Article

Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder

Department of Psychiatry, Osaka University Graduate School of Medicine, D3, 2-2, Yamadaoka, Suita, 565-0871, Osaka, Japan. .
Molecular Autism (Impact Factor: 5.41). 05/2011; 2(1):9. DOI: 10.1186/2040-2392-2-9
Source: PubMed

ABSTRACT

The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN) 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD.
We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays.
The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells.
Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients.

Full-text

Available from: Kazutaka Ohi
RESEARCH Open Access
Gene expression analysis in lymphoblasts derived
from patients with autism spectrum disorder
Yuka Yasuda
1,2
, Ryota Hashimoto
1,2,3*
, Hidenaga Yamamori
1,4
, Kazutaka Ohi
1,2
, Motoyuki Fukumoto
1,2
,
Satomi Umeda-Yano
4
, Ikuko Mohri
3,5
, Akira Ito
4
, Masako Taniike
3,5
and Masatoshi Takeda
1,3
Abstract
Background: The autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that result in
severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal
communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare
mutations of some genes, such as neuroligin (NLGN) 3/4, neurexin (NRXN)1,SHANK3, MeCP2 and NHE9, have been
reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression
levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD.
Methods: We measured mRNA expres sion levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in
lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with
schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction
assays.
Results: The mRNA expres sion levels of NLGN3 and SHANK3 normalized by b-actin or TBP were significantly
decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA
expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were
not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not
quantitatively measurable in lymphoblastoid cells.
Conclusions: Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in
lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the
possibility that decreased mRNA expression levels of these genes m ight be involved in the pathophysiology of
ASD in a substantial population of ASD patients.
Background
Autism spectrum disorder (ASD), also known as perva-
sive developmental disorder (PDD), is defined as severe
and pervasive impairments i n the development of reci-
procal social interaction an d verbal and nonver bal com-
munication skills. These disorders are also characterized
by stereotypical behavior, interests and activities. The
lifetime morbidity rate of ASD is 0.2% to 1.0% across
studies [1]. In addition, twin and family studies of ASD
have demonstrated a high heritability of approximately
90% [2], indicating that ASD is a heterogeneous condi-
tion that is likely to result from the combined effects of
multiple genetic factors interacting with environmental
factors. Recent genetic studies have identified several
vulner ability loci and genetic mutations that cause ASD.
One of the most striking revelations is the important
role of genes that encode proteins at the neuronal
synapse [3].
Rare mutations in the neuroligin 3 (NLGN3) and neu-
roligin 4 (NLGN4) genes, which map to chromosomes
Xq13 and Xp22.3, h ave bee n report ed in some pa tien ts
with ASD and other neurodevelopmental impair ments
[4-8]. A particular mutation of NL GN 3 (Arg451Cys) is
known to cause a defect in protein processing of
NLGN3 [9]. In addition, a particular mutation of
NLGN4 (1186insT) c auses a frameshif t mutation that
leads to premature termination of NLGN4 (D396X),
resulting in a loss of 421 amino acids (51% o f the
* Correspondence: hashimor@psy.med.osaka-u.ac.jp
1
Department of Psychiatry, Osaka University Graduate School of Medicine,
D3, 2-2, Yamadaoka, Suita, 565-0871, Osaka, Japan
Full list of author information is available at the end of the article
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
© 2011 Yasuda et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creative commons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Page 1
protein) [4]. Neuroligins, which are postsynaptically
localized cell adhesion molecules, play a crucial role in
organizing excitatory glutamatergic and inhibitory
GABAergic synapses in the mammalian brain by inter-
acting with presynaptic b-neurexins (NRXN ), thereby
triggering the formation o f functional presynaptic struc-
tures in contacting axons [10]. Mutations of the neur-
exin 1 (NRXN1) g ene, at the chromosome locus 2q32,
have been found in individuals with ASD [11-14].
Furthermore, de novo copy number variation a nalysis
revealed deletion of the NRXN1-containing gene region
in ASD [15]. The binding of NRXN1 and NLGN genes
mediates synaptic development [16]. Interestingly, a
mutation of NLGN3 results in a disruption of the ability
to bind to NRXN [9]. In addition, neuroligins interact
with a postsynaptic scaffolding protein, SHANK3, which
is also implicated in ASD [17] and is located on the
telomeric terminal of chromosome 22q13.3. Shank pro-
teins couple neurotransmitter receptors, ion channels
and other membrane proteins to the actin cy toskeleton
and G protein-coupled signaling pathways, and they also
play a role in synapse formation and dendritic spine
maturation [18]. Deletion or translo cation of the geno-
mic locus, which includes the SHANK3 gene, and de
novo mutations of the SH ANK3 gene result in prema-
ture stop codons and have been found in ASD
[17,19,20].
In a study of consanguineous autism families, Morrow
et al. [21] observed a relati onship betw een ASD and
alterations in the sodium/hydrogen exchanger 9 (NHE9)
gene. Specifically, they found a nonsense mutation in
patients with ASD t hat is a heterozygous CGA-t o-TGA
transition, changing arginine 423 to a stop codon [21].
The NHE9 gene is located on chromosome 3q24 and i s
one of the families of Na
+
/H
+
exchangers that regulate
ion flux across membranes [22]. Rett syndrome is
another PDD, and the methyl-CpG-binding protein 2
(MeCP2) g ene is a causal gene for Rett syndrome.
MeCP2 is a transcriptional repressor that binds to
methylated CpG d inucleotides generally located at gene
promoters and recruits histon e deacetylase 1 and other
proteins involved in chromatin repression [23]. De novo
mutations of t he MeCP 2 gene loca ted on chr omosome
Xp28 occur in 80% of female patients with Rett syn-
drome [24]. Some evidence of dysregulation of the phos-
phatidylinositol 3-kinase (PI3K)/AKT pathway is
implicated in ASD, despit e the fact that no mutation
which causes ASD has been reported in association with
the AKT1 gene. The expre ssion and phosphorylation
and/or activation of AKT were found to be decreased in
the autistic brain [25]. T he PTEN gene (phosphatase
and tensin homolog deleted on chromosome 10) is a
major negative regulator of the PI3K/AKT pathway, and
PTEN mutations have been linked to ASD [26].
Recently, several studies have suggested that lympho-
blasto id cells can be used to detect biologically plausible
correlations between candi date genes and neuropsychia-
tric diseases, including Rett syndrome [27], nonspecific
X-linked mental retardation [28], bipolar disorder [29],
fragile X syndrome [30,31] and dup(15q) [32]. In the
present study, we compared mRNA expression levels of
various genes in blood-derived lymphoblastoid cells
from individuals with ASD and healthy controls.
Methods
Participants
We obtained mRNA samples from patients with ASD,
patients with schizophrenia and healthy controls from
the research bioresource of the Human Brain Phenotype
Consortium in Japan (http://www.sp-web.sakura.ne.jp/
consortium.html). The ASD cohort consisted of
35 patients with ASD and healthy controls (Table 1).
Patient s with ASD and patients with schizophrenia were
rec ruited from bot h outpatient and inpatient services at
OsakaUniversityHospital.Each ASD patient was diag-
nosed by at least two trained child psychiatrists and/or
child neurologists according to the Diagnostic and
Statistical Manual of Mental Disorders, Fourth Edition-
Text Revision (DSM-IV-TR) crite ria based on un struc-
tured or semistructured behavioral observations of the
patients and interviews with the patients and their par-
ents or caregivers. During the interview, the Pervasive
Developmental Disorders Autism Society Japan Rating
Scale(PARS)[33]andtheJapaneseversionofthe
Aspergers Questionn aire [34] were used to assist in the
evaluation of ASD-specific behaviors and symptoms.
PARS is a semistructured interview that is composed of
Table 1 Demographic information for the ASD and
control cohorts
a
Demographics ASD
(n = 35)
Controls
(n = 35)
P value
Sex, M/F 27/8 26/9 c
2
= 0.078 (1, N =
70), P = 0.78
Mean age, years ( ± SD) 12.9 (12.4) 34.8 (9.7) U = 86, P = 0.60 ×
10
-9
, Z = -6.19
Age range, years 3 to 63 21 to 65
Number of ASD
(with IQ < 70)
35 (11) 0
Number of Autism
(with IQ < 70)
20 (10) -
Number of Aspergers
syndrome (with IQ < 70)
11 (0) -
Number with PDD-NOS
(with IQ < 70)
4 (1) -
ASD: autism spectrum disorder, M: male, F: female, IQ: intelligence quotient;
PDD-NOS: pervasive developmental disorder not otherwise specified. Data are
means ± SD unless otherwise specified. Differences in clinical characteristics
were analyzed using the c
2
test for gender and the Mann-Whitney U test for
age.
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
Page 2 of 8
Page 2
57 quest ions i n eight domains of the characteristics of
children with PDD, which was de velo ped by the Autism
Society Japan. The clinicians who diagnosed the indivi-
duals w ere trained in the use of PARS. Twenty indivi-
duals met the full criteria for autistic disorder, 11 met the
criteria for Asperger syndrome and four for PDD-not
otherwise specified (PDD-NOS). Among the patients
with ASD, 11 had a low intelligence quotient (IQ) ( < 70).
The schizophrenia cohort consisted of 45 patients with
schizophrenia and 45 age- and sex-matched healthy con-
trols (Table 2). Each patient with schizophrenia received
a consensus diagnosis by at least two trained psychiatrists
according to the DSM-IV-TR criteria using the struc-
tured clinical interview (SCID) for DSM-IV.
A detailed description of healthy controls was given in
previous reports [35,36]. Briefly, controls were biologi-
cally unrelated Japanese participants. Healthy controls
were screened using the SCID for the Diagnostic and
Statistical Manual, Fourth Edition , Axis I Disorders,
Non-Patient version (SCID-I/NP) and were excl uded if
they (1) had neurological or medical conditions that
could potentially affect the central nervous system, (2)
had any psychiatric diseases and/or received psychiatric
medication, (3) had first- or second-degree relatives with
psychiatric disease or (4) presented with an IQ < 70. IQ
data were collected using the Japanese version of the
full-scale Wechsler Adult Intelligence Scale (WAIS)-III
or the full-scale Wechsler Intelligence Scale for Chil-
dren-Third Edition (WISC-III) [37,38].
Following description of the study, written info rmed
consent was obtained from each individua l (or, when
appropriate, his/her guardians). This study was carried
out in accordance with the World Medical Associations
Declaration of H elsinki and was approved by the ethics
committee at Osaka University.
Immortalization of lymphocytes and RNA extraction
Isolation of lymphocytes from blood and lymphocyte
immortalization using Epstein-Barr virus (EBV) were
entrusted to SRL of Tokyo, Japan. Immortalized,
patient-derived lymphocytes were grown in culture
media supplemented with 20% fetal bovine serum. Total
RNA was extracted from cell pellets using the RNeasy
Mini Kit (Qiagen K.K., Tokyo, Japan). The total RNA
yield was determined by absorbance at 260 nm, and RNA
quality was analyzed using agarose gel electrophoresis.
DNase treatment and reverse transcriptase reaction
Total RNA was treated with DNase to remove contami-
nating genomic DNA using DNase Treatment &
Removal Reagents (Ambion, Austin, TX, USA) according
to the manufac turers protocol. Total RNA (10 μg) trea-
ted with DNase was used in a 50-μL reverse transcriptase
reaction to synthesize cDNA with t he SuperSc ript First-
Strand Synthesis System for RT-PCR (Invitrogen, Carls-
bad, C A, USA) according to the manufacturers protocol.
Briefly, total RNA (10 μg) was denatured with 1 mM
deoxyribonucleotide triphosphat e (dNTP) and 5 ng/μL
random hexamers at 65°C for 5 minutes. After the addi-
tion of 10xRT buffer (20 mM Tris-HCl (pH 8.4) and
50 mM KCl final concentration; Invitrogen), MgCl
2
(5 mM final concentration), dithiothreitol (10 mM final
concentration), RNaseOUT Recombinant Ribonuclease
Inhibitor (100 U; Invitrogen) and SuperScript III Reverse
Transcriptase (125 U; Invitrogen), the reaction mixture
was incubated at 25°C for 10 minutes, at 42°C for
40 minu tes and at 70°C for 15 minutes. RNase H (5 U)
was added to the reaction mixture and incubated at 37°C
for 20 minutes to stop the reaction.
Real-time quantitative RT-PCR
The Pre-Developed TaqMan Assay Reagent kit (Applied
Biosystems, Foster City, CA, USA) was used to measure
mRNA expression levels of NLGN3, NLGN4, NRXN1,
SHANK3, MeCP2, NHE9, AKT1 and housekee ping genes
(b-actin and TBP). Primers were purchased from Applied
Biosystems (gene name: assay ID, transcript ID, target
region; NLGN3: Hs01043809_m1, NM_181303.1, Exon4-
5; NLGN4: Hs00535592_m1, NM_020742.2, Exon1-2;
NRXN1: Hs00985123_m1, NM_001135659.1, Exon22-23;
SHANK3: H s01586468_m1, NM_001080420.1, Exon22-
23; MECP2: Hs00172845_m1, NM_004992.3, Exon2-3;
NHE9: Hs00543518_m1, NM_173653.3, Exon7-8; AKT1:
Hs00920503_m1, NM_001014432.1, Exon13-14; b-actin:
4326315E, NM_001101, no region indicated; TBP:
4326 322E, NM_003194, no region indicated). Expression
levels of these genes were measured by real- time quanti-
tative reverse transcriptase polymerase chain reaction
(qRT-PCR) using an ABI Prism 7900 Sequence Detection
System (Applied Biosystems) with a 384-well format as
previously described [39,40]. Each 20-μLPCRreaction
contained 6 μL of cDNA, 900 nM concentra tions of each
primer, a 250 nM concentration of probe an d 10 μLof
Table 2 Demographic information for schizophrenia and
control cohorts
a
Demographics Schizophrenia
(n = 45)
Controls
(n = 45)
P value
Sex, M/F 26/19 26/19 c
2
= 0 (1, N = 90),
P = 1.0
Mean age, years
( ± SD)
37.9 (1.6) 38.1 (1.7) U = 988.5, P = 0.9,
Z = -0.2
Age range, years 21 to 65 21 to 65
Estimated premorbid
IQ (JART50)
100.8 (9.3) 105.4 (8.4) U = 687, P =
0.009, Z = -2.6
M: mal e, F: female, IQ: intelligence quotient; JART50: Japanese Adult Reading
Test: Japanese version of the National Adult Reading Test. Data are means ±
SD unl ess otherwise specified. Differences in clinic al characteristics were
analyzed using the c
2
test for gender and the Mann-Whitney U test for age.
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
Page 3 of 8
Page 3
TaqMan Universal PCR Master Mix containing Ampli-
Taq Gold DNA Polymerase and AmpErase Uracil N-gl y-
cosylase (all from Applied Biosystems), as well as dNTP
with deoxyuridine triphosphate, passive refere nce and
optimized buffer components. The PCR cycling condi-
tions were 50°C f or 2 minutes, 95°C f or 10 minutes,
40 cycles of 95°C for 15 seconds and 59°C or 60°C for 1
minute. PCR data were obtained by using Sequence
Detector software (SDS version 2.1; Applied Biosystems)
and quantified using a standard cu rve method. This
software plotted the real-time fluorescence intensity and
selected the threshold within the linear phase of the
amplicon profile. The software plotted a standard curve
of the c ycle at threshold (C
t
) (where the fluorescence
generated within a reaction crossed the t hreshold) ver-
sus the quantity of RNA. All samples were measured
using a single plate per target gene, and their C
t
values
were in the linear range of the standard curve. Sample
quantities were predicted by C
t
values. Experiments
were typically perfor med three times in triplica te, and
each gene e xpression level was taken as the average of
three independent experiments. The individual expres-
sion level of each target gene normalized by a house-
keeping gene (raw target gene expression level divided
by raw housekeeping gene expression level) was used
for statistical analysis.
Statistical analyses
Statistical analyses were carried out using SPSS for Win-
dows ve rsion 16.0 software (SPSS Japan Inc., Tokyo,
Japan). Group comparisons of demographic data were
performed using the c
2
test for one ca tegorical variable
(sex) or the Mann-Whitney U test for continuous vari-
ables as appropriate. Differences in mRNA transcript
levels between the g roups were analyzed using the
Mann-Whitney U test. The Bonferroni correction for
multiple tests was applied to assess the mRNA tran-
script levels on the number of genes (five). All P values
reported are based on two-tailed tests. Stat istica l signifi-
cance was defined as P < 0.05.
Results
Standard curves for the seven target genes ( NLGN3,
NLGN4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1)
and the two housekeeping genes (b-actin and TBP) were
prepared using serial d ilutions (1:4) of pooled cDNA
from 300 ng of total RNA derived from immortalized
lymphoblasts (Figure 1). The R
2
values of the standard
curves were more than 0.99 (NLGN3, MeCP2, NHE9,
AKT1, b-actin and TBP), 0.87 (SHANK3), 0.64 (NRXN1)
and 0.63 (NLGN4). Although the SHANK3 gene expr es-
sion was relatively low, it was measurable in our sample.
On the other hand, we did not further analyze NLGN4
and NRXN1 gene expressi on, as the expression levels of
thetwogenesweretoolowtoquantifyusingthis
method.
Using immortalized lymphoblastoid cells from 35 indi-
viduals with ASD and 35 controls, we quantified the
mRNA expression levels of the NLGN3, SHANK3,
NHE9, MeCP2 and AKT1 genes normalized by two
housekeeping genes, b-actin and TBP (Figure 2). The
mRNA expression levels of the NLGN3 gene normalized
by b-actin or TBP were decreased by 35% or 26%,
respectively, in individual s with ASD (b-actin : P =
Figure 1 Standard curves for target genes and housekeeping
genes. Standard curves for NLGN3, NLGN4, NRXN1, SHANK3, MeCP2,
NHE9, AKT1 and two housekeeping genes (b-actin and TBP). The
highest quantity represents an amount of cDNA prepared from 300
ng of total RNA in the polymerase chain reaction.
Figure 2 Expression analysis of NLGN3, SHANK3, NHE9, MeCP2
and AKT1 in autism spectrum disorder. Mean relative mRNA
expression level scores normalized by housekeeping gene b-actin
(a) or TBP (b) in the autism spectrum disorder (ASD) group and the
control group are shown. Bars represent the standard error of the
mean. Differences between the groups in expression levels of the
five genes were analyzed by using the Mann-Whitney U test. Post
hoc comparisons were performed by using the Bonferroni
correction. **P < 0.01 and ***P < 0.001.
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
Page 4 of 8
Page 4
0.00024; TBP: P = 0.00089). The mRNA expression
levels of the SHANK3 gene normalized by b-actin or
TBP were also decreased in individuals with ASD by
39% or 40%, respectively (b-actin: P = 0.000036; TBP:
P = 0.0061). The mRNA expression leve ls of the NHE9
gene were increased by 24% (P = 0.052: normalized by
b-actin) and 39% (P = 0.048: normalized by TBP). There
was no significant difference in mRNA expression levels
of the Me CP2 gene normalized by b-actin or TBP
between the two groups (P >0.1).ThemRNAexpres-
sion levels of the AKT1 gene were decreased by 11%
(P = 0.03: normalized by b-actin); however, those levels
were not altered when normalized by TBP (P =0.45).
After correction for multiple tests, mRNA expression
levels of NLGN3 and SHANK3 remained significantly
lower in individuals with ASD than in healthy controls
(NLGN3: co rrected P = 0.001 2, normalized by b-actin,
corrected P = 0.0045, normalized by TBP; SHANK3: cor-
rected P = 0.00018, normalized by b-actin, corrected P =
0.03, normalized by TBP). However, the altered expres-
sion level of NHE9 or AKT1 was no longer significant
after the correction for multiple tests (P > 0.1).
We next measured NLGN3 and SHANK3 mRNA
expr
ession levels in immortal ized lymphoblastoid cells
from 45 patients with schizophrenia and 45 healthy con-
trols to examine the disease specific ity of the differential
expression levels between patients and healthy controls
(Figure 3). We found that the mRNA expression levels
for these two genes normalized by b-actin or TBP were
not significantly different between patients with schizo-
phrenia and healthy controls (P > 0.2). These results
suggest t hat reduced lev els of NLGN3 and SHANK3
mRNA expression might be associated with ASD but
not with schizophrenia.
Discussion
In this study, we found that the mRNA expression levels
of NLGN3 and SHANK3 were significantly lower in indi-
viduals with ASD than in healthy controls. Mutations of
causal genes are rare, and they have been found to be
associated with specific types of ASD. Our findings sug-
gest that not only rare mutat ions of the causa l genes
but also functional alterations in the transcriptional
activity of these genes might be associated with the
pathophysiology of ASD. The NLGN3 and SHANK3
genes are synapse-re lated genes and were found to be
affected in ASD, whereas other genes, including NHE9
and MeCP2, do not play major ro les at the synaps e and
were not found to be affected in ASD. These findings
suggest that impairments in synaptic function might be
associated with the pathophysiology of ASD.
Reduced expression of the NLGN3 and SHANK3
genes in lymphoblasts of individuals with ASD is consis-
tent with previous reports indicating that mutations of
these genes cause reduc ed expression or loss of function
of the protein. Since the NLGN3 gene is located in chro-
mosome X, there may be expressional difference
between genders. However, no significant difference of
NLGN3 gene expression normalized by b-acti n or TBP
was observed with regard to gender in h ealthy controls
or individuals with ASD (P >0.05).Thismightbedue
to inactivation of one X chromo some in females [41].
There are several possibilities that might explain the
reduced expression of the NLGN3 and SHANK3 genes
in ASD. First, our sporadic ASD cases might have muta-
tions, polymorphisms or copy number variations in the
NLGN3 or SHANK3 genes, which could result in
reduced expression of these genes. Second, mutations or
polymorphisms in genes th at regulate the expression of
NLGN3 or SHANK3 might co ntribute t o the observed
reductioninexpressionoftheNLGN3 or SHANK3
gen es. To our knowledge, although the regulation of
NLGN3 by other genes has not been reported, there are
some reports in the literature describing the regulation of
SHANK3 gene expression. For example, SHANK3 expres-
sion is regulated by DNA methylation [42,43]. In addition,
SHANK3 is one of the predicted targets of dysregulated
microRNA (miRNA), and altered miRNA expression levels
werefoundinpostmortembrainfromautismpatients
[44]. Further epige netic analyses might elucidate the
mechanisms of reduced SHANK3 expression.
Some findings of gene expression in lymphoblastoid
cell lines are in conflict with those of previous studies.
For example, Beri et al. [42] repor ted that SHANK3 is
not expressed in EBV-transformed human lymphoblas-
toid cell lines in an investigation of tissue-specific
Figure 3 Expression ana lysis of the NLGN3 and SHANK3 genes
in patients with schizophrenia. Mean relative mRNA expression
level scores normalized by housekeeping gene b-actin (a) or TBP (b)
in the schizophrenia group and the control group are shown. Bars
represent standard error of the mean. Differences in expression
levels of the two genes between the groups were analyzed by
using the Mann-Whitney U test.
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
Page 5 of 8
Page 5
SHANK3 ge ne expression and DNA methylation. By
using lymphoblastoid cells from autism patients, Talebi -
zadeh et al. [8] det ected novel splice i soforms of
NLGN4. There are methodological differences between
previous studies and our study. SHANK3 gene expres-
sion in the previous study [42] was analyzed by using a
conventional RT-PCR method; however, we measured
the expression levels of SHANK3 gene by using a real-
time qRT-PCR method (the TaqMan method). Further-
more, the expression level of SHANK3 wa s relatively
low, which is shown in the standard curve in Fi gure 1.
It is possible that the sensitivity of our real-time qRT-
PCR method to detect the SHANK3 gene expression
level might be higher than that of a conventional RT-
PCR method. On the other hand, we could not quanti-
tatively measure the NLGN4 and NRXN1 genes by using
the real-time qRT-PCR method. However, there were
slight expressions of these ge nes in lymphoblastoid cell
lines when we used a large quantity of cDNA for the
real-time qRT-PCR (Figure 1). Unfortunately, the small
expression levels of these genes made it impossible to
quantitatively measure the gene expressions in our sam-
ple. This may explain possible discrepancies of the gene
expression findings of previous studies and our results.
There are several limitations of this study. First, our
positive results might have arisen from sample bi as due
to non-age-matched samples, although the Japanese are
a relatively homogeneo us population, so the use of non-
age-matched samples is unlikely to explain our findings.
Second, our s ample size might not be small f or type I
errors b ut small for type II errors. There is a possibility
of type II errors in mRNA expression differences of
NHE9, MEC P2 and AKT1 between individuals with
ASD and healthy controls and expression differences of
NLGN3 and SHANK3 between individuals with schizo-
phrenia and healthy controls. In particular, NHE9 might
be increased in individuals with ASD, as the expression
level of NHE9 was marginally significant before correc-
tion for multiple testing. Thus, replication studies using
a larger sample size are needed before a fir m conclusion
can be drawn. Third, we did not perform a mutation
search for the examined genes in our sample to replicate
the association between the examined genes and ASD
and how the causal or risk variants of the genes regulate
the gene expression. As the previous evidence for candi-
date genes of ASD are based on rare mutations and/or
copy number variations of the genes, it might be diffi-
cult to find a mutation in our 35 individuals with ASD
for analysis of the variant effects on the gene expression
in this study. A mutation search study of these candi-
date genes should be done in future studies . Fourth, the
IQ scores in the ASD group were lower than those in
the healthy control group, so reduced gene expression
could be related to lower IQ. However, lower expression
of the NLGN3 or SHANK3 genes was not found in indi-
viduals with schizophrenia who had lower premorbid IQ
scores, and no expression difference was observed in
individuals with ASD and mental retardation versus
individuals with ASD but without mental retardation
(data not shown). Taken together, the reduced gene
expression in ASD might be specific to ASD, although
other neuropsychiatric diseases, such as attention-defi-
cit/hyperactivity disorder, mental retardation, major
depression and bipolar disorder, should be examined in
future studie s. The ASD cases in this study were consis-
tent with idiopathic autism diagnosed on the basis of
clinical features. We did not include individuals with
Rett syndrome and the other syndromic autisms, such as
multiple sclerosis, which could explain why we did not
find altered expressio n of MeCP2 in this cohort. Our
results suggest that the MeCP2 gene may not be associated
with the com mon pathology of ASD, while NLGN3 and
SHANK3 may b e. Because lymphoblastoid cell lines are
not neuronal cells, some of our findings might not reflect
the pathophysiology in ASD brains. Further studies inves-
tigating these limitations are warranted.
Conclusions
Our study reveals reduced levels of NLGN3 and
SHANK3 mRNA expression in lymphoblastoid cell lines
derived from individuals with ASD, but not from those
of individuals with schizophrenia. These results are con-
sistent with findings that rare mutations of these genes
in specific cases cause loss of function, suggesting that
reduction of NL GN3 and SHANK3 mRNA expression
could b e relate d to the pathophysiol ogy of ASD in a
substantial population of patients. Although there a re
several limitations present in this study, lymphoblastoid
cell lines may still allow investigation of the pathophy-
siology of ASD. Further analyses are required, such as a
mutation analysis of the NLGN3 and SHANK3 genes
and the genes regulating their expression, in additio n to
studies designed to elucidate the mechanisms of this
reduced expression.
Abbreviations
ASD, autism spectrum disorder; DSM-IV-TR, Diagnostic and Statistical Manual
of Mental Disorders, Fourth Edition-Text Revision; F, female; IQ, intelligence
quotient; JART50, Japanese Adult Reading Test; M, male; MeCP2, methyl-CpG-
binding protein 2; NHE9, sodium/hydrogen exchanger 9; NLGN, neuroligin;
NRXN, neurexin; PARS, Pervasive Developmental Disorders Autism Society
Japan Rating Scale; PDD, pervasive developmental disorder; PDD-NOS,
pervasive developmental disorder not otherwise specified; SCID, structured
clinical interview; SCID-I/NP, Diagnostic and Statistical Manu al, Fourth Edition,
Axis I Disorders, Non-Patient version; SD, standard deviation; WAIS-III,
Wechsler Adult Intelligence Scale-III; WISC-III, Wechsler Intelligence Scale for
Children-Third Edition.
Acknowledgements
This work was funded in part by Grants-in-Aid from the Japanese Ministry of
Health, Labor and Welfare (H19-kokoro-002, H22-seishin-ippan-001, H22-
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
Page 6 of 8
Page 6
rinken-ippan-002, H22-sinkei-ippan-001); the Japanese Ministry of Education,
Culture, Sports, Science and Technology (18689030, 22390225); CREST (Core
Research for Evolutionary Science and Technology) of JST (Japan Science
and Technology Agency), Grant-in-Aid for Scientific Research on Priority
Areas, Research on the Pathomechanisms of Brain Disorders, from the
Ministry of Education, Culture, Sports, Science, and Technology (18023045);
the Japan Foundation for Neuroscience and Mental Health and the Meiji
Yasuda Mental Health Foundation; and the Osaka University Program for the
Support of Networking among Present and Future Researchers. The study
sponsors had no further role in the study design; the collection, analysis and
interpretation of data; the writing of the report; or the decision to submit
the manuscript for publication.
Author details
1
Department of Psychiatry, Osaka University Graduate School of Medicine,
D3, 2-2, Yamadaoka, Suita, 565-0871, Osaka, Japan.
2
CREST (Core Research for
Evolutionary Science and Technology) of JST (Japan Science and Technology
Agency), 4-1-8, Honcho, Kawaguchi, Saitama, 332-0112, Japan.
3
Molecular
Research Center for Childrens Mental Development, United Graduate School
of Child Development, Osaka University, Kanazawa University and
Hamamatsu University School of Medicine, D3, 2-2, Yamadaoka, Suita, Osaka,
565-0871, Japan.
4
Department of Molecular Neuropsychiatry, Osaka
University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka, 565-
0871, Japan.
5
Division of Developmental Neuroscience, United Graduate
School of Child Development, Osaka University, Kanazawa University and
Hamamatsu University School of Medicine, 2-2, Yamadaoka, Suita, Osaka,
565-0871, Japan.
Authors contributions
RH supervised the entire project; collected the data; wrote the manuscript;
was critically involved in the design, analysis and interpretation of the data;
and was responsible for performing the literature review. YY was critically
involved in the collection and analysis of the data, contributed to the
editing of the final manuscript and contributed intellectually to the
interpretation of the data. HY, SU and AI were involved in the mRNA
measurements and collection of the majority of the data. KO, MF, IM, MTan
and MTak were heavily involved in the collection of the majority of the data
and contributed intellectually to the interpretation of the data. All authors
reviewed the manuscript before submission and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 9 March 2011 Accepted: 26 May 2011 Published: 26 May 2011
References
1. Levy SE, Mandell DS, Schultz RT: Autism. Lancet 2009, 374:1627-1638.
2. Lichtenstein P, Carlström E, Råstam M, Gillberg C, Anckarsäter H: The
genetics of autism spectrum disorders and related neuropsychiatric
disorders in childhood. Am J Psychiatry 2010, 167:1357-1363.
3. Walsh CA, Morrow EM, Rubenstein JL: Autism and brain development. Cell
2008, 135:396-400.
4. Jamain S, Quach H, Betancur C, Råstam M, Colineaux C, Gillberg IC,
Soderstrom H, Giros B, Leboyer M, Gillberg C, Bourgeron T: Paris Autism
Research International Sibpair Study: Mutations of the X-linked genes
encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nat
Genet 2003, 34:27-29.
5. Laumonnier F, Bonnet-Brilhault F, Gomot M, Blanc R, David A, Moizard MP,
Raynaud M, Ronce N, Lemonnier E, Calvas P, Laudier B, Chelly J, Fryns JP,
Ropers HH, Hamel BC, Andres C, Barthélémy C, Moraine C, Briault S: X-linked
mental retardation and autism are associated with a mutation in the NLGN4
gene, a member of the neuroligin family. Am J Hum Genet 2004, 74:552-557.
6. Lawson-Yuen A, Saldivar JS, Sommer S, Picker J: Familial deletion within
NLGN4 associated with autism and Tourette syndrome. Eur J Hum Genet
2008, 16:614-618.
7. Yan J, Oliveira G, Coutinho A, Yang C, Feng J, Katz C, Sram J, Bockholt A,
Jones IR, Craddock N, Cook EH Jr, Vicente A, Sommer SS: Analysis of the
neuroligin 3 and 4 genes in autism and other neuropsychiatric patients.
Mol Psychiatry 2005, 10:329-332.
8. Talebizadeh Z, Lam DY, Theodoro MF, Bittel DC, Lushington GH, Butler MG:
Novel splice isoforms for NLGN3 and NLGN4 with possible implications
in autism. J Med Genet 2006, 43:e21.
9. Comoletti D, De Jaco A, Jennings LL, Flynn RE, Gaietta G, Tsigelny I,
Ellisman MH, Taylor P: The Arg451Cys-neuroligin-3 mutation associated
with autism reveals a defect in protein processing. J Neurosci 2004,
24:4889-4893.
10. Craig AM, Kang Y: Neurexin-neuroligin signaling in synapse development.
Curr Opin Neurobiol 2007, 17:43-52.
11. Feng J, Schroer R, Yan J, Song W, Yang C, Bockholt A, Cook EH Jr,
Skinner C, Schwartz CE, Sommer SS: High frequency of neurexin 1β signal
peptide structural variants in patients with autism. Neurosci Lett 2006,
409:10-13.
12. Kim HG, Kishikawa S, Higgins AW, Seong IS, Donovan DJ, Shen Y, Lally E,
Weiss LA, Najm J, Kutsche K, Descartes M, Holt L, Braddock S, Troxell R,
Kaplan L, Volkmar F, Klin A, Tsatsanis K, Harris DJ, Noens I, Pauls DL,
Daly MJ, MacDonald ME, Morton CC, Quade BJ, Gusella JF: Disruption of
neurexin 1 associated with autism spectrum disorder. Am J Hum Genet
2008, 82:199-207.
13. Autism Genome Project Consortium, Szatmari P, Paterson AD,
Zwaigenbaum L, Roberts W, Brian J, Liu XQ, Vincent JB, Skaug JL,
Thompson AP, Senman L, Feuk L, Qian C, Bryson SE, Jones MB, Marshall CR,
Scherer SW, Vieland VJ, Bartlett C, Mangin LV, Goedken R, Segre A, Pericak-
Vance MA, Cuccaro ML, Gilbert JR, Wright HH, Abramson RK, Betancur C,
Bourgeron T, Gillberg C, et al: Mapping autism risk loci using genetic
linkage and chromosomal rearrangements. Nat Genet 2007, 39
:319-328.
14.
Yan J, Noltner K, Feng J, Li W, Schroer R, Skinner C, Zeng W, Schwartz CE,
Sommer SS: Neurexin 1α structural variants associated with autism.
Neurosci Lett 2008, 438:368-370.
15. Glessner JT, Wang K, Cai G, Korvatska O, Kim CE, Wood S, Zhang H, Estes A,
Brune CW, Bradfield JP, Imielinski M, Frackelton EC, Reichert J, Crawford EL,
Munson J, Sleiman PM, Chiavacci R, Annaiah K, Thomas K, Hou C,
Glaberson W, Flory J, Otieno F, Garris M, Soorya L, Klei L, Piven J, Meyer KJ,
Anagnostou E, Sakurai T, et al: Autism genome-wide copy number
variation reveals ubiquitin and neuronal genes. Nature 2009, 459:569-573.
16. Dean C, Dresbach T: Neuroligins and neurexins: linking cell adhesion,
synapse formation and cognitive function. Trends Neurosci 2006, 29:21-29.
17. Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F,
Nygren G, Rastam M, Gillberg IC, Anckarsäter H, Sponheim E, Goubran-
Botros H, Delorme R, Chabane N, Mouren-Simeoni MC, de Mas P, Bieth E,
Rogé B, Héron D, Burglen L, Gillberg C, Leboyer M, Bourgeron T: Mutations
in the gene encoding the synaptic scaffolding protein SHANK3 are
associated with autism spectrum disorders. Nat Genet 2007, 39:25-27.
18. Vessey JP, Karra D: More than just synaptic building blocks: scaffolding
proteins of the post-synaptic density regulate dendritic patterning. J
Neurochem 2007, 102:324-332.
19. Bonaglia MC, Giorda R, Borgatti R, Felisari G, Gagliardi C, Selicorni A,
Zuffardi O: Disruption of the ProSAP2 gene in a t(12;22)(q24.1;q13.3) is
associated with the 22q13.3 deletion syndrome. Am J Hum Genet 2001,
69:261-268.
20. Moessner R, Marshall CR, Sutcliffe JS, Skaug J, Pinto D, Vincent J,
Zwaigenbaum L, Fernandez B, Roberts W, Szatmari P, Scherer SW:
Contribution of SHANK3 mutations to autism spectrum disorder. Am J
Hum Genet 2007, 81:1289-1297.
21. Morrow EM, Yoo SY, Flavell SW, Kim TK, Lin Y, Hill RS, Mukaddes NM,
Balkhy S, Gascon G, Hashmi A, Al-Saad S, Ware J, Joseph RM, Greenblatt R,
Gleason D, Ertelt JA, Apse KA, Bodell A, Partlow JN, Barry B, Yao H,
Markianos K, Ferland RJ, Greenberg ME, Walsh CA: Identifying autism loci
and genes by tracing recent shared ancestry. Science 2008, 321:218-223.
22. Nakamura N, Tanaka S, Teko Y, Mitsui K, Kanazawa H: Four Na
+
/H
+
exchanger isoforms are distributed to Golgi and post-Golgi
compartments and are involved in organelle pH regulation. J Biol Chem
2005, 280:1561-1572.
23. Chahrour M, Zoghbi HY: The story of Rett syndrome: from clinic to
neurobiology. Neuron 2007, 56:422-437.
24. Renieri A, Meloni I, Longo I, Ariani F, Mari F, Pescucci C, Cambi F: Rett
syndrome: the complex nature of a monogenic disease. J Mol Med 2003,
81:346-354.
25. Sheikh AM, Malik M, Wen G, Chauhan A, Chauhan V, Gong CX, Liu F,
Brown WT, Li X: BDNF-Akt-Bcl2 antiapoptotic signaling pathway is
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
Page 7 of 8
Page 7
compromised in the brain of autistic subjects. J Neurosci Res
88:2641-2647.
26. Zhou J, Blundell J, Ogawa S, Kwon CH, Zhang W, Sinton C, Powell CM,
Parada LF: Pharmacological inhibition of mTORC1 suppresses anatomical,
cellular, and behavioral abnormalities in neural-specific Pten knock-out
mice. J Neurosci 2009, 29:1773-1783.
27. Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu T: Loss of silent-
chromatin looping and impaired imprinting of DLX5 in Rett syndrome.
Nat Genet 2005, 37:31-40.
28. Meloni I, Muscettola M, Raynaud M, Longo I, Bruttini M, Moizard MP,
Gomot M, Chelly J, des Portes V, Fryns JP, Ropers HH, Magi B, Bellan C,
Volpi N, Yntema HG, Lewis SE, Schaffer JE, Renieri A: FACL4, encoding fatty
acid-CoA ligase 4, is mutated in nonspecific X-linked mental retardation.
Nat Genet 2002, 30:436-440.
29. Iwamoto K, Kakiuchi C, Bundo M, Ikeda K, Kato T: Molecular
characterization of bipolar disorder by comparing gene expression
profiles of postmortem brains of major mental disorders. Mol Psychiatry
2004, 9:406-416.
30. Brown V, Jin P, Ceman S, Darnell JC, ODonnell WT, Tenenbaum SA, Jin X,
Feng Y, Wilkinson KD, Keene JD, Darnell RB, Warren ST: Microarray
identification of FMRP-associated brain mRNAs and altered mRNA
translational profiles in fragile X syndrome. Cell 2001, 107:477-487.
31. Nishimura Y, Martin CL, Vazquez-Lopez A, Spence SJ, Alvarez-Retuerto AI,
Sigman M, Steindler C, Pellegrini S, Schanen NC, Warren ST, Geschwind DH:
Genome-wide expression profiling of lymphoblastoid cell lines
distinguishes different forms of autism and reveals shared pathways.
Hum Mol Genet 2007, 16:1682-1698.
32. Baron CA, Tepper CG, Liu SY, Davis RR, Wang NJ, Schanen NC, Gregg JP:
Genomic and functional profiling of duplicated chromosome 15 cell
lines reveal regulatory alterations in UBE3A-associated ubiquitin-
proteasome pathway processes. Hum Mol Genet 2006, 15:853-869.
33. Yamada A, Suzuki M, Kato M, Tanaka S, Shindo T, Taketani K, Akechi T,
Furukawa TA: Emotional distress and its correlates among parents of
children with pervasive developmental disorders. Psychiatry Clin Neurosci
2007, 61:651-657.
34. Wakabayashi A, Tojo Y, Baron-Cohen S, Wheelwright S: [The Autism-
Spectrum Quotient (AQ) Japanese version: evidence from high-
functioning clinical group and normal adults] [in Japanese]. Shinrigaku
Kenkyu 2004, 75:78-84.
35. Yasuda Y, Hashimoto R, Ohi K, Fukumoto M, Takamura H, Iike N, Yoshida T,
Hayashi N, Takahashi H, Yamamori H, Morihara T, Tagami S, Okochi M,
Tanaka T, Kudo T, Kamino K, Ishii R, Iwase M, Kazui H, Takeda M:
Association study of KIBRA gene with memory performance in a
Japanese population. World J Biol Psychiatry 2010, 11:852-857.
36. Hashimoto R, Ohi K, Yasuda Y, Fukumoto M, Iwase M, Iike N, Azechi M,
Ikezawa K, Takaya M, Takahashi H, Yamamori H, Okochi T, Tanimukai H,
Tagami S, Morihara T, Okochi M, Tanaka T, Kudo T, Kazui H, Iwata N,
Takeda M: The impact of a genome-wide supported psychosis variant in
the ZNF804A gene on memory function in schizophrenia. Am J Med
Genet B Neuropsychiatr Genet 2010, 153B:1459-1464.
37. Committee JW-IP: Japanese Wechsler Intelligence Scale for Children Tokyo:
Nihon Bunka Kagakusha; 1998.
38. Wechsler D: Wechsler Intelligence Scale for Children-Third Edition Manual New
York: Psychological Corp; 1991.
39. Hashimoto R, Straub RE, Weickert CS, Hyde TM, Kleinman JE,
Weinberger DR: Expression analysis of neuregulin-1 in the dorsolateral
prefrontal cortex in schizophrenia. Mol Psychiatry 2004, 9:299-307.
40. Chiba S, Hashimoto R, Hattori S, Yohda M, Lipska B, Weinberger DR,
Kunugi H: Effect of antipsychotic drugs on DISC1 and dysbindin
expression in mouse frontal cortex and hippocampus. J Neural Transm
2006, 113:1337-1346.
41. Willard HF: X chromosome inactivation and X-linked mental retardation.
Am J Med Genet 1996, 64:21-26.
42. Beri S, Tonna N, Menozzi G, Bonaglia MC, Sala C, Giorda R: DNA
methylation regulates tissue-specific expression of Shank3. J Neurochem
2007, 101:1380-1391.
43. Maunakea AK, Nagarajan RP, Bilenky M, Ballinger TJ, DSouza C, Fouse SD,
Johnson BE, Hong C, Nielsen C, Zhao Y, Turecki G, Delaney A, Varhol R,
Thiessen N, Shchors K, Heine VM, Rowitch DH, Xing X, Fiore C,
Schillebeeckx M, Jones SJ, Haussler D, Marra MA, Hirst M, Wang T,
Costello JF: Conserved role of intragenic DNA methylation in regulating
alternative promoters. Nature 2010, 466:253-257.
44. Abu-Elneel K, Liu T, Gazzaniga FS, Nishimura Y, Wall DP, Geschwind DH,
Lao K, Kosik KS: Heterogeneous dysregulation of microRNAs across the
autism spectrum. Neurogenetics 2008, 9:153-161.
doi:10.1186/2040-2392-2-9
Cite this article as: Yasuda et al.: Gene expression analysis in
lymphoblasts derived from patients with autism spectrum disorder.
Molecular Autism 2011 2:9.
Submit your next manuscript to BioMed Central
and take full advantage of:
Convenient online submission
Thorough peer review
No space constraints or color figure charges
Immediate publication on acceptance
Inclusion in PubMed, CAS, Scopus and Google Scholar
Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Yasuda et al. Molecular Autism 2011, 2:9
http://www.molecularautism.com/content/2/1/9
Page 8 of 8
Page 8
  • Source
    • "In addition, the Autism Diagnostic Interview-Revised (ADI-R) [28], the Pervasive Developmental Disorders Autism Society Japan Rating Scale (PARS) [29], Page 3 of 8 Yasuda et al. Ann Gen Psychiatry (2016) 15:8 and the AQ-J [30] were used to evaluate ASD-specific behaviors and symptoms, as previously described [27]. Patients were recruited at Osaka University Hospital. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: The Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) recently included sensory processing abnormalities in the diagnostic criteria for individuals with autism spectrum disorder (ASD). However, there is no standard method for evaluating sensory abnormalities in individuals with ASD. Methods: Fifteen individuals with ASD and 15 age- and sex-matched controls were enrolled in this study. We compared objective pain sensitivity by measuring the pain detection threshold and pain tolerance to three different stimuli (electricity, heat, and cold). Then, we compared both subjective pain sensitivity, assessed by the visual analog scale (VAS), and quality of pain, assessed by the short-form McGill Pain Questionnaire (SF-MPQ), to determine the maximum tolerable pain intensities of each stimulation. Results: The pain detection threshold and pain tolerance of individuals with ASD were not impaired, indicating that there were no differences in the somatic perception of pain between groups. However, individuals with ASD were hyposensitive to subjective pain intensity compared to controls (VAS; electrical: p = 0.044, cold: p = 0.011, heat: p = 0.042) and hyposensitive to affective aspects of pain sensitivity (SF-MPQ; electrical: p = 0.0071, cold: p = 0.042). Conclusions: Our results suggest that the cognitive pathways for pain processing are impaired in ASD and, furthermore, that our methodology can be used to assess pain sensitivity in individuals with ASD. Further investigations into sensory abnormalities in individuals with ASD are needed to clarify the pathophysiologic processes that may alter sensory processing in this disorder.
    Preview · Article · Dec 2016 · Annals of General Psychiatry
  • Source
    • "The patients were diagnosed by at least two trained child psychiatrists and/or child neurologists according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5). The assessments were performed using unstructured or semi-structured behavioral observation and interviews with the patients and their parents or caregivers [16]. Additionally, the participants met the ASD criteria of the Autism Diagnostic Interview-Revised (ADI-R) [17]. "
    [Show abstract] [Hide abstract] ABSTRACT: Autism spectrum disorder is a neurodevelopmental disorder characterized by impairments in social interactions, reduced verbal communication abilities, stereotyped repetitive behaviors, and restricted interests. It is a complex condition caused by genetic and environmental factors; the high heritability of this disorder supports the presence of a significant genetic contribution. Many studies have suggested that copy-number variants contribute to the etiology of autism spectrum disorder. Recently, copy-number variants of the nephronophthisis 1 gene have been reported in patients with autism spectrum disorder. To the best of our knowledge, only six autism spectrum disorder cases with duplications of the nephronophthisis 1 gene have been reported. These patients exhibited intellectual dysfunction, including verbal dysfunction in one patient, below-average verbal intellectual ability in one patient, and intellectual disability in four patients. In this study, we identified nephronophthisis 1 duplications in two unrelated Japanese patients with autism spectrum disorder using a high-resolution single-nucleotide polymorphism array. This report is the first to describe a nephronophthisis 1 duplication in an autism spectrum disorder patient with an average verbal intelligence quotient and an average performance intelligence quotient. However, the second autism spectrum disorder patient with a nephronophthisis 1 duplication had a below-average performance intelligence quotient. Neither patient exhibited physical dysfunction, motor developmental delay, or neurological abnormalities. This study supports the clinical observation of nephronophthisis 1 duplication in autism spectrum disorder cases and might contribute to our understanding of the clinical phenotype that arises from this duplication.
    Full-text · Article · Aug 2014 · Annals of General Psychiatry
  • Source
    • "This technology has been used in several post-mortem brain studies of psychiatric disorders, including schizophrenia, bipolar disorder, and autism891011. Since there are several limitations to the use of the post-mortem brain tissue in gene expression studies, and the study of fresh brain tissue from living psychiatric patients is impractical at the present time, several studies have reported the use of peripheral blood cells and lymphoblastoid cell lines (LCL) as surrogates for brain tissue in the gene expression studies of mental disorders1213141516. In addition, a moderate correlation of gene expression between peripheral blood cells and brain tissue in humans has been reported, supporting the usefulness of peripheral blood cells in the gene expression studies for psychiatric research [17]. "
    [Show abstract] [Hide abstract] ABSTRACT: Comparative gene expression profiling analysis is useful in discovering differentially expressed genes associated with various diseases, including mental disorders. Autism spectrum disorders (ASD) are a group of complex childhood-onset neurodevelopmental and genetic disorders characterized by deficits in language development and verbal communication, impaired reciprocal social interaction, and the presence of repetitive behaviors or restricted interests. The study aimed to identify novel genes associated with the pathogenesis of ASD. We conducted comparative total gene expression profiling analysis of lymphoblastoid cell lines (LCL) between 16 male patients with ASD and 16 male control subjects to screen differentially expressed genes associated with ASD. We verified one of the differentially expressed genes, FOXP1, using real-time quantitative PCR (RT-qPCR) in a sample of 83 male patients and 83 male controls that included the initial 16 male patients and male controls, respectively. A total of 252 differentially expressed probe sets representing 202 genes were detected between the two groups, including 89 up- and 113 downregulated genes in the ASD group. RT-qPCR verified significant elevation of the FOXP1 gene transcript of LCL in a sample of 83 male patients (10.46 +/- 11.34) compared with 83 male controls (5.17 +/- 8.20, P = 0.001). Comparative gene expression profiling analysis of LCL is useful in discovering novel genetic markers associated with ASD. Elevated gene expression of FOXP1 might contribute to the pathogenesis of ASD.Clinical trial registration: Identifier: NCT00494754.
    Full-text · Article · Jul 2013 · Molecular Autism
Show more