1
Role of an Atypical Cadherin Gene, Cdh23 in Prepulse Inhibition and
Implication of CDH23 in Schizophrenia
Shabeesh Balan
1
, Tetsuo Ohnishi
1
, Akiko Watanabe
1
, Hisako Ohba
1
, Yoshimi Iwayama
1
,
Manabu Toyoshima
1
, Tomonori Hara
1
,
Yasuko Hisano
1
, Yuki Miyasaka
2,3
, Tomoko Toyota
1
,
Chie Shimamoto-Mitsuyama
1
, Motoko Maekawa
1,4
, Shusuke Numata
5
, Tetsuro Ohmori
5
,
Tomomi Shimogori
6
, Yoshiaki Kikkawa
2
, Takeshi Hayashi
7
, Takeo Yoshikawa*,
1
1 Laboratory for Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama,
Japan
2 Deafness Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo,
Japan
3 Division of Experimental Animals, Graduate School of Medicine, Nagoya University,
Nagoya, Japan
4 Department of Biological Science, Graduate School of Humanities and Science,
Ochanomizu University, Tokyo, Japan
5 Department of Psychiatry, Institute of Biomedical Science, Tokushima University Graduate
School, Tokushima, Japan
6 Laboratory for Molecular Mechanisms of Brain Development, RIKEN Center for Brain
Science, Wako, Saitama, Japan
7 Agricultural Artificial Intelligence (AI) Research Office, Research Center for Agricultural
Information Technology, National Agriculture and Food Research Organization (NARO),
Tokyo, Japan
Running title: Cdh23 and prepulse inhibition
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2
*Correspondence:
Takeo Yoshikawa, MD, PhD
Laboratory for Molecular Psychiatry
RIKEN Center for Brain Science
2-1 Hirosawa, Wako, Saitama 351-0198
Japan
Tel: +81(Japan)-48-467-5968
Fax: +81(Japan)-48-467-7462
E-mail: takeo.yoshikawa@riken.jp
Abstract: 187 words
Main text: 3876 words
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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3
Abstract
We previously identified quantitative trait loci (QTL) for prepulse inhibition (PPI), an
endophenotype of schizophrenia, on mouse chromosome 10 and reported Fabp7 as a
candidate gene from an analysis of F2 mice from inbred strains with high (C57BL/6N; B6)
and low (C3H/HeN; C3H) PPI levels. Here, we reanalyzed the previously reported QTLs
with increased marker density. The highest LOD score (26.66) peaked at a synonymous
coding and splice-site variant, c.753G>A (rs257098870), in the Cdh23 gene on chromosome
10; the c.753G (C3H) allele showed a PPI-lowering effect. Bayesian multiple QTL
mapping also supported the same variant with a posterior probability of 1. Thus, we
engineered the c.753G (C3H) allele into the B6 genetic background, which led to dampened
PPI. We also revealed an e-QTL (expression-QTL) effect imparted by the c.753G>A variant
for the Cdh23 expression in the brain. In a human study, a homologous variant (c.753G>A;
rs769896655) in CDH23 showed a nominally significant enrichment in individuals with
schizophrenia. We also identified multiple potentially deleterious CDH23 variants in
individuals with schizophrenia. Collectively, the present study reveals a PPI-regulating
Cdh23 variant and a possible contribution of CDH23 to schizophrenia susceptibility.
Keywords: prepulse inhibition, quantitative trait locus, Cdh23 (CDH23), schizophrenia,
hearing loss
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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4
Introduction
Deciphering neurobiological correlates for behavioral phenotypes in terms of genetic
predispositions that affect molecular, cellular and circuit-level functions is crucial for
understanding the pathogenesis of psychiatric disorders.
1
However, polygenicity and the
inherent limitations of psychiatric diagnosis based on the subjective experiences of patients
have impeded this endeavor.
2, 3
With efforts to classify psychiatric disorders based on the
dimensions of observable behavioral and neurobiological measures, endophenotype-based
approaches for elucidating genetic liability have attracted interest because these approaches
could mitigate clinical heterogeneity.
4, 5
Among the behavioral endophenotypes, the prepulse inhibition (PPI) of acoustic
startle response, a reflection of sensorimotor gating, has been consistently reported to be
dampened in psychiatric disorders, particularly in schizophrenia.
6, 7
As a robust and heritable
endophenotype in schizophrenia,
8-11
the genes/variants conferring the risk of dampened PPI
can help to elucidate the genetic architecture of schizophrenia.
12, 13
We have previously
performed a large-scale quantitative trait loci (QTL) analysis for PPI and mapped six major
loci through an analysis of 1,010 F2 mice derived by crossing selected inbred mouse strains
with high (C57BL/6NCrlCrlj: B6Nj) and low (C3H/HeNCrlCrlj: C3HNj) PPI.
14
Among these
six loci, the chromosome-10 QTL showed the highest logarithm of odds (LOD) score, and we
identified fatty acid-binding protein 7 (Fabp7) as one of the candidate genes in the PPI
regulation and schizophrenia pathogenesis.
14, 15
However, this high LOD score cannot be
explained by a single gene, considering the low phenotypic variance attributed to the
individual genes/markers, suggesting the presence of additional causative gene(s).
16
Therefore, in the current study, we aimed to (i) delineate additional gene(s) that regulate the
PPI phenotype by reanalyzing the QTL with higher marker density, (ii) validate the candidate
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.29.360180doi: bioRxiv preprint
5
gene variant(s) by analyzing causal allele knock-in mice, and (iii) examine the potential role
of candidate gene in schizophrenia susceptibility.
Materials and Methods
Animals
All animal experiments were approved by the Animal Ethics Committee at RIKEN. For the
QTL analysis, we genotyped F2 mice from our previous study.
14
For other experiments,
C57BL/6NCrl (B6N) and C3H/HeNCrl (C3HN) mice were used (Japan's Charles River
Laboratories, Yokohama, Japan). Note that the previously used mouse strains
C57BL/6NCrlCrlj (B6Nj) and C3H/HeNCrlCrlj (C3HNj) are no longer available because
their supply ended in 2014.
16
Human DNA Samples
Studies involving human subjects were approved by the Human Ethics Committee at RIKEN.
All participants in the genetic studies gave informed written consent. For resequencing the all
protein-coding exons of the CDH23 gene, a total of 1,200 individuals with schizophrenia
(diagnosed according to DSM-IV) of Japanese descent (657 men, mean age 49.1±14.0 years;
543 women, mean age 51.1±14.4 years) were used. For subsequent genotyping, an additional
811 individuals with schizophrenia were included, for a total of 2,011 affected individuals
(1,111 men, mean age 47.2 ± 14.1 years; 901 women, mean age 49.2 ± 14.7 years), along
with 2,170 healthy controls (889 men, mean age 39.2 ±13.8 years; 1,281 women, mean age
44.6 ± 14.1 years). The samples were recruited from the Honshu region of Japan (the main
island), where the population fall into a single genetic cluster.
17, 18
Additionally, we also used
genetic data of 8,380 healthy Japanese controls from the Integrative Japanese Genome
Variation Database (iJGVD) by Tohoku Medical Megabank Organization (ToMMo)
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.29.360180doi: bioRxiv preprint