METHODS OF SCREENING FOR ULCERATIVE COLITIS BY DETECTING AN INTERLEUKIN-1 RECEPTOR ANTAGONIST POLYMORPHISM

Abstract: A novel, differential association between allele 2 of the variable number of tandem repeats polymorphism at intron 2 of the human IL-1 receptor antagonist gene and ulcerative colitis in humans of Jewish ancestry has been discovered. In accordance with the present invention, there is provided methods of screening for ulcerative colitis in human subjects of Jewish ancestry comprising detecing the presence or absence of nucleic acid of the subject encoding allele 2 of the variable number of tandem repeats polymorphism at intron 2 of the human IL-1 receptor antagonist gene, wherein the presence of nucleic acid encoding allele 2 is indicative of ulcerative colitis. Kits useful for screening for ulcerative colitis in human subjects of Jewish ancestry are also provided.

Description: METHODS OF SCREENING FOR ULCERATIVE COLITIS BY DETECTING AN INTERLEUKIN-1 RECEPTOR ANTAGONIST POLYMORPHISM BACKGROUND OF THE INVENTION A. Inflammatory Bowel Disease Inflammatory bowel disease (IBD) is the collective term used to describe two chronic, idiopathic gastrointestinal disorders, ulcerative colitis ("UC") and Crohn's disease ("CD"). Although the diseases have distinct pathophysiological characteristics, they are frequently considered together due to several clinical and therapeutic similarities. Several other types of inflammatory conditions ofthe bowel having known infectious, toxic or ischemic etiology, such as irritable bowel syndrome, infectious diarrhea, rectal bleeding, radiation colitis, and the like, may mimic IBD acutely, because the mucosa ofthe small and large intestines reacts in a similar way to a large number of different insults. However, if the disease progression is monitored over time, they can be distinguished from IBD by their failure to cause a chronic relapsing and remitting syndrome. IBD occurs world-wide and is reported to afflict as many as two million people. The course and prognosis of IBD is widely variable. Onset has been documented at all ages; however, IBD predominately begins in young adulthood. The three most common presenting symptoms of IBD are diarrhea, abdominal pain, and fever. The diarrhea may range from mild to severe and is often accompanied by urgency and frequency. In UC, the diarrhea is usually bloody and may contain mucus and purulent matter as well. Anemia and weight loss are additional common signs of IBD. Ten to fifteen percent of all patients with IBD will require surgery over a 10- year period. The risk for the development of cancer is increased in patients with IBD as well, particularly in those with UC. The longer the duration of disease, the higher the risk of developing carcinoma. Patients with UC regularly undergo cancer surveillance by endoscopy after ten years of disease. Reports of an increasing occurrence of psychological problems, including anxiety and depression, are perhaps not surprising secondary effects of what is often a debilitating disease that occurs in people in the prime of life. B. The Caused of IBD are Unknown Although the etiology of IBD is unknown, a number of studies have suggested that genetics is important in a person's susceptibility to IBD and that the immune system is responsible for mediating the tissue damage in these diseases. Generally speaking, a failure to down regulate the normal self-limited inflammatory response ofthe bowel is characteristic of IBD, but it remains unclear what initiates the pathogenic processes and how it may differ, if at all, in UC and CD. It has also been suggested that a primary abnormality ofthe immune system and its regulation might serve as primary initiating factors, or that the disease process might be initiated by an infectious agent and the injury is then perpetuated through immune-mediated or other processes. Although the mucosal injury observed during episodes of acute disease can resemble the effects of any of a number of recognized infectious agent, no transmissible infectious agent has been consistently identified with IBD. Autoimmunity has also been suggested in the pathogenesis of IBD. Evidence to suggest this hypothesis is based on the existence of circulating antibodies that react with unknown alimentary tract antigens of both human and animal origin. For example, human fetal and adult colonic, biliary, skin and vascular epithelial cells, epithelial cell associated components from murine small intestine, rat and human colonic epithelial giycoproteins, intestinal bacterial polysaccharide, and antigens from germ-free rat feces have been described to react with sera from patients with IBD. Other studies demonstrated an increased local IgG response in the colonic mucosa of patients with IBD and other colonic inflammations. The mechanism of this IgG response, the specific local antigens involved, and the role of these antibodies are unknown. C. Need for Objective Diagnostic Tools Inflammatory bowel disease poses a clinical and scientific challenge to physicians and researchers. To date most ofthe diagnostic tools for IBD are quite subjective. Diagnosis depends upon a host of procedures aimed at confirming the suspected diagnosis. The initial symptoms are often confused for non-chronic bowel disorders by physicians unfamiliar with IBD, because the ucosa ofthe small and large intestines reacts in a similar way to a large number of different insults. Consequently, IBD often goes mistreated and undiagnosed until the disease shows its chronicity which results in referral ofthe patient to a specialist. The imprecise and subjective nature of endoscopic and radiologic examination can result in a misdiagnosis or indeterminate diagnosis even when the IBD is suspected. Unfortunately, the patient must often suffer as the disease progresses before a definitive diagnosis can be made. In many patients, though, the diagnosis of IBD must still be regarded as indeterminate The differentiation between the types of IBD, ulcerative colitis and Crohn's disease, carries important prognostic and therapeutic implications. For example, when colectomy is indicated, the type of IBD involved determines which surgical options are appropriate. Surgery (total colectomy) does represent a cure for the symptoms of UC, though a dramatic one. In CD, surgery is never curative. Continent procedures such as the ileorectal pull-through (mucosal proctectomy) or the Kock pouch may be desirable in UC, but are contraindicated in CD. Thus, IBD and quite often its treatment affects the lifestyle and functional capabilities of those afflicted. Treatment courses often result in adverse physiologic manifestations which must be balanced against the therapeutic benefit. Any intervention which can improve patients' toleration of their disease and therapeutic program is welcome. The availability of diagnostic methods that would readily distinguish UC from CD as well as other inflammatory disorders ofthe bowel would represent a major clinical advance which would aid in therapeutic management of IBD and the design of more specific treatment modalities. In addition specific detection ofthe disease in prospective parents can be useful in genetic counseling. Accordingly, there has existed a need for convenient and reliable methods of screening for IBD for diagnostic, prognostic and therapeutic purposes. BRIEF DESCRIPTION OF THE INVENTION A powerful association has been discovered in Jews between ulcerative colitis ("UC") and the presence of allele 2 ofthe variable number of tandem repeats ("VNTR") polymorphism at intron 2 ofthe human interleukin-1 receptor antagonist ("LL-lra") gene. This association provides the basis for convenient and reliable methods of screening Jewish patients for UC, and distinguishing UC from Crohn's disease ("CD") and other inflammatory diseases ofthe bowel The present invention provides novel methods of screening for UC in Jews which comprise detecting the presence or absence of nucleic acid encoding allele 2 of the VNTR polymoφhism at intron 2 ofthe IL-lra gene, wherein the presence of nucleic acid encoding allele 2 indicates UC. These novel methods do not depend upon the presentation of clinical symptoms or the activity ofthe disease and provide a more sensitive method of screening for UC within this sub-population than is provided by prior art methods. Thus, the present invention has both prognostic and diagnostic value. Nucleic acid encoding allele 2 of VNTR polymoφhism ofthe IL-lra gene can be detected in accordance with the present invention by amplifying the genomic DNA of a Jewish subject which encodes at least the portion intron 2 ofthe DL-lra gene encoding the VNTR allele and identifying the allele present. Allele 2 can be identified by assaying the nucleic acid sequence at intron 2 ofthe IL-lra gene of a subject for defining characteristics of allele 2 and comparing the results to a positive and or negative control. Defining characteristics of nucleic acid encoding allele 2 include, for example, size, sequence, homology to a probe, and the like. Thus there are an array of different formats in which the methods ofthe present invention can be performed. Primers suitable for use in amplifying nucleic acid encoding intron 2 of the IL-lra gene are provided herein Also provided by the present invention are kits for screening for UC in Jews by detecting the presence or absence of allele 2 ofthe VNTR polymoφhism at intron 2 ofthe IL-lra gene. Such kits include reagents, primers, sequencing markers, positive and negative controls and the like, which are useful in the practice ofthe present invention. DETAILED DESCRIPTION OF THE INVENTION Although the clinical features of ulcerative colitis ("UC") and Crohn's disease ("CD") have been well characterized, diagnosis of these disease is often a protracted and expensive undertaking that requires invasive and unpleasant procedures. The etiology and pathogenesis of these chronic, relapsing inflammatory bowel diseases ("IBD") remain unknown. Several factors have been implicated as possible initiating events in IBD, including environmental agents, bacterial or viral organisms, bacterial cell wall components or ingested toxins, and dietary products. These factors, capable of initiating an inflammatory response in genetically predisposed individuals, may initiate a sequence of chronic immunological processes that are not appropriately down-regulated. These events peφetuate a prolonged cellular and humoral immune response that leads to subsequent tissue injury. The search for a specific genetic marker which would provide a non-invasive, convenient and reliable method of diagnosing IBD has been frustrated by the apparent heterogeneity of these diseases. Certainly, there can be no group more troubled by this than people of Jewish ancestry, particularly Caucasians of Jewish ancestry, who as a group, show the highest reported risk of developing EBD than any other ethnic group. While UC and CD are both found worldwide, they occur in the highest frequency in North America and Northern Europe where the incidence ranges from 2 to 15 and the prevalence 35-150 per 1000,000 for UC, and 2-7 and 30-100 per 100,000 for CD, respectively. The incidence of IBD is highest among Caucasians, lower in blacks, and lowest in Asians. For Caucasians, the most consistent observation has been that Ashkenazi Jewish populations has been shown to be at a higher risk of developing IBD than other ethnic groups. A differential association between UC and the presence of allele 2 ofthe variable number tandem repeat polymoφhism at intron 2 ofthe interleukin- 1 receptor antagonist gene in human subjects of Jewish ancestry has been discovered. This association provides the basis for convenient and reliable methods of screening for UC in human subjects of Jewish ancestry, providing valuable information in the diagnosis of EBD and the determination of susceptibility to UC. Interleukin- 1 ("IL-1") is a pro-inflammatory cytokine produced predominately by activated macrophages. JL-1 induces fever, release of acute phase reactants, production of arachidonic acid metabolites, and activation of other immune cells. The cytokine interleukin- 1 receptor antagonist ("EL- Ira") is a potent anti- inflammatory protein that appears to play a role in several chronic inflammatory bowel diseases including IBD. The inflammatory response to IL-1 is modulated by IL-lra, a 22 kilodalton protein secreted by the same cells that produce JL-1. The IL- lra is structurally related to IL-lα and EL-lβ and competes with these molecules for occupancy of IL-1 cell surface receptors. Since IL-lra does not trigger signal transduction, it acts as a competitive inhibitor. It may therefore play a crucial role in many IL-1 mediated diseases, acting as an important endogenous regulator of inflammation. The gene encoding IL-lra is located on human chromosome 2 (2ql3-14). The published gene sequence of IL-lra shows four copies of an 86-base pair sequence in intron 2 ofthe IL-lra gene. See, Lennard, A. et al., Cytokine 4:83-89 (1992), incoφorated herein by reference in its entirety. Recently, a variable length polymoφhism was reported at intron 2 ofthe IL-lra gene, comprising five alleles. See, Tarlow, J., et. al., Human Genetics 91 :403-404 (1993), incoφorated herein by reference in its entirety. This polymoφhism is referred to herein as the yariable number of tandem repeats ("VNTR") polymoφhism ofthe IL-lra gene. Alleles 1 through 5 (A1-A5) ofthe VNTR polymoφhism are characterized by the number of copies ofthe 86-base pair sequence present at the loci as described in Table 1. These alleles can be easily distinguished from one another on the basis of their unique lengths, by direct sequencing and the like. For example, primers flanking this region ofthe VNTR polymorphism can be used to amplify the polymoφhic region by PCR. The PCR products can then be analyzed by electrophoresis to determine the allele present. The primers defined as SEQ ID NO 1 and SEQ ID NO 2 have been used to amplify genomic DNA encoding the VNTR polymoφhism of IL- lra and the five alleles characterized as follows in Table 1: Table I. Five alleles identified from DNA amplified from 70 unrelated individuals Tarlow, J., et. al., Human Genetics 91:403-404 (1993). Allele Frequency Size (bp) Number of repeats Al 0.736 410 4 A2 0.214 240 2 A3 0.036 500 5 A4 0.007 325 3 A5 0.007 595 6 While investigators have observed a moderate association (Odds ratio ~2) between allele 2 ofthe VNTR polymoφhism of IL-lra and UC patients in Caucasian populations from North America and Europe, this association was too weak to be of diagnostic or prognostic value (allele 2 of EL-1 ra present in 35% of UC versus 24% of controls). See, Mansfield, J. et al., Gastroenterology 106:637-642 (1994). For the first time, it has been discovered that allele 2 ofthe VNTR polymorphism at intron 2 of IL-lra can be used as a diagnostic and prognostic genetic marker of UC in humans of Jewish ancestry. Jewish UC patients have a significantly increased frequency of allele 2 compared with Jewish controls (Odds ratio = 5), while non-Jewish UC patients have a frequency of allele 2 similar to non- Jewish controls. More specifically, when the genotype at the IL-lra locus was determined for unrelated Jewish and non-Jewish Caucasian human subjects with UC (n=106), CD (n=l 58), and controls (n=l 14) by PCR using the primers and procedures described below in greater detail, it was determined that the frequency of individuals carrying the allele 2 was significantly increased in the UC compared with controls (p=0.04; OR=l .7; 95% CI=1.0-3.1). CD patients had a similar frequency as controls (p=0.66). However, when this population was divided into Jewish and non- Jewish groups, a significant difference was only observed in Jews (p=0.003; OR=5.0; 95%CI=1.5- 17.5), but not in non- Jews (p=0.85) as reported in detail in Table 2. The OR of 5 is the highest reported in any population. Table 2. Ethnic Groups UC CD Controls N . %of 2 N %of2 N %of2 Total Caucasian 106* 58.5 158* 47.5 114* 44.7 Jews 39 76.9 68 48.5 25 40.0 Non-Jews 66 48.5 88 46.6 81 46.9 * unknown ethnicity for 1 UC, 2 CD, and 8 controls The χ2 test was used for the statistical test. Both Odds Ratio (OR) and its 95% confidence interval (Cl) were calculated to measure the strength of associations. Thus, in accordance with the present invention, there is provided methods of screening for UC in a human subject of Jewish ancestry, comprising detecting the presence or absence of nucleic acid encoding allele 2 of VNTR polymoφhism at intron 2 ofthe DL-lra gene in said subject, wherein the presence of nucleic acid encoding allele 2 is indicative of UC. Nucleic acid of a subject which is suitable for screening in accordance with the present invention may be derived from any nucleated cell sample, and preferably from peripheral mononuclear blood cells. "A human subject of Jewish ancestry" or "a person of Jewish ancestry" refers to a person having had at least one biologically-related grandparent who is Jewish. Preferably, the human subject is Caucasian of Jewish ancestry. Even more preferably, the human subject is an Caucasian of Ashkenazi Jewish ancestry. Detecting the presence or absence of nucleic acid encoding allele 2 ofthe VNTR polymoφhism of the IL-lra gene may be accomplished by determining whether or not genomic DNA a the human subject possesses a defining characteristic of nucleic acid encoding allele 2 ofthe VNTR polymoφhism of the IL-lra gene. One of skill in the art will understand that there are many means available to make such a determination, e.g., electrophoresis, automated sequencing, allele-specific oligonucleotide probing, differential restriction endonuclease digestion, ligase- mediated gene detection, and the like. For example, genomic DNA encoding at least intron 2 ofthe IL-lra human gene can be isolated from nucleated cells of a human subject of Jewish ancestry and assayed for such characteristics as size, specific sequence, number of sequence repeats, ability to hybridize with a labeled probe under specific hybridization parameters, ability to hybridize with an antibody specific for a particular allele and the like, and then compared to a positive control which defines the same characteristic for allele 2 ofthe VNTR polymoφhism and/or a negative control which defines the same characteristic for an allele ofthe VNTR polymoφhism not known to be associated with UC in persons of Jewish ancestry. A positive control for allele 2 ofthe VNTR polymoφhism ofthe IL-lra gene using the primers described herein for PCR amplification of genomic DNA and fragment length as the defining characteristic, a fragment of 240 base pairs would be a positive control and a negative control would be a fragments of 325, 410, 500 and/or 595 base pairs. Of course, the greatest likelihood of accurately detecting the presence of allele 2 ofthe VNTR polymoφhism ofthe IL-lra gene in this example would be to compare the results ofthe assay to the positive and negative control. To increase the accuracy of detecting allele 2 ofthe VNTR polymoφhism, the control should be subjected to the same test procedures and parameters as the nucleic acid ofthe subject being assayed. Likewise, assays to detect the presence or absence of nucleic acid encoding allele 2 ofthe VNTR polymoφhism should be calibrated against a standard. For example, in a presently preferred embodiment using fragment length as the defining characteristic of alleles ofthe VNTR polymoφhism, a positive control (a nucleic acid sequence known to encode allele 2 ofthe VNTR polymoφhism) is amplified and electrophoresed using the same reagents, primers and parameters as that used for the nucleic acid ofthe subject being tested. A sequencing marker equal in size to the control is also subjected to electrophoresis using the same reagents and parameters as those used with the test and control nucleic acid. Sequencing markers useful in the practice ofthe present invention are available from a variety of commercial sources. Genomic DNA of a subject encoding the sequence of interest at intron 2 of the IL-lra gene can be amplified to make detection ofthe VNTR allele easier. Amplification of nucleic acid may be achieved using conventional methods, see, e.g., Maniatis, et al., Molecular Cloning: A Laboratory Manual 187-210 (Cold Spring Harbour Laboratory, 1982) which is incoφorated herein by reference. Amplification, however, is preferably accomplished via the polymerase chain reaction ("PCR") method disclosed by U.S. Pat. Nos. 4,698,195 and 4,800,159, the respective contents of which are incoφorated herein by reference. Application of PCR to detect alleles ofthe VNTR polymoφhism of DL-lra gene requires less DNA and is faster than standard Southern blotting and hybridization techniques. Thus, oligonucleotide primer pairs can be constructed that allow enzymatic amplification of a subject's nucleic acid that encodes the VNTR polymoφhism at intron 2 ofthe IL-lra gene. The amplified nucleic acid can then be assayed to detect the presence or absence of allele 2. Primer pairs suitable for use in the practice ofthe present invention are linear oligonucleotides ranging in length from about ten to about thirty nucleotides in length. One ofthe primers in the pair should be complementary to a nucleotide sequence upstream ofthe nucleic acid encoding the VNTR polymoφhism at intron 2 ofthe IL-lra gene targeted for amplification. The other primer should be complementary to a sequence located down stream of this target site. Preferably, the primers suitable for use in the present invention are specific for amplification of nucleic acid encoding VNTR polymoφhism at intron 2 ofthe IL-lra gene and do not prime amplification of nucleic acid which does not encode VNTR polymoφhism of the IL-lra gene. The sequences complementary to the primer pairs may be separated by as many nucleotides as the PCR technique and the other technique(s) for detecting the presence or absence of VNTR polymoφhism will allow, provided that an appropriate control is used. Primers suitable for use in amplifying genomic DNA encoding the VNTR polymoφhism at intron 2 ofthe DL-lra gene can be constructed using the oligonucleotide primer sequences described herein as well as the genomic sequence ofthe IL-lra gene provided at Lennard, A. et al., Cytokine 4:83-89 (1992) and incoφorated herein by reference . A pair of primers suitable for use in the practice ofthe present invention is set forth in SEQ ID NOS 1 and 2. These primers are suitable for use in amplifying genomic DNA encoding the alleles ofthe VNTR polymorphism of intron 2 ofthe IL- Ira gene, and may be used as a pair or each in combination with another suitable primer. The novel methods for screening for UC and for distinguishing UC from CD disclosed herein include the use of traditional diagnostic tests for UC in combination with the detection of nucleic acid of a subject encoding allele 2 ofthe VNTR polymoφhism of DL-lra. Thus, for example, a positive test for HLA DR2 and/or a positive pANCA status may be used in combination with detecting the presence of nucleic acid encoding allele 2 ofthe VNTR polymoφhism. Kits for use in screening for UC in human subjects of Jewish ancestry are also provided by the present invention. Such kits can include all or some ofthe positive controls, negative controls, reagents, primers, sequencing markers, probes and antibodies described herein for determining the presence or absence of nucleic acid encoding allele 2 ofthe VNTR polymoφhism ofthe IL-lra gene Kits ofthe present invention may contain, for example, nucleic acid encoding allele 2 and/or alleles 1,3,4 and 5 ofthe VNTR polymorphism ofthe IL-lra gene, the nucleic acid sequence of any one or more of these alleles, schedules ofthe number and type of nucleotide sequence repeats and characteristics of one or more of these alleles, one or more labeled oligonucleotide probes specific for one or more alleles ofthe VNTR alleles, one or more primers for amplification of nucleic acid encoding the VNTR polymoφhism at intron 2 ofthe IL-lra gene, reagents commonly used for amplification, polymerase, antibody specific for, or which binds particular VNTR alleles and combinations of any ofthe above. As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like. A presently preferred embodiment ofthe inventive kits for use in screening for UC comprises DNA encoding allele 2 ofthe VNTR polymoφhism ofthe IL-lra gene in Tris-EDTA buffer solution preferably kept at 4°C. Another embodiment ofthe inventive kits for use in screening for UC further comprises one or more primers specific for amplification of nucleic acid encoding the VNTR polymoφhism at intron 2 ofthe IL-lra gene , for example, primers selected from the group comprising SEQ ID NO 1 and SEQ ID NO 2. Yet another embodiment ofthe inventive kits for use in screening for UC further comprises sequencing markers ranging in size from about 100 to about 600 base pairs. EXAMPLES Blood samples were obtained from 106 human patients diagnosed as having ulcerative colitis ("UC"), 158 human patients diagnosed as having Crohn's disease ("CD"), and 114 healthy humans ("controls"). The control group was selected to be ethnically and socioeconomically matched to the UC and CD patients. An individual was used as control only if he/she did not have inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, or other recognized autoimmune diseases. The distribution of age, gender, and ethnicity (Jewish/non- Jewish) were comparable between patients and controls. A patient was considered to be Jewish if he/she was of Jewish ancestry, i.e., at least one ofthe patient's biological grandparents was Jewish. All patients and controls analyzed were Caucasians. A. Isolation of Genomic DNA Genomic DNA is isolated from blood samples of each patient and each ofthe control group by methods well known in the art, for example, by methods described in J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989), incoφorated herein by reference. B. Amplification Of DNA Encoding Intron 2 ofthe IL-lra Gene Bv PCR The polymerase chain reaction ("PCR") was performed to amplify genomic DNA encoding intron 2 ofthe IL-lra gene. The primers identified as SEQ ID NOs. 1 and 2 were used. The PCR reaction is performed in a total volume of 20μl using 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.15 μM primers, 200 μM each dATP, dCTP, dGTP, dTTP; 100 to 300 ng genomic, and two units Taq DNA polymerase. PCR conditions are as follows: Denaturing ofthe nucleic acid sample in the first cycle of amplification is at 96 °C for one minute, followed by annealing primers at 60 °C for one minute, and polymerization at 70 °C for two minutes. Twenty nine subsequent cycles of amplification were carried out, denaturing at 94 °C for one minute, followed by annealing primers at 60 °C for one minute, and polymerization at 70°C for two minutes. After the final round of amplification, the final PCR products are analyzed on a 2% agarose gel. Electrophoresis of Amplified Genomic DNA Encoding Intron 2 ofthe IL-lra Gene 20 μl amplified DNA is electrophoresed on a 2% agarose gel and stained with ethidium bromide. Gels are run with a 100 base pair ladder control DNA sequencing marker to size fragments. Although extra fragments may shadow the specific DNA bands, results are unambiguously inteφretable, and confirmed by comparison to nucleic acid derived from human patients known to encode the specified alleles at Intron 2 ofthe IL-lra gene and subjected to the same amplification and electrophoresis procedures described above. Although the invention has been described with reference to presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit ofthe invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (I) APPLICANT: CEDARS-SINAI MEDICAL CENTER, et al. (ii) TITLE OF INVENTION: METHODS OF SCREENING FOR ULCERATIVE COLITIS BY DETECTING AN INTERLEUKIN- 1 RECEPTOR ANTAGONIST POLYMORPHISM (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Pretty, Schroeder & Poplawski (B) STREET: 444 South Flower Street, Suite 2000 © CITY: Los Angeles (D) STATE: California (E) COUNTRY: U.S.A. (F) ZIP: 90071 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible © OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: © CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Fujita, Sharon (B) REGISTRATION NUMBER: 38,459 © REFERENCE/DOCKET NUMBER: FP07 37833 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 213-622-7700 (B) TELEFAX: 213-489-4210 (2) INFORMATION FOR SEQ DD NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL. NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : CTC AGC AAC A CTCCTAT 17 (2) INFORMATION FOR SEQ ID NO.2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO.2: TCCTGGTCTG CAGGTAA 17

Claims: We claim. 1. A method of screening for ulcerative colitis in a human subject of Jewish ancestry, comprising detecting the presence or absence of nucleic acid encoding allele 2 ofthe VNTR polymoφhism at intron 2 ofthe interleukin- 1 receptor antagonist gene in said subject, wherein the presence of nucleic acid encoding allele 2 is indicative of ulcerative colitis. 2. A method of screening for ulcerative colitis in a human of Jewish ancestry, comprising: a.) isolating genomic DNA of said subject, wherein said genomic DNA encodes at least intron 2 ofthe interleukin- 1 receptor antagonist gene, and b.) detecting the presence or absence of a nucleic acid sequence encoding allele 2 ofthe interleukin- 1 receptor antagonist gene, wherein the presence of said nucleic acid sequence is indicative of ulcerative colitis. 3. The method of claim 2, wherein said detecting comprises amplifying at least the genomic DNA encoding intron 2 ofthe interleukin- 1 receptor antagonist gene and identifying the allele encoded by said amplified DNA. 4. The method of claim 3, wherein identifying comprises subjecting the amplified DNA to electrophoresis and comparing the results to a positive control. 5. The method of claim 4, wherein identifying further comprises comparing the results of electrophoresis to a negative control. 6. The method of claim 5, wherein amplifying comprises use of one or more primers specific for amplification of genomic DNA encoding interleukin- 1 receptor antagonist alleles. 7. The method of claim 6, wherein at least one primer is selected form the group consisting of SEQ ID NO 1 and SEQ ID NO 2. 8. The method of claim 2, wherein detecting comprises sequencing the DNA encoding the VNTR allele ofthe interleukin- 1 receptor antagonist gene and identifying the VNTR allele present.

Inventors: COMINELLI FABIO, PIZARRO THERESA, ROTTER JEROME I, YANG HUIYING, COMINELLI, FABIO, PIZARRO, THERESA, ROTTER, JEROME, I., YANG, HUIYING
Applicants: CEDARS SINAI MEDICAL CENTER, UNIV VIRGINIA, CEDARS-SINAI MEDICAL CENTER, UNIVERSITY OF VIRGINIA PATENT FOUNDATION
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1