The Schizophrenia Variant V1282F in SCN2A Causes Functional Impairment of NaV1.2

Research Article

J Schizophr Res. 2021; 7(1): 1038.

The Schizophrenia Variant V1282F in SCN2A Causes Functional Impairment of NaV1.2

Kohlnhofer B, Liu Y, Woodruff G, Lovenberg T, Bonaventure P and Harrington AW*

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, USA

*Corresponding author: Anthony W Harrington, Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA

Received: March 17, 2021; Accepted: April 01, 2021; Published: April 08, 2021


Neuropsychiatric disorders such as schizophrenia are challenging to treat due to the biological complexity of the disease and the lack of knowledge of the underlying pathophysiology. Whole exome and genome sequencing studies have identified disease-linked rare variants in patients with large effect size. Here, we functionally characterize the schizophrenia linked variant V1282F in SCN2A, encoding the sodium channel Nav1.2. This variant was introduced into isogenic lines of hiPSCs using CRISPR/CAS9 genome editing tools. hiPSCs were then differentiated into cortical neurons to understand how the variant and gene may be contributing to disease. We observed a significant (~25%) decrease in sodium current in the V1282F neurons compared to control neurons, suggesting the mutation is causing a loss-of-channel function. These results were supported by recordings in recombinant cells overexpressing either the mutant or wildtype Nav1.2, with the mutant channel having significantly (~75%) lower current amplitude than wildtype. We hypothesize that this phenotype may contribute to disease either through the direct loss of neuronal activity or through subsequent abnormal neurodevelopment.


Elucidating novel therapeutic targets for schizophrenia has been difficult due to the lack of knowledge of the fundamental biology and pathophysiology of this complex disease. Additionally, there is poor predictive validity in current pre-clinical models. Factoring into these problems is the lack of human model systems and our poor understanding of the disease genetics. Genetics are a major contributor to schizophrenia, as the heritability of schizophrenia is estimated to be 79% from twin studies [1]. With the recent advances in genome sequencing, we now have insight into genes that may be contributing to schizophrenia and that have the potential to be novel pharmaceutical targets. Genome Wide Association Studies (GWAS) and whole genome or exome sequencing efforts have identified variants in schizophrenia patients, presumed to be disease causing but it is not clear what the functional effects of these variants are, if any. Most of the GWAS-identified common variants are within non-coding regions, so the field resorts to investigating rare coding variants identified through whole genome or exome sequencing. To transition from the bench to the bedside, it is necessary to first understand how individual patient risk variants affect gene or gene product function and how this contributes to disease.

Many of the genes that contain variants for schizophrenia overlap with those that contain variants for Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) [2]. The gene that harbors the greatest combined number of variants identified in ASD, ID, and schizophrenia patient populations is the voltage-gated Sodium Channel Alpha Subunit 2 (SCN2A). SCN2A encodes for the sodium channel Nav1.2, which is critical for the initiation and propagation of action potentials [3]. In fact, in excess of a hundred unique de novo mutations in SCN2A have been identified and with hundreds of children born each year having mutations in SCN2A [4,5]. These variants have been identified through whole genome or exome sequencing studies and although extremely rare, they are expected to have high penetrance. SCN2A variants have also been identified in patients with Infantile Epileptic Encephalopathy (IEE) and Benign Infantile Familial Seizures (BIFS), where the contribution of variants to disease is better understood and those variants that have been functionally analyzed have been found to be gain-of-function [6-11]. Through bioinformatic modeling, multiple SCN2A variants identified in neuropsychiatric patients were predicted to be loss-of-function and recently a few have been functionally evaluated using overexpression studies in HEK293 cells. Eleven unique ASD variants and three unique ID variants were found to either functionally inactive or inhibit channel function [12,13]. In mice, SCN2A knockout is embryonic lethal and multiple groups have found that SCN2A+/- mice display autistic and schizophrenia-like phenotypes [14-16].

While schizophrenia-linked mutations in SCN2A are less common, mutations have been identified through whole genome or exome sequencing, including one that leads to loss of a splice site (c.2150 A>G), one that generates a nonsense mutation (E169X), and one that creates a missense mutation (V1282F) [2,17]. However, none of them have been functionally investigated [2,17-19]. To date, there are no reports on the functionality or effect of SCN2A variants associated with schizophrenia. In this study, we investigated one coding variant, V1282F (Chr2: 166226804, (G/T)), located in the third transmembrane segment of the third domain. This is a missense variant that has been identified in two unrelated patients and is absent in the control dataset [17]. To investigate this variant in a biologically relevant system, we used human induced pluripotent stem cell (hiPSC) derived cortical neurons and introduced the variant using CRISPR/CAS9 genome editing tools to analyze the variant under endogenous expression levels. We found that the V1282F variant causes a significant decrease in Na+ channel current density. V1282F mutant channels exogenously expressed in HEK293 cells further showed lower current amplitudes than Wildtype (WT) channels, consistent with the findings in hiPSC-derived neurons and suggesting a loss-of-channel function associated with the V1282F mutant. To our knowledge, this is the first report of the functional impact of a SCN2A variant identified in a schizophrenia patient.


hiPSC maintenance and genome editing

The healthy control hiPSC line GM25430 (CVB) was obtained from the Coriell Institute (Camden, NJ). All hiPSC lines were maintained in mTeSR on vitronectin coated plates passaged using ReLeSR (Stem Cell Technologies, Vancouver, Canada). To generate SCN2AWT/V1282F and SCN2AV1282F/V1282F hiPSC lines, a guide RNA (gcatactcacATCAACAATC) was designed to target exon 19 near chromosome position: 166226804(G/T) using the guide design tool from and a SSODN donor template was designed using 50bp homology arms incorporating the (G/T) variant (G T T T T C A A G T G T A T T T T A C C A A T G C C T G G T G C T G G C T A G A C T T C C T G A T T (T) T T G A T G T G A G T A T G C T G C A C T T T G C T G C T T T A T T C A T T G G C A T A T A T G T). Genome editing was performed using the Alt-R-CRISPR system (IDT, Coralville, IA). Alt-R S.p. HiFi Cas9 Nuclease V3, Alt-R CRISPR-Cas9 crRNA, Alt-R CRISPR-Cas9 tracrRNA (ATTO 550), and SSODN were nucleofected into hiPSCs using the P3 Primary Cell 4D-Nucleofector X kit and the Amaxa 4D-Nucleofector X Unit (Lonza, Basel, Switzerland) setting CB-150. 24 hours post-nucleofection ATTO 550+ single cells were FACS sorted and plated. Isogenic clones were screened for the variant by amplifying the DNA using Polymerase Chain Reaction (PCR) and primers flanking the variant (Primer F: aggagttcctgcaaatgagttaccc and Primer R: aatgttttgaggcatcctctcactg) followed by Sanger sequencing.

Neural precursor cell differentiation and neuron differentiation

hiPSCs were differentiated into Neural Precursor Cells (NPCs) over the course of three weeks using the STEMdiff Neural Induction Media + SMADi Kit and protocol (STEMCELL Technologies) on 0.01% Poly-L-ornithine and Laminin (ThermoFisher, Waltham, MA) coated plates. NPCs were cultured in neural maintenance media: 0.5X DMEM/F12 + Glutamax, Neurobasal , 0.5X N2 supplement, 0.5X B27 Supplement , 0.5X Glutamax, 0.5X IT-S, 0.25X 2-Mercaptoethanol (Gibco-ThermoFisher, Waltham, MA), Pen- Strep (Corning, Corning, NY) and NEAA (Hyclone Laboratories Inc- ThermoFisher, Waltham, MA), and 20ng/ml FGF2 (R&D Systems, Minneapolis, MN). NPCs were differentiated into neurons by withdrawing FGF2 once confluent. After three weeks, cells were replated for downstream assays and 24 hours later treated with 1uM cytosine arabinoside (ARA-C) (Sigma, St. Louis, MO) for 72 hours to eliminate proliferating cells. After ARA-C treatment the media was switched to neuronal maturation media containing Brain Phys (STEMCELL Technologies), 1X PenStrep, 1X N2 supplement, 1X B27 Supplement, 20ng/uL BDNF (Peprotech, Rocky Hill, NJ), 20ng/ uL GDNF (Peprotech), 1mM cAMP (Sigma, St. Louis, MO), 1ug/ mL Laminin, and 200nM Ascorbic Acid (Sigma). Neurons were matured for an additional month with weekly half media changes. All assays were performed at eight weeks after FGF2 withdrawal unless indicated.

RNA isolation, cDNA synthesis and qRT-PCR

RNA was isolated from hiPSCs or two-month neuronal cultures using a RNeasy Mini Kit and samples were DNase treated on column (Qiagen, Hilden, Germany). cDNA was synthesized using SuperScript IV First-Strand Synthesis System (Invitrogen, Carlsbad, CA) and qRT-PCR was performed using TaqMan assays (ThermoFisher, Waltham, MA) and analyzed on a QuantStudio Real Time PCR System (Applied Biosystems, Foster City, CA). TaqMan assays: OCT4: Hs00999634_gH, SCN2A: Hs01109871_m1, SCN3A: Hs00366902_m1, SCN8A: Hs00274075_m1, and GAPDH: Hs02786624_g1. Data were normalized to GAPDH expression levels. Data were plotted as mean ±SEM. Statistical analysis was performed using a one-way ANOVA and results were considered significant at p-value <0.05 (GraphPad Prism).


NPCs were fixed in 4% Paraformaldehyde (PFA) for five minutes and two-month neuronal cultures were fixed in with 4% PFA for 30 minutes. Samples were permeabilized and blocked in 0.5% PB-Triton and 5% BSA for one hour. Primary antibodies: anti- Nav1.2, Cat#: ASC-002 (Alomone Labs, Jerusalem, Israel); MAP2, Cat#: AB5392 (Abcam, Cambridge, United Kingdom); SYP, Cat#: AB14692 (Abcam); SOX2, Cat#: AB97959 (Abcam); NES, Cat#: ab22035 (Abcam) were incubated at 4°C overnight and secondary antibodies were incubated at room temperature for one hour. Images were captured on an Opera Phenix high-content imager at 20x magnification (PerkinElmer, Waltham, MA) and analyzed using their Columbus Image Data Storage and Analysis System software. Synapse density was quantified by having the software find puncta in the area surrounding the neurites. Images were analyzed from two lines from each genotype, three differentiations, with at least six wells with five images from each well. Data were plotted as mean ±SEM. Statistical analysis was performed using a one-way ANOVA and results were considered significant at p-value <0.05 (GraphPad Prism).

SCN2A pulldown and simple western blot- protein simple wes/jess platform

Nav1.2 membrane protein was isolated from two-month neurons by preincubation of the neurons with the 100ug/ml leupeptin (Sigma) for 30 minutes at 37°C, biotinylation of membrane proteins in 1.5mg/ ml sulfo-NHS-SS-biotin (Sigma) for 20 minutes at 4°C rotating, cell lysis, and pull-down of the biotinylated protein using neutravidinagarose beads (Pierce, Waltham, MA). Both membrane Nav1.2 and total Nav1.2 samples were probed using Nav1.2 ASC-002 (Alomone Labs) at 1:500 via western using the Jess High Molecular Weight 66- 440 kDa Separation Module (Protein Simple, San Jose, CA). Total Nav1.2 samples were normalized to total protein using the Protein Simple- Total Protein Assay (Protein Simple) and membrane Nav1.2 was normalized to input Nav1.2 protein. Data were plotted as mean ±SEM. Statistical analysis was performed using a one-way ANOVA and results were considered significant at p-value <0.05 (GraphPad Prism).


For manual patch clamp recording, neurons were cultured at a density of 3×105 cells on 15mm glass coverslips placed in a chamber on the stage of an inverted microscope. Upon formation of the whole-cell conformation, the cell was constantly perfused with an extracellular solution containing (in mM): 74.5 NaCl, 74.5 choline Cl, 2.5 BaCl2, 2 KCl, 1 MgCl2, 5 glucose, 10 HEPES, 0.3 CdCl2, 3 4-aminopyridine, pH 7.4, 310 mOsm/L. The pipette electrode was filled with an intracellular solution containing (in mM): 115 Cs methanesulfonate, 20 CsCl, 3 NaCl, 4 MgATP, 0.3 Na2GTP, 10 EGTA and 20 HEPES, pH 7.2, 290 mOsm/L. Currents were measured by whole-cell patch clamp using an Axopatch 200B amplifier and pClamp 11 software (Molecular Devices, San Jose, CA), digitized at 20 kHz and lowpass filtered at 5 kHz. Series resistance was 75% compensated. Currents were leak subtracted using a P/4 protocol.

For automated Syncropatch electrophysiology (Nanion, Germany), cDNA of the a subunit of the human Nav1.2 WT and V1282F mutant was transfected into HEK293 cells (76 μg each per T75 flask). Transfected cells were kept at 37°C for the first day and moved to 30°C for the second day before experiment. Single-hole chips with medium resistance were used. The extracellular solution contained (in mM): 149 NaCl, 2.8 CaCl2, 4 KCl, 1 MgCl2, 5 glucose, 10 HEPES, pH 7.4, 310 mOsm/L. The intracellular solution contained (in mM): 110 CsF, 10 NaCl, 10 KCl, 10 EGTA, 10 HEPES, pH7.2, 280 mOsm/L. Currents were digitized at 20 kHz and lowpass filtered at 5 kHz. Series resistance was automatically compensated. Currents were leak subtracted.

The voltage protocol was the same for both manual and automated patch clamp. From a holding potential of -100 mV, a 100-ms preconditioning pulse ranging from -100 mV to +40 mV (in 10 mV increments) was followed by a 10 ms test pulse to 0 mV and subsequent return to -100 mV. The time interval between preconditioning pulses was five seconds. The peak current amplitudes during the preconditioning pulses were used to calculate the voltage dependence of activation (I-V/G-V). The peak current amplitudes during the test pulses were used to calculate the current density and voltage dependence of inactivation. All electrophysiological recordings were performed at room temperature.

Electrophysiology data analysis

V1/2, the voltage at which the conductance is 50% of the maximum value, was obtained by fitting the conductance-voltage (G-V) relationship to a Boltzmann function. Kinetic parameters were obtained by fitting the data with a single exponential function. Data fitting and statistical analyses (two-tailed Student’s t-test or two-way ANOVA as indicated) were performed using Origin (Northampton, MA, USA). Results were considered statistically significant at p-value <0.05. Experimental data are reported as mean ±SEM, which result from independent measurements on n different cells.


Generation and characterization of isogenic SCN2A V1282F hiPSCs and neurons

To investigate whether the SCN2AV1282F/V1282F variant has any effect in hiPSC-derived cortical neurons, we used CRISPR/CAS9 genome editing tools to introduce the (G/T) variant into control hiPSCs. The (G/T) variant is in exon 19 and results in the substitution of a valine residue for a phenylalanine residue in the third segment of the third transmembrane domain in Nav1.2 (Figure 1A). This variant has been identified in two schizophrenia patients, is absent from the control EVS population and statistics suggest this is a nonrandom association by the Fischer’s exact test, 2-tailed p-value=0.034 [17]. However, the functional effects of V1282F are unknown.