Pericentromeric Region Of Chromosome Research Articles

The Number of Meiotic Double-Strand Breaks Influences Crossover Distribution in Arabidopsis.

Meiotic recombination generates genetic diversity and ensures proper chromosome segregation. Recombination is initiated by the programmed formation of double-strand breaks (DSBs) in chromosomal DNA by DNA Topoisomerase VI-A Subunit (SPO11), a topoisomerase-like enzyme. Repair of some DSBs leads to the formation of crossovers (COs). In most organisms, including plants, the number of DSBs greatly exceeds the number of COs and which DSBs become CO sites is tightly controlled. The CO landscape is affected by DNA sequence and epigenome features of chromosomes as well as by global mechanisms controlling recombination dynamics. The latter are poorly understood and their effects on CO distribution are not well elucidated. To study how recombination dynamics affects CO distribution, we engineered Arabidopsis thaliana plants to carry hypomorphic alleles of SPO11-1 Two independent transgenic lines showed ∼30% and 40% reductions in DSB numbers, which were commensurate with the dosage of the SPO11-1 transcript. The reduction in DSB number resulted in proportional, although smaller, reductions of the number of COs. Most interestingly, CO distribution along the chromosomes was dramatically altered, with substantially fewer COs forming in pericentromeric chromosome regions. These results indicate that SPO11 activity, and the resulting DSB numbers are major factors shaping the CO landscape.

Satellite RNA Increases DNA Damage and Accelerates Tumor Formation in Mouse Models of Pancreatic Cancer.

Highly repetitive tandem arrays such as satellite sequences in the centromeric and pericentromeric regions of chromosomes, which were previously considered to be silent, are actively transcribed in various biological processes, including cancers. In the pancreas, this aberrant expression occurs even in Kras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To determine the biological role of satellite RNAs in carcinogenesis in vivo, we constructed mouse major satellite (MajSAT) RNA-expressing transgenic mice. However, these transgenic mice did not show spontaneous malignant tumor formation under normal breeding. Importantly, however, DNA damage was increased in pancreatic tissues induced by caerulein treatment or high-fat diet, which may be due to impaired nuclear localization of Y-Box Binding Protein 1 (YBX1), a component of the DNA damage repair machinery. In addition, when crossed with pancreas-specific Kras-mutant mice, MajSAT RNA expression resulted in an earlier increase in PanIN formation. These results suggest that aberrant MajSAT RNA expression accelerates oncogenesis by increasing the probability of a second driver mutation, thus accelerating cells to exit from the breakthrough phase to the expansion phase.Implications: Aberrant expression of satellite RNAs accelerates oncogenesis through a mechanism involving increased DNA damage. Mol Cancer Res; 16(8); 1255-62. ©2018 AACR.

The genetic architecture of genome-wide recombination rate variation in allopolyploid wheat revealed by nested association mapping.

SummaryRecombination affects the fate of alleles in populations by imposing constraints on the reshuffling of genetic information. Understanding the genetic basis of these constraints is critical for manipulating the recombination process to improve the resolution of genetic mapping, and reducing the negative effects of linkage drag and deleterious genetic load in breeding. Using sequence‐based genotyping of a wheat nested association mapping (NAM) population of 2,100 recombinant inbred lines created by crossing 29 diverse lines, we mapped QTL affecting the distribution and frequency of 102 000 crossovers (CO). Genome‐wide recombination rate variation was mostly defined by rare alleles with small effects together explaining up to 48.6% of variation. Most QTL were additive and showed predominantly trans‐acting effects. The QTL affecting the proximal COs also acted additively without increasing the frequency of distal COs. We showed that the regions with decreased recombination carry more single nucleotide polymorphisms (SNPs) with possible deleterious effects than the regions with a high recombination rate. Therefore, our study offers insights into the genetic basis of recombination rate variation in wheat and its effect on the distribution of deleterious SNPs across the genome. The identified trans‐acting additive QTL can be utilized to manipulate CO frequency and distribution in the large polyploid wheat genome opening the possibility to improve the efficiency of gene pyramiding and reducing the deleterious genetic load in the low‐recombining pericentromeric regions of chromosomes.

Fluorescence in situ Hybridization Karyotype Analysis of Seven Platycodon grandiflorum (Jacq.) A. DC. Cultivars

Platycodon grandiflorum has long been cultivated for its medicinal properties and economic value, prompting crop improvement programs that have spurred the development of new cultivars. However, limited cytogenetic information is available for this species, especially for different P. grandiflorum cultivars. Karyotype analysis via fluorescence in situ hybridization (FISH) provides essential information about chromosome count and the localization of DNA sequences, thus providing a chromosome-level understanding of genome structure. Here, we carried out triple-color FISH karyotype analysis of various P. grandiflorum (Jacq.) A. DC. cultivars using 5S and 45S ribosomal DNA (rDNA) and telomeric repeat probes. The karyotype is composed of 2n = 18 chromosomes consisting of seven pairs of metacentric chromosomes, one pair of submetacentric chromosomes, and one pair of subtelocentric chromosomes. The chromosome length ranges from 1.92 to 4.98 μm. One pair of 5S rDNA loci was observed on either the intercalary or pericentromeric regions of chromosome 3. All cultivars showed 45S rDNA signals on chromosome 9, and in two closely related cultivars, one additional pair of 5S rDNAs co-localized with these 45S rDNAs. Arabidopsis -type telomeric repeats were observed in most telomeric regions of the chromosomes in all seven cultivars. Our data lay the foundation for future cytogenetic studies of P. grandiflorum focusing on breeding and the elucidation of its genome structure.

Recurrent establishment of de novo centromeres in the pericentromeric region of maize chromosome 3.

Centromeres can arise de novo from non-centromeric regions, which are often called "neocentromeres." Neocentromere formation provides the best evidence for the concept that centromere function is not determined by the underlying DNA sequences, but controlled by poorly understood epigenetic mechanisms. Numerous neocentromeres have been reported in several plant and animal species. However, it has been elusive how and why a specific chromosomal region is chosen to be a new centromere during the neocentromere activation events. We report recurrent establishment of neocentromeres in a pericentromeric region of chromosome 3 in maize (Zea mays). This latent region is located in the short arm and is only 2Mb away from the centromere (Cen3) of chromosome 3. At least three independent neocentromere activation events, which were likely induced by different mechanisms, occurred within this latent region. We mapped the binding domains of CENH3, the centromere-specific H3 histone variant, of the three neocentromeres and analyzed the genomic and epigenomic features associated with Cen3, the de novo centromeres and an inactivated centromere derived from an ancestral chromosome. Our results indicate that lack of genes and transcription and a relatively high level of DNA methylation in this pericentromeric region may provide a favorable chromatin environment for neocentromere activation.

Karyological characterization and identification of four repetitive element groups (the 18S - 28S rRNA gene, telomeric sequences, microsatellite repeat motifs, Rex retroelements) of the Asian swamp eel (Monopterus albus).

Among teleost fishes, Asian swamp eel (Monopterus albus Zuiew, 1793) possesses the lowest chromosome number, 2n = 24. To characterize the chromosome constitution and investigate the genome organization of repetitive sequences in M. albus, karyotyping and chromosome mapping were performed with the 18S – 28S rRNA gene, telomeric repeats, microsatellite repeat motifs, and Rex retroelements. The 18S – 28S rRNA genes were observed to the pericentromeric region of chromosome 4 at the same position with large propidium iodide and C-positive bands, suggesting that the molecular structure of the pericentromeric regions of chromosome 4 has evolved in a concerted manner with amplification of the 18S – 28S rRNA genes. (TTAGGG)n sequences were found at the telomeric ends of all chromosomes. Eight of 19 microsatellite repeat motifs were dispersedly mapped on different chromosomes suggesting the independent amplification of microsatellite repeat motifs in M. albus. Monopterus albus Rex1 (MALRex1) was observed at interstitial sites of all chromosomes and in the pericentromeric regions of most chromosomes whereas MALRex3 was scattered and localized to all chromosomes and MALRex6 to several chromosomes. This suggests that these retroelements were independently amplified or lost in M. albus. Among MALRexs (MALRex1, MALRex3, and MALRex6), MALRex6 showed higher interspecific sequence divergences from other teleost species in comparison. This suggests that the divergence of Rex6 sequences of M. albus might have occurred a relatively long time ago.

HGSNAT has a TATA-less promoter with multiple starts of transcription

Acetyl-CoA:α-glucosaminide N-acetyltransferase (N-acetyltransferase) is a lysosomal membrane enzyme that catalyzes a key step in the lysosomal degradation of heparan sulfate. Its deficiency causes Sanfilippo syndrome type IIIC (Mucopolysaccharidosis type IIIC, MPS IIIC). Here we characterize the promoter region of HGSNAT, the gene encoding N-acetyltransferase, which is located in the pericentromeric region of chromosome 8.We show that HGSNAT transcription is driven by a TATA-less promoter whose key elements are contained within the 1054bp region upstream of exon 1. About 400 bases of the region's 3′-prime end overlap with an unmethylated CpG island. Reduced reporter activities from promoter serial deletion constructs suggested strong regulatory elements at positions −101 to −20bp and −1073 to −716bp of the downstream initiation codon (DS-ATG). Targeted mutagenesis of the first Specificity protein 1-A (Sp1-A) of the six in silico-predicted Sp1 sites in the region flanking the major transcription start sites (TSSs, +50/−101) led to a 55% decrease of reporter activity, while inactivation of each of Sp1-B and Sp1-C resulted in its almost two-fold increase. The binding of Sp1 to the region was confirmed by chromatin immunoprecipitation (ChIP). Overall, this confirms that Sp1 is important for regulation of the HGSNAT promoter.Promoter fragments in antisense orientation (constructs pGL4 −20/−1305 and pGL4 +50/−1305) led to reporter activities of about 50% of the pGL4 −1305/−20 activity, implying divergent initiation of transcription at the promoter. We identified two main TSSs at positions +1 and −15 from DS-ATG using Rapid amplification of cDNA ends (5′RACE). Transcripts initiating at the TSSs thus contain only DS-ATG. Five patients from our MPS IIIC cohort (n=23) carried the rs4523300 promoter variant and one the rs149596192 promoter variant. Both variants lowered the expression of the reporter down to 68% and 59%, respectively. However, white blood cell (WBC) N-acetyltransferase activities in individuals carrying the variants did not significantly differ from homozygotes for the wild-type alleles, suggesting only a partial impact of transcriptional regulation on N-acetyltransferase activities in vivo.

DNA demethylation caused by 5-Aza-2'-deoxycytidine induces mitotic alterations and aneuploidy.

Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation.Here, we report that the DNA hypomethylating drug 5-aza-2′-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduction of 5-Methyl Cytosine at the pericentromeric region of chromosomes correlates with aneuploidy and mitotic defects.Our results suggest that DNA hypomethylation leads to aneuploidy by altering the DNA methylation landscape at the centromere that is necessary to ensure proper chromosomes segregation by recruiting the proteins necessary to build up a functional kinetochore.

Genome-Wide DNA Methylation Analysis Identifies Novel Hypomethylated Non-Pericentromeric Genes with Potential Clinical Implications in ICF Syndrome.

Introduction and ResultsImmunodeficiency, centromeric instability and facial anomalies syndrome (ICF) is a rare autosomal recessive disease, characterized by severe hypomethylation in pericentromeric regions of chromosomes (1, 16 and 9), marked immunodeficiency and facial anomalies. The majority of ICF patients present mutations in the DNMT3B gene, affecting the DNA methyltransferase activity of the protein. In the present study, we have used the Infinium 450K DNA methylation array to evaluate the methylation level of 450,000 CpGs in lymphoblastoid cell lines and untrasformed fibroblasts derived from ICF patients and healthy donors. Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein. We identified 181 novel differentially methylated positions (DMPs) including subtelomeric and intrachromosomic regions, outside the classical ICF-related pericentromeric hypomethylated positions. Interestingly, these sites were mainly located in intergenic regions and inside the CpG islands. Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues.ConclusionsIn conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B. A high percentage of the DMPs are located in the subtelomeric regions, indicating a specific role of DNMT3B in methylating these chromosomal sites. Therefore, we provide further evidence that hypomethylation in specific non-pericentromeric regions of chromosomes might be involved in the molecular pathogenesis of ICF syndrome. The detection of DNA hypomethylation at BOLL, SYCP2 and NCRNA00221 may pave the way for the development of specific clinical biomarkers with the aim to facilitate the identification of ICF patients.

Histone H3.3 maintains genome integrity during mammalian development.

Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.

Comparative analysis of DNA sequences of regions of X-chromosome attachment to the nuclear envelope of nurse cells Anopheles messeae Fall.

Polytene chromosomes of ovarian nurse cells of Anopheles mosquitoes form strong contacts with the nuclear envelope. The presence of contacts, their position at nurse cell chromosomes, and their morphological features are species-specific in malaria mosquitoes. It is important to determine the nature of these interspecies differences in the nuclear architecture, both to understand the function of the nucleus and to assess the role of the spatial organization of chromosomes in evolution. Using dot-blot hybridization, we compared DNA sequences of the clone library from the X-chromosome attachment region to the nuclear envelope of ovarian nurse cells of Anopheles messeae with DNA-probes: (1) of the X-chromosome attachment region of An. atroparvus, (2) of the 3R chromosome attachment region ofAn. messeae, and (3) of the chromosome 2 pericentromeric region of An. messeae, without expressed contacts with the nuclear envelope. It has been shown that the chromosome attachment regions have a significantly higher number of homologous DNA sequences as compared with the pericentromeric region of chromosome 2. Sequences that are common for attachment regions are largely potentially able to participate in the formation of chromatin loop domains and to interact with some nucleus frameworks, according to the analysis in the ChrClass program. The obtained results support the important role of DNA in the formation of strong chromosomal attachments to the nuclear envelope in nurse cells of Anopheles mosquitoes.

The genetics of folate metabolism and maternal risk of birth of a child with Down syndrome and associated congenital heart defects.

Almost 15 years ago it was hypothesized that polymorphisms of genes encoding enzymes involved in folate metabolism could lead to aberrant methylation of peri-centromeric regions of chromosome 21, favoring its abnormal segregation during maternal meiosis. Subsequently, more than 50 small case-control studies investigated whether or not maternal polymorphisms of folate pathway genes could be risk factors for the birth of a child with Down syndrome (DS), yielding conflicting and inconclusive results. However, recent meta-analyses of those studies suggest that at least three of those polymorphisms, namely MTHFR 677C>T, MTRR 66A>G, and RFC1 80G>A, are likely to act as maternal risk factors for the birth of a child with trisomy 21, revealing also complex gene-nutrient interactions. A large-cohort study also revealed that lack of maternal folic acid supplementation at peri-conception resulted in increased risk for a DS birth due to errors occurred at maternal meiosis II in the aging oocyte, and it was shown that the methylation status of chromosome 21 peri-centromeric regions could favor recombination errors during meiosis leading to its malsegregation. In this regard, two recent case-control studies revealed association of maternal polymorphisms or haplotypes of the DNMT3B gene, coding for an enzyme required for the regulation of DNA methylation at centromeric and peri-centromeric regions of human chromosomes, with risk of having a birth with DS. Furthermore, congenital heart defects (CHD) are found in almost a half of DS births, and increasing evidence points to a possible contribution of lack of folic acid supplementation at peri-conception, maternal polymorphisms of folate pathway genes, and resulting epigenetic modifications of several genes, at the basis of their occurrence. This review summarizes available case-control studies and literature meta-analyses in order to provide a critical and up to date overview of what we currently know in this field.

Targeted Resequencing of the Pericentromere of Chromosome 2 Linked to Constitutional Delay of Growth and Puberty

Constitutional delay of growth and puberty (CDGP) is the most common cause of pubertal delay. CDGP is defined as the proportion of the normal population who experience pubertal onset at least 2 SD later than the population mean, representing 2.3% of all adolescents. While adolescents with CDGP spontaneously enter puberty, they are at risk for short stature, decreased bone mineral density, and psychosocial problems. Genetic factors contribute heavily to the timing of puberty, but the vast majority of CDGP cases remain biologically unexplained, and there is no definitive test to distinguish CDGP from pathological absence of puberty during adolescence. Recently, we published a study identifying significant linkage between a locus at the pericentromeric region of chromosome 2 (chr 2) and CDGP in Finnish families. To investigate this region for causal variation, we sequenced chr 2 between the genomic coordinates of 79–124 Mb (genome build GRCh37) in the proband and affected parent of the 13 families contributing most to this linkage signal. One gene, DNAH6, harbored 6 protein-altering low-frequency variants (< 6% in the Finnish population) in 10 of the CDGP probands. We sequenced an additional 135 unrelated Finnish CDGP subjects and utilized the unique Sequencing Initiative Suomi (SISu) population reference exome set to show that while 5 of these variants were present in the CDGP set, they were also present in the Finnish population at similar frequencies. Additional variants in the targeted region could not be prioritized for follow-up, possibly due to gaps in sequencing coverage or lack of functional knowledge of non-genic genomic regions. Thus, despite having a well-characterized sample collection from a genetically homogeneous population with a large population-based reference sequence dataset, we were unable to pinpoint variation in the linked region predisposing delayed puberty. This study highlights the difficulties of detecting genetic variants under linkage regions for complex traits and suggests that advancements in annotation of gene function and regulatory regions of the genome will be critical for solving the genetic background of complex phenotypes like CDGP.

Rapid and reliable identification of tomato fruit weight and locule number loci by QTL-seq.

Bulk segregant analysis coupled with whole genome sequencing is a powerful approach and cost-effective method to identify loci controlling fruit traits in tomato. Domestication of fruit and vegetable crops was accompanied by selection for weight of the edible parts. Increases in fruit weight are controlled by multiple quantitative trait loci (QTL). To date, only two fruit weight genes have been cloned and a third has been fine-mapped. Genes that control locule number also impact fruit weight and two of them are known. To efficiently identify additional tomato fruit weight (FW) and locule number (LC) loci, six F2 populations were generated from crosses between closely related tomato accessions for which the alleles of the cloned FW and LC genes were known. We employed the bulk segregant approach coupled to whole genome sequencing (QTL-seq) which led to the identification of three highly significant and newly mapped FW QTL. fw11.2 was located in the distal part of chromosome 11 above the known loci fas and fw11.3; fw1.1 in the pericentromeric region of chromosome 1; and fw3.3 located ~1.6Mb below the known fruit weight gene, SlKLUH/FW3.2. In addition, we mapped three LCQTL (lcn2.4, lcn5.1, and lcn6.1) although their significance was generally low. To confirm the location of the gene underlying fw11.2, we developed additional markers and conducted progeny tests. These results allowed us to narrow down the fw11.2 QTL to a region of ~750kb corresponding to 66 candidate genes. Our research approach provided a cost-effective and time-efficient method for the identification of additional genes involved in FW and LC that could be used for both fruit development studies and crop improvement programs.

Specific features in linear and spatial organizations of pericentromeric heterochromatin regions in polytene chromosomes of the closely related species Drosophila virilis and D. kanekoi (Diptera: Drosophilidae).

Heterochromatin plays an important role in the spatial arrangement and evolution of the eukaryotic genetic apparatus. The closely related species Drosophila virilis (phyla virilis) and D. kanekoi (phyla montana) differ in the amount of heterochromatin along the chromosomes as well as by the presence of the metacentric chromosome 2, which emerged as a result of a pericentric inversion during speciation, in the D. kanekoi karyotype. The purpose of this study was to establish if chromosome rearrangements have any influence on the linear redistribution of centromeric heterochromatin in polytene chromosomes and the spatial organization of chromosomes in the nuclei of nurse cell. We have microdissected the chromocenter of D. virilis salivary gland polytene chromosomes; obtained a DNA library of this region (DvirIII); and hybridized (FISH) DvirIII to the salivary gland and nurse cell polytene chromosomes of D. virilis and D. kanekoi. We demonstrated that DvirIII localizes to the pericentromeric heterochromatin regions of all chromosomes and peritelomeric region of chromosome 5 in both species. Unlike D. virilis, the DvirIII signal in D. kanekoi chromosomes is detectable in the telomeric region of chromosome 2. We have also conducted a 3D FISH of DvirIII probe to the D. virilis and D. kanekoi nurse cell chromosomes. In particular, the DvirIII signal in D. virilis was observed in the local chromocenter at one pole of the nucleus, while the signal belonging to the telomeric region of chromosome 5 was detectable at the other pole. In contrast, in D. kanekoi there exist two separate DvirIII-positive regions. One of these regions belongs to the pericentromeric region of chromosome 2 and the other, to pericentromeric regions of the remaining chromosomes. These results suggest that chromosome rearrangements play an important role in the redistribution of heterochromatin DNA sequences in the genome, representing a speciation mechanism, which, in general, could also affect the chromosome orientation in the 3D nuclear space.

Maternal risk for down syndrome and polymorphisms in the promoter region of the DNMT3B gene: a case-control study.

Epigenetic changes leading to improper methylation of the pericentromeric region of chromosome 21 may contribute to the nondisjunction of this chromosome. Polymorphisms in the DNA Methyltransferase 3B (DNMT3B) gene, one of the crucial gene of the folate metabolism, affects the activity of the enzyme and increases the susceptibility of nondisjunction in mothers of Down syndrome children (MDS). Considering this hypothesis we investigated the association of single nucleotide polymorphisms in the promoter region of the DNMT3B gene (rs1569686 -579G>T; rs2424913 -149C>T) with a predisposition of mothers to deliver a Down syndrome (DS) child. The study was performed on DNA samples from 150 MDS and 172 control mothers. Transmission disequilibrium tests were performed on 103 DS trio families. Genotyping was done using a polymerase chain reaction-restriction fragment length polymorphism method. With respect to the single nucleotide polymorphisms studied, no significant difference was observed in the genotypes and alleles frequency distributions between MDS and control mothers. The frequency of the DNMT3B-579G allele was, respectively, 0.34 in MDS and 0.33 in control mothers whereas the frequency of the DNMT3B-149C allele was respectively 0.31 in MDS and 0.26 in control mothers. No significant deviation in genotypic combinations as well as in transmission disequilibrium tests analysis was observed. However, a strong linkage disequilibrium was observed with significant differences in the distribution of G-T and G-C haplotypes among case and control mothers. Although the above studied polymorphisms of DNMT3B may not be an independent risk factor it might be possible that certain allelic combinations (G-T) are. This finding suggests that DNMT3B might be a maternal risk factor for DS in our Indian cohort. Replication studies are required to confirm these findings.

Species-specific localization of DNA from pericentromeric heterochromatin on polytene chromosomes in the salivary gland cells and 3D-nuclear organization nurse cells in Drosophila virilis and Drosophila kanekoi (Diptera: Drosophilidae)

Microdissection of the chromocenter of D. virilis salivary gland polytene chromosomes has been carried out and the region-specific. DNA library (DvirIII) has been obtained. FISH was used for DvirIII hybridization with salivary gland polytene chromosomes and ovarian nurse cells of D. virilis and D. kanekoi. Localization of DvirIII in the pericentromeric regions of chromosomes and in the telomeric region of chromosome 5 was observed in both species. Moreover, species specificity in the localization of DNA sequences of DvirIII in some chromosomal regions was detected. In order to study the three-dimensional organization of pericentromeric heterochromatin region of polytene chromosomes of ovarian nurse cells of D. virilis and D. kanekoi, 3D FISH DvirIII was performed with nurse cells of these species. As a result, species specificity in the distribution of DvirIII signals in the nuclear space was revealed. Namely, the signal was detected in the local chromocenter at one pole of the nucleus in D. virilis, while the signal from the telomeric region of chromosome 5 was detected on another pole. At the same time, DvirIII signals in D. kanekoi are localized in two separate areas in the nucleus: the first belongs to the pericentromeric region of chromosome 2 and another to pericentromeric regions of the remaining chromosomes.

Two Cases of Multiple Endocrine Neoplasia Type II with RET Mutaion

Multiple endocrine neoplasia type 2(MEN2) is a rare autosomal dominant inherited disorder characterized by the presence of medullary thyroid carcinoma, pheochromocytoma and other hyperplasia and/or neoplasia of different endocrine tissues in a single patient. MEN 2 is caused by germline mutations in the RET proto-oncogene is located on the pericentromeric region of chromosome 10 (10q11.2). We present our experience with two rare cases of MEN 2, an 11-years-old girl and a 10-years-old boy. Their parents had medullary thyroid carcinoma and genetic analysis showed the missense mutation of RET. They were screened for mutations in the RET proto-oncogene and RET mutations were found at codons 634 and 641. They were asymptomatic state but the girl had prophylactic total thyroidectomy. Children of families with RET mutations may develop early cancers and require prophylactic thyroidectomy before eight years. pISSN 2233-5250 / eISSN 2233-4580 http://dx.doi.org/10.15264/cpho.2014.21.2.163 Clin Pediatr Hematol Oncol 2014;21:163∼167

Addressing unmet clinical needs: FISHing for bile duct cancer.

Addressing unmet clinical needs: FISHing for bile duct cancer.

The Clinicopathologic Significance of Centromere 17 Copy Number Alterations in Invasive Breast Carcinoma

The human epidermal growth factor receptor 2 (HER2) oncoprotein is overexpressed in about 18% to 20% of breast cancers, and HER2 gene amplification is responsible for protein overexpression in most cases. A small subset of cases show an increase in centromere 17 copy numbers without HER2 amplification, commonly referred to as polysomy. Although HER2 overexpression is an established adverse prognostic factor in breast cancer, there are conflicting results about adverse prognostic factors in cases with increased numbers of centromere 17 copies without HER2 amplification. Whereas most patients with increased centromere 17 copy numbers are HER2 2+ by immunohistochemistry, a minority are 3+. Currently, patients with increase in centromere 17 copy numbers without HER2 amplification are treated similar to patients who show neither amplification nor polysomy. Recent studies have demonstrated that what is commonly referred to as chromosome 17 polysomy is not true polysomy of chromosome 17, but often due to CEP17 duplication or amplification, or due to gains or amplification in the pericentromeric region of chromosome 17. Therefore, it is believed that use of CEP17 to correct HER2 copy numbers might not be ideal and may give misleading results. The case presented highlights a conundrum where, by absolute HER2 copy numbers, the case would be HER2 amplified, and by HER2/CEP17 ratio it is not amplified. In this context, we discuss HER2 amplification and centromere 17 copy number alterations in invasive breast cancer.