RESEARCH ARTICLE

Neurexin Gene Family Variants as Risk Factors for Autism

Spectrum Disorder
Jia Wang , Jianhua Gong , Li Li, Yanlin Chen, Lingfei Liu, HuaiTing Gu, Xiu Luo, Fang Hou,
Jiajia Zhang, and Ranran Song

Increasing evidence suggests that abnormal synaptic function leads to neuronal developmental disorders and is an
important component of the etiology of autism spectrum disorder (ASD). Neurexins are presynaptic cell-adhesion
molecules that affect the function of synapses and mediate the conduction of nerve signals. Thus, neurexins are
attractive candidate genes for autism. Since gene families have greater power to reveal genetic association than single
genes, we designed this case-control study to investigate six genetic variants in three neurexin genes (NRXN1,
NRXN2, and NRXN3) in a Chinese population including 529 ASD patients and 1,923 healthy controls. We found that
two SNPs were significantly associated with ASD after false discovery rate (FDR) adjustment for multiple comparisons.
The NRXN2 rs12273892 polymorphism T allele and AT genotype were significantly associated with increased risk of
ASD (respectively: OR 5 1.328, 95% CI 5 1.133–1.557, P < 0.001; OR 5 1.528; 95% CI 5 1.249–1.868, P < 0.001). The
dominant model showed the same association (OR 5 1.495, 95% CI 5 1.231–1.816, P < 0.001). The NRXN3
rs12879016 polymorphism played a significant role in ASD susceptibility under the dominant model (OR 5 0.747,
95% CI5 0.615–0.908, P 5 0.023), with the same trend detected for the G allele and GT genotype (respectively:
OR 5 0.811, 95% CI 5 0.699–0.941, P 5 0.036; OR 5 0.755, 95% CI 5 0.615–0.928, P 5 0.035). In conclusion, this study
supports the importance of two genetic variants in the neurexin gene family in ASD susceptibility in China. Autism
Res 2018, 11: 37–43. VC 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Lay Summary: Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is highly heritable, and stud-
ies have found a number of candidate genes that might contribute to ASD. Neurexins are presynaptic cell-adhesion
molecules that affect the function of synapses and mediate the conduction of nerve signals, and they play an impor-
tant role in normal brain development and become candidate genes for autism. The purpose of our study is to
explore the association between variants of the neurexins gene family and ASD in a Chinese population through a
case-control study.

Keywords: autism spectrum disorder; synapses; gene family; neurexins; single-nucleotide polymorphism; Chinese

Introduction identified individuals with ASD with an average preva-

Autism spectrum disorder (ASD) is a wide range of neu-
rodevelopmental disorders whose core symptoms
include pervasive and severe impairments in socializa-
tion and communication, restricted interests, and repet-
itive or unusual behaviors [Levy, Mandell, & Schultz,
2009]. According to estimates from the American Cen-
ters for Disease Control and Prevention (CDC) Autism
and Developmental Disabilities Monitoring Network
2000–2012, the prevalence of ASD has risen from 1 in
150 to 1 in 68 [Christensen et al., 2016; States, A.S.S.U.,
2014]. Studies in Asia, Europe, and North America have
lence of between 1% and 2% [Matsuishi et al., 1987;
Bailey et al., 1995; Icasiano, Hewson, Machet, Cooper,
& Marshall, 2004; Parner et al., 2011; Christensen et al.,
2016; Noroozi et al., 2016], and the prevalence of ASD
shows a clear upward trend. Although ASD has received
increasing attention, its etiology remains unclear [Levy
et al., 2009].
Family and early twin studies estimate that genetic
factors (heritability) can account for 90% of ASD cases
[Lichtenstein, Carlstrom, Rastam, Gillberg, & Anckarsater,
2010; Folstein & Rutter, 1977; Bailey et al., 1995; Freitag,
2008], making it the most heritable of all mental

From the Department of Maternal and Child Health and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430030, China (J.W., L.L., H.G., X.L., F.H., R.S.); Maternity and Children Health Care Hos-
pital of Luohu District, Shenzhen 518019, China (J.G., L.L., Y.C.); Department of Epidemiology and Biostatistics, Arnold School of Public Health,
University of South Carolina, Columbia, SC 29208 (J.Z.)
Jia Wang and Jianhua Gong have contributed equally to this work.
Received May 01, 2017; accepted for publication October 02, 2017
Address for correspondence and reprints: Ranran Song, Department of Maternal and Child Health and MOE Key Lab of Environment and Health,
School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, No 13 Hangkong Road, Wuhan, Hubei, China,
E-mail: songranran@hust.edu.cn
Published online 16 October 2017 in Wiley Online Library (wileyonlinelibrary.com)
DOI: 10.1002/aur.1881
C 2017 International Society for Autism Research, Wiley Periodicals, Inc.
V

INSAR Autism Research 11: 37–43, 2018 37

disorders. Thus, etiological research on ASD predomi-
nantly concentrates on genetic factors [Hallmayer et al.,
2011], and the study of candidate susceptibility genes pro-
vides an opportunity to better understand ASD. ASD is a
complex multigenic disorder. Genetic studies have esti-
mated that approximately 400–1,000 genes are involved
in ASD susceptibility [Geschwind & State, 2015]. A recent
genome-wide association (GWAS) study [C et al., 2017]
identified 18 new candidate ASD-risk genes. Many of the
identified genes encode synaptic proteins [Pinto et al.,
2010], which indicates that synaptic dysfunction might be
critical in autism.
The neurexins are a family of synaptic adhesion pro-
teins encoded by paralogous genes (NRXN13) that play
key roles in synaptic function [Tabuchi & Sudhof,
2002]. The neurexins are predominantly presynaptic
cell-adhesion molecules (CAMs) expressed in neurons.
Their binding with neuroligins located on post-synaptic
membranes plays an important role during synaptic for-
mation [Ichtchenko et al., 1995]. This protein family
participates in cell recognition and cell adhesion, cell
signal transduction, neurotransmitter release, and func-
tional synaptic formation. Neurexins alter the function
and development of synapses, which is relevant to
autism in humans [Hu et al., 2012]. Studies have identi-
fied mutations in the genes encoding neurexins as a
cause of ASD [Sudhof, 2008]. Rare NRXN1 deletions
[Marshall et al., 2008; Glessner et al., 2009], chromo-
somal abnormalities, and gene variants involving
2p16.3/NRXN1 have been identified in ASD [Kim et al.,
2008; Yan et al., 2008; Sommer, Feng, & Yan, 2011;
Zahir et al., 2008]. Liu et al. reported a statistically sig-
nificant association of NRXN1 with risk of autism in a
Han Chinese population [Liu et al., 2012]. There is also
evidence for a potential role of NRXN2 in autism, as
reported in a boy with a frameshift mutation within
NRXN2 exon 12 [Gauthier et al., 2011]. This mutation
resulted in a truncated Nrxn2a protein that lacked the
binding sites for the established postsynaptic binding
partners. Then, a 570-kb de novo deletion of 24 genes
at chromosome 11q13.1, including NRXN2, was found
in a 21-year-old man with a clinical phenotype includ-
ing autistic traits [Mohrmann, Gillessen-Kaesbach, Sie-
bert, Caliebe, & Hellenbroich, 2011]. Hemizygous
deletions involving NRXN3 might be involved in the
manifestation of an ASD phenotype [Vaags et al., 2012].
However, most studies generally focus on one of the
neurexins. The strength of single gene to explain the
etiology of diseases is limited. To improve the power of
detecting genetic associations with diseases, more stud-
ies need to address gene families [Ylisaukko-oja et al.,
2005], gene networks [Shao et al., 2016] or pathways
[Betancur, Sakurai, & Buxbaum, 2009]. A gene family
shares some common features that together might
38 Wang et al./Genetic risk for autism spectrum disorder
contribute to the pathogenesis of disease in a way that
is easier to detect than that of a single gene.
Here, we explore the association of the neurexin gene
family (NRXN1–3) with autism. Few studies have
addressed the neurexin genes family as a whole, espe-
cially in the Chinese population. We conducted this
case-control study and tested for the association
between variants in neurexin genes and ASD.
Materials and Methods
Subjects
A total of 529 ASD patients (average age of 8.24 years)
participated in this study. They were recruited from the
Maternal and Child Care Service Centre in Zhuhai city
(China), the Luohu district, Shenzhen city (China), the
Mental Health Center in Wuhan city (China), and the
Special Children’s Education Agencies in Guangzhou,
Suzhou and Wuhan, between July 2010 and July 2016.
The inclusion criteria for ASD patients were based on
the Diagnostic and Statistical Manual of Mental Disor-
ders-4th Edition (DSM-IV), and psychiatrists interviewed
all patients. This study was approved by the Ethics
Committee of Tongji Medical College of Huazhong
University of Science and Technology. The control data
came from a GWAS study in a healthy Chinese popula-
tion (average age of 61.38 years). Controls were
matched to the cases according to gender.
Identification of Candidate SNPs and Genotyping
To screen for candidate SNPs in the neurexins, we first
extracted some SNPs (splicing regulation, post-translation,
protein-coding or transcriptional regulation) from the F-
SNP database (http://compbio.cs.queensu.ca/F-SNP). Next,
these SNPs were filtered to include only those with minor
allele frequency (MAF) >5% of Han Chinese in Beijing
from the HapMap database (http://hapmap.ncbi.nlm.nih.
gov/). Then, SNAP Pairwise (http://www.broadinstitute.
org/mpg/snap/ldsearchpw.php) was used to test the link-
age disequilibrium (LD). If the SNPs had a strong LD with
each other (r2  0.80), they were considered redundant.
As a result, six SNPs remained for further analysis
(rs1045881 and rs12998798 in NRXN1; rs12273892 in
NRXN2; and rs12879016, rs2270964, and rs929921 in
NRXN3). We extracted genomic DNA from oral swab sam-
ples using a QIAamp DNA Investigator Kit DP56504 (QIA-
GEN, Beijing, China) according to the manufacturer’s
instructions. The DNA concentration and optical density
were evaluated using a Nano-Drop 1000 spectrophotome-
ter (Thermo Fisher Scientific, Waltham, MA). Genotyping
was performed at the BIO MIAO BIOLOGICAL Corpora-
tion (Beijing, China) on the Sequenom MassARRAY plat-
form (San Diego, CA) using the manufacturer’s protocol.
PCR primers and termination mixes for multiplexed
INSAR
Table 1. Basic Information of SNPs in Study Association Analysis between Individual SNPs and ASD Risk
Detection
The genotyping call rates of all six included SNPs were
Gene rs MA MAFa MAFb rate(%) P Power(%)
>95%. All six SNPs conformed to Hardy-Weinberg equi-
NRXN1 rs1045881 A 0.148 0.116 98.4 0.559 61.9 librium (P > 0.05). The MAFs of the six SNPs were simi-
NRXN1 rs11885824 A 0.309 0.356 98.0 0.399 89.8
lar to those in the HapMap database of Han Chinese in
NRXN2 rs12273892 T 0.207 0.192 96.8 0.255 78.3
NRXN3 rs12879016 G 0.340 0.411 98.0 0.966 90.7 Beijing, China. The statistical power for detecting the
NRXN3 rs2270964 A 0.415 0.465 98.6 0.077 90.9 effects of the SNPs ranged from 61.9% to 90.9% (Table 1).
NRXN3 rs929921 C 0.309 0.258 99.3 0.677 85.2 As shown in Table 2, when adjusted for gender, two SNPs
were significantly associated with ASD risk. The T allele
MA minor allele; MAF minor allele frequency.
a and the AT genotype of the rs12273892 polymorphism in
The minimum allele frequency of the SNP in the control group.
b
The minimum allele frequency of the SNPs in the Hapmap database NRXN2 were significantly associated with increased risk of
in the Chinese Beijing Han population; P Hardy-Weinberg equilibrium ASD (respectively: OR 5 1.328, 95% CI 5 1.133–1.557,
test. P < 0.001; OR 5 1.528, 95% CI 5 1.249–1.868, P < 0.001),
as was the dominant model (OR 5 1.495, 95% CI 5 1.231–
1.816, P < 0.001). The NRXN3 rs12879016 polymorphism
assays were designed using the MassARRAY Assay played a significant protective role in ASD susceptibility
Designer software (v3.1). Then, the mass of the extended under the dominant model (OR 5 0.747, 95% CI 5 0.615–
primer was determined using a MALDI-TOF mass spec- 0.908, P 5 0.023). The same trend was found for the G
trometer. Mass ARRAY Type 4.0 software was used to ana- allele and GT genotype (respectively: OR 5 0.811, 95%
lyze the resulting genotype. CI 5 0.699–0.941, P 5 0.036; OR 5 0.755, 95% CI 5 0.615–
0.928, P 5 0.035). However, we found no evidence of an
Statistical Analysis
association of other SNPs with ASD under heterozygous,
Hardy–Weinberg equilibrium for genotypes was calcu- additive or dominant models after LR.
lated by a goodness-of-fit v2 test in controls. The differ-
ences in the distribution of genotype frequencies Discussion
between cases and controls were assessed by Pearson’s
v2 test. Unconditional logistic regression (LR) using In our case-control study, we investigated whether the
additive, dominant, recessive and genotype models for NRXN gene family was associated with ASD risk in a Chi-
each SNP were executed in association analysis. Odds nese population. Our results suggest that rs12273892 in
ratios (ORs) and 95% confidence intervals (CIs) were NRXN2 and rs12879016 in NRXN3 are significantly asso-
estimated for the links between the SNPs and ASD sus- ciated with the risk of ASD in the Chinese population.
ceptibility, suggesting that variant alleles were the risk Individuals who carried the rs12273892 T allele (TT/AT)
alleles. To control for the false discovery rate (FDR), the had higher risk of ASD than AA carriers. However, indi-
Benjamin-Hochberg method was used to adjust the P viduals who carried the rs12879016 G allele (GG/GT)
values for multiple tests within the univariate LR analy- had less risk of ASD than TT carriers.
sis. The statistical power to detect the effects of the The integrity of synaptic function is essential for neu-
SNPs was calculated by Power v3.0.0 [Garcia-Closas & ral physiology [Zoghbi, 2003]. Changes in the structure
Lubin, 1999]. For example, for SNPs with MAF of 0.116, of synaptic connections and abnormalities in synaptic
the power of the sample size to detect an OR of 1.50 plasticity, density, and morphology, as well as synaptic
was 61.9%. All of the above statistical analyses were car- signal transmission, play important roles in the etiology
ried out with SPSS software v13.0 and Power v3.0.0, and pathogenesis of ASD [Blanpied & Ehlers, 2004].
and all P-values were two-tailed with a statistical signifi- Neurexins are presynaptic cell-adhesion molecules
cance level set at 0.05. encoded by paralogous genes (NRXN1–3). Each neurex-
ins gene encodes two major subtype variants, a (long)
and b (short) [Tabuchi & Sudhof, 2002]. The neurexins
Results
Subject Characteristics proteins can interact with neuroligins to form Ca21-
activated heterophilic trans-synaptic compounds. Their
A sample of 529 children with autism (441 boys and 63 interactions induce the formation of synapse-like struc-
girls, 25 without gender information; 8.24 6 3.16 years) tures and are crucial for neurotransmitter release, func-
and 1,923 controls (1,683 males and 240 females; tional synaptic formation, and other processes [Graf,
61.38 6 8.51 years) were used in the analysis. The ASD Zhang, Jin, Linhoff, & Craig, 2004; Varoqueaux et al.,
and controls were matched according gender (male:fe- 2006]. In neurexin-knockout mice, the synaptic release
male ratio of 7:1). is decreased and synaptic transmission is damaged,

INSAR Wang et al./Genetic risk for autism spectrum disorder 39

Table 2. Association Analysis between Individual SNP and ASD Risk
Gene SNP Genotype Case Control OR(95%CI) P FDR-P

NRXN1 rs1045881 G 877 3276 1

A 163 570 1.070 (0.883–1.295) 0.490 0.700
GG 367 1392 1
AG 143 492 1.102 (0.886–1.372) 0.383 0.638
AA 10 39 0.973 (0.481–1.967) 0.938 0.938
Dom 1.093 (0.883–1.353) 0.415 0.655
Rec 0.947 (0.470–1.910) 0.880 0.943
rs11885824 C 733 2656 1
A 305 1190 0.930 (0.801–1.079) 0.337 0.674
CC 258 925 1
CA 217 806 0.965 (0.787–1.183) 0.734 0.847
AA 44 192 0.822 (0.576-1.172) 0.279 0.761
Dom 0.938 (0.772–1.138) 0.515 0.702
Rec 0.835 (0.593–1.177) 0.303 0.699
NRXN2 rs12273892 A 785 3048 1
T 273 798 1.328 (1.133–1.557) <0.001 <0.001
AA 283 1216 1
AT 219 616 1.528 (1.249–1.868) <0.001 <0.001
TT 27 91 1.275 (0.814–1.997) 0.289 0.723
Dom 1.495 (1.231–1.816) <0.001 <0.001
Rec 0.835 (0.593–1.177) 0.724 0.869
NRXN3 rs12879016 T 731 2538 1
G 305 1308 0.811 (0.699–0.941) 0.006 0.036
TT 263 837 1
GT 205 864 0.755 (0.615–0.928) 0.007 0.035
GG 50 222 0.717 (0.512–1.004) 0.053 0.227
Dom 0.747 (0.615–0.908) 0.003 0.023
Rec 0.819 (0.592–1.131) 0.225 0.675
rs2270964 C 595 2250 1
A 447 1596 1.057 (0.922–1.212) 0.423 0.635
CC 168 677 1
CA 259 896 1.165 (0.936–1.449) 0.171 0.641
AA 94 350 1.082 (0.815–1.437) 0.585 0.763
Dom 1.142 (0.929–1.403) 0.208 0.693
Rec 0.989 (0.769–1.272) 0.934 0.966
rs929921 T 740 2656 1
C 310 1190 0.936 (0.807–1.085) 0.381 0.672
TT 264 921 1
TC 212 814 0.909 (0.741–1.114) 0.357 0.669
CC 49 188 0.909 (0.645–1.281) 0.587 0.734
Dom 0.909 (0.749–1.102) 0.331 0.709
Rec 0.950 (0.683–1.322) 0.761 0.846

Dom, dominant model; Rec, recessive model.

indicating that neurexins play an important role in
neurodevelopmental disorders [Zhang et al., 2005; Sons
et al., 2006].
NRXN2 is located on chromosome 11q13.1. Recent
studies have discovered genetic mutations affecting
NRXN2 in autism patients [Mohrmann et al., 2011]. We
found that the NRXN2 rs12273892 T/A polymorphism
were significantly associated with ASD risk in our popu-
lation. Fast SNP and F-SNP predict that rs12273892
directly affects protein coding. SIFT [Kumar, Henikoff,
& Ng, 2009] and PolyPhen [Adzhubei et al., 2010] pre-
dict that the SNP rs12273892 T/A polymorphism affects
protein coding, causing the amino acid change L81Q,
located in the laminin G-like 1 domain of the
Neurexin-2-b protein. NRXN2 deletion is reported to
disrupt the membrane anchor, leading to an absence of
the C-terminal trans-membrane and cytoplasmic
domains [Gauthier et al., 2011]. An NRXN2 mutation
has also been reported that removes laminin/neurexin/
sex hormone-binding globulin (LNS) domains, the
binding site for NLGNs [Gauthier et al., 2011]. Dysfunc-
tion of membrane structure and synaptic cell-adhesion
molecules affects synapse function and signal transmis-
sion, leading to brain-developmental disorders.
NRXN3 is located on chromosome 14q31. It is highly
expressed in the central nervous system. Mutations in
NRXN3 have been associated with schizophrenia in a
Han Chinese population [Hu et al., 2013] and have

40 Wang et al./Genetic risk for autism spectrum disorder INSAR

been linked to ASD [Vaags et al., 2012]. Some NRXN3
genotypes might induce synaptic dysfunction, thus
affecting brain development [Dean & Dresbach, 2006].
Our results show that carriers of the G allele (GG/GT)
of rs12879016 had less risk of ASD than TT carriers.
According to Fast SNP and F-SNP, the NRXN3
rs12879016 polymorphism could regulate its expression
and transcription. This SNP is within the 30 untrans-
lated region (UTR) and might have a role in the tran-
scription of this gene. Here, we found a trend toward
an association between NRNX3 and ASD, while the
mechanistic connection between the NRNX3 and ASD
remains to be tested.
As an important trans-membrane neural cell adhesion
molecule, NRXN1 links postsynaptic membrane pro-
teins to promote effective neurotransmission, which is
thought to be an important risk factor for ASD [Arac
et al., 2007; Pardo & Eberhart, 2007]. Although it has
been reported in many previous studies, we found no
significant association between NRXN1 and ASD. The
heterogeneity of ASD may be one of the reasons for this
discrepancy. More studies are needed to confirm our
findings because the Han Chinese population is a differ-
ent race from those in which the NRXN1 associations
have been reported.
In summary, we tested whether six neurexin gene
variants were associated with ASD susceptibility in
China. To our knowledge, this is the first study to
address NRXN1–NRXN3 and ASD susceptibility in a Chi-
nese population. We identified that two novel SNPs
(rs12273892 and rs12879016) were associated with ASD
in a Chinese population. The NRXN2 rs12273892 vari-
ant increased the risk of ASD, while the NRXN3
rs12879016 variant decreased the risk. Exciting recent
research has suggested that genetic variants in candi-
date autism genes alter brain function and structure
[Voineskos et al., 2011; Minshew & Keller, 2010], which
are linked to behavior outcomes [Shailesh, Gupta, Sif, &
Ouhtit, 2016; Shao et al., 2014]. Therefore, the explora-
tion of candidate autism genes is likely to improve our
understanding of ASD.
There are some limitations to this study. First, the
method we used to screen candidate SNPs was not suffi-
ciently rigorous, and we did not cover all SNPs likely to
affect neurexin gene function. Second, the sample size
was limited, and we did not exactly match our controls
to our cases. We used public controls taken from GWAS
data in a healthy population. Human genotypes gener-
ally do not change with age, and the differences that
come from age could be diluted by the large sample
size of controls. We matched the patients and controls
by gender, and we therefore consider our findings valid.
Third, we lacked experimental data (in vitro or in vivo)
to support the putative associations between these SNPs
and autism. Studies with larger sample sizes are needed
INSAR Wang et al./Genetic risk for autism spectrum disorder
to confirm our results, and further functional studies
are warranted.
Acknowledgments
This work was supported by Deep Science and Technol-
ogy Innovation Fund from Shenzhen (JCYJ2016042
8095110571) and the Fundamental Research Funds for
the Central Universities (HUST: 2015TS096). The
authors wish to thank all the children, parents, and
schools who participated in this project.
Conflict of Interest
All the authors declare that they have no potential
sources of conflict of interest. The corresponding author
ensures that all co-authors agree with the submission of
the paper.
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