Clinical and functional investigation of
10
missense mutations and a novel
frameshift insertion mutation of the
gene for copper-zinc
superoxide
disrnutase in UK families with
amyotrophic lateral sclerosis
R.W. Orrell, MD;
J.J. Habgood, BSc; I. Gardiner, BSc; A.W. King, BSc; F.A. Bowe, PhD; R.A. Hallewell,
PhD; S.L. Marklund, MD;
J. Greenwood, RGN; R.J.M. Lane, MD; and J. deBelleroche, PhD
Article
abstract-Mutations of the gene SOD-1, which encodes the enzyme copper-zinc superoxide dismutase, occur in
patients with a familial form of amyotrophic lateral sclerosis (ALS). We investigated 71 families with more than one
individual affected by
ALS for clinical features and SOD-1 mutations. Mutations were identified in 14 families, indicating
the presence of
SOD-1 mutations in around 20% of this population. There were 10 different heterozygote missense point
mutations in eight different codons, and a novel two-base frameshift insertion (132insTT), which leads
to substitution of
aspartic acid for glutamic acid at codon 132, and a premature stop codon at 133, with predicted truncation
of the protein.
SOD enzyme activity was reduced
to around 50% of normal in individuals with SOD-1 mutations, and may be a useful
predictor for the presence of these mutations. A predilection
for disease onset in the lower limbs appears to be a
distinguishing feature of familial ALS with
SOD-1 mutations, and accords with findings in transgenic mouse models. In
general, the finding of an
SOD-1 mutation does not accurately predict a prognosis or disease severity.
NEUROLOGY
1997;48:746-751
Amyotrophic lateral sclerosis (ALS), or motor neuron
disease, is a neurodegenerative disease, affecting primarily
upper and lower motor neurons, leading to
variable degrees of spasticity, weakness, and wasting
of the muscles of the limbs, with additional involvement
of the muscles affecting speech, swallowing,
and respirati0n.l The mean age of onset is
58
years, and median survival from first symptom to
death
36 months, but with considerable variation
around these means.2 The incidence of ALS is reported
to be around
0.6 to 2.6 per 100,000 population
Following linkage studies in the larger families
indicating a locus on chromosome
21 in the region
q21.1
to q22.3,9 Rosen et al.lo identified mutations of
the
SOD-1 gene that encodes the enzyme superoxide
dismutase (SOD).11J2T o date more than 30 different
mutations have been identified.13 We have investigated
families with ALS for mutations
of SOD-1,
with particular regard to the immediate implications
in providing genetic and prognostic advice to patients
and families. We present the correlation
of the
clinical presentation of disease with the mutation.
per year, and prevalence
2 to 8 per 100,000.193-Afi
proximately 5 to 10% of patients have another affected
individual in the family,2.3za7n d in larger famdominant
mode, affecting several generations.7 The
presentation of the disease in these patients with
familial disease is generally indistinguishable from
'-
Methods. Patients and families were assessed from
throughout the
UK, recording clinical details and pedigree
information. Patients and families were referred
by genone
individual in the family being affected by ALS, as part
of a wider study collecting families for genetic linkage
analvsis. Families where DNA was available from at least
ilies the inheritance
usuallY follOws an autosoma' eral practitioners and physicians on the basis of more than
that in patients with sporadic disease.s
one individual were included in the study, without prior
From the Departments of Biochemistry (Drs. Orrell and deBelleroche, and
J.J. Habgood, I. Gardiner, J. Greenwood, and A.W. King) and Clinical Neuroscience
(Drs. Orrell and Lane), Charing Cross and Westminster Medical School, London, England; the Department of Biochemistry (Drs. Bowe and
Hallewell), Imperial College
of Science, Technology and Medicine, London, England; and the Department of Clinical Chemistry (Dr. Marklund), Umea
University Hospital, Umea, Sweden.
Supported by the
Motor Neurone Disease Association, The American ALS Association, The Special Trustees of Charing Cross and Westminster Hospitals,
Wellcome Trust Grant 038968, The Swedish Natural Science Research Council, and the Council of Vasterbotten County, Sweden.
Received May 6, 1996. Accepted in final form August 27, 1996.
Address correspondence and reprint requests to Dr. Richard
W. Orrell, Department of Neurology, University of Rochester Medical Center, 601 Elmwood
Avenue, Box 673, Rochester,
NY 14642.
746
Copyright 0 1997 by the American Academy of Neurology
knowledge of the presence or absence of
SOD-1 mutations.
The project had the approval of the Riverside Research
Ethics Committee.
DNA was extracted from lymphocytes
of whole blood using standard methods. The five
exons14 of
SOD-1 were amplified by polymerase chain reaction
(PCR) using biotinylated primers.1°J1J5J6T he reaction
mixture included
1 pg of genomic DNA in a 100 pl reaction
volume with
5 ~1 DMSO, 1.5 mM MgCl,, 10 p1 lox PCR
buffer
IV (Advanced Biotechnologies Ltd) [lox buffer IV
contains
200 mM (NH,),SO,, 750 mM Tris-HC1 (pH 9.01,
0.1% Tween],
200 FM dNTPs (Advanced Biotechnologies),
2.5
units Taq polymerase, 0.5 pM of biotinylated and nonbiotinylated
primers. The reaction mixture was overlaid
with mineral oil before thermal cycling (Hybaid Omnigene
thermal cycler), initial denaturation at
95 "C for 2 min, 32
cycles of annealing at
55 "C for 1 min, extension at 72 "C
for 2 min, strand separation at
95 "C for 1 min, and a final
extension
at 72 "G for 6 minutes. The protocol was optimized
for individual primers and samples, in particular
the cycling conditions were varied, and DMSO was sometimes
omitted. For the mutation of exon
5 described here
the primers
5' AGT GAT TAC TTG ACA GCC CA and 5'
TTC TAC AGC TAG CAG GAT AAC were used. The amplified
product was separated on Dynabeads
M-280 Streptavadin
(Dynal, Norway). The purified single-stranded product
was sequenced using the dideoxy chain termination
method, with Sequenase (Amersham), labeled with
35SdATP, electrophoresed on a
6% polyacrylamide gel (National
Diagnostics Sequagel-61, exposed to radiosensitive
film, and the sequence read from the developed film.15
Sense and antisense sequences were read, and the mutation
confirmed with restriction enzymes where appropriate.
Whole blood
(10 ml) was collected
in EDTA, and the erythrocytes separated by centrifugation,
removal of plasma, and washed in
10 ml isotonic
saline. The samples were stored at
-70 "C prior to further
analysis, using a spectrophotometric assay of the disproportionation
of
the superoxide anion radical obtained from
KO,, as described.17Js The assay was performed directly on
hemolysates without prior precipitation of hemoglobin,
and the SOD enzyme activity was expressed as Units per
mg hemoglobin (U/mgHb). Daily controls of a human hemolysate
stored at
-70°C were used, and no change in
activity of the control was seen over
a period of 2 years.
SOD mutation analysis.
SOD enzyme activity.
Results.
Clinical details and DNA from affected family
members with
ALS were obtained from 71 families with
more than one individual affected with ALS. Forty-four
patients from
41 families were assessed personally by
R.W.O. SOD enzyme activity was measured in 12 normal
individuals with no clinical evidence or family history
of
ALS,
40 individuals with ALS but no other affected family
member (SALS), and
30 individuals with a family history
of ALS (FALS) (figure
1). The mean SOD activity in the
normal samples was
56.1 Ifr 1.4 (SE) U/mgHb, which did
not differ from the sporadic
ALS group (mean 59.9 Ifr 0.9
U/mgHb), but a subgroup of seven individuals with familial
ALS had reduced SOD activity, ranging from
31.7% to
63.5%
of normal, mean 28.7 ? 2.1 U/mgHb. This reduction
in SOD activity was statistically significant on
t test ( p <
0.001).
All cases with reduced SOD enzyme activity
showed mutations of
SOD-1. The remaining familial pa-
8o
l
70
60
50
40
5
30
n
20
10
3
3
I
.-
.>-
Q)
c
N
0
.
' $
.0
.
0
*
Figure 1.
SOD enzyme activity measured in erythrocytes
from unaffected individuals with no family history of ALS
(Normal), individuals with ALS and no family history
of
the condition (SALS), and patients with familial ALS
(FALS). Those with FALS and mutations of
SOD-1
(SOD-1 FALS) have a significant reduction of SOD activity
(p
< 0.001).
tients had SOD activity within the normal range (mean
55.2
It 1.4 U/mgHb) with no evidence of SOD-1 mutations.
The clinically normal and sporadic ALS individuals were
not examined for
SOD-1 mutations in this study.
SOD-1
mutations were identified in 14 of 71 families
(table), indicating the presence of these mutations in
around
20% of this population. These consisted of 10 different
missense point mutations in eight different codons.
The point mutations lead to
a single base change, and
substitution of a single amino acid in an otherwise normal
protein. The mutations are all heterozygote mutations,
with a copy of both the normal and mutation containing
sequence being present. Details of some of these mutations
have been
reported.16.16~18.19~zI1n, z3a~dzd4i tion, one novel
two-base frameshift insertion mutation was identified at
codon
132 (132insTT). The two-base-pair insertion leads to
substitution of aspartic acid for glutamic acid at codon
132,
and a stop codon at
133, with a predicted truncation of the
transcribed protein. Figure 2a illustrates sequencing
of the
sense strand, and Figure 2b sequencing of the antisense
strand, the insertion (TT) being confirmed when sequenced
in the reverse direction.
The majority of the mutations (8 of
11) are so far unique
to this group of families. In families with
SOD-1 mutations
the mean age at onset was
45 2 2 (SE) years (median 46,
range
24-72 years), mean duration of disease 4.0 2 0.8
years (median
3.0, range 0.3-20 years), and mean age at
death
52 ? 2 (median 52, range 26-76 years). Age at onset
was defined as the time
of first onset of symptom$, and
March
1997
NEUROLOGY 48 747
Table
The 11 mutations of
SOD-1 identified in a total of 14 families with ALS
Effect on coding SOD enzyme activity Number
of Abbreviated
Exon Codon Nucleotide change sequence
% normal (U/mgHb) families Reference notation
2
4
4
4
4
4
4
4
5
5
5
48
93
93
100
101
101
108
113
125
132
149
CAT-CAG
GGT-CGT
GGT-GTT
GAA-GGA
GAT-GGT
GAT-AAT
GGA-GTA
ATT-ACT
GAC-CAC
insertion
of TT
ATT-ACT
His-Gln
Gly- Arg
GI y-Val
Glu-Gly
Asp-Gly
Asp-Asn
Gly -Val
Ile-Thr
Asp-His
Asp at 132
Stop at 133
Ile-Thr
-
31.7% (17.8)
54.2% (30.4)
63.5% (35.6)
-
-
-
50.6% (28.4)
52.8% (29.6)
46.3% (26.0)
53.3% (29.9)
1
1
1
2
1
1
1
3
1
1
1
19
18,20
21
10,16
16
22,23
24
10,15,16,25,26,27
19
described here
H48Q
G93R
G93V
E lOOG
DlOlG
DlOlN
G108V
I113T
D125H
132insTT
19,27 I149T
_ _
- not measured as sample unavailable.
duration of disease the time from onset to death. The male:
female sex ratio was 1.2:l. The ethnic background was
British in
13 families, and Indian in one family. Clinical
data are restricted due to the difficulties in determining
the precise age at onset, duration, age at death, and symptoms
in individuals who may have died several decades
ago, or who are remote members of the family, and only
reliable information is included in these results.
The age at onset of disease, disease duration, and age at
death, plotted for each mutation type, are presented in
figures 3 and
4. Age of onset varied within and between
families with the same mutation, e.g., by up to 27 years
for
DlOlN (see figure 3). Disease duration also varied within
and between families, and was most marked for G93R
(2-12 years) and I113T (2.5-20 years)I5 (see figure
4). Age
at death showed similar variation between and within
families, e.g., by up
to 24 years for H48Q and 27 years for
DlOlN (see figure 3). Adequate information on site of disease
onset was available for 24 patients, with onset in the
lower limbs in 83%, the upper limbs in 17%; no patients
presented with bulbar symptoms. The heterozygote mutations
were found in two families with only two siblings
affected in one generation (I113T and D125H). In the first
instance one parent died age 49 years in war, the other age
60 years of accidental causes, and in the second instance
one parent died age 68 years of carcinomatosis, the other
age
68 years of myocardial infarction. Mutations were also
present in 12 more typical families with two or three affected
generations, although the majority of multigeneration
families had
no evidence of SOD-1 mutations.
Discussion.
We identified SOB-2 mutations in 14
of 71 (19.7%) of the families investigated. The ascertainment
of the families was opportunistic and may
be open
to bias; for example, toward larger families,
and the 20% proportion must be considered in this
context. Nevertheless, this may be seen as an indica-
Figure
2.
(A) Illustration of 132insTT
mutation (sense).
(B) Illustration of
132insTT mutation (antisense): a is
normal,
b is mutant sequence. G
=
guanine,
A = adenine, T = thymine,
C
= cytosine. The mutation is a heterozygote,
and the effects
of the frameshift
are apparent, with displacement
of the mutant sequence
by two bases.
748
NEUROLOGY
48 March 1997
I
tn
% 70 -
60
-
%
E
E
$'
40 -
'ii;
30 -
50
-
b
tn
s 20
-
c)
I:
0 1 ,
I , , , I I , , , I 1
87o0
1 1 TI 1
I
$ 2 0 1
lo
i
P
.?)
SOD-I mutation type
Figure 3. Age at onset of symptoms
o f M S for individual
SOD-1
mutations, (two families are indicated for 1113T),
and age at death from
ALS for individual SOD-1 mutations
(two families are indicated for ElOOG, and three for
1113T). Each vertical column represents an affected family
with the mutation. Open triangles indicate the age of individuals
who were still living at the time of analysis.
tion
of the chances of a patient with familial ALS
who
presents to a neurologist having an SOD-I mutation.
The mutations were in families with typical
autosomal dominant involvement, and also in two
families with only two individuals affected in one
generati~n.'T~h e penetrance of these mutations
is
age-dependent and variable; incomplete penetrance
could account for some apparently sporadic case
~ . ~If th~e p, a~ren~ts i,n t~hes~e t wo families had lived
longer they might have manifested the disease. The
commonest mutation was I113T, which is identified
with variable p e n e t r a n ~ e . ~ ~ J ~ , ~ ~ , ~ ~
This is the first report
of an exonic insertion mutation
of
SOD-1 with frameshift. The mutation
132insTT (insertion of an additional TT in codon
132) causes a frameshift, with the normal sequence
displaced by two bases, resulting in
a disruption of
translation of the remaining sequence. The translation
of
codon 132 is altered from glutamic acid to
aspartic acid, and a stop codon
(TAA) is generated at
the next codon (133) (see figure 2a). The predicted
mutant protein would have 132 amino acids, the normal
length of the protein being 153 amino acids.
I
' I I
SOD-I
mutation type
Figure
4. Duration of disease (or survival) for individual
SOD-1
mutations. Each vertical column represents an affected
family with the mutation. Open triangles indicate
the individuals who were still living at the time
of analysis.
(Two families are indicated for 1113T.)
These terminal amino acids include a region
of the
active-site loop and a region involved in dimer contact
(the active enzyme being a dimeric metalloenzyme).
ll The position
of the stop codon is close to that
of
the previously reported premature stop codon at
131 in a family with ALS.28 In this mutation, there
was a two-base deletion in codon 126 (TTG
to
This frameshift leads to
a change in the terminal 5
amino acids (codons 126-130)
of the predicted protein,
prior
to the premature stop codon at 131. The
mutation 132insTT also results in a change
of the
terminal amino acid, and
it is uncertain in these two
instances whether any toxic property of the mutant
protein is related to the altered terminal amino acids,
or the predicted premature truncation. This
raises the question
of how a similar disease results
from both the mutations leading
to predicted truncation
of
the protein and the more commonly described
point mutations. The point mutations all lead to single
amino acid substitutions in an otherwise normal
protein, largely affecting the structural regions."
We consistently found
a reduction in erythrocyte
SOD
enzyme activity in the families with SOD-I mutations.
Whether this reflects the pathogenic mechanism
has been
c o n t r ~ v e r s i a l . ~C~uJr~re~n~t ~e~v~i-~
dence, particularly including studies in transgenic
mice that overexpress human mutant
SOD-1 and
have no deficiency in
SOD activity,31 indicates that
disease is primarily due
to the gain of a toxic function
by the enzyme. The reduced
SOD enzyme activity
may reflect instability of the mutant protein in
erythrocyte^,^^,^^ and, as such, may not account
for
the primary pathophysiology
of the disease. Nevertheless,
this reduced
SOD activity on functional assay
may serve as a simpler and more rapid screening
method as a preliminary
to complete sequencing for
the detection of
SOD-1 mutations in those seeking
genetic counseling.
March
1997
NEUROLOGY 48 749
Generally there is no clear correlation between
a
specific mutation and the duration of disease or age
at death, both of which may be measures of disease
severity. Hence it is difficult to provide advice to
individual patients on disease prognosis based on the
finding of a mutation. Individual families may appear
to
follow a pattern, and we have previously
reported the reduced age
at onset in the family with
a G93R mutation,18 which was also distinguished by
the reduction of SOD enzyme activity to 30%
(a dominant
negative effect). The finding of prolonged survival
of mean 17 years in
a Japanese family with the
H46R mutation of
SOD-1 with 80% preservation of
SOD
enzyme may support an influence of
enzyme activity on the course of the disease. However,
there was SOD activity of only 61% in one
affected family member32; the mutant SOD lacked
significant enzyme and the hypothesis
that SOD enzyme activity influences the severity of
the disease lacks substantial support
at present. The
mutation A4V
is also associated with a more aggressive
course
of disease, with reduced disease duration
or survival (mean 1.2
Ifi. 0.2 [SEI years) and reduction
of mean erythrocyte SOD activity to 58% nor-
It
is not possible to distinguish reliably individual
patients with
SOD-1 mutations from patients with
familial
ALS without mutations on clinical grounds,
However, in our series the majority had onset in the
lower limbs, while for ALS in general, the onset is
distributed among the lower limbs (41%), upper
limbs (34%), and bulbar region (24%), with
a similar
distribution in familial ALS of all types.2 This is of
interest as transgenic mice carrying
a human SOD-1
mutation develop a motor neuron disease that commences
in the limbs, especially the lower limbs.31
These animals also have filamentous inclusions in
neurons and axons in the late stages of the disease,35
in common with the human disease. The pathogenic
mechanism by which these mutations cause the disease
is uncertain, but the predilection for the motor
neurons that are the largest and longest, with high
metabolic activity, makes the common involvement
of neurofilaments an attractive hypothesi~.l~J~>~~
For the patients and families with the disease the
two immediate issues relating to
SOD-1 are treatment
and genetic counseling. The ultimate goal of
this field of research is
to determine the pathogenesis
of
ALS, and to develop appropriate treatments or
preventive strategies. The immediate response to the
finding of
SOD-1 mutations and reduced SOD enzyme
activity was
to suggest free radical scavenging
agents such
as vitamins E and C, or other medications
such as ~elegi l ineH.~o~w ever, if the disease
is
due to other mechanisms, the potential benefits or
adverse effects of these when used over prolonged
periods of time are unknown.12 Again, based on the
finding of reduced SOD enzyme activity, SOD was
given using an intrathecal route with implanted
pump,38 although the patient had advanced disease
at the time of treatment.
ma1.34
750
NEUROLOGY 48 March 1997
Genetic counseling is possible, based on the autosoma1
dominant pattern of inheritance in larger families,
although the disease onset and duration
is unpredictable.
(In regions of Sweden and Finland the
mutation, or polym~rphi smD,~90~A may be inherited
in an autosomal recessive fashion, the heterozygotes
being asymptomatic carriers and only the homozygotes
manifesting the disease.40) “Predictive testing”
for the mutation is possible, but with many of the
anxieties associated with an adult-onset disease, an
individual with
a mutation may lead an otherwise
normal life without developing
ALS. In the absence
of the mutation, the individual from the
SOD-1 family
will still have a probability of developing the disease
similar to that in the rest of the population,
which is approximately l:l,000.3
Our finding of
SOD-1 mutations in around 20% of
families with
ALS concords with that found in North
A m e r i ~ a .T~h~e .g~en~et ic cause in the remaining 80%
of families and the 98% of
ALS patients in general
remains
to be determined, but further investigation
of the clinicopathologic correlations of these
SOD1
mutations in patients with ALS may give clues
to
the common pathogenic mechanisms in
ALS. If specific
treatments or preventive strategies are available
for
SOD-1 ALS, mutation testing may be indicated
but, at present, the indications are largely
research-based.
Acknowledgments
We thank all the patients and their families who have participated
in this project. We thank the many physicians who have
collaborated in providing clinical information and support, in particular
Professor Nigel Leigh and his team at the Institute of
Psychiatry, London, and the Motor Neurone Disease Association
and its network of care advisors. We acknowledge the use of the
resources of the MRC Human Gene Mapping Project.
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