Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.

• MeSH
• Arabidopsis genetika   MeSH
• duplikace genu * MeSH
• exprese genu MeSH
• genom rostlinný MeSH
• geny rRNA * MeSH
• nestabilita genomu MeSH
• odolnost vůči nemocem genetika   MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné geny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To assess the prognostic relevance of activating mutations of FLT3 gene on outcome of allogeneic transplantations in AML patients, we performed an analysis of all patients with FLT3 mutations registered in the Czech Acute Leukemia Clinical Register (ALERT) from 2003 till the end of 2005. Within the mentioned period 170 patients with AML of median age 56 years (23-77) were investigated for FLT3 mutation, within them 36 cases (21%) with FLT3 mutations (32 FLT3 ITD and 4 FLT3 D835) were found. Out of FLT3 ITD positive patients 13 had allogeneic transplantation, 20 patients with mutations of FLT3 were treated with chemotherapy without transplantation. Results of the treatment of these patients were compared with the results of the group of patients without FLT3 mutation, which was according to other characteristics identical with the group of patients with FLT3 mutations (n=134). Median overall survival (OS) was significantly shorter for patients with FLT3 ITD (34.8 weeks) than for those without FLT3 mutations (67.7 weeks; P=0.028). Median OS of patients with FLT3 ITD who had allogeneic transplantation was 42.5 weeks; median OS of patients with FLT3 ITD treated only with chemotherapy was 29.6 weeks (P=0.362). After allogeneic transplantation, median OS of FLT3 mutations negative patients was similar to FLT3 ITD positive patients (46.7 versus 42.5 weeks; P=0.443). Our results suggest that at present there is no strong evidence that FLT3 status alone should influence the decision to proceed to allogeneic transplantation in AML patients. Decision to proceed to alogeneic transplantation should not be based on the FLT3 status only, but it should also consider other prognostic factors.

• MeSH
• akutní nemoc MeSH
• časové faktory MeSH
• dospělí MeSH
• duplikace genu MeSH
• homologní transplantace MeSH
• Kaplanův-Meierův odhad MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• myeloidní leukemie farmakoterapie   genetika   chirurgie   MeSH
• prognóza MeSH
• registrace statistika a číselné údaje   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tandemové repetitivní sekvence MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Geografické názvy
• Česká republika MeSH

Alu-mediated tandem duplication of exons 4 and 5 (g.15815_22218dup6404) is a novel mutation that has been detected in the LAMP2 gene (Xq24). This exon copy number variation was found in two brothers with the typical phenotype of Danon disease, including characteristic myocardial changes on magnetic resonance imaging. The 6.4 kb duplication was identified in both boys by a combination of exon dosage qPCR analyses and duplication breakpoint/junction mapping. The rearrangement results in a plethora of abnormal LAMP2 splicing variants and also in use of likely cryptic splice sites in the 3' terminus of LAMP2 gene. Although we found minute amounts of normal LAMP2B and LAMP2A mRNAs, no protein was detectable in peripheral blood leukocytes by flow cytometry in both brothers. Uniquely, the fraction of LAMP2-deficient granulocytes (0.06%) assessed by flow cytometry in the patients' asymptomatic mother substantially differed from the random distribution of X-chromosome inactivation in her leukocytes. This discrepancy was later explained by molecular genetic methods as a consequence of mosaic distribution of the mutation in her somatic tissues. Altogether, we report a novel and mosaically distributed exon copy number rearrangement in the LAMP2 gene and comment on obstacles this genetic setup presents to the overall cellular and molecular diagnostic algorithm of Danon disease. Our observations of the mosaicism in the asymptomatic mother suggest that similarly affected females could be a potentially under-diagnosed Danon disease carrier group and that LAMP2 flow cytometry, because of its supreme sensitivity, can be an efficient method for pedigree screening.

• MeSH
• dospělí MeSH
• duplikace genu * MeSH
• exony * MeSH
• fenotyp MeSH
• glykogenóza typu IIb diagnóza   genetika   MeSH
• granulocyty cytologie   MeSH
• leukocyty cytologie   MeSH
• lidé MeSH
• magnetická rezonanční tomografie MeSH
• membránový protein 2 asociovaný s lyzozomy genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mozaicismus MeSH
• mutace MeSH
• myokard patologie   MeSH
• průtoková cytometrie MeSH
• rodokmen MeSH
• sourozenci MeSH
• tkáňová distribuce MeSH
• variabilita počtu kopií segmentů DNA MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

• MeSH
• alternativní sestřih * MeSH
• exony genetika   MeSH
• genetická variace MeSH
• geny BRCA1 * MeSH
• geny BRCA2 * MeSH
• komplementární DNA MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• místa sestřihu RNA * MeSH
• mutace MeSH
• nádory prsu genetika   MeSH
• nádory vaječníků genetika   MeSH
• počítačová simulace MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Patients with acute myeloid leukaemia (AML) positive for internal tandem duplication (ITD) mutations of FLT3 have poor outcomes. Quizartinib, an oral, highly potent, selective, type 2 FLT3 inhibitor, plus chemotherapy showed antitumour activity with an acceptable safety profile in patients with FLT3-ITD-positive newly diagnosed AML. The aim of the study was to compare the effect of quizartinib versus placebo on overall survival in patients with FLT3-ITD-positive newly diagnosed AML aged 18-75 years. METHODS: We conducted a randomised, double-blind, placebo-controlled, phase 3 trial comparing quizartinib and placebo in combination with chemotherapy in induction and consolidation, followed by quizartinib or placebo single-agent continuation, in patients with FLT3-ITD-positive newly diagnosed AML at 193 hospitals and clinics in 26 countries in Europe; North America; and Asia, Australia, and South America. Patients aged 18-75 years were eligible. Patients were randomly assigned (1:1) to the quizartinib group or the placebo group by an independent biostatistician through an interactive web and voice response system, stratified by region, age, and white blood cell count at diagnosis. Patients, investigators, funders, and contract research organisations were masked to treatments assigned. Induction therapy comprised a standard 7 + 3 induction regimen of cytarabine 100 mg/m2 per day (or 200 mg/m2 per day allowed if institutional or local standard) by continuous intravenous infusion from day 1 to day 7 and anthracycline (daunorubicin 60 mg/m2 per day or idarubicin 12 mg/m2 per day) by intravenous infusion on days 1, 2, and 3, then quizartinib 40 mg orally or placebo once per day, starting on day 8, for 14 days. Patients with complete remission or complete remission with incomplete neutrophil or platelet recovery received standard consolidation with high-dose cytarabine plus quizartinib (40 mg per day orally) or placebo, allogeneic haematopoietic cell transplantation (allo-HCT), or both as consolidation therapy, followed by continuation of single-agent quizartinib or placebo for up to 3 years. The primary outcome was overall survival, defined as time from randomisation until death from any cause and assessed in the intention-to-treat population. Safety was evaluated in all patients who received at least one dose of quizartinib or placebo. This study is registered with ClinicalTrials.gov (NCT02668653). FINDINGS: Between Sept 27, 2016, and Aug 14, 2019, 3468 patients with AML were screened and 539 patients (294 [55%] male patients and 245 [45%] female patients) with FLT3-ITD-positive AML were included and randomly assigned to the quizartinib group (n=268) or placebo group (n=271). 148 (55%) of 268 patients in the quizartinib group and 168 (62%) of 271 patients in the placebo group discontinued the study, primarily because of death (133 [90%] of 148 in the quizartinib group vs 158 [94%] of 168 in the placebo group) or withdrawal of consent (13 [9%] of 148 in the quizartinib group vs 9 [5%] of 168 in the placebo group). Median age was 56 years (range 20-75, IQR 46·0-65·0). At a median follow-up of 39·2 months (IQR 31·9-45·8), median overall survival was 31·9 months (95% CI 21·0-not estimable) for quizartinib versus 15·1 months (13·2-26·2) for placebo (hazard ratio 0·78, 95% CI 0·62-0·98, p=0·032). Similar proportions of patients in the quizartinib and placebo groups had at least one adverse event (264 [100%] of 265 in the quizartinib group and 265 [99%] of 268 in the placebo group) and one grade 3 or higher adverse event (244 [92%] of 265 in the quizartinib group and 240 [90%] of 268 in the placebo group). The most common grade 3 or 4 adverse events were febrile neutropenia, hypokalaemia, and pneumonia in both groups and neutropenia in the quizartinib group. INTERPRETATION: The addition of quizartinib to standard chemotherapy with or without allo-HCT, followed by continuation monotherapy for up to 3 years, resulted in improved overall survival in adults aged 18-75 years with FLT3-ITD-positive newly diagnosed AML. Based on the results from the QuANTUM-First trial, quizartinib provides a new, effective, and generally well tolerated treatment option for adult patients with FLT3-ITD-positive newly diagnosed AML. FUNDING: Daiichi Sankyo.

• MeSH
• akutní myeloidní leukemie * farmakoterapie   genetika   MeSH
• benzothiazoly * terapeutické užití   MeSH
• cytarabin MeSH
• dospělí MeSH
• dvojitá slepá metoda MeSH
• fenylmočovinové sloučeniny * terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   MeSH
• senioři MeSH
• tyrosinkinasa 3 podobná fms antagonisté a inhibitory   genetika   MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze III MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH

BACKGROUND: Acipenseriformes is a basal lineage of ray-finned fishes and comprise 27 extant species of sturgeons and paddlefishes. They are characterized by several specific genomic features as broad ploidy variation, high chromosome numbers, presence of numerous microchromosomes and propensity to interspecific hybridization. The presumed palaeotetraploidy of the American paddlefish was recently validated by molecular phylogeny and Hox genes analyses. A whole genome duplication in the paddlefish lineage was estimated at approximately 42 Mya and was found to be independent from several genome duplications evidenced in its sister lineage, i.e. sturgeons. We tested the ploidy status of available chromosomal markers after the expected rediploidization. Further we tested, whether paralogs of Hox gene clusters originated from this paddlefish specific genome duplication are cytogenetically distinguishable. RESULTS: We found that both paralogs HoxA alpha and beta were distinguishable without any overlapping of the hybridization signal - each on one pair of large metacentric chromosomes. Of the HoxD, only the beta paralog was unequivocally identified, whereas the alpha paralog did not work and yielded only an inconclusive diffuse signal. Chromosomal markers on three diverse ploidy levels reflecting different stages of rediploidization were identified: quadruplets retaining their ancestral tetraploid condition, semi-quadruplets still reflecting the ancestral tetraploidy with clear signs of advanced rediploidization, doublets were diploidized with ancestral tetraploidy already blurred. Also some of the available microsatellite data exhibited diploid allelic band patterns at their loci whereas another locus showed more than two alleles. CONCLUSIONS: Our exhaustive staining of paddlefish chromosomes combined with cytogenetic mapping of ribosomal genes and Hox paralogs and with microsatellite data, brings a closer look at results of the process of rediploidization in the course of paddlefish genome evolution. We show a partial rediploidization represented by a complex mosaic structure comparable with segmental paleotetraploidy revealed in sturgeons (Acipenseridae). Sturgeons and paddlefishes with their high propensity for whole genome duplication thus offer suitable animal model systems to further explore evolutionary processes that were shaping the early evolution of all vertebrates.

• MeSH
• diploidie * MeSH
• duplikace genu * MeSH
• genomika * MeSH
• genotypizační techniky MeSH
• hybridizace in situ fluorescenční * MeSH
• mikrosatelitní repetice genetika   MeSH
• ribozomy genetika   MeSH
• rybí proteiny genetika   MeSH
• ryby genetika   MeSH
• sekvenční homologie nukleových kyselin * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation.

• MeSH
• Arabidopsis genetika   MeSH
• DNA bakterií genetika   MeSH
• duplikace genu genetika   MeSH
• inzerční mutageneze genetika   MeSH
• proteiny huseníčku genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML), with internal tandem duplications (ITDs) accounting for up to 30% of its mutations. To analyze the impact of individual ITDs on the expression profile of immature myeloid cells, we have established 32D cell lines expressing nine different FLT3/ITDs isolated from AML patients and subjected them to whole genome expression profiling and 2DE/LC/MS proteomics. Our data indicate that in comparison to the controls, FLT3/ITD-positive 32D cells exhibit less mature expression profiles resembling early hematopoietic progenitors. Moreover, our results suggest that there exist biological differences among individual ITD variants.

• MeSH
• akutní myeloidní leukemie genetika   patologie   MeSH
• blastická krize genetika   patologie   MeSH
• buněčná diferenciace MeSH
• buněčné dělení MeSH
• exony MeSH
• genetická variace MeSH
• klonování DNA MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• Retroviridae genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese MeSH
• tandemové repetitivní sekvence genetika   MeSH
• tyrosinkinasa 3 podobná fms genetika   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

To analyze the influence of distinct combinations of molecular aberrations on outcome after allogeneic hematopoietic stem cell transplantation (HSCT) for cytogenetically normal acute myeloid leukemia (CN-AML), a retrospective registry analysis was performed on 702 adults undergoing HSCT in first complete remission (CR). Patients were grouped according to presence or absence of NPM1 mutations (NPM1(mut)) and FLT3 internal tandem duplications (FLT3-ITD). Double-negative patients were evaluated for mutations of the CCAAT/enhancer binding protein α gene (CEBPα). The influence of genotypes on relapse, non-relapse mortality, leukemia-free survival (LFS) and overall survival (OS), and a prognostic classification combining NPM1/FLT3-ITD profile and classical risk factors were calculated. Two-year OS from HSCT was 81 ± 5% in NPM1(mut)/FLT3(wt), 75 ± 3% in NPM1(wt)/FLT3(wt), 66 ± 3% in NPM1(mut)/FLT3-ITD, and 54 ± 7% in NPM1(wt)/FLT3-ITD (P = .003). Analysis of CEBPα among patients with NPM1(wt)/FLT3(wt) revealed excellent results both in patients with CEBPα(mut) and with a triple negative genotype (2-year OS: 100%/77 ± 3%). In a Cox-model of predefined variables, age, FLT3-ITD and >1 course of chemotherapy to reach CR were risk factors associated with inferior outcome, regardless of NPM1 mutational status, variations of transplant protocols, or development of graft-versus-host disease. In a prognostic risk classification, 2-year OS/LFS rates were 88 ± 3%/79 ± 4% without any, 77 ± 2%/73 ± 3% with one, and 53 ± 4%/50 ± 4 with ≥2 risk factors (P = .003/.002).

• MeSH
• akutní myeloidní leukemie klasifikace   genetika   mortalita   terapie   MeSH
• cytogenetické vyšetření MeSH
• dospělí MeSH
• genotyp MeSH
• homologní transplantace MeSH
• indukce remise MeSH
• lidé středního věku MeSH
• lidé MeSH
• míra přežití MeSH
• následné studie MeSH
• nemoc štěpu proti hostiteli MeSH
• prognóza MeSH
• retrospektivní studie MeSH
• staging nádorů MeSH
• tandemové repetitivní sekvence genetika   MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• tyrosinkinasa 3 podobná fms genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Akutní myeloidní leukémie (AML) je klonálním maligním onemocněním krvetvorby. Byly identifikovány molekulární markery, dovolující stratifikovat AML pacienty do prognostických skupin, s návazností na cílenou terapii. Interní tandemové duplikace genu FLT3(FLT3/ITD) jsou nepříznivým prognostickým faktorem u AML. Nicméně patogeneze FLT3/ITD- indukovaného fenotypu AML blastů není dosud uspokojivě rozřešena. Expresní profilování rozsáhlého souboru 32D linií nesoucích autentické FLT3/ITD mutanty původem z lidských blastů pomocí RNA micoarrays a 2DE LC/MS proteomiky umožní identifikovat charakteristické rysy FLT3/ITD, nicméně může odhalit i drobné, ač biologicky významné rozdíly mezi typovými FLT3/ITD mutacemi. Korelace výsledků získaných na myším 32D systémus lidským materiálem primárních AML blastů umožní identifikovat biologicky relevantní prognostické markery vyskytující se v kontextu FLT3/ITD, s potenciálním využitím v diagnostice a cílené terapii pacientů s AML.; Acute myeloid leukemia (AML) is a malignant clonal disorder of hematopoiesis. Molecular markers have been identified allowing to stratify AML patients into prognostic subgroups, with relevance to the therapy. Internal tandem duplications of FLT3 gene (FLT3/ITD) represent an unfavorable prognostic marker in AML. Despite this fact, the pathogenesis of the FLT3/ITD-induced phenotype has not been elucidated to satisfaction. Expression profiling of a large set of 32D cell lines carrying authentic FLT3/ITDs derived from human AML blasts, using the microarray technology, allows to identify characteristic features of FLT3/ITDs. Nevertheless, it might disclose subtle, yet biologically important differences between typical FLT3/ITD mutants. Correlation of the data obtained on the murine 32D system with the data obtained on authentic human AML blasts might reveal novel biologically relevant prognostic markers with potential usage in diagnostics and tailored treatment of patients with AML.

• MeSH
• akutní myeloidní leukemie patofyziologie   MeSH
• genetické inženýrství MeSH
• mutace MeSH
• proteomika MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• signální transdukce MeSH
• tandemové repetitivní sekvence MeSH
• tyrosinkinasa 3 podobná fms MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• hematologie a transfuzní lékařství
• onkologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR