A pyrosequencing assay for the quantitative methylation analysis of the PCDHB gene cluster, the major factor in neuroblastoma methylator phenotype

Abstract

Epigenetic alterations are hallmarks of cancer and powerful biomarkers, whose clinical utilization is made difficult by the absence of standardization and of common methods of data interpretation. The coordinate methylation of many loci in cancer is defined as 'CpG island methylator phenotype' (CIMP) and identifies clinically distinct groups of patients. In neuroblastoma (NB), CIMP is defined by a methylation signature, which includes different loci, but its predictive power on outcome is entirely recapitulated by the PCDHB cluster only. We have developed a robust and cost-effective pyrosequencing-based assay that could facilitate the clinical application of CIMP in NB. This assay permits the unbiased simultaneous amplification and sequencing of 17 out of 19 genes of the PCDHB cluster for quantitative methylation analysis, taking into account all the sequence variations. As some of these variations were at CpG doublets, we bypassed the data interpretation conducted by the methylation analysis software to assign the corrected methylation value at these sites. The final result of the assay is the mean methylation level of 17 gene fragments in the protocadherin B cluster (PCDHB) cluster. We have utilized this assay to compare the methylation levels of the PCDHB cluster between high-risk and very low-risk NB patients, confirming the predictive value of CIMP. Our results demonstrate that the pyrosequencing-based assay herein described is a powerful instrument for the analysis of this gene cluster that may simplify the data comparison between different laboratories and, in perspective, could facilitate its clinical application. Furthermore, our results demonstrate that, in principle, pyrosequencing can be efficiently utilized for the methylation analysis of gene clusters with high internal homologies.

DNA Methylation Fingerprint of Neuroblastoma Reveals New Biological and Clinical Insights

“…Recent genome-wide studies are starting to reveal a role for DNA methylation outside such genomic contexts [12,41,[47][48]. However, the great majority of studies that have investigated DNA methylation in neuroblastoma were focused significantly on the analysis of CpG islands and promoter regions of genes with potential prognostic relevance [20][21][22][23][24][25][26][27][28][29]. Such studies have rarely provided insights into DNA methylation changes outside these well-described sites.…”

“…In fact, low-risk neuroblastoma showed mean methylation levels similar to normal adult tissues, specifically to adult cerebellum. Previous studies have shown that the simultaneous methylation of multiple CGIs, the CpG island methylator phenotype (CIMP), is associated with poor prognosis in neuroblastoma [21][22][23]. However, in neuroblastoma the CIMP is exemplified by the methylation state of the PCDHB gene family (32)(33)(34).…”

“…Survival analysis showed that all seven CpGs were significantly associated with event-free (EFS; p < 0.001) and overall survival (OS; p < 0.005) (Figure 4, Supplementary Table 6). We performed similar survival analysis using clinical and biological parameters (INSS, MYCN status and patient's age at diagnosis) as well as differentially methylated CpGs associated with PCDHB genes and other genes with reported prognostic value in neuroblastoma [21][22][23]. INSS and age of patients performed similarly to CpGs identified in our study, being statistically significantly associated with both EFS (p < 0.001, for both) and OS (p < 0.005, for both; Figure 4 & Supplementary Figure 6).…”

“…The great majority of studies that have investigated DNA methylation in neuroblastoma covered only a small fraction of the methylome of this tumor, specifically CpG islands (CGIs) and promoter regions, and were mainly focused on the analysis of specific genes of potential prognostic relevance [20][21][22][23][24][25][26][27][28][29]. Such studies rarely provide insights into DNA methylation changes outside these canonical sites.…”

Aberrantly methylated DNA regions lead to low activation of CD4+ T-cells in IgA nephropathy

“…The PCRs were performed using IMMOLASE TM DNA Polymerase (Bioline, Aurogene) according to the manufacturer's instructions. The resulting amplicons were subjected to pyrosequencing analysis to confirm the appropriateness of the result considering the influence of the annealing temperature on the possible preferential amplification of the methylated compared with unmethylated template [20] and considering the presence of degenerated bases in the pyrosequencing primers [21]. The sequence of all primers, the PCR amplification conditions, and the sequence of the pyrosequenced fragments are indicated in Supplementary Table S5.…”