Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation

Abstract Background: Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. This suggests other predisposition defects that may support the oncogenic transformation of cells, e.g. via impaired DNA-repair. Our study consequently aims to investigate the impact of increased methylation of intron 2 of RAD9A in cancer patients which may be associated with oncogenic transformation. Methods: We performed an epimutation screen of RAD9A and other candidate genes ( APC , CDKN2A , EFNA5 , and TP53 ) using bisulfite pyrosequencing and deep bisulfite sequencing (DBS) in skin fibroblasts of 20 patients with primary cancer in childhood and second primary cancer (2N) later in life, 20 matched patients with only one primary cancer (1N) in childhood and 20 matched cancer-free (0N) controls. Furthermore, we analyzed leukemia cancer samples, tumor cell lines, EBV lymphoblasts and FaDu subclones. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotype SNP-array experiments were performed. Data were analyzed using the Kruskal-Wallis rank-sum test, Benjamini-Hochberg procedure, REML and R-scripts. Results: Four 1N patients and one 2N patient displayed elevated mean methylation levels (>10%) in intron 2 of RAD9A . DBS of RAD9A in these patients revealed >2% hypermethylated alleles consistent with relevant epimutations. We found RAD9A hypermethylation in the bone marrow of patients with pre-ALL (pre-acute lymphoblastic leukemia), AML (acute myeloid leukemia), NHL (non-Hodgkin lymphoma), PBL (plasmablastic lymphoma) and EBV-(Epstein Barr virus) transformed lymphoblastoid cells. Molecular karyotyping of AML samples with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. In generated FaDu subclones with hypermethylated RAD9A, we found a homozygous inactivation of CHD2, SPATA8 , SMARCA1 and a 302 kb duplication including genes deregulated in cancer. The detected aberrations proved to influence cell viability. RAD9A methylation was not affected by radiation or the chemotherapeutical daunorubicin.Conclusion: The analysis of patient samples, cell lines and subclones suggest a connection between methylation levels of the RAD9A intron 2 locus and inactivation or amplification of important genes and survival of the cells. We propose that RAD9A epimutations may have an impact on leukemia, tumorigenesis, cancer progression and can potentially serve as a valuable biomarker.