Autism, a pervasive neurodevelopmental disorder manifested by deficits in social behavior and interpersonal communication, and by stereotyped, repetitive behaviors, is inexplicably biased towards males by a ratio of ∼4∶1, with no clear understanding of whether or how the sex hormones may play a role in autism susceptibility. Here, we show that male and female hormones differentially regulate the expression of a novel autism candidate gene, retinoic acid-related orphan receptor-alpha (RORA) in a neuronal cell line, SH-SY5Y. In addition, we demonstrate that RORA transcriptionally regulates aromatase, an enzyme that converts testosterone to estrogen. We further show that aromatase protein is significantly reduced in the frontal cortex of autistic subjects relative to sex- and age-matched controls, and is strongly correlated with RORA protein levels in the brain. These results indicate that RORA has the potential to be under both negative and positive feedback regulation by male and female hormones, respectively, through one of its transcriptional targets, aromatase, and further suggest a mechanism for introducing sex bias in autism.

Although differences in DNA methylation have been associated with both syndromic and idiopathic autism, differential methylation has not been examined previously with respect to sex differences. The goals of this study were to (1) identify differences in the DNA methylation profiles of lymphoblastoid cell lines derived from a subgroup of severely affected individuals with idiopathic autism and their respective sex-matched siblings, (2) describe autism spectrum disorder–relevant pathways and functions that may be impacted by differentially methylated genes, and (3) investigate sex-dependent differences in methylation patterns and signaling pathways. Our results revealed significant differences in DNA methylation in cells from individuals with idiopathic autism spectrum disorders and from their unaffected sex-matched siblings. The samples were divided either by sex or by separation into discovery and validation groups. The genes in differentially methylated regions were statistically enriched in autism susceptibility genes and canonical pathways commonly associated with autism spectrum disorders, including synaptogenesis, semaphorin, and mammalian target of rapamycin signaling pathways. Differentially methylated region–associated genes in females were additionally associated with pathways that implicate mitochondrial dysfunction and metabolic disorders that may offer some protection against autism spectrum disorders. Further investigations of sex differences are required to develop a fuller understanding of the pathobiology, gene regulatory mechanisms, and differential susceptibility of males and females toward autism spectrum disorders. Lay abstract This study investigates altered DNA methylation that may contribute to autism spectrum disorders. DNA methylation is an epigenetic mechanism for regulating the level at which genes are expressed, and is thus complementary to genetics and gene expression analyses which look at the variations in gene structure and gene products in cells. Here, we identify DNA methylation differences between autistic and sex-matched non-autistic siblings, focusing on a subgroup of severely affected individuals with language impairment to reduce the clinical heterogeneity among the cases. Our results show significant differentially methylated genes between the sibling groups that are enriched in autism risk genes as well as in signaling and biochemical pathways previously associated with the pathobiology of autism spectrum disorders. Moreover, we show for the first time that these differences are in part sex dependent, with differentially methylated genes in females associated with pathways that implicate mitochondrial dysfunction and metabolic disorders that may offer some protection to females against autism spectrum disorders. Further investigations of sex differences are required to develop a fuller understanding of the pathobiology, gene regulatory mechanisms, and differential susceptibility of males and females toward autism spectrum disorders.