Genetic and functional characterization of the P/Q calcium channel

in episodic ataxia with epilepsy

Sanjeev Rajakulendran, Tracey D Graves, Robyn W Labrum, Dimitrios Kotzadimitriou, Louise H Eunson,

Mary B Davis, Rosalyn Davies, Nicholas W Wood, Dimitri M Kullmann, Michael G Hanna, and Stephanie

Schorge,

Supplemental data: S1 and S2

 Supplemental data S1: List of references reporting mutations in CACNA1A associated with EA2.

References

BERTHOLON, P., CHABRIER, S., RIANT, F., TOURNIER-LASSERVE, E. & PEYRON, R. 2009. Episodic
ataxia type 2: unusual aspects in clinical and genetic presentation. Special emphasis in childhood. J
Neurol Neurosurg Psychiatry, 80, 1289-92.
CRICCHI, F., DI LORENZO, C., GRIECO, G. S., RENGO, C., CARDINALE, A., RACANIELLO, M.,
SANTORELLI, F. M., NAPPI, G., PIERELLI, F. & CASALI, C. 2007. Early-onset progressive ataxia
associated with the first CACNA1A mutation identified within the I-II loop. J Neurol Sci, 254, 69-71.
CUENCA-LEON, E., BANCHS, I., SERRA, S. A., LATORRE, P., FERNANDEZ-CASTILLO, N.,
COROMINAS, R., VALVERDE, M. A., VOLPINI, V., FERNANDEZ-FERNANDEZ, J. M.,
MACAYA, A. & CORMAND, B. 2009. Late-onset episodic ataxia type 2 associated with a novel loss-
of-function mutation in the CACNA1A gene. J Neurol Sci, 280, 10-4.
DENIER, C., DUCROS, A., DURR, A., EYMARD, B., CHASSANDE, B. & TOURNIER-LASSERVE, E.
2001. Missense CACNA1A mutation causing episodic ataxia type 2. Arch Neurol, 58, 292-5.
DENIER, C., DUCROS, A., VAHEDI, K., JOUTEL, A., THIERRY, P., RITZ, A., CASTELNOVO, G.,
DEONNA, T., GERARD, P., DEVOIZE, J. L., GAYOU, A., PERROUTY, B., SOISSON, T.,
AUTRET, A., WARTER, J. M., VIGHETTO, A., VAN BOGAERT, P., ALAMOWITCH, S.,
ROULLET, E. & TOURNIER-LASSERVE, E. 1999. High prevalence of CACNA1A truncations and
broader clinical spectrum in episodic ataxia type 2. Neurology, 52, 1816-21.
EUNSON, L. H., GRAVES, T. D. & HANNA, M. G. 2005. New calcium channel mutations predict aberrant
RNA splicing in episodic ataxia. Neurology, 65, 308-10.
Author. 1999. Detection of a novel missense mutation and second recurrent mutation in the CACNA1A gene in
individuals with EA-2 and FHM. Hum Genet, Sep, p.3.
GRAVES, T. D., IMBRICI, P., KORS, E. E., TERWINDT, G. M., EUNSON, L. H., FRANTS, R. R., HAAN, J.,
FERRARI, M. D., GOADSBY, P. J., HANNA, M. G., VAN DEN MAAGDENBERG, A. M. &
KULLMANN, D. M. 2008. Premature stop codons in a facilitating EF-hand splice variant of Ca(V)2.1
cause episodic ataxia type 2. Neurobiol Dis.
GUERIN, A. A., FEIGENBAUM, A., DONNER, E. J. & YOON, G. 2008. Stepwise developmental regression
associated with novel CACNA1A mutation. Pediatr Neurol, 39, 363-4.
GUIDA, S., TRETTEL, F., PAGNUTTI, S., MANTUANO, E., TOTTENE, A., VENEZIANO, L., FELLIN, T.,
SPADARO, M., STAUDERMAN, K., WILLIAMS, M., VOLSEN, S., OPHOFF, R., FRANTS, R.,
JODICE, C., FRONTALI, M. & PIETROBON, D. 2001. 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, 68, 759-64.
IMBRICI, P., EUNSON, L. H., GRAVES, T. D., BHATIA, K. P., WADIA, N. H., KULLMANN, D. M. &
HANNA, M. G. 2005. Late-onset episodic ataxia type 2 due to an in-frame insertion in CACNA1A.
Neurology, 65, 944-6.
IMBRICI, P., JAFFE, S. L., EUNSON, L. H., DAVIES, N. P., HERD, C., ROBERTSON, R., KULLMANN, D.
M. & HANNA, M. G. 2004. Dysfunction of the brain calcium channel CaV2.1 in absence epilepsy and
episodic ataxia. Brain, 127, 2682-92.
JEN, J., KIM, G. W. & BALOH, R. W. 2004. Clinical spectrum of episodic ataxia type 2. Neurology, 62, 17-22.
JEN, J., WAN, J., GRAVES, M., YU, H., MOCK, A. F., COULIN, C. J., KIM, G., YUE, Q., PAPAZIAN, D. M.
& BALOH, R. W. 2001. Loss-of-function EA2 mutations are associated with impaired neuromuscular
transmission. Neurology, 57, 1843-8.
JEN, J., YUE, Q., NELSON, S. F., YU, H., LITT, M., NUTT, J. & BALOH, R. W. 1999. A novel nonsense
mutation in CACNA1A causes episodic ataxia and hemiplegia. Neurology, 53, 34-7.
JODICE, C., MANTUANO, E., VENEZIANO, L., TRETTEL, F., SABBADINI, G., CALANDRIELLO, L.,
FRANCIA, A., SPADARO, M., PIERELLI, F., SALVI, F., OPHOFF, R. A., FRANTS, R. R. &
FRONTALI, M. 1997. 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, 6, 1973-8.
JOUVENCEAU, A., EUNSON, L. H., SPAUSCHUS, A., RAMESH, V., ZUBERI, S. M., KULLMANN, D. M.
& HANNA, M. G. 2001. Human epilepsy associated with dysfunction of the brain P/Q-type calcium
channel. Lancet, 358, 801-7.
KAUNISTO, M. A., HARNO, H., KALLELA, M., SOMER, H., SALLINEN, R., HAMALAINEN, E.,
MIETTINEN, P. J., VESA, J., ORPANA, A., PALOTIE, A., FARKKILA, M. & WESSMAN, M. 2004.
Novel splice site CACNA1A mutation causing episodic ataxia type 2. Neurogenetics, 5, 69-73.
KRISHNAN, A. V., BOSTOCK, H., IP, J., HAYES, M., WATSON, S. & KIERNAN, M. C. 2008. Axonal
function in a family with episodic ataxia type 2 due to a novel mutation. J Neurol, 255, 750-5.
LABRUM, R. W., RAJAKULENDRAN, S., GRAVES, T. D., EUNSON, L. H., BEVAN, R., SWEENEY, M.
G., HAMMANS, S. R., TUBRIDY, N., BRITTON, T., CARR, L. J., OSTERGAARD, J. R.,
KENNEDY, C. R., AL-MEMAR, A., KULLMANN, D. M., SCHORGE, S., TEMPLE, K., DAVIS, M.
B. & HANNA, M. G. 2009. Large scale calcium channel gene rearrangements in episodic ataxia and
hemiplegic migraine: implications for diagnostic testing. J Med Genet, 46, 786-91.
MANTUANO, E., VENEZIANO, L., SPADARO, M., GIUNTI, P., GUIDA, S., LEGGIO, M. G., VERRIELLO,
L., WOOD, N., JODICE, C. & FRONTALI, M. 2004. Clusters of non-truncating mutations of P/Q type
Ca2+ channel subunit Ca(v)2.1 causing episodic ataxia 2. J Med Genet, 41, e82.
MARTI, S., BALOH, R. W., JEN, J. C., STRAUMANN, D. & JUNG, H. H. 2008. Progressive cerebellar ataxia
with variable episodic symptoms--phenotypic diversity of R1668W CACNA1A mutation. Eur Neurol,
60, 16-20.
MATSUYAMA, Z., MURASE, M., SHIMIZU, H., AOKI, Y., HAYASHI, M., HOZUMI, I. & INUZUKA, T.
2003. A novel insertion mutation of acetazolamide-responsive episodic ataxia in a Japanese family. J
Neurol Sci, 210, 91-3.
OPHOFF, R. A., TERWINDT, G. M., VERGOUWE, M. N., VAN EIJK, R., OEFNER, P. J., HOFFMAN, S.
M., LAMERDIN, J. E., MOHRENWEISER, H. W., BULMAN, D. E., FERRARI, M., HAAN, J.,
LINDHOUT, D., VAN OMMEN, G. J., HOFKER, M. H., FERRARI, M. D. & FRANTS, R. R. 1996.
Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel
gene CACNL1A4. Cell, 87, 543-52.
RIANT, F., LESCOAT, C., VAHEDI, K., KAPHAN, E., TOUTAIN, A., SOISSON, T., WIENER-VACHER, S.
R. & TOURNIER-LASSERVE, E. 2009. Identification of CACNA1A large deletions in four patients
with episodic ataxia. Neurogenetics.
RIANT, F., MOURTADA, R., SAUGIER-VEBER, P. & TOURNIER-LASSERVE, E. 2008. Large CACNA1A
deletion in a family with episodic ataxia type 2. Arch Neurol, 65, 817-20.
ROBBINS, M. S., LIPTON, R. B., LAURETA, E. C. & GROSBERG, B. M. 2009. CACNA1A nonsense
mutation is associated with basilar-type migraine and episodic ataxia type 2. Headache, 49, 1042-6.
ROUBERTIE, A., ECHENNE, B., LEYDET, J., SOETE, S., KRAMS, B., RIVIER, F., RIANT, F. &
TOURNIER-LASSERVE, E. 2008. Benign paroxysmal tonic upgaze, benign paroxysmal torticollis,
episodic ataxia and CACNA1A mutation in a family. J Neurol, 255, 1600-2.
SCOGGAN, K. A., CHANDRA, T., NELSON, R., HAHN, A. F. & BULMAN, D. E. 2001. Identification of two
novel mutations in the CACNA1A gene responsible for episodic ataxia type 2. J Med Genet, 38, 249-
53.
SCOGGAN, K. A., FRIEDMAN, J. H. & BULMAN, D. E. 2006. CACNA1A mutation in a EA-2 patient
responsive to acetazolamide and valproic acid. Can J Neurol Sci, 33, 68-72.
SELF, J., MERCER, C., BOON, E. M., MURUGAVEL, M., SHAWKAT, F., HAMMANS, S., HODGKINS, P.,
GRIFFITHS, H. & LOTERY, A. 2009. Infantile nystagmus and late onset ataxia associated with a
CACNA1A mutation in the intracellular loop between s4 and s5 of domain 3. Eye.
SPACEY, S. D., HILDEBRAND, M. E., MATEREK, L. A., BIRD, T. D. & SNUTCH, T. P. 2004. Functional
implications of a novel EA2 mutation in the P/Q-type calcium channel. Ann Neurol, 56, 213-20.
SPACEY, S. D., MATEREK, L. A., SZCZYGIELSKI, B. I. & BIRD, T. D. 2005. Two novel CACNA1A gene
mutations associated with episodic ataxia type 2 and interictal dystonia. Arch Neurol, 62, 314-6.
STRUPP, M., KALLA, R., DICHGANS, M., FREILINGER, T., GLASAUER, S. & BRANDT, T. 2004.
Treatment of episodic ataxia type 2 with the potassium channel blocker 4-aminopyridine. Neurology,
62, 1623-5.
SUBRAMONY, S. H., SCHOTT, K., RAIKE, R. S., CALLAHAN, J., LANGFORD, L. R., CHRISTOVA, P. S.,
ANDERSON, J. H. & GOMEZ, C. M. 2003. Novel CACNA1A mutation causes febrile episodic ataxia
with interictal cerebellar deficits. Ann Neurol, 54, 725-31.
TONELLI, A., D'ANGELO, M. G., SALATI, R., VILLA, L., GERMINASI, C., FRATTINI, T., MEOLA, G.,
TURCONI, A. C., BRESOLIN, N. & BASSI, M. T. 2005. Early onset, non fluctuating spinocerebellar
ataxia and a novel missense mutation in CACNA1A gene. J Neurol Sci.
VAN DEN MAAGDENBERG, A. M., KORS, E. E., BRUNT, E. R., VAN PAESSCHEN, W., PASCUAL, J.,
RAVINE, D., KEELING, S., VANMOLKOT, K. R., VERMEULEN, F. L., TERWINDT, G. M.,
HAAN, J., FRANTS, R. R. & FERRARI, M. D. 2002. Episodic ataxia type 2. Three novel truncating
mutations and one novel missense mutation in the CACNA1A gene. J Neurol, 249, 1515-9.
VENEZIANO, L., GUIDA, S., MANTUANO, E., BERNARD, P., TARANTINO, P., BOCCONE, L.,
HISAMA, F. M., CARRERA, P., JODICE, C. & FRONTALI, M. 2009. Newly characterised 5' and 3'
regions of CACNA1A gene harbour mutations associated with Familial Hemiplegic Migraine and
Episodic Ataxia. J Neurol Sci, 276, 31-7.
WAN, J., CARR, J. R., BALOH, R. W. & JEN, J. C. 2005. Nonconsensus intronic mutations cause episodic
ataxia. Ann Neurol, 57, 131-5.
YUE, Q., JEN, J. C., NELSON, S. F. & BALOH, R. W. 1997. Progressive ataxia due to a missense mutation in a
calcium-channel gene. Am J Hum Genet, 61, 1078-87.
YUE, Q., JEN, J. C., THWE, M. M., NELSON, S. F. & BALOH, R. W. 1998. De novo mutation in CACNA1A
caused acetazolamide-responsive episodic ataxia. Am J Med Genet, 77, 298-301.
ZAFEIRIOU, D. I., LEHMANN-HORN, F., VARGIAMI, E., TEFLIOUDI, E., VERVERI, A. & JURKAT-

ROTT, K. 2008. Episodic ataxia type 2 showing ictal hyperhidrosis with hypothermia and interictal chromic

diarrhea due to a novel CACNA1A mutation. Eur J Paediatr Neurol.

Supplemental data S2

Clinical data for the 15 patients reported in this study. Additional clinical data on patients 16 and 17 is reported
in Labrum et al, 2009.

Patient 1
Patient 1 experienced ‘funny turns’ between the ages of 2 weeks and 20 months. Since the age of 2 years, she
experienced monthly episodes of unsteadiness on her feet with abnormal eye movements. At the age of 2 years
and 6 months, she had two seizures during which she became stiff and lost consciousness. The episodes lasted 12
and 20 minutes respectively. She was placed on Carbamazepine and has had no seizures since. She was also
commenced on Acetazolamide which has reduced the frequency of her episodes of ataxia. Examination revealed
gait ataxia but no nystagmus. Brain MRI and EEG were both normal. There was no family history of EA or
epilepsy.

Patient 2
From the age of 18 months, patient 2 experienced episodes of unsteadiness on her feet and poor coordination.
These episodes improved in frequency and severity without medication. She had several generalized tonic clonic
seizures as a child but these stopped by the age of 6 years. She continues to experience absence seizures every
few months. Interictal neurological examination was normal. Her MRI brain study and EEG were both normal.
There was no family history of EA or epilepsy.

Patient 3
Patient 3’s attacks started at the age of 12 years during which she became suddenly unsteady on her feet and
clumsy. These episodes lasted several hours and occurred once a week. Initial treatment with Acetazolamide
reduced the frequency of attacks to once a month but the effect was short-lived. Although she has not had any
seizures, her EEG demonstrated frankly epileptiform (spike wave) changes. Interictal neurological examination
was normal. Her mother and maternal grandfather also had attacks of EA. Her father suffers from generalized
tonic clonic seizures.

Patient 4
Between the ages of 1 and 2, patient 4 had several febrile convulsions. From the age of 4 years, she had seizures
suggestive of temporal lobe epilepsy in which she would become unresponsive and look strange with a blank
expression on her face. An EEG at 6 years of age demonstrated generalized spike and slow wave complexes with
rhythmical delta activity over the left temporal region. MRI brain was normal. Starting at the age of 12 years, she
experienced stereotyped attacks of unsteadiness on her feet, vertigo, tiredness and nausea and vomiting, triggered
by exercise and excitement. Acetazolamide greatly reduced the frequency of these attacks. Interictal neurological
examination was normal. There was no family history of EA or epilepsy.
Patient 5
Episodes of ataxia began between the ages of 2 and 3years during which he became unsteady on his feet and
would vomit. Sleep helped in attack resolution. These attacks initially occurred every 3 weeks but
Acetazolamide successfully reduced the frequency. Over the years, the patient also experienced intense vertigo
during an attack. He was well in between attacks. Interictal examination demonstrated nystagmus. Patient 5 did
not have seizures, although an EEG revealed paroxysmal generalized slow wave bursts with some 3Hz spike
wave discharges. There was no family history of EA or epilepsy.

Patient 6
Typical attacks of EA comprising unsteadiness and vertigo began at the age of 6 years in patient 6. These attacks
were precipitated by tiredness and responded well to Acetazolamide. He was normal in between attacks.
Interictal examination demonstrated downbeating nystagmus only with no other cerebellar signs. In addition, he
suffers from absence seizures. His EEG demonstrated a 3Hz spike and wave pattern. There was no family history
of EA or epilepsy.

Patient 7
Patient 7 developed episodes of ataxia towards the end of her first year of life. The features of these attacks
included profound unsteadiness on her feet, dysarthria, vomiting, dizziness, mild weakness and paraesthesia of
the left hand and foot. Interictal examination revealed an intention tremor, dysarthria and a broad based gait. At
eight months, she developed episodes during which her eyes looked vacant and rolled accompanied sometimes
by a fall and a scream. These attacks were initially abolished by sodium valproate. At 8 years, she developed
focal seizures affecting her left arm and leg with consciousness intact. Her EEG showed multifocal abnormalities
with both generalized and focal interictal discharges with an emphasis on the right parietal region close to an
area of white matter abnormality detected on her brain MRI. There was no relevant family history.

Patient 8
From the age of 2 years, patient 8 experienced attacks of dizziness, clumsiness in his arms, slurred speech and
unsteadiness on his feet with no obvious precipitants. There was no nausea. Acetazolamide improved his attack
frequency. Interictal neurological examination was normal. His father, brother and sister also had similar EA
attacks. He experienced several generalized tonic-clonic seizures. His EEG demonstrated generalized
epileptiform activity with a degree of photosensitivity. There was no family history of epilepsy.

Patient 9
Patient 9’s first episode of ataxia was aged 21 years; attacks were stereotyped and manifested as unsteadiness on
her feet, vertigo and nausea with vomiting. She was normal in between attacks which have now been completely
abolished by Acetazolamide. Interictal neurological examination was normal. In addition, she suffers from
generalized tonic clonic seizures which tend to be nocturnal. Her EEG is normal. Her seizures are well controlled
on Lamotrigine and Carbamazepine. There is no family history of EA or epilepsy.
Patient 10
From the age of 8 years, patient 10 experienced sudden attacks of unsteadiness, profuse vomiting and abnormal
eye movements. He was normal in between attacks with a normal interictal neurological examination. His
epilepsy began at the age of 18 months with generalized tonic clonic seizures initially, followed by drop attacks,
myoclonic jerks and absences seizures. A series of EEG recordings demonstrated generalized 2-3 Hz spike wave
complexes. There was no family history of EA or epilepsy.

Patient 11
Episodes of unsteadiness, vertigo, diplopia and oscillopsia began at the age of 51 years and lasted up to a few
hours. He was normal in between attacks. Interictal examination demonstrated down beating nystagmus only
with no other cerebellar signs. Acetazolamide reduced the intensity and frequency of these attacks. In addition,
Patient 11 experienced several generalized tonic-clonic seizures. His interictal EEG demonstrated bi-hemispheric
sharp waves and spike and wave discharges. There was no family history of EA, but his daughter has juvenile
myoclonic epilepsy.

Patient 12
Patient 12 experienced lifelong attacks of unsteadiness, slurred speech, nausea, vertigo and imbalance lasting
several hours and usually precipitated by tiredness and exertion. A migrainous headache was also part of the
attack symptom complex. Acetazolamide reduced the frequency of attacks. When examined during an attack, she
was found to have dysarthria, dysmetria, dysdiadochokinesis but no nystagmus. As a child she had several
different types of seizures, including tonic, absence and complex partial seizures. In addition she has also had
frequent nocturnal myoclonic jerks. Several EEG’s have been normal. There was no family history of EA or
epilepsy.

Patient 13
Episodes of ataxia began at the age of 10 years. Attacks consisted of slurred speech, unsteadiness on his feet,
vomiting, headache and numbness of his left hand which lasted up to 24 hours. Interictal examination at the age
of 10 years was normal. Five other members of the family over 3 generations were also affected. Patient13’s
mother had interictal nystagmus on examination. Although there was no history of epilepsy, Patient13’s EEG
demonstrated epileptiform spike wave discharges.

Patient 14
Patient 14’s attacks of EA began at the age of 1 year. They were characterized by a sudden onset of profound
unsteadiness on his feet and dysarthria precipitated usually by exertion. Acetazolamide virtually abolished these
attacks. Interictal examination revealed a mild dysarthria, an ataxic gait and a mild right-sided hemiparesis which
was present from birth. In addition, from the age of six months, he developed frequent episodes characterized by
jerky movements of his arms and legs, incontinence and a vacancy of facial expression with his eyes rolling up.
These were considered to be epileptic in origin and resolved after a year. His EEG was normal. There was no
family history of EA or epilepsy.
Patient 15
Attacks of EA began at the age of 2 years and consisted of unsteadiness on her feet, upper limb clumsiness and
dysarthria. There were no precipitants for these attacks which lasted up to several days. Interictal examination at
the age of 21 years was remarkable for the presence of generalized chorea in additional to mild cerebellar signs
including dysmetria, dysdiadochokinesis. There was no nystagmus. Patient 15 had several generalized tonic-
clonic seizures which started at the age of 14 years. An EEG demonstrated generalized paroxysmal sharp bursts
with evidence of photosensitivity. There was no family history of EA or epilepsy.