Int J Clin Exp Pathol 2016;9(8):8622-8626 www.ijcep.com /ISSN:1936-2625/IJCEP0009658

Original Article Functional implication of KCNJ10 polymorphism in childhood

Jing Zhang1*, Xueqin Xiong2*, Jinqiu Wang3, Jianmin Du4, Zhanli Liu4, Liqiong Chen4, Xianmei Huang4, Chunming Jiang4

1Department of Pediatrics, Maternity and Children Hospital, Tangshan 063000, Hebei Province, China; 2Department of Pediatrics Neurology, Wuhan Medical Care Center for Women and Children, Wuhan 430016, Hubei Province, China; 3Department of Pediatrics Neurology, Guangxi Maternity Hospital, Nanning 530000 Guangxi Province, China; 4Department of Pediatrics, Hangzhou First People’s Hospital, Hangzhou 310003, Zhejiang Province, China. *Equal contributors. Received April 28, 2015; Accepted June 22, 2015; Epub August 1, 2016; Published August 15, 2016

Abstract: As a commonly occurred brain dysfunction, epilepsy has a sudden onset and is believed to be related with abnormal potassium level and firing pattern of brain neurons. The genetic factor has been implicated under the pathogenesis of epilepsy especially in children patients. KCNJ10 gene locates on 1q22-q23 and encodes for one important rectifying . This study thus aimed to investigate the correlation be- tween KCNJ10 gene polymorphism and childhood epilepsy. A total of 90 core families consisting of Chinese epilepsy children (Han ethical group) were analyzed with their parents for single nucleotide polymorphism (SNP) at loci rs1186679 using PCR-restriction fragment length polymorphism (RFLP). The genotype frequency distribution at loci sr1186679 follows Hardy-Weinberg equilibrium. Haplotype relative risk (HRR) analysis revealed correlation between sr1186679 loci and childhood epilepsy (P<0.005). Transmission disequilibrium test (TDT) showed significant dif- ference in the genotype frequency distribution of between the transmissions from heterozygous parents to affected children and that in the transmission of unrelated alleles. There is a potential relationship between sr1186679 loci of KCNJ10 gene and childhood epilepsy in Chinese Han people.

Keywords: Genetics of epilepsy, gene polymorphism, restriction fragment length polymorphism, KCNJ10 gene

Introduction involve calcium-dependent sodium influx and potassium efflux [4, 5], the role of related potas- Caused by abnormal synchronized hyperpolar- sium channel is worth further studies. ization firing of brain neurons, epilepsy is mani- fested with sudden onset and temporary brain KCNJ10 gene locates chromosome 1q22-q23 dysfunction. More than half epilepsy patients and encodes for one inwardly rectifying potas- had their first onset during childhood, suggest- sium channels, which can be activated by ing the involvement of genetic factors in the endogenous adenosine tri-phosphate (ATP) and pathogenesis. Traditional genetic research us- helps to maintain the cytosolic potassium level ing whole genome screening has revealed so- of glial cells in the brain [6, 7]. This study the- me evidences of genetic predisposing factors refore investigated the relationship between of epilepsy [1, 2]. With advancement of genom- KCNJ10 gene polymorphism and childhood ic and proteomic techniques, epilepsy-related epilepsy. such as γ2 subunit of GABAA receptor, KCNQ2/3 potassium channel, SCN1A/B genes Materials and methods have been identified. Recent studies have established the participation of calcium ions Research objects in certain phosphorylation/de-phosphorylation mechanisms in the systemic epilepsy onset We recruited 90 core families, which consist- with fever and seizure [3]. As these processes ed of Chinese childhood epilepsy patients and KCNJ10 gene and child epilepsy

Figure 1. Sequencing results of restrictive digestion.

Table 1. Genotype frequency distribution at instruction. Extraction products were measu- loci rs1186679 red using ultraviolet spectrometer for concen- The genotypic tration and purity, and were stored at -20°C for frequency Chi-square P further use. value value AA AG GG PCR amplification of target gene fragments Transmitted 94 76 9 1.234 0.038 Non-transmitted 7 38 46 Specific designed primers (Forward: 5’-TAG GAA AGG GCT CAG CGT AG-3’; Reverse: 5’-CCT AAG ACG GGG AAA GAA GC-3’) were used to amplify Table 2. DTT analysis at loci rs1186679 target loci of KCNJ10 gene using PCR buffered Transmitted Chi-square Allele P value mix (Invitrogen, US). The amplification parame- frequency value ters were: pre-denature for 5 min at 94°C; 94°C G 15 1.203 0.026 denature for 1 min, 60°C annealing for 1min and 72°C elongation for 3 min, repeated for 30 cycles; ended with 72°C elongation for 3 min. their biological parents (all in Han ethical group). Peripheral venous blood (5 mL) were PCR products were separated by 1% agarose drawn and stored at -20°C for extracting ge- gel electrophoresis. nomic DNA. This study has been pre-approved Restriction enzyme digestion by the ethical committee of our hospital. Written consents have been obtained from all PCR products were processed by restriction children’s guardians. enzymes according to the cutting pattern and Genomic DNA extraction were further separated by agarose gel electro- phoresis. For those 300 bp length fragments, 2 mL venous blood was extracted for genomic three genotypes have been identified: (1) uncut DNA using a whole-blood DNA extraction kit homozygous (A/A); (2) heterozygous with cut- (Tiangen Biochem, China) following manual ting (A/G) and homozygous (G/G).

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Statistical analysis Discussion

Using chi-square method, we firstly tested if Epilepsy had an averaged incidence at about the distribution of genotype frequency follows 7.0% in china [5], making it one of most com- Hardy-Weinberg equilibrium. Then haplotype monly occurred neurological diseases and ca- relative risk (HRR) and transmission disequilib- using heavy burdens for patient family and the rium test (TDT) were performed. In HRR analy- whole social health care system. It is believed sis, non-transmitted alleles in patient’s parents that epilepsy, especially those with childhood were established as a reference for the calcula- onset, has certain genetic basis under multiple tion of disease allele transmission. TDT was genes, as it show certain family concentrated based on the linkage analysis in a disequilibri- pattern from epidemiology study. Besides the um scenario to observe the probability of trans- genetic factor, other influences from environ- mission of predisposing genes. SPSS 13.0 so- ment or postnatal diseases may also cause epi- ftware package was used to perform all ana- lepsy. Overall speaking, epilepsy has a very lysis. complex pathogenesis mechanism and no sin- gle gene has been confirmed to play a critical Results role in epilepsy. Current genetic studies mainly compared the clinical patients and family his- PCR amplification tory to determine the epidemiology. Recent reports performed correlation analysis between Extracted DNA had a satisfactory concentra- and childhood epilepsy risk tion, with 260/280 ratio at 1.7~1.9, suggesting gene factors using the third generation of sin- a high purity. Target gene KCNJ10 fragments gle nucleotide polymorphism (SNP) approach were amplified by PCR and sequenced at loci [1, 5]. With advancement of proteomic and sr1186679 as shown in Figure 1. genomic methods, data analysis related with Hardy-Weinberg equilibrium epilepsy predisposing factors has been exten- sively explored. With the help of bioinformatics, We analyzed the genotypes of both parents and epilepsy gene microarray data have been pro- affected children using Hardy-Weinberg equilib- cesses to obtain some genes/loci that were rium test. The obtained P value is larger than crucial, such as those encoding voltage gat- 0.05, suggesting that the frequency distribu- ed calcium/sodium/potassium channel and lig- tion of genotype basically follow Hard-Weinberg and-receptor signal pathways. equilibrium Locates within 1q22-23, KCNJ10 gene encodes Allele frequency analysis for one transmembrane inwardly rectifying po- tassium channel that can be activated by ade- We further employed HRR method to compare nosine tri-phosphate (ATP) which is widely dis- the allele frequency distribution between pa- tributed and evolutionally conserved among all tients and their families. Results showed sig- mammalian cells, thereby playing a certain role nificant relationship between loci rs1186679 in buffering intracellular potassium level in and childhood epilepsy due to significant differ- brain glial cells. This study employed both TDT ence of allele frequency (P<0.05), as shown in and HRR linkage analysis to test one certain Table 1. SNP loci (rs1186670) of KCNJ10 gene and found a close correlation of these loci with Transmission disequilibrium test childhood epilepsy.

Within all core families that we studied, TDT Studies have found prominent expression of method was applied to analyze the transmis- various potassium channels in GABA-ergic sion pattern for those at least one of parents interneurons within cerebral cortex and hippo- had heterozygous genotype. Results showed campus [6-10]. As those neurons have certain significant difference between frequencies of roles in inhibiting activity of pyramidal neurons two alleles that were transmitted from parents [6-8], it is highly likely that potassium channels to their children (Table 2). Our study thus sug- participate in the epilepsy onset under certain gested the correlation between rs1886679 conditions. Recent studies have found the pos- and childhood epilepsy. sible correlation between potassium channel

8624 Int J Clin Exp Pathol 2016;9(8):8622-8626 KCNJ10 gene and child epilepsy and selective serotonin reuptake inhibitor and potassium ions. Our future work therefore (SSRI) targets. It is well known that serotonin will be concentrated in the dynamic change of (5-HT) mainly modulates the potassium chan- cytosolic potassium level during the pathogen- nel open in an attempt to regulate the excitabil- esis process of epilepsy, thereby identifying ity of neurons, thereby playing a certain role in direct evidence and potential drug targets for epilepsy onset [9, 10]. Animals studies showed clinical treatment. that potassium channel gene knockout mice, when treated with glutamate receptor agonist On the other hand, the correlation between (such as digenic acid) and GABA receptor Cyp450 gene polymorphism and refractory epi- antagonist (pentylenetetrazole, PTZ) to induce lepsy has been established. For example, one epilepsy onset, had more severe disease, high- study analyzed the SNP pattern of Cyp3A4 er mortality rates and maximal onset strength gene in Chinese Han children with refractory compared to controlled animals [11-13], sug- epilepsy using PCR-RFLP technique. They found gesting the involvement of potassium channel that 7% of mutant heterozygous in certain loci in epilepsy onset. Further electrophysiological on affected children, but not in any of epilepsy experiments found that those potassium chan- children who had satisfactory response to med- nel gene knockout mice had significantly higher ications. Further statistical analysis found sig- frequency and amplitude of bilateral spike firing nificantly higher genotype/mutant allele fre- compared to those in wild type mice. Moreover, quency at Cyp3A4-18A polymorphism loci, c-fox, a biochemical marker for neuronal activi- compared to those patients who are responded ty, showed elevated expression in those knock- to drugs. Their results suggested certain co- out mice. All these results support the role of rrelation between Cyr3A4-18A gene polymor- potassium channel in suppressing neuronal phism and drug resistance of epilepsy, indicat- excitability and thus may work as a target for ing the potential significance of genetic diagno- epilepsy onset [14, 15]. sis in optimizing treatment plan.

It is once believed that one inducible endoge- In summary, this study found possible relation- nous mechanism existed in the brain that can ship between sr1186679 loci of KCNJ10 gene protect neurons from damages from ischemia. and childhood epilepsy in Chinese Han people, This mechanism may consist of potassium thus further suggesting the role of potassium channels as supported by previous studies in channel in the pathogenesis of epilepsy. Our cerebral ischemia models [16-18]. Based on results suggest one possible genetic diagnosis the interaction between cerebral ischemia and method for early identification of childhood epilepsy, it is believed that potassium channel onset epilepsy and are worth for further explo- plays a certain role in epilepsy onset, but with ration regarding the gene polymorphism, chan- unclear mechanisms so far. Recent studies nel function and epilepsy pathology. showed some inhibitory function of potassium channels in hippocampal GABAergic neurons Acknowledgements [19-21]. Based on numerous in vivo and in vitro studies, potassium channel may indirectly in- The study was supported by the Zhejiang Sci- duce the abnormal firing of neurons via its mod- ence and Technology Project (No. 2010KYB- ulation on neuronal excitability, thereby playing 085), The Medical and Health Technology a role in epilepsy onset. Projects of Hangzhou (No. 2010A002) and Hangzhou Key Disciplines Fund. Although great achievements have been ob- tained regarding pathogenesis of epilepsy, the Disclosure of conflict of interest detailed mechanism underlying this neural dis- order is still unclear. Past studies and our result None. both point to the potential involvement of potassium channel. With further in-depth work, Address correspondence to: Dr. Chunming Jiang, especially those for pathological mechanisms, Department of Pediatrics, Hangzhou First People’s related genes and drug target screening, clini- Hospital, 261 Huansha Road, Hangzhou 310003, cal treatment of epilepsy will be benefited from Zhejiang Province, China. Tel: +86-571-56005690; basic researches. So far little has been known Fax: +86-571-56005690; E-mail: kewuzhijiren00@ about the direct relationship between epilepsy 163.com

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