CACNA1B Mutation Is Linked to Unique Myoclonus Dystonia Syndrome

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Genetic architecture of dystonia

Groen, J.L.

Publication date 2014

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Citation for published version (APA): Groen, J. L. (2014). Genetic architecture of dystonia.

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CACNA1B mutation is linked to unique myoclonus dystonia syndrome

Justus L Groen*, Arturo Andrade*, Katja Ritz, Hamid Jalalzadeh, Martin Haagmans, Ted Bradley, Peter Nürnberg, Dineke Verbeek, Sylvia Denome, Raoul C.M. Hennekam, Diane Lipscombe, Frank Baas** & Marina A.J. Tijssen** * shared first;** shared last

Submitted

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abstract

Exome sequencing combined with linkage analysis in a 3-generation pedigree with an unique familial Myoclonus-Dystonia syndrome (M-D) identified a mutation in the CACNA1B gene

coding for the neuronal voltage-gated calcium channel CaV2.2. In tsA201 cells, a human cell

line, CaV2.2 calcium channel currents were larger in cells transiently expressing mutant human

CaV2.2 channels compared to wild-type. This electrophysiological property is consistent with the observed hyperexcitability clinical phenotype.

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introduction

Myoclonus-Dystonia syndrome (M-D, MIM #159900) is a rare hyperkinetic movement disorder with myoclonic symptoms combined with dystonia, mainly of the upper part of the body.1 Psychiatric comorbidity has been described and symptoms often respond to alcohol ingestion.2-4 In as much as 21-85% of M-D cases mutations in the SGCE gene (epsilon-sarcoglycan, DYT11, OMIM159900) can be identified.5 Previ- ously, we described a SGCE-negative 3-generation pedigree (five affected members) with an unique familial M-D syndrome.6 The phenotype included cervical and axial dystonia at rest, writer’s cramp and action-induced foot dystonia. Myoclonic jerks were detected in legs and arms during rest and action. On EMG semirhythmic bursts of 40–200ms were seen. Additional symptoms, not previously described in M-D, were high-frequency continuous myoclonus in the legs while standing causing unsteadi- ness. Coherences analysis of the EMG signals suggested an afferent (sensory) cortical input with a frequency around 12Hz. Eye movement recordings indicated cerebel- lar pathology. Also present, and not previously linked to M-D, were cardiac arrhythmias and attacks of painful cramps in upper and lower limbs in 3 of 5 affected family members. By combining exome sequencing with linkage analysis in this pedigree we identified a dominant mutation in theCACNA1B gene (MIM 601012) encoding the

neuronal voltage-gated calcium (CaV) channel CaV2.2. Electrophysiological analyses

of mutant and wild-type human CaV2.2 channels in a mammalian cell lines suggest that this mutation results in larger calcium current density in cells expressing mutant

CaV2.2 compared to wild-type controls. These functional analyses of cloned CaV2.2 channels are consistent with a gain-of-function phenotype.

methods

Patients Pedigree and presentation of affected members were previously described.6 (Figure 1) The study was approved by the Medical Ethical Committee and investigations were performed with informed consent. Recently, the single unaffected brother (III-4) presented with a sudden onset, fixed dystonic posture of the left ankle and wrist, consistent with functional dystonia. No myoclonic jerks or orthostatic myoclonus were noticed. All patients were examined by a movement disorder specialist (MAJT).

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Figure 1. Pedigree of m-d family with r1389h mutation in cacna1b. Unaffected and affected family members are indicated by clear symbols and blackened symbols, respectively. Diagonal bars through the symbols denote deceased individuals. * denote examined and included in linkage studies. In individual III-2 exome sequencing was performed.

Genotyping We performed linkage analysis in this three generation family and combined results with the exome data, obtained in the index case (III-2). Linkage analysis was performed using the Affymetrix GeneChip Human Mapping 250K Nsp Array in 5 affected and one unaffected sibling from 3 generations (Figure 1: II-2, III-2, III-3, III-4, III-6, IV-6) providing a maximum LOD score of 1.2. Linkage analysis was performed with the program ALLEGRO making use of a reduced marker panel of approximately 20000 SNPs. Autosomal dominant mode of inheritance with full penetrance was assumed with a disease allele frequency of 0.0001. For the 13 linked regions, again calculations with ALLEGRO was performed with all SNPs from 250K-Chip to determine more accurately the limiting SNPs of the linkage regions. Exome sequencing (NimbleGen SeqCap EZ Exome v2 Sequence Capture) was performed on a SOLiD v4 platform (Applied Biosystems), with 50 bases forward and 35 bases reverse reads. Colour space reads were mapped to the reference human genome (hg18) with Bioscope software version 1.3 (Life Technologies). Duplicate reads were removed prior to variant calling using MarkDuplicates from the Picard software (picard.sourceforge.net/index.shtml). Reads were realigned around known ‘indels’ with the Genome Analysis Toolkit (Broad Institute). Variants were called using the GATK’s Unified Genotyper. GATK’s VariantAnnotator was used to anno- tate variant calls in preparation of filtering. Non-synonymous variants in coding regions that are not present in dbSNP 130 were identified with ANNOVAR. SNV population frequency was examined with TaqMan Custom Endpoint Genotyping assays (ABI). Identified variants were confirmed by Sanger sequencing and their frequency in general population was investigated in 760 healthy controls from the Dutch Blood

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Bank with TaqMan SNP genotyping assays (Applied Biosystems, Life Technologies). Variant R1389H, situated in the extracellular domain (IIIS5 through IIIS6), was investigated in 47 patients with similar clinical features of dystonia. The three exons (exon 26, 27 and 28) that code for this extracellular domain were screened for the pres- ence of novel variations by Sanger sequencing.

Electrophysiological analyses of human-derived CaV2.2 clones

Full-length human CaV2.2 cDNA (ORF 7094bp, pSAD442-1) was cloned from human brain and spinal cord and inserted into pcDNA6 mammalian expression vector. pSAD442-1 contains alternate exons [+e10a, +18a, D19a, +e31a, +e37b, +e46].7 The R1389H mutant was generated using standard mutagenesis methods. Wild-type b and mutant hCaV2.2 were transiently expressed in human tsA201 cells with CaV 3, α d CaV 2 -1 and enhanced green fluorescent protein cDNAs (eGFP; BD Bioscience) as described previously.8 Calcium channel currents were recorded 24 hours after transfection. cDNA concentrations were measured and run on agarose gels to ensure these were identical between mutant and wild-type transfections. Recording from wild-

type and mutant CaV2.2 clones were interspersed and the experimenter blind to clone identity over the recording period. Recordings were obtained from 7 separate transfections for both WT and R1389H cDNAs. The identity of WT and R1389H clones were revealed post analysis. Standard whole cell patch clamp recording was used to 8 compare calcium channel currents. External solution contained: 1 mM CaCl2, 4 mM

MgCl2, 10 mM HEPES, 135 mM tetraethylammonium (TEA) chloride, pH adjusted to 7.2 with TEAOH. Internal solution contained: 126 mM CsCl, 10 mM EGTA, 1 mM EDTA, 10 mM HEPES, 4 mM MgATP, pH 7.2 with CsOH. Calcium currents were evoked by voltage-steps and currents leak subtracted on-line using a P/-4 protocol. Data were sampled at 20 kHz and filtered at 2 kHz (-3 dB). All recordings were obtained at room temperature.

results

CACNA1B R1389H Linkage analysis indentify 16 regions shared by all affected, together spanning 150 MB. In these regions 5 rare heterozygous missense SNV were identified by exome sequencing and confirmed in the proband by Sanger sequencing. Segregation with disease within the family was seen for SNVs in the genes CACNA1B, VPS13D and SPTAN1. These SNVs were not present in 760 ethnically matched controls (n=1012 for CACNA1B). Because the clinical presentation of myoclonic jerks and dystonia in combination with arrhythmias in this family pointed to a possible channelopathy, we focused our investigations on the CACNA1B variant. G4166A is located in exon 28 of CACNA1B and causes an amino acid change from Arginine to Histidine at residue 1389 (R1389H). This amino acid is part of the putative extracellular loop between the S5 and S6 α-helices of domain III. R1389 is highly conserved across species including C. elegans (15 species) and it is predicted to be essential for normal channel function

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(Align GVGD Class C25 (GV: 0.00 – GD: 28.82), SIFT 0,00 and PolyPhen humdiv 1.0).

The CACNA1B gene encodes CaV2.2, the main of pore-forming alpha-1 subunit of the multi-subunit voltage-gated calcium channel complex that generates N-type currents in neurons. In an attempt to identify additional cases with mutations in CAC- NA1B the III-S5 through III-S6 region was Sanger sequenced in 47 patients with M-D phenotype showing wild-type R1389 in all subjects and lack of (other) missense SNVs in this region.

Larger calcium current density in cells expressing mutant CaV2.2

To assess the functional consequences of the R1389H mutation on CaV channel

activity we expressed wild-type and mutant human CaV2.2 channels in the mammalian tsA201 cell line and compared calcium channel currents using whole cell patch recording. Calcium currents in cells expressing R1389H mutant were on average ~ 2-fold larger compared to control currents recorded under identical conditions (Fig- ure 2A-C). The steady state current (I) is the product of single channel current ampli-

tude (i), the number of channels (N) and the single channel open probability (Po).

The voltage-dependence of single channel Po obtained from measuring tail current amplitudes for wild-type and R1389H mutants are not significantly different (Figure 2D). Channel inactivation curves obtained with 2 second pre-pulses were also not distinguishable between wild-type and R1389H channel currents (Figure 2E). The instantaneous current-voltage relationships calculated from the average peak current voltage plot (Figure 2F), illustrate that currents through wild-type and R1389H mutant channels reverse direction at close to the same voltages (54 mV and 52 mV) but with different slopes.

Figure 2. calcium channel currents are significantly larger in cells expressing

r1389h compared to wild-type human cav2.2 (hcav2.2). (A - F) Biophysical properties of calcium channel currents recorded from tsA201 cells

expressing hCaV2.2 together with CaVb3 and CaVα2δ-1. Whole cell currents were recorded 24 hours after transfection using 1 mM calcium as the charge carrier. (A) Calcium channel currents were evoked by voltage steps of 10 mV increments applied from a holding potential of -80 mV. Deactivation tail currents were measured at -60 mV. Currents recorded

from cells expressing wild-type and R1389H hCaV2.2 cDNAs are shown superimposed. (B) Peak calcium channel current densities at evoked at 10 mV from individual cells (open symbols) expressing WT and R1389H

CaV2.2 shown together with averaged current density and SEM (solid symbols). Averaged peak current densities at +10 mV were: 97.9 ± 15.0 pA/pF in 22 cells expressing R1389H and 55.1 ± 9.1 pA/pF in 29 cells

expressing WT CaV2.2 (p = 0.014, Student’s t-test). (C) Current-voltage

(I-V) relationships of wild-type and R1389H hCaV2.2 are compared. Peak current densities are plotted against test potential. Currents were elicited by step depolarizations from -60 mV to 80 mV in 10 mV increments from a holding potential of -80 mV. I-V relationships were fit to data from

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10 AB -300 C -60 120 (pA/pF) -80 -250 80 WT -200 40 R1389H -150 -60 -40 20-20 40 80 -100 Vm (mV) 50 pA/pF -50 5ms Currentdensity (pA/pF) WT(26) R1389H(21) -120 WT

R1389H DTest Potential EPre-pulse Potential F

-60 -80 -80 100 WT(9) Potential(mV) WT(26) -40-60-20 20 40 1.0 R1389H(21) 1.0 R1389H (11) 0.8 0.8

0.6 0.6 WT 0.4 0.4 -200

0.2 pulse) (test I/Imax 0.2 I/Imax (at -60 mV) -60 (at I/Imax 0.0 0.0 R1389H -300 (pA/pF) -40-60-20 0 20 40 60 80 -100 -80 -60 -40 -20 0 Test Potential Pre-pulse potential (mV) (mV)

individual cells using the product of the Boltzmann function and a straight

line for estimates of activation mid-point (V1/2), slope factor (k) and

reversal potential (Vrev). Average ± SE for WT (n = 26) and R1389H (n = 21)

for V1/2: 0.85 ± 1.03 mV and -0.18 ± 0.98 mV (p = 0.47); k: 5.8 ± 0.5 mV and

4.8 ± 0.2 (p = 0.06); and Vrev: 54.2 ± 0.8 mV and 51.6 ± 1.2 mV (p = 0.08). (D) Activation plots obtained from the same cells in C, obtained from tail current amplitudes at –60 mV at the end of 25 ms duration test pulses.

Normalized tail currents at -60 mV (I/Imax) plotted against test pulse were

fit with Boltzmann functions to estimate V1/2 and k. Average ± SE for WT

(n = 26) and R1389H (n = 21) were for V1/2: 13.6 ± 0.9 mV and 12.6 ± 1.1 mV (p = 0.47) and k: 11.6 ± 0.4 mV and 10.7 ± 0.3 mV (p = 0.09) Stimulation protocols used to generate I-V and activation curves are shown. (E) Steady state inactivation relationships from normalized peak current amplitudes at 0 mV following 2 s pre-pulse steps applied in 10 mV increments to potentials between -110 mV and 0 mV. Boltzmann functions were fit to each

data set and average ± SE values for WT (n = 9) and R1389H (n = 11) for V1/2: -51.4 ± 3.8 mV and -48.0 ± 1.8 mV (p = 0.40) and k: 8.3 ± 0.5 mV and 8.4 ± 0.8 mV (p = 0.88). F) Instantaneous I-V derived from average current-voltage relationship illustrates that current flow through open channels is Ohmic.

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discussion

We report a missense variant in CACNA1B (MIM 601012) in a 3-generation family with a M-D syndrome characterized by progressive myoclonic jerks, dystonia and an action-induced lower limb myoclonus combined with cardiac arrhythmias. Clinical data is most consistent with a subcortical origin of symptoms, with possible cerebel-

lar involvement. CACNA1B codes for the pore-forming subunit of presynaptic CaV2.2

channels that together with CaV2.1 (CACNA1A) control depolarization-induced calcium entry and transmitter release from the majority of presynaptic terminals at mammalian synapses.

α d b CaV2.2 forms a complex with CaV 2 and CaV subunits localizing to active zones of presynaptic nerve terminals throughout the brain and nervous system. Together with

CaV2.1, the activity of CaV2.2 controls the amount of transmitter released in response to depolarization of the presynaptic terminal. At most synapses, the concerted action

of CaV2.1 and CaV2.2 channels regulate depolarization-dependent presynaptic cal-

cium entry, while at other synapses presynaptic CaV2.1 or CaV2.2 channels control calcium entry exclusively 9;10. Our functional analyses predict a gain of function phenotype that could increase the amount of transmitter release at excitatory and/or inhibitory synapses for the same size depolarization, at terminals containing mutant

H1389-CaV2.2 channels.

The R1389H mutation in CACNA1B conserves the positive charge (at neutral pH) consistent with the preservation of basic channel and protein function and with the high frequency of arginine to histidine-disease causing mutations. Our heterologous

expression studies in a human cell line suggest that mutant CaV2.2 channels are either at higher density on the cell surface or that more current flows through single ion

channels compared to wild-type. A change in single CaV2.2 channel PO is less likely because the voltage-dependence and channel kinetics are not significantly different

between mutant and wild-type CaV2.2 channels. H1389 could impact the rate of ion

movement through the CaV2.2 ion pore, especially because of its proximity to the ion pore, but any alteration in ion flow rates would have to preserve ion selectivity based

on the similarities between CaV2.2 channel reversal potentials that are only slightly different between R1389 and H1389 channels (2.5 mV). Differences in the density

of functional CaV2.2 channels on the cell surface can result from altered interactions with auxiliary subunits, increased rate of trafficking to, stability on, or decreased rate of removal from the plasma membrane.

Mutations in the closely related CACNA1A gene are linked to different autosomal dominant neurologic disorders including familial hemiplegic migraine (FHM), epi- sodic ataxia type 2 (EA-2), and spinocerebellar ataxia type 6 (SCA-6). Surprisingly, CACNA1B has not until now not been linked to a human mendelian disorders. Com- mon variants in CACNA1B are associated with cerebral infarction 11, bipolar disorders

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and schizophrenia 12;13. Here, we show that a missense SNV in an extracellular region

of the CaV2.2 channel causes an unique M-D syndrome associated with cardiac arrhythmias.

references

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