Content uploaded by Takashi Fujisawa
Author content
All content in this area was uploaded by Takashi Fujisawa on Nov 28, 2016
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
November 2016 | Volume 7 | Article 1841
ORIGINAL RESEARCH
published: 16 November 2016
doi: 10.3389/fpsyt.2016.00184
Frontiers in Psychiatry | www.frontiersin.org
Edited by:
Ashok Mysore,
St. John’s Medical College Hospital,
India
Reviewed by:
Kürs¸at Altınbas¸,
Çanakkale Onsekiz Mart University,
Turkey
Meera Purushottam,
National Institute of Mental Health
and Neurosciences, India
*Correspondence:
Kazuyuki Shinohara
kazuyuki@nagasaki-u.ac.jp
Specialty section:
This article was submitted to Child
and Adolescent Psychiatry,
a section of the journal
Frontiers in Psychiatry
Received: 05July2016
Accepted: 31October2016
Published: 16November2016
Citation:
FujisawaTX, NishitaniS, IwanagaR,
MatsuzakiJ, KawasakiC, TochigiM,
SasakiT, KatoN and ShinoharaK
(2016) Association of Aryl
Hydrocarbon Receptor-Related Gene
Variants with the Severity of Autism
Spectrum Disorders.
Front. Psychiatry 7:184.
doi: 10.3389/fpsyt.2016.00184
Association of Aryl Hydrocarbon
Receptor-Related Gene Variants
with the Severity of Autism
Spectrum Disorders
Takashi X. Fujisawa1,2, Shota Nishitani1, Ryoichiro Iwanaga3, Junko Matsuzaki4,
Chisato Kawasaki5, Mamoru Tochigi6, Tsukasa Sasaki7, Nobumasa Kato8 and
Kazuyuki Shinohara1*
1 Department of Neurobiology and Behavior, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan,
2 Research Center for Child Mental Development, University of Fukui, Fukui, Japan, 3 Department of Occupational Therapy,
Graduate School of Health Sciences, Nagasaki University, Nagasaki, Japan, 4 Nagasaki Municipal Welfare Center for the
Handicapped, Nagasaki, Japan, 5 Sasebo Child Development Center, Sasebo, Japan, 6 Department of Neuropsychiatr y,
Teikyo University School of Medicine, Tokyo, Japan, 7 Department of Physical and Health Education, Graduate School of
Education, The University of Tokyo, Tokyo, Japan, 8 Medical Institute of Developmental Disabilities Research, Showa
University, Tokyo, Japan
Exposure to environmental chemicals, such as dioxin, is known to have adverse effects on
the homeostasis of gonadal steroids, thereby potentially altering the sexual differentiation
of the brain to express autistic traits. Dioxin-like chemicals act on the aryl hydrocarbon
receptor (AhR), polymorphisms, and mutations of AhR-related gene may exert patholog-
ical influences on sexual differentiation of the brain, causing autistic traits. To ascertain
the relationship between AhR-related gene polymorphisms and autism susceptibility, we
identified genotypes of them in patients and controls and determined whether there are
different gene and genotype distributions between both groups. In addition, to clarify the
relationships between the polymorphisms and the severity of autism, we compared the
two genotypes of AhR-related genes (rs2066853, rs2228099) with the severity of autistic
symptoms. Although no statistically significant difference was found between autism
spectrum disorder (ASD) patients and control individuals for the genotypic distribution
of any of the polymorphisms studied herein, a significant difference in the total score of
severity was observed in rs2228099 polymorphism, suggesting that the polymorphism
modifies the severity of ASD symptoms but not ASD susceptibility. Moreover, we found
that a significant difference in the social communication score of severity was observed.
These results suggest that the rs2228099 polymorphism is possibly associated with the
severity of social communication impairment among the diverse ASD symptoms.
Keywords: autism spectrum disorder, aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator,
polymorphism, social communication, severity
Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; ASD, autism spectrum
disorder; CARS, Childhood Autism Rating Scale; DSM, diagnostic and statistical manual of mental disorders; EMB, extreme
male brain; IQ, intelligence quotient; PBDE, polybrominated diphenyl ether; PCDD/Fs, polychlorinated dibenzo-p-dioxins and
dibenzofurans; PCB, polychlorinated biphenyl; SNP, single nucleotide polymorphisms; TCDD, tetrachlorodibenzo-p-dioxin.
2
Fujisawa et al. Autism Spectrum Disorders and AhR-Related Polymorphisms
Frontiers in Psychiatry | www.frontiersin.org November 2016 | Volume 7 | Article 184
INTRODUCTION
Reports of the incidence of autism spectrum disorder (ASD) –
characterized by two core symptoms: communication and social
decits and xed or repetitive behavior (1) – have been increasing
in recent years (2). e prevalence of ASD rose from 1 per 5,000
children in 1975 to 1 per 110 children in 2009 in the United States
(3). Current estimates of the break up of possible reasons are as
follows: about 25%, attributed to diagnostic accretion; 15%, to the
growing awareness of ASD; 10%, to advanced parental age; and
4%, to geographic clustering. However, for the remaining 46% of
the cases, the underlying reasons remain unclear (3). Although a
strong genetic contribution to ASD has been suggested by many
previous studies (4, 5), the syndrome has many features that are
not well explained by genetic factors alone (6). erefore, some
researchers have considered projecting ASD as a multifactorial
disorder with both genetic and environmental inuences (7–9).
e “extreme male brain” (EMB) theory is one of the lead-
ing hypotheses for explaining the mechanism of ASD (10, 11).
e EMB theory suggests that exposure to imbalanced levels of
gonadal steroids (androgen and estrogen) could exert a patho-
logical inuence on the sexual dierentiation of the brain during
the fetal period, which may cause ASD traits in such individuals.
Prenatal gonadal steroid levels in the amniotic uid are correlated
with ASD traits in children at 12 and 24months of age (12, 13).
Exposure to environmental chemicals, especially dioxin-like
chemicals, is known to have adverse eects on the homeostasis
of gonadal steroids, thereby altering the sexual dierentiation of
the brain to express ASD traits. Dioxin-like chemicals, such as
tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated dibenzo-
p-dioxins and dibenzofurans (PCDD/Fs), and some polychlorin-
ated biphenyls (PCBs), act on the aryl hydrocarbon receptor
(AhR) (14, 15). Recent accumulating evidence suggests that
ligand-activated AhR might alter both estrogen and androgen
signals (16, 17). ese ndings further support that dioxin-like
chemical exposure during the fetal period may exert pathologi-
cal inuences on sexual dierentiation of the brain, causing ASD
traits (18).
Epidemiological studies have shown that PCB exposure at
low levels can exert adverse clinical and subclinical eects on
sociocognitive functions (19–23). Depending on geographical
location, children might be exposed varying background levels
of toxic environmental chemicals. Whether the adverse eects are
expressed or not depends on inherent individual vulnerability to
the environmental chemicals. erefore, with regard to dioxin-
like chemicals in particular, the vulnerability could be modied
by an individual’s receptor (AhR)-related gene polymorphisms.
Numerous studies have investigated the association between
AhR-related gene polymorphisms and reproductive system
diseases, such as endometriosis or infertility (24–27), because
these diseases are regarded as complex traits in which genetic and
environmental factors contribute to the disease phenotype (28).
Various studies on AhR-related gene polymorphisms, as explored
by a recent meta-analysis on endometriosis risk in Asian popula-
tions (27), have considered AhR Arg554Lys and AhR nuclear
translocator (ARNT) Val189Val as potential candidates (24–27).
Although it may be hypothesized that these two polymorphisms
contribute to the ASD phenotype by modulating vulnerability to
the environmental chemicals, little evidence is available on the
relationship between ASD and AhR-related gene polymorphisms
in humans, with the exception of the investigation on ARNT2
polymorphisms (29). erefore, the current study aimed to
determine whether polymorphisms of AhR-related genes (AhR
Arg554Lys and ARNT Val189Val) contribute to ASD susceptibil-
ity and/or severity.
First, to ascertain the relationship between the two AhR-related
gene polymorphisms and ASD susceptibility, we identied these
genotypes in patients and controls and determined whether there
are dierent gene and genotype distributions between the two
groups. Second, to clarify the relationships between the polymor-
phisms and the severity of ASD, we compared the genotypes of
AhR-related genes with the severity of ASD symptoms using the
Childhood Autism Rating Scale (CARS) (30). Finally, we applied
factor analysis to the CARS scale and tried to identify several
core symptoms, such as social communication, stereotypies, and
sensory abnormalities (31). We also tried to evaluate the relation-
ship between the severity of these symptoms and the AhR-related
gene polymorphisms because the disease severity is not always
consistent across symptoms; rather, the relative severity of dier-
ent symptoms varies among individual cases (32, 33).
MATERIALS AND METHODS
Participants
Ninety-ve children and adults with ASD participated in the
present study. Participants with ASD were recruited in two dif-
ferent geographical regions, Tokyo and Nagasaki, in Japan. e
participants in Tokyo consisted of 68 ASD patients (58 males and
10 females, mean age= 12.43 ± 7.7years) and were recruited
from the outpatient clinics of the Department of Psychiatry in
the University of Tokyo Hospital. e participants in Nagasaki
consisted of 27 ASD patients (26 male and 1 female patients,
mean age = 11.35 ± 3.2 years), and they were recruited from
two day-care facilities in Nagasaki prefecture for patients with
developmental disorders. In both areas, the diagnoses were made
by two or more senior pediatric-psychiatric clinicians through
structured interviews and reviews of clinical records according
to the DSM-IV criteria (34). Among the 68 patients in Tokyo, 66
were diagnosed with autistic disorder and 2 with Asperger’s disor-
der. Among the 27 patients in Nagasaki, 17 were diagnosed with
autistic disorder and 10 with Asperger’s disorder. Both patient
groups excluded individuals with pervasive developmental
disorder, not otherwise specied (PDD-NOS). Participants with
severe intellectual disability were excluded if they had a full-scale
intelligence quotient (FSIQ) <50 on the Wechsler Intelligence
Scale for Children (35), Wechsler Adult Intelligence Scale (36),
or the Tanaka–Binet Intelligence Scale (a Japanese revised version
of the Stanford–Binet Intelligence Scale) (37).
Additionally, 527 adults (332 men and 195 women patients,
mean age = 40.9 ± 9.7 years) were recruited as control par-
ticipants from the nearby community around the University
of Tokyo Hospital, without any psychiatric disorder disturbing
their work function. e Mini-International Neuropsychiatric
3
Fujisawa et al. Autism Spectrum Disorders and AhR-Related Polymorphisms
Frontiers in Psychiatry | www.frontiersin.org November 2016 | Volume 7 | Article 184
Interview (MINI) (38) and other surveys were administered in
the recruitment of controls to exclude those who had current
or lifetime history of mental disorders. ese individuals were
recruited from the community through advertisements as well as
an online solicitation.
e race/ethnicity of all participants was Japanese. Potential
participants were also excluded if they had any history of
substance abuse, recent substance use, head trauma with loss
of consciousness, signicant fetal exposure to alcohol or drugs,
perinatal or neonatal complications, and neurological disorders
or medical conditions.
e present study was approved by the Ethical Committees
of the University of Tokyo Graduate School of Medicine and the
Nagasaki University Graduate School of Biomedical Sciences. All
participants or parents of the aected individuals provided writ-
ten informed consent prior to their participation in this study.
e experimental protocol was conducted in accordance with the
Declaration of Helsinki.
Assessment of the Severity of ASD
e severity of ASD was assessed on the basis of the Japanese ver-
sion of the CARS (30). e assessment by CARS was performed at
the same time as genomic sampling in this study. e CARS is a
behavior-based clinical scale developed by observation and inter-
action with ASD patients. e scale has been reported to have
a high degree of internal consistency, inter-rater and test–retest
reliability, high criterion-related validity, and good discriminant
validity (39). e severity was rated for 15 items (“Relationship
to People,” “Imitation,” “Emotional Response,” “Body Use,”
“Object Use,” “Adaptation to Change,” “Visual Response,”
“Listening Response,” “Taste, Smell, Touch Response and Use,”
“Fear and Nervousness,” “Verbal Communication,” “Non-verbal
Communication,” “Activity Level,” “Level and Consistency of
Intellectual Response,” and “General Impressions”) on a scale of
1 (normal for child’s age) to 4 (severely abnormal) in units of 0.5.
In this study, experienced clinical psychologists rated the subjects
based on behavioral observation and parental reports.
Genotyping
Genomic DNA was extracted from the peripheral blood using the
standard phenol–chloroform method for set A and from the oral
mucosa of the participants using the QIAamp DNA Micro Kit
(Qiagen, Tokyo, Japan) in set B. All participants were genotyped
by real-time polymerase chain reaction (PCR) analysis using
Roche LightCycler 480 II (Roche Diagnostics, Tokyo, Japan) for
the following two single nucleotide polymorphisms (SNPs): AhR
codon 554 in exon 10 (G/A, Arg to Lys, rs2066853) and ARNT
codon 189 in exon 7 (G/C, silent mutation, rs2066853) (24–28).
Reactions were performed in 5-μl reactions, each containing 5ng
genomic DNA, 2.75μl HPLC water, 0.25μl of each TaqMan probe
(Applied Biosystems, Foster City, CA, USA), and 2.5μl TaqMan
PCR Master Mix (Applied Biosystems, Foster City, CA, USA).
e PCR cycling conditions consisted of a 10-min cycle at 95°C,
followed by 60 cycles of 95°C for 30s and 60°C for 30 s. Five
microliters of HPLC water and Mater Mix were used as a negative
PCR control in each amplication. Allele calling was performed
using LightCycler CW 1.5 soware (Roche Diagnostics).
Data Analysis
Analyses proceeded in four steps. First, the chi-squared test was
used to investigate the relationship between each AhR-related
gene polymorphism and susceptibility to ASD. Next, analysis
of variance (ANOVA) was used to compare the severity of ASD
among AhR-related gene polymorphisms. ird, to assess the
severity corresponding to several core behavioral symptoms
of ASD, factor analysis with Varimax rotation for CARS was
performed, and the factor score was calculated by regression
method for each subject. Finally, ANOVA was also used to assess
the eects of AhR-related gene polymorphisms for each severity
of discriminative behavioral symptoms of ASD identied by the
prior factor analysis. e chi-squared test, factor analysis, and
multinomial logistic regression analysis were performed using
IBM SPSS 20.0 for Windows (Statistical Package for the Social
Sciences; IBM). e ANOVA was performed using Anovakun
soware (version 4.8.0.1) in the R soware space (version 3.2.0.
for Windows, R2).
RESULTS
Genotypes of AhR-Related Genes and
Susceptibility to and Severity of ASD
e genotype and allele frequencies of AhR codon 554 and ARNT
codon 189 are shown in Tab le 1 . Fourteen samples with ARNT
codon 189 in healthy participants were excluded from the data
because the signal failed to be detected as a result of misamplica-
tion. e genotype distributions were in Hardy–Weinberg equi-
librium (p>0.05). Comparisons between genotype groups did
not demonstrate statistically signicant dierences with regard
to children’s age, sex, or IQ level. To investigate the relationship
between each AhR-related gene polymorphism and susceptibility
to ASD, the chi-squared test was used to evaluate the genotype
distribution according to developmental status. No statistically
signicant association was observed between any of the polymor-
phisms and susceptibility to ASD.
Next, to investigate the relationship between each AhR-related
gene polymorphism and the severity of ASD, one-way ANOVA
was used for the total CARS score of ASD patients as the dependent
variable and the genotype of each AhR-related gene polymorphism
as the independent variable. ere was a statistically signicant
dierence between genotype groups for ARNT polymorphism
(rs2228099), as determined by ANOVA [F(2,92)=5.69, p<0.01,
eect size f=0.352, power=0.865]. Holm’s sequentially rejective
Bonferroni posthoc test revealed that the total CARS score of the
GG genotype was statistically signicantly higher than those of
the GC genotypes [t(92)=3.17, p<0.05], whereas there were no
statistically signicant dierences in score between the CC geno-
type and both genotype groups [GC-CC: t(92)=2.18; GG-CC:
t(92)=0.10]. Additionally, there were no statistically signicant
dierences between genotype groups for AhR polymorphism
(rs2066853) [F(2,92)=0.26]. Taken together, these results suggest
1 http://riseki.php.xdomain.jp.
2 https://www.r-project.org/.
TABLE 2 | Factor loadings from factor analysis with Varimax rotationa, mean, and SD of the 15 items of the CARS.
CARS Item Factor M (SD)
Social communication Sensory and emotional response Stereotypies
Verbal communication 0.833 2.46 (0.8)
Non-verbal communication 0.563 2.37 (0.7)
Imitation 0.561 2.03 (0.8)
Visual response 0.457 0.456 2.08 (0.8)
Relating to people 0.447 0.446 2.70 (0.7)
Level and consistency of intellectual response 0.419 2.55 (0.7)
Activity level 0.642 2.16 (0.7)
Object use 0.459 0.539 1.98 (0.7)
Emotional response 0.511 2.68 (0.7)
Taste, smell, touch response and use 0.505 1.97 (0.6)
Listening response 0.504 2.12 (0.6)
Fear or nervousness 0.360 2.34 (0.6)
General impressions 0.365 0.732 2.94 (0.6)
Adaptation to change 0.549 2.36 (0.6)
Body use 0.396 0.391 0.430 2.34 (0.5)
aOnly factor loadings >0.35 are reported.
TABLE 1 | Genotype and allele frequencies of AhR and ARNT polymorphisms.
ASD Control ASD Control
Genotype n%n% Allele n%n%
AhR (rs2066853)
GG 24 25.3 160 30.4 G 102 53.7 579 54.9
GA 54 56.8 259 49.1 A 88 46.3 475 45.1
AA 17 17.9 108 20.5
Total 95 100.0 527 100.0 190 100.0 1054 100.0
ARNT (rs2228099)
GG 39 41.1 189 35.9 G 120 63.2 624 60.8
GC 42 44.2 246 46.7 C 70 36.8 402 39.2
CC 14 14.7 78 14.8
Total 95 100.0 513a100.0 190 100.0 1026a100.0
a14 samples with ARNT polymorphism from control participants were excluded from the data because the signal failed to be detected as a result of misamplification.
4
Fujisawa et al. Autism Spectrum Disorders and AhR-Related Polymorphisms
Frontiers in Psychiatry | www.frontiersin.org November 2016 | Volume 7 | Article 184
that ARNT polymorphism modied the severity of ASD among
the AhR-related genes examined in the current study.
Factor Analysis of CARS
To assess the severity according to several core behavioral symp-
toms of ASD, factor analysis with Varimax rotation was performed
for the CARS data. e analysis produced three factors with
eigenvalues greater than one. ese factors accounted for 55.1% of
the common variance. Tab l e 2 shows the factor loadings and the
descriptive statistics for each item of the CARS data. e rst fac-
tor was “social communication,” which consisted of “verbal com-
munication,” “non-verbal communication,” “imitation,” “visual
response,” “relating to people,” and “level and consistency,” and
assessed the prociency of social communication and reciprocity.
e second factor was “sensory and emotional response,” which
consisted of “activity level,” “object use,” “emotional response,”
“taste, smell, touch, and response,” “listening response,” and
“fear or nervousness,” and assessed abnormalities of sensory and
emotional responses. e third factor was “stereotypies,” which
consisted of “total impression,” “adaptation to change,” and “body
use” and assessed restricted, repetitive patterns of behavior. ese
three factors well recapitulated the core behavioral symptoms of
ASD, as described by the DSM-5, and the results were consistent
to those of a similar previous study applying a factor analysis to
CARS data (31). erefore, we used the factor score for each of the
three factors in the association analysis between the genotype of
AhR-related genes and the severity of each of the three behavioral
symptoms of ASD.
Genotypes of AhR-Related Genes and the
Severity of the Core Behavioral Symptoms
of ASD
To investigate relationship between each AhR-related gene poly-
morphism and the severity of several of the core symptoms of ASD,
one-way ANOVA was performed for the factor score of CARS of
ASD patients as the dependent variable, with the genotype of each
AhR-related gene polymorphism as the independent variable.
Similar to the result for the total score, there were no statisti-
cally signicant associations between factor scores and genotype
TABLE 3 | Genotype frequencies of AhR and ARNT polymorphisms.
Social communication Sensory and emotional response Stereotypies
AhR (rs2066853)
Genotype GG GA AA GG GA AA GG GA AA
n24 54 17 24 54 17 24 54 17
CARS score M −0.11 0.06 −0.04 −0.01 −0.05 0.16 −0.04 −0.07 0.28
SD (0.9) (0.9) (0.9) (1.0) (0.8) (0.6) (0.8) (0.8) (1.0)
ANOVA F0.35 0.40 1.15
p0.704 0.670 0.320
Effect size f0.088 0.094 0.158
Power 0.107 0.116 0.255
ARNT (rs2228099)
Genotype GG GC CC GG GC CC GG GC CC
n39 42 14 39 42 14 39 42 14
CARS score M 0.32 −0.29 −0.01 0.13 −0.17 0.15 0.02 −0.13 0.34
SD (1.0) (0.7) (0.9) (0.8) (0.6) (1.1) (0.8) (0.8) (0.8)
ANOVAaF5.29 1.68 1.72
p0.007* 0.191 0.185
Effect size f0.339 0.191 0.193
Power 0.838 0.356 0.363
aThe statistical threshold was set at corrected *p<0.05 (0.05/6=0.0083…) with the Bonferroni adjustment for multiple comparisons.
5
Fujisawa et al. Autism Spectrum Disorders and AhR-Related Polymorphisms
Frontiers in Psychiatry | www.frontiersin.org November 2016 | Volume 7 | Article 184
groups for AhR polymorphism (rs2066853) (Tab l e 3 ). However,
for ARNT polymorphism (rs2228099), a signicant dierence
was observed for the factor score of the “social communication”
factor, but not for the “sensory and emotional response” factor
or “stereotypies” factor. Holm’s sequentially rejective Bonferroni
posthoc test revealed that the factor score of the GG genotype
was statistically signicantly higher than that of the GC geno-
types [t(92)=3.25, p<0.01], whereas there were no statistically
signicant dierences in score between CC genotype and both
genotype groups [GC-CC: t(92)=1.24; GG-CC: t(92) = 1.09]
(Figure1).
DISCUSSION
In this study, no statistically signicant dierence was found
between ASD patients and control individuals for the geno-
typic distribution of any of the polymorphisms studied herein.
However, a signicant dierence in the severity score, especially
for the symptom of “social communication,” was observed in
ARNT codon 189 polymorphism, suggesting that the ARNT
polymorphism modies the severity of ASD symptoms but not
susceptibility to ASD.
A large twin population study estimated that environmental
factors common to twins explain about 55% of the liability to
ASD, while genetic factors explain 35% (9). As one of the envi-
ronmental factors for the liability to ASD, the possible involve-
ment of dioxin and/or dioxin-like environmental chemicals
was investigated. Although many environmental chemicals
aect neurodevelopment in humans, we focused on dioxin and
dioxin-like chemicals because we have shown that higher levels of
dioxin-like PCBs in the cord blood appear as a manifestation of
ASD-like behaviors in 4-month-old infants (40), and also because
maternal exposure to such environmental chemicals possibly dis-
rupts fetal gonadal hormone balances, which could lead to EMB
(12, 13). e current study investigated the eects of AhR-related
gene polymorphisms on ASD susceptibility and/or severity to
determine the relationship between possible vulnerability to
dioxin and dioxin-like PCBs and ASD susceptibility and/or sever-
ity because dioxin and dioxin-like PCBs at low levels have spread
almost uniformly throughout the country. AhR-related gene
polymorphisms have been found to underlie physical diseases
such as breast cancer and endometriosis, but no report is available
on the eect of these polymorphisms on mental disorders. To
the best of our knowledge, the present study is the rst report
to clarify the association between ARNT, an AhR-related gene
polymorphism, and the severity of ASD symptoms.
e current study could not clarify the mechanism underlying
how the ARNT polymorphism modies ASD severity in terms of
social communication since no functional analysis of the ARNT
polymorphism was carried out. However, a possible explanation
is that this polymorphism might alter the gonadal hormone
balance in the prenatal period through alterations in the AhR
signaling pathway and could thus aect ASD severity. It is well
known that the sexual dierentiation of the human brain depends
on prenatal exposure levels of androgens (41), and according to
the EMB hypothesis, gonadal hormone imbalances make the
autistic brain develop beyond that of the typical male (10, 11). In
fact, evidence in favor of the positive association between autistic
symptomatology and the levels of fetal testosterone has been found
(13, 42). Although there is no evidence for a direct interaction
between fetal testosterone and the AhR signaling pathway, the
AhR–ARNT heterodimer has been reported to have estrogenic
functions in the absence of estrogen (16). erefore, some sort
of functional variant induced by ARNT polymorphisms might
alter the prenatal exposure levels of gonadal hormone and have
adverse eects on sexual dierentiation of the brain.
Our preliminary results showed a signicant association
between the severity of ASD and polymorphism at ARNT codon
189, which results in a silent mutation (Val189Val). Although
the exact molecular and physiological mechanisms underlying
this eect remain unknown, a recent study suggested that silent
mutations may contribute to mental disorders (43). erefore, to
FIGURE 1 | Three factor scores of CARS (mean±SE) for each
polymorphism of AhR-related genes. The statistical threshold was
corrected with Holm’s sequentially rejective Bonferroni adjustment for multiple
comparisons. (A) AhR (rs2066853) and (B) ARNT (rs2228099). Note:
**p<0.01.
6
Fujisawa et al. Autism Spectrum Disorders and AhR-Related Polymorphisms
Frontiers in Psychiatry | www.frontiersin.org November 2016 | Volume 7 | Article 184
clarify the possible meaning of this association, further genetic
analyses are necessary. Such analyses should particularly address
the interaction with other genetic polymorphisms, both upstream
and downstream from rs2228099, which may interfere with splic-
ing and/or ARNT mRNA stability.
One of the most interesting ndings in the current study is
that the ARNT polymorphism is specically associated with the
severity of social communication impairment among the diverse
ASD symptoms. e diversity of ASD symptoms is an obstacle
for elucidating the pathology and etiology, and the core behav-
ioral symptoms dening ASD are genetically heterogeneous in
that there are no overlapping genes acting on any of these traits.
New, ecient models have been proposed to describe the diverse
symptoms by evaluating the severity of the major components
of impairments (44, 45). According to this strategy, we surmised
that the ARNT polymorphism correlated with the severity of
social communication diculties but not with rigid and repeti-
tive behaviors.
Several limitations of the present study should be noted and
taken into consideration in future studies. First, the main limita-
tion is the relatively small patient group. We did not observe a
signicant association between genetic SNPs and susceptibility to
ASD in this study, and one possible explanation is the small sample
number, as a large number of subjects are needed for case–control
studies based on the frequency distribution. erefore, studies
involving a larger number of subjects are essential to generalize
our results. Second, the number and age of subjects between the
cases and control groups were not well matched. It is possible that
the risk modulation by AhR-related gene polymorphism depends
on the fetal environment and exposure of the mother, which
would be expected to vary across dierent time periods. e
disease risk imparted by the allele would therefore be expected
to vary in dierent age groups. ird, although we have used the
MINI to ensure the quality of the controls, it is important to note
that the MINI does not necessarily exclude ASD. In addition,
although the controls also need to be technically screened for
family history of ASD or developmental disorders, MINI does
not exclude individuals with unidentied Asperger’s or broader
autism phenotypes, which is a key concern in the recruitment
of controls in this study. In this regard, tools such as the Social
Responsiveness Scale (SRS) may have provided a better index
(46). is heterogeneity of our sample may be another possible
explanation for our negative ndings between groups described
above. Finally, we used only one clinical scale (CARS) to assess
the severity of ASD because we did not obtain full data for any
other clinical scale. e ratings by CARS are not invariant across
the life span (e.g., non-verbal ability in a 4-year-old individual
may not be as severe as that in a 20-year-old individual), although
the majority of our clinical samples consisted of children aged
18years and younger, and the genotype groups were conrmed
to not be dierent with regard to age. However, the heterogeneity
in age within our ASD samples could introduce a bias for severity
assessment with respect to genetics, and it would also the aect
factor analysis process. An informative measure such as SRS
adjusted for age may have thus been more useful as a severity
measure for purposes of this study (46). erefore, future studies
are needed to assess various aspects of behavioral symptoms of
broad social communication using other established scales and to
clarify the contribution of ARNT gene polymorphism to aspects
of social communication.
CONCLUSION
In conclusion, the current results showed that individuals
with the ARNT GG genotype had more severely impaired
social communication than those with GC genotype in ASD,
indicating that the dierences in social functioning in ASD
patients may be modulated by ARNT variants. Considering
that ARNT is a component of AhR cascades, vulnerability to
environment chemicals, especially dioxin-like chemicals may
aect the severity of impaired social communication, although
the functional analysis of ARNT gene polymorphism remains to
7
Fujisawa et al. Autism Spectrum Disorders and AhR-Related Polymorphisms
Frontiers in Psychiatry | www.frontiersin.org November 2016 | Volume 7 | Article 184
be performed. To identify the neuronal mechanism underlying
this eect, combining the present experimental paradigm with
neurophysiological indicators of brain activities is warranted in
future studies.
AUTHOR CONTRIBUTIONS
TF and SN were involved in conducting the experiment, analyz-
ing and interpreting data, and draing the article. RI, JM, and
CK were involved in recruiting the participants and diagnosing
the participants with ASD. MT, TS, and NK were involved in
conducting the experiment, analyzing and interpreting data, and
revising the article. KS conceived of the study, participated in
its design and coordination, and draed the manuscript. All the
authors have read and approved the nal manuscript.
FUNDING
is work was supported by a Grant-in-Aid for Scientic Research
(C) from the Ministry of Education, Culture, Sports, Science
and Technology (MEXT) of Japan (KAKENHI: grant numbers
25461774 to KS, grant number 15K01753 to TF).
REFERENCES
1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental
Disorders (DSM-5). 5th ed. Washington, DC: APA (2013).
2. Boyle CA, Boulet S, Schieve LA, Cohen RA, Blumberg SJ, Yeargin-Allsopp M,
etal. Trends in the prevalence of developmental disabilities in US children,
1997-2008. Pediatrics (2011) 127(6):1034–42. doi:10.1542/peds.2010-2989
3. Weintraub K. e prevalence puzzle: autism counts. Nature (2011)
479(7371):22–4. doi:10.1038/479022a
4. Santangelo SL, Tsatsanis K. What is known about autism: genes, brain,
and behavior. Am J Pharmacogenomics (2005) 5(2):71–92. doi:10.2165/
00129785-200505020-00001
5. Freitag CM. e genetics of autistic disorders and its clinical relevance: a review
of the literature. Mol Psychiatry (2007) 12(1):2–22. doi:10.1038/sj.mp.4001896
6. Gillberg C, Coleman M. e Biology of the Autistic Syndromes (Clinics in
Developmental Medicine). Cambridge, UK: Cambridge University Press
(2000).
7. Tabor HK, Risch NJ, Myers RM. Candidate-gene approaches for studying
complex genetic traits: practical considerations. Nat Rev Genet (2002)
3(5):391–7. doi:10.1038/nrg796
8. Veenstra-Vanderweele J, Christian SL, Cook EH Jr. Autism as a paradigmatic
complex genetic disorder. Annu Rev Genomics Hum Genet (2004) 5:379–405.
doi:10.1146/annurev.genom.5.061903.180050
9. Hallmayer J, Cleveland S, Torres A, Phillips J, Cohen B, Torigoe T, et al.
Genetic heritability and shared environmental factors among twin pairs
with autism. Arch Gen Psychiatry (2011) 68(11):1095–102. doi:10.1001/
archgenpsychiatry.2011.76
10. Baron-Cohen S. e extreme male brain theory of autism. Trends Cogn Sci
(2002) 6(6):248–54. doi:10.1016/S1364-6613(02)01904-6
11. Baron-Cohen S, Knickmeyer RC, Belmonte MK. Sex dierences in the
brain: implications for explaining autism. Science (2005) 310(5749):819–23.
doi:10.1126/science.1115455
12. Lutchmaya S, Baron-Cohen S, Raggatt P. Foetal testosterone and eye contact
in 12 month old infants. Infant Behav Dev (2002) 25:327–35. doi:10.1016/
S0163-6383(02)00094-2
13. Auyeung B, Baron-Cohen S, Ashwin E, Knickmeyer R, Taylor K,
Hackett G, et al. Fetal testosterone predicts sexually dierentiated
childhood behavior in girls and in boys. Psychol Sci (2009) 20(2):144–8.
doi:10.1111/j.1467-9280.2009.02279.x
14. Helmig S, Seelinger JU, Döhrel J, Schneider J. RNA expressions of AHR,
ARNT and CYP1B1 are inuenced by AHR Arg554Lys polymorphism. Mol
Genet Metab (2011) 104(1–2):180–4. doi:10.1016/j.ymgme.2011.06.009
15. Wahl M, Guenther R, Yang L, Bergman A, Straehle U, Strack S, et al.
Polybrominated diphenyl ethers and arylhydrocarbon receptor agonists: dif-
ferent toxicity and target gene expression. Toxicol Lett (2010) 198(2):119–26.
doi:10.1016/j.toxlet.2010.06.001
16. Ohtake F, Takeyama K, Matsumoto T, Kitagawa H, Yamamoto Y, Nohara K,
et al. Modulation of oestrogen receptor signalling by association with the
activated dioxin receptor. Nature (2003) 423(6939):545–50. doi:10.1038/
nature01606
17. Del Pino Sans J, Clements KJ, Suvorov A, Krishnan S, Adams HL, PetersenSL.
Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin may
alter LH release patterns by abolishing sex dierences in GABA/glutamate
cell number and modifying the transcriptome of the male anteroventral
periventricular nucleus. Neuroscience (2016) 329:239–53. doi:10.1016/
j.neuroscience.2016.04.051
18. Ohtake F, Fujii-Kuriyama Y, Kato S. AhR acts as an E3 ubiquitin ligase to mod-
ulate steroid receptor functions. Biochem Pharmacol (2009) 77(4):474–84.
doi:10.1016/j.bcp.2008.08.034
19. Needleman HL. e future challenge of lead toxicity. Environ Health Perspect
(1990) 89:85–9. doi:10.1289/ehp.908985
20. Winneke G. Developmental aspects of environmental neurotoxicology: lessons
from lead and polychlorinated biphenyls. J Neurol Sci (2011) 308(1–2):9–15.
doi:10.1016/j.jns.2011.05.020
21. Nishijo M, Tai PT, Nakagawa H, Maruzeni S, Anh NT, Luong HV, etal. Impact
of perinatal dioxin exposure on infant growth: a cross-sectional and longi-
tudinal studies in dioxin-contaminated areas in Vietnam. PLoS One (2012)
7(7):e40273. doi:10.1371/journal.pone.0040273
22. Nowack N, Wittsiepe J, Kasper-Sonnenberg M, Wilhelm M, Schölmerich A.
Inuence of low-level prenatal exposure to PCDD/Fs and PCBs on empathiz-
ing, systemizing and autistic traits: results from the Duisburg birth cohort
study. PLoS One (2015) 10(6):e0129906. doi:10.1371/journal.pone.0129906
23. Tran NN, Pham TT, Ozawa K, Nishijo M, Nguyen AT, Tran TQ, etal. Impacts
of perinatal dioxin exposure on motor coordination and higher cognitive
development in Vietnamese preschool children: a ve-year follow-up. PLoS
One (2016) 11(1):e0147655. doi:10.1371/journal.pone.0147655
24. Tsuchiya M, Katoh T, Motoyama H, Sasaki H, Tsugane S, Ikenoue T. Analysis
of the AhR, ARNT, and AhRR gene polymorphisms: genetic contribution
to endometriosis susceptibility and severity. Fertil Steril (2005) 84(2):454–8.
doi:10.1016/j.fertnstert.2005.01.130
25. Wu CH, Guo CY, Yang JG, Tsai HD, Chang YJ, Tsai PC, etal. Polymorphisms
of dioxin receptor complex components and detoxication-related genes
jointly confer susceptibility to advanced-stage endometriosis in the
taiwanese han population. Am J Reprod Immunol (2012) 67(2):160–8.
doi:10.1111/j.1600-0897.2011.01077.x
26. Merisalu A, Punab M, Altmäe S, Haller K, Tiido T, Peters M, et al. e
contribution of genetic variations of aryl hydrocarbon receptor pathway
genes to male factor infertility. Fertil Steril (2007) 88(4):854–9. doi:10.1016/
j.fertnstert.2006.12.041
27. Zheng NN, Bi YP, Zheng Y, Zheng RH. Meta-analysis of the association of
AhR Arg554Lys, AhRR Pro185Ala, and ARNT Val189Val polymorphisms
and endometriosis risk in Asians. J Assist Reprod Genet (2015) 32(7):1135–44.
doi:10.1007/s10815-015-0505-3
28. Kennedy S. e genetics of endometriosis. Eur J Obstet Gynecol Reprod Biol
(1999) 82(2):129–33. doi:10.1016/S0301-2115(98)00213-9
29. Di Napoli A, Warrier V, Baron-Cohen S, Chakrabarti B. Genetic variant
rs17225178 in the ARNT2 gene is associated with Asperger syndrome. Mol
Autism (2015) 6:9. doi:10.1186/s13229-015-0009-0
30. Schopler E, Reichler RJ, DeVellis RF, Daly K. Toward objective classication of
childhood autism: childhood autism rating scale (CARS). J Autism Dev Disord
(1980) 10(1):91–103. doi:10.1007/BF02408436
31. Magyar CI, Pandol V. Factor structure evaluation of the childhood autism
rating scale. J Autism Dev Disord (2007) 37(9):1787–94. doi:10.1007/
s10803-006-0313-9
8
Fujisawa et al. Autism Spectrum Disorders and AhR-Related Polymorphisms
Frontiers in Psychiatry | www.frontiersin.org November 2016 | Volume 7 | Article 184
32. Ronald A, Happé F, Bolton P, Butcher LM, Price TS, Wheelwright S, etal.
Phenotypic and genetic overlap between autistic traits at the extremes of the
general population. J Am Acad Child Adolesc Psychiatry (2006) 45(10):1206–
14. doi:10.1097/01.chi.0000230165.54117.41
33. Happé F, Ronald A, Plomin R. Time to give up on a single explanation for
autism. Nat Neurosci (2006) 9(10):1218–20. doi:10.1038/nn1770
34. American Psychiatric Association. Diagnostic and Statistical Manual of Mental
Disorders (DSM-IV). 4th ed. Washington, DC: APA (1994).
35. Wechsler D. Wechsler Intelligence Scale for Children. 3rd ed. San Antonio, TX:
Psychological Corporation (1991).
36. Wechsler D. Wechsler Adult Intelligence Scale. 3rd ed. San Antonio, TX:
Psychological Corporation (1997).
37. Tanaka Institute for Educational Research. Tanaka Binet Chinou Kensa V
[Tanaka-Binet Intelligence Scale V]. Tokyo: Taken Publishing (2003). (in
Japanese).
38. Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E, etal.
e Mini-International Neuropsychiatric Interview (MINI): the development
and validation of a structured diagnostic psychiatric interview for DSM-IV
and ICD-10. J Clin Psychiatry (1998) 59:22–33.
39. Parks LK, Hill DE, oma RJ, Euler MJ, Lewine JD, Yeo RA. Neural correlates
of communication skill and symptom severity in autism: a voxel-based mor-
phometry study. Res Autism Spectr Disord (2009) 3(2):444–54. doi:10.1016/j.
rasd.2008.09.004
40. Doi H, Nishitani S, Fujisawa TX, Nagai T, Kakeyama M, Maeda T, et al.
Prenatal exposure to a polychlorinated biphenyl (PCB) congener inuences
xation duration on biological motion at 4-months-old: a preliminary study.
PLoS One (2013) 8(3):e59196. doi:10.1371/journal.pone.0059196
41. McCarthy MM, Arnold AP. Reframing sexual dierentiation of the brain. Nat
Neurosci (2011) 14(6):677–83. doi:10.1038/nn.2834
42. Auyeung B, Taylor K, Hackett G, Baron-Cohen S. Foetal testosterone and
autistic traits in 18 to 24-month-old children. Mol Auti sm (2010) 1(1):11.
doi:10.1186/2040-2392-1-11
43. Takata A, Ionita-Laza I, Gogos JA, Xu B, Karayiorgou M. De novo synon-
ymous mutations in regulatory elements contribute to the genetic etiology
of autism and schizophrenia. Neuron (2016) 89(5):940–7. doi:10.1016/
j.neuron.2016.02.024
44. Gebregziabher M, Shotwell MS, Charles JM, Nicholas JS. Comparison of
methods for identifying phenotype subgroups using categorical features data
with application to autism spectrum disorder. Comput Stat Data Anal (2012)
56(1):114–25. doi:10.1016/j.csda.2011.06.014
45. Bitsika V, Sharpley CF, Orapeleng S. An exploratory analysis of the use of
cognitive, adaptive and behavioural indices for cluster analysis of ASD
subgroups. J Intellect Disabil Res (2008) 52(11):973–85. doi:10.1111/
j.1365-2788.2008.01123.x
46. Constantino JN, Gruber CP. e Social Responsiveness Scale Manual. 2nd ed.
Los Angeles: Western Psychological Services (2012).
Conict of Interest Statement: e authors declare that the research was con-
ducted in the absence of any commercial or nancial relationships that could be
construed as a potential conict of interest.
Copyright © 2016 Fujisawa, Nishitani, Iwanaga, Matsuzaki, Kawasaki, Tochigi,
Sasaki, Kato and Shinohara. is is an open-access article distributed under the
terms of the Creative Commons Attribution License (CC BY). e use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms.
