Nine patients with KCNQ2-related neonatal seizures and functional studies of two missense variants

Patients and clinical data collection

The institutional review board of the Faculty of Medicine, Chulalongkorn University approved this study (IRB No. 264/62) which follows the Declaration of Helsinki Guidelines and all subsequent amendments. Written informed consents were obtained from parents or legal guardians of the participants. From June 2016 to December 2020, we studied 104 patients with infantile-onset pharmaco-resistant epilepsy, defined as failure of adequate trials of two antiepileptic drugs20 who underwent exome or genome sequencing at King Chulalongkorn Memorial Hospital. The cohort is described in the authors’ previous work21. Nine patients were found to harbor pathogenic or likely pathogenic variants in the KCNQ2 gene. The detailed demographic data and clinical characteristics were collected. The diagnosis of benign/self-limited neonatal epilepsy (OMIM#121200) or KCNQ2-related developmental and epileptic encephalopathy (DEE; OMIM#613720) weremade based on ILAE Classification and Definition of Epilepsy Syndromes with Onset in Neonates and Infants22,22.

Exome, genome sequencing, bioinformatics and variant prioritization

Trio exome or genome was performed for all nine families. Genomic DNA was isolated from peripheral blood leucocytes. For exome sequencing, the DNA was enriched by SureSelect Human All Exon V5 kits (Agilent Technologies, Santa Clara, CA) and sent to Macrogen Inc., Seoul, South Korea. Illumina HiSeq 2000 Sequencer was used with a target output of 6 GB. For genome sequencing, DNA was sent to Beijing Genomics Institute (BGI), China. Sequence reads in FASTQ sequencing files were aligned to the Human Reference Genome hg19 from UCSC using Burrows-Wheeler Alignment (BWA) software (bio-bwa.sourceforge.net/). Single nucleotide variants (SNVs) and small insertions/ deletions (indels) were detected by GATK Haplotypecaller and annotated by dbSNP&1000G.

A list of 728 genes associated with Genetic Epilepsy Syndrome according to Genomics England PanelApp Version 2.2 (nhsgms-panelapp.genomicsengland.co.uk/panels/402/v2.2) were used for the first step of analysis. The analysis included gene list outside the “green entities” with high levels of evidence for disease association. In silico analysis including SIFT (sift.jcvi.org/); Polyphen-2, (genetics.bwh.harvard.edu/pph2/); M-CAP (bejerano.stanford.edu/mcap/); CADD (cadd.gs.washington.edu/); recommended pathogenicity threshold > 20) were also used to predict variants’ pathogenicity. Variants were considered novel if they were not previously reported in Genome Aggregation Database (gnomAD V3.1.2; gnomad.broadinstitute.org/), ClinVar (www.ncbi.nlm.nih.gov/clinvar/), not documented in PubMed scientific literature, and were not identified in our in-house Thai reference exome database (T-REx)23. Variants were classified according to the recommendation of American College of Medical Genetics and Genomics (ACMG)24.

Plasmid construction, protein expression and localization in HEK293 cells

pcDNA3.1 + /KV7.2-DYK (#NM_004518.6) and pcDNA3.1 + /KV7.3-DYK (# NM_004519.4) were synthesized by Genscript (Piscataway, New Jersey, USA). cDNAKv7.2 was subcloned into the pcDNA3.1/CT-GFP-TOPO Vector (Invitrogen, Waltham, Massachusetts, USA), resulting in a C-terminal GFP, used to identify transfected cells. The two novel mutations, c.774C > G p.(N258K) and c.836G > A p.(G279D), were introduced using QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, California, USA). Mutagenic primers are listed in Supplemental Table S1. All plasmids were verified using Sanger sequencing.

HEK 293 cells were transiently transfected with 2.5 μg of either wild-type, p.(N258K), or p.(G279D) Kv7.2 for homotetrameric experiments using Lipofectamine 3000 (Invitrogen). For heterotetrameric experiments, different types of Kv7.2 were co-transfected with wild-type Kv7.3 (wild-type Kv7.2/Kv7.3, p.(N258K) Kv7.2/Kv7.3, p.(G279D) Kv7.2/Kv7.3, wild-type Kv7.2/p.(N258K) Kv7.2/Kv7.3, wild-type Kv7.2/p.(G279D) Kv7.2/Kv7.3) at a 1:1 ratio (1.25g: 1.25g) or 1:1:2 ratio (0.625g: 0.625 g: 1.25 g) and assayed 48- or 72- hours post transfection.

Western blot analysis was used to determine Kv7.2 expressions in transfected HEK293 cells. Cells were scraped, centrifuged and resuspended. Membrane protein and cytoplasmic protein were extracted using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher). The membrane protein and cytoplasmic protein were separated based on their hydrophobicity using two different buffers, a permeabilization buffer and a solubilization buffer. After that, 20 ug of denatured proteins were separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Invitrogen). Transferred membranes were blocked for 1 h at room temperature (RT) in 5% nonfat dry milk and incubated overnight at 4 °C with monoclonal anti-Kv7.2 Rabbit mAb (1:500) (Cell Signaling Danvers, Massachusetts, USA) in 5% Bovine serum albumin (BSA) and then with monoclonal anti-GAPDH Rabbit (1:500) (Cell Signaling) in 5% BSA as a loading control. The membranes were then incubated for 2 h at RT with Anti-rabbit IgG, horseradish peroxidase (HRP)-linked Antibody (1:1000) (Cell Signaling) in 5% BSA. Specific protein bands were visualized using the ImageQuant Las4000 chemi-image (GE Healthcare).

For immunofluorescence, cells were fixed with 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.2% Triton X-100 for 20 min (heterotetrameric channels), and then blocked with 1% BSA for 1 h at RT. Cells were labeled overnight at 4 °C with polyclonal anti- KCNQ3 (1:800) (Invitrogen# PA1-930) in 0.1% BSA, then incubated with a Donkey Anti-Rabbit IgG H&L(1:500) (Alexa Fluor 647, abcam) in 0.1% BSA for 2 h at RT and mounted using ProLong Gold Antifade Mountant (Invitrogen# P36934). Confocal images were obtained using a Zeiss axio observer z1 microscope equipped with a 63 × oil immersion lens. The Zen 3.4 (blue edition) software was used for image analysis.

Electrophysiological and potassium (K +) gating properties data acquisition and analysis

At 48-h post transfections, cells were sorted using fluorescence activated cell sorting (FACS) and seeded onto Poly-D-lysine (2 mg/mL) pre-coated 35 mm petri dishes. Potassium currents were investigated by standard whole-cell patch-clamp technique, using an Axopatch 200-B amplifier (Axon Instruments, Inc, Melbourne, Australia), controlled by a Digidata 1440A digitizer. The pCLAMP software (Version 10, Axon Instruments, Inc) was used for data acquisition and analysis. The extracellular solution contained (in mM) the following: 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.3–7.4 titrated with NaOH; osmolality 315–320 mOsm/Kg. Microelectrodes (borosilicate glass capillaries BF150-86-10, Sutter/USA) had resistances of 1–3 MΩ when pulled with intracellular solution contained (in mM) the following: 140 K-gluconate, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES, and 10 Mg‐ATP, pH 7.3–7.4 titrated with KOH; osmolality 285–290 mOsm/Kg. Cells were held at –80 mV, voltage steps of 10 mV increment during 1.3 s from − 110 mV to + 50 mV and tail currents were recorded at a potential of − 40 mV.

Electrophysiological properties were demonstrated by representative raw M-current traces, current–voltage (I–V) curves were used to estimate the conductance and reversal potential, and M-current density was used to estimate electrical capacitance16. It was calculated as peak current (pA) at + 50 mV divided by the cell capacitance (pF). K + gating properties were investigated by the half-activating voltage (V1/2) detected depolarization of channels, the slope conductance (k) of channels. There were calculated by the Boltzmann function: I = 1/[1 + exp(V½–V)/k], V½ = half-activating voltage, k = slope, at tail current amplitudes at − 40 mV. The membrane resistance (Rm) was calculated according to Ohm’s law (V = IR), I is the current, V is the voltage and R is the resistance detected the resistance of the cell membrane when ions flow through it21. The membrane time constant (Tau) were calculated by Rm·Cm, Cm is membrane capacitance detected time of change membrane potential25.

Data analysis

Data are expressed as means ± S.E.M. Statistical analyses were carried out using SPSS and GraphPad Prism 9.4.0. One-way ANOVA followed by Tukey’s post-hoc test was used to determine the statistical difference of experimental responses from untransfected cells and transfected cells, as well as the responses between wild type and mutants. The numbers of samples (n) have been indicated in the figure legends. Statistical significance was defined by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Read more here: Source link