Nijmegen Breakage Syndrome Research Articles

Instabilidade cromossómica e imunodeficiência - associação essencial no diagnóstico de Síndrome de Nijmegen

Introduction: Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder that belongs to the group of chromosomal instability syndromes, more prevalent in Central and Eastern Europe.Clinical case: We describe the case of a 14-month-old boy, born to Ukrainian parents, that presented at birth with microcephaly, small to gestational age and dysmorphic facial characteristics, which became more obvious with age. The complementary study revealed spontaneous and induced chromosomal instability, with normal fetal haemoglobin and α-fetoprotein and cellular immunodeficiency. These features suggested NBS, confirmed by the identification of the founder mutation (657del5) in homozygous state, common in patients of Slavic origin.Discussion: NBS should be considered in the presence of microcephaly, typical facial features, growth retardation, chromosomal instability and immunodeficiency. The prognostic is poor due to the occurrence of frequent infections and the high incidence of cancer. Multidisciplinary follow-up and avoidance of radiation exposition are crucial.

Effects of Pulsed Electromagnetic Fields on Breast Cancer Cell Line MCF 7 Using Absorption Spectroscopy.

We present an analysis of the effects of pulsed electromagnetic fields (PEMF) with 3.3 MHz carrier frequency and modulated by audio resonant frequencies on the MCF-7 breast cancer cell line in vitro using absorption spectroscopy. This involves a fluorescence dye called PrestoBlue™ Cell Viability Reagent and a spectrophotometry to test the viability of MCF-7 breast cancer cells under different PEMF treatment conditions in terms of the cell absorption values. The DNA molecule of the MCF-7 breast cancer cells has an electric dipole property that renders it sensitive and reactive to applied electromagnetic fields. Resonant frequencies derived from four genes mutated in MCF-7 breast cancer cells [rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR), peroxisome proliferator-activated receptor (PPARG), Nijmegen breakage syndrome 1 (NBN) and checkpoint kinase 2 (CHEK2)] were applied in generating square pulsed electromagnetic waves. Effects were monitored through measurement of absorption of the samples with PrestoBlue™, and the significance of the treatment was determined using the t-test. There was a significant effect on MCF-7 cells after treatment with PEMF at the resonant frequencies of the following genes for specific durations of exposure: RICTOR for 10 min, PPARG for 10 min, NBN for 15 min, and CHEK2 for 5 min.

Recommendations for Childhood Cancer Screening and Surveillance in DNA Repair Disorders.

DNA repair syndromes are heterogeneous disorders caused by pathogenic variants in genes encoding proteins key in DNA replication and/or the cellular response to DNA damage. The majority of these syndromes are inherited in an autosomal-recessive manner, but autosomal-dominant and X-linked recessive disorders also exist. The clinical features of patients with DNA repair syndromes are highly varied and dependent on the underlying genetic cause. Notably, all patients have elevated risks of syndrome-associated cancers, and many of these cancers present in childhood. Although it is clear that the risk of cancer is increased, there are limited data defining the true incidence of cancer and almost no evidence-based approaches to cancer surveillance in patients with DNA repair disorders. This article is the product of the October 2016 AACR Childhood Cancer Predisposition Workshop, which brought together experts from around the world to discuss and develop cancer surveillance guidelines for children with cancer-prone disorders. Herein, we focus on the more common of the rare DNA repair disorders: ataxia telangiectasia, Bloom syndrome, Fanconi anemia, dyskeratosis congenita, Nijmegen breakage syndrome, Rothmund-Thomson syndrome, and Xeroderma pigmentosum. Dedicated syndrome registries and a combination of basic science and clinical research have led to important insights into the underlying biology of these disorders. Given the rarity of these disorders, it is recommended that centralized centers of excellence be involved directly or through consultation in caring for patients with heritable DNA repair syndromes. Clin Cancer Res; 23(11); e23-e31. ©2017 AACRSee all articles in the online-only CCR Pediatric Oncology Series.

Contribution of Double-strand Break Repair Gene Nijmegen Breakage Syndrome 1 Genotypes, Gender Difference and Smoking Status to Taiwanese Lung Cancer

Nijmegen breakage syndrome 1 (NBS1) is a component of MRE11/RAD50/NBS1 complex (MRN) that plays a critical role in the cellular response to DNA damage and maintenance of chromosomal integrity. Failure in DNA damage response affects the level of cell survival, increases the frequency of gene mutation or chromosomal instability and other cellular phenotypic abnormalities, which are the important mechanisms of carcinogenesis. However, the contribution of variant NBS1 genotypes to lung cancer is not known. The current study aimed to evaluate the contribution of the common variant NBS1 Glu185Gln (rs1805794, E185Q) genotypes to the risk of lung cancer. The contributions of the NBS1 Glu185Gln genotypes to lung cancer risk were investigated among 358 patients with lung cancer and 716 age- and gender-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). GG, CG and CC NBS1 Glu185Gln genotype percentages were 45.2%, 43.9% and 10.9% in the patient group and 46.1%, 45.1% and 8.8% in the non-cancer control group, respectively (p for trend=0.5423). Analysis of allelic frequency distributions showed that the C allele of NBS1 Glu185Gln did not increase lung cancer susceptibility (p=0.4916). Interestingly, the CC genotypes at NBS1 Glu185Gln enhanced the risk of lung cancer among the males adjusted odds ratio (aOR)=1.85, 95% confidence interval (CI)=1.12-2.83 and among the smokers (aOR=1.76, 95% CI=1.09-2.64) but not among the females and non-smokers. The CC genotype of NBS1 Glu185Gln may increase lung cancer risk only for males and smokers and may serve as a practical marker for early detective and predictive purposes of lung cancer.

Outcome of hematopoietic cell transplantation for DNA double-strand break repair disorders

Rare DNA breakage repair disorders predispose to infection and lymphoreticular malignancies. Hematopoietic cell transplantation (HCT) is curative, but coadministered chemotherapy or radiotherapy is damaging because of systemic radiosensitivity. We collected HCT outcome data for Nijmegen breakage syndrome, DNA ligase IV deficiency, Cernunnos-XRCC4-like factor (Cernunnos-XLF) deficiency, and ataxia-telangiectasia (AT). Data from 38 centers worldwide, including indication, donor, conditioning regimen, graft-versus-host disease, and outcome, were analyzed. Conditioning was classified as myeloablative conditioning (MAC) if it contained radiotherapy or alkylators and reduced-intensity conditioning (RIC) if no alkylators and/or 150mg/m2 fludarabine or less and 40mg/kg cyclophosphamide or less were used. Fifty-five new, 14 updated, and 18 previously published patients were analyzed. Median age at HCT was 48months (range, 1.5-552months). Twenty-nine patients underwent transplantation for infection, 21 had malignancy, 13 had bone marrow failure, 13 received pre-emptive transplantation, 5 had multiple indications, and 6 had no information. Twenty-two received MAC, 59 received RIC, and 4 were infused; information was unavailable for 2 patients. Seventy-three of 77 patients with DNA ligase IV deficiency, Cernunnos-XLF deficiency, or Nijmegen breakage syndrome received conditioning. Survival was 53 (69%) of 77 and was worse for those receiving MAC than for those receiving RIC (P=.006). Most deaths occurred early after transplantation, suggesting poor tolerance of conditioning. Survival in patients with AT was 25%. Forty-one (49%) of 83 patients experienced acute GvHD, which was less frequent in those receiving RIC compared with those receiving MAC (26/56 [46%] vs 12/21 [57%], P=.45). Median follow-up was 35months (range, 2-168months). No secondary malignancies were reported during 15years of follow-up. Growth and developmental delay remained after HCT; immune-mediated complications resolved. RIC HCT resolves DNA repair disorder-associated immunodeficiency. Long-term follow-up is required for secondary malignancy surveillance. Routine HCT for AT is not recommended.

Parkin regulates translesion DNA synthesis in response to UV radiation.

Deficiency of Parkin is a major cause of early-onset Parkinson's disease (PD). Notably, PD patients also exhibit a significantly higher risk in melanoma and other skin tumors, while the mechanism remains largely unknown. In this study, we show that depletion of Parkin causes compromised cell viability and genome stability after ultraviolet (UV) radiation. We demonstrate that Parkin promotes efficient Rad18-dependent proliferating cell nuclear antigen (PCNA) monoubiquitination by facilitating the formation of Replication protein A (RPA)-coated ssDNA upon UV radiation. Furthermore, Parkin is found to physically interact with NBS1 (Nijmegen breakage syndrome 1), and to be required for optimal recruitment of NBS1 and DNA polymerase eta (Polη) to UV-induced damage sites. Consequently, depletion of Parkin leads to increased UV-induced mutagenesis. These findings unveil an important role of Parkin in protecting genome stability through positively regulating translesion DNA synthesis (TLS) upon UV damage, providing a novel mechanistic link between Parkin deficiency and predisposition to skin cancers in PD patients.

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms.

Case report: rapid and durable response to PDGFR targeted therapy in a child with refractory multiple infantile myofibromatosis and a heterozygous germline mutation of the PDGFRB gene

BackgroundInfantile myofibromatosis belongs to a family of soft tissue tumors. The majority of these tumors have benign behavior but resistant and malignant courses are known, namely in tumors with visceral involvement. The standard of care is surgical resection. Observations suggest that low dose chemotherapy is beneficial. The treatment of resistant or relapsed patients with multifocal disease remains challenging. Patients that harbor an actionable mutation in the kinase domain are potential subjects for targeted tyrosine kinase inhibitor therapy.Case presentationAn infant boy with inborn generalized infantile myofibromatosis that included bone, intracranial, soft tissue and visceral involvement was treated according to recent recommendations with low dose chemotherapy. The presence of a partial but temporary response led to a second line of treatment with six cycles of chemotherapy, which achieved a partial response again but was followed by severe toxicity. The generalized progression of the disease was observed later. Genetic analyses were performed and revealed a PDGFRB gene c.1681C>A missense heterozygous germline mutation, high PDGFRβ phosphokinase activity within the tumor and the heterozygous germline Slavic Nijmegen breakage syndrome 657del5 mutation in the NBN gene. Targeted treatment with sunitinib, the PDGFRβ inhibitor, plus low dose vinblastine led to an unexpected and durable response without toxicities or limitations to daily life activities. The presence of the Slavic NBN gene mutation limited standard chemotherapy dosing due to severe toxicities. Sister of the patient suffred from skull base tumor with same genotype and histology. The same targeted therapy led to similar quick and durable response.ConclusionProgressive and resistant incurable infantile myofibromatosis can be successfully treated with the new approach described herein. Detailed insights into the biology of the patient’s tumor and genome are necessary to understand the mechanisms of activity of less toxic and effective drugs except for up to date population-based chemotherapy regimens.

An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations.

Nbs1 is one of the genes responsible for Nijmegen breakage syndrome, which is marked with high radiosensitivity. In human NBS1 (hNBS1), Q185E polymorphism is known as the factor to cancer risks, although its DSB repair defect has not been addressed. Here we investigated the genetic variations in medaka (Oryzias latipes) wild populations, and found 40 nonsynonymous single nucleotide polymorphisms (SNPs) in medaka nbs1 (olnbs1) gene within 5 inbred strains. A mutation to histidine in Q170 residue in olNbs1, which corresponds to Q185 residue of hNBS1, was widely distributed in the closed colonies derived from the eastern Korean population of medaka. Overexpression of H170 type olNbs1 in medaka cultured cell lines resulted in the increased accumulation of olNbs1 at laser-induced DSB sites. Autophosphorylation of DNA-dependent protein kinase at T2609 was suppressed after the γ-ray irradiation, which was followed by prolonged formation of γ-H2AX foci and delayed DSB repair. These findings suggested that the nonsynonymous SNP (Q170H) in olnbs1, which induced DSB repair defects, is specifically distributed in the eastern Korean population of medaka. Furthermore, examination using the variation within wild populations might provide a novel method to characterize a driving force to spread the disease risk alleles.

DNA Damage Response in Human Stem Cells and Neural Descendants.

Glial cells are crucial for the normal function of neurons and are intricately involved in the pathogenesis of neurodegenerative diseases as well as neurologic malignancies. A deeper understanding of the mechanisms by which glial cells influence the development of such pathologies will undoubtedly lead to new and improved therapeutic approaches. Commercially available human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), both of which can be differentiated into neural progenitors (NPs) and various neural cell lineages, have become widely used as sources for producing normal human central nervous system (CNS) cells. A better understanding of the DNA damage response (DDR) that occurs in these cells after therapeutic ionizing radiation (IR) and chemotherapy is essential for assessing the effects on healthy human brain.Neurodegenerative features associated with conditions such as ataxia telangiectasia and Nijmegen breakage syndrome highlight the importance of DNA double strand break (DSB) repair pathways in maintaining genomic integrity in cells of the CNS. Similarly, the development of brain tumors is also intricately linked to DNA repair. The importance of ATM and the other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, ATR and DNA-PKcs, is not fully defined in either CNS developmental or pathological states. While their roles are relatively well established in the DDR of proliferating cells, our recent work has demonstrated that these processes exhibit spatiotemporal evolution during cell differentiation. This chapter discusses and explores various laboratory techniques for investigating the role of ATM in hESCs and differentiated neural cells.

Circulating T Cells of Patients with Nijmegen Breakage Syndrome Show Signs of Senescence

PurposeThe Nijmegen breakage syndrome (NBS) is an inherited genetic disorder characterized by a typical facial appearance, microcephaly, growth retardation, immunodeficiency, and a strong predisposition to malignancies, especially of lymphoid origin. NBS patients have a mutation in the NBN gene which involves the repair of DNA double-strand breaks (DSBs). Here we studied the peripheral T cell compartment of NBS patients with a focus on immunological senescence.MethodsThe absolute numbers and frequencies of the different T cell subsets were determined in NBS patients from young age till adulthood and compared to age-matched healthy individuals (HI). In addition, we determined the expression of senescent T cell markers and the signal joint T cell receptor excision circles (sjTRECs) content.ResultsOur results demonstrate that NBS patients have reduced T cell numbers. NBS patients showed lower numbers of αβ+ T cells, but normal γδ+ T cell numbers compared to HI. Concerning the αβ+ T cells, both CD4+ as well as CD8+ T cells were excessively reduced in numbers compared to aged-matched HI. In addition, NBS patients showed higher frequencies of the more differentiated T cells expressing the senescent cell marker CD57 and did not express co-stimulatory molecule CD28. These effects were already present in the youngest age group. Furthermore, NBS patients showed lower sjTREC content in their T cells possibly indicative of a lower thymic output.ConclusionsWe conclude that circulating T cells from NBS patients show signs of a senescent phenotype which is already present from young age on and which might explain their T cell immune deficiency.

The Slavic NBN Founder Mutation: A Role for Reproductive Fitness?

The vast majority of patients with Nijmegen Breakage Syndrome (NBS) are of Slavic origin and carry a deleterious deletion (c.657del5; rs587776650) in the NBN gene on chromosome 8q21. This mutation is essentially confined to Slavic populations and may thus be considered a Slavic founder mutation. Notably, not a single parenthood of a homozygous c.657del5 carrier has been reported to date, while heterozygous carriers do reproduce but have an increased cancer risk. These observations seem to conflict with the considerable carrier frequency of c.657del5 of 0.5% to 1% as observed in different Slavic populations because deleterious mutations would be eliminated quite rapidly by purifying selection. Therefore, we propose that heterozygous c.657del5 carriers have increased reproductive success, i.e., that the mutation confers heterozygote advantage. In fact, in our cohort study of the reproductive history of 24 NBS pedigrees from the Czech Republic, we observed that female carriers gave birth to more children on average than female non-carriers, while no such reproductive differences were observed for males. We also estimate that c.657del5 likely occurred less than 300 generations ago, thus supporting the view that the original mutation predated the historic split and subsequent spread of the ‘Slavic people’. We surmise that the higher fertility of female c.657del5 carriers reflects a lower miscarriage rate in these women, thereby reflecting the role of the NBN gene product, nibrin, in the repair of DNA double strand breaks and their processing in immune gene rearrangements, telomere maintenance, and meiotic recombination, akin to the previously described role of the DNA repair genes BRCA1 and BRCA2.

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. DisclosuresNo relevant conflicts of interest to declare.

Successful Treatment of Splenomegaly and Pancytopenia with Rituximab in a Patient with Nijmegen Breakage Syndrome

: Nijmegen breakage syndrome is a rare autosomal recessive congenital disorder causing chromosomal instability, characterized by short stature, microcephaly, distinctive facial features, recurrent respiratory tract infections, an increased risk of cancer, intellectual disability, and other health problems. People with Nijmegen breakage syndrome have immunodeficiency. Some patients with ataxia telangiectasia-like syndromes (about 10%) have decreased serum IgA and IgG levels with normal or raised IgM level, a phenotype reminiscent of hyper IgM syndrome, which is due to class switch recombination defect. The case presented in this report was an eight-year-old female with related parents, who had been admitted to the hospital several times with recurrent infections (pneumonia and sinusitis). In immunological workups, she had high IgM, low IgG, and IgA levels. According to high α-fetoprotein level and her microcephaly, Nijmegen breakage syndrome was suggested. She was receiving IVIg monthly for two years, when she developed hypersplenism and pancytopenia. Her bone marrow aspiration and biopsy was reported normal twice. The patient underwent Rituximab therapy (375mg/m2) weekly for four weeks, with good response and improvement of splenomegaly and pancytopenia. Class switch recombination defects should be considered in patients with ataxia telangiectasia variants, especially when they have hyper IgM phenotype, and if they present lymphoproliferation, Rituximab therapy could be an effective treatment.

High Expression of MRE11-RAD50-NBS1 Is Associated with Poor Prognosis and Chemoresistance in Gastric Cancer.

The MRN complex of meiotic recombination 11 (MRE11), DNA repair protein Rad50 (RAD50) and Nijmegen breakage syndrome 1 (NBS1) proteins coordinate the detection and repair of DNA double-strand breaks (DSBs). DNA DSB repair-dependent chemoresistance likely has an effect on the treatment of human cancer. We investigated the expression of MRN complex in human gastric cancer (GC) tissues using immunohistochemistry and analyzed its clinical significance and prognostic relevance. The expression of MRN complex was significantly associated with clinical factors including poorer prognosis and negatively associated with the expression of DNA damage marker phosphorylated H2A histone family, member X (γH2AX) in the nucleus. In the biopsy specimens, low expression of MRE11 correlated with good response to chemotherapy and surgical resection after down-staging by chemotherapy. Furthermore, the expression levels of MRE11 and RAD50 were independent predictors of surgical resection after chemotherapy. The high expression of MRN complex constituents could be a predictor for poor prognosis and chemoresistance in GC.

The frequency of incidental findings in expanded carrier screening

To examine the frequency of incidental findings in expanded carrier screening - diseases where carriers may also have clinical health implications (CHI). Expanded carrier screening (ECS) identifies couples at risk for transmitting a genetic condition to their offspring. Patients may be counseled during the informed consent process that carriers of recessive conditions usually do not experience symptoms. Yet, there are several conditions on ECS panels where carriers may have CHI. We analyzed Counsyl laboratory’s experience of over 346,790 expanded carrier screens to calculate this frequency. Individuals were tested for carrier status in up to 108 genes by either targeted genotyping (TG) of up to 417 sites or next-generation sequencing (NGS) of the exons and selected introns of the chosen genes. Carrier frequencies for each condition were calculated as the higher of TG or NGS frequencies. Five autosomal recessive diseases were identified for their association with CHI: Nijmegen breakage syndrome (NBN) for cancer risk; ataxia-telangiectasia (ATM) for breast cancer risk; pseudocholinesterase deficiency (BCHE) for increased risk for prolonged period of breathing paralysis after receiving choline ester drugs; hereditary thymine-uraciluria (DPYD) for risk for toxicity following 5-fluorouracil chemotherapy treatment; and factor XI deficiency (F11) for risk for bleeding problems. Carrier frequencies for each condition were tabulated per ethnic group, weighted by US Census Data 2010, and summated to represent the ethnic distribution in USA. 346,790 patients indicating “routine carrier testing” as the reason for testing were selected from a total population of 430,584 (all indications). From this subset, 308,668 (89%) received TG and 38,122 (11%) received NGS test. The carrier frequencies for each condition were 1/280 (ATM), 1/24 (BCHE), 1/109 (DPYD), 1/150 (F11), and 1/560 (NBN). This summates to an overall frequency of 6.2% for CHI on ECS. BCHE accounted for 67% of the frequency of CHI and is more common in individuals of Ashkenazi Jewish (4.5%) and European descent (4.9%) than those of African (1.0%) or Asian descent (1.2%). 6.2% of individuals (1 in 16) screened for these diseases will have an incidental finding - where carriers identified may experience CHI. Pre-test counseling, informed consent, reporting, and post-test counseling should educate patients and providers regarding this possibility.

Nijmegen-Breakage Syndrome; Two Siblings Presenting with Different Phenotypes

The Nijmegen Breakage syndrome (NBS) is characterized by chromosomal instability, combined immunodeficiency, and distinctive physical features. We present two siblings with NBS presenting with strikingly different manifestations. The proband is a 6-year-old female with short stature, microcephaly, hepatosplenomegaly, rectovaginal fistula, anal atresia, an ectopic kidney, recurrent fevers and otitis media. A 7-year-old brother has developmental delay, failure to thrive, and microcephaly without recurring infections. Both patients have hypogammaglobulinemia, B cell lymphopenia and reduced phytohaemagglutinin-induced lymphocyte proliferation. Both siblings are homozygous for the c.657_661delACAAA (p.Lys219Asnfs*16) mutation in the NBN (NBS1) gene.

Non-Hodgkin lymphoma and pre-existing conditions: spectrum, clinical characteristics and outcome in 213 children and adolescents.

Children and adolescents with pre-existing conditions such as DNA repair defects or other primary immunodeficiencies have an increased risk of non-Hodgkin lymphoma. However, large-scale data on patients with non-Hodgkin lymphoma and their entire spectrum of pre-existing conditions are scarce. A retrospective multinational study was conducted by means of questionnaires sent out to the national study groups or centers, by the two largest consortia in childhood non-Hodgkin lymphoma, the European Intergroup for Childhood non-Hodgkin Lymphoma, and the international Berlin-Frankfurt-Münster Study Group. The study identified 213 patients with non-Hodgkin lymphoma and a pre-existing condition. Four subcategories were established: a) cancer predisposition syndromes (n=124, 58%); b) primary immunodeficiencies not further specified (n=27, 13%); c) genetic diseases with no increased cancer risk (n=40, 19%); and d) non-classifiable conditions (n=22, 10%). Seventy-nine of 124 (64%) cancer predispositions were reported in groups with more than 20 patients: ataxia telangiectasia (n=32), Nijmegen breakage syndrome (n=26), constitutional mismatch repair deficiency (n=21). For the 151 patients with a known cancer risk, 5-year event-free survival and overall survival rates were 40%±4% and 51%±4%, respectively. Five-year cumulative incidences of progression/relapse and treatment-related death as a first event were 22%±4% and 24%±4%, respectively. Ten-year incidence of second malignancy was 24%±5% and 7-year overall survival of the 21 patients with a second malignancy was 41%±11%. Patients with non-Hodgkin lymphoma and pre-existing conditions have an inferior survival rate with a large proportion of therapy-related deaths compared to patients with non-Hodgkin lymphoma and no pre-existing conditions. They may require special vigilance when receiving standard or modified/reduced-intensity chemotherapy or when undergoing allogeneic stem cell transplantation.

Chromosomal Instability and Molecular Defects in Induced Pluripotent Stem Cells from Nijmegen Breakage Syndrome Patients

Nijmegen breakage syndrome (NBS) results from theabsence of the NBS1 protein, responsible for detection of DNA double-strand breaks (DSBs). NBS is characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Here, we show successful reprogramming ofNBS fibroblasts into induced pluripotent stem cells (NBS-iPSCs). Our data suggest a strong selection for karyotypically normal fibroblasts to go throughthe reprogramming process. NBS-iPSCs then acquire numerous chromosomal aberrations and show a delayed response to DSB induction. Furthermore, NBS-iPSCs display slower growth, mitotic inhibition, a reduced apoptotic response tostress, and abnormal cell-cycle-related gene expression. Importantly, NBS neural progenitor cells (NBS-NPCs) show downregulation of neural developmental genes, which seems to be mediated by P53. Our results demonstrate the importance of NBS1 in early human development, shed light on the molecular mechanisms underlying this severe syndrome, and further expand our knowledge of the genomic stress cells experience during the reprogramming process.

Abstract 2428: Genetic aberrations in the DNA repair pathway among children with Philadelphia chromosome positive leukemias

Abstract Background. Aberrations in the DNA repair pathway among children with leukemia are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. Germline aberrations of the DNA-repair machinery are crucial for generating chromosomal instability and occurrence of acute leukemia in children. In order to better understand this mechanism, we studied a specific subgroup of childhood leukemia, e.g. t(9;22) or chromosome Philadelphia positive (Ph+) acute lymphoblastic and chronic myeloid leukemias. We addressed our research to specific genes (Nijmegen Breakage Syndrome (NBS1) and Fanconi Anemia) involved in the DNA repair pathway, in the attempt to identify mutations and aberrant expressions, predisposing and/or cooperating with the leukemogenic process. Materials and Methods. We analyzed diagnostic and remission samples of 12 children: 8 with a Ph+ acute lymphoblastic leukemia (ALL) and 4 with a chronic myeloid leukemia (CML) diagnosed and treated at our institution from 1999 to 2013. We performed RT-PCR analyses to detect mutations in exons 3-6 of the NBS1 gene, followed by a direct sequencing analysis (BMR - Padova - Italy). The status of FANCD2 and PALB2 genes was studied by a multiplex ligation-dependent probe amplification (MLPA) using the Salsa MLPA P057-025R FANCD2-PALB2 Probemix and SALSA MLPA EK1 kit (MRC-Holland, Amsterdam, the Netherlands). Samples from five healthy donors (HDs) were used as wild-type controls. For data elaboration, we used Coffalyser.Net software for MLPA. Moreover we performed a real-time PCR amplification of BRCA1 (exons 14-15) and BRCA2 (exons 15-16) genes, respectively, calculating the median fold-changes (MFC) in patients comparing with HDs. Results. We detected the NBS1 E185Q polymorphism (c533G&amp;gt;C, rs1805794) in both diagnostic and remission samples in 3 out of 12 children with a Ph positive acute leukemia (2 ALL, 1 CML). We also found two pairs of deletions in FANCD2 (Δ32-35; Δ35-38) and PALB2 (Δ2; Δ2-6) in 4 out of 12 Ph positive cases (2 ALL and 2 CML). These aberrations were mutually exclusive. Thus a total of 7 cases (58%) showed an aberrant pathway. Moreover we determined that the MFC of BRCA1 expression among children with Ph positive ALL and CML was slightly higher than HDs (1,388 vs 0,433 and 1,625 vs 0,451, respectively). Surprisingly the MFC of BRCA2 expression was strongly higher than HDs in both children with Ph positive ALL (5,924 vs 0,002) and CML (54,504 vs 0,256). Conclusion. Our preliminary findings strongly suggest that germline mutations or deletions in association with aberrant expression of specific genes that regulate the DNA repair machinery may contribute to the chromosomal instability in children with Ph positive leukemias. These data will be confirmed in a larger population of different subtypes of pediatric leukemia. Citation Format: Nellina Andriano, Paola Bonaccorso, Valeria Iachelli, Manuela La Rosa, Emanuela Cannata, Luca Lo Nigro. Genetic aberrations in the DNA repair pathway among children with Philadelphia chromosome positive leukemias. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2428.