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

The ch12q13 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 cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome wide association studies (GWAS) have identified loci for sleep-related traits, they do not directly uncover the underlying causal variants and corresponding effector genes. The majority of such variants reside in non-coding regions and are therefore presumed to impact cis-regulatory elements. Our previously reported 'variant-to-gene mapping' effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia 'WDR90' GWAS locus. However, importantly that effort did not characterize the corresponding underlying causal variant. Specifically, our previous 3D genomic datasets nominated a shortlist of three neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium within an intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. We sought to investigate the influence of these SNPs collectively and then individually on PIG-Q modulation to pinpoint the causal "regulatory" variant. Starting with gross level perturbation, deletion of the entire region in NPCs via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from individual luciferase reporter assays for each SNP in iPSCs revealed that the region with the rs3752495 risk allele induced a ~2.5-fold increase in luciferase expression. Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by ~2-fold versus the non-risk allele. In conclusion, our variant-to-function approach and in vitro validation implicates rs3752495 as a causal insomnia variant embedded within WDR90 while modulating the expression of the distally located PIG-Q.

3D genomic features across >50 diverse cell types reveal insights into the genomic architecture of childhood obesity.

The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the BDNF, ADCY3, TMEM18 and FTO loci in skeletal muscle myotubes and the pancreatic beta-cell line, EndoC-BH1. One novel implicated effector gene, ALKAL2 - an inflammation-responsive gene in nerve nociceptors - was observed at the key TMEM18 locus across multiple immune cell types. Interestingly, this observation was also supported through colocalization analysis using expression quantitative trait loci (eQTL) derived from the Genotype-Tissue Expression (GTEx) dataset, supporting an inflammatory and neurologic component to the pathogenesis of childhood obesity. Our comprehensive appraisal of 3D genomic datasets generated in a myriad of different cell types provides genomic insights into pediatric obesity pathogenesis.

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

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.

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome wide association studies (GWAS) have been crucial for the identification of loci associated with sleep traits and disorders, the method itself does not directly uncover the underlying causal variants and corresponding effector genes. The overwhelming majority of such variants reside in non-coding regions and are therefore presumed to impact the activity of cis-regulatory elements, such as enhancers. Our previously reported 'variant-to-gene mapping' effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia 'WDR90' locus. However, importantly that effort did not characterize the corresponding underlying causal variant at this GWAS signal. Specifically, our genome-wide ATAC-seq and high-resolution promoter-focused Capture C datasets generated in this cell setting brought our attention to a shortlist of three tightly neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium in a candidate intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. The objective of this study was to investigate the influence of the proxy SNPs collectively and then individually on PIG-Q modulation and to pinpoint the causal "regulatory" variant among the three SNPs. Starting at a gross level perturbation, deletion of the entire region harboring all three SNPs in human iPSC-derived neural progenitor cells via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from more refined individual luciferase reporter assays for each of the three SNPs in iPSCs revealed that the intronic region with the rs3752495 risk allele induced a ~2.5-fold increase in luciferase expression (n=10). Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by ~2-fold compared to the non-risk allele. In conclusion, our variant-to-function approach and subsequent in vitro validation implicates rs3752495 as a causal insomnia risk variant embedded at the WDR90-PIG-Q locus.

Strategies to identify causal common genetic variants and corresponding effector genes for paediatric obesity.

BACKGROUND: Childhood obesity rates are on the rise, but there are currently no effective therapies available to slow or halt their progression. Although environmental and lifestyle factors have been implicated in its pathogenesis, childhood obesity is considered a complex disorder with a clear genetic component. Intense genome-wide association study (GWAS) efforts through large-scale collaborations have enabled the discovery of genetic loci robustly associated with childhood obesity beyond the classic FTO locus. That said, GWAS itself does not pinpoint the actual underlying causal effector genes, but rather just yields association signals in the genome. OBJECTIVE: This review aims to outline what has been elucidated thus far on the genetic aetiology of commong childhood obesity and to describe strategies to identify and validate both causal common genetic variants and their corresponding effector genes. RESULTS: Relevant cell types for molecular studies can be identified by gene set enrichment analysis and considering known biology of obesity-related physiological processes. Putatively causal single nucleotide polymorphisms (SNPs) can be identified by several methods including statistical fine mapping and 'assay for transposase accessible chromatin sequencing' (ATAC-seq). Variant to gene mapping can then nominate effector genes likely regulated by cis-regulatory elements harbouring putatively causal SNPs. A SNP's cis-regulatory activity can be functionally validated by several in vitro methods including luciferase assay and CRISPR approaches. These CRISPR approaches can also be used to investigate how dysregulatn of effector genes may confer obesity risk. CONCLUSION: Uncovering the causative genes related to GWAS signals and elucidating their functional contributions to paediatric obesity with these strategies will deepen our understanding of this disease and serve better treatment outcomes.

Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant complex traits.

The hypothalamus regulates metabolic homeostasis by influencing behavior and endocrine systems. Given its role governing key traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function could yield insights into disease pathogenesis. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. Next, we integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. Our results reveal common target genes for these traits, potentially affecting core developmental pathways. Our atlas will enable future efforts to determine hypothalamic mechanisms influencing disease susceptibility.

Genetic Determinants of Childhood Obesity.

Obesity represents a major health burden to both developed and developing countries. Furthermore, the incidence of obesity is increasing in children. Obesity contributes substantially to mortality in the United States by increasing the risk for type 2 diabetes, cardiovascular-related diseases, and other comorbidities. Despite environmental changes over past decades, including increases in high-calorie foods and sedentary lifestyles, there is very clear evidence of a genetic predisposition to obesity risk. Childhood obesity cases can be categorized in one of two ways: syndromic or non-syndromic. Syndromic obesity includes disorders such as Prader-Willi syndrome, Bardet-Biedl syndrome, and Alström syndrome. Non-syndromic cases of obesity can be further separated into rarer instances of monogenic obesity and much more common forms of polygenic obesity. The advent of genome-wide association studies (GWAS) and next-generation sequencing has driven significant advances in our understanding of the genetic contribution to childhood obesity. Many rare and common genetic variants have been shown to contribute to the heritability in obesity, although the molecular mechanisms underlying most of these variants remain unclear. An important caveat of GWAS efforts is that they do not strictly represent gene target discoveries, rather simply the uncovering of robust genetic signals. One clear example of this is with progress in understanding the key obesity signal harbored within an intronic region of the FTO gene. It has been shown that the non-coding region in which the variant actually resides in fact influences the expression of genes distal to FTO instead, specifically IRX3 and IRX5. Such discoveries suggest that associated non-coding variants can be embedded within or next to one gene, but commonly influence the expression of other, more distal effector genes. Advances in genetics and genomics are therefore contributing to a deeper understanding of childhood obesity, allowing for development of clinical tools and therapeutic agents.