Autism spectrum disorder (ASD) is characterized by phenotypic heterogeneity and a complex genetic architecture which includes distinctive epigenetic patterns. We report differential DNA methylation patterns associated with ASD in South African children. An exploratory whole‐epigenome methylation screen using the Illumina 450 K MethylationArray identified differentially methylated CpG sites between ASD and controls that mapped to 898 genes ( P ≤ 0.05) which were enriched for nine canonical pathways converging on mitochondrial metabolism and protein ubiquitination. Targeted Next Generation Bisulfite Sequencing of 27 genes confirmed differential methylation between ASD and control in our cohort. DNA pyrosequencing of two of these genes, the mitochondrial enzyme Propionyl‐CoA Carboxylase subunit Beta ( PCCB ) and Protocadherin Alpha 12 ( PCDHA12 ), revealed a wide range of methylation levels (9–49% and 0–54%, respectively) in both ASD and controls. Three CpG loci were differentially methylated in PCCB ( P ≤ 0.05), while PCDHA12 , previously linked to ASD, had two significantly different CpG sites ( P ≤ 0.001) between ASD and control. Differentially methylated CpGs were hypomethylated in ASD. Metabolomic analysis of urinary organic acids revealed that three metabolites, 3‐hydroxy‐3‐methylglutaric acid ( P = 0.008), 3‐methyglutaconic acid ( P = 0.018), and ethylmalonic acid ( P = 0.043) were significantly elevated in individuals with ASD. These metabolites are directly linked to mitochondrial respiratory chain disorders, with a putative link to PCCB , consistent with impaired mitochondrial function. Our data support an association between DNA methylation and mitochondrial dysfunction in the etiology of ASD. Autism Res 2020, 13 : 1079‐1093. © 2020 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals, Inc. Lay Summary Epigenetic changes are chemical modifications of DNA which can change gene function. DNA methylation, a type of epigenetic modification, is linked to autism. We examined DNA methylation in South African children with autism and identified mitochondrial genes associated with autism. Mitochondria are power‐suppliers in cells and mitochondrial genes are essential to metabolism and energy production, which are important for brain cells during development. Our findings suggest that some individuals with ASD also have mitochondrial dysfunction.

Autism spectrum disorder (ASD) is a complex disorder that is underpinned by numerous dysregulated biological pathways, including pathways that affect mitochondrial function. Epigenetic mechanisms contribute to this dysregulation and DNA methylation is an important factor in the etiology of ASD. We measured DNA methylation of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), as well as five genes involved in regulating mitochondrial homeostasis to examine mitochondrial dysfunction in an ASD cohort of South African children. Using targeted Next Generation bisulfite sequencing, we found differential methylation (p < 0.05) at six key genes converging on mitochondrial biogenesis, fission and fusion in ASD, namely PGC-1α, STOML2, MFN2, FIS1, OPA1, and GABPA. PGC-1α, the transcriptional regulator of biogenesis, was significantly hypermethylated at eight CpG sites in the gene promoter, one of which contained a putative binding site for CAMP response binding element 1 (CREB1) (p = 1 × 10–6). Mitochondrial DNA (mtDNA) copy number, a marker of mitochondrial function, was elevated (p = 0.002) in ASD compared to controls and correlated significantly with DNA methylation at the PGC-1α promoter and there was a positive correlation between methylation at PGC-1α CpG#1 and mtDNA copy number (Spearman’s r = 0.2, n = 49, p = 0.04) in ASD. Furthermore, DNA methylation at PGC-1α CpG#1 and mtDNA copy number correlated significantly (p < 0.05) with levels of urinary organic acids associated with mitochondrial dysfunction, oxidative stress, and neuroendocrinology. Our data show differential methylation in ASD at six key genes converging on PGC-1α-dependent regulation of mitochondrial biogenesis and function. We demonstrate that methylation at the PGC-1α promoter is associated with elevated mtDNA copy number and metabolomic evidence of mitochondrial dysfunction in ASD. This highlights an unexplored role for DNA methylation in regulating specific pathways involved in mitochondrial biogenesis, fission and fusion contributing to mitochondrial dysfunction in ASD.

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with extensive genetic and aetiological heterogeneity. While the underlying molecular mechanisms involved remain unclear, significant progress has been facilitated by recent advances in high-throughput transcriptomic, epigenomic and proteomic technologies. Here, we review recently published ASD proteomic data and compare proteomic functional enrichment signatures with those of transcriptomic and epigenomic data. We identify canonical pathways that are consistently implicated in ASD molecular data and find an enrichment of pathways involved in mitochondrial metabolism and neurogenesis. We identify a subset of differentially expressed proteins that are supported by ASD transcriptomic and DNA methylation data. Furthermore, these differentially expressed proteins are enriched for disease phenotype pathways associated with ASD aetiology. These proteins converge on protein–protein interaction networks that regulate cell proliferation and differentiation, metabolism, and inflammation, which demonstrates a link between canonical pathways, biological processes and the ASD phenotype. This review highlights how proteomics can uncover potential molecular mechanisms to explain a link between mitochondrial dysfunction and neurodevelopmental pathology.

Molecular autism research is evolving toward a biopsychosocial framework that is more informed by autistic experiences. In this context, research aims are moving away from correcting external autistic behaviors and toward alleviating internal distress. Autism Spectrum Conditions (ASCs) are associated with high rates of depression, suicidality and other comorbid psychopathologies, but this relationship is poorly understood. Here, we integrate emerging characterizations of internal autistic experiences within a molecular framework to yield insight into the prevalence of psychopathology in ASC. We demonstrate that descriptions of social camouflaging and autistic burnout resonate closely with the accepted definitions for early life stress (ELS) and chronic adolescent stress (CAS). We propose that social camouflaging could be considered a distinct form of CAS that contributes to allostatic overload, culminating in a pathophysiological state that is experienced as autistic burnout. Autistic burnout is thought to contribute to psychopathology via psychological and physiological mechanisms, but these remain largely unexplored by molecular researchers. Building on converging fields in molecular neuroscience, we discuss the substantial evidence implicating mitochondrial dysfunction in ASC to propose a novel role for mitochondrial allostatic load in the relationship between autism and psychopathology. An interplay between mitochondrial, neuroimmune and neuroendocrine signaling is increasingly implicated in stress-related psychopathologies, and these molecular players are also associated with neurodevelopmental, neurophysiological and neurochemical aspects of ASC. Together, this suggests an increased exposure and underlying molecular susceptibility to ELS that increases the risk of psychopathology in ASC. This article describes an integrative framework shaped by autistic experiences that highlights novel avenues for molecular research into mechanisms that directly affect the quality of life and wellbeing of autistic individuals. Moreover, this framework emphasizes the need for increased access to diagnoses, accommodations, and resources to improve mental health outcomes in autism.

BackgroundAutism Spectrum Disorder (ASD) is a complex disorder that is underpinned by numerous dysregulated biological pathways, including canonical mitochondrial pathways. Epigenetic mechanisms contribute to this dysregulation and DNA methylation is an important factor in the aetiology of ASD. We examined the relationship between DNA methylation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), an essential transcriptional regulator of mitochondrial homeostasis, and mitochondrial dysfunction in an ASD cohort of South African children.ResultsUsing targeted Next Generation bisulfite sequencing, we found 12 highly variable CpG sites in PGC-1α that were significantly differentially methylated (p<0.05) between ASD (n = 55) and controls (n = 44). In ASD, eight CpG sites were hypermethylated in the PGC-1α promotor with a putative binding site for CAMP response binding element 1 (CREB1) spanning one of these CpG sites (p = 1 × 10−6). Mitochondrial DNA (mtDNA) copy number, a marker of mitochondrial function, was elevated (p = 0.002) in ASD compared to controls and correlated significantly with DNA methylation at the PGC-1α promoter. There was a positive correlation between methylation at PGC-1α at CpG#1 and mtDNA copy number (Spearman’s r = 0.2, n = 49, p = 0.04) in ASD, but a negative correlation between methylation at PGC-1α at CpG#4 promoter and mtDNA copy number in controls (Spearman’s r = −0.4, n = 42, p = 0.045). While there was no relationship between mtDNA deletions and PGC-1α methylation in ASD, mtDNA deletions correlated negatively with methylation at PGC-1α at CpG#4 (Spearman’s r = −0.4, n = 42, p = 0.032) in controls. Furthermore, levels of urinary organic acids associated with mitochondrial dysfunction correlated significantly (p<0.05) with DNA methylation at PGC-1α CpG#1 and mtDNA copy number in ASD (n= 20) and controls (n= 13) with many of these metabolites involved in altered redox homeostasis and neuroendocrinology.ConclusionsThese data show an association between PGC-1α promoter methylation, elevated mtDNA copy number and metabolomic evidence of mitochondrial dysfunction in ASD. This highlights an unexplored link between DNA methylation and mitochondrial dysfunction in ASD.