Chromosome Breakage Research Articles

Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress

Chickpea (Cicer arietinum L.) is a bisexual and self-pollinated legume. It improves the soil fertility through its natural ability to fix atmospheric nitrogen with its symbiotic bacteria. Salinity is one of the most important abiotic stress factors affecting plant growth. γ-radiation is a very effective tool for inducing mutations in many plants. This study evaluated the γ-radiation effect on germination, cell division and plant growth of first-generation plants. Seeds of seven chickpea varieties were irradiated with γ-radiation doses ranging between 50 Gy and 600 Gy. Non-significant differences in germination percentage were recorded for seeds exposed to 50 Gy, 100 Gy, and 200 Gy of γ-radiation in comparison to the corresponding controls except ILC 484. The mitotic index (MI) of root cells increased at the low doses of 50 Gy, 100 Gy and 200 Gy comparing and reduced at the higher doses in all chickpea varieties to the control. All doses of γ-radiation induced a variable range of chromosomal abnormalities; the most common were bridges, laggard chromosomes, stickiness at metaphase, chromosome breaks, micronuclei and binucleate cells. The 300 Gy to 600 Gy doses induced degradation of nuclear membranes. The salinity treatments at 25 mM NaCl and 60 mM NaCl reduced seedling’s growth of all cultivars. The dose of 100 Gy alleviated the impact of salinity at a concentration of 25 mM NaCl for all varieties, except FLIP 84-188 and FLIP 97-263. The 60 mM NaCl treatment significantly reduced early growth of all cultivars and its effect was not alleviated by the γ-radiation.

The impact of monosomies, trisomies and segmental aneuploidies on chromosomal stability.

Aneuploidy and chromosomal instability are both commonly found in cancer. Chromosomal instability leads to karyotype heterogeneity in tumors and is associated with therapy resistance, metastasis and poor prognosis. It has been hypothesized that aneuploidy per se is sufficient to drive CIN, however due to limited models and heterogenous results, it has remained controversial which aspects of aneuploidy can drive CIN. In this study we systematically tested the impact of different types of aneuploidies on the induction of CIN. We generated a plethora of isogenic aneuploid clones harboring whole chromosome or segmental aneuploidies in human p53-deficient RPE-1 cells. We observed increased segregation errors in cells harboring trisomies that strongly correlated to the number of gained genes. Strikingly, we found that clones harboring only monosomies do not induce a CIN phenotype. Finally, we found that an initial chromosome breakage event and subsequent fusion can instigate breakage-fusion-bridge cycles. By investigating the impact of monosomies, trisomies and segmental aneuploidies on chromosomal instability we further deciphered the complex relationship between aneuploidy and CIN.

P-549 Paraclinical and spermogram evaluation of infertile males with balanced chromosomal rearrangements referred to Royan institute from 2009-2021

Abstract Study question Whether balanced chromosomal rearrangements are associated with altered semen parameters/increased DNA Fragmentation Index (DFI), are the common breakpoints related to male infertility in patients ? Summary answer Balanced chromosomal rearrangements is associated with abnormal semen parameters and increased DFI in carriers. Chromosomes 9,1,3 showed the highest incidence of chromosomal rearrangement What is known already Among many factors, balanced chromosomal rearrangements are thought to be one of the major genetic factors influencing male infertility which can cause disorders related to spermogram, DNA fragmentation, increased risk of miscarriage and ART failure. Finding common chromosomal breakpoints in infertile men carrying balanced chromosomal translocations is significant since these patients usually possess normal clinical features. Numerous studies have been performed on the various chromosome translocations. However, there has not been any report on the common breakpoints in balanced chromosomal abnormalities related to male infertility in Iran. Study design, size, duration This cross-sectional study performed on 25000 patients’ files admitted to the Royan institute during the period 2009- 2021.Infertile men carrying balanced chromosomal rearrangements were included as case group. Sperm parameters, DFI and common chromosomal break points were evaluated Participants/materials, setting, methods 25000 files of patients were reviewed. 252 infertile men carrying balanced chromosomal rearrangements and 252 men with normal karyotype were included (case and control group, respectively). Sperm concentration, motility (CASA), sperm morphology (Papanicolaou Staining),DFI (SCSA) and cytogenetic analysis of peripheral blood lymphocytes (G-banding) were evaluated. Numerical chromosomal abnormalities, deletions, marker chromosomes, alcohol or drug addiction, smoking, chemotherapy and radiation, varicocele and exposure to environmental pollution were excluded. The common chromosomal breakpoints in translocations were investigated. Main results and the role of chance Sperm concentration, total motility and normal morphology were significantly lower in balanced chromosomal rearrangements carriers compared to control group(p < 0.05). DFI in patient was significantly higher in comparison to control group(p < 0.05). Among different kind of chromosomal rearrangement carriers’, sperm concentration and total motility were lower in Robertsonian translocation carriers comparing to the Reciprocal translocation and inversion ones. The reciprocal translocation and inversion carriers did not differ regarding to sperm concentration and total motility. Meanwhile, normal morphology was lower in the translocation compared to inversion carriers. No difference was observed in DFI among carriers of balanced chromosomal rearrangements. Among case group, inversion accounted for the largest share, followed by Reciprocal, Robertsonian translocation and insertion. Group C had the highest frequency in reciprocal translocation, inversion and insertion among the chromosomal groups. The highest balanced translocation prevalence was related to three chromosomes 9,3, and1. 9q12,1p36,3p11 were the most frequent breakpoints among them. Chromosome 9 had the largest contribution to inversions,22 to reciprocal translocations and14 to Robertsonian translocations. In the Robertsonian translocation, group D showed the highest frequency. Interestingly, patients carrying normal variations such as pericentric inversion of chromosome 9showed a significant alteration in sperm parameters, therefore normal variations showed a possible relationship to infertility. Limitations, reasons for caution In order to achieve stronger statistical results on DFI, a larger sample size would be more helpful. Wider implications of the findings The results may be useful to identify the most common breakpoints and consequently genes related to male infertility and can suggest appropriate molecular mechanism for their infertility. Trial registration number not applicable

A chromosome-level genome assembly and intestinal transcriptome of Trypoxylus dichotomus (Coleoptera: Scarabaeidae) to understand its lignocellulose digestion ability.

Lignocellulose, as the key structural component of plant biomass, is a recalcitrant structure, difficult to degrade. The traditional management of plant waste, including landfill and incineration, usually causes serious environmental pollution and health problems. Interestingly, the xylophagous beetle, Trypoxylus dichotomus, can decompose lignocellulosic biomass. However, the genomics around the digestion mechanism of this beetle remain to be elucidated. Here, we assembled the genome of T. dichotomus, showing that the draft genome size of T. dichotomus is 636.27 Mb, with 95.37% scaffolds anchored onto 10 chromosomes. Phylogenetic results indicated that a divergent evolution between the ancestors of T. dichotomus and the closely related scarabaeid species Onthophagus taurus occurred in the early Cretaceous (120 million years ago). Through gene family evolution analysis, we found 67 rapidly evolving gene families, within which there were 2 digestive gene families (encoding Trypsin and Enoyl-(Acyl carrier protein) reductase) that have experienced significant expansion, indicating that they may contribute to the high degradation efficiency of lignocellulose in T. dichotomus. Additionally, events of chromosome breakage and rearrangement were observed by synteny analysis during the evolution of T. dichotomus due to chromosomes 6 and 8 of T. dichotomus being intersected with chromosomes 2 and 10 of Tribolium castaneum, respectively. Furthermore, the comparative transcriptome analyses of larval guts showed that the digestion-related genes were more commonly expressed in the midgut or mushroom residue group than the hindgut or sawdust group. This study reports the well-assembled and annotated genome of T. dichotomus, providing genomic and transcriptomic bases for further understanding the functional and evolutionary mechanisms of lignocellulose digestion in T. dichotomus.

The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF for such EJ. However, weakening XLF via disrupting interaction interfaces causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

Evaluation of the Possible Protective Effect of Fisetin against Cyclophosphamide-induced Genotoxicity in Bone Marrow and Spleen Cells of Male Rats

Fisetin is a plant flavonoid found in strawberries and other fruits and vegetables such as apples, persimmons, and onions. It has many pharmacological effects like anti-inflammatory, antioxidant, cardioprotective, neuroprotective, and anti-carcinogenicity which are attributed to its ability to reduce oxidative stress which considers the main reason for different disease conditions. Genotoxicity refers to the genetic material destruction within the cell which can be caused by different chemicals as well as radiation. The present study evaluates the effect of orally-administered fisetin daily for seven constitutive days on genotoxicity induced by cyclophosphamide in rats’ bone marrow and spleen cells. Results showed that fisetin exhibited a non-significant increase in total chromosomal aberrations, mitotic index, and micronucleus appearance in comparison with the same parameters in control group rats (p>0.05); and it produces protection when administered before cyclophosphamide by causing significant decrease (p 0.05) in the total chromosomal aberrations, chromatid breaks, ring chromosome, and chromosomal breaks in BM cells and total chromosomal aberrations and chromosomal breaks in spleen cells were shown. In conclusion, fisetin has no genotoxic effect on bone marrow and spleen cells when orally administered alone to rats, and it exhibits some protection against cyclophosphamide-induced genotoxicity.

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events

At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote’s cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.

PB1914: CYTOGENETIC FINDINGS IN 18 CHILDREN WITH MYELODYSPLASTIC SYNDROMES BY KARYOTYPIC AND FISH ANALYSIS

Background: Pediatric myelodysplastic syndromes (MDS) are rare disorders characterized by hematopoietic cell dysfunction, an increased risk of AML and a poor prognosis. Although it is known that cytogenetic abnormalities are seen in approximately half of children diagnosed with MDS, their detailed cytogenetic profile has not been elucidated yet due to the rarity of the disease. Aims: We tried to detect chromosomal abnormalities in pediatric patients with MDS, including submicroscopic aberrations such as gene alterations associated with the disease and possible malignant transformation by karyotypic and molecular cytogenetic analysis (fluorescence in situ hybridization, FISH) to contribute to the elucidation of the cytogenetic profile of pediatric MDS and the investigation of the pathogenesis of the disease. Methods: In this study 18 children with MDS are included. Karyotypic analysis was performed on 24h, 48h and 72h bone marrow (BM) cultures using GTG-banding. Karyotypic analysis was carried out on 25 at least metaphases. FISH analysis was performed on cultured BM cells using IVD probes for centromere (CEP) of chromosomes 7 and 8 and the chromosomal regions 7q22, 7q31, 8q24(MYC), 11q22.3(ATM), 11q23(MLL), and 17p13(TP53). For each sample at least 200 interphase cells (iFISH) and 5 metaphases (mFISH) were analyzed to increase sensitivity. Normal cut-off was estimated at 3%. Results: The sex ratio was 1,25 (10 M/8 F) and the mean age was 9,42 years (range 0,4y - 19y). Eleven patients had RCC, 5 MDS-EB and 2 t-MDS. Normal karyotypes were detected in 10 patients (55,6%), 6 patients with RCC, 3 with MDS-EB and 1 with t-MDS. Abnormal karyotypes were found in 8 patients (44,4%), 4 with RCC, 2 with MDS-EB and 1 with t-MDS. Monosomy 7 was observed in 2/18 patients (>70% of metaphases), +8 in 2/18 patients (5-6.6% of metaphases), inv(17)(p11.2q21) in 1/18 patients (18.75% of metaphases), complex karyotypes in 2/18 patients (31.3-73.2% of metaphases including abnormalities of 1q, 2q, 4q, 5q, 16q, 18 and Xp), in one patient after SCT in the transformation of MDS into s-AML and in the other at diagnosis with t-MDS) and chromatid and chromosome breaks in 1/18 patients, (8,6-9,6% of metaphases) in 2 different BM samples taken with an interval of 3 months, which were also found in his brother’s BM karyotype. FISH revealed -7 in 2/18 patients confirming their karyotypes, loss of one p53 allele in 3/18 patients at low frequencies (6,5%, 4,5%, 3,5%) all with hypocellular RCC and transfusion-dependency that underwent SCT, +8 in 2/18 (in 8% and 2,7% of cells) confirming trisomy 8 in their karyotypes and loss of one MLL allele and MLL rearrangement in a patient that was transformed to s-AML in 4,4% of cells. A normal pattern of hybridization was observed for the above probes in the rest cases and for MYC and ATM genes in all the examined cases. Summary/Conclusion: Karyotypic aberrations were detected in 44,4% of children including -7, +8, inv(17), complex karyotypes and chromatid and chromosome breaks, revealing that other chromosome aberrations except -7/del(7q) and trisomy 8 are characterized this disease. Clonal chromosome abnormalities can be detected at low frequencies in the karyotypes of pediatric MDS patients so except from iFISH, mFISH is also needed especially in cases with small clones or submicroscopic translocations. The low frequency of submicroscopic aberrations such as p53 deletions observed in 3 cases of hypocellular RCC must be confirmed in larger studies by molecular analysis as they may play a potential role in MDS pathogenic evolution.

P814: CLINICAL CHARACTERISTICS AND GENE MUTATION ANALYSIS OF 148 CHILDREN WITH FANCONI ANEMIA IN CHINA

Background: Fanconi anemia (FA) is a rare autosomal recessive, X-linked (FANCB) or autosomal dominant (FANCR/RAD51) disease of bone marrow failure. Pathogenesis is mainly caused by gene mutation related to the FA pathway. At present, at least 23 genes have been discovered to play a role in the FA pathway. The gene mutations of FA have been reported in more and more countries. In China, there no large-scale cases have been reported. Aims: In this study, we analyzed the clinical characteristics of 148 FA children from 20 provinces in China from October 2003 to October 2021. We explored the relationship between genotype and phenotype in 105 children with FA genotyping results. This is the largest series of subtyped Chinese Fanconi anemia patients to date, and the results will be helpful for future clinical management. Methods: A total of 148 Fanconi anemia patients from 140 families were diagnosed by clinical phenotype, family history, chromosome breakage test induced by mitomycin C, Single cell gel electrophoresis experiment, targeted - sequence, whole exon sequencing, and MLPA method from October 2003 to October 2021. Since there were no genetic test results for the children before 2010, only the 105 patients after 2010 had gene sequencing results, which allowed genotyping. DNA damage repair defects were detected by MMC-induced Single cell gel electrophoresis and chromosome breakage experiments. Targeted-seq was used to identify the FA mutations of the patients. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large fragment deletions in patients with negative results in the targeted-seq but conforming to the FA by MMC-induced chromosome breakage experiments test and clinical manifestations. We performed WES of DNA samples from five patients and their parents. Results: 1) The most common subtype of FA in China is FA-A, followed by FA-D2 and FA-P。2) The most common deformities in FA-A patients are finger deformities and skin pigmentation, while heart deformities are more common in FA-D2/B/G/I/P subtypes. 3) The common mutations of FANCA are exon23c.2101A>G, exon28c.2778 + 1G>A, exon34c.3348 + 1G>A, exon21c.1844delC, and exon30 C2941T>G, which are different from Korea and Japan. 4) Homozygous nonsense mutation of exon32 c.3188G>A and exon29 c.2851C>T in FA-A likely benign, but the homozygous splicing mutation in exon29c.2852 + 1G>T is harmful. 4) Children with splicing and Del mutations of FANCD1 gene had poor clinical prognosis, while children with homozygous mutations of exon10c. 1792A>G is likely benign. 5) Cases of initial diagnosis of MDS or poor disease progression tend to occur in FA-B/C/E/G/J/L/M and FA-S subtypes. 6)Chromosome 1, 3, 7, and 8 abnormalities and mutations of SF3B1 P53 are related to disease progression. Summary/Conclusion: We made a comprehensive description and analysis of the clinical characteristics and mutant genes of Children with FA in China. We proposed the mutation sites related to clinical prognosis and the cytogenetic abnormalities of disease progression, providing new data for our comprehensive understanding of FA. Our data will be useful for FA future menagement.

P614: SUBCLONAL ARCHITECTURE OF CHROMOSOMES REVEALED BY SINGLE-CELL ANALYSIS OF GENE EXPRESSION IN A PATIENT WITH CLONAL EVOLUTION OF RELAPSING/REFRACTORY CLL

Background: Chronic lymphocytic leukemia (CLL) manifests by remarkable intraclonal heterogeneity of genomic defects, especially in patients with relapsed/refractory disease, often connected with complex karyotype. In cancer cells, chromosomal changes are often multiple and complex. Aberrations can be present only in subclones and often underlie disease refractoriness. Using the bulk analysis such as WGS, WES, or genomic array to precisely determine the co-occurrence of aberrations in the individual cell provides limited information. Aims: To perform an in-depth, single-cell RNA sequencing (scRNAseq) study of subclonal changes associated with relapsed/refractory CLL. Methods: A bulk analysis of DNA from separated malignant cells using comprehensive NGS panel LYNX (PMID 34082072) was performed on a representative CLL case at three time-points (TPs): at dg (TP1, month 0, Rai II), before first therapy (TP2, month 19) and during relapse (TP3, month 59). Simultaneously, the transcriptome was analyzed using scRNAseq (Chromium system, 10x Genomics) in 2330 cells (TP1), 2397 cells (TP2), and 1902 cells (TP3). A detailed data analysis was carried out with the Seurat R package. In addition, we used the InferCNV tool to detect the chromosomal aberrations in every tested cell. We identified multiple chromosomal changes and compared the findings with the results of bulk DNA analysis using a genomic array (ThermoFisher Scientific) and FISH. Results: We detected a number of aberrations that were already present at TP1, including del13q, del11q, mutations in NOTCH1, RPS15, ZMYM3, and minor mutations in TP53 and ATM. At TP2, additional changes comprised mutations in PIM1, XPO1, and del(3q). Between TP2 and TP3, the patient underwent several treatment lines (FCR, BR, COP) without an apparent therapeutic effect. The cells from TP3 carried complex changes, including additional mutations in BIRC3 and RB1. Unsupervised clustering of scRNAseq gene expression data defined major clones bearing aberrations (losses on chromosomes 1, 9, 13, 17, 18). Moreover, these clones were composed of cells with additional aberrations defining a number of separate subclones. The proportion of subclones was typically under the detection limit of the genomic array and showed extreme intraclonal heterogeneity of the relapsed/refractory CLL case. Summary/Conclusion: We showcase how genomic alterations influence the expression of affected genes. Such fact can be exploited to reconstruct chromosomal disruptions in individual cells using scRNAseq and to uncover the subclonal architecture of the disease. Grants: MH-CZ_AZV_NU20-08-00314, MH-CZ_AZV_NV19-03-00091, MEYS-CZ_MUNI/A/1330/2021, MH-CZ_RVO_65269705.

Reactive oxygen species accumulation is synchronised with growth inhibition of temperature-sensitive recAts polA Escherichia coli

When combined with recombinase defects, chromosome breakage and double-strand break repair deficiencies render cells inviable. However, cells are viable when an SOS response occurs in recAts polA cells in Escherichia coli. Here, we aimed to elucidate the underlying mechanisms of this process. Transposon mutagenesis revealed that the hslO gene, a redox chaperone Hsp33 involved in reactive oxidative species (ROS) metabolism, was required for the suppression of recAts polA lethality at a restricted temperature. Recently, it has been reported that lethal treatments trigger ROS accumulation. We also found that recAts polA cells accumulated ROS at the restricted temperature. A catalase addition to the medium alleviates the temperature sensitivity of recAts polA cells and decreases ROS accumulation. These results suggest that the SOS response and hslO manage oxidative insult to an acceptable level in cells with oxidative damage and rescue cell growth. Overall, ROS might regulate several cellular processes.

Abstract 6196: Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma

Abstract Fanconi anemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink (ICL) repair resulting in chromosome breakage. The FA repair pathway comprises at least 22 FANC proteins including BRCA1 and BRCA2 and protects against carcinogenic endogenous and exogenous aldehydes. Individuals with FA are hundreds to thousands-fold more likely to develop head and neck (HNSCC), esophageal and anogenital squamous cell carcinomas (SCCs) with a median onset age of 31 years. The aggressive nature of these tumors and poor patient tolerance of platinum and radiation-based therapy have been associated with short survival in FA. Molecular studies of SCCs from individuals with FA (FA SCCs) have been limited, and it is unclear how they relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or human papillomavirus (HPV) infection. Here, by sequencing FA SCCs, we demonstrate that the primary genomic signature of FA-deficiency is the presence of a high number of structural variants (SVs). SVs are enriched for small deletions, unbalanced translocations, and fold-back inversions that arise in the context of TP53 loss. The SV breakpoints preferentially localize to early replicating regions, common fragile sites, tandem repeats, and SINE elements. SVs are often connected forming complex rearrangements. Resultant genomic instability underlies elevated copy number alteration (CNA) rates of key HNSCC-associated genes, including PIK3CA, MYC, CSMD1, PTPRD, YAP1, MXD4, and EGFR. In contrast to sporadic HNSCC, we find no evidence of HPV infection in FA HNSCC, although positive cases were identified in gynecologic tumors. A murine allograft model of FA pathway-deficient SCC was enriched in SVs, exhibited dramatic tumor growth advantage, more rapid epithelial-to-mesenchymal transition, and enhanced autonomous inflammatory signaling when compared to an FA pathway-proficient model. In light of the protective role of the FA pathway against SV formation uncovered here, and recent findings of FA pathway insufficiency in the setting of increased formaldehyde load resulting in hematopoietic stem cell failure and carcinogenesis, we propose that high copy-number instability in sporadic HNSCC may result from functional overload of the FA pathway by endogenous and exogenous DNA crosslinking agents. Our work lays the foundation for improved FA patient treatment and demonstrates that FA SCC is a powerful model to study tumorigenesis resulting from DNA crosslinking damage. Citation Format: Andrew L. Webster, Mathijs A. Sanders, Krupa Patel, Ralf Dietrich, Raymond J. Noonan, Francis P. Lach, Ryan R. White, Audrey M. Goldfarb, Kevin Hadi, Matthew M. Edwards, Frank X. Donovan, Moonjung Jung, Sunandini Sridhar, Olivier Fedrigo, Huasong Tian, Joel Rosiene, Thomas Heineman, Jennifer Kennedy, Lorenzo Bean, Rasim O. Rosti, Rebecca Tryon, Ashlyn-Maree Gonzalez, Allana Rosenberg, Ji-Dung Luo, Thomas Carrol, Eunike Velleuer, Jeff C. Rastatter, Susanne I. Wells, Jordi Surrallés, Grover Bagby, Margaret L. MacMillan, John E. Wagner, Maria Cancio, Farid Boulad, Theresa Scognamiglio, Roger Vaughan, Amnon Koren, Marcin Imielinski, Settara Chandrasekharappa, Arleen D. Auerbach, Bhuvanesh Singh, David Kutler, Peter J. Campbell, Agata Smogorzewska. Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6196.

Abstract 2172: Tumor break load is a biologically relevant characteristic of genomic instability with prognostic value for patients with localized colorectal cancer

Abstract Background: Genomic instability is a common feature of colorectal cancer (CRC). More than 80% of CRCs exhibit chromosomal instability (CIN) and 15-20% microsatellite instability (MSI). MSI results in a high ‘tumor mutational burden’ (TMB) and is associated with relatively good prognosis in localized CRC. However, for the large group of CIN CRC patients with localized disease it remains unclear who has a relatively good prognosis and who is at high risk of disease recurrence. CIN is characterized by excessive somatic copy number aberrations (SCNAs), which is often summarized as the ‘fraction genome altered’ (FGA). In addition, CIN is also characterized by excessive chromosomal rearrangement structural variants (SVs). We quantitated the chromosomal breaks as the ‘Tumor Break Load’ (TBL) to investigate the biological impact and clinical relevance of chromosomal breakage in colorectal cancer. Aim: To investigate the biological impact and clinical relevance of TBL in CRC. Data and Methods: We used SCNA, RNA-Seq, and Disease-Free Survival (DFS) data from 633 CRC samples from The Cancer Genome Atlas project. For validation purposes, we used SCNA and DFS data from an independent series of 72 CRC samples. TBL was calculated as the sum of SCNA-associated chromosomal breaks. We used the 25% and 75% quantiles of the TBL distribution as predefined high and low TBL groups to train an RNA expression-based classifier that identifies TBL-associated expression phenotypes as an indicator of biological impact. Results: Within the group of microsatellite stable (MSS) CRC samples there was a large variety in TBL (median TBL of 54 [1-308]). TBL was poorly correlated with TMB (R2: 0.06) and FGA (R2: 0.07). Using RNA expression data as a phenotypic representation of genomic instability, MSI and MSS CRC samples could be classified with high accuracy (AUC: 0.99, p < 0.01), confirming the well-known impact of MSI on tumor biology. Using the same methodology within the group of MSS samples, the TBL could also be classified with high accuracy (AUC: 0.88; p < 0.01). Moreover, high TBL was associated with poor DFS in stage II-III localized MSS CRC (HR: 6.1; p = 0.007). This result was validated in an independent data series of 72 untreated stage II-III localized MSS CRC patients (HR: 3.0; p < 0.02). Conclusion: TBL is a characteristic of chromosomal instability that is largely independent from TMB and FGA, with significant impact on tumor biology. Our data indicate that the TBL may be used as a valuable prognostic biomarker for DFS in patients with localized colorectal cancer. Citation Format: Soufyan Lakbir, Jaap Heringa, Gerrit A. Meijer, Sanne Abeln, Remond J. Fijneman. Tumor break load is a biologically relevant characteristic of genomic instability with prognostic value for patients with localized colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2172.

The relevance of chromatin architecture to genome rearrangements in Drosophila.

DNA within chromosomes in the nucleus is non-randomly organized into chromosome territories, compartments and topologically associated domains (TADs). Chromosomal rearrangements have the potential to alter chromatin organization and modify gene expression leading to selection against these structural variants. Drosophila pseudoobscura has a wealth of naturally occurring gene arrangements that were generated by overlapping inversion mutations caused by two chromosomal breaks that rejoin the central region in reverse order. Unlike humans, Drosophila inversion heterozygotes do not have negative effects associated with crossing over during meiosis because males use achiasmate mechanisms for proper segregation, and aberrant recombinant meiotic products generated in females are lost in polar bodies. As a result, Drosophila populations are found to harbour extensive inversion polymorphisms. It is not clear, however, whether chromatin architecture constrains which inversions breakpoints persist in populations. We mapped the breakpoints of seven inversions in D. pseudoobscura to the TAD map to determine if persisting inversion breakpoints are more likely to occur at boundaries between TADs. Our results show that breakpoints occur at TAD boundaries more than expected by chance. Some breakpoints may alter gene expression within TADs supporting the hypothesis that position effects contribute to inversion establishment. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Epigenetics and Congenital Heart Diseases.

Congenital heart disease (CHD) is a frequent occurrence, with a prevalence rate of almost 1% in the general population. However, the pathophysiology of the anomalous heart development is still unclear in most patients screened. A definitive genetic origin, be it single-point mutation or larger chromosomal disruptions, only explains about 35% of identified cases. The precisely choreographed embryology of the heart relies on timed activation of developmental molecular cascades, spatially and temporally regulated through epigenetic regulation: chromatin conformation, DNA priming through methylation patterns, and spatial accessibility to transcription factors. This multi-level regulatory network is eminently susceptible to outside disruption, resulting in faulty cardiac development. Similarly, the heart is unique in its dynamic development: growth is intrinsically related to mechanical stimulation, and disruption of the intrauterine environment will have a direct impact on fetal embryology. These two converging axes offer new areas of research to characterize the cardiac epigenetic regulation and identify points of fragility in order to counteract its teratogenic consequences.

Effect of Triclosan and Silver Nanoparticles on DNA Damage Investigated with DNA-Based Biosensor.

Triclosan (TCS) is a broad-spectrum antimicrobial agent widely used in personal care, healthcare, and clinical practice. One of the most important aspects of toxicological profiling of compounds is their interaction with DNA. In human cells, TCS causes a significant reduction in DNA methylation. The involvement of TCS in chromosomal aberrations, DNA damage, and strand breaks, as well as DNA damage from TCS degradation products, was reported. AgNPs share similarities with TCS in terms of antimicrobial properties, enter the body after exposure, and are used even together with TCS in oral care products. Therefore, their mutual effect on the DNA is of interest. In this study, the electrochemical behavior of TCS on a glassy carbon electrode (GCE) and the biosensor with salmon sperm dsDNA (DNA/GCE), DNA damage by TCS present in phosphate buffer solution pH 7.4 and an additional effect of the immobilized AgNP layer on such DNA damage have been investigated. Two different sizes of AgNPs (about 15 and 37 nm) were tested. Using square-wave voltammetric signals of nucleobases, the portion of survived DNA was 64% in the presence of 15 nm AgNPs compared to 55% in its absence. The protective effect of AgNPs on DNA against TCS-induced DNA damage was found.

A glimpse into the past: phylogenesis and protein domain analysis of the group XIV of C-type lectins in vertebrates

BackgroundThe group XIV of C-type lectin domain-containing proteins (CTLDcps) is one of the seventeen groups of CTLDcps discovered in mammals and composed by four members: CD93, Clec14A, CD248 and Thrombomodulin, which have shown to be important players in cancer and vascular biology. Although these proteins belong to the same family, their phylogenetic relationship has never been dissected. To resolve their evolution and characterize their protein domain composition we investigated CTLDcp genes in gnathostomes and cyclostomes and, by means of phylogenetic approaches as well as synteny analyses, we inferred an evolutionary scheme that attempts to unravel their evolution in modern vertebrates.ResultsHere, we evidenced the paralogy of the group XIV of CTLDcps in gnathostomes and discovered that a gene loss of CD248 and Clec14A occurred in different vertebrate groups, with CD248 being lost due to chromosome disruption in birds, while Clec14A loss in monotremes and marsupials did not involve chromosome rearrangements. Moreover, employing genome annotations of different lampreys as well as one hagfish species, we investigated the origin and evolution of modern group XIV of CTLDcps. Furthermore, we carefully retrieved and annotated gnathostome CTLDcp domains, pointed out important differences in domain composition between gnathostome classes, and assessed codon substitution rate of each domain by analyzing nonsynonymous (Ka) over synonymous (Ks) substitutions using one representative species per gnathostome order.ConclusionsCTLDcps appeared with the advent of early vertebrates after a whole genome duplication followed by a sporadic tandem duplication. These duplication events gave rise to three CTLDcps in the ancestral vertebrate that underwent further duplications caused by the independent polyploidizations that characterized the evolution of cyclostomes and gnathostomes. Importantly, our analyses of CTLDcps in gnathostomes revealed critical inter-class differences in both extracellular and intracellular domains, which might help the interpretation of experimental results and the understanding of differences between animal models.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

Homologous recombination deficiency predicts response to platinum-based neoadjuvant chemotherapy in early-stage triple-negative breast cancer patients: Secondary analysis of the neocart randomized clinical trial.

e12606 Background: We recently performed the NeoCART trial to compare six cycles of docetaxel plus carboplatin (DCb) with four cycles of epirubicin/cyclophosphamide followed by four cycles of docetaxel (EC-D) in the neoadjuvant setting of triple-negative breast cancer (TNBC). The present study assessed the homologous recombination deficiency (HRD) score, stromal tumor-infiltrating lymphocytes (TILs), and Ki67 as predictors of pathological complete response (pCR) in the NeoCART trial cohort. Methods: Participants in the NeoCART trial (NCT03154749) were randomly assigned, at a 1:1 ratio, to an experimental DCb for 6 cycles (DCb group) or an EC for 4 cycles followed by docetaxel for 4 cycles group (EC-D group). Pretherapeutic formalin-fixed, paraffin-embedded tumor tissues were assessed retrospectively for DNA extraction and sequencing. Sequencing was performed using the Illumina HiSeq4000 platform. The HRD score was calculated based on the genome-wide allele-specific copy number result, and it was composed of three parts: 1) loss of heterozygosity (LOH): the number of segments >=15 Mb in length (but not covering the entire chromosome), mCN =0 and tCN > 0; 2) telomeric allelic imbalance (TAI): the number of segments with allelic imbalances (mCN!= tCN - mCN) extending to the telomeric end of a chromosome; 3) large-scale state transitions (LST), the number of chromosomal breaks between adjacent segments of at least 10 Mb, with a distance between them not larger than 3 Mb. Results: The primary and secondary endpoints, including pCR, event-free survival, overall survival, breast-conserving surgery rates, and treatment-related toxicities, were published previously. HRD testing was attempted on baseline FFPE tumor samples for 54 patients in the NeoCART cohort and was successfully completed for 43 patients (79.6%). It was unavailable for 11 patients due to inadequate tissue or the quality filter. Patients with HRD measurable tumors received DCb in 22 cases and EC-D in 21 cases. TP53 (93.0%), PIK3CA (27.9%), PTEN (11.6%), ATRX (9.3%), NF1 (9.3%), PIK3R1 (9.3%) and PKHD1 (9.3%) were the most frequently mutated genes. No significant association between BRCA1/2 mutation status and pCR was observed in the general population or the two treatment arms. Patients in the DCb group who achieved pCR had a higher HRD score (p =0.04), higher stromal TILs (p =0.02), and a higher Ki67 index (p =0.04) than non-pCR patients. RCB 0/1 was associated with higher stromal TILs (P =0.05) and a higher Ki67 index (P =0.008). However, no correlation between HRD score and pCR was observed in the EC-D group. Conclusions: PCR in TNBC patients correlated with a higher HRD score, higher stromal TILs, and a higher Ki67 index on carboplatin-based neoadjuvant chemotherapy regardless of BRCA status. Clinical trial information: NCT03154749.

Whole genome sequencing of HER2+ metastatic breast cancer and CNA comparison between long term survivor and short-term survivor.

e13019 Background: The introduction of anti-HER2 therapies such as trastuzumab for HER2+ metastatic breast cancer (MBC) has led to significant improvements to disease progression. We and others have reported long-term durable complete response to trastuzumab in patients with HER2+ MBC. We hypothesise that genomic copy number alteration (CNA) and tumour mutational burden (TMB) may act as a prognostic measure of predicting response to trastuzumab in long-term survivors (LTS) compared to short-term survivors (STS). Using whole-genome sequencing (WGS) to detect these alterations, we aim to identify the molecular characteristics of primary and metastatic lesions in patients with metastatic HER2+ breast cancer. Methods: WGS was performed on samples from two patients from a HER2+ MBC cohort; a of long-term survivor (PFS 27 months; OS > 60 months) and a short-term survivor (PFS 4 months; OS < 14 months). Primary tumours, adjacent normal tissue and metastases were sequenced at a mean depth of 60X. Reads were aligned on the hg38 reference genome using Burrows-Wheeler Aligner (bwa), CNA were detected using Control-FREEC to evaluate total CNA burden. Single nucleotide variants were detected using GATK Mutect2 to calculate TMB. Results: Analysis of DNA chromosome disruption (fraction of the genome amplified/deleted) revealed a higher overall fraction of disrupted genome in the LTS primary tumour and metastasis compared with the STS (0.061 and 0.064 vs 0.031 and 0.047, respectively). Further delineation of the distribution of CNA burden in all genomes identified chr1 as the most altered in the STS samples whereas chr17 was the most disrupted in the LTS samples. HER2 and CDK12 were amplified in both LTS and STS samples (4 copies in LTS primary tumour and metastasis, 18 and 14 copies in STS primary tumour and metastasis respectively). A high TMB was estimated in both samples but was higher in the STS primary tumour than in the LTS one (161 vs 142, respectively). TMB was increased in LTS metastatic lesions. NRAS mutation was found in LTS primary tumour but was lost in the metastasis. Conclusions: This pilot study highlights the potential for whole-genome sequencing to identify CNA and TMB in HER2+ MBC in patients who display long-term and short-term survival. Further samples from this cohort need to be studied to investigate the relationship between CNA burden and HER2+ MBC patient survival.