In this study, we identified five de novo mutational genes associated with five ASD children among 122 clinically diagnosed autistic children. This is similar to results from previous studies of coding-sequence mutations making up 5–10% of ASD patients3. These genes include ASH1L, SCN2A, GIGYF2, NAA15, and DDX3X. They has been reported to be associated with ASD in the previous studies21–27. We then carried out plasma, PBMCs proteomics, and plasma metabolomics studies in these ASD children and healthy controls. The results showed that total plasma and PBMCs proteins, total plasma metabolites, were not well distinguished between the two groups, but DEPs and differential metabolites between these two groups were able to distinguish the two groups. Here, we focus on these DEPs, differential metabolites, and their related mechanisms.
By plasma proteomics analysis, 13 DEPs were identified. Consistent with the previous studies, these DEPs identified in the blood were mainly involved in complement and coagulation cascades, inflammatory and immune responses. Altered complement proteins in the blood from patients with ASD have been broadly reported, including changes in protein expression levels15,28−31 and post-translational modifications32,33. Complement C5 was also found to be up-regulated in the PBMCs of children with ASD in our previous study11. Here, C1QA, C1QB, C1S, and C2 were significantly up-regulated in the plasma of children with ASD, whereas COLEC10 was down-regulated. To the best of our knowledge, C1S, C2, and COLEC10 were first reported to be associated with ASD. The expression of most complement proteins is up-regulated in the periphery of autistic patients, suggesting that the complement pathway may be activated in the periphery of autistic patients. However, the expression trends of complement proteins in the brain of autistic patient are not completely consistent34,35. On the other hand, beyond its involvement with innate immune responses, complement proteins has been increasingly implicated in playing an important role in neurodevelopment, including neurogenesis, neuronal migration, and synaptic remodeling34–36. Together, the changes in the complement system in peripheral blood, PBMCs, and brain of ASD patients highlights that this system may play a key role in the pathogenesis of ASD and is worthy of further study.
Accumulating evidence suggests a potential role of the immune system in the pathophysiology of ASD during the pre, neo-, and postnatal periods13,37. In this study, 7 plasma DEPs and 36 PBMCs DEPs were involved in the immune system, further supporting the association between immunity and ASD pathogenesis. Ten DEPs of PBMCs were associated with interleukin-1 (IL-1), together with our previous study (IL-12) 11 and other studies38,39, suggesting that IL-1 and IL-12 might be key inflammatory cytokines in peripheral blood and blood mononuclear cells.
Mitochondrial dysfunction has also been implicated in immune dysregulation in ASD 40. In this study, the expression of three proteins (NDUFA7, NDUFA11, and NDUFA13) related to energy metabolism were increased in PBMCs of autistic children. These results were similar to previous observations in PBMCs 11 and LCL cells 41, different tissues and organs42, T cell, NK cell, and monocyte 43 in children with autism. Activation and proliferation of microglia and astrocytes have also been observed in the brains of ASD subjects44. Interestingly, transcriptome analysis between autistic brains and normal brains identified discrete modules by gene co-expression network analysis: a neuronal module and a module enriched for immune genes and glial markers45. This may explain the uniqueness of mitochondrial dysfunction in ASD and how autistic children may have nontraditional mitochondrial diseases. After all, mitochondrial dysfunction has been reported by postmortem brain tissue examinations on ASD subjects and reduction of ETC (Electron transport chain) complexes has also been observed in different brain regions of autistic children46–48.
Consistent with our previous study11, the main pathways associated with DEPs of PBMCs have also involved in proteasome, ubiquitin mediated proteolysis, and protein processing in the endoplasmic reticulum, which strongly suggest that endoplasmic reticulum stress (ER) being linked to the PBMCs of children with ASD. ER stress occurs when the amount of unfolded proteins in the ER reach an unmanageable level, triggering the unfolded protein response (UPR)49. Under excess or chronic ER stress, cell apoptosis is induced to eliminate unhealthy cells50. Here, 6 DEPs were associated with necroptosis. Similarly, LCLs from children with autism were shown to be more sensitive to necrosis than their non-autistic siblings51. Thus, necroptosis may be associated with the ER stress in the PBMCs of ASD subjects. Moreover, increase in ER stress has been observed in the brains of autistic children52,53 and those of ASD model mice54. The mutation of ASD related genes and the aggregation of their encoded proteins 11,52 and oxidative stress may be responsible for ER stress in children with ASD 52. On the other hand, the proteostasis network machinery plays a role in the establishment, maintenance, and plasticity of stable and dynamic dendritic arbors. Ubiquitin-proteasome system is required for developmental dendritic pruning55,56.
Interestingly, among the DEPs of PBMS, three other genes have also been linked to ASD in addition to DDX3X, including SLC9A957, CSDE158, and CCT459. Among them, The SLC9A9 gene encodes Na+ /H+ transpteron-9 endometrium protein (NHE9), which has been shown to be related to endocytosis, protein ubiquitination, and phagosome57. CSDE1 encodes RNA-binding proteins that may be involved in translation-coupled mRNA conversion and is associated with neurodevelopment and neuropsychiatric disorders58. The CCT4 gene encodes a molecular chaperone that assists in protein folding during ATP hydrolysis60. These results further support that protein folding and ER stress may be associated with the pathogenesis of ASD, and that at least some children with ASD carry more than two risk genes in this study.
By metabolomics analysis, the differential metabolites in the plasma between cases and controls were mainly involved in amino acid (alanine, aspartate and glutamate, arginine biosynthesis, and D-glutamine and D-glutamate), vitamin (nicotinate and nicotinamide, and vitamin B6), and lipid (glycerophospholipid and sphingolipid) metabolism. Nitrogen metabolism, N-Glycan biosynthesis, and neomycin, kanamycin and gentamicin biosynthesis were also involved.
In the present study, among the altered amino acids, L-Glutamate was down-regulated in plasma of controls while L-Glutamine was increased in plasma of autistic children. Although previous studies showed that the altered levels of glutamate and glutamine in the blood of autistic patients were inconsistent 61–66, these data support the current view that excitatory/inhibitory imbalance, especially the abnormality of the excitatory neurotransmitter glutamate, is one of the pathogenesis of ASD 65,67. Indeed, it has been reported that glutamate signals in the anterior cingulate cortex and cerebellum of ASD patients were significantly decreased68. More recently, the gut metabolites involved in alanine, aspartate, and glutamate metabolic pathways were reported to be significantly lower in children with ASD, which was associated with differences in the abundance of gut microbiota related to D-Glutamine and D-glutamate metabolism, suggesting that the gut microbiota might contribute to abnormal glutamate metabolism in autistic children69. Besides, similar with the present study, aspartate has been observed to be decreased in the fecal ASD subjects70, while ornithine was observed to be increased in the blood of children with ASD64.
Our results showed that three differential metabolites were also involved in the nicotinate and nicotinamide metabolism pathway. Niacin (NA), also known as Vitamin B3 and nicotinic acid, can be biosynthetically converted into nicotinamide adenine dinucleotide (NAD). NAD has a variety of biological functions and plays a central role in redox reactions71. Nicotinate and nicotinamide metabolism have been reported to be altered in the prefrontal cortex72, urine73, and blood74 of ASD individuals, and associated with microbiota transfer therapy (MTT) of autistic children74. Nicotinamide is derived from tryptophan, while abnormal tryptophan metabolism has been observed in children with ASD73,75−77. In addition, three differential metabolites (O-Phospho-4-hydroxy-L-threonine, Pyridoxamine, and 4-Pyridoxate) involved in Vitamin B6 metabolism were found to be decreased in autistic children. Reduced levels of Vitamin B6 have been observed in the urine of autistic children78. It is the main cofactor of biological reactions and is important for the synthesis of neurotransmitters and trans-sulfuration. Its deficiency is related to oxidative stress, high blood homocysteine and hypomethylation in children with AD78. In general, the lack of vitamin B group in children with ASD may be caused by nutritional deficiency, poor absorption or alteration in gut microbiota74,79,80, contributing to the pathogenesis of ASD.
The brain is particularly enriched in lipids with a diverse lipid composition compared to other tissues81. Glycerophospholipids are critical components of neuronal membranes and myelin, and principal regulators of synaptic function81. Sphingolipid is involved in neuronal differentiation, synaptic transmission in neuronal-glial connections, and myelin stability. Disturbance of their metabolism has been linked to various neuropsychiatric diseases include autism82, Rett syndrome83, and ASD84.
Finally, integrating omics data, glycerophospholipid and N-linked glycosylation metabolism was associated with the DEPs and differential metabolites. N-linked glycosylation is important in brain structure and function. The extracellular glycans and glycoconjugates may contribute to the etiology and pathogenesis of pervasive neurodevelopmental disorders include idiopathic ASDs. Glycobiology related genes were implicated in ASD. Mutations in glycogenes associated with ASD affect the downstream steps of N-glycan biosynthesis85.