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Contents lists available at ScienceDirect

Neuroscience Research
journal homepage: www.elsevier.com/locate/neures

Two genetic variants of CD38 in subjects with autism spectrum disorder andcontrols
Toshio Munesue a,b,c,e,j,k,1 , Shigeru Yokoyama a,f,j,1 , Kazuhiko Nakamura l,1 , Ayyappan Anitha l,1 ,Kazuo Yamada m,1 , Kenshi Hayashi d,f,g , Tomoya Asaka n , Hong-Xiang Liu a,f , Duo Jin a,f ,Keita Koizumi a,b,f,k , Mohammad Saharul Islam a,f,j , Jian-Jun Huang a,f , Wen-Jie Ma a,f,j , Uh-Hyun Kim o ,Sun-Jun Kim p , Keunwan Park q , Dongsup Kim q , Mitsuru Kikuchi c,e,r , Yasuki Ono e , Hideo Nakatani e ,Shiro Suda l , Taishi Miyachi l , Hirokazu Hirai a,s , Alla Salmina t , Yu A. Pichugina f,u ,Andrei A. Soumarokov u , Nori Takei k,l,v , Norio Mori k,l,v , Masatsugu Tsujii k,w , Toshiro Sugiyama a,x ,Kunimasa Yagi g , Masakazu Yamagishi g , Tsukasa Sasaki y,z , Hidenori Yamasue j,z , Nobumasa Kato j,z,A ,Ryota Hashimoto k,B , Masako Taniike k,B , Yutaka Hayashi h , Junichiro Hamada h , Shioto Suzuki i ,Akishi Ooi i , Mami Noda C , Yuko Kamiyama C , Mizuho A. Kido D , Olga Lopatina a,f,j,t , Minako Hashii f,j ,Sarwat Amina a,f , Fabio Malavasi E , Eric J. Huang F , Jiasheng Zhang F , Nobuaki Shimizu G ,Takeo Yoshikawa m , Akihiro Matsushima n , Yoshio Minabe a,b,e,k,r , Haruhiro Higashida a,b,f,j,k,∗a

Kanazawa University 21st Century Center of Excellence (COE) Program on Innovative Brain Science on Development, Learning and Memory, Kanazawa 920-8640, Japan
Osaka-Hamamatsu-Kanazawa Universities Joint Research Centers, Kanazawa University Center for Child Mental Development, Kanazawa 920-8640, Japanc
Department of Child Psychiatry, Kanazawa University Hospital, Kanazawa 920-8641, Japand
Department of Clinical Laboratory, Kanazawa University Hospital, Kanazawa 920-8641, Japane
Department of Psychiatry and Neurobiology, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japanf
Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japang
Department of Internal Medicine, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japanh
Department of Neurosurgery, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japani
Department of Molecular and Cellular Pathology, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, JapanjCore Research for Evolutional Science and Technology, Tokyo 102-0075, Japank
United Graduate School of Child Development, Osaka-Kanazawa-Hamamatsu Universities, Osaka 565-0871, Japanl
Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japanm
Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama 351-0198, Japann
Nanao National Hospital, Nanao 920-8531, Japano
Department of Biochemistry, Chonbuk National University Medical School, Jeonju, Republic of Koreap
Department of Pediatrics, Chonbuk National University Medical School, Jeonju, Republic of Koreaq
Bio and Brain Engineering, Korea Advanced Institute of Science and Technology, Republic of Korear
Hokuriku Innovation Cluster for Health Science, Kanazawa 920-8640, Japans
Department of Neurophysiology, Gunma University Graduate School of Medicine, Gunma 371-8511, Japant
Department of Biochemistry and Medical Chemistry, Krasnoyarsk State Medical University, Krasnoyarsk 660022, RussiauDepartment of Psychiatry, Krasnoyarsk State Medical University Krasnoyarsk 660022, Russiav
Osaka-Hamamatsu-Kanazawa Universities Joint Research Centers, Hamamatsu University Center for Child Mental Development, Medical, Hamamatsu 431-3197, Japanw
Faculty of Sociology, Chukyo University, Toyota, Aichi 470-0393, Japanx
Aichi Children’s Health and Medical Center, Aichi 474-8710, JapanyOfﬁce for Mental Health Support, Division for Counseling and Support, University of Tokyo, Tokyo 113-0033, Japanz
Department of Neuropsychiatry, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, JapanA
Department of Psychiatry, Showa University School of Medicine, Tokyo 157-8577, Japan
B
Osaka-Hamamatsu-Kanazawa Universities Joint Research Centers, Molecular Research Center for Children’s Mental Development, Osaka University, Osaka 565-0871, Japan
C
Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, JapanD
Department of Oral Anatomy and Cell Biology, Graduate School of Dental Science, Kyushu University, Fukuoka 812-8582, Japan
E
Laboratory of Immunogenetics, Department of Genetics, Biology and Biochemistry and CeRMS, University of Torino Medical School, Torino 10126, Italy
F
Department of Pathology, University of California San Francisco and Pathology Service, Veterans Affair Medical Center, San Francisco, CA 94121, USA
G
Institute of Nature and Environmental Technology, Kanazawa University, Kanazawa 920-1192, Japanb∗ Corresponding author at: Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan.
1

E-mail address: haruhiro@med.kanazawa-u.ac.jp (H. Higashida).
These authors contributed equally to the work.

0168-0102/$ – see front matter © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
doi:10.1016/j.neures.2010.03.004

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a r t i c l e

i n f oArticle history:
Received 3 December 2009
Received in revised form 4 March 2010
Accepted 8 March 2010
Keywords:
CD38
Oxytocin
Mutation
Polymorphism
AutismHigh-functioning autisma b s t r a c t
The neurobiological basis of autism spectrum disorder (ASD) remains poorly understood. Given the roleof CD38 in social recognition through oxytocin (OT) release, we hypothesized that CD38 may play arole in the etiology of ASD. Here, we ﬁrst examined the immunohistochemical expression of CD38 in
the hypothalamus of post-mortem brains of non-ASD subjects and found that CD38 was colocalizedwith OT in secretory neurons. In studies of the association between CD38 and autism, we analyzed 10
single nucleotide polymorphisms (SNPs) and mutations of CD38 by re-sequencing DNAs mainly froma case–control study in Japan, and Caucasian cases mainly recruited to the Autism Genetic Resource
Exchange (AGRE). The SNPs of CD38, rs6449197 (p < 0.040) and rs3796863 (p < 0.005) showed signiﬁcantassociations with a subset of ASD (IQ > 70; designated as high-functioning autism (HFA)) in the U.S.
104 AGRE family trios, but not with Japanese 188 HFA subjects. A mutation that caused tryptophan toreplace arginine at amino acid residue 140 (R140W; (rs1800561, 4693C>T)) was found in 0.6–4.6% of
the Japanese population and was associated with ASD in the smaller case–control study. The SNP wasclustered in pedigrees in which the fathers and brothers of T-allele-carrier probands had ASD or ASD
traits. In this cohort OT plasma levels were lower in subjects with the T allele than in those without. One
proband with the T allele who was taking nasal OT spray showed relief of symptoms. The two variant
CD38 poloymorphysms tested may be of interest with regard of the pathophysiology of ASD.
© 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental disease manifesting in childhood but extending through to adulthood.
The disorder is more common than previously supposed, with aworldwide frequency of >0.6% (Honda et al., 2005; Baird et al., 2006;
Williams et al., 2006). The region with the maximum reported rate
(3% of births) is the Nagoya/Hamamatsu region in Japan (Sumi etal., 2006). ASD can be sporadic or familial and is far more common
in males than in females (Zhao et al., 2007). Because ASD is etiologically heterogeneous and forms a continuum, it is likely to involve
many genes (Sutcliffe, 2008; Levitt and Campbell, 2009). De novo
mutations and copy number variations (CNVs) are reported in asmall fraction of ASD cases (Sebat et al., 2007; Glessner et al., 2009),
but common variants also underlie the disease (Wang et al., 2009),
and a uniﬁed mechanism for both forms of genetic inheritance has
been proposed (Zhao et al., 2007).
Oxytocin (OT) is secreted into the brain by hypothalamic neuronal dendrites and plays important roles in social recognition
and memory (Insel and Fernald, 2004; Takayanagi et al., 2005;
Donaldson and Young, 2008; Neumann, 2008). This hormone mediates behavioral effects, such as pair bonding, mate guarding, andparental care in rodents (Ferguson et al., 2000; Ludwig and Leng,
2006; Campbell, 2008) and may be involved in romantic love, trust,and fear in humans (Kosfeld et al., 2005; Zeki, 2007; Domes et al.,2007; Ditzen et al., 2009). Recently, evidence has accumulating tosuggest that the polymorphisms of multiple OT-related genes areassociated with ASD (Wu et al., 2005; Jacob et al., 2007; Ebstein etal., 2009; Gregory et al., 2009; Wermter et al., 2010).
Peripheral or nasal administration of OT facilitates social recognition and trust in healthy humans (Guastella et al., 2008a,b, 2010;
Ditzen et al., 2009) and increases eye contact and recognition in
autistic subjects (Hollander et al., 2007; Yamasue et al., 2009). Thoseobservations are based mostly on a small number of administrations, and the effects of long-term OT treatment on human social
behavior in ASD patients is unknown.
Human CD38 is a type II transmembrane antigen (Malavasi etal., 2008). The CD38 gene consists of 8 exons on 4p15 (Nakagawara
et al., 1995) and spans a genomic stretch of 70.51 kb. The mRNA
contains 1227 bases, and single nucleotide polymorphisms (SNPs)
have been reported (Nata et al., 1997; Yagui et al., 1998; Ebstein etal., 2009; see Fig. 2A). CD38 has been studied extensively because
it is a reliable negative prognostic marker for chronic lymphocytic
leukemia (Deaglio et al., 2008). CD38 is expressed in the brain(Lee, 2001; Higashida et al., 2007) and can catalyze the formationof cyclic ADP-ribose (cADPR) from NAD+ (Lee, 2001; Guse, 2005;
Malavasi et al., 2008). cADPR mobilizes Ca2+ from intracellular Ca2+
stores, thus acting as a second messenger (Lee, 2001). Little was
known about the CD38-dependent cADPR/Ca2+ signaling pathway
in the brain until recent studies in our laboratory. They showedthat CD38 regulates OT secretion in the mouse hypothalamus andposterior pituitary, which is critical for mouse social behavior (Jinet al., 2007; Liu et al., 2008). The precise role of CD38 in the human
hypothalamus, however, has not been clariﬁed.
As OT seems to be an important factor for the understanding of
ASD, we examined the relationships among human CD38 polymorphisms and mutations in Japanese, Korean, and Caucasian subjects.
We identiﬁed two functional polymorphisms in subgroups: ASD
and high-functioning autism (HFA) based on IQ (>70 classiﬁed as
HFA). We measured the carrier’s serum OT levels and examinedeach subject for ASD. Here, we also discuss the possibility of treating ASD patients who have a SNP that lowers OT levels by intranasal
administration of OT.
2. Materials and methods
2.1. CD38 expression and immunohistochemistry
We measured CD38 mRNA levels by the semi-quantitative orreal-time quantitative RT-PCR method (Jin et al., 2007) using commercially available total RNAs from various regions of the humanbrain. Control human brain tissues were obtained from archival
blocks in the Departments of Pathology at the University of California San Francisco and Kanazawa University Graduate School of
Medicine. The use of this tissue followed the institutional guidelines
established by the Committee on Human Research (CHR) in both
universities. The immunoﬂuorescent stainings for human CD38 and
OT were performed according to the procedures described previously (Zhang et al., 2007). Brieﬂy, sections of the hypothalamus
were treated with antigen retrieval protocol (0.01 M citrate acidbuffer pH 6.0, plus heating for 121 ◦ C for 5 min). The sections werethen incubated with primary antibody against human CD38 (1:50,
sc-7325, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and OT(1:500, AB911, Chemicon, Billerica, MA, USA) overnight at room
temperature, followed by washing in Tris-buffered saline (TBS), andincubation with secondary antibodies that were conjugated with
Alexa 488 or Alexa 568 (1:300, A11001 and A11011; Invitrogen)for 1 h at room temperature. Images for the co-localization of CD38
and OT in the paraventricular nucleus of the hypothalamus were

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Table 1
Data sets for the 4 case–control study cohorts.
Data seta

Cohort 1
Subjects, age (range), male/female

Cohort 2
Subjects, age (range), male/female

Cohort 3
Subjects

Cohort 4
Subjects, male/female

Cases (Probands)
Controls
Trio families
Family member
HFA subjects
Usage/analysis

29, 22.8 ± 7.6 (12–44), 23/6
315, 34.1 ± 4.3 (8–75), 171/144
3
25, 53.0 ± 4.5 (21–84), 15/10–
Intronic SNP
Exonic mutation
Family-based association
OT/AVP measurement

301, 11.9 ± 6.7 (3–64), 263/38
417, 28.6 ± 14.4 (5–65), 229/188
334
297, 39.2 ± 15.5 (3–93), 143/154
188
–
R140W
HFA association

16
150

263, 263/0
–
252
–
104
Intronic SNP
R140W
HFS association

–
–
R140W

a
Cohort 1 was from the Kanazawa area, Japan; Cohort 2, the Nagoya/Hamamatsu, Tokyo, and Osaka areas, Japan; Cohort 3, Jeonju, Korea; Cohort 4, study of 252 trio
samples from the Autism Genetic Resource Exchange comprising 252 U.S., 7 Russian, and 4 Italian ASD patients. Age ± SEM years (year range).

captured using a Leica confocal microscope (TCS SP, Bannockburn,IL, USA) and imported into the Photoshop software.
2.2. Participants
The participants consisted of cohorts organized by the OsakaHamamatsu-Kanazawa University Joint Research Centers for Child
Mental Development, the Kanazawa University COE Program, theCore Research for Evolutional Science and Technology Program inJapan, which includes DNA samples collected at the Universityof Tokyo (Table 1). Three hundred and ﬁfty-seven ASD subjects
were recruited from outpatient psychiatry or pediatric clinics ofeach university hospital. All subjects fullﬁlled the DSM-IV criteria
for autistic disorder. The diagnoses were made by two experienced child psychiatrists through interviews and clinical record
reviews, and the subjects had no apparent physical anomalies.
We also recruited patients’ parents, grandparents, siblings, andother relatives from 322 families. The controls consisted of unrelated healthy Japanese volunteers—315 from the ﬁrst cohort and
417 from the second. We recruited adult controls mainly fromamong hospital and facility staff and medical schools, and agematched children as controls. In the Japanese cohorts, all subjects
resided in Kanazawa, Hamamatsu/Nagoya, Tokyo, or Osaka, Japan,
and all patients and controls were Japanese with no non-Japaneseparents or grandparents. Two experienced child psychiatrists independently conﬁrmed the diagnosis of ASD for most patients bysemi-structured behavior observation and interviews with the subjects and their parents. At the interview with the parents, which
was helpful in the evaluation of autism-speciﬁc behaviors andsymptoms, the examiner used one of the following instruments:
the Asperger Syndrome Diagnostic Interview (Gillberg et al., 2001),
Autism Diagnostic Interview-Revised (ADI-R; Lord et al., 1994),
Pervasive Developmental Disorders Autism Society Japan Rating
Scale (2006), or Diagnostic Interview for Social and Communication
Disorders (Wing et al., 2002).

In addition, for a third case–control study, we recruited 16 male
ASD patients and 150 non-ASD male controls from Jeonju University Hospital in Korea. For a fourth study, we recruited 252 families
from the Autism Genetic Resource Exchange (http://www.agre.org;
AGRE cohort; Geschwind et al., 2001). Additional selection criteria
required that (i) there be no possible non-idiopathic autism ﬂags
and (ii) all the trios be Whites, with the exclusion of Hispanic andLatino races (Anitha et al., 2008). Seven Russian male patients from
Krasnoyarsk State Medical University Hospital, and 4 lymphoblastoid cells from Italian male ASD patients from Trino University
Medical Hospital were also included in the study. These subjects
met the DSM-IV or ADI-R criteria for autistic disorder.
For the HFA group, the U.S. autistic offspring of 104 trios (patientplus two parents) among the 252 AGRE trios, who had IQ > 70, wereconsidered. In the second cohort, we selected 188 trio families asJapanese HFA cases (Table 1).
Using the Autism-Spectrum Quotient (AQ) (Baron-Cohen et al.,2006; Munesue et al., 2008), we evaluated members of families inwhich older subjects performed self-evaluation by recalling how
they behaved in their 20 s. Subjects of autism traits in 3 kindreds
was deﬁned by AQ scores, above the average (>27) but less than thehigher level (<32), during interviews by two psychiatrists.
This study was approved by the ethics committees of Kanazawa
University, Hamamatsu Medical University, University of Tokyo,
Osaka University, RIKEN, and the other participating institutes.
2.3. Marker selection
The genomic structure of CD38 is based on the University of California, Santa Cruz, March 2006 draft assembly ofthe human genome (http://www.genome.ucsc.edu). We selected
SNPs using information from the International HapMap Project(http://www.hapmap.org) and the National Center for Biotechnology Information (NCBI dbSNP: http://www.ncbi.nlm.nih.gov/SNP).
Initially, all the SNPs with MAF > 0.1 were selected. Tags, which

Table 2
Oligonucleotides. Designed for ampliﬁcation of coding sequences including 60–100 bp of ﬂanking intronic sequences. Primers were
followings.
Exon
1
2
3
4
5
6
7
8

Up


Down


5 -AGGGAAACAGAGAAAAGGCAAGTGU-3
5 -GGCATATAATAGATGCTTCC-3
5 -GACATGCTAAATTGATCTCAG-3
5 -TCCACTATGACTGAACAGCC-3
5 -CTTAACCAGCTATTGCTAAG-3
5 -TCTGCCTGCTGGTTGTTGAG-3
5 -CCCAACAGCCTCTTAACTTT-3
5 -AGCGAATTGGACGACAGATG-3

5 -GGCCAGCTGCTCCTGAAAG-3
5 -TGGACCTATGAATTGTTACC-3
5 -CAGCAGAAGTCACTCTGTTC-3
5 -AGCACTGACTGAGTAACG-3
5 -ACTGTGATATTTGCAACAGG-3
5 -TCCTGAGTCAATTTGTTCCC-3
5 -ATCACCAGAGGTTGCCAT-3
5 -CATTGACCTTATTGTGGAGG-3

Usually, we used the following temperatures: 15 min at 95 ◦ C, 35 cycles of 30 s at 95 ◦ C, 30 s at annealing temperature at 52 ◦ C, 5 min at 72 ◦ C
for extension followed by a ﬁnal extension of 10 min at 72 ◦ C. SNPs of these samples were examined by the methods described.

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Table 3
Sequence information for SNPs1-10 and R140W.
SNP

Sequence

StrandON GENEUCSC No

SNP01 (rs3796878)
SNP02 (rs3796875)
SNP03 (rs6449197)
SNP04 (rs11574927)
SNP05 (rs10805347)
SNP06 (rs3796863)
SNP07 (rs1130169)
SNP08 (rs13137313)
SNP09 (rs3733593)
SNP10 (rs3733593)
R140W (rs1800561)

TTGCTCTGTTCCCAGGTTGGTCCTCC[A/G]CATACCTCCCACTGCAGGATCCTGG
GTTTTTCAAGAGTCCTAAGACAAAGA[A/G]GAAAGGAAGAAGCAGAGAAGCCATG
CAGGTTGAGGAAATTTTATTTCTAAT[C/T]TGCTCAGTGTTTTTTTCATCACAAGAAAATTGTGTACCCCAATTCAGTAGT[A/G]AAACTACTACCGGGAACATCGGGAAATTAACATTTCAGAATTTTATGATCT[A/G]ATATTATGGTTCAAGCACTTGAAACGGGAGGGGAGCTATCCATGCCACCTG[A/C]TGGTCAAAAAAACAGCAGGAGCAGCTGTACCCTTCCT ACAGATAGTCAAAC[C/T]ATAAACTTCATGGTCATGGGTCATGAAATAAACCATATGTGTTGAACAAAG[A/G]ATTAATAAATTAATTTGAGACTCAA
ATCTTGAACAAAATCGCCTAACCTTTC[C/T]GAACTTCAACTTCCTTGCCACTCCT
CTGCCTCCGAATTCCATAGTTTCCAC[C/T]GCCTTGCGCTACTTGCATTCTCTGATTTGGCCCATCAGTTCACACAGGTCCAG[C/T]GGGACATGTTCACCCTGGAGGACAC

−
−
+
+
+
−
+
+
+
−
+

T/C
T/C
C/T
A/G
A/G
T/G
C/T
A/G
C/T
G/A
C/T

15,394,757
15,396,312
15,424,020
15,449,341
15,449,937
15,459,084
15,459,783
15,461.066
15,461,202
15,463,823
15,435,656

could capture the common allelic variants with r2 > 0.8 by pairwise tagging, were picked from this set using Haploview v4.0
(http://www.broad.mit.edu/mpg/haploview).

(Applied Biosystems). Fig. 2A shows the SNPs and mutations analyzed in this study and their locations.

2.5. Enzyme immunoassay for OT and vasopressin
2.4. Genetic analysis
We isolated genomic DNA from venous blood samples using
the standard phenol/chloroform method (Easy-DNA kit, Invitrogen, Carlsbad, CA). We ampliﬁed CD38 exons (Tables 2 and 3) andﬂanking introns (Table 3) by PCR (Taq PCR Core Kit, Qiagen, Hilden,
Germany). We used Assay-on-Demand SNP genotyping products(Applied Biosystems, Foster City, CA) to score SNPs based on the
TaqMan assay method described previously (Anitha et al., 2008). An
ABI 7900 Sequence Detection System (SDS) was used to determine
genotypes and analyses were performed with SDS v2.0 software

Blood samples for measuring OT and vasopressin concentrations were collected in two hospitals in the Kanazawa area between
10:00 and 12:00 or 15:00 and18:00 o’clock from subjects who had
been asked to fast for the previous 2 h. Qualiﬁed lab technicians
drew 10 ml of blood from an arm vein into heparinized tubes inless than 15 min. The samples were centrifuged at 0 ◦ C at 2600 × g
for 15 min and the plasma was separated off, divided into 2 tubes,
and stored at −80 ◦ C. We performed the peptide assay for OT andvasopressin (AVP) as described previously (Jin et al., 2007; Liu etal., 2008).

Fig. 1. Immunohistochemical analysis of CD38 (A, D) and oxytocin (B, E) in the human brain. Cell montages of panels were taken from the paraventricular nucleus (PVN)
in the hypothalamus (A–C) and insular cortex (D–F) of autopsy subjects from the USA. Arrowheads indicate extensive colabeling with OT. The insets in panels are enlarged
images of neurons showing coexpression of CD38 and OT. Scale bars: 40 ␮m in C and 8 ␮m in insert.

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5Fig. 2. Genome structure, SNPs, and mutations of CD38. (A) Genomic structure of CD38 and locations of SNPs in introns (upper) and exons (lower). Exons are indicated byboxes, with translated regions in closed boxes and untranslated regions in open boxes. Numbering of the nucleotides starts at the A of ATG and refers to GenBank Accession
number D84284. (B) The sequence trace was derived from a DNA sample of a 4693C/T heterozygote. (C) Amino acid sequence of CD38 showing conservation of R at the 140th
amino acid among different species, except for rodents and rabbits. Sequences were obtained through the accession numbers NM001775, AY555148, NM175798, AF117714,
AF272974, NM013127, NM007646, D30048, and M85206/M37644 for the indicated species.

2.6. Statistics
We analyzed the data using one- or two-way ANOVA, as appropriate. The criterion for signiﬁcance in all cases was p < 0.05.
PedCheck program v1.1 (http://watson.hgen.pitt.edu) was used to
identify and eliminate all Mendelian inheritance inconsistencies
in the trio genotype data. Markers were tested for association by family-based association test (FBAT), using FBAT v2.0.3(http://www.biostat.harvard.edu/∼fbat/).
3. Results
The highest level of CD38 mRNA expression was detected in
the human hypothalamus, and we detected substantial expres-sion in the frontal cortex, amygdala, and cerebellum (data notshown). CD38 immunoreactivity was detected in the hypothalamus of the two Japanese brains (data not shown). In brains ofsamples from USA (Fig. 1), double immunohistochemical staining revealed high levels of CD38 immunoreactivity in many cells
in the paraventricular nucleus of the hypothalamus and showed
extensive colabeling with OT (Yamashita et al., 2002), whilemuch lower CD38 expression levels and little or no detectable
OT were observed in the insular cortex, which served as a control. These results suggested that CD38 may have an importantrole in OT release in the human hypothalamus, as in the mice
(Jin et al., 2007; Liu et al., 2008). Based on this new information
about the human brain, we set out to examine the human CD38
gene.

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Table 4
FBAT analysis of CD38 SNPs in AGRE trios.
MarkerAllele252 triosars3796878

A
G
A
G
T
C
A
G
A
G
A
C
T
C
A
G
T
C
T
C

rs3796875
rs6449197
rs11574927
rs10805347
rs3796863
rs1130169
rs13137313
rs17476066
rs3733593

HFA trios

Families

Frequency

Z-Scorep-Value

Familiesa

Frequency

Z-Scorep-Valueb

119
119
171
171
79
79
111
111
171
171
160
160
195
195
168
168
167
167
178
178

0.163
0.837
0.659
0.341
0.103
0.897
0.830
0.170
0.321
0.679
0.277
0.723
0.503
0.497
0.742
0.258
0.714
0.286
0.451
0.549

0.253
−0.253−0.7400.740−0.6470.647
0.947−0.947−0.6140.614−1.7061.706
1.004−1.0040.765−0.7650.697−0.6970.962−0.962

0.801

49
49
78
78
30
30
55
55
71
71
74
74
85
85
72
72
69
69
74
74

0.161
0.839
0.640
0.360
0.103
0.897
0.834
0.166
0.309
0.691
0.270
0.730
0.517
0.483
0.726
0.274
0.714
0.286
0.428
0.572

0.640
−0.640−0.5940.594−2.0582.058
1.861−1.861−0.6630.663−2.8002.800
0.659−0.6590.426−0.4260.655−0.6550.887−0.8870.522

p-Value after multimarker testing

0.459
0.518
0.344
0.539
0.088
0.315
0.445
0.486
0.336
0.295

0.552
0.040
0.063
0.508
0.005
0.510
0.670
0.513
0.375
0.053

a

Informative families.
b
Signiﬁcant p-values are indicated in bold italic.

3.1. Intronic SNP analysis in Japanese and U.S. subjects
An association study for 10 intronic SNPs shown in Fig. 2 was
ﬁrst performed in a case–control study in a Japanese population(29 ASD subjects and 315 controls, the ﬁrst cohort in the Table 1).
No signiﬁcant association with ASD was found for these SNPs (datanot shown).
Next, we analyzed U.S. ASD DNA samples (cohort 4). FBAT was
performed for the whole set of 252 trios in the AGRE samples. Again,
none of the SNPs showed signiﬁcant associations, except rs3796863(SNP06) with a tendency toward association (p = 0.088; Table 4).
Therefore, we further analyzed this SNP for the U.S. HFA subgroupof 104 trios in our AGRE samples (cohort 4). In the FBAT of HFA trios,
rs6449197 (p = 0.040) and rs3796863 (p = 0.005) showed signiﬁcantassociations; a tendency for association (p = 0.053) was found after
multimarker testing (Table 4).
Unlike the U.S. cases, no association was detected in 188
Japanese HFA trio cases selected from cohort 2 (p = 0.228).
One-way ANOVA showed a signiﬁcant variation in the distribution of ADI-R C scores (restricted, repetitive, and stereotyped
patterns of behavior) between the C/C, C/A, and A/A genotypes of

SNP06 of CD38, in 252 trios (p = 0.013) and HFA trios (p = 0.0067)(Fig. 3). Following post hoc pairwise comparison with the Bonferroni method, the variations in the distribution of ADI-R C between
the C/C and C/A groups were found to be signiﬁcant at the 0.05- and0.01-levels, in the 252 and HFA trios, respectively.
Linkage disequilibrium (LD) analysis identiﬁed three haplotype
blocks across the CD38 gene in 104 trios in AGRE samples, withthe ﬁrst block comprising SNPs01-05, the second block comprising SNPs06 and 07, and the third block comprising SNPs08 and 09(Fig. 4). The results of haplotype transmission disequilibrium test
(TDT) for the HFA trios are shown in Table 5. The associations of
haplotypes in the three haploblocks were examined based on the
LD structure of CD38. The ﬁrst haploblock including SNPs01-05,
showed a weak tendency for association (p = 0.055; Table 5). The
haplotypes GGCAG (p = 0.022) and GGTAG (p = 0.034) of this block
showed signiﬁcant associations; however, this was not signiﬁcantby permutation (permutation p = 0.157 for GGCAG and permutation p = 0.271 for GGTAG). The GGCAG haplotype, with the C alleleof SNP03, showed overtransmission (62.61%). Overtransmission(51.58%) of the C allele of SNP03 was also observed in single SNP
TDT.

Table 5
Haplotype associations of SNPs belonging to the three LD blocks of CD38 in HFA trios.
Block

Haplotypea

Frequency

T (%)

Individual
p-value

Permutationb
p-value

Block 1 (SNPs 1-5)

GACAA
GGCAG
GACGG
AACAG
GGTAG
GACAG

0.304
0.248
0.163
0.153
0.099
0.02

49.41
62.61
39.68
54.39
31.25
62.65

0.914
0.022
0.102
0.508
0.034
0.456

1
0.157
0.565
0.998
0.271
0.994

CC
AT
CT

0.476
0.287
0.23

48.69
37.05
69.18

0.781
0.015
0.0007

1
0.12
0.005

0.001

AT
AC
GT

0.441
0.286
0.274

54.53
46.7
47.73

0.351
0.543
0.67

0.979
0.999
1

0.638

Block 2 (SNPs 6-7)

Block 3 (SNPs 8-9)Blockp-value

0.055

T (%): Transmitted/(transmitted + untransmitted). Signiﬁcant values (p < 0.05) are indicated in bold italicaAll possible combinations of haplotypes with frequency > 0.01.
b
10,000 permutations.

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codon 264. Two others mutations [C4092T (SNP14) and A5346C(rs1800051)] were synonymous.
A weak association was detected for rs1800561 (R140W) in
adult ASD patients (average age = 22.8 ± 7.6 years old; allele frequency = 0.052) compared with controls (average age 34.1 ± 4.3
years old; allele frequency = 0.006) (p < 0.05) in cohort 1. Therefore, we focused only on the rs1800561 (R140W) polymorphism
in case–control cohort 2 (Table 1) from 3 Japanese sites and genotyped in the same platform. Of 301 Japanese ASD subjects with theaverage age of 11.9 ± 6.7 years old, 13 male (but no female) patientswere heterozygous for R140W (allelic frequency, 0.022). In 417
unscreened control subjects without ASD, 10 males and 7 females
were heterozygous for the mutation and one female was homozygous (allelic frequency, 0.023). We failed to replicate the associationin the larger sample set (cohort 2) (2 = 1.20, p < 0.3). Furthermore,
although we detected the SNP in 5 of the 150 Korean controls, it was
not detected it among the 16 Korean patients (cohort 3 in Table 1)or the 263 Caucasian patients (cohort 4 in Table 1).
3.3. rs1800561 (R140W) SNP in families

Fig. 3. Comparison of the distribution of ADI-R C scores of autistic individuals acrossthe C/C, C/A, and A/A genotypes of rs3796863 in 252 trios (A) and HFA trios (B). Signiﬁcant variation was observed in the distribution of ADI-R C scores between thethree groups, in the 252 trios and HFA trios. The variation in the distribution of ADIR C between the C/C and C/A groups was signiﬁcant at the 0.05- and 0.01-levelsin the 252 and HFA trios, following post hoc pairwise comparison with Bonferroni’s
method. One-way analysis of variance (ANOVA), followed by post hoc pairwise comparison with Bonferroni’s test, was used to examine the variability in the distributionof ADI-R phenotypic data (ADI-R A, ADI-R BV, ADI-R C, ADI-R D) across the homozygous and heterozygous genotypes of SNPs that showed signiﬁcant associations insingle SNP TDT.

The 2nd haploblock including SNPs06 and 07, showed a strong
association (p = 0.001) in the HFA subgroup (Fig. 4 and Table 5). In
this block, the haplotypes AT (p = 0.015) and CT (p = 0.0007) showedsigniﬁcant associations; the association shown by CT remained signiﬁcant (permutation p = 0.005), while that of AT was not signiﬁcant(permutation p = 0.12), by permutation. There was an overtransmission (69.18%) of the CT haplotype, with the C allele of SNP06.
The C allele of SNP06 also showed overtransmission (54.52%) in
single SNP TDT.
The haplotypes of the 3rd haploblock, which included SNPs 08
and 09, did not show any association with HFA. None of the haplotypes in the 252 trios showed signiﬁcant association.
3.2. Mutation analysis
Next, we performed mutation and/or exonic SNP analysis in
CD38 (Fig. 2A) in cohort 1. The C3139T polymorphism in exon 1
led to an arginine-to-cysteine substitution at codon 47, the C4693T(rs1800561) polymorphism in exon 3 that led to an arginine-totryptophan substitution at codon 140 (R140W), and the C6900T
polymorphism in exon 7 led to a serine-to-leucine substitution at

In the course of our studies of the rs1800561 SNP (R140W),
we identiﬁed three families (cohort 1) in which ASD appeared torelatively segregate as a dominant trait (Fig. 5). In these families,
fathers, brothers, and other relatives of 3 probands (two autistic(2-II-1 and 3-III-2) and one Asperger (1-III-1)) showed clinically
identiﬁed ASD or exhibited ASD traits. AQ scores for the brothers of
two probands (1-III-2 and 3-III-1) fulﬁlled the criteria (score >28)
for ASD (Asperger disorder).
Twenty-eight family members were available (Fig. 5, not all are
shown), and the R140W heterozygous SNP was found in 18 subjects
whose ages ranged from 22 to 86 years old. Out of them, 8 carriers
were clinically diagnosed as ASD or with ASD traits (44%). In thesepedigrees, no ASD subjects were found without this mutant SNP.
Next, we examined whether mRNA from the mutant allele isexpressed in the patients. We prepared blood RNA samples fromone subject with the C/C genotype and 3 subjects with C/T genotype. cDNA with 4693C has the MspA1I restriction site. The RT-PCR
products from homozygous 4693C/C and heterozygous C/T subjects were digested by MspA1I. RT-PCR products from the C/C
subject gave two (digested) bands, while those from the C/T subjects gave 3 bands with an additional undigested one (data notshown). Furthermore, sequencing of RT-PCR products of the C/Tsamples conﬁrmed the existence of the SNP. These results showthat the mutant (W140) allele was transcribed and expressed inthe 3 probands.
3.4. Plasma OT and vasopressin levels
Plasma OT levels of ASD probands with the W140 allele(79.3 ± 14.9 pg/ml; n = 3) were signiﬁcantly lower than those of ASD
subjects without the W140 allele in cohort 1 (147.7 ± 15.0 pg/ml;
p < 0.01, n = 26; Fig. 6). The OT levels in ASD patients with theW140 allele were signiﬁcantly lower than those in control subjects with the R140 allele (198.2 ± 24.7 pg/ml; n = 100; p < 0.01).
Only one control subject with the W140 allele was available forplasma OT measurement, and the value was 174.7 pg/ml. The OT
levels in ASD patients with the R140 allele were almost equivalent
to those in control subjects with the R140 allele, suggesting thatallele (C/T) status may be a unique determinant for the plasma OTlevel among multiple confounding factors. In contrast, plasma AVPlevels were slightly higher in the probands with the W140 allele
than in ASD patients with the R140 allele (38.9 ± 3.8 pg/ml, n = 3,
vs. 26.9 ± 5.0 pg/ml, n = 26; p < 0.05) (Fig. 6).
Finally, one proband (3-III-2) aged 23 years, diagnosed with
autism at the age of 3 years and 9 months, began nasal OT adminis-

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Fig. 4. LD structure of CD38 based on D values calculated from HFA trios. A linkage disequilibrium (LD) plot was constructed using the D (linkage disequilibrium coefﬁcient;
Ranade, 2001) pairwise LD values between markers, estimated using the Haploview software. Based on the LD structure of the gene, haplotype associations were examined;
all the haplotypes with frequency >0.01 were included for the association test.

Fig. 5. Pedigrees of the 3 ASD probands carrying the W140 allele. Squares and circles represent male and female family members, respectively. Black squares represent thosewith ASD or ASD trait. A slash mark through symbols indicates the subject is deceased. The allele status is indicated under the symbols: N/N, two normal alleles; R140W/N,
one mutant and one normal allele. Gray symbols indicate undetermined (no DNA available for analysis). The subjects are identiﬁed by Arabic numbers, and the generation
by Roman numerals. The arrow indicates the proband. (*) Autism trait; (#) Autism; (A) Asperger disorder.

Fig. 6. Plasma oxytocin and vasopressin levels in ASD subjects. Bar graph (A) and Scatchard plot (B) of plasma concentrations of OT and AVP levels in 29 ASD patients incohort 1 with (red) or without (green) the W140 allele. Mean ± S.E.M. *p < 0.05; **p < 0.01 (one-way ANOVA).

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9Fig. 7. Molecular structure of CD38. The interactions between R140 (A) or W140 (B) and nearby residues in CD38 protein are shown. Hydrogen bonds are shown as dashedlines. The protein residues are colored as follows: C, blue except the 140 residue (yellow); N, blue; O, red. R140 and W140 are packed into the helix, and the packing
conformation seems stable. The W140 protein has an outward conformation and fewer interacting residues than the wild-type protein. (C) Distribution of distance and angle
between domains. The distance between Q171 and S213 and the angle between Q171, G113 (hinge), and S213 are plotted based on domain analysis. The R140 structuresare shown as black dots and the W140 as red dots. Each point representing a conformation is from MD simulation (5–9 ns). The mutant structure has a more the open
conformation than the wild-type and a slightly larger degree of variation. (D) Solvent-accessible surface areas. The active site properties are signiﬁcantly different between
the mutant and wild-type. (E) Distribution of distance and angle between domains during 5–9 ns MD simulation as in (C): R140, black; W140, red; K140, green; and G140,
blue. K140 shows almost the same distribution as the wild-type. G140 shows a slightly different distribution, but less than W140.

tration twice a day at home with parental assistance in June 2008.
The immediate effect of OT was obvious after the ﬁrst trial, in which
he was no longer boisterous after awaking early in the morning.
This quieting on awaking has been maintained for more than 12
months. He showed improvements in eye contact behavior withsmiling and answering to yes/no questions in his daily life. There
were no signiﬁcant adverse effects.
4. Discussion
Here, we demonstrated that one rs3796863 (C > A) SNP of CD38
showed signiﬁcant association with U.S. but not Japanese highﬁunctioning autism (HFA) patients, with a cutoff at IQ > 70. Basedon the results of SNP- and haplotype-TDT analyses, the A alleleof rs3796863 of CD38 may be considered a protective allele andthe C allele as a risk allele for U.S. HFA cases. As the allele frequency is about 0.3, this variant is common. Very recently, common
variants on 5p14.1 between neural cadherin 10 and 9 have beenreported to associate with ASD (Wang et al., 2009). As rs3796863 is
an intronic SNP, the functional importance of this SNP remains to
be determined. There were signiﬁcant variations in the distributionof ADI-R C scores (restricted, repetitive, and stereotyped patternsof behavior) between the C/C, C/A, and A/A genotypes of this SNP
in 252 trios and HFA trios. The second haploblock of CD38, which
included rs3796863, showed a signiﬁcant association with HFA.
The association shown by the CT haplotype of second haploblock
remained signiﬁcant by permutation analysis. This common variant (Arking et al., 2008) may contribute to the genetic susceptibilityof HFA, in addition to other susceptiblity genes for HFA (Weiss andArking, 2009; Wermter et al., 2010).
SNP (rs1800561) of CD38 was reported in Japanese (allele frequency, 0.035) and Han Chinese (0.01) but not in European or
African control populations in the online SNP database. However,recently, it was detected in Polish Caucasians, where 3 healthy
controls out of 500 yielded an allele frequency of 0.003, and 21
W140 carriers were found among 439 B-cell chronic leukocytic
leukemia patients (frequency, 0.024) (Jamroziak et al., 2009). An
Italian study indicated one carrier among 25 healthy controls (frequency, 0.02; cohort 4) (Mallone et al., 2001). We found 68 carriersof the T genotype among 1384 Japanese, and they included controls, ASD patients, and family members of ASD patients. We also
detected this genotype in 5 of 150 Koreans non-ASD controls withdiabetes (frequency, 0.017; cohort 3) (Table 1), indicating that thepolymorphism is more common among Asians than Caucasians.

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The biological relevance of the inheritance patterns of the
R140W allele was unclear in the context of ASD. The male offspring of W140 carriers seem to have a higher risk of ASD thanfemales. Why the effect of the W140 variant on development of ASD
varies with sex is unknown, but females may have more protective
factors and/or weaker risk factors related to female hormones or
OT. Plasma OT levels were lower in W140 allele ASD carriers than
R140 ASD carriers. However, taking into account the results that
the W140 allele is not associated with ASD in general population,
we can only say that the W140 allele is deemed to have a role indecreased plasma OT levels, but not in AVP levels, regardless of disease status. This scenario is expected from our prior observation in
Cd38 knockout mice (Jin et al., 2007), in which the plasma OT but
not AVP level was differentially decreased.
Our ﬁnding that one proband (3-III-2 in Fig. 5) with the W140
allele had been receiving intranasal OT was unexpected. His social
behavior showed some improvement after the ﬁrst administration of OT, and this improvement has been maintained for morethan 12 months. The observed effects are in accordance with thosereported previously for OT: improved mind- or emotion-reading
and social memory, increased eye contact, and positive communication (Hollander et al., 2007; Domes et al., 2007; Guastella etal., 2008a,b, 2010). However, to our knowledge, this is the ﬁrst
report of a long-term therapeutic effect of OT on the social deﬁcits
in ASD, suggesting that it may beneﬁt a broader group of patients,
disorders, or typical adults and individuals carrying this rare allele.
We conducted a preliminary structural analysis of CD38 andvarious mutant proteins by Dyndom and MD simulation to analyze domain motion (Hayward and Lee, 2002) (Fig. 7). W140-CD38
had a completely different conformation than R140-CD38 (Fig. 7A
and B). The charge change at the packed site caused by the R → W
substitution was likely the primary reason for the outward conformation of the mutant protein. The mutant structure is more open
and has a slightly larger degree of variation (Fig. 7C and E). Fig. 7Dshows the solvent-accessible surface area of the active site of themutant protein altered by the closure motion, signiﬁcantly changing its properties. Thus, W140 CD38 showed changes that may
affect the substrate binding afﬁnity and eventually enzyme activity.
The amino acid substitution can cause severe perturbations of thepredicted protein structure in comparison with wild-type human(R140), rabbit (K140), and mouse (G140) CD38. In CHO cells, in fact,
the W140-CD38 protein possesses only one third of the ADP-ribosyl
cyclase activity of R140-CD38 (Yagui et al., 1998). Moreover, social
amnesia was not rescued by local re-expression of human W140CD38 in the hypothalamus in the Cd38 null mice (Jin et al., 2007).
Taken together, these observations indicate functional abnormalityof W140-CD38.
In conclusion, despite their statistical limitations, our results
suggest that the rs3796863 SNP may contribute to genetic susceptibility to HFA in U.S. but not necessarily in Japanese subjects (at least
to the limits of our current analysis). Our results call for functionaland expression assay to assess the biological effects of the variant.
Furthermore, the W140 allele could be a potential risk factor for asubset of Japanese ASD patients, i.e., males with low blood OT levels. Patients in this subgroup are candidates for a clinical trial of OT
treatment, although further systematic case–control investigations
are required to verify its effects.

Acknowledgments
This study was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. We
thank Takako Ohbayashi, Michiko Hoshii, and Shizuka Aikawa
for technical assistance. We thank D.A. Brown for discussion, theAGRE for samples, and are grateful for fundings from Fondazione

Guido Berlucchi and Compagnia di SanPaolo and the Japan-Russian
Bilateral Science Promotion Program of the Japan Society for the
Promotion of Science.

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Please cite this article in press as: Munesue, T., et al., Two genetic variants of CD38 in subjects with autism spectrum disorder andcontrols. Neurosci. Res. (2010), doi:10.1016/j.neures.2010.03.004