WO2015124921A1 - Methods and uses for determining the presence of inflammatory bowel disease - Google Patents

Methods and uses for determining the presence of inflammatory bowel disease Download PDF

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Publication number
WO2015124921A1
WO2015124921A1 PCT/GB2015/050464 GB2015050464W WO2015124921A1 WO 2015124921 A1 WO2015124921 A1 WO 2015124921A1 GB 2015050464 W GB2015050464 W GB 2015050464W WO 2015124921 A1 WO2015124921 A1 WO 2015124921A1
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individual
methylation
cpg sites
inflammatory bowel
disease
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PCT/GB2015/050464
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French (fr)
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Alexander Adams
Nicholas T VENTHAM
Nicholas A KENNEDY
Elaine NIMMO
Jack Satsangi
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The University Court Of The University Of Edinburgh
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Publication of WO2015124921A1 publication Critical patent/WO2015124921A1/en

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    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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 relates to methods and uses for determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in an individual, and for predicting the likelihood of an individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis).
  • Inflammatory Bowel Disease such as Crohn's Disease or Ulcerative Colitis
  • Inflammatory Bowel Disease is a group of inflammatory conditions of the colon and small intestine, the principal types of which are the conditions Crohn's Disease and Ulcerative Colitis.
  • Crohn's Disease is a chronic inflammatory condition of the gastrointestinal tract, which is also known as Crohn's Syndrome or regional enteritis. It affects approximately 1 in 200 of the Western population (Busch et a/., 2013, Aliment. Pharmacol. Ther., 10.11 1 1/apt.12528), and patients most-commonly present with symptoms of the disorder in childhood or in early adult life (Sonnenberg, 2010, Inflamm. Bowel Dis., 16:452-7).
  • Inflammatory Bowel Disease typically include abdominal pain, diarrhea, weight loss, mouth ulcers, fever, joint pains, fatigue and malaise. Diagnosis is made on the basis of: clinical suspicion; blood tests showing anemia; malnutrition; blood and stool tests which provide non-specific evidence of inflammatory disease; endoscopic appearances of stomach and colon; and histological examination of biopsies taken at endoscopy.
  • Inflammatory Bowel Disease and, particularly, Crohn's Disease has an increasingly-large economic impact due to costly medication, frequent hospitalisations and surgeries, and loss of productivity (van der Valk er a/., 2012, Gut, 10.1136/gutjnl-2012-303376).
  • Inflammatory Bowel Disease such as Crohn's Disease and Ulcerative Colitis
  • Inflammatory Bowel Disease is associated with a wide range of clinical symptoms, many of which are also present in other, unrelated gastrointestinal disorders, it can prove difficult to identify whether an individual has the disease or not based purely on the symptoms presented.
  • diagnostic biomarker that is sensitive and specific and which permits identification of Inflammatory Bowel Disease (such as Crohn's Disease and Ulcerative Colitis) in an individual.
  • those skilled in the art of medicine may identify individuals suspected of having such disease, but often be unable to reach a determinative diagnosis based on symptomology.
  • Inflammatory Bowel Disease such as Crohn's Disease and Ulcerative Colitis
  • the inventors' findings thus provide both diagnostic and prognostic information in relation to those disorder and, for the first time, allow methods for identifying the presence of Inflammatory Bowel Disease (such as Crohn's Disease and Ulcerative Colitis).
  • the invention provides a method for determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in an individual, comprising the steps of: - providing a sample comprising leukocyte DNA from the individual;
  • each CpG site is selected from those in Table 3;
  • Inflammatory Bowel Disease such as Crohn's Disease or Ulcerative Colitis
  • Inflammatory Bowel Disease is a group of inflammatory conditions of the colon and small intestine, the principal types of which are the conditions: Crohn's Disease and Ulcerative Colitis. Thus, by "Inflammatory Bowel Disease” we include the conditions Crohn's Disease and Ulcerative Colitis. As discussed herein, it is preferred that the Inflammatory Bowel Disease is selected from Crohn's Disease and Ulcerative Colitis; most preferably, the invention relates to Crohn's Disease.
  • the first aspect the invention provides a method for determining the presence of Crohn's Disease in an individual, comprising the steps of:
  • Crohn's Disease is a chronic inflammatory condition of the gastrointestinal tract, which is also known as Crohn's Syndrome or regional enteritis. It can affect any part or structure of the gastrointestinal tract from the mouth to the anus, for example one or more of: the mouth; oesophagus; stomach; small intestine (which comprises the duodenum, jejunum and ileum); large intestine (which comprises the cecum, colon and rectum); and the anus.
  • Crohn's Disease and Ulcerative Colitis are associated with similar symptoms. Medical practitioners generally distinguish between the two conditions on the basis of the location and nature of the inflammatory changes. As discussed above, Crohn's Disease can affect any part or structure of the gastrointestinal tract - by contrast, Ulcerative Colitis is restricted to the colon and the rectum. Additionally, Crohn's Disease affects the full thickness of the bowel wall ("transmural lesions"), whilst Ulcerative Colitis is restricted to the mucosa (i.e. the epithelial lining of the gut). Crohn's Disease and Ulcerative Colitis are associated with a wide range of symptoms that are known to those skilled in the arts of medicine and pharmacy, and which often present in re-occurring periods of appearance and remission. Individuals with Crohn's Disease or Ulcerative Colitis generally present with one or more symptom selected from: one or more gastrointestinal symptom; one or more systemic symptom; and/or one or more extra- intestinal symptom.
  • the one or more gastrointestinal symptom is selected from: diarrhea; bloody diarrhea; intestinal stenosis; vomiting; nausea; ulcerative colitis; primary sclerosing cholangitis; abdominal pain; difficulty swallowing; abscesses, mouth ulcers; itching and/or fistulization of the anal area; small bowel bacterial overgrowth syndrome; orofacial granulomatosis; pyostomatitis vegetans; recurrent aphthous stomatitis; geographic tongue; migratory stomatitis; and/or incontinence.
  • the one or more systemic symptom is selected from: weight loss; malaise; fatigue and/or stunted growth.
  • the one or more extra-intestinal symptom is selected from: fever; joint pain; inflammation of the eye (including episcleritis and uveitis), which can lead to blindness; gallstones; rheumatologic disease (including seronegative spondyloarthropathy, arthritis, enthesitis, ankylosing spondylitis and/or sacroiliitis); erythema nodosum; pyoderma gangrenosum; blood clots (including deep venous thrombosis); autoimmune hemolytic anemia; anemia; osteoporosis; neurological complications (including seizures, stroke, myopathy, peripheral neuropathy, headaches, and/or depression).
  • individuals having Crohn's Disease or Ulcerative Colitis display one or more of the following symptoms: abdominal pain; diarrhea; weight loss; mouth ulcers; fever; joint pain; fatigue; and malaise.
  • Crohn's Disease and Ulcerative Colitis are associated with a wide range of clinical symptoms, many of which are also present in other, unrelated gastrointestinal disorders, it can prove difficult to identify whether an individual has Crohn's Disease or not (or Ulcerative Colitis or not) based purely on the symptoms presented. Thus, those skilled in medicine may identify individuals suspected of having Crohn's Disease or Ulcerative Colitis, but be unable to reach a determinative diagnosis based on symptomology. In the method of the first aspect of the invention, it is preferred that the individual is one which is suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis).
  • such an individual preferably displays one or more symptom of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), as discussed above.
  • Inflammatory Bowel Disease such as Crohn's Disease or Ulcerative Colitis
  • those skilled in the art will be capable of identifying such individuals, which is typically established based on known approaches, such as the identification of clinical symptoms (as discussed above); blood tests showing the presence of anemia; evidence of malnutrition; blood and stool tests showing nonspecific evidence of inflammatory disease; endoscopic appearance of the stomach and colon; and histological examination of biopsies taken at endoscopy.
  • the individual is one which is suspected of having Crohn's Disease and such an individual preferably displays one or more symptom of Crohn's Disease, as discussed above.
  • leukocytes are white cells of the immune system which circulate in the bloodstream (and which are also known as white blood cells).
  • Leukocytes include granulocytes (i.e. polymorphonuclear leukocytes, which include neutrophils, basophils and eosinophils) and agranulocytes (i.e. mononuclear granulocytes, which include lymphocytes, monocytes, and macrophages).
  • the leukocyte of the present invention is one or more cell type selected from the group consisting of: granulocytes; agranulocytes; neutrophils; basophils; eosinophils; lymphocytes; monocytes; and/or macrophages.
  • leukocyte DNA we mean the DNA present in a leukocyte.
  • the DNA is genomic DNA.
  • Methods for isolating and/or purifying leukocyte DNA are known in the art (as described, for example, in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press), and include those described herein and in the accompanying Examples.
  • CpG site refers to a dinucleotide within a polynucleotide sequence, in which a cytosine nucleotide and a guanine nucleotide are arranged next to one another in a linear sequence and are covalently joined to one another by a phosphodiester bond. Whilst a CpG site may be present in any type of polynucleotide sequence in the leukocytes DNA, it is preferred that the CpG site is present in genomic leukocyte DNA.
  • methylation state at a CpG site refers to the presence or absence of a methyl group bound to the cytosine nucleotide of the CpG site, which thereby forms 5- methylcytosine (commonly abbreviated to "5-mCyt").
  • Methylation of CpG sites is a well- recognised modification of cellular DNA and is known to contribute to and influence cellular processes, such as the regulation of gene expression and nucleic acid replication.
  • a CpG site in a polynucleotide sequence may or may not be methylated. Where multiple CpG sites are present in a polynucleotide sequence, then some, none, or all of those sites may be methylated.
  • CpG site refers to the corresponding nucleotide sequence and site on both the sense- and anti-sense strands
  • methylation state refers to the methylation state of that CpG site on the sense- and the anti-sense strand.
  • determining the methylation state we mean determining whether a CpG site has a methyl group bound (i.e. is methylated), or not (i.e. is un-methylated). It will be appreciated that the methylation state of a CpG site can be determined by assaying for the physical presence of the methyl group at that site. Assays for doing so are well known and include those discussed herein and in the accompanying Examples. Methylation of polynucleotide sequences is known to have a functional effect on cellular processes and can, for example, lead to altered expression of associated genes by affecting their translation. For example, hypo-methylation (i.e.
  • Methylation state may also be secondary to other factors which influence gene expression (see, for example, Stadler et al., 201 1 , Nature, 480:490). Methylation state is therefore a cellular mechanism for regulating gene expression. Changes in methylation state is therefore thought to alter gene expression.
  • the presence of methylation at a particular CpG site or gene could be determined by measuring the presence and/or level of expression and/or transcription of an associated gene.
  • Methods for determining the transcription of a gene and/or gene expression are well known to those in the art of biochemistry and involve detecting and/or measuring the presence and/or concentration of an associated gene product such as mRNA or protein. Methods of detecting and/or measuring the concentration of mRNA and/or protein are well known to those skilled in the art; see, for example, the methods described in the accompanying Examples and in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press. Differential expression (up-regulation or down regulation) of a gene, or lack thereof, can be determined by any suitable means known to a skilled person.
  • the step of determining the methylation state at two or more CpG sites in the leukocyte DNA is performed by determining the physical presence of a methyl group at the two or more CpG sites.
  • the invention provides a method for predicting the likelihood of an individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), comprising the steps of: - providing a sample comprising leukocyte DNA from the individual;
  • each CpG site is selected from those in Table 3;
  • the individual is one which is known not to have Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), or which is not suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis).
  • Inflammatory Bowel Disease such as Crohn's Disease or Ulcerative Colitis
  • such an individual preferably does not display one or more of the symptoms of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) discussed herein.
  • contracting Inflammatory Bowel Disease we include the meaning that the individual develops Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) and presents with one or more symptom as discussed herein. It will be appreciated that development of Inflammatory Bowel Disease in an individual not previously afflicted with the disorder can occur at any time during the lifetime of that individual. Whilst the causes of Inflammatory Bowel Disease are not completely understood, a number of different factors are likely to be involved, including: genetic disposition to the disorder, the presence or development of autoimmunity or other immune dysfunction; microbial infection; or environmental factors.
  • predicting the likelihood of an individual contracting Inflammatory Bowel Disease we include the meaning that an indication of the predisposition of that individual to the disorder, and/or an indication of the risk of that individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) over a given time period, such as the lifetime of the individual.
  • the risk may be calculated as the percentage chance of the individual contracting Inflammatory Bowel Disease within a given time period - for example, a 5% chance of development within 5 years; or a 10% chance of development within 5 years; or a 20% chance of development within 5 years; or a 30% chance of development within 5 years; or a 40% chance of development within 5 years; or a 50% chance of development within 5 years; or a 60% chance of development within 5 years; a 70% chance of development within 5 years; an 80% chance of development within 5 years; a 90% chance of development within 5 years; or a 95% chance of development within 5 years; or a 100% chance of development within 5 years.
  • the second aspect of the invention provides a method for predicting the likelihood of an individual contracting Crohn's Disease, comprising the steps of:
  • each CpG site is selected from those in Table 3;
  • the individual is one which is known not to have Crohn's Disease, or which is not suspected of having Crohn's Disease.
  • such an individual preferably does not display one or more of the symptoms of Crohn's Disease discussed herein.
  • contracting Crohn's Disease we include the meaning that the individual develops Crohn's Disease and presents with one or more symptom of Crohn's Disease discussed herein. It will be appreciated that development of Crohn's Disease in an individual not previously afflicted with the disorder can occur at any time during the lifetime of that individual. Whilst the causes of Crohn's Disease are not completely understood, a number of different factors are likely to be involved, including: genetic disposition to the disorder, the presence or development of autoimmunity or other immune dysfunction; microbial infection; or environmental factors.
  • predicting the likelihood of an individual contracting Crohn's Disease we include the meaning that an indication of the predisposition of that individual to the disorder, and/or an indication of the risk of that individual contracting Crohn's Disease over a given time period, such as the lifetime of the individual.
  • the risk may be calculated as the percentage chance of the individual contracting Crohn's Disease within a given time period - for example, a 5% chance of development within 5 years; or a 10% chance of development within 5 years; or a 20% chance of development within 5 years; or a 30% chance of development within 5 years; or a 40% chance of development within 5 years; or a 50% chance of development within 5 years; or a 60% chance of development within 5 years; a 70% chance of development within 5 years; an 80% chance of development within 5 years; a 90% chance of development within 5 years; or a 95% chance of development within 5 years; or a 100% chance of development within 5 years.
  • the preferred method of the second aspect of the invention provides a method of prognosis of Crohn's Disease in an individual.
  • the invention provides a method for selecting a treatment for an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), comprising the steps of: providing a sample comprising leukocyte DNA from the individual;
  • each CpG site is selected from those in Table 3;
  • an individual suspected of having Inflammatory Bowel Disease we include an individual presenting with one or more symptom associated with Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), as discussed herein.
  • Inflammatory Bowel Disease cannot be cured, anti-Inflammatory Bowel Disease treatments are known and used to manage the disorder and alleviate the associated symptoms. However, as discussed above, because Inflammatory Bowel Disease is associated with a wide range of clinical, non-determinative symptoms, it can prove difficult to identify individuals actually having the disorder and thereby those in need of (i.e. who would benefit from) anti-Inflammatory Bowel Disease treatment.
  • anti-Inflammatory Bowel Disease treatment is aggressive, associated with patient side-effects and expensive if treatment is continued long-term. It is therefore undesirable for all individuals displaying an Inflammatory Bowel Disease symptom to be subjected to such treatments, particularly if it is unclear if Inflammatory Bowel Disease is actually present.
  • the present invention allows the presence of Inflammatory Bowel Disease in an individual to be determined so that an appropriate and necessary treatment can then be selected.
  • the step of selecting a treatment comprises selecting an anti-Inflammatory Bowel Disease treatment.
  • the step of selecting a treatment comprises selecting a treatment other than an anti-Inflammatory Bowel Disease treatment.
  • the step of selecting an anti-Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) treatment comprises selecting one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
  • the method of the third aspect of the invention further comprises the step of treating the individual with the selected treatment.
  • the third aspect of the invention provides a method for selecting a treatment for an individual suspected of having Crohn's Disease, comprising the steps of:
  • each CpG site is selected from those in Table 3;
  • an individual suspected of having Crohn's Disease we include an individual presenting with one or more symptom associated with Crohn's Disease, as discussed herein. Whilst Crohn's Disease cannot be cured, anti-Crohn's Disease treatments are known and used to manage the disorder and alleviate the associated symptoms. However, as discussed above, because Crohn's Disease is associated with a wide range of clinical, non-determinative symptoms, it can prove difficult to identify individuals actually having the disorder and thereby those in need of (i.e. who would benefit from) anti-Crohn's Disease treatment.
  • anti-Crohn's Disease treatment is aggressive, associated with patient side-effects and expensive if treatment is continued long-term. It is therefore undesirable for all individuals displaying a Crohn's Disease symptom to be subjected to such treatments, particularly if it is unclear if Crohn's Disease is actually present.
  • the present invention allows the presence of Crohn's Disease in an individual to be determined so that an appropriate and necessary treatment can then be selected.
  • the step of selecting a treatment comprises selecting an anti-Crohn's Disease treatment.
  • the step of selecting a treatment comprises selecting a treatment other than an anti-Crohn's Disease treatment.
  • the step of selecting an anti-Crohn's Disease treatment comprises selecting one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
  • the method of the third aspect of the invention further comprises the step of treating the individual with the selected treatment.
  • the invention provides a method for predicting the response to anti- Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) treatment of an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), comprising the steps of: - providing a sample comprising leukocyte DNA from the individual;
  • each CpG site is selected from those in Table 3;
  • an individual suspected of having Inflammatory Bowel Disease we include an individual presenting with one or more symptom associated with Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) discussed herein.
  • the anti-Inflammatory Bowel Disease treatment comprises one or more treatment selected from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
  • the present invention permits the prediction of whether the anti-Inflammatory Bowel Disease treatment will have a beneficial therapeutic effect on the individual.
  • the anti-Inflammatory Bowel Disease treatment can be predicted to have a beneficial therapeutic effect on the individual (for example, by alleviating one or more symptom of the disorder; and/or improving the quality of life of the individual; and/or avoiding death and/or hospitalisation and/or surgery of the individual; and/or resulting in remission of the disorder).
  • the anti-Inflammatory Bowel Disease treatment can be predicted not to have a beneficial therapeutic effect on the individual (for example, by failing to alleviate one or more symptom of the disorder; and/or failing to improve the quality of life of the individual; and/or failing to avoid death and/or hospitalisation and/or surgery of the individual; and/or failing to cause remission of the disorder).
  • an anti-Inflammatory Bowel Disease treatment can be selected and, more preferably, used to treat the individual.
  • anti-Inflammatory Bowel Disease treatment comprises one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
  • an anti-Inflammatory Bowel Disease treatment will not be selected and the individual will not be treated with it.
  • the fourth aspect of the invention provides a method for predicting the response to anti-Crohn's Disease treatment of an individual suspected of having Crohn's Disease, comprising the steps of: providing a sample comprising leukocyte DNA from the individual;
  • each CpG site is selected from those in Table 3;
  • an individual suspected of having Crohn's Disease we include an individual presenting with one or more symptom associated with Crohn's Disease discussed herein.
  • the anti-Crohn's Disease treatment comprises one or more treatment selected from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
  • the present invention permits the prediction of whether the anti-Crohn's Disease treatment will have a beneficial therapeutic effect on the individual.
  • the anti-Crohn's Disease treatment can be predicted to have a beneficial therapeutic effect on the individual (for example, by alleviating one or more symptom of the disorder; and/or improving the quality of life of the individual; and/or avoiding death and/or hospitalisation and/or surgery of the individual; and/or resulting in remission of the disorder).
  • the anti-Crohn's Disease treatment can be predicted not to have a beneficial therapeutic effect on the individual (for example, by failing to alleviate one or more symptom of the disorder; and/or failing to improve the quality of life of the individual; and/or failing to avoid death and/or hospitalisation and/or surgery of the individual; and/or failing to cause remission of the disorder).
  • an anti-Crohn's Disease treatment can be selected and, more preferably, used to treat the individual.
  • anti- Crohn's Disease treatment comprises one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
  • an anti-Crohn's Disease treatment will not be selected and the individual will not be treated with it.
  • the methylation state of the one or more CpG sites listed in Table 3 is determined using a method selected from the group comprising: bisulfite conversion and DNA sequencing (for example, using pyrosequencing or Sanger sequencing); methylation- specific restriction enzyme analysis; COBRA analysis; methylation-specific PCR; Methyl Light analysis; HeavyMethyl analysis; Quantitative Methylation analysis; Ms-SNuPE analysis.
  • the methylation state of the one or more CpG sites listed in Table 3 is determined using one or more relevant probe from those listed in Table 3.
  • the individual is a child (and preferably a human child).
  • it is preferred that the individual is an adult (and preferably a human adult).
  • an adult we include an individual that has reached sexual maturity.
  • a child we include an individual between the age of birth and adulthood which has not reached sexual maturity.
  • the individual Whilst it is preferred that the individual is a human, the individual may also be a non-human mammal ⁇ i.e. any mammal other than a human), such as, a horse, cow, goat, sheep, pig, dog, cat, rabbit, mouse or rat.
  • a non-human mammal i.e. any mammal other than a human
  • a human individual between the age of birth and 18 years of age, for example: 1 year of age; or 2 years of age; or 3 years of age; or 4 years of age; or 5 years of age; or 6 years of age; or 7 years of age; or 8 years of age; or 9 years of age; or 10 years of age; or 11 years of age; or 12 years of age; or 13 years of age; or 14 years of age; or 15 years of age; or 16 years of age; or 17 years of age; or 18 years of age.
  • the individual is a human adult
  • the step of determining the methylation state comprises determining the methylation state at three or more CpG sites in the leukocyte DNA, for example, four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more, or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or more, or 110 or more, or 120 or more, or 130 or more, or 140 or more, or 150 or more, or 160 or more, or 165, CpG sites in the leukocyte DNA.
  • the step of determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
  • the step of determining the presence of Crohn's Disease in the individual comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
  • the step of predicting the likelihood of the individual contracting Inflammatory Bowel Disease comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites the leukocyte DNA from one or more control individual.
  • the step of predicting the likelihood of the individual contracting Crohn's Disease comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites the leukocyte DNA from one or more control individual.
  • the invention generally relates to Inflammatory Bowel Diseases (such as Crohn's Disease or Ulcerative Colitis). It will be appreciated that comparison with a control in the preferred methods of the invention allows the presence or likelihood of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) to be determined.
  • Inflammatory Bowel Diseases such as Crohn's Disease or Ulcerative Colitis
  • the one or more control individual has Inflammatory Bowel Disease or, alternatively, the one or more control individual does not have Inflammatory Bowel Disease.
  • the one or more control individual comprises one or more individual with Inflammatory Bowel Disease and one or more individual which does not have Inflammatory Bowel Disease.
  • the one or more control individual has Ulcerative Colitis or, alternatively, the one or more control individual does not have Ulcerative Colitis.
  • the one or more control individual comprises one or more individual with Ulcerative Colitis and one or more individual which does not have Ulcerative Colitis.
  • the invention relates to Crohn's Disease.
  • the one or more control individual has Crohn's Disease.
  • the one or more control individual does not have Crohn's Disease.
  • the one or more control individual comprises one or more individual with Crohn's Disease and one or more individual which does not have Crohn's Disease. It will be appreciated that comparison with a control in the preferred methods of the invention allows the presence or likelihood of Crohn's Disease to be determined.
  • the presence of Crohn's Disease in the individual is identified, or Crohn's Disease is predicted, when: the two or more CpG sites in the individual are methylated in the same way as the corresponding CpG sites in a control which has Crohn's Disease; and/or when the two or more CpG sites in the individual are differently methylated as the corresponding CpG sites in a control which does not have Crohn's Disease.
  • the presence of Crohn's Disease in the individual is not identified, or Crohn's Disease is not predicted, when: the two or more CpG sites in the individual are differently methylated as the corresponding CpG sites in a control which has Crohn's Disease; and/or when the two or more CpG sites in the individual are methylated in the same way as the corresponding CpG sites in a control which does not have Crohn's Disease.
  • the control individual will be one that is appropriately matched with the individual being tested - for example, in terms of being the same sex and/or of similar age and/or smoking status.
  • the CpG sites may be "hyper-methylated” as compared to the control (i.e. have a greater level of methylation) or be "hypo-methylated” (i.e. have a lower level of methylation) compared to the control.
  • the accompanying Examples describe exemplary approaches for determining the presence of Inflammatory Bowel Disease (and, in particular, Crohn's Disease and/or Ulcerative Colitis) in an individual based on the methylation state of two or more CpG sites in the individual, as required by the methods of the invention.
  • LDA linear discriminant analysis
  • the CpG sites are associated with a gene selected from those listed in Table 4. In other words, in that embodiment of the invention, all of the CpG sites are associated with a single gene selected from those in Table 4.
  • the CpG sites are associated with two or more genes selected from those listed in Table 4, for example, three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more genes selected from those listed in Table 4.
  • each of the CpG sites is associated with a different gene selected from those listed in Table 4.
  • each CpG sites is associated with a different gene, each of which is selected from the genes in Table 4.
  • the methylation state of the CpG sites associated with the genes listed in Table 4 is determined using one or more relevant probe from those listed in Table 6.
  • CpG site is within a region (i.e. a sequence) of the genome that is within or is adjacent to that gene. It will be appreciated that the CpG site could be functionally associated with the expression of the gene within which it is found, and/or with other genes in the genetic region.
  • each CpG site is located in a regulatory region of the associated gene.
  • a gene comprises a coding region and regulatory regions such as a promoter and/or other sequences which normally govern expression.
  • each CpG site is located in a regulatory region selected from the group comprising: promoter; enhancer; exon; intron; and terminator.
  • the CpG site is within 1000 base pairs (bp) of the coding region of the gene, and is preferably, within 900bp or 800bp, or 700bp, or 600bp or 500bp or 400bp, or 300bp, or 200bp or 100bp, of the coding region of the gene.
  • genetic coordinates and gene names refer to the All coordinates refer to the publically-available Genome Reference Consortium Human Build 37 (aka GRCh37 & hg19), which is found at the following web address: http://www.ncbi.nlm.nih.gov/assembly/GCF 000001405.13/.
  • the CpG sites are located in a genetic region that is a Differentially Methylated Region (abbreviated to "DMR").
  • DMR Differentially Methylated Region
  • DMRs are genomic regions which have different methylation statuses among multiple samples (for example, between tissues, cells, individuals or other sample types).
  • DMRs were defined using a modification of the lasso technique employed by the ChAMP pipeline (Morris ei al. (2014), Bioinformatics 30(3): 428-430 and at: http://www2.cancer.ucl.ac.uk/medicalqenomics/champ/).
  • a DMRs is a genetic region in which: all probes were found to have disease- associated methylation differences at the significance level FDR corrected p value ⁇ 0.05 and shared the same direction of change in methylation in association with disease; and have a minimum size of three CpG sites and no maximum.
  • DMRs were identified using an algorithm based on a modified version of ChAMP (Morris ei al. (2014), Bioinformatics 30(3): 428-430, and at: http://www2.cancer.ucl.ac.uk/medicalqenomics/champ/).
  • the distance that the algorithm looks for adjacent CpG sites is variable and based on the local density of probes covering the region.
  • the CpG sites are within a genetic region selected from those listed in Table 5 (all of which are DMRs).
  • the CpG sites are within two or more genetic regions selected from those listed in Table 5, for example: three or more; or four or more; or five or more; or six or more; or seven or more; or eight or more, or nine or more, or ten or more, genetic regions selected from Table 5.
  • each of the CpG sites is associated with a different genetic locus selected from those listed in Table 5.
  • the methylation state of the one or more CpG sites associated with the genetic regions listed in Table 5 is determined using one or more relevant probe from those listed in Table 7.
  • the CpG sites are selected from the group comprising:
  • the two or more CpG sites are selected from the group comprising:
  • VMP1 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544
  • MY01 E 15:59588622
  • the CpG sites are associated with one or more gene selected from the following: ANKRD11 (i.e. cg16525838, cg16710656); or
  • ARHGEF3 i.e. (cg04389058);
  • CDK6 (/.e.cgl 5732164, cg14100946, cg06688763); or
  • CSMD3 (/.e.cg02292450, cg23676042, cg20323509); or
  • GNAS (/.e.cg26791489, cg04019914, cg11357538, cg10011623, cg01748573, cg17334845, cg26767990, cg19592829, cg09123158, cg19140375, cg11669839, cg14482474, cg06200857, cg08091561 , cg22860367, cg07947033, cg04677683, eg 15160445, cg25326570, cg23249369); or
  • GPRIN3 (/.e.cg02734358); or
  • HEATR2 ( .e.cg1047271 1);
  • 1TGB2 (/.e.cgl 41 2356, cg22699620, cg21006727, cg13315706); or MIR1973 (/.e.cg22914762); or
  • NDUFS4 ( .e.cgl 2351310);
  • NMUR1 (/.e.cgl 9077400, cg19733463, cg20608294, cg01616956); or PWWP2B (/.e.cgl 5995714, cg021 16864, cg07733247, cg16784468, cg06427772, cg1 1229101 , cg11579421 , cg25303150, cg24085039); or RPS6KA2 (/.e.cgl 1599721 , cg05691806, cg09430664, cg17501210); or
  • SEPT9 (/.e.cg05626616, cg01749539, cg06791979, cg02442640, eg 17922695, g 14843920); or
  • TNFSF10 (/.e.cg01059398); or
  • TNS1 (/.e.cg12338137); or
  • VMP1 (/.e.cgl 6936953, cg12054453, cg01409343, cg18942579, cg24174557); or
  • CpG sites are associated with the following combinations of genes:
  • VMP1 cg12054453
  • SEPT9 cg01749539
  • VMP1 cg12054453
  • ITGB2 cg13315706
  • VMP1 eg 12054453
  • NA cg09304397
  • VMP1 eg 16936953
  • YWHAE cg06219337
  • the gene is selected from the group comprising: TOLLIP; SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2; SBN02; TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9; SPARC; SUB1 ; TRAM2; KCNAB2; BBS9; AGL; FRMD4A; F BP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3; PALM; ICA1 ; BAHCC1 ; LRRC47; cg01101459; cg02719954; cg04086394; cg12807764; cg19445588;
  • two or more genes are selected from the group comprising:
  • the CpG sites are selected from the group comprising:
  • VMP1 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544
  • the two or more CpG sites are selected from the group comprising: - 17:75473969 (SEPT9) and 6: 166970252 (RPS6KA2); or
  • VMP1 15:59588622
  • the gene is selected from the group comprising:
  • TNFSF10 TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9; SPARC; SUB1 ; TRAM2; KCNAB2;
  • BBS9 AGL; ICA1 ; FRMD4A; FKBP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3; PALM;
  • the CpG sites are associated with one or more gene selected from the following: RPS6KA2; SBN02; VMP1 ; cg12992827; KCNAB2; cg07398517; BBS9; AGL; ICA1 ; FRMD4A; FKBP5; TNFSF10; CALHM1 ; cg25114611 ; AIM2; BCL3; cg01101459; cg02719954; cg04086394; cg09349128; cg12807764; cg19445588; cg19821297; cg20519581 ; CPVL; CSGALNACT1 ; GPRIN3.
  • the two or more genes are selected from the group comprising: cg25114611 & AIM2; cg251 14611 & BCL3; cg2511461 1 & cg01101459; cg2511461 1 & cg02719954; cg251 14611 & cg04086394; cg251 14611 & cg09349128; cg25114611 & cg12807764; cg251 14611 & cg19445588; cg25114611 & cg19821297; cg25114611 & cg20519581 ; cg25114611 & CPVL; cg25114611 & CSGALNACT1; cg25114611 & GPRIN3; cg251 14611 & ICA
  • the CpG sites are associated with one or more gene selected from the following: RPS6KA2; SBN02; cg09349128; VMP1 ; cg12992827; cg251 1461 1 ; KCNAB2; cg07398517; BBS9; AGL; ICA1 ; cg19821297; FRMD4A; FKBP5; AIM2; BCL3; cg12582317; cg00053916; PHF21A; ANO10; GPRIN3; PALM; ITGB2; ICA1 ; BAHCC1 ; LRRC47.
  • the two or more genes are selected from the group comprising: AIM2 & BCL3; AIM2 & cg12582317; AIM2 & FKBP5; cg07398517 & BCL3; cg09349128 & cg00053916; cg09349128 & FKBP5; cg09349128 & FRMD4A; cg09349128 & PHF21A; cg12992827 & ANO10; cg12992827 & BCL3; cg12992827 & FRMD4A; cg19821297 & PHF21A; cg25114611 & BCL3; cg251 14611 & GPRIN3; FRMD4A & PALM; GPRIN3 & ITGB2; ICA1 & cg09349128; PALM; GPRIN3 & ITGB2; ICA1 & cg
  • the sample is selected from the group comprising: blood; serum; plasma; intestinal cells; biopsy; stool.
  • the sample is a blood sample.
  • Methods for isolating and/or purifying relevant cells from such samples are well known in the art and include density centrifugation and magnetic cell sorting.
  • the leukocyte DNA is obtained from the sample by a method comprising the steps of: (i) cell lysis; (ii) removal of cell membranes and/or cellular RNA and/or cellular protein; (iii) purification of DNA (for example, by extraction using phenol-chloroform; or using column- based extraction; or using ethanol-precipitation).
  • Suitable kits for obtaining DNA from leukocytes are commercially-available and include the "AllPrep DNA/RNA kit" (Qiagen), and "Nucleon kit” (GE Healthcare).
  • the methylation state is determined by a method selected from the group comprising: bisulfite conversion and DNA sequencing; methylation-specific restriction enzyme analysis; COBRA analysis; methylation-specific PCR; MethylLight analysis; HeavyMethyl analysis; Quantitative Methylation analysis; Ms-SNuPE analysis.
  • bisulfite conversion and DNA sequencing we include a technique in which short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment, and either completely sequenced (OIek & Walter, Nat Genet. 1997 17:275-6, 1997), subjected to one or more primer extension reactions (Gonzalgo & Jones, Nucleic Acids Res., 25:2529-31 , 1997; WO 95/00669; U.S. Patent No. 6,251 ,594) to analyse individual cytosine positions, or treated by enzymatic digestion (Xiong & Laird, Nucleic Acids Res., 25:2532-4, 1997). Sequencing may be performed using techniques known in the art, such as Sanger sequencing or pyrosequencing.
  • methylation-specific restriction enzyme analysis we include a process by which restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is used; for example, the method described by Sadri & Hornsby (Nucl. Acids Res. 24:5058- 5059, 1996), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997).
  • COBRA combined Bisulfite Restriction Analysis
  • COBRA analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific gene loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite- treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831 , 1992).
  • PCR amplification of the bisulfite converted DNA is then performed using primers specific for the CpG sites of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labelled hybridization probes.
  • Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels.
  • this technique can be reliably applied to DNA obtained from micro-dissected paraffin- embedded tissue samples.
  • MSP methylation-specific PCR
  • DNA is modified by sodium bisulfite converting all un-methylated, but not methylated cytosines to uracil, and subsequently amplified with primers specific for methylated versus un-methylated DNA.
  • MSP requires only small quantities of DNA, is sensitive to 0.1 % methylated alleles of a given CpG site, and can be performed on DNA extracted from paraffin- embedded samples.
  • MethylLightTM analysis which is also referred to as the MethylLight assay, we include the art-recognized fluorescence-based real-time PCR technique described by Eads et al., (Cancer Res. 59:2302-2306, 1999).
  • the MethylLight assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight process begins with a mixed sample of genomic DNA that is converted, in a sodium bisulfite reaction, to a mixed pool of methylation-dependent sequence differences according to standard procedures (the bisulfite process converts un- methylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" (with PCR primers that overlap known CpG dinucleotides) reaction. Sequence discrimination can occur both at the level of the amplification process and at the level of the fluorescence detection process.
  • TaqMan fluorescence-based real-time PCR
  • the MethyLight assay may be used as a quantitative test for methylation patterns in the genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization.
  • the PCR reaction provides for a methylation specific amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site.
  • An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides.
  • a qualitative test for genomic methylation is achieved by probing of the biased PCR pool with either control oligonucleotides that do not "cover" known methylation sites (a fluorescence-based version of the HeavyMethylTM and MSP techniques), or with oligonucleotides covering potential methylation sites.
  • the MethyLight process can by used with any suitable probes, e.g. "TaqMan” or Lightcycler.
  • double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using TaqMan probes; e.g., with MSP primers and/ or HeavyMethyl blocker oligonucleotides and TaqMan probe.
  • the TaqMan probe is dual-labelled with fluorescent "reporter” and "quencher” molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about 10 ° C higher temperature in the PCR cycle than the forward or reverse primers.
  • TaqMan probe This allows the TaqMan probe to remain fully hybridized during the PCR annealing/extension step. As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5' to 3' endonuclease activity will then displace the TaqMan probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system.
  • HeavyMethyl analysis we include an assay, wherein methylation specific blocking probes (also referred to herein as blockers) covering CpG positions between, or covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample.
  • QM quantitative Methylation
  • the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site.
  • An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides.
  • a qualitative test for genomic methylation is achieved by probing of the biased PCR pool with either control oligonucleotides that do not "cover" known methylation sites (a fluorescence- based version of the HeavyMethyl and MSP techniques), or with oligonucleotides covering potential methylation sites.
  • the QM process can be used with any suitable probes, e.g. "TaqMan” or Lightcycler, in the amplification process.
  • any suitable probes e.g. "TaqMan” or Lightcycler
  • double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and the TaqMan probe.
  • the TaqMan probe is dual-labelled with fluorescent "reporter” and “quencher” molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about 10 ° C higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan probe to remain fully hybridized during the PCR annealing/extension step.
  • Taq polymerase As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan probe. The Taq polymerase 5' to 3' endonuclease activity will then displace the TaqMan probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system.
  • Typical reagents for QM analysis may include, but are not limited to: PCR primers for specific gene (or bisulfite treated DNA sequence or CpG site); TaqMan or Lightcycler probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.
  • Ms-SNuPE Metal-sensitive Single Nucleotide Primer Extension
  • the Ms-SnuPE technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo & Jones (Nucleic Acids Res. 25:2529-2531 , 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert un-methylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest. Small amounts of DNA can be analyzed (e.g., micro-dissected pathology sections), and it avoids utilization of restriction enzymes for determining the methylation state at CpG sites.
  • arrays of nucleic acid probes can be used to extract sequence and/or methylation state information from, for example, nucleic acid samples.
  • the samples are exposed to the probes under conditions that allow hybridisation, and the arrays are then scanned to determine to which probes the sample molecules have hybridised.
  • Sequence information can be obtained by careful probe selection and using algorithms to compare patterns of hybridisation and non-hybridisation.
  • a preferred microarray for use in the methods of the present invention is the lllumina 450k microarray (http://www.illumina.com/products/methylation 450 beadchip kits.ilmn).
  • That array permits the determination of methylation state by binding bisulphite-converted DNA and returning a colour signal based on the sequence differences caused by bisulphite conversion depending on starting methylation state. Interpretation of the signal allows one to calculate the proportion of strands of input DNA which were methylated and un- methylated at each probe location.
  • the methods of the invention further comprise the step of selecting a treatment for the individual and/or treating the individual with the selected treatment, as discussed herein.
  • a treatment for the individual such as Crohn's Disease or Ulcerative Colitis
  • the inventors' findings mean that the methylation state of leukocyte DNA can be used to determine the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in an individual and/or predict the likelihood of an individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis).
  • further aspects of the invention relate to the use of leukocyte DNA from an individual in such approaches.
  • the invention provides a use of leukocyte DNA from an individual, for determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual.
  • the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual on the basis of the methylation state.
  • the fifth aspect of the invention provides a use of leukocyte DNA from an individual, for determining the presence of Crohn's Disease in the individual - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and determining the presence of Crohn's Disease in the individual on the basis of the methylation state. More preferably, the use comprises the methods of the first aspect of the invention as described above.
  • the invention provides a use of leukocyte DNA from an individual, for predicting the likelihood of the individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis).
  • the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the likelihood of the individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) on the basis of the methylation state.
  • the sixth aspect of the invention provides a use of leukocyte DNA from an individual, for predicting the likelihood of the individual contracting Crohn's Disease - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the likelihood of the individual contracting Crohn's Disease on the basis of the methylation state. More preferably, the use comprises the methods of the second aspect of the invention as described above.
  • the invention provides a use of leukocyte DNA from an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), for selecting a treatment for the individual.
  • the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and selecting a treatment for the individual on the basis of the methylation state.
  • the seventh aspect of the invention provides a use of leukocyte DNA from an individual suspected of having Crohn's Disease, for selecting a treatment for the individual - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and selecting a treatment for the individual on the basis of the methylation state.
  • such treatments are as discussed herein.
  • the use comprises the methods of the third aspect of the invention as described above.
  • the invention provides a use of leukocyte DNA from an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) for predicting the response to anti-Crohn's Disease therapy of the individual.
  • the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the response to anti-Inflammatory Bowel Disease therapy of the individual on the basis of the methylation state.
  • the eighth aspect of the invention provides a use of leukocyte DNA from an individual suspected of having Crohn's Disease for predicting the response to anti-Crohn's Disease therapy of the individual - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the response to anti-Crohn's Disease therapy of the individual on the basis of the methylation state.
  • such treatments are as discussed herein.
  • the use comprises the methods of the fourth aspect of the invention as described above.
  • the invention provides a kit for performing a method or use according to the invention, the kit comprising one or more reagent for determining the methylation state at two or more CpG sites in leukocyte DNA, wherein each CpG site is selected from those in Table 3.
  • the CpG sites are associated with a gene selected from those listed in Table 4.
  • all of the CpG sites are associated with a single gene selected from those in Table 4.
  • the CpG sites are associated with two or more genes selected from those listed in Table 4, for example, three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more genes selected from those listed in Table 4. More preferably, in the ninth aspect of the invention, each of the CpG sites is associated with a different gene selected from those listed in Table 4. In other words, in that embodiment of the invention, each CpG sites is associated with a different gene, each of which is selected from the genes in Table 4.
  • the methylation state of the CpG sites associated with the genes listed in Table 4 is determined using one or more relevant probe from those listed in Table 6.
  • invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
  • Bisulfite conversion is a process by which cytosine residues are converted to uracil, but methylated cytosine residues are not. Such processes are well known in the art and described herein.
  • test DNA is diluted and denatured, and then incubated with sodium bisulphite which converts un-methylated cytosine to uracil, but does not alter methylated cytosine.
  • sodium bisulphite which converts un-methylated cytosine to uracil, but does not alter methylated cytosine.
  • the DNA is treated with a desulphonation reagent, and can then be subjected to further analysis to determine the presence of cytosine and uracil residues.
  • the kit of this embodiment of the invention is suitable for a Polymerase Chain Reaction (PCR)-based determination of methylation state, as discussed above.
  • PCR Polymerase Chain Reaction
  • PCR amplification will replace uracil with thymine such that, when the amplified sequence is read, all un-methylated cytosines are read as thymine, and all detected cytosines were methylated.
  • PCR is described in Saiki et al. (1988, Science, 239: 487-491).
  • Approaches for designing oligonucleotide primers suitable for PCR-based amplification and related PCR-based approaches are well known in the art.
  • the invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
  • oligonucleotide primers for methylation-specific PCR amplification of the polynucleotide sequence at a first CpG site selected from those listed in Table
  • an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation-specific PCR amplification.
  • kits of that embodiment of the invention further comprise:
  • an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the first CpG site when the first CpG site is not methylated
  • an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the second CpG site when the first CpG site is not methylated.
  • kits of this embodiment of the invention are suitable for determination of the methylation state using Methylation-specific PCR-based approaches, such as the MethylLight or HeavyMethyl or MS-SNuPE approaches, as discussed above.
  • MethyLight and HeavyMethyl processes comprise methylation- specific blocking probes (also referred to herein as blockers) covering CpG positions between, or covered by, the amplification primers to enable methylation-specific selective amplification of a nucleic acid sample.
  • methylation- specific blocking probes also referred to herein as blockers
  • an oligonucleotide capable of preventing and/or reducing PCR amplification we include such blocking probes.
  • oligonucleotide probe of the invention By “selectively binding” we include the ability of an oligonucleotide probe of the invention to anneal (i.e. bind) selectively to the one or more polynucleotide sequence generated by methylation-specific PCR amplification and to do so more strongly than to polynucleotide sequences not generated by methylation-specific PCR amplification.
  • the oligonucleotide probe may bind at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or 1000-fold more strongly to the one or more polynucleotide sequence generated by methylation-specific PCR amplification than to polynucleotide sequences not generated by methylation-specific PCR amplification.
  • an oligonucleotide probe to bind to a polynucleotide target will depend on the particular conditions to which they are subjected. For example, those skilled in the art would understand that the pH, temperature and/or salt concentration used may affect the manner in which an oligonucleotide probe selectively binds to a polynucleotide target.
  • the stringency of the conditions can be used to determine the strength of binding between the oligonucleotide probe and polynucleotide target (as described, for example, in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press).
  • the invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises: one or more reagent for bisulfite conversion of the leukocyte DNA;
  • an oligonucleotide primer for determining the polynucleotide sequence at a first CpG site selected from those listed in Table 3;
  • kits of this embodiment of the invention is suitable for determination of the methylation state by DNA sequencing, as discussed above. DNA sequencing is well known to those skilled in the art and described, for example, in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press).
  • the invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises: - one or more reagent for bisulfite conversion of the leukocyte DNA;
  • kits of this embodiment of the invention is suitable for determination of the methylation state by methylation-specific restriction enzyme analysis or COBRA analysis, as discussed above.
  • kits of the invention comprise one or more probe selected from those listed in Tables 3 and/or 6 and/or 7 and/or 8.
  • kits of the invention further comprise one or more control sample.
  • the invention generally relates to Inflammatory Bowel Diseases (such as Crohn's Disease or Ulcerative Colitis).
  • the one or more control sample is preferably a DNA sample which is indicative of the presence of Inflammatory Bowel Disease and/or a DNA sample which is indicative of the absence of Inflammatory Bowel Disease.
  • the one or more control sample comprises leukocyte DNA in which the methylation state of the first CpG site and/or the second CpG site is indicative of the presence of Inflammatory Bowel Disease in an individual.
  • the one or more control sample may be leukocyte DNA in which the first CpG site and/or the second CpG site is methylated, or in which the first CpG site and/or the second CpG site is un-methylated.
  • the one or more control sample comprises leukocyte DNA from a control individual which has Inflammatory Bowel Disease and/or leukocyte DNA from a control individual which does not have Inflammatory Bowel Disease. Such control individuals are discussed herein.
  • the kit of the invention relates to Ulcerative Colitis
  • the one or more control sample is preferably a DNA sample which is indicative of the presence of Ulcerative Colitis and/or a DNA sample which is indicative of the absence of Ulcerative Colitis.
  • the one or more control sample comprises leukocyte DNA in which the methylation state of the first CpG site and/or the second CpG site is indicative of the presence of Ulcerative Colitis in an individual.
  • the one or more control sample may be leukocyte DNA in which the first CpG site and/or the second CpG site is methylated, or in which the first CpG site and/or the second CpG site is un-methylated.
  • the one or more control sample comprises leukocyte DNA from a control individual which has Ulcerative Colitis and/or leukocyte DNA from a control individual which does not have Ulcerative Colitis. Such control individuals are discussed herein.
  • the invention relates to Crohn's Disease.
  • the one or more control sample is preferably a DNA sample which is indicative of the presence of Crohn's Disease and/or a DNA sample which is indicative of the absence of Crohn's Disease.
  • the one or more control sample comprises leukocyte DNA in which the methylation state of the first CpG site and/or the second CpG site is indicative of the presence of Crohn's Disease in an individual.
  • the one or more control sample may be leukocyte DNA in which the first CpG site and/or the second CpG site is methylated, or in which the first CpG site and/or the second CpG site is un-methylated.
  • the one or more control sample comprises leukocyte DNA from a control individual which has Crohn's Disease and/or leukocyte DNA from a control individual which does not have Crohn's Disease. Such control individuals are discussed herein.
  • the one or more reagent for bisulfite conversion of the leukocyte DNA comprises:
  • the individual is a child and the gene is selected from the group comprising: TOLLIP; SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2;
  • the gene is: TOLLIP.
  • two or more genes are selected from the group comprising: TOLLIP and RPS6KA2; or SEPT9 and RPS6KA2; or TOLLIP and MY01E; or RPS6KA2 and YWHAE; or RPS6KA2 and cg0930439; or VMP1 and MY01 E; or YWHAE and ARHGEF3; or cg251 1461 1 and AIM2; or cg2511461 1 and BCL3; or cg2511461 1 and cg01101459; or cg2511461 1 and cg02719954; or cg25114611 and cg04086394; or cg251 14611 and cg09349128; or cg25114611 and cg12807764; or cg25114611 and cg19445588; or cg251 1461 1 and cg19821297; or cg2511
  • the individual is an adult and the CpG sites are selected from the group comprising: SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2; SBN02; TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9; SPARC; SUB1 ; TRAM2; KCNAB2; BBS9; AGL; ICA1 ; FRMD4A; FKBP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3; PALM; ICA1 ; BAHCC1 ; LRRC47; cg01101459; cg02719954; cg04086394; cg12807764
  • the two or more genes are selected from the group comprising: SEPT9 and RPS6KA2; or RPS6KA2 and YWHAE; or RPS6KA2 and cg0930439; or VMPI and MY01 E; or YWHAE and ARHGEF3; or cg251 14611 and AIM2; or cg25114611 and BCL3; or cg25114611 and cg01101459; or cg25114611 and cg02719954; or cg25114611 and cg04086394; or cg25114611 and cg09349128; or cg2511461 1 and cg12807764; or cg251 1461 1 and cg19445588; or cg251 1461 1 and cg19821297; or cg25114611 and cg20519581 ; or cg251 1461 1 and
  • Figure 2 (A) 7 examples of separation by diagnosis in considering M values (log2[proportion of methylated probes/proportion of unmethylated probes]) for pairs of lllumina 450K probes. Column headings give Probe 1 v Probe 2 and (Sensitivity, specificity) of models based on these probe pairs in discovery (left) and replication (right) cohorts. Results for paediatric discovery and paediatric replication cohorts are shown in the top and bottom rows respectively. (B) Replication in adults (20v20) by pyrosequencing of methylation differences in highly significant probes from the combined paediatric analysis.
  • Figure 3 Schema for the selection of targets for further study, showing examples and total numbers for each set.
  • GWAS risk loci correspond to all CD and IBD results from GWAS meta-analysis. 1 Inclusion within epigenome-wide significance and differentially methylated regions sets based on individual probe significance surviving Bonferroni correction, and being identified as a DMR by the modified ChAMP algorithm respectively (Methods). VMP1/MIR21 shown at the intersection of all three sets.
  • FIG. 4 (A and B) Schematics of VMP1 and MIR21 showing the distribution of lllumina 450k probes coloured by significance in the combined paediatric data (Black - Bonferroni corrected P ⁇ 0.05, Grey - FDR corrected P ⁇ 0.05, unfilled - non-significant). Plotted as (A) histogram by position across VMP1 (B) Position and log-io P value around MIR21. (pri- mir21 - primary transcript, mir21 - mature transcript). (C) Methylation results across 15 lllumina 450k probes within VMP1 for all paediatric samples. X axis shows hg19/GRCh37 coordinates (not to scale), shaded region corresponds to DMR in (B).
  • Figure 5 (A) Replication of MIR21 hypomethylation in CD in 172 adults at cg16936953 by pyrosequencing. (B) Increased leucocyte pri-mir21 mRNA in CD measured by qPCR. (C) Microarray data showing significantly increased pri-mir21 mRNA in response to inflammation in CD and UC. VMP1 increased in CD, but not UC or control 3 .
  • Figure 6 Experimental data showing M values for preferred models of the invention.
  • Figure 7 Experimental data showing M values for preferred models of the invention - Crohn's Disease vs. Control.
  • Figure 8 Experimental data showing M values for preferred models of the invention - Ulcerative Colitis vs. Control.
  • Table 1 lllumina 450k probes with significant (FDR adjusted P ⁇ 0.05) methylation differences in the discovery cohort which replicated in the second cohort (P ⁇ 0.05).
  • Table 2 Differentially Methylated Regions - Top DMRs from combined paediatric lllumina 450k data ranked by (A) most significant individual probe, (B) density of DMRs in a region, (C) proximity to GWAS SNPs. Coordinates refer to hg19/GRCh37. (up arrow - Hypermethylated, down arrow - Hypomethylated, dash - Intergenic probe)
  • Table 3 Details of the CpG sites and probes used in the present invention.
  • the "probe start”, “probe end” and “probe target” numbers indicate the coordinates of the probe target.
  • the term “NA” indicates that the CpG site and probe is not currently known to be associated with a gene.
  • NA indicates that the CpG site and probe is not currently known to be associated with a gene.
  • Table 5 Details of the Differentially Methylated Regions used in the present invention.
  • the "DMR start”, “DMR stop” and “coordinate” numbers indicate the coordinates of the probe target; specifically, the coordinate of one probe within the DMR.
  • the term “NA” or a blank gene symbol indicates that the DMR is not currently known to be associated with a gene.
  • Table 6 Details of the gene probes used in the present invention.
  • Table 7 Details of the Differentially Methylated Regions and probes used in the present invention.
  • TMEM49 is a synonym for VMP1.
  • Table 8 lllumina 450k probes with significant (FDR adjusted P ⁇ 0.05) methylation differences in the following cohorts:
  • NA - adult Ulcerative Colitis.
  • the term "NA” indicates that the CpG site and probe is not currently known to be associated with a gene.
  • the present invention is also described by reference to the following numbered paragraphs, which relates to particularly preferred embodiments of the invention:
  • a method for determining the presence of Crohn's Disease in an individual comprising the steps of:
  • each CpG site is selected from those in Table 3;
  • a method for predicting the likelihood of an individual contracting Crohn's Disease comprising the steps of:
  • each CpG site is selected from those in Table 3;
  • a method for selecting a treatment for an individual suspected of having Crohn's Disease comprising the steps of:
  • a method for predicting the response to anti-Crohn's Disease treatment of an individual suspected of having Crohn's Disease comprising the steps of:
  • the step of determining the methylation state comprises determining the methylation state at three or more CpG sites in the leukocyte DNA, for example, four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more, or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or more, or 110 or more, or 120 or more, or 130 or more, or 140 or more, or 150 or more, or 160 or more, or 165, CpG sites in the leukocyte DNA.
  • step of determining the presence of Crohn's Disease in the individual comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
  • step of predicting the likelihood of the individual contracting Crohn's Disease comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
  • CpG sites are associated with two or more genes selected from those listed in Table 4, for example, three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more genes selected from those listed in Table 4.
  • each of the CpG sites is associated with a different gene selected from those listed in Table 4.
  • each CpG site is located in a regulatory region of the associated gene.
  • CpG sites are within two or more genetic loci selected from those listed in Table 5, for example, three or more, or four or more, or five or more; or six or more, or seven or more, or eight or more, or nine or more, or ten or more, genetic loci selected from Table 5.
  • each of the CpG sites is associated with a different genetic locus selected from those listed in Table 5.
  • the CpG sites are selected from the group comprising:
  • VMP1 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544
  • VMP1 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544
  • MY01 E 15:59588622
  • the gene is selected from the group comprising: TOLLIP; SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2.
  • the CpG sites are selected from the group comprising:
  • VMP1 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544
  • MY01E 15:59588622
  • the gene is selected from the group comprising: SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3: 101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2.
  • the two or more genes are selected from the group comprising:
  • the sample is selected from the group consisting of: a blood sample; a serum sample; a plasma sample; a sample of intestinal cells; a biopsy; a stool sample.
  • the leukocyte DNA is obtained from the sample by a method comprising the steps of: (i) cell lysis; (ii) removal of cell membranes and/or cellular RNA and/or cellular protein; (iii) purification of DNA.
  • methylation state is determined by a method selected from the group comprising: bisulfite conversion and DNA sequencing; methylation-specific restriction enzyme analysis; COBRA analysis; methylation-specific PCR; MethylLight analysis; HeavyMethyl analysis; Quantitative Methylation analysis; Ms-SNuPE analysis.
  • kits for performing a method according to any of paragraphs 1 to 33 to a use according to any of Claims 34 to 37 comprising one or more reagent for determining the methylation state at two or more CpG sites in leukocyte DNA, wherein each CpG site is selected from those in Table 3.
  • the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
  • the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
  • an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation- specific PCR amplification.
  • kit of paragraph 40 further comprising:
  • an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the first CpG site when the first CpG site is not methylated
  • an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the second CpG site when the first CpG site is not methylated.
  • the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
  • oligonucleotide primer for determining the polynucleotide sequence at a first CpG site selected from those listed in Table 3;
  • the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
  • an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation-specific digestion.
  • kit of any of paragraphs 38 to 43 further comprising one or more control sample comprising leukocyte DNA in which the first CpG site and/or the second CpG site is methylated.
  • kit of any of paragraphs 38 to 43 further comprising one or more control sample comprising leukocyte DNA in which the first CpG site and/or the second CpG site is not methylated.
  • kit of any of paragraphs 39 to 45, wherein the one or more reagent for bisulfite conversion of the leukocyte DNA comprises:
  • Probe ID Symbol Chromosome Probed Sequence Probe start Probe End Probe Target cg12054453 V P1 17 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 57915717 57915766 57915717 cg 7501210 RPS6KA2 6 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 166970204 166970253 166970252 eg 12992827 NA 3 CCTCTGCCATGCATCAG I I I I I I CTTGGTTGGCCACTGATTAATAATCATCG 101901234 101901283 101901234 eg 16936953 VMP1 17 GAGCCTCCAGGTACTCCTGAAATGGCTTCTGCAGAGATGGACCTATGCCG 57915665 57915714 57915665 cg04389058 ARHGEF3 3 CGTGCCTTCTGCTGAGGCTGAGCTGGAATTGA I I I I I GTTTATTCCAAAAT
  • cg09588020 WASF2 1 CGGTACAGATCCTATCCCTGGGGTGTTCATAACCAGGACAGATCAGATTC 27755803 27755852 27755803 eg 12662084 KIF13A 6 AGGAAGTTTGCTTCCCTCACCAAGGTATTAGCTTTAACCAGCTGCTCTCG 17809078 17809127 17809126 cg03725573 ZBTB 6 11 GTAGCCAGCCCTGGAAGGCCTGAATGCACAAACACAGTGCCCTTTCACCG 113962901 113962950 113962901 cg25653947 NA 8 ACATACAGGGGATCATGTGGGGGTGGTGCTCCCCGGTGCCCGCACCATCG 144443217 144443266 144443217 cg27361520 IL18 AP 2 CGCCTTGCTCTCAGCAGACAGCCAGGTCAGCATCAGCAGCCAGGTGGTAG 103038171 103038220 103038171 cg16739178 NA 16
  • Probe ID Gene Symbol Chromosome Coordinates Probed Sequence Probe Start Probe End Probe eg 12054453 VMP1 17 57915717 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 57915717 57915766 57915 eg 12992827 NA 3 101901234 CCTCTGCCATGCATCAG I I I I CTTGGTTGGCCACTGATTAATAATCATCG 10 901234 101901283 10190 cg01749539 Sept 9 17 75473969 GAACACGGTTGTGCAAGGATCTGTCTGGGTCCCTGCTTTCCATTCTTTCG 75473969 75474018 75473 cg26599989 TOLLIP 11 1297087 CGGATGTAAACCCACTGATAACGGACAGAAAGAGAATGCCCACAAGTGGG 1297039 1297088 12970 cg16936953 V P1 17 57915665 GAGCCTCCAGGTACTCCTGAAATGGCTTCTGCAGAGATGG
  • Probe ID Gene Symbol Chromosome Coordinate Probed Sequence Coordinate Coordinate Coordin eg 16936953 TME 49 17 57915665 GAGCCTCCAGGTACTCCTGAAATGGCTTCTGCAGAGATGGACCTATGCCG 57915665 57915714 579156 cg12054453 TMEM49 17 57915717 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 57915717 57915766 579157 cg01409343 TMEM49 17 57915740 CGC I I I I I GTGGTGAAGCTTCTGCCGTTGAGCCTCCAGGTACTCCTGAAAT 57915692 57915741 579157 cg18942579 TMEM49 17 57915773 GCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACTTTAATCCG 57915725 57915774 579 57 cg02560388 2 11969958 CGTGAGGCAGAATCCCCTGAAGGACT
  • OVOL1 11 65561705 CGCAGGAAGCCATGGTCTCTGCTCTGGGGGTCTGTCAAGTGGCCCATGTC 65561657 65561706 655617 cg09181559 OVOL1 11 65561733 CGCGAGTGGCATTACCTTCATCTTGGTGCAGGAAGCCATGGTCTCTGC 65561685 65561734 655617 eg 15453482 OVOL1 11 65561749 CGGGGGCGTGCATGTAGACCTGCGTCCAGCTCATGAGACATGGCACAAAT 65561749 65561798 655617 cg03062944 10 6183455 ATCCGGGATTGAACAACGAACTCGCCACAGCCGTGCTGGTCAGGGAAACG 6183455 6183504 618345 cg04 89187 10 6183528 CGCCCTGTTCTATCGCCGCATGGATCTGCAGAGGCAGCCGTTCCTGCCGG 6183528 6183577 618352 e
  • TNFSF12- cg10479431 TNFSF13 17 7461421 CGGCCTCCTTGGCCTAGGAGGCAGACTTCCTCCTGCGGGGAAATCCCTTC 7461421 7461470 746142
  • TNFSF12- cg05514680 TNFSF13 17 7461556 CGCCCCACCTCCCAGCCAGGGCCTGTGCCAGCACCTGCTGAATGTCCGAG 7461508 7461557 746155 cg23161218 TNFSF13 17 7461638 CGGGATTCCTCCCCAGCACACAGGGTTCCGGGTTCCCGTGTGCCTGCTGA 7461590 7461639 746163 cg13358186 TNFSF13 17 7461775 TTGCCAATTTCAGCACAGGGAGTAGTGCAGGCCTTATTCCAACACACCCG 746 727 7461776 746177 cg03607916 2 58478696 CGATGCTGCCGCAGGGTCTGAGGATGAGGCTGGAGCCGCAGCGGGAACCG 58478696 58478745 584786 cg25724323 2 58478735 CGGCAGCATCGGCTACAACTGCGGCTCCTGGCCAGACCCCGGCCTCAGCG
  • cg04682802 BTBD3 20 1 1898478 CGGAGCACTGGCTGTCTGACTCCATCTGCAGGGCTGTAATACCTACTCTC 11898478 11898527 118984 cg05822633 BTBD3 20 11898531 CGGAGGAGAGTAGGTATTACAGCCCTGCAGATGGAGTCAGACAGCCAGTG 11898483 11898532 118985 cg01444716 BTBD3 20 11898557 CGGCTGAACAGACTCACGCAGCTCCAGCCCATCTTGCTGACCTAATTCAG 11898557 11898606 118985 cg01513913 14 1.06E+08 GACACATTCCTCAGCCATCACTAAGACCCCTGGTTTGTTCAGGCATCTCG 1.06E+08 1.06E+08 1.06E+ eg 13074055 14 1.06E+08 CGCACCCAACCTGAGTCCCA I I I I I CCAAAGGCATCGGAAAATCCACAGAG 1.06E+
  • the lllumina 450K platform was applied to assess epigenome-wide methylation profiles in circulating leucocyte DNA in newly diagnosed and established paediatric CD cohorts. Data were corrected for differential neutrophil/mononuclear cell counts. Targeted replication was performed in an adult CD cohort using pyrosequencing. Methylation changes were correlated with gene expression in blood and intestinal mucosa. Results
  • lllumina 450k DNA methylation analysis was initially performed in a paediatric discovery cohort (stage 1) and then later in a paediatric replication cohort (stage 2).
  • the discovery cohort included newly diagnosed cases of paediatric CD and the replication cohort established cases of paediatric CD. Controls for both cohorts were drawn from children who underwent colonoscopy for gastrointestinal symptoms, who had no pathology.
  • the Bacteria in Inflammatory bowel disease in Scottish Children Undergoing Investigation before Treatment (BISCUIT) study provided peripheral blood leucocyte DNA for the discovery cohort from 18 treatment naive, newly diagnosed patients and matched non-diseased controls from Aberdeen, Glasgow and Dundee. Controls had been rigorously investigated for gastrointestinal symptoms but did not have or subsequently develop any organic gastrointestinal pathology including IBD.
  • the replication cohort comprised DNA samples from children with established CD supplied by the Paediatric-onset Inflammatory bowel disease Cohort and Treatment Study (PICTS), 22 analysed against a second set of controls from the BISCUIT study. Within both cohorts patients and controls were matched for age and gender.
  • Peripheral blood leucocyte DNA was bisulphite converted and analysed using the lllumina Human Methylation 450k platform (lllumina, San Diego, USA) 23 with cases and controls distributed across chips. Probes were filtered to remove any with a detection P value of ⁇ 0.01 , those from sex chromosomes and those that had single nucleotide polymorphisms (SNPs) with a minor allele frequency of >0.01 in the European population in the 1000 Genomes Project. 24 Samples were removed if there was a gender mismatch or if more than 5% of probes failed. In the first cohort all samples passed QC, but 1 case and 4 controls were excluded from the final analysis as they lacked contemporaneous full blood count data. In the second cohort, one sample failed QC due to gender mismatch.
  • SNPs single nucleotide polymorphisms
  • DMRs were defined using a modification of the lasso technique employed by the ChAMP pipeline (Morris ef al. (2014), Bioinformatics 30(3): 428-430 and at: http://www2.cancer.ucl.ac.uk/medicalgenomics/champ/), which defines a DMR as a genetic region (size defined by the probe density at that location within the genome) of 3 or more probes, all with FDR corrected P ⁇ 0.05. We added the constraint that all probes should share the same direction of change in methylation.
  • DNA was bisulphite converted with EZ-96 DNA MethylationTM Kits (Zymo Research, Irvine, USA), assays were designed using PyroMark Assay Design Software V2.0.1.15 (Qiagen, Dusseldorf, Germany) and primers were ordered from Sigma-Aldrich (St. Louis, USA). Sequencing was performed on a Pyromark Q96 ID machine (Qiagen) and analysed in R V3.0.1. VMP1 and MIR21 expression
  • MIR21 primary transcript (pri-mir21 ) was assayed by qPCR (Figure 5B).
  • Sample collection was performed using SAHSC BioResource ethical approval (REC Reference 06/S1101/16), with all patients and controls giving written, informed consent.
  • Suitable CD patients were prospectively recruited from gastroenterology clinic and endoscopy lists, and healthy controls recruited from volunteers.
  • Blood samples were taken using a 21 gauge butterfly needle and 9ml K3 EDTA vacuette® (Greiner, Germany) and stored at 4°C for up to 2 hours.
  • Total RNA was then extracted from 1.5ml whole blood using QIAamp RNA blood mini kit (Qiagen), and stored at -80°c.
  • cDNA was converted using Superscript® ViloTM cDNA synthesis kits (Invitrogen, Carlsbad, USA) and analysed on a Corbett Rotor- Gene 6000 (Qiagen) with DyNAmo Flash SYBR green reagent (Thermo Scientific, Waltham, USA).
  • Expression of pri-miR21 was normalised to reference gene TBP) after initial optimization against 4 reference genes ⁇ GAPDH, TBP, SDHA and ACTB), and analysed by the AACt method in R. Expression in intestinal biopsies as described previously. 31
  • Models were derived using the paediatric discovery cohort and tested on the paediatric replication cohort. Probes were selected based on significance in the discovery cohort (Table 1 ) not the combined analysis. Linear discriminant analysis was performed on M values (post-QC and normalisation for neutrophil : lymphocyte ratio) in R 3.0.1 , using the Ida function in the 'MASS' package. 32
  • VMP1 has five highly significant probes within 50kb of GWAS SNP rs1292053 and accounts for a large proportion of all probes at low P value thresholds, resulting in very high percentages of colocalization at low P values (50% for P values ⁇ 1x10 12 within 50kb), we therefore only show P values up to 10 "7 in figure 1 C to not overshadow the effect. Exclusion of VMP1 from analysis (by using a distance threshold of 45kb) preserves the trend seen in figure 1C. Due to intrinsic design factors in methylation and genotyping platforms there is a positive correlation between density of methylation probes and distance to GWAS SNPs (not shown). To help control for these effects probes were grouped into 1kb bins and considered by the lowest P value they contain.
  • Candidate biomarkers were identified from a preliminary analysis of the replication cohort. Using the discovery cohort as a learning set, multiple models were constructed to predict the presence of CD based on age, sex and methylation of two CpG sites using linear discriminant analysis. Mean sensitivities and specificities of all possible models were 95% and 96% respectively, with 19% of models achieving 100% sensitivity and specificity.
  • this gene may represent an interesting target for further investigation as a paediatric specific marker, or provide insight into biological differences between adult-onset and paediatric CD.
  • VMP1 Five probes within VMP1 reached the highest significance level, 4 of which lie within a DMR ( Figure 4). These probes are clustered at the 3' end of VMP1, around the 1 1 th exon, within 50kb of a GWAS SNP (rs1292053).
  • the DMR is directly adjacent to the transcription start site and promoter region for the primary transcript of MIR21 (pri-miR21 ).
  • VMP1 encodes a transmembrane protein located in the Golgi apparatus, endoplasmic reticulum and vacuoles with high degrees of expression in the intestine, kidney, ovary and placenta. 34 There is high trans-species conservation of VMP1, and it is noteworthy that expression induces autophagy through interactions with BECN1.
  • MIR21 was one of the earliest described microRNAs and has been implicated in numerous cancers, including IBD-associated colorectal cancer. 37 The mature sequence is produced from a precursor overlapping with the 3' end of VMP1. This region is highly conserved, exhibits DNAse I hypersensitivity and is associated with the promoter associated histone marks H3K4Me1 and H3K4Me3. 38
  • MIR21 has a known role in T-cell differentiation and development. 39-42 Increased expression of MIR21 in active IBD and IBD-associated dysplasia has been described elsewhere 43 44 and MIR21 knock-out mice have been shown to be protected from DSS- induced colitis. 45
  • ATG16L1 can be regulated by multiple microRNAs, with resulting effects on autophagy 49 50 - particularly interesting with respect to our data as ATG16L1 contains an MIR21 target motif. 51 Epigenome-wide significance
  • the Bonferroni correction for multiple testing is overly conservative, as it ignores the correlation of methylation between neighbouring probes.
  • the Benjamini-Hochberg FDR of 5% for inclusion of probes in analysis for wider patterns beyond individual CpG significance is necessary to ensure sufficient data is included - though it may be considered insufficiently discriminating for reporting individual CpGs. Linear Discriminant Analysis
  • methylation states of specific DNA sequences have found use as biomarkers, with some notable successes in cancer, 52,53 based on sensitively detecting abnormal methylation states, such as methylation at a tumour suppressor CpG Island.
  • the results of our linear discriminant analysis of the methylation results serve as a proof of concept for the development of diagnostic biomarkers in complex diseases. Future work should also seek to establish links with other clinical outcomes such as response to treatment and disease course.
  • DNA methylation Whilst the differences in DNA methylation are the composite effects of several factors including shifts in immune cell populations, environmental exposures, variations in the microbiome and altered expression linked to genetic variation, they are eminently suitable as biomarkers. In addition, DNA methylation is chemically stable, amplifiable and can be tested cheaply and easily.
  • Example 3 outlines an exemplary method for performing Linear Discriminant Analysis.
  • Figure 6 provides experimental data showing M values for preferred models of the invention. Strengths and limitations
  • the present study provides impetus for further analysis of alterations of leucocyte DNA methylation in IBD and other complex diseases, with many targets emerging for further study.
  • the strength and reproducibility of our findings compare favourably with epigenetic data generated to date in IBD and other complex diseases 10,12 and also with the results of theoretical modelling based on predicted disease-associated methylation patterns.
  • the magnitude and variance of observed methylation changes in whole blood contrasts with models used to predict required group sizes. 9
  • These data may inform future study design in CD and other complex diseases.
  • the enrichment of methylation differences near to previously described risk loci from GWAS raises the possibility that epigenetic modifications may help identify specific points within large susceptibility loci where genetic and biological variation overlap.
  • ARHGEF3 3 57041402 -0.74 6.5x10- 7 -0.48 3.1x10 "7 -0.07 -0.56 3.7x10- 12 1.7 ⁇ 10- 6
  • Top DMRs from combined paediatric lilumina 450k data ranked by (A) most significant individual probe, (B) density of DMRs in a region, (C) proximity to GWAS SNPs. Coordinates refer to hg19/GRCh37. (up arrow - Hypermethylated, down arrow - Hypomethylated, dash - Intergenic probe) References
  • Ventham NT a, Kennedy N a, Nimmo ER, et al. Beyond gene discovery in inflammatory bowel disease: the emerging role of epigenetics. Gastroenterology 2013; 145:293-308.

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Abstract

The present invention relates to methods and uses for determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in an individual, and predicting the likelihood of an individual contracting the disease. Kits for performing the methods and uses of the invention are also disclosed.

Description

METHODS AND USES FOR DETERMINING THE PRESENCE OF INFLAMMATORY BOWEL DISEASE
The present invention relates to methods and uses for determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in an individual, and for predicting the likelihood of an individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis).
Inflammatory Bowel Disease is a group of inflammatory conditions of the colon and small intestine, the principal types of which are the conditions Crohn's Disease and Ulcerative Colitis. Crohn's Disease is a chronic inflammatory condition of the gastrointestinal tract, which is also known as Crohn's Syndrome or regional enteritis. It affects approximately 1 in 200 of the Western population (Busch et a/., 2013, Aliment. Pharmacol. Ther., 10.11 1 1/apt.12528), and patients most-commonly present with symptoms of the disorder in childhood or in early adult life (Sonnenberg, 2010, Inflamm. Bowel Dis., 16:452-7).
The symptoms associated with Inflammatory Bowel Disease (and, particularly, Crohn's Disease) typically include abdominal pain, diarrhea, weight loss, mouth ulcers, fever, joint pains, fatigue and malaise. Diagnosis is made on the basis of: clinical suspicion; blood tests showing anemia; malnutrition; blood and stool tests which provide non-specific evidence of inflammatory disease; endoscopic appearances of stomach and colon; and histological examination of biopsies taken at endoscopy.
Inflammatory Bowel Disease (and, particularly, Crohn's Disease) has an increasingly-large economic impact due to costly medication, frequent hospitalisations and surgeries, and loss of productivity (van der Valk er a/., 2012, Gut, 10.1136/gutjnl-2012-303376).
Because Inflammatory Bowel Disease (such as Crohn's Disease and Ulcerative Colitis) is associated with a wide range of clinical symptoms, many of which are also present in other, unrelated gastrointestinal disorders, it can prove difficult to identify whether an individual has the disease or not based purely on the symptoms presented. At present, there is no diagnostic biomarker that is sensitive and specific and which permits identification of Inflammatory Bowel Disease (such as Crohn's Disease and Ulcerative Colitis) in an individual. Thus, those skilled in the art of medicine may identify individuals suspected of having such disease, but often be unable to reach a determinative diagnosis based on symptomology. The difficulties associated with reaching a determinative diagnosis mean that many cases of Inflammatory Bowel Disease (such as Crohn's Disease and Ulcerative Colitis) will go untreated or, more commonly, individuals with an unrelated disorder will be treated anyway. Since the treatments are costly, aggressive and associated with serious side- effects, that is undesirable.
Against that background, the present inventors have surprisingly identified that the presence of, and predisposition to, Inflammatory Bowel Disease (such as Crohn's Disease and Ulcerative Colitis) effectively correlates with the methylation state of certain sites in the genome of affected individuals.
The inventors' findings thus provide both diagnostic and prognostic information in relation to those disorder and, for the first time, allow methods for identifying the presence of Inflammatory Bowel Disease (such as Crohn's Disease and Ulcerative Colitis).
Accordingly, in a first aspect the invention provides a method for determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in an individual, comprising the steps of: - providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual on the basis of the methylation state at the two or more CpG sites.
Inflammatory Bowel Disease is a group of inflammatory conditions of the colon and small intestine, the principal types of which are the conditions: Crohn's Disease and Ulcerative Colitis. Thus, by "Inflammatory Bowel Disease" we include the conditions Crohn's Disease and Ulcerative Colitis. As discussed herein, it is preferred that the Inflammatory Bowel Disease is selected from Crohn's Disease and Ulcerative Colitis; most preferably, the invention relates to Crohn's Disease.
Preferably, the first aspect the invention provides a method for determining the presence of Crohn's Disease in an individual, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual; - determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- determining the presence of Crohn's Disease in the individual on the basis of the methylation state at the two or more CpG sites.
Crohn's Disease is a chronic inflammatory condition of the gastrointestinal tract, which is also known as Crohn's Syndrome or regional enteritis. It can affect any part or structure of the gastrointestinal tract from the mouth to the anus, for example one or more of: the mouth; oesophagus; stomach; small intestine (which comprises the duodenum, jejunum and ileum); large intestine (which comprises the cecum, colon and rectum); and the anus.
Crohn's Disease and Ulcerative Colitis are associated with similar symptoms. Medical practitioners generally distinguish between the two conditions on the basis of the location and nature of the inflammatory changes. As discussed above, Crohn's Disease can affect any part or structure of the gastrointestinal tract - by contrast, Ulcerative Colitis is restricted to the colon and the rectum. Additionally, Crohn's Disease affects the full thickness of the bowel wall ("transmural lesions"), whilst Ulcerative Colitis is restricted to the mucosa (i.e. the epithelial lining of the gut). Crohn's Disease and Ulcerative Colitis are associated with a wide range of symptoms that are known to those skilled in the arts of medicine and pharmacy, and which often present in re-occurring periods of appearance and remission. Individuals with Crohn's Disease or Ulcerative Colitis generally present with one or more symptom selected from: one or more gastrointestinal symptom; one or more systemic symptom; and/or one or more extra- intestinal symptom.
The one or more gastrointestinal symptom is selected from: diarrhea; bloody diarrhea; intestinal stenosis; vomiting; nausea; ulcerative colitis; primary sclerosing cholangitis; abdominal pain; difficulty swallowing; abscesses, mouth ulcers; itching and/or fistulization of the anal area; small bowel bacterial overgrowth syndrome; orofacial granulomatosis; pyostomatitis vegetans; recurrent aphthous stomatitis; geographic tongue; migratory stomatitis; and/or incontinence.
The one or more systemic symptom is selected from: weight loss; malaise; fatigue and/or stunted growth. The one or more extra-intestinal symptom is selected from: fever; joint pain; inflammation of the eye (including episcleritis and uveitis), which can lead to blindness; gallstones; rheumatologic disease (including seronegative spondyloarthropathy, arthritis, enthesitis, ankylosing spondylitis and/or sacroiliitis); erythema nodosum; pyoderma gangrenosum; blood clots (including deep venous thrombosis); autoimmune hemolytic anemia; anemia; osteoporosis; neurological complications (including seizures, stroke, myopathy, peripheral neuropathy, headaches, and/or depression).
Typically, individuals having Crohn's Disease or Ulcerative Colitis display one or more of the following symptoms: abdominal pain; diarrhea; weight loss; mouth ulcers; fever; joint pain; fatigue; and malaise.
Because Crohn's Disease and Ulcerative Colitis are associated with a wide range of clinical symptoms, many of which are also present in other, unrelated gastrointestinal disorders, it can prove difficult to identify whether an individual has Crohn's Disease or not (or Ulcerative Colitis or not) based purely on the symptoms presented. Thus, those skilled in medicine may identify individuals suspected of having Crohn's Disease or Ulcerative Colitis, but be unable to reach a determinative diagnosis based on symptomology. In the method of the first aspect of the invention, it is preferred that the individual is one which is suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis). For example, such an individual preferably displays one or more symptom of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), as discussed above. As discussed above, those skilled in the art will be capable of identifying such individuals, which is typically established based on known approaches, such as the identification of clinical symptoms (as discussed above); blood tests showing the presence of anemia; evidence of malnutrition; blood and stool tests showing nonspecific evidence of inflammatory disease; endoscopic appearance of the stomach and colon; and histological examination of biopsies taken at endoscopy. In the method of the first aspect of the invention, it is preferred that the individual is one which is suspected of having Crohn's Disease and such an individual preferably displays one or more symptom of Crohn's Disease, as discussed above.
The invention is based on the determination of the methylation state of leukocyte DNA from the individual. As will be well understood by those skilled in the art, leukocytes are white cells of the immune system which circulate in the bloodstream (and which are also known as white blood cells). Leukocytes include granulocytes (i.e. polymorphonuclear leukocytes, which include neutrophils, basophils and eosinophils) and agranulocytes (i.e. mononuclear granulocytes, which include lymphocytes, monocytes, and macrophages). Accordingly, it is preferred that the leukocyte of the present invention is one or more cell type selected from the group consisting of: granulocytes; agranulocytes; neutrophils; basophils; eosinophils; lymphocytes; monocytes; and/or macrophages.
Thus, by "leukocyte DNA", we mean the DNA present in a leukocyte. Preferably the DNA is genomic DNA. Methods for isolating and/or purifying leukocyte DNA are known in the art (as described, for example, in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press), and include those described herein and in the accompanying Examples.
The term "CpG site" refers to a dinucleotide within a polynucleotide sequence, in which a cytosine nucleotide and a guanine nucleotide are arranged next to one another in a linear sequence and are covalently joined to one another by a phosphodiester bond. Whilst a CpG site may be present in any type of polynucleotide sequence in the leukocytes DNA, it is preferred that the CpG site is present in genomic leukocyte DNA.
The term "methylation state at a CpG site" refers to the presence or absence of a methyl group bound to the cytosine nucleotide of the CpG site, which thereby forms 5- methylcytosine (commonly abbreviated to "5-mCyt"). Methylation of CpG sites is a well- recognised modification of cellular DNA and is known to contribute to and influence cellular processes, such as the regulation of gene expression and nucleic acid replication. A CpG site in a polynucleotide sequence may or may not be methylated. Where multiple CpG sites are present in a polynucleotide sequence, then some, none, or all of those sites may be methylated.
In the context of the present invention, where a polynucleotide sequence is double- stranded, the "CpG site" refers to the corresponding nucleotide sequence and site on both the sense- and anti-sense strands, and "methylation state" refers to the methylation state of that CpG site on the sense- and the anti-sense strand.
By "determining the methylation state" we mean determining whether a CpG site has a methyl group bound (i.e. is methylated), or not (i.e. is un-methylated). It will be appreciated that the methylation state of a CpG site can be determined by assaying for the physical presence of the methyl group at that site. Assays for doing so are well known and include those discussed herein and in the accompanying Examples. Methylation of polynucleotide sequences is known to have a functional effect on cellular processes and can, for example, lead to altered expression of associated genes by affecting their translation. For example, hypo-methylation (i.e. reduced levels of methylation) correlates with higher expression of the associated genes, and hyper- methylation (i.e. increased levels of methylation) of a CpG site correlates with lower expression of the associated genes. Methylation state may also be secondary to other factors which influence gene expression (see, for example, Stadler et al., 201 1 , Nature, 480:490). Methylation state is therefore a cellular mechanism for regulating gene expression. Changes in methylation state is therefore thought to alter gene expression. In an alternative embodiment of the invention, the presence of methylation at a particular CpG site or gene could be determined by measuring the presence and/or level of expression and/or transcription of an associated gene.
Methods for determining the transcription of a gene and/or gene expression are well known to those in the art of biochemistry and involve detecting and/or measuring the presence and/or concentration of an associated gene product such as mRNA or protein. Methods of detecting and/or measuring the concentration of mRNA and/or protein are well known to those skilled in the art; see, for example, the methods described in the accompanying Examples and in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press. Differential expression (up-regulation or down regulation) of a gene, or lack thereof, can be determined by any suitable means known to a skilled person. Differential expression may be determined to a p value of at least less than 0.05 (p = < 0.05), for example, at least <0.04, <0.03, <0.02, <0.01 , <0.009, <0.005, <0.001 , <0.0001 , <0.00001 or at least <0.000001.
It is preferred that, particularly in relation to the methods of first, second, third and/or fourth aspects of the invention, the step of determining the methylation state at two or more CpG sites in the leukocyte DNA is performed by determining the physical presence of a methyl group at the two or more CpG sites. In a second aspect, the invention provides a method for predicting the likelihood of an individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), comprising the steps of: - providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- predicting the likelihood of the individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) on the basis of the methylation state at the two or more CpG sites.
In that method of the second aspect of the invention, it is preferred that the individual is one which is known not to have Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), or which is not suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis). For example, such an individual preferably does not display one or more of the symptoms of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) discussed herein.
By "contracting Inflammatory Bowel Disease" we include the meaning that the individual develops Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) and presents with one or more symptom as discussed herein. It will be appreciated that development of Inflammatory Bowel Disease in an individual not previously afflicted with the disorder can occur at any time during the lifetime of that individual. Whilst the causes of Inflammatory Bowel Disease are not completely understood, a number of different factors are likely to be involved, including: genetic disposition to the disorder, the presence or development of autoimmunity or other immune dysfunction; microbial infection; or environmental factors.
Thus, by "predicting the likelihood of an individual contracting Inflammatory Bowel Disease" we include the meaning that an indication of the predisposition of that individual to the disorder, and/or an indication of the risk of that individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) over a given time period, such as the lifetime of the individual. The risk may be calculated as the percentage chance of the individual contracting Inflammatory Bowel Disease within a given time period - for example, a 5% chance of development within 5 years; or a 10% chance of development within 5 years; or a 20% chance of development within 5 years; or a 30% chance of development within 5 years; or a 40% chance of development within 5 years; or a 50% chance of development within 5 years; or a 60% chance of development within 5 years; a 70% chance of development within 5 years; an 80% chance of development within 5 years; a 90% chance of development within 5 years; or a 95% chance of development within 5 years; or a 100% chance of development within 5 years.
Preferably, the second aspect of the invention provides a method for predicting the likelihood of an individual contracting Crohn's Disease, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- predicting the likelihood of the individual contracting Crohn's Disease on the basis of the methylation state at the two or more CpG sites. In that preferred method of the second aspect of the invention, it is preferred that the individual is one which is known not to have Crohn's Disease, or which is not suspected of having Crohn's Disease. For example, such an individual preferably does not display one or more of the symptoms of Crohn's Disease discussed herein. By "contracting Crohn's Disease" we include the meaning that the individual develops Crohn's Disease and presents with one or more symptom of Crohn's Disease discussed herein. It will be appreciated that development of Crohn's Disease in an individual not previously afflicted with the disorder can occur at any time during the lifetime of that individual. Whilst the causes of Crohn's Disease are not completely understood, a number of different factors are likely to be involved, including: genetic disposition to the disorder, the presence or development of autoimmunity or other immune dysfunction; microbial infection; or environmental factors.
Thus, by "predicting the likelihood of an individual contracting Crohn's Disease" we include the meaning that an indication of the predisposition of that individual to the disorder, and/or an indication of the risk of that individual contracting Crohn's Disease over a given time period, such as the lifetime of the individual. The risk may be calculated as the percentage chance of the individual contracting Crohn's Disease within a given time period - for example, a 5% chance of development within 5 years; or a 10% chance of development within 5 years; or a 20% chance of development within 5 years; or a 30% chance of development within 5 years; or a 40% chance of development within 5 years; or a 50% chance of development within 5 years; or a 60% chance of development within 5 years; a 70% chance of development within 5 years; an 80% chance of development within 5 years; a 90% chance of development within 5 years; or a 95% chance of development within 5 years; or a 100% chance of development within 5 years. Thus, the preferred method of the second aspect of the invention provides a method of prognosis of Crohn's Disease in an individual.
In a third aspect, the invention provides a method for selecting a treatment for an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), comprising the steps of: providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3;
- determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual on the basis of the methylation state at the two or more CpG sites; and
- selecting a treatment for the individual. By "an individual suspected of having Inflammatory Bowel Disease" we include an individual presenting with one or more symptom associated with Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), as discussed herein.
Whilst Inflammatory Bowel Disease cannot be cured, anti-Inflammatory Bowel Disease treatments are known and used to manage the disorder and alleviate the associated symptoms. However, as discussed above, because Inflammatory Bowel Disease is associated with a wide range of clinical, non-determinative symptoms, it can prove difficult to identify individuals actually having the disorder and thereby those in need of (i.e. who would benefit from) anti-Inflammatory Bowel Disease treatment.
Current anti-Inflammatory Bowel Disease treatments rely heavily on immunosuppression. Corticosteroids are administered to effectively induce remission of Inflammatory Bowel Disease but are not used to maintain remission. Thiopurines and/or methotrexate are administered to maintain remission. Over the last decade, it has become increasingly common for biological agents (such as antibody therapies) to be administered to induce and maintain remission, such as Adalimimab or Infliximab (both of which are antibody therapies capable of inhibiting TNF activity). All such treatments carry the risk of drug toxicity which can lead to undesirable and unpleasant side-effects in the treated individual.
In addition to those treatments, individuals with Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) will also require at least one intestinal operation to manage the disorder.
Thus, anti-Inflammatory Bowel Disease treatment is aggressive, associated with patient side-effects and expensive if treatment is continued long-term. It is therefore undesirable for all individuals displaying an Inflammatory Bowel Disease symptom to be subjected to such treatments, particularly if it is unclear if Inflammatory Bowel Disease is actually present. The present invention allows the presence of Inflammatory Bowel Disease in an individual to be determined so that an appropriate and necessary treatment can then be selected.
Preferably, where the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) is identified in the individual, the step of selecting a treatment comprises selecting an anti-Inflammatory Bowel Disease treatment. Alternatively, where the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) is not identified in the individual, the step of selecting a treatment comprises selecting a treatment other than an anti-Inflammatory Bowel Disease treatment. More preferably, the step of selecting an anti-Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) treatment comprises selecting one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation. In a preferred embodiment, the method of the third aspect of the invention further comprises the step of treating the individual with the selected treatment.
Preferably, the third aspect of the invention provides a method for selecting a treatment for an individual suspected of having Crohn's Disease, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3;
- determining the presence of Crohn's Disease in the individual on the basis of the methylation state at the two or more CpG sites; and
selecting a treatment for the individual. By "an individual suspected of having Crohn's Disease" we include an individual presenting with one or more symptom associated with Crohn's Disease, as discussed herein. Whilst Crohn's Disease cannot be cured, anti-Crohn's Disease treatments are known and used to manage the disorder and alleviate the associated symptoms. However, as discussed above, because Crohn's Disease is associated with a wide range of clinical, non-determinative symptoms, it can prove difficult to identify individuals actually having the disorder and thereby those in need of (i.e. who would benefit from) anti-Crohn's Disease treatment.
Current anti-Crohn's Disease treatments rely heavily on immunosuppression. Corticosteroids are administered to effectively induce remission of Crohn's Disease but are not used to maintain remission. Thiopurines and/or methotrexate are administered to maintain remission. Over the last decade, it has become increasingly common for biological agents (such as antibody therapies) to be administered to induce and maintain remission, such as Adalimimab or Infliximab (both of which are antibody therapies capable of inhibiting TNF activity). All such treatments carry the risk of drug toxicity which can lead to undesirable and unpleasant side-effects in the treated individual.
In addition to those treatments, most (approximately 75%) individuals with Crohn's Disease will also require at least one intestinal operation to manage the disorder.
Thus, anti-Crohn's Disease treatment is aggressive, associated with patient side-effects and expensive if treatment is continued long-term. It is therefore undesirable for all individuals displaying a Crohn's Disease symptom to be subjected to such treatments, particularly if it is unclear if Crohn's Disease is actually present. The present invention allows the presence of Crohn's Disease in an individual to be determined so that an appropriate and necessary treatment can then be selected.
Preferably, where the presence of Crohn's Disease is identified in the individual, the step of selecting a treatment comprises selecting an anti-Crohn's Disease treatment. Alternatively, where the presence of Crohn's Disease is not identified in the individual, the step of selecting a treatment comprises selecting a treatment other than an anti-Crohn's Disease treatment. More preferably, the step of selecting an anti-Crohn's Disease treatment comprises selecting one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation. In a preferred embodiment, the method of the third aspect of the invention further comprises the step of treating the individual with the selected treatment. In a fourth aspect, the invention provides a method for predicting the response to anti- Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) treatment of an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), comprising the steps of: - providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3;
- determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual on the basis of the methylation state at the two or more CpG sites; and
- predicting the response to anti-Inflammatory Bowel Disease treatment of the individual.
By "an individual suspected of having Inflammatory Bowel Disease" we include an individual presenting with one or more symptom associated with Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) discussed herein.
In the method of the fourth aspect of the invention, the anti-Inflammatory Bowel Disease treatment comprises one or more treatment selected from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
It will be appreciated that, by determining whether an individual has Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), the present invention permits the prediction of whether the anti-Inflammatory Bowel Disease treatment will have a beneficial therapeutic effect on the individual. For example, where Inflammatory Bowel Disease is identified in the individual, the anti- Inflammatory Bowel Disease treatment can be predicted to have a beneficial therapeutic effect on the individual (for example, by alleviating one or more symptom of the disorder; and/or improving the quality of life of the individual; and/or avoiding death and/or hospitalisation and/or surgery of the individual; and/or resulting in remission of the disorder). Conversely, where Inflammatory Bowel Disease is not identified in the individual, the anti-Inflammatory Bowel Disease treatment can be predicted not to have a beneficial therapeutic effect on the individual (for example, by failing to alleviate one or more symptom of the disorder; and/or failing to improve the quality of life of the individual; and/or failing to avoid death and/or hospitalisation and/or surgery of the individual; and/or failing to cause remission of the disorder).
The invention therefore allows decisions to be made regarding the treatment of an individual. Preferably, where the anti-Inflammatory Bowel Disease treatment is predicted to have a beneficial therapeutic effect on the individual, an anti-Inflammatory Bowel Disease treatment can be selected and, more preferably, used to treat the individual. As discussed above, anti-Inflammatory Bowel Disease treatment comprises one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation. Alternatively, where the anti-Inflammatory Bowel Disease treatment is predicted to have a beneficial therapeutic effect on the individual, an anti-Inflammatory Bowel Disease treatment will not be selected and the individual will not be treated with it.
Preferably, the fourth aspect of the invention provides a method for predicting the response to anti-Crohn's Disease treatment of an individual suspected of having Crohn's Disease, comprising the steps of: providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3;
- determining the presence of Crohn's Disease in the individual on the basis of the methylation state at the two or more CpG sites; and
- predicting the response to anti-Crohn's Disease treatment of the individual.
By "an individual suspected of having Crohn's Disease" we include an individual presenting with one or more symptom associated with Crohn's Disease discussed herein.
In that preferred method of the fourth aspect of the invention, the anti-Crohn's Disease treatment comprises one or more treatment selected from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation.
It will be appreciated that, by determining whether an individual has Crohn's Disease, the present invention permits the prediction of whether the anti-Crohn's Disease treatment will have a beneficial therapeutic effect on the individual. For example, where Crohn's Disease is identified in the individual, the anti-Crohn's Disease treatment can be predicted to have a beneficial therapeutic effect on the individual (for example, by alleviating one or more symptom of the disorder; and/or improving the quality of life of the individual; and/or avoiding death and/or hospitalisation and/or surgery of the individual; and/or resulting in remission of the disorder). Conversely, where Crohn's Disease is not identified in the individual, the anti-Crohn's Disease treatment can be predicted not to have a beneficial therapeutic effect on the individual (for example, by failing to alleviate one or more symptom of the disorder; and/or failing to improve the quality of life of the individual; and/or failing to avoid death and/or hospitalisation and/or surgery of the individual; and/or failing to cause remission of the disorder).
The invention therefore allows decisions to be made regarding the treatment of an individual. Preferably, where the anti-Crohn's Disease treatment is predicted to have a beneficial therapeutic effect on the individual, an anti-Crohn's Disease treatment can be selected and, more preferably, used to treat the individual. As discussed above, anti- Crohn's Disease treatment comprises one or more treatment from the group comprising: immunosuppressive therapy; corticosteroids; thiopurines; methotrexate; an anti-TNF agent, such as Adalimimab or Infliximab; intestinal operation. Alternatively, where the anti- Crohn's Disease treatment is predicted to have a beneficial therapeutic effect on the individual, an anti-Crohn's Disease treatment will not be selected and the individual will not be treated with it.
In an embodiment, the methylation state of the one or more CpG sites listed in Table 3 is determined using a method selected from the group comprising: bisulfite conversion and DNA sequencing (for example, using pyrosequencing or Sanger sequencing); methylation- specific restriction enzyme analysis; COBRA analysis; methylation-specific PCR; Methyl Light analysis; HeavyMethyl analysis; Quantitative Methylation analysis; Ms-SNuPE analysis. Such methods are known to those skilled in the art and are discussed further below.
In an embodiment, the methylation state of the one or more CpG sites listed in Table 3 is determined using one or more relevant probe from those listed in Table 3. In a preferred embodiment of the invention, the individual is a child (and preferably a human child). Alternatively, it is preferred that the individual is an adult (and preferably a human adult). By an adult, we include an individual that has reached sexual maturity. By a child, we include an individual between the age of birth and adulthood which has not reached sexual maturity.
Whilst it is preferred that the individual is a human, the individual may also be a non-human mammal {i.e. any mammal other than a human), such as, a horse, cow, goat, sheep, pig, dog, cat, rabbit, mouse or rat. Where the individual is a human child, we mean a human individual between the age of birth and 18 years of age, for example: 1 year of age; or 2 years of age; or 3 years of age; or 4 years of age; or 5 years of age; or 6 years of age; or 7 years of age; or 8 years of age; or 9 years of age; or 10 years of age; or 11 years of age; or 12 years of age; or 13 years of age; or 14 years of age; or 15 years of age; or 16 years of age; or 17 years of age; or 18 years of age.
Where the individual is a human adult, we mean a human individual between the ages of 18 years of age and death, for example: 19 years of age; or 20 years of age; or 30 years of age; or 40 years of age; or 50 years of age; or 60 years of age; or 70 years of age; or 80 years of age; or 90 years of age; or 100 years of age; or older.
Preferably, the step of determining the methylation state comprises determining the methylation state at three or more CpG sites in the leukocyte DNA, for example, four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more, or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or more, or 110 or more, or 120 or more, or 130 or more, or 140 or more, or 150 or more, or 160 or more, or 165, CpG sites in the leukocyte DNA. In the method of the first, third and fourth aspects of the invention, it is preferred that the step of determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual. In the preferred methods of the first, third and fourth aspects of the invention, it is preferred that the step of determining the presence of Crohn's Disease in the individual comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
In the method of the second aspect of the invention, it is preferred that the step of predicting the likelihood of the individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites the leukocyte DNA from one or more control individual. In the preferred method of the second aspect of the invention, it is preferred that the step of predicting the likelihood of the individual contracting Crohn's Disease comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites the leukocyte DNA from one or more control individual.
The skilled person will be capable of selecting an appropriate control for the methods of the invention based on the teaching herein and his common general knowledge.
As discussed herein, the invention generally relates to Inflammatory Bowel Diseases (such as Crohn's Disease or Ulcerative Colitis). It will be appreciated that comparison with a control in the preferred methods of the invention allows the presence or likelihood of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) to be determined.
Where the invention relates to Inflammatory Bowel Disease, the one or more control individual has Inflammatory Bowel Disease or, alternatively, the one or more control individual does not have Inflammatory Bowel Disease. In one embodiment, the one or more control individual comprises one or more individual with Inflammatory Bowel Disease and one or more individual which does not have Inflammatory Bowel Disease.
For example, where the invention relates to Ulcerative Colitis, the one or more control individual has Ulcerative Colitis or, alternatively, the one or more control individual does not have Ulcerative Colitis. In one embodiment, the one or more control individual comprises one or more individual with Ulcerative Colitis and one or more individual which does not have Ulcerative Colitis. As discussed herein, it is preferred that the invention relates to Crohn's Disease. In those preferred aspects of the invention, it is preferable that the one or more control individual has Crohn's Disease. Alternatively, the one or more control individual does not have Crohn's Disease. In one embodiment, the one or more control individual comprises one or more individual with Crohn's Disease and one or more individual which does not have Crohn's Disease. It will be appreciated that comparison with a control in the preferred methods of the invention allows the presence or likelihood of Crohn's Disease to be determined.
For example, the presence of Crohn's Disease in the individual is identified, or Crohn's Disease is predicted, when: the two or more CpG sites in the individual are methylated in the same way as the corresponding CpG sites in a control which has Crohn's Disease; and/or when the two or more CpG sites in the individual are differently methylated as the corresponding CpG sites in a control which does not have Crohn's Disease.
Similarly, the presence of Crohn's Disease in the individual is not identified, or Crohn's Disease is not predicted, when: the two or more CpG sites in the individual are differently methylated as the corresponding CpG sites in a control which has Crohn's Disease; and/or when the two or more CpG sites in the individual are methylated in the same way as the corresponding CpG sites in a control which does not have Crohn's Disease. Preferably, the control individual will be one that is appropriately matched with the individual being tested - for example, in terms of being the same sex and/or of similar age and/or smoking status.
By "differently methylated" we mean that the methylation state at one or more of the CpG sites of the individual being tested is different from that in the control. For example, the CpG sites may be "hyper-methylated" as compared to the control (i.e. have a greater level of methylation) or be "hypo-methylated" (i.e. have a lower level of methylation) compared to the control. The accompanying Examples describe exemplary approaches for determining the presence of Inflammatory Bowel Disease (and, in particular, Crohn's Disease and/or Ulcerative Colitis) in an individual based on the methylation state of two or more CpG sites in the individual, as required by the methods of the invention. Various mathematical methods known in the art may be used to analyse the experimental data generated from the determination of the methylation state of two or more CpG sites in an individual. For example, linear classification and/or machine learning techniques may be used. Examples of such techniques include: generalized linear model or logistic (or probit) regression; multivariate multiple regression (which may be used to predict multiple outcome variables, such as chance of hospitalisation/surgery/extraintestinal symptoms); multinomial logistic regression (which may be used to classify into more groups, such as Crohn's, Ulcerative colitis, irritable bowel syndrome & other disorders); linear discriminant analysis (LDA). Preferably, LDA is used, as discussed in the accompanying Examples.
In a preferred embodiment, the CpG sites are associated with a gene selected from those listed in Table 4. In other words, in that embodiment of the invention, all of the CpG sites are associated with a single gene selected from those in Table 4.
In another preferred embodiment, the CpG sites are associated with two or more genes selected from those listed in Table 4, for example, three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more genes selected from those listed in Table 4.
More preferably, each of the CpG sites is associated with a different gene selected from those listed in Table 4. In other words, in that embodiment of the invention, each CpG sites is associated with a different gene, each of which is selected from the genes in Table 4.
Preferably, the methylation state of the CpG sites associated with the genes listed in Table 4 is determined using one or more relevant probe from those listed in Table 6.
By "associated with a gene" we include the meaning that the CpG site is within a region (i.e. a sequence) of the genome that is within or is adjacent to that gene. It will be appreciated that the CpG site could be functionally associated with the expression of the gene within which it is found, and/or with other genes in the genetic region.
It is preferred that each CpG site is located in a regulatory region of the associated gene. As is well known, a gene comprises a coding region and regulatory regions such as a promoter and/or other sequences which normally govern expression. Preferably, each CpG site is located in a regulatory region selected from the group comprising: promoter; enhancer; exon; intron; and terminator. Preferably, the CpG site is within 1000 base pairs (bp) of the coding region of the gene, and is preferably, within 900bp or 800bp, or 700bp, or 600bp or 500bp or 400bp, or 300bp, or 200bp or 100bp, of the coding region of the gene. For the purpose of the present invention, genetic coordinates and gene names refer to the All coordinates refer to the publically-available Genome Reference Consortium Human Build 37 (aka GRCh37 & hg19), which is found at the following web address: http://www.ncbi.nlm.nih.gov/assembly/GCF 000001405.13/. Preferably, the CpG sites are located in a genetic region that is a Differentially Methylated Region (abbreviated to "DMR").
The concept of DMRs is well-known to those in the art. DMRs are genomic regions which have different methylation statuses among multiple samples (for example, between tissues, cells, individuals or other sample types).
In the present invention, DMRs were defined using a modification of the lasso technique employed by the ChAMP pipeline (Morris ei al. (2014), Bioinformatics 30(3): 428-430 and at: http://www2.cancer.ucl.ac.uk/medicalqenomics/champ/).
In particular, a DMRs is a genetic region in which: all probes were found to have disease- associated methylation differences at the significance level FDR corrected p value<0.05 and shared the same direction of change in methylation in association with disease; and have a minimum size of three CpG sites and no maximum. DMRs were identified using an algorithm based on a modified version of ChAMP (Morris ei al. (2014), Bioinformatics 30(3): 428-430, and at: http://www2.cancer.ucl.ac.uk/medicalqenomics/champ/). In that method, the distance that the algorithm looks for adjacent CpG sites is variable and based on the local density of probes covering the region. Preferably, the CpG sites are within a genetic region selected from those listed in Table 5 (all of which are DMRs).
Preferably, the CpG sites are within two or more genetic regions selected from those listed in Table 5, for example: three or more; or four or more; or five or more; or six or more; or seven or more; or eight or more, or nine or more, or ten or more, genetic regions selected from Table 5. Preferably, each of the CpG sites is associated with a different genetic locus selected from those listed in Table 5. Preferably, the methylation state of the one or more CpG sites associated with the genetic regions listed in Table 5 is determined using one or more relevant probe from those listed in Table 7.
Preferably, when the individual is a child, the CpG sites are selected from the group comprising:
- 11 :1297087 (TOLLIP); and
- 17:75473969 (SEPT9); and
- 6:166970252 (RPS6KA2); and
- 15:59588622 (MY01 E); and
- 17: 1278466 (YWHAE) ; and
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1); and
- 17:57918262 (MIR21 ); and
- 3:57041402 (ARHGEF3); and
- 3:101901234; and
- 4:87752504 (SLC10A6); and
- 22:50327986; and
- 7:797592 (HEATR2); and
- 2:235580461 ; and
- 21 :46341054 (ITGB2).
More preferably, the two or more CpG sites are selected from the group comprising:
- 1 1 : 1297087 (TOLLI P) and 6: 166970252 (RPS6KA2); or
- 17:75473969 (SEPT9) and 6:166970252 (RPS6KA2); or
- 11 : 1297087 (TOLLI P) and 15: 59588622 (M Y01 E); or
- 6:166970252 (RPS6KA2); and 7: 1278466 (YWHAE); or - 6:166970252 (RPS6KA2); and cg0930439; or
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1 ), and 15:59588622 (MY01 E); or
- 17: 1278466 (YWHAE) and 3:57041402 (ARHGEF3).
It is particularly preferred that the CpG sites are associated with one or more gene selected from the following: ANKRD11 (i.e. cg16525838, cg16710656); or
ARHGEF3 i.e. (cg04389058); or
ATP9A (/.e.cg07339236); or
CDK6 (/.e.cgl 5732164, cg14100946, cg06688763); or
CSMD3 (/.e.cg02292450, cg23676042, cg20323509); or
D2HGDH (/'.e.cg24743237, cg13613174); or
GNAS (/.e.cg26791489, cg04019914, cg11357538, cg10011623, cg01748573, cg17334845, cg26767990, cg19592829, cg09123158, cg19140375, cg11669839, cg14482474, cg06200857, cg08091561 , cg22860367, cg07947033, cg04677683, eg 15160445, cg25326570, cg23249369); or
GPRIN3 (/.e.cg02734358); or
HEATR2 ( .e.cg1047271 1); or
HK2 ( .e.cg27049094); or
1TGB2 (/.e.cgl 41 2356, cg22699620, cg21006727, cg13315706); or MIR1973 (/.e.cg22914762); or
MIR21 (/.e.cg27023597); or
MY01 E (/.e.cg08423142); or
NDUFS4 ( .e.cgl 2351310); or
NMUR1 (/.e.cgl 9077400, cg19733463, cg20608294, cg01616956); or PWWP2B (/.e.cgl 5995714, cg021 16864, cg07733247, cg16784468, cg06427772, cg1 1229101 , cg11579421 , cg25303150, cg24085039); or RPS6KA2 (/.e.cgl 1599721 , cg05691806, cg09430664, cg17501210); or
SEPT9 (/.e.cg05626616, cg01749539, cg06791979, cg02442640, eg 17922695, g 14843920); or
SOCS3 (/.e.cg18181703); or
TNFSF10 (/.e.cg01059398); or
TNS1 (/.e.cg12338137); or
TOLLIP (/.e.cg26599989); or
VMP1 (/.e.cgl 6936953, cg12054453, cg01409343, cg18942579, cg24174557); or
YWHAE (/.e.cg06219337); or
ZBTB16 (/.e.cg22768358, cg10096321 , cg03725573, cg06493289); or Unannotated gene (/.e.cg09304397, cg05740793, cg27534567, cg21653586, cg09349128, cg12992827). It is further preferred that the CpG sites are associated with the following combinations of genes:
- ARHGEF3 (cg04389058) and NA (cg09304397); or
- ARHGEF3 (cg04389058) and TOLLIP (cg26599989); or
- ARHGEF3 (cg04389058) and YWHAE (cg06219337); or
- CDK6 (cg14100946) and NA (cg05740793); or
- CSMD3 (cg02292450) and MIR1973 (cg22914762); or
- CSMD3 (cg20323509) and MIR1973 (cg22914762); or
- CSMD3 (cg02292450) and NA (cg27534567); or
- CSMD3 (cg20323509) and NA (cg05740793); or
- GNAS (eg 10011623) and NA (cg27534567); or
- GNAS (cg01748573) and NA (cg27534567); or
- GNAS (cg26767990) and NA (cg05740793); or
- GPRIN3 (cg02734358) and D2HGDH (cg24743237); or
- GPRIN3 (cg02734358) and NA (cg05740793); or
- GPRIN3 (cg02734358) and NDUFS4 (cg12351310); or
- MIR1973 (cg22914762) and GNAS (cg01748573); or
- MIR1973 (cg22914762) and TNS1 (cg12338137); or
- MIR21 (cg27023597) and NA (cg21653586); or
- PWWP2B (cg07733247) and NA (cg05740793); or
- RPS6KA2 (cg17501210) and SEPT9 (cg01749539); or
- RPS6KA2 (cg17501210) and ANKRD11 (cg16525838); or
- RPS6KA2 (cg17501210) and ATP9A (cg07339236); or
- RPS6KA2 (cg17501210) and HEATR2 (cg10472711); or
- RPS6KA2 (cg17501210) and HK2 (cg27049094); or
- RPS6KA2 (eg 17501210) and NA (cg09304397); or
- RPS6KA2 (cg17501210) and NA (cg09349128); or
- RPS6KA2 (cg17501210) and NA (cg12992827); or
- RPS6KA2 (cg17501210) and NMUR1 (cg01616956); or
- RPS6KA2 (cg17501210) and TOLLIP (cg26599989); or
- RPS6KA2 (cg17501210) and YWHAE (cg06219337); or
- RPS6KA2 (eg 17501210) and ZBTB16 (cg22768358); or
- SOCS3 (cg18181703) and D2HGDH (cg13613174); or
- TNFSF10 (cg01059398) and NA (cg21653586); or
- TOLLIP (cg26599989) and MY01 E (cg08423142); or - TOLLIP (cg26599989) and NA (cg09304397); or
- VMP1 (cg12054453) and SEPT9 (cg01749539); or
- VMP1 (cg12054453) and ITGB2 (cg13315706); or
- VMP1 (cg12054453) and MY01 E (cg08423142); or
- VMP1 (cg16936953) and MY01 E (cg08423142); or
- VMP1 (eg 12054453) and NA (cg09304397); or
- VMP1 (cg16936953) and NA (cg09304397); or
- VMP1 (cg12054453) and TOLLIP (cg26599989); or
- VMP1 (cg16936953) and TOLLIP (cg26599989); or
- VMP1 (cg12054453) and YWHAE (cg06219337); or
- VMP1 (eg 16936953) and YWHAE (cg06219337).
The accompanying Examples and Figures (particularly Figures 6, 7 and 8) provide experimental data relating M values for preferred models of the invention.
Preferably, when the individual is a child, the gene is selected from the group comprising: TOLLIP; SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2; SBN02; TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9; SPARC; SUB1 ; TRAM2; KCNAB2; BBS9; AGL; FRMD4A; F BP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3; PALM; ICA1 ; BAHCC1 ; LRRC47; cg01101459; cg02719954; cg04086394; cg12807764; cg19445588; cg20519581 ; cg09349128; cg12992827; cg25114611 ; cg07398517; cg19821297; cg12582317; cg00053916. Most preferably, the gene is: TOLLIP.
Preferably, when the individual is a child, two or more genes are selected from the group comprising:
- TOLLIP and RPS6KA2; or
- SEPT9 and RPS6KA2; or
- TOLLIP and MY01 E; or
- RPS6KA2 and YWHAE; or
- RPS6KA2 and cg0930439; or
- VMP1 and MY01 E; or
- YWHAE and ARHGEF3; or
- cg25114611 and AIM2; or
- cg25114611 and BCL3; or - cg25114611 and cg01 101459; or
- cg25114611 and cg02719954; or
- cg25114611 and cg04086394; or
- cg2511461 1 and cg09349128; or - cg25114611 and cg12807764; or
- cg25114611 and cg19445588; or
- cg25114611 and cg19821297; or
- cg25114611 and cg20519581 ; or
- cg2511461 1 and CPVL; or - cg25114611 and CSGALNACT1 ; or
- cg2511461 1 and GPRIN3; or
- cg25114611 and ICA1 ; or
- cg25114611 and MY01 E; or
- cg25114611 and NEDD9; or
- cg25114611 and SPARC; or
- cg2511461 1 and SUB1 ; or
- cg25114611 and TNFSF10; or
- cg25114611 and TRAM2; or
- AIM2 and BCL3; or
- AIM2 and eg 12582317; or
- AIM2 and FKBP5; or
- cg07398517 and BCL3; or
- cg09349128 and cg00053916; or
- cg09349128 and FKBP5; or
- cg09349128 and FRMD4A; or
- cg09349128 and PHF21A; or
- eg 12992827 and ANO10; or
- eg 12992827 and BCL3; or
- eg 12992827 and FRMD4A; or - cg19821297 and PHF21A; or
- FRMD4A and PALM; or
- GPRIN3 and ITGB2; or
- ICA1 and cg09349128; or
- PALM and BAHCC1 ; or
- PALM and KCNAB2; or
- PALM and LRRC47. Preferably, when the individual is an adult, the CpG sites are selected from the group comprising:
- 17:75473969 (SEPT9); and
- 6:166970252 (RPS6 A2); and
- 15:59588622 (MY01 E); and
- 17:1278466 (YWHAE); and
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1 ); and
- 17:57918262 (MIR21); and
- 3:57041402 (ARHGEF3); and
- 3:101901234; and
- 4:87752504 (SLC10A6); and
- 22:50327986; and
- 7:797592 (HEATR2); and
- 2:235580461 ; and
- 21 :46341054 (ITGB2).
More preferably, the two or more CpG sites are selected from the group comprising: - 17:75473969 (SEPT9) and 6: 166970252 (RPS6KA2); or
- 6: 166970252 (RPS6KA2); and 17: 1278466 (YWHAE); or
- 6:166970252 (RPS6KA2); and cg0930439; or
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1), and 15:59588622 (MYC-1 E); or
- 17:1278466 (YWHAE) and 3:57041402 (ARHGEF3).
Preferably, when the individual is an adult, the gene is selected from the group comprising:
SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2; SBN02;
TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9; SPARC; SUB1 ; TRAM2; KCNAB2;
BBS9; AGL; ICA1 ; FRMD4A; FKBP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3; PALM;
ICA1 ; BAHCC1 ; LRRC47; cg01101459; cg02719954; cg04086394; cg12807764; cg19445588; cg20519581 ; cg09349128; cg12992827; cg25114611; cg07398517; cg19821297; cg12582317; cg00053916. More preferably, in the methods of the invention the two or more genes are selected from the group comprising:
- SEPT9 and RPS6KA2; or
- RPS6KA2 and YWHAE; or
- RPS6KA2 and cg0930439; or
- VMP1 and MY01 E; or
- YWHAE and ARHGEF3.
- cg25114611 and AIM2; or
- cg2511461 1 and BCL3; or
- cg25114611 and cg01101459; or
- cg25114611 and cg02719954; or
- cg25114611 and cg04086394; or
- cg25114611 and cg09349128; or
- cg25114611 and cg12807764; or
- cg2511461 1 and eg 19445588; or
- cg25114611 and cg19821297; or
- cg25114611 and cg20519581 ; or
- cg25114611 and CPVL; or
- cg25114611 and CSGALNACT1 ; or
- cg25114611 and GPRIN3; or
- cg25114611 and ICA1 ; or
- cg25114611 and MY01E; or
- cg25114611 and NEDD9; or
- cg2511461 1 and SPARC; or
- cg2511461 1 and SUB1 ; or
- cg25114611 and TNFSF10; or
- cg25114611 and TRAM2; or
- AIM2 and BCL3; or
- AIM2 and cg12582317; or
- AIM2 and FKBP5; or
- cg07398517 and BCL3; or
- cg09349128 and cg00053916; or
- cg09349128 and FKBP5; or
- cg09349128 and FRMD4A; or
- cg09349128 and PHF21A; or
- eg 12992827 and ANO10; or - eg 12992827 and BCL3; or
- cg12992827 and FRMD4A; or
- cg19821297 and PHF21A; or
- FRMD4A and PALM; or
- GPRIN3 and ITGB2; or
- ICA1 and cg09349128; or
- PALM and BAHCC1 ; or
- PALM and KCNAB2; or
- PALM and LRRC47.
When the invention relates to Crohn's Disease, it is particularly preferred that the CpG sites are associated with one or more gene selected from the following: RPS6KA2; SBN02; VMP1 ; cg12992827; KCNAB2; cg07398517; BBS9; AGL; ICA1 ; FRMD4A; FKBP5; TNFSF10; CALHM1 ; cg25114611 ; AIM2; BCL3; cg01101459; cg02719954; cg04086394; cg09349128; cg12807764; cg19445588; cg19821297; cg20519581 ; CPVL; CSGALNACT1 ; GPRIN3.
More preferably, when the invention relates to Crohn's Disease, it is particularly preferred that the two or more genes are selected from the group comprising: cg25114611 & AIM2; cg251 14611 & BCL3; cg2511461 1 & cg01101459; cg2511461 1 & cg02719954; cg251 14611 & cg04086394; cg251 14611 & cg09349128; cg25114611 & cg12807764; cg251 14611 & cg19445588; cg25114611 & cg19821297; cg25114611 & cg20519581 ; cg25114611 & CPVL; cg25114611 & CSGALNACT1; cg25114611 & GPRIN3; cg251 14611 & ICA1 ; cg25114611 & MY01 E; cg2511461 1 & NEDD9; cg25114611 & SPARC; cg25114611 & SUB1 ; cg25114611 & TNFSF10; cg25114611 & TRAM2.
When the invention relates to Ulcerative Colitis, it is particularly preferred that the CpG sites are associated with one or more gene selected from the following: RPS6KA2; SBN02; cg09349128; VMP1 ; cg12992827; cg251 1461 1 ; KCNAB2; cg07398517; BBS9; AGL; ICA1 ; cg19821297; FRMD4A; FKBP5; AIM2; BCL3; cg12582317; cg00053916; PHF21A; ANO10; GPRIN3; PALM; ITGB2; ICA1 ; BAHCC1 ; LRRC47.
More preferably, when the invention relates to Ulcerative Colitis, it is particularly preferred that the two or more genes are selected from the group comprising: AIM2 & BCL3; AIM2 & cg12582317; AIM2 & FKBP5; cg07398517 & BCL3; cg09349128 & cg00053916; cg09349128 & FKBP5; cg09349128 & FRMD4A; cg09349128 & PHF21A; cg12992827 & ANO10; cg12992827 & BCL3; cg12992827 & FRMD4A; cg19821297 & PHF21A; cg25114611 & BCL3; cg251 14611 & GPRIN3; FRMD4A & PALM; GPRIN3 & ITGB2; ICA1 & cg09349128; PALM & BAHCC1 ; PALM & KCNAB2; PALM & LRRC47.
Preferably, the sample is selected from the group comprising: blood; serum; plasma; intestinal cells; biopsy; stool. Preferably, the sample is a blood sample. Methods for isolating and/or purifying relevant cells from such samples are well known in the art and include density centrifugation and magnetic cell sorting.
Methods for obtaining DNA from leukocytes are well known in the art. Preferably, the leukocyte DNA is obtained from the sample by a method comprising the steps of: (i) cell lysis; (ii) removal of cell membranes and/or cellular RNA and/or cellular protein; (iii) purification of DNA (for example, by extraction using phenol-chloroform; or using column- based extraction; or using ethanol-precipitation). Suitable kits for obtaining DNA from leukocytes are commercially-available and include the "AllPrep DNA/RNA kit" (Qiagen), and "Nucleon kit" (GE Healthcare).
Methods for determining methylation state are known in the art. Preferably, the methylation state is determined by a method selected from the group comprising: bisulfite conversion and DNA sequencing; methylation-specific restriction enzyme analysis; COBRA analysis; methylation-specific PCR; MethylLight analysis; HeavyMethyl analysis; Quantitative Methylation analysis; Ms-SNuPE analysis.
By "bisulfite conversion and DNA sequencing" we include a technique in which short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment, and either completely sequenced (OIek & Walter, Nat Genet. 1997 17:275-6, 1997), subjected to one or more primer extension reactions (Gonzalgo & Jones, Nucleic Acids Res., 25:2529-31 , 1997; WO 95/00669; U.S. Patent No. 6,251 ,594) to analyse individual cytosine positions, or treated by enzymatic digestion (Xiong & Laird, Nucleic Acids Res., 25:2532-4, 1997). Sequencing may be performed using techniques known in the art, such as Sanger sequencing or pyrosequencing.
Detection by hybridisation has also been described in the art (OIek et al., WO 99/28498). Additionally, use of the bisulfite technique for methylation detection with respect to individual genes has been described (Grigg & Clark, Bioessays, 16:431 -6, 1994; Zeschnigk M, et al., Hum Mol Genet, 6:387-95, 1997; Feil R, et al., Nucleic Acids Res., 22:695-, 1994; Martin V, et al., Gene, 157:261 - 4, 1995; WO 9746705 and WO 9515373). By "methylation-specific restriction enzyme analysis" we include a process by which restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is used; for example, the method described by Sadri & Hornsby (Nucl. Acids Res. 24:5058- 5059, 1996), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997).
By "Combined Bisulfite Restriction Analysis (COBRA) analysis" we include the art- recognized methylation assay described by Xiong & Laird (Nucleic Acids Res. 25:2532- 2534, 1997).
COBRA analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific gene loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite- treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831 , 1992). PCR amplification of the bisulfite converted DNA is then performed using primers specific for the CpG sites of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labelled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, this technique can be reliably applied to DNA obtained from micro-dissected paraffin- embedded tissue samples.
By "methylation-specific PCR (MSP)" we include the art-recognized methylation assay described by Herman et al. (Proc. Natl. Acad. Sci. USA 93:9821 - 9826, 1996), and by US Patent No. 5,786,146. MSP allows for assessing the methylation state of virtually any group of CpG sites within a CpG site, independent of the use of methylation-sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821 -9826, 1996; US Patent No. 5,786, 146). Briefly, DNA is modified by sodium bisulfite converting all un-methylated, but not methylated cytosines to uracil, and subsequently amplified with primers specific for methylated versus un-methylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1 % methylated alleles of a given CpG site, and can be performed on DNA extracted from paraffin- embedded samples. By "MethylLight™ analysis", which is also referred to as the MethylLight assay, we include the art-recognized fluorescence-based real-time PCR technique described by Eads et al., (Cancer Res. 59:2302-2306, 1999).
The MethylLight assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight process begins with a mixed sample of genomic DNA that is converted, in a sodium bisulfite reaction, to a mixed pool of methylation-dependent sequence differences according to standard procedures (the bisulfite process converts un- methylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" (with PCR primers that overlap known CpG dinucleotides) reaction. Sequence discrimination can occur both at the level of the amplification process and at the level of the fluorescence detection process.
The MethyLight assay may be used as a quantitative test for methylation patterns in the genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction provides for a methylation specific amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing of the biased PCR pool with either control oligonucleotides that do not "cover" known methylation sites (a fluorescence-based version of the HeavyMethyl™ and MSP techniques), or with oligonucleotides covering potential methylation sites.
The MethyLight process can by used with any suitable probes, e.g. "TaqMan" or Lightcycler. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using TaqMan probes; e.g., with MSP primers and/ or HeavyMethyl blocker oligonucleotides and TaqMan probe. The TaqMan probe is dual-labelled with fluorescent "reporter" and "quencher" molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about 10°C higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan probe to remain fully hybridized during the PCR annealing/extension step. As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5' to 3' endonuclease activity will then displace the TaqMan probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system. By "HeavyMethyl analysis" we include an assay, wherein methylation specific blocking probes (also referred to herein as blockers) covering CpG positions between, or covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample. By "Quantitative Methylation (QM) analysis" we include the test for methylation patterns in genomic DNA samples, wherein sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing of the biased PCR pool with either control oligonucleotides that do not "cover" known methylation sites (a fluorescence- based version of the HeavyMethyl and MSP techniques), or with oligonucleotides covering potential methylation sites.
The QM process can be used with any suitable probes, e.g. "TaqMan" or Lightcycler, in the amplification process. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and the TaqMan probe. The TaqMan probe is dual-labelled with fluorescent "reporter" and "quencher" molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about 10°C higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan probe to remain fully hybridized during the PCR annealing/extension step. As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan probe. The Taq polymerase 5' to 3' endonuclease activity will then displace the TaqMan probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system. Typical reagents (e.g., as might be found in a typical QM- based kit) for QM analysis may include, but are not limited to: PCR primers for specific gene (or bisulfite treated DNA sequence or CpG site); TaqMan or Lightcycler probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase. By "Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) analysis", which is also referred to as the Ms-SNuPE technique, we include the art-recognized assay described by Gonzalgo & Jones (Nucleic Acids Res. 25:2529-2531 , 1997). The Ms-SnuPE technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo & Jones (Nucleic Acids Res. 25:2529-2531 , 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert un-methylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest. Small amounts of DNA can be analyzed (e.g., micro-dissected pathology sections), and it avoids utilization of restriction enzymes for determining the methylation state at CpG sites.
It will be appreciated that biological chips or arrays are useful in a variety of screening techniques for obtaining information about either the probes or target molecules. For example, arrays of nucleic acid probes can be used to extract sequence and/or methylation state information from, for example, nucleic acid samples. The samples are exposed to the probes under conditions that allow hybridisation, and the arrays are then scanned to determine to which probes the sample molecules have hybridised. Sequence information can be obtained by careful probe selection and using algorithms to compare patterns of hybridisation and non-hybridisation. A preferred microarray for use in the methods of the present invention is the lllumina 450k microarray (http://www.illumina.com/products/methylation 450 beadchip kits.ilmn). That array permits the determination of methylation state by binding bisulphite-converted DNA and returning a colour signal based on the sequence differences caused by bisulphite conversion depending on starting methylation state. Interpretation of the signal allows one to calculate the proportion of strands of input DNA which were methylated and un- methylated at each probe location.
Preferably, the methods of the invention further comprise the step of selecting a treatment for the individual and/or treating the individual with the selected treatment, as discussed herein. As discussed above, the inventors' findings mean that the methylation state of leukocyte DNA can be used to determine the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in an individual and/or predict the likelihood of an individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis). Accordingly, further aspects of the invention relate to the use of leukocyte DNA from an individual in such approaches.
Therefore, in a fifth aspect, the invention provides a use of leukocyte DNA from an individual, for determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual. Preferably, the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and determining the presence of Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) in the individual on the basis of the methylation state. Most preferably, the fifth aspect of the invention provides a use of leukocyte DNA from an individual, for determining the presence of Crohn's Disease in the individual - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and determining the presence of Crohn's Disease in the individual on the basis of the methylation state. More preferably, the use comprises the methods of the first aspect of the invention as described above.
In a sixth aspect, the invention provides a use of leukocyte DNA from an individual, for predicting the likelihood of the individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis). Preferably, the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the likelihood of the individual contracting Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) on the basis of the methylation state. Most preferably, the sixth aspect of the invention provides a use of leukocyte DNA from an individual, for predicting the likelihood of the individual contracting Crohn's Disease - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the likelihood of the individual contracting Crohn's Disease on the basis of the methylation state. More preferably, the use comprises the methods of the second aspect of the invention as described above.
In a seventh aspect, the invention provides a use of leukocyte DNA from an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis), for selecting a treatment for the individual. Preferably, the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and selecting a treatment for the individual on the basis of the methylation state. Most preferably, the seventh aspect of the invention provides a use of leukocyte DNA from an individual suspected of having Crohn's Disease, for selecting a treatment for the individual - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and selecting a treatment for the individual on the basis of the methylation state. Preferably, such treatments are as discussed herein. More preferably, the use comprises the methods of the third aspect of the invention as described above.
In an eighth aspect, the invention provides a use of leukocyte DNA from an individual suspected of having Inflammatory Bowel Disease (such as Crohn's Disease or Ulcerative Colitis) for predicting the response to anti-Crohn's Disease therapy of the individual. Preferably, the use comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the response to anti-Inflammatory Bowel Disease therapy of the individual on the basis of the methylation state. Most preferably, the eighth aspect of the invention provides a use of leukocyte DNA from an individual suspected of having Crohn's Disease for predicting the response to anti-Crohn's Disease therapy of the individual - that preferably comprises determining the methylation state at two or more CpG sites in the leukocyte DNA, and predicting the response to anti-Crohn's Disease therapy of the individual on the basis of the methylation state. Preferably, such treatments are as discussed herein. More preferably, the use comprises the methods of the fourth aspect of the invention as described above.
In a ninth aspect, the invention provides a kit for performing a method or use according to the invention, the kit comprising one or more reagent for determining the methylation state at two or more CpG sites in leukocyte DNA, wherein each CpG site is selected from those in Table 3.
Preferably, in the ninth aspect of the invention, the CpG sites are associated with a gene selected from those listed in Table 4. In other words, in that embodiment of the invention, all of the CpG sites are associated with a single gene selected from those in Table 4.
In another preferred embodiment of the ninth aspect of the invention, the CpG sites are associated with two or more genes selected from those listed in Table 4, for example, three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more genes selected from those listed in Table 4. More preferably, in the ninth aspect of the invention, each of the CpG sites is associated with a different gene selected from those listed in Table 4. In other words, in that embodiment of the invention, each CpG sites is associated with a different gene, each of which is selected from the genes in Table 4.
Preferably, in the ninth aspect of the invention, the methylation state of the CpG sites associated with the genes listed in Table 4 is determined using one or more relevant probe from those listed in Table 6. In a preferred embodiment, invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- a pair of oligonucleotide primers for PCR amplification of the polynucleotide sequence at a first CpG site selected from those listed in Table 3; and
- a pair of oligonucleotide primers for PCR amplification of the polynucleotide sequence at a second CpG site selected from those listed in Table 3. Bisulfite conversion is a process by which cytosine residues are converted to uracil, but methylated cytosine residues are not. Such processes are well known in the art and described herein.
In brief, test DNA is diluted and denatured, and then incubated with sodium bisulphite which converts un-methylated cytosine to uracil, but does not alter methylated cytosine. Once completed, the DNA is treated with a desulphonation reagent, and can then be subjected to further analysis to determine the presence of cytosine and uracil residues.
It will be appreciated that the kit of this embodiment of the invention is suitable for a Polymerase Chain Reaction (PCR)-based determination of methylation state, as discussed above. In that approach PCR amplification will replace uracil with thymine such that, when the amplified sequence is read, all un-methylated cytosines are read as thymine, and all detected cytosines were methylated. PCR is described in Saiki et al. (1988, Science, 239: 487-491). Approaches for designing oligonucleotide primers suitable for PCR-based amplification and related PCR-based approaches are well known in the art. In a further embodiment, the invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- two or more oligonucleotide primers for methylation-specific PCR amplification of the polynucleotide sequence at a first CpG site selected from those listed in Table
3;
- two or more oligonucleotide primers for methylation-specific PCR amplification of the polynucleotide sequence at a second CpG site selected from those listed in Table 3; and
- optionally, an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation-specific PCR amplification.
Preferably, the kits of that embodiment of the invention further comprise:
- an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the first CpG site when the first CpG site is not methylated; and
an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the second CpG site when the first CpG site is not methylated.
It will be appreciated that the kits of this embodiment of the invention are suitable for determination of the methylation state using Methylation-specific PCR-based approaches, such as the MethylLight or HeavyMethyl or MS-SNuPE approaches, as discussed above. As discussed, in the MethyLight and HeavyMethyl processes comprise methylation- specific blocking probes (also referred to herein as blockers) covering CpG positions between, or covered by, the amplification primers to enable methylation-specific selective amplification of a nucleic acid sample. Accordingly, by "an oligonucleotide capable of preventing and/or reducing PCR amplification", we include such blocking probes. A person skilled in the art of molecular biology would be capable of designing and testing such probes, in line with the teaching above and their common general knowledge. By "selectively binding" we include the ability of an oligonucleotide probe of the invention to anneal (i.e. bind) selectively to the one or more polynucleotide sequence generated by methylation-specific PCR amplification and to do so more strongly than to polynucleotide sequences not generated by methylation-specific PCR amplification. For example, the oligonucleotide probe may bind at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or 1000-fold more strongly to the one or more polynucleotide sequence generated by methylation-specific PCR amplification than to polynucleotide sequences not generated by methylation-specific PCR amplification.
It will be understood by those skilled in the art that the ability of an oligonucleotide probe to bind to a polynucleotide target will depend on the particular conditions to which they are subjected. For example, those skilled in the art would understand that the pH, temperature and/or salt concentration used may affect the manner in which an oligonucleotide probe selectively binds to a polynucleotide target. The stringency of the conditions can be used to determine the strength of binding between the oligonucleotide probe and polynucleotide target (as described, for example, in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press). In a further embodiment, the invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises: one or more reagent for bisulfite conversion of the leukocyte DNA;
- an oligonucleotide primer for determining the polynucleotide sequence at a first CpG site selected from those listed in Table 3; and
- an oligonucleotide primer for determining the polynucleotide sequence at a second CpG site selected from those listed in Table 3. It will be appreciated that the kit of this embodiment of the invention is suitable for determination of the methylation state by DNA sequencing, as discussed above. DNA sequencing is well known to those skilled in the art and described, for example, in Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory Press). In a still further embodiment, the invention provides a kit wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises: - one or more reagent for bisulfite conversion of the leukocyte DNA;
- a restriction enzyme capable of methylation-specific digestion of the polynucleotide sequence at a first CpG site selected from those listed in Table 3;
- a restriction enzyme capable of methylation-specific digestion of the polynucleotide sequence at a second CpG site selected from those listed in Table 3;
- optionally, an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation-specific digestion. It will be appreciated that the kit of this embodiment of the invention is suitable for determination of the methylation state by methylation-specific restriction enzyme analysis or COBRA analysis, as discussed above.
Preferably, the kits of the invention comprise one or more probe selected from those listed in Tables 3 and/or 6 and/or 7 and/or 8.
Preferably, the kits of the invention further comprise one or more control sample.
As discussed herein, the invention generally relates to Inflammatory Bowel Diseases (such as Crohn's Disease or Ulcerative Colitis). Where the kit of the invention relates to Inflammatory Bowel Disease, the one or more control sample is preferably a DNA sample which is indicative of the presence of Inflammatory Bowel Disease and/or a DNA sample which is indicative of the absence of Inflammatory Bowel Disease. In such an embodiment, it is preferred that the one or more control sample comprises leukocyte DNA in which the methylation state of the first CpG site and/or the second CpG site is indicative of the presence of Inflammatory Bowel Disease in an individual. Depending on the particular CpG sites concerned, the one or more control sample may be leukocyte DNA in which the first CpG site and/or the second CpG site is methylated, or in which the first CpG site and/or the second CpG site is un-methylated. In one embodiment, the one or more control sample comprises leukocyte DNA from a control individual which has Inflammatory Bowel Disease and/or leukocyte DNA from a control individual which does not have Inflammatory Bowel Disease. Such control individuals are discussed herein. Where the kit of the invention relates to Ulcerative Colitis, the one or more control sample is preferably a DNA sample which is indicative of the presence of Ulcerative Colitis and/or a DNA sample which is indicative of the absence of Ulcerative Colitis. In such an embodiment, it is preferred that the one or more control sample comprises leukocyte DNA in which the methylation state of the first CpG site and/or the second CpG site is indicative of the presence of Ulcerative Colitis in an individual. Depending on the particular CpG sites concerned, the one or more control sample may be leukocyte DNA in which the first CpG site and/or the second CpG site is methylated, or in which the first CpG site and/or the second CpG site is un-methylated. In one embodiment, the one or more control sample comprises leukocyte DNA from a control individual which has Ulcerative Colitis and/or leukocyte DNA from a control individual which does not have Ulcerative Colitis. Such control individuals are discussed herein. As discussed herein, it is preferred that the invention relates to Crohn's Disease. Where the kit of the invention relates to Crohn's Disease, the one or more control sample is preferably a DNA sample which is indicative of the presence of Crohn's Disease and/or a DNA sample which is indicative of the absence of Crohn's Disease. In such an embodiment, preferably the one or more control sample comprises leukocyte DNA in which the methylation state of the first CpG site and/or the second CpG site is indicative of the presence of Crohn's Disease in an individual. Depending on the particular CpG sites concerned, the one or more control sample may be leukocyte DNA in which the first CpG site and/or the second CpG site is methylated, or in which the first CpG site and/or the second CpG site is un-methylated. In one embodiment, the one or more control sample comprises leukocyte DNA from a control individual which has Crohn's Disease and/or leukocyte DNA from a control individual which does not have Crohn's Disease. Such control individuals are discussed herein. Preferably, the one or more reagent for bisulfite conversion of the leukocyte DNA comprises:
a reagent for denaturation of leukocyte DNA;
a reagent for sulfonation of leukocyte DNA;
a reagent for desulfonation of leukocyte DNA; and
- a reagent for isolation or purification of bisulfite converted leukocyte DNA. Reagents and kits for performing bisulfite conversion are well known in the art, and include the "EZ DNA Methylation" kit and ΈΖ-96 DNA Methylation" kit (both from Zymo Research Corp., USA). Preferably, in the ninth aspect of the invention, the individual is a child and the gene is selected from the group comprising: TOLLIP; SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2;
2:235580461 ; ITGB2; SBN02; TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9;
SPARC; SUB1 ; TRAM2; KCNAB2; BBS9; AGL; FRMD4A; FKBP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3; PALM; ICA1 ; BAHCC1 ; LRRC47; cg01 101459; cg02719954; cg04086394; cg12807764; cg19445588; cg20519581 ; cg09349128; cg12992827; cg25114611 ; cg07398517; cg19821297; cg12582317; cg00053916. Most preferably, the gene is: TOLLIP. Preferably, when the individual is a child, two or more genes are selected from the group comprising: TOLLIP and RPS6KA2; or SEPT9 and RPS6KA2; or TOLLIP and MY01E; or RPS6KA2 and YWHAE; or RPS6KA2 and cg0930439; or VMP1 and MY01 E; or YWHAE and ARHGEF3; or cg251 1461 1 and AIM2; or cg2511461 1 and BCL3; or cg2511461 1 and cg01101459; or cg2511461 1 and cg02719954; or cg25114611 and cg04086394; or cg251 14611 and cg09349128; or cg25114611 and cg12807764; or cg25114611 and cg19445588; or cg251 1461 1 and cg19821297; or cg25114611 and cg20519581 ; or cg251 14611 and CPVL; or cg251 14611 and CSGALNACT1 ; or cg25114611 and GPRIN3; or cg251 1461 1 and ICA1 ; or cg25114611 and MY01 E; or cg25114611 and NEDD9; or cg25114611 and SPARC; or cg25114611 and SUB1 ; or cg25114611 and TNFSF10; or cg251 14611 and TRAM2; or AIM2 and BCL3; or AIM2 and cg12582317; or AIM2 and FKBP5; or cg07398517 and BCL3; or cg09349128 and cg00053916; or cg09349128 and FKBP5; or cg09349128 and FRMD4A; or cg09349128 and PHF21A; or cg12992827 and ANO10; or cg12992827 and BCL3; or cg12992827 and FRMD4A; or cg19821297 and PHF21A; or FRMD4A and PALM; or GPRIN3 and ITGB2; or ICA1 and cg09349128; or PALM and BAHCC1 ; or PALM and KCNAB2; or PALM and LRRC47.
Preferably, in the ninth aspect of the invention, the individual is an adult and the CpG sites are selected from the group comprising: SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2; SBN02; TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9; SPARC; SUB1 ; TRAM2; KCNAB2; BBS9; AGL; ICA1 ; FRMD4A; FKBP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3; PALM; ICA1 ; BAHCC1 ; LRRC47; cg01101459; cg02719954; cg04086394; cg12807764; cg19445588; cg20519581 ; cg09349128; cg12992827; cg2511461 1 ; cg07398517; cg19821297; cg12582317; cg00053916.
More preferably, in the methods of the invention the two or more genes are selected from the group comprising: SEPT9 and RPS6KA2; or RPS6KA2 and YWHAE; or RPS6KA2 and cg0930439; or VMPI and MY01 E; or YWHAE and ARHGEF3; or cg251 14611 and AIM2; or cg25114611 and BCL3; or cg25114611 and cg01101459; or cg25114611 and cg02719954; or cg25114611 and cg04086394; or cg25114611 and cg09349128; or cg2511461 1 and cg12807764; or cg251 1461 1 and cg19445588; or cg251 1461 1 and cg19821297; or cg25114611 and cg20519581 ; or cg251 1461 1 and CPVL; or cg25114611 and CSGALNACT1 ; or cg25114611 and GPRIN3; or cg251 4611 and ICA1 ; or cg251146 1 and MY01 E; or cg25114611 and NEDD9; or cg25114611 and SPARC; or cg251 1461 1 and SUB1 ; or cg25114611 and TNFSF10; or cg25114611 and TRAM2; or AIM2 and BCL3; or AIM2 and cg 2582317; or AIM2 and FKBP5; or cg07398517 and BCL3; or cg09349128 and cg00053916; or cg09349128 and FKBP5; or cg09349128 and FRMD4A; or cg09349128 and PHF21A; or cg12992827 and ANO10; or cg12992827 and BCL3; or cg12992827 and FRMD4A; or cg19821297 and PHF21A; or FRMD4A and PALM; or GPRIN3 and ITGB2; or lCAI and cg09349128; or PALM and BAHCC1 ; or PALM and KCNAB2; or PALM and LRRC47.
The listing or discussion in this specification of an apparently prior-published document should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
Figure 1: (A) Schematic of study design, n=CD samples : control samples (B) Comparison of log2 fold-change of lllumina 450k probes with nominal P values <0.05 in the discovery cohort (n=53610) and their replication. (C) The minimum P value from lllumina 450k data in the combined paediatric data within a 1kb region is correlated with chance of being in proximity to GWAS SNPs. (D) Manhattan plot of -Log-io P values from lllumina 450k data in the combined paediatric datasets. Horizontal line at Bonferroni corrected P<0.05 (165 probes). Asterisks denote probes with FDR corrected P<0.05 in the discovery cohort (Table 1), with annotated gene symbol where applicable.. Figure 2: (A) 7 examples of separation by diagnosis in considering M values (log2[proportion of methylated probes/proportion of unmethylated probes]) for pairs of lllumina 450K probes. Column headings give Probe 1 v Probe 2 and (Sensitivity, specificity) of models based on these probe pairs in discovery (left) and replication (right) cohorts. Results for paediatric discovery and paediatric replication cohorts are shown in the top and bottom rows respectively. (B) Replication in adults (20v20) by pyrosequencing of methylation differences in highly significant probes from the combined paediatric analysis. Figure 3: Schema for the selection of targets for further study, showing examples and total numbers for each set. GWAS risk loci correspond to all CD and IBD results from GWAS meta-analysis.1 Inclusion within epigenome-wide significance and differentially methylated regions sets based on individual probe significance surviving Bonferroni correction, and being identified as a DMR by the modified ChAMP algorithm respectively (Methods). VMP1/MIR21 shown at the intersection of all three sets.
Figure 4: (A and B) Schematics of VMP1 and MIR21 showing the distribution of lllumina 450k probes coloured by significance in the combined paediatric data (Black - Bonferroni corrected P<0.05, Grey - FDR corrected P <0.05, unfilled - non-significant). Plotted as (A) histogram by position across VMP1 (B) Position and log-io P value around MIR21. (pri- mir21 - primary transcript, mir21 - mature transcript). (C) Methylation results across 15 lllumina 450k probes within VMP1 for all paediatric samples. X axis shows hg19/GRCh37 coordinates (not to scale), shaded region corresponds to DMR in (B). Figure 5: (A) Replication of MIR21 hypomethylation in CD in 172 adults at cg16936953 by pyrosequencing. (B) Increased leucocyte pri-mir21 mRNA in CD measured by qPCR. (C) Microarray data showing significantly increased pri-mir21 mRNA in response to inflammation in CD and UC. VMP1 increased in CD, but not UC or control 3 . Figure 6: Experimental data showing M values for preferred models of the invention.
Figure 7: Experimental data showing M values for preferred models of the invention - Crohn's Disease vs. Control. Figure 8: Experimental data showing M values for preferred models of the invention - Ulcerative Colitis vs. Control. Table 1 : lllumina 450k probes with significant (FDR adjusted P<0.05) methylation differences in the discovery cohort which replicated in the second cohort (P<0.05).
Table 2: Differentially Methylated Regions - Top DMRs from combined paediatric lllumina 450k data ranked by (A) most significant individual probe, (B) density of DMRs in a region, (C) proximity to GWAS SNPs. Coordinates refer to hg19/GRCh37. (up arrow - Hypermethylated, down arrow - Hypomethylated, dash - Intergenic probe)
Table 3: Details of the CpG sites and probes used in the present invention. The "probe start", "probe end" and "probe target" numbers indicate the coordinates of the probe target. The term "NA" indicates that the CpG site and probe is not currently known to be associated with a gene.
Table 4: Details of the genes used in the present invention. The term "NA" indicates that the CpG site and probe is not currently known to be associated with a gene.
Table 5: Details of the Differentially Methylated Regions used in the present invention. The "DMR start", "DMR stop" and "coordinate" numbers indicate the coordinates of the probe target; specifically, the coordinate of one probe within the DMR. The term "NA" or a blank gene symbol indicates that the DMR is not currently known to be associated with a gene.
Table 6: Details of the gene probes used in the present invention. Table 7: Details of the Differentially Methylated Regions and probes used in the present invention. Note: TMEM49 is a synonym for VMP1.
Table 8: lllumina 450k probes with significant (FDR adjusted P<0.05) methylation differences in the following cohorts:
- paediatric Crohn's Disease;
- adult Crohn's Disease;
- adult Inflammatory Bowel Disease; and
- adult Ulcerative Colitis. The term "NA" indicates that the CpG site and probe is not currently known to be associated with a gene. The present invention is also described by reference to the following numbered paragraphs, which relates to particularly preferred embodiments of the invention:
1 . A method for determining the presence of Crohn's Disease in an individual, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- determining the presence of Crohn's Disease in the individual on the basis of the methylation state at the two or more CpG sites.
2. A method for predicting the likelihood of an individual contracting Crohn's Disease, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- predicting the likelihood of the individual contracting Crohn's Disease on the basis of the methylation state at the two or more CpG sites.
3. A method for selecting a treatment for an individual suspected of having Crohn's Disease, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual; - determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3;
determining the presence of Crohn's Disease in the individual on the basis of the methylation state at the two or more CpG sites; and - selecting a treatment for the individual.
4. A method for predicting the response to anti-Crohn's Disease treatment of an individual suspected of having Crohn's Disease, comprising the steps of:
providing a sample comprising leukocyte DNA from the individual; - determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; - determining the presence of Crohn's Disease in the individual on the basis of the methylation state at the two or more CpG sites; and
- predicting the response to anti-Crohn's Disease treatment of the individual.
The method according to any preceding paragraph wherein the individual is a child. The method according to any preceding paragraph wherein the individual is an adult.
The method according to any preceding paragraph wherein the individual is a human.
The method of any preceding paragraph, wherein the step of determining the methylation state comprises determining the methylation state at three or more CpG sites in the leukocyte DNA, for example, four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more, or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or more, or 110 or more, or 120 or more, or 130 or more, or 140 or more, or 150 or more, or 160 or more, or 165, CpG sites in the leukocyte DNA.
The method according to any of paragraphs 1 and 3 to 8, wherein the step of determining the presence of Crohn's Disease in the individual comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
The method according to any of paragraphs 2 and 5 to 8, wherein the step of predicting the likelihood of the individual contracting Crohn's Disease comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
The method according to paragraph 9 or 10, wherein the one or more control individual has Crohn's Disease. The method according to paragraph 9 or 10, wherein the one or more control individual does not have Crohn's Disease.
The method of any preceding paragraph, wherein the CpG sites are associated with a gene selected from those listed in Table 4.
The method of any preceding paragraph, wherein the CpG sites are associated with two or more genes selected from those listed in Table 4, for example, three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more genes selected from those listed in Table 4.
The method of any preceding paragraph, wherein each of the CpG sites is associated with a different gene selected from those listed in Table 4.
The method according to any preceding claim, wherein each CpG site is located in a regulatory region of the associated gene.
The method of any of any preceding paragraph, wherein the CpG sites are located in a defined genetic region.
The method of any of paragraphs to 1 to 12, wherein the CpG sites are within a genetic locus selected from those listed in Table 5.
The method of paragraph 18, wherein the CpG sites are within two or more genetic loci selected from those listed in Table 5, for example, three or more, or four or more, or five or more; or six or more, or seven or more, or eight or more, or nine or more, or ten or more, genetic loci selected from Table 5.
The method of paragraph 18, wherein each of the CpG sites is associated with a different genetic locus selected from those listed in Table 5.
The method according to any preceding paragraph wherein, preferably when the individual is a child, the CpG sites are selected from the group comprising:
- 1 1 :1297087 (TOLLIP); and
- 17:75473969 (SEPT9); and
- 6:166970252 (RPS6KA2); and - 15:59588622 (MY01 E); and
- 17:1278466 (YWHAE); and
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1 ); and
- 17:57918262 (MIR21); and
- 3:57041402 (ARHGEF3); and
- 3:101901234; and
- 4:87752504 (SLC10A6); and
- 22:50327986; and
- 7:797592 (HEATR2); and
- 2:235580461 ; and
- 21 :46341054 (ITGB2).
The method according to paragraph 21 wherein the two or more CpG sites are selected from the group comprising:
- 11 :1297087 (TOLLI P) and 6: 166970252 (RPS6KA2) ; or
- 17:75473969 (SEPT9) and 6: 166970252 (RPS6KA2); or
- 11 :1297087 (TOLLIP) and 15:59588622 (MY01 E); or
- 6: 166970252 (RPS6KA2); and 17: 1278466 (YWHAE); or
- 6:166970252 (RPS6KA2); and cg0930439; or
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1 ), and 15:59588622 (MY01 E); or
- 17:1278466 (YWHAE) and 3:57041402 (ARHGEF3).
The method according to any preceding paragraph wherein, preferably when the individual is a child, the gene is selected from the group comprising: TOLLIP; SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2.
The method according to any of paragraphs 21 to 23 wherein the gene is: TOLLIP.
The method according to paragraph 23 wherein, preferably when the individual a child, two or more genes are selected from the group comprising:
- TOLLIP and RPS6KA2; or
- SEPT9 and RPS6KA2; or
- TOLLIP and MY01 E; or - RPS6KA2 and YWHAE; or
- RPS6KA2 and cg0930439; or
- VMP1 and MY01 E; or
- YWHAE and ARHGEF3.
The method according to any preceding paragraph wherein, preferably when the individual is an adult, the CpG sites are selected from the group comprising:
- 17:75473969 (SEPT9); and
- 6:166970252 (RPS6KA2); and
- 15:59588622 (MY01 E); and
- 17:1278466 (YWHAE); and
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1); and
- 17:57918262 (MIR21); and
- 3:57041402 (ARHGEF3); and
- 3:101901234; and
- 4:87752504 (SLC10A6); and
- 22:50327986; and
- 7:797592 (HEATR2); and
- 2:235580461 ; and
- 21 :46341054 (ITGB2).
The method according to paragraph 26 wherein the two or more CpG sites are selected from the group comprising:
- 17:75473969 (SEPT9) and 6: 166970252 (RPS6KA2); or
- 6:166970252 (RPS6KA2); and 17: 1278466 (YWHAE); or
- 6:166970252 (RPS6KA2); and cg0930439; or
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1), and 15:59588622 (MY01E); or
- 17:1278466 (YWHAE) and 3:57041402 (ARHGEF3).
The method according to any preceding paragraph wherein, preferably when the individual is an adult, the gene is selected from the group comprising: SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3; 3: 101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2. The method according to paragraph 28 wherein the two or more genes are selected from the group comprising:
- SEPT9 and RPS6 A2; or
- RPS6KA2 and YWHAE; or
- RPS6KA2 and cg0930439; or
- VMP1 and MY01 E; or
- YWHAE and ARHGEF3. The method according to any preceding paragraph, wherein the sample is selected from the group consisting of: a blood sample; a serum sample; a plasma sample; a sample of intestinal cells; a biopsy; a stool sample.
The method according to any preceding paragraph, wherein the leukocyte DNA is obtained from the sample by a method comprising the steps of: (i) cell lysis; (ii) removal of cell membranes and/or cellular RNA and/or cellular protein; (iii) purification of DNA.
The method according to any preceding paragraph, wherein methylation state is determined by a method selected from the group comprising: bisulfite conversion and DNA sequencing; methylation-specific restriction enzyme analysis; COBRA analysis; methylation-specific PCR; MethylLight analysis; HeavyMethyl analysis; Quantitative Methylation analysis; Ms-SNuPE analysis.
The method of any of paragraphs 3 and 5 to 32, further comprising the step of treating the individual with the selected treatment.
Use of leukocyte DNA from an individual, for determining the presence of Crohn's Disease in the individual.
Use of leukocyte DNA from an individual, for predicting the likelihood of the individual contracting Crohn's Disease.
Use of leukocyte DNA from an individual suspected of having Crohn's Disease, for selecting a treatment for the individual.
Use of leukocyte DNA from an individual suspected of having Crohn's Disease for predicting the response to anti-Crohn's Disease therapy of the individual.
A kit for performing a method according to any of paragraphs 1 to 33 to a use according to any of Claims 34 to 37, the kit comprising one or more reagent for determining the methylation state at two or more CpG sites in leukocyte DNA, wherein each CpG site is selected from those in Table 3.
The kit of paragraph 38, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- a pair of oligonucleotide primers for PCR amplification of the polynucleotide sequence at a first CpG site selected from those listed in Table 3; and
- a pair of oligonucleotide primers for PCR amplification of the polynucleotide sequence at a second CpG site selected from those listed in Table 3.
The kit of paragraph 38, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- two or more oligonucleotide primers for methylation-specific PCR amplification of the polynucleotide sequence at a first CpG site selected from those listed in Table 3;
- two or more oligonucleotide primers for methylation-specific PCR amplification of the polynucleotide sequence at a second CpG site selected from those listed in Table 3; and
- optionally, an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation- specific PCR amplification.
The kit of paragraph 40, further comprising:
an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the first CpG site when the first CpG site is not methylated; and
- an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the second CpG site when the first CpG site is not methylated.
The kit of paragraph 38 or 39, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA; - an oligonucleotide primer for determining the polynucleotide sequence at a first CpG site selected from those listed in Table 3; and
- an oligonucleotide primer for determining the polynucleotide sequence at a second CpG site selected from those listed in Table 3.
The kit of paragraph 38 or 39, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- a restriction enzyme capable of methylation-specific digestion of the polynucleotide sequence at a first CpG site selected from those listed in Table 3;
- a restriction enzyme capable of methylation-specific digestion of the polynucleotide sequence at a second CpG site selected from those listed in Table 3;
- optionally, an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation- specific digestion.
The kit of any of paragraphs 38 to 43, further comprising one or more control sample comprising leukocyte DNA in which the first CpG site and/or the second CpG site is methylated.
The kit of any of paragraphs 38 to 43, further comprising one or more control sample comprising leukocyte DNA in which the first CpG site and/or the second CpG site is not methylated.
The kit of any of paragraphs 39 to 45, wherein the one or more reagent for bisulfite conversion of the leukocyte DNA comprises:
- a reagent for denaturation of leukocyte DNA;
- a reagent for sulfonation of leukocyte DNA;
- a reagent for desulfonation of leukocyte DNA; and
- a reagent for isolation or purification of bisulfite converted leukocyte DNA. 47. A method or a use or a kit substantially as described or claimed herein, with reference to the accompanying description and drawings. Table 3 - CpG sites and CpG probe sequences
Gene
Probe ID Symbol Chromosome Probed Sequence Probe start Probe End Probe Target cg12054453 V P1 17 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 57915717 57915766 57915717 cg 7501210 RPS6KA2 6 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 166970204 166970253 166970252 eg 12992827 NA 3 CCTCTGCCATGCATCAG I I I I CTTGGTTGGCCACTGATTAATAATCATCG 101901234 101901283 101901234 eg 16936953 VMP1 17 GAGCCTCCAGGTACTCCTGAAATGGCTTCTGCAGAGATGGACCTATGCCG 57915665 57915714 57915665 cg04389058 ARHGEF3 3 CGTGCCTTCTGCTGAGGCTGAGCTGGAATTGA I I I I GTTTATTCCAAAAT 57041402 57041451 57041402 cg27049094 HK2 2 CACTGTACGTAGTCTGTCTTTGCC I I I I CAAAGCTGTGGGGGTTATGCCG 75067668 75067717 75067716 cg00382138 CFI 4 CACCTCCCTCAGCTCTTTAATGGGAAGGCCATTTTTTGGTTTCAGTTACG 110723299 110723348 110723299 cg27310092 NA 1 ACTGTACTTTAATGTGCTGTTTGACTCAGCAGTTAACCTCCTGTGATACG 151945615 151945664 151945663 cg02560388 NA 2 CGTGAGGCAGAATCCCCTGAAGGACTTCTTAAAACAGATAACCCGGCCCC 11969910 11969959 11969958 cg20995564 ZEB2 2 GCGACTGTGCAGGTCCCAGACCCAGGAAGCTGGCCCTGTGTATGTTATCG 145171987 145172036 145172035 eg 18942579 VMP1 17 GCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACTTTAATCCG 57915725 57915774 57915773 cg04987734 CDC42BPB 14 CGCTTCCTGCCTCGCATGTGTTTGCGTTGAGAAGTTTGCCACAGGGGAAT 103415825 1034 5874 103415873 cg02003183 CDC42BPB 14 CGCTCCCGTTCCTGCGCACGCAGGCCCGTATGCATGTCTGTTCATACGCA 103415882 103415931 103415882 cg13155421 NA 3 CCCTGGCTGGTGTTTCATGTCCACCAGCTCTGCGTTCAGCACAGCACTCG 105716988 105717037 105717036 cg01671681 PLCH1 3 CCCATTGCACTTACTATCTCCACTTGTAAGGAATGGGCCCCACCTCAACG 155421735 155421784 155421735 cg18181703 SOCS3 17 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG 76354573 76354622 76354621 eg 19821297 NA 19 TGGTTTCCAGGAACAGCTGGGCCCAGCCTAGGTTGTACTGCTGGGAAGCG 12889981 12890030 12890029 cg23966214 SA D14 17 CGAGGAATACACACAGCACACAATGGACAGCCAAACCTCTATGATGCAAC 48203188 48203237 48203188 cg18860310 SLC10A6 4 CGGCCAAAGCTGTTGTTTGTACACATGAGCAAGGGCTGCTGACTACATGA 87752456 87752505 87752504 cg24531955 LOXL2 8 CGTGTCTGTGTTTCCTTTGACCCACAGCACCTGGGCCCTGAGCAGCAGGC 23154643 23154692 23154691 cg01059398 TNFSF10 3 CGGGTTGACGTGAGGTGCTGTGGTTATTCCAAGAATGATAATTAATACGA 172235808 172235857 172235808 cg03746015 CLEC16A 16 CGTGTGCA I I I I ACTTCCTGGTGATTTACGAGAACTCACTAATGAGAAAC 11141000 11141049 11141000 cg22768358 ZBTB16 11 CGTGGAAAAACTGTGTACGTCTCAAATTAAAAGCAGCTCTTGCCCCGCTT 13947148 113947197 113947148 cg05740793 NA 11 GCCCATTTCTTGCCACCTCATGGGCTACACCTTGACCTAACGTCTTTACG 10531043 10531092 10531091 cg25517015 SLC27A1 19 CGCCCAGCGTGAACTGCTTTGAACCATATTGTCTACTTGACTTTCAGACA 17584020 17584069 17584020 cg09349128 NA 22 CGCTGAAGAAGTCTCAGGGTGCCTCACCTCACCCAGCCCCCAGGTCAGCT 50327938 50327987 50327986
cg00506299 RFTN1 3 CGGTGAGCTGCTTCATCCCAAATTGACCATATGCTTGGAGTCTGTGAAAT 16469079 16469128 16469127 cg19653 17 NA 12 GGCAGTACAGACCCGTTTCCCAGGAGGAGCGGGCCAGGATGTGTGTGGCG 132654924 132654973 132654924 cg08423142 MY01E 15 CAACATTACAAACCAAAACAGATCTG I I I I CCTACTGACCTCAATTTGCG 59588574 59588623 59588622 cg26599989 TOLLIP 11 CGGATGTAAACCCACTGATAACGGACAGAAAGAGAATGCCCACAAGTGGG 1297039 1297088 1297087 eg 10780778 NA 6 AAGGAGGATCATCTGCCTGGGTGATCCTGGATCACTGAGCCATGATAACG 26351915 26351964 26351915 eg 10472711 HEATR2 7 CGAGAGGTGGGGCTGTTGACAGTCA I I I I I G IAAAC I G I I I I AAGAAACC 797592 797641 797592 cg1741 1016 NA 2 CAGCACAAACAATGCAGGGTGACTCATCGCCTGTGACTAACAGGCCTGCG 47100912 47100961 47100912 cg07398517 NA 3 CGGGTAGCATTCAGTACAGGTTGATTTCTGCTGCTAC I I I I ACTGGCAAA 30327579 30327628 30327579 eg 10096321 ZBTB16 11 TCTCGCCAAAGACCTTCCAGCAGATTCTGGAGTATGCATATACAGCCACG 113934246 113934295 113934294 cg22529645 LRRC47 1 GTCACCTGCACG I I I I GCCAAAGTCTGATCAGTCACGGTCATAGAAAACG 3704559 3704608 3704559 cg02805025 ASB4 7 CGAGCTGACCTGAAATAAGACCTGACCCAGAACTTCTCTAGGGCAGAATA 95155053 95155102 95155053 cg27039118 TRPS1 8 CGTGGTTGGCTCCCATGGAAGATTTACATTGTTAAAGTAAACTTGTGTGG 116575854 116575903 116575902 cg01409343 V P1 17 CGC I I I I GTGGTGAAGCTTCTGCCGTTGAGCCTCCAGGTACTCCTGAAAT 57915692 57915741 57915740 cg06981309 PLSCR1 3 CGAGTGAGGTTACAGAGCAAAAGAGGGAGACATTCTTAGAGATGAAGATG 146260954 146261003 146260954 eg 16300565 ZFR 5 CGTGCTTTAAAACTCGTTTCAGACAGTTTGTCTGAACATGAGAAGAACAA 32388617 32388666 32388665 cg06219337 YWHAE 17 TCTCCAAACATTCTACGACCAGTGCTTTGTACCAGTTTGTACAAATAACG 1278418 1278467 1278466 eg 10574006 NA 5 CGGTCTGGTCACCAAGCTGTTGTCACAAAATCCACC I I I I ATGGTCTGCC 123965051 123965100 123965051 eg 12338137 TNS1 2 CGAAGTCCCCAGCCCCAGTTGGCCTCTGGGATAI I I I I GGCTTTCTCTGG 218701429 218701478 218701429 cg08692676 TBPL1 6 CGTGCAATGTCTGACTCACCACTTAATAGCTATCAGAACTTGGGCAAGTT 134299546 134299595 134299594 cg02716826 NA 9 GGCCAGACACCCAGGACCCAAATAACCTAATAATGGCTCATTTGCCTCCG 33446984 33447033 33447032 cg05316065 GSDMC 8 CGAGCCAAAGAAGTTGTTCCAGCTAGAGTTGCTGGGAATCCATGAATCAA 130798959 130799008 130799007 eg 18376497 INPP4B 4 CGGAAGGCTGAAGGGGGAGATAATGAGCTAAAGAACAACTTTCACCACCC 143488574 143488623 143488622 cg04222728 KIAA1549 7 CGCACCCATTTGTTGGCTTGATATCAATGACTGTTTTTGCTGCAAGGCAG 138549617 138549666 138549665 eg 18349022 ZSCAN25 7 GAGGAGCAGCGAGAGAGAGCCCACAGGGGCTGCCAGCAGAGCGCACTGCG 99222411 99222460 99222459 eg 16525838 ANKRD11 16 TGCAGGACAGGAGCTCCGCACTTCTGCAAAGCCAGAGTGGAGACAACACG 89440412 89440461 89440460 eg 13745870 NA 3 GACACCAGAGGCTCC I I I I GAGGTCTGATACTGCAGGCTGGGATCTGACG 57094763 57094812 57094763 cg02734358 GPRIN3 4 ACTTCCCATGTGAAGCTGATGCACTTGTGA I I I I GTCC I AA TCA'I TTTCG 90227026 90227075 90227074 eg 11645762 ZBTB12 6 CGTGCGCTTCGCCCACAAGCCTGCCATTAGGCGGCACCTCAAGGAGCAAC 31867771 31867820 31867819
cg09588020 WASF2 1 CGGTACAGATCCTATCCCTGGGGTGTTCATAACCAGGACAGATCAGATTC 27755803 27755852 27755803 eg 12662084 KIF13A 6 AGGAAGTTTGCTTCCCTCACCAAGGTATTAGCTTTAACCAGCTGCTCTCG 17809078 17809127 17809126 cg03725573 ZBTB 6 11 GTAGCCAGCCCTGGAAGGCCTGAATGCACAAACACAGTGCCCTTTCACCG 113962901 113962950 113962901 cg25653947 NA 8 ACATACAGGGGATCATGTGGGGGTGGTGCTCCCCGGTGCCCGCACCATCG 144443217 144443266 144443217 cg27361520 IL18 AP 2 CGCCTTGCTCTCAGCAGACAGCCAGGTCAGCATCAGCAGCCAGGTGGTAG 103038171 103038220 103038171 cg16739178 NA 16 CGCCCATGCCCCCGCCCCCGCCATCTGCTCAGAGGCCAAAAGTCGCTGGG 85470626 85470675 85470674 cg25385322 FIS1 7 CGGGGTCCCCACCTCACCTTGAGCCGGTAGTTCCCCACGGCCAGGTAGAA 100884094 00884143 00884094 cg24743237 D2HGDH 2 GCAGCATTTATAAAGTCTAGAGTAGAGTACGGTCATCTCCTAGGCCTTCG 242702932 242702981 242702932 eg 16622517 NA 4 ACACTTTCTCTTTGATTAGGCTTTCCTC I I I I GCAAGCTAATATCACTCG 63075650 63075699 63075650 cg15174812 NA 1 TTTCTGCAACAGCTAAGGACTGCAAAACCCCACTCTGCATCAACTGAACG 566124 566173 566172 cg02295856 MAPK8IP3 16 CGCCGACCCACAGGGCACAATGGTCTACAAAGTTGGAAATGAAGGTCTGC 1758887 1758936 1758935 cg07339236 ATP9A 20 TCACAATCTATTGAC I I I I AAAACACCAACAACACTGTTTAAAAAGCACG 50312442 50312491 50312490 eg 16292768 CLU 8 TGAATGAAATGCAACAGCCGAAAGTGCTTGGGAAATGCCTGCAAATGCCG 27467735 27467784 27467783 cg03940776 SYNJ2 6 CGGCCACTTGTTGAGGCTAATTTATGCTGCCAGAGGGAGCTGGCATGTGT 158490013 158490062 158490013 cg25953130 ARID5B 10 I I I IC I CAAAGC I I I A I AC I I I I CACTGTGCTCCTAGCAACCAACCAACG 63753550 63753599 63753550 eg 04465154 NA 8 CCACACAGGAAGCAGGAGCACAGGTATAATAAGTCAAAAGCCAGGTCTCG 9045558 9045607 9045558 cg06493289 ZBTB16 11 GGCGGAGGACCTGGATGACCTGCTGTATGCGGCCGAGATCCTGGAGATCG 113934307 113934356 113934355 eg 15466952 NA 1 CAACCTTGATACTCTGTTTCTCACTCAGCAGCACAACAAGGGCAAATACG 65889807 65889856 65889855 cg06653632 SLC15A4 12 TGGCAGGCCACGTGAACTGAAGGCTGAGGCCACATGAACTGAAGGCTGCG 129281396 129281445 129281444 cg13613174 D2HGDH 2 AAGGTAAAAATGCCACACCTGCCTGGGGTGCCCATCACGAGTGGGTCTCG 242702833 242702882 242702881 eg 13410000 BLM 15 CGGGTAATGCTGCCCACAGCCATCACTGTAAACATGCCGGCCCCATGGTT 91357521 91357570 91357521 cg21653586 NA 11 TGCTTGGTTGCAA I I I I I CATCTTTCCCTTGCGGTACTATATCTATTGCG 10530588 10530637 10530636 eg 14880894 CNOT6L 4 TAACTCCTTCCCTTCATTTAGCTGCACTGCTTTCCCACACAGCAGTTTCG 78697811 78697860 78697859 cg08620426 NA 16 CGCTCGCACCCCAGGGCACCAGCCCCCACCACCCACACTGCAGGTCCTTG 29606982 29607031 29607030 cg03665078 TNFAIP8 5 AAACATCTGGAAAATATCAGTGTTGCAGAGCCTCCCGGAAACTGACCTCG 118689961 118690010 118689961 cg01 161042 ZFYVE28 4 TTTGAGACACAAAAGCCAGGATGTCAGGCCCTGTGGCCCCCTGGGTGCCG 2322004 2322053 2322052 cg26914296 OIT3 10 CGACCACTGTACCACATGTCTTGAGAGAGAAGAGGATGTTGACATGGGTG 74673175 74673224 74673223 cg00459243 ZBTB12 6 CGGCCACACTGGCCTCCAGCACGTTCTCGGCCGTGGTCTTGCCGTGTTGC 31867726 31867775 31867726
cg03546163 FKBP5 6 CGGAGGGCTTATTCTATGTAAATAGTTGAAAGGAACTGGATAAGACTGTA 35654315 35654364 35654363 cg24998981 CTDP1 18 TCCAGGCAGCACCGTCTGTGGCTTTCTCTAAGGCAAGGATTTACCTGCCG 77441810 77441859 77441810 cg0466691 1 LSP1 11 CTGCCCTTGAGGTCCTTCCCTTGGCCTGCAGAAGGGCAGCCCCAGCATCG 1872756 1872805 1872756 cg27534567 NA 1 ATGGGGGCTTCAACCGGGAGTACTACTCGATTGTCAACGTCAAGGAGTCG 568536 568585 568536 cg04757345 C4orf29 4 CAACAGCTGCTATGGTTACACATCTTTAATCACATAGTTGCCTACTGCCG 128918835 128918884 128918835 cg207821 17 SLC25A13 7 CGCAGAGTCTCGGTGAGAGAGACCTTAGTAGAGGATAAAATGTCCTTGGG 95865583 95865632 95865631 cg24749265 TRE L5P 6 CCCCCATAGTACAGAATAGAGAGCACACA I I I I GGATCAAAATAAACACG 41216090 41216139 41216090 cg071 19168 SPTB 14 GTTTGTAAAATCCACTTGTGAATTACCAGAGCCCTCCACTCAGCAGCACG 65225253 65225302 65225253 eg 12510999 RBKS 2 CGCTGAATGAGTCACTACCCAAACTGCCGAGGGACCCTAACAGTTACAGC 28022223 28022272 28022223 cg20944315 NA 1 GATGTGGGCACCGCTCACCAGCAACCACATGCTCAGACATTAGAAAAGCG 200839412 200839461 200839460 cg12351310 NDUFS4 5 CTGCCTTTATTAAACCACAAAAATGCCACTGGCCTTAGTAATTACTTCCG 52937992 52938041 52937992 eg 14599823 NA 5 CGGGGCTGGATTTGGGAAAGGCACATATTCGATGATGACATAAATTCTGG 77973731 77973780 77973731 cg1 1249283 JAK3 19 CGGGGCTCTGATCCTATTATCCTAAGAACACTGGCCCAAGTCTCTGCATT 17960179 17960228 17960227 cg09304397 NA 2 CGGGCACCAGGCCTCTGCATCTTTCTCATGTGGGTTTGGTTATCAGTGCT 235580413 235580462 235580461 eg 10045881 CHI3L2 1 GCTTCTTCTGGGATACACATTCTCTAGGTC I I I I ATCCACTGAGGTTTCG 111770291 11 1770340 1 1 1770291 cg00548098 NA 16 CGATCAGTGGCTCACTTGTGTGACTCATCTGTGGGTCCGGCCCTTCATTC 11295619 11295668 1 1295667 eg 15900052 HS2ST1 1 TCTGAGACTGTGAAAAACTTAATCATAAGCAGTTACTTAACTGTGCCACG 87404379 87404428 87404379 cg07243548 VAV3 1 AAACACCACTG CTTTG I I I I CTTAACTGATACCTGTAATATATTTCCACG 108231 1 12 108231 161 108231 160 cg09631059 TSPAN4 11 AT I I I A I lAACCACG I CACGUA I I I I ATCTGTGTGGGTCTGCCAGCACG 856894 856943 856942 cg27307975 LIPC 15 CTCCTCTCTTTATTGTACCAAGATCTGTTTCCTTCACGTTTCACCCAACG 58797791 58797840 58797839 eg 14898177 CACNA1 C 12 CGGCTATCACAGGTGTGCTGGGCCCCTGACGTCGAGGGATTTAGGG I I I I 2486634 2486683 2486634 cg26573274 NA 13 CGCCCCCACGGCTCCCCGACAGCTCCGCCACGCCCTCCCCACGGCTCTCC 1 10386104 110386153 1 10386152 cg23842572 PRIP 17 ACAGAGACAGAGCCCAAGAATAGAGGCACACGGGGAAGTAGACAACATCG 17030253 17030302 17030253 eg 10207609 CD36 7 TGACTTGCTGAGTTTCCCCTAACATATTCTCAGATGCTGAACTGAGACCG 80267619 80267668 80267619 eg 13775629 PRF1 10 CGGCACCTGCACCCTCTGTGAAAATGCCCTACAGGAGGGCACCCTCCAGC 72360400 72360449 72360448 eg 16710656 ANKRD1 1 16 GCCACCAGCGAAGGATGAGCTGAAGCCAGTGCTTGGTCAGCACCACACCG 89381968 89382017 89381968 cg04345034 NA 15 CGCCCCCCGGCGAGGCAGTCCCAGGCCCCGACATTTCAGCTGCATGTGGT 70797389 70797438 70797389 cg27023597 MIR21 17 TGTGTCTGTTGAAACCAGAGTACATGCTAGAAAACATTAACACAGATACG 57918262 5791831 1 57918262
cg01749539 Sept9 17 GAACACGGTTGTGCAAGGATCTGTCTGGGTCCCTGCTTTCCATTCTTTCG 75473969 75474018 75473969 cg27209729 NRXN2 11 GCAGGAATGACCCAGTGAATGATCCAACCCTTTCCGAGCTCAAGATTTCG 64428925 64428974 64428925 cg02795981 Z IZ1 10 CGGGACTCTGGGCCTCATACATGTTAGGCACTTACACTTAACCCTCACGG 81045119 81045168 81045119 cg26894079 CLMP 11 CGGATTCGCTGCCAGTAATACACAATGGGCTCTGTGCCAGAGGATGACTC 122954435 122954484 122954435 cg25001190 NFIA 1 CGGGACTCGGAATAAAAAACACATTTGGAAATAAAATAAAGGTTAAGCTC 61668787 61668836 61668835 cg02192117 TNRC18 7 CGCACCTTCTGCGCTCGCTGGGAGCTGCCTGACTTCTGCACGCCGAGGCT 5458671 5458720 5458719 cg02991558 NA 11 GACAGATGGGAGGGCAGTGAGCACTCCCTCTTTAAAACAACATTTAATCG 4128469 14128518 114128517 cg09137630 ZC3H4 19 CCAGGAAGTCACATGGTGCCTCTGTCTCCAGATTTATCACTGTTGACACG 47618017 47618066 47618017 cg10472395 RORA 15 CGAAAGGGCACCCCCCACTGAGTTCCAAAGAGCCGTTGCCCCTTAAAGTG 61070548 61070597 61070548 cg07677157 NA 12 CGGTGATCCATAGCAAAAGAGCCTATGAGTCAGGTGATGATCCATTCATC 66050880 66050929 66050928 cgO8508455 PTDSS2 11 CGGGGAGCCTGAAGAGACAGAACGAGTAGATGCCATGTGGCTTCCAGGAA 470429 470478 470429 cg04444771 NA 10 CTTCCTGTTCAGGTTAAAAACCCCAAAGGCTTAAGAI I I I ATCATTATCG 45473664 45473713 45473712 cg07949141 ABCB9 12 CGCTAAGGCCTGCAAGTTCAAAGGCTCTGAGGTGGGAACCAACTTGGCCA 123451874 123451923 123451874 cg22515589 BAHCC1 17 TGGCCCAGGGCTGCCTGAGAGTCTGGTCCCTGGGCCTGGGAGTGGCTGCG 79426432 79426481 79426432 cg06192883 MY05C 15 CGCAGGGAGGGGCAGGACTGTTTCCATGGCTTCCTACACTGTGTCCTGAG 52554123 52554172 52554171 cg01726890 NA 7 CGGCAGAATGAGGGTCTTGCAGTTGCCTTACTTGCTGAAACTCTGAAGTG 38370826 38370875 38370874 cg16395183 HHAT 1 TGATGCAAGCACTCCCTTGGATGCTCTGGCTGGTGACTCACTAGGTCACG 210771651 210771700 210771699 cg13800005 NA 7 CGGAAAGCACAAAAGCTCAGAACAGAA TAGTGATCAAAATGGATTAATG 39629242 39629291 39629290 cg05041061 BAHCC1 17 TGGATGGCACACGAGCGCGCTGTGGGCACAAGGTGTCAGCGGCCGGCACG 79426049 79426098 79426049 cg25522181 CIA01 2 ATTGCCTGTTTCCTCCCCAGGGCTGGTTCAGGAACCTGAGCCAACCTGCG 96933235 96933284 96933283 cg01636910 BCL10 1 TGTTCCAGATGCTGTGTCACACAGTTTACAATGTTCCCACTTAACCTTCG 85740751 85740800 85740751 cg15032960 RAB22A 20 CGGAACCTGTGTGCAGGCTATGACAAGATGATAGGGCTGAGGAAGAAACA 56888488 56888537 56888536 cg10864794 ARSB 5 GAGACTGTTTCTAAGGGCATTTGATCACTCCTGACCCTGTTCTTCCCTCG 78203122 78203171 78203170 cg01616956 NMUR1 2 GTGGTGCACCCACTCCAGGCCAGGTCCATGGTGACGCGGGCCCATGTGCG 232393196 232393245 232393196 cg05567440 NA 11 CAGCTGAGAGACATTTTTTAAGAGCAAAATCCATGCATGGCCAGGCCTCG 61772344 61772393 61772344 cg26470501 BCL3 19 TAGGACCCTGATCACTTGTGGCCCCACATAACTGAGTACCCAGAAACCCG 45252955 45253004 45252955 cg13993179 RALBP1 18 CTCTGCCTGTGACAGCACTGATTCGGCCAAGGGGCAGGCACTGATCCACG 9474234 9474283 9474234 cg24632582 NA 15 ATGCTCCATCTCTTGACTGGGACCCACAGAGCTCTCCGTAGCTTCCCTCG 41233653 41233702 41233701
cg25391820 NA 5 CGGCTGTCACAGCGTGTTCATAAAGGAAGGTTATGAGAGCTATAAAACCA 138608471 138608520 138608519 eg 15872437 ITGA9 3 ATGACCATGCCTCAACTTCCAGGTAGTGCTAGAGCTGCTGGACAGCTACG 37804282 37804331 37804282 cg18352710 PRF1 10 AACAGGGGCTCCTGCTCCTCGCTTTGTGAGGACAGCCAGTGGCCTCCACG 72363168 72363217 72363216 cg15011409 ICA 5 19 CCAGGAGTGCGCTGCTCTCGGGAAGGCATCCCATGGCCTGAGCAGCAGCG 10405178 10405227 10405226 cg26101890 NA 2 CGCGTGCTGGCCTGCGAGCACTGCACACAGCCCTGGTTCCTGCCTGCGCC 228185348 228185397 228185396 cg01303372 BAG3 10 CGCACAGGAACTTGCTCAGCCACAACTGCAACTTGCTGGAAATACGAAGG 121415141 121415190 121415189 cg1 848003 LRRC47 1 AACGCTTTCCCGTGGGCCACGGGGCTTCCTGGGCATCTTTGTGGCACACG 3704513 3704562 3704513 cg27469606 SBN02 19 CGGTCCCAGCCTGGGCTGCAGCTCCTGCTACCTCCTGGGCCGGAACCAAA 154485 1154534 154485 cg05304366 EIF2AK4 15 CGCCCCTTGCTACTTTGTTCTGAGAACGC I I I I I GGGCTCTCGCACAGGC 40226905 40226954 40226905 cg12627844 VPS54 2 TATCAATTACAGCTACTGGTAACCTATCATAAGCAATGATGAGCACTACG 64244952 64245001 64245000 cg17042439 PTGFRN 1 CTC I I I I CTGCATGTCAAGTTCTGAGCGCGGACATGTTTACCAGCACACG 117529724 117529773 117529772 cg20477259 TNF 6 GGTACAGGCCCTCTGATGGCACCACCAGCTGGTTATCTCTCAGCTCCACG 31544960 31545009 31544960 cg07094298 TNIP2 4 CGGGAAGCCCCGGGTGACTCAGCTGAGTTCAGGGCTGAGAAGTGACACTT 2748026 2748075 2748026 cg22914762 MIR1973 4 TTCATACAAGTCCCTATTTAGGGAACAAGCGATTATGCTACCTTTGCACG 117220908 117220957 117220908 eg 11793449 NA 17 TCCCAGCCATCCCTGGGTCTAGGATCCAGAATGCTCAATCCTGGATGTCG 76313872 76313921 76313872 cg06065549 NA 20 ATTTAAAAAACAAATTGCGTGCTTGCATACTCGGCATCCCCTCTCCCTCG 57427395 57427444 57427443 eg 13134297 NA 7 CTGAACGCTCATCTGGTTCCTGTTCTTGTGGTGTTTATAGCCCAGAAGCG 30737556 30737605 30737556 cg20655099 TNRC18 7 CCTGCCAGGCAGCAGGTTCTGGGCCACAAGAGGCCATAACCATGAGGACG 5394841 5394890 5394889 cg0841 886 FU 16 AGCCTCTGTTCCTCCTGGACATAGGAAGGTCCCAAGCTTAGTATCCCACG 70513782 70513831 70513830 cg20359445 IKZF4 12 TCCCCAAGGCCTAATCAAGATGTCACCATAGGCTCCCACTGTCCAAGTCG 56415591 56415640 56415591 cg204 2356 FOXK2 17 TTCAACAACAGCCTATTTGTATGTCCACAAACAACGCAAACAAGTGGCCG 80519873 80519922 80519873 cg24174557 VMP1 17 CGGACAAGAAGAGAAAGGGG I I I I CCCAAGCTGCAGAAAGAGGTTATGTT 57903496 57903545 57903544 eg 18990407 EPHB3 3 GTTTGCCAAGGAGATCGACGTGTCCTGCGTCAAGATCGAGGAGGTGATCG 184297332 184297381 184297380 cg23598089 ATP2B4 1 CTGGACAACTACTATCATCACCACCTGGGGACACCAATCATCGTGACACG 203652031 203652080 203652079 cg04656070 TRPS1 8 GCTGGATAATAACTGATGAAAGTACTTTTTTAAGTCTAACCCAAACCACG 116661015 116661064 116661063 eg 19459791 NA 15 TGGAGCAAATATAGGTCTAGACACGTAGGGCCTGTGAGG I I I I CCTGACG 65362974 65363023 65363022 cg06914505 NA 15 CGGTTCCGAACTGC I I I I I GTCTCTGGGCTCAAGTTAGAGAACCTTGCAA 39464601 39464650 39464601
Table 3 continued - CpG sites and CpG probes
Probe ID Chr Position Gene Symbol Probe Sequence
eg 17501210 6 166970252 RPS6KA2 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC eg 18608055 19 1130866 SBN02 TAATAATGAGGGAGTGCTTGCAGCTTTCAGGCTTCAAGAACGCAGCCTCG cg09349128 22 50327986 NA CGCTGAAGAAGTCTCAGGGTGCCTCACCTCACCCAGCCCCCAGGTCAGCT eg 12170787 19 1130965 SBN02 CGCTGAGGGCACCTGCTAGAAGTGAAGGCTTCGAGGGCTCATGCACCTGG eg 16936953 17 57915665 VMP1 GAGCCTCCAGGTACTCCTGAAATGGCTTCTGCAGAGATGGACCTATGCCG eg 12992827 3 101901234 NA CCTCTGCCATGCATCAG I I I I CTTGGTTGGCCACTGATTAATAATCATCG cg25114611 6 35696870 NA GCGCAGTGCCCTGAGGGATGCTTTGTGCCTGGAGAGTGCCAGGACAGGCG cg02448796 1 6101339 KCNAB2 CGCAGGCACAGGCAGTACCCCAGCAGCCCATCAGTCTTGGGAAGCCAGAC eg 12054453 17 57915717 VMP1 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT cg07398517 3 30327579 NA CGGGTAGCATTCAGTACAGGTTGATTTCTGCTGCTAC I I I I ACTGGCAAA eg 13619623 7 33637324 BBS9 TGATGCCACAAATGTCCCAGAGCTAGTGCAGGTTCCCCTCCTTCCTATCG eg 16724148 1 100326338 AGL ATAATCCCGACATGGGCAATCTTAAGCTACCAACACGACAACATTGAACG cg26804423 7 8201134 ICA1 TTGTCAAGCTTACAGTTTCATTACATTTGTTAACATCCCATTGACCAACG eg 19821297 19 12890029 NA TGGTTTCCAGGAACAGCTGGGCCCAGCCTAGGTTGTACTGCTGGGAAGCG cg22959742 10 13913931 FRMD4A CGGGGCTGTGGCTTATCCTGGCTTCACCACTTCCTAGCTGAGTGTGATCC cg03546163 6 35654363 FKBP5 CGGAGGGCTTATTCTATGTAAATAGTTGAAAGGAACTGGATAAGACTGTA cg01059398 3 172235808 TNFSF10 CGGGTTGACGTGAGGTGCTGTGGTTATTCCAAGAATGATAATTAATACGA cg26955383 10 105218660 CALHM1 CTCCTGGCTGGGACCAACAGAGCTCAGAGCAGAGGCTGAGGTCACTGTCG eg 10636246 1 159046973 AI 2 CGACACCCTCAAGGGAGGAGTGCAGGCACTCAAAGATTTGAGTCACAGGC eg 18942579 17 57915773 V P1 GCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACTTTAATCCG cg01101459 1 234871477 NA CGCGGGCCTGGCCTGTGTGTTCATCCAAGTTGCTGCAATC I I I I CTTGTT eg 17953136 2 20232577 LAPTM4A CGGGGTGAGTAAGTAGTTGGGTATAAGATCTGAAC I I I I CATCTGCAGAG cg26020069 6 52382441 TRA 2 CGGCCACCAGGCTCACAGCCCCGCAGCCTGCTGCCCACTGACTGGAGCAA cg02716826 9 33447032 NA GGCCAGACACCCAGGACCCAAATAACCTAATAATGGCTCATTTGCCTCCG cg25132241 14 92396859 FBLN5 TTCCTCTCTGGGCCTGCGTAGTCCCAATGCCCTCAGCAGCTAAAGAATCG cg27087650 19 45255796 BCL3 GCCACCGAGCCCAGCTTACAGCTTTACTTTGCCA I I I I CTATTCTAGTCG cg23172671 1 203482523 NA TGGGTTCTTTAGGGTGTGTCTCCAGCTTGAAGTGTGCCTCACAGTAGACG cg26470501 19 45252955 BCL3 TAGGACCCTGATCACTTGTGGCCCCACATAACTGAGTACCCAGAAACCCG eg 12670943 22 43006541 POLDIP3 CAAGGCTGCCACACTGAACTGCTTGGAGGCAGCTGACAGAAAGCTATCCG
cg07035242 1 1 336263 UBIAD1 CGGGTGGTAGTGGAAGAGGTAAGTAGCCAGATTCTAAATATGCTTTGAAG cg17781958 17 79428404 BAHCC1 CGGCCCCTGCCCAGCCCCAGCTATGTGCACCCGGCCCTTGTGGGCAAGGA cg02734358 4 90227074 GPRIN3 ACTTCCCATGTGAAGCTGATGCACTTGTGATTTTGTCCTAATCATTTTCG cg25533551 19 48823178 CCDC114 CTACTGGTATCCAGTGAGTAGAAGCCATGGACACTGCTTGGCATCCTACG eg 1 1849692 10 103875969 LDB1 TGATACACATTTCAACCTGTGATATATCTTTTTTCCAAGACGGAGTCTCG cg12582317 17 55822272 NA CGCGGCAACAGGAAGGTGAGAACCTTGTTGCGCACGATGCCACGGTCCTC eg 17588003 17 5138696 SCIMP CGAGGGGACCCAACTCAGTCAAAATGCTCCACTGGCCACATTTCTCAAGG cg23740758 6 11324433 NEDD9 CGGCAGTGTCAGGAGAGAGCCACAAAATGAAGGCAAGGGAATCACGGAAG cg25653947 8 144443217 NA ACATACAGGGGATCATGTGGGGGTGGTGCTCCCCGGTGCCCGCACCATCG cg27243685 21 43642366 ABCG1 CGGAGGATGTGTTTCCAGAGAGCTGGTCTATTTCAGACTGACAGGCCACT cg09007354 1 54100163 GLIS1 CGCTCAGCTGTGTCAAGGCTGCTTACAAGACAGACAAAATGAGGCCTGAG eg 11832534 1 3563998 WRAP73 CCAGATCCAGCACATCGAGTGGTCGGCAGACTCGCTCTTCATCCTGTGCG eg 16805291 7 36022575 NA CGCCATTCAGCAGTGGGATTTTATTTTGTTTCAGAAATACTCCAGGGCCG cg04987734 14 103415873 CDC42BPB CGCTTCCTGCCTCGCATGTGTTTGCGTTGAGAAGTTTGCCACAGGGGAAT eg 12669088 12 25541364 NA GCTGAATTGCCCACATTAGCATTTATGGTAGAGACACACCTCCTTTATCG cg04465154 8 9045558 NA CCACACAGGAAGCAGGAGCACAGGTATAATAAGTCAAAAGCCAGGTCTCG cg09018739 16 57180107 CPNE2 CGCCCACGATGATGATGGACATGGGCAGCTTGGAAGCCTGCACCACGGCA cg10180440 10 102325486 NA CGCGGTGGTTTAGAAAGGACTCGTGAATATAGAACACTCCTTGGCATGAA cg00053916 8 37457329 NA ACCCTGGCCTGTCTCGCAGGGTCATCCTGAGAAATAGCTGATGTCAGACG eg 13585930 10 72027357 NPFFR1 TCTGTGAATTAGGACCAGGCTTTGGTGTGTGTTTCCCCAGGCTTGCTTCG cg01145119 8 144441955 NA GGGTCAGGCGGGGCCCTCAGTGGCCCTCTTCCTGTGGGTGACCCAGGTCG cg05768620 11 71752971 NUMA1 CGCAGGCCTGTACTCCAGGGCCAGAATCTGCCTGGGCTTCTTGTTTGGGT cg22448090 1 200978598 KIF21 B TCCCGGGGTAACAGAGGTACAGATGTGACAGCCCTCAATCTTCTCCTTCG cg02719954 8 23830907 NA CATGGTAGCTTGTCTTCAAAGATGACCCCCCTGCATAGAATCATGTGTCG cg11393173 1 116369577 NA CTCAATTACCCTGTTCCTTCTGTTACACTATCATTGGCTCTGTCTATACG cg11396411 7 33679233 NA GGAGGCAGTGAGTCCGGAGTAGACAACATAACACAATTATTGTTATTCCG cg1 515347 1 159047163 AIM2 TTCCCGGAATCGGGGTGCCACCCTTTTCTTGACTAAAGATGGTTAATCCG cg02543993 7 5736195 RNF216 TTTGGTGAAGAGCGGCCGAAGCGAGACCATAGGACACGATCTTGGGCACG cg00382138 4 110723299 CFI CACCTCCCTCAGCTCTTTAATGGGAAGGCCATTTTTTGGTTTCAGTTACG cg00826902 1 3563954 WRAP73 TGTGCGCCATGTACAAGCGAGGGCTGGTGCAGGTGTGTGCTGCCGTGTCG cg01799015 19 707791 PALM CTCACCCATCTCCAAGCTGTGTGACCCTGGGTCTGTTTCTCAACCTCTCG cg03393889 16 2094700 NTHL1 CGGTGGACAGCATCCTGCAGACAGATGATGCCACGCTGGGCAAGCTCATC cg25422678 2 28304502 BRE CGCTGAGAAGGGCCCAGTGTGAATTAGGCTAGTGGAAATGCAGACTCTTA
cg02650017 17 47301614 PHOSPH01 GGGAAACGGCTGCAGATGTGCGCCTCCACCTGCAACAGGTGCTGAAGTCG cg03128029 2 203143288 NOP58 CGGGACAACAGAGTGATCGGGTTTATTTACTGTAATTGACTGCAGTAGGT cg20228731 7 130646051 LINC-PINT CG G G CTATTG G CTAAATG AAGG C AATG CGTGTGGAAGTCTGATTTGCCAC cg24430034 13 110386176 NA CGCTCCCCAAGTGCTGACCACGCGCGCCCCCACGGCTCCCCGACAGCTCC eg 12424624 3 46718941 ALS2CL GTGTAGCCTACGAGCATGACACTGGAGTGCAGGGCTCCATTAGACATGCG cg27128761 5 151055650 SPARC AGCCCCTGTAAGTACCTAAATATCATGTCTTCTTGTTTTCATTCCTACCG cg00441899 4 3432483 RGS12 GAGACTGCCTCTTCCAGATCTGGGCGGTGCCCTCCTGCGCGAGGGAGACG eg 13354241 2 64447091 NA CGTTCACATATGTATTGAGCAGCTAGTAAGTGTCGGGCACTCTTCTAGGT cg02297838 13 92002454 NA AAGGCAATTTAGTCCATGTGTACCTTTTAGAGGAAATCTTCACATCCACG cg21846903 17 26697281 VTN TTCTAGCTCAGTGCCTGGCAAGCTGGGCTCTGGTCTCCCTGAAGTCTCCG eg 14556762 1 170532470 NA CTAGGATTTCTATTCTCTTTTTTATTCTCTGTAAAATTGAAATGAACTCG cg08392125 2 108427720 NA CGGGAGGGAGCACAGTTTGCAGCCTGTGACCATGGCATACACTGGCTATT cg06846976 16 2121682 TSC2 CGCCACCACAGCCCCAGCGCCTCTGACGCCCTGAGCCTCATGGGCCGTGG cg15551881 9 123688715 TRAF1 CGGCAGCCTGCGATTCTCAACCAGGGGAACTCTCACTGTGCTTTCTGAGA cg26307105 19 17861017 FCH01 GACCTCTGAGTCAGCTGGCAATGTGCTGTGTAACCTTGGGCAAGTCTCCG cg22753611 6 17472892 CAP2 CGGCTACTGCCCGTGCTGGATGTAGATGCTGGTTAACACACAATAAAACA cg07148145 7 99437259 CYP3A43 CGCACAAAGAATAAGGTTAAGAGCAGAGGTTGGATAGGTGAAAGAAAGAG eg 17980786 3 32933637 TRIM71 GTGCCCTCCCAGCCATACAGATGCCAGGCTTGTGCATATCATGCCCCACG cg00490406 1 159046773 AIM2 CGCAGATGACTTCTTGATACACTTTGTGACTAAGGGGCCTGGTGATGACA cg18721397 5 32584912 SUB1 CGAGGAAACCAAAGTTAACGCCCTATGGAGAGGATATTTCCTGTTACAGC cg04036920 11 33562503 KIAA1549L CCTTTCTCATTCTTCCTTTTCTTTAAAATTCCCCATTCTTGGGGTTTACG cg20364632 6 49636226 NA CGGTGTGAGGCAGCTTATTTACCAGGAAATGAAAGATCTATGTGTGTCTT eg 10549973 14 58863097 TOMM20L AGGGCACGCAGGTGCAGTGCTTTCGATCCGCACAGGTAGCTCAGTAGACG cg07835482 2 182656091 NA CGGGGCCATCTGTGCAACTTAAACACAATGGCAACCTATGCAGGTGGTTG cg03523676 14 24540235 CPNE6 CGGGTTAGTTTGAGTGAGTACATGTGTGACAAGGACAGCCCAGTGTTCCT cg08289839 13 111318640 CARS2 CGGTGTCGGTGGAGTTTCTAATCAGAACAAACGCCTGCCGCCCTCACTTC cg20141 08 1 165907859 NA GATGAATTTTCAAACACTCTTACAGCTACTTCTTACAACACCGTCTCACG cg24469729 7 27160520 HOXA3 ACAGTCCTCCTTTGCCACTCTGCAATCCAGTTTCCTGGAGCAAAGCTACG cg26599989 11 1297087 TOLLIP CGGATGTAAACCCACTGATAACGGACAGAAAGAGAATGCCCACAAGTGGG cg20995564 2 145172035 ZEB2 G CGACTGTGCAGGTCC CAGACCCAG GAAGCTGGC CCTGTGTATGTTATCG eg 15466952 1 65889855 NA CAACCTTGATACTCTGTTTCTCACTCAGCAGCACAACAAGGGCAAATACG cg07879825 7 69358880 AUTS2 CGCCACTCTTGAATTTCAAAATGAGATGGTTCAGTGTTGTGCAAACTAAG cg14176339 17 79244542 SLC38A10 AAAAGGGACTTGGTCCAGTCAGCCTGGGGAACCCTGGGTTCACCAGGACG
eg 18860310 4 87752504 SLC10A6 CGGCCAAAGCTGTTGTTTGTACACATGAGCAAGGGCTGCTGACTACATGA eg 12269535 6 43142014 SRF CCTCGTCCTAGGGGACAGGTGTCCATCCTCACTTTCCTGATAGAAGACCG eg 14722693 8 19436451 CSGALNACT1 TCCTTCCTTACATCATACTTTCTCTTGGAGTTCTTGCTACGTTGACCACG cg23761815 10 73083123 SLC29A3 CGGGACAGCCCAAGGCTGGTATGATGTTCTATGGCATTAGGAACCCAGTT eg 10005565 2 235578952 NA TGAATGCACACGATTGCCTTAAGTGGAATCCAGGGACTTTCCCTCGGTCG cg21727145 15 101458127 LRRK1 CGCATGCCTAATGGCTAGAATTAAATGCTGGGAATATAACGAGCTCAGCA cg05316065 8 130799007 GSDMC CGAGCCAAAGAAGTTGTTCCAGCTAGAGTTGCTGGGAATCCATGAATCAA cg06192883 15 52554171 MY05C CGCAGGGAGGGGCAGGACTGTTTCCATGGCTTCCTACACTGTGTCCTGAG cg27023597 17 57918262 MIR21 TGTGTCTGTTGAAACCAGAGTACATGCTAGAAAACATTAACACAGATACG eg 12807764 5 146864669 NA CGATTGCAAAAGCAACCCACAGCATCGTAGAAAATGTAGAAAATGGAGCT cg11847933 1 227130394 ADCK3 CGTATGTGTATTTTTACACAGCAGCGTGGACATGGGTATGATTTGTGGAG cg15310871 8 20077936 ATP6V1 B2 CGCGGAGCAATGCAGAAGCAGCAGCTAATGCTTTGCAGAGTCTCGAGGGT eg 13447080 8 131054408 NA CGGGGTGCAGGGTTTGCATTATTAGCACTCAAGTTTGAGACCCACAGGTT cg08423142 15 59588622 MY01 E CAACATTACAAACCAAAACAGATCTGTTTTCCTACTGACCTCAATTTGCG cg03998636 13 111210121 RAB20 GCCGCCTTCCAAGCTGGCTCTTCAAGGGCCAGGTGCTGGGGATGCAAACG cg25001190 1 61668835 NFIA CGGGACTCGGAATAAAAAACACATTTGGAAATAAAATAAAGGTTAAGCTC eg 13558682 1 3705262 LRRC47 CGCCATCCCATTTCTGAGCCATGCTGCCCTTACCCGTGTCTGAGCAGCTT cg22768358 11 113947148 ZBTB16 CGTGGAAAAACTGTGTACGTCTCAAATTAAAAGCAGCTCTTGCCCCGCTT eg 19445588 12 116904641 NA CGGACGGGCATTACTCATGGGCTTAACTGGAATGTTTAGGAAGCAGATGT eg 15082870 7 36022841 NA AGGGAATACCACGTTTGGCTTACCATTCAGCCTGTTAACGTTTGTTCGCG cg02976843 17 2843257 RAP1GAP2 CGCTGGACAACTTCCTTCAGATCAAACAAGGAACTACAGGACTCGGGTGT cg02569509 7 142423070 NA CGCAACACAGCCAACTCAGGCTGAGGACATTTGTAATCAATGTCATTTTC cg00371134 1 77912654 AK5 CTTTCAACATCAAGCACAAGTGTTTCCTGCAGAGCCGGGAGGCAAACCCG cg27269962 7 127540997 SND1 CGGCTGTTAAGCAGACACAGCAGGAGAAGTTTAGAAGTCAGGAGTTTCAT eg 11058932 7 130372167 TSGA13 CGGGTCTTGTCTGCCTCTTAAGCTGTGAGTCCCTTAAAAGTGGAAATGTA cg08549335 7 30387954 ZNRF2 CATTGGCTCTTCACTTGTTGCTTGGTGTTTTCCTTAGTCTTCTGAGATCG cg23665802 13 92002338 NA CGTTCTGCTACAATTGACTGATAACACTTGAAGTGTAGTCTGAACAGTAA cg26663590 16 28959310 NA CGGTGCACTCAGCATTGATCTCAGGGCAGCTGCTCCCTGACCTGCTTCCC cg03940776 6 158490013 SYNJ2 CGGCCACTTGTTGAGGCTAATTTATGCTGCCAGAGGGAGCTGGCATGTGT cg05399785 1 3564031 WRAP73 CCTTCAGATCCTTCAGCTGTACACGTGCCTAGACCAGATCCAGCACATCG cg13316148 2 191916269 STAT4 AATGTTGTTGACCTCTCTTGTCTACTGTGTCTCCTTTTGCACTTGTGTCG cg03951877 6 13120250 PHACTR1 GGAGATGCACCCTGCCTGTCACATCAAATCGACCTGCTGGATGAAATACG cg14761417 7 130636860 LINC-PINT AGCTCCTGGCCCCTGTTTCTGCTTTCTCTAGCAGTTTCCTGGCAAAAGCG
cg09912552 8 134250937 NDRG1 TCTGGTGGAGACAAATGAGGTCTATTACGTGGGTGCCCTCTCCAAAGGCG cg21075678 7 137557164 NA CGCCACAGCTCATAGCCAAGCTGCCTTCCTGCTCAGGCCTCTGAGGGTGA eg 18376497 4 143488622 INPP4B CGGAAGGCTGAAGGGGGAGATAATGAGCTAAAGAACAACTTTCACCACCC cg17411016 2 47100912 NA CAGCACAAACAATGCAGGGTGACTCATCGCCTGTGACTAACAGGCCTGCG cg02782634 17 57916643 VMP1 CGCCCGGCCAGAGCACTGT I I I I I TTAATGGCCTTGCACTCTTCTTATGG cg06160572 12 123892724 SETD8 CGTCTCAACAGGCGCTCACTGAAGTGTATGAATATTTTTTAAAAAGGTTT cg27365342 6 12881417 PHACTR1 CGGCAGATGTAAAAAAGGAAGGACAGTGGGGGCTCTTGTTGCCATGGTGA cg06710464 17 79047695 BAIAP2 CGCTCACGGTTAAGCCCGTGTATCACTTATTTGTCTCAAGTGCTTATGGA eg 18181703 17 76354621 SOCS3 CGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGG cg02349866 15 90391877 AP3S2 CGTGGCATAGCTGGTTTCATTAATTCCTAGGAAAGGGCTTCAGAAAATAA cg26227957 1 19547285 EMC1 CACTGCCCATGAGGGTCTCACCTGCTTTGTTCTGTTGGGATCTCGGGGCG cg23752007 7 37429500 EL 01 CGGTGATTAATTTTTGTTTCAAAGAGCAGCTCTCCCTAATGAGGGTTGCA cg05398036 1 186504948 NA CGTGAAGTACATGCCACTTTCTCAGTAGGAAAGACTGTTGGCAGACTGAC eg 14237301 16 28506477 APOBR GGCACCTTTGTCTCCTACCTCCTGGGAGATGCAGTCCCCACTGTAGAGCG cg25368647 5 176736591 MXD3 CTAGGCCCTATTCTAGGCACTGGGGGCTGTGGCACACAAAGACCTATGCG cg04086394 1 193082764 NA CGGCAAGGTCTCAGCTGAATCTTGGTGGAAACTTCACTTCTGTCAATGAC cg01839860 5 138957422 UBE2D2 GTTACTGACACCCTCTTCTGTAACACTAATCACACATGTACCGCAGTACG eg 16755922 17 80536214 FOXK2 CGCTCAATTTGGAAAGCCTTTGGGGGAAAGGTAATTGCTCAGAATTGCAA cg22749855 17 76353952 SOCS3 CGGGGAGAAGGGCTTGGGGTGACCTGAAGGGAACCATCCTGGTACCCCAC cg06086933 1 100326491 AGL GTTGCTAAATCTTTATTAGCACATCACTGGACTTGAGCAGATATCAGTCG eg 13951069 21 43919801 SLC37A1 AGAAACGAAGTACAGAGGAACCGTGGCCACTGTTGCCCTCCACTCTCTCG cg24386894 8 8863572 ERI1 CGGATGCTGATGCAAATGTAAGTATGTATTTCGTAAACTAATTTTGTCTT cg05544807 2 254759 6 DN T3A ACTCTGGGCCTTCCCTGGATTTCTCATTTTCCTGTTTGCAAGGTTGGTCG eg 15928106 7 130646078 LINC-PINT TTAGGATATAAGACGCCTGCCCTGAAGGTGGCAAATCAGACTTCCACACG eg 12053291 12 125282342 SCARB1 CGGCTAAGGAGGCCTGGGAGGACAAAGAAAAATGGTGCAGAACAGCATCC eg 19742686 19 10823913 QTRT1 CGCTCTGGCCTCTGTGGGAATCACACTGGGCTGACCTGGCATTGGGAGAG eg 10472711 7 797592 HEATR2 CGAGAGGTGGGGCTGTTGACAGTCATTTTTGTAAACTGTTTTAAGAAACC eg 13270236 6 42658437 UBR2 C GTGTAAAAAG G G AAG AG G G C AGAG GAG AAC ACAAAAG AAG AATTATAG G cg07793033 16 85256423 NA CGGGCCTCCTCTGCAGCAGAAGTTTGGATCCATGCGGAGCTTCCTTCATA cg01409343 17 57915740 VMP1 CGCTTTTGTGGTGAAGCTTCTGCCGTTGAGCCTCCAGGTACTCCTGAAAT cg02003183 14 103415882 CDC42BPB CGCTCCCGTTCCTGCGCACGCAGGCCCGTATGCATGTCTGTTCATACGCA cg21211645 3 45590039 LARS2 CGGTCTGGCTGTTGGGAAGATGGCCAGGAATGGACTCATACCATTGGCAC cg02600394 4 48136234 TXK ATGGTAGCCCCTTCTGCGGGGAGCACACAACAGTCTTCAGTTCTTCTGCG
eg 14427590 17 60695089 NA GAAACTTCTTTGCAAAGCACAAGTGCTAGAGCCCCTTCTCTCTGCTATCG eg 16663980 1 158809049 MNDA CGTTCTCTGAGGCCTATATGGACGTGTGTCTTGCCTGGGCTTATGTGTGT cg20519581 20 48959844 NA CCCAGGGTGCACCTGCTGCCTCAGTGTTGTGTATGCTGTGGCTCAAATCG eg 11679124 10 14058944 FRMD4A CGAGCTGAACAGCCTGCAAGGATGCTGATGGGCCTTGTCCTTGATGTCCA cg23842572 17 17030253 MPRIP ACAGAGACAGAGCCCAAGAATAGAGGCACACGGGGAAGTAGACAACATCG cg15321908 3 53187213 NA CGTGGGAGCTGGTATCACTGACTGTCCCCCAAATGTGCCTCCCCTGGATT cg25445707 3 46719086 ALS2CL GTCCACTCCCAGCTCCTGCTGTCAGGGAGAAGCAGCATGCAGGGACAGCG cg05941027 17 61774174 LIMD2 TCAGCAGAGATGGTGCCCCGGCCTGGATCCTCCCAAGTGTGAAGTGAGCG cg27106909 16 30106897 YPEL3 TTCCCCATGGATGCAACCTGAGGCCTCCTCACTGAGTCAAAACCGAGACG eg 17969271 5 171430188 FBXW11 GCAGAAATTGATGCTATCACTTTAGGAAAGGCGATCCTGCCTCCTCCACG cg22688566 17 27459835 MY018A ATGGCAAATATTCACCAGAGTGGAGCCTACTTCTTTAAGAATTGGAATCG cg03546806 12 114297307 RB 19 CGATATCTTCTAAGCCAAAGTCAGGCAAAGCTCTGGAGAAGAAAATGGCT cg20510033 1 8960134 NA CGGAAATTCAGTGATGAGTAACTCAGAGGAGCGGTTAGAACTTAGGGCTT eg 11095027 11 1297066 TOLLIP CGGACAGAAAGAGAATGCCCACAAGTGGGTCTTCTGTGGAAGATGCAGAA eg 12394201 11 43942418 NA AAGTGGGAGCAGCCCTGCAGGGAATGCACTCAATTGCCTAATAGTTCTCG eg 16739178 16 85470674 NA CGCCCATGCCCCCGCCCCCGCCATCTGCTCAGAGGCCAAAAGTCGCTGGG cg13410000 15 91357521 BLM CGGGTAATGCTGCCCACAGCCATCACTGTAAACATGCCGGCCCCATGGTT cg02593766 17 48608677 NA CGGGACAACATCACAGGCAGAGTTTACTCTGTGGGAAGCACCTATATATG cg02538248 16 75045643 ZNRF1 TGATAAGGGCTGACTTGCAAGCTAGCTGCCATCCATCTTAATATCTGACG eg 19405484 8 29513210 NA GCACCTTCTTCTTTCCTTGAAGGCTTGTGACTCATAGCTGCTGTGTAACG cg21416544 12 8986988 A2 L1 ATGTTTCTTGTGCAAGAGCTCAGTCACAGGGTACTGAGACTGGTCTGACG cg06724236 14 67853825 PLEK2 CGCCTAATGGCTGTTCTAGCCTTTCCAGGTTTGTAACATGAAGATGGGGA eg 10722217 3 14347415 NA CGAGGCAGTGACTTCTCATTTTTATGGCTGTGTGGTACGCCATAACCAAA cg27310092 1 151945663 NA ACTGTACTTTAATGTGCTGTTTGACTCAGCAGTTAACCTCCTGTGATACG cg21190595 11 3071167 CARS TGCACACGAATGCCTTTCTCTGCTTTGCTTAGTGTGGGGAAAGCCCTTCG eg 13442606 11 3071269 CARS CGGCCCAGTGGGGCATTCTTGCTGCTGCAGCTGTGCACTCCATTCTGACT cg02508743 8 56903623 LYN CGTGGTAGTACTGCCGGCGTGTCTCTATTTGTTGGTATATTCACATCTCT cg23669 18 16 1538347 PTX4 AGGGTGGCCTCATGAGTGCTTTACGAGGCCCAAACAGGAAGCACCCAGCG cg06541968 2 240431144 NA CTGTCTCCTGCTCCACCCTGTAAGAAATGCCTGCTTTCAATTGCCTTCCG eg 18307303 5 158757456 IL12B CGGAGAGTCCAATGGCCCTGAAACAGATGTTGTTTCTTCTGCTGCTGTTG cg08952844 2 88313351 NA CGGTTTCACCCTGGAGGGCCAAAAGTTCCAATAACCAAGTAAAGCCCATG eg 16642695 8 67343633 ADHFE1 CGTCAGGTATAGTTTTGTTTGGGGGAAGAAAGAAACAGACCCATAAGATC cg00657780 16 50285254 NA ATGAAATGACATTACTTCCCGGTACTTAAAACAGCCTCTGGCACTCATCG
eg 12866551 10 20019641 NA TCCAGTTCCCGTAAACTGGATTGACAAAGGCACAGTTACCACTTCCCTCG cg07723258 17 38176064 MED24 GAGCCGGGAAGGTGGTACTCACGGTGGTGAAGGGCATGAACTGCAGGACG cg19588519 10 125817817 NA CTGCAAGTGGCAGACACTGGACTATCAGGCATTTCCTTCTTCTTTTGCCG cg20813374 6 35657180 FKBP5 GCCAGGTTAGCCCTAGGAAGCAACCCCATTAAGTCTAAGATGCCCAGTCG cg26712428 10 105218771 CALHM1 CGCCTCCACCTCCGGCGCTAGCACCTGCTAGAGACCAGCTTTCTGTGGGT cg26182406 14 105491309 NA CGGCTGGGGTCACAGCATC CTGTGTCTCATGAG G C ATC AGCTG AGCTCAC cg04287259 1 9730401 PIK3CD CGGTGATGATGTGTGGTCTATTTGTAAATTGAGTGCAGCACATCTAATAT cg00851028 1 234905772 NA CGGAAGGTGGCTCTGCAGATCCCCTCAGATGATGCACGCTGTGGACCCGA cg14515811 9 129088215 MVB12B CGCCCAGCCAGGCACCCCAGGCAGAGGCCAGCGTGTGCAAGATTTGAAAG cg00483891 2 13 098007 CCDC115 C G G CAC ATACCAG ATATATCTG GCTTCAG G G CTTTTGTC CTG G CTG GTTC eg 12535090 11 20071677 NAV2 GCAAGCGAGAGCTTCCGCTGCCCCAGATCAAGTGGCAGGGTTTAAGGTCG cg20697417 1 41786797 NA GGCCGGTGGGCTCTAGGGAGTCCCACTTCTCTGCCAGCCAAGTGAGAACG cg20671910 1 151262619 ZNF687 CTCTGACATCTACCCCTGCTCTCCTAGTCAGTGAAAAAGTTCCCCTGTCG cg07683636 2 219940977 NHEJ1 GGGAAGGCCAATTCCCTGTGGGCCTGTCAACATCAACTTCAGTTCTCTCG cg09481056 16 2339260 ABCA3 TGCTGTGCCTGAGGTAGAGCATGAGAAATTCCAGTTGCTCCATTCAAACG cg07168939 8 143763412 PSCA C AG C AAAG G CTG C AG CTTG AACTG C GTG G ATG ACTCAC AG GACTACTACG cg08032483 16 1495363 NA CGCGTTCAGACCCAACACACTCGGACACCTTGGGGCCCTGTGAGGGCTAA eg 15357118 2 128927972 UGGT1 CGCTGTGCAGGAAGTGTACATCACCTTTGTTTGAAGTTGTCACATGCTCT cg05554192 11 17515836 USH1 C CGGATCAGCCTTGAGAGAGGCCACACTACACACACCAGATGGCATCCTTG eg 16277071 17 75537317 NA GTTAATTGAAAAAGGCTCTTTGTCTGTCTTCAGCTGTCAGCTCACATGCG eg 14016363 7 100486297 NA CGAGAGTCAGGAGCCCTGTGGAAGTGCTTTTATTAGCAGTAAGGCTGATC eg 14534148 6 170559869 NA CGGAGAGGCACTGTGCCTCGGTGTCTCCATTTCAACCTGGGTTTCATAAA cg00159243 12 109023799 SELPLG CGCCGCTCCGGGCCTTGCTTGCTAATCAGTGTGATGGCACCGTTAGAGCC cg02481950 16 21665002 NA CTACTGCCACTGCCAAACACTAGGTGCCATCAGCCCTGTTGTCAGCATCG eg 16291589 20 60231 8 LRRN4 CGGGGCAAGCACTGAGAAGGAGTCCAGATTCTCTGAGTGTCATTTGCCAT eg 16395183 1 210771699 HHAT TGATGCAAGCACTCCCTTGGATGCTCTGGCTGGTGACTCACTAGGTCACG cg02147592 19 4090388 MAP2 2 CGCTGCTGTGTGTGGTCTCAGAGGCTCTGCTTCCTTAGGTTACAAAACAA cg27470978 8 38704972 TACC1 CGGAAAGCTAAAAATTGCTTAGTGAGAAACAGGGATTGTTGCTAGTCCTG cg08571738 2 2605398 NA CGGTAGTCTTTCTCAATCTGTTTCTTGTCAGGGAATGCCTAATTGGGCTT cg24707889 21 46341304 ITGB2 CGCCCTCACATGCAGGTGGATGTCAATGCGTGCACGCACCCAGGTTTTCT eg 16246188 11 113947203 ZBTB16 CGGCTCTAAGCGGGGCAAGAGCTGCTTTTAATTTGAGACGTACACAGTTT cg04255937 12 122269675 SETD1 B CGGACGCAGGCGGCCGGAGCCTCTGGTATCTCAGCTTGTGTCAAGCTTGT cg07539709 17 80545454 FOXK2 TGTAGATTTCCTGGGTGTGGCCTGGAAGCGGCCTGAAACGGCGGGAGCCG
cg19991780 3 128533372 RAB7A CGCCTTCTAGAACAAAGGTAAAATCACTAGGACAGATCTGTTAGGGTTTT eg 12680205 10 80303482 NA CGTCCAAGGTGGCACAGTGGCAGTGAGAAAGAAGGGCAGGCACAGCTGCA eg 19674091 15 90643766 IDH2 TGTTTCTGGTAAGTTAGAGCTTGGGGCAGTGCGGACCAGTTCTGTGAACG cg24527461 17 79419366 BAHCC1 CGGCTCTTCCACAGACCCCTGGCCCTGCCACTTGGTCTCGCTGCTGGCCG cg25059899 18 60904328 BCL2 TCTTGCCTCTGGGAAATACATAAAATGCCTAGCAACAGTTCCTGGTGGCG cg25953130 10 63753550 ARID5B TTTTCTCAAAGCTTTATACTTTTCACTGTGCTCCTAGCAACCAACCAACG cg24394336 1 17962705 ARHGEF10L CGGTGACAATGATGTGTCTGTGGCCGCATTCCTCAAACCAGAGTCACGTA cg06291107 20 36157675 BLCAP CGGCTTTGAGAAGTTAAGTTTGAAATGTCGGGGCAACATGCAGGCAGAGA cg02112168 14 45579561 NA TGGCTGTTTCGGGGCTACAATGGCTTACAAAGCCTGAAAGTCAGAACACG cg23298114 8 118933411 EXT1 GCTGCATTTCCTGAGAGTAATGACTAATAAAGGCTTTGGGCAAAGCCTCG eg 18518074 11 64642316 EHD1 TGCTGACTTGTTAGTGAAAACTGTGCAAGCCTAGGTCACCATTCAGTTCG cg20981615 4 48136280 TXK TGCGGTGCTCTACTCACAAAAACACATCTTTCAACTGAAATCATAGTTCG cg08446900 17 38501519 RARA GGAGACGCTTATCAGGCGGCCGCTGGGCTAGGGGCCTTCTTCCGCTGCCG cg12067024 1 153387689 S100A7A TGTTTCTCCCTCTCCCAGTTTCCCATATGACGGCAATATTACTTTTAGCG cg08791347 10 13831250 FRMD4A GGTATCTTCCCTGGCTGATGGAGACAGCAGCTGCTACTTCCTGTCACTCG cg07372520 1 180086434 NA CGGAGGGTCCTTCCACTGCAGCACAAAGTGGGGCACTTGCAGTCACATCG cg25364972 2 217075573 NA GATCAAACACACCATCAAAGAATTACACACATATCCTTGTGGCCACATCG cg01589587 14 76002440 BATF AGTGTGTTTATTTGTTTTCCAGCCTGACTTCCAACTGACTGTAAGGTTCG cg01153613 6 139928856 NA ACCCCACCCCTTAGTTGGGCAAACAACCTAAACCCTCAGGAGAAACTTCG eg 14088282 1 222003220 NA TCTCTAGCACTTGTTACAGTGATTCATATCCTGGATACGCATGTGAATCG cg19868691 2 219744597 WNT10A TAAATAAACTATCTCAACGGCCACTTCTAGGGTTAAAAGTCTCTCTAACG cg07267600 12 2750053 CACNA1C CGTCCACCCCCTCAGGGGTGAGTCAGCTCAGCCTGCACGACTCCAGCCCT cg00138407 3 47386505 KLHL18 TGCCACTCTTCTTGGTGGGGGCTCCTAGCTGTGGCTGGGGGCTCCAGGCG cg10158598 7 47344579 TNS3 CGCTCACTCACGCTGTGGGGATGTCAGAGAGCCCCATCGGACCCAAATCC cg01126162 10 5734985 FA 208B CGCTGAGTGCGAAGGAGACCCGAGAAAATGGGCCTATTCTGTCTAGTAAC eg 12728588 1 36025489 NCDN CATTTCTGCCACCTTCTCAGGGGAAATGGCTCCTGTGTCTCGCCGCTCCG eg 10975863 14 68830704 RAD51 B GGTGGGAAGGCCGCTTCTGGCTGGACTGCAGGCTTGTTTGCCCACACGCG cg24129923 17 7814251 CHD3 CCAGCTGACCTGTGATGAAGGACCCTGCAGGCATCTGGCTGTAATTGGCG cg11551560 15 70528789 NA CGCTCAGTGGCCAAGCCTCCTGAGCCACAGACGTTGGGGTGAACCTGGCC eg 13300580 1 27440539 SLC9A1 AGTTCACAGAAAACCTGGGCACCATCCTGATCTTTGCCGTGGTGGGCACG cg06721473 3 50355416 HYAL2 GGAGTGGGCAGAGCCCCCAGGAAGCCCAGGAGGGCATCCATACCAGCTCG eg 10013455 10 134400191 INPP5A CGCCATCACCACGAGGCTCTGGCGCACGTATCCGTAACCAGCCATTTCAC cg05901196 10 61664954 CCDC6 CGCCTCATCACTGCTATAGGGGCCTTTCTAAACGCCTGCCACACCAGGAG
cg01526748 3 191930926 FGF12 CACAAGAAGGTTCCTCACAGAACGTGCTTACGCTTCTGGGCAAGAAAACG cg04523589 3 48265146 CAMP CGGTCCTCGGATGCTAACCTCTACCGCCTCCTGGACCTGGACCCCAGGCC cg09786420 1 154583389 ADAR CGGTGATGTTCTAGCTCTTGACTTGGGTAGTGGCTACATATGTTTGCTTT cg04074945 11 46071833 PHF21A C GG C AG G AG AAAG G CATAG G GACTAATAC C ACTGG GTCTTGTTG AAG AAA cg03963853 16 4732369 MGRN1 TGGCCACGGGTCATTCGTGGTTCCCCTGGAGCCTTGCGGTGTATAGAGCG cg01599714 8 142285223 NA CGAGAGAGGAGTTATCCCATAACCTCGTGGTCCCCCATTTTCTGTGACGA cg24067911 6 16729610 ATXN1 CGCTGGCTTTCTTTGA TGTTTTCTCATCTAAACTTGGGAGCTGCTGGC cg01616956 2 232393196 NMUR1 GTGGTGCACCCACTCCAGGCCAGGTCCATGGTGACGCGGGCCCATGTGCG cg21121843 4 3203982 HTT CTTGTTCTGAGTCACCCTAGATTTGGGACATTCATTCGCCACCAGTACCG eg 14849578 12 125282480 SCARB1 CGGATCAGTGATCCCACTGCCGGGCATCCCCCCAGAGGAAATGAAATCAG cg23044270 3 43659607 ANO10 CGTCTGTCTACATCTGTGTAACTCCCCAAGGCAGCTGCCCTAGGTACAAG cg17821158 19 4090395 MAP2K2 CGGGCGACGCTGCTGTGTGTGGTCTCAGAGGCTCTGCTTCCTTAGGTTAC cg03295554 11 128395450 ETS1 CGGCTGATCCAGAACCAGTTTATGACTCACAGATGTTGATTTACTGGTAT cg03819286 16 4673974 MGRN1 ATGGCGCTGCGGGCAAGATTGCAAGAATCGCTTTCAGAATGAAAACCTCG cg22985172 6 151258337 MTHFD1 L CGGTCTCCCATTTCATCAGGGCCTTCTTTGCCAAGTGATCTTGAAACACG cg03466342 12 52753899 KRT85 CGCCAGCTCTGCATTGGACAGTCCCCACCTGCACCCAGCAAGGGGGCATC cg05931265 11 122527736 UBASH3B CGGCTCAAGTCTCAGGGGCTGGAACTCTGCCTAAGAGGAAGCCAAGGCCC cg21066748 19 46005805 NA CGTTACAGGGCACCTGCTGTGTGCCAGGTCTTAGCCTCACAGTCATGAAC cg22690339 6 38249061 BTBD9 CGGTAATTTACAAACTGAATAGATAATCTAGAGTTGGTTGTAGCCACACA eg 15028232 1 234871410 NA GCTTTGCTTTTGGAAAGGCGAGCAAAACGCCTCTGACGTATTCCCATACG cg26572392 10 24496943 KIAA1217 AAGTATGGTCACCAGACTAACCATTGTCC I ΤΊ T TTACCCCTCCTGTGACG eg 1 1888982 8 116231420 NA TGCAGTGTTCAGAATAGGCTGCATTTGTTGCTACCACCCTTGTTTCCTCG cg08539067 3 49395985 GPX1 AAGCCGGAAGGAGCAGCAGGCATGTCTGGTCTGGCCCAGGCAACCAGGCG eg 14081226 10 5563419 NA TTTTAAACAGCGTTGCTTAGTGCAAAGTGACTCACAGAGGACCAGGACCG cg22881435 8 37732086 RAB11 FIP1 CGGTTCAGACTTGGACTCTGGCTCAGCTTCTGGTTCTGTGATCTGCACAT cg21281007 12 5939611 AN02 CCATCCTCTTTCCTAACACAGTACATCCATTTACTTACAATGCGGCTGCG cg04782146 16 85112370 KIAA0513 CGGTGCTATTCCCACCCGGCAATAAGGACTTCTTCTGAGGGATGGTTCCG cg07058694 12 111909145 ATXN2 CTTTATGTCTTATCTTAATCCACACAGGTGGTCTGCATGTAAGGGCCTCG cg08181850 16 17200244 XYLT1 TTTCTCAGAAAGCCCCTATGATCTCTTTTACAGACTCTATGGTGGCCACG
Table 4 - Genes CNOT6L 4 APK8IP3 16
5 CTDP1 18 IR1973 4
Gene Symbol or D2HGDH 2 IR21 17
Probe Target
17 Coordinate Chromosome EIF2AK4 15 PRIP
EPHB3 3 MY01 E 15
ABCB9 12
ANKRD11 FIS1 7 MY05C 15
16
F BP5 6 11295667 16
ARHGEF3 3
FOXK2 17 38370874 7
ARID5B 10
FUK 16 11969958 2
ARSB 5
GPRIN3 4 33447032 9
ASB4 7
GSD C 8 114128517 11
ATP2B4 1
HEATR2 7 70797389 15
ATP9A 20
HHAT 1 45473712 10
BAG3 10
HK2 2 9045558 8
BAHCC1 17
HS2ST1 1 61772344 11
BCL10 1
ICAM5 19 10531091 11
BCL3 19
IKZF4 12 57427443 20
BL 15
IL18RAP 2 39464601 15
C4orf29 4
INPP4B 4 30327579 3
CACNA1C 12
ITGA9 3 66050928 12
CD36 7
JAK3 19 29607030 16
CDC42BPB 14
CFI 4 KIAA1549 7 235580461 2
KIF13A 6 50327986 22
CHI3L2 1
LIPC 15 123965051 5
CIA01 2
LOXL2 8 26351915 6
CLEC16A 16
LRRC47 1 76313872 17
CLMP 11
LSP1 11 101901234 3
CLU 8
30737556 7 NMUR1 2 TBPL1 6
105717036 3 NRXN2 11 TNF 6
57094763 3 OIT3 10 TNFAIP8 5
39629290 7 PLCH1 3 TNFSF10 3
77973731 5 PLSCR1 3 TNIP2 4
566172 1 PRF1 10 TNRC18 7
65889855 1 PTDSS2 11 TNS1 2
63075650 4 PTGFRN 1 TOLLIP 11
85470674 16 RAB22A 20 TREML5P 6
47100912 2 RALBP1 18 TRPS1 8
65363022 15 RBKS 2 TSPAN4 11
132654924 12 RFTN1 3 VAV3 1
12890029 19 RORA 15 VMP1 17
200839460 1 RPS6KA2 6 VPS54 2
10530636 11 SAMD14 17 WASF2 1
41233701 15 SBN02 19 YWHAE 17
138608519 5 SEPT_9 17 ZBTB12 6
144443217 8 SLC10A6 4 ZBTB16 11
228185396 2 SLC15A4 12 ZC3H4 19
110386152 13 SLC25A13 7 ZEB2 2
151945663 1 SLC27A1 19 ZFR 5
568536 1 SOCS3 17 ZFYVE28 4
NDUFS4 5 SPTB 14 ZMIZ1 10
NFIA 1 SYNJ2 6 ZSCAN25 7
Table 4 continued - Genes BCL3 19 45252955 NTHL1 16 2094700
POLDIP3 22 43006541 BRE 2 28304502
UBIAD1 1 11336263 PHOSPH01 17 47301614
BAHCC1 17 79428404 NOP58 2 203143288
GPRIN3 4 90227074 LINC-PINT 7 130646051
CCDC114 19 48823178 NA 13 110386176
LDB1 10 103875969 ALS2CL 3 46718941
NA 17 55822272 SPARC 5 151055650
SCiMP 17 5138696 RGS12 4 3432483 NEDD9 6 11324433 NA 2 64447091 NA 8 144443217 NA 13 92002454
ABCG1 21 43642366 VTN 17 26697281 GLIS1 1 54100163 NA 1 170532470 WRAP73 1 3563998 NA 2 108427720 NA 7 36022575 TSC2 16 2121682
CDC42BPB 14 103415873 TRAF1 9 123688715
NA 12 25541364 FCH01 19 17861017
NA 8 9045558 CAP2 6 17472892
CPNE2 16 57180107 CYP3A43 7 99437259
NA 10 102325486 TRIM71 3 32933637
NA 8 37457329 AIM2 1 159046773
NPFFR1 10 72027357 SUB1 5 32584912 NA 8 144441955 KIAA1549L 11 33562503
NUMA1 11 71752971 NA 6 49636226
KIF21 B 1 200978598 TOMM20L 14 58863097
NA 8 23830907 NA 2 182656091
NA 1 116369577 CPNE6 14 24540235
NA 7 33679233 CARS2 13 111318640
AIM2 1 159047163 NA 1 165907859
RNF216 7 5736195 HOXA3 7 27160520
CFI 4 110723299 TOLLiP 11 1297087
WRAP73 1 3563954 ZEB2 2 145172035 PALM 19 707791 NA 1 65889855
Figure imgf000070_0001
AUTS2 7 69358880 PHACTR1 6 13120250 LARS2 3 45590039
SLC38A10 17 79244542 LINC-PINT 7 130636860 TXK 4 48136234
SLC10A6 4 87752504 NDRG1 8 134250937 NA 17 60695089
SRF 6 43142014 NA 7 137557164 NDA 1 158809049
CSGALNACT1 8 19436451 INPP4B 4 143488622 NA 20 48959844
SLC29A3 10 73083123 NA 2 47100912 FRMD4A 10 14058944 A 2 235578952 VMP1 17 57916643 MPRIP 17 17030253
LRRK1 15 101458127 SETD8 12 123892724 NA 3 53187213
GSDMC 8 130799007 PHACTR1 6 12881417 ALS2CL 3 46719086
MY05C 15 52554171 BAIAP2 17 79047695 LIMD2 17 61774174
MIR21 17 57918262 SOCS3 17 76354621 YPEL3 16 30106897
NA 5 146864669 AP3S2 15 90391877 FBXW11 5 171430188
ADCK3 1 227130394 EMC1 1 19547285 MY018A 17 27459835
ATP6V1 B2 8 20077936 ELM01 7 37429500 RBM19 12 114297307
NA 8 131054408 NA 1 186504948 NA 1 8960134
MY01 E 15 59588622 APOBR 16 28506477 TOLLIP 11 1297066
RAB20 13 111210121 MXD3 5 176736591 NA 11 43942418
NFIA 1 61668835 NA 1 193082764 NA 16 85470674
LRRC47 1 3705262 UBE2D2 5 138957422 BLM 15 91357521
ZBTB16 11 1 3947148 FOXK2 17 80536214 NA 17 48608677
NA 12 116904641 SOCS3 17 76353952 ZNRF1 16 75045643
NA 7 36022841 AGL 1 100326491 NA 8 29513210
RAP1 GAP2 17 2843257 SLC37A1 21 43919801 A2ML1 12 8986988
NA 7 142423070 ERI1 8 8863572 PLEK2 14 67853825
AK5 1 77912654 DNMT3A 2 25475916 NA 3 14347415
SND1 7 127540997 LINC-PINT 7 130646078 NA 1 151945663
TSGA13 7 130372167 SCARB1 12 125282342 CARS 11 3071167
ZNRF2 7 30387954 QTRT1 19 10823913 CARS 11 3071269
NA 13 92002338 HEATR2 7 797592 LYN 8 56903623
NA 16 28959310 UBR2 6 42658437 PTX4 16 1538347
SYNJ2 6 158490013 NA 16 85256423 NA 2 240431144
WRAP73 1 3564031 VMP1 17 57915740 IL12B 5 158757456
STAT4 2 191916269 CDC42BPB 14 103415882 NA 2 88313351
ADHFE1 8 67343633 SETD1 B 12 122269675 INPP5A 10 134400191
NA 16 50285254 FOXK2 17 80545454 CCDC6 10 61664954
NA 10 20019641 RAB7A 3 128533372 FGF12 3 191930926
MED24 17 38176064 NA 10 80303482 CAMP 3 48265146
NA 10 125817817 IDH2 15 90643766 ADAR 1 154583389
FKBP5 6 35657180 BAHCC1 17 79419366 PHF21A 11 46071833
CALHM1 10 105218771 BCL2 18 60904328 MGRN1 16 4732369
NA 14 105491309 ARID5B 10 63753550 NA 8 142285223
PIK3CD 1 9730401 ARHGEF10L 1 17962705 ATXN1 6 16729610
NA 1 234905772 BLCAP 20 36157675 NMUR1 2 232393196
MVB12B 9 129088215 NA 14 45579561 HTT 4 3203982
CCDC115 2 131098007 EXT1 8 1 18933411 SCARB1 12 125282480
NAV2 11 20071677 EHD1 11 64642316 ANO10 3 43659607
NA 1 41786797 TXK 4 48136280 MAP2K2 19 4090395
ZNF687 1 151262619 RARA 17 38501519 ETS1 11 128395450
NHEJ1 2 219940977 S100A7A 1 153387689 GRN1 16 4673974
ABCA3 16 2339260 FRMD4A 10 13831250 MTHFD1 L 6 151258337
PSCA 8 143763412 NA 1 180086434 KRT85 12 52753899
NA 16 1495363 NA 2 217075573 UBASH3B 11 122527736
UGGT1 2 128927972 BATF 14 76002440 NA 19 46005805
USH1 C 11 17515836 NA 6 139928856 BTBD9 6 38249061
NA 17 75537317 NA 1 222003220 NA 1 234871410
NA 7 100486297 WNT10A 2 219744597 KIAA1217 10 24496943
NA 6 170559869 CACNA1 C 12 2750053 NA 8 116231420
SELPLG 12 109023799 KLHL18 3 47386505 GPX1 3 49395985
NA 16 21665002 TNS3 7 47344579 NA 10 5563419
LRRN4 20 6023178 FAM208B 10 5734985 RAB11 FIP1 8 37732086
HHAT 1 210771699 NCDN 1 36025489 AN02 12 5939611
MAP2K2 19 4090388 RAD51 B 14 68830704 KIAA0513 16 85112370
TACC1 8 38704972 CHD3 17 7814251 ATXN2 12 111909145
NA 2 2605398 NA 15 70528789 XYLT1 16 17200244
ITGB2 21 46341304 SLC9A1 1 27440539 NFATC3 16 68117991
ZBTB16 11 113947203 HYAL2 3 50355416 JADE2 5 133866097
Figure imgf000073_0001
5
Table 5 - Differentially Methylated Regions
DMR no. Gene Symbol Chromosome Coordinate DMR start DMR stop DMR size (bases)
1 ATAD3C 1 1396125 1396079 1396215 136
2 1 98513986 98512985 98515389 2404
3 2 6910518 6910391 6910758 367
4 2 9235898 9235771 9236099 328
5 2 11969958 1 1969951 11970205 254
6 CXCR1 2 219030752 219030741 219030919 178
7 3 164924675 164924548 164924903 355
8 EPHB3 3 184297380 184297196 184297706 510
9 RNU5E 5 80529067 80529017 80529225 208
10 TMED7-TICAM2 5 114938389 114938314 114938534 220
11 TRIM10 6 30124240 30124136 30124848 712
12 6 32765314 32765187 32765479 292
13 HLA-DPA1 6 33039396 33039292 33039585 293
14 6 68599196 68599153 68599407 254
15 C7orf50 7 1095595 1095591 1095741 150
16 MAD1L1 7 1986245 1986141 1986438 297
17 IGF2BP3 7 23387365 23387261 23387500 239
18 7 73241878 73240877 73243029 2152
19 ATXN7L1 7 105319437 105319382 105319662 280
20 ATP6V0E2 7 149570036 14957001 1 149570188 177
21 KIAA0146 8 48572447 48572392 48572600 208
22 C8orf56 8 104153753 104153752 104 53902 150
23 PFKP 10 3138534 3138401 3138609 208
24 ClOor lO 10 45473712 45473025 45474399 1374
26 C10orf26 10 104535990 104535831 104536079 248
27 FA 160B1 10 116636907 116636879 116637087 208
28 PWWP2B 10 134211908 134211782 1342 1983 201
29 11 2308362 2308360 2308614 254
30 CCND2 12 4384890 4384804 4384965 161
31 GRASP 12 52404134 52403950 52404345 395
32 14 106329652 106328606 106330653 2047
33 MAGEL2 15 23894198 23894197 23894299 102
34 CLCN7 16 1521676 1521433 1521860 427
35 PMM2 16 8942906 8942189 8943839 1650
36 CETP 16 56995856 56995751 56995945 194
37 CBFA2T3 16 89002930 89002243 89004328 2085
38 C17orf91 17 1617291 1617288 1617522 234
39 ZMYND15 17 4648682 4648476 4648684 208
TNFSF12-
40 TNFSF13 17 7461421 7460951 7462462 1511
41 MFAP4 17 19290755 19290657 19290759 102
42 RAB34 17 27045048 27045038 27045188 150
43 TMEM49 17 57915665 57915561 579 5877 316
44 TIMP2 17 76886742 76886731 76886939 208
45 RPTOR 17 78800806 78800692 78800881 189
46 FOX 2 17 80545272 80545197 80545397 200 B3GNTL1 17 80924969 80924785 80925287 502
KANK2 19 11289376 11289274 1 1289451 177
BTBD3 20 11898478 1898389 11898646 257
RBM9 22 36236649 36236545 36236836 291
CYB5R3 22 43042879 43042192 43043597 1405
LRRC47 1 3704460 3704438 3704588 150
TNFRSF25 1 6526073 6526003 65261 19 116
PI 3CD 1 9788715 9788623 9788861 238
RUNX3 1 25291989 25291938 25292123 185
MAP3K6 1 27683345 27683299 27683432 133
LCK 1 32717002 32716274 32717689 1415
PTGFRN 1 117529478 1 17528902 117530726 1824
1 153762201 153762074 153762371 297
LAX1 1 203734478 203734455 203734663 208
2 58478696 58478606 58478795 189
2 105735756 105735562 105735816 254
NMUR1 2 232389985 232389881 232390175 294
2 241644166 241643165 241645241 2076
3 25728479 25728244 25728606 362
ZBTB47 3 42705828 42705389 42706823 1434
3 66633255 66633247 66633501 254
DHFRL1 3 93780328 93780253 93780403 150
LOC100130872 4 1198278 1198174 1198457 283
ZFYVE28 4 2321957 2321948 2322156 208
5 969939 969772 970066 294
CLPT 1 L 5 1342170 1341986 1342384 398
MEF2C 5 88018481 88018420 88018628 208
6 209809 209585 209936 351
6 1595099 1594098 1596742 2644
ATXN1 6 16306683 16305379 16307269 1890
HCG18 6 30292258 30292183 30292367 184
PPP1 R10 6 30569156 30567908 30569873 1965
TUBB 6 30691683 30691499 30692079 580
LTA 6 31540461 31540310 31540550 240
TNF 6 31544931 31544747 31545742 995
C6orf27 6 31743769 31743665 31743924 259
SLC44A4 6 31833199 31832778 31833383 605
ZBTB12 6 31867698 31867661 31868282 621
SKIV2L 6 31937228 31937171 31937539 368
TNXB 6 32038097 32037941 32038149 208
HSD17B8 6 33173482 33173407 33173576 169
6 85824216 85824041 85824343 302
PLEKHG1 6 151125848 151125744 151125989 245
RPS6 A2 6 166908758 166908573 166908781 208
6 169284285 169284195 169284434 239
7 175423 1 4422 176968 2546 AD1 L1 7 2151681 2151497 2151972 475 94 7 2764599 2764359 2764726 367
95 WIPI2 7 5272064 5271558 5273414 1856
96 CDK6 7 92238207 92237179 92238613 1434
97 7 93474059 93473942 93474285 343
98 CSMD3 8 1 14389388 114389284 114389583 299
99 LARP4B 10 858817 858548 859107 559
100 10 6183455 6182454 6184576 2122
101 ARID5B 10 63809098 63808994 63809274 280
102 PWWP2B 10 134222445 134222176 134222776 600
103 PWWP2B 10 134230041 134229929 134231602 1673
104 TNNT3 11 1945564 1945532 1945682 150
105 11 2021658 2021323 2023387 2064
106 EHD1 11 64635728 64635670 64635878 208
107 OVOL1 11 65561705 65561601 65561853 252
108 COR01B 11 67206263 67206079 67206618 539
109 TNFRSF1A 12 6442954 6442850 6443149 299
1 10 PTPN6 12 7066563 7066459 7066768 309
111 12 19557320 19557193 19557470 277
112 LRP1 12 57588243 57588040 57588347 307
113 TMC03 13 1 14172890 114172835 114173043 208
114 13 114908876 114907875 114910334 2459
115 C1 orf64 14 98444417 98444313 98444521 208
116 14 106306830 106306802 106307056 254
117 AXIN1 16 367990 367886 368170 284
1 18 16 8985593 8985375 8987025 1650
119 MAZ 16 29820429 29820385 29820535 150
120 CORQ1A 16 30198370 30198186 30198693 507
120 COR01A 16 30198505 30198186 30198693 507
121 16 85479053 85478805 85479059 254
122 FA 38A 16 88832476 88832372 88832676 304
123 ANKFY1 17 4079262 4079158 4079445 287
124 UNCI 3D 17 73824396 73823052 73824486 1434
125 Sept9 17 75451779 75451595 75451993 398
126 Sept9 17 75473895 75473865 75474073 208
127 TMC8 17 76129402 76129400 76129550 150
128 RPTOR 17 78925278 78924988 78925462 474
129 TBCD 17 80823240 80823056 80823424 368
130 ATG4D 19 10662621 10662478 10662725 247
131 RINL 19 39369196 39368618 39370070 1452
132 RPL13AP5 19 49993217 49993050 49993292 242
133 VPS 16 20 2844678 2844574 2844817 243
134 GNASAS 20 57414596 57414454 57414653 199
135 GNASAS 20 57425979 57425977 57426771 794
136 GNAS 20 57463615 57463238 57463707 469
137 ITGB2 21 46349059 46347756 46349378 1622 Table 6 - Gene probes
Probe ID Gene Symbol Chromosome Coordinates Probed Sequence Probe Start Probe End Probe eg 12054453 VMP1 17 57915717 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 57915717 57915766 57915 eg 12992827 NA 3 101901234 CCTCTGCCATGCATCAG I I I I CTTGGTTGGCCACTGATTAATAATCATCG 10 901234 101901283 10190 cg01749539 Sept 9 17 75473969 GAACACGGTTGTGCAAGGATCTGTCTGGGTCCCTGCTTTCCATTCTTTCG 75473969 75474018 75473 cg26599989 TOLLIP 11 1297087 CGGATGTAAACCCACTGATAACGGACAGAAAGAGAATGCCCACAAGTGGG 1297039 1297088 12970 cg16936953 V P1 17 57915665 GAGCCTCCAGGTACTCCTGAAATGGCTTCTGCAGAGATGGACCTATGCCG 57915665 57915714 57915 cg17501210 RPS6KA2 6 166970252 CGCCCGATTCAGACAGCTGGACTCAGAGGGATTCTGCTCCACAGAGAAAC 166970204 166970253 16697 eg 18860310 SLC10A6 4 87752504 CGGCCAAAGCTGTTGTTTGTACACATGAGCAAGGGCTGCTGACTACATGA 87752456 87752505 87752 cg09645475 FAM1 10A 20 815316 CGGAGGTTCTAAGAAGTTAAGCCCC I I I I CGAAGTCACAGTCAGACAATG 815316 815365 81531 cg06219337 YWHAE 17 1278466 TCTCCAAACATTCTACGACCAGTGCTTTGTACCAGTTTGTACAAATAACG 1278418 1278467 12784 cg08423142 MY01 E 15 59588622 CAACATTACAAACCAAAACAGATCTG I I I I CCTACTGACCTCAATTTGCG 59588574 59588623 59588 cg04389058 ARHGEF3 3 57041402 CGTGCCTTCTGCTGAGGCTGAGCTGGAATTGA I I I I GTTTATTCCAAAAT 57041402 57041451 57041 cg13315706 ITGB2 21 46341054 TGTTCTTCTTCCTAATGGGTGCCCTTTCTGCCTGGCGCTCCTGGAGGCCG 46341054 46341103 46341 cg09349128 NA 22 50327986 CGCTGAAGAAGTCTCAGGGTGCCTCACCTCACCCAGCCCCCAGGTCAGCT 50327938 50327987 50327 cg1047271 1 HEATR2 7 797592 CGAGAGGTGGGGCTGTTGACAGTCA I I I I I IAAAC I I I I I AAGAAACC 797592 797641 79759 cg09304397 NA 2 235580461 CGGGCACCAGGCCTCTGCATCTTTCTCATGTGGGTTTGGTTATCAGTGCT 235580413 235580462 23558
5
10
Table 7 - "D R" probes
Probe start Probe End Probe T
Probe ID Gene Symbol Chromosome Coordinate Probed Sequence Coordinate Coordinate Coordin eg 16936953 TME 49 17 57915665 GAGCCTCCAGGTACTCCTGAAATGGCTTCTGCAGAGATGGACCTATGCCG 57915665 57915714 579156 cg12054453 TMEM49 17 57915717 CGGCAGAAGCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACT 57915717 57915766 579157 cg01409343 TMEM49 17 57915740 CGC I I I I GTGGTGAAGCTTCTGCCGTTGAGCCTCCAGGTACTCCTGAAAT 57915692 57915741 579157 cg18942579 TMEM49 17 57915773 GCTTCACCACAAAAGCGAAATGGGCACACCACAGGTAAGACTTTAATCCG 57915725 57915774 579 57 cg02560388 2 11969958 CGTGAGGCAGAATCCCCTGAAGGACTTCTTAAAACAGATAACCCGGCCCC 11969910 11969959 119699 cg10018519 2 11970078 TGTATTGCTCAGCTCACATCTGACTTTAATTTGGCCTGGCCAGAGCAACG 11970030 11970079 119700 cg01876978 2 11970145 TCAAGCGAGCGCCTGTACTGGGTTTCCAAGCTCC I I I I CAGGAGTGGCCG 11970145 11970194 119701 cg23277830 LRRC47 1 3704460 GTGGGCCCGATGCACCTGTCCTGGGTCAGACGAAGGCTGAGAACCCATCG 3704460 3704509 370446 cg17848003 LRRC47 1 3704513 AACGCTTTCCCGTGGGCCACGGGGCTTCCTGGGCATCTTTGTGGCACACG 3704513 3704562 370451 cg22529645 LRRC47 1 3704559 GTCACCTGCACG I I I I GCCAAAGTCTGATCAGTCACGGTCATAGAAAACG 3704559 3704608 370455 cg10917426 ZBTB12 6 31867698 CGCGGCTAGCGGATGAGGACGTTAATCTCGGCCACACTGGCCTCCAGCAC 31867698 31867747 318676 cg08975528 ZBTB12 6 31867700 ACGTGCTGGAGGCCAGTGTGGCCGAGATTAACGTCCTCATCCGCTAGCCG 31867700 31867749 318677 cg16463880 ZBTB12 6 31867707 GCCGAGAACGTGCTGGAGGCCAGTGTGGCCGAGATTAACGTCCTCATCCG 31867707 31867756 318677 cg00459243 ZBTB12 6 31867726 CGGCCACACTGGCCTCCAGCACGTTCTCGGCCGTGGTCTTGCCGTGTTGC 31867726 31867775 318677 cg14734916 ZBTB12 6 31867747 CATTAGGCGGCACCTCAAGGAGCAACACGGCAAGACCACGGCCGAGAACG 31867747 31867796 318677 cg14562426 ZBTB12 6 31867757 ACAAGCCTGCCATTAGGCGGCACCTCAAGGAGCAACACGGCAAGACCACG 31867757 31867806 318677 cg06636203 ZBTB12 6 31867788 CGGCACCTCAAGGAGCAACACGGCAAGACCACGGCCGAGAACGTGCTGGA 31867740 31867789 318677 eg 11645762 ZBTB12 6 31867819 CGTGCGCTTCGCCCACAAGCCTGCCATTAGGCGGCACCTCAAGGAGCAAC 31867771 31867820 318678 cg09788778 ZBTB12 6 31867822 GCTCCTTGAGGTGCCGCCTAATGGCAGGCTTGTGGGCGAAGCGCACGTCG 3 867774 31867823 3 8678 cg25470384 ZBTB12 6 31867836 CGCTGCTCCTACTGCGACGTGCGCTTCGCCCACAAGCCTGCCATTAGGCG 31867788 31867837 3 8678 cg00805874 ZBTB12 6 31867847 CGCGGCCCTACCGCTGCTCCTACTGCGACGTGCGCTTCGCCCACAAGCCT 31867799 31867848 318678 cg04603811 ZBTB12 6 31867873 CGACCACCTCAACCTGCACTCGGGAGCGCGGCCCTACCGCTGCTCCTACT 31867825 31867874 3 8678 cg25861453 ZBTB12 6 31867906 CGGCAAGTGCTTCACACAGAAGTCCACCCTTCACGACCACCTCAACCTGC 31867858 31867907 318679 cg25013586 ZBTB12 6 31867915 CGGCATCTGCGGCAAGTGCTTCACACAGAAGTCCACCCTTCACGACCACC 31867867 31867916 318679 cg13127825 ZBTB12 6 31867919 CGTGCGGCATCTGCGGCAAGTGCTTCACACAGAAGTCCACCCTTCACGAC 31867871 31867920 318679 cg05680710 ZBTB12 6 31868144 CAGCAGCGGTGGCCATGGCTGCCCGGGGGGCGGGGGGCAGCCTGGGGGCG 31868144 31868193 318681
cg07910050 ZBTB12 6 31868162 CGCCCCCCGGGCAGCCATGGCCACCGCTGCTGCTTCCACCAGGCCCGAGG 31868162 3186821 1 318681 cg17766150 ZBTB12 6 31868169 GGGGCCACCTCGGGCCTGGTGGAAGCAGCAGCGGTGGCCATGGCTGCCCG 31868169 31868218 318681 cg06169557 ZBTB12 6 31868207 AGGGCCTGCTGTTGATTCCCGGAGGCCGGGCCAGCGTGGGGGCCACCTCG 31868207 31868256 318682 cg22488278 ZFYVE28 4 2321957 CGGTCCAAGTTCAGAGGTCCGTCCGCATAGACCACGAGGCCACTGCATTT 2321957 2322006 232195 cg01 161042 ZFYVE28 4 2322052 TTTGAGACACAAAAGCCAGGATGTCAGGCCCTGTGGCCCCCTGGGTGCCG 2322004 2322053 232205 cg12811871 ZFYVE28 4 2322078 GGCCGTGCTGGGACAGTGGCGCTGCAGAGTCCCAGCGGGCGTGG I I I I CG 2322078 2322127 232207 cg05626616 Sept9 17 75473895 TACCCACGTCGACTCTCAGAAAGGTTCTGATTTGGACAGTAACTGACACG 75473895 75473944 754738 cg01749539 Sept9 17 75473969 GAACACGGTTGTGCAAGGATCTGTCTGGGTCCCTGCTTTCCATTCTTTCG 75473969 75474018 754739 cg06791979 Sept9 17 75474070 CGGCAGCTGCCCCGTTGAGATCCCCATCGCACCTCCAGGCCTCGCCCTGC 75474022 75474071 754740
Cg04444771 C10orf10 10 45473712 CTTCCTGTTCAGGTTAAAAACCCCAAAGGCTTAAGA I I I I ATCATTATCG 45473664 45473713 454737 cg17186163 C10orf10 10 45474317 CGAGGGCCAAAAATAGCCTGCTCGGAAGGCTGTCTTAGGAAAACAGCTTG 45474317 45474366 454743 cg02076355 C10orf10 10 45474372 CAAAAATAGCCTGCTCGGAAGGCTGTCTTAGGAAAACAGCTTGGTGGGCG 45474324 45474373 454743 eg 14027204 PTGFRN 1 1.18E+08 CGGGCTCCTGTCCTGTCTCATCGGGTACTGCAGCTCCCACTGGTGTTGTA 1.18E+08 1.18E+08 1.18E+ eg 15937641 PTGFRN 1 1.18E+08 TTG I I I I AAAATATCACTGTCCTCTTCTTGCCCCAATTGCTCCTAGAACG 1.18E+08 1.18E+08 1.18E+ eg 17042439 PTGFRN 1 1.18E+08 CTC I I I I CTGCATGTCAAGTTCTGAGCGCGGACATGTTTACCAGCACACG 1.18E+08 1.18E+08 1. 8E+ cg24687529 PTGFRN 1 1.18E+08 CGGAGTTCTTTATCTTCAGTGAGAATGTGCCTGCCCGCCTGAGAGCCAGC 1.18E+08 1.18E+08 1.18E+ cg11708721 PTGFRN 1 1.18E+08 CGCGGAAGCTGGCTCTCAGGCGGGCAGGCACATTCTCACTGAAGATAAAG 1.18E+08 1. 8E+08 1.18E+ cg01360627 TNF 6 31544931 CGCCGGGCCAATGCCCTCCTGGCCAATGGCGTGGAGCTGAGAGATAACCA 31544931 31544980 315449 cg23384708 TNF 6 31544934 AGCTGGTTATCTCTCAGCTCCACGCCATTGGCCAGGAGGGCATTGGCCCG 31544934 31544983 315449 cg20477259 TNF 6 31544960 GGTACAGGCCCTCTGATGGCACCACCAGCTGGTTATCTCTCAGCTCCACG 31544960 31545009 315449 cg0963 172 TNF 6 31545252 CGGCCCGACTATCTCGACTTTGCCGAGTCTGGGCAGGTCTACTTTGGGAT 31545252 31545301 315452 cg05952498 TNF 6 31545257 CAATGATCCCAAAGTAGACCTGCCCAGACTCGGCAAAGTCGAGATAGTCG 31545257 3 545306 315452 eg 15989608 TNF 6 3 545321 CGTCCTCCTCACAGGGCAATGATCCCAAAGTAGACCTGCCCAGACTCGGC 31545273 31545322 315453 cg26736341 TNF 6 3 545342 TTTGGGATCATTGCCCTGTGAGGAGGACGAACATCCAACCTTCCCAAACG 31545294 31545343 315453 cg04472685 TNF 6 31545473 CGAAGTGGTGGTCTTGTTGCTTAAAGTTCTAAGCTTGGGTTCCGACCCTA 31545425 31545474 315454 eg 18990407 EPHB3 3 1.84E+08 GTTTGCCAAGGAGATCGACGTGTCCTGCGTCAAGATCGAGGAGGTGATCG 1.84E+08 1.84E+08 1.84E+0 eg 15334250 EPHB3 3 1.84E+08 CGGCACACTTCCCCAAATTCCCCTGCAGGGCACAGGGCACAAGCTATGAC 1.84E+08 1.84E+08 1.84E+0 cg272 4730 EPHB3 3 1.84E+08 CGGCGGCCAGGCTGTTTCAGTCGACCACGGCACACTTCCCCAAATTCCCC 1.84E+08 1.84E+08 1.84E+0
cg07801894 UNX3 1 25291989 ACTCCTTCTTAATTAAGGCCAGCATTGGCCTGGGCGGGCTAGGAGCCCCG 25291941 25291990 252919 cg14425908 RUNX3 1 25291991 CGCGGGGCTCCTAGCCCGCCCAGGCCAATGCTGGCCTTAATTAAGAAGGA 25291943 25291992 252919 eg 12459932 RUNX3 1 252920 8 CCAGGGGCGAGGTGGCATGGCAGGGAGCTCTCCGGCCGCTGGTGGATCCG 25292018 25292067 252920 cg2391B689 RUNX3 1 25292034 GGGAGTCAGCCGCAGCCCAGGGGCGAGGTGGCATGGCAGGGAGCTCTCCG 25292034 25292083 252920 eg 11585280 RUNX3 1 25292059 CGCCCCTGGGCTGCGGCTGACTCCCTGCCATTGTCCACCACCATTGTTGC 25292059 25292108 252920 cg06037693 RUNX3 1 25292072 CGGCTGACTCCCTGCCATTGTCCACCACCATTGTTGCCAGGGAGAGGTGT 25292072 25292121 252920 cg09115713 FAM38A 16 88832476 CGCTGCCGACTGAGAACACAGCCGTGCGCTGACTCGCATGTGATGTCTCT 88832428 88832477 888324 cg03831847 FA 38A 16 88832485 CGGCACCCACGCTGCCGACTGAGAACACAGCCGTGCGCTGACTCGCATGT 88832437 88832486 888324 cg08798862 FAM38A 16 88832532 CGCTGCTGGGCCACGCTGCCCAGTTTAGTCAAACGCCAATCGCAATTTGG 88832532 88832581 888325 cg05635114 FAM38A 16 88832572 CG CAATTTG GTGTGCAGGTGTTTTTAAAATGAGATTGATTTAAATCAGCA 88832572 88832621 888325 cg06247108 FAM38A 16 88832667 GCAGACGGCATGAGGGCCTCATCCAATCAGCTGAAGGCAGGGGAGTGTCG 88832619 88832668 888326 cg09693588 3 66633255 GGCTCGTCTCCAGCTTTGTGTGGGCAGATGCTGTGCCAGCGCCCAGGCCG 66633207 66633256 666332 cg05068866 3 66633374 CGCTCCACTAGTGTGAGAAAGCCACAACACAGCACGGAGGGAACCATTCA 66633374 66633423 666333 cg14630001 3 66633408 CAGCTCAAATAATACGCTCCACTAGTGTGAGAAAGCCACAACACAGCACG 66633360 66633409 666334 cg02292450 CSMD3 8 1.14E+08 CGCTCGAACTTCCGGCCATTGGAGCGGATGTAGGGTTTGCAGGGTTTCCG 1.1 E+08 1.14E+08 1.14E+ cg23676042 CSMD3 8 1.14E+08 GGCTCCACGGTTTG I I I I I GTATCCTCGGCTGGCCCTTCGGTCTCTCGCG 1.14E+08 1.14E+08 1.14E+ cg20323509 CS D3 8 1.14E+08 CGGTTTGTTTTTGTATCCTCGGCTGGCCCTTCGGTCTCTCGCGCGCTCGA 1.14E+08 1.14E+08 1.14E+ cg00619505 TMC03 13 1. 4E+08 GAGATGAGTCTGTGGGATGCAAAGGAGCCACGGCCACCCCACATCTTCCG 1.14E+08 1.14E+08 1.14E+ cg08289375 TMC03 13 1.14E+08 GGAAGATGTGGGGTGGCCGTGGCTCCTTTGCATCCCACAGACTCATCTCG 1.14E+08 1.14E+08 1.14E+ cg15208672 TMC03 13 1.14E+08 CGCTGATCTGTGAAGCCTCCGCATGGACAATATTAGTTGGGAGGCAATTT 1.14E+08 1.14E+08 1.1 E+ cg15264991 PTPN6 12 7066563 CGGGGACAATGCCTGCCCTGGCAACGTTTGTTGAATGACAAACGGATGTA 7066563 7066612 706656 cg24437859 PTPN6 12 7066614 GGACAATGCCTGCCCTGGCAACGTTTGTTGAATGACAAACGGATGTACCG 7066566 7066615 706661 cg11700959 PTPN6 12 7066664 GTGAAGTGGCTGGCCAGGCCTCACCACCTGTTGGTGGTTGATCTGAGACG 7066616 7066665 706666 cg00335908 CBFA2T3 16 89002930 CGGGTCTGGGCAGCTCAGAGCCTCCTCGGCTGCCCCTGAGGCTGAGGTCG 89002930 89002979 890029 cg06460328 CBFA2T3 16 89003037 CGGCCGCCTGCCTCTGCGTTTCCCGCCCGCTTAGCCACCCAGGGCCGGTT 89002989 89003038 890030 cg02187822 CBFA2T3 16 89003166 CGTGGGACCTCACTGAGAGCCAGATGTCACTGGGAAGGAAAACAAAGATG 89003118 89003167 890031 cg00762678 CBFA2T3 16 89003235 CGCCTGTGCTGTGGCAATGAGTTTCCTGTGGCAATAAAACAGAAGTTCAG 89003187 89003236 890032 cg05557991 CBFA2T3 16 89003641 TCGTCCACCTCGTGGCTCAGTCTCTCTGGGAAAGCCTGCGTCAGTGAACG 89003641 89003690 890036
cg21380181 LOC100130872 4 1198278 TGGAGCTGGTGCGTGGGTCGCTCACAGTAACAATGCGAACGGTGAACACG 1198278 1198327 119827 cg18406106 LOC100130872 4 1198308 CACCGGAGGCTGCACCTAGGGAGCCCCGTGTGGAGCTGGTGCGTGGGTCG 1198308 1198357 119830 cg05821186 LOC 100130872 4 1198353 AACAAAAGATGAACAAGGGAACCGTGGCCACCAGG I I I IAACACACACCG 1198353 1 98402 119835 cg08418872 TNFRSF1A 12 6442954 CGGTGAAGGAGCCGCTCTCACACTCCCTGCAGTCCGTATCCTGCCCCGGG 6442954 6443003 644295 cg23752651 TNFRSF1A 12 6442966 TGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGGAGTGTGAGAGCG 6442966 6443015 644296 eg 14009561 TNFRSF1A 12 6443045 CGGCCCCATTCACAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGG 6442997 6443046 644304 cg01140143 HLA-DPA1 6 33039396 CGTCCTGTTCTGCAGACGCGTATAAGTCACAGAAGGAAACACAAGTGACA 33039396 33039445 330393 cg02904996 HLA-DPA1 6 33039412 CGCGTATAAGTCACAGAAGGAAACACAAGTGACAGGACAACAGCAGGTTC 33039412 33039461 330394 cg10921592 HLA-DPA1 6 33039414 CTGAACCTGCTGTTGTCCTGTCACTTGTGTTTCCTTCTGTGACTTATACG 33039414 33039463 330394 cg05366160 HLA-DPA1 6 33039481 CGTAGGTATACACAAGATGCGACCAAACAGCACATCAGGGAAGGCTTCCT 33039481 33039530 330394 cg13947735 B3GNTL1 17 80924969 CGGTAGGGAAGTCCACCTAACGGCCTCATCTGAACTTTGCATCTGCAAAG 80924969 80925018 809249 cg26628069 B3GNTL1 17 80925036 CGCCCACATTCTGAGATTGGGGGTGAGGACACCAACATATCA I I I I GGGG 80925036 80925085 809250 cg 4524754 B3GNTL1 17 80925103 CGTGGGCTCCATGGTGTCCCCCCAAAATGATATGTTGGTGTCCTCACCCC 80925055 80925104 809251 cg20953257 KANK2 19 11289349 TGGGCACTCGGCCTGTGCTCTTCTCAGGCTGTGTAAGGGAGGCTACCTCG 11289301 11289350 112893 cg22807822 KA K2 19 11289365 CGTCACCCCCGGGGTCCGAGGTAGCCTCCCTTACACAGCCTGAGAAGAGC 11289317 11289366 112893 cg17107691 KANK2 19 11289376 GCTGTGTAAGGGAGGCTACCTCGGACCCCGGGGGTGACGAAGACGATTCG 11289328 11289377 112893 cg24082526 RPL13AP5 19 49993125 TCTGAGTCC I I I I GCCCTTGTCTCCCACAGGTCCTGGTGCTTGATGGTCG 49993077 49993126 499931 cg10380760 RPL13AP5 19 49993143 CGGCCCAGGAGATGGCCTCGACCATCAAGCACCAGGACCTGTGGGAGACA 49993095 49993144 499931 cg23390488 RPL13AP5 19 49993150 CACAGGTCCTGGTGCTTGATGGTCGAGGCCATCTCCTGGGCCGCCTGGCG 49993102 49993151 499931 cg15531369 RPL13AP5 19 49993157 CGTGGCTAAACAGGTACTGCTGGGTAAGTCGCTGCTCGTGGCCCCTCTGT 49993157 49993206 499931 cg27321942 RPL13AP5 19 49993217 TGAGACTCAAACGAAAGATGGTGAATGTTGCTCATCACTGACCTCCAGCG 49993217 49993266 499932 cg07499142 PIK3CD 1 9788715 CGAAACAGGCAGACGGTCTAGTGAGTTTCAGCACTTGCTGGGAACAGGCG 9788667 9788716 978871 cg12033075 PIK3CD 1 9788767 ATGGGGAAAGCCGTGCGTGCGCGTTATTTATTTAAGTGCGCCTGTGTGCG 9788719 9788768 978876 cg03971555 PIK3CD 1 9788769 CGCGCACACAGGCGCACTTAAATAAATAACGCGCACGCACGGCTTTCCCC 9788721 9788770 978876 eg 12054318 GNASAS 20 57414529 CGGGAGCCTGCGCCCACCTGCCCAAGTACTGGACCTGGGGCTAGCTTGCC 57414529 57414578 574145 cg25268451 GNASAS 20 57414539 CGCCCACCTGCCCAAGTACTGGACCTGGGGCTAGCTTGCCGCTTGCTCCT 57414539 57414588 574145 cg1 1996914 GNASAS 20 57414578 CGGCAAGCTAGCCCCAGGTCCAGTACTTGGGCAGGTGGGCGCAGGCTCCC 57414530 57414579 574145 cg21330323 GNASAS 20 57414596 GCCCAAGTACTGGACCTGGGGCTAGCTTGCCGCTTGCTCCTTGCCACCCG 57414548 57414597 574145
cg27220660 MAZ 16 29820429 GCGCAGCCACAAGGTCTTGTCTCCAGCTCCTGGGGCAGGTGGAGTACACG 29820381 29820430 298204 cg03523538 AZ 16 29820460 CGGGTAGCAGAGAAAGCTGCCTTCAGTCAGACTCACCGGTTAACTGGGTT 29820460 29820509 298204 cg27338089 MAZ 16 29820515 CAGAGAAAGCTGCCTTCAGTCAGACTCACCGGTTAACTGGGTTGAGACCG 29820467 29820516 298205 cg07126783 RPTOR 17 78800767 CGCCAGGGCAGTACGCAGATGCGTGCCCAGGTGTGAGTTTAAAAAAGAAA 78800719 78800768 788007 cg16638092 RPTOR 17 78800774 TTTAAACTCACACCTGGGCACGCATCTGCGTACTGCCCTGGCGACTGACG 78800726 78800775 788007 cg08939850 RPTOR 17 78800806 CTGCCCTGGCGACTGACGTGAGCATGAAGGGAGCTTTAGTCAGCCGCTCG 78800758 78800807 788008 cg03910874 6 209712 CAGGCAGCCTTTCCTGCTGGTCCCAGTGCTTCTCGGAGGGCACCATCACG 209712 209761 209712 cg05869491 6 209763 CGGCAGGCAGCCTTTCCTGCTGGTCCCAGTGCTTCTCGGAGGGCACCATC 209715 209764 209763 eg 15253304 6 209809 CCGTAGTCAA I I I I I I GATTTCCCCATCCACCAAGAAACGCTCCCTTACG 209809 209858 209809 cg03303325 TBCD 17 80823240 CGCTGAGGGTCCCTCCACATCCACCCCATCGTGGGAGGGAGGCTCAGTGC 80823240 80823289 808232 cg16538568 TBCD 17 80823310 CGGGGAGACACCCCCTGCCCACAGCCACCCTCTCTGGGCTATGTGGGAGG 80823310 80823359 808233 cg23352492 TBCD 17 80823397 TATGTGGGAGGTGCCCACGTTTCCACGCCTTCCTCGTGGAGGGTGTGGCG 80823349 80823398 808233 cg11477010 ANKFY1 17 4079262 CGGTCCCTGACCAAGGACCCCGCCTTCCTGGGGCCCGGCATGACAGATGA 4079214 4079263 407926 cg12077963 ANKFY1 17 4079306 CGAGGCCGATTCCCTACGACTTTGGGACCCAGCTCCGCCTCCTTGGCTGC 4079306 4079355 407930 cg07697428 ANKFY1 17 4079341 AGACTGATGAACACGAGGCCGATTCCCTACGACTTTGGGACCCAGCTCCG 4079293 4079342 407934 cg26182859 1 1.54E+08 GGAGCTCGCTCTGGGGTAGACACGCATGAAAGCCGGTGTGAAATGCAGCG 1.54E+08 1.54E+08 1.54E+ cg09762021 1 1.54E+08 GCATGAAAGCCGGTGTGAAATGCAGCGCCCTGGATTGAACATCAGAGACG 1.54E+08 1.54E+08 1.54E+ eg 12067764 1 1.54E+08 TTTGAAGCCCTTATGTCTATGGTAATCTGTTACAGCCCCCGCAGGAAACG 1.54E+08 1.54E+08 1 54E+ cg09889350 CETP 16 56995813 CGGAACATGAGGCTCTTCCGGGCAGCAGGGTCTTCCTGGCCCCAGAGATT 56995765 56995814 569958 cg12564453 CETP 16 56995840 CGGGCTCCAGGCTGAACGGCTCGGGCCACTTACACACCACTGCCTGATAA 56995840 56995889 569958 cg16660091 CETP 16 56995856 CGGCTCGGGCCACTTACACACCACTGCCTGATAACCATGCTGGCTGCCAC 56995856 56995905 569958 cg15377174 PPP1R10 6 30568625 CGGTGGTGCTGGTCTGCCTTTGCCAACTATAGCCAGTCTGGAGACTTGAT 30568577 30568626 305686 eg 16586538 PPP1R10 6 30569105 CGTTCCTGGACTGCTGAAGAGGAGACCCAGTTGGCTTCAC I I I I I GAGAA 30569057 30569106 305691 eg 19200440 PPP1 R10 6 30569156 CGCACCAGTGATCAGAAAACCCCCAGGAACCCAAGCAAGTGGGAACTGAG 30569156 30569205 305691 eg 12701674 13 1.15E+08 TGGTTGTCCTGAGCTGGCACCTTCGGCACATGCGCACATGCTCTCACACG 1.15E+08 1.15E+08 1.15E+ cg246351 12 13 1.15E+08 GCAAAATTCCTGAACACGTAATGCTTGGTTGTCCTGAGCTGGCACCTTCG 1.15E+08 1.15E+08 1.15E+ cg05799596 13 1.15E+08 TGTGTTTGGACGCACATACACCACTGTGTGGCAGTCGCCTGTGGCATTCG 1.15E+08 1.15E+08 1.15E+ cg26791489 GNAS 20 57463330 CGGCTCACCTGGGCACATGGTGAGCCGCGGAACTGGCAGGCACCAAACAT 57463330 57463379 574633
cg04019914 GNAS 20 57463357 CTGGGATACGGTTTATTCATTCAATAAATGTTTGGTGCCTGCCAGTTCCG 57463357 57463406 574633 cg11357538 GNAS 20 57463397 CGGTTTATTCATTCAATAAATGTTTGGTGCCTGCCAGTTCCGCGGCTCAC 57463349 57463398 574633 cg10011623 GNAS 20 57463527 CGGCGGGACACTTGAGAGTTTTGAAAGAACCCTCAAACAAGTTCACGTTC 57463527 57463576 574635 cg01748573 GNAS 20 57463530 ACGGAACGTGAACTTGTTTGAGGGTTCTTTCAAAACTCTCAAGTGTCCCG 57463530 57463579 574635 eg 17334845 GNAS 20 57463572 CGTGAACTTGTTTGAGGGTTCTTTCAAAACTCTCAAGTGTCCCGCCGCAC 57463524 57463573 574635 cg26767990 GNAS 20 57463615 CGCCCTGGGGGCTCTGGAACAGAGGGAAAAGTAGTTTACGGAACGTGAAC 57463567 57463616 574636 cg18935491 GNASAS 20 57425979 CGGTTTCCGGTCTGACGCCCCCTGCTCATTCGCCAGGCAGCCTTGATTGG 57425979 57426028 574259 cg24203465 GNASAS 20 57425986 CGGTCTGACGCCCCCTGCTCATTCGCCAGGCAGCCTTGATTGGCATGACC 57425986 57426035 574259 cg26102503 GNASAS 20 57425994 CGCCCCCTGCTCATTCGCCAGGCAGCCTTGATTGGCATGACCCCTGCCCA 57425994 57426043 574259 cg03344105 GNASAS 20 57426131 CGGCTTAACCACTTGAGCATCCACTGAATGGCCCAACTGGGTCCCAACAC 57426083 57426132 574261 cg19592829 GNAS 20 57426215 GAACACAGTTGGACCCTGGGAAACGTTTGGGATCCCCCAAAGATATATCG 57426215 57426264 574262 cg09123158 GNAS 20 57426264 GATATATCTTTGGGGGATCCCAAACGTTTCCCAGGGTCCAACTGTGTTCG 57426216 57426265 574262 cg19140375 GNAS 20 57426274 CGGAAGGAGGCGAACACAGTTGGACCCTGGGAAACGTTTGGGATCCCCCA 57426226 57426275 574262 eg 11669839 GNAS 20 57426322 CGGTGCCTGTGGTACCCTGGCTCTAAGAGGGATGCATTAGACTAAACGGG 57426322 57426371 574263 cg14482474 GNAS 20 57426383 ACCCTGGCTCTAAGAGGGATGCATTAGACTAAACGGGATCGCAACTTGCG 57426335 57426384 574263 cg06200857 GNAS 20 57426420 GCTGGATGAGTTACACAGGGCGGGCTGCAGCAAGCAGGGCGCACGGCACG 57426420 57426469 574264 cg08091561 GNAS 20 57426425 GGGAGGCTGGATGAGTTACACAGGGCGGGCTGCAGCAAGCAGGGCGCACG 57426425 57426474 574264 cg22860367 GNAS 20 57426538 CGTGGCTCGCCAATCTGTCTCACTGGTGAGACCGACCTGATGCGCACCTG 57426538 57426587 574265 cg07947033 GNAS 20 57426545 AGATGACCCCCTGCCTCCTGCCCAGTTGCTCTGTGCCTAGACGTGGCTCG 57426497 57426546 574265 cg04677683 GNAS 20 57426743 TGTGGGCATTTACCTTTGGCTAGCTCTGAGCTCACCGCAGCATGGTAGCG 57426695 57426744 574267 cg15160445 GNAS 20 57426749 CGCTCCCGCTACCATGCTGCGGTGAGCTCAGAGCTAGCCAAAGGTAAATG 57426701 57426750 574267 cg25326570 GNAS 20 57426757 TTTGGCTAGCTCTGAGCTCACCGCAGCATGGTAGCGGGAGCGCAGCTGCG 57426709 57426758 574267 cg23249369 GNAS 20 57426759 CGCGCAGCTGCGCTCCCGCTACCATGCTGCGGTGAGCTCAGAGCTAGCCA 57426711 57426760 574267 cg13106215 14 1.06E+08 CTGAGGGCTGGGCGGGGCTGGGACACGCAGCACACTCCAGGCCCAGAACG 1.06E+08 1.06E+08 1.06E+ cg04991452 14 1.06E+08 CGCCCCCCAACATCCTCCTGATGTGGCTGGAGGACCAGCGTGAGGTGAAC 1.06E+08 1.06E+08 1.06E+ cg19571360 14 1.06E+08 ACGAGGCCAGCAGGTTCAGGGAAAGCTTGACGGGTGCCTGCGCAGCTGCG 1.06E+08 1.06E+08 1.06E+ cg09857617 SKIV2L 6 31937228 CGGCTGGCTGGGGAGAACCTGCCCAGGCTGAGTGCATCCAGTCCTCACCC 31937228 31937277 319372 cg00423167 SKIV2L 6 31937355 GCAGGGCTCTCAGGGACCCCTGAGGGCCTGGTGGTCCGCTGCATTCAGCG 31937307 31937356 319373
eg 10874097 STK19 6 31937470 CGGCCAGCCTCTACACCCAGTGAATGCCCCATGTAAAAACATGATGATAA 31937470 31937519 319374 cg15995714 PWWP2B 10 1.34E+08 GACCTCACACGGAGGGGTGCCCAAGTGTGTCACAGCCATGCGTCCTGGCG 1.34E+08 1.34E+08 1.34E+ cg02116864 PWWP2B 10 1.34E+08 CGCATGGCTGTGACACACTTGGGCACCCCTCCGTGTGAGGTCCTGATGGA 1.34E+08 1.34E+08 1.34E+ cg07733247 PWWP2B 10 1.34E+08 GCTGTGACACACTTGGGCACCCCTCCGTGTGAGGTCCTGATGGAGGTGCG 1.34E+08 1.34E+08 1.34E+ cg23468927 COR01B 11 67206263 CTGCGGGAGGCCTACGTGCCCAGCAAGCAGCGGGACCTGAAGATCAGCCG 67206263 67206312 672062 eg 12044599 COR0 B 11 67206308 GAGGAGTGGGTGAGCGGGCGGGATGCCGACCCGATCCTCATCTCACTGCG 67206308 67206357 672063 cg02740606 COR01B 11 67206418 CGGACCTCTTCCAGGATGATCTGTACCCCGACACAGCCGGGCCCGAGGCA 67206370 67206419 672064 eg 17690322 COR01B 11 67206434 CGGCCCCCTGCCCAGTCGGACCTCTTCCAGGATGATCTGTACCCCGACAC 67206386 67206435 672064 cg27395666 1 98513986 CGGCAGCACTCGCAGCAGAACTGACGCTCTGAGCTCTAACCAAAGCTCTC 98513986 98514035 985139 cg04288975 1 98514064 CGAGAACACCATTTCCTACGAGACCACTGCCGCTGGGTGCATATTCAATT 98514064 98514113 985140 cg19361032 1 98514282 CGGAGGCTTAGGTCCGCATTAAGTACCTTTGTTCATCGACTCCTGCAAGC 98514282 98514331 985142 cg22158212 1 98514388 TTAGAATCTACATTTCTCTCG CG CCC CAATAACTAGAGGTGTG 1 1 1 1 GCG 98514340 98514389 985143 cg02442640 Sept9 17 75451779 CGGACATGAGGATTGTGTCTTGTCGATCCACGTCCTGAGTTCCCAGACGT 75451779 75451828 754517 cg17922695 Sept9 17 75451809 CGTCCTGAGTTCCCAGACGTCATAGGTGCTTGCTCAACGAGTGTTTGAAT 75451809 75451858 754518 cg14843920 Sept9 17 75451932 CGGGATAGCCTCACAGAAACCACAAACTATTTGGATAAAAGTTGAAAATC 75451884 75451933 754519 cg17893934 LARP4B 10 858817 AAAACCGTACGTCTGGGAGGGGTCGCAGAGCGCTGTGTTAACCACAAACG 858817 858866 858817 cg06296597 LARP4B 10 858900 CAGACGTACGG 1 1 1 1 CACTGAGGAGACTTGGGGGGAGTGCTCTGCTCTCG 858852 858901 858900 cg08876481 LARP4B 10 858923 CGGCCATGGGGAAGCGTCTCAGCCGAGAGCAGAGCACTCCCCCCAAGTCT 858875 858924 858923 cg01861740 7 2764486 CGGGAAGGATTTCCAAACGTCAGGGTTAAGCATGGGAGCGATTCTCGTCC 2764486 2764535 276448 cg06624267 7 2764503 GGGCGCGTGAGAGTCTCACAATGACAATGTCCGGGAAGGATTTCCAAACG 2764455 2764504 276450 cg03366574 7 2764599 CCGCCAGGACAGAGAAGCCGGTACCCCAGCGGGTGGTCTGC I 1 1 I CTGCG 2764551 2764600 276459 cg04699519 ATXN1 6 16306096 GTGAGGGTCTC 1 1 1 1 1 CTCTGCCTCCCTCTGCCTCACTCTCTTGCTATCG 16306096 16306145 163060 cg 8455405 ATXN1 6 16306552 CGCTGCCTCTACTTGCCTACATTAGACCGGCCTTCAATGCAAATCTTAAC 16306552 16306601 163065 cg03680932 ATXN1 6 16306683 CGGCGCCAGAGAGCCGCAAACTGGAGAAGTCAGAAGACGAACCACCTTTG 16306635 16306684 163066 eg 18584387 CCND2 12 4384879 AGCTCCAAAACGGGGAGTCGGAGAAGTGCGCAGCAGCCGCGGCAGCTCCG 4384879 4384928 438487 eg 07066369 CCND2 12 4384888 GAGTTAGAGTGCGCGAAGGAGTAGCCAAAGGGAGCGTCGCGGAGCTGCCG 4384840 4384889 438488 cg17580045 CCND2 12 4384890 GGGTGAACTACAGCTCCAAAACGGGGAGTCGGAGAAGTGCGCAGCAGCCG 4384890 4384939 438489 eg 13325231 CLPT 1 L 5 1342170 CGCGTCCTGAGAACTCGGCACAGGTGTGGGCGCCTACAGCCGAAAGCAAA 1342170 1342219 13421
cg07493874 CLPTM1 L 5 13421 2 G U M GCTTTCGGCTGTAGGCGCCCACACCTGTGCCGAGTTCTCAGGACG 1342172 342221 134217 cg14733637 CLPTM1 L 5 1342200 CCTGAGATGGAGGCAGGAAGTGCGGGCCG I I I I GCTTTCGGCTGTAGGCG 1342200 1342249 134220 cg03162779 11 2308362 CGTGATGCCGTGTAACTTCCTGTTGCTTCTTAGAC I I I I ACTCACTCGTT 2308314 2308363 230836 eg 16059978 11 2308487 GCTGGTTGTGTGCAGTGAAGGCCACGTGTGGTCCTAACTGCTCTATCACG 2308487 2308536 230848 eg 11821865 11 2308511 CCGGGTCCTTCTTCCTCTTTGGGCCGTGATAGAGCAGTTAGGACCACACG 2308463 2308512 230851 cg21619547 DHFRL1 3 93780328 GGAGGTGTTAACCGCTGCTGTCATGTTTCTTTTGCTAAACTGCATCGTCG 93780328 93780377 937803 cg23126947 NSUN3 3 93780364 TTCTTGGCCTGGCTTCCTGGCGTAGCCAGCAAGTTCGGAGGTGTTAACCG 93780364 93780413 937803 eg 15999104 NSUN3 3 93780377 GAATTTCGCGGCATTCTTGGCCTGGCTTCCTGGCGTAGCCAGCAAGTTCG 93780377 93780426 937803 cg03431067 AXIN1 16 367990 CGGCACACGGGTGCATCTGTAGCCCACGGCACTTGGTGCGTCTGTAGCCC 367990 368039 367990 cg03660377 AXIN1 16 368048 GTGCATCTGTAGCCCACGGCACTTGGTGCGTCTGTAGCCCACAGCACACG 368000 368049 368048 cg03316628 AXIN1 16 368066 CGGCACATGGCGTGTCTGTCTTGGCACGCACTGGAAGCACGTGCCCTGCC 368066 368115 368066 cg16306259 6 1595099 TGTACA I I I I GAATGCTAATTTCTAAGTCCTGGGCCTGCCTCAGTTGTCG 1595099 1595148 159509 cg02278728 6 1595575 CGCACCCACATCTAAGGCAGCCAGGATGGTCGGAAAGTGTCTGCCAGCAG 1595575 1595624 159557 cg06399596 6 1595676 GGGGATCCCCAGGGGCCCCATCAGCCCAGGGCTGCTCCTCAGGATGGCCG 1595628 1595677 159567 cg23388085 6 1595741 AACTGGAAGTAGACCCGGGGTGCCCGGACCCGCTGTAGGGGAGGCAGACG 1595741 1595790 159574 cg08506869 KIAA0146 8 48572447 CGGAGTCAGGCCCGGCTGTTTCATTCTAGGTCACTGTCAACTTAAGCAGC 48572399 48572448 485724 cg20190772 KIAA0146 8 48572496 CGTGAGTTATGGGAGCAGTTCTTAGCAGTCCACCAAGAATTCCCGGAATC 48572448 48572497 485724 cg25257799 KIAA0146 8 48572557 GCTCAGTTTCATAGCTACACGAATGAAGGACAGCGAGAGCACAGCAAACG 48572557 48572606 485725 cg12956598 ATG4D 19 10662582 AGTGAGTGTCGCGGTTTCCCACCCATGATGCCCAGGCACAGCTCGCAACG 10662582 10662631 106625 cg23002708 ATG4D 19 10662587 AGTACAGTGAGTGTCGCGGTTTCCCACCCATGATGCCCAGGCACAGCTCG 10662587 10662636 106625 cg05648345 ATG4D 19 10662621 CGCGGTTTCCCACCCATGATGCCCAGGCACAGCTCGCAACGCAGGAGTTC 10662573 10662622 106626 cg13798621 ATAD3C 1 1396125 CAGTCGAACTGCTCGGGGTGGCAGCTGGCCAGGATCAGCATGAATCTGCG 1396125 1396174 139612 eg 13487474 ATAD3C 1 1396160 AGTGGACCATCACGTCGATGCAGGCATTGATGGCCCAGTCGAACTGCTCG 1396160 1396209 139616 cg23016632 ATAD3C 1 1396169 CGACTGGGCCATCAATGCCTGCATCGACGTGATGGTCCACTTCGACCTGC 1396169 1396218 139616 cg11599721 RPS6KA2 6 1.67E+08 GGGCTCAAGATCACATAATAGTCCCAGAAACACATTCTCATTTAGTGCCG 1.67E+08 1.67E+08 1.67E+ cg05691806 RPS6KA2 6 1.67E+08 CGGTATTCTTTCTGTGCACAGAAATGAAAACTAAAGCTGAAAGAGCCTAA 1.67E+08 1.67E+08 1.67E+ cg09430664 RPS6KA2 6 1.67E+08 CGGCAACAGCCAGCCCTGCCTCGGGCTCGAGAGTGACACAGCAGTTCTGG 1.67E+08 1.67E+08 1.67E+ cg14112356 ITGB2 21 46348443 GCAGTGGGAGATAATGCCCTCATCTACCAAAACAAGATCTGCCACCTTCG 46348395 46348444 463484
cg22699620 ITGB2 21 46348691 CGTGGCTCCGTTTGGGCAGAAACCATGAATTGAACGGGAAAAGAAATATG 46348643 46348692 463486 cg21006727 ITGB2 21 46349059 CGGGGACAACACAGGTGTGTTCAGAGACAAGGCGGGTCATTAGATTTGAT 46349011 46349060 463490 cg10923018 C6orf27 6 31743769 CGCCTCGGGGCTGCACTTCATGCCCTGCAGGTGAGAACAGGGTTGGGTTG 31743721 31743770 317437 cg15318662 C6orf27 6 31743775 CGCCAGCGCCTCGGGGCTGCACTTCATGCCCTGCAGGTGAGAACAGGGTT 31743727 31743776 317437 cg26656884 C6orf27 6 31743820 CGGGAGACCGTGGTGGCAGCCAGGGCCCTTGACCACACCCTGGCTCGCCA 31743772 31743821 317438 cg 9248564 CYB5 3 22 43042879 CGGCTGTGAGTGCTCAGCGACCCTGACAGGCTGACAGGTACAGTCTGCCG 43042879 43042928 430428 cg2719 921 CYB5R3 22 43042910 GGGACAGTAGGAAAGCCCGGCAGACTGTACCTGTCAGCCTGTCAGGGTCG 43042862 430429 1 430429 cg01194538 CYB5R3 22 43043270 TGAATGGCTGAGCAGTCCTCTGTAAGGGGCAGAGCACTGCCTGGGAGTCG 43043270 43043319 430432 cg10360071 2 1.06E+08 CGGGCAGAGGCAGGGTGTTGGCCAAGGTCACGGCCCCAGAGACCACAATA 1.06E+08 1.06E+08 1 06E+ cg12495283 2 1.06E+08 GCGGTGAGTGCGCGTGCTCAGGGCCCTCCTGGGGCTGCATGGAGGCCTCG 1.06E+08 1.06E+08 1 06E+ cg08019058 2 1.06E+08 CAGAGACGACAACTGAGTCACGTAAGGGAGACGCGCAGTGGATGCCCACG 1.06E+08 1.06E+08 1 06E+ cg21330577 TUBB 6 30691683 CGAGCTCTCACAGTGCCGGAACTCACCCAGCAGGTCTTCGATGCCAAGAA 30691683 30691732 306916 cg26470641 TUBB 6 30691760 CGGCCGATACCTCACCGTGGCTGCTGTCTTCCGTGGTCGGATGTCCATGA 30691760 30691809 306917 cg27500784 TUBB 6 30691764 TCCTTCATGGACATCCGACCACGGAAGACAGCAGCCACGGTGAGGTATCG 30691764 30691813 306917 cg00070870 TUBB 6 30691895 CGGCTGTCTTGACATTGTTGGGGATCCATTCCACAAAGTAGCTGCTGTTC 30691847 30691896 306918 cg06038367 COR01A 16 30198370 GGAGGCTCATGGCTTCTGACACTGTGGGGAACGTGCAGGTTGTGACAACG 30198322 30198371 301983 cg27583010 COR01A 16 30198505 CGCGCTTGTCACGGCAGGAGGTACAAATGAGGCCTCCATCTCGGCTCCAG 30 98457 30198506 301985 cg06749872 COR01A 16 30198509 AGATGGCGACTCACAGCTACGACAGTGCCTTTGCGGGGCTCGATGATGCG 30198509 30198558 301985 cg22582875 FOXK2 17 80545272 TTCTGACTGTAACAA I I I IAGC I I I I GTCTGAGGCAATGTTTGCATTTCG 80545224 80545273 805452 cg14173033 FOXK2 17 80545310 GTTTGCATTTCGAAGCTGCAGGAAATCTAGTCATGCTGGGGAAGTGGTCG 80545262 80545311 805453 cg021 16768 FOXK2 17 80545322 CGGCCGACTCCACGACCACTTCCCCAGCATGACTAGATTTCCTGCAGCTT 80545274 80545323 805453 cg25425537 2 6910518 TCCAGGGTCTGAGGCTCTTCCTTCCTGGCCCTAGAATGGCGTCTCTTGCG 6910518 6910567 691051 cg02738156 2 6910588 TTCTCCGAAACAGGAAATTGTTGTACATTCAGACACACCATAAATACACG 69 0588 6910637 691058 cg0093 181 2 6910631 CGGAGAAAATAGTTCCTTCCTGGAAGGGCTGTTCTGTATTTGCACACTTT 6910631 6910680 69 063 cg06053959 C17orf91 17 1617291 CGCCCTCACCTGGCTGAGCCGCAGTAGTTCTTCAGTGGCAAGCTTTATGT 1617243 1617292 161729 cg26697882 C17orf91 17 1617363 CGCTGACCCACTCGCTGGCCATCAGGGTCCTGCCCCAGTAGACCTCTGAG 1617363 1617412 161736 cg00956971 C 7orf91 17 1617375 CGCTGGCCATCAGGGTCCTGCCCCAGTAGACCTCTGAGATTCTAATGCAG 1617375 1617424 161737 eg 06927305 C 7orf91 17 1617447 CGGGCCCAGATCAGAGCTGCAGCCCTGCATTAGAATCTCAGAGGTCTACT 161 399 1617448 161 44
cg22516975 C17orf91 17 1617465 TAATGCAGGGCTGCAGCTCTGATCTGGGCCCGCGGAAGCTGTTAACCTCG 1617417 1617466 161746 cg25618559 2 2.42E+08 GGAATGTGGGGCACCCACCTGACTGACGACATGGAGGCCCGGGAGCAGCG 2.42E+08 2.42E+08 2.42E+ eg 11564239 2 2.42E+08 CGTCAGTCAGGTGGGTGCCCCACATTCCCGTGGGTGTGCAGAGGCTTCTC 2.42E+08 2.42E+08 2.42E+ eg 12565788 2 2.42E+08 AGTCAGGTGGGTGCCCCACATTCCCGTGGGTGTGCAGAGGCTTCTCACCG 2.42E+08 2.42E+08 2.42E+ eg 11683242 LCK 1 32716557 CGGACACACACTCAGCTTATGGTGAGAGGCAAACAGCACCTCAGCACCAC 32716509 32716558 327165 cg05350315 LCK 1 32716961 CAGCCAGGTTAGGCCAGGAGGACCATGTGAATGGGGCCAGAGGGCTCCCG 32716913 32716962 327169 eg 17078393 LCK 1 32717002 GGGCTCCCGGGCTGGGCAGGTAAGGAGCGCTGGTATTGGGGGCGCAGGCG 32716954 32717003 327170 cg24831051 EF2C 5 88018481 TCCTTCAGAAAGTCGCATGCGCTTGACTGAGGGACTTTCCCTTTCGTCCG 88018433 88018482 880184 cg17245135 MEF2C 5 88018524 CGGTGATCCTCTCGGTCGCTCCCGTCGTACGAACTGCTACAGCTGCTCAA 88018524 88018573 880185 cg0604081 1 EF2C 5 88018605 CGCCACGAGGCGGGGAGATCTCCTGTTGACAGCTTGAGCAGCTGTAGCAG 88018557 88018606 880186 cg27572068 3 25728371 CGGACCCGGAGCCGCCATGCCCAAGTGCCCCAAGTGCGACAAGGAAGTTT 25728371 25728420 257283 cg25509643 3 25728428 ACTTCAGGCAGGGCCGAGGCAAGTCCTTGCCCAGAGAGGTCACCCTCTCG 25728428 25728477 257284 cg11115005 3 25728479 CGCACTTCAGGCAGGGCCGAGGCAAGTCCTTGCCCAGAGAGGTCACCCTC 25728431 25728480 257284 cg01157951 LTA 6 31540399 CGACAGAGAAGGGGACAAGATGCAGTCAGAGAAACCCCAAGGTGAGCAGA 31540351 31540400 315403 cg22318806 LTA 6 3154041 1 GGGGTTTCTCTGACTGCATCTTGTCCCCTTCTCTGTCGATCTCTCTCTCG 31540363 31540412 315404 eg 13815684 LTA 6 31540440 GAGAGAGACAGTGAGCGGGGCGGGGCACGCGGCGGAAGACAGACCTCCCG 31540440 31540489 315404 cg17709873 LTA 6 31540456 TCTCGGGGGTCGGGGGGTGCTCTCTCCCAGGGCGGGAGGTCTGTCTTCCG 31540408 31540457 315404 cg16280132 LTA 6 31540459 CGGCGGAAGACAGACCTCCCGCCCTGGGAGAGAGCACCCCCCGACCCCCG 31540411 31540460 315404 cg26348243 LTA 6 31540461 CGCGGCGGAAGACAGACCTCCCGCCCTGGGAGAGAGCACCCCCCGACCCC 31540413 31540462 315404 cg12699756 HSD17B8 6 33173482 GACAAACTATGCAGCATCCAAGGCTGGAGTGATTGGGCTGACCCAGACCG 33173434 33173483 331734 cg01970322 HSD17B8 6 33173489 CGGGCTGCGGTCTGGGTCAGCCCAATCACTCCAGCCTTGGATGCTGCATA 33173441 33173490 331734 eg 17066452 HSD17B8 6 33173501 AAGGCTGGAGTGATTGGGCTGACCCAGACCGCAGCCCGGGAGCTTGGACG 33173453 33173502 331735 cg14427668 ATXN7L1 7 1.05E+08 CGGAACAGAGATCCTGGCAGGCAGATCATGCTGGCAGCCCACGAGGCATT 1.05E+08 1.05E+08 1.05E+ cg00423035 ATXN7L1 7 1.05E+08 TCCTTATCCAAACGGCAAGGCAGCCTCCCCGGGATGTGTAAGTGTGAGCG 1.05E+08 1.05E+08 1.05E+ eg 18527241 ATXN7L1 7 1.05E+08 TGGCCAGGGCCCTCATCCAGGAGCCAGGAAACATCTACTACCACGAGTCG 1.05E+08 1.05E+08 1.05E+ cg05803296 ATXN7L1 7 1.05E+08 GTAGTAGATGTTTCCTGGCTCCTGGATGAGGGCCCTGGCCAGTCAGAACG 1.05E+08 1.05E+08 1.05E+ cg02827175 MAD1 L1 7 1986245 CGCCTGCTCGCTCTGCAAGTGAAAGATTTCTGTTGAGTCAATGATTGCAC 1986197 1986246 198624 cg16993108 MAD1 L1 7 1986301 TGCTGCTACCCAGCGCGGTTAAGCGGAAGAGCCGGCTGGCGGGTGCAGCG 1986253 1986302 198630
cg01952989 MAD1 L1 7 1986334 CGGGGACCTGCCCTGCCCGTCCCAGCATTCTCCCGCTGCACCCGCCAGCC 1986286 1986335 198633 eg 13706058 6 85824168 CGCCGCTCTGTGTAATTAATTGCTGCGAGAGGAAGCGGACCAATGGCGCC 85824120 85824169 858241 cg10439246 6 85824196 CGCGTTTGCTGTGGTCCGCGTCAAAACACGCCGCTCTGTGTAATTAATTG 85824148 85824197 858241 eg 11452329 6 85824216 CGCGTATCTGAGAGCAACGTCTTTATCATCAGCACGGTGCGTTCCCTGCA 85824216 85824265 858242 cg02775414 SLC44A4 6 31832962 AGTCGGGCCCTAGACTTAGTTTAGTGCTCTGCTCTTGCCATCTTGAAACG 31832962 31833011 318329 cg09153713 SLC44A4 6 31833028 CGTGGGTGTGCTGTGCTCAGTCGGGCCCTAGACTTAGTTTAGTGCTCTGC 31832980 31833029 318330 cg07705820 SLC44A4 6 31833104 CGCATGAGTAGCATGAACGCATTTTTGGCTGAGACACAGAAATTCTTCCC 31833104 31833153 318331 cg07546508 SLC44A4 6 31833199 GTTCTGGTGCCCTTTGGTCTAGACACCCCCATCCTGGTCCAGGCAGCTCG 31833199 31833248 318331 cg0146121 1 16 85478908 CGCCTCTCCACTGTGCTAGCAGCAGTGGCGGCCACACCTGGTGTCAGCCT 85478860 85478909 854789 cg09363128 16 85478932 CGCTGGAGCAGGTAGCAGCACACTCGCCTCTCCACTGTGCTAGCAGCAGT 85478884 85478933 854789 cg06841648 16 85479053 AGCTCAACAGAATCTGGGCCTGCACCTGTGAGGGGTCTGGGGACTGGCCG 85479053 85479102 854790 cg10789824 RINL 19 39369196 AGACATTGCTTATTGGAGACAAATTAAAAACAAAAACAACTAACAATCCG 39369196 39369245 393691 cg05886321 SIRT2 19 39369335 TGTGGGCCTCTGAATATAACCCACACCCAGCGTAGGGGGAGTCTGAGCCG 39369335 39369384 393693 eg 15581429 SIRT2 19 39369353 CGCTGGGTGTGGGTTATATTCAGAGGCCCACACCCACACTGGGGTAGGTT 39369353 39369402 393693 cg09050670 CLCN7 16 1521617 CGCTGAGCACGGTGCCTGTCACAAACTACGCTCTGAAGAAACGTGAGTCG 1521617 1521666 152161 cg05413628 CLCN7 16 1521656 CCAG I I I I CCCGTCTGCGAAAAAGTTAACGCATGCATAGCGCTGAGCACG 1521656 152 705 152165 cg08810397 CLCN7 16 1521676 CGCATGCATAGCGCTGAGCACGGTGCCTGTCACAAACTACGCTCTGAAGA 1521628 1521677 152167 cg06961439 ATP6V0E2 7 1.5E+08 GTGGGGACTGCGCCTCCCTATGTCATGAAGCCAGGGAATATGTGTGGCCG 1.5E+08 1.5E+08 1.5E+0 cg27527018 ATP6V0E2 7 1.5E+08 CGGCCGGCCACACATATTCCCTGGCTTCATGACATAGGGAGGCGCAGTCC 1.5E+08 1.5E+08 1.5E+0 cg08634133 ATP6V0E2 7 1.5E+08 GAATATGTGTGGCCGGCCGCTTGGGGTAGCACTTGTTGCTAGAGATACCG 1.5E+08 1.5E+08 1.5E+0 cg09469432 ATP6V0E2 7 1.5E+08 CGCCAGAGAAGAAGGGATATGTGAGATCCAAACGCCGAAGGAGGAAAGCA 1.5E+08 1.5E+08 1.5E+0 cg24227081 ATP6V0E2 7 1.5E+08 TTTCTTCCATCAGGGCTGGAAGAGACCACGGAGCCTGCTTTCCTCCTTCG 1 5E+08 1.5E+08 1.5E+0 cg16012294 MAD1 L1 7 2151681 CGGCTGCTCTGGGACTTGTTTATTCGTTCACTGGTTGAAGGACATCTGTA 2151633 2151682 215168 cg05030518 MAD1 L1 7 2151737 CATACAAATATGGCATCTTTGGCTCATGGAGGTGCATTTGGGACTCACCG 2151737 2151786 215173 cg24655669 MAD1 L1 7 2151788 CGTCATACAAATATGGCATCTTTGGCTCATGGAGGTGCATTTGGGACTCA 2151740 2151789 215178 eg 14378925 EHD1 11 64635728 TGGGCTTCAGTATTTCTCGCCCTAGCTCTCCATCACACAGGTTTCCAACG 64635680 64635729 646357 cg07019638 EHD1 11 64635774 AAATGCTGAGGAACGGAGGCGCCTCTGTGCTCCACTGGAAAACCCTTCCG 64635774 64635823 646357 cg04038246 EHD1 11 64635846 CGCCCACACGAACCACGTACTTGACTCAAAACTGCCCCGGGTGGAACAAC 64635846 64635895 646358
eg 13912753 OVOL1 11 65561705 CGCAGGAAGCCATGGTCTCTGCTCTGGGGGTCTGTCAAGTGGCCCATGTC 65561657 65561706 655617 cg09181559 OVOL1 11 65561733 CGCGAGTGGCATTACCTTCATCTTGGTGCGCAGGAAGCCATGGTCTCTGC 65561685 65561734 655617 eg 15453482 OVOL1 11 65561749 CGGGGGCGTGCATGTAGACCTGCGTCCAGCTCATGAGACATGGCACAAAT 65561749 65561798 655617 cg03062944 10 6183455 ATCCGGGATTGAACAACGAACTCGCCACAGCCGTGCTGGTCAGGGAAACG 6183455 6183504 618345 cg04 89187 10 6183528 CGCCCTGTTCTATCGCCGCATGGATCTGCAGAGGCAGCCGTTCCTGCCGG 6183528 6183577 618352 eg 18264092 10 6183575 ATAACTCAGAGTGGTTCTGTGTTGTCCTTCGCGGAAGGCAAATCCACCCG 6183575 6183624 618357 eg 10351914 MFAP4 17 9290690 CGGGCAAAGTACTGCAGACGTTAACTCCCTGCTGGCTCCAACTGTTCCCT 19290690 19290739 192906 eg 13030582 MFAP4 17 19290708 CGTCTGCAGTACTTTGCCCGACAGGACTAATGAGACACAGGTGGTCCCCT 19290660 19290709 192907 cg15119221 MFAP4 17 19290755 GCCAGGGGGCCCACAGGTCTAGCAGGGTGGGTGGCAGATGGCGGATAGCG 19290755 19290804 192907 eg 15227982 C10orf26 10 1.05E+08 ACATCCTCCTCAGTATTCCAGTGCAGCTGTCTGAAG I I I I I I CTGCTGCG 1 05E+08 1.05E+08 1.05E+ cg25104397 C10orf26 10 1.05E+08 CGTGACATATCTCAGACGCAGCAAAGCCGAAGCTGTCTGCCAAGGGGGAA 1.05E+08 1.05E+08 1.05E+ cg23247968 C10orf26 10 1.05E+08 GTAAACAGAGTCCTGGGACCGAGCAGGCACACTGTCTCCTGCTCTCCTCG 1.05E+08 1.05E+08 1.05E+ eg 14939082 C10orf26 10 1.05E+08 CGGTCCCAGGACTCTGTTTACTTTGTCTGCTTTGCTAAAGAAGGCCGGTG 1.05E+08 1.05E+08 1.05E+ cg23202468 ZBTB47 3 42705828 GGCCTTGAGCATGTTGGAGAAGCGGAAGCGCTGGCCACACACGTCGCACG 42705828 42705877 427058 cg06181470 ZBTB47 3 42705925 GTGCGTGCGCCTGGGGCTGGGTTGGGGGCAGGCCTGGGGCAGCGGGGACG 42705925 42705974 427059 cg03540622 ZBTB47 3 42706066 CGGGTGCACGGGTGCAGCTCGGCAGCTAGTTGTTGGCGTTCATCCTCCCG 42706066 42706115 427060 cg13613891 ZBTB47 3 42706106 GTGGGTCCCTCCTGGAGTGGGCCCTGACTCCAGGCCCCAGCGGGTGCACG 42706106 42706155 427061
TNFSF12- cg10479431 TNFSF13 17 7461421 CGGCCTCCTTGGCCTAGGAGGCAGACTTCCTCCTGCGGGGAAATCCCTTC 7461421 7461470 746142
TNFSF12- cg05514680 TNFSF13 17 7461556 CGCCCCACCTCCCAGCCAGGGCCTGTGCCAGCACCTGCTGAATGTCCGAG 7461508 7461557 746155 cg23161218 TNFSF13 17 7461638 CGGGATTCCTCCCCAGCACACAGGGTTCCGGGTTCCCGTGTGCCTGCTGA 7461590 7461639 746163 cg13358186 TNFSF13 17 7461775 TTGCCAATTTCAGCACAGGGAGTAGTGCAGGCCTTATTCCAACACACCCG 746 727 7461776 746177 cg03607916 2 58478696 CGATGCTGCCGCAGGGTCTGAGGATGAGGCTGGAGCCGCAGCGGGAACCG 58478696 58478745 584786 cg25724323 2 58478735 CGGCAGCATCGGCTACAACTGCGGCTCCTGGCCAGACCCCGGCCTCAGCG 58478735 58478784 584787 cg09026213 2 58478744 CGGCTACAACTGCGGCTCCTGGCCAGACCCCGGCCTCAGCGCAAGCTCCA 58478744 58478793 584787 cg06958535 LAX1 1 2.04E+08 GATTCTCCCTGAGCCACCTCACTTGGAAGCACCATGTCCGGATGAGATCG 2.04E+08 2.04E+08 2.04E+ cg12022621 LAX1 1 2.04E+08 GCCAAGTTGACCCAACATACAAAAGCAACAGGTTTGGTTCTACCTGGACG 2.04E+08 2.04E+08 2.04E+ cg00942920 LAX1 1 2.04E+08 CGTCAGGCCAAGTTGACCCAACATACAAAAGCAACAGGTTTGGTTCTACC 2.04E+08 2.04E+08 2.04E+ eg 13094252 C8orf56 8 1.04E+08 ATCCATTGGCCACCTGGTGCCCCTCTCTCCCTGGATTTATACACAAGGCG 1.04E+08 1.04E+08 1.04E+
cg16314639 C8orf56 8 1.04E+08 TGGTGCCCCTCTCTCCCTGGATTTATACACAAGGCGCCTGCCTGGGATCG 1.04E+08 1.04E+08 1.04E+ cg23816247 BAALC 8 1 04E+08 CGCGGGCTTCAAAGACCAGAATCCCAGTTTAGGTCAAAATAAATGCACCC 1.04E+08 1.04E+08 1.04E+ cg23001725 VPS 16 20 2844678 CGGAGTAAGAGACACCAGGCGTGTCCCCCAGCTTCTGGTTAATGGCTCGA 2844630 2844679 284467 cg23719322 VPS 16 20 2844709 CGGCCACAACCATAGGCTCGTGCAGCAATGTCGGAGTAAGAGACACCAGG 2844661 2844710 284470 cg04068601 VPS 16 20 2844713 CGGAGCTGGCCATCAAGGTGTGGGTGCCCAGCCCTCCACAGACACTCTGA 2844713 2844762 284471 cg02556649 TNNT3 11 945564 CGCCCACACCCGCCCACGCCTGCCCGGGCACGATGGAGACGCCCACAGAC 1945516 1945565 194556 cg02821464 TNNT3 11 1945607 GCGTGAACGCGCTCCCCAGTTGAGTGGGTGCCAGGGATTCCAGGGCAGCG 1945607 1945656 194560 cg11654118 TNNT3 11 945648 CACTCAGGACTCCTCACAGGACAGCCCTGCCTGCTAAGGTTGCGTGAACG 1945648 1945697 194564 cg01856069 7 93474069 AGAATTCTCAGTTTCGCCGGAGGTTTACTGTGTCGGTGTTCCCTTGCGCG 93474021 93474070 934740 cg21700582 7 93474119 CGCGGGATCAGGAATTTCAACCTAGAGAACCCAGCGGAACGGGAAATCAG 93474119 93474168 934741 cg01381374 7 93474158 GCGGTGACTCGCGGGATCAGGAATTTCAACCTAGAGAACCCAGCGGAACG 93474110 93474159 934741 cg24211657 TIMP2 17 76886742 CGGGAGTTTCAGAACATATGCTAAATGCGAGAGGCCCACCCGGGGTAAAT 76886694 76886743 768867 cg12125614 TI P2 17 76886835 CGGCACAGTCAGATGATCCGAGACCAGCAGCAGCAGATCTTACAGCAAAA 76886787 76886836 768868 cg04397137 TIMP2 17 76886912 TGGGGAAGTGGTGCTGAGCTCAGATCCTCTCTCCCTTTCCTGGGCTCTCG 76886864 76886913 768869 cg1 720554 6 1.69E+08 ATTACTTGTGCAAGGTAGAAC I I I I CAGAACCCCAGCGTCGGCAGTGCCG 1.69E+08 1.69E+08 1.69E+ eg 10213762 6 1.69E+08 CGGCGGACCACGGGTTTAGGCTCACCGCGCAGTCCCAATACAACTCTATT 1 69E+08 1.69E+08 1.69E+ eg 12550496 6 1.69E+08 GGCAGTCGGAAAAGCAAGTTCTGCGGCGGACCACGGGTTTAGGCTCACCG 1.69E+08 1.69E+08 1.69E+ cg2 6111 0 TICAM2 5 1.15E+08 CGGTTTGTCTGGTCTGTAAGGAGCAAACACGGTGGAATTCCTTTCCAGAT 1.15E+08 1.15E+08 1.15E+ cg08675743 TICAM2 5 1.15E+08 CGAGACTATCTTAGGGAACCAGGAGAGTCGCTGTACCTCTGAAAGATCTA 1.15E+08 1.15E+08 1.15E+ cg15395441 TICAM2 5 1.15E+08 CGGCCCTCTCACTTGACTACTGCAGCCAACCATACCCCTGATTAGGGGGT 1.15E+08 1.15E+08 1.15E+ cg00928816 ARID5B 10 63809098 CGCTGAGCCTCGCAGCTCGCATTCGGAGGGAAGCTGACATCCACACCAAG 63809098 63809147 638090 cg20746552 ARID5B 10 63809108 CGCAGCTCGCATTCGGAGGGAAGCTGACATCCACACCAAGTCGAGACTTC 63809108 63809157 638091 cg16389209 ARID5B 10 63809121 CGGAGGGAAGCTGACATCCACACCAAGTCGAGACTTCCAGGGATGTGGCC 63809121 63809170 638091 cg07520810 ARID5B 10 63809149 CGAGACTTCCAGGGATGTGGCCGGGGAGCAGTCACATGCTGTAGCTTTCA 63809149 63809198 638091 cg 6401465 ARID5B 10 63809170 CGGGGAGCAGTCACATGCTGTAGCTTTCATGAGCACAGGCATCAGTCAGG 63809170 63809219 638091 cg09523782 WIPI2 7 5272064 CCACTTGCACCTCTGCCTAATGCAGATGTTGTGTGTGCCGCACACCAGCG 5272064 5272113 527206 cg20099458 WIPI2 7 5272275 CGCCTGCGTCTTGTGGTGCAAGGCCAGAGGGCTCTCTCTAGAACCTGACC 5272275 5272324 527227 cg27021327 WIPI2 7 5272697 TTAGCCCTGACCCAGGCAAGCACAGGGCTTCCCGTGGTCTGCAGGGAACG 5272697 5272746 527269
cg04682802 BTBD3 20 1 1898478 CGGAGCACTGGCTGTCTGACTCCATCTGCAGGGCTGTAATACCTACTCTC 11898478 11898527 118984 cg05822633 BTBD3 20 11898531 CGGAGGAGAGTAGGTATTACAGCCCTGCAGATGGAGTCAGACAGCCAGTG 11898483 11898532 118985 cg01444716 BTBD3 20 11898557 CGGCTGAACAGACTCACGCAGCTCCAGCCCATCTTGCTGACCTAATTCAG 11898557 11898606 118985 cg01513913 14 1.06E+08 GACACATTCCTCAGCCATCACTAAGACCCCTGGTTTGTTCAGGCATCTCG 1.06E+08 1.06E+08 1.06E+ eg 13074055 14 1.06E+08 CGCACCCAACCTGAGTCCCA I I I I CCAAAGGCATCGGAAAATCCACAGAG 1.06E+08 1.06E+08 1.06E+ cg23594345 14 1.06E+08 GGCCGACAGTGGTCTGGCTTCTGAGGGGTCAGGCCAGAATGTGGGGTACG 1.06E+08 1.06E+08 1.06E+ cg01208318 14 1.06E+08 CGGCCTGGGAAGTCCAACTGCAAGCAGACGGCTGCTAAGTCACCCCCAGG 1.06E+08 1.06E+08 1.06E+ cg18396660 6 68599196 CGAGGTTGATTGATCAGTTAGGGTAAGCCTGTTACAATGACAGAATGTTG 68599196 68599245 685991 cg22261402 6 68599280 CGGCTAGTTCCTGTCTTAGCTGATTAGCCAACCTTGCAACATTCTGTCAT 68599232 68599281 685992 cg26410148 6 68599358 AAAGACATTCTAAGGTCCTCTGAGCAGGCTGCACCATGGTCAAGCCATCG 68599358 68599407 685993 cg07060794 IGF2BP3 7 23387365 CGCCCACTATCAGAATCGATGTCCACCGTAAAGAAAATGCGGGGGCTGCT 23387317 23387366 233873 cg03240301 IGF2BP3 7 23387383 CGAGAGCAGTAAGCGTGTATTCACAGTGGAGTCTGTAATTACCAAATGCA 23387383 23387432 233873 cg17209188 IGF2BP3 7 23387396 AGCCCTATGACTTTGCATTTGGTAATTACAGACTCCACTGTGAATACACG 23387396 23387445 233873 cg07406498 5 969899 CGTGCTTTGGTGGCTGTCGTGCTTTAACTGCCCCATTTCTGCTCTTGGGG 969851 969900 969899 cg13674369 5 969918 GCAGTTAAAGCACGACAGCCACCAAAGCACGAAAGGGGCTCTGCACATCG 969870 969919 969918 cg04131890 5 969939 CGCAGTTGAGCTCGTGGTGCCCGATGTGCAGAGCCCCTTTCGTGCTTTGG 969891 969940 969939 cg23260525 FAM160B1 10 1.17E+08 CGGGCTCAGAGCACCCCTGTTCTTAGAGGAACAATCAATGGGAAGCAAAG 1.17E+08 1.17E+08 1.17E+ cg25233709 FAM160B1 10 1.17E+08 CGGGAACTGTTGAGAGGCCTGGGTACACCTTTGCTTCCCATTGATTGTTC 1.17E+08 1.17E+08 1.17E+ cg05388821 FAM160B1 10 1.17E+08 CGGCGCCGCCCGAGGGTTCAGGTCAAGCACGAGTTTCCAAATTCATTCAA 1.17E+08 1.17E+08 1.17E+ cg24343322 RPTOR 17 78925172 CGGCCCAGACCCTCCAGTGCTTCCTTCAGAGTGGGAGCCTGTGCTGCACT 78925172 78925221 789251 cg25057221 RPTOR 17 78925232 TCCAGTGCTTCCTTCAGAGTGGGAGCCTGTGCTGCACTTCAGGGGTCTCG 78925184 78925233 789252 eg 18425700 RPTOR 17 78925278 GGCACAGCCTTCGGGGACTGGGGCCTGGAGCTCTTACGTCTGGCGAGACG 78925278 78925327 789252 cg22455527 7 73241878 CGGGCTTTGGGAAGAGGGCCAGATGCAGGCCTAGAGGTAAATTTCCTTGT 73241878 73241927 732418 cg04663842 7 73242001 GCTTTGTTGATGCCAAGAGACCGTTGGCTCAGTCCCACCCACCTGCTGCG 73242001 73242050 732420 cg128561 14 7 73242028 TGCCTTTGCTGGTG I I I 1 I CCCGCAGCAGGTGGGTGGGACTGAGCCAACG 73241980 73242029 732420 cg10073842 MAGEL2 15 23894198 1 1 1 1 GTCACTCTGGACTCCTGCCGTAGGCTGCCAACAGCCTATTTAAACG 23894198 23894247 238941 cg25 35755 MAGEL2 15 23894248 TTTAAATAGGCTGTTGGCAGCCTACGGCAGGAGTCCAGAGTGACAAAACG 23894200 23894249 238942 cg01152488 AGEL2 15 23894273 CGTCCTCATCTGCCCCCTGGTGGCACG 1 1 1 1 GTCACTCTGGACTCCTGCC 23894225 23894274 238942
cg07244268 3 1.65E+08 CCTTGTTCCACCCACAGTTGTTATAAATTTGCTGCAGCTGCTTCTTAACG 1.65E+08 1.65E+08 1.65E+ cg26066486 3 1.65E+08 CGGCTCCAAGCTCATTCTCTCCAGCAGCTGGCAACTGGCCGGTCGCTGGG 1.65E+08 1.65E+08 1.65E+ eg 16982917 3 1.65E+08 TGCCAGCTGCTGGAGAGAATGAGCTTGGAGCCGCAGGGCTTAATCCCGCG 1 65E+08 1.65E+08 1.65E+ cg20610452 2 9235898 CCCTGCCCCTGTCGGGCTGACAGCTGAGAGATCGGGAAGTGCTCCCTGCG 9235850 9235899 923589 cg24231854 2 9235924 AGAGATCGGGAAGTGCTCCCTGCGGCCGCCCAGGCCTGCTTGATATTCCG 9235876 9235925 923592 cg04575202 2 9235972 CGGAGAGGGCGGCAGCAGCGTTCAGAATCCTTAGATGAACCTGCCTGGCG 9235924 9235973 923597 cg08065963 16 8985593 CCAGGTCCCATAGTGTCTCCCTTGGGCTTTGCTCACAGCACGAGCCACCG 8985545 8985594 898559 cg05946118 16 8985638 CACCGCGCCTGGCTGCTCTTCTCA I I 1 1 GAAACAAGACTGCTTCAGCTCG 8985590 8985639 898563 cg16455376 16 8985720 CTGTGGGGTGTGGATGCCATCTGTTGACAGCATTCGGAGTCTTCACAGCG 8985720 8985769 898572 cg04444027 USP7 16 8986092 AAGACACCTTAATGTTTGTTCAAAGACAAACCCACAGCGAACGGCTCTCG 8986044 8986093 898609 cg01601926 USP7 16 8986308 CGGCCTTGCCTTCCAGCTCCGTGGCACGGTTTCCTGGTCTTTGGGCCAGT 8986260 8986309 898630 cg04945945 TMC8 17 76129402 CGGGGGCTAACAAGATCATATATGGAAAGCACTTGAGCAGAGCCTGGTGC 76129402 76129451 761294 cg05637296 TMC8 17 76129475 AAAGCACTTGAGCAGAGCCTGGTGCTCACCCGGGCTGGGTATCTTCGTCG 76129427 76129476 761294 cg26003388 TMC8 17 76129533 CGGGCTGAGGAGGTAGGCCAGGCGGATGCTGTACACGGAGCTGCTCTCCG 76129533 76129582 761295 eg 19077400 NMUR1 2 2.32E+08 GGTCTCTCGGAAGCGGCTGGACATGAGGCTATAGAGCACGGGGTTGGCCG 2.32E+08 2.32E+08 2.32E+ cg 9733463 NMUR1 2 2.32E+08 GAGCGTCGTGTCACAGTGGACAGATGGCCTGCACCTGGCCTTCCAGCACG 2.32E+08 2.32E+08 2.32E+ cg20608294 NMUR1 2 2.32E+08 GCACGTGCTGGAAGGCCAGGTGCAGGCCATCTGTCCACTGTGACACGACG 2.32E+08 2.32E+08 2.32E+ cg14522800 ZMYND15 17 4648566 AGTGCCAGCCTTCTTCACCGAGAGCAGCGAGTACAGCTGTGTGATGGACG 4648518 4648567 464856 cg23350904 ZMYND15 17 4648580 CGCCATGGTCTGGCCGTCCATCACACAGCTGTACTCGCTGCTCTCGGTGA 4648532 4648581 464858 cg24122498 ZMYND15 17 4648682 CGCAGACCCTTACCAGGACATGCAGTTGTCGGCCGCTCTGAGGCGAAAGG 4648634 4648683 464868 cg06130562 P M2 16 8942906 AGGCAGGAGTGCAGGGCAGCCTGGGCTCCCCTGGCCCCAGACTCAGGTCG 8942906 8942955 894290 cg27018309 P M2 16 8943122 CACTTTAATGTGCTACTCTTCACTTTGGAAGGTCATTACAGTATCATTCG 8943122 8943171 894312 cg02435083 16 8943436 CACAGCAGCAACCCTCCATCTCCGGTTAAAACAAGTTTGAGTAAGACTCG 8943436 8943485 894343 cg07056079 6 32765314 GCAAAGAATTCGCTCAGGA 1 1 1 1 GCCTGAAGCTGCACTTCCGGGGTCTCG 32765314 32765363 327653 cg01423251 6 32765322 TCCTGGCAGCAAAGAATTCGCTCAGGA 1 1 1 1 GCCTGAAGCTGCACTTCCG 32765322 32765371 327653 cg23090653 6 32765352 CGCTCAGGA I 1 1 I GCCTGAAGCTGCACTTCCGGGGTCTCGCTTCATCAGA 32765304 32765353 327653 cg19559587 LRP1 12 57588144 GCTGGAAGAGCACGCCCTTCACGCCCAGGCGGAAGTAGCTGCTGCAGTCG 57588144 57588193 575881 cg21913159 LRP1 12 57588171 CGTAGCAGAGTGAGGTCCGCTCGCAGGGCTGGAAGAGCACGCCCTTCACG 57588171 57588220 575881
eg 13038544 LRP1 12 57588198 TGGCGCCATCACACACCCAGCTGGGTGCGTAGCAGAGTGAGGTCCGCTCG 57588198 57588247 57588 cg21702971 LRP1 12 57588243 CGCCATCACACACCCAGCTGGGTGCGTAGCAGAGTGAGGTCCGCTCGCAG 57588195 57588244 575882 eg 15732164 CD 6 7 92237376 CGGGTATCTTCAGAACTTTAACCTAAGCACATCAACGGAA I I 1 1 I CTAGG 92237376 92237425 922373 eg 14 00946 CDK6 7 92237896 CGCCTGAGAGATGCGGGGTAGACAGCTTCACACAGGGCAGCTGCTACCGC 92237896 92237945 922378 cg06688763 CDK6 7 92238207 CGCGCCCGGAAGGCTTTCAAGTGGGAATCAAG 1 1 1 1 GAGCACGAGCAAAT 92238207 92238256 922382 eg 16784468 PWWP2B 10 1.34E+08 CGGAGCCCCAGGTGTCAGCCCTGCTTCCATCGCCCCCCACCTGAGCCCAC 1.34E+08 1.34E+08 1.34E+ cg06427772 PWWP2B 10 1.34E+08 CGCAGCCGGCCAGAAGGCTCACAGGGTCCCCGAACAGAGATCGGAAGCTC 1.34E+08 1.34E+08 1.34E+ cg11229101 PWWP2B 10 1.34E+08 AGGCCTGGTTCCTCCATGGGACCCCAGCATGCGGGCAGCAGGTGAGGCCG 1.34E+08 1.34E+08 1.34E+ cg03860038 10 1.34E+08 TGTCCCTGTCGGGGCTGACGGGGCCTGAGTCCCCTGGGCACTGCTGGGCG 1.34E+08 1.34E+08 1.34E+ cg18420781 TRIM10 6 30124240 CCTTCTCACTTTCTTTCTGTTAGTCTGGCTCAGAGTTAGACGGTCCTACG 30124240 30124289 301242 cg25981998 TRIM10 6 30124248 CCCTCCTTCCTTCTCACTTTCTTTCTGTTAGTCTGGCTCAGAGTTAGACG 30124248 30124297 301242 cg11079936 TRIM10 6 30124299 AATTAATAGCCAACAACAGTATTTATCTTGACCCTGTGGTATCTGGCTCG 30124299 30124348 301242 cg22118082 TRIM10 6 30124647 CCACAGAGCGCAGGTGGCCATGAAGCCTGGGTTTAGGAGTGGCCTCCACG 30124647 30124696 301246 cg12251145 TRIM10 6 30124744 GAGGCCTGGGGTTCTCTTACCCAGAAACATCTTCATCTCCCTCTGCAGCG 30124696 30124745 301247 eg 00550080 TRIM10 6 30124792 GTGCCCCTAGATGTGAAACCAGAAAGTGCCGGAAACCGGTGGCTGTGTCG 30124792 30124841 301247 cg15768138 CXCR1 2 2.19E+08 CTGGAGAGTCCTGGAGCTTTGAGGGGCTCTTTAGGGGCCATCTGGGTTCG 2.19E+08 2.19E+08 2.19E+ cg15908708 CXCR1 2 2. 9E+08 CGGTGACTTTCCAAAGGCTTTCAGACTTCTCTGGAGAGTCCTGGAGCTTT 2.19E+08 2.19E+08 2.19E+ cg13519373 CXCR1 2 2.19E+08 CCTAGCATCCAGTCTTCATTCCATCCTGATTCTACCATTAATAAGTTTCG 2.19E+08 2.19E+08 2.19E+ cg21330949 12 19557320 CGCCCTAAAAGAGCTGGGCAACCCAGGCCACAGGGGAAGGCCTTAACCCT 19557272 19557321 195573 cg20894640 12 19557334 CCCCTGTGGCCTGGGTTGCCCAGCTC 1 1 1 1 AGGGCGTGTGTGGCATGGCG 19557286 19557335 195573 cg14287022 12 19557343 CGCGATGGCCGCCATGCCACACACGCCCTAAAAGAGCTGGGCAACCCAGG 19557295 19557344 195573 cg02627403 UNC13D 17 73823769 AGACAGATGGCCCAATCCCCTGCCCACCACAGCAGC 1 1 1 1 CTGAGAGGCG 73823721 73823770 738237 cg12407791 UNCI 3D 17 73824354 GCCCCTCGCTGCAGTCAGGACAGTGTCCCAGCAGCACTCTGTCTGTTACG 73824354 73824403 738243 cg07010633 UNC13D 17 73824396 CGCTGCAGTCAGGACAGTGTCCCAGCAGCACTCTGTCTGTTACGCGACCC 73824348 73824397 738243 cg14242936 GRASP 12 52404134 TCCCCCAGCAGGCGAGGATCTGGAGCAGCTCCAGACCATTGTGTCCAGCG 52404086 52404135 524041 cg19374752 GRASP 12 52404151 ATCTGGAGCAGCTCCAGACCATTGTGTCCAGCGGGCAGGCCTTCAGTGCG 52404103 52404152 524041 cg06611426 GRASP 12 52404161 CGCCCCTTCCCGCACTGAAGGCCTGCCCGCTGGACACAATGGTCTGGAGC 52404113 52404162 524041 cg20327845 PFKP 10 3138418 CGCGTGCTCCAGGAATACTCTGAAAGCTGATGCCGTTAGGGGTGCATGGT 3138418 3138467 313841
cg26644674 PFKP 10 3138505 GGGTGCATGGTTGACTCCTGGGCCTGAGACGGCCTCAGGATGCTGCAGCG 3138457 3138506 313850 cg04188920 PFKP 10 3138534 CGGCCTCAGGATGCTGCAGCGGCCTTGGCATCCTCTTCAGCAGGGTGGCG 3138486 3138535 313853 cg19177307 11 2021658 CGGGCGGTGAGACTGAAGGGGAAGCCTCCAGAAATACACATGTGCTATGC 2021658 2021707 202165 cg16574793 11 2022324 TTGCCTGCAGAAACATCCCGGGTCAACAGGCCAGGCACCGCATTGGTTCG 2022324 2022373 202232 cg09452478 11 2022386 GCCTGGCCTGTTGACCCGGGATGTTTCTGCAGGCAAACCACAGGTAGCCG 2022338 2022387 202238 cg02084087 TNFRSF25 1 6526049 ATCGGAAAGGGCGGCCAACCCAGCCCCCAGTGAGGCTTGGAGTGGAGACG 6526001 6526050 652604 eg 15205428 TNFRSF25 1 6526051 CGGAAAGGGCGGCCAACCCAGCCCCCAGTGAGGCTTGGAGTGGAGACGCG 6526003 6526052 652605 cg11756870 TNFRSF25 1 6526073 TGAGACGCAGGAGGCTAGGGGCAGCTTTGAGCCCTGTCCCAAGAGCCTCG 6526073 6526122 652607 cg11592082 AP3K6 1 27683345 CGCCTGGGGCCTGCCCAGAGCCGGGCACCCTACCCCCGCACTTTGGGGTC 27683297 27683346 276833 cg09230763 MAP3K6 1 27683351 CGGCACCGCCTGGGGCCTGCCCAGAGCCGGGCACCCTACCCCCGCACTTT 27683303 27683352 276833 cg11705208 MAP3K6 1 27683386 CGGCGCCTGCCTGCCCCTGTCCCGCCTGGCCCCCACCGGCCCCATTCGAT 27683386 27683435 276833 cg18377014 C7orf50 7 1095595 GGGGTGACGTCCCCAAACTGGGCTGCCACTCTGTGACAGCCAAAGCCACG 1095547 1095596 109559 cg18315380 C7orf50 7 1095666 CGCCAGGGTCCAGAGGACGGGGTTCTTCAGGATCGTGGTCCTCCACACCC 1095618 1095667 109566 eg 19340455 C7orf50 7 1095720 GGGCCAGCACAAGCTCCAGACAGGATGCACCTGCTCCAAGCACAGTGGCG 1095720 1095769 109572 cg10418114 PLEKHG1 6 1.51 E+08 CGCTTCATGTCATTGATATGCCAGGCGACTCGCTGCATTGTGTCTATAGC 1.51 E+08 1.51 E+08 1.51 E+ cg26700102 PLE HG1 6 1.51 E+08 CGGACCGCGTGCTCGTGTTTCCGCTTCATGTCATTGATATGCCAGGCGAC 1.51 E+08 1.51 E+08 1.51 E+ cg21668166 PLEKHG1 6 1.51 E+08 CGGGCTCACCTGTAACCGGACCGCGTGCTCGTGTTTCCGCTTCATGTCAT 1.51 E+08 1.51 E+08 1.51 E+ cg17627829 HCG18 6 30292258 CGGAGTCGGCAAACTACGGTCTACTATCTGTCTTCGTATACGTCAAAAGC 30292258 30292307 302922 cg01842890 HCG18 6 30292264 CTATCGCTTGAAAAATACCGGATATTTCATTATTATCCTGTCCGGAGTCG 30292216 30292265 302922 cg04651621 HCG18 6 30292292 CGAAGACAGATAGTAGACCGTAGTTTGCCGACTCCGGACAGGATAATAAT 30292244 30292293 302922 eg 04451770 ENTPD1 10 97515372 CGCAGCGTCTCCTGCAGGAAACTCATCGCTTGGGTCACCTGTTGCTCTTT 97515372 97515421 975153 eg 12497543 ENTPD1 10 97515377 AGAGCAAAGAGCAACAGGTGACCCAAGCGATGAGTTTCCTGCAGGAGACG 97515377 97515426 975153 cg10381771 ENTPD1 10 97515398 CGCTTGGGTCACCTGTTGCTCTTTGCTCTAATGAGCCTTGAGAAAGGATT 97515398 97515447 975153 eg 16062483 C14orf64 14 98444417 GGCGAGAGCCGGAGGGGCTGCACTGCACCGAGCTTGCACAGGGCGAGGCG 98444417 98444466 984444 eg 16278496 C14orf64 14 98444476 CTTGAGCCAAGCGTTTAGTCCTCAGCTGAACTGCTGCCAGCCTGCGCACG 98444476 98444525 984444 cg11798182 C14orf64 14 98444513 CGCTTGGCTCAAGTTCAACACGGCTGCCAAGATTTGGCAACCAGAGAGTT 98444513 98444562 984445 cg25472539 TNXB 6 32037978 CGGCCGGCATCCAGAGGGGTGACAGTGACAGAGCGCTCATGGCCCTCCAC 32037978 32038027 320379 eg 16318036 TNXB 6 32038045 TCCTGAGGGCCAGTTCGACTC I I I I GTGGTCCAGTTCAAGGACAAAGACG 32038045 32038094 320380
cg23760574 TNXB 6 32038097 CGGTTCCTGAGGGCCAGTTCGACTC I I I TGTGGTCCAGTTCAAGGACAAA 32038049 32038098 320380 eg 11579421 PWWP2B 10 1.34E+08 CGGGCAGGAACCACACGGGAGCCATATGTTAAATCACGTGACTTGTGGAT 1.34E+08 1.34E+08 1.34E+ cg25303150 PWWP2B 10 1.34E+08 TTTAACATATGGCTCCCGTGTGGTTCCTGCCCGCTCCGTGTGGGCTACCG 1.34E+08 1.34E+08 1.34E+ cg24085039 PWWP2B 10 1.34E+08 TCCGTGTGGGCTACCGCTGGGAGTGCACGGGCAGGATGGAGGGACACTCG 1.34E+08 1.34E+08 1.34E+ cg04816394 RNU5E 5 80529067 CCTGCCTTCAAGGGTACAGCTGTGGGGGCCGGTGCGCCCGGAGGTCTACG 80529019 80529068 805290 eg 12827530 RNU5E 5 80529121 CGGCCCGACCAGCTCGCCCTGCATACACTTCTTGGCTGTGTGCGCTCAGC 80529121 80529170 805291 cg23425533 RNU5E 5 80529187 CTGCATACACTTCTTGGCTGTGTGCGCTCAGCAGGACGTGGGAGGCTCCG 80529139 80529188 805291 cg25042710 7 175423 CGGGAAAAATGAGAGATGCCGAGAC I I I I GCCAATGCAAACCTCTCTGCA 175375 175424 175423 cg18990588 7 175713 CGTGAAGAAACAGACTAAAGGACTTTGTTGCAAGCTACAGTCATGAAGAA 175713 175762 175713 eg 19894980 7 175967 AGGAACCATGATGCAGATCTTCTGCATACCCATAAGCTAAGCGGGCAGCG 175967 176016 175967 cg00615377 RBM9 22 36236649 TCTCCCAACTCCAGAAGCAGGCCTGCTGGAGAATGGCAGTTTGGTGTACG 36236601 36236650 362366 cg26196531 RB 9 22 36236656 ACTCCAGAAGCAGGCCTGCTGGAGAATGGCAGTTTGGTGTACGCTGTGCG 36236608 36236657 362366 cg15352367 RBM9 22 36236732 CGCCATGTGCTGGCATA I I I I AAGGATGCGATGATGGAATTAATTATATA 36236732 36236781 362367 cg08839210 RAB34 17 27045048 GGGAGAGCAGCCTGGGGAGTCCCTATCTGGATAGGCTCCAGCCTGGGTCG 27045048 27045097 270450 cg03452174 RAB34 17 27045113 CGGCTGCGCCTTCATGGGGACCCTTACCCAGCCGAATTGGTGATGGGGAA 27045113 27045162 270451 cg08032476 RAB34 17 27045176 CGCAGCAGTCTCCGATTCCCCATCACCAATTCGGCTGGGTAAGGGTCCCC 27045128 27045177 270451
EXAMPLES
Example 1: Experimental data Epigenetic Changes in Crohn's Disease
Summary Methods
The lllumina 450K platform was applied to assess epigenome-wide methylation profiles in circulating leucocyte DNA in newly diagnosed and established paediatric CD cohorts. Data were corrected for differential neutrophil/mononuclear cell counts. Targeted replication was performed in an adult CD cohort using pyrosequencing. Methylation changes were correlated with gene expression in blood and intestinal mucosa. Results
We identified 165 individual CpG sites with methylation alterations achieving epigenome- wide significance after Bonferroni correction (P<1.1x107); 138 differently methylated regions (DMR) of unidirectional methylation change, as well as highly significant enrichment of methylation changes around GWAS SNPs (PO.0001). Two-locus discriminant analysis in the discovery cohort predicted disease in the paediatric replication cohort with presence 94% sensitivity, and 100% specificity.
The outstanding discovery implicates the MIR1/VMP1 locus - specifically a DMR containing the most significant individual probes (P=1.2x10"14) only 47kb from GWAS SNP rs1292053. We confirm hypomethylation of MIR21 in adults (P=6.6x10"5, n=172), and show increased mRNA expression in leucocytes (P<0.005, n=66) and in the inflamed intestine (P=1.4x10"6, n=99).
Conclusion
We demonstrate highly significant and replicable differences in DNA methylation in CD. The data strongly implicate known GWAS loci, with compelling evidence implicating MIR21. The predictive accuracy of discriminant analysis provides a potential avenue for clinical translation. introduction
The last decade has seen tremendous success in identifying genetic loci associated with inherited susceptibility to CD, with 140 loci identified in the most recent GWAS meta- analysis.1 However, these determinants collectively explain only 13.6%1 of disease variability, and the biological variation each confers is unclear. The importance of non- inherited factors in pathogenesis has been highlighted by studies on the increasing incidence of CD, especially in children2 3 and in the developing world,4 5 and by a greater understanding of the effects of gut microbiota and diet on risk.6 7 A critical objective for CD research is to characterise the interaction between genetic and environmental factors.
Epigenetic alteration has emerged as a potential mechanism through which these interactions may occur.8 Developments allowing rapid assaying of cytosine methylation at nearly 5*106 positions and insights into confounding effects in study design9 have provided the impetus to build upon promising pilot data from previous generation technology to allow epigenome-wide association studies (EWAS) to become a valuable complement to the more mature GWAS.10 Epigenome mapping has been used in the study of diseases such as rheumatoid arthritis and type 2 diabetes.11-13
Whilst the relationship between methylation and gene expression and function is incompletely understood, relevant modifying influences include age, ethnicity, smoking, gut microbiota and diet.14-17 DNA-binding factors can directly influence methylation, and in turn, altering methylation can directly influence expression.18 19 We hypothesised that identification of altered levels of methylation which are significantly associated with disease state, whether predating or following disease, offers the potential for discovering new pathways integral to the disease process and for predicting disease status.
Materials And Methods
Study design
lllumina 450k DNA methylation analysis was initially performed in a paediatric discovery cohort (stage 1) and then later in a paediatric replication cohort (stage 2). The discovery cohort included newly diagnosed cases of paediatric CD and the replication cohort established cases of paediatric CD. Controls for both cohorts were drawn from children who underwent colonoscopy for gastrointestinal symptoms, who had no pathology.
The final results are derived from a single joint analysis of these two paediatric cohorts.9 Three tiers of statistical correction for multiple testing were applied to these results to minimise the risk of false discoveries - these involved calculation of Benjamini-Hochberg false discovery rate (FDR)20; the more stringent Bonferroni correction for reporting highly significant individual probes, with further validation of the latter by permutation analysis. Linear discriminant analysis was applied to data generated in the discovery cohort to identify biomarker candidates which were then confirmed in the replication cohort.
The most significant CpG sites implicated by the combined paediatric analysis were assessed by pyrosequencing in an adult cohort with established CD, and disease- associated expression changes were explored (Figure 1A).
Paediatric Patient Selection and Ethics
Paediatric samples were collected from centres across Scotland. The Bacteria in Inflammatory bowel disease in Scottish Children Undergoing Investigation before Treatment (BISCUIT) study provided peripheral blood leucocyte DNA for the discovery cohort from 18 treatment naive, newly diagnosed patients and matched non-diseased controls from Aberdeen, Glasgow and Dundee. Controls had been rigorously investigated for gastrointestinal symptoms but did not have or subsequently develop any organic gastrointestinal pathology including IBD.21 The replication cohort comprised DNA samples from children with established CD supplied by the Paediatric-onset Inflammatory bowel disease Cohort and Treatment Study (PICTS),22 analysed against a second set of controls from the BISCUIT study. Within both cohorts patients and controls were matched for age and gender. The BISCUIT study was approved by the North of Scotland Research Ethics Committee (09/S0802/24) and PICTS by ethics committees at participating centres (Edinburgh, Glasgow, Aberdeen and Dundee - LREC 2002/6/18). Written informed consent was obtained from the parents of all participating children. Informed assent was also obtained from older children capable of understanding the nature of the study. Genome-Wide Methylation Profiling
Peripheral blood leucocyte DNA was bisulphite converted and analysed using the lllumina Human Methylation 450k platform (lllumina, San Diego, USA)23 with cases and controls distributed across chips. Probes were filtered to remove any with a detection P value of ≥0.01 , those from sex chromosomes and those that had single nucleotide polymorphisms (SNPs) with a minor allele frequency of >0.01 in the European population in the 1000 Genomes Project.24 Samples were removed if there was a gender mismatch or if more than 5% of probes failed. In the first cohort all samples passed QC, but 1 case and 4 controls were excluded from the final analysis as they lacked contemporaneous full blood count data. In the second cohort, one sample failed QC due to gender mismatch.
Data were corrected using background removal and quantile normalisation in the lumi R package25 26 followed by beta mixture quantile dilation (BMIQ).27 Batch effects were controlled for using ComBAT.28 Analysis of the methylation status of cases vs. controls was performed using M values (log2 [proportion of methylated probes/proportion of unmethylated probes]) and linear modelling in limma.29 In order to adjust for the differential white cell count, the proportion of neutrophils on a full blood count taken at a time near the DNA sample was used as a covariate in the linear models.
We used the widely accepted Bonferroni correction to define epigenome-wide significance for individual CpG sites. To validate this approach we performed a permutation test30 (n=1000), repeating the combined paediatric analysis on the post-QC data with the diagnosis for each sample permuted randomly but retaining the correct numbers of cases and controls. The smallest P value from each permutation was recorded, with the 5th percentile equalling 2.00x10"7, marginally less conservative than the Bonferroni correction (equivalent to P<1.1x10-7). Differently Methylated Regions
DMRs were defined using a modification of the lasso technique employed by the ChAMP pipeline (Morris ef al. (2014), Bioinformatics 30(3): 428-430 and at: http://www2.cancer.ucl.ac.uk/medicalgenomics/champ/), which defines a DMR as a genetic region (size defined by the probe density at that location within the genome) of 3 or more probes, all with FDR corrected P<0.05. We added the constraint that all probes should share the same direction of change in methylation.
Replication of Methylation Findings in Adults
20 whole-blood DNA samples from adults with CD were recruited from gastroenterology clinic while their disease was quiescent, with 20 healthy controls collected during the same time period (LREC 2000/4/192). All adult pyrosequencing replication was performed on this cohort of 40, apart from VMP1/MIR21 (Figure 5A) which was tested in an extended replication cohort of 87 adults with CD and 85 healthy controls, of which the smaller group was a subset.
DNA was bisulphite converted with EZ-96 DNA Methylation™ Kits (Zymo Research, Irvine, USA), assays were designed using PyroMark Assay Design Software V2.0.1.15 (Qiagen, Dusseldorf, Germany) and primers were ordered from Sigma-Aldrich (St. Louis, USA). Sequencing was performed on a Pyromark Q96 ID machine (Qiagen) and analysed in R V3.0.1. VMP1 and MIR21 expression
MIR21 primary transcript (pri-mir21 ) was assayed by qPCR (Figure 5B). Sample collection was performed using SAHSC BioResource ethical approval (REC Reference 06/S1101/16), with all patients and controls giving written, informed consent. Suitable CD patients were prospectively recruited from gastroenterology clinic and endoscopy lists, and healthy controls recruited from volunteers. Blood samples were taken using a 21 gauge butterfly needle and 9ml K3 EDTA vacuette® (Greiner, Germany) and stored at 4°C for up to 2 hours. Total RNA was then extracted from 1.5ml whole blood using QIAamp RNA blood mini kit (Qiagen), and stored at -80°c. cDNA was converted using Superscript® Vilo™ cDNA synthesis kits (Invitrogen, Carlsbad, USA) and analysed on a Corbett Rotor- Gene 6000 (Qiagen) with DyNAmo Flash SYBR green reagent (Thermo Scientific, Waltham, USA). Expression of pri-miR21 was normalised to reference gene TBP) after initial optimization against 4 reference genes {GAPDH, TBP, SDHA and ACTB), and analysed by the AACt method in R. Expression in intestinal biopsies as described previously.31
Pathway analysis
Analysis identified pathways from the IPA (Ingenuity Systems, www.ingenuity.com) library of canonical pathways that were most significant to the dataset. Methylation sites that met the P-value cut-off of 0.001 and were associated with a canonical pathway in the Ingenuity Knowledge Base were considered for the analysis. Fisher's exact test was used to calculate a P-value determining the probability that the association between the genes in the dataset and the canonical pathway was explained by chance alone.
Linear discriminant analysis
Models were derived using the paediatric discovery cohort and tested on the paediatric replication cohort. Probes were selected based on significance in the discovery cohort (Table 1 ) not the combined analysis. Linear discriminant analysis was performed on M values (post-QC and normalisation for neutrophil : lymphocyte ratio) in R 3.0.1 , using the Ida function in the 'MASS' package.32
GWAS colocalization
VMP1 has five highly significant probes within 50kb of GWAS SNP rs1292053 and accounts for a large proportion of all probes at low P value thresholds, resulting in very high percentages of colocalization at low P values (50% for P values <1x10 12 within 50kb), we therefore only show P values up to 10"7 in figure 1 C to not overshadow the effect. Exclusion of VMP1 from analysis (by using a distance threshold of 45kb) preserves the trend seen in figure 1C. Due to intrinsic design factors in methylation and genotyping platforms there is a positive correlation between density of methylation probes and distance to GWAS SNPs (not shown). To help control for these effects probes were grouped into 1kb bins and considered by the lowest P value they contain.
Results
Paediatric lllumina 450k
A preliminary analysis of the discovery cohort was performed identifying 16 probes with FDR corrected P<0.05, 14 of which achieved nominal significance in the replication cohort to go forward as candidate biomarkers, including VMP1, TOLLIP and SEPT9 (Figure 1 B&C, Table 1). In the replication cohort there were a greater number of significant results, possibly due to larger group size and epigenetic effects of longer disease duration. Correlation between cohorts was high, with 83.3% of probes reaching nominal significance in the discovery cohort showing the same direction of change in the replication cohort (Figure 1 C). Final analysis was based on data from the two paediatric cohorts combined9 which, after Bonferroni correction for multiple testing, identified 165 CpGs with epigenome-wide significance. At these probes we observed absolute differences in mean methylation between CD and control groups of up to 16% (mean 6%), with 40% also showing a difference in variance (P<0.05, with the larger variance being in CD at 83% of these). 30,550 CpGs were significant at a FDR <0.05, which were used for DMR analysis.
Defining Differentially Methylated Regions
138 DMRs were identified - regions where all probes survived FDR correction and shared a single direction of change. By best or combined probe significance, the most significant DMR was VMP1/MIR21 with all probes surviving Bonferroni correction; this DMR was also the closest to a GWAS SNP (47kb). Five regions are noteworthy for the presence of multiple DMRs (Table 2B); the highest density was a 15kb sequence centred around TNF containing 3 DMRs. The HLA region of chromosome 6 contained 18 of the 138 identified DMRs. Colocalization with GWAS
Significant colocalization of methylation changes with GWAS SNPs was found by Chi- square test across a range of distance thresholds in line with previously published work,33 P<0.0001 for Bonferroni significant probes within 50kb of GWAS SNPs (Figure 1 D). This relationship remained significant if VMP1/MIR21 was excluded from analysis.
Ingenuity Pathway Analysis
Ingenuity pathway analysis of the combined dataset demonstrated enrichment of methylation changes in host defence pathways involved in NK cell signalling, NF B activation by viruses and dysregulation of macrophages, fibroblasts and endothelial cells in rheumatoid arthritis.
Deriving Biomarkers with Linear Discriminant Analysis
Candidate biomarkers were identified from a preliminary analysis of the replication cohort. Using the discovery cohort as a learning set, multiple models were constructed to predict the presence of CD based on age, sex and methylation of two CpG sites using linear discriminant analysis. Mean sensitivities and specificities of all possible models were 95% and 96% respectively, with 19% of models achieving 100% sensitivity and specificity.
In testing the predictive accuracy in the paediatric replication cohort, the best model, based on methylation of RPS6KA2 and TOLLIP maintained 94% sensitivity and 100% (Figure 2A). This model was not an outlier, with many others showing high predictive accuracy and validating this approach (Figure 2A).
Adult replication with pyrosequencing
Pyrosequencing assays were designed for a selection of significant probes surviving Bonferroni correction in the combined paediatric data. Methylation changes were assayed in a group of 20 adults with CD and 20 controls in multiple CpGs across 9 genes with resultant P values between 0.004 and 2x10"5 (Figure 2B). As with the paediatric data, the commonest finding was of hypomethylation with increased variance, and combining methylation results at two probes achieved strong separation between control and disease groups. The only tested gene where a significant finding in children did not replicate in the adult cohort was TOLLIP. Given the significance of differences found in multiple probes in both paediatric cohorts and its function as an inhibitory adaptor for Toll-like receptors, this gene may represent an interesting target for further investigation as a paediatric specific marker, or provide insight into biological differences between adult-onset and paediatric CD.
Interpretation and target selection
To highlight genes for further study we considered three criteria - the significance of individual CpGs, clustering of CpGs into DMRs, and colocalization of methylation changes with risk loci identified by GWAS. Genes which scored highly in multiple categories were given the highest priority for further investigation (Figure 3), with VMP1/MIR21 emerging as the strongest candidate.
VMP1/MIR21
Five probes within VMP1 reached the highest significance level, 4 of which lie within a DMR (Figure 4). These probes are clustered at the 3' end of VMP1, around the 1 1th exon, within 50kb of a GWAS SNP (rs1292053). The DMR is directly adjacent to the transcription start site and promoter region for the primary transcript of MIR21 (pri-miR21 ).
We confirmed CD-associated hypomethylation of this region in blood by pyrosequencing in 172 adults (P=6.6*10-5, Figure 5A). qPCR for pri-miR21 in 43 adults with CD and 23 healthy controls demonstrated an increase in expression in CD (P<0.005, Figure 5B). Analysis of previously published data31 demonstrated altered expression of MIR21 (P=1.4x10~6) and VMP1 (P=2.6x10~3) in biopsies from inflamed versus uninflamed mucosa in CD but not controls (Figure 5C).
Discussion
Principal findings
This study establishes a significant and highly replicable pattern of DNA methylation associated with paediatric CD, with further replication in adults for many of the most significant (p<10"1°) paediatric results. We show a significant enrichment of methylation changes in proximity to GWAS risk loci, offering a novel approach to exploring the biological variation associated with common genetic variants. As such, this study provides an important confirmation of the validity and feasibility of methylation screening in complex disease, and complements the emerging evidence implicating epigenetic alterations in inflammatory bowel disease and other complex immune-mediated diseases. We have derived biomarkers which show remarkable accuracy in determining the presence of Crohn's disease. The magnitude and variance of methylation changes in whole blood may inform future study design in CD and other complex diseases. The discovery of epigenetic alterations within the VMP1/MIR21 locus emerges as the strongest individual result. Further confirmation of altered MIR21 methylation in CD by pyrosequencing in adults is augmented with data showing increased expression of MIR21 in blood in CD and increased expression of MIR21 in inflamed intestinal biopsies in CD but not controls.
VMP1 and MIR21
VMP1 encodes a transmembrane protein located in the Golgi apparatus, endoplasmic reticulum and vacuoles with high degrees of expression in the intestine, kidney, ovary and placenta.34 There is high trans-species conservation of VMP1, and it is noteworthy that expression induces autophagy through interactions with BECN1.Z6 m
MIR21 was one of the earliest described microRNAs and has been implicated in numerous cancers, including IBD-associated colorectal cancer.37 The mature sequence is produced from a precursor overlapping with the 3' end of VMP1. This region is highly conserved, exhibits DNAse I hypersensitivity and is associated with the promoter associated histone marks H3K4Me1 and H3K4Me3.38
MIR21 has a known role in T-cell differentiation and development.39-42 Increased expression of MIR21 in active IBD and IBD-associated dysplasia has been described elsewhere43 44 and MIR21 knock-out mice have been shown to be protected from DSS- induced colitis.45
There is a growing body of evidence for numerous microRNAs being involved in CD such as the regulation of NOD2 by microRNAs4847 and NOD2 genotype influencing IL-23 production in dendritic cells by regulation of MIR29 production48. Recent work has shown ATG16L1 can be regulated by multiple microRNAs, with resulting effects on autophagy49 50 - particularly interesting with respect to our data as ATG16L1 contains an MIR21 target motif.51 Epigenome-wide significance
The Bonferroni correction for multiple testing is overly conservative, as it ignores the correlation of methylation between neighbouring probes. The result of our permutation test, that if methylation and disease state are unrelated the chance of finding at least one result stronger than P=2x10"7 is 5%, is slightly less conservative, but we utilize the Bonferroni correction as there is not yet a consensus on the limit of epigenome-wide significance for DNA methylation arrays. The establishment of such a consensus should be a priority for future work. The Benjamini-Hochberg FDR of 5% for inclusion of probes in analysis for wider patterns beyond individual CpG significance is necessary to ensure sufficient data is included - though it may be considered insufficiently discriminating for reporting individual CpGs. Linear Discriminant Analysis
The methylation states of specific DNA sequences have found use as biomarkers, with some notable successes in cancer,52,53 based on sensitively detecting abnormal methylation states, such as methylation at a tumour suppressor CpG Island. The results of our linear discriminant analysis of the methylation results serve as a proof of concept for the development of diagnostic biomarkers in complex diseases. Future work should also seek to establish links with other clinical outcomes such as response to treatment and disease course.
The mean sensitivities of all possible two-probe combinations in the discovery (95% and 96%) and replication (78% and 83%) paediatric cohorts strongly refutes the possibility of the best model being a statistical aberration. The use of children who required colonoscopy to rule out inflammatory bowel disease as controls precisely models the clinical scenario in which a diagnostic biomarker would find use. CD-specific methylation patterns weaken with increased age and were absent in the elderly (data not shown), possibly due to the accumulation of confounding factors such as environmental exposure, comorbidity and polypharmacy, or inherent effects of aging on methylation.17
Whilst the differences in DNA methylation are the composite effects of several factors including shifts in immune cell populations, environmental exposures, variations in the microbiome and altered expression linked to genetic variation, they are eminently suitable as biomarkers. In addition, DNA methylation is chemically stable, amplifiable and can be tested cheaply and easily.
Example 3 outlines an exemplary method for performing Linear Discriminant Analysis.
Figure 6 provides experimental data showing M values for preferred models of the invention. Strengths and limitations
The present study provides impetus for further analysis of alterations of leucocyte DNA methylation in IBD and other complex diseases, with many targets emerging for further study. In comparison with GWAS data, we show highly reproducible and significant disease-associated methylation changes using a modest number of samples. Indeed, the strength and reproducibility of our findings compare favourably with epigenetic data generated to date in IBD and other complex diseases10,12 and also with the results of theoretical modelling based on predicted disease-associated methylation patterns. In particular the magnitude and variance of observed methylation changes in whole blood contrasts with models used to predict required group sizes.9 These data may inform future study design in CD and other complex diseases. The enrichment of methylation differences near to previously described risk loci from GWAS raises the possibility that epigenetic modifications may help identify specific points within large susceptibility loci where genetic and biological variation overlap.
Whilst the combined factors of moderate study size and conservative correction for multiple testing may well contribute to false negatives (type II error), the reproducibility in independent cohorts and level of statistical significance provide confidence in our findings. The emerging evidence of a role for MIR21 in IBD from other approaches enhances the biological plausibility of this finding and strengthens the case for using EWAS in CD and other complex diseases.
Parallels may be drawn with the early linkage and association studies in IBD, which allowed modelling of the genetic architecture, and delivered 'low-hanging fruit' in terms of NOD2 and HLA associations,54-56 findings that have subsequently been unequivocally replicated in large scale experiments.
Study design
Studies in children have the advantage of reducing the influence of age, comorbidity, polypharmacy, smoking, and environmental factors which could confound epigenetic changes. The focus on circulating leucocytes in IBD is strongly supported by scientific evidence of immune dysregulation, the well-recognised clinical extra-intestinal manifestations, and the recent evidence of an encouraging response to autologous bone marrow transplant in refractory disease.57 Methylation at numerous sites has also been shown to influence PBMC response to stimulation of Toll-like receptors ex-vivo with multiple ligands.15 Ease of access to blood is clearly advantageous in biomarker discovery. In the epigenome-wide analysis we chose to use contemporaneous clinical full blood count data to correct for the proportion of neutrophils within the whole blood, rather than cell separation. This strategy is feasible for transiational studies, especially high throughput clinical investigations. Estimates of leucocyte subpopulations were made by comparing methylation to a reference dataset with available flow cytometry data;5859 and we were able to validate this approach in a comparison of the estimated cell population with differential cell count.
Conclusion
Overall these observations serve to highlight the need to integrate methylation, genetic and expression data in future studies of the pathogenesis of complex diseases, and provide insight into potential mechanisms involved in gene-environmental interaction. There are exciting and immediate implications for early clinical translation; the discovery of easily accessible biomarkers in peripheral blood to predict disease susceptibility, progression or response to therapy and the potential for new therapeutic targets.
Future studies should evaluate altered methylation and expression at these sites, including MIR21 , both in whole blood and specific cell types, prior to initiation of disease and in association with environmental factors to better understand causality.
Table 1. Illumina 450k probes with significant (FDR adjusted P<0.05) methylation differences in the discovery cohort which replicated in the second cohort (P<0.05).
Cohort 1 Cohort 2 Combined
Chr:Coord LogFC Bonferroni
Gene P value LogFC P value mdB LogFC P value
(GRCh37) corrected
17:5791571
VMP1 -1.54 2.3x10'8 -1.20 1.4x10"7 -0.14 -1.33 1.2x 0 4 5.5x109
7
6:16697025
RPS6KA2 -1.30 1.4x10'7 -0.84 4.6x10"8 -0.10 -1.04 5.5x 0-1" 2.4x10-8
2
- 3:10190123
-1.32 2.4x10"3 -0.85 3.3x 0"7 -0.14 -1.03 7.3x 0-1" 3.3x10-a 4
17:5791566
VMP1 -1.39 1.4x10-7 -1.05 9.6x10-7 -0.16 -1.18 6.4x10- 3 2.9x10
5
ARHGEF3 3:57041402 -0.74 6.5x10-7 -0.48 3.1x10"7 -0.07 -0.56 3.7x10-12 1.7Χ10-6
SLC10A6 4:87752504 -1.09 1.9x10'7 -0.74 1.9x10-" -0.11 -0.87 4,4x10" 0 2.0x10""
22:5032798
- -0.99 1.5X10-6 -0.62 9.0x10"5 -0.09 -0.76 7.3x10-10 3.3x1 O^
6
15:5958862
MY01 E -0.99 4.5x10'7 -0.46 1.1 x10-" -0.04 -0.66 9.2x10-10 4.1 x10""
2
TOLLIP 11 :1297087 0.44 6.4x108 0.32 2.8x10"5 0.05 0.34 9.7x10" 0 4.3x10""
HEATR2 7:797592 -0.67 1.7Χ10-6 -0.54 1.7x10"" -0.08 -0.59 1.2x10-9 5.4x10""
YWHAE 17:1278466 0.52 3.8x10'7 0.26 3.8x10-" 0.03 0.35 3.2x10"9 0.0014
2:23558046
- 1 0.49 1.8X10"6 0.27 1.7x10 " 0.04 0.33 3.4x10"8 0.015
17:7547396
SEPT9 0.72 003
9 5.8x10-8 0.25 0. 0.03 0.41 4.5x10-8 0.02
21 :4634105 1.43x10'
ITGB2 0.52 1.2X10-6 0.20 0.007 0.05 0.32 0.06
4
A Log Fold-Change. B mean difference. All probes with combined Bonferroni corrected P values <0.05 (equivalent to P<1 ,1x10"7) are listed in Table 3.
Table 2. Differentially Methylated Regions
A P (Bonferroni
Location Coordinates Genes P value
corrected)
1 17q 57915561 MIR21 , VMP1 1.22 x10-14 5.50 x10-9
1 2ρ 11969951 LPIN1 3.45 x10 1 1.55 x10"5 ΐ 1 ρ 3704438 LRRC47 2.17 x10"9 0.001 ΐ 6ρ 31867661 ΖΒΤΒ12 6.00 x10-9 0.0027 τ 4ρ 2321948 ZFYVE28 1.69 x10-8 0.008 τ 17q 75473865 SEPT9 4.51 x108 0.02 i 10q 45473025 C10orf10, RASSF4 5.40 x10"8 0.024 τ 1 ρ 117528902 PTGFRN 9.56 x10"8 0.043 τ 6ρ 31544747 TNF 9.74 Χ10-8 0.044 ϊ 3q 184297196 EPHB3 1.06 x10'7 0.047
B Location Coordinates Genes DMRs Region
6p 31540310 TNF, LTA, LST1 3 14.7kb
10q 134211782 PWWP2B 3 19.8kb
20q 57414454 G AS 3 49.2kb
17q 75451595 SEPT9 2 22.5kb
6q 166876721 RPS6KA2 2 32.0kb
C Closest
Location Coordinates Genes Distance
SNP
1 17q 57915561 MIR21 , VMP1 rs 1292053 47.8kb r 11 p 1945532 TNNT3 rs907611 71.5kb t 2q 241643165 - rs3749171 74.5kb ΐ 11q 65561601 OVOL1 rs2231884 94.8kb
T 10p 6182454 - rs12722515 102.3kb i 2q 219030741 CXCR1 rs2382817 120.4kb
T 11p 2021323 - rs907611 148.3kb i 1p 1396079 ATAD3C rs12103 148.7kb
T 19p 10662478 ATG4D, MIR1238 rs11879191 149.7kb
Top DMRs from combined paediatric lilumina 450k data ranked by (A) most significant individual probe, (B) density of DMRs in a region, (C) proximity to GWAS SNPs. Coordinates refer to hg19/GRCh37. (up arrow - Hypermethylated, down arrow - Hypomethylated, dash - Intergenic probe) References
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Epigenetic Changes in Inflammatory Bowel Disease, Ulcerative Colitis and Crohn's Disease Summary
Methods, Results and Conclusions
The approach and methods described in Example 1 were applied to assess epigenome- wide methylation profiles in circulating leucocyte DNA in the following cohorts:
- paediatric Crohn's Disease;
- adult Crohn's Disease;
- adult Inflammatory Bowel Disease; and
- adult Ulcerative Colitis.
Results are shown in Table 8 and Figures 7 and 8.
The results show CpG sites with methylation alterations achieving epigenome-wide significance after Bonferroni correction (P<1.1x10"7) in each of paediatric Crohn's Disease, adult Crohn's Disease, adult Inflammatory Bowel Disease, and adult Ulcerative Colitis. Additionally, there is a strong correlation between the results obtained for each cohort - in particular, many of the same CpG sites with methylation alterations achieving epigenome- wide significance are found in each of the individual cohorts. That correlation is shown in the final four columns of Table 8, which show the "Rank" importance of each particular CpG site in each cohort. As an example, the CpG at Position 166970252 of Chromosome 6 (identified using Probe cg17501210) associates with the RPS6KA2 gene - methylation alterations at that CpG site achieves epigenome-wide significance in:
- paediatric Crohn's Disease (where it is ranked as second most-significant site);
- adult Crohn's Disease (ranked as the top most-significant site);
- adult Inflammatory Bowel Disease (ranked as the top most-significant site); and
- adult Ulcerative Colitis (ranked as the ninth most-significant site).
Accordingly, we demonstrate highly significant and replicable differences in DNA methylation in each of paediatric Crohn's Disease, adult Crohn's Disease, adult Inflammatory Bowel Disease, and adult Ulcerative Colitis. The predictive accuracy of discriminant analysis provides a potential avenue for clinical translation for each of those diseases. Table 8 - Illumina 450k probes and genes with significant methylation differences in the following cohorts: paediatric Crohn's Disease; adult Crohn's Disease; adult Inflammatory Bowel Disease; and adult Ulcerative Colitis.
P Value (Uncorrected) Rank
ProbelD Chr Position GeneSymbol Paediatric CD Adult IBD Adult CD Adult UC Paediatric CD Adult IBD Adult CD Adult UC cg17501210 6 166970252 RPS6KA2 4.47x10"15 6.85x10" 8.51x10" 4.78x10-12 2 1 1 9 eg 18608055 19 1130866 SBN02 5.04x109 2.01x10" 4.46x10' 6.28x10-14 29 2 2 3 cg09349128 22 50327986 NA 3.00x10"9 9.15x10" 1.04x10" 5.05x10-1 20 3 6 15 cg12170787 19 1130965 SBN02 1.61X10-8 1.06x10" 1.95x10- 7.51x10-11 39 4 3 17 eg 16936953 17 57915665 VMP1 2.65x 0"13 2.39x10" 1.32x10" 4.26x10" 2 4 5 5 7 eg 12992827 3 101901234 NA 1.59x10"13 5.54x10" 1.21x10" 3.12x10-14 3 6 7 1 cg2511461 1 6 35696870 NA 3.05x10"7 7.23x10" 1.89x10" 4.26x10"14 102 7 12 2 cg02448796 1 6101339 KCNAB2 3.46X10- 5.38x10 7.12x10- 2.92x10 10 2005 8 10 24 eg 12054453 17 57915717 VMP1 1.97x10"15 1.45x10" 1.03x10" 4.10X10-8 1 9 4 78 cg07398517 3 30327579 NA 4.44x10"9 1.83x10" 1.59x10" 2.90x10" 0 27 10 21 23 cg13619623 7 33637324 BBS9 9.79x10^ 2.63x10 9.76x10 1.01x10 11 13925 11 19 10 eg 16724148 1 100326338 AGL 5.26x10-2 7.21 10" 2.13x10" 9.56x10" 45887 12 65 19 cg26804423 7 8201 134 ICA1 9.02Χ10-3 9.08x10" 4.44x10" 3.22x10"11 13175 13 16 14 eg 19821297 19 12890029 NA 1.91x10"10 2.36x10 7.25x10 4.72x10 12 9 14 17 8 cg22959742 10 13913931 FRMD4A 3.24X10-3 2.43x10" 1.44x10" 1.92x10"12 6879 15 60 6 cg03546163 6 35654363 FKBP5 7.93x10"8 2.83x10' 1.98x10" 7.55x10 10 59 16 13 29 cg01059398 3 172235808 TNFSF10 3.28x10"10 4.99x10' 2.06x10" 7.85x10 13 12 17 64 5 cg26955383 10 105218660 CALHM1 4.17Χ102 5.24x10" 2.25x10" 2.66x10"10 38423 18 25 21 eg 10636246 1 159046973 AIM2 2.58x10"4 7.36x10' 1.00x10" 1.15X10-7 1736 19 32 106 eg 18942579 17 57915773 VMP1 8.23x10"10 9.51x10" 2.20x10" 1.94x10"9 15 20 24 38 cg01101459 1 234871477 NA 1.10X10"4 1.47x10' 6.92x10" 4.44x10-9 1145 21 30 47 eg 17953136 2 20232577 LAPTM4A 2.20x10 2.14x10' 5.17x10" 3.62x1010 24081 22 48 25 cg26020069 6 52382441 TRA 2 2.42x10"4 3.29x10- 1.80x10" 1.11x10-8 1683 23 38 54 cg02716826 9 33447032 NA 2.56x10"9 3.57x10" 3.57x10- 4.51x10"8 17 24 15 79 cg25132241 14 92396859 FBLN5 5.61 Χ10"1 3.64x10 2.91x10" 2.61Χ10"6 2545 25 8 280 cg27087650 9 45255796 BCL3 2.33x10-3 3.96x10' 5.80x10 1.22x10'11 5666 26 247 11 cg23172671 1 203482523 NA 2.25X10-3 4.96x10' 9.60x10" 6.84x10"9 5548 27 54 52
cg26470501 19 45252955 BCL3 4.08x10"7 5.17x10" 2.89x10"10 2.08x10"8 114 28 72 66 eg 12670943 22 43006541 POLDIP3 1.68x10-2 5.35x10" 2.78x10"12 1.54X10'7 20023 29 26 120 cg07035242 1 11336263 UBIAD1 8.45x102 6.16x10" 3.35x10-10 7.48x10-8 65975 30 75 94 cg 7781958 17 79428404 BAHCC1 2.61x10-e 7.70x10" 1.89X10-12 2.01 x107 240 31 23 130 cg02734358 4 90227074 GPRIN3 3.20x10 s 1.08x10 9.78X10"9 1.26x10 10 45 32 164 20 cg25533551 9 48823 78 CCDC1 14 1.24x10-2 1.18x10" 1.25x10"9 2.41x10 " 16243 33 98 13 eg 11849692 10 103875969 LDB1 4.19X10-3 1.43x10" 11 2.20x10 10 1.19x107 8005 34 66 109 eg 1258231 17 55822272 NA 8.92x10"3 1.46x10" 2.31x10-10 4.93x10"8 13076 35 68 82 cg17588003 17 5138696 SCIMP 1.87Χ10"4 1.57x10" 2.17X10"1 1 2.21x10"7 1482 36 41 132 cg23740758 6 11324433 NEDD9 9.69x10 2.15x10- 1 1.38x10-'° 3.48X10-8 3419 37 58 75 cg25653947 8 144443217 NA 1.76x10"7 2.24x10" 5.58x10-11 9.79x10"8 78 38 49 100 cg27243685 21 43642366 ABCG1 1.21 103 2.30x10" 3.47x10"7 1.06X10"9 3865 39 394 34 cg09007354 1 54 00163 GLIS1 8.95X10-2 2.83x10" 11 4.34x10'9 2.77X10"10 69012 40 138 22 eg 11832534 1 3563998 WRAP73 3.21 x10-5 2.83x10" 1.76x10"12 2.48x10"7 669 41 22 137 eg 16805291 7 36022575 NA 2.57x10-2 3.63x10" 7.29x10"14 9.92X10-8 26892 42 11 102 cg04987734 14 103415873 CDC42BPB 2.17x10 s 4.01x10" 11 1.25X10-12 2.15X10-6 41 43 20 264 eg 12669088 12 25541364 NA 8.1 1Χ10-1 4.81χΐσ 1.56Χ10-9 3.66x10-8 381132 44 104 76 cg04465154 8 9045558 NA 1.28x10"7 5.09x10" 4.73x10-10 4.06x10-9 70 45 80 46 cg09018739 16 57180107 CPNE2 6.64x10"4 6.59x10- 1 1.75X10-1 1 1.24Χ10"7 2779 46 37 112 eg 10180440 10 02325486 NA 2.13x10-5 6.72x10- 1 1.80x10"9 3.69x10-10 569 47 112 26 cg000539 6 8 37457329 NA 3.43x10 6.94x10" 1 1.44x 0"9 2.34x10"8 33142 48 100 69 cg13585930 10 72027357 NPFFR1 2.49x10-6 7. 9x10- 1 1.72x10 s 9.97x10"8 232 49 109 103 cg01 45119 8 44441955 NA 5.37x10"5 7.20x10" 3.44x10-13 8.42x10-6 828 50 14 387 cg05768620 11 71752971 NUMA1 3.84x10"1 7.99x10" 1.70x10'9 1.12x10" 214939 51 106 889 cg22448090 1 200978598 KIF21 B 7.40Χ10-3 9.96x10" 1 1.44x108 3.00X10 7 1 1622 52 181 150 cg02719954 8 23830907 NA 1.56x10-2 1.02x10" 1.63x10"7 5.09x10-9 19023 53 318 50 cg11393173 1 116369577 NA 2.23x10"5 1.14x10" 6.36x10"10 5.86x10-9 578 54 86 51 cg113964 1 7 33679233 NA 2.86x10 1.16x10" 6.62x10-10 8.95x10 s 29008 55 88 98 eg 17515347 1 159047163 AI 2 2.88x10" 1.23x10" 6.43x10"8 1.23x10-13 1836 56 255 4 cg02543993 7 5736195 RNF216 9.14x10"3 1.36x10" 3.70x10"10 2.05x10"7 13275 57 77 131 cg00382138 4 110723299 CFI 3.27x10 11 1.52x10" 7.34x10 " 1.84x10-7 7 58 18 126 cg00826902 1 3563954 WRAP73 1.00x10"· 1.56x10" 4.93x1011 1.94x10-8 1092 59 47 65 cg01799015 19 707791 PALM 2.23x10^ 1.60x10" 3.15x10"9 8.34x10 10 223 60 126 31
cg03393889 16 2094700 NTHL1 2.81x10 1 1.77x10 2.28x10"9 1.43X10-6 169201 61 116 227 cg25422678 2 28304502 BRE 6.19X10-1 2.04x10" 2.04x10-11 6.04x10-6 309814 62 39 354 cg02650017 17 47301614 PHOSPH01 1.92x10"7 2.27x10" 9.74x1 ο-8 2.91x10"7 82 63 282 147 cg03128029 2 203143288 NOP58 3.42x10'5 2.29x10 1.99X10"8 1.37x10 694 64 198 59 cg20228731 7 130646051 LINC-PINT 9.85x104 2.59x10- 7.29x10"10 6.70x10-11 3450 65 90 16 cg24430034 13 110386176 NA 8.19X10"7 2.75x10" 1.68X10-9 5.36x105 144 66 105 692 eg 12424624 3 46718941 ALS2CL 2.49x10"1 2.76x10 2.48x10 11 1.70x106 154341 67 42 489 cg27128761 5 151055650 SPARC 5.03x10"1 3.12x10" 7.43x10"8 1.57x10-9 264797 68 264 37 cg00441899 4 3432483 RGS12 4.36x102 3.12x10" 5.90x109 1.21x10-7 39769 69 146 110 cg13354241 2 64447091 NA 1.09x10"3 3.14x10- 1.28x10"10 1.83x10^ 3638 70 57 250 cg02297838 13 92002454 NA 1.09x1a3 3.68x10" 6.39x10"9 1.88x105 3650 71 149 502 cg21846903 17 26697281 VTN 7.22x10"1 3.89x10" 3.70x10-" 5.54x10^ 348608 72 45 342 eg 14556762 1 170532470 NA 1.03Χ10"4 3.94x10" 3.13x10"10 1.40X10-5 1109 73 73 462 cg08392125 2 108427720 NA 4.76x102 4.10x10" 3.66x10'9 3.07x107 42466 74 131 151 cg06846976 16 2121682 TSC2 6.98X10"3 4.39x10" 6.60x10"11 2.54X105 11173 75 51 547 cg15551881 9 123688715 TRAF1 1.84x10-5 4.43x10" 1.04x10 " 2.22x10-7 535 76 33 133 cg26307105 19 17861017 FCH01 1.89x10-3 4.44x10" 1.92X10"8 1.05x10"9 5001 77 195 33 cg22753611 6 17472892 CAP2 1.66X10"4 4.57x10" 4.09Χ10-8 1.26X10-8 1397 78 225 56 cg07148145 7 99437259 CYP3A43 5.07X10-4 4.63x10" 3.93x10-11 5.02x107 2405 79 46 167 eg 17980786 3 32933637 TRIM71 8.62x10"4 4.80x10" 2.48x10"10 5.42x10"7 3199 80 70 170 cg00490406 1 159046773 AIM2 3.05x1 ο 5 5.22x10" 2.73x10 10 3.10x10 653 81 71 72 cg18721397 5 32584912 SUB1 1.03x10-3 5.40x10" 2.08x10"9 4.74x10-9 3537 82 113 48 cg04036920 11 33562503 KIAA1549L 5.55X102 5.42x10" 2.80x10"11 1.56x10"9 47826 83 44 36 cg20364632 6 49636226 NA 5.03x1ο 2 5.44x10' 1.39x10 10 1.35Χ10 5 44266 84 59 456 eg 10549973 14 58863097 TOMM20L 9.40x1 a1 5.69x10" 1.60x10-10 5.47x10"4 427849 85 61 1700 cg07835482 2 182656091 NA 8.12x10 5.77x10" 1.25X10"7 1.99x10"7 63967 86 299 128 cg03523676 14 24540235 CPNE6 8.61x10 5.78x10" 1.63x10 11 1.43x10-6 66929 87 35 226 cg08289839 13 11 1318640 CARS2 1.80X10-4 5.81x10- 2.09x1 a11 9.68x10-7 1463 88 40 209 cg20141108 1 165907859 NA 7.13Χ102 5.96x10" 4.23x10"10 2.49x10 57878 89 79 70 cg24469729 7 27160520 HOXA3 3.49x10-2 6.86x10" 6.26x10-12 3.43x10 33527 90 29 599 cg26599989 1 1 1297087 TOLLIP 8.18x10-7 7.41x1 a 9.19x10"12 4.81x10 143 91 31 664 cg20995564 2 145172035 ZEB2 2.58x10"9 8.49x10" 1.78x10"9 1.30x10-11 18 92 111 12 eg 15466952 1 65889855 NA 3.80Χ10"6 9.36x10" 1.70x10"11 1.10x10 274 93 36 430
cg07879825 7 69358880 AUTS2 2.97x10"1 1.00x10* 3.03x107 2.09x10"9 176452 94 377 39 eg 14176339 17 79244542 SLC38A10 1.55x102 1.06x109 5.95x10 9 2.88x107 18928 95 147 146 eg 18860310 4 87752504 SLC10A6 4.97X10-8 1.18x10-9 2.39x10-10 3.00x10-9 49 96 69 42 eg 12269535 6 43142014 SRF 4.99x10"7 1.18x10* 8.88x10* 1.68x10 e 120 97 162 241
CSGALNACT
eg 14722693 8 19436451 1 4.30X10-6 1.24X10-9 1.56x107 3.65x10"9 294 98 313 45 cg23761815 10 73083123 SLC29A3 4.08X10-3 1.29X 0-9 6.07X10-8 5.28X10-8 7894 99 251 84 eg 10005565 2 235578952 NA 4.89x10"1 1.41x10-9 2.50x10-7 3.48x10-9 259238 100 357 44 cg21727145 15 101458127 LRRK1 1.28x10 1.47x10* 9.04x10-10 2.33x10-7 91738 101 93 134 cg05316065 8 130799007 GSDMC 3.17x10* 1.48x10* 5.68x10-" 1.34x10* 22 102 50 222 cg06192883 15 52554171 MY05C 2.17x10* 1.56x10* 7.21x10"10 2.18x10* 216 104 89 267 cg27023597 17 57918262 MIR21 5.42x10* 1.63x 0"9 3.60x10* 3.08x10-7 50 105 130 152 eg 12807764 5 146864669 NA 8.85X10-2 1.87x10"9 3.48x10'7 1.76X10-8 68414 06 395 63 eg 11847933 1 227130394 ADCK3 2.45x10* 1.87x10* 3.18x10-8 3.77x1 ο-6 5830 107 213 307 cg15310871 8 20077936 ATP6V1 B2 2.63x10-2 1.96x10* 1.64x10"7 1.17x10 27294 108 320 55 eg 13447080 8 131054408 NA 1.31X10-3 2.10x10-° 4.51x107 1.53x10* 4045 109 424 233 cg08423142 15 59588622 MY01 E 4.07x10"9 2.15x10* 2.88x10·3 1.93x10* 25 110 122 64 cg03998636 13 111210121 RAB20 1.62x10"5 2.38x10-9 1.98x10 1.32x10* 504 111 197 57 cg25001 190 1 61668835 NFIA 1.76x107 2.59x10 9 2.12Χ10 7 7.06x10"10 79 112 342 28 eg 13558682 1 3705262 LRRC47 3.65x10-1 2.59x10* 1.46Χ10"6 1.17x10-7 206810 113 547 108 cg22768358 11 1 13947148 ZBTB16 1.78X10-9 2.62x10* 1.00x10"7 9.38x10* 16 114 284 842 eg 19445588 12 1 16904641 NA 1.05x10 1 2.65x10* 1.25x107 3.45x10* 78520 115 300 43 eg 15082870 7 36022841 NA 3.31 x10"3 2.71x10* 9.78x10"10 7.75x10-7 6972 116 94 194 cg02976843 17 2843257 RAP1GAP2 1.51x10"1 2.73x10* 2.28x10-9 1.34x10"7 104149 117 115 115 cg02569509 7 142423070 NA 2.50x10* 2.85x10* 5.31Χ10-6 4.67x10* 591 1 118 766 80 cg00371134 1 77912654 AK5 3.29x10* 2.88x10* 1.09x10-7 1.14x10* 6953 119 287 218 cg27269962 7 127540997 SND1 4.16x10"1 2.89x10* 4.40x10-s 9.34x10-7 229147 120 727 207 eg 11058932 7 130372167 TSGA13 5.41 x10-1 2.94x10-9 4.50x10"9 2.49x10"7 279736 121 140 138 cg08549335 7 30387954 ZNRF2 4.87x10-4 3.03x 0"9 8.13X10-11 1.03x10* 2351 122 53 415 cg23665802 13 92002338 NA 7.22x10-4 3.11x10* 1.08X10-7 1.24x10* 2906 123 286 444 cg26663590 16 28959310 NA 7.74x10-2 3.25x10* 2.94x10-12 1.12x10* 61540 124 27 217 cg03940776 6 158490013 SYNJ2 5.82x10-8 3.46x10* 6.90x10 3.26x107 53 125 259 155 cg05399785 1 3564031 WRAP73 3.23x10* 3.52x10"9 2.12x10 5.00x10* 673 126 201 49 cg13316148 2 191916269 STAT4 1.78x10* 3.60x10-9 2.19x10-9 8.96x10"11 4811 127 114 18
cg03951877 6 13120250 PHACTR1 9.16x105 3.62x10"9 2.80x10" 1 4.78x10"5 1049 128 43 661 cg14761417 7 130636860 LINC-PINT 1.26x10'2 3.66x10-9 6.64x10"7 1.51x105 16412 129 456 474 cg09912552 8 134250937 NDRG1 3.19x10"1 3.75x10-9 4.82x10'9 2.66x10-7 186601 130 142 143 cg21075678 137557164 NA 5.71x10"1 3.78x10"9 1.53x10"9 1.30x10-" 291598 131 102 934 eg 18376497 4 143488622 INPP4B 3.09x10-9 3.79x10-9 4.02x10"7 2.61x10^ 21 132 408 281 cg17411016 2 47100912 NA 5.04x10"9 3.99x10-9 7.85x10'9 2.84x10"7 30 133 155 144 cg02782634 17 57916643 VMP1 5.63x10-6 4.06x109 1.07x10 " 9.58Χ10-6 319 134 34 402 cg06160572 12 123892724 SETD8 3.53x10-1 4.20x10"9 1.17x10-8 5.35Χ10-6 201699 135 169 336 cg27365342 6 12881417 PHACTR1 2.59x10-2 4.22x10-9 4.26x10"2 5.54x10"4 27037 136 28 1712 cg06710464 17 79047695 BAIAP2 3.36x103 4.89x 0"9 4.24x10 s 4.40x10"7 7035 137 228 163 cg18181703 17 76354621 S0CS3 4.56x10"10 5.16x10-9 3.88x10"4 2.58x10 13 138 9 3708 cg02349866 15 90391877 AP3S2 3.42x10"3 5.60x10-9 8.82x10-9 7.23Χ10-8 7110 139 159 93 cg26227957 1 19547285 EMC1 3.77X10-3 5.93x10"9 3.45x10"7 9.87x10-'° 7516 140 393 32 cg23752007 7 37429500 ELM01 5.03X10-2 6.11x109 3.50x10-9 5.93Χ10-6 44243 141 129 348 cg05398036 1 186504948 NA 2.00x10"4 6.17X10-9 4.68x10-8 1.43x10-5 1527 142 231 466 eg 14237301 16 28506477 APOBR 1.88x10" 6. 8x109 1.26x10-8 8.19X10"7 1486 143 171 198 cg25368647 5 176736591 MXD3 1.09x10"5 6.34x 0-9 1.10x 0-7 5.76X10-8 425 44 288 86 cg04086394 1 193082764 NA 9.03x10" 6.36x10-9 8.55x10"7 5.08x10 3281 145 481 83 cg01839860 5 138957422 UBE2D2 1.33x10 1 6.39x109 1.60X10'7 9.54x10 94598 146 314 400 eg 16755922 17 80536214 FOXK2 9.05x10-4 6.80x10-9 9.00x10 2.23x10 3287 147 274 67 cg22749855 17 76353952 SOCS3 7.57x10"5 6.89x10 9 3.68x10"10 6.24X10-5 953 148 76 717 cg06086933 1 100326491 AGL 3.56x10-2 7.21x109 4.55x1 O^ 6.83x10-7 34027 149 735 187 cg13951069 21 43919801 SLC37A1 9.06x10-3 7.27x10-9 1.07X10-6 6.02x10-7 13215 150 507 178 cg24386894 8 8863572 ERI1 1.63x10" 7.55x10 s 5.60x10^ 1.38x10 1387 151 778 60 cg05544807 2 25475916 DN T3A 1.29x10 7.78x10"9 3.77x10^ 1.42x10" 16625 152 221 969 eg 15928106 7 130646078 LINC-PINT 1.50x10-2 7.86x10 9 2.37x10"7 8.23x10 10 18514 153 351 30 eg 12053291 12 125282342 SCARB1 1.44Χ10-4 8.27x10"9 1.78x10-8 1.76x10-5 1308 154 190 495 cg 9742686 19 10823913 QTRT1 1.44x10" 8.41x10"9 1.32x10-8 2.32x10" 100201 155 1 7 1177 cg1047271 1 7 797592 HEATR2 3.49X10-8 8.49x10-9 2.40x10"7 2.50x 0-9 46 156 353 40 eg 13270236 6 42658437 UBR2 3.83x10 8.56x10-9 4.06x10"9 3.40x10 36018 157 133 300 cg07793033 16 85256423 NA 1.90x10-3 8.67x10"9 1.64x10"7 6.43x10-7 5010 158 319 181 cgO 1409343 17 57915740 VMP1 1.58x 08 8.92x10"9 1.30x10 6.96x10"7 38 159 173 188 cg02003183 14 03415882 CDC42BPB 6.69x107 9.10x109 1 .00X10"10 8.67Χ10 5 134 160 55 819
cg2121 1645 3 45590039 LARS2 2.50x10-2 9.22x10-9 2.85x10"6 3.1 1x10-7 26344 161 644 154 cg02600394 4 48136234 TXK 2.22x10"6 9.39x10"9 2.23x10-6 1.06x10"7 221 162 606 104 eg 14427590 17 60695089 NA 2.75x10-2 9.45x109 1.83Χ10"8 7.20x10-6 28173 163 191 373 eg 16663980 1 158809049 MNDA 2.52x10"1 9.54x 0 9 8.86x10"9 8.36x10"7 155659 164 161 199 cg20519581 20 48959844 NA 5.46x10"6 9.60x10"9 2.52x107 5.44x10 10 315 165 359 27 cg1 1679124 10 14058944 FRMD4A 9.38x10"4 1.07X10-8 3.61x10"7 2.39x10"6 3354 166 398 272 cg23842572 17 17030253 PRIP 4.37x10"8 1.08x10 s 4.30x10"9 2.81x10-4 47 167 136 1276 eg 15321908 3 53187213 NA 8.61x10 s 1.12Χ10"8 1.30X10"8 1.83Χ10·6 1009 168 172 251 cg25445707 3 46719086 ALS2CL 3.77x10" 1.14x10-8 1.15x10"8 1.66X10"6 212250 169 168 239 cg05941027 17 61774174 LIMD2 1.77X10"1 1.22x10-8 2.97x10"8 4.03x10"5 444 170 209 629 cg27106909 16 30106897 YPEL3 9.33Χ102 1.31x10 B 2.10Χ10"7 3.44x10"· 71341 171 341 1401 eg 17969271 5 171430188 FBXW1 1 3.09x10-3 1.33x10-8 1.36x10-8 4.62X10-4 6687 172 178 1590 cg22688566 17 27459835 MY018A 3.20x10'1 1.33x10"8 1.14X10"8 9.19X10"7 186975 73 167 205 cg03546806 12 1 14297307 RB 19 2.55x10 1 1.34x10 s 4.16x10^ 8.33X10"6 157055 74 226 385 cg20510033 1 8960134 NA 6.15X10"3 1.41x10-a 8.16X10 9 2.96x10^ 10295 175 156 71 eg 1 1095027 1 1 1297066 TOLLIP 9.23x10"6 1.46x10 8 1 .74X10 10 1.25X10"4 389 176 62 924 eg 12394201 1 1 43942418 NA 9.64x10^ 1 .47x10 s 1.38x10^ 1.60x10^ 3409 177 537 61 eg 16739178 16 85470674 NA 6.24x10^ 1 .49x10"8 2.74x10-9 6.26x10"5 330 178 121 718 eg 13410000 15 91357521 BLM 1.56x10"5 1 .51x10"8 5.45x10"8 1.81x10"5 495 179 774 498 cg02593766 1 48608677 NA 3.93x10-1 1.60Χ10"8 6.72x10-8 2.17x10^ 219245 180 257 265 cg02538248 16 75045643 ZNRF1 3.80x10^ 1 .62X10-8 1.86x 0"8 3.54x10"5 275 181 192 605 eg 19405484 8 29513210 NA 8.45x10-5 1 .75x10"8 1.92x10-7 4.81x10"4 1004 182 334 1623 cg21416544 12 8986988 A2ML1 5.10x10-1 1 .84X10-8 1.72x10"7 3.40x10"7 267519 183 324 157 cg06724236 14 67853825 PLEK2 1.97Χ10"1 1 .85x10 s 3.29x10 B 7.04X10-8 128473 184 216 371 eg 10722217 3 14347415 NA 7.37x10"1 1 .87X10-8 2.92x10"9 8.79X10 5 353780 185 123 822 cg27310092 1 151945663 NA 2.54x10"10 1 .88x10-8 5.61x10 s 1 .04x10 1 1 186 145 417 cg21190595 1 1 3071 167 CARS 2.41x10'5 1 .89x10-8 1.73X10-6 1.47x 0-7 592 187 568 1 18 cg13442606 1 1 3071269 CARS 1.1 1x10 1 .92Χ10 8 1.02X10-6 3.31x10"7 8 926 188 500 156 cg02508743 8 56903623 LYN 3.26x10-5 1.93X10"8 4.32x10-9 3.62x10-5 675 189 137 609 cg236691 18 16 1538347 PTX4 3.84X10-2 1 .96X10 8 2.53x10"8 4.95x10"7 36087 190 205 166 cg06541968 2 240431 144 NA 7.14x10"1 2.04x1 Or8 9.80x10 10 1.95X10"1 345571 191 95 1 102 eg 18307303 5 158757456 IL12B 4.12x10-1 2.08x10-8 2.02X10-6 5.09x10"7 227176 192 588 168 cg08952844 2 88313351 NA 6.02x10 1 2.09x10"8 6.00x10"8 5.00x10-6 303249 193 250 328
eg 16642695 8 67343633 ADHFE1 7.42x10"1 2.13x10 8 6.51 x10^ 8.24x10^ 355615 194 826 383 cg00657780 16 50285254 NA 3.42x10-1 2.14x10"8 3.19X10-9 5.15x10 196779 195 127 169 eg 12866551 10 20019641 NA 1.09x10"3 2.25x10 8 2.52Χ10 5 6.50x10 3648 196 1273 183 cg07723258 17 38176064 ED24 8.88x10"2 2.27x10-8 4.75x10"8 2.99x10 s 6861 1 197 235 573 cg19588519 10 125817817 NA 1.13x10"1 2.30x10 8 1.26x10-8 8.60X10"6 83168 198 170 390 cg20813374 6 35657180 F BP5 1.04x10-= 2.30x10"8 6.05x10"8 2.27x10s 412 199 803 269 cg26712428 10 105218771 CALHM1 4.66x10-2 2.37x10"8 4.89x10-8 7.60x10 41849 200 237 193 cg26182406 14 105491309 NA 4.57X10-4 2.37x1 0-8 8.50x10 7 3.33X10-6 2280 201 479 298 cg04287259 1 9730401 PIK3CD 1.16x10"5 2.39x10"8 1 .92Χ10-8 2.83X10"8 435 202 196 287 cg00851028 1 234905772 NA 5.09x10"4 2.39x10^ 1.71x10-9 1.59X10-4 2415 203 107 1021 cg1451581 1 9 129088215 MVB12B 1.77x10"1 2.68x10 8 9.47X108 1.31X10-4 1 18261 204 279 936 cg00483891 2 131098007 CCDC115 6.00x10 s 2.74x10^ 5.63x10 2.97x10 870 205 244 148 eg 12535090 11 20071677 NAV2 6.50x10-3 2.81X10-8 5.92x10-10 2.02Χ10-4 10665 206 83 1 1 17 cg20697417 1 41786797 NA 5.61x10-2 2.81 x10-« 1 .80X10"10 9.00x10 s 48163 207 63 829 cg20671910 1 151262619 ZNF687 9.81x10 s 2.84x10^ 2.30x10"8 1.32Χ10-4 1078 208 204 938 cg07683636 2 219940977 NHEJ1 7.29x10-2 2.95x10-8 5.14X10-7 5.45Χ10-6 58777 209 437 341 cg09481056 16 2339260 ABCA3 6.02x10-4 2.98x1 0 8 2.93x10 6.80x10 2640 210 370 186 cg07168939 8 143763412 PSCA 5.36x10" 3.06x10 s 1.72x10 7 3.21 x10^ 2480 21 1 325 295 cg08032483 16 1495363 NA 1.85x10"1 3.15x 0-8 2.93x10 s 3.30x1 ο-6 122165 212 1328 296 cg15357118 2 128927972 UGGT1 3.26x10"3 3.21 x10-8 7.05X10 8 8.35x1 ο-6 6916 213 260 386 cg05554192 11 17515836 USH1C 6.31x10 3.21 x10 8 6.84x10 9 1 .99x10" 52706 214 151 1 1 10 cg16277071 17 75537317 NA 1.08x10 3.30x10-8 6.21x10"10 3.64x10"5 80125 215 85 612 eg 14016363 7 100486297 NA 2.01 Χ10·4 3.31 x10 8 1 .25X10-8 1.69χ10"5 1531 216 525 486 eg 14534148 6 170559869 NA 2.16x10"3 3.33x10"8 6.85x10 2.25x10^ 5431 217 458 268 cg00159243 12 109023799 SELPLG 1.67x103 3.39x10-8 1 .19x10 1.49Χ10-6 4649 218 295 231 cg02481950 16 21665002 NA 7.19x10 3.51 x10"8 2.20x10 3.47χ10-5 58229 219 203 600 eg 16291589 20 6023178 L RN4 8.73x10-1 3.63x10"8 3.52x10^ 9.52x10^ 403482 220 682 99 cg16395183 1 210771699 HHAT 4.80x10"7 3.64x10-8 3.16X10-8 2.65x10 1 18 221 659 142 cg02147592 19 4090388 MAP2K2 2.78x10"3 3.67X10-8 1.76Χ10-8 7.34x10 s 6255 222 189 760 cg27470978 8 38704972 TACC1 8.68x10 3.67Χ10-8 2.28x10"9 4.47χ10-5 67368 223 1 17 649 cg08571738 2 2605398 NA 2.45x10" 3.71 x10 5.37x10^ 3.72x1 152272 224 243 304 cg24707889 21 46341304 ITGB2 5.37x10 s 3.76x10"8 2.50x10 7.03x10 829 225 358 189 eg 16246188 11 113947203 ZBTB16 1.40x10^ 3.78x10-8 2.26x10"6 1.55Χ10-4 183 226 610 1005
cg04255937 12 122269675 SETD1 B 1.55x1a4 3.83X10-8 1.01X10"8 1.41x10^ 1352 227 165 967 cg07539709 17 80545454 FOXK2 2.12x10"1 3.97x10* 8.1 7X10'7 2.99x107 136215 228 474 149 cg19991780 3 128533372 RAB7A 6.93x10-2 3.98x10-8 7.67X10-6 5.72x10'7 56593 229 870 174 eg 12680205 10 80303482 NA 3.98x10"6 4.04x10"8 3.02X10"9 5.86x10* 285 230 124 347 eg 19674091 15 90643766 IDH2 9.87X10-6 4.14x10* 2.07x107 1.11x10* 404 231 340 216 cg24527461 17 79419366 BAHCC1 1.08x10* 4.15X10-8 9.72x10* 2.18x10* 421 232 936 266 cg25059899 18 60904328 BCL2 4.55x10"1 4.21x10-8 4.86x10* 1.23X10-4 244982 233 236 918 cg25953130 10 63753550 ARID5B 7.08x107 4.28x10* 1.00x10"10 1.14x10* 138 234 56 2398 cg24394336 1 17962705 ARHGEF10L 8.98x10-2 4.31x10* 2.84x10* 7.60x10* 69181 235 1314 95 cg06291107 20 36157675 BLCAP 5.19X10 2 4.34x10 s 1.54x10* 2.14x10* 45390 236 558 263 cg02112168 14 45579561 NA 1.83x101 4.37x10 s 5.63x107 3.32x10* 121535 237 444 73 cg232981 14 8 118933411 EXT1 3.05x10-3 4.38x10"8 9.15x10* 3.18x10* 6634 238 276 586 eg 18518074 11 64642316 EHD1 9.35X10-3 4.57x10-8 3.79x10-8 1.59x10"7 13499 239 222 123 cg20981615 4 48136280 TXK 5.28x10* 4.61x10 8 4.40x107 7.13x10* 311 240 420 757 cg08446900 17 38501519 RARA 7.60x10"1 4.67x10-8 9.32x10-6 3.45x10-4 362197 241 925 1403 eg 12067024 1 153387689 S100A7A 2.98x10 1 4.69x10* 1.95Χ10"5 8.57x10* 176897 242 1157 97 cg08791347 10 13831250 FRMD4A 1.50x10-1 4.85x10* 5.47x10* 2.47x107 103817 243 775 136 cg07372520 1 180086434 NA 7.64x10* 4.87x10-8 1.70x107 1.80x10* 11845 244 322 496 cg25364972 2 217075573 NA 5.71x10"1 4.94x10* 5.99x10* 1.60x10* 291293 245 798 237 cgO 1589587 14 76002440 BATF 3.00x10-1 4.96x10* 4.24x10"7 1.65x10^ 178034 246 415 1030 cg01153613 6 139928856 NA 1.93x10-1 5.17x10* 4.16x10"7 1.91x10* 126521 247 413 255 eg 14088282 1 222003220 NA 1.07x10* 5.30x10* 2.67x10* 3.99x10"7 3596 248 632 160 eg 19868691 2 219744597 WNT10A 7.75x10"1 5.31 X10"8 1.29x10-7 2.09x10* 367859 249 302 3296 cg07267600 12 2750053 CACNA1 C 2.95x10* 5.37x10-8 4.51x10* 6.91x10* 6508 250 733 367 cg00138407 3 47386505 KLHL18 1.49x10"1 5.58x10"8 1.50x10* 7.81x10-" 103213 251 184 2002 cg10158598 7 47344579 TNS3 1.62x10-2 5.69x10-8 2.74x10 7 2.51x10"· 19459 252 366 1219 cg01126162 10 5734985 FAM208B 6.55x102 5.76x10* 3.68x10* 1.00x10* 54219 253 219 2243 cg12728588 1 36025489 NCDN 1.97x103 5.91x10* 9.52x10* 2.67X10"1 5130 254 163 1254 eg 10975863 14 68830704 RAD51 B 7.86x10-2 5.98x10* 1.06x10* 2.36x10* 62347 255 505 271 cg24129923 17 7814251 CHD3 2.08x10* 6.05x10* 9.75x10"7 5.73x10"7 5304 256 495 175 cg11551560 15 70528789 NA 5.63x10^ 6.23x10* 5.95x107 1.24X10 7 2549 257 448 111 eg 13300580 1 27440539 SLC9A1 2.95x10^ 6.27x10* 1.47x10-7 6.78x10* 1849 258 311 744 cg06721473 3 50355416 HYAL2 4.12X10-1 6.35x10-8 7.45x10"7 9.70x10* 227230 259 468 405
cg10013455 10 134400191 INPP5A 1.32x10"3 6.41x10"8 6.63x10-6 5.84X10-6 4067 260 831 346 cg05901 196 10 61664954 CCDC6 4.35x10"1 6.59X10-8 3.60x10"6 2.41x10^ 236888 261 686 273 cgO 1526748 3 191930926 FGF12 4.94x102 6.59x10^ 6.24x105 5.98x108 43634 262 1696 87 cg04523589 3 48265146 CAMP 1.86x10"3 6.63x10-* 4.01Χ10 9 8.68X10-6 4948 263 132 391 cg09786420 1 154583389 ADAR 1.58X10-1 6.78X10"8 2.34x107 6.55x10"7 107771 264 349 184 cg04074945 11 46071833 PHF21A 9.94x1 σ3 6.88Χ10-8 2.09Χ10 5 5.46x10-8 14077 265 1 185 85 cg03963853 16 4732369 MGRN1 2.10x10"1 6.91X10-8 1.38X10"4 7.87x10"7 135163 266 2253 197 cg01599714 8 142285223 NA 2.03x10 1 6.97X10-8 1.23X10-6 8.89X10-6 131401 267 524 395 cg24067911 6 16729610 ATXN1 3.12X10-2 7.01X1 0-8 8.74x10 1.37x10-5 30900 268 905 459 cg01616956 2 232393196 NMUR1 3.80x10"7 7.04X10-8 1.75x107 4.83x10"4 109 269 329 1626 cg21121843 4 3203982 HTT 1.48X10"3 7.28x10 6.32x10-6 1.80X10-6 431 270 816 247 eg 14849578 12 125282480 SCARB1 5.59x10-3 7.69X10-8 2.69x10'9 1.50x10^ 9696 271 120 989 cg23044270 3 43659607 ANO10 7.80x10-3 7.78X10-8 1.88x10-5 1.91 10-7 12024 272 1 147 127 cg17821 158 19 4090395 MAP2K2 3.27x10 7.82x10 4.44X10-6 1.69X10-6 6920 273 731 243 cg03295554 11 128395450 ETS1 1.27x10"1 7.89x10 5.95x10-'° 5.84x10^ 90952 274 84 1750 cg03819286 16 4673974 MGRN1 1.32x10-2 8.03x10-8 5.14x10'9 4.25x10-5 16978 275 143 639 cg22985172 6 151258337 MTHFD1 L 3.23X10 3 8.11x 0 1.42X10-6 6.68x10"7 6865 276 542 185 cg03466342 12 52753899 KRT85 4.70x10-3 8.14x10 3.54x10 5.46x10-5 8633 277 218 698 cg05931265 11 122527736 UBASH3B 9.53x10 1 8.14x10 2.81X10-6 7.12X10"4 432601 278 641 1928 cg2 066748 19 46005805 NA 1.23Χ102 8.20Χ10-8 3.33x10"7 4.14x10 16134 279 390 312 cg22690339 6 38249061 BTBD9 3.48x10-1 8.23X10-8 4.93x10-8 2.53x10"* 199671 280 239 1222 eg 15028232 1 234871410 NA 1.47x10'1 8.33x10 3.60x105 4.68x10^ 101886 281 1416 321 cg26572392 10 24496943 KIAA1217 2.77x103 8.39x10 8 1.39x109 1.33x10^ 6251 282 99 943 cg11888982 8 1 16231420 NA 6.34x10-2 8.45x10-8 1.70x10 s 2.05x10s 52870 283 1119 260 cg08539067 3 49395985 GPX1 4.08x10 8.46x10 9.09x10'7 1.13X10-5 7888 284 486 435 eg 14081226 10 5563419 NA 2.21 x10"1 8.66x10-8 3.97X10-6 1.20x10-5 140713 285 707 441 cg22881435 8 37732086 RAB11 FIP1 1.28X10"6 8.67x10 4.38X10-6 3.99x10-5 178 286 724 627 cg21281007 12 5939611 AN02 7.78x10 5 8.81x10 8.85x10^ 1.91x10-* 966 287 273 1097 cg04782146 16 85112370 KIAA0513 3.10X10"1 8.84x10 2.65x10"9 1.11x10-5 182768 288 119 432 cg07058694 12 1 1 1909145 ATXN2 1.51x10"1 8.87Χ10-8 5.33x10"7 6.14X10-6 104258 289 440 358 cg0818 850 16 17200244 XYLT1 9.26x10 9.03x10 2.11x10 1.16X10-3 13402 290 200 2420 cg04202511 16 681 17991 NFATC3 2.48x10-1 9.06X10 8 1.29X10-6 8.55x10"7 153695 291 529 201 cg0829511 1 5 133866097 JADE2 4.91X102 9.08x10 1.46x10 5.53x105 43447 292 182 702
cg23039250 1 223308238 TLR5 4.30x10"5 9.20Χ10-8 3.76x107 9.82x109 757 293 401 53 cg05279866 8 37378355 NA 7.57x10^ 9.51 x10"8 4.74x10 5.64x10'7 2986 294 234 1 2 eg 17890233 7 6465684 DAG LB 1.47x10-2 9.70x10-8 1.20x10"5 1.69X10-6 18332 295 1006 242 cg23902264 1 241815413 WDR64 7.73x10"· 9.74x10"8 3.56x10 s 8.33Χ108 3019 296 1410 96 cg03541887 1 248903323 LYPD8 2.45x10 1 9.74X10-8 3.11x10 s 3.08x10 s 152329 297 212 578 cg05914060 11 20478861 PRMT3 7.30x10"5 9.81X10-8 1.52X10-6 1.31X10-7 944 298 556 114 cg21477985 12 56732427 IL23A 3.62x10"1 1.00x10-7 3.63x107 8.26x10"6 205787 299 399 384 cg02797539 17 72740524 RAB37 3.75x1 O"1 1.02x107 4.42x10-8 8.18x1 O^ 21 1059 300 229 381 cg15112006 12 93568577 NA 1.40x10"1 1.06x10-7 2.45X10-6 3.29x10 s 98307 301 621 590 cg24002003 15 101668143 NA 3.56x10"4 1.07X107 5.44x10"7 3.76x10'5 2033 302 441 615 eg 11969064 8 1845598 ARHGEF10 2.00x10 1 1.08X10'7 6.04x10 s 1.44x107 130109 303 1676 1 17
Example 3: Linear Discriminant Analysis (LDA)
In the present invention, Linear Discriminant Analysis (LDA) was used to predict the likelihood of a sample coming from a person with Crohn's disease.
An overview of the LDA technique, and how it may be performed as part of the present invention, is presented below.
Techniques other than LDA can be used to perform such predictions in the present invention. In particular the following may be used: linear classification; and machine learning techniques. Examples of such techniques include: generalized linear model or logistic (or probit) regression; multivariate multiple regression (which may be used to predict multiple outcome variables, such as chance of hospitalisation/surgery/extraintestinal symptoms); multinomial logistic regression (which may be used to classify into more groups, such as Crohn's, Ulcerative colitis, irritable bowel syndrome & other disorders).
The LDA technique In LDA, each sample is associated with certain observations. In the present invention, the observations include at least two methylation values, but additional information may also be included (for example, including further methylation values, patient age or a binary classifier of the patient gender, smoking status etc.). Each sample is classified as belonging to one of two groups {i.e. Crohn's and non-Crohn's), which are assigned the values "1 " and "0".
A collection of samples (known as "a training set" or "a discovery cohort") is initially examined, in which the group each sample belongs to is known. That cohort is used to calculate the numbers on which a model is based - that model can then be applied to a further set where the diagnosis is known (to test its accuracy) and/or to samples where the diagnosis is unknown.
Central to classifying any one sample into one of the two groups is a linear combination of the observations (methylation values). A linear combination is an expression which multiplies each observation by a constant and sums the results - i.e. LC= (observation x constant-i) + (observation x constant.2). The constants are calculated using the training set, to maximise the ratio of the difference between group means, and the standard deviation within the groups - in practice, the mean value of the linear combination will be as different as possible for Crohn's compared to non-Crohn's, but the variation within the Crohn's group and the variation within the non- Crohn's group will be as small as possible. The technique is well-established (see, for example, Fisher RA, The use of multiple measurements in taxonomic problems (1936), Annals of Eugenics, 7(2): 179-188.
Examples are shown below.
M values for the probes cg17501210 (RPS6KA2) and eg 26599989 (TOLLIP) in a cohort of children. The value for each probe overlap between the two groups, but in considering the M values of both probes, the groups can be seen to separate.
Δ Non-Crohn's
M values • Crohn's
0.2 0.4 0.6 0.8 1.2
cg26599989 M value
Calculating the linear combination of the observations for each sample transforms the data into one dimension. A threshold can be calculated to determine which group a sample belongs to based on the linear combination of its observations (-2.08 in the example), or all values can be shifted by this value to set the threshold to zero.
Δ Non-Crohn's
Figure imgf000127_0001
-6 -4 -2 0 2 4
Value of linear combination
In this case, as the model is very good, the two groups do not overlap. However, to give more useful information, it may be beneficial to calculate the probability that the diagnosis is Crohn's, given the value of the linear discriminant function. The steps involved in calculating probability are known to those skilled in the art - computer packages are available for such calculations (such as the predict. Ida function in the R package, MASS).
Δ Non-Crohn's
Probability of Crohn's • Crohn's
Figure imgf000127_0002
-4 -2 0 2 4 6
Value of linear discriminant function
This gives more useful information than a simple positive or negative result as the result of a threshold when the value of the linear discriminant function is near zero.
Constants are calculated from the learning set. As the test is performed on more individuals where the diagnosis is known, the constants may be adjusted to improve the accuracy of the model. Exemplary data - The following pages provide exemplary data obtained from the experiments of Example 1 and demonstrate how LDA is used in the present invention.
1. Experimental data
genel probel gene2 probe2 sens spec ppv npv scalingl scaling2 mean a mean b mean c m
1 TOLLIP cg26599989 RPS6KA2 cg17501210 0.941176 1 1 0.947368 3.941008 -2.01668 0.290418 0.616618 2.698046 1.3
2 SEPT9 cgO 1749539 RPS6KA2 cg17501210 0.941176 0.944444 0.941176 0.944444 2.939254 -1.84716 2.539978 3.15555 2.698046 1.3
3 TOLLIP cg26599989 MY01E cg08423142 0.941176 0.944444 0.941176 0.944444 3.332436 -2.31909 0.290418 0.616618 3.713528 2.7
4 RPS6KA2 cg17501210 YWHAE cg06219337 0.941176 0.944444 0.941176 0.944444 -1.59582 3.213322 2.698046 1.329427 2.073447 2.5
5 RPS6KA2 cg17501210 NA cg09304397 0.941176 0.944444 0.941176 0.944444 -2.06218 3.514457 2.698046 1.329427 1.150233 1.5
6 VMP1 eg 12054453 MY01 E cg08423142 0.882353 1 1 0.9 -1.22375 -1.47336 -0.95168 -2.47721 3.713528 2.7
7 YWHAE cg06219337 ARHGEF3 cg04389058 0.941176 0.888889 0.888889 0.941176 2.912356 -2.47722 2.073447 2.553819 1.383525 0.6
8 ARHGEF3 eg 04389058 NA cg09304397 0.941176 0.888889 0.888889 0.941176 -3.05786 2.24377 1.383525 0.630266 1.150233 1.5
9 TOLLIP cg26599989 ARHGEF3 cg04389058 0.882353 0.944444 0.9375 0.894737 3.862936 -3.34344 0.290418 0.616618 1.383525 0.6
10 VMP1 eg 12054453 TOLLIP cg26599989 0.823529 1 1 0.857143 -1.68697 3.732692 -0.95168 -2.47721 0.290418 0.6
11 VMP1 eg 12054453 ITGB2 eg 13315706 0.823529 1 1 0.857143 -1.26453 3.402054 -0.95168 -2.47721 -0.86043 -0.
12 VMP1 eg 12054453 NA cg09304397 0.823529 1 1 0.857143 -1.56597 1.844683 -0.95168 -2.47721 1.150233 1.5
13 TOLLIP cg26599989 VMP1 eg 16936953 0.823529 1 1 0.857143 3.345292 -1.59823 0.290418 0.616618 0.377681 -0.
14 VMP1 eg 16936953 MY01E cg08423142 0.823529 1 1 0.857143 -1.1164 -1.63468 0.377681 -0.99497 3.713528 2.7
15 NA eg 12992827 RPS6KA2 cg17501210 0.882353 0.888889 0.882353 0.888889 -1.44062 -1.29368 1.837089 0.416899 2.698046 1.3
16 TOLLIP cg26599989 NA cg09304397 0.882353 0.888889 0.882353 0.888889 3.420445 2.902505 0.290418 0.616618 1.150233 1.5
17 V P1 eg 12054453 SEPT9 cg01749539 0.823529 0.944444 0.933333 0.85 -1.50765 2.761355 -0.95168 -2.47721 2.539978 3.
18 VMP1 eg 12054453 YWHAE cg06219337 0.823529 0.944444 0.933333 0.85 -1.29164 2.900395 -0.95168 -2.47721 2.073447 2.5
19 VMP1 eg 16936953 YWHAE cg06219337 0.823529 0.944444 0.933333 0.85 -1.24404 3.235064 0.377681 -0.99497 2.073447 2.5
20 V P1 eg 16936953 NA cg09304397 0.823529 0.944444 0.933333 0.85 -1.54392 2.350898 0.377681 -0.99497 1.150233 1.5
21 RPS6KA2 eg 17501210 NA cg09349128 0.823529 0.944444 0.933333 0.85 -1.55353 -1.16812 2.698046 1.329427 -1.19916 -2.
22 RPS6KA2 cg17501210 HEATR2 eg 10472711 0.823529 0.944444 0.933333 0.85 -1.5552 -1.50824 2.698046 1.329427 1.34885 0.5
23 VMP1 eg 12054453 ARHGEF3 cg04389058 0.764706 1 1 0.818182 -1.09368 -1.82821 -0.95168 -2.47721 1.383525 0.6
24 VMP1 eg 16936953 RPS6KA2 cg17501210 0.764706 1 1 0.818182 -0.94326 -1.46965 0.377681 -0.99497 2.698046 1.3
25 RPS6KA2 cg17501210 ARHGEF3 cg04389058 0.764706 1 1 0.818182 -1.36883 -1.96681 2.698046 1.329427 1.383525 0.6
26 TOLLIP cg26599989 SLC10A6 eg 18860310 0.823529 0.888889 0.875 0.842105 4.004723 -2.06647 0.290418 0.616618 2.010164 0.7
27 TOLLIP cg26599989 HEATR2 eg 10472711 0.823529 0.888889 0.875 0.842105 4.544029 -2.88366 0.290418 0.616618 1.34885 0.5
28 V P1 eg 12054453 VMP1 eg 16936953 0.764706 0.944444 0.928571 0.809524 -1.58662 -0.33175 -0.95168 -2.47721 0.377681 -0.
29 V P1 eg 12054453 RPS6KA2 eg 17501210 0.764706 0.944444 0.928571 0.809524 -1.08611 -1.35579 -0.95168 -2.47721 2.698046 1.3
30 VMP1 eg 12054453 NA cg09349128 0.764706 0.944444 0.928571 0.809524 -1.33945 -0.88034 -0.95168 -2.47721 -1.19916 -2.
31 VMP1 eg 16936953 ITGB2 eg 13315706 0.764706 0.944444 0.928571 0.809524 -1.24353 3.657794 0.377681 -0.99497 -0.86043 -0.
32 VMP1 eg 16936953 NA cg09349128 0.764706 0.944444 0.928571 0.809524 -1.19411 -1.19015 0.377681 -0.99497 -1.19916 -2.
33 V P1 eg 12054453 SLC10A6 eg 18860310 0.705882 1 1 0.782609 -1.13396 -1.22895 -0.95168 -2.47721 2.010164 0.7
34 VMP1 eg 12054453 HEATR2 eg 10472711 0.705882 1 1 0.782609 -1.35694 -1.14541 -0.95168 -2.47721 1.34885 0.5
35 VMP1 eg 16936953 HEATR2 eg 10472711 0.705882 1 1 0.782609 -1.19798 -1.51578 0.377681 -0.99497 1.34885 0.5
36 SEPT9 cg01749539 ARHGEF3 eg 04389058 0.882353 0.777778 0.789474 0.875 2.765214 -2.92883 2.539978 3.15555 1.383525 0.6
37 NA cg12992827 SEPT9 cgO 1749539 0.823529 0.833333 0.823529 0.833333 -1.71657 2.296938 1.837089 0.416899 2.539978 3.
38 NA eg 12992827 TOLLIP cg26599989 0.823529 0.833333 0.823529 0.833333 -2.11519 4.083486 1.837089 0.416899 0.290418 0.6
39 TOLLIP cg26599989 YWHAE cg06219337 0.823529 0.833333 0.823529 0.833333 2.726623 4.18014 0.290418 0.616618 2.073447 2.5
40 VMP1 eg 12054453 NA eg 12992827 0.764706 0.888889 0.866667 0.8 -0.88966 -1.43551 -0.95168 -2.47721 1.837089 0.4
41 NA cg12992827 VMP1 eg 16936953 0.764706 0.888889 0.866667 0.8 -1.58356 -0.81006 1.837089 0.416899 0.377681 -0.
42 ARHGEF3 cg04389058 HEATR2 cg10472711 0.764706 0.888889 0.866667 0.8 -2.55645 -1.27952 1.383525 0.630266 1.34885 0.5
43 SEPT9 cg0 749539 VMP1 eg 16936953 0.705882 0.944444 0.923077 0.772727 3.158488 -1.6169 2.539978 3.15555 0.377681 -0.
44 VMP1 eg 16936953 SLC10A6 cg18860310 0.705882 0.944444 0.923077 0.772727 -1.09138 -1.44348 0.377681 -0.99497 2.010164 0.7
45 VMP1 eg 16936953 ARHGEF3 cg04389058 0.705882 0.944444 0.923077 0.772727 -0.93279 -2.31712 0.377681 -0.99497 1.383525 0.6
46 RPS6KA2 cg17501210 FA 110A cg09645475 0.705882 0.944444 0.923077 0.772727 -1.72948 3.444598 2.698046 1.329427 -1.60709
47 SEPT9 cg01749539 MY01E cg08423142 0.882353 0.722222 0.75 0.866667 2.715845 -2.08215 2.539978 3.15555 3.713528 2.7
48 MY01 E cg08423142 NA cg09304397 0.882353 0.722222 0.75 0.866667 -2.28277 2.654033 3.713528 2.763641 1.150233 1.5
49 NA eg 12992827 NA cg09304397 0.823529 0. // / / /8 0.777778 0.823529 -1 89888 1.59851 1.837089 0.416899 1.150233 1.5
50 SEPT9 cg01749539 TOLLIP cg26599989 0.823529 0.777778 0.777778 0.823529 3.022355 2.497763 2.539978 3.15555 0.290418 0.6
51 TOLLIP cg26599989 ITGB2 eg 13315706 0.823529 0.777778 0.777778 0.823529 3.280168 4.466861 0.290418 0.616618 -0.86043 -0.
52 YWHAE cg06219337 NA cg09304397 0.823529 0.777778 0.777778 0.823529 4.25228 1.66682 2.073447 2.553819 1.150233 1.5
53 MY01 E cg08423142 HEATR2 eg 10472711 0.823529 0.777778 0.777778 0.823529 -1.75071 -1.56523 3.713528 2.763641 1.34885 0.5
54 NA cg09349128 NA cg09304397 0.823529 0.777778 0.777778 0.823529 -1.79235 2.556281 -1.19916 -2.29872 1.150233 1.5
55 NA eg 12992827 Y01E cg08423142 0.764706 0.833333 0.8125 0.789474 -1.58302 -1.1569 1.837089 0.416899 3.713528 2.7
56 NA eg 12992827 ARHGEF3 cg04389058 0.764706 0.833333 0.8125 0.789474 -1.49107 -1.64926 1.837089 0.416899 1.383525 0.6
57 NA cg12992827 NA cg09349128 0.764706 0.833333 0.8125 0.789474 -2.3452 0.181844 1.837089 0.416899 -1.19916 -2.
58 NA eg 12992827 HEATR2 eg 10472711 0.764706 0.833333 0.8125 0.789474 -2.08811 -0.20372 1.837089 0.416899 1.34885 0.5
59 SEPT9 cg01 49539 HEATR2 eg 10472711 0.764706 0.833333 0 8125 0.789474 3.165815 -2.5014 2.539978 3.15555 1.34885 0.5
60 TOLLIP cg26599989 NA cg09349128 0.764706 0.833333 0.8125 0.789474 5.018556 -2.28948 0.290418 0.616618 -1.19916 -2.
61 V P1 eg 16936953 FAM110A cg09645475 0.764706 0.833333 0.8125 0.789474 -1.33475 3.246965 0.377681 -0.99497 -1.60709
62 RPS6KA2 cg17501210 MY01E cg08423142 0.764706 0.833333 0.8125 0.789474 -1.46634 -1.29896 2.698046 1.329427 3.713528 2.7
63 YWHAE eg06219337 HEATR2 eg 1047271 1 0.764706 0.833333 0.8125 0.789474 3.680396 -2.07081 2.073447 2.553819 1.34885 0.5
64 ARHGEF3 eg 04389058 NA cg09349128 0.764706 0.833333 0.8125 0.789474 -2.50607 -1.02967 1.383525 0.630266 -1.19916 -2.
65 RPS6KA2 cg17501210 ITGB2 cg13315706 0.705882 0.888889 0.857143 0.761905 -1.50159 3.348179 2.698046 1.329427 -0.86043 -0.
66 TOLLIP cg26599989 FA 110A cg09645475 0.823529 0.722222 0.736842 0.8125 3.159982 3.984736 0.290418 0.616618 -1.60709
67 Y01 E cg08423142 ARHGEF3 cg04389058 0.823529 0.722222 0.736842 0.8125 -1.48643 -2.32594 3.713528 2.763641 1.383525 0.6
68 NA eg 12992827 FAM110A cg09645475 0.764706 0.777778 0.764706 0.777778 -1.65353 2.694296 1.837089 0.416899 -1.60709
69 NA eg 12992827 YWHAE cg06219337 0.764706 0.777778 0.764706 0.777778 -1.61182 2.575125 1.837089 0.416899 2.073447 2.5
70 SEPT9 cg01749539 SLC10A6 eg 18860310 0.764706 0.777778 0.764706 0.777778 2.604983 -1.71135 2.539978 3.15555 2.010164 0.7
71 SEPT9 cg01749539 NA cg09349128 0.764706 0.777778 0.764706 0.777778 2.717099 -1.61245 2.539978 3.15555 -1.19916 -2.
72 SEPT9 cg01 49539 NA cg09304397 0.764706 0. /////8 0.764706 0.777778 3.055906 1.848765 2.539978 3.15555 1.150233 1.5
73 SLC10A6 eg 18860310 YWHAE cg06219337 0.764706 0.777778 0.764706 0.777778 -1.60307 3.367051 2.010164 0.782997 2.073447 2.5
74 SLC10A6 eg 18860310 HEATR2 cg1047271 1 0.764706 0.777778 0.764706 0.777778 -1.92435 -0.52288 2.010164 0.782997 1.34885 0.5
75 SLC10A6 cg18860310 NA cg09304397 0.764706 0.777778 0.764706 0.777778 -1.86361 1.813013 2.010164 0.782997 1.150233 1.5
76 YWHAE cg06219337 NA cg09349128 0.764706 0.777778 0.764706 0.777778 3.420759 -1.41896 2.073447 2.553819 -1.19916 -2.
77 VMP1 eg 12054453 FAM1 10A cg09645475 0.705882 0.833333 0.8 0.75 -1.36795 3.044155 -0.95168 -2.47721 -1.60709
78 RPS6KA2 eg 17501210 SLC10A6 cg18860310 0.705882 0.833333 0.8 0.75 -1.45193 -1.35303 2.698046 1.329427 2.010164 0.7
79 SLC10A6 cg18860310 MY01 E cg08423142 0.705882 0.833333 0.8 0.75 -1.44334 -1.60016 2.010164 0.782997 3.713528 2.7
80 NA cg09349128 HEATR2 cg10472711 0.705882 0.833333 0.8 0.75 -1.2472 -1.6351 -1.19916 -2.29872 1.34885 0.5
81 YWHAE cg06219337 MY01E cg08423142 0.823529 0.666667 0.7 0.8 3.035353 -1.74078 2.073447 2.553819 3.713528 2.7
82 Y01 E cg08423142 ITGB2 eg 133 5706 0.823529 0.666667 0.7 0.8 -1.81661 3.663792 3.713528 2.763641 -0.86043 -0.
83 SEPT9 cgO 1749539 YWHAE cg06219337 0.764706 0.722222 0.722222 0.764706 2.079383 3.262307 2.539978 3.15555 2.073447 2.5
84 HEATR2 eg 10472711 NA cg09304397 0.764706 0.722222 0.722222 0.764706 -2.41024 2.519061 1.34885 0.540018 1.150233 1.5
85 NA eg 12992827 SLC10A6 eg 18860310 0.705882 0.777778 0.75 0.736842 -1.69123 -0.6327 1.837089 0.416899 2.010164 0.7
86 NA eg 12992827 ITGB2 cg13315706 0.705882 0.777778 0.75 0.736842 -1.48237 2.561252 1.837089 0.416899 -0.86043 -0.
87 SLC10A6 eg 18860310 ARHGEF3 cg04389058 0 705882 0.777778 0.75 0.736842 -1.23526 -2.08834 2.010164 0.782997 1.383525 0.6
88 FAM110A cg09645475 MY01 E cg08423142 0.705882 0.777778 0.75 0.736842 3.556101 -2.08609 -1.60709 -1.108 3.713528 2.7
89 FAM110A cg09645475 HEATR2 cg1047271 1 0.705882 0.777778 0.75 0.736842 3.457804 -2.11062 -1.60709 -1.108 1.34885 0.5
90 ARHGEF3 cg04389058 ITGB2 cg13315706 0.823529 0.611111 0.666667 0.785714 -2.2943 3.236406 1.383525 0.630266 -0.86043 -0.
91 SLC10A6 eg 18860310 ITGB2 cg13315706 0.764706 0.666667 0.684211 0.75 -1.30226 3.069463 2.010164 0.782997 -0.86043 -0.
92 FAM1 10A cg09645475 YWHAE cg062 9337 0.764706 0.666667 0.684211 0.75 2.915815 3.238404 -1.60709 -1.108 2.073447 2.5
93 ITGB2 eg 13315706 HEATR2 eg 10472711 0.764706 0.666667 0.684211 0.75 3.591739 -1.66863 -0.86043 -0.32912 1.34885 0.5
94 FAM110A cg09645475 ARHGEF3 eg 04389058 0.705882 0.722222 0.705882 0.722222 3.183853 -2.70013 -1.60709 -1.108 1.383525 0.6
95 FAM110A cg09645475 NA cg09349128 0.705882 0.722222 0.705882 0.722222 3.172559 -1.44114 -1.60709 -1.108 -1.19916 -2.
96 YWHAE cg06219337 ITGB2 cg13315706 0.705882 0 722222 0.705882 0.722222 3.320761 3.710152 2.073447 2.553819 -0.86043 -0.
97 MY01 E cg08423142 NA cg09349128 0.705882 0 722222 0.705882 0.722222 -1.75002 -1.21255 3.713528 2.763641 -1.19916 -2.
98 SLC10A6 eg 18860310 NA cg09349128 0.647059 0.777778 0.733333 0.7 -1.60783 -0.80825 2.010164 0.782997 -1.19916 -2.
99 SEPT9 cg01749539 ITGB2 eg 13315706 0.764706 0.611111 0.65 0.733333 2.410479 3.830239 2.539978 3.15555 -0.86043 -0.
100 SLC10A6 eg 18860310 FAM110A cg09645475 0.647059 0.722222 0.6875 0.684211 -1.55827 2.882917 2.010164 0.782997 -1.60709
101 ITGB2 cg13315706 NA cg09349128 0.647059 0.722222 0.6875 0.684211 3.559295 -1.21762 -0.86043 -0.32912 -1.19916 -2.
102 SEPT9 cg01749539 FA 110A cg09645475 0.705882 0.611111 0.631579 0.6875 2.184586 3.009803 2.539978 3.15555 -1.60709
103 FAM110A cg09645475 ITGB2 cg13315706 0.705882 0.611111 0.631579 0.6875 3.159974 3.79949 -1.60709 -1.108 -0.86043 -0.
104 FAM110A cg09645475 NA cg09304397 0.705882 0.611111 0.631579 0.6875 3.965283 1.710965 -1.60709 -1.108 1.150233 1.5
105 ITGB2 eg 13315706 NA cg09304397 0.705882 0.555556 0.6 0.666667 4.655098 0.924636 -0.86043 -0.32912 1.150233 1.5
PR0BE1 PROBE2
Disease FALSE mean a mean c
TRUE mean b mean d
2. LDA on experimental data
Model
Details
genel probel gene2 probe2 sensitivity specificity
TOLLIP cg26599989 RPS6KA2 cgl7501210 0.94 1 scalingl scaling2 mean a mean b mean c mean d
3.94 -2.02 0.29 0.62 2.70 1.33
PROBE1 PROBE2 Prior Probabilities
on-Crohn's mean a mean c Non-Crohn's
Crohn's mean b mean d Crohn's
Example Values
M Values
Linear
Crohn's probel probe2 combination Centred on zero Probability of Croh
TRUE 0.89 2.01 -0.54 1.54 1.00
TRUE 1.06 1.29 1.59 3.66 1.00
TRUE 0.52 1.59 -1.17 0.91 0.99
FALSE 0.37 3.16 -4.89 -2.81 0.00
TRUE 0.72 2.34 -1.89 0.18 0.83
FALSE 0.17 2.65 -4.69 -2.61 0.00
TRUE 1.09 1.00 2.28 4.36 1.00
FALSE 0.26 2.43 -3.89 -1.81 0.00
TRUE 0.46 1.34 -0.89 1.19 1.00
FALSE 0.05 2.91 -5.68 -3.60 0.00
TRUE 0.40 1.05 -0.53 1.55 1.00
FALSE 0.30 2.57 -4.01 -1.93 0.00
TRUE 0.44 1.24 -0.79 1.29 1.00
FALSE 0.39 3.41 -5.33 -3.26 0.00
TRUE 0.62 1.14 0.15 2.22 1.00
FALSE 0.45 2.51 -3.29 -1.21 0.02
TRUE 0.58 2.12 -1.98 0.09 0.78
FALSE 0.27 2.74 -4.48 -2.40 0.00
TRUE 0.51 1.36 -0.75 1.33 1.00
TRUE 0.72 1.81 -0.83 1.25 1.00
FALSE 0.38 2.08 -2.69 -0.61 0.17
TRUE 0.31 0.50 0.21 2.29 1.00
FALSE 0.22 2.82 -4.81 -2.73 0.00
TRUE 0.56 1.55 -0.93 1.14 1.00
FALSE 0.38 2.95 -4.44 -2.36 0.00
TRUE 0.43 0.45 0.77 2.85 1.00
FALSE 0.22 2.34 -3.83 -1.75 0.00
TRUE 0.63 1.04 0.39 2.47 1.00
FALSE 0.35 2.83 -4.32 -2.24 0.00
TRUE 0.55 0.76 0.65 2.72 1.00
FALSE 0.25 2.37 -3.80 -1.73 0.00
Attached Figure 6 provides M values for preferred models of the invention.

Claims

A method for determining the presence of Inflammatory Bowel Disease in an individual, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- determining the presence of Inflammatory Bowel Disease in the individual on the basis of the methylation state at the two or more CpG sites.
A method for predicting the likelihood of an individual contracting Inflammatory Bowel Disease, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3; and
- predicting the likelihood of the individual contracting Inflammatory Bowel Disease on the basis of the methylation state at the two or more CpG sites.
A method for selecting a treatment for an individual suspected of having Inflammatory Bowel Disease, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual;
- determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3;
determining the presence of Inflammatory Bowel Disease in the individual on the basis of the methylation state at the two or more CpG sites; and
selecting a treatment for the individual.
A method for predicting the response to anti-Inflammatory Bowel Disease treatment of an individual suspected of having Inflammatory Bowel Disease, comprising the steps of:
- providing a sample comprising leukocyte DNA from the individual; - determining the methylation state at two or more CpG sites in the leukocyte DNA, wherein each CpG site is selected from those in Table 3;
determining the presence of Inflammatory Bowel Disease in the individual on the basis of the methylation state at the two or more CpG sites; and
- predicting the response to anti-Inflammatory Bowel Disease treatment of the individual.
The method according to any preceding claim wherein the individual is a child.
The method according to any preceding claim wherein the individual is an adult.
The method according to any preceding claim wherein the individual is a human.
The method of any preceding claim, wherein the step of determining the methylation state comprises determining the methylation state at three or more CpG sites in the leukocyte DNA, for example, four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more, or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or more, or 110 or more, or 120 or more, or 130 or more, or 140 or more, or 150 or more, or 160 or more, or 165, CpG sites in the leukocyte DNA.
The method according to any of Claims 1 and 3 to 8, wherein the step of determining the presence of Inflammatory Bowel Disease in the individual comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
The method according to any of Claims 2 and 5 to 8, wherein the step of predicting the likelihood of the individual contracting Inflammatory Bowel Disease comprises comparing the methylation state of the two or more CpG sites in the individual with the methylation state of corresponding CpG sites in the leukocyte DNA from one or more control individual.
1 1. The method according to Claim 9 or 10, wherein the one or more control individual has Inflammatory Bowel Disease.
12. The method according to Claim 9 or 10, wherein the one or more control individual does not have Inflammatory Bowel Disease.
13. The method of any preceding claim, wherein the CpG sites are associated with a gene selected from those listed in Table 4. 14. The method of any preceding claim, wherein the CpG sites are associated with two or more genes selected from those listed in Table 4, for example, three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more genes selected from those listed in Table 4.
15. The method of any preceding claim, wherein each of the CpG sites is associated with a different gene selected from those listed in Table 4.
16. The method according to any preceding claim, wherein each CpG site is located in a regulatory region of the associated gene.
17. The method of any of any preceding claim, wherein the CpG sites are located in a defined genetic region. 18. The method of any of Claims to 1 to 12, wherein the CpG sites are within a genetic locus selected from those listed in Table 5.
19. The method of Claim 18, wherein the CpG sites are within two or more genetic loci selected from those listed in Table 5, for example, three or more, or four or more, or five or more; or six or more, or seven or more, or eight or more, or nine or more, or ten or more, genetic loci selected from Table 5.
20. The method of Claim 18, wherein each of the CpG sites is associated with a different genetic locus selected from those listed in Table 5.
21. The method according to any preceding claim wherein, preferably when the individual is a child, the CpG sites are selected from the group comprising: - 1 1 :1297087 (TOLLIP); and
- 17:75473969 (SEPT9); and
- 6:166970252 (RPS6KA2); and
- 15:59588622 (MY01 E); and
- 17: 1278466 (YWHAE); and
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1); and
- 17:57918262 (MIR21); and
- 3:57041402 (ARHGEF3); and
- 3:101901234; and
- 4:87752504 (SLC10A6); and
- 22:50327986; and
- 7:797592 (HEATR2); and
- 2:235580461 ; and
- 21 :46341054 (ITGB2).
The method according to Claim 21 wherein the two or more CpG sites are selected from the group comprising:
- 11 :1297087 (TOLLIP) and 6:166970252 (RPS6KA2); or
- 17:75473969 (SEPT9) and 6: 166970252 (RPS6KA2); or
- 11 :1297087 (TOLLI P) and 15: 59588622 (MY01 E); or
- 6:166970252 (RPS6KA2); and 17: 278466 (YWHAE); or
- 6:166970252 (RPS6KA2); and cg0930439; or
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1 ), and 15:59588622 ( Y01 E); or
- 17:1278466 (YWHAE) and 3:57041402 (ARHGEF3).
The method according to any preceding claim wherein, preferably when the individual is a child, the gene is selected from the group comprising: TOLLiP SEPT9; RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3 3: 101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2; SBN02 TNFSF10; CALHM1 ; CPVL; CSGALNACT1; NEDD9; SPARC; SUB1; TRAM2 KCNAB2; BBS9; AGL; FRMD4A; FKBP5; AIM2; BCL3; PHF21A; ANO10; GPRIN3 PALM; ICA1 ; BAHCC1 ; LRRC47; cg01101459; cg02719954; cg04086394 cg12807764; cg19445588; cg20519581 ; cg09349128; cg12992827; cg25114611 cg07398517; cg19821297; cg12582317; cg00053916. The method according to any of Claims 21 to 23 wherein the gene is: TOLLIP.
The method according to Claim 23 wherein, preferably when the individual child, two or more genes are selected from the group comprising:
- TOLLIP and RPS6KA2; or
- SEPT9 and RPS6KA2; or
- TOLLIP and MY01 E; or
- RPS6KA2 and YWHAE; or
- RPS6KA2 and cg0930439; or
- VMP1 and MY01 E; or
- YWHAE and ARHGEF3; or
- cg25114611 and AIM2; or
- cg25114611 and BCL3; or
- cg25114611 and cg01101459; or
- cg25114611 and cg02719954; or
- cg25114611 and cg04086394; or
- cg25114611 and cg09349128; or
- cg25114611 and cg12807764; or
- cg25114611 and cg19445588; or
- cg25114611 and cg19821297; or
- cg25114611 and cg20519581 ; or
- cg25114611 and CPVL; or
- cg25114611 and CSGALNACT1 ; or
- cg2511461 1 and GPRIN3; or
- cg2511461 1 and ICA1 ; or
- cg2511461 1 and MY01 E; or
- cg251 46 1 and NEDD9; or
- cg25114611 and SPARC; or
- cg25114611 and SUB1 ; or
- cg251 4611 and TNFSF10; or
- cg25114611 and TRAM2; or
- AIM2 and BCL3; or
- AIM2 and cg12582317; or
- AIM2 and FKBP5; or
- cg07398517 and BCL3; or
- cg09349128 and cg00053916; or cg09349128 and FKBP5; or
cg09349128 and FRMD4A; or
cg09349128 and PHF21A; or
eg 12992827 and ANO10; or
cg12992827 and BCL3; or
cg12992827 and FRMD4A; or
cg19821297 and PHF21A; or
FRMD4A and PALM; or
GPRIN3 and ITGB2; or
ICA1 and cg09349128; or
PALM and BAHCC1 ; or
PALM and KCNAB2; or
PALM and LRRC47.
The method according to any preceding claim wherein, preferably when the individual is an adult, the CpG sites are selected from the group comprising:
- 17:75473969 (SEPT9); and
- 6:166970252 (RPS6KA2); and
- 15:59588622 (MY01 E); and
- 17:1278466 (YWHAE); and
- 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1); and
- 17:57918262 (MIR21 ); and
3 57041402 (ARHGEF3); and
3 101901234; and
87752504 (SLC10A6); and
- 22:50327986; and
- 7:797592 (HEATR2); and
- 2:235580461 ; and
- 21 :46341054 (ITGB2).
The method according to Claim 26 wherein the two or more CpG sites are selected from the group comprising:
- 17:75473969 (SEPT9) and 6:166970252 (RPS6KA2); or
- 6:166970252 (RPS6KA2); and 17:1278466 (YWHAE); or
- 6:166970252 (RPS6KA2); and cg0930439; or - 17:57915717 and/or 17:57915665 and/or 17:57915773 and/or 17:57915740 and/or 17:57903544 (VMP1 ), and 15:59588622 (MY01 E); or
- 17:1278466 (YWHAE) and 3:57041402 (ARHGEF3).
The method according to any preceding claim wherein, preferably when the individual is an adult, the gene is selected from the group comprising: SEPT9 RPS6KA2; MY01 E; YWHAE; cg0930439; VMP1 ; MIR21 ; ARHGEF3 3:101901234; SLC10A6; 22:50327986; HEATR2; 2:235580461 ; ITGB2; SBN02 TNFSF10; CALHM1 ; CPVL; CSGALNACT1 ; NEDD9; SPARC; SUB1 ; TRAM2 KCNAB2; BBS9; AGL; ICA1 ; FRMD4A; FKBP5; AIM2; BCL3; PHF21A; ANO10 GPRIN3; PALM; ICA1 ; BAHCC1 ; LRRC47; cg01101459; cg02719954 cg04086394; cg12807764; cg19445588; cg20519581 ; cg09349128; cg12992827 cg25114611 ; cg07398517; cg19821297; cg12582317; cg00053916.
The method according to Claim 28 wherein the two or more genes are selected from the group comprising:
- SEPT9 and RPS6KA2; or
- RPS6KA2 and YWHAE; or
- RPS6KA2 and cg0930439; or
- VMP1 and MY01 E; or
- YWHAE and ARHGEF3.
- cg25114611 and AIM2; or
- cg25114611 and BCL3; or
- cg2511461 and cg01 101459; or
- cg251 1461 and cg02719954; or
- cg25114611 and cg04086394; or
- cg25114611 and cg09349128; or
- cg25114611 and cg12807764; or
- cg25114611 and cg19445588; or
- cg25114611 and cg19821297; or
- cg25114611 and cg20519581 ; or
- cg25114611 and CPVL; or
- cg25114611 and CSGALNACT1 ; or
- cg25114611 and GPRIN3; or
- cg25114611 and ICA1 ; or
- cg25114611 and MY01E; or - cg251 14611 and NEDD9; or
- cg251 1461 1 and SPARC; or
- cg251 14611 and SUB1 ; or
- cg251 14611 and TNFSF10; or
- cg251 1461 1 and TRAM2; or
- AIM2 and BCL3; or
- AIM2 and cg12582317; or
- AIM2 and F BP5; or
- cg07398517 and BCL3; or
- cg09349128 and cg00053916; or
- cg09349128 and FKBP5; or
- cg09349128 and FRMD4A; or
- cg09349128 and PHF21A; or
- cg12992827 and ANO10; or
- eg 12992827 and BCL3; or
- eg 12992827 and FRMD4A; or
- cg19821297 and PHF21A; or
- FRMD4A and PALM; or
- GPRIN3 and ITGB2; or
- ICA1 and cg09349128; or
- PALM and BAHCC1 ; or
- PALM and KCNAB2; or
- PALM and LRRC47.
The method according to any preceding claim, wherein the sample is selected from the group consisting of: a blood sample; a serum sample; a plasma sample; a sample of intestinal cells; a biopsy; a stool sample.
The method according to any preceding claim, wherein the leukocyte DNA is obtained from the sample by a method comprising the steps of: (i) cell lysis; (ii) removal of cell membranes and/or cellular RNA and/or cellular protein; (iii) purification of DNA.
The method according to any preceding claim, wherein methyiation state is determined by a method selected from the group comprising: bisulfite conversion and DNA sequencing; methylation-specific restriction enzyme analysis; COBRA analysis; methylation-specific PCR; MethylLight analysis; HeavyMethyl analysis; Quantitative Methylation analysis; Ms-SNuPE analysis.
33. The method of any of Claims 3 and 5 to 32, further comprising the step of treating the individual with the selected treatment.
34. Use of leukocyte DNA from an individual, for determining the presence of Inflammatory Bowel Disease in the individual. 35. Use of leukocyte DNA from an individual, for predicting the likelihood of the individual contracting Inflammatory Bowel Disease.
36. Use of leukocyte DNA from an individual suspected of having Inflammatory Bowel Disease, for selecting a treatment for the individual.
37. Use of leukocyte DNA from an individual suspected of having Inflammatory Bowel Disease for predicting the response to anti-Inflammatory Bowel Disease therapy of the individual. 38. A kit for performing a method according to any of Claims 1 to 33 to a use according to any of Claims 34 to 37, the kit comprising one or more reagent for determining the methylation state at two or more CpG sites in leukocyte DNA, wherein each CpG site is selected from those in Table 3. 39. The kit of Claim 38, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- a pair of oligonucleotide primers for PCR amplification of the polynucleotide sequence at a first CpG site selected from those listed in Table 3; and
- a pair of oligonucleotide primers for PCR amplification of the polynucleotide sequence at a second CpG site selected from those listed in Table 3. 40. The kit of Claim 38, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA; - two or more oligonucleotide primers for methylation-specific PCR amplification of the polynucleotide sequence at a first CpG site selected from those listed in Table 3;
- two or more oligonucleotide primers for methylation-specific PCR amplification of the polynucleotide sequence at a second CpG site selected from those listed in Table 3; and
- optionally, an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation- specific PCR amplification.
The kit of Claim 40, further comprising:
- an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the first CpG site when the first CpG site is not methylated; and
- an oligonucleotide capable of preventing and/or reducing PCR amplification of the polynucleotide sequence at the second CpG site when the first CpG site is not methylated.
The kit of Claim 38 or 39, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- an oligonucleotide primer for determining the polynucleotide sequence at a first CpG site selected from those listed in Table 3; and
- an oligonucleotide primer for determining the polynucleotide sequence at a second CpG site selected from those listed in Table 3.
The kit of Claim 38 or 39, wherein the one or more reagent for determining the methylation state at two or more CpG sites in the leukocyte DNA comprises:
- one or more reagent for bisulfite conversion of the leukocyte DNA;
- a restriction enzyme capable of methylation-specific digestion of the polynucleotide sequence at a first CpG site selected from those listed in Table 3;
- a restriction enzyme capable of methylation-specific digestion of the polynucleotide sequence at a second CpG site selected from those listed in Table 3; optionally, an oligonucleotide probe capable of selectively binding to one or more of the polynucleotide sequences generated by methylation- specific digestion.
44. The kit of any of Claims 38 to 43, further comprising one or more control sample comprising leukocyte DNA in which the first CpG site and/or the second CpG site is methylated.
45. The kit of any of Claims 38 to 43, further comprising one or more control sample comprising leukocyte DNA in which the first CpG site and/or the second CpG site is not methylated.
46. The kit of any of Claims 39 to 45, wherein the one or more reagent for bisulfite conversion of the leukocyte DNA comprises:
a reagent for denaturation of leukocyte DNA;
- a reagent for sulfonation of leukocyte DNA;
- a reagent for desulfonation of leukocyte DNA; and
- a reagent for isolation or purification of bisulfite converted leukocyte DNA.
47. A method according to any of Claims 1 to 33, or a use according to any of Claims 34 to 37, or a kit according to any of Claims 38 to 46, wherein the Inflammatory Bowel Disease is selected from: Crohn's Disease or Ulcerative Colitis.
48. A method or a use or a kit substantially as claimed herein, with reference to the accompanying description and drawings.
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CN113462767A (en) * 2021-07-23 2021-10-01 新开源晶锐(广州)生物医药科技有限公司 FKBP5 gene methylation detection primer and kit based on pyrosequencing technology

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