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. 2023 Mar 2;31:100456. doi: 10.1016/j.ensci.2023.100456

A neurodevelopmental disorder caused by a dysfunctional CACNA1A allele

Audra A Kramer a,⁎, Daniel F Bennett a, Kristin W Barañano b, Roger A Bannister a,⁎
PMCID: PMC10020665  PMID: 36938367

Abstract

P/Q-type Ca2+ flux into nerve terminals via CaV2.1 channels is essential for neurotransmitter release at neuromuscular junctions and nearly all central synapses. Mutations in CACNA1A, the gene encoding CaV2.1, cause a spectrum of pediatric neurological disorders. We have identified a patient harboring an autosomal-dominant de novo frameshift-causing nucleotide duplication in CACNA1A (c.5018dupG). The duplicated guanine precipitated 43 residues of altered amino acid sequence beginning with a glutamine to serine substitution in CaV2.1 at position 1674 ending with a premature stop codon (CaV2.1 p.Gln1674Serfs*43). The patient presented with episodic downbeat vertical nystagmus, hypotonia, ataxia, developmental delay and febrile seizures. In patch-clamp experiments, no Ba2+ current was observed in tsA-201 cells expressing CaV2.1 p.Gln1674Serfs*43 with β4 and α2δ-1 auxiliary subunits. The ablation of divalent flux in response to depolarization was likely attributable to the inability of CaV2.1 p.Gln1674Serfs*43 to form a complete channel pore. Our results suggest that the pathology resulting from this frameshift-inducing nucleotide duplication is a consequence of an effective haploinsufficiency.

Keywords: CACNA1A, CaV2.1, α1A, P/Q-type, ataxia, Developmental delay

Highlights

  • •

    CACNA1A encodes the P/Q-type CaV2.1 voltage-gated calcium channel.

  • •

    A patient presented with ataxia, developmental delay and febrile seizures.

  • •

    The patient harbored a nucleotide duplication in CACNA1A.

  • •

    The c.5018dupG variant caused a frame shift and an early stop codon.

  • •

    The resultant CaV2.1 p.Gln1674Serfs*43 channel was non-functional.

1. Introduction

The CACNA1A gene encodes for the pore-forming subunit of the P/Q-type voltage-gated Ca2+ channel, CaV2.1 [1,2]. CaV2.1 is highly expressed in Purkinje neurons [[2], [3], [4]] and at the neuromuscular junction [5,6]. At these synapses, Ca2+ influx through CaV2.1 supports action potential-triggered neurotransmitter release [5,[7], [8], [9]]. Missense point mutations in CACNA1A cause a wide range of clinical symptoms including Familial Hemiplegic Migraine type 1 (FHM1) [[9], [10], [11], [12], [13], [14], [15], [16]], Episodic Ataxia type 2 (EA2) [[17], [18], [19]] and epilepsy [[20], [21], [22]], as described in detail the Online Mendelian Inheritance in Man website (OMIM 601011). These point mutations, which typically result in a single amino acid substitution in CaV2.1, can alter channel gating, ion selectivity and expression and are typically categorized as either Gain-of-Function (GOF) or Loss-of-Function (LOF) [[23], [24], [25], [26], [27], [28], [29], [30]]. However, some point mutations in CaV2.1 have been reported to have mixed effects on channel function [[31], [32], [33], [34]].

While missense mutations in CACNA1A can result in syndromes which vary in severity, perhaps the most severe clinical cases stem from frameshifts that lead to stretches of altered amino acid sequence and/or premature termination of translation [[35], [36], [37], [38]]. ClinVar lists 97 known frameshift variants, all of which are pathogenic save eight variants with altered sequence beginning after residue 2220 (https://www.ncbi.nlm.nih.gov/clinvar/?term=CACNA1A[gene). Here, we describe a patient with developmental delay, episodic vertical nystagmus, hypotonia and febrile seizures, resultant from an autosomal-dominant de novo frameshift-causing guanine duplication in CACNA1A (c.5018dupG). The duplicated guanine led to an altered amino acid sequence beginning with a glutamine to serine substitution at position 1674 and ending with a premature stop codon 43 residues downstream (p.Gln1674Serfs*43). Electrophysiological analysis of CaV2.1 p.Gln1674Serfs*43 indicated that this frameshift precludes the ability of CaV2.1 to form a functional channel within the plasma membrane.

2. Materials and methods

2.1. Compliance with ethics of experimentation

Informed consent for this case report publication was obtained and is on record. The study was performed in accordance with the principles of the World Medical Declaration of Helsinki. No animals were used in this study.

2.2. Genetic analysis

Genomic DNA was obtained via buccal swab. The extracted DNA was enriched for the complete coding regions and splice-site junctions for most genes of the human genome using a proprietary capture system developed by GeneDx for next-generation sequencing with CNV calling (NGS-CNV). The enriched targets were simultaneously sequenced with paired-end reads on an Illumina platform. Bi-directional sequence reads were assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome build GRCh37/UCSC hg19. Data were filtered and analyzed with a custom-developed analysis tool (XomeAnalyzer) for the identification of sequence variants and most deletions/duplications involving three or more coding exons [39].

2.3. Health and safety

All laboratory procedures were approved by University of Maryland Baltimore Environmental Health and Safety (00005440).

2.4. Molecular biology

The GFP-CaV2.1 p.Gln1674Serfs*43 mutant construct was derived from the plasmid GFP-CaV2.1 (NM_001127221.1), known alternatively as EGFP-FLAG-α1A BI-1 (V1) [40]. To generate GFP-CaV2.1 p.Gln1674Serfs*43, a guanine at bp 5018 of the sequence encoding CaV2.1 (Δ10A (-V + G), 16+17+, Δ17A (-VEA), −31* (-NP), 37a (EFa), 43+44+, Δ47) was introduced into the plasmid by Genscript, Inc. (Pistacataway, NJ) using proprietary methods. The EGFP tag was located at the amino-terminus of the CaV2.1 coding sequence, which allowed visualization of the translated protein product in the likely case of premature truncation. The integrity of the novel construct was guaranteed by Genscript, Inc. and confirmed by restriction digests upon receipt.

2.5. Cell culture and transfection

tsA-201 cells (American Type Culture Collection, Manassas, VA) were cultured and transfected using Lipofectamine 2000 (Invitrogen, Waltham, MA) as described previously [34,41]. The transfection mixture contained expression plasmids encoding GFP-CaV2.1 or GFP-CaV2.1 p.Gln1674Serfs*43 with rat β4 and rabbit α2δ-1 auxiliary subunits (1 μg of each cDNA per dish). The day following transfection, cells were trypsinized, re-plated onto 35 mm Primaria-treated plastic culture dishes (BD Falcon, Franklin Lakes, NJ) and transferred to a 30 °C humified incubator. Successfully transfected (i.e., EGFP-positive) cells were used in experiments ∼24 h later.

2.6. Whole-cell patch clamp electrophysiology

Electrophysiological experiments were performed at room temperature. Borosilicate pipettes (2.5–4.0 MΩ) were filled with a solution containing (mM): 140 Cs-Aspartate, 10 Cs2-EGTA, 5 MgCl2 and 10 HEPES, pH 7.4 with CsOH. The bath solution contained (mM): 145 tetraethylammonium-Cl, 10 BaCl2, 10 HEPES and pH 7.4 with tetraethylammonium-OH; 10 mM Ba2+ was used in the external solution to ensure the detection of even the smallest divalent currents supported by GFP-CaV2.1 p.Gln1674Serfs*43. Electronic compensation was used to reduce the effective series resistance. Leak and capacitive currents were corrected with an -P/4 online subtraction protocol. Filtering was at 5–10 kHz and digitization was at 50–100 kHz. Cell membrane capacitance (Cm) was determined by integration of a transient from −80 mV to −70 mV using Clampex 10.6 (Molecular Devices, San Jose, CA) and was used to normalize current amplitudes (pA/pF). The mean value of Cm without compensation was 25.1 ± 2.3 pF (n = 16). The time constant for decay of the whole-cell capacity transient (τm) was reduced as much as possible using the analog compensation circuit of the amplifier; the mean values of τm and Ra were 106.5 ± 9.1 μs and 4.6 ± 0.4 MΩ, respectively. Data were obtained from two transfections for each condition; unpaired experiments were performed over two days with recordings for both conditions collected on each day. Three additional control cells were not included in analysis due to a high amplitude of current.

2.7. Analysis

SigmaPlot (version 14.0, Systat Software, Inc., Chicago, IL) were used for data analysis and for construction of figures. All data are presented as mean ± SEM. Statistical comparisons were made by unpaired, two-tailed t-test, with P < 0.05 considered significant.

3. Results

3.1. Clinical presentation

The proband is a 4.5-year-old female who was initially evaluated at 2.5 years of age. She was born at term weighing 7 pounds, 11 oz following an unremarkable gestation and delivery. In the first months of life, she had episodes of uprolling of the eyes and of downbeating jerk nystagmus. At 9 months of age, her EEG was normal though brain MRI indicated non-specific T2/FLAIR hyperintensities in the superior periventricular region. Febrile seizures occurred at 16 and 18 months of age.

Gross motor delays were noted in infancy, and as her motor skills progressed, hypotonia, imbalance and truncal titubation were also noted. She began walking independently at 26 months of age. She had mild delays in her expressive language but age-appropriate visual motor problem solving. She had mild issues with oral motor dysphagia. A figure 8 head rolling stereotypy was noted. On the most recent examination at 4 years of age, she had esotropia without apparent nystagmus, mild central hypotonia, no dysmetria or tremor and a mildly wide-based gait. Formal neuropsychological including administration of the WPPSI-IV revealed high average full-scale IQ.

She did not have paroxysmal episodes consistent with episodic ataxia, but her gait unsteadiness was noted to worsen with fatigue. A trial of acetazolamide did not appear to improve her neurological symptoms. Occasional episodes of upward eye-rolling continued to occur. Routine EEG at 4 years of age demonstrated some non-specific slowing but no epileptiform discharges. She demonstrated marked improvement in her motor skills coinciding with bouts of intensive therapies. The patient's clinical presentation is summarized in Table 1.

Table 1.

Clinical phenotype of the female patient carrying the c.5018dupG/p.Gln1674Serfs*43 variant.

phenotype age
age 2.5 years 4 years
febrile seizures + −
headaches − +
paroxysmal upgaze + +
myoclonus − +
fig. 8 stereotypy + −
nystagmus + +
esotropia − +
gross motor delay + mild
hypotonia + mild
imbalance + mild
truncal titubation + −
language delay + −
oral motor dysphagia + −
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3.2. Genetic analysis

Genetic testing of the proband and unaffected parents revealed an autosomal-dominant de novo mutation at c.5018dupG in exon 32 of CACNA1A. The guanine duplication induced a frameshift which results in an initial glutamine to serine substitution in CaV2.1 at position 1674 followed by a 43 amino-acid stretch of altered protein sequence terminated by a premature stop codon (p.Gln1674Serfs*43). The predicted functional consequence of the frameshift was total channel LOF due to protein truncation.

3.3. CaV2.1 p.Gln1674Serfs*43 is a non-functional channel

To test whether the frameshift beginning at position 1674 causes CaV2.1 channel LOF, whole-cell Ba2+ currents were recorded from tsA-201 cells co-expressing either GFP-fused wild-type CaV2.1 or GFP-fused CaV2.1 p.Gln1674Serfs*43 with auxiliary β4 and α2δ-1 subunits. In this set of experiments, only GFP-CaV2.1 supported inward currents when 10 mM Ba2+ was used as the charge carrier (Fig. 1A and B). A comparison of the current-voltage relationships for GFP-CaV2.1 and GFP-CaV2.1 p.Gln1674Serfs*43 confirmed the absence of any P/Q-type divalent current in the frameshift mutant beyond background recording noise (Ipeak = −23.9 ± 0.5 pA/pF, n = 6 vs. -2.3 ± 0.5 pA/pF at +20 mV, n = 10, respectively; p = 3 × 10−6; Fig. 1C).

Fig. 1.

Fig. 1

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CaV2.1 p.Gln1674Serfs*43 does not form a functional channel. Ba2+ current families recorded from tsA-201 cells expressing GFP-CaV2.1 (A) or GFP-CaV2.1 p.Gln1674Serfs*43 (B) with auxiliary β4 and α2δ-1 subunits. Currents were elicited by a 25 ms step depolarizations from −80 mV to indicated test potentials; the repolarization voltage was −40 mV (illustrated in A-top). (C) Comparison of GFP-CaV2.1 (●; n = 6) and GFP-CaV2.1 p.Gln1674Serfs*43 (○; n = 10) average peak current-voltage relationships. Currents were evoked at 0.1 Hz by test potentials ranging from −50 mV through +80 mV in 10 mV increments. Amplitudes were normalized by capacitance (pA/pF).

4. Discussion

In this study, we have described a CACNA1A-related disorder caused by a guanine duplication at position c.5018. The patient presented with episodic downbeat vertical nystagmus, hypotonia, ataxia, developmental delay and febrile seizures (Table 1). The de novo frameshift mutation resulted in a glutamine to serine replacement at residue 1674 followed by incorrectly-translated 43-residue amino acid stretch (p.Gln1674Serfs*43). Predictably, the presence of mistranslated sequence resulted in a non-functional channel (Fig. 1).

Three phenotypes have been typically associated with pathogenic variants in CACNA1A: FHM1, EA2 and spinocerebellar ataxia type 6 (SCA6). FHM1 has been thought to be associated with GOF variants, while EA2 is more associated with LOF variants. SCA6 is caused by a pathogenic polyglutamine expansion that results in toxic aggregation of the mutant protein in cerebellar Purkinje cells [[42], [43], [44]]. More recently, the spectrum of CACNA1A-related disorders has expanded through whole-exome sequencing to include individuals with either epileptic encephalopathy [[45], [46], [47]], epilepsy [48] or congenital ataxia [49,50]. Rather than an EA2 phenotype, our patient presents with a picture of congenital ataxia which manifests clinically as hypotonia, episodic downbeating nystagmus and gait ataxia without epilepsy or significant cognitive impairment. Her eye-rolling episodes are likely to represent a form fruste of paroxysmal tonic upgaze similar to other cases arising from CaV2.1 LOF [51,52]. Thus, this clinical report expands the spectrum of described cases of CACNA1A-related neurodevelopmental disorder.

Multiple explanations are possible for the pathological effects of p.Gln1674Serfs*43 on the level of a neuron. The most straightforward explanation is a functional haploinsufficiency consequential to the inability of CaV2.1 p.Gln1674Serfs*43 to form a functional channel. One would predict that a substantial reduction of CaV2.1 channels at central synapses as result of the functional ablation of one allele would impair neurosecretion by reducing Ca2+ flux into axon terminals in response to a single action potential or trains of stimuli [53]. Another plausible mechanism by which haploinsufficiency may influence synaptic efficacy is by opening up membrane “slots” normally reserved for CaV2.1 to either CaV2.2 or CaV2.3 [54,55] as the probability of synaptic vesicle release at some synapses is preferentially driven by Ca2+ flux via CaV2.1 [56]. In this regard, compensatory substitution of the CaV2.2 and CaV2.3 isoforms would alter the Ca2+ flux profile at synapses which typically rely on CaV2.1 for the Ca2+ influx that triggers neurotransmitter release. While either of these mechanisms (or others) may be applicable, the systemic impact of altered neurotransmission is undoubtedly complex and dependent on multiple variables. For example, conditional knockout of CACNA1A in GABAergic cortical interneurons caused epileptic EEG waveforms through the removal of GABAergic input onto cortical pyramidal cells [57]. Another consideration is that the detrimental effects of p.Gln1674Serfs*43 might be a consequence of a toxic GOF instead of a simple functional haploinsufficency because CaV2.1 (+/−) mice appear to be largely unaffected by the ablation of only one allele [58]. In this context, the pathological effect of the truncated protein may be related to cellular stress secondary to the unfolded protein response and/or protein aggregation similar to that observed in cases of SCA6. Studies utilizing tissue from SCA6 mouse models and SCA6 patients have indicated accumulation of the expanded CaV2.1 channel in lysosomes, thereby shifting the cell to a pro-apoptotic state [42,59,60].

5. Conclusions

We have described a young girl presenting with episodic downbeat vertical nystagmus, hypotonia, ataxia, developmental delay and febrile seizures. This syndrome was caused by a de novo autosomal-dominant guanine duplication c.5018dupG in CACNA1A. The additional guanine caused a frameshift with early termination which ultimately ablated CaV2.1 channel function (p.Gln1674Serfs*43), suggesting effective haploinsufficiency as the basis of the disorder.

Funding

This work was supported by the National Institutes of Health grant R01-NS103777 and a pilot grant from the National Ataxia Foundation (both to R.A.B.). A.A.K. was supported by National Institutes of Health grant T32-AR007592.

Author contributions

A.A.K. performed research, analyzed data and wrote the paper. D.F.B. performed research. K.W.B. cared for and evaluated the patient, analyzed genetic data, and wrote the paper. R.A.B. designed research, analyzed data and wrote the paper. All authors read, revised and approved the final manuscript. This work was prepared while R.A.B. was employed at the University of Maryland Baltimore. The opinions expressed in this article are the author's own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services or the United States government.

CRediT authorship contribution statement

Audra A. Kramer: Conceptualization, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization, Project administration. Daniel F. Bennett: Investigation. Kristin W. Barañano: Conceptualization, Validation, Formal analysis, Writing – review & editing, Project administration. Roger A. Bannister: Conceptualization, Validation, Formal analysis, Writing – original draft, Writing – review & editing, Visualization, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Acknowledgements

We thank Drs. Y.-Q. Cao, B.A. Adams and W.A. Sather for the gifts of the parent EGFP-CaV2.1, β4 and α2δ-1 constructs, respectively.

Contributor Information

Audra A. Kramer, Email: audra.kramer@som.umaryland.edu.

Kristin W. Barañano, Email: kwb@jhmi.edu.

Roger A. Bannister, Email: rbannister@som.umaryland.edu.

Data availability

Genetic information obtained during the current study is publicly available in the ClinVar database. Expression plasmids are available from the corresponding authors upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Genetic information obtained during the current study is publicly available in the ClinVar database. Expression plasmids are available from the corresponding authors upon reasonable request.

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