Sign In
Individual Sign In
Create an Account
Institutional Sign In
OpenAthens Shibboleth
April 2003

Phenotypes of Spinocerebellar Ataxia Type 6 and Familial Hemiplegic Migraine Caused by a Unique CACNA1A Missense Mutation in Patients From a Large Family

Author Affiliations

From UnIGENe, IBMC, Porto (Ms Alonso, and Mr Coelho and Drs Sequeiros and Silveira); Laboratório de Genética Médica, ICBAS, Universidade do Porto (Ms Alonso and Drs Sequeiros and Silveira), and Serviço de Neurologia, HGSA (Drs Barros and Tuna), Porto; and Serviço de Neurologia, Hospital São Sebastião, Feira (Dr Coutinho), Portugal.


Copyright 2003 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.2003

Arch Neurol. 2003;60(4):610-614. doi:10.1001/archneur.60.4.610

Background  Different mutations in the α1A-subunit of the brain P/Q-type calcium channel gene (CACNA1A) are responsible for familial hemiplegic migraine (FHM), episodic ataxia type 2, and spinocerebellar ataxia type 6 (SCA6). Missense and splice site mutations have been found in FHM and episodic ataxia type 2, respectively, whereas a CAG repeat in the CACNA1A gene was found expanded in patients with SCA6.

Objective  To identify the disease causing mutation in a large family of patients with phenotypes of hemiplegic migraine with or without cerebellar signs or permanent cerebellar ataxia without migraine inherited in a dominant manner.

Patients and Methods  We examined 15 patients from a large family identified through a systematic survey of hereditary ataxias being conducted in Portugal. Linkage analysis was performed with CACNA1A gene markers, and mutation analysis was performed by single strand conformational polymorphism analysis and sequencing.

Results  Genetic linkage analysis with CACNA1A intragenic markers showed positive LOD scores. The maximal LOD score was obtained with the polymorphic CAG repeat (Zmax = 4.47, θ = 0). By single-strand conformational polymorphism analysis, a shift in exon 13 of the CACNA1A gene was detected in all patients. A G-to-A substitution was then identified, resulting in an arginine-to-glutamine change at codon 583 of this calcium channel α1A-subunit.

Conclusions  The disease-causing mutation in this family was identified, showing that a unique mutation in the CACNA1A gene causes several phenotypes, including those of SCA6 and FHM, thus suggesting that SCA6 and FHM are not only allelic diseases but are the same disorder with a large phenotypic variability.

FAMILIAL HEMIPLEGIC migraine (FHM) is a subtype of migraine with aura showing autosomal dominant inheritance. Episodes of FHM are characterized by some degree of hemiparesis occasionally associated with other symptoms, such as fever, drowsiness, confusion, or coma, which can be prolonged for days or weeks. Onset usually occurs during childhood or adolescence, although later onset has been reported.1 Permanent neurological signs of the disease are present in some patients, most often nystagmus and ataxia. Genetic heterogeneity of FHM has been established. A significant number of FHM families show genetic linkage to chromosome 19p13,2 including all those with cerebellar signs.26 Some FHM families without cerebellar signs have been assigned to chromosome 1q, whereas others are not linked to any known loci.7

Episodic ataxia is a dominantly inherited paroxysmal cerebellar neurological disorder characterized by episodes of cerebellar ataxia, often accompanied by nausea, vertigo, and headache. Episodic ataxia type 1 (EA1) presents interictal myokymia during and between episodes due to mutations in a potassium voltage-gated channel gene, located on chromosome 12.8,9 Patients with episodic ataxia type 2 (EA2) show interictal nystagmus, and the disease gene maps to chromosome 19p.10 Migraine with or without aura may be present in some patients from EA2 families.11,12

Spinocerebellar ataxias (SCAs) are progressive neurodegenerative disorders characterized by late-onset gait ataxia and dysarthria. Seven dominantly inherited SCAs are caused by polyglutamine expansions: SCA1-2, Machado-Joseph disease, SCA6, SCA7, SCA17, and dentatorubropallidoluysian atrophy.1323

The gene responsible for FHM, EA2, and SCA6 encodes an α1A-subunit of the brain P/Q-type calcium channel and is located on chromosome 19p13. Missense and splice site mutations have been found in FHM and EA2, respectively, whereas a CAG repeat in the CACNA1A gene was expanded in patients with SCA6.2,21 A G293R missense mutation in the CACNA1A gene is also responsible for progressive cerebellar ataxia.24

To improve knowledge of the underlying mechanism involved in hemiplegic migraine and progressive cerebellar ataxia, we studied a large family in which patients presented with phenotypes of either hemiplegic migraine or progressive cerebellar ataxia, performed genetic linkage analysis with chromosome 19p13 markers, and performed mutation screening in the CACNA1A gene.


We studied a Portuguese family ascertained during a systematic, population-based survey of hereditary ataxias and spastic paraplegias, initiated in 1993 and covering half of the Portuguese population (5.6 million people).25 This family consisted of 17 patients with hemiplegic migraine and/or progressive cerebellar ataxia in 4 consecutive generations. Fifteen patients were clinically examined by one of us (P.C., J.B., or A.T.) (Table 1). Age at onset ranged from 3 to 23 years for migraine episodes (mean, 13.4 ± 7.2 years) and from 16 to 50 years for cerebellar ataxia (mean, 31.7 ± 11.5 years). The age at examination varied from 8 to 71 years. Clinical manifestations were pleomorphic, including episodes of altered consciousness precipitated by minor head trauma, focal neurological deficits precipitated or not by minor head trauma, and migraine without aura, besides progressive late-onset cerebellar ataxia in a few patients. One of these patients (III-3) was studied by brain magnetic resonance imaging, which showed atrophy of the cerebellum.

Table 1. 
Image not available
Clinical Features of the Patients

Peripheral blood samples were collected from patients and their relatives after written informed consent was obtained. Genomic DNA was obtained from peripheral blood leukocytes by standard techniques.26 Molecular analyses of genetic markers, exons, and intronic sequences of the CACNA1A gene were performed by polymerase chain reaction (PCR) amplification using the published primer sequences.2,21 The PCR was performed with 1µM of each primer, 200µM deoxynucleotides, 1mM magnesium chloride, 10mM Tris (pH 9.0), 50mM potassium chloride, 1 U of Taq polymerase, and 2% formamide in a final volume of 12.5 µL. The PCR products of markers were radioactively labeled and analyzed on 6% polyacrylamide gels. Allele sizes were determined by comparing migration relative to an M13 sequencing ladder.

Polymorphic markers, within a 4-centimorgan (M) interval containing the CACNA1A gene tel-D19S840-19S1150-(CAG)n-D19S226-cen, according to the Fondation Jean Dausset Centre d'Études des Polymorphismes (Paris, France) database, were selected for linkage analysis. Markers D19S1150 and the polymorphic CAG repeat are intragenic. Analysis was performed with the LINKAGE27 software program version 5.22. The disease was considered autosomal dominant with incomplete penetrance (95%) and with a disease gene frequency of 0.0001. The PCR products of exons and intronic sequences of the CACNA1A gene were screened for molecular variants by single strand conformational polymorphism analysis28 and electrophoresis in ×0.5 Mutation Detection Enhancement gels (BioWhittaker Molecular Applications, Rockland, Me) at 4°C. Conformational changes were confirmed by sequencing with Thermo Sequenase cycle-sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Restriction analysis of exon 13 was performed by PCR amplification, and products were digested with the BanII restriction enzyme (New England BioLabs, Beverly, Mass) according to manufacturer instructions.


The size of the CAG repeat, in the 3′ end of the CACNA1A gene, responsible for SCA6 was determined in all 25 subjects available for this study. The repeat size in 12 patients, 9 at-risk individuals, and 4 spouses, ranged from 7 to 14 units, which is within the normal allele interval for SCA6. Further screening excluded SCA1, SCA2, Machado-Joseph disease, SCA7, SCA8, SCA10, SCA12, SCA17, and dentatorubropallidoluysian atrophy expansion.

Linkage analysis with polymorphic markers D19S840, D19S1150, the polymorphic CAG repeat, and D19S226 gave positive LOD scores (Table 2). The maximal LOD score was obtained with the intragenic CAG repeat (Zmax = 4.47, θ = 0). Haplotype construction with chromosome 19p markers showed a common haplotype shared by all patients of this family (Figure 1), except patient II-7, in whom a recombination occurred between the CAG repeat and marker D19S226, located 3 cM from the CACNA1A gene (Fondation Jean Dausset CEPH database). His affected offspring shared the same recombined haplotype.

Table 2. 
Image not available
Linkage Relationships Between the Disease Locus and Chromosome 19p13 Markers
Figure 1.
Image not available

Pedigree and haplotypes of the described family. Black circles and squares indicate affected individuals with progressive cerebellar ataxia and hemiplegic migraine; right side black symbols, patients with hemiplegic migraine; and left side black symbols, patients with progressive cerebellar ataxia. Haplotypes of 4 genetic markers spanning 4 centimorgans within the CACNA1A gene are shown. The additional 2-allele marker CACNA1Ant2023 polymorphism is also represented. The haplotype that segregates with the disease is boxed, and the inferred haplotypes are bracketed.

Mutation detection was performed by single strand conformational polymorphism analysis after PCR amplification of each exon and exon-intron boundaries. A mobility variant was detected in the exon 13 fragment that showed a 3-band pattern (Figure 2A). By direct sequencing, a G-to-A substitution at position 2023 was identified (Figure 2B). This substitution produces an arginine-to-glutamine change at codon 583 in the CACNA1A gene. By restriction analysis with BanII restriction enzyme, this mutation was excluded in 100 control chromosomes from the Portuguese general population. After BanII digestion, fragments of 123, 122, and 67 base pairs (bp) were detected for healthy individuals, whereas in the patients an additional band of 245 bp was also present, resulting from the loss of a restriction site on the mutated allele.

Figure 2.
Image not available

Single-strand conformational polymorphism (SSCP) and sequencing of exon 13. A, Polymerase chain reaction products were analyzed on Mutation Detection Enhancement gel by SSCP. A 3-band pattern shift was detected in all patients and in an at-risk individual. B, Sequencing of exon 13 fragment in 2 patients and 2 healthy relatives. A G-to-A base substitution was detected at the 2023 position, causing an arginine-to-glutamine change in the CACNA1A protein. Individuals are identified according to the family tree.


In this study, we describe the first family to our knowledge in which patients presented phenotypes of hemiplegic migraine with or without cerebellar signs or permanent progressive cerebellar ataxia without migraine due to a unique missense mutation in the CACNA1A gene. The disease locus in this family showed strong linkage to intragenic markers in this gene. By mutation analysis, we identified an R583Q substitution in all available patients. This mutation had first been described in 2 affected members from a family with hemiplegic migraine and ataxia.3 We described a large family with 17 patients who presented with high clinical variability due to this R583Q mutation.

The α1A-subunit of the P/Q-type calcium channel gene is composed of 4 homologous domains (I-IV), each containing 6 putative transmembrane segments (S1-S6) and a pore-forming segment between S5 and S6.2 The missense mutation identified in this family is located in the S4 transmembrane segment of protein domain II, which is thought to be the voltage sensor of the channel.

Mutation R583Q replaces a conserved, polar, positively charged arginine by a neutral glutamine, which can increase hydrophobicity and reduce polarity in this voltage sensor segment. This mutation causes a shift in the activation and inactivation voltage dependence of the channel to more negative potentials.29 The hyperpolarization shift increases intracellular calcium levels by altering P/Q-type calcium channel activity at weak depolarizations in mutants with this substitution.29 Channel recovery from inactivation in R583Q mutants is slower, which can lead to an accumulation of inactivated channels during rapid depolarizations.29 Another FHM mutation due to an arginine-to-glutamine substitution also located in the S4 transmembrane segment, but of protein domain I at codon 192, also causes an excess of intracellular calcium due to altered gating properties.30 The abnormal calcium influx, mostly during high neuronal activity, would explain the paroxysmal character of FHM and the precipitation of episodes by sensory or emotional stimuli.29 Calcium overload causes excessive release of excitotoxic neurotransmitters such as glutamate, which can lead neurons to apoptotic death.

In this family, the mean age at onset for hemiplegic migraine symptoms was in the second decade and approximately 20 years earlier than that for the cerebellar signs. This onset of migraine symptoms is close to that reported in other clinical descriptions of FHM due to mutations in the CACNA1A gene.6 The 2 patients previously described as having mutation R583Q began migraine episodes at 17 and 40 years, respectively, whereas cerebellar signs were first noticed in both patients when they were in their 60s.3

Emotional stress was the most frequent triggering factor of hemiplegic migraine in families with mutations in the CACNA1A gene as described in a previous study.6 In the present family, patients with hemiplegic migraine did not refer to emotional stress as a triggering factor, whereas minor head trauma was referred to in approximately 4 patients (44%). However, this family is unique in which cerebellar progressive ataxia was also triggered by mild head trauma.

Expansion of a CAG repeat in the CACNA1A gene causes not only SCA6 but also EA2 phenotypes in patients from the same family.31,32 On the other hand, in a family described by Yue et al,24 a point mutation in this gene originates severe progressive ataxia in some patients and episodic ataxia in others. Moreover, some families had members with either hemiplegic migraine accompanied by cerebellar signs or episodic ataxia with headache due to a point mutation in the CACNA1A gene.3,4,33,34 In this family, we found patients who only had symptoms of progressive cerebellar ataxia, patients affected by hemiplegic migraine only, and patients with both hemiplegic migraine and symptoms of progressive cerebellar ataxia. Thus, the R583Q mutation causes phenotypes of SCA6 and FHM. These results, in addition to those referred to herein,31,32 suggest that EA2, SCA6, and FHM are not only allelic diseases but are the same disorder with a large phenotypic variability. The presence of several different phenotypes strongly suggests the involvement of modifying polymorphisms in either this or other genes.

Mutations in the α1A-subunit orthologous mouse gene are responsible for 2 phenotypes: the tottering (tg) and the leaner (tgla). The tgla mice phenotype presents severe progressive ataxia caused by a mutation in a splicing consensus sequence, which gives rise to CACNA1A aberrant transcripts.35 On the other hand, the tg mutant mice phenotype is caused by an amino acid substitution in the pore-forming region of mice α1A protein domain II.35 This mutant expresses a milder phenotype and shows less functional changes.36 The tg and tgla mutated channels exhibit a reduced calcium influx in Purkinje cells.3638

In conclusion, the mutation R583Q in the CACNA1A gene causes a large variety of clinical phenotypes, including hemiplegic migraine, permanent ataxia, and coma. Mutations not only in the pore-forming segments but also in the voltage sensor transmembrane segments alter the gating properties of neuronal P/Q-type calcium channels, causing alterations in calcium influx through neurons.

Back to top
Article Information

Corresponding author and reprints: Isabel Silveira, PhD, UnIGENe, IBMC, Rua do Campo Alegre 823, 4150-180, Porto, Portugal (e-mail:

Accepted for publication July 12, 2002.

Author contributions: Study concept and design (Drs Silveira and Coutinho); acquisition of data (Ms Alonso, Drs Barros, Tuna, Silveira, and Coutinho, and Mr Coelho); analysis and interpretation of data (Ms Alonso, Drs Barros, Tuna, Sequeiros, Silveira, and Coutinho, and Mr Coelho); drafting of the manuscript (Ms Alonso, Drs Barros, Tuna, Silveira, and Coutinho, and Mr Coelho); critical revision of the manuscript for important intellectual content (Ms Alonso and Drs Sequeiros, Silveira, and Coutinho); obtained funding (Dr Silveira); administrative, technical, and material support (Ms Alonso, Drs Barros and Tuna, and Mr Coelho); study supervision (Drs Sequeiros, Silveira, and Coutinho).

This study was supported by grants PRAXIS/P/SAU/13226/1998 and POCTI/32643/ESP/2000 and the Financiamento Plurianual de Unidades de Investigação from Fundação para a Ciência e Tecnologia, Lisbon, Portugal. Ms Alonso and Mr Coelho are recipients of scholarships from the Fundação para a Ciência e Tecnologia.

We thank family members for their cooperation and António Amorim, PhD, for providing control DNA samples.

Headache Classification Committee of the International Headache Society Classification and diagnostic criteria for headache disorders, cranial neuralgias and facial pain. Cephalalgia.1988;8 Suppl 7:1-96.
Ophoff  RATerwindt  GMVergouwe  MN  et al Familial hemiplegic migraine and episodic ataxia type 2 are caused by mutations in the Ca2+ channel gene CACNL1A4Cell.1996;87:543-552.
Battistini  SStenirri  SPiatti  M  et al A new CACNA1A gene mutation in acetazolamide-responsive familial hemiplegic migraine and ataxia. Neurology.1999;53:38-43.
Vahedi  KDenier  CDucros  A  et al CACNA1A gene de novo mutation causing hemiplegic migraine, coma, and cerebellar atrophy. Neurology.2000;55:1040-1042.
Ducros  ADenier  CJoutel  A  et al Recurrence of the T666M calcium channel CACNA1A gene mutation in familial hemiplegic migraine with progressive cerebellar ataxia. Am J Hum Genet.1999;64:89-98.
Ducros  ADenier  CJoutel  A  et al The clinical spectrum of familial hemiplegic migraine associated with mutations in a neuronal calcium channel. N Engl J Med.2001;345:17-24.
Ducros  AJoutel  AVahedi  K  et al Mapping of a second locus for familial hemiplegic migraine to 1q21-q23 and evidence of further heterogeneity. Ann Neurol.1997;42:885-890.
Litt  MKramer  PBrowne  D  et al A gene for episodic ataxia/myokymia maps to chromosome 12p13. Am J Hum Genet.1994;55:702-709.
Browne  DLGancher  STNutt  JG  et al Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1Nat Genet.1994;8:136-140.
von Brederlow  BHahn  AFKoopman  WJEbers  GCBulman  DE Mapping the gene for acetazolamide responsive hereditary paroxysmal cerebellar ataxia to chromosome 19p. Hum Mol Genet.1995;4:279-284.
Jen  JYue  QNelson  SF  et al A novel nonsense mutation in CACNA1A causes episodic ataxia and hemiplegia. Neurology.1999;53:34-37.
Friend  KLCrimmins  DPhan  TG  et al Detection of a novel missense mutation and second recurrent mutation in the CACNA1A gene in individuals with EA2 and FHM. Hum Genet.1999;105:261-265.
Orr  HTChung  MBanfi  S  et al Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat Genet.1993;4:221-226.
Koide  RIkeuchi  TOnodera  O  et al Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA). Nat Genet.1994;6:9-13.
Nagafuchi  SYanagisawa  HSato  K  et al Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p. Nat Genet.1994;6:14-18.
Kawaguchi  YOkamoto  TTaniwaki  M  et al CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nat Genet.1994;8:221-228.
Sanpei  KTakano  HIgarashi  S  et al Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT. Nat Genet.1996;14:277-284.
Pulst  SMNechiporuk  ANechiporuk  T  et al Moderate expansion of normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nat Genet.1996;14:269-276.
Imbert  GSaudau  FYvert  G  et al Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats. Nat Genet.1996;14:285-291.
David  GAbbas  NStevanin  G  et al Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nat Genet.1997;17:65-70.
Zhuchenko  OBailey  JBonnen  P  et al Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel. Nat Genet.1997;15:62-69.
Koide  RKobayashi  SShimohata  T  et al A neurological disease caused by an expanded CAG trinucleotide repeat in the TATA-binding protein gene: a new polyglutamine disease? Hum Mol Genet.1999;8:2047-2053.
Nakamura  KJeong  SYUchihara  T  et al SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein. Hum Mol Genet.2001;10:1441-1448.
Yue  QJen  JCNelson  SFBaloh  RW Progressive ataxia due to a missense mutation in a calcium-channel gene. Am J Hum Genet.1997;61:1078-1087.
Silva  MCCoutinho  PPinheiro  CDNeves  JMSerrano  P Hereditary ataxias and spastic paraplegias: methodological aspects of a prevalence study in Portugal. J Clin Epidemiol.1997;50:1377-1384.
Sambrook  JFritsch  EFManiatis  T Molecular Cloning: A Laboratory Manual.  Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
Lathrop  GMLalouel  JM Easy calculations of lod scores and genetic risks on small computers. Am J Hum Genet.1984;36:460-465.
Orita  MSuzuki  YSekiya  THayashi  K Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics.1989;5:874-879.
Kraus  RLSinnegger  MJKoschak  A  et al Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics. J Biol Chem.2000;275:9239-9243.
Hans  MLuvisetto  SWilliams  ME  et al Functional consequences of mutations in the α1A calcium channel subunit linked to familial hemiplegic migraine. J Neurosci.1999;19:1610-1619.
Geschwind  DHPerlman  SFigueroa  KPKarrim  JBaloh  RWPulst  SM Spinocerebellar ataxia type 6: frequency of the mutation and genotype-phenotype correlations. Neurology.1997;49:1247-1251.
Jodice  CMantuano  EVeneziano  L  et al Episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6) due to CAG repeat expansion in the CACNA1A gene on chromosome 19p. Hum Mol Genet.1997;6:1973-1978.
Guida  STrettel  FPagnutti  S  et al Complete loss of P/Q calcium channel activity caused by a CACNA1A missense mutation carried by patients with episodic ataxia type 2. Am J Hum Genet.2001;68:759-764.
Scoggan  KAChandra  TNelson  RHahn  AFBulman  DE Identification of two novel mutations in the CACNA1A gene responsible for episodic ataxia type 2. J Med Genet.2001;38:249-253.
Fletcher  CFLutz  CMO'Sullivan  TN  et al Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell.1996;87:607-617.
Wakamori  MYamazaki  KMatsunodaira  H  et al Single tottering mutation responsible for the neuropathic phenotype of the P-type calcium channel. J Biol Chem.1998;273:34857-34867.
Lorenzon  NMLutz  CMFrankel  WNBeam  KG Altered calcium channel currents in Purkinje cells of the neurological mutant mouse leanerJ Neurosci.1998;18:4482-4489.
Dove  LSAbbott  LCGriffith  WH Whole-cell and single-channel analysis of P-type calcium currents in cerebellar Purkinje cells of leaner mutant mice. J Neurosci.1998;18:7687-7699.