Background: Genome-wide association studies have discovered blocks of common variants—likely transcriptional-regulatory—associated with major depressive disorder (MDD), though the functional subset and their biological impacts remain unknown. Likewise, why depression occurs in females more frequently than males is unclear. We therefore tested the hypothesis that risk-associated functional variants interact with sex and produce greater impact in female brains. Methods: We developed techniques to directly measure regulatory variant activity and sex interactions using massively parallel reporter assays in the mouse brain in vivo, in a cell type–specific manner, and applied these approaches to measure activity of >1000 variants from >30 MDD loci. Results: We identified extensive sex-by-allele effects in mature hippocampal neurons, suggesting that sex-differentiated impacts of genetic risk may underlie sex bias in disease. Unbiased informatics approaches indicated that functional MDD variants recurrently disrupt a number of transcription factor binding motifs, including those of sex hormone receptors. We confirmed a role for the latter by performing massively parallel reporter assays in neonatal mice on the day of birth (during a sex-differentiating hormone surge) and hormonally quiescent juveniles. Conclusions: Our study provides novel insights into the influence of age, biological sex, and cell type on regulatory variant function and provides a framework for in vivo parallel assays to functionally define interactions between organismal variables such as sex and regulatory variation. Moreover, we experimentally demonstrate that a portion of the sex differences seen in MDD occurrence may be a product of sex-differentiated effects at associated regulatory variants.

Original languageEnglish
Pages (from-to)466-478
Number of pages13
JournalBiological Psychiatry
Issue number6
StatePublished - Sep 15 2023


  • Hippocampus
  • Major depressive disorder
  • Noncoding variation
  • Reporter assay
  • Sex differences
  • Transcription factors


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