DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

<div><p></p><p>A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, <i>THBS1</i> CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these <i>THBS1</i> sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a <i>THBS1</i> CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.</p></div

Combined bisulfite restriction analysis (COBRA) of <i>THBS1</i> in pediatric renal tumors.

<p>Using the same PCR products as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062233#pone-0062233-g003" target="_blank">Figure 3</a>, COBRA analysis was performed by digesting with the HpyCH4IV enzyme. The HpyCH4IV site is equivalent to CpG18 (chr15:37,660,642-37,660,645/hg18) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062233#pone-0062233-g003" target="_blank">Figure 3</a>. The positions of bands representing methylated and unmethylated DNA are indicated by arrows. As the control of HpyCH4IV digestion, 0, 50, and 100% methylated DNA were loaded on the same gel. WT: Wilms’ tumor.</p

Frequency of <i>THBS1</i> methylation by COBRA.

<p>Frequency of <i>THBS1</i> methylation by COBRA.</p

MassARRAY analysis of methylation in <i>THBS1</i>.

<p>In addition to the 6 cases for each tumor group, 9 and 21 cases of CMN and Wilms’ tumor (WT), respectively, were analyzed for DNA methylation of the <i>THBS1</i> CpG site, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062233#pone-0062233-g002" target="_blank">Figure 2A</a>. Different colored of circles mark the position of CpG within the sequence (straight line) and the levels of methylation are shown in color (red, low methylation level; yellow, high methylation level). Gray circles represent the unanalyzed CpG sites. WT: Wilms’ tumor.</p

The number of hyper- and hypomethylated genes in comparison with non-neoplastic kidney.

<p>Hypermethylation: difference of average β-value >0.3. Hypomethylation: difference of average β-value<−0.3.</p

Hierarchical cluster analysis of methylation level of CpG analyzed by MassARRAY.

<p>(A) Based on the results indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062233#pone-0062233-g001" target="_blank">Figure 1</a>, 8 genes were selected as described in the text and analyzed by MassARRAY. Two-way hierarchical cluster analysis was performed using the CpG methylation average of all CpG that have passed QC from each gene. Two cases (RTK4 and CCSK3) showed failed analysis of some CpG sites, and were excluded from hierarchical analysis. (B) Four genes were further selected, and cluster analysis using the methylation average was performed as in (A). CCSK3 was successfully analyzed in all four genes, and included in this analysis.</p