Expression Changes in Epigenetic Gene Pathways Associated With One-Carbon Nutritional Metabolites in Maternal Blood From Pregnancies Resulting in Autism and Non-Typical Neurodevelopment

Abstract

The prenatal period is a critical window for the development of autism spectrum disorder (ASD). The relationship between prenatal nutrients and gestational gene expression in mothers of children later diagnosed with ASD or non-typical development (Non-TD) is poorly understood. Maternal blood collected prospectively during pregnancy provides insights into the effects of nutrition, particularly one-carbon metabolites, on gene pathways and neurodevelopment. Genome-wide transcriptomes were measured with microarrays in 300 maternal blood samples in Markers of Autism Risk in Babies-Learning Early Signs. Sixteen different one-carbon metabolites, including folic acid, betaine, 5'-methyltretrahydrofolate (5-MeTHF), and dimethylglycine (DMG) were measured. Differential expression analysis and weighted gene correlation network analysis (WGCNA) were used to compare gene expression between children later diagnosed as typical development (TD), Non-TD and ASD, and to one-carbon metabolites. Using differential gene expression analysis, six transcripts (TGR-AS1, SQSTM1, HLA-C, and RFESD) were associated with child outcomes (ASD, Non-TD, and TD) with genome-wide significance. Genes nominally differentially expressed between ASD and TD significantly overlapped with seven high confidence ASD genes. WGCNA identified co-expressed gene modules significantly correlated with 5-MeTHF, folic acid, DMG, and betaine. A module enriched in DNA methylation functions showed a suggestive protective association with folic acid/5-MeTHF concentrations and ASD risk. Maternal plasma betaine and DMG concentrations were associated with a block of co-expressed genes enriched for adaptive immune, histone modification, and RNA processing functions. These results suggest that the prenatal maternal blood transcriptome is a sensitive indicator of gestational one-carbon metabolite status and changes relevant to children's later neurodevelopmental outcomes. LAY SUMMARY: Pregnancy is a time when maternal nutrition could interact with genetic risk for autism spectrum disorder. Blood samples collected during pregnancy from mothers who had a prior child with autism were examined for gene expression and nutrient metabolites, then compared to the diagnosis of the child at age three. Expression differences in gene pathways related to the immune system and gene regulation were observed for pregnancies of children with autism and non-typical neurodevelopment and were associated with maternal nutrients.

Keywords: autism spectrum disorder; maternal blood; neurodevelopment; nutrition; one-carbon metabolites; prenatal; transcriptome.

Figure 1

Identification and function of ASD associated and Non‐TD associated differentially expressed genes in maternal peripheral blood. Differential expression analysis was performed in maternal peripheral blood transcriptomes (n = 300) after adjustment for surrogate variables. (A) Identification of 1,912 differentially expressed genes (2,012 transcripts, P‐value <0.05) compared between children diagnosed as ASD (n = 67) and TD (n = 154). (B) Identification of 1,919 differential expressed genes (1,987 transcripts, P‐value <0.05) compared between children diagnosed as Non‐TD (n = 79) and TD (n = 154). Two transcripts located at RFESD and TRG‐AS1 were genome‐wide significant in the Non‐TD to TD comparison (Table S2). (C) Venn diagram represents the overlap in differentially expressed transcripts (unadjusted P <0.05) identified in ASD to TD versus Non‐TD to TD comparisons, which was greater than expected by random using a Fisher's exact test (P‐value <0.001***). (D) Gene ontology (GO) and pathway analysis was performed on the 218 transcripts differentially expressed in both ASD‐TD and Non‐TD‐TD comparisons, with significant enrichments (Fisher's exact test, FDR P‐value <0.05). In contrast, the differentially expressed transcripts uniquely associating with either ASD or Non‐TD were not significantly enriched for any GO terms.

Figure 2

Co‐expression network modules with demographic factors and maternal peripheral blood one‐carbon metabolites. (A) Heatmap of Z‐scores of modules eigengenes with sample covariates with 27 co‐expression network modules on all 300 maternal blood samples. Each row represents a different module eigengene and each column is the associated trait, which include child clinical outcome, demographic factors, and maternal blood metabolite concentrations. The matrix was calculated by Pearson correlation and P‐values adjusted for the total number of comparisons. Color represents the direction (red, positive correlation; blue, negative correlation) and intensity reflects the significance. (^ unadjusted P‐value <0.05 and FDR adjusted P‐value >0.05; * FDR adjusted P‐value <0.05). (B) Number of transcripts and hub genes from all 27 co‐expressed modules are listed.

Figure 3

“Greenyellow” module was positively associated with diagnosis and negatively associated with folic acid and 5‐MeTHF. (A) “Greenyellow” module eigengene was significantly associated with child diagnosis (one‐way ANOVA, unadjusted P‐value <0.05). Greenyellow eigengene values were higher in maternal blood from ASD pregnancies than TD or Non‐TD pregnancies. (B) “Greenyellow” module eigengene level was significantly negatively associated with 5‐MeTHF concentrations in maternal blood (ANOVA, P‐value <0.05). (C) Bar graph shows gene ontology (GO) and pathway significant enrichments from the 224 transcripts in “greenyellow” module (Table S8). (D) Transcripts (16857547, 16867905, and 16867910) from MBD3L3‐5 genes encoding proteins involved in methylation‐CpG binding functions were significantly negatively associated with “greenyellow” module eigengene.

Figure 4

Eight weighted gene co‐expression modules associated with maternal betaine and DMG concentrations were strongly clustered. (A) Unsupervised hierarchical clustering dendrogram was performed with module eigengenes, betaine and DMG. The height of each node represents the intergroup dissimilarity. Similar nodes clustered together under one branch. (B) Unsupervised hierarchical clustering adjacency heatmap, with color and intensity representing the degree of correlation (dark, high; light, low correlation). Black box indicates the block of eight weighted gene co‐expression modules associated with betaine and DMG concentrations.

Figure 5

Imputed cell type proportions in material peripheral blood associated with demographic factors and maternal nutrients. (A) Barplot of each cell type mean estimated proportion separated by children diagnosis outcomes using peripheral blood reference panel in CIBERSORT. (B) Heatmap of correlation between sample demographic factors and maternal nutrients with cell type proportions. Each row represents a cell type proportion and columns represent traits, including child diagnostic outcome, demographic factors, and maternal blood nutrient concentrations. P‐values adjusted for the total number of comparisons. Color represents the direction (red, positive correlation; blue, negative correlation) and intensity reflects the significance, *P‐value <0.05 after FDR correction.