Frequency Of Translocations Research Articles

Nanopore Resistive Pulse Sensing with Multiple Alpha-Hemolysin Pores Improves the Detection Limit of Microrna

The concentration profile of microRNAs in body fluids has been related to a variety of diseases, including specific cancers. By conjugation with a complementary DNA probe, specific miRNA species can be enumerated by nanopore resistive pulsing. This is possible because a miRNA-DNA probe duplex temporarily blocks a nanopore of ∼1.5 nm diameter, causing a decrease in the electrical current by displacing electrolyte ions. Under the influence of the electrophoretic driving force, the duplex dissociates after ∼10-1000 ms, and both the miRNA and the DNA molecules transit through the pore. The pore is now open again and can capture another duplex, with the frequency of the duplex pore translocations corresponding to the miRNA concentration in the solution. Biological porins are attractive because of their reproducible self-assembly, but the lipid bilayer matrix prevents the application of voltages >120 mV. It has previously been shown that the electrophoretic driving force can also be enhanced by an electrolyte concentration gradient of 0.5 / 4 M KCl, but few pulses were observed below 1 nM duplex concentration. Here, we demonstrate that the frequency can be further increased by incorporating multiple pores in the same bilayer. With an electrolyte gradient of 0.2 / 1 M KCl, it was possible to perform resistive pulse sensing with up to 100 alpha-hemolysin pores in an aperture-suspended DPhPC bilayer. We characterize the effect of electrolyte salt gradients on the stability of bilayer-incorporated alpha-hemolysin and quantify the resistive pulses for duplex concentrations in the range 0.01-100 nM. The achieved ∼10-fold increase in frequency, with respect to single-pore sensing with a 0.5 / 4 M KCl gradient, demonstrates the potential of multi-pore sensing to achieve miRNA detection at clinically relevant concentrations.

Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and -independent mechanisms

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.

Assessment of working environment and personal dosimeter-wearing compliance of industrial radiographers based on chromosome aberration frequencies

Industrial radiographers are exposed to relatively higher doses of radiation than other radiation-exposed workers in South Korea. The objective of our study was to investigate the impact of specific occupational conditions on chromosome aberration frequency and evaluate dosimeter-wearing compliance of industrial radiographers in Korea. We studied individual and occupational characteristics of 120 industrial radiographers working in South Korea and evaluated the frequency of dicentrics and translocations in chromosomes to estimate radiation exposure. The association between working conditions and chromosome aberration frequencies was assessed by Poisson regression analysis after adjusting for confounding factors. Legal personal dosimeter-wearing compliance among workers was investigated by correlation analysis between recorded dose and chromosome aberration frequency. Daily average number of radiographic films used in the last six months was associated with dicentrics frequency. Workers performing site radiography showed significantly higher translocation frequency than those working predominantly in shielded enclosures. The correlation between chromosome aberration frequency and recorded dose was higher in workers in the radiography occupation since 2012 (new workers) than other veteran workers. Our study found that site radiography could affect actual radiation exposure to workers. Controlling these working conditions and making an effort to improve personal dosimeter-wearing compliance among veteran workers as well as new workers may be necessary to reduce radiation exposure as much as possible in their workplace.

Detection of Simple, Complex, and Clonal Chromosome Translocations Induced by Internal Radioiodine Exposure: A Cytogenetic Follow-Up Case Study after 25 Years

Here, we report the findings of a 25-year cytogenetic follow-up study on a male patient who received 2 rounds of radioiodine treatment within a span of 26 months (1.78 GBq in 1992 and 14.5 GBq in 1994). The patient was 34 years old with a body mass index of 25 at the time of the first radioiodine treatment. Multicolor FISH and multicolor banding (mBAND) techniques performed on the patient detected inter- and intrachromosomal exchanges. Although the frequency of chromosome translocations remained essentially the same as reported in our earlier study (0.09/cell), the percentage of reciprocal (balanced) translocations increased from 54.38 to 80.30% in the current study. In addition to simple chromosome translocations, complex exchanges (0.29%) involving more than 2 chromosomes were detected for the first time in this patient. Strikingly, a clonal translocation involving chromosomes 14 and 15, t(14p;15q), was found in 7 of the 677 cells examined (1.03%). The presence of complex and clonal translocations indicates the onset of chromosomal instability induced by internal radioiodine exposure. mBAND analysis using probes specific for chromosomes 1, 2, 4, 5, and 10 revealed 5 inversions in a total of 717 cells (0.69%), and this inversion frequency is several-fold higher than the baseline frequency reported in healthy individuals using the classical G-banding technique. Collectively, our study suggests that stable chromosome aberrations such as translocations and inversions can be useful not only for retrospective biodosimetry but also for long-term monitoring of chromosomal instability caused by past radioiodine exposure.

Single-Molecule Translocation Conformational Sensing of Multiarm DNA Concatemers Using Glass Capillary Nanopore.

Glass capillary-based nanopore is exploited for single-molecule conformational sensing of multiarm DNA concatemers during translocation. Both translocation frequency and orientation preference were found related with the number of arms of the DNA concatemers.

Chromosome Aberrations Produced by Mestranol in Human Lymphocyte Cultures

Abstract In this investigation, the genotoxic properties of mestranol were examined in vitro. Human lymphocyte cultures were exposed for 72 h to mestranol at concentrations of 7.5, 15 and 30 µg/g. The genotoxic effects of the chemosterilant were assessed by numerical and structural chromosome aberrations. Mestranol induced certain genotoxic effects in human lymphocytes. There was a dose-dependent significant (p<0.01) increase in the number of numerical aberrations in comparison to the control, but without significant differences (p>0.05) between the doses applied. Further, structural aberrations increased significantly (p<0.01) in the presence of mestranol, being most frequent in cultures exposed to the highest mestranol dose. The frequency of Robertsonian translocations increased significantly only in cultures treated with mestranol at concentration of 30 µg/g in comparison both with the control (p<0.01) and the lowest chemosterilant dose (p<0.01). There were significant differences (p<0.01) in the levels of chromosome gaps and fragments compared to Robertsonian translocations, whilst the frequencies between gaps and fragments were not significantly different (p>0.05).

Comprehensive Analysis of MYC Translocations in Multiple Myeloma By Whole Genome Sequencing and Whole Transcriptome Sequencing

Background 8q24 translocations leading to overexpression of MYC are an established prognostic marker in multiple myeloma (MM). Currently FISH (fluorescence in situ hybridization) on CD138+ enriched cell population is the standard diagnostic approach to evaluate the presence of 8q24 translocations. Due to the heterogeneity of breakpoints and technical issues the design of FISH probes is challenging and so far no single FISH assay is capable of detecting each translocation. Aims (1) Evaluation of the frequency of 8q24 translocations in MM by whole genome sequencing (WGS). (2) Determination of the breakpoints on 8q24 and partners. (3) Correlation of WGS data with FISH and MYC expression determined by whole transcriptome sequencing (WTS). Patient cohort and methods CD138+ cell fractions were selected by MACS from bone marrow aspirate samples of 264 patients diagnosed with MM. FISH, WGS and WTS were performed in all cases. For WGS, 151bp paired-end sequences where generated on NovaSeq 6000 machines (Illumina, San Diego, CA). All reported p-values are two-sided and were considered significant at p<0.05. For gene expression (GE) analysis by WTS, estimated gene counts were normalized and the resulting log2 counts per million were used as a proxy of gene expression in each sample. For artefact exclusion, structural variants were checked against 4386 cases covering the spectrum of hematological malignancies. Results In 91/264 (34%) of cases, at least one rearrangement involving the MYC locus (MYCr) was detected by WGS. In 18 of these samples (20%), >1 MYCr was present (114 MYCr in total). Out of these 91 patients, in 32 (35%) the MYCr had been identified by FISH, in 46 cases (51%) it was not detected due to the heterogeneity of breakpoints, while in 13 (14%) patients FISH could not be evaluated (e.g. due to insufficient patient material). Of the 114 MYCr encountered in WGS, 42 involved one of the immunoglobulin loci (IGH n=25, IGK n=9, IGL n=8). The remaining 72 MYCr involved other rare partners. In 29 of these rearrangements, as well as in four complex rearrangements involving IGH or IGK, recurrent rare partners were identified, comprising 1p12/FAM46C (n=6), 6p24.3/BMP6 (n=10), 6q21/FOXO3 (n=4), 7p21.3 (n=3), 11q13/CCND1 (n=5), 20q11.22 (n=5). 43 MYCr involved non-recurrent (single) rare partners, for 4 of these a MYCr was also detected by FISH. The MYCr detected were rather complex: only 34 (30%) showed a simple reciprocal translocation (IGH n=7, IGL n=2, IGK n=4, rare partners n=21), 60 (53%) showed more complex rearrangements (IGH n=12, IGL n=4, IGK n=2, rare partners n=42) and in 20 cases (18%) at least one additional chromosome was involved (IGH n=6, IGL n=3, IGK n=2, rare partners n=9). In 80% of MYCr, breakpoints were located between genomic positions 128.203.605 and 129.375.490 encompassing the pre-described MYC surrounding locus PVT1. IGH-MYC rearrangements showed a tendency to cluster towards the centromere. MYCr involving rare partners showed the broadest breakpoint spectrum and clustered in both directions of the hotspot (Fig 1A). Regarding expression of MYC, all cases showed an overexpression (median GE: 6.9 vs 4.5 in normal controls). Median GE was similar in cases with Ig partners (IGH: 7.1, IGL: 6.7, IGK: 6.6) and non Ig partners (6.8) and also in cases with MYCr detected by FISH (7.0) and cases in which it was not detected by FISH (6.5). Analysis of additional chromosomal aberrations revealed that hyperdiploidy was significantly more frequent in MYCr (n=68/91, 75% vs n=76/173, 44%; p=0.001), while t(11;14) was found significantly less frequent (n=11/91, 12% vs n=49/173, 28%; p=0.003) (Fig 1B). No associations were found between MYCr and other frequent chromosomal abnormalities. Furthermore, molecular mutations frequently occurring in MM (ATM, BRAF, KRAS, NRAS, TP53, IRF4) were analyzed, revealing that patients with MYCr were significantly less frequently associated with mutations in the IRF4 gene (MYCr patients n=1/91; non-MYCr patients n=13/173; p =0.028) (Fig 1C). Conclusions (1) WGS detects ~3x more MYCr compared to FISH. (2) The complexity on the genomic level of MYCr is high, therefore the detection with targeted assays is limited while WGS allows a more comprehensive analysis. (3) MYC expression in cases with MYCr with non Ig partners is comparably high as for Ig-MYC translocations. (4) MYCr are associated with hyperdiploidy, whereas t(11;14) and IRF4 mutations were detected at a lower frequency. Disclosures Seliger: MLL Munich Leukemia Laboratory: Employment. Geirhos:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Lack of Significant Differences in Somatic Alterations between MGUS, SMM and Symptomatic Multiple Myeloma: A Result from Comprehensive Genomic Profiling Study

Somatic alterations including single nucleotide variants (SNVs), copy number alterations (CNAs) and structural variants (SVs) are an important component and the hallmark of Multiple Myeloma (MM) with certain alterations having clinical implications. Recently, the chronological order of such events was explained in detailed studies however previous study has compared the frequency and clonality of such events in early stage plasma cell disorder (MGUS or SMM) and their comparison with MM.Here we performed whole genome (WGS) and RNA sequencing (RNAseq) on 67 samples (8 Unique paired samples from patients with MGUS and when progressed to MM, 10 paired samples at SMM stage and then progressed to MM, as well as non-progressor SMM (NP-SMM) patients who have not progressed after at least 5 years of follow-up. In addition, we profiled CNAs and their clonality level in 164 MGUS, 147 SMM and 336 newly diagnosed MM patients to construct the clonal structure and frequency of somatic alterations.We did not observe significant difference in overall frequency of mutations at MGUS (median=5662 SNVs, IQR= [5252 - 7864]) and SMM stage (median=5308 SNVs, IQR=[4236 - 6125] ) and at progression to MM (median= 5983 SNVs, IQR=[5097 - 8916]). Overall mutational load increased from precursor stage to MM by only 5% with no recurrent change and the difference was not statistically significant (p > 0.05). Similar trend was also observed for SVs [median=36, IQR= [25.25 -40.75] at MGUS, median=34, IQR= [12-52] at SMM, median=27, IQR= [27-41] at MM]. In fact, some of the samples had lower SNV and SV load in MM compared to precursor stages even though that had increased cancer cell fraction. Copy number analysis on paired samples also showed >90% similarity only with few non-recurrent focal CNAs. We have compared WGS data from non-progressor SMM with progressor SMM and newly-diagnosed MM. The non-progressor SMM patients showed identical CNAs and not significantly different Ig translocation frequencies to both other groups. Except for NRAS mutations, mutation frequency in NP-SMM was similar to others. Finally, by using data from 647 patients at three stages (MGUS, SMM and MM), we compared the frequency of CNAs at each time point. Similar to MM, we observed almost equal frequency of hyperdiploid and non-hyperdiploid SMM cases. In hyperdiploid SMM, clonal copy number gain was observed in 95% of patients involving two of the three chromosomes 9, 15 and 19. Moreover, a similar pattern was observed in other hyperdiploid chromosomes including chromosomes 3,5,7,11 and 21. The observed event frequency and clonality levels in SMM were identical to one observed in MM as well as MGUS. Non-hyperdiploid SMM group had early and clonal events targeting chromosome 13 deletion with gain of 1q, 11 and/or 9 at the same frequency level as in MM. The only significant difference was observed in subclonal deletions targeting some of the infrequent deletion events observed in HMM and NHMM. In conclusion, our data from longitudinal analysis of paired precursor and symptomatic MM samples as well as cross sectional data highlight lack of significant differences in genomic changes between precursor conditions and symptomatic myeloma. This lack of progressive and unique genomic change suggests the need for additional transcriptomic, epigenomic or tumor micro environment changes to accelerate the tumor development. Disclosures Bolli: Celgene: Honoraria; Gilead: Other: travel expenses; Novartis: Honoraria. Moreau:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Munshi:Celgene: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy.

Chromosome territory formation attenuates the translocation potential of cells.

The formation and spatial arrangement of chromosome territories (CTs) in interphase has been posited to influence the outcome and frequency of genomic translocations. This is supported by correlations between the frequency of inter-chromosomal contacts and translocation events in myriad systems. However, it remains unclear if CT formation itself influences the translocation potential of cells. We address this question in Drosophila cells by modulating the level of Condensin II, which regulates CT organization. Using whole-chromosome Oligopaints to identify genomic rearrangements, we find that increased contact frequencies between chromosomes due to Condensin II knockdown leads to an increased propensity to form translocations following DNA damage. Moreover, Condensin II over-expression is sufficient to drive spatial separation of CTs and attenuate the translocation potential of cells. Together, these results provide the first causal evidence that proper CT formation can protect the genome from potentially deleterious translocations in the presence of DNA damage.

Investigation of the cumulative number of chromosome aberrations induced by three consecutive CT examinations in eight patients.

In our previous study, we found that chromosomes were damaged by the radiation exposure from a single computed tomography (CT) examination, based on an increased number of dicentric chromosomes (Dics) formed in peripheral blood lymphocytes after a CT examination. We then investigated whether a cumulative increase in the frequency of Dics and chromosome translocations (Trs) formation could be observed during three consecutive CT examinations performed over the course of 3–4 years, using lymphocytes in peripheral bloods of eight patients (five males and three females; age range 27–77 years; mean age, 64 years). The effective radiation dose per CT examination estimated from the computational dosimetry system was 22.0–73.5 mSv, and the average dose per case was 40.5 mSv. The frequency of Dics formation significantly increased after a CT examination and tended to decrease before the next examination. Unlike Dics analysis, we found no significant increase in the frequency of Trs formation before and after the CT examination, and we observed no tendency for the frequency to decrease before the next CT examination. The frequency of Trs formation was higher than that of Dics formation regardless of CT examination. Furthermore, neither analysis of Dics nor Trs showed a cumulative increase in the frequency of formation following three consecutive CT examinations.

Interpretation of FISH Results in the Case of Nonuniform Internal Radiation Exposure of Human Body with the Use of Model Approach

The fluorescence in situ hybridization (FISH) technique allows for the estimation of the number of stable chromosomal aberrations (translocations) in human blood T-lymphocytes decades after radiation exposure. Therefore, it is used for retrospective assessment of irradiation doses received by red bone marrow (RBM). Cytogenetic analysis of individuals exposed on the Techa River in the 1950s to 89,90Sr, locally irradiating RBM, showed that dose assessment based on FISH data led to underestimation of the actual dose estimated from measurements of whole-body 90Sr content. The purpose of current study was to evaluate the dose–effect relationship and the frequency of translocations per 1 Gy of RBM irradiation dose and T-lymphocytes dose for donors living in Techa riverside villages (n = 178; translocation yield = 2389; number of genome equivalents GE = 96 446). Irradiation doses were estimated using the latest version of the Techa River dosimetry system and the T-cell irradiation model. Statistical analysis showed that the frequency of translocations per 1000 GE per 1 Gy of RBM dose was 8.0 ± 0.7; this value was statistically significantly lower than the value estimated for Sellafield radiation workers under external exposure (11.6 ± 1.6). At the same time, the use of T-cell and their progenitor doses led to re-evaluation (increase) in the slope of the dose–response curve to 11.8 ± 1.6 per 1 Gy per 1000 GE, which agreed well with the published data. The obtained estimates of the background frequencies of translocations also were close to the published values ​​for unexposed donors. These findings demonstrate the legitimacy of using a model approach for estimation of irradiation doses in T-lymphocytes and their progenitors and the need to take into account complex T-cell dynamics in interpretation of cytogenetic data for the purposes of biodosimetry.

The nucleoskeleton protein IFFO1 immobilizes broken DNA and suppresses chromosome translocation during tumorigenesis.

Chromosome translocation is a major cause of the onset and progression of diverse types of cancers. However, the mechanisms underlying this process remain poorly understood. Here, we identified a non-homologous end-joining protein, IFFO1, which structurally forms a heterotetramer with XRCC4. IFFO1 is recruited to the sites of DNA damage by XRCC4 and promotes the repair of DNA double-strand breaks in a parallel pathway with XLF. Interestingly, IFFO1 interacts with lamin A/C, forming an interior nucleoskeleton. Inactivating IFFO1 or its interaction with XRCC4 or lamin A/C leads to increases in both the mobility of broken ends and the frequency of chromosome translocation. Importantly, the destruction of this nucleoskeleton accounts for the elevated frequency of chromosome translocation in many types of cancer cells. Our results reveal that the lamin A/C-IFFO1-constituted nucleoskeleton prevents chromosome translocation by immobilizing broken DNA ends during tumorigenesis.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Abstract 5176: Topological characterization of chromosome territories 9 and 22 and BCR-ABL1 genes in bone marrow CD34+ cells

Abstract During interphase, chromosomes occupy a distinct, limited space within the nucleus, referred to as chromosome territory (CT). It has been postulated that the non-random CT’s spatial organization within the cell nucleus contributes to determining the outcome of chromosomal translocations. Comparative analysis of the spatial arrangement of translocation partners and their frequency of translocation suggest that translocations occur preferentially among proximally positioned genome regions. The consistence appearance of t(9;22) in pluripotent hematopoietic stem cells, apart from being etiological factor for chronic myeloid leukemia (CML), is a clear example of what is considered a non-random chromosome aberration. In order to highlight the importance of spatial proximity and topological features in determining the t(9;22) formation, we evaluated these elements using three-dimensional fluorescence in situ hybridization and 3D Structure Illumination Microscopy. This analysis was carried out in CD34+ cells from 5 CML patients in complete response, in Mobilized Bone Marrow (MBM) from 5 healthy donors, and also in peripheral blood mononuclear cells (PBMC). 3D reconstructions from at least 25 cells per sample were analyzed. We determined the nucleus’ and CTs’ volume, the distance between BCR and ABL (BA) genes and their CTs, and the distances between them and the nuclear center. We found that nuclear volumes were significantly larger in CD34 + cells from MBM and CML patients compared to PBMC controls. Likewise, the absolute CTs volume analysis shows a major volume of CT9 compared to CT22. In order to avoid the bias of size differences due to nuclear volumes, we normalized the CTs’ volumes and expressed them as a percentage of the nuclear volume in each studied population, however it does not show a significant variation. After normalizing the distance between the CTs a To determine the distance between BA genes and their CTs we calculated the nearest distance between the mass center of CT9 and CT22. Shorter distances were observed between BA from MBM and CML patients in complete response compared to PBMC samples. After normalizing the distance between the CTs and BA genes against the radius of the nucleus. We observed that the location of CT22 tended towards the center, whereas that of CT9 towards the nuclear periphery. Meanwhile BA genes were located closer to the nuclear center than the respective CTs’ geometrical center. Given that CTs are discrete chromatin domains, histone modifications might have a significant role in dictating the topological CTs characteristics. Therefore, we have mapped some relevant histone modifications like H3K9ac and H3K27me3 across CT9 and 22 using 3D immuno-FISH. The enrichment of H3K27me3 seems to be greater in CT22 from samples of CML patients in complete response. These are preliminary results and we pretend increase the sample size to delineate conclusive results. Note: This abstract was not presented at the meeting. Citation Format: Eunice Fabián-Morales, Yameli Lizeth Rodríguez Torres, David G. Vallejo Escamilla, Rodrigo González-Barrios, Alfredo De La Torre Luján, Alejandro López-Saavedra, Clementina Castro-Hernández, Luis A Herrera Montalvo. Topological characterization of chromosome territories 9 and 22 and BCR-ABL1 genes in bone marrow CD34+ cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5176.

Abstract 2571: Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations

Abstract The faithful repair of DNA double strand breaks (DSBs) is crucial for genome integrity, and aberrant repair can result in chromosomal translocations, including those associated with infant leukemia. Evidence from our lab and others suggests that environmental and dietary compounds such as bioflavonoids have the potential to promote chromosomal translocations in utero. DSBs can be repaired by either classical non-homologous end joining (C-NHEJ) that is mostly accurate or by microhomology-mediated alternative end joining (Alt-EJ) that is more error prone and can cause chromosomal translocations. Ku70/80, Ligase 4 (LIG4), X-Ray Repair Cross-Complementing factor 4 (XRCC4), and XRCC4 like factor (XLF) are central to C-NHEJ. Poly ADP Ribose 1 (PARP 1) and C terminal binding protein interacting protein (CtIP) participate in Alt-EJ. CtIP favors Alt-EJ pathway by promoting the resection of DNA ends to uncover microhomologies supporting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in the C-NHEJ repair pathway may promote the use of the other more error-prone pathway such as Alt-EJ. The purpose of the study is to understand the relationship between bioflavonoid exposure, induction of DNA repair proteins, and repair pathway choice to understand the underlying mechanisms of bioflavonoid-induced infant leukemias. Embryonic stem (ES) cells were exposed to genistein 1 hr at doses 0μM-100μM based on determined LD50 values. Following exposure, cells were recovered 4hr, 24hr and 48 hr, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 were measured by densitometry and analyzed using one way ANOVA. CRISPR-Cas9 was used to downregulate C-NHEJ DNA repair protein XRCC4. XRCC4-/- cells will subsequently be exposed to genistein, recovered, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 will measured by densitometry. Protein levels before and after gene editing of XRCC4 will be compared and analyzed using two way ANOVA. We have downregulated the XRCC4 gene in ES cells and anticipate that following exposure to genistein in the absence of XRCC4, we will detect elevated chromosomal translocation frequency in those stem cells by stimulating the Alt-EJ pathway. This research will be highly impactful in understanding how bioflavonoid-induced DNA damage is repaired by cells and whether these compounds promote genome instability analogous to that observed in infant leukemia. Citation Format: Anindita Ghosh, Christine A. Richardson. Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2571.

Diversified Chromosome Rearrangements Detected in a Wheat‒Dasypyrum breviaristatum Substitution Line Induced by Gamma-Ray Irradiation.

To determine the composition of chromosome aberrations in a wheat‒Dasypyrum breviaristatum substitution line with seeds treated by a dose of gamma-rays (200 Gy), sequential non-denaturing fluorescence in situ hybridization (ND-FISH) with multiple oligonucleotide probes was used to screen individual plants of the mutagenized progenies. We identified 122 types of chromosome rearrangements, including centromeric, telomeric, and intercalary chromosome translocations from a total of 772 M1 and 872 M2 plants. The frequency of reciprocal translocations between B- and D-chromosomes was higher than that between A- and D-chromosomes. Eight translocations between D. breviaristatum and wheat chromosomes were also detected. The 13 stable plants with multiple chromosome translocations displayed novel agronomic traits. The newly developed materials will enhance wheat breeding programs through wheat‒Dasypyrum introgression and also facilitate future studies on the genetic and epigenetic effects of translocations in wheat genomics.

PS1431 IDENTIFICATION OF THE TRUE HYPERDIPLOID MULTIPLE MYELOMA SUBSET BY COMBINING CONVENTIONAL KARYOTYPING AND FISH ANALYSIS

Background:Prognostic evaluation of Multiple Myeloma (MM) by FISH is focused on high risk (HR) cytogenetic aberrations, namely t(4;14), t(14;16) and deletion of 17p13. Hyperdiploid MM (HRD‐MM), instead, that accounts for about 50% of newly diagnosed MM, is not routinely investigated because multiple probes are required to test over the conventional markers. However, in the era of novel agents, the identification of hyperdiploidy may be helpful in MM prognostication since trisomic MM is responsive to lenalidomide treatment and retains a favourable outcome.Aims:The aim of our study was to elaborate a flow chart of FISH prognostic assessment of MM that, beside the analysis of conventional high risk abnormalities, allows to identify those cases with favourable prognosis within hyperdiploid MM.Methods:Bone marrow of 292 patients affected by newly diagnosed MM was studied by conventional karyotype (CK) and on separated plasmacells by FISH for HR cytogenetic aberrations including t(4;14), t(14;16), 17p13 deletion and 1q21 gain. Patient's characteristics including ISS stage, symptoms at diagnosis, type of treatment and OS were collected.Results:Among our entire cohort, CK was productive in 76 patients and based on the number of chromosome of clone (47–57 chromosomes) 50 patients (66%) were classified as hyperdiploid. All patients received treatment with novel agents, including bortezomib (99%), lenalidomide (70%) or both (70%).Of note, cytogenetic classification of HRD‐MM, beyond the definition of ploidy, includes a heterogeneous group characterized not only by numerical abnormalities, but also by several structural aberrations. Therefore, even the ploidy level was not enough to characterize hyperdiploid MM. As a matter of fact, within HRD‐MM patients, high risk chromosomal changes were detected in 26/50 (52%) cases, including 21 (42%) with gain 1q, one with t(4;14) and 8 (16%) with 17p deletion. Finally, 20 (40%) patients displayed an IgH translocation. Major features associated to decreased OS were trisomy 19 (32 vs 62 months, p = 0.0194), gain 1q (30 vs 56 months, p = 0.0371), and IgH translocations (33 vs 57 months, p = 0.0285) (Figure 1A to C).Provided that definition of HRD‐MM is based on trisomies of odd chromosomes, we further classified our patients according to the number of trisomies in cases with > 5 trisomies (trisomic HRD‐MM, T‐HRD‐MM) and cases with <5 trisomies (non trisomic HRD‐MM, N‐HRD‐MM). The group with > 5 trisomies (n = 26) was characterized by lower rates of overall FISH alterations (<2, 92% vs 31%, p = 0.0001). More in detail, lower frequencies of IgH translocations (23% vs 75%, p = 0.0014) and HR FISH changes (5% vs 50%, p = 0.0023) were detected. Even monosomy 13 was less represented in this subset of patients (15% vs 69%, p = 0.008) as compared to N‐HRD‐MM. Interestingly, concomitant trisomy of 9, 11 and 15 chromosomes was described in 53% of T‐HRD‐MM patients but it was completely absent in N‐HRD MM group (p = 0.0002), indicating that in our cohort these three numerical changes represent the most powerful signature to identify HRD MM. Finally, T‐HRD MM subset displayed better outcome towards N‐HRD MM (57 vs 32 months, p = 0.001, Figure 1D).Summary/Conclusion:In our cohort of patients the combination of banding analysis and FISH for HR aberrations identify some genetic features that correlate with the presence of true hyperdiploid clone. In fact, less than two FISH aberrations, absence of HR aberrations and IgH translocation and the concomitant presence of trisomies of chromosome 9, 11 and 15 identify the hyperdiploid MM subset characterized by a favourable outcome.image

The repetitive DNA landscape in Avena (Poaceae): chromosome and genome evolution defined by major repeat classes in whole-genome sequence reads

BackgroundRepetitive DNA motifs – not coding genetic information and repeated millions to hundreds of times – make up the majority of many genomes. Here, we identify the nature, abundance and organization of all the repetitive DNA families in oats (Avena sativa, 2n = 6x = 42, AACCDD), a recognized health-food, and its wild relatives.ResultsWhole-genome sequencing followed by k-mer and RepeatExplorer graph-based clustering analyses enabled assessment of repetitive DNA composition in common oat and its wild relatives’ genomes. Fluorescence in situ hybridization (FISH)-based karyotypes are developed to understand chromosome and repetitive sequence evolution of common oat. We show that some 200 repeated DNA motifs make up 70% of the Avena genome, with less than 20 families making up 20% of the total. Retroelements represent the major component, with Ty3/Gypsy elements representing more than 40% of all the DNA, nearly three times more abundant than Ty1/Copia elements. DNA transposons are about 5% of the total, while tandemly repeated, satellite DNA sequences fit into 55 families and represent about 2% of the genome. The Avena species are monophyletic, but both bioinformatic comparisons of repeats in the different genomes, and in situ hybridization to metaphase chromosomes from the hexaploid species, shows that some repeat families are specific to individual genomes, or the A and D genomes together. Notably, there are terminal regions of many chromosomes showing different repeat families from the rest of the chromosome, suggesting presence of translocations between the genomes.ConclusionsThe relatively small number of repeat families shows there are evolutionary constraints on their nature and amplification, with mechanisms leading to homogenization, while repeat characterization is useful in providing genome markers and to assist with future assemblies of this large genome (c. 4100 Mb in the diploid). The frequency of inter-genomic translocations suggests optimum strategies to exploit genetic variation from diploid oats for improvement of the hexaploid may differ from those used widely in bread wheat.

Effect of Radiation Exposures on Fetal Hematopoietic Cells

It has generally been thought that fetuses are highly sensitive to radiation-induced cancer. Epidemiologic case-control studies indicated that X-ray exposures given to pregnant women (on the order of 1 cGy) could have increased the risk of developing childhood leukemia and solid cancers in the offspring. The authors wished to re-consider this observation. Atomic bomb survivors who were exposed in utero were found to show almost no increase in the frequency of translocations in their blood lymphocytes when the survivors were examined at around 40 years of age. Subsequent animal studies revealed that tissue stem cells in embryos/fetuses may or may not retain radiation-induced chromosome damage depending on the developmental stage at the time of irradiation. Our data are compatible with the model that radiation effects can be recorded only when an exposure occurs after the stem cells have settled in their appropriate niche, and that a small fraction of fetal hematopoietic stem cells began making long-term contributions to the lymphoid cell pool in both mice and humans. It remains to be established whether or not the increased risk of childhood leukemia and other childhood cancers was caused by fetal X-ray exposures of about 1 cGy.

DSB structure impacts DNA recombination leading to class switching and chromosomal translocations in human B cells.

Class switch recombination (CSR) requires activation-induced cytidine deaminase (AID) to trigger DNA double strand breaks (DSBs) at the immunoglobulin heavy chain (IGH) in B cells. Joining of AID-dependent DSBs within IGH facilitate CSR and effective humoral immunity, but ligation to DSBs in non-IGH chromosomes leads to chromosomal translocations. Thus, the mechanism by which AID-dependent DSBs are repaired requires careful examination. The random activity of AID in IGH leads to a spectrum of DSB structures. In this report, we investigated how DSB structure impacts end-joining leading to CSR and chromosomal translocations in human B cells, for which models of CSR are inefficient and not readily available. Using CRISPR/Cas9 to model AID-dependent DSBs in IGH and non-IGH genes, we found that DSBs with 5’ and 3’ overhangs led to increased processing during end-joining compared to blunt DSBs. We observed that 5’ overhangs were removed and 3’ overhangs were filled in at recombination junctions, suggesting that different subsets of enzymes are required for repair based on DSB polarity. Surprisingly, while Cas9-mediated switching preferentially utilized NHEJ regardless of DSB structure, A-EJ strongly preferred repairing blunt DSBs leading to translocations in the absence of NHEJ. We found that DSB polarity influenced frequency of Cas9-mediated switching and translocations more than overhang length. Lastly, recombination junctions from staggered DSBs exhibited templated insertions, suggesting iterative resection and filling in during repair. Our results demonstrate that DSB structure biases repair towards NHEJ or A-EJ to complete recombination leading to CSR and translocations, thus helping to elucidate the mechanism of genome rearrangements in human B cells.