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

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. Overall design: 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.