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Cardiomyopathy (CMP) is a heritable genetic disorder. Protein-coding variants account for 20-30% of cases. The contribution of variants in non-coding DNA elements that regulate gene expression has not been explored. We performed whole-genome sequencing (WGS) of 228 unrelated CMP families. Besides pathogenic protein-coding variants in known CMP genes, 5% cases harbored rare loss-of-function variants in novel cardiac genes, with NRAP and FHOD3 being strong candidates. WGS also revealed a high burden of high-risk variants in promoters and enhancers of CMP genes in an additional 20% cases (Odds ratio 2.14, 95% CI 1.60-2.86, p=5.26×10−7 vs 1326 controls) with genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) specifically enriched for regulatory variants (False discovery rate

Background Gene expression differences between species are driven by both cis and trans effects. Whereas cis effects are caused by genetic variants located on the same DNA molecule as the target gene, trans effects are due to genetic variants that affect diffusible elements. Previous studies have mostly assessed the impact of cis and trans effects at the gene level. However, how cis and trans effects differentially impact regulatory elements such as enhancers and promoters remains poorly understood. Here, we use massively parallel reporter assays to directly measure the transcriptional outputs of thousands of individual regulatory elements in embryonic stem cells and measure cis and trans effects between human and mouse. Results Our approach reveals that cis effects are widespread across transcribed regulatory elements, and the strongest cis effects are associated with the disruption of motifs recognized by strong transcriptional activators. Conversely, we find that trans effects are rare but stronger in enhancers than promoters and are associated with a subset of transcription factors that are differentially expressed between human and mouse. While we find that cis-trans compensation is common within promoters, we do not see evidence of widespread cis-trans compensation at enhancers. Cis-trans compensation is inversely correlated with enhancer redundancy, suggesting that such compensation may often occur across multiple enhancers. Conclusions Our results highlight differences in the mode of evolution between promoters and enhancers in complex mammalian genomes and indicate that studying the evolution of individual regulatory elements is pivotal to understand the tempo and mode of gene expression evolution.

Gene expression differences between species are driven by bothcisandtranseffects. Whereasciseffects are caused by genetic variants in close proximity to the target gene,transeffects are due to distal genetic variants that affect diffusible elements such as transcription factors. Previous studies have mostly assessed the impact ofcisandtranseffects at the gene level. However, howcisandtranseffects differentially impact regulatory elements such as enhancers and promoters remains poorly understood. Here, we used massively parallel reporter assays to directly measurecisandtranseffects between human and mouse embryonic stem cells at thousands of individual regulatory elements. Our approach revealed thatciseffects are widespread across regulatory elements, and the strongestciseffects are associated with the disruption of motifs recognized by strong transcriptional activators. Conversely, we found thattranseffects are rare but stronger in enhancers than promoters, and can be attributed to a subset of transcription factors that are differentially expressed between human and mouse. While previous studies have found extensive co-occurrence ofcisandtranseffects in opposite directions that stabilize gene expression throughout evolution, we find thatcis-transcompensation is uncommon within individual regulatory elements. Thus, our results are consistent with a model wherein compensatorycis-transeffects at the gene level are explained bycisandtranseffects that separately impact several regulatory elements rather thancis-transeffects that occur simultaneously within a single regulatory element. Together, these results indicate that studying the evolution of individual regulatory elements is pivotal to understand the tempo and mode of gene expression evolution.