Research

Plots of ASD prevalence by sex and diagram illustrating effect of sex-differential biology on impact of ASD risk factors
Males are diagnosed with ASD more frequently than females in the US (A), a pattern that is consistent across ASD prevalence studies (B). (C) Hypothesis: Sex-differential biology modulates the impact of genetic and other risk factors such that one sex is sensitized and/or the other is protected from risk
Source: (A) adapted from CDC ADDM Network, 2018, MMWR, data in (B) from Fombonne 2009, Ped Res.

Autism Spectrum Disorder (ASD) is currently diagnosed 3-5 times more frequently in males than females. This skew is one of the most consistent, yet mechanistically unexplained, features of ASD. Many other neurodevelopmental and neuropsychiatric conditions such as ADHD, Tourette Syndrome, schizophrenia, and major depression, also show striking sex differences in their prevalence or presentation that are similarly unaccounted for.

In contrast, evidence from human genetics research is clear: genetic variants, including common, inherited variants of individual small effect and rare, de novo variants of large effect, are significantly associated with risk for neuropsychiatric and developmental disorders. Many genetic loci and individual genes involved in this risk have been identified in recent years, and many more will be discovered in the years to come as sample sizes grow. But, a statistical link between a gene to a trait is only a first step toward understanding disease mechanisms, and much work is needed to understand what these genes do and what happens when they are disrupted.

Our goals:

The Werling Lab is interested in investigating the key neurobiological mechanisms involved in the etiology of ASD and other neuropsychiatric disorders, including the dimensions of genetic variation, development, and sex-differential biology, as well as the interactions between them. The long-term goal of our research program is to uncover fundamental causal pathways in both sexes that will facilitate treatment development and benefit affected individuals and their families.

Approach:

Genome-scale analyses are especially powerful for unbiased discovery, and so we apply genome-wide genetics, functional genomics, and bioinformatics approaches (e.g. RNA-seq, single cell analyses, eQTLs) in human tissue and model systems to identify and characterize the mechanisms involved in sex-differential and disorder-associated neurobiology.

Research areas

Diagram illustrating approach for characterizing sex differences in human brain
Characterizing sex differences in transcriptomic patterns in human brain tissue across development and their intersections with known disease biology may identify processes most likely involved in sex-differential risk mechanisms.

Sex differences in human brain tissue across development

How do male and female human brains differ from one another, and could these differences influence disease risk? How do sex differences in brain biology change across development and the human lifespan? Transcriptomics from human brain tissue can give us a molecular-level readout of the active biological processes in brain tissue from particular regions of the brain and at different stages of development. Comparing male and female gene expression at a genomic scale can identify sets of genes, and their associated functions, that differ by sex. We can then investigate how these sex differences intersect with genes implicated in disease risk or pathology.

Scatterplot of gene expression differences by sex and ASD status
Genes associated with astrocyte function are more likely to have higher expression in neurotypical males than females (x-axis), and in ASD than controls (y-axis), while neuronal genes do not show this pattern.
Source: Image adapted from Werling et al. 2016, Nat Commun

Sex differences in human brain cells

Earlier studies comparing gene expression in male and female brain tissue show greater expression of genes associated with microglia, astrocytes, and immune-related functions in males compared with females (Werling et al. 2016, Nat Commun). A similar set of genes are elevated in the autistic brain regardless of sex (Parikshak, Swarup, Belgard et al. 2016, Nature), suggesting that these glial cell types could be involved in ASD’s sex-differential risk. However, from tissue-level expression, we cannot distinguish male-female differences in relative cell number, function, or identity. Single-cell technologies allow us to investigate these possibilities to determine if, and how, specific neural cell types contribute to sex-differential risk.

Scatterplot of results from category-wide association study in ASD families
Category-wide association study of de novo variants across the genome in 519 ASD cases and sibling controls demonstrates the importance of multiple testing correction in genome-scale association analyses.
Source: Werling, Brand, An, Stone, Zhu et al. 2018, Nat Genet

Genetic risk for neuropsychiatric disorders

Identifying genetic loci that are robustly associated with neuropsychiatric conditions requires genome-wide analysis with stringent statistical significance thresholds to account for multiple testing. Genome- and exome-wide association studies have been incredibly fruitful for identifying disease-associated loci and genes in recent years. We are interested in continued gene and locus discovery work for ASD and other neuropsychiatric disorders, including focused efforts to find genetic risk loci on the sex chromosomes. Then, as gene discovery continues, follow up work is needed to understand the individual and collective roles of the implicated genes in disease etiology. Do different risk genes affect a set of common biological pathways? How are the downstream effects of mutations in risk genes similar to each other, and how do they differ? Does sex-differential biology interact with all risk genes in a similar way?

Diagram illustrating approach for characterizing sex differences in rodent brain
Characterizing sex differences in transcriptomic patterns in rodent brain tissue and cells will set the stage for using the rodent model to investigate mechanisms of sex-differential risk and protection.

Model systems: Sex-differential and ASD-associated biology

The experiments required to determine how, at a mechanistic level, sex-differential processes or cell types shape brain development, function, and risk for disease will require a model system. In vivo rodent models can be used to generate construct-valid models of human genetic risk, produce multiple neural cell types, including glia, release endogenous sex steroid hormones, and follow a developmental time course analogous to humans. However, it is currently not clear how well rodents recapitulate human sex differences in gene expression and/or cell types. So, we are working to characterize sex differences in rodent brain tissue and cells in order to explore the validity of the rodent model system for studying the mechanisms behind sex-differential disease risk. We can also use rodent models of ASD-associated risk loci to explore how sex interacts with genetic risk pathways within the same organism, as well as to characterize phenotypic similarities and differences between different loci.