 6Departments of
Neurology and Medical and Molecular Genetics, Guy’s King’s and
St. Thomas’ School of Medicine, and Institute of Psychiatry, King’s
College London, United Kingdom.
Received Jul 15, 2002, and in revised form Apr 7, 2003. Accepted
for publication Apr 7, 2003.
Address correspondence to Dr Al-Chalabi, Department of Neurology, PO41, Institute of Psychiatry, De Crespigny Park, London SE5
8AF, UK. E-mail: ammar@iop.kcl.ac.uk
130
© 2003 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
for 2 to 7% of all cases, but no other genes clearly
associated with typical disease have yet been identified.1,2 There is considerable phenotypic heterogeneity
in ALS, even within families, but no definite phenotypic modifier genes have been found.
The ciliary neurotrophic factor (CNTF) gene codes
for a 200 –amino acid protein that plays an important
role in maintenance and regeneration of the adult nervous system.3 It prolongs neuronal survival of various
culture systems4 and also improves survival in the pmn
and wobbler mutant mouse models of motor neuron
disease,5,6 even though these mice do not have an obvious defect in CNTF or its receptor. Conversely, mice
with disruption of CNTF or its receptor do develop motor neuron degeneration.7 These observations led to the
unsuccessful use of CNTF in therapeutic clinical trials in
ALS on the basis that increased levels improve survival
regardless of the underlying cause of disease.8 –10
In humans, a null allele of CNTF is generated by a
G to A intronic point mutation that results in a new
splice site, a reduction in polypeptide length from 200
to 62 (only the first 39 are part of the normal protein),
and prevention of expression of normal protein.11 Between 1 and 2.3% of the population may be homozygous for this null mutant but are not at increased risk
for neurological disease, including ALS.11–14 Some
studies have shown differences in CNTF protein and
CNTF receptor mRNA expression in spinal cord from
patients with ALS compared with normal controls,15
suggesting that this pathway may be involved in ALS
pathogenesis. It is possible that possession of the null
allele influences ALS once the disease process has
started because motor neurons cannot be protected or
rescued from the disease-causing insult. In support of
such an idea, anecdotal reports suggest that the null
allele may be associated with a younger onset and more
rapid disease.16,17 CNTF genotype therefore is an interesting candidate as a phenotypic modifier, particularly for disease onset and duration. We have tested
this hypothesis in two populations of patients with
sporadic ALS and a second group with familial ALS,
homozygous for the D90A mutation of SOD1, using
for comparison a set of neurologically normal controls.
Subjects and Methods
Clinical Resource
Subjects were seen in tertiary referral clinics in the United
States, United Kingdom, and Scandinavia. The diagnosis of
ALS was made by a consultant neurologist after exclusion of
other conditions. Demographic data, age of symptom onset,
site of first weakness, time to referral, and disease duration
from first symptom were recorded. In a subset of patients
from the United States, rate of progression also was recorded,
as assessed by repeated ALS functional rating scale and
forced vital capacity assessments. Controls were anonymous
blood donors from the United Kingdom.
Genotyping
Genotypes were determined as previously described.12
Statistics
The ␹2 test was used to test the association of ALS with the
null allele of CNTF. Analysis of variance (ANOVA) was
used to compare means of continuous variables. Kaplan–
Meier analysis (K-M) and Cox regression were used to test
potential continuous and categorical predictors of age of onset and survival. SPSS statistical software (version 10.0.7;
SPSS, Chicago, IL) and SAS version 8.1 (SAS Institute,
Cary, NC) were used for all statistical analysis.
Results
CNTF genotypes were obtained for 636 subjects; 217
were neurologically normal controls, 167 were from
the United Kingdom, and 50 were from Scandinavia.
There were also 400 subjects with ALS, of whom 351
had sporadic ALS and 49 had familial ALS, homozygous for the D90A mutation of SOD1, which is prevalent in Scandinavia. The remaining 19 people were
homozygous for the D90A mutation but were unaffected by ALS as of January 1, 2000.
For the subjects with sporadic disease, the age of onset distribution, gender ratio, and site of onset were
similar to those reported in other retrospective studies.
Mean age of onset was 53.8 years, 64% were men, and
the first symptoms were in the bulbar region in 31%.
CNTF genotype frequencies were in Hardy–Weinberg equilibrium for controls, sporadic ALS, and familial ALS (Table), and comparison of the sporadic ALS
genotypes and controls confirmed there was no association of the null allele with ALS (␹2 ⫽ 0.11, p ⫽
0.95). In contrast with the expected findings, there was
no significant difference in mean or median age of onset for different CNTF genotypes (ANOVA p ⫽ 0.26;
K-M log rank 4.0, p ⫽ 0.14), and homozygosity for
CNTF null mutant tended to delay disease onset (Fig
1). In addition, there was no significant difference in
survival for different CNTF genotypes (K-M log rank
1.5, p ⫽ 0.47; Fig 2) or when controlling for known
prognostic factors using multivariate regression analysis
with available data (Cox model p ⫽ 0.44). For 47 subjects with sporadic ALS, there was no significant difference in disease progression for different CNTF genotypes ( p ⫽ 0.38).
For the Scandinavian D90A homozygous subjects
with symptoms, genotype frequencies were in agreement with Hardy–Weinberg proportions (␹2 1.56, p ⫽
0.21), and there was no association between the null
CNTF allele and ALS (␹2 1.19, p ⫽ 0.55). Mean age
of onset of paresis was 47.5 years, and mean age of the
unaffected was 49 years. There was no significant difference in mean or median age of onset for different
CNTF genotypes (ANOVA p ⫽ 0.73; K-M log rank
0.49, p ⫽ 0.78). Median survival could not be calcu-
Al-Chalabi et al: CNTF and ALS
131
Table.1 Sporadic and Homozygous D90A ALS
Test Values for
Each Model (P)
Genotypes
Sporadic ALS
SALS (n)
Controls (n)
NN
NM
MM
Total
260
84
7
351
122
42
3
167
Bulbar onset (n)
63
25
1
89
Limb onset (n)
Male (n)
145
168
45
52
5
5
195
225
91
54
32
53
2
64
125
55
56
68
223
36
74
41
5
31
302
173
53
5
231
34
15
0
49
36
8
13
10
1
1
50
19
Female (n)
Mean age of onset
(yr)
Median age of onset
(yr)
No. of subjects
Median survival
(mo)
No. of subjects
Homozygous D90A
ALS
D90A ALS (n)
Controls (n)
D90A disease-free
subjects (n)
Mean age of onset
(yr)
Mean age of
disease-free D90A
subjects at January 1, 2000 (yr)
Median age of onset
(yr)
47
48
N/A
54
46
40
45
50
N/A
Test
Dominant
Recessive
1. No association of cases
with CNTF null mutant
2. Case alleles in HardyWeinberg equilibrium
3. Random assortment of
alleles by site of onset
␹2
0.805
0.59 (one tailed)
␹2
0.52
0.67 (two tailed)
4. Random assortment of
alleles by gender
␹2
0.70
1.00 (two tailed)
Null Hypothesis
5. No differences in mean
ages of onset
6. No differences in median
ages of onset
␹
2
Test
Values
(P)
0.92
ANOVA
0.26
Log rank
0.14
7. No differences in survival
Log rank
0.47
1. No association of cases
with CNTF null mutant
␹2
0.77
0.51 (one tailed)
0.21
2. Case alleles in Hardy–
Weinberg equilibrium
3. No differences in mean
ages of onset
4. No differences in mean
ages of disease-free D90A
subjects
5. No differences in median
ages of onset
␹2
0.21
ANOVA
0.73
ANOVA
0.11
Log rank
0.78
The wild-type allele is coded as N, the null mutant as M. Numbers for each genotype and phenotypic category are shown with the statistical
test used and p value obtained. Dominant model refers to possession of at least one M allele; recessive model refers to possession of two. For
MM genotype, there are low numbers, and the Fisher exact test was used. In all D90A cases included, subjects were homozygous for the D90A
mutation of SOD1. There was no evidence for an effect of the CNTF null mutant allele on ALS phenotype in sporadic or D90A ALS.
ALS ⫽ amyotrophic lateral sclerosis; CNTF ⫽ ciliary neurotrophic factor; SALS ⫽ sporadic ALS; ANOVA ⫽ analysis of variance; N/A ⫽ not
applicable.
lated because of the long survival of people with this
mutation. There was no difference in survival between
homozygous wild-type and heterozygous subjects (K-M
log rank 0.2, p ⫽ 0.66).
Discussion
Studies using recombinant mice suggest that CNTF is
not required for motor neuron development but has a
role in maintenance of motor neuron survival in
adults.18 Mice null for CNTF develop normally but in
adulthood experience progressive loss of motor neurons
accompanied by mild weakness. In contrast, mice null
for the CNTF receptor gene either die in the perinatal
period or have severe motor neuron loss.7 Mice transgenic for the G93A SOD1 mutation develop motor
neuron degeneration significantly earlier if they are also
132
Annals of Neurology
Vol 54
No 1
July 2003
CNTF deficient,17 suggesting that CNTF can act as a
disease modifier.
Direct evidence that CNTF might act as a diseasemodifying gene in ALS comes from two anecdotal reports. In one study of 154 subjects, the 3 homozygous
for the null mutant CNTF allele had a younger onset
of motor neuron diseases.16 In the other, subjects from
a family with SOD1-mediated ALS had an age of onset
and disease duration related to CNTF genotype.17 A
man with a V148G SOD1 mutation had a young onset of ALS at 25 years and a rapid disease course with
death by 11 months. He was homozygous for the
CNTF null mutant allele. His sister and mother also
carried the SOD1 mutation but were not homozygous
for the null mutant CNTF allele. They were disease
free at 35 and 54 years of age, respectively.
If CNTF is acting as a disease modifier in ALS, various dominance relationships between the null mutant
and wild-type alleles are possible. The most likely in a
loss of function mutation is that the mutant is recessive
to wild type, but a dose effect or even overdominance,
in which the heterozygote has a phenotype not seen in
the homozygotes, is possible. Levels of CNTF postmortem in human sciatic nerve are, in fact, determined
mainly by gene dose such that heterozygotes have approximately half the level of CNTF as wild-type homozygotes.19 This would suggest that heterozygotes
should have a phenotype intermediate between the two
homozygotes. It is possible that any effect might be
masked by the natural heterogeneity of ALS or because
sporadic ALS could be different in some way from familial ALS. We therefore included a population of 49
subjects with ALS caused by homozygosity for the
D90A mutation of SOD1.20 This SOD1 mutation is
important because it has arisen on a genetically homogeneous background, and subjects with ALS related to
this mutation have a uniform phenotype, with very
slowly progressive ascending paresis and a mean survival time of 14 years. This makes it ideal for the study
of phenotypic modifiers, because deviation would be
more obvious.
In the two sporadic ALS groups we studied, there
was no significant difference in age of onset for the
three CNTF genotypes, and, for the five null homozygotes with data, there was actually a later age of onset
than for the other two genotypes, although this was
not statistically significant. The possibility that the null
allele only has an effect which is mild and recessive
Fig 2. Kaplan–Meier curve of survival for the three ciliary
neurotrophic factor (CNTF) genotypes. There is no significant
difference in survival between any groups. N is the wild-type
and M the null mutant allele. Cross marks represent subjects
who were alive at the censor date and therefore do not reduce
the proportion surviving. Numbers of subjects: NN 173, NM
53, MM 5.
cannot be excluded by this or previous studies but
seems unlikely given the later age of onset for homozygotes that we have observed. Because subjects homozygous for the null mutant constitute at most 2.3% of
the population, to have sufficient power to detect a
subtle effect would require a study of thousands.
These findings suggest that the null allele of CNTF
does not have a major modifying effect on ALS onset,
rate of progression, or duration.
This work was supported by a Medical Research Council Clinician
Scientist Fellowship (A.A.-C.); the Medical Research Council
(V.K.H.); a Wellcome Trust Fellowship (M.R.T.); the MNDA,
ALSA, MDA, Project ALS and the Angel Fund; and the National
Institutes of Health (NINDS, NIA; R.B.).
References
Fig 1. Kaplan–Meier curve of age of onset for the three different ciliary neurotrophic factor (CNTF) genotypes. There is no
significant difference in age of onset between any groups. N is
the wild-type and M the null mutant allele. Numbers of subjects: NN 223, NM 74, MM 5. The marked step-like shape
for the MM genotype survival curve reflects the low number of
subjects in this group.
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