Potential Role of STAG1 Mutations in Genetic Predisposition to Childhood Hemato-Oncological Diseases

Background. Genetic predisposition has been recently assessed in about 5-10% of pediatric tumors, including leukemias. Among genetic syndromes associated with an increased risk of developing leukemia, a possible link has been proposed for Cohesinopathies, like Cornelia de Lange Syndrome (CdLS), caused by mutations in Cohesin family genes. Somatic mutation in these genes, known for their fundamental role in cell cycle and DNA repair mechanisms, are described in myeloid malignancies (10-20% of AML, 50% of DS-AMKL, 5-15% of MDS and 10% of MPN) and solid tumours. We previously described the first germline variant of NIPBL in a CdL case with acute lymphoblastic leukemia (ALL). The identification of germline variants can provide new insights in the pathogenesis of hemato-oncological diseases.

Objectives. This study aims to define the association of germline variants of the Cohesin family genes with predisposition to pediatric hemato-oncological diseases, including ALL. Functional assays have been performed on a STAG1 unique variant.

Methods. We set up an NGS capture-base DNA panel, including 40 genes associated with 8 pathways associated to ALL predisposition, including Cohesin genes. Patients' bone marrow (BM) diagnostic samples have been analyzed together with a germline tissue (BM at remission or buccal brush). Bioinformatic analysis has been carried out by Sophia DDM software (Variant Fraction >5%; Coverage at least 500X; Variant Allele Frequency 1%) and variants were annotated as certainly pathogenic, potentially pathogenic or VUS.

A Lymphoblastoid Cell Line (LCL) was obtained from PB of a MDS patient, then used for functional studies.

Genomic stability and X-ray induced DNA-damage repair were evaluated in the STAG1-mutated LCL by Sister Chromatids Exchange (SCE) assay, cell cycle analysis (Propidium Iodide) and pH2AX staining.

Results. Overall, 117 consecutive pediatric ALL patients and sporadic cases with familial recurrency of cancer or other hematological disorders have been analyzed through the NGS panel. A total of 225 mutations were identified in ALL, distributed as it follows: 77 variants in Transcription factors, 68 in Signaling-associated genes, 56 variants in RAS pathway, 44 in chromatin modifier genes, 38 variants in B-cell differentiation genes, 30 in DNA repair genes and 16 variants in genes associated to predisposition. Overall, 12/117 unique variants (5%) were found in Cohesins family genes; 11/12 were germline, while a single somatic variant was found in STAG2 in the same position as a germline variant. In details, 3 mutations were found in BRD2 gene while 2 variants were identified in STAG1, STAG2 and NIPBL genes, respectively. Only 1 variant was detected in SMC1A, SMC3 and BRD2. No mutations were detected in HDAC8 and RAD21 genes.

Interestingly, the Arg1167Gln variant in STAG1 is located in a highly conserved region involved in solid tumors. A STAG1 variant (Arg1187Gln) was identified in a pediatric MDS patient, which was then further characterized in the LCL. First, we evaluated the effects on chromosomal stability, and we observed a higher number of abnormal chromatids exchanges in mutated LCL (STAG1 patient mean = 4.8 exchanges/metaphase compared to four control LCLs mean = 3.05; p <0.0001), as shown in the attached figure. Differences in cell cycle phases between STAG1-mutated LCL and controls, even after X-ray irradiation (3Gy - 6Gy), are not significant. Mutated cells showed a significantly lower capability to repair DNA after an ionizing radiation. γH2AX phosphorylation status of STAG1 mutated LCL appears higher also in basal condition (T0: 1.7X, p <0.01; T24: 2.2X, p <0.0001; T48: 2.4X, p <0.0001; MFI STAG1 over MFI control LCLs) and it remains at higher levels at 48 hours after the irradiation (T48: 3.7X, p<0.001 [3Gy]; 4.1X, p<0.0001 [6Gy]; MFI STAG1 over MFI control LCLs). These data are confirmed by the ratio of the STAG1-mutated LCL median MFI over controls (T48/T0: 3.42 vs 0.92, p <0.0001 [3Gy]; T48/T0: 2.33 vs 0.64, p <0.01 [6Gy]).

Conclusion. In pediatric hematological diseases, NGS screening showed several variants among Cohesin genes, with either a potentially pathogenic or unknown significance.

Our study confirms a considerable effect of a STAG1 germline variant on genetic instability that leads to oncogenesis, opening new scenarios for Cohesins' contribution to genetic predisposition to leukemias.

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