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Identification of Candidate Genes for Generalized Tonic–Clonic Seizures in Noda Epileptic Rat

Abstract

The Noda epileptic rat (NER) exhibits generalized tonic–clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

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Acknowledgements

This study was supported in part by JSPS KAKENHI Grant Numbers JP12680810 to KK and JP20240042 to TS, and by Japan Epilepsy Research Foundation to YO. We are thankful to the National Bio Resource Project–Rat (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing NER/Kyo rats. We are grateful to C. Yamane, K. Kumafuji, and Z. Cui for technical assistance in animal breeding and care, and to H. Yamazoe for phenotyping of backcross progeny.

Author information

Correspondence to Takashi Kuramoto or Tadao Serikawa.

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Conflict of interest

Takashi Kuramoto, Birger Voigt, Satoshi Nakanishi, Kazuhiro Kitada, Tadashi Nakamura, Kaori Wakamatsu, Minako Yoshihara, Mikita Suyama, Risa Uemura, Miyuu Tanaka, Mitsuru Kuwamura, Saki Shimizu, Yukihiro Ohno, Masashi Sasa and Tadao Serikawa declare that they have no conflict of interest.

Ethical Approval

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. All applicable international and institutional guidelines for the care and use of laboratory animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not describe any studies with human participants performed by any of the authors.

Additional information

Edited by Stephen Maxson.

Electronic Supplementary Material

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Supplementary Table 1. Survey of ERV insertion in Phf24 among 169 laboratory rat strains (XLSX 29 KB)

Supplementary Table 2. 268 polymorphic microsatellite markers used for genetic analysis and development of congenic strains (XLSX 32 KB)

Supplementary Table 3. Genetic linkage analysis for GTCS in (F344×NER)F1×NER backcross progeny (XLSX 28 KB)

Supplementary Figure 1. Gene expression levels in the NF-Chr1e/5d congenic strain (open bars) and NER (filled bars) are indicated for each gene. Gene symbol and probe number are indicated in the upper part of each panel. CxL, left cortex; CxR, right cortex; Amy, amygdala; Hip, hippocampus. *; p < 0.05, **; p < 0.01. A-1, Genes located on segment 1e were down-regulated in NER. Cckbr, cholecystokinin B receptor; Clpb, ClpB homolog, mitochondrial AAA ATPase chaperonin; Dkk1, dickkopf WNT signaling pathway inhibitor 1; Dnhd1, dynein heavy chain domain 1; Hbb, hemoglobin β; Hbb-b1, hemoglobin β adult major chain; Neu3, sialidase 3, membrane sialidase; Nucb2, nucleobindin 2; Olfml1, olfactomedin-like 1; Olr59, olfactory receptor 59; Psma1, proteasome subunit α1; Rnf141, ring finger protein 141; St5, suppression of tumorigenicity 5; Tmem41b, transmembrane protein 41B; Zfp143, zinc finger protein 143. A-2, Genes located in segment 1e that were up-regulated in NER. Dennd5a, DENN/MADD domain–containing 5A; Fam168a, family with sequence similarity 168, member A; Fchsd2, FCH and double SH3 domains 2; Inppl1, inositol polyphosphate phosphatase-like 1; Lipt2, lipoyl(octanoyl) transferase 2; LOC689064, β-globin; Mical2, microtubule associated monooxygenase, calponin and LIM domain containing 2; Mrvi1, murine retrovirus integration site 1 homolog; Rab6a, RAB6A, member of RAS oncogene family; Smpd1, sphingomyelin phosphodiesterase 1, acid lysosomal; Tpp1, tripeptidyl peptidase 1. A-3, Genes located in segment 1e that were tissue-specifically up- or down- regulated in NER. Lmo1, LIM domain only 1; Syt17, synaptotagmin XVII; Ucp2, uncoupling protein 2, mitochondrial, proton carrier. B-1, Genes located in segment 5d that were down-regulated in NER. Foxe1, forkhead box E1; Nudt2, nucleoside diphosphate linked moiety X-type motif 2; Phf24, PHD finger protein 24; Vcp, valosin-containing protein; Dnajb5, DnaJ (Hsp40) homolog, subfamily B, member 5. B-2, Genes located in segment 5d that were up-regulated in NER. Grin3a, glutamate receptor, ionotropic, N-methyl-D-aspartate 3A; RGD1309821, similar to KIAA1161 protein; Sec61b, Sec61 translocon β subunit; Grhpr, glyoxylate reductase/hydroxypyruvate reductase. B-3, Genes located in segment 5d that were tissue-specifically up- or down- regulated in NER. Dcaf10, DDB1- and CUL4-associated factor 10; Gabbr2, γ-aminobutyric acid B receptor 2 (PPTX 207 KB)

Supplementary Figure 2. Sequence similarity between ERV in intron 2 of Phf24 to the rat ERV K families. A, Dot plots of ERV in Pfh24 of NER and its two rat ERV K families were created using the BLAST2 algorithm. The ERV of the NER Phf24 gene is plotted on the x-axis; the y-axis represents RnERVK8c (left) and RnERVK-8e (right). The rnERV8c sequence was obtained from Repbase (http://www.girinst.org/about/repbase.html). The accession number of the RnERVK-8e sequence was EF532341, and an 8359-bp sequence (from nucleotides 303 to 8662) was subjected to the alignment. B, Sequence similarity among three ERV sequences. Regions exhibiting significantly high similarity between two sequences are indicated by dotted lines, and sequences exhibiting significant similarity with RNLTR8A (Repbase) are indicated by black boxes. Four complete open reading frames encoding the retroviral gag, prt, pol, and env genes of RnERVK-8e are indicated by open boxes (PPTX 90 KB)

Supplementary Video 1. GTCS of NER. GTCS observed in 7-month-old female NERs (MP4 249030 KB)

Supplementary Video 1. GTCS of NER. GTCS observed in 7-month-old female NERs (MP4 249030 KB)

Supplementary Video 2. GTCS of HER. GTCS observed in 6-month-old female HERs (MP4 78886 KB)

Supplementary Video 2. GTCS of HER. GTCS observed in 6-month-old female HERs (MP4 78886 KB)

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Kuramoto, T., Voigt, B., Nakanishi, S. et al. Identification of Candidate Genes for Generalized Tonic–Clonic Seizures in Noda Epileptic Rat. Behav Genet 47, 609–619 (2017) doi:10.1007/s10519-017-9870-2

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Keywords

  • Cckbr
  • Generalized tonic–clonic seizures
  • Noda epileptic rat
  • Phf24
  • St5