Detecting colorectal neoplasia

Abstract

Provided herein is technology for colorectal neoplasia screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of colorectal neoplasia in 1) individuals at, older or younger than 50 years of age, or 2) individuals having Lynch Syndrome.

We claim: 
     
         1 . A method of characterizing a biological sample, the method comprising:
 determining a methylation profile in at least one differentially methylated region (DMR) of a DNA sample obtained from a subject having or suspected of having colorectal neoplasia by treating the sample with a reagent that modifies DNA in a methylation-specific manner;   wherein the at least one DMR is from NDRG4 and/or ARHGEF4.   
     
     
         2 . The method of  claim 1 , wherein the at least one DMR is from NDRG4. 
     
     
         3 . The method of  claim 1 , wherein the at least one DMR is from ARHGEF4. 
     
     
         4 . The method of  claim 1 , wherein the at least one DMR is from NDRG4 and ARHGEF4. 
     
     
         5 . The method of  claim 1 , wherein the biological sample is a stool sample, a tissue sample, a blood sample, or a urine sample. 
     
     
         6 . The method of  claim 5 , wherein the tissue sample comprises at least one of stomach tissue, pancreatic tissue, bile duct/liver tissue, and/or colorectal tissue. 
     
     
         7 . The method of  claim 1 , wherein the at least one DMR comprises an increased methylation percentage as compared to a control DNA sample, or an increased hypermethylation ratio as compared to a control DNA sample. 
     
     
         8 . The method of  claim 7 , wherein the control sample is from a human subject that does not have colorectal neoplasia. 
     
     
         9 . The method of  claim 1 , wherein the reagent that modifies DNA in a methylation-specific manner is a borane reducing agent. 
     
     
         10 . The method of  claim 1 , wherein the reagent that modifies DNA in a methylation-specific manner comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. 
     
     
         11 . The method of  claim 1 , wherein determining the methylation profile of at least one DMR comprises amplifying at least a portion of the DMR using a set of primers for NDRG4 comprising SEQ ID NOs: 35 and 36. 
     
     
         12 . The method of  claim 1 , wherein determining the methylation profile of at least one DMR comprises amplifying at least a portion of the DMR using a set of primers for ARHGEF4 comprising SEQ ID NOs: 17 and 18. 
     
     
         13 . The method of  claim 1 , wherein determining the methylation profile of at least one DMR comprises performing at least one of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and/or bisulfite genomic sequencing PCR. 
     
     
         14 . The method of  claim 1 , wherein determining the methylation profile of at least one DMR comprises determining the presence or absence of methylation at one or more CpG sites. 
     
     
         15 . The method of  claim 14 , wherein the one or more CpG sites are present in a coding region, a non-coding region, and/or a regulatory region of NDRG4 and/or ARHGEF4. 
     
     
         16 . The method of  claim 1 , wherein determining the methylation profile of at least one DMR comprises determining a methylation frequency. 
     
     
         17 . The method of  claim 1 , wherein determining the methylation profile of at least one DMR comprises determining a methylation pattern. 
     
     
         18 . The method of  claim 1 , wherein the subject is a human subject. 
     
     
         19 . The method of  claim 1 , wherein the biological sample is obtained from the subject, and wherein the method further comprises extracting the DNA sample from the biological sample.