Identification and Analysis of ZIC-Related Genes in Cerebellum of Autism Spectrum Disorders

Abstract

Objective: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with significant genetic heterogeneity. The ZIC gene family can regulate neurodevelopment, especially in the cerebellum, and has been implicated in ASD-like behaviors in mice. We performed bioinformatic analysis to identify the ZIC gene family in the ASD cerebellum.

Methods: We explored the roles of ZIC family genes in ASD by investigating (i) the association of ZIC genes with ASD risk genes from the Simons Foundation Autism Research Initiative (SFARI) database and ZIC genes in the brain regions of the Human Protein Atlas (HPA) database; (ii) co-expressed gene networks of genes positively and negatively correlated with ZIC1, ZIC2, and ZIC3, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and receiver operating characteristic (ROC) curve analysis of genes in these networks; and (iii) the relationship between ZIC1, ZIC2, ZIC3, and their related genes with cerebellar immune cells and stromal cells in ASD patients.

Results: (i) ZIC1, ZIC2, and ZIC3 were associated with neurodevelopmental disorders and risk genes related to ASD in the human cerebellum and (ii) ZIC1, ZIC2, and ZIC3 were highly expressed in the cerebellum, which may play a pathogenic role by affecting neuronal development and the cerebellar internal environment in patients with ASD, including immune cells, astrocytes, and endothelial cells. (iii) OLFM3, SLC27A4, GRB2, TMED1, NR2F1, and STRBP are closely related to ZIC1, ZIC2, and ZIC3 in ASD cerebellum and have good diagnostic accuracy. (iv) ASD mice in the maternal immune activation model demonstrated that Zic3 and Nr2f1 levels were decreased in the immune-activated cerebellum.

Conclusion: Our study supports the role of ZIC1, ZIC2, and ZIC3 in ASD pathogenesis and provides potential targets for early and accurate prediction of ASD.

Keywords: ZIC1; ZIC2; ZIC3; autism; biomarkers; cerebellum.

Figure 1

ZIC1, ZIC2 and ZIC3 are associated with ASD and highly expressed in the cerebellum. (A) Network of human ASD risk gene network associated with ZIC family genes in the cerebellum. (B) Network of human ASD risk genes associated with ZIC1, ZIC2 and ZIC3 in the cerebellum from the gene-disease association database in NetworkAnalyst. (C) The expression of ZIC1, ZIC2 and ZIC3 in the brain regions of the GTEx human brain RNA-Seq dataset in HPA.

Figure 2

Networks of genes positively and negatively associated with ZIC1, ZIC2 and ZIC3. The first up-regulated and the down-regulated network of genes associated with ZIC1 (A), ZIC2 (B) and ZIC3 (C) in the cerebellum of ASD patients.

Figure 3

Evaluation of ZIC1, ZIC2 and ZIC3 associated genes in cerebellum as biomarkers for ASD. (A) Venn diagrams of the genes with AUC > 0.7 and genes positively or negatively associate with ZIC1 (A), ZIC2 (B) and ZIC3 (C). ROC analysis for OLFM3 and SLC27A4 from ZIC1 networks (D), GRB2 and TMED1 from ZIC2 networks (E), NR2F1 and STRBP from ZIC3 networks (F).

Figure 4

Validation of ZIC1, ZIC2 and ZIC3 associated genes in cerebellum, NPC and neurons of ASD patients. ROC analysis of OLFM3, SLC27A4, GRB2, TMED1, NR2F1 and STRBP in cerebellum of ASD patients from GSE38322 (A), and iPSC derived NPC (B) and neurons (C) of ASD patients from GSE142670.

Figure 5

ZIC1, ZIC2 and ZIC3 associated genes are related to immune cells in cerebellum of ASD. Pearson’s correlation coefficient was utilized to analyze the correlations between genes and xCell scores from the datasets GSE28521(A) and GSE38322(B) using the R package PerformanceAnalytics. (*P<0.05, **P<0.01, ***P<0.001).

Figure 6

Decreased expression of Zic3 and Nr2f1 in the cerebellum of ASD mice of MIA model. qPCR for Zic1, Zic2, Zic3 (A) and Zic3 associated genes, Nr2f1, Strbp (B), in the cerebellum of ASD mice of MIA model (n = 3, *p < 0.05, **p < 0.01). Representative data from three independent experiments were shown.