WO2014195306A1 - Methods and kits for diagnosing type 2 diabetes - Google Patents
Methods and kits for diagnosing type 2 diabetes Download PDFInfo
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- WO2014195306A1 WO2014195306A1 PCT/EP2014/061470 EP2014061470W WO2014195306A1 WO 2014195306 A1 WO2014195306 A1 WO 2014195306A1 EP 2014061470 W EP2014061470 W EP 2014061470W WO 2014195306 A1 WO2014195306 A1 WO 2014195306A1
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- methylation
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Definitions
- the present invention provides methods and kits for diagnosing type 2 diabetes by determining the methylation pattern in the KCNJ11 gene promoter in samples of peripheral blood DNA.
- Type 2 diabetes is the most common form of diabetes and is currently becoming a fast-growing pandemic. Type 2 diabetes is influenced by lifestyle factors, such as age, obesity and pregnancy, but also has a strong genetic background (Ridderstrale M and Groop L. 2009. Molecular and Cellular Endocrinology, 297: 10-17). Furthermore, recent studies propose that specific changes in the epigenome are associated with the onset and progression of diabetes (Pinney SE and Simmons RA. 2010. Trends in Endocrinology & Metabolism, 21 :223-229; Pirola L., et al. 2010. Nature Reviews Endocrinology, 6:665-675; Wren JD and Garner HR. 2005. Journal of Biomedicine and Biotechnology, 2005: 104-112; Slomko H., et al. 2012. Endocrinology, 153: 1025-1030; Bouchard L., et al. 2010. Diabetes Care, 33:2436-2441).
- DNA methylation and histone modifications are the main molecular events that initiate and sustain epigenetic modifications. Only few studies so far have documented aberrant DNA methylation events in type 2 diabetes.
- WO 2012/097903 Al and Volkmar M., et al. disclose a method for predicting, prognosing or diagnosing type 2 diabetes comprising the measurement of the methylation status of one or more CpG sites from different genes in a sample. Said method discloses 276 CpG dinucleotides differentially methylated in pancreatic islet samples of type 2 diabetic patients corresponding to 254 genes. However, said alterations are not detected in blood samples.
- the present invention relates to a method for diagnosing type 2 diabetes in a subject comprising:
- step (ii) comparing the methylation pattern of said one or more CpG site(s) obtained in step (i) with the methylation pattern of said CpG site(s) in a reference sample,
- the present invention relates to a method of treatment of type 2 diabetes in a subject comprising the administration to said subject of an antidiabetic agent wherein said subject shows
- the present invention relates to the use of an antidiabetic agent for the manufacture of a medicament for the treatment of type 2 diabetes of a subject, wherein a biological sample from said subject shows
- the present invention relates to a method of treatment of type 2 diabetes of a subject comprising administering an antidiabetic agent, wherein a biological sample from said subject shows
- the present invention relates to a kit comprising at least one oligonucleotide or polynucleotide capable of hybridizing to at least one sequence of a CpG site of the KCNJll gene promoter selected from those CpG site(s) as defined in Table 1 and 2 in a methylation-specific manner.
- the present invention relates to a nucleic acid comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene promoter and wherein the contiguous base sequence comprises at least one CpG site selected from those CpG sites as defined in Tables 1 and 2, a nucleic acid comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene promoter wherein the position corresponding to the C within the at least one CpG site is a uracile or a polynucleotide which specifically hybridizes to any of said nucleic acids.
- the present invention relates to the use of the kit of the invention or of the nucleic acid of the invention for diagnosing type 2 diabetes in a subject.
- Figure 1 ROC curve of type 2 diabetes status according to DNA methylation of CpGs 14-20 in the KCNJll promoter in peripheral blood DNA.
- FIG. 1 Box plot of methylation values of CpGs 11-28 in the KCNJll promoter in lean controls, overweight controls, subjects with prediabetes (prediabetics), type 1 diabetic (T1DM) and type 2 diabetic (T2DM) patients, in peripheral blood DNA.
- prediabetes prediabetes
- T1DM type 1 diabetic
- T2DM type 2 diabetic
- the authors of the present invention have developed a method for diagnosing type 2 diabetes in a patient based in the determination of the DNA methylation pattern in the promoter region of the KCNJll gene in a sample of said patient followed by the comparison of said methylation pattern with the methylation pattern from a reference sample.
- This method has the advantage that it is simpler and less invasive because it can be carried out in peripheral blood rather than in a biopsy, and it is independent of the fasting status of the patient. Furthermore, this method is more specific than the tests based on plasma glucose or glycated blood, and is more comfortable for the patient than an oral glucose tolerance test.
- the authors of the present invention have identified a specific methylation pattern related to type 2 diabetes in the KCNJ11 gene promoter in peripheral blood. Specifically, 25 CpG sites out of 27 analyzed presented different methylation values (p ⁇ 0.0001) between type 2 diabetic patients and overweight controls. In addition, the study of lean controls, prediabetic subjects and type 1 diabetic patients revealed that the KCNJ11 gene promoter methylation pattern found in type 2 diabetic patients was unique to type 2 diabetes. Thus, this differentially methylated pattern could be used as a potential biomarker of type 2 diabetes. Consequently, the method developed by the authors of the present invention allows a differential diagnosis between type 2 diabetes and type 1 diabetes or prediabetes.
- the invention relates to a method for diagnosing type 2 diabetes in a subject (hereinafter referred to as the "diagnostic method of the invention"), comprising:
- step (ii) comparing the methylation pattern of said one or more CpG site(s) obtained in step (i) with the methylation pattern of said CpG site(s) in a reference sample,
- determining the diagnosis refers both to the process of attempting to determine and/or identify a possible disease in a subject, i.e. the diagnostic procedure, and to the opinion reached by this process, i.e. the diagnostic opinion. As such, it can also be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made.
- diagnosis of type 2 diabetes although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as suffering from type 2 diabetes.
- Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
- Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- the p- values are, preferably, 0.05, 0.01, 0.005 or lower.
- type 2 diabetes or “type 2 diabetes mellitus” or “noninsulin- dependent diabetes mellitus (NIDDM)” or “adult-onset diabetes”, as used herein, refers to relates to a disease characterized by an inappropriate increase in blood glucose levels, which generates chronic complications as it affects large and small vessels and nerves.
- the underlying disorder in this disease is the difficulty for insulin action (in the form of a loss of tissue sensitivity to this hormone), which is called insulin resistance, and an inadequate secretion of insulin by the beta cells responsible for their production, the beta cells, in the pancreas.
- faulty insulin action frequently translates into an increase in cholesterol and/or triglyceride levels.
- the actual criteria for diagnosis of type 2 diabetes are:
- test should be performed in a laboratory using a method that is NGSP certified and standardized to the DCCT assay; or
- FPG fasting plasma glucose
- 126 mg/dL 7.0 mmol/L
- Fasting is defined as no caloric intake for at least 8 h; or 2 h plasma glucose (PG) > 200mg/dL (l l . lmmol/L) during an oral glucose tolerance test (OGTT).
- OGTT oral glucose tolerance test
- the test should be performed as described by the World Health Organization, using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water; or
- the type 2 diabetes is gestational diabetes. This is a type of type 2 diabetes which may first be diagnosed during pregnancy as gestational diabetes. For this reason, women with a history of gestational diabetes have a greatly increased subsequently risk of having diabetes and should be followed up with subsequent screening for the development of diabetes.
- gestational diabetes or "gestational diabetes mellitus” or “GDM”, as used herein, refers to a condition wherein women without being previously diagnosed with diabetes have high levels of glucose in plasma during pregnancy. Formally, it is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. This definition recognizes the possibility that a woman may have previously undiagnosed diabetes type 1 or 2, or may have developed diabetes type 1 or 2 coincidentally with pregnancy. The fact that the symptoms subside after pregnancy is irrelevant to the diagnosis. A woman is diagnosed with gestational diabetes when glucose intolerance continues beyond 24-28 weeks of gestation. Generally, gestational diabetes shows few symptoms and is most often diagnosed by screening during pregnancy, where diagnostic tests detect high levels of glucose in plasma or serum.
- gestational diabetes As with diabetes mellitus during pregnancy, babies born to mothers with untreated gestational diabetes typically have a higher risk for problems such as an increased size for gestational age (which can lead to complications during delivery), low levels of sugar in blood and jaundice. If untreated, it can also cause seizures or stillbirth. Gestational diabetes is a treatable condition and women who have adequate control of glucose levels can effectively decrease these risks.
- the type 2 diabetes is gestational diabetes.
- subject refers to an individual, such as a human, a nonhuman primate (e.g. chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs.
- a nonhuman primate e.g. chimpanzees and other apes and monkey species
- farm animals such as birds, fish, cattle, sheep, pigs, goats and horses
- domestic mammals such as dogs and cats
- laboratory animals including rodents, such as mice, rats and guinea pigs.
- mice a particular age or sex.
- the subject is a mammal.
- the subject is a human.
- the subject has not been treated prior to the determination with any antidiabetic drugs.
- the first method of the invention comprises the determination of the methylation pattern in one or more CpG site(s) of the KCNJ11 gene promoter selected from those CpG sites as defined in Tables 1 and 2 in a biological sample of said subject containing genetic material
- DNA methylation refers to a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides. DNA methylation at the 5 position of cytosine has the specific effect of reducing gene expression and has been found in every vertebrate examined. In adult non-gamete cells, DNA methylation typically occurs in a CpG site.
- CpG site refers to regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length.
- CpG is shorthand for "C-phosphate-G", that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA.
- CpG notation is used to distinguish this linear sequence from the CG base-pairing of cytosine and guanine. Cytosines in CpG dinucleotides can be methylated to form 5- methylcytosine.
- methylation pattern refers to, but is not limited to, the presence or absence of methylation of one or more nucleotides. Thereby said one or more nucleotides are comprised in a single nucleic acid molecule. Said one or more nucleotides have the ability of being methylated or being non-methylated.
- methylation status may also be used, wherein only a single nucleotide is considered.
- a methylation pattern can be quantified, wherein it is considered over more than one nucleic acid molecule.
- KCNJH relates to the potassium inwardly- rectifying channel, subfamily J, member 1 1 gene, with accession number (ENST00000339994).
- the KCNJII gene may also be referred to as BIR, HHF2, PHHI, IKATP, TNDM3 or KIR6.2.
- KCNJII encodes the ATP-sensitive potassium channel subunit Kir 6.2 which together with the sulfonylurea receptor SUR1 forms an octameric complex that regulates glucose-stimulated insulin secretion in pancreatic ⁇ cells (Ridderstrale and Groop, 2009, Molecular and Cellular Endocrinology, 297: 10-7).
- promoter refers to a region of DNA that initiates transcription of a particular gene. Promoters are located near the genes they transcribe, on the same strand and upstream on the DNA, and can be about 100-1000 base pairs long. In the particular case of the KCNJII gene promoter, it starts at -2424 bp according to the ATG position for the KCNJII gene and ends at -1720 bp. As defined in the present invention, the promoter zone covers a 1,000 bp area upstream of the first exon of the human KCNJII gene.
- sample refers to biological material isolated from a subject.
- the biological sample contains any biological material suitable for detecting the desired methylation pattern in one or more CpG site(s) and can comprise cell and/or non-cell material of the subject.
- the sample comprises genetic material, e.g., DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA, heterogeneous nuclear RNA (hnRNA), mRNA, etc., from the subject under study.
- the genetic material is DNA.
- the DNA is genomic DNA.
- the DNA is circulating DNA.
- the sample can be isolated from any suitable tissue or biological fluid such as, for example blood, saliva, plasma, serum, urine, cerebrospinal liquid (CSF), feces, a buccal or buccal-pharyngeal swab, a surgical specimen, a specimen obtained from a biopsy, and a tissue sample embedded in paraffin.
- tissue or biological fluid such as, for example blood, saliva, plasma, serum, urine, cerebrospinal liquid (CSF), feces, a buccal or buccal-pharyngeal swab, a surgical specimen, a specimen obtained from a biopsy, and a tissue sample embedded in paraffin.
- CSF cerebrospinal liquid
- feces feces
- buccal or buccal-pharyngeal swab a surgical specimen
- a specimen obtained from a biopsy and a specimen obtained from a biopsy.
- the sample is selected from blood, serum, plasma, hair, urine and saliva.
- determination of the methylation pattern in a CpG site refers to the determination of the methylation status of a particular CpG site.
- the determination of the methylation pattern in a CpG site can be performed by means of multiple processes known by the person skilled in the art.
- the nucleic acid is extracted from cells which are present in a biological fluid (e.g., whole blood, saliva, synovial fluid, etc.) as an initial step, and, in such cases, the total nucleic acid extracted from said samples would represent the working material suitable for subsequent analysis. Isolating the nucleic acid of the sample can be performed by standard methods known by the person skilled in the art.
- the methylation pattern of one or more CpG site(s) is determined.
- the analysis of the methylation pattern present in one or several of the CpG sites disclosed herein in a subject's nucleic acid can be done by any method or technique capable of measuring the methylation pattern present in a CpG site. For instance, one may detect SNPs in the first method of the invention by performing by a method selected from the group consisting of Methylation-Specific PCR (MSP), an enrichment-based method (e.g.
- MSP Methylation-Specific PCR
- MeDIP, MBD-seq and MethylCap bisulfite sequencing and bisulfite-based method (e.g. RRBS, Infmium, GoldenGate, COBRA, MSP, MethyLight) and a restriction-digestion method (e.g., MRE-seq, or HELP assay), ChIP- on-chip assay, or differential-conversion, differential restriction, differential weight of the DNA methylated CpG site(s).
- RRBS Infmium, GoldenGate, COBRA, MSP, MethyLight
- a restriction-digestion method e.g., MRE-seq, or HELP assay
- ChIP- on-chip assay or differential-conversion, differential restriction, differential weight of the DNA methylated CpG site(s).
- the genomic DNA sample is chemically treated in such a way that all of the unmethylated cytosine bases are modified to uracil bases, or another base which is dissimilar to cytosine in terms of base pairing behaviour, while the 5-methylcytosine bases remain unchanged.
- modify means the conversion of an unmethylated cytosine to another nucleotide which will distinguish the unmethylated from the methylated cytosine.
- the conversion of unmethylated, but not methylated, cytosine bases within the DNA sample is conducted with a converting agent.
- converting agent or "converting reagent”, as used herein, relates to a reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
- the converting agent is preferably a bisulfite such as disulfite or hydrogen sulfite.
- other agents that similarly modify unmethylated cytosine, but not methylated cytosine can also be used in the method of the invention, such as hydrogen sulfite.
- reaction is performed according to standard procedures (Frommer et al, 1992, Proc Natl Acad Sci USA 89: 1827-31; Olek, 1996, Nucleic Acids Res 24:5064-6; EP 1394172). It is also possible to conduct the conversion enzymatically, e.g by use of methylation specific cytidine deaminases.
- the sample has been treated with a reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
- the agent used for modifying unmethylated cytosine is sodium bisulfite.
- the region containing the one or more CpG site(s) is amplified using primers allowing to distinguish the unmethylated sequence (wherein the cytosine of CpG site is converted into uracil) of the methylated sequence (wherein the cytosine of the CpG site remains cytosine).
- amplification methods rely on an enzymatic chain reaction such as, for example, a polymerase chain reaction (PCR), Ligase Chain Reaction (LCR), Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, and NASBA.
- RNA molecules there are strand displacement amplification (SDA), transcription mediated amplification (TMA), and QP-amplification, etc.; this list is merely illustrative and in no way limiting.
- SDA strand displacement amplification
- TMA transcription mediated amplification
- QP-amplification etc.; this list is merely illustrative and in no way limiting.
- MSP methylation specific PCR method
- US 5, 786,146 which combines bisulfite treatment and allele-specific PCR (see e.g. US 5,137,806, US 5,595,890, US 5,639,611).
- Uracil is recognized as a thymine by Taq polymerase and therefore upon PCR, the resultant product contains cytosine only at the position where 5- methylcytosine occurs in the starting template DNA.
- oligonucleotides or polynucleotides capable of specifically hybridizing to a bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter are described in the context of the kit of the invention, and are used with the same meaning in the context of the method of treatment according to the invention.
- the amplification products are then detected according to standard procedures in the art.
- the amplified nucleic acid may be determined or detected by standard analytical methods known to the person skilled in the art and described e.g. in Sambrook et al, 2001 (cited at supra). There may be also further purification steps before the target nucleic acid is detected e.g. a precipitation step.
- the detection methods may include but are not limited to the binding or intercalating of specific dyes as ethidium bromide which intercalates into the double-stranded DNA and changes its fluorescence thereafter.
- the purified nucleic acids may also be separated by electrophoretic methods optionally after a restriction digest and visualized thereafter.
- probe-based assays which exploit the oligonucleotide hybridization to specific sequences and subsequent detection of the hybrid. It is also possible to sequence the target nucleic acid after further steps known to the expert in the field. Other methods apply a diversity of nucleic acid sequences to a silicon chip to which specific probes are bound and yield a signal when a complementary sequences bind.
- the amplified nucleic acid is detected using the MassARRAY® platform (Sequenom, Inc.; San Diego, CA, USA) by a combination of RNA base-specific cleavage (MassCLEAVETM) and MALDI-TOF mass spectrometry.
- MassCLEAVETM RNA base-specific cleavage
- MALDI-TOF mass spectrometry.
- Base-specific cleavage generates a defined experimental mass signal pattern of the sample.
- the experimental pattern is subsequently compared to an in silico reference mass signal pattern derived from an individual or set of reference sequences. Differences between the expected and the observed mass signal pattern are interpreted and enable identification of sequence variations.
- the nucleic acid amplification is carried out by real time PCR and real time probes are used to detect the presence of the extension product.
- real time probes e.g. Lightcycler, Taqman, Scorpio, Sunrise, Molecular Beacon or Eclipse probes. Details concerning structure or detection of these probes are known in the state of the art.
- the methylation pattern of the nucleic acid can be confirmed by restriction enzyme digestion and Southern blot analysis.
- methylation sensitive restriction endonucleases which can be used to detect CpG methylation include Smal, Sacll, Eagl, Mspl, Hpall, BstXJl and BssHll, for example.
- the CpG sites are defined using the distance in base pair from the CpG site to the transcription start site for the KCNJ11 gene.
- Table 2 List of CpG sites with specific differential hypermethylation in peripheral blood DNA samples
- Table 4 List of CpG sites whose mean is hypermethylated in peripheral blood DNA samples of type 2 diabetic patients
- the first method of the invention comprises the comparison of the methylation pattern of said one or more CpG site(s) obtained in step (i) with the methylation pattern of said CpG site(s) in a reference sample.
- the invention provides some specific CpG site(s) which are significantly associated with the diagnosis of type 2 diabetes in a subject.
- the determination of the methylation pattern comprises the determination of the methylation pattern of all the CpG sites of Tables 1 and 2.
- reference sample means a sample obtained from a pool of healthy subjects which does not have a disease state or particular phenotype.
- the reference sample may comprise tissue or blood (or the correspondent) samples from subjects which do not suffer type 2 diabetes or which do not have a history of type 2 diabetes.
- the reference sample is a sample of subjects matched on age and body mass index to the subject analysed.
- the subject has not received an antidiabetic treatment prior to obtaining the sample.
- the reference sample is a sample from a type 1 diabetic patients.
- the reference sample is a sample from a prediabetic subjects,
- the determination of the methylation pattern comprises the determination of the methylation pattern of all CpG sites of Table 3, wherein if the mean level of methylation of said CpG sites of Table 3 is decreased in a subject with respect to the reference sample is indicative that the subject suffers from type 2 diabetes.
- the determination of the methylation pattern comprises:
- the level of methylation of one or more CpG site(s) is increased when the level of methylation of said one or more CpG site(s) in a sample is higher than in the reference sample.
- the level of methylation of one or more CpG site(s) is considered to be higher than in the reference sample when they are at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more higher than in the reference sample.
- the level of methylation of one or more CpG site(s) is decreased when the level of methylation of said one or more CpG site(s) in a sample is lower than a reference value.
- the level of methylation of one or more CpG site(s) is considered to be lower than in the reference sample when it is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150% or more lower than in the reference sample.
- mean level of methylation refers to the mean or average level of methylation in said region, which may be calculated as the sum of the methylation levels of all CpG sites within said region divided by the number of CpG sites in the region. In the particular case of the present embodiments, the mean level of methylation is calculated for all CpG sites of Table 3, Table 4 or Table 5.
- the mean level of methylation of all CpG sites of Table 3, Table 4 or Table 5 is increased when the mean level of methylation of said all CpG sites in a sample is higher than in the reference sample.
- the mean level of methylation of all CpG sites is considered to be higher than in the reference sample when it is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more higher than in the reference sample.
- the mean level of methylation of all CpG sites of Table 3, Table 4 or Table 5 is decreased when the mean level of methylation of said all CpG sites in a sample is lower than a reference value.
- the mean level of methylation of one or more CpG site(s) is considered to be lower than in the reference sample when it is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more lower than in the reference sample.
- Antidiabetic agent for use in the treatment of type 2 diabetes in another aspect, relates to an antidiabetic agent for use in the treatment of type 2 diabetes of a subject (hereinafter referred to as the "antidiabetic agent of the invention"), wherein a biological sample from said subject shows
- the present invention relates to the use of an antidiabetic agent for the manufacture of a medicament for the treatment of type 2 diabetes of a subject, wherein a biological sample from said subject shows a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
- the present invention relates to a method of treatment of type 2 diabetes of a subject comprising administering an antidiabetic agent, wherein a biological sample from said subject shows
- said subject shows a decrease in the mean level of methylation of all CpG sites of Table 3 with respect to the reference sample.
- said subject shows an increase in the mean level of methylation of all CpG sites of Table 4 with respect to the reference sample, or an increase in the mean level of methylation of all CpG sites of Table 5 with respect to the reference sample, or a combination thereof.
- the type 2 diabetes is gestational diabetes.
- the diagnostic method of the invention can be applied herein in determining the level of methylation one or more CpG site(s) of the KCNJ11 gene promoter with respect to a reference sample.
- insulin drug refers to compounds that which show antiglycaemic activity or agents which sensitize insulin activity.
- these compounds include, without limitation, insulin and functionally equivalent variants thereof, secretagogues of insulin such as the sulfonylureas (tolbutamide, chlorpropamide, glipicide, glibenclamide, glicazide, glipentide, glimepiride, glibenclamide, glipizide, gliquidone, glisentide, glimepride and such like) and metiglinides (repaglinide, nateglinide, mitiglinide and such like), reducing agents of liver glucose production (biguanides and, in particular, metformin and buformin), agents which cause carbohydrate decrease such as a-glucosidase inhibitors (acarbose, miglitol or voglibose), agents that increase peripheral use of glucose such as thi
- “Functionally equivalent variant of insulin” is understood as all those polypeptides resulting from the elimination, insertion or modification of at least one amino acid with respect to the insulin sequence and which substantially maintains the same properties as the insulin it comes from. Insulin activity can be determined by methods widely known by those skilled in the art such as normoglycaemic clamping or the measurement of glycosylated proteins in serum (Bunn et al, Diabetes, 1981, 30:613-617).
- Functionally equivalent variants of insulin include, without limitation, the des-pentapeptide (B26-B30)-Phe B25 -a-carboxamide]insulin, Asp B1 ° insulin (disclosed in US4992417), Lys B28 -Pro B29 insulin Lys B28 -Pro B29 and the hexameric variant thereof (disclosed in US5474978 and US5514646), formulations of insulin and protamine (US5650486), acylated Lys B28 -Pro B29 insulin (US5922675), and compositions of stabilized insulin such as those disclosed in US5952297, US6034054 and US6211144, superactive analogs of insulin, monomeric insulins, hepatospecific insulins, insulin lispro (Humalog®), insulin lispro formulated with insulin lispro protamine (marketed as Humalog ®50/50TM, Humalog® 75/25TM), NPH insulin or insulin isophane human (marketed as Humulin
- treatment includes any process, action, application, therapy, or the like, wherein a subject, including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject, or ameliorating at least one symptom of the disease or disorder under treatment.
- a suitable treatment of type 2 is based on an anti-diabetic agent therapy.
- anti-diabetic agent and its particulars have been described in detail in the context of the diagnostic method of the invention and are used with the same meaning in the context of the method of treatment according to the invention.
- Type 2 diabetes is initially managed by increasing exercise and dietary modification in order to lower blood glucose levels.
- anti-diabetic treatment or “treatment of type 2 diabetes”, as used herein, refers to an anti-diabetic agent used to lower blood glucose levels.
- the present invention relates to a kit (hereinafter referred to as the "kit of the invention"), comprising at least one oligonucleotide or polynucleotide capable of hybridizing in a methylation-specific manner to at least one sequence of a CpG site of the KCNJll gene promoter selected from those CpG site(s) as defined in Table 1 and 2.
- kit of the invention comprising at least one oligonucleotide or polynucleotide capable of hybridizing in a methylation-specific manner to at least one sequence of a CpG site of the KCNJll gene promoter selected from those CpG site(s) as defined in Table 1 and 2.
- kits include various reagents for use in accordance with the present invention in suitable containers and packaging materials, including tubes, vials, and shrink-wrapped and blow-moulded packages. Additionally, the kits of the invention can contain instructions for the simultaneous, sequential or separate use of the different components which are in the kit. Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like. Additionally or alternatively, the media can contain Internet addresses that provide said instructions.
- Materials suitable for inclusion in an exemplary kit in accordance with the present invention comprise one or more of the following: at least one oligonucleotide or polynucleotide capable of hybridizing to at least one sequence of a CpG site of the KCNJll gene promoter selected from those CpG site(s) as defined in Table 1 and 2 in a methylation-specific manner, reagents capable of amplifying a specific sequence domain in either genomic DNA or cDNA without the requirement of performing PCR; reagents required to discriminate between the various possible alleles in the sequence domains amplified by PCR or non-PCR amplification (e.g., restriction endonucleases, oligonucleotide that anneal preferentially to methylated or to unmethylated CpG sites, including those modified to contain enzymes or fluorescent chemical groups that amplify the signal from the oligonucleotide and make discrimination of methylated or unmethylated CpG sites more robust); or reagents required
- oligonucleotide refers to a short, single-stranded DNA or RNA molecule, with up to 13 bases in length.
- the oligonucleotides of the invention are preferably DNA molecules of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or 13 bases in length.
- polynucleotide refers to single-stranded DNA or RNA molecules, of more than 13 bases in length.
- the polynucleotides of the invention are preferably DNA molecules of at least 14, at least 15, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more bases in length.
- the at least one oligonucleotide or polynucleotide capable of hybridizing to at least one sequence of a CpG site of the KCNJ11 gene promoter selected from those CpG site(s) as defined in Table 1 and 2 in a methylation-specific manner are used as a primer to amplify the region containing said CpG site(s).
- oligonucleotide or polynucleotide can also be used as a probe for detecting said methylated or unmethylated CpG site(s).
- hybridizing refers to the capacity of an oligonucleotide or polynucleotide of recognizing specifically the sequence of a CpG site.
- hybridization is the process of combining two complementary single-stranded nucleic acid molecules, or molecules with a high degree of similarity, and allowing them to form a single double- stranded molecule through base pairing. Normally, the hybridization occurs under high stringent conditions or moderately stringent conditions.
- the "similarity" between two nucleic acid molecules is determined by comparing the nucleotide sequence of one molecule to the nucleotide sequence of a second molecule.
- Variants according to the present invention include nucleotide sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar or identical to the sequence of the CpG site.
- the degree of identity between two nucleic acid molecules is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
- the identity between two amino acid sequences is preferably determined by using the BLASTN algorithm (BLAST Manual, Altschul et al, 1990, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol Biol 215:403-10).
- “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
- stringent conditions typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Fico 11/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50%> formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1 %> SDS, and 10% dextran sulfate
- formamide for example, 50% (v/v) formamide
- Modely stringent conditions may be identified as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above.
- washing solution and hybridization conditions e.g., temperature, ionic strength and % SDS
- An example of moderately stringent conditions is overnight incubation at 37°C.
- the kit comprises a first oligonucleotide or polynucleotide capable of specifically hybridizing to a bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is methylated, and at least one oligonucleotide or polynucleotide capable of specifically hybridizing to the same bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is unmethylated.
- oligonucleotides or polynucleotides used in the invention for amplification of the CpG-containing nucleic acid in the sample, after bisulfite modification, specifically distinguish between untreated DNA, methylated and non- methylated DNA.
- primers for the non- methylated DNA preferably have a T in the 3' CG pair to distinguish it from the C retained in methylated DNA, and the complement is designed for the antisense primer.
- Primers usually contain relatively few Cs or Gs in the sequence since the Cs will be absent in the sense primer and the Gs absent in the antisense primer (cytosine becomes modified to uracil, which is amplified as thymidine in the amplification product). Accordingly, for hybridization to a methylated CpG site, primers for the methylated DNA preferably have a C in the 3' CG pair.
- the kit comprises oligonucleotides or polynucleotides capable of hybridizing to all CpG sites of the KCNJl l gene promoter as defined in Table 1 and 2 in a methylation-specific manner.
- the kit comprises at least one oligonucleotide or polynucleotide selected from the group consisting of sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
- the kit of the invention further comprises one or more reagents for converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
- the one or more reagents capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties is a bisulfite, preferably sodium bisulfite.
- the reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties is metabisulfite, preferably sodium metabisulfite.
- the present invention relates to a nucleic acid (hereinafter referred to as the "first polynucleotide of the invention") comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene and wherein the contiguous base sequence comprises at least one CpG site selected from those CpG sites as defined in Tables 1 and 2.
- the first polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJll gene.
- the present invention relates to a nucleic acid (hereinafter referred to as the "second polynucleotide of the invention"), comprising at least 9 contiguous nucleotides of the promoter region of the KCNJ11 gene promoter wherein the position corresponding to the C within the at least one CpG site selected from those CpG sites as defined in Tables 1 and 2 is a uracile.
- the second polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJ11 gene.
- the present invention relates to a nucleic acid (hereinafter referred to as the "third polynucleotide of the invention") comprising a polynucleotide which specifically hybridizes to any of the first and second nucleic acids of the invention.
- the third polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides.
- nucleic acids of the invention have been described in detail in the context of the methods and kits of the invention and are used with the same meaning in the context of the uses according to the invention. Diagnostic uses of the invention
- kits and nucleic acids of the invention are particularly useful in the diagnosis of type 2 diabetes in a subject.
- the present invention relates to the use of the kit of the invention (hereinafter referred to as the "first use of the invention") for diagnosing type 2 diabetes in a subject.
- the type 2 diabetes is gestational diabetes.
- the kit comprises a first oligonucleotide or polynucleotide capable of specifically hybridizing to a bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is methylated, and at least one oligonucleotide or polynucleotide capable of specifically hybridizing to the same bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJll gene promoter when said CpG site is unmethylated.
- the kit comprises oligonucleotides or polynucleotides capable of hybridizing to all CpG sites of the KCNJl l gene promoter as defined in Table 1 and 2 in a methylation-specific manner.
- the kit comprises at least one oligonucleotide or polynucleotide selected from the group consisting of sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
- the kit of the invention further comprises one or more reagents for converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
- the one or more reagents for converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties is a bisulfite, preferably sodium bisulfite.
- the present invention relates to the use of the first polynucleotide of the invention (hereinafter referred to as the "second use of the invention") for diagnosing type 2 diabetes in a subject.
- the type 2 diabetes is gestational diabetes.
- the first nucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJll gene.
- the present invention relates to the use of the second polynucleotide of the invention (hereinafter referred to as the "third use of the invention"), for diagnosing type 2 diabetes in a subject.
- the type 2 diabetes is gestational diabetes.
- the second nucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJll gene.
- the present invention relates to the use of the third polynucleotide of the invention (hereinafter referred to as the "fourth use of the invention"), for diagnosing type 2 diabetes in a subject.
- the type 2 diabetes is gestational diabetes.
- Sequenom's MassARRAY platform was used to perform quantitative DNA methylation analysis (Ehrich M et al. 2005. Proc Natl Acad Sci U S A, 102: 15785- 15790). This system utilizes MALDI-TOF mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). A detectable pattern is then analyzed for methylation status. PCR primers for the amplification of the promoters of KCNJl 1 gene were designed using Epidesigner (Sequenom). When possible, amplicons were designed to cover CpG islands in the same region as the 5' UTR.
- PCR reactions were carried out in 5 ⁇ total volume with 10 ng/ml bisulfite- treated DNA, 0.2 units TaqONA polymerase (Sequenom), lx supplied Taq buffer, and 200 mM PCR primers. Amplification was done as follows: preactivation of 95°C for 15 min, 45 cycles consisting of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s, finishing with a 72°C incubation for 4 min.
- Dephosphorylation of unincorporated dNTPs was performed by adding 1.7 ml of H 2 0 and 0.3 units of shrimp alkaline phosphatase (Sequenom), incubating at 37°C for 40 min and then at 85° C for 10 min, to deactivate the enzyme.
- the MassCLEAVE biochemistry was performed as follows: in vivo transcription and RNA cleavage was achieved by adding 2 ⁇ of PCR product to 5 ⁇ of transcription/cleavage reaction and incubating at 37°C for 3 h.
- the transcription/cleavage reaction contains 27 units of T7 RNA&DNA polymerase (Sequenom), 0.64x of T7 R&DNA polymerase buffer, 0.22 ⁇ T Cleavage Mix (Sequenom), 3.14 mM DTT, 3.21 ⁇ H 2 0, and 0.09 mg/ml RNaseA (Sequenom).
- the reactions were additionally diluted with 20 ml H 2 0 and conditioned with 6 mg of CLEAN Resin (Sequenom) for optimal mass-spectra analysis.
- Adiponectin, leptin and insulin were quantified from serum samples by ELISA (Mercodia), according to the manufacturer's instructions. Cytokines were measured from serum samples using CBA Human Inflammatory Cytokines kit (BD Bioscience), following the manufacturer ' s instructions. Two-color flow cytometric analysis was performed using LSRFortessa (BD bioscience). Data were acquired and analyzed using FACS Diva and FCAP Array 1.01 Softwares.
- Descriptive data are presented as the mean and standard deviation (SD) for continuous outcomes, or number and percentage (%) for categorical outcomes.
- SD standard deviation
- cytokines cytokines
- HOMA-IR insulin
- leptin adiponectin
- adiponectin adiponectin
- Student's t test was used for the comparison of the rest of continuous outcomes and Chi-square test for categorical outcomes.
- Correlation between DNA methylation values and covariates that were significantly different between type 2 diabetic patients and overweight controls was assessed by Spearman's rank correlation coefficient. Adjustment by disease status (i.e, being case or control) was done. Bonferroni correction was used for multiple comparisons. All significance tests were 2-tailed and values of p ⁇ 0.05 were considered significant. All analyses were conducted using the statistical software package Stata version 11.
- Total cholesterol and fasting adiponectin were lower in cases as compared to controls (total cholesterol mean values of 4.77 mmol/L vs 5.25 mmol/L, and fasting adiponectin mean values of 7.0 ⁇ g/mL vs 10.0 ⁇ g/mL, p ⁇ 0.0001).
- Epidemiological studies have shown that higher adiponectin levels in serum are associated with a lower risk of type 2 diabetes (Fagerberg B., et al. 2011. J Intern Med, 269:636-643).
- IL 12 52.8 ⁇ 58.3 pg/mL in cases vs 29.7 ⁇ 37.4 pg/mL in controls, p ⁇ 0.0001).
- Type 2 diabetic patients were in optimal glycemic control (mean glycated hemoglobin 5.8%).
- Triglycerides (mmol/L) 1.43 ⁇ 0.89 1.31 ⁇ 0.75 0.34
- Table 7 Demographic and clinical characteristics of type 2 diabetic patients and age- and BMI-matched controls. Values shown are means ⁇ SD, unless otherwise indicated. P values were calculated with the t test for quantitative variables or Chi- square test for categorical ones, except for HOMA-IR, fasting insulin, fasting leptin and fasting adiponectin, where non-parametric Mann- Whitney U test was applied. HOMA-IR was calculated as [Insulin mUI/1 x Glycemia: (mmol/l)/22.5]. Quantitative DNA Methylation analysis in peripheral blood identifies a full distinct pattern in the KCNJ11 promoter in type 2 diabetic patients as compared to age- and BMI-matched controls.
- Methylation levels in DNA from whole blood of 186 subjects were obtained for 27 CpG sites covering 1,000 bp upstream of the first exon of the human KCNJ11 gene.
- the heat map showing the values of methylation (%) for each CpG site analyzed revealed a unique and compelling differentiated pattern of methylation between type 2 diabetic patients and controls in the KCNJ11 promoter (data not shown). Strikingly, 25 CpG sites out of 27 analyzed (93%) presented significantly different methylation values (p ⁇ 0.0001) amongst cases and controls (Table 8).
- the first 13 CpGs were hypomethylated in type 2 diabetic patients (methylation average 4.0 %, 95% CI 3.8-4.2) as compared to controls (methylation average 26.0%, 95% CI 25.0-27.1, p ⁇ 0.0001). Furthermore, there was a complete degree of methylation (100%) in the area comprised between -1898 to -1847 bp upstream the transcription start site (TSS) position for the KCNJll gene in all type 2 diabetic patients. The area comprised between -1781 and -1778 upstream the TSS was also hypermethylated in all type 2 diabetic patients (mean methylation of 94.5% in cases vs 11.0 % in controls).
- Table 9 Spearman correlations between DNA methylation and laboratory values from type 2 diabetic patients and age- and BMI-matched controls. Quantitative DNA Methylation analysis of the KCNJ11 promoter in peripheral blood of lean controls, subjects with prediabetes and type 1 diabetic patients.
- IFG denotes impaired fasting glucose.
- IGT denotes impaired glucose tolerance. Values are expressed as mean (SD), unless otherwise indicated.
- prediabetic subjects were 66.8 years old on average and had a mean BMI of 29.3, a mean fasting glucose of 5.68 mmol/L and mean glucose post OGTT (oral glucose tolerance test) of 8.44 mmol/L.
- Type 1 diabetic patients had a mean fasting glucose of 10.99 mmol/L and a mean glycated hemoglobin of 9.4%.
- the pattern found in type 2 diabetic patients is unique to type 2 diabetic patients and does not appear gradually with diabetic status since it is absent in prediabetic subjects. Indeed, prediabetic subjects, lean controls, overweight controls and type 1 diabetic patients all have a similar methylation pattern, especially in CpGs 11-28 ( Figure 2).
- Type 2 diabetic patients however, presented a hypomethylation of CpGs 1- 10 and a hypermethylation of GpGs 14-20 and CpGs 24-25. This pattern does not appear to be secondary to changes in weight or age. Moreover, a chronic effect of alteration in glucose homeostasis is not responsible for this pattern since type 1 diabetic patients did not present it, either.
Abstract
The invention relates to methods for diagnosing type 2 diabetes as well as antidiabetic agents for use in the treatment of type 2 diabetes. The invention relates as well to kits and nucleic acids for carrying out the diagnostic methods.
Description
METHODS AND KITS FOR DIAGNOSING TYPE 2 DIABETES
FIELD OF THE INVENTION The present invention provides methods and kits for diagnosing type 2 diabetes by determining the methylation pattern in the KCNJ11 gene promoter in samples of peripheral blood DNA.
BACKGROUND OF THE FNVENTION
Type 2 diabetes is the most common form of diabetes and is currently becoming a fast-growing pandemic. Type 2 diabetes is influenced by lifestyle factors, such as age, obesity and pregnancy, but also has a strong genetic background (Ridderstrale M and Groop L. 2009. Molecular and Cellular Endocrinology, 297: 10-17). Furthermore, recent studies propose that specific changes in the epigenome are associated with the onset and progression of diabetes (Pinney SE and Simmons RA. 2010. Trends in Endocrinology & Metabolism, 21 :223-229; Pirola L., et al. 2010. Nature Reviews Endocrinology, 6:665-675; Wren JD and Garner HR. 2005. Journal of Biomedicine and Biotechnology, 2005: 104-112; Slomko H., et al. 2012. Endocrinology, 153: 1025-1030; Bouchard L., et al. 2010. Diabetes Care, 33:2436-2441).
DNA methylation and histone modifications are the main molecular events that initiate and sustain epigenetic modifications. Only few studies so far have documented aberrant DNA methylation events in type 2 diabetes.
WO 2012/097903 Al and Volkmar M., et al. (Volkmar M., et al. 2012. EMBO J., 31(6): 1405-1426) disclose a method for predicting, prognosing or diagnosing type 2 diabetes comprising the measurement of the methylation status of one or more CpG sites from different genes in a sample. Said method discloses 276 CpG dinucleotides differentially methylated in pancreatic islet samples of type 2 diabetic patients corresponding to 254 genes. However, said alterations are not detected in blood samples.
Barring a laboratory error, such patients are likely to have test results near the margins of the threshold for a diagnosis and remain without a definite diagnostic. Thus,
there is a need in the art for alternative and accurate methods for diagnosing type 2 diabetes that do not depend on the fasting status of the subject and on laboratory inaccuracies. SUMMARY OF THE INVENTION
In a first aspect, the present invention relates to a method for diagnosing type 2 diabetes in a subject comprising:
(i) determining the methylation pattern in one or more CpG site(s) of the KCNJ11 gene promoter selected from those CpG sites as defined in Tables 1 and 2 in a biological sample of said subject containing genetic material, and
(ii) comparing the methylation pattern of said one or more CpG site(s) obtained in step (i) with the methylation pattern of said CpG site(s) in a reference sample,
wherein
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 in a subject with respect to the reference sample or
b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 in a subject with respect to the reference sample or
c) a combination of a) and b)
is indicative that the subject suffers from type 2 diabetes.
In another aspect, the present invention relates to a method of treatment of type 2 diabetes in a subject comprising the administration to said subject of an antidiabetic agent wherein said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
In another aspect, the present invention relates to the use of an antidiabetic agent for the manufacture of a medicament for the treatment of type 2 diabetes of a subject, wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of
Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
In another aspect, the present invention relates to a method of treatment of type 2 diabetes of a subject comprising administering an antidiabetic agent, wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
In another aspect, the present invention relates to a kit comprising at least one oligonucleotide or polynucleotide capable of hybridizing to at least one sequence of a CpG site of the KCNJll gene promoter selected from those CpG site(s) as defined in Table 1 and 2 in a methylation-specific manner.
In another aspect, the present invention relates to a nucleic acid comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene promoter and wherein the contiguous base sequence comprises at least one CpG site selected from those CpG sites as defined in Tables 1 and 2, a nucleic acid comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene promoter wherein the position corresponding to the C within the at least one CpG site is a uracile or a polynucleotide which specifically hybridizes to any of said nucleic acids.
In another aspect, the present invention relates to the use of the kit of the invention or of the nucleic acid of the invention for diagnosing type 2 diabetes in a subject.
LEGENDS TO THE FIGURES
Figure 1. ROC curve of type 2 diabetes status according to DNA methylation of CpGs 14-20 in the KCNJll promoter in peripheral blood DNA.
Figure 2. Box plot of methylation values of CpGs 11-28 in the KCNJll promoter in lean controls, overweight controls, subjects with prediabetes (prediabetics), type 1 diabetic (T1DM) and type 2 diabetic (T2DM) patients, in peripheral blood DNA. DETAILED DESCRIPTION OF THE INVENTION
The authors of the present invention have developed a method for diagnosing type 2 diabetes in a patient based in the determination of the DNA methylation pattern in the promoter region of the KCNJll gene in a sample of said patient followed by the comparison of said methylation pattern with the methylation pattern from a reference sample. This method has the advantage that it is simpler and less invasive because it can
be carried out in peripheral blood rather than in a biopsy, and it is independent of the fasting status of the patient. Furthermore, this method is more specific than the tests based on plasma glucose or glycated blood, and is more comfortable for the patient than an oral glucose tolerance test.
The authors of the present invention have identified a specific methylation pattern related to type 2 diabetes in the KCNJ11 gene promoter in peripheral blood. Specifically, 25 CpG sites out of 27 analyzed presented different methylation values (p<0.0001) between type 2 diabetic patients and overweight controls. In addition, the study of lean controls, prediabetic subjects and type 1 diabetic patients revealed that the KCNJ11 gene promoter methylation pattern found in type 2 diabetic patients was unique to type 2 diabetes. Thus, this differentially methylated pattern could be used as a potential biomarker of type 2 diabetes. Consequently, the method developed by the authors of the present invention allows a differential diagnosis between type 2 diabetes and type 1 diabetes or prediabetes.
Method for diagnosing type 2 diabetes
In a first aspect, the invention relates to a method for diagnosing type 2 diabetes in a subject (hereinafter referred to as the "diagnostic method of the invention"), comprising:
(i) determining the methylation pattern in one or more CpG site(s) of the KCNJ11 gene promoter selected from those CpG sites as defined in Tables 1 and 2 in a biological sample of said subject containing genetic material, and
(ii) comparing the methylation pattern of said one or more CpG site(s) obtained in step (i) with the methylation pattern of said CpG site(s) in a reference sample,
wherein
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 in a subject with respect to the reference sample or
b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 in a subject with respect to the reference sample or
c) a combination of a) and b)
is indicative that the subject suffers from type 2 diabetes.
The term "determining the diagnosis", as used herein, refers both to the process of attempting to determine and/or identify a possible disease in a subject, i.e. the diagnostic procedure, and to the opinion reached by this process, i.e. the diagnostic opinion. As such, it can also be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. As will be understood by those skilled in the art, the diagnosis of type 2 diabetes, although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as suffering from type 2 diabetes. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p- values are, preferably, 0.05, 0.01, 0.005 or lower.
The term "type 2 diabetes" or "type 2 diabetes mellitus" or "noninsulin- dependent diabetes mellitus (NIDDM)" or "adult-onset diabetes", as used herein, refers to relates to a disease characterized by an inappropriate increase in blood glucose levels, which generates chronic complications as it affects large and small vessels and nerves. The underlying disorder in this disease is the difficulty for insulin action (in the form of a loss of tissue sensitivity to this hormone), which is called insulin resistance, and an inadequate secretion of insulin by the beta cells responsible for their production, the beta cells, in the pancreas. In addition to increasing glucose concentration, faulty insulin action frequently translates into an increase in cholesterol and/or triglyceride levels.
The actual criteria for diagnosis of type 2 diabetes are:
AIC > 6.5%. The test should be performed in a laboratory using a method that is NGSP certified and standardized to the DCCT assay; or
fasting plasma glucose (FPG) > 126 mg/dL (7.0 mmol/L). Fasting is defined as no caloric intake for at least 8 h; or
2 h plasma glucose (PG) > 200mg/dL (l l . lmmol/L) during an oral glucose tolerance test (OGTT). The test should be performed as described by the World Health Organization, using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water; or
- in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose > 200 mg/dL (11.1 mmol/L)
In the absence of unequivocal hyperglycemia according to the first three criteria, results should be confirmed by repeat testing.
However, in practice, a large portion of the diabetic population remains unaware of their condition. On another hand, since there is preanalytic and analytic variability of all the tests, it is also possible that when a test whose result was above the diagnostic threshold is repeated, the second value will be below the diagnostic cut point. This is least likely for Ale (hemoglobin Ale), somewhat more likely for FPG, and most likely for the 2 h plasma glucose. Barring a laboratory error, such patients are likely to have test results near the margins of the threshold for a diagnosis and remain without a definite diagnostic.
In a preferred embodiment, the type 2 diabetes is gestational diabetes. This is a type of type 2 diabetes which may first be diagnosed during pregnancy as gestational diabetes. For this reason, women with a history of gestational diabetes have a greatly increased subsequently risk of having diabetes and should be followed up with subsequent screening for the development of diabetes.
The term "gestational diabetes" or "gestational diabetes mellitus" or "GDM", as used herein, refers to a condition wherein women without being previously diagnosed with diabetes have high levels of glucose in plasma during pregnancy. Formally, it is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. This definition recognizes the possibility that a woman may have previously undiagnosed diabetes type 1 or 2, or may have developed diabetes type 1 or 2 coincidentally with pregnancy. The fact that the symptoms subside after pregnancy is irrelevant to the diagnosis. A woman is diagnosed with gestational diabetes when glucose intolerance continues beyond 24-28 weeks of gestation. Generally, gestational diabetes shows few symptoms and is most often diagnosed by screening during pregnancy, where diagnostic tests detect high levels of glucose in plasma or serum. As
with diabetes mellitus during pregnancy, babies born to mothers with untreated gestational diabetes typically have a higher risk for problems such as an increased size for gestational age (which can lead to complications during delivery), low levels of sugar in blood and jaundice. If untreated, it can also cause seizures or stillbirth. Gestational diabetes is a treatable condition and women who have adequate control of glucose levels can effectively decrease these risks.
In a particular embodiment, the type 2 diabetes is gestational diabetes.
The term "subject", as used herein, refers to an individual, such as a human, a nonhuman primate (e.g. chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote a particular age or sex. In a particular embodiment of the invention, the subject is a mammal. In a preferred embodiment of the invention, the subject is a human. In another embodiment, the subject has not been treated prior to the determination with any antidiabetic drugs.
In a first step, the first method of the invention comprises the determination of the methylation pattern in one or more CpG site(s) of the KCNJ11 gene promoter selected from those CpG sites as defined in Tables 1 and 2 in a biological sample of said subject containing genetic material
The term "DNA methylation", as used herein, refers to a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides. DNA methylation at the 5 position of cytosine has the specific effect of reducing gene expression and has been found in every vertebrate examined. In adult non-gamete cells, DNA methylation typically occurs in a CpG site.
The term "CpG site", as used herein, refers to regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. "CpG" is shorthand for "C-phosphate-G", that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA. The "CpG" notation is used to distinguish this linear sequence from the CG base-pairing of cytosine and guanine. Cytosines in CpG dinucleotides can be methylated to form 5- methylcytosine.
The term "methylation pattern", as used herein, refers to, but is not limited to, the presence or absence of methylation of one or more nucleotides. Thereby said one or more nucleotides are comprised in a single nucleic acid molecule. Said one or more nucleotides have the ability of being methylated or being non-methylated. The term "methylation status" may also be used, wherein only a single nucleotide is considered. A methylation pattern can be quantified, wherein it is considered over more than one nucleic acid molecule.
The term "KCNJH" gene, as used herein, relates to the potassium inwardly- rectifying channel, subfamily J, member 1 1 gene, with accession number (ENST00000339994). The KCNJII gene may also be referred to as BIR, HHF2, PHHI, IKATP, TNDM3 or KIR6.2. KCNJII encodes the ATP-sensitive potassium channel subunit Kir 6.2 which together with the sulfonylurea receptor SUR1 forms an octameric complex that regulates glucose-stimulated insulin secretion in pancreatic β cells (Ridderstrale and Groop, 2009, Molecular and Cellular Endocrinology, 297: 10-7).
The term "promoter", as used herein, refers to a region of DNA that initiates transcription of a particular gene. Promoters are located near the genes they transcribe, on the same strand and upstream on the DNA, and can be about 100-1000 base pairs long. In the particular case of the KCNJII gene promoter, it starts at -2424 bp according to the ATG position for the KCNJII gene and ends at -1720 bp. As defined in the present invention, the promoter zone covers a 1,000 bp area upstream of the first exon of the human KCNJII gene.
The term "sample" or "biological sample", as used herein, refers to biological material isolated from a subject. The biological sample contains any biological material suitable for detecting the desired methylation pattern in one or more CpG site(s) and can comprise cell and/or non-cell material of the subject. In the present invention, the sample comprises genetic material, e.g., DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA, heterogeneous nuclear RNA (hnRNA), mRNA, etc., from the subject under study. In a particular embodiment, the genetic material is DNA. In a preferred embodiment the DNA is genomic DNA. In another preferred embodiment, the DNA is circulating DNA. The sample can be isolated from any suitable tissue or biological fluid such as, for example blood, saliva, plasma, serum, urine, cerebrospinal liquid (CSF), feces, a buccal or buccal-pharyngeal swab, a surgical specimen, a
specimen obtained from a biopsy, and a tissue sample embedded in paraffin. Methods for isolating cell and tissue samples are well known to those skilled in the art. In a particular embodiment, the sample is selected from the group consisting of blood, urine, saliva, serum, plasma, a buccal or buccal-pharyngeal swab, hair, a surgical specimen, and a specimen obtained from a biopsy. In preferred embodiment, the sample is selected from blood, serum, plasma, hair, urine and saliva.
The term "determination of the methylation pattern in a CpG site", as used herein, refers to the determination of the methylation status of a particular CpG site. The determination of the methylation pattern in a CpG site can be performed by means of multiple processes known by the person skilled in the art.
In some embodiments, for example, when the determination of the methylation pattern of a particular CpG site is carried out in a sample from whole blood, it may be used directly for the determination of said particular CpG site. In other embodiments, the nucleic acid is extracted from cells which are present in a biological fluid (e.g., whole blood, saliva, synovial fluid, etc.) as an initial step, and, in such cases, the total nucleic acid extracted from said samples would represent the working material suitable for subsequent analysis. Isolating the nucleic acid of the sample can be performed by standard methods known by the person skilled in the art. Said methods can be found, for example, in Sambrook et al, 2001, "Molecular cloning: a Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3 and in the commonly used QIAamp DNA mini kit protocol by Qiagen.
After isolating and amplifying (if necessary) the nucleic acid, the methylation pattern of one or more CpG site(s) is determined. Those skilled in the art will readily recognize that the analysis of the methylation pattern present in one or several of the CpG sites disclosed herein in a subject's nucleic acid can be done by any method or technique capable of measuring the methylation pattern present in a CpG site. For instance, one may detect SNPs in the first method of the invention by performing by a method selected from the group consisting of Methylation-Specific PCR (MSP), an enrichment-based method (e.g. MeDIP, MBD-seq and MethylCap), bisulfite sequencing and bisulfite-based method (e.g. RRBS, Infmium, GoldenGate, COBRA, MSP, MethyLight) and a restriction-digestion method (e.g., MRE-seq, or HELP assay), ChIP-
on-chip assay, or differential-conversion, differential restriction, differential weight of the DNA methylated CpG site(s).
When perfoming bisulfite sequencing and bisulfite-based method, the genomic DNA sample is chemically treated in such a way that all of the unmethylated cytosine bases are modified to uracil bases, or another base which is dissimilar to cytosine in terms of base pairing behaviour, while the 5-methylcytosine bases remain unchanged. The term "modify", as used herein, means the conversion of an unmethylated cytosine to another nucleotide which will distinguish the unmethylated from the methylated cytosine. The conversion of unmethylated, but not methylated, cytosine bases within the DNA sample is conducted with a converting agent. The term "converting agent" or "converting reagent", as used herein, relates to a reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. The converting agent is preferably a bisulfite such as disulfite or hydrogen sulfite. However, other agents that similarly modify unmethylated cytosine, but not methylated cytosine can also be used in the method of the invention, such as hydrogen sulfite. The reaction is performed according to standard procedures (Frommer et al, 1992, Proc Natl Acad Sci USA 89: 1827-31; Olek, 1996, Nucleic Acids Res 24:5064-6; EP 1394172). It is also possible to conduct the conversion enzymatically, e.g by use of methylation specific cytidine deaminases.
In a preferred embodiment of the diagnostic method of the invention, the sample has been treated with a reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In a more preferred embodiment, the agent used for modifying unmethylated cytosine is sodium bisulfite.
Once the DNA has been treated with a bisulfite, the region containing the one or more CpG site(s) is amplified using primers allowing to distinguish the unmethylated sequence (wherein the cytosine of CpG site is converted into uracil) of the methylated sequence (wherein the cytosine of the CpG site remains cytosine). Many amplification methods rely on an enzymatic chain reaction such as, for example, a polymerase chain reaction (PCR), Ligase Chain Reaction (LCR), Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, and NASBA. Further, there are strand displacement amplification (SDA), transcription mediated amplification (TMA), and
QP-amplification, etc.; this list is merely illustrative and in no way limiting. Methods for amplifying nucleic acid are described in Sambrook et al, 2001 (cited at supra). Particularly preferred amplification methods according to the invention are the methylation specific PCR method (MSP), disclosed in US 5, 786,146 which combines bisulfite treatment and allele-specific PCR (see e.g. US 5,137,806, US 5,595,890, US 5,639,611). Uracil is recognized as a thymine by Taq polymerase and therefore upon PCR, the resultant product contains cytosine only at the position where 5- methylcytosine occurs in the starting template DNA.
Details and particulars of the oligonucleotides or polynucleotides capable of specifically hybridizing to a bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter are described in the context of the kit of the invention, and are used with the same meaning in the context of the method of treatment according to the invention.
The amplification products are then detected according to standard procedures in the art. The amplified nucleic acid may be determined or detected by standard analytical methods known to the person skilled in the art and described e.g. in Sambrook et al, 2001 (cited at supra). There may be also further purification steps before the target nucleic acid is detected e.g. a precipitation step. The detection methods may include but are not limited to the binding or intercalating of specific dyes as ethidium bromide which intercalates into the double-stranded DNA and changes its fluorescence thereafter. The purified nucleic acids may also be separated by electrophoretic methods optionally after a restriction digest and visualized thereafter. There are also probe-based assays which exploit the oligonucleotide hybridization to specific sequences and subsequent detection of the hybrid. It is also possible to sequence the target nucleic acid after further steps known to the expert in the field. Other methods apply a diversity of nucleic acid sequences to a silicon chip to which specific probes are bound and yield a signal when a complementary sequences bind.
In a particularly preferred embodiment of the invention, the amplified nucleic acid is detected using the MassARRAY® platform (Sequenom, Inc.; San Diego, CA, USA) by a combination of RNA base-specific cleavage (MassCLEAVE™) and MALDI-TOF mass spectrometry. Base-specific cleavage generates a defined experimental mass signal pattern of the sample. For analysis, the experimental pattern is
subsequently compared to an in silico reference mass signal pattern derived from an individual or set of reference sequences. Differences between the expected and the observed mass signal pattern are interpreted and enable identification of sequence variations.
In another particular embodiment, the nucleic acid amplification is carried out by real time PCR and real time probes are used to detect the presence of the extension product. Several versions of real time probes are known, e.g. Lightcycler, Taqman, Scorpio, Sunrise, Molecular Beacon or Eclipse probes. Details concerning structure or detection of these probes are known in the state of the art.
Alternatively, the methylation pattern of the nucleic acid can be confirmed by restriction enzyme digestion and Southern blot analysis. Examples of methylation sensitive restriction endonucleases which can be used to detect CpG methylation include Smal, Sacll, Eagl, Mspl, Hpall, BstXJl and BssHll, for example.
In the following Tables 1 to 5, the CpG sites are defined using the distance in base pair from the CpG site to the transcription start site for the KCNJ11 gene.
Table 1 : List of CpG sites with specific differential hypomethylation in peripheral blood DNA samples.
CpG site Position (bp)
CpG 7 -2174
CpG 14 -1898
CpG 15 -1895
CpG 16 -1885
CpG 17 -1873
CpG 18 -1853
CpG 19 -1851
CpG 20 -1847
CpG 21 -1840
CpG 23 -1789
CpG 24 -1781
CpG 25 -1778
Table 2: List of CpG sites with specific differential hypermethylation in peripheral blood DNA samples
Table 3. List of CpG sites whose mean is hypomethylated in peripheral blood DNA samples of type 2 diabetic patients
CpG site Position (bp)
CpG 7 -2174
CpG 14 -1898
CpG 15 -1895
CpG 16 -1885
CpG 17 -1873
CpG 18 -1853
CpG 19 -1851
CpG 20 -1847
CpG 21 -1840
CpG 23 -1789
CpG 24 -1781
CpG 25 -1778
Table 4. List of CpG sites whose mean is hypermethylated in peripheral blood DNA samples of type 2 diabetic patients
Table 5. List of CpG sites whose mean is hypermethylated in peripheral blood DNA samples of type 2 diabetic patients. In a second step, the first method of the invention comprises the comparison of the methylation pattern of said one or more CpG site(s) obtained in step (i) with the methylation pattern of said CpG site(s) in a reference sample.
In this connection, the invention provides some specific CpG site(s) which are significantly associated with the diagnosis of type 2 diabetes in a subject. Thus,
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 in a subject with respect to the reference sample or
b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 in a subject with respect to the reference sample or
c) a combination of a) and b)
is indicative that the subject suffers from type 2 diabetes.
In a particular embodiment, the determination of the methylation pattern comprises the determination of the methylation pattern of all the CpG sites of Tables 1 and 2.
The term "reference sample", as used herein, means a sample obtained from a pool of healthy subjects which does not have a disease state or particular phenotype. For example, the reference sample may comprise tissue or blood (or the correspondent) samples from subjects which do not suffer type 2 diabetes or which do not have a history of type 2 diabetes.
In a particular embodiment of the invention, the reference sample is a sample of subjects matched on age and body mass index to the subject analysed. In a preferred embodiment, the subject has not received an antidiabetic treatment prior to obtaining the sample. In another example, the reference sample is a sample from a type 1 diabetic patients. In another embodiment, the reference sample is a sample from a prediabetic subjects,
In another particular embodiment of the diagnostic method of the invention, the determination of the methylation pattern comprises the determination of the methylation pattern of all CpG sites of Table 3, wherein if the mean level of methylation of said CpG sites of Table 3 is decreased in a subject with respect to the reference sample is indicative that the subject suffers from type 2 diabetes.
In another particular embodiment of the diagnostic method of the invention, the determination of the methylation pattern comprises:
a) the determination of the methylation pattern of all CpG sites of Table 4, b) the determination of the methylation pattern of all CpG sites of Table 5 or c) a combination of a) and b)
wherein if the mean level of methylation of said CpG sites of Table 4, Table 5 or the combination thereof is increased in a subject with respect to the reference sample is indicative that the subject suffers from type 2 diabetes.
According to the present invention, the level of methylation of one or more CpG site(s) is increased when the level of methylation of said one or more CpG site(s) in a sample is higher than in the reference sample. The level of methylation of one or more CpG site(s) is considered to be higher than in the reference sample when they are at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more higher than in the reference sample.
Likewise, in the context of the present invention, the level of methylation of one or more CpG site(s) is decreased when the level of methylation of said one or more CpG site(s) in a sample is lower than a reference value. The level of methylation of one or more CpG site(s) is considered to be lower than in the reference sample when it is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150% or more lower than in the reference sample.
The term "mean level of methylation", as used herein to refer to a genetic region, refers to the mean or average level of methylation in said region, which may be calculated as the sum of the methylation levels of all CpG sites within said region divided by the number of CpG sites in the region. In the particular case of the present embodiments, the mean level of methylation is calculated for all CpG sites of Table 3, Table 4 or Table 5.
According to the present invention, the mean level of methylation of all CpG sites of Table 3, Table 4 or Table 5 is increased when the mean level of methylation of said all CpG sites in a sample is higher than in the reference sample. The mean level of methylation of all CpG sites is considered to be higher than in the reference sample when it is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least
85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more higher than in the reference sample.
Likewise, in the context of the present invention, the mean level of methylation of all CpG sites of Table 3, Table 4 or Table 5 is decreased when the mean level of methylation of said all CpG sites in a sample is lower than a reference value. The mean level of methylation of one or more CpG site(s) is considered to be lower than in the reference sample when it is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more lower than in the reference sample.
Antidiabetic agent for use in the treatment of type 2 diabetes In another aspect, the present invention relates to an antidiabetic agent for use in the treatment of type 2 diabetes of a subject (hereinafter referred to as the "antidiabetic agent of the invention"), wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
In another aspect, the present invention relates to the use of an antidiabetic agent for the manufacture of a medicament for the treatment of type 2 diabetes of a subject, wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
In another aspect, the present invention relates to a method of treatment of type 2 diabetes of a subject comprising administering an antidiabetic agent, wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
In a particular embodiment of the method of treatment of the invention, said subject shows a decrease in the mean level of methylation of all CpG sites of Table 3 with respect to the reference sample.
In another particular embodiment, said subject shows an increase in the mean level of methylation of all CpG sites of Table 4 with respect to the reference sample, or an increase in the mean level of methylation of all CpG sites of Table 5 with respect to the reference sample, or a combination thereof.
In another particular embodiment, the type 2 diabetes is gestational diabetes.
The terms "subject", "type 2 diabetes", "gestational diabetes", "DNA methylation", "biological sample", "CpG site", and "CpG site", and their particulars have been described in detail in the context of the diagnostic method of the invention and are used with the same meaning in the context of the method of treatment according to the invention.
The person skilled in the art will understand that the diagnostic method of the invention can be applied herein in determining the level of methylation one or more CpG site(s) of the KCNJ11 gene promoter with respect to a reference sample.
The term "antidiabetic drug" or "antidiabetic agent", as used herein, refers to compounds that which show antiglycaemic activity or agents which sensitize insulin activity. These compounds include, without limitation, insulin and functionally equivalent variants thereof, secretagogues of insulin such as the sulfonylureas (tolbutamide, chlorpropamide, glipicide, glibenclamide, glicazide, glipentide, glimepiride, glibenclamide, glipizide, gliquidone, glisentide, glimepride and such like) and metiglinides (repaglinide, nateglinide, mitiglinide and such like), reducing agents of liver glucose production (biguanides and, in particular, metformin and buformin), agents which cause carbohydrate decrease such as a-glucosidase inhibitors (acarbose, miglitol or voglibose), agents that increase peripheral use of glucose such as thiazolidinediones (rosiglitazone, pioglitazone and such like), GLP- or a GLP-1 mimetic (Byetta-Exanatide, Liraglutinide, CJC-1131 (ConjuChem, Exanatide-LAR (Amylin), BIM-51077, ZP-10, the compounds described in WO 00/07617 and such like), exendin, secretin, DPP-IV inhibitors (sitagliptin, saxagliptin, denagliptin, vildagliptin, ALS-2- 0426, ARI- 2243, BI-A, BI-B, SYR-322, MP-513, DP-893, RO-0730699 and such like), SGLT-2 inhibitors (dapagliflozin, and sergliflozin, AVE2268, T- 1095 and such like) and peptides that cause an increase in glucose production (amlintide, pramlintide, exendin), compounds with GLP-1 activity (glucagon- like peptide 1), inhibitors of the protein tyrosine phosphatase IB, dipeptidyl peptidase inhibitors, secretagogues of insulin; fatty acid oxidation inhibitors, A2 antagonists, c-jun terminal kinase inhibitors, insulin; insulin mimetics, glycogen phosphorylase inhibitors, VPAC2 receptor agonists, glucokinase inhibitors, etc.).
"Functionally equivalent variant of insulin", as used in the present invention, is understood as all those polypeptides resulting from the elimination, insertion or modification of at least one amino acid with respect to the insulin sequence and which substantially maintains the same properties as the insulin it comes from. Insulin activity can be determined by methods widely known by those skilled in the art such as normoglycaemic clamping or the measurement of glycosylated proteins in serum (Bunn et al, Diabetes, 1981, 30:613-617). Functionally equivalent variants of insulin include, without limitation, the des-pentapeptide (B26-B30)-PheB25-a-carboxamide]insulin, AspB1° insulin (disclosed in US4992417), LysB28-ProB29 insulin LysB28-ProB29 and the hexameric variant thereof (disclosed in US5474978 and US5514646), formulations of insulin and protamine (US5650486), acylated LysB28-ProB29 insulin (US5922675), and compositions of stabilized insulin such as those disclosed in US5952297, US6034054 and US6211144, superactive analogs of insulin, monomeric insulins, hepatospecific insulins, insulin lispro (Humalog®), insulin lispro formulated with insulin lispro protamine (marketed as Humalog ®50/50™, Humalog® 75/25™), NPH insulin or insulin isophane human (marketed as Humulin®), regular insulin, NPH insulin combined with regular insulin (US5547929), insulin zinc, insulin glargine, glulisine (APidra), insulin Aspart (Novomix), insulin detemir (levemir), biota, LP- 100, novarapid, insulin zinc suspension (slow and ultraslow), GLP-I (1-36) amide, GLP-I (73-7) insulinotropin (disclosed in US5614492), LY-315902 (Lilly), GLP-I (7-36)- NH2), AL-401 (Autoimmune), and compositions such as those disclosed in US4579730, US4849405, US4963526, US5642868, US5763396, US5824638, US5843866, US6153632, US6191105, and WO 85/05029.
The term "treatment" or "therapy" includes any process, action, application, therapy, or the like, wherein a subject, including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject, or ameliorating at least one symptom of the disease or disorder under treatment. In the particular case of the subject of the present invention, a suitable treatment of type 2 is based on an anti-diabetic agent therapy. The terms "anti-diabetic agent" and its particulars have been described in detail in the context of the diagnostic method of the invention and are used with the same meaning in the context of the method of treatment according to the invention.
Type 2 diabetes is initially managed by increasing exercise and dietary modification in order to lower blood glucose levels. When this is not achieved, subjects suffering from type 2 diabetes are put on anti-diabetic treatment. The term "anti-diabetic treatment" or "treatment of type 2 diabetes", as used herein, refers to an anti-diabetic agent used to lower blood glucose levels.
Kits of the invention
In another aspect, the present invention relates to a kit (hereinafter referred to as the "kit of the invention"), comprising at least one oligonucleotide or polynucleotide capable of hybridizing in a methylation-specific manner to at least one sequence of a CpG site of the KCNJll gene promoter selected from those CpG site(s) as defined in Table 1 and 2.
The terms "CpG site", "KCNJll gene", and "promoter", and their particulars have been described in detail in the context of the diagnostic method of the invention and are used with the same meaning in the context of the kit according to the invention.
Suitable kits include various reagents for use in accordance with the present invention in suitable containers and packaging materials, including tubes, vials, and shrink-wrapped and blow-moulded packages. Additionally, the kits of the invention can contain instructions for the simultaneous, sequential or separate use of the different components which are in the kit. Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like. Additionally or alternatively, the media can contain Internet addresses that provide said instructions.
Materials suitable for inclusion in an exemplary kit in accordance with the present invention comprise one or more of the following: at least one oligonucleotide or polynucleotide capable of hybridizing to at least one sequence of a CpG site of the KCNJll gene promoter selected from those CpG site(s) as defined in Table 1 and 2 in a methylation-specific manner, reagents capable of amplifying a specific sequence domain in either genomic DNA or cDNA without the requirement of performing PCR; reagents required to discriminate between the various possible alleles in the sequence
domains amplified by PCR or non-PCR amplification (e.g., restriction endonucleases, oligonucleotide that anneal preferentially to methylated or to unmethylated CpG sites, including those modified to contain enzymes or fluorescent chemical groups that amplify the signal from the oligonucleotide and make discrimination of methylated or unmethylated CpG sites more robust); or reagents required to physically separate products derived from the various amplified regions (e.g. agarose or polyacrylamide and a buffer to be used in electrophoresis, HPLC columns, SSCP gels, formamide gels or a matrix support for MALDI-TOF).
The term "oligonucleotide", as used herein, refers to a short, single-stranded DNA or RNA molecule, with up to 13 bases in length. The oligonucleotides of the invention are preferably DNA molecules of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or 13 bases in length.
The term "polynucleotide", as used herein, refers to single-stranded DNA or RNA molecules, of more than 13 bases in length. The polynucleotides of the invention are preferably DNA molecules of at least 14, at least 15, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more bases in length.
As used in the kit of the invention, the at least one oligonucleotide or polynucleotide capable of hybridizing to at least one sequence of a CpG site of the KCNJ11 gene promoter selected from those CpG site(s) as defined in Table 1 and 2 in a methylation-specific manner are used as a primer to amplify the region containing said CpG site(s). Alternatively, oligonucleotide or polynucleotide can also be used as a probe for detecting said methylated or unmethylated CpG site(s).
The term "capable of hybridizing" or "capable of specifically hybridizing", as used herein, refers to the capacity of an oligonucleotide or polynucleotide of recognizing specifically the sequence of a CpG site. As used herein, "hybridization" is the process of combining two complementary single-stranded nucleic acid molecules, or molecules with a high degree of similarity, and allowing them to form a single double- stranded molecule through base pairing. Normally, the hybridization occurs under high stringent conditions or moderately stringent conditions.
As known in the art, the "similarity" between two nucleic acid molecules is determined by comparing the nucleotide sequence of one molecule to the nucleotide sequence of a second molecule. Variants according to the present invention include nucleotide sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar or identical to the sequence of the CpG site. The degree of identity between two nucleic acid molecules is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTN algorithm (BLAST Manual, Altschul et al, 1990, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol Biol 215:403-10).
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
The term "stringent conditions" or "high stringency conditions", as used herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Fico 11/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50%> formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1 %> SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2xSSC (sodium
chloride/sodium citrate) and 50% formamide, followed by a high- stringency wash consisting of O. lxSSC containing EDTA at 55 °C.
"Moderately stringent conditions" may be identified as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C. in a solution comprising: 20% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10%> dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in lxSSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
In a particular embodiment, the kit comprises a first oligonucleotide or polynucleotide capable of specifically hybridizing to a bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is methylated, and at least one oligonucleotide or polynucleotide capable of specifically hybridizing to the same bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is unmethylated.
The oligonucleotides or polynucleotides, i.e. primers, used in the invention for amplification of the CpG-containing nucleic acid in the sample, after bisulfite modification, specifically distinguish between untreated DNA, methylated and non- methylated DNA. For hybridization to an unmethylated CpG site, primers for the non- methylated DNA preferably have a T in the 3' CG pair to distinguish it from the C retained in methylated DNA, and the complement is designed for the antisense primer. Primers usually contain relatively few Cs or Gs in the sequence since the Cs will be absent in the sense primer and the Gs absent in the antisense primer (cytosine becomes modified to uracil, which is amplified as thymidine in the amplification product). Accordingly, for hybridization to a methylated CpG site, primers for the methylated DNA preferably have a C in the 3' CG pair.
In a preferred embodiment, the kit comprises oligonucleotides or polynucleotides capable of hybridizing to all CpG sites of the KCNJl l gene promoter as defined in Table 1 and 2 in a methylation-specific manner.
In another preferred embodiment, the kit comprises at least one oligonucleotide or polynucleotide selected from the group consisting of sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
In another particular embodiment, the kit of the invention further comprises one or more reagents for converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In a preferred embodiment, the one or more reagents capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties is a bisulfite, preferably sodium bisulfite. In another embodiment, the reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties is metabisulfite, preferably sodium metabisulfite.
The term "converting reagent" and its particulars have been described in detail in the context of the diagnostic method of the invention and are used with the same meaning in the context of the kit according to the invention.
Polynucleotides of the invention
In another aspect, the present invention relates to a nucleic acid (hereinafter referred to as the "first polynucleotide of the invention") comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene and wherein the contiguous base sequence comprises at least one CpG site selected from those CpG sites as defined in Tables 1 and 2.
In a particular embodiment, the first polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJll gene.
In another aspect, the present invention relates to a nucleic acid (hereinafter referred to as the "second polynucleotide of the invention"), comprising at least 9 contiguous nucleotides of the promoter region of the KCNJ11 gene promoter wherein the position corresponding to the C within the at least one CpG site selected from those CpG sites as defined in Tables 1 and 2 is a uracile.
In a particular embodiment, the second polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJ11 gene.
In another aspect, the present invention relates to a nucleic acid (hereinafter referred to as the "third polynucleotide of the invention") comprising a polynucleotide which specifically hybridizes to any of the first and second nucleic acids of the invention. In a particular embodiment, the third polynucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more nucleotides.
The terms and particulars of the nucleic acids of the invention have been described in detail in the context of the methods and kits of the invention and are used with the same meaning in the context of the uses according to the invention. Diagnostic uses of the invention
The person skilled in the art will understand that the kits and nucleic acids of the invention are particularly useful in the diagnosis of type 2 diabetes in a subject. Thus, in another aspect, the present invention relates to the use of the kit of the invention (hereinafter referred to as the "first use of the invention") for diagnosing type 2 diabetes in a subject.
In a particular embodiment, the type 2 diabetes is gestational diabetes.
In another particular embodiment, the kit comprises a first oligonucleotide or polynucleotide capable of specifically hybridizing to a bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is methylated, and at least one oligonucleotide or polynucleotide capable of specifically hybridizing to the same bisulfite-treated polynucleotide comprising at least
one sequence of a CpG site of the KCNJll gene promoter when said CpG site is unmethylated.
In a preferred embodiment, the kit comprises oligonucleotides or polynucleotides capable of hybridizing to all CpG sites of the KCNJl l gene promoter as defined in Table 1 and 2 in a methylation-specific manner.
In another preferred embodiment, the kit comprises at least one oligonucleotide or polynucleotide selected from the group consisting of sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
In another particular embodiment, the kit of the invention further comprises one or more reagents for converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In a preferred embodiment, the one or more reagents for converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties is a bisulfite, preferably sodium bisulfite.
In another aspect, the present invention relates to the use of the first polynucleotide of the invention (hereinafter referred to as the "second use of the invention") for diagnosing type 2 diabetes in a subject.
In a particular embodiment, the type 2 diabetes is gestational diabetes.
In another particular embodiment, the first nucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJll gene.
In another aspect, the present invention relates to the use of the second polynucleotide of the invention (hereinafter referred to as the "third use of the invention"), for diagnosing type 2 diabetes in a subject.
In a particular embodiment, the type 2 diabetes is gestational diabetes.
In another particular embodiment, the second nucleotide of the invention comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30 or more contiguous nucleotides of the promoter region of the KCNJll gene.
In another aspect, the present invention relates to the use of the third polynucleotide of the invention (hereinafter referred to as the "fourth use of the invention"), for diagnosing type 2 diabetes in a subject.
In a particular embodiment, the type 2 diabetes is gestational diabetes.
The terms and particulars of the first, second, third and fourth uses of the invention have been described in detail in the context of the methods, kits and nucleotides of the invention and are used with the same meaning in the context of the uses according to the invention.
The invention is described by way of the following examples which ;
construed as merely illustrative and not limitative of the scope of the invention.
EXAMPLE 1 MATERIALS AND METHODS
Study design and subjects included
We conducted a case-control study to identify DNA methylation differences in KCNJ11 promoter from peripheral blood DNA. Cases (n = 93) were defined as patients suffering from type 2 diabetes without any pharmacological treatment that were treated only by diet. All cases were recruited from the same primary health care center. After recruitment, randomly selected controls from the Biobank Hospital Clinic-IDIBAPS; Barcelona, Spain (http://www.clinicbiobanc.org/en_index.html) were chosen. Controls (n = 93) were matched on age and BMI to cases to control for factors (such as age and BMI) that could possibly influence changes in DNA methylation patterns. For each case, one control was sought. None of the controls suffered from type 2 diabetes or prediabetes. Diagnosis of type 2 diabetes and prediabetes was done following ADA recommendations (Standards of Medical Care in Diabetes—2012. Diabetes Care 2011; 35(Supplement_l):Sl l-S63). DNA methylation of KCNJ11 in peripheral blood DNA was studied for all subjects (93 cases and 93 overweight controls). The methylation
profile of KCNJl 1 promoter was later studied in lean controls (n = 15), prediabetic subjects (n = 40), also matched on age and BMI to cases, and type 1 diabetic patients (n = 21), who were also selected from the Biobank. This study was approved by the ethics committees of the Hospital Clinic and the Biobank, and complies with all laws and international ethics guidelines outlined in the Declaration of Helsinki. All human subjects provided written, informed consent.
DNA methylation analysis
Sequenom's MassARRAY platform was used to perform quantitative DNA methylation analysis (Ehrich M et al. 2005. Proc Natl Acad Sci U S A, 102: 15785- 15790). This system utilizes MALDI-TOF mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). A detectable pattern is then analyzed for methylation status. PCR primers for the amplification of the promoters of KCNJl 1 gene were designed using Epidesigner (Sequenom). When possible, amplicons were designed to cover CpG islands in the same region as the 5' UTR. For each reverse primer, an additional T7 promoter tag for in vivo transcription with an 8 bp insert (for prevention of abortive cycling and constant 5' fragment for RNAseA reaction) was added, as well as a 10-mer tag on the forward primer to adjust for melting-temperature differences. The primers used appear in Table 6, wherein capital letters indicate the actual sequence for the primers.
Table 6. Primers used in this study.
PCR reactions were carried out in 5 μΐ total volume with 10 ng/ml bisulfite- treated DNA, 0.2 units TaqONA polymerase (Sequenom), lx supplied Taq buffer, and 200 mM PCR primers. Amplification was done as follows: preactivation of 95°C for 15 min, 45 cycles consisting of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s, finishing with a 72°C incubation for 4 min. Dephosphorylation of unincorporated dNTPs was performed by adding 1.7 ml of H20 and 0.3 units of shrimp alkaline phosphatase (Sequenom), incubating at 37°C for 40 min and then at 85° C for 10 min, to deactivate the enzyme. The MassCLEAVE biochemistry was performed as follows: in vivo transcription and RNA cleavage was achieved by adding 2 μΐ of PCR product to 5 μΐ of transcription/cleavage reaction and incubating at 37°C for 3 h. The transcription/cleavage reaction contains 27 units of T7 RNA&DNA polymerase (Sequenom), 0.64x of T7 R&DNA polymerase buffer, 0.22 μΐ T Cleavage Mix (Sequenom), 3.14 mM DTT, 3.21 μΐ H20, and 0.09 mg/ml RNaseA (Sequenom). The reactions were additionally diluted with 20 ml H20 and conditioned with 6 mg of CLEAN Resin (Sequenom) for optimal mass-spectra analysis.
Hormone and cytokine measurements
Adiponectin, leptin and insulin were quantified from serum samples by ELISA (Mercodia), according to the manufacturer's instructions. Cytokines were measured from serum samples using CBA Human Inflammatory Cytokines kit (BD Bioscience), following the manufacturer's instructions. Two-color flow cytometric analysis was performed using LSRFortessa (BD bioscience). Data were acquired and analyzed using FACS Diva and FCAP Array 1.01 Softwares.
Statistical analysis
Descriptive data are presented as the mean and standard deviation (SD) for continuous outcomes, or number and percentage (%) for categorical outcomes. The methylation values (in %), cytokines, HOMA-IR, insulin, leptin and adiponectin were compared using non-parametric Mann- Whitney U test because normality and equality of variance could not be assumed. Student's t test was used for the comparison of the rest of continuous outcomes and Chi-square test for categorical outcomes. Correlation
between DNA methylation values and covariates that were significantly different between type 2 diabetic patients and overweight controls (fasting glucose, HOMA-IR, fasting adiponectin, total cholesterol, IL-10 and IL-12) was assessed by Spearman's rank correlation coefficient. Adjustment by disease status (i.e, being case or control) was done. Bonferroni correction was used for multiple comparisons. All significance tests were 2-tailed and values of p < 0.05 were considered significant. All analyses were conducted using the statistical software package Stata version 11.
RESULTS
Clinical characteristics of the type 2 diabetic patients and age- and BMI-matched controls
The study of peripheral blood DNA methylation pattern for KCNJ11 promoter was done in 186 subjects, half of them affected by type 2 diabetes (cases). Their baseline characteristics are summarized in Table 7. The majority of all the patients (65%) were overweight, 31% were obese and 4% (only cases) presented a normal weight. Mean age of all patients was 68 years and there were no significant differences in gender (67% were men in the group of cases vs 54% in the group of controls). Total cholesterol and fasting adiponectin were lower in cases as compared to controls (total cholesterol mean values of 4.77 mmol/L vs 5.25 mmol/L, and fasting adiponectin mean values of 7.0 μg/mL vs 10.0 μg/mL, p<0.0001). Epidemiological studies have shown that higher adiponectin levels in serum are associated with a lower risk of type 2 diabetes (Fagerberg B., et al. 2011. J Intern Med, 269:636-643). HOMA-IR was higher in cases (2.6 vs 1.8 in controls, p=0.0001). From the cytokines analyzed, significant differences were found for IL 10 (4.1±3.0 pg/mL in cases vs 5.2±3.7 pg/mL in controls, p=0.04) and IL 12 (52.8±58.3 pg/mL in cases vs 29.7±37.4 pg/mL in controls, p<0.0001).
Lower levels of the anti- inflammatory IL 10 were found in type 2 diabetic patients, which is consistent with previous research that showed that low levels of IL 10 are associated with type 2 diabetes (Bluher M., et al. 2005. Exp Clin Endocrinol Diabetes, 113:534-537; Charles BA., et al. 2011. J Clin Endocrinol Metab, 96:E2018- 2022).
IL-12 serum levels were higher in type 2 diabetic patients than in controls. A recent study showed that elevated serum IL-12 was present at the onset of type 2 diabetes, and that further increases in IL-12 correlated with endothelial dysfunction and cardiovascular disease progression (Mishra M., et al. 2011. Diabetes Res Clin Pract, 94:255-261).
Type 2 diabetic patients were in optimal glycemic control (mean glycated hemoglobin 5.8%).
Variable Type 2 Overweight P Value
diabetic Controls
patients (n=93)
(n=93)
Demographic characteristics
Age, yr 69.1±9.2 66.6±11.7 Matching
variable
BMI, kg/m2 29.2±3.7 28.8±2.5 Matching
variable
Male sex, (%) 66.7 53.8 0.07
Duration of diabetes, yr 6.3±5.9
Laboratory values
Fasting glucose, (mmol/L) 6.36±1.17 4.59±0.35 0.0001
Glycated hemoglobin, (%) 5.8±0.6
Fasting insulin , (pmol/L) 55.61±28.59 52.43±21.05 0.39
HOMA-IR 2.6±1.5 1.8±0.7 0.0001
Fasting leptin, (ng/mL) 18.0±16.7 25.4±26.8 0.07
Fasting adiponectin, ^g/mL) 7.0±3.8 10.0±4.2 O.OOOl
Alanine aminotransferase (ALT), 13.5±7.9 14.6±7.3 0.49
(IU/liter)
Aspartate aminotransferase 16.6±8.2 19.0±6.0 0.14
(AST), (IU/liter)
Total cholesterol (mmol/L) 4.77±1.03 5.25±1.08 0.002
LDL cholesterol (mmol/L) 2.84±0.85 2.87±0.76 0.78
HDL cholesterol (mmol/L) 1.31±0.33 1.36±0.35 0.26
Triglycerides (mmol/L) 1.43±0.89 1.31±0.75 0.34
Table 7. Demographic and clinical characteristics of type 2 diabetic patients and age- and BMI-matched controls. Values shown are means ±SD, unless otherwise indicated. P values were calculated with the t test for quantitative variables or Chi-
square test for categorical ones, except for HOMA-IR, fasting insulin, fasting leptin and fasting adiponectin, where non-parametric Mann- Whitney U test was applied. HOMA-IR was calculated as [Insulin mUI/1 x Glycemia: (mmol/l)/22.5]. Quantitative DNA Methylation analysis in peripheral blood identifies a full distinct pattern in the KCNJ11 promoter in type 2 diabetic patients as compared to age- and BMI-matched controls.
Methylation levels in DNA from whole blood of 186 subjects were obtained for 27 CpG sites covering 1,000 bp upstream of the first exon of the human KCNJ11 gene. The heat map showing the values of methylation (%) for each CpG site analyzed revealed a unique and compelling differentiated pattern of methylation between type 2 diabetic patients and controls in the KCNJ11 promoter (data not shown). Strikingly, 25 CpG sites out of 27 analyzed (93%) presented significantly different methylation values (p<0.0001) amongst cases and controls (Table 8).
Table 8. Peripheral blood DNA methylation values (in %) for each CpG site analyzed in the KCNJll promoter in type 2 diabetic patients and age- and BMI-matched controls. Values are means ± SD. P values were calculated using the Mann- Whitney U test. Statistical significance was set at p<(0.05/27=0.0018) using Bonferroni correction. *** denotes P value less than 0.0001.† CpG dinucleotides have been numbered relative to ATG. % CpG dinucleotide position has been determined according to the ATG position for the KCNJll gene (ENST00000339994).
The first 13 CpGs were hypomethylated in type 2 diabetic patients (methylation average 4.0 %, 95% CI 3.8-4.2) as compared to controls (methylation average 26.0%, 95% CI 25.0-27.1, p<0.0001). Furthermore, there was a complete degree of methylation (100%) in the area comprised between -1898 to -1847 bp upstream the transcription start site (TSS) position for the KCNJll gene in all type 2 diabetic patients. The area comprised between -1781 and -1778 upstream the TSS was also hypermethylated in all type 2 diabetic patients (mean methylation of 94.5% in cases vs 11.0 % in controls).
To validate the results, the DNA methylation analysis of the KCNJll promoter were repeated in a subset of 12 different age- and BMI-matched controls, and the results were similar to those previously found in controls (data not shown).
Thus, we determined that DNA methylation patterns in the promoter studied using peripheral blood were different in type 2 diabetic patients as compared to controls, and those differences were of special relevance in the case of the KCNJll promoter. Indeed, the Area Under Curve of distinguishing a type 2 diabetic patient from an age- and BMI-matched control was equal to 100% if the methylation values of CpGs 14 to 20 were equal to or higher than 95.7% (Figure 1).
Spearman correlation coefficients were calculated using the DNA methylation values of the CpG islands from the promoter and factors significantly different between type 2 diabetic patients and controls (Table 9). Fasting glucose, fasting adiponectin, total cholesterol and IL 12 serum levels were significantly associated with methylation values in the KCNJll promoter. When adjusting for disease status (i.e, being case or control), the unique factors that were significantly associated with methylation values in the KCNJll promoter were adiponectin in controls (r=0.23, p=0.03, in CpGs 1-10) and IL 12 in cases (r=0.21, p=0.049, in CpGs 11-28) (data not shown).
Table 9. Spearman correlations between DNA methylation and laboratory values from type 2 diabetic patients and age- and BMI-matched controls.
Quantitative DNA Methylation analysis of the KCNJ11 promoter in peripheral blood of lean controls, subjects with prediabetes and type 1 diabetic patients.
Next, we focused on the study of the KNCJ11 promoter in different subgroups of subjects in order to detect how and in which direction (hypo or hypermethylation) the methylation pattern may vary in relation to the phenotype. We studied lean controls over 50 years of age, in order to determine the methylation pattern in healthy weight controls. We also investigated prediabetic individuals matched for BMI and age with the type 2 diabetic patients, to see whether the type 2 diabetic-associated methylation pattern appeared gradually with diabetic status. Finally, we examined type 1 diabetic patients with more than 5 years of evolution to discern whether the epigenetic mark found depended on a chronic effect of hyperglycemia. Their clinical characteristics are shown in Tables 10, 11 and 12.
Prediabetic Fasting glucose 2 hour plasma BMI Age Male
subjects (mmol/L) glucose in the gender
(N= 40) 75 gr- OGTT (%)
(mmol/L)
IFG 6.36 (0.23) 6.84 (1.06) 29.5 (0.93) 64.9 (4.36) 60%
n= 10
IFG +IGT 5.98 (0.35) 9.12 (1.02) 28.9 (0.93) 67.1 (5.80) 87 %
n= 15
IGT 4.93 (0.37) 8.82 (0.61) 29.6 (1.29) 67.8 (5.42) 60 %
n=15
Table 10. Clinical characteristics of subjects with prediabetes. IFG denotes impaired fasting glucose. IGT denotes impaired glucose tolerance. Values are expressed as mean (SD), unless otherwise indicated.
Table 11. Clinical characteristics of type 1 diabetic patients. Values are
Table 12. Clinical characteristics of lean controls. Values are expressed as mean (SD), unless otherwise indicated.
Briefly, prediabetic subjects were 66.8 years old on average and had a mean BMI of 29.3, a mean fasting glucose of 5.68 mmol/L and mean glucose post OGTT (oral glucose tolerance test) of 8.44 mmol/L. Type 1 diabetic patients had a mean fasting glucose of 10.99 mmol/L and a mean glycated hemoglobin of 9.4%. Surprisingly, the pattern found in type 2 diabetic patients is unique to type 2 diabetic patients and does not appear gradually with diabetic status since it is absent in prediabetic subjects. Indeed, prediabetic subjects, lean controls, overweight controls and type 1 diabetic patients all have a similar methylation pattern, especially in CpGs 11-28 (Figure 2). Type 2 diabetic patients, however, presented a hypomethylation of CpGs 1- 10 and a hypermethylation of GpGs 14-20 and CpGs 24-25. This pattern does not appear to be secondary to changes in weight or age. Moreover, a chronic effect of alteration in glucose homeostasis is not responsible for this pattern since type 1 diabetic patients did not present it, either.
CONCLUSIONS - The authors have identified a methylation pattern specific to patients with type 2 diabetes in DNA taken from peripheral blood. Said methylation pattern was present in all type 2 diabetic patients and was not present in type 1 diabetic patients, prediabetic subjects,
Genetic background was not a determinant, either, since all patients studied were unrelated. Family history of diabetes was present in 49% of type 2 diabetic patients and in 31% of their matched controls, but that did not have an impact on the methylation differences found.
None of the type 2 diabetic patients were on any pharmacological therapy for diabetes. Thus, no confounding effect of antidiabetic drugs or insulin therapy was possible.
Claims
A method for diagnosing type 2 diabetes in a subject comprising:
(i) determining the methylation pattern in one or more CpG site(s) of the KCNJ11 gene promoter selected from those CpG sites as defined in Tables 1 and 2 in a biological sample of said subject containing genetic material, and
(ii) comparing the methylation pattern of said one or more CpG site(s) obtained in step (i) with the methylation pattern of said CpG site(s) in a reference sample,
wherein
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 in a subject with respect to the reference sample or
b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 in a subject with respect to the reference sample or
c) a combination of a) and b)
is indicative that the subject suffers from type 2 diabetes.
The method according to claim 1 wherein the determination of the methylation pattern comprises the determination of the methylation pattern of all CpG sites of Table 3, wherein a decrease in the mean level of methylation of said CpG sites of Table 3 in a subject with respect to the reference sample is indicative that the subject suffers from type 2 diabetes.
The method according to any of claims 1 or 2 wherein the determination of the methylation pattern comprises:
a) the determination of the methylation pattern of all CpG sites of Table 4,
b) the determination of the methylation pattern of all CpG sites of Table 5 or
c) a combination of a) and b).
and wherein if the mean level of methylation of said CpG sites of Table 4, of Table 5 or the combination thereof is increased in a subject with respect to the reference sample is indicative that the subject suffers from type 2 diabetes.
4. The method according to claim 1 wherein the determination of the methylation pattern comprises the determination of the methylation pattern of all the CpG sites of Tables 1 and 2.
5. The method according to any of claims 1 to 4, wherein the methylation pattern is measured by a method selected from the group consisting of Methylation-Specific PCR (MSP), an enrichment-based method (e.g. MeDIP, MBD-seq and MethylCap), bisulfite sequencing and bisulfite-based method (e.g. RRBS, Infinium, GoldenGate, COBRA, MSP, MethyLight) and a restriction-digestion method (e.g., MRE-seq), or differential-conversion, differential restriction, differential weight of the DNA methylated CpG site(s) of the KCNJ11 gene promoter in a biological sample containing genetic material as compared to the reference sample.
6. The method according to any of claims 1 to 5 wherein the reference sample is a sample of subjects matched on age and body mass index to the subject analysed.
7. The method according to any of claims 1 to 6 wherein the subject has not received an antidiabetic treatment prior to obtaining the sample.
8. The method according to any of claims 1 to 7 wherein the biological sample containing genetic material is peripheral blood, plasma or serum.
9. The method according to any of claims 1 to 8 wherein the sample has been treated with a reagent capable of converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
The method according to any of claims 1 to 9 wherein the type 2 diabetes is gestational diabetes.
An antidiabetic agent for use in the treatment of type 2 diabetes of a subject wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
Use of an antidiabetic agent for the manufacture of a medicament for the treatment of type 2 diabetes of a subject, wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
A method of treatment of type 2 diabetes of a subject comprising administering an antidiabetic agent, wherein a biological sample from said subject shows
a) a decrease in the level of methylation of one or more CpG site(s) selected from Table 1 with respect to the reference sample, b) an increase in the level of methylation of one or more CpG site(s) selected from Table 2 with respect to the reference sample, c) a decrease in the mean level of methylation of the CpG sites of Table 3,
d) an increase in the mean level of methylation of said CpG sites of Table 4,
e) an increase in the mean level of methylation of said CpG sites of Table 5 or
f) a combination of any one of a), b), c), d) and e).
Antidiabetic agent according to claim 11, use of an antidiabetic agent according to claim 12, or method of treatment according to claim 13, wherein the type 2 diabetes is gestational diabetes.
A kit comprising at least one oligonucleotide or polynucleotide capable of hybridizing to at least one sequence of a CpG site of the KCNJ11 gene promoter selected from those CpG site(s) as defined in Table 1 and 2 in a methylation- specific manner.
The kit according to claim 15 comprising a first oligonucleotide or polynucleotide capable of specifically hybridizing to a bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is methylated, and at least one oligonucleotide or polynucleotide capable of specifically hybridizing to the same bisulfite-treated polynucleotide comprising at least one sequence of a CpG site of the KCNJ11 gene promoter when said CpG site is unmethylated.
The kit according to claims 15 or 16 which comprises oligonucleotides or polynucleotides capable of hybridizing to at all CpG sites of the KCNJll gene promoter as defined in Table 1 and 2 in a methylation-specific manner.
The kit according to any of claims 15 to 17 further comprising one or more reagents for converting an unmethylated cytosine to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
A nucleic acid selected from the group consisting of
(i) A nucleic acid comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene and wherein the contiguous base sequence comprises at least one CpG site selected from those CpG sites as defined in Tables 1 and 2,
(ii) a nucleic acid comprising at least 9 contiguous nucleotides of the promoter region of the KCNJll gene promoter wherein the position corresponding to the C within the at least one CpG site selected from those CpG sites as defined in Tables 1 and 2 is a uracil and
(iii) a polynucleotide which specifically hybridizes to any of said nucleic acids.
Use of a kit according to any of claims 15 to 18 or of a nucleic acid according claim 19 for diagnosing type 2 diabetes in a subject.
21. Use according to claim 20 wherein the type 2 diabetes is gestational diabetes.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017003166A1 (en) * | 2015-06-29 | 2017-01-05 | 고려대학교 산학협력단 | Composition for early diagnosis of diabetes using metabolome analysis |
KR101995835B1 (en) * | 2018-09-20 | 2019-07-08 | 대한민국 | Composition and method for diagnosing type 2 diabetes using ELOVL fatty acid elongase 5 gene |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6200756B1 (en) * | 1996-06-03 | 2001-03-13 | The Johns Hopkins University School Of Medicine | Methods for identifying methylation patterns in a CpG-containing nucleic acid |
WO2002018632A2 (en) * | 2000-09-01 | 2002-03-07 | Epigenomics Ag | Method for determining the degree of methylation of defined cytosines in genomic dna in the sequence context 5'-cpg-3' |
EP2097538A2 (en) * | 2006-12-07 | 2009-09-09 | Switchgear Genomics | Transcriptional regulatory elements of biological pathways, tools, and methods |
WO2012097903A1 (en) * | 2011-01-20 | 2012-07-26 | Université Libre de Bruxelles | Methylation patterns of type 2 diabetes patients |
-
2013
- 2013-06-03 ES ES201330814A patent/ES2524194B1/en not_active Withdrawn - After Issue
-
2014
- 2014-06-03 WO PCT/EP2014/061470 patent/WO2014195306A1/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6200756B1 (en) * | 1996-06-03 | 2001-03-13 | The Johns Hopkins University School Of Medicine | Methods for identifying methylation patterns in a CpG-containing nucleic acid |
WO2002018632A2 (en) * | 2000-09-01 | 2002-03-07 | Epigenomics Ag | Method for determining the degree of methylation of defined cytosines in genomic dna in the sequence context 5'-cpg-3' |
EP2097538A2 (en) * | 2006-12-07 | 2009-09-09 | Switchgear Genomics | Transcriptional regulatory elements of biological pathways, tools, and methods |
WO2012097903A1 (en) * | 2011-01-20 | 2012-07-26 | Université Libre de Bruxelles | Methylation patterns of type 2 diabetes patients |
Non-Patent Citations (16)
Title |
---|
BJÖRN ÞÓR AÐALSTEINSSON: "Investigation of DNA methylation in type 2 diabetes genetic risk loci.", 1 April 2012 (2012-04-01), pages Frontpage - 68, 29 pps Supplement., XP055133892, Retrieved from the Internet <URL:http://skemman.is/stream/get/1946/11873/29967/1/Final.pdf> [retrieved on 20140808] * |
DATABASE EMBL [online] 18 June 2002 (2002-06-18), "Homo sapiens chromosome 11, clone RP1-239B22, complete sequence.", XP002728379, retrieved from EBI Database accession no. AC124798 * |
DATABASE EMBL [online] 5 April 2011 (2011-04-05), "Sequence 5512 from Patent EP2097538.", XP002728378, retrieved from EBI Database accession no. JA022308 * |
DATABASE GenBank [online] 17 February 2013 (2013-02-17), "Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11), transcript variant 1, mRNA", XP002728377, retrieved from NCBI Database accession no. NM_000525.3 * |
DATABASE Geneseq [online] 12 July 2002 (2002-07-12), "Oligonucleotide for detecting cytosine methylation SEQ ID NO 287.", XP002728381, retrieved from EBI Database accession no. ABQ13696 * |
DATABASE Geneseq [online] 12 July 2002 (2002-07-12), "Oligonucleotide for detecting cytosine methylation SEQ ID NO 288.", XP002728380, retrieved from EBI Database accession no. ABQ13697 * |
ELIANA RENDA ET AL: "Treatment of gestational diabetes: oral hypoglycemic agents or insulin?", JOURNAL OF PRENATAL MEDICINE, 1 July 2011 (2011-07-01), Italy, pages 63 - 64, XP055133769, Retrieved from the Internet <URL:http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3279167&tool=pmcentrez&rendertype=abstract> * |
HANNAH R ELLIOTT ET AL: "Migration and DNA methylation: a comparison of methylation patterns in type 2 diabetes susceptibility genes between indians and europeans", JOURNAL OF DIABETES RESEARCH AND CLINICAL METABOLISM, vol. 2, no. 1, 6, 6 February 2013 (2013-02-06), pages 1 - 11, XP055132999, DOI: 10.7243/2050-0866-2-6 * |
INZUCCHI S E: "Oral Antihyperglycemic Therapy for Type 2 Diabetes Scientific Review", JAMA THE JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, AMERICAN MEDICAL ASSOCIATION, US, vol. 287, no. 3, 1 January 2002 (2002-01-01), pages 360 - 372, XP002990855, ISSN: 0098-7484, DOI: 10.1001/JAMA.287.3.360 * |
J. L. FERNÁNDEZ-MORERA ET AL: "The Possible Role of Epigenetics in Gestational Diabetes: Cause, Consequence, or Both", OBSTETRICS AND GYNECOLOGY INTERNATIONAL, vol. 21, no. 4, 605163, 1 January 2010 (2010-01-01), pages 1 - 7, XP055133832, ISSN: 1687-9589, DOI: 10.1074/jbc.M800128200 * |
LIJUAN YANG ET AL: "Association between KCNJ11 gene polymorphisms and risk of type 2 diabetes mellitus in East Asian populations: a meta-analysis in 42,573 individuals", MOLECULAR BIOLOGY REPORTS ; AN INTERNATIONAL JOURNAL ON MOLECULAR AND CELLULAR BIOLOGY, vol. 39, no. 1, 15 May 2011 (2011-05-15), KLUWER ACADEMIC PUBLISHERS, DO, pages 645 - 659, XP019981566, ISSN: 1573-4978, DOI: 10.1007/S11033-011-0782-6 * |
M. EHRICH ET AL: "Introduction to EpiTYPER for Quantitative DNA Methylation Analysis Using the MassARRAY' System Page 1 Introduction to EpiTYPER for Quantitative DNA Methylation Analysis Using the MassARRAY", 18 July 2006 (2006-07-18), XP055134012, Retrieved from the Internet <URL:http://epicore.med.cornell.edu/files/epityper.pdf> [retrieved on 20140808] * |
SHAAT N ET AL: "Association of the E23K polymorphism in the KCNJ11 gene with gestational diabetes mellitus", DIABETOLOGIA ; CLINICAL AND EXPERIMENTAL DIABETES AND METABOLISM, vol. 48, no. 12, 1 December 2005 (2005-12-01), SPRINGER, BERLIN, DE, pages 2544 - 2551, XP019322420, ISSN: 1432-0428, DOI: 10.1007/S00125-005-0035-0 * |
T. A. DAYEH ET AL: "Identification of CpG-SNPs associated with type 2 diabetes and differential DNA methylation in human pancreatic islets", DIABETOLOGIA, vol. 56, no. 5, 6 March 2013 (2013-03-06), pages 1036 - 1046, XP055133035, ISSN: 0012-186X, DOI: 10.1007/s00125-012-2815-7 * |
TOPEROFF GIDON ET AL: "Genome-wide survey reveals predisposing diabetes type 2-related DNA methylation variations in human peripheral blood", HUMAN MOLECULAR GENETICS, vol. 21, no. 2, January 2012 (2012-01-01), pages 371 - 383, XP002728382 * |
VERONICA LANG ET AL: "The Molecular Genetics of Sulfonylurea Receptors in the Pathogenesis and Treatment of Insulin Secretory Disorders and Type 2 Diabetes", CURRENT DIABETES REPORTS, CURRENT SCIENCE INC, NEW YORK, vol. 11, no. 6, 4 October 2011 (2011-10-04), pages 543 - 551, XP019971896, ISSN: 1539-0829, DOI: 10.1007/S11892-011-0233-8 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017003166A1 (en) * | 2015-06-29 | 2017-01-05 | 고려대학교 산학협력단 | Composition for early diagnosis of diabetes using metabolome analysis |
KR101995835B1 (en) * | 2018-09-20 | 2019-07-08 | 대한민국 | Composition and method for diagnosing type 2 diabetes using ELOVL fatty acid elongase 5 gene |
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