Identification
of Six Novel SOD1 Gene Mutations in Familial Amyotrophic Lateral
Sclerosis
Y.
Boukaftane, J. Khoris, B. Moulard, F. Salachas, V. Meininger,
A. Malafosse,
W.
Camu and G.A. Rouleau
Abstract:
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative
disease characterized by the premature death of motor neurons.
In approximately 10% of the cases the disease is inherited
as autosomal dominant trait (FALS). It has been found that
mutations in the Cu/Zn superoxide dismutase gene (SOD1) are
responsible for approximately 15% of FALS kindreds. We screened
affected individuals from 70 unrelated FALS kindreds and identified
10 mutations, 6 of which are novel. Surprisingly, we have
found a mutation in exon 3, which includes most of the active
site loop and Zn2+ binding sites, a region where no previous
SOD1 mutations have been found. Our data increase the number
of different SOD1 mutations causing FALS to 55, a significant
fraction of the 154 amino acids of this relatively small protein.
Amyotrophic lateral sclerosis (ALS) is a fatal neurological
disorder characterized by progressive degeneration of large
motor neurons in the motor cortex, brainstem and spinal cord.
The mean age at onset of the disease is 45 years with a mean
survival of 3 years.1,2 ALS occurs in two clinically
indistinguishable forms, sporadic (SALS; 90%) and familial (FALS;
10%). FALS is usually inherited as autosomal dominant trait3
though a few kindreds show autosomal recessive inheritance.4,5
Approximately
15% of all dominant FALS are caused by a defect in the cytosolic
Cu/Zn superoxide dismutase gene (SOD1) localized to human
chromosome 21q22.1.6,7 To date forty-five different
missense mutations affecting 34 codons, one nonsense mutation,
one deletion mutation causing a frameshift and two intronic
mutation in intron 4 have been described in the SOD1 gene.8
All the SOD1 mutations are dominant except for the Asp90Ala
mutation which is thought to be recessive.5,9 In
order to study the effect of these mutations and to gain insight
into the mechanisms leading to motor neuron degeneration animal
models of FALS have been generated using mutant SOD1 transgenes.10,12
We
have screened genomic DNA from 70 unrelated patients for mutations
in the SOD1 gene by PCR-SSCP. Here, we report six new and
unpublished SOD1 gene mutations found in six unrelated patients.
PATIENTS
AND METHODS
Patients
Blood
was collected from 70 unrelated patients with FALS. These
FALS kindreds were of Canadian and French origins. The El
Can
J Neurol Sci 1998; 25: 192
Escorial
diagnostic criteria were used.13 Genomic DNA was
prepared from blood using standard procedures.
Primer
design, SSCP analysis and direct sequencing
To
improve the PCR amplification of SOD1 gene exons for SSCP
screening, we designed new intronic primers for exons 1 to
3 (Table 1). PCR amplification of exons 4 and 5 was performed
using primers as previously described.14
The
50 µl amplification reaction contained 100 ng of genomic
DNA, 1X PCR buffer (Promega), 100 ng of each primer, 50 µM
of dCTP, 50 µM of dGTP, 50 µM of dTTP, 25 µM
of dATP, 12.5 µM of (a-35S)-dATP (1,000 mCi/mmol,
Amersham) and 1.5 units of Taq polymerase (Promega). All SOD1
gene exons were amplified using thirty cycles of amplification:
30 s at 94°C, 30 s at 59°C (62°C for exon 2)
and 45 s at 72°C followed by a 5-min extension at 72°C.
The
single-strand conformational polymorphism (SSCP) analysis15
was performed as described by Michaud et al.16
except for using an acrylamide concentration of 8% in
the gels. In order to maximize the mutation detection, we
also used the 0.5X Hydrolink MDE gel supplemented with or
without 5% glycerol. Electrophoretic migration at 6V and autoradiology
were performed for 16 and 18 hours, respectively. Any difference
in migration between patient and control samples was noted
as positive. To confirm the SSCP results, symmetric direct
sequencing method17,18 using modified T7 polymerase
(sequenase, Amersham) was used as described by Brody et al.19
All exons were sequenced at least once on each strand.
RESULTS
In
our hands the new oligonucleotide primers designed to amplify
exons 1-3 (Table 1) gave clearer and more intense bands
on SSCP than those described in reference 14. Genomic DNA
from 70 unrelated FALS affected patients and from 26 normal
controls were screened for mutations in SOD1 gene in all 5
exons using primers from the flanking intron sequences. PCR-SSCP
analysis of all DNA samples showed variant bands in 10 unrelated
families (14.3%). We had previously screened 300 controls
for mutations in these exons; none were found.
Direct
sequencing of PCR-amplified DNA from the 10 FALS individuals
with SSCP variants confirmed the presence of mutations in
all cases. We identified 6 new mutations of which 5 were missense
nucleotide substitutions and one 3 bp deletion in intron 4
(Table 2). Two other known mutations were found in
four patients (Asp90Ala and Ile113Thr).5,20 All
affected patients with the SOD1 mutations are heterozygotes
except for Leu84Phe and Asp90Ala amino acid substitutions
where patients were homozygous. The 3 bp (CTT) deletion detected
in intron 4 is 30 bp downstream of exon-intron splice junction.
DISCUSSION
All
5 exons of the SOD1 gene from 70 unrelated FALS affected patients
were screened for mutations using SSCP. Using our SSCP protocol,
which involves testing of DNA fragments smaller than 300 bp
using many different gel conditions, we expect to have
Table
1: Amplified DNA length, annealing temperature and oligonucleotide
primer sequences for PCR amplification of Human SOD1 gene
exons.
detected
over 90% of all mutations, suggesting that few, if any, were
missed in our screen. Therefore, 14.3% of our FALS families
showed a defect in the SOD1 gene, confirming the relatively
low prevalence of SOD1 mutations found in previous studies.8,22
We
found three new mutations in exon 2 replacing Glu 21 by Gly,
Leu 38 by Arg and Glu 49 by Lys. At the position 21, a Glu21Lys
mutation was previously reported in one sporadic case of ALS.23
The SOD1 protein of all compared species in the Figure
contain the Glu 21, except chicken and nematodes, showing
that this amino acid is highly conserved. Based on the bovine
and yeast crystallographic SOD1 protein structure21 Glu 21
is part of a b strand. Thus the Glu21Gly mutation may destabilize
the SOD1 structure. As predicted for Leu38Val mutation,21
Leu38Arg would be expected to destabilize protein folding
by altering a Greek Key. The bovine and yeast crystallographic
SOD1 protein structure21 show that Glu 49 is involved in dimer
contact or Cu2+ binding. While the amino acid Glu 49 is not
highly conserved between species, this substitution constitutes
a significant amino acid change from a negatively charged
(Glu) to a positively charged (Lys) amino acid.
In
exon 4 we found another novel mutation, Leu84Phe, and two
previously published mutations Asp90Ala and Ile113Thr in six
unrelated families. One of our families carrying an Asp90Ala
mutation contains two affected patients and two normal individuals
who are homozygotes and heterozygotes, respectively for the
mutation (B. Moulard and Y. Boukaftane, in preparation). This
finding supports a recessive inheritance mechanism for the
Asp90Ala mutation.5,9 The Leu 84 amino acid is
conserved in all known mammalian SOD1 genes (Figure),
and it is a part of fourteen conserved amino acids region
which would have an important role in SOD1 function. Surprisingly,
the woman patient carrying the Leu84Phe mutation is homozygote.
She died at the age of 43 years, 3 years after the onset of
the disease. She has a 48-year-old normal sister found also
to be homozygous for the same mutation.
We
also found a deletion of three nucleotides 30 bp downstream
of the exon-intron splice junction in intron 4. The consequence
of this mutation is unknown. The patient and his parents have
died and no cell line is available to test for splicing errors
or other biological effects. However, the mutation is not
seen in 600 control chromosomes.
We
found one kindred showing a missense mutation (CTA to CGA;
Leu67Arg) in exon 3 which encodes the Zn-binding loop of the
active site. This mutation replaces a predicted Leu at position
67 by an Arg, introducing a voluminous amino acid with a positive
charge, which constitutes a major structural change. The Zn2+
ion is known to be a potential neurotoxin.24-26 Therefore,
the alteration of SOD1 role in the binding of Zn2+ may affect
its homeostasis leading to neurodegeneration. We have found
two different mutations involving the Zn2+ binding site (Leu67Arg
and Leu84Phe), in two unrelated FALS patients suggesting a
possible role of Zn2+ ions in the development of ALS.
This
study increases the number of known mutations causing FALS
to 55 involving 37 different codons. The fact that we have
found 5 new missense mutations suggest there are other as
yet undiscovered SOD1 mutations in FALS. This represents a
surprisingly large number of gain of function mutations for
such a small protein.
Abbreviations
ALS,
Amyotrophic lateral sclerosis; FALS, familial ALS; SOD1, Cu/Zn
superoxide dismutase1; SALS, sporadic ALS; SSCP, single strand
conformational polymorphism.
ACKNOWLEDGEMENTS
We
thank members of FALS kindreds for their cooperation. G.A.R.
is supported by the Medical Research Council of Canada. The
work was supported by the Muscular Dystrophy Association (U.S.A.),
the ALS Association, the Association Française contre
le Myopathie and the Association pour la recherche sur la
sclérose laterale amyotrophique.
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Can
J Neurol Sci 1998; 25: 196
From
the Centre for Research in Neuroscience, McGill University,
and the Montreal General Hospital Research Institute, Montreal,
Canada, (Y.B., J.K., G.A.R.); Laboratoire de Médecine
Expérimentale, Institut de Biologie, Montpelier, France
(B.M.); Service de Neurologie, division Mazarin, Hôpital
de la Salpétrière, Paris, France (F.S., V.M.);
Division de Neuropsychiatrie, Hôpital Belle-Idée,
Genève, Suisse (A.M.); Département de physiopathologie
Neuromusculaire, Institut de Biologie, Montpelier, France
(W.C.).
Received
June 4, 1998. Accepted in final form June 5, 1998.
Reprint
requests to: G.A. Rouleau: Rm L7 224, Department of Neurology,
Montreal General Hospital, 1650 Cedar Avenue, Montreal,
Quebec, Canada, H3G 1A4
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Can.
J. Neurol. Sci. 1998; 25: 192-196
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