Molecular and cytogenetic analyses of cryptic species within the Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae) grouping: species delimitations, karyotypic evolution and intraspecific diversification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of Long-Term Outcomes, Cytogenetic and Molecular Responses with Imatinib Mesylate in Early and Late Chronic-Phase Chronic Myeloid Leukemia: A Report from a Single Institute

Here we compare the management and survival outcomes of chronic myeloid leukemia (CML) patients who had early or late imatinib mesylate (IM) therapy. The cytogenetic and molecular responses of 189 CML patients were analyzed. Of this group, 121 patients were classified as the early chronic phase (ECP) group and started IM within 12 months of diagnosis. The other 68 patients were classified as the late chronic phase (LCP) group who had been treated with interferon (IFN)-alpha-2 and crossed over to IM more than 12 months after diagnosis. The overall rates of complete cytogenetic response (CCyR) and major molecular response (MMR) at last follow-up were 83.6 and 78.1% in the ECP and LCP groups, respectively. The CCyR rates were 89.3 (for ECP patients) versus 73.5% (for LCP patients; p < 0.0001). At last follow-up, 82.4% ECP and 64.2% LCP patients had achieved an MMR (p < 0.0001). No significant differences were noted between the two groups with regard to survival outcomes. Our experience reveals that IM is an effective rescue therapy in most CML LCP patients who are intolerant or in whom IFN-alpha therapy fails. Such therapeutic options should be considered in LCP patients, particularly in countries where IM may not be available. Copyright (C) 2012 S. Karger AG, Basel

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 Sao 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. (C) 2011 Elsevier Inc. All rights reserved.

Cytogenetic findings in pediatric radiation-induced atypical meningioma after treatment of medulloblastoma: case report and review of the literature

Ionizing radiation is the most recognized risk factor for meningioma in pediatric long-term cancer survivors. Information in this rare setting is exceptional. We report the clinical and cytogenetic findings in a radiation-induced atypical meningioma following treatment for desmoplastic medulloblastoma in a child. This is the second study to describe the cytogenetic aspects on radiation-induced meningiomas in children. Chromosome banding analysis revealed a 46, XX, t(1;3)(p22;q12), del(1)(p?)[8]/46, XX[12]. Loss of chromosome 1p as a consequence of irradiation has been proposed to be more important in the development of secondary meningiomas in adults. Deletions in the short arm of chromosome 1 also appear to be a shared feature in both pediatric cases so far analyzed.

The impact of polymorphisms in P-gp, DNA repair and folic acid metabolism genes in newly diagnosed multiple myeloma patients treated with thalidomide plus dexamethasone, with or without bortezomib

The principle aim of this study was to investigate biological predictors of response and resistance to multiple myeloma treatment. Two hypothesis had been proposed as responsible of responsiveness: SNPs in DNA repair and Folate pathway, and P-gp dependent efflux. As a first objective, panel of SNPs in DNA repair and Folate pathway genes, were analyzed. It was a retrospective study in a group of 454, previously untreated, MM patients enrolled in a randomized phase III open-label study. Results show that some SNPs in Folate pathway are correlated with response to MM treatment. MTR genotype was associated with favorable response in the overall population of MM patients. However, this relation, disappear after adjustment for treatment response. When poor responder includes very good partial response, partial response and stable/progressive disease MTFHR rs1801131 genotype was associated with poor response to therapy. This relation - unlike in MTR – was still significant after adjustment for treatment response. Identification of this genetic variant in MM patients could be used as an independent prognostic factor for therapeutic outcome in the clinical practice. In the second objective, basic disposition characteristics of bortezomib was investigated. We demonstrated that bortezomib is a P-gp substrate in a bi-directional transport study. We obtain apparent permeability rate values that together with solubility values can have a crucial implication in better understanding of bortezomib pharmacokinetics with respect to the importance of membrane transporters. Subsequently, in view of the importance of P-gp for bortezomib responsiveness a panel of SNPs in ABCB1 gene - coding for P-gp - were analyzed. In particular we analyzed five SNPs, none of them however correlated with treatment responsiveness. However, we found a significant association between ABCB1 variants and cytogenetic abnormalities. In particular, deletion of chromosome 17 and t(4;14) translocation were present in patients harboring rs60023214 and rs2038502 variants respectively.

Impact of additional cytogenetic aberrations at diagnosis on prognosis of CML: long-term observation of 1151 patients from the randomized CML Study IV

The prognostic relevance of additional cytogenetic findings at diagnosis of chronic myeloid leukemia (CML) is unclear. The impact of additional cytogenetic findings at diagnosis on time to complete cytogenetic (CCR) and major molecular remission (MMR) and progression-free (PFS) and overall survival (OS) was analyzed using data from 1151 Philadelphia chromosome-positive (Ph(+)) CML patients randomized to the German CML Study IV. At diagnosis, 1003 of 1151 patients (87%) had standard t(9;22)(q34;q11) only, 69 patients (6.0%) had variant t(v;22), and 79 (6.9%) additional cytogenetic aberrations (ACAs). Of these, 38 patients (3.3%) lacked the Y chromosome (-Y) and 41 patients (3.6%) had ACAs except -Y; 16 of these (1.4%) were major route (second Philadelphia [Ph] chromosome, trisomy 8, isochromosome 17q, or trisomy 19) and 25 minor route (all other) ACAs. After a median observation time of 5.3 years for patients with t(9;22), t(v;22), -Y, minor- and major-route ACAs, the 5-year PFS was 90%, 81%, 88%, 96%, and 50%, and the 5-year OS was 92%, 87%, 91%, 96%, and 53%, respectively. In patients with major-route ACAs, the times to CCR and MMR were longer and PFS and OS were shorter (P < .001) than in patients with standard t(9;22). We conclude that major-route ACAs at diagnosis are associated with a negative impact on survival and signify progression to the accelerated phase and blast crisis.

Early molecular and cytogenetic response is predictive for long-term progression-free and overall survival in chronic myeloid leukemia (CML)

In the face of competing first-line treatment options for CML, early prediction of prognosis on imatinib is desirable to assure favorable survival or otherwise consider the use of a second-generation tyrosine kinase inhibitor (TKI). A total of 1303 newly diagnosed imatinib-treated patients (pts) were investigated to correlate molecular and cytogenetic response at 3 and 6 months with progression-free and overall survival (PFS, OS). The persistence of BCR-ABL transcript levels >10% according to the international scale (BCR-ABL(IS)) at 3 months separated a high-risk group (28% of pts; 5-year OS: 87%) from a group with >1-10% BCR-ABL(IS) (41% of pts; 5-year OS: 94%; P=0.012) and from a group with 1% BCR-ABL(IS) (31% of pts; 5-year OS: 97%; P=0.004). Cytogenetics identified high-risk pts by >35% Philadelphia chromosome-positive metaphases (Ph+, 27% of pts; 5-year OS: 87%) compared with 35% Ph+ (73% of pts; 5-year OS: 95%; P=0.036). At 6 months, >1% BCR-ABL(IS) (37% of pts; 5-year OS: 89%) was associated with inferior survival compared with 1% (63% of pts; 5-year OS: 97%; P<0.001) and correspondingly >0% Ph+ (34% of pts; 5-year OS: 91%) compared with 0% Ph+ (66% of pts; 5-year OS: 97%; P=0.015). Treatment optimization is recommended for pts missing these landmarks.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

BACKGROUND: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. METHODS: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. RESULTS: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. CONCLUSIONS: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution

Chromosome painting in placental mammalians illustrates that genome evolution is marked by chromosomal synteny conservation and that the association of chromosomes 3 and 21 may be the largest widely conserved syntenic block known for mammals. We studied intrachromosomal rearrangements of the syntenic block 3/21 by using probes derived from chromosomal subregions with a resolution of up to 10–15 Mbp. We demonstrate that the rearrangements visualized by chromosome painting, mostly translocations, are only a fraction of the actual chromosomal changes that have occurred during evolution. The ancestral segment order for both primates and carnivores is still found in some species in both orders. From the ancestral primate/carnivore condition an inversion is needed to derive the pig homolog, and a fission of chromosome 21 and a pericentric inversion is needed to derive the Bornean orangutan condition. Two overlapping inversions in the chromosome 3 homolog then would lead to the chromosome form found in humans and African apes. This reconstruction of the origin of human chromosome 3 contrasts with the generally accepted scenario derived from chromosome banding in which it was proposed that only one pericentric inversion was needed. From the ancestral form for Old World primates (now found in the Bornean orangutan) a pericentric inversion and centromere shift leads to the chromosome ancestral for all Old World monkeys. Intrachromosomal rearrangements, as shown here, make up a set of potentially plentiful and informative markers that can be used for phylogenetic reconstruction and a more refined comparative mapping of the genome.

Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders.

Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD). Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma. other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis. To date, the critical genomic segment and candidate genes involved in these deletions have not been identified. In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization. As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3. In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1). Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment. Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster. This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD.

Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.

Deletion of the TP53 gene on chromosome 17p13.1 is the prognostic factor associated with the shortest survival in CLL. We used array-based comparative genomic hybridisation (arrayCGH) to identify additional DNA copy number changes in peripheral blood samples from 74 LRF CLL4 trial patients, 37 with >or=5% and 37 without TP53-deleted cells. ArrayCGH reliably detected deletions on 17p, including the TP53 locus, in cases with >or=50%TP53-deleted cells detected by fluorescence in situ hybridisation, plus seven additional cases with deleted regions on 17p excluding TP53. Losses on chromosomal regions 18p and/or 20p were found exclusively in cases with >or=5%TP53-deleted cells (por=5%TP53-deleted cases (p=0.02). In particular, amplification of 2p and deletion of 6q were both more frequent. Cases with >20%TP53-deleted cells had the worst prognosis in the LRF CLL4 trial.

Abnormalities on 1q and 7q are associated with poor outcome in sporadic Burkitt's lymphoma. A cytogenetic and comparative genomic hybridization study.

Comparative genomic hybridization (CGH) studies have demonstrated a high incidence of chromosomal imbalances in non-Hodgkin's lymphoma. However, the information on the genomic imbalances in Burkitt's Lymphoma (BL) is scanty. Conventional cytogenetics was performed in 34 cases, and long-distance PCR for t(8;14) was performed in 18 cases. A total of 170 changes were present with a median of four changes per case (range 1-22). Gains of chromosomal material (143) were more frequent than amplifications (5) or losses (22). The most frequent aberrations were gains on chromosomes 12q (26%), Xq (22%), 22q (20%), 20q (17%) and 9q (15%). Losses predominantly involved chromosomes 13q (17%) and 4q (9%). High-level amplifications were present in the regions 1q23-31 (three cases), 6p12-p25 and 8p22-p23. Upon comparing BL vs Burkitt's cell leukemia (BCL), the latter had more changes (mean 4.3 +/- 2.2) than BL (mean 2.7 +/- 3.2). In addition, BCL cases showed more frequently gains on 8q, 9q, 14q, 20q, and 20q, 9q, 8q and 14q, as well as losses on 13q and 4q. Concerning outcome, the presence of abnormalities on 1q (ascertained either by cytogenetics or by CGH), and imbalances on 7q (P=0.01) were associated with a short survival.

Lake Nabugabo: a potential reservoir for mukene (Rastrineobola argentea)

"Mukene"Rastrineobola argentea occurs in lakes Victoria, Kyoga, Nabugabo and the Victoria Nile. On lakes Victoria and Kyoga the commercial fishery is well developed. While some fishing occurs at isolated places on the Victoria Nile, the species is yet to be exploited on lake Nabugabo although the potential exists. Experimental fishing (light fishing and beach seining) was conducted on Lake Nabugabo based at the Lake Nabugabo Holiday and Conference Center. Specimens obtained were analyzed for biological and fisheries parameters. Mukene on Lake Nabugabo still grows to a large adult size compared to the Lake Victoria populations. Size at first maturity at 42mmSL is close to that observed on Lake Victoria. Peak breeding occurred between August and January. Unlike on Lake Victoria, R.argentea in Lake Nabugabo is not infested by the cestode Ligula intestinalis that is common in these fishes. Catch rates of 9.8 kg/lamp/hr by the lampara net on this lake compare favorably with many parts of Lake Victoria and on Lake Kyoga where a commercial fishery exists. The amount of by-catch species in the catches however is high and does not therefore favour the establishment of the fishery on this lake. Human exploitation of mukene on Lake Nabugabo would also affect the Nile perch fishery which depends on the species as a source of food.

Cytogenetic characterization of the Passiflora edulis Sims x Passiflora cincinnata Mast. interspecific hybrid and its parents.

The genus Passiflora L. consists of approximately 530 widely distributed species, including Passiflora edulis, which has drawn interest because of its commercial and agronomic value. Passiflora cincinnata is another important species owing to its long flowering period and resistance or tolerance to diseases and pests. In the present study, the meiotic segregation and pollen viability of an interspecific hybrid (P. edulis x P. cincinnata) and its parents were analyzed. The genomic contents were characterized using chromomycin A3 (CMA3)/40-60-diamidino-2-phenylindole (DAPI) staining, fluorescent in situ hybridization with 5S/45S ribosomal DNA (rDNA), genomic in situ hybridization (GISH), and inter-simple sequence repeat (ISSR) markers. The results indicated the diploid chromosome number for the parents and interspecific hybrid was 2n = 18. We also observed regular meiosis, one pair of S rDNA sites, and two pairs of 45S rDNA sites that colocalized with two pairs of CMA3 /DAPI- bands. The GISH data revealed three distinct chromosomal groups in the hybrid. The genetic origins of the interspecific hybrid, and its relationship with its parents were also confirmed using ISSR markers.

Selection of efficient rhizobial symbionts for Cratylia argentea in the cerrado biome.

Cratylia argentea é uma leguminosa arbustiva nativa do cerrado, com alto potencial para produção de forragem e recuperação de áreas degradadas. Este trabalho teve como objetivo isolar, caracterizar e selecionar estirpes de rizóbios eficientes na simbiose com C. argentea. As bactérias foram isoladas de nódulos de plantas com 12 meses de idade, cultivadas em solo de cerrado, em vasos. Foram obtidas 25 estirpes, que apresentaram alta variabilidade em relação às características morfológicas e simbióticas. Sementes de C. argentea foram plantadas em potes contendo 5kg de solo de cerrado, e mantidos em casa de vegetação. Os tratamentos consistiram em 25 isolados, controle (sem N e sem inoculação) e adubação nitrogenada (5mgN·planta-1·semana-1), em quatro repetições. As plantas foram colhidas 150 dias após o plantio para avaliar o número e a massa seca de nódulos, a massa seca e concentração de nitrogênio da parte área e das raízes. Foram calculadas as eficiências relativas e simbióticas. Treze estirpes se destacaram quanto ao acúmulo de massa seca (até 65,8%) e acúmulo de nitrogênio (até 76%), da parte aérea, em relação ao tratamento controle. Dentre estas, as estirpes 4 e 22 apresentaram maiores eficiências simbióticas (em torno de 20%). Portanto, estas estirpes apresentaram potencial para formulação de inoculantes, visando otimizar o processo simbiótico rizóbio-Cratylia e a incorporação de biomassa e nutrientes em sistema produtivos no cerrado.