In this study, and following the model of strand asymmetry replication of mtDNA, evidence for mtDNA damage 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 following 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-dihydro-2′-deoxyguanosine (8oxodG)[1, 38, 39], is effectively repaired and/or removed from the template , inducing predominantly G→T transitions by mispairing with A during DNA replication [41–43]; (ii) higher frequency 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 autism, 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 transitions 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 children 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 disorders, in which deletion ≥ 60% is required to demonstrate a mitochondrial defect [47, 48], it is suggested that the extent 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, regardless 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 children 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 incidence of deletions when compared to age-matched groups. This might indicate a compensatory mechanism, by which a lower replicative rate might prevent additional 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 accumulate mtDNA deletions was transmitted paternally. It should be noted that paternal mtDNA deletions are not inherited but, accumulation of deletions (or the predisposition 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 families 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 considered for a proper interpretation of the results and consequences 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 significance at the α = 0.001 level minimizing type I errors, 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 identified by M.I.N.D. developmental pediatricians. Nevertheless, 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, although the outcomes reported here for PBMC may represent 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 between peripheral immune cells and the central nervous system, and that disruption of this process during early life may condition inflammatory responses as well as behavioral changes that persist during adulthood [51–54]. Finally, inferences about a cause-effect association between ROS-mediated mtDNA damage and typical autism are intricate because this is cross-sectional, and not a longitudinal study. In addition, several factors influence expression of mtDNA damage, for example, nuclear genetic backgrounds , mtDNA heteroplasmy in tissues , 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 tissues of apparently healthy individuals [12, 48, 59–61], or in patients with other disorders not necessarily linked to autism, such as, inherited mutations in nuclear genes [62, 63], neurodegenerative disorders [62, 64], cancer , and diabetes . 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 (maternally inherited) consistent with ROS-mediated damage (based on sequence variants), and presenting a predisposition to accumulate damage (deletions) similar to that of their fathers.