KR20210125148A - Compositions for diagnosis of diabetes or diabetic complications using circulating cell free DNA and method thereof - Google Patents

Abstract

The present invention provides a composition for diagnosing diabetes or diabetic complications, which can significantly predict or grasp the early diagnosis and disease degree of a patient with diabetes or diabetic complications. In addition, the present invention can analyze the mitochondrial DNA (mtDNA) change of circulating cell-free DNA (ccf-DNA) and thus enable liquid biopsy without collecting invasive tissue.

Description

Compositions for diagnosis of diabetes or diabetic complications using circulating cell free DNA and method thereof

The present invention provides a composition for diagnosing type 2 diabetes or type 2 diabetic complications, a kit for diagnosing type 2 diabetes or type 2 diabetic complications, including the composition, and information for diagnosing the type 2 diabetes or type 2 diabetic complications provides a method of providing

Diabetes mellitus is a metabolic disease in which high blood sugar levels persist for a long period of time, and is generally classified into type 1 diabetes, type 2 diabetes, gestational diabetes, and the like. Type 1 diabetes is caused by the pancreas not making enough insulin, also called “insulin-dependent diabetes” or “juvenile diabetes”. Type 2 diabetes is characterized by insulin resistance, which prevents cells from responding properly to insulin. All other forms of impaired glucose tolerance that first occur or are diagnosed during pregnancy are called gestational diabetes.

The prevalence of diabetes is greatly increasing due to the increase in the elderly population and changes in lifestyle, and it is estimated that the number of diabetes patients worldwide will reach 300 million by 2025. Diabetes treatment is broadly divided into diet, exercise, and drug therapy, and it is determined according to the patient's condition. Since it is almost impossible to cure diabetes after the onset of diabetes, secondary prevention is focused on improving symptoms and preventing acute and chronic complications. In other words, since the prevention and treatment of complications is more important than the disease itself, the purpose of management is always to maintain blood sugar and prevent complications.

Therefore, there is a need for technology for early diagnosis of diabetes and its complications.

Republic of Korea Patent No. 10-1168052

One object of the present invention is to provide a composition for diagnosing type 2 diabetes or type 2 diabetes complications, comprising an agent for detecting mitochondrial DNA (mtDNA) levels in circulating cell-free DNA (ccf-DNA) will do

Another object of the present invention is to provide a kit for diagnosing type 2 diabetes or complications of type 2 diabetes, comprising the composition.

Another object of the present invention is the step of isolating ccf-DNA from a biological sample isolated from a suspected subject. Measuring the level of mitochondrial DNA (mtDNA) by performing PCR with a primer set consisting of a nucleotide sequence and comparing the expression level of mtDNA measured in the above step with a normal control sample, Type 2 diabetes or An object of the present invention is to provide a method of providing information for diagnosing complications of type 2 diabetes.

In order to achieve the above object, the present invention is for diagnosing type 2 diabetes or type 2 diabetic complications, including an agent for detecting the mitochondrial DNA (mtDNA) level in circulating cell-free DNA (ccf-DNA) A composition is provided.

As used herein, the term “diabetes” refers to a type of metabolic disease such as insufficient insulin secretion or failure to function normally, and is a disease characterized by a high blood glucose concentration. Diabetes is divided into type 1 and type 2, and type 1 diabetes is a disease caused by the inability to produce insulin at all. Type 2 diabetes mellitus, in which insulin is relatively insufficient, is characterized by insulin resistance (insulin resistance; the inability of cells to effectively burn glucose due to a lack of insulin function to lower blood sugar). In the present invention, diabetes specifically refers to type 2 diabetes.

The "diabetic complications" of the present invention may be diseases induced by chronic inflammation induced by diabetes, and may be caused by chronic inflammation induced by AIM2 inflammasome.

Chronic inflammation is associated with insulin resistance and impaired glucose homeostasis in type 2 diabetes. Inflammasomes are multiprotein complexes that activate caspase-1 and downstream immune responses, including maturation and secretion of interleukin-1β (IL-1) and IL-18 in immune cells such as macrophages.

In the present invention, the inflammation induced by the AIM2 inflammasome causes psoriasis, dermatitis and nephropathy and nephropathy. In addition, it is known that the expression of AIM2 is induced in diabetic nephropathy or non-diabetic nephropathy patients. Therefore, the diabetic complication in the present invention may be specifically diabetic nephropathy.

The “diabetic nephropathy” refers to kidney disease induced by diabetes, and includes all of glomerulosclerosis, vascular lesion and tubulointerstitial disease, but in particular refers to diabetic glomerulosclerosis. .

As used herein, the term “diagnosis” means identifying the presence or characteristics of a pathological condition. For the purpose of the present invention, diagnosis is to determine whether type 2 diabetes or type 2 diabetic complications occur, and whether type 2 diabetes or type 2 diabetic complications develop can be confirmed early.

In the present invention, “circulating cell-free DNA (ccf-DNA)” is a fragment of DNA that does not exist in the cell nucleus and floats in the blood. Also called cell-free DNA, it is found in plasma and gaseous body fluids. do. The use of ccf-DNA allows liquid biopsies without the collection of invasive tissue.

In the present invention, "mitochondrial DNA (mtDNA)" is DNA present in mitochondria in the cytoplasm of a cell. Specifically, in the present invention, the mtDNA may be a human NADH dehydrogenase 1 gene.

In the present invention, the agent for detecting the human NADH dehydrogenase 1 gene may be a primer set consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. In addition, the agent for measuring the mtDNA level may be a primer pair, a probe, or an antisense oligonucleotide that specifically binds to the gene.

In an example of the present invention, changes in mtDNA levels in type 2 diabetes patients were analyzed. mtDNA was analyzed in the plasma of 141 patients with type 2 diabetes and 22 controls. mtDNA levels were determined by quantification of the copy number of ccf-DNA to mtDNA in plasma. mtDNA levels were significantly higher in patients with type 2 diabetes mellitus (T2D) compared to controls. Next, for the diagnosis of type 2 diabetes, the correlation between HbA1c, a hyperglycemic index, and mtDNA was confirmed. There was a slight correlation between elevated mtDNA levels and HbA1c levels in the plasma of type 2 diabetic patients ( FIG. 5 ). On the other hand, the nDNA level in the plasma of type 2 diabetic patients was lower than the mtDNA level ( FIG. 6 ). These results indicate that mtDNA levels are increased in patients with type 2 diabetes.

In other words, it was confirmed that the mitochondrial gene NADH dehydrogenase 1 gene was increased in circulating cell-free DNA (ccf-DNA) of type 2 diabetes patients, using the cell-free circulating DNA ( An agent that measures the mitochondrial gene NADH dehydrogenase 1 gene level in circulating cell-free DNA, ccf-DNA) can be used for the diagnosis of type 2 diabetes.

IL-1β is an important pro-inflammatory cytokine that is regulated by the inflammasome during chronic inflammation in patients with type 2 diabetes. To investigate the role of mtDNA in chronic inflammation in patients with type 2 diabetes, the effect of increased mtDNA level on IL-1β levels was analyzed. IL-1β levels were measured in plasma of 141 type 2 diabetic patients and 22 controls.

As a result of the analysis, it was confirmed that the IL-1β level was significantly higher in type 2 diabetes patients compared to the control group, and it was confirmed that the high mtDNA level and the IL-1β level had a weak correlation. Through the above results, it was confirmed that high mtDNA levels are associated with chronic inflammation mediated by IL-1β in type 2 diabetes patients.

In addition, it was confirmed that mtDNA of type 2 diabetes patients induces AIM2 inflammatory activation in macrophages.

Therefore, it is possible to diagnose complications due to chronic inflammation caused by type 2 diabetes by analyzing the increase in mtDNA.

Another aspect of the present invention provides a kit for diagnosing type 2 diabetes or complications of type 2 diabetes.

The diagnostic kit may further include one or more other component compositions, solutions, or devices suitable for the assay method.

In another aspect of the present invention, separating circulating cell-free DNA (ccf-DNA) from a biological sample isolated from a suspected individual, SEQ ID NO: 1 and SEQ ID NO: 2 from the ccf-DNA isolated above Measuring the level of mitochondrial DNA (mtDNA) by performing PCR with a primer set consisting of a nucleotide sequence of Type 2 diabetes or A method of providing information for diagnosing complications of type 2 diabetes is provided.

In the present invention, the description of "circulating cell-free DNA (ccf-DNA)" and "mitochondrial DNA (mtDNA)" is the same as described above.

As used herein, the term “biological sample” refers to a tissue, cell, whole blood, serum, plasma, saliva, cerebrospinal fluid, or urine sample with a different gene expression level due to the onset of type 2 diabetes or type 2 diabetes complications. includes

In an embodiment of the present invention, plasma was used as a biological sample. Accordingly, the biological sample of the present invention may preferably be plasma.

Since the expression level of all of the mitochondrial DNA (mtDNA) is changed in an individual with diabetes as compared to a normal control group, when the level is changed, diabetes can be diagnosed.

Specifically, when the level of mtDNA in the isolated biological sample of a subject suspected of type 2 diabetes or type 2 diabetic complications is higher than the level of mtDNA in a normal control sample, it can be determined as type 2 diabetes or type 2 diabetic complications. .

The present invention, by providing a composition for diagnosing diabetes or diabetic complications, it is possible to significantly predict or grasp the degree of disease and early diagnosis of diabetes or diabetic complications. In addition, by analyzing the mitochondrial gene change of the found circulating cell-free DNA (ccf-DNA), it is possible to perform a liquid biopsy without collecting invasive tissue.

1 is a measurement of circulating cell-free mitochondria (ccf-DNA) levels in plasma in 141 patients with type 2 diabetes mellitus (T2D) and 22 people in a control group. (A) The single-stranded DNA (ssDNA) concentration was measured. **p<0.001 two-tailed Student's t-test; Cohen's d effect size d = 1.84. (B) The double-stranded DNA (dsDNA) concentration was measured. **p<0.001 two-tailed Student's t-test; Cohen's d effect size d = 0.69.
Figure 2 shows the results of quantitative PCR analysis of mitochondrial DNA (mtDNA) levels in plasma in 141 patients with type 2 diabetes (T2D) and 22 people in the control group. **p<0.001 two-tailed Student's t-test.
3 is a result of analyzing the level of IL-1β in plasma in 141 patients with type 2 diabetes mellitus (T2D) and 22 patients in a control group. **p<0.001 two-tailed Student's t-test.
4 is a result of analyzing absent in melanoma 2 (AIM2) inflasome activation induced by mtDNA of diabetic patients in macrophages. (a) Caspase in LPS-primed WT BMDM stimulated with poly(dA:dT) and ccf-DNA (ccf-DNA#1 and ccf-DNA#2) or poly(dA:dT) and vehicle control (control) These are the results of immunoblot analysis for -1 and IL-1β. For immunoblots, β-actin was used as a loading control. (b) IL in LPS-primed WT BMDM stimulated with poly(dA:dT) and ccf-DNA (ccf-DNA#1 and ccf-DNA#2) or poly(dA:dT) and vehicle control (control) The secretion of -1β, IL-18 and TNF-α was quantified. (c) IL-1β and IL-18 secretion in LPS-primed WT BMDM stimulated with ATP, flagellin or MDP and ccf-DNA (ccf-DNA #1 and ccf-DNA #2) or vehicle control (control). it will be analyzed (d) Immunofluorescence images in LPS-primed WT BMDM (100 cells total in 15 individual images per group) and caspase recruitment domain (ASC) speck formation (white arrows) (ASC for each mouse) quantification of speck-positive cells). (n = 6 mice per group). They were stimulated with poly(dA:dT) and ccf-DNA, or poly(dA:dT) and vehicle controls (controls). Scale bar, 20 μm
5 is a result of analyzing the correlation between HbA1c level, which is a hyperglycemic index, and mtDNA level in a type 2 diabetes patient. R=0.279, P=0.0004 Spearman correlation coefficient analysis, black line is linear regression (r ² =0.0298).
6 shows the results of quantitative PCR analysis of ccf-mtDNA and ccf-nDNA levels in plasma in 141 patients with type 2 diabetes mellitus (T2D) and 22 people in a control group. **p<0.001 two-tailed Student's t-test.
7 is a result of analyzing the correlation between IL-1β levels and ccf-mtDNA levels in type 2 diabetes patients. R=0.1404, P=0.0484 Spearman correlation coefficient analysis, black line is linear regression (r ² =0.1107).
8 is a result showing that caspase-1-dependent IL-1β and IL-18 secretion is required in absent in melanoma 2 (AIM2) inflasome activation induced by mtDNA. (a) Immunoblot analysis of IL-1β and IL-18 in BMDMs pre-treated with poly (dA: dT) and selective caspase-1 inhibitors (Z-VAD, 10 μM, 1 h) prior to ccf-DNA stimulation after LPS incubation is a result Beta actin was used as a loading control. (b) IL-1β, IL-18 and TNF-α in BMDMs pre-treated with poly (dA: dT) and selective caspase-1 inhibitors (Z-VAD, 10 μM, 1 h) prior to ccf-DNA stimulation after LPS incubation. to quantify the secretion of

Hereinafter, the present invention will be described in more detail through Examples according to the present invention and Comparative Examples not according to the present invention, but the scope of the present invention is not limited by the Examples presented below.

Materials and Methods

1. Research subject

Human subjects studies were conducted according to the Helsinki Declaration. The protocol of this study was approved by the Institutional Review Committee of Pusan National University Hospital (20100024). Subjects submitted informed consent prior to enrollment. Subjects were recruited from the Diabetic Kidney Disease Study (DKDS). A total of 172 patients were enrolled in the outpatient clinic from February 2010 to February 2012, of which 147 were type 2 diabetes patients and the remaining 25 were healthy control patients. All enrolled patients had estimated glomerular filtration rates (eGFR) of 60 ml/min/1.73 m or more and serum creatinine less than 1.2 mg/dl, with relatively preserved renal function. In addition, it had a sufficient washout period for renin-angiotensin system (RAS) inhibitors. Patients had no history of RAS inhibitors at enrollment or had a washout period of at least 2 months on these drugs prior to enrollment. After registration, medical history and anthropometric measurements were performed at the initial clinic visit, and blood samples were collected at the same time. Nine of 172 patients were excluded from analysis due to insufficient baseline plasma or spot urine samples. The 163 patients included in this study consisted of 141 type 2 diabetic patients and 22 healthy controls.

2. Isolation of circulating cell-free DNA (ccf-DNA) from plasma

Plasma was collected in EDTA-coated tubes. Plasma samples were stored at -80 °C. Ccf-DNA was isolated from plasma (100 μl) using a Maxwell® RSC instrument (AS4500, Promega) and a Maxwell® RSC ccf-DNA plasma kit (AS1480, Promega). In the final elution step, the isolated ccf-DNA was eluted using 1×TE buffer (50 μl) according to the manufacturer's manual.

3. Quantification of the level of ccf-DNA in plasma

Quantification of dsDNA and single-stranded DNA (ssDNA) concentrations in ccf-DNA was measured by fluorescence detection using a Quantus-Fluorometer (E6150, Promega). The concentration of dsDNA was measured using the QuantiFluor® dsDNA system (E2670, Promega), and the concentration of ssDNA was measured using the QuantiFluor® ssDNA system (E3190, Promega).

4. Quantification of mtDNA and nDNA levels in ccf-DNA

As a standard for mtDNA copy number, a cDNA clone of human nicotinamide adenine dinucleotide (reduced) (NADH) dehydrogenase 1, MTND1) was used. For standard of nuclear DNA (nuclearDNA, nDNA) copy number, human hemoglobin, beta (HBB) cDNA ORF clone (Myc-DDK tagged) was used. Concentrations were converted to copy numbers using a DNA copy number calculator using the formula of Equation 1.

10 μl mixture containing 5 μl ccf-DNA and gene-specific primer set was mixed with 10 μl of 2×SYBR Green PCR Master Mix, and real-time polymerase chain reaction (RT) using an ABI PRISM 7300 real-time PCR system. -PCR) quantification was performed.

The nucleotide sequence of the gene-specific primer set is shown in Table 1.

gene forward primer reverse primer human NADH dehydrogenase 1 gene (mtDNA), 5'-ATACCCATGGCCAACCTCCT-3'
(SEQ ID NO: 1) 5'-GGGCCTTTGCGTAGTTGTAT-3'
(SEQ ID NO:2) human-globin (nuclear DNA) 5'-GTGCACCTGACTCCTGAGGAGA-3'
(SEQ ID NO: 3) 5'-CCTTGATACCAACCTGCCCAG-3'
(SEQ ID NO: 4)

To detect mtDNA, RT-PCR analysis was performed as follows.

The denaturation step was initiated at 95° C. for 10 minutes, and then 50 cycles of 30 seconds at 95° C., 30 seconds at 60° C. and 30 seconds at 72° C. were repeated, followed by PCR.

The level of mtDNA in all plasma assays was calculated in copies per microliter of plasma based on Equation 2 below.

where C is the DNA level in plasma (copy/microliter plasma); Q is the amount of DNA (copy) determined by the sequence detector in PCR; VDNA is a total volume of 50 μl of plasma ccf-DNA solution obtained after isolation; VPCR was performed in a volume of 5 μl of the plasma ccf-DNA solution used for PCR; VEXT is 100 μl of plasma extraction.

5. Cell Culture

All mouse experimental protocols were approved by the Institutional Animal Care and Use Committee of Soonchunhyang University (protocol number: SCH18-0032). For primary mouse bone marrow-derived macrophages (BMDM), bone marrow collected from wild-type (WT) mice (male, 8-10 weeks old) femurs and tibia was plated in sterile Petri dishes and 10% (v/v) It was cultured for 7 days in DMEM medium containing heat inactive fetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/ml streptomycin, and conditioned medium from 25% (v/v) mouse L929 fibroblasts.

For AIM2 inflammasome activation, LPS-primed wild-type (WT) bone marrow-derived macrophages (BMDM) were mixed with poly(dA:dT) (1 mg/ml, 3 time) was transfected.

For NLRP3 inflammasome activation, LPS-primed WT BMDMs were treated with ATP (2 mM, 0.5 h). For NLRC4 inflammatory enzyme activation, LPS-primed WT BMDM was transfected with flagellin (5 μg/ml, 6 hours) using Lipofectamine with a plus reagent according to the manufacturer's instructions. For NLRP1 inflammasome activation, LPS-primed WT BMDM was treated with MDP (5 μg/ml, 16 h).

For cytokine analysis, cell supernatants and cell lysates were collected and analyzed for levels of IL-1β, IL-18 and TNF-α using an ELISA kit.

6. Immunoblot Analysis

WT BMDM was harvested and lysed in NP40 cell lysis buffer. The lysate was centrifuged at 15,300×g for 10 min at 4°C to obtain the supernatant. The Bradford assay was applied to determine the protein concentration in the supernatant. Proteins were electrophoresed on NuPAGE 4-12% Bis-Tris gels and transferred to protran nitrocellulose membranes. Membranes were blocked in TBS-T (TBS and 1% v/v Tween-20) with 5% (w/v) bovine serum albumin (BSA) for 30 minutes at 25°C. Membranes were incubated with primary antibody diluted in 1% (w/v) BSA in TBS-T for 16 h at 4 °C and horseradish peroxidase (HRP) conjugated secondary antibody (rabbit IgG-HRP and Mouse m-IgG BP-HRP) was diluted 1:2500 in TBS-T and incubated at 25° C. for 30 minutes. Immunoreactive bands were detected using a SuperSignalWest Pico chemiluminescent substrate

7. Cytokine analysis

Supernatants from BMDM were prepared for mouse IL-1β (MLB00C, R&D Systems), mouse IL-18 (7625, R&D Systems), and mouse TNF-α (MTA00B, R&D Systems) according to the manufacturer's instructions. measured. Plasma from human subjects was measured for human IL-1β (DLB50, R&D Systems) according to the manufacturer's instructions.

8. ASC oligomerization and ASC spec formation

WT BMDM was seeded on chamber slides. After LPS and poly(dA:dT) stimulation, cells were fixed with 4% paraformaldehyde followed by polyclonal ASC antibody (ADI-905-173-100, Enzo Life Sciences) and FITC goat anti-rabbit (IgG) for 16 h. and incubated with Secondary antibody (ab6717, Abcam, Cambridge, MA, USA) was followed by DAPI (P36962, Thermo Fisher Scientific) staining for 1 hour. ASC specs were analyzed using a Zeiss LSM880 laser scanning confocal. Microscopy was used and quantified using ImageJ software v1.52a (NIH, Bethesda, MD, USA). The graph in FIG. 4D shows the quantification of the percentage of ASC speck positive cells for each mouse.

9. Statistical Analysis

All data are combined mean SD from three independent experiments. All statistical tests were analyzed using a statistical software package (GraphPad Prism version 4.0, GraphPad software) for comparison of the two groups using two-tailed Student's t-test and analysis of variance (ANOVA) (post-hoc comparison using Dunnett test).

Correlation tests were analyzed using the Spearman correlation coefficient (Spearman's R) for comparison of the two groups. The strength of the difference was analyzed using Cohen's effect size (d) for comparison of the two groups. Linear regression analysis (r ² ) was analyzed by comparing the two groups, and p-values less than 0.05 were considered statistically significant.

<Result>

1. Increased plasma ccf-DNA levels in patients with type 2 diabetes

To investigate the role of ccf-mtDNA in patients with type 2 diabetes, total ccf-DNA levels were analyzed in plasma isolated from controls of patients with type 2 diabetes. Total ccf-DNA including single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) was isolated from plasma of type 2 diabetes patients. ccf-DNA was isolated from the plasma of 141 patients with type 2 diabetes and 22 healthy subjects without type 2 diabetes as a control (Table 2).

The table is the baseline characteristics of non-diabetic controls and patients with type 2 diabetes.

control
n=22 Type 2 diabetes patients n=141 p-value age, year 51.6 ± 6.0 56.5 ± 10.7 0.003 Gender, Male (%) 11 (50.0) 60 (42.6) 0.672 ccf-mtDNA, copy number (1 × 10 ³ )/μl 0.1 ± 0.01 1.91 ± 0.17 <0.01 IL-1β, pg/μl 30.27 ± 0.12 38.53 ± 0.71 <0.001 HbA1c (%) 0 (0.0) 8.1 ± 1.99 1.000 high blood pressure, present 0 (0.0) 103 (75.2) 1.000 body mass index (BMI), kg/m ² 23.0 ± 2.6 23.5 ± 3.5 0.464 Systolic blood pressure, mmHg 119.1 ± 15.1 124.0 ± 13.6 0.129 diastolic blood pressure, mmHg 74.1 ± 8.3 77.3 ± 9.4 0.124 Hemoglobin, g/dL 14.7 ± 1.7 13.5 ± 1.7 0.001 Urea nitrogen, mg/dL 14.2 (11.5-16.4) 15.9 (12.8-20.4) 0.039 Creatine, mg/dL 0.83 ± 0.14 0.81 ± 0.20 0.580 Albumin, g/dL 4.49 ± 0.28 4.43 ± 0.42 0.522 Aspartate Aninotransferase (AST), IU/L 19.5 (18.0, 23.8) 19.0 (16.0, 25.0) 0.459 Alanine aninotransferase (ALT), IU/L 16.5 (13.0, 23.0) 20.0 (16.0, 27.3) 0.044 Total Bilirubin, mg/dL 1.09 ± 0.38 0.65 ± 0.26 <0.001 Total cholesterol, mg/dL 195.0 ± 34.5 178.7 ± 37.6 0.059 Triglycerides, mg/dL 120.5
(60.0, 141.0) 142.0
(91.5, 216.5) 0.020 Uric acid, mg/dL 5.02 ± 0.88 4.76 ± 1.33 0.367 Calcium, mg/dL 9.42 ± 0.45 9.35 ± 0.43 0.465 Phosphorus, mg/dL 3.45 ± 0.58 3.69 ± 0.58 0.077 Estimation of glomerular filtration rate (eGFR), ml/min/1.73 m ² 101.2
(89.7, 110.2) 99.2
(87.6, 109.9) 0.690 C-reactive protein, mg/L 0.04 (0.03, 0.08) 0.07 (0.03, 0.15) 0.193

After ccf-DNA was isolated from plasma, the concentrations of ssDNA and dsDNA in plasma were quantified to measure DNA levels. The ssDNA and dsDNA concentrations were relatively high in plasma of type 2 diabetic patients (T2D) compared to healthy subjects (control) ( FIGS. 1A and B). In the above results, the level of ccf-DNA was increased in the plasma of type 2 diabetic patients.

2. Increased mtDNA Levels in Plasma of Patients with Type 2 Diabetes

Next, we analyzed changes in mtDNA levels in patients with type 2 diabetes. mtDNA was analyzed in the plasma of 141 patients with type 2 diabetes and 22 controls. mtDNA levels were determined by quantification of the copy number of ccf-DNA to mtDNA in plasma. mtDNA levels were significantly higher in type 2 diabetic patients (T2D) compared to controls ( FIG. 2 ).

Next, for the diagnosis of type 2 diabetes, the correlation between HbA1c, a hyperglycemic index, and mtDNA was confirmed. There was a slight correlation between elevated mtDNA levels and HbA1c levels in the plasma of type 2 diabetic patients ( FIG. 5 ). On the other hand, the nDNA level in the plasma of type 2 diabetic patients was lower than the mtDNA level ( FIG. 6 ). These results indicate that mtDNA levels are increased in patients with type 2 diabetes.

3. Increased levels of interleukin-1β (IL-1β) in plasma of patients with type 2 diabetes

IL-1β is an important pro-inflammatory cytokine that is regulated by the inflammasome during chronic inflammation in patients with type 2 diabetes. To investigate the role of mtDNA in chronic inflammation in patients with type 2 diabetes, the effect of increased mtDNA level on IL-1β levels was analyzed. IL-1β levels were measured in plasma of 141 type 2 diabetic patients and 22 controls. As a result of the analysis, it was confirmed that the IL-1β level was significantly higher in type 2 diabetes patients compared to the control group ( FIG. 3 ).

Next, the correlation between high mtDNA level and IL-1β level in type 2 diabetes patients was confirmed. As a result of the analysis, it was confirmed that the high mtDNA level and the IL-1β level had a weak correlation ( FIG. 7 ). Through the above results, it was confirmed that high mtDNA levels are associated with chronic inflammation mediated by IL-1β in type 2 diabetes patients.

4. Induction of AIM2 Imflamasome Activation in Macrophages of mtDNA in Patients with Type 2 Diabetes

To investigate the molecular target of ccf-mtDNA in the regulation of chronic inflammation in type 2 diabetes, we examined ccf-DNA with high mtDNA levels from patients with type 2 diabetes to confirm that it can induce AIM2 inflasome activation in macrophages. did. Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages using ccf-DNA of type 2 diabetes patients and poly(dA:dT), an AIM2 inflammasome activator , BMDMs) were analyzed for caspase-1 activation, IL-1β and IL-18 secretion.

ccf-DNA (ccf-DNA #1 and ccf-DNA # 2) isolated from two independent type 2 diabetic patients was compared to vehicle controls in response to poly(dA:dT) stimulation. -1) and the expression of cleaved IL-1β were significantly higher. However, the expression of pro-IL-1β did not change compared to the vehicle control ( FIG. 4A ).

Consistently, ccf-DNA (ccf-DNA #1 and ccf-DNA #2) from two independent patients with type 2 diabetes mellitus showed significantly higher IL in response to poly(dA:dT) compared to vehicle controls. -1β and IL-18 secretion were exhibited ( FIG. 4b ), whereas the secretion of tumor necrosis factor-α (TNF-α) was not changed ( FIG. 4b ).

Next, it was confirmed whether the caspase-1 activation-dependent IL-1β and IL-18 secretion, which are important for the mtDNA-induced AIM2 inflammasome activation, were observed. Caspase-1 activation and IL-1β and IL-18 secretion were assayed in BMDMs pre-treated with poly(dA:dT) and selective caspase-1 inhibitor (Z-VAD) prior to ccf-DNA stimulation after LPS incubation. Z-VAD inhibited caspase-1 activation, IL-1β cleavage and secretion of IL-1β and IL-18 in response to ccf-DNA and poly(dA:dT) stimulation compared to vehicle control, and TNF-α did not change ( FIG. 8 ). These results show that ccf-DNA-induced AIM2 inflammasome activation is required for caspase-1-dependent IL-1β and IL-18 secretion.

In contrast, ccf-DNA (ccf-DNA #1 and ccf-DNA # 2) is either ATP (NLRP3 inflammasome activator) or flagellin (NLRC4 inflammasome activator) or mura, referring to Figure 4c. It did not affect the secretion of IL-1 and IL-18 in reaction with miley peptide (muramyldipeptide, MDP), (NLRP1 inflammasome activator).

We also investigated whether ccf-DNA promotes ASC stain formation, which is required for NLRP3-dependent caspase-1 activation in type 2 diabetes. The ccf-DNA of type 2 diabetic patients had significantly higher formation of ASC plaques induced by LPS and poly(dA:dT) stimulation compared with vehicle controls (Fig. 4d). These results confirmed that mtDNA of type 2 diabetes patients induced AIM2 inflammatory activation in macrophages.

<110> Sonchhunhyang University Industry Academy Cooperation Foundation <120> Compositions for diagnosis of diabetes or diabetic complications using circulating cell-free DNA and method thereof <130> DP20190360 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human NADH dehydrogenase 1 gene (mtDNA) forward primer <400> 1 atacccatgg ccaacctcct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human NADH dehydrogenase 1 gene (mtDNA) reverse primer <400> 2 gggcctttgc gtagttgtat 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> human beta globin (nuclear DNA) forward primer <400> 3 gtgcacctga ctcctgagga ga 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> human beta globin (nuclear DNA) reverse primer <400> 4 ccttgatacc aacctgccca g 21

Claims (8)

A composition for diagnosing type 2 diabetes or complications of type 2 diabetes, comprising an agent for detecting the level of mitochondrial DNA (mtDNA) in circulating cell-free DNA (ccf-DNA). The diagnostic composition of claim 1, wherein the mtDNA is a human NADH dehydrogenase 1 gene. The diagnostic composition according to claim 2, wherein the agent for detecting the human NADH dehydrogenase 1 gene is a primer set consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. The composition for diagnosis according to claim 1, wherein the complication is diabetic nephropathy. According to claim 1,
The agent for measuring the mtDNA level is a primer pair, a probe, or an antisense oligonucleotide that specifically binds to the gene, characterized in that the diagnostic composition. A kit for diagnosing type 2 diabetes or type 2 diabetes complications, comprising the composition according to claim 1. separating circulating cell-free DNA (ccf-DNA) from the biological sample isolated from the suspect;
Measuring the level of mitochondrial DNA (mtDNA) by performing PCR with a primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the ccf-DNA isolated in the above step; and
A method of providing information for diagnosing type 2 diabetes or complications of type 2 diabetes, comprising comparing the expression level of mtDNA measured in the above step with a normal control sample. The method of claim 7, wherein the biological sample is plasma.

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