HK1140520B - Genetic markers and arrays containing the same for predicting risk of diabetic kidney and use thereof - Google Patents

Description

Genetic marker and chip for predicting diabetic nephropathy risk and application thereof

Technical Field

The present invention relates to a method and a chip for detecting a patient of Chinese descent suffering from, likely to develop into or suspected to suffer from nephropathy using one or more markers, in particular a method and a chip for detecting a patient of Chinese descent suffering from, likely to develop into or suspected to suffer from nephropathy using one or more genetic polymorphism markers selected from the group consisting of the genes ACE, AGT, ALR2 and TNF-a, provided that the genotype of ALR2 is not used alone.

Background

Diabetic nephropathy is a leading cause of morbidity and mortality in diabetic patients. With the prevalence of diabetes in developed and developing countries, diabetes has now become the leading cause of End-stage renal disease (ESRD), accounting for 40-50% of all new patients treated with renal replacement (Ritz E, RychlikI, Locatelli F, Halimi S, End-stage renal failure in type 2 diabetes: a media catastrophe of world dimensions, Am J Kidney Dis, 1999; 34: 795-. China is one of the three countries with the most diabetic patients, and it is estimated that by 2025, diabetic patients will increase to 4000 ten thousand and this increase will occur mainly in the middle-aged population (Chan J.C.N, NgM.C.Y, Critchley J.A.J.H, Lee S.C, Cockram C.S, Diabetes mellitis-obesity structural from a Chinese curative, Diabetes Research and clinical practical Practicality, 2001; 54: S19-27), the incidence of young Diabetes and childhood obesity and metabolic syndrome is the main cause of this condition (Chan J.C.N, Ng M.C.Y, Less lean Diabetes mellitus structural syndromes in Chi, Diabetes J.C.N, 2003; N.M.C.Y, C.S. 103, C.M.C.D., C.D. 107, C.C.D. 1. C.D. and C.D.C.D. C.D. D. C.D. D. C.D. C. D. C. D. C. C.D. D. C. D. C. D. C. D. C. D. C. D. C. D., leung D, Cheung R.C.K, Cheung M, So W.Y et al, The longterm effects of angiotensin converting enzyme inhibition and metabolism on carbon dioxide and residual outer monomers in hypertonic Type 2 diabetes patents, Kidney International, 2000; 57: 590-600). In a study of the world health organization of the Multinational Diabetes cardiovascular disease (WHO-MSVDD), it was found that Asian, and particularly Chinese and Japanese patients had a higher incidence of ESRD than white type II Diabetes patients (Morrish N.J., Wangs, Stevens L.K, Fuller J.H, Keen H, Mortality and houses of death in the WHO Multi surface of Vascular Diseases in Diabetes, 2001; 44: S14-21).

In contrast to the death of cardiovascular disease in the majority of the white diabetic population, the leading cause of death in Chinese descent diabetic patients is end-stage renal disease (ESRD) (Chan J.C.N, Cheng C.K, Cheng MYF, Swaminathan R, Critchley J.A.J.H, CockramC.S, Abnormal album as a predictor of mortality and renal Impatientin Chinese Patients with NIDDM, Diabetes Care, 1995; 18: 1013-. These findings have recently been confirmed by WHO-MSVDD (Morrish N.J., Wang S, Stevens L.K, Fuller J.H, Keen H, Mortality and consumers of death in the WHOMULATION Survey of Vascular Diseases in Diabetes, 2001; 44: S14-21). Consistent with these findings, it has been demonstrated that allelic frequencies and haplotypes of disease-associated genes differ from one another, which may lead to ethnic differences in susceptibility to disease development (Ng M, Wang Y, So W, Cheng S, Visvikis S, Zee R et al, ethical differences in the line disease and distribution of single nuclear genes in 35 cardiac genes for cardiac diseases, Genomics, 2003: in press; Young RP, Thomas G.N, Crithley J.A.J.H, Tomlisnone B, Woo K.S, Sanderson J.E, International genes in coronary syndromes in molecular biology in 25 genes, strain of related genes: 1998; Journal of growth factor in plant diseases in 25 genes: 1998). With the development of socioeconomic, renal failure and cardiovascular disease will become prevalent in our more and more elderly population (Chan J.C.N, Ng M.C.Y., Critchley J.A.J.H, Lee S.C., Cockram C.S, Diabetes mellitis-a specific mechanical change from a genetic choice, Diabetes Research and Clinical Practice, 2001; 54: S19-27) in view of the impact of the duration of the disease on the co-morbidities, the increased incidence of the young and middle-aged diabetic population, and the ethnic preference for the development of diabetic nephropathy.

We have previously reported a high incidence of 30% to 50% renal disease and The predictive value of a urinary marker of renal death and degeneration in diabetic patients of Chinese descent (Chan J.C.N, Cheng J.C.K, Laue. M.C, Woo J, Swaminathan R, Cockram C.S, The Metabolic hormone in Hong Kong tea-The inter-shift amplitude complex by structural modification, Diabetes Care, 1996; 19: 953-9). We have reported an independent relationship between insulin resistance and Diabetic nephropathy in patients of Chinese descent excluding hypertension and hyperlipidemia (Chan J.C.N, Tomlinson B, Nichols M.G, Swaminathan R, Cheung C.K, Cockram CS, Albumin, insulin resistance and dyslipaedia in Chinese Patients with non-insulin-dependent diabetes (NIDDM), diabetes Medicine, 1996; 13: 150-55) and their close relationship with Obesity, Albuminuria, and blood glucose metabolism disorders, respectively (Lee Z, Critchley J, Ko G.T, Anderson P, ThomN, Young R, et, Obesity and diabetes mellitus 182, Hong Yang Kong 3, Kong 3: Re 178). Family-based Research and isolation analysis (The Diabetes Control and compatibility Research Group, marketing of long ligands in microorganisms with microorganisms in The Diabetes Control and compatibility trial, Diabetes, 1997; 46: 1829-1839) and genome scanning have demonstrated that genetic factors play a strong role in The development of diabetic nephropathy (Imperosure G, Hanson RL, Pet D, Kobes S, BennettP, Knowl W, silicon pair linking analysis for genetic genes for genetic compatibility analysis, microorganism compatibility analysis with microorganisms in microorganisms with 2 microorganisms, genes groups, 1998; plant type, catalog W, catalog of 2000, Diabetes mellitus, beer. The angiotensin system (RAS) plays a key role in the regulatory system and in renal hemodynamics and tissue growth (Cooper M, Pathogenesis, prevention and treatment of diabetes, Lancet, 1998; 352: 213-9). The TT genotype of the AGT M235T polymorphism, the ACE I/D polymorphism D allele are related to nephropathy in diabetic patients of Caucasian, Japanese and Chinese blood systems (Fujisawa T, Ikegami H, Kawaguchhi Y, Hamada Y, Ueda H, Shintani M, et al, Meta analysis of infection/deletion polymorphism of infection I converting enzyme with Diabetes mellitus, 1998; 41: 47-53; Young R.P, Chan J.C.N, Poon E, Crithley J.A.J.H, Cockram C.S, associates beta.gene amplification and mutation, 9: 7, molecular dynamics, 1997 J.193, molecular dynamics, 1997, molecular dynamics, 65: 7, molecular dynamics, 1997, 9: 7, molecular dynamics, 1997, molecular dynamics, 3: 7, molecular dynamics. The cytokine secreted by adipocytes, i.e., Tumor necrosis factor a (TNF-a), is a factor that correlates obesity with insulin resistance (Hotamisoil GS, Spiegelman BM, Tumor neofactor: a key component of the obesity-Diabetes links, Diabetes, 1994; 43: 1271-8), while insulin resistance is an important feature of renal patients, including Chinese diabetics (Chan J.C.N, Tomlinson B, Nichols M.G, Swaminathan R, Cheng C.K, Cockram C.S, Albumin, insulin resistance and Diabetes in Chinepathies with non-insulin-dependent Diabetes (NIDDM), Diabetes, 13: 150-55). Recent Japanese studies have shown that Elevated serum TNF-a levels are associated with kidney disease in type II diabetic patients (Moriwaiky, Yamamoto T, Shibutani Y, Aoki E, Tsutsumi Z, Takahashi S, et al, expressed levels of interleukin 18 and tumor necrosis factor alpha in serum of disorders with type 2 diabetes mellitus: related with diabetes photopathology, Metabolim: Clinical and Experimental, 2003; 52: 605-8). In this regard, G-308A polymorphisms in The TNF-a gene promoter region have been reported to be associated with Obesity, insulin resistance (Dalziel B, Goskby A, Richman R, Bryson J, Caterson I, Association of TNF alpha-308G/A promoter with insulin resistance in vivo, Obesity Research, 2002; 10: 401-7) and enhanced TNF-a gene transcription activity (Kroeger K, Carville K, Abraham L, The-308 promoter activity transcripts, molecular immunology, 1997; 34: 391-99). Aldose reductase (ALR2) is the most critical enzyme of the polyol pathway, which leads to increased oxidative stress and changes in the intracellular environment, leading to diabetic microangiopathy (Hodgkinson A, Sondergard K, Yang B, Cross D, Millward B, Demaine A, Aldose reduction expression induced by hyperglycemic Indianal neuropathies, Kidney International, 2001; 60: 211-8). The z-2 allele of the ALR2 gene 5' - (CA), the C-106T polymorphic T allele, have been shown to increase the risk of developing renal disease in Type I, II diabetic patients, including patients of Chinese descent (Wang Y, NgM, Lee S, So W, Tong C, Cockram C, et al, photoplastic genetic disorders of two enzyme products with a novel neuropathology and a novel arthritis in Type 2 Diabetes, Diabetes Care, 2003; 26: 2410-5). The genetic factors disclosed above further interact with metabolic, hemodynamic and growth factors, leading to proteinuria and progressive failure of renal function (Parving H.H, Tarnow L, Rossing P, Genetics of metabolic neuropathology, Journal of American Society of neuropathology, 1996; 7: 2509-17).

Although there have been reports on the relationship of these 5 genetic markers to diabetic complications in caucasians and Japanese, there have been no consistent reports on the Chinese ancestral diabetic population. To date, there has been no report showing that these genetic factors have an interactive effect on the development of diabetic complications, including diabetic nephropathy.

The prospect of using genomics lies in its potential use, which can be used to identify patients at risk for earlier and targeted intervention to ensure physical health and reduce the impact of fatal diseases such as diabetes (Collins F, Green E, Guttmacher A, GuyerMobottunNHGRI, A vision for the future of genetics research. A blue for the genetics, Nature, 2003; 422: 835-47). In a study to identify genetic factors in patients at high risk for diabetic complications in China, we first reported that the AGT TT genotype was associated with Diabetes and that it, together with the ACE D allele, produced a synergistic effect on the development of Diabetes (Young RP, Chan J.C.N, Poon E, Critchley J.A.J.H, Cockram C.S, associates Weeenalbum album and angiotensinogen T235 and angiotensins conversion enzyme insert/deletion polymorphins in Chinese NIDDMPATITES, Diabetes Care, 1997; 21: 431-7). Similarly, we first reported that the ALR2TT genotype was associated with the risk of diabetic nephropathy in patients of Chinese descent (Wang Y, Ng M, Lee S, So W, Tong C, Cockram C, et al, photopheretic genetic associations of two times of aldose reduction genes with neuropathound retting Type 2 Diabetes, Diabetes Care, 2003; 26: 2410-5).

Summary of The Invention

Accordingly, the present invention relates to a method of detecting a subject of Chinese descent who has, is likely to develop, or is suspected of having a kidney disease, the method comprising the steps of:

detecting whether a sample from a diabetic patient contains at least one of the following polymorphic sequences: an I/D genotype of an ACE gene, an M235T genotype of an AGT gene, a (CA) n-5' (z-2) genotype of an ALR2 gene, a C106T genotype of a promoter region of an ALR2 gene, a G-308A genotype of a TNF-a gene, and complementary sequences thereof, provided that the genotype of ALR2 is not used alone, wherein the presence of a polymorphic sequence indicates that the diabetic patient has, will likely develop, or is suspected of having a renal disease.

In one embodiment of the present invention, the above method further comprises the step of taking a sample from the patient. The patient preferably suffers from type II diabetes, and the sample taken is preferably blood.

The invention also relates to a chip for detecting that a Chinese ancestral diabetic patient has, is likely to develop, or is suspected of having a nephropathy, the chip comprising at least one of the following polymorphic sequences: the I/D genotype of ACE gene, the M235T genotype of AGT gene, the (CA) n-5' (z-2) genotype of ALR2 gene, the C106T genotype of the promoter region of ALR2 gene, the G-308A genotype of TNF-a gene, and complementary sequences thereof, provided that the genotype of ALR2 is not used alone.

In the present invention, the I/D polymorphism preferably contains the DD genotype, and the G-308A polymorphism preferably contains the GG genotype.

The invention also relates to a kit for detecting that a Chinese ancestral diabetic patient has, will likely develop, or is suspected of having kidney disease. The kit generally comprises a chip as defined herein and instructional materials directing the treatment of the sample with the chip. Preferably, the kit contains a device for taking a sample from a patient.

Brief description of the drawings

FIG. 1 shows the probability of developing nephropathy in 711 patients with type II diabetes of Chinese descent who have a different number of risk genotypes, including the TT genotype of the AGT gene, the DD/DI genotype of the ACE gene, the GG genotype of the TNF- α gene, the TT genotype of the AGT gene, the x/z-2o or z-2/z-2 genotype of the ALR2 gene, and the CT/TT genotype of the ALR2 gene.

FIG. 2 shows a Kaplan-Meier plot of end-point events in renal disease in carriers of the ACE gene I/D genotype in 947 Chinese descent type II diabetics.

FIG. 3 shows a Kaplan-Meier plot of patient primary composite endpoint event (Panel A) or end-stage renal disease endpoint event (Panel B) and total lethal (Panel C). The patients were treated with a regimen focusing on the implementation of periodic monitoring, treatment and enhancement of patient compliance on the target (intervention group) and a regimen based on general clinical care (control group) by means of a multidisciplinary team, wherein patients in the intervention group were treated at all times under the direction of the physician, whereas patient compliance in the control group was unstable.

Detailed description of the invention

Genetic, epidemiological and experimental studies support such an assertion: diabetic nephropathy involves a variety of biochemical pathways (Cooper M, Pathogenesis, prevention and treatment of metabolic neuropathiy, Lancet, 1998; 352: 213-9). Based on international and regional, clinical and experimental evidence, we consider that the TT genotype of AGT gene M235T, the DD/ID genotype of ACE gene I/D, the GG genotype of TNF-alpha gene G-308A, the X/z-2 or z-2/z-2 (any allele except z-2) and the CT/TT genotype of ALR2 genes are potential risk genotypes of Chinese ancestral diabetes patients suffering from nephropathy.

Definition of

Unless specifically stated otherwise in the present invention, the term "AGT gene M235T" or "AGT M235T" refers to "M235T genotype of AGT gene"; the term "ACE gene I/D" or "ACE I/D polymorphism" has the same meaning as "I/D gene of ACE gene"; the term "TNF- α gene GG" is equivalent to "GG genotype of TNF- α gene G-308A"; the term "TNF-alpha gene G-308A" or "G-308A polymorphism in the promoter region of the TNF-alpha gene" or "TNF-alpha G-308A" or "TNF alpha G308A polymorphism" refers to the "G-308A genotype of the TNF-alpha gene"; the term "z-2 allele of aldose reductase 5 ' - (CA) n" or "ALR 2(CA) n-5 ' (z-2)" is equivalent to "the (z-2) genotype of ALR2 gene 5 ' - (CA) repeat sequence"; the term "T allele of the C-106T polymorphism of aldose reductase (ALR 2)" is equivalent to "ALR 2TT genotype, ALR2 gene TT" or "ALR 2 gene CT/TT genotype"; the term "a C-106T polymorphism of ALR 2" refers to "a C-106 genotype of the ALR2 gene in the promoter region".

The method for detecting the nephropathy of the Chinese ancestral diabetes patient comprises the following steps: detecting whether a sample from a diabetic patient contains at least one of the following polymorphic sequences: an I/D genotype of an ACE gene, an M235T genotype of an AGT gene, a (CA) n-5' (z-2) genotype of an ALR2 gene, a C106T genotype of a promoter region of an ALR2 gene, a G-308A genotype of a TNF-a gene, and complementary sequences thereof, provided that the genotype of ALR2 is not used alone, wherein the presence of a polymorphic sequence indicates that the diabetic patient has, will likely develop, or is suspected of having a renal disease.

Polymorphisms used as genetic markers in the above-described methods can be identified by:

(a) extraction of genomic DNA from a patient

(b) Amplifying ACE gene, AGT, TNF-alpha G-308A polymorphism gene, aldose reductase (ALR2) CA repetitive sequence and C106T promoter of ALR2 gene in genome DNA by using PCR method with genome DNA as template; and

(c) identifying the product of step (b) using gel separation or sequencing.

In the present invention, the genomic DNA may be extracted from a body fluid of a patient, such as blood and urine, preferably blood.

Although the detection method of the present invention can be applied to any diabetic patient, the method is preferably applied to type II diabetic patients. The invention is particularly suitable for the diabetes patients of Chinese ancestry.

All four candidate genes of this study may cause the development of diabetic nephropathy through possible metabolic pathways. AGT and ACE are important components of the RAS that cause hypertension and abnormal tissue growth (Cooper M, Patherisis, preservation and treatment of metabolic neuropathology, Lancet, 1998; 352: 213-9; ringer J, Beige J, Kunz R, Distler A, Sharma A, Genetic variants of the metabolic aminopeptidase system, metabolic neuropathology and hypertension, Diabetologia, 1997; 40: 193-9). The cytokine TNF- α makes a correlation between obesity and insulin resistance (Hotamisigil GS, Spiegelman BM, Tumor Diabetes factor: a key component of the obesity-Diabetes links, Diabetes, 1994; 43: 1271-8), and has been reported to show that its serum levels are positively associated with diabetic nephropathy (Moriwaki Y, Yamamoto T, Shibutani Y, Aoki E, Tsutsumi Z, Takahashi S, et al, evolved leaves of interleukin 18 and Diabetes factor alpha in server of tissues with type 2 Diabetes mellitus: translation with Diabetes mellitus, pathway, Metatism: Clinical and Experimental, 2003; 52: 8). ALR2 causes the development of microvascular disease through intercellular accumulation of sorbitol esters and increased oxidative stress under hyperglycemic conditions (Chung S, Ho E, Lam K, Chung S, restriction of polyol pathway to diabetes-induced oxidative stress, Journal of American Society of neurology, 2003; 14: S233-6).

The discovery of these genotypes in the Chinese ancestral population was based on a series of lateral, prospective and case-controlled studies, with results consistent with phenotypic characteristics of diabetic nephropathy patients who are more obese, have higher blood pressure, and have more poor lipid and glucose regulation than patients who do not have diabetic nephropathy. Large-scale randomized clinical studies have demonstrated beneficial Effects on the treatment of diabetic proteinuria and ESRD development by inhibiting RASD, enhancing glycemic and blood pressure regulation (Brenner B.M, Cooper M.E, De Zeeuw D, Keane W.F, Mitch W.E, Parving H.H et al, Effects of Losartan on repeat and cardiac outlook in tissues with type 2 diabetes and photopathicity, New England Journal of Medicine, 2001; 345: 861-9; UKPDS, Intensive blood glucose control with subcutaneous tissues orientation and reaction of tissue with Medical treatment and design of reactions with properties 2 diabetes and reaction of reactions of tissues with type 2 diabetes (UKPDS 33), Lance-1998; experiment of culture of filtration, purity of culture of 2 diabetes mellitus, Japan. Recent studies have shown that combined treatment with ACE and ALR2 inhibitors can produce a synergistic effect to improve neurological function in diabetic rats (Cotter M, Mirrlees D, Cameron N, neurovacular interactions between enzyme activity and angiotensins converting enzymeinhibitation in diabetes rates, European Journal of Pharmacology, 2001; 417: 223-30). In summary, the identification of these genetic factors and their interactions (and the associated microchip technology that improves the efficiency of simultaneous screening of risk genotypes for the same patient) allows the screening of high-risk individuals for intensive and targeted treatment thereof to reduce the risk of complications. In this regard, relying on a multidisciplinary team, we have demonstrated that multiple approaches focusing on periodic monitoring of goals, treatment, and enhanced patient compliance can reduce the risk of death and end-stage renal disease (ESRD) in chinese descent diabetic patients by 40-70%. (FIG. 3). The advantages of the disease control scheme and its cost effectiveness should be further enhanced due to the ever increasing risk of patients carrying these genotypes.

After adjusting for age and gender, we found that the risk of renal disease progressively increased significantly with increasing number of risk genotypes. Patients carrying 3 or more risk genotypes make up 66% of the total patients, and the risk of diabetic nephropathy is increased by 1.8-2.0 fold compared to those carrying 0 or1 risk genotype.

We provide raw data demonstrating the prognostic role of DD genotype on ESRD formation, TNF- α on diabetic nephropathy, and in particular in obese diabetic patients. More importantly, these data demonstrate that the interaction between these 5 genotypes has a prognostic role in the development of diabetic nephropathy.

It will be appreciated that one or more of the above mentioned genotypes may be used to prepare a chip which may be used together with other known clinical, biochemical and genetic chips to predict the risk of diabetic complications, including nephropathy, in diabetic patients of chinese descent. These genotypes or their equivalent chips can be used to detect a patient's risk of diabetes and/or diabetic nephropathy, and thus to alter the risk using multiple methods including intensity monitoring, pharmacological and non-pharmacological therapies.

In order to practice the present invention, it is convenient to use the chip defined in the present invention as a kit for detecting that a diabetic patient of chinese ancestry has, will likely develop or is suspected of having a renal disease. The kit comprises the chip and a pair of primers for amplifying genes ACE, AGT, ALR2 or TNF-alpha. In one embodiment of the kit of the invention, the primer is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10.

the present invention also provides a kit comprising a chip containing at least one of the following polymorphic sequences and any probe designed based on the sequence as a control: the I/D genotype of ACE gene, the M235T genotype of AGT gene, the (CA) n-5' (z-2) genotype of ALR2 gene, the C106T genotype of promoter region of ALR2 gene, the G-308A genotype of TNF-a gene, and complementary sequences thereof.

The invention will be further described by the following examples.

Example 1

Identification of the genotypes of the five polymorphic sequences

Preparation of the human genome

Approximately 10ml EDTA blood samples were collected from each patient, and after overnight lysis of the cells with SDS and proteinase K, genomic DNA was extracted with benzene and chloroform. The DNA particles were then dissolved in 1 XTE buffer. The quantity and quality of the extracted DNA was determined by measuring the optical density at 260nm and 280 nm. The extracted DNA was stored at 4 ℃ for the next genotyping.

PCR conditions for ACE gene

The reaction is carried out according to the method of modified Rigat (Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Souberer F, An interruption deletion polymorphism in I converting enzyme gene encoding for halogen of the vacuum of the server enzyme levels, Journal of Clinical Investigation, 1990; 86: 1343-. GeneAmp PCR System 9700(ABI) was used in standard PCR buffer (50mM KCl, 10mM Tris-HCl, pH 8.3, 3mM MgCl)₂150ng of DNA template was amplified in 0.2mM each of dNTPs) (ABI), 5pmol each of primer concentration, 0.6U (Amersham) of Taq polymerase, and 20. mu.l in total. The cycling conditions were as follows: the denaturation was started at 94 ℃ for 2min, 94 ℃ for 1min, 58 ℃ for 1min, and 72 ℃ for 2min for 30 cycles, and finally the extension was continued at 72 ℃ for 5 min. The sequences of the primers are as follows:

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

the PCR product was a 190bp and 490bp fragment, the former containing no insertional allele (insertinalle) gene and the latter containing an insertional allele.

PCR conditions for angiotensin Gene

The reaction was carried out according to the method of Russ (Russ A, Maerz W, Ruzicka V, Stein U, Gross W, Rapid detection of the hyper arranged Met235 → Thr alleof the Human angiotensinogen gene, Human Molecular Genetics, 1994; 2: 609-10). Using GeneAmp PCR System 9700(ABI) in standard PCR buffer (50mM KCl, 10mM Tris-HCl, pH 8.3, 1.5mM MgCl)₂200ng of DNA template were amplified in 50uM of dNTP (ABI), 0.3uM of each primer concentration, and 0.75U of Taq polymerase (Amersham). The sequences of the primers are as follows:

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

the cycling conditions were as follows: denaturation at 90 deg.C for 3 min; 94 ℃ 1min 68 ℃ 1min., 72 ℃ 1min., 10 cycles; followed by 90 ℃ 30sec, 68 ℃ 1min., 72 ℃ 30sec., 30 cycles, and finally 72 ℃ extension for 10 min.

The PCR product was digested with 5UTth 111I (Promega) enzyme overnight at 65 ℃. The digested fragments were separated by electrophoresis in a 3% agarose gel. The pure methionine allele appeared as an undigested 165bp single band pattern, and threonine appeared as 141bp and 24bp band patterns.

PCR conditions for TNF- α G-308A polymorphism

The reaction was carried out according to the method described by Wilson et al (Wilson A, di G)Iovine F, Blakemore A, Duff G, Single base polymorphism in the Human tumor factor alpha detection by NcoI restriction of PCR products, Human Molecular Genetics, 1992; 1: 535). The reaction was performed using the GeneAmp PCR System 9700(ABI) in a final volume of 20. mu.l, containing 100ng of DNA template, 50mM KCl, 10mM Tris-HCl, pH 8.3, 2.5mM MgCl₂0.2mM of each dNTP (Boehringer-Mannheim, Germany). The primer concentrations were 0.5mM each and 0.05U of Taq polymerase (Boehringer-Mannheim, Germany). The sequences of the primers are as follows:

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

the cycling conditions were as follows: denaturation was started at 95 ℃ for 3min, 95 ℃ for 1min, 58 ℃ for 1min, 72 ℃ for 1min for 35 cycles. Finally, extension was carried out at 72 ℃ for 10 min. The PCR amplification product was digested with 10UNcoI enzyme (Promega) overnight at 37 ℃. The digested fragments were separated by electrophoresis in a 3% agarose gel. The A allele appeared as an undigested 165bp band pattern and the G allele appeared as 87bp and 20bp band patterns.

Aldose reductase (ALR2) CA repeat PCR conditions

The region containing the dinucleotide CA repeat was amplified by PCR as described by Ko et al (Ko B.C.B, Lam K.S.L, Watt N.M.S, Chung S.S.M, An (A-C) ligated nucleotide repeat polynucleotide marker at the 5' end of the amplified produced gene is associated with amplified with oligonucleotide on set di-acidic repeat in NIDDM primers, Diabetes, 1995; 44: 727-32) using primers flanking the 138bp region.

The sequence of the sense strand used for the reaction was:

5’-GAA TCT TAA CAT GCT CTG AAC C-3’ SEQ ID NO.7

the antisense strand sequence is:

Arpr25’-GCC CAG CCC TAT ACC TAG T-3’. SEQ ID NO.8

add 1M 13 tail (5'-CAC GAC GTT GTAAAA CGA C-3') to the 5 ' end of the sense Strand primer for Infrared fluorescence labeling

PCR was performed in a buffer prepared according to the formulation provided, in a total volume of 4. mu.l, containing 1ng of DNA template, 2.5mM MgCl₂0.2mM each dNTP, 0.1 pmol/. mu.l each primer, 0.15 pmol/. mu.l IRD 800-labeled M13 forward (-29) primer, 0.15U Taq polymerase (Amplitaq, Perkin-Elmer/Cetus, Norwalk, CT). The circulation conditions are as follows: initial denaturation at 94 ℃ for 3min, 94 ℃ for 1min, 57 ℃ for 1min, 72 ℃ for 1min for 35 cycles, and final extension at 72 ℃ for 10 min.

The amplified PCR product was heated at 95 ℃ for 5min, loaded on a 5.5% polyacrylamide gel, and electrophoretically separated in 0.8 XTBE in a Li-COR DNA Analyser (Li-COR, Lincoln, NE) at a constant power of 75W and a temperature of 55 ℃. The size of the allele was obtained by comparison with the 23(CA) repeat sequence carrying the ALR2 gene, as presented by the Dr.Shiro Maeda of Shiga University of Medical Science of Japan, in which the 23(CA) repeat sequence of the ALR2 gene was present.

PCR conditions for ALR2 Gene C106T promoter

The reaction was carried out according to the method described by Kao YL et al (Kao Y, Donaghue K, Chan A, Knight J, Silink M, A novel polymorphism in the enzyme reaction region is a linear associated with a metabolic reaction with a metabolic reactivity index and an amino substituents with type 1 Diabetes, 1999; 48: 1338-40). Reactions were performed using the GeneAmp PCR System 9700(ABI) in a final volume of 20. mu.l containing 100ng DNA template, 50mM KCl, 10mM Tris-HCl, pH 8.3, 2mM MgCl₂dNTPs 0.2mM each) (Boehringer-Mannheim, Germany). The primer concentrations were 0.5 pmol/. mu.l each and 0.5U for Taq polymerase (Boehringer-Mannheim, Germany). The sequences of the primers are as follows:

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

the cycling conditions were as follows: initial 94 ℃ denaturation 3min., 94 ℃ 1min., 57 ℃ 1min., 72 ℃ 1min, for 35 cycles, and final 72 ℃ extension 10 min. The PCR amplification product was digested with 5U of BfaI (New England Biolabs, Beverly, Mass.) enzyme overnight at 37 ℃. The digested fragments were separated by electrophoresis in a 3.5% agarose gel. The C allele is expressed as fragments of 206bp and 57bp, and the fragments of 206bp are further cut into fragments of 147bp and 59bp to obtain the T allele.

Example 2

Relationship between nephropathy and intergenic interactions

711 cases (303 men and 408 women, age 63.1 + -11.1 years) of diabetes mellitus type II patients of Chinese descent tested the AGT gene M235T, ACE (I/D), TNF-alpha gene G-308A, ALR2 gene 5' - (CA)_nAnd the promoter C-106T polymorphism. Patients with a history of diabetes over ten years, plasma creatinine less than 100 μmol/l and immediate urine Albumin Creatinine Ratio (ACR) less than 3.5mg/mmol were selected as controls (n. 388). Patients with plasma creatinine greater than 150 μmol/l or ACR no less than 25mg/mmol are considered to have renal disease (n 323). The irregular data including triglycerides, ACR were logarithmically transformed and statistically analyzed using a social statistical package (Version 10.0, SPSS Inc, Chicago). Continuous variables are expressed as mean ± SD or geometric mean (x/÷ inverse log) SD, as appropriate. Comparative analysis between populations was performed using the independency Sample T-test and the analysis of covariates. Analysis of allelic and genotype frequencies and metabolic abnormalities and complications associated with different types of diabetic patients using the chi-square assayAnd (4) percent. The probability (OR) that patients with different numbers of risk genotypes with a Confidence Interval (CI) of 95% are likely to suffer from diabetic nephropathy is calculated, and a P-value of less than 0.05 (2-tailed) is considered to be a high probability. Patients with kidney disease are more elderly men than patients without kidney disease. After age and gender correction, patients with renal disease are more obese and have a higher Body Mass Index (BMI), waist-to-hip ratio (WHR) and higher blood pressure than patients without renal disease. They also have a poorer fat profile, higher serum Total Cholesterol (TC), Triglycerides (TG), lower HDL-C and are more susceptible to sensory neurological disorders, retinopathy, peripheral vascular disease, cardiovascular disease. Table 1 shows the clinical and biochemical parameters of patients with renal disease compared to patients without renal disease in 711 patients with type II diabetes of Chinese descent. Data are expressed as mean ± SD or geometric mean (x/÷ antilogarithm) SD, and data for patients with renal disease and patients without renal disease are compared using Independent Sample T-test values, and the percentage of diabetic complications in both study groups is compared using chi-square measurements.

TABLE 1

P values obtained by covariant analysis after correcting age and gender

Table 2 summarizes the distribution of these 5 types of genotypes in patients with or without renal disease. Patients with renal disease had a higher frequency of z-2 of ALR2 gene 5' - (CA) n (24.1% vs. 18.6%, P ═ 0.01) and the T allele of ALR2 gene C-106T (25.8% vs. 21.4%, P ═ 0.05) than patients without renal disease by comparing the genotype or allele frequency of patients with or without renal disease by the chi-square assay. ALR 25' - (CA)_nz-2 allele carriers had higher ACR levels (17.8 x/12.3 vs.12.6 x/12.9 mg/mmol, P ═ 0.062) and retinopathy percentage (47.4% vs.37.4%, P ═ 0.009) than non-z-2 allele carriers. DD/DI radical of ACE I/DGenotype carriers had higher levels of TC than genotype II carriers (5.7 ± 1.4vs 5.5 ± 1.3mmol/l, P ═ 0.047). Obese GG genotype carriers of TNF-alpha gene G-308A had higher levels of ACR (22.9 x/11.5 vs 10.7 x/13.2 mg/mmol, P < 0.001) and plasma creatinine levels (125 ± 95vs.108 ± 75 μmol/l, P ═ 0.005) than non-obese patients after age and gender correction

TABLE 2

x ═ any (CA) n allele other than the z-2 allele;

y-any (CA) n allele other than the z +6 allele;

p-0.01, P-0.09, P-0.07 when comparing the combined genotype frequencies of ALR2 genes x/z-2o or z-2/z-2, y/z +6or z +6/z +6 and CT/TT, respectively, in renal and non-renal patients;

+ P ═ 0.01, + + P ═ 0.04, + + P ═ 0.05, when renal and non-renal patients, ALR2 gene z-2, z +6 and T allele frequencies were compared, respectively.

Table 3 summarizes the distribution of the number of genotypes in the patient population. Of 711 type II diabetics of chinese descent 64 (9.0%) had 0 or1 risk genotype, 176 (24.8%) had 2 risk genotypes, 290 (40.8%) had 3 risk genotypes, and 181 (25.5%) had 4 or 5 risk genotypes. Patients with 2, 3 and more than 4 risk genotypes (trend P ═ 0.006) had increased rates of renal disease from 1.4 (95% CI 0.8-2.4, P ═ 0.3) to 1.8 (95% CI 1.1-1.3, P ═ 0.03) and 2.0 (95% CI 1.1-3.6, P ═ 0.02), respectively, compared to patients with no more than 1 risk genotype (fig. 2).

TABLE 3

Example 3

Relationship between TNF-alpha GG genotype and nephropathy

Table 4 shows the effect of the interaction between the G-308A polymorphism of the TNF- α gene and obesity on the development of renal disease in type II diabetes patients of Chinese descent, wherein "Ref" represents the reference group, which is a non-obese GA/AA carrier. Obese patients with the GG genotype had a 1.9-fold increased risk of renal disease (95% CI: 1.1-3.2, P ═ 0.012).

TABLE 4

Table 5 shows a multivariate logistic regression analysis for examining the effect of the interaction between TNF-. alpha.gene G-308A and obesity on renal disease after correction of interfering factors such as age, sex, microvascular blood glucose, hypertension, hyperlipidemia, retinopathy, neuropathy and peripheral vascular disease. In table 5, the total percentage corrected is 75.2%, depending on the variables: code for renal disease 1; independent variables include age, gender (code 1 in men), HbAlc, FPG and the presence of hyperglycemia, hypertension and retinopathy, neuropathy and peripheral vascular disease, ischemic heart disease and cerebrovascular disease (code 1). The interaction between obesity and the polymorphism of the TNF-alpha gene G-308A is: GG-/obese- (code ═ 0), GG-/obese + (code ═ 1), GG +/obese- (code ═ 2), and GG +/obese + (code ═ 3).

TABLE 5

Example 4

Relationship between ACE II/DD genotype and diabetic nephropathy

In an expanded set of 947 Chinese diabetic patients with an average 4.0 + -1.4 years history, we examined the effect of ACE II/DD genotype on the development of ESRD or renal events (requiring dialysis or > 500. mu. mol/l plasma creatinine or > 150. mu. mol/l plasma creatinine baseline) defined as renal death. Of the 947 patients, 62 patients had a renal endpoint event.

Table 6 shows the frequency of ACE I/D polymorphism genotypes and alleles in 947 chinese diabetic patients with or without the occurrence of a renal endpoint event, defined as twice the plasma creatinine baseline value or the need for renal dialysis after a mean period of 4.0 years. We used the chi-square to compare the genotype and allele frequencies of the occurrence and non-occurrence of end-point events of renal disease.

As can be seen from table 6, the former group of patients had higher DD genotype (19.4% vs.9.8%, P ═ 0.031) and D allele frequency (41.2% vs.30.2%, P ═ 0.011) than patients who did not develop end-point events of renal disease. Kaplan-Meier analysis showed a clear difference in cumulative renal survival rates among patients carrying the genotypes II (23 patients with renal end-point events 460), DI (27 patients with renal end-point events 388), DD (12 patients with renal end-point events 99). (log-rank P ═ 0.019) (fig. 2).

TABLE 6

P＝0.031，P value 0.011 when genotype and allele frequency comparisons were made between patients with and without end-point events of renal disease, respectively.

Table 7 shows the multivariate Cox-regression analysis used to detect the prognostic factor of renal endpoint events in 947 chinese ancestral type II diabetic patients, where "a" represents patients who initially developed microalbuminuria or macroalbuminuria; "b" represents a DI genotype carrier corresponding to a genotype II carrier, and "c" represents a DD genotype carrier corresponding to a genotype II carrier. Dependent variables include: kidney death or renal event (code ═ 1); the independent variables include: age, gender (for males, code ═ 1), time of onset of diabetes, log values of SBP, DBP, TC, TG, HDL-C, LDL-C, occurrence of complications (code ═ 1), such as nephropathy, retinopathy, neuropathy and peripheral vasculopathy occurring at baseline, and ACE gene I/D polymorphisms (code ═ 1vs. ii for DI and 2vs. ii for DD).

In multivariate Cox-regression analysis, the occurrence of end-point events in renal disease has been significantly affected by I/D polymorphisms that contain a DD genotype with a significant deleterious effect (DD vs.ii, corrected risk ratio of 3.4, 95% CI 1.6-7.3, P ═ 0.002). Other independent prognostic factors include prolonged onset of diabetes, high systolic blood pressure, triglycerides, initial onset of nephropathy and retinopathy.

TABLE 7

Example 5

Relationship between genotype of ALR2 and diabetic nephropathy

In another group of 738 patients with type II diabetes of Chinese descent [ age 55.5. + -. 13 years, confirmed history of 5.7. + -. 5.7 years, mean. + -. SD ], only 21.5% suffered from nephropathy (DN), only 8% from retinopathy (DR), and 53.1% from no complications (UC). The urinary AER was higher in CT/TT genotype carriers (N267) than in CC genotype carriers. (N ═ 471) (30.2 x/7.2 vs.21.9 x/6.9 μ g/min, P ═ 0.03). This difference is still evident after correction of interfering variables such as age, duration of diabetes, blood pressure and hemoglobin Alc (P ═ 0.04).

Because the duration of the disease is the major determinant of diabetic microvascular complications (Rogus J.J, Warram J.H, Krolewski A.S, Genetic students of late Diabetes compatibility. the overloaded organism of Diabetes mellitus development before the onset of Diabetes mellitus, Diabetes, 2002; 51: 1655-cake 1662), patients with Diabetes less than 5 years old (n 300) were excluded from the following analysis, and the remaining patients (n 438) were divided into four subgroups: 159 patients (36.3%) had only DN, 66 patients (15.1%) had only DR, 121 patients (27.6%) had DN and DR, and 92 patients (21%) had no complications. Univariate analysis indicated a higher frequency of z-2 alleles (25.7% vs.16.9%, OR 1.7, 95% CI 1.0-2.8, P ═ 0.03), T alleles (26.4% vs.18.5%, OR 1.6, 95% CI 1.0-2.7, P ═ 0.04) in the DN and DR groups relative to the UC groups.

Age, sex, time to disease onset, BP, metabolic index and three ALR2 genotypes (z +6 carried, z-2 carried and CT/TT carried) were used as independent variables, UC group was used as control (code ═ 0), z-2 carried (OR2.64, 95% CI 1.02-5.83) and CT/TT genotypes (OR2.48, 95% CI 1.19-5.19) and age (OR 1.06, 95% CI 1.02-1.10), BP (OR1.04, 95% CI 1.02-1.06), HbAlc (OR 1.23, 95% CI male (OR 2.25, 95% CI1.10-4.61) were all independent risk factors for predicting DN, DR with a prediction accuracy of 76.9%.

Claims (9)

1. A chip for detecting that a diabetic patient of chinese ancestry has, will likely develop, or is suspected of having a kidney disease, the chip comprising a polymorphic sequence: the M235T genotype of AGT gene, the (CA) n-5' (z-2) genotype of ALR2 gene and the C106T genotype of ALR2 gene promoter region, or their complementary sequences.

2. The chip of claim 1, further comprising a polymorphic sequence of the G-308A genotype of the TNF-a gene or a complementary sequence thereof.

3. The chip of claim 2, wherein the G-308A genotype comprises a GG genotype.

4. The chip of any one of claims 1-3, wherein the patient is a patient who has, will likely develop, or is suspected of having type II diabetes.

5. A kit for detecting a diabetic patient of Chinese descent who has, will likely develop, or is suspected of having a kidney disease, comprising

Primers for amplifying M235T genotype of AGT gene, (CA) n-5' (z-2) genotype of ALR2 gene and C106T genotype of promoter region of ALR2 gene; and

instructional material directing how to detect a patient who is determined to have, or to be at risk of developing, or suspected of having, a renal disorder.

6. The kit of claim 5, further comprising primers that amplify the G-308A genotype of the TNF- α gene.

7. The kit of claim 6, wherein the G-308A genotype comprises a GG genotype.

8. The kit of claim 6, wherein the primers used for amplification are: amplifying the M235T genotype of AGT gene using SEQ ID NO.3 and SEQ ID NO. 4; SEQ ID No.5 and SEQ ID No.6 used for amplifying the G-308A genotype of the TNF-alpha gene; amplifying the (CA) n-5' (z-2) genotype of the ALR2 gene using SEQ ID No.7 and SEQ ID No. 8; or the SEQ ID NO.9 and SEQ ID NO.10 used for amplifying the C106T genotype of the promoter region of the ALR2 gene.

9. The kit of any one of claims 5-8, wherein the patient is a patient who has, is likely to develop, or is suspected of having type II diabetes.