Genes influenced by MEF2C contribute to neurodevelopmental disease via gene expression changes that affect multiple types of cortical excitatory neurons

Myocyte enhancer factor 2 C (MEF2C) is an important transcription factor during neurodevelopment. Mutation or deletion of MEF2C causes intellectual disability (ID) and common variants within MEF2C are associated with cognitive function and schizophrenia risk. We investigated if genes influenced by MEF2C during neurodevelopment are enriched for genes associated with neurodevelopmental phenotypes, and if this can be leveraged to identify biological mechanisms and individual brain cell types affected. We used a set of 1,052 genes that were differentially expressed in the adult mouse brain following early embryonic deletion of Mef2c in excitatory cortical neurons. Using GWAS data, we found these differentially expressed genes (DEGs) to be enriched for genes associated with schizophrenia, intelligence and educational attainment but not autism spectrum disorder (ASD). Using sequencing data from trios studies, we found these DEGs to be enriched for genes containing de novo mutations reported in ASD and ID, but not schizophrenia. Using single cell RNA-seq data, we identified that a number of different excitatory glutamatergic neurons in the cortex were enriched for these DEGs including deep layer pyramidal cells and cells in the retrosplenial cortex, entorhinal cortex and subiculum. These data indicate that genes influenced by MEF2C during neurodevelopment contribute to cognitive function and risk of neurodevelopmental disorders. Within excitatory neurons, common SNPs in these genes contribute to cognition and SZ risk via an effect on synaptic function based on gene ontology analysis. In contrast, rare mutations contribute to earlier onset ASD and ID via an effect on cell morphogenesis. Author Summary Schizophrenia is a complex disorder caused by many genes. Current drugs for schizophrenia are only partially effective and do not treat cognitive deficits, which are key factors for explaining disability. Here we take an individual gene identified in genetic studies of schizophrenia and cognition called MEF2C, which on its own is a very important regulator of brain development. We use data from a mouse study where MEF2C has been stopped from functioning or knocked out during brain development. The effect of that knock out has been measured when the mice reach adulthood, in the form of a set of differentially expressed genes (DEGs) from the somatosensory cortex. We found that this set of DEGs contains more genes than expected by chance that are associated with schizophrenia and cognition or contain rare new (de novo) mutations reported in autism and intellectual disability. Using gene expression data from single brain cells, we identified that a number of specific excitatory glutamatergic neurons in the cortex were enriched for these DEGs. This study provides evidence that the molecular mechanisms that underpin schizophrenia and cognitive function include disruption of cell types influenced by MEF2C.

variants within MEF2C are associated with cognitive function and schizophrenia risk. We investigated 23 if genes influenced by MEF2C during neurodevelopment are enriched for genes associated with 24 neurodevelopmental phenotypes, and if this can be leveraged to identify biological mechanisms and 25 individual brain cell types affected. We used a set of 1,052 genes that were differentially expressed 26 in the adult mouse brain following early embryonic deletion of Mef2c in excitatory cortical neurons.

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Using GWAS data, we found these differentially expressed genes (DEGs) to be enriched for genes 28 associated with schizophrenia, intelligence and educational attainment but not autism spectrum 29 disorder (ASD). Using sequencing data from trios studies, we found these DEGs to be enriched for

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Comparison of association signals at MEF2C in GWAS data 131 We reviewed the largest published GWAS, which report that SNPs at the MEF2C locus are genome-132 wide significant for SZ, IQ and EA but not ASD (S1 Figure A- We used a set of 1,055 DEGs (listed in S1 Table) based on an RNA-seq study that captured the 151 transcriptional changes in adult male mouse brain that result from the early embryonic deletion of 152 Mef2c in cortical and hippocampal excitatory neurons (13). RNA-seq was performed on mRNA 153 isolated from the somatosensory cortex of Mef2c cKO or control littermates (13). These genes that 154 were differentially expressed upon cKO of MEF2C were enriched for genes associated with SZ 155 (P=1.70x10 -06 ), IQ (P =9.88x10 -08 ) and EA (P=2.24x10 -10 ) but not ASD (P=0.247; see Table 1). Brain-156 expressed genes are a major contributor to these phenotypes. It is possible that the enrichment 157 detected here could be due to the MEF2C gene-set representing a set of brain-expressed genes.
158 However, the MEF2C enrichment was robust to the inclusion in the analyses of both 'brain-159 expressed' (n=14,243) and 'brain-elevated' (n=1,424) gene-sets as covariates (S2 Table). To examine 160 if the enrichment we detect for SZ, IQ and EA is a property of polygenic phenotypes in general, we 161 obtained GWAS summary statistics for ten phenotypes and tested the MEF2C gene-set for 162 enrichment in each one. These were a child-onset psychiatric disorders, other brain-related 163 disorders, non-brain related diseases, and height. No enrichment was detected for any of the ten 164 phenotypes (S3 Table).

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By performing enrichment analysis on gene expression data from scRNA-seq of the mouse nervous 211 system, we sought to detect individual cell types that are enriched for genes that are differentially 212 expressed upon cKO of MEF2C. We used two scRNA-seq data-sets that included 265 cell types from 213 the mouse nervous system (28) and 565 cell types from the mouse brain (29). After Bonferroni 214 correcting for the 830 cell types tested, we identified 31 significantly enriched cell types in both 215 data-sets (62 cell types in total with p<6.03x10 -5 ). These results are summarized in Table 4 and full   216  details are provided in S9-10 Tables. 217 Mef2c in the animal model, the majority of cell types affected are glutamatergic excitatory neurons.

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The advantage of these large scRNA-seq resources is that we can identify some of the specific genes that contribute to ASD and ID, based on analysis of rare mutations. Therefore, it is reasonable 318 to conclude that the specific glutamatergic excitatory neurons in the cortex, which we find to be 319 enriched for these MEF2C DEGs, are fundamental to the pathophysiology of neurodevelopmental 320 disorders. orthologue. This resulted in a gene-set of 1,055 genes (465 up-regulated and 590 down-regulated; 334 S1 Table). Sets of 'brain-expressed' genes (n=14,243) and 'brain-elevated' genes (n=1,424) were 335 sourced from the Human Protein Atlas (https://www.proteinatlas.org/humanproteome/brain) and 336 used as covariates in analyses. Brain-elevated genes are those that show an elevated expression in 337 brain compared to other tissue types.