Missense CACNA1A Mutation Causing Episodic Ataxia Type 2

OBSERVATION Missense CACNA1A Mutation Causing Episodic Ataxia Type 2

Christian Denier, MD; Anne Ducros, MD, PhD; Alexandra Durr, MD, PhD; Bruno Eymard, MD; Be´ne´dicte Chassande, MD; Elisabeth Tournier-Lasserve, MD

Objectives: To characterize the nature of CACNA1A mu- Results: A CACNA1A missense mutation, Glu 1757 Lys, tation in a previously unreported family with episodic ataxia was identified. It was absent in 200 control chromo- type 2 (EA2) and to better delineate EA2 clinical features. somes. It is predicted to result in an amino acid substitution at a highly phylogenetically conserved position, Background: Episodic ataxia type 2 is an autosomal domi- within a domain that plays a major role in the function nant disorder characterized by the recurrence of acetazol- of the channel. amide-responsive spells of cerebellar ataxia, usually starting during childhood or adolescence. The mutated gene, Conclusions: The Glu 1757 Lys missense mutation is CACNA1A, is located on chromosome 19 and encodes likely to be pathogenic, causing episodic ataxia within a the ␣1A subunit voltage-dependent calcium channel. So family whose phenotype is indistinguishable from EA2 far, most CACNA1A mutations detected in patients with except for a slightly later age of onset. These data strongly EA2 have led to a truncated CACNA1A protein, whereas suggest that additional work is needed to fully establish missense mutations cause familial hemiplegic migraine. genotype/phenotype correlations for CACNA1A mutations. Methods: All 47 exons of CACNA1A were screened by a combination of single-strand conformer polymorphism and sequencing analysis. Arch Neurol. 2001;58:292-295

UTOSOMAL dominant epi- permanent gait ataxia. In a minority of pa- sodic ataxias are a clini- tients, permanent symptoms are severe, cally and genetically het- and some patients may be wheelchair con- erogeneous group of fined.2,8 Cerebral imaging often reveals a conditions characterized vermian cerebellar atrophy. byA recurrent paroxysmal attacks of cerebellar ataxia starting during childhood or For editorial comment adolescence. Episodic ataxia type 1, a con- see page 179 dition characterized by the association of brief ataxic spells and interictal myokym- Episodic ataxia type 2 is caused by mu- ias, is caused by mutations within a voltage- tations within the ␣1A subunit of a P/Q- gated potassium channel gene, KCNA1. Epi- type voltage-dependent calcium channel sodic ataxia type 2 (EA2) is an autosomal gene, CACNA1A.6 P/Q type channels, which Institut National de la Sante´ dominant paroxysmal cerebellar ataxia, are expressed throughout the brain and at et de la Re´che´rche Me´dicale characterized by acetazolamide-respon- the neuromuscular junction, are impli- (INSERM) EPI 99-21, Faculte´ sive recurrent attacks of unsteadiness, lack cated in the control of membrane excit- de Me´decine Lariboisie`re of limb coordination, and dysarthria, of- ability and neurotransmitter release.10 So far, (Drs Denier, Ducros and ten provoked by emotional or physical 11 EA2 mutations have been reported, most Tournier-Lasserve), INSERM stress.1 Other symptoms during attacks in- of them leading to a truncated CACNA1A U289 (Dr Durr), Service de clude vertigo or dizziness, visual distur- protein.6-9 Interestingly, distinct types of Neurologie du Pr J-Y, Delattre, bances (diplopia or oscillopsia), and head- CACNA1A mutations have been reported Hoˆpital de la Salpeˆtrie`re, ache.1-9 Attacks last from several minutes to in other autosomal dominant neurologi- (Drs Eymard and Chassande), and Laboratoire de a few hours or days. Clinical onset occurs cal conditions. Chromosome 19–linked fa- Cytoge´ne´tique, Hoˆpital usually during childhood or adoles- milial hemiplegic migraine is caused by mis- 1-9 6,11-13 Lariboisie`re cence. Findings of interictal neurologi- sense mutations. Small expansions of (Dr Tournier-Lasserve), cal examination usually disclose a gaze- the CAG repeat located within the 3Ј cod- Paris, France. evoked nystagmus and sometimes a mild ing end of CACNA1A cause spinocerebel-

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 lar ataxia type 6,14 a late-onset, moderate to severe progressive cerebellar ataxia, without paroxysmal event. I 1 2 However, these strong genotype/phenotype correlations

may not be absolute. Two families with a permanent pro- II gressive cerebellar ataxia, associated with paroxysmal ataxic 1 234 5 6 episodes, and 1 family with pure episodic ataxia were shown to harbor CAG repeat expansions.15-16 More recently, a III 4 2 4 2 CACNA1A missense mutation was shown to cause a se- 1 2 vere progressive cerebellar ataxia with early onset in sev- 17 IV eral members of a family. 123 We report herein a missense CACNA1A mutation causing episodic ataxia within a family whose pheno- Figure 1. Family pedigree. Squares indicate male family members; circles, female; open symbols, unaffected individuals; solid symbols, affected; type is indistinguishable from EA2 except for a slightly diamond-shaped open symbols with enclosed numbers, number of later onset. unaffected children whose sex was not specified.

REPORT OF CASES cal examination results disclosed a gaze-evoked nystagmus and a mild statokinetic cerebellar ataxia. PEDIGREE OTHER AFFECTED FAMILY MEMBERS This family included 4 symptomatic members (Figure 1, individuals I-2, II-1, II-5, and III-2). Detailed clinical in- The proband reported that his father (individual II-1, Fig- formation was obtained from patient III-2 and 2 of his 3 ure 1) experienced similar attacks of episodic ataxia since sons who were clinically examined. Clinical informa- his 40s, with an average frequency initially close to twice tion regarding patients I-2, II-1, II-5 was obtained from a month and increasing with age. Attacks usually lasted patient III-2 (Figure 1). 2 hours, precipitated by physical exercise or emotional stress, and disappeared with sleep. Acetazolamide treat- PROBAND ment had never been tried. He had not developed any progressive severe ataxia or gait disorder by the time of Proband III-2 (Figure 1), a 53-year-old man, experi- his death at age 76 years (of prostate cancer). The pro- enced recurrent episodes of paroxysmal cerebellar ataxia band’s grandmother (Figure 1, I-2) and paternal aunt (Fig- since he was 40 years old. His medical history was un- ure 1, II-5) also exhibited paroxysmal attacks of gener- remarkable except for a strabismus, which needed sur- alized ataxia with late onset (after age 30 years ) and gical repair at age 22 years. Ataxic spells were strongly without permanent severe gait disorders. stereotyped. Onset was sudden with brief bilateral par- The proband had 3 sons, aged 12 (Figure 1, IV-1), esthesias in upper and lower extremities, diffuse weak- 20 (Figure 1, IV-2), and 21 (Figure 1, IV-3) years. All 3 ness, and heat sensations rapidly followed by general- were asymptomatic. Findings from clinical examina- ized ataxic symptoms. Attacks always included severe tion of patients IV-1 and IV-2 were normal. Patient IV-3 truncal and limb ataxia with dysarthria, vertigo, and os- was not examined. cillopsia and diplopia sometimes associated with nau- sea, vomiting, and blurred vision. The patient reported GENETIC ANALYSIS headaches fulfilling International Headache Society cri- teria for migraine without aura, both during and be- Samples of DNA from the proband (Figure 1, III-2) and tween ataxic spells. Duration of ataxic episodes usually 2 of his 3 sons (Figure 1, IV-1 and IV-2) were extracted ranged from half an hour to 4 hours. They were precipi- from peripheral blood using standard procedures. In ad- tated by emotional and physical stress and spontane- dition, DNA samples from 100 unrelated healthy sub- ously resolved with rest or sleeping. jects (white French individuals, to match the proband This patient suffered from 3 to 4 attacks per month, family) were also available for the study. All 47 exons of up to 1 per day in stressful periods. He first presented to CACNA1A were screened using a combination of single- us in 1996 at age 50. Findings of interictal examination strand conformer polymorphism18 and sequencing analy- disclosed an isolated, gaze-evoked nystagmus. The re- sis as previously described.11 mainder of his neurological examination results were normal. Brain magnetic resonance imaging revealed a RESULTS moderate vermian cerebellar atrophy. Findings of elec- troencephalographic and electromyelographic studies A missense mutation was identified within exon 35 in were normal. The patient began treatment with 250 mg the proband’s DNA. This G→A substitution at codon 1757 of acetazolamide twice a day, and reported a marked de- (GAA→AAA; Figure 2 and Figure 3) leads to the re- crease in severity and frequency of attacks (once a month) placement of a highly conserved preexisting glutamic acid during 1 year, but no improvement on isolated mi- for a lysine. This mutation was absent in the panel of 200 graine episodes. In 1997, he stopped treatment during 2 normal chromosomes, as well as in the 2 asymptomatic months and experienced an outbreak of attacks; fre- sons of the proband (Figure 1, IV-1 and IV-2). The num- quency of these attacks decreased with the reinstate- ber of CAG repeats at the 3Ј coding end of the gene was ment of treatment. Two years later, interictal neurologi- in the normal range (7/13).

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Extracellular

Membrane S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6

Intracellular

N C

Figure 2. Schematic structure of the ␣1A calcium channel subunit coded by CACNA1A and location of the missense episodic ataxia type 2–causing mutation. The pore-forming ␣1A subunit includes 4 homologous domains (I-IV), each of which contains 6 membrane-spanning segments (S1-S6; see “Comment” section). Glu 1757 Lys, located within the pore loop connecting S5 and S6 segments of the fourth domain, is indicated by a black star.

WT C C A A G C T T C C C C G G age 30 years. However, clinical onset after age 30 years 2 MT C C A A G C T T N C C C G G has been reported in a few members of families with EA2. CACNA1A screening revealed a missense mutation, Glu 1757 Lys, while most previously described EA2 mutations led to a truncated CACNA1A protein.6-9 Mul- tiple arguments strongly suggest that this amino acid substitution caused the disease of our patient. First, it was not detected in 200 control chromosomes, strongly sug- gesting 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 delin- eated 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 del- eterious. In addition, glutamic acid at codon 1757 is a highly conserved amino acid from Drosophila to man. For Figure 3. Sequence chromatogram. Reverse strand chromatogram is shown. these reasons, despite the fact that DNA from other mem- The arrow indicates the heterozygous mutation. WT indicates wild type; MT, bers was not available for cosegregation analysis, we think mutated; and N, nucleotide substitution. that this missense mutation most likely caused the disease observed in this family. COMMENT To our knowledge, there is only 1 family harboring a CACNA1A missense mutation, although not affected Clinical manifestations observed within affected mem- with familial hemiplegic migraine. This family included bers of this family (namely, recurrent paroxysmal acet- 8 affected members who suffered from a severe progres- azolamide-responsive attacks of generalized cerebellar sive cerebellar ataxia,17 which confined some of them to ataxia associated with interictal permanent cerebellar wheelchairs by their 40s. Interestingly, 2 of these mem- symptoms as well as the cerebellar atrophy evident on bers had, in addition to this severe progressive ataxia, ac- magnetic resonance imaging) are strongly suggestive of etazolamide-resistant paroxysmal episodes of vertigo and EA2. None of these family members suffered from ataxia. Mutation in this family substituted an un- hemiplegic migraine. The only subtle difference from pre- charged glycine for a positively charged arginine within viously reported families with EA2 was a later age of on- the pore-loop of the first domain of CACNA1A. set. Whereas in most patients with EA2 clinical onset oc- Although in most cases families with EA2 harbor curs during childhood or adolescence, initial symptoms truncating mutations in CACNA1A whereas in familial in the 4 affected members of this family occurred after hemiplegic migraine missense mutations occur, the fam-

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 ily reported herein is an example of overlap between epi- episodic ataxia type 2 are caused by mutations in the Ca2+ channel gene CACNL1A4. sodic neurological conditions due to CACNA1A mis- Cell. 1996;87:543-552. 7. Yue Q, Jen JC, Thwe MM, Nelson SF, Baloh RW. De novo mutation in CACNA1A- sense mutations. These data strongly suggest that caused acetazolamide-responsive episodic ataxia. Am J Med Genet. 1998;77: additional work is needed to fully establish genotype/ 298-301. phenotype correlations. The mechanisms leading from 8. Denier C, Ducros A, Vahedi K, et al. High prevalence of CACNA1A truncations these various types of mutations to these phenotypes are and broader clinical spectrum in episodic ataxia type 2. Neurology. 1999;52: 1816-1821. not understood at present, and electrophysiological stud- 9. Jen J, Yue Q, Nelson SF, et al. A novel nonsense mutation in CACNA1A causes ies are strongly needed. episodic ataxia and hemiplegia. Neurology. 1999;53:34-37. 10. Catterall WA. Stucture and function of voltage-gated ion channels. Annu Rev Bio- Accepted for publication August 8, 2000. chem. 1995;64:493-531. This work was supported by INSERM, Paris, France. 11. Ducros A, Denier C, Joutel A, et al. Recurrence of the T666M calcium channel CACNA1A gene mutation in familial hemiplegic migraine with progressive cer- Dr Denier is a recipient of a fellowship from Fonda- ebellar ataxia. Am J Hum Genet. 1999;64:89-98. tion pour la Recherche Me´dicale, Paris. 12. Carrera P, Piatti M, Stenirri S, et al. Genetic heterogeneity in Italian families with Corresponding author: Elisabeth Tournier-Lasserve, familial hemiplegic migraine. Neurology. 1999;53:26-33. MD, Inserm EPI 99-21, Faculte´deMe´decine Lariboisie`re, 13. Battistini S, Stenirri S, Piatti M, et al. A new CACNA1A gene mutation in acetazolamide-responsive familial hemiplegic migraine and ataxia. Neurology. 1999; 10, Avenue de Verdun, 75010 Paris, France (e-mail: 53:38-43. [email protected]). 14. Zhuchenko O, Bailey J, Bonnen P, et al. Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the ␣1A-voltage dependent calcium channel. Nat Genet. 1997;15:62-69. REFERENCES 15. Geschwind DH, Perlman S, Figueroa KP, Karrim J, Baloh RW, Pulst SM. Spino- cerebellar ataxia type 6: frequency of the mutation and genotype-phenotype cor- 1. Griggs RC, Moxley RT, Lafrance RA, Mc Quillen J. Hereditary paroxysmal ataxia: relations. Neurology. 1997;49:1247-1251. response to acetazolamide. Neurology. 1978;28:1259-1264. 16. Jodice C, Mantuano E, Veneziano L, et al. Episodic ataxia type 2 (EA2) and spi- 2. Baloh RW, Yue Q, Furman JM, Nelson SF. Familial episodic ataxia: clinical het- nocerebellar ataxia type 6 (SCA6) due to CAG repeat expansion in the CACNA1A erogeneity in four families linked to chromosome 19. Ann Neurol. 1997;41:8- gene on chromosome 19p. Hum Mol Genet. 1997;6:1973-1978. 16. 17. Yue Q, Jen JC, Nelson SF, Baloh RW. Progressive ataxia due to a missense mu- 3. Kramer PL, Yue Q, Gancher ST, et al. A locus for the nystagmus-associated form tation in a calcium-channel gene. Am J Hum Genet. 1997;61:1078-1087. of episodic ataxia maps to an 11 cM region on chromosome 19p. Am J Hum 18. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymor- Genet. 1995;57:182-185. phisms of human DNA by gel electrophoresis as single-strand conformation poly- 4. Teh BT, Silburn P, Lindblad K, et al. Familial periodic cerebellar ataxia without morphisms. Proc Natl Acad SciUSA.1989;86:2766-2770. myokima maps to a 19 cM region on 19p13. Am J Hum Genet. 1995;56:1443- 19. Heinemann SH, Terlau H, Stuhmer W, Imoto K, Numa S. Calcium channel char- 1449. acteristics conferred on sodium channel by single mutations. Nature. 1992;356: 5. Von Brederlow B, Hahn AF, Koopman WJ, Ebers GC, Bulman DE. Mapping the 441-443. gene for acetazolamide-responsive hereditary paroxysmal cerebellar ataxia to chro- 20. Yang J, Ellinor P, Sather WA, Zhang JF, Tsien RW. Molecular determinants of mosome 19p. Hum Mol Genet 1995;4:279-284. Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature. 1993;366: 6. Ophoff RA, Terwindt GM, Vergouwe MN, et al. Familial hemiplegic migraine and 158-161.

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