Aberrant promoter methylation of <i>PPP1R3C</i> and <i>EFHD1</i> in plasma of colorectal cancer patients

<jats:title>Abstract</jats:title><jats:p>Aberrant <jats:styled-content style="fixed-case">DNA</jats:styled-content> methylation is a common epigenetic alteration involved in colorectal cancer (<jats:styled-content style="fixed-case">CRC</jats:styled-content>). In our previous study, we performed methylated <jats:styled-content style="fixed-case">DNA</jats:styled-content> immunoprecipitation‐on‐chip analysis combined with gene re‐expression analysis by 5‐aza‐2′‐deoxycytidine treatment, to identify methylation genes in <jats:styled-content style="fixed-case">CRC</jats:styled-content> genome widely. Among these genes, 12 genes showed aberrant hypermethylation frequently in >75% of 149 <jats:styled-content style="fixed-case">CRC</jats:styled-content> samples but did not in normal samples. In this study, we aim to find out any of these methylation genes to be utilized for <jats:styled-content style="fixed-case">CRC</jats:styled-content> detection using plasma <jats:styled-content style="fixed-case">DNA</jats:styled-content> samples. Primers for methylation‐specific <jats:styled-content style="fixed-case">PCR</jats:styled-content> and pyrosequencing were designed for seven of the 12 genes. Among them, <jats:italic><jats:styled-content style="fixed-case">PPP</jats:styled-content>1R3C</jats:italic> and <jats:italic><jats:styled-content style="fixed-case">EFHD</jats:styled-content>1</jats:italic> were rarely hypermethylated in peripheral blood cells, but frequently hypermethylated in 24 <jats:styled-content style="fixed-case">CRC</jats:styled-content> tissue samples and their corresponding plasma samples. In plasma samples, <jats:italic><jats:styled-content style="fixed-case">PPP</jats:styled-content>1R3C</jats:italic> was methylated in 81% (97/120) of <jats:styled-content style="fixed-case">CRC</jats:styled-content> patients, but only in 19% (18/96) of noncancer patients (<jats:italic>P</jats:italic> = 6 × 10<jats:sup>−20</jats:sup>, Fisher's exact test). In combined analysis with <jats:italic><jats:styled-content style="fixed-case">EFHD</jats:styled-content>1</jats:italic>, both genes were methylated in 53% (64/120) of <jats:styled-content style="fixed-case">CRC</jats:styled-content> patients, but only in 4% (4/96) of noncancer patients (<jats:italic>P</jats:italic> = 2 × 10<jats:sup>−16</jats:sup>), giving high specificity of 96%. At least one of the two genes was methylated in 90% (108/120) of <jats:styled-content style="fixed-case">CRC</jats:styled-content> patients, and 36% (35/96) of control patients, giving high sensitivity of 90%. Compared with low sensitivity of carcinoembryonic antigen (17% at stage I, 40% at stage II) and <jats:styled-content style="fixed-case">CA</jats:styled-content>19‐9 (0% at stage I, 13% at stage II) for early‐stage <jats:styled-content style="fixed-case">CRC</jats:styled-content>s, sensitivity of aberrant methylation was significantly higher: <jats:italic><jats:styled-content style="fixed-case">PPP</jats:styled-content>1R3C</jats:italic> methylation at 92% (11/12) for stage I and 77% (23/30) for stage II, and methylation of at least one gene at 100% (12/12) for stage I and 87% (26/30) for stage II. <jats:italic><jats:styled-content style="fixed-case">PPP</jats:styled-content>1R3C</jats:italic> methylation or its combined use of <jats:italic><jats:styled-content style="fixed-case">EFHD</jats:styled-content>1</jats:italic> methylation was highly positive in <jats:styled-content style="fixed-case">CRC</jats:styled-content> plasma samples, and they might be useful in detection of <jats:styled-content style="fixed-case">CRC</jats:styled-content>, especially for early‐stage <jats:styled-content style="fixed-case">CRC</jats:styled-content>s.</jats:p>