Genetic Lesion Associated With Cancer

Abstract

The invention comprises methods for identifying mutations within the 3′UTR of genes that lead to increased risk or probability of developing cancer.

What is claimed is: 
     
         1 . An isolated polynucleotide molecule comprising of between 10-50 bases of which at least 10 contiguous bases including a polymorphic site are from SEQ ID NO: 21 in which the nucleotide at position 4 of SEQ ID NO: 21 is not a uracil (U) or thymine (T). 
     
     
         2 . The isolated polynucleotide molecule of  claim 1  wherein the nucleotide at position 4 is a guanine (G). 
     
     
         3 . An isolated polynucleotide molecule that is complementary to the isolated polynucleotide molecule of  claim 1 . 
     
     
         4 . An isolated polynucleotide molecule comprising a 3′ untranslated region (UTR) of KRAS, wherein said polynucleotide contains a single nucleotide polymorphism (SNP) within a Let-7 Complementary Site (LCS) that modulates the binding efficacy of said let-7 family miRNA molecule. 
     
     
         5 . The isolated polynucleotide molecule of  claim 4  wherein the SNP occurs at position 4 of LCS6 and wherein the nucleotide at position 4 is a guanine (G). 
     
     
         6 . An isolated polynucleotide molecule that is complementary to the isolated polynucleotide molecule of  claim 4 . 
     
     
         7 . An isolated and purified polynucleotide comprising a sequence of at least 20 nucleotides of a KRAS allele, wherein said polynucleotide contains at least one mutation relative to KRAS shown in SEQ ID NO: 24, said mutation comprising a uracil (U) or thymine (T) to guanine (G) transition at nucleotide 3377 as shown in SEQ ID NO: 24. 
     
     
         8 . An isolated and purified polynucleotide comprising a sequence of at least 20 nucleotides of a KRAS allele, wherein said polynucleotide contains at least one mutation relative to KRAS shown in SEQ ID NO: 25, said mutation comprising a uracil (U) or thymine (T) to guanine (G) transition at nucleotide 3253 as shown in SEQ ID NO: 25. 
     
     
         9 . An isolated polynucleotide selected from the group consisting of:
 (a) a nucleotide sequence comprising SEQ ID NO: 21;   (b) a fragment of said nucleotide sequence, provided that the fragment includes a polymorphic site in said polymorphic sequence;   (c) a complimentary nucleotide sequence comprising a sequence complementary to SEQ ID NO: 21; and   (d) a fragment of said complementary nucleotide sequence, provided that the fragment includes a polymorphic site in said polymorphic sequence.   
     
     
         10 . A method of detecting the LCS6 SNP in a KRAS polynucleotide, the method comprising:
 (a) obtaining a sample of KRAS polynucleotide;   (b) amplifying said KRAS polynucleotide sample by polymerase chain reaction (PCR);   (c) digesting the PCR product of (a) with one or more restriction enzyme(s);   (d) separating the fragments of (b) by gel electrophoresis; and   (e) comparing the pattern of fragment migration of the polynucleotide sample to a control sample, wherein any change from the control pattern indicates the presence of a SNP in the polynucleotide.   
     
     
         11 . The method of  claim 6 , wherein said control sample comprises SEQ ID NO: 15. 
     
     
         12 . A method of identifying a mutation within a Let-7 Complementary Site (LCS) of a test polynucleotide, the method comprising:
 (a) contacting said test polynucleotide to a let-7 family miRNA molecule;   (b) assessing the binding efficacy of said let-7 family miRNA molecule to said test polynucleotide; and   (c) comparing the binding efficacy of said let-7 family miRNA molecule to said test polynucleotide to the binding efficacy of said let-7 family miRNA molecule to a control polynucleotide, wherein an alternation in the binding efficacy to said test polynucleotide compared to said control polynucleotide indicates the presence of a mutation in said test polynucleotide.   
     
     
         13 . A method of identifying a subject at risk for developing a cell proliferative disease, comprising detecting a mutation in the 3′ untranslated region (UTR) of RAS in a patient sample wherein the presence of a mutation indicates greater risk of developing a cell proliferative disease. 
     
     
         14 . A method of predicting the onset of developing a cell proliferative disease in a subject at risk for developing a cell proliferative disease, comprising detecting a mutation in the 3′ untranslated region (UTR) of RAS in a patient sample wherein the presence of a mutation indicates an earlier onset of developing a cell proliferative disease. 
     
     
         15 . The method of  claim 13  or  14 , wherein said mutation modulates the binding efficacy of at least one miRNA. 
     
     
         16 . The method of  claim 13  or  14 , wherein said cell proliferative disorder is cancer. 
     
     
         17 . The method of  claim 16 , wherein said cancer is lung cancer. 
     
     
         18 . The method of  claim 16 , wherein said cancer is a lung, an ovarian, a breast, a uterine, a head and neck, a pancreatic, or a colon cancer. 
     
     
         19 . The method of  claim 13  or  14 , wherein said RAS is HRAS, KRAS, or NRAS. 
     
     
         20 . The method of  claim 14 , wherein said miRNA belongs to the let-7 family of miRNA molecules. 
     
     
         21 . The method of  claim 13  or  14 , wherein said mutation is a single nucleotide polymorphism (SNP). 
     
     
         22 . The method of  claim 13  or  14 , wherein said mutation is a deletion, insertion, inversion, substitution, frameshift, or recombination. 
     
     
         23 . The method of  claim 13  or  14 , wherein said mutation occurs within a let-7 complemetary site (LCS). 
     
     
         24 . The mutation of  claim 14 , wherein said LCS is LCS6. 
     
     
         25 . The method of  claim 13  or  14 , wherein said mutation is a SNP at position 4 of LCS6 of KRAS. 
     
     
         26 . The method of  claim 13  or  14 , wherein said mutation is a SNP comprising a uracil (U) or thymine (T) to guanine (G) transition at position 4 of LCS6 of KRAS. 
     
     
         27 . The method of  claim 13  or  14 , wherein said mutation occurs within a methylated genomic sequence. 
     
     
         28 . The method of  claim 13  or  14 , wherein said mutation occurs within an unmethylated genomic sequence. 
     
     
         29 . A method of identifying a subject at risk for developing a cell proliferative disease, comprising detecting a mutation in the 3′ untranslated region (UTR) of RAS that modulates the binding efficacy of a let-7 family miRNA molecule, wherein the presence of a mutation indicates greater risk of developing a cell proliferative disease.