Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2 (RNA-Seq)

The ch12q13 obesity locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via an influence on cis-regulation within the genomic region. We implicated rs7132908 as a putative causal variant at this locus leveraging a combination of our inhouse 3D genomic data, public domain datasets, and several computational approaches. Using a luciferase reporter assay in human primary astrocytes, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. Motivated by this finding, we went on to generate isogenic human embryonic stem cell lines homozygous for either rs7132908 allele with CRISPR-Cas9 homology-directed repair to assess changes in gene expression due to genotype and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. We observed that the rs7132908 obesity risk allele influenced the expression of FAIM2 along with other genes, decreased the proportion of neurons produced during differentiation, up-regulated cell death gene sets, and conversely down-regulated neuron differentiation gene sets. We have therefore functionally validated rs7132908 as a causal obesity variant which temporally regulates nearby effector genes at the ch12q13 locus and influences neurodevelopment and survival. Overall design: First, to identify putative cis-regulatory elements harboring childhood obesity GWAS variants and implicate their effector genes, we performed RNA-seq using a human primary astrocyte cell line. During downstream analyses, this data was used in combination with ATAC-seq and Hi-C in the same cell type, following our established 'variant-to-gene' mapping approach. Then, to investigate the cis-regulatory effect of rs7132908 genotype on gene expression, we established H9 ESC lines homozygous for the rs7132908 obesity risk A allele with CRISPR-Cas9 homology-directed repair while the parent line is homozygous for the non-risk G allele. We performed RNA-seq on these ESCs as well as hypothalamic neural prgenitors derived from these ESCs after 14 days of differentiation. We identified genes significantly differentially expressed due to rs7132908 in each cell type. We also obtained primary human pediatric hypothalamus tissue from 4 donors from the NIH NeuroBiobank to serve as a reference to validate the identity of our ESC-derived hypothalamic neural progenitors. We compared average TPMs from each homozygous rs7132908 non-risk G allele hypothalamic neural progenitor sample to average TPMs from each donor homozygous for the rs7132908 non-risk G allele.