Disease-associated non-coding variants alter NKX2-5 DNA-binding affinity.
Disease-associated non-coding variants alter NKX2-5 DNA-binding affinity.
复制标题
疾病相关的非编码变异会改变 NKX2-5 DNA 结合亲和力。
DOI：
10.1016/j.bbagrm.2023.194906
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Rodríguez-Martínez,JoséA
中科院分区：
文献类型：
--
作者：
Peña-Martínez,EdwinG;Rivera-Madera,Alejandro;Pomales-Matos,DiegoA;Sanabria-Alberto,Leandro;Rosario-Cañuelas,BrittanyM;Rodríguez-Ríos,JessicaM;Carrasquillo-Dones,EmanuelA;Rodríguez-Martínez,JoséA
全基因组关联研究(Gwas)已经在非编码基因组中定位了超过90%的与疾病或性状相关的变异，如调控元件(CRE)。非编码单核苷酸多态(SNPs)是一种基因组变异，它可以改变DNA结合调节蛋白，如转录因子(TF)与基因组相互作用的方式，并调节基因表达。Nkx2-5是心脏正常发育所必需的转铁蛋白，影响其功能的突变与先天性心脏病(CHDS)有关。然而，建立非编码基因组变异与人类疾病之间的因果机制仍然具有挑战性。为了应对这一挑战，我们使用基于位置权重矩阵(PWM)的预测模型确定了8475个SNPs，这些SNP预测会改变NKX2-5DNA结合。五个变种被优先进行体外验证；其中四个与影响心血管健康的特征和疾病有关。使用纯化的重组NKX2-5同源结构域，用凝胶迁移率改变分析(EMSA)评价这些突变体对NKX2-5结合的影响。构建结合曲线以确定变异等位基因和参考等位基因之间结合的变化。与参考基因组相比，突变体rs7350789、rs7719885、rs747334和rs3892630增加了结合亲和力，而rs61216514降低了NKX2-5的结合。我们的发现表明，不同的TF-DNA结合亲和力可能是建立致病变异原因机制的关键。
Genome-wide association studies (GWAS) have mapped over 90 % of disease- or trait-associated variants within the non-coding genome, likecis-regulatory elements (CREs). Non-coding single nucleotide polymorphisms (SNPs) are genomic variants that can change how DNA-binding regulatory proteins, like transcription factors (TFs), interact with the genome and regulate gene expression. NKX2–5 is a TF essential for proper heart development, and mutations affecting its function have been associated with congenital heart diseases (CHDs). However, establishing a causal mechanism between non-coding genomic variants and human disease remains challenging. To address this challenge, we identified 8475 SNPs predicted to alter NKX2-5 DNA-binding using a position weight matrix (PWM)-based predictive model. Five variants were prioritized for in vitro validation; four of them are associated with traits and diseases that impact cardiovascular health. The impact of these variants on NKX2-5 binding was evaluated with electrophoretic mobility shift assay (EMSA) using purified recombinant NKX2-5 homeodomain. Binding curves were constructed to determine changes in binding between variant and reference alleles. Variants rs7350789, rs7719885, rs747334, and rs3892630 increased binding affinity, whereas rs61216514 decreased binding by NKX2-5 when compared to the reference genome. Our findings suggest that differential TF-DNA binding affinity can be key in establishing a causal mechanism of pathogenic variants.