Hindawi Publishing Corporation
Autism Research and Treatment
Volume 2012, Article ID 205362, 4 pages
DecreasedLevelsof EGFinPlasma of ChildrenwithAutism
CharityOnore,1,2Judy Vande Water,2,3andPaulAshwood1,2
1Department of Medical Microbiology and Immunology, University of California, Davis, USA
2The M.I.N.D. Institute, University of California, Davis, Sacramento, CA 95817, USA
3Division of Rheumatology, Allergy and Clinical Immunology, University of California, Davis, USA
Correspondence should be addressed to Paul Ashwood, firstname.lastname@example.org
Received 14 September 2011; Revised 3 November 2011; Accepted 16 December 2011
Academic Editor: Antonio M. Persico
Copyright © 2012 Charity Onore et al.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder estimated to affect 1 in 110 children in the U.S., yet the
pathology of this disorder is not fully understood. Abnormal levels of several growth factors have been demonstrated in adults
with ASD, including epidermal growth factor (EGF) and hepatocyte growth factor (HGF). Both of these growth factors serve
important roles in neurodevelopment and immune function. In this study, concentrations of EGF and HGF were assessed in the
plasma of 49 children with ASD aged 2–4 years old, and 31 typically developing controls of a similar age as part of the Autism
Phenome Project (APP). Levels of EGF were significantly reduced in the ASD group compared to typically developing controls
(P = 0.003). There were no significant differences in HGF levels in young children with ASD and typically developing controls.
EGF plays an important role in regulating neural growth, proliferation, differentiation and migration, and reduced levels of this
molecule may negatively impact neurodevelopment in young children with ASD.
Autism Spectrum Disorder (ASD) is a developmental disor-
der characterized by impairments in social interaction and
communication, and the presence of restricted behaviors or
interests . According to the most current CDC estimate,
ASD affects 1 in 110 children in the US , yet the patho-
physiology of the disorder is largely unknown. Recently, sev-
eral growth factors have been found to be dysregulated in a
substantial proportion of adults with ASD .
the processes of neuronal growth, differentiation, and pro-
liferation, as well as regulating neuronal survival, neuronal
migration, and the formation or elimination of synapses .
In addition to their central function in regulating neuro-
development, current literature has also illustrated the dual
nature of many growth factors as immune modulators and
highlighted their involvement in crosstalk between the im-
mune system and the central nervous system (CNS) [4–7].
Many studies suggest the presence of aberrant immune
activity in ASD, in the CNS [8, 9] and in the periphery
[10–12], which may be influenced by atypical growth factor
activity. Growth factor dysregulation may contribute to ASD
pathology by directly affecting CNS development, and/or by
augmenting immune function.
Epidermal growth factor (EGF) and hepatocyte growth
factor (HGF) are both involved in the growth and prolifera-
tion of several cell types, including neurons and glia of the
CNS. EGF is present at high levels in the central nervous
system (CNS) and plays a critical role in controlling pro-
liferation and differentiation of nervous tissue during neu-
rogenesis [13, 14]. In addition to the function of EGF as a
CNS growth factor, it is a central factor in promoting wound
healing. EGF is expressed at sites of injury and inhibits the
activity of nitric oxide synthase, preventing inflammation
[4, 15]. EGF deficiency results in several neurological,
gastrointestinal, dermal, and pulmonary abnormalities in
animal models . An increased frequency of EGF single
nucleotide polymorphisms has been reported in ASD, as well
as lower plasma EGF levels in adults with autism [17, 18].
and an immune modulator. HGF can modulate immune
2Autism Research and Treatment
responses by signaling through the MET receptor on antigen
presenting cells, which results in a tolerogenic phenotype
with reduced proinflammatory cytokine production and
cellular activity . HGF is also essential for normal neu-
rodevelopment, and disruption of HGF signaling results in
complex alterations in GABAergic neuron development in
the forebrain of animal models . Lower levels of HGF
in sera of autistic adults have been described , as well
as decreased expression of the HGF receptor in postmortem
brain samples .
To determine if there exists a differential profile for
peripheral blood growth factor levels in ASD, we analyzed
plasma EGF and HGF in well-characterized young children
aged 2–4 years old with a diagnosis of ASD and unrelated
typically developing children who were frequency-matched
for age. In addition, levels of plasma EGF and HGF were
investigated for any associations with clinical behavioral and
2.Methods and Materials
2.1. Subjects and Behavioral Assessments. Eighty study par-
ticipants aged between 2–4 years of age were recruited as
part of the APP . Participants consisted of 49 children
with ASD (median age of 2.88 years, interquartile range
2.66–3.41 years, 42 males) and 31 typically developing (TD)
children (median age of 2.96 years, interquartile range
2.85–3.27, 20 males). Diagnostic instruments included the
Autism Diagnostic Observation Schedule-Generic (ADOS-
G)  and the Autism Diagnostic Interview-Revised (ADI-
R) . All diagnostic assessments were conducted or
directly observed by trained, licensed clinical psychologists
who specialize in autism and had been trained according
to research standards for these tools. Inclusion criteria for
ASD were taken from the diagnostic definition of ASD
in young children formulated and agreed upon by the
Collaborative Programs of Excellence in Autism. Inclusion
criteria for TD controls included developmental scores
within two standard deviations of the mean on all subscales
of the MSEL. Exclusion criteria for TD controls included
a diagnosis of mental retardation, pervasive developmental
disorder or specific language impairment, or any known
developmental, neurological, or behavioral problems. TD
children were screened and excluded for autism with the
Social Communication Questionnaire (scores > 11) (SCQ—
Lifetime Edition) .
2.2. Measurement of EGF. Peripheral blood was collected in
acid-citrate-dextrose Vacutainers (BD Biosciences, San Jose,
CA). The plasma fraction was immediately harvested by
centrifugation and stored as aliquots at −80◦C until the date
of assay. Plasma levels of EGF were determined by Human
Systems, Minneapolis, MN). Plasma levels of HGF were
according to the protocols provided by the manufacturer,
and all samples were assayed in duplicate. Optical density
was measured on a Wallac Victor3 multilabel-plate reader
(PerkinElmer, Boston, MA) at 450nm.
2.3. Statistical Analysis. Statistical analysis to compare levels
of growth factors between ASD and TD groups was con-
ducted with unpaired Student’s t-test. All analyses were con-
ducted with GraphPad Prism statistical software (GraphPad
Software Inc., San Diego, CA).
Plasma levels of EGF were approximately 3-fold lower in
the ASD group (23.1 ± 6.2pg/mL) compared with the TD
group(76.9±20.0pg/mL)(P = 0.003).PlasmalevelsofHGF
were not statistically different between children with ASD
(423.5 ± 20.9pg/mL) and TD controls (436.6 ± 19.2pg/mL)
In this study, we investigated the levels of EGF and HGF,
two growth factors involved in neurodevelopment. Previous
studies have found decreased levels of EGF in high function-
ing adults with autism , but no studies have looked at
children with ASD who are close to the onset of the disorder.
We found that levels of plasma EGF were significantly
reduced in young children with ASD as compared with
similarly age-matched typically developing control children.
This finding is consistent with that seen in adults with high
functioning autism and may suggest that a deficiency in EGF
is persistent throughout the time course of ASD.
Current research has demonstrated that EGF is involved
in growth, differentiation, and maintenance of several tissues
including the CNS and the gastrointestinal tract (GI) .
reported in ASD . In the CNS, EGF serves as a potent
neurotrophic factor, and in vivo studies have demonstrated
the effect of EGF in promoting proliferation, differentiation,
survival, and migration of multipotent neural progenitor
cells and the differentiation of these cells into astrocytes and
neurons . In this paper, we were limited to investigating
EGF in plasma and not in cerebral spinal fluid. However,
EGF rapidly transports across the blood-brain barrier ,
suggesting peripheral EGF levels could be representative of
levels in the CNS that may impact neurodevelopment.
In the GI tract, EGF is necessary for normal development
of the intestinal mucosa, and mice deficient in EGF receptor
suffer from symptoms similar to those of necrotizing ente-
rocolitis, with gradual destruction of villi, become severely
malnourished, and typically die before postnatal day 8
. EGF promotes wound healing in animal models of
ulcerative colitis [29, 30]. Although GI symptoms affect a
large proportion of children with ASD, the exact nature and
extent of GI inflammation in ASD are still controversial [31–
34]. Interestingly, preliminary investigations in this study
show that EGF levels are associated with increased bloating
in children with ASD, as well as with raw, standard, and
percentile rank scores on the Peabody picture vocabulary
Autism Research and Treatment3
P = 0.0032
Figure 1: Plasma levels of EGF and HGF. Levels of EGF and HGF in peripheral blood plasma from ASD participants and similarly aged
typically developing controls as measured by ELISA (data is shown as mean and SEM). EGF levels are significantly lower in ASD subjects in
comparison to typically developing controls (P = 0.003). HGF levels did not differ significantly between the two groups. Significance was
determined by two-tailed unpaired Student’s t-test.
test-III and with the composite and nonverbal development
quotient of the Mullen’s test (P < 0.05, data not shown).
However, the meaning of this data is not clear and we will
validate these findings in a larger replication cohort.
It has been previously reported that HGF was decreased
find a significant difference between children with ASD and
typically developing children for plasma HGF levels. This
maybe dueto agedifferencesbetween the participants of our
study and those in the previous reports. To our knowledge,
the relationship between age and serum HGF levels has not
been thoroughly established, but there is evidence that levels
may decrease with age .
Collectively, our data suggest that reduced levels of EGF
research to better elucidate the relationship between this
potent growth factor and ASD pathophysiology.
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