WO2014067447A1

WO2014067447A1 - Novel biomarkers for diabetic kidney diseases and use thereof

Info

Publication number: WO2014067447A1
Application number: PCT/CN2013/086133
Authority: WO
Inventors: Ronald Ching Wan Ma; Wing Yee So; Juliana Chung Ngor Chan
Original assignee: The Chinese University Of Hong Kong; Hospital Authority
Priority date: 2012-10-30
Filing date: 2013-10-29
Publication date: 2014-05-08
Also published as: CN105189778A; HK1213604A1

Abstract

Disclosed are methods, kits or arrays for detecting, preventing or treating a condition in a subject. The methods comprise a polymorphic sequence as a biomarker in a sample from the subject, thereby determining that the subject is suffering from, at risk for, or suspected of suffering from diabetic kidney complications, a diabetic kidney disease, or cardiovascular disease. Disclosed also are methods or use of the polymorphic sequence for treating diabetic kidney complications, a diabetic kidney disease, or cardiovascular disease. In particular, the polymorphisms are selected from the group consisting of rs10493064 in DLGAP3 gene, rs10199869 in CRIM1 gene, and rs16865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rs1241486 in STXBP6 gene and rs1241499 in STXBP6 gene.

Description

Novel biomarkers for diabetic kidney diseases and use thereof

FIELD OF THE INVENTION

The present invention relates to methods, kits and arrays for diagnosing or detecting a genetic susceptibility for a condition in a subject, in particular for diabetic kidney complications.

BACKGROUND OF THE INVENTION

Asia is in the midst of an epidemic of diabetes [1, 2]. According to the International Diabetes Federation (IDF) and World Health Organization (WHO), 285 million people worldwide are currently affected by diabetes, of which 60% are from Asia [3]. Latest epidemiological figures from China suggest that 10%, i.e. 100 million people are affected, with a significant proportion of young adults already affected with the disease [4]. With increasingly young age of onset, the financial implications due to productivity loss and health care expenditures are colossal. In 2004, total direct spending on diabetes in China was estimated at 57,649 billion RMB (US$6.97billion) or 7.57% of national total healthcare expenditure [5]. As a result, prevention of diabetes and diabetic complications has been identified as a top healthcare priority in China.

In Asian population, there is increased risk of diabetic kidney complications compared to European or other ethnic groups [6, 7]. In Chinese diabetic kidney disease with albuminuria, which reflects widespread vascular damage, is a major predictor for end-stage renal failure, cardiovascular complications and death, and a major contributor to the increased healthcare burden associated with diabetes. Diabetic retinopathy is the leading cause of blindness in developed countries, and share similar pathogenetic pathways with diabetic nephropathy. Apart from age, sex and disease duration, conventional risk factors such as blood pressure, abnormal lipids, obesity and hyperglycemia interact with diabetic proteinuria to give rise to cardio-renal complications with an annual event rate of 2-5% [8].

Diabetic nephropathy and diabetic kidney complications show moderate to high heritability, though only few genetic factors have been consistently shown to be associated with diabetic kidney disease [9, 10] or diabetic retinopathy [11]. Identification of genetic factors predicting these complications can facilitate early identification of high risk subjects for treatment, as well as provide novel targets for drug treatment. Traditional strategies to identify genetic factors for diabetic complications have mainly utilized the candidate gene approach. Using this approach, earlier studies have led to identification of polymorphisms of the aldose reductase gene as being associated with diabetic nephropathy [12] and diabetic retinopathy [12, 13]. Other studies have supported a role for the advanced glycation end products (RAGE) [14] gene as well as the angiotensin I converting enzyme gene [15] as being implicated in the development of diabetic kidney complications. Some of these genetic factors have also been found to be associated with presence of diabetic eye complications. Importantly, only few studies were performed in the Chinese population, and furthermore, most studies are limited by having involved only relatively modest sample sizes [10].

Another traditional approach is by linkage analysis using large affected families. Studies to date, predominantly in European populations, have identified several common linkage peaks associated with kidney complications in type 2 diabetes, including those on chromosome 18q22-23, 7q35-35, 7pl5, and 10q26 [9]. Importantly, variants within some of these regions have been consistently shown to be associated with diabetic kidney complications, as in the case for polymorphisms within the Engulfment and Cell Motility 1 (ELMOl) gene on chromosome 7pl4 [9, 10].

More recently, the use of genome wide-association studies (GWAS) has revolutionized the discovery of genetic markers associated with different common polygenic disorders. This approach has already led to the successful identification of more than 60 loci being associated with type 2 diabetes, often giving rise to novel biological insights [16]. Although this powerful approach has successfully identified several genetic loci associated with renal impairment in non-diabetic populations [17-19], there has only been few studies conducted in patients with diabetic kidney complications, and only very few published studies have been carried out for kidney complications in type 2 diabetes, albeit in African Americans or other populations [20].

In Hong Kong Chinese type 2 diabetic patients, 40% were non-obese (body mass index: BMI<25 kg/m²) and had low plasma C peptide level (which reflect pancreatic β cell reserve) associated with high glycated haemoglobin (Ale) [21]. In these diabetic patients, low BMI was associated with increased risk of renal dysfunction [8] which strongly predicted CVD and related death [22]. Using the Hong Kong Diabetes Registry with an enrolment of 8000 Chinese diabetic patients since 1995, more than 3000 patients have died or developed a major clinical event (cancer, renal failure, heart disease, stroke) [23] [24]. With the onset of hyperglycemia, there is perturbation of the metabolic milieu, cellular growth and sodium and water homeostasis resulting in widespread endothelial dysfunction and increased thrombotic tendency. Using a panel of candidate genes known to increase cardiovascular risks, we have demonstrated the complex phenotype-genotype interactions in predicting renal disease [25]. Using a prospective cohort of 1500 type 2 diabetic patients followed up for a mean period of 8 years, we were able to demonstrate the additive effects of these risk variants with 5-10 fold increased risk for CVD in subjects with 10 or more risk variants especially in those with low BMI. Yet, attainment of multiple treatment goals including blood glucose, blood pressure, blood lipids and use of RAS inhibitors can reducer risk of cardio-renal endpoints in type 2 diabetic patients by 50-70% [26] [27].

SUMMARY

One aspect disclosed herein is directed to a method for detecting or diagnosing a condition in a subject, comprising

obtaining a nucleic acid molecule from a sample of the subject,

determining whether at least one polymorphic sequence is present in said sample, wherein said polymorphic sequence is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and about 2kb of the flanking region thereof, PDE11A gene and 2kb of the flanking region thereof, NELL1 and 2kb of the flanking region thereof, MPPED2 and 2kb of the flanking region thereof, MON2 and 2kb of the flanking region thereof, STXBP6 and 2kb of the flanking region thereof, aldose reductase (ALR2) and 2kb of the flanking region thereof, protein kinase C beta (PRKCB1) and 2kb of the flanking region thereof,

and wherein the presence of a polymorphic sequence indicates that the subject is suffering from, at risk for, or suspected of suffering from the condition selected from the group consisting of diabetic kidney complications, diabetic kidney disease, diabetic nephropathy, or diabetic end-stage renal disease, cardiovascular disease (CVD) due to type 2 diabetes or type 1 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction.

In one embodiment of the method disclosed herein, the polymorphic sequence detected in the method is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDEllA gene and 2kb of the flanking region thereof.

In another embodiment, the polymorphic sequence detected in the method disclosed herein is

at least one polymorphism selected from the group consisting of rs 10493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDEllA gene, rsl459865 in NELLl gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rsl241499 in STXBP6 gene and/or

at least one risk variant selected from the group consisting of the risk variants (SNPs) listed in Table A.

In some embodiments of the method disclosed herein, the subject is Asian or Chinese descent.

In other embodiments of the method disclosed herein, the sample from the subject detected in the method described herein is blood, saliva, or other tissues.

In some embodiments of the method disclosed herein, the nucleic acid molecule detected in the method is selected from the group consisting of DNA, RNA, mRNA.

Another aspect disclosed herein is directed to an array or diagnostic kit for diagnosing a condition in a subject comprising,

a reagent for detecting at least one polymorphic sequence in a sample of the subject, wherein said polymorphic sequence is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDEllA gene and 2kb of the flanking region thereof, NELLl and 2kb of the flanking region thereof MPPED2 and 2kb of the flanking region thereof MON2 and 2kb of the flanking region thereof STXBP6 and 2kb of the flanking region thereof aldose reductase (ALR2) and 2kb of the flanking region thereof protein kinase C beta ( PRKCB1 ) and 2kb of the flanking region thereof, and

and instructions for use of the kit, comprising an direction, which suggests that the presence of a polymorphic sequence indicate that the subject is suffering from, at risk for, or suspected of suffering from the condition selected from the group consisting of diabetic kidney complications, diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction.

In an embodiment of the array or kit disclosed herein, reagent comprised therein is selected from the group consisting of reagents used in PCR-based technology, at least one primer for extension of multiplex products with detection by MALDI-TOF mass spectroscopy on a MassARRAY platform, one or more probes for hybridizing said polymorphic sequence.

In one embodiment of the array or kit disclosed herein, the polymorphic sequence detected with the array or kit is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDE11A gene and 2kb of the flanking region thereof.

In another embodiment, the polymorphic sequence detected with the array or kit disclosed herein is

at least one polymorphism selected from the group consisting of rs 10493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rsl241499 in STXBP6 gene and/or

In a still embodiment of the array or kit disclosed herein, the polymorphic sequence detected with the array or kit is at least one or two or all genetic variants selected from the group consisting of the genetic variant rs 10493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIM1 gene, and the genetic variant rsl6865645 of PDEllA gene.

In some embodiments of the array or kit disclosed herein, the subject is Asian or Chinese descent.

In other embodiments of the array or kit disclosed herein, the sample from the subject detected in the method described herein is blood, saliva, or other tissues.

In some embodiments of the array or kit disclosed herein, the nucleic acid molecule detected with the array or kit is selected from the group consisting of DNA, RNA, mRNA.

In some embodiments of the array or kit disclosed herein, they are useful for performing the method disclosed herein. Yet another aspect disclosed herein provides a method for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction. The method for treating or preventing the condition as disclosed herein comprises administering to the subject a compound counteracting the effect of any said polymorphism in the subject.

In an embodiment of the method for the treatment of the condition of the subject, the compound comprises agents for inhibiting at least one of the SNPs disclosed herein selected from a group consisting of an inhibitory RNA, an antibody, an anti-sense nucleic acid, or other agent for inhibiting the SNPs disclosed herein, as well as agents or drugs for reduction of blood pressure, glucose control, lipid parameters, and those for modulating the renin-angiotensin system.

Another aspect disclosed herein provides use of a reagent for inhibiting at least one, two, or three the SNPs or polymorphisms selected from the group consisting of rsl 0493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene and rsl 241499 in STXBP6 gene, in preparation of the medicament for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction.

Still another aspect disclosed herein provides use of a reagent for inhibiting at least one, two, or three the SNPs or polymorphisms selected from the group consisting of rsl 0493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene and rsl241499 in STXBP6 gene, for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows manhattan plot from genome-wide association study for diabetic kidney disease.

Figure 2 shows association of DLGAP3 rs7555884 with CKD (Dominant model).

Figure 3 shows association of PDE11A rsl6865645 with CKD (Dominant model).

Figure 4 shows association of CRIM1 rs2666136 with CKD (Dominant model).

Figure 5 shows joint analysis of DLGAP3 rs7555884, PDE11A rsl6865645, and CRIM1 rs2666136 for association with diabetic kidney disease, in which the age, sex and DM duration are adjusted. Compared to subjects who do not carry any risk variants at these 3 loci (N=512), subjects who carry 1, 2 or 3 risk variants (line 1, 2 and 3, respectively) have progressively increased risk of developing diabetic kidney disease during the follow-up period with HR of 1.214 (CI 0.979-1.51), HR 1.493 (CI 1.204-1.851) and HR 1.699 (CI 1.306-2.21), respectively.

Figure 6 shows additive effect of DLGAP3 rs7555884, PDE11A rsl6865645, and CRIM1 rs2666136 for association with diabetic kidney disease (dominant model), which includes adjustment for multiple baseline clinical variables including gender, age of patient, duration of diabetes, smoking status, BMI, HbAlc, systolic blood pressure, diastolic blood pressure, presence of diabetic retinopathy, peripheral neuropathy, peripheral vascular disease, history of coronary heart disease, history of stroke, baseline eGFR, use of lipid-lowering agents, use of antihypertensive agents, use of ACEI/ARBs, use of oral glucose-lowering agents, use of insulin, Ln triglyceride, Ln baseline albumin/creatinine ratio and baseline LDL levels.

Figure 7 shows interaction between the number of genetic risk variants and achievement of Ale, BP and cholesterol treatment targets for risk of developing diabetic kidney disease in a prospective cohort of 3469 Chinese subjects with type 2 diabetes.

DETAILED DESCRIPTIONS

Definitions

As used herein the terms "patient", "individual" and "subject", which can be alternately used, are not limited to human beings, but are intended to include all vertebrate animals in addition to human beings. In some embodiments disclosed herein, the terms "patient", "individual" and "subject" refer to Asian ancestry, including Chinese ancestry or descent. In other embodiments, the patient is a patient of diabetes, such as a patient of type 2 diabetes of Chinese ancestry.

As used herein, "gene" means any amount of nucleic acid material that is sufficient to encode a transcript or protein having the function desired. Thus, it includes, but is not limited to, genomic DNA, cDNA, RNA, and nucleic acid that are otherwise genetically engineered to achieve a desired level of expression under desired conditions. Accordingly, it includes fusion genes (encoding fusion proteins), intact genomic genes, and DNA sequences fused to heterologous promoters, operators, enhancers, and/or other transcription regulating sequences. The term refers to an entirety containing entire transcribed region and all regulatory regions of a gene. The transcribed region of a gene including all exon and intron sequences of a gene including alternatively spliced exons and introns so the transcribed region of a gene contains in addition to polypeptide encoding region of a gene also regulatory and 5' and 3' untranslated regions present in transcribed RNA.

As used herein, the terms "polymorphic sequence", "single nucleotide polymorphism", "SNP" and "genetic polymorphism" is a DNA sequence variation or a genetic variant that occurs when a nucleotide, e.g., adenine (A), thymine (T), cytosine (C), or guanine (G), in the genome sequence is altered to another nucleotide. SNPs are occasional variations in DNA sequence; the vast majority of the DNA sequence is identical among all humans, especially in a particular ancestry or descent. SNPs or other variants may also be found in genomic regions that do not contain genes. They represent a genomic hot spot responsible for the genetic variability among humans. The term SNP can be alternately used with the term "genetic variant" or "variant" which is present at a particular genetic locus in at least one individual in a population and that differs from the corresponding reference type in the vast majority of the DNA sequence.

As used in this application, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Reference throughout this specification to "one embodiment", or "an embodiment", or "in another embodiment", or "some embodiments", or "in certain embodiments" means that a particular referent feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases "in one embodiment", or "in an embodiment", or "in another embodiment", or "in some embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The inventors of the present application have completed the first genome-wide association for diabetic kidney disease in Chinese diabetic patients, and identified a panel of genetic markers associated with development of diabetic kidney complications. These markers provide a means by which subjects at risk of diabetic kidney complications can be identified at early stage of disease, and be targeted for intensive treatment to prevent disease progression. In addition to disease prediction and personalized medicine, these markers also provide novel targets for future drug development for treatment and prevention of diabetic kidney and other vascular complications.

The inventors of the present application surprisingly found that some genetic variants, such as those in DLGAP3, CRIMl, PDEllA, MPPED2, MON2 and STXBP6 and their flanking region of about 2kb, can be used individually or in a combination of at least two thereof to:

Prioritize risk stratification for personalized treatment to reduce risk of diabetes- associated co-morbidities including but not limited to diabetic kidney complications and CVD, in subjects with or at risk of diabetes;

Predict risk for diabetic kidney disease, diabetic nephropathy, end-stage renal disease or need for dialysis in a subject followed by intensive treatment and use of pharmacological treatments early (notably insulin, RAS inhibitors, statins, lipid-lowering agents, anti-hypertensive agents) with regular monitoring for attainment of treatment goals to reduce risk of these life- threatening events;

Predict risk for CVD in at risk subjects followed by intensive lifestyle modification to reduce modifiable risk factors (such as smoking, obesity, lipids, blood pressure) and use pharmacological treatments early (notably insulin, RAS inhibitors, statins, lipid-lowering agents, anti-hypertensive agents) with regular monitoring for attainment of treatment goals to reduce risk of these life- threatening events; and/or Identify subjects who may benefit from pharmacological agents which modulate the pathways of DLGAP3, CRIMl, PDEllA, MPPED2, MON2 or STZBP6 to reduce the risk of diabetes-related complications.

Accordingly, one aspect disclosed herein is directed to a method for detecting or diagnosing a condition in a subject, comprising

obtaining a nucleic acid molecule from a sample of the subject,

determining whether at least one polymorphic sequence is present in said sample, wherein said polymorphic sequence is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIMl gene and 2kb of the flanking region thereof, PDEllA gene and 2kb of the flanking region thereof, NELLl and 2kb of the flanking region thereof, MPPED2 and 2kb of the flanking region thereof, MON2 and 2kb of the flanking region thereof, STXBP6 and 2kb of the flanking region thereof, aldose reductase (ALR2) and 2kb of the flanking region thereof, protein kinase C beta ( PRKCB1 ) and 2kb of the flanking region thereof,

and wherein the presence of a polymorphic sequence indicates that the subject is suffering from, at risk for, or suspected of suffering from the condition selected from the group consisting of diabetic kidney complications, diabetic kidney disease, diabetic nephropathy, or diabetic end-stage renal disease, cardiovascular disease (CVD) due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction.

In one embodiment of the method disclosed herein, the polymorphic sequence detected in the method is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIMl gene and 2kb of the flanking region thereof, PDEllA gene and 2kb of the flanking region thereof.

at least one polymorphism selected from the group consisting of rs 10493064 in DLGAP3 gene, rsl0199869 in CRIMl gene, and rsl6865645 in PDEllA gene, rsl459865 in NELLl gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rs 1241499 in STXBP6 gene and/or at least one risk variant selected from the group consisting of the risk variants (SNPs) listed in Table A.

Table A

No SNP Gene Chr

1 rs5063 NPPA 664G>A 1

2 rs 10493064 DLGAP3 1

3 rs7555884 DLGAP3 1

4 rs699 AGT 235M>T 1

5 rs2666136 CRIM1 2

6 rsl6865645 PDE11A 2

7 rsl438040 PDE11A 2

8 rsl801282 PPARG Prol2Ala (125C>G) 3

9 rs5186 AGTR1 1166A>C 3

ADD1 Gly460Ala

10 rs4961 4

(1566G>T)

11 rsl062535 ITGA2 873 G>A 5

ADRB2 Argl6Gly

12 rs 1042713 5

(285A>G)

13 rs7756992 CDKAL1 6

14 rs909253 LTA 1069A>G 6

15 rsl041981 LTA Thr26Asn 6

16 rs 1800629 TNF-alpha 6

17 rsALR2 ALR2(CA)n 7

18 rs854560 PON1 Met55Leu 7

19 rs7493 PON2 7

20 rs759853 ALR2 7

21 rs 1800779 NOS3 -922A>G 7

NOS3 Glu298Asp

22 rsl799983 7

(1187G>T)

23 rs328 LPL 8

24 rs4994 ADRB3 8

25 rsl 1026146 NELL1 11

26 rsl459865 NELL1 11

27 rs5443 GNB3 825C>T 12

28 rs7953138 MON2 12

29 rsl800588 LIPC 15

30 rs3760106 PRKCB1 16

31 rs2575390 PRKCB1 16

32 rs7404928 PRKCB1 16

33 rs4787733 PRKCB1 16

34 rsl799752 ACE I/D 17 In a still embodiment of the method disclosed herein, the polymorphic sequence detected in the method is at least one or two or all genetic variants selected from the group consisting of the genetic variant rsl0493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIM1 gene, and the genetic variant rsl6865645 of PDEllA gene.

In an embodiment, the method disclosed herein further comprises a step for obtaining the sample containing at least one polynucleotide from the subject. In other embodiment, the method can be performed in vitro where the sample has been collected and is outside of the subject. In some embodiment, the nucleic acid is obtained by extracting and/or purifying it from the sample of the subject in vitro.

and instructions for use of the kit, comprising an direction, which suggests that the presence of a polymorphic sequence indicates that the subject is suffering from, at risk for, or suspected of suffering from the condition selected from the group consisting of diabetic kidney complications, diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction. In an embodiment of the array or kit disclosed herein, reagent comprised therein is selected from the group consisting of reagents used in PCR-based technology, at least one primer for extension of multiplex products with detection by MALDI-TOF mass spectroscopy on a MassARRAY platform, one or more probes for hybridizing said polymorphic sequence.

In one embodiment of the array or kit disclosed herein, the polymorphic sequence detected by the array or kit is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDE11A gene and 2kb of the flanking region thereof.

In another embodiment, the polymorphic sequence detected by the array or kit disclosed herein is:

at least on, two or three of the polymorphisms selected from the group consisting of rsl0493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rsl241499 in STXBP6 gene, and/or

In a still embodiment of the array or kit disclosed herein, the polymorphic sequence detected by the array or kit is at least one or two or all genetic variants selected from the group consisting of the genetic variant rs 10493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIM1 gene, and the genetic variant rsl6865645 of PDEllA gene.

In other embodiments of the method disclosed herein, the sample from the subject detected by the array or kit described herein is blood, saliva, or other tissues.

In some embodiments of the array or kit disclosed herein, the nucleic acid molecule detected by the array or kit is selected from the group consisting of DNA, RNA, mRNA.

In some embodiments, kits (e.g. diagnostic or reagent kits) or arrays disclosed herein comprise reagents or materials, and optionally protocols or instructions for assessing one or more SNPs to make a risk assessment, diagnosis or prognosis of a diabetes related complications and optimized therapeutic suggestions. Useful reagents and materials for kits can comprise, but are not limited to at least one agent selected from the group consisting of PCR primers, hybridization probes and primers as described herein (e.g., labeled probes or primers), allele-specific oligonucleotides, reagents for genotyping SNP markers, reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), DNA polymerases, RNA polymerases, DNA ligases, marker enzymes, antibodies which bind to altered or to non-altered (native) a polypeptide, means for amplification of nucleic acids fragments from one or more SNPs selected from, means for analyzing the nucleic acid sequence of one or more diabetes-complications or cardiovascular disease due to type 2 diabetes or ESRD related SNPs, or means for analyzing the sequence of one or more amino acid residues of polypeptides encoded by genes comprising such SNPs, etc.

In one embodiment, the kits or arrays for diagnosing susceptibility to a diabetes-related complication or cardiovascular disease due to type 2 diabetes in a subject comprise primers and reagents for detecting the nucleotides present in one or more SNP markers selected from the genetic variant rsl0493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIM1 gene, and the genetic variant rsl6865645 of PDE11A gene in individual's nucleic acid.

Yet another aspect disclosed herein provides a method for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction. The method for treating or preventing the condition as disclosed herein comprises administering to the subject a compound counteracting the effect of any polymorphism or SNP disclosed herein in the subject.

In an embodiment of the method for the treatment of the condition of the subject, the compound comprises agents for inhibiting at least one of the SNPs disclosed herein selected from a group consisting of a inhibitory RNA, an antibody, an anti-sense nucleic acid, or other agent for inhibiting the SNPs disclosed herein, as well as agents or drugs for reduction of blood pressure, glucose control, lipid parameters, and those for modulating the renin-angiotensin system.

In one embodiment of the method disclosed herein, the polymorphic sequence is within at least one gene selected from the group consisting of DLGAP3 gene, CRIM1 gene, PDE11A gene, NELL1 gene, MPPED2 gene, MON2 gene, STXBP6 gene, aldose reductase (ALR2) gene, protein kinase C beta (PRKCB1 ) gene, and 2kb of the flanking region thereof; alternatively, DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDE11A gene and 2kb of the flanking region thereof. In another embodiment of the method disclosed herein, the polymorphic sequence is at least one polymorphism selected from the group consisting of rs 10493064 in

DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in

NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rs 1241499 in STXBP6 gene and/or

In a still embodiment of the method disclosed herein, the polymorphic sequence is at least one or two or all genetic variants selected from the group consisting of the genetic variant rsl0493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIM1 gene, and the genetic variant rsl6865645 of PDE11A gene.

In some embodiments of the method disclosed herein, the nucleic acid molecule is selected from the group consisting of DNA, cDNA, RNA, mRNA.

Another aspect disclosed herein provides use of a reagent for inhibiting at least one, two, or three the SNPs or polymorphisms selected from the group consisting of rs 10493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene and rs 1241499 in STXBP6 gene, in preparation of the medicament for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction.

Still another aspect disclosed herein provides use of a reagent for inhibiting at least one, two, or three the SNPs or polymorphisms selected from the group consisting of rs 10493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene and rsl241499 in STXBP6 gene, for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction.

In one embodiment of the use disclosed herein, the polymorphic sequence in the use is within at least one gene selected from the group consisting of DLGAP3 gene, CRIM1 gene, PDE11A gene, NELL1 gene, MPPED2 gene, MON2 gene, STXBP6 gene, aldose reductase (ALR2) gene, protein kinase C beta (PRKCB1) gene, and 2kb of the flanking region thereof; alternatively, DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDE11A gene and 2kb of the flanking region thereof.

In another embodiment of the use disclosed herein, the polymorphic sequence is

at least one polymorphism selected from the group consisting of rs 10493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rs 1241499 in STXBP6 gene and/or

In a still embodiment of the use disclosed herein, the polymorphic sequence is at least one or two or all genetic variants selected from the group consisting of the genetic variant rsl0493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIM1 gene, and the genetic variant rsl6865645 of PDE11A gene.

In some embodiments of the use disclosed herein, the subject is Asian or Chinese descent.

In other embodiments of the use disclosed herein, the sample from the subject is blood, saliva, or other tissues.

Although the numerical chromosomal position of a SNP may still change upon annotating the current human genome build, the SNP identification information such as variable alleles and flanking nucleotide sequences assigned to a SNP will remain the same. Those skilled in the art will readily recognize that the analysis of the nucleotides present in one or more SNPs set forth herein in an individual's nucleic acid can be done by any method or technique capable of determining nucleotides present in a polymorphic site using the sequence information assigned in prior art to the rs IDs of the SNPs listed disclosed herein. It is also appreciated that in the art the nucleotides present in polymorphisms can be determined from either nucleic acid strand or from both strands.

Preparation of Samples

The presence of genetic variants or SNPs in the relevant genes disclosed herein is determined in sample of a subject.

The nucleic acid sequence can be DNA or RNA. In some embodiments, for the assay of genomic DNA, virtually any biological sample containing genomic DNA (e.g. not pure red blood cells) can be used. For example, and without limitation, genomic DNA can be conveniently extracted or purified from blood, semen, saliva, tears, urine, fecal material, sweat, buccal cells, skin or hair. For assays using cDNA or mRNA, the target nucleic acid must be obtained from cells or tissues that express the target sequence.

Many of the methods described herein require the amplification of DNA from target samples. This can be accomplished by any method known in the art but preferably is by the polymerase chain reaction (PCR). Optimization of conditions for conducting PCR should be determined for each reaction and can be accomplished without undue experimentation by one of ordinary skill in the art. In general, methods for conducting PCR can be found in Ausbel et al., eds., Short Protocols in Molecular Biology, 3.sup.rd ed., Wiley, 1995; and Innis et al., eds., PCR Protocols, Academic Press, 1990.

Other amplification methods include the ligase chain reaction (LCR) (see, Wu and Wallace, Genomics, 4:560-569, 1989; Landegren et al., Science, 241 :1077-1080, 1988), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173-1177, 1989), self- sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874-1878, 1990), and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produces both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

Polymorphism Detection

The polymorphism detection is to determine which form of the polymorphism is present in individuals for diagnostic or epidemiological purposes. The detection technology comprises sequencing, PCR-based technology, or methods involving primer extension of multiplex products with detection by MALDI-TOF mass spectroscopy on a MassARRAY platform (Sequenom, San Diego, California). Several methods have been developed to detect known SNPs. Many of these assays have been reviewed by Landegren et al., Genome Res., 8:769-776, 1998, and will only be briefly reviewed here.

One type of assay has been termed an array hybridization assay, an example of which is the multiplexed allele-specific diagnostic assay (MASDA) (U.S. Pat. No. 5,834,181; Shuber et al., Hum. Molec. Genet., 6:337-347, 1997).

High throughput screening for SNPs that affects restriction sites can be achieved by Microtiter Array Diagonal Gel Electrophoresis (MADGE) (Day and Humphries, Anal. Biochem., 222:389-395, 1994).

Additional assays for SNPs depend on mismatch distinction by polymerases and ligases. This has allowed the use of PCR for the rapid detection of single base changes in DNA by using specifically designed oligonucleotides in a method variously called PCR amplification of specific alleles (PASA) (Sommer et al., Mayo Clin. Proc, 64: 1361-1372 1989; Sarker et al., Anal. Biochem. 1990), allele-specific amplification (ASA), allele-specific PCR, and amplification refractory mutation system (ARMS) (Newton et al., Nuc. Acids Res., 1989; Nichols et al., Genomics, 1989; Wu et al., Proc. Natl. Acad. Sci. USA, 1989).

In another method for the detection of SNPs termed minisequencing, the target-dependent addition by a polymerase of a specific nucleotide immediately downstream (3') to a single primer is used to determine which allele is present (U.S. Pat. No. 5,846,710).

The above discussion of methods for the detection of SNPs is exemplary only and is not intended to be exhaustive. Those of ordinary skill in the art will be able to envision other methods for detection of SNPs that are within the scope and spirit of the present invention. These methods can be used individually or combinatively for the detection of the SNPs disclosed herein.

SNPs

In the present invention, at least one gene and/or its relevant region of the genes selected from the group consisting of DLGAP3 gene [Chromosome 1 NC_000001.10] CRIM1 gene [Chromosome 2 NC_000002.11] PDE11A gene [Chromosome 2 NC_000002.11] NELL1 gene [Chromosome 11 NC_000011.9] MPPED2 gene [Chromosome 11 NC_000011.9] MON2 gene [Chromosome 12 NC_000012.11] STXBP6 gene [Chromosome 14 NC_000014.8], and/or their the flanking regions of about 2kb, is selected to determine the polymorphisms or SNPs disclosed herein, such as rsl0493064 in DLGAP3 gene, rsl0199869 in CRIMl gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rsl241499 in STXBP6 gene and/or he risk variants (SNPs) listed in Table A, for determining the susceptibility to the diseases, disorders or conditions as described herein. The methods, kits and arrays as described herein relate to detection of SNPs at genetic loci of rsl0493064 in DLGAP3 gene, rsl0199869 in CRIMl gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rsl241499 in STXBP6 gene and/or he risk variants (SNPs) listed in Table A. In one embodiment, the methods, kits and arrays as described herein relate to detection of SNPs at the genetic loci comprising at least one, at least two or at least three of: rsl0493064 in DLGAP3 gene, rsl0199869 in CRIMl gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rsl241499 in STXBP6 gene and he risk variants (SNPs) listed in Table A.

The SNP is associated with a genetic predisposition for a disease, condition or disorder comprising but not limited to non-insulin dependent diabetes, insulin dependent diabetes, end stage renal disease due to non-insulin dependent diabetes, hypertension due to non-insulin dependent diabetes, end stage renal disease due to hypertension of non-insulin dependent diabetes, atherosclerotic peripheral vascular disease due to non-insulin dependent diabetes, and end stage renal disease due to insulin dependent diabetes.

Although the methods or kits disclosed herein can be performed in sample obtained from a subject with or without diabetes, it is preferred to perform them in a subject who is suffering from Type 2 diabetes. The subject can be Asian, including Chinese descent.

The risk of developing end stage renal disease increases with increasing number of risk SNPs within the genes and relevant regions thereof. For example, compared to subjects who do not carry any risk variants at these 3 loci, i.e. DLGAP3 rs7555884, PDE11A rsl6865645, and CRIMl rs2666136 (N=512) , subjects who carry 1, 2 or 3 risk variants (line 1, 2 and 3, respectively) have progressively increased risk of developing diabetic kidney disease during the follow-up period with HR of 1.214 (CI 0.979-1.51), HR 1.493 (CI 1.204-1.851) and HR 1.699 (CI 1.306-2.21), respectively. It is understood that one or more genotypes mentioned above can be used to develop arrays that are used in conjunction with other known clinical, biochemical and genetic for predicting the risk of diabetic complications including nephropathy and ESRD in diabetic patients of Chinese ancestry or other ethnic groups in the world, and these genotypes or equivalent arrays thereof can be used to identify at risk subjects for diabetes and/or diabetic ESRD for risk modification using a multifaceted approach including intensive monitoring, pharmacological and non-pharmacological therapy.

The present invention will be further described with the following Examples.

EXAMPLE METHODS

The biosamples of blood samples of the subjects were collected for genotyping of DNA to detect variants or SNPs as mentioned below. DNA was extracted from leukocytes as per standard protocol. Genotyping of genetic variants of interest were carried out . Genome-wide association study was performed by genotyping with the Illumina 610 Quad array. Additional genotyping of variants for replication by de novo genotyping was performed using methods involving primer extension of multiplex products with detection by MALDI-TOF mass spectroscopy on a MassARRAY platform (Sequenom, USA).

Genome wide association study for progression to diabetic kidney disease

Using the Hong Kong Diabetes Registry, a nested case-control study cohort was identified. Four hundred Chinese subjects with type 2 diabetes free of diabetic kidney disease (Diabetic kidney disease defined as diabetes with eGFR< 60ml/kg/m² with no history of other significant renal diseases) were identified. Controls were defined as type 2 diabetic cases who remained free of diabetic kidney disease after median duration of follow-up of 8 years. Age-and gender-matched T2DM cases who progressed to diabetic kidney disease were identified as cases. Genotyping was performed using the Illumina 610Quad array. Baseline characteristics of the study participants are listed in Table 1 :

Table 1. Baseline characteristics of subjects in the discovery cohort for a genome-wide association study for development of diabetic kidney complications No DKD (n=194) With DKD (n=194) P value

Age (years) 62(54 to 68) 63(54 to 69) 0.700Φ

Male sex 52.6%(102) 49.0%(95) 0.477†

Body mass index (kg/m²) 24.9(22.6 to 27.1) 24.5(22.3 to 27.1) 0. 827φ

Systolic BP (mmHg) 135(121 to 150) 143(129 to 160) <0.001φ

Diastolic BP (mmHg) 78(70 to 84) 80(73 to 89) 0.001 t

HbAi_c (%) 7.4(6.7 to 8.9) 8.2(7.0 to 10.0) Ο.ΟΟΙφ

LDL cholesterol (mmol/L) 3.30(2.80 to 3.90) 3.40(2.90 to 4.00) 0.523Φ

HDL cholesterol (mmol/L) 1.23(1.02 to 1.49) 1.16(0.96 to 1.37) 0.040Φ

Triglyceride (mmol/L) 1.18(0.84 to 1.98) 1.57(1.13 to 2.59) <0.001φ

Total cholesterol (mmol/L) 5.3(4.6 to 6.1) 5.6(4.7 to 6.3) 0.036Φ

Haemoglobin (g/dL) 14.1(13.0 to 15.2) 13.7(12.7 to 14.7) 0.026Φ

White blood cell count (xl0⁹/L) 6.9(6.0 to 7.8) 7.8(6.5 to 8.5) <0.001φ

ACR (mg/mmol) 1.85(0.85 to 5.60) 15.81(3.73 to 113.87) <0.001φ

Baseline eGFR (ml/min per 110.7(96.8 to

93.2(74.3 to 112.0) <0.001φ 1.73m²) 133.1)

Baseline eGFR 60 -90ml/min per

12.9%(25) 43.3%(84) <0.001† 1.73m²

Follow-up eGFR (ml/min per

95.5(82.6 to 113.0) 56.9(42.3 to 80.0) <0.001φ 1.73m²)

Duration of diabetes (years) 10(2 to 15) 9(3 to 14) 0.869Φ

Duration of follow-up (years) 8(6 to 9) 4(3 to 6) <0.001φ

†, Derived from Chi-square text; φ, Wilcoxon Two-Sample

Median (25^th to 75^th quartiles) or %(N);f ,

Table 2 Gene regions associated with development of diabetic kidney disease identified from

upstream .

Table 3. Known biological function of newly identified gene regions

Key SNP Chr Gene Known Biological Function rs 10493064 1 molecular organization of synapses and neuronal cell signaling.

rsl0199869 2 encoded protein is highly basic, may play a role in short-interfering-RNA-mediated gene silencing

rsl6865645 2 encodes a member of the PDE protein superfamily. Mutations in this gene are a cause of Cushing disease and adrenocortical hyperplasia rsl459865 11 encodes a cytoplasmic protein that contains epidermal growth factor (EGF)-like repeats, may be involved in cell growth regulation and differentiation.

rs559519 11 encodes a metallophosphoesterase 6 rs7953138 12 MON2 MON2 homolog

7 rsl241486 14 STXBP6 may be involved in regulating SNARE complex

formation

A total of 8 novel genomic regions showed suggestive association with development of diabetic kidney disease using a threshold of p<10^~5. The identified variants, along with a suitable proxy with r >0.8, were genotyped in a replication cohort of 4701 consecutive cases of Chinese T2DM from the Hong Kong Diabetes Registry, of which 1011 patients developed diabetic kidney disease during follow-up.

Table 4. Clinical characteristics of replication cohort

Data were expressed as mean + SD or median (25th-75th percentiles) or number (%) as appropriate.

Incident DKD Free of DKD P value

Baseline characteristics

(N = 1011) (N = 3690)

Male sex 440 (43.5%) 1680 (45.5%) 0.256

Age (year) 64 + 11 53 + 12 <0.001

Age at onset 54 + 12 47 + 12 <0.001

Duration of diabetes (year) 9.5 + 7.1 5.7 + 5.8 <0.001

Disease follow up time (year) 3.8 + 2.7 9.0 + 3.1 <0.001

Body mass index (kg/m2) 25.2 + 4.0 24.9 + 4.0 0.831

Waist circumference (cm)

Men 88.5 + 9.3 87.9 + 9.8 0.215

Women 85.8 + 9.9 82.8 + 9.8 <0.001

Systolic BP (mmHg) 143.4 + 20.8 131.2 + 18.6 <0.001

Diastolic BP (mmHg) 76.6 + 11.9 75.4 + 10.2 0.019

HbAlc (%) 8.1 + 1.9 7.6 + 1.8 <0.001

LDL cholesterol (mmol/L) 3.32 + 1.02 3.24 + 0.93 0.078

HDL cholesterol (mmol/L) 1.28 + 0.37 1.32 + 0.38 0.008

Total cholesterol 5.37 + 1.16 5.24 + 1.09 0.001

Triglyceride (mmol/L) 1.50 (1.07-2.25) 1.29 (0.91-1.95) <0.001 71.6 (16.6- 12.6 (6.9-36.6) <0.001

AER (ug/min)

287.4)

eGFR (ml/min per 1.73m2) 90.74 + 24.4 118.3 + 28.8 <0.001

Hypertension 572 (56.5%) 1235 (33.5%) <0.001

Retinopathy 427 (42.2%) 660 (17.9%) <0.001

Sensory neuropathy 386 (38.2%) 685 (18.6%) <0.001

Peripheral arterial disease 91 (9.0%) 121 (3.3%) <0.001

History of stroke 67 (6.6%) 105 (2.8%) <0.001

History of coronary heart 108 (10.7%) 140 (3.8%) <0.001

disease

Smoker 366 (36.2%) 1228 (33.3%) 0.086

Alcohol drinker 204 (20.5%) 721 (19.8%) 0.626

Drug use at baseline

Lipid lowering drugs 180 (17.8%) 420 (11.4%) <0.001

ACE inhibitor or angiotensin 351 (34.7%) 549 (14.9%) <0.001

receptor blockers

Other antihypertensive drugs 536 (53.0%) 987 (26.7%) <0.001

Oral antidiabetic drugs 711 (70.3%) 1366 (37.0%) <0.001

Insulin 284 (28.1%) 455 (12.3%) <0.001

DLGPA3, CRIMl and PDEllA genetic variants predict development of diabetic kidney disease or decline in kidney function among Chinese subjects with type 2 diabetes

In a prospective cohort involving Chinese subjects with type 2 diabetes (N=4701), genetic variants in DLGPA3, CRIMl and PDEl lA were found to identify those at risk of developing diabetic kidney complications and renal dysfunction (figures 2-4).

In an analysis including a combination of the above genetic variants, we identified additive effect of the number of genetic variants in predicting increased risk of subsequent diabetic kidney complications among patients with type 2 diabetes.

As shown in Figure 5, compared to subjects who do not carry any risk variants at these 3 loci (N=512), subjects who carry 1, 2 or 3 risk variants have progressively increased risk of developing diabetic kidney disease during the follow-up period with HR of 1.214 (CI 0.979-1.51), HR 1.493 (CI 1.204-1.851) and HR 1.699 (CI 1.306-2.21), respectively. This demonstrates the additive effect of the genetic variants in prediction of subsequent risk of diabetic kidney complications among Chinese patients with T2 DM.

In regression models in which traditional risk factors for diabetic kidney complications were included, this independent association between the described genetic factors and subsequent risk of diabetic kidney disease remains very strong (Figure 6). This regression model includes adjustment for multiple baseline clinical variables including gender, age of patient, duration of diabetes, smoking status, BMI, HbAlc, systolic blood pressure, diastolic blood pressure, presence of diabetic retinopathy, peripheral neuropathy, peripheral vascular disease, history of coronary heart disease, history of stroke, baseline eGFR, use of lipid-lowering agents, use of antihypertensive agents, use of ACEI/ARBs, use of oral glucose-lowering agents, use of insulin, Ln triglyceride, Ln baseline albumin/creatinine ratio and baseline LDL levels.

Figure 7 showed Interaction between the number of genetic risk variants and achievement of Ale, BP and cholesterol treatment targets for risk of developing diabetic kidney disease in a prospective cohort of 3469 Chinese subjects with type 2 diabetes.

We examined the interaction between genetic variants including DLGAP3 rs7555884, PDE11A rsl6865645 and CRIMl rs2666136 with achievement of clinical risk factor targets on the risk of developing chronic kidney disease (CKD) in a prospective cohort of 3469 subjects with type 2 diabetes. During the follow-up period, 880 subjects progressed to chronic kidney disease. Subjects were divided into tertiles based on the number of the risk variants present (8-20 variants, 21-23 variants and 24-36 variants) and also according to the number of risk factor treatment targets achieved (HbAlc <7 , blood pressure <130/80, LDL-cholesterol < 2.6mmol/l). Compared to the reference group (8-20 risk variants, 2-3 treatment targets achieved), subjects with the same number of risk variants but only 1 treatment target achieved or none achieved have significantly elevated risk of developing CKD (HR 1.82 and HR 2.268, respectively, p<0.001). Increasing number of risk variants was associated with increased risk of developing CKD, except in the group with 2-3 treatment targets achieved. The risk of developing CKD was ameliorated among subjects with 8-20 risk variants, or 21-23 risk variants, when compared to the reference group, if 2-3 treatment targets are achieved.

This analysis highlights the interactive effect of genetic variants and clinical risk factors control, pointing to the reduced risk of developing kidney complications, despite the presence of increased genetic risk, if appropriate intensive management to control clinical risk factors is implemented. This highlights the utility to use the genetic markers to empower healthcare professionals as well as patients to strive to achieve better risk factor management in order to reduce the risk of developing diabetic kidney complications.

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Claims

CLAIMS What is claimed is:

1. A method for detecting or diagnosing a condition in a subject, comprising

obtaining a nucleic acid molecule from a sample of the subject,

and wherein the presence of a polymorphic sequence indicates that the subject is suffering from, at risk for, or suspected of suffering from the condition selected from the group consisting of diabetic kidney complications, diabetic kidney disease, diabetic nephropathy, or diabetic end-stage renal disease.

2. The method of claim 1, wherein said polymorphic sequence is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIMl gene and 2kb of the flanking region thereof, PDEllA gene and 2kb of the flanking region thereof.

3. The method of claim 1, wherein said polymorphic sequence is

at least one polymorphism selected from the group consisting of rs 10493064 in DLGAP3 gene, rsl0199869 in CRIMl gene, and rsl6865645 in PDEllA gene, rsl459865 in NELLl gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene, rsl241499 in STXBP6 gene and/or

4. The method of claim 2, wherein said polymorphic sequence is at least one or two or all genetic variants selected from the group consisting of the genetic variant rs 10493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIMl gene, and the genetic variant rsl6865645 of PDEllA gene.

5. The method of any one of claims 1 to 4, wherein said subject is Asian or Chinese descent.

6. The method of any one of claims 1 to 5, wherein said sample from the subject is blood, saliva, or other tissues.

7. The method of any one of claims 1 to 5, wherein said nucleotic acid molecule is selected from the group consisting of DNA, RNA, mRNA.

8. An array or diagnostic kit for or diagnosing a condition in a subject comprising, a reagent for detecting at least one polymorphic sequence in a sample of the subject, wherein said polymorphic sequence is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDEllA gene and 2kb of the flanking region thereof, NELLl and 2kb of the flanking region thereof MPPED2 and 2kb of the flanking region thereof MON2 and 2kb of the flanking region thereof STXBP6 and 2kb of the flanking region thereof aldose reductase (ALR2) and 2kb of the flanking region thereof protein kinase C beta ( PRKCB1 ) and 2kb of the flanking region thereof, and

and instructions for use of the kit, wherein the presence of a polymorphic sequence indicates that the subject is suffering from, at risk for, or suspected of suffering from the condition selected from the group consisting of diabetic kidney complications, diabetic kidney disease, diabetic nephropathy, or diabetic end-stage renal disease.

9. The array or kit of claim 8, wherein said reagent is selected from the group consisting of reagents used in PCR-based technology, at least one primer for extension of multiplex products with detection by MALDI-TOF mass spectroscopy on a MassARRAY platform, one or more probes for hybridizing said polymorphic sequence.

10. The array or kit of claim 9, wherein said polymorphic sequence is within at least one gene selected from the group consisting of DLGAP3 gene and 2kb of the flanking region thereof, CRIM1 gene and 2kb of the flanking region thereof, PDEllA gene and 2kb of the flanking region thereof.

11. The array or kit of claim 8, wherein said polymorphic sequence is

12. The array or kit of claim 8, wherein said polymorphic sequence is at least one or two or all genetic variants selected from the group consisting of the genetic variant rs 10493064 of the DLGPAP3 gene, the genetic variant of rsl0199869 of the CRIM1 gene, and the genetic variant rsl6865645 of PDEllA gene.

13. The array or kit of any one of claims 8 to 12, wherein said subject is Asian or Chinese descent.

14. The array or kit of any one of claims 8 to 13, wherein said sample from the subject is blood, saliva, or other tissues.

15. The array or kit of any one of claims 8 to 14, wherein said nucleotic acid molecule is selected from the group consisting of DNA, RNA, mRNA.

16. A method for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction, comprising administering to the subject a compound counteracting the effect of any said polymorphism in the subject.

17. The method of claim 16, wherein the compound comprises agents for inhibiting at least one of the SNPs selected from a group consisting of an inhibitory RNA, an antibody, an anti- sense nucleic acid, or other agent for inhibiting the SNPs, as well as agents or drugs for reduction of blood pressure, glucose control, lipid parameters, and those for modulating the renin-angiotensin system.

18. Use of a reagent for inhibiting at least one, two, or three the SNPs or polymorphisms selected from the group consisting of rsl0493064 in DLGAP3 gene, rsl0199869 in CRIM1 gene, and rsl6865645 in PDE11A gene, rsl459865 in NELL1 gene, rs559519 in MPPED2 gene, rs7953138 in MON2 gene, rsl241486 in STXBP6 gene and rsl241499 in STXBP6 gene, for treating or preventing a condition in a subject selected from the group consisting of diabetic kidney complications comprising diabetic kidney disease, diabetic nephropathy, diabetic end-stage renal disease, cardiovascular disease due to type 2 diabetes, such as atherosclerotic peripheral vascular disease, hypertension, ischemic cardiomyopathy, and myocardial infarction.

19. The use of claim 18, wherein the reagent comprises agents for inhibiting at least one of the SNPs or polymorphisms selected from a group consisting of an inhibitory RNA, an antibody, an anti-sense nucleic acid, or other agent for inhibiting the SNPs, as well as agents or drugs for reduction of blood pressure, glucose control, lipid parameters, and those for modulating the renin- angiotensin system.