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WO1999023255A1 - Methodes d'identification et de diagnostic d'affections intestinales inflammatoires ou de leurs sous-types au moyen du locus de la nramp - Google Patents

Methodes d'identification et de diagnostic d'affections intestinales inflammatoires ou de leurs sous-types au moyen du locus de la nramp Download PDF

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Publication number
WO1999023255A1
WO1999023255A1 PCT/US1998/022993 US9822993W WO9923255A1 WO 1999023255 A1 WO1999023255 A1 WO 1999023255A1 US 9822993 W US9822993 W US 9822993W WO 9923255 A1 WO9923255 A1 WO 9923255A1
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Prior art keywords
nramp
locus
nucleic acid
kit
dna
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Inventor
Jerome I. Rotter
Huiying Yang
Allan B. Dietz
Holly L. Niebergs
Susan Galandiuk
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University of Louisville
Cedars Sinai Medical Center
Mayo Foundation for Medical Education and Research
University of Louisville Research Foundation ULRF
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University of Louisville
Cedars Sinai Medical Center
Mayo Foundation for Medical Education and Research
University of Louisville Research Foundation ULRF
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Priority to AU12884/99A priority Critical patent/AU1288499A/en
Publication of WO1999023255A1 publication Critical patent/WO1999023255A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • 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.
  • IBD Inflammatory Bowel Disease
  • CD Crohn's Disease
  • UC Ulcerative Colitis
  • 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.
  • 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.
  • NRAMP Natural Resistance- Associated Protein
  • Contingent procedures such as the ileorectal pull-through (mucosal proctectomy) or the Kock pouch may be desirable in UC, but are contraindicated in CD.
  • 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.
  • NRAMP Natural Resistance-Associated Macrophage Protein
  • D2S173 and D2S104 have 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.
  • NRAMP Natural Resistance-Associated Macrophage Protein
  • IBD including UC and CD presently afflicts as many as two million people world-wide.
  • IBD including UC and CD presently afflicts as many as two million people world-wide.
  • UC UC
  • CD presently afflicts as many as two million people world-wide.
  • 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.
  • 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.
  • NRAMP natural resistance-associated protein
  • 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.
  • NRAMP natural resistance-associated macrophage protein
  • 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.
  • 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.
  • 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 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.
  • This invention arose from a desire by the inventors to improve on prior art technology for diagnosing IBD, particularly UC and CD.
  • 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.
  • IBD inflammatory bowel disease
  • UC Ulcerative Colitis
  • CD Crohn's Disease
  • 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.
  • IBD, CD and UC 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 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 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 polymo ⁇ hism 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 polymo ⁇ hism 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.
  • IBD inflammatory bowel disease
  • UC Ulcerative Colitis
  • CD Crohn's Disease
  • 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 polymo ⁇ hism 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.
  • the polymo ⁇ hism 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.
  • the polymo ⁇ hism is located in the 5' regulatory region of an NRAMP locus, and in another in a satellite region of the NRAMP locus.
  • the polymorphism 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 polymo ⁇ hisms may be detected by methods known in the art. More particularly the polymo ⁇ hism 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 polymo ⁇ hism- 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 polymo ⁇ hism.
  • 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.
  • the NRAMP locus polymo ⁇ hism- 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 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 polymo ⁇ hism at a functional site of a natural resistance-associated macrophage protein (NRAMP) locus in the nucleic acid from the subject, comparing the found polymo ⁇ hism 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.
  • the inflammatory disease is Ulcerative Colitis
  • the NRAMP locus comprises the satellite region of the NRAMP locus
  • 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 polymo ⁇ hism 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 polymo ⁇ hism.
  • NRAMP natural resistance-associated macrophage protein
  • the Inflammatory Bowel Disease comprises Ulcerative Colitis
  • 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.
  • kits 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.
  • 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.
  • 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.
  • NRAMP Natural Resistance- Associated Macrophage Protein
  • 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).
  • subject refers to any animal, particularly a mammal, more particularly a human.
  • diagnosisd with having an inflammatory bowel disease refers to any diagnoses, whether based on symptoms, empirical data or the like, whether tentative or definitive.
  • allelic combination means the same as a "haplotype,” i.e. is referring to particular combination of alleles for at least two loci.
  • locus means a physical location, place or position occupied by a particular gene on a chromosome.
  • 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.
  • 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.
  • susceptibility refers to a subject having any increased chance or risk, i.e.
  • 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 polymo ⁇ hism and an increased chance of acquiring a disease or condition, i.e. whether a subject is susceptible to acquiring a disease or condition.
  • screening also refers to methods of diagnosis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • phrases "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 inco ⁇ orated by reference.
  • 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) inco ⁇ orated herein by reference; and Sambrook et al. and Mullis et al.. both of which have been inco ⁇ orated by reference.
  • PCR polymerase chain reaction
  • 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 polymo ⁇ hisms 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.
  • Inflammatory Bowel Disease has been classified into the broad categories of Crohn's Disease (CD) and ulcerative colitis (UC).
  • CD also called regional enteritis
  • CD 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.
  • 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.
  • CD Crohn's disease
  • 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 inco ⁇ orated herein by reference) .
  • Ulcerative colitis 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.
  • UC and especially long-standing, extensive disease is associated with an increased risk of colon carcinoma.
  • UC ulcerative colitis
  • Table 1 Characteristics that serve to distinguish CD from UC are summarized in Table 1 below, which is taken from Rubin and Farber, supra (1994).
  • 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.
  • a characteristic clinical feature is perforating or fistulizing disease; or an obstructive symptom secondary to small bowel stenosis or stricture.
  • a characteristic endoscopic feature is a deep linear or serpiginous ulceration; a discrete ulcer in normal-appearing mucosa; cobblestoning; or discontinuous or asymmetric inflammation.
  • a characteristic radiographic feature is segmental disease (skip lesion); a small bowel or colon stricture; stenosis or fistula.
  • 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.
  • CD and UC Clinical, endoscopic, and histopathologic criteria, as discussed above, have been developed to classify patients into one or the other category
  • overlap between CD and UC also has been demonstrated at a variety of levels by clinical, immunological and genetic studies, for example.
  • 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 polymo ⁇ hism 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.
  • 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.
  • 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.
  • 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 polymo ⁇ hisms or alleles associated with UC or other d ⁇ sease(s) or condition(s) and comparing the results to positive and/or negative controls
  • 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 databank
  • 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.
  • NCBI human genome project
  • NIH human genome project
  • 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 inco ⁇ orated herein by reference.
  • 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.
  • CHLC Cooperative Human Linkage Center
  • micro satellites are characterized by a series of GACT tetranucleotide sequence repeats.
  • 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.
  • 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 polymo ⁇ hisms 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 polymo ⁇ hic 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 inco ⁇ orated herein by reference.
  • 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.
  • NRAMP Natural Resistance-Associated Macrophage Protein
  • 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 NR ⁇ MP locus.
  • 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 prefe ⁇ ed embodiment, the kit is directed to Ulcerative Colitis (UC).
  • UC Ulcerative Colitis
  • 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.
  • the kit of the invention may contain, for example, nucleic acid encoding NRAMP alleles or polymo ⁇ hisms 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 polym
  • 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 polymo ⁇ hisms associated with UC and/or CD.
  • kits 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.
  • a buffered solution e.g., a Tris-EDTA buffer solution
  • Other NRAMP alleles may be utilized by themselves in the kit or in combination with the D2S434 allele.
  • 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.
  • Genomic DNA 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.
  • the PCR reactions were carried out in a 10 ⁇ l volume containing 100 ng of genomic DNA (see Example 1), 70 ⁇ M of each dNTP, 1.0 ⁇ M of each primer, 2-2.5 U Taq DNA polymerase, 1 ⁇ Ci of [ 32 P] ⁇ -dCTP (3000 Ci/mmol), 1.5 mM MgCl 2 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).
  • PCR products were resolved by polyacrylamide gel electrophoresis (PAGE).
  • PAGE polyacrylamide gel electrophoresis
  • 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.
  • 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 1)
  • 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 ⁇ in both concordant affected and concordant unaffected groups, but a alter of ⁇ in the discordant group.
  • 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.
  • 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 ( ⁇ ).
  • the proportion of haplotypes shared identical-by-descent between the pairs ( ⁇ ) 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.
  • 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.
  • the D2S434 allele is characterized by repeats of (GATA) tone. 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.
  • 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.
  • Primer A TAAATCACTAGCCTTTGCCG (SEQ.ID NO:2)
  • Primer B GCCATCTGTACTGTTCCCAG (SEQ.ID NO:3)
  • D2S1323 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.
  • the D2S1323 marker is characterized by repeats of (GATA) tone, 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.
  • 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 crosatelhte maikei s at or near the
  • 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 inco ⁇ orated by reference
  • 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 hnka ⁇ e analyses which do not assume a mode of inheritance were employed A statistically significant linkage between the maiker D2S434 and Ulceiative Colitis
  • 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
  • Example 11 Sib pair Analysis foi D2S434 and D2S1323 Haplotypes and UC
  • 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
  • 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
  • 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.
  • ADNRAMPU 5 ' -TGG ACC CAG GAA ACA TCG bp65
  • the RT-PCR is a 50 ⁇ l 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 ⁇ l of total RNA, 20 pmol of each primer, and 1.5 mM MgCL, added.
  • the antisense primer (ADNRAMPD) was used as the gene specific primer for the RT.
  • This RT-PCR yields an expected amplification fragment of 1404 bp.
  • 20-30 ⁇ l 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.
  • GCTGGCCGC ⁇ TATC GGGCCTCAC ⁇ ACCT ⁇ GCTGGCCGCACCCTACCTGSGCCT ⁇ X- ⁇
  • N represents undetermined nucleotide.
  • Top sequence is mutated NRAMP.
  • the mutant sequence contains a deletion at 970 to 1340 with respect to NRAMP (GENBANK).
  • CAC- ⁇ A TT ⁇ CC ⁇ C7G CC ⁇ GM3AGGGCr ⁇
  • Top sequence is mutated NRAMP.
  • Mutant sequence contains at least two inserts which are absent from the NRAMP sequence (GENBANK.).
  • Tahle 15 Mutated NRAMP DNA Sequence
  • CTTCTTCCTC TTCCTCGATA ACTACGGGCT GCGGAAGCTG GGAAGCTTTT TTTGGACTCC 780
  • Mutant sequence includes an insert of 400 or more bp than the non-mutant NRAMP sequence (GENBANK).
  • 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.
  • RNA of the patients allows a better understanding of the role of NRAMP in the pathogenesis of disease.
  • 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.
  • 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.
  • an IBD such as Ulcerative Colitis or Crohn's Disease or subtype(s) thereof

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Abstract

L'invention concerne des méthodes d'identification, de diagnostic et de dépistage d'affections intestinales inflammatoires, en particulier la rectocolite hémorragique, la maladie de Crohn et leurs sous-types, lesquelles méthodes consistent à identifier des allèles et des polymorphismes associés à une réponse biologique en relation avec une affection intestinale inflammatoire, notamment la rectocolite hémorragique. Un procédé permet de déterminer si une thérapie modifiant le niveau ou la fonctionnalité de la protéine associée à la résistance naturelle (NRAMP) est efficace pour traiter une affection intestinale inflammatoire, telle que la rectocolite hémorragique, la maladie de Crohn ou leurs sous-types. L'invention concerne également un kit permettant de déterminer une sensibilité aux affections intestinales inflammatoires, comprenant de l'ADN issu des zones fonctionnelles (gène de structure) ou des zones satellites du locus de la protéine associée à la résistance naturelle, leurs oligonucléotides ou acides nucléiques antisens.
PCT/US1998/022993 1997-10-31 1998-10-30 Methodes d'identification et de diagnostic d'affections intestinales inflammatoires ou de leurs sous-types au moyen du locus de la nramp Ceased WO1999023255A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2806739A1 (fr) * 2000-03-27 2001-09-28 Fond Jean Dausset Ceph Genes impliques dans les maladies inflammatoires de l'intestin et leur utilisation
WO2003052412A3 (fr) * 2001-12-17 2004-02-26 Oxagen Ltd Liaison genetique
WO2012068229A1 (fr) * 2010-11-16 2012-05-24 The Trustees Of The University Of Pennsylvania Compositions et méthodes de traitement des infections à alphavirus
RU2601132C1 (ru) * 2015-11-27 2016-10-27 федеральное государственное автономное образовательное учреждение высшего образования "Московский физико-технический институт (государственный университет)" (МФТИ) Набор синтетических олигонуклеотидов для диагностики болезни крона и неспецифического язвенного колита путем выявления маркерных участков бактериальной днк методом полимеразной цепной реакции

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961866B (zh) * 2021-02-25 2023-05-09 云南农业大学 一种三七中的PnNramp3基因及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021941A1 (fr) * 1994-02-11 1995-08-17 Cedars-Sinai Medical Center Procedes de diagnostic de la rectocolite hemorragique et la maladie de crohn
WO1997025445A1 (fr) * 1996-01-12 1997-07-17 Cedars-Sinai Medical Center Techniques de depistage de la rectocolite hemorragique par detection d'un polymorphisme d'un antagoniste du recepteur de l'interleukine-1
WO1997039146A1 (fr) * 1996-04-12 1997-10-23 Cedars-Sinai Medical Center Procede d'identification et de diagnostic d'affections intestinales inflammatoires non caracteristiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021941A1 (fr) * 1994-02-11 1995-08-17 Cedars-Sinai Medical Center Procedes de diagnostic de la rectocolite hemorragique et la maladie de crohn
WO1997025445A1 (fr) * 1996-01-12 1997-07-17 Cedars-Sinai Medical Center Techniques de depistage de la rectocolite hemorragique par detection d'un polymorphisme d'un antagoniste du recepteur de l'interleukine-1
WO1997039146A1 (fr) * 1996-04-12 1997-10-23 Cedars-Sinai Medical Center Procede d'identification et de diagnostic d'affections intestinales inflammatoires non caracteristiques

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BIOQUE G ET AL: "ALLELIC POLYMORPHISM IN IL-1BETA AND IL-1 RECEPTOR ANTAGONIST (IL-1RA) GENES IN INFLAMMATORY BOWEL DISEASE", CLINICAL AND EXPERIMENTAL IMMUNOLOGY, vol. 102, no. 2, 1995, pages 379 - 383, XP000673099 *
DEGROEN P C ET AL.: "Are there genetic associations of MLH1 and NRAMP with primary sclerosing cholangitis and inflammatory bowel disease ?", HEPATOLOGY, vol. 26, no. 4 Part 2, 1997, pages 1483, XP002094174 *
HOFMEISTER A ET AL.: "The natural resistance-associated macrophage protein gene is associated with Crohn's disease", SURGERY, vol. 122, no. 2, 1997, pages 173 - 179, XP002094173 *
MANSFIELD J C ET AL: "NOVEL GENETIC ASSOCIATION BETWEEN ULCERATIVE COLITIS AND THE ANTI- INFLAMMATORY CYTOKINE INTERLEUKIN-1 RECEPTOR ANTAGONIST", GASTROENTEROLOGY, vol. 106, no. 3, March 1994 (1994-03-01), pages 637 - 642, XP000673119 *
NEIBERGS H L ET AL.: "Linkage analysis supports NRAMP as a major susceptibility locus for ulcerative colitis", AMERICAN JOURNAL OF HUMAN GENETICS, vol. 61, no. 4 Suppl., 1997, pages 280, XP002094171 *
STOKKERS P C F ET AL.: "A polymorphism in the NRAMP1-gene in relation to IBD", GASTROENTEROLOGY, vol. 112, no. 4 Suppl., 1997, pages A1097, XP002094172 *
YANG H ET AL: "INTERCELLULAR ADHESION MOLECULE 1 GENE ASSOCIATIONS WITH IMMUNOLOGIC SUBSETS OF INFLAMMATORY BOWEL DISEASE", GASTROENTEROLOGY, vol. 109, no. 2, August 1995 (1995-08-01), pages 440 - 448, XP002040021 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2806739A1 (fr) * 2000-03-27 2001-09-28 Fond Jean Dausset Ceph Genes impliques dans les maladies inflammatoires de l'intestin et leur utilisation
WO2001072822A3 (fr) * 2000-03-27 2002-07-18 Fond Jean Dausset Ceph Genes impliques dans les maladies inflammatoires de l"intestin et leur utilisation
US7592437B2 (en) 2000-03-27 2009-09-22 Fondation Jean Dausset-Ceph Genes involved in intestinal inflammatory diseases and use thereof
US8137915B2 (en) 2000-03-27 2012-03-20 Fondation Jean Dausset-Ceph Genes involved in intestinal inflammatory diseases and use thereof
WO2003052412A3 (fr) * 2001-12-17 2004-02-26 Oxagen Ltd Liaison genetique
WO2012068229A1 (fr) * 2010-11-16 2012-05-24 The Trustees Of The University Of Pennsylvania Compositions et méthodes de traitement des infections à alphavirus
RU2601132C1 (ru) * 2015-11-27 2016-10-27 федеральное государственное автономное образовательное учреждение высшего образования "Московский физико-технический институт (государственный университет)" (МФТИ) Набор синтетических олигонуклеотидов для диагностики болезни крона и неспецифического язвенного колита путем выявления маркерных участков бактериальной днк методом полимеразной цепной реакции

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