Identification of candidate genes for generalized tonic–clonic seizures in Noda Epileptic Rat

The Noda epileptic rat (NER) exhibits generalized tonic–clonic seizures (GTCS). A genetic linkage analysis demonstrated two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. In single-locus congenic lines, neither wild-type Ner1 nor Ner3 allele suppressed GTCS, but both wild-type alleles suppressed GTCS when they were combined in double-locus congenic lines. Global expression analysis showed that Cckbr and St5 locating within Ner1 and Phf24 locating within Ner3 were significantly　downregulated. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome. PHF24 protein was almost absent in the NER brain. It has been known that the Phf24 gene encodes Gαi-interacting protein which is involved in GABAB receptor signaling pathway. Together, we concluded that Cckbr, St5, and Phf24 were strong candidate genes for GTCS in NER. NER (6 and 18 weeks of age, n=6 each) and GTCS-resistant NF-Chr1e/5d congenic (6 and 18 weeks of age, n=6 each) were used for gene expression analysis. Brain sampling was conducted under interictal conditions with no observable seizures for at least 2 h before sampling. Rats were deeply anesthetized with pentobarbital (80 mg/kg, i.p.), and brains were removed and chilled in ice-cold saline. The amygdala, cortex (Left and Right) and hippocampus were then dissected immediately and stored in RNAlater (Qiagen, Tokyo, Japan). Total RNA was isolated using ISOGEN II (Nippon Gene Co., Ltd, Tokyo, Japan). To reduce variations, RNA samples from 3 rats in each group were pooled. Two-color microarray-based gene expression analyses were conducted using SurePrint G3 Rat GE 8×60K Microarray Kit (Agilent Technologies, Santa Clara, CA, USA). Mean values with standard deviation were calculated in each probe and compared between NER and the age-matched NF-Chr1e/5d. Differences in gene expression levels were evaluated by t-test. To eliminate the Type 1 false positive errors, we interpreted that the P value less than 0.01 was significant.

野田癫痫大鼠（Noda epileptic rat, NER）会出现全身性强直-阵挛发作（generalized tonic–clonic seizures, GTCS）。遗传连锁分析显示，存在两个与GTCS相关的基因座：位于1号染色体的Ner1，以及位于5号染色体的Ner3。在单基因座同源导入近交系中，野生型Ner1与Ner3等位基因均无法抑制GTCS，但当二者结合构建双基因座同源导入近交系时，两种野生型等位基因均可抑制GTCS。全基因组表达分析显示，定位于Ner1区域的Cckbr与St5，以及定位于Ner3区域的Phf24均出现显著表达下调。从头细菌人工染色体（BAC）测序发现，NER基因组中Phf24基因的第2内含子区域存在一段内源性逆转录病毒序列的插入。NER大脑中几乎检测不到PHF24蛋白。已知Phf24基因编码Gαi相互作用蛋白，该蛋白参与GABAB受体信号通路。综上，本研究认定Cckbr、St5与Phf24为NER中GTCS的强势候选基因。
本研究选用6周龄与18周龄的NER（每组n=6）以及抗GTCS的NF-Chr1e/5d同源导入近交系大鼠（6周龄与18周龄，每组n=6）进行基因表达分析。脑组织采样于发作间期进行：采样前至少2小时未观察到癫痫发作。大鼠经戊巴比妥（80 mg/kg，腹腔注射）深度麻醉后，取脑并置于冰冷生理盐水中冷却。随后立即分离杏仁核、左右大脑皮层以及海马体，将组织储存在RNAlater试剂（Qiagen，日本东京）中。采用ISOGEN II试剂（日本东京Nippon Gene有限公司）提取总RNA。为减少实验误差，将每组3只大鼠的RNA样本进行混合。采用SurePrint G3大鼠基因表达8×60K微阵列试剂盒（安捷伦科技，美国加利福尼亚州圣克拉拉）进行双色微阵列基因表达分析。对每个探针的信号值计算平均值与标准差，并将NER与同年龄段的NF-Chr1e/5d大鼠进行比较。采用t检验评估基因表达水平的差异。为排除I类假阳性误差，本研究将P值小于0.01认定为具有统计学显著性。