家族性肾性糖尿基因与功能诊断方法建立及临床应用

发布时间：2018-05-16 22:34

本文选题：家族性肾性糖尿 + 钠葡萄糖共转运子2　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究背景家族性肾性糖尿主要表现为血糖正常,而尿糖阳性,是编码肾脏近端小管刷状缘钠葡萄糖共转运子 2(sodium glucose cotransporter 2,SGLT2)的 SLC5A2 基因突变所致。近年来,SGLT2抑制剂作为新型降糖药,因其长期肾脏和心脏保护作用广受关注。但其先天异常所致的肾性糖尿,作为重要的肾小管转运蛋白缺陷疾病,并未受到足够的重视,国内外报告的病例数均有限,缺乏定量的体内功能试验,对其调节基因、及与近端小管其他物质转运关系的研究也较少。研究目的1.建立家族性肾性糖尿的基因诊断方法,包括体外功能试验用于验证新变异的致病性;建立遗传性肾小管转运子异常疾病筛查的高通量测序方案Tubule Panel,筛查其它肾性糖尿相关基因及可能的遗传学关联;2.通过肾糖阈值测定,定量评估肾性糖尿严重程度;3.在基因确诊的患者中,评估肾小管其他物质转运功能,为探讨不同转运子之间可能的相互作用提供线索。研究方法1.家族性肾性糖尿基因诊断方法的建立和体外验证:对临床怀疑家族性肾性糖尿的患者,外周血提取DNA,对SLC5A2基因14个外显子进行PCR扩增后测序,与GenBank中标准序列进行比对。参考文献,设计并定制遗传性肾小管转运子疾病筛查的高通量测序方案Tubule Panel(Thermo Fisher Scientific公司)。将候选患者的基因组DNA,通过多重PCR扩增建立文库,经过乳化PCR将文库连接至Ion微球颗粒制备模板,使用测序仪(PGM)测序,根据测得变异的频率、位置和变异类型进行筛选,通过一代测序验证,确定候选基因。SLC5A2基因新突变位点体外功能验证:将野生型SLC5A2编码序列插入pEGFP-N1质粒和pEGFP-C1质粒,通过定点突变获得突变型质粒。通过脂质体转染至HEK-293T细胞中。使用共聚焦显微镜观察SGLT2-EGFP融合蛋白在细胞内的定位,明确SGLT2是否表达及表达位置。保留SLC5A2编码序列终止密码子,插入pEGFP-N1质粒,在HEK-293T细胞中表达野生型和突变型SGLT2蛋白,使用流式细胞仪检测细胞对带有荧光基团的葡萄糖类似物(2-NBDG)的摄取效率,评价突变是否影响蛋白的转运功能。2.建立口服葡萄糖测定肾糖阈的方法:患者空腹口服无水葡萄糖75g,测定服糖前(0h)、服糖后0.5h、1、1.5h、2h、3h和4h的血糖值及4h内尿糖排出总量,编程通过三次样条插值拟合血糖随时间变化曲线,通过数值逼近方法估算肾糖阈值,定量评估肾脏对葡萄糖重吸收功能。3.评价确诊患者近端肾小管转运其他物质功能:取血、尿标本,计算尿酸排泄分数及24h尿酸排泄量;行磷廓清试验,测定肾小管对磷重吸收率和磷廓清指数;进行尿氨基酸定性检测。研究结果1.家族性肾性糖尿基因诊断方法的建立和体外验证:共收集10个家族性肾性糖尿家系,共测得SLC5A2基因13种变异,其中有7种新变异(3种错义变异A212S、G484D、R564W,1 种无义变异 W649X,1 种移码变异 p.Ser592CysfsTer6,1种剪接位点变异c.574+lGC,1个内含子变异c.469-124_469-107del)。成功建立了遗传性肾小管转运子异常疾病高通量测序Tubule Panel。并在本组患者中,观察到编码MAP17蛋白的PDZK1IP1存在新变异p.Asn26ThrfsTer39;2.SGLT2体外功能试验验证:成功建立了 HEK-293T细胞体外表达SGLT2体系。观察到三种错义变异的SGLT2-EGFP融合蛋白在细胞内定位。与野生型SGLT2类似,A212S突变型所编码的SGLT2在细胞膜上呈点状分布;而G484D、R564W没有在细胞膜表达。流式细胞术观察到错义变异及无义变异(A212S、G484D、R564W、W649X)突变型编码的SGLT2蛋白,转运荧光葡萄糖类似物2-NBDG的功能均显著下降(分别为野生型的15.4%,19.3%,19.5%,21.5%)。3.口服葡萄糖测定肾糖阈值方法的建立:成功建立了口服葡萄糖测定肾糖阈值的计算工具,测得4位确诊的家族性肾性糖尿患者肾糖阈(分别为0.7、2.4、6.2和9.1mmol/L)。2位患者(2/4)的肾小管磷重吸收率和1位患者(1/4)的磷廓清指数超过正常上限;10位患者中9位接受了尿氨基酸定性检查,其中有1位尿氨基酸定性弱阳性,1位可疑阳性。尿酸排泄分数、24h尿酸排泄量(n=4)与健康受试者(n=22)无显著差异。小结:本实验条件下1.建立了家族性肾性糖尿的基因及临床诊断方法。观察到SLC5A2的7种新变异,并通过体外功能试验确认了其中错义变异和无义变异的致病性。2.建立了遗传性肾小管转运子异常疾病高通量测序Tubule Panel,观察到编码MAP17蛋白的PDZK1IP1存在新变异;3.建立了口服葡萄糖测定肾糖阈的方法,观察到部分家族性肾性糖尿患者SLC5A2突变可能同时存在近端小管对磷和氨基酸的转运异常。
[Abstract]:Background familial renal diabetes is mainly characterized by normal blood glucose and positive urine sugar, which is the result of the SLC5A2 gene mutation that encodes the renal proximal tubule marginal sodium glucose co transporter 2 (sodium glucose cotransporter 2, SGLT2). In recent years, SGLT2 inhibitors have been widely used as a new type of hypoglycemic agents for their long-term renal and cardiac protection. However, renal diabetes caused by its congenital abnormalities, as an important disease of renal tubular transporter deficiency, has not been paid enough attention. The number of cases reported at home and abroad is limited, lack of quantitative body function test, and few studies on its regulatory genes and the relationship with other substances in proximal tubules. 1. research aims to establish a family. Genetic diagnosis of renal diabetes, including in vitro functional tests to verify the pathogenicity of the new variants; to establish a high throughput sequencing scheme Tubule Panel for the screening of abnormal renal tubular transporters, screening other renal related diabetes related genes and possible genetic associations; 2. the quantitative assessment of renal sugar by renal glucose threshold determination The degree of urinary severity; 3. in the patients with gene diagnosis, the evaluation of the other substance transport function of renal tubules provides a clue to explore the possible interaction between different transporters. Method the establishment and in vitro validation of the method of diagnosis of 1. familial renal diabetic genes: for patients with clinically suspected familial diabetic nephropathy, peripheral blood extracts DNA, and SLC 14 exons of the 5A2 gene were sequenced by PCR amplification and compared with the standard sequence in GenBank. Reference was made to design and customize the high throughput sequencing scheme Tubule Panel (Thermo Fisher Scientific company) for genetic renal tubular transporter screening. The candidate patient's gene group DNA was established by multiplex PCR amplification and emulsified. PCR connects the library to the Ion microspheres to prepare the template, and uses the sequencing instrument (PGM) to sequence. According to the frequency, location and variation type of the mutation, the function verification of the new mutation site of the candidate gene.SLC5A2 gene is confirmed by one generation sequencing. The wild type SLC5A2 coding sequence is inserted into the pEGFP-N1 plasmid and the pEGFP-C1 plasmid. The mutant plasmids were transfected into HEK-293T cells through liposomes. The localization of SGLT2-EGFP fusion protein in the cells was observed by confocal microscopy, and the expression and location of SGLT2 were determined. The SLC5A2 coding sequence was retained and the pEGFP-N1 particles were inserted into the pEGFP-N1 granules, and the wild type and mutation were expressed in the HEK-293T cells. Type SGLT2 protein, using flow cytometry to detect the uptake efficiency of glucose analogues (2-NBDG) with fluorescent groups, evaluate whether mutation affects the transport function of protein.2., establish oral glucose threshold for oral glucose determination by oral administration of oral glucose 75g, 0h, 1,1.5h, 2h, 3H and 4h after taking sugar (0h). The blood sugar value and the total excretion of urine sugar in 4H were fitted by three spline interpolation to fit the curve of blood sugar with time. The renal sugar threshold was estimated by numerical approximation method. The function of renal tubule transport in proximal renal tubules was evaluated by the renal glucose reabsorption function.3.. Blood collection, urine specimen, uric acid excretion fraction and 24h urine were calculated. Acid excretion; phosphorus clearance test, determination of phosphorus reabsorption rate and phosphorus clearance index in renal tubules; qualitative detection of urinary amino acids. The establishment and in vitro validation of 1. familial renal diabetic gene diagnosis methods: a total of 10 familial renal diabetic families were collected, and 13 mutations of SLC5A2 gene were measured, of which 7 new variants (3 kinds of errors) were detected. A212S, G484D, R564W, 1 kinds of non sense variant W649X, 1 transcoding mutation p.Ser592CysfsTer6,1 splice site variant c.574+lGC, 1 intron variant c.469-124_469-107del). The hereditary renal tubular transporter was successfully established by high throughput sequencing Tubule Panel. and the PDZK1I of MAP17 protein was observed in this group. P1 has a new variant p.Asn26ThrfsTer39; 2.SGLT2 in vitro functional test verified that the SGLT2 system expressed in vitro of HEK-293T cells was successfully established. The SGLT2-EGFP fusion protein of three missense variants was located in the cell. Similar to the wild type SGLT2, the SGLT2 encoded by the A212S mutation was punctuated on the cell membrane; G484D, R564W was not Expression of the cell membrane. Flow cytometry was used to observe the mutational and nonsense mutation (A212S, G484D, R564W, W649X) mutated SGLT2 protein, and the function of transport fluorescent glucose analogue 2-NBDG decreased significantly (15.4%, 19.3%, 19.5%, 21.5%).3. oral glucose threshold method for the determination of the kidney sugar threshold method: successful establishment of the method. The rate of renal glucose threshold (0.7,2.4,6.2 and 9.1mmol/L).2 patients (2/4) in 4 patients with familial renal diabetes mellitus (2/4) was measured by oral glucose, and the phosphorus clearance index of renal tubules and 1 patients (1/4) were higher than the normal upper limit, and 9 of the 10 patients received the qualitative examination of urinary amino acids, including 1 The urinary amino acids were weak positive, 1 suspected positive. Uric acid excretion score, 24h uric acid excretion (n=4) and healthy subjects (n=22) were not significantly different. Summary: under the conditions of 1., the gene and clinical diagnosis of familial renal diabetes were established. 7 new variations of SLC5A2 were observed and the missense was confirmed by the function test in vitro. The pathogenicity of variant and nonsense variant of.2. established a hereditary renal tubular transporter with high throughput sequencing of Tubule Panel, and a new variation in the PDZK1IP1 of the encoded MAP17 protein was observed. 3. the method for the determination of the renal sugar threshold by oral glucose was established and the SLC5A2 mutation in some familial renal glucuria patients may have a small proximal end. Transshipment of phosphorus and amino acids is abnormal.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692

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