Compositions for detecting epigenetic modifications
A method of detecting a predisposition to, or the incidence of, cancer in a sample comprises detecting an epigenetic change in at least one gene selected from an NDRG4/NDRG2 subfamily gene, GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, wherein detection of the epigenetic change is indicative of a predisposition to, or the incidence of, cancer. Also described are pharmacogenetic methods for determining suitable treatment regimens for cancer and methods for treating cancer patients, based around selection of the patients according to the methods of the invention. The present invention is also concerned with improved methods of collecting, processing and analyzing samples, in particular body fluid samples. These methods may be useful in diagnosing, staging or otherwise characterizing various diseases. The invention also relates to methods for identifying, diagnosing, staging or otherwise characterizing cancers, in particular gastrointestinal cancers such as colorectal cancers, gastric cancers and oesophageal cancers. The methods of the invention relate, inter alia, to isolating and analyzing the human DNA component from faecal samples and blood-based samples.
1. A composition comprising a reaction mixture, the reaction mixture comprising:
sample DNA that is treated with a bisulfite reagent that modifies unmethylated DNA to form treated sample DNA, wherein the sample DNA is from a human;
NDRG4-specific primers that hybridize to a target sequence in the treated sample DNA that is within a promoter region of a human NDRG4 gene that corresponds to positions 1-1000 of methylated and bisulfite-treated sequence SEQ ID NO: 524;
amplified DNA obtained by amplifying the treated sample DNA using the NDRG4-specific primers; and
a fluorophore-labeled NDRG4-specific probe hybridizable to a site in the amplified DNA that is within a sequence that corresponds to positions 1-1000 of methylated and bisulfite-treated sequence SEQ ID NO: 524.
2. The composition of claim 1 , wherein the NDRG4-specific primers hybridize to CpG dinucleotides in a target sequence that is within positions 1-1000 of SEQ ID NO:524.
3. The composition of claim 1 , wherein the fluorophore-labeled NDRG4-specific probe comprises a quencher moiety.
4. The composition of claim 1 , wherein the fluorophore-labeled NDRG4-specific probe hybridizes to CpG dinucleotides in the amplified DNA.
5. The composition of claim 1 , further comprising a DNA polymerase comprising a 5′-3′ exonuclease activity.
6. The composition of claim 1 , wherein the sample DNA is obtained by a method comprising a step of hybridizing DNA from the sample to an oligonucleotide capture probe and isolating DNA hybridized to the oligonucleotide capture probe.
7. The composition of claim 1 , wherein the sample DNA is from a sample comprising one or more of a tissue sample, a fecal sample and/or a bodily fluid sample.
8. The composition of claim 7 , wherein the bodily fluid sample comprises a blood sample and/or a plasma sample.