Dynamic effects of genetic variation on gene expression revealed following hypoxic stress in cardiomyocytes

Cardiovascular disease (CVD), which ultimately damages heart muscle, is a leading cause of death worldwide (WHO, 2018). CVD encompasses a range of pathologies including myocardial infarction (MI), where ischemia or a lack of oxygen delivery to energy-demanding cardiomyocytes results in cellular stress, irreparable damage, and cell death. Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (Nikpay et al., 2015), MI, and heart failure (Shah et al., 2020), indicating the potential contribution of specific genetic variants to disease risk. Most disease-associated loci do not localize within coding regions of the genome, often making inference about the molecular mechanisms of disease challenging. That said, because most GWAS loci fall within non-coding regions, these variants are thought to have a role in regulating gene expression. One of the main goals of the Genotype-Tissue Expression (GTEx) project has been to bridge the gap between genotype and organismal level phenotypes by identifying associations between genetic variants and intermediate molecular level phenotypes such as gene expression levels (GTEx Consortium et al., 2017). The GTEx project has identified tens of thousands of expression quantitative trait loci (eQTLs); namely, variants that are associated with changes in gene expression levels, across dozens of tissues including ventricular and atrial samples from the heart. However, the eQTLs reported by GTEx explain a modest proportion of GWAS loci, and while increasing the diversity of tissues and sample sizes will enable further insight, orthogonal approaches also need to be considered.

It is becoming increasingly evident that many genetic variants that are not associated with gene expression levels at steady state, may be found to impact dynamic programs of gene expression in specific contexts. This includes specific developmental stages (Cuomo et al., 2020; Strober et al., 2019), or specific exposure to an environmental stimulus such as endoplasmic reticulum stress (Dombroski et al., 2010), hormone treatment (Maranville et al., 2011), radiation-induced cell death (Smirnov et al., 2012), vitamin D exposure (Kariuki et al., 2016), drug-induced cardiotoxicity (Knowles et al., 2018), and response to infection (Alasoo et al., 2018; Barreiro et al., 2012; Çalışkan et al., 2015; Kim-Hellmuth et al., 2017; Manry et al., 2017; Nédélec et al., 2016). The studies of context-specific dynamic eQTLs highlight the need to determine the effects of genetic variants in the relevant environment. Therefore, if we are to fully understand the effects of genetic variation on disease, we must assay disease-relevant cell types and disease-relevant perturbations. Most of the aforementioned studies were performed in whole blood or immune cells, which means that there are many cell types and disease-relevant states that have yet to be explored.

With advances in pluripotent stem cell technology, we can now generate otherwise largely inaccessible human cell types through directed differentiation of induced pluripotent stem cells (iPSCs) reprogrammed from easily accessible tissues such as fibroblasts or B-cells. One of the advantages of iPSC-derived cell types as a model system is that the environment can be controlled, and thus we can specifically test for genetic effects on molecular phenotypes in response to controlled perturbation. This is particularly useful for studies of complex diseases such as CVD, which result from a combination of both genetic and environmental factors.

The heart is a complex tissue consisting of multiple cell types, yet the bulk of the volume of the heart is comprised of cardiomyocytes (Donovan et al., 2019; Pinto et al., 2016), which are particularly susceptible to oxygen deprivation given their high metabolic activity. iPSC-derived cardiomyocytes (iPSC-CMs) have been shown to be a useful model for studying genetic effects on various cardiovascular traits and diseases, as well for studying gene regulation (Banovich et al., 2018; Benaglio et al., 2019; Brodehl et al., 2019; Burridge et al., 2016; de la Roche et al., 2019; Ma et al., 2018; McDermott-Roe et al., 2019; Panopoulos et al., 2017; Pavlovic et al., 2018; Ward and Gilad, 2019).

In humans, coronary artery disease can lead to MI (Dzau et al., 2006) which results in ischemia and a lack of oxygen delivery to energy-demanding cardiomyocytes. Given the inability of cardiomyocytes to regenerate, this cellular stress ultimately leads to tissue damage. Advances in treatment for MI, such as surgery to restore blood flow and oxygen to occluded arteries, have improved clinical outcomes. However, a rapid increase in oxygen levels post-MI can generate reactive oxygen species leading to ischemia-reperfusion (I/R) injury (Giordano, 2005). Both MI and I/R injury can thus ultimately influence the amount of damage in the heart. iPSC-CMs allow us to mimic the I/R injury process in vitro by manipulating the oxygen levels that cardiomyocytes are exposed to in vivo.

We thus designed a study aimed at developing an understanding of the genetic determinants of the response to a universal cellular stress, oxygen deprivation, in a disease-relevant cell type, mimicking a disease-relevant process. To do so, we established an in vitro model of oxygen deprivation (hypoxia) and re-oxygenation in a panel of iPSC-CMs from 15 genotyped individuals (Banovich et al., 2018). We collected data for three molecular level phenotypes: gene expression, chromatin accessibility, and DNA methylation to understand both the genetic and regulatory responses to this cellular stress. This framework allowed us to identify eQTLs that are not evident at steady state, and assess their association with complex traits and disease.

We differentiated iPSC-CMs from iPSCs of 15 Yoruba individuals that were part of the HapMap project (Banovich et al., 2018). To obtain a measure of variance associated with the differentiation process, and to more effectively account for batch effects, we replicated the iPSC-CM differentiation from three individuals three times, yielding 21 differentiation experiments in total. The proportion of iPSC-CMs in each cell culture was enriched by metabolic purification (see Materials and methods). On Day 20, cardiomyocyte differentiation cultures from each individual were split into sub-cultures for each of the four subsequent oxygen conditions and for assessment of cardiomyocyte purity (Figure 1—figure supplement 1). iPSC-CMs were matured by electrical pulsing and maintenance in cell culture for 30 days. On Day 30 (+/- 1 day), the median cardiomyocyte purity from one representative sub-culture across individuals was 81% (40–97% range), determined by flow cytometry as the proportion of cells that were positive for the cardiac-specific marker, TNNT2 (Figure 1—figure supplement 2; Supplementary file 1; see Materials and methods).

We studied the response of the iPSC-CMs to hypoxia and re-oxygenation (Figure 1A). To do so, we first cultured the iPSC-CMs at oxygen levels that are close to physiological oxygen levels (10% oxygen - Condition A) for 7 days. We then subjected the iPSC-CMs to 6 hours of hypoxia (1% oxygen - Condition B), followed by re-oxygenation for 6 hours (10% oxygen - Condition C), or 24 hours (10% oxygen - Condition D) as previously described (Ward and Gilad, 2019). Oxygen levels were reproducibly controlled in cell culture (Figure 1B, Supplementary file 1). In order to determine whether the cardiomyocytes were affected by the changes in oxygen levels, we measured the enzymatic activity of released lactate dehydrogenase throughout the experiment, as a proxy for cytotoxicity. We also measured released BNP, a clinical marker of heart failure (Maeda et al., 1998). As expected, both cytotoxicity (p=0.01, Figure 1—figure supplement 3A–B) and BNP (p=5×10⁻⁶, Figure 1—figure supplement 3C) levels increased following hypoxia and long-term re-oxygenation.

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With this system established, we sought to understand the contribution of the global gene regulatory response to the molecular and cellular response to hypoxia and re-oxygenation. To do so, we collected global gene expression data (using RNA-seq; n = 15 individuals), chromatin accessibility data (using ATAC-seq; n = 14), and DNA methylation data (using the EPIC arrays; n = 13; Figure 1C) in each condition. With these data we studied both the gene regulatory response to oxygen perturbation, as well as the interaction of the response with the underlying genotype of the assayed individuals.

Gene expression changes in response to hypoxia and re-oxygenation

We first sought to identify those genes important for regulating the response by analyzing the gene expression (RNA-seq) data. We processed samples in batches as described in Supplementary file 1 and mapped and filtered sequencing reads to prevent allelic mapping biases (Figure 2—figure supplement 1; Supplementary file 1; see Materials and methods; van de Geijn et al., 2015). We observed that one sample (18852A) was a clear outlier when comparing read counts for 18,226 autosomal genes across all samples, and thus excluded it from further analysis (Figure 2—figure supplement 2). We filtered out genes with low expression levels (see Materials and methods) to yield a final set with data from 12,347 expressed genes (see Materials and methods). We performed a number of correlation-based analyses using the data from the technical replicates (Figure 2—figure supplement 3), and confirmed that the quality of the data is high and that, in line with our flow cytometry data, our iPSC-CMs express a range of cardiomyocyte marker genes across conditions including MYH7 and TNNT2 (Figure 2—figure supplement 4).

We took advantage of the fact that we have replicate experiments from three individuals to correct the data for unwanted variation (see Materials and methods; Risso et al., 2014). Following this procedure, our samples clustered both by oxygen level and individual (Figure 2—figure supplement 5). To identify genes that respond to hypoxia and re-oxygenation, we first tested for differential expression between pairs of conditions using a linear model with a fixed effect for ‘condition’, a random effect for ‘individual’, and four unwanted factors of variation, learned from the data, as covariates. At an FDR of 10%, we identified thousands of genes that are differentially expressed between conditions (A vs. B = 4,983, B vs. C = 6,311; B vs. D = 6,792; A vs. D = 2,835; Figure 2 and Figure 2—figure supplement 6A). In order to identify a single set of genes which respond to hypoxia across conditions, we used Cormotif (Wei et al., 2015) to jointly model pairs of tests. This approach led to the classification of 2,113 genes (17% of all expressed genes) as responding to hypoxia, which we term ‘response genes’ (Figure 2, Figure 2—figure supplement 6B–C), and 9,949 genes that do not change their expression level throughout the experiment which we term ‘non-response genes’. Response genes are enriched for genes previously identified to respond to hypoxia in a Caucasian population of individuals (Chi-squared test; p<2.2×10⁻¹⁶; Ward and Gilad, 2019), and are highly enriched for gene ontologies in transcription-related processes (see Materials and methods, modified Fisher’s exact test; p=1×10⁻¹⁹).

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Dynamic eQTLs are revealed following hypoxia

Having established that oxygen stress initiates a transcriptional response affecting thousands of genes, we sought to identify eQTLs, either before or after oxygen stress. Using the combined haplotype test (CHT), an approach that leverages allele-specific information in small sample sizes (see Materials and methods; van de Geijn et al., 2015), we identified 1,886 SNPs which associate with gene expression (eSNPs) in at least one condition resulting in 1,573 genes with eQTLs (eGenes) in at least one condition (q-value <0.1; A: 613; B: 564; C: 564 and D: 464; Figure 3A–B; Supplementary file 2). Given that cardiomyocytes were split from a single differentiation culture for each condition, we do not expect cell composition to bias the identification of eQTLs in each condition. Indeed, the distribution of eQTL effect sizes is similar across conditions (Figure 3—figure supplement 1A), and the eQTL effect size values are correlated across conditions (Figure 3—figure supplement 1B). In addition, we included the same number of individuals in each condition, RNA extraction batches included all conditions from a given individual, RIN scores are similar across conditions, and the number of sequencing reads are similar across conditions (Supplementary file 1). Together, these results suggest that our overall power to detect eQTLs is similar across conditions. We refer to the 613 eGenes identified in condition A as ‘baseline eGenes’.

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Our goal was to identify dynamic eQTLs, which are either revealed or suppressed as the cells transition between conditions. Due to the small sample size of our study, we have incomplete power to detect eQTLs in any condition; thus, a naive comparison of eQTLs classified as ‘significant’ across conditions will result in an over-estimation of the number of dynamic eQTLs. Indeed, the p-values of genes whose expression levels are not significantly associated with a SNP in any condition deviate from the expected uniform distribution (Kolmogorov–Smirnov test; p<2.2×10⁻¹⁶; Figure 3—figure supplement 2A). To address this challenge, we first considered eQTLs identified using a q-value <0.1 in at least one condition, and visualized the p-value distributions of the corresponding eQTL associations in all other conditions. These p-value distributions are expected to be uniform if we had complete power to detect eQTLs in any condition (because in that theoretical case, even a naive comparison of eQTLs classified as ‘significant’ across conditions will result in the identification of true condition-specific eQTLs). Due to incomplete power, this is clearly not the case (KS test; p<2.2×10⁻¹⁶; Figure 3—figure supplement 2B); however, this distribution allowed us to choose a lenient secondary p-value-based cutoff, where values deviate from the uniform distribution, to classify dynamic QTLs (p=0.15; Figure 3—figure supplement 2B).

We specifically focused on two dynamic scenarios. First, we defined suppressed eQTLs, as eQTLs that are identified in condition A at a q-value <0.1 but not in any of the other conditions, with a p-value greater than 0.15 (37 instances; Figure 3C). Second, we defined induced eQTLs as eQTLs identified in conditions B, C, or D at a q-value <0.1, but not in A, with a p-value greater than 0.15 (330 instances; Figure 3C). This set of 367 dynamic eQTLs corresponds to 328 unique dynamic eGenes (see Materials and methods) and includes ZNF845 and RFC2 (Figure 3E) as well as PPARGC1, C8orf82 and CELF1 (Figure 3—figure supplement 3). While our choice of the particular statistical cutoffs is somewhat arbitrary, we can evaluate the false discovery rate associated with our chosen cutoff. Based on the p-value distributions of the corresponding eQTL associations in all other conditions, we estimate that our approach to classify dynamic eQTLs is associated with a false discovery rate of 48%. The relatively high FDR associated with our choice of statistical cutoffs does not indicate that these loci are not eQTLs; rather it means that if we had a larger sample size, roughly half of our dynamic eQTLs should have been classified as eQTLs in more conditions, potentially in all of them. As expected, using a more stringent p-value cutoff of 0.9, we find fewer dynamic eQTLs (19) and a lower false discovery rate of 7%.

We next wanted to determine whether the dynamic eQTLs we identified in iPSC-CMs are also eQTLs in primary heart tissue. To do so, we compared our 367 dynamic eSNPs and eSNPs regulating the same gene in all four conditions (‘shared eQTLs’, n = 20) to eQTLs identified in left ventricle heart tissue and 49 tissues assayed from hundreds of individuals in the GTEx study (GTEx Consortium et al., 2017). Thirty-two of 326 dynamic eSNP-eGene pairs tested in GTEx are eQTLs in heart tissue (9.8%), and 140 are eQTLs in at least one other assayed tissue (42.9%) demonstrating that many of these genes have context-dependent inter-individual variation in expression. Six of 19 shared eSNP-eGene pairs tested in GTEx are eQTLs in heart tissue (31.6%), and six are an eQTL in at least one other tissue (31.6%). Shared eSNPs are therefore more likely to be eSNPs in heart tissue than dynamic eSNPs (Chi-squared test; p=0.01). To further investigate eQTL concordance, we compared the eQTL effect size of our dynamic and shared eQTLs to the eQTL effect size in heart tissue determined by the GTEx consortium. Shared eQTLs overlapping heart tissue eQTLs (n = 6) have a higher concordance of effect than dynamic eQTLs overlapping heart tissue eQTLs (n = 32; Spearman correlation = 0.73 vs. 0.12) suggesting that our perturbation study has revealed novel eQTL effects.

Chromatin accessibility changes following hypoxia and re-oxygenation

We next asked whether the hundreds of transcription factor expression changes following hypoxic stress are accompanied by global chromatin accessibility changes. To examine this, we performed ATAC-seq experiments in all four conditions to identify regions of open chromatin (we were only able to collect these data from 14 of the 15 individuals; Figure 1—figure supplement 1, see Materials and methods). We filtered the ATAC-seq reads to include only those reads that map unambiguously to the nuclear genome (see Materials and methods, Figure 4—figure supplement 1). We identified a set of open chromatin regions in each sample, and merged samples across individuals within each condition. Genomic regions identified as accessible in each condition were then merged to yield a set of 128,672 open chromatin regions across conditions (with a median length of 312 bp). Regions with low read counts were filtered out, resulting in a final set of 110,128 regions. Analysis of various metrics revealed the data to be of good quality (Figure 4—figure supplements 2–3).

We sought to identify chromatin regions that are differentially accessible across pairs of conditions. Using a sensitive adaptive shrinkage-based approach with a False Sign Rate of 10% (Stephens, 2017), we could not detect changes in accessibility between baseline and hypoxia across 110,128 regions; however, we identified 831 differentially accessible regions (DARs) between hypoxia and short-term re-oxygenation (BC-DARs; 429 regions with increased accessibility and 402 with decreased accessibility), and 71 DARs between hypoxia and long-term re-oxygenation (BD-DARs; Figure 4A). Despite the fact that we do not identify any DARs between normoxia and hypoxia, there is a strong anti-correlation in the effect size between the normoxia (A) and hypoxia (B) conditions, and the hypoxia (B) and re-oxygenation (C) conditions across regions (Spearman correlation = −0.62; sign test p=4.6×10⁻¹⁴; Figure 4B) suggesting minor changes in accessibility in response to hypoxia that do not meet our significance threshold. Conversely, there is a strong correlation in effect sizes between hypoxia and short-term re-oxygenation (BC-DARs), and hypoxia and long-term re-oxygenation (BD-DARs; Spearman correlation = 0.74; Figure 4C), and 59 of the 71 BD-DARs are amongst the 831 BC-DARs (83%), suggesting that most regions have returned to baseline levels of accessibility by the first re-oxygenation condition. This includes a region within the intron of the FOXO1 gene, a master regulator of the oxidative stress response (Figure 4D). We therefore considered the 831 BC-DARs, henceforth DARs, in further analysis. These DARs represent 0.8% of the total number of accessible regions.

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Genomic features associated with differentially accessible regions (DARs)

We next wanted to determine what distinguishes DARs from constitutively accessible regions (CARs). To do so, we investigated three classes of genomic features: (1) promoter- and enhancer-associated marks, (2) transcription factor binding locations, and (3) underlying DNA sequence features. We found that DARs are more likely to overlap TSS than constitutively accessible regions (43% overlap vs. 11% overlap; chi-squared test, p<2×10⁻¹⁶; Figure 5A) suggesting that DARs may be involved in the gene regulatory response. Indeed, DARs are more likely to coincide with active histone marks in left ventricle heart tissue than constitutively accessible regions (H3K4me3: 57% overlap DARs vs. 24% overlap constitutively accessible regions; chi-squared test; p<2.2×10⁻¹⁶; H3K4me1: 86% overlap DARs vs. 52% overlap constitutively accessible regions; p<2.2×10⁻¹⁶; Figure 5B).

Figure 5

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To determine whether sequence-specific hypoxia-responsive transcription factors associate with differentially accessible chromatin, we integrated DARs with published chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) data for the well-studied hypoxia-inducible factors HIF1α and HIF2α (Schödel et al., 2011). A total of 234 of the 356 HIF1α ChIP-seq binding sites (66%), and 150 of the 301 HIF2α binding sites (50%) overlap with our set of 110,128 accessible chromatin regions. We found that these HIF1α and HIF2α binding sites are more likely to overlap the 831 differentially accessible regions than the 109,275 constitutively accessible regions (Fisher test; p=0.03; Figure 5C).

We next took an unbiased approach to identify transcription factor binding motifs that are enriched in DARs compared to all accessible regions using MEME-ChIP software (see Materials and methods). We found two motifs to be enriched in DARs compared to all regions (Figure 5D). Motif 1 (p=2×10⁻²) is recognized by HTF4 and TFE2, both of which are non-response genes in our system. Motif 2 (p=6.2×10⁻⁴²) is posited to be recognized by ZN770, E2F3, and E2F4. Both ZN770 and E2F4 are response genes in our system. DARs arise between the hypoxia and re-oxygenation conditions, and E2F4 expression increases following re-oxygenation, suggesting that it may be involved in the response. To test this hypothesis, we obtained a published ChIP-seq data set for E2F4 (Lee et al., 2011), and overlapped the 16,245 E2F4-bound regions with DARs and constitutively accessible regions. E2F4-bound regions are significantly enriched in DARs compared to constitutively accessible regions (Chi-squared test; p<2.2×10⁻¹⁶; Figure 5D). E2F4 is important for survival following ischemia in neurons, and has been suggested to be an anti-apoptotic factor in cardiomyocytes (Dingar et al., 2012; Iyirhiaro et al., 2014).

To identify additional sequence features that associate with DARs, we asked whether transposable elements (TE), a potential source of regulatory sequence subjected to chromatin-level regulation, are enriched in these sites (Du et al., 2016; He et al., 2019). We found that while three of the main TE classes, LINEs, LTRs, and DNA elements are similarly enriched in DARs compared to constitutively accessible regions; SINEs are specifically enriched in DARs (p=6.5×10⁻⁶; Figure 5E). There is an enrichment of both Alu and MIR SINE family members in DARs (p=3×10⁻⁵ and p=0.006). AluS elements, and the AluSq and AluSp sub-families, are particularly enriched within the Alu family (p=0.007 and p=0.02 respectively). A different cellular stress, heat shock, has previously been shown to remodel chromatin accessibility at Alu elements in cervical cancer cells (Kim et al., 2001).

As Alu and MIR TE sequences are notably CpG dense (Medstrand et al., 2002), we next asked about the enrichment of CpG-dense CpG islands (CGIs) in our differentially accessible regions. We found that CpG islands are enriched in DARs compared to constitutively accessible regions, whether these regions fall within 1 kb of TSS, which are typically enriched for CGIs, or not (p<2.2×10⁻¹⁶; Figure 5F).

DNA methylation state at stress-responsive genes and chromatin regions

Genes with CGI promoters are thought to allow flexibility in TSS choice compared to genes without CGI promoters (Carninci et al., 2006), and to allow for the rapid induction of gene expression in response to stimuli (Ramirez-Carrozzi et al., 2009). We therefore asked whether this promoter feature is enriched in the stress response genes. Indeed, we find that response genes are more likely to have CGI promoters than non-response genes (Chi-squared test; p=0.002).

Given the enrichment of CpG islands in gene promoters and chromatin regions that are responsive to stress, we asked whether this feature corresponded to differences in CpG DNA methylation levels in these same regions. We measured global DNA methylation levels at 766,658 CpG sites in all conditions from 13 of our individuals (no data was collected from two of the 15 individuals), together with 23 replicate samples from three individuals (see Materials and methods; Supplementary file 1). We found the expected bimodal distribution of DNA methylation Beta-values across CpGs (Beta-values represent the ratio of intensities between the methylated and unmethylated alleles; Figure 6—figure supplement 1A). Additional analyses indicated the data to be of good quality (Figure 6—figure supplements 1–2).

To determine whether steady-state DNA methylation levels mark genes or regions that will change their expression level in response to stress, we investigated baseline DNA methylation levels in the promoters of genes classified as response genes and non-response genes, as well as accessible regions and DARs. To do so, we assessed the DNA methylation level at CpGs within 200 bp upstream of the TSS in the baseline condition (condition A). The majority of the assayed CpGs were hypomethylated with a median Beta-value of less than 0.2 across genes and regions (Figure 6A). While there is no difference in median DNA methylation levels between response and non-response genes, we found that the median DNA methylation level is lower in DARs compared to all accessible regions (p<2.2×10⁻¹⁶). These data suggest that responsive chromatin regions may have specific epigenetic profiles which poise them for rapid response to stress.

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Dynamic eQTLs associate with traits and disease

Finally, we wanted to determine whether any of the dynamic eQTL SNPs or genes that we identified might also be associated with complex traits or disease. To maximize the number of potentially phenotypically-relevant genomic loci, we performed three independent analyses. We first took an unbiased, SNP-based approach by searching within a catalog of genetic variants associated with a variety of traits assayed in thousands of GWAS for overlap with our dynamic eSNPs (Buniello et al., 2019). By intersecting these two data sets, we found one induced dynamic eSNP (rs8053350) that is also associated with a measured phenotype in GWAS – varicose veins (Supplementary file 1; Figure 7—figure supplement 1A-B).

Given that our eQTL data are from the Yoruba population from West Africa and that most GWAS studies are performed in European populations, which have an inherently different LD structure, we may not expect to see strong overlap at the level of individual variants. We therefore next took an orthogonal gene-based approach, using the same GWAS catalog, to specifically investigate the genes implicated in three phenotypes associated with cardiovascular function or response to oxygen deprivation: MI, heart failure, and stroke (see Materials and methods). If the expression of these CVD genes is found to vary across individuals following hypoxia, it suggests that they may be important for mediating disease risk. Indeed, we found that six of our dynamic eGenes are implicated in these three disease states by GWAS gene mapping approaches (Supplementary file 1). This list includes the DNA damage and apoptosis factor ZC3HC1, which is implicated in MI and stroke (Figure 7—figure supplement 1C–D). Importantly, ZC3HC1 is not an eGene in LV or AA, but the SNP-gene pair is an eQTL in other tissues.

Lastly, we performed an in-depth SNP-based analysis using full summary statistics for coronary artery disease (CAD) and MI from the CARDIoGRAMplusC4D Consortium (Stitziel et al., 2016; Nikpay et al., 2015; Webb et al., 2017). We tested whether the lead eSNP from our set of dynamic eQTLs is also associated with CAD or MI and identified two loci (Supplementary file 1). This set includes the eSNP (rs12588981) for the dynamic eQTL EIF2B2 which is nominally associated with CAD (GWAS association p=4.7×10⁻⁵) and is in high and statistically significant LD with the lead GWAS variant (GWAS association p=9.9×10⁻⁸) at this locus (rs3832966; R² = 0.96, p<0.001; Figure 7; Figure 7—figure supplement 2). Given that we have incomplete power to detect dynamic eQTLs, we also used the same GWAS dataset to investigate whether any of the significant eQTL SNPs in any condition are associated with CAD or MI. We determined that eSNP (rs8105092), which regulates CARM1 in the hypoxic condition (condition B), is nominally associated with MI (GWAS association p=1.79×10⁻⁵) and in moderate, yet significant LD, with the lead GWAS variant (GWAS association p=2.8×10⁻⁹) at this locus (rs4804142; R² = 0.32, p<0.001; Figure 7—figure supplement 3), and is not an eGene in LV or AA. A handful of the eQTLs that we highlight here were tested for association with gene expression by the GTEx consortium, yet are not eQTLs in heart tissue. These results suggest that perturbation studies in relevant cell types can give insight into the molecular basis for the genetic association with complex traits and disease, which might not be gleaned from the study of post-mortem tissues.

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Studying gene expression across individuals in response to stress can reveal latent effects of genetic variation, which may contribute to higher-order phenotypes and disease. In order to understand the effects of genetic variation in a disease-relevant cell type and a disease-relevant process, we differentiated cardiomyocytes from a panel of genotyped individuals, and subjected them to hypoxia and re-oxygenation. We found hundreds of eQTLs that are revealed or suppressed following hypoxic stress (dynamic eQTLs), several of which have been associated with phenotypes measured in GWAS.

Sequencing data have been deposited in GEO under accession codes GSE144426.

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   ID GSE144426. Dynamic effects of genetic variation on gene expression revealed following hypoxic stress in cardiomyocytes.

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144426

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Author details

1. Michelle C Ward

   1. Department of Medicine, University of Chicago, Chicago, United States
   2. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United States

   Present address

   Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Project administration

   Contributed equally with

   Nicholas E Banovich

   For correspondence

   miward@utmb.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1485-320X

2. Nicholas E Banovich

   1. Department of Human Genetics, University of Chicago, Chicago, United States
   2. Integrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, United States

   Present address

   Integrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Michelle C Ward

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2604-3247

3. Abhishek Sarkar

   Department of Human Genetics, University of Chicago, Chicago, United States

   Contribution

   Formal analysis, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4636-9255

4. Matthew Stephens

   1. Department of Human Genetics, University of Chicago, Chicago, United States
   2. Department of Statistics, University of Chicago, Chicago, United States

   Contribution

   Supervision

   Competing interests

   No competing interests declared

5. Yoav Gilad

   1. Department of Medicine, University of Chicago, Chicago, United States
   2. Department of Human Genetics, University of Chicago, Chicago, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - review and editing

   For correspondence

   gilad@uchicago.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8284-8926

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