Substitutions of the NBS1 gene and clinicopathological characteristics of young breast cancer patients

The purpose of this study was to determine characteristics potentially related to NBS1 mutations and polymorphisms in young (≤50 years of age) breast cancer patients. Blood from 80 breast cancer patients was collected. NBS1 mutations c.657_661del, p.R215W, p.I171V, and polymorphisms c.8360G>C, c.30537G>C were genotyped by the PCR-RFLP method. Two-sided Chi-square test was used for univariate analysis and logistic regression analysis was used to evaluate the odds ratio. No carriers of the c.657_661del, p.R215W and p.I171V mutations were found. NBS1 c.8360G>C logistic regression analysis showed that GC and CC genotypes compared with GG genotype had decreased risk of low grade tumour, 2.885-fold (OR = 2.885, 95% CI 0.173–0.735, P = 0.005) and 2.186-fold (OR = 2.186, 95% CI 0.188–0.888, P = 0.024), respectively. 8360 CC genotype (OR = 3.034, 95% CI 0.156–0.778, P = 0.010) significantly increased the chances of HER2 amplification compared to GG genotype. NBS1 8360 GC genotype had a higher risk for breast cancer progression (OR = 1.673, 95% CI 0.233–0.915, P = 0.027). The homozygote 8360 CC carriers had approximately a six times higher risk for the disease progress (OR = 5.946, 95% CI 0.098–0.585, P = 0.002). The prevalence of triple negative breast cancer type was significantly higher in individuals with NBS1 8360 CC genotype (OR = 2.186, 95% CI 0.188–0.888, P = 0.024). Regarding c.30537G>C polymorphism, none of the genotypes had a significant influence on pathological characteristics. NBS1 gene c.8360G>C polymorphism might be associated with breast cancer aggressiveness in young breast cancer patients.

The p53 Slavic Gene Mutation Nijmegen Breakage Syndrome

Nijmegen breakage syndrome is a rare autosomal congenital disorder. It originates from mutations in the NBS1 gene located in band 8q21, which encodes for the DNA double strand repair protein Nibrin. Immunodeficiency with increased susceptibility to infection. It is a condition characterized by short stature, an unusually small head size (microcephaly), distinctive facial features, recurrent respiratory tract infections, an increased risk of cancer, intellectual disability, and other health problems. People with this condition typically grow slowly during infancy and early childhood. After this period of slow growth, affected individuals grow at a normal rate but remain shorter than their peers. Microcephaly is apparent from birth in the majority of affected individuals. The head does not grow at the same rate as the rest of the body, so it appears that the head is getting smaller as the body grows (progressive microcephaly).Individuals with Nijmegen breakage syndrome have distinctive facial features that include a sloping forehead, a prominent nose, large ears, a small jaw, and outside corners of the eyes that point upward (up slanting palpebral fissures). These facial features typically become apparent by age 3. Keywords: Nijmegen breakage syndrome; Chromosomal instability; Immunodeficiency; Microcephaly

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 NBS1 (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 NBS1 gene (exons 3-6). The status of FANCD2 and PALB2 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 BRCA1 (exons 14-15) and BRCA2 (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 BRCA1 and BRCA2 in 8 and 10 cases, respectively, showing a statically significant difference (p<0.0001; p<0.0048, respectively) vs HDs. We identified in 7 cases (44%) a missense variant mutation (G>C;Glu185Gln; SNP-rs1805794) in NBS1 gene. Deletions or duplications for FANCD2 and PALB2 genes were not found. In the 8 cases with Ph+ B-ALL, we detected an overexpression of BRCA1 and BRCA2 in 4 and 7, respectively, showing a statistically significant difference (p<0.0084 and p<0.0009, respectively) vs HDs. In 3 cases with CML we found the same pattern. Analysis of NBS1 showed a missense variant mutation (G>C;Glu185Gln) in 2 cases (25%) with B-ALL and in 1 patients with CML, respectively. Analyses of FANCD2 showed deletions in 3 cases with B-ALL and 2 children with CML; PALB2 resulted deleted in 2 cases with B-ALL and 2 cases with CML. Interestingly, NBS1 mutations were detected in remission samples, showing a germline genomic aberration, as well. Surprisingly, deletions of FANCD2 and PALB2 were found only in leukemic samples. In 11 cases with t(1;19) positive ALL, we observed statically significant difference in BRCA1 (p<0.0293) and BRCA2 (p<0.0705) expression vs HDs, respectively. Analyzing NBS1, we detected missense variant mutation (G>C;Glu185Gln) in 4 patients (36%); in one case we detected an NBS1 exon 4 deletion. We found FANCD2 exon-1 duplication in 3 patients; in 4 cases we detected PALB2 genomic aberrations (3 duplication and one exon-1 deletion). Among 10 cases with B-ALL without known translocations, we observed statically significant difference in BRCA1 (p<0.0031) and BRCA2 (p<0.125) expression vs HDs. We found 3 patients (30%) with a missense variant mutation (G>C;Glu185Gln) in NBS1. We also detected in 4 cases different mutations in FANCD2 and PALB2 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 BRCA1 and BRCA2 overexpression. Conclusion Our findings strongly suggest that the DNA repair machinery is frequently disrupted in childhood ALL. The significance of BRCA1 and BRCA2 overexpression in all subgroups is still unclear. NBS1 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 FANCD2 and PALB2 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.

Cernunnos/XLF Deficiency: A Syndromic Primary Immunodeficiency

Artemis, DNA ligase IV, DNA protein kinase catalytic subunit, and Cernunnos/XLF genes in nonhomologous end joining pathways of DNA repair mechanisms have been identified as responsible for radiosensitive SCID. Here, we present a 3-year-old girl patient with severe growth retardation, bird-like face, recurrent perianal abscess, pancytopenia, and polydactyly. Firstly, she was thought as Fanconi anemia and spontaneous DNA breaks were seen on chromosomal analysis. After that DEB test was found to be normal and Fanconi anemia was excluded. Because of that she had low IgG and IgA levels, normal IgM level, and absence of B cells in peripheral blood; she was considered as primary immunodeficiency, Nijmegen breakage syndrome. A mutation in NBS1 gene was not found; then Cernunnos/XLF deficiency was investigated due to clinical similarities with previously reported cases. Homozygous mutation in Cernunnos/XLF gene (NHEJ1) was identified. She is now on regular IVIG prophylaxis and has no new infection. Fully matched donor screening is in progress for bone marrow transplantation which is curative treatment of the disease. In conclusion, the patients with microcephaly, bird-like face, and severe growth retardation should be evaluated for hypogammaglobulinemia and primary immunodeficiency diseases.

657del5 mutation of the NBS1 gene in myelodysplastic syndrome

Myelodysplastic syndromes (MDS) are clonal hematologic stem cell disorders with an as yet unknown molecular pathology. Genetic instability has been proposed as a cause of MDS. Mutations in the NBS1 gene, whose product nibrin (p95) is involved in DNA damage repair and cell-cycle control, might be associated with an elevated predisposition to the development of MDS. The aim of the study was to examine truncating 5 bp deletion (657del5), the most frequent NBS1 gene mutation in Slavic populations, in MDS patients. Among 71 MDS patients, we found one case that was heterozygous for the NBS1 657del5 mutation. To the best of our knowledge, this is the first report of a NBS1 mutation in MDS.