Field of the Invention

This invention relates to screening for ulcerative colitis (UC). In particular, the invention relates to a new biomarker for UC. The present invention also relates to a method for diagnosing and/or prognosing UC.

Background of the Invention

Inflammatory bowel disease (IBD), of which ulcerative colitis is the most common form, costs the health care system in the UK alone about £1 billion per year, and costs in the US being in excess of $1.7 billion per year. IBD combined with colorectal cancer costs the UK economy £8 billion per year (G. Williams et al., Gut 56 Suppl 1, 1 (2007).

Currently there is no molecular diagnostic test available for UC. At present, the diagnosis of UC involves multiple visits to a patient's own doctor, blood and stool tests followed by a referral to a hospital gastroenterology department for a colonoscopy. The colonoscopy will visually inspect the bowel and allow the collection of biopsy samples from around the colon and small bowel. These are then sent along with blood samples for further testing. Histological assessment provides a strong indicatory as to the disease phenotype and the blood results provide a rough idea as to the inflammatory status of the individual. There are problems with all of the current tests and retrospective studies of colectomy specimens have shown that 3 to 23% of patients undergoing surgery for presumed UC will ultimately be diagnosed with Crohn's disease. This misdiagnosis and subsequent surgical treatment has major otherwise avoidable complications. There is also no gold standard in determining the disease activity, which makes drug responses difficult to assess. A large proportion of colitis patients go on to develop colorectal cancer and to date no prognostic biomarkers have been verified. Increased surveillance using colonoscopy is the standard mode of practise.

It is therefore important to utilise the modern molecular biological methodologies to help diagnose the disease phenotype, help drug management and predict disease outcome.

Summary of the Invention

The present inventors have identified a number of genes which are abnormally expressed in ulcerative colitis patients. In particular, the inventors have identified that the FAM5C gene is significantly under-expressed in patients with UC compared to healthy controls. The Inventors have found that FAM5C is under-expressed in both inflamed and non-inflamed tissue. This means that FAM5C may be used as a biomarker for UC which is not clinically active, i.e. in which there is no current inflammation at the time a patient or individual of interest is assessed or a sample taken. Accordingly, this observation can be used in the diagnosis and/or prognosis of UC by screening samples from a patient.

Accordingly, the present invention provides a method for diagnosing or prognosing ulcerative colitis (UC) comprising determining the level of FAM5C in a sample obtained from a patient, wherein under-expression of FAM5C is indicative of the presence of UC or a risk of developing UC.

The invention further provides a method for distinguishing between UC and Crohn's Disease (CD), comprising determining the level of FAM5C in a sample obtained from a patient, wherein under-expression of FAM5C is indicative of the presence of UC or a risk of developing UC, rather than CD.

The invention further provides a method for diagnosing or prognosing colon cancer comprising determining the level of FAM5C in a sample obtained from a patient, wherein under-expression of FAM5C is indicative of the presence of colon cancer or a risk of developing colon cancer.

The invention also provides a test kit for use in a method according to the invention, comprising one or more reagents for determining the level of FAM5C and instructions for carrying out a method of diagnosing and/or prognosing UC.

Description of the Figures

Figure 1 Graph of FAM5C expression against location within the bowel in healthy controls (squares) compared with inflamed (closed circles) and non-inflamed (open circle) UC patients. FAM5C is abnormally under-expressed throughout the colon in patients with UC - both in non-inflamed and inflamed biopsies.

Figure 2 A - graph of FAM5C expression (measured by qPCR) in healthy controls (HC) compared with UC patients (UC). B - graph of data from second cohort (n=5, non inflamed biopsies) showing expression levels (by qPCR) of CLDN8, HOXD11, FAM5C, HOXD10 and HOXD13 in healthy controls (white bars) compared with UC patients (grey bars). Figure 3 A - graph comparing expression of FAM5C in the ascending colon in healthy controls (HC), patients with left-sided UC and patients with pancolitis. Non-inflamed samples shown in open circles, inflamed samples in closed circles. B - graph of FAM5C expression against DEFA5 expression in the descending colon. C - graph of FAM5C expression against S100A8 expression in the descending colon.

Figure 4 A - graph of FAM5C expression at different locations within the bowel in healthy controls (HC) (triangle) compared to patients with UC (circle) and patients with Crohn's disease (CD) (square). B - graph of FAM5C expression in the descending colon in HC compared with patients with UC or CD. Non-inflamed samples shown in open symbols, inflamed samples in closed symbols.

Figure 5 A - graph comparing FAM5C mRNA levels in healthy controls (HC) to patients with UC. B - graph comparing FAM5C DNA methylation levels in HC to patients with UC.

Figure 6 A - graph comparing DEFA6 expression against location within the bowel in healthy controls (square) compared with UC patients (circle). B - graph comparing

HOXD11 expression against location within the bowel in healthy controls (square) compared with UC patients (circle). C - graph comparing S100A8 expression against location within the bowel in healthy controls (square) compared with UC patients (circle).

Figure 7 A - graph of CLDN8 expression at different locations within the bowel in healthy controls (HC) (triangle) compared to patients with UC (circle) and patients with Crohn's disease (CD) (square). B - graph comparing expression of CLDN8 in HC with patients with UC or CD (inflamed samples in closed symbols, non-inflamed samples in open symbol). C - graph of CLDN8 expression against DEFA5 expression in the rectum.

Figure 8 Graph comparing FAM5C, CLDN8, HOXD 11 , DEF A6 and S 100A8 mRNA levels in healthy controls to patients with colon tumours.

Figure 9 A - Protein level of FAM5C in biopsies taken from the descending colon from UC patients with low mRNA expression was determined by Western blotting and compared against HC. FAM5C positive (NCI-H716) and negative (HT29) colonic epithelial cell lines were used as controls. B - Relative FAM5C protein levels (using relative luminenscence FAM5C/ACTIN) within the descending colon of the UC patients (n=3) was compared to HC (n=2). Figure 10 FAM5C expression and relationship to on-going inflammation. Dot plots of FAM5C Log ₂ expression in HCs, non-inflamed UC (NIUC, inflammation score =

0), inflamed UC (IUC, inflammation score >1) in: A - Rectum (NIUC=9, IUC=1 1, HC=26), B - Descending colon (NIUC=8, IUC=12, HC=17), C - Ascending colon (NIUC=3, IUC=13, HC=13) and D - Terminal ileum (NIUC=10, IUC=2, HC=13), separated according to their histological status. E - In the rectum, the UC biopsies with the lowest expression of FAM5C in the colon, graded as being non inflamed, are highlighted (1-3), with their respective histological images.

Figure 11 There is no relationship between the expression markers of colonic

inflammation and FAM5C within the colon. A - Correlation analysis of the Log ₂ expression of FAM5C in the descending colon with the Log ₂ expression of known markers of colonic inflammation, DEFA5 (Paneth cell specific defensins 5), CLDN8 (claudin 8), IL-8 and S100A8 (interleukin 8 and SI 00 calcium binding protein A8) in UC patients. R ² correlation coefficients a p values demonstrate no significant correlation between the probes. B and C - qPCR verification of high expression of DEFA5 and S100A8 in the descending colon of UC patients identified as having high expression in the initial dataset and HCs from the same anatomical location. *** p<0.001.

Figure 12 FAM5C expression is not influenced by treatment. Dot plot of FAM5C Log ₂ expression in the rectum in HC (n=26) and UC patients on no medical treatment (n=3), 5- ASA alone (n=14), 5-ASA plus AZA (n=3) and CD patients who are on treatment (5-ASA ± AZA) (n=4). *** p<0.001; ns, non-significant. (5-ASA, 5-aminosalicylates; AZA, azathioprine.) Figure 13 Longitudinal studies into FAM5C expression. Low expression of FAM5C in patients with previously documented low FAM5C expression was verified by qPCR using repeat biopsies taken from the: A - Rectum, UC (n=5) and HCs (n=4); B - Descending colon, UC (n=5) and HCs (n=4). Results expressed as Mean ± SEM, **p<0.01, ***p<0.001. Figure 14 Cellular localisation of FAM5C in the lower gastrointestinal tract. A - Immunohistochemistry of FAM5C performed on resection specimens from HC patients in the terminal ileum (i-ii) and colonic mucosa (iii) {black arrows denote FAM5C expression). B - Electron micrographs of the brush border epithelium in a human colonic epithelial cell using anti-FAM5C and immunogold secondary antibodies (i-iii). (ii-iii) Immunogold staining of the microvilli shown in (i) {black box) at higher magnification {black arrows and numbered 1-4) Abbreviations: MV, microvilli; Cy, cytoplasm; JC, epithelial cell junctional complex; Nu, nucleus. C - Subcellular fractionation of the NCI-H716 adenocarcinoma epithelial cell line followed by Western blotting with anti-FAM5C, anti-GPR75 (a mitochondrial protein) and anti-vinculin (an epithelial cytoskeletal protein). PNS, post nuclear supernatant. D - Separation of the soluble (S, supernatant) and insoluble (P, pellet) material in the 24% sucrose fraction (F) by ultracentrifugation, followed by western blotting of FAM5C and vinculin. E - Confocal microscopy of the NCI-H716 epithelial cell line stained with FAM5C and vinculin.

Figure 15 Confocal microscopy demonstrates no co-localisaton of FAM5C with the mitochondrial marker, GPR75. Confocal microscopy using the NCI-H716 epithelial cell line shows no evidence of any co-localisation of FAM5C with the mitochondrial marker, GPR75. Figure 16 Methylation of the FAM5C promoter in the distal colon. A - Quantification of FAM5C Log ₂ relative expression in the rectum in re-biopsied UC (n=5) patients and additional HC (n=4) patients by qPCR. B - Percentage (%) methylation status of the FAM5C promoter in UC (n=5) and HC (n=4) rectal biopsies and in circulating leukocytes in the same UC (n=4) and HC (n=4) patients. ***p<0.001; **p<0.01; ns, non-significant.

Figure 17 Hypermethylation of FAM5C appears to be associated with low mRNA expression. Regression analysis between FAM5C Log ₂ relative expression from qPCR verification and FAM5C percentage (%) methylation in the rectum in re-biopsied UC (n=5) patients and additional HCs (n=4) demonstrating a significant inverse relationship

(r ² =0.6991, p=0.005).

Detailed Description of the Invention

The present invention provides methods for the diagnosis and/or prognosis of ulcerative colitis (UC) in a patient or individual of interest. These methods involve determining the level of a biomarker for UC in a sample from the patient or individual of interest. The biomarker for UC is Family with Sequence Similarity 5, member C (FAM5C). In some embodiments of the invention, the methods use diagnostic and/or prognostic chips. FAM5C

FAM5C was originally identified in the mouse brain as a gene that is induced by bone morphogenic protein and retinoic acid signalling (BRINP3 gene). Studies have also shown that FAM5C localises to the mitochondria. Levels of FAM5C have been shown to be increased in human pituitary tumours, and FAM5C polymorphisms have been shown to be associated with myocardial infarction.

The Inventors have now found that the level of FAM5C in individuals may be used to aid the diagnosis of UC. In particular, the Inventors have found that the level of FAM5Cis significantly reduced in bowel samples taken from patients suffering from UC. The Inventors have also shown that the under-expression of FAM5C is not dependent on inflammation, observing the under-expression of FAM5C in samples taken from non-inflamed regions of the bowel in patients suffering from UC. Accordingly, under-expression of FAM5C is indicative of the presence of UC or a risk of developing UC. It is possible to provide a method for diagnosing and/or prognosing UC in a patient or individual of interest comprising determining the level of FAM5C in a sample obtained from the patient or individual. Thus, the diagnosis and/or prognosis of UC may be achieved by determining the level of FAM5C in a sample obtained from the patient or individual of interest and thereby diagnosing UC. A decreased level of FAM5C in the sample, such as a decreased level when compared to the level in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC, such as Crohn's Disease (CD), may reflect the presence of UC in the patient or individual of interest.

According to the invention, it is therefore possible to diagnose whether a patient or individual has UC, and also to predict or prognose the risk of the patient or individual developing UC in the future. It is also possible to monitor disease progression of UC.

Monitoring of disease progression typically involves repeated testing during therapy, such that an accurate measure of disease activity and/or drug responsiveness can been monitored. Repeated testing may, for example, be carried out on a weekly, monthly, every two months, every three months, every four months, every six months, yearly, or otherwise as appropriate to a given individual or patient. As FAM5C is not under expressed in individuals suffering from CD, it is possible to use the level of FAM5C to differentiate between UC and CD.

The Inventors have found that FAM5C levels are decreased in samples taken from colon tumours. Lower levels of FAM5C is indicative of a patient and/or individual being at risk of developing colorectal cancer, and so may be considered as a risk factor for colorectal cancer. Patients and/or individuals identified as at risk may require more frequent screening to monitor their condition. Accordingly, it is possible to use the level of FAM5C to diagnose and/or prognose colon cancer. Therefore, the invention provides a method for diagnosing or prognosing colon cancer comprising determining the level of FAM5C in a sample obtained from a patient, wherein under-expression of FAM5C is indicative of the presence of colon cancer or a risk of developing colon cancer.

The FAM5C gene is hypermethylated in samples taken from patients or individuals suffering from UC. Measuring the methylation of the FAM5C gene may also be used in the diagnosis and/or prognosis of UC.

The FAM5C gene may have the nucleotide sequence of SEQ ID NO: 1. The FAM5C gene may be a variant of SEQ ID NO: 1 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the nucleotide sequence of SEQ ID NO: 1.

The FAM5C protein may have the amino acid sequence of SEQ ID NO: 2. The FAM5C protein may be a variant of SEQ ID NO: 1 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 2.

The level of FAM5C in a sample encompasses the mass of FAM5C in the sample, the molar level of FAM5C in the sample, the concentration of FAM5C in the sample, the molarity of FAM5C in the sample. This level may be given in any appropriate units. For example, the concentration of FAM5C may be given in pg/ml, ng/ml or μg/ml. The mass of FAM5C may be given in pg, ng or μg.

The level of FAM5C may be measured directly. For example, the FAM5C itself may be detected in the sample and the level of the FAM5C in the sample may be assessed.

The level of FAM5C may be measured indirectly. For example, an agent or label that binds to FAM5C may be used to detect and/or quantify the FAM5C. The level of that agent or label may then be assessed as an indicator of the level of FAM5C in the sample.

The level of FAM5C in a sample of interest may be compared with the level of FAM5C in another sample as described herein. Typically, the level of FAM5C in a sample of interest is compared with the level of FAM5C is a sample from a healthy individual, a sample from an individual who does not suffer from UC or a sample from an individual who suffers from a form of IBD other than UC, such as Crohn' s Disease (CD). In such a method, the actual levels of FAM5C, such as the mass, molar amount, concentration or molarity of the FAM5C in the samples may be assessed. The levels of FAM5C in the samples may be measured directly or indirectly as described herein. The level of FAM5C may be compared with that in another sample without quantifying the mass, molar amount, concentration or molarity of the FAM5C. Thus, the level of FAM5C in a sample according to the invention may be assessed as a relative level, such as a relative mass, relative molar amount, relative concentration or relative molarity of the FAM5C based on a comparison between two or more samples.

The level of FAM5C in a sample obtained from the patient or individual of interest may be determined using any appropriate technique. Standard methods known in the art may be used to assay the level of FAM5C. These methods may involve using an agent for the detection of FAM5C and/or an agent for the determination of the level of FAM5C. Any method that allows for the detecting of FAM5C and the quantification, or relative

quantification of the FAM5C may be used. The presence and/or level of FAM5C may be determined using techniques that involve binding an antibody or other ligand to the FAM5C. Such an antibody or other ligand may be labelled and that label may be detected to detect and/or quantify the FAM5C in the sample. Such techniques may include Enzyme-linked Immunosorbant Assay (ELISA) and immunofluorescence. The level of FAM5C may be determined using mass spectrometry.

Agents for the detection of or for the determination of the level of FAM5C may be used to determine the level of FAM5C in a sample obtained from the patient or individual of interest. Such agents typically bind to FAM5C. Such agents may bind specifically to

FAM5C. The agent for the detection of or for the determination of the level of FAM5C may be an antibody or other binding agent specific for FAM5C. By specific, it will be understood that the agent or antibody binds to the molecule of interest, in this case FAM5C, with no significant cross-reactivity to any other molecule, particularly any other protein. For example, an agent or antibody that is specific for FAM5C will show no significant cross- reactivity with human neutrophil elastase. Cross-reactivity may be assessed by any suitable method. Cross-reactivity of an agent or antibody for FAM5C with a molecule other than FAM5C may be considered significant if the agent or antibody binds to the other molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to FAM5C. An agent or antibody that is specific for FAM5C may bind to an other molecule such as human neutrophil elastase at less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that it binds to FAM5C. Preferably, the agent or antibody binds to the other molecule at less than 20%, less than 15%, less than 10% or less than 5%, less than 2% or less than 1% the strength that it binds to FAM5C.

An antibody used in the method of the invention may be a whole antibody or a fragment thereof which is capable of binding to FAM5C e.g. Fab or F(ab)2 fragments. The antibody may be monoclonal or polyclonal. The antibody may be produced by any suitable method known in the art. For example, polyclonal antibodies may be obtained by

immunising a mammal, typically a rabbit or a mouse, with FAM5C under suitable conditions and isolating antibody molecules from, for example, the serum of said mammal. Monoclonal antibodies may be obtained by hybridoma or recombinant methods.

Hybridoma methods may involve immunising a mammal, typically a rabbit or a mouse, with FAM5C under suitable conditions, then harvesting the spleen cells of said mammal and fusing them with myeloma cells. The mixture of fused cells is then diluted and clones are grown from single parent cells. The antibodies secreted by the different clones are then tested for their ability to bind to FAM5C, and the most productive and stable clone is then grown in culture medium to a high volume. The secreted antibody is collected and purified.

Recombinant methods may involve the cloning into phage or yeast of different immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences. Those sequences which give rise to antibodies which bind to FAM5C may be selected and the sequences cloned into, for example, a bacterial cell line, for production. Typically the antibody is a mammalian antibody, such as a primate, human, rodent (e.g. mouse or rat), rabbit, ovine, porcine, equine or camel antibody. The antibody may be a camelid antibody or shark antibody. The antibody may be a nanobody. The antibody can be any class or isotype of antibody, for example IgM, but is preferably IgG. The antibody may be a humanised antibody.

The antibody or fragment may be associated with other moieties, such as linkers which may be used to join together 2 or more fragments or antibodies. Such linkers may be chemical linkers or can be present in the form of a fusion protein with the fragment or whole antibody. The linkers may thus be used to join together whole antibodies or fragments which have the same or different binding specificities, e.g. that can bind the same or different polymorphisms. The antibody may be a bispecific antibody which is able to bind to two different antigens, typically any two of the polymorphisms mentioned herein. The antibody may be a 'diabody' formed by joining two variable domains back to back. In the case where the antibodies used in the method are present in any of the above forms which have different antigen binding sites of different specificities then these different specificities are typically to polymorphisms at different positions or on different proteins. In one embodiment the antibody is a chimeric antibody comprising sequence from different natural antibodies, for example a humanised antibody.

Methods to assess a level of FAM5C may involve contacting a sample with an agent or antibody capable of binding specifically to FAM5C. Such methods may include dipstick assays and Enzyme-linked Immunosorbant Assay (ELISA). Other immunoassay types may also be used to assess FAM5C levels. Typically dipsticks comprise one or more antibodies or proteins that specifically bind FAM5C. If more than one antibody is present, the antibodies preferably have different non-overlapping determinants such that they may bind to FAM5C simultaneously.

ELISA is a heterogeneous, solid phase assay that requires the separation of reagents. ELISA is typically carried out using the sandwich technique or the competitive technique. The sandwich technique requires two antibodies. The first specifically binds FAM5C and is bound to a solid support. The second antibody is bound to a marker, typically an enzyme conjugate. A substrate for the enzyme is used to quantify the FAM5C -antibody complex and hence the level of FAM5C in a sample. The antigen competitive inhibition assay also typically requires a FAM5C -specific antibody bound to a support. A FAM5C -enzyme conjugate is added to the sample (containing FAM5C) to be assayed. Competitive inhibition between the FAM5C -enzyme conjugate and unlabeled FAM5C allows quantification of the level of FAM5C in a sample. The solid supports for ELISA reactions preferably contain wells.

Antibodies capable of binding specifically to FAM5C may be used in methods of immunofluorescence to detect the presence of FAM5C and hence in methods of diagnosing UC according to the present invention.

The present invention may also employ methods of determining the level of FAM5C that do not comprise antibodies. Quantitative assays may be based on measuring the mRNA levels of FAM5C and any additional biomarkers. Appropriate assays include microarrays, bead arrays, quantitative PCR (qPCR) and any other applicable methodology. Microarrays may take the form of prognostic and/or diagnostic chips, with one or more probes for FAM5C immobilised on a solid support. Quantitative assays may also be based on measuring FAM5C protein levels in a sample. High Performance Liquid Chromatography (FIPLC) separation and fluorescence detection may be used as a method of determining the level of FAM5C. FIPLC apparatus and methods as described previously may be used (Tsikas D et al. J Chromatogr B Biomed Sci Appl 1998; 705 : 174-6) Separation during FIPLC is typically carried out on the basis of size or charge. Prior to HPLC, endogenous amino acids and an internal standard L-homoarginine are typically added to assay samples and these are phase extracted on CBA cartridges (Varian, Harbor City, CA). Amino acids within the samples are preferably derivatized with o-phthalaldehyde (OP A). The accuracy and precision of the assay is preferably determined within quality control samples for all amino acids. Other methods of determining the level of FAM5C that do not comprise antibodies include mass spectrometry. Mass spectrometric methods may include, for example, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), surface-enhanced laser desorption/ionization mass spectrometry (SELDI MS), time of flight mass spectrometry (TOF MS) and liquid chromatography mass spectrometry (LC MS).

Other biomarkers for UC

The methods of the invention comprise detecting or determining the level of one or more biomarkers for UC. A biomarker is a characteristic or molecule that can be objectively measured and evaluated as an indicator of a biologic processes or state, such as UC.

Different biomarkers for UC may increase or decrease in level when UC is present.

The preferred biomarker is FAM5C. The level of FAM5C in a sample from a patient or individual of interest may be used to determine whether that patient or individual has UC. As discussed above, the level of FAM5C may be assessed by any known method. A decreased level of FAM5C as described herein indicates that the patient or individual of interest may have UC, or that the patient or individual at interest is at risk of developing UC.

According to the present invention, a method for diagnosing UC may comprise determining the level of FAM5C in a sample from a patient or individual of interest and also detecting or determining the level of one or more additional biomarkers for UC and/or inflammation.

Additional biomarkers for UC include CLDN8, HOXD 1 1, DEFA6 and S 100A8. Other biomarkers for UC may be used. Biomarkers for inflammation, and other general markers for IBD may also be used. A method for diagnosing UC may comprise determining the level of one or more of these additional biomarkers in addition to determining the level of FAM5C.

The level of CLDN8 and/or HOXDl 1 is typically decreased in individuals suffering from UC compared with the level of CLDN8 and/or HOXDl 1 in healthy individuals, individuals who do not suffer from UC, or from individuals suffering from a form of IBD other than UC, such as Crohn's Disease (CD). Accordingly, under-expression of CLDN8 and/or HOXDl 1 is indicative of the presence of UC or a risk of developing UC.

The level of DEFA6 and/or S 100A8 is typically increased in individuals suffering from UC compared with the level of DEFA6 and/or S 100A8 in healthy individuals, individuals who do not suffer from UC, or from individuals suffering from a form of IBD other than UC, such as Crohn's Disease (CD). Accordingly, over-expression of DEFA6 and/or S 100A8 is indicative of the presence of UC or a risk of developing UC.

The one or more additional biomarkers may be used in any combination with

FAM5C. For instance, FAM5C may be used with one or more of CLDN8, HOXDl 1, DEF A6 and S 100 A8, two or more of CLDN8, HOXD 1 1 , DEF A6 and S 100 A8, three or more of CLDN8, HOXDl 1, DEFA6 and S 100A8 or all of CLDN8, HOXDl 1, DEFA6 and

S 100A8. In particular, FAM5C may be used with CLDN8, HOXDl 1, DEFA6 or S100A8, with CLDN8 and HOXDl 1, with CLDN8 and DEFA6, with CLDN8 and S 100A8, with HOXDl 1 and DEFA6, with HOXDl 1 and S 100A8, with DEFA6 and S 100A8, with CLDN8, HOXD 1 1 and DEF A6, with CLDN8, HOXD 1 1 and S 100 A8, with HOXD 1 1 , DEF A6 and S 100A8, or with CLDN8, DEFA6 and S 100A8.

The CLDN8 gene may have the nucleotide sequence of SEQ ID NO: 3. The CLDN8 gene may be a variant of SEQ ID NO: 3 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the nucleotide sequence of SEQ ID NO: 3.

The CLDN8 protein may have the amino acid sequence of SEQ ID NO: 4. The CLDN8 protein may be a variant of SEQ ID NO: 4 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 4.

The HOXDl 1 gene may have the nucleotide sequence of SEQ ID NO: 5. The

HOXDl 1 gene may be a variant of SEQ ID NO: 5 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the nucleotide sequence of SEQ ID NO: 5. The HOXD1 1 protein may have the amino acid sequence of SEQ ID NO: 6. The HOXD1 1 protein may be a variant of SEQ ID NO: 6 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 6.

The DEFA6 gene may have the nucleotide sequence of SEQ ID NO: 7. The DEFA6 gene may be a variant of SEQ ID NO: 7 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the nucleotide sequence of SEQ ID NO: 7.

The DEFA6 protein may have the amino acid sequence of SEQ ID NO: 8. The DEFA6 protein may be a variant of SEQ ID NO: 8 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 8.

The S100A8 gene may have the nucleotide sequence of SEQ ID NO: 9. The S100A8 gene may be a variant of SEQ ID NO: 9 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the nucleotide sequence of SEQ ID NO: 9.

The S 100A8 protein may have the amino acid sequence of SEQ ID NO: 10. The S 100A8 protein may be a variant of SEQ ID NO: 10 that shares at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 10.

The one or more additional biomarkers may be detected or the level of the one or more additional biomarkers determined using any of the techniques disclosed herein for the determination of the level of FAM5C. Agents or antibodies specific for the one or more additional biomarkers may be used in such techniques. The one or more additional biomarkers may, for example, be detected, or the level of the one or more additional biomarkers determined using ELISA, immunofluorescence, UPLC, or mass spectrometry. Mass spectrometric methods may include, for example, MALDI MS, SELDI MS, TOF MS, LC MS.

The one or more additional biomarkers may be detected or the level of the one or more additional biomarkers determined in the same sample obtained from the patient or individual of interest in which the level of FAM5C is determined. The one or more additional biomarkers may be detected or the level of the one or more additional biomarkers determined in a different sample obtained from the patient or individual of interest in which the level of FAM5C is determined. The one or more additional biomarkers may be detected or the level of the one or more additional biomarkers determined at the same time as the level of FAM5C is determined. In particular, the one or more additional biomarkers may be detected or the level of the one or more additional biomarkers determined during the same process as the level of FAM5C is determined. Alternatively, the one or more additional biomarkers may be detected or the level of the one or more additional biomarkers determined before or after the level of FAM5C is determined, or in a separate process. The level of FAM5C may be determined first, with the one or more additional biomarkers detected or the level of the one or more additional biomarkers determined afterwards. The subsequent detection of the one or more additional biomarkers detected or the subsequent determination of the level of the one or more additional biomarkers may be used to confirm the diagnosis and/or prognosis using FAM5C.

The one or more additional biomarkers may be detected or the level of the one or more additional biomarkers determined by the same or different methodology as is used to determine the level of F AM5 C .

The method for diagnosing and/or prognosing UC of the present invention may involve determining the level of FAM5C in a sample obtained from a patient and determining the level of one or more biomarkers for UC and/or inflammation in the sample in one step.

The method for diagnosing and/or prognosing UC of the present invention may involve determining the level of FAM5C in a sample obtained from a patient and determining the level of one or more biomarkers for UC and/or inflammation in the sample in two or more steps.

According to the present invention, a method for diagnosing and/or prognosing UC may comprise determining the level of FAM5C for UC in a sample from a patient or individual of interest. Thus, the diagnosis and/or prognosis of UC may be achieved by determining the level of FAM5C in a sample obtained from the patient or individual of interest and thereby diagnosing UC in the patient or individual of interest.

Such a method will include FAM5C, and may in addition include any of the other biomarkers for UC described herein. The one or more additional biomarker for UC may be selected from CLDN8, HOXD1 1, DEFA6 and S 100A8.

The "leveF of a biomarker for UC will be understood to have the equivalent meaning as the "leveF of FAM5C as discussed above. Any of the meanings or definitions of an level discussed above in relation to FAM5C also apply to other biomarkers of UC as discussed herein. The level of any biomarker of UC may be determined using any of the methods described above for determining the level of FAM5C.

The discussion above in relation FAM5C also applies to other biomarkers of UC. Agents for the detection of a biomarker of UC may be the same types of agents described above in relation to FAM5C. Equivalent agents may be used to detect other biomarkers of UC. For example, any antibody to a biomarker of UC may be used in the same way described above for antibodies to FAM5C. The same types of antibodies described above in relation to FAM5C may be used to detect other biomarkers of UC. Such agents or antibodies may be specific to a biomarker of UC as defined above in relation to FAM5C. The methods described above for the detection or determining the level of FAM5C may be used to detect or determine the level of other biomarkers of UC.

Sample obtained from a patient or individual of interest

The invention is typically carried out in vitro on a sample obtained from a patient or individual of interest. The sample may be from the gastrointestinal tract, particularly the bowel, or may be a faecal/stool sample, serum or plasma sample, or any other appropriate body tissue or fluid. The sample typically comprises a tissue sample from the bowel of the individual of interest. Said tissue sample may have been obtained from a biopsy performed during a colonoscopy. Typically the tissue sample size is at least lmg, at least 5mg, at least lOmg, at least 20mg, at least 30mg, at least 40mg, at least 50mg or at least lOOmg. Tissue samples of may be obtained by standard endoscopic pinch biopsies. The colonoscopy may be either part of the routine monitoring of a patient or individual, or as a diagnostic for UC.

Samples may be obtained from any location in the bowel. In preferred embodiments, the sample is taken from the rectum, sigmoid colon, ascending colon or descending colon. The sample may be taken from an area of the bowel that is inflamed, or from an area of the bowel that is not inflamed at the time of sampling. The under-expression of FAM5C is independent of inflammation.

The sample may be processed prior to being assayed, for example by centrifugation.

The sample may also be stored prior to assay, for example below -80°C.

The sample obtained from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC, such as Crohn' s

Disease (CD) may be obtained as outlined above.

When the level of FAM5C in the sample obtained from the patient or individual of interest is compared with the level of FAM5C in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC, such as Crohn' s Disease (CD), the sample from the a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC, such as Crohn's Disease (CD) is preferably a sample of the same body fluid. The control sample may also be of the same volume.

Comparative methods

In the methods of the invention, the level of a biomarker such as FAM5C in a sample from an individual or patient of interest may be compared with the level of said biomarker in one or more other samples. This comparison may be with a control sample. A suitable control sample is from a comparable source, such as a gender and/or age-matched individual.

Preferably the control individual does not have UC.

A suitable control sample is from a source that is not expected to have elevated levels of the biomarker such as FAM5C. A suitable control sample may be a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC, such as Crohn's Disease (CD).

The methods of the invention allow for the comparison of the level of biomarker such as FAM5C in different samples. These methods allow for the determination of the relative level of a biomarker such as FAM5C in a sample from a patient or individual of interest when compared to a control.

If the level of a biomarker for UC such as FAM5C in the sample from the individual or patient of interest is less than that in the control sample, such as a sample from a healthy individual, this indicates that the individual or patient of interest is likely to have UC, or to develop UC. This may indicate a diagnosis or prognosis of UC or may suggest the presence of UC or a risk of developing of UC. Such a diagnosis or suggestion may lead to further testing for UC.

Some biomarkers, such as FAM5C, have a reduced level in UC. For those biomarkers, if the level of a biomarker in the sample from the patient or individual of interest is lower than that in the control sample, such as a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC, this indicates that the patient or individual of interest is likely to have UC or to be at risk of UC. This may indicate a diagnosis or prognosis of UC for the individual or patient or may suggest the presence of UC. Such a diagnosis or suggestion may lead to further testing for UC. Other biomarkers have an increased level in UC. If the level of such a biomarker for UC in the sample from the patient or individual of interest is significantly greater than that in the control sample, such as a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC, this indicates that the patient or individual of interest is likely to have UC or be at risk of UC..

A significant increase or decrease may be assessed by standard statistical methods. Z scores and p values may be used to assess whether an increase or decrease in biomarker expression is significant compared to one or more control or reference samples. To assess the significance of the standardised deviation of the patient sample expression values from the comparison group average, a Z-score is calculated, using equation 7, for each probe-set.

F sample - ra

^ = (equation 1)

σ

where: E _sam pie is the expression level of the patient sample; and μ and σ are the mean and standard deviation of the expression levels of the samples in the comparison group. The comparison group may be a control or reference group, for example, one or more healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC. From these Z-scores, p-values are calculated by determining the likelihood that the standardised expression value of the probe-set, in the patient sample, would be selected at random from a normal distribution with a mean of 0 and standard deviation of 1. These p-values can be used to assess the significance. According to the present invention, the increase or decrease in level of at least one biomarker for UC which is associated with a risk of UC may be a value of p<0.05, p<0.01, p<0.005, pO.001, p<0.0005 or pO.0001.

According to the present invention, the increase or decrease in level of at least one biomarker for UC which is associated with a risk of UC may be an increase or decrease of at least 1.5 fold and typically by at least 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold or 10 fold relative to the level of the same biomarker(s) for UC in a control sample, such as a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC.

Different levels of increase or decrease in biomarker expression may be observed in different regions within the gastrointestinal tract, particularly the bowel. The level of decrease in FAM5C expression may differ depending on from where the sample is taken, for example, whether the sample is taken from the rectum, the sigmoid colon, the ascending colon or the descending colon. For example, typical values for the FAM5C levels found in the ascending colon, descending colon and rectum of a healthy individual are shown in Table 1 below.

Table 1 - representative levels of FAM5C in samples from the ascending colon, descending colon and rectum of a healthy individual.

The level of decrease in CLDN8 expression may differ depending on from where the sample is taken, for example, whether the sample is taken from the rectum, the sigmoid colon, the ascending colon or the descending colon. The level of decrease in HOXDl 1 may differ depending on from where the sample is taken, for example, whether the sample is taken from the rectum, the sigmoid colon, the ascending colon or the descending colon. For example, typical values for the CLDN8 levels found in the ascending colon, descending colon and rectum of a healthy individual are shown in Table 2 below.

Table 2 - representative levels of CLDN8 in samples from the ascending colon, descending colon and rectum of a healthy individual.

The level of increase in DEFA6 expression may differ depending on from where the sample is taken, for example, whether the sample is taken from the rectum, the sigmoid colon, the ascending colon or the descending colon. The level of increase in S 100A8 expression may differ depending on from where the sample is taken, for example, whether the sample is taken from the rectum, the sigmoid colon, the ascending colon or the descending colon. For example, typical values for the DEFA6 levels found in the ascending colon, descending colon and rectum of a healthy individual are shown in Table 3 below.

Table 3 - representative levels of DEFA6 in samples from the ascending colon, descending colon and rectum of a healthy individual.

The level of FAM5C in the descending colon of a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC may be more than 7.5, typically between 7.5 and 8.5 and preferably between 7.7 and 8.3.

The level of FAM5C in a patient or individual with UC or at risk of developing UC may be less than 7.5, is typically between 6.5 and 7.5 and preferably between 7.0 and 7.3.

According to the present invention, the decrease in FAM5C level which is associated with a risk of UC may be an decrease of at least 1.5 fold and typically at least 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold or 10 fold relative to the level of FAM5C in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC.

The decrease in FAM5C level which is associated with a risk of UC may be a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to the level of FAM5C in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC.

The decrease in FAM5C level which is associated with a risk of UC may be calculated using Z scores and p-value cut offs compared to one or more control or reference group, for example, one or more healthy individual, an individual who does not suffer from

UC, or from an individual suffering from a form of IBD other than UC can be used to assess the significance. According to the present invention, the decrease in level of FAM5C which is associated with a risk of UC may be a value of p<0.05, p<0.01, p<0.005, p<0.001, p<0.0005 or pO.0001. According to the present invention, the decrease in CLDN8 level which is associated with a risk of UC is an decrease of at least 1.5 fold and typically at least 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold or 10 fold relative to the level of CLDN8 in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC.

The decrease in CLDN8 level which is associated with a risk of UC may be calculated using Z scores and p-value cut offs compared to one or more control or reference group, for example, one or more healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC can be used to assess the significance. According to the present invention, the decrease in level of CLDN8 which is associated with a risk of UC may be a value of p<0.05, p<0.01, p<0.005, p<0.001, p<0.0005 or pO.0001.

According to the present invention, the decrease in HOXDl 1 level which is associated with a risk of UC is an decrease of at least 1.5 fold and typically at least 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold or 10 fold relative to the level of

HOXDl 1 in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC.

The decrease in HOXDl 1 level which is associated with a risk of UC may be calculated using Z scores and p-value cut offs compared to one or more control or reference group, for example, one or more healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC can be used to assess the significance. According to the present invention, the decrease in level of HOXDl 1 which is associated with a risk of UC may be a value of p<0.05, p<0.01, p<0.005, p<0.001, p<0.0005 or pO.0001.

According to the present invention, the increase in DEFA6 level which is associated with a risk of UC is an increase of at least 1.5 fold and typically at least 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold or 10 fold relative to the level of DEFA6 in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC.

The increase in DEFA6 level which is associated with a risk of UC may be calculated using Z scores and p-value cut offs compared to one or more control or reference group, for example, one or more healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC can be used to assess the significance. According to the present invention, the increase in level of DEFA6 which is associated with a risk of UC may be a value of p<0.05, p<0.01, p<0.005, p<0.001, p<0.0005 or p<0.0001.

According to the present invention, the increase in S 100A8 level which is associated with a risk of UC is an increase of at least 1.5 fold and typically at least 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold or 10 fold relative to the level of S I 00 A8 in a sample from a healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC.

The increase in S 100A8 level which is associated with a risk of UC may be calculated using Z scores and p-value cut offs compared to one or more control or reference group, for example, one or more healthy individual, an individual who does not suffer from UC, or from an individual suffering from a form of IBD other than UC can be used to assess the significance. According to the present invention, the increase in level of S 100A8 which is associated with a risk of UC may be a value of p<0.05, p<0.01, p<0.005, pO.001, p<0.0005 or pO.0001.

In order to take account of this variation, the level of FAM5C may be expressed as a multiple of the median for unaffected individuals (MoM). The level of other biomarkers for UC may be expressed in the same way. For example, if the median biomarker level is 2.5 μιηοΙ/L, the biomarker level of a patient or individual who is found to have 5.0 μιηοΙ/L biomarker is twice that of the median (5.0/2.5) or 2.0 MoM. Similarly, the biomarker level of a patient or individual who is found to have 1.25 μιηοΙ/L is half that of the median (1.25/2.5) or 0.5 MoM.

According to the present invention, the level of FAM5C level which is associated with a risk of UC may be expressed as an MoM of less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.05 or less than 0.01.

According to the present invention, the level of CLDN8 level which is associated with a risk of UC may be expressed as an MoM of less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.05 or less than 0.01. According to the present invention, the level of HOXD1 1 level which is associated with a risk of UC may be expressed as an MoM of less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.05 or less than 0.01.

According to the present invention, the level of DEFA6 level which is associated with a risk of UC may be expressed as an MoM of at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 3.0, at least 4.0, at least 5.0 or at least 10.0. According to the present invention, the level of S 100A8 level which is associated with a risk of UC may be expressed as an MoM of at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 3.0, at least 4.0, at least 5.0 or at least 10.0.

The sample obtained from a patient or individual of interest may also be tested to determine the level of one or more housekeeping genes. The level of the one or more housekeeping genes may be used as an internal reference point for the sample. For example the level of the one or more housekeeping genes may provide information on the total level of material, within the sample. This would allow data to be normalised, and comparisons made between different samples, which, for example, may be of different concentrations.

Examples of housekeeping genes useful in the present invention include GAPDH, ACTG1, RPL41, RPS27 and ACTB.

Further diagnostic methods

The methods of the invention allow for a diagnosis for UC based on a method carried out on a patient or individual of interest. The methods of the invention may be used in combination with other methods for detecting or diagnosing UC.

These additional tests may be carried out at the same time, before or after the level of

FAM5C is determined. For example, a colonoscopy may be carried out to examine the bowel, and biopsy samples obtained. A biopsy sample may then be tested for FAM5C.

Examples of such additional diagnostic tests that may be used are blood and stool tests, colonoscopy and histological analysis of biopsy samples. Such other methods may involve detection of other biomarkers of UC. Diagnostic and Prognostic Chips

The invention provides diagnostic and or prognostic chips for use in diagnosing and/or prognosing UC. These chips will have probes for FAM5C attached to their surface that can be used to detect and determine the level of FAM5C in a sample. These probes may be immobilised on the chip by any appropriate means. The probes may be any appropriate means to bind FAM5C specifically to the surface of the chip. The probes may be antibodies or oligonucleotides, including fragments of FAM5C. One or more different probes for FAM5C may be included on the same chip. For example, multiple oligonucleotides or antibodies may be used. The discussion above in relation to FAM5C also applies to other biomarkers of UC. Agents for the detection of a biomarker of UC may be the same types of agents described above in relation to the particular biomarker for UC, FAM5C. Equivalent agents may be used to detect other biomarkers of UC. For example, any antibody to a biomarker of UC may be used in the same way described above for antibodies to FAM5C. The same types of antibodies described above in relation to FAM5C may be used to detect other biomarkers of UC. Such agents or antibodies may be specific to a biomarker of UC as defined above in relation to the specific biomarker FAM5C. The methods described above for the detection or determining the level of FAM5C may be used to detect or determine the level of other biomarkers of UC.

When using a diagnostic and/or prognostic chip of the invention, biopsies may be obtained during routine endoscopy (for example, from the rectum, sigmoid colon, descending colon and/or ascending colon). The biopsy samples may be placed into RNA stabilization reagent before being stored at room temperature for 24 hours. The samples may then be frozen at -80°C for up to one year.

The samples may then be lysed and homogenised using appropriate buffers and equipment. Proteins may be removed from the sample and the total RNA extracted. DNA may be removed by digestion using an RNase free DNase enzyme. Total RNA may be converted into cDNA and the level of FAM5C determined using the appropriate method. The level of the one or more additional biomarkers for UC and one or more housekeeping genes may also be determined. The appropriate method for determining the level of cDNA may be selected from spot based and bead based microarrays, as well as q PCR assays.

Results may be quantitated and normalised to the housekeeper genes and compared with a standard cohort to determine the changes in expression the FAM5C gene and any additional biomarkers for UC.

Kits

The invention provides test kits for use in the methods of the invention. The invention provides a test kit for use in a method of diagnosing or prognosing UC or colon cancer according to the present invention, comprising one or more reagents for determining the level of FAM5C and instructions for carrying out a method of diagnosing and/or prognosing UC or colon cancer.

Such kits typically comprise an reagent for the determination of the level of FAM5C. Such kits typically comprise instructions for carrying out a suitable method for diagnosing UC comprising determining the level of FAM5C in a sample from the patient or individual of interest.

The test kit may also include one or more reagent for determining the level of one or more additional biomarkers for UC and/or inflammation. The one or more additional biomarkers for UC and/or inflammation may be selected from CLDN8, HOXD1 1, DEFA6 and S100A8. One or more reagents for two or more, three or more, or all of CLDN8, HOXD1 1, DEFA6 and S 100A8 may be included in the test kit of the invention.

Any reagent for the detection of a biomarker of UC may be used. Suitable reagents are described herein. The one or more reagents for the detection of a biomarker of UC may be selected from an antibody or fragment thereof, nucleic acid probe, or primer/primer pair as described herein. The detection reagent may be immobilised on a test chip.

The test kit of the invention may comprise a chip comprising one or more probes for FAM5C. The one or more probes for FAM5C may be immobilised on the surface of the chip. Any appropriate means may be used to immobilise the one or more probes for FAM5C on the surface of the chip. The chip may be a diagnostic or prognostic chip for UC. The chip may additionally contain one or more probes for other biomarkers of UC. The probes may be selected from antibodies, nucleic acids (including both DNA and RNA) and any other suitable probe materials. Other suitable reagents include primers and primer pairs for FAM5C. Such primers and primer pairs may be immobilised on a chip of the invention.

The test kit may comprise a single chip comprising one or more probes or reagents for

FAM5C and one or more probes or reagents for the one or more additional biomarkers for UC and/or inflammation. The test kit may comprise a chip comprising one or more probes for FAM5C and a different chip comprising one or more probes for the one or more additional biomarkers for UC and/or inflammation. The level of FAM5C may be measured alongside the level or concentration of other biomarkers for UC, such as CLDN8, HOXD1 1, DEFA6 and S 100A8. One or more reagents for the detection of one or more additional biomarkers for UC or agents for determining the level of one or more additional biomarkers for UC may be included in the kit. The one or more reagents for the detection of the one or more additional biomarkers may be an antibody or fragment thereof, or a nucleic acid probe, as described herein.

In one embodiment, the chip of the invention contains one or more probes or reagents for FAM5C and the one or more other biomarkers of UC and/or inflammation of the invention (CLDN8, HOXD1 1, DEFA6 and S100A8), and optionally includes one or more probes or reagents for other biomarkers of UC, CD, IBD or inflammation, but no probes or reagents for any other biomarkers.

The kits of the invention may additionally comprise means for the measurement of other laboratory or clinical parameters.

The kits of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the methods of the invention to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means to isolate the at least one biomarker for UC from a sample or a support on which quantitative reactions can be done.

The kits of the invention may, optionally, comprise instructions to enable the kit to be used in the method of the invention or details regarding which individuals the method may be carried out upon.

The following Examples illustrate the invention.

Examples

Example 1

Tissue collection

Endoscopic pinch biopsies (Radial Jaw™ 4 2.8mm Single-Use Biopsy Forceps, Boston Scientific Corporation, USA) were obtained from macroscopically normal mucosa of the terminal ileum, ascending colon, descending colon and rectum. Where possible samples were taken from all locations. One biopsy was stored in RNAlater stabilization reagent (Qiagen) at -80°C, and the other placed in 4% formaldehyde (CellPath, Newtown, UK) for histological evaluation.

Blood samples were taken from the study participants at the time of endoscopy. A blood serum sample was taken for measurement of C-Reactive protein (CRP) and albumin (SST II Advance Ref: 367954, 5 ml tube, BD Vacutainer®, New Jersey, USA). A further EDTA blood sample tube was taken for genomic DNA and measurement of the platelet count, white cell count, neutrophil count, haemoglobin and Erythrocyte Sedimentation Rate (ESR) (K2E 7.2mg Ref: 367839, 4 ml tube, BD Vacutainer®, New Jersey, USA) for the assessment of systemic inflammatory status. RNA extraction

Biopsies (-25 mg) were lysed in a 300 μΐ RNeasy Fibrous Tissue kit (Qiagen, GmbH) RLT buffer and 0.14 M β-mercaptoethanol (β-ΜΕ) (Sigma- Aldrich, St. Louis, USA), and then homogenised by centrifugation at 10,000 g through a Qiashredder column (Qiagen, Hilden, Germany). Protein was removed by incubation for 10 minutes at 55°C with 10 μΐ Proteinase K (20 mg/ml) (>600 mAU/ml) (Qiagen). Total RNA was extracted following the manufacturers protocol using the RNeasy Mini spin column with DNA removal using RNase free DNase digestion (Qiagen). RNA was eluted in 30 μΐ of RNAase free water. Samples were included if they had an optical density ratio reading of 1.8-2.0 OD ₂ 6o/OD ₂ 8o and >1.8 OD ₂ 6o/OD ₂₃₀ using a NanoDrop ND-1000 spectrophotometer (Fisher Scientific,

Loughborough, UK), as well as a RNA concentration greater than 50 ng/μΐ.

Microarray analysis

For each biopsy sample, 500 ng of total RNA was amplified and purified using the Illumina TotalPrep-96 RNA Amplification kit (Ambion, UK), according to the

manufacturer' s instructions. Biotin-Labelled cRNA was then normalized to a concentration of 150 ng/μΐ and 750 ng was hybridised to Illumina Human-12 v4 beadarrays (Illumina, CA, USA) 16 hours at 58°C. Following hybridisation, beadarrays were washed and stained with streptavidin-Cy3 (GE Healthcare, UK). Beadarrays were scanned using the Beadarray reader and image data was then processed using Genome Studio software (Illumina, CA, USA).

Results

Intestinal barrier dysfunction and an abnormal immune response to colonic microbial flora are thought to play an important role in the pathogenesis of UC. mRNA profiles of macroscopically non-inflamed mucosa from the colon in patients with UC, Crohn' s disease (CD) and control subjects without gastrointestinal disease (HC) were investigated, to identify genes that might be implicated in the pathogenesis of the disease.

Mucosal biopsies were taken from 24 quiescent UC patients, 14 CD patients and 27 HCs undergoing routine colonoscopy. Patients were on no treatment or on 5-aminisalicylates ± azathioprine. Paired biopsies at the same site were taken from macroscopically non- inflamed mucosa in the ascending and descending colon, and the rectum for histology and RNA extraction. cRNA was hybridised to Illumina HumanHT vl2.0 Expression Beadchips. Array expression data were log transformed and normalised. Only probes with a detection p- value <0.01 were analysed. Differential gene expression analysis between groups (using p<0.05 FDR correction) and outlier analysis (p<0.005, fold change (FC)≥1.5) were performed using customised software at each location. Figurel shows that FAM5C is abnormally under-expressed throughout the colon in patients with UC compared to healthy controls. This decrease in the level of FAM5C is observed in samples from both inflamed and non-inflamed biopsies of UC patients. Results were verified by qPCR and candidate molecules examined in an independent UC cohort (Figure 2). Figure 3 A also shows that FAM5C expression is reduced in non-inflamed mucosal samples from UC patients compared to samples from healthy controls, i.e. that FAM5C expression is low in UC patients even in tissue where the disease has not yet macroscopically developed.

In group comparisons, of the 26261 expressed probes, Family with Sequence

Similarity 5, member C (FAM5C) was the only gene to be significantly under-expressed in UC both in the rectum (FC=-1.58, p=0.0008) and descending colon (FC=-1.64, p=0.001 1). Outlier analysis showed that FAM5C was also grossly under-expressed in the ascending colon, in 37.5% of UC patients, indicating that its expression is abnormal throughout the colon. Expression of FAM5C was not correlated with known markers of inflammation, IL-8, S 100A8, DEFA5, DEFA6 (Figure 3B shows lack of correlation with DEFA6 expression. Figure 3C shows lack of correlation with S 100A8 expression). FAM5C was not under- expressed in the mucosa of CD patients at any point along the colon (Figure 4). Thus, low expression of FAM5C appears specific for UC. Comparison analysis of data from the second cohort of non-inflamed rectal biopsies (FC=-1.68, p=0.0073) and qPCR (p<0.001) verified the under-expression of FAM5C in UC.

Genome-wide DNA methylation in UC patients has recently been conducted to establish a "methylome" for UC (Hasler et al. (2012) Genome Res. 22(1 1): 2130 - 2137). This methylome was mined, and it has been found that FAM5C DNA is hypermethylated in UC patients (Figure 5B). This may play a role in the decreased expression of FAM5C observed in UC patients. Consistent with this, FAM5C mRNA levels are decreased in UC patients compared with healthy controls (Figure 5A).

The microarray analysis which identified FAM5C as a biomarker for UC also identified other genes which are differentially expressed in UC patients compared with healthy controls. As shown in Figures 6, A and C, DEFA6 and S 100A8 levels are increased in UC patients compared with healthy controls. Figure 6B shows that HOXD1 1 levels are decreased in UC patients compared with healthy controls. Figures 7 A and B show that CLDN8 is also under-expressed in UC patients compared with healthy controls and patients with CD. However, the under-expression of CLDN8 is correlated with inflammation. Figure 7C shows the correlation between CLDN8 expression and inflammation biomarker DEFA6,

mRNA microarray analysis was also conducted using samples obtained from patients with colon tumours (Figure 8). FAM5C, CLDN8 and HOXD11 levels were found to be significantly decreased in samples taken from colon adenomas compared to samples taken from the colons of healthy controls. In contrast, DEFA6 and S100A8 expression levels were found to be significantly increased in samples taken from colon adenomas compared to samples taken from the colons of healthy controls.

The Inventors have made the first observations linking the under-expression of FAM5C, which appears to be manifest in non-inflamed bowel, to the pathogenesis of UC and colon tumours.

Example 2

This Example demonstrates the following: (a) attenuated expression of FAM5C in UC was independent of inflammation, unrelated to phenotype or treatment, and remained low at rebiopsy approximately 23 months later, and (b) FAM5C is localised to the brush border of the colonic epithelium and expression is influenced by DNA methylation within its promoter.

FAM5C protein expression

UC patients with reduced FAM5C mRNA expression also demonstrated

approximately 70% lower levels of protein within the descending colonic biopsies compared to HC (Figure 9 A and B). FAM5C expression and relationship to on-going inflammation

FAM5C expression in UC was unrelated to the presence or absence of microscopic inflammation (Figure 1 and Figures 10A-D). Rectal biopsies from the three lowest expressers of FAM5C were shown to be devoid of any signs of inflammation (Figure 10E, 1-3). FAM5C expression was not correlated with previously identified markers of colonic inflammation (upregulated genes IL-8, S100A8, DEFA5 and downregulated CLDN8) (Figure 11 and Figure 3B and C). FAM5C association with disease phenotype and treatment

To assess whether low FAM5C expression was associated with a particular disease phenotype, biopsies obtained from the ascending colon of UC patients with either pan- or left-sided colitis were compared (see above; Figure 3 A). Although overall the expression levels compared to HC were lower in individuals with a history of pan-colitis (p<0.0008) as opposed to patients with left-sided disease (p=0.183) (see above; Figure 3 A), FAM5C levels were not consistent with phenotype as normal expression was observed in 30% of patients with pan-colitis and low in two out of seven individuals with left-sided colitis. To address the possible effects of treatment on FAM5C expression, patients were separated into subgroups according to their medication at the time biopsies were taken. The results illustrate that treatment has no noticeable impact on FAM5C expression (Figure 12).

Longitudinal studies into FAM5C expression

To ascertain whether low expression levels of FAM5C in UC were stable over time, patients with attenuated expression, were re-biopsied after a period of 21-23 months. Low levels were maintained in both the rectum (p=0.0001) and the descending colon (p=0.0072) in all patients re-biopsied (Figures 13 A and 13B). These patients had all remained in clinical remission over this time period.

Cellular localisation ofFAM5C in the lower gastrointestinal tract

Immunohistochemistry, western blotting, subcellular fractionation, immunogold labelling and confocal microscopy were utilised to establish the subcellular location of FAM5C.

Immunohistochemistry was performed on resected ileal and colonic tissue from subjects without inflammatory bowel disease and with normal histology. FAM5C was observed to be located in the brush border region of ileal and colonic epithelial cells (Figure 14A). Electron microscopy immunogold staining localised FAM5C to the region of the brush border microvilli just under the luminal plasma membrane of human colonic epithelial cells (Figure 14B). Minimal staining was seen elsewhere within cells.

The NCI-H716 cell line, a human colorectal adenocarcinoma were used as they were found to have the highest level of expression of FAM5C in the Cancer Cell Line

Encyclopaedia (CCLE) (GEO dataset GSE36133) (Barretina J, Caponigro G, Stransky N, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012;483 :603-607.). After fractionation on continuous sucrose gradients, the protein was identified in the fractions by western blotting. It was mainly found in the region of the gradient normally occupied by the light membrane fraction (Dawson J, Bryant MG, Bloom SR, et al. Subcellular fractionation studies of human rectal mucosa: localization of the mucosal peptide hormones. Clin Sci (Lond) 1980;59:457-462.) (Figure 14C). The mitochondrial marker GRP75 showed no overlap with FAM5C (Figure 14C). Confocal microscopy confirmed the lack of association between FAM5C staining and mitochondria identified with an antibody to GPR75 (Figure 15). It was possible that FAM5C migrated from the cytosol into the denser fractions as part of a macromolecular complex, and evidence for this was its co-location with vinculin, a component of the actin cytoskeleton. To distinguish this from a membrane location we subjected a FAM5C-positive fraction to an additional centrifugation step in which membranes would not sediment at their buoyant density and would remain in suspension, whereas macromolecular protein complexes would sediment into the pellet. FAM5C and vinculin were found almost exclusively in the pellet, indicative of their association with large protein complexes (Figure 14D). Confocal microscopy demonstrates co-localisation of FAM5C and vinculin just under the plasma membrane of the NCI-H716 cell line, providing additional evidence of a direct association with the

cytoskeleton (Figure 14E).

Methylation of the FAM5C promoter in the distal colon - Materials and Methods for DNA methylation and pyrosequencing

Endoscopic pinch biopsies from five UC and four HC were stored in RNAlater stabilization reagent (Qiagen) at -80°C. DNA was extracted from intestinal biopsies and blood leukocytes using the DNeasy Blood and Tissue kit (Qiagen) with 500 ng DNA bisulfite-converted using the EZ-96 DNA Methylation™ Kit (Deep Well Format) (Zymo Research, CA, USA). Sequencing and polymerase chain reaction (PCR) primers were designed using the PyroMark™ Assay Design Software 2.0 (Qiagen/Biotage, Uppsala, Sweden) and are shown in Table 4 below:

FAM5C PYROSEQUENCING PRIMERS

FAM5C

CpG Product size

Forward primer (5'-3') Reverse primer (5'-3') island (bp) region

Region 1 [Biotin] -

TGGGATTTGGGAGGGTTGTT CCCACTCCCCAAATAAACCACT 233 (SEQ ID NO: 13)

(SEQ ID NO: 14)

Region 2 [Biotin] - TGGGATTTGGGAGGGTTGTT AACCAATTCCCCCACTCC

243 (SEQ ID NO: 15) (SEQ ID NO: 16)

FAM5C PCR AMPLICON SEQUENCING PRIMERS (5'-3')

Region 1 GTTTTTTTGTTTAAGT

(SEQ ID NO: 17)

Region 2 CCCCAAATAAACCACTAC

(SEQ ID NO: 18)

Table 4 - Primer table for quantitative PCR (qPCR) verification and pyrosequencing. Forward and reverse primers (5 '-3 ') used for FAM5C qPCR verification, including the product size (bp, base pairs). PCR and sequencing primers (5'-3 ') used for pyrosequencing FAM5C are documented with the biotinylated primers marked clearly with [Biotin]. Primers for pyrosequencing were designed within a CpG island located in the promoter region of FAM5C (Region 1 and Region 2) in patients with UC (n=5) and HCs (n=4).

Bisulfite-modified DNA was amplified by PCR using the primers of Table 4 with GoTaq® Hot Start Polymerase and reagents (Promega, WI, USA) and dNTP Mix (Eurogentec,

Hampshire, UK) in a 50 μΐ reaction volume. A 30 μΐ aliquot of the PCR product was

transferred to a PSQ™ HS 96 plate (Qiagen/Biotage) and pyrosequencing was performed following the manufacturer's protocol utilising a PyroMark Q96 Vacuum Prep Workstation and a PyroMark™ (Qiagen/Biotage) Q96 MD System. DNA methylation levels were quantified using Pyro Q-CpG Software (Qiagen/Biotage). Percentage methylation was analysed using GraphPad Prism 4 for Windows (GraphPad Software Inc., La Jolla, USA).

Methylation of the FAM5C promoter in the distal colon - Results Prior GWAS in UC revealed no significant association with the FAM5C region

(Anderson CA, Boucher G, Lees CW, et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat Genet

201 1 ;43 :246-252; Johnson AD, Handsaker RE, Pulit SL, et al. SNAP: a web-based tool for identification and annotation of proxy SNPs using HapMap. Bioinformatics 2008;24:2938- 2939.). A recent microarray study on the intestinal mucosa of monozygotic twins discordant for UC demonstrated that FAM5C expression was significantly reduced in the affected as compared with the unaffected twin (Hasler R, Feng Z, Backdahl L, et al. A functional methylome map of ulcerative colitis. Genome Res 2012;22:2130-2137.). These findings suggest that alterations in FAM5C expression are not attributable to genetic inheritance and could therefore result from epigenetic modifications. DNA methylation is a major regulator of gene expression and has been previously shown to be abnormal in UC patients (Wheeler JM, Kim HC, Efstathiou JA, et al. Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer. Gut

2001 ;48:367-371 ; Hasler R, Feng Z, Backdahl L, et al. A functional methylome map of ulcerative colitis. Genome Res 2012;22:2130-2137; Cooke J, Zhang H, Greger L, et al.

Mucosal genome-wide methylation changes in inflammatory bowel disease. Inflamm Bowel Dis 2012; 18:2128-2137.). FAM5C contains a CpG rich sequence ('CpG island') (chrl :

190446552-190447574) in the promoter region. The methylation status of the FAM5C promoter region was determined in the rectal mucosa of FAM5C low UC patients (n=5) and HC (n=4) as well as in circulating leukocytes from the same individuals (Figure 16). FAM5C expression was also quantitated by qPCR in the same rectal biopsy specimens. UC patients expressing FAM5C at low levels in the rectum (Figure 16A) had significantly increased methylation in the promoter region (p<0.01) (Figure 16B). There was an inverse relationship between FAM5C expression and methylation (r ² =0.6991, p=0.005) (Figure 17). There was no significant increase in the extent of FAM5C promoter methylation in the circulating leukocyte population in UC patients indicating a possible tissue-specific modification.

Sequence Information

Gene sequences (Excluding introns) SEQ ID NO: 1

FAM5C chrl: 190066797-190446759

>NM_199051.1

ttcgtggctctctgtgtaactggtttttacttggttacatgccaccaggaactgctg attattttaaagtcctactaccctgcagctcactacttt accttgatttggaagatcatggaatatctatttgaatcctggatgtatttttctcacagt cttcttgcttcctgaaatttcctctggtgttgaggg aaagctgagagaatgaaggctctaaatccccagtggaagcatgatatggcgaagcagagc tggtgctgaattgttctctctgatggctc tatgggagtggatagcactgagtcttcattgctgggttttagcggttgctgctgtttcgg atcagcatgccacaagccccttcgactggct cctctctgataagggacccttccatcgctcacaggaatacacagattttgtggacagaag ccggcagggatttagcacaagatacaag atatacagggagtttggccgctggaaagtaaataaccttgcagttgagagaagaaatttc cttggctctcctctgcctcttgcccctgaatt cttccgcaacataagacttttgggacgtcgacctacccttcagcaaatcacagaaaacct tatcaagaaatatgggacacatttcttgcta tctgctactctgggaggagaggagtcactcacaatttttgtggacaagcggaagttgagc aaacgagctgaaggaagtgattccacca ccaatagctcttcggtcactctggagacgctacatcagctagccgcttcttatttcattg acagggacagcacccttcggagacttcacca cattcaaattgcatccactgccataaaggtaacagaaacacggactggtcctcttggctg cagtaactatgacaacctagattctgtcagt tctgttctggttcagagtcctgagaataagattcagttgcaagggcttcaagtacttctc ccagactatcttcaggaacgttttgtacaagca gctttgagctacattgcttgcaattcagagggagagtttatctgcaaggaaaatgactgc tggtgtcactgtggtcccaaatttccagaat gcaactgcccctccatggacattcaagccatggaagagaatcttcttcgaataactgaaa cctggaaagcttacaacagtgactttgag gaatcagatgaattcaagttatttatgaaaaggctacctatgaattatttcctcaacaca tctactataatgcatttgtggacaatggattcta attttcagcgccgttatgaacaactggagaacagcatgaaacaacttttcctaaaggcgc agaaaattgtacacaagctttttagccttag caagaggtgtcataaacaacccctcatcagcctgccaagacaaagaacctcaacctactg gcttactcgcatccagtcttttctctactg caatgagaacggcctcctaggcagcttttcagaagagacgcactcgtgcacgtgtccgaa tgaccaggtggtctgcaccgcgttcctg ccctgcacagtgggagacgcctctgcctgcctgacatgcgcaccagacaaccgcacccgc tgcggcacctgcaacaccggctacat gctcagccaggggctctgcaagcctgaagtcgccgagtccaccgatcactatattggctt tgaaactgacctgcaagatctcgagatg aaatatctgctgcagaaaacggacagacgaatagaagtccatgccatttttatcagcaat gacatgcgcctcaatagctggtttgatccct cctggcgtaagcggatgctcctcaccttgaagagcaataagtacaagtcaagtctggtcc atatgattttgggtctctctttacagatttgc ttaactaaaaacagcaccttggagccagtgttggctgtttatgtcaatcccttcggaggc agccactctgagagctggtttatgcctgtga atgaaaacagctttccagactgggagcggactaagttggacctacccctgcagtgttata actggacattaactctggggaacaaatgg aagacattttttgagacagtacacatctacctgagaagtcgcatcaagtccaatggtccc aatggtaatgagagcatttactatgaacctc tggagtttattgacccttcccggaacctgggctatatgaaaatcaataacattcaagtgt ttggctacagcatgcactttgaccctgaagca attcgggacctgattttgcagctggactacccctatactcagggatcccaggattcagca cttttgcaactactagagatcagagaccgt gtaaataaactctccccacctggtcagcgtcgtctagatcttttctcttgcttgcttcgt catagactcaagctgtctactagtgaggtggtg aggatccaatctgctctgcaggcgtttaatgccaaattgccaaacacaatggattatgac acgaccaaattatgtagttaaccataaatgt caagcacaacccaaaatcttgaaggagtttttacagtgcttttgtggaacagtttatgtt tggaagagtaaatttaaattgtcttttcaatatct gtcttatatcagtcaataacattggatggcaatttacacacatgaacttgctgacaatga atatattatacagcagttttggtttatgaatgac ataaatactgacaccagtctagaagacattctactttttacaataaatttcatttgtaat tttatatgttccgtggcaatgcttttgtgcattacat cctctagagggaacataaaaagataccaataaaattttgtagctgaacagttatt

Protein Sequence SEQ ID NO: 2

> P_950252 length=766

MIWRSRAGAELFSLMALWEWIALSLHCWVLAVAAVSDQHATSPFDWLLSDKGPFH RSQEYTDFVDRSRQGFSTRYKIYREFGRWKVN LAVERR FLGSPLPLAPEFFRNIRL LGRRPTLQQITE LIKKYGTHFLLSATLGGEESLTIFVDKRKLSKRAEGSDSTTNSSSV TLETLHQLAASYFIDRDSTLRRLHHIQIASTAIKVTETRTGPLGCSNYD LDSVSSVLV QSPE KIQLQGLQVLLPDYLQERFVQAALSYIACNSEGEFICKE DCWCHCGPKFPEC NCP SMDIQ AMEE LLRITETWK A YNSDFEE SDEFKLFMKRLPMN YFLNT S TIMUL WT MD S F QRRYEQLENSMKQLFLK AQKI VHKLF SL SKRCHKQPLI SLPRQRT S T YWLTR IQSFLYC ENGLLGSFSEETHSCTCP DQVVCTAFLPCTVGDASACLTCAPD RTRCG TCNTGYMLSQGLCKPEVAESTDHYIGFETDLQDLEMKYLLQKTDRRIEVHAIFIS D MRLNSWFDPSWRKRMLLTLKS KYKSSLVHMILGLSLQICLTKNSTLEPVLAVYVN PFGGSHSESWFMPVNENSFPDWERTKLDLPLQCYNWTLTLG KWKTFFETVHIYLR SRIKSNGPNG ESIYYEPLEFIDPSR LGYMKINNIQVFGYSMHFDPEAIRDLILQLDY PYTQGSQDSALLQLLEIRDRVNKLSPPGQRRLDLFSCLLRHRLKLSTSEVVRIQSALQ AFNAKLPNTMDYDTTKLCS

CLDN8 chr21:31,586,324-31,588,469 SEQ ID NO: 3

> M_199328.2

gatttgtaagtttacctgttgcagccaatagcagggccatctcagccagccagcact ggatactatctggccagaagtagcaaagcagc tcttatttgaaaaaccactgggttccgagttcattactacaggaaaaactgttctcttct gtggcacagagaaccctgcttcaaagcagaa gtagcagttccggagtccagctggctaaaactcatcccagaggataatggcaacccatgc cttagaaatcgctgggctgtttcttggtg gtgttggaatggtgggcacagtggctgtcactgtcatgcctcagtggagagtgtcggcct tcattgaaaacaacatcgtggtttttgaaa acttctgggaaggactgtggatgaattgcgtgaggcaggctaacatcaggatgcagtgca aaatctatgattccctgctggctctttctc cggacctacaggcagccagaggactgatgtgtgctgcttccgtgatgtccttcttggctt tcatgatggccatccttggcatgaaatgca ccaggtgcacgggggacaatgagaaggtgaaggctcacattctgctgacggctggaatca tcttcatcatcacgggcatggtggtgct catccctgtgagctgggttgccaatgccatcatcagagatttctataactcaatagtgaa tgttgcccaaaaacgtgagcttggagaagct ctctacttaggatggaccacggcactggtgctgattgttggaggagctctgttctgctgc gttttttgttgcaacgaaaagagcagtagct acagatactcgataccttcccatcgcacaacccaaaaaagttatcacaccggaaagaagt caccgagcgtctactccagaagtcagta tgtgtagttgtgtatgtttttttaactttactataaagccatgcaaatgacaaaaatcta tattactttctcaaaatggaccccaaagaaactttg atttactgttcttaactgcctaatcttaattacaggaactgtgcatcagctatttatgat tctataagctatttcagcagaatgagatattaaacc caatgctttgattgttctagaaagtatagtaatttgttttctaaggtggttcaagcatct actctttttatcatttacttcaaaatgacattgctaaa gactgcattattttactactgtaatttctccacgacatagcattatgtacatagatgagt gtaacatttatatctcacatagagacatgcttatat ggttttatttaaaatgaaatgccagtccattacactgaataaatagaactcaactattgc ttttcagggaaatcatggatagggttgaagaa ggttactattaattgtttaaaaacagcttagggattaatgtcctccatttataatgaaga ttaaaatgaaggctttaatcagcattgtaaagga aattgaatggctttctgatatgctgttttttagcctaggagttagaaatcctaacttctt tatcctcttctcccagaggctttttttttcttgtgtatt aaattaacatttttaaaaagcagatattttgtcaaggggctttgcattcaaactgctttt ccagggctatactcagaagaaagataaaagtgt gatctaagaaaaagtgatggttttaggaaagtgaaaatatttttgtttttgtatttgaag aagaatgatgcattttgacaagaaatcatatatgt atggatatattttaataagtatttgagtacagactttgaggtttcatcaatataaataaa agagcagaaaaatatgtcttggttttcatttgctta ccaaaaaaacaacaacaaaaaaagttgtcctttgagaacttcacctgctcctatgtgggt acctgagtcaaaattgtcatttttgttctgtga aaaataaatttccttcttgtaccatttctgtttagttttactaaaatctgtaaatactgt atttttctgtttattccaaatttgatgaaactgacaatcc aatttgaaagtttgtgtcgacgtctgtctagcttaaatgaatgtgttctatttgctttat acatttatattaataaattgtacatttttctaattatttg aa Protein Sequence SEQ ID NO: 4

> P_955360 length=225

MATHALEIAGLFLGGVGMVGTVAVTVMPQWRVSAFIENNIVVFE FWEGLWMNCV RQANIRMQCKIYDSLLALSPDLQAARGLMCAASVMSFLAFMMAILGMKCTRCTGD EKVKAHILLTAGIIFIITGMVVLIPVSWVANAIIRDFYNSIVNVAQKRELGEALYLGW TTALVLIVGGALFCCVFCC EKSSSYRYSIPSHRTTQKSYHTGKKSPSVYSRSQYV

HOXD11 chr2: 176,972,084-176,974,316 SEQ ID NO: 5

> M_021 192.2

atgaacgactttgacgagtgcggccagagcgcagccagcatgtacctgccgggctgc gcctactatgtggccccgtctgacttcgcta gcaagccttcgttcctttcccaaccgtcgtcctgccagatgactttcccctactcttcca acctggctccgcacgtccagcccgtgcgcg aagtggccttccgcgactacggcctggagcgcgccaagtggccgtaccgcggcggcggcg gcggcggcagcgcggggggcgg cagcagcgggggcggccccggcgggggcggcggcggcgcggggggctacgctccctacta cgcggcggcggcggcggcgg ctgcggcggccgcggcggccgaggaggcggccatgcaacgcgagcttctcccgcccgcgg gccgccggccggacgtgctcttc aaggcgcctgagccggtgtgcgctgcgccggggccgccgcacggccccgcgggcgccgcc tccaacttctacagcgcggtggg ccgcaatggcatcttgccacagggcttcgaccagttctacgaggcagcgcccgggccccc gttcgccgggccgcagcccccgccg ccacccgcgccgccacagcccgagggcgcagccgacaagggcgaccccaggaccggggct ggtggcggcgggggcagtccct gcaccaaggcgacccctggctcggagcccaagggggcagcagaaggcagcggtggcgacg gcgagggccccccgggagagg cgggggccgagaagagcagcagcgcagttgccccccagcggtcccggaaaaagcgctgtc cctataccaagtaccagatccgcg aactggaacgcgagtttttctttaacgtgtacataaacaaagagaaaagacttcaactct ctcggatgctcaacctcactgaccggcaag tcaaaatctggttccagaatcgcaggatgaaagaaaagaaactgaacagagaccgtctgc agtatttcactggaaaccccttattttgag agctccaggaagcgccctcaccccagccccactcacccaccctccttcccaccagcctgc tctccgcaggcccactgtccttgggttt aatgacgtctcttctctgtggaacttcacgattccttcccacggtcaactcgggacctcc cagcgaccactgcagcctgcggacgaggc cgggacttggccgagcggatcctaataaggggaaaatggtaaatgcaaacgtcccgttac aattttaccgccagtgtgctgtcgttccc cctccccctctccgagtcctcgtggggacacggcggggtctgtaggaagttgggccgggt tgggggttgctagaaggcgctggtgttt tgctctgagttttaagagatcccttccttcctcttcggtgaatgcaggttatttaaactt tgggaaatgtacttttagtctgtcatatcaa

Protein Sequence SEQ ID NO: 6

> P_067015 length=338

MNDFDECGQSAASMYLPGCAYYVAPSDFASKPSFLSQPSSCQMTFPYSS LAPHVQP VREVAFRD YGLERAKWP YRGGGGGGS AGGGS SGGGPGGGGGGAGGYAPYYAAAA AAAAAAAAAEEAAMQRELLPPAGRRPDVLFKAPEPVCAAPGPPHGPAGAAS FYSA VGRNGILPQGFDQFYEAAPGPPFAGPQPPPPPAPPQPEGAADKGDPRTGAGGGGGSP CTKATPGSEPKGAAEGSGGDGEGPPGEAGAEKSSSAVAPQRSRKKRCPYTKYQIREL EREFFFNVYINKEKRLQLSRMLM.TDRQVKIWFQ RRMKEKKL RDRLQYFTG PL F

DEFA6 chr8:6,782,216-6,783,598 SEQ ID NO: 7

> M_001926.3

acacatctgctcctgctctctctcctccagcgaccctagccatgagaaccctcacca tcctcactgctgttctcctcgtggccctccaggc caaggctgagccactccaagctgaggatgatccactgcaggcaaaagcttatgaggctga tgcccaggagcagcgtggggcaaatg accaggactttgccgtctcctttgcagaggatgcaagctcaagtcttagagctttgggct caacaagggctttcacttgccattgcagaa ggtcctgttattcaacagaatattcctatgggacctgcactgtcatgggtattaaccaca gattctgctgcctctgagggatgagaacaga gagaaatatattcataatttactttatgacctagaaggaaactgtcgtgtgtcctataca ttgccatcaactttgtttcctcatctcaaata aagtcctttcagcaagttaaaaaaaaaaa

Protein sequence SEQ ID NO: 8

> P_067015 length=338

MNDFDECGQSAASMYLPGCAYYVAPSDFASKPSFLSQPSSCQMTFPYSS LAPHVQP VREVAFRD YGLERAKWPYRGGGGGGSAGGGSSGGGPGGGGGGAGGYAPYYAAAA AAAAAAAAAEEAAMQRELLPPAGRRPDVLFKAPEPVCAAPGPPHGPAGAAS FYSA VGRNGILPQGFDQFYEAAPGPPFAGPQPPPPPAPPQPEGAADKGDPRTGAGGGGGSP CTKATPGSEPKGAAEGSGGDGEGPPGEAGAEKS S S AVAPQRSRKKRCP YTKYQIREL EREFFFNVYINKEKRLQLSRMLM.TDRQVKIWFQ RRMKEKKL RDRLQYFTG PL F

S100A8 chrl: 153,362,508-153,363,664 SEQ ID NO:9

> M_002964.4

gagaaaccagagactgtagcaactctggcagggagaagctgtctctgatggcctgaa gctgtgggcagctggccaagcctaaccgc tataaaaaggagctgcctctcagccctgcatgtctcttgtcagctgtctttcagaagacc tggtggggcaagtccgtgggcatcatgttg accgagctggagaaagccttgaactctatcatcgacgtctaccacaagtactccctgata aaggggaatttccatgccgtctacaggga tgacctgaagaaattgctagagaccgagtgtcctcagtatatcaggaaaaagggtgcaga cgtctggttcaaagagttggatatcaaca ctgatggtgcagttaacttccaggagttcctcattctggtgataaagatgggcgtggcag cccacaaaaaaagccatgaagaaagcca caaagagtagctgagttactgggcccagaggctgggcccctggacatgtacctgcagaat aataaagtcatcaatacctcaaaaaaaa aa Protein sequence SEQ ID NO: 10

>NP_002955 length=93

MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIRKKGADVWFKE LDINTDGA V F QEFLIL VIKMGV A AHKK SHEE SHKE