Dissecting The Dna Repair Machinery In Biological Subgroups Of Childhood Acute Lymphoblastic Leukemia

Background . Aberrations in the DNA repair pathway among children with acute lymphoblastic leukemia (ALL) are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis and direct patient care. Germline aberrations of the DNA-repair machinery are crucial for generating chromosomal instability and occurrence of acute leukemia. In order to better understand this mechanism, we addressed our research to NBS 1 (Nijmegen Breakage Syndrome) gene and Fanconi Anemia/BRCA1 and BRCA2 pathway, in the attempt to identify mutations and aberrant expression, predisposing and/or cooperating with the leukemogenic process, among biological subgroups of childhood ALL. Materials and Methods . We analyzed 48 diagnostic samples from children with ALL treated at our institution from 2000 to 2010: 11 cases with chromosome Philadelphia positive (Ph+) leukemias (8 ALL and 3 CML), 15 with t(12;21), 11 with t(1;19) positive ALL, respectively and 10 samples with B-ALL without known translocations. We also analyzed three cases of relapse. In Ph+ subgroup, we analyzed remission samples in order to detect germline mutations. We performed RT-PCR and sequencing analyses to detect mutations in NBS 1 gene (exons 3-6). The status of FANCD 2 and PALB 2 genes was studied by a multiplex ligation-dependent probe amplification (MLPA). Samples from healthy donors (HDs) were used as wild-type control. For data elaboration, we used Coffalyser.Net software for MLPA. We performed Sybr-Green Real-Time PCR amplification of BRCA 1 (exons 14-15) and BRCA 2 (exons 15-16) genes, respectively, calculating the expression in patients with method 2^-ΔΔC comparing with HDs. Results . Among the 15 cases with t(12;21) positive ALL, we found overexpression of BRCA 1 and BRCA 2 in 8 and 10 cases, respectively, showing a statically significant difference (p C;Glu185Gln; SNP-rs1805794) in NBS 1 gene. Deletions or duplications for FANCD 2 and PALB 2 genes were not found. In the 8 cases with Ph+ B-ALL, we detected an overexpression of BRCA 1 and BRCA 2 in 4 and 7, respectively, showing a statistically significant difference (p NBS 1 showed a missense variant mutation (Gu003eC;Glu185Gln) in 2 cases (25%) with B-ALL and in 1 patients with CML, respectively. Analyses of FANCD 2 showed deletions in 3 cases with B-ALL and 2 children with CML; PALB 2 resulted deleted in 2 cases with B-ALL and 2 cases with CML. Interestingly, NBS 1 mutations were detected in remission samples, showing a germline genomic aberration, as well. Surprisingly, deletions of FANCD 2 and PALB 2 were found only in leukemic samples. In 11 cases with t(1;19) positive ALL, we observed statically significant difference in BRCA 1 (p BRCA 2 (p NBS 1, we detected missense variant mutation (Gu003eC;Glu185Gln) in 4 patients (36%); in one case we detected an NBS 1 exon 4 deletion. We found FANCD 2 exon-1 duplication in 3 patients; in 4 cases we detected PALB 2 genomic aberrations (3 duplication and one exon-1 deletion). Among 10 cases with B-ALL without known translocations, we observed statically significant difference in BRCA 1 (p BRCA 2 (p C;Glu185Gln) in NBS 1. We also detected in 4 cases different mutations in FANCD 2 and PALB 2 genes (deletion in exons 9-30 and duplication in exons 1-2-29, respectively). Notably, the relapsed case within the latter subgroup, at diagnosis showed genomic aberrations in all the selected genes in association with BRCA 1 and BRCA 2 overexpression. Conclusion Our findings strongly suggest that the DNA repair machinery is frequently disrupted in childhood ALL. The significance of BRCA 1 and BRCA 2 overexpression in all subgroups is still unclear. NBS 1 showed a missense mutation in a range from 25% to 45% of children with ALL. Interestingly, these mutations were found at germline level in cases with Ph+ leukemias, suggesting a predisposition profile. Aberrations of FANCD 2 and PALB 2 genes were mostly detected in the subgroup of children without known translocation. Finally the role in leukemic predisposition and the clinical impact of these genomic aberrations need to be more elucidated in a larger population. Disclosures No relevant conflicts of interest to declare.