Centromeric Region Of Chromosome Research Articles

Characteristics of the karyotype of Ukrainian buffalos

The article presents the results of studying the karyotype of the Ukrainian buffalo population. With the help of routine and GTG methods of analysis of metaphase plates of chromosomes, it was established that in the studied animals the diploid chromosome set was equal to fifty chromosomes (2n=50), which consisted of 5 pairs of meta- and submetacentric and 19 pairs of acrocentric autosomes and one pair (XX) or (XY) sex chromosomes. The total number of chromosome arms (FN) was 60, which correspond to animals of the water buffalo species (Bubalus bubalis), a subspecies of the river buffalo (B. b. Bubalis). Cytogenetic control revealed wide intraspecies limits of spontaneous somatic mutagenesis: absence of constitutive chromosomal disorders; the frequency of metaphases with aneuploidy, the average value (M±m) of which was equal to 10.5±0.13%, polyploidy (M±m=0.7±0.25%), asynchronous separation of the centromeric regions of chromosomes - (M±m=5.3±2.00), chromosomal breaks (M±m=0.7±0.24%), the proportion of cells with micronuclei (M±m=2.5±0.39‰), binucleated cells (M±m=2.6±0.32‰) and mitotic index (M±m=4.8±0.65‰). It has been established that Ukrainian river buffaloes are characterized by high karyotype stability.

Gross Chromosomal Rearrangement at Centromeres.

Centromeres play essential roles in the faithful segregation of chromosomes. CENP-A, the centromere-specific histone H3 variant, and heterochromatin characterized by di- or tri-methylation of histone H3 9th lysine (H3K9) are the hallmarks of centromere chromatin. Contrary to the epigenetic marks, DNA sequences underlying the centromere region of chromosomes are not well conserved through evolution. However, centromeres consist of repetitive sequences in many eukaryotes, including animals, plants, and a subset of fungi, including fission yeast. Advances in long-read sequencing techniques have uncovered the complete sequence of human centromeres containing more than thousands of alpha satellite repeats and other types of repetitive sequences. Not only tandem but also inverted repeats are present at a centromere. DNA recombination between centromere repeats can result in gross chromosomal rearrangement (GCR), such as translocation and isochromosome formation. CENP-A chromatin and heterochromatin suppress the centromeric GCR. The key player of homologous recombination, Rad51, safeguards centromere integrity through conservative noncrossover recombination between centromere repeats. In contrast to Rad51-dependent recombination, Rad52-mediated single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) lead to centromeric GCR. This review summarizes recent findings on the role of centromere and recombination proteins in maintaining centromere integrity and discusses how GCR occurs at centromeres.

CYTOGENETIC VARIABILITY OF THE HUTSUL BREED OF HORSES

Goal. Study of features of karyotypic variability of Hutsul horses. Methods. Cytogenetic analysis, micronucleus test. Results. The average rate of genomic disorders (aneuploidy) was 10.8%, with a range of values of this variability of 8.8%–11.1% in animals of the LLC "Krai Neba" and the National Nature Park "Hutsulshchyna" with a statistically insignificant difference between the mean values. The multiple increase in the number of chromosomes, polyploidy, was in the range of 1.1%–2.0%, which does not exceed the species of this variability. The magnitude of the asynchronous divergence of the centromeric regions of the chromosomes was 2.0%–6.0%, which corresponds to the spontaneous level for horses as a whole (2.2%–9.1%). Structural chromosome abnormalities (chromosomal breaks) in the horses of the Krai Neba LLC farm were not detected, and in the animals of the Hutsulshchyna National Nature Park they were 1.1%. The range of variability of lymphocytes with a micronucleus for animals of two farms was 4.0–4.2‰. The number of dinuclear lymphocytes (6.2‰) exceeded the mitotic index (4.3‰) with an insignificant difference in the average values, which indicates the absence of radionuclide contamination of the territory. Conclusions. The average number of aneuploid cells – 10.8%, polyploid – 1.4%, asynchronous divergence of the centromeric regions of chromosomes – 3.9% and the proportion of chromosomal breaks – 1.1%. The proportion of lymphocytes with a micronucleus – 4.0–4.2–4, dinuclear lymphocytes – 6.2‰ and the mitotic index – 4.3‰, which indicates the stability of the karyotype of the studied horses.

The association of centromere amplification and response to trastuzumab in HER2+ metastatic breast cancer.

1016 Background: Chromosomal instability and copy number aberration (CNA) burden can be associated with patients' survival in some cancers. Centromeres, chromosomal regions crucial for genomic stability, can be found altered by CNA in tumours but remain largely unexplored due to sequencing technologies. Using whole-genome sequencing (WGS) data from cases of long term, never relapsed HER2+ metastatic breast cancer (MBC) patients, also referred as exceptional responders (ExRs), we aim to better characterise the CNA profiles and underlying long-term survival of this “exceptional” cohort and investigate the centromeric and pericentromeric association with prognosis and treatment response. Methods: Two hundred and forty-three HER2+ MBC patients were enrolled in the HER2 Patients Project Database from St Vincent University Hospital, Dublin, Ireland. Eighty-five HER2+ MBC patients were identified as exceptional survivors (ExS) with an OS > 60 months (range 60-248 months), of which 28 never-relapsed and responded exceptionally (ExR) to trastuzumab. Patients with an OS < 60 months (N=158, range 0.2-59 months) were identified as non responders (NR). WGS was performed on 13 ExR tumours (primary or metastases) and matching control at a mean depth of 60X and 30X, respectively. Control-FREEC and CNVkit were used to characterise the CNA profiles and estimate the CNA burden. Samples from 10 NR patients were used for comparison with the ExR. Results: WGS analysis revealed that ExR samples are more impacted by gain rather than loss of copy overall, with a median fraction altered by gain of 0.24. No significant difference was observed between the ExR and NR in terms of CNA burden. However, a large fragment on chromosome 9 was amplified in 92% of ExR (12/13) which corresponded to the centromere (chr9q11) and a large heterochromatic block (chr9q12). A higher copy number status of this centromeric region was detected in the ExR, with a gain of at least 1 additional copy compared to the NR tumoral samples (P<0.001). The dichotomisation into high versus low copy number of the centromeric region of chromosome 9 was also observed in centromeres of chromosome 17 (P=0.009) and chromosome 19 (P<0.001). No case of polysomy was detected in our cohort and genes located near the centromeres, such as ERBB2 (amplified in both ExR and NR), were independent of the centromeres copy-number status. Conclusions: The identification of the genomic aberrations of these metastatic patients, treated with trastuzumab who never relapsed, increases our understanding of the mechanisms involved in MBC progression. Our results suggest that the centromere co-amplification of chr9q11-q12, chr17p11.1-q11.1 and chr19p11-q11 stratifies patients according to their OS. CNA status of the centromeric regions of chromosomes 9, 17 and 19 may therefore represent a novel prognostic predictor to trastuzumab response and new outcomes for patients.

Establishment and inheritance of minichromosomes from Arabidopsis haploid induction

Minichromosomes are small, sometimes circular, rearranged chromosomes consisting of one centromere and short chromosomal arms formed by treatments that break DNA, including plant transformation. Minichromosomes have the potential to serve as vectors to quickly move valuable genes across a wide range of germplasm, including into adapted crop varieties. To realize this potential, minichromosomes must be reliably generated, easily manipulated, and stably inherited. Here we show a reliable method for minichromosome formation in haploids resulting from CENH3-mediated genome elimination, a process that generates genome instability and karyotypic novelty specifically on one parental genome. First, we identified 2 out of 260 haploids, each containing a single-copy minichromosome originating from centromeric regions of chromosomes 1 and 3, respectively. The chromosome 1 minichromosome we characterized did not pair at meiosis but displayed consistent transmission over nine selfing generations. Next, we demonstrated that CENH3-based haploid induction can produce minichromosomes in a targeted manner. Haploid inducers carrying a selectable pericentromeric marker were used to isolate additional chromosome-specific minichromosomes, which occurred in 3 out of 163 haploids. Our findings document the formation of heritable, rearranged chromosomes, and we provide a method for convenient minichromosome production.

Cytogenetic and Molecular Characterization of Eigenmannia aff. desantanai (Gymnotiformes: Sternopygidae): A First Report of System of Sex Chromosomes ZW1W2/ZZ in Gymnotiformes.

Gymnotiformes a monophyletic group of fish endemic to the Neotropics, represent an important component of the freshwater ichthyofauna that presents relevant taxonomic problems. Thus, in view of the morphological complexity involving Eigenmannia (Gymnotiformes) fish species, this study aimed to characterize Eigenmannia aff. desantanai of the upper Paraguay River basin through cytogenetic and molecular analyses, to help in the correct identification and delimitation of species. This study reports a multiple sex system of the type ZW1W2/ZZ, with 2n = 31 for females and 2n = 30 for males. A single pair of chromosomes carrying the nucleolar organizing regions (NORs) was detected. The heterochromatin was colocated in NOR sites and mainly located in the centromeric regions of chromosomes. Besides that, individual sequences COI from the specimens of E. aff. desantanai were obtained, totalizing three haplotypes. The distance p between the haplotypes in E. aff. desantanai, ranged from 0.2% to 7.1%. Species delimitation tests indicated the existence of two possible operational taxonomic units of E. aff. desantanai. Thus, this study reports a new multiple sex system in Gymnotiformes and these specimens previously identified as E. aff. desantanai may belong to two distinct species.

KARYOTYPAL VARIABILITY OF UKRAINIAN BROWN DAIRY BREED COWS

KARYOTYPAL VARIABILITY OF UKRAINIAN BROWN DAIRY BREED COWS

Overexpression of HMGB3 and its prognostic value in breast cancer.

High mobility group protein B3 (HMGB3) is abundantly expressed in a number of malignancies, contributing to tumor cell growth and predicting poor outcomes. More research on the connection between HMGB3 and breast cancer is needed. The prognostic significance of HMGB3 in breast cancer was examined and validated in this study. Using The Cancer Genome Atlas (TCGA) database RNA sequencing and clinical data, we investigated the associations between HMGB3 expression and tumor mutations, prognosis, and immune infiltration in breast cancer. The Gene Expression Profiling Interactive Analysis (GEPIA), Tumor Immune Estimation Resource (TIMER), breast cancer gene-expression miner (bc-GenExMiner), UALCAN, OncoLnc, cBio Cancer Genomics Portal (cBioPortal), and LinkedOmics databases were applied to examine the levels of expression, mutation, coexpression, and immune correlation of HMGB3 in breast cancer. cBioPortal and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) were used for coexpression and enrichment analyses, respectively. Experimental tests and a separate cohort of breast cancer patients in our center were used for validation. To determine independent risk factors affecting breast carcinoma prognosis, multivariate Cox regression analysis was performed. The Kaplan-Meier method was applied to analyze the connection between HMGB3 expression and overall survival time in breast cancer. Pan-cancer investigation using the GEPIA and UALCAN databases revealed a high level of HMGB3 expression in different malignancies, including breast cancer. HMGB3 might be a potential diagnostic biomarker, according to the receiver operating characteristic (ROC) curve (AUC=0.932). And immunohistochemistry confirmed higher HMGB3 protein expression in breast cancer tissues in clinical samples. Experimental tests also showed that breast cancer cells have higher expression of HMGB3, and knockdown of HMGB3 can promote the proliferation of breast cancer cells and increase sensitivity to chemotherapy. Human epidermal growth factor receptor 2 (HER2), Nottingham Prognostic Index (NPI), basal-like status, nodal status (N+), triple-negative status, and Scarff-Bloom-Richardson (SBR) grade all showed positive correlations with HMGB3 expression. Conversely, HMGB3 expression was negatively associated with the expression of estrogen receptor (ER) and progesterone receptor (PR) in breast cancer. Breast cancer patients with high HMGB3 expression had poor overall survival, which was validated by an analysis of a separate cohort of breast cancer patients in our center. Cox regression analysis identified high HMGB3 expression as an independently associated risk factor for breast carcinoma. The amount of immunological infiltration was substantially linked with the high expression of HMGB3. The chromosome centromeric region, ATPase activity, and the cell cycle are critical areas where HMGB3 is involved, according to enrichment analysis. Therefore, we suspected that HMGB3 might be a potential biomarker for detecting and treating breast carcinoma. Breast cancer tissues had higher HMGB3 expression than normal breast tissues. HMGB3 overexpression may serve as an indicator for poor breast cancer outcomes.

Хромосомна нестабільність чистопородних і кросбредних корів молочного напряму продуктивності

The article presents the results of research on the karyotype characteristics variability in purebred and crossbred dairy cows. The material for the research was peripheral blood samples of purebred firstborn cows of the Ukrainian red-spotted dairy and Ukrainian black-spotted dairy breeds, as well as crossbred cows obtained from crossing Ukrainian red-spotted with Montbeliard bulls (DG “Nyva” SE of the Institute of Animal Breeding and Genetics named after M. V. Zubets NAAS. Preparation of cytogenetic preparations, analysis of morphology, identification and classification of chromosome aberrations were carried out according to generally accepted methods. We performed cell analysis with the Axiostar plus microscope (Carl Zeiss, Germany) under immersion magnification of 1000 times and took microphotographs. In all studies, the frequency of aberrant metaphases and the spectrum of chromosomal aberrations were determined as parameters of chromosomal instability. The following signs were taken into account: the frequency of aneuploid and polyploid cells, cells with premature separation of the centromeric regions of chromosomes (CRC), cells with structural aberrations of chromosomes (breaks, fragments and associations of non-homologous chromosomes). As a result of the analysis of karyotypes of firstborn cows of purebred and crossbred origin, it was established that the proportion of diploid cells in the norm is on average 85%. The remaining almost 15% are somatic cells with numerical and structural abnormalities. Crossbred cows have significantly higher frequencies (P<0.001) of aneuploid (one and a half times), polyploid (by 27%), structural aberrations of chromosomes (by 20%) than in purebred cows of the Ukrainian red-spotted and black-spotted dairy breeds. 15–20% more cells containing chromosomes with breaks and fragments were also found in crossbred firstborns. The results of the cytogenetic study indicate greater chromosomal instability in crossbred cows compared to purebred cows. One of the reasons for this phenomenon may be the influence of breeding methods, in particular crossbreeding.

Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes

ABSTRACT The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in recent years sparked the development of transgenic technologies with the potential to be used as novel vector control tools. These technologies rely on genome editing that confer traits able to affect vectorial capacity. This can be achieved by either reducing the mosquito population or by making mosquitoes refractory to the parasite infection. For any genetically modified organism that is regarded for release, molecular characterization of the transgene and flanking sites are essential for their safety assessment and post-release monitoring. Despite great advancements, Whole-Genome Sequencing data are still subject to limitations due to the presence of repetitive and unannotated DNA sequences. Faced with this challenge, we describe a number of techniques that were used to identify the genomic location of a transgene in the male bias mosquito strain Ag(PMB)1 considered for potential field application. While the initial inverse PCR identified the most likely insertion site on Chromosome 3 R 36D, reassessment of the data showed a high repetitiveness in those sequences and multiple genomic locations as potential insertion sites of the transgene. Here we used a combination of DNA sequencing analysis and in-situ hybridization to clearly identify the integration of the transgene in a poorly annotated centromeric region of Chromosome 2 R 19D. This study emphasizes the need for accuracy in sequencing data for the genome of organisms of medical importance such as Anopheles mosquitoes and other tools available that can support genomic locations of transgenes.

High Expression Levels of CDK1 and CDC20 in Patients With Lung Squamous Cell Carcinoma are Associated With Worse Prognosis.

Purpose: Progress related to the early detection and molecular targeted therapy of lung squamous cell carcinoma (LUSC) remains limited. The goal of our study was to identify key candidate indicators of LUSC. Methods: Three microarray datasets (GSE33532, GSE30219 and GSE19188) were applied to find differentially expressed genes (DEGs). Functional enrichment analyses of DEGs were carried out, and their protein-protein interaction (PPI) network was established. Hub genes were chosen from the PPI network according to their degree scores. Then, overall survival (OS) analyses of hub genes were carried out using Kaplan-Meier plotter, and their GSEA analyses were performed. Public databases were used to verify the expression patterns of CDK1 and CDC20. Furthermore, basic experiments were performed to verify our findings. Results: A total of 1,366 DEGs were identified, containing 669 downregulated and 697 upregulated DEGs. These DEGs were primarily enriched in cell cycle, chromosome centromeric region and nuclear division. Seventeen hub genes were selected from PPI network. Survival analyses demonstrated that CDK1 and CDC20 were closely associated with OS. GSEA analyses revealed that cell cycle, DNA replication, and mismatch repair were associated with CDK1 expression, while spliceosome, RNA degradation and cell cycle were correlated with CDC20 expression. Based on The Cancer Genome Atlas (TCGA) and The Human Protein Atlas (THPA) databases, CDK1 and CDC20 were upregulated in LUSC at the mRNA and protein levels. Moreover, basic experiments also supported the obvious upregulation of CDK1 and CDC20 in LUSC. Conclusion: Our study suggests and validates that CDK1 and CDC20 are potential therapeutic targets and prognostic biomarkers of LUSC.

Impact of Infrequently Mutated Genes on Prognosis in 531 Untreated CLL Patients

Background: In a previous multicentre study, 55 recurrent aberrations were identified in 538 chronic lymphocytic leukemia (CLL) cases including 278 pre-treatment samples (Landau et al., Nature 2015). Some of these aberrations are frequent and their impact on prognosis in CLL is well analyzed. However limited data exist on the rarely mutated genes.Aim: Characterize 531 untreated CLL patients in terms of infrequently mutated genes and impact on prognosis.Patients and Methods: 531 CLL patients at diagnosis or without prior treatment were screened for gene mutations by a targeted amplicon sequencing approach: ASXL1, ATM, BIRC3, BRAF, BTK, EGR2, FBXW7, KRAS, MAP2K1, MYD88, NRAS, NFKBIE, NOTCH1, PLCG2, POT1, SAMHD1, SF3B1, SRSF2, TP53, U2AF1, XPO1, and ZRSR2 . The mean overall read depth for all genes was 2,496 (range: 305-13,078). IGHV mutational status was analyzed in all cases. IGHV unmutated status (IGHV-U) was defined by sequence identity ≥98%. Chromosome banding analysis and interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 were performed in all cases. Male:female ratio was 1.6:1 and median age was 66 years (range: 30 - 87 years). In all cases data on time to first treatment (TTT) and overall survival (OS) was available with a median follow-up of 6 years, each.Results: Overall 223 (42%) mutated cases comprising 343 mutations were detected in 19/22 analyzed genes. The frequencies of the gene mutations were as follows: NOTCH1 (12%), ATM (9%), SF3B1 (9%), TP53 (7%), NFKBIE (5%), XPO1 (4%), BIRC3 (3%), MYD88 (2%), POT1 (2%), EGR2 (2%), FBXW7 (2%), KRAS (2%), BRAF (1%), NRAS (1%), ASXL1 (<1%), MAP2K1 (<1%), SAMHD1 (<1%), U2AF1 (<1%), and ZRSR2 (<1%). No mutations were detected in SRSF2 and, as expected in untreated CLL cases, also not in BTK and PLCG2 .We restricted the statistical analyses on infrequently mutated genes (≤5%) found in at least 10 mutated cases. Most of the gene mutations were associated with IGHV-U: NFKBIE (10% vs. 2%, p<0.001), POT1 (6% vs. 1%, p<0.001), EGR2 (4% vs. 1%, p=0.005), and XPO1 (11% vs. 1%, p<0.001). MYD88 was found only in IGHV-M (3% vs. 0%, p=0.017) and was associated with del(13q) sole (3% vs. 1%, p=0.049). NOTCH1 mutated vs. wild-type (wt) cases showed significantly higher frequencies of mutations in NFKBIE (11% vs. 4%, p=0.029), XPO1 (12% vs. 3%, p=0.004) and KRAS (8% vs. 1%, p=0.002). Accordingly, following genes were highly mutated in cases with trisomy 12 cases vs. without: NFKBIE (10% vs. 4%, p=0.037), KRAS (9% vs. 1%, p<0.001), BIRC3 (10% vs. 2%, p=0.003), and FBXW7 (10% vs. 1%, p<0.001). In contrast, most of the mutations were rare in patients with del(13q) sole: NFKBIE (2% vs. 7%, p=0.008), FBXW7 (0% vs. 4%, p=0.014), and KRAS (0% vs. 3%, p=0.005). Interestingly, EGR2 mutations were associated with del(11q) (7% vs. 1%, p=0.016), XPO1 with ATM (13% vs. 4%, p=0.011) and NFKBIE mutations (15% vs. 4%, p=0.021), and BIRC3 with FBXW7 mutations (18% vs. 3%, p=0.045).In Kaplan-Meier analyses, the infrequently mutated genes NFKBIE (4 vs. 8 years, p=0.006), POT1 (3 vs. 8 years, p=0.012), EGR2 (1 vs. 8 years, p=0.020), and XPO1 (3 vs. 8 years, p<0.001) showed significant shorter TTT vs. wt cases, but no effect on OS. Whereas BIRC3 showed only impact on OS (5 year-OS: 61% vs. 88%, p=0.002). Cox regression analysis was performed with following covariates: genes with at least 2% mutation frequency, IGHV status, FISH and cytogenetic aberrations. In multivariate analysis for TTT following markers showed significant effect: IGHV-M (RR=relative risk: 0.3, p<0.001), del(11q) (RR: 2.1, p=0.001), trisomy 12 (RR: 2.1, p<0.001) and SF3B1 mutation (RR: 1.9; p=0.001). The rarely mutated genes NFKBIE, POT1, EGR2 and XPO1 that were significant in univariate analysis showed no independent impact. For OS BIRC3 (RR: 4.1, p<0.001) besides IGHV-M (RR: 0.6, p=0.031) and del(17p) (RR: 5.3, P=0.002) retained significant effect. None of the other rarely mutated genes showed statistical significance already in univariate analysis.Conclusion: In 42% of 531 untreated CLL patients, mutations can be found in 19 distinct genes. Patients with the rarely mutated genes NFKBIE, POT1, EGR2 and XPO1 show reduced time to treatment, whereas BIRC3 mutations are associated independently with shorter overall survival. Even though most of the rare gene mutations analyzed here have no strong impact on prognosis, some of them might be interesting for targeted therapy approaches in the near future. DisclosuresJeromin:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clonal Evolution of Molecular and Cytogenetic Aberrations in 80 CLL Patients

Background: In recent years, the knowledge on the genomic landscape of chronic lymphocytic leukemia (CLL) has dramatically increased. However, the clinical course is highly variable and understanding the clonal evolution is just in the beginnings.Aim: To characterize mutational and cytogenetic changes during the course of CLL.Patients and Methods: 80 CLL cases at diagnosis or without prior treatment (time-point A) presenting with persistence of disease at the follow-up evaluation (time-point B) were selected. The time lag was 1-10 years (mean and median: 5 years). All cases were screened at both time-points for following gene mutations by a targeted amplicon sequencing approach: ASXL1, ATM, BIRC3, BRAF, BTK, EGR2, FBXW7, KRAS, MAP2K1, MYD88, NRAS, NFKBIE, NOTCH1, PLCG2, POT1, SAMHD1, SF3B1, SRSF2, TP53, U2AF1, XPO1, and ZRSR2 . The mean overall read depth for all genes was 2,588 (range: 324 - 11,445). Allele frequency of 3% was preset as cut-off for positivity. IGHV mutational status was analyzed in all cases at time-point A. IGHV unmutated status (IGHV-U, sequence identity ≥ 98%) was detected in 55% of cases. Interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 was performed in 65/80 cases at both time-points. Male:female ratio was 1.9:1 and median age was 59 years (range: 38 - 80 years).Results: 46% (37/80) of the patients presented with mutational alterations at follow-up. Overall, 46 changes of the mutation status in 14 genes were present with following frequencies: TP53 30%, NOTCH1 15%, BIRC3 11%, ATM 9%, SF3B1 9%, NRAS 7%, and POT1, BRAF , EGR2, KRAS, MYD88, NFKBIE, XPO1 and ZRSR2 <4%, each.Diverse routes of changes were detected. Nine patients (24%) were unmutated at time-point A and showed acquisition of mutations at time-point B, 4 (11%) presented with no changes, and 1 case (3%) lost the known mutation at follow-up. The remaining cases (62%) showed diverse combinations of acquisition, loss and stability of mutations. Out of the 26 cases with acquired mutations during the disease course the mutations were retrospectively detected in 5 patients (19%) 1-9 years (mean: 4 years) prior to follow-up analysis at an allele frequency of 1-2%.Clonal evolution of gene mutations was associated with the presence of following markers at time-point A: trisomy 12 (77% vs. 40%; p=0.030), IGHV-U (57% vs. 33%, p=0.044), SF3B1 mutations (73% vs. 40%, p=0.024) and begin of therapy before time-point B (60% vs. 25%, p=0.003).In 65 cases, clonal evolution regarding FISH could be analyzed. At a frequency of 26% (17/65) patients showed cytogenetical evolution over a period of 2-9 years after first analysis (mean and median: 5 years). Overall, 21 changes were detected at following frequencies: del(17p) 47%, del(13q) 35%, del(11q) 29%, and trisomy 12 12%. Clonal evolution regarding FISH was associated with the presence of following markers at time-point A: IGHV-U (42% vs. 7%, p=0.002), NOTCH1 mutations (54% vs. 19%, p=0.029) and begin of therapy before time-point B (37% vs. 11%, p=0.024).For the three most frequently evolved mutations TP53, NOTCH1 and BIRC3 single associations were analyzed. Gain of TP53 mutations was associated with clonal evolution in FISH (47% vs. 4%. P<0.001), mainly due to the association with the evolution of del(17p) (88% vs. 5%, p<0.001). Moreover, it was more frequent if therapy was applied before time-point B (27% vs. 3%, p=0.006), in IGHV-U cases (27% vs. 6%, p=0.016), and if SF3B1 mutations at time-point A were present (40% vs. 12%, p=0.020). Interestingly, SF3B1 mutated cases seem to be prone to acquire TP53 mutations especially if patients carry IGHV-U status: 50% (5/10). Clonal evolution of NOTCH1 mutations was associated with clonal evolution in FISH in general (24% vs. 2%, p=0.015) and clonal evolution of BIRC3 mutations was associated with del(11q) at time-point A (23% vs. 3%, p=0.028).Conclusion: 46% of the analyzed cases showed clonal evolution of gene mutations. 62% of these presented with complex pathways suggesting the need for analysis at follow-up not only of already known mutations. Both molecular and cytogenetical evolution were associated with IGHV unmutated status and need for start of therapy. Particularly IGHV unmutated cases with SF3B1 mutations have a high risk to evolve TP53 mutations resulting in therapeutical consequences. DisclosuresJeromin:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mutational Pattern and Clonal Hierarchy in MDS with Monosomy 7

Background: Monosomy 7 (-7) is a typical abnormality in MDS with a frequency of ~ 10%. -7 either occurs as the sole abnormality (~4%), accompanied by one additional abnormality (~1%) or in a complex karyotype (~5%). A detailed molecular analysis of MDS with -7 as the sole abnormality (-7 sole) has not been performed to date.Aims: Evaluation of molecular mutations in MDS with -7 sole and analysis of the clonal hierarchy.Patient cohorts and methods: 100 patients (pts) with MDS at diagnosis harboring -7 sole in chromosome banding analysis (59 male, 41 female) with available cytomorphology and material for mutation screening were selected. Median age was 70 years (range: 21-89 years). 86 pts had de novo and 14 pts therapy-related MDS. Blast count was <5% in 29 MDS and ≥5% in 71 MDS pts. 97 pts were also analyzed by interphase FISH using a probe for the centromeric region of chromosome 7. Targeted sequencing of 29 genes commonly mutated in myeloid malignancies was performed in all 100 pts (Illumina, San Diego, CA). Variants of unknown significance were excluded from statistical analysis.Results: The most frequently mutated genes were: ASXL1 (42%), SETBP1 (31%), U2AF1 (29%), DNTM3A (27%), RUNX1 (17%), ETV6 (14%), EZH2 (14%), TET2 (12%), SRSF2 (10%), CBL (9%), PTPN11 (9%), NF1 (6%), CSF3R (4%), JAK2 (4%), TP53 (3%), KRAS (3%), NRAS (3%), and MPL (3%). Genes mutated in 2% or less of pts were: GATA2, IDH1, IDH2, KIT, CALR, ETNK1, NPM1, SF3B1, and WT1 . No mutations were observed in CSNK1A1 and GATA1 (figure 1).RUNX1 mutations were significantly more frequent in t-MDS as compared to de novo MDS (43% vs 13%, p=0.02), while ASXL1 mutations were more frequent in MDS with blast count <5% (60% vs 41%, p=0.04).A median of 2 mutations per case were observed (range 0-6), with 94 pts harboring at least 1 mutation. No association between age and the number of mutations was observed. However, mutations in ETV6, SETBP1, U2AF1 and PTPN11 were associated with a higher number of mutations, while this was not the case for other gene mutations. In pts with 0-2 mutations ETV6, SETBP1, U2AF1 and PTPN11 were mutated in 4%, 15%, 14%, and 2%, respectively, while the respective mutation frequencies were 27%, 49%, 47%, and 19% in pts with >2 mutations (for all comparisons p<0.01).The DNMT3A mutation frequency was low in ASXL1 mutated pts as compared to ASXL1 wild type (wt) pts (8% vs 42%; p<0.0001), while RUNX1 mutations were more frequent in ASXL1 mutated pts (28% vs 10%, p=0.03). U2AF1 and ETV6 mutations frequently co-occurred as 67% of ETV6 mutated pts also harboured a U2AF1 mutation (p=0.002) while only 21% of ETV6 wt pts showed U2AF1 mutations. In addition, the following mutation combinations were more frequent than expected U2AF1 / ETV6 (p=0.002), RUNX1 / EZH2 (p=0.04), EZH2 / TET2 (p=0.04), SETBP1 / U2AF1 (p=0.01), SETBP1 / PTPN11 (p=0.02), and U2AF1 / TP53 (p=0.02), while the following mutation combinations co-occurred less frequently than expected: EZH2 / SETPB1 (p=0.05), RUNX1 / SETBP1 (p=0.02), and SRSF2 / U2AF1 (p=0.02).For clonal architecture analysis the clone size of cells harboring -7 was related to the mutation loads of the 11 most frequently mutated genes. The proportion of cells harboring -7 determined by FISH analyses varied between 6% and 95% (median: 57%). The -7 clone size was significantly correlated to the mutation loads of DNMT3A (r=0.67, p<0.001), RUNX1 (r=0.65, p=0.003), PTPN11 (r=0.71, p=0.02), and NF1 (r=0.81, p=0.01) indicating that -7 and the respective mutation were present together in the same clone. The mutation loads of the following mutations were significantly correlated: ASXL1 / PTPN11 (r=0.95, p=0.01), ASXL1 / U2AF1 (r=0.58, p=0.04), ETV6 / SETBP1 (r=0.91, p=0.005), and ETV6 / U2AF1 (r=0.87, p=0.03). Estimated based on clone size and mutation load -7 was present in the main clone (MC) in 61% of cases and in the subclone (SC) in 39%. Most mutations occurred in the MC as well as in the SC at comparable frequencies (ASXL1, EZH2, RUNX1, SETBP1, SRSF2). Mutations in ETV6 and PTPN11 were present slightly more frequently in a SC only (64% and 60%), while mutations in CBL, DNMT3A, TET2 and U2AF1 were found rarely in a SC only (33%, 18%, 18% and 17%) (figure 2).Conclusions: A broad spectrum of molecular mutations is present in MDS with -7 sole with the highest mutation frequencies in ASXL1, SETBP1, U2AF1, DNMT3A and RUNX1 . No clear hierarchy of -7 and mutations was identified, however, in most cases if mutated DNMT3A, TET2 and U2AF1 are present in the main clone. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Peculiarities of karyotype of the Ukrainian aboriginal Hutsul breed of horse

Saving biodiversity and assessment of the genetic diversity of local breeds of domestic animals as priority subjects of protection in agrobiocenoses is one of the global challenges facing humanity in the 21st century. Hutsul horse is one of the oldest horse breeds in Ukraine, which according to the gene pool subject of horses in Ukraine belongs to the group “Local (mountain and ponies)” of the I category, which is already on the verge of extinction, and according to the FAO classification it is considered to be a subject of the gene pool threatened with extinction. Since the breeding chromosomal polymorphism of horses is insufficiently studied, it is timely to carry out a cytogenetic analysis of the characteristics of spontaneous mutagenesis in Hutsul horses. Karyotypic variability of Hutsul breed horses was determined using the methods of cytogenetic analysis and micronucleus test. The paper presents the results of cytogenetic analysis and micronucleus test of karyotypic variability of Hutsul breed horses. Asynchronous divergence of centromere regions of chromosomes occurs as a result of premature replication of centromere regions of heterochromatin associated with centromere activity. In the studied Hutsul horses, the magnitude of this variability corresponds to the spontaneous level for horses as a whole (2.2-9.1 %). No structural chromosomal abnormalities (chromosomal breaks) were revealed in Hutsul breed horses at Krai Neba LLC, and in the animals of the Hutsulshchina National Nature Park, the percentage of metaphase plates with chromosomal breaks was low (1.1 %), which indicates the stability of the karyotype of the studied animals. The results of the micronucleus test showed that the proportion of lymphocytes with micronuclei in the animals at the both farms was practically the same - 4.0-4.2. It can be concluded based on the data obtained that the animals under study were in ecologically clean conditions relative to the level of radionuclide contamination and were characterized by karyotype stability and reduced sensitivity to mutagenic factors of various nature.

A placental trisomy 2 detected by NIPT evolved in a fetal small Supernumerary Marker Chromosome (sSMC)

BackgroundNon-invasive prenatal testing (NIPT) is a rapidly developing and widely used method in the prenatal screening. Recently, the widespread use of the NIPT caused a neglecting of the limitations of this technology.Case presentationThe 38-year-old woman underwent amniocentesis because of a high risk of trisomy 2 revealed by the genome-wide Non-Invasive Prenatal Test (NIPT). The invasive prenatal diagnosis revealed the mosaicism for a small supernumerary marker chromosome sSMC derived from chromosome 2. Interphase fluorescence in situ hybridization (FISH) on uncultured amniocytes revealed three signals of centromere 2 in 30% of the cells. GTG-banded metaphases revealed abnormal karyotype (47,XX,+mar[21]/46,XX[19]) and was confirmed by array comparative genomic hybridization (aCGH). Cytogenetic analyses (FISH, aCGH, karyotype) on fetal skin biopsies were performed and confirmed the genomic gain of the centromeric region of chromosome 2. In the placenta, three cell lines were detected: a normal cell line, a cell line with trisomy 2 and a third one with only the sSMC.ConclusionWhole-genome Non-Invasive Prenatal Testing allows not only the identification of common fetal trisomies but also diagnosis of rare chromosomal abnormalities. Especially in such cases, it is extremely important to perform not only NIPT verification on a sample of material other than trophoblast, but also to apply appropriate research methods. Such conduct allows detailed analysis of the detected aberration, thus appropriate clinical validity.

Cell-cycle phospho-regulation of the kinetochore.

The kinetochore is a mega-dalton protein assembly that forms within centromeric regions of chromosomes and directs their segregation during cell division. Here we review cell cycle-mediated phosphorylation events at the kinetochore, with a focus on the budding yeast Saccharomyces cerevisiae and the insight gained from forced associations of kinases and phosphatases. The point centromeres found in the budding yeast S. cerevisiae are one of the simplest such structures found in eukaryotes. The S. cerevisiae kinetochore comprises a single nucleosome, containing a centromere-specific H3 variant Cse4CENP-A, bound to a set of kinetochore proteins that connect to a single microtubule. Despite the simplicity of the budding yeast kinetochore, the proteins are mostly homologous with their mammalian counterparts. In some cases, human proteins can complement their yeast orthologs. Like its mammalian equivalent, the regulation of the budding yeast kinetochore is complex: integrating signals from the cell cycle, checkpoints, error correction, and stress pathways. The regulatory signals from these diverse pathways are integrated at the kinetochore by post-translational modifications, notably phosphorylation and dephosphorylation, to control chromosome segregation. Here we highlight the complex interplay between the activity of the different cell-cycle kinases and phosphatases at the kinetochore, emphasizing how much more we have to understand this essential structure.

CHARACTERIZATION OF HUMAN CHROMOSOMAL CONSTITUTIVE HETEROCHROMATIN

Heterochromatin of centromeric chromosome regions contains late replicating, largely repetitive DNA. It is suggested that heterochromatin participates in chromosome pairing, crossing-over and in chromosome disjunction control (1,3). Centromeric heterochromatin, a variety of heterochromatin, is a tightly packed form of DNA.Centromeric heterochromatin is a constituent in the formation ofactive centromeres in most higher-order organisms; the domain exists on both mitotic and interphase chromosomes. (4,5,6,8) Centromeric heterochromatin is usually formed on alpha satellite DNA in humans; however, there have been cases where centric heterochromatin and centromeres have formed on originally euchromatin domains lacking alpha satellite DNA; this usually happens as a result of a chromosome breakage event and the formed centromere is called a neocentromere.

Abstract P4-09-09: Characteristics of stage 1 to 3 breast cancer patients with amplification of centromere of chromosome 17

Abstract Background: Amplification of the centromeric region of chromosome 17 (CEP17) as measured by In Situ Hybridization (ISH) of the CEP17 probe is used clinically as part of the ISH assay for HER2 status determination in breast cancer. The value of amplification of CEP17 beyond its use in the HER2 ISH test and characteristics of cases bearing CEP17 amplification, have not been fully explored. Methods: A retrospective review of charts of patients with stage 1 to 3 breast cancer that had a dual probe HER2/ CEP17 FISH test in our cancer center during an eight-year period was performed. Data on demographic, and cancer-specific characteristics of the included patients were extracted. The group of patients with an amplified CEP17 defined as mean copy number ≥3 per nucleus was compared with the group without amplification. Results: Two hundred and two patients were included in the analysis. Amplification of CEP17 was observed in 36 patients (17.8%). All patients in the amplified group had a concomitant amplification of HER2 (mean copy number ≥3 per nucleus). In the CEP17 non-amplified group 78 of 166 patients (47%) had an amplified HER2 status. More patients in the amplified group had a clinical HER2+ status according to the 3-protein classifier (33.3% versus 11.5% in the non-amplified group) and less patients in the amplified group had a clinical ER+/ HER2- status (63.9% versus 81.8% in the non-amplified group, x2 p=0.0003). Other significant differences between the amplified and CEP17 non-amplified groups were observed in their lymph node (LN) status (55.6% of patients in the amplified group versus 36.8% in the non-amplified group were lymph node positive, p= 0.04) and in the nuclear heterogeneity component of grade (90.9% of patients in the amplified group were nuclear grade 3 versus 66.9% in the non-amplified group, p= 0.02). There were no statistically significant differences between the groups in overall stage, grade, menopause status or histology. Conclusion: Patients with amplification of CEP17 had a co-amplified HER2 and were more commonly HER2+, LN positive and grade 3 in the nuclear component of grade. Citation Format: Ioannis Voutsadakis, Vanessa Davies. Characteristics of stage 1 to 3 breast cancer patients with amplification of centromere of chromosome 17 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-09.

Amplification of Chromosome 17 Centromere (CEP17) in Breast Cancer Patients with a Result of HER2 2± by Immunohistochemistry

Background: Amplification of the centromeric region of chromosome 17 (CEP17) as measured by In Situ Hybridization (ISH) of the CEP17 probe is used clinically as part of the ISH assay for HER2 status determination in breast cancer. The value of amplification of CEP17 beyond its use in the HER2 ISH test has not been fully explored.Methods: A retrospective review of patients with breast cancer that had a dual probe HER2/CEP17 FISH test during an eight-year period was performed. Data on demographic and cancer-specific characteristics of the included patients were extracted. The group of patients with an amplified CEP17 defined as mean copy number of ≥3 per nucleus was compared with the group without amplification.Results: Two hundred and twelve patients were eligible and included in the analysis. Amplification of CEP17 was observed in 39 patients (18.4%). All patients in the amplified group had a concomitant amplification of HER2 (mean copy number ≥3 per nucleus). In the CEP17 non-amplified group 82 of 172 patients (47.7%) had an amplified HER2 status. More patients in the amplified group had a clinical HER2+ status according to the 3-protein classifier (30.8% versus 12.3% in the non-amplified group) and fewer patients in the amplified group had a clinical ER+/HER2- status (66.7% versus 81.3% in the non-amplified group, x2 p = .01). Other significant differences between the amplified and CEP17 non-amplified groups were observed in their lymph node (LN) status (56.4% of patients in the amplified group versus 38.8% in the non-amplified group were lymph node positive, p = .04) and in the nuclear heterogeneity component of grade (91.2% of patients in the amplified group were nuclear grade 3 versus 67.1% in the non-amplified group, p = .005). There were no statistically significant differences between the groups in overall stage, grade, menopause status or histology. Recurrence Free Survival (RFS) was shorter in stage I to III patients with an amplified CEP17 compared with the non-amplified group.Conclusion: Patients with amplification of CEP17 had a co-amplified HER2 and were more commonly HER2+, LN positive and grade 3 in the nuclear component of grade.