From the Department of Medical Biology, Amsterdam University Medical Centers, Academic Medical Center, the Netherlands (A.F.v.O., F.M.B., K.v.D., P.B., V.M.C.).
Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, the Netherlands (J.L., J.Z., D.P., A.A.F.d.V.).
Circ Res. 2020 Jul 3;127(2):229-243. doi: 10.1161/CIRCRESAHA.119.316006. Epub 2020 Apr 6.
Genome-wide association studies have identified a large number of common variants (single-nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located mainly in noncoding regions of the genome and likely include variants that modulate the function of transcriptional regulatory elements (REs) such as enhancers. However, the actual REs modulated by variants and the target genes of such REs remain to be identified. Thus, the biological mechanisms by which genetic variation promotes AF has thus far remained largely unexplored.
To identify REs in genome-wide association study loci that are influenced by AF-associated variants.
We screened 2.45 Mbp of human genomic DNA containing 12 strongly AF-associated loci for RE activity using self-transcribing active regulatory region sequencing and a recently generated monoclonal line of conditionally immortalized rat atrial myocytes. We identified 444 potential REs, 55 of which contain AF-associated variants (<10). Subsequently, using an adaptation of the self-transcribing active regulatory region sequencing approach, we identified 24 variant REs with allele-specific regulatory activity. By mining available chromatin conformation data, the possible target genes of these REs were mapped. To define the physiological function and target genes of such REs, we deleted the orthologue of an RE containing noncoding variants in the (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) locus of the mouse genome. Mice heterozygous for the RE deletion showed bradycardia, sinus node dysfunction, and selective loss of expression.
We have identified REs at multiple genetic loci for AF and found that loss of an RE at the locus results in sinus node dysfunction and reduced gene expression. Our approach can be broadly applied to facilitate the identification of human disease-relevant REs and target genes at cardiovascular genome-wide association studies loci.
全基因组关联研究已经确定了大量与心房颤动（AF）相关的常见变体（单核苷酸多态性）。这些变体主要位于基因组的非编码区域，可能包括调节转录调控元件（REs）如增强子功能的变体。然而，变体调节的实际 REs 和这些 REs 的靶基因仍有待确定。因此，遗传变异促进 AF 的生物学机制迄今在很大程度上仍未得到探索。
鉴定全基因组关联研究位点中受 AF 相关变体影响的 REs。
我们使用自我转录活性调控区测序和最近生成的条件永生化大鼠心房肌细胞单克隆系，筛选包含 12 个强烈与 AF 相关的位点的 2.45 Mbp 人类基因组 DNA，以检测 RE 活性。我们鉴定了 444 个潜在的 RE，其中 55 个含有 AF 相关变体（<10）。随后，我们使用自我转录活性调控区测序方法的一种改编，鉴定了 24 个具有等位基因特异性调控活性的变体 RE。通过挖掘可用的染色质构象数据，映射了这些 RE 的可能靶基因。为了定义这些 RE 的生理功能和靶基因，我们删除了小鼠基因组中 （钾/钠离子超极化激活环核苷酸门控通道 4） 位点含非编码变体的 RE 同源物。RE 缺失杂合子的小鼠表现出心动过缓、窦房结功能障碍和 表达选择性丧失。
我们已经确定了多个与 AF 相关的遗传位点的 RE，并发现 基因座中 RE 的缺失导致窦房结功能障碍和基因表达减少。我们的方法可以广泛应用于促进心血管全基因组关联研究位点的人类疾病相关 RE 和靶基因的鉴定。
