Additional Chromosome Research Articles

(Not) getting what you want: frustration and emotion regulation in children with sex chromosome trisomies.

The presence of an additional X or Y chromosome (sex chromosome trisomies, SCT) is associated with an increased risk for neurodevelopmental difficulties, including socio-emotional problems, across the life span. Studying emotion regulation in young children with SCT could signal deviations in emotional development that serve as risk markers to guide clinical care. This study explored the presence and variety of emotion regulation strategies in 75 SCT children and 81 population-based controls, aged 1-7 years, during a frustration-inducing event in which physiological (heart rate) and observational data (behavioral responses) were collected. Children with SCT were equally physiologically aroused by the event as compared to controls. However, they showed more emotion regulation difficulties in terms of behavior compared to controls that were not explicable in terms of differences in general intellectual functioning. Specifically, they had a more limited range of behavioral alternatives and tended to rely longer on inefficient strategies with increasing age. The field of practice should be made aware of these early risk findings regarding emotion regulation in SCT, which may potentially lay the foundation for later socio-emotional problems, given the significant impact of emotion regulation on child and adult mental health outcomes. The current results may help to design tailored interventions to reduce the impact of the additional sex chromosome on adaptive functioning, psychopathology, and quality of life.

The hereditary N363K POLE exonuclease mutant extends PPAP tumor spectrum to glioblastomas by causing DNA damage and aneuploidy in addition to increased mismatch mutagenicity.

The exonuclease domain of DNA polymerases epsilon's catalytic subunit (POLE) removes misincorporated nucleotides, called proofreading. POLE-exonuclease mutations cause colorectal- and endometrial cancers with an extreme burden of single nucleotide substitutions. We recently reported that particularly the hereditary POLE exonuclease mutation N363K predisposes in addition to aggressive giant cell glioblastomas. We knocked-in this mutation homozygously into human cell lines and compared its properties to knock-ins of the likewise hereditary POLE L424V mutation and to a complete proofreading-inactivating mutation (exo-null). We found that N363K cells have higher mutation rates as both L424V- or exo-null mutant cells. In contrast to L424V cells, N363K cells expose a growth defect, replication stress and DNA damage. In non-transformed cells, these burdens lead to aneuploidy but macroscopically normal nuclei. In contrast, transformed N363K cells phenocopy the enlarged and disorganized nuclei of giant cell glioblastomas. Taken together, our data characterize a POLE exonuclease domain mutant that not only causes single nucleotide hypermutation, but in addition DNA damage and chromosome instability, leading to an extended tumor spectrum. Our results expand the understanding of the polymerase exonuclease domain and suggest that an assessment of both the mutational potential and the genetic instability might refine classification and treatment of POLE-mutated tumors.

Psychological capital, quality of life, and well-being in mother caregivers of individuals with down syndrome.

Caused by an error in cell division that produces an additional chromosome 21, Down syndrome (DS) is one of the most common developmental disorders in the world. This study aims to analyze the relationship between psychological capital, quality of life and well-being of caregivers of individuals with Down syndrome (DS). The participants were 98 caregivers (mothers, M = 52.13, SD = 11.39) of individuals with Down syndrome. The instruments used were the Psychological Capital Questionnaire (measuring self-efficacy, resilience, optimism, and hope), Quality of Life Questionnaire (including social support, general satisfaction, physical/psychological health, absence of excessive workload/free time), and Psychological Wellbeing Scale, investigating the following dimensions: self-acceptance, positive relationships with others, autonomy, environmental mastery, purpose in life, and personal growth. The mediation analysis showed that self-efficacy, hope, and resilience are positively associated to quality of life, and optimism is positively associated to well-being. The total effects of psychological capital on well-being are positive and significant and quality of life mediates the relationship between psychological capital and well-being. These results show that psychological capital is an important inner resource for caregivers of DS individuals and must be improved through support services, so that caregivers have a higher perception of the quality of life and implicitly of well-being.

Identification of a Monosomic Alien Chromosome Addition Line Responsible for the Purple Color Trait in Heading Chinese Cabbage

Purple heading Chinese cabbage has become popular in recent years due to its attractive color and health benefits. However, purple varieties remain rare, and the regulation mechanism of anthocyanin accumulation in Chinese cabbage is still largely unknown. By introducing the purple color trait from Brassica juncea, a new purple heading Chinese cabbage cultivar (18M-245) was generated with deep purple leaves at both the seedling and adult stages. Anthocyanin accumulation in 18M-245 increased when grown at low temperatures. FISH and genotyping results showed that the purple trait was caused by an alien chromosome addition line derived from the Brassica B genome. The LDOX coding gene BjuB014115 from the addition line was highly expressed in 18M-245, consistent with the results of anthocyanin accumulation. Meanwhile, several MYB and bHLH transcriptional factors from the Brassica A genome were found to directly bind to the promoter of BjuB014115, suggesting that interactions between the Brassica A and B genomes are involved in the regulatory network of anthocyanin biosynthesis in Chinese cabbage. Our results provide new insights into the regulation mechanism of anthocyanin biosynthesis in purple heading Chinese cabbage.

Studies of Oat-Maize Hybrids Tolerance to Soil Drought Stress

The ontogenesis and yield formation in crop plants are modified by environmental conditions. Due to climatic change detected over two decades, the harmful influence of abiotic factors is increasing. One of the most threatening issues reducing plant productivity is drought stress. The strength of plant response to water shortages could differ depending on the strength of the drought stress, type of crop, genetic background, presence of additional stresses, and stage of plant development. There are examples of sexual hybridization between crop plants like oat (Avena sativa L.) and maize (Zea mays L.) with which stable fertile hybrids were generated. Additional maize chromosomes in oat plants (oat × maize addition, OMA) often infer morphological and physiological (e.g., PS II photosystem activity and chlorophyll production) changes modulated by the interaction of certain maize chromosomes added to the oat genome. The aim of the research was to evaluate the chosen physiological, biochemical, and agronomic parameters of OMA plants subjected to soil drought. Analysis of variance indicated that the main effects of genotype as well as treatment × genotype interaction were significant for all the traits studied (photosynthetic pigment content, selected PSII indices, mass of stem, number of grains/plant, mass of grains/plant). Most of the examined lines severely reduced PSII photosystem parameters, pigment content, and yield-related traits under drought stress. The results indicated that two lines (9 and 78b) retained high yielding potential under drought stress compared to commercial cv. Bingo.

Association of Supernumerary Sex Chromosome Aneuploidies With Venous Thromboembolism

An increased risk of venous thromboembolism (VTE) has been reported in men with an additional sex chromosome. The association between other sex chromosome aneuploidies and VTE is not well characterized. To determine if sex chromosome aneuploidy is associated with VTE. Retrospective cohort study of sex chromosome aneuploidy and VTE, performed by analyzing X- and Y-chromosome dosage and VTE incidence in 642 544 individuals from 2 population-scale biobanks: the US Geisinger MyCode Community Health Initiative (N = 154 519) and the UK Biobank (N = 488 025); analysis was limited to participants self-identified as White because of inadequate sample sizes for other race and ethnicity groups. A total of 108 461 unrelated MyCode participants with electronic health record follow-up ranging from September 1996 to December 2020 and 418 725 unrelated British and Irish UK Biobank participants who attended the baseline assessment between March 2006 and October 2010, with follow-up extending to November 2020, were included in analyses of VTE. Sex chromosome aneuploidies. Individuals with 1 primary inpatient VTE diagnosis, 2 primary outpatient VTE diagnoses, or a self-reported VTE diagnosis were defined as VTE cases. P values were adjusted for multiple comparisons. Identification of sex chromosome aneuploidy was undertaken among 642 544 individuals aged 18 to 90 years. Identification of a diagnosis of VTE was undertaken among 108 461 unrelated MyCode participants (65 565 [60.5%] female; mean age at last visit, 58.0 [SD, 17.6] years; median follow-up, 15.3 [IQR, 9.7] years) and among 418 725 unrelated UK Biobank participants (224 695 [53.7%] female; mean age at baseline interview, 56.9 [SD, 8.0] years; median follow-up, 12.0 [IQR, 1.6] years). Among MyCode participants, during 10 years of follow-up, 17 incident VTE events per 1353 person-years were detected among those with supernumerary sex chromosome aneuploidy (1.3% per person-year) compared with 2060 per 816 682 person-years among those with 46,XX or 46,XY (0.25% per person-year) (hazard ratio, 5.4 [95% CI, 3.4-8.7]; 10-year risk difference, 8.8% [95% CI, 4.2%-14.0%]; P < .001). Among UK Biobank participants, during 10 years of follow-up, 16 incident VTE events per 3803 person-years were detected among those with supernumerary sex chromosome aneuploidy (0.42% per person-year) compared with 4491 per 3 970 467 person-years among those with 46,XX or 46,XY (0.11% per person-year) (hazard ratio, 4.1 [95% CI, 2.5-6.7]; 10-year risk difference, 3.7% [95% CI, 1.4%-5.9%]; P < .001). Adults with supernumerary sex chromosome aneuploidies compared with 2 sex chromosomes had a small but statistically significant increased risk of VTE. Further research is needed to understand the clinical implications of this association.

The Chromosome Counts Database (CCDB).

For decades, plant biologists have been interested in the determination and documentation of chromosome numbers for extant taxa. This central cytological character has been used as an important phylogenetic marker and as an indicator for major genomic events such as polyploidy and dysploidy. Due to their significance and the relative ease by which chromosome numbers can be obtained, chromosome numbers have been extensively recorded across the plant kingdom and documented in a wide variety of resources. This makes the collection process a wearing task, often leading to partial data retrieval. In 2015, the Chromosome Counts Database (CCDB) was assembled, being an online unified community resource. This database compiles dozens of different chromosome counts sources, of which a significant portion had been unavailable before in a digitized, searchable format. The vast amount of data assembled in CCDB has already enabled a large number of analyses to examine the evolution of different plant hierarchies, as well as the application of various follow-up analyses, such as ploidy-level inference using chromEvol. CCDB ( http://ccdb.tau.ac.il/ ) encourages data sharing among the botanical community and is expected to continue expanding as additional chromosome numbers are recorded.

The Impact of Cytogenetic Aberrations in the Clonal Evolution of Chronic Myeloid Leukemia: A Single-Center Experience Among 450 Turkish Patients (Cohort Study)

Chronic myeloid leukemia (CML) is a clonal hematologic disorder characterized by t(9;22) translocation, in which cytogenetic aberrations can occur in Ph(+) and (-) clones. These aberrations develop due to clonal evolution as well as treatment and they have prognostic significance. They are grouped as major and minor route anomalies in terms of their effects on prognostic parameters, such as treatment response, overall survival (OS), disease stage, complete cytogenetic response (CCyR), and major molecular response (MMR). It is stated that major route anomalies have unfavorable prognostic effects compared to minor route anomalies. We aimed to investigate the frequency and prognostic effects of cytogenetic anomalies detected in Ph(+) and (-) clones. In this study, we retrospectively analyzed the cytogenetic results of 450 patients diagnosed with CML between 2005 and 2020. We detected cytogenetic aberrations in Ph-positive and negative clones in 41 of 450 patients. The most common anomalies were trisomy 8 (+8), additional Ph chromosome (+Ph), and loss of chromosome Y. Rarely, aneuploidy of the Y chromosome, dup (22), +11, and +6 were seen in CML patients. We observed that these identified aberrations negatively affected MMR and CCyR, and generally resulted in changing imatinib treatment for second-generation tyrosine kinase activity inhibitors. Our results are compatible with the literature. We suggest that cytogenetic aberrations detected in Ph(+) and (-) clones should be a warning sign in terms of treatment and require close observation. The use of cytogenetic methods for the identification of these anomalies is also important.

Trisomy 8 is Associated with Favorable Outcome in the Patients with Myelodysplastic Syndromes Treated with Allogeneic Hematopoietic Stem Cell Transplantation

Favorable prognostic significance of sole trisomy 8 and its associations with additional chromosome aberrations was confirmed in 7 adult and 3 pediatric patients with myelodysplastic syndromes treated with hematopoietic stem cell transplantation (HSCT). The group of comparison included 10 MDS patients with sole monosomy 7 or 5 chromosome and those within complex karyotypes (CK). Cytogenetic investigations were carried out according to standard GTG and multi-colored fluorescence in situ hybridization (mFISH) techniques. Our data revealed significant difference in overall survival (OS) between the tested and control groups (p=0.045) thus being additional argument reinforcing the concept of favorable prognosis of trisomy 8 in HSCT-treated MDS patients. Eight of ten patients (5 with sole trisomy 8 and three with more complex karyotypes) are alive. Of the deceased patients, one had CK trisomy 8 was associated with poor-prognostic monosomy 7. In accordance with experimental findings Sloand et al., 2007, this favorable effect of trisomy 8 in MDS patients might be linked with inhibition of programmed cell death with anti-apoptotic proteins, including myc, which are activated in these cases and needs additional in-depth studies.

Directed Evolution Detects Supernumerary Centric Chromosomes Conferring Resistance to Azoles in Candida auris.

Candida auris exhibits resistance to multiple antifungal drug classes and sterilization agents, posing threats to the immunocompromised worldwide. Among the four major geographical clades, the East Asian clade 2 isolates of C. auris are mostly drug susceptible. In this study, we experimentally evolved one such drug-susceptible isolate for multiple generations in the presence of the antifungal compound fluconazole and analyzed changes in the karyotype, DNA sequence, and gene expression profiles in three evolved drug-resistant isolates. Next-generation sequencing and electrophoretic karyotyping confirm the presence of segmental aneuploidy as supernumerary chromosomes originating from centromere-inclusive chromosomal duplication events in two such cases. A 638-kb region and a 675-kb region, both of which originated from chromosome 5 and contained its centromere region, are instances of supernumerary chromosome formation identified in two evolved fluconazole-resistant isolates. Loss of the supernumerary chromosomes from the drug-resistant isolates results in a complete reversal of fluconazole susceptibility. Transcriptome analysis of the third isolate identified overexpression of drug efflux pumps as a possible non-aneuploidy-driven mechanism of drug resistance. Together, this study reveals how both aneuploidy-driven and aneuploidy-independent mechanisms may operate in parallel in an evolving population of C. auris in the presence of an antifungal drug, in spite of starting from the same strain grown under similar conditions, to attain various levels of fluconazole resistance. IMPORTANCE Fungal pathogens develop drug resistance through multiple pathways by acquiring gene mutations, increasing the copy number of genes, or altering gene expression. In this study, we attempt to understand the mechanisms of drug resistance in the recently emerged superbug, C. auris. One approach to studying this aspect is identifying various mechanisms operating in drug-resistant clinical isolates. An alternative approach is to evolve a drug-susceptible isolate in the presence of an antifungal compound and trace the changes that result in drug resistance. Here, we evolve a drug-susceptible isolate of C. auris in the laboratory in the presence of a widely used antifungal compound, fluconazole. In addition to the already known changes like overexpression of drug efflux pumps, this study identifies a novel mechanism of azole resistance by the emergence of additional chromosomes through segmental duplication of chromosomal regions, including centromeres. The centric supernumerary chromosome helps stable amplification of a set of genes with an extra copy to confer fluconazole resistance.

Prospective Evaluation of Sorafenib Combined with Chemotherapy in Newly Diagnosed Adult Core-Binding Factor Acute Myeloid Leukemia: An Open-Label, Randomized Controlled, Multicenter Phase II Trial

Objective Core-binding factor acute myeloid leukemia (CBF-AML) includes AML with t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22) chromosomal rearrangements, leading to the RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes, respectively. Although CBF-AML is considered to have a favorable prognosis relative to other AML subtypes, up to 40% of these patients experience relapse. Recent studies demonstrate achievement of complete molecular remission (CMR) at the end of treatment significantly associated with a reduced risk of relapse. A high frequency of mutations and/or high expression of KIT gene is observed in CBF-AML. Sorafenib is a first-generation type-II multitargeted tyrosine kinase receptor inhibitor (TKI) that suppresses various signaling pathways associated with the development of AML, such as RTK (FLT3, c-KIT). Thus the purpose of this study is to evaluate the safety and efficacy of sorafenib combined with chemotherapy in adult newly diagnosed CBF-AML. Methods Patients were randomly assigned (1:1) to control group (chemotherapy alone) or sorafenib group (sorafenib combined with chemotherapy). For control group, patients received one cycle of induction therapy with idarubicin (12 mg/m² on days 1-3) plus cytarabine (100 mg/m² on days 1-7), followed by one cycle of idarubicin (8 mg/m² on days 1-3) plus cytarabine (2 g/m² q12h on days 1-3) and two cycles of high-dose cytarabine consolidation therapy (2 g/m² q12h on days 1-3). For sorafenib group, patients were treated with chemotherapy plus sorafenib at 400 mg twice daily on days 8-21 for induction cycle, and on days 1-21 for each consolidation, and as maintenance for 12 months. Allogeneic or autologous HSCT were implemented based on the measurable residual disease (MRD) level and donor availability after 4 cycles of therapy. MRD levels were serially monitored for RUNX1-RUNX1T1 or CBFB-MYH11 transcripts by real-time quantitative polymerase chain reaction. Results were expressed as a [fusion gene/ABL1] ×100 transcript ratio. Major molecular response (MMR) and complete molecular remission (CMR) are defined as transcript ratio < 0.1% and 0.001%, respectively. The primary endpoint was CMR after 4 cycles of therapy in bone marrow (BM). Results Between January 2020 and March 2022, 64 patients were enrolled and randomly assigned to sorafenib group (n=32) or control group (n=32). There is no significant difference between the two groups about age, gender, CBF subtype, BM blast percentage, additional chromosome abnormalities and gene mutations at diagnosis. The hematological complete remission in the sorafenib and control group were 96.9% and 94.1%, respectively (p=.592). Following three cycles of therapy, MMR was observed in 79.3% of the sorafenib group and 46.9% of the control group (p =.009). Sorafenib group also had a significantly higher CMR than control group (62.1% vs 28.1%, p =.009). After four cycles of therapy, MMR increased to 100% in the sorafenib group and 70.8% in the control group (p =.019), whileCMR was also more frequently achieved in the sorafenib group than that in the control group (90.9% vs 54.2%, p =.006). There was no significant difference of grades 3-4 adverse events between the two groups. Grade 4 neutropenia (neutrophil count less than 0.5×10⁹ cells per L) and thrombocytopenia (platelet count less than 20×10⁹ per L) were observed in all cases. The median duration of neutropenia was 12d (range, 6-25d) in the sorafenib group and 10d (range, 5-22d) in the control group (p =.063) , while the median duration of thrombocytopenia was 12d (range, 5-33d) and 9d (range, 6-19d) (p =.052), respectively.The most common grade 3-4 non-hematological adverse events in sorafenib and control group were pneumonia (31.3% vs 57.4%, p=.019), mucositis (21.9% vs 24.1%, p =.816), blood stream infection (37.5% vs 31.5%, p =.568). Conclusion Sorafenib combined with chemotherapy significantly increased the CMR after 4 cycles of therapy, however, the incidence of haematological and non-haematological adverse events did not apparently increased. The impact of sorafenib combined with chemotherapy on relapse and survival of CBF-AML need further following-up. Keywords: Sorafenib, CBF-AML, CMR, adverse events

Chromosome 7 Myeloid Tumor Suppressor Genes Cooperate in Chemotherapy Resistance

Loss of all or part of chromosome 7 [-7/del(7q)] is common in myeloid neoplasms and is associated with chemoresistance. -7/del(7q) occurs in de novo and therapy-related myeloid neoplasms (t-MN), high-risk secondary malignancies arising after prior exposure to chemotherapy or radiation. We previously reported that deficiency of CUX1, a 7q-encoded tumor suppressor gene, promotes hematopoietic stem and progenitor cell (HSPC) drug resistance, enabling HSPCs to persist during genotoxic insult, expand, and transform to t-MN. To comprehensively identify additional chromosome 7 genes putatively involved in drug resistance, we mined 13 chemotherapy resistance CRISPR-Cas9 genome-wide knockout screens performed in human cell lines. We included screens with daunorubicin, commonly used in the treatment of de novo acute myeloid leukemia, as well as screens with cisplatin and etoposide, chemotherapeutics that predispose to t-MN. Using loose thresholds for inclusion, we identified 113 genes that are potentially selected for by chemotherapy when edited. We prioritized candidates for validation based on those genes that were recurrent hits in more than one screen and genes with high expression levels in human HSPCs. Of the 9 top candidates tested in subsequent in vitro assays, we confirmed four genes to promote resistance when CRISPR-edited. This work yielded two 7q genes known to promote drug resistance when deficient, EZH2 and KMT2C/MLL3, validating our approach. We also identified novel drug resistance phenotypes for AHR and LUC7L2 deficiency. We demonstrate that combined deficiency in EZH2 and CUX1 leads to increased drug resistance in vitro and in vivo, to an extent greater than loss of either single gene alone. Transcriptome profiling additionally supports a genetic interaction between EZH2 and CUX1. In summary, chromosome 7 harbors multiple genes involved in chemotherapy resistance, supporting the concept of 7q as a contiguous gene syndrome. Furthermore, we reveal a previously unknown genetic interaction between the 7q genes CUX1 and EZH2. A refined understanding of the molecular pathways driving del(7q) pathogenesis and drug resistance will enable development of therapies designed to counter these treatment barriers.

Cytogenetical and hematological analysis of chronic myelogenous leukemia patients with a novel case 52XX, t (1;9;22) (q23.3; q34; q11.2), +6, +8, i(9) (q10;q10), +18,+19,+21+der22 t(9;22)(q34;q11).

(9;22) (q34; q11) translocation is appear in above ninety percent of chronic myelogenous leukemia patients while variant/complex translocations were observed in almost 5% to 8% chronic myelogenous leukemia (CML) positive cases. Gleevec (Imatinib Mesylate) is the first choice breakpoint cluster region (BCR)/ABL targeted oral therapy that produced a complete response almost in 71% to 80% of patients affected with CML. A complete blood count (CBC) of 37 patients was done during diagnosis, however only 21 showed abnormal CBC values which were selected for the study. Karyotyping study using bone marrow samples was performed on 21 CML patients for the conformation of 9;22, however, fluorescence in situ hybridisation was performed for the detection of the BCR-ABL fusion gene of 15 patients. Out of 21, 17 patients showed Ph-positive (9;22) (q34; q11) translocation. Sixteen CML patients showed standard translocation however only CML patients showed a three-way variant/complex translocation with six additional chromosomes, 52XX, t(1;9;22) (q23.3;q34;q11),+6,+8, i(9)(q10;q10), +18,+19,+21 + der22 t(9;22)(q34;q11)). Here we report we report a novel case of six additional chromosomes with the three-way translocation of 52XX, t(1;9;22) (q23.3;q34;q11),+6,+8, i(9)(q10;q10), +18,+19,+21 + der22 t(9;22)(q34;q11) in blast phase.

113. Optical Genome Mapping: Clinical validation and diagnostic utility for cytogenomic analysis of Hematological Neoplasms

Hematological neoplasms are predominantly defined by chromosomal aberrations that includes structural variants (SVs) and copy number variations (CNVs). The current standard-of-care (SOC) methods [karyotyping, fluorescence in-situ hybridization (FISH), and chromosomal microarrays (CMA)] employed for the detection of SVs and CNVs are labor intensive, time and cost-prohibitive, and often does not reveal the genetic complexity of the tumor. Optical genome mapping (OGM) is an emerging technology that can detect all classes of SVs in a single assay. We report the results from our clinical validation (in a CLIA setting) of the OGM technique for hematological neoplasms. The study included 94 sample runs (including replicates) using 71 well characterized unique samples (61 hematological neoplasms and 10 controls). The analytical performance showed a sensitivity of 98.7%, specificity of 100%, accuracy of 98.7%, PPV of 100% and NPV of 98%, that included the detection of 61 aneuploidies, 35 deletions, 28 translocations, 11 duplications/amplifications, 15 insertions/additional material not identified with karyotyping, 12 marker chromosomes, 1 each of ring chromosome, inversion and isochromosome. OGM showed high (robust) technical and analytical reproducibility with a limit of detection of 5% allele fraction for the variant classes evaluated in the study. In addition, OGM demonstrated higher resolution to refine breakpoints, identify the additional material, marker and ring chromosomes. This first CLIA validation report demonstrates the technical and analytical advantages of OGM compared to the current SOC methods used for chromosomal characterization, and we recommend OGM as a potential first-tier cytogenetic test for the evaluation of hematological neoplasms. Hematological neoplasms are predominantly defined by chromosomal aberrations that includes structural variants (SVs) and copy number variations (CNVs). The current standard-of-care (SOC) methods [karyotyping, fluorescence in-situ hybridization (FISH), and chromosomal microarrays (CMA)] employed for the detection of SVs and CNVs are labor intensive, time and cost-prohibitive, and often does not reveal the genetic complexity of the tumor. Optical genome mapping (OGM) is an emerging technology that can detect all classes of SVs in a single assay. We report the results from our clinical validation (in a CLIA setting) of the OGM technique for hematological neoplasms. The study included 94 sample runs (including replicates) using 71 well characterized unique samples (61 hematological neoplasms and 10 controls). The analytical performance showed a sensitivity of 98.7%, specificity of 100%, accuracy of 98.7%, PPV of 100% and NPV of 98%, that included the detection of 61 aneuploidies, 35 deletions, 28 translocations, 11 duplications/amplifications, 15 insertions/additional material not identified with karyotyping, 12 marker chromosomes, 1 each of ring chromosome, inversion and isochromosome. OGM showed high (robust) technical and analytical reproducibility with a limit of detection of 5% allele fraction for the variant classes evaluated in the study. In addition, OGM demonstrated higher resolution to refine breakpoints, identify the additional material, marker and ring chromosomes. This first CLIA validation report demonstrates the technical and analytical advantages of OGM compared to the current SOC methods used for chromosomal characterization, and we recommend OGM as a potential first-tier cytogenetic test for the evaluation of hematological neoplasms.

Chromosomal instability (CIN) in HAP1 cell lines revealed by multiplex fluorescence in situ hybridisation (M-FISH)

BackgroundHAP1, a near-haploid human leukemic cancer cell line is often used in combination with CRISPR-Cas9 gene editing technology for genetic screens. HAP1 carries the Philadelphia chromosome (Ph) and an additional ~ 30 Mb fragment of chromosome 15 inserted into chromosome 19. The potential use of an in vitro cell line as a model system in biomedical research studies depends on its ability to maintain genome stability. Being a cancer cell line with a near-haploid genome, HAP1 is prone to genetic instability, which is further compounded by its tendency to diploidise in culture spontaneously. Moreover, CRISPR-Cas9 gene editing coupled with prolonged in-vitro cell culturing has the potential to induce unintended ‘off-target’ cytogenetic mutations.To gain an insight into chromosomal instability (CIN) and karyotype heterogeneity, 19 HAP1 cell lines were cytogenetically characterised, 17 of which were near-haploids and two double-haploids, using multiplex fluorescence in situ hybridisation (M-FISH), at single cell resolution. We focused on novel numerical (N) and structural (S) CIN and discussed the potential causal factors for the observed instability. For each cell line we examined its ploidy, gene editing status and its length of in-vitro cell culturing.ResultsSixteen of the 19 cell lines had been gene edited with passage numbers ranging from 10 to 35. Diploidisation in 17 near-haploid cell lines ranged from 4 to 35% and percentage of N- and S-CIN in [1n] and [2n] metaphases ranged from 7 to 50% with two cell lines showing no CIN. Percentage of cells with CIN in the two double-haploid cell lines were 96% and 100% respectively. The most common S-CIN observed was deletion followed by translocation of both types, non-reciprocal and Robertsonian. Interestingly, we observed a prevalence of S-CIN associated with chromosome 13 in both near-and double-haploid cell lines, with a high incidence of Robertsonian translocation involving chromosome 13. Furthermore, locus-specific BAC (bacterial artificial chromosome) FISH enabled us to show for the first time that the additional chromosome 15 fragment is inserted into the p-arm rather than the q-arm of chromosome 19 of the HAP1 genome.ConclusionOur study revealed a high incidence of CIN leading to karyotype heterogeneity in majority of the HAP1 cell lines with the number of chromosomal aberrations varying between cell lines. A noteworthy observation was the high frequency of structural chromosomal aberrations associated with chromosome 13. We showed that CRISPR-Cas9 gene editing technology in combination with spontaneous diploidisation and prolonged in-vitro cell culturing is potentially instrumental in inducing further chromosomal rearrangements in the HAP1 cell lines with existing CIN. We highlight the importance of maintaining cell lines at low passage and the need for regular monitoring to prevent implications in downstream applications. Our study also established that the additional fragment of chromosome 15 in the HAP1 genome is inserted into chromosome 19p rather than 19q.

Whole genome sequencing of Moraxella bovis strains from North America reveals two genotypes with different genetic determinants

BackgroundMoraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi.ResultsPhylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins.ConclusionsThere are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study.

Flow karyotyping of wheat-Aegilops additions facilitate dissecting the genomes of Ae. biuncialis and Ae. geniculata into individual chromosomes.

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

The SPARC complex defines RNAPII promoters in Trypanosoma brucei.

Kinetoplastids are a highly divergent lineage of eukaryotes with unusual mechanisms for regulating gene expression. We previously surveyed 65 putative chromatin factors in the kinetoplastid Trypanosoma brucei. Our analyses revealed that the predicted histone methyltransferase SET27 and the Chromodomain protein CRD1 are tightly concentrated at RNAPII transcription start regions (TSRs). Here, we report that SET27 and CRD1, together with four previously uncharacterized constituents, form the SET27 promoter-associated regulatory complex (SPARC), which is specifically enriched at TSRs. SET27 loss leads to aberrant RNAPII recruitment to promoter sites, accumulation of polyadenylated transcripts upstream of normal transcription start sites, and conversion of some normally unidirectional promoters to bidirectional promoters. Transcriptome analysis in the absence of SET27 revealed upregulated mRNA expression in the vicinity of SPARC peaks within the main body of chromosomes in addition to derepression of genes encoding variant surface glycoproteins (VSGs) located in subtelomeric regions. These analyses uncover a novel chromatin-associated complex required to establish accurate promoter position and directionality.

A novel neo-sex chromosome in Sylvietta brachyura (Macrosphenidae) adds to the extraordinary avian sex chromosome diversity among Sylvioidea songbirds.

We report the discovery of a novel neo-sex chromosome in an African warbler, Sylvietta brachyura (northern crombec; Macrosphenidae). This species is part of the Sylvioidea superfamily, where four separate autosome-sex chromosome translocation events have previously been discovered via comparative genomics of 11 of the 22 families in this clade. Our discovery here resulted from analyses of genomic data of single species-representatives from three additional Sylvioidea families (Macrosphenidae, Pycnonotidae and Leiothrichidae). In all three species, we confirmed the translocation of a part of chromosome 4A to the sex chromosomes, which originated basally in Sylvioidea. In S. brachyura, we found that a part of chromosome 8 has been translocated to the sex chromosomes, forming a unique neo-sex chromosome in this lineage. Furthermore, the non-recombining part of 4A in S. brachyura is smaller than in other Sylvioidea species, which suggests that recombination continued along this region after the fusion event in the Sylvioidea ancestor. These findings reveal additional sex chromosome diversity among the Sylvioidea, where five separate translocation events are now confirmed.

Addition of Chromosome 17 Polysomy and HER2 Amplification Status Improves the Accuracy of Clinicopathological Factor-Based Progression Risk Stratification and Tumor Grading of Non-Muscle-Invasive Bladder Cancer.

Simple SummaryThe addition of chromosome 17 polysomy/HER2 amplification status to the updated EAU and AUA scores improves their accuracy, allowing molecular reclassification of EAU high-risk NMIBCs and G2 tumors. Based on this, we propose to reclassify the non-HER2 amplified, non-polysomic EAU 3–4 (high- and very high-risk) cases to the EAU 2 (intermediate) risk group to prevent unnecessarily strict follow-up and treatment for these patients. Furthermore, to classify Chr17 polysomic and/or HER2 amplified G2 tumors as high-grade (HG) and non-HER2 amplified, non-polysomic G2 tumors as low-grade (LG) NMIBCs (G1 and G3 tumors remain graded as low- and high-grade, respectively). Thus, the implementation of Chr17 polysomy/HER2 amplification testing would provide an immediate and simple solution to further refine the prognostic risk assessment of NMIBCs in the uro-oncology practice.Progression of non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive disease (MIBC) significantly worsens life expectancy. Its risk can be assessed by clinicopathological factors according to international guidelines. However, additional molecular markers are needed to refine and improve the prediction. Therefore, in the present study, we aimed to predict the progression of NMIBCs to MIBC by assessing p53 expression, polysomy of chromosome 17 (Chr17) and HER2 status in the tissue specimens of the tumors of 90 NMIBC patients. Median follow-up was 77 months (range 2–158). Patients with Chr17 polysomy or HER2 gene amplification had a higher rate of disease progression (hazard ratio: 7.44; p < 0.001 and 4.04; p = 0.033, respectively; univariate Cox regression). Multivariable Cox regression models demonstrated that the addition of either Chr17 polysomy or HER2 gene amplification status to the European Association of Urology (EAU) progression risk score increases the c-index (from 0.741/EAU/ to 0.793 and 0.755, respectively), indicating that Chr17 polysomy/HER2 amplification status information improves the accuracy of the EAU risk table in predicting disease progression. HER2/Chr17 in situ hybridization can be used to select non-progressive cases not requiring strict follow-up, by reclassifying non-HER2-amplified, non-polysomic NMIBCs from the high- and very high-risk groups of EAU to the intermediate-risk group.