Title: Sex-differentially expressed genes in prenatal human cortex intersect with genes dysregulated in autism cortex
Legend: Sex-differential patterns of gene expression in the prenatal cortex. Plots of sex-differential expression effects in two, independent data sets (BrainVar and UCLA) for the top 200 sex-differentially expressed genes (sex-DEGs) by p-value identified from a meta-analysis of these data sets (A) and for all X chromosome genes, excluding the XIST transcript (B). Plotted points come from differential expression models that include all surrogate variables estimated by the num.sv() function in svaseq, plus age (days), batch, RIN, PMI, intermediate progenitor cell proportion (Model #1). Correlation coefficients and p-values from correlation between log2(fold change) for the displayed gene set between the two data sets are shown. Model #2 includes the same covariates as Model #1, but uses a reduced set of surrogate variables selected by sex separation on the first principal component of expression (BrainVar: 8; UCLA: 11 SVs). Inset in (A) displays the top 200 sex-DEGs, including Y chromosome genes and XIST. (C) Heatmap of gene set enrichment results (Fisher’s exact test) for sets of sex-DEGs with male- and female-biased expression and false discovery rate ≤ 0.1 or within the top 200 sex-DEGs by p-value. Top: Sex-DEGs from Model #1. Bottom: Sex-DEGs from Model #2. Sex-DEGs are tested for enrichment for neural cell type-associated genes, neuropsychiatric disorder risk genes from genetic studies, and gene co-expression modules with evidence of altered expression in the autistic brain. AD, Alzheimer’s disease; ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; ASDG, modules from Gandal et al, Nature, 2022; ASDP, modules from Parikshak et al, Nature, 2016; DD, developmental delay; EA, educational attainment; EPDD, epilepsy with developmental delay; FC, fold change; F, female; M, male; MDD, major depressive disorder; MS, multiple sclerosis; PD, Parkinson’s disease; SCZ, schizophrenia.
Citation: Kissel LT, Pochareddy S, An J-Y, Roeder K, Sestan N, Sanders SJ, Werling DM. Sex-differentially expressed genes in the developing and ASD cortex implicate glial function in sex-differential ASD risk. INSAR 2023 Annual Meeting; May 3-6, 2023; Stockholm, Sweden.
Abstract: Background: Autism spectrum disorder (ASD) has a consistent 4:1 male prevalence, suggesting a role for sex-differential biology in risk. Defining molecular differences between males and females in the brain will advance understanding of how sex impacts neurodevelopment and disorder risk. Previous studies of human adult cortex show subtle male-skewed expression of glial-associated genes that are coordinately elevated in the ASD brain, but it is unknown whether this sex-differential pattern emerges in early cortical development or is preserved in ASD brain.Objectives: We sought to quantify sex-differential gene expression in two under-studied biological contexts relevant to ASD: neurotypical prenatal development, and in ASD cases. By comparing sex-differentially expressed genes (sex-DEGs) with ASD risk genes and affected molecular pathways, we aimed to identify putative mechanisms involved in sex-differential ASD risk. Methods: We analyzed sex-differential expression and co-expression patterns in prenatal dorsolateral prefrontal cortex (dlPFC) from BrainVar (14-21 post-conception weeks, PCW; 39 male, 46 female) and in cortical tissue from an independent, published data set generated at UCLA (14-21 PCW; 101 male, 85 female). We tested sex-DEGs for enrichment of risk genes for ASD and other diagnoses, ASD-associated expression changes, and cell type marker genes. To identify sex differences preserved or altered in ASD, we also quantified sex-differential expression in published RNA-seq data from frontal cortex of ASD (34 male, 9 female) and control individuals (37 male, 8 female). Results: In BrainVar, we observe 69 significant sex-DEGs at FDR≤0.1 (30 X chr, 19 Y chr, 20 autosomal) and an extended set of 227 sex-DEGs at p≤0.05 and fold change >1.2. ASD risk genes from exome sequencing are not enriched among either set of sex-DEGs, but the extended set of male-skewed genes are enriched for ASD-upregulated, microglial- and immune-associated CTX.M19 (OR=6.75, p=0.01; Fisher’s exact test) and CD11 (OR=5.5, p=0.008) modules. Three co-expression modules evident in both sexes are enriched for both ASD risk genes and either vascular or neuronal cell markers, while sex-specific modules are not enriched for ASD risk genes. Similar to BrainVar, sex-DEGs from the UCLA data set do not overlap with ASD risk genes, while male-biased DEGs show trending enrichment for the CTX.M19 module (OR=3.86, unadjusted p=0.05), implicating elevated expression of CTX.M19 genes in both ASD pathology and male-typical cortical neurodevelopment. In the ASD brain, we find 38 sex-DEGs at FDR≤0.1 (13 X chr, 23 Y chr, 2 autosomal) and 50 sex-DEGs (21 X chr, 23 Y, 6 autosomal) in controls. Among extended sets of sex-DEGs, we observe greater male-biased expression in ASD (ASD: 239 male-biased DEGs, controls: 89), and greater female bias in controls (controls: 217 female-biased DEGs, ASD: 154). ASD-specific, male-biased DEGs are enriched for astrocyte markers (OR=23.39, p=1.54E-32) and ASD-upregulated, astrocyte-associated CTX.M9 (OR=8.20, p=2.36E-24) and CD4 (OR=13.42, p=7.66E-44) modules. Conclusions: Our analyses confirm fetal male-biased expression of ASD-upregulated glial- and immune-associated genes in two large, independent data sets, and we observe male-biased expression of astrocyte-associated genes specific to ASD cases. Collectively, these results are consistent with a role for glial and/or immune function in sex-differential ASD risk mechanisms.
About the Lab: Donna Werling is interested in characterizing sex-differential risk mechanisms in autism spectrum disorder (ASD). During her doctoral work in the laboratory of Dan Geschwind at the University of California, Los Angeles, Werling used functional genomics, human genetics and bioinformatics approaches to understand the relationship between sex and genetic risk in ASD. Visit The Werling Lab for more information.
Investigator: Donna Werling, PhD