WO2025109148A1

WO2025109148A1 - Diagnosis of inflammatory bowel disease by dna methylation analysis

Info

Publication number: WO2025109148A1
Application number: PCT/EP2024/083248
Authority: WO
Inventors: Jack Satsangi; Alexandra NOBLE; Alex Adams
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2023-11-24
Filing date: 2024-11-22
Publication date: 2025-05-30
Anticipated expiration: 2026-05-24
Also published as: GB202318002D0

Abstract

The invention relates to methods for diagnosing inflammatory bowel disease (IBD) and for distinguishing between common gastrointestinal disease (principally functional bowel disease/irritable bowel syndrome and coeliac disease) and IBD, especially in children and in subjects with normal levels of C-reactive protein. The methods are based on measuring the methylation level of at least one CpG site in at least one gene.

Description

DIAGNOSIS OF INFLAMMATORY BOWEL DISEASE BY DNA METHYLATION ANALYSIS

Field of the invention

The invention relates to methods for diagnosing inflammatory bowel disease (IBD) and for distinguishing between common gastrointestinal disease (principally functional bowel disease/irritable bowel syndrome and coeliac disease) and IBD, especially in children and in subjects with normal levels of C-reactive protein.

Background of the invention

Inflammatory diseases, such as inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are debilitating chronic inflammatory conditions of the gastrointestinal tract with a relapsing-remitting course. Crohn’s disease and ulcerative colitis are common diseases affecting an estimate of between 0.8 and 1% of the UK population.

The diagnosis of IBD involves assessment of symptoms, together with endoscopic and radiological investigations; these are complemented by the use of certain biomarkers. For example, measurements of faecal calprotectin in stool samples may be used to try and detect IBD, and differentiate from functional disease. However, clinical experience shows that the use of stool samples is not well accepted in clinical settings, in particular in children, and low compliance with the test is common. There is also inter-individual variability and diurnal variation in calprotectin measurements. The test is less accurate in inflammatory bowel disease in the small intestine than in colonic disease. From all these perspectives, and also ease of handling in the laboratory, a diagnosis method that relied on a blood sample rather than a stool sample would be preferred. Clinicians may also use serum C-reactive protein (CRP) measurements to attempt to differentiate IBD from functional disease. However, a significant portion of patients with IBD (up to 50% in Crohn’s disease and higher in ulcerative colitis) do not have elevated CRP levels at diagnosis.

In view of these drawbacks, the current gold standard for diagnosis of IBD (in both children and adults) is endoscopy with histology. It can be appreciated that endoscopsies and biopsies are invasive and costly, and especially inconvenient for the diagnosis of children. There is therefore a need in the art to provide improved diagnostic methods for IBD which are more cost-effective, reliable and/or non-invasive and which further avoid the current drawbacks seen with faecal calprotectin and CRP.

Summary of the invention The present inventors have determined that methylation levels of VMP1, RPS6KA2, CFI and ARHGEF3 are indicative of inflammatory bowel disease (IBD). Moreover, the inventors have determined that these markers can be used to effectively diagnose ulcerative colitis, distinguish between IBD and coeliac disease and detect IBD even in patients who are CRP negative. Accordingly, in a first aspect there is provided a method of diagnosing an inflammatory bowel disease in a subject comprising a step of measuring the methylation level of at least one site in at least one gene selected from the group consisting of VMP1, RPS6KA2, CFI and ARHGEF3 in a biological sample obtained from the subject.

In a second aspect, there is provided a method for ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease comprising a step of measuring the methylation level of at least one site in at least one gene selected from the group consisting of VMP1, RPS6KA2, CFI and ARHGEF3 in a biological sample obtained from the subject.

In a third aspect, there is provided a method of treating inflammatory bowel disease in a subject, the method comprising: a) diagnosing the subject with inflammatory disease by the method of the invention; and b) treating the subject with suitable treatment.

In a fourth aspect, there is provided a compound for use in a method of treating an inflammatory bowel disease in a subject, the method comprising diagnosing the subject with an inflammatory bowel disease by the method of the invention.

Brief Description of the Drawings

Figure 1(A) shows a 2-dimensional tsne plot computed from the 4-sites used in the diagnostic model from the Oxford and Cambridge cohort coloured by disease type.

Figure 1(B) shows the ROC curve and AUC from the logistic regression of the 4-sites trained in Scottish children and tested in Oxford & Cambridge IBD samples.

Figure 1(C) shows the ROC curve and AUC of the 4- site model fitted to an externally validated CD cohort from North America (RISK cohort)

Figure 1(D) shows the ROC curve and AUC of the 4-site model fitted to a subset population of the Oxford and Cambridge cohort who are CRP positive compared to non -IBD controls.

Figure 1(E) shows the ROC curve and AUC of the 4-site model fitted to a subset population of the Oxford and Cambridge cohort who are CRP negative compared to non -IBD controls.

Figure 1(F) shows the ROC curve and AUC of the 4-site model fitted to cohort of paediatric coeliac patients vs non -coeliac controls. Figure 1(G) shows the ROC curve and AUC of the 4-site model fitted to a UK adult IBD cohort at diagnosis (IBD BIOM).

Figure 2 shows a ROC curve showing the diagnostic performance of the 4-site model in patients that are faecal calprotectin positive. F calpro positive vs controls AUC: 0.759; 95% CI: 0.6764-0.8407.

Figure 3 shows a ROC curve showing the diagnostic performance of the 4-site model in patients that are faecal calprotectin negative (stool concentration of less than 250 pg/g). F calpro negative (less than 250 pg/g) vs controls AUC0.722; 95% CI: 0.5797-0.8635.

Figure 4 shows a ROC curve showing the diagnostic performance of the 4-site model in patients that are faecal calprotectin negative (stool concentration of less than 50 pg/g). F calpro negative (less than 50 pg/g) vs controls AUC: 0.738; 95% CI: 0.5304-0.9454.

Detailed description of the preferred embodiments

General Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

In general, the term "comprising" is intended to mean including but not limited to. For example, the phrase “A method comprising a step of measuring methylation" should be interpreted to mean that the method comprises a step of measuring methylation, but the method may comprise further steps.

In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of’. The term “consisting of' is intended to be limiting. For example, the phrase “A method consisting of a step of measuring methylation” should be understood to mean that the method consists of a step of measuring methylation and no further steps.

In some embodiments of the invention, the word “comprising ” is replaced with the phrase “consisting essentially of” . The term “consisting essentially of’ means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.

The term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error. Optionally, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x. The singular forms “a ”, “an ”, and “the ” include plural referents unless the content clearly dictates otherwise.

For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotides or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid at the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions /total number of positions in the reference sequence x 100).

Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 1. If the test sequence is shorter than SEQ ID NO: 1, the gaps or missing positions should be considered to be nonidentical positions.

The skilled person is aware of different computer programs that are available to align two sequences. For instance, an alignment between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the two amino acid or nucleic acid sequences are aligned using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Measurement of methylation levels

The present invention provides methods of diagnosing an inflammatory bowel disease and methods of ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease comprising a step of measuring the methylation level of at least one site in at least one gene.

DNA methylation is an epigenetic mark that plays an essential role in regulating gene expression. DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5- methylcytosine by DNA methyltransferase (DNMT) enzymes. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide, resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands. DNA methylation often occurs at sites such as CpG sites (sites comprising a CG dinucleotide), which are DNA methylations regions in promoters known to regulate gene expression through transcriptional silencing of the corresponding gene. DNA methylation at CpG sites is crucial for gene expression and tissue-specific processes.

The sites will generally be methylated or not methylated, and the level of methylation at a site (the number of gene copies comprising a methyl group at a particular site in DNA in a biological sample) can correlate to a disease state.

The methylation level at the at least one site in the at least one gene may be measured using any suitable technique.

For example, DNA methylation can be detected using PCR -based amplification fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), melting point PCR, pyrosequencing, bisulphite sequence (BS-seq) or protocols that employ a combination.

The methylation level can also be analysed using high performance liquid chromatographyultraviolet (HPLC-UV) (Kuo et al , Nucleic Acids Res. 1980;8:4763-4776). This assay allows for the quantification of deoxycytidine (dC) and methylated cytosines (5 mC) present in a hydrolysed DNA sample. Briefly, the DNA must be hydrolysed into its constituent nucleoside bases, the 5 mC and dC bases separated chromatographically, and then the fractions measured. Then, the 5 mC/dC ratio can be calculated for each sample, and this can be compared between the experimental and control samples.

Another suitable assay for analysing the methylation level of a gene is Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). LC-MS/MS has been validated for detecting levels of methylation levels ranging from 0.05%-10%, and it can confidently detect differences between samples as small as -0.25% of the total cytosine residues (Song et al.; Anal. Chem. 2005;77:504-510). There are several commercially available kits, all enzyme-linked immunosorbent assay (ELISA) based, that enable the quick assessment of DNA methylation levels.

The LUMA (luminometric methylation assay) technique (Karimi et al.; Exp. Cell Res. 2006;312:1989-1995) utilizes a combination of two DNA restriction digest reactions performed in parallel and subsequent pyrosequencing reactions to fill-in the protruding ends of the digested DNA strands.

Next generation sequencing methods, such as methods based on the Illumina technology (like 27K, 450K and EPIC arrays) as well as twist methods are also suitable.

In preferred embodiments the methylation level is assessed using nanopore sequencing, such as Oxford nanopore sequencing. This method is preferred as it provides several advantages, such as a higher genomic coverage with lower GC bias, higher number of CpG positions called at lower read depth and simplified haplotype phasing of methylated bases using long reads.

Inflammatory bowel disease

Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC), and IBD refers to disorders characterised by chronic inflammation of the GI tract including CD and UC. Ulcerative colitis is an inflammatory bowel disease that causes inflammation and ulcers in the colon and rectum. Inflammatory bowel disease and/or ulcerative colitis may be identified using a combination of endoscopy, colonoscopy and imaging studies such as contrast radiography, magnetic resonance imaging, and computed tomography.

The inflammatory bowel disease may be ulcerative colitis. For example, the method may be a method of diagnosing ulcerative colitis in a subject comprising a step of measuring the methylation level of at least one site in at least one gene selected from the group consisting of VMP1, RPS6KA2, CFI and ARHGEF3 in a biological sample obtained from the subject. The method may further comprise a step of determining that the subject is likely to have ulcerative colitis if the methylation level of the at least one site in the at least one gene in the biological sample is significantly different to the methylation level of the at least one site in the at least one gene in the reference.

Diagnosing/distinguishing The methods of the invention may be a method of diagnosing an inflammatory bowel disease, or a method of ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease.

The phrase “a method of diagnosing an inflammatory bowel disease”, refers to a method that is able to at least contribute to a clinician’s diagnosis of inflammatory bowel disease. The methods need not always result in a diagnosis of inflammatory bowel disease. For example, the methods may be used to determine that the subject is unlikely to have inflammatory bowel disease. In addition, the methods may be used simply to identify a subset of subjects who are very unlikely to have inflammatory bowel disease, thereby allowing clinicians to avoid subjecting that subset of subjects to unnecessary and potentially invasive diagnostic steps. Similarly, the methods may be used to identify a subsets of patients that have markers associated with inflammatory bowel disease and therefore may have inflammatory bowel disease, and for whom it is worth performing additional tests to further investigate whether they have inflammatory bowel disease. The methods may be used to identify patients who would typically be diagnosed as not having an inflammatory bowel disease because they are negative for CRP (i.e. concentration of less than 5 mg/1 of C-reactive protein in their blood).

Thus, the methods may further comprise a step of confirming that the subject has inflammatory bowel disease by carrying out further testing, such as endoscopy, colonoscopy and/or imaging studies (such as contrast radiography, magnetic resonance imaging, or computed tomography). Of course, the fact that the methods can predict inflammatory bowel disease or rule out inflammatory bowel disease with a reasonable likelihood can help to ensure that fewer patients are subjected to invasive (and expensive) testing for inflammatory bowel disease that is not required.

The phrase “a method for ruling out inflammatory bowel disease”, refers to a method that is able to determine that a subset of subjects that have symptoms of inflammatory bowel disease do not in fact have markers associated with inflammatory bowel disease and therefore are unlikely to have inflammatory bowel disease. Such methods may be used by clinicians to identify subjects with symptoms of inflammatory bowel disease who do not have inflammatory bowel disease without subjecting them to invasive (and expensive) testing for inflammatory bowel disease that is not required.

Similarly, “a method of diagnosing ulcerative colitis” refers to a method that is able to at least contribute to a clinician’s diagnosis of ulcerative colitis. The methods need not always result in a diagnosis of ulcerative colitis. For example, the methods may be used to determine that the subject is unlikely to have ulcerative colitis. In addition, the methods may be used simply to identify a subset of subjects who are very unlikely to have ulcerative colitis, thereby allowing clinicians to avoid subjecting that subset of subjects to unnecessary and potentially invasive diagnostic steps. Similarly, the methods may be used to identify a subsets of patients that have markers associated with ulcerative colitis and therefore may have ulcerative colitis, and for whom it is worth performing additional tests to further investigate whether they have ulcerative colitis. The method of diagnosing ulcerative colitis may further comprise a step of confirming that the subject has ulcerative colitis disease by carrying out further testing, such as endoscopy, colonoscopy and/or imaging studies (such as contrast radiography, magnetic resonance imaging, or computed tomography).

At least one gene

The methods of the invention comprise measuring methylation at at least one site in at least one gene.

The at least one gene may comprise VMP1. VMP1 is also known as EPG3, HSPC292, TANG05, TDC1 and TMEM49. The sequence of VMP1 is available under NG_051107.1 and provided here as SEQ ID NO: 1. The analysis of the methylation level of VMP1 thus may require the analysis of the methylation level of a sequence with an identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 1. A suitable at least one site in VMP1 corresponds to the CpG site starting at nucleotide 130,703 in SEQ ID NO: 1.

The at least one gene may comprise RPS6KA2. RPS6KA2 is also known as RSK, HU-2, RSK3, p90RSK2, p90-RSK3, pp90RSK3, MAPKAPK1C, S6K-alpha, and S6K-alpha2. The sequence of RPS6KA2 is available under NP_001006933.3 and provided here as SEQ ID NO: 2. The analysis of the methylation level of RPS6KA2 thus may require the analysis of the methylation level of a sequence with an identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 2. A suitable at least one site in RPSKA2 corresponds to the CpG site starting at nucleotide 694,001 in SEQ ID NO: 2.

The at least one gene may comprise CFI. CFI is also known as FI, IF, KAF, AHUS3, ARMD13, C3BINA, C3b-INA. The sequence of CFI is available under NC_000004.12 and provided here as SEQ ID NO: 3. The analysis of the methylation level of CFI thus may require the analysis of the methylation level of a sequence with an identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 3. A suitable at least one site in CFI corresponds to the CpG site starting at nucleotide 71,162 in SEQ ID NO: 3. The at least one gene may comprise ARHGEF3. ARHGEF3 is also known as GEF3, STA3 and XPLN. The sequence of ARHGEF3 is available under NC_000003.12 and provided here as SEQ ID NO: 4. The analysis of the methylation level of ARHGEF3 thus may require the analysis of the methylation level of a sequence with an identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 4. A suitable at least one site in ARHGEF3 corresponds to the CpG site starting at nucleotide 279,955 in SEQ ID NO: 4.

In a preferred embodiment, the at least one gene comprises VMP1 and RPS6KA2. In a further embodiment, the least one gene comprises CFI and/or ARHGEF3. For example, the at least one gene may consist of VMP1 and RPS6KA2. Similarly, the at least one gene may comprise VMP1, RPS6KA2 and CFI. The at least one gene may comprise VMP1, RPS6KA2 and ARHGEF3. The at least one gene may comprise VMP1, RPS6KA2, CFI and ARHGEF3. The at least one gene may consist of VMP1, RPS6KA2, CFI and ARHGEF3.

The at least one gene may comprise further genes whose methylation level correlates with IBD. For example, additional suitable genes may include ZBTB16, SLC15A4, TNF, DIABLO, ZFYVE28, RUNX3, HGF, TOLLIP, ITGB2, PIEZOI, PRF1, ZBTB12, PRF1, CD247, CDETP, UBASH3A, WRAP73, SBNO2, TNSF10, BCL3, ICA1, FGD6, COL11A2, UIMC1, KIAA0513, GBX1, GALNT11, CCDC85C, TXLNB, AP1B1, EMX20S, PRSS23, SYTL2, LOC150776, CRHR1, GBF1, CTSZ, ANUBL1, BRE, SOCS3, FKBP5, CALHM1, NLRC5, TRAPPC2L, NLRC5, SRF, VPS26B, LUN, FRMD4A, UBE2D2, WDR8, ITGB2, TXK, CRELD2, CLU, and/or HOOK2.

In some embodiments, the at least one gene consists of 100 or fewer, 50 or fewer, 25 or fewer, 10 or fewer, or 6 or fewer genes. In a preferred embodiment, the at least one gene consists of 25 or fewer genes.

At least one site

The methods of the invention comprising measuring the methylation level of at least one site of at least one gene.

The method may comprise measuring the methylation level of the at least one gene across the whole gene, or at a subset of potential methylation sites. For example, the method may comprise measuring the methylation level of the at least one gene at all CpG sites in the at least one gene. The method may comprise measuring the methylation level at 10 or fewer, 5 or fewer, or 2 or fewer sites in the at least one gene. Preferably, the method comprises measuring the methylation level at one CpG site in the at least one gene. A suitable CpG site on VMP1 includes Chrl7 5791517. A suitable CpG site on RPS6KA2 includes Chr6 16697052.

It is within the abilities of the person skilled in the art to identify suitable methylation sites in a gene. For example, the skilled person would understand that methylation commonly occurs at CpG sites, which may be identified simply by examining the sequence (for example the sequence of SEQ ID NO: 1-4) and locating CG dinucleotides in that sequence.

The method may comprise measuring the methylation level at one CpG site in VMP1, at one CpG site in RPS6KA2, at one CpG site in ARHGEF3, and/or at one CpG site in CFI. The method may comprise measuring the methylation level at one CpG site in VMP1 and one CpG site in RPS6KA2. The method may comprise measuring the methylation level at one CpG site in VMP1 and one CpG site in RPS6KA2, wherein the one CpG site in VMP1 corresponds to the CpG site starting at nucleotide 130,703 in SEQ ID NO: 1 and the one CpGsite in RPS6KA2 corresponds to the CpG site starting at nucleotide 694,001 in SEQ ID NO: 2, optionally wherein the subject has a concentration of less than 5 mg/1 of C-reactive protein in their blood or serum, optionally wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has an AUC of greater than 0.85, an accuracy of greater than 0.75, and/or a PPV of greater than 0.8. The method may comprise measuring the methylation level at one CpG site in VMP1, one CpG site in RPS6KA2, one CpG site in ARHGEF3 and one CpG site in CFI. The method may comprise measuring the methylation level at one CpG site in VMP1, one CpG site in RPS6KA2, one CpG site in CFI and one CpG site in ARHGEF3, wherein the one CpG site in VMP1 corresponds to the CpG site starting at nucleotide 130,703 in SEQ ID NO: 1, the one CpG site in RPS6KA2 is corresponds to the CpG site starting at nucleotide 694,001 in SEQ ID NO: 2, the one CpG site in CFI corresponds to the CpG site starting at nucleotide 71,162 in SEQ ID NO: 3, and the one CpG site in ARHGEF3 corresponds to the CpG site starting at nucleotide 279,955 in SEQ ID NO: 4, optionally wherein the subject has a concentration of less than 5 mg/1 of C- reactive protein in their blood or serum, optionally wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has an AUC of greater than 0.85, an accuracy of greater than 0.75, and/or a PPV of greater than 0.8.

In any of the methods described herein, the subject may have:

(i) a stool concentration of at least 250 pg/g calprotectin, optionally wherein the subject is an adult; or

(ii) a stool concentration of less than 250 pg/g calprotectin, optionally less than 50 pg/g calprotectin, and further optionally wherein the subject is an adult. DNA methylation markers as described herein may be at least one CpG site in at least one gene, wherein the at least one gene comprises VMP1 and CFI, and preferably wherein the at least one gene further comprises ARHGEF3 and RPS6KA2, and pereferably wherein the at least one CpG site in VMP1 corresponds to the CpG site starting at nucleotide 130,703 in SEQ ID NO: 1, the at least one CpG site in CFI corresponds to the CpG site starting at nucleotide 694,001 in SEQ ID NO: 2, the at least one CpG site in ARHGEF3 corresponds to the CpG site starting at nucleotide 71,162 in SEQ ID NO: 3, and the at least one CpG site in RPS6KA2 corresponds to the CpG site starting at nucleotide 279,955 in SEQ ID NO: 4. The invention also provides a variety of methods, each comprising any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (or any range derivable therein) of a variety of steps and in no particular order, including methods of the following: measuring in a sample; analyzing a sample; assessing a sample; evaluating a sample; measuring nucleic acids in a sample; assessing nucleic acids in a sample; detecting nucleic acids in a sample; measuring methylation in nucleic acids in a sample; analyzing nucleic acids in a sample; assessing nucleic acids in a sample; measuring methylation at one or more CpG dinucleotides in a sample; detecting methylation at one or more CpG dinucleotides in a sample; assaying methylation at one or more CpG dinucleotides in a sample; assessing methylation at one or more CpG dinucleotides in a sample; measuring a methylation status in a sample; assaying a methylation status in a sample; detecting methylation status in a sample; determining methylation status in a sample; identifying methylation status in a sample; measuring one or more DNA methylation markers in a sample; assessing one or more DNA methylation markers in a sample; detecting one or more DNA methylation markers in a sample; measuring the presence of methylation at one or more markers in a sample; detecting the presence of methylation at one or more markers in a sample; assessing the presence of methylation at one or more markers in a sample; assaying the presence of one of more markers in a sample; measuring one or more DNA methylation markers in a sample but excluding the measuring of one or more other DNA methylation markers in the sample; assessing one or more DNA methylation markers in a sample but excluding the assessing of one or more other DNA methylation markers in the sample; analyzing one or more DNA methylation markers in a sample but excluding the analyzing of one or more other DNA methylation markers in the sample; detecting one or more DNA methylation markers in a sample but excluding the detecting of one or more other DNA methylation markers in the sample; measuring methylation status in nucleic acids from a sample from a sample of an individual suspected of, or at risk for, having inflammatory bowel disease; detecting methylation status in nucleic acids from a sample from tissue from an individual suspected of, or at risk for, having inflammatory bowel disease; analyzing methylation status in nucleic acids from a sample from tissue from an individual from the individual suspected of, or at risk for, having inflammatory bowel disease; assessing methylation status in nucleic acids from a sample from tissue from an individual suspected of, or at risk for, having inflammatory bowel disease; measuring methylation at one or more CpG dinucleotides in a sample but excluding the measuring of methylation at one or more CpG dinucleotides in the sample; assessing methylation at one or more CpG dinucleotides in a sample but excluding the assessing of methylation at one or more CpG dinucleotides in the sample; analyzing methylation at one or more CpG dinucleotides in a sample but excluding the analyzing of methylation at one or more CpG dinucleotides in the sample; detecting methylation at one or more CpG dinucleotides in a sample but excluding the detecting of methylation at one or more CpG dinucleotides in the sample; measuring methylation at one or more CpG dinucleotides in nucleic acids from a sample from tissue from an individual suspected of, or at risk for, having inflammatory bowel disease; detecting methylation at one or more CpG dinucleotides in nucleic acids from a sample from tissue from an individual suspected of, or at risk for, having inflammatory bowel disease; analyzing methylation at one or more CpG dinucleotides in nucleic acids from a sample from tissue from an individual suspected of, or at risk for, having inflammatory bowel disease; assessing methylation at one or more CpG dinucleotides in nucleic acids from a sample from tissue from an individual suspected of, or at risk for, having inflammatory bowel disease; treating an individual for inflammatory bowel disease when the individual has been determined to have a methylation status at one or more methylation markers; treating an individual for inflammatory bowel disease when the individual has been determined to have methylation at one or more CpG dinucleotides; wherein any of the aforementioned methods, or any other methods encompassed by the disclosure, may comprise any one or more of the following method steps: measuring methylation status, wherein the measuring identifies the methylation status of two or more markers, or four or more markers, from nucleic acids in a sample; measuring methylation status, wherein the measuring identifies the presence of two or more markers, or four or more markers, from nucleic acids in a sample; measuring the presence of two or more methylation markers, or four or more methylation markers, from a sample; providing DNA from a sample; providing nucleic acids from a sample; determining whether one or more methylation markers from nucleic acids from a sample are methylated; measuring whether one or more methylation markers from nucleic acids from a sample are methylated; performing a sequencing step on nucleic acids from a sample; determining a sequence of nucleic acids from a sample; performing bisulphite conversion on one or more markers; performing bisulphite conversion on one or more CpG dinucleotides; hybridizing DNA to an array comprising probes capable of determining methylated versus non-methylated markers; hybridizing DNA to an array comprising probes capable of determining methylated versus non-methylated CpG dinucleotides; hybridizing DNA to an array comprising probes capable of discriminating between methylated and non-methylated markers; hybridizing DNA to an array comprising probes capable of discriminating between methylated and non-methylated CpG dinucleotides; performing an amplification step on sequence from nucleic acids from a sample; performing an amplification step on sequence from nucleic acids using methylation-specific primers; performing amplification of sequence comprising one or more regions suspected of having methylation or in need of determination of a methylation status; performing PCR on sequence comprising one or more regions suspected of having methylation or in need of determination of a methylation status; performing a capturing step; performing a binding step; performing a purification step; performing a capturing step comprising binding of polynucleotides comprising one or more methylation markers to binding molecules specific to the one or more methylation markers and collecting complexes thereof; identifying inflammatory bowel disease obtaining a sample from an individual; obtaining DNA from a sample from an individual; administering a treatment to an individual; providing DNA from a sample; determining whether one or more methylation markers from a panel of methylation markers comprises a specific sequence; and/or obtaining data that identifies whether each one of a group of methylation markers from a panel comprises a specific sequence.

Moreover, in some aspects of the invention, an individual who is administered a therapy or treatment has been subjected to any of the methods and steps described herein.

Subject

The subject who is diagnosed using the methods of the invention is a mammal for example a primate, rodent (including mice and rats), or other common laboratory, domestic or agricultural animal, including but not limited to rabbits, dogs, cats, horses, cows, sheep, goats, pigs etc. Preferably the subject is a human.

The subject may be a child. The inventors have shown that the methods of the invention work particularly well for children. The child may be less than 18 years of age, for example less than 14 years, less than 13 years, less than 12 years, less than 11 years, less than 10 years, less than 9 years, less than 8 years, less than 7 years, less than 6 years, less than 5 years, less than 4 years, less than 3 years, less than 2 years, or less than 1 year old.

The subject may have symptoms of IBD, such as diarrhea, abdominal pain, or flatulence. In other embodiments, the subject does not have any physical symptoms of IBD. In these embodiments, the methods of the invention may be useful for assessing whether said subject is likely to develop symptoms of IBD.

The subject may have a low concentration of C-reactive protein (CRP) in the blood. CRP is a homopentameric acute -phase inflammatory protein. CRP exhibits elevated expression during inflammatory conditions including IBD. CRP is the most widely used serum indicator of inflammation in IBD. Increased levels of CRP help differentiate mucosal active disease from quiescent IBD. Generally, CRP levels of 10-40 mg/1 are found in cases of mild inflammation or viral infections. Severe active inflammation or bacterial infection will typically generate CRP levels of 50-200 mg/1, and very high levels of >200-250 mg/1 are only found in severe conditions and burns. However, many cases of inflammatory bowel disease (particularly ulcerative colitis) are not associated with elevated CRP levels, and the present Examples demonstrate that the methods of the invention may be used to detect inflammatory bowel disease even in subjects who do not have elevated CRP levels.

Thus, in some embodiments, the subject may have a blood concentration of CRP of >5mg/l, >4mg/l, >3mg/l, >2mg/l, >lmg/l, or less than 0.5 mg/1. Thus, in some embodiments, the subject may have a blood concentration of CRP of <5mg/l, <4mg/l, <3mg/l, <2mg/l, <lmg/l, or less than 0.5 mg/1. Optionally, the level of C-reactive protein in the subject’s blood or serum is determined by taking a sample from the subject and quantifying the level of CRP in the sample. Optionally, the methods comprise a step of measuring the CRP level in a sample obtained from the subject. Methods for quantifying the amount of CRP in blood are well known in the art and include, for example, the latex agglutination method (Singer et al.', Am J Clin Pathol. 1957 Dec;28(6):611-7).

Preferably, where the subject is determined to have a particular CRP level, the level of CRP has been determined at the same time as the method of the invention or within the previous month prior to the method of the invention being performed.

Sample

The methylation levels of the genes are determined in a biological sample obtained from the subject. Determining the methylation levels may be performed entirely in vitro. The method may further comprise a step of obtaining a sample (biological sample) from the subject.

The sample (biological sample) can be any sample that contains DNA from the subject, for example saliva, fecal material, urine, blood or serum. In preferred embodiments the sample is blood, preferably whole blood. The sample may be serum. Methods of obtaining such samples from the subject are well known in the art.

Comparing the methylation level to a reference

The methods of the invention may comprise a step of comparing the methylation level of the at least one site in the at least one gene in the biological sample with the methylation level of the at least one site in the at least one gene in a reference. For example, if the at least one gene comprises VMP1 and RPS6KA2, the method may comprise a step of comparing the methylation level of VMP1 and the methylation level of RPS6KA2 in a sample from the subject, with the methylation level of VMP1 and the methylation level of RPS6KA2 respectively in a reference. Similarly, if the at least one gene comprises VMP1, RPS6KA2, CFI and ARHGEF3, then the method may comprise a step of comparing the methylation level of VMP1, the methylation level of RPS6KA2, the methylation level of CFI and the methylation level of ARHGEF3 in a sample from the subject with the methylation level of VMP1, the methylation level of RPS6KA2, the methylation level of CFI and the methylation level of ARHGEF3 respectively in a reference.

The reference may be a cut-off level. For example, the user of the method may find that the methylation level of a gene is changed if it is significantly different to a pre-defined value. The pre-defined value the methylation level of the same at least one gene in a healthy person. Similarly, the pre-defined value may be an average of the methylation level of the same gene in multiple samples from multiple healthy people.

The reference may be a sample from a reference subject, wherein the reference subject does not have inflammatory bowel disease or is a healthy person.

The healthy person may be a person who has not had any symptoms of IB D or one who has been determined to be free of IB (i.e. has not symptoms of IBD and has never been diagnosed with IBD). The healthy person may have a different disorder, such as coeliac disease, or may be believed to be free of any disease.

The healthy person or reference subject may be a person who is matched for one or more characteristics to the subject. Matching the healthy person or reference subject to the subject can improve the accuracy of the methods of the invention. For example, the healthy person or reference subject may be matched for sex, ethnicity, age or any other characteristic known in the art. Optionally, the healthy person or reference subject is matched for sex, ethnicity and age. The healthy person or reference subject may be matched for sex if the healthy person has the same biological (birth) sex as the subject. The healthy person or reference subject may be matched for ethnicity if it falls within the same one of the following ethnic groups as the subject, (i) white, (ii) Asian, (iii) Black, Caribbean or African, (iv) mixed, or (v) other. The healthy person or reference subject may be matched for age if it falls within the same one of the following age ranges as the subject, (i) 0-12 years, (ii) 13-18 years, (iii) 19-30 years, (iv) 41 to 50 years, (v) 51 to 65 years, or (vi) 65 years +. Optionally, the healthy person or reference subject is matched for age and sex.

The methods may comprise a step of determining that the subject is likely to have inflammatory bowel disease if the methylation level of the at least one site in the at least one gene in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference. Similarly, the methods may comprise a step of determining that the subject is likely to have ulcerative colitis if the methylation level of the at least one site in the at least one gene in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference. Similarly, the methods may comprise a step of determining that the subject is unlikely to have an inflammatory bowel disease if the methylation level of the at least one site in the at least one gene in the biological sample is not significantly different to and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference. Similarly, the methods may comprise a step of determining that the subject is unlikely to have ulcerative colitis if the methylation level of the at least one site in the at least one gene in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference.

A methylation level at at least one site in at least one gene may be considered to be significantly different to the methylation level of the at least one site in at least one gene in the reference, if the methylation level differs by at least 1 , at least 2, or at least 3 standard deviations compared to the methylation level of the at least one site in at least one gene in the reference.

A methylation level at at least one site in at least one gene may be considered to be lower than the methylation level of the at least one site in the at least one gene in the reference, if the methylation level is 1 , at least 2 or at least 3 standard deviations lower than the methylation level of the at least one site in the at least one gene in the reference.

Optionally, the methylation level of the at least one site in the at least one gene in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference if there is an at least 2%, or at least 3% difference or decrease. Optionally, the at least one site in at least one gene comprises a site in RPS6KA2 and the methylation level of the at least one site in RPS6KA2 in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in RPS6KA2 in the reference if the methylation level of RPS6KA2 in the biological sample is at least 5% lower or different to (in the biological sample). Optionally, the at least one site in at least one gene comprises a site in VMP1 and the methylation level of the at least one site in VMP1 in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in VMP1 in the reference if the methylation level of VMP1 in the biological sample is at least 5% lower or different to (in the biological sample). Optionally, the at least one site in at least one gene comprises a site in ARHGEF3 and the methylation level of the at least one site in ARHGEF3 in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in ARHGEF3 in the reference if the methylation level of ARHGEF3 in the biological sample is at least 2.5% lower or different to (in the biological sample). Optionally, the at least one site in at least one gene comprises a site in CFI and the methylation level of the at least one site in CFI in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in CFI in the reference if the methylation level of CFI in the biological sample is at least 7% lower or different to (in the biological sample).

Optionally, the methylation level of the at least one site in the at least one gene in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference if there is a less than 4%, or less than 2% difference or decrease. Optionally, the at least one site in at least one gene comprises a site in RPS6KA2 and the methylation level of the at least one site in RPS6KA2 in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in RPS6KA2 in the reference if the methylation level of RPS6KA2 in the biological sample is less than 5% lower or different to. Optionally, the at least one site in at least one gene comprises a site in VMP1 and the methylation level of the at least one site in VMP1 in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in VMP1 in the reference if the methylation level of VMP1 in the biological sample is less than 5% lower or different to.

Optionally, the at least one site in at least one gene comprises a site in ARHGEF3 and the methylation level of the at least one site in ARHGEF3 in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in ARHGEF3 in the reference if the methylation level of ARHGEF3 in the biological sample is not at least 2.5% lower or different to. Optionally, the at least one site in at least one gene comprises a site in CFI and the methylation level of the at least one site in CFI in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in CFI in the reference if the methylation level of CFI in the biological sample is not at least 7% lower or different to.

Accuracy and sensitivity of the methods

The method may be a method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease which has an AUC of greater than 0.8, greater than 0.85, or greater than 0.9. The AUC may be less than 1.0. The method may be a method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease which has an accuracy of greater than 0.6, greater than 0.65, or greater than 0.7. The accuracy may be less than 1.0 The method may be a method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease which has a PPV of greater than 0.85, greater than 0.9, or greater than 0.95. The PPV may be less than 1.00.

The AUC can be calculated by plotting a receiver operating curve (ROC) in which the true positive rate (sensitivity) is plotted against the false positive rate (specificity). The AUC is the two dimensional area underneath the ROC. If the user wishes to determine the AUC of their method, they may (subject to patient consent) monitor whether the inflammatory bowel disease and/or ulcerative colitis diagnosis was later confirmed using diagnosis methods such as endoscopy, colonoscopy or imaging methods and therefore whether the method gave a correct diagnosis, a false positive or a false negative. Typically, this analysis of the method would be carried out when seeking approval to use the method for diagnostic purposes. It is sufficient to perform this analysis on a subset of patients to determine an average AUC for the method.

Similarly, the accuracy level may be calculated using the formula: Accuracy = (number of true positives + number of true negatives)/(true positive + true negative + false positive + false negative). PPV may be calculated using the formula: PPV = true positive/ (true positive + false positive).

Other method steps

As noted above, the methods may further comprise a step of obtaining the sample from the subject. In addition, the method may comprise a step of isolating DNA within the sample. Methods for isolating DNA from a sample, such as a blood sample, are well known in the art and include, for example, organic extraction, chelex extraction, and solid phase extraction.

Commercial kits for DNA extraction, such as the Qiagen ™ Puregene Blood core Kit C, are available.

Treatment

The invention further provides a method of treating an inflammatory bowel disease in a subject, the method comprising: a) diagnosing the subject with an inflammatory bowel disease by the method of the invention; and b) treating the subject with a suitable treatment.

Similarly, the invention provides a compound for use in a method of treating an inflammatory bowel disease in a subject, the method comprising diagnosing the subject with an inflammatory disease by the method of the invention and administering the compound to the subject.

Treatments for IBD/compounds suitable for treating IBD are known in the art. They include, for example, anti-inflammatories such as aminosalicylates, mesalamine (Delzicol, Rowasa, others), balsalazide (Colazal) and olsalazine (Dipentum). Suitable immunosuppressant drugs include azathioprine (Azasan, Imuran), mercaptopurine (Purinethol, Purixan) and methotrexate (Trexall). Treatment may also be with biologies, such as infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), certolizumab (Cimzia), vedolizumab (Entyvio), ustekinumab (Stelara), and risankizumab (Skyrizi).

It will be understood that a treatment will be required only if the subject is found to have IBD, for example ulcerative colitis.

Kits

The invention further provides kits for use in the methods of the invention. The kits may comprise reagents to detect the methylation status of one or more genes selected from the group consisting of VMP1, RPS6KA2, CFI and/or ARHGEF3, preferably VMP1 and RPS6KA2, most preferably all of VMP1, RPS6KA2, CFI and ARHGEF3.

The kit may contain reagents for the detection of fewer than 100 genes (for example fewer than 90 genes, fewer than 80 genes, fewer than 70 genes, fewer than 60 genes, fewer than 50 genes, fewer than 40 genes, fewer than 30 genes, fewer than 20 genes, fewer than 10 genes, or fewer than 5 genes.

The kit may further contain reagents suitable for measuring the concentration of CRP in a blood sample, or a serum sample.

The kit may further comprise reagents suitable for contain reagents for extracting DNA from the biological sample.

Examples Example 1 - Methods and materials

Oxford and Cambridge paediatric IBP inception cohort

A total of 86 IBD patients (33 CD, 31 UC, 22 IBDU/VEOIBD) with a median age of 12 years (0.9-17.5 y) were prospectively recruited from gastroenterology appointments at the John Radcliffe Hospital, Oxford and Cambridge, United Kingdom. The majority of children were of white Northern European ancestry. All children were aged less than 18 years of age at diagnosis and were diagnosed with IBD in line with the (modified) Porto criteria. Blood samples were taking using EDTA vacutainer tubes, and stored at -80°C until used.

CRP measurements were recorded from patients where levels had been measured within a month of the blood sample being obtained. Faecal calprotectin levels were not available in this cohort. A total of 30 non-IBD controls were also included with a median age of 9.5 (2.2-17.3 y). These patients were symptomatic with no IBD on evaluation. Ethical approval for this study was obtained from the Y orkshire & The Humber Sheffield Research Ethics Committee Oxford Gastrointestinal (GI) cohort (2 I/YH/0206). For the Cambridge Cohort, ethical approval was obtained from the local research committee (REC 12/EE/0482 and REC 17/EE/0265) and patients were recruited after informed patient and/or carer consent as appropriate.

DNA extraction and Genome -wide methylation profiling

Genomic DNAs was extracted from peripheral blood sample collected in EDTA by the Qiagen Puregene Blood core Kit C for Oxford samples and then DNA Blood Mini Kit for Cambridge samples. For methylation arrays 500 ng of DNA was randomised onto 96-well plates before shipment to UCL Genomics (London, UK). DNA was then bisulphite converted and analysed with the HumandMethylation850K platform (Illumina, San Diego, CA, USA).

Data processing and statistical analysis

Idat files were processed using pipelines from minfi 10 in R (R foundation for Statistical Computing, Vienna). Firstly, failed probes (detection P value >0.01 in at least 50% of the samples) or probes found on the sex chromosomes were removed. Probes containing SNPs with a minor allele frequency of >0.01 in the European population in the 1000 Genomes Project were also removed. The raw data were background adjusted and corrected from dye colour bias, and quantile normalised. Batch effects were adjusted for both slide and position using the tool ComBat. Beta values were calculated for each of the CpG sites defined as the ratio of the methylated probe intensity (M)/the sum of the overall intensity of the unmethylated probe (U) + methylated probe (M). Estimated cell proportions (CD4+, CD8+, T cells, natural killer, B cells, monocytes and granulocytes) were computed using the Houseman algorithm. Biomarker validation and 4-site based diagnostic model

The most significantly differentially methylated sites in the discovery cohort were selected for analysis as potential biomarkers. The top 4 non -redundant sites were taken forward as the 4-site diagnostic logistic regression model. This was initially trained in a cohort on normalised beta values from a paediatric CD cohort from Scotland (n=36 CD patients and n=36 controls) then tested in the Oxford and Cambridge samples. A 3 -dimensional tsne plot was created for a visual analogue of the data using the Oxford and Cambridge cohort (Figure 1A). Publicly available methylation data from the RISK cohort consisting of paediatric CD patients from North America was accessed at NCIB GEO database (accession GSE11261) to test accuracy of the model in a non-UK based diseased cohort. The model was then subjected to further testing in a paediatric Coeliac cohort (N= 36 with a median age of 7.7 y) and matched controls (N= 35 with a median age of 7.6 y). The model was also tested in the IBD-BIOM adult inception cohort from Scotland (n=240 IBD and n=190 controls). The diagnostic value of the four probes was also tested in a large cohort of mostly middle-aged rheumatoid arthritis patients who were recruited before initiation of treatment (n=354 cases, n=337 controls) 8 (accession GSE42861). Performance of the models was defined via AUC, ROC curves, accuracy, specificity, sensitivity, PPV and NPV. (Table 1) Further iterations of the model were also performed including assessing performance using only RPS6KA2 and VMP1 (Table 2), performance looking solely at UC patients using all four probes of the model (Table 3) and performance of only RPS6KA2 and VMP1 in only UC patients (Table 4).

Determining methylation levels

Genomic DNA is isolated from a peripheral blood sample using the PureLink Genomic DNA kit (Thermo Fisher Scientific) as per manufacturer’s recommended protocol. From this extraction, Ipg of high molecular weight DNA is taken forward for methylation analysis using Oxford Nanopore Technology (ONT). Library preparation is carried out using the Ligation Sequencing Kit (SQK-LSK114) as per manufacture’s recommended protocol. The prepared library is then loaded onto a Minion flow cell (RIO.2.1) (ONT). To maximise the efficiency of the sequencing process, the method of adaptive sampling is employed. In this approach, a predefined bed file specifying the sequencing targets, which includes the four genes of interest, is preloaded into the computer before initiating the sequencing run. The Sequencing continues for 24-48 hours, or until the intended coverage has been achieved. The methylation status is determined from fast5 files at all modified bases using the remora pipeline, which aligns the files to the human reference genome. Subsequently, a script is executed to calculate the average methylation (ranging between 0-1) at RPS6KA2, VMP1, ARHGEF3 and CFI. The resulting numeric values representing the methylation levels at these four sites are input into a web-based calculator tool designed for implementing a general linear model. This tool generates an outcome, which is accompanied by an estimate of prediction and an interpretation description providing insights into automated diagnosis.

Example 2 - Derivation and validation of a 4-site methylation biomarker in peripheral blood with high diagnostic accuracy in paediatric inflammatory bowel disease

The application of -omics technology offers important opportunities for biomarker discovery to personalise management of patients with inflammatory bowel disease (IBD). In previous work, we initially reported a characteristic profile of genome-wide DNA methylation in peripheral blood leucocytes from children with IBD at diagnosis defining the IBD methylome. This characteristic pattern of genome -wide alterations was replicated in inception cohorts of adult patients in UK and Scandinavia, and this profile was also replicated in adult patients with medically refractory Crohn's disease (CD), requiring resection surgery. Genetic influence, active inflammation and environmental exposures are implicated in shaping the methylome for IBD.

These data were further validated in children by meta-analysis to derive a model of four methylation markers with high diagnostic accuracy. The inventors have demonstrated that this model has a calculated accuracy comparable to that of faecal calprotectin or any single bloodbased biomarker for IBD in UK-based and North American cohorts. The model is specific to IBD in children with gastrointestinal symptoms, distinguishes IBD from coeliac disease; and, of particular note, is accurate even in children with IBD with no CRP (C-reactive protein) elevation at presentation.

This epigenetic signature provides the basis for a rapid blood test as an alternative or complement to baseline tests currently available. The methylation signature allows for stratification of symptomatic individuals, particularly children, for endoscopic investigation at presentation.

Firstly, the inventors re-analysed the markers with discrimination ability discovered in the epigenome -wide association analysis (EWAS) of a cohort of paediatric CD patients from Scotland. The analysis was corrected for age, sex, IBD disease type, and cell type proportions to ensure methylation sites picked were independent of confounding variables. Four single methylation sites were selected from the following genes, RPS6KA2, VMP1, CF1 and ARHGEF3. Of the 40-100 sites, the two methylation sites within the RPS6KA2 and VMP1 locus (neighbouring RPS6KB1), contributed the most weight in the model, however increasing to the four probes optimised performance. A logistic regression model consisting of methylation of these 4-sites was then tested in a validation cohort of patients from Oxford and Cambridge. Following receiver operating characteristic (ROC) area under the curve 105 (AUC) analyses the model demonstrated an AUC of 0.912 (95% CI: 0.86-0.96) (Accuracy 0.74 and PPV 0.98) (Figure, B and Table 1).

To further validate the model, the inventors compared children who were CRP -positive (>5 mg/1; n=32) and CRP -negative (<5 mg/1; n=49) at presentation against non-IBD controls. In the CRP- positive group, we found an AUC of 0.99 (95% CI: 0.99-1) (accuracy 0.88, PPV 0.89) (Figure ID and Table 1). Within the CRP -negative group an AUC of 0.90 was observed against controls (95% CI: 0.83-0.96) (accuracy 0.82 and PPV 0.85) (Figure IE and Table 1). The inventors then externally validated the model using the available methylation data at the baseline timepoint of study entry for the North American paediatric RISK cohort (164 children with CD, 74 controls 6). In this cohort an AUC of 0.93 (95% CI: 0.90-0.96) (accuracy 0.87 and PPV 0.81) (Figure 1C and Table 1) was demonstrated.

At the second time point (1-3 year following up after diagnosis), accuracy was markedly reduced, with AUC of 0.66 (95% CI: 0.59-0.74) (accuracy 0.69 and PPV 0.63) demonstrating the reduced utility in diagnosis after treatment initiated, and highlighting the importance of active disease in the methylation signature.

To confirm the specificity for IBD, the inventors tested their prediction model in a comparator cohort of paediatric patients diagnosed with coeliac disease and a further group of non-coeliac, non-IBD controls. This resulted in an observed AUC of 0.55 (95% CI: 0.44-0.71) (accuracy 0.55 and PPV 0.55) (Figure IF and Table 1). To investigate the utility in adults at presentation, the inventors analysed a large adult IBD inception cohort (IBD-BIOM consortium) for accuracy of diagnosis with our model, a more modest accuracy than in children was noted - AUC 0.74 (95% CI: 0.68-0.81) (accuracy 0.71 and PPV 0.73) was observed (Figure 1G). The model does not discriminate between adult rheumatoid arthritis patients and controls with an AUC 0.64 (95% CI 0.60-0.68) (accuracy 0.622 and PPV 0.63).

The predictive accuracy (0.91-0.93) of our proposed 4-site based diagnostic model in children offers promising implications for clinical practise, with similar predictive power in both CD and UC. The diagnostic accuracy similar to that reported for faecal calprotectin, currently the most commonly used biomarker in this context. Emerging technology, notably Oxford Nanopore or next generational sequencing could allow development of a rapid point of care test utilising methylation sites for diagnosis of patients, in whom need for investigation is uncertain - particularly in the important sub-group who present with normal values of CRP.

The model is specific for children with inflammatory bowel disease when compared with coeliac disease and compared with symptomatic children with no demonstrable pathology on investigation. By utilising this non-invasive approach, clinicians can identify children who may forego invasive investigations at presentation.

Table 1 - Diagnosis of IBD based on analysis of VMP1, RPS6KA2, CFI and ARHGEF3

Table 2 -Diagnosis of IBD based on analysis of VMP1 and RPS6KA2

Table 3 -Diagnosis of UC based on analysis of VMP1, RPS6KA2, CFI and ARHGEF3

Table 4 - Diagnosis of UC based on analysis of VMP1 and RPS6KA2

Example 2 — Calprotectin

Calprotectin is a biomarker detectable in stool samples. A stool concentration of calprotectin (i.e. faecal calprotectin/ ’F calpro’) above a threshold of 250 pg/g is currently the most accurate diagnostic test for IBD available in the clinic.

Background: We had previously found our methylation-based biomarker to produce an AUC of 0.748 (95% CI 0.685-0.82) in adults at diagnosis. In this analysis, we utilised an adult cohort who have active disease (duration of disease varies per individual) that are about to initiate a biological therapy (EPICC-UK study-baseline timepoint).

Methodology:

Defining F calpro positive

A patient was defined as positive when having greater than 250 ug/g at baseline (active disease, prior to initiation of biologic).

Defining F calpro negative

We choose two thresholds of negativity:

1. Strict threshold of less than 50 pg/g at baseline (active disease, prior to initiation of biologic)

2. The more standardised clinical approach of less than 250 pg/g at baseline (active disease, prior to initiation of biologic).

A total of N= 55, 29.8% of patients were classified as F calpro positive based on the standard clinical approach of greater than 250 pg/g.

A total of N=8, 4.3% of patients were classified as F calpro negative based on the strict threshold.

A total of N=27, 14.6% of patients were classified as F calpro negative based on the standardised clinical approach.

We had a total of N=31 symptomatic/non-symptomatic controls included in the comparator group.

NOTE: 94 patients did not give stool samples (51.0% of the total cohort).

Results:

Figure 2: F calpro positive vs controls

AUC: 0.759

95% CI: 0.6764-0.8407 Figure 3: F calpro negative (250 pg/g) vs controls

AUC:0.722

95% CI: 0.5797-0.8635

Figure 4: F calpro negative (250 pg/g) vs controls

AUC:0.722

95% CI: 0.5797-0.8635

ASPECTS OF THE INVENTION

1. A method of diagnosing an inflammatory bowel disease in a subject comprising a step of measuring the methylation level of at least one site in at least one gene selected from the group consisting of VMP1, RPS6KA2, CFI and ARHGEF3 in a biological sample obtained from the subject.

2. A method for ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease comprising a step of measuring the methylation level of at least one site in at least one gene selected from the group consisting of VMP1, RPS6KA2, CFI and ARHGEF3 in a biological sample obtained from the subject.

3. The method of aspect 1 or 2, wherein the at least one gene comprises VMP1 and/or RPS6KA2.

4. The method of any one of the preceding aspects, wherein the at least one gene comprises CFI.

5. The method of any one of the preceding aspects, wherein the at least one gene comprises ARHGEF3.

6. The method of any one of the preceding aspects, wherein the at least one site in the at least one gene comprises at least one CpG site in the at least one gene.

7. The method of any one of the preceding aspects, wherein the at least one site in at least one gene comprises at least one CpG site in VMP1 and at least one CpG site in RPS6KA2.

8. The method of aspect 7, wherein the at least one site in at least one gene comprises one CpG site in VMP1 and one CpG site in RPS6KA2.

9. The method of any one of the preceding aspects, wherein the at least one site in at least one gene comprises at least one CpG site in CFI.

10. The method of aspect 9, wherein the at least one site in at least one gene comprises one CpG site in CFI. 11. The method of any one of the preceding aspects, wherein the at least one site in at least one gene comprises at least one CpG site in ARHGEF3.

12. The method of aspect 11, wherein the at least one site in at least one gene comprises one CpG site in ARHGEF3.

13. The method of any one of the preceding aspects, further comprising a step of comparing the methylation level of the at least one site in the at least one gene in the biological sample with the methylation level of the at least one site in the at least one gene in a reference.

14. The method of aspect 13, wherein the reference is a cut-off or a sample from a reference subject, wherein the reference subject does not have inflammatory bowel disease, or is a healthy person.

15. The method of aspect 13 or 14, further comprising a step of determining that the subject is likely to have inflammatory bowel disease if the methylation level of the at least one site in the at least one gene in the biological sample is significantly different to and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference.

16. The method of any one of aspects 13 to 15, further comprising a step of determining that the subject is unlikely to have inflammatory bowel disease if the methylation level of the at least one site in the at least one gene in the biological sample is not significantly different to and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference.

17. The method of any one of the preceding aspects, wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has an AUC of greater than 0.8, greater than 0.85, or greater than 0.9.

18. The method of any one of the preceding aspects, wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has an accuracy of greater than 0.6, greater than 0.65, or greater than 0.7.

19. The method of any one of the preceding aspects, wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has a PPV of greater than 0.85, greater than 0.9, or greater than 0.95. 20. The method of any one of the preceding aspects, wherein the inflammatory bowel disease is ulcerative colitis.

21. The method of aspect 20, further comprising a step of determining that the subject is likely to have ulcerative colitis if the methylation level of the at least one site in the at least one gene in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference.

22. The method of aspect 20 or 21, further comprising a step of determining that the subject is unlikely to have ulcerative colitis if the methylation level of the at least one site in the at least one gene in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference.

23. The method of any one of the preceding aspects, wherein the subject has a concentration of less than 5 mg/1 of C-reactive protein in their blood or serum.

24. The method of aspect 23, wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has an AUC of greater than 0.85, an accuracy of greater than 0.75, and/or a PPV if greater than 0.8.

25. The method of any one of the preceding aspects, wherein the methylation level of fewer than 100 genes, fewer than 50 genes, fewer than 25 genes, or fewer than 10 genes is measured.

26. The method of any one of the preceding aspects, wherein the methylation level of the at least one site in the at least one gene is determined by one or more techniques selected from the group consisting of nucleic acid amplification, polymerase chain reaction (PCR), next generation sequencing, methylation specific PCR, bisulfite pyrosequencing, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, and microarray technology.

27. The method of any one of the preceding aspects, wherein the biological sample is whole blood, serum or saliva.

28. The method of aspect 27, wherein the biological sample is whole blood.

29. The method of any one of the preceding aspects, wherein the subject and/or the reference subject is human. 30. The method of aspect 29, wherein the subject and/or the reference subject is 0 to 18 years of age.

31. The method of any one of the preceding aspects, wherein the subject and/or the reference subject has symptoms of inflammatory bowel disease.

32. The method of any one of the preceding claims, wherein the subject:

(i) has a stool concentration of at least 250 pg/g calprotectin; or

(ii) has a stool concentration of less than 250 pg/g calprotectin, optionally less than 50 pg/g calprotectin.

33. A method of treating inflammatory bowel disease in a subject, the method comprising: a) diagnosing the subject with inflammatory bowel disease by the method of any one of aspects 1 or 3 to 32; and b) treating the subject with suitable treatment.

34. The method of aspect 33, wherein the suitable treatment is an anti-inflammatory or immunosuppressant drug.

35. A compound for use in a method of treating inflammatory bowel disease in a subject, the method comprising diagnosing the subject with inflammatory disease by the method of any one of aspects 1 or 3 to 32 and administering the compound.

36. The compound for use of aspect 35, wherein the compound is an anti-inflammatory or immunosuppressant drug.

37. The method or compound for use of any one of aspects 33 to 36, wherein the inflammatory bowel disease is ulcerative colitis.

Claims

1. A method of diagnosing an inflammatory bowel disease in a subject comprising a step of measuring the methylation level of at least one site in at least one gene in a biological sample obtained from the subject, wherein the at least one site in at least one gene comprises one CpG site in VMP1 and one CpG site in RPS6KA2, wherein the subject has a concentration of less than 5 mg/1 of C-reactive protein in their blood or serum.

2. A method for ruling out inflammatory bowel disease in a subject having symptoms of inflammatory bowel disease comprising a step of measuring the methylation level of at least one site in at least one gene in a biological sample obtained from the subject, wherein the at least one site in at least one gene comprises one CpG site in VMP1 and one CpG site in RPS6KA2, wherein the subject has a concentration of less than 5 mg/1 of C-reactive protein in their blood or serum.

3. The method according to claim 1 or claim 2, wherein the one CpG site in VMP1 corresponds to the CpG site starting at nucleotide 130,703 in SEQ ID NO: 1 and the one CpG site in RPS6KA2 corresponds to the CpG site starting at nucleotide 694,001 in SEQ ID NO: 2.

4. The method according to any one of claims 1 to 3, wherein the step of measuring the methylation level of at least one site in at least one gene further comprises measuring the methylation level at one CpG site in CFI and one CpG site in ARHGEF3, optionally wherein the one CpG site in CFI corresponds to the CpG site starting at nucleotide 71,162 in SEQ ID NO: 3, and the one CpG site in ARHGEF3 corresponds to the CpG site starting at nucleotide 279,955 in SEQ ID NO: 4.

5. The method of any one of the preceding claims, further comprising a step of comparing the methylation level of the at least one site in the at least one gene in the biological sample with the methylation level of the at least one site in the at least one gene in a reference, optionally wherein the reference is a cut-off or a sample from a reference subject, and wherein the reference subject does not have inflammatory bowel disease, or is a healthy person.

6. The method of claim 5, further comprising a step of:

(i) determining that the subject is likely to have inflammatory bowel disease if the methylation level of the at least one site in the at least one gene in the biological sample is significantly different to and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference; and/or (ii) determining that the subject is unlikely to have inflammatory bowel disease if the methylation level of the at least one site in the at least one gene in the biological sample is not significantly different to and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference.

7. The method of any one of the preceding claims, wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has:

(i) an AUC of greater than 0.8, greater than 0.85, or greater than 0.9;

(ii) an accuracy of greater than 0.6, greater than 0.65, or greater than 0.7; and/or

(iii) a PPV of greater than 0.85, greater than 0.9, or greater than 0.95.

8. The method of any one of the preceding claims, wherein the inflammatory bowel disease is ulcerative colitis, optionally wherein the method further comprises:

(i) a step of determining that the subject is likely to have ulcerative colitis if the methylation level of the at least one site in the at least one gene in the biological sample is significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference; and/or

(ii) a step of determining that the subject is unlikely to have ulcerative colitis if the methylation level of the at least one site in the at least one gene in the biological sample is not significantly different and/or significantly decreased compared to the methylation level of the at least one site in the at least one gene in the reference.

9. The method of any one of the preceding claims, wherein the method of diagnosing an inflammatory bowel disease or ruling out inflammatory bowel disease has an AUC of greater than 0.85, an accuracy of greater than 0.75, and/or a PPV of greater than 0.8.

10. The method of any one of the preceding claims, wherein:

(i) the methylation level of fewer than 100 genes, fewer than 50 genes, fewer than 25 genes, or fewer than 10 genes is measured; and/or

(ii) the methylation level of the at least one site in the at least one gene is determined by one or more techniques selected from the group consisting of nucleic acid amplification, polymerase chain reaction (PCR), next generation sequencing, methylation specific PCR, bisulfite pyrosequencing, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, and microarray technology.

11. The method of any one of the preceding claims, wherein the biological sample is whole blood, serum or saliva.

12. The method of any one of the preceding claims, wherein the subject and/or the reference subject:

(i) is human;

(ii) is 0 to 18 years of age; and/or

(iii) has symptoms of inflammatory bowel disease.

13. The method of any one of the preceding claims, wherein the subject:

(i) has a stool concentration of at least 250 pg/g calprotectin; or

14. A method of treating inflammatory bowel disease in a subject, the method comprising: a) diagnosing the subject with inflammatory bowel disease by the method of any one of claims 1 or 3 to 13; and b) treating the subject with suitable treatment.

15. The method of claim 14, wherein the suitable treatment is an anti-inflammatory or immunosuppressant drug.

16. A compound for use in a method of treating inflammatory bowel disease in a subject, the method comprising diagnosing the subject with inflammatory disease by the method of any one of claims 1 or 3 to 13 and administering the compound.

17. The compound for use of claim 16, wherein the compound is an anti-inflammatory or immunosuppressant drug.

18. The method or compound for use of any one of claims 14 to 17, wherein the inflammatory bowel disease is ulcerative colitis.