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WO1997039147A1 - Procedes d'identification et de diagnostic d'une maladie intestinale inflammatoire - Google Patents

Procedes d'identification et de diagnostic d'une maladie intestinale inflammatoire Download PDF

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WO1997039147A1
WO1997039147A1 PCT/US1997/006039 US9706039W WO9739147A1 WO 1997039147 A1 WO1997039147 A1 WO 1997039147A1 US 9706039 W US9706039 W US 9706039W WO 9739147 A1 WO9739147 A1 WO 9739147A1
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tnf
locus
polymorphism
nucleic acid
inflammatory bowel
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PCT/US1997/006039
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WO1997039147B1 (fr
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Scott E. Plevy
Huiying Yang
Stephan R. Targan
Jerome I. Rotter
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Cedars-Sinai Medical Center
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Priority to AU24561/97A priority Critical patent/AU2456197A/en
Publication of WO1997039147A1 publication Critical patent/WO1997039147A1/fr
Publication of WO1997039147B1 publication Critical patent/WO1997039147B1/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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates generally to the field of identifying, diagnosing and screening for inflammatory bowel disease, and more specifically, to methods of identifying, diagnosing and screening for Crohn's Disease.
  • IBD Inflammatory Bowel Disease
  • UC ulcerative colitis
  • CD Crohn's disease
  • Diagnosis depends upon a host of procedures aimed at confirming the suspected diagnosis of CD.
  • the initial symptoms are often confused for non-chronic bowel disorders by physicians unfamiliar with IBD. Consequently, IBD often goes mistreated and undiagnosed until the disease shows its chronicity which results in referral of the patient to a specialist.
  • the imprecise and subjective nature of endoscopic and radiologic examination can result in a misdiagnosis between UC and CD, indeterminate diagnosis of CD.
  • the physician has no means to identify and diagnose clinical subtypes of CD.
  • Histological examination does provide greater certainty of an accurate diagnosis, but the problems of differentiating between UC and CD based on histological findings are often underestimated.
  • the epithelial cell granuloma which is often accorded a key role in the diagnosis of CD, is only found in about 20% of biopsy specimens taken from patients diagnosed with CD. The patient often suffers as the disease progresses before a definitive diagnosis can be made.
  • the selective identification of CD as opposed to UC or other inflammatory conditions of the intestines carries important prognostic and therapeutic implications. For example, when colectomy is indicated, the type of IBD involved determines which surgical options are appropriate.
  • Tumor necrosis factor- ⁇ is a T-cell and monocyte derived cytokine. It can mediate immunological and inflammatory events in Crohn's disease. Increased TNF- ⁇ messenger RNA levels are found in lamina intestinal mononuclear cells (LPMC) from inflamed CD mucosa. Spontaneous TNF- ⁇ protein production has been measured using a reversed ELISA spot assay and TNF- ⁇ mRNA levels has been measured using competitive reverse transcriptase-polymerase chain reaction (RT-PCR) from LPMC from CD, UC and control mucosa.
  • RT-PCR competitive reverse transcriptase-polymerase chain reaction
  • Elevated levels of TNF- ⁇ mRNA and increased numbers of TNF- ⁇ producing cells are found exclusively in LPMC from inflamed CD, but not in uninflamed CD, inflamed or uninflamed UC or normal intestinal mucosa.
  • the present invention provides methods of identifying, diagnosing and screening for IBD, particularly Crohn's Disease (CD) , comprising identifying alleles or an allelic combination and polymorphisms associated with a biological response related to an inflammatory bowel disease.
  • the invention further provides for a method of determining whether a therapy which decreases the levels of TNF- ⁇ would be effective in treating an inflammatory bowel disease.
  • the invention also provides for an assay system for screening for the diagnosis or susceptibility to inflammatory bowel disease.
  • Figure 1 is a schematic of a portion of Chromosome 6 demonstrating the mapping of the TNF locus; specifically, the microsatellite loci approximate the TNF- ⁇ and TNF- ⁇ loci.
  • FIG. 2A demonstrates that total TNF production from lectin
  • Figure 2C demonstrates that patients who had the TNF haplotype a2blc2d4el, when compared patients who had none of these individual alleles, had increased TNF responses to Con A and
  • Figure 3 demonstrates that a particular allele within a microsatellite haplotype is associated with increased TNF production in CD.
  • Figure 4 demonstrates that the TNF microsatellite haplotype a2blc2d4el is not linked to all TNF- ⁇ dysregulated allelic variations in CD patients.
  • the present invention provides methods of identifying, diagnosing and screening for inflammatory bowel disease, particularly Crohn's Disease (CD) .
  • These methods include identifying an inflammatory bowel disease or a subtype thereof in a subject comprising: detecting an allele or an allelic combination at a TNF locus by analyzing a nucleic acid from a subject; identifying a biological response related to the inflammatory bowel disease; and, identifying a correlation between the allele or allelic combination and the biological response.
  • a further embodiment includes a method of diagnosing an inflammatory bowel disease in a subject comprising: detecting an allele or an allelic combination at a TNF locus by analyzing a nucleic acid from a subject; and, using a correlation found by the method of claim l to diagnose the inflammatory bowel disease.
  • a further embodiment includes a method of screening for a susceptibility to an inflammatory bowel disease in a subject comprising: detecting an allele or an allelic combination at a TNF locus by analyzing a nucleic acid from a subject; and, using a correlation found by the method of claim 1 to evaluate the susceptibility of the subject for the inflammatory bowel disease.
  • the inflammatory disease is Crohn's Disease
  • the TNF locus is the TNF- ⁇ locus
  • the TNF- ⁇ locus allele is the d4 allele.
  • the invention also provides for a method of identifying an inflammatory bowel disease or a subtype thereof in a subject comprising: detecting a polymorphism at a TNF locus by analyzing a nuc l eic acid from a subject; identifying a biological respo a related to the inflammatory bowel disease; and, identifyinc a correlation between the polymorphism and the biological response.
  • a further embodiment includes a method of diagnosing an inflammatory bowel disease in a subject comprising: detecting a polymorphism at a TNF locus by analyzing a nucleic acid from a subject; and, using a correlation found by the method of claim 1 to diagnose the inflammatory bowel disease.
  • a further embodiment includes a method of screening for a susceptibility to an inflammatory bowel disease in a subject comprising: detecting a polymorphism at a TNF locus by analyzing a nucleic acid from a subject; using a correlation found by the method of claim 1 to evaluate the susceptibility of the subject for the inflammatory bowel disease.
  • the polymorphism can comprise any nucleotide substitution, addition, deletion or combination thereof.
  • the nucleotide substitution comprises a substitution in the 5' regulatory region of a TNF- ⁇ loci.
  • the nucleotide substitution in said TNF- ⁇ loci 5• regulatory region is selected from the group consisting of a guanosine nucleotide substitution for adenosine at the -238 position and a guanosine nucleotide substitution for adenosine at the -308 position.
  • a preferred embodiment is a method of screening for a susceptibility to Crohn's Disease in a subject comprising detecting a d4 allele at a TNF locus by analyzing a nucleic acid from the subject.
  • the invention further provides for a method of determining whether a therapy which decreases the levels of TNF- ⁇ would be effective in treating an inflammatory bowel disease.
  • the invention also provides for an assay system for screening for a susceptibility to an inflammatory bowel disease in a subject comprising nucleic acid encoding a d4 allele.
  • the assay system further comprising one or more nucleic acid primers specific for amplification of nucleic acid encoding a d4 allele.
  • the assay system screens for Crohn's Disease.
  • the methods of the invention can be further used to identify new genetic marker correlations that can identify new subtypes and new categories of various inflammatory bowel diseases, particularly new clinical subtypes of Crohn's disease.
  • inflammatory bowel disease means any disease of the bowels with an infectious, inflammatory, autoimmune or hyperimmune component or response, acute or chronic, including ulcerative colitis and Crohn's disease (CD) .
  • the term "subject" refers to any animal, particularly a mammal, more particularly a human.
  • diagnosis 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.
  • TNF locus refers to any DNA or chromosomal segment encoding for a TNF or which is structurally or functionally associated with or influencing the expression of any TNF gene.
  • the term "related to” refers to a finding that the probability that at least two occurrences happen together or two things co-exist are greater than would be expected by chance alone.
  • the term "susceptibility" refers to a subject having any increased chance or risk, i.e. increased probability, of an occurrence, such as acquiring a disease or condition, over what would be expected by chance alone.
  • the term “screening” refers to any evaluation of a subject for susceptibility to a disease or condition, for example, by identifying any correlation between a genetic profile, an allele, an allelic combination, or a polymorphism and an increased chance of acquiring a disease or condition, i.e. whether a subject is susceptible to acquiring a disease or condition. The term screening also refers to methods of diagnosis.
  • 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 detecting can also include the use of isotopic or non-isotopic detection, such as fluorescent detection or enzyme labeled- oligonucleotides or a 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.
  • the term "increase in the levels of a cytokine” means any increase in the expression of a cytokine, such as TNFs.
  • increases in the level of TNF expression includes increases in TNF- ⁇ and TNF- ⁇ expression. The increases can be due to greater rates of mRNA transcription, greater rates of mRNA translation, longer mRNA or protein half-lifes and the like.
  • a therapy which decreases the levels of TNF- ⁇ means any therapy which decreases the expression, effective concentration or bioavailability of the cytokine.
  • a therapy can decrease the level of TNF expression by decreasing rates of mRNA transcription or translation.
  • the therapy can shorten mRNA or protein half- life.
  • the therapy can neutralize the cytokine's activity, increase its clearance, compartmentalize it, and the like.
  • the therapy can indirectly effect the levels of the cytokine by decreasing the numbers or activity of cell which secrete the cytokine.
  • allele means alternative genes 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.
  • 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 can be either single-stranded or double-stranded.
  • a particularly useful nucleic acid is genomic DNA, complementary DNA or messenger RNA.
  • the methods and assay systems of the invention employ nucleic acid.
  • Nucleic acid of a subject which is suitable for manipulation, such as for diagnoses or screening, in accordance with the present invention may be derived from any nucleated cell sample, and preferably from peripheral mononuclear blood cells.
  • the term "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 an oligonucleotide probe or primer will form a hydrogen bonded, sequence-specific association with, i.e. specifically 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 incorporated by reference.
  • the term "amplification” refers to the generation of specific DNA sequences using specific oligonucleotide primers through the use of techniques generally described as polymerase chain reaction (PCR) , which are well known in the art, described for example in PCR Technology, Erlich, Ed., Stockton Press, New York, N.Y, (1989) incorporated herein by reference; and, Sambrook et al . and Mullis et al . , both of which have been incorporated by reference.
  • the term “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.
  • Detecting or identifying the presence or absence of nucleic acid of a subject encoding TNF alleles and polymorphisms associated with CD may be accomplished by any means.
  • 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.
  • Inflammatory bowel disease has been classified into the broad categories of Crohn's Disease (CD) and ulcerative colitis (UC) .
  • CD also called regional enteritis, is a disease of chronic inflammation that can involve any part of the gastrointestinal tract.
  • CD commonly involves the duodenum and stomach, and more rarely the esophagus and oral cavity.
  • CD Crohn's disease
  • 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.
  • the inflammation associated with CD involves 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 also is discontinuous in that segments of inflamed tissue, known as “skip lesions,” are separated by apparently normal intestine.
  • skip lesions segments of inflamed tissue
  • linear ulcerations, edema, and inflammation of the intervening tissue lead to a "cobblestone" appearance of the intestinal mucosa, which is distinctive of CD.
  • a hallmark of CD is the presence of discrete aggregations of inflammatory cells, known as granulomas, which are generally found in the submucosa.
  • CD cases display the typical discrete granulomas, while others show a diffuse granulomatous reaction or nonspecific transmural inflammation.
  • the presence of discrete granulomas is indicative of CD, although the absence granulomas also is consistent with the 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 incorporated 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.
  • the manif ⁇ "tations of UC vary widely.
  • 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 (Rubin and Farber, supra , 1994) .
  • patient with Crohn's disease is synonymous with the term "a subject diagnosed with having an inflammatory bowel disease” wherein 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; or a skip lesion, including histologic rectal sparing in the absence of local therapy.
  • Patients with chronic inflammatory bowel disease generally are characterized as having either Crohn's disease or ulcerative colitis to describe specific patterns of disease, to predict outcomes based on expected natural histories, and to help guide medical and surgical treatment strategies.
  • Clinical, endoscopic, and histopathologic criteria as discussed above, have been developed to classify patients into one or the other category.
  • 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 that such clinical subtypes of CD can be diagnosed by identifying a TNF locus allele or allelic combination or a TNF polymorphism that is correlated with a biological response related to a inflammatory bowel disease.
  • the invention further provides for a method of determining whether a therapy which decreases the levels of TNF- ⁇ would be effective in treating an inflammatory bowel disease.
  • the present invention provides non-invasive methods to diagnose, screen for and distinguish clinical subtypes of CD from UC, and assay systems to accomplish the same.
  • the invention also demonstrates, using nonparametric linkage methods, that there is a genetic linkage of CD to the TNF locus (nonparametric statistics are statistical calculations that are not based on any prior assumptions with respect to the variable and the probability distribution of the data) .
  • nonparametric statistics are statistical calculations that are not based on any prior assumptions with respect to the variable and the probability distribution of the data.
  • Example I demonstrates an analysis of 29 multiplex families by several nonparametric linkage methods supporting linkage of CD to the TNF locus. The results demonstrate that at least one gene involved in the pathogenesis of CD is located on the short arm of chromosome 6 which contains the human major histocompatibility complex (MHC) .
  • MHC human major histocompatibility complex
  • kits for screening for CD comprising detecting the presence or absence of nucleic acid of a subject encoding TNF alleles and polymorphisms associated with CD, wherein the presence of nucleic acid encoding the allele or the polymorphism is indicative, i.e. predictive, of CD.
  • the alleles can be TNF microsatellite alleles.
  • TNF microsatellite alleles associated with CD include the a2, bl, c2, d4 and el TNF microsatellite alleles.
  • Nucleic acid encoding TNF alleles and polymorphisms associated with CD can be identified or detected in accordance with the present invention by amplifying the nucleic acid and identifying the TNF alleles and polymorphisms.
  • TNF alleles can be identified by assaying nucleic acid of a subject for defining characteristics of TNF polymorphisms or alleles associated with CD and comparing the results to a positive and or negative control. Defining characteristics of nucleic acid encoding TNF alleles and polymorphisms associated with CD include, 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 region of the chromosome, such as from the MHC and the TNF loci.
  • One skilled in the art would be able to identify primers suitable for use in amplifying and sequencing any particular nucleic acid using published sequence databanks.
  • the map and sequence of the human MHC and TNF locus is available from Genbank as part of the human genome project, NCBI, NIH, easily searchable, for example, on the internet world wide web at http://www.ncbi.nlra.nih.gov. NCBI's database is herein incorporated by reference in its entirety.
  • Primers suitable for use in hybridizing, amplifying or sequencing nucleic acid encoding TNF locus DNA, TNF alleles, particularly the d4 allele, are also provided in Plevy, S.E., et al . , PCT US95/06107, WO 95/31575, which is herein incorporated by reference in its entirety.
  • Methods for identifying, isolating, manipulating, sequencing and analyzing DNA from patient samples are well known in the art, for example, see Molecular Cloning, 2nd Ed., Sambrook, J. , et al . , Eds., Cold Spring Harbor Laboratory Press, Plainview, NY (1989); and, The Polymerase Chain Reaction, Mullis, K.B., et al . , Eds., Maple Press Co., York, PA, each of which are incorporated herein by reference in their entirety.
  • An allele or polymorphism at a polymorphic locus can be identified or detected by a variety of methods including assays using the polymerase chain reaction (PCR) .
  • Oligonucleotide hybridization such as allele-specific hybridization (see Mullis et al., Id . , incorporated by reference) , denaturing gradient gel electrophoresis (see, for example, Innis 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.
  • TNF microsatellites are characterized by a series of CA/GT or CT/CA dinucleotide sequence repeats.
  • Figure 1 depicts a map of the TNF microsatellite loci at the TNF locus.
  • Nedospasov S.A., et al ., ; Jongeneel, C.V. , et al . ,- Udalova, I.A., et al . ; and, Pociot, F. , et al . , as indicated above, where each has been incorporated by reference.
  • Primer pairs suitable for use in the practice of the present invention are linear oligonucleotides ranging in length from about ten to about thirty nucleotides in length.
  • One of the primers in the pair should be complementary to a nucleotide sequence upstream of the nucleic acid encoding the TNF locus targeted for amplification.
  • the other primer should be complementary to a sequence located down stream of this target site.
  • the primers suitable for use in the present invention are specific for amplification of nucleic acid encoding the TNF locus do not prime amplification of nucleic acid which does not encode the TNF locus.
  • the sequence 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 TNF alleles or polymorphisms associated with CD will allow, provided that an appropriate control is used. For example, if the presence or absence of nucleic acid of a subject encoding the TNF locus associated with CD 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 polymorphic size differences (by inclusion, for example, of polymorphic size differences which may be present in regions flanking the TNF locus) .
  • Primers suitable for use in amplifying nucleic acid encoding the TNF locus can be constructed using the oligonucleotide primer sequences described in Plevy, S.E., et al., PCT US95/06107, WO 95/31575, the cell lines described in Udalova, I.A., et al . , Genomics , 16:180-186 (1993) and deposited with the ASHI and the CEPH, and the map and sequence of the TNF locus available from Genbank, NIH (all references have been incorporated herein by reference) . Assay systems for use in screening for CD and distinguishing CD from UC are also provided by the present invention.
  • Such assay systems can 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 TNF alleles or polymorphisms associated with CD.
  • Assay systems of the present invention may contain, for example: nucleic acid encoding TNF alleles or polymorphisms associated with CD; nucleic acid encoding TNF locus, including TNF alleles, not known to be associated with CD; the nucleic acid sequence of TNF alleles or TNF polymorphisms; one or more labeled oligonucleotide probes specific for a particular TNF locus allele or polymorphism; one or more primers for amplification of nucleic acid encoding TNF DNA, such as TNF alleles or polymorphisms; reagents commonly used for amplification; polymerases such as DNA or reverse transcriptase DNA polymerase; antibody specific for, or which binds particular TNF alleles or TNF polymorphisms; and, combinations of any of the above.
  • these suggested assay system components may be packaged in a manner customary for use by those of skill in the art.
  • these suggested assay system components may be provided in solution or as a liquid dispersion or the like.
  • the assay system of the invention can be in the form of semi-automated or entirely automated systems, including robotic systems, for the diagnosis or for the evaluation for a susceptibility to disease, particularly CD.
  • a presently preferred embodiment of the inventive assay systems for use in screening for CD or distinguishing CD from UC comprises DNA encoding two or more TNF alleles or polymorphisms associated with CD in Tris-EDTA buffer solution preferably kept at 4°C or lyophilized.
  • TNF locus alleles can be selected from a group comprising the a2, bl, c2, d4 and el TNF microsatellite alleles.
  • Another embodiment of the inventive assay systems for use in screening for CD or distinguishing CD from UC further comprises one or more primers specific for amplification of nucleic acid encoding TNF microsatellite alleles, for example, primers as described in Plevy, S.E., et al . , PCT US95/06107, WO 95/31575 (which has been incorporated by reference) .
  • Yet another embodiment of the inventive assay systems for use in screening for CD or distinguishing CD from UC further comprises sequencing markers ranging in size from about 80 to 200 base pairs.
  • EXAMPLE I To demonstrate a genetic linkage of CD to the TNF locus, the invention provides for an analysis of 29 multiplex families by several nonparametric linkage methods. A genetic association has been reported in CD using groups of polymorphic microsatellite markers at or near the TNF locus within the MHC on the short arm of human chromosome 6. However, a showing of a "genetic association" cannot distinguish between the possibilities that the MHC region actually contains genes that contribute to the pathogenesis of CD or simply whether the "association" results from population stratification. To distinguish between these possibilities, a linkage study was performed which tests whether a disease cosegregates in families with the marker locus and therefore is independent of specific alleles.
  • Genomic DNA from 29 families with two or more siblings affected with CD was obtained from the IBD cell bank at Cedars-Sinai Medical Center, Los Angeles, CA.
  • TNF microsatellite alleles at five loci: a, b, c, d, e were typed by polymerase chain reaction (PCR), see Plevy, S.E., et al . , PCT US95/06107, WO 95/31575, which is herein incorporated by reference in its entirety. Alleles were interpreted independently by two investigators blinded to diagnosis and f amily pedigrees. As there is no clear pattern of Mendelian inheritance for CD, nonparametric linkage analyses which do not assume a mode of inheritance were employed.
  • SIBPAL Sib-Pair Linkage Program
  • SIBPAL Sib-Pair Linkage Program
  • Version 2.6 authored by Tran, L.D., et al., Louisiana State University Medical Center, New La
  • the program package S.A.G.E. (1994) or Statistical Analysis for Genetic Epidemiology, Release 2.2, Dept. Of Biometry and Genetics, LSU Medical Center, is supported by a U.S. Public Health Service Resource Grant 1 P41 RR03655, from the Division of Research Resources) .
  • affected sibpairs 44 share one or two TNF microsatellite haplotypes, which is greater than the expected value of 35.25 at the 0.005 significance level.
  • TNF levels in CD have a genetic component
  • experiments determined that there is a heterogeneity in TNF production between UC and CD patients and between different allelic combinations, or haplotypes, of CD.
  • total TNF production from lectin (Con A) and phorbol ester-activated PBMC of CD patients is as a whole greater than TNF production in UC patients.
  • the TNF haplotype A2B1C2D4E1 correlates with increases in TNF- ⁇ protein production in patients with CD.
  • the present invention demonstrates that the TNF haplotype A2B1C2D4E1 defines a subtype of CD.
  • Total TNF and TNF- ⁇ bioactivity were measured at various time points using PBMC from CD and UC patients.
  • the PBMC were activated with optimal concentrations of LPS, PHA and concanavalin A (Con A) plus the phorbol ester phorbol myristic acid (PMA) .
  • PBMC are isolated from peripheral blood using Percoll Gradient and immediately cultured at 2xl0 6 cells per ml. in RPMI with 10% fetal calf serum (FCS).
  • TNF- ⁇ and TNF- ⁇ bioactivity are determined at intervals from 24 to 60 hours. Following activation, the cells are isolated and then stored in aliquots until assayed at minus 70°C. Total TNF bioactivity is determined by L929 fibrobiast cytotoxicity assay.
  • TNF production from PBMC activated with Con A and PMA for 24 hours was higher in patients with CD than UC.
  • TNF production from UC patients was about 10 3 picograms per ml, while TNF production from CD patients was about 4xl0 3 picograms per ml.
  • Other stimuli showed no differences, suggesting that responsiveness to phorbol ester can define a unique characteristic in CD.
  • results from patients who had the TNF haplotype a2blc2d4el were compared to the results from patients who had none of the individual alleles. As shown in Figure 2C, this comparison defined a more homogeneous subtype of patient that had increased TNF responses to Con A and PMA stimulation.
  • TNF production from CD patients without any alleles from the haplotype a2blc2d4el was about lxlO 3 picograms per ml, while TNF production from CD patients with haplotype a2blc2d4el was about 4xl0 3 picograms per ml.
  • the CD group as a whole had a greater TNF secretion than the UC group.
  • the subgroup of CD patients with the TNF allelic combination a2blc2d4el appeared to account for the increased TNF production in CD.
  • these data support a method of determining whether a therapy which decreases the levels of TNF- ⁇ would be effective in treating an inflammatory bowel disease.
  • the method would comprise identifying a TNF locus allelic combination, such as the TNF allelic combination a2blc2d4el, by analyzing a nucleic acid from a subject diagnosed with having an inflammatory bowel disease.
  • the increased TNF- ⁇ production in a subset of CD patients also correlated with the presence of the TNF allelic combination a2blc2d4el, and particularly the d4 allele expressed in the "d" locus.
  • these data support a method of determining whether a therapy which decreases the levels of TNF- ⁇ would be effective in treating an inflammatory bowel disease.
  • the method would comprise identifying a TNF allele, such as the d4 allele, by analyzing a nucleic acid from a subject diagnosed with having an inflammatory bowel disease. Confirming the presence of an allele known to have a correlation with increased levels of TNF- ⁇ and IBD allows an evaluation of the effectiveness of the therapy. The evaluation would effect diagnosis, selection of patients, election of treatment modalities and prognosis after treatment to decrease TNF levels.
  • TNF polymorphisms exist in functionally significant regions of the TNF locus. Two polymorphisms have been described in the 5' regulatory region of the human TNF- ⁇ gene. One has guanosine nucleotide substitution for adenosine at the -238 position and another allele has the same substitution at the -308 position. These single base changes were analyzed in eight CD patients with the TNF haplotype a2blc2d4el. It was found that neither of the guanosine nucleotide substitution alleles is linked to the a2blc2d4el haplotype.
  • these results demonstrate novel methods to define new polymorphisms in TNF locus gene regulatory regions. Furthermore, these data support a method of determining whether a therapy which decreases the levels of TNF- ⁇ would be effective in treating an inflammatory bowel disease.
  • the method would comprise identifying a TNF polymorphism, such as the TNF- ⁇ guanosine nucleotide substitution for adenosine at the -238 position and the -308 position, by analyzing a nucleic acid from a subject diagnosed with having an inflammatory bowel disease. Confirming the presence of a TNF polymorphism known to have a correlation with increased levels of TNF- ⁇ and IBD allows an evaluation of the effectiveness of the therapy. The evaluation would effect diagnosis, selection of patients, election of treatment modalities and prognosis after treatment to decrease TNF levels.

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Abstract

L'invention concerne des procédés destinés à identifier, à diagnostiquer et à rechercher par criblage une maladie intestinale inflammatoire, en particulier, la maladie de Crohn (CD), consistant à identifier des allèles et des polymorphismes associés à une réaction biologique relative à une maladie intestinale inflammatoire. Elle concerne également un procédé destiné à déterminer si une thérapie diminuant les niveaux de TNF-α serait efficace pour traiter une maladie intestinale inflammatoire. Elle concerne également une méthode de détermination et de recherche d'une sensibilité à une maladie intestinale inflammatoire.
PCT/US1997/006039 1996-04-12 1997-04-11 Procedes d'identification et de diagnostic d'une maladie intestinale inflammatoire WO1997039147A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2336431A (en) * 1998-04-17 1999-10-20 Johnson & Johnson Medical Ltd Method of analysis of chronic ulcers
WO2003031651A3 (fr) * 2001-10-10 2003-11-27 Oxagen Ltd Maladie intestinale inflammatoire

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031575A1 (fr) * 1994-05-17 1995-11-23 Cedars-Sinai Medical Center Procedes pour depister la maladie de crohn a l'aide d'alleles microsatellites du facteur de necrose tumorale

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031575A1 (fr) * 1994-05-17 1995-11-23 Cedars-Sinai Medical Center Procedes pour depister la maladie de crohn a l'aide d'alleles microsatellites du facteur de necrose tumorale

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOUMA G ET AL: "Distribution of four polymorphisms in the tumour necrosis factor (TNF) genes in patients with inflammatory bowel disease (IBD)", CLINICAL AND EXPERIMENTAL IMMUNOLOGY, vol. 103, no. 3, March 1996 (1996-03-01), pages 391 - 6, XP002038832 *
BOUMA G ET AL: "Secretion of tumour necrosis factor alpha and lymphotoxin alpha in relation to polymorphisms in the TNF genes and HLA-D alleles. Relevance for inflammatory bowel disease", SCANDINAVIAN JOURNAL OF IMMUNOLOGY, vol. 43, no. 4, April 1996 (1996-04-01), pages 456 - 63, XP002038831 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2336431A (en) * 1998-04-17 1999-10-20 Johnson & Johnson Medical Ltd Method of analysis of chronic ulcers
WO1999054499A1 (fr) * 1998-04-17 1999-10-28 Johnson & Johnson Medical Limited Procede d'analyse de plaies chroniques
GB2336431B (en) * 1998-04-17 2003-06-25 Johnson & Johnson Medical Ltd Method of analysis of chronic wounds
WO2003031651A3 (fr) * 2001-10-10 2003-11-27 Oxagen Ltd Maladie intestinale inflammatoire

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