Mitochondrial Dysfunction in Autism Spectrum

Disorder: Unique Abnormalities and Targeted

Treatments
Richard E. Frye, MD, PhD*,†

Several lines of evidence implicate mitochondria in the pathophysiology of autism spectrum

disorder (ASD). In this review, we outline some of the evidence supporting this notion, as well
as discuss novel abnormalities in mitochondrial function that appear to be related to ASD,
and treatments that both target mitochondria and have evidence of usefulness in the treat-
ment of ASD in clinical trials. A suspicion of the mitochondrion’s involvement in ASD can be
traced back to 1985 when lactic acidosis was noted in a subset of children with ASD. A large
population-based study in 2007 confirmed this notion and found that a subset of children with
ASD (»4%) could be diagnosed with a definite mitochondrial disease. Further studies sug-
gested that children with ASD and mitochondrial disease may have certain characteristics
such as fatigability, gastrointestinal disorders, unusual types of neurodevelopmental regres-
sion, seizures/epilepsy, and motor delay. Further research examining biomarkers of mito-
chondrial dysfunction and electron transport chain activity suggest that abnormalities of
mitochondrial function could affect a much higher number of children with ASD, perhaps up
to 80%. Recent research has identified a type of dysfunction of mitochondria in which the
activity of the electron transport chain is significantly increased. This novel type of mitochon-
drial dysfunction may be associated with environmental exposures and neurodevelopmental
regression. Several treatments that target mitochondria appear to have evidence for use in
children with ASD, including cofactors such as L-Carnitine and the ketogenic diet. Although
the understanding of the involvement of mitochondria in ASD is evolving, the mitochondrion
is clearly a novel molecular target which can be helpful in understanding the etiology of ASD
and treatments that may improve function of children with ASD.
Semin Pediatr Neurol 35:100829 © 2020 The Author(s). Published by Elsevier Inc. This is an
open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction complicated by the fact that the disorder itself is defined by

purely behavioral criteria which continue to evolve as the

R ecent estimates suggest that autism spectrum disorder

(ASD) may affect about 2% of children in the United
States with the prevalence continuing to increase.1 Under-
Diagnostic Statistical Manual of Mental Disorders (DSM) is
revised.2 The diagnosis continues to be defined based on
behavior, which is, in part, due to a lack of understanding of
standing ASD from a biologic and neurologic point of view is the underlying biological processes that drive ASD.3 As a
consequence of this behavioral approach and the lack of
From the *Division of Neurology, Section on Neurodevelopmental Disorders,
understanding the biology, the development of quantitative
Barrow Neurological Institute at Phoenix Children’s Hospital, Phoenix, AZ. objective biomarkers to assist in the diagnosis and manage-
y
Department of Child Health, University of Arizona College of Medicine  ment of ASD is still evolving.4
Phoenix, Phoenix, AZ. One of the reasons that the underlying biology of ASD is
Conflict of Interest: The authors have no conflicts of interest to declare. difficult to understand is the fact that it involves the interac-
Funding: There was no funding for this article.
Address reprint requests to Richard E. Frye, MD, PhD, Phoenix Children’s
tion between many biological systems rather than abnormali-
Hospital, 1919 E Thomas St, Phoenix, AZ 85016 ties in one well-defined system or biological mechanism.3,5
E-mail: [email protected] Because of this, ASD does not follow the rules of well-known

https://doi.org/10.1016/j.spen.2020.100829 1
1071-9091/11/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
2 R.E. Frye

diseases. The most troubling case is for genetics of ASD. that NDR and gastrointestinal complaints had higher preva-
While the idea that ASD is genetic is driven by the high heri- lence in children with ASD with mitochondrial disease as
tability rate of ASD, empirical studies continue to demon- compared to the general ASD population and also found
strate that it is the great minority of children with ASD have a that seizures/epilepsy and motor delay were also associated
clear genetic etiology.6 One explanation is that the genetics with mitochondrial disease in ASD.11 Other reviews point
are just more complicated than previously assumed, so we out that individuals with ASD share other clinical abnormal-
just do not fully understand it yet. However, studies which ities commonly seen in mitochondrial disease such as devel-
show that 69% of sibling with ASD and genetic disorders opmental delays, ataxia, muscle weakness, peripheral
have different genetic mutations7 and the fact that most neuropathy, endocrinology abnormalities, and failure to
genetic mutation identified are de novo8,9 suggests that thrive (Fig. 1).16
inherited genetic mutations are not driving the high heritabil-
ity rate. Other studies have noted that the etiology of ASD is
highly likely driven by genetic-environmental interactions
with each contributing about equally.10 ASD Is Associated With Unique
The insight into the possible genetic-environmental interac- Disorders of Mitochondrial
tions as a cause of ASD provides a clue to the possible biologi-
cal underpinnings of ASD. Interestingly, one of the major
Metabolism
physiological abnormalities found in children with ASD is A meta-analysis demonstrated that the overall prevalence of
atypical function of mitochondria.11,12 This is remarkably classical mitochondrial disease is 5% when considering the 3
interesting since mitochondria are very vulnerable to environ- large published studies on the prevalence of mitochondrial dis-
mental factors. This makes the mitochondrion a potentially ease in ASD.11 However, the same meta-analysis demonstrated
key mediator of environmental-genetic interactions. Here we that the prevalence of abnormal biomarkers of mitochondrial
described some of the abnormalities identified in mitochon- function were much higher than the expected 5% of those
drial function and how they may be related to environmental with mitochondrial disease. Indeed, elevations in lactate, pyru-
triggers. vate, lactate-to-pyruvate ratio, alanine, creatine kinase, ammo-
nia, aspartate aminotransferase, and alanine aminotransferase
was found in 31%, 14%, 28%, 8%, 47%, 35%, 45%, and 7%,
respectively, and low carnitine was found in 80%, of children
Brief History of Mitochondrial with ASD. Since many of these studies were based on the use
of normative values to define abnormalities, the meta-analysis
Disorders in ASD also examined studies which used contemporaneous typically
The notion that ASD is associated with a disorder of mitochon- developing control groups. Such controlled studies verified
drial function can be traced back to before mitochondrial dis- that those with ASD as a group demonstrate abnormalities in
ease itself was well defined. Specifically, in 1985 Mary lactic acid, pyruvic acid, carnitine, creatine kinase, and transa-
Coleman at Georgetown University found that 5% of children minases as compared to typically developing contemporaneous
with ASD demonstrated lactic acidosis and proposed that they controls. To investigate the possibility that perhaps these bio-
may suffer from a defect in carbohydrate metabolism linked to markers were spuriously elevated, one study looked at the
the pyruvate dehydrogenase complex.13 Perhaps the strongest repeatability of these abnormalities. Abnormalities could be
evidence supporting the notion that mitochondrial disease is confirmed on a repeat blood draw about 50% of the time.
associated with ASD was a population-based study of Portugal However, even eliminating those individuals without consis-
and the Azores which screened over 300,000 school-age chil- tent abnormalities, the prevalence of elevations in lactic acid
dren for ASD and investigated co-morbid medical condi- was 16%.18 This study also looked at 2 other biomarkers of
tions.14 Lactic acidosis was found in 20% of the cases of ASD mitochondrial dysfunction including the alanine-to-lysine ratio
that underwent medical investigation with 46% of the individ- and acyl-carnitine elevations, which were found to be repeat-
uals that underwent further investigation for lactic acidosis edly abnormal in 16% and 24% of the children with ASD
being diagnosed with a definite mitochondrial respiratory examined.18
chain disorder, resulting in a prevalence of 4.2% of the overall These biomarker studies suggest the prevalence of abnor-
ASD sample having a diagnosis of a definite mitochondrial mal mitochondrial function is much higher than the preva-
respiratory chain disorder. lence of classic mitochondrial disease, perhaps around 30%-
Additional case studies and series continued to document 50%. However, other studies which directly measured func-
the association with mitochondrial disease and ASD with tion of the electron transport chain (ETC) found much high
one particularly large case series pointing out that children rates of abnormalities. For example, in 2 studies of immune
with ASD and mitochondrial disease had high rates of fati- cells, abnormal ETC complex activity was found in 80% of
gability, gastrointestinal disorders, and unusual types of lymphocytes19 and granulocytes.20 Using the buccal swab
neurodevelopmental regression (NDR) including multiple technique to examine ETC complex I and IV and citrate syn-
regressions or regression later than commonly associated thase activity, 2 studies suggested that 65%21 and 62%22 of
with ASD.15 A meta-analysis of the case reports and series individuals with ASD demonstrated mitochondrial enzyme
of children with ASD and mitochondrial disease confirmed activity outside the normal range. Thus, the prevalence of
Mitochondrial Dysfunction in Autism 3

Figure 1 Important characteistics of mitochondrial dysfunction associated with autism spectrum disorder (ASD). The
upper left circle provides a summary of the biochemical mitochondrial abnormalities identified in children with ASD.
Other boxes summarize biomarkers, genetic abnormalities, clinical symptoms, and treatments studied for mitochon-
drial dysfunction associated with ASD [MD = Classic mitochondrial disease; Electron transport chain complex I (C1),
Complex II (C2), Complex III (C3), Complex IV (C4) and Complex V (C5); CS, citrate synthase; FAOD, fatty acid oxi-
dation defect]. From ref.17 with permission.

abnormalities in mitochondrial function in individuals with gyrus,25 buccal swabs enzymology,22 and lymphoblastoid
ASD appears to be much higher than the prevalence of those cell lines (LCLs) using high-resolution respirometry.26
diagnosed with classical mitochondrial disease, although the We have developed a model of atypical mitochondrial func-
exact prevalence of mitochondrial dysfunction is not exactly tion in LCLs derived from children with ASD. Using the Sea-
clear. In addition to the abnormalities mentioned above, horse XF96 respirometer, we have shown that about one-third
other ETC abnormalities and biomarkers of abnormal mito- of LCLs derived from children with ASD repeatedly show ele-
chondrial function have been reported, as discussed in our vated respiratory rates, approximately 200% of controls, for
recent review.12 Those, more common abnormalities are respiratory parameters associated with adenosine triphosphate
summarized in Figure 1. Below we will discuss an interesting production.27-29 We have also demonstrated the consequences
unique type of mitochondrial dysfunction that has been of elevated respiratory rates; specifically, this subset of ASD
uncovered in emerging research. LCLs are more sensitive to acute exposure to reactive oxygen
species (ROS) such that respiratory rates drop precipitously
with acute increases in ROS. We hypothesized these changes
Mitochondrial Overactivity represent an adaptive response to previous environmental
Classic mitochondrial disease is defined, in part, by severe exposures, a phenomenon known as mitoplasticity.30 To sup-
deficits in ETC complex activity. However, as more cases of port this hypothesis, we demonstrated that elevated respiratory
children with ASD and symptoms of mitochondrial abnor- rates can be induced in LCLs with prolonged exposure (96
malities were investigated, a slightly different pattern of ETC hours) to mild ROS, a microenvironment that simulates the
abnormalities has emerged. In the first case report of his effect of environmental toxicants on mitochondria.30 Addition-
kind, a girl with Leigh syndrome and decreased ETC activity ally, we have demonstrated that this subset of LCLs respond
due to a heteroplasmic (blood 82%; muscle 86%) G8363A differently to environmental agents associated with ASD,
tRNALys mutation was reported. Interestingly, her brother including trichloroacetaldehyde hydrate31 and ethylmercury,32
who had ASD but not Leigh syndrome harbored the same and enteric short chain fatty acids propionate33 and butyrate,34
mutations with a lower heteroplasmy (blood 60%; muscle as compared to ASD LCLs which do not demonstrate these
61%) but instead of demonstrating lower than normal ETC high respiratory rates at baseline.
complex activity he demonstrated markedly higher than nor-
mal activity in ETC Complex I.23 Following the notion that
elevations in ETC complex activity may be abnormal and
related to ASD, 5 patents with ASD with elevations in ETC
Mitochondrial Dysfunction May Be
Complex IV activity (»200% normal) in muscle was Associated With Neurodevelopmental
reported in a case series.24 Subsequently this association Regression
between significant elevations in ETC Complex IV activity Individuals with mitochondrial disease are susceptible to
and ASD has been reported in fresh frozen superior temporal NDR, especially with illness.36 Thus, NDR may be the
4 R.E. Frye

Figure 2 Seahorse profiles demonstrating the elevated respiratory rates (oxygen consumption rate [OCR]) for individu-
als with autism spectrum disorder (ASD) and neurodevelopmental regression (NDR); (A) Maximal OCR is significantly
elevated for those with ASD and NDR (ASD/NDR) as compared to those without ASD (non-ASD) and those with ASD
but without NDR (ASD/No-NDR); (B) Maximal OCR is also elevated in a twin with ASD and NDR as compared to the
twin without NDR but with ASD. Figure from Singh et al.35

hallmark of mitochondrial dysfunction in ASD. For example, children with ASD, such as L-Carnitine and the Ketogenic
Shoffner et al37 showed that the majority of children with diet (KD). Some of these treatments are well known to child
ASD and mitochondrial disease developed ASD symptoms neurologists because of their use in other neurological
after a sudden rapid NDR associated with a fever and a meta- disorders.
analysis found that NDR was more common in children with
ASD and mitochondrial disease than in the general ASD pou-
lation.11 This is consistent with our in vitro LCL model of Cofactor Supplementation
mitochondrial dysfunction in ASD where a subset of LCLs One of the mainstays for treatment of mitochondrial disease
with mitochondrial dysfunction is found to be very sensitive is the use of specific cofactors. One of the advantages of using
to increases in physiological stress.27-29 Interestingly, in a these types of treatments is that they are overall well tolerated
recent study, we have demonstrated that respiratory rates are without significant adverse effects and no serious or long-
elevated in peripheral blood mononuclear cells from individ- term adverse effects. Many cofactors are B-vitamins which,
uals with ASD and NDR as compared to those with ASD being water soluble, are eliminated from the body by the kid-
without NDR (Fig. 2).35 Thus, NDR may be a hallmark of neys and cannot build up in the body. Table 1 outlines some
abnormal mitochondrial physiology in ASD. of the commonly used co-factors for mitochondrial disease.
As we have reviewed previously,12 several studies have
examined cofactors that are commonly used for the treat-
ment of mitochondrial disease in the treatment of general
Potential Treatments for ASD without considering whether the individuals with ASD
Mitochondrial Dysfunction in have mitochondrial disease or dysfunction. Perhaps the best
studied cofactor is L-Carnitine. Two medium sized (n = 30,
ASD 30) double-blind placebo-controlled studies using L-Carni-
Several treatments for mitochondrial disorders overlap with tine treatment (50 mg/kg/d for 3 months and 100 mg/kg/d
treatments which have been studied in clinical trials in for 6 months) found that scores on the Childhood Autism
Mitochondrial Dysfunction in Autism 5

Table 1 Cofactors for Treatment of Mitochondrial Disease

Vitamin Dose Adverse Effects Function

Electron transport chain support

Co-enzyme Q10 5-30 mg/kg/d, 1-2x/d Appetite loss, nausea, Energy carrier between
(Reduced): Ubiquinol diarrhea at high doses complex I and III and
complex II and III
Co-enzyme Q10 10-30 mg/kg/d; 1-2x/d
(Oxidized): Ubiquinone
Electron carrier support
Niacin (B3) 50-100 mg given daily Flushing Reaction Nicotinamide adenine
dinucleotide (NAD)
precursor
Riboflavin (B2) 100-400 mg given daily Nausea at High Doses Flavin adenine dinucleotide
(FAD) precursor
Energy Storage
Creatine monohydrate 100 mg/kg/d; 1-3x/d Increased urination. High-energy phosphate
buffer. Precursor to
phosphocreatine
Fatty acid oxidation support
L-carnitine or 30-120 mg/kg/d, 1-3x/d Stool loose/fishy smell Carrier of long chain
acetyl-L-carnitine fatty acids
Biotin (B7) 5-10 mg/d given daily None Cofactor for carboxylase
enzymes
Mitochondria
l enzyme .co-factors
Thiamine (B1) 50-100 mg given daily None Cofactor for citric
acid cycle enzymes
Pantothenic Acid (B5) 5-1200 mg/d, 1-3x/d Diarrhea at high doses Precursor to coenzyme A
Pyridoxine (B6) 200 mg given daily Cofactor for over 100 enzymes
Biotin (B7) As above None Cofactor for carboxylase enzymes
Alpha-lipoic acid 50-200 mg given daily Headache, paresthesia, Cofactor for citric acid
rash, muscle cramps cycle enzymes
Antioxidants
Co-Enzyme Q10 As above As above Targets ETC oxidative stress
L-carnitine As above As above Scavenger of organic acids
Vitamin E 200-400 IU given daily Bleeding at high doses Protects cell membranes
Vitamin C 100-500 mg given daily Diarrhea at high doses Protects iron and copper
Redox metabolism support
Methylcobalamin (B12) 5-2000 mcg every 1-3 days Hyperactivity, sleep Supports methylation and
disruption folate cycles and glutathione
production
Reduced folate (B9) Folinic acid 400-800 None Supports methylation
ug daily and folate cycles
N-acetyl-L-cysteine (NAC) 10-70 mg/kg/d. 1-3x/d Diarrhea at high doses Precursor to glutathione
Zinc 10-40 mg daily Suppresses iron and Supports superoxide dismutase
copper absorption
Central folate support
Folinic Acid / Leucovorin 0.5-4mg/kg/day. 1-3x/day Hyperactivity Supports adequate folate
Calcium (B9) levels in the brain
From ref. 54 with permission.

Rating Scale (CARS) improved with L-Carnitine as compared improvements in behavior and hyperactivity before correc-
to placebo38,39 with 1 study finding that greater symptomatic tion for multiple comparisons and improvements in language
improvement was correlated with a greater increase in blood correlated with post-treatment blood carnitine levels.40 Chil-
carnitine levels.39 A small (n = 10) 8-week open-label trial of dren with ASD and genetic mutations in the carnitine path-
L-Carnitine used particularly high doses (up to 400 mg/kg/d way also appear to response to L-Carnitine. For example, a
in 3 divided doses, maximum of 6000 mg/d) with only 1 boy with ASD, multiple NDRs and a mutation in the TMLHE
child transiently stopping treatment and 3 children remain- gene causing a deficiency in carnitine biosynthesis41 and a
ing on a relatively lower dose (200 mg/kg/d) because of odor- girl with a mutation in SCL22A5, the organic cation trans-
ous loose stools. Several parental rated measures showed port type 2, resulting in a systemic primary carnitine
6 R.E. Frye

deficiency,42 both responded clinically to L-Carnitine supple- relationship between ETC Complex I and IV was positively
mentation. Thus, L-Carnitine is a promising treatment for modulated by folate supplementation, while the relationship
children with ASD. For child neurologists, it is always impor- between Complex I and citrate synthase was positively mod-
tant to remember that children with ASD could have an ulated by both cobalamin and folate supplementation. In the
underlying defect in carnitine metabolism, especially when only study of its kind, Legido et al56 conducted a small
using a medication such as valproic acid where co-treatment (n = 11) open-label cross-over study for children with ASD
with L-Carnitine is recommended.43 and mitochondrial ETC abnormalities. Children were treated
Coenzyme Q10 has been studied in several open-label for 3 months with a mitochondrial cocktail containing L-Car-
studies. Coenzyme Q10 in the form of ubiquinol 50 mg nitine, Coenzyme Q10, and alpha-lipoic acid. The cocktail
twice a day for 3 months resulted in parental reports of improved mitochondrial function as well as ASD behavior.
improvements in communication, interactions, sleep, and Discontinuation of the cocktail resulted in worsening of
food rejection for those who attained a Coenzyme Q10 levels behavior.
above 2.5 umol/L in a small (n = 24) open-label study.44
Ubiquinol 30-60 mg was found to improve markers of oxida-
tive stress and CARS scores in a rather large (n = 90) open- The Ketogenic Diet
label study.45 The KD has been studied for ASD as well as epilepsy and
Other supplements that support the mitochondria that mitochondrial disease. The KD has an important role in treat-
have been studied in small studies include reduced nicotin- ing drug-resistant epilepsy, a category of epilepsy that is
amide adenine dinucleotide,46 thiamine tetrahydrofurfuryl more common in children with ASD,57 and it has been noted
disulfide,47 and biotin.48 A combination nutritional supple- as an safe and potentially effective treatment for epilepsy in
ment that included L-Carnitine, Coenzyme Q10 as well other children with ASD.43 In a parent survey of epilepsy treat-
supplements to support the mitochondria, redox and folate ments for ASD, the KD was rated highest for providing seiz-
pathways has demonstrated improvement in ASD symptoms ures control, above the rating of antiepileptic drugs, and was
as reported by the Parental Global Impressions-Revised in 2 reported to have positive effects on core and related ASD
controlled studies.49,50 Beyond this review of mitochondrial symptom unlike antiepileptic drugs which tended to have
specific cofactors is the important role of treatment of redox negative effects on core and related ASD symptoms.58
abnormalities, most preferable treated with methylcobala- The KD has been studied for ASD in 2 uncontrolled and 1
min51 and/or N-Acetyl-Cysteine,52 and folate metabolism53 controlled open-label clinical trials and the modified Atkin’s
in children with ASD. diet (MAD) has been studied in 1 controlled open-label trial.
Only 2 studies have directly examined the effect of mito- The outcomes for these studies are outlined in Table 2. Most
chondrial supplements on mitochondrial function in ASD. In of the studies applied the diet for 3 months60-62 while 1
a large (n = 127) natural history study, our group examined applied the diet for 6 months.59 Of the studies that reported
the effect of various supplements taken by children with ASD providing dietary education,60,61 50 of 82 families (61%)
on citrate synthase as well as ETC Complex I and IV using decided to start the diet after dietary education. Of the 95
the buccal swab technique in children with ASD with and patients that started with the KD or MAD diet, 60 (63%)
without identified mitochondrial disease.55 For those with were able to tolerate the diet. Outcomes could be categorized
mitochondrial disease folate and antioxidants increased ETC in the 3 KD studies, 1 using the CARS59 and 2 using the
Complex I and citrate synthase activity, respectively, while ADOS.60,61 Most commonly (40%), there was a moderate
fatty acid supplementation increased ETC Complex I activity improvement with 58% of the children that tolerated the diet
for the overall group. We also looked at the correlation demonstrating a moderate or better improvement. There was
between mitochondrial enzymes and found that the only 1 study where worsening in outcomes was seen. The

Table 2 Outcomes From Studies on the Ketogenic (Evangeliou, Lee, Mu) and Modified Atkin’s (El-Rashidy) Diet for Treating
Autism Spectrum Disorder
Evangeliou59 *Lee60 Mu61 El-Rashidy62 Combined

Diet education 46 36
Diet started 30 27 (59%) 23 (64%) 15 50/82 (61%)
Diet tolerated 23 (77%) 23/30 (77%)
Complied with diet 18 (60%) 15 (56%) 17 (74%) 10 (67%) 60/95 (63%)
Improvement
Significant 2 (11%) 2 (13%) 5 (29%) 9/50 (18%)
Moderate 8 (44%) 6 (40%) 6 (35%) 20/50 (40%)
Mild 8 (44%) 2 (13%) 6 (35%) 16/50 (32%)
Worsened 0 (0%) 4 (27%) 0 (0%) 4/50 (8%)
*In Lee % Change in ADOS-2 score was used to rate improvement, with <0% = worsening, 0%-20% = mild improvement, 20%-40% = moderate
improvement, and >40% = significant improvement.
Mitochondrial Dysfunction in Autism 7

only study of the MAD found that it was superior for decreas- mitochondrial DNA deletions.87 Effectiveness of the KD was
ing CARS scores as compared to a control group which did shown in mouse,88,89 Drosophila,90 and cell line91,92 models
not have any special dietary change. However, a comparison of human mitochondrial disorders.
group on the gluten-free casein-free diet showed equivalent The KD is a promising therapy that clearly needs more
improvement to the MAD.62 Only 1 study reported any study for its use in individuals with ASD. Given the fact that
adverse effect which only occurred during the initiation of both clinical and animal models support the potentially ben-
the diet. This is consistent with our survey of the use of the efit of the KD in ASD, it should be considered carefully, espe-
KD and MAD for seizures in ASD where few adverse effects cially in children with ASD with comorbid epilepsy and/or
were reported.58 Two studies used biomarkers to better mitochondrial dysfunction. The major limitation appears to
understand the physiology of the KD. One study found that be the ability to tolerate the diet but in those that can tolerate
improvement in ADOS scores, particularly social affect, was the KD, symptoms of sociability appear to improve in more
related to lower baseline high density lipoprotein and albu- than half. Overall, dietary therapies are difficult to imple-
min.60 In a unique open-label controlled study, metabolic ment. For other dietary treatments, outcomes are related to
patterns were assessed in blood using plasma gas chroma- the ability of the family to implement the diet adequately.93
tography-mass spectrometry, inductively coupled plasma- Thus, it is important to counsel the families on a commit-
mass spectrometry and proton nuclear magnetic resonance ment to the KD before implementing as if it is not possible
spectroscopy61; an increase in chromium, creatine and a for the family to implement the diet adequately, the expected
decrease in ornithine, acetoacetate, cesium and N-acetyl- outcome may not be obtainable and other therapeutic
serotonin across the treatment period correlated with better options may be a better choice.
outcomes.
Both genetic and environmentally induced mouse models
of ASD show positive response to the KD. The KD improved
sociability and repetitive behaviors in the EL mouse model of Consideration of Treatment for Mitochondrial
ASD, primarily in female mice.63 The BTBR mouse demon- Dysfunction in ASD
strated improved sociability and repetitive behaviors when About 5% of children with ASD are affected by classically
treated with the KD with these effects dissociable from anti- defined mitochondrial disease,11 while a much larger per-
seizure effects of the KD.64 The KD was shown to improve centage of children with ASD have some amount of mito-
sociability and repetitive behaviors in two environmentally chondrial dysfunction.11 This raises 2 questions. First, how
induced models of ASD, male mice with maternal immune is mitochondrial dysfunction defined and, second, to what
activation (MIA)-induced ASD65 and mice with prenatal val- extent should mitochondrial dysfunction be treated.12
proic acid exposure induced ASD.66 The mechanism of the Previous publications have outlined guidance for the
KD in ASD may be multifold with evidence for modulation workup for mitochondrial disease in ASD.11,94 The use of
of cerebral glucose metabolism,67 bypassing a ETC Complex the buccal swab technique to measure enzymology has
1 defect,69 upregulating mitochondrial genes,69 improving helped the work-up21,22,55,95-97 but this technique does have
mitochondrial morphology,70 increasing mitochondrial turn- its limitation, especially in regards to the need for more vali-
over,71 normalizing the microbiome,72 modulating the corti- dation studies. Another important nuance in the way mito-
cal excitatory-to-inhibitory balance,73 and improving white chondrial disease is defined. While most studies use the
matter brain development.74 Modified Walker Criteria which requires very low ETC activ-
The KD has been proposed as a treatment for mitochondrial ity or a known genetic mutation, other criteria outlined by
disease, although controlled studies are lacking. Existing clini- Morava et al98 concentrate on symptoms and biomarker
cal studies are limited to case reports and series. The KD has abnormalities.16 Clearly more work needs to be done in
been best studied in children with pyruvate dehydrogenase defining more exact criteria for defining both mitochondrial
complex deficiency: one case series found a positive effect on disease and dysfunction in children with ASD. However,
ataxia, sleep, speech and language and social functioning75 until agreed upon criteria are in place, the diagnosis of mito-
while another case series found that initiation of the KD early chondrial disease and mitochondrial dysfunction must
in life or with greater carbohydrate restriction improved lon- involve clinical judgment along with repeatable biomarkers
gevity and mental development.76 Other case reports have of abnormal mitochondrial function which includes abnor-
found a benefit of the KD in children with a m.5559A>G mal overactivity of the ETC.
mutation,77 BCS1lrelated mitochondrial disease78 and ETC It is always important to look for a genetic cause for mito-
Complex I defect associated with Ohtahara79 or Landau-Kleff- chondrial disease and dysfunction. However, only approxi-
ner80 syndromes, mitochondrial polymerase gamma gene mately 25% of children with ASD and classically defined
mutations in a women81 and child,82 a women with mitochon- mitochondrial disease have a clear genetic mutation to explain
drial encephalopathy with lactic acidosis and stroke-like epi- their mitochondrial disease.11 This means that 75% of children
sodes,83 and 2 patients with combined oxidative with ASD and mitochondrial disease do not have an identified
phosphorylation deficiency-10.84 However, the KD did not mutation to explain their classically defined mitochondrial dis-
seem to help patients with SUCLA285 or BOLA386 mutations ease. Furthermore, genetic disorders which are associated with
and the MAD was detrimental in patients with mitochondrial ASD but not classically associated with mitochondrial disease,
myopathy with progressive external ophthalmoplegia with including Down syndrome, Rett syndrome, Phelan-McDermid
8 R.E. Frye

Syndrome,96 15q11-13 duplication,99 and others100 sometime Sequencing in Children With Autism Spectrum Disorder. Jama
involved mitochondrial dysfunction and respond to mitochon- 314:895-903, 2015
7. Yuen RK, Thiruvahindrapuram B, Merico D, et al: Whole-genome
drial interventions.54 Thus, in our previous publication, we
sequencing of quartet families with autism spectrum disorder. Nat
recommended a trial of mitochondrial-targeted treatments Med 21:185-191, 2015
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for the genetic causes of the mitochondrial abnormality is Novo Mutations in Developmental and Epileptic Encephalopathies.
ongoing.54 For children with ASD, it is important to realize Am J Hum Genet 101:664-685, 2017
that finding a genetic explanation is unlikely and withholding 9. Sanders SJ, Murtha MT, Gupta AR, et al: De novo mutations revealed
by whole-exome sequencing are strongly associated with autism.
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is evident just because a gene abnormality cannot be found to 10. Hallmayer J, Cleveland S, Torres A, et al: Genetic heritability and
explain the mitochondrial dysfunction may deprive a child shared environmental factors among twin pairs with autism. Arch Gen
with a severe neurodevelopmental disability a treatment that Psychiatry 68:1095-1102, 2011
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which are safe and have a low incidence of adverse effects. For 17:290-314, 2012
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