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

Danuta Galetzka; Julia Böck; Lukas Wagner; Marcus Dittrich; Olesja Sinizyn; Marco Ludwig; Heidi Rossmann; Claudia Spix; Markus Radsak; Peter Scholz-Kreisel; Johanna Mirsch; Matthias Linke; Walburgis Brenner; Manuela Marron; Alicia Poplawski; Thomas Haaf; Heinz Schmidberger; Dirk Prawitt

doi:10.17179/excli2021-4482

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

EXCLI J. 2022 Jan 7:21:117-143. doi: 10.17179/excli2021-4482. eCollection 2022.

Authors

Danuta Galetzka¹, Julia Böck^{2

3}, Lukas Wagner⁴, Marcus Dittrich⁵, Olesja Sinizyn¹, Marco Ludwig⁶, Heidi Rossmann⁷, Claudia Spix⁸, Markus Radsak⁹, Peter Scholz-Kreisel¹⁰, Johanna Mirsch¹¹, Matthias Linke¹², Walburgis Brenner¹³, Manuela Marron¹⁴, Alicia Poplawski¹⁵, Thomas Haaf², Heinz Schmidberger¹, Dirk Prawitt⁴

Affiliations

¹ Department of Radiation Oncology and Radiation Therapy, University Medical Centre, Mainz, Germany.
² Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany.
³ Institute of Pathology, Julius Maximilians University, Würzburg, Germany.
⁴ Center for Pediatrics and Adolescent Medicine, University Medical Centre, Mainz, Germany.
⁵ Bioinformatics Department, Julius Maximilians University, Würzburg, Germany.
⁶ DRK Medical Centre, Alzey, Germany.
⁷ Institute of Clinical Chemistry and Laboratory Medicine, University Medical Centre, Mainz, Germany.
⁸ Division of Childhood Cancer Epidemiology, Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Centre, Mainz, Germany.
⁹ Department of Hematology, University Medical Centre, Mainz, Germany.
¹⁰ Federal Office of Radiation, Neuherberg, Germany.
¹¹ Radiation Biology and DNA Repair, Technical University of Darmstadt, Germany.
¹² Institute of Human Genetics, University Medical Centre, Mainz, Germany.
¹³ Department of Obstetrics and Women's Health, University Medical Centre, Mainz, Germany.
¹⁴ Leibniz Institute for Prevention Research and Epidemiology - BIPS, Bremen, Germany.
¹⁵ Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Centre, Mainz, Germany.

Abstract

Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. However, risk factors might trigger the oncogenic transformation of cells. Among other regulatory signals, hypermethylation of RAD9A intron 2 is responsible for the increased expression of RAD9A protein, which may play a role in oncogenic transformation. Here, we analyzed the RAD9A intron 2 methylation in primary 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 in childhood (1N) and 20 matched cancer-free controls (0N), using bisulfite pyrosequencing and deep bisulfite sequencing (DBS). Four 1N patients and one 2N patient displayed elevated mean methylation levels (≥ 10 %) of RAD9A. DBS revealed ≥ 2 % hypermethylated alleles of RAD9A, indicative for constitutive mosaic epimutations. Bone marrow samples of NHL and AML tumor patients (n=74), EBV (Epstein Barr Virus) lymphoblasts (n=6), tumor cell lines (n=5) and FaDu subclones (n=13) were analyzed to substantiate our findings. We find a broad spectrum of tumor entities with an aberrant methylation of RAD9A. We detected a significant difference in mean methylation of RAD9A for NHL versus AML patients (p ≤0.025). Molecular karyotyping of AML samples during therapy with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotyping SNP-array experiments using generated FaDu subclones suggest that hypermethylation of RAD9A intron 2 is associated with genomic imbalances in regions with tumor-relevant genes and survival of the cells. In conclusion, this is the very first study of RAD9A intron 2 methylation in childhood cancer and Leukemia. RAD9A epimutations may have an impact on leukemia and tumorigenesis and can potentially serve as a biomarker.

Keywords: DNA repair; RAD9A; childhood cancer; hypermethylation; leukemia; regular body cells; somatic mosaicism.