Genetic lesion associated with cancer

Abstract

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

1 . An isolated polynucleotide molecule comprising 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 - 11 . (canceled) 
     
     
         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 complementary 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 comprising assaying for the presence of a uracil or thymine to guanine transition at position 4 of LCS6 of KRAS, wherein the transition modulates the binding efficacy of a let-7 family miRNA molecule. 
     
     
         30 . A method of determining a cancer prognosis, the method comprising assaying for the presence of a uracil or thymine to guanine transition at position 4 of LCS6 of KRAS, wherein the presence of the transition indicates a poor prognosis. 
     
     
         31 . A method of determining responsiveness to a form of cancer treatment, the method comprising assaying for the presence of a uracil or thymine to guanine transition a position 4 of LCS6 of KRAS, wherein the presence of the transition predicts whether the cancer is resistant or responsive to the form of treatment. 
     
     
         32 . The method of  claim 31 , wherein the presence of the transition predicts that the cancer is resistant to the form of treatment, and thereby indicates a poor prognosis. 
     
     
         33 . The method of  claim 31 , wherein the presence of the transition predicts that the cancer is responsive to the form of treatment, and thereby indicates an improved prognosis. 
     
     
         34 . 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.   
     
     
         35 . The method of  claim 34 , wherein said control sample comprises SEQ ID NO: 15.