Frequency Of Rearrangements Research Articles

Multiple genome analysis of Candida glabrata clinical isolates renders new insights into genetic diversity and drug resistance determinants.

The emergence of drug resistance significantly hampers the treatment of human infections, including those caused by fungal pathogens such as Candida species. Candida glabrata ranks as the second most common cause of candidiasis worldwide, supported by rapid acquisition of resistance to azole and echinocandin antifungals frequently prompted by single nucleotide polymorphisms (SNPs) in resistance associated genes, such as PDR1 (azole resistance) or FKS1/2 (echinocandin resistance). To determine the frequency of polymorphisms and genome rearrangements as the possible genetic basis of C. glabrata drug resistance, we assessed genomic variation across 94 globally distributed isolates with distinct resistance phenotypes, whose sequence is deposited in GenBank. The genomes of three additional clinical isolates were sequenced, in this study, including two azole resistant strains that did not display Gain-Of-Function (GOF) mutations in the transcription factor encoding gene PDR1. Genomic variations in susceptible isolates were used to screen out variants arising from genome diversity and to identify variants exclusive to resistant isolates. More than half of the azole or echinocandin resistant isolates do not possess exclusive polymorphisms in PDR1 or FKS1/2, respectively, providing evidence of alternative genetic basis of antifungal resistance. We also identified copy number variations consistently affecting a subset of chromosomes. Overall, our analysis of the genomic and phenotypic variation across isolates allowed to pinpoint, in a genome-wide scale, genetic changes enriched specifically in antifungal resistant strains, which provides a first step to identify additional determinants of antifungal resistance. Specifically, regarding the newly sequenced strains, a set of mutations/genes are proposed to underlie the observed unconventional azole resistance phenotype.

Novel gene rearrangement pattern in mitochondrial genome of Ooencyrtusplautus Huang & Noyes, 1994: new gene order in Encyrtidae (Hymenoptera, Chalcidoidea).

Studies of mitochondrial genomes have a wide range of applications in phylogeny, population genetics, and evolutionary biology. In this study, we sequenced and analyzed the mitochondrial genome of Ooencyrtusplautus Huang & Noyes, 1994 (Hymenoptera, Encyrtidae). The nearly complete mitogenome of O.plautus was 15,730 bp in size, including 13 PCGs (protein-coding genes), 22 tRNAs, 2 rRNAs, and a nearly complete control region. The nucleotide composition was significantly biased toward adenine and thymine, with an A + T content of 84.6%. We used the reference sequence of Chouioiacunea and calculated the Ka/Ks ratio for each set of PCGs. The highest value of the Ka/Ks ratio within 13 PCGs was found in nad2 with 1.1, suggesting that they were subjected to positive selection. This phenomenon was first discovered in Encyrtidae. Compared with other encyrtid mitogenomes, a translocation of trnW was found in O.plautus, which was the first of its kind to be reported in Encyrtidae. Comparing with ancestral arrangement pattern, wasps reflect extensive gene rearrangements. Although these insects have a high frequency of gene rearrangement, species from the same family and genus tend to have similar gene sequences. As the number of sequenced mitochondrial genomes in Chalcidoidea increases, we summarize some of the rules of gene rearrangement in Chalcidoidea, that is four gene clusters with frequent gene rearrangements. Ten mitogenomes were included to reconstruct the phylogenetic trees of Encyrtidae based on both 13 PCGs (nucleotides of protein coding genes) and AA matrix (amino acids of protein coding genes) using the maximum likelihood and Bayesian inference methods. The phylogenetic tree reconstructed by Bayesian inference based on AA data set showed that Aenasiusarizonensis and Metaphycuseriococci formed a clade representing Tetracneminae. The remaining six species formed a monophyletic clade representing Encyrtinae. In Encyrtinae, Encyrtus forms a monophyletic clade as a sister group to the clade formed by O.plautus and Diaphorencyrtusaligarhensis. Encyrtussasakii and Encyrtusrhodooccisiae were most closely related species in this monophyletic clade. In addition, gene rearrangements can provide a valuable information for molecular phylogenetic reconstruction. These results enhance our understanding of phylogenetic relationships among Encyrtidae.

Genetic and phenotypic characterisation of HIV-associated aggressive B-cell non-Hodgkin lymphomas, which do not occur specifically in this population: diagnostic and prognostic implications.

The frequency of aggressive subtypes of B-cell non-Hodgkin lymphoma (B-NHL), such as high-grade B-cell lymphomas (HGBL) with MYC and BCL2 and/or BCL6 rearrangement (HGBL-DH/TH) or Burkitt-like lymphoma (BL) with 11q aberration, is not well known in the HIV setting. We aimed to characterise HIV-associated aggressive B-NHL according to the 2017 WHO criteria, and to identify genotypic and phenotypic features with prognostic impact. Seventy-five HIV-associated aggressive B-NHL were studied by immunohistochemistry (CD10, BCL2, BCL6, MUM1, MYC, and CD30), EBV-encoded RNAs (EBERs), and fluorescence in situ hybridisation (FISH) to evaluate the status of the MYC, BCL2, and BCL6 genes and chromosome 11q. The 2017 WHO classification criteria and the Hans algorithm, for the cell-of-origin classification of diffuse large B-cell lymphomas (DLBCL), were applied. In DLBCL cases, the frequencies of MYC and BCL6 rearrangements (14.9 and 27.7%, respectively) were similar to those described in HIV-negative patients, but BCL2 rearrangements were infrequent (4.3%). MYC expression was identified in 23.4% of DLBCL cases, and coexpression of MYC and BCL2 in 13.0%, which was associated with a worse prognosis. As for BL cases, the expression of MUM1 (30.4%) conferred a worse prognosis. Finally, the prevalence of HGBL-DH/TH and BL-like with 11q aberration are reported in the HIV setting. The phenotypic and genotypic characteristics of HIV-associated aggressive B-NHL are similar to those of the general population, except for the low frequency of BCL2 rearrangements in DLBCL. MYC and BCL2 coexpression in DLBCL, and MUM-1 expression in BL, have a negative prognostic impact on HIV-infected individuals.

Genomic landscape of lung cancer in the young.

BackgroundLung cancer in the young is a rare entity of great interest due to the high frequency of targetable mutations. In this study, we explored the genomic landscape of non-small cell lung cancer (NSCLC) in young patients and compared it with genetic alterations in older patients.MethodsComparative study of the genomic profile of NSCLC young (≤40 years old) vs older patients (>40 years old) from Instituto Nacional de Enfermedades Neoplásicas (INEN) in Lima, Peru. Archival paraffin-embedded tumor samples were profiled with FoundationOne CDx assay to identify short variants alterations (insertions and deletions), copy number variations (CNV), tumor mutational burden and microsatellite instability in 324 driver genes and rearrangements in 28 commonly rearranged genes. A targetable alteration was defined as any alteration in a driver oncogene for which an FDA approved therapy existed at the time of study enrollment.ResultsOverall, 62 tumors were profiled, 32 from young and 30 from older patients. All clinicopathological features (smoking status, clinical stage, and histology) were similar between groups, except for gender (65.6% of females in the younger group vs 40% in the older group, P=0.043). At least one actionable mutation was present in 84.4% and 83.3% in younger and older patients, respectively. Alteration rates in the main genes were: BRAF, 3.1%(n=1) vs 0%; EGFR, 46.9% (n=15) vs 43.3% (n=13); ERBB2, 12.5% (n=4) vs 16.7% (n=5); KRAS, 15.6% (n=5) vs 16.7% (n=5); ALK, 6.3% (n=2) vs 3.3% (n=1); RET, 0.0% vs 3.3% (n=1); ROS1, 3.1% (n=1) vs 3.3% (n=1); NTRK1, 0.0% vs 3.3% (n=1) and MET, 3.1% (n=1) vs 13.3% (n=4). Mean TMB was 4.04 Mut/Mb (SD ± 3.98) for young vs 8.06 Mut/Mb (SD ± 9.84) for older patients (P=0.016). There were not significant differences in CNV, frequency of gene rearrangements, or microsatellites instability.ConclusionNSCLC in the young in our cohort was characterized by a high frequency of actionable genetic aberrations and a low TMB, which was also true for our older patients. The enrichment of actionable mutations in young patients described in other reports might be attributed to differences in the etiology and clinicopathological characteristics between younger and older patients and therefore not be applicable to all populations.

BCR-ABL1 Gene Mutation in Acute Lymphoblastic Leukemia

Objective:Â Determination of frequency of BCR-ABL1 gene mutation in Acute Lymphoblastic Leukemia. Methodology: This cross sectional study was carried out at the Department of Molecular Haematology, Armed Forces Institute of Pathology between January 2018 to March 2021. All newly diagnosed patients of Acute Lymphoblastic Leukemia were included in the study while patients already on treatment and with secondary leukemia were excluded from the study. Blood counts were done and various parameters such as total leukocyte count, hemoglobin, and platelet count were calculated. Results: 623 patients were included in the study. BCR-ABL gene was positive in 105 (16.8%) patients. A total of 96 (92%) were positive for BCR ABL p190 rearrangement and 9 (8%) were positive for BCR ABL p210 rearrangement. Â Mean age of patients was 21 years. The frequency of BCR ABL gene was positive in 72% males and 28% females. BCR-ABL positive ALL was associated with higher TLC count. CNS involvement was more common in Acute Lymphoblastic Leukemia with BCR-ABL. The likelihood of post-induction remission decreased with age. Conclusion: In our study population, a frequency of 16.8% patients were BCR ABL1 positive in acute lymphoblastic leukemia. BCR- ABL gene mutation in ALL is more prevalent in young adults. The frequency of p190 rearrangement is more common than p210 BCR-ABL rearrangement. BCR-ABL positive ALL is more prevalent in males than in females.

121P Spectrum of druggable gene fusions in microsatellite-unstable colorectal tumors

Colorectal carcinomas (CRCs) with high-level microsatellite instability (MSI-H) often contain gene fusions, which involve receptor tyrosine kinases. This study included 105 MSI-H CRCs. NTRK1/2/3, ALK, ROS1 and RET gene fusions were analyzed by PCR test for 5’/3’-end unbalanced expression, which is capable of detecting all translocation variants. The type of rearrangements was subsequently determined by variant-specific PCR for common fusions, and, wherever necessary, by targeted RNA next-generation sequencing (NGS). NTRK1/2/3 translocations were the most common, being detected in 8/105 (8%) CRCs (TPM3-NTRK1 (T8;N10): n = 2; ETV6-NTRK2 (E5;N15): n = 2; ETV6-NTRK3 (E5;N15): n = 4). There were 2 tumors with RET rearrangements (CCDC6-RET (C1;R12) and NCOA4-RET (N9del501;R12), respectively) and 1 instance of SPTBN1-ALK (S7;A20) chimera. 76/105 (72%) CRCs were negative for KRAS, NRAS or BRAF mutations; gene rearrangements were detected in 10 (13%) of these tumors. 29/105 (28%) carcinomas carried activating lesions in RAS/RAF oncogenes; one of these CRCs had both ETV6-NTRK3 (E5;N15) translocation and BRAF V600E substitution. Microsatellite-unstable CRCs have high frequency of druggable gene rearrangements, with NTRK1/2/3 deserving particular attention.

Host translesion polymerases are required for viral genome integrity

Human cells encode up to 15 DNA polymerases with specialized functions in chromosomal DNA synthesis and damage repair. In contrast, complex DNA viruses, such as those of the herpesviridae family, encode a single B-family DNA polymerase. This disparity raises the possibility that DNA viruses may rely on host polymerases for synthesis through complex DNA geometries. We tested the importance of error-prone Y-family polymerases involved in translesion synthesis (TLS) to human cytomegalovirus (HCMV) infection. We find most Y-family polymerases involved in the nucleotide insertion and bypass of lesions restrict HCMV genome synthesis and replication. In contrast, other TLS polymerases, such as the polymerase ζ complex, which extends past lesions, was required for optimal genome synthesis and replication. Depletion of either the polζ complex or the suite of insertion polymerases demonstrate that TLS polymerases suppress the frequency of viral genome rearrangements, particularly at GC-rich sites and repeat sequences. Moreover, while distinct from HCMV, replication of the related herpes simplex virus type 1 is impacted by host TLS polymerases, suggesting a broader requirement for host polymerases for DNA virus replication. These findings reveal an unexpected role for host DNA polymerases in ensuring viral genome stability.

Occurrence of cytogenetic effects under the action of epimutagen in winter wheat

Research on studying the features of 0.01%, 0.05%, 0.10% and 0.50% epimutagen Triton-X-305 impact at the cellular level, which means the way of identifying the viability of pollen in plants of the first generation, and cytological analysis of chromosomal aberrations in the cells of the primary meristem of germinal roots during germination of bread winter wheat seeds have been conducted. Research in this direction allows us to significantly improve the monitoring of same type of substances in the environment and to forecast the nature of their action at the DNA-level. Genotypes Podolyanka and Spivanka created by Ukrainian selection and varieties of French selection Altigo, Courtot, Lyrik, Flamenko have been studied. These genotypes were selected in order to characterize possible genotype-mutagenic interactions for a compound of complex hereditary pattern for a range of concentrations with maximum contrast taking into account the possible high site-specific effect. The main purpose of this research was to identify the specificity of impact of the agent Triton X-305 at the cellular level and identify parameters fully showing the effect of this substance on the subsequent induction of biodiversity and the enhancement of hereditary variability at the cellular level. Such indicators as pollen sterility effected by various concentrations, the total frequency of chromosomal rearrangements, the spectrum of chromosomal rearrangements, including fragments and double fragments, single and double bridges, micronuclei and lagging chromosomes have been investigated. The ratio of fragments to bridges as an indicator of the active factor nature, the number of cells with two or more rearrangements as an indicator of genetic toxicity of this substance has been established. As a result, a significantly weaker effect of the epimutagen on the overall frequency of chromosomal rearrangement has been shown unlike other factors of a mutagenic nature, at the same time changing the ratio of the obtained aberrations in favour of micronuclei and lagging chromosomes. In addition, other parameters are determined as more meaningful for identifying a specific agent of impact, and the differences in concentrations are less contrasting. There is also a less significantly decreasing fertility, however, this parameter is highly dependent on the source material. In the future, we intend to assess the variability, primarily of a hereditary nature, by the way of a visual analysis of the obtained material in subsequent generations, as well as through biochemical analysis, yield qualities analysis and morphometry of the obtained material.

Clinical application of next-generation sequencing-based monitoring of minimal residual disease in childhood acute lymphoblastic leukemia.

Next-generation sequencing (NGS) is an emerging technology that can comprehensively assess the diversity of the immune system. We explored the feasibility of NGS in detecting minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) based on immunoglobulin and T cell receptor. Bone marrow samples were collected pre- and post-treatment with pediatric ALL admitted to Shenzhen Children's Hospital from February 1st, 2020 to January 31st, 2021. We analyzed the MRD detected by NGS, multiparametric flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR), and analyzed risk factors of positive NGS-MRD at the end of B-ALL induction chemotherapy. A total of paired 236 bone marrow samples were collected from 64 children with ALL (58 B-ALL and 6T-ALL). The decrease in the clonal rearrangement frequency of IGH, IGK, and IGL was generally consistent after treatment. Positive MRD was detected in 57.5% (77/134) of B-ALL and 80% (12/15) of T-ALL by NGS after chemotherapy, which was higher than those detected by MFC and RQ-PCR. In B-ALL patients, MRD results detected by NGS were consistent with MFC (r = 0.708, p < 0.001) and RQ-PCR (r = 0.618, p < 0.001). At the end of induction, NGS-MRD of 40.4% B-ALL was > 0.01% and multivariate analysis indicated that ≧2 clonal rearrangement sequences before treatment were an independent factor of negative NGS-MRD. NGS is more sensitive than MFC and RQ-PCR for MRD measurement. B-ALL children with ≧2 clonal rearrangements detected by NGS before treatment are difficult to switch to negative MRD after chemotherapy.

ТРЁХЦВЕТНый FISH-МЕТОД: ТРАНСЛОКАЦИИ В КУЛЬТУРАХ ЛИМФОЦИТОВ ПЕРИФЕРИЧЕСКОЙ КРОВИ ДО И ПОСЛЕ ЛОКАЛЬНОГО Гамма-ОБЛУЧЕНИЯ ПО ПОВОДУ РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ

Purpose: Study of chromosomal aberration yields with a three-color FISH method in the peripheral blood lymphocyte cultures of patients with breast cancer before and after local irradiation. Material and methods: Venous blood was obtained from 24 patients aged from 33 to 79 years which suffering from cancer of the left or right breast after the implementation of a sectoral or radical resection of an affected organ with subsequent local adjuvant distant radiation therapy (DRT) to the scar area. Also it could be preceded by adjuvant polychimotherapy (PCT). A linear accelerator of the Trilogy system based on the Clinac iX platform was used for local irradiation. SOD was installed at 50 Gy per 25 fractions (daily 2 Gy). venous blood sampling produced to PCT and/or DRT and after 1 and/or 3 months after irradiation. A set of DNA probes to 1, 4 and 12 pairs of chromosomes was used for three-color FISH staining. Results: Levels of translocations, dicentrics and acentrics (per 100 cells) in peripheral blood lymphocyte cultures of patients up to DRT did not differ significantly with and without PCT and were amounted to average 0.63 ± 0.10; 0.10 ± 0.04 and 0.17 ± 0.06, respectively. The frequency of these chromosome rearrangements significantly increased after radiation therapy by an average of up to 4.62 ± 0.46; 1.48 ± 0.19 and 0.95 ± 0.14. The differences between 1 and 3 months of taking material after irradiation were absent. One of the patients, unlike the rest, was cytogenetically examined 11 years after a similar local irradiation, and the frequencies of translocations and dicentrics in her lymphocyte culture were 4.56 and 0.19 (per 100 cells), respectively, while they were varied from 1.63 to 10.00 for translocations and from 0.27 (0 in one patient) to 4.06 for dicentrics for other patients. Conclusion: When using a three-color FISH method there was a significant increase in the frequencies of translocations, dicentrics and acentrics in peripheral blood lymphocyte cultures of patients after DRT of breast cancer. At the same time there were more translocations than dicentrics and, apparently, they remain at the initial level for a long time. The frequency of translocations per 1 cell with translocations in the peripheral blood lymphocyte cultures in most cases exceeds 1 after local DRT of the breast cancer. The correlation between average physical doses to the whole body and cytogenetical estimates in individual patients was statistically insignificant.

Interspecies genome divergence is predominantly due to frequent small scale rearrangements in Eucalyptus.

Synteny, the ordering of sequences within homologous chromosomes, must be maintained within the genomes of sexually reproducing species for the sharing of alleles and production of viable, reproducing offspring. However, when the genomes of closely related species are compared, a loss of synteny is often observed. Unequal homologous recombination is the primary mechanism behind synteny loss, occurring more often in transposon rich regions, and resulting in the formation of chromosomal rearrangements. To examine patterns of synteny among three closely related, interbreeding, and wild Eucalyptus species, we assembled their genomes using long-read DNA sequencing and de novo assembly. We identify syntenic and rearranged regions between these genomes and estimate that ~48% of our genomes remain syntenic while ~36% is rearranged. We observed that rearrangements highly fragment microsynteny. Our results suggest that synteny between these species is primarily lost through small-scale rearrangements, not through sequence loss, gain, or sequence divergence. Further examination of identified rearrangements suggests that rearrangements may be altering the phenotypes of Eucalyptus species. Our study also underscores that the use of single reference genomes in genomic variation studies could lead to reference bias, especially given the scale at which we show potentially adaptive loci have highly diverged, deleted, duplicated and/or rearranged. This study provides an unbiased framework to look at potential speciation and adaptive loci among a rapidly radiating foundation species of woodland trees that are free from selective breeding seen in most crop species.

Repetitive DNA Sequences in the Human Y Chromosome and Male Infertility.

The male-specific Y chromosome, which is well known for its diverse and complex repetitive sequences, has different sizes, genome structures, contents and evolutionary trajectories from other chromosomes and is of great significance for testis development and function. The large number of repetitive sequences and palindrome structure of the Y chromosome play an important role in maintaining the stability of male sex determining genes, although they can also cause non-allelic homologous recombination within the chromosome. Deletion of certain Y chromosome sequences will lead to spermatogenesis disorders and male infertility. And Y chromosome genes are also involved in the occurrence of reproductive system cancers and can increase the susceptibility of other tumors. In addition, the Y chromosome has very special value in the personal identification and parentage testing of male-related cases in forensic medicine because of its unique paternal genetic characteristics. In view of the extremely high frequency and complexity of gene rearrangements and the limitations of sequencing technology, the analysis of Y chromosome sequences and the study of Y-gene function still have many unsolved problems. This article will introduce the structure and repetitive sequence of the Y chromosome, summarize the correlation between Y chromosome various sequence deletions and male infertility for understanding the repetitive sequence of Y chromosome more systematically, in order to provide research motivation for further explore of the molecules mechanism of Y-deletion and male infertility and theoretical foundations for the transformation of basic research into applications in clinical medicine and forensic medicine.

High early death rates, treatment resistance, and short survival of Black adolescents and young adults with AML

AbstractSurvival of patients with acute myeloid leukemia (AML) is inversely associated with age, but the impact of race on outcomes of adolescent and young adult (AYA; range, 18-39 years) patients is unknown. We compared survival of 89 non-Hispanic Black and 566 non-Hispanic White AYA patients with AML treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols. Samples of 327 patients (50 Black and 277 White) were analyzed via targeted sequencing. Integrated genomic profiling was performed on select longitudinal samples. Black patients had worse outcomes, especially those aged 18 to 29 years, who had a higher early death rate (16% vs 3%; P=.002), lower complete remission rate (66% vs 83%; P=.01), and decreased overall survival (OS; 5-year rates: 22% vs 51%; P<.001) compared with White patients. Survival disparities persisted across cytogenetic groups: Black patients aged 18 to 29 years with non–core-binding factor (CBF)-AML had worse OS than White patients (5-year rates: 12% vs 44%; P<.001), including patients with cytogenetically normal AML (13% vs 50%; P<.003). Genetic features differed, including lower frequencies of normal karyotypes and NPM1 and biallelic CEBPA mutations, and higher frequencies of CBF rearrangements and ASXL1, BCOR, and KRAS mutations in Black patients. Integrated genomic analysis identified both known and novel somatic variants, and relative clonal stability at relapse. Reduced response rates to induction chemotherapy and leukemic clone persistence suggest a need for different treatment intensities and/or modalities in Black AYA patients with AML. Higher early death rates suggest a delay in diagnosis and treatment, calling for systematic changes to patient care.

Multiple Stochastic Parameters Influence Genome Dynamics in a Heterozygous Diploid Eukaryotic Model.

The heterozygous diploid genome of Candida albicans displays frequent genomic rearrangements, in particular loss-of-heterozygosity (LOH) events, which can be seen on all eight chromosomes and affect both laboratory and clinical strains. LOHs, which are often the consequence of DNA damage repair, can be observed upon stresses reminiscent of the host environment, and result in homozygous regions of various sizes depending on the molecular mechanisms at their origins. Recent studies have shed light on the biological importance of these frequent and ubiquitous LOH events in C. albicans. In diploid Saccharomyces cerevisiae, LOH facilitates the passage of recessive beneficial mutations through Haldane’s sieve, allowing rapid evolutionary adaptation. This also appears to be true in C. albicans, where the full potential of an adaptive mutation is often only observed upon LOH, as illustrated in the case of antifungal resistance and niche adaptation. To understand the genome-wide dynamics of LOH events in C. albicans, we constructed a collection of 15 strains, each one carrying a LOH reporter system on a different chromosome arm. This system involves the insertion of two fluorescent marker genes in a neutral genomic region on both homologs, allowing spontaneous LOH events to be detected by monitoring the loss of one of the fluorescent markers using flow cytometry. Using this collection, we observed significant LOH frequency differences between genomic loci in standard laboratory growth conditions; however, we further demonstrated that comparable heterogeneity was also observed for a given genomic locus between independent strains. Additionally, upon exposure to stress, three outcomes could be observed in C. albicans, where individual strains displayed increases, decreases, or no effect of stress in terms of LOH frequency. Our results argue against a general stress response triggering overall genome instability. Indeed, we showed that the heterogeneity of LOH frequency in C. albicans is present at various levels, inter-strain, intra-strain, and inter-chromosomes, suggesting that LOH events may occur stochastically within a cell, though the genetic background potentially impacts genome stability in terms of LOH throughout the genome in both basal and stress conditions. This heterogeneity in terms of genome stability may serve as an important adaptive strategy for the predominantly clonal human opportunistic pathogen C. albicans, by quickly generating a wide spectrum of genetic variation combinations potentially permitting subsistence in a rapidly evolving environment.

Establishment of a Cre-loxP System Based on a Leaky LAC4 Promoter and an Unstable panARS Element in Kluyveromyces marxianus.

The Cre-loxP system produces structural variations, such as deletion, duplication, inversion and translocation, at specific loci and induces chromosomal rearrangements in the genome. To achieve chromosomal rearrangements in Kluyveromyces marxianus, the positions and sequences of centromeres were identified in this species for the first time. Next, a Cre-loxP system was established in K. marxianus. In this system, the Cre recombinase was expressed from a leaky LAC4 promoter in a plasmid to alleviate the cytotoxicity of Cre, and the unstable plasmid contained a panARS element to facilitate the clearance of the plasmid from the cells. By using LAC4 as a reporter gene, the recombination frequencies between loxP sites or loxPsym sites were 99% and 73%, respectively. A K. marxianus strain containing 16 loxPsym sites in the genome was constructed. The recombination frequency of large-scale chromosomal rearrangements between 16 loxPsym sites was up to 38.9%. Our study provides valuable information and tools for studying chromosomal structures and functions in K. marxianus.

Abstract 2286: Profiling the complex rearrangement landscape of sarcoma

Abstract Genomic rearrangements are key mutational processes in a number of bone and soft tissue tumors (sarcomas). These events are used for both disease classification and as prognostic and predictive biomarkers. However, the rearrangement architecture and mutational processes giving rise to many of these events remain poorly characterized. Recent data indicate that sarcomas show particularly high frequencies of complex structural rearrangements, including patterns which do not fit those of known mutational mechanisms. As the largest whole genome sequencing (WGS) dataset of sarcomas to date, the Genomics England (GE) 100,000 Genomes Project represents a unique cohort in which to examine these events. Structural variants (SVs) were identified using five SV callers (Manta, Delly, LUMPY, SvABA and GRIDSS) in WGS data from 1330 GE samples. SVs were filtered to retain only those passing the quality filter, and to remove variants present in either matched germline samples or &amp;gt;1% of a panel of normal variants. Caller performance was evaluated by comparison of distances between SV and copy number call breakpoints generated by Battenberg. SVs identified by at least two callers were taken forward as high-quality calls. Chromothriptic events, extrachromosomal DNA (ecDNA) and breakage-fusion-bridges were identified using established algorithms. In keeping with previous data, the prevalence of structural variants varies by tumor type, with particularly high numbers of SVs observed in liposarcoma and osteosarcoma. Many tumor types included notable outlier samples; these were more likely to show evidence of chromothripsis, as identified by a dedicated chromothripsis caller. Liposarcomas show the highest frequency of chromothripsis, as described previously. In addition, we have examined some tumor types at higher granularity than previously available, demonstrating directly that the prevalence of chromothripsis varies by histological subtype. For example, chromothriptic events are identified in 100% of well-differentiated liposarcomas, but less than 10% of myxoid liposarcomas. Similarly, the presence of ecDNA varies by tumor type, with the novel observation of particularly high rates in angiosarcoma. Despite recent advances in histological classification, the survival for patients with sarcoma has remained largely static for over 40 years. Determination of the mutational processes generating these rearrangements will shed light on the pathogenesis of these tumors. Citation Format: Sara Waise, Tom Lesluyes, Jonas Demeulemeester, Maxime Tarabichi, Sarcoma Genomics England Clinical Interpretation Partnership, Nischalan Pillay, Adrienne M. Flanagan, Peter Van Loo. Profiling the complex rearrangement landscape of sarcoma [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 2286.

14. Four-way translocation in a patient with mild clitoromegaly

Balanced reciprocal translocations are frequent chromosomal rearrangements between two nonhomologous chromosomes with an approximate incidence of 1/500 and can be inherited or de novo. Individuals carrying these rearrangements are usually phenotypically normal but with increased risk for infertility, miscarriages, stillbirths, and abnormal liveborn offspring. More complex rearrangements such as double reciprocal, triple reciprocal, and three-way balanced translocations are rare events. Complex constitutional rearrangements involving more than three chromosomes are extremely rare and a recent review of the literature described less than 15 patients. We report here a one-day-old full-term female with prominent labial folds and enlarged clitoris that was referred for cytogenetic analysis. Chromosome studies demonstrated the appearance of either two reciprocal translocations or a single four-way translocation. Metaphase FISH and SNP microarray analyses were performed on the patient to determine the complexity of the rearrangement. FISH analysis confirmed the presence of a four-way translocation without deletions or duplications at the breakpoints as determined by SNP array. Further, the SNP array detected a 4.5 Mb deletion of 1p31.1 and a triplication of 1q43 characterized as a variant of unknown significance (VUS). Parental peripheral blood has been requested to determine the origin of the rearrangement, the 1p31.1 deletion, and the 1q34 VUS and these results will be presented accordingly.

Treacle Sticks the Nucleolar Responses to DNA Damage Together.

The importance of chromatin environment for DNA repair has gained increasing recognition in recent years. The nucleolus is the largest sub-compartment within the nucleus: it has distinct biophysical properties, selective protein retention, and houses the specialized ribosomal RNA genes (collectively referred to as rDNA) with a unique chromatin composition. These genes have high transcriptional activity and a repetitive nature, making them susceptible to DNA damage and resulting in the highest frequency of rearrangements across the genome. A distinct DNA damage response (DDR) secures the fidelity of this genomic region, the so-called nucleolar DDR (n-DDR). The composition of the n-DDR reflects the characteristics of nucleolar chromatin with the nucleolar protein Treacle (also referred to as TCOF1) as a central coordinator retaining several well-characterized DDR proteins in the nucleolus. In this review, we bring together data on the structure of Treacle, its known functions in ribosome biogenesis, and its involvement in multiple branches of the n-DDR to discuss their interconnection. Furthermore, we discuss how the functions of Treacle in ribosome biogenesis and in the n-DDR may contribute to Treacher Collins Syndrome, a disease caused by mutations in Treacle. Finally, we outline outstanding questions that need to be addressed for a more comprehensive understanding of Treacle, the n-DDR, and the coordination of ribosome biogenesis and DNA repair.

CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana.

The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.

CRLF2 overexpression results in reduced B-cell differentiation and upregulated E2F signaling in the Dp16 mouse model of Down syndrome

Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.