WO1999023255A1 - Methods of identifying and diagnosing inflammatory bowel disease or subtypes with the nramp locus - Google Patents

Abstract

Methods of identifying, diagnosing and screening for inflammatory bowel disease (IBD), particularly ulcerative colitis (UC), Crohn's disease and their subtypes, comprise identifying alleles and polymorphisms associated with a biological response related to an inflammatory bowel disease, such as UC. A method determines whether a therapy which alters the level or functionality of NRAMP is effective in treating an inflammatory bowel disease, UC, CD or their subtypes. A kit for screening for susceptibility to inflammatory bowel diseases comprises DNA from the functional (structural gene) or satellite regions of the NRAMP locus, oligonucleotides thereof or antisense nucleic acids thereto.

Description

METHODS OF IDENTIFYING AND DIAGNOSING INFLAMMATORY BOWEL DISEASE OR SUBTYPES WITH THE NRAMP LOCUS

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates generally to the identification of alleles, and diagnosis of, and screening for, inflammatory bowel disease (IBD), including Crohn's Disease (CD), Ulcerative Colitis (UC), and its subtypes. Description of the Background Inflammatory Bowel Disease (IBD) is a collective term used to describe two chronic, idiopathic inflammatory diseases of the gastrointestinal tract: ulcerative colitis (UC) and Crohn's disease (CD). IBD occurs world-wide and is reported to afflict as many as two million people. UC and CD are grouped together because of their overlapping clinical, etiologic, and pathogenic features. Furthermore, some clinical data have provided evidence that CD may be a heterogenous group of disorders, although the etiologies and pathogeneses of these CD subtypes still remain unknown. Even less is known about the heterogeneity and subtyping of UC. From a therapeutic and prognostic standpoint, however, it is as important to distinguish between UC and CD as well as amongst their subtypes. This will make it possible to distinguish chronic from non-chronic inflammatory diseases of the bowel. The heterogeneity underlying UC or CD may be reflected in the CD and UC patients' variable responses to a particular treatment strategy. The availability of methods of diagnosing IBD, and particularly UC and/or clinical subtypes of UC would represent a major clinical advance that would aid in the therapeutic and prophylactic treatment of these diseases or conditions and would provide a basis for designing treatment modalities that are specific to a particular disease subtype.

Unfortunately, no methods for identifying a gene(s) associated with IBD, UC and the like, or of stratifying the diseases into clinical subtypes are presently available, which would allow the design of custom tailored treatment strategies. Thus, there is a need for a method that will permit diagnosing IBD, UC and clinical subtypes of UC to advance the clinical diagnosis of the diseases and conditions as well as to permit better therapeutic management of the patient.

Although the causes of UC and CD remain unknown, there is overall agreement that a person's genetic heritage plays an important role in his/her susceptibility to IBD, and that the immune system is responsible for mediating tissue damage in these diseases. Generally speaking, one characteristic of IBD is a failure to down-regulate the normal, self-limited inflammatory responses in the bowel. A wide range of immunologic abnormalities and dysfunctions have been reported to be associated with these disorders. However, before this invention none had been linked with specific Natural Resistance- Associated Protein (NRAMP) alleles or polymoφhisms. Nor had the possibility been entertained that the NRAMP locus might be diagnostic or prognostic value.

At present, a battery of laboratory, radiological, and endoscopic evaluations need to be combined to derive a diagnosis of IBD and to assess the extent and severity of the disease. In addition, the differentiation of UC from CD and other types of inflammatory conditions of the bowel, such as irritable bowel syndrome, infectious diarrhea, rectal bleeding, radiation colitis, and the like, is also difficult, because the mucosa of the small and large intestines react in a similar way to a large number of different conditions. Once other conditions, such as infectious-types of bowel disorders, have been ruled out a final diagnosis of IBD must often be made on the basis of the progression of the disease. In many patients a diagnosis must still be regarded as indeterminate because of the overlapping features of UC and CD, particularly with CD of the colon. Thus, a great need exists for novel methods for identifying IBD, CD, UC and diagnose their clinical subtypes.

To date, no objective diagnostic tools for the identification and diagnosis of IBD, UC, CD and their subtypes are commercially available. Diagnosis depends upon a host of procedures aimed at confirming a suspected diagnosis of any of these diseases or conditions. The initial symptoms are often confused for non-chronic bowel disorders by physicians unfamiliar with IBD and, consequently, it often goes mistreated and undiagnosed until the disease shows its chronicity which results in a referral of the patient to a specialist. The imprecise and subjective nature of endoscopic and radiologic examination often results in a misdiagnosis between UC and CD, or an indeterminate diagnosis of UC. A physician has at this point no means for identifying and diagnosing clinical subtypes of many of these conditions, particularly UC,

Although histological examination provides some certainty of an accurate diagnosis, it is not that accurate when it comes to differentiating between UC and CD. At the present time, there is no single histological criterion which definitively identifies CD, UC or their subtypes. For example, the epithelial cell granuloma, which is often accorded a key role in the diagnosis of CD, is only found in about 20% of biopsy specimens taken from patients diagnosed with CD. The patient often suffers as the disease progresses before a definitive diagnosis can be made. The selective identification of UC, as opposed to CD or other inflammatory conditions of the intestines, carries important prognostic and therapeutic implications. For example, when colectomy is indicated, the type of IBD involved determines which surgical options are appropriate. While in UC total colectomy surgery represents somewhat of a cure, in CD surgery it is never curative. Contingent procedures such as the ileorectal pull-through (mucosal proctectomy) or the Kock pouch may be desirable in UC, but are contraindicated in CD.

Although previous studies have provided some evidence that genetic factors may contribute to the pathogenesis of IBD, it is clearly not transmitted in a simple Mendelian pattern. This has been suggested to be due to genetic heterogeneity (mutations in any one of several genes resulting in identical phenotypes), multi-locus inheritance (more than one distinct locus influences a single phenotype), and multifactorial inheritance (the phenotype is determined by both genetic and environmental factors). Identifying genes involved in the etiology of IBD would aid tremendously in understanding the cause of the disease and might expand treatment options. The macrophage is a large phagocytic cell derived from the blood monocyte, which also functions as an antigen-presenting cell and can mediate antibody-dependent-cell medicated cytotoxicity. Macrophage hyperactivity to intestinal flora has been implicated in the pathogenesis of IBD. A population study by one of the inventors recently found an association of the Natural Resistance-Associated Macrophage Protein (NRAMP) gene with IBD. NRAMP functions in macrophage regulation and confers resistance or susceptibility to intracellular pathogens, especially of the gastrointestinal tract. NRAMP appears to be critical for the bacteriostatic activity of human mononuclear cells. One mutation in the murine NRAMP gene was shown to decrease the resistance to infection by intracellular pathogens. For instance, the similarity of Crohn's Disease to intestinal tuberculosis, a granulomatous enteritis in cattle (Johne's disease), and the intestinal lesions observed in a mouse model of IBD, have evoked interest in the possibility that these two conditions may have a common etiology. The pathogen responsible for Johne's disease is Mycobacterium paratuberculosis. Patients with Crohn's Disease exhibit a diminished lymphoproliferative response to mycobacterium Paratuberculosis compared to healthy individuals, suggesting that they are also less resistant to this pathogen. In many instances, an inability to effectively control an infection produced by intracellular pathogens may result in hyper reactivity towards other bacteria of the intestinal flora. A recent study showed that patients with active IBD evidence an extremely low tolerance or absence of tolerance to their autologous intestinal flora. This observation parallels the finding of an increased level of mononuclear cell activity in the intestinal lamina propria of IBD patients. At the same time, the intestinal lesions in mice that resemble the inflammatory lesions of Crohn's disease patients have been found to be caused by numerous bacterial and fungal species.

A physical assignment of the NRAMP gene and microsatellite genetic markers, D2S173 and D2S104 has placed the three loci within a 150,000 base pair yeast artificial chromosome. Linkage assignments of D2S173, D2S104, D2S434, and D2S1323 place all four loci within 0.06 centimorgans. This is shown in Table 2 below, which is a schematic of a portion of Chromosome 2 demonstrating the mapping of the NRAMP locus, and more specifically the microsatellite loci that approximate the NRAMP locus.

An innate susceptibility or resistance to various mycobacteria has been correlated with differences in the Natural Resistance-Associated Macrophage Protein (NRAMP) gene in mice. It has been found, moreover, that a susceptibility to infection is caused by a single glycine-to-aspartic am ino acid substitution at nucleotide 169 of NRAMP. This mutation affects the capacity of macrophages to destroy ingested intracellular pathogens during early infection with respect to wild type macrophages, which are superior in their functional and phenotypic activation. NRAMP, the human homologue gene, is expressed in monocytes/macrophages as well as in polymorphonuclear neutrophils. Although various studies have shown pleiotropic effects of NRAMP on macrophage activation, its specific function remains unknown. NRAMP has recently been isolated and mapped to chromosome 2q35.

IBD including UC and CD presently afflicts as many as two million people world-wide. There is, thus, a great need for convenient and reliable methods of screening populations for IBD, particularly for distinguishing CD from UC for diagnostic, prognostic and therapeutic purposes, and for finding and diagnosing their subtypes. Of particular importance would be the finding of, and making available, genetic markers and the corresponding immunological markers that would readily distinguish between these diseases or conditions and help identify their subtypes, either independent of or in combination with existing diagnostic tools. This would represent a major clinical advance which would aid the therapeutic management of each of these conditions and the design of more specific treatment modalities.

SUMMARY OF THE INVENTION The present invention relates to a method for identifying novel allele(s) or allelic combination(s) in the natural resistance-associated protein (NRAMP) locus, which evidence a statistically significant correlation with one or more biological response(s) exhibited by subjects known to be afflicted with a disease(s) or condition(s) such as IBD, particularly Crohn's Disease (CD), Ulcerative Colitis (UC) or their subtypes. Once found, the correlation(s) may be applied to the identification, diagnosis and screening of populations for, the disease(s) and/or condition(s) described above. Specific methods encompassed by this technology are briefly described below.

A method of identifying an inflammatory bowel disease (IBD) or subtype thereof, comprises selecting a population comprised of subjects having at least one biological response associated with IBD or a subtype thereof, obtaining nucleic acid from the subjects in the selected population, detecting a polymorphism at a natural resistance-associated macrophage protein (NRAMP) locus in the nucleic acid from the subjects, establishing whether a statistically significant correlation exists between the thus found polymorphism and at least one of the biological responses, and identifying an IBD or subtype thereof when the existence of a statistically significant correlation is established. This method may be applied to identifying an IBD, CD or UC subtype by selecting a population comprised of subjects having at least one biological response associated with the disease(s) or condition(s), and then applying the method described above to the nucleic acid of the thus selected population, and identifying a subtype of either of the diseases(s) or condition(s) when the existence of a statistically significant correlation is established. A method for diagnosing an IBD or subtype thereof, comprises selecting a subject exhibiting at least one biological response associated with IBD of subtype thereof, detecting a polymorphism at a site of an NRAMP locus in the nucleic acid from the subject, comparing the found polymorphism with that of IBD or subtype DNA controls found by the method described above until a statistically significant correlation with a biological response is found, and diagnosing IBD or a subtype thereof when a match with an IBD or subtype control is found. In one preferred embodiment the method of the invention may be applied to diagnosing an UC subtype by applying it to a subject exhibiting at least one biological response associated with UC.

A method of screening a population for susceptibility to IBD or subtype thereof, comprises diagnosing the disease(s) or condition(s) or subtype thereof by applying the method described above to nucleic acid obtained from members of a predetermined population, and selecting the members of the population which show a statistically significant correlation with the biological response(s) as exhibiting a susceptibility to IBD or subtype thereof. In one preferred embodiment, this method may be applied to screening a population for a susceptibility to UC or another specific disease or condition by applying it to a population suspected of being afflicted by the disease or condition. Still another embodiment utilizes the satellite region of the NRAMP locus for identifying a polymorphism, and making a diagnosis if a subject's satellite region has a similar number and size of repeats as a control.

A method of determining the effectiveness of a therapy for treating inflammatory bowel disease, e.g. by altering the level or function of a natural resistance-associated macrophage protein (NRAMP), comprises detecting a polymorphism at a functional site within an NRAMP locus in a subject's nucleic acid, and determining that the therapy is effective using the correlation described above. This method can determine when NRAMP mRNA or protein production or NRAMP function or levels no longer exhibit a statistically significant correlation with the polymorphism.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention arose from a desire by the inventors to improve on prior art technology for diagnosing IBD, particularly UC and CD. Prior to this invention, the artisan had a long list of symptoms which had been exhibited by one or the other patients afflicted with the diseases. A prior study by one of the inventors proposed an association of the markers utilized in the present exemplary disclosure with CD. See, Hofmeister et al, Surgery 122 (2): 175 (1997). Because such an association may be due to an unrecognized stratification of the population or, alternatively, to a minor effect of the gene, the inventors conducted the present work and established a true linkage between one of the markers and UC, but not CD. This was clearly an unexpected result based on what was known in the art at the time of the invention.

The present inventors surmised that a mutation in the NRAMP gene may cause a susceptibility to inflammatory bowel disease (IBD), and more particularly to either Ulcerative Colitis (UC), Crohn's Disease (CD) or their subtypes. To test this hypothesis the inventors set out to investigate genetic differences in markers adjacent to the NRAMP gene, and to analyze their possible linkage with a specific IBD population comprised of sib pairs otherwise diagnosed with UC, CD or those having symptoms of both. The inventors trusted that such finding would be helpful in distinguishing Crohn's Disease from Ulcerative Colitis.

In addition, because IBD, CD and UC, among others, are chronic inflammatory diseases, the inventors surmised that any abnormalities in the NRAMP locus may produce a defective gene product, causing susceptibility to unknown pathogens leading to chronic inflammation. This invention, therefore, is applicable to other known auto-immune diseases such as those which are known to have an association with IBD, UC and/or CD. Examples of these auto-immune diseases are thyroiditis and multiple sclerosis. Other auto-immune diseases are lupus erythematosus, rheumatoid arthritis, subacute and bacterial endocarditis, etc. The identification of genes involved in complex disorders such as IBD is challenging. The modest nature of each gene's effect in multi-locus diseases presents difficulties in reproducibly identifying loci by linkage analysis. A genetic linkage of the NRAMP markers with, for example, UC was established by analyzing a large number of sib pairs and controls. The identification of a genetic marker(s) that reliably distinguish(es) between Ulcerative Colitis and Crohn's Disease is especially valuable to the 10% to 15% of patients for whom a definitive diagnosis of either Crohn's Disease or Ulcerative Colitis cannot be established by conventional methods.

The present invention provides a method for identifying an inflammatory bowel disease (IBD), and particularly Ulcerative Colitis (UC) and Crohn's Disease (CD) and their subtypes in a subject by selecting a population comprised of subjects having at least one biological response associated with IBD or a subtype thereof, obtaining nucleic acid from the subjects in the selected population, detecting a polymoφhism at a natural resistance-associated macrophage protein (NRAMP) locus in the nucleic acid from the subjects, establishing whether a statistically significant correlation exists between the thus found polymoφhism and at least one of the biological responses, and identifying an IBD or subtype thereof when the existence of a statistically significant correlation is established. One preferred NRAMP locus comprises a functional NRAMP locus, and a more preferred functional NRAMP locus comprises a 5' regulatory region of the NRAMP locus. Still another preferred NRAMP locus comprises a satellite NRAMP locus, and still more preferred locus comprises a satellite fragment vicinal to the 5' end of the functional NRAMP locus. A most preferred haplotype is that corresponding to the D2S434 allele which was shown to be linked to Ulcerative Colitis in a statistically significant manner. Yet another preferred embodiment targets a population otherwise diagnosed with UC in the search for further loci which are linked to different subtypes of this disease or condition in a statistically significant manner.

The polymoφhism may comprises a mutation such as one or more nucleotide substitutions, additions, deletions and/or combination thereof. The deletions and additions are generally of a few nucleotides, for example up to 5, sometimes 10 nucleotides, although sometimes a stop codon may truncate the remainder of the sequence, in which case a longer nucleotide segment is deleted. In one preferred case, the polymoφhism comprises a nucleotide substitution which may be at a functional or a satellite region of the NRAMP locus. Where the polymorphism is located at a functional site of the NRAMP locus, functional variations are, most likely, observed. In a preferred embodiment, the polymoφhism is located in the 5' regulatory region of an NRAMP locus, and in another in a satellite region of the NRAMP locus. When occurring in the satellite region the polymorphism may be located vicinal to the 3' or the 5" end of an NRAMP locus, and most preferably it corresponds to the D2S434 haplotype. Another preferred embodiment is when the biological response corresponds to an alteration in the expression of the NRAMP gene with respect to the general population, as measured by NRAMP mRNA or protein production. Still another when the biological response corresponds to an increase in the expression of the NRAMP gene with respect to a general population, as measured by NRAMP blood levels as compared to the general population.

The polymoφhisms may be detected by methods known in the art. More particularly the polymoφhism may be detected by isolating a fragment of the nucleic acid comprising the NRAMP locus from the sample, contacting the nucleic acid fragment with an NRAMP polymorphism-specific oligonucleotide probe under conditions effective to hybridize the nucleic acid and the polymoφhism- specific oligonucleotide probe, detecting the presence of any hybrid formed, and taking the presence of any hybrid as an indication of the existence of a polymoφhism. The nucleic acid may be isolated from a patient's biological sample by methods known in the art, one of which is by enzymatic amplification. Others, however, may also be utilized. In general, the NRAMP locus polymoφhism- specific probes/primers are designed to flank a targeted locus. That is, one is targeted to an oligonucleotide up-stream, and the other to an oligonucleotide down-stream of the locus desired to be mapped. The probes need not be of any specific length but are, generally, at least about 8 nucleotides long, and seldom contain more than about 60 nucleotides. However, other lengths may also utilized. The probes may comprise oligonucleotides of the functional region of, or satellite region to, an NRAMP locus. The hybridization of the nucleic acid with the primers/probes and formation of a complementary strand of DNA and amplification by PCR, as is known in the art. The thus resulting DNA segment may be identified by restriction endonucleases and size separation, and comparison to length markers, by sequencing or by other methods known in the art.

The thus obtained patterns may then be compared to known positive and negative controls for the diagnosis of a single patient or for the screening of a larger population.

The above method may be applied to diagnosing an inflammatory bowel disease (IBD) or subtype thereof by selecting a subject exhibiting at least one biological response associated with IBD of subtype thereof, detecting a polymoφhism at a functional site of a natural resistance-associated macrophage protein (NRAMP) locus in the nucleic acid from the subject, comparing the found polymoφhism with that of IBD or subtype DNA controls found by the method described above to have a statistically significant correlation with a biological response, and diagnosing IBD or a subtype thereof when a match with an IBD or subtype control is found. This method may be applied to separate populations of individual diseases if so desired, particularly to UC, and by practicing any of the different embodiments described above.

The above method may also be applied to screening a population for a susceptibility to an IBD or subtype thereof by diagnosing IBD or subtype thereof by applying the method described above to nucleic acid samples obtained from members of a predetermined population, and selecting the members of the population which show a statistically significant correlation with the biological response as exhibiting a susceptibility to IBD or subtype thereof. Clearly all the different modes described above may be incoφorated into this screening method. In particularly preferred embodiments, the inflammatory disease is Ulcerative Colitis, the NRAMP locus comprises the satellite region of the NRAMP locus, and the NRAMP locus allele is the D2S434 allele. The method of the invention may also be applied to determining the effectiveness of a therapy to alter the level or function of a natural resistance-associated macrophage protein (NRAMP) for treating inflammatory bowel disease (IBD) or one of its subtypes by detecting a polymoφhism at a functional site within an NRAMP locus in a nucleic acid from a subject, and determining that the therapy is effective using the correlation found by the method described above with respect to NRAMP mRNA or protein production, or to NRAMP blood levels, if the NRAMP mRNA or protein production or the NRAMP function and/or blood level is altered and/or no longer exhibits a statistically significant correlation with the polymoφhism. In preferred applications of this method, the Inflammatory Bowel Disease comprises Ulcerative Colitis, and the polymorphism occurs in the satellite region of the NRAMP locus, a still more preferred embodiment as applied to determining the effectiveness of a treatment for Ulcerative Colitis in a subject utilizes a locus which corresponds to the D2S434 allele at an NRAMP locus.

Another aspect of the invention relates to an Inflammatory Bowel Disease kit, which comprises a nucleic acid comprising a functional and/or satellite region of a natural resistance-associated macrophage protein (NRAMP) locus, oligonucleotides thereof or anti-sense nucleic acids thereto, and instructions for its use in the practice of this invention. The kit's nucleic acid may comprise a 5' regulatory region of an NRAMP locus, oligonucleotides thereof or anti-sense nucleic acids thereto. In a most preferred embodiment, the nucleic acid comprises a satellite region to an NRAMP locus, and more preferably a satellite region vicinal to the 5' or the 3' terminus of the functional gene. In addition, the kit may also comprise means for amplifying the nucleic acid, such as primers/probes, restriction endonuclease and other enzymes for conducting PCR, and the like. The nucleic acid may be about 8 to 60, preferably 12 to 30, and more preferably about 15 to 20 nucleotide long, although other lengths are also contemplated, a preferred kit is one intended for the diagnosis of ulcerative colitis (UC) and, therefore, will comprise a nucleic acid comprising functional and/or satellite regions of a Natural Resistance- Associated Macrophage Protein (NRAMP) locus, and still more preferably the DNA will correspond to that of at least a portion of the D2S434 allele of the NRAMP locus.

By following the inventors' teaching, an artisan may apply the method of the invention to the identification of new statistically significant genetic marker correlations which, in turn, will help identify new subtypes and/or categories of Inflammatory Bowel Diseases, particularly new clinical subtypes of Ulcerative Colitis and Crohn's Disease. In the following description, a variety of technologies are mentioned which are known to those skilled in the art of molecular genetics, microbiology, pathology and general biology. The relevant portions of the publications, patents and other materials setting forth such known technologies and the techniques utilized to implement them to which reference is made are incorporated herein by reference. Unless defined otherwise, all technical and scientific terms used herein have a meaning which is commonly understood by those of skill in the art to which this invention pertains. The description that follows utilizes terms of art in the field of biotechnology. In order to provide a clear and consistent understanding of the disclosure provided by this patent and its claims, including the scope to be given such terms, the following definitions are provided. As used herein, the term "inflammatory bowel disease" means any disease of the bowels with an infectious, inflammatory, autoimmune or hyper immune component or response, acute or chronic, including ulcerative colitis and Crohn's disease (CD). The term "subject" refers to any animal, particularly a mammal, more particularly a human. The term "diagnosed with having an inflammatory bowel disease" refers to any diagnoses, whether based on symptoms, empirical data or the like, whether tentative or definitive. The term "allelic combination" means the same as a "haplotype," i.e. is referring to particular combination of alleles for at least two loci. The term "locus" means a physical location, place or position occupied by a particular gene on a chromosome. The term "NRAMP locus" refers to any DNA or chromosomal segment encoding for a NRAMP or which is structurally or functionally associated with or influencing the expression of any NRAMP gene. The term "related to" refers to a finding that the probability that at least two occurrences happen together or two things co-exist is greater than would be expected by chance alone. The term "susceptibility" refers to a subject having any increased chance or risk, i.e. increased probability, of an occurrence, such as acquiring a disease or condition, over what would be expected by chance alone. The term "screening" refers to any evaluation of a subject for susceptibility to a disease or condition, for example, by identifying any correlation between a genetic profile, an allele, an allelic combination, or a polymoφhism and an increased chance of acquiring a disease or condition, i.e. whether a subject is susceptible to acquiring a disease or condition. The term screening also refers to methods of diagnosis. The terms "identifying" and "detecting" refer to any means of detecting or identifying, for example, including hybridization with specific primers or probes and detection of such specific hybridizations, DNA sequencing, antibody binding and detection, or the like. The detection includes isotopic or non-isotopic detection, such as fluorescent, phosphorescent or radioactive detection, or the use of enzyme labeled-oligonucleotides or of an hybridization protection assay. The term "biological response" refers to any cellular, neurological, chemical, inflammatory, immunologic or pathologic biological response, process or reaction by the subject. The response, process or reaction can be chemical, cellular, neurological, psychological or the like. The phrase "'alter the levels or function of NRAMP" means any alteration in the expression of the NRAMP gene, whether in amount of mRNA or protein or protein function and immunogenicity. For example, an increases in the level of NRAMP expression includes increases in NRAMP expression. The increases can be due to greater rates of mRNA transcription, greater rates of mRNA translation, longer mRNA or protein half-lifes and the like. The phrase "a therapy which alters the levels or function of NRAMP" means any therapy which alters the expression, effective concentration, bioavailability or ability to perform its intrinsic function(s) of the NRAMP protein. For example, a therapy may alter the level of NRAMP gene expression by decreasing or increasing rates or changes in the splicing pattern(s) of mRNA transcription and/or translation. The therapy may shorten the half life of the mRNA or the protein. The therapy may also neutralize the NRAMP' s activity, increase its clearance, compartmentalize it, and the like. The therapy may indirectly affect NRAMPS levels by decreasing the number or activity of cells which secrete NRAMPS, such as macrophages. The term "allele" means alternative form of the gene that occupy the same chromosomal locus, with an alternative gene including any modification or variation of a gene. The term "polymorphism" means the occurrence of two or more forms, such as the different forms of a nucleic acid in individuals of the same species. For example, the different form can be a difference in DNA sequences between individuals due to a substitution, a deletion or an addition. The term "nucleic acid" as used herein means a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A nucleic acid may be either single-stranded or double-stranded. Particularly useful nucleic acids for practicing methods of the invention are genomic DNA, complementary DNA and/or messenger RNA. The assay and kit of the invention employ nucleic acids. Nucleic acid of a subject which is suitable for manipulation, such as for diagnoses or screening, in accordance with the present invention may be derived from any nucleated cell sample, and preferably from peripheral mononuclear blood cells. The phrase "under conditions suitable for formation of a specific hybrid" means any set of physical conditions such as temperature, or chemical conditions such as pH, salt concentration, wherein oligonucleotide probe(s)/primer(s) form a hydrogen bonded, sequence-specific association with or selectively hybridize to, the DNA target sequence such as an allele, to which the probe or primer nucleotide sequence is complementary. Defining such parameters and conditions is routine to one skilled in the art, and for example is described in Sambrook et al. and Mullis et al. , both of which have been incoφorated by reference. The term "amplification" refers to the generation of specific DNA sequences using specific oligonucleotide primers through the use of techniques generally described as polymerase chain reaction (PCR), which are well known in the art, described for example in PCR Technology, Erlich, Ed. , Stockton Press, New York, NY, (1989) incoφorated herein by reference; and Sambrook et al. and Mullis et al.. both of which have been incoφorated by reference. The terms "assay system" means any kit or automated system, any form or combination that the reagents of the methods described herein can be combined, formulated or utilized. The assay system includes any combination of methods and reagents, manual or automated, including robotic systems, for the diagnosis of IBD, specifically CD, and for screening for a susceptibility to CD.

The detecting or identification of the presence or absence of a subject's nucleic acid which encodes NRAMP alleles and/or polymoφhisms associated with IBD, UC, CD and their subtypes may be accomplished by all means known in the art. One of skill in the art will know of the many means available to make such a determination, including electrophoresis, automated sequencing, allele-specific oligonucleotide probing, differential restriction endonuclease digestion, ligase-mediated gene detection, and the like. In terms of their symptomatology, Inflammatory Bowel Disease has been classified into the broad categories of Crohn's Disease (CD) and ulcerative colitis (UC). CD, also called regional enteritis, is a disease of chronic inflammation that may afflict any portion of the gastrointestinal tract, although the most commonly affected are the distal portion of the small intestine (ileum) and the cecum. In some cases, this disease may be confined to the small intestine, colon or anorectal region. CD occasionally involves the duodenum and stomach, and more rarely the esophagus and oral cavity. The variable clinical manifestations of CD are, in part, a result of the varying anatomic localization of the disease. The most frequent symptoms of CD are abdominal pain, diarrhea and recurrent fever. CD is commonly associated with intestinal obstruction or fistula, which is an abnormal passage between diseased loops of bowel, for example. CD also includes complications such as inflammation of the eye, joints and skin, liver disease, kidney stones or amyloidosis. In addition, CD is associated with an increased risk of intestinal cancer. Several features are characteristic of the pathology of CD, such as the inflammation which is often associated with CD, and known as transmural inflammation, involving all layers of the bowel wall. Thickening and edema, for example, typically also appear throughout the bowel wall, with fibrosis also present in long-standing disease. The inflammation characteristic of CD is also discontinuous, and segments of inflamed tissue, known as "skip lesions," are separated by apparently normal intestine. Furthermore, linear ulcerations, edema, and inflammation of the intervening tissue lead to a "cobblestone" appearance of the intestinal mucosa, which is distinctive of CD. A further hallmark of CD is the presence of discrete aggregations of inflammatory cells, known as granulomas, which are generally found in the submucosa. About half of the CD cases display typical discrete granulomas, while others show a diffuse granulomatous reaction or non-specific transmural inflammation. As a result, the presence of discrete granulomas is indicative of CD, although the absence granulomas also is consistent with the disease. Thus, transmural or discontinuous inflammation, rather than the presence of granulomas, is a preferred diagnostic indicator of CD (Rubin and Farber, Pathology (Second Edition) Philadelphia, J. B. Lippincott Company (1994), which is incoφorated herein by reference) .

Ulcerative colitis (UC) is a disease of the large intestine characterized by chronic diarrhea with cramping abdominal pain, rectal bleeding, and loose discharges of blood, pus and mucus, and of widely varying manifestations, a pattern of exacerbations and remissions typifies the clinical course of most UC patients (70%), although continuous symptoms without remission are present in some patients with UC. Local and systemic complications of UC include arthritis, eye inflammation such as uveitis, skin ulcers and liver disease. In addition, UC and especially long-standing, extensive disease is associated with an increased risk of colon carcinoma.

In comparison with CD, which is a patchy disease with frequent sparing of the rectum, UC is characterized by a continuous inflammation of the colon that usually is more severe distally than proximally. The inflammation in UC is superficial in that it is usually limited to the mucosal layer and is characterized by an acute inflammatory infiltrate with neutrophils and crypt abscesses. In contrast, CD affects the entire thickness of the bowel wall with granulomas often, although not always, present. Disease that terminates at the ileocecal valve, or in the colon distal to it, is indicative of ulcerative colitis, while involvement of the terminal ileum, a cobblestone-like appearance, discrete ulcers or fistulas suggest CD. Characteristics that serve to distinguish CD from UC are summarized in Table 1 below, which is taken from Rubin and Farber, supra (1994).

As used herein, a patient afflicted with "Crohn's Disease" is synonymous with the phrase a subject diagnosed with an "Inflammatory Bowel Disease" , where the IBD is CD, and means a patient having a characteristic feature from at least two of the following categories: clinical, endoscopic, radiographic and histopathologic. As used herein, a characteristic clinical feature is perforating or fistulizing disease; or an obstructive symptom secondary to small bowel stenosis or stricture. As used herein, a characteristic endoscopic feature is a deep linear or serpiginous ulceration; a discrete ulcer in normal-appearing mucosa; cobblestoning; or discontinuous or asymmetric inflammation. As used herein, a characteristic radiographic feature is segmental disease (skip lesion); a small bowel or colon stricture; stenosis or fistula. As used herein, a characteristic histopathologic feature is submucosal or transmural inflammation; multiple granulomas; marked focal cryptitis or focal chronic inflammatory infiltration within and between biopsies, 01 a skip lesion, including histologic rectal sparing in the absence of local therapy. Patients with chronic inflammatory bowel disease are generally characterized as having either Crohn's Disease or Ulcerative Colitis to describe specific patterns of disease, to predict outcomes based on expected natural histories, and to help guide medical and surgical treatment strategies. Clinical, endoscopic, and histopathologic criteria, as discussed above, have been developed to classify patients into one or the other category However, overlap between CD and UC also has been demonstrated at a variety of levels by clinical, immunological and genetic studies, for example. Furthermore, CD and UC each can encompass a number of distinct conditions affecting the gastrointestinal tract, with different clinical subtypes being classified together as CD or UC because they present with similar symptoms.

One embodiment of the present invention is directed to the discovery of genetic correlations that will help diagnose such clinical UC or CD subtypes by identifying a NRAMP locus allele or allelic combination or an NRAMP polymoφhism that is correlated with a biological response related to a inflammatory bowel disease. The invention further provides a method of determining whether a therapy which alters the level or of NRAMP is also effective m treating a specific type ot Inflammatory Bowel Disease.

In summary, the present invention provides non-invasive methods to diagnose, screen for, and distinguish clinical subtypes of CD from UC, as well as for ascertaining whether a specific therapy directed to one of these diseases or conditions is effective, and a kit to practice the methods. The inventors have demonstrated, using non-parametnc linkage methods, that there is a genetic linkage of UC to the NRAMP locus. Non-parametnc statistics are statistical calculations that are based on no prior assumptions with respect to the variable and the probability distribution of the data. Previously, a "genetic association" between the NRAMP locus and Chron Disease (CD) had been shown by one of the inventors by means of a study of a population of unrelated subjects No coi ι elation was shown in that study between the NRAMP locus and UC The examples demonstiate an analysis of a large number of Caucasian multiplex families by several non-parametnc linkage methods supporting linkage of UC to the NRAMP locus. The results demonstrate that at least one gene involved in the pathogenesis of UC is located on the long arm of chromosome 2, which does not contain the human major histocompatibility complex (MHC), another aiea of the genome which has been linked with IBD. The present invention allows the screening for UC by detecting the presence or absence in a subject's nucleic acid of segments encoding NRAMP alleles and polymorphisms associated with IBD, CD, or UC, wherein the presence of nucleic acid encoding specific alleles or the poly oiphisms is indicative, i.e. predictive, of a specific disease The alleles may be NRAMP microsatellite alleles, and a presently preferred NRAMP microsatellite allele associated with UC includes the D2S434 NRAMP microsatellite allele. Further nucleic acids encoding NRAMP alleles and polymoiphisms associated with UC and the other diseases or conditions may be identified or detected in accordance with the present invention by amplifying the nucleic acid and identifying the NRAMP alleles and polymoi phisms by assaying the subject's nucleic acid for NRAMP polymoφhisms or alleles associated with UC or other dιsease(s) or condition(s) and comparing the results to positive and/or negative controls Among the defining characteristics of the nucleic acids encoding NRAMP alleles and polymorphisms associated with UC are, for example, size, sequence, type of sequence repeats, and the like One skilled in the art would be able to isolate and sequence DNA from any legion of the chromosome, such as from the functional and satellite DNA of the NRAMP locus One skilled in the art would be able to identify other primers suitable for use in amplifying and sequencing any particular nucleic acid using published sequence databanks. For example, the map and sequence of the human NRAMP locus is available from GENBANK as part of the human genome project, NCBI, NIH, easily searchable, for example, at http://www.ncbi.nlm.nih.gov. The relevant portion of the NCBI's database is herein incoφorated by reference. Examples of primers suitable for use in hybridizing, amplifying or sequencing nucleic acid encoding NRAMP locus DNA, NRAMP alleles, particularly the D2S434 allele. are also provided by GENBANK and Hofmeister et al., Surgery 122(2); 173 (1997), the relevant portion of which is incoφorated herein by reference. Methods for identifying, isolating, manipulating, sequencing and analyzing DNA from patient samples are well known in the art, for example (See, Molecular Cloning, 2nd.. Ed., Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, Plainview, NY (1989); The Polymerase Chain Reaction, Mullis, et al., Eds. , Maple Press Co., York, PA (19 ), the relevant portions of which are incoφorated herein by reference. An allele or polymorphism at a locus, such as the NRAMP locus, may be identified or detected by a variety of methods including assays using the polymerase chain reaction (PCR). Oligonucleotide hybridization, such as allele-specific hybridization (See, Mullis et al., above), denaturing gradient gel electrophoresis (See, for example, Irmis et al., Eds., PCR Protocols: a Guide to Methods and Application, Academic Press, Inc. , San Diego, CA (1990)) and restriction fragment length polymorphism based methods (Sambrook, supra, (1989)), which are well known in the art and encompassed within the invention. The human NRAMP locus micro satellites were mapped, characterized and reported by Murray et al.. Cooperative Human Linkage Center (CHLC), Univ. of Iowa, Dpt. Pediatrics, Iowa City, IA 5542. and GENBANK, the relevant portions of which are incorporated herein by reference. These micro satellites are characterized by a series of GACT tetranucleotide sequence repeats. For a more detailed description of the NRAMP microsatellite loci and the NRAMP locus, see Murray et al. and Hofmeister et al. and Table 1, above, which depicts the location of several alleles with respect to the NRAMP microsatellite loci at the NRAMP locus. Primer/probe pairs suitable for use in the practice of the present invention are linear oligonucleotides ranging in length from about 8-10 to about 30-60 nucleotide in length. One of the primers/probes in the pair should be complementary to a nucleotide sequence upstream of the nucleic acid encoding the NRAMP locus targeted for amplification, whereas the other should be complementary to a sequence located down stream of this target site. Suitable primers for use in the present invention are specific for amplification of the nucleic acid encoding an NRAMP locus and, generally, do not prime amplification of nucleic acid which does not encode the NRAMP locus. Examples of primers/probes are provided in the examples, which were applied in the experimental representation of this invention. The sequences of the probe/primer pairs may be separated by as many nucleotide as the PCR technique and the other technique(s) for detecting the presence or absence of NRAMP alleles or polymoφhisms associated with UC will allow, provided that appropriate controls are used. For example, if the presence or absence of nucleic acid of a subject encoding the NRAMP locus associated with UC is detected on the basis of size, then the primers used for amplification must not include amplification of nucleic acid flanking the allele which would interfere with the ability to detect polymoφhic size differences, for example by inclusion of polymorphic size differences which may be present in regions flanking the NRAMP locus. Primers suitable for use in amplifying nucleic acid encoding the NRAMP locus may be constructed using the oligonucleotide primer sequences described by Murray et al., above, and deposited with the CHLC and GENBANK, and the map and sequence of the NRAMP locus available from GENBANK, NIH, the relevant contents of which are incoφorated herein by reference.

This patent also provides an Inflammatory Bowel Disease kit with nucleic acid comprising a DNA encoding functional or satellite regions of a Natural Resistance-Associated Macrophage Protein (NRAMP) locus, oligonucleotides thereof and anti-sense nucleic acids thereto, and instructions for use of the DNA in accordance with this invention to find further alleles, or to determine whether a specific sample's DNA matches available controls. In a preferred embodiment, the nucleic acid comprises a portion of a functional region of an NRAMP locus, and still more preferred within the functional region of the NRAMP locus comprises a portion of the 5' regulatory region of an NRΛMP locus. In another preferred embodiment, the nucleic acid comprises a portion of a satellite region to an NRAMP locus, and more preferably a satellite region vicinal to either the 3' or 5' end of the NRAMP functional region or both. The kit may, in addition, include means for amplifying the nucleic acid, such as enzymes, buffers, and other necessary components. The oligonucleotides provided with the kit may be about 8 to 60, preferably about 12 to 30, and still more preferably about 15 to 20, nucleotide long. However, oligonucleotides of lesser or greater length may also be utilized. In a most prefeπed embodiment, the kit is directed to Ulcerative Colitis (UC). and it contains a nucleic acid comprising a DNA corresponding to the d2s434 allele. The kits provided here are suitable for screening for IBD, including CD and UC, and for distinguishing the one from the other as well as setting apart different subtypes of these diseases. The present kit may include all or some of the positive controls, negative controls, reagents, primers, sequencing markers, probes and antibodies described herein for determining the presence or absence of nucleic acid encoding NRAMP alleles or polymorphisms associated with the diseases involved. As described above, the kit of the invention may contain, for example, nucleic acid encoding NRAMP alleles or polymoφhisms associated with UC (or CD or their subtypes), nucleic acid encoding the functional and/or satellite portions of the NRAMP locus or segments thereof, including NRAMP alleles not known to be associated with UC (or CD or their subtypes), nucleic acid sequences of NRAMP alleles or NRAMP polymorphisms, labeled or unlabeled oligonucleotide probes specific for particular a NRAMP locus allele or polymorphism, primers for amplification of nucleic acid encoding NRAMP such as NRAMP alleles or polymorphisms, reagents commonly used for DNA or RNA amplification, polymerases such as DNA or reverse transcriptase DNA polymerase, restriction endonuclease enzymes, antibody specific for, or which binds particular NRAMP alleles or NRAMP polymoφhisms, and others.

The kit components may be packaged in a manner customary for storage, shipment and use by those of skill in the art. For example, the kit components may be provided in solid and/or lyophilized and/or vacuum dried form, in solution, as a liquid dispersion, and other available forms. The kit may be provided in the form of a semi-automated or entirely automated systems, including a robotic system, for the diagnosis and/or evaluation of a susceptibility to IBD disease, and more particularly to establish a difference between UC and CD and/or their subtypes, a presently preferred embodiment of the of the kit is for use in screening for UC and/or distinguishing CD from UC, and it employs DNA corresponding to the D2S434 allele, and may also contain DNA encoding other NRAMP alleles or polymoφhisms associated with UC and/or CD. The components of the kit may be provided in a buffered solution, e.g., a Tris-EDTA buffer solution, which is preferably kept at 4°C and/or lyophilized until ready for use. Other NRAMP alleles may be utilized by themselves in the kit or in combination with the D2S434 allele. In yet another preferred embodiment, the kit also contains sequencing markers ranging in size from about 80 to 200 base pairs, although other size markers may also be included as needed.

The invention will now be describe in greater detail by reference to the following examples, which are intended to illustrate, without limiting, the present invention.

EXAMPLES

Example 1: Source of Human Nucleic Acid

Nucleic acid was extracted from peripheral blood lymphocytes. Genomic DNA for a large number of Caucasian multiplex families (two or more siblings) from Southern California was obtained from the IBD cell bank at Cedars-Sinai Medical Center, Los Angeles, CA.

Example 2: DNA Markers

The microsatellite D2S434 and D2S1323 DNAs were obtained from a public database. (Cooperative Human Linkage Center by Murray et al. , the University of Iowa, Department of Pediatrics, Iowa City, IA 52242, USA, GENBANK.) They were chosen as markers for NRAMP due to their close linkage to DNA markers D2S104 (recombination fraction (θ) = 0 for D2S434, θ =0.06 for D2S1323) and D2S173 (θ = 0, θ=0.06 for D2S434 and D2S1323, respectively), which had previously been mapped within 150,000 base pairs of the NRAMP gene, and for their ease of genotyping (CHLC and GENBANK; see also Liu et al. Am. J. Hum. Genet. 1995; 56:845-53.) Physical assignments of NRAMP, D2S173, and D2S104 placed all three loci within a 150,000 base pair in the yeast artificial chromosome. Linkage assignments of D2S173, D2S104, D2S434, and D2S1323 placed all four loci within 0.06 centimorgans. The data are shown in Table 2 below.

Table 2: Physical and Linkage Assignments of NRAMP and Related Loci

Physical assignment 1.5 Mbp NRAMP D2S173 D2S104 centromere telomere

Linkage assignment < 0.1cM NRAMP D2S173 D2S104 D2S434 and D2S1323

Example 3: PCR Conditions for DNA Amplification

The PCR reactions were carried out in a 10 μl volume containing 100 ng of genomic DNA (see Example 1), 70 μM of each dNTP, 1.0 μM of each primer, 2-2.5 U Taq DNA polymerase, 1 μCi of [³²P]α-dCTP (3000 Ci/mmol), 1.5 mM MgCl₂ and magnesium-free storage buffci (5 mM KCl, 1 mM Tris-HCl, 1 % Triton^*; Promega Corporation, Madison WI). All reactions were carried out for 25 cycles (94°C for 30 s, at an annealing temperature of 53 °C [D2S1323] or 57°C [D2S434] for 30s, and 72°C for 1 min). The thus obtained PCR products were resolved by polyacrylamide gel electrophoresis (PAGE). The band shifts observed on autoradiographs were scored by two independent observers unaware of patient identity or the group being analyzed. The D2S434 PCR reaction produced nucleotide fragments of recognizable lengths, and the D2S1323 PCR reaction also produced nucleotide fragments of recognizable lengths.

The linkage of the NRAMP markers with Ulcerative Colitis was tested as described in the examples below.

Example 4: Statistical Program: Sibpal Program In The SAGE Package

The SIBPAL program in the SAGE package utilized for the affected sib pair analysis was S.A.G.E. (1994), Statistical Analysis for Genetic Epidemiology, Release 2.2 (Department of Epidemiology and Biostatistics, Rammelkamp Center for Education and Research, MetroHealth Campus. Case Western Reserve University, Cleveland, OH). A two point linkage analysis was performed using the SIBPAL subroutine program (version

2.7) of S.A.G.E. See, Tran, L.D. , Elston, R. C, Keats, B. J. B. , and Wilson, A. F. (1994), Sib-pair linkage program (SIBPAL) User's Guide (Part of the S.A.G.E. Release 2.2 documentation (2.7 SIBPAL update), Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH). This computerized sib-pair analysis program applies the algorithm described by Amos with modifications. It estimates the proportion (π) of alleles the sib-pair shares identical-by-descent at that locus. See, Amos, C. I. (1988), Robust Method for Detection of Genetic Linkage for Data from Extended Families and Pedigrees, Ph. D. Dissertation, Louisiana State University Medical Center, New Orleans, LA.

The alleles were given numerical assignments, beginning with 1, for the smallest PCR- amplified nucleotide fragment. Seven alleles were observed with D2S434 (1-7) and two alleles with D2S1323 (1 & 2) in the tested population. Allele frequencies were similar among all groups for both markers. The observed mean proportion of alleles shared by sib-pairs was compared separately for concordant affected, concordant unaffected, and discordant sib-pairs with expected 0.5 by z-test. If the linkage between disease and locus exists, we expect to observe a significant increase of π in both concordant affected and concordant unaffected groups, but a alter of π in the discordant group.

In this program, when the parents were not informative, an estimated proportion of allele sharing was used in the test. For example, if one parent was heterozygous and another parent was homozygous and both affected sibs had identical haplotypes, the haplotype from the homozygous parent had 50% probability being identical-by-descent and 50% probability not identical-by-descent from the same ancestral chromosomal. In such case, an average sharing is estimated, i.e. (0.5x1 +0.5x2)/2=0.75. Therefore, in this analysis, all sib-pairs are utilized , including affected concordant, discordant, and unaffected concordant, and fully informative and partially informative.

Example 5: Statistical Method: Linear Regression Linkage Test

A linear regression analysis was also conducted with the SIBPAL program to assess the prediction of the differences in disease status according to the degree of sharing th marker alleles. See, Example 4 above. In this method, all types of sib-pairs are used and the affected individuals have a trait value 1, while the unaffected have a trait value zero. The y-axis is the squared difference between the pairs (Y) and the x-axis is the proportion of haplotypes shared identical-by-descent between the pairs (π). For a qualitative trait, Y varies between 0 and 1 , that is Y = 0 for sibs with the same trait value, i.e. both affected and unaffected, and Y = 1 for sibs with different trait values. The proportion of haplotypes shared identical-by-descent between the pairs (π) ranges from 0 to 1. that is 0 for sharing none, 0.5 for sharing 1, 1 for sharing two, and the values in between are derived from estimation by using noninformative parents.

If there were no linkage, a horizontal line would be observed, indicating that the degree of clinical concordance would be independent of haplotype sharing. A significantly negative regression coefficient β of the dependent variable (phenotype) Y on the independent variable (genotype) π was taken as evidence for linkage. That is, the more alleles that are shared by a sib-pair, the more alike their phenotypes are. This inverse relationship is expressed by a negative regression slope in this analysis.

Example 6: D2S434 NRAMP Satellite Allele Seven alleles of the NRAMP satellite marker were studied in the multiplex Caucasian families with two or more sibs affected with either Ulcerative Colitis (UC), Crohn's Disease (CD) or with both. The D2S434 alleles are located on chromosome 2q. The base pair sequence of the satellite region corresponding to the NRAMP marker D2S434 was deposited with the Cooperative Human Linkage Center by Murray et al., the University of Iowa, Department of Pediatrics, Iowa City, IA 52242, USA, and with GENBANK, and is provided in Table 3 below.

Table 3: D2S434 NRAMP Allele Sequence

AAAGCTCTGG GGCCTTGAGT AAATCACTAG CCTTTGCCGG TCTCJACTT TCTTATCTCA

61 GGAATACACC ACCAGCCCTT GCTACTCTGC AGAACTTCGT AGAAGAACAT GTAAGAGTAT 121 GGATGTGGAA ATCATAGCCA ACTTCCTACT TGGTGGGATA CCTG.JAGAAG ACTCAGA3AT

181 GGCAGATAGA TAGATAGATA GATAGATAGA TAGATAGATA GATAGATAGA TAGATAGATA

241 GATAGAAAGA AAAACAGATC ATCTCCCAGA TTTCTGGGAA CAGTΛCAGAT GGGAGATGTT

301 CTTCCTCAGT TTACTCATGA GCATAGTCTT ACTTTTAAGG CGGTΛTT ( SEQ . ID NO : l )

The D2S434 allele is characterized by repeats of (GATA)„. where n is the number of repeats of the tetranucleotide units. Seven alleles were identified, ranging in size from 262 base pairs to 286 base pairs. The allele fragment sizes for D2S434 are shown in Table 4 below.

Table 4: D2S434 Allele Fragment Sizes Allele No. Fragment Size

1 262 bp

2 266 bp 3 270 bp 4 274 bp 5 278 bp 6 282 bp 7 286 bp

In the present linkage studies, the GATA4G12 (d2S434) DNA segment of allele 1 was utilized for the PCR amplification of microsatellite DNA. This segment has 262 base pairs. Two primers, A and B, were obtained based on the sequence of this probe, and were designed so that they would hybridize to sequences upstream and downstream of the desired or target NRAMP satellite DNA. The two primers have the sequences shown in Table 5 below.

Table 5: D2S434 Primer Sequences

Primer A: TAAATCACTAGCCTTTGCCG (SEQ.ID NO:2) Primer B: GCCATCTGTACTGTTCCCAG (SEQ.ID NO:3)

Example 7: 2DS1323 NRAMP Satellite Allele

A second microsatellite region near the NRAMP locus, D2S1323, was studied in the same population of multiplex Caucasian families with two or more sibs affected with either Ulcerative Colitis (UC) or Crohn's Disease (CD). The D2S1323 marker is located on chromosome 2q. The sequence of the satellite region corresponding to NRAMP marker D2S1323 was deposited with the Cooperative Human Linkage Center by Murray et al., the University of Iowa, Department of Pediatrics, Iowa City, IA 52242, USA and is shown in Table 6 below.

Table 6: D2S1323 NRAMP Allele

1 GGGACTACAG GCACCCACCA CCACGCCCGG CTAATTTNGT TTTTGTATTT TTAGTAGAGA

61 AGGAGTTTCA CTGTGTTAGC CAGGATGGTC TTGATCTCCT GATCTTGTGA TΌOGCCCACC

121 TTGGCCTCCC AAAGTGCTGG GATTAC TGG GTGAGCCACC GCAC CGGCT GTCAGGAGTT 181 TTTTGTATGT TTCCATGTTT CTCTTCACAT TCAGCACCAG AATGGTCTGG GAAGCCATGG 241 AGTAATGTGG TTATGGGAGG CATCTTCTTA GT^{^}ACTCATT CAT1 TTCA TTCATTCATT 301 CATTTATTCA TTCATTTGCT CATTCATTCA TT ^{^}CACTAAC TTAT' TGATT CAGGAGACAG 361 AAAAGACTTG CTAATGCAGT GCCTGGCAAA TAAGTGGTGC TCCACAAATA TTTGTTGAAT 421 TAATGATTGG ATGAAGGGAA ATGCTATAAA GA-.AAACCTT TAAT^"A GGA ACTCTTTTGA 481 CATTATATCA ATATTCAATC TGCTACATTT AGAACCTGCC AGAG TCTCTG CIGCAAATTC 541 TATTTCGAAG GCCGCAACAA CTTGGCCAGG AA^" ACATTAC CACT AAATT TTGCGIAGAC 601 ATCCAC (SEQ.ID NO:4)

The D2S1323 marker is characterized by repeats of (GATA)„, where n is the number of repeated tetranucleotide units. Two alleles were identified, one of 324 base pairs and one of 328 base pairs. The allele fragment sizes for D2S1323 are shown in Table 7 below.

Table 7: D2S1323 Allele Fragment Sizes

Allele No. Fragment Size

1 324 bp

2 328 bp

In the present linkage studies, the GAAT1B02 DNA segment of allele 1 was utilized for PCR amplification. Allele 1 has a fragment size of 324 base pairs. Two primers, C and D, were obtained based on the sequence of this probe, and were designed so that they would h\ bridize to sequences upstream and downstream of the desired or target NRAMP satellite DNA The primers have the sequences shown m Table 8 below

Table 8: D2S1323 Primer Sequences

Primer Sequence

C ACATTCAGCACCAGAATGGT (SEQ ID NO 5)

D ATTTGCAGCAGAGACTCTGG (SEQ ID NO 6)

Example 8: Isolation & Identification of Patients' Alleles

The primers were individually reacted with the DNA isolated from the patients and amplified by PCR methods standard in the art See, for example Maniatis et al , supra After completion of the PCR cycles, the reaction was stopped, and the DNA isolated by methods known in the art See, Maniatis et al , supra In addition, the assessment of the level, sequence and length of mRNA and NRAMP protein is also undertaken by methods known in the ait, as is the detection of the NRAMP protein with antibodies, and the assessment of its function See, for example, Maniatis et al , supra

Example 9: Non-parametric Statistical Analysis Shows Linkage of an IBD to the NRAMP Locus A genetic association in CD with two poh morphic miciosatelhte maikei s at or near the

NRAMP locus on human chromosome 2 away from the MIIC locus was previously leported by one of the present inventors A simple showing of a "genetic association" is unable to distinguish whether the NRAMP region actually contains genes that contnbute to the pathogenesis of UC or whether the "association" results from simple population stratification The present linkage study, utilizing several non-parametnc linkage methods, was performed to distinguish between these two possibilities This study tests whether a disease co-segregates in families with the marker locus and, therefore, is independent ot specific alleles T he genetic markers tested include D2S434 and D2S1323 which were withm 1 mb of the NRAMP gene on chromosome 2 Interestingly, the present study found a statistically significant linkage for one of the alleles with UC, not CD

Genomic DNA from Caucasian multiplex families (two or more siblings) from Southern California was obtained from the IBD cell bank at Cedars-Sinai Medical Centei , Los Angeles, CA NRAMP microsatellite alleles at tw o loci, D2S434 and D2S1323, weie typed by polymerase chain reaction (PCR) See, Plevy, et al , PCT US95/06107 WO 95/31575, the relevant portion of which is herein incoφorated by reference As indicated in Table 2 above, the tested genetic markers D2S434 and D2S1323 are within 1 Mbp of the NRAMP gene The alleles were interpieted independently by two investigators blinded to diagnosis and family pedigi ees Since there is no clear pattern of Mendehan inheritance for UC or CD, non-parametnc hnkaαe analyses which do not assume a mode of inheritance were employed A statistically significant linkage between the maiker D2S434 and Ulceiative Colitis was observed The mean proportion of alleles shaied m affected concordant sibpans (n = 19) was 0 61 (p=0 022), in discordant sibpairs (n = 57) was 0 48 (p =0 29), and in concordant unaffected sibpairs (n=49) was 0 55 (p=0 09) Figure not shown

Furthermore, a linear regression analysis of the phenotypic difference -\ ersus the proportion of alleles shared by identity-by-descent yielded a slope of -0 30 (p=0 039) In contrast, no linkage was found between this region and the Crohn's Disease population The vertical axis represented the difference between the phenotypes between the sibs (Figuie not shown) If the sibs have the same phenotype the difference is zero, whereas if they have diffei ent phenotypes the difference is one If there is linkage between a disease and the marker, e g gene or allele, the diffeience would be small (it would approach zero) These results identify a statisticalh significant linkage between UC and the NRAMP locus

Thus, an analysis of Caucasian multiplex families by several non-parametnc linkage methods demonstrated a statistically significant linkage between UC and the NRAMP locus

Example 10: D2S1323 Shows No Independent Linkage to UC

A second NRAMP polymoiphism, D2S1321 was also found in the satellite region of the NRAMP locus withm 1 mb of the NRAMP locus NRAMP haplotype D2S 1323 w as found by the

32 length of the PCR amplified genomic DNA with hybndization of P '-end labeled allele-specific oligonucleotides, as described in Plevy, S E , et al , PCT US95/06107, WO 95/31575 which is herem incoφorated m its entirety by this reference This marker with two alleles was studied by the non- parametnc statistical analysis of Example 4 above, in the same population of Caucasian multiplex families from Southern California The results indicated that the marker D2S1323 gave little or no information in most families This maiker, therefoie, appeals to be unmtormative

Example 11: Sib pair Analysis foi D2S434 and D2S1323 Haplotypes and UC

The linkage between haploty pes and ulceia e colitis w as studied in a population of Caucasian multiplex families from Southern California as dtsu ibed above The results obtained for both haplotypes show that among concordant sibpairs the mean piopoition of alleles sh ued (n = 18) was 0 61 (p=0 03), in discordant sibpairs (n = 57) it was 0 54 (p = 1 0), and in concordant unaffected sibpairs (n=41) it was 0 64 (p=0 002) Thus the D2S434 & D2S1323 haplotypes in aflected and unaffected concordant sibpairs exhibited results consistent with linkage

Example 12: Sib pair Analysis for D2S434 and D2S1323 Haplotypes and CD In a study similar to that described for Example 13 the linkage between haplotypes and

Crohn's Disease was studied in a population of 108 Caucasian multiplex families from Southern California (90 CD, 40 UC and 22 that had both) 1 he results \ lelded by the linkage analysis show that among concordant sibpairs the mean piopoition of alleles sh ired (n = 64) was 0 14 while in discordant sibpairs (n= 128) it was 0 51 , and in concordant unaffected sibpan s (n=41) it was 0 54 (p^O 002) These results show little evidence of linkage with the D2S434 & D2S1323 h iplotypes and Crohn's Disease

Example 13: Isolation of Nucleic Acid from Patients' Samples

A number of samples fiom individuals with the D2S434 and D2S1323 NRAMP genotypes/haplotypes associated with disease phenotvpes eie analyzed for altciations in the coding sequence expressed in circulating while blood cells See Hotmeistei et al Suιguyl22 (2) 173 (1997), the entire contents of which are incorporated herein by reference to enable the patients' and allele information.

White blood cell samples were obtained from the patients described in Hofmeister et al. (1997), from the Liver Transplant Unit at the Mayo Clinic, Rochester. MN. The cell pellets were maintained at -70°C and transported on dry ice until prepared for analysis. The white blood cell pellets were used to simultaneously isolate DNA and RNA using TRIzol reagent (Gibco, BRL Catalog #15596-018) per manufacturer's directions. The quantity and purity of the RNA and DNA samples were analyzed by spectrophotometry .

Example 14: Reverse Transcription of Patients' RNA The RNA isolated from these samples was subjected to a reverse transcriptase-polymerase chain reaction (RT-PCR). Each RNA was analyzed in an RT-PCR using primers specific for NRAMP. The primers used are shown in Table 9 below.

Table 9: Primers Sequences & NRAMP Gene Locations Primer Sequence SEQ.ID NO: Location

ADNRAMPU 5 ' -TGG ACC CAG GAA ACA TCG bp65 ADNRAMPD 5' -CAA GGA GCC CAT ACA GGA bpl451

These primers were designed based on the previously published NRA P sequence (Accession

Number D38171, GENBANK - CHLC).

The RT-PCR is a 50 μl one rube RT-PCR and was performed according to the manufacturer's instructions (Boehringer Mannheim, Titan RT-PCR, catalog #1855476), and consisted of supplied components with 2 μl of total RNA, 20 pmol of each primer, and 1.5 mM MgCL, added. In this instance the antisense primer (ADNRAMPD) was used as the gene specific primer for the RT. The

Reaction was performed in a Perkin Elmer 9600, as shown in Tables 14-16 below.

Table 10: Initial PCR Cycles & Conditions

Temperature Duration Number of Cycles (°C) (min) (n)

50 30 min 95 2 min

The following three steps make up one cycle, which was repeated 20 times as shown in Table

11 below. Table 11: PCR Conditions for Next 20 PCR Cycles

Temperature Duration Number of Cycles

20

94 30 sec 59 30 sec 68 3 min 40 sec lowered by 5 sec every cycle

The following three steps make up one cycle, the cycle is repeated 15 times as shown in Table

12 below.

Table 12: Final PCR Cycle & Conditions

Temperature Duration Number of Cycles

15

94 30 sec 59 30 sec 68 2 min

68 7 min

This RT-PCR yields an expected amplification fragment of 1404 bp. After amplification 20-30 μl of the reaction mixture was electrophoresed on a 1 % agarose gel containing ethidium bromide. Products were visualized with a UV light source and documented by photography. Using control samples, a single positive band of the expected size was seen. However, clinical samples analyzed contained multiple bands with the most prominent band greater than 400 bp larger than predicted. Remaining material from the RT-PCR was used to clone the fragments. The fragments were cloned using the TA Cloning Kit (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. The isolated clones were sequenced at the Mayo Core Facility.

Example 15: Three Mutated NRAMP DNA Sequences & Wild Type NRAMP DNA Sequences

The following are 3 DNA sequences which wei e obtained from various patients. Their sequences differ from the NRAMP DNA sequence as show n in Tables 13-15 below. Table 13: Mutated and Non-mutated NRAMP DNA Sequences

Sequence A and Wild-type NRAMP

GAATCGGCCpATGTGAACCGAATGTTGATGTAAGAGGCAGGGCACTCGGCTGCGGATGGGTAACAGGGCGTGGGCTGGCA, 10 20 30 40 50 60 70 80

CACTTACTTGCACCAGTGCCCAGAGAGGGGGTGCAGGCTGAGGAGCTGCCCAGAGCACCGCTCACACTCCCAGAGTACCT,

90 100 110 120 130 140 150 160

GAAGTCGGCATTTC-AATGACAGGTGACAAGGGTCCCCAAAGGCTAAGCGGGTCCAGCTATGGTTCCATCTCCAGCCCGAG

170 180 190 200 210 220 230 240

CAGCCCGACCAGCCCAGGGCCTiCGGCAAGCACCTCCCAG GAGACCTACCTGAGTGAGAAGATCCCCATGCCAGACACAA

250 260 270 280 290 300 310 320

AACCGGGCACCTTC^GCCTGCGGAAGCTATGGGCCTTCACGGGGCCTGGCTTCCTCATGAGCATTGCTT CCTGGACCCA,

330 340 350 360 370 380 390 400

10 20 30 CO 50 60

! I I I I I

ATCAGNCrTC^ySCTGrcGCCGTGGCGGGAπ GδAAA ΛT GAGTCΛGATCT O^GGCTGGCGCCG GGCG^

I I 1 I I I I I

410 420 430 440 450 460 470 480

GGGCΓTGCTCTGCCACKXΣACICSΠΌCAC^

I I 1 I I ! I I

«90 500 S10 520 530 540 550 560

ISO 160 170 180 190 200 210 220

I I I I I I 1 I

ACCC^JNGGTGCCXrG ΛCCGTCCTCTGGCT

ACCcτAAGcrιcccccGCACc<rrccτc α»rrcACC^

I I I I I I 1 I

570 580 590 600 610 620 630 640

230 240 250 260 270 280 290 300

I I I I I I I I _

ACGGCCATIC«TTCAAT GC CTΑUKRRGGACGAA CCC^^

ACGGCCAITGCATIOA CTGCTCTCAGCΓGGACGAATCC^

I I I I I I I I

650 660 670 630 690 700 710 720

390 400 410 420 430 440 4S0 460

I I I I I I I I

CCTTTGCCrATC.AGTA!Kn JrGGC&3^^

I I I I I I I I

810 820 830 840 850 860 870 880

I 890 I900 I 910 I 920 X930 I940 I950 I 960 550

I

CCTGGTCAAC

CCTGCTCA3u?TCICGAGACATACACCSGGCrcGCX^^

I I I I I I I I

970 980 990 1000 1010 1020 1030 104O

αA α3cccτCTcα rcα rAτcA ^

OU» TGCσrrC-^CΛTCrGTGCC*ACAGCAGCCTCCACGACT

1130 1140 1150 1160 1170 1180 1190 1200

OTG ^TπACCAKXXΪGGCGTGATCCTGGGC^^

I I I I I I I I

1210 1220 1230 1240 1250 1260 1270 1280

560 570

GG ICTTCCTGAOG ICTGCGGTββ

T»KGGCTGGGCXGAK5\ ACCATGACGGGCACCTA

I I I I I I I I

1290 1300 1310 1320 1330 1340 1350 1360

740 750 760 770 7B0 7S0 800 810

I I I I . I I I I

TCACCA 3flX CCACCCTαTGCAGGAσTT^ TCACCAOΛTCCCCλCCCTCATGCA«ACTT^

I I I I I 1 I I

1530 1540 1550 1560 1570 1580 15S0 1600

GTCTGCGCOO λACCTCTACl GTGGTC^iGCT

I I f I I I I I

1610 1620 1630 1640 16S0 1660 1670 1680

900 510 920 930 940 950 960 970

I I I I I I I I

GCTGGCCGCλα TATC GGGCCTCACκACCT^ GCTGGCCGCACCCTACCTGSGCCT^X-^^

I I I ! I I I I

1690 1700 1710 1720 1730 1740 1750 1760

980 990 1000 1C10

I I • I I

A^GCTCCl-J ACC^TICCTtfJ'ATGG<λ'l'- ri ; (SEQ.ID NO: 10)

AC GCTCCCACCAtt^ΥlU:i\πAT«^^^^^

I I I I I I I I _Λ

1770 1780 1790 1800 1810 1820 1830 1840

<sccroσcrrcGGAGτακAτCTAT(yutx^^ ^•

I I I I I I I- I

1850 1860' 1870 1880 1890 1900 1910 1920

U3C U40 I960 960 1970 198C 1130 2000

ttlO 3030 2090 3040 2050 20C0 3β?0 30*0

2»70 JICO 2190 MOO '22*0 ^l _iΑζ '333,, ^

ΪT» ^T^ττMCTΛTCTλTTTfcrπAM^TITCAGV»ΘOQΛ7Λ903TC

2450 »ft> 2270 UtO 229D 33O0 2310 2120 4330 23 0 Hit '23βC 2Ϊ7D 3380 2390 24CC

ττe ΛCJ^τcoo«χym:τccAi CC αrτx!r^

34M 2500 251C 2530 2S30 2540 2 3 35⁽0

WJJ MMX (SEQ.ID NO: 9)

3570

N represents undetermined nucleotide.

Top sequence is mutated NRAMP.

Lower sequence is wild type NRAMP.

The mutant sequence contains a deletion at 970 to 1340 with respect to NRAMP (GENBANK).

Table 14; Mutated and Non-mutated NRAMP DNA Sequences

Sequence B and Wild-type NRAMP Fragment

GAATCGGCCGATGTGAACCGAATGTTGATGTAAGAGGCAGGGCACTCGGCTGCGGATGGGTAACAGGGCC 3j ^{'■ '}TTTTGGGCG' GCTGGC. 10 20 30 40 50 60 70 0

CAC-πA=TT∞CCλC7G CCλGM3AGGGCrø^

I I I I I I I I

90 100 110 120 130 140 150 160

ty*CTCGGCΛ7TrCAAT(y£A«zrGACλAG»π^

170 B0 190 ^l200 210 220 230 240

CAGCCMACCACCTCfcGGGCαcαKAAeCACCrCCttCau»αW

I I I i I I 1 1

250 260 270 280 290 300 310 320

10 20 30 40 50 60

I I I I I I

* • ♦ • • • •••■ •«•• •■

AλCO_ GCλCCTTC« CTGCGCyAGCT;CGG^

I I I ! I I I I

33C 340 350 350 370 380 390 400

ISO 160 17C 110 «β «•«-. 3-ιH 22t>

490 560 UC »0 '130 540 'gss Sfβ

310 3 0 35: 260 170 110 2M 30β

AαLJϋuaτt^sαx^^g:^gτ^τMϋcaiλcc^

590 iM WO too < UO 63C >40

>1C 13C 336 140 259 MO 379 166 I _J ! I * I I ! xoxscΛrπicgtTiu yi ji'cscΛCCTWicα** ^βO ββO 610 i-B 680 7«C 710 730

390 400 41C 420 430 440 450 460

I i i i I I I I

CTTglYS TV.¹¹. ICC _AU^ ACT CKGCTQS∞AA5CTSSJl*CL^ι:'': V.¹ lV_MSA I ^,l^,:'atAΛCCA'TTATSS£CT3SA

:TCC&TOg AC3GCCrSCtX»»GCrGSftR3Cl ^^^{^}^ i I I • I ^• I I X

730 740 75C 760 "70 780 790 800

470 (83 490 500 510 520 530 540 i __ι_ i i i i L__ i ccnφ∞ct«gACTAWTGCTGGC∞σιgr:xasc^^

I I I I I I I I

810 820 530 840 8S0 860 870 BEO

550 560 S70 580 590 600 610 620

I I I I I I I I

TGCGcx jiccαaascrscr&CArxrcyπcG^

I B90 I900 I910 I920 I930 I940 I950 I960

630 640 650 660 670 680 690 700

I I I I I I ^• I ^' I ccTGGTCλAπcrcsAαABiTαλccαrax∞

710 720 730 740 750 760 770 78C

I I I I I I I I ceaτcsccc:ά L tt.ΥiλτcλτcaCc: Λ^ ccAicGCccι^ι :cc.. LT. AτciθJu:cτ:^^

I I I I I I I I

1050 1060 1070 iQBO 1090 I CO 1110 1120 790 800 εio S2C 830 640 850 S60

1 ! I

CA3GCTGcσrrcAftCArcτ37x^cyA ΛCA3 crc: Cϊ ;λCTAC3CCAA iSATCrrCCCC iATGAΛCAACG iCCACC8TG0C :

ΓACG CAAOATCTTCO CATOAACAACCCCACCOIGOC

I I i I

1130 1140 1150 1160 1170 1180 1190 1200

870 680 390 900 910 920 930 940

I I ⁱ !^• I I 1 I

CCTGCYVCATTTACCAGGXOTGAGCG GGTTCXK ^^

CGΓGOACATTTA CAGGGSG I I

1210 122C

cccrrGATC'i'^GGcr cc^πujs c gcsqeecTCTAeATπgwccAT

I

:230 1240 1250 1260 1270

1030 1040 1050 1060 1070 1080 1090 1100 I

Asστcτcc?CGC«gτa»X3iGλscrccAα^^

AGgrcTCcraκπGcτκ»3^t cc_A^

1110 1120 1130 1140 1150 1160 1170 1180 I I

TGCASTGGTCACGCT I I I I ΪTGCTCGXGGC ITσTCTTCCGG I

T3CGGTGG CACC TTCG I Γ CTCACCCC TCCTGCGCCAΓCCTC

I I I I I

1360 1370 1380 1390 1400 1410 1420 1430

1190 1200 1210 1220 1220 1240 1250 1260

I I I i I I I I

GACCTGAGGGALTlUlUK^βCCrCSATGATCTSCTCAλCGTKτKΛGAGCCTSC XGTGCTGCCCAT

6AcxrsA3ccacττ^groϊθscn AT(^τcτ^^

I I I I I ! I I

1440 1450 1460 1470 1460 1490 1500 1510

1270 12SS :2S0 13C0 131C 1320 1330 134 C

I i : i

C r ACCTTC^CCAC ATσCCCλCCCTCATGCAGα rSAACAAOGTCSTCACCTCrrCCΛT A c rαicGττcA ykacATG c»cccrrcA syiGGAcπ y^A TGGCSTgCTO ACAASCTCCaU ' ' 'eCATeA

I

1520 1520 1540 1550 1S70 1560 LS90

1350 1360 1370 13S0 1390 1400 1410 1420

I I I i I I I i ταrτGCTλcπ r«xκx^7CAACcτcτAcrr^

TCCTG IAC7rCTGC»XλTCAAC C7ACπT i i i : i I i i

1600 161C 1620 1630 1640 1650 166C 1670 1430 1440 1450 1460 1470 1480 1490 1500 I I I I I I I I

I 1680 I 1690 11700 I 1710 I 1720 I 1730 I 1740 I 1750

1510 1520 1530 1S40,

„ I I I x

T^C^^G^cc c^⁽CTcaaccAcA iv-r iCG« : U-iu (SEQ. ID NO:^' 12)

M3CM9SA

I 1.840 I1850 I1860 I1870 I1880 I1890 I1900 I1910

GGATGGAGjTGGGAC-AGTTCpGAGACCAGCCAACCTGGGGGCTTTAGGGACCTGCTGTTTCCjTAGCGCAGCGATGTGATTACC (SEQ . ID 1920 1930 1940 1950 1960 1970 1980 1990 v_jn -i i _\

Top sequence is mutated NRAMP.

Lower sequence is wild type NRAMP. _mΛ»,_{n IPEMD} ^

Mutant sequence contains at least two inserts which are absent from the NRAMP sequence (GENBANK.).

Tahle 15: Mutated NRAMP DNA Sequence

Sequence C

CTCAGGCTGG CGCCGTGGCG GGATTCAAAC TTCTCTGGGT GCTGCTCTGG GCCACCGTGT 60

TGGGCTTGCT CTGCCAGCGA CTGGCTGCAC GTCTGGGCGT GGTGACAGGC AAGGACTTGG 120

GCGAGGTCTG CCATCTCTAC TACCCTAAGG TGCCCCGCAC CGTCCTCTGG CTGACCATCG 180

AGCTAGCCAT TGTGGGCTCC GACATGCAGG AAGTCATCGG CACGGCCATT GCATTCAATC 240

TGCTCTCAGC TGGACGGTAC CACCCCAGTG TACCCCAACT CTTCAGGCCA GGCAGAGAAC 300

AGCTGCTGCT ACTTCCCCCC CTAACCAGTC CCTCCCAGAG TCTATTTTAT CCTGCTGTCC 360

CCTCTGAAGC AGGGCTGCTG CCCTGTTTTC CAGAAATGTA AAGTGACTTG TCTAAAGTCA 420

CACAGATGTG AGTCATGCAG GACTTTGGGA CTGCAGCCCC AAACTCCCTG CTGCGCCGGG 480

TGCCAGGTCT CTCCTCTAGC TCTGCCCTGC CTCGACTGTT CTATGCCACA CTCCCACTCC 540

CCTTGCCCTA GCTGTCTGGG GGCGCTTAGG GTCCTGCTCC CAGGAGGCCA GATTCCTGTC 600

TCCAAGCCCT GAGGCTCCGT AGGAGTTAGA GACCCCTGGA CCAAGCTGGG CTGACCCGGG 660

CCACTCTGGT TTCAGAATCC CACTCTGGGG TGGCGTCCTC ATCACCATCG TGGACACCTT 720

CTTCTTCCTC TTCCTCGATA ACTACGGGCT GCGGAAGCTG GGAAGCTTTT TTTGGACTCC 780

TTATAACCAT TATGGCCTTG ACCTTTGGCT ATGAGTATGT GGTGGCGCGT CCTGAGCAGG 840

GAGCGCTTCT TCGGGGCCTG TTCCTGCCCT CGTGCCCGGG CTGCGGCCAC CCCGAGCTGC 900

TGCAGGCGGT GGGCATTGTT GGCGCCATCA TCATGCCCCA CAACATCTAC CTGCACTCGG 960

CCCTGGTCAA GTCTCGAGAG ATAGACCGGG CCCGCCGAGC GGACATCAGA GAAGCCAACA 1020

TGTACTTCCT GATTGAGGCC ACCATCGCCC TGTCCGTCTC CTTTATCATC AACCTCTTTG 1080

TCATGGCTGT CTTTGGGCAG GCCTTCTACC AGAAAACCAA CCAGGCTGCG TTCAACATCT 1140

GTGCCAACAG CAGCCTCCAC GACTACGCCA AGATCTTCCC CATGAACAAC GCCACCGTGG 1200

CCGTGGACAT TTACCAGGGG GGCGTGATCC TGGGCTGCCT GTTCGGCCCC GCGGCCCTCT 1260

ACATCTGGGC CATAGGTCTC CTGGCGGCTG GGCAGAGCTC CACCATGACG GGCACCTACG 1320

CGGGACAGTT CGTGATGGAG GGCTTCCTGA GGCTGCGGTG GTCACGCTTC GCCCGTGTCC 1380

TCCTCACCCG CTCCTGCGCC ATCCTGCCCA CCGTGCTCGT GGCTGTCTTC CGGGACCTGA 1440

GGGACTTGTC GGGCCTCAAT GATCTGCTCA ACGTGCTGCA GAGCCTGCTG CTCCCGTTCG 1500

CCGTGCTGCC CATCCTCACG TTCACCAGCA TGCCCACCCT CATGCAGGAG TTTGCCAATG 1560

GCCTGCTGAA CAAGGTCGTC ACCTCTTCCA TCATGGTGCT AGTCTGCGCC ATCAACCTCT 1620

ACTTCGTGGT CAGCTATCTG CCCAGCCTGC CCCACCCTGC CTACTTCGGC CTTGCGGCCT 1680

TGCTGGCCGC AGCCTACCTG GGCCTCAGCA CCTACCTGGT CTGGACCTGT TGCCTTGCCC 1740

ACGGAGCCAC CTTTCTGGCC CACAGCTCCC ACCACCACTT CCTGTATGGG CTCCTTG 1797 (Seq. ID No: 13)

Mutant sequence includes an insert of 400 or more bp than the non-mutant NRAMP sequence (GENBANK).

Example 16: Results and Conclusions

The prominent band from the clones of these patients were shown to contain the following alterations.

(1) An in-frame insert of approximately 400 bp.

(2) Deletions of fragments of coding sequences.

In either case, these mutations are likely to cause the expression of an abnormal NRAMP protein.

Controls were performed to show that the results obtained did not result from a primer-induced phenomenon, from an artifact resulting from genomic DNA amplification, from post-isolation handling of RNA, or from dependent on annealing temperature or magnesium concentration. The tests were repeated with similar results on numerous samples from patients exhibiting the indicated alleles, with controls only showing the predicted NRAMP band.

The identification of altered cDNA obtained from RNA of the patients allows a better understanding of the role of NRAMP in the pathogenesis of disease. These results indicate that some of the patients have deletions, some have additions and still others have mutations. These alterations in the mRNA sequences of the alleles studied present an excellent opportunity for diagnosing amongst the general population which individuals have a genetic predispositions for IBD, CD, UC and their subtypes. More generally, these alterations in the mRNAs corresponding to patients to NRAMP alleles shown to exhibit a statistically significant linkage to these diseases may be applied to screening entire populations, including families which are known to transmit it, for the presence of a genetically inherited propensity to the diseases. This information, in turn, may be utilized to develop and implement new therapies as well as to implement known therapies, at an earlier stage of the disease(s) or condition(s).

The present method may rely on the above exemplified RT-PCR procedure applied to a patient's nucleic acid to assess the presence of any abnormalities in the NRAMP locus. In addiiton, since abnormal transcripts generally result in abnormal proteins, the present method may also rely on antibodies specific to NRAMP to either determine whether the protein is present, e.g., on the cell surface, given that an incorrectly spliced mRNA may result in a defective protein incapable of passing through the cell wall. The early detection of a propensity to these disease(s) or condition(s) may also permit therapeutic advances, for example, by replacement of the abnormal protein with normal protein or a normally expressible transgene(s).

The present invention provides a novel method to define new polymorphisms associated with, or linked to, the NRAMP locus gene in a statistically significant manner. The method identifies an NRAMP polymorphism by analyzing a nucleic acid from a subject diagnosed with or suspected of having an Inflammatory Bowel Disease, and establishes whether it is linked to one of the types of IBD in a statistically significant manner. The invention's confirmation of the presence of a NRAMP polymorphism known to have a statistically significant correlation with an IBD such as Ulcerative Colitis or Crohn's Disease or subtype(s) thereof, or with at least one IBD symptom or biological response, allows a significantly more precise diagnosis and prognosis, and a better election of treatment modalities after treatment.

Claims

1. A method of identifying an inflammatory bowel disease (IBD) or subtype thereof, comprising selecting a population comprised of pairs of related subjects having at least one biological response associated with IBD or a subtype thereof; obtaining nucleic acid from the subjects in the selected population; detecting a polymorphism at a natural resistance-associated macrophage protein (NRAMP) locus in the nucleic acid from the subjects; establishing whether a statistically significant correlation exists between the thus found polymorphism and at least one of the biological responses; and identifying an IBD or subtype thereof when the existence of a statistically significant correlation is established.

2. A method of identifying an ulcerative colitis (UC) subtype, comprising selecting a population comprised of subjects having at least one biological response associated with UC; applying the method of claim 1 to the nucleic acid of the thus selected population; and identifying an UC subtype when the existence of a statistically significant correlation is established.

3. A method of diagnosing an inflammatory bowel disease (IBD) or subtype thereof, comprising selecting a subject exhibiting at least one biological response associated with IBD of subtype thereof; detecting a polymorphism at a functional site of a natural resistance-associated macrophage protein (NRAMP) locus in the nucleic acid from the subject; comparing the found polymorphism with that of IBD or subtype DNA controls found by the method of claim 1 to have a statistically significant correlation with a biological response; and diagnosing IBD or a subtype thereof when a match with an IBD or subtype control is found.

4. A method of diagnosing an ulcerative colitis (UC) subtype, comprising selecting a subject exhibiting at least one biological response associated with UC; applying the method of claim 3 to the nucleic acid of the subject; and diagnosing an UC subtype when a match with an UC subtype control is found.

5. A method of screening a population for a susceptibility to inflammatory bowel disease (IBD) or subtype thereof, comprising diagnosing IBD or subtype thereof by applying the method of claim 4 to nucleic acid obtained from members of a predetermined population; and selecting the members of the population which show a statistically significant correlation with the biological response as exhibiting a susceptibility to IBD or subtype thereof.

6. The method of claim 1 , wherein the polymorphism comprises a mutation selected from the group consisting of nucleotide substitutions, additions, deletions and combination thereof.

7. The method of claim 6, wherein the polymorphism comprises a nucleotide substitution; and the nucleotide substitution comprises a substitution at a site selected from the group consisting of a functional NRAMP locus and a satellite NRAMP locus.

8. The method of claim 1, wherein the polymoφhism is located at a functional site of the NRAMP locus.

9. The method of claim 8, wherein the polymorphism at a functional site of the NRAMP locus is located in the 5' regulatory region of an NRAMP locus.

10. The method of claim 7, wherein the polymorphism is located at a satellite site to the NRAMP locus.

11. The method of claim 10, wherein the polymoφhism in the satellite region of the NRAMP locus is located vicinal to the 3' end of an NRAMP locus.

12. The method of claim 10, wherein the polymoφhism in the satellite region of the NRAMP locus is located vicinal to the 5' end of an NRAMP locus.

13. The method of claim 10, wherein the NRAMP locus satellite DNA corresponds to the D2S434 allele.

14. The method of claim 1 , wherein the biological response comprises an increase in the expression of the NRAMP gene with respect to a general population, as measured by NRAMP mRNA or protein production.

15. The method of claim 1, wherein the biological response comprises an increase in the expression of the NRAMP gene with respect to a general population, as measured by NRAMP blood levels.

16. The method of claim 1, wherein the polymorphism is detected by isolating a fragment of the nucleic acid comprising the NRAMP locus from the sample; contacting the nucleic acid fragment with an NRAMP polymoφhism-specific oligonucleotide probe under conditions effective to hybridize the nucleic acid and the polymoφhism-specific oligonucleotide probe; detecting the presence of any hybrid formed; and taking the presence of any hybrid as an indication of the existence of a polymoφhism.

17. The method of claim 16, wherein the nucleic acid is isolated by enzymatic amplification.

18. The method of claim 16, further comprising enzymatically restricting the nucleic acid.

19. A method of determining the effectiveness of a therapy to alter the serum level of a natural resistance-associated macrophage protein (NRAMP) for treating inflammatory bowel disease, comprising detecting a polymoφhism at a functional site within an NRAMP locus in a nucleic acid from a subject; applying a therapy to the patient; and determining whether die therapy is effective using the correlation found by the method of claim 3, with respect to NRAMP mRNA or protein production, length or sequence and/or NRAMP blood level or function, if the NRAMP mRNA or protein production, length or sequence and/or the NRAMP blood level or function are altered and/or no longer exhibit(s) a statistically significant correlation with the polymoφhism.

20. The method of claim 19, wherein the inflammatory bowel disease comprises ulcerative colitis.

21. An inflammatory bowel disease kit, comprising a nucleic acid comprising a DNA selected from the group consisting of those encoding a functional or satellite region of a natural resistance-associated macrophage protein (NRAMP) locus, oligonucleotides thereof and antisense nucleic acids thereto; and instructions for its use.

22. The kit of claim 21, wherein the nucleic acid comprises a fragment of a functional region of an NRAMP locus.

23. The kit of claim 22, wherein the functional region of the NRAMP locus comprises a 5' regulatory region of an NRAMP locus.

24. The kit of claim 21, wherein the nucleic acid comprises a fragment of a satellite region to an NRAMP locus.

25. The kit of claim 24, wherein the fragment of the NRAMP satellite region comprises DNA vicinal to the 3' end of the NRAMP functional region.

26. The kit of claim 24, wherein the fragment of the NRAMP satellite region comprises DNA vicinal to the 5' end of the NRAMP functional region.

27. The kit of claim 24, wherein the NRAMP satellite region comprises DNA corresponding to a fragment of a satellite region of an NRAMP D2S434 allele.

28. The kit of claim 21, further comprising means for amplifying the nucleic acid.

29. The kit of claim 21, wherein the DNA is about 8 to 60 nucleotide long.

30. The kit of claim 29, wherein the DNA is about 12 to 30 nucleotide long.

31. The kit of claim 30, wherein the DNA is about 15 to 20 nucleotide long.

32. An ulcerative colitis (UC) kit, comprising a nucleic acid comprising a DNA corresponding to a satellite region of a natural resistance- associated macrophage protein (NRAMP) D2S434 allele; and instructions for its use.

33. The kit of claim 32, further comprising means for amplifying the nucleic acid.

34. The kit of claim 32, wherein the DNA is about 8 to 60 nucleotide long.

35. The kit of claim 34, wherein the DNA is about 12 to 30 nucleotide long.

36. An ulcerative colitis (UC) kit, comprising a nucleic acid comprising a DNA selected from the group consisting of those encoding a fragment of a functional region to a natural resistance-associated macrophage protein (NRAMP) D2S434 allele; and instructions for its use.

37. The kit of claim 36, further comprising means for amplifying the nucleic acid.

38. The kit of claim 37, wherein the DNA is about 8 to 60 nucleotide long.

39. The kit of claim 38, wherein the DNA is about 12 to 30 nucleotide long.

40. The kit of claim 39, wherein the DNA is about 15 to 20 nucleotide long.