Chia-Yi Lee, Ashvin Ravi, Tzu-Chieh Huang, Briana Wyman, Ziyu Liu et al.
Genome-wide association studies (GWAS) for Alzheimer’s disease (AD) predominantly implicate non-coding genetic variants that are presumed to regulate gene expression. Yet, around half of GWAS variants do not coincide with known regulatory variants, such as expression quantitative trait loci (eQTLs). AD GWAS loci are enriched for microglia-specific regulatory regions, but the causal variants and mechanisms remain unresolved. To connect risk variants to microglial function, we evaluated the regulatory activity of AD GWAS variants, microglial eQTLs, and chromatin accessibility QTLs (caQTLs) by screening 11,550 candidate regulatory sequences (CRSs) with a massively parallel reporter assay (MPRA). Whether prioritized by GWAS or eQTLs, variants are equally likely to predict active CRS and expression-modulating variants (emVars) by MPRA, yet there were clear systematic differences, particularly in that predicted target genes of GWAS loci, but not eQTLs, are enriched for AD relevant biology. Notably, only 7% of all emVars were shared between two distinct sources of human microglia cells, human induced pluripotent stem cell (hiPSC)-derived microglia (iMGLs) and immortalized HMC3 cells, underscoring the importance of cellular context in evaluating regulatory activity. Our findings establish the first large-scale microglial-context MPRA, define a microglial regulome underlying AD genetic risk, and provide a functional framework to refine noncoding variant interpretation.
Chia-Yi Lee, Ashvin Ravi, Tzu-Chieh Huang, Briana Wyman, Ziyu Liu, Ariana Chriss, Jack Humphrey, David A. Knowles, Chris Cotsapas, Laura M. Huckins, Towfique Raj, Kristen J. Brennand. Regulatory landscape of Alzheimer’s disease variants in human microglia. 2025-12-20. https://doi.org/10.1101/2025.10.13.25337902
A pooled second-generation lentiviral MPRA tested both alleles of candidate regulatory variants nominated from Alzheimer’s disease GWAS fine-mapping, microglial eQTLs, and caQTLs in the immortalized human HMC3 microglial cell line. Reporter DNA and RNA barcodes were collected after 72 hours and analyzed for allelic activity.
A pooled second-generation lentiviral MPRA tested both alleles of candidate regulatory variants nominated from Alzheimer’s disease GWAS fine-mapping, microglial eQTLs, and caQTLs in mature hiPSC-derived microglia-like cells (iMGLs). Reporter DNA and RNA barcodes were collected after 72 hours and analyzed for allelic activity.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 7 files (ZIP)HMC3_MPRA_AllelicActivity.tsv.gziMGL_MPRA_AllelicActivity.tsv.gziMGL_MPRA_consensus_table.tsvMPRA_library.Rmdpaper_v2.pdfrepository_README.mdSupplementary_Tables_AD_MPRA.xlsx