肾脏阴离子转运相关基因变异与肾结石发病风险的关系研究

发布时间：2018-04-18 22:16

本文选题：肾结石 + SLC26A6蛋白　；参考：《辽宁大学》2017年硕士论文

【摘要】：肾结石是由遗传因素和饮食环境等因素引起的一种常见的、多发的复杂疾病,具有较高的复发率,形成机制尚不明确,目前缺乏较为有效的预防和治疗措施。草酸钙结石是目前最为常见的肾结石类型,约占全部肾结石种类的80%以上。草酸是生物体中的一种常见代谢产物,能够使矿质元素的生物利用率降低,在人体中容易与钙离子结合,形成草酸钙,进而导致肾结石疾病的发生。尿中焦磷酸是重要的结晶形成抑制物,可降低尿中钙、磷等离子的过饱和状态,在肾小管中可结合到磷酸钙表面,抑制结晶进一步形成。SLC26A6蛋白是一个多功能的阴离子转运蛋白,尤其在转运草酸阴离子的过程中发挥着重要的作用。进行性关节强直基因同源物ANKH(progressive ankylosis gene homolog,Ank/ANKH)是一种无机焦磷酸盐的转运蛋白(inorganic pyrophosphate transport regulator),能够在肾脏表达,在集合管的腔面和基底面,向肾小管和肾间质转运焦磷酸。推测这两种蛋白可能在肾结石形成过程中发挥重要作用。本文首先以SLC26A6基因和ANKH基因为对象,联合采用几种基因致病型SNP预测生物信息学软件,分别预测出两种基因可能导致疾病的SNPs。然后以肾结石疾病患者和健康人的血液样本进行基因型检测,以验证预测的两种基因的致病型SNPs是否与肾结石的发病相关,并研究其基因多态性与肾结石疾病发病风险的关系。我们从肾结石疾病患者和健康人的血液中提取人全基因组DNA,并以其为PCR扩增模板,通过等位基因特异性PCR及基因测序方法对SLC26A6基因中经生物信息学软件预测得到的1个候选SNP以及ANKH基因中的14个可能的致病候选SNPs进行基因定型。相关分析发现,在SLC26A6基因中,肾结石患者nsSNP rs184187143的C等位基因率显著高于无肾结石病史以及结石疾病家族史的健康人对照样本(OR 6.1,95%CI 1.36-27.38,p=0.007)。而所选择验证的ANKH基因中14个位点的SNPs与东北地区汉族草酸钙肾结石发病的风险并无明显关系。最后,我们利用分子动力学方法对SLC26A6基因致病nsSNP rs184187143导致肾结石患病风险增加的分子机制进行进一步研究,结果发现SLC26A6基因致病nsSNP rs184187143位点处,等位基因G到C的变化(导致氨基酸从甘氨酸到精氨酸的突变)可能通过影响蛋白的二级结构,影响其草酸离子转运功能,从而与肾结石发病风险相关。综上,本课题首次证明了SLC26A6基因多态性变异nsSNP rs184187143影响中国东北地区汉族草酸钙肾结石发病风险,暗示草酸转运过程的异常可能贡献于肾结石的形成。这一研究成果为肾结石疾病的复杂分子机制的研究提供了新的思路。
[Abstract]:Kidney stone is a common and complex disease caused by genetic factors and dietary environment. It has a high recurrence rate and its formation mechanism is not clear. At present, it is lack of effective prevention and treatment measures.Calcium oxalate stone is the most common type of renal calculi, accounting for more than 80% of all renal calculi.Oxalic acid is a common metabolite in organism, which can reduce the bioavailability of mineral elements, bind easily with calcium ion in human body, form calcium oxalate, and lead to the occurrence of kidney stone disease.Urinary pyrophosphoric acid is an important inhibitor of crystallization formation, which can reduce the supersaturated state of calcium and phosphorus plasma in urine. It can bind to the surface of calcium phosphate in renal tubules and inhibit the further formation of crystallization. SLC26A6 protein is a multifunctional anion transporter.Especially in the process of oxalic acid anion transport plays an important role.The progressive ankylosis gene congener, ANKH(progressive ankylosis gene homologue, is an inorganic pyrophosphate transport protein, the organic pyrophosphate transport regulator, that can be expressed in the kidney and transport pyrophosphoric acid to the renal tubules and the interstitial surface in the lumen and basal plane of the collecting duct.It is speculated that these two proteins may play an important role in the formation of renal calculi.In this paper, SLC26A6 gene and ANKH gene were used to predict SLC26A6 gene and ANKH gene respectively by using several gene pathogenic SNP predictive bioinformatics software.Then genotypes were detected in the blood samples of patients with renal stone disease and healthy people to verify whether the predicted pathogenic SNPs of the two genes was related to the pathogenesis of renal calculi and to study the relationship between the gene polymorphism and the risk of renal stone disease.We extracted human genomic DNA from the blood of patients with kidney stone disease and healthy people and used it as a template for PCR amplification.Allele-specific PCR and gene sequencing were used to identify one candidate SNP and 14 possible pathogenicity candidate SNPs in SLC26A6 gene and ANKH gene predicted by bioinformatics software.Correlation analysis showed that the C allele rate of nsSNP rs184187143 in patients with renal calculi was significantly higher than that in healthy controls with no history of renal calculi and family history of calculi.However, there was no significant correlation between SNPs of 14 loci of the selected ANKH gene and the risk of calcium oxalate nephrolithiasis in Northeast China.Finally, we further studied the molecular mechanism of the increase in the risk of renal calculi caused by SLC26A6 gene pathogenic nsSNP rs184187143 by molecular dynamics. The results showed that the site of SLC26A6 gene pathogenic nsSNP rs184187143 was found.The change of allele G to C (leading to the mutation of amino acid from glycine to arginine) may affect the oxalate ion transport function of the protein by affecting its secondary structure, which may be related to the risk of renal calculi.In conclusion, this study for the first time proved that SLC26A6 gene polymorphism nsSNP rs184187143 affects the risk of calcium oxalate nephrolithiasis in Northeast China, suggesting that the abnormal oxalic acid transport process may contribute to the formation of renal calculi.This study provides a new idea for the study of complex molecular mechanism of renal stone disease.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.4

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