Functional Single Nucleotide Polymorphisms (SNPs) in the Genes Encoding the Human Deoxyribonuclease (DNase) Family Potentially Relevant to Autoimmunity

Objective: To continue our previous investigations, we have extensively investigated the function of the 61, 41, and 35 non-synonymous single nucleotide polymorphisms (SNPs) in the human genes encoding DNASE1, DNASE1L3, and DNASE2, respectively, potentially relevant to autoimmune diseases. Methods: The site-directed mutagenesis was employed to amino acid–substituted constructs corresponding to each SNP. The COS-7 cells were transfected with each vector and DNase activity was assayed by the single radial enzyme diffusion method. By using PolyPhen-2, changes in the DNase function of each non-synonymous SNP were predicted. Genotyping of all the non-synonymous SNPs was performed in 14 different populations including 3 ethnic groups using the polymerase chain reaction followed by the restriction fragment length polymorphism method. Results: Expression analysis demonstrated these SNPs to be classified into four categories with regard to the effect on DNase activity: SNPs not affecting the activity level, ones reducing it, ones abolishing it, and ones elevating it. In particular, 9, 5, and 4 SNPs producing a loss-of-function variant of the enzymes in DNASE1, DNASE1L3, and DNASE2, respectively, were confirmed. SNPs producing DNase loss of function can be estimated by PolyPhen-2 to be “probably damaging” with a high accuracy of prediction. Almost all of these functional SNPs producing a loss of function or substantially low activity-harboring forms exhibited a mono-allelic distribution in all of the populations. Conclusion: A minor allele of functional SNPs, despite the remarkably low genetic heterogeneity of the SNPs, might be a genetic risk factor for autoimmune diseases.研究目的：为延续前期研究工作，本研究针对分别编码DNASE1、DNASE1L3与DNASE2的人类基因中61、41及35个非同义单核苷酸多态性（non-synonymous single nucleotide polymorphism, SNP，以下简称SNPs）展开系统性研究，上述位点或与自身免疫性疾病存在潜在关联。 研究方法：本研究采用定点诱变技术构建对应各SNP的氨基酸替换重组载体。将各载体转染至COS-7细胞中，通过单径向酶扩散法检测脱氧核糖核酸酶（DNase）活性。借助PolyPhen-2工具，预测各非同义SNP对DNase功能的影响。本研究采用聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）法，对涵盖3个族群的14个不同人群中的所有非同义SNPs进行基因分型。 研究结果：表达分析结果显示，根据对DNase活性的影响，上述SNPs可分为四类：不影响活性水平的SNP、降低活性的SNP、完全丧失活性的SNP以及提升活性的SNP。其中，在DNASE1、DNASE1L3与DNASE2基因中，分别证实存在9个、5个及4个可导致对应酶功能丧失的SNP。经PolyPhen-2预测，可导致DNase功能丧失的SNPs多被归类为"可能具有致病性"，且预测准确率较高。上述所有可导致酶功能丧失或活性显著降低的功能性SNPs，在所有研究人群中均呈现单等位基因分布特征。 研究结论：尽管上述SNPs的遗传异质性极低，但其中功能性SNPs的次要等位基因或可作为自身免疫性疾病的遗传风险因子。