Centromeric Region Of Chromosome Research Articles

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.

FEATURES OF THE KARYOTYPE OF WHITE-HEADED COWS OF UKRAINIAN BREED

The White-Headed Ukrainian breed of cattle belongs to breeds of a dairy direction of productivity and belongs to small disappearing breeds. According to calculations, in accordance with the FAO recommendations, the White-Headed Ukrainian breed of cattle is at risk. The aim of the work was to establish the karyotype variability of White-Headed Ukrainian cows, whose animals belong to closed gene pool herds. Cytogenetic monitoring showed that the quantitative chromosome abnormalities, in particular aneuploidy, in White-Headed Ukrainian cows of two groups according to the purpose of the cow (selection nucleus and production group) did not exceed the spontaneous level of this variability (1.5-8.3%) and was 6.0% and 7.0% respectively. A multiple increase in the number of chromosomes (polyploidy) was not detected. The average value of metaphase plates with asynchronous differences in the centromeric regions of chromosomes was 1.0% and corresponded to the level of this variability characteristic of the species Bos taurus. The frequency of structural chromosome abnormalities (chromosomal breaks) in animals of the two groups was 2.3% and 2.0%, respectively, which corresponds to the norm characteristic of cattle with spontaneous mutagenesis. The results of the micronucleus test showed that the proportion of lymphocytes with micronuclei in animals of the two groups was in the range of 4.8 - 5.0%, dinuclear lymphocytes 5.6-6.4 %, and the proportion of dividing cells (mitotic index) was 2.8-3.8 %. The frequency of cells with aneuploidy in cows of the Ukrainian Black-Spotted dairy breed of this farm was established higher than the spontaneous level. The difference between the mean values on this basis in cows of White-Headed Ukrainian and Ukrainian Black-Spotted dairy breeds was statistically significant at P> 0.99. In cows of the Ukrainian Black-Spotted dairy breed under conditions of intensive use, an increase in the frequency of metaphases with aneuploidy was observed, which was not manifested in cows of the White-Headed Ukrainian breed.

CENP-A nucleosome clusters form rosette-like structures around HJURP during G1

CENP-A is an essential histone H3 variant that epigenetically marks the centromeric region of chromosomes. Here we show that CENP-A nucleosomes form characteristic clusters during the G1 phase of the cell cycle. 2D and 3D super-resolution microscopy and segmentation analysis reveal that these clusters encompass a globular rosette-like structure, which evolves into a more compact structure in late G1. The rosette-like clusters contain numerous CENP-A molecules and form a large cellular structure of ∼250–300 nm diameter with remarkably similar shapes for each centromere. Co-localization analysis shows that HJURP, the CENP-A chaperone, is located in the center of the rosette and serves as a nucleation point. The discovery of an HJURP-mediated CENP-A nucleation in human cells and its structural description provide important insights into the mechanism of CENP-A deposition and the organization of CENP-A chromatin in the centromeric region.

The effect of an alternative chromosome 17 probe on fluorescence in situ hybridization for the assessment of HER2 amplification in invasive breast cancer.

Fluorescent in situ hybridization (FISH) is commonly used to determine the ratio of human epidermal growth factor receptor 2 (HER2) to centromere enumeration probe for chromosome 17 (CEP17), which further determines HER2 gene status in breast cancer. However, due to copy number alteration in CEP17, inaccurate diagnoses can occur. The current study was performed to investigate the diagnostic value of an alternative CEP17 reference probe for HER2 status in invasive breast cancer. A higher-order repeat in the centromeric region of chromosome 17 was identified and an alternative probe (SCEP17) was subsequently prepared. Karyotype analysis of peripheral blood was used to detect SCEP17 probe specificity. Using a HER2/CEP17 probe, karyotype analysis revealed two strong green signals at the centromere of chromosome 17 and one weaker signal at the other centromere. However, two strong hybridization signals at the centromere of chromosome 17 were observed when the HER2/SCEP17 probe was used. In the 425 patients with invasive breast cancer, no statistical difference was observed between HER2/SCEP17 and HER2/CEP17 when detecting HER2 gene amplification (P=0.157). However, in terms of copy number, the SCEP17 probe exhibited a reduced number compared with the conventional CEP17 probe (P<0.001). In conclusion, the HER2/SCEP17 probe may lead to increased accuracy HER2 status assessment in invasive breast cancer. However, a further large-scale and prospective clinical trial is required for confirmation of the potential benefits of using the HER2/SCEP17 probe.

Transcriptional silencing of centromere repeats by heterochromatin safeguards chromosome integrity.

The centromere region of chromosomes consists of repetitive DNA sequences, and is, therefore, one of the fragile sites of chromosomes in many eukaryotes. In the core region, the histone H3 variant CENP-A forms centromere-specific nucleosomes that are required for kinetochore formation. In the pericentromeric region, histone H3 is methylated at lysine 9 (H3K9) and heterochromatin is formed. The transcription of pericentromeric repeats by RNA polymerase II is strictly repressed by heterochromatin. However, the role of the transcriptional silencing of the pericentromeric repeats remains largely unclear. Here, we focus on the chromosomal rearrangements that occur at the repetitive centromeres, and highlight our recent studies showing that transcriptional silencing by heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres in fission yeast. Inactivation of the Clr4 methyltransferase, which is essential for the H3K9 methylation, increased GCRs with breakpoints located in centromeric repeats. However, mutations in RNA polymerase II or the transcription factor Tfs1/TFIIS, which promotes restart of RNA polymerase II following its backtracking, reduced the GCRs that occur in the absence of Clr4, demonstrating that heterochromatin suppresses GCRs by repressing the Tfs1-dependent transcription. We also discuss how the transcriptional restart gives rise to chromosomal rearrangements at centromeres.

Chromosome 17 centromere amplification and chromosomal instability (CIN) in breast cancer: Pathogenic and therapeutic implications.

Chromosomal instability (CIN) is present in variable degrees in a significant percentage (up to 90%) of cancers and often portends adverse outcomes. However, it has not been incorporated in clinical practice as a prognostic marker due to the lack of standardization and proof of clinical utility of assays to measure it, as well as uncertainties regarding optimal cut-offs. Amplification of the centromeric region of chromosome 17 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 in cases with intermediate (2+) result of HER2 protein expression by immunohistochemistry. CEP17 amplification concerns the centromeric area and rarely extends beyond it to involve polysomy of the whole chromosome. The association of CEP17 amplification with generalized CIN remains uncertain. Such association, if confirmed, could be an opportunity for a practical and clinically validated test of CIN in breast cancer. This paper explores the association of CIN with centromere 17 amplification and with centromere function in general, as well as the pathophysiology of centromeres/kinetochore function during mitosis that underlies their relationship with CIN in cancer and in breast cancer in particular.

Cytogenetic studies in three octopods, Octopusminor, Amphioctopusfangsiao, and Cistopuschinensis from the coast of China.

To provide markers to identify chromosomes in the genome of octopods, chromosomes of three octopus species were subjected to NOR/C-banding. In addition, we examined their genome size (C value) to submit it to the Animal Genome Size Database. Silver staining revealed that the number of Ag-nucleoli was 2 (Octopusminor (Sasaki, 1920)), 2 (Amphioctopusfangsiao (d’Orbigny, 1839)) and 1 (Cistopuschinensis Zheng et al., 2012), respectively, and the number of Ag-nucleoli visible was the same as that of Ag-NORs on metaphase plates in the same species. In all analyzed metaphases, Ag-NORs were mainly located terminally on the long arms of chromosomes 3 (3rd) of O.minor and on the short arms of chromosomes 4 (4th) of A.fangsiao, whereas only one of the chromosomes 23 (23rd) was found Ag-NORs of C.chinensis. C-bands were localized predominantly in the centromeric regions of chromosomes in the three species, while other conspicuous stable C-bands were observed in terminal regions, including the Ag-NORs. That means these three chromosome pairs (3rd, 4th and 23rd) could be considered species-specific cytogenetic markers. The mean C values of O.minor, A.fangsiao and C.chinensis were 7.81±0.39 pg (0.070 pg per unit length), 8.31±0.18 pg (0.068 pg per unit length) and 5.29±0.10 pg (0.038 pg per unit length), respectively, and results showed that C values of the three species were not proportional to the relative length of the chromosomes. These cytogenetic characteristics will provide more theoretical foundation for further researches on chromosome evolution in octopods.

Genome-wide association study provides insights into genes related with horn development in Nelore beef cattle

The causal mutation for polledness in Nelore (Bos taurus indicus) breed seems to have appeared first in Brazil in 1957. The expression of the polled trait is known to be ruled by a few groups of alleles in taurine breeds; however, the genetic basis of this trait in indicine cattle is still unclear. The aim of this study was to identify genomic regions associated with the hornless trait in a commercial Nelore population. A total of 107,294 animals had phenotypes recorded and 2,238 were genotyped/imputed for 777k SNP. The weighted single-step approach for genome-wide association study (WssGWAS) was used to estimate the SNP effects and variances accounted for by 1 Mb sliding SNP windows. A centromeric region of chromosome 1 with 3.11 Mb size (BTA1: 878,631–3,987,104 bp) was found to be associated with hornless in the studied population. A total of 28 protein-coding genes are mapped in this region, including the taurine Polled locus and the IFNAR1, IFNAR2, IFNGR2, KRTAP11-1, MIS18A, OLIG1, OLIG2, and SOD1 genes, which expression can be related to the horn formation as described in literature. The functional enrichment analysis by DAVID tool revealed cytokine-cytokine receptor interaction, JAK-STAT signaling, natural killer cell mediated cytotoxicity, and osteoclast differentiation pathways as significant (P < 0.05). In addition, a runs of homozygosity (ROH) analysis identified a ROH island in polled animals with 2.47 Mb inside the region identified by WssGWAS. Polledness in Nelore cattle is associated with one region in the genome with 3.1 Mb size in chromosome 1. Several genes are harbored in this region, and they may act together in the determination of the polled/horned phenotype. Fine mapping the locus responsible for polled trait in Nelore breed and the identification of the molecular mechanisms regulating the horn growth deserve further investigation.

Partition of Repeat-Induced Point Mutations Reveals Structural Aspects of Homologous DNA-DNA Pairing

In some fungi, a premeiotic process known as repeat-induced point mutation (RIP) can accurately identify and mutate nearly all gene-sized DNA repeats present in the haploid germline nuclei. Studies in Neurospora crassa have suggested that RIP detects sequence homology directly between intact DNA double helices, without strand separation and without the participation of RecA-like proteins. Those studies used the aggregated number of RIP mutations as a simple quantitative measure of RIP activity. Additional structural information about homologous DNA-DNA pairing during RIP can be extracted by analyzing spatial distributions of RIP mutations converted into profiles of partitioned RIP propensity (PRP). Further analysis shows that PRP is strongly affected by the topological configuration and the relative positioning of the participating DNA segments. Most notably, pairs of closely positioned repeats produce very distinct PRP profiles depending on whether these repeats are present in the direct or the inverted orientation. Such an effect can be attributed to a topology-dependent redistribution of the supercoiling stress created by the predicted limited untwisting of the DNA segments during pairing. This and other results raise a possibility that such pairing-induced fluctuations in DNA supercoiling can modulate the overall structure and properties of repetitive DNA. Such effects can be particularly strong in the context of long tandem-repeat arrays that are typically present in the pericentromeric and centromeric regions of chromosomes in many species of plants, fungi, and animals, including humans.

A study of constitutive heterochromatin and NOR banding in three species of Indian major carps from the State of Haryana, India

Cytogenetic survey of fishes becomes increasingly important to establish chromosomal relation between the teleosts, to have a glimpse of the relation between chromosomal evolution and differentiation of vertebrate species. In the present study, Chromosome banding studies was done in three species of carps i.e. Catla catla (Hamilton, 1822), Labeo rohita (Hamilton, 1822) and Cirrhinus mrigala (Hamilton, 1822). Diploid chromosome number 50 was observed in all 3 species of carps. The chromosomes of C. catla, L. rohita and C. mrigala showed constitutive heterochromatin at telomeric and centromeric regions of chromosomes. The Ag-NOR (Argyrophilic-Nucleolus Organizer Region) bands were observed on homologous chromosome pair number 11 in C. catla, 15th chromosome pair in L. rohita. In C. mrigala, the Ag- NOR staining elucidated the presence of darkly stained NORs on the terminal region of the long arms of one of the chromosome. Another homologue of this chromosome pair could not be localized due to scattering of chromosomes. The results depict that variation in ecological conditions with time due to human activities can not only affect the chromosomal number but also chromosomal morphology.&#x0D;

Molecular cytogenetic of the Amoy croaker, Argyrosomus amoyensis (Teleostei, Sciaenidae)

The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fluorescence in situ hybridization (FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18S and 5S rDNA probes, and a self-genomic in situ hybridization procedure (Self-GISH). The karyotype of A. amoyensis comprised 2n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions (NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation (AgNO3) staining and denaturation-propidium iodide (DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole (DAPI) staining, and was confirmed by FISH with 18S rDNA probes. The 5S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with different intensities, but internal telomeric sequences (ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specific chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18S rDNA are conserved, the distribution of 5S rDNA varies dynamically among sciaenid species. Thus, the 5S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be effective cytotaxonomic markers in Sciaenidae.

Performance of the UroVysion® FISH assay for the diagnosis of malignant effusions using two cutoff strategies.

The cytological examination of cavity fluids has limited sensitivity in the diagnosis of malignancy. Aneuploidy, which is commonly observed in neoplastic cells, could potentially be used as an ancillary diagnostic tool. To evaluate the detection of aneuploid cells in cavitary effusion samples using the fluorescence in situ hybridization (FISH) assay UroVysion ® with some adaptations and two different cutoff strategies. Seventy samples of pleural or peritoneal fluid with positive (n = 40), negative (n = 15), or suspicious (n = 15) oncotic cytology were subjected to FISH assay with the multitarget UroVysion ® kit, which is composed of probes that hybridize to the centromeric region of chromosomes 3, 7, and 17 and to the locus 9p21. FISH performance was evaluated using two different cutoffs: (1) the manufacturer's cutoff (M‐FISH) and 2) a proposed cutoff (P‐FISH). Using M‐FISH, the diagnostic sensitivity was 57.1%, specificity 87.5%, and accuracy 60.0%; with P‐FISH, the sensitivity was 87.3%, specificity 71.4%, and accuracy 85.7%. When combined with cytology, the sensitivity, specificity, and accuracy were 88.0%, 83.3%, and 87.8%, respectively. Malignant cells presented a predominance of chromosomal gains. The UroVysion ® test using the P‐FISH cutoff was effective in demonstrating aneuploid cells in all malignant effusions, confirming the diagnosis of malignancy even in cases with suspicious cytology.

Analysis of Chromosome 3, 7 and 8 Centromeric Regions in Bronchial Lavage Specimens by FISH.

Multiple genetic changes are observed in malignant tumors but are rare or absent in benign conditions. Aneuploidy is the most common feature of solid tumors including lung cancer and diagnosis of malignant tumors is possible through detection of aneuploidy. The aim of this study was to investigate chromosomal abnormalities in cells from non-small cell lung cancer patients obtained bronchoscopically and to evaluate the suitability of fluorescence in situ hybridization (FISH). Bronchial lavage samples of 17 non-small cell lung cancer (NSCLC) patients were evaluated with four-color FISH using deoxyribonucleic acid (DNA) probes specific for the centromere regions of chromosomes 3, 7 and 8. tested specimens were first hybridized with probes, then visualized under fluorescence microscobe and captured with device's camera. High number of aneuploidic cells were detected in all the samples. Increased or decreased abnormal copies or chromosomes 3, 7 and 8 were obserced in all the 17 patients. Aneuploidy of chromosome 3 (21.35%) was higher than those of chromosome 7 (9.06%) and chromosome 8 (15.47%). Moreover, our results were significant for monosomy and trisomy of chromosome 3, trisomy of chromosome 7, nullisomy, monosomy and trisomy of, chromosome 8 (p< 0.05). It has been observed that FISH is a useful technique for detection of aneuploidy in bronchial lavage samples obtained by bronchoscopy. Interphase cells were evaluated without cell culturing with this method and high number of tumor cells were enumerated rapidly. Our study has demonstrated that, FISH technique may be used successfully in detection of chromosome number abnormalities in NSCLC patients and may facilitate evaluation of genetic abnormalities.

The presence of PD-1 positive tumor infiltrating lymphocytes in triple negative breast cancers is associated with a favorable outcome of disease.

Triple negative breast cancer patients have a poor course of disease not least because of limited treatment options however immunotherapy by targeting the PD-1/PD-L1 checkpoint system is a promising strategy to improve the outcome. Here we systematically investigated the expression of PD-1 on tumor infiltrating lymphocytes and PD-L1 on both tumor and infiltrated immune cells. Moreover, the PD-L1 gene status in tumor cells was assessed.103 tissue microarray samples derived from triple negative breast cancer specimens were immunohistochemically stained against PD-1 and PD-L1. Dual marker fluorescence in-situ hybridization was applied to the PD-L1 gene and centromere region of chromosome 9. The disease free and overall survival rates were determined as a function of the PD-1/PD-L1 status.A slight gain of the PD-L1 gene region was found in 55% of all samples but an elevated PD-L1/cen9 ratio was rather rare (7%). An increased gene dose is not associated with an enhanced protein expression and the PD-L1 expression only weakly correlates with the amount of immune cell infiltration. Instead, we found an association of PD-L1 expression on tumor and immune cells, respectively. Notably, the PD-1 expression on immune cells is associated with a favorable disease free and overall survival. PD-1 expression indicates an enhanced immunological anti-tumor activity and represents a favorable prognostic impact. A deeper understanding of factors that affect the regulation and function of the PD-1/PD-L1 system is required to establish predictive variables and to utilize the system for therapeutic intervention of triple negative breast cancer patients.

X-derived marker chromosome in patient with mosaic Turner syndrome and Dandy-Walker syndrome: a case report

BackgroundSmall supernumerary marker chromosomes can be derived from autosomes and sex chromosomes and can accompany chromosome pathologies, such as Turner syndrome.Case presentationHere, we present a case report of a patient with mosaic Turner syndrome and Dandy-Walker syndrome carrying a marker chromosome. We showed the presence of the marker chromosome in 33.8% of blood cells. FISH of the probe derived from the marker chromosome by microdissection revealed that it originated from the centromeric region of chromosome X. Additionally, we showed no telomeric sequences and no XIST sequence in the marker chromosome. This is the first report of these two syndromes accompanied by the presence of a marker chromosome.ConclusionMarker chromosome was X-derived and originated from centromeric region. Patient has mild symptoms but there is no XIST gene in marker chromosome.Trial registrationCPG137. Registered 03 March 2017.

Interplay between the phosphatase PHLPP1 and E3 ligase RNF41 stimulates proper kinetochore assembly via the outer-kinetochore protein SGT1

Kinetochores link chromosomes to spindle microtubules and are essential for accurate chromosome segregation during cell division. Kinetochores assemble at the centromeric region of chromosomes as a multiprotein complex. However, the molecular mechanisms of kinetochore assembly have not yet been fully elucidated. In this study, we identified pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) as a regulatory phosphatase that facilitates proper kinetochore assembly. We found that PHLPP1 interacted with the essential outer-kinetochore protein SGT1 and stabilized its protein levels. Loss of PHLPP1 from cells led to SGT1 degradation and thereby caused defective kinetochore assembly. We also found that the ring finger protein 41 (RNF41) as an E3 ligase ubiquitinated and degraded SGT1 in a phosphorylation-dependent manner. PHLPP1 dephosphorylated SGT1 at four conserved residues (Ser-17, Ser-249, Ser-289, and Thr-233) and thereby prevented SGT1 from associating with RNF41, in turn, countering SGT1 degradation. Importantly, depletion of RNF41 or expression of a non-phosphorylatable SGT1 mutant rescued the kinetochore defects caused by the loss of PHLPP1. Taken together, our results suggest that PHLPP1 plays an important role in the assembly of kinetochores by counteracting RNF41-mediated SGT1 degradation.

Immunodeficiency, centromeric heterochromatin instability of chromosomes 1, 9, and 16, and facial anomalies: the ICF syndrome

Instability of the heterochromatic centromeric regions of chromosomes 1 associated with immunodeficiency was found in a 3 and half months old girl. The case was referred to Department of Genetics, Global Reference Laboratory, Metropolis Healthcare Ltd, Mumbai with the suspicion of Downs Syndrome for chromosomal karyotyping. This patient had facial anomalies in addition to combined immunodeficiency and chromosomal instability. Stretching of the heterochromatic centromeric regions of chromosomes 1 and homologous and non-homologous associations of these regions were the most common cytogenetic findings in this patient. Multi-branched configurations and whole arm deletions of chromosomes 1 were also found. Comparing clinical and chromosomal data we conclude that the patient was suffering from immunodeficiency, centromeric heterochromatin instability and facial syndrome. The chromosomal karyotyping report was showing instability around vicinity of chromosome 1 and various abnormalities around vicinity of both chromosomes 1 were found in form of random breakages of chromosome 1, fragile sites, deletions/duplications of small and long arm, extra copies of chromosome 1 with rosette formations, exchange of arms and partial aneuploidies of chromosome 1. Further, the investigations regarding the immune status revealed that the level of IgM (5.98 mg/dl), IgA (&lt;6.16mg/dl) and IgG (92.10 mg/dl) subgroup of immunoglobulin was very low. The results were consistent with The Immunodeficiency, Centromeric region instability, Facial anomalies (ICF) syndrome. Second sample from the patient for molecular studies could not be collected and performed since the patient failed to survive after 3 and half months.

Satellite alpha transcripts as a biomarker to predict the development of breast cancer with bilateral or multiple primary malignancies.

e23202 Background: The impaired stability of chromosomal structure promotes the abnormal segregation of chromosomes, increasing the risk of carcinogenesis. Chromosomal stability during segregation is managed by appropriate methylation in the centromeric region of chromosomes. Satellite alpha transcripts (SatA) is a non-coding RNA transcribed from the centromere, which could be involved in this machinery. We reported that overexpression of SatA led to chromosomal instability, which was observed in not only cancer regions (CR) but also noncancerous regions (NCR). It was enhanced in NCR of patients with multiple primary malignancies (MPM), suggesting the involvement of SatA in genomic damage in regions where multiple cancers may arise. In this study, we determined whether SatA is involved in breast cancer development with bilateral or MPM. Methods: We constructed lentiviral vectors expressing SatA and control viruses, which were used to infect human mammary epithelial cells. SatA levels were determined by real-time quantitative PCR (qRT-PCR) and copy number alterations were evaluated by array comparative genomic hybridization (CGH). Then, we investigated the levels of SatA in clinical specimens in 135 breast cancer (BC) patients including 128 tumors and 83 matched normal tissues. Results: qRT-PCR showed higher expression levels of SatA in cells infected with SatA virus than in the control. Array CGH analysis indicated frequent copy number alterations at several specific chromosomes in infected cells, despite no alterations in the control. Among the 135 patients, 24 had bilateral breast cancer (BBC) and 18 had MPM. SatA was much higher in CR in BBC and in MPM than in SBC. SatA was also higher in NCR in MPM than without MPM. Considering human epidermal growth factor receptor type2 (HER2), hormone receptor (HR), family history and other factors, the levels of SatA in CR were a significant independent factor predicting the development of BBC patients. Conclusions: Chromosomal instability induced by SatA is involved in the development of BBC and BC with MPM.

Overexpression of satellite alpha transcript in breast cancer patients to impair specific chromosomes.

e23216 Background: Impairment of stability in chromosomal structure facilitates abnormal segregation of chromosomes, increasing the risk of carcinogenesis. Chromosomal stability during segregation is managed by appropriate methylation in the centromere region of chromosomes, which is associated with heterochromatin structures. Insufficient methylation, or demethylation, results in chromosomal instability. Satellite alpha transcript (Sat-A) is non-coding RNA transcribed from satellite alpha repetitive sequences at centromere, which is overexpressed by demethylated status of centromere. We have reported that increased levels of Sat-A were observed in patients with colorectal cancer, resulting in chromosomal instability. Difference levels of demethylation were reported in breast cancer patients but their association with Sat-A expression and chromosomal instability are not well known. In this study, we determined if Sat-A was involved in the chromosomal instability of breast cancer. Methods: We constructed lentiviral vectors expressing Sat-A and control viruses, which were infected in human mammary epithelial cells. The expression levels of Sat-A were determined by qRT-PCR and copy number alterations were evaluated by array CGH. Demethylation levels of satellite alpha sequences were determined by Methylight PCR. Then, these results were verified by using clinical specimens of 45 breast cancer patients. Results: Array CGH analysis revealed that copy number alterations were frequently seen at specific chromosomes of 8q and 20q in infected cells, despite no alteration in control, which was consistent with results in the clinical specimens. Patients with high levels of Sat-A expression showed more alterations than patients with low. Regression analysis exhibited that levels of Sat-A expression were significantly collated with demethylation levels of satellite alpha sequences. Conclusions: Overexpression of Sat-A impaired stability of chromosome 8q and 20q in human mammary epithelial cells, which was also recognized in clinical specimens of breast cancer patients. Chromosomal instability induced by Sat-A is involved in the development of breast cancer.

The Hypermethylated Regions in Avian Chromosomes

Chromosomal locations and amounts of 5-methylcytosine-rich chromosome regions were detected in the karyotypes of 13 bird species by indirect immunofluorescence using a monoclonal anti-5-methylcytosine antibody. These species belong to 7 orders and 10 families of modern (Neognathae) and primitive (Palaeognathae) birds and are characterized by macro- and microchromosomes as well as ZW sex chromosomes. In all 13 species, the hypermethylated chromosome segments are confined to constitutive heterochromatin. The chromosomal locations of hypermethylated DNA regions in the karyotypes are constant and species-specific. There is no general rule with regard to the distribution of these hypermethylated chromosome regions in the genomes of birds. In most instances, hypermethylated segments are located in the centromeric regions of chromosomes, but in the sex chromosomes, these can also be found in telomeric and interstitial postitions. In most of the species studied, the centromeric heterochromatin in many, if not all, of the microchromosomes is hypermethylated. However, in one species, the only detectable hypermethylated heterochromatic regions are located in one pair of macroautosomes and in the Z sex chromosome, but none of the microchromosomes contains visible quantities of 5-methylcytosine. The analysis of 5-methylcytosine-rich chromosome regions can be very helpful for the comparative cytogenetics of closely related species or subspecies. It also reflects the dynamic evolutionary process operating in the highly repetitive DNA of eukaryotic chromosomes.