A new homozygous CACNB2 mutation has functional relevance and supports a role for calcium channels in autism spectrum disorder

A new homozygous CACNB2 mutation has functional relevance and supports a role for calcium channels in autism spectrum disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research A new homozygous CACNB2 mutation has functional relevance and supports a role for calcium channels in autism spectrum disorder Claudio Graziano, Patrick Despang, Flavia Palombo, Giulia Severi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.18548/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jun, 2020 Read the published version in Journal of Autism and Developmental Disorders → Version 1 posted You are reading this latest preprint version Abstract Background Diagnostic yield in patients with autism spectrum disorder (ASD) has improved over the last years, thanks to the introduction of whole genome arrays and next generation sequencing, but etiology is still unknown for the majority of cases. Among distinct cellular pathways, evidence implicating dysregulation of cellular calcium homeostasis in ASD pathogenesis has been accumulating, and specific mutations in voltage-gated calcium channels found in patients with autism were shown to be functionally relevant. Methods Whole exome sequencing and Sanger sequencing were performed to identify and confirm variants in a girl with ASD, global developmental delay and precocious puberty, born of first-degree cousins. Site-directed mutagenesis was used to generate a human Ca V β 2d calcium channel subunit carrying a CACNB2 mutation. Whole-cell patch-clamp recordings were performed to reveal functional effects of mutant Ca V β 2d on Ba 2+ -currents mediated by L-type (Ca V 1.2) calcium channels in transiently transfected HEK-293 cells. Results In an ASD patient, we identified a rare homozygous variant (p.Arg70Cys) in the CACNB2 gene coding for the auxiliary Ca V β 2 subunit of voltage-gated calcium channels. In a recombinant system, the Ca V β 2 variant, which was not previously associated to ASD, was found to alter Ca V 1.2 calcium channel function by significantly affecting activation and inactivation of whole-cell Ba 2+ -currents. Limitations Although the evidence of CACNB2 involvement in ASD is slowly accumulating, the number of reported patients is very limited. Deep clinical phenotyping and functional studies in larger sets of subjects will be instrumental to fully understand the penetrance and outcome of CACNB2 variants. Conclusions The p.Arg70Cys variant in CACNB2 shows functional consequences similar to other ASD-associated Ca V β 2 mutations. These results support the idea of CACNB2 variations contributing to the development of ASD and hint to a rare form of Mendelian recessive autism with possible specific comorbidities. Cellular & Molecular Neuroscience Molecular Genetics Autism spectrum disorders Calcium channel genes CACNB2 Intellectual disability Precocious puberty Consanguinity Figures Figure 1 Figure 2 Background Autism spectrum disorder (ASD) is a complex neurodevelopmental condition affecting about 1 in 60 individuals [ 1 ]. It is defined by dysfunctions in social interaction and communication, stereotypic behaviors and sensory integration problems. ASD can be isolated and, in such cases, predominantly follows a polygenic pattern of inheritance caused by several weakly penetrant genetic variants that incrementally enhance susceptibility [ 2 , 3 ]. Common genetic variants have been implicated by genome-wide association studies, with the identification of polygenic risk scores [ 4 , 5 ]. On the other hand, ASD is a frequent feature of rare multisystemic neurodevelopmental disorders, where Intellectual Disability (ID) and epilepsy are common comorbidities [ 6 ]. A substantial subset of these patients show rare de novo copy number or single nucleotide variants (CNVs and SNVs) [ 7 – 9 ]. The contribution of recessive mutations has been less characterized and was addressed in a recent large-scale sequencing study, which demonstrated an excess of biallelic mutations justifying approximately 5% of ASD cases [ 10 ]. Monogenic “recessive” ASD is expected to be very rare but should be enriched in children of consanguineous marriages. The introduction of next generation sequencing technologies offers the opportunity to test large cohorts of ASD patients and the results are helping to delineate the pathophysiological pathways which lead to this disorder. Hundreds of genes have been implicated in the pathogenesis of ASD, but the impacted pathways often cluster into three major functional groups: chromatin structure, transcription factors and calcium signaling [ 11 ]. Voltage-gated calcium channels (VGCCs) are heteromultimeric protein complexes consisting of up to four different subunits: a pore-forming α1 subunit (Ca V 1.x – Ca V 3.x) and an auxiliary α 2 δ- (α 2 δ 1−4 ), β- (Ca V β 1−4 ) and in some tissues γ-subunit (γ 1−8 ) [ 12 , 13 ]. Among other functions, Ca V β subunits modulate the channel activation and inactivation [ 14 , 15 ]. The first calcium channel gene that was reported as mutated in a form of monogenic syndromic autism was CACNA1C , which is the cause of Timothy syndrome, characterized by arrhythmia, hand/foot anomalies and high prevalence of ASD [ 16 ]. Since then other VGCC subunits have been associated with ASD (reviewed in [ 17 ]). Concerning to Ca V β subunits, CACNB2 variants showed genome-wide suggestive significance in siblings with autism [ 18 ]. Furthermore, CACNB2 was found as a risk locus for five major psychiatric disorders including ASD [ 19 ]. We previously reported three rare heterozygous CACNB2 missense variants in patients with autism, altering the kinetic parameters of recombinant L-type calcium channels similarly to the CACNA1C mutations associated with Timothy syndrome [ 20 ]. Furthermore, an N-terminal de novo missense mutation (p.Val2Asp) in CACNB2 was recently discovered in a whole-genome sequencing study of ASD individuals and it was considered clinically relevant [ 21 ]. In the present work, we report the identification of a CACNB2 homozygous rare variant in a girl with ASD, global developmental delay and precocious puberty via whole exome sequencing (WES). Functional characterization by whole-cell patch-clamp revealed a decelerated inactivation behavior of L-type calcium channels (LTCCs) similar to the previously described CACNB2 mutations suggesting a common feature among ASD-associated Ca V β 2 mutations. Methods Clinical characteristics of the proband An 11-year-old girl received a diagnosis of global developmental delay and ASD at the age of four. She is the first child of consanguineous parents (first-degree cousins) from Bangladesh. She was born at term after an uneventful pregnancy. Birth weight was 3255g. Motor development was normal, she had normal growth and no facial dysmorphisms. She underwent a neuropsychiatric evaluation at the age of four for absent speech. The Denver Scale showed a severe developmental delay, mainly affecting the areas of language and socialization. She was administered ADOS (Autism Diagnostic Observational Schedule) and CARS (Childhood Autism Rating Scale) and met diagnostic criteria for ASD. Brain imaging and EEG were normal. She also underwent a cardiac evaluation with ECG recordings, which did not show abnormalities of heart rhythm; she had normal hearing and normal ophthalmic evaluation. She had sleep disturbances with frequent awakenings during the night and a severe constipation. Blood karyotype, FRAXA analysis, MECP2 sequencing and MLPA, chromosomal microarray, urine and plasma metabolic investigations, were normal. At the age of 6 years and 6 months, progression of breast development (B3 according to Tanner stages) and advanced bone age (8 years 10 months) were noticed. Pubertal response of LH (LH peak > 5 IU/L ) to standard GnRH test and increased uterine length ( 40 mm ) at ultrasound confirmed the diagnosis of central precocious puberty. MR imaging of hypothalamus-pituitary region was normal. Therapy with GnRH analogues was started at the age of seven years and two months, and was interrupted after three years. One year after interruption (at 11), she had menarche. At the last evaluation (11 years), speech was absent and the sleeping pattern was described as slightly more regular. She still showed relevant deficits in the areas of social interaction, communication, and range of interests and activities, confirming the diagnosis of ASD (severity level 3: "Requiring very substantial support”) in association with a severe ID (DSM-5 criteria). Parents do not show cognitive/behavioral problems and family history is unremarkable in this respect. Mother’s menarche was at 13 years. The father was a heavy smoker, had a gastric lymphoma at 34 years and an acute myocardial infarction at 37: coronary angiography showed an occlusion of the left circumflex artery and he was subjected to primary angioplasty and placement of a bare-metal stent; ECG-holter was performed during follow-up and it was normal. Whole Exome Sequencing Genomic DNA was extracted from peripheral blood with the QIAamp DNA Blood Mini (Qiagen, Venlo, Netherlands). Targeted capture and enrichment was performed using the Nextera Rapid Capture Exome kit (Illumina Inc., San Diego, CA) and library was sequenced as 100-bp paired-end reads on Illumina HiScanSQ (Illumina). Generated reads were treated following a general pipeline elsewhere described [22] including alignment with BWA [23] to the reference genome hg19, realignment and base quality score recalibration with GATK [24] and duplicate removal with Picard Tools (https://broadinstitute.github.io/picard). Alignment and coverage statistics were collected with SAMtools and GATK. Variants were called and filtered by quality with GATK HaplotypeCaller and Variant Quality Score Recalibration, and then annotated with Ensembl Variant Effect Predictor (www.ensembl.org/info/docs/tools/vep/index.html). H 3 M 2 [25] was used for the identification of ROHs from WES alignments. Candidate disease-causing variants were defined as variants with potential to alter the protein product (missense, nonsense, small insertion/deletions and splicing-affecting variants) with allele frequency lower than 0.01 and not seen in homozygous state in gnomAD database [26]. The selected variant in CACNB2 was confirmed by Sanger sequencing and tested in parents. DNA constructs and site-directed mutagenesis For functional analysis, the p.Arg70Cys variant was introduced in human Ca V β 2d (NM_201596.2) by site-directed mutagenesis (Stratagene QuikChange Kit) and verified by sequencing. EGFP was used as reporter gene, which was co-expressed together with the β 2 -subunit by the bicistronic pIRES2-EGFP vector (Clontech). Primer pairs for the mutagenesis were the following: p.Arg70Cys forward primer 5'-gccgaaccctggcaaacaaaactatttgaggtagtatca-3'; reverse primer 5'-tgatactacctcaaatagttttgtttgccagggttcggc-3'. Since the N-termini of Ca V β 2 vary in sequence and length among the different splice variants (Ca V β 2a-e ) and this has been shown to impact LTCC modulation [27,28], we used a Ca V β 2d backbone as in our previous study [20], thus comparing wild-type (WT) Ca V β 2d_WT and mutant Ca V β 2d_R70C . Cell culture and transfection HEK-293 cells were grown in Petri dishes in Dulbecco’s modified Eagle medium (Gibco Thermo Fisher, Waltham, MA, USA) supplemented with 10% FCS (Biochrom GmbH, Berlin Germany). Cells were routinely passaged twice a week and incubated at 37°C und 5% CO 2 growth conditions. HEK-293 cells were transfected with human calcium-channel subunits and EGFP using a standard calcium phosphate method [29]. For whole-cell recordings, HEK-293 cells were transfected with a 1 : 0.5 : 1.5 ratio of Ca V 1.2 (α1c 77 ) [30], either a WT or a mutant β 2d -subunit and an α 2 δ 1 -subunit [31]. Whole-cell patch-clamp recordings Whole-cell recordings of EGFP-positive cells were obtained 48–72 h after transfection. Immediately prior to recording, cells kept in 35-mm culture dishes were washed at room temperature (19–23°C) with bath solution. The bath solution contained (in mM) 10 BaCl 2 , 1 MgCl 2 , 10 HEPES, 65 CsCl, 40 TEA-Cl, 10 Glucose (pH 7.3 with TEA-OH) and the pipette solution (in mM) 140 CsCl, 10 EGTA, 9 HEPES, 1 MgCl 2 ,4 MgATP (pH 7.3 with CsOH). Patch pipettes made from borosilicate glass (1.7 mm diameter and 0.283 mm wall thickness, Hilgenberg GmbH, Malsfeld, Germany) were pulled using a Sutter Instrument P-97 horizontal puller and fire-polished using a Narishige MF-83 microforge (Narishige Scientific Instrument Lab, Tokyo, Japan). Pipette resistance was 3-5 MΩ. Currents were elicited by applying 500 ms long test potentials of -40 mV to +50 mV from a holding potential of -80 mV using Clampex software pClamp 10 and an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Currents were sampled at 10 kHz and filtered at 2 kHz. Data were analyzed using Clampfit10.3 (Molecular Devices, Sunnyvale, CA, USA) and GraphPad 6 Prism software. For voltage dependence of activation data were fitted by combined Ohm and Boltzmann relation according to Karmazinova and Lacinova [32]. To obtain the time-course of activation ( τ act ) a first order exponential function was used to fit to the current traces. Statistical analyses Data are shown as mean ± SEM and were analyzed using Student’s unpaired two-sided t-test. Differences were considered statistically significant if p < 0.05. Results The proband belonged to a cohort of 50 individuals, all children of consanguineous parents, affected by heterogeneous disorders. According to our local protocol for consanguinity, WES experiment was performed in the proband only. Mean coverage was 66.8 X with the 90.7% of the bases covered. No pathogenic/likely pathogenic variants remained after filtering, but a rare and potentially disruptive homozygous missense variant in CACNB2 (NM_201597.2:c.208C>T:p.Arg70Cys) was selected for further investigation. It was confirmed by Sanger sequencing and parents were found to be heterozygous carriers (Supplementary Figures 1a-b). It is a very rare variant (only three alleles were reported in the gnomAD database, none in the South Asian population). The pathogenic computational verdict is supportive, because of six pathogenic predictions from DANN, GERP, MutationTaster, PROVEAN, FATHMM-MKL and SIFT vs three benign predictions from FATHMM, LRT and MutationAssessor. Some CACNB2 heterozygous variants have been described in families with Brugada syndrome [33,34], while others were associated with ASD [20]. The present CACNB2 variant was deposited in ClinVar with identification number 545669, as a variant of unknown significance according to the American College of Medical Genetics (ACMG) guidelines [35]. Since our study design was a “proband only” WES, we could not evaluate the presence of de novo variants. However, no pathogenic/likely pathogenic heterozygous variants (according to ACMG guidelines) were identified in genes known to cause neurodevelopmental disorders. In order to investigate the effect of the p.Arg70Cys variant, we expressed human Ca V β 2d_WT or Ca V β 2d_R70C together with Ca V 1.2 and Ca V α 2 δ 1 in HEK-293 cells and performed whole-cell patch-clamp recordings. Exemplary traces of Ba 2+ -currents are shown in Figure 1a. Our analysis revealed no differences in current density (Figure 1b), but a significantly lower activation time constant ( τ act ) at various test potential in the presence of Ca V β 2d_R70C compared to Ca V β 2d_WT (Figure 2a). Furthermore, time-dependent inactivation at 150 ms was significantly reduced by Ca V β 2d_R70C (Figure 2b). Accordingly, the inactivation time constant ( τ inact ) was increased for Ca V β 2d­_R70C compared to Ca V β 2d­_WT (not shown). In summary, the p.Arg70Cys mutation shows functional consequences on Ca V 1.2-mediated Ba 2+ -currents similarly to other ASD-associated Ca V β 2 mutations as we described previously [20]. Discussion More than 100 genes and genomic regions have been associated with ASD [6], suggesting a genetic architecture with many genes involved, each accounting for a small fraction of cases. Several lines of evidence indicate calcium signaling as one of the most significant pathways for ASD [17,36,37] and mutations in at least one gene encoding a calcium channel ( CACNA1C ) have been linked to a syndrome with a high prevalence of ASD [16]. We provide further evidence that CACNB2 variation may be involved in the pathogenesis of ASD: a young girl with ASD, child of consanguineous parents, was found to carry a rare homozygous missense variant in CACNB2 , which was predicted pathogenic by software tools. We proved that this variant has a functional impact since it alters the kinetic parameters of currents carried by recombinant L-type calcium channels. In a recent study on recessive ASD, CACNB2 was among 409 distinct genes that harbored biallelic, damaging missense mutations in cases but not in controls. It is instructive to note that also in this study one single gene ( AMT ) was independently hit in multiple families, confirming how rare the involvement of each independent gene is [10]. Unfortunately, no additional clinical data were reported for the subject mutated in CACNB2 , therefore it was not possible to know if relevant comorbidities were present. Our female patient showed ID, which is the most common associated feature in ASD patients, and precocious puberty. An early timing of puberty is a specific feature of some disorders characterized by ID [38], such as imprinting disorders and chromosome defects [39,40]. Our proband did not have features evocative of an imprinting defect (such as IUGR and small stature) and chromosomal microarray excluded CNVs. Moreover, she had normal brain imaging and no familiarity for early onset of puberty (maternal age of menarche was 13 years). To our knowledge, no variants in calcium channels have ever been associated to precocious puberty. However, given that calcium signaling is involved in essential cellular functions that span multiple tissues and physiological systems, including the reproductive axis through gonadotropin-releasing hormone neurons [41], CACNB2 variants may indeed play a role in puberty progression. Only the identification of further patients carrying homozygous mutations in CACNB2 will give the opportunity to clarify this issue. Ca V β subunits are the only cytosolic subunit of high voltage-activated (HVA) VGCCs and serve, besides other functions, as main modulator of activation and inactivation of the channel [14,15,42]. They consist of variable N- and C-termini and conserved SH3- and GK-domains, separated by a variable HOOK-domain [43]. HOOK-domain and N-terminus have a major impact on voltage-dependent inactivation of HVA VGCCs [27,44–47]. The herein described p.Arg70Cys mutation significantly attenuates inactivation of LTCCs, similar to the previously described ASD-associated mutations p.Gly167Ser and p.Ser197Phe [20]. Given these data, it is tempting to speculate that ASD-associated mutations in Ca V β-subunits cause similar functional effects, i.e. enhanced calcium influx due to lowered channel inactivation, reminiscent of CACNA1C mutations in the Ca V 1.2 subunit of LTCCs causing Timothy syndrome [16]. Timothy syndrome is characterized by abnormalities of heart rhythm and a high co-occurrence of ASD. Some mutations were identified in patients presenting only with non-syndromic long QT syndrome [48], whereas no CACNA1C mutations were reported, to our knowledge, in patients with an isolated neurodevelopmental disorder. The dissimilarities in genotype-phenotype correlation for calcium-channel disorders might be determined by diverse factors, ranging from the impact of the mutation on channel activity (gain of function vs loss of function vs a dominant-negative effect), to the extensive alternative splicing, with many distinct transcripts exhibiting different biophysical properties and expression profiles. For instance, the classical Timothy syndrome phenotype results from the presence of the p.Gly406Arg pathogenic variant in exon 8A, an exon contained in a specific splice variant of CACNA1C found in approximately 20% of all cardiac mRNAs [16]. Two individuals with atypical Timothy syndrome had been reported with pathogenic variants (p.Gly402Ser and p.Gly406Arg) in exon 8 of an alternate splice form that represents 80% of all cardiac mRNAs [49]. Dominant mutations in CACNB2 have been identified in a small subset of individuals with Brugada syndrome, a familial cardiac arrhythmia [33,34]; none of the reported patients had autism or other neurodevelopmental disorders. Data from the Genotype-Tissue Expression (GTEx) project indicate that the p.Arg70Cys variant in exon 2a of CACNB2 is present in a transcript with higher expression in brain than in heart tissue, whereas the transcript with highest heart tissue expression does not include exon 2a (Supplementary Figure 1c). This expression pattern suggests a possible explanation for the absence of heart rhythm abnormalities in the present family. As the expansion of personalized medicine proceeds, with increasing potential for interpretation of genomic data, the early identification of molecular and genetic defects can lead to a better clinical refinement and the possibility of applying timely targeted therapies. Most importantly, it is crucial to carry out the functional characterization of proteins and mutations, to make progress from gene identification to function [50]. Therefore, developing models for specific mutations allows to better define the underlying molecular defects, exemplified in the present case by a significantly lower current activation time of a calcium channel. Limitations The major limit of this study is that the analysis was performed on a single subject. The evidence of CACNB2 involvement in ASD is slowly accumulating, but the number of reported patients is very limited. Although knowledge on recessive causes of ASD is lacking, they are expected to be rare, and the majority of large-scale studies evaluated polygenic inheritance or de novo variants. Deep clinical phenotyping and functional studies in larger sets of subjects will be instrumental to fully understand the penetrance and outcome of CACNB2 variants. Ba 2+ is not the physiological charge carrier trough L-type VGCCs, but it was used here for two main reasons: (1) Ca 2+ -dependent effects on channel gating, like calcium-dependent inactivation, might be difficult to discriminate from other Ca 2+ -independent effects, e.g. voltage-dependent inactivation; (2) using Ba 2+ allows for comparison with findings from our previous study [20]. Conclusions In conclusion, biallelic CACNB2 variants may define a rare form of recessive ASD, which is comorbid with Intellectual Disability and, possibly, precocious puberty. The electrophysiological phenotype is similar to that of other Ca V β 2 variants described earlier and thus suggests common features of ASD-associated Ca V β 2 mutations, reminiscent of Timothy syndrome mutations in CACNA1C . List of Abbreviations ADOS: autism diagnostic observational schedule ACMG: American College of Medical Genetics ASD: autism spectrum disorder CARS: childhood autism rating scale CNV: copy number variant HVA: high voltage-activated ID: intellectual disability LTCC: L-type calcium channel SNV: single nucleotide variant VGCC: voltage-gated calcium channel WES: whole exome sequencing Declarations Ethics approval and consent to participate Parents provided consent according to the IRB protocol 3206/2016 at Policlinico S. Orsola-Malpighi (Bologna, Italy). Consent for publication Consent to participate included consent for publication. Availability of data and materials The datasets analysed during the current study are available from the corresponding authors on reasonable request. Competing interests The authors declare no competing interests. Funding This work was partially supported by Telethon Grant n. GGP15171 to EB. Authors' contributions CG conceived and designed the study, analyzed the data, and wrote the paper. PD and JM performed the electrophysiological studies and contributed to the writing of the paper. FP, TP, FI performed the sequencing and bioinformatic analyses. GS, AP, AC provided the patient data. EB supervised the research and co-wrote the paper. All authors read and approved the final manuscript. Acknowledgements We thank the patient and her parents who contributed to the study. References Baio J, Wiggins L, Christensen DL, Maenner MJ, Daniels J, Warren Z, et al. Prevalence of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2014. Morb Mortal Wkly Rep Surveill Summ Wash DC 2002. 2018;67:1–23. de la Torre-Ubieta L, Won H, Stein JL, Geschwind DH. Advancing the understanding of autism disease mechanisms through genetics. Nat Med. 2016;22:345–61. Weiner DJ, Wigdor EM, Ripke S, Walters RK, Kosmicki JA, Grove J, et al. Polygenic transmission disequilibrium confirms that common and rare variation act additively to create risk for autism spectrum disorders. Nat Genet. 2017;49:978–85. Gaugler T, Klei L, Sanders SJ, Bodea CA, Goldberg AP, Lee AB, et al. Most genetic risk for autism resides with common variation. Nat Genet. 2014;46:881–5. Grove J, Ripke S, Als TD, Mattheisen M, Walters RK, Won H, et al. Identification of common genetic risk variants for autism spectrum disorder. Nat Genet. 2019;51:431–44. Betancur C. Etiological heterogeneity in autism spectrum disorders: more than 100 genetic and genomic disorders and still counting. Brain Res. 2011;1380:42–77. Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, et al. Strong association of de novo copy number mutations with autism. Science. 2007;316:445–9. Pinto D, Pagnamenta AT, Klei L, Anney R, Merico D, Regan R, et al. Functional impact of global rare copy number variation in autism spectrum disorders. Nature. 2010;466:368–72. Iossifov I, O’Roak BJ, Sanders SJ, Ronemus M, Krumm N, Levy D, et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014;515:216–21. Doan RN, Lim ET, De Rubeis S, Betancur C, Cutler DJ, Chiocchetti AG, et al. Recessive gene disruptions in autism spectrum disorder. Nat Genet. 2019;51:1092–8. Nguyen RL, Medvedeva YV, Ayyagari TE, Schmunk G, Gargus JJ. Intracellular calcium dysregulation in autism spectrum disorder: An analysis of converging organelle signaling pathways. Biochim Biophys Acta Mol Cell Res. 2018;1865:1718–32. Catterall WA, Goldin AL, Waxman SG. International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev. 2005;57:397–409. Buraei Z, Yang J. The ß subunit of voltage-gated Ca2+ channels. Physiol Rev. 2010;90:1461–506. Birnbaumer L, Qin N, Olcese R, Tareilus E, Platano D, Costantin J, et al. Structures and functions of calcium channel beta subunits. J Bioenerg Biomembr. 1998;30:357–75. Buraei Z, Yang J. Structure and function of the β subunit of voltage-gated Ca 2+ channels. Biochim Biophys Acta. 2013;1828:1530–40. Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, et al. Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell. 2004;119:19–31. Breitenkamp AF, Matthes J, Herzig S. Voltage-gated Calcium Channels and Autism Spectrum Disorders. Curr Mol Pharmacol. 2015;8:123–32. Trikalinos TA, Karvouni A, Zintzaras E, Ylisaukko-oja T, Peltonen L, Järvelä I, et al. A heterogeneity-based genome search meta-analysis for autism-spectrum disorders. Mol Psychiatry. 2006;11:29–36. Cross-Disorder Group of the Psychiatric Genomics Consortium. Identification of risk loci with shared effects on five major psychiatric disorders: a genome-wide analysis. Lancet Lond Engl. 2013;381:1371–9. Breitenkamp AFS, Matthes J, Nass RD, Sinzig J, Lehmkuhl G, Nürnberg P, et al. Rare mutations of CACNB2 found in autism spectrum disease-affected families alter calcium channel function. PloS One. 2014;9:e95579. Yuen RKC, Thiruvahindrapuram B, Merico D, Walker S, Tammimies K, Hoang N, et al. Whole-genome sequencing of quartet families with autism spectrum disorder. Nat Med. 2015;21:185–91. Bonora E, Bianco F, Cordeddu L, Bamshad M, Francescatto L, Dowless D, et al. Mutations in RAD21 disrupt regulation of APOB in patients with chronic intestinal pseudo-obstruction. Gastroenterology. 2015;148:771-782.e11. Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinforma Oxf Engl. 2010;26:589–95. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491–8. Magi A, Tattini L, Palombo F, Benelli M, Gialluisi A, Giusti B, et al. H3M2 : detection of runs of homozygosity from whole-exome sequencing data. Bioinformatics. 2014;30:2852–9. gnomAD [Internet]. 2019 [cited 2019 Nov 24]. Available from: https://gnomad.broadinstitute.org/ Herzig S, Khan IFY, Gründemann D, Matthes J, Ludwig A, Michels G, et al. Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. FASEB J Off Publ Fed Am Soc Exp Biol. 2007;21:1527–38. Miranda-Laferte E, Ewers D, Guzman RE, Jordan N, Schmidt S, Hidalgo P. The N-terminal domain tethers the voltage-gated calcium channel β2e-subunit to the plasma membrane via electrostatic and hydrophobic interactions. J Biol Chem. 2014;289:10387–98. Koch P, Herzig S, Matthes J. An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells. J Pharmacol Toxicol Methods. 2016;82:20–5. Soldatov NM, Bouron A, Reuter H. Different voltage-dependent inhibition by dihydropyridines of human Ca2+ channel splice variants. J Biol Chem. 1995;270:10540–3. Schleithoff L, Mehrke G, Reutlinger B, Lehmann-Horn F. Genomic structure and functional expression of a human alpha(2)/delta calcium channel subunit gene (CACNA2). Genomics. 1999;61:201–9. Karmažínová M, Lacinová L. Removal of the outermost arginine in IVS4 segment of the Ca(V)3.1 channel affects amplitude but not voltage dependence of gating current. Gen Physiol Biophys. 2010;29:419–23. Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC, Aizawa Y, et al. Loss-of-function mutations in the cardiac calcium channel underlie a new clinical entity characterized by ST-segment elevation, short QT intervals, and sudden cardiac death. Circulation. 2007;115:442–9. Cordeiro JM, Marieb M, Pfeiffer R, Calloe K, Burashnikov E, Antzelevitch C. Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome. J Mol Cell Cardiol. 2009;46:695–703. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med Off J Am Coll Med Genet. 2015;17:405–24. Lu AT-H, Dai X, Martinez-Agosto JA, Cantor RM. Support for calcium channel gene defects in autism spectrum disorders. Mol Autism. 2012;3:18. Bonora E, Graziano C, Minopoli F, Bacchelli E, Magini P, Diquigiovanni C, et al. Maternally inherited genetic variants of CADPS2 are present in autism spectrum disorders and intellectual disability patients. EMBO Mol Med. 2014;6:795–809. Latronico AC, Brito VN, Carel J-C. Causes, diagnosis, and treatment of central precocious puberty. Lancet Diabetes Endocrinol. 2016;4:265–74. Severi G, Bernardini L, Briuglia S, Bigoni S, Buldrini B, Magini P, et al. New patients with Temple syndrome caused by 14q32 deletion: Genotype-phenotype correlations and risk of thyroid cancer. Am J Med Genet A. 2016;170A:162–9. Giorda R, Bonaglia MC, Beri S, Fichera M, Novara F, Magini P, et al. Complex segmental duplications mediate a recurrent dup(X)(p11.22-p11.23) associated with mental retardation, speech delay, and EEG anomalies in males and females. Am J Hum Genet. 2009;85:394–400. Spergel DJ. Calcium and small-conductance calcium-activated potassium channels in gonadotropin-releasing hormone neurons before, during, and after puberty. Endocrinology. 2007;148:2383–90. Lacerda AE, Kim HS, Ruth P, Perez-Reyes E, Flockerzi V, Hofmann F, et al. Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature. 1991;352:527–30. Van Petegem F, Clark KA, Chatelain FC, Minor DL. Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature. 2004;429:671–5. Stotz SC, Jarvis SE, Zamponi GW. Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels. J Physiol. 2004;554:263–73. He J-Y, Zhang W, He L-C, Cao Y-X. Imperatorin induces vasodilatation possibly via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+ influx and release. Eur J Pharmacol. 2007;573:170–5. Jangsangthong W, Kuzmenkina E, Khan IFY, Matthes J, Hullin R, Herzig S. Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits. Pflugers Arch. 2010;459:399–411. Miranda-Laferte E, Schmidt S, Jara AC, Neely A, Hidalgo P. A short polybasic segment between the two conserved domains of the β2a-subunit modulates the rate of inactivation of R-type calcium channel. J Biol Chem. 2012;287:32588–97. Wemhöner K, Friedrich C, Stallmeyer B, Coffey AJ, Grace A, Zumhagen S, et al. Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome. J Mol Cell Cardiol. 2015;80:186–95. Splawski I, Timothy KW, Decher N, Kumar P, Sachse FB, Beggs AH, et al. Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations. Proc Natl Acad Sci U S A. 2005;102:8089–96; discussion 8086-8088. Vissers LELM, Veltman JA. Standardized phenotyping enhances Mendelian disease gene identification. Nat Genet. 2015;47:1222–4. Cite Share Download PDF Status: Published Journal Publication published 06 Jun, 2020 Read the published version in Journal of Autism and Developmental Disorders → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9167","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":243870,"identity":"22147c29-ee39-454b-a260-0d09d32c2fb6","order_by":1,"name":"Claudio Graziano","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDACdiDmMYByEoCYH4gPgwRxamFG1yLZANWCUw8zuoEGByCCOK3hb2Z+JvGmwIZBPvrw4w8Pau7IGd/IPXi4oIJBxh6HFonDbGaScwzSGAzPpRkYJBx7Zmx2Iy/h8IwzuB1mwMxgJs1jcJjBsIfBICGB7XDiths5Bod52/BpYf8G1cL+4UDCv8OJm2cQ1MIDsUWeh8ewIbHtcOIGCQJaJA7zFFsC/cJjwMNTzJDYd9hY4swbA6BfJHh4DuAIsfb2jTfe/LGRk+9h3/zxx7fDcvztOcafCyps7NkbcFgDBTwGaGZK4FcPAvIEzBwFo2AUjIIRDACgJFApHmr0TgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3875-6869","institution":"Azienda Ospedaliero-Universitaria di Bologna Policlinico Sant'Orsola-Malpighi","correspondingAuthor":true,"prefix":"","firstName":"Claudio","middleName":"","lastName":"Graziano","suffix":""},{"id":243871,"identity":"a47d70bf-bf3b-444d-aa30-b6ce640e762e","order_by":2,"name":"Patrick Despang","email":"","orcid":"","institution":"Universitat zu Koln","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Despang","suffix":""},{"id":243872,"identity":"88093cbc-7642-44eb-9e94-852de409874f","order_by":3,"name":"Flavia Palombo","email":"","orcid":"","institution":"IRCCS Istituto Delle Scienze Neurologiche di Bologna","correspondingAuthor":false,"prefix":"","firstName":"Flavia","middleName":"","lastName":"Palombo","suffix":""},{"id":243873,"identity":"f9d8c32c-d83b-4ad4-9b63-03bef8957980","order_by":4,"name":"Giulia Severi","email":"","orcid":"","institution":"Azienda Ospedaliero-Universitaria di Bologna Policlinico Sant'Orsola-Malpighi","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Severi","suffix":""},{"id":243874,"identity":"cb54ed74-a780-4fed-8ea9-4d0b08a0ddde","order_by":5,"name":"Annio Posar","email":"","orcid":"","institution":"IRCCS Istituto Delle Scienze Neurologiche di Bologna","correspondingAuthor":false,"prefix":"","firstName":"Annio","middleName":"","lastName":"Posar","suffix":""},{"id":243875,"identity":"806e9386-792c-40d8-b4e9-bb20f055917f","order_by":6,"name":"Alessandra Cassio","email":"","orcid":"","institution":"Azienda Ospedaliero-Universitaria di Bologna Policlinico Sant'Orsola-Malpighi","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Cassio","suffix":""},{"id":243876,"identity":"54649dc3-abf3-47e6-b7f9-f5b9dcf6a26a","order_by":7,"name":"Tommaso Pippucci","email":"","orcid":"","institution":"Azienda Ospedaliero-Universitaria di Bologna Policlinico Sant'Orsola-Malpighi","correspondingAuthor":false,"prefix":"","firstName":"Tommaso","middleName":"","lastName":"Pippucci","suffix":""},{"id":243877,"identity":"962b0278-bf1b-4dc7-9a0c-9b851ae33270","order_by":8,"name":"Federica Isidori","email":"","orcid":"","institution":"Azienda Ospedaliero-Universitaria di Bologna Policlinico Sant'Orsola-Malpighi","correspondingAuthor":false,"prefix":"","firstName":"Federica","middleName":"","lastName":"Isidori","suffix":""},{"id":243878,"identity":"9159eb28-d0c6-4a17-9f88-e5ee675569ba","order_by":9,"name":"Jan Matthes","email":"","orcid":"","institution":"Universitat zu Koln","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Matthes","suffix":""},{"id":243879,"identity":"af87a664-c5b9-4917-8d3b-666f8de4fd62","order_by":10,"name":"Elena Bonora","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDACdjB5AML5UMHAwCYBZCTg08KMpIVxxhmYFnx6kLUwc7YBSZAWfNbwMzMfe/CD4Y6cOXvvw8+M8w7n8Uk3H2B4+AO3FslmtnTDHoZnxpY9x42lC7cdLmaTOZaA12EGh3nMJHgYDiduuJHGID1z2+HENokcA7xa7A/zf5P8A9HC/Jt3DhFaDJh52KShtrBJ8zYQoUXiMJuZtIzBM2ODM8fYLGccS09sA/rlQEIabi387c3PJN9U3JEzON7GfONDjXXi/NnNBx/+sMGtBeo8NP4BQhpGwSgYBaNgFOAHAKUVT5VSi4h+AAAAAElFTkSuQmCC","orcid":"","institution":"Azienda Ospedaliero-Universitaria di Bologna Policlinico Sant'Orsola-Malpighi","correspondingAuthor":true,"prefix":"","firstName":"Elena","middleName":"","lastName":"Bonora","suffix":""}],"badges":[],"createdAt":"2019-12-06 12:42:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.18548/v1","doiUrl":"https://doi.org/10.21203/rs.2.18548/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10803-020-04551-y","type":"published","date":"2020-06-06T21:09:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":251280,"identity":"28004bed-c450-4dcd-9172-c83749d4494b","added_by":"auto","created_at":"2019-12-11 22:09:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87737,"visible":true,"origin":"","legend":"(a) Representative whole-cell Ba2+-currents recorded of HEK-293 cells expressing CaV1.2/α2δ1 and either CaVβ2d_WT (left) or the ASD-associated CaVβ2d_R70C (right). (b) I-V curve of CaVβ2d_WT (n=8) and CaVβ2d_R70C (n=13). Currents were elicited from -40 to +50 mV test potentials in 10 mV increments (Holding potential: -80 mV; charge carrier: 10 mM Ba2+).","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/8f2f9760-246b-45da-ad0a-6f9037e69fa0/v1/Figure 1.png"},{"id":251285,"identity":"9e04608b-c826-4d9e-89c1-7f4a46e6db01","added_by":"auto","created_at":"2019-12-11 22:09:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110881,"visible":true,"origin":"","legend":"(a) Activation time constants (τact) showed a significant reduction for CaVβ2d_R70C (n=10) compared to CaVβ2d_WT (n=6) at various test potentials. (b) Time-dependent inactivation was analyzed as the remaining fraction of whole-cell current that has not been inactivated after 150 ms of depolarization. CaVβ2d_R70C (n=12) displayed a significant reduction at positive test potentials compared to CaVβ2d_WT (n=7) (Holding potential: -80 mV; charge carrier: 10 mM Ba2+). *: p\u003c0.05 in Student’s t-test.","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/8f2f9760-246b-45da-ad0a-6f9037e69fa0/v1/Figure 2.png"},{"id":15665961,"identity":"8878de82-2aa5-4127-a4b0-ce53beaaa51d","added_by":"auto","created_at":"2021-11-18 13:34:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":520785,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9167/v1/231308c7-61b5-43e5-befd-365842d292a9.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eA new homozygous \u003cem\u003eCACNB2\u003c/em\u003e mutation has functional relevance and supports a role for calcium channels in autism spectrum disorder\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eAutism spectrum disorder (ASD) is a complex neurodevelopmental condition affecting about 1 in 60 individuals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is defined by dysfunctions in social interaction and communication, stereotypic behaviors and sensory integration problems. ASD can be isolated and, in such cases, predominantly follows a polygenic pattern of inheritance caused by several weakly penetrant genetic variants that incrementally enhance susceptibility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Common genetic variants have been implicated by genome-wide association studies, with the identification of polygenic risk scores [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. On the other hand, ASD is a frequent feature of rare multisystemic neurodevelopmental disorders, where Intellectual Disability (ID) and epilepsy are common comorbidities [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A substantial subset of these patients show rare de novo copy number or single nucleotide variants (CNVs and SNVs) [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The contribution of recessive mutations has been less characterized and was addressed in a recent large-scale sequencing study, which demonstrated an excess of biallelic mutations justifying approximately 5% of ASD cases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Monogenic \u0026ldquo;recessive\u0026rdquo; ASD is expected to be very rare but should be enriched in children of consanguineous marriages.\u003c/p\u003e \u003cp\u003eThe introduction of next generation sequencing technologies offers the opportunity to test large cohorts of ASD patients and the results are helping to delineate the pathophysiological pathways which lead to this disorder. Hundreds of genes have been implicated in the pathogenesis of ASD, but the impacted pathways often cluster into three major functional groups: chromatin structure, transcription factors and calcium signaling [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVoltage-gated calcium channels (VGCCs) are heteromultimeric protein complexes consisting of up to four different subunits: a pore-forming α1 subunit (Ca\u003csub\u003eV\u003c/sub\u003e1.x \u0026ndash; Ca\u003csub\u003eV\u003c/sub\u003e3.x) and an auxiliary α\u003csub\u003e2\u003c/sub\u003eδ- (α\u003csub\u003e2\u003c/sub\u003eδ\u003csub\u003e1\u0026minus;4\u003c/sub\u003e), β- (Ca\u003csub\u003eV\u003c/sub\u003eβ\u003csub\u003e1\u0026minus;4\u003c/sub\u003e) and in some tissues γ-subunit (γ\u003csub\u003e1\u0026minus;8\u003c/sub\u003e) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Among other functions, Ca\u003csub\u003eV\u003c/sub\u003eβ subunits modulate the channel activation and inactivation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The first calcium channel gene that was reported as mutated in a form of monogenic syndromic autism was \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNA1C\u003c/span\u003e, which is the cause of Timothy syndrome, characterized by arrhythmia, hand/foot anomalies and high prevalence of ASD [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Since then other VGCC subunits have been associated with ASD (reviewed in [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]). Concerning to Ca\u003csub\u003eV\u003c/sub\u003eβ subunits, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNB2\u003c/span\u003e variants showed genome-wide suggestive significance in siblings with autism [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNB2\u003c/span\u003e was found as a risk locus for five major psychiatric disorders including ASD [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We previously reported three rare heterozygous \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNB2\u003c/span\u003e missense variants in patients with autism, altering the kinetic parameters of recombinant L-type calcium channels similarly to the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNA1C\u003c/span\u003e mutations associated with Timothy syndrome [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, an N-terminal de novo missense mutation (p.Val2Asp) in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNB2\u003c/span\u003e was recently discovered in a whole-genome sequencing study of ASD individuals and it was considered clinically relevant [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present work, we report the identification of a \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNB2\u003c/span\u003e homozygous rare variant in a girl with ASD, global developmental delay and precocious puberty via whole exome sequencing (WES). Functional characterization by whole-cell patch-clamp revealed a decelerated inactivation behavior of L-type calcium channels (LTCCs) similar to the previously described \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eCACNB2\u003c/span\u003e mutations suggesting a common feature among ASD-associated Ca\u003csub\u003eV\u003c/sub\u003eβ\u003csub\u003e2\u003c/sub\u003e mutations.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eClinical characteristics of the proband\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn 11-year-old girl received a diagnosis of global developmental delay and ASD at the age of four. She is the first child of consanguineous parents (first-degree cousins) from Bangladesh. She was born at term after an uneventful pregnancy. Birth weight was 3255g. Motor development was normal, she had normal growth and no facial dysmorphisms. She underwent a neuropsychiatric evaluation at the age of four for absent speech. The Denver Scale showed a severe developmental delay, mainly affecting the areas of language and socialization. She was administered ADOS (Autism Diagnostic Observational Schedule) and CARS (Childhood Autism Rating Scale) and met diagnostic criteria for ASD. Brain imaging and EEG were normal. She also underwent a cardiac evaluation with ECG recordings, which did not show abnormalities of heart rhythm; she had normal hearing and normal ophthalmic evaluation. She had sleep disturbances with frequent awakenings during the night and a severe constipation.\u003c/p\u003e\n\u003cp\u003eBlood karyotype, FRAXA analysis, \u003cem\u003eMECP2\u003c/em\u003e sequencing and MLPA, chromosomal microarray, urine and plasma metabolic investigations, were normal.\u003c/p\u003e\n\u003cp\u003eAt the age of 6 years and 6 months,\u0026nbsp; progression of\u0026nbsp; breast development (B3 according to Tanner stages) and advanced bone age (8 years 10 months) were noticed. Pubertal response of LH (LH peak \u0026gt; 5 IU/L ) to standard GnRH test\u0026nbsp; and increased uterine length \u003cem\u003e\u0026nbsp;(\u003c/em\u003e40 mm ) at ultrasound\u0026nbsp; confirmed the\u0026nbsp; diagnosis of central precocious puberty. MR imaging of hypothalamus-pituitary region was normal.\u003c/p\u003e\n\u003cp\u003eTherapy with GnRH analogues was started at the age of seven years and two months, and was interrupted after three years. One year after interruption (at 11), she had menarche.\u003c/p\u003e\n\u003cp\u003eAt the last evaluation (11 years), speech was absent and the sleeping pattern was described as slightly more regular. She still showed relevant deficits in the areas of social interaction, communication, and range of interests and activities, confirming the diagnosis of ASD (severity level 3: \"Requiring very substantial support\u0026rdquo;) in association with a severe ID (DSM-5 criteria).\u003c/p\u003e\n\u003cp\u003eParents do not show cognitive/behavioral problems and family history is unremarkable in this respect. Mother\u0026rsquo;s menarche was at 13 years. The father was a heavy smoker, had a gastric lymphoma at 34 years and an acute myocardial infarction at 37: coronary angiography showed an occlusion of the left circumflex artery and he was subjected to primary angioplasty and placement of a bare-metal stent; ECG-holter was performed during follow-up and it was normal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole Exome Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from peripheral blood with the QIAamp DNA Blood Mini (Qiagen, Venlo, Netherlands). Targeted capture and enrichment was performed using the Nextera Rapid Capture Exome kit (Illumina Inc., San Diego, CA) and library was sequenced as 100-bp paired-end reads on Illumina HiScanSQ (Illumina).\u003c/p\u003e\n\u003cp\u003eGenerated reads were treated following a general pipeline elsewhere described [22] including alignment with BWA [23] to the reference genome hg19, realignment and base quality score recalibration with GATK [24] and duplicate removal with Picard Tools (https://broadinstitute.github.io/picard). Alignment and coverage statistics were collected with SAMtools and GATK. Variants were called and filtered by quality with GATK HaplotypeCaller and Variant Quality Score Recalibration, and then annotated with Ensembl Variant Effect Predictor (www.ensembl.org/info/docs/tools/vep/index.html).\u003c/p\u003e\n\u003cp\u003eH\u003csup\u003e3\u003c/sup\u003eM\u003csup\u003e2\u003c/sup\u003e [25] was used for the identification of ROHs from WES alignments. Candidate disease-causing variants were defined as variants with potential to alter the protein product (missense, nonsense, small insertion/deletions and splicing-affecting variants) with allele frequency lower than 0.01 and not seen in homozygous state in gnomAD database [26]. The selected variant in \u003cem\u003eCACNB2\u003c/em\u003e was confirmed by Sanger sequencing and tested in parents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDNA constructs and site-directed mutagenesis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor functional analysis, the p.Arg70Cys variant was introduced in human Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d\u003c/sub\u003e (NM_201596.2) by site-directed mutagenesis (Stratagene QuikChange Kit) and verified by sequencing. EGFP was used as reporter gene, which was co-expressed together with the \u0026beta;\u003csub\u003e2\u003c/sub\u003e-subunit by the bicistronic pIRES2-EGFP vector (Clontech). Primer pairs for the mutagenesis were the following: p.Arg70Cys forward primer 5'-gccgaaccctggcaaacaaaactatttgaggtagtatca-3'; reverse primer 5'-tgatactacctcaaatagttttgtttgccagggttcggc-3'.\u003c/p\u003e\n\u003cp\u003eSince the N-termini of Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2\u003c/sub\u003e vary in sequence and length among the different splice variants (Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2a-e\u003c/sub\u003e) and this has been shown to impact LTCC modulation [27,28], we used a Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d\u003c/sub\u003e backbone as in our previous study [20], thus comparing wild-type (WT) Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d_WT\u003c/sub\u003e and mutant Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d_R70C\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell culture and transfection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK-293 cells were grown in Petri dishes in Dulbecco\u0026rsquo;s modified Eagle medium (Gibco Thermo Fisher, Waltham, MA, USA) supplemented with 10% FCS (Biochrom GmbH, Berlin Germany). Cells were routinely passaged twice a week and incubated at 37\u0026deg;C und 5% CO\u003csub\u003e2\u003c/sub\u003e growth conditions.\u003c/p\u003e\n\u003cp\u003eHEK-293 cells were transfected with human calcium-channel subunits and EGFP using a standard calcium phosphate method [29]. For whole-cell recordings, HEK-293 cells were transfected with a 1 : 0.5 : 1.5 ratio of Ca\u003csub\u003eV\u003c/sub\u003e1.2 (\u0026alpha;1c\u003csub\u003e77\u003c/sub\u003e) [30], either a WT or a mutant \u0026beta;\u003csub\u003e2d\u003c/sub\u003e-subunit and an \u0026alpha;\u003csub\u003e2\u003c/sub\u003e\u0026delta;\u003csub\u003e1\u003c/sub\u003e-subunit [31].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWhole-cell patch-clamp recordings\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-cell recordings of EGFP-positive cells were obtained 48\u0026ndash;72 h after transfection. Immediately prior to recording, cells kept in 35-mm culture dishes were washed at room temperature (19\u0026ndash;23\u0026deg;C) with bath solution. The bath solution contained (in mM) 10 BaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 10 HEPES, 65 CsCl, 40 TEA-Cl, 10 Glucose (pH 7.3 with TEA-OH) and the pipette solution (in mM) 140 CsCl, 10 EGTA, 9 HEPES, 1 MgCl\u003csub\u003e2\u003c/sub\u003e,4 MgATP (pH 7.3 with CsOH). Patch pipettes made from borosilicate glass (1.7 mm diameter and 0.283 mm wall thickness, Hilgenberg GmbH, Malsfeld, Germany) were pulled using a Sutter Instrument P-97 horizontal puller and fire-polished using a Narishige MF-83 microforge (Narishige Scientific Instrument Lab, Tokyo, Japan). Pipette resistance was 3-5 M\u0026Omega;. Currents were elicited by applying 500 ms long test potentials of -40 mV to +50 mV from a holding potential of -80 mV using Clampex software pClamp 10 and an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Currents were sampled at 10 kHz and filtered at 2 kHz.\u003c/p\u003e\n\u003cp\u003eData were analyzed using Clampfit10.3 (Molecular Devices, Sunnyvale, CA, USA) and GraphPad 6 Prism software. For voltage dependence of activation data were fitted by combined Ohm and Boltzmann relation \u0026nbsp;according to Karmazinova and Lacinova [32]. To obtain the time-course of activation (\u003cstrong\u003e\u0026tau;\u003c/strong\u003e\u003csub\u003eact\u003c/sub\u003e) a first order exponential function was used to fit to the current traces.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analyses \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are shown as mean \u0026plusmn; SEM and were analyzed using Student\u0026rsquo;s unpaired two-sided t-test. Differences were considered statistically significant if p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe proband belonged to a cohort of 50 individuals, all children of consanguineous parents, affected by heterogeneous disorders. According to our local protocol for consanguinity, WES experiment was performed in the proband only. Mean coverage was 66.8 X with the 90.7% of the bases covered. No pathogenic/likely pathogenic variants remained after filtering, but a rare and potentially disruptive homozygous missense variant in \u003cem\u003eCACNB2\u003c/em\u003e (NM_201597.2:c.208C\u0026gt;T:p.Arg70Cys) was selected for further investigation. It was confirmed by Sanger sequencing and parents were found to be heterozygous carriers (Supplementary Figures 1a-b). It is a very rare variant (only three alleles were reported in the gnomAD database, none in the South Asian population). The pathogenic computational verdict is supportive, because of six pathogenic predictions from DANN, GERP, MutationTaster, PROVEAN, FATHMM-MKL and SIFT vs three benign predictions from FATHMM, LRT and MutationAssessor. Some \u003cem\u003eCACNB2\u003c/em\u003e heterozygous variants have been described in families with Brugada syndrome [33,34], while others were associated with ASD [20]. The present \u003cem\u003eCACNB2\u003c/em\u003e variant was deposited in ClinVar with identification number 545669, as a variant of unknown significance according to the American College of Medical Genetics (ACMG) guidelines [35].\u003c/p\u003e\n\u003cp\u003eSince our study design was a \u0026ldquo;proband only\u0026rdquo; WES, we could not evaluate the presence of de novo variants. However, no pathogenic/likely pathogenic heterozygous variants (according to ACMG guidelines) were identified in genes known to cause neurodevelopmental disorders.\u003c/p\u003e\n\u003cp\u003eIn order to investigate the effect of the p.Arg70Cys variant, we expressed human Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d_WT\u003c/sub\u003e or Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d_R70C \u003c/sub\u003etogether with Ca\u003csub\u003eV\u003c/sub\u003e1.2 and Ca\u003csub\u003eV\u003c/sub\u003e\u0026alpha;\u003csub\u003e2\u003c/sub\u003e\u0026delta;\u003csub\u003e1\u003c/sub\u003e in HEK-293 cells and performed whole-cell patch-clamp recordings. Exemplary traces of Ba\u003csup\u003e2+\u003c/sup\u003e-currents are shown in Figure 1a.\u003c/p\u003e\n\u003cp\u003eOur analysis revealed no differences in current density (Figure 1b), but a significantly lower activation time constant (\u003cstrong\u003e\u0026tau;\u003c/strong\u003e\u003csub\u003eact\u003c/sub\u003e) at various test potential in the presence of Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d_R70C\u003c/sub\u003e compared to Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d_WT\u003c/sub\u003e (Figure 2a). Furthermore, time-dependent inactivation at 150 ms was significantly reduced by Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d_R70C \u003c/sub\u003e(Figure 2b). Accordingly, the inactivation time constant (\u003cstrong\u003e\u0026tau;\u003c/strong\u003e\u003csub\u003einact\u003c/sub\u003e) was increased for Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d\u0026shy;_R70C\u003c/sub\u003e compared to Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2d\u0026shy;_WT\u003c/sub\u003e (not shown). In summary, the p.Arg70Cys mutation shows functional consequences on Ca\u003csub\u003eV\u003c/sub\u003e1.2-mediated Ba\u003csup\u003e2+\u003c/sup\u003e-currents similarly to other ASD-associated Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2\u003c/sub\u003e mutations as we described previously [20].\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMore than 100 genes and genomic regions have been associated with ASD [6], suggesting a genetic architecture with many genes involved, each accounting for a small fraction of cases. Several lines of evidence indicate calcium signaling as one of the most significant pathways for ASD [17,36,37] and mutations in at least one gene encoding a calcium channel (\u003cem\u003eCACNA1C\u003c/em\u003e) have been linked to a syndrome with a high prevalence of ASD [16]. We provide further evidence that \u003cem\u003eCACNB2\u003c/em\u003e variation may be involved in the pathogenesis of ASD: a young girl with ASD, child of consanguineous parents, was found to carry a rare homozygous missense variant in \u003cem\u003eCACNB2\u003c/em\u003e, which was predicted pathogenic by software tools. We proved that this variant has a functional impact since it alters the kinetic parameters of currents carried by recombinant L-type calcium channels.\u003c/p\u003e\n\u003cp\u003eIn a recent study on recessive ASD, \u003cem\u003eCACNB2\u003c/em\u003e was among 409 distinct genes that harbored biallelic, damaging missense mutations in cases but not in controls. It is instructive to note that also in this study one single gene (\u003cem\u003eAMT\u003c/em\u003e) was independently hit in multiple families, confirming how rare the involvement of each independent gene is [10]. Unfortunately, no additional clinical data were reported for the subject mutated in \u003cem\u003eCACNB2\u003c/em\u003e, therefore it was not possible to know if relevant comorbidities were present.\u003c/p\u003e\n\u003cp\u003eOur female patient showed ID, which is the most common associated feature in ASD patients, and precocious puberty. An early timing of puberty is a specific feature of some disorders characterized by ID [38], such as imprinting disorders and chromosome defects [39,40]. Our proband did not have features evocative of an imprinting defect (such as IUGR and small stature) and chromosomal microarray excluded CNVs. Moreover, she had normal brain imaging and no familiarity for early onset of puberty (maternal age of menarche was 13 years). To our knowledge, no variants in calcium channels have ever been associated to precocious puberty. However, given that calcium signaling is involved in essential cellular functions that span multiple tissues and physiological systems, including the reproductive axis through gonadotropin-releasing hormone neurons [41], \u003cem\u003eCACNB2\u003c/em\u003e variants may indeed play a role in puberty progression. Only the identification of further patients carrying homozygous mutations in \u003cem\u003eCACNB2 \u003c/em\u003ewill give the opportunity to clarify this issue.\u003c/p\u003e\n\u003cp\u003eCa\u003csub\u003eV\u003c/sub\u003e\u0026beta; subunits are the only cytosolic subunit of high voltage-activated (HVA) VGCCs and serve, besides other functions, as main modulator of activation and inactivation of the channel [14,15,42]. They consist of variable N- and C-termini and conserved SH3- and GK-domains, separated by a variable HOOK-domain [43]. HOOK-domain and N-terminus have a major impact on voltage-dependent inactivation of HVA VGCCs [27,44\u0026ndash;47]. The herein described p.Arg70Cys mutation significantly attenuates inactivation of LTCCs, similar to the previously described ASD-associated mutations p.Gly167Ser and p.Ser197Phe [20]. Given these data, it is tempting to speculate that ASD-associated mutations in Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;-subunits cause similar functional effects, i.e. enhanced calcium influx due to lowered channel inactivation, reminiscent of \u003cem\u003eCACNA1C\u003c/em\u003e mutations in the Ca\u003csub\u003eV\u003c/sub\u003e1.2 subunit of LTCCs causing Timothy syndrome [16].\u003c/p\u003e\n\u003cp\u003eTimothy syndrome is characterized by abnormalities of heart rhythm and a high co-occurrence of ASD. Some mutations were identified in patients presenting only with non-syndromic long QT syndrome [48], whereas no \u003cem\u003eCACNA1C\u003c/em\u003e mutations were reported, to our knowledge, in patients with an isolated neurodevelopmental disorder. The dissimilarities in genotype-phenotype correlation for calcium-channel disorders might be determined by diverse factors, ranging from the impact of the mutation on channel activity (gain of function vs loss of function vs a dominant-negative effect), to the extensive alternative splicing, with many distinct transcripts exhibiting different biophysical properties and expression profiles. For instance, the classical Timothy syndrome phenotype results from the presence of the p.Gly406Arg pathogenic variant in exon 8A, an exon contained in a specific splice variant of \u003cem\u003eCACNA1C\u003c/em\u003e found in approximately 20% of all cardiac mRNAs [16]. Two individuals with atypical Timothy syndrome had been reported with pathogenic variants (p.Gly402Ser and p.Gly406Arg) in exon 8 of an alternate splice form that represents 80% of all cardiac mRNAs [49].\u003c/p\u003e\n\u003cp\u003eDominant mutations in \u003cem\u003eCACNB2\u003c/em\u003e have been identified in a small subset of individuals with Brugada syndrome, a familial cardiac arrhythmia [33,34]; none of the reported patients had autism or other neurodevelopmental disorders. Data from the Genotype-Tissue Expression (GTEx) project indicate that the p.Arg70Cys variant in exon 2a of \u003cem\u003eCACNB2\u003c/em\u003e is present in a transcript with higher expression in brain than in heart tissue, whereas the transcript with highest heart tissue expression does not include exon 2a (Supplementary Figure 1c). This expression pattern suggests a possible explanation for the absence of heart rhythm abnormalities in the present family.\u003c/p\u003e\n\u003cp\u003eAs the expansion of personalized medicine proceeds, with increasing potential for interpretation of genomic data, the early identification of molecular and genetic defects can lead to a better clinical refinement and the possibility of applying timely targeted therapies. Most importantly, it is crucial to carry out the functional characterization of proteins and mutations, to make progress from gene identification to function [50]. Therefore, developing models for specific mutations allows to better define the underlying molecular defects, exemplified in the present case by a significantly lower current activation time of a calcium channel.\u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003eThe major limit of this study is that the analysis was performed on a single subject. The evidence of \u003cem\u003eCACNB2\u003c/em\u003e involvement in ASD is slowly accumulating, but the number of reported patients is very limited. Although knowledge on recessive causes of ASD is lacking, they are expected to be rare, and the majority of large-scale studies evaluated polygenic inheritance or de novo variants. Deep clinical phenotyping and functional studies in larger sets of subjects will be instrumental to fully understand the penetrance and outcome of \u003cem\u003eCACNB2\u003c/em\u003e variants.\u003c/p\u003e\n\u003cp\u003eBa\u003csup\u003e2+\u003c/sup\u003e is not the physiological charge carrier trough L-type VGCCs, but it was used here for two main reasons: (1) Ca\u003csup\u003e2+\u003c/sup\u003e-dependent effects on channel gating, like calcium-dependent inactivation, might be difficult to discriminate from other Ca\u003csup\u003e2+\u003c/sup\u003e-independent effects, e.g. voltage-dependent inactivation; (2) using Ba\u003csup\u003e2+\u003c/sup\u003e allows for comparison with findings from our previous study [20].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, biallelic\u003cem\u003e CACNB2\u003c/em\u003e variants may define a rare form of recessive ASD, which is comorbid with Intellectual Disability and, possibly, precocious puberty. The electrophysiological phenotype is similar to that of other Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2\u003c/sub\u003e variants described earlier and thus suggests common features of ASD-associated Ca\u003csub\u003eV\u003c/sub\u003e\u0026beta;\u003csub\u003e2\u003c/sub\u003e mutations, reminiscent of Timothy syndrome mutations in \u003cem\u003eCACNA1C\u003c/em\u003e.\u003c/p\u003e"},{"header":"List of Abbreviations","content":"\u003cul\u003e\n\u003cli\u003eADOS: autism diagnostic observational schedule\u003c/li\u003e\n\u003cli\u003eACMG: American College of Medical Genetics\u003c/li\u003e\n\u003cli\u003eASD: autism spectrum disorder\u003c/li\u003e\n\u003cli\u003eCARS: childhood autism rating scale\u003c/li\u003e\n\u003cli\u003eCNV: copy number variant\u003c/li\u003e\n\u003cli\u003eHVA: high voltage-activated\u003c/li\u003e\n\u003cli\u003eID: intellectual disability\u003c/li\u003e\n\u003cli\u003eLTCC: L-type calcium channel\u003c/li\u003e\n\u003cli\u003eSNV: single nucleotide variant\u003c/li\u003e\n\u003cli\u003eVGCC: voltage-gated calcium channel\u003c/li\u003e\n\u003cli\u003eWES: whole exome sequencing\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParents provided consent according to the IRB protocol 3206/2016 at Policlinico S. Orsola-Malpighi (Bologna, Italy).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent to participate included consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analysed during the current study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by Telethon Grant n. GGP15171 to EB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCG conceived and designed the study, analyzed the data, and wrote the paper. PD and JM performed the electrophysiological studies and contributed to the writing of the paper. FP, TP, FI performed the sequencing and bioinformatic analyses. GS, AP, AC provided the patient data. EB supervised the research and co-wrote the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patient and her parents who contributed to the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaio J, Wiggins L, Christensen DL, Maenner MJ, Daniels J, Warren Z, et al. Prevalence of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2014. Morb Mortal Wkly Rep Surveill Summ Wash DC 2002. 2018;67:1\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003ede la Torre-Ubieta L, Won H, Stein JL, Geschwind DH. Advancing the understanding of autism disease mechanisms through genetics. Nat Med. 2016;22:345\u0026ndash;61.\u003c/li\u003e\n\u003cli\u003eWeiner DJ, Wigdor EM, Ripke S, Walters RK, Kosmicki JA, Grove J, et al. Polygenic transmission disequilibrium confirms that common and rare variation act additively to create risk for autism spectrum disorders. Nat Genet. 2017;49:978\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eGaugler T, Klei L, Sanders SJ, Bodea CA, Goldberg AP, Lee AB, et al. Most genetic risk for autism resides with common variation. Nat Genet. 2014;46:881\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eGrove J, Ripke S, Als TD, Mattheisen M, Walters RK, Won H, et al. Identification of common genetic risk variants for autism spectrum disorder. Nat Genet. 2019;51:431\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eBetancur C. Etiological heterogeneity in autism spectrum disorders: more than 100 genetic and genomic disorders and still counting. Brain Res. 2011;1380:42\u0026ndash;77.\u003c/li\u003e\n\u003cli\u003eSebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, et al. Strong association of de novo copy number mutations with autism. Science. 2007;316:445\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003ePinto D, Pagnamenta AT, Klei L, Anney R, Merico D, Regan R, et al. Functional impact of global rare copy number variation in autism spectrum disorders. Nature. 2010;466:368\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eIossifov I, O\u0026rsquo;Roak BJ, Sanders SJ, Ronemus M, Krumm N, Levy D, et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014;515:216\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eDoan RN, Lim ET, De Rubeis S, Betancur C, Cutler DJ, Chiocchetti AG, et al. Recessive gene disruptions in autism spectrum disorder. Nat Genet. 2019;51:1092\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eNguyen RL, Medvedeva YV, Ayyagari TE, Schmunk G, Gargus JJ. Intracellular calcium dysregulation in autism spectrum disorder: An analysis of converging organelle signaling pathways. Biochim Biophys Acta Mol Cell Res. 2018;1865:1718\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eCatterall WA, Goldin AL, Waxman SG. International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev. 2005;57:397\u0026ndash;409.\u003c/li\u003e\n\u003cli\u003eBuraei Z, Yang J. The \u0026szlig; subunit of voltage-gated Ca2+ channels. Physiol Rev. 2010;90:1461\u0026ndash;506.\u003c/li\u003e\n\u003cli\u003eBirnbaumer L, Qin N, Olcese R, Tareilus E, Platano D, Costantin J, et al. Structures and functions of calcium channel beta subunits. J Bioenerg Biomembr. 1998;30:357\u0026ndash;75.\u003c/li\u003e\n\u003cli\u003eBuraei Z, Yang J. Structure and function of the \u0026beta; subunit of voltage-gated Ca\u003csup\u003e2+\u003c/sup\u003e channels. Biochim Biophys Acta. 2013;1828:1530\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eSplawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, et al. Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell. 2004;119:19\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eBreitenkamp AF, Matthes J, Herzig S. Voltage-gated Calcium Channels and Autism Spectrum Disorders. Curr Mol Pharmacol. 2015;8:123\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eTrikalinos TA, Karvouni A, Zintzaras E, Ylisaukko-oja T, Peltonen L, J\u0026auml;rvel\u0026auml; I, et al. A heterogeneity-based genome search meta-analysis for autism-spectrum disorders. Mol Psychiatry. 2006;11:29\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eCross-Disorder Group of the Psychiatric Genomics Consortium. Identification of risk loci with shared effects on five major psychiatric disorders: a genome-wide analysis. Lancet Lond Engl. 2013;381:1371\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eBreitenkamp AFS, Matthes J, Nass RD, Sinzig J, Lehmkuhl G, N\u0026uuml;rnberg P, et al. Rare mutations of CACNB2 found in autism spectrum disease-affected families alter calcium channel function. PloS One. 2014;9:e95579.\u003c/li\u003e\n\u003cli\u003eYuen RKC, Thiruvahindrapuram B, Merico D, Walker S, Tammimies K, Hoang N, et al. Whole-genome sequencing of quartet families with autism spectrum disorder. Nat Med. 2015;21:185\u0026ndash;91.\u003c/li\u003e\n\u003cli\u003eBonora E, Bianco F, Cordeddu L, Bamshad M, Francescatto L, Dowless D, et al. Mutations in RAD21 disrupt regulation of APOB in patients with chronic intestinal pseudo-obstruction. Gastroenterology. 2015;148:771-782.e11.\u003c/li\u003e\n\u003cli\u003eLi H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinforma Oxf Engl. 2010;26:589\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eDePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eMagi A, Tattini L, Palombo F, Benelli M, Gialluisi A, Giusti B, et al. H3M2 : detection of runs of homozygosity from whole-exome sequencing data. Bioinformatics. 2014;30:2852\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003egnomAD [Internet]. 2019 [cited 2019 Nov 24]. Available from: https://gnomad.broadinstitute.org/\u003c/li\u003e\n\u003cli\u003eHerzig S, Khan IFY, Gr\u0026uuml;ndemann D, Matthes J, Ludwig A, Michels G, et al. Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. FASEB J Off Publ Fed Am Soc Exp Biol. 2007;21:1527\u0026ndash;38.\u003c/li\u003e\n\u003cli\u003eMiranda-Laferte E, Ewers D, Guzman RE, Jordan N, Schmidt S, Hidalgo P. The N-terminal domain tethers the voltage-gated calcium channel \u0026beta;2e-subunit to the plasma membrane via electrostatic and hydrophobic interactions. J Biol Chem. 2014;289:10387\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003eKoch P, Herzig S, Matthes J. An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells. J Pharmacol Toxicol Methods. 2016;82:20\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eSoldatov NM, Bouron A, Reuter H. Different voltage-dependent inhibition by dihydropyridines of human Ca2+ channel splice variants. J Biol Chem. 1995;270:10540\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eSchleithoff L, Mehrke G, Reutlinger B, Lehmann-Horn F. Genomic structure and functional expression of a human alpha(2)/delta calcium channel subunit gene (CACNA2). Genomics. 1999;61:201\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eKarmaž\u0026iacute;nov\u0026aacute; M, Lacinov\u0026aacute; L. Removal of the outermost arginine in IVS4 segment of the Ca(V)3.1 channel affects amplitude but not voltage dependence of gating current. Gen Physiol Biophys. 2010;29:419\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eAntzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC, Aizawa Y, et al. Loss-of-function mutations in the cardiac calcium channel underlie a new clinical entity characterized by ST-segment elevation, short QT intervals, and sudden cardiac death. Circulation. 2007;115:442\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eCordeiro JM, Marieb M, Pfeiffer R, Calloe K, Burashnikov E, Antzelevitch C. Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome. J Mol Cell Cardiol. 2009;46:695\u0026ndash;703.\u003c/li\u003e\n\u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med Off J Am Coll Med Genet. 2015;17:405\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eLu AT-H, Dai X, Martinez-Agosto JA, Cantor RM. Support for calcium channel gene defects in autism spectrum disorders. Mol Autism. 2012;3:18.\u003c/li\u003e\n\u003cli\u003eBonora E, Graziano C, Minopoli F, Bacchelli E, Magini P, Diquigiovanni C, et al. Maternally inherited genetic variants of CADPS2 are present in autism spectrum disorders and intellectual disability patients. EMBO Mol Med. 2014;6:795\u0026ndash;809.\u003c/li\u003e\n\u003cli\u003eLatronico AC, Brito VN, Carel J-C. Causes, diagnosis, and treatment of central precocious puberty. Lancet Diabetes Endocrinol. 2016;4:265\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eSeveri G, Bernardini L, Briuglia S, Bigoni S, Buldrini B, Magini P, et al. New patients with Temple syndrome caused by 14q32 deletion: Genotype-phenotype correlations and risk of thyroid cancer. Am J Med Genet A. 2016;170A:162\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eGiorda R, Bonaglia MC, Beri S, Fichera M, Novara F, Magini P, et al. Complex segmental duplications mediate a recurrent dup(X)(p11.22-p11.23) associated with mental retardation, speech delay, and EEG anomalies in males and females. Am J Hum Genet. 2009;85:394\u0026ndash;400.\u003c/li\u003e\n\u003cli\u003eSpergel DJ. Calcium and small-conductance calcium-activated potassium channels in gonadotropin-releasing hormone neurons before, during, and after puberty. Endocrinology. 2007;148:2383\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eLacerda AE, Kim HS, Ruth P, Perez-Reyes E, Flockerzi V, Hofmann F, et al. Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature. 1991;352:527\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eVan Petegem F, Clark KA, Chatelain FC, Minor DL. Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature. 2004;429:671\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eStotz SC, Jarvis SE, Zamponi GW. Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels. J Physiol. 2004;554:263\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003eHe J-Y, Zhang W, He L-C, Cao Y-X. Imperatorin induces vasodilatation possibly via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+ influx and release. Eur J Pharmacol. 2007;573:170\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eJangsangthong W, Kuzmenkina E, Khan IFY, Matthes J, Hullin R, Herzig S. Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits. Pflugers Arch. 2010;459:399\u0026ndash;411.\u003c/li\u003e\n\u003cli\u003eMiranda-Laferte E, Schmidt S, Jara AC, Neely A, Hidalgo P. A short polybasic segment between the two conserved domains of the \u0026beta;2a-subunit modulates the rate of inactivation of R-type calcium channel. J Biol Chem. 2012;287:32588\u0026ndash;97.\u003c/li\u003e\n\u003cli\u003eWemh\u0026ouml;ner K, Friedrich C, Stallmeyer B, Coffey AJ, Grace A, Zumhagen S, et al. Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome. J Mol Cell Cardiol. 2015;80:186\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eSplawski I, Timothy KW, Decher N, Kumar P, Sachse FB, Beggs AH, et al. Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations. Proc Natl Acad Sci U S A. 2005;102:8089\u0026ndash;96; discussion 8086-8088.\u003c/li\u003e\n\u003cli\u003eVissers LELM, Veltman JA. Standardized phenotyping enhances Mendelian disease gene identification. Nat Genet. 2015;47:1222\u0026ndash;4.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Autism spectrum disorders, Calcium channel genes, CACNB2, Intellectual disability, Precocious puberty, Consanguinity","lastPublishedDoi":"10.21203/rs.2.18548/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.18548/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eDiagnostic yield in patients with autism spectrum disorder (ASD) has improved over the last years, thanks to the introduction of whole genome arrays and next generation sequencing, but etiology is still unknown for the majority of cases. Among distinct cellular pathways, evidence implicating dysregulation of cellular calcium homeostasis in ASD pathogenesis has been accumulating, and specific mutations in voltage-gated calcium channels found in patients with autism were shown to be functionally relevant.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWhole exome sequencing and Sanger sequencing were performed to identify and confirm variants in a girl with ASD, global developmental delay and precocious puberty, born of first-degree cousins. Site-directed mutagenesis was used to generate a human Ca\u003csub\u003eV\u003c/sub\u003eβ\u003csub\u003e2d\u003c/sub\u003e calcium channel subunit carrying a \u003cem\u003eCACNB2\u003c/em\u003e mutation. Whole-cell patch-clamp recordings were performed to reveal functional effects of mutant Ca\u003csub\u003eV\u003c/sub\u003eβ\u003csub\u003e2d \u003c/sub\u003eon Ba\u003csup\u003e2+\u003c/sup\u003e-currents mediated by L-type (Ca\u003csub\u003eV\u003c/sub\u003e1.2) calcium channels in transiently transfected HEK-293 cells.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eIn an ASD patient, we identified a rare homozygous variant (p.Arg70Cys) in the \u003cem\u003eCACNB2 \u003c/em\u003egene coding for the auxiliary Ca\u003csub\u003eV\u003c/sub\u003eβ\u003csub\u003e2\u003c/sub\u003esubunit of voltage-gated calcium channels. In a recombinant system, the Ca\u003csub\u003eV\u003c/sub\u003eβ\u003csub\u003e2\u003c/sub\u003e variant, which was not previously associated to ASD, was found to alter Ca\u003csub\u003eV\u003c/sub\u003e1.2 calcium channel function by significantly affecting activation and inactivation of whole-cell Ba\u003csup\u003e2+\u003c/sup\u003e-currents.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAlthough the evidence of \u003cem\u003eCACNB2\u003c/em\u003e involvement in ASD is slowly accumulating, the number of reported patients is very limited. Deep clinical phenotyping and functional studies in larger sets of subjects will be instrumental to fully understand the penetrance and outcome of \u003cem\u003eCACNB2\u003c/em\u003e variants.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe p.Arg70Cys variant in \u003cem\u003eCACNB2\u003c/em\u003e shows functional consequences similar to other ASD-associated Ca\u003csub\u003eV\u003c/sub\u003eβ\u003csub\u003e2\u003c/sub\u003e mutations. These results support the idea of \u003cem\u003eCACNB2\u003c/em\u003e variations contributing to the development of ASD and hint to a rare form of Mendelian recessive autism with possible specific comorbidities.\u003c/p\u003e","manuscriptTitle":"A new homozygous CACNB2 mutation has functional relevance and supports a role for calcium channels in autism spectrum disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-12-11 22:09:05","doi":"10.21203/rs.2.18548/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b0e0cf2-d6db-44f7-8cad-dd4569bf3b31","owner":[],"postedDate":"December 11th, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41349,"name":"Cellular \u0026 Molecular Neuroscience"},{"id":41350,"name":"Molecular Genetics"}],"tags":[],"updatedAt":"2021-07-22T21:09:30+00:00","versionOfRecord":{"articleIdentity":"rs-9167","link":"https://doi.org/10.1007/s10803-020-04551-y","journal":{"identity":"journal-of-autism-and-developmental-disorders","isVorOnly":false,"title":"Journal of Autism and Developmental Disorders"},"publishedOn":"2020-06-06 21:09:30","publishedOnDateReadable":"June 6th, 2020"},"versionCreatedAt":"2019-12-11 22:09:05","video":"","vorDoi":"10.1007/s10803-020-04551-y","vorDoiUrl":"https://doi.org/10.1007/s10803-020-04551-y","workflowStages":[]},"version":"v1","identity":"rs-9167","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-9167","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}