The two patients are siblings. The elder sister is 3 years old, and she has suffered from epilepsy since the age of 7 months. The younger brother is 1 year 8 months old. He has had epilepsy since the age of 6 months, and the seizure semiology was similar to that of his elder sister. Their parents were healthy, and their grandfather had a cerebral contusion at 40 years old and has had secondary epilepsy since then.
Genomic DNA was extracted from peripheral blood samples from the two patients and their parents using standard protocol. DNA libraries were prepared using a Joy Orient DNA Library Preparation Kit (Joy Orient Translational Medicine Research Center Co. Ltd., Beijing, China), in which platform-specific adaptors and unique DNA indexes are ligated. The libraries were tested for enrichment by quantitative polymerase chain reaction (qPCR) and for size distribution and concentration using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA). Targeted next-generation sequencing was performed using a SeqCap Clinical Exome sequencing panel (Roche AG., Basel, Switzerland) customized by Joy Orient, which targeted 3,372 genes that are potentially associated with 4,213 known diseases with Mendelian inheritance by capturing 7,465,978 bp of targeted exon regions using 91,867 probes. A HiSeq 2500 sequencer was used to sequence the samples as instructed by protocols (version 3, Illumina, Inc., San Diego, California). Raw image files were processed by the BclToFastq (Illumina) for base calling and generating the raw data. The low-quality variations were filtered out using the quality score >= 20 (Q20). The sequencing reads were aligned to the National Center for Biotechnology Information (NCBI) human reference genome version hg19 using BWA. Samtools and Pindel were used to screen single-nucleotide polymorphism (SNP), insertion, and deletion mutations of the sequence. All genetic variants were screened by pathogenicity, mode of inheritance, and clinical phenotypes, and we identified two variants, c.601G > A and c.626T > C, in the KCNJ10 alleles, respectively, that are potential disease causing in the patients.
To predict the possible impact of missense mutations on the structure and function of Kir4.1, four bioinformatic tools were used: PolyPhen-2 (polymorphism phenotyping, v2), PROVEAN (protein variation effect analyzer, v1.1.3), SIFT, and MutationTaster. The degree of amino acid conservation of KCNJ10 was evaluated with Clustal Omega.
Human KCNJ10 cDNA was cloned, fused with a human influenza hemagglutinin (HA) tag on its N terminus and inserted into a pCDH-CMV-MCS-EF1-copGFP vector (System Biosciences, CD511B-1). This KCNJ10-HA served as a template into which the c.601G > A or c.626T > C variant was introduced using a KOD-plus-mutagenesis kit (SMK-101, TOYOBO). The integrity of all the constructs was verified by Sanger sequencing.
Chinese hamster ovary (CHO) cells were plated in poly-d-lysine-coated sterile glass coverslips and cultured in Dulbecco's modified Eagle medium (DMEM)/F-12 (Gibco, 11320033) with 10% fetal bovine serum (FBS) (FS101-02, TransGen Biotech, China). After 60% confluence was reached, CHO cells were transfected with the wild-type (WT), c.601G > A, and c.626T > C KCNJ10 plasmids or co-transfected with WT and c.601G > A, WT and c.626T > C, and c.601G > A and c.626T > C plasmids using TurboFect transfection reagents. Cell transfected with empty vector (mock transfected) served as a control. Twelve hours after transfection, patch clamp experiments were conducted on fluorescing cells under ultraviolet light. Typically, we used a bathing solution (containing (mM) 40 KCl, 100 NaCl, 1.8 CaCl2, 0.53 MgCl2, 5.5 glucose, and 5.5 HEPES-KOH (pH 7.4)) and an intracellular solution [containing (mM) 140 KCl, 5 K2 ATP, 1 MgCl2, 5 EGTA, and 5 HEPES-KOH (pH 7.3)]. Currents were recorded with voltage steps from -120 to +40 mV in 10-mV steps, with a holding potential of 0 mV. Current signals were amplified using a Multiclamp 700B amplifier (Axon Instruments), digitized using a 1440A digidata, and recorded with Clampex 10.6. Data were analyzed using MiniAnalysis (Synaptosoft). Each average steady-state current was measured at the end of 300 ms during the voltage step-pulses duration.
293T cells plated in poly-d-lysine-coated dishes (60 mm) were transfected at 70% confluence with 6 mug of WT or mutant KCNJ10 plasmids. Twenty-four hours later, cell surface protein biotinylation was performed using a commercial biotinylation kit. Briefly, cells were incubated with Sulfo-NHS-LC-biotin (Thermo Scientific, 21335) (0.5 mg/ml) for 20 min at 4 C. The reaction was quenched, and the cells were scraped for lysis. Following cell lysis, the protein supernatant was collected and then incubated with immobilized streptavidin agarose (Thermo Scientific, 20347) overnight at 4 C. Biotinylated proteins were eluted with sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blot analysis. Nonbiotinylated samples were used as a negative control (input).
293T cells plated in six-well cell culture plates were transfected with the empty vector, WT, or mutant KCNJ10 plasmids for 24 h. Total mRNA was extracted using TRIzol reagent according to the manufacturer's instructions and then reverse transcribed to cDNA by a PrimeScript reverse transcription reagent kit (TaKaRa, RR047A). The expression of KCNJ10 was detected by FastStart Universal SYBR Green Master (Rox) (Roche, 04913914001) on a LightCycler  480 System (Roche). Primers for KCNJ10-HA were forward 5'-CTGAAAAGCTCAAGTTGGAGGA-3', reverse 5'-GTAATCTGGAACATCGTATGGGTAG-3'; primers for internal control ACTB were 5'-TGACGTGGACATCCGCAAAG-3', reverse 5'-CTGGAAGGTGGACAGCGAGG-3'. All samples were assayed in triplicate in an optical 96-well reaction plate. Data analysis was performed by the comparative Ct method, and beta-actin served as the reference gene. The results were expressed as the ratio of Kir4.1-HA mRNA level to beta-actin mRNA level.
293T cells transfected with WT or 601G > A KCNJ10 plasmids were incubated for 6 h with 0.1% dimethylsulfoxide (DMSO), lysosomal inhibitors (100 mug/ml leupeptin (Sigma, 62070) and 50 mM of NH4Cl) or proteasomal inhibitors [10 muM of MG132 (MCE, HY-13259)]. After treatment, cells were lysed and subjected to western blot analysis.
Statistical analyses were performed with GraphPad Prism 6 (GraphPad Software Inc., La Jolla, USA). Data distribution was assessed by a Kolmogorov-Smirnov nonparametric test of equality. Differences among multiple means were assessed, as indicated, by one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test. Error bars represent standard error of the mean (SEM). Null hypotheses were rejected at the 0.05 level.