Interinstitutional Posgrade in Science and Technology (PICYT), Research Center of Technology and Design Assistance of Jalisco State, (CIATEJ A.C.), Guadalajara, Mexico.
Laboratory of Regulatory SNPs, Personalized Medicine National Laboratory (LAMPER), Pharmaceutical and Medical Biotechnology, Central Unit, CIATEJ A.C., National Council of Science and Technology (CONACYT), Guadalajara, Mexico.
Int J Immunogenet. 2020 Aug;47(4):332-341. doi: 10.1111/iji.12475. Epub 2020 Jan 29.
The prediction of regulatory single nucleotide polymorphisms (rSNPs) in proximal promoters of disease-related genes could be a useful tool for personalized medicine in both patient stratification and customized therapy. Using our previously reported method of rSNPs prediction (currently a software called SNPClinic v.1.0) as well as with PredictSNP tool, we performed in silico prediction of regulatory SNPs in the antimicrobial peptide human β-defensin 1 gene in three human cell lines from 1,000 Genomes Project (1kGP), namely A549 (epithelial cell line), HL-60 (neutrophils) and T 1 (lymphocytes). These predictions were run in a proximal pseudo-promoter comprising all common alleles on each polymorphic site according to the 1,000 Genomes Project data (1kGP: ALL). Plasmid vectors containing either the major or the minor allele of a putative rSNP rs5743417 (categorized as regulatory by SNPClinic and confirmed by PredictSNP) and a non-rSNP negative control were transfected to lung A549 human epithelial cell line. We assessed functionality of rSNPs by qPCR using the Pfaffl method. In A549 cells, minor allele of the SNP rs5743417 G→A showed a significant reduction in gene expression, diminishing DEFB1 transcription by 33% when compared with the G major allele (p-value = .03). SNP rs5743417 minor allele has high frequency in Gambians (8%, 1kGP population: GWD) and Afro-Americans (3.3%, 1kGP population: ASW). This SNP alters three transcription factors binding sites (TFBSs) comprising SREBP2 (sterols and haematopoietic pathways), CREB1 (cAMP, insulin and TNF pathways) and JUND (apoptosis, senescence and stress pathways) in the proximal promoter of DEFB1. Further in silico analysis reveals that this SNP also overlaps with GS1-24F4.2, a lincRNA gene complementary to the X Kell blood group related 5 (XKR5) mRNA. The potential clinical impact of the altered constitutive expression of DEFB1 caused by rSNP rs5743417 in DEFB1-associated diseases as tuberculosis, COPD, asthma, cystic fibrosis and cancer in African and Afro-American populations deserves further research.
预测疾病相关基因近端启动子中的调控单核苷酸多态性（rSNP）可能成为个体化医学中患者分层和定制治疗的有用工具。我们使用先前报道的 rSNP 预测方法（目前称为 SNPClinic v.1.0 软件）和 PredictSNP 工具，对来自 1000 基因组计划（1kGP）的三种人类细胞系中的抗菌肽人β-防御素 1 基因的调控 SNP 进行了计算机预测，这三种细胞系分别为 A549（上皮细胞系）、HL-60（中性粒细胞）和 T1（淋巴细胞）。这些预测是在一个近端伪启动子中进行的，该启动子包含根据 1000 基因组计划数据（1kGP：ALL）每个多态性位点的所有常见等位基因。包含假定 rSNP rs5743417 的主要或次要等位基因的质粒载体（根据 SNPClinic 分类为调控，并通过 PredictSNP 证实）和非 rSNP 阴性对照被转染到肺 A549 人上皮细胞系。我们使用 Pfaffl 方法通过 qPCR 评估 rSNP 的功能。在 A549 细胞中，SNP rs5743417 的 G→A 次要等位基因显示出基因表达的显著减少，与 G 主要等位基因相比，DEFB1 转录减少了 33%（p 值=0.03）。SNP rs5743417 的次要等位基因在冈比亚人（8%，1kGP 人群：GWD）和非裔美国人（3.3%，1kGP 人群：ASW）中具有高频率。该 SNP 改变了近端 DEFB1 启动子中三个转录因子结合位点（TFBS），包括 SREBP2（固醇和造血途径）、CREB1（cAMP、胰岛素和 TNF 途径）和 JUND（凋亡、衰老和应激途径）。进一步的计算机分析表明，该 SNP 还与 GS1-24F4.2 重叠，后者是与 X 型 Kell 血型相关 5（XKR5）mRNA 互补的 lincRNA 基因。rSNP rs5743417 在 DEFB1 相关疾病（如肺结核、COPD、哮喘、囊性纤维化和癌症）中引起的 DEFB1 组成型表达改变的潜在临床影响值得进一步研究。
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