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Missense CACNA1A Mutation Causing Episodic Ataxia Type 2
Christian Denier, MD;
Anne Ducros, MD, PhD;
Alexandra Durr, MD, PhD;
Bruno Eymard, MD;
Bénédicte Chassande, MD;
Elisabeth Tournier-Lasserve, MD
Arch Neurol. 2001;58:292-295.
ABSTRACT
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Objectives To characterize the nature of CACNA1A mutation in a previously
unreported family with episodic ataxia type 2 (EA2) and to better delineate
EA2 clinical features.
Background Episodic ataxia type 2 is an autosomal dominant disorder characterized
by the recurrence of acetazolamide-responsive spells of cerebellar ataxia,
usually starting during childhood or adolescence. The mutated gene, CACNA1A, is located on chromosome 19 and encodes the  1A subunit
voltage-dependent calcium channel. So far, most CACNA1A mutations
detected in patients with EA2 have led to a truncated CACNA1A
protein, whereas missense mutations cause familial hemiplegic migraine.
Methods All 47 exons of CACNA1A were screened by a combination
of single-strand conformer polymorphism and sequencing analysis.
Results A CACNA1A missense mutation, Glu 1757 Lys,
was identified. It was absent in 200 control chromosomes. It is predicted
to result in an amino acid substitution at a highly phylogenetically conserved
position, within a domain that plays a major role in the function of the channel.
Conclusions The Glu 1757 Lys missense mutation is likely to be pathogenic, causing
episodic ataxia within a family whose phenotype is indistinguishable from
EA2 except for a slightly later age of onset. These data strongly suggest
that additional work is needed to fully establish genotype/phenotype correlations
for CACNA1A mutations.
INTRODUCTION
AUTOSOMAL dominant episodic ataxias are a clinically and genetically
heterogeneous group of conditions characterized by recurrent paroxysmal attacks
of cerebellar ataxia starting during childhood or adolescence. Episodic ataxia
type 1, a condition characterized by the association of brief ataxic spells
and interictal myokymias, is caused by mutations within a voltage-gated potassium
channel gene, KCNA1. Episodic ataxia type 2 (EA2)
is an autosomal dominant paroxysmal cerebellar ataxia, characterized by acetazolamide-responsive
recurrent attacks of unsteadiness, lack of limb coordination, and dysarthria,
often provoked by emotional or physical stress.1
Other symptoms during attacks include vertigo or dizziness, visual disturbances
(diplopia or oscillopsia), and headache.1, 2, 3, 4, 5, 6, 7, 8, 9
Attacks last from several minutes to a few hours or days. Clinical onset occurs
usually during childhood or adolescence.1, 2, 3, 4, 5, 6, 7, 8, 9
Findings of interictal neurological examination usually disclose a gaze-evoked
nystagmus and sometimes a mild permanent gait ataxia. In a minority of patients,
permanent symptoms are severe, and some patients may be wheelchair confined.2, 8 Cerebral imaging often reveals a vermian
cerebellar atrophy.
Episodic ataxia type 2 is caused by mutations within the 1A subunit
of a P/Q-type voltage-dependent calcium channel gene, CACNA1A.6 P/Q type channels, which are expressed
throughout the brain and at the neuromuscular junction, are implicated in
the control of membrane excitability and neurotransmitter release.10 So far, 11 EA2 mutations have been reported, most
of them leading to a truncated CACNA1A protein.6, 7, 8, 9 Interestingly,
distinct types of CACNA1A mutations have been reported
in other autosomal dominant neurological conditions. Chromosome 19–linked
familial hemiplegic migraine is caused by missense mutations.6, 11, 12, 13
Small expansions of the CAG repeat located within the 3' coding end
of CACNA1A cause spinocerebellar ataxia type 6,14 a late-onset, moderate to severe progressive cerebellar
ataxia, without paroxysmal event. However, these strong genotype/phenotype
correlations may not be absolute. Two families with a permanent progressive
cerebellar ataxia, associated with paroxysmal ataxic episodes, and 1 family
with pure episodic ataxia were shown to harbor CAG repeat expansions.15, 16 More recently, a CACNA1A missense mutation was shown to cause a severe progressive cerebellar
ataxia with early onset in several members of a family.17
We report herein a missense CACNA1A mutation
causing episodic ataxia within a family whose phenotype is indistinguishable
from EA2 except for a slightly later onset.
REPORT OF CASES
PEDIGREE
This family included 4 symptomatic members (Figure 1, individuals I-2, II-1, II-5, and III-2). Detailed clinical
information was obtained from patient III-2 and 2 of his 3 sons who were clinically
examined. Clinical information regarding patients I-2, II-1, II-5 was obtained
from patient III-2 (Figure 1).
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Figure 1. Family pedigree. Squares indicate
male family members; circles, female; open symbols, unaffected individuals;
solid symbols, affected; diamond-shaped open symbols with enclosed numbers,
number of unaffected children whose sex was not specified.
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PROBAND
Proband III-2 (Figure 1),
a 53-year-old man, experienced recurrent episodes of paroxysmal cerebellar
ataxia since he was 40 years old. His medical history was unremarkable except
for a strabismus, which needed surgical repair at age 22 years. Ataxic spells
were strongly stereotyped. Onset was sudden with brief bilateral paresthesias
in upper and lower extremities, diffuse weakness, and heat sensations rapidly
followed by generalized ataxic symptoms. Attacks always included severe truncal
and limb ataxia with dysarthria, vertigo, and oscillopsia and diplopia sometimes
associated with nausea, vomiting, and blurred vision. The patient reported
headaches fulfilling International Headache Society criteria for migraine
without aura, both during and between ataxic spells. Duration of ataxic episodes
usually ranged from half an hour to 4 hours. They were precipitated by emotional
and physical stress and spontaneously resolved with rest or sleeping.
This patient suffered from 3 to 4 attacks per month, up to 1 per day
in stressful periods. He first presented to us in 1996 at age 50. Findings
of interictal examination disclosed an isolated, gaze-evoked nystagmus. The
remainder of his neurological examination results were normal. Brain magnetic
resonance imaging revealed a moderate vermian cerebellar atrophy. Findings
of electroencephalographic and electromyelographic studies were normal. The
patient began treatment with 250 mg of acetazolamide twice a day, and reported
a marked decrease in severity and frequency of attacks (once a month) during
1 year, but no improvement on isolated migraine episodes. In 1997, he stopped
treatment during 2 months and experienced an outbreak of attacks; frequency
of these attacks decreased with the reinstatement of treatment. Two years
later, interictal neurological examination results disclosed a gaze-evoked
nystagmus and a mild statokinetic cerebellar ataxia.
OTHER AFFECTED FAMILY MEMBERS
The proband reported that his father (individual II-1, Figure 1) experienced similar attacks of episodic ataxia since his
40s, with an average frequency initially close to twice a month and increasing
with age. Attacks usually lasted 2 hours, precipitated by physical exercise
or emotional stress, and disappeared with sleep. Acetazolamide treatment had
never been tried. He had not developed any progressive severe ataxia or gait
disorder by the time of his death at age 76 years (of prostate cancer). The
proband's grandmother (Figure 1,
I-2) and paternal aunt (Figure 1,
II-5) also exhibited paroxysmal attacks of generalized ataxia with late onset
(after age 30 years ) and without permanent severe gait disorders.
The proband had 3 sons, aged 12 (Figure
1, IV-1), 20 (Figure 1, IV-2), and 21 (Figure 1, IV-3) years.
All 3 were asymptomatic. Findings from clinical examination of patients IV-1
and IV-2 were normal. Patient IV-3 was not examined.
GENETIC ANALYSIS
Samples of DNA from the proband (Figure
1, III-2) and 2 of his 3 sons (Figure 1, IV-1 and IV-2) were extracted from peripheral blood using
standard procedures. In addition, DNA samples from 100 unrelated healthy subjects
(white French individuals, to match the proband family) were also available
for the study. All 47 exons of CACNA1A were screened
using a combination of single-strand conformer polymorphism18
and sequencing analysis as previously described.11
RESULTS
A missense mutation was identified within exon 35 in the proband's DNA.
This G A substitution at codon 1757 (GAAAAA; Figure 2 and Figure 3)
leads to the replacement of a highly conserved preexisting glutamic acid for
a lysine. This mutation was absent in the panel of 200 normal chromosomes,
as well as in the 2 asymptomatic sons of the proband (Figure 1, IV-1 and IV-2). The number of CAG repeats at the 3'
coding end of the gene was in the normal range (7/13).
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Figure 3. Sequence chromatogram. Reverse
strand chromatogram is shown. The arrow indicates the heterozygous mutation.
WT indicates wild type; MT, mutated; and N, nucleotide substitution.
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COMMENT
Clinical manifestations observed within affected members of this family
(namely, recurrent paroxysmal acetazolamide-responsive attacks of generalized
cerebellar ataxia associated with interictal permanent cerebellar symptoms
as well as the cerebellar atrophy evident on magnetic resonance imaging) are
strongly suggestive of EA2. None of these family members suffered from hemiplegic
migraine. The only subtle difference from previously reported families with
EA2 was a later age of onset. Whereas in most patients with EA2 clinical onset
occurs during childhood or adolescence, initial symptoms in the 4 affected
members of this family occurred after age 30 years. However, clinical onset
after age 30 years has been reported in a few members of families with EA2.2
CACNA1A screening revealed a missense mutation,
Glu 1757 Lys, while most previously described EA2 mutations led to a truncated CACNA1A protein.6, 7, 8, 9
Multiple arguments strongly suggest that this amino acid substitution caused
the disease of our patient. First, it was not detected in 200 control chromosomes,
strongly suggesting that it is not a rare polymorphism. Second, this mutation
affects a highly conserved amino acid located within the pore loop, which
plays a major role in the function of the channel pore.
The 1A calcium channel subunit encoded by CACNA1A is formed by 4 homologous domains (Figure 2). Each domain contains 6 membrane-spanning segments (S1-S6).
The central pore of the channel is delineated by the 4 pore-loop regions,
which interconnect the fifth and sixth segment membrane spanning each domain.
The glutamates located within each pore loop are key players for calcium selectivity.19, 20 Substitution within this pore loop
of a negatively charged glutamic acid for a positively charged lysine would
most likely be very deleterious. In addition, glutamic acid at codon 1757
is a highly conserved amino acid from Drosophila
to man. For these reasons, despite the fact that DNA from other members was
not available for cosegregation analysis, we think that this missense mutation
most likely caused the disease observed in this family.
To our knowledge, there is only 1 family harboring a CACNA1A missense mutation, although not affected with familial hemiplegic
migraine. This family included 8 affected members who suffered from a severe
progressive cerebellar ataxia,17 which confined
some of them to wheelchairs by their 40s. Interestingly, 2 of these members
had, in addition to this severe progressive ataxia, acetazolamide-resistant
paroxysmal episodes of vertigo and ataxia. Mutation in this family substituted
an uncharged glycine for a positively charged arginine within the pore-loop
of the first domain of CACNA1A.
Although in most cases families with EA2 harbor truncating mutations
in CACNA1A whereas in familial hemiplegic migraine
missense mutations occur, the family reported herein is an example of overlap
between episodic neurological conditions due to CACNA1A missense mutations. These data strongly suggest that additional work
is needed to fully establish genotype/phenotype correlations. The mechanisms
leading from these various types of mutations to these phenotypes are not
understood at present, and electrophysiological studies are strongly needed.
AUTHOR INFORMATION
Accepted for publication August 8, 2000.
This work was supported by INSERM, Paris, France.
Dr Denier is a recipient of a fellowship from Fondation pour la Recherche
Médicale, Paris.
Institut National de la Santé et de la Réchérche
Médicale (INSERM) EPI 99-21, Faculté de Médecine Lariboisière
(Drs Denier, Ducros and Tournier-Lasserve), INSERM U289 (Dr Durr), Service
de Neurologie du Pr J-Y, Delattre, Hôpital de la Salpêtrière,
(Drs Eymard and Chassande), and Laboratoire de Cytogénétique,
Hôpital Lariboisière (Dr Tournier-Lasserve), Paris, France.
Corresponding author: Elisabeth Tournier-Lasserve, MD, Inserm EPI
99-21, Faculté de Médecine Lariboisière, 10, Avenue de
Verdun, 75010 Paris, France (e-mail: elisabeth.tournier{at}lrb.ap-hop-paris.fr).
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