Variant-to-gene mapping in human microglial cell models with CRISPR validation implicates RTFDC1 at the Alzheimer's disease 'CASS4' locus.

Abstract

Alzheimer's disease (AD) research has principally focused on neurons due to their role in neurodegeneration. In contrast, recent studies suggest that genetic mechanisms drive microglia to prolonged inflammation in AD brains, exacerbating neurodegeneration. Indeed, recent genome-wide association studies (GWAS) of AD have identified multiple loci near genes related to microglial function, such as TREM2 and CR1. However, GWAS does not have the sensitivity to identify causal variants or effector genes. We used a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models to map interactions between GWAS-implicated variants and their putative effector genes. We then validated an observed interaction at the 'CASS4' locus using CRISPR-Cas9 genome editing. Improving on the relatively low resolution of available Hi-C data, we employed high resolution Capture-C to characterize the physical genome-wide interactions of all human promoters. We performed this in the human microglial cell line HMC3 and human iPSC-derived microglia (iMg). We confirmed the enhancer activity of a SNP we elected to pursue and its associated regulatory element using dual-luciferase assays. To confirm the regulatory interaction with the Capture-C implicated effector gene, we used lentiviral CRISPR-Cas9 to delete an approximately 300bp region containing the SNP in HMC3 cells. We then confirmed changes in RNA and protein expression using RNA-seq and Western blotting, respectively. Variant-to-gene mapping in both microglial cell models identified 67 putative effector genes (51 coding) across both cell types, with 14 observed in both models. We identified a novel proxy SNP, rs6024870 (r2 = 0.93 to sentinel SNP rs6014724), at the 'CASS4' locus, which coincided with open chromatin and directly contacted the promoter of RTFDC1, a gene not previously implicated in AD. Deletion of the putative enhancer region harboring rs6024870 by CRISPR-Cas9 in HMC3 reduced the expression of RTFDC1 at both the mRNA and protein level. We also note that CASS4 levels were modestly influenced by this CRISPR-mediated perturbation. We implicate RTFDC1 as a novel effector gene at the AD 'CASS4' GWAS locus. Further efforts will characterize the phenotypic effect of this variant in microglial cell models, including on inflammation and phagocytic activity.