Page created by Sam Simpson
Brain (1997), 120, 1723–1737

Phenotypic heterogeneity in motor neuron disease
patients with CuZn-superoxide dismutase mutations
in Scandinavia
P. M. Andersen,1 P. Nilsson,2 M.-L. Keränen,5 L. Forsgren,1 J. Hägglund,3 M. Karlsborg,6
L.-O. Ronnevi,4 O. Gredal7 and S. L. Marklund2

Departments of 1Neurology and 2Clinical Chemistry, Umeå            Correspondence to: Dr Peter M. Andersen, Department of
University Hospital, Umeå, 3Department of Neurology,               Neurology, Umeå University Hospital, S-901 85 Umeå,
Mälar Hospital, Eskilstuna, and 4Department of Neurology,         Sweden
Karolinska Hospital, Stockholm, Sweden, and 5Department
of Neurology, Lapland Central Hospital, Rovaniemi,
Finland, 6Department of Neurology, Hvidovre Hospital and
7Department of Neurology, Glostrup Hospital, Copenhagen,


Four-hundred and fifty-one blood samples from Scandinavian          and lower motor neuron systems affected. The patients
patients with motor neuron disease were analysed for                showed different sites of onset, though the progressive bulbar
mutations in the CuZn-superoxide dismutase gene. Forty-one          palsy form of the disease appears to be rare among patients
(9.6%) of the 427 patients with the amyotrophic lateral             with a CuZn-superoxide dismutase mutation. The progression
sclerosis (ALS) form of the disease were found to have a            of motor signs and symptoms followed the same basic pattern
disease-associated mutation, and 14 of these patients were          in patients with different mutations. Extra-motor system
apparently sporadic cases. A mutation was found in 12 of            symptoms were frequent among patients with a CuZn-
the 51 families with recognized familial ALS. The five different    superoxide dismutase mutation. The results suggest that
mutations found (Ala4Val, Val14Gly, Asp76Tyr, Asp90Ala,             patients with mutations in the CuZn-superoxide dismutase
Gly127insTGGG) have different genetic characteristics and           gene constitute one disease entity. The Val14Gly and Asp76Tyr
are associated with very variable phenotypes spanning from          mutations have not been reported before, and the latter is
rapidly progressing disease with only lower motor neuron            the first mutation to be found in exon 3 of the CuZn-
signs to very slowly progressing disease with both the upper        superoxide dismutase gene.

Keywords: Asp76Tyr CuZn-SOD mutation; Asp90Ala CuZn-SOD mutation; amyotrophic lateral sclerosis; progressive
bulbar palsy

Abbreviations: ALS 5 amyotrophic lateral sclerosis; CuZn-SOD 5 copper- and zinc-containing superoxide dismutase;
FALS 5 familial ALS; Hb 5 haemoglobin; PBP 5 progressive bulbar palsy; PCR 5 polymerase chain reaction; SALS 5
sporadic ALS; SSCP 5 single strand conformation polymorphism

The term motor neuron disease encompasses a variety of              cord, and loss of the motor neurons in these nuclei. This is
neurodegenerative diseases of which amyotrophic lateral             clinically manifested by hyperreflexia, clonus, spasticity and
sclerosis (ALS) is the most common. Since the work of               progressive paresis and wasting of the skeletal muscles with
Charcot and Joffroy (1869), ALS has come to define a fatal,         fasciculations relatively sparing the oculomotor and sphincter
progressive disease involving loss of motor neurons in              muscles (Brooks, 1994).
the cerebral cortex, degeneration of the descending motor              Most epidemiological studies have found that ~35% of
pathways to the motor nuclei in the brainstem and spinal            cases begin in the lower limbs, 30–35% in the upper
© Oxford University Press 1997
1724      P. M. Andersen et al.

Table 1 Number of samples analysed from each country and subdivided into hereditary disposition for motor neuron
Country                                       Denmark             Finland            Norway              Sweden              Total

Motor neuron disease patients analysed* (n)   27                  107                43                  274                 451
Those with ALS only
  SALS                                        25 (1)               80 (9)†           37                  213 (4)             355 (14)
  FALS                                         2 (2)               21 (9)             4                   45 (16)             72 (27)

Number of patients with a missense or non-sense SOD mutation is shown in parenthesis. *Includes in addition to patients with ALS also
patients with Kennedy’s Syndrome (11), primary lateral sclerosis (four), motor neuron disease–parkinsonism and/or motor neuron
disease–dementia complex (seven), monomelic motor neuron disease (one) and Hiryama’s disease (one). None of these carried a CuZn-
SOD mutation. †Of the nine sporadic cases with a SOD mutation found in Finland, seven were homozygous and two were heterozygous
for the Asp90Ala mutation. SALS 5 sporadic ALS; FALS 5 familial ALS.

extremities, 3–5% have truncal onset and in 18–33% the               by DNA sequence analysis performed on plasmid-cloned
disease starts as a progressive bulbar palsy (PBP) (Kristensen       PCR products. At least two clones showing the same PCR–
and Melgaard, 1977; Rosati et al., 1977; Forsgren et al.,            SSCP bands as the genomic DNA from the patient were
1983; Gubbay et al., 1985; Haverkamp et al., 1995). It               sequenced. With informed consent, samples were collected
has been disputed whether patients with PBP constitute a             from relatives of the patients with a CuZn-SOD mutation.
nosologic entity distinct from patients with limb and truncal        These samples were analysed either by PCR–SSCP or by a
onset. In 1830, Sir Charles Bell described a patient with            restriction enzyme test, as the Asp90Ala mutation introduces
paresis of the tongue and limbs, in 1848–50 Aran detailed            a new site for the restriction enzyme Fnu4H1 (Andersen
adult patients with asymmetric limb onset, progressive               et al., 1996).
wasting and paresis, and in 1860 Duchenne described patients            The SSCP technique is reported to have a sensitivity of
with ‘progressive muscular paralysis of the tongue, palate and       80–90% (Sheffield et al., 1993). To increase the likelihood
lips’. Finally, Charcot (1881) applied the name amyotrophic          of finding patients with mutations, we also measured the
lateral sclerosis to the motor neuron syndrome with lesions          CuZn-SOD activity in erythrocytes as described previously
of both the upper and lower motor neurons, with the                  (Marklund, 1976, 1985). The assay was performed directly
syndromes of progressive muscular atrophy and PBP being              on haemolysates without prior precipitation of haemo-
considered as separate entities. Supporting the idea that PBP        globin (Hb), and the SOD enzyme activity was expressed in
may be a separate entity is a female preponderance and               U/mg Hb.
significantly higher mean age of onset compared with patients           The case records of the patients with a CuZn-SOD mutation
with onset in other parts of the body (Kristensen and                were scrutinized, and the family history and pedigree were
Melgaard, 1977; Rosati et al., 1977; Forsgren et al., 1983;          documented by interviewing patients and relatives, and by
Haverkamp et al., 1995).                                             inspection of official records. The study received approval
   Since the observation in 1993 that some familial cases of         from the ethical committees of the universities of
ALS are associated with mutations in the gene encoding the           Copenhagen, Denmark; Oulu, Finland and Umeå, Sweden.
free radical scavenging enzyme CuZn-superoxide dismutase
(CuZn-SOD) (Rosen et al.,1993), we have analysed blood
samples from Scandinavian patients with motor neuron
disease for mutations in the CuZn-SOD gene. We present               Results
here the results of our detailed phenotypic and genealogical         From the spring of 1993 to the end of 1996 we collected a
studies of the patients with different CuZn-SOD mutations.           total of 451 samples from approximately a quarter of all
                                                                     adult motor neuron disease cases in the four countries during
                                                                     that period. Of the analysed samples from ALS cases (Table
Patients and methods                                                 1), 355 were sporadic ALS (SALS) cases and 72 (16.9%)
Blood samples were collected according to a standardized             were familial ALS (FALS) cases from 51 families. A CuZn-
protocol (Andersen et al., 1995), with informed consent,             SOD mutation was found in 44 (10.3%) samples. Three
from patients with adult-onset motor neuron disease living           (AAC139→AAT, GCT140→GCA, CAA153→CAG) were
in Denmark, Finland, Norway and Sweden. All samples                  silent mutations (mutations not causing a change in the amino
were screened for mutations in the CuZn-SOD gene using               acid sequence of the final polypeptide), each found in one
a polymerase chain reaction (PCR) single-stranded                    ALS patient. In 41 patients five different mutations were
conformational polymorphism (SSCP) technique as described            found, of which four were missense mutations (replacement
previously (Andersen et al., 1995). All five exons of the            of one amino acid with another amino acid) and one a
CuZn-SOD gene were analysed. In samples with an abnormal             nonsense mutation (replacement of an amino acid codon with
SSCP band pattern, the existence of a mutation was confirmed         a stop codon), and can be presumed to be disease causative.
Phenotypic heterogeneity in motor neuron disease          1725

The Ala4Val (A4V) mutation                                           Six months after onset the patient complained of dyspnoea
The Ala4Val CuZn-SOD mutation was found in a large                   and aphagia, and a gastrostomy was performed. At the time
family originating in central Sweden. Part of the pedigree is        of his death, ,8 months after onset of the first symptom, the
shown in Fig. 1A.                                                    patient could still walk but the arms and tongue were wasted;
   II-2 developed a rapidly progressive motor neuron disease         the left Achilles reflex could be elicited but otherwise there
at the age of 55 years, beginning in the hand and soon               was areflexia. As in all earlier examinations, the Babinski
involving the whole upper left extremity with initial shoulder       sign could not be elicited. Oculomotor function and sphincter
pain. Death came 11 months after onset of the first symptom.         functions remained intact.
   III-4 developed suddenly, at the age of 68 years, ripplings          SSCP analysis on lymphocytes obtained from IV-4 revealed
in the left facial muscles and progressive dysarthria followed       an aberrant band pattern in exon 1. Subsequent nucleotide
by dysphagia and a progressive left facial nerve palsy. Ten          sequencing revealed a heterozygous missense C to T exchange
months after onset and 1 month before her death, she                 in base pair position 94 in codon 4, resulting in an alanin to
had severe dysarthria, tongue atrophy with fasciculations,           valin (Ala4Val; A4V) substitution. Samples from eight family
atrophic paralytic facial muscles, paralysis of the left recurrent   members with a 50% risk of having inherited the mutant
laryngeal nerve, severely paretic and wasted upper                   allele were also analysed, and two were found to have
extremities, especially distally. There was only slight paresis      inherited the mutation, including an off-spring of III-6. They
of the lower limbs. The deep tendon reflexes were very weak          are 56 and 54 years of age, and do not show symptoms of
or absent in all four limbs. The Babinski response could not         motor neuron disease. The enzyme activity in the three
be elicited. Cutaneous sensation and neuro-ophtalmological           individuals with the Ala4Val mutation was found to be
                                                                     decreased to ~50% (22.9, 23.3 and 26.0 U/mg Hb) of control
examination were normal. EMG showed neurogenic atrophy
                                                                     subjects (Table 2).
in all four limbs.
   III-6 noticed right foot dorsal flexion paresis at the age of
50 years. Six months later she had a severe flaccid paresis
of the right leg, apparently without the left leg or of the
                                                                     The Val14Gly (V14G) mutation
                                                                     The Val14Gly CuZn-SOD mutation was found in a family
upper extremities being affected. Cutaneous sensation was
                                                                     whose paternal line originates in central Sweden and the
intact. No fasciculations were registered, and no signs of upper
                                                                     maternal line in Finland. The patient is the only known case
motor neuron involvement. Spinal myelography revealed no
                                                                     of motor neuron disease in his family and he was considered
significant abnormalities. EMG showed neurogenic atrophy.
                                                                     a SALS case. The grandparents all lived to an advanced age
Ten months from onset the patient had a flaccid paraparesis,
                                                                     and died rather suddenly. The father died of myocardial
and distal paresis and wasting in the right arm. The left arm
                                                                     infarction at the age of 51 years, and the mother shows no
was unaffected until 3 months before her death, 17 months            symptoms of motor neuron disease at the age of 68 years. A
after onset of the first symptom. Autopsy confirmed the              sister is healthy at the age of 40 years.
diagnosis.                                                              The patient developed a sense of fatigue in both legs at
   In IV-4, illness began at the age of 61 years with sudden         the age of 39 years. Some months later, lower back fatigue
disturbed motor control of his right hand. A progressive             and episodes of severe neuralgic pain in the hips began.
stroke was suspected, and he was initially treated with              Later he noted stiff calves and muscle twitches in both legs.
infusion of heparin but a CT-scan of the brain was normal.           In the ensuing months he had unprovoked episodes of
The initial examination revealed intact cranial nerves,              chilliness of up to 3–4 h duration. Initially, paresis developed
fasciculations on both shoulders and on the right thigh, a           distally in the legs, but it soon ascended to the thighs. Six
slight paresis of shoulder elevators and paresis of the right        months after noticing fatigue he needed to walk with the aid
hand but no paresis in the lower limbs. The right brachioradial      of a stick. A spinal cord MRI scan was unremarkable, as
reflex was absent. The other deep tendon reflexes were               was a spinal fluid examination, except that the protein content
normal. Decreased pain sensation was noted in the right              was raised to 900 mg/l. EMG, 7 months from onset, revealed
forearm and hand. Within 1 month dysphagia developed and             neurogenic changes in the lower extremities only. Motor and
the left arm became paretic. Three months after onset,               sensory conduction studies were normal. Repeated
dysarthria was noted. On re-examination, the upper                   neurological examinations revealed involvement of both
extremities showed a flaccid paresis with distal wasting             lower and upper motor neuron systems, but Babinski signs
and paresis of the neck extensors, lingual fasciculations,           were not found on any examination. After 10 months, paresis
diminished or absent deep tendon reflexes in the arms,               appeared first in the left arm and later in the right arm, and
predominantly on the right side, but reflexes in the legs were       progressive urgency of micturition also developed. Sixteen
normal. Sensation of vibration was normal. EMG revealed              months from the initial symptoms, a reduced sense of
neurogenic changes in the tongue and in both arms but not            vibration was noted in both feet, otherwise sensibility has
in the legs. Five months after onset, paresis developed in the       remained intact. The patient is now severely tetraparetic with
right leg, and the patellar reflexes were absent. The right          dysphagia and dysphonia, but no dysarthria. The patient has
Achilles reflex was weak while that on the left was brisk.           not shown mental impairment or affective lability.
1726     P. M. Andersen et al.

  Analysis of a blood sample from the patient showed a        I-1 and I-2 died of unknown causes aged 57 and 47 years,
reduced CuZn-SOD activity of 30.3 U/mg Hb in erythrocytes     respectively. They raised four children, of whom II-1 died at
(control subjects 52.8 6 5.86 U/mg Hb, n 5 96) but the        the age of 60 years of a heart disease, II-2 at the age of 52
SSCP analysis only revealed a faint, easily overlooked,       years (unknown cause) and II-3 at the age of 77 years of a
aberrant band in exon 1 (Fig. 2A) the SSCPs for exons 2–5     stroke. None of them had children. Born in 1868, II-4 enjoyed
being normal. Nucleotide sequencing of exon 1 revealed a      a healthy life until, at the age of 67 years, he complained
heterozygous missense T to G exchange in position 124 in      of pharyngeal irritation and copious phlegm, recovering
codon 14, resulting in a valin to glycin (Val14Gly; V14G)     completely within a few weeks. The sense of irritation in the
substitution (Fig. 2B). This example shows that analysis of   pharynx returned ~6 months later, and he soon developed
erythrocyte SOD activity in addition to screening by PCR–     dysphonia and dysphagia causing drooling. He had no
SSCP increases the likelihood of detecting CuZn-SOD           complaints about the extremities. Physical examination the
mutations.                                                    following month revealed no signs of infection, but the deep
                                                              tendon reflexes in the extremities were very brisk. During
The Asp76Tyr (D76Y) mutation                                  the following months dysarthria developed and the dysphonia
Two cases of motor neuron disease have been found in this     progressed, but the patient could still communicate by writing.
Danish family with the Asp76Tyr mutation (see Fig. 1B).       His personality changed. He became self-centred, stubborn,
Phenotypic heterogeneity in motor neuron disease                1727

with affective lability but no symptoms of dementia.                      whistle and his tongue was atrophic with ‘fibrillations’.
Dyspnoea developed, and shortly before his death ~15 months               Fibrillations were not found in the extremities. The abdominal
after the first pharyngeal symptoms, it was noted that he had             reflexes were absent. The musculature in the extremities was
difficulties in walking, particularly with the right leg. Pupil           weak and wasted with brisk tendon reflexes in all four
and eye movements were normal, the patient could not                      extremities mainly on the right side, and clonus was noted

Table 2 Main features of the five CuZn-SOD mutations

                                        Ala4Val          Val14Gly         Asp76Tyr         Asp90Ala          Asp90Ala         Gly127ins4TGGG

Zygosity                                Hetero           Hetero           Hetero           Homo              Hetero           Hetero
Exon                                     1                1                3                4                 4                5
Patients identified (n)                  4                 1                2              44                 2                3
Patients genotyped (n)                   1                 1                1              34                 2                2
Uniform phenotype                       No               –                No               Yes               Yes              No
Site of onset                           B,UE,LE          LE               B,LE             LE                B                UE,LE
Mean age at onset in years (n)          58.5 (4)         39 (1)           58 (2)           44,8 (44)         64 (2)           50 (3)
Onset range (years)                     50–68            39               49–67            20–94             54–74            41–59
Mean survival time in years (n)          0.9 (4)         –                –                14.2 (15)          2.5 (2)          2.8 (3)
Asymptomatic individuals (n)             2                 0                1              11*                1†               4
CuZn-SOD activity in erythrocyte        24.1 6 1.7 (3)   30.2 (1)         27.4 6 0.6 (2)   49.1 6 5.9 (44)   43.6 6 4.3 (2)   21.2 6 2.4 (4)
   hemolysate mean 6 SD (U/mg Hb)
Geographical area                       Central          Central          Denmark          Finland           Northern         Denmark
                                        Sweden           Sweden                            Sweden            Finland

Location of onset: B 5 bulbar; UE 5 upper extremity; LE 5 lower extremity; T 5 truncal (none found in this study). *Seven of them are in
their twenties or thirties (children of homozygous patients married to heterozygous spouses). †An Asp90Ala heterozygous sister of one of the two
cases with sporadic PBP. Additional 115 Asp90Ala heterozygous individual blood-relatives of the Asp90Ala homozygous patients were also
found; none of these have developed motor neuron disease (mean age 56 6 15 years). ‡The CuZn-SOD activity in 96 blood relatives without a
CuZn-SOD mutation was 52.8 6 86 U/mg Hb.

                 Fig. 1 Pedigrees of the families with the Ala4Val (A), Asp76Tyr (B) and Gly127insTGGG (C)
                 mutations. Pedigrees of families with the Asp90Ala mutation have been published earlier (Andersen
                 et al., 1995, 1996). The fates of II-4–11 in the Ala4Val family are unknown. Individuals are indicated
                 by generations and pedigree numbers. Circle 5 female; square5 male; diamond 5 unspecified sex;
                 filled symbols 5 affected; partly filled symbol 5 possible affected (unconfirmed); open symbols 5
                 unaffected; symbols with horizontal lines 5 dementia; oblique line 5 deceased individual;
                 arrow 5 index patient. Minor changes have been made to the pedigrees for confidentiality.
1728      P. M. Andersen et al.

Fig. 2 PCR-SSCP and DNA sequence analysis of samples from patients with the Val14Gly and Asp76Tyr mutations. The analyses were
performed as described under Patients and methods. (A) PCR–SSCP analysis of exon 1 of a Val14Gly sample and a control; (B) Exon 1
DNA sequence analysis of the Val14Gly sample showing the missense mutation GTG→GGG causing the Val14Gly substitution; (C)
PCR–SSCP analysis of exon 3 of the two Asp76Tyr samples, a control sample and a sample lacking DNA; (D) Exon 3 DNA sequence
analysis showing the GAT→TAT missense mutation causing the Asp76Tyr substitution.

in both ankles. The Babinski sign was found on the left side.       The disease progressed with slowly ascending paresis. Five
No tremor was found, and cutaneous sensation was intact.            years from onset, he required a walking stick. At 6 years he
   IV-5, the grandchild of II-4, noted instability of his right     had developed a complete drop-foot on the right side, and
ankle at the age of 49 years. The following year he complained      after 7 years he first experienced symptoms in the upper
of right knee pain and of being unable to stand on the tip of       extremities in the form of slowly progressive paresis and
his right foot. An examination 1 year later revealed intact         wasting of the small muscles of the right hand. Twelve years
cranial nerves and upper extremities. Extensive wasting was         after onset, he could no longer walk or stand on his feet.
found only in the right lower limb affecting both the extensor      The same year an insulin-dependent diabetes mellitus was
and flexor muscles, and being most severe distally. No paresis      diagnosed. EMG performed 13 years after onset showed the
was found in the left leg. No fasciculations were found. The        classical signs of neurogenic lesions in all four limbs. A
patellar reflexes were bilaterally brisk, the right more than       physical examination found no signs of cognitive disturbance
the left. The Achilles reflex could not be evoked on the right      and the cranial nerves were intact. The patient was severely
side but was brisk on the left side. The plantar response on        tetraparetic with extensive muscular wasting mainly distally
the right side was extensor, but normal on the left. The            and on the right side of the body. There was surprising
abdominal reflexes were present bilaterally. Cutaneous              strength in the shoulder muscles. The deep tendon reflexes
sensibility and sphincter functions were found intact. Signs        were absent, plantar reflexes were ‘normal’, and cutaneous
of ataxia were not found on this or later examinations. A           sensation and proprioception were intact. The following year
spinal fluid examination was unremarkable as was a spinal           dysphagia developed. An examination revealed slight paresis
myelography. EMG of the right tibial anterior muscle showed         and atrophy of the tongue with fasciculations, but the
neurogenic changes. Motor and sensory nerve conduction              pharyngeal reflexes were normal. Since then, dysphonia and
studies were normal.                                                dyspnoea have appeared but the patient still lives at home
   Three years after onset in the right leg he experienced          15 years after onset of the first symptom, using an electric
instability and later weakness starting distally in the left leg.   wheelchair controlled by his left hand.
Phenotypic heterogeneity in motor neuron disease           1729

   His mother, III-4, died at the age of 84 years after having    the Asp90Ala mutation. These two patients both presented
suffered from dementia, allegedly due to arteriosclerosis in      with PBP, the male patient dying ,2 years after disease
the last 6 years of her life. According to available records,     onset, at the age of 56 years, and the female patient died 3
her cardinal symptom was progressive loss of memory. No           years after disease onset, at the age of 77 years. A 72-
information has been found suggesting that she ever               year-old sister of the male patient was also found to be
developed signs of affective lability like those of her father.   heterozygous for the Asp90Ala mutation. She shows no
An examination 2 weeks prior to her death showed no paresis       signs of motor neuron disease. The basis for the existence
or wasting. She died of sepsis a week after a femur amputation.   of adult-onset recessive ALS with patients homozygous
   III-2 and III-5 were both demented with a clinical picture     for the Asp90Ala CuZn-SOD mutation is a CuZn-SOD
very similar to III-4 and they died aged 77 and 84 years,         polymorphism (Kirjarinta et al. 1969; Beckman, 1973). The
respectively. They did not show signs or symptoms of motor        allele frequency in most of Finland is ~0.5–1%, but in
neuron disease, and none of their six children (aged 45–70        northern Finland and in northern Sweden frequencies as high
years) had developed neurological symptoms. III-7 died,           as 2.5–2.7% have been found (see Andersen et al., 1996).
aged 71 years, of a ruptured aneurysm of the aorta without        The two sporadic cases with PBP both came from northern
prior signs of motor neuron disease, and III-11 died at the       Finland where the allele frequency is ~1%, i.e. 2% of
age of 72 years after suffering from repeated cerebrovascular     the population in this area should be heterozygous for the
insults. Neither showed signs of intellectual impairment          Asp90Ala mutation, and it may be a coincident finding.
before death. III-10 is alive without signs of motor neuron          The enzyme activity of individuals homo- or heterozygous
disease or dementia. Genealogical studies of the family of        for the Asp90Ala mutation is reduced by ~7% in both groups
III-3 revealed no cases of motor neuron disease among his         compared with neurological control subjects and blood-
parents, eight siblings or their children.                        relatives without a CuZn-SOD mutation (Table 2; Andersen
   SSCP analysis of a sample from IV-5 showed an aberrant         et al., 1995).
band in exon 3 (Fig. 2C), and nucleotide sequencing revealed
the patient to be heterozygous for a missense G to T exchange
in position 695 in codon 76 (Fig. 2D) resulting in an aspartic    The Gly127insTGGG mutation
acid to tyrosin (Asp76Tyr; D76Y) substitution. The mutation       Three definitive cases of motor neuron disease have been
was also found in a middle-aged healthy relative of IV-5 (not     found in the Danish family with this mutation (see Fig. 1C).
shown in the pedigree for reasons of confidentiality). III-10        I-1 died in 1893, at the age of 46 years, of ‘nerve
and the children of III-2 and III-5 do not carry the mutation.    inflammation’ and may have had motor neuron disease. II-2
   The enzyme activity in the two individuals with the            died at the age of 35 years after an abortion, and II-1 died
Asp76Tyr mutation was found to be decreased to ~50% (27.0         at the age of 92 years of ‘old age’. III-2 and III-6 both died
and 27.8 U/mg Hb, respectively) of control subjects (Table 2).    at the age of 72 years of myocardial infarction and ventricular
                                                                  cancer, respectively. III-1 and III-7 are both alive (aged 89 and
                                                                  81 years, respectively) without signs of motor neuron disease.
The Asp90Ala (D90A) mutation                                         III-5 developed progressive wasting and paresis at the age
Of the 451 samples tested, 34 were found to be homozygous         of 50 years, starting in the left calf. Examined 1 year later,
for the Asp90Ala CuZn-SOD mutation, which we have earlier         the patient had a paraparesis. The left Achilles reflex was
reported (Andersen et al., 1995). Including deceased ALS          absent and the right was diminished, the left patellar reflex
patients, relatives of the 34 whom we have shown to be            was weak and the right considered normal. There were no
homozygous for the Asp90Ala mutation, a total of 44 patients      Babinski signs. The upper extremities had normal strength
have been found showing a characteristic phenotype with           with symmetrically very brisk reflexes. The cranial nerves
asymmetrical onset distally in the lower extremities with         were intact. Re-examined 9 months later, the patient walked
slowly ascending paresis. Details of the phenotype of 36 of       with difficulty and had severe paresis and extensive wasting
these patients have been reported earlier (Andersen et al.,       of both upper extremities. Abundant fasciculations were
1996). However, among the additional eight patients, signs        found all over the body. The tongue was considered normal,
of slight ataxia have been noted in four patients. The 44         but a left-sided paresis of the buccator muscle was found.
homozygous patients (10 apparent SALS cases and 34 FALS           Cutaneous sensation was normal. The deep tendon reflexes
cases taken from 10 families) were all born in northern           and Babinski signs were absent. EMG showed neurogenic
Sweden or in Finland. Among their relatives, we have found        atrophy. Dysphagia developed 3 months prior to her death
115 individuals to be heterozygous for the Asp90Ala mutation      30 months after onset.
as well as 68 individuals without a CuZn-SOD mutation.               IV-13 first noticed weakness in the left arm at the age of
None of these have demonstrated symptoms of motor                 59 years. An examination 3 months later revealed intact
neuron disease.                                                   cranial nerves, paresis of the left shoulder with inablity to
   Of the 80 blood samples obtained from cases of sporadic        lift the left arm above the head. Cutaneous sensation was
motor neuron disease living in Finland, seven were found to       normal, but the sense of vibration was decreased in the left
be homozygous (described above) and two heterozygous for          hand and left foot. The muscle tone was normal. The right
1730      P. M. Andersen et al.

upper extremity and the lower extremities had normal strength     we have found five different CuZn-SOD mutations with
and showed no atrophy. The deep tendon reflexes were very         novel features:
brisk in all four extremities, a Babinski response was found
on the right side only, and there was a tendency for clonus
in the left ankle. The abdominal reflexes were present.           The phenotypes
No fasciculations were noticed at this time. EMG showed           Whereas the 44 patients homozygous for Asp90Ala showed
neurogenic atrophy with denervation, polyphasia and               a characteristic uniform phenotype with insidious onset,
spontaneous activity with fasciculations. MRI of the cervical     slowly ascending paresis beginning distally in the lower
spine revealed no significant abnormalities. Six months after     extremities and long survival, patients with the other four
first symptom she could no longer rise from a squatting           mutations showed diverging phenotypes. The four patients
position. Generalized myalgia and cramps appeared. Re-            with the Ala4Val mutation all had sudden onset (suggesting
examined 9 months after onset revealed generalized                the existence of some kind of trigger factor) and showed
symmetrical hyperreflexia, extensive fasciculations, paresis      rapid disease progression, but the sites of onset differed. The
and wasting of the left arm (more proximal than distal). No       three patients with Gly127insTGGG exhibited different sites
paresis in the right arm. Twelve months after onset increased     of onset, but the disease progression rate was slower than
muscle tone was noted in both lower and the upper left            for the patients with the Ala4Val mutation. In the Asp76Tyr
extremity, atrophy of the small hand muscles bilaterally, very    family, one patient had bulbar onset and rapid progression,
brisk deep reflexes in the right arm and both legs but only       while his daughter never showed symptoms of motor neuron
weak reflexes in the left arm. Neither clonus nor the Babinski    disease and the grandchild developed a slowly ascending
sign were found on this or later examinations. Fifteen months     paresis very similar to that of the patients homozygous for
after onset, the patient could still elevate her shoulders, but   Asp90Ala. Strikingly, even on repeated examinations and at
the arms were severely paretic. A predominantly left-sided        different stages of the disease, the patients with Ala4Val only
paraparesis and dysphagia developed, but articulation was         showed signs of lesion to the lower motor neuron system.
intact for several months. In the last months of life the         For the other mutations, signs of lesions to the descending
patient was severely handicapped with generalized wasting,        motor pathways were found in all patients except for III-4
tetraparalysis and universal areflexia. Bladder function          (Gly127insTGGG) who only had lower motor neuron signs.
remained intact during the 32-month-long disease period.          None of the patients developed severe spasticity and, judging
   IV-16 developed a progressive motor neuron disease similar     from published descriptions of patients with other CuZn-
to his sister IV-13 with the exceptions that the first symptoms   SOD mutations, spasticity appears to be uncommon among
were distal paresis in the left lower limb with severe myalgia,   patients with a CuZn-SOD mutation. The greatest difference
and the second limb to be clinically affected was the upper       among patients with the different mutations is the rate of
right extremity. He died at the age of 44 years after a disease   disease progression; the four patients with the Ala4Val
duration of 41 months.                                            mutation had a mean survival time of only 0.9 years compared
   DNA analysis of samples from IV-13 and IV-16 revealed          with 14.2 years for 15 deceased patients homozygous for
an insertion of four base pairs TGGG after base pair 1450         Asp90Ala (Table 2).
in codon 127 causing a frameshift and the introduction of            Urgency of micturition and/or difficulty initiating urination
five new amino acids ending with a premature stop codon in        is a common symptom in patients homozygous for Asp90Ala
position 133. The CuZn-SOD subunit is thereby truncated           (Andersen et al., 1996) and was also reported by the single
by 21 amino acids compared with the wild-type polypeptide         patient with the Val14Gly mutation. Four of the patients
of 153 amino acids. The genetic details of this mutation is       with the Asp90Ala mutation exhibited slight ataxia indicating
being published (Hansen et al., 1997). Four of the five           the involvement of the posterior columns and cerebellum.
siblings of IV-13 and IV-16 (aged 45–67 years) have also          None of the patients with the Ala4Val, Asp76Tyr or
inherited the mutation but are without motor neuron disease.      Gly127insTGGG mutations had similar findings. A preparetic
The CuZn-SOD activity in erythrocytes was measured in             phase characterized by myalgia, muscle cramps and painful
four individuals with the mutation and was found to be            parestheses were only reported consistently by patients
decreased to ~40% (24.5, 21.1, 19.1 and 20.1 U/mg Hb) of          homozygous for the Asp90Ala mutation and the single patient
control subjects (Table 2).                                       with the Val14Gly mutation. The existence of a preparetic
                                                                  phase of months to years duration supports the theory (Swash
                                                                  and Ingram, 1988) that there is a long preclinical period
Discussion                                                        of relative tolerance and compensation. Several patients
The present study is the first large epidemiological study of     demonstrated signs and symptoms of autonomic, bladder,
the frequency of CuZn-SOD mutations in an unselected              cerebellar and/or sensory involvement indicating that in
population of motor neuron disease patients. A smaller study      patients with CuZn-SOD mutations the disease is not confined
of 67 ALS patients has been reported earlier from Scotland        to the voluntary motor system. When present, these features
(Jones et al., 1995). In the comparatively small motor neuron     may distinguish patients with a CuZn-SOD mutation from
disease population in Scandinavia (23.3 million inhabitants)      other motor neuron disease patients.
Phenotypic heterogeneity in motor neuron disease             1731

Table 3 Order of progression of signs and symptoms in motor neuron disease patients with a CuZn-SOD mutation based
on patients with the Ala4Val, Val14Gly, Asp76Tyr, Asp90Ala and Gly127insTGGG mutations (not all symptoms and signs
are found in all patients)
  (i) Symptoms of myalgia and/or cramps. Increased deep tendon reflexes may be found locally, but there is no wasting, paresis or
 (ii) Symptoms of ‘instability’ and/or weakness. The only sign found is that of brisk reflexes not only in the region of complaint but also
      proximal to it, and perhaps in other, still asymptomatic regions.
(iii) Focal, asymmetric paresis develops with some wasting. The deep tendon reflexes in the affected region are diminished or absent. In
      other regions the reflexes are brisk. Superficial reflexes are intact and the plantar response flexor.
(iv) The paresis and wasting progress to include the complete extremity, and the first symptoms appear in another region. In the primary
      affected extremity, the reflexes will be diminished or absent, in the other regions the deep tendon reflexes will be brisk.
      Fasciculations appear not only in the primary affected region but also in nearby myotomes. The abdominal reflexes are absent, and
      the plantar responses may be extensor.
 (v) Generalized wasting with tetraparesis with diminished or, more likely, areflexia in most regions. No plantar response can be elicited.

   Involvement of the posterior columns and spinocerebellar             reported (Kaplan et al., 1996), and two FALS families,
tracts is a common pathoanatomical finding in ALS patients              one homozygous the other heterozygous for the Asp90Ala
though it is clinically most often silent (Engel et al., 1959;          mutation, have been found in different locations in France
Hirano et al., 1967; Averback et al., 1982). Autopsies have             (Dr W. Camu, personal communication). These findings
revealed, in addition to loss of anterior horn cells, involvement       suggest that, in the area where the Asp90Ala polymorphism
of the posterior columns and spinocerebellar tracts in patients         exists in Finland and northern Sweden, there may also be an
with the Ala4Thr (Takahashi et al., 1994), Ile113Thr (Orrell            unknown factor reducing the susceptibility of individuals
et al., 1995b), Glu100Gly (Ince et al., 1996) and Ala4Val               with the Asp90Ala mutation to the deleterious effect of
(Shibata et al., 1996) mutations. Thus, patients with the               the mutated CuZn-SOD molecule; thus only individuals
Ala4Val, Val14Gly and Gly127insTGGG mutations conform                   homozygous for the mutation develop ALS. This resistance
to Type II and patients homozygous for the Asp90Ala                     factor may also account for Scandinavian Asp90Ala patients
mutation conform to Type III according to the FALS                      having a far more homogeneous phenotype compared with
classification of Horton et al. (1976).                                 patients with the Asp90Ala mutation from other locations,
   The patients had asymmetrical and focal onset of paresis             or, indeed, compared with nearly all other reported CuZn-
and wasting as dominant symptoms in common. Development                 SOD mutations. This resistance factor may also contribute
of new symptoms and signs followed a stereotyped pattern                to the very long disease duration in Scandinavian patients
(Table 3). With disease progression, the signs of lesion to             homozygous for the Asp90Ala mutation. Several of these
the descending motor pathways may be masked by advanced                 patients moved before onset of ALS to other parts of Sweden,
muscular atrophy. Also in common was the absence of                     where they lived under different conditions, but the phenotype
bedsores. Only two patients developed small bedsores after              of the patients who moved is similar to patients who remained,
long immobilization, supporting the idea that the skin is               suggesting this tentative protective factor may be of genetic
involved in the ALS disease process (Kolde et al., 1996).               origin or from influences early in life, or both. This idea is
                                                                        supported by the fact that the Finnish population is very
                                                                        homogeneous and genetically different from most other
Dominantly and recessively inherited ALS with                           populations of Europe (Nevanlinna, 1972).
the Asp90Ala mutation
The relatively uniform phenotype of the 44 patients
homozygous for the Asp90Ala mutation is noteworthy. Our
earlier finding of two patients with PBP heterozygous for the           Sporadic cases and disease penetrance
Asp90Ala mutation suggests that either they were coincidental           The Val14Gly was found in an apparently sporadic case.
carriers of the Asp90Ala allele or that there are other factors         Genealogical studies failed to find any other family members
shaping the phenotype of individuals with a CuZn-SOD                    with a neurodegenerative disease. The widespread Asp90Ala
mutation. Recently, five patients of non-Scandinavian descent           polymorphism in Finland and northern Sweden associated
heterozygous for the Asp90Ala mutation have been found in               with ALS in homozygous form makes it possible that
Belgium with variable phenotypes (Robberecht et al., 1996),             apparently sporadic cases have a genetic aetiology. Seven
none having a phenotype similar to our earlier reported                 of the 80 sporadic motor neuron disease cases analysed in
homo- and heterozygous patients. Also, a SALS case                      Finland were homozygous for the Asp90Ala mutation, and a
heterozygous for the Asp90Ala mutation has been found in                further three apparently sporadic cases were found in Sweden
England (Jackson et al., 1996), a North American Caucasian              (Table 1). Since we have found Asp90Ala homozygous
FALS family homozygous for the Asp90Ala mutation with                   patients with late onset of ALS, we cannot exclude the
rapidly progressing disease and ataxia has recently been                possibility that homozygosity for Asp90Ala mutation may
1732      P. M. Andersen et al.

be associated with complete penetrance for ALS; it then          Ala4Val mutation showing only symptoms and signs of a
becomes a matter of the patient surviving other diseases.        rapidly progressive lower motor neuron disease, but autopsy
   The other three mutations found show dominant                 revealed degeneration of the corticospinal tracts in three of
inheritance, the Ala4Val with high penetrance, but the           the American cases (Shibata et al., 1996). Since members of
pedigree is too small to conclude that the penetrance is         the Swedish family are known to have emigrated to North
complete. In the family with the Gly127insTGGG mutation,         America, it is possible that the American patients are
III-4 was considered a SALS case because her mother died         descendants of Swedish emigrants (1.2 million Swedes
early, at the age of 35 years, without symptoms of ALS.          emigrated to North America in the last century).
Four asymptomatic carriers of the Gly127insTGGG allele
were found in this family aged between 45 and 67 years.
The sample is too small to assess whether or not the             Progressive bulbar palsy
Gly127insTGGG mutation is associated with decreased              PBP appears to be a very rare phenotype in patients with a
penetrance.                                                      CuZn-SOD mutation (Radunovic and Leigh, 1996). We
   The ALS patient with the Asp76Tyr mutation was                reported two cases of PBP heterozygous for the Asp90Ala
considered sporadic until the discovery of reduced CuZn-         mutation (Andersen et al., 1995), but due to the high
SOD activity and subsequently the Asp76Tyr mutation              frequency of the Asp90Ala allele in northern Finland these
prompted us to investigate the family history. No sample is      findings may have been coincidental. In the present study,
available from II-4 to prove that he did carry Asp76Tyr. The     the detailed case records leave no doubt that III-4 in the
possibility exists that his grandchild IV-5 may be a new         Ala4Val-family and II-4 of the Asp76Tyr-family both
mutation and that the occurrence of motor neuron disease in      presented with PBP, even though samples are not available
II-4 and IV-5 may be independent events. In the case records     to prove that they actually did carry a SOD mutation. III-4
of II-4, it is stated that no other cases are known in the       (Ala4Val) is an obligate gene carrier. The present report is
family. None of his six children developed motor neuron          the first of an Ala4Val patient with PBP. Of the 57 CuZn-
disease. The only living child is III-10 and she does not        SOD mutations reported in ALS patients (Fig. 3), PBP has
carry the Asp76Tyr allele. III-2, -4 and -5 all developed a      been reported only in a Japanese family with the Val148Ile
similar demential disease in their seventies, with progressive   mutation (Ikeda et al., 1995a), and a German family with
loss of memory as the cardinal symptom. We suggest that          the Ile151Thr mutation (Kostrzewa et al., 1996). This is
the Val14Gly and Asp76Tyr mutations may be associated            surprising, since epidemiological studies show that PBP
with decreased penetrance as was found previously in cases       comprises ~18–33% of all ALS cases (Kristensen and
with the Ile113Thr mutation in Australia (Suthers et al.,        Melgaard, 1977; Rosati et al., 1977; Forsgren et al., 1983;
1994), Scotland (Jones et al., 1995) and North America           Gubbay et al., 1985; Haverkamp et al., 1995). Of the ALS
(Deng et al., 1995), and in one case each with the Glu21Lys      samples analysed in this study, 19.4% were from patients
(Jones et al., 1994a), Asp101Asn (Jones et al., 1994b) and       with PBP.
Gly16Ser (Kawamata et al., 1995).                                   The tendency of patients with a CuZn-SOD mutation to
   The possibility exists that Asp76Tyr may in fact have         develop the first symptom in the neurons innervating the
complete penetrance and that the dementia found in III-2,        limbs rather than the neurons innervating the labio-glosso-
-4 and -5 may be another manifestation of the mutation. A        pharyngeal muscles is striking and accords with findings in
few families where different generations have developed          mice carrying different ALS-associated human CuZn-SOD
either motor neuron disease or dementia have been reported       mutations inserted into the genome (Gurney et al., 1994;
(Hudson, 1981; Frecker et al., 1990; Gunnarson et al., 1991).    Ripps et al., 1995; Wong et al., 1995). The neurons
However, no biological material is available from any of         innervating the limbs, particularly the lower limbs, are also
these three. None of the living children of III-2 and III-5      the neurons with the longest axons and therefore the ones
carries the Asp76Tyr mutation. The present data do not allow     with the largest metabolic demand and should be more
the conclusion that Asp76Tyr is associated with dementia.        susceptible to interference with the axonal transportation
We reported earlier that Asp90Ala is not associated with         mechanism.
dementia (Andersen et al., 1996).

                                                                 The CuZn-SOD mutations
The Ala4Val mutation                                             While the Ala4Val (Deng et al., 1993), Asp90Ala (Andersen
The Ala4Val mutation is the most commonly found CuZn-            et al., 1995) and Gly127insTGGG (Hansen et al., 1997)
SOD mutation associated with motor neuron disease,               mutations have all been reported previously, the Val14Gly
accounting for about a fifth of all SOD-associated FALS          and Asp76Tyr mutations are novel discoveries. Fifty-seven
families in the USA (Rosen et al., 1994), but has not            different mutations at 42 codons of the 153 codons defining
previously been reported outside North America. The              the CuZn-SOD subunit (the CuZn-SOD enzyme is a dimer
phenotype of the American patients (Rosen et al., 1994) is       consisting of two identical subunits) have now been reported
similar to the phenotype in the Swedish family with the          in cases with ALS (Fig. 3). Of the mutations, 10 occur
Phenotypic heterogeneity in motor neuron disease              1733

among the 23 codons of exon 1, seven among the 32 codons       one hand and the phenotype and prognosis with the different
of exon 2, 20 among the 39 codons of exon 4, and 17 among      mutations on the other (Table 2), supports the theory that the
the 35 codons of exon 5. Two mutations are located in the      various CuZn-SOD mutations cause ALS by the mutated
intron between exon 4 and 5, leading to changes in splice      CuZn-SOD protein gaining an adverse property, rather than
sites and the final sequence of the polypeptide. The lack of   by loss of enzyme activity (Borchelt et al., 1994; Esteban
association between the genotype and enzyme activity on        et al., 1994; Gurney et al., 1994; Tsuda et al., 1994; Andersen
                                                               et al., 1995; Bowling et al., 1995; Rabizadeh et al., 1995;
                                                               reviewed by Radunovic and Leigh, 1996). The uneven
                                                               distribution of mutations may reflect the differences in
                                                               importance of changes in amino acid residues in different
                                                               regions in causing the gain of this toxic property. It may also
                                                               partially reflect a difference in mutation rates in different
                                                               parts of the gene. Indeed, in our study with screening of all
                                                               five exons with SSCP in ALS cases, we have found three
                                                               silent mutations in exon 5, which may be a mutation hot-
                                                               spot. It should be noted that the GCT140→GCA silent
                                                               mutation was found in a FALS case and also in the 40-year-
                                                               old healthy son of a deceased FALS patient from the
                                                               same family (pedigree not shown). This silent mutation was
                                                               reported earlier (Hosler et al., 1996). We cannot exclude
                                                               the possibility that these silent mutations are involved in the
                                                               pathogenesis of the disease.
                                                                  Val14 has previously been found mutated to a Met in cases
                                                               of FALS (Deng et al., 1995). It forms part of a cluster of
                                                               hydrophobic packing interactions closing off one end of the
                                                               β-barrel of the CuZn-SOD molecule (Deng et al., 1995). It
                                                               is highly conserved but not sequence-invariant, suggesting
                                                               that its interactions are of structural importance. It has tight
                                                               interactions with Ile35, Leu38, His43 and Leu144, the latter
                                                               three being involved in mutations associated with ALS. The
                                                               remaining 50% of SOD activity in erythrocytes of the
                                                               heterozygous Val14Gly patient is indicative of a reduced
                                                               stability caused by the mutation.
                                                                  The Asp76Tyr mutation is the first reported among the 24
                                                               codons of exon 3. The negatively charged Asp76 is located
                                                               in the ‘Zn sub-loop’(Getzoff et al., 1989) on the surface

                                                               Fig. 3 Schematic illustration of the reported mutations in the five
                                                               exons of the CuZn-SOD gene. The numbers on the left show the
                                                               positions of the silent mutations; on the right side of the column
                                                               the positions of the mutated loci with the amino acid residues are
                                                               shown in the three-letter code. The black sections between the
                                                               exons illustrate the four introns of the CuZn-SOD gene, with the
                                                               two mutations with base pair change (bp) shown in intron 4. The
                                                               residue numbers of the first and last amino acids in each exon are
                                                               shown in small numbers inside the boxes. *Indicates the
                                                               mutations described in this report. The other data were compiled
                                                               from Aoki et al., 1993; Deng et al., 1993, 1995; Rosen et al.,
                                                               1993; Elshafey et al., 1994; Esteban et al., 1994; Hirano et al.,
                                                               1994; Jones et al., 1994 a, b; Kostrzewa et al., 1994; Nakano
                                                               et al., 1994; Pramatarova et al., 1994, 1995; Andersen et al.,
                                                               1995; Aoki et al., 1995; Calder et al., 1995; Enayat et al., 1995;
                                                               Ikeda et al., 1995a, b; Kawamata et al., 1995; Moulard et al.,
                                                               1995; Nakashima et al., 1995; Orrell et al., 1995a, b, 1997 a, b;
                                                               Sapp et al., 1995; Yulug et al., 1995; Hosler et al., 1996;
                                                               Kostrzewa et al., 1996; Morita et al., 1996; Robberecht et al.,
                                                               1996; Siddique et al., 1996; Bereznai et al., 1997; Hansen et al.,
                                                               1997; Watanabe et al., 1997.
1734      P. M. Andersen et al.

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electrostatic contacts with the positively charged Lys128 in        225]. Nat Genet 1993; 5: 323–4.
the ‘electrostatic channel’ loop (Getzoff et al., 1989). This       Aoki M, Abe K, Houi K, Ogasawara M, Matsubara Y, Kobayashi
interaction may be important for the joining of these two           T, et al. Variance of age at onset in a Japanese family with
loops, which together form the active site channel (Tainer          amyotrophic lateral sclerosis associated with a novel Cu/Zn
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(Bordo et al., 1994) and at position 128 the positively             Aran FA. Researches sur une maladie non encore décrite du système
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