Treatments for Biomedical Abnormalities Associated with Autism Spectrum Disorder

Abstract

Recent studies point to the effectiveness of novel treatments that address physiological abnormalities associated with autism spectrum disorder (ASD). This is significant because safe and effective treatments for ASD remain limited. These physiological abnormalities as well as studies addressing treatments of these abnormalities are reviewed in this article. Treatments commonly used to treat mitochondrial disease have been found to improve both core and associated ASD symptoms. Double-blind, placebo-controlled (DBPC) studies have investigated l-carnitine and a multivitamin containing B vitamins, antioxidants, vitamin E, and co-enzyme Q10 while non-blinded studies have investigated ubiquinol. Controlled and uncontrolled studies using folinic acid, a reduced form of folate, have reported marked improvements in core and associated ASD symptoms in some children with ASD and folate related pathway abnormities. Treatments that could address redox metabolism abnormalities include methylcobalamin with and without folinic acid in open-label studies and vitamin C and N-acetyl-l-cysteine in DBPC studies. These studies have reported improved core and associated ASD symptoms with these treatments. Lastly, both open-label and DBPC studies have reported improvements in core and associated ASD symptoms with tetrahydrobiopterin. Overall, these treatments were generally well-tolerated without significant adverse effects for most children, although we review the reported adverse effects in detail. This review provides evidence for potentially safe and effective treatments for core and associated symptoms of ASD that target underlying known physiological abnormalities associated with ASD. Further research is needed to define subgroups of children with ASD in which these treatments may be most effective as well as confirm their efficacy in DBPC, large-scale multicenter studies.

Full text

PEDIATRICS
REVIEW ARTICLE
published: 27 June 2014
doi: 10.3389/fped.2014.00066
Treatments for biomedical abnormalities associated with
autism spectrum disorder
Richard Eugene Frye1* and Daniel A. Rossignol 2
1Department of Pediatrics, Arkansas Children’s Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA
2Rossignol Medical Center, Irvine, CA, USA
Edited by:
Roberto Canitano, University Hospital
of Siena, Italy
Reviewed by:
Andrew Walter Zimmerman, Harvard
Medical School, USA
Robert Hendren, University of
California San Francisco, USA
*Correspondence:
Richard Eugene Frye, Arkansas
Children’s Hospital Research Institute,
Slot 512-41B, 13 Children’sWay, Little
Rock, AR 72202, USA
e-mail: refrye@uams.edu
Recent studies point to the effectiveness of novel treatments that address physiological
abnormalities associated with autism spectrum disorder (ASD).This is significant because
safe and effective treatments for ASD remain limited. These physiological abnormalities
as well as studies addressing treatments of these abnormalities are reviewed in this arti-
cle.Treatments commonly used to treat mitochondrial disease have been found to improve
both core and associated ASD symptoms. Double-blind, placebo-controlled (DBPC) studies
have investigated L-carnitine and a multivitamin containing B vitamins, antioxidants, vitamin
E, and co-enzyme Q10 while non-blinded studies have investigated ubiquinol. Controlled
and uncontrolled studies using folinic acid, a reduced form of folate, have reported marked
improvements in core and associated ASD symptoms in some children with ASD and folate
related pathway abnormities.Treatments that could address redox metabolism abnormali-
ties include methylcobalamin with and without folinic acid in open-label studies and vitamin
C and N-acetyl-L-cysteine in DBPC studies. These studies have reported improved core
and associated ASD symptoms with these treatments. Lastly, both open-label and DBPC
studies have reported improvements in core and associated ASD symptoms with tetrahy-
drobiopterin. Overall, these treatments were generally well-tolerated without significant
adverse effects for most children, although we review the reported adverse effects in
detail. This review provides evidence for potentially safe and effective treatments for core
and associated symptoms of ASD that target underlying known physiological abnormalities
associated with ASD. Further research is needed to define subgroups of children with ASD
in which these treatments may be most effective as well as confirm their efficacy in DBPC,
large-scale multicenter studies.
Keywords: autism spectrum disorders, mitochondria, folate receptor alpha, folinic acid, folate metabolism, redox
regulation, oxidative stress, tetrahydrobiopterin
BACKGROUND
The autism spectrum disorders (ASD) are a group of behav-
iorally defined neurodevelopmental disorders with lifelong con-
sequences. They are defined by impairments in communication
and social interaction along with restrictive and repetitive behav-
iors (1). The definition of ASD has recently undergone revision.
Previously, the Diagnostic Statistical Manual (DSM) Version IV
Text Revision divided ASD into several diagnoses including autis-
tic disorder, Asperger syndrome, and pervasive developmental
disorder-not otherwise specified. The new revision of the DSM
now does not differentiate between these ASD subtypes and con-
siders communication and social impairments together in one
symptom class (2). Complicating this change is the fact that over
the past several decades, most research has used a framework from
the former DSM versions.
Autism spectrum disorder has been recently estimated to affect
1 out of 68 individuals in the United States (3) with four times
more males than females being affected (4). Over the past two
decades, the prevalence of the ASDs has grown dramatically,
although the reasons for this increase are continually debated.
Despite decades of research on ASD, identification of the causes
of and treatments for ASD remain limited. The standard-of-care
treatment for ASD is behavioral therapy that requires full-time
engagement of a one-on-one therapist typically requiring many
years of treatment, and recent reviews have pointed out that con-
trolled studies on commonly used behavior therapies are generally
lacking (5). The only medical treatments approved by the United
States of America Food and Drug Administration for ASD are
antipsychotic medications. However, these medications only treat
a symptom associated with ASD, irritability, but not any core ASD
symptom. In children, these medications can be associated with
significant adverse effects, including detrimental changes in body
weight as well as triglyceride, cholesterol, and blood glucose con-
centrations within a short time (6) and they also increase the risk
of type 2 diabetes (7). In some studies, the percentage of chil-
dren experiencing these side effects is quite high. For example,
one recent study reported that 87% of ASD children had side
effects with risperidone, including drowsiness, weight gain, and
rhinorrhea (8).
A great majority of ASD research has concentrated on genetic
causes of ASD (9) despite the fact that inherited single gene and
chromosomal defects are only found in the minority of cases
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Frye and Rossignol Treatments for biomedical abnormalities associated with autism
(10). In fact, several recent studies that have conducted genome
wide searches for common genetic defects across large samples of
ASD children have only identified rare de novo mutations, thereby
pointing to acquired mutations and/or mutations secondary to
errors in DNA maintenance rather than inherited genetic syn-
dromes (11,12). As research in the field of ASD continues, it
is becoming clear that the etiology of most ASD cases involves
complicated interactions between genetic predisposition and envi-
ronmental exposures or triggers. Indeed,a recent study of dizygotic
twins estimated that the environment contributes a greater per-
centage of the risk of developing autistic disorder as compared to
genetic factors (13). Another study of over two million children
reported that environmental risk factors accounted for approx-
imately 50% of ASD risk (14). Recent reviews have outlined the
many environmental factors that are associated w ithASD and have
described how polymorphisms in specific genes can combine with
the environment to cause neurodevelopmental problems (15).
Recent studies have suggested that ASD is associated with
impairments in basic physiological processes such as redox (16)
and mitochondrial (9) metabolism as well as abnormalities in
regulating essential metabolites such as folate (17), tetrahydro-
biopterin (18–20), glutathione (21–23), cholesterol (24), carnitine
(25–28), and branch chain amino acids (29). Although many of
these studies have based their findings on peripheral markers of
abnormal metabolism, many studies have documented some of
these same abnormalities in the brain of individuals with ASD,
including mitochondrial dysfunction and oxidative stress (30)
and one study has demonstrated a link between oxidative stress,
inflammation, and mitochondrial dysfunction in the brain of
individuals with ASD (23). Interestingly, several of these phys-
iological abnormalities are also observed in genetic syndromes
associated with ASD. For example, mitochondrial dysfunction is
prevalent in both idiopathic ASD (31) and is associated with Rett
syndrome (32–34), PTEN mutations (35), Phelan-McDermid syn-
drome (36), 15q11-q13 duplication syndrome (37,38), Angelman
syndrome (39), Septo-optic dysplasia (40), and Down syndrome
(41,42).
Identifying the metabolic or physiological abnormalities asso-
ciated with ASD is important, as treatments for such abnormalities
may be possible. Thus, a better understanding of these abnormali-
ties may allow for the development of novel treatments for children
with ASD. Below the evidence for metabolic abnormalities related
to ASD that may be amenable to treatment arediscussed along w ith
the evidence of potential treatments for these disorders. Figure 1
provides a summary of the pathways and demonstrates which
pathways are targeted by the better studied treatments. In addi-
tion, a section on the common adverse effects of these treatments
follows the discussion of treatments.
REVIEW OF TREATABLE CONDITIONS AND THEIR POTENTIAL
TREATMENTS
MITOCHONDRIAL DYSFUNCTION
Recent studies suggested that 30–50% of children with ASD pos-
sess biomarkers consistent with mitochondrial dysfunction (31,
43) and that the prevalence of abnormal mitochondrial function
in immune cells derived from children with ASD is exceedingly
high (44,45). Mitochondrial dysfunction has been demonstrated
FIGURE 1 | Pathways affected in autism spectrum disorder that are
discussed in this article as well as the treatments discussed with their
points of action. Pathways are outlined in blue while treatments are
outlined in green. Oxidative stress is outlined in red and the red arrows
demonstrate how it can negatively influence metabolic pathways. Certain
pathways such as glutathione and tetrahydrobiopterin pathways have an
antioxidant effect and a reciprocal relationship with oxidative stress such
that they can improve oxidative stress but at the same time oxidative stress
has a direct detrimental effect on them. Mitochondrial dysfunction and
oxidative stress have mutually negative effects on each other such that
oxidative stress causes mitochondrial dysfunction while mitochondrial
dysfunction worsens oxidative stress. Dihydrofolate reductase (DHFR) is
colored in red since polymorphisms in this gene, that are commonly seen in
individuals with autism, have a detrimental effect on the reduction of folic
acid such that the entry of folic acid into the folate cycle is decreased.
Folinic acid enters the folate cycle without requiring this enzyme. Similarly
the folate receptor alpha can be impaired in individuals with autism by
autoantibodies and by mitochondrial dysfunction. In such cases, folinic acid
can cross the blood–brain barrier by the reduced folate carrier. Methionine
synthase (MS) connects the folate and methylation cycles and requires
methylcobalamin as a cofactor.
in the postmortem ASD brain (23,30,46–49) and in animal mod-
els of ASD (50). Novel types of mitochondrial dysfunction have
been described in children with ASD (28,51,52) and in cell lines
derived from children with ASD (53,54). Several studies sug-
gest that children with ASD and mitochondrial dysfunction have
more severe behavioral and cognitive disabilities compared with
children who have ASD but without mitochondrial dysfunction
(55–57). Interestingly, a recent review of all of the known pub-
lished cases of mitochondrial disease and ASD demonstrated that
only about 25% had a known genetic mutation that could account
for their mitochondrial disease (31).
Treatments that are typically used for patients with mito-
chondrial disease have been shown to improve functioning in
some children with ASD (31). Several studies, including two
double-blind, placebo-controlled (DBPC) studies (58,59) and
case reports (25,37,60–63) have reported improvements in
core and associated ASD behaviors with l-carnitine treatment.
Two DBPC studies using a multivitamin containing B vitamins,
antioxidants, vitamin E, and co-enzyme Q10 reported various
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Frye and Rossignol Treatments for biomedical abnormalities associated with autism
improvements in ASD symptoms compared to placebo (64,65).
Several other antioxidants (66), including vitamin C (67), methyl-
cobalamin (68–70), N-acetyl-l-cysteine (71–73), ubiquinol (74),
and carnosine (75), have also reported to demonstrate significant
improvements in ASD behaviors and may function to improve
mitochondrial function.
Thus, many treatments that are believed to improve mitochon-
drial function have been shown to be helpful for some children
with ASD. However, none of these studies have specifically selected
children with mitochondrial dysfunction or disease to study, so
it is difficult to know if individuals with ASD and mitochondr-
ial dysfunction would benefit the most from these treatments or
whether these treatments are effective for a wider group of chil-
dren with ASD. One study did demonstrate that the multivitamin
used for treatment resulted in improvements in biomarkers of
energy metabolism (as well as oxidative stress) suggesting that
the effect of the multivitamin may have been at least partially
related to improvements in mitochondrial function (65). Clearly,
this is a fertile area for research but there remain several compli-
cations that could impede moving forward in a systematic way.
For example, given the inconsistency in the prevalence estimates
of mitochondrial disease and dysfunction across studies (ranging
from about 5–80%), the notion that mitochondrial abnormalities
are even associated with ASD is somewhat controversial. This may
be, in part, due to the unclear distinction between mitochondrial
disease and dysfunction. However, even the lower bound of the
prevalence estimate of 5% is significant, as mitochondrial disease
is only believed to affect <0.1% of individuals in the general popu-
lation and given the current high prevalence of ASD,a disorder that
affects even 5% of individuals with ASD would add up to millions
of individuals who have the potential to have a treatable metabolic
abnormality. Other complicating factors include the fact that there
are many treatments for mitochondrial disease and these treat-
ments have not been well-studied (76). Hopefully, the increased
interest in treatments for mitochondrial disease will help improve
our knowledge of how to best treat mitochondrial disease so that
such information can be applied to children who have mitochon-
drial disease and dysfunction with ASD. Other recent approaches
include the in vitro assessment of compounds that may improve
mitochondrial function in individuals with ASD (53).
FOLATE METABOLISM
Several lines of evidence point to abnormalities in folate metabo-
lism in ASD. Several genetic polymorphisms in key enzymes in the
folate pathway have been associated with ASD. These abnormali-
ties can cause decreased production of 5-methyltetrahydrofolate,
impair the production of folate cycle metabolites and decrease
folate transport across the blood–brain barrier and into neu-
rons. Indeed, genetic polymorphisms in methylenetetrahydrofo-
late reductase (22,77–85), dihydrofolate reductase (86) and the
reduced folate carrier (22) have been associated with ASD.
Perhaps the most significant abnormalities in folate metabo-
lism associated with ASD are autoantibodies to the folate receptor
alpha (FRα). Folate is transported across the blood–brain barrier
by an energy-dependent receptor-mediated system that utilizes the
FRα(87). Autoantibodies can bind to the FRαand greatly impair
its function. These autoantibodies have been linked to cerebral
folate deficiency (CFD). Many cases of CFD carry a diagnosis of
ASD (88–94) and other individuals with CFD are diagnosed with
Rett syndrome, a disorder closely related to ASD within the perva-
sive developmental disorder spectrum (95–97). Given that the FRα
folate transport system is energy-dependent and consumes ATP, it
is not surprising that a wide variety of mitochondrial diseases (91,
94,97–102) and novel forms of mitochondrial dysfunction related
to ASD (52) have been associated with CFD. Recently, Frye et al.
(17) reported that 60% and 44% of 93 children with ASD were
positive for the blocking and binding FRαautoantibody, respec-
tively. This hig h rate of FRαautoantibody positivity was confirmed
by Ramaekers et al. (103) who compared 75 ASD children to 30
non-autistic controls with developmental delay. The blocking FRα
autoantibody was positive in 47% of children with ASD but in only
3% of the control children.
Many children with ASD and CFD have marked improve-
ments in clinical status when treated with folinic acid – a reduced
form of folate that can cross the blood–brain barrier using the
reduced folate carrier rather than the FRαtransport system. Sev-
eral case reports (89) and case series (90,91) have described
neurological, behavioral, and cognitive improvements in children
with documented CFD and ASD. One case series of five children
with CFD and low-functioning autism with neurological deficits
found complete recovery from ASD symptoms with the use of
folinic acid in one child and substantial improvements in com-
munication in two other children (90). In another study of 23
children with low-functioning regressive ASD and CFD, 2 younger
children demonstrated full recovery from ASD and neurologi-
cal symptoms, 3 older children demonstrated improvements in
neurological deficits but not in ASD symptoms, and the remain-
der demonstrated improvements in neurological symptoms and
partial improvements in some ASD symptoms with folinic acid;
the most prominent improvement was in communication (91).
Recently, in a controlled open-label study, Frye et al. (17) demon-
strated that ASD children who were positive for at least one of
the FRαautoantibodies experienced significant improvements in
verbal communication, receptive and expressive language, atten-
tion, and stereotypical behavior with high-dose (2 mg/kg/day in
two divided doses; maximum 50 mg/day) folinic acid treatment
with very few adverse effects reported.
Thus, there are several lines of converging evidence suggesting
that abnormalities in folate metabolism are associated with ASD.
Evidence for treatment of these disorders is somewhat limited but
it is growing. For example, treatment studies have mostly concen-
trated on the subset of children with ASD who also possess the
FRαautoantibodies. These studies have only examined one form
of reduced folate, folinic acid, and have only examined treatment
response in limited studies. Thus, large DBPC studies would be
very helpful for documenting efficacy of this potentially safe and
effective treatment. In addition, the role of other abnormalities
in the folate pathway beside FRαautoantibodies, such as genetic
polymorphisms, in treatment response needs to be investigated. It
might also be important to investigate the role of treatment with
other forms of folate besides folinic acid, but it might also be wise
to concentrate research on one particular form of folate for the
time being so as to optimize the generalizability of research stud-
ies in order to have a more solid understanding of the role of folate
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Frye and Rossignol Treatments for biomedical abnormalities associated with autism
metabolism in ASD. Given the ubiquitous role of folate in many
metabolic pathways and the fact that it has a role in preventingASD
during the preconception and prenatal periods (104), this line of
research has significant potential for being a novel treatment for
many children with ASD.
REDOX METABOLISM
Several lines of evidence support the notion that some children
with ASD have abnormal redox metabolism. Two case-control
studies have reported that redox metabolism in children with ASD
is abnormal compared to unaffected control children (22,105).
This includes a significant decrease in reduced glutathione (GSH),
the major intracellular antioxidant, and mechanism for detoxifica-
tion, as well as a significant increase in the oxidized disulfide form
of glutathione (GSSG). The notion that abnormal glutathione
metabolism could lead to oxidative damage is consistent with stud-
ies which demonstrate oxidative damage to proteins and DNA
in peripheral blood mononuclear cells and postmortem brain
from ASD individuals (23,30,106), particularly in cortical regions
associated with speech, emotion, and social behavior (30,107).
Treatments for oxidative stress have been shown to be of benefit
for children with ASD. In children with ASD, studies have demon-
strated that glutathione metabolism can be improved with subcu-
taneously injected methylcobalamin and oral folinic acid (69,105),
a vitamin and mineral supplement that includes antioxidants,
co-enzyme Q10, and B vitamins (65) and tetrahydrobiopterin
(20). Interestingly, recent DBPC studies have demonstrated that
N-acetyl-l-cysteine, a supplement that provides a precursor to
glutathione, was effective in improving symptoms and behav-
iors associated with ASD (72,73). However, glutathione was not
measured in these two studies.
Small (64,67), medium (72,73), and large (108) sizedDPBC t ri-
als and small and medium-sized open-label clinical trials (68,70)
demonstrate that novel treatments for children with ASD, which
can address oxidative stress are associated with improvements in
core ASD symptoms (68,70,72), sleep and gastrointestinal symp-
toms (64), hyperactivity, tantruming, and parental impression of
general functioning (108), sensory-motor symptoms (67), and
irritability (72,73). These novel treatments include N-acetyl-l-
cysteine (72,73), methylcobalamin with (69,70) and without (68)
oral folinic acid, vitamin C (67), and a vitamin and mineral supple-
ment that includes antioxidants, co-enzyme Q10, and B vitamins
(64,65).
Several other treatments that have antioxidant properties (66),
including carnosine (75), have also been reported to significantly
improve ASD behaviors, suggesting that treatment of oxidative
stress could be beneficial for children with ASD. Many antioxi-
dants can also help improve mitochondrial function (31), sug-
gesting that clinical improvements with antioxidants may occur
through a reduction of oxidative stress and/or an improvement in
mitochondrial function.
These studies suggest that treatments that address oxidative
stress may improve core and associated symptoms of ASD. Fur-
thermore, these treatments are generally regarded as safe with
a low prevalence of adverse effects. Unfortunately many studies
that have looked at antioxidants and treatments that potentially
support the redox pathway did not use biomarkers to measure
redox metabolism status in the participants or the effect of treat-
ment on redox pathways. Including biomarkers in future studies
could provide important information regarding which patients
may respond to treatments that address redox metabolism and can
help identify the most effective treatments. Since there are many
treatments used to address oxidative stress and redox metabolism
abnormalities in clinical practice and in research studies, the most
effective treatments need to be carefully studied in DBPC studies to
document their efficacy and effectiveness. Overall, the treatments
discussed above have shown some promising results and deserve
further study.
TETRAHYDROBIOPTERIN METABOLISM
Tetrahydrobiopterin (BH4) is a naturally occurring molecule that
is an essential cofactor for several critical metabolic pathways,
including those responsible for the production of monoamine
neurotransmitters, the breakdown of phenylalanine, and the pro-
duction of nitric oxide (19). BH4is readily oxidized by reactive
species, leading it to be destroyed in the disorders where oxida-
tive stress is prominent such asASD (18). Abnormalities in several
BH4related metabolic pathways or in the products of these path-
ways have been noted in some individuals with ASD, and the
cerebrospinal fluid concentration of BH4has been reported to
be depressed in some individuals with ASD (19). Clinical tri-
als conducted over the past 25years have reported encouraging
results using sapropterin, a synthetic form of BH4, to treat children
with ASD (19). Three controlled (109–111) and several open-label
trials have documented improvements in communication, cogni-
tive ability,adaptability, social abilities, and verbal expression with
sapropterin treatment in ASD, especially in children younger than
5 years of age and in those who are relatively higher functioning at
the beginning of the trial (19).
Frye has shown that the ratio of serum citrulline-to-methionine
is related to the BH4concentration in the cerebrospinal fluid,
suggesting that abnormalities in both oxidative stress and nitric
oxide metabolism may be related to central BH4deficiency (18).
More recently, Frye et al. demonstrated, in an open-label study,
that sapropterin treatment improves redox metabolism and fun-
damentally alters BH4metabolism in children with ASD. Interest-
ingly, serum biomarkers of nitric oxide metabolism were found
to predict response to sapropterin treatment in children with
ASD (20), thereby suggesting that the therapeutic effect of BH4
supplementation may be specific to its effect on nitric oxide
metabolism.
The potential positive effects on nitric oxide metabolism by
BH4supplementation could be significant for several reasons. The
literature supports an association between ASD and abnormalities
in nitric oxide metabolism. Indeed studies have documented alter-
ations in nitric oxide synthase genes in children with ASD (112,
113). In the context of low BH4concentrations, nitric oxide syn-
thase produces peroxynitrite,an unstable reactive nitrogen species
that can result in oxidative cellular damage. Indeed, nitrotyrosine,
a biomarker of reactive nitrogen species, has been shown to be
increased in multiple tissues in children with ASD, including the
brain (22,23,107,114,115). Thus, BH4supplementation could
help stabilize nitric oxide synthase as well as act as an antioxidant
and improve monoamine neurotransmitter production. Further
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Frye and Rossignol Treatments for biomedical abnormalities associated with autism
DBPC studies using biomarkers of metabolic pathways related to
BH4metabolism will be needed to determine which children with
ASD will most benefit from formulations of BH4supplementation
like sapropterin.
POTENTIAL ADVERSE EFFECTS
Although many of the treatments discussed within this manu-
script are considered safe and are generally well-tolerated, it is
important to understand that these treatments are not without
potential adverse effects. In general, these treatments are without
serious adverse effects but some children may not tolerate all treat-
ments well. Systematic and controlled studies are best at providing
data on adverse effects, so the true adverse effects of the supple-
ments discussed will only be based on the limited treatments that
have been studied in such a fashion. It is also important to under-
stand that because of the complicated nature of the effects of these
treatments, they should only be used under the care of a medical
professional with appropriate expertise and experience.
Controlled studies for treatments that address mitochondr-
ial disorders include l-carnitine and a multivitamin with various
mitochondrial supplements. In one small DBPC study, there were
no significant adverse events reported in the 16 children treated
with l-carnitine (59) while a second small DBPC trial reported
no differences between the adverse effects reported by the treat-
ment and placebo groups; notably, more patients in the placebo
group withdrew from the study because of adverse effects (58).
Thus, there is no data to suggest that l-carnitine has any significant
adverse effects. In the large DBPC multivitamin study, about equal
numbers of children in the treatment and placebo groups with-
drew from the study because of behavior or gastrointestinal issues
(65). In another small DBPC study, the investigators noted that
two children began to have nausea and emesis when they started
receiving the treatment at nighttime on an empty stomach (64).
This adverse effect resolved when the timing of the treatment was
adjusted. Thus, with proper dosing of this multivitamin, it appears
rather safe and well-tolerated.
Controlled studies for folate pathway abnormalities only
include folinic acid. In a medium-sized, open-label controlled
study, 44 children with ASD and the FRαautoantibody were
treated with high-dose folinic acid (2 mg/kg/day in two divided
doses; maximum 50 mg/day) and four children discontinued the
treatment because of an adverse effect (17). Of the four children
who discontinued the treatment, three children, all being concur-
rently treated with risperidone, demonstrated increased irritability
soon after starting the high-dose folinic acid while the other child
experienced increased insomnia and gastroesophageal reflux after
6 weeks of treatment. Since there was no placebo in this study,
the significance of these adverse effects is difficult to determine.
For example, it is not clear whether this was related to concurrent
risperidone treatment or was related to a baseline high irritabil-
ity resulting in the needed for risperidone. All other participants
completed the trial without significant adverse effects. Due to the
timing of the adverse events in the children on risperidone in this
trial, to be safe, the authors suggested caution when using folinic
acid in children already on antipsychotic medications.
Clinical studies for treatments that could address redox
metabolism include N-acetyl-l-cysteine, methylcobalamin,
methylcobalamin combined with oral folinic acid and a multivi-
tamin (as previous mentioned). One small open-label study that
provided 25–30 µg/kg/day (1500 µg/day maximum) of methyl-
cobalamin to 13 patients found no adverse effects (68) while a
medium-sized, open-label trial that provided 75 µg/kg subcuta-
neously injected methylcobalamin given every 3 days along with
twice daily oral low-dose (800 µg/day) folinic acid to 44 children
noted some mild adverse effects (69,70). Four children discontin-
ued the treatment, two because their parents were uncomfortable
given injections and two because of hyperactivity and reduced
sleep. The most common adverse effect in the participants that
remained in the study was hyperactivity, which resolved with a
decrease in the folinic acid to 400 µg/day. Lastly, two medium
sized, DBPC studies examined N-acetyl-l-cysteine, one as a pri-
mary treatment and another as an add-on to risperidone. The
trial that used N-acetyl-l-cysteine as a primary treatment noted
no significant differences in adverse events between the treatment
and placebo groups, although both groups demonstrated a high
rate of gastrointestinal symptoms and one participant in the active
treatment phase required termination due to increased agitation
(72). In the add-on study, one patient in the active treatment group
withdrew due to severe sedation (73). In this latter study, adverse
effects were not compared statistically between groups, but most
adverse effects were mild and had a low prevalence. Such adverse
effects included constipation, increased appetite, fatigue, nervous-
ness, and daytime drowsiness. Lastly, a small DPBC study using
vitamin C did not report any adverse effects from the treatment
(67). Thus, there are several relatively safe and well-tolerated treat-
ments for addressing abnormal redox metabolism, but there does
appear to be a low rate of adverse effects, reinforcing the notion
that a medical professional should guide treatment.
Three DBPC studies, one small (110), one medium (111),
and one medium-to-large (109) sized, were conducted using
sapropterin as a treatment for ASD. None of these studies have
reported a higher prevalence of adverse effects in the treatment
group as compared to the placebo group and none of these stud-
ies attributed any dropouts to the treatment. Thus, sapropterin
appears to be a well-tolerated treatment.
DISCUSSION
One advantage of the treatments outlined above is that the physi-
ological mechanisms that they address are known and biomarkers
are available to identify children who may respond to these treat-
ments. Preliminary studies suggest that there are a substantial
number of ASD children with these metabolic abnormalities. For
example, mitochondrial abnormalities may be seen in 5–80% of
children with ASD (31,43–45,53,54) and FRαautoantibodies
may be found in 47% (103) to 75% (17) of children with ASD.
Clearly,further studies will be required to clarify the percentage of
these subgroups.
Further large-scale, multicenter DBPC clinical trials are needed
for these promising treatments in order to document the efficacy
and define the subgroups that best respond to these treatments. As
more treatable disorders are documented and as data accumulates
to demonstrate the efficacy of treatments for these disorders, clini-
cal algorithms to approach the work-up for a child with ASD need
to be developed by a consensus of experts. Indeed, developing
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Frye and Rossignol Treatments for biomedical abnormalities associated with autism
guidelines will be the next step for applying many of these sci-
entific findings. Clearly many children with ASD may be able to
benefit from such treatments, which are focused on improving
dysfunctional physiology. Given the fact that no approved medical
treatment exists which addresses the underlying pathophysiology
or core symptoms of ASD,these treatments could make a substan-
tial difference in the lives of children with ASD and their families.
With the high prevalence of ASD, treatments that successfully treat
even only a fraction of children affected with ASD would trans-
late into substantial benefits for millions of individuals with ASD
and their families. In summary, it appears that many of these treat-
ments may provide benefit for a substantial proportion of children
with ASD.
REFERENCES
1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental
Disorders. Washington, DC: American Psychiatric Association (1994).
2. Volkmar FR, McPartland JC. From Kanner to DSM-5: autism as an evolving
diagnostic concept. Annu Rev Clin Psychol (2013) 10:193–212. doi:10.1146/
annurev-clinpsy-032813-153710
3. Developmental Disabilities Monitoring Network Surveillance 2010 Year Prin-
cipal Investigators; Centers for Disease Control and Prevention. Prevalence of
autism spectrum disorder among children aged 8 years – autism and develop-
mental disabilities monitoring network, 11 sites, United States, 2010. MMWR
Surveill Summ (2014) 63:1–21.
4. Autism and Developmental Disabilities Monitoring Network SurveillanceYear
2002 Principal Investigators; Centers for Disease Control and Prevention.
Prevalence of autism spectrum disorders – autism and developmental disabil-
ities monitoring network, 14 sites, United States, 2002. MMWR Surveill Summ
(2007) 56:12–28.
5. Reichow B, Barton EE, Boyd BA, Hume K. Early intensive behavioral inter-
vention (EIBI) for young children with autism spectrum disorders (ASD).
Cochrane Database Syst Rev (2012) 10:CD009260. doi:10.1002/14651858.
CD009260.pub2
6. Correll CU, Manu P, Olshanskiy V, Napolitano B, Kane JM, Malhotra AK.
Cardiometabolic risk of second-generation antipsychotic medications dur-
ing first-time use in children and adolescents. JAMA (2009) 302:1765–73.
doi:10.1001/jama.2009.1549
7. Bobo WV, Cooper WO, Stein CM, Olfson M, Graham D, Daugherty J, et al.
Antipsychotics and the risk of type 2 diabetes mellitus in children and youth.
JAMA Psychiatry (2013) 70:1067–75. doi:10.1001/jamapsychiatry.2013.2053
8. Boon-YasidhiV, Jearnarongr it P, Tulayapichitchock P, Tarugsa J.Adverse effects
of risperidone in children with autism spectrum disorders in a naturalistic
clinical setting at Siriraj Hospital, Thailand. Psychiatry J (2014) 2014:136158.
doi:10.1155/2014/136158
9. Rossignol DA, Fr ye RE. A review of research trends in physiological abnor-
malities in autism spectrum disorders: immune dysregulation, inflammation,
oxidative stress, mitochondrial dysfunction and environmental toxicant expo-
sures. Mol Psychiatry (2012) 17:389–401. doi:10.1038/mp.2011.165
10. Schaefer GB, Mendelsohn NJ. Genetics evaluation for the etiologic diagnosis
of autism spectrum disorders. Genet Med (2008) 10:4–12. doi:10.1097/GIM.
0b013e31815efdd7
11. Neale BM, Kou Y, Liu L, Ma’ayan A, Samocha KE, Sabo A, et al. Patterns and
rates of exonic de novo mutations in autism spectrum disorders. Nature (2012)
485:242–5. doi:10.1038/nature11011
12. Sanders SJ, Murtha MT, Gupta AR, Murdoch JD, Raubeson MJ, Willsey AJ,
et al. De novo mutations revealed by whole-exome sequencing are strongly
associated with autism. Nature (2012) 485:237–41. doi:10.1038/nature10945
13. Hallmayer J, Cleveland S, Torres A, Phillips J, Cohen B, Torigoe T, et al.
Genetic heritability and shared environmental factors among twin pairs
with autism. Arch Gen Psychiatry (2011) 68:1095–102. doi:10.1001/
archgenpsychiatry.2011.76
14. Sandin S, Lichtenstein P, Kuja-Halkola R, Larsson H, Hultman CM, Reichen-
berg A. The familial risk of autism. JAMA (2014) 311:1770–7. doi:10.1001/
jama.2014.4144
15. Rossignol DA, Genuis SJ, Frye RE. Environmental toxicants and autism
spectrum disorders: a systematic review. Transl Psychiatry (2014) 4:e360.
doi:10.1038/tp.2014.4
16. Frye RE, James SJ. Metabolic pathology of autism in relation to redox metab-
olism. Biomark Med (2014) 8:321–30. doi:10.2217/bmm.13.158
17. Frye RE, Sequeira JM, Quadros EV, James SJ, Rossignol DA. Cerebral folate
receptor autoantibodies in autism spectrum disorder. Mol Psychiatry (2013)
18:369–81. doi:10.1038/mp.2011.175
18. Frye RE. Central tetrahydrobiopterin concentration in neurodevelopmental
disorders. Front Neurosci (2010) 4:52. doi:10.3389/fnins.2010.00052
19. Frye RE, Huffman LC, Elliott GR. Tetrahydrobiopterin as a novel therapeutic
intervention for autism. Neurotherapeutics (2010) 7:241–9. doi:10.1016/j.nurt.
2010.05.004
20. Frye RE, Delatorre R, Taylor HB, Slattery J, Melnyk S, Chowdhury N, et al.
Metabolic effects of sapropterin treatment in autism spectrum disorder: a pre-
liminary study. Transl Psychiatry (2013) 3:e237. doi:10.1038/tp.2013.14
21. Evangeliou A, Vlachonikolis I, Mihailidou H, Spilioti M, Skarpalezou A,
Makaronas N, et al. Application of a ketogenic diet in children with autis-
tic behavior: pilot study. J Child Neurol (2003) 18:113–8. doi:10.1177/
08830738030180020501
22. James SJ, Melnyk S, Jernigan S, Cleves MA, Halsted CH, Wong DH, et al.
Metabolic endophenotype and related genotypes are associated with oxidative
stress in children with autism. Am J Med Genet B Neuropsychiatr Genet (2006)
141B:947–56. doi:10.1002/ajmg.b.30366
23. Rose S, Melnyk S, Pavliv O, Bai S, Nick TG, Frye RE, et al. Evidence of oxidative
damage and inflammation associated with low glutathione redox status in the
autism brain. Transl Psychiatry (2012) 2:e134. doi:10.1038/tp.2012.61
24. Tierney E, Bukelis I, Thompson RE, Ahmed K, Aneja A, Kratz L, et al. Abnor-
malities of cholesterol metabolism in autism spectrum disorders. Am J Med
Genet B Neuropsychiatr Genet (2006) 141B:666–8. doi:10.1002/ajmg.b.30368
25. Gargus JJ, Lerner MA. Familial autism with primary carnitine deficiency, sud-
den death, hypotonia and hypochromic anemia. Am J Human Gen (1997)
61:A98.
26. Filipek PA, Juranek J, Nguyen MT, Cummings C, Gargus JJ. Relative carni-
tine deficiency in autism. J Autism Dev Disord (2004) 34:615–23. doi:10.1007/
s10803-004- 5283-1
27. Celestino-Soper PB, Violante S, Crawford EL, Luo R, Lionel AC, Delaby E, et al.
A common X-linked inborn error of carnitine biosynthesis may be a risk fac-
tor for nondysmorphic autism. Proc Natl Acad Sci U S A (2012) 109:7974–81.
doi:10.1073/pnas.1120210109
28. Frye RE, Melnyk S, Macfabe DF. Unique acyl-carnitine profiles are potential
biomarkers for acquired mitochondrial disease in autism spectrum disorder.
Transl Psychiatry (2013) 3:e220. doi:10.1038/tp.2012.143
29. Novarino G, El-Fishawy P, Kayserili H, Meguid NA, Scott EM, Schroth J, et al.
Mutations in BCKD-kinase lead to a potentially treatable form of autism with
epilepsy. Science (2012) 338:394–7. doi:10.1126/science.1224631
30. Rossignol DA, Frye RE. Evidence linking oxidative stress, mitochondrial dys-
function, and inflammation in the brain of individuals with autism. Front
Physiol (2014) 5:150. doi:10.3389/fphys.2014.00150
31. Rossignol DA, Frye RE. Mitochondrial dysfunction in autism spectrum disor-
ders: a systematic review and meta-analysis. Mol Psychiatry (2012) 17:290–314.
doi:10.1038/mp.2010.136
32. Condie J, Goldstein J, Wainwright MS. Acquired microcephaly, regression
of milestones, mitochondrial dysfunction, and episodic rigidity in a 46,XY
male with a de novo MECP2 gene mutation. J Child Neurol (2010) 25:633–6.
doi:10.1177/0883073809342004
33. Gibson JH, Slobedman B, Kaipananickal H, Williamson SL, Minchenko D, El-
Osta A, et al. Downstream targets of methyl CpG binding protein 2 and their
abnormal expression in the frontal cortex of the human Rett syndrome brain.
BMC Neurosci (2010) 11:53. doi:10.1186/1471-2202- 11-53
34. Grosser E, Hirt U, Janc OA, Menzfeld C, Fischer M, Kempkes B, et al. Oxidative
burden and mitochondrial dysfunction in a mouse model of Rett syndrome.
Neurobiol Dis (2012) 48:102–14. doi:10.1016/j.nbd.2012.06.007
35. Napoli E, Ross-Inta C, Wong S, Hung C, Fujisawa Y, Sakaguchi D, et al. Mito-
chondrial dysfunction in Pten haplo-insufficient mice with social deficits and
repetitive behavior: interplay betweenP tenand p53. PLoS One (2012) 7:e42504.
doi:10.1371/journal.pone.0042504
36. Frye RE. Mitochondrial disease in 22q13 duplication syndrome. J Child Neurol
(2012) 27:942–9. doi:10.1177/0883073811429858
Frontiers in Pediatrics | Child and Neurodevelopmental Psychiatry June 2014 | Volume 2 | Article 66 | 6

Frye and Rossignol Treatments for biomedical abnormalities associated with autism
37. Filipek PA, Juranek J, Smith M, Mays LZ, Ramos ER, Bocian M, et al. Mito-
chondrial dysfunction in autistic patients with 15q inverted duplication. Ann
Neurol (2003) 53:801–4. doi:10.1002/ana.10596
38. Frye RE. 15q11.2-13 Duplication, mitochondrial dysfunction, and devel-
opmental disorders. J Child Neurol (2009) 24:1316–20. doi:10.1177/
0883073809333531
39. Su H, Fan W, Coskun PE, Vesa J, Gold JA, Jiang YH, et al. Mitochon-
drial dysfunction in CA1 hippocampal neurons of the UBE3A deficient
mouse model for Angelman syndrome. Neurosci Lett (2011) 487:129–33.
doi:10.1016/j.neulet.2009.06.079
40. Schuelke M, Krude H, Finckh B, Mayatepek E, Janssen A, Schmelz M, et al.
Septo-optic dysplasia associated with a new mitochondrial cytochrome b muta-
tion. Ann Neurol (2002) 51:388–92. doi:10.1002/ana.10151
41. Pallardo FV, Lloret A, Lebel M, D’Ischia M, Cogger VC, Le Couteur DG, et al.
Mitochondrial dysfunction in some oxidative stress-related genetic diseases:
ataxia-Telangiectasia, Down syndrome, Fanconi anaemia and Werner syn-
drome. Biogerontology (2010) 11:401–19. doi:10.1007/s10522-010-9269-4
42. Pagano G, Castello G. Oxidative stress and mitochondrial dysfunction in
Down syndrome. Adv Exp Med Biol (2012) 724:291–9. doi:10.1007/978-1-
4614-0653- 2_22
43. Frye RE. Biomarker of abnormal energy metabolism in children with autism
spectrum disorder. N A J Med Sci (2012) 5:141–7.
44. Giulivi C, Zhang YF, Omanska-Klusek A, Ross-Inta C, Wong S, Hertz-Picciotto
I, et al. Mitochondrial dysfunction in autism. JAMA (2010) 304:2389–96.
doi:10.1001/jama.2010.1706
45. Napoli E, Wong S, Hertz-Picciotto I, Giulivi C. Deficits in bioenergetics and
impaired immune response in granulocytes from children with autism. Pedi-
atrics (2014) 133:e1405–10. doi:10.1542/peds.2013-1545
46. Chauhan A, Gu F, Essa MM, Wegiel J, Kaur K, Brown WT, et al. Brain region-
specific deficit in mitochondrial electron transport chain complexes in children
with autism. J Neurochem (2011) 117:209–20. doi:10.1111/j.1471-4159.2011.
07189.x
47. Anitha A, NakamuraK, Thanseem I, Yamada K, IwayamaY, Toyota T,et al. Brain
region-specific altered expression and association of mitochondria-related
genes in autism. Mol Autism (2012) 3:12. doi:10.1186/2040-2392-3-12
48. Anitha A, Nakamura K, Thanseem I, Matsuzaki H, Miyachi T, Tsujii M, et al.
Downregulation of the expression of mitochondrial electron transport com-
plex genes in autism brains. Brain Pathol (2013) 23:294–302. doi:10.1111/bpa.
12002
49. Tang G, Gutierrez Rios P, Kuo SH, Akman HO, Rosoklija G, Tanji K, et al.
Mitochondrial abnormalities in temporal lobe of autistic brain. Neurobiol Dis
(2013) 54:349–61. doi:10.1016/j.nbd.2013.01.006
50. Kriaucionis S, Paterson A, Curtis J, Guy J, Macleod N, Bird A. Gene expres-
sion analysis exposes mitochondrial abnormalities in a mouse model of Rett
syndrome. Mol Cell Biol (2006) 26:5033–42. doi:10.1128/MCB.01665-05
51. Graf WD, Marin-Garcia J, Gao HG, Pizzo S, Naviaux RK, Markusic D, et al.
Autism associated with the mitochondrial DNA G8363A transfer RNA(Lys)
mutation. J Child Neurol (2000) 15:357–61. doi:10.1177/088307380001500601
52. Frye RE, Naviaux RK. Autistic disorder with complex IV overactivity: a new
mitochondrial syndrome. J Pediatr Neurol (2011) 9:427–34. doi:10.3233/JPN-
2001-0507
53. Rose S, Frye RE, Slattery J,Wynne R, Tippett M,Melnyk S, et al. Oxidative stress
induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell
lines. Transl Psychiatry (2014) 4:e377. doi:10.1038/tp.2014.15
54. Rose S, Frye RE, Slattery J, Wynne R, Tippett M, Pavliv O, et al. Oxidative
stress induces mitochondrial dysfunction in a subset of autism lymphoblas-
toid cell lines in a well-matched case control cohort. PLoSO ne (2014) 9:e85436.
doi:10.1371/journal.pone.0085436
55. Minshew NJ, Goldstein G, Dombrowski SM, Panchalingam K, Pettegrew JW.
A preliminary 31P MRS study of autism: evidence for under synthesis and
increased degradation of brain membranes. Biol Psychiatry (1993) 33:762–73.
doi:10.1016/0006-3223(93)90017- 8
56. Mostafa GA, El-Gamal HA, El-Wakkad ASE, El-Shorbagy OE, Hamza MM.
Polyunsaturated fatty acids, carnitine and lactate as biological markers of brain
energy in autistic children. Int J Child Neuropsychiatry (2005) 2:179–88.
57. Frye RE, Delatorre R, Taylor H, Slattery J, Melnyk S,Chowdhury N, et al. Redox
metabolism abnormalities in autistic children associated with mitochondrial
disease. Transl Psychiatry (2013) 3:e273. doi:10.1038/tp.2013.51
58. Geier DA, Kern JK, Davis G, King PG, Adams JB, Young JL, et al. A prospec-
tive double-blind, randomized clinical trial of levocarnitine to treat autism
spectrum disorders. Med Sci Monit (2011) 17:I15–23. doi:10.12659/MSM.
881792
59. Fahmy SF, El-Hamamsy MH, Zaki OK, Badary OA. L-carnitine supplementa-
tion improves the behavioral symptoms in autistic children. Res Autism Spectr
Disord (2013) 7:159–66. doi:10.1016/j.rasd.2012.07.006
60. Poling JS, Frye RE, Shoffner J, Zimmerman AW. Developmental regression
and mitochondrial dysfunction in a child with autism. J Child Neurol (2006)
21:170–2. doi:10.1177/08830738060210021401
61. Gargus JJ, Imtiaz F. Mitochondrial energy-deficient endophenotype in autism.
Am J Biochem Biotech (2008) 4:198–207. doi:10.3844/ajbbsp.2008.198.207
62. Pastural E, Ritchie S, LuY, Jin W, Kavianpour A, Khine Su-Myat K, et al. Novel
plasma phospholipid biomarkers of autism: mitochondrial dysfunction as a
putative causative mechanism. Prostaglandins Leukot Essent Fatty Acids (2009)
81:253–64. doi:10.1016/j.plefa.2009.06.003
63. Ezugha H, Goldenthal M, Valencia I, Anderson CE, Legido A,Marks H. 5q14.3
Deletion manifesting as mitochondrial disease and autism: case report. J Child
Neurol (2010) 25:1232–5. doi:10.1177/0883073809361165
64. Adams JB, Holloway C. Pilot study of a moderate dose multivitamin/mineral
supplement for children with autistic spectrum disorder. J Altern Complement
Med (2004) 10:1033–9. doi:10.1089/acm.2004.10.1033
65. Adams JB, Audhya T, McDonough-Means S, Rubin RA, Quig D, Geis E, et al.
Effect of a vitamin/mineral supplement on children and adults with autism.
BMC Pediatr (2011) 11:111. doi:10.1186/1471- 2431-11-111
66. Rossignol DA. Novel and emerging treatments for autism spectrum disorders:
a systematic review. Ann Clin Psychiatr y (2009) 21:213–36.
67. Dolske MC, Spollen J, McKay S, Lancashire E, Tolbert L. A preliminary trial of
ascorbic acid as supplemental therapy for autism. Prog Neuropsychopharmacol
Biol Psychiatry (1993) 17:765–74. doi:10.1016/0278-5846(93)90058-Z
68. Nakano K, Noda N, TachikawaE, Urano MAN, Takazawa M, Nakayama T, et al.
A preliminary study of methylcobalamin therapy in autism. J Tokyo Womens
Med Univ (2005) 75:64–9. doi:10.1016/j.chc.2013.03.002
69. James SJ, Melnyk S, Fuchs G, Reid T, Jernigan S, Pavliv O, et al. Efficacy of
methylcobalamin and folinic acid treatment on glutathione redox status in
children with autism. Am J Clin Nutr (2009) 89:425–30. doi:10.3945/ajcn.2008.
26615
70. Frye RE, Melnyk S, Fuchs G,Reid T, Jernigan S, Pavliv O, et al. Effectiveness of
methylcobalamin and folinic acid treatment on adaptive behavior in children
with autistic disorder is related to glutathione redox status. Autism Res Treat
(2013) 2013:609705. doi:10.1155/2013/609705
71. Ghanizadeh A, Derakhshan N. N-acetylcysteine for treatment of autism, a case
report. J Res Med Sci (2012) 17:985–7.
72. Hardan AY, Fung LK, Libove RA, Obukhanych TV, Nair S, Herzenberg LA, et al.
A randomized controlled pilot trial of oral N-acetylcysteine in children with
autism. Biol Psychiatry (2012) 71:956–61. doi:10.1016/j.biopsych.2012.01.014
73. Ghanizadeh A, Moghimi-Sarani E. A randomized double blind placebo con-
trolled clinical trial of N-acetylcysteine added to risperidone for treating autistic
disorders. BMC Psychiatry (2013) 13:196. doi:10.1186/1471- 244X-13-196
74. Gvozdjakova A, KucharskaJ, Ostatnikova D, Babinska K, Nakladal D, Crane FL.
Ubiquinol improves symptoms in children with autism. Oxid Med Cell Longev
(2014) 2014:798957. doi:10.1155/2014/798957
75. Chez MG, Buchanan CP, Aimonovitch MC, Becker M, Schaefer K, Black C,
et al. Double-blind, placebo-controlled study of L-carnosine supplementation
in children with autistic spectrum disorders. J Child Neurol (2002) 17:833–7.
doi:10.1177/08830738020170111501
76. Avula S, Parikh S, Demarest S, Kurz J, GropmanA. Treatment of mitochondrial
disorders. Curr Treat Options Neurol (2014) 16:292. doi:10.1007/s11940-014-
0292-7
77. Boris M, Goldblatt A, Galanko J,James SJ. Association of MTHFR gene variants
with autism. J Am Phys Surg (2004) 9:106–8.
78. Goin-Kochel RP, Porter AE, Peters SU, Shinawi M, Sahoo T, Beaudet AL. The
MTHFR 677C–>T polymorphism and behaviors in children with autism:
exploratory genotype-phenotype correlations. Autism Res (2009) 2:98–108.
doi:10.1002/aur.70
79. Mohammad NS, Jain JM, ChintakindiKP, Singh RP, Naik U,Akella RR. Aberra-
tions in folate metabolic pathway and altered susceptibility toautism. Psychiatr
Genet (2009) 19:171–6. doi:10.1097/YPG.0b013e32832cebd2
www.frontiersin.org June 2014 | Volume 2 | Article 66 | 7

Frye and Rossignol Treatments for biomedical abnormalities associated with autism
80. Pasca SP, Dronca E, Kaucsar T, Craciun EC, Endreffy E, Ferencz BK, et al. One
carbon metabolism disturbances and the C677T MTHFR gene polymorphism
in children with autism spectrum disorders. J Cell Mol Med (2009) 13:4229–38.
doi:10.1111/j.1582-4934.2008.00463.x
81. Liu X, Solehdin F, Cohen IL, Gonzalez MG, Jenkins EC, Lewis ME, et al.
Population- and family-based studies associate the MTHFR gene with idio-
pathic autism in simplex families. J Autism Dev Disord (2011) 41:938–44.
doi:10.1007/s10803-010- 1120-x
82. Frustaci A, Neri M, Cesario A, Adams JB, Domenici E, Dalla Bernardina B, et al.
Oxidative stress-related biomarkers in autism: systematic review and meta-
analyses. Free Radic Biol Med (2012) 52:2128–41. doi:10.1016/j.freeradbiomed.
2012.03.011
83. Guo T,Chen H, Liu B, Ji W, Yang C. Methylenetetrahydrofolate reductase poly-
morphisms C677T and risk of autism in the Chinese Han population. Genet
Test Mol Biomarkers (2012) 16:968–73. doi:10.1089/gtmb.2012.0091
84. Schmidt RJ, Tancredi DJ, Ozonoff S, Hansen RL, Hartiala J, Allayee H, et al.
Maternal periconceptional folic acid intake and risk of autism spectrum disor-
ders and developmental delay in the CHARGE (childhood autism risks from
genetics and environment) case-control study. Am J Clin Nutr (2012) 96:80–9.
doi:10.3945/ajcn.110.004416
85. Pu D, ShenY, Wu J. Association between MTHFR gene polymorphisms and the
risk of autism spectrum disorders: a meta-analysis. Autism Res (2013) 6:384–92.
doi:10.1002/aur.1300
86. Adams M, Lucock M, Stuart J, Fardell S, Baker K, Ng X. Preliminary evi-
dence for involvement of the folate gene polymorphism 19bp deletion-DHFR
in occurrence of autism. Neurosci Lett (2007) 422:24–9. doi:10.1016/j.neulet.
2007.05.025
87. Wollack JB, Makori B,Ahlawat S,Koneru R, Picinich SC, Smith A, et al. Char-
acterization of folate uptake by choroid plexus epithelial cells in a rat primary
culture model. J Neurochem (2008) 104:1494–503. doi:10.1111/j.1471-4159.
2007.05095.x
88. Ramaekers VT, Blau N. Cerebral folate deficiency. Dev Med Child Neurol (2004)
46:843–51. doi:10.1111/j.1469-8749.2004.tb00451.x
89. Moretti P, Sahoo T, Hyland K, Bottiglieri T, Peters S, Del Gaudio D,
et al. Cerebral folate deficiency with developmental delay, autism, and
response to folinic acid. Neurology (2005) 64:1088–90. doi:10.1212/01.WNL.
0000154641.08211.B7
90. Ramaekers VT, Rothenberg SP, Sequeira JM, Opladen T, Blau N, Quadros EV,
et al. Autoantibodies to folate receptors in the cerebral folate deficiency syn-
drome. N Engl J Med (2005) 352:1985–91. doi:10.1056/NEJMoa043160
91. Ramaekers VT, Blau N, Sequeira JM, Nassogne MC, Quadros EV. Folate recep-
tor autoimmunity and cerebral folate deficiency in low-functioning autism
with neurological deficits. Neuropediatrics (2007) 38:276–81. doi:10.1055/s-
2008-1065354
92. Moretti P, Peters SU, Del Gaudio D, Sahoo T, Hyland K, Bottiglieri T, et al.
Brief report: autistic symptoms, developmental regression, mental retardation,
epilepsy, and dyskinesias in CNS folate deficiency. J Autism Dev Disord (2008)
38:1170–7. doi:10.1007/s10803-007-0492-z
93. RamaekersVT, Sequeira JM, Blau N, Quadros EV. A milk-free diet downregu-
lates folate receptor autoimmunity in cerebral folate deficiency syndrome. Dev
Med Child Neurol (2008) 50:346–52. doi:10.1111/j.1469-8749.2008.02053.x
94. Shoffner J, Hyams L, Langley GN, Cossette S, Mylacraine L, Dale J, et al. Fever
plus mitochondrial disease could be risk factors for autistic regression. J Child
Neurol (2010) 25:429–34. doi:10.1177/0883073809342128
95. Ramaekers VT, Hansen SI, Holm J, Opladen T, Senderek J, Hausler M, et al.
Reduced folate transport to the CNS in female Rett patients. Neurology (2003)
61:506–15. doi:10.1212/01.WNL.0000078939.64774.1B
96. Ramaekers VT, Sequeira JM, Artuch R, Blau N, Temudo T, Ormazabal A, et al.
Folate receptor autoantibodies and spinal fluid 5-methyltetrahydrofolate defi-
ciency in Rett syndrome. Neuropediatrics (2007) 38:179–83. doi:10.1055/s-
2007-991148
97. Perez-Duenas B, Ormazabal A, Toma C, Torrico B, Cormand B, Serrano M,
et al. Cerebral folate deficiency syndromes in childhood: clinical, analytical,
and etiologic aspects. Arch Neurol (2011) 68:615–21. doi:10.1001/archneurol.
2011.80
98. Allen RJ, Dimauro S, Coulter DL, Papadimitriou A, Rothenberg SP. Kearns-
Sayre syndrome with reduced plasma and cerebrospinalfluid folate. Ann Neurol
(1983) 13:679–82. doi:10.1002/ana.410130620
99. Pineda M, Ormazabal A, Lopez-Gallardo E, Nascimento A, Solano A, Her-
rero MD, et al. Cerebral folate deficiency and leukoencephalopathy caused by
a mitochondrial DNA deletion. Ann Neurol (2006) 59:394–8. doi:10.1002/ana.
20746
100. Garcia-Cazorla A, Quadros EV, Nascimento A, Garcia-Silva MT, Briones P,
Montoya J, et al. Mitochondrial diseases associated with cerebral folate defi-
ciency. Neurology (2008) 70:1360–2. doi:10.1212/01.wnl.0000309223.98616.e4
101. Hasselmann O, Blau N, Ramaekers VT, Quadros EV, Sequeira JM, Weissert M.
Cerebral folate deficiency and CNS inflammatory markers in Alpers disease.
Mol Genet Metab (2010) 99:58–61. doi:10.1016/j.ymgme.2009.08.005
102. Frye RE, Rossignol DA. Mitochondrial dysfunction can connect the diverse
medical symptoms associated with autism spectrum disorders. Pediatr Res
(2011) 69:41R–7R. doi:10.1203/PDR.0b013e318212f16b
103. Ramaekers VT, Quadros EV,Sequeira JM. Role of folate receptor autoantibodies
in infantile autism. Mol Psychiatry (2013) 18:270–1. doi:10.1038/mp.2012.22
104. Suren P,Roth C, Bresnahan M, Haugen M, Hornig M, Hirtz D, et al. Association
between maternal use of folic acid supplements and risk of autism spectrum
disorders in children. JAMA (2013) 309:570–7. doi:10.1001/jama.2012.155925
105. James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW, et al. Metabolic
biomarkers of increased oxidative stress and impaired methylation capacity in
children with autism. Am J Clin Nutr (2004) 80:1611–7.
106. Rose S, Melnyk S, Trusty TA, Pavliv O, Seidel L, Li J, et al. Intracellu-
lar and extracellular redox status and free radical generation in primary
immune cells from children with autism. AutismRes Treat (2012) 2012:986519.
doi:10.1155/2012/986519
107. Sajdel-Sulkowska EM, Xu M, McGinnis W, Koibuchi N. Brain region-specific
changes in oxidative stress and neurotrophin levels in autism spectrum disor-
ders (ASD). Cerebellum (2011) 10:43–8. doi:10.1007/s12311-010-0223-4
108. Adams JB, Audhya T, McDonough-Means S. Nutritional and metabolic status
of children with autism vs. neuro-typical children, and the association with
autism severity. Nutr Metab (2011) 8:34. doi:10.1186/1743-7075-8- 34
109. Nareuse H, Hayash TI, Takesada M, Nakane A, Yamazaki K, Noguchi T, et al.
Therapeutic effect of tetrahydrobiopterin in infantile autism. Proc Jpn Acad
(1987) 63:231–3. doi:10.2183/pjab.63.231
110. Danfors T, von Knorring AL, Hartvig P, Langstrom B, Moulder R, Stromberg
B, et al. Tetrahydrobiopterin in the treatment of children with autistic disor-
der: a double-blind placebo-controlled crossoverstudy. J Clin Psychopharmacol
(2005) 25:485–9. doi:10.1097/01.jcp.0000177667.35016.e9
111. Klaiman C, Huffman L, Masaki L, Elliott GR. Tetrahydrobiopterin as a treat-
ment for autism spectrum disorders: a double-blind, placebo-controlled trial.
J Child Adolesc Psychopharmacol (2013) 23:320–8. doi:10.1089/cap.2012.0127
112. Kim HW, Cho SC, Kim JW, Cho IH, Kim SA, Park M, et al. Family-based
association study between NOS-I and -IIA polymorphisms and autism spec-
trum disorders in Korean trios. Am J Med Genet B Neuropsychiatr Genet (2009)
150B:300–6. doi:10.1002/ajmg.b.30798
113. Delorme R, Betancur C, Scheid I, Anckarsater H, Chaste P, Jamain S, et al.
Mutation screening of NOS1AP gene in a large sample of psychiatric patients
and controls. BMC Med Genet (2010) 11:108. doi:10.1186/1471- 2350-11-108
114. Lakshmi Priya MD, Geetha A.A biochemical study on the level of proteins and
their percentage of nitration in the hair and nail of autistic children. Clin Chim
Acta (2011) 412:1036–42. doi:10.1016/j.cca.2011.02.021
115. Melnyk S, Fuchs GJ, Schulz E, Lopez M, Kahler SG, Fussell JJ, et al. Meta-
bolic imbalance associated with methylation dysregulation and oxidative
damage in children with autism. J Autism Dev Disord (2012) 42:367–77.
doi:10.1007/s10803-011- 1260-7
Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 09 May 2014; accepted: 09 June 2014; published online: 27 June 2014.
Citation: Frye RE and Rossignol DA (2014) Treatments for biomedical abnor-
malities associated with autism spectrum disorder. Front. Pediatr. 2:66. doi:
10.3389/fped.2014.00066
This article was submitted to Child and Neurodevelopmental Psychiatry, a section of
the journal Frontiers in Pediatrics.
Copyright © 2014 Frye and Rossignol. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribution
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are credited and that the original publication in this journal is cited, in accordance with
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Frontiers in Pediatrics | Child and Neurodevelopmental Psychiatry June 2014 | Volume 2 | Article 66 | 8