Biallelic Variants in CNPY3, Encoding an Endoplasmic Reticulum Chaperone, Cause Early-Onset Epileptic Encephalopathy  Hiroki Mutoh, Mitsuhiro Kato, Tenpei Akita, Takuma Shibata, Hiroyuki Wakamoto, Hiroko Ikeda, Hiroki Kitaura, Kazushi Aoto, Mitsuko Nakashima, Tianying Wang, Chihiro Ohba, Satoko Miyatake, Noriko Miyake, Akiyoshi Kakita, Kensuke Miyake, Atsuo Fukuda, Naomichi Matsumoto, Hirotomo Saitsu  The American Journal of Human Genetics  Volume 102, Issue 2, Pages 321-329 (February 2018) DOI: 10.1016/j.ajhg.2018.01.004 Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 1 Biallelic CNPY3 Variants in Individuals with WS (A) Familial pedigrees of three individuals with CNPY3 variants. The segregation of each variant is shown. (B) CNPY3 transcripts (the UTR and coding region are represented as open and filled rectangles, respectively). Five mRNA transcripts of CNPY3 are registered in RefSeq. Human CNPY3 is 278 aa in length (GenBank: NP_006577.2 translated from GenBank: NM_006586.4) and contains an N-terminal signal sequence, a domain of unknown function (DUF3456), and a putative C-terminal endoplasmic reticulum (ER) retention signal.8 The three variants identified in our individuals were a canonical splice-site variant (c.495+1G>A), a missense variant (c.373G>C) that substituted the evolutionarily conserved glycine residue in the DUF3456 domain, and a frameshift variant (c.702_720dup) that removed the putative ER retention signal conserved among mammals (highlighted in green). Homologous sequences were aligned with CLUSTALW. (C) Top: immunoblot analysis using anti-human CNPY3 (HPA016560 against 140–249 aa of human CNPY3; Sigma Aldrich) and anti-β-actin (Abcam) antibodies. The amount of CNPY3 was severely lower in LCLs derived from two affected individuals than in control LCLs. Bottom: the amount of β-actin was similar in all individuals. Data of replicated experiments are shown. The American Journal of Human Genetics 2018 102, 321-329DOI: (10.1016/j.ajhg.2018.01.004) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 2 Brain MRI of Individuals with CNPY3 Variants Individual 1 at 7 years (A and B), individual 2 at 4 years (C and D), and individual 3 at 4 months (E and F). Axial T2-weighted images (A, C, and E) show mildly enlarged lateral ventricles in all individuals. Individuals 1 and 2 show a deformed cranium with left occipital flatness, caused by a bedridden posture and facing toward the left. Individual 3 shows cavum septi pellucidi and cavum vergae, which are usually considered normal variants. Coronal T2-weighted images (B, D, and F) show malrotation of the left, right, and bilateral hippocampus in individuals 1, 2, and 3, respectively (white arrows). The American Journal of Human Genetics 2018 102, 321-329DOI: (10.1016/j.ajhg.2018.01.004) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 3 Physical and Behavioral Characteristics of Cnpy3−/− Mice (A) Physical appearance of Cnpy3+/+, Cnpy3+/−, and Cnpy3−/− littermate mice at 20 days of age. (B) Differences in body weight (left) and rectal temperature (right) between Cnpy3+/+ (white circles; n = 15), Cnpy3+/− (gray circles; n = 16), and Cnpy3−/− mice (black circles; n = 11). (C) Representative movement trajectories of Cnpy3+/+, Cnpy3+/−, and Cnpy3−/− mice during the open field test for 5 min. (D) Comparisons of the total distances traveled (left), number of entries into the center area (middle), and proportions of the time spent in corners (right) during the test period by Cnpy3+/+ (white bar; n = 11), Cnpy3+/− (gray bar; n = 12), and Cnpy3−/− mice (black bar; n = 10). (E) Representative hindlimb positions and corresponding scores of the hindlimb clasping test. (F) Comparison of the times spent in the score 3 position during the 30-s test period of Cnpy3+/+ (white bar; n = 11), Cnpy3+/− (gray bar; n = 19), and Cnpy3−/− mice (black bar; n = 8). ∗∗∗∗p < 0.0001 by one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM. The American Journal of Human Genetics 2018 102, 321-329DOI: (10.1016/j.ajhg.2018.01.004) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 4 Enhancement in Beta-Band Power in Resting EEG of Cnpy3−/− Mice (A) Representative EEG traces during normal waking states from Cnpy3+/+ (top) and Cnpy3−/− (bottom) mice. The sections underlined in red (left) are shown to the right on an expanded scale. (B) Power density spectra of EEG traces in (A). The densities were normalized to the total signal power. (C) Averaged spectra of EEG traces from Cnpy3+/+ (black line; n = 8) and Cnpy3−/− (red line; n = 9) mice. The inset indicates a magnified view of the spectra in the range of 15–50 Hz. Data are presented as means ± SEM. ∗∗∗p < 0.001 at 27 Hz by the Mann-Whitney test. The American Journal of Human Genetics 2018 102, 321-329DOI: (10.1016/j.ajhg.2018.01.004) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 5 Enhancement and Leftward Shift of the Beta-Band Power Hump on EEG during Pilocarpine-Induced Seizure Development (A and B) Representative EEG traces during pilocarpine-induced seizure development for 90 min (top) and spectrograms of the traces (bottom), indicating the time evolution of power spectra from Cnpy3+/+ (A) and Cnpy3−/− (B) mice. Pilocarpine was administered intraperitoneally at points 1 (first 100 mg/kg at 5 min), 2 (second 100 mg/kg at 35 min), and 3 (third 100 mg/kg at 65 min). The white arrow in the spectrogram in (A) indicates the emergence of a beta-band hump 20 min after the first pilocarpine injection in a Cnpy3+/+ mouse. The arrow in the spectrogram in (B) indicates significant enhancement of the hump in a Cnpy3−/− mouse at the corresponding time. (C) Average power spectra of EEG during the seizures over the groups of Cnpy3+/+ (black line; n = 6) and Cnpy3−/− (red line; n = 7) mice were calculated and aligned at 5-min intervals. Power densities were normalized to the total power. Timing of pilocarpine injections was the same as in (A) and (B). Black and red arrows indicate the peaks of beta-band humps in Cnpy3+/+ and Cnpy3−/− mice, respectively, 20 min after the first pilocarpine injection. The humps gradually enlarged, and the peaks shifted to the slow beta-band range afterward in both types of mice. Data are presented as means ± SEM. (D) Time courses of the peak frequency of the spectral hump in the range of 10–40 Hz during seizures in Cnpy3+/+ (black line and circles; n = 6) and Cnpy3−/− (red line and circles; n = 7) mice. ∗p < 0.05, ∗∗p < 0.001 by the Mann-Whitney test. Data are presented as means ± SEM. The American Journal of Human Genetics 2018 102, 321-329DOI: (10.1016/j.ajhg.2018.01.004) Copyright © 2018 American Society of Human Genetics Terms and Conditions