
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.22.609230
preprint
1
Article
Variant-to-function mapping of late-onset Alzheimer’s disease GWAS signals in human microglial cell models implicates RTFDC1 at the CASS4 locus
Burton Elizabeth A. http://orcid.org/0000-0002-8376-9282

Argenziano Mariana
Cook Kieona
Ridler Molly
Lu Sumei
Su Chun
Manduchi Elisabetta
Littleton Sheridan H. http://orcid.org/0000-0002-4322-4069

Leonard Michelle E.
Hodge Kenyaita M.
Wang Li-San http://orcid.org/0000-0002-3684-0031

Schellenberg Gerard D. http://orcid.org/0000-0003-1115-2475

Johnson Matthew E.
Pahl Matthew C.
Pippin James A.
Wells Andrew D.
Anderson Stewart A.
Brown Christopher D.
Grant Struan F.A. http://orcid.org/0000-0003-2025-5302

Chesi Alessandra http://orcid.org/0000-0002-0954-7446

22 8 2024
2024.08.22.609230https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.22.609230
nihpp-2024.08.22.609230.pdf
ABSTRACT

Late-onset Alzheimer’s disease (LOAD) research has principally focused on neurons over the years due to their known role in the production of amyloid beta plaques and neurofibrillary tangles. In contrast, recent genomic studies of LOAD have implicated microglia as culprits of the prolonged inflammation exacerbating the neurodegeneration observed in patient brains. Indeed, recent LOAD genome-wide association studies (GWAS) have reported multiple loci near genes related to microglial function, including TREM2 , ABI3 , and CR1 . However, GWAS alone cannot pinpoint underlying causal variants or effector genes at such loci, as most signals reside in non-coding regions of the genome and could presumably confer their influence frequently via long-range regulatory interactions. We elected to carry out a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models (iPSC-derived microglia and HMC3) in order to physically map interactions between LOAD GWAS-implicated candidate causal variants and their corresponding putative effector genes. Notably, we observed consistent evidence that rs6024870 at the GWAS CASS4 locus contacted the promoter of nearby gene, RTFDC1 . We subsequently observed a directionallly consistent decrease in RTFDC1 expression with the the protective minor A allele of rs6024870 via both luciferase assays in HMC3 cells and expression studies in primary human microglia. Through CRISPR-Cas9-mediated deletion of the putative regulatory region harboring rs6024870 in HMC3 cells, we observed increased pro-inflammatory cytokine secretion and decreased DNA double strand break repair related, at least in part, to RTFDC1 expression levels. Our variant-to-function approach therefore reveals that the rs6024870-harboring regulatory element at the LOAD ‘ CASS4’ GWAS locus influences both microglial inflammatory capacity and DNA damage resolution, along with cumulative evidence implicating RTFDC1 as a novel candidate effector gene.
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