We envision that the identification of new biomarkers, risk genes and related molecular pathways may contribute to the early diagnosis of ASD and to the improvement of clinical and pharmacological treatment of this disease. The results of this study validated that six DEGs were differentially expressed in autistic patients. For methylation markers, it may have more clinical diagnostic significance, especially for MDM2.
This study further supported the possible role of protein ubiquitination in neuropathology as a pathological mechanism associated with autism (Fatemi et al., 2012). Signal recognition particle (SRP) is a ribonucleic acid (Rnp) complex that mediates the targeting of nascent signal sequence-carrying polypeptides, including membrane and secreted proteins, to the ER surface (Keenan, Freymann, Stroud, & Walter, 2001). ER stress and apoptosis may play an important role in the pathogenesis of autism (Dong, Zielke, Yeh, & Yang, 2018). The Sphingolipid metabolism pathway is altered in a biologically similar maternal immune activation (MIA) mouse model to ASD (Naviaux et al., 2014). In addition, the PPARγ agonist pioglitazone has been used in clinical trials, suggesting that PPAR may be a target for ASD drug therapy (Ghaleiha et al., 2015). Selective activation of PPARα and PPARγ plays a neuroprotective role by reducing oxidative stress and neuroinflammation, which is an important link in the pathophysiological process of ASD (Barone et al., 2019).
Among the key genes identified in the PPI network, the differently expressed MDM2 had been validated by other ASD study (Chow et al., 2012). The E3 ubiquitin ligase MDM2 promoted ubiquitination of itself, overexpression also caused genomic instability (Cao et al., 2019). In the ASD model, dysregulated MDM 2 prevented myocyte enhancer factor 2-induced PSD-95 ubiquitination and synaptic elimination (Tsai et al., 2012). TATA-binding protein (TBP) is a ubiquitinated substrate by a special E3 ligase in vivo and in vitro, which helps to activate gene expression in animal cells (Li, Martinez, Hu, Liu, & Tjian, 2015; Ravarani et al., 2020). In the neuroprotective proteins of ASD, the expression of TBP is relatively reduced, which is consistent with our experimental results (Grigg et al., 2020). RPL4 is involved in the regulation of splicing and transcription networks in human neuronal development by RBFOX 1 (Fogel et al., 2012). Unfortunately, however, no studies had been found on the relationship between RPL4, RPL5, RPS13, RPS14, and FAU and ASD.
Our comparison of DNA methylation modification differences between ASD and controls enabled us to identify specific methylation markers associated with ASD. Among the methylation markers we identified, the key factor MDM 2 was modified by methylation. MDM2 may serve as a new and promising molecule for research and targeted therapy of ASD. RUNX2, as a candidate gene for ASD, is involved in abnormal brain rhythm and language deficits (Benitez-Burraco, Lattanzi, & Murphy, 2016; Benitez-Burraco & Murphy, 2016). IMMP2L is associated with susceptibility to autism in Caucasian populations (Maestrini et al., 2010). IMMP2L is not only associated with autism, but also with diagnosable autism-related diseases (Muhle, Trentacoste, & Rapin, 2004). Logistic regression analysis showed that MDM2, RUNX2 and IMMP2L had a better role in the differential diagnosis of ASD. These results suggested that MDM2, RUNX2 and IMMP2L had great application prospects in the diagnosis of ASD and deserve further study.
Our study also had some limitations. First, our analytical data were derived from public databases, and there was not much sample data for validation experiments, and larger clinical samples needed to validate important analytical results in the follow-up. Second, we had not explored the mechanism of methylation markers and need further in-depth study.
In summary, we report the association of methylation modifications and gene expression. Our results will need to be followed up to determine the diagnostic role of methylation markers. These results may provide new insights into the biological mechanisms and diagnostic markers of ASD.