WO2013045902A1

WO2013045902A1 - Predicting thiopurine hypermethylation

Info

Publication number: WO2013045902A1
Application number: PCT/GB2012/052356
Authority: WO
Inventors: Anthony Marin MARINAKI; Paul Andrew BLAKER; Jeremy David Sanderson
Original assignee: Guy's And St. Thomas' Nhs Foundation Trust
Priority date: 2011-09-28
Filing date: 2012-09-24
Publication date: 2013-04-04
Also published as: GB201116664D0

Abstract

Single nucleotide polymorphisms in the genes encodingcatecholamine-O- methyltransferase (COMT), molybdenum cofactor sulfurase (MOCOS) and ATP- binding cassette protein B5 (ABCB5) are associated with the likelihood of a patient suffering from thiopurine hypermethylation during 6-mercaptopurine therapy, for example, for treatment of inflammatory bowel disease. A method for predicting thiopurine hypermethylationin response to a 6-mercaptopurinedrug or prodrug in an individual in need thereof comprises: (a) genotyping the individual at a polymorphic site in at least a catecholamine-O-methyltransferase (COMT) gene, a molybdenum cofactor sulfurase (MOCOS) gene, and/or an ATP-binding cassette protein B5 (ABCB5) gene; and (b) determining the presence or absence of a variant allele at said polymorphic site;wherein the presence or absence of the variant allele at said polymorphic site is indicative of a likelihood of thiopurine hypermethylation. A method of developing a treatment regimefor an individual in need of a 6-mercaptopurine drug or prodrugincludes predicting thiopurine hypermethylation and planning a course of treatment or a dosage regime based on the results thereof. A method of treating an individual in need of a6- mercaptopurine drug or prodrug, includes: (a) predicting thiopurine hypermethylation in response to the 6-mercaptopurine drug or prodrugin the individual; and (b) treating the individual based on the results of the prediction.

Description

PREDICTING THIOPURINE HYPERMETHYLATION

The present invention relates to predicting thiopurine hypermethylation, and in particular to pharmacogenetic markers predicting the likelihood of thiopurine hypermethylation in a patient in need of a 6-mercaptopurine drug.

The thiopurines, 6-mercaptopurine (6-MP) and its pro-drug Azathioprine (AZA), are the first line immunomodulators used in the management of myriad inflammatory conditions, including Crohn's disease (CD) and ulcerative colitis (UC). In the body, 6-MP may be converted to either thioguanine nucleotides (TGNs) or 6- methylmercaptopurine (6-MEMP) (see Figure 1 ). TGNs are the principle

metabolites leading to immunosuppression, whereas 6-MEMP has been linked to hepatotoxicity and other side effects. The conversion of 6-MP to 6-MEMP is catalysed by the polymorphic methyltransferase enzyme thiopurine-S- methyltransferase (TPMT). Pre-treatment measurement of TPMT activity is recommended to allow safe dose rationalisation of thiopurines, since low levels of this enzyme are associated with myelotoxicity at standard drug doses.

As shown in Figure 1 , several major enzyme pathways are involved in the metabolism of 6-MP. Xanthine oxidase (XO) converts 6-MP to 6-thiouric acid (TUA). Hypoxanthine phosphoribosyl transferase (HPRT) converts 6-MP to 6- thioinosine-5'-monophosphate (TIMP), which is a precursor to 6-TGNs. Thiopurine methyltransferase (TPMT) catalyses the S-methylation of 6-MP to 6-MeMP. Thus, 6-MP is enzymatically converted to various metabolites, including 6-thioguanine (6-TG) and 6-TGNs, which are the presumptive active metabolites mediating the effects of AZA 6-MP drug therapy.

AZA and 6-MP are currently prescribed on empirical dosing based on weight and pre-treatment measurement of TPMT levels (Mowat et al. (2011 ) Gut 60, 571 -607). In approximately 15% of patients with normal (wildtype) TPMT levels established on treatment with thiopurines, 6-MEMP is produced in preference to TGNs. This phenotype, known as thiopurine hypermethylation, is associated with treatment resistance, drug toxicity, hepatotoxicity and other side effects and is not predicted by measurement of TPMT activity alone. However, thiopurine hypermethylation may be circumvented with the addition of another drug called allopurinol following dose reduction of the thiopurine (Ansari et al. (2008) Aliment. Pharmacol. Ther. 28, 5 734-41 ).

The interplay of the pathways described above is genetically determined and creates a highly individualised response to AZA 6-MP drug therapy. The population frequency distribution of TPMT enzyme is trimodal, with the majority of individuals 10 (89%) having high activity, 11 % having intermediate activity, and about 1 in 300 (0.33%) having undetectable activity (Weinshilboum & Sladek (1980) Amer. J. Hum. Genet. 32, 651 -662).

The applicant's earlier PCT application, published as WO 2009/047001 and incorporated herein by reference, discloses methods for predicting tolerance

15 associated with 6-MP drug treatment of an immune-mediated gastrointestinal

disorder such as inflammatory bowel disease. In particular the methods include genotyping a patient at a polymorphic site in at least a xanthine dehydrogenase (XDH) gene, a molybdenum cofactor sulfurase (MOCOS) gene and/or an aldehyde oxidase (AO) gene. The single nucleotide polymorphism (SNP) XDH

20 837C>T and the SNP MOCOS 2107A>C genotypes were found to protect against side-effects to azathioprine. The SNP AOX 3404A>G was found to be associated with a lack of response to azathioprine.

To date, no genetic markers predicting thiopurine hypermethylation have been identified.

25 According to a first aspect of the present invention, there is provided a method for predicting thiopurine hypermethylation in response to a 6-mercaptopurine drug or prodrug in an individual in need thereof, the method comprising: (a) genotyping the individual at a polymorphic site in at least a catecholamine-O-methyltransferase (COMT) gene, a molybdenum cofactor sulfurase (MOCOS) gene, and/or an ATP-

30 binding cassette protein B5 (ABCB5) gene; and (b) determining the presence or absence of a variant allele at said polymorphic site, wherein the presence or absence of the variant allele at said polymorphic site is indicative of a likelihood of thiopurine hypermethylation. As will be appreciated by the skilled person, the genotyping is generally carried out on a sample obtained from the individual.

The individual may have an inflammatory condition, such as an immune-mediated gastrointestinal disorder.

The immune-mediated gastrointestinal disorder may be an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis.

In an embodiment, the gene is a COMT gene.

The method may comprise testing for a low activity variant of COMT, wherein the presence of a low activity COMT gene is indicative of protection from thiopurine hypermethylation. The polymorphic site may comprise the COMT rs9332377C>T variant allele and/or the COMT rs4646316C>T variant allele.

The genotype may be rs9332377 T/T or C/T and may be indicative of protection from thiopurine hypermethylation. Additionally/alternatively the genotype may be rs4646316 C/C and may be indicative of protection from thiopurine

hypermethylation.

In an embodiment, the gene is a MOCOS gene. The polymorphic site may be the MOCOS 2600T>C (rs1057251 ) variant allele. The genotype may be rs1057251 T/C or C/C, and may be indicative of protection from thiopurine hypermethylation.

In an embodiment, the gene is an ABCB5 gene.

The polymorphic site may be ABCB5 rs2301641 (K115E) A>G variant allele.

The genotype may be rs2301641 G/G and may be indicative of a high risk of thiopurine hypermethylation.

The genotype may be determined by a method including DNA sequencing, PCR, real time PCR, allele specific PCR, restriction endonuclease genotyping, or microarray-based genotyping. In certain embodiments at least two of the genes or polymorphic sites are genotyped. At least three of the genes or polymorphic sites may be genotyped.

In preferred embodiments, the individual is also tested for activity of thiopurine-S- methyltransferase prior to commencing treatment.

In preferred embodiments, levels of 6-methylmercaptopurine are also measured.

The drug may be 6-mercaptopurine, azathioprine, 6-thioguanine, and/or 6-methylmercaptopurine riboside.

The drug may further comprise allopurinol.

According to a second aspect of the present invention, there is provided a method of developing a treatment regime including predicting thiopurine hypermethylation using a method as set out above and planning a course of treatment or a dosage regime based on the results thereof. According to a third aspect of the present invention, there is provided a method of treating an individual in need of a 6-mercaptopurine drug or prodrug, including: (a) predicting thiopurine hypermethylation in response to the 6-mercaptopurine drug or prodrug in the individual using a method as described above; and (b) treating the individual based on the results of the prediction.

Preferred embodiments are described below, by way of example only, with reference to the accompanying drawing, in which: Figure 1 shows the metabolism of azathioprine and 6-mercaptopurine. 6- mercaptopurine metabolic pathways are indicated by solid arrows; dashed arrows indicate putative products of dephosphorylation to nucleotides and further catabolism to nucleobases (HPRT=hypoxanthine phosphoribodyltransferase; TMPT=thiopurine methyltransferase; XO=xanthine oxidase; IMPD=inosine monophosphate dehydrogenase; GMPS=guanosine monophosphate synthetase).

As used herein, the term "6-mercaptopurine drug" or "6-MP drug" includes any drug that can be metabolised to an active 6-MP metabolite that has therapeutic efficacy such as 6-TG. Exemplary 6-MP drugs as defined herein include 6-MP and AZA. As illustrated in Figure 1 , both 6-MP and AZA can be metabolised to 6- MP metabolites such as the exemplary 6-MP metabolites shown, including 6-TG, 6-MeMP, and 6-TUA (Lennard (1992) Eur. J. Clin. Pharmacol. 43, 329 339).

Other 6-MP drugs include, for example, 6-methylmercaptopurine riboside and 6- TG. 6-TG is a particularly useful 6-MP drug in patients having high TPMT activity. Patients exhibiting high TPMT activity are expected more easily to convert 6-MP drugs such as 6-MP and AZA to 6-MeMP (see Figure 1 ). As indicated above, high levels of 6-MeMP are associated with hepatotoxicity . Therefore, patients with high TPMT activity can be more susceptible to toxic effects of 6-MP drug therapy. By administering 6-TG, which is an active 6-MP metabolite associated with

therapeutic efficacy, the toxicity that can be associated with conversion of 6-MP to 6-MeMP is bypassed. As used herein, the term "6-thioguanine" or "6-TG" includes 6-thioguanine or analogues thereof, including molecules having the same base structure, for example, 6-thioguanine ribonucleoside, 6-thioguanine ribonucleotide mono-, di- and tri-phosphate, 6-thioguanine deoxyribonucleoside and 6-thioguanine deoxyribonucleotide mono, di, and triphosphate. The term "6-TG" also includes derivatives of 6-thioguanine, including chemical modifications of 6-TG, so long as the structure of the 6-TG base is preserved. As used herein, the term "6-methylmercaptopurine" or "6-MeMP" includes 6- methylmercaptopurine or analogues thereof, including analogues having the same base structure, for example, 6-methylmercaptopurine ribonucleoside, 6- methylmercaptopurine ribonucleotide mono-, di-, and tri-phosphate, 6- methylmercaptopurine deoxyribonucleoside, and 6-methylmercaptopurine deoxyribonucleotide mono-, di- and tri-phosphate. The term "6-MeMP" also includes derivatives of 6-methylmercaptopurine, including chemical modifications of 6-MeMP, so long as the structure of the 6-MeMP base is preserved.

As used herein, the term "6-mercaptopurine metabolite" includes a product derived from 6-MP in a biological system. Exemplary 6-MP metabolites are shown in Figure 1 and include 6-TG, 6-MeMP and 6-TUA and analogues thereof. For example, 6-MP metabolites include 6-TG bases such as 6-TG, 6-thioguanosine mono-, di- and tri-phosphate; 6-MeMP bases such as 6-methylmercaptopurine and 6-methylthioinosine monophosphate; 6-thioxanthosine (6-TX) bases such as 6- thioxanthosine monophosphate; 6-TUA; and 6-MP bases such as 6-MP and 6- thioinosine monophosphate. The immunosuppressive properties of 6-MP are believed to be mediated via the intracellular transformation of 6-MP to its active metabolites such as 6-TG and 6-MMP nucleotides. Furthermore, 6-MP metabolites such as 6-TG and 6-MMP were found to correlate with therapeutic efficacy and toxicity associated with 6-MP drug treatment of patients with inflamatory bowel disease. The term "polymorphism" includes the occurrence of two or more genetically determined alternative sequences or alleles in a population. A "polymorphic site" includes the locus at which divergence occurs. Preferred polymorphic sites have at least two alleles, each occurring at a particular frequency in a population. A polymorphic locus may be as small as one base pair (single nucleotide

polymorphism, or SNP). The first identified allele is arbitrarily designated as the reference allele, and other alleles are designated as alternative alleles, "variant alleles," or "variances." The alleles occurring most frequently in a selected population is sometimes referred to as the "wild-type" allele. Diploid organisms may be homozygous or heterozygous for the variant alleles. The variant allele may or may not produce an observable physical or biochemical characteristic

("phenotype") in an individual carrying the variant allele. For example, a variant allele may alter the enzymatic activity of a protein encoded by a gene of interest. An "SNP" occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). A SNP usually arises due to substitution of one nucleotide for another at the polymorphic site. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.

The present applicant has identified a method of determining, prior to

commencement of therapy with a 6-MP drug, whether a patient is likely to suffer from the toxic side effects of thiopurine hypermethylation. If thiopurine

hypermethylation is predicted, an alternative treatment regime may be proposed.

The applicant has focussed on SNPs found in the genes encoding catecholamine- O-methyltransferase (COMT), molybdenum cofactor sulfurase (MOCOS) and ATP- binding cassette protein B5 (ABCB5). It has found that by assessing an individual patient's genotype for these particular SNPs, a prediction can be made regarding the likelihood of that patient suffering from purine hypermethylation during 6-MP therapy.

5 The applicant has identified two SNPs in the COMT gene and a separate SNP in the MOCOS gene that predict protection from the phenotype of thiopurine hypermethylation. The applicant has also identified a single nucleotide

polymorphism in the ABCB5 gene that predicts the occurrence of thiopurine hypermethylation.

10

Catecholamine-O-Methyltransferase ( COMT)

COMT, an enzyme that degrades catecholamines, was the first S- adenosyltransferase (SAM)-dependent methyltransferase to be structurally characterised. The level of COMT enzyme activity is genetically polymorphic with

15 a tri-modal distribution of low, intermediate and high activities in red cells. Two

relatively common SNPs that are associated with reduced enzyme activity have been described in the COMT gene: rs9332377 A and rs4646316 G, both of which contain a low-activity synonymous COMT variant, rs4818 that is associated with an 11 -18 fold reduction in COMT activity (Nackley et al. (2006) Science 314,1930-

20 33). In children receiving cisplatin therapy these SNPs were associated with

ototoxicity (Ross et al. (2009) Nat. Genet. 41 ,1345-49). It was hypothesised that reduced COMT activity resulted in increased cisplatin toxicity through a relative increase in SAM pools available for other methylation reactions (Ross et al. (2009); Ochoa et al. (2009) Arch. Med. Res. 40,54-8).

25

It can be hypothesised that reduced COMT activity will lead to a greater pool of SAM available for TPMT methylation reactions and thereby contribute to the phenotype of thiopurine hypermethylation. Prior to the work of the present applicant, no studies existed to show a correlation between COMT activity and 30 thiopurine hypermethylation. Molybdenum Cofactor Sulfurase (MOCOS)

The degradation of 6-MP occurs through two pathways; firstly via TPMT to form 6- MeMP and secondly by a hydroxylation catalysed by xanthine oxidase (XO) and aldehyde oxidase (AO) (see Figure 1 ). A molybdenum cofactor is essential for the action of XO and AO, which if deficient leads to severe neurodegeneration and early infant death. The specific adaptation of molybdenum cofactor for XO and AO requires the action of molybdenum cofactor sulfurase (MOCOS). The MOCOS gene is subject to genetic polymorphism and thereby may explain some of the variation in thiopurine metabolism. In a retrospective study of 192 patients with inflammatory bowel disease an SNP in exon 11 of the MOCOS gene (2107A>C, Asn703His), which leads to an amino acid substitution, was associated with a reduction in AZA adverse reactions in a recessive model (Smith et al. (2009) Aliment. Pharmacol. Ther. 30,375-84). However functional studies did not find significant changes in metabolite concentrations with this polymorphism (Kudo et al. (2008) Pharmacogenet. Genomics 18, 243-51 ). Prior to the present work there was no data to correlate the presence of SNPs in the MOCOS gene with thiopurine hypermethylation.

ATP-Binding Cassette Protein B5 (ABCB5) I MDR5

ABCB5 is a member of the ATP-binding cassette (ABC) superfamily of transporters that function in the ATP-dependent transport of functionally diverse molecules (Moitra et al. (2011 ) PLoS ONE 6, 1 -12). Evolutionary analysis suggests these proteins have roles in regulating membrane potential and efflux transport of drugs and cellular xenobiotics (Frank (2009) Leukaemia Res. 33, 1305-5). ABCB5 has also been implicated in cancers resistant to chemotherapy. The most common form of ABCB5, ABCB5 beta, contains 10 non-synonymous SNPs including K115E (rs2301641 ), which changes a positively charged amino acid to a negatively charged one. The amino acid change occurs in the cytoplasmic loop and is predicted to adversely affect protein signalling (Moitra et al. (2011 )). In the applicant's own cohort of 192 patients receiving AZA for inflammatory bowel disease the K115E SNP was associated with a lack of clinical response (p=0.02, OR 2.43, 95% CI 1 .145-5.17) and a trend towards a higher risk of adverse events.

There is no evidence in the current literature linking any specific SNP in the COMT, MOCOS or ABCB5 genes with the development of purine hypermethylation in a patient receiving 6-MP drug therapy.

Current thiopurine dosing paradigms are based on weight and pre-treatment ex- vivo measurement of TPMT activity. This strategy does not take into account inter- individual genetic variation in other enzymes which may affect the methylation of thiopurines. Identification of the genetic factors underlying such variation provides a rational basis for the use of combination treatment with a thiopurine and allopurinol. At present the decision to start combination therapy is based on measurement of thiopurine metabolites after a patient has been established on thiopurine monotherapy. The ability to predict which patients are likely to hypermethylate, will allow the use of early combination therapy to circumvent treatment non-response and side effects. This may obviate the need to start second line immunosuppressants such as methotrexate, which are less proven and have greater overall toxicity. Prior to the work of the present applicant, no reliable markers of thiopurine hypermethylation had been identified.

It has been proposed that thiopurine hypermethylation occurs as a result of factors that affect methylation flux (Roberts et al. (2010) Pharmacogenomics 11 ,1505-8). Since all methyltransferase enzymes require S-adenosyl-transferase (SAM) as a co-substrate, and generate S-adenosyl-homocystine (SAH) as a reaction product, which inhibits methyltransferase enzymes, changes in SAM to SAH ratios may directly affect the activity of TPMT. Without being limited to theory, it is

hypothesised that polymorphisms in other methyltransferase enzymes affect the concentration of available SAM and SAH pools and thereby affect TPMT activity in vivo. In addition to factors which affect methylation flux, polymorphisms in other enzymes involved in thiopurine metabolism, such as MOCOS, may also influence levels of 6-MeMP. In contrast with the prior art studies mentioned above, the present applicant has identified a strong association between the COMT rs9332377 C>T and rs4646316 C>T SNPs, the MOCOS rs1057251 T>C SNP, and the ABCB5 rs2301641 A>G SNP and the likelihood of development of thiopurine hypermethylation during 6-MP chemotherapy. Pre-treatment testing for these markers of toxicity will allow an informed decision regarding dose and/or combination therapy with allopurinol to be made.

For example, patients with a COMT rs9332377 C/C genotype, a COMT rs4646316 C/T or TAT genotype, a MOCOS T/T genotype and/or an ABCB5 rs2031641 G/G genotype are at increased risk of thiopurine hypermethylation. These patients can be prescribed a low dose 6-MP or AZA combination therapy with allopurinol from the outset.

Between 45,000 and 60,000 new patients in the UK are started on thiopurine therapy annually. Pre-treatment testing for markers that predict thiopurine hypermethylation, which is estimated to affect approximately 8,000 of these patients, will allow the early use of combination therapy with a low dose of AZA or 6-MP and allopurinol. This is likely to translate into a reduction in drug side effects and treatment non-response. Several methods of carrying out the pre-treatment testing are possible. For example, the genotyping could be carried out using nucleic acid sequencing techniques, PCR, real time PCR, allele specific PCR, electrophoretic analysis, restriction endonuclease genotyping or microarray-based genotyping, alone or in combination. There are many genotyping techniques known in the art that may be adapted for use with the presently disclosed methods. Enzymatic amplification of nucleic acid from a subject can be conveniently used to obtain nucleic acid for subsequent analysis. The presence or absence of a variant allele can also be determined directly from the subject's nucleic acid without enzymatic amplification.

A sample of material containing nucleic acid is routinely obtained from subjects. Such a sample may include any biological matter from which nucleic acid can be prepared. As non-limiting examples, the sample can be whole blood, plasma, saliva, cheek swab, or other bodily fluid or tissue that contains nucleic acid. In an embodiment, the method can use a sample of whole blood, which can be obtained readily by non-invasive means and used to prepare genomic DNA. In another embodiment, genotyping involves amplification of a subject's nucleic acid using PCR. In yet another embodiment, PCR amplification is performed using one or more fluorescently labelled primers. In a further embodiment, PCR amplification is performed using one or more labelled or unlabelled primers that contain a DNA minor groove binder.

Any of a variety of different primers can be used to amplify a subject's nucleic acid by PCR. As understood by one skilled in the art, additional primers for PCR analysis can be designed based on the sequence flanking the polymorphic site(s) of interest. As a non-limiting example, a sequence primer can contain from about 15 to about 30 nucleotides of a sequence upstream or downstream of the polymorphic site of interest. Such primers generally are designed to have sufficient guanine and cytosine content to attain a high melting temperature which allows for a stable annealing step in the amplification reaction. Several computer programs, such as Primer Select, are available to aid in the design of PCR primers.

A Taqman® allelic discrimination assay available from Applied Biosystems can be useful for genotyping an individual at a polymorphic site and thereby determining the presence or absence of a variant allele. In a Taqman® allelic discrimination assay, a specific fluorescent dye-labelled probe for each allele is constructed. The probes contain different fluorescent reporter dyes such as FAM and VIC to differentiate amplification of each allele. In addition, each probe has a quencher dye at one end which quenches fluorescence by fluorescence resonance energy transfer. During PCR, each probe anneals specifically to complementary sequences in the nucleic acid from the subject. The 5' nuclease activity of Taq polymerase is used to cleave only probe that hybridises to the allele. Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter dye. Thus, the fluorescence signal generated by PCR amplification indicates which alleles are present in the sample. Mismatches between a probe and allele reduce the efficiency of both probe hybridisation and cleavage by Taq polymerase, resulting in little to no fluorescent signal. Those skilled in the art understand that improved specificity in allelic discrimination assays can be achieved by conjugating a DNA minor groove binder group to a DNA probe as described, e.g., in Kutyavin et al, Nuc. Acids Research 28:655-661 (2000). Minor groove binders include, but are not limited to, compounds such as dihydrocyclopyrroloindole tripeptide. Sequence analysis can also be useful for genotyping a subject at a polymorphic site. A variant allele can be detected by sequence analysis using the appropriate primers, which are designed based on the sequence flanking the polymorphic site of interest, as is known by those skilled in the art. As a non-limiting example, a sequence primer can contain from about 15 to about 30 nucleotides of a sequence that corresponds to a sequence about 40 to about 400 base pairs upstream or downstream of the polymorphic site of interest. Such primers are generally designed to have sufficient guanine and cytosine content to attain a high melting temperature which allows for a stable annealing step in the sequencing reaction. The term "sequence analysis" means any manual or automated process by which the order of nucleotides in a nucleic acid is determined. As an example, sequence analysis can be used to determine the nucleotide sequence of a sample of DNA. The term sequence analysis encompasses, without limitation, chemical and enzymatic methods such as dideoxy enzymatic methods including, for example, Maxam-Gilbert and Sanger sequencing as well as variations thereof. The term sequence analysis further encompasses, but is not limited to, capillary array DNA sequencing, which relies on capillary electrophoresis and laser-induced fluorescence detection and can be performed using instruments such as the MegaBACE 1000 or ABI 3700. As additional non-limiting examples, the term sequence analysis encompasses thermal cycle sequencing Sears et al. (1992) Biotechniques 13, 626-633); solid-phase sequencing (Zimmerman et al. (1992) Methods Mol. Cell. Biol. 3, 39-42); and sequencing with mass spectrometry, such as matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS; Fu et al. (1998) Nat. Biotech. 16, 381 -384). The term sequence analysis further includes, but is not limited to, sequencing by hybridization (SBH), which relies on an array of all possible short oligonucleotides to identify a segment of sequence (Chee et al. (1996) Science 274, 610-614; Drmanac et al. (1993) Science 260, 1649-1652; and Drmanac et al. (1998) Nat. Biotech. 16, 54-58). One skilled in the art understands that these and additional variations are

encompassed by the term sequence analysis as defined herein. Electrophoretic analysis also can be useful in genotyping a subject according to the methods of the present invention. "Electrophoretic analysis" as used herein in reference to one or more nucleic acids such as amplified fragments means a process whereby charged molecules are moved through a stationary medium under the influence of an electric field. Electrophoretic migration separates nucleic acids primarily on the basis of their charge, which is in proportion to their size, with smaller molecules migrating more quickly. The term electrophoretic analysis includes, without limitation, analysis using slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, or capillary electrophoresis.

Capillary electrophoretic analysis generally occurs inside a small-diameter (50-100 m) quartz capillary in the presence of high (kilovolt-level) separating voltages with separation times of a few minutes. Using capillary electrophoretic analysis, nucleic acids are conveniently detected by UV absorption or fluorescent labeling, and single-base resolution can be obtained on fragments up to several hundred base pairs. Such methods of electrophoretic analysis, and variations thereof, are well known in the art. Restriction fragment length polymorphism (RFLP) analysis can also be useful for genotyping a subject at a polymorphic site. As used herein, "restriction fragment length polymorphism analysis" includes any method for distinguishing polymorphic alleles using a restriction enzyme. One skilled in the art understands that the use of RFLP analysis depends upon an enzyme that can differentiate a variant allele from a wild-type or other allele at a polymorphic site.

In addition, allele-specific oligonucleotide hybridisation can be useful for

genotyping a subject in the methods of the present invention. Allele-specific oligonucleotide hybridisation is based on the use of a labelled oligonucleotide probe having a sequence perfectly complementary, for example, to the sequence encompassing the variant allele. Under appropriate conditions, the variant allele- specific probe hybridises to a nucleic acid containing the variant allele but does not hybridise to the one or more other alleles, which have one or more nucleotide mismatches as compared to the probe. If desired, a second allele-specific oligonucleotide probe that matches an alternate (for example, wild-type) allele can also be used. Similarly, the technique of allele-specific oligonucleotide

amplification can be used to selectively amplify, for example, a variant allele by using an allele-specific oligonucleotide primer that is perfectly complementary to the nucleotide sequence of the variant allele but which has one or more

mismatches as compared to other alleles. One skilled in the art understands that the one or more nucleotide mismatches that distinguish between the variant allele and other alleles are often located in the centre of an allele-specific oligonucleotide primer to be used in the allele-specific oligonucleotide hybridisation. In contrast, an allele-specific oligonucleotide primer to be used in PCR amplification generally contains the one or more nucleotide mismatches that distinguish between the variant and other alleles at the 3' end of the primer.

A heteroduplex mobility assay (HMA) is another well-known assay that can be used for genotyping at a polymorphic site in embodiments of methods of the present invention. HMA is useful for detecting the presence of a variant allele since a DNA duplex carrying a mismatch has reduced mobility in a polyacrylamide gel compared to the mobility of a perfectly base-paired duplex (Delwart et al. (1993) Science 262, 1257-1261 ; White et al. (1992) Genomics 12, 301 - 306).

The technique of single strand conformational polymorphism (SSCP) can also be useful for genotyping at a polymorphic site in the methods of the present invention. This technique is used to detect variant alleles based on differences in the secondary structure of single-stranded DNA that produce an altered

electrophoretic mobility upon non-denaturing gel electrophoresis. Variant alleles are detected by comparison of the electrophoretic pattern of the test fragment to corresponding standard fragments containing known alleles.

Denaturing gradient gel electrophoresis (DGGE) can be useful in the methods of the present invention. In DGGE, double-stranded DNA is moved by

electrophoresis through a gel containing an increasing concentration of denaturant; double-stranded fragments made up of mismatched alleles have segments that melt more rapidly, causing such fragments to migrate differently as compared to perfectly complementary sequences.

Other molecular methods useful for genotyping a subject at a polymorphic site are known in the art and useful in the methods of the present invention. Such well- known genotyping approaches include, without limitation, automated sequencing and RNAase mismatch techniques. Furthermore, one skilled in the art

understands that, where the presence or absence of multiple variant alleles is to be determined, individual variant alleles can be detected by any combination of molecular methods. In addition, one skilled in the art understands that multiple variant alleles can be detected in individual reactions or in a single reaction (a "multiplex" assay).

Further information regarding possible methods are described in the applicant's earlier patent publication, WO 2009/047001 , the contents of which are

incorporated herein by reference. Genetic markers predictive of thiopurine hypermethylation are of immense clinical benefit. This phenotype is associated with treatment non-response, hepatotoxicity and other side effects. Therefore the ability to predict hypermethylation before the start of treatment will allow individualisation of treatment and an opportunity to start combination treatment with allopurinol, which appears to circumvent thiopurine hypermethylation. This would not only improve the quality of life of patients starting treatment with a thiopurine, but will also be cost-effective by reducing hospital admissions and the number of outpatient visits required to manage continuing active disease and drug toxicity. EXAMPLES

Example 1

In a prospective cohort of 168 patients, predominantly with inflammatory bowel disease, treated with thiopurines, the applicant has identified an association between the COMT rs9332377 C>T and rs4646316 C>T and the development of thiopurine hypermethylation. The haplotypes of rs9332377 T and rs4646316 C, which encode a low-activity synonymous COMT variant, rs4818, showed a trend towards protection from thiopurine hypermethylation (rs9332377 T, p=0.0178, rs4646316 C, p=0.033).

Table 1 - COMT rs9332377 C>T genotype and thiopurine hypermethylation (6-

MeMP:TGN>11 :1 and MeMP <5000)

Fisher's exact test

Two-tailed P value = 0.0178

OR 0.350 (0.141 -0.870) Table 2 - COMT rs4646316 C>T genotype and thiopurine hypermethylation (6- MeMP:TGN>11 :1 and MeMP <5000)

Fisher's exact test

Two-tailed P value = 0.033

OR 0.417 (95% CI 0.189-0.919)

This is the first time a statistically significant association has been shown between the presence of low activity COMT variants and protection from thiopurine hypermethylation. This phenotype is predicted to occur in up to 15% of patients started on either AZA or 6-MP and is associated with treatment non-response, hepatotoxicity and other side effects. Pre-treatment detection for low activity COMT variants will assist clinicians in determining which patients are likely to be protected from thiopurine hypermethylation and therefore less likely to require combination treatment with a reduced dose of AZA or 6-MP and allopurinol.

Example 2

In a prospective cohort of 168 patients, predominantly with inflammatory bowel disease, treated with thiopurines, we have identified an association between the presence of MOCOS 2600T>C (rs1057251 ) and protection from thiopurine hypermethylation (p=0.031 ). Table 3 - MOCOS rs1057251 (Val867Ala)

Fisher's exact test

Two-tailed P value = 0.031

OR 0.243 (95% CI 0.069-0.862)

When the patients were separated by MeMP above and below 5000 the association was even stronger.

Table 4 - MOCOS rs1057251 (Val867Ala)

Fisher's exact test

Two-tailed P value = 0.027

OR 0.209 (95% CI 0.047-0.924)

This is the first time that the presence of the rs1057251 C genotype has been shown to confer significant protection from thiopurine hypermethylation.

Example 3

In a prospective study of 168 patients, predominantly with inflammatory bowel disease, treated with thiopurines, we have identified an association between the presence of ABCB5 SNP K115E (rs2301641 A>G) and high levels of thiopurine hypermethylation. Table 5 - ABCB5 rs2301641 (K115E)

Fisher's exact test

Two-tailed P value = 0.0098

OR 4.3 (95% CM .412-13.093)

The association was strengthened when patients were separated by levels of 6- MeMP above or below 5000 alone.

Table 6

Fisher's exact test

Two-tailed P value = 0.0065

OR 4.119 (95% CI 1 .522-11 .147)

This is the first time that the presence of the rs23016141 (K115E) G/G genotype has been shown to confer a significant chance of thiopurine hypermethylation. Since high levels of 6-MeMP have been associated with thiopurine hepatotoxicity and other side effects, the ability to predict this phenotype using the ABCB5 K115E SNP, will allow the early use of combination therapy with a low dose thiopurine and allopurinol. This will circumvent unnecessary drug toxicity and reduce the need to switch patients to potentially more toxic and less proven alternative immunosuppressants.

The sequences of all of the SNPs disclosed herein are available from the National Center for Biotechnology Information's database of short Genetic Variations, dbSNP: www.ncbi.nlm.nih.gov/snp

The disclosures in GB 1116664.2, from which the present application claims priority, and in the accompanying Abstract, are incorporated herein by reference.

Claims

1 . A method for predicting thiopurine hypermethylation in response to a 6- mercaptopurine drug or prodrug in an individual in need thereof, the method comprising:

(a) genotyping the individual at a polymorphic site in at least a

catecholamine-O-methyltransferase (COMT) gene, a molybdenum cofactor sulfurase (MOCOS) gene, and/or an ATP-binding cassette protein B5 (ABCB5) gene; and

(b) determining the presence or absence of a variant allele at said polymorphic site;

wherein the presence or absence of the variant allele at said polymorphic site is indicative of a likelihood of thiopurine hypermethylation.

2. A method as claimed in claim 1 , wherein the individual has an inflammatory condition.

3. A method as claimed in claim 2, wherein the condition is an immune- mediated gastrointestinal disorder.

4. A method as claimed in claim 3, wherein the immune-mediated

gastrointestinal disorder is an inflammatory bowel disease.

5. A method as claimed in claim 4, wherein the disorder is Crohn's disease.

6. A method as claimed in claim 4, wherein the disorder is ulcerative colitis.

7. A method as claimed in any preceding claim, wherein the gene is a COMT gene.

8. A method as claimed in claim 7, comprising testing for a low activity variant of COMT, wherein the presence of a low activity COMT variant is indicative of protection from thiopurine hypermethylation.

9. A method as claimed in claim 7 or 8, wherein the polymorphic site comprises the COMT rs9332377C>T variant allele.

10. A method as claimed in claim 7, 8 or 9, wherein the polymorphic site comprises the COMT rs4646316C>T variant allele.

11 . A method as claimed in any of claims 7 to 10, wherein the genotype is rs9332377 T/T and is indicative of protection from thiopurine hypermethylation.

12. A method as claimed in any of claims 7 to 11 , wherein the genotype is rs9332377 C/T and is indicative of protection from thiopurine hypermethylation.

13. A method as claimed in any of claims 7 to 12, wherein the genotype is rs4646316 C/C and is indicative of protection from thiopurine hypermethylation.

14. A method as claimed in any preceding claim, wherein the gene is a

MOCOS gene.

15. A method as claimed in claim 14, wherein the polymorphic site is the MOCOS 2600T>C (rs1057251 ) variant allele.

16. A method as claimed in claim 14 or 15, wherein the genotype is rs1057251 T/C, and is indicative of protection from thiopurine hypermethylation.

17. A method as claimed in claim 14, 15 or 16, wherein the genotype is rs1057251 C/C, and is indicative of protection from thiopurine hypermethylation.

18. A method as claimed in any preceding claim, wherein the gene is an ABCB5 gene.

19. A method as claimed in claim 18, wherein the polymorphic site is ABCB5 5 rs2301641 (K115E) A>G variant allele.

20. A method as claimed in claim 18 or 19, wherein the genotype is rs2301641 G/G and is indicative of a high risk of thiopurine hypermethylation.

10 21 . A method as claimed in any preceding claim, wherein the genotype is

determined by a method including nucleic acid sequencing.

22. A method as claimed in claim 21 , wherein the nucleic acid sequencing is DNA sequencing.

15

23. A method as claimed in any preceding claim, wherein the genotype is determined by a method including PCR.

24. A method as claimed in claim 23, wherein the PCR includes real time PCR.

20

25. A method as claimed in claim 23 or 24, wherein the PCR includes allele- specific PCR.

26. A method as claimed in any preceding claim, wherein the genotype is 25 determined by a method including restriction endonudease genotyping.

27. A method as claimed in any preceding claim, wherein the genotype is determined by a method including microarray-based genotyping.

30 28. A method as claimed in any preceding claim, wherein at least two of the genes are genotyped.

29. A method as claimed in any preceding claim, wherein at least two of the polymorphic sites are genotyped.

30. A method as claimed in any preceding claim, wherein at least three of the 5 genes are genotyped.

31 . A method as claimed in any preceding claim, wherein at least three of the polymorphic sites are genotyped.

10 32. A method as claimed in any preceding claim, wherein the individual is

tested for activity of thiopurine-S-methyltransferase prior to commencing treatment.

33. A method as claimed in any preceding claim, wherein levels of 6- methylmercaptopurine are measured prior to commencing treatment.

15

34. A method as claimed in any preceding claim, wherein the drug is 6- mercaptopurine.

35. A method as claimed in any of claims 1 to 33, wherein the drug is 6- 20 azathioprine.

36. A method as claimed in any of claims 1 to 33, wherein the drug is 6- thioguanine.

25 37. A method as claimed in any of claims 1 to 33, wherein the drug is 6- methylmercaptopurine riboside.

38. A method as claimed in any preceding claim, wherein the drug further comprises allopurinol.

30

39. A method of developing a treatment regime for an individual in need of a 6- mercaptopurine drug or prodrug including predicting thiopurine hypermethylation using a method as claimed in any preceding claim and planning a course of treatment or a dosage regime based on the results thereof.

40. A method of treating an individual in need of a 6-mercaptopurine drug or prodrug, including:

(a) predicting thiopurine hypermethylation in response to the 6- mercaptopurine drug or prodrug in the individual using a method as claimed in any of claims 1 to 38;

(b) treating the individual based on the results of the prediction.