Genome wide association studies are enriched for interacting genes

doi:10.21203/rs.3.rs-5189487/v2

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Genome wide association studies are enriched for interacting genes

https://doi.org/10.21203/rs.3.rs-5189487/v2

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Background: With recent advances in single cell technology, high-throughput methods provide unique insight into disease mechanisms and more importantly, cell type origin. Here, we used multi-omics data to understand how genetic variants from genome-wide association studies influence development of disease. We show in principle how to use genetic algorithms with normal, matching pairs of single-nucleus RNA- and ATAC-seq, genome annotations, and protein-protein interaction data to describe the genes and cell types collectively and their contribution to increased risk.

Results: We used genetic algorithms to measure fitness of gene-cell set proposals against a series of objective functions that capture data and annotations. The highest information objective function captured protein-protein interactions. We observed significantly greater fitness scores and subgraph sizes in foreground vs.matching sets of control variants. Furthermore, our model reliably identified known targets and ligand-receptor pairs, consistent with prior studies.

Conclusions: Our findings suggested that application of genetic algorithms to association studies can generate a coherent cellular model of risk from a set of susceptibility variants. Further, we showed, using breast cancer as an example, that such variants have a greater number of physical interactions than expected due to chance.

GWAS

genetic algorithms

variant prioritization

multi-omics

breast cancer

complex disease

etiology

susceptibility

gene network.

The primary goal of genome-wide association studies (GWAS) is to catalog and translate genetic variants to uncover disease mechanisms [1–3]. Over the past twenty years, researchers leveraged GWAS to pinpoint specific genomic regions for further investigation [4, 5]. However, one of the challenges of interpreting GWAS is that 95% of single nucleotide polymorphisms (SNPs) fall outside of the protein coding region [6–8]. Depending on the linkage disequilibrium (LD) structure, anywhere from one to hundreds of non-functional SNPs may be associated with a disease at a single locus [9]. Thus, identification of the causal variant and gene poses great difficulty.

Considerable work has gone into analyzing and interpreting GWAS data [2, 4–6, 9–12]. FunciSNP [6] and HaploReg [13] were developed to identify candidate functional SNPs in non-coding regions by integrating biofeatures such as SNPs with high LD, epigenomic data, and DNA-binding factors. The impact of functional SNPs has been tested through in vitro multi-tissue expression quantitative trait loci (eQTL) to find gene associations [10].

More recently, machine learning approaches through aggregation of multi-omics data were developed to improve prioritization [8, 11]. Mountjoy et al. developed a locus-to-gene (L2G) pipeline that integrates QTL, gene distance, and pathogenicity predictions to rank likely causal genes [11]. While their method provides statistical evidence for prioritization, they don’t account for cell type specificity [11]. The use of single-cell sequencing technology provided unique insights into molecular mechanisms. Corces et al. used bulk and single-cell assay for transposase-accessible chromatin sequencing (ATAC-seq) data to identify cell type specific open chromatin to prioritize gene and cell type of noncoding GWAS loci in neurodegenerative diseases [8]. Zhang et al. developed single-cell disease relevance score (scDRS), which exploits single-cell RNA sequencing (scRNA-seq) data and associates disease specific expression signatures with specific cell populations [14].

One of our prior studies connected SNPs to genes encoding both ligands and their cognate receptors [12]. The existence of ligand receptor pairs in GWAS implies intercellular communication as part of susceptibility – highlighting the potential role of other cell types besides the cell-of-origin [12]. What is currently lacking from attempts to integrate single-cell omics and GWAS data is that multiple independent genetic signals may produce similar cellular effects through protein interaction networks.

Our hypothesis is that variants associated with cancer affect interacting proteins and cell types to promote disease initiation. Based on this hypothesis, we predict that accounting for physical interaction of susceptibility genes will increase sensitivity and accuracy. Here, we use genetic algorithms (GA) to integrate breast cancer (BCa) GWAS with interaction data, single-nucleus RNA-seq (snRNA-seq) data, single-nucleus ATAC-seq (snATAC-seq) data, and genome annotations to prioritize gene and cell type at each locus.

GWAS data. We obtained BCa variants from NHGRI-EBI GWAS Catalog [15, 16]. That data was derived from cases and controls of European ancestry from studies using the Breast Cancer Association Consortium (BCAC) [17] and Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) [18] (Table 1). We identified the most recent BCa GWAS [16] that expanded on previous BCAC GWAS [19–21]. We performed LD expansion using LDlinkR [22] for the European population. Due to genetic drift, the Finnish population was excluded. We selected proxy SNPs with MAF ≥ 5%, R² ≥ 0.6, D′ ≥ 0.9.

Single-nuclei data. We identified published data with normal breast tissue, including matching pairs of samples for snRNA-seq (GSE168836) and snATAC-seq (GSE168837) [23] (Table 1, Fig. 1A). We applied sctransform [24] for cell-to-cell normalization and variance stabilization on the RNA dataset and used provided scripts (process_atac.R) from [23] to acquire the peak matrix by cell type.

Identification of candidate genes. We used protein coding genes and lncRNAs from the 10X Genomics human reference “refdata-gex-GRCh38-2020-A” [23]. To identify candidate genes (“nearby gene set”), we defined a window size using the minimum and maximum chromosome positions from the lead SNP and its proxies. We expanded the window by 200 kb on each side to account for adjacent genes and imposed a minimum of five genes up- and downstream for each lead SNP.

Genetic algorithm. The GA model consists of five steps: 1) generate a population of 1,000 random proposals (potential solution); 2) score proposal “fitness” as the average of all objective functions (OFs); 3) select pairs of proposals for mating with probability proportional to fitness rank; 4) introduce mutations for gene and cell type; 5) repeat steps 2–4 for 200 generations (Fig. 1B).

Initiation of proposals. The number of proposal elements (gene-cell type combinations) is equal to the number of lead SNPs. Each element consists of a lead SNP, a gene, and a cell type. We randomly select a gene from nearby gene sets and cell type using the cell labels from the snRNA- and snATAC-seq data [23].

Building objective functions (OFs). We created OFs (names italicized throughout the text) using external data sources for gene and cell type prioritization (Fig. 1A). For gene prioritization, these functions are: isMAGMAgene, isCancerGene, protein-protein interaction (isPPI), lncRNA and protein interaction (isLPI), and isPromoter. For cell type prioritization, these OFs capture: non-cell type specific ATAC peaks - isCommonATAC and and cell type specific peaks - isMarkerATAC. For data that inform on both gene and cell type, these functions are: isMarkerGene, isMarkerPPI, intracellular PPI (isIntraPPI) and intercellular PPI (isInterPPI). These datasets and their relationships to the OFs are described in Table 1 and Fig. 1A.

Conversion of breast data sources to boolean values in OFs. All OFs were scored as boolean values on each proposal element. For isMAGMAgene, we obtained a set of genes from Multi-marker Analysis of Genomic Annotation (MAGMA) database [25] based on the 2013 UK biobank 460k release for BCa, which uses GWAS summary statistics to identify genes strongly associated with the phenotype [14]. We scored loci as positive when the proposed gene is from the MAGMA gene set. For isCancerGene, we combined BCa associated gene mutations and gene fusions from COSMIC Cancer Gene Census [26] to curate the cancer gene set. We scored based on membership in this set, similar to isMAGMAgene. For isPPI, we selected protein-protein interactions with experimental evidence > 0 from STRING v11.5 [27]. We scored isPPI by identifying proposed genes at different loci found in STRING. We scored isLPI by identifying proposed genes at different loci found in LncBook [28]. In isPromoter, we identified variants in promoter regions (defined as 1 kb upstream and 100 bp downstream of transcription start site (TSS)) using the 10X Genomics human reference genome. We scored loci that reside in a promoter region of the proposed gene. In isCommonATAC, we used the peak matrix described in “GWAS and Single-nuclei data.” Since enhancers bind regulatory factors in the regions immediately flanking open chromatin, we identified peaks that contain SNPs with cell type annotation. SNPs in ATAC-seq peaks found in multiple cell types were labeled as “common.” In isMarkerATAC, we identified cell type specific peaks [29] at FDR ≤ 0.05 and log₂ fold change ≥ 0.25. In isMarkerGene, we used the count matrix data, as previously described in “GWAS and Single-nuclei data,” to identify gene expression markers for each cell type [30] using a different cell type as the background. We selected genes at p ≤ 0.05 and log₂ fold change ≥ 0.25. In isMarkerPPI, a combination of isPPI and isMarkerGene, we scored as positive when two conditions were met: 1) both proposed genes participate in PPI, 2) the proposed cell type is a valid cell type marker. In isInterPPI, we filtered for genes found in CellTalkDB [31], a database of ligand-receptor interactions. We scored as positive any two loci with proposed genes in a ligand-receptor interaction and the cell types are heterogeneous. In contrast, we removed CellTalkDB genes to curate isIntraPPI. For isIntraPPI, we scored similarly to isInterPPI, but the proposed cell type must be the same.

Selection. To identify parent proposals for the next generation, we use a fitness rank proportional selection method. To accomplish this, we compute fitness score as the arithmetic mean of all OFs. We rank proposals from highest to lowest and divide them into five equal groups (Group 1 being the highest rank). We sample 100 proposals without replacement using group probability for mating and crossover. We replace a Group 5 proposal at random with the top proposal (“elite”) in the current generation. During crossover, we select 50% proposal elements (loci) at random from the first proposal, then select the complementary half from the second proposal. We combine the results to construct a child proposal. For each of the 100 parent proposal pairs, we generate 10 child proposals for the next generation, for a total of 1,000.

Mutation. We implemented a 1% mutation rate on gene and cell type for each child proposal. For gene mutation, we randomly selected a gene from the nearby gene set to replace the current gene. For cell type, we similarly selected a random cell label to replace the current one.

Termination of the algorithm. We repeated steps 2–4 until the fitness score variance < 1% for 10 generations (Fig. 2A, B), empirically determined to be 123 generations. We rounded this number up to 200 for all subsequent trials.

Curation of control SNPs. We used vSampler v1.2.1 [32] to generate control variants matching BCa SNPs (Supplemental Fig. 1). We used the following parameters: MAF (± 0.05), distance to closest TSS (± 100 kb), gene density (± 20 in ± 200 kb), number of proxy SNPs in LD (± 75 for R² > 0.8), and enabled sampling across chromosome (Supplemental Fig. 2A). For computed parameters, we selected a value two standard deviations away from the mean. Using the GWAS lead SNPs as a model, we identified 10 matched control variants for each locus. We randomly selected 10 matching sets, each set mirrors 176 of the 206 BCa variants. Thirty SNPs were excluded as insufficiently matching our criteria. We observed low similarity between the candidate gene lists in the control sets and BCa GWAS (Supplemental Fig. 2B). We chose this many controls to estimate the variation or noise inherent in a set of variants of equal size.

OF enrichment calculation. We calculated enrichment of an OF in BCa as the posterior probability of observing the fraction of positives in the OF compared to control. We defined enrichment exclusion of zero from the 95% range of credible differences.

Optimization of gene-cell proposals against breast data using GA

To describe the mechanisms of cancer risk based on population genetics of BCa, we acquired 206 lead variants of European ancestry [16, 19–21] (Table 1). For each variant, we identified proxy SNPs in LD plus candidate genes (described in Methods). These SNPs were within 200 kb of 2,292 genes of which 51% (n = 1,175) were protein-coding. To better understand these SNPs in the context of normal breast, we identified matching pairs of samples for snRNA-seq and snATAC-seq [23] (Table 1, Fig. 1A). Within these data, cells were divided into 10 clusters: hormone receptor-positive and -negative luminal cells, basal cells, blood and lymphatic endothelial cells, vascular accessory cells, adipocytes, fibroblasts, myeloid, and lymphoid cells.

The biggest challenge is the large number of combinations of hypotheses for every locus. In this study, there are at least 10²⁰⁶ combinations of plausible solutions when considering only genes. We chose GA to identify the most plausible gene and cell set (“proposal”) based on diverse evidence sources. The evidence sources for gene and cell type prioritization are captured in a set of named objective functions described in the Methods and Table 1.

Table 1

Data sets used in this study.
Data Set	Source	Description
BCa GWAS (integrated references)	Zhang et al., 2020 [16], Michailidou et al., 2017 [19], Garcia-Closas et al., 2013 [20], Milne et al., 2017 [21]	The following study accessions were used: GCST001930, GCST010098, GCST010099, GCST010100, GCST004988, GCST005076, GCST005077, and GCST005075.
snRNA-seq	Raths et al., 2023 [23]	66,926 nuclei from 9 cis-gender females (GSE168836).
snATAC-seq	Raths et al., 2023 [23]	27,459 nuclei from 9 cis-gender females (GSE168837).
Marker gene		This study, reanalysis of gene expression marker set at cell type resolution from Raths et al., 2023 [23].
MAGMA gene	Zhang et al., 2022 [14]	MAGMA, a set of putative disease genes from the 2013 UK biobank 460k release for BCa, consisting of 1,000 genes.
Cancer gene	Sondka et al., 2018 [26]	COSMIC cancer gene consensus is a collection of gene mutation and fusion implicated in cancer.
Protein-protein interactions	Szklarczyk et al., 2023 [27]	STRING (string-db.org), is a database of protein-protein interaction with experimental evidence.
LncRNA and protein interactions	Li et al., 2023 [28]	LncBook is a comprehensive resource of human lncRNA-protein interaction.
Promoter regions	10X Genomics	Promoter region extracted from prebuilt GRCh38 genome reference version 2020-A [23].
Common/marker ATAC peaks set		This study, reanalysis of open chromatin peak set at cell type resolution from Raths et al., 2023 [23].

We optimized for 200 generations and then analyzed the proposals in the last generation (Gen200) to assess the result (Fig. 2A, B). We observed that information was distributed unevenly between OFs: the mean score for isCancerGene, isLPI, isPromoter, and isInterPPI were less than 0.1 (Fig. 2C), whereas isPPI had the highest score (0.941). The remaining OFs had scores ranging from 0.410 to 0.832. When compared to other proposals, the elite proposal did not have top scores in all OFs. We asked whether consensus solutions might have a higher score than the elite proposal. To do this, we identified the top gene and cell type for all loci across 1,000 Gen200 proposals. Surprisingly, we observed a fitness score of 0.433 for the consensus – an improvement over the elite proposal (0.429). We observed no change for isMAGMAgene, isCancerGene, isPPI, and isPromoter between the consensus and elite proposal. However, we did observe higher OF scores for isMarkerGene, isMarkerPPI, isIntraPPI, isInterPPI, isCommonATAC and isMarkerATAC, and lower OF scores for isLPI in the consensus compared to the elite proposal. This result suggests the existence of multiple, mutually exclusive, but equally stable solutions preserved only in the consensus proposal.

GA identifies known targets

We compared genes discovered in the consensus against L2G [11] and a naive nearest gene classifier (distance from TSS). L2G outputs the likelihood a gene is causal for the SNP (L2G score) based on distance, molecular QTL, chromatin interaction and variant pathogenicity. We identified the same SNPs across the dataset and selected the gene with the highest L2G score. Of the 175 common loci, we observed 46.8% (n = 82) with shared prediction between L2G and consensus. Across all three models, 68 loci shared the same gene. In total, 77.7% (136 out of 175 loci) L2G genes were the nearest gene to the SNP, so we did not expect our model to have high concordance with L2G because we did not include a gene distance OF. While gene distance to SNP is worth consideration, it has been reported that the nearest gene to the SNP is affected only 15% of the time [33]. In contrast, in our predictions, 41.7% (86 out of 206 loci) were the nearest gene, an intermediate value between these two figures.

The identification of high confidence gene and cell type calls are essential for downstream analysis. We performed a power calculation to determine the threshold for identifying high confidence calls. To do this, we selected a threshold where 80% of high confidence L2G SNPs with the same gene prediction as the consensus (L2G ≥ 0.7) are detected (949 proposals) (Fig. 3A). We used this same threshold to identify high confidence cell types (Fig. 3B). The number of loci with a high confidence call in gene and cell type are 147 and 118 out of 206 respectively. At lead SNP rs10941679, we found the top gene and cell type was FGF10 and “fibroblast” in Gen200 (Fig. 3C, D). Compared to L2G, MRPS30 (L2G = 0.542) was ranked higher than FGF10 (L2G = 0.145) for the same SNP due to support from the QTL and distance modules [11]. Interestingly, eQTL analysis with rs10941679 revealed changes in gene expression levels for MRPS30 and FGF10 in MCF7 and BT474 BCa cell lines [34]. In our model, we observed shared evidence (isMAGMAgene and isPPI) for both genes. However, FGF10 had isMarkerGene as additional evidence. This result highlights the ability of our model to account for complex interactions and mechanisms.

Contribution of individual OFs to overall fitness

We assessed each OF’s contribution to fitness by comparing information content between Gen0 and Gen200. To do this, we computed the posterior probability of observing an OF score in Gen200 given Gen0. We also computed the effect size (ES) as the median difference between the two distributions. We found that the most informative OFs were isPPI (ES = 0.744) and isIntraPPI (ES = 0.870). We expected isMAGMAgene, which captures gene expression as a function of GWAS, to be the most informative OF. Although informative, isMAGMAgene yielded a lower score (ES = 0.359) than the top OF. In contrast, isCancerGene was not informative (ES = 0.106). IsLPI was also not informative (ES = 0.008), possibly due to a low number of lncRNA in the consensus (n = 5). For cell type prioritization, we observed isCommonATAC (ES = 0.438) and isMarkerATAC (ES = 0.398) to be informative, as expected.

We next investigated the information content on a locus-by-locus basis. To accomplish this, we counted all loci with OF support in Gen0 and Gen200. We used Kolmogorov-Smirnov (KS) to test whether these observations derive from the same theoretical distribution (KS test p = 2.20 x 10^− 16). In Gen0, we observed 54.8% loci (n = 113) without OF support. In contrast, every locus had at least one supporting OF in Gen200. Additionally, Gen200 had more supporting OFs per locus (µ = 4.76, SD = 1.64) when compared to Gen0 (µ = 0.8, SD = 1.04). Taken together, five OFs had evidence for a large number of positive loci (isMAGMAgene, isPPI, isCommonATAC, isMarkerATAC, and isIntraPPI).

BCa GWAS loci are enriched in associations with breast-specific assays

We reasoned that, according to our hypothesis, in which GWAS variants interact to link common cell types and pathways, there should be a greater number of associations both with disease relevant data and between loci. To test these predictions, we first evaluated whether the solutions discovered by GA had higher fitness than those from equivalent sets of randomly selected variants. Second, we analyzed the network properties of BCa GWAS relative to these control sets.

We repeated GA as before with our 10 control sets. To capture random variation in stable solutions we ran nine additional models for BCa and each control set, each with a different initial population, (10 BCa and 100 control GA runs) (Fig. 4A). In Gen200, we computed the posterior probability of observing the BCa fitness scores given the control distribution (BCa: µ = 0.415, SD = 1.83 x 10^− 3; control: µ = 0.330, SD = 8.77 x 10^− 3). We observed the BCa fitness scores were significantly higher compared to the control by assessing the probability that the mean difference is zero or less (p = 0.041). Thus, our model is able to distinguish between BCa and randomly chosen SNPs. Moreover, the higher fitness score reveals the potential for true biological associations between BCa GWAS and breast derived multi-omics data.

If a higher fitness score in BCa is driven by its associations with breast-specific data, we predict that the BCa and control set fitness scores should also be driven by different OFs. To test this prediction, we computed the posterior probability of observing positive OFs in BCa given the control set (Fig. 4B). We observed isMAGMAgene, isCommonATAC, and isMarkerATAC higher in BCa than control (ES greater than zero, p < 0.05). We expected isMAGMAgene to outperform in BCa compared to the control group (ES = 0.314) as it’s derived from breast expression. The enrichment of isCommonATAC and isMarkerATAC relative to control suggests that BCa SNPs are associated with normal breast cell types. In contrast, isPromoter, isLPI, isMarkerGene, isMarkerPPI, and isInterPPI were indistinguishable between the BCa and control set when assessing the frequency of ES greater than zero (p ≥ 0.05) (Fig. 4B). Surprisingly, we observed isIntraPPI (p = 0.073) and isPPI (p = 0.074) had a small ES when comparing the BCa to the control set, 0.057 and 0.039 respectively. The result shows that even randomly selected SNPs have a high PPI score.

Given the enriched OFs in BCa, we asked how individual loci contributed to increased fitness over control. To address this, we measured the information content at all BCa loci. We computed the number of OF support for the 176 BCa loci used to match the control sets. We identified the consensus gene and cell type in Gen200 for the 10 BCa GA runs and the 10 matching SNPs from the 10 control GA runs (total of 10 x 10 = 100 runs) and computed the number OF support for each of the 110 GA runs. We used the Wilcoxon rank-sum test to identify differences between OF support by comparing the two distributions. After multiple hypothesis correction, we observed 61.4% (n = 108) BCa loci with higher OF support than control (p ≤ 0.05, ES > 0). In contrast, we observed 8.5% (n = 15) BCa loci with lower OF support than control (p ≤ 0.05, ES < 0). Our analysis of the result demonstrates a majority of loci in BCa have higher OF support than due to chance alone, and provides critical information about lack of support for other loci. This procedure can be used to measure the benefit of OFs, and to exclude non-informative loci from downstream analysis.

By curating a set of control SNPs, we identified the most informative OFs (isMAGMAgene, isCommonATAC, and isMarkerATAC) that distinguish BCa from control. These OFs corresponded to the breast specific data. We anticipated that isPPI would be an informative OF, but despite its overall importance to the outcome for BCa and control (OF mean = 0.94 vs. 0.90 controls) our analysis revealed no significant difference. It is possible that including all interaction experimental evidence (interaction score > 0) from STRING in our isPPI OF may not be stringent enough. It is also possible the quality of interactions as measured in network size is better in BCa than control, and we explore this next.

GWAS variants are enriched for larger networks

Based on our OF enrichment analysis (Fig. 4B), PPI failed to distinguish between the BCa and control set. This finding did not support our hypothesis that molecular interaction mechanisms are embedded within GWAS. If the control set represents variants without any true associations to breast data, then we predict BCa will have larger PPI network sizes. To test our prediction, we identified all PPI (interaction score ≥ 0.4) for the 10 BCa and 100 control GA run. We observed no significant difference in the number of subgraphs between the two groups (KS test p = 0.633) (Fig. 5A). Next, we computed the number of genes per subgraph and observed the control having fewer genes in their largest subgraph (µ = 7.95, SD = 3.52) when compared to the BCa sets (µ = 28.6, SD = 3.5). We used KS to test whether these observations derive from the same theoretical distribution (KS test p = 2.132 x 10^− 14). Additionally, we downsampled the BCa (n = 176) to adjust for the additional 30 SNPs that we excluded in making the control sets. We observed BCa (µ = 21.9, SD = 7.61) still had more genes in their largest subgraph compared to controls (KS = 4.71 x 10^− 8) (Fig. 5B, C). The result strongly supports the conclusion that genes selected in BCa GWAS have a larger PPI network than expected due to chance, consistent with our hypothesis that GWAS variants are functionally connected.

Reconstruction of cellular interaction from the consensus proposal

Earlier, we found the surprising result that the consensus proposal scored higher than the highest scoring elite proposal. We speculated that competing subsets of loci in different proposals produce more than one family of stable solutions. To quantify diversity of the Gen200 proposal set, we computed the Gini-Simpson index for the 206 loci in the 10 BCa GA runs. We selected loci with low diversity (Gini-Simpson index ≤ 0.5 and gene count ≤ 2) that produced the same gene predictions across multiple independent runs. Of the 118 high confidence BCa SNPs, we identified 26 loci with PPI. We constructed a projection of the protein interaction network which consisted of 6 subgraphs – the largest having a total of 12 genes (subgraph 1) (Fig. 6).

We constructed a map that links genetic variants to gene and cell type. To accomplish this, we annotated predicted cell type on the PPI network graph from Fig. 6. The largest subgraph included basal, luminal hormone receptor positive and negative, fibroblast, adipocytes and blood endothelial, and lymphatic cell types. This result shows in principle how an interpretable model of GWAS can be constructed from the consensus proposal.

We introduced a framework that leverages single-nucleus multi-omics, genome annotations and interaction data to prioritize gene and cell type for GWAS loci. As proof of principle, we selected BCa for study because of availability of public data, in particular matching single-nucleus multi-omics for normal breast. Our method considered all BCa GWAS loci as a single proposal rather than individually. We employed GA to evaluate, score, and modify proposals based on OFs that capture mechanisms such as disruption of promoters, open chromatin, and PPI. We applied this method to BCa and recovered known target genes. We showed BCa loci were enriched in association with OF in BCa multi-omic data and PPI when compared to equivalent sets of randomly selected variants. These analyses provided support for our hypothesis that interactions between proteins encoded at GWAS loci are an important feature of genetic association studies.

We note several limitations of our work. First, we grouped lead SNPs with proxies under the lead SNP term. In our model, the GA could use OF support from more than one SNP under the lead SNP term – masking the causal SNP. This limitation could be addressed by allowing the GA to fit data to proxy SNPs as a third parameter the way we fit nearby genes and cell type in this study.

Second, our model utilized 11 OFs. As we noted at the end, our largest subgraph was not as coherent with respect to cell type. Although it is beyond the scope of this work, we have reason to believe that future inclusion of transcription factor networks will greatly enhance the overall coherence of our models. Nonetheless, the results presented here represent the best explanation of BCa risk given the data we used. We expect the solutions to evolve as additional data and OFs are introduced.

Third, the model does not account for the independent risk of histological subtypes. As discussed above, this may contribute to competing optimizations within each proposal set. We plan to address these shortcomings in future analyses.

These findings suggest that our framework is able to uncover molecular mechanisms embedded in GWAS. Future studies using GA or other artificial intelligence approaches explicitly modeling molecular interactions between loci have great potential to provide novel insight for GWAS in mediating risk.

BCa Breast cancer

BCAC Breast Cancer Association Consortium

Bp Base pairs

CIMBA Consortium of Investigators of Modifiers of BRCA1/2

COSMIC The Catalog Of Somatic Mutations In human Cancer

ES Effect Size

eQTL Expression quantitative trait loci

GA Genetic algorithm

Gen0 Initial population

Gen200 Final population

GWAS Genome-wide association studies

Kb Kilobases

KS Kolmogorov-Smirnov

L2G Locus-to-gene

LD Linkage disequilibrium

LPI LncRNA-protein interaction

MAGMA Multi-marker Analysis of Genomic Annotation

MAF Minor allele frequency

OF Objective function

PPI Protein-protein interaction

snATAC-seq Single-nucleus RNA sequencing

snRNA-seq Single-nucleus assay for transposase-accessible chromatin sequencing

SD Standard deviation

SNP Single nucleotide polymorphism

STRING Search Tool for Recurring Instances of Neighboring Genes

TSS Transcription start site

UK United Kingdom

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The data supporting the conclusions of this article are available in the Zenodo repository under https://zenodo.org/records/13851449.
All code for producing the analyses and figures herein are included in this fully reproducible manuscript in R markdown format. R markdown files are available from our repository on the distributed version control site, Github: https://github.com/Junkdnalab/Inherited_Risk_GA.
Further information and requests for resources and analyses should be directed to and will be fulfilled by the lead contact, Dennis J. Hazelett, Ph.D. (Dennis.Hazelett at csmc dot edu)

Competing interests

There are no financial and non-financial competing interests declared by authors.

Funding

The authors would like to acknowledge funding from the NIH to Jason Moore R01LM010098 and U01AG066833

Authors’ contributions

D.H. is the Principal Investigator of the study who developed the hypotheses, designed the study, and participated in every stage of the manuscript development. D.H., S.C., and P.N. conducted the experiments and analyses. P.N., I.S., and D.H. drafted and reviewed the final manuscript. All authors approved the final version of the manuscript.

Acknowledgements

The authors would like to thank Drs. Jason Moore, Paul Pharoah, and Ryan Urbanowicz for helpful discussions.

Visscher PM, Wray NR, Zhang Q, Sklar P, McCarthy MI, Brown MA, Yang J (2017) 10 years of GWAS discovery: Biology, function, and translation. Am J Hum Genet 101:5–22
Abdellaoui A, Yengo L, Verweij KJH, Visscher PM (2023) 15 years of GWAS discovery: Realizing the promise. Am J Hum Genet 110:179–194
Bressan E, Reed X, Bansal V, et al (2023) The Foundational Data Initiative for Parkinson Disease: Enabling efficient translation from genetic maps to mechanism. Cell Genom 3:100261
Gallagher MD, Chen-Plotkin AS (2018) The post-GWAS era: From association to function. Am J Hum Genet 102:717–730
Hazelett DJ, Conti DV, Han Y, Al Olama AA, Easton D, Eeles RA, Kote-Jarai Z, Haiman CA, Coetzee GA (2016) Reducing GWAS complexity. Cell Cycle 15:22–24
Coetzee SG, Rhie SK, Berman BP, Coetzee GA, Noushmehr H (2012) FunciSNP: an R/bioconductor tool integrating functional non-coding data sets with genetic association studies to identify candidate regulatory SNPs. Nucleic Acids Res 40:e139
Nasser J, Bergman DT, Fulco CP, et al (2021) Genome-wide enhancer maps link risk variants to disease genes. Nature 593:238–243
Corces MR, Shcherbina A, Kundu S, et al (2020) Single-cell epigenomic analyses implicate candidate causal variants at inherited risk loci for Alzheimer’s and Parkinson's diseases. Nat Genet 52:1158–1168
Zhu C, Baumgarten N, Wu M, et al (2023) CVD-associated SNPs with regulatory potential reveal novel non-coding disease genes. Hum Genomics 17:69
GTEx Consortium, Laboratory, Data Analysis &Coordinating Center (LDACC)-Analysis Working Group, Statistical Methods groups-Analysis Working Group, et al (2017) Genetic effects on gene expression across human tissues. Nature 550:204–213
Mountjoy E, Schmidt EM, Carmona M, et al (2021) An open approach to systematically prioritize causal variants and genes at all published human GWAS trait-associated loci. Nat Genet 53:1527–1533
Hazelett DJ, Rhie SK, Gaddis M, et al (2014) Comprehensive functional annotation of 77 prostate cancer risk loci. PLoS Genet 10:e1004102
Ward LD, Kellis M (2016) HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease. Nucleic Acids Res 44:D877–81
Zhang MJ, Hou K, Dey KK, et al (2022) Polygenic enrichment distinguishes disease associations of individual cells in single-cell RNA-seq data. Nat Genet 54:1572–1580
Buniello A, MacArthur JAL, Cerezo M, et al (2019) The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019. Nucleic Acids Res 47:D1005–D1012
Zhang H, Ahearn TU, Lecarpentier J, et al (2020) Genome-wide association study identifies 32 novel breast cancer susceptibility loci from overall and subtype-specific analyses. Nat Genet 52:572–581
Breast Cancer Association Consortium (2006) Commonly studied single-nucleotide polymorphisms and breast cancer: results from the Breast Cancer Association Consortium. J Natl Cancer Inst 98:1382–1396
Couch FJ, Wang X, McGuffog L, et al (2013) Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk. PLoS Genet. https://doi.org/10.1371/journal.pgen.1003212
Michailidou K, Lindström S, Dennis J, et al (2017) Association analysis identifies 65 new breast cancer risk loci. Nature 551:92–94
Garcia-Closas M, Couch FJ, Lindstrom S, et al (2013) Genome-wide association studies identify four ER negative-specific breast cancer risk loci. Nat Genet 45:392–8, 398e1–2
Milne RL, Kuchenbaecker KB, Michailidou K, et al (2017) Identification of ten variants associated with risk of estrogen-receptor-negative breast cancer. Nature Genetics 49:1767–1778
Myers TA, Chanock SJ, Machiela MJ (2020) LDlinkR: An R package for rapidly calculating linkage disequilibrium statistics in diverse populations. Front Genet 11:157
Raths F, Karimzadeh M, Ing N, et al (2023) The molecular consequences of androgen activity in the human breast. Cell Genom 3:100272
Hafemeister C, Satija R (2019) Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol 20:296
de Leeuw CA, Mooij JM, Heskes T, Posthuma D (2015) MAGMA: generalized gene-set analysis of GWAS data. PLoS Comput Biol 11:e1004219
Sondka Z, Bamford S, Cole CG, Ward SA, Dunham I, Forbes SA (2018) The COSMIC Cancer Gene Census: describing genetic dysfunction across all human cancers. Nat Rev Cancer 18:696–705
Szklarczyk D, Kirsch R, Koutrouli M, et al (2023) The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res 51:D638–D646
Ma L, Cao J, Liu L, Du Q, Li Z, Zou D, Bajic VB, Zhang Z (2019) LncBook: a curated knowledgebase of human long non-coding RNAs. Nucleic Acids Res 47:D128–D134
Granja JM, Corces MR, Pierce SE, Bagdatli ST, Choudhry H, Chang HY, Greenleaf WJ (2021) ArchR is a scalable software package for integrative single-cell chromatin accessibility analysis. Nat Genet 53:403–411
Butler A, Hoffman P, Smibert P, Papalexi E, Satija R (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36:411–420
Shao X, Liao J, Li C, Lu X, Cheng J, Fan X (2021) CellTalkDB: a manually curated database of ligand-receptor interactions in humans and mice. Brief Bioinform 22:bbaa269
Huang D, Wang Z, Zhou Y, Liang Q, Sham PC, Yao H, Li MJ (2021) vSampler: fast and annotation-based matched variant sampling tool. Bioinformatics 37:1915–1917
Yao L, Shen H, Laird PW, Farnham PJ, Berman BP (2015) Inferring regulatory element landscapes and transcription factor networks from cancer methylomes. Genome Biol 16:105
Ghoussaini M, French JD, Michailidou K, et al (2016) Evidence that the 5p12 variant rs10941679 confers susceptibility to estrogen-receptor-positive breast cancer through FGF10 and MRPS30 regulation. Am J Hum Genet 99:903–911

No competing interests reported.

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Genome wide association studies are enriched for interacting genes

Status:

Version 2

Abstract

Figures

Background

Methods

Results

Optimization of gene-cell proposals against breast data using GA

GA identifies known targets

Contribution of individual OFs to overall fitness

BCa GWAS loci are enriched in associations with breast-specific assays

GWAS variants are enriched for larger networks

Reconstruction of cellular interaction from the consensus proposal

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 2