Karyotypic Data Research Articles

Intraspecific Karyotypic Variability Among Accessions ofPsoralea corylifoliaL.-A Medicinally Important Plant

Psoralea corylifolia L. is a traditional healer used in skin diseases of human beings. The karyotypes of 8 accessions of P. corylifolia L. were examined to explore the intrapopulation chromosome structural variations. Although all the accessions exhibited a chromosome number of 2n=22, they could be differentiated by their karyotype formulae and quantitative parameters of the karyotypes. Significant differences were noted among accessions in the number of metacentric, submetacentric and subtelocentric chromosomes, chromosome length, total haploid complement, arm ratio and centromeric index. Accession IC 111239 showed highest tendency towards karyotype symmetry, whereas accession IC 11228 presented the most asymmetrical nature confirming more heterogeneity. The present investigation also traces for the first time structural chromosomal polymorphism among P. corylifolia accessions, and emphasizes the possible mechanisms underlying the origin and establishment of this variation. The widespread karyotypic variability seems to have originated through repeated interchanges involving a number of non-homologous chromosomes. Phenetic representation of genetic relationship on the basis of karyotype data separated the accessions in two groups, which reveal intraspecific chromosome polymorphism.

Genome size and chromosome number in velvet worms (Onychophora)

The Onychophora (velvet worms) represents a small group of invertebrates (~180 valid species), which is commonly united with Tardigrada and Arthropoda in a clade called Panarthropoda. As with the majority of invertebrate taxa, genome size data are very limited for the Onychophora, with only one previously published estimate. Here we use both flow cytometry and Feulgen image analysis densitometry to provide genome size estimates for seven species of velvet worms from both major subgroups, Peripatidae and Peripatopsidae, along with karyotype data for each species. Genome sizes in these species range from roughly 5-19 pg, with densitometric estimates being slightly larger than those obtained by flow cytometry for all species. Chromosome numbers range from 2n = 8 to 2n = 54. No relationship is evident between genome size, chromosome number, or reproductive mode. Various avenues for future genomic research are presented based on these results.

Karyotype of Nannospalax ehrenbergi (Nehring 1898) (Rodentia: Spalacidae) in the Mosul Province, Iraq

The karyotypes of mole rats Nannospalax ehrenbergi (Nehring 1897) from two closely located populations from Mosul province, Iraq, were investigated. The karyotype of this form was 2n=52, NF= 76, NFa=72 which consists of 11 pairs of metacentric/submetacentric autosomes, and 14 pairs of acrocentric autosomes. The X chromosome was large and metacentric, whereas the Y chromosome was small and acrocentric. The species occurrence in Iraq, based on literature overview and the present study, was updated. Karyotypic data for the Nannospalax ehrenbergi from Iraq are presented for the first time.

Monoclonal B-Cell Lymphocytosis Exhibiting Immunophenotypic Features Consistent with Marginal Zone Origin: What Is This Entity?

AbstractAbstract 1587Monoclonal B-cell lymphocytosis (MBL) with an immunophenotype consistent with marginal zone origin (MBL-MZ), that can be either CD5− or CD5+ but atypical for CLL, and also lacking an IGH/CCND1 translocation, is an increasingly recognised entity with poorly understood biological background and clinical significance. In particular, it is not yet clarified whether it represents a precursor state to one of the distinct lymphoma entities recognized by WHO as deriving from MZ cells or whether it constitutes a novel entity, likely with similar ontogeny. To obtain insight into this issue we retrospectively evaluated a series of 71 patients (male/female: 35/36, median age: 73.3 years) with lymphocytosis (median lymphocyte count: 5.77 × 109/l) detected incidentally on a routine blood test. No case had lymphadenopathy, organomegaly or any clinical features to suggest a concurrent marginal zone lymphoma. Hemoglobin and platelet counts were normal in all cases; 15/57 (26%) cases had paraproteinemia. Peripheral blood immunophenotyping revealed the presence of a clonal B-cell population with Matutes score <2 in all cases. Individual markers were expressed as follows: CD5: 15/71, CD23: 7/70, CD79β: 60/64, FMC7: 50/67, CD49d: 35/35, CD38: 6/53. In 8/45 cases assessed with CT-scan and/or ultrasound, borderline splenomegaly was observed. Histopathological examination of the bone marrow biopsy (BMB) was available in 11 cases and demonstrated mostly mixed patterns of neoplastic lymphocytic inflitration from small B cells. Karyotype data were available in 66 cases; 48/66 (72.7%) had abnormal karyotype. Main cytogenetic findings are as follows: (1) translocations: n=16 cases of which 3 carried t(2;7)(p11;q21/22); (2) isochromosome 17q: n=8; (3) trisomy 12: n=8; (4) del(7q): n=7; (5) trisomy 3: n=4. Immunogenetic analysis revealed overusage of the IGHV4–34 gene (15/63, 23.8%). Notably, the IGHV1–2 gene was utilized by a single case, thus sharply contrasting (p<0.0001) splenic marginal-zone lymphoma (SMZL) with a reported frequency of IGHV1–2 in excess of 30%. Seven of 63 rearrangements (11.1%) carried IGHV genes with no somatic mutations, whereas the remainder (56/63, 88.9%) exhibited some impact of somatic hypermutation (SHM), ranging from minimal to (mostly) pronounced. Overall, cases of the present study exhibited a significantly (p<0.005) higher SHM load compared to SMZL. With a median follow-up of 4.9 years (0.8–20), 54 cases (group A) remain stable with no signs of progression. The remaining 17 cases (group B) have MBL along with clinical or histopathologic evidence of lymphoma. In particular: (i) a female with MBL as an incidental finding, also carrying t(2;7), was eventually diagnosed with gastric MALT lymphoma at +11 months from presentation; immunogenetic analysis confirmed clonal identity between the MBL and the lymphoma; (ii) 2 cases developed lymphadenopathy; (iii) a single case developed diffuse large B cell lymphoma of the skin; and, (iv) 13 cases developed splenomegaly and, thus, can be considered as either SMZL or splenic lymphoma/leukemia unclassifiable (SLLU). Groups A and B did not differ in terms of demographics, diagnostic blood counts (including clonal MBL count) and SHM status; the only difference concerned cytogenetic profiles, with i(17)(q10) and del(7q) being almost exclusive to group A. In conclusion, we demonstrate that MBL-MZ can be the presenting feature of occult MZ lymphoma, most frequently SMZL/SLLU. However, in a sizeable proportion of cases, MBL-MZ remains stable over time with no evidence of organ involvement and distinct immunogenetic features from SMZL, thereby raising the possibility that it might represent a newcomer to the spectrum of B-cell lymphoproliferations of MZ origin. Disclosures:No relevant conflicts of interest to declare.

Frequency and Prognostic Relevance of Gene Mutations in Pediatric AML Patients At First Relapse.

AbstractAbstract 2480Relapsed pediatric AML patients respond poorly to conventional re-induction therapy and as a consequence long term survival rates of these patients are low (up to 36%). Accurate characterization and risk group stratification at first relapse may facilitate personalized, targeted re-induction therapies for these patients and ultimately improve outcome. Gene mutations provide putative targets in personalized treatment (e.g. FLT3/ITD, KIT or RAS mutations) and their incidence and prognostic relevance in pediatric AML at first relapse remains to be elucidated. Changes in mutational status occur during therapy; hence, assessment of mutations at first relapse is warranted to establish the accurate mutation status of the leukemic cells before the start of salvage treatment.Mutational status at first relapse as well as clinical and karyotypic data were retrospectively analyzed in a large set (n = 240) of relapsed non-FAB M3, non-Down syndrome AML patients younger than 19 years. The majority of patients (88%) were uniformly treated at first relapse according to the ‘Relapsed AML 2001/01' protocol of the International Berlin-Frankfurt-Münster (BFM) study group that involved two re-induction courses of chemotherapy with FLAG as standard treatment with or without liposomal daunorubicin (randomization), followed by allogeneic stem cell transplantation in most cases. Other patients received FLAG based (5%) or other high dose cytarabin based therapy (6%).We screened the relapse samples for hotspot mutations in a selected panel of genes (FLT3, WT1, KIT, N-RAS, K-RAS, NPM1) relevant for AML and found one or more mutations in 139 out of 240 patients (57.9%). Gene mutations were mutually exclusive in 73 out of 139 patients carrying mutations (52.5%), while in 66 out of 139 patients (47.5%) two or more gene mutations were observed. FLT3/ITD mutations coincided with 50% of NPM1 and 53% of WT1 exon 7 mutated cases (p=.003 and p<.0001 respectively). The frequencies of mutations at first relapse are summarized in the Table below including their impact on event free and overall survival, according to uni-variate analysis. In multivariate analyses, we included mutations and other variables (e.g. FAB type, WBC) with a uni-variate P value below 0.2 to exclude confounding factors. From these analyses, three independent factors significantly increased the risk of a second relapse (RFS after first relapse diagnosis); WT1 mutations (HR=11.2, P<.0001), WT1 single nucleotide variants (HR 3.4, P=.003) and FAB type M7 (HR= 2.2, P=.027). Different independent factors were associated with dismal overall survival after first relapse including FLT3/ITD mutations (HR=2.2, P=.028) and high WBC (HR=3.2, P=.004) at first relapse.In conclusion, mutations in the studied panel of genes in this large cohort of pediatric relapsed AML patients were frequent. Overall, we detected receptor tyrosine kinase mutations in more than 25% of the patients, indicating the large proportion of relapsed patients that is eligible for targeted receptor tyrosine kinase inhibitor treatment. The gene mutations studied here provide strong prognostic factors for dismal outcome after first relapse. The relevance of these markers in predicting outcome should be validated in a prospective setting. Accurate risk group stratification of relapsed AML patients should be based on clinical and molecular characterization performed at first relapse. In addition, mutations in drugable genes can identify patients that are eligible for personalized, targeted re-induction strategies.TableUni-variate analysis of the association of relevant gene mutations in pediatric AML at first relapse with outcome after 1st relapse, ranked according to frequency.FrequencyRFS after 1st relapseOS after 1st relapseHazard ratioCIPHazard ratioCIPFLT3/ITD17.8%1.20.6–2.30.5861.40.9–2.40.153WT1 mutations10.2%5.02.0–12.40.0012.00.9–4.10.077WT1 SNV10.2%2.01.1–3.80.0251.00.6–1.70.989KIT9.0%0.50.2–1.30.1370.60.3–1.10.114KRAS7.5%1.60.8–3.30.1760.80.4–1.50.422NRAS c12/136.4%0.60.2–1.90.3761.20.6–2.20.658NPM15.7%0.20.1–1.70.1560.70.3–1.80.519NRAS c611.9%0.10.0–3970.5112.50.9–6.80.072ITD = internal tandem duplication, SNV = single nucleotide variant, c = codon, CI = 95% confidence interval Disclosures:No relevant conflicts of interest to declare.

The Common Deleted Region in the Short ARM of Chromosome 17 in CLL Includes the MAP2K4 GENE

AbstractAbstract 4562As part of a preliminary study, we show the advantages of whole genome screening of CLL patient samples and highlight several issues. Firstly, 17p rearrangements appear to be far more complex than a simple loss of TP53; secondly, commercially available TP53 FISH probe sets may miss important genetic events for a significant number of CLL patients. Finally we show the possible role of the tumour suppressor MAP2K4 in the pathogenesis of CLL.Specific genome aberrations are recognised prognostic factors in CLL. FISH based genome risk stratifications have been used in clinical decision-making for over a decade. However molecular karyotyping is gaining acceptance as an alternative that not only overcomes the limitations of FISH but also provides a comprehensive whole genome scan. Indeed, several recent studies (Rinaldi et al., BJH 2011, Parker et al., Leukemia 2011, Quilette et al, Blood 2011) using different array platforms have revealed novel clinically relevant cryptic genome aberrations. Here we compare FISH and molecular karyotyping data of 50 diagnostic and 26 follow up samples from CLL patients.Since the loss of the TP53 gene was associated with resistance to chemotherapy, a short survival and a poor prognosis (Dohner et al, NEJM, 2000) CLL patients are routinely screened by FISH to assess its presence. Here we present 6 cases out of the 76 (5 presentation and 1 follow up), that have a deletion of the short arm of chromosome 17 (17p) by whole genome scanning with the 8×60K Agilent array platform. In 4 of the 6 cases the genome loss involved nearly the entire 17p and as expected, they were shown to carry TP53 deletion, when tested by commercial FISH probes. The remaining two cases were found to carry much smaller deletions of 17p. In one of these aCGH found a 5Mb loss within the 17p13.1 cytoband, at address chr17: 10.2–15.2. This region houses many genes, including MAP2K4, but does not include the TP53 (genome address hg19 chr17: 7.57–7.59), although FISH with commercial probes indicated TP53 loss. In the second case, the aCGH analysis identified a cryptic 120Kb loss housing the entire MAP2K4 at 17p12. As expected, commercial FISH probe analysis failed to detect any TP53 changes. However, FISH screening with BACs probes RP11–170H18 (covering the MAP2K4) and RP11–89D11 (TP53) confirmed the array results in both cases.The tumour suppressor gene MAP2K4 (mitogen-activated protein kinase kinase 4) was revealed by our aCGH study to represent the common deleted region in all cases with 17p loss. It was identified in 6 out of 76 CLL samples (8%) by the 60K genomic arrays. MAP2K4 is a putative tumor suppressor gene frequently found to be deleted in various cancer types, including solid tumors, ovarian and breast cancer (Teng DH, et al Cancer Res 1997, Davis SJ, et al BMC Cancer 2011), but to our knowledge has not been described in hematological malignancy. Further work is required to assess the frequency, type and clinical relevance of the genetic aberrations within the short arm of chromosome 17 in CLL patients. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic Analysis of Lagria villosa (Coleoptera, Tenebrionidae): Emphasis on the Mechanism of Association of the Xyp Sex Chromosomes

The Xy_p sex determination mechanism is the system most frequent and ancestral to Coleoptera. Moreover, the presence of argyrophilous material associated with the sex bivalent is described as being responsible for the maintenance and association of these chromosomes. There are no karyotype data available regarding the genus Lagria and no consensus in the literature regarding the argyrophilous material present in the lumen of sex bivalent. Therefore, the aim of this work was to investigate the mechanism of sex chromosome bivalent association in Lagria villosa by analyzing the argyrophilous nature of the material present in the Xy_p lumen. It was also intended to characterize L. villosa cytogenetically. The analysis of meiotic cells showed 2n = 18 = 16+Xy_p for males and 2n = 18 = 16+XX in females and the meiotic formula was 2n = 8^II+Xy_p. The C-banding showed blocks of pericentromeric heterochromatin in all chromosomes except in the y_p chromosome. In these regions, the use of fluorochromes revealed the presence of heterochromatin containing GC rich DNA sequences. The study of synaptonemal complex showed a gradual increase in the electron-density of the axial elements of the sex chromosomes and their association with strongly electron-dense material. The pepsin pretreatment revealed that the material impregnated by silver is protein.

The First Cytogenetic Characterization of Atemnids: Pseudoscorpions with the Highest Chromosome Numbers (Arachnida: Pseudoscorpiones)

The karyotypes of pseudoscorpions of the family Atemnidae (Arachnida: Pseudoscorpiones) were studied for the first time. Karyotype data for 7 species have been obtained. The diploid chromosome numbers of most species considerably exceed the numbers reported in pseudoscorpions so far, with males ranging between 65 and 143. In spite of this, the sex chromosome system of atemnids is characterized by the same features that are found in the majority of other pseudoscorpions with an X0 system; the X chromosome is metacentric and is the largest chromosome or one of the largest chromosomes of the karyotype. Male meiotic cells of Atemnus politus contain 1 or 2 autosome multivalents; most specimens had 2 multivalents. The multivalents were composed of 4, 6, 8 or 10 chromosomes. Multivalent number and structure was consistent within each of the studied individuals. The same number of chromosomes in all of the males examined suggests that multivalents are generated by reciprocal translocations. The high diversity of multivalents suggests considerable range of translocation heterozygosity in the studied population.

Revisando el estatus taxonómico y la partición ecológica de los monos nocturnos del género Aotus en el noroccidente de Colombia, con notas sobre Aotus zonalis Goldman, 1914

As part of the project Programa Regional de Biodiversidad, subprograma Linea Base de Conoci- miento de la Biodiversidad del Choco , designed to document the mammalian fauna of the Biogeographic Choco, museum voucher specimens from the Colombian Chocoan Region deposited in American institutions have been taxonomically assessed in the past three years. Herein, we review the morphological variation, distribution patterns, and taxonomy of night monkeys in the genus Aotus from northwestern Colombia deposited at the Field Museum of Natural History (FMNH), and GIS based analyses and Maxent modeling are used to define the geographic extent and ecological limiting factors of the analyzed taxa. Our Principal Component Analysis showed high variation in skull morphology among Aotus from northwestern Colombia. However, differences observed in both discrete and morphometric analyses of the interorbital region of A. zonalis (more depressed than that in the grographically adjacent A. griseimembra and A. lemurinus ) appear as good diagnostic characters for this taxon. Our analyses on the ecological variation associated with collecting localities of Aotus specimens support the geographic subdivision previously proposed based on karyotypic data. Based on obtained models of potential distribution we define the location and extent of potential contact zones among species of Aotus from northwestern Colombia.

Fish Karyome: A karyological information network database of Indian Fishes

‘Fish Karyome’, a database on karyological information of Indian fishes have been developed that serves as central source for karyotype data about Indian fishes compiled from the published literature. Fish Karyome has been intended to serve as a liaison tool for the researchers and contains karyological information about 171 out of 2438 finfish species reported in India and is publically available via World Wide Web. The database provides information on chromosome number, morphology, sex chromosomes, karyotype formula and cytogenetic markers etc. Additionally, it also provides the phenotypic information that includes species name, its classification, and locality of sample collection, common name, local name, sex, geographical distribution, and IUCN Red list status. Besides, fish and karyotype images, references for 171 finfish species have been included in the database. Fish Karyome has been developed using SQL Server 2008, a relational database management system, Microsoft's ASP.NET-2008 and Macromedia's FLASH Technology under Windows 7 operating environment. The system also enables users to input new information and images into the database, search and view the information and images of interest using various search options. Fish Karyome has wide range of applications in species characterization and identification, sex determination, chromosomal mapping, karyo-evolution and systematics of fishes.

Cytogenetic and flow cytometry data expand knowledge of genome evolution in three Coffea species

Karyotype and nuclear 2C-value data are considered important in taxonomic and evolutionary approaches in Coffea. Still, new methods are needed to further support such studies, especially to determine the progenitors of Coffea arabica. In this work, new cytogenetic and flow cytometry data were used to compare Coffea arabica, Coffea canephora and Coffea congensis. These data corroborate the hypothesis that C. canephora and C. congensis originated from a single ancestor, whose basic chromosome number was x = 11. In agreement with the observations of other authors, the karyotype and mean 2C-values confirm that C. arabica is a true allotetraploid originating from two diploid Coffea species with similar genomes. Although C. canephora and C. congensis have been considered potential progenitors of C. arabica, karyotype comparison revealed that only one of these species may be parental to C. arabica. These accurate cytogenetic and flow cytometry data contribute to expand our knowledge of the Coffea genome, as well as of possible progenitors of C. arabica.

Molecular Insights into X;BTA5 Chromosome Rearrangements in the Tribe Antilopini (Bovidae)

For a clade that includes Antilope, Gazella,Nanger and Eudorcas (Antilopinae), X;BTA5 translocation is a synapomorphy. Using a combination of fluorescence in situ hybridization (FISH) probes and polymerase chain reaction techniques, we provide (i) the first insight into the X;BTA5 architecture which differs in the species under study: Antilope cervicapra (genus Antilope), Gazella leptoceros (genus Gazella) and Nanger dama ruficollis (genus Nanger), (ii) determination of interstitial satellite DNA at the X;BTA5 junctions, and (iii) determination of repetitive sequences occupying constitutive heterochromatin of Xp arms in the studied species. The distribution of 2 repetitive DNA families in the centromeric regions of all chromosomes has been investigated by FISH with probes representing satellite I and satellite II DNA in all studied species. In this context, we discuss a markedly smaller centromere in the BTA5 (Y2) unfused chromosomes in males in the XY1Y2 determining system in comparison with other acrocentrics. An analysis of karyotypic data described in current published studies revealed a disparity with the data determined by FISH. In this report, we document chromosomal fusions in the 3 species mentioned resulting from FISH with painting probes prepared from cattle (Bos taurus). The number and chromosomal location of nucleolus organizer regions were determined by FISH. In the present study, we emphasize the importance of chromosomal rearrangement verification, particularly, if they are used for phylogenetic analysis.

Karyotype analysis in some species of Consolida (Ranunculaceae) from Iran

AbstractConsolida (dc.) S. F. Gray belongs to Ranunculaceae. The genus includes about 52 species worldwide. Here we report the diploid chromosome number and chromosome size and morphology for six Consolida species. For C. anthoroidea, C. leptocarpa, C. paradoxa and C. rugulosa the diploid chromosome number is reported for the first time. All investigated species have a diploid chromosome number of 2n = 2x = 16, except for C. persica having 2n = 2x = 14. The karyotypes of all six taxa are asymmetric, consisting of all four major chromosome types: metacentric, submetacentric, subtelocentric and telocentric chromosome type. However, considering the karyotype formula, all six species could be distinguished. In all taxa, metacentric chromosome pair 1 possesses a satellite. The only exception is C. rugulosa having an additional satellite positioned on metacentric chromosome pair 2. Karyotype data allow the separation of Aconitella from Consolida. Karyotype data plus morphological evidence support the reduction of C. paradoxa to formae level of C. rugulosa. (© 2013 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Distribution of 45S rDNA sites in chromosomes of plants: Structural and evolutionary implications

Background45S rDNA sites are the most widely documented chromosomal regions in eukaryotes. The analysis of the distribution of these sites along the chromosome in several genera has suggested some bias in their distribution. In order to evaluate if these loci are in fact non-randomly distributed and what is the influence of some chromosomal and karyotypic features on the distribution of these sites, a database was built with the position and number of 45S rDNA sites obtained by FISH together with other karyotypic data from 846 plant species.ResultsIn angiosperms the most frequent numbers of sites per diploid karyotype were two and four, suggesting that in spite of the wide dispersion capacity of these sequences the number of rDNA sites tends to be restricted. The sites showed a preferential distribution on the short arms, mainly in the terminal regions. Curiously, these sites were frequently found on the short arms of acrocentric chromosomes where they usually occupy the whole arm. The trend to occupy the terminal region is especially evident in holokinetic chromosomes, where all of them were terminally located. In polyploids there is a trend towards reduction in the number of sites per monoploid complement. In gymnosperms, however, the distribution of rDNA sites varied strongly among the sampled families.ConclusionsThe location of 45S rDNA sites do not vary randomly, occurring preferentially on the short arm and in the terminal region of chromosomes in angiosperms. The meaning of this preferential location is not known, but some hypotheses are considered and the observed trends are discussed.

Cytogenetic Risk Remains a Major Predictor of Outcome in Pediatric AML and ALL Treated with Allogeneic Stem Cell Transplantation,

Abstract 3524 Background:Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) each represent a heterogeneous group of disorders resulting from diverse mechanisms of leukemogenesis. Cytogenetic abnormalities are considered reliable risk-stratification tools in both AML and ALL. Most studies assessing clinical outcome according to cytogenetic risk consider patients treated with conventional intensive chemotherapy. The impact of cytogenetics on outcome in patients treated uniformly with hematopoietic stem cell transplantation (HSCT), especially in the pediatric group, is not well studied. We evaluated whether allogeneic HSCT has an impact on outcome as predicted by cytogenetic risk at diagnosis. Methods:Bone marrow karyotyping and FISH data from 61 pediatric AML patients and 37 pediatric patients with ALL was reviewed and patients divided into two groups according to their cytogenetic risk: one group combined patients with favorable or intermediate risk and the second group comprised patients with adverse-risk cytogenetics. Cytogenetic risk classification was according to WHO and MRC cytogenetic criteria. The AML cohort included 47 (77%) patients treated in their first remission (CR1) while most non-CR1 AML patients were treated with HSCT in CR2. Of the ALL patients, 18 (49%) were treated with HSCT in CR1. The outcome of patients with either favorable/intermediate cytogenetic risk or adverse cytogenetics was compared for AML and ALL patients, first including all patients, then including only CR1 patients. The median age of patients was 8 years (range: 9 months to 13 years). Results:Patients with ALL and favorable / intermediate cytogenetics had significantly longer overall survival (OS) and event-free survival (EFS) as compared with the adverse-risk group (P=0.02 and P=0.03 for OS and EFS, respectively). A similar trend was observed when only CR1 ALL patients were evaluated, although their number is small. Notably, separation between the CR1 ALL and CR2 ALL groups was stronger when only Philadelphia chromosome positive and MLL positive patients were considered (P=0.01 for OS and EFS for all patients; P=0.003 for OS and EFS for CR1patients only). Also for pediatric AML, combined favorable / intermediate-risk cytogenetics was associated with significantly longer OS (P=0.03) and EFS (P=0.04) when all patients were included (Fig. 1). A similar trend was obtained when only CR1 AML patients were considered (P=0.03 for both OS and EFS). Conclusions:This study suggests that stratification of AML and ALL in pediatric patients according to their cytogenetic risk remains relevant even when treated with HSCT and irrespective if patients were treated in CR1 or CR2 after relapse. In particular, MLL gene rearrangements and the Philadelphia chromosome remain poor prognostic factors in pediatric ALL despite HSCT treatment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

MOLECULAR Karyotyping In CLL - COST Neutral Alternative to FISH SCREENING for Detection of Clinically Relevant Genome Imbalances,

Abstract 3561Specific genome aberrations are recognised prognostic factors in CLL. FISH-based genome risk classifications used in clinical decision making for over a decade. However molecular karyotyping is gaining acceptance as an alternative, albeit cost and skill demanding, that not only overcomes the limitations of FISH but provides comprehensive whole genome scanning. Indeed, several recent studies (Rinaldi et al., BJH 2011, Parker et al., Leukemia 2011, Quilette et al, Blood 2011) using different array platforms have revealed novel clinically relevant cryptic genome aberrations. Here we compare FISH and molecular karyotyping data of 40 diagnostic and follow up samples from 22 CLL patients. The whole genome screening (WGS) was performed with 8×60K oligonucleotide arrays (Agilent) using DNA from mononuclear fraction of p.blood/bone marrow samples as well as from fixed chromosome preparations and commercial reference DNA. Following manufacturerÕs protocols and bio-informatics routines (Z score and ADM1/2 algorithms) WGS was done at a cost comparable to the expense of routine FISH screening for 13q14, p53, ATM loss and trisomy 12. Genome array screening identified clonal imbalances recognised by FISH if present above 13%. In addition, WGS provided novel information in 73% of the samples, the results of which are summarised as follows (see figure below): (i) The 13q genome loss found in 10/13 presentation samples was stratified by size and location resulting in a positive assessment for 40% of the cases positive by FISH for del 13q14 as class II/type 2 deletions. These are known to be associated with high risk for disease progression; (ii) A common deleted region of 206.2Kb at @ 50556688–50699677 (hg19) was found to encompass the DLEU2 gene and is seen in 5/13 presentation samples with 13q loss; (iii) In 3 out of 13 cases FISH-positive for 13q loss, disease progression was accompanied by a proximal extension of the deletion to include the RB1 gene region thus acquiring the status of class II/type 2 deletion (high risk) (iv) Genome loss involving IGH sequences at 14q32.32, consistent with B cell type VDJ gene rearrangements was detected in all samples, while deletions within IG kappa and IG lambda were found in 5/25 patients; (v) Cryptic deletion of the ATM gene region was detected in 2 samples undetected by FISH; (vi) Total genome imbalances (TGA) in presentation samples varied between 3 and 17 with higher levels found in samples with p53 loss and class II, type 2 13q loss; (vii) Persistent disease was found to be associated with increase of TGA, some of which appeared to be non-random, such as loss of the whole or part of chromosome 19 and deletions of 4q21 region. [Display omitted] In conclusion, the application of 8×60K genome arrays provides established and growing genome-wide clinically relevant information as a cost neutral assay. A strategy by which array interrogation is carried out at presentation, followed by therapy response monitoring using FISH on a selected marker in CLL is a plausible alternative to routine practice. In cases where FISH has provided evidence for persistent disease, array screening will assess the genome damage, especially if carried on CD19(+)cell isolates, to assist treatment decisions. Disclosures:No relevant conflicts of interest to declare.

Demographic processes in the montane Atlantic rainforest: Molecular and cytogenetic evidence from the endemic frog Proceratophrys boiei

Historical climatic refugia predict genetic diversity in lowland endemics of the Brazilian Atlantic rainforest. Yet, available data reveal distinct biological responses to the Last Glacial Maximum (LGM) conditions across species of different altitudinal ranges. We show that species occupying Brazil’s montane forests were significantly less affected by LGM conditions relative to lowland specialists, but that pre-Pleistocene tectonics greatly influenced their geographic variation. Our conclusions are based on palaeoclimatic distribution models, molecular sequences of the cytochrome b, 16S, and RAG-1 genes, and karyotype data for the endemic frog Proceratophrys boiei. DNA and chromosomal data identify in P. boiei at least two broadly divergent phylogroups, which have not been distinguished morphologically. Cytogenetic results also indicate an area of hybridization in southern São Paulo. The location of the phylogeographic break broadly matches the location of a NW–SE fault, which underwent reactivation in the Neogene and led to remarkable landscape changes in southeastern Brazil. Our results point to different mechanisms underpinning diversity patterns in lowland versus montane tropical taxa, and help us to understand the processes responsible for the large number of narrow endemics currently observed in montane areas of the southern Atlantic forest hotspot.

Chromosomal complements of some Atlantic Blennioidei and Gobioidei species (Perciformes)

A remarkable degree of chromosomal conservatism (2n=48, FN=48) has been identified in several families of Perciformes. However, some families exhibit greater karyotypic diversity, although there is still scant information on the Atlantic species. In addition to a review of karyotypic data available for representatives of the suborders Blennioidei and Gobioidei, we have performed chromosomal analyses on Atlantic species of the families Blenniidae, Ophioblennius trinitatis Miranda-Ribeiro, 1919 (2n=46; FN=64) and Scartella cristata (Linnaeus, 1758) (2n=48; FN=50), Labrisomidae, Labrisomus nuchipinnis (Quoy &amp; Gaimard, 1824) (2n=48; FN=50) and Gobiidae, Bathygobius soporator (Valenciennes, 1837) (2n=48; FN=56). Besides variations in chromosome number and karyotype formulas, Ag-NOR sites, albeit unique, were located in different positions and/or chromosome pairs for the species analyzed. On the other hand, the heterochromatic pattern was more conservative, distributed predominantly in the centromeric/pericentromeric regions of the four species. Data already available for Gobiidae, Blenniidae and Labrisomidae show greater intra- and interspecific karyotypic diversification when compared to other groups of Perciformes, where higher uniformity is found for various chromosome characteristics. Evolutionary dynamism displayed by these two families is likely associated with population fractionation resulting from unique biological characteristics, such as lower mobility and/or specific environmental requirements.

Chromosomal complements of some Atlantic Blennioidei and Gobioidei species (Perciformes).

A remarkable degree of chromosomal conservatism (2n=48, FN=48) has been identified in several families of Perciformes. However, some families exhibit greater karyotypic diversity, although there is still scant information on the Atlantic species. In addition to a review of karyotypic data available for representatives of the suborders Blennioidei and Gobioidei, we have performed chromosomal analyses on Atlantic species of the families Blenniidae, Ophioblennius trinitatis Miranda-Ribeiro, 1919 (2n=46; FN=64) and Scartella cristata (Linnaeus, 1758)(2n=48; FN=50), Labrisomidae, Labrisomus nuchipinnis (Quoy & Gaimard, 1824)(2n=48; FN=50) and Gobiidae, Bathygobius soporator (Valenciennes, 1837)(2n=48; FN=56). Besides variations in chromosome number and karyotype formulas, Ag-NOR sites, albeit unique, were located in different positions and/or chromosome pairs for the species analyzed. On the other hand, the heterochromatic pattern was more conservative, distributed predominantly in the centromeric/pericentromeric regions of the four species. Data already available for Gobiidae, Blenniidae and Labrisomidae show greater intra- and interspecific karyotypic diversification when compared to other groups of Perciformes, where higher uniformity is found for various chromosome characteristics. Evolutionary dynamism displayed by these two families is likely associated with population fractionation resulting from unique biological characteristics, such as lower mobility and/or specific environmental requirements.

The utility of fluorescence in situ hybridization analysis in diagnosing myelodysplastic syndromes is limited to cases with karyotype failure

Fluorescence in situ hybridization (FISH) is being used increasingly in cytogenetic diagnosis of myelodysplastic syndromes (MDS). However, the utility of FISH in this role has not been well-defined. A total of 249 de novo MDS patients were submitted to karyotyping and FISH analysis for −5/del(5)(q31), −7/del(7)(q31), +8, −17/i(17)(q10), del(20)(q12), and −Y. Of the 234 patients with available karyotypic data, 143 cases (60.9%) demonstrated normal karyotype and 91 cases (39.1%) showed abnormal karyotype. FISH confirmed R-banding findings in 96.6% (226/234) of samples with successful karyotyping and detected cytogenetic abnormalities in 46.7% (7/15) of cases with karyotype failure. Of the 3.4% (8/234) patients showing discrepancies between FISH and R-banding, FISH revealed cytogenetic abnormalities in four patients with normal karyotypes and four patients with complex karyotypes. These results highlight FISH analysis has limited value in MDS cases with successful karyotyping and is only informative in MDS cases with karyotype failure.