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Refractory anaemia with ring sideroblasts (RARS) and marked thrombocytosis (RARS-T) is a provisional entity in the World Health Organisation 2008 classification and has previously been shown to have a high proportion of JAK2V617F (Janus Kinase 2) and SF3B1 (Splicing Factor 3B subunit 1) mutations. The purpose of the present study was to analyse the frequency of SF3B1 mutations in a large cohort of 111 patients with RARS-T and 33 patients with RARS and to explore the prognostic impact of SF3B1 mutational status on RARS-T. The frequency of SF3B1 mutations in RARS-T (96/111, 86.5%) and RARS (28/33, 84.8%) was similar. In RARS-T, median survival was better in SF3B1-mutated patients than in SF3B1-non-mutated patients (6.9 and 3.3 years, respectively, P=0.003). RARS can be differentiated from RARS-T by the frequency of JAK2V617F (0% vs 48.6%). In RARS-T patients, SF3B1 (P=0.021) and JAK2 mutations (P=0.016) were independent factors for a better prognosis. Altogether, our results confirm that RARS-T is an independent entity that should be recognised by the next World Health Organisation classification. The assessment of SF3B1 mutations is of prognostic interest in RARS-T patients. Younger age, JAK2V617F and SF3B1 mutations are the main predicting factors for survival in RARS-T. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented discussing detection of the activating mutation V617F in the Janus kinase 2 (JAK2).

Real-world data have revealed that a substantial portion of patients with myelodysplastic syndromes (MDS) does not respond to epigenetic therapy with hypomethylating agents (HMAs). The cellular and molecular reasons for this resistance to the demethylating agent and biomarkers that would be able to predict the treatment refractoriness are largely unknown. In this study, we shed light on this enigma by characterizing the epigenomic profiles of patients with MDS treated with azacitidine. Our approach provides a comprehensive view of the evolving DNA methylation architecture of the disease and holds great potential for advancing our understanding of MDS treatment responses to HMAs., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Genome assembly is one of the most important problems in computational genomics. Here, we suggest addressing an issue that arises in homology-based scaffolding, that is, when linking and ordering contigs to obtain larger pseudo-chromosomes by means of a second incomplete assembly of a related species. The idea is to use alignments of binned regions in one contig to find the most homologous contig in the other assembly. We show that ordering the contigs of the other assembly can be expressed by a new string problem, the longest run subsequence problem (LRS). We show that LRS is NP-hard and present reduction rules and two algorithmic approaches that, together, are able to solve large instances of LRS to provable optimality. All data used in the experiments as well as our source code are freely available. We demonstrate its usefulness within an existing larger scaffolding approach by solving realistic instances resulting from partial Arabidopsis thaliana assemblies in short computation time.

Azacitidine is the first drug to demonstrate a survival benefit for patients with MDS. However, only half of patients respond and almost all patients eventually relapse. Limited and conflicting data are available on predictive factors influencing response. We analyzed 128 patients from two institutions with MDS or AML treated with azacitidine to identify prognostic indicators. Genetic mutations in ASXL1, RUNX1, DNMT3A, IDH1, IDH2, TET2, TP53, NRAS, KRAS, FLT3, KMT2A-PTD, EZH2, SF3B1, and SRSF2 were assessed by next-generation sequencing. With a median follow up of 5.6 years median survival was 1.3 years with a response rate of 49%. The only variable with significant influence on response was del(20q). All 6 patients responded ( p = 0.012) but survival was not improved. No other clinical, cytogenetic or molecular marker for response or survival was identified. Interestingly, patients from poor-risk groups as high-risk cytogenetics (55%), t-MDS/AML (54%), TP53 mutated (48%) or relapsed after chemotherapy (60%) showed a high response rate. Factors associated with shorter survival were low platelets, AML vs. MDS, therapy-related disease, TP53 and KMT2A - PTD . In multivariate analysis anemia, platelets, FLT3 -ITD, and therapy-related disease remained in the model. Poor-risk factors such as del(7q)/-7, complex karyotype, ASXL1, RUNX1, EZH2, and TP53 did not show an independent impact. Thus, no clear biomarker for response and survival can be identified. Although a number of publications on predictive markers for response to AZA exist, results are inconsistent and improved response rates did not translate to improved survival. Here, we provide a comprehensive overview comparing the studies published to date., Competing Interests: CONFLICTS OF INTEREST None.

Background: Aberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown., Methods: We screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors., Results: We identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML., Conclusions: Our study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.

Objective: To examine mitochondrial gene expression in hematopoietic progenitor cells of patients with myelodysplastic syndromes (MDS). Mitochondrial pathology in MDS is suggested by abnormal mitochondrial iron accumulation, increased apoptosis, and a high frequency of acquired mitochondrial DNA mutations in bone marrow cells., Materials and Methods: Mitochondrial gene expression was measured by real-time reverse transcription polymerase chain reaction in CD34(+) bone marrow cells from 37 patients with MDS (22 refractory cytopenia with multilineage dysplasia, 9 refractory anemia with excess of blasts [5-9% marrow blasts], 6 refractory anemia with excess of blasts [10-19% marrow blasts]), 14 cases of acute myeloid leukemia with myelodysplasia-related changes (acute myeloid leukemia-MDS), and 9 normal controls. Relative quantification was achieved by using specific plasmid standards and 18S ribosomal RNA. Genes were selected to represent the multi-enzyme complexes I to IV of the mitochondrial respiratory chain: nicotinamide adenine dinucleotide dehydrogenase subunit 3 (subunit of complex I), succinate dehydrogenase B (complex II), cytochrome B (complex III), and cytochrome-c-oxidase subunit I (complex IV)., Results: Expression of mitochondrial-encoded genes was significantly reduced in patients with MDS and acute myeloid leukemia-MDS compared to normal controls, while mitochondrial DNA copy number was increased rather than diminished. An age-related decrease in mitochondrial gene expression was observed in MDS patients as well as controls. However, this effect was less pronounced than the MDS-related effect. Besides an overall decrease in mitochondrial gene expression, MDS patients displayed a stochiometric imbalance of mitochondrial-encoded genes, assessed in relation to the nuclear-encoded succinate dehydrogenase B., Conclusions: Our results show dysregulated mitochondrial gene expression that goes beyond a simple age-related effect and is compatible with the putative role of mitochondrial dysfunction in MDS pathophysiology. However, it remains unclear whether the problem arises from primary lesions in the mitochondria, i.e., mitochondrial DNA mutations, or as a result of changes in the cell nucleus., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

This study evaluates response-adapted treatment of chronic myelogenous leukaemia (CML) in chronic phase using molecular response criteria. bcr-abl/G6PDH ratios were assessed by Light-Cycler quantitative real-time polymerase chain reaction (PCR( in 277 peripheral blood samples from 33 patients, before and every 3 months during therapy. Sixty-six per cent (22/33) of the patients fulfiled our molecular response criterion of > or = 1 log decrease in bcr-abl transcript after 6 or > or = 2 log decrease after 9 and every following 3 months. Dose escalation was necessary for 33% (11/33) of the patients. Of these, 54% (6/11) achieved a reduction of bcr-abl mRNA by > or = 2 log (n = 3) or > or = 3 log (n = 3) with 800 mg Imatinib. Forty-five per cent (5/11) showed insufficient molecular response with 800 mg Imatinib and received Nilotinib. In conclusion, the assessment of molecular response permits an individual patient-tailored treatment of CML in first chronic phase, resulting in the majority of patients achieving a major molecular response after 2 years of therapy., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Objective: To determine the frequency and spectrum of somatic mutations of mitochondrial DNA (mtDNA) in bone marrow of patients with myelodysplastic syndrome (MDS)., Materials and Methods: Analysis included 104 patients with MDS (24 refractory anemia, 32 refractory anemia with ringed sideroblasts, 34 refractory anemia with excess of blasts, 7 refractory anemia with excess of blasts in transformation to acute leukemia, and 7 chronic myelo-monocytic leukemia), 3 patients with acute myeloid leukemia from MDS, and 36 patients with myeloproliferative disease (23 chronic myeloid leukemia, 9 polycythemia vera, 4 idiopathic myelofibrosis). Mutation scanning was performed using heteroduplex analysis with denaturing high-performance liquid chromatography (dHPLC). The entire mitochondrial genome was amplified in 67 overlapping polymerase chain reaction fragments carefully optimized regarding DNA melting profiles. Abnormal dHPLC findings were confirmed by DNA sequencing., Results: Heteroplasmic mtDNA mutations, mostly transitions, were identified in 56% of MDS and 44% of myeloproliferative disorders patients. In MDS, mutation frequency increased with age and more-advanced disease. Mutational spectra showed no hot spots and were similar in different types of MDS. Heteroplasmic mutations generally did not represent known polymorphisms, and about half of them affected conserved amino acids or nucleotides. Mutations were less frequent in protein encoding genes (50 per 10(6) base pairs) than other mitochondrial genes (transfer RNAs, ribosomal RNAs, and control region; about 80 per 10(6) base pairs)., Conclusions: As mitochondria often show ultrastructural abnormalities in MDS, including pathological iron accumulation, mitochondrial dysfunction may contribute to MDS pathology. We found a high frequency of acquired mtDNA mutations in MDS. However, their functional importance remains unclear, considering that genotype correlates poorly with phenotype in mitochondrial diseases. The clonally expanded mtDNA mutations in MDS support the concept of age-related damage to mtDNA in hematopoietic stem cells.

Introduction: Increased numbers of mitochondria in differentiated thyroid cancer and, most strikingly, mutations in human mitochondrial DNA (mtDNA) in older people have led to speculation that mtDNA mutations might contribute to aging or accumulate in postmitotic tissues with age. Mutation analyses of mtDNA in papillary (PTCs) and follicular (FTCs) thyroid carcinomas have been limited to date. The significance and frequency of mtDNA mutations in PTC and FTC are therefore controversial, as is age dependence., Methods: We analyzed eight sample pairs of PTC and six of FTC tissue with the corresponding normal thyroid tissue. DNA was extracted from frozen and formaldehyde-fixed tissue using the QIAmp Tissue Kit. Sequence differences in the mtDNA between tumor and normal tissue were detected using appropriate polymerase chain reaction (PCR) products for heteroduplex analysis in a denaturing high performance liquid chromatography (HPLC) Wave System (Transgenomic). Mutations were confirmed and identified by sequencing the PCR products of conspicuous chromatograms. The samples were obtained from 346 patients with PTC and 105 patients with FTC. We analyzed the whole mitochondrial genome from seven PTC and three FTC tumors along with the corresponding normal thyroid tissue. 3/7 PTC samples showed two heteroplasmic mutations and one polymorphism; all 3 FTCs showed homoplasmic and/or heteroplasmic mutations., Results: All but one of these tumors are well documented in the mitochondrial database MITOMAP. MtDNA mutations were found in all three patients aged 45 years and older. There was no correlation, however, in this small group to clinical prognostic factors for recurrence and especially for survival in differentiated thyroid carcinomas, such as histology, tumor size, lymph node metastases, distant metastases, and gender, most likely because of the short follow-up. While univariate analysis of the findings in the whole cohort of 346 patients with PTC suggested that age is a significant prognostic factor for survival (P = 0.0237) but not for recurrence (P = 0.65), this was not the case in the 105 patients with FTC., Conclusions: Although we found accumulation of mutations in two older patients with PTC and one patient with FTC (all three patients older than 45 years had mtDNA mutations), the low frequency of these mutations in the small group of 10 analyzed patients did not correlate with statistically validated clinical prognosticators for recurrence or survival, especially not with age. The low power of our data are therefore not able to support or refute the hypothesis that these mtDNA mutations are related to age-dependent tumor progression in the thyroid or that they "may be involved in thyroid tumorigenesis."

Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.

Myelodysplastic syndromes (MDS) are comprised of a heterogeneous group of stem cell disorders characterized by ineffective hematopoiesis and susceptibility to transform to acute myeloid leukemia. The molecular pathways underlying disease initiation and evolution are still largely unknown. We recently demonstrated that acquired mutations in PTPN11 are a major event in JMML and occur with variable prevalence in children with other hematologic malignancies, including MDS. Here, we investigated contribution of PTPN11 mutations to adult MDS and CMML pathogenesis. Our results indicate that PTPN11 lesions might play a role in adult MDS/CMML pathogenesis but do not represent a major molecular event.

In a patient with refractory anemia with excess blasts (RAEB), a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly, the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However, PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays, CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis, thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient.

By combining the advantages of capillary electrophoresis and temperature gradient gel electrophoresis, a method was developed to detect point mutations in polymerase chain reaction (PCR) fragments. Increasing and decreasing temporal temperature gradients were established by means of a computer-controlled Peltier module. Native and denaturing conditions were achieved by cooling to 25 degrees C and heating to 70 degrees C, respectively, a thermostating liquid surrounding the capillary. To separate nucleic acid fragments, a sieving media, containing 4% linear polyacrylamide, 1 x Tris borate EDTA buffer (TBE) and 6 M urea, was found appropriate. Renewal of the sieving matrix before each run significantly improved the reproducibility of fragment separation. The ability of this capillary electrophoresis system to detect point mutations is demonstrated with the human prion-protein gene.

Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (CJD) are caused by point mutations or octarepeat insertions in the prion protein (PrP) gene. In the present work a method was established that is appropriate for a thorough screening for mutations in the PrP gene and is generally applicable to screenings of any given gene. Temperature gradient gel electrophoresis (TGGE) was modified at two critical steps by UV cross-linking of the DNA strands and by replacing the spatial gradient with a temporal one. The shift of a DNA band in temporal temperature gradient gel electrophoresis (tTGGE) due to a mutation can be calculated as a function of the position of the mutation in the sequence. Appropriate DNA fragments were selected for polymerase chain reaction (PCR) amplification and analysis by tTGGE on the basis that the predicted band shifts amount to more than 10% of the migration distance for all possible mutations. The accuracy of the prediction was tested experimentally with ten known mutations in the human PrP gene, and quantitative agreement between theory and experiment was achieved. Thus, this screening method is also a suitable means to verify the absence of mutations in a given gene segment.

Studies on prions involve nucleic acid chemistry under two headings: i) do infectious prion particles contain nucleic acids? ii) is it possible by a simple procedure to screen the prion protein (PrP)-gene for mutations? The return refocusing gel electrophoresis technique was developed to detect by sensitive silver staining homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all kinds of nucleic acids from a length of 13 nucleotides up to several thousand could be recovered and detected with a yield over 90%. Despite extensive nuclease digestion some small polynucleotides remained. The results define clear restrictions for a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be smaller than 80 nucleotides in length at a particle-to-infectivity ratio near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To detect mutations in the PrP-gene, either known mutations from human prion diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole PrP-ORF. It will be shown that a combination of PCR and temperature-gradient gel electrophoresis fulfils these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated depending on the position of the mutation. The theoretical results were tested with the mutations known so far.

In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.