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Interplay between maternal Slc6a4 mutation and prenatal stress: A possible mechanism for autistic behavior development

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The low activity allele of the maternal polymorphism, 5HTTLPR, in the serotonin transporter, SLC6A4, coupled with prenatal stress is reported to increase the risk for children to develop autism spectrum disorder (ASD). Similarly, maternal Slc6a4 knock-out and prenatal stress in rodents results in offspring demonstrating ASD-like characteristics. The present study uses an integrative genomics approach to explore mechanistic changes in early brain development in mouse embryos exposed to this maternal gene-environment phenomenon. Restraint stress was applied to pregnant Slc6a4+/+ and Slc6a4+/− mice and post-stress embryonic brains were assessed for whole genome level profiling of methylome, transcriptome and miRNA using Next Generation Sequencing. Embryos of stressed Slc6a4+/+ dams exhibited significantly altered methylation profiles and differential expression of 157 miRNAs and 1009 genes affecting neuron development and cellular adhesion pathways, which may function as a coping mechanism to prenatal stress. In striking contrast, the response of embryos of stressed Slc6a4+/− dams was found to be attenuated, shown by significantly reduced numbers of differentially expressed genes (458) and miRNA (0) and genome hypermethylation. This attenuated response may pose increased risks on typical brain development resulting in development of ASD-like characteristics in offspring of mothers with deficits in serotonin related pathways during stressful pregnancies.
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SCIENTIFIC RePoRTS | 7: 8735 | DOI:10.1038/s41598-017-07405-3
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Interplay between maternal Slc6a4
mutation and prenatal stress: a
possible mechanism for autistic
behavior development
Calvin P. Sjaarda1,2, Patrick Hecht3, Amy J. M. McNaughton1,2, Audrina Zhou1,2, Melissa L.
Hudson1,2, Matt J. Will4, Garth Smith5,6, Muhammad Ayub1, Ping Liang7, Nansheng Chen8,
David Beversdorf
3,9 & Xudong Liu1,2
The low activity allele of the maternal polymorphism, 5HTTLPR, in the serotonin transporter, SLC6A4,
coupled with prenatal stress is reported to increase the risk for children to develop autism spectrum
disorder (ASD). Similarly, maternal Slc6a4 knock-out and prenatal stress in rodents results in ospring
demonstrating ASD-like characteristics. The present study uses an integrative genomics approach to
explore mechanistic changes in early brain development in mouse embryos exposed to this maternal
gene-environment phenomenon. Restraint stress was applied to pregnant Slc6a4+/+ and Slc6a4+/
mice and post-stress embryonic brains were assessed for whole genome level proling of methylome,
transcriptome and miRNA using Next Generation Sequencing. Embryos of stressed Slc6a4+/+ dams
exhibited signicantly altered methylation proles and dierential expression of 157 miRNAs and 1009
genes aecting neuron development and cellular adhesion pathways, which may function as a coping
mechanism to prenatal stress. In striking contrast, the response of embryos of stressed Slc6a4+/ dams
was found to be attenuated, shown by signicantly reduced numbers of dierentially expressed genes
(458) and miRNA (0) and genome hypermethylation. This attenuated response may pose increased
risks on typical brain development resulting in development of ASD-like characteristics in ospring of
mothers with decits in serotonin related pathways during stressful pregnancies.
Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition characterised by decits in two
core domains: impairments in social communication and restricted and repetitive behaviors1. Although early
reports on the prevalence of autism described a severe condition aecting 4.5 cases per 10,000 children in the
1960s2, recent studies report a spectrum disorder aecting 1 in 68 children3. ere is no consensus in the scien-
tic community about the main cause for this increase, while much is ascribed to new administrative classica-
tions, policy and practice changes and increased awareness4, 5, it is also commonly agreed that environmental
and genetic factors play important roles6. ASD has a complex etiology with large genetic and signicant envi-
ronmental components with prenatal maternal stress7 having been shown to be an important non-genetic factor
associated with development of ASD.
Serotonin (5-HT) functions as a neurotransmitter in the central nervous system (CNS) where it is involved
in a range of behaviors and psychological processes including mood, appetite, anxiety, social interactions, and
hormone release. Studies have reported increased whole blood 5-HT levels in individuals with ASD810 causing
1Department of Psychiatry, Queen’s University, Kingston, Ontario, Canada. 2Queen’s Genomics Lab at Ongwanada
(QGLO), Ongwanada Resource Center, Kingston, Ontario, Canada. 3Interdisciplinary Neuroscience Program,
University of Missouri, Columbia, Missouri, USA. 4Psychological Sciences and Bond Life Sciences Center, University
of Missouri, Columbia, Missouri, USA. 5Department of Pediatrics, Queen’s University, Kingston, Ontario, Canada.
6Child Development Centre, Hotel Dieu Hospital, Kingston, Ontario, Canada. 7Department of Biological Sciences,
Brock University, St. Catharines, Ontario, Canada. 8Department of Molecular Biology and Biochemistry, Simon Fraser
University, Burnaby, British Columbia, Canada. 9Departments of Radiology, Neurology, and Psychological Sciences,
and the Thompson Center for Autism and Neurodevelopmental Disorders, and William and Nancy Thompson
Endowed Chair in Radiology, University of Missouri, Columbia, Missouri, USA. Correspondence and requests for
materials should be addressed to X.L. (email: liux@queensu.ca)
Received: 10 February 2017
Accepted: 23 June 2017
Published: xx xx xxxx
OPEN
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a negative feedback response in the CNS, resulting in a 5-HT decit in certain target areas in the brain1113. is
has been demonstrated by positron emission tomography scans showing reduced 5-HT synthesis and changes in
receptor density in the cortex and thalamus of autistic individuals1416. A polymorphism investigated extensively
is an allelic variant, 5-HTTLPR, within the promoter region of the SLC6A4 gene which codes for the serotonin
transporter (SERT) protein. e 5-HTTLPR is a functional polymorphism located ~1 kb upstream of the tran-
scription start site and consists of a long (L), higher activity allele and a short (S), lower activity allele17. Cells with
the L/L genotypes of 5-HTTLPR have 1.9–2.2 times higher 5-HT uptake than cells with one or two copies of the
S allele18. Individuals carrying an S allele demonstrate greater amygdalar neuronal activity in response to negative
environmental cues and fearful stimuli19 and are more susceptible to anxiety and stress20. Multiple studies have
reported association of 5-HTTLPR with individuals with ASD2124.
Maternal stress causing adverse psychopathological outcomes in the ospring has been reported for many
psychological stressors2527. Our early studies on humans suggest that prenatal maternal stress can cause cogni-
tive, behavioral, physical and emotional problems in the ospring including ASD7. Many factors can modulate
the impact of prenatal maternal stress on development of ASD including timing of stress7, 28, coping mechanism
of the mother29, 30, and genetic susceptibility of the mother and embryo30.
Our more recent study reported that the maternal 5-HTTLPR polymorphism in SLC6A4, coupled with pre-
natal stress, may signicantly aect the risk for ospring to develop ASD31. Specically, mothers of children with
ASD and who carry the stress-susceptible, short allele of the 5-HTTLPR locus had higher incidences and severity
of stress during their aected pregnancies. is conclusion was based on two observations: mothers carrying
the short allele of the 5-HTTLPR loci reported more stressors compared to mothers homozygous for the long
allele during their aected pregnancy, and the same mothers reported no signicant stress exposure during their
pregnancies resulting in typically developing children, regardless of maternal genotype31. ese results suggest
that the relationship between this allele and history of stress exposure in ASD is specic to ASD and is not sim-
ply due to increased recall of stressors during pregnancy in the presence of this allele. is interaction has been
modeled in rodents, since Slc6a4+/ rodents under adverse environmental conditions develop behavioral changes
including acoustic startle, learned helplessness32, and increased fear, anxiety and depression that are consistent
with humans carrying a short allele of the 5-HTTLPR locus18, 3335. Evidence in mice models indicate that when
dams with a heterozygous knockout in the Slc6a4 gene are exposed to stress, their ospring have increased risk
to demonstrate increased anxiety and ASD-like characteristics including decreased social interaction and social
interest36. In addition, reports have shown that the homozygous or heterozygous knockout in the Slc6a4 and/
or prenatal stress in the dam aects the epigenetic signatures and transcriptome proles in newborn and adult
ospring3739. In short, mothers with aberrant serotonin function (via reduced SERT expression in humans and
Slc6a4 gene knockout in mice) respond dierently to stress, the dierential stress response is passed to the devel-
oping embryo, which increases their risk of developing ASD and ASD-like characteristics. In this study, using
integrative genomics approaches, we describe the transcriptome, miRNA and methylome response of developing
embryos to maternal cues stemming from the interplay between maternal genotype and prenatal stress.
Methods and Materials
Animal model. Slc6a4/ male mice were bred with wild-type (WT: Slc6a4+/+) female mice on a C57BL/6J
background (Jackson Laboratories, Bar Harbor, ME). ese mice contain a neomycin selection cassette that
replaced a DNA segment containing exon 2 in the Slc6a4 gene. Mice with a heterozygous knockout (KO) of
the Slc6a4 gene have a 50% reduction in SERT density, but similar serotonin transport when compared with
WT mice, while homozygous KO mice have no SERT and no serotonin transport33. e behaviors of mice with
reduced or absent Slc6a4 expression (homozygous and heterozygous KO) has been reviewed in detail by Murphy
et al.; but briey include: an increase in anxiety, depression, startle response, and response to stress, as well as
reduced learning capacity in aggression tests which may be related to decits in other social interactions tasks32.
WT female ospring underwent another round of breeding with male Slc6a4+/ mice to generate the experi-
mental dams and sires used in this study. Experimental dams (Slc6a4+/+ and Slc6a4+/) were housed in pairs of
two and bred with WT males and identication of a vaginal plug was marked embryonic day 0.5 (E0.5). ree
pregnant mice were randomly selected from each genotype for the stress group and subjected to acute restraint
stress on E12.5, as described in the Supplementary Information. All experimental procedures were approved by
and conducted in accordance with the University of Missouri Institutional Animal Care and Use Committee and
the University of Missouri Internal Review.
No statistical methods were used to pre-determine sample sizes, but we followed best practices in RNA-seq
which stipulates three biological replicates as the minimum for inferential analysis40. Placenta and embryonic
brain tissue was collected from E13.5 mice and used as a source of genomic DNA and total RNA. We collected
tissue from 3 placentas and 3 embryos for each experimental maternal condition (WT control, SERT control,
WT stress, SERT stress) for a total of 12 placenta and 12 embryo brain samples (Table1). Dams and embryos
were genotyped for the Slc6a4 allele with the EZ Fast Tissue/Tail PCR Genotyping Kit as per manufacturer’s
instructions (EZ BioResearch). Tail clippings were collected from mice at the time of weaning and from embry-
onic tissue at the time of tissue collection. e following primers were used for the PCR reaction: Forward:
5-AATGGTGAGGAGTGGTGGAG-3; Wild-type Reverse: 5-CCTAGATACCAGGCCCACAA-3; Knockout
Reverse: 5-GCCAGAGGCCACTTGTGTAG-3. Embryos had a 50% chance of inheriting the knockout Slc6a4
from their mother; to avoid embryo genotype from confounding the eects of the interaction between maternal
genotype and environmental stress, Slc6a4+/ embryos were removed from the study (sample E72L1: the rst
embryo in the le uterine horn (L1) obtained from dam 72 (E72) was removed). To aid in dierentiating between
groups of samples in this manuscript, embryos growing within Slc6a4+/+ dams are referred to as WT embryos
and within Slc6a4+/ dams are called SERT embryos even though all embryos were determined to be Slc6a4+/+.
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Library preparation and sequencing. Nucleic acids were collected from placenta and embryo brains and
used as the template for methylome, transcriptome and miRNA library construction, followed by sequencing on
the Ion Proton System (ermosher Scientic, Carlsbad, CA). Tissue collection, nucleic acid isolation, library
preparation and sequencing are described in the Supplementary Information. For each sample, we generated
19.40 ± 1.03 million mapped reads for methylome libraries, 18.86 ± 2.10 million reads for transcriptome libraries
and 4.77 ± 0.13 million reads for miRNA libraries.
Bioinformatic analysis. DNA methylation was assessed using R-based pipelines MEDIPS41 and
MethylAction42 with parameters described in the Supplementary Information. Dierentially methylated regions
(DMRs) called by both pipelines were used in the downstream analysis. One sample with low enrichment of
methylated DNA was removed from further methylome analysis (Table1). Genomic features and context were
resolved using the R package Goldmine43 using default arguments. e global methylation index was determined
as area under the curve using the trapezoidal approximation.
Transcriptome and miRNA raw reads were imported into Partek Flow version 5.0.16.1113 and analyzed with
the WT pipeline for Ion Torrent pipeline and microRNA Bowtie pipeline respectively. Dierentially expressed
genes (DEGs) and miRNA were selected by p-value 0.05, fold change >1.5 or <−1.5 and average coverage
of 15 normalized reads based on assessment of ERCC RNA Spike-In Control Mixes (Supplementary Fig.S1).
A subset of DEGs was validated by qPCR as described in the Supplementary Information and Supplementary
TableS1. Gene ontology (GO) pathway analyses were carried out using WEB-based GEne SeT AnaLysis Toolkit
(http://bioinfo.vanderbilt.edu/webgestalt/login.php/)44, 45 using the mouse genome as the reference, signicance
p-value <0.05 with the Benjamini & Hochberg multiple test adjustment. TargetScan7.0 was used to discover
genes targeted by dierentially expressed microRNA (DE miRNA) (http://www.targetscan.org/mmu_71/)46. We
downloaded the Summary Counts, default predictions les and extracted the 157 mouse miRNAs that were
dierentially expressed in our study with their potential mRNA targets. e number of targets was reduced by
removal of all mRNA that were not dierentially expressed in our study. Interactions between miRNA and their
targeted genes were networked using Cytoscape47.
Statistics. Statistical signicance was calculated using IBM SPSS Statistics Version 24 soware. Genome
methylation index was compared using one-way ANOVA followed by Tukey post-hoc test. A test for homogene-
ity of variances did not show any signicant variance between groups (df1 = 3, df2 = 6, p = 0.170). Correlation of
gene expression measured by qPCR and RNA-seq was determined using Pearson correlation coecient. Data are
indicated as mean ± standard deviation. Data distribution was assumed to be normal, but this was not formally
tested.
Data availability. Embryo brain sample metadata is provided in Table1. DMRs are listed in Supplementary
TableS2A–C, DEGs are listed in Supplementary TableS3AC and DE miRNAs are listed in Supplementary
TableS4A,B.
Results
To aid in dierentiating between groups of samples in this manuscript, embryos growing within Slc6a4+/+ dams
are referred to as WT embryos and within Slc6a4+/ dams are called SERT embryos even though all embryos were
determined to be Slc6a4+/+.
Methylome proling of embryo brain tissues. Without stress, SERT control embryos displayed 2,398
DMRs (77.5% hypomethylated) compared with the WT embryos (Supplementary TableS2A), though the overall
Sample Slc6a4 genotype
of mother Treatment Slc6a4 genotype
of embryo Sex of
embryo
E74L1 +/+Control +/+F
E59R5 +/+Stress +/+F
E69R7 +/Stress +/+M
E77L1 +/+Control +/+F
E72L1 +/Control +/M
E67R9 +/+Stress +/+M
E75L1 +/Control +/+F
E81R9 +/Stress +/+F
E23L1 +/+Control +/+M
E48R5 +/Control +/+F
E18R2 +/+Stress +/+M
E83L6 +/Stress +/+F
Table 1. Sample summary of embryo brain tissue. I. Sample identier: E74L1 refers to the rst embryo in
the le uterine horn (L1) obtained from dam 74 (E74) II. E72L1: removed from study because embryo was
heterozygous for Slc6a4. III. E23L1: was removed from methylome analysis because library was not enriched for
methylated DNA.
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global methylation index was not signicantly dierent between the two groups (WT control area under the
curve (AUC) = 116.47 ± 4.57, n = 2; SERT control AUC = 102.41 ± 25.48, n = 2) (Fig.1A). When mothers were
subjected to the stress paradigm, WT embryos displayed 844 DMRs (95.3% hypermethylated) compared to
embryos without stress (Supplementary TableS2B) but no signicant change in the global methylation index
(WT stress AUC = 118.41 ± 22.41, n = 3) was observed, while SERT embryos displayed signicantly increased
global methylation (SERT stress AUC = 170.54 ± 19.55, n = 3) (Fig.1A) due to the presence of 13,512 DMRs
(93.7% hypermethylated) (Supplementary TableS2C).
Annotation of DMRs based on its genomic context illustrated that most DMRs in the SERT control groups
map to intergenic (58.81%) and intronic (23.68%) regions of the genome when compared with the WT control
group (Fig.1B). DMRs found in embryos under stress were present in higher proportions in the promoter (WT
7.75%, SERT 6.89%), exon (WT 17.14%, SERT 13.00%) and 3 end (WT 9.86, SERT 8.37) when compared with
their respective embryos without stress (Fig.1B).
Transcriptome profiling. Whole transcriptome profiling of the embryo’s brains revealed there were
zero DEGs in the comparison between the SERT and WT control embryos. When mothers were subjected to
the stress paradigm, WT embryos responded by dierentially expression of 1,009 genes (61.5% upregulated)
(Supplementary TableS3A) and the SERT embryos differentially expressed 458 genes (53.1% upregulated)
(Supplementary TableS3B). ere were 257 DEGs shared in the WT stress and SERT stress embryos (Fig.2A).
e genotype x environment interaction (SERT stress versus WT stress) identied 149 genes that were dieren-
tially expressed between the two groups (Supplementary TableS3C). RNA-seq expression levels were validated by
Figure 1. Embryo’s methylation prole is impacted by maternal eect. (A) e global methylation index
quanties genome wide methylation level for areas with similar CpG island density. SERT embryos exposed
to prenatal stress display signicant increase in genome methylation (one-way ANOVA F (3,6) = 5.92,
p = 0.032 followed by Tukey post-hoc test p = 0.038). (B) Gene annotation links the genomic ranges containing
dierentially methylated regions (DMRs) to the gene model contexts. Control: the dierence between maternal
Slc6a4+/ (SERT) embryos and wild-type (WT) embryos without stress, WT: the eect of stress on the WT
embryos, and SERT: the eect of stress on SERT embryos.
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quantitative real-time PCR (qPCR) for 12 DEGs reecting various levels of expression, fold change and genomic
context (Fig.2B: Pearson R = 0.995, p < 0.001, R2 = 0.9899).
DEG lists were submitted to a GO pathway mapping algorithm which demonstrated that many overrepre-
sented pathways were similar in the WT and SERT embryos in response to stress (Fig.3). Generally, both groups
downregulated genes involved in neuron projection and dierentiation and upregulated genes involved in extra-
cellular matrix and adhesion; however, the number of aected genes in these pathways are much higher in the
WT response than the SERT response to stress. Overrepresentation of ribosome pathways occurs only in the
SERT response to stress.
To qualify communication between mother and embryo via dierential gene expression in the placenta, the
placenta transcriptomes were sequenced. ere was no clustering of related samples due to genotype or environ-
mental stress (Supplementary Fig.S2) and consequently, few genes were dierentially expressed due to genotype
or environmental conditions. In the absence of stress, only one gene (Xaf1) was dierentially expressed in the
SERT placenta compared with the WT placenta (fold change = 2.19, p = 1.06E-5).
miRNA proling. Comparison between miRNA expression in the SERT control versus WT control yielded
no DE miRNA. When mothers were exposed to the stress paradigm, WT embryo demonstrated 157 DE miRNAs
between control and stress groups, only 6 were upregulated (Supplementary TableS4A). Similar comparison
between the SERT embryos under stress with the SERT controls yielded no DE miRNA. e gene x environment
interaction (SERT stress versus WT stress) identied two DE miRNAs (Supplementary TableS4B).
Integration of mRNA, miRNA and methylome datasets. Selection of DMRs that overlapped with
DEGs detected 52 regions in 50 genes in the WT stress embryos and 246 regions in 146 genes in the SERT stress
embryos. Retaining only genes listed in the SFARI and/or AutismKB databases identied 19 DMRs in 17 dier-
ent ASD genes in the WT stress group and 67 DMRs in 29 ASD genes in the SERT stress group. ere were 10
DMRs in 10 WT genes and 45 DMRs in 23 SERT genes whose methylation prole correlated with the expected
change in gene expression (Table2). All 55 DMRs were hypermethylated and expression of the associated genes
are down-regulated.
Target prediction of DE miRNAs found 13 miRNAs (1224-5p, 134-5p, 135a-5p, 154-3p, 16-5p, 21c, 292a-
5p, 299a-3p, 331-3p, 376b-3p, 495-3p, 760-3p, 874-3p) (Fig.4A) that target many of the DEG in the WT stress
embryos (Fig.4B). Filtering the list of targeted DEGs to include only genes that are listed on the SFARI and/or
Figure 2. Maternal genotype and prenatal stress aect the transcriptome of developing embryos. (A) Venn
diagram showing overlap between dierentially expressed genes (DEGs) in wild-type (WT) and maternal
Slc6a4+/ (SERT) embryos in response to stress. (B) Validation of subset of DEGs identied by RNA-seq using
qPCR correlated with fold change from RNA-seq data (Pearson R = 0.995, p < 0.001, R2 = 0.9899).
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AutismKB identied 69 genes that are targeted by the 13 DE miRNA (Fig.5A). Pathway analysis of the 69 targeted
genes show similar over- and underrepresented pathways as seen with the entire list of WT stress DEGs (Fig.5B).
Discussion
Animal models have been used to investigate the neurobiological, physiological, and behavioral consequences
of reductions in SERT expression. Heterozygous knockout mice for the serotonin transporter gene, Slc6a4+/,
resemble humans homozygous with the short 5HTTLPR allele regarding SERT expression and function32 allow-
ing for predictive assessments across species. ese mice display a multitude of abnormalities in their physiolog-
ical makeup including response to stress with elevated levels of corticosterone, adrenocorticotropic hormone,
and epinephrine32, 48, 49 as well as in their behavioral phenotype including increased anxiety, acoustic startle, and
learned helplessness32 which are consistent with observations made in human carriers of the short 5 HTTLPR
Figure 3. Analysis of gene ontology for biological processes and cellular component for downregulated (le
panel) and upregulated (right panel) genes from WT embryos in response to stress (top panel), SERT embryos
in response to stress (middle panel), and the gene x environment interaction (bottom panel).
Genes that are dierentially expressed and have dierential methylation
WT Cadps, Chd7, Mtss1, Nbea, Nrxn2, Nrxn3, Plekha6, Slc4a8, Stox2, Vta1
SERT Amph (3), Ank2 (3), Apc (2), Atp2b2 (2), Camta1 (5), Ccdc88c (2), Celf5 (2), Celsr3, Chst10 (2), Clcn3 (2), Dlg4
(2), Kif5a, Mtss1 (4), Myo16, Nmnat2, Pcdh9, Phactr1, Plxna4 (2), Rere (3), Sema5b, Slc6a1 (2), Sqle, Syn3
Table 2. Integration of DEGs and DMRs datasets from embryos in WT or SERT dams in response to prenatal
stress identies genes that are dierentially expressed and have dierential methylation. I. Parenthesis indicate
the number of DMRs found within and/or surrounding each gene. II. List reects genes that are: listed in SFARI
and/or AutismKB databases, dierentially expressed (fold change 1.5 or ≤−1.5, p 0.05) and dierentially
methylated (p 0.05) in the WT or SERT embryos in response to prenatal stress.
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allele. Furthermore, the ospring of Slc6a4+/ mice exposed to stress demonstrate ASD-like characteristics
including increased anxiety37 and decreased social interaction and social interest36, similar the increased risk
of developing ASD in human children resulting from maternal polymorphism in the promoter region of the
SLC6A4 gene when combined with prenatal stress31.
e present study investigates mechanisms behind this phenomenon by examining epigenetic and transcrip-
tomic proles of mice embryo brains resulting from the interplay between maternal Slc6a4 genotype and expo-
sure to prenatal stress. We discuss the eect of maternal genotype in the absence of environmental stressors,
the typical response to stress by embryos in Slc6a4+/+ dams and the attenuated response to stress of embryos in
Slc6a4+/ dams.
Eect of maternal genotype on embryo’s brain in control environment. Comparison of the WT
and SERT control embryos demonstrated that Slc6a4+/ maternal genotype resulted in regional hypomethylation
of many regions of the genome when compared with embryos from Slc6a4+/+ maternal genotype. Genome meth-
ylation is performed by the DNA methyltransferase enzymes (Dnmt1, Dnmt3a and Dnmt3b). In addition, the
UHRF1 protein interacts with the DNMT1 enzyme at the replication fork and disruption of the DNMT1-UHRF1
interaction results in massive genomic hypomethylation50. ough individually the reduced expression of Dnmt1
(1.27 fold, p = 0.08), Dnmt3b (1.33 fold, p = 0.22), and Uhrf1 (1.49 fold, p = 0.03) were not statistically
signicant, we speculate that together the reduction in expression of these 3 genes may play a role in the regional
hypomethylation seen in the SERT embryos. e DMRs in the SERT control embryos did not have a direct eect
on gene or miRNA expression likely due to most DMRs being in intronic and intergenic regions which are usually
less signicant for impacting transcription than DMRs in promoter or exonic regions. Hypomethylation plays
an important role in cancer development because hypomethylation causes genome instability resulting in an
increased loss of heterozygosity and oncogene activation50, 51. Similarly, hypomethylation of SERT embryos may
have an indirect eect on the brain development though genome instability.
To qualify communication between mother and embryo via dierential gene expression in the placenta,
the placenta transcriptomes were sequenced. In the absence of stress, only one gene (Xaf1) was dierentially
expressed in the SERT placenta compared with the WT placenta. XAF1 has a role during implantation52, its
Figure 4. Expression level and targets of dierentially expressed (DE) miRNA. (A) Number of reads mapping
to the 13 DE miRNAs (p 0.05) that are predicted to target DE mRNA (p 0.05) in the wild-type (WT)
embryos response to stress. (B) Cytoscape map illustrating the network of DE miRNA and their predicted
DEGs.
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aberrant expression leads to pregnancy complications53. and the maternal Slc6a4+/ genotype has been shown
to aect Xaf1 expression in the ospring in mice37. Ospring from Slc6a4+/ dams have a y percent chance of
receiving one short 5HTTLPR allele and these embryos were removed from further analysis to minimize con-
founders of the maternal eect. We only found one of the six embryos (17%) collected from Slc6a4+/ dams with
the Slc6a4+/ genotype suggesting that these embryos may have reduced viability compared with WT embryos
potentially resulting from aberrant XAF expression in the placenta. While the interpretation is speculative, these
observations warrant further study to dene a role for increased expression of Xaf1 in the placenta of Slc6a4+/
dams and viability of Slc6a4+/ mice compared to WT mice in utero.
Typical embryo response to prenatal stress in Slc6a4+/+ mother. e typical embryo response to
prenatal stress is demonstrated by WT stress embryos compared with WT control embryos. We expected that
stress would have a physiological eect on the mother and the stress would be communicated to the embryo
through the placenta. e placenta forms a barrier between the mother and the embryo controlling and limiting
the movement of stress induced hormones54. In contrast to studies showing that stress caused dierential expres-
sion of several genes in the placenta55, 56, this study did not uncover DEGs in the placenta in response to stress.
Other potential maternal cues that were not considered in this report include maternal antibodies57, maternal-fetal
HPA axis (hormones) and uterine artery resistance (blood ow)58 (Fig.6). e WT embryo response to prenatal
stress is quite striking as seen by dramatic changes in the methylome, transcriptome and miRNA proles. e
transcriptomic response involved 621 upregulated genes over representing pathways involved in the extracellular
matrix and cell adhesion, and 388 downregulated genes associated with neuron development and projection.
Other studies have also shown that prenatal stress has been associated with decreased neuron proliferation59 and
dendrite growth60 in specic regions of the brain. We speculate that a coping mechanism utilized by a WT embryo
when confronted with stress involves strengthening existing cellular interactions by increased expression of genes
involved in cell adhesion and the extracellular matrix while simultaneously reducing genes involved in neuron
growth and development that may result in erroneous connectivity.
Figure 5. Autism spectrum disorder (ASD)-related DEGs targeted by DE miRNA. (A) List of 13 DE miRNA
(p 0.05) and their DEGs gene targets (p 0.05) in the WT embryos in response to stress. (B) Analysis of gene
ontology for biological process and cellular component for targeted, ASD-related DEGs for downregulated (le
panel) and upregulated (right panel) genes.
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Most DMRs found in the WT stress embryos compared with WT control embryo were hypermethylated
and many located in gene exonic and regulatory regions, indicating that they are involved in controlling gene
transcription. Likewise, other studies have reported an increase in methylation due to prenatal stress in targeted
region of the genome including the glucocorticoid receptor gene (Nr3c1)61 and the brain-derived neurotrophic
factor (Bdnf)62. e mechanism behind the hypermethylation is unknown because there was no change in Dnmt1,
Dnmt3a, or Dnmt3b expression. e list of ASD gene related DMRs include Cadps, Chd7, Mtss1, Nbea, Nrxn2,
Nrxn3, Plekha6, Slc4a8, Stox2, and Vta1, where each DMR and DEG interaction is characterised by hypermeth-
ylation and decreased gene expression. Chromodomain Helicase DNA Binding Protein 7 (CHD7) is a protein
involved in helicase activity and chromatin modeling. Mutations in the CHD7 gene are responsible for the devel-
opment of most cases of CHARGE syndrome63, an ASD syndromic subtype64. e products of the neurexin genes
(Nrxn2 and Nrxn3) function as cell adhesion molecules in the formation of neuronal synapses and are important
for the development of ASD in mice65 and humans66.
e WT response to stress also involves post-transcriptional gene regulation by miRNAs. miR-21c is one
of 3 miRNAs upregulated in the WT stress embryos and one of its predicted targets is Chd7. For Chd7, the
two epigenetic mechanisms, hypermethylation of the genomic Chd7 gene and increased expression of miR-21c,
work together to reduce gene expression. For other gene targets, like Nrxn2, hypermethylation and decreased
miR-874-3p expression appear to be working against each other. A single miRNA can target 200 transcripts and
multiple miRNA can act upon a single transcript67, so interactions involving methylation status, miRNA expres-
sion and gene expression are complex. ere are reports describing miRNA that are dysregulated in response to
stress68 and in individuals with ASD69, but there is little overlap between these studies indicating that the role of
miRNA in ASD requires more investigation69.
Atypical embryo response to prenatal stress in Slc6a4+/ mother. e SERT embryos responded
to stress by signicant increase in genome methylation and reduced transcriptional response illustrated by the
decreased number of DEGs and absence of DE miRNA. e extensive genome-wide hypermethylation may be
a result of genome instability associated with hypomethylation in the SERT control, and resulted in silencing
of genes involved in the “coping” mechanism described in the WT response. Many of the DEGs and aected
pathways are similar in the SERT and WT stress responses, however the number of DEGs associated with each
shared pathway was always less in SERT embryos, indicating the coping mechanism may be attenuated in the
SERT embryos responding to prenatal stress. Interestingly, one aected pathway unique to the SERT embryos is
the ribosome pathway, with 30 genes being overexpressed only in the SERT stress embryos. Overexpression of
ribosomal RNA and proteins has been described in cancers70, 71, though their roles beyond protein synthesis is
not well understood. ere is little research connecting overexpression of ribosomal protein and developmental
disorders, but recently Smagin et al.72 described how chronic social defeat stress leads to development of anxiety
and depression in male mice and is accompanied by upregulation of many ribosomal genes in the hypothalamus.
Figure 6. Non-genetic, in-utero factors that may contribute to development of neurodevelopmental disorders.
A mother persistently encounters challenging environmental conditions, maternal factors impact how she
relates with these conditions and impact how the stress is translated to her developing ospring. Genetically
equivalent embryos face varying environmental conditions depending on the maternal environment and
maternal response to her environment. Variables reported in this study using a mouse model are indicated
by boxes with solid line, variables prevalent in the literature and could be tested in a mouse model and/or in
humans are indicated by boxes with dotted line, and a variable that is not relevant for a mouse in laboratory
conditions but salient for ASD-risk in humans is shown by an asterisk.
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Limitations. We found variation within the SERT embryo group, specically one SERT embryo that appeared
to respond to stress comparable to the WT embryo, reducing the signicance of the gene vs environment compar-
ison. We propose that this embryo may be receiving dierent cues from the mother73 and despite the combined
risk of maternal genotype and prenatal stress, that this embryo is developing typically. Second, the size of a E13.5
embryo made brain dissection from the head dicult, and some trends presented may represent tissue surround-
ing the brain. ird, this small study presents a snapshot of the eect of maternal genotype and prenatal stress
on embryonic development. A larger sample size with multiple timepoints during embryo development would
provide a timeline demonstrating how embryos cope with acute or chronic stress.
Conclusion
Based on the results and trends of this study, we propose a generalized epigenetic mechanism behind the devel-
opment of ASD-like characteristics in embryos developing in Slc6a4+/ dams exposed to prenatal stress. Since
all the embryos were genetically equivalent, all embryonic changes that occur are likely a result of maternal eect
stemming from maternal SERT genotype and maternal stress (Fig.6). Initially, embryos in Slc6a4+/ mice demon-
strated regional hypomethylation compared to the WT mice, which we speculate may be due to reduced expres-
sion of embryonic methyltransferases and their related machinery74. e DMRs do not have a direct eect on
expression of miRNA or mRNA in the embryo; however, we speculate that the regional hypomethylation may act
indirectly in contributing to reduced embryo resilience to maternal eect as seen by the attenuated response to
prenatal stress. e typical embryonic response to stress (i.e. embryo with a WT mother) is large and organized,
involving increased methylation in transcriptionally relevant regions of many genes and dierential expression
of many mRNA and miRNA. is response may constitute a coping mechanism allowing for correct neural
development and dierentiation during adverse conditions. On the other hand, the response of an embryo with
a Slc6a4+/ mother is diminished or sluggish; they have signicant increase in genome methylation and signif-
icantly reduced transcriptomic and miRNA response. Jones et al. reported that embryos in the same maternal
environment described in this study develop into mice displaying ASD-like characteristics36. We speculate that
the transcriptomic and epigenetic changes reported in this study, specically altered gene expression involved in
neuron development, may eect typical brain development contributing to the development of ASD-like char-
acteristics described by Jones et al.36. is study highlights the importance of gene/environment interactions, the
unresolved role of maternal eect on embryo development, and generally contributes to our understanding of the
intricacy underlying the development of complex disorders like ASD.
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Acknowledgements
is work was supported by funding from Ongwanada’s operating fund (X.L.) and private donations.
Author Contributions
C.S. conducted experiments, data analysis and wrote the manuscript; P.H. performed the mouse model and tissue
collection experiments; A.M. performed methylome sequencing; A.Z. analyzed methylome data; M.H., M.W.,
G.S., M.A., P.L. and N.C. supported data analysis and manuscript writing; D.B. and X.L. conceived and supervised
the project, and supported data analysis and manuscript writing.
Additional Information
Supplementary information accompanies this paper at doi:10.1038/s41598-017-07405-3
Competing Interests: e authors declare that they have no competing interests.
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... Previous work explored the miRNA gene profile, expression, and methylation profile in the brains of the offspring of this SERT-het/stress model in mice, revealing a striking attenuation of the gene expression and miRNA changes in response to stress in the brains of the SERT-het/stress offspring mice in contrast to response to prenatal stress in the brains of SERT-wt mice (78). Significantly increased global methylation was observed in SERThet/stress offspring brains, and there were more upregulated miRNA in stressed control mice as compared to wt, but not for SERT-het/stress compared to SERT-wt. ...
... Similarly, there were fewer upregulated genes when SERT-het/stress was compared to SERT-wt than when stressed control mice were compared to wt. Therefore, with increased methylation (generally suppressing gene expression), and decreased miRNA and overall expression, it appears that the typical epigenetic response to stress in offspring brains is blunted in the presence of maternal SERThet (78). ...
... We focused on highimpact variants. See Table 1 for grouping and sample sizes c . a Exhibited a stress-dependent expression pattern in rodent brain samples from embryos exposed to prenatal stress (78). b Has been reported in association with maternal stress (71,85). ...
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This manuscript reviews the mechanisms that contribute to the production of the autism spectrum disorder (ASD), especially the genetic and immunological components. Knowing the participating elements and mechanisms are essential to establish preventive measures and look for early markers. The ASD can have subtle or devastating manifestations, and exerting immunomodulatory actions could be useful in the management of these patients. There seems to be different environmental insults that may act as triggers in genetically predisposed subjects; these insults can promote an inflammatory response in which interleukin-6 could participate actively at the level of neural stem cells and progenitors. The degree of involvement in neurogenesis and astrogenesis, and therefore, the observed clinical spectrum will depend on two facts that alter the neural circuits, including the brain region that loses proper input or output connectivity due to abnormal migration of a group of neurons, and the astrocytic survival.
... Another research investigated the effects of stress on fetuses being carried by mothers possessing stress susceptible genes. It was concluded that the fetuses of stress-exposed mothers who possess the 5-HTTLPR polymorphism gene, were more likely to develop autism 29 . Even though many studies have been conducted to understand the etiology of ASD, the underlying complex transcriptomic circuitry of it is still unclear. ...
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Rapid progress is being made in the development of next-generation sequencing (NGS) technologies, allowing repeated findings of new genes and a more in-depth analysis of genetic polymorphisms behind the pathogenesis of a disease. In a field such as psychiatry, characteristic of vague and highly variable somatic manifestations, these technologies have brought great advances towards diagnosing various psychiatric and mental disorders, identifying high-risk individuals and towards more effective corresponding treatment. Psychiatry has the difficult task of diagnosing and treating mental disorders without being able to invariably and definitively establish the properties of its illness. This calls for diagnostic technologies that go beyond the traditional ways of gene manipulation to more advanced methods mainly focusing on new gene polymorphism discoveries, one of them being NGS. This enables the identification of hundreds of common and rare genetic variations contributing to behavioral and psychological conditions. Clinical NGS has been useful to detect copy number and single nucleotide variants and to identify structural rearrangements that have been challenging for standard bioinformatics algorithms. The main objective of this article is to review the recent applications of NGS in the diagnosis of major psychiatric disorders, and hence gauge the extent of its impact in the field. A comprehensive PubMed search was conducted and papers published from 2013-2018 were included, using the keywords, "schizophrenia" or "bipolar disorder" or "depressive disorder" or "attention deficit disorder" or "autism spectrum disorder" and "next-generation sequencing"
... Therefore, any insult, including pathological conditions, environmental factors, medication, and/or epigenetics during pregnancy, may significantly affect its orchestration, resulting in suboptimal in utero conditions. Specifically, polymorphisms of its decisive components, including SERT [59,60], OCT3 [61], TPH [62], IDO [63] and MAO [64], have all been linked to poor mental health outcomes. Additionally, environmental factors (including prenatal stress, diet, and smoking) modify MAO-A expression and function [64]; the KYN pathway is particularly susceptible to inflammation [65] and hypoxia [66,67] during pregnancy. ...
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... This suggests that this specific maternal genotype together with prenatal stress increases offspring's risk of ASD [298]. In support, mouse studies showed that a combination of maternal stress and maternal 5-HTT genotype affects epigenetic mechanisms in the embryo resulting in social deficits but not anxiety-like behavior [299,300]. Moreover, maternal TPH2 functional haplotypes influence maternal depression during and after pregnancy [301]. ...
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The negative effects of prenatal stress on child wellbeing have been extensively documented. Here we consider a radically different perspective—that prenatal stress promotes postnatal developmental plasticity (Pluess M, Belsky J, Develop Psychopathol 23:29–38, 2011). We begin by outlining the differential-susceptibility hypothesis. Next, we describe two separate sets of evidence, one indicating (1) that heightened behavioral and physiological reactivity are markers of increased susceptibility (i.e., greater responsiveness to both positive and negative developmental experiences and exposures) and the other (2) prenatal stress is associated with heightened behavioral and physiological reactivity. After considering this indirect evidence consistent with the prenatal-programming-of-postnatal-plasticity hypothesis, we summarize the results of an experimental rodent study which manipulated prenatal stress and the quality of the postnatal environment via cross-fostering. We then consider a number of potential mechanisms which might instantiate prenatal-stress effects on developmental plasticity (e.g., microbiota, placental transmission). We conclude by outlining future directions for research.
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Problem/condition: Autism spectrum disorder (ASD). Period covered: 2012. Description of system: The Autism and Developmental Disabilities Monitoring (ADDM) Network is an active surveillance system that provides estimates of the prevalence and characteristics of ASD among children aged 8 years whose parents or guardians reside in 11 ADDM Network sites in the United States (Arkansas, Arizona, Colorado, Georgia, Maryland, Missouri, New Jersey, North Carolina, South Carolina, Utah, and Wisconsin). Surveillance to determine ASD case status is conducted in two phases. The first phase consists of screening and abstracting comprehensive evaluations performed by professional service providers in the community. Data sources identified for record review are categorized as either 1) education source type, including developmental evaluations to determine eligibility for special education services or 2) health care source type, including diagnostic and developmental evaluations. The second phase involves the review of all abstracted evaluations by trained clinicians to determine ASD surveillance case status. A child meets the surveillance case definition for ASD if one or more comprehensive evaluations of that child completed by a qualified professional describes behaviors that are consistent with the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision diagnostic criteria for any of the following conditions: autistic disorder, pervasive developmental disorder-not otherwise specified (including atypical autism), or Asperger disorder. This report provides ASD prevalence estimates for children aged 8 years living in catchment areas of the ADDM Network sites in 2012, overall and stratified by sex, race/ethnicity, and the type of source records (education and health records versus health records only). In addition, this report describes the proportion of children with ASD with a score consistent with intellectual disability on a standardized intellectual ability test, the age at which the earliest known comprehensive evaluation was performed, the proportion of children with a previous ASD diagnosis, the specific type of ASD diagnosis, and any special education eligibility classification. Results: For 2012, the combined estimated prevalence of ASD among the 11 ADDM Network sites was 14.5 per 1,000 (one in 69) children aged 8 years. Estimated prevalence was significantly higher among boys aged 8 years (23.4 per 1,000) than among girls aged 8 years (5.2 per 1,000). Estimated ASD prevalence was significantly higher among non-Hispanic white children aged 8 years (15.3 per 1,000) compared with non-Hispanic black children (13.1 per 1,000), and Hispanic (10.2 per 1,000) children aged 8 years. Estimated prevalence varied widely among the 11 ADDM Network sites, ranging from 8.2 per 1,000 children aged 8 years (in the area of the Maryland site where only health care records were reviewed) to 24.6 per 1,000 children aged 8 years (in New Jersey, where both education and health care records were reviewed). Estimated prevalence was higher in surveillance sites where education records and health records were reviewed compared with sites where health records only were reviewed (17.1 per 1,000 and 10.4 per 1,000 children aged 8 years, respectively; p<0.05). Among children identified with ASD by the ADDM Network, 82% had a previous ASD diagnosis or educational classification; this did not vary by sex or between non-Hispanic white and non-Hispanic black children. A lower percentage of Hispanic children (78%) had a previous ASD diagnosis or classification compared with non-Hispanic white children (82%) and with non-Hispanic black children (84%). The median age at earliest known comprehensive evaluation was 40 months, and 43% of children had received an earliest known comprehensive evaluation by age 36 months. The percentage of children with an earliest known comprehensive evaluation by age 36 months was similar for boys and girls, but was higher for non-Hispanic white children (45%) compared with non-Hispanic black children (40%) and Hispanic children (39%). Interpretation: Overall estimated ASD prevalence was 14.5 per 1,000 children aged 8 years in the ADDM Network sites in 2012. The higher estimated prevalence among sites that reviewed both education and health records suggests the role of special education systems in providing comprehensive evaluations and services to children with developmental disabilities. Disparities by race/ethnicity in estimated ASD prevalence, particularly for Hispanic children, as well as disparities in the age of earliest comprehensive evaluation and presence of a previous ASD diagnosis or classification, suggest that access to treatment and services might be lacking or delayed for some children. Public health action: The ADDM Network will continue to monitor the prevalence and characteristics of ASD among children aged 8 years living in selected sites across the United States. Recommendations from the ADDM Network include enhancing strategies to 1) lower the age of first evaluation of ASD by community providers in accordance with the Healthy People 2020 goal that children with ASD are evaluated by age 36 months and begin receiving community-based support and services by age 48 months; 2) reduce disparities by race/ethnicity in identified ASD prevalence, the age of first comprehensive evaluation, and presence of a previous ASD diagnosis or classification; and 3) assess the effect on ASD prevalence of the revised ASD diagnostic criteria published in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition.
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Problem/condition: Autism spectrum disorder (ASD). Period covered: 2012. Description of system: The Autism and Developmental Disabilities Monitoring (ADDM) Network is an active surveillance system that provides estimates of the prevalence and characteristics of ASD among children aged 8 years whose parents or guardians reside in 11 ADDM Network sites in the United States (Arkansas, Arizona, Colorado, Georgia, Maryland, Missouri, New Jersey, North Carolina, South Carolina, Utah, and Wisconsin). Surveillance to determine ASD case status is conducted in two phases. The first phase consists of screening and abstracting comprehensive evaluations performed by professional service providers in the community. Data sources identified for record review are categorized as either 1) education source type, including developmental evaluations to determine eligibility for special education services or 2) health care source type, including diagnostic and developmental evaluations. The second phase involves the review of all abstracted evaluations by trained clinicians to determine ASD surveillance case status. A child meets the surveillance case definition for ASD if one or more comprehensive evaluations of that child completed by a qualified professional describes behaviors that are consistent with the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision diagnostic criteria for any of the following conditions: autistic disorder, pervasive developmental disorder-not otherwise specified (including atypical autism), or Asperger disorder. This report provides ASD prevalence estimates for children aged 8 years living in catchment areas of the ADDM Network sites in 2012, overall and stratified by sex, race/ethnicity, and the type of source records (education and health records versus health records only). In addition, this report describes the proportion of children with ASD with a score consistent with intellectual disability on a standardized intellectual ability test, the age at which the earliest known comprehensive evaluation was performed, the proportion of children with a previous ASD diagnosis, the specific type of ASD diagnosis, and any special education eligibility classification. Results: For 2012, the combined estimated prevalence of ASD among the 11 ADDM Network sites was 14.6 per 1,000 (one in 68) children aged 8 years. Estimated prevalence was significantly higher among boys aged 8 years (23.6 per 1,000) than among girls aged 8 years (5.3 per 1,000). Estimated ASD prevalence was significantly higher among non-Hispanic white children aged 8 years (15.5 per 1,000) compared with non-Hispanic black children (13.2 per 1,000), and Hispanic (10.1 per 1,000) children aged 8 years. Estimated prevalence varied widely among the 11 ADDM Network sites, ranging from 8.2 per 1,000 children aged 8 years (in the area of the Maryland site where only health care records were reviewed) to 24.6 per 1,000 children aged 8 years (in New Jersey, where both education and health care records were reviewed). Estimated prevalence was higher in surveillance sites where education records and health records were reviewed compared with sites where health records only were reviewed (17.1 per 1,000 and 10.7 per 1,000 children aged 8 years, respectively; p<0.05). Among children identified with ASD by the ADDM Network, 82% had a previous ASD diagnosis or educational classification; this did not vary by sex or between non-Hispanic white and non-Hispanic black children. A lower percentage of Hispanic children (78%) had a previous ASD diagnosis or classification compared with non-Hispanic white children (82%) and with non-Hispanic black children (84%). The median age at earliest known comprehensive evaluation was 40 months, and 43% of children had received an earliest known comprehensive evaluation by age 36 months. The percentage of children with an earliest known comprehensive evaluation by age 36 months was similar for boys and girls, but was higher for non-Hispanic white children (45%) compared with non-Hispanic black children (40%) and Hispanic children (39%). Interpretation: Overall estimated ASD prevalence was 14.6 per 1,000 children aged 8 years in the ADDM Network sites in 2012. The higher estimated prevalence among sites that reviewed both education and health records suggests the role of special education systems in providing comprehensive evaluations and services to children with developmental disabilities. Disparities by race/ethnicity in estimated ASD prevalence, particularly for Hispanic children, as well as disparities in the age of earliest comprehensive evaluation and presence of a previous ASD diagnosis or classification, suggest that access to treatment and services might be lacking or delayed for some children. Public health action: The ADDM Network will continue to monitor the prevalence and characteristics of ASD among children aged 8 years living in selected sites across the United States. Recommendations from the ADDM Network include enhancing strategies to 1) lower the age of first evaluation of ASD by community providers in accordance with the Healthy People 2020 goal that children with ASD are evaluated by age 36 months and begin receiving community-based support and services by age 48 months; 2) reduce disparities by race/ethnicity in identified ASD prevalence, the age of first comprehensive evaluation, and presence of a previous ASD diagnosis or classification; and 3) assess the effect on ASD prevalence of the revised ASD diagnostic criteria published in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition.
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Chronic social defeat stress leads to the development of anxiety- and depression-like states in male mice and is accompanied by numerous molecular changes in brain. The influence of 21-day period of social stress on ribosomal gene expression in five brain regions was studied using the RNA-Seq database. Most Rps, Rpl, Mprs , and Mprl genes were upregulated in the hypothalamus and downregulated in the hippocampus, which may indicate ribosomal dysfunction following chronic social defeat stress. There were no differentially expressed ribosomal genes in the ventral tegmental area, midbrain raphe nuclei, or striatum. This approach may be used to identify a pharmacological treatment of ribosome biogenesis abnormalities in the brain of patients with “ribosomopathies.”
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Background: Autism is a multi-factorial condition where a single risk factor can unlikely provide comprehensive explanation for the disease origin. Moreover, due to the complexity of risk factors interplay, traditional statistics is often unable to explain the core of the problem due to the strong inherent non-linearity of relationships. The aim of this study was to assess the frequency of twenty-seven potential risk factors related to pregnancy and peri-post natal period. Methods: The mothers of forty-five autistic children and of 68 typical developing children completed a careful interview. 24 siblings of 19 autistic children formed an internal control group. Results: A higher prevalence of potential risk factors was observed in twenty-two and fifteen factors in external control and internal control group respectively. For six of them, the difference in prevalence was statistically significant. Specialized Artificial Neural Networks (ANNs) discriminated between autism and control subjects with 80.19% global accuracy when the data set was pre-processed with TWIST system selecting 16 out of 27 variables. Logistic regression applied to 27 variables gave unsatisfactory results with global accuracy of 46%. Conclusions: Pregnancy factors play an important role in autism development. ANNs are able to build up a predictive model, which could represent the basis for a diagnostic screening tool.Pediatric Research (2015); doi:10.1038/pr.2015.222.
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Stress exposure during gestation is implicated in several neuropsychiatric conditions, including autism spectrum disorder (ASD). Previous research showed that prenatal stress increases risk for ASD with peak exposure during the end of the second and the beginning of the third trimester. However, exposures to prenatal stress do not always result in ASD, suggesting that other factors may interact with environmental stressors to increase ASD risk. The present study examined a maternal genetic variation in the promoter region of the serotonin transporter gene (5-HTTLPR) affecting stress tolerance and its interaction with the effect of environmental stressors on risk for ASD. Two independent cohorts of mothers of ASD children recruited by the University of Missouri and Queen's University were surveyed regarding the prenatal environment and genotyping on 5-HTTLPR was performed to explore this relationship. In both samples, mothers of children with ASD carrying the stress susceptible short allele variant of 5-HTTLPR experienced a greater number of stressors and greater stress severity when compared to mothers carrying the long allele variant. The temporal peak of stressors during gestation in these mothers was consistent with previous findings. Additionally, increased exposure to prenatal stress was not reported in the pregnancies of typically developing siblings from the same mothers, regardless of maternal genotype, suggesting against the possibility that the short allele might increase the recall of stress during pregnancy. The present study provides further evidence of a specific maternal polymorphism that may affect the risk for ASD with exposure to prenatal stress. Autism Res 2016. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.