SHORT REPORTOpen Access
Evidence of reactive oxygen species-mediated
damage to mitochondrial DNA in children with
Eleonora Napoli1, Sarah Wong1and Cecilia Giulivi1,2*
Background: The mitochondrial genome (mtDNA) is particularly susceptible to damage mediated by reactive
oxygen species (ROS). Although elevated ROS production and elevated biomarkers of oxidative stress have been
found in tissues from children with autism spectrum disorders, evidence for damage to mtDNA is lacking.
Findings: mtDNA deletions were evaluated in peripheral blood monocytic cells (PBMC) isolated from 2–5 year old
children with full autism (AU; n = 67), and typically developing children (TD; n = 46) and their parents enrolled in
the CHildhood Autism Risk from Genes and Environment study (CHARGE) at University of California Davis. Sequence
variants were evaluated in mtDNA segments from AU and TD children (n = 10; each) and their mothers
representing 31.2% coverage of the entire human mitochondrial genome. Increased mtDNA damage in AU children
was evidenced by (i) higher frequency of mtDNA deletions (2-fold), (ii) higher number of GC→AT transitions (2.4-fold),
being GC preferred sites for oxidative damage, and (iii) higher frequency of G,C,T→A transitions (1.6-fold) suggesting a
higher incidence of polymerase gamma incorporating mainly A at bypassed apurinic/apyrimidinic sites, probably
originated from oxidative stress. The last two outcomes were identical to their mothers suggesting the inheritance of a
template consistent with increased oxidative damage, whereas the frequency of mtDNA deletions in AU children was
similar to that of their fathers.
Conclusions: These results suggest that a combination of genetic and epigenetic factors, taking place during perinatal
periods, results in a mtDNA template in children with autism similar to that expected for older individuals.
Keywords: Autism, Mitochondria, Mitochondrial DNA, Oxidative damage, Bioenergetics
The human mitochondrial genome is a 16.5-kb circular,
double stranded DNA that encodes thirteen polypeptides
of the mitochondrial respiratory chain, twenty-two transfer
RNAs and two ribosomal RNAs required for protein syn-
thesis. The mitochondrial DNA (mtDNA) consists of a
heavy (H) and a light (L) strand, in accord with its G and T
base composition. mtDNA is particularly susceptible to
mutations because of the high level of reactive oxygen spe-
cies (ROS) (including superoxide anion, hydrogen perox-
ide, hydroxyl radical and peroxynitrite) generation in this
organelle [1,2], and lack of introns or histones, coupled
with a low level of DNA repair .
Damage to mtDNA, elicited by ROS continually gener-
ated in mitochondria, may result from defective replica-
tion and/or repair of mtDNA of primary (genetic)  or
secondary (for example, oxidative damage to single base
pairs inflicted by ROS) [5,6] origins. ROS-mediated dam-
age is characterized by a variety of lesions to DNA in
general [7,8] and to mtDNA in particular , including
single- and double-stranded DNA breaks, abasic sites,
and oxidized bases (reviewed in [10-13]). Considering
that mtDNA replication occurs as a coupled leading and
lagging strand replication pathway, the H strand DNA
exists for extended periods in the single-stranded form
in this asymmetric mode of mtDNA replication, favoring
damage to the exposed bases not protected by the com-
plementary DNA strand . In this regard, mtDNA
* Correspondence: firstname.lastname@example.org
1Department of Molecular Biosciences, University of California, One Shields
Ave, 1120 Haring Hall, Davis, CA 95616, USA
2Medical Investigations of Neurodevelopmental Disorders (M.I.N.D.) Institute,
University of California, Davis, CA 95616, USA
© 2013 Napoli et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Napoli et al. Molecular Autism 2013, 4:2
damage is asymmetric resulting in the majority of single
and multiple mtDNA deletions occurring between the
OHand OL. In addition, cytosine deamination to
uracil would be expected to be an asymmetric process
because it occurs > 100-fold more rapidly in single-
stranded DNA, as it would be when mtDNA is temporar-
ily exposed during ongoing replication and transcription
. Increased oxidative stress has been claimed as one of
the features present in autism [15-19], although its precise
role in the etiology of autism is still undefined. In support
of this hypothesis, higher rates of mitochondrial hydrogen
peroxide production (accompanied by lower activities in
the pyruvate dehydrogenase complex, Complex I alone, or
in combination with other Complexes) from lymphocytic
mitochondria  and increased markers of oxidative
stress (for example, increased oxidized glutathione)
[19-22] had been reported in samples from individuals
There are limited studies characterizing mtDNA from
children with full autism, and none evaluating the puta-
tive ROS-mediated damage to mtDNA in the index
child. We evaluated the occurrence of ROS-mediated
damage to mtDNA in children who met the criteria for
presenting full-syndrome autism and age-matched and
genetically unrelated typically developing children (TD)
without a clinical diagnosis of autism or developmental
delays, recruited by the CHildhood Autism Risk from
Genes and Environment (CHARGE) Study at the Uni-
versity of California (UC) Davis . To minimize inva-
sive procedures, we evaluated the quality of mtDNA
(deletions and sequence variants) in peripheral blood
monocytic cells (PBMC) from children with typical aut-
ism to ascertain if (i) the diagnosis of autism segregated
with a pattern consistent of increased oxidative damage
to mtDNA and (ii) if the putative mtDNA damage was
de novo (when compared to their parents living in the
same household) or inherited (if outcomes followed pa-
ternal and/or maternal patterns), when compared to par-
ents of TD or AU children. To our knowledge, there are
no systematic and comprehensive studies aimed at in-
vestigating the role of ROS-mediated damage to mtDNA
in children with autism.
Clinical selection of individuals and diagnosis
The CHARGE study is an epidemiologic case control in-
vestigation launched by the UC Davis Center for Chil-
dren’s Environmental Health that has been enrolling
families through the Medical Investigations of Neurode-
velopmental Disorders (M.I.N.D.) Institute since 2003.
The focus of the CHARGE study is on modifiable factors
in autism etiology and markers of biological dysregula-
tion that may provide mechanistic clues.
Families had been recruited from three groups: chil-
dren diagnosed with autism, children diagnosed with de-
velopmental delay but not an autism spectrum disorder
(ASD) or autism, and children from the general popula-
tion. These children are 24 to 60 months old, and reside
with a biological parent in a well-defined catchment area
of over 22 counties in northern California and parts of
Los Angeles County. Cases are recruited through the
California Department of Developmental Services system,
M.I.N.D. Institute clinics, clinician referrals, and self-
referrals. General population controls are sampled from
birth files with frequency-matching to the projected distribu-
tion of sex, age, and geographic area among cases of autism.
Environmental, lifestyle, reproductive, maternal med-
ical, and detailed demographic information is collected
through an extensive telephone interview with the pri-
mary caregiver. Participants classify themselves into race
and ethnicity categories identical to those used in the
US Census. Diagnoses are confirmed through clinical
examinations using the Autism Diagnostic Inventory-
Revised (ADI-R) , and the Autism Diagnostic Obser-
vation Schedule (ADOS) . The ADI-R provides a
standardized, semi-structured interview and a diagnostic
algorithm for the Diagnostic and Statistical Manual of
Mental Disorders-Fourth Edition (Text Revision) (DSM-
IV-TR) and the International Statistical Classification of
Diseases and Related Health Problems 10th Revision
(ICD-10) definitions of autism . The ADOS is a
semi-structured, standardized assessment in which the
researcher observes the social interaction, communication,
play, and imaginative use of materials for children sus-
pected of having autism and ASD. The final CHARGE
study diagnosis is defined as meeting criteria on the com-
munication, social, and repetitive behavior domains of the
ADI-R and scoring at or above the cutoff for autistic dis-
order on the ADOS (module 1, 2 or 3). The Social Com-
munication Questionnaire is used to screen for ASD
among those recruited as developmentally delayed, or as
general population controls. Children who score above
the screening cutoff are fully assessed using the ADI-R
and ADOS. The developmental and adaptive functions of
all children are evaluated with the Mullen Scales of Early
Learning  and the Vineland Adaptive Behavior Scales
, and children scoring 71 or above on both these scales
who do not have an autism spectrum disorder qualify as TD.
The child’s medical history is taken and a developmental-
behavioral pediatrician conducts an examination for
physical or neurological abnormalities. Further details on
the CHARGE study protocols are published elsewhere .
For the studies on mitochondrial DNA, we sampled 67
children with full-syndrome autism and 46 classified as
TD; all of these children were genetically unrelated, both
within and between diagnostic groups. We also attempted
to achieve comparable age, sex, and race/ethnicity in the
Napoli et al. Molecular Autism 2013, 4:2
Page 2 of 8
TD and AU groups. Demographic and clinical data from
these groups are presented in Additional file 1: Tables S1
and S2. A computer-generated random sampling of 10
TD and 10 AU children was used for mtDNA sequencing.
The study protocol follows the ethical guidelines of the
Declaration of Helsinki  and was approved by the In-
stitutional Review Board of the UC Davis School of Medi-
cine. All subjects enrolled in the study had written
informed consent provided by their parents and self-
assented to participate if developmentally able.
DNA purification, PCR amplification and sequencing
Blood samples (approximately 8 ml) were collected in a
BD vacutainer CPT tube (BD Biosciences, catalog number
362753; San Jose, CA, USA). CPT tubes were kept on ice
until ready to use. The CPT tubes were centrifuged at
1650 g for 15 minutes at 22°C. The plasma layer was
removed and stored at −80°C. The lymphocytes were
transferred to another tube, washed according to the
manufacturer’s specifications and resuspended in 600
μl of buffer A (in mM, 220 sucrose, 50 KCl, 10
KH2PO4, 5 MgCl2, 1 EGTA and 10 HEPES, pH 7.5).
Genomic DNA was extracted from isolated lympho-
cytes using Puregene kit (catalog number 158388) from
Qiagen (Valencia, CA, USA). Each DNA concentration
was determined by triplicate measurements of the ab-
sorbance at 260 nm using a Tecan Quantiplate plate
reader (Grödig, Austria). DNA was diluted to 0.63 ng/μl
and served as stock DNA template for both qPCR and se-
quencing. To determine sequence variants in mtDNA, nt
2995 to 5570, 7960 to 9867 and 14732 to 15419 (5,172 bp
out of 16,569 bp, for a 31.2% coverage of the entire human
mitochondrial genome [Genbank: NC_012920] including
the following protein-encoding genes: ATP6/8, ND1 and
ND2, and parts of CYTB, COX2 and COX3) were PCR-
amplified from 10 samples randomly selected from
children with AU and 10 TD children in 1 to 3-kb
overlapping fragments, and the PCR products were
completely sequenced. The use of large PCR products
excluded the possibility that nuclear pseudogenes
could complicate this analysis . The sequences
obtained were first compared to those recorded in exten-
sive mitochondrial databanks [31,32]. To distinguish som-
atic mutations from rare germline variants, we determined
the variations present in samples from randomly selected
age-matched TD children. It is important to notice that
the sequencing was performed to look for evidence of
ROS-mediated damage and not to identify or claim any
pathogenic mutation associated with autism. Sequencing
primers were obtained from reference . Four sets of se-
quencing primers were used:
13f - E, 7960–7979
13r - E, 8641–8621
14f - E, 8563–8581
14r - E, 9231–9212
15f - E, 9181–9198
15r - E, 9867–9848
24f - H, 14732–14752
24r - H, 15419–15400
For sequencing, PCR amplification was done using the
Qiagen Taq DNA polymerase (catalog number Q201203;
Valencia, CA, USA) consisting of 2.5 μl of 10x Buffer (Qia-
gen catalog number Q201203; Valencia, CA, USA), 1.7 μl
of 25 mM MgCl2 (Qiagen catalog number Q201203;
Valencia, CA, USA), 0.5 μl of 10 mM dNTP mix (Invi-
trogen catalog number 10297–018; Grand Island, NY,
USA) , 0.5 μl of each of the two primers, 10 μl of sam-
ple with 6 ng total of DNA, 0.2 μl of Taq enzyme and
8.1 μl of MilliQ water for the total reaction volume of
24 μl. The following cycling conditions were used: 94°C
for 3 minutes, 10 cycles of 94°C for 15 s, 65°C for 30 s
(with 1°C decrease at each cycle), and 72°C for 40 s, fol-
lowing with 30 cycles of 94°C for 15 s, 55°C for 30 s, and
72°C for 40 s. Final elongation was done for 5 minutes at
72°C with a 15°C forever-hold step. PCR products were
run on 1.3% agarose gel, excised and purified using Qia-
quick Gel Extraction Kit (Qiagen, catalog number 28704,
Valencia, CA, USA) according to manufacturer’s instruc-
tions, and submitted for sequencing to the UC Sequencing
Core Facility on the UC Davis campus. Final readouts
Analyzer software, and alignments of sequences were
done through Invitrogen’s Vector NTI software.
Evaluation of mtDNA deletions
The majority of mtDNA deletions involve the major arc of
the mitochondrial genome between the origin of the heavy
strand replication (nucleotides 110 to 441) and the origin
of the light strand replication (nucleotides 5721 to 5798)
. In the majority of patients with single and multiple
mtDNA deletions, the ND4 (mitochondrial gene encoding
for the ND4 subunit of Complex I) and/or CYTB (mito-
chondrial gene encoding for cytochrome b) genes present
deletions whereas the ND1 (mitochondrial gene encoding
subunit ND1 in Complex I) is rarely deleted; therefore, we
evaluated the ratios of ND4/ND1 and CYTB/ND1 gene
copy number with dual-labeled probes to detect mtDNA
microdeletions  in mtDNA from PBMC from TD chil-
dren (n = 46) and children with autism (n = 67). Changes
in mtDNA copy number were evaluated by dual-labeled
probes using quantitative (q) PCR. The gene copy number
of cytochrome b, ND1 and ND4 were normalized by a
single-copy nuclear gene (pyruvate kinase) as explained in
detail before . mtDNA deletions were considered if
the Z-scores were < −2SD, where the means and SD were
obtained withTD values for each age and sex group.
Napoli et al. Molecular Autism 2013, 4:2
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Experiments were run in triplicate and repeated three times
in independent experiments. The percentage of individuals
with mtDNA deletions was calculated using the Z-scores.
The Z-scores were calculated as (xi-mean)/SD for each
group, in which the mean and SD were obtained from TD
children (for comparison of children), from TD mothers (for
comparison of mothers) and TD fathers (for comparison of
fathers). The cutoff for considering an outcome as either
high or low was > 2SD or <−2SD respectively. The chi-
square test was utilized to evaluate significance in the distri-
bution of frequencies between groups.
Deletions in mtDNA of TD and AU children were evalu-
ated by qPCR using the mitochondrial gene ratios of
CYTB/ND1 and ND4/ND1. The percentage of TD chil-
dren (n = 46) with deletions (deletion = Z-score < −2SD)
encoding for CYTB and ND4 was 8.7% and 6.5%, respect-
ively (Table 1). In samples from AU children (n = 67),
these outcomes were significantly higher by 2.4- and
2.3-fold, respectively (Table 1). In both groups, TD and
AU children, the frequency of deletions at genes
located closer to OH(CYTB) relative to those located
closer to the OL(ND4) was 1.3- and 1.4-fold, respectively,
with no difference between the groups. The higher inci-
dence of individuals with CYTB deletions vs. ND4 ones
was also observed in all parents, regardless of sex or diag-
nosis of child (Table 1, last row). This strand asymmetry
of mtDNA deletions was suggestive of ROS-mediated
damage to the single-stranded state of the H strand during
the asynchronous mtDNA replication.
The extent of the mtDNA deletions at CYTB in AU
children was 14 ± 1%, 1.5-fold greater than the corre-
sponding TD values (9 ± 1%, P < 0.005) and was similar
to that of older individuals, regardless of sex or the diag-
nosis of the child (16 ± 2%, P < 0.01).
To discern between de novo (acquired) vs. inherited
deletions (from either maternal mtDNA or parental
gDNA-inherited mechanisms that favor accumulation of
deletions in the mtDNA), deletions in both segments of
the mtDNA were evaluated in the parents of TD and
AU children. The percentage of fathers of AU children
with mtDNA deletions at the segments encoding for
CYTB and ND4 was higher than for those of TD chil-
dren (1.4-fold and 1.9-fold respectively), following the
pattern of AU children when compared to TD children
for both genes. In contrast, mothers of AU children pre-
sented a low incidence of deletions at both segments when
compared to mothers of TD children (50%) (Table 1) sug-
gesting lower mtDNA replication.
The ratio of parents of TD children to TD children
with mtDNA deletions was 1.2-fold in the segment en-
coding for CYTB (Table 1). This increase suggested an
age-dependent accumulation of mtDNA deletions. Age-
dependent mtDNA deletions are thought to accumulate
at lower levels in human mitotic tissues than post mi-
totic ones because they could be lost by negative selec-
tion in rapidly dividing cells, by a higher tendency of the
cells to undergo apoptosis, mitochondria being able to
replicate independently of the cell cycle (avoiding being
diluted out by cell division) and/or by preferential repli-
cation of deleted mtDNA .
To determine sequence variants that were ascribed to
ROS-mediated damage in mtDNA from AU and TD
children, 31.2% of the entire human mtDNA was
sequenced encompassing the following protein-encoding
genes: ATP6/8, ND1 and ND2, and parts of CYTB,
COX2 and COX3. A relatively higher frequency of tran-
sition mutations has been reported in aerobic organisms
(from 60% to 68%) [36,37] consistent with the transitions
observed in this study for both children regardless of
diagnosis (Table 2). Transitions (90% of base variants)
affecting GC and AT pairs in TD children were 44.4%
and 55.6% (Table 2). This pattern of base substitutions
(90.0% transitions with 55.6% of them converting an AT
to a GC pair) resembles that of the human mtDNA se-
quence polymorphisms reported in the MITOMAP data-
base, which shows an enormous preponderance of
transitions (88%) over transversions, with a nucleotide bias
approximating that of the genome (that is, the genome is
56% A + T, and 58% of the transitions A-T→G-C). In AU
Table 1 Percentage of TD and AU children and their parents with mtDNA deletions
Mitochondrial gene Individuals with mtDNA deletions
CYTB deletions, %8.720.9 < 0.00110.8 6.1 0.0210.8 14.6*0.030
ND4 deletions, %6.514.9 < 0.0018.14.1 0.025.4 10.2**0.009
CYTB/ND4 fold change, % 18.104.22.168.52.01.4
Individuals with a Z-score < −2SD were considered as having deletions. The mean and SD utilized for the Z-scores were obtained from TD values from each of the
groups (child, mother, father). The significance shown in the Table is for each AU vs. TD comparison. *P = 0.017 and **P = 0.030 vs. mothers of AU children.
mtDNA mitochondrial DNA; TD typically developing; AU full autism.
Napoli et al. Molecular Autism 2013, 4:2
Page 4 of 8
children, transitions affecting GC and AT pairs were
64.7% and 35.3% indicating that CG pairs were replaced
2.5 times more often than AT pairs, which was an oppos-
ite trend to TD children (Table 2). The percentage of
(G,C,T)→A transitions to the total number of transitions
in AU children was 1.6 times that in TD children (Table 2).
To ascertain if the sequence variants obtained with
mtDNA from AU children were de novo or the result of
maternal inheritance, the same segments were analyzed
in their mothers. Consistent with the maternal inherit-
ance of mtDNA, all variants found in children were also
observed in their mothers, regardless of diagnosis.
In this study, and following the model of strand asym-
metry replication of mtDNA, evidence for mtDNA dam-
age has been observed in all individuals, regardless of
age and/or diagnosis. The higher frequency of deletions
at the segment encoding for CYTB compared to ND4
(Table 1, last row) attests for an asymmetric damage of
the mtDNA when the H-strand is exposed in the single-
stranded state during replication.
If the hypothesis that higher oxidative damage to
mtDNA was occurring in a more exacerbated form in
PBMC from AU than from TD children, then the fol-
lowing results would be expected: (i) higher percentage
of GC transitions over AT ones because when the
well-known marker of oxidative stress, 8-oxo-7,8-dihy-
dro-20-deoxyguanosine (8oxodG)[1,38,39], is effectively
repaired and/or removed from the template , indu-
cing predominantly G→T transitions by mispairing
with A during DNA replication [41-43]; (ii) higher fre-
quency of mtDNA deletions, especially at the segment
encoding for CYTB compared to ND4; and (iii) higher
frequency of G,C,T→A transitions because when Polγ
bypasses apurinic/apyrimidinic (AP) sites, formed as a
result of spontaneous deamination or oxidative lesions,
incorporating mainly adenine at these positions .
In favor of the model of higher oxidative stress in aut-
ism, a higher frequency of GC transitions over AT ones
(2 vs. 0.8) (Table 2), higher frequency of deletions (by
2-fold) (Table 1), and higher number of G,C,T→A
transitions (1.3-fold those in TD children) (Table 2)
were observed in AU children. The lack of G→T tran-
sitions could be explained, considering that these types
of transitions are rarely observed in both in vivo and
in vitro somatic sets of mtDNA point mutations
[45,46]. The extent of the deletions in AU children was
1.6-fold of that in TD children, and was similar to that
of all parents, suggesting more damage to their mtDNA.
However, given that the percentages observed in AU chil-
dren were similar to those observed in older individuals in
general, and that these percentages are usually much
lower than are seen in patients with mitochondrial disor-
ders, in which deletion ≥ 60% is required to demonstrate a
mitochondrial defect [47,48], it is suggested that the ex-
tent of the deletions do not seem to be pathogenic per se.
The sequence variants observed in all children were
explained by the maternal inheritance of mtDNA, re-
gardless of diagnosis (Table 2). However, given that these
outcomes were different between TD child-mother vs.
AU child-mother, it suggests that mothers of AU chil-
dren share a DNA template consistent with a model of
higher oxidative stress-mediated damage. It is interesting
to note that mothers of AU children, although having
more damaged mtDNA, also presented the lowest inci-
dence of deletions when compared to age-matched
groups. This might indicate a compensatory mechanism,
by which a lower replicative rate might prevent add-
itional accumulation of deletions.
Considering that a higher percentage of AU children
exhibited mtDNA deletions compared to TD children,
and that this pattern was also present in fathers of AU
children, it is likely that a genetic predisposition to accu-
mulate mtDNA deletions was transmitted paternally. It
should be noted that paternal mtDNA deletions are not
inherited but, accumulation of deletions (or the predispos-
ition to accumulate deletions) resulting from increased
ROS production, defective antioxidant/repair system, or
defective clearance of damaged mitochondria, could be
transmitted from either parent. Alternatively, exposure to
epigenetic factors different from those to which families of
TD children are exposed, or identical to those of TD fam-
ilies but perceived with a different genetic susceptibility
, may have resulted in the increased mtDNA deletions
observed in AU children and their fathers.
This study has several limitations that need to be con-
sidered for a proper interpretation of the results and con-
sequences for the field of autism. First, the number of
individuals on which mtDNA sequencing was performed
was relatively small, although significant differences were
observed between TD child-mother and AU child-mother.
Second, the comparisons made in this study reached a sig-
nificance at the α = 0.001 level minimizing type I errors,
Table 2 Summary of outcomes evaluated in mtDNA from
TD and AU children
Sequence variants, number1731
Transitions, %100 90
Transversions, %0 10
Type of transitions
GC transitions, %64.7*44.4*0.005
AT transitions, %35.3* 55.6*
G,C,T → A, % of all other transitions 89*56*0.001
*See text for discussion of these results.
Napoli et al. Molecular Autism 2013, 4:2
Page 5 of 8
whereas type II errors were reduced by increasing the
number of observations per group, limited only by the
availability of samples. Third, children in this study had
not been previously diagnosed with a genetic syndrome,
nor had any indications of genetic syndromes been identi-
fied by M.I.N.D. developmental pediatricians. Neverthe-
less, defects (other than deletions) in genes other than
those tested could have been present in these samples as
recently reported in other studies of ASD . Fourth, al-
though the outcomes reported here for PBMC may repre-
sent those present in other cells more relevant to autism
(for example, neurons), it is important to consider that the
neuroimmune response is characterized by cross-talk be-
tween peripheral immune cells and the central nervous
system, and that disruption of this process during early life
may condition inflammatory responses as well as behav-
ioral changes that persist during adulthood [51-54]. Fi-
nally, inferences about a cause-effect association between
ROS-mediated mtDNA damage and typical autism are
intricate because this is cross-sectional, and not a lon-
gitudinal study. In addition, several factors influence
expression of mtDNA damage, for example, nuclear
genetic backgrounds , mtDNA heteroplasmy in tis-
sues , energy thresholds for a given tissue/organ
, and epigenetic factors , in both affected and
general healthy populations. Multiple mtDNA deletions
for example, may accumulate with age in post-mitotic tis-
sues of apparently healthy individuals [12,48,59-61], or in
patients with other disorders not necessarily linked to aut-
ism, such as, inherited mutations in nuclear genes [62,63],
neurodegenerative disorders [62,64], cancer , and dia-
betes . Nevertheless, our study showed that mtDNA
in children with autism is more damaged than in age-,
sex-, and race-matched TD children, and is more similar
to that of older individuals, with a mtDNA template (ma-
ternally inherited) consistent with ROS-mediated damage
(based on sequence variants), and presenting a predispos-
ition to accumulate damage (deletions) similar to that of
Several scenarios may result in increased mtDNA damage,
among them: (i) higher oxidative stress not accompanied
by antioxidant defenses and/or repair/maintenance of
mtDNA; (ii) lower ability to clear mitochondria with
damaged mtDNA; (iii) replicative advantage of deleted
mtDNA over wild-type mtDNA, or (iv) a combination of
any of the aforementioned possibilities.
The fact that mothers of AU children have a template
with an array consistent with increased mtDNA damage,
and that fathers of AU children accumulate more dele-
tions than fathers of TD children, seems to point to a
combination of these factors in addition to the age of
onset for these events. In this regard, changes in mtDNA
copy number and/or deletions seem to be age-dependent,
for the clinical onset of mtDNA depletion is typically in
infancy or early childhood whereas multiple deletions sel-
dom present before adolescence. A genetic background, in
combination with other genetic (originated from parental
genomic DNA or maternal mtDNA) and/or epigenetic
factors, for example, dysfunctional electron transport
chain , low levels of antioxidant enzymes bound to
mtDNA , environmental factors  and dietary defi-
ciencies , acting additively or synergistically may lead
to more damaged mtDNA during vulnerable windows
such as the perinatal periods. This altered process may be
causative per se, or may set up the stage for a heightened
susceptibility for further insults, which may ultimately
alter an appropriate development of energy status and in-
crease autism risk. Structural instability of mtDNA, con-
sisting either of large-scale rearrangements, tissue-specific
depletion or deletions, is a major cause of mitochondrial
dysfunction and disease in humans , and possibly in
children with autism.
Additional file 1: Additional material for this study has been
provided under Additional documentation. This file includes all
demographics of subjects utilized in this study, sequence variants data
and associated references.
ADI-R: Autism Diagnostic Inventory-Revised; ADOS: Autism Diagnostic
Observation Schedule; AP: Apurinic/apyrimidinic sites; 8oxodG: 8-oxo-7,8-
dihydro-2′-deoxyguanosine; ASD: Autism spectrum disorders; AU: Full autism;
DSM-IV-TR: Diagnostic and Statistical Manual of Mental Disorders-Fourth
Edition (Text Revision); H: Heavy; ICD-10: International Statistical Classification
of Diseases and Related Health Problems 10th Revision; L: Light;
mtDNA: Mitochondrial DNA; mtSSB: Mitochondrial single stranded-DNA
binding proteins; PBMC: Peripheral blood monocytic cells; PCR: Polymerase
chain reaction; Polγ: Mitochondrial DNA polymerase gamma; ROS: Reactive
oxygen species; TD: Typically developing.
The authors of this publication declare that they have no conflicting financial
interest in relation to the work described.
EN has been involved in statistical analyses of the data and drafted the
manuscript; SW carried out all experiments and helped to draft the
manuscript; CG conceived the study, contributed to the analysis and
interpretation of data, and revised it critically for important intellectual
content. All authors have given final approval of the version to be published.
We wish to express our gratitude to the children and their families who
participated in this study; Ms Alicja Omanska-Klusek for technical assistance;
Dr Flora Tassone for providing all samples utilized in this study; Dr Irva Hertz-
Picciotto for providing diagnostic, sociodemographic, and comorbidity data,
and Dr Isaac Pessah for providing input at early stages of the study. This
study was performed under the funding from the 2008 M.I.N.D. Institute Pilot
Research Grant, University of California Davis, #2444 Autism Speaks, and
NIEHS R01-ES011269, NIEHS R01-ES015359 and NIEHS R01-ES020392. The
abovementioned funding agencies were not responsible for the design and
conduct of the study, collection, management, analysis or interpretation of
the data, or the preparation, review, or approval of this manuscript.
Napoli et al. Molecular Autism 2013, 4:2
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Received: 23 October 2012 Accepted: 4 January 2013
Published: 25 January 2013
1.Giulivi C, Cadenas E: The role of mitochondrial glutathione in DNA base
oxidation. Biochim Biophys Acta 1998, 1366:265–274.
2.Giulivi C, Boveris A, Cadenas E: The steady-state concentrations of oxygen
radicals in mitochondria. In Reactive Oxygen Species in Biological Systems:
An Interdisciplinary Approach. Volume 77. Edited by Gilbert DL. Colton:
Kluwer Academic/Plenum Publishers; 1997:77–102.
3.Lin MT, Beal MF: Mitochondrial dysfunction and oxidative stress in
neurodegenerative diseases. Nature 2006, 443:787–795.
4.Zeviani M, Spinazzola A, Carelli V: Nuclear genes in mitochondrial
disorders. Curr Opin Genet Dev 2003, 13:262–270.
5.Albers DS, Beal MF: Mitochondrial dysfunction and oxidative stress in
aging and neurodegenerative disease. J Neural Transm Suppl 2000,
6.Lezza AMS, Mecocci P, Cormio A, Beal MF, Cherubini A, Cantatore P, Senin
U, Gadaleta MN: Mitochondrial DNA 4977 bp deletion and OH8dG levels
correlate in the brain of aged subjects but not Alzheimer's disease
patients. FASEB J 1999, 13:1083–1088.
7.Reid TM, Feig DI, Loeb LA: Mutagenesis by metal-induced oxygen
radicals. Environ Health Perspect 1994, 102(Suppl 3):57–61.
8.Kreutzer DA, Essigmann JM: Oxidized, deaminated cytosines are a source
of C –> T transitions in vivo. Proc Natl Acad Sci USA 1998, 95:3578–3582.
9. Khrapko K, Coller HA, Andre PC, Li XC, Hanekamp JS, Thilly WG:
Mitochondrial mutational spectra in human cells and tissues. Proc Natl
Acad Sci USA 1997, 94:13798–13803.
10.Croteau DL, Bohr VA: Repair of oxidative damage to nuclear and
mitochondrial DNA in mammalian cells. J Biol Chem 1997, 272:25409–25412.
11.Beckman KB, Ames BN: Oxidative decay of DNA. J Biol Chem 1997,
12.Chabi B, Mousson de Camaret B, Chevrollier A, Boisgard S, Stepien G:
Random mtDNA deletions and functional consequence in aged human
skeletal muscle. Biochem Biophys Res Commun 2005, 332:542–549.
13.Srivastava S, Moraes CT: Double-strand breaks of mouse muscle mtDNA
promote large deletions similar to multiple mtDNA deletions in humans.
Hum Mol Genet 2005, 14:893–902.
14.Lindahl T: Instability and decay of the primary structure of DNA. Nature
15. Giulivi C, Zhang YF, Omanska-Klusek A, Ross-Inta C, Wong S, Hertz-Picciotto
I, Tassone F, Pessah IN: Mitochondrial dysfunction in autism. JAMA 2010,
16.Chauhan A, Audhya T, Chauhan V: Brain region-specific glutathione redox
imbalance in autism. Neurochem Res 2012, 37:1681–1689.
17. Ming X, Cheh MA, Yochum CL, Halladay AK, Wagner GC: Evidence of
oxidative stress in autism derived from animal models. Am J Biochem
Biotechnol 2008, 4:218–225.
18.Napoli E, Ross-Inta C, Wong S, Hung C, Fujisawa Y, Sakaguchi D, Angelastro
J, Omanska-Klusek A, Schoenfeld R, Giulivi C: Mitochondrial dysfunction in
Pten haplo-insufficient mice with social deficits and repetitive behavior:
interplay between Pten and p53. PLoS One 2012, 7:e42504.
19. James SJ, Rose S, Melnyk S, Jernigan S, Blossom S, Pavliv O, Gaylor DW:
Cellular and mitochondrial glutathione redox imbalance in
lymphoblastoid cells derived from children with autism. FASEB J 2009,
20.James SJ, Melnyk S, Fuchs G, Reid T, Jernigan S, Pavliv O, Hubanks A, Gaylor
DW: Efficacy of methylcobalamin and folinic acid treatment on
glutathione redox status in children with autism. Am J Clin Nutr 2009,
21.Geier DA, Kern JK, Garver CR, Adams JB, Audhya T, Nataf R, Geier MR:
Biomarkers of environmental toxicity and susceptibility in autism.
J Neurol Sci 2009, 280:101–108.
22.Ming X, Johnson WG, Stenroos ES, Mars A, Lambert GH, Buyske S: Genetic
variant of glutathione peroxidase 1 in autism. Brain Dev 2010, 32:105–109.
23.Hertz-Picciotto I, Croen LA, Hansen R, Jones CR, van de Water J, Pessah IN:
The CHARGE study: an epidemiologic investigation of genetic and
environmental factors contributing to autism. Environ Health Perspect
24.Lord C, Rutter M, Le Couteur A: Autism Diagnostic Interview-Revised: a
revised version of a diagnostic interview for caregivers of individuals
with possible pervasive developmental disorders. J Autism Dev Disord
Lord C, Risi S, Lambrecht L, Cook EH Jr, Leventhal BL, DiLavore PC, Pickles A,
Rutter M: The autism diagnostic observation schedule-generic: a
standard measure of social and communication deficits associated with
the spectrum of autism. J Autism Dev Disord 2000, 30:205–223.
Association AP: Diagnostic and statistical manual of mental disorders DSM-IV-TR
(text revision). 4th edition. Washington, DC: American Psychiatric Press; 2000.
Mullen EM: Mullen Scales of Early Learning. Circle Pines, MN: American
Guidance Service; 1995.
Sparrow SS, Cicchetti DV: Diagnostic uses of the vineland adaptive
behavior scales. J Pediatr Psychol 1985, 10:215–225.
World Medical Association: Declaration of Helsinki: ethical principles for
medical research involving human subjects. J Postgrad Med [serial online]
2002, 48(3):206–208. http://www.jpgmonline.com/text.asp?2002/48/3/206/
103 [cited 2013 Jan 22].
Parfait B, Rustin P, Munnich A, Rotig A: Co-amplification of nuclear
pseudogenes and assessment of heteroplasmy of mitochondrial DNA
mutations. Biochem Biophys Res Commun 1998, 247:57–59.
Brandon MC, Lott MT, Nguyen KC, Spolim S, Navathe SB, Baldi P, Wallace
DC: MITOMAP: a human mitochondrial genome database–2004 update.
Nucleic Acids Res 2005, 33:D611–D613.
Ingman M, Gyllensten U: mtDB: Human Mitochondrial Genome Database,
a resource for population genetics and medical sciences. Nucleic Acids
Res 2006, 34:D749–D751.
Taylor RW, Taylor GA, Durham SE, Turnbull DM: The determination of
complete human mitochondrial DNA sequences in single cells:
implications for the study of somatic mitochondrial DNA point
mutations. Nucleic Acids Res 2001, 29:E74–74.
He L, Chinnery PF, Durham SE, Blakely EL, Wardell TM, Borthwick GM, Taylor
RW, Turnbull DM: Detection and quantification of mitochondrial DNA
deletions in individual cells by real-time PCR. Nucleic Acids Res 2002, 30:e68.
Yowe DL, Ames BN: Quantitation of age-related mitochondrial DNA
deletions in rat tissues shows that their pattern of accumulation differs
from that of humans. Gene 1998, 209:23–30.
Morton BR, Bi IV, McMullen MD, Gaut BS: Variation in mutation dynamics
across the maize genome as a function of regional and flanking base
composition. Genetics 2006, 172:569–577.
Zhang F, Zhao Z: The influence of neighboring-nucleotide composition
on single nucleotide polymorphisms (SNPs) in the mouse genome and
its comparison with human SNPs. Genomics 2004, 84:785–795.
Giulivi C, Boveris A, Cadenas E: Hydroxyl radical generation during
mitochondrial electron transfer and the formation of 8-
hydroxydesoxyguanosine in mitochondrial DNA. Arch Biochem Biophys
Loft S, Poulsen HE: Markers of oxidative damage to DNA: antioxidants
and molecular damage. Methods Enzymol 1999, 300:166–184.
Wong TS, Rajagopalan S, Townsley FM, Freund SM, Petrovich M, Loakes D,
Fersht AR: Physical and functional interactions between human
mitochondrial single-stranded DNA-binding protein and tumour
suppressor p53. Nucleic Acids Res 2009, 37:568–581.
Le Page F, Guy A, Cadet J, Sarasin A, Gentil A: Repair and mutagenic
potency of 8-oxoG:A and 8-oxoG:C base pairs in mammalian cells.
Nucleic Acids Res 1998, 26:1276–1281.
Shibutani S, Takeshita M, Grollman AP: Insertion of specific bases during DNA
synthesis past the oxidation-damaged base 8-oxodG. Nature 1991, 349:431–434.
Wood ML, Dizdaroglu M, Gajewski E, Essigmann JM: Mechanistic studies of
ionizing radiation and oxidative mutagenesis: genetic effects of a single
8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique
site in a viral genome. Biochemistry 1990, 29:7024–7032.
Pinz KG, Shibutani S, Bogenhagen DF: Action of mitochondrial DNA
polymerase gamma at sites of base loss or oxidative damage. J Biol
Chem 1995, 270:9202–9206.
Marcelino LA, Thilly WG: Mitochondrial mutagenesis in human cells and
tissues. Mutat Res 1999, 434:177–203.
Marcelino LA, Andre PC, Khrapko K, Coller HA, Griffith J, Thilly WG:
Chemically induced mutations in mitochondrial DNA of human cells:
mutational spectrum of N-methyl-N'-nitro-N-nitrosoguanidine. Cancer Res
Holt IJ, Harding AE, Cooper JM, Schapira AH, Toscano A, Clark JB, Morgan-
Hughes JA: Mitochondrial myopathies: clinical and biochemical features
Napoli et al. Molecular Autism 2013, 4:2
Page 7 of 8
of 30 patients with major deletions of muscle mitochondrial DNA. Ann Download full-text
Neurol 1989, 26:699–708.
Cortopassi GA, Shibata D, Soong NW, Arnheim N: A pattern of
accumulation of a somatic deletion of mitochondrial DNA in aging
human tissues. Proc Natl Acad Sci USA 1992, 89:7370–7374.
Shelton JF, Hertz-Picciotto I, Pessah IN: Tipping the balance of autism risk:
potential mechanisms linking pesticides and autism. Environ Health
Perspect 2012, 120:944–951.
Pinto D, Pagnamenta AT, Klei L, Anney R, Merico D, Regan R, Conroy J,
Magalhaes TR, Correia C, Abrahams BS, et al: Functional impact of global
rare copy number variation in autism spectrum disorders. Nature 2010,
Shanks N, Windle RJ, Perks PA, Harbuz MS, Jessop DS, Ingram CD, Lightman
SL: Early-life exposure to endotoxin alters hypothalamic-pituitary-adrenal
function and predisposition to inflammation. Proc Natl Acad Sci USA 2000,
Boisse L, Mouihate A, Ellis S, Pittman QJ: Long-term alterations in
neuroimmune responses after neonatal exposure to lipopolysaccharide.
J Neurosci 2004, 24:4928–4934.
Ellis S, Mouihate A, Pittman QJ: Early life immune challenge alters innate
immune responses to lipopolysaccharide: implications for host defense
as adults. FASEB J 2005, 19:1519–1521.
Spencer SJ, Heida JG, Pittman QJ: Early life immune challenge–effects on
behavioural indices of adult rat fear and anxiety. Behav Brain Res 2005,
Hao H, Morrison LE, Moraes CT: Suppression of a mitochondrial tRNA
gene mutation phenotype associated with changes in the nuclear
background. Hum Mol Genet 1999, 8:1117–1124.
Betts J, Jaros E, Perry RH, Schaefer AM, Taylor RW, Abdel-All Z, Lightowlers
RN, Turnbull DM: Molecular neuropathology of MELAS: Level of
heteroplasmy in individual neurones and evidence of extensive vascular
involvement. Neuropathol Appl Neurobiol 2006, 32:359–373.
Davey GP, Peuchen S, Clark JB: Energy thresholds in brain mitochondria.
Potential involvement in neurodegeneration. J Biol Chem 1998,
Meng XM, Zhu DM, Ruan DY, She JQ, Luo L: Effects of chronic lead
exposure on 1H MRS of hippocampus and frontal lobes in children.
Neurology 2005, 64:1644–1647.
Wang Y, Michikawa Y, Mallidis C, Bai Y, Woodhouse L, Yarasheski KE, Miller
CA, Askanas V, Engel WK, Bhasin S, Attardi G: Muscle-specific mutations
accumulate with aging in critical human mtDNA control sites for
replication. Proc Natl Acad Sci USA 2001, 98:4022–4027.
Lee HC, Pang CY, Hsu HS, Wei YH: Differential accumulations of 4,977 bp
deletion in mitochondrial DNA of various tissues in human ageing.
Biochim Biophys Acta 1994, 1226:37–43.
Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, Beal MF, Wallace DC:
Mitochondrial DNA deletions in human brain: regional variability and
increase with advanced age. Nat Genet 1992, 2:324–329.
Melberg A, Nennesmo I, Moslemi AR, Kollberg G, Luoma P, Suomalainen A,
Holme E, Oldfors A: Alzheimer pathology associated with POLG1
mutation, multiple mtDNA deletions, and APOE4/4: premature ageing or
just coincidence? Acta Neuropathol 2005, 110:315–316.
Komulainen T, Hinttala R, Karppa M, Pajunen L, Finnila S, Tuominen H,
Rantala H, Hassinen I, Majamaa K, Uusimaa J: POLG1 p.R722H mutation
associated with multiple mtDNA deletions and a neurological
phenotype. BMC Neurol 2010, 10:29.
Napoli E, Wong S, Hung C, Ross-Inta C, Bomdica P, Giulivi C: Defective
mitochondrial disulfide relay system, altered mitochondrial morphology
and function in Huntington's disease. Hum Mol Genet 2012. doi:10.1093/
hmg/dds503. epub 11/29/2012.
Yin PH, Lee HC, Chau GY, Wu YT, Li SH, Lui WY, Wei YH, Liu TY, Chi CW:
Alteration of the copy number and deletion of mitochondrial DNA in
human hepatocellular carcinoma. Br J Cancer 2004, 90:2390–2396.
Whittaker RG, Schaefer AM, McFarland R, Taylor RW, Walker M, Turnbull DM:
Prevalence and progression of diabetes in mitochondrial disease.
Diabetologia 2007, 50:2085–2089.
67.Kienhofer J, Haussler DJ, Ruckelshausen F, Muessig E, Weber K, Pimentel D,
Ullrich V, Burkle A, Bachschmid MM: Association of mitochondrial
antioxidant enzymes with mitochondrial DNA as integral nucleoid
constituents. FASEB J 2009, 23:2034–2044.
Schmidt RJ, Hansen RL, Hartiala J, Allayee H, Schmidt LC, Tancredi DJ,
Tassone F, Hertz-Picciotto I: Prenatal vitamins, one-carbon metabolism
gene variants, and risk for autism. Epidemiology 2011, 22:476–485.
Cite this article as: Napoli et al.: Evidence of reactive oxygen species-
mediated damage to mitochondrial DNA in children with typical autism.
Molecular Autism 2013 4:2.
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