Hypermethylation of RAD9A intron 2 in childhood cancer patients and tumor cell lines

Abstract

Background Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or unhealthy lifestyle. The prenatal origins hypothesis postulates a role for (epi)genetic mutations which occur stochastically in rapidly dividing cells. This study aims to investigate the impact of adverse methylation in tumor relevant genes in former childhood cancer patients which may be associated with an enhanced risk to develop primary secondary cancers Methods We performed an epimutation screen of several candidate genes (APC, CDKN2A, EFNA5, RAD9A, and TP53), in skin fibroblasts of 20 patients with a primary cancer in childhood and subsequent second primary cancer (2N), 20 matched patients with only one primary cancer in childhood (1N), 20 cancer free controls (0N) and unrelated leukemia cancer samples, using bisulfite pyrosequencing and deep bisulfite sequencing. radiation, colony formation assays, cell proliferation, PCR and molecular karyotype SNP-array, experiments were performed to further characterize RAD9A hypermethylation in fibroblasts and FaDu sub clones. Data were analyzed using the Kruskal-Wallis rank sum test and Benjamini-Hochberg procedure, the linear mixed-effects model was fit using REML and R-scripts. Results Four 1N patients and one 2N patient displayed elevated (10%) mean methylation levels of RAD9A intron 2. Deep bisulfite sequencing of RAD9A in these patients revealed 2% allele methylation errors (defined as alleles with >60% methylated CpGs). We found RAD9A hypermethylation in bone marrow of patients with pre-ALL (pre-acute lymphoblastic leukaemia), AML (acute myloid leukaemia), and plasmablastic lymphoma (PBL), and in EBV-(Epstein Barr virus) transformed lymphoblastoid cells. RAD9A methylation in fibroblasts or tumor cells (FaDu) was not affected by in vitro aging, or DNA damage induced by radiation or the chemotherapeutical daunorubicin. Molecular karyotyping of FaDu sub clones revealed a homozygous inactivation of CHD2, SPATA8 and SMARCA1, in clones with hypermethylation in RAD9A. Conclusion We propose that constitutive RAD9A epimutations may have arisen early in development, predisposing the compromised cells to tumorigenesis and increased childhood cancer risk. Analyses of tumor cell clones with high methylation levels of RAD9A suggest a connection between methylation levels of the RAD9A intron 2 locus and a homozygous inactivation of important genes