Clinical impact of genomic analysis in children with B-acute lymphoblastic leukemia: A pilot study in Slovakia.

Acute lymphoblastic leukemia (ALL) belongs to a genetically heterogeneous disease associated with a wide range of chromosomal and molecular changes. Determining these changes at the time of diagnosis can help the therapeutic decision, and contributes to the prediction of patients' clinical outcomes. A part of B-ALL (B-other) lacks cytogenetic abnormalities with clinical relevance for prognosis. Our first goal was to retrospectively review genetic results of patients from 2013-2017 and identify number of B-other patients in Slovak population. The second goal was to implement single nucleotide polymorphism (SNP) array analysis to improve the diagnosis and risk stratification. In this study we reviewed 133 B-ALL patients. We found that nearly 40% of them (52 cases) belonged to the B-other ALL group. Eighteen B-other ALL patients were subjected to the analysis using SNP-array. Overall, we identified 126 cytogenomic changes and in 4 patients the SNP array revealed clinically relevant markers of adverse prognosis and high relapse risk. Integrating identified genetic changes into clinical practice can bring improvement of prognosis assessment for children with ALL in Slovakia.

Diagnosis and treatment of juvenile myelomonocytic leukemia in Slovak Republic: novel approaches.

Juvenile myelomonocytic leukemia (JMML) is a rare, aggressive clonal myeloproliferative disorder of infancy and early childhood caused by oncogenic mutations in genes involved in the Ras pathway. Long-term survival has only been achieved with hematopoietic stem cell transplantation (HSCT), being able to cure more than 50% patients. To manage the disease before HSCT remains an important issue with constant searching for optimal treatment modalities. According to several retrospective analyses, azacitidine (AZA) induced clinical and molecular responses in patients with relapsed JMML pre-transplant and post-transplant, suggesting its use as a promising "bridging" therapy before HSCT. In this paper we report our first consecutive cohort of patients with JMML treated at our institution as well as our experience with the diagnosis, novel treatment and management of these patients before the HSCT. We present 6 patients with JMML, harboring different somatic mutations (PTPN11 and NRAS), with distinct clinical features; 3 of them had been treated with AZA 75 mg/m2 i.v. on days 1 to 7 of a 28-day cycle before the HSCT. Response to therapy was evaluated after each cycle in accordance with the International response criteria. One patient had a progression of splenomegaly during the treatment and after three cycles he was urgently transplanted. At the present, he is remaining in complete remission 3 years after HSCT. Two patients showed impressive response following the first cycle of the therapy with a regression of splenomegaly and monocyte count, normalized leukocytes, platelets and absent blasts in peripheral blood. The treatment was well tolerated with no adverse effect recorded. The clinical activity and favorable toxicity of AZA in JMML provide a rationale for its use as a "bridging" therapy before HSCT. Prospective trials with accompanying translational studies are required to provide further information regarding individual factors that may direct the most appropriate choice of pretransplantation therapy.

Pilot Study of the Occurrence of Somatic Mutations in Ciliary Signalling Pathways as a Contribution Factor to Autosomal Dominant Polycystic Kidney Development.

Autosomal-dominant polycystic kidney disease (ADPKD) is an inherited disease that results in multiple kidney cysts, and it is a common cause of end-stage renal disease. Recent studies have shown that disease progression can be slowed by simultaneous disruption of the primary cilium and polycystins. The exact genetic mechanism of this process is still unknown. The aim of the present study was to characterize the mutation profile of ciliary signalling pathways in the renal epithelial cells of ADPKD patients. In our study, we performed an analysis of 110 genes encoding the components of Sonic Hedgehog, Hippo, Notch, Wnt and planar cell polarity signalling (PCP) by targeted next-generation sequencing. We analysed 10 formalin-fixed, paraffinembedded (FFPE) tissue samples of patients with ADPKD. We identified a unique mutation profile in each of the analysed ADPKD samples, which was characterized by the presence of pathogenic variants in eight to 11 genes involved in different signalling pathways. Despite the significant genetic heterogeneity of ADPKD, we detected five genes whose genetic variants affected most ADPKD samples. The pathogenic variants in NCOR2 and LRP2 genes were present in all analysed samples of ADPKD. In addition, eight out of 10 samples showed a pathogenic variant in the MAML2 and FAT4 genes, and six out of 10 samples in the CELSR1 gene. In our study, we identified the signalling molecules that may contribute to the cystogenesis and may represent potential targets for the development of new ADPKD treatments.

The asymmetric meiosis in pentaploid dogroses (Rosa sect. Caninae) is associated with a skewed distribution of rRNA gene families in the gametes.

In pentaploid dogroses, Rosa section Caninae (2n=5x=35), the pollen transmits one basic genome (x=7) derived from the seven segregating bivalents, whereas the egg transmits four basic genomes (4x=28) one set derived from the segregation of seven bivalents and three sets of univalent-forming chromosomes. Chromosomes from all five genomes carry 18-5.8-26S nuclear ribosomal DNA (rDNA) sites. This mode of sexual reproduction, known as permanent odd polyploidy, can potentially lead to the independent evolution of rDNA on bivalent- and univalent-forming chromosomes. To test this hypothesis, we analyzed rRNA gene families in pollen and somatic leaf tissue of R. canina, R. rubiginosa and R. dumalis. Six major rRNA gene families (alpha, beta, beta' gamma, delta and epsilon) were identified based on several highly polymorphic sites in the internal transcribed spacers (ITSs). At least two of the major rRNA gene families were found in each species indicating that rDNAs have not been homogenized across subgenomes. A comparison of ITS1 sequences from leaf and pollen showed differences: the shared beta rRNA gene family was more abundant among pollen clones compared to leaf clones and must constitute a major part of the rDNA loci on bivalent-forming chromosomes. The gamma and delta families were underrepresented in pollen genomes and are probably located predominantly (or solely) on the univalents. The results support the hypothesis that pentaploid dogroses inherited a bivalent-forming genome from a common proto-canina ancestor, a likely donor of the beta rDNA family. Allopolyploidy with distantly related species is likely to have driven evolution of Rosa section Caninae.

Preferential elimination of repeated DNA sequences from the paternal, Nicotiana tomentosiformis genome donor of a synthetic, allotetraploid tobacco.

Nicotiana tabacum (tobacco, 2n = 4x = 48) is a natural allotetraploid combining two ancestral genomes closely related to modern Nicotiana sylvestris and Nicotiana tomentosiformis. Here we examine the immediate consequences of allopolyploidy on genome evolution using 20 S4-generation plants derived from a single synthetic, S0 plant made by Burk in 1973 (Th37). Using molecular and cytogenetic methods we analysed 14 middle and highly repetitive sequences that together total approximately 4% of the genome. Two repeats related to endogenous geminiviruses (GRD5) and pararetroviruses (NtoEPRV), and two classes of satellite repeats (NTRS, A1/A2) were partially or completely eliminated at variable frequency (25-60%). These sequences are all from the N. tomentosiformis parent. Genomic in situ hybridization revealed additivity in chromosome numbers in two plants (2n = 48), while a third was aneuploid for an N. tomentosiformis-origin chromosome (2n = 49). Two plants had homozygous translocations between chromosomes of the S- and T-genomes. * The data demonstrate that genetic changes in synthetic tobacco were fast, targeted to the paternal N. tomentosiformis-donated genome, and some of the changes showed concordance with changes that presumably occurred during evolution of natural tobacco.

Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana.

An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.