一例HSD11B2新突变位点所致的AME及新型生物学标志物与2型糖尿病DKD进展相关性初探

发布时间：2018-04-01 14:17

本文选题：表象性盐皮质激素增多征　切入点：11β-羟化类固醇脱氢酶2　出处：《重庆医科大学》2017年博士论文

【摘要】：目的:表象性盐皮质激素增多征(AME)是一种由11β-羟化类固醇脱氢酶2(HSD11B2)突变所致的罕见继发性高血压。本研究旨在报道一例由HSD11B2新的突变位点所致的AME,并对突变位点进行功能验证,以探索其致病原因。方法:采用尿皮质醇/皮质素比值评估体内11β-HSD2的活性。采集患者及一级亲属的外周血细胞,进行基因测序。在体外,将野生型或位点突变型11β-HSD2质粒转染进HEK293FT,并通过酶促反应验证突变位点对酶活性的影响。分子对接试验通过Modeller 9.15以及Autodock Vina 1.1.2软件完成。结果:患者表现为高血压、低血钾、低血浆醛固酮、低血浆肾素以及尿皮质醇/皮质素比例明显增高等特征。二代测序发现,患者HSD11B2基因存在一个错义突变,即第5外显子1088位的胞嘧啶突变为胸腺嘧啶,导致363位的氨基酸由亮氨酸变为脯氨酸。一代测序验证了上述突变,并且发现其父母均为该位点单等位基因突变的杂合携带子。体外研究中发现,p.L363P突变型11β-HSD2酶活性明显受抑制。分子对接提示该位点突变后使11β-HSD2与其底物皮质醇结合的亲和力降低,从而影响了酶的活性。结论:本研究报道了一例因HSD11B2新的突变位点所致的AME,并且该突变位点的致病性在于其改变了1β-HSD2与底物的亲和力,从而使酶活性下降。目的:本研究旨在通过一项前瞻性队列研究,在伴有微量蛋白尿的糖尿病人群中,从基线横断面及前瞻性分析两个角度,探讨糖尿病肾病(DKD)新型生物学标志物(包括s TNFR1、s TNFR2、RBP)与肾功能变化的关系,寻找能够较好反映DKD肾功能变化的标志物。方法:研究对象为160名伴有微量白蛋白尿的糖尿病患者,在基线以及随访6月后,监测患者的一般情况、肾功能情况,以及s TNFR1、s TNFR2、RBP等DKD早期预测指标的水平。采用相关分析及多元线性回归分析上述指标与尿白蛋白/肌酐(UACR),24小时尿蛋白定量(UAE),估算肾小球滤过率(e GFRcr-cys)等肾功能指标的相关性。结果:在基线时,RBP、s TNFR1、s TNFR2与e GFRcr-cys均显著性相关(R值分别为-0.523,-0.334,-0.392),采用多元线性回归分析矫正后,上述相关性仍存在。基线RBP、s TNFR1、s TNFR2与6个月后的e GFRcr-cys均呈独立相关(R值分别为-0.254,-0.323,-0.401);6个月后,RBP变化值(△RBP)与△e GFRcr-cys密切相关(R=-0.699,P0.001),采用多元线性回归分析矫正年龄、病程、BMI、腰围、臀围、血压等混杂因素后,△RBP与△e GFRcr-cys仍然显著相关。结论:基线s TNFR1、s TNFR2、RBP均能独立预测DKD患者6个月后的肾功能情况;RBP增加与e GFR下降独立相关。
[Abstract]:Objective: HSD11B2 is a rare secondary hypertension caused by 11 尾 -hydroxysteroid dehydrogenase (HSD11B2) mutation. Methods: the activity of 11 尾 -HSD2 in vivo was evaluated by the ratio of urinary cortisol to cortisol. Peripheral blood cells of patients and their first-degree relatives were collected and sequenced. The wild-type or locus mutant 11 尾 -HSD2 plasmid was transfected into HEK293FT2.The effect of mutation site on enzyme activity was verified by enzymatic reaction. The molecular docking test was performed by Modeller 9.15 and Autodock Vina 1.1.2.Results: the patients showed hypertension and hypokalemia. Low plasma aldosterone, low plasma renin and urinary cortisol / cortisol ratio were significantly increased. The second generation sequencing showed that there was a missense mutation in the HSD11B2 gene, that is, the cytosine mutation at position 1088 of exon 5 was thymine. The amino acid at position 363 changed from leucine to proline. The first generation sequencing confirmed the mutation. It was also found that both parents were heterozygous carriers of the single allelic mutation at this locus. In vitro studies showed that the enzyme activity of 11 尾 -HSD2 mutated L363P was significantly inhibited. The molecular docking indicated that the affinity of 11 尾 -HSD2 to cortisol was decreased after the mutation. Conclusion: a new mutation site of HSD11B2 was reported in this study. The pathogenicity of the mutant site is that it changes the affinity of 1 尾 -HSD2 to the substrate. Objective: to investigate the baseline cross-sectional and prospective analysis of diabetic patients with microalbuminuria through a prospective cohort study. To investigate the relationship between the changes of renal function and the changes of renal function in diabetic nephropathy patients (including s TNFR1 / s TNFR2rRBP). Methods: 160 diabetic patients with microalbuminuria were studied. At baseline and 6 months after follow-up, the patient's general condition, renal function, and renal function were monitored. The levels of early DKD predictors such as s TNFR1 and s TNFR2RBP. The correlation analysis and multivariate linear regression analysis were used to estimate the renal function indexes such as urinary albumin / creatinine creatinine 24 hour urinary protein quantitative UAEI, and the glomerular filtration rate (GFRcr-cys). Results: there were significant correlation R values between TNFR2 and e GFRcr-cys at baseline. The values of TNFR2 and e GFRcr-cys were -0.523 ~ 0.334U ~ (-0.392g), respectively. After correction by multivariate linear regression analysis, there was no significant correlation between RBP / TNFR _ (1) and e GFRcr-cys. The correlation between baseline RBPs TNFR1s TNFR2 and e GFRcr-cys after 6 months were -0.254 and 0.323- 0.401, respectively. After 6 months, there was a close correlation between RBPs and e GFRcr-cys. Multiple linear regression analysis was used to correct the age, the course of disease, waist circumference, hip circumference, hip circumference, and so on. Conclusion: baseline s TNFR1 / s TNFR2RBP can independently predict renal function in patients with DKD after 6 months. There is an independent correlation between the increase of RBP and the decrease of e GFR.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R544.1

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