Methods for detecting a polymorphism in the nfkb1 gene promoter

Abstract

The present invention discloses a functional relationship between a recognized disease condition and a polymorphism in the nucleotide factor kappa B promoter (NFKB1). This relationship provides a platform for methods of altering promoter activity and for determining similar relationships between specific pathologies and identified polymorphisms. A statistically significant risk of developing ulcerative colitis was shown to be correlated with the presence of an ATTG insertion/deletion in the NFKB1 promoter and is likely to apply also to a variety of other inflammatory diseases.

1 . An isolated polynucleotide probe for detecting a functional polymorphism in a human NFKB1 promoter, comprising a nucleic acid segment that binds to the functional polymorphic allele position in the promoter but does not bind to wildtype NFKB promoter allele. 
     
     
         2 . An isolated polynucleotide probe for detecting an ATTG D allele polymorphism of a human NFKB1 promoter, wherein said probe specifically binds to the ATTG D allele but exhibits little or no binding to the ATTG W allele of the NFKB1 promoter. 
     
     
         3 . An isolated polynucleotide probe for detecting an ATTG W polymorphism in a mammalian NFKB1 promoter, wherein said probe specifically binds to the ATTG W allele of said promoter but exhibits little or no binding to the ATTG D allele of the NFKB1 promoter. 
     
     
         4 . The isolated polynucleotide probe of  claim 2 , which has an oligonucleotide sequence of about 10-30 nucleotides complementary to a NFKB1 promoter region comprising an ATTG D allele. 
     
     
         5 . The isolated polynucleotide probe of  claim 2  wherein the ATTG D allele polymorphism is a −94 ins/del ATTG in SEQ ID NO:1. 
     
     
         6 . The isolated polynucleotide probe of  claim 2  selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. 
     
     
         7 . The isolated polynucleotide probe of  claim 3  that has the sequence of SEQ ID NO:2. 
     
     
         8 . The isolated polynucleotide probe of  claim 2  or  claim 3  further comprising a detectable label. 
     
     
         9 . The isolated polynucleotide probe of  claim 8  wherein the detectable label is selected from the group consisting of a fluorescent, colorimetric, radio-, and antibody label. 
     
     
         10 . The isolated polynucleotide probe of  claim 9  wherein the label is a radioactive label. 
     
     
         11 . The isolated oligonucleotide probe of  claim 3  wherein the promoter is a human NFKB1 promoter. 
     
     
         12 . The isolated oligonucleotide probe of  claim 2  wherein the ATTG-D allele is an ATTG singlet polymorphism at position −94 of SEQ ID NO:1. 
     
     
         13 . The isolated oligonucleotide probe of  claim 3  wherein the ATTG-W allele is a duplet polymorphism at position −94 of SEQ ID NO:1. 
     
     
         14 - 21 . (canceled) 
     
     
         22 . A mammalian NFKB1 promoter binding protein that selectively binds to NFKB1 −94insATTG duplet promoter but exhibits little or no binding to the NFKB1 −94delATTG singlet allele or to other NFKB1 binding sites. 
     
     
         23 . The NFKB1 promoter binding protein identified as NFKB1-PIP 
     
     
         24 . A kit for detecting a −94ins/del ATTG polymorphism in a NFKB1 promoter, said kit comprising at least one oligonucleotide probe that selectively hybridizes to a −94delATTG, optionally at least one oligonucleotide probe that selectively hybridizes to a −94insATTG polymorphism, and directions for use. 
     
     
         25 . The kit of  claim 24  wherein the oligonucleotide probe is detectably labeled. 
     
     
         26 . The kit of  claim 25  wherein the detectable label is a fluorescent, radio-, antibody or colorimetric label. 
     
     
         27 . The kit of  claim 25  wherein the oligonucleotide probe for detecting the −94delATTG polymorphism is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. 
     
     
         28 . The kit of  claim 25  wherein the oligonucleotide probe for detecting the −94insATTG polymorphism is a probe having the sequence of SEQ ID NO:5. 
     
     
         29 . A method of determining risk or presence of an inflammatory disease in an individual, comprising determining binding of mammalian nuclear proteins to a functional polymorphism sequence in an NFKB1 promoter gene wherein the functional polymorphism sequence is C/C or C/G at exon 1+252 of NFKB1 and wherein said nuclear proteins exhibit decreased binding compared with binding to corresponding wildtype allele of NFKB1. 
     
     
         30 . A method of determining risk or presence of inflammatory bowel disease in an individual, comprising determining binding of mammalian nuclear protein NFKB1-PIP to the −94delATTG allele in NFKB1 promoter, wherein decreased binding of the NFKB1-PIP compared with wildtype −94ins/delATTG polymorphism is indicative of inflammatory bowel disease. 
     
     
         31 . The method of  claim 29  wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis. 
     
     
         32 . The method of  claim 29  wherein decreased binding of NFKB1-PIP to the −94delATTG allele compared to binding to wildtype −94ins/delATTG allele is indicative of increased risk of inflammatory bowel disease. 
     
     
         33 . The method of  claim 31  wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis. 
     
     
         34 . A method for determining the presence of a −94delATTG polymorphism in an individual homozygous or heterozygous for the polymorphism, comprising amplifying genomic DNA with 5′ to 3′ forward and reverse primers designed to bracket positions −91 to −94 in SEQ ID NO:1, cleaving the product by enzyme digestion and identifying the −94 del ATTG polymorphism by selective hybridization with a detectable oligonucleotide probe. 
     
     
         35 . The detection method of  claim 33  wherein the forward primer and reverse primers are selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15. 
     
     
         36 . An isolated human NFKB1 promoter gene comprising a nucleic acid sequence that selectively binds to SEQ ID NO:4. 
     
     
         37 . The promoter of  claim 35  that selectively binds to SEQ ID NO:5. 
     
     
         38 . The promoter of  claim 35  that comprises a −94del ATTG deletion in SEQ ID NO:1. 
     
     
         39 . The promoter of  claim 35  that comprises an ATTG (SEQ ID NO:1 from position −91 to position −96) insertion/deletion. 
     
     
         40 . The promoter gene of  claim 35  consisting essentially of SEQ ID NO: 1 and complements thereof. 
     
     
         41 . An isolated oligonucleotide comprising a contiguous nucleotide sequence of human NKFB1 promoter comprising an ATTG singlet as a polymorphism −94ins/de1ATTG of SEQ ID NO:1 wherein said sequence maintains NKFB1 promoter function. 
     
     
         42 . A method of treating an individual having symptoms of inflammatory bowel disease, comprising administering to said individual a pharmaceutically effective composition that promotes increased binding of nuclear proteins to NKFB1 promoter, wherein the increased binding is comparable to binding of nuclear proteins to wildtype NKFB1 promoter. 
     
     
         43 . A pharmaceutical composition effective to enhance or promote binding of nuclear proteins to a NKFB1 promoter having an ATTG deletion, said composition comprising a nuclear binding polypeptide modified to bind at about 15-20 by 5′ or 3′ of position −94 of SEQ ID NO:1.