ORIGINAL RESEARCH
published: 08 March 2022
doi: 10.3389/fpsyt.2022.858238
Contribution of CACNA1H Variants in
Autism Spectrum Disorder
Susceptibility
Marta Viggiano 1 , Tiziano D’Andrea 2 , Cinzia Cameli 1 , Annio Posar 3,4 , Paola Visconti 3 ,
Maria Cristina Scaduto 3 , Roberta Colucci 3,5 , Magali J. Rochat 6 , Fabiola Ceroni 1 ,
Giorgio Milazzo 1 , Sergio Fucile 2,7 , Elena Maestrini 1* and Elena Bacchelli 1*
1
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy, 2 Department of Physiology and
Pharmacology, Sapienza University, Rome, Italy, 3 Unità Operativa Semplice d’Istituto (UOSI) Disturbi dello Spettro Autistico,
Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Istituto delle Scienze Neurologiche di Bologna, Bologna, Italy,
4
Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy, 5 Department of Medical and
Surgical Sciences, University of Bologna, Bologna, Italy, 6 Functional and Molecular Neuroimaging Unit, Istituto di Ricovero e
Cura a Carattere Scientifico (IRCCS) Istituto delle Scienze Neurologiche di Bologna, Bologna, Italy, 7 Istituto di Ricovero e
Cura a Carattere Scientifico (IRCCS) Neuromed, Pozzilli, Italy
Edited by:
Sara Calderoni,
Stella Maris Foundation (IRCCS), Italy
Reviewed by:
John Jay Gargus,
University of California, Irvine,
United States
Andreas G. Chiocchetti,
University Hospital Frankfurt, Germany
*Correspondence:
Elena Bacchelli
elena.bacchelli@unibo.it
Elena Maestrini
elena.maestrini@unibo.it
Specialty section:
This article was submitted to
Child and Adolescent Psychiatry,
a section of the journal
Frontiers in Psychiatry
Received: 19 January 2022
Accepted: 08 February 2022
Published: 08 March 2022
Citation:
Viggiano M, D’Andrea T, Cameli C,
Posar A, Visconti P, Scaduto MC,
Colucci R, Rochat MJ, Ceroni F,
Milazzo G, Fucile S, Maestrini E and
Bacchelli E (2022) Contribution of
CACNA1H Variants in Autism
Spectrum Disorder Susceptibility.
Front. Psychiatry 13:858238.
doi: 10.3389/fpsyt.2022.858238
Frontiers in Psychiatry | www.frontiersin.org
Autism Spectrum Disorder (ASD) is a highly heterogeneous neuropsychiatric disorder
with a strong genetic component. The genetic architecture is complex, consisting of
a combination of common low-risk and more penetrant rare variants. Voltage-gated
calcium channels (VGCCs or Cav ) genes have been implicated as high-confidence
susceptibility genes for ASD, in accordance with the relevant role of calcium signaling in
neuronal function. In order to further investigate the involvement of VGCCs rare variants
in ASD susceptibility, we performed whole genome sequencing analysis in a cohort of
105 families, composed of 124 ASD individuals, 210 parents and 58 unaffected siblings.
We identified 53 rare inherited damaging variants in Cav genes, including genes coding
for the principal subunit and genes coding for the auxiliary subunits, in 40 ASD families.
Interestingly, biallelic rare damaging missense variants were detected in the CACNA1H
gene, coding for the T-type Cav 3.2 channel, in ASD probands from two different families.
Thus, to clarify the role of these CACNA1H variants on calcium channel activity we
performed electrophysiological analysis using whole-cell patch clamp technology. Three
out of four tested variants were shown to mildly affect Cav 3.2 channel current density
and activation properties, possibly leading to a dysregulation of intracellular Ca2+ ions
homeostasis, thus altering calcium-dependent neuronal processes and contributing
to ASD etiology in these families. Our results provide further support for the role of
CACNA1H in neurodevelopmental disorders and suggest that rare CACNA1H variants
may be involved in ASD development, providing a high-risk genetic background.
Keywords: ASD, rare variants, VGCCs, CACNA1H, Cav 3.2, calcium channel
INTRODUCTION
Autism Spectrum Disorder (ASD) is a group of clinically heterogeneous neurodevelopmental
disorders with a prevalence of >1% (1), characterized by impairments in communication and social
interaction, and the presence of repetitive and restrictive behaviors (2).
ASD is a multifactorial disorder, with a strong genetic component and an estimated heritability
of 60–90% (3, 4). The genetic architecture is highly heterogeneous and consists of a complex
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CACNA1H Variants in ASD Susceptibility
The sample includes 21 multiplex and 84 simplex families, for
a total of 98 males and 26 females with ASD, as well as 210 parents
and 58 unaffected siblings. DNA samples were extracted from
whole blood.
Individuals with ASD were assessed using a set of standardized
diagnostic instruments to evaluate the ASD phenotype (ADOS,
CARS and M-CHAT), to assess developmental/cognitive levels
(PEP-3, Leiter-R, Griffith Scales, or Wechsler Scales) and adaptive
behavior (Vineland Adaptive Behavior Scale, VABS); clinical
signs such as mimicry, hyperactivity, sensory abnormalities
and symptoms onset were also evaluated. Moreover, probands
underwent EEG and MRI. Subclinical features in relatives
were assessed using the Social and Communication Disorders
Checklist and The Broad Autism Phenotype Questionnaire.
All participants provided a written informed consent to
participate to this study. This study was approved by the local
Ethical Committee (Comitato Etico di Area Vasta Emilia Centro
(CE-AVEC); code CE 14060). All research was performed in
accordance with the relevant guidelines and regulations.
interplay of rare deleterious variants and common low-risk
alleles. The discovery of rare, highly penetrant, variants in
a proportion of cases (10–25%) (5), contributed to the
identification of numerous candidate genes, showing functional
convergence on a small set of common pathological pathways.
Among these, calcium signaling has been consistently implicated
in the molecular bases of ASD and associated comorbidities
(6–8). It represents a universal and versatile pathway involved
in a wide range of cellular processes including synaptic
plasticity, by modulating neurotransmitter release and shaping
of the synaptic membrane composition (9). Intracellular calcium
concentration is mainly but not exclusively regulated by voltagegated calcium channels (VGCCs or Cav channels), a family
of calcium channels that allow the influx of ions into the
cell in response to voltage changes, regulating intracellular
calcium concentration and initiating a variety of calciumdependent processes, such as exocytosis and neurotransmitter
release (10). VGCCs variants have been indicated as a shared risk
factor for several neuropsychiatric disorders (11). In particular,
perturbation of intracellular calcium homeostasis caused by
disruption of VGCCs genes has been associated to increased ASD
susceptibility (8, 9, 12).
In order to investigate the contribution of VGCCs to ASD,
we analyzed the sequence of the whole genome in a cohort
of 105 families comprising 124 individuals with a diagnosis
of ASD, and we looked for rare coding damaging variants in
genes encoding for VGCCs. We evaluated variants in genes
for the VGCCs α1 principal subunit (CACNA1A, CACNA1B,
CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G,
CACNA1H, CACNA1I, CACNA1S), as well as genes coding for
the auxiliary subunits (CACNA2D1, CACNA2D2, CACNA2D3,
CACNA2D4, CACNB1, CACNB2, CACNB3 and CACNB4).
Auxiliary subunits have also been implicated in ASD risk,
due to their important role in the regulation of channel
biophysical properties and targeting of the α1 subunit to the
cell membrane (13).
Interestingly, we identified biallelic variants in the CACNA1H
gene in two unrelated ASD families. CACNA1H encodes the
Cav 3.2 channel, belonging to the low-voltage-activated (LVA)
T-type Cav channels subfamily, that is widely expressed in
mammalian tissues, including brain where is involved in
the regulation of neuronal firing (14). CACNA1H missense
variants have been previously identified in ASD individuals
and implicated in the ASD phenotype (15). Thus, we used
heterologous expression of mutated Cav 3.2 channels in
mammalian cells to perform electrophysiological analysis of the
identified CACNA1H variants, to functionally characterize their
impact and to assess if the ASD phenotype could be explained by
the combined effect of the two mutations in the gene.
Whole Genome Sequencing Analysis
Whole genome sequencing (WGS) was performed at New
York Genome Center. Quality controls, alignment and variant
calling were carried out according to the pipeline developed
by the Center for Common Disease Genomics project (https://
github.com/CCDG/Pipeline-Standardization/blob/master/
PipelineStandard.md).
Variant annotation was performed with ANNOVAR, using
RefSeq for gene-based annotation (Genome Build hg38).
Annotated variants were filtered in order to retain only coding
and splicing variants, and to remove low-quality variants
[Coverage (DP) < 10 and Genome Quality (GQ) < 20]. To
select rare variants, a minor allele frequency (MAF) threshold ≤
1% in Genome Aggregation Database (gnomAD, https://gnomad.
broadinstitute.org/) was chosen. Specifically, population allele
frequencies for variants were obtained from the non-neurological
subset of gnomAD v.2.1 and the entire data set of gnomAD v.3.0,
which represents a large collection of individuals of different
ancestry. Variants were further filtered according to their exonic
function, excluding synonymous variants and highlighting
deleterious variants, including Likely Gene Disrupting (LGD)
and damaging missense variants. LGD variants consist of stopgain, stop-loss, frameshift and splicing variants, while damaging
missense variants were defined according to CADD score (16),
assuming CADD score value ≥ 15 as damaging threshold.
To identify variants acting under a recessive inheritance
model, homozygous, hemizygous and compound heterozygous
variants in probands were selected.
Ultra-rare variants were obtained by further filtering variants
according to their MAF in the same previously used data sets, but
retaining only variants having MAF ≤0.1%.
Genes previously associated with ASD were defined using
the SFARI Gene database and its scoring system, including
four categories: S (syndromic), 1 (high confidence), 2 (strong
candidate) and 3 (suggestive evidence) (https://gene.sfari.org/,
Release: 2021 Q3).
MATERIALS AND METHODS
Cohort
Our cohort consists of 105 families with ASD, recruited at
the UOSI Disturbi dello Spettro Autistico, IRCCS Istituto delle
Scienze Neurologiche (Bologna, Italy).
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Functional Analysis of ASD Variants on
Cav 3.2 Protein Activity
cover slides (8 x 104 cells/ml) and, after 24 h, transiently
transfected using Lipofectamine 3000 (Invitrogen) according to
the manufacturer’s protocol, and adding 0.5 µg of plasmid
DNA subtype per well. Recordings were carried out 24–36 h
following transfection. Electrophysiological experiments were
performed using the whole-cell configuration of the patchclamp technique. Recordings were obtained using a HEKA
EPC800 amplifier, Digidata 1322A analog-to-digital converter,
and pClamp 10 software (Molecular Devices, Union City, CA).
Data were filtered at 2 kHz and digitized at 5 kHz. Normal
external solution contained: 140 mM NaCl, 2.8 mM KCl, 2 mM
MgCl2 , 2 mM CaCl2 , 10 mM HEPES, and 10 mM glucose (pH
7.4; 300 mosM). The internal pipette solution contained: 140 mM
CsCl, 5 mM BAPTA, 2 mM Mg-ATP, and 10 mM HEPES (pH
7.4; 300 mOsm). Borosilicate glass pipettes were pulled with a
Narishige puller to a typical pipette resistance of 3–4 MΩ. Cell
capacitance was measured for each cell and access resistance
compensated to 70%.
Generation of WT and Mutant Plasmid Constructs
Recombinant WT protein expression construct for CACNA1H
was generated by cloning the coding sequence (NM_021098.3),
in frame with the 3xFLAG epitope, in the p3xFLAG-CMV-10
mammalian expression vector (Sigma-Aldrich). CACNA1H
coding sequence was subcloned into p3xFLAG-CMV-10
from a1Ha-pcDNA3 plasmid (Addgene plasmid #45809;
http://n2t.net/addgene:45809; RRID:Addgene_45809) (17).
WGS identified mutations [p.(Lys785Met), p.(Pro849Ser),
p.(Pro2124Leu), p.(Ser2338Phe)] were introduced to the
WT CACNA1H coding sequence by multi-step site-directed
mutagenesis, performing whole-plasmid PCR reactions (primers
are reported in Supplementary Materials) with Herculase II
Fusion DNA Polymerase (Agilent Technologies, Inc.). Sanger
sequencing was performed to check both WT and mutant
coding sequences cloned (BigDye Terminator Cycle Sequencing
kit-ThermoFisher Scientific).
Data Acquisition and Analysis
The current-voltage protocol stepped the cell membrane
potential from −120 mV to test potentials starting at −110 mV
and increasing to 20 mV in 10 mV increments. Test potentials
were 100 ms in duration, and the membrane potential was
returned to −120 mV for 10 s between acquisitions to allow
complete recovery from inactivation. Peak inward Ca2+ currents
were plotted as a function of the test potential to generate
current-voltage relations (I-V). The peak currents were also
normalized by the individual cell capacitance measurement for
the comparison of current densities. Mean current densityV relations were fit with a modified form of the Boltzmann
equation, where Ipeak = (V – Erev) Gmax (1/1 + exp (Vh –
V)/S)), and Erev is the reversal potential, Vh is the half-activation
potential, Gmax is the maximum slope conductance, and S is the
slope factor that is inversely proportional to the effective gating
charge. To assess the voltage dependence of inactivation, the cell
membrane was stepped from a holding potential of −120 mV
to conditioning potentials 1 s in duration between −120 and
−60 mV in 10 mV increments before proceeding to a test
potential of −40 mV for 100 ms, from which the resulting inward
Ca2+ currents were analyzed. The voltage of half-inactivation
(Vi) was estimated from Boltzmann fits of I/Imax vs. voltage
where I/Imax = 1/(1 + exp (z∗ (V – Vi)/25.6)). Clampfit 10 was
used to analyse all data obtained in Clampex (Molecular Devices).
Current kinetics were evaluated at −40 mV test potential by
measuring the time from basal to peak current, and by fitting the
current decay with a single exponential equation. Fits of the IV relations, activation and inactivation curves, and decays were
carried out in SigmaPlot (Jandel Scientific). Data are presented as
the means ± S.E. Statistical tests was done with one-way analysis
of variance (ANOVA).
Transient Transfection and Immunofluorescence (IF)
Assay
Human Embryonic Kidney-293T (HEK-293T) cells were grown
on collagen coated glass coverslips in 6-well plate in Dulbecco’s
Modified Eagle’s Medium (DMEM) high glucose (Sigma-Aldrich)
supplemented with 10% fetal bovine serum and 0.05 mg/ml
penicillin-streptomycin (DMEM-complete medium), at 37◦ C in
a 5% CO2 humidified atmosphere.
Cells were transiently transfected with 3 µg of p3xFLAGCACNA1H plasmid constructs using 0.3 µl of PolyEthylenImine
(PEI) per 1 µg of plasmid DNA in DMEM + L-glutamine (1mM).
Ninety minutes after transfection, serum deprived medium
was replaced with DMEM-complete medium and cells were
maintained at 37◦ C in a 5% CO2 humidified atmosphere. GFPcoding plasmid was used as positive transfection control, while
a p3xFLAG-CMV-10 empty vector and a 3xFLAG plasmid
encoding the 3xFLAG-ABCC3 transmembrane fusion protein
were used as IF negative and positive control respectively.
Forty-eight hours after transfection, cells were fixed for 15’
with 4% paraformaldehyde in PBS, washed three times in PBS
and incubated with blocking solution (4% normal donkey serum
and 0.05% tween in PBS) for 30’ at room temperature (RT).
Then, cells were stained with mouse anti-FLAG M2 antibody
(1:200; Sigma-Aldrich) for 1.5 h at RT. After three 0.05% tweenPBS washing step, cells were incubated with goat anti-mouse
Cy3 antibody (1:400, Jackson ImmunoResearch) for 1 hour at
RT. Samples were washed with PBS and nuclei were stained
with 1 µg/ml Hoechst (Sigma-Aldrich). Coverslips were finally
mounted on glass slides (glycerol PBS 9:1, pH = 8.5–9.0)
and image acquisitions were taken by Nikon 90i wide-field
fluorescence microscope. RAW images were processed into TIF
files using ImageJ open-source software.
RESULTS
Electrophysiological Experiments
Electrophysiology
Identification of CACNA1H Biallelic
Variants From WGS
HEK-293 cells were grown in DMEM supplemented with 10%
heat-inactivated FBS and 1% penicillin-streptomycin, at 37◦ C
in a 5% CO2 humidified atmosphere. Cells were plated on
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We performed WGS analysis in a cohort of 105 ASD families,
including 124 ASD individuals, 210 parents and 58 unaffected
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CACNA1H Variants in ASD Susceptibility
of proline 849 with a serine (NP_066921.2:p.Pro849Ser)
(Figure 1B). The two non-synonymous variants identified
in the twins (22.3 and 22.4) are located in the last exon
of the gene (exon 35), causing respectively the amino
acid change Pro2124Leu and Ser2338Phe (NP_066921.2)
in the cytoplasmic C-terminal region of the protein
(Figure 1B). The biallelic condition was not shared with
the unaffected sister (22.5), who inherited only the paternal
variant (Figure 1A).
In both families, no clear pathogenic sequence variants were
identified from WGS analysis. Among ultra-rare (MAF ≤ 0.1%)
variants of uncertain significance (VUS) emerged from WGS
analysis in the two families, the most interesting variants were
one de novo missense variant in PRSS2 and 9 inherited missense
variants predicted to be damaging in SFARI genes (SFARI score 2
and 3) in proband 105.3, and one de novo novel missense variant
in ARFGEF3 and 20 inherited ultra-rare potentially deleterious
variants (2 LGD and 18 damaging missense variants) in SFARI
genes (with SFARI score 1, 2 and 3) in probands 22.3 and 22.4
(Supplementary Table 2).
Additional heterozygous deleterious ultra-rare variants
in CACNA1H were identified in other 9 ASD individuals of
our cohort, one of them was also shared with an unaffected
sister (Supplementary Table 1). No biallelic variants in
CACNA1H were detected in parents or unaffected siblings of
our sample.
siblings. Within WGS data, we explored the presence of rare
coding damaging variants in voltage-gated calcium channels
(VGCCs) genes, to investigate their role in ASD development
in the affected individuals of our cohort. Specifically, we
looked for damaging variants with MAF ≤ 0.1% (ultra-rare
variants) that could potentially act under a dominant model of
inheritance and variants meeting a less stringent threshold of
MAF ≤ 1% (rare variants), possibly acting under a recessive
inheritance model (homozygous, hemizygous and compound
heterozygous variants).
We identified 53 ultra-rare damaging variants in 17 VGCCs
genes in 41 ASD individuals (Supplementary Table 1). All
these variants were inherited from unaffected parents and
no de novo variants were identified. Among recessive-acting
variants, 2 hemizygous variants in CACNA1F and 4 compound
heterozygous variants in CACNA1H emerged from our analysis
(Table 1). The presence of recessive-acting mutations in two
VGCCs genes led us to evaluate the hypothesis that recessive
model could be a shared mechanism of action of variants
affecting voltage-gated calcium channels. Given the classification
of CACNA1H as a strong ASD candidate gene in the SFARI
Gene database (SFARI score = 2, https://gene.sfari.org/database/
human-gene/CACNA1H), we decided to further investigate the
role of biallelic variants in this gene.
Compound heterozygous missense variants in CACNA1H
were present in 3 ASD individuals belonging to 2 unrelated ASD
families. In particular, two CACNA1H SNVs, one inherited from
the father and one inherited from the mother, were identified
in two female monozygotic ASD twins (indicated as proband
22.3 and proband 22.4), and other two CACNA1H variants, one
paternally and one maternally inherited, were identified in the
male proband of a trio (indicated as proband 105.3) (Figure 1A).
All identified variants were rare coding non-synonymous
variants predicted to be damaging for the normal protein
function (CADD score ≥15) (16) (Table 1). Proband 105.3
paternal and maternal variants are located within CACNA1H
exons 10 and 11, respectively. The maternal substitution
A > T causes the substitution of a lysine with methionine
(NP_066921.2:p.Lys785Met) in the cytoplasmic loop linking
protein domains I and II; the paternal change C > T
affects the last amino acid of the transmembrane segment
2 of the protein domain II, leading to the substitution
Clinical Characterization of ASD
Individuals Carrying CACNA1H Biallelic
Variants
The two probands of family 22 are 9.9-year-old monozygotic
female twins, born from consanguineous parents coming from
Bangladesh. Family history is positive for ASD in one maternal
first cousin. Twins were born at 33 weeks of pregnancy through
cesarean delivery due to premature rupture of membrane.
They were hospitalized due to prematurity, respiratory distress,
hyperbilirubinemia, and feeding problems. Motor and language
development were delayed for both. They showed earlyonset atypical socio-communicative skills, restricted/repetitive
interests and activities, and sensory abnormalities. Both twins
were diagnosed with ASD at the age of 40 months using ADOS2 [severity level 3, according to DSM-5 (2)]. Twin 22.3 had 4
TABLE 1 | Rare recessive-acting damaging variants in VGCCs genes identified in our WGS data set.
Proband ID (sex) Gene (SFARI score)
Genomic change (hg38)
Amino acid change
105.3 (M)
NC_000016.10:g.1204361A>T
NP_066921.2: p.(Lys785Met)
24.7
NC_000016.10:g.1205207C>T
NP_066921.2: p.(Pro849Ser)
18.22
NC_000016.10:g.1220303C>T
NP_066921.2: p.(Pro2124Leu)
NC_000016.10:g.1220945C>T
22.3-22.4 (F-F)
CACNA1H (2)
CACNA1H (2)
CADD Inheritance
score
dbSNP
Total MAF
(gnomAD
v.3.0)
Maternal
rs28365117
0.00300000
Paternal
rs370675810
0.00009770
19.03
Maternal
rs372453886
0.00006978
NP_066921.2: p.(Ser2338Phe)
16.99
Paternal
rs757713867
0.00002792
5.3 (M)
CACNA1F (3)
NC_000023.11:g.49211360C>T
NP_005174.2: p.(Ala1419Thr)
18.34
Maternal
rs782741094
0.00020000
112.3 (M)
CACNA1F (3)
NC_000023.11:g.49211983C>T
NP_005174.2: p.(Gly1350Ser)
24.7
Maternal
rs782780521
0.00002839
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FIGURE 1 | Family 22 and 105 CACNA1H variants. (A) Family segregations and gene location of CACNA1H biallelic variants. Filled shapes indicate ASD individuals.
*DNA was not available for individual 105.4. UCSC hg38 Genome Browser screenshot shows the location of biallelic variants within the CACNA1H gene. (B)
Schematic of the CACNA1H protein channel (Cav 3.2). Cav 3.2 channel consists of the single α1 pore-forming subunit of about 260 kDa, organized in four homologous
domain each composed of six transmembrane segments (S1–S6). Within each domain, the arginine/lysine-rich S4 segment represents the voltage-sensing region of
the channel, while the extracellular loop linking S5 and S6 segments (P loop) ensures the ion conductivity and selectivity of the channel (14, 18). Protein visualization
was generated using Protter–visualize proteoforms (19).
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FIGURE 2 | Immunofluorescence (IF) assay of 3xFLAG-Cav 3.2. IF assay was performed in HEK-293T cells, by transiently transfecting cells with p3xFLAG-CACNA1H
plasmid constructs. An empty vector (EV) was used as negative control, while 3xFLAG-ABCC3 transmembrane fusion protein was used as positive control. Mouse
anti-FLAG M2 antibody was used for detection of the recombinant proteins. Goat anti-mouse Cy3 antibody (red signal) and Hoechst dye (blue signal) were employed
to detect anti-FLAG antibody and nuclei respectively.
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FIGURE 3 | Lys785Met is a gain of function Cav 3.2 subtype. (A) Representative current traces recorded from two HEK-293 cells transiently transfected with the WT
(left) or Lys785Met (right) CACNA1H plasmid construct. The currents were elicited by step depolarizations from a holding potential of −120 mV to various test
potentials. (B) Representative current traces recorded from three HEK-293 cells transiently transfected with the Pro2124Leu (left) or Ser2338Phe (middle) or
(Continued)
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FIGURE 3 | Pro849Ser (right) plasmid construct, same protocol as (A). (C) Mean activation curves for WT, Pro2124Leu and Ser2338Phe plasmid constructs
transfected cells, as indicated. Solid lines represent data fit to the Boltzmann equation (Vh values are −45.5, −49.3 and −43.4 mV for WT, Pro2124Leu and
Ser2338Phe, respectively). Data were averaged from 16, 19 and 17 cells, for WT, Pro2124Leu and Ser2338Phe, respectively. (D) Mean activation curves for WT
(same data as C), Pro849Ser and Lys785Met plasmid constructs transfected cells, as indicated. Solid lines represent data fit to the Boltzmann equation (Vh values are
−45.5, −49.5 and −50.1 mV for WT, Pro849Ser and Lys785Met, respectively). Data were averaged from 16 (same cells as C), 16 and 14 cells, for WT, Pro849Ser
and Lys785Met, respectively. (E) Normalized mean activation curves for WT, Pro2124Leu and Ser2338Phe plasmid constructs transfected cells, same data as (C).
Solid lines represent data fit to the activation Boltzmann equation. (F) Normalized mean activation curves for WT, Pro849Ser and Lys785Met plasmid constructs
transfected cells, same data as (D). Solid lines represent data fit to the activation Boltzmann equation. (G) Inactivation open probability-voltage relationships for WT,
Pro2124Leu and Ser2338Phe plasmid constructs transfected cells. Solid lines represent data fit to the inactivation Boltzmann equation (Vi values are −78.9, −79.5
and −78.2 for WT, Pro2124Leu and Ser2338Phe, respectively). No significant differences were detected. (H) Inactivation open probability-voltage relationships for WT,
Pro849Ser and Lys785Met plasmid constructs transfected cells. Solid lines represent data fit to the inactivation Boltzmann equation (Vi values are −78.9, −75.3 and
−77.1 mV for WT, Pro849Ser and Lys785Met, respectively). No significant differences were detected.
cellular localization among them and compared to the positive
control (Figure 2).
To highlight possible functional effects due to identified
SNVs, electrophysiological experiments were performed using
the whole-cell patch-clamp technique. Plasmid encoding mutant
3xFLAG-Cav 3.2, along with plasmid encoding the WT isoform,
were transfected in HEK-293 cells, and the resulting voltageactivated Ca2+ currents were recorded and analyzed. Cells
transfected with plasmid DNA coding for the mutant proteins
exhibited inward currents in response to depolarization steps,
similarly to WT protein (Figures 3A,B). Loss of function was
not observed in any mutant channel, while current densities
(Figures 3C,D) and activation properties (Figures 3E,F) were
differently modulated by distinct mutations. Specifically, cells
transfected with plasmid DNA encoding the Lys785Met-mutant
Cav 3.2 exhibited a larger mean current density (Figures 3D,
4A), and a left shift in the activation I-V curves, with a more
hyperpolarized Vh value of −50 ± 1 mV, compared to the WT
Vh value of −45 ± 2 mV (Figure 4B; p = 0.037). The inactivation
properties were unaffected by the mutations (Figures 3G,H).
The kinetics of inward currents, elicited by a −40 mV
depolarization step, were differently affected by the mutations:
in comparison with the WT values, currents mediated by the
Ser2338Phe, Pro849Ser and Lys785Met mutant channels showed
a slower time to peak (Figure 4C), and those mediated by the
Pro849Ser isoform also decayed more rapidly (Figure 4D). To
evaluate the impact of these altered parameters on the channel
function, we measured the total charge transfer (i.e., the total
amount of Ca2+ ions entering the cells) induced by a −40 mV
depolarization step, at different time points (2, 10 and 100 ms):
a significant increase was observed at all time points only in
cells transfected with the Lys785Met -mutant Cav 3.2 expression
plasmid (Figure 4E).
febrile convulsions (from 18 months to 5 years of age), followed
by two apparently generalized convulsive seizures without fever
(the last one was a status epilepticus). EEG showed focal (right
mid-posterior) and diffuse paroxysmal abnormalities. Seizures
remitted with topiramate treatment. Twin 22.4 had no seizures
and her EEG was normal. Intellectual disability was present in
both twins: severe for twin 22.3 and moderate for twin 22.4.
Neurological examination showed lack of speech and stereotypies
in both. Array-CGH showed no pathogenetic copy number
variants. Brain MRI (1,5 Tesla) was normal for both twins.
The proband of family 105 is a 8.5-year-old boy, born
from non-consanguineous Italian parents. Family history is
positive for learning disability in one paternal first cousin.
Pregnancy, delivery and neonatal period were normal. Motor
development milestones were acquired regularly, while language
was delayed. At 1 year of age, social communication deficits
and restricted/repetitive interests and activities as well as sensory
abnormalities became evident. At 40 months of age, he was
diagnosed with ASD through ADOS-2 [severity level 3, according
to DSM-5 (2)]. A moderate intellectual disability was associated.
Neurological examination showed speech delay and stereotypies
with upper and lower arms. Array-CGH showed no pathogenic
copy number variants. Molecular analysis for fragile X syndrome
was negative. EEG showed frequent multifocal paroxysmal
abnormalities (spike-waves) in the left central and in the right
centro-temporal regions, slightly increased in the early stages
of sleep. The boy never presented epileptic seizures. Brain MRI
(1,5 Tesla) was normal. Additional clinical data are reported in
Supplementary Table 3.
Functional Characterization of CACNA1H
Biallelic Variants
In order to perform functional analysis to clarify the effect
of the CACNA1H variants on the calcium channel activity,
we generated 3xFLAG-Cav 3.2 wild-type (WT) and 4 3xFLAGCav 3.2 mutant recombinant proteins, corresponding to paternal
and maternal mutations identified in families 22 and 105.
Recombinant proteins expression in mammalian cells and
their cellular localization were assessed by immunofluorescence
(IF) assay in HEK-293T cells. As illustrated in Figure 2,
by merging Hoechst and Cy3 signals, indicating nuclei and
recombinant proteins respectively, WT and mutant recombinant
proteins were expressed in our cell system and correctly localize
at the cell membrane, with no considerable differences in
Frontiers in Psychiatry | www.frontiersin.org
DISCUSSION
Voltage-gated calcium channels (VGCCs or Cav channels) are
transmembrane protein mediating calcium ions influx into
excitable cells upon depolarization of the cell membrane. The
family of VGCCs includes distinct types of channels, differing
in electrophysiological properties. High-voltage-activated (HVA)
calcium channels consist of Cav 1 and Cav 2 subfamilies, while
low-voltage-activated (LVA) calcium channels consist of Cav 3
subfamily exclusively. The principal functional subunit of Cav
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CACNA1H Variants in ASD Susceptibility
FIGURE 4 | Activation and kinetic parameters of Lys785Met, Ser2338Phe and Pro849Ser Cav 3.2 subtypes are different from WT. (A) Histogram representing the
mean conductance density of WT and mutant channels expressed in HEK-293 cells, measured at −40 mV test potential for each Cav 3.2 subtype, as indicated. Mean
values were averaged from 16, 15, 13, 12 and 10 cells, from left to right. The conductance density was significantly higher for Lys785Met mutant, as compared to WT
(a, p < 0.001). (B) Histogram representing the mean Vh value measured at −40 mV test potential for each Cav 3.2 subtype, as indicated. Same cells as (A). The Vh
value was significantly higher for Lys785Met mutant, as compared to WT (b, p = 0.037). (C) Left, histogram representing the mean values of time to peak measured at
−40 mV test potential for each Cav 3.2 subtype, as indicated. Mean values were averaged from 10, 13, 10, 18 and 15 cells, from left to right. The time to peak was
(Continued)
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FIGURE 4 | significantly higher for Ser2338Phe, Pro849Ser and Lys785Met mutants, as compared to WT (c, p = 0.002; d, p = 0.002; e, p = 0.003). (D) Histogram
representing the mean values of exponential τ decay measured at −40 mV test potential for each Cav 3.2 subtype, as indicated. Same cells as (C). The time to peak
was significantly higher for Pro849Ser mutant, as compared to WT (f, p = 0.039). (E) Histograms representing the mean values of charge transfer measured at
−40 mV test potential for each Cav 3.2 subtype, as indicated, at different current times (left, 2 ms; center, 10 ms; right, 100 ms). Same cells as (C). The charge transfer
value was significantly higher only for Lys785Met mutant, at each time point, as compared to WT (g, p = 0.004; h, p = 0.010; i, p < 0.001).
The CACNA1H gene encodes the LVA T-type calcium channel
Cav 3.2, that is widely distributed in excitable cells, including
brain where it is highly expressed in thalamus, hippocampus,
amygdala and putamen. Cav 3.2 regulates intracellular calcium
concentration, playing important roles in neuronal firing and in
neurotransmitter release (14, 30, 31).
CACNA1H is reported to be a strong candidate for
ASD as both de novo and inherited rare variants in
CACNA1H were identified in individuals with ASD and
other neurodevelopmental disorders (15, 32–40), and functional
analysis showed a significant effect of CACNA1H variants on
channel function. Specifically, Splawski et al. tested five missense
variants identified in a sample of 461 ASD individuals, and
detected a decreased activity in mutant channels, implicating
CACNA1H missense variants in ASD risk with loss-of-function
mechanism and incomplete penetrance (15).
We identified biallelic variants in the CACNA1H gene in
3 ASD individuals (2 monozygotic twins and an unrelated
proband) from two families. Probands 22.3-22.4 biallelic variants
(Ser2338Phe and Pro2124Leu) affect the cytoplasmic C-terminal
domain of the channel, not directly implicated in ion transport
and channel functionality but with a putative regulatory role
by interacting with Sintaxin-1A (41); proband 105.3 variants
(Pro849Ser and Lys785Met) are located in protein regions
expected to have a greater effect on the T-type calcium channel
function. Indeed, several variants affecting the same channel
region were previously identified and functionally tested, with
some of them showing a functional effect (40). Specifically,
proband 105.3 paternal Pro849Ser variant is located in the S2
segment of the second protein domain, in which two gain
of function mutations were already identified in individuals
with Childhood Absence Epilepsy (42, 43). Moreover, the
maternal Lys785Met mutation is located in the cytosolic III loop, that is an important regulator of channel function,
contributing to the regulation of its gating properties and surface
expression (44, 45). Several variants were identified in this
channel region in individuals affected by idiopathic generalized
epilepsy, neuromuscular disorder and chronic pain, with most
of them occurring in the region surrounding Lys785Met variant,
some of which showing gain of function effect (40).
We functionally characterized these four CACNA1H variants
to clarify their effect on the Cav 3.2 channel activity. No decrease
in the voltage-gated Ca2+ conductance was registered for any
of the tested variants, thus excluding loss of function effects.
In contrast, a clear gain of function effect was observed for the
Lys785Met mutant channel, which exhibited a higher functional
expression in HEK293 cells, along with a significantly more
hyperpolarized Vh value, indicating a higher open probability
than WT at the same potential. These properties conferred the
channel is the pore-forming α1 subunit, encoded by the
CACNA1A to CACNA1I and CACNA1S genes. A single α1
subunit constitutes the functional form of LVA or Cav 3 channels,
while HVA (Cav 1 and Cav 2) channels require the presence of
α2 δ and β auxiliary subunits, encoded by the four CACNA2D1-4
genes and the four CACNB1-4 genes respectively. The α1 subunit
co-assemblies with one of four α2 δ and one of four β subunits,
forming HVA multiprotein functional complex (20, 21). Even if
the main properties of the channel are determined by the poreforming unit both in LVA and HVA channels, in the latter group
biophysical properties and the pore-forming unit targeting at
the cell membrane are profoundly modulated by the auxiliary
subunits (13, 22–24).
By mediating Ca2+ entry, VGCCs are involved in multiple
processes critical for cellular function, thus their dysfunction
is associated with a wide range of different diseases, including
neuropsychiatric disorders. VGCCs have been consistently
implicated in schizophrenia, ADHD, ASD, epilepsy, bipolar
disorder, anxiety and major depressive disorder (MDD),
implicating Cav channels altered function in dysregulation of
calcium signaling, postsynaptic function, synaptic plasticity and
gene transcription (10, 21, 25, 26). Specifically, several studies
investigated the role of both rare and common variants in the α1
and the auxiliary subunits of VGCCs genes in ASD, highlighting
the implication of voltage-dependent calcium channels in the
disease susceptibility (12). Moreover, 13 out of 18 VGCCs genes
are included in the SFARI Gene database (https://gene.sfari.
org/), a curated list of genes implicated in ASD susceptibility.
Specifically, 4 VGCCs genes (CACNA1A, CACNA1C,
CACNA1E and CACNA2D3) are reported to be clearly
implicated in ASD (SFARI 1), while other 3 genes (CACNA1D,
CACNA1H and CACNB2) are defined as strong ASD candidate
genes (SFARI 2).
In order to investigate the role of Cav channels in ASD
etiology, we looked for rare coding damaging variants in VGCCs
genes in WGS data of 105 ASD families, which include 124 ASD
individuals. We identified 53 ultra-rare damaging variants in
VGCCs genes, none of them was de novo. Interestingly, about
one third of ASD individuals of this cohort (41 out of 124)
had a deleterious variant in at least one VGCCs gene, with 10
ASD individuals carrying deleterious variants in two or more
VGCCs genes. Moreover, four rare damaging biallelic variants
were detected in the CACNA1H gene in two ASD families.
No biallelic variants in CACNA1H have been previously
reported in individuals with ASD. However, CACNA1H
compound heterozygous variants have been previously identified
in neuromuscular disorders, for which functional analysis
showed mild but significant changes on T-type channel activity
that are consistent with a loss of channel function (27–29).
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CACNA1H Variants in ASD Susceptibility
individual(s), and minor(s)’ legal guardian/next of kin, for the
publication of any potentially identifiable images or data included
in this article.
ability to transfer, upon activation, a larger electrical charge
to the Lys785Met mutant channel when compared to WT,
suggesting an increased Ca2+ entry in neurons expressing this
mutant channel. A detailed kinetic analysis revealed that three
out of four mutants (Ser2338Phe, Pro849Ser and Lys785Met)
exhibited altered activation kinetics. Therefore, in family 22
only one CACNA1H variant caused mild alterations in channel
properties, while both variants in family 105 showed a functional
effect, even if with variable intensity and acting on different
channel properties. It is thus likely that the identified variants
in CACNA1H may lead to subtle dysregulation of intracellular
Ca2+ concentration, thereby altering neuronal cell signaling and
gene transcription. Our results suggest that CACNA1H variants
influence the ASD phenotype with incomplete penetrance,
driving individual susceptibility over the ASD threshold, together
with other risk variants. Indeed, in both families the probands
inherited additional rare damaging variants in other highconfidence ASD genes. Since only one of the twins of family
22 had fever triggered epilepsy, we might hypothesize that
CACNA1H variants could also contribute to epilepsy, but with
variable penetrance.
In conclusion, in the present study we investigated the
involvement of VGCCs rare variants in ASD susceptibility.
This led to the identification of CACNA1H biallelic mutations
in probands from two families. Functional characterization by
patch-clamp revealed that three out of four tested variants
cause mild alterations of the Cav 3.2 channel properties. Our
results provide additional support to previous studies implicating
CACNA1H in neurodevelopmental disorders and suggest that
CACNA1H mutations may be involved in the pathophysiology
of ASD.
AUTHOR CONTRIBUTIONS
MV participated to study design, performed WGS data
bioinformatic analysis, performed cloning experiments,
immunofluorescence assay, wrote the manuscript, and prepared
figures and tables. TD’A performed electrophysiological analyses
and contributed to figures preparation. CC participated to study
design and contributed to WGS data bioinformatic analysis. AP,
PV, MS, RC, and MR collected the samples and performed the
clinical characterization of patients. FC participated to study
design and contributed to manuscript writing. GM designed
and supervised cloning experiments and immunofluorescence
assay. SF supervised electrophysiological, data analyses and
contributed to manuscript writing. EM participated to study
design, supervised all analyses and contributed to manuscript
writing. EB participated to study design, performed WGS data
bioinformatic analysis, supervised all analyses and contributed
to manuscript writing. All authors read and approved the
final manuscript.
FUNDING
This research was funded by Italian Ministry of Health,
grant number GR-2013-02357561, and by RFO (University of
Bologna). WGS data were generated at the New York Genome
Center with funds provided by NHGRI Grant 3UM1HG008901.
The Centers for Common Disease Genomics are funded by the
National Human Genome Research Institute and the National
Heart, Lung, and Blood Institute.
DATA AVAILABILITY STATEMENT
The datasets that support the findings of this study can be found
in the Supplementary Material of this article. Requests to access
the raw data should be directed to elena.bacchelli@unibo.it.
ACKNOWLEDGMENTS
We gratefully acknowledge all the subjects who have participated
in the study. We also thank Dr. Sara De Fanti and Dr. Marco
Sazzini for providing Qubit Fluorometer device.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by Comitato Etico di Area Vasta Emilia Centro (CEAVEC); code CE 14060. Written informed consent to participate
in this study was provided by the participants’ legal guardian/next
of kin. Written informed consent was obtained from the
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fpsyt.
2022.858238/full#supplementary-material
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