Karyotypic Changes Research Articles

Molecular characterization of complex chromosomal rearrangement: First report of novel t(7;12) (q11;q22) as part of a complex karyotype in de novo AML-M2 case

The strong association of diagnostic karyotype with clinical outcome has made cytogenetics one of the most valuable diagnostic and prognostic tools for acute myeloid leukemia (AML) till today. Complex chromosomal findings are reported to be seen in nearly 10–15% of adult AMLs and are generally associated with poor outcome. In the current report, we present the results of hematologic, immunophenotypic, cytogenetic, chromosomal microarray and molecular analyses of a 60-year-old female patient diagnosed with AML-M2. Cytogenetic analysis revealed complex chromosomal findings involving seven different chromosomes. However, cytogenetic analyses were not able to precisely unveil all karyotypic changes, hence chromosomal microarray was used for further characterization. The most interesting observation was identification of a t(7;12) (q11;q22) as part of this complex karyotype. To the best of our knowledge, this is the first report of identification of novel t(7;12) (q11;q22) as part of a complex karyotype in de novo AML-M2.

Centromere Strength Provides the Cell Biological Basis for Meiotic Drive and Karyotype Evolution in Mice

Mammalian karyotypes (number and structure of chromosomes) can vary dramatically over short evolutionary time frames. There are examples of massive karyotype conversion, from mostly telocentric (centromere terminal) to mostly metacentric (centromere internal), in 10(2)-10(5) years. These changes typically reflect rapid fixation of Robertsonian (Rb) fusions, a common chromosomal rearrangement that joins two telocentric chromosomes at their centromeres to create one metacentric. Fixation of Rb fusions can be explained by meiotic drive: biased chromosome segregation during female meiosis in violation of Mendel's first law. However, there is no mechanistic explanation of why fusions would preferentially segregate to the egg in some populations, leading to fixation and karyotype change, while other populations preferentially eliminate the fusions and maintain a telocentric karyotype. Here we show, using both laboratory models and wild mice, that differences in centromere strength predict the direction of drive. Stronger centromeres, manifested by increased kinetochore protein levels and altered interactions with spindle microtubules, are preferentially retained in the egg. We find that fusions preferentially segregate to the polar body in laboratory mouse strains when the fusion centromeres are weaker than those of telocentrics. Conversely, fusion centromeres are stronger relative to telocentrics in natural house mouse populations that have changed karyotype by accumulating metacentric fusions. Our findings suggest that natural variation in centromere strength explains how the direction of drive can switch between populations. They also provide a cell biological basis of centromere drive and karyotype evolution.

Recent changes in the distribution of carboxylesterase genes and associated chromosomal rearrangements in Greek populations of the tobacco aphidMyzus persicae nicotianae

We present data on the frequency of amplified E4 and FE4 carboxylesterase genes in Myzus persicae s.l. clones collected during the years 2002–2007 and 2012 in Greece. Most clones were of the tobacco aphid, Myzus persicae nicotianae. Samples from 2012 were genotyped with microsatellite DNA markers and a number of them were karyotyped. Aphid clones with amplified FE4 genes predominated in all years, whereas E4 was present in only 3.5% of all samples and always occurred in clones with FE4. Most of the clones examined showed high carboxylesterase activity levels (R2 resistant category). The results showed marked changes in the frequencies of the two carboxylesterase genes in the tobacco aphid populations compared to published data that were collected in Greece in the mid 1990s, when E4 was recorded on its own in 20% of all samples and in 32% of samples from tobacco. A parallel change in karyotype was also observed because the A1,3 translocation, which had a worldwide association with amplified E4 genes in the 1990s, was not detected in the clones analyzed in 2012. Possible causes for these changes are discussed, although selection as a result of pest management practices appears to be the major one. Novel chromosomal rearrangements were also found in M. persicae nicotianae clones. These rearrangements could be a result of clastogenic effects of nicotine, which could persist because of the holocentric nature of aphid chromosomes. The results are discussed in relation to rapid evolution events that have taken place in the tobacco aphid in Greece during the last two decades. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113, 455–470.

Effects of prolonged in vitro culture and cryopreservation on viability, DNA fragmentation, chromosome stability and ultrastructure of bovine cells from amniotic fluid and umbilical cord.

The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long-term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33±5.58%, 90.56±4.40% and 81.90±3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2±8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.

Mitotic DNA damages induced by carbon–ion radiation incur additional chromosomal breaks in polyploidy

Compared with low linear energy transfer (LET) radiation, carbon–ion radiation has been proved to induce high frequency of more complex DNA damages, including DNA double strands (DSBs) and non-DSB clustered DNA lesions. Chemotherapeutic drug doxorubicin has been reported to elicit additional H2AX phosphorylation in polyploidy. Here, we investigated whether mitotic DNA damage induced by high-LET carbon–ion radiation could play the same role. We demonstrate that impairment of post-mitotic G1 and S arrest and abrogation of post-mitotic G2-M checkpoint failed to prevent mis-replication of damaged DNA and mis-separation of chromosomes. Meanwhile, mitotic slippage only nocodazole-related, cytokinesis failure and cell fusion collectively contributed to the formation of binucleated cells. Chk1 and Cdh1 activation was inhibited when polyploidy emerged in force, both of which are critical components for mitotic exit and cytokinesis. Carbon–ion radiation irrelevant of nocodazole incurred additional DNA breaks in polyploidy, manifesting as structural and numerical karyotype changes. The proliferation of cells given pre-synchronization and radiation was completely inhibited and cells were intensely apoptotic. Since increased chromosomal damage resulted in extensive H2AX phosphorylation during polyploidy, we propose that the additional γ-H2AX during polyploidy incurred by carbon–ion radiation provides a final opportunity for these dangerous and chromosomally unstable cells to be eliminated.

Evidence for meiotic drive as an explanation for karyotype changes in fishes

The process of preferential chromosome segregation during meiosis has been suggested to be responsible for the predominance of certain chromosome types in the karyotypes of mammals, birds and insects. We developed an extensive analysis of the fixation of mono- or bibrachial chromosomes in the karyotypes of the large Actinopterygii fish group, a key link in the evolution of terrestrial vertebrates, in order to investigate the generality of meiotic drive in determining karyotypic macrotrends. Unlike mammals, fishes have markedly undergone several types of preferential chromosomal rearrangements throughout evolution. Data from the analyzed orders indicate a prevalence of karyotypes with few (<33%) or many (>66%) acrocentric chromosomes and a low number of karyotypes with balanced numbers of mono- and bi-brachial elements. Parallel trends towards a higher number of karyotypes with prevalence of monobrachial chromosomes occurred in phylogenetically close orders (e.g. Perciformes and Tetraodontiformes, and in the order Mugiliformes) and in clades with prevalence of bibrachial elements (e.g. Characiformes, Gymnotiformes, Siluriformes, and Cypriniformes). Some orders where fewer species were available for study, such as Atheriniformes and Anguilliformes, showed karyotype assemblages where both trends were present. Our results strongly suggest a primary role of meiotic drive in karyotypic evolution as indicated by the accumulation of monobrachial chromosomes in Perciformes and Cypriniformes, or bibrachial chromosomes in Siluriformes and Characiformes. Further examinations of the interaction between life history traits, environmental characteristics, and the fixation of chromosomal rearrangements would be exceedingly valuable.

Investigation of IGF2, Hedgehog and fusion gene expression profiles in pediatric sarcomas

The childhood sarcomas are malignant tumors with high mortality rates. They are divided into two genetic categories: a category without distinct pattern karyotypic changes and the other category showing unique translocations that originate gene rearrangements. This category includes rhabdomyosarcoma (RMS), Ewing's sarcoma (ES) and synovial sarcoma (SS). Diverse studies have related development genes, such as; IGF2, IHH, PTCH1 and GLI1 and sarcomatogenesis. ObjectiveTo characterize the RMS, ES and SS rearrangements, we quantify the expression of IGF2 IHH, PTCH1 and GLI1 genes and correlate molecular data with clinical parameters of patients. DesignWe analyzed 29 RMS, 10 SS and 60 ES tumor samples by RT-PCR (polymerase chain reaction-reverse transcription) and qPCR (quantitative PCR). ResultsAmong the samples of ARMS, 50% had rearrangements of PAX3/7–FOXO1, 60% of ES samples were EWS–FLI1 positive and 90% of SS samples were positive for SS18–SSX1/2. In relation to the control reference samples (QPCR Human Reference Total RNA-Stratagene, Human Skeletal Muscle Total RNA-Ambion, Universal RNA Human Normal Tissues-Ambion), RMS samples showed a high IGF2 gene expression (p<0.0001). Moreover, ES samples showed a low IGF2 gene expression (p<0.0001) and high IHH (p<0.0001), PTCH1 (p=0.0173) and GLI1 (p=0.0113) gene expressions. ConclusionsThe molecular characterization of IGF and Hedgehog pathway in these pediatric sarcomas may collaborate to enable a better understanding of the biological behavior of these neoplasms.

Differentiation of umbilical cord lining membrane-derived mesenchymal stem cells into endothelial-like cells.

Stem cell therapy for the treatment of vascular-related diseases through functional revascularization is one of the most important research areas in tissue engineering. The aim of this study was to investigate the in vitro differentiation of umbilical CL-MSC into endothelial lineage cells. In this study, isolated cells were characterized for expression of MSC-specific markers and osteogenic and adipogenic differentiation. They were induced to differentiate into endothelial-like cells and then examined for expression of the endothelial-specific markers, karyotype, and functional behavior of cells. Isolated cells expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. After endothelial differentiation, they expressed CD31, vWF, VE-cadherin, VEGFR1, and VEGFR2 at both mRNA and protein level, but their morphological changes were not apparent when compared with those of undifferentiated cells. There were no significant changes in karyotype of differentiated cells. Furthermore, angiogenesis assay and LDL uptake assay showed that differentiated cells were able to form the capillary-like structures and uptake LDL, respectively. The results indicated that umbilical CL-MSC could differentiate into functional endothelial-like cells. Also, they are suitable for basic and clinical studies to cure several vascular-related diseases.

Rapid chromosomal evolution in a morphologically cryptic radiation

Cryptic species occur within most of the major taxonomic divisions, and a current challenge is to determine why some lineages have more cryptic species than others. It is expected that cryptic species are more common in groups where there are life histories or genetic architectures that promote speciation in the absence of apparent morphological differentiation. Chromosomal rearrangements have the potential to lead to post-zygotic isolation and might be an important factor leading to cryptic species. Here we investigate the potential role of chromosomal change in driving speciation in the karyotypically diverse scale insect genus Apiomorpha, focussing on four species placed in the same species group (the A. minor species group Gullan, 1984). Using mitochondrial and nuclear DNA sequence data, we find that Apiomorpha minor is not monophyletic and consists of at least nine cryptic species. Diploid chromosome counts range from 2n=4 to 2n=84 across the four currently recognized species, and some of the chromosomal variation exists in the absence of other genetic or host use differences, consistent with karyotypic changes being involved in lineage divergence and the generation of cryptic species.

Well-plate mechanical confinement platform for studies of mechanical mutagenesis

Limited space for cell division, perhaps similar to the compressed microenvironment of a growing tumor, has been shown to induce phenotypic and karyotypic changes to a cell during mitosis. To expand understanding of this missegregation of chromosomes in aberrant multi-daughter or asymmetric cell divisions, we present a simple technique for subjecting mammalian cells to adjustable levels of confinement which allows subsequent interrogation of intracellular molecular components using high resolution confocal imaging. PDMS micropatterned confinement structures of subcellular height with neighboring taller media reservoir channels were secured on top of confluent cells with a custom compression well-plate system. The system improved ease of use over previous devices since confined cells could be initially grown on glass coverslips in a 12-well plate, and subsequently be imaged by high resolution confocal imaging, or during compression by live cell imaging. Live cell imaging showed a significant increase in abnormal divisions of confined cells across three different cell lines (HeLa, A375, and A549). Immunofluoresecence stains revealed a significant increase in cell diameter and chromosome area of confined cells, but no significant increase in centrosome-centromere distance upon division when compared to unconfined cells. The developed system could open up studies more broadly on confinement effects on mitotic processes, and increase the throughput of such studies.

Karyotypic Changes through Dysploidy Persist Longer over Evolutionary Time than Polyploid Changes

Chromosome evolution has been demonstrated to have profound effects on diversification rates and speciation in angiosperms. While polyploidy has predated some major radiations in plants, it has also been related to decreased diversification rates. There has been comparatively little attention to the evolutionary role of gains and losses of single chromosomes, which may or not entail changes in the DNA content (then called aneuploidy or dysploidy, respectively). In this study we investigate the role of chromosome number transitions and of possible associated genome size changes in angiosperm evolution. We model the tempo and mode of chromosome number evolution and its possible correlation with patterns of cladogenesis in 15 angiosperm clades. Inferred polyploid transitions are distributed more frequently towards recent times than single chromosome gains and losses. This is likely because the latter events do not entail changes in DNA content and are probably due to fission or fusion events (dysploidy), as revealed by an analysis of the relationship between genome size and chromosome number. Our results support the general pattern that recently originated polyploids fail to persist, and suggest that dysploidy may have comparatively longer-term persistence than polyploidy. Changes in chromosome number associated with dysploidy were typically observed across the phylogenies based on a chi-square analysis, consistent with these changes being neutral with respect to diversification.

Outcome of Children with Marked Changes in Maternal Screening Tests and Normal Karyotype

Objective: To investigate whether chromosomally normal fetuses with marked changes in maternal serum markers and first trimester ultrasound NT marker have an increased risk of congenital anomalies or delayed development at 2 years of age. Methods: Screening tests of 5257 pregnant women were analyzed during a one-year period. Significant changes in biochemical and/or ultrasound markers were documented in 138 pregnant women, whereas positive risk calculation for chromosomal anomalies was evident in 74 of them, who were included in our study. Postnatal study group included 35 children born from mothers with marked changes in screening tests. Results: Among the 74 pregnant women, a structural or genetic abnormality was diagnosed in 16 cases (21.6%), fetal death occurred in 12 cases (16.2%) and child was healthy at the age of 2 years in 31 cases (41.9%). In 3/4 of the cases, a pathology was diagnosed prenatally, while the remaining 1/4 were discovered postnatally. Four children had with congenital anomalies and/or syndromes: two had congenital heart disease – atrial septal defect and ventricular septal defect with patent ductus arteriosus, one Silver-Russell syndrome and one congenital adrenal hyperplasia. It was not possible to get the final information about outcome in 15 cases (20.3%). Conclusions: Children born to these mothers should be actively followed by a pediatrician or clinical geneticist for additional investigations after birth as they have a risk of 5.4% of having a congenital or genetic abnormality.

Prognostic Significance Of The Cytogenetic Evolution After The Hematopoietic Stem Cell Transplantation In Adult Acute Myeloid Leukemia

BackgroundCytogenetic abnormalities at diagnosis have significant impacts on disease outcome and clonal evolution at cytogenetic level is considered to be associated with relapse and refractoriness to chemotherapy. Relapse of AML was reported to be associated with cytogenetic clonal evolution in 40% of patients who have received chemotherapy. However, little is known about the association of cytogenetic clonal evolution and relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study was conducted to see the karyotypic changes of the AML relapsed after allo-HSCT and to see whether the presence of the cytogenetic evolution has a prognostic impact. Patients and MethodsWe retrospectively reviewed patients diagnosed as AML who underwent allo-HSCT for the first time at our institute from Jan. 2006 to Dec. 2012 consecutively and analyzed the cytogenetic evolution patterns in patient at relapse of AML after HSCT. ResultsThree hundred and thirty patients were included in this study. We identified fifty-seven (17%) relapsed in bone marrow after HSCT. We excluded two patients, because of that one patient was not performed bone marrow aspiration after relapse and the other received auto-PBSCT after allo-HSCT due to an engraftment failure. So, we analyzed the bone marrow karyotypic changes before and after transplantation of 55 patients. Thirty-eight (69%) were male, and median age at allo-HSCT was 56 years (range, 21-82). Eight (14%) are still alive. Thirty-four (61%) received umbilical cord blood transplantation (UCBT), ten (18%) did related HSCT, and eleven (20%) did unrelated HSCT. Eleven (20%) had normal karyotype, fourteen (25%) had one chromosomal abnormality, eleven (20%) had two abnormalities, and nineteen (35%) had more than three abnormalities before HSCT. Median observation time after HSCT was 16 months (1-75). Thirty (55%) showed gain or loss of chromosomal abnormalities from original one (group1), sixteen (29%) showed no karyotypic change between HSCT, and nine (16%) showed totally different karyotype. We defined the cytogenetic evolution group as group 1, and the others as group 2. Median day of relapse after HSCT was not different between group 1 and 2 (4 months (range 1-73), 6 months (range 1-26), P=0.29). Shorter survival time after relapse was observed in group 1 (5.9% vs. 16.8% at 2 year post-relapse, P= 0.04) (figure. 1). [Display omitted] ConclusionAlthough durations from HSCT to relapse were comparable between group 1 and 2, survival time after relapse was significantly shorter in group 1 than in group 2, which indicates that cytogenetic clonal evolution may confer resistant characters against treatments, and needs novel therapeutic applications. Disclosures:No relevant conflicts of interest to declare.

GATA2 Mutations In Nonsyndromic Pediatric Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is rare in children. Certain inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia, severe congenital neutropenia and Shwachman Diamond syndrome, markedly increase the risk of MDS during childhood. However, the genetic factor(s) underlying sporadic pediatric MDS are unknown. Germline mutations in GATA2, a hematopoietic transcription factor, explain four MDS-predisposing conditions: monocytopenia and mycobacterial infection (MonoMAC); dendritic cell, monocyte, B and NK cell lymphoid deficiency (DCML); primary lymphedema with myelodysplasia progressing to acute myeloid leukemia (Emberger syndrome); and a subset of familial MDS. Cases of pediatric MDS have been observed in some of the reported pedigrees. In addition, three individuals have been reported with large, de novo deletions encompassing GATA2 and surrounding genes and manifesting developmental delay, intellectual disability and dysmorphic features alongside their hematologic abnormalities. We identified a novel GATA2 splice site variant (c.1018-2A>C) in a teenager with MDS, WHO classification refractory cytopenia of childhood (RCC). Although he was found to have monocytopenia and B and NK cell deficiencies, he had no history of infections associated with MonoMAC or pertinent family history. We, therefore, hypothesized that mutations in GATA2 might be present in additional cases of pediatric MDS that were neither associated with an IBMFS nor relevant personal or family history.Two Baylor College of Medicine biology studies open to children with hematologic disease were queried for patients with the diagnosis of MDS. Exclusion criteria included treatment-related MDS, diagnosis of an IBMFS, prior diagnosis of severe aplastic anemia or infections suspicious for MonoMAC or DCML, and known or suspected family history of a GATA2-associated disorder. Cases lacking a pre-hematopoietic stem cell transplantation (HSCT) tissue sample available for study were also excluded. In addition to the patient described above, six children were identified who met eligibility criteria. DNA was isolated from banked peripheral blood or bone marrow cells and GATA2 sequencing performed, including upstream and intronic regulatory regions. Array comparative genomic hybridization was also performed on one sample that lacked GATA2 sequence variants, but was notable for complete absence of heterozygosity (AOH), including 6 polymorphic sites with minor allele frequencies of 0.20 or greater. Pertinent clinical and laboratory features were extracted by medical record review blinded to GATA2 status.We found heterozygous GATA2 mutations in three of the six additional patient samples. Thus, four of this seven patient, pediatric MDS cohort had mutated GATA2. Two of the newly identified mutations were splice site variants: a previously described c.1018-1G>A and a novel variant altering the exon 7 splice site acceptor (c.1114-1G>C). The third mutation was a de novo 3.1-3.3 Mb deletion encompassing the entire GATA2 locus and contiguous genes, and was established to be germline by analysis of skin fibroblasts. Notably, the patient had normal neurocognitive development and was without dysmorphic features. Their ages of presentation were 5, 9, 12 and 15 years. With the exception of the initial case, peripheral blood T and B cell phenotyping was not obtained prior to HSCT. Monocytopenia of less than 200/µL was present in five of seven patients, three of whom had a GATA2 mutation. All four GATA2 mutation cases had RCC and three of the four had monosomy 7 at diagnosis. In contrast, the three cases lacking GATA2 mutation presented with the MDS classification refractory anemia with excess blasts (RAEB-2), with either a normal karyotype, complex karyotypic changes or chromosome 13.q12q14 deletion.GATA2 mutation may explain a significant portion of sporadic, seemingly nonsyndromic pediatric MDS, particularly cases with monosomy 7. Evaluation of larger cohorts is warranted to ascertain the true prevalence. Although this cohort is small, we recommend GATA2 sequencing be performed as part of the initial evaluation of pediatric MDS as the identification of a germline mutation has critical implications for related donor selection and genetic counseling. AOH in GATA2 sequencing should prompt deletion analysis, even in cases without infections, dysmorphic features or neurocognitive impairment. Disclosures:No relevant conflicts of interest to declare.

Genomic Architecture and Treatment Response In Pediatric Acute Myeloid Leukemia: A Report From The Children's Oncology Group

BackgroundChildhood acute myeloid leukemia (AML) is characterized by chromosomal instability and requires intensive therapy for cure. Here we present a large-scale study from 3 Children's Oncology Group (COG) trials to define the genomic architectural profiles of pediatric AML and to describe whole-arm gains and losses and focal rearrangements using SNP microarrays, accounting for mutation status, non-neoplastic cell infiltration and aneuploidy. We also investigate the association between somatic copy number aberrations (CNAs) and event free survival (EFS). Patients and MethodsA total of 505 matched tumor-remission samples from 459 children with de novo AML were obtained from the COG studies AAML-0531, AAML-03P1, and CCG-2961. 254 paired tumor-remission samples were genotyped on the Affymetrix SNP 6.0 chip at the University of Washington, Seattle, WA, and 251 on the Illumina 2.5M OmniQuad at the Children's Hospital of Philadelphia, PA. All genotyping output was converted to log R ratio and B-allele frequency values and annotations updated to hg19, GRCh37. Illumina intensities were additionally corrected for patterns of genomic wave.A matched allele-specific copy number analysis of tumors (ASCAT) was performed using ASCAT 2.2. A total of 246 (98%) Illumina and 247 (97%) Affymetrix samples passed quality control criteria. ASCAT profiles of patients genotyped on both platforms were manually reviewed and those with highest false CNA calls excluded. Samples with a low signal-to-noise ratio were either eliminated or visually annotated. All CNA segments were manually inspected and false positives removed. The resulting 452 ASCAT profiles were stratified into the risk categories 1) favorable, e.g. inv(16), t(16;16), t(8;21), NPM1, and CEBPα, 2) standard, e.g. normal karyotype, +8, +21, +22, del(7q), del(9q), abnormal 11q23, or other structural changes, and 3) poor, e.g. -5, -7, del(5q), or FLT3/ITD+. GISTIC 2.0 was used to identify genomic areas with significant recurrent aberrations, e.g. amplifications, deletions, and copy-neutral loss-of-heterozygosity (CN-LOH) events. Finally, we investigated whether profiles of genomic instability in the AML genome are predictive of 3 year EFS by using the total number of CNAs as a measure for allelic imbalance. ResultsThe inter-platform concordance for samples genotyped on the two platforms was very high. On average leukemic samples acquire 1.14 somatic CNAs, with a mean of 1.1, 1.3, and 0.8 in the favorable, standard, and poor risk groups respectively (Fig 1). CN-LOH events are observed in 14% (n = 64) of the patients, with 28% involving chromosome 13, and others involving the arms of 11p (23%), 1p (11%), 9p (8%), 7q (6%), 19q (6%), and 3q (5%). Known mutations in AML were enriched in recurrent focal CNA regions, as shown by amplifications on 17q24 (*TK1), 1q32, 3q28, 11q23 (*MLL), 6q27 (*DLL1), 2q32.1, and 4q35.2 (Fig 2). Focal CN-LOH regions were confined to 11p15.5 (*NUP98, *PICALM, *WT1), 1p36.3 (*RUNX3, *NRAS), 9p24.3 (*MLLT3), 3q25.3, 6p23 and 7q35 (*MLL3). Deletions include 7q36.1 (*MLL3, *EZH2), 16p13.11 (*MYH11), 9q21.32, 11p13 (*WT1), 2q37.1 (*IDH1, *DNMT3A), 10p12.31 (*MLLT10), 11q23.3 (*MLL), 16q22.1 (*CBFB), and 1p36.3 (*RUNX3). The association between CNA status and 3 year EFS approached statistical significance for all patients (Table 1), and EFS was significantly lower in standard risk patients with CNAs (51% no CNA vs 34% with CNAs, P 0.03). CNA status did not alter the event risk in the other risk groups. [Display omitted] [Display omitted] Table 1Prognostic Properties of CNAsRisk categoryCNATotal3yr EFSLogrank POverall021554% ± 7%0.0871+23547% ± 7%Favorable08066% ± 11%0.8091+8765% ± 10%Standard08051% ± 11%0.0301+10034% ± 10%Poor04842% ± 15%0.3761+4137% ± 15% ConclusionsThe number of CNAs occurring in this cohort is lower than previously reported. However, the presence of somatic CNAs in standard risk patients is significantly associated with a worse treatment outcome that is similar to the poor risk group. This study confirms the established regions of CNA enrichment in pediatric AML and identifies novel regions that may involve driving mediators of tumor fitness and/or acquired resistance to targeted therapies. Disclosures:No relevant conflicts of interest to declare.

Unstable genomes elevate transcriptome dynamics

The challenge of identifying common expression signatures in cancer is well known, however the reason behind this is largely unclear. Traditionally variation in expression signatures has been attributed to technological problems, however recent evidence suggests that chromosome instability (CIN) and resultant karyotypic heterogeneity may be a large contributing factor. Using a well-defined model of immortalization, we systematically compared the pattern of genome alteration and expression dynamics during somatic evolution. Co-measurement of global gene expression and karyotypic alteration throughout the immortalization process reveals that karyotype changes influence gene expression as major structural and numerical karyotypic alterations result in large gene expression deviation. Replicate samples from stages with stable genomes are more similar to each other than are replicate samples with karyotypic heterogeneity. Karyotypic and gene expression change during immortalization is dynamic as each stage of progression has a unique expression pattern. This was further verified by comparing global expression in two replicates grown in one flask with known karyotypes. Replicates with higher karyotypic instability were found to be less similar than replicates with stable karyotypes. This data illustrates the karyotype, transcriptome, and transcriptome determined pathways are in constant flux during somatic cellular evolution (particularly during the macroevolutionary phase) and this flux is an inextricable feature of CIN and essential for cancer formation. The findings presented here underscore the importance of understanding the evolutionary process of cancer in order to design improved treatment modalities.

Extensive chromosome conservatism in Atlantic butterflyfishes, genus Chaetodon Linnaeus, 1758: Implications for the high hybridization success

Chaetodontidae is a family of marine butterflyfishes phylogenetically derived within the Perciformes, whose representatives are important members of coral reef ecosystems worldwide. Biological aspects of Chaetodontidae have been intensively studied, except for chromosomal analyses. Although previous reports indicate a conserved perciform-like karyotype in butterflyfishes, it remains unclear if this pattern extends to the chromosomal microstructure. New cytogenetic data are presented for two Chaetodontidae species (Chaetodon striatus and Chaetodon ocellatus) from the Western Atlantic, including karyotyping, C-banding, Ag-NOR, CMA3/DAPI staining, and two-color-FISH for mapping of 18S and 5S ribosomal genes. All populations of both species shared a karyotype with 2n=48 acrocentric chromosomes, with pericentromeric C-positive heterochromatin and 5S and 18S rDNA located in the same region on the long arms of pairs 10 and 21, respectively. The cytogenetic similarities within and between both Chaetodon species reinforce their remarkable stability also in the chromosome microstructure. Therefore, speciation in this genus was not followed by significant karyotypic changes. Both ecological and chromosomal properties, combined with recent diversification, might be responsible for the apparent karyotype stasis and high hybridization levels found in marine butterflyfishes.

Transposable Elements: Powerful Contributors to Angiosperm Evolution and Diversity

Transposable elements (TEs) are a dominant feature of most flowering plant genomes. Together with other accepted facilitators of evolution, accumulating data indicate that TEs can explain much about their rapid evolution and diversification. Genome size in angiosperms is highly correlated with TE content and the overwhelming bulk (>80%) of large genomes can be composed of TEs. Among retro-TEs, long terminal repeats (LTRs) are abundant, whereas DNA-TEs, which are often less abundant than retro-TEs, are more active. Much adaptive or evolutionary potential in angiosperms is due to the activity of TEs (active TE-Thrust), resulting in an extraordinary array of genetic changes, including gene modifications, duplications, altered expression patterns, and exaptation to create novel genes, with occasional gene disruption. TEs implicated in the earliest origins of the angiosperms include the exapted Mustang, Sleeper, and Fhy3/Far1 gene families. Passive TE-Thrust can create a high degree of adaptive or evolutionary potential by engendering ectopic recombination events resulting in deletions, duplications, and karyotypic changes. TE activity can also alter epigenetic patterning, including that governing endosperm development, thus promoting reproductive isolation. Continuing evolution of long-lived resprouter angiosperms, together with genetic variation in their multiple meristems, indicates that TEs can facilitate somatic evolution in addition to germ line evolution. Critical to their success, angiosperms have a high frequency of polyploidy and hybridization, with resultant increased TE activity and introgression, and beneficial gene duplication. Together with traditional explanations, the enhanced genomic plasticity facilitated by TE-Thrust, suggests a more complete and satisfactory explanation for Darwin’s “abominable mystery”: the spectacular success of the angiosperms.

Extra-low-frequency magnetic fields alter cancer cells through metabolic restriction

Background: Biological effects of extra-low-frequency (ELF) magnetic fields (MFs) have lacked a credible mechanism of interaction between MFs and living material. Objectives: To examine the effect of ELF-MFs on cancer cells. Methods: Five cancer cell lines were exposed to ELF-MFs within the range of 0.025–5 µT, and the cells were examined for karyotype changes after 6 d. Results: All cancer cells lines lost chromosomes from MF exposure, with a mostly flat dose-response. Constant MF exposures for three weeks allow a rising return to the baseline, unperturbed karyotypes. From this point, small MF increases or decreases are again capable of inducing karyotype contractions (KCs). Our data suggest that the KCs are caused by MF interference with mitochondria’s adenosine triphosphate synthase (ATPS), compensated by the action of adenosine monophosphate-activated protein kinase (AMPK). The effects of MFs are similar to those of the ATPS inhibitor, oligomycin. They are amplified by metformin, an AMPK stimulator, and attenuated by resistin, an AMPK inhibitor. Over environmental MFs, KCs of various cancer cell lines show exceptionally wide and flat dose-responses, except for those of erythroleukemia cells, which display a progressive rise from 0.025 to 0.4 µT. Conclusions: The biological effects of MFs are connected to an alteration in the structure of water that impedes the flux of protons in ATPS channels. These results may be environmentally important, in view of the central roles played in human physiology by ATPS and AMPK, particularly in their links to diabetes, cancer and longevity.

Acellular amniotic membrane: an appropriate scaffold for fibroblast proliferation

Various materials have been investigated as possible skin substitutes to repair skin defects such as burns. Because of its unique characteristics, acellular amniotic membrane seems to provide a good scaffold for cell cultures. To investigate the proliferation of fibroblasts on an amniotic membrane scaffold, and the preparation of a temporary skin substitute using this method. Neonatal foreskin tissue was harvested after circumcision and used for isolation of skin fibroblasts. The skin sample was refrigerated in cell-culture solution, and later treated with trypsin, minced, and incubated in the same solution at 37 °C with in an atmosphere of 95% O2 /5% CO2 . The confluent cultures were treated with trypsin, and fibroblasts were subcultured up to the 10th passage. Cells were tested for microbial contamination, presentation of major histocompatibility complex, and karyotype changes. Amniotic membrane was harvested after elective caesarean section from donors who had been screened for infection. The membrane was washed and then subjected to three freeze-thaw cycles, before having the cells removed. The fibroblasts were seeded onto the scaffold, and after 24 h, the prepared skin substitute was ready. This was examined under electron microscopy. The skin substitute showed excellent growth of fibroblasts on the amniotic membrane scaffold. Fibroblasts had excellent adherence to and viability on the acellular amniotic membrane, which seems to provide an acceptable temporary skin substitute that can be used for wounds.