Novel method for DNA methylation analysis using high-performance liquid chromatography and its clinical application

Abstract

The aim of this study was to develop a new methodology that is suitable for DNA methylation diagnostics and to demonstrate its clinical applicability. We developed a new anion-exchange column for high-performance liquid chromatography (HPLC) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. The DNA methylation levels of synthetic DNA were quantified accurately and reproducibly within 10 minutes without time-consuming pretreatment of PCR products, and the measured values were unaffected by the distribution of methylated CpG within the synthetic DNA fragments. When the DNA methylation status of the FAM150A gene, a marker of the CpG island methylator phenotype specific to clear cell renal cell carcinoma (ccRCC), was examined in 98 patients with ccRCC, bulk specimens of tumorous tissue including cancer cells showing DNA methylation of the FAM150A gene were easily identifiable by simply viewing the differentiated chromatograms, even when the cancer cell content was low. Sixteen ccRCC showing DNA methylation more frequently exhibited clinicopathological parameters reflecting tumor aggressiveness (ie, a larger diameter, higher histological grade, vascular involvement, renal vein tumor thrombi, infiltrating growth, tumor necrosis, renal pelvis invasion and higher pathological TNM stage), and had significantly lower recurrence-free and overall survival rates. These data indicate that HPLC analysis using this newly developed anion-exchange column could be a powerful tool for DNA methylation diagnostics, including prognostication of patients with cancers, in a clinical setting.

Keywords: CpG island methylator phenotype; DNA methylation diagnostics; anion-exchange column for high-performance liquid chromatography; clear cell renal cell carcinoma; prognostication.

Figure 1

Schematic view of the newly developed packing material with electrostatic and hydrophobic properties for the high‐performance liquid chromatography column

Figure 2

Separation of PCR products amplified from the synthetic DNA fragments by the newly developed anion‐exchange high‐performance liquid chromatography method. A, Chromatograms of synthetic DNA fragments 1, 3, 5, 6, 7, 8, 9 and 10 corresponding to the sequences after bisulfite modification of 0%, 25% 50%, 75% and 100% methylated promoter regions of the FAM150A gene (Table 1). Flow rate, 1.0 mL/min; buffer A, 25 mmol/L Tris‐HCl, pH 7.5; buffer B, 1 mol/L NH ₄(SO ₄)₂ in buffer A; linear gradient from 40%‐100% buffer B in 10 minutes; column temperature, 70°C; injection volume, 5 μL. B, Enlarged view of the chromatograms of (A); retention time: 7‐8.4 minutes. C, Linearity of the decrease in the retention time associated with an increase in the methylation level from 0% to 100%. D, Within‐run reproducibility test using PCR products obtained from Fragments 2, 3 and 4 corresponding to the sequences after bisulfite modification of 20%, 25% and 30% methylated promoter regions of the FAM150A gene (Table 1). Error bar: SD

Figure 3

High‐performance liquid chromatography (HPLC) analysis using the newly developed anion‐exchange column for tissue specimens of clear cell renal cell carcinoma (ccRCC). A, Representative chromatograms. PCR products of 384 bp encompassing the promoter region of the FAM150A gene from the 98 ccRCC were subjected to HPLC analysis. Case 3 ccRCC showed a single peak with retention times similar to the unmethylated DNA control. In Cases 84 and 89 ccRCC, a single peak with a shoulder was observed. In Case 91 ccRCC, a bimodal peak pattern consisting of 2 peaks which had retention times similar to the unmethylated and methylated DNA was observed. Chromatograms of all of the 98 ccRCC examined are shown in Figure S1. B, Differential processing of the chromatograms. The differential patterns of chromatograms in (A) are shown in this panel. In Case 3, a chromatogram showing a “single peak” pattern was converted to a differential curve with 1 upward convex portion. In Cases 84 and 89, chromatograms showing a “single peak with a shoulder” pattern were converted to differential curves with 2 upward convex portions. In Case 91, a “bimodal peak” pattern chromatogram was converted to a differential curve with 2 upward convex portions

Figure 4

Kaplan‐Meier survival curves of patients with clear cell renal cell carcinoma (ccRCC) showing the “one upward convex pattern” and the “two upward convex pattern.” A, Recurrence‐free survival was examined in patients for whom curative resection of both primary and metastatic lesions (if present) had been performed (n = 84). B, Overall survival was examined for all patients (n = 98)