DETECTING HEPATOCELLULAR CARCINOMA
Provided herein is technology for hepatocellular carcinoma screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of hepatocellular carcinoma.
1 . A method for characterizing a biological sample comprising:
measuring a methylation level of a CpG site for one or more genes selected from either
ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1 in a biological sample of a human individual through
treating genomic DNA in the biological sample with bisulfite;
amplifying the bisulfite-treated genomic DNA using a set of primers for the selected one or more genes; and
determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR.
2 . The method of claim 1 further comprising
comparing the methylation level to a methylation level of a corresponding set of genes in control samples without HCC; and
determining that the individual has HCC when the methylation level measured in the one or more genes is different (e.g., higher) than the methylation level measured in the respective_control samples.
3 . The method of claim 1 wherein the following set of primers for the selected one or more genes are used if the biological sample is a tissue sample:
for ACP1 a set of primers consisting of SEQ ID NOS: 10 and 11 or SEQ ID NOS: 58 and 59,
for BDH1 a set of primers consisting of SEQ ID NOS: 13 and 14 or SEQ ID NOS: 61 and 62,
for Chr12.133 a set of primers consisting of SEQ ID NOS: 25 and 26 or SEQ ID NOS: 49 and 50,
for CLEC11A a set of primers consisting of SEQ ID NOS: 7 and 8 or SEQ ID NOS: 52 and 53,
for DAB2IP a set of primers consisting of SEQ ID NOS: 4 and 5 or SEQ ID NOS: 46 and 47,
for DBNL a set of primers consisting of SEQ ID NOS: 34 and 35 or SEQ ID NOS: 82 and 83,
for EMX1 a set of primers consisting of SEQ ID NOS: 16 and 17 or SEQ ID NOS: 91 and 92,
for EFNB2 a set of primers consisting of SEQ ID NOS: 37 and 38 or SEQ ID NOS: 64 and 65,
for HOXA1 a set of primers consisting of SEQ ID NOS: 28 and 29 or SEQ ID NOS: 88 and 89,
for LRRC4 a set of primers consisting of SEQ ID NOS: 40 and 41 or SEQ ID NOS: 85 and 86,
for SPINT2 a set of primers consisting of SEQ ID NOS: 31 and 32 or SEQ ID NOS: 55 and 56,
for TSPYL5 a set of primers consisting of SEQ ID NOS: 1 and 2 or SEQ ID NOS: 43 and 44,
for CCNJ_3707 a set of primers consisting of SEQ ID NOS: 67 and 68,
for CCNJ_3124 a set of primers consisting of SEQ ID NOS: 70 and 71,
for PFKP a set of primers consisting of SEQ ID NOS: 73 and 74,
for SCRN1 a set of primers consisting of SEQ ID NOS: 76 and 77, and
for ECE1 a set of primers consisting of SEQ ID NOS: 79 and 80.
4 . The method of claim 1 , wherein the biological sample is a plasma sample or a tissue sample.
5 . The method of claim 1 , wherein said CpG site is present in a coding region or a regulatory region.
6 . The method of claim 1 , wherein said measuring the methylation level a CpG site for one or more genes comprises a determination selected from the group consisting of determining the methylation score of said CpG site and determining the methylation frequency of said CpG site.
7 . The method of claim 1 , wherein the one or more genes is Chr12.133, CLEC11A, EMX1, HOXA1, and CCNJ_3707.
8 . A method for characterizing a plasma sample comprising:
(a) measuring a methylation level of a CpG site for two or more genes selected from ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1 in a plasma sample of a human individual through
treating genomic DNA in the biological sample with bisulfite;
amplifying the bisulfite-treated genomic DNA using the following set of primers for the selected two or more genes:
for ACP1 a set of primers consisting of SEQ ID NOS: 10 and 11 or SEQ ID NOS: 58 and 59,
for BDH1 a set of primers consisting of SEQ ID NOS: 13 and 14 or SEQ ID NOS: 61 and 62,
for Chr12.133 a set of primers consisting of SEQ ID NOS: 25 and 26 or SEQ ID NOS: 49 and 50,
for CLEC11A a set of primers consisting of SEQ ID NOS: 7 and 8 or SEQ ID NOS: 52 and 53,
for DAB2IP a set of primers consisting of SEQ ID NOS: 4 and 5 or SEQ ID NOS: 46 and 47,
for DBNL a set of primers consisting of SEQ ID NOS: 34 and 35 or SEQ ID NOS: 82 and 83,
for EMX1 a set of primers consisting of SEQ ID NOS: 16 and 17 or SEQ ID NOS: 91 and 92,
for EFNB2 a set of primers consisting of SEQ ID NOS: 37 and 38 or SEQ ID NOS: 64 and 65,
for HOXA1 a set of primers consisting of SEQ ID NOS: 28 and 29 or SEQ ID NOS: 88 and 89,
for LRRC4 a set of primers consisting of SEQ ID NOS: 40 and 41 or SEQ ID NOS: 85 and 86,
for SPINT2 a set of primers consisting of SEQ ID NOS: 31 and 32 or SEQ ID NOS: 55 and 56,
for TSPYL5 a set of primers consisting of SEQ ID NOS: 1 and 2 or SEQ ID NOS: 43 and 44,
for CCNJ_3707 a set of primers consisting of SEQ ID NOS: 67 and 68,
for CCNJ_3124 a set of primers consisting of SEQ ID NOS: 70 and 71,
for PFKP a set of primers consisting of SEQ ID NOS: 73 and 74,
for SCRN1 a set of primers consisting of SEQ ID NOS: 76 and 77, and
for ECE1 a set of primers consisting of SEQ ID NOS: 79 and 80; and
determining the methylation level of the CpG site by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR;
(b) comparing the methylation level to a methylation level of a corresponding set of genes in control samples without stomach cancer; and
(c) determining that the individual has HCC when the methylation level measured in the two or more genes is different (e.g., higher) than the methylation level measured in the respective_control samples.
9 . The method of claim 8 , wherein said CpG site is present in a coding region or a regulatory region.
10 . The method of claim 8 , wherein said measuring the methylation level a CpG site for two or more genes comprises a determination selected from the group consisting of determining the methylation score of said CpG site and determining the methylation frequency of said CpG site.
11 . The method of claim 8 , wherein the two or more genes is Chr12.133, CLEC11A, EMX1, HOXA1, and CCNJ_3707.
12 . A kit comprising two or more of:
1) a bisulfite reagent;
2) a control nucleic acid comprising a sequence from a DMR selected from ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1, and having a methylation state associated with a subject who does not have a cancer;
3) one or more of the following set of primers for ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1:
for ACP1 a set of primers consisting of SEQ ID NOS: 10 and 11 or SEQ ID NOS: 58 and 59,
for BDH1 a set of primers consisting of SEQ ID NOS: 13 and 14 or SEQ ID NOS: 61 and 62,
for Chr12.133 a set of primers consisting of SEQ ID NOS: 25 and 26 or SEQ ID NOS: 49 and 50,
for CLEC11A a set of primers consisting of SEQ ID NOS: 7 and 8 or SEQ ID NOS: 52 and 53,
for DAB2IP a set of primers consisting of SEQ ID NOS: 4 and 5 or SEQ ID NOS: 46 and 47,
for DBNL a set of primers consisting of SEQ ID NOS: 34 and 35 or SEQ ID NOS: 82 and 83,
for EMX1 a set of primers consisting of SEQ ID NOS: 16 and 17 or SEQ ID NOS: 91 and 92,
for EFNB2 a set of primers consisting of SEQ ID NOS: 37 and 38 or SEQ ID NOS: 64 and 65,
for HOXA1 a set of primers consisting of SEQ ID NOS: 28 and 29 or SEQ ID NOS: 88 and 89,
for LRRC4 a set of primers consisting of SEQ ID NOS: 40 and 41 or SEQ ID NOS: 85 and 86,
for SPINT2 a set of primers consisting of SEQ ID NOS: 31 and 32 or SEQ ID NOS: 55 and 56,
for TSPYL5 a set of primers consisting of SEQ ID NOS: 1 and 2 or SEQ ID NOS: 43 and 44,
for CCNJ_3707 a set of primers consisting of SEQ ID NOS: 67 and 68,
for CCNJ_3124 a set of primers consisting of SEQ ID NOS: 70 and 71,
for PFKP a set of primers consisting of SEQ ID NOS: 73 and 74,
for SCRN1 a set of primers consisting of SEQ ID NOS: 76 and 77, and
for ECE1 a set of primers consisting of SEQ ID NOS: 79 and 80.