本文選題：家族性腎性糖尿 + 鈉葡萄糖共轉(zhuǎn)運子2�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：研究背景家族性腎性糖尿主要表現(xiàn)為血糖正常,而尿糖陽性,是編碼腎臟近端小管刷狀緣鈉葡萄糖共轉(zhuǎn)運子 2(sodium glucose cotransporter 2,SGLT2)的 SLC5A2 基因突變所致。近年來,SGLT2抑制劑作為新型降糖藥,因其長期腎臟和心臟保護作用廣受關(guān)注。但其先天異常所致的腎性糖尿,作為重要的腎小管轉(zhuǎn)運蛋白缺陷疾病,并未受到足夠的重視,國內(nèi)外報告的病例數(shù)均有限,缺乏定量的體內(nèi)功能試驗,對其調(diào)節(jié)基因、及與近端小管其他物質(zhì)轉(zhuǎn)運關(guān)系的研究也較少。研究目的1.建立家族性腎性糖尿的基因診斷方法,包括體外功能試驗用于驗證新變異的致病性;建立遺傳性腎小管轉(zhuǎn)運子異常疾病篩查的高通量測序方案Tubule Panel,篩查其它腎性糖尿相關(guān)基因及可能的遺傳學(xué)關(guān)聯(lián);2.通過腎糖閾值測定,定量評估腎性糖尿嚴重程度;3.在基因確診的患者中,評估腎小管其他物質(zhì)轉(zhuǎn)運功能,為探討不同轉(zhuǎn)運子之間可能的相互作用提供線索。研究方法1.家族性腎性糖尿基因診斷方法的建立和體外驗證:對臨床懷疑家族性腎性糖尿的患者,外周血提取DNA,對SLC5A2基因14個外顯子進行PCR擴增后測序,與GenBank中標準序列進行比對。參考文獻,設(shè)計并定制遺傳性腎小管轉(zhuǎn)運子疾病篩查的高通量測序方案Tubule Panel(Thermo Fisher Scientific公司)。將候選患者的基因組DNA,通過多重PCR擴增建立文庫,經(jīng)過乳化PCR將文庫連接至Ion微球顆粒制備模板,使用測序儀(PGM)測序,根據(jù)測得變異的頻率、位置和變異類型進行篩選,通過一代測序驗證,確定候選基因。SLC5A2基因新突變位點體外功能驗證:將野生型SLC5A2編碼序列插入pEGFP-N1質(zhì)粒和pEGFP-C1質(zhì)粒,通過定點突變獲得突變型質(zhì)粒。通過脂質(zhì)體轉(zhuǎn)染至HEK-293T細胞中。使用共聚焦顯微鏡觀察SGLT2-EGFP融合蛋白在細胞內(nèi)的定位,明確SGLT2是否表達及表達位置。保留SLC5A2編碼序列終止密碼子,插入pEGFP-N1質(zhì)粒,在HEK-293T細胞中表達野生型和突變型SGLT2蛋白,使用流式細胞儀檢測細胞對帶有熒光基團的葡萄糖類似物(2-NBDG)的攝取效率,評價突變是否影響蛋白的轉(zhuǎn)運功能。2.建立口服葡萄糖測定腎糖閾的方法:患者空腹口服無水葡萄糖75g,測定服糖前(0h)、服糖后0.5h、1、1.5h、2h、3h和4h的血糖值及4h內(nèi)尿糖排出總量,編程通過三次樣條插值擬合血糖隨時間變化曲線,通過數(shù)值逼近方法估算腎糖閾值,定量評估腎臟對葡萄糖重吸收功能。3.評價確診患者近端腎小管轉(zhuǎn)運其他物質(zhì)功能:取血、尿標本,計算尿酸排泄分數(shù)及24h尿酸排泄量;行磷廓清試驗,測定腎小管對磷重吸收率和磷廓清指數(shù);進行尿氨基酸定性檢測。研究結(jié)果1.家族性腎性糖尿基因診斷方法的建立和體外驗證:共收集10個家族性腎性糖尿家系,共測得SLC5A2基因13種變異,其中有7種新變異(3種錯義變異A212S、G484D、R564W,1 種無義變異 W649X,1 種移碼變異 p.Ser592CysfsTer6,1種剪接位點變異c.574+lGC,1個內(nèi)含子變異c.469-124_469-107del)。成功建立了遺傳性腎小管轉(zhuǎn)運子異常疾病高通量測序Tubule Panel。并在本組患者中,觀察到編碼MAP17蛋白的PDZK1IP1存在新變異p.Asn26ThrfsTer39;2.SGLT2體外功能試驗驗證:成功建立了 HEK-293T細胞體外表達SGLT2體系。觀察到三種錯義變異的SGLT2-EGFP融合蛋白在細胞內(nèi)定位。與野生型SGLT2類似,A212S突變型所編碼的SGLT2在細胞膜上呈點狀分布;而G484D、R564W沒有在細胞膜表達。流式細胞術(shù)觀察到錯義變異及無義變異(A212S、G484D、R564W、W649X)突變型編碼的SGLT2蛋白,轉(zhuǎn)運熒光葡萄糖類似物2-NBDG的功能均顯著下降(分別為野生型的15.4%,19.3%,19.5%,21.5%)。3.口服葡萄糖測定腎糖閾值方法的建立:成功建立了口服葡萄糖測定腎糖閾值的計算工具,測得4位確診的家族性腎性糖尿患者腎糖閾(分別為0.7、2.4、6.2和9.1mmol/L)。2位患者(2/4)的腎小管磷重吸收率和1位患者(1/4)的磷廓清指數(shù)超過正常上限;10位患者中9位接受了尿氨基酸定性檢查,其中有1位尿氨基酸定性弱陽性,1位可疑陽性。尿酸排泄分數(shù)、24h尿酸排泄量(n=4)與健康受試者(n=22)無顯著差異。小結(jié):本實驗條件下1.建立了家族性腎性糖尿的基因及臨床診斷方法。觀察到SLC5A2的7種新變異,并通過體外功能試驗確認了其中錯義變異和無義變異的致病性。2.建立了遺傳性腎小管轉(zhuǎn)運子異常疾病高通量測序Tubule Panel,觀察到編碼MAP17蛋白的PDZK1IP1存在新變異;3.建立了口服葡萄糖測定腎糖閾的方法,觀察到部分家族性腎性糖尿患者SLC5A2突變可能同時存在近端小管對磷和氨基酸的轉(zhuǎn)運異常。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R692

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