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. 2010 Feb 15;588(Pt 11):1905–1913. doi: 10.1113/jphysiol.2009.186437

Genetic and functional characterisation of the P/Q calcium channel in episodic ataxia with epilepsy

Sanjeev Rajakulendran 1, Tracey D Graves 1, Robyn W Labrum 1, Dimitrios Kotzadimitriou 1, Louise Eunson 1, Mary B Davis 1, Rosalyn Davies 1, Nicholas W Wood 1, Dimitri M Kullmann 1, Michael G Hanna 1, Stephanie Schorge 1
PMCID: PMC2901979  PMID: 20156848

Abstract

Mutations in CACNA1A, which encodes the principal subunit of the P/Q calcium channel, underlie episodic ataxia type 2 (EA2). In addition, some patients with episodic ataxia complicated by epilepsy have been shown to harbour CACNA1A mutations, raising the possibility that P/Q channel dysfunction may be linked to human epilepsy. We undertook a review of all published CACNA1A EA2 cases and this showed that 7% have epilepsy – representing a sevenfold increased epilepsy risk compared to the background population risk (P < 0.001). We also studied a series of 17 individuals with episodic ataxia accompanied by epilepsy and/or clearly epileptiform electroencephalograms (EEGs). We screened the entire coding region of CACNA1A for point mutations and rearrangements to determine if genetic variation in the gene is associated with the epilepsy phenotype, and measured the functional impact of all missense variations on heterologously expressed P/Q channels. We identified two large scale deletions and two new missense mutations in CACNA1A. When expressed, L621R had little detectable effect on P/Q channel function, while the other missense change, G540R, caused an approximately 30% reduction in current density. In nine patients we also identified the previously reported non-synonymous coding variants (E921D and E993V) which also resulted in impairment of P/Q channel function. Taken together, 12 of the 17 patients have genetic changes which decrease P/Q channel function. We conclude that variants in the coding region of CACNA1A that confer a loss of P/Q-type channel function are associated with episodic ataxia and epilepsy. Our data suggest that functional stratification of all variants, including common polymorphisms, rare variants and novel mutations, may provide new insights into the mechanisms of channelopathies.

Introduction

The CACNA1A gene encodes the pore-forming subunit of the P/Q calcium channel, one of the principal channels supporting neurotransmitter release in the mammalian central nervous system (Westenbroek et al. 1995; Catterall, 1999). Dominant mutations in CACNA1A (MIM: 601011) are associated with episodic ataxia type 2 (EA2; MIM: 108500), familial hemiplegic migraine type 1(FHM1; MIM: 141500) and spinocerebellar ataxia type 6 (SCA6; MIM: 183086). Many genetic changes predicted to result in a null allele of CACNA1A including nonsense mutations (Ophoff et al. 1996), deletions (Riant et al. 2008; Labrum et al. 2009) and missense mutations (Guida et al. 2001; Jouvenceau et al. 2001; Wappl et al. 2002; Imbrici et al. 2004; Jen et al. 2004) cause EA2.

Several mice with recessive mutations in CACNA1A exhibit ataxia and spike-wave epilepsy and have long been considered a model of absence epilepsy (Noebels & Sidman, 1979; Fletcher et al. 1996; Lorenzon et al. 1998; Fletcher & Frankel, 1999; Zwingman et al. 2001; Miki et al. 2008). It is possible that the calcium channel gene may also contribute to the development of epilepsy in humans and this is suggested by the observation that several individuals in whom episodic ataxia is complicated by epilepsy have mutations in CACNA1A (Jouvenceau et al. 2001; Imbrici et al. 2004; Jen et al. 2004, 2007). In this report we have reviewed all 230 published genetically confirmed cases of EA2 and observed that 7% are associated with seizures. In contrast the prevalence of epilepsy in the general population is only 0.5–1% (Sander, 2003) suggesting that patients with EA2 have a sevenfold increased risk of developing epilepsy (P < 0.001; binomial test). However, the molecular pathophysiology of a possible relationship between variation in CACNA1A and human epilepsy remains unclear (Chioza et al. 2001; Khosravani & Zamponi, 2006).

We have previously reported an association between P/Q channel dysfunction induced by pathogenic mutations in CACNA1A and a clinical phenotype in which patients experience a combination of episodic ataxia and epilepsy (Jouvenceau et al. 2001; Imbrici et al. 2004). Here we describe a series of 17 patients with a similar phenotype of episodic ataxia and epilepsy and/or a clearly epileptiform EEG. We have conducted a detailed clinical, genetic and functional characterisation of variability in the P/Q calcium channel in these patients.

Although initially we identified new mutations in CACNA1A that could explain the phenotype of EA with epilepsy in some of these individuals, our genetic data also raise the question whether rare and common polymorphisms in the gene that reduce P/Q channel function may also increase the risk of epilepsy. Since CACNA1A encodes an ion channel, we were able to assess the gross functional impact of each amino acid change found in our patients with voltage-clamp recordings.

In total, we report that 12 of 17 patients had genetic changes predicted to reduce P/Q channel function. In addition, our data highlight the importance of functionally characterising known variants in candidate ion channel genes in determining pathogenicity. The data presented suggest that genetic variations in CACNA1A that confer a reduction in P/Q calcium channel function are associated with the syndrome of episodic ataxia with epilepsy and may partly explain the sevenfold increased epilepsy risk.

Methods

Patient data and sequencing

The study was approved by the local ethics committee. Patients referred from around the UK were screened for a clinical history (e.g. age of onset, symptoms and duration of attack, response to acetazolamide) similar to that seen in EA2, and those who had epilepsy or an epileptiform EEG were included. Patients with a clear alternative aetiology for seizures or metabolic causes of episodic ataxia were excluded. We originally identified 17 patients meeting these criteria. In 2 of the 17 patients we identified large scale deletions in CACNA1A (see reference Labrum et al. 2009 for genetic and clinical details). Clinical data for all 17 patients are summarised in Table 2, and descriptions are given in the online Supplemental Material.

Table 2.

Patient data and clinical summary (details in online Supplemental Material S2)

Episodic Ataxia
 Epilepsy
Patient Sex Onset (years) EA features IN ACZ response FH Seizures EEG Additional features Genetics
1 F <1 Ataxia; abnormal eye movements N Good N GTCS Normal — —
2 F 1.5 Ataxia; tremor N Not tested N GTCS, absence Normal — —
3 F 12 Ataxia; poor coordination N Good Y None Spike wave epileptiform EEG — —
4 F 12 Ataxia; vertigo; vomiting N Excellent N TLE, FC Generalized SWD; focal slowing, left temporal emphasis — G540R
5 M 2 Ataxia; vertigo; vomiting Y Good N None 3 Hz SWD — L621R
6 M 6 Ataxia; vertigo Y Good N Absence 3 Hz SWD LD G1105S
7 F 1 Ataxia; dizziness; dysarthria N Not tested N CPS, SPS Generalized and focal interictal discharges, right parietal emphasis Alternating hemiplegia E921D E993V
8 M 2 Ataxia; dysarthria; dizziness N Good Y GTCS, Generalized epileptiform activity; mild photosensitivity — E921D E993V
9 F 21 Ataxia; vomiting; vertigo N Excellent N GTCS, CPS Normal — E921D E993V
10 M 8 Ataxia; abnormal eye movements; dizziness N Not tested N Myoclonic astatic Generalised 2–3 Hz spike wave activity Autism; LD E921D E993V
11 M 50 Ataxia; vertigo; vomiting; oscillopsia Y Good Y GTCS Burst of sharp waves over frontotemporal region; SWD — E921D E993V
12 F <1 Ataxia; vertigo; dysarthria; vomiting N Good N Absence, CPS Normal LD E921D E993V
13 M 10 Ataxia; vomiting; dysarthria N Not tested Y CPS SWD: Irregular slow — E921D E993V
14 M 1 Ataxia; dysarthria N Excellent N GTCS Normal — E921D E993V
15 F 2 Ataxia; dysarthria N Not tested N GTCS Paroxysmal bursts bilaterally; photosensitivity Chorea E921D E993V, E1018K
16 F 5 Ataxia; vertigo; dysarthria Y No N None Epileptiform EEG — Deletion of exon 27
17 M 10 Ataxia; vertigo; vomiting Y Good N Absence 3Hz SWD — Deletion of exons 39–47
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IN, interictal nystagmus; FH, family history; GTCS, generalised tonic-clonic seizures; TLE, temporal lobe epilepsy; FC, febrile convulsions; SPS, simple partial seizures; CPS, complex partial seizures; LD, learning difficulties.

Genetic characterisation of patients

DNA was extracted from peripheral blood; the coding exons and flanking sequences of CACNA1A were sequenced using standard methods. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out according to established protocols (CACNA1A P279 A1MRC Holland) on all patients in this study. To characterise segregation of variants on exon 19, an amplicon spanning the variants in the exon was cloned into pCR2.1TOPO (Invitrogen), and individual colonies were analysed for the presence of each SNP.

Molecular biology

Mutations were introduced into CACNA1A cDNA in the pMT2LF mammalian expression vector by site-directed mutagenesis. Sequences are available upon request. Human embryonic kidney cells (HEK293) were grown in standard conditions and transfected using Lipofectamine 2000 (Invitrogen) with CACNA1A together with the auxiliary subunits, β4 and α2δ (a gift from A. Dolphin, University College London) in a ratio of 1.5:1:1. Green fluorescent protein (GFP) was co-transfected as a reporter (total DNA: 4 μg). Following transfection, human embryonic kidney (HEK) cells were incubated at 28°C for 48 h to increase cell viability prior to recording.

Electrophysiology

Standard whole-cell patch clamp recordings were performed at room temperature. The external solution contained (in mm): NaCl, 135; KCl, 4; MgCl2, 1; BaCl2, 10; Hepes, 10; pH 7.3. The pipette solution contained (in mm): CsCl, 150; Hepes, 10; and EGTA, 10; pH 7.3. Cells with series resistance above 10 MΩ were discarded. To control variability, recordings from WT CACNA1A cells were routinely intercalated with mutant CACNA1A. Leak subtracted currents were recorded using –P/4 protocol with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Data were sampled at 20 kHz and filtered at 5 kHz, and acquired and analysed using LabView software (v. 8.0, National Instruments, Austin, TX, USA) with in-house programs (DMK). Graphs were plotted using SigmaPlot software (v. 8.0, Systat Software Inc., San Jose, CA, USA).

Statistics

Whole cell currents were compared using Student's t test (Microsoft Excel and SigmaPlot v. 8.0). All data are given as mean ±s.e.m.

Results

Literature review of EA2 and epilepsy

A review of the literature (see Supplemental Material S1) indicates that a total of 73 mutations identified in 230 individuals with EA2 have been reported to date. In addition, a further 27 affected individuals are likely to harbour mutations in CACNA1A as they are members of families with genetically confirmed EA2 but have not been tested themselves. A total of 17 individuals or 7.4% of genetically confirmed individuals with EA2 (Table 1) also have epilepsy, or 6.6% of those clinically affected with EA2. Both figures are higher than expected from the prevalence of epilepsy in the general population, which is 0.5–1.0% (Sander, 2003). If individuals with clearly epileptiform abnormal EEGs are included in the analysis, then at least 10.4% of genetically confirmed or 9.3% of clinically affected EA2 individuals have abnormal EEGs. Because most individuals with EA2 have not had EEGs, these figures represent the minimum prevalence. These data suggest that individuals with EA2 have a sevenfold increased risk of epilepsy (P < 0.001; binomial test).

Table 1.

Mutations in CACNA1A associated with EA2 and either epilepsy or an abnormal EEG

CACNA1A mutation Phenotype Number of individuals with epilepsy Reference
E147K EA2; absence seizures 5/5 (Imbrici et al. 2004)
Deletion of exon 6 EA2; epilepsy; cognitive impairment 2/2 (Riant et al. 2009)
G638D EA2; abnormal EEG 1/1 (Cuenca-Leon et al. 2009)
I712V EA2; seizures; mental retardation 1/1 (Guerin et al. 2008)
2278–9 del AG (exon 19) EA2; generalised epilepsy 3/4 (Jen et al. 2004)
g→a donor splice site (3977 + 1) EA2; abnormal EEG 2/2 (Eunson et al. 2005)
Deletion of exon 27 EA2; abnormal EEG 1 (Labrum et al. 2009)
R1820X EA2; absence seizures, cognitive impairment 2 (Jouvenceau et al. 2001; Strupp et al. 2004)
3′ acceptor splice site (intron 36) EA2; epilepsy 3/17 (Kaunisto et al. 2004)
Deletion of GAGT at donor splice site → skipping of exon 41 EA2; abnormal EEG 3/5 (Zasorin et al. 1983; Baloh et al. 1997; Wan et al. 2005)
Deletion of exons 39–47 EA2; absence seizures 1 (Labrum et al. 2009)
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Genetic results of patients with EA and epilepsy

Seventeen patients with a phenotype of episodic ataxia with epilepsy or epileptiform EEGs were selected for this study. Two of the patients have been previously reported to harbour large deletions in CACNA1A when screened for genetic rearrangements using MLPA (see patients 16 and 17 in Table 2 and Labrum et al. 2009). The clinical and genetic features of all 17 patients are summarised in Table 2, and the full clinical details are described in the Supplemental Material S2. Missense mutations were identified in 2 out of 17 patients. In addition, four non-synonymous polymorphisms were identified in the cohort; three of these (E921D, E993V and E1018K) are located in exon 19 and the fourth (G1105S) in exon 20. The positions of all non-synonymous coding variants identified in the patients are indicated in Fig. 1.

Figure 1. Distribution of coding variants in CACNA1A in the predicted transmembrane topology of the calcium channel α-subunit.

Figure 1

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The SNARE interaction site in II–III interlinker is shown.

Identification of two new missense mutations in CACNA1A

Complete sequencing of the coding region of CACNA1A in the 15 patients in whom no rearrangements were identified revealed two new missense mutations. In patient 4, a G to C transversion in exon 12 at nucleotide 1910 of CACNA1A leads to the substitution of an arginine for glycine at position 540 (G540R) in the S2 segment of domain II of the channel (Fig. 1). The mutation was also identified in the patient's clinically unaffected father. In patient 5, a T to G transversion in exon 14 at position 2144 of CACNAIA was identified. This predicts the substitution of arginine for leucine at position 621 of the protein (L621R). This represents a radical amino acid change in a leucine-rich region in the S5 segment of domain II of the channel, which forms part of the selectivity filter (Fig. 1). Both the G540R and L621R mutations were absent in a panel of over 300 control chromosomes and both mutated residues exhibit a high degree of evolutionary conservation.

Functional expression of new missense mutations

When expressed in HEK cells, G540R caused a significant (P= 0.02; Student's t test) reduction in peak current density compared to WT channel (WT: 9.1 ± 0.9 pA pF−1, n= 10; G540R: 5.4 ± 0.4 pA pF−1, n= 5). In contrast, expression of the other new variant, L621R, did not have a significant effect on calcium channel function. Neither mutation altered the rate of activation or inactivation of P/Q channels (Fig. 2; Table 3).

Figure 2. Functional characterisation of new missense mutations in CACNA1A.

Figure 2

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Representative traces (top) from wild-type and mutant P/Q channels (at +10 mV), showing peak inward barium currents (middle) and normalised tail currents (bottom), in response to depolarising steps from −80 mV to +60 mV. Scale bar, 100 pA and 50 ms.

Table 3.

Whole cell parameters for HEK cells expressing P/Q channels with coding variants

WT G540R L621R E921D:E993V E1018K G1105S
τact (ms) 0.53 ± 0.07 0.79 ± 0.12 0.60 ± 0.06 0.80 ± 0.13 0.74 ± 0.22 0.50 ± 0.07
Inactivation 0.83 ± 0.05 0.74 ± 0.03 0.74 ± 0.02 0.72 ± 0.03 0.79 ± 0.05 0.77 ± 0.05
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Inactivation is expressed as percentage of peak current remaining at 175 ms after the step. All values are means ±s.e.m. No values are significantly different. Inactivation was measured at a test pulse of +10 mV; n≥ 5 for WT and all mutants.

Two non-synonymous variants in CACNA1A co-segregate in nine patients and reduce P/Q function

We also identified four previously reported non-synonymous SNPs (dbSNP, NCBI) in the patients. Three of these occur in exon 19, and two, E921D (rs16022) and E993V (rs16023), were present in nine of the patients – 9/17 patients vs. 30/188 control chromosomes (Fig. 1 and Table 2). To determine whether these two coding variants resided on the same ancestral chromosome we amplified a region spanning both E921D and E993V in the patients, cloned the product, and isolated and sequenced individual copies. In all cases both E921D and E993V were found to be located on the same allele. We also confirmed the cis configuration of the two variants in healthy control chromosomes obtained from the 1958 Wellcome Trust Case Control Cohort (WTCCC).

Because they occurred on the same allele, we determined the combined functional impact of E921D and E993V together on the P/Q channel. The pair of SNPs conferred both a reduction in calcium current density (E921D:E993V: 5.5 ± 0.8, n= 11; WT: 9.1 ± 0.9 pA pF−1, n= 10, P= 0.03), and an approximately 7 mV depolarising shift in channel activation (V1/2max E921D:E993V = 8.3 ± 0.8; WT = 1.5 ± 0.6 mV; P < 0.00001). Both of these effects are consistent with a loss of function.

Functional expression of remaining known non-synonymous CACNA1A SNPs in patients

In addition to E921D and E993V, patient 15 also carried the E1018K (rs16024) variant. This variant is also located in exon 19 (Fig. 1) but did not segregate with the E921D and E993V, and is likely to be derived from a separate ancestral chromosome. The final variant detected in our patient series, G1105S identified in patient F, is in exon 20 (Fig. 1) and segregation analysis could not be carried out due to the distance between exons 19 and 20 in CACNA1A.

E1018K conferred a 9 mV depolarising shift in channel activation (V1/2max E1018K = 10.3 ± 1.3; P= 0.0016) suggesting a loss of function. This variant also produced a small non-significant reduction in calcium current density (E1018K: 6.9 ± 1.2 pA pF−1, n= 6, P= 0.17). In contrast the final variant, G1105S (rs16027), present in patient 6, conferred a small gain of function on heterologously expressed P/Q channels (G1105S, current density: 14.0 ± 2.1 pA pF−1, n= 9, P= 0.04) (Fig. 3).

Figure 3. Functional characterisation of non-synonymous SNPs in CACNA1A.

Figure 3

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Representative traces (top) from wild-type and mutant P/Q channels (at +10 mV), showing peak inward barium currents (middle) and normalised tail currents (bottom), in response to depolarising steps from −80 mV to +60 mV. Scale bar, 100 pA and 50 ms.

Taken together, these data suggest that the known polymorphisms in CACNA1A exert significant and different effects on P/Q function in patients and in healthy controls. However as both G1105S and E1018K were only present in individual patients, our genetic data are not sufficient to determine whether these variants play a role in developing episodic ataxia and epilepsy.

Summary of results

In summary, out of a series of 17 patients with a phenotype of episodic ataxia with epilepsy, large deletions in CACNA1A were identified in two patients; a further two patients (patients 4 and 5) harboured new missense mutations, of which only one (G540R) detectably altered P/Q channel function although on genetic grounds alone G540R has features of pathogenic mutations. Nine additional individuals carried the two allelic exon 19 SNPs (E921D and E993V). Our data show that 12 of 17 patients with episodic ataxia and epilepsy carry genetic variants in CACNA1A that result either in a deletion or reduction in P/Q channel function.

Discussion

The phenotype of episodic ataxia with epilepsy

We have described the clinical features of the largest series of patients with a phenotype of episodic ataxia with clinical seizure episodes and/or abnormal epileptiform EEG findings. The phenotype of these patients is consistent with EA2 and similar to previously reported patients with EA2 and epilepsy in whom mutations in CACNA1A were identified (Jouvenceau et al. 2001; Imbrici et al. 2004). The duration of the attacks was generally hours and the patients did not exhibit myokymia typical of episodic ataxia type 1 (EA1). In addition when tested there was often a good or excellent response to acetazolamide. There was no family history in 13 of the 17 cases. Alternative metabolic causes of episodic ataxia were not found.

Although the present study describes 17 patients, we suggest that the clinical syndrome of episodic ataxia with epilepsy may be under-recognised and that ataxia may be inappropriately attributed to side-effects of anti-convulsants.

Identification of large deletions and new missense mutations in CACNA1A

Two patients had deletions that involved at least one exon, consistent with haploinsufficiency of CACNA1A. The previously reported R1820X mutation identified in a patient with episodic ataxia and epilepsy (Jouvenceau et al. 2001) is also consistent with a null allele of CACNA1A in this syndrome, bringing the total number to three patients.

Patients 4 and 5 harboured new missense mutations: G540R and L621R, respectively. Both changes were absent in a panel of over 300 control chromosomes and altered highly conserved positions of the channel peptide. G540R reduced P/Q-type channel function consistent with pathogenicity. However, this mutation segregated with the proband's unaffected father suggesting either reduced penetrance or that, on its own, the G540R change is insufficient to cause the disease. In contrast, L621R had no detectable impact on P/Q function despite exhibiting genetic features suggestive of pathogenicity. However, we cannot exclude effects on neuronal functions not apparent in the HEK cell expression system. In addition, mutations can affect channel function in a number of different ways including altering subcellular trafficking, protein folding and subsequent expression at the plasma membrane, or impair interactions with other proteins and intracellular signalling cascades.

Common and rare polymorphisms also impair P/Q-type function

Two polymorphisms, E921D and E993V which are in cis in CACNA1A, were identified in nine patients. These SNPs reduced P/Q calcium channel function and their presence may predispose individuals to episodic ataxia with epilepsy. The E1018K variant present in patient 15, who also has the E921D and E993V changes, also resulted in impairment of P/Q channel function. If E1018K did contribute to development of disease then patient 15, who carried all three exon 19 variants, might be predicted to have a more severe phenotype. However this was not observed to be the case.

Likewise G1105S was only found in one of our patients. Our functional data do not support a role for this variant in predisposing to episodic ataxia and epilepsy, as unlike most episodic ataxia mutations this polymorphism exerts a gain of function in expressed P/Q channels.

Exon 19 SNPs and migraine

Recently, the two coding variants that co-segregate on exon 19 have been investigated for their frequencies in migraine (Cuenca-Leon et al. 2008; D’Onofrio et al. 2009; Gerola et al. 2009). In this case the authors found that when the two coding variants were separated they associated with disease; however a follow-up paper did not find a significant relationship (Gerola et al. 2009), and so any association is not likely to be strong. No reports of the frequencies of these exon 19 non-synonymous variants in epilepsy are available, but it is possible that these variants contribute to the risk of developing epilepsy without ataxia, as they are present in the general population in 10–15% of chromosomes (dbSNP rs16022), suggesting many people are carry them without developing ataxia.

Insights into pathophysiology of EA and epilepsy

While EA2 is generally associated with mutations which profoundly affect the P/Q channel, the results in this study suggest that less severe perturbations of calcium channel function may also predispose individuals to EA with epilepsy. The G540R mutation and the exon 19 SNPs all induced changes in channel function similar to those reported for the E147K mutation that segregated with episodic ataxia and epilepsy in a three generation family (Imbrici et al. 2004).

Our data also reveal similarities to functional data from mice with mutations in CACNA1A that develop ataxia and seizures. For example, the recently characterised tg-4J allele of tottering (V581A) induced a 7 mV positive shift in the voltage-dependence of activation (Miki et al. 2008). A similar change was conferred by the E921D and E993V pair of variants that are increased in our patients (7 mV shift and loss in current density). The rocker mutation (T1310K), which is also associated with ataxia and epilepsy in homozygous mice, reduces P/Q current density only by ∼25% without altering voltage dependence of activation (Kodama et al. 2006). These data suggest that in mice, relatively modest changes in P/Q channel function are sufficient (when homozygous) to cause ataxia and epilepsy.

Interestingly all three exon 19 SNPs (E921D, E993V and E1018K) are located in the long interlinker between repeats II and III of the α subunit. This repeat contains critical sites for G-protein modulation, channel phosphorylation and importantly SNARE protein interaction, aberrations of which may contribute to disease pathophysiology.

Functional characterisation of variations in candidate genes

The findings in this study highlight the importance of functional characterisation of variants in candidate genes in establishing both disease susceptibility and causality, especially with respect to missense variations in sporadic cases. For example we would have predicted that L621R would be a pathological mutation based on such criteria as sequence homology, conservation, and absence from a control population. However functionally there was no detectable change in P/Q channels harbouring L621R and thus its role in disease causation remains uncertain. In a similar vein, although three of the coding variants (E1108K, E921D:E993V) are also present in healthy controls, the finding that they induced a loss of P/Q-type channel function may have been missed if only new mutations in CACNA1A were functionally expressed.

In our experiments, all HEK cells were incubated at 28°C following transfection with either WT or mutant DNA constructs. Incubation at this lower temperature promotes the expression of calcium channels (Jeng et al. 2008), and consequently our recordings may underestimate the deficits caused by the variants. In addition, it has been shown in heterologous systems that mutant P/Q channels can cause a dominant loss-of-function effect when co-expressed with WT channels (Jouvenceau et al. 2001; Jeng et al. 2008). We did not test for interactions with WT channels, which could represent an addition means of reducing channel function beyond what we observed. However, neither of these additional tests would change the main conclusion of the work, that a large proportion of patients with episodic ataxia and epilepsy have genetic changes in CACNA1A that lead to loss-of-function in P/Q channels.

Conclusions

We have presented a series of 17 patients with a core clinical phenotype characterised by episodic ataxia complicated by seizure disorder. We have provided evidence that genetic variation conferring loss of P/Q calcium channel function is associated with the phenotype in 12 of these patients. However we recognise that genetic variation in the coding region of CACNA1A cannot be the only predisposing factor since five of our patients with an indistinguishable clinical phenotype did not harbour any loss of function variants in the gene. In these cases, either variations in non-coding regions of CACNA1A or other genes are likely to influence their phenotype. Three out of the five patients were sporadic cases which precludes linkage analysis to determine susceptibility loci.

Finally, further work examining variation in CACNA1A in patients with other types of epilepsy may also help determine whether there is a more general role for CACNA1A loss of function variations in conferring risk in generalised epilepsy.

Acknowledgments

This work was funded by the MRC, the Brain Research Trust and Action Medical Research. S.S. is funded by the Worshipful Company of Pewterers. S.R. was funded by a Clinical Research Training Fellowship from the Wellcome Trust. M.G.H. is Director of the MRC Centre for Neuromuscular Diseases MRC Centre grant (G0601943). T.D.G. holds an Action Medical Research Training Fellowship and was previously funded by the Guarantors of Brain. T.D.G., S.R. and M.G.H. receive support from CINCH (NIH RU54 RR019482, NINDS/ORD). R.L. and M.D. receive support from NHNN. This work was undertaken at UCLH/UCL, which received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. We wish to thank all referring clinicians, S. J. Wallace, C. Downie, D. Cameron, J. Duncan, T. Davies, C. Constantinescu, R. McWilliam and S. Zuberi. The authors declare no conflict of interest.

Glossary

Abbreviations

EA

episodic ataxia

EA1

episodic ataxia type 1

EA2

episodic ataxia type 2

SNP

single nucleotide polymorphism

Author contributions

Conception and design of the experiments: S.R., T.D.G., M.B.D., N.W., D.M.K., M.G.H., S.S. Collection, analysis and interpretation of data: S.R., T.D.G., R.L., L.E., D.K., R.D., D.M.K., M.G.H., S.S. Drafting the article or revising it critically for important intellectual content: S.R., T.D.G., D.M.K., M.G.H., S.S. All experiments were carried out at the Institute of Neurology, University College London, UK.

Supplemental material

Supplemental data S1

Supplemental data S2

tjp0588-1905-SD1.pdf (33.7KB, pdf)

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