Recurrent Germline Variant in the Cohesin Complex Gene <i>RAD21</i> Predisposes Children to Lymphoblastic Leukemia and Lymphoma (2021)

Abstract Introduction: Cohesin complex genes are commonly mutated in cancer particularly in myeloid malignancies. Yet patients with germline mutations in cohesin genes, leading to cohesinopathies like Cornelia-de-Lange syndrome (CdLS) are generally not known to be tumor-prone. The complex plays a major role in chromosome alignment and segregation (Uhlmann, Nature Reviews Molecular Cell Biology, 2016), homologous recombination-driven DNA repair (Ström et al., Molecular Cell, 2004) and regulation of gene expression (Busslinger et al., Nature, 2017). To deepen the understanding of cohesin variants in cancer predisposition, we performed TRIO Sequencing in two independent pediatric cancer cohorts. Thereby, we identified a novel recurrent heterozygous germline variant in the cohesin gene RAD21 not described in CdLS patients , located in the binding domain of the cofactors WAPL and PDS5B . Methods: Whole exome sequencing (WES) in a TRIO (child-parent datasets) setting was carried out in two independent, unselected cancer cohorts (TRIO-D, n=158 (Wagener et al., European Journal of Human Genetics, 2021) and TRIO-DD, n=60). To investigate the oncogenic potential of the novel RAD21 variant molecular and functional assessment was performed focusing on potential implications on the complex. Results: The newly identified RAD21 variant at amino acid position 298 resulting in a Proline to Serine (p.P298S) and a Proline to Alanine exchange, respectively, (p.P298A) is only rarely mutated in the general population (gnomAD database n=118,479; RAD21 p.P298S MAF &amp;lt;10 -6 and RAD21 p.P298A MAF &amp;lt;10 -5). While both patients did not show any signs of CdLS, they both have a remarkable family history of cancer. Patient 1 (13y) was diagnosed with T-cell acute lymphoblastic leukemia (T-ALL) whose father had died from breast cancer (41y), while patient 2 (2y) presented with precursor B-cell lymphoblastic lymphoma (pB-LBL) whose uncle had died from pediatric cancer of unknown subtype (8y). To assess the influence of RAD21 p.P298S/A on the binding capacity of the complex, RAD21 variants and the wildtype (WT) were cloned and transfected into HEK293T cells, respectively. Immunoprecipitation analysis of RAD21 with the cofactors WAPL and PDS5B showed no differential binding between the WT and the variants, suggesting that RAD21 p.P298S/A does not impact the formation of the complex. Nevertheless, on a transcriptional level 83 genes were significantly differentially expressed in RAD21 p.P298S and p.P298A compared to the wildtype (fc&amp;gt;1.5, adj. p-value &amp;lt;0.05) with enrichment of genes in p53 signaling pathways. We further observed an increased number of γH2AX and 53BP1 co-localized foci compared to the WT (p≤0.01; Student's t-test). In line, following ionizing radiation, primary patients' samples showed increased cell cycle arrest at G2/M cell-cycle stage compared to a healthy control (p.P298S: p=0.0049 [6Gy]; p=0.0026 [10Gy]; p.P298A: p=0.0054 [6Gy]; p=0.0006 [10Gy]; Student's t-test). For cross-validation of the germline variant RAD21 p.P298S/A and its potential role in pediatric lymphoblastic malignancies, we analysed a third cohort of 150 children with relapsed ALL (IntReALL) for RAD21 p.P298S/A. We again identified RAD21 p.P298A in a boy (12y) with B-cell precursor acute lymphoblastic leukemia. To compare our data to a non-pediatric cancer setting, a cohort of 2300 young adults (&amp;lt;51 years) with cancer was mined (MASTER program). Here, one patient carrying RAD21 p.P298A with a solid tumor was identified. Therefore, amongst all cohorts, RAD21 p.P298S/A was found to be enriched in pediatric vs. adult cancers (3/479 vs. 1/2299; Fisher's exact test; p=0.018). Conclusion: Taken together, we present for the first time the potential role of RAD21 germline variants in pediatric lymphoblastic malignancies. This may shed new light on the many roles of the cohesin complex and its implication outside the typical syndromal presentation. Disclosures No relevant conflicts of interest to declare.