CN1683555A - 应用遗传标记和芯片预测糖尿病肾病危险的方法 - Google Patents
应用遗传标记和芯片预测糖尿病肾病危险的方法 Download PDFInfo
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Abstract
公开了检测中国血统糖尿病患者已患有、将有可能发展成或疑似患有肾病的方法。该方法包括检测来自糖尿病患者的样品中是否含有选自ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5’(z-2)基因型,ALR2基因启动子区的C106T基因型,TNF-a基因的G-308A基因型,以及其互补序列多态性序列的至少一个多态性序列,条件是ALR2的基因型不单独使用,其中多态性序列的存在表明该糖尿病患者已患有、将有可能发展成、或者疑似患有肾病。本发明还提供了可用来检测中国糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的芯片。
Description
发明领域
本发明涉及使用一个或多个标记来检测中国血统糖尿病患者已患有、将有可能发展成或者疑似患有肾病的一种方法和芯片,特别是使用选自基因ACE,AGT,ALR2和TNF-a的一个或多个遗传多态性标记检测中国血统糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的一种方法和芯片,条件是不单独使用ALR2的基因型。
背景技术
糖尿病肾病是糖尿病患者的主要致病、致死原因。随着糖尿病在发达国家与发展中国家的流行,糖尿病目前已成为晚期肾病(ESRD)的最主要的病因,占所有使用肾脏替换治疗的新患者的40-50%(Ritz E,RychlikI,Locatelli F,Halimi S,End-stage renal failure in type 2 diabetes:a medicalcatastrophe of worldwide dimensions,Am J Kidney Dis,1999;34:795-808)。中国是拥有最多糖尿病患者的三个国家之一,估计到2025年,糖尿病患者将增加到4000万人且该增长主要发生在中年人群中(Chan J.C.N,NgM.C.Y,Critchley J.A.J.H,Lee S.C,Cockram C.S,Diabetes mellitus-aspecial medical challenge from a Chinese perspective,Diabetes Research andClinical Practice,2001;54:S19-27),年轻型糖尿病与幼年肥胖症以及新陈代谢综合症发病率的增高是导致这种状况的出现主要原因(Chan J.C.N,Ng M.C.Y,Lessons learned from young onset diabetes in China,CurrentDiabetes Report,2003;3:101-107;Chan J.C.N,Cheung C.K,CheungM.Y.F,Swaminathan R,Critchley J.A.J.H,Cockram C.S,Abnormalalbuminuria as a predictor of mortality and renal impairment in Chinesepatients with NIDDM,Diabetes Care,1995;18:1013-1014;Chan J.C.N,Ko G.T.C,Leung D,Cheung R.C.K,Cheung M,So W.Y等,The longterm effects of angiotensin converting enzyme inhibition and metaboliccontrol on cardiovascular and renal outcomes in hypertensive Type 2 diabeticpatients,Kidney International,2000;57:590-600)。在世界卫生组织多国糖尿病心血管病(WHO-MSVDD)的研究中发现,亚洲,特别是中国、日本患者比白人II型糖尿病患者有更高的ESRD发病率(Morrish N.J,WangS,Stevens L.K,Fuller J.H,Keen H,Mortality and causes of death in theWHO Multinational Survey of Vascular Diseases in Diabetes,Diabetologia,2001;44:S14-21)。
与白人糖尿患者群中大多数患者死于心血管疾病形成对照的是,中国血统糖尿病患者致死的主要原因是晚期肾病(ESRD)(Chan J.C.N,Cheung C.K,Cheung MYF,Swaminathan R,Critchley J.A.J.H,CockramC.S,Abnormal albuminuria as a predictor of mortality and renal impairmentin Chinese patients with NIDDM,Diabetes Care,1995;18:1013-1014)。这些发现最近已被WHO-MSVDD证实(Morrish N.J,Wang S,Stevens L.K,Fuller J.H,Keen H,Mortality and causes of death in the WHOMultinational Survey of Vascular Diseases in Diabetes,Diabetologia,2001;44:S14-21)。与这些发现一致,现已经证实疾病相关基因的等位基因频率和单元型具有种族间差别,这可能导致了疾病形成敏感性方面出现种族差异(Ng M,Wang Y,So W,Cheng S,Visvikis S,Zee R等,Ethnicdifferences in the linkage disequilibrium and distribution of single nucleotidepolymorphisms in 35 candidate genes for cardiovascular diseases,Genomics,2003:in press;Young RP,Thomas G.N,Critchley J.A.J.H,TomlisnonB,Woo K.S,Sanderson J.E,Interethnic differences in coronary heartdiseaes mortality in 25 populations:associations with the angiotensinconverting enzyme DD genotype frequency,Journal of Cardiovascular Risk,1998;5:303-7)。考虑到疾病的持续时间对并发疾病的影响、年青与中年糖尿患者人群发病率的升高和发生糖尿病肾病的种族偏好,随着社会经济的发展,肾功能衰竭与心血管疾病将在我们越来越多的年青人群中流行(Chan J.C.N,Ng M.C.Y,Critchley J.A.J.H,Lee S.C,Cockram C.S,Diabetes mellitus-a special medical challenge from a Chineseperspective,Diabetes Research and Clinical Practice,2001;54:S19-27)。
以前我们报道过肾病有30%到50%的高发病率和中国血统糖尿病患者肾功能死亡与退化的尿标记的预测值(Chan J.C.N,CheungJ.C.K,LauE.M.C,Woo J,Swaminathan R,Cockram C.S,The Metabolic Syndromein Hong Kong Chinese-the inter-relationships amongst its componentsanalysed by structural equation modeling,Diabetes Care,1996;19:953-9)。我们已报道了在排除高血压与高血脂的情况下中国血统患者胰岛素抗性与糖尿病肾病之间的独立关系(Chan J.C.N,Tomlinson B,Nicholls M.G,Swaminathan R,Cheung C.K,Cockram CS,Albuminuria,insulin resistanceand dyslipidaemia in Chinese patients with non-insulin-dependent diabetes(NIDDM),Diabetic Medicine,1996;13:150-55)以及其分别与肥胖症、白蛋白尿症、血糖代谢异常之间的密切关系(Lee Z,Critchley J,Ko G.T,Anderson P,Thomas N,Young R,et al,Obesity and cardiovascular riskfactors in Hong Kong Chinese,Obesity Reviews,2002;3:178-182)。以家族为基础的研究与隔离分析(The Diabetes Control and ComplicationsTrial Research Group,Clustering of long term complicatiohs in families withdiabetes in the diabetes control and complication trial,Diabetes,1997;46:1829-1839)和基因组扫描已经证实了遗传因素在糖尿病肾病形成过程中起到了很强的作用(Imperatore G,Hanson RL,Pettitt D,Kobes S,BennettP,Knowler W,Sib pair linkage analysis for susceptibility genes formicrovascular complications among Pima Indians with tyoe 2 diabetes.PimaDiabetes Gene Group,Diabetes,1998;47:821-30;Imperatore G,KnowlerW,Pettitt D,Kobes S,Bennett P,Hanson R,Segregation analysis of diabeticnephropathy in Pima Indians,Diabetes,2000;49:1049-56)。血管紧张素系统(RAS)在调节系统与肾脏血流动力学和细胞组织生长方面起关键作用(Cooper M,Pathogenesis,prevention and treatment of diabeticnephropathy,Lancet,1998;352:213-9)。AGT M235T多态性TT基因型、ACE I/D多态性D等位基因与白种人血统、日本人血统和中国人血统糖尿病患者患肾病有关系(Fujisawa T,Ikegami H,Kawaguchhi Y,Hamada Y,Ueda H,Shintani M,et al,Meta analysis of association ofinsertion/deletion polymorphism of angiotensin I converting enzyme genewith diabetic nephropathy and retinopathy,Diabetologia,1998;41:47-53;Young R.P,Chan J.C.N,Poon E,Critchley J.A.J.H,Cockram C.S,Associations between albuminuria and angiotensinogen T235 and angiotensinconverting enzyme insertion/deletion polymorphims in Chinese NIDDMpatients,Diabetes Care,1997;21:431-7;Ringel J,Beige J,Kunz R,Distler A,Sharma A,Genetic variants of the renin angiotensin system,diabetic nephropathy and hypertension,Diabetologia,1997;40:193-9)。由脂肪细胞分泌的细胞因子,即肿瘤坏死因子a(TNF-a)是肥胖症与胰岛素抗性的关联因子(Hotamisligil GS,Spiegelman BM,Tumor necrosisfactor:a key component of the obesity-diabetes link,Diabetes,1994;43:1271-8),而胰岛素抗性是包括中国糖尿病患者在内的肾病患者的一个重要特征(Chan J.C.N,Tomlinson B,Nicholls M.G,Swaminathan R,Cheung C.K,Cockram C.S,Albuminuria,insulin resistance anddyslipidaemia in Chinese patients with non-insulin-dependent diabetes(NIDDM),Diabetic Medicine,1996;13:150-55)。最近日本的研究表明血清TNF-a水平的升高与II型糖尿病患者的肾病有确定的关系(MoriwakiY,Yamamoto T,Shibutani Y,Aoki E,Tsutsumi Z,Takahashi S,et al,Elevated levels of interleukin 18 and tumor necrosis factor alpha in serum ofpatients with type 2 diabetes mellitus:relationship with diabeticnephropathy,Metabolism:Clinical and Experimental,2003;52:605-8)。关于这一点,已有报道TNF-a基因启动子区域的G-308A多态性与肥胖症、胰岛素抗性(Dalziel B,Goskby A,Richman R,Bryson J,Caterson I,Association of TNF alpha-308 G/A promoter polymorphism with insulinresistance in obesity,Obesity Research,2002; 10:401-7)及TNF-a基因转录活性增强有关(Kroeger K,Carville K,Abraham L,The-308 tumornecrosis factor alpha promoter polymorphism effects transcription,MolecularImmunolgy,1997;34:391-99)。醛糖还原酶(ALR2)是多醇路径最关键的酶,多醇路径可以导致氧化压力增加和细胞内环境改变,从而引起糖尿病微血管病(Hodgkinson A,Sondergaard K,Yang B,Cross D,Millward B,Demaine A,Aldose reductase expression is induced by hyperglycemia in
diabetic nephropathy,Kidney International,2001;60:211-8)。已经表明ALR2基因5’-(CA)的z-2等位基因、C-106T多态性T等位基因使包括中国血统患者在内的I,II型糖尿病患者患肾病的危险性增加(Wang Y,NgM,Lee S,So W,Tong C,Cockram C,et al,Phenotypic heterogeneityassociations of two aldose reductase gene polymorphisms with nephropathyand retinopathy in Type 2 diabetes,Diabetes Care,2003;26:2410-5)。上述公开的遗传因子进一步地与代谢的、血液动力学的和生长因子作用,致使肾功能蛋白尿和逐渐衰竭(Parving H.H,Tarnow L,Rossing P,Genetics of diabetic nephropathy,Journal of American Society ofNephrology,1996;7:2509-17)。
尽管已经有关于这5个遗传标记与白种人和日本人糖尿病并发症的关系的报道,但对于中国血统糖尿病患者群还没有相一致的报道。到目前为止,还没有报道表明这些遗传因子对糖尿病并发症,包括糖尿病肾病的形成产生交互作用效应。
应用基因组学的前景在于它的潜在用途,它可以用来鉴别危险性患者以期较早地和有目地干预,从而确保身体健康和减少诸如糖尿病之类致死性疾病的影响(Collins F,Green E,Guttmacher A,GuyerMobotuNHGRI,A vision for the future of genomics research.A blueprint forthe genomic era,Nature,2003;422:835-47)。在鉴定中国糖尿病患者并发症高危患者的遗传因子的研究中,我们第一个报道AGT TT基因型与糖尿病有关以及其与ACE D等位基因一起对糖尿病的形成产生协同作用(Young RP,Chan J.C.N,Poon E,Critchley J.A.J.H,Cockram C.S,Associations between albuminuria and angiotensinogen T235 and angiotensinconverting enzyme insertion/deletion polymorphims in Chinese NIDDMpatients,Diabetes Care,1997;21:431-7)。同样,我们第一个报道了ALR2 TT基因型与中国血统患者患糖尿病患肾病的危险性有关(Wang Y,Ng M,Lee S,So W,Tong C,Cockram C,et al,Phenotypic heterogeneityassociations of two aldose reductase gene polymorphisms with nephropathyand retinopathy in Type 2 diabetes,Diabetes Care,2003;26:2410-5)。
发明概要
因此,本发明涉及到一种检测中国血统糖尿病患者已患有、将可能发展成、或者疑似患有肾病的方法,该方法包括如下步骤:
检测来自糖尿病患者的样品中是否含有下列多态性序列中的至少一种:ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5’(z-2)基因型,ALR2基因启动子区的C106T基因型,TNF-a基因的G-308A基因型,以及其互补序列,条件是不单独使用ALR2的基因型,其中多态性序列的存在表明该糖尿病患者已患有、将有可能发展成、或者疑似患有肾病。
在本发明的一个实施方案中,上述方法还包括从患者中取样的步骤。该患者优选患有II型糖尿病,取得的样品优选为血液。
本发明还涉及到用来检测中国血统糖尿病患者已患有、将可能发展成、或者疑似患有肾病的芯片,该芯片包括下列多态性序列中的至少一种:ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5’(z-2)基因型,ALR2基因启动子区的Cl06T基因型,TNF-a基因的G-308A基因型,以及其互补序列,条件是不单独使用ALR2的基因型。
在本发明中,I/D多态性优选含有DD基因型,G-308A多态性优选含有GG基因型。
本发明还涉及到用于检测中国血统糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的试剂盒。试剂盒一般包括本发明所定义的芯片和指导用此芯片处理样品的指导性材料。优选地,该试剂盒含有从患者中采取样品的装置。
附图的简要说明
图1显示了711例具有不同数目危险基因型的中国血统II型糖尿病患者患肾病的几率,这些基因型包括AGT基因的TT基因型,ACE基因的DD/DI基因型,TNF-α基因的GG基因型,AGT基因的TT基因型,ALR2基因的x/z-2 o或z-2/z-2基因型,ALR2基因的CT/TT基因型。
图2显示了947例中国血统II型糖尿患者中ACE基因I/D基因型携带者发生肾病终点事件的Kaplan-Meier曲线图。
图3显示了患者初级复合终点事件(图3A)或晚期肾病终点事件(图3B)和全致死(图3C)的Kaplan-Meier曲线图。依托多学科团队,按照注重对目标实施定期监控、治疗和增强患者依从性的方案(干扰组)与基于通常的临床护理的方案(对照组)处理上述患者,其中干扰组的患者一直在医生的指导下治疗,而对照组的患者依从性不稳定。
发明的详细描述
遗传学、流行病学与试验研究支持了这样一种主张:糖尿病肾病涉及到多种生物化学途径(Cooper M,Pathogenesis,prevention and treatmentof diabetic nephropathy,Lancet,1998;352:213-9)。基于国际和地区的、临床与实验的证据,我们认为AGT基因M235T的TT基因型,ACE基因I/D的DD/ID基因型,TNF-α基因G-308A的GG基因型,ALR2基因x/z-2或z-2/z-2(z=z-2之外的任何等位基因)和CT/TT基因型都是中国血统糖尿病患者患肾病的潜在危险基因型。
定义
除非在发明中特别指出,术语“AGT基因M235T”或“AGT M235T”是指“AGT基因的M235T基因型”;术语“ACE基因I/D”或者“ACE I/D多态性”与“ACE基因的I/D基因”含意相同;术语“TNF-α基因GG”等同于“TNF-α基因G-308A的GG基因型”;术语″TNF-α基因G-308A”或“TNF-α基因启动子区域的G-308A多态性“或“TNF-α G-308A″或″TNFα G308A多态性”是指“TNF-α基因的G-308A遗传型”;术语“醛醣还原酶5’-(CA)n的z-2等位基因”或者“ALR2(CA)n-5’(z-2)″等同于“ALR2基因5’-(CA)重复序列的(z-2)基因型”;术语“醛醣还原酶(ALR2)的C-106T多态性的T等位基因”等同于“ALR2 TT基因型,ALR2基因TT”或者“ALR2基因CT/TT基因型”;术语″一个ALR2的C-106T多态性”是指“启动子区域的ALR2基因的一个C-106基因型”。
本发明用来检测中国血统糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的方法包括如下步骤:检测来自糖尿病患者的样品中是否含有下列多态性序列中的至少一种:ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5’(z-2)基因型,ALR2基因启动子区的C106T基因型,TNF-a基因的G-308A基因型,以及其互补序列,条件是不单独使用ALR2的基因型,其中多态性序列的存在表明该糖尿病患者已患有、将有可能发展成、或者疑似患有肾病。
在上述方法中用作遗传标记的多态性可以通过以下步骤予以鉴别:
(a)从患者中提取基因组DNA
(b)以基因组DNA为模板用PCR方法扩增基因组DNA中的ACE基因,AGT,TNF-αG-308A多态性基因,醛醣还原酶(ALR2)CA重复序列,ALR2基因的C106T启动子;和
(c)应用凝胶分离或测序鉴别(b)步骤中的产物。
在本发明中,基因组DNA可以从患者的体液中抽提,如血液和尿液,优选为血液。
尽管本发明的检测方法可以应用于任何一糖尿病患者,但是该方法优选用于II型糖尿病患者。中国血统糖尿病患者特别适合于本发明。
本研究的四个候选基因均可通过可能的代谢途径引起糖尿病肾病的形成。AGT与ACE是导致高血压和组织异常生长的RAS的重要组成元件(Cooper M,Pathogenesis,prevention and treatment of diabeticnephropathy,Lancet,1998;352:213-9;Ringel J,Beige J,Kunz R,Distler A,Sharma A,Genetic variants of the renin angiotensin system,diabetic nephropathy and hypertension,Diabetologia,1997;40:193-9)。细胞因子TNF-α使肥胖症与胰岛素抗性间产生关联(Hotamisligil GS,Spiegelman BM,Tumor necrosis factor:a key component of theobesity-diabetes link,Diabetes,1994;43:1271-8),有报道表明其血清水平确定地与糖尿病肾病相关(Moriwaki Y,Yamamoto T,Shibutani Y,Aoki E,Tsutsumi Z,Takahashi S,et al,Elevated levels of interleukin 18 andtumor necrosis factor alpha in serum of patients with type 2 diabetesmellitus:relationship with diabetic nephropathy,Metabolism:Clinical andExperimental,2003;52:605-8)。通过山梨醇酯的细胞间积累和高血糖条件下的氧化压力增加,ALR2引起微血管疾病的发生(Chung S,Ho E,Lam K,Chung S,Contribution of polyol pathway to diabetes-inducedoxidative stress,Journal of American Society of Nephrology,2003;14:S233-6).
在中国血统人群中这些基因型的发现是基于一系列横向的、预期的和病例对照研究,结果与糖尿病肾病患者的表型特征一致,其中的糖尿病肾病患者比未患糖尿病肾病的患者更肥胖、血压更高,具有更多不良的脂和血糖调控。大规模随机临床研究已经证实抑制RASD,加强血糖和血压调控对治疗糖尿病蛋白尿病和ESRD形成具有有益效果(Brenner B.M,Cooper M.E,De Zeeuw D,Keane W.F,Mitch W.E,Parving H.H等,Effects of Losartan on renal and cardiovascular outcomes in patients withtype 2 diabetes and nephropathy,New England Journal of Medicine,2001;345:861-9;UKPDS,Intensive blood glucose control with sulphonylureas orinsulin compared with conventional treatment and risk of complications inpatients with type 2 diabetes(UKPDS 33),Lancet,1998;352:837-53;Adler A.I,Stratton I.M,Neil H.A,Yudkin J.S,Matthews D.R,Cull C.A等,Association of systolic blood pressure with macrovascular andmicrovascular complications of type 2 diabetes(UKPDS 36):prospectiveobservational study,British Medical Journal,2000;321:412-9)。最近的研究表明,组合使用ACE、ALR2抑制剂治疗能产生协同作用从而改善糖尿病大鼠的神经功能(Cotter M,Mirrlees D,Cameron N,Neurovascularinteractions between aldose reductase and angiotensin converting enzymeinhibition in diabetic rats,European Journal of Pharmacology,2001;417:223-30)。总之,对这些遗传因子及其交互作用的鉴定(和提高对同一患者危险基因型的同时筛选效率的相关微芯片技术)可筛选出高危个体以对其进行强化与有目的治疗从而减少并发症危险。关于这一点,依赖多学科团队,我们已经证实注重对目标实施定期监控、治疗和增强患者的依从性的多重方法可以使中国血统糖尿患者死亡和患晚期肾病(ESRD)的危险减少40-70%。(图3)。由于携带这些基因型的患者的危险性不断地增加,该疾病调控方案及其花费有效性的优点应该还会得到进一步的提高。
在对年龄与性别进行调整后,我们发现患肾病的危险性随着危险基因型数目的增加而渐进式地显著增加。携带有3种或者更多危险基因型的患者占总患者的66%,与携带有0种或者1种危险基因型的那些患者相比,这些患者患糖尿病肾病的危险性增加了1.8-2.0倍。
我们提供了原始数据证实了DD基因型对ESRD形成、TNF-α对糖尿病肾病,特别是对肥胖性糖尿患者的糖尿病肾病的前兆性作用。更重要的是这些数据证实了这5种基因型间的交互作用对糖尿病肾病形成具有前兆性作用。
能够理解以上所提到的一种或者多种基因型可以用于制备芯片,该芯片可以与其它已知的临床的、生物化学的与遗传学的芯片一起用来预测中国血统糖尿病患者中糖尿病并发症,包括肾病发生的危险性。这些基因型或其等效芯片可以用于检测患者患糖尿病和/或糖尿病肾病的危险性,从而使用包括强度监测、药理学与非药理学疗法在内的多重方法来改变危险性。
为了实施本发明,使用本发明定义的芯片作为试剂盒来检测中国血统糖尿病患者已患有、将有可能发展或疑似患有肾病是方便的。该试剂盒包括上述芯片和扩增基因ACE、AGT、ALR2或TNF-α的一对引物。在本发明试剂盒的一个实施方案中,该引物选自从SEQ ID NO:1到SEQID NO:10。
此发明还提供了含有一种芯片的试剂盒,该芯片含有下列多态性序列中的至少一种和根据该序列设计的作为对照的任意探针:ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5’(z-2)基因型,ALR2基因启动子区的C106T基因型,TNF-a基因的G-308A基因型,以及其互补序列。
本发明将通过以下实施例作进行进一步的描述。
实施例1
五个多态性序列基因型的鉴定
人类基因组的制备
从每个患者中收集大约10ml EDTA血样,用SDS和蛋白酶K过夜裂解细胞后,用苯和氯仿抽提得到基因组DNA。然后将DNA颗粒溶解到1X TE缓冲液中。通过检测260nm和280nm处的光密度测定抽提的DNA数量和质量。将抽提的DNA在4℃下贮藏以备下次基因型分析。
ACE基因的PCR条件
反应依据改进的Rigat的方法进行(Rigat B,Hubert C,Alhenc-Gelas F,Cambien F,Corvol P,Soubrier F,An insertion deletion polymorphism inangiotensin I converting enzyme gene accounting for half the variance ofserum enzyme levels,Journal of Clinical Investigation,1990;86:1343-1346)。使用GeneAmp PCR System 9700(ABI)在标准PCR缓冲液(50mM KCl,10mM Tris-HCl,pH 8.3,3mM MgCl2,dNTP各0.2mM)(ABI)中扩增150ng DNA模板,引物浓度为各5pmol,Taq聚合酶为0.6U(Amersham),总体积为20μl。循环条件如下:开始在94℃下变性2min,94℃ 1min,58℃ 1min,72℃ 2min,共30个循环,最后在72℃下延伸5min。引物的序列如下:
5’CTG GAG ACC ACT CCC ATC CTT TCT 3’ SEQ ID NO.1
5’GAT GTG GCC ATC ACA TTC GTC AGA T 3’ SEQ ID NO.2
PCR产物是190bp和490bp的片断,前者不含有插入等位(insertionallele)基因,后者含有插入等位基因。
血管紧张素基因的PCR条件
反应依照Russ的方法进行(Russ A,Maerz W,Ruzicka V,Stein U,Gross W,Rapid detection of the hypertension associated Met235→Thr alleleof the human angiotensinogen gene,Human Molecular Genetics,1994;2:609-10)。使用GeneAmp PCR System 9700(ABI)在标准PCR缓冲液(50mMKCl,10mM Tris-HCl,pH 8.3,,1.5mM MgCl2,dNTP各50uM)(ABI)中扩增200ng DNA模板,引物浓度为各0.3uM,Taq聚合酶为0.75U(Amersham)。引物的序列如下:
5’-CAG GGT GCT GTC CAC ACT GGA CCC C-3’ SEQ ID NO.3
5’-CCG TTT GTG CAG GGC CTG GCT CTC T-3’ SEQ ID NO.4
循环条件如下:90℃变性3min;94℃ 1min 68℃ 1min.,72℃ 1min.,10个循环;之后为90℃ 30sec,68℃ 1min.,72℃ 30sec.,30个循环,最后72℃延伸10min。
PCR产物用5UTth 111 I(Promega)酶在65℃消化过夜。消化片段在3%的琼脂糖凝胶中通过电泳进行分离。纯的蛋氨酸等位基因表现为未消化的165bp单带带型,苏氨酸呈现为141bp与24bp带型。
TNF-αG-308A多态性的PCR条件
依据Wilson等描述的方法进行反应(Wilson A,di Giovine F,Blakemore A,Duff G,Single base polymorphism in the human tumornecrosis factor alpha gene detectable by NcoI restriction of PCR product,Human Molecular Genetics,1992;1:535)。使用GeneAmp PCR System 9700(ABI)进行反应,终体积为20μl,包含100ng DNA模板,50mM KCl,10mMTris-HCl,pH 8.3,2.5mM MgCl2,0.2mM每种dNTP(Boehringer-Mannheim,Germany)。引物浓度为各0.5mM,Taq聚合酶为0.05U(Boehringer-Mannheim,Germany)。引物的序列如下:
5’-AGG CAA TAG GTT TTG AGG GCC AT-3’ SEQ ID NO.5
5’-TCC TCC CTG CTG CTC CGA TTC CG-3’ SEQ ID NO.6
循环条件如下:开始在95℃变性3min,95℃ 1min,58℃ 1min,72℃ 1min,共35个循环。最后在72℃延伸10min。PCR扩增产物用10UNcoI酶(Promega)在37℃消化过夜。消化片段在3%的琼脂糖凝胶中通过电泳进行分离。A等位基因表现为未消化的165bp带型,G等位基因呈现为87bp与20bp带型。
醛醣还原酶(ALR2)CA重复序列PCR条件
含有二核苷酸CA重复序列的区域通过用Ko等所描述的PCR方法(Ko B.C.B,Lam K.S.L,Wat N.M.S,Chung S.S.M,An(A-C)ndinucleotide repeat polymorphic marker at the 5′end of the aldose reductasegene is associated with early onset diabetic retinopathy in NIDDM patients,Diabetes,1995;44:727-32)进行扩增,所使用的引物侧翼与138bp的区域连接。
反应所用的有义链的序列是:
5’-GAA TCT TAA CAT GCT CTG AAC C-3’ SEQ ID NO.7
反义链序列是:
Arpr2 5’-GCC CAG CCC TAT ACC TAG T-3’. SEQ ID NO.8
将1个M13尾(5’-CAC GAC GTT GTAAAA CGA C-3’)加到有意义链引物5’末端用来标记红外荧光
PCR在按照提供的配方制备的缓冲液中进行,总体积为4μl,包含1ng DNA模板,2.5mM MgCl2,dNTP各0.2mM,引物各0.1pmol/μl,0.15pmol/μl IRD800标记的M13正向(-29)引物,0.15U Taq聚合酶(Amplitaq,Perkin-Elmer/Cetus,Norwalk,CT)。循环条件为:初始94℃变性3min,94℃ 1min.,57℃ 1min,72℃ 1min,共35个循环,最后72℃延伸10min。
扩增出的PCR产物在95℃加热5min,然后上样到5.5%的聚丙烯酰胺凝胶上,在0.8X TBE电泳液中用Li-COR DNA Analyser(Li-COR,Lincoln,NE)电泳分离,功率恒定为75W,温度为55℃。通过与携带有ALR2基因的23(CA)重复序列比较的得到等位基因的大小,其中的ALR2基因的23(CA)重复序列由Shiga University of Medical Science of Japan的Dr.Shiro Maeda赠送。
ALR2基因C106T启动子的PCR条件
反应依据Kao YL等描述的方法进行(Kao Y,Donaghue K,Chan A,Knight J,Silink M,A novel polymorphism in the aldose reductase genepromoter region is strongly associated with diabetic retinopathy inadolescents with type 1 diabetes,Diabetes,1999;48:1338-40)。使用GeneAmp PCR System 9700(ABI)进行反应,最终体积为20μl,包含100ngDNA模板,50mM KCl,10mM Tris-HCl,pH 8.3,2mM MgCl2,dNTP各0.2mM)(Boehringer-Mannheim,Germany)。引物浓度为各0.5pmol/μl,Taq聚合酶为0.5U(Boehringer-Mannheim,Germany)。引物的序列如下:
5’-CCT TTC TGC CAC GCG GGG CGC GGG-3’ SEQ ID NO.9
5’-CAT GGC TGC TGC GCT CCC CAG-3’ SEQ ID NO.10
循环条件如下:初始94℃变性3min.,94℃ 1min.,57℃ 1min.,72℃1min,共35个循环,最后72℃延伸10min。PCR扩增产物用5U BfaI(NewEngland Biolabs,Beverly,MA)酶在37℃消化过夜。消化片段在3.5%的琼脂糖凝胶中通过电泳进行分离。C等位基因表现为206bp与57bp的片断,206bp片段进一步切割成147bp与59bp片段而得到T等位基因。
实施例2
肾病与基因间交互作用的关系
我们检测了711例(303男,408女,年龄63.1±11.1岁)中国血统II型糖尿病患者的AGT基因M235T,ACE(I/D),TNF-α基因G-308A,ALR2基因5’-(CA)n和启动子C-106T多态性的交互作用情况。选择有超过十年糖尿病病史,血浆肌氨酸酐小于100μmol/l以及即时尿清蛋白肌氨酸酐比率(spot urine albumi creatinine ratio)(ACR)小于3.5mg/mmol的患者作为对照(n=388)。血浆肌氨酸酐大于150μmol/l或者ACR不小于25mg/mmol的患者认为患有肾病(n=323)。将包括甘油三酸酯、ACR在内的不规则数据进行对数转换,用社会学统计包(Version 10.0,SPSS Inc,Chicago)进行统计学分析。如果合适,连续变量用平均值±SD或者几何学平均值(×/÷反对数)SD表示。应用Independence Sample T-测试与共变异数分析进行群体间的比较分析。用卡方测定来分析等位基因与基因型频率以及不同类型糖尿病患者相关的代谢异常与并发症的百分率。计算置信区间(CI)为95%的具有不同数目危险基因型的患者可能患糖尿病肾病的几率(OR),P值小于0.05(2-尾)可认为几率很大。患有肾病的患者与未患肾病的患者相比,年纪大的男性居多。校正年龄和性别后,患有肾病的患者比未患肾病的患者更肥胖和有更高的体重指数(BMI)、腰-臀比(WHR)和更高的血压。他们还具有更不良的脂肪分布,更高的血清总胆固醇(TC)、甘油三酸酯(TG),更低的HDL-C和更容易患感官神经疾病、视网膜病,周边血管病变、心血管疾病。表1显示了711例中国血统II型糖尿病患者中患肾病者与未患肾病的临床与生物化学参数比较。数据表示为平均值±SD或者几何平均值(×/÷反对数)SD,患肾病的患者和未患肾病的患者的数据用Independent Sample T-测试值进行比较,两研究组的糖尿病并发症的百分率用卡方测定值比较。
表1
研究组 | P值 | 校正后的P值* | ||
未患肾病(N=323) | 患肾病(N=388) | |||
性别(%男性)年龄(年)糖尿病发病时间(年)体重指数(kg/m2)腰高比收缩压(mmHg)舒张压(mmHg)糖化血红蛋白(%)禁食血浆葡萄糖(mmol/l)总胆固醇(mmol/l)甘油三酸酯(mmol/l)a高密度脂蛋白-C(mmol/l)低密度脂蛋白-C(mmol/l)血浆肌氨酸酐(μmol/l)清蛋白肌氨酸酐比率(mg/mmol)a肥胖(%)高血压(%)血脂异常(%)视网膜病(%)神经病(%)周边血管病变(%)缺血性心脏病(%)脑血管疾病(%)RAS抑止剂的应用(%) | 30.060.6±10.116.0±1.623.9±3.20.87±0.06131±1872±107.6±1.38.3±2.85.2±1.01.1×/÷1.71.4±0.43.20±0.8472.4±14.31.1×/÷2.034.546.752.025.118.62.85.93.726.9 | 53.165.1±11.68.2±5.425.5±4.00.91±0.07153±2383±128.1±2.19.5±4.45.9±1.51.9×/÷1.91.2±0.33.73±1.21152.6±105.4128.8×/÷2.947.981.283.054.643.813.412.48.579.9 | <0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.0010.0030.009<0.001 | ---<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.0010.0190.098- |
*校正年龄与性别后,应用共变异数分析得到的P值
表2总结了这5种类型基因型在患有或未患有肾病的患者中的分布情况。通过卡方测定对患有或未患有肾病的患者的基因型或者等位基因频率进行比较,患肾病的患者比未患肾病的患者具有更高频率的ALR2基因5’-(CA)n的z-2(24.1%vs.18.6%,P=0.01)和ALR2基因C-106T的T等位基因(25.8%vs.21.4%,P=0.05)。ALR2 5’-(CA)n z-2等位基因携带者比非z-2等位基因携带者具有更高的ACR水平(17.8×/÷12.3 vs.12.6×/÷12.9mg/mmol,P=0.062)和视网膜病百分率(47.4%vs.37.4%,P=0.009)。ACE I/D的DD/DI基因型携带者比II基因型携带者具有更高水平的TC水平(5.7±1.4 vs 5.5±1.3mmol/l,P=0.047)。在对年龄与性别进行校正后,TNF-α基因G-308A的肥胖性GG基因型携带者比非肥胖性患者具有更高水平的ACR(22.9×/÷11.5 vs 10.7×/÷13.2mg/mmol,P<0.001)和血浆肌氨酸酐水平(125±95 vs.108±75μmol/l,P=0.005)
表2
基因多态性的基因型 | 基因型频率(%) | 基因多态性的等位基因 | 等位基因频率(%) | ||
未患肾病(N=323) | 患肾病(N=388) | 未患肾病(N=646) | 患肾病(N=776) | ||
AGT基因M235TTTTMMM | 70.926.92.2 | 72.925.51.5 | AGT基因M235TTM | 84.415.6 | 85.714.3 |
ACE基因I/DIIDIDD | 45.841.512.7 | 46.441.811.9 | ACE基因I/DID | 66.633.4 | 67.332.7 |
TNF-α基因 TNF-α基因
G-308AGGGAAA | 80.818.30.9 | 84.015.50.5 | G-308AGA | 89.910.1 | 91.88.2 |
ALR2基因5’-(CA)nx/xx/z-2z-2/z-2 | 67.228.54.3 | 57.736.35.9* | ALR2基因5’-(CA)nxz-2 | 81.418.6 | 75.924.1+ |
ALR2基因5’-(CA)ny/yy/z+6z+6/z+6 | 89.99.30.9 | 93.36.70** | ALR2基因5’-(CA)nyz+6 | 94.45.6 | 96.63.4++ |
ALR2基因C-106TCCCTTT | 63.530.36.2 | 56.735.18.2*** | ALR2基因C-106TCT | 78.621.4 | 74.225.8+++ |
x=除了z-2等位基因以外的任何(CA)n等位基因;
y=除了z+6等位基因以外的任何(cA)n等位基因;
*P=0.01,**P=0.09,***P=0.07,当分别比较肾病患者与非肾病患者ALR2基因x/z-2 o或z-2/z-2,y/z+6 or或z+6/z+6与CT/TT的组合基因型频率时;
+P=0.01,++P=0.04,+++P=0.05,,当当分别比较肾病患者与非肾病患者ALR2基因z-2,z+6与T等位基因频率时。
表3总结了患者群中基因型数目的分布。在711个中国血统II型糖尿病患者中,64(9.0%)有0或1个危险基因型,176(24.8%)有2个危险基因型,290(40.8%)有3个危险基因型,181(25.5%)有4或5个危险基因型。与不多于1个危险基因型的患者相比,有2、3和大于4个危险基因型(趋势值P=0.006)的患者患肾病的几率分别从1.4(95%CI 0.8-2.4,P=0.3)增加到1.8(95%CI 1.1-1.3,P=0.03)和2.0(95%CI 1.1-3.6,P=0.02)(图2)。
表3
危险基因型的数目 | 未患肾病 | 患肾病 | 总群N(%) |
0危险基因型1危险基因型2危险基因型3危险基因型4危险基因型5危险基因型总计 | 631881255815323 | 225881658721388 | 8(1.1)56(7.9)176(24.8)290(40.8)145(20.4)36(5.1)711 |
实施例3
TNF-α GG基因型与肾病的关系
表4显示了在中国血统II型糖尿病患者中TNF-α基因的G-308A多态性与肥胖间的交互作用对肾病形成的影响,其中“Ref”代表参考组,该组是非肥胖GA/AA携带者。拥有GG基因型的肥胖患者患肾病的危险性升高了1.9倍(95%CI:1.1-3.2,P=0.012)。
表4
组 | 频率数.(%) | 糖尿病肾病的危险性 |
未患肾病 患肾病 | OR 95%CI P值 | |
GG-/非肥胖(Ref.)GG-/肥胖GG+/非肥胖GG+/肥胖 | 40(12.4) 38(9.8)22(6.8) 24(6.2)172(53.1) 164(42.3)89(27.6) 162(41.8) | 11.2 0.55-2.38 0.7101.0 0.62-1.65 0.9701.9 1.15-3.20 0.012 |
表5显示了在校正干扰因素如年龄、性别、微血管血糖、高血压、高血脂、视网膜病、神经病与周边血管病变后用来检测TNF-α基因G-308A与肥胖间的交互作用对肾病的影响的多元逻辑斯蒂回归分析。在表5中,校正的总百分率为75.2%,依变量:肾病的code=1;自变量包括年龄、性别(男性的code=1)、HbA1c、FPG和存在的高血糖、高血压及视网膜病、神经病与周边血管病变、缺血性心脏病与脑血管疾病(code=1)。肥胖与TNF-α基因G-308A多态性间的交互作用为:GG-/肥胖-(code=0),GG-/肥胖+(code=1),GG+/肥胖-(code=2),GG+/肥胖+(code=3)。
表5
独立预测因子 | 患糖尿病肾病的危险性 | ||
几率 | 95%CI | P值 | |
年龄(年)雄性禁食血浆葡萄糖(mmol/l)高血脂高血压视网膜病神经病周边血管病变GG-/肥胖+vs GG-/肥胖-GG+/肥胖-vs GG-/肥胖- | 1.022.61.12.83.22.42.12.81.41.2 | 1.002-1.041.78-3.711.03-1.161.86-4.182.19-4.811.62-3.511.36-3.221.21-6.590.57-3.260.64-2.15 | 0.033<0.0010.002<0.001<0.001<0.0010.0010.0170.4820.614 |
实施例4
ACE II/DD基因型与糖尿病肾病的关系
在病史平均为4.0±1.4年的947名中国血统糖尿病患者的扩增组中,我们检测了ACE II/DD基因型对定义为肾脏死亡的ESRD或肾脏事件(需要透析或血浆肌氨酸酐>500μmol/l或血浆肌氨酸酐基准值的两倍>150μmol/l)形成的影响。在947名患者中,62名患者发生了肾脏终点事件。
表6显示了在947名中国糖尿病患者中的ACE I/D多态性基因型和等位基因的频率,这些患者有或没有发生肾脏终点事件,此终点事件定义为血浆肌氨酸酐基准值的两倍或者在4.0年的平均期限后仍需要肾透析。我们应用卡方对发生和未发生肾病终点事件的基因型和等位基因频率进行了比较。
从表6可以看出,前一组患者比没有发生肾病终点事件的患者具有更高的DD基因型(19.4%vs.9.8%,P=0.031)和D等位基因频率(41.2%vs.30.2%,P=0.011)。Kaplan-Meier分析表明了在携带II(460中23个患者发生肾脏终点事件),DI(388中27个患者演化出肾脏终点事件),DD(99中12个患者发生肾脏终点事件)基因型的患者中累积性肾脏存活率(cumulative renal survival rate)有明显的不同。(log-rank P=0.019)(图2)。
表6
肾病终点事件的发生状况 | ||
非发生率 | 发生率 | |
基因型频率(%)IIDIDD基因型数目 | 49.440.89.8885 | 37.143.519.462 |
等位基因频率(%)ID等位基因总数 | 69.830.21771 | 58.841.22124 |
P=0.031,P value=0.011当分别在发生和没有发生肾病终点事件的患者间进行基因型和等位基因频率比较时。
表7显示了用来检测947名中国血统II型糖尿病患者中的肾脏终点事件的前兆因子的多元Cox-回归分析,在其中,“a”代表在初始时出现微量或巨量蛋白尿的患者;“b”代表与II基因型携带者相应的DI基因型携带者,“c”代表与II基因型携带者相应的DD基因型携带者。依变量包括:肾脏死亡或肾脏事件(code=1);自变量包括:年龄,性别(对男性,code=1),糖尿病发病时间,SBP,DBP,TC,TG、HDL-C、LDL-C的对数值,并发症的出现(code=1),如在基线处出现的肾病、视网膜病、神经病与周边血管病变,以及ACE基因I/D多态性(对于DI,code=1 vs.II,对于DD,code=2 vs.II)。
在多元Cox-回归分析中,肾病终点事件的出现一直显著地受到I/D多态性的影响,该I/D基因型含有一个具有明显有害效果的DD基因型(DD vs.II,校正的危险比率为3.4,95%CI 1.6-7.3,P=0.002)。其它独立的前兆因子包括长期患糖尿病、高收缩血压、甘油三酸酯、在初始时发生肾病和视网膜病。
表7
独立变量(基线值) | β相关系数 | 危险比率* | 95%CI | P值 |
糖尿病发病时间(年)血收缩压(mmHg)甘油三酸酯对数值(mmol/l)糖尿病肾病的出现a视网膜病的出现ACE基因I/DbDI基因型携带者基因I/DcDD基因型携带者基因I/Dc | 0.0500.0142.0633.5790.8500.5951.223 | 1.051.017.8735.82.341.813.40 | 1.01-1.091.00-1.032.24-27.74.84-265.21.27-4.300.99-3.311.59-7.27 | 0.0130.0120.001<0.0010.0060.0530.002 |
实施例5
ALR2的基因型与糖尿病肾病的关系
在有738个中国血统II型糖尿病患者的另一组中[年龄55.5±13岁,确诊病史为5.7±5.7年,平均值±SD],仅有21.5%患肾病(DN),仅有8%患有视网膜病(DR),53.1%未患并发症(UC)。CT/TT基因型携带者(N=267)的尿AER比CC基因型携带者高。(N=471)(30.2×/÷7.2 vs.21.9×/÷6.9μg/min,P=0.03)。在校正干扰变量,如年龄、糖尿病持续时间、血压和血红蛋白A1c后,该差异还是很明显(P=0.04)。
因为疾病的持续期是糖尿病微血管并发症的主要决定因素(Rogus J.J,Warram J.H,Krolewski A.S,Genetic studies of late diabetic complications.The overlooked importance of diabetes duration before complication onset,Diabetes,2002;51:1655-1662),因此患糖尿病时间不足5年的患者(n=300)在接下来的分析中被排除,将剩余的患者(n=438)分成四个亚组:159名患者(36.3%)仅具有DN,66名患者(15.1%)仅具有DR,121名患者(27.6%)具有DN和DR,92名患者(21%)没有并发症。单变量分析表明相对于UC组,DN和DR组的z-2等位基因(25.7%vs.16.9%,OR 1.7,95%CI 1.0-2.8,P=0.03)、T等位基因(26.4%vs.18.5%,OR 1.6,95%CI 1.0-2.7,P=0.04)的频率较高。
采用年龄、性别、疾病发病时间、BP、代谢指数和三个ALR2的基因型(携带的z+6,携带的z-2和携带的CT/TT)作为自变量,UC组作为对照(code=0),携带的z-2(OR2.64,95%CI 1.02-5.83)和CT/TT基因型(OR2.48,95%CI 1.19-5.19)以及年龄(OR 1.06,95%CI 1.02-1.10),BP(OR1.04,95%CI 1.02-1.06),HbAlc(OR 1.23,95%CI 1.03-1.46),log TG(OR20.1,95%CI 3.73-107.7)和雄性(OR 2.25,95%CI 1.10-4.61)都是预测DN、DR的独立的危险因子,该预测正确率达76.9%。
Claims (18)
1.一种检测中国血统糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的方法,包括如下步骤:
检测来自糖尿病患者的样品中是否含有下列多态性序列中的至少一种:ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5′(z-2)基因型,ALR2基因启动子区的C106T基因型,TNF-a基因的G-308A基因型,以及其互补序列,条件是不单独使用ALR2的基因型,其中多态性序列的存在表明该糖尿病患者已患有、将有可能发展成、或者疑似患有肾病。
2.根据权利要求1所述的方法,还包括从患者中取样的步骤。
3.根据权利要求2所述的方法,其中所取样品为血样。
4.根据权利要求1至3任一项所述的方法,该方法还包括用PCR方法扩增基因ACE、AGT、ALR2或TNF-α的步骤。
5.根据权利要求4所述的方法,其中扩增所用的引物为:扩增ACE基因使用的SEQ ID NO.1与SEQ ID NO.2;扩增AGT基因使用的SEQ IDNO.3与SEQ ID NO.4;扩增TNF-α基因使用的SEQ ID NO.5与SEQ IDNO.6;扩增ALR2基因使用的SEQ ID NO.7与SEQ ID NO.8或SEQ IDNO.9与EQ ID NO.10。
6.根据权利要求1至5任一项所述的方法,其中所述患者为已患有、将有可能发展成、或者疑似患有II型糖尿病的患者。
7.根据权利要求1至6任一项所述的方法,其中所述的I/D基因型含有DD基因型。
8.根据权利要求1至7任一项所述的方法,其中所述的G-308A基因型含有GG基因型。
9.检测中国血统糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的芯片,该芯片包括下列多态性序列中的至少一种:ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5’(z-2)基因型,ALR2基因启动子区的C106T基因型,TNF-a基因的G-308A基因型,以及其互补序列。
10.根据权利要求9所述的芯片,其中所述的患者是已患有、将有可能发展成、或者疑似患有II型糖尿病的患者。
11.根据权利要求9至10任一项所述的芯片,其中所述的I/D基因型含有DD基因型。
12.根据权利要求9至10任一项所述的芯片,其中所述的G-308A基因型含有GG基因型。
13.检测中国血统糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的试剂盒,包括:
芯片,其包括以下多态性序列中的至少一种:ACE基因的I/D基因型,AGT基因的M235T基因型,ALR2基因的(CA)n-5’(z-2)基因型,ALR2基因启动子区的C106T基因型,TNF-a基因的G-308A基因型,以及其互补序列;以及
指导性材料,其指导如何检测糖尿病患者已患有、或有可能发展成或疑似患有肾病。
14.根据权利要求13所述的试剂盒,其中所述的患者是已患有、将有可能发展成、或者疑似患有II型糖尿病的患者。
15.根据权利要求13或14所述的试剂盒,其中所述的I/D基因型含有DD基因型。
16.根据权利要求13到15任一项所述的试剂盒,其中所述的G-308A基因型含有GG基因型。
17.检测中国血统糖尿病患者已患有、将有可能发展成、或者疑似患有肾病的试剂盒,包括
扩增基因ACE、AGT、ALR2或TNF-α的引物;以及
指导如何检测糖尿病患者已患有、或有可能发展成或疑似患有肾病的指导性材料。
18.根据权利要求17所述的试剂盒,其中扩增所用的引物为:扩增ACE基因使用的SEQ ID NO.1与SEQ ID NO.2;扩增AGT基因使用的SEQ ID NO.3与SEQ ID NO.4;扩增TNF-α基因使用的SEQ ID NO.5与SEQ ID NO.6;扩增ALR2基因使用的SEQ ID NO.7与SEQ ID NO.8或SEQ ID NO.9与EQ ID NO.10。
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CN117954098A (zh) * | 2024-03-26 | 2024-04-30 | 天津医科大学朱宪彝纪念医院(天津医科大学代谢病医院、天津代谢病防治中心) | 一种糖尿病肾病鉴别诊断模型与系统 |
CN117954098B (zh) * | 2024-03-26 | 2024-06-07 | 天津医科大学朱宪彝纪念医院(天津医科大学代谢病医院、天津代谢病防治中心) | 一种糖尿病肾病鉴别诊断系统 |
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WO2009150550A2 (en) * | 2008-06-13 | 2009-12-17 | Prognomix, Inc. | Genetic component of complications in type 2 diabetes |
US20140023635A1 (en) * | 2010-12-21 | 2014-01-23 | Prognomix, Inc. | Single nucleotide polymorphisms and genes associated with t2d-related complications |
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CN117954098A (zh) * | 2024-03-26 | 2024-04-30 | 天津医科大学朱宪彝纪念医院(天津医科大学代谢病医院、天津代谢病防治中心) | 一种糖尿病肾病鉴别诊断模型与系统 |
CN117954098B (zh) * | 2024-03-26 | 2024-06-07 | 天津医科大学朱宪彝纪念医院(天津医科大学代谢病医院、天津代谢病防治中心) | 一种糖尿病肾病鉴别诊断系统 |
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