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  including alignment with BWA  to the reference genome hg19, realignment and base quality score recalibration with GATK  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).
H3M2  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 . 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 CaVβ2d (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 β2-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 CaVβ2 vary in sequence and length among the different splice variants (CaVβ2a-e) and this has been shown to impact LTCC modulation [27,28], we used a CaVβ2d backbone as in our previous study , thus comparing wild-type (WT) CaVβ2d_WT and mutant CaVβ2d_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% CO2 growth conditions.
HEK-293 cells were transfected with human calcium-channel subunits and EGFP using a standard calcium phosphate method . For whole-cell recordings, HEK-293 cells were transfected with a 1 : 0.5 : 1.5 ratio of CaV1.2 (α1c77) , either a WT or a mutant β2d-subunit and an α2δ1-subunit .
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 BaCl2, 1 MgCl2, 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 MgCl2,4 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 . To obtain the time-course of activation (τact) a first order exponential function was used to fit to the current traces.
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.