Compositions and Methods of Diagnosing Diseases and Disorders Associated with Cell Death by Detecting Hypo-methylated DNA in Bodily Fluids

Cross-Reference to Related Applications

[0002] This non-provisional application is related to International Application Number PCT/US2012/043747 filed on June 22, 2012; US patent application 14/127,906 (now US 10,125,394 issued on November 13, 2018); US Patent Application 11/321,090 “Pharmaceutical Compositions and Methods For Restoring Beta Cell Mass and Function” filed on December 30, 2005 (now US Patent 11/321,090); US provisional patent application 63/271,217 “Compositions and Methods of Diagnosing Diseases and Disorders Associated with Cell Death by Detecting Hypo-methylated DNA in Bodily Fluids” filed on October 24, 2021 and US provisional patent application 63/271,660 “Compositions and Methods of Diagnosing Diseases and Disorders Associated with Cell Death by Detecting Hypo-methylated DNA in Saliva” filed on October 25, 2021, the entirety of each of which is hereby incorporated by reference.

[0003] This non-provisional application furthermore claims priority to the aforementioned US provisional patent applications 63/271,217 “Compositions and Methods of Diagnosing Diseases and Disorders Associated with Cell Death by Detecting Hypo-methylated DNA in Bodily Fluids” filed on October 24, 2021 and US provisional patent application 63/271,660 “Compositions and Methods of Diagnosing Diseases and Disorders Associated with Cell Death by Detecting Hypo-methylated DNA in Saliva” filed on October 25, 2021.

Field of the Invention

[0004] The invention relates to compositions and methods for detecting hypo-methylated DNA in bodily fluids and in particular urine and saliva as well as methods for treatment following early detection.

Federal Research Statement

[0005] None.

Background of the Invention

[0006] The p cell loss that leads to diabetes is silent. In type 1 diabetes mellitus (T1D), killing of cells and subsequent presentation with hyperglycemia takes weeks in the nonobese diabetic (NOD) mouse model of T1D and possibly years in humans (Akirav et al., 2008, Diabetes 57:2883-2888).

Hyperglycemia occurs when the majority of p cells have been destroyed, and by that time, only limited therapeutic options are available (Bluestone et al., 2010, Nature 464: 1293-1300; Waldron-Lynch et al., 2009, Endocrinol. Metab. Clin. North Am. 38:303-317). Accordingly, early detection of ongoing p cell death would allow for earlier interventions at a time before the development of hyperglycemia, when a more significant p cell mass is present.

[0007] Epigenetic modifications of DNA are used by various different types of cells to control gene expression, including tissue-specific gene expression. Examples of epigenetic modifications affecting gene expression include histone acetylation/deacetylation and DNA methylation (Klose et al., 2006, Trends Biochem. Sci. 31 :89-97; Bartke et al., 2010, Cell 143:470-484; Wang et al., 2007, Trends Mol. Med. 13:373-380). Methylation of DNA occurs at CpG dinucleotide sites, and this modification maintains a transcriptionally repressive chromatin configuration (Miranda et al., 2007, J. Cell Physiol. 213 :384-390). Conversely, demethylation of CpG dinucleotide sites allows a transcriptionally permissive configuration (Id). Beta cells, but not other cell types, express insulin, and thus are the only known cells to maintain transcriptionally-permissive hypomethylated copies of their insulin genes. Therefore, the presence of hypomethylated insulin gene DNA outside of the pancreas of a subject correlated with the release of hypomethylated insulin gene DNA from dead and dying (e.g., apoptotic) p cells is a strong indication of beta cell death that could lead to hyperglycemia and eventually diabetes.

[0008] As disclosed herein, a source of hypomethylated insulin gene DNA from p cells is the urine from individuals who are experiencing the destruction of their p cells. Generally, DNA that is present in urine has crossed the renal barrier. In other words, DNA in urine is transrenal. The presence of transrenal DNA has been explained through the apoptosis phenomenon. More specifically, during cell death most of the nuclear DNA is converted into nucleosomes and oligomers (Umansky, S. R., et al. [1982], "In vivo DNA degradation of thymocytes of gamma-irradiated or hydrocortisone-treated rats"; Biochim. Biophys. Acta 655:9-17), which are finally digested by macrophages or neighboring cells. However, a portion of this degraded DNA escapes phagocytic metabolism, and can be found in the bloodstream (Lichtenstein, A. V., et al. [2001], "Circulating nucleic acids and apoptosis;" Ann NY Acad Sci, 945:239-249), and also inurine. The present invention addresses the detection of beta cell-specific epigenetic modifications that are detectable in bodily fluids such as plasma, saliva and urine following the destruction of beta cells.

Summary of the Invention

[0009] The present invention incorporates the disclosure of International Patent Application Publication WO 2012/178007, which relates the discovery that the presence of hypomethylated beta cell genomic DNA outside of the pancreas of a subject is indicative of cell death and adds methods of detecting hypomethylated DNA in bodily fluids and in particular, urine. [0010] Described herein are methods for the in vivo detection of cell death in autoimmune and chemically- induced diabetes in mice, in humans, in human tissues, and in serum from patients with T1D. The methods identify a methylated CpG dinucleotide in insulin DNA that is derived exclusively from cells. The data described herein indicate that the method provides a biomarker for detecting beta cell loss in prediabetic subjects during progression of diabetes, as well as in subjects with new-onset T1D. Demethylation of CpG sites in the insulin promoter has previously been identified (Kuroda et al., 2009, PLoS. ONE 4:e6953), consistent with the notion that methylation of promoters is a mechanism for controlling tissue-specific gene expression. However, the studies described herein targeted differentially methylated CpG dinucleotides in the Insl gene in mice and the Ins gene in humans. Although not wishing to be bound by any particular theory, the conservation of demethylation of this sequence across species is consistent with the explanation that its methylation plays an active role in the regulation of insulin gene transcription. In addition, via sequencing, it was shown that CpG sites both upstream and downstream of the CpG at +177 are also equally demethylated in beta cell DNA, implicating the entire region in gene regulation. Sequence analyses revealed that unlike human Ins, which was completely demethylated in primary cells, murine Ins 1 was demethylated in 75% of the CpG sites studied from murine beta cells isolated from MIP-GFP+ mice. Acute beta cell death in vivo was detected, as indicated by the presence of cell-derived demethylated DNA after streptozotocin (STZ) treatment. The fact that hyperglycemia was not observed at the eight-hour time-point demonstrates the ability of the present method to detect beta cell death before frank hyperglycemia occurs. This conclusion is supported by the histomorphic analysis of the percentage of nucleated cells in the islet, which revealed a drop in the percentage of DAPI-positive, insulinpositive cells, consistent with the explanation that DNA material is released to the surrounding tissues and can be detected in the circulation. [0011] The measure of cell death demonstrated continued release of demethylated insulin DNA after the appearance of frank hyperglycemia, but at a reduced level compared with prediabetic (i.e., 14-week-old) mice. The decline in p cell-derived DNA after the onset of hyperglycemia is consistent with the explanation that the relative abundance of demethylated insulin DNA in the circulation may be reduced because of a total loss of cell mass. For example, a higher percentage of cells may be destroyed after diagnosis with hyperglycemia than before diagnosis, but fewer cells actually may be destroyed (Sherryet al., 2006, Diabetes 55:3238-3245; Basadonna et al., 1988, Am. J. Surg. 156:191-193; Berney et al., 2006, Am. J. Transplant. 6:1704-1711).

[0012] In various embodiments, the invention is a method of detecting hypomethylated p cell insulin gene DNA in a urine sample of a subject including the steps of: obtaining a urine or saliva sample from the subject, where the urine or saliva sample contains p cell insulin gene DNA; determining the methylation status of at least one of the CpG dinucleotides in the p cell insulin gene DNA, where when at least one of the CpGdi nucleotides in the cell insulin gene DNA is determined to be unmethylated, the hypomethylated cell insulin gene DNA is detected.

[0013] In various other embodiments, the invention is a method of measuring the level of p cell death by detecting hypomethylated p cell insulin gene DNA in a urine or saliva sample, where the amount of hypomethylated p cell insulin gene DNA is quantified, and where a higher amount of hypomethylated p cell insulin gene DNA indicates a higher level of cell death.

[0014] In one embodiment, the invention is a method of diagnosing a subject with a disease or disorder associated with p cell death by detecting hypomethylated cell insulin gene DNA in the subject's urine or saliva, where when hypomethylated p cell insulin gene DNA is detected, a disease or disorder associated with cell death in the subject is diagnosed. In various embodiments, the disease or disorder diagnosableby the methods of the invention include pre-diabetes mellitus, diabetes mellitus, diabetes mellitus type 1, diabetes mellitus type 2, and gestational diabetes.

[0015] In another embodiment, the invention is a method of assessing the severity of a disease or disorder associated with p cell death in a subject by detecting hypomethylated p cell insulin gene DNA in the subject's urine or saliva, where the amount of hypomethylated p cell insulin gene DNA is quantified, and where a higher quantity of hypomethylated p cell insulin gene DNA indicates a greater severity of the disease or disorder in the subject. In various embodiments, the assessment of the severity of the disease or disorder diagnosable by the methods of the invention include pre-diabetes mellitus, diabetes mellitus, diabetes mellitus type 1, diabetes mellitus type 2, and gestational diabetes.

[0016] In a further embodiment, the invention is a method of monitoring the progression of a disease or disorder associated with p cell death in a subject by detecting hypomethylated p cell insulin gene DNA in the subject's urine or saliva or other bodily fluids, where when the amount of hypomethylated p cell insulin gene DNA detectedat a first time point is different than the amount of hypomethylated p cell insulin gene DNA detected at a second time point, the difference in the amount of hypomethylated p cell insulin gene DNA is an indicator of the progression of the disease or disorder associated with cell death in the subject. In various embodiments, the disease or disorder diagnosable by the methods of the invention include pre-diabetes mellitus, diabetes mellitus, diabetes mellitus type 1, diabetes mellitus type 2, and gestational diabetes. [0017] In one embodiment, the invention is a method of monitoring the effect of a therapeutic regimen on a disease or disorder associated with p cell death in a subject by detecting hypomethylated cell insulin gene DNA in the subject's urine or saliva or other bodily fluids, where when the amount of hypomethylated p cell insulin gene DNA detected before therapeutic regimen is applied is different than the amount of hypomethylated p cell insulin gene DNA detected during or after the therapeutic regimen is applied, thedifference in the amount of hypomethylated p cell insulin gene DNA is an indicator of the effect of the therapeutic regimen on the disease or disorder associated with p cell death in the subject. In various embodiments, the disease or disorder diagnosable by the methods of the invention include pre-diabetesmellitus, diabetes mellitus, diabetes mellitus type 1, diabetes mellitus type 2, and gestational diabetes.

[0018] In another embodiment, the invention is a method of assessing the post-operative prognosis of a p cell transplant, islet transplant, or pancreas transplant by detecting hypomethylated p cell insulin gene DNA in a subject, where the amount of hypomethylated p cell insulin gene DNA in the subject's urine, or saliva or other bodily fluids is quantified, and the amount of hypomethylated p cell insulin gene DNA is a measure of the prognosis of a p cell transplant, islet transplant, or pancreas transplant subject.

[0019] In one embodiment, the invention is a kit for detecting hypomethylated p cell insulin gene DNA in a urine, or saliva or other bodily fluids sample of a subject, including: at least one reagent or device for isolating p cell insulin gene DNA from the urine or saliva or other bodily fluids sample; at least one reagent or device for determining the methylation status of the p cell insulin gene DNA isolated from the urine or saliva or other bodily fluids sample; at least one comparator; and instructions for the preparation, performance, and analysis of the determination of methylation statusof the p cell insulin gene DNA isolated from the urine or saliva or other bodily fluids sample. [0020] In another embodiment, the invention is a composition comprising a biomarker, where the biomarker comprises an isolated hypomethylated p cell insulin gene, or fragment thereof, where the isolated hypomethylated cell insulin gene, or fragment thereof, was isolated from a urine or saliva or other bodily fluids sample obtained from a subject.

[0021] In a further embodiment, the invention is a composition comprising an amplicon, where the amplicon was produced by polymerase chain reaction (PCR) using at least one primer that hybridizes toa template comprising an isolated hypomethylated p cell insulin gene, or fragment thereof, where the isolated hypomethylated p cell insulin gene was isolated from a urine or saliva or other bodily fluids sample obtained from a subject. In some embodiments, the at least one primer has a nucleic acid sequence selected from the group consisting of: SEQ 10 NO: 1 and 2. In other embodiments, the at least one primer has a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 8-11.

[0022] In a further embodiment, the invention is a method of treating beta cell death following early detection via analysis of bodily fluids by administering pharmaceuticals such as Lisofyloline and Exenatide or similar drugs. Furthermore, beta cell death can be reversed by transplanting islets or the entire pancreas. Reversing beta cell death following early detection prevents the onset of diabetes.

[0023] While the invention involves early detection of beta cell death by analyzing several types of bodily fluids including urine and saliva, for simplicity and ease of understanding, the present invention is described and claimed in terms of urine with the assumption that the methods disclosed herein also apply to other bodily fluids. Brief Description of the Drawings

[0024] Figure 1, comprising Figures 1A-1B, is a schematic depicting DNA sequences from PTC3 and PMJ cell lines and non- p cells having a differentially methylated CpG dinucleotide in the Insl gene. Figure 1A is a representation of unmodified DNA sequence of murine Insl gene depicting the position of the differentially methylated CpG dinucleotide (arrow, upper region) and a comparison of bisulfite treated genomic DNA from either the PTC3 or PMJ cell line, demonstrating the nucleotide modification of CpG dinucleotides due to demethylation at position 523393278 (lower region). Figure IB is a representation of the sequence analysis of product amplified in the first-step PCR. The sequence of 15 clones from murine p cells and 8 clones from murine liver cells are shown (o indicates demethylated cytosines; • indicates methylated cytosines). The locations of the methylation sites from the transcription start site are indicated. The primers of the second-step PCR were specific for methylated/demethylated cytosine at nucleotide position + 177, corresponding to nucleotide 52339278.

[0025] Figure 2 is a schematic depicting the methods used to identify differentially methylated DNA using real-time PCR. Bisulfite-treated purified DNA from tissues, cells, or serum was purified and used in the first-step, methylation- insensitive reaction. The products were gel-purified and used as a template in a second-step reaction with methylation-specific primers.

[0026] Figure 3, comprising Figures 3A-3D, depicts the results of experiments demonstrating that demethylated Ins 1 gene DNA is enriched in primary islets and FACS-sorted primary insulin-positive cells.

Figure 3A is a graph depicting the ratio of demethylated:methylated DNA in primary murine tissues. The cycle differences were normalized to the cycle difference of kidney DNA. The data are froma single experiment representative of more than five experiments. Figure 3B is a FACS plot analysis showing the presence of insulin-positive and insulin-negative cells sorted from dispersed islets. Figure 3C is a graph depicting the demethylated:methylated DNA levels in the sorted cell population (shown in Figure 3B). The insulin-positive cell cycle difference was normalized to the insulin-negative cell cycle difference. Data are from a single experiment representative of two experiments. Figure 3D is a graph depicting DNA from the first-step reactions from sorted cells and from islet-derived non- cells, which were mixed in ratios of 1:1, 1:10, and 1:100 and then added to the second-step reaction. The relationship between the ratio of DNA and the demethylation index is shown (r2 = 0.96; P = 0.0038).

[0027] Figure 4, comprising Figures 4A-4D, depicts the results of experiments demonstrating the increase of demethylated Ins 1 gene DNA in the serum after STZ treatment of mice. Figure 4A is a graph depicting blood glucose levels of untreated and STZ-injected BALB/c mice (n=6 animals per group)

*P < 0.05; ±P < 0.02 vs.prediabetic mice.

[0028] Figure 4B is a graph depicting the demethylation index of the nested PCR performed on DNA from sera of the BALB/c mice. Between 16 and 18 mice were analyzed at each time point. The sera fromtwo mice were pooled for analysis. *P < 0.05. The box-and-whisker plots show the minimum and maximum values. Figure 4C is a graph depicting the histomorphic analysis of DAPI-positive, insulin- positive cells in the islets of the STZ-treated mice shown in B. *P < 0.0001; ±P < 0.002. Figure 4D is a series of images of representative islets of STZ-treated mice, stained for DAPI and insulin.

[0029] Figure 5, comprising Figures 5A-5E, depicts the results of experiments demonstrating the increase in serum-derived demethylated Insl gene DNA in prediabetic NOD mice with impaired glucose tolerance. Figure 5A is a graph depicting IPGTT data for prediabetic NOD mice at various ages (n > 5 per group). Note that the fasting glucose (at t = 0) is similar at all time-points. Figure 5B is a graph depicting the area under the curve of IPGTT data from Figure 5A. *P < 0.05. Figure 5C is a graph depicting the demethylation index measured with DNA from the sera of prediabetic (week 7-14) and diabetic NOD mice. P = 0.0002 by ANOVA; **P < 0.01; *P < 0.05; n=5, 5, 5, 7, and 5 mice/group. The box-and-whisker plots show the minimum and maximum values. Figure 5D is a graph depicting the results of an experiment where pancreata and serum were harvested from mice at the indicated ages (n = 5 mice per time point) for measurement of insulin content. Figure 5E is a graph depicting the relationship between pancreatic insulin content and demethylation index in individual mice. Two measurements from each mouse are plotted (r2 = 0.28; P < 0.05). In this experiment, pancreata and serum were harvested from mice at the indicated ages (n = 5 mice per time point) for measurement of demethylation index. The insulin content and demethylation index in 11- and 15-wk-old mice were compared with 7-wk-old mice.

*P < 0.05; **P < 0.02 by post hoc analysis of ANOVA.

[0030] Figure 6, comprising Figures 6A-6E, depicts the results of an analysis of insulin DNA sequences in human tissues and sera. Figure 6A is an illustration of the unmodified DNA sequence in human Ins gene showing the preserved CpG pair at nucleotide positions +273 and +399 identified in the UCSC Genome Browser (genome.ucsc.edu/cgi-bin/hg Gateway). Figure 6B is an illustration depicting the sequence data of the first-step PCR showing methylation DNA patterns in primary human kidney and whole islets. The arrow shows the presence of demethylated CpG found in human islets at nucleotide position +399 (at position 2182036, site of the reverse primer). Note the two peaks in human islets representing both demethylated and methylated forms from cells and non-cells in the islets. Figure 6C is an illustration depicting the sequence analysis of product amplified in the first-step PCR from sorted human cells and kidney. The sequence of 10 clones from human cells and 12 clones from human kidney cells are shown (o indicates demethylated cytosines; • indicates methylated cytosines). The base pairs are indicated downstream from the transcription start site. The primers of the second-step PCR were specific for methylated/demethylated cytosine at nucleotide position +273 and +399. Figure 6D is a graph depicting DNA isolated from human kidney, liver, and islets and analyzed by nested PCR. Synthetic DNA was also analyzed in these reactions. Each dot represents a separate isolation and analysis of tissue DNA. The demethylation index was significantly greater with DNA from islets compared with liver and kidney.

***P < 0.00 1. Figure 6E is a graph showing the demethylation index of DNA isolated from five subjects with recent-onset T1D (•) and from six healthy control subjects (■). The demethylation index was significantly higher in patients with T1D (P = 0.017, Mann- Whitney U test).

[0031] Figure 7 depicts melting curves from the 2nd step PCR reactions. Real- time PCR data for methylated (left) and demethylated (right) dependent primers of PMJ and PTC3. The upper graphs depict amplification plots. The lower graphs depict melting curves. The primer specific for the demethylated sequence shows lower Ct values than the primer for the methylated sequence with DNA from PTC3 cells, whereas the opposite is seen with DNA from PMJ. A single experiment representative of three independent experiments is shown.

[0032] Figure 8 is a table depicting the primer sequences and PCR conditions used for studies of murine Ins 1.

[0033] Figure 9 is a table depicting the primer sequences and PCR conditions used for studies of human

Ins. [0034] Figure 10 is a table depicting the primer sequences used for cloning and sequencing of murine Ins 1.

[0035] Figure 11 presents a flow chart showing the current normal progression of beta cell death leading to symptoms and onset of diabetes and administration of insulin to combat the disease.

[0036] Figure 12 presents a flow chart showing early detection of Juvenile Diabetes and the potential to stop and reverse beta cell death following early detection.

[0037] Figure 13 presents a flow chart showing early detection of Diabetes After Age 35 and the potential to stop and reverse beta cell death following early detection.

[0038] The invention relates to compositions and methods for detecting hypomethylated DNA in urine and correlating the detection of hypomethylated DNA with p cell death. Thus, the invention relates to compositions and methods useful for assessing the extent of methylation of cell DNA, for detecting the presence of hypomethylated p cell DNA in the urine or saliva or other bodily fluids of a subject as an indicator of cell death, for assessing the level of hypomethylated p cell DNA present in the urine or saliva or other bodily fluids of a subject as a measure of cell death, for diagnosing a disease or disorder associated with p cell death, for monitoring the progression of a disease or disorder associated with p cell death, for assessing the severity of a disease or disorderassociated with cell death, for selecting a treatment regimen to treat a disease or disorder associated with p cell death, for assessing the postoperative prognosis of a p cell transplant, islet transplant, or pancreas transplant subject, and for monitoring the effect of a treatment of a disease or disorder associated with cell death. [0039] In various embodiments, the disease or disorder associated with p cell death is pre-diabetes mellitus, diabetes mellitus, diabetes mellitus type 1, diabetes mellitus type 2, or gestational diabetes. In one embodiment, the hypomethylated cell DNA is an insulin gene or fragment thereof, such as, but not limited to the human Insl gene. In various embodiments, the hypomethylated insulin DNA is hypomethylated within at least one of a regulatory region, an intron, an exon, a non-coding region, or a coding region.

Detailed Description of the Invention

[0040] The invention relates to the correlation between the presence of p cell-derived hypomethylated DNA in a subject's urine or saliva or other bodily fluids and p cell death. As used herein "hypomethylated" means that the extent of methylation of a target nucleic acid (such as genomic DNA) is lower than it could be (i.e., a DNA or DNA fragment in which many or most of the CpG dinucleotides are not methylated). By way of a non-limiting example, a hypomethylated nucleic acid is a nucleic acid that is less methylated than it could be, because less than all of the potential methylation sites of the nucleic acid are methylated. By way of another non-limiting example, a hypomethylated nucleic acid, such as the insulin gene, is a nucleic acid that is less methylated in a cell type that expresses the nucleic acid (e.g., cells), as compared with a cell type that does not express the nucleic acid (e.g., liver cell). Thus, by way of one non-limiting example, a hypomethylated p cell insulin gene has less than all of the potential methylation sites methylated and isless methylated as compared with a liver cell insulin DNA.

[0041] In various embodiments, the invention relates to compositions and methods useful for assessing the extent of methylation of p cell-derived DNA, for diagnosing a disease or disorder associated with cell death, for monitoring the progression of a disease or disorder associated with p cell death, for assessing the severity of a disease or disorder associated with p cell death, for selecting a treatment regimen to treat a disease or disorder associated with p cell death, for assessing the post-operative prognosis of a p cell transplant, islet transplant, or pancreas transplant subject, or for monitoring the effect of a treatment of a disease or disorder associated with p cell death, or for any combination thereof.

[0042] It is an advantage of the present invention that p cell death can be detected non-invasively and earlier in the pathological process than other available methods for detecting diseases and disorders associated with p cell death, thereby allowing for earlier diagnosis and therapeutic intervention of the pathologic process. In some embodiments, the disease or disorder associated with p cell death is prediabetes mellitus, diabetes mellitus, diabetes mellitus type 1, diabetes mellitus type 2, or gestational diabetes.

[0043] In one embodiment, the hypomethylated p cell-derived DNA is insulin gene DNA (such as, murine INS1 or human INS), or a fragment thereof. In various embodiments, the hypomethylated insulin gene DNA is hypomethylated within a regulatory region, an intron, an exon, a non-coding region, or a coding region, or a combination thereof.

[0044] In various embodiments, the hypomethylated insulin gene DNA is unmethylated at one or more of the CpG dinucleotides at nucleotide positions 255, 273, 303, 329, 364, 370, 396, and 399 of the human insulin gene (INS). [0045] As stated above, the invention includes methods that are useful for assessing whether a subject has hypomethylated p cell-derived DNA in a subject's urine or saliva or other bodily fluids. Various methods of the invention involve detecting hypomethylated cell insulin gene DNA in a subject's urine or saliva or other bodily fluids, including the steps of: obtaining a urine or saliva or other bodily fluids sample from the subject that contains p cell-derived insulin gene DNA; determining the methylation status of at least one of the CpG dinucleotides of the insulin gene DNA, wherein when at least one of the CpG dinucleotides of the insulin DNA is determined to be unmethylated, hypomethylated cell insulin DNA is detected.

[0046] The invention contemplates various methods for determining the extent of DNA methylation, including those that include: 1) a methylation- specific polymerase chain reaction (PCR) approach; 2) a methylation-specific DNA microarray approach (the term "microarray" refers broadly to both "DNA microarrays" and "DNA chip(s)," and encompasses all art-recognized solid supports, and all art- recognized methods for affixing nucleic acid molecules thereto); 3) a bisulfite sequencing approach; 4) a pyrosequencing of bisulfite treated DNA approach; 5) or combinations thereof. Information obtained from the methods of the invention described herein (e.g., methylation status) can be used alone, or in combination with other information (e.g., disease status, disease history, vital signs, blood chemistry, etc.) from the subject or from the biological sample obtained from the subject. The information obtained from the methods of the invention described herein can also be stored in a manipulatable database that can be used for the analysis, diagnosis, prognosis, monitoring, assessment, treatment planning, treatment selection and treatment modification of diseases and disorders associated with p cell death.

Thus, the invention also includes such databases and their methods of use. [0047] In various embodiments, the invention also relates to compositions and methods useful for detecting methylated DNA derived from p cells or other cell types or from non-cellular sources for use as assay controls, (e.g., comparator or reference samples), for assessing the relative level of DNA hypomethylation. For example, the invention contemplates that the extent of methylation of hypomethylated cell insulin gene DNA can be compared with the extent of methylation of the insulin gene DNA from a comparator cell type which does not express insulin (i.e., the insulin gene is not hypomethylated). Non-limiting examples of comparator cell types useful in the methods of the invention include liver cells and kidney cells. Controls of the inventive method also include an expected normal background methylation value for the subject, a historical normal background methylation valuefor the subject, an expected normal background methylation value for a population to which the subject is a member, or a historical normal background methylation value for a population to which the subjectis a member.

[0048] As discussed above, the invention determines the methylation status of insulin gene DNA in urine, saliva or other bodily fluids. In certain embodiments of the invention, a urine sample can be assessed by the method of the invention without undergoing further processing. However, where DNA is present in minute amounts inthe urine, urine samples can be collected and thereafter concentrated by any means that does not affect the detection of DNA present in the sample. For example, in various embodiments, the method of the invention substantially isolates nucleic acids from a sample of urine, and comprises:

[0049] a) selecting an an ion exchange material which effectively adsorbs said target nucleic acids or proteinous complexes thereof. For example, the methods of the invention can utilize commercially available anion exchange materials. Either strong or weak anion exchangers may be employed. A preferred weak exchanger can be one in which primary, secondary, or tertiary amine groups (i.e., protonatable amines) provide the exchange sites. The strong base anion exchanger has quaternary ammonium groups (i.e., not protonatable and always positively charged) as the exchange sites. Both exchangers can be selected in relation to their respective absorption and elution ionic strengths and/or pH for the nucleic acid being separated. Purification by anion exchange chromatography is described in EP 0 268 946 Bl which is incorporated by reference herein. The material which is commercially available under the designation Q-Sepharose™ is a particularly suitable for the methodsof the invention. Q- Sepharose™, can be a strong anion exchanger based on a highly cross-linked, bead formed 6% agarose matrix, with a mean particle size of 90 mm. The Q-Sepharose™ can be stable in all commonly used aqueous buffers with the recommended pH of 2-12 and recommended working flow rate of 300-500 cm/h. In other preferred embodiments, the anion-exchange medium can be selected from sepharose- based quaternary ammonium anion exchange medium such as Q-filters or Q-resin. Thechromatographic support material for the anion charge used in the instant methods can be a modified porous inorganic material. As inorganic support materials, there may be used materials such as silica gel, diatomaceous earth, glass, aluminium oxides, titanium oxides, zirconium oxides, hydroxyapatite, and as organic support materials, such as dextrane, agarose, acrylic amide, polystyrene resins, or copolymers of the monomeric building blocks of the polymers mentioned. The nucleic acids can also bepurified by anion exchange materials based on: Polystyrene/DVB, such as Poros™ 20 for medium pressure chromatography, and Poros™ 50 HQ, ( BioPerseptive, Cambridge, U.S.A.); DEAE Sepharose™, and DEAE Sephadex™ (Pharmacia, Sweden); DEAE Spherodex™, and DEAE Spherosil™, (Biosepra, France).

[0050] b) applying urine containing nucleic acids or their proteinous complexes to the selected anion exchange material, and said nucleic acids or their complexes becoming adsorbed to said column material. The contact and subsequent adsorption onto the resin can take place by simple mixing of the anion exchange media with the body fluid, with the optional addition of a solvent, buffer or other diluent, in a suitable sample container such as a glass or plastic tube, or vessel commonly used for handling biological specimens. This simple mixing referred to as batch processing, can be allowed to take place for a period of time sufficiently long enough to allow for binding of the nucleoprotein to the media, preferably 10 to 40 min. The media/complex can then be separated from the remainder of the sample/liquid by decanting, centrifugation, filtration or other mechanical means;

[0051] c) optionally washing said anion exchange material with an aqueous solution of a salt at which the nucleic acids remain bound to said anion exchange material, said washing being of sufficient volumeand ionic strength to wash the non-binding or weakly binding components through the anion-exchange material. In some embodiments, the resin can be washed with 2x SSC (300 mM NaCI/30 mM sodium citrate (pH 7.0). Preferred ranges of the salt solutions are 300-600 nM NaCI/30 mM sodium citrate (pH 7.0) In other preferred embodiments, the resin can be washed with 300 mM LiCI/10 mM NaOAc(pH 5.2). Preferred ranges of the salt solutions are 300-600 mM LiCI/10 mM NaOAc (pH 5.2); and

[0052] d) eluting the bound nucleic acids by passing through said anion exchange material an aqueous solution of increasing ionic strength to remove in succession proteins that are not bound or are weakly bound to the anion-exchange material and said nucleic acids of increasing molecular weight from the column. In some preferred embodiments, both proteins and high and low molecular weight nucleicacids (as low as 10 base pairs) can be selectively eluted from the resin stepwise with the salt solution of concentrations from 300 mM to 2.0 M of NaCI and finally with 2.0 M guanidine isothiocyanate. In other preferred embodiments, LiCI solutions in the concentration range of 300 mM to 2.0 M of LiCI are used for stepwise elution.

[0053] In some embodiments, the nucleic acids isolated by the methods of the present invention may be in double-stranded or single-stranded form. In some embodiments, the urine, can be pre-filtered through a membrane and supplemented with 10 mM EDTA (pH 8.0) and 10 mM Tris-HCL (pH 8.0) prior to adsorption onto the anion-exchange medium. Commercial sources for filtration devices include Pa II- Filtron (Northborough, Mass.), Millipore (Bedford, Mass.), and Amicon (Danvers, Mass.). The filtration devices may be used with the methods of the instant invention such as a flat plate device, spiral wound cartridge, hollow fiber, tubular or single sheet device, open-channel device, etc. The surface area of the filtration membrane used can depend on the amount of nucleic acid to be purified. The membrane may be of a low-binding material to minimize adsorptive losses and is preferably durable, cleanable, and chemically compatible with the buffers to be used. A number of suitable membranes are commercially available, including, e.g., cellulose acetate, polysulfone, polyethersulfone, and polyvinylidene difluoride. Preferably, the membrane material is polysulfone or polyethersulfone.

[0054] In other embodiments of the invention, a urine sample, can be supplemented with EDTA and Tris-HCL buffer (pH 8.0) and digested with proteinases, such as for example Proteinase K, prior to adsorption onto the anion exchange medium.

[0055] In certain embodiments of the invention, the anion-exchange medium can be immobilized on an individualized carrier wherein such a carrier is a column, cartridge or portable filtering system which can be used for transport or storage of the medium/nucleoprotein bound complex. In some embodiments, the nucleic acid/anion exchange is maintained in storage for up to three weeks.

[0056] The invention also contemplates a kit with solid carrier capable of adsorbing the nucleic acids containing in a sample of urine. The kit also can comprise components necessary for processing a urine sample according to the invention. These include, in particular, reagents, also in concentrated form for final mixing by the user, chromatographic materials for the separation of the nucleic acids, aqueous solutions (buffers, optionally also in concentrated form for final adjusting by the user) or chromatographic materials for desalting nucleic acids which have been eluted with sodium chloride.

[0057] Preferably, the reagent kit contains additional means for purifying nucleic acids which comprise, for example, inorganic and/or organic carriers and optionally solutions, excipients and/or accessories. Such agents are known from the prior art (for example WO 95/01359) and are commercially available. Inorganic components of carriers may be, for example, porous or non-porous metal oxides or mixed metal oxides, e.g. aluminium oxide, titanium dioxide, iron oxide or zirconium dioxide, silica gels, materials based on glass, e.g. modified or unmodified glass particles or ground glass, quartz, zeolite or mixtures of one or more of the above-mentioned substances. On the other hand, a carrier may also contain organic ingredients which may be selected, for example, from latex particles optionally modifiedwith functional groups, synthetic polymers such as polyethylene, polypropylene, polyvinylidene fluoride, particularly ultra high molecular polyethylene or HD-polyethylene, or mixtures of one or more of the above-mentioned substances.

[0058] In addition, a kit according to the invention may also contain excipients such as, for example, a protease such as proteinase K, or enzymes and other agents for manipulating nucleic acids, e.g. at least one amplification primer, and enzymes suitable for amplifying nucleic acids, e.g. DNase, a nucleic acid polymerase and/or at least one restriction endonuclease. DNA is subject to degradation by DNases present in bodily fluids, such as urine. Thus, in certain embodiments, it is advantageous to inhibit DNase activity to prevent or reduce the degradation of DNA while in urine so that sufficiently large sequences are available for detection by known methods of DNA detection such as those described below. In one embodiment, samples of urine are taken when the urine has been held in the bladder for less than 12 hours, in a specific embodiment the urine is held in the bladder for less than 5 hours, more preferable for less than 2 hours. Collecting and analyzing a urine sample before it has been held in thebladder for a long period of time reduces the exposure of DNA to the any DNase present in the urine.

[0059] In another embodiment of the present invention, after collection, the urine sample is treated using one or more methods of inhibiting DNase activity. Methods of inhibiting DNase activity include, but are not limited to, the use of ethylenediaminetetraacetic acid (EDTA), guanidine-HCl, GITC (Guanidine isothiocyanate), N-lauroylsarcosine, Na-dodecylsulphate (SDS), high salt concentration and heat inactivation of DNase.

[0060] In another embodiment, after collection, the urine sample is treated with an adsorbent that traps DNA, after which the adsorbent is removed from the sample, rinsed and treated to release the trapped DNA for detection and analysis. This method not only isolates DNA from the urine sample, but, when used with some adsorbents, including, but not limited to Hybond™ N membranes (Amersham Pharmacia Biotech Ltd., Piscataway, N.J.) protects the DNA from degradation by DNase activity.

[0061] In some cases, the amount of DNA in a urine sample is relatively limited. Therefore, the invention encompasses embodiments wherein sensitivity of detection is increased by any method known in the art for increasing the number of copies of target nucleic acids (e.g., copies of nucleic acids comprising methylated or unmethylated nucleotides).

[0062] In various embodiments, hybridization methods, such as Southern analysis, can be used (see Current Protocols in Molecular Biology, 2012, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements). For example, methylation-specific restriction enzymes can be used to digest DNA, cleaving at specific sites depending upon methylation status, followed by hybridization with a nucleic acid probe. A "nucleic acid probe," as used herein, can be a DNA probe or an RNA probe; the nucleic acid probe can contain at least one polymorphism of interest, as described herein. The probe can be, forexample, the gene, a gene fragment (e.g., one or more exons), a vector comprising the gene, a probe or primer, etc. For representative examples of use of nucleic acid probes, see, for example, U.S. Pat. Nos. 5,288,611 and 4,851 ,330, which are incorporated herein.

[0063] A preferred probe for detecting DNA is a labeled nucleic acid probe capable of hybridizing to target DNA. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate target DNA. The hybridization sample is maintained under conditions which are sufficient to allow specific hybridizationof the nucleic acid probe to DNA. Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, as appropriate. In a preferred embodiment, the hybridization conditions for specific hybridization are high stringency. Specific hybridization, if present, is then detected using standard methods. More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of the presence of the target DNA of interest.

[0064] Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described herein. A PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, Tor U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, 1994, Nielsen et al., Bioconjugate Chemistry 5: 1). A PNA probe can be designed to specifically hybridize to a target nucleic acid sequence. Hybridization of a PNA probe to a target nucleic acid sequence is indicative of the presence of the target nucleic acid.

[0065] In another embodiment of the methods of the invention, analysis by methylation sensitive restriction enzymes can be used to detect the methylation status of a target nucleic acid, if the methylation status results in the creation or elimination of a restriction site. Typically, such a restrictionfragment length polymorphism (RFLP) analysis is conducted as described (see Current Protocols in Molecular Biology, supra). The digestion pattern of the relevant fragments indicates the presence or absence of methylation.

[0066] Various methods are available for determining the methylation status of a target nucleic acid. (See, for example, Rapley and Harbron, 2011, Molecular Analysis and Genome Discovery, John Wiley & Sons; Tollefsbol, 2010, Handbook of Epigenetics: The New Molecular and Medical Genetics, Academic Press). For example, direct sequence analysis can be used in the methods of the invention to detect the methylation status of a target nucleic acid. For example, bisulfite-treated DNA utilizing PCR and standard dideoxynucleotide DNA sequencing can directly determine nucleotides that are resistant to bisulfite conversion. (See, for example, Frommer et al., 1992, PNAS 89:1827-1831). Briefly, in an example direct sequencing method, primers are designed that are strand-specific as well as bisulfite-specific (e.g., primers containing non-CpG cytosines so that they are not complementary to non- bisulfite-treated DNA), flanking the potential methylation site. Such primers will amplify both methylated and unmethylated sequences. Pyrosequencing can also be used in the methods of the invention to detect the methylation status of a target nucleic acid. Briefly, in an example pyrosequencing method, followingPCR amplification of the region of interest, pyrosequencing is used to determine the bisulfite-convertedsequence of specific CpG dinucleotide sites in the target nucleic. (See, for example, Tost et al., 2003, BioTechniques 35:152- 156; Wong et al., 2006, 41:734-739).

[0067] A microarray methylation assay can also be used in the methods of the invention to detect the methylation status of a target nucleic acid. Briefly, target nucleic acids are treated with bisulfite, amplified, hybridized to probes, labeled and detected. (See, for example, Wang and Petronis, 2008, DNA Methylation Microarrays: Experimental Design and Statistical Analysis; Weisenberger et al., 2008, Comprehensive DNA Methylation Analysis on the lliumina Infinium Assay Platform).

[0068] In various embodiments, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also known as "Genechips," have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092, which are incorporated herein. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al., Science, 251:767-777 (1991), Pirrung eta!., U.S. Pat. No. 5,143,854 (see also PCT ApplicationNo. WO 90/15070) and Fodor et al., PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein.

[0069] Methylation specific PCR can also be used in the methods of the invention to detect the methylation status of a target nucleic acid. Briefly, sets of PCR primers are designed that will hybridize specifically to either methylated nucleotides or unmethylated nucleotides, after their modification by bisulfite treatment. (See, for example, Ymyev, 2007, PCR Primer Design, Volume 402, Chapter 19, Humana Press; Esteller, 2005, DNA Methylation: Approaches, Methods, and Applications, CRC Press). Non-limiting examples of primers useful in the methods of the invention included the primers exemplified by SEQ ID NOS: 1-11. For example, in various embodiments, the amplicons of the invention are produced in PCR reaction using at least one of the primers exemplified by SEQ ID NOS: 1 and 2. In other embodiments, the amplicons of the invention are produced in PCR reaction using at least one of the primers exemplified by SEQ ID NOS: 2, 3, 4 and 5. In some embodiments, the amplicons of the invention are produced in PCR reaction using at least one of the primers exemplified by SEQ ID NOS: 6 and 7. In other embodiments, the amplicons of the invention are produced in PCR reaction using at least one of the primers exemplified by SEQ ID NOS: 8, 9, 10 and 11.

[0070] As stated above, the invention also pertains to kits useful in the methods of the invention described elsewhere herein. Such kits comprise components useful in any of the methods described herein, including for example, hybridization probes or primers (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes, allele-specific oligonucleotides, means for amplification of a subject's nucleic acid, means for analyzing a subject's nucleic acid, negative comparator standards, positive comparator standards, and instructional materials. For example, in one embodiment, the kit comprises components useful for analysis of the methylation status of nucleic acidsin a biological sample obtained from a subject outside of the subject's pancreas.

[0071] A variety of kits having different components are contemplated by the current invention. Generally, the invention provides a kit comprising a component for detecting or quantifying methylation status of a nucleic acid obtained from the subject. In another embodiment, as discussed above, a kit comprises a component for collecting urine. In various embodiments the kit comprises components and reagents for processing urine for analysis (e.g., substantially purifying nucleic acids). In various embodiments, a kit comprises instructions for use of the kit contents.

[0072] In a further embodiment of the present invention, the early detection of beta cell death inherent in the present invention allows for various treatments/procedures that can stop and even reverse beta cell death. These treatments include without limitation administering drugs such as Lisoffylline and Exenatide as well as changes in diet and exercise and islet and pancreas transplants. In the current state, diabetes is not detected until certain symptoms are experienced. Generally, when the symptoms arise, the only treatment available is insulin which can be expensive and include certain unpleasant side effects. Early detection opens up a wide spectrum of potential treatments to stop and even reverse beta cell death.

[0073] Figure 11 shows the normal course of events related to beta cell death, development of symptoms and treatment with insulin for Juvenile Diabetes and Adult-Onset Diabetes. Beta cell death begins to occur far in advance of the development of symptoms.

[0074] Figure 12 shows how early detection associated with the present invention well in advance of symptoms arising can lead to alternate and less onerous treatments than relying on insulin for Juvenile Diabetes.

[0075] Figure 13 shows how early detection associated with the present invention well in advance of symptoms arising can lead to alternate and less onerous treatments than relying on insulin for Adult

Diabetes. Examples

[0076] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident asa result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0077] Example 1: Detection of p cell death in diabetes using differentially methylated circulating DNA

[0078] The present method useful in mice was also useful for detecting circulating p cell-derived DNA in humans. Uniform demethylation of CpG sites within the insulin gene in human cells and methylation in non- cells was found. Tissue analysis findings were consistent with this finding from the sequence analysis. The average demethylation index was significantly greater in subjects with new-onset T1D, in whom cell death occurs, than in healthy control subjects. The materials and methods employed in these experiments are now described. Female NOD/LtJ, MIP-GFP NOD, and BALB/c mice were obtained from The Jackson Laboratory and maintained under pathogen-free conditions. Seven- week-old NOD mice were screened for hyperglycemia every 2 weeks and were diagnosed with diabetes when two consecutive glucose levels >200mg/dL were measured in whole blood from the tail vein using a Bayer Glucometer Elite XL. The animal care protocol was approved by Yale University's Animal Use Committee. [0079] H uman Subjects. Tissues were obtained from the pathology laboratory at Yale New Haven Hospital. Serum was collected from healthy control subjects and from individuals with recent-onset (i.e., within the first 1-1/2 y) T1D participating in a clinical trial (NCT 00378508). Institutional Review Board approval was obtained for the collection of tissues and sera, and informed consent was obtainedfrom subjects for the collection of sera.

[0080] STZ Treatment. Eight-week-old BALB/c mice received a single i.p. injection of 200 mg/kg of STZ. Blood glucose levels were measured at 8 hours and 24 hours after STZ treatment. At designated time points, mice were killed and serum and pancreas were collected for further analysis.

[0081] Insulin Content of Pancreas

[0082] Whole pancreas was snap-frozen in liquid nitrogen (Best et al., 1939, J. Physiol 97:107-119).

Insulin was extracted with precooled (-20° C acid-ethanol, and the insulin content was measured with a mouse insulin ELISA kit (Crystal Chem, Downers Grove, IL).

[0083] DNA Collection and Bisulfite Treatment. For isolation of purified p cells, islets were isolated from NOD/SCID mice, and single cell suspensions were prepared by collagenase digestion. The cells were stained intracellularly with guinea pig anti-insulin antibodies, followed by a secondary FITC- conjugated donkey anti-guinea pig antibody. The stained cells were then FACS-sorted into either insulin-positive or insulin-negative fractions. Other p cells were isolated from islets from NOD MIP-GFP mice, and insulinpositive cells were sorted on the basis of GFP fluorescence. Purified human cells were isolated from dissociated islets that were permeabilized and stained with FluoZin-3-AM (Jayaraman, 2011, Curr.

Protoc. Cytom. 55:6.27.1-6.27-16). The cells were sorted by gating on the upper 16% of the stained cells. DNA from tissue, cells, and serum samples was purified using the Qiagen QIAamp DNA Blood Kit following the manufacturer-recommended protocol. Synthetic unmethylated and methylated DNA was purchased from Millipore (Billerica, MA). Purified DNA was quantitated using a NanoDrop 2000 spectrophotometer. DNA was then subjected to bisulfite treatment and purified on a DNA binding column to remove excessive bisulfite reagent using the Zymo EZ DNA Methylation Kit.

[0084] First-Step PCR and Gel Extraction. A methylation-independent reaction was carried out to increase the amount of DNA template for PCR analysis. The forward and reverse primers and melting temperatures for the murine and human insulin genes are listed in Figure 8 and Figure 9. For the reaction, bisulfite-treated DNA template was added to Zymo Tag Premix. The PCR conditions for murine and human reactions are given in Figure 8 and Figure 9. The PCR products were excised from a 3% agarose gel. Negative controls without DNA did not yield products in the first-step reaction. Inceiiain experiments, the purified product was sequenced at Yale University's Keck Biotechnology Research Laboratory.

[0085] Cloning and Sequencing of Insulin DNA. PCR products obtained using methylation-independent primers (from sorted p cells, pancreatic islet cells, and control tissue, either kidney or liver) were purified using a Qiagen PCR Purification Kit and ligated via TOPO-TA cloning into the pCR2.1-TOPO vector (Invitrogen, Grand Island, NY). For the mouse sequence, primers outside the region in the nested PCR reactions (Figure 10) were used to increase the number of CG sites. Competent TOP-1 0 bacteria cells were transformed with the products of TOPO ligation and streaked onto agar plates (ampicillin- resistance). After overnight incubation at 37°C, between 12 and 40 colonies from each ligation were picked with clean pipette tips and individually inoculated into 96-well plates. After culture, the bacteria were lysed and used as template DNA for real-time PCR with SYBR Green with the methylationindependent primers. Productive ligations were identified based on Ct values and melting points. The PCR products were sequenced by the Keck Biotechnology Research Laboratory.

[0086] Nested Methylation-Dependent Real-Time PCR. Gel-purified PCR products were used as a template for a quantitative PCR with primers specific for demethylated and methylated insulin 1 DNA.

The conditions for the reaction with SYBR Green (Qiagen) and primers are listed in Figure 8. The reaction was performed on an iQ-5 multicolor real-time PCR system (Bio-Rad), and the Ct cycle was determined for reactions with the demethylated and methylated primer pairs (Figure 8). The relative abundance of demethylated DNA was expressed using the following equation: demethylation index=imethylated cycle number)- (ctemethylatect cycle number). In some experiments (Figure 3A and Figure3C), the ratio of the demethylation index between tissues is presented. The second-step reaction Ct values were between 15 and 40.

[0087] Immunofluorescence. Pancreas was resected and fixed for 24 hours in 2% PFA, then placed in a sucrose gradient and snap-frozen in liquid nitrogen. Noncontiguous 14-p.m pancreatic sections were stained with antibodies to insulin (Invitrogen) and DAPL The bound anti-insulin antibody was detected by immunofluorescent secondary antibodies (Jackson Immunoresearch). The slides were analyzed by fluorescence microscopy using an Olympus BX-51 microscope. Image analysis and postprocessing were performed using ImageJ (rsb.info.nih.gov/ij/). Numbers of single- and dual- color-labeled cells were counted using functions in ImageJ (colocalization, watershed, and analyze particles) (Collins, 2007, Biotechniques 43 (Suppl. l):25-30).

[0088] Statistical Analyses. Data are expressed as mean± SEM. The differences between means and the effects of treatments were analyzed by one-way ANOVA with Tukey's post hoc test using Prism 5 (GraphPad software) to identify the significance (P < 0.05) for all pairs of combinations. Non-normally distributed data were analyzed using nonparametric tests. The results of this example are now described.

[0089] Methylation-Specific Primers Can Detect Differentially Methylated Insl Gene DNA from QTC3 and PMJ Murine Cell Lines.

[0090] To identify differentially methylated CpG dinucleotides present in the Insl gene in p cells, the methylation patterns of the Ins 1 gene in the glucose- responsive murine insulinoma cell line TC3 were examined (Poitout et al., 1995, Diabetes 44:306-313). As a non- cell control, the PMJ macrophage cell line was used. DNA from both cell types was extracted and subjected to bisulfite treatment as described below. A differentially methylated CpG dinucleotide at position NUCL:52339278 (genome.ucsc.edu/cgi- bin/hgGateway, Feb 2009 GRCh37/hgl9) on chromosome 19 was identified, corresponding to the CpG in position+ 177 downstream from the Insl transcription start site, which was demethylated in TC3 cells and methylated in control PMJ cells (Figure IA). This CpG dinucleotide is located in the coding region of the insulin mRNA residing in the proinsulin protein and is evolutionarily conserved in mouse and human insulin genes.

[0091] To verify the tissue specificity of demethylation at this site, the frequency of demethylated and methylated CpG sites was determined in products of the methylation-insensitive PCR from bisulfite- treated DNA from sorted murine insulin-positive cells isolated from MIP-GFP mice and from liver (Figure IB). The majority of the sites were demethylated in DNA from p cells. The CpG site at + 177 was demethylated in 13 of 15 clones isolated from p cells, but in 0 of 8 clones isolated from liver the CpG site was methylated (P < 0.001). It was found that 25% of the 105 sites, or 33% of the clones, showed methylated cytosines in at least one of the seven CpG sites analyzed. In contrast, 86% of the 56 sites analyzed from liver were methylated. The relatively low amounts of circulating DNA in the serum posed a challenge for detecting cell-specific DNA species. Thus, a nested PCR was designed in which insulin DNA with methylation-insensitive primers was first amplified between a region spanning the CpG dinucleotide of interest, followed by a second reaction with methylation-specific primers capable of differentiating p cell-derived and non- cell-derived insulin DNA (Figure 2A and Figure 8). The first PCR generated a product of204 bp that was gel-extracted to improve real-time PCR efficiency. This first-step product was used as template in a second PCR with methylation site-specific primers. Real-time PCR analysis showed a 256-fold (eight-cycle) increase in demethylated DNA levels relative to methylated DNA levels in bisulfite-treated DNA from TC3 cells with a single melting peak (Figure 2B). An exact inverse ratio was observed in the non- cell line PMJ, in which PCR product from methylation-dependent primers was observed eight cycles earlier than PCR product from methylation-independent primers. The identity of the PCR products was verified by sequencing. Taken together, these data indicate the presence of a unique differentially methylated CpG dinucleotide in the coding region of the Ins 1 gene, and demonstrate the ability to detect differentially methylated DNA from either a p cell-like or non-p cell-like origin by methylation-specific quantitative PCR analysis.

[0092] Demethylated Insl DNA Is Enriched in Primary Murine Islets and Cell-Sorted Insulin-Positive Cells.

[0093] To assess the assay's ability to detect methylation-specific modification of DNA from primary murine tissues, kidney, liver, brain, and islet tissues were collected from NOD/SCID mice, which, unlike WT NOD mice, do not develop insulitis or p cell destruction. DNA was extracted and treated with bisulfite, followed by the nested PCR analysis described above. Methylation-specific primers demonstrated a> 12-fold increase in demethylated DNA in the crude islet preparations compared with liver, kidney, and brain (Figure 3A).

[0094] To confirm that p cells were the primary source of the demethylated insulin DNA in our nested PCR, murine islets were dissociated into single cells and stained for insulin. Insulin-positive p cells and insulin-negative cells were sorted by FACS (Figure 3B), and the DNA was isolated and treated as described above. There was a 45-fold increase in demethylated DNA in the insulin-positive cell fraction compared with insulin-negative cells from islets (Figure 3C). Product sequencing revealed an identical demethylated modification in insulin-positive islet cells as in the PTC3 cell line, whereas the non- cell fraction demonstrated a methylated CpG dinucleotide, as observed in the PMJ cell line.

[0095] The ratio between the two DNA species was next analyzed by mixing demethylated DNA (derived from p cells) and methylated DNA (derived from non-p cells) and measuring the difference in cycle threshold (Ct) values detected (Figure 3D). The difference in the Ct values of the methylated and demethylated products of the second-step PCR were characterized using the demethylation index as below, which corresponds to quantitative differences in the quantity of DNA. There was a linear relationship between the log ratio of p cell-derived and non-P cell-derived DNA and a demethylation index between 100:1 and 1:100 (r2 = 0.957; P < 0.01), suggesting that it is possible to measure the quantitative differences in the DNA species over this wide range.

[0096] Circulating Demethylated Ins DNA Is Increased in Streptozotocin-Treated BALB/c Mice.

[0097] To determine whether the assay can detect p cell death in vivo, serum was collected from BALB/c mice before and after treatment with high-dose (200 mg/kg) streptozotocin (STZ), and the DNAwas isolated, processed, and analyzed as described above. The STZ-treated mice demonstrated increased glucose levels at 24 hours after STZ injection, indicating acute injury to cells (P < 0.001) (Figure 4A).

Despite a modest decline in glucose levels at 8 hours after treatment (P < 0.05), most likely reflecting loss of cell membrane integrity and release of insulin granules, there was a 2.6-fold increase inthe demethylation index at 8 hours (P < 0.05) and a 3.8-fold increase at 24 hours (P < 0.02) (Figure 3C).

[0098] The percentage of nucleated cells in the islets after STZ treatment was studied and a reduced percentage ofDAPI- positive, insulin-positive cells staining in the islets at 8 hours after STZ treatment was found (UnTx = 55.1% vs. t8 = 41.3%; P < 0.002) (Figure 3D). A further reduction in the percentage ofDAPI- positive, insulin-positive cells was found at 24 hours after STZ treatment (Figure 3C), which corresponded to the peak in circulating demethylated DNA and increased baseline glucose levels (UnTx = 55.1% vs. Iz4 = 32.8%; P < 0.0001) (Figure 3B). Taken together, these data indicate the ability ofmethylation-specific real-time PCR to detect demethylated DNA derived from damaged p cells in the serum of STZ-treated mice.

[0099] Circulating Demethylated Insl DNA Is Elevated in Prediabetic NOD Mice.

[0100] Next assessed was whether chronic cell destruction could be detected in the NOD mouse model of spontaneous diabetes, a model of chronic autoimmunity in human T1D. NOD mice were challenged with an i.p. glucose tolerance test (IPGTT) beginning at 7 weeks of age, during which basal glucose levels were normal, and extending through the development of overt hyperglycemia (Figure 5A).The IPGTTs revealed subtle changes in glucose tolerance beginning at 9 weeks of age that were statistically significantly different from the 7-week response only at 14 weeks (P< 0.05) (Figure 5B). The fasting glucose levels remained normal at all time-points (Figures 5A-5B). The demethylation index increased significantly before the decline in insulin levels and before the increase in fasting glucose levels (P = 0.0002) (Figure 5C). At 14-15 weeks, the median demethylation index was increased by 21 fold (range, 3.2- to 211-fold; n=12) compared with the average of7-week-old mice (P< 0.01) (Figures 5C-5D). In 16- to 24-week-old mice with overt hyperglycemia, the index declined but was still elevated compared with that in the 7- week-old NOD mice (P < 0.05). The range of increase in demethylation indices in the prediabetic mice was broad, possibly related to individual differences. To understand the relationships between p cell mass and the demethylation index, the relationship between total pancreatic insulin content and the demethylation index was investigated in a separate experiment with prediabetic NOD mice. A decline in pancreatic insulin content with age was found thatwas statistically lower at 15 week compared with 7-week-old NOD mice (P < 0.05). At the same time, the demethylation index increased by 13-fold at 11 weeks compared with 7 weeks (P < 0.05), and by 14 fold at 15 weeks (P < 0.01) (Figure 5D). To analyze the relationship between pancreatic insulin content and the demethylation index in individual mice, these two parameters were compared and found to be significantly correlated (H= 0.28; P < 0.05) (Figure 5E). Taken together, these data show alink between an increased demethylation index and cell loss.

[0101] Demethylated Ins DNA Is Increased in Human Islets and in Serum from Patients with New-Onset T1D.

[0102] A similar strategy was used to analyze demethylated insulin DNA in human tissues. Primers for the first step and nested PCR reactions were prepared from the analogous sequences in human INS on chromosome 11 (Figure 6A and Figure 8). Total DNA was isolated and used in the first-step PCR after bisulfite treatment. The products of the first-step PCR were sequence and two peaks in the CpG site at nucleotide 2182036 (genome.ucsc.edu/cgi-bin/hgGateway; Feb 2009 GRCh37/hgl9) in position +399 downstream from the transcription start site in the DNA from human islets were identified. This double peak corresponds to methylated and demethylated cytosines. Only a single peak, corresponding to methylated cytosine, was found in human kidney DNA (Figures 6A-6B).

[0103] Primary insulin-positive human p cells were sorted from dissociated islets by staining with the zinc selective dye FluoZin-3-AM and products of the first- step reaction from these cells were cloned, and the sequences compared with kidney cells (Jayamaran, 2011, Curr. Protoc. Cytom. 55:6.27.1- 6.27.16). All of the clones (10 of 10) exhibited purified cells demethylated at bp 273 and 399 in the insulin gene, compared with the CpG sites 0 of 12 clones from kidney being methylated (P < 0.001) (Figure 6C). Moreover, CpG sites were rarely demethylated in kidney (<25% of clones), and none oftheclones from kidney exhibited demethylation at all of the CpG sites, whereas all sites but one were demethylated in all 10 clones sequenced from human p cells.

[0104] The demethylation index in DNA isolated from islets, kidney, and liver as well as in unmethylated and methylated synthetic DNA was compared (Figure 6D). A significant increase in the demethylation index in islets (P < 0.001) compared with liver (57-fold) and kidney (91-fold) was found. The demethylation index with islet DNA (0.729 ± 0.05) was similar to the demethylation index with synthetic unmethylated DNA (0.70 ± 0.03). The identity of products was verified by sequencing. The average interassay coefficient of variation from three separate analyses of this tissue DNA was 21.7% ±6.4. The demethylation index in serum samples from patients with T1D (n = 5; mean age, 10.8 ± 1.02 years; range, 8-14 years) within the first year (mean duration of T1D, 7.0 ± 1.30 months; range, 4-11 months) after diagnosis with healthy control subjects who were age-matched were compared, because demethylation might have been affected by islet growth in children (Figure 6E). The demethylation index was significantly higher in the patients with Tl D (P < 0.02), and the average demethylation index inthe nondiabetic subjects was similar to the index with DNA isolated from liver or kidney.

[0105] A similar analysis with second-step PCR primers that target bp +329 was also conducted. Analysis with this primer pair resulted in overall lower demethylation indices, but we found a similar significant increase in the demethylation index (4.42 x 10' ⁴ ± 2.07 x 10' ⁴ vs. 2.37 x 10' ⁶ ± 1.81 x 10' ⁶ ) in this second cohort of subjects with recent-onset (i.e., first 1-1/2 y) T1D (n = 12) compared with healthy control subjects (n = 11; P = 0.0 15).

[0106] Demethylation Index in Human Patients with Recent Onset of Disease.

[0107] The demethylation index of 43 subjects with recent onset (i.e. within 1 year of diagnosis) of disease was compared with the demethylation index of 13 healthy control subjects. The demethylation index was significantly higher in the subjects with disease and there was an inverse relationship between the demethylation index and the insulin secretory response to a mixed meal in these subjects (Figure 11). In addition the coefficient of variation among repeated (4) sampling from 3 healthy control individuals was determined; the CV's ranged from 9.6%-12.8%.

[0108] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the mi without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.