Upregulated expression of BATF2 and FCGR1A in Israeli children with ASD confirms findings in Japanese adults with ASD
The upregulated expression levels of both BATF2 and FCGR1A in our Israeli cohort of whole blood samples from children with ASD (Fig. 1) corroborate findings from a recent Japanese RNA-seq study in whole blood from adults with ASD17. To our knowledge our study provides the first validation of a genome-wide RNA-seq study with ASD blood samples. Our literature search of RNA-seq studies (Table 3; see Methods) revealed large variation in findings from earlier transcriptomic studies (using either RNA-seq or RNA microarray technologies) in whole blood samples of individuals with ASD compared to matched NT controls. Notably, each of the dysregulated genes listed in our literature survey was mentioned in only a single study (or a single meta-analysis in regard to the meta-analysis by Lee et al., 201918). Therefore, our current study appears to be the first validation of any dysregulated gene in blood samples from individuals diagnosed with ASD. Moreover, our validation was done on children with ASD (Table 1), in contrast to adults with ASD in the mentioned Japanese study17.
Our RNA-seq detected SERPING1 as the top upregulated gene in whole blood from children with ASD (FD=3.4962; Padj=0.0072; Table 2). SERPING1 was among the upregulated genes in whole blood from Japanese adults with ASD17. Yet, our real-time PCR experiments could not validate this finding in the entire whole blood samples (Supplementary Fig. S1). Likewise, SERPING1 expression was similar in PBMC-derived RNA samples from children with ASD and NT children (Supplementary Table S2). Nonetheless, our real-time PCR experiments in PBMC-derived RNA samples (our U.S. cohort) indicated upregulated expression of SERPING1 in children with ASD compared with their NT siblings (Supplementary Table S2). These findings exemplify an advantage of including NT siblings of children with ASD in autism research studies.
Our findings thus suggest a central role in ASD for the upregulated genes identified, independent of age and ethnicity. Further studies, including transcriptomic studies with brain tissues from ASD animal models, are needed to elucidate the relevance of the dysregulated genes for ASD behavioral scores (Fig. 2 & Fig. 3) and their implications for ASD phenotypes.
Dysregulated ASD genes and cancer
All four genes that were found in our study to present dysregulated mRNA transcript levels in blood from children with ASD have been investigated mostly in the context of cancer. Both BATF2 and FCGR1A, detected in our study as upregulated in blood samples of children with ASD, code for cancer protective proteins. BATF2 was shown to have an antitumor effect in a mouse model through upregulation of IL-12 p40 in tumor-associated macrophages, leading to CD8+ T-cell activation and tumor accumulation19. Among other cancers, BATF2 was demonstrated as a tumor suppressor of gastric cancer20, glioblastoma21, and esophageal squamous cell carcinoma22. Higher tumor FCGR1A expression correlated with improved prognosis in laryngeal cancer23, as well as in cervical cancer and melanoma, in which it was associated with increased tumor infiltration of CD4+ and CD8+ T cells and dendritic cells24.
Additionally, higher expression levels of both ISG15 and MT2A, found in this study as downregulated in blood samples from children with ASD, were reported to be associated with worse cancer prognosis. The protein coded by ISG15 (interferon-stimulated gene 15 ubiquitin like modifier) was implicated in autophagy, exosome secretion, DNA repair, and immune modulation pathways; it is also a known tumor promoter by suppressing immune cell tumor infiltration25. ISG15 was shown to drive tumorigenesis and metabolic plasticity of pancreatic cancer, suggesting that its inhibition may be a treatment option for pancreatic cancer26. Higher ISG15 expression was also associated with poor prognosis in breast cancer27. The protein coded by MT2A, metallothionein 2A, is the major metallothionein in humans, and serves as a chelator of intracellular zinc ions and protects cells against free radicals. MT2A is upregulated in most cancers, and contributes to their chemotherapy resistance by chelation of zinc and platinum-containing drugs and by its action on p53 zinc-dependent activity. MT2A upregulation results in p53 misfolding secondary to zinc chelation, while low cellular MT2A levels allow proper p53 function as a genome stability guardian28,29. Lastly, downregulated C1 Inhibitor (encoded by SERPING1) was shown to increase cancer risk30,31. Hence, the upregulated SERPING1 observed in sub-cohorts of this study may also contribute to reduced cancer risk in children with ASD.
Taken together, the upregulation of both BATF2 and FCGR1A, and the downregulation of both ISG15 and MT2A, as we detected in blood samples from children with ASD, all suggest a reduced risk of cancer. Indeed, a huge reduction in cancer risk (OR=0.06; 95% CI: 0.02, 0.19; p<0.0001) was reported among children with ASD aged 0 to 14 years compared with matched controls 32. These authors compared cancer rates in 1,837 individuals with ASD and in 9,336 controls in the registry of the University of Iowa Hospitals and Clinics. They observed that the large gap in cancer rates between individuals with ASD and controls was lower at older ages, being only 2-fold less among individuals with ASD aged above 55 years compared with controls.
Our findings on upregulated BATF2 and FCGR1A, and downregulated ISG15 and MT2A (or possibly some of these genes) in children with ASD thus seem to agree with the findings of the Darbro et al.201632 epidemiologic survey. Albeit, we did not identify similarly large studies on cancer risk among children with ASD. The only other epidemiologic study reporting reduced cancer risk among individuals with ASD was smaller (91 individuals with ASD and 6,186 sex- and birth-year controls), and was based on death records, thus on older individuals. For all ages combined, it reported a 4.3-fold reduced risk of death from metastatic cancer compared with controls33. However, an earlier study reported 1.95-fold higher cancer incidence among males with ASD based on a Taiwanese cancer registry; the elevated cancer risk was particularly high (3.58-fold) for individuals with ASD aged 15-19 years34. Additionally, higher cancer mortality (OR=1.80) among individuals with ASD was reported in a study on premature mortality35. Yet, the latter study did not include breakdown of death by age. Hence the cancer risk among individuals with ASD remains controversial. Epidemiologic studies with larger cohorts are required to assess the cancer risk among children with ASD compared with NT children.
Dysregulated ASD genes and immunity
Among the common phenotypic features observed in ASD is innate immune system dysregulation, leading to a chronic pro-inflammatory state6,36. The innate immune pathways affected in ASD include signaling mediated via cytokines, hepatocyte growth factor receptor, microglia, and the complement system. These suggest a role for aberrant immune function in the broad ASD phenotypes37. A recent RNA-seq study of whole blood from adults with ASD found dysregulated transcription of genes implicated in innate and adaptive immunity. These included upregulated expression of BATF2 and FCGR1A17, as confirmed in our current study of whole blood from children with ASD. The consequences to the immune system, of dysregulation in children with ASD of the four genes observed in our study (Fig. 1), is discussed in the above section. Notably, BATF2 was shown to promote inflammation in response to lipopolysaccharides or infection38, while ISG15 is known to promote anti-inflammatory pathways39,40. Thus, the upregulation of BATF2 mRNA, as well as the downregulation of ISG15 mRNA observed in our study supports the involvement of the pro-inflammatory phenotypes that have often been observed in ASD6,7,36.
Lack of validation of the genes that were dysregulated in whole blood RNA-seq, in PBMC samples
Our real-time PCR experiments did not validate any of the 10 dysregulated genes detected by our RNA-seq of whole blood (Israeli cohort; Table 1) in RNA extracted from PBMCs of a second cohort of children with ASD and NT controls (U.S. cohort; Table 1). Nonetheless, comparing children with ASD to their NT siblings indicated upregulated SERPING1 in the PBMCs of those with ASD (Supplementary Table S2). The real-time PCR findings from the PBMC samples, as presented in Supplementary Fig. S2 and compared with Fig. 1, suggest that the source of the other dysregulated transcripts detected in our RNA-seq of whole blood RNA (Table 2) mostly represent neutrophil RNA. This is because these cells (which represent the major source of blood-derived RNA) are depleted during isolation of PBMCs from whole blood (neutrophils have higher density than PBMCs, and are removed during PBMC separation, together with erythrocytes). This conclusion is rational considering that neutrophils are the key players in inflammation41,42, and that individuals with ASD often display pro-inflammatory phenotypes6,7,36. Indeed, inflammatory signaling and reactive oxygen species mediators were shown to be upregulated in neutrophils of children with ASD43.
Correlations of whole blood gene expression levels with serum endocannabinoids
Recent years have seen growing interest in studying the use of cannabinoid drugs for treating behavioral and social deficits of individuals with ASD44. The endocannabinoid system was reported to be dysregulated in various animal models of ASD45. Children with ASD were reported to have lower serum endocannabinoids16, and rare mutations in endocannabinoid pathway genes were implicated in some persons with ASD46. Circulating endocannabinoids are derived from multiple tissues47. However, plasma endocannabinoid levels were demonstrated to reflect brain concentrations48. Hence, the correlations reported here for blood expression of some of the dysregulated genes with serum endocannabinoids (Fig. 4) can shed light on the pathophysiology of ASD. However, studies in animal models of ASD, which allow measurements of endocannabinoid levels and transcriptomics in brain tissues during different stages of pre- and postnatal development49,50, are required for exploring these correlations. The endocannabinoid PEA was reported to display anti-inflammatory13,51, antiepileptic52, and antineuropathic53 properties. The LY6E cell surface protein was shown to be upregulated by several inflammatory cytokines, including interferons, TNF-alpha, and IL-1 alpha54. As expected, we found a strong negative correlation between the anti-inflammatory endocannabinoid PEA and the pro-inflammatory transcript LY6E in NT children (Supplementary Table S3, r=-0.73, p=0.0004). This expected negative correlation was not observed in children with ASD, thus demonstrating another example of dysregulated immunity in ASD. Further studies are needed to clarify the relation of the dysregulated blood transcriptomics to the reduced serum endocannabinoids observed in children with ASD16.
Our current study has several limitations. First, the small cohorts do not enable separate analysis for males and females. As all the participants in the RNA-seq discovery cohort and the majority of the participants in the entire Israeli cohort were male, the relevance of our findings to female children with ASD needs to be clarified. Second, the whole blood samples (Israeli cohort) and PBMC samples (U.S. cohort) were collected separately, prior to conducting the RNA-seq project; hence, we did not have both whole blood and freshly separated PBMCs from the same individuals. Since the RNA-seq project, its analysis, and real-time PCR validation experiments were conducted from October 2020 to September 2021, recruitment of the children was affected by the Covid-19 pandemic and related lockdowns. Additionally, the children with ASD in the U.S. cohort were on average four years younger than those of the Israeli cohort (Table 1). Hence, our findings on lack of validation for the whole blood dysregulated genes in PBMC samples from children with ASD require confirmation with a study that compares whole blood and PBMC derived RNAs from the same participants. Lastly, the relevance of our transcriptomic findings for early ASD diagnosis is uncertain, as only a few children in both the Israeli and the U.S. cohorts were under age 4 years, the most crucial period for early ASD diagnosis10,11.
In light of the above mentioned reservations, we conclude that validation in larger cohorts, which will ideally include blood samples from younger children, both males and females, are essential for assessing the potential diagnostic and prognostic values of the dysregulated genes detected in our current study. The implications for the upregulated blood transcription of BATF2 and FCGR1A, and downregulated transcription of ISG15 and MT2A, for the distinctive immune system phenotypes in autism, as well as the controversial published findings on lower cancer risk among children with ASD, merit further studies.