Genome characterization and CRISPR-Cas9 editing of a human neocentromere.

The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

TNFRSF13B gene mutation in familial acute myeloid leukemia: A new piece in the complex scenario of hereditary predisposition?

TNFRSF13B mutations are widely associated with common variable immunodeficiency. TNFRSF13B was recently counted among relevant genes associated with childhood-onset of hematological malignancies; nonetheless, its role in acute myeloid leukemia (AML) remains unexplored. We report the study of a family with two cases of AML, sharing a germline TNFRSF13B mutation favoring the formation of a more stable complex with its ligand TNFSF13: a positive regulator of AML-initiating cells. Our data turn the spotlight onto the TNFRSF13B role in AML onset, inserting a new fragment into the complex scenario of a hereditary predisposition to myeloid neoplasms.

IRF4 Gene Expression on the Trail of Molecular Response: Looking at Chronic Myeloid Leukemia from Another Perspective.

INTRODUCTION: Interferon regulatory factor 4 (IRF4) is a transcriptional factor with a key role in the modulation of inflammation and immune surveillance. The IRF4 gene is downregulated in Philadelphia-negative myeloproliferative neoplasms, and its expression is associated with prognosis and response to treatment. METHODS: We evaluated the IRF4 expression kinetics during tyrosine kinase inhibitor (TKI) treatment in a cohort of 116 chronic myeloid leukemia (CML) patients to elucidate its role in the disease course. RESULTS: A relationship between the IRF4 expression and the disease burden was observed at various disease stages. A correlation analysis between the International Scale (IS) and IRF4 values confirmed this close association. A significant increase is detected after 3 months of TKI treatment. Patients achieving an early molecular response (EMR) had higher IRF4 values at both diagnosis and after 3 months of therapy as compared to those failing the EMR target. Patients achieving treatment-free remission did not show IRF4 fluctuations during monitoring, while a decreased IRF4 expression emerged at the time of molecular relapse. CONCLUSION: Our data seem to confirm the relevance of IRF4 in the pathogenesis of CML, suggesting a pivotal role at the disease onset and a predictive value during the CML course.

The Role of NLRP3, a Star of Excellence in Myeloproliferative Neoplasms.

Nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) is the most widely investigated inflammasome member whose overactivation can be a driver of several carcinomas. It is activated in response to different signals and plays an important role in metabolic disorders and inflammatory and autoimmune diseases. NLRP3 belongs to the pattern recognition receptors (PRRs) family, expressed in numerous immune cells, and it plays its primary function in myeloid cells. NLRP3 has a crucial role in myeloproliferative neoplasms (MPNs), considered to be the diseases best studied in the inflammasome context. The investigation of the NLRP3 inflammasome complex is a new horizon to explore, and inhibiting IL-1ß or NLRP3 could be a helpful cancer-related therapeutic strategy to improve the existing protocols.

IKAROS in Acute Leukemia: A Positive Influencer or a Mean Hater?

One key process that controls leukemogenesis is the regulation of oncogenic gene expression by transcription factors acting as tumor suppressors. Understanding this intricate mechanism is crucial to elucidating leukemia pathophysiology and discovering new targeted treatments. In this review, we make a brief overview of the physiological role of IKAROS and the molecular pathway that contributes to acute leukemia pathogenesis through IKZF1 gene lesions. IKAROS is a zinc finger transcription factor of the Krüppel family that acts as the main character during hematopoiesis and leukemogenesis. It can activate or repress tumor suppressors or oncogenes, regulating the survival and proliferation of leukemic cells. More than 70% of Ph+ and Ph-like cases of acute lymphoblastic leukemia exhibit IKZF1 gene variants, which are linked to worse treatment outcomes in both childhood and adult B-cell precursor acute lymphoblastic leukemia. In the last few years, much evidence supporting IKAROS involvement in myeloid differentiation has been reported, suggesting that loss of IKZF1 might also be a determinant of oncogenesis in acute myeloid leukemia. Considering the complicated "social" network that IKAROS manages in hematopoietic cells, we aim to focus on its involvement and the numerous alterations of molecular pathways it can support in acute leukemias.

Epitranscriptomics in Normal and Malignant Hematopoiesis.

Epitranscriptomics analyze the biochemical modifications borne by RNA and their downstream influence. From this point of view, epitranscriptomics represent a new layer for the control of genetic information and can affect a variety of molecular processes including the cell cycle and the differentiation. In physiological conditions, hematopoiesis is a tightly regulated process that produces differentiated blood cells starting from hematopoietic stem cells. Alteration of this process can occur at different levels in the pathway that leads from the genetic information to the phenotypic manifestation producing malignant hematopoiesis. This review focuses on the role of epitranscriptomic events that are known to be implicated in normal and malignant hematopoiesis, opening a new pathophysiological and therapeutic scenario. Moreover, an evolutionary vision of this mechanism will be provided.

Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology.

We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations. We demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.

Myelodysplastic syndrome with 5q deletion following IgM monoclonal gammopathy, showing gene mutation MYD88 L265P.

Patients affected by monoclonal gammopathy of undetermined significance (MGUS) very rarely develop a myelodysplastic syndrome (MDS). However, it was also demonstrated that MGUS patients had a significantly increased risk of developing MDS compared to the general population. We report a case of 5q-syndrome following a MGUS IgMk with mutation of MYD88 L256P. To our knowledge, this is the first case of del(5q) MDS following MGUS IgMk with the MYD88 L256P mutation in which there is coexistence of the markers of the two clonal diseases, but as an expression of distinct pathological features.

ADAMTS2 gene dysregulation in T/myeloid mixed phenotype acute leukemia.

BACKGROUND: Mixed phenotype acute leukemias (MPAL) include acute leukemias with blasts that express antigens of more than one lineage, with no clear evidence of myeloid or lymphoid lineage differentiation. T/myeloid (T/My) MPAL not otherwise specified (NOS) is a rare leukemia that expresses both T and myeloid antigens, accounting for less than 1% of all leukemias but 89% of T/My MPAL. From a molecular point of view, very limited data are available on T/My MPAL NOS. CASE PRESENTATION: In this report we describe a T/My MPAL NOS case with a complex rearrangement involving chromosomes 5 and 14, resulting in overexpression of the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) gene due to its juxtaposition to the T cell receptor delta (TRD) gene segment. CONCLUSION: Detailed molecular cytogenetic characterization of the complex rearrangement in the reported T/My MPAL case allowed us to observe ADAMTS2 gene overexpression, identifying a molecular marker that may be useful for monitoring minimal residual disease. To our knowledge, this is the first evidence of gene dysregulation due to a chromosomal rearrangement in T/My MPAL NOS.

Venetoclax-based treatment in acute myeloid leukemia: an unexpected bonus on the path to allogeneic hematopoietic stem cell transplant?

Despite the approval of new drugs, the inclusion of -omics-derived data and the integration of machine learning in both the diagnostic and therapeutic process, the prognosis of acute myeloid leukemia (AML) remains dismal. The curative path is still aimed at achieving a successful allogeneic hematopoietic stem cell transplant (HSCT) in most patients. Nevertheless, access to this procedure is limited to eligible patients. Moreover, post-HSCT outcomes are influenced by AML heterogeneity and patient-related factors. The rise of venetoclax (VEN)-based combinations as standard of care in the treatment of older or unfit AML patients, together with their peculiar management profile, has led researchers to evaluate the feasibility of this approach in patients proceeding toward HSCT. We reviewed the available evidence to weigh up the advantages and pitfalls of this new therapeutic strategy.

Gene expression profiling of chronic myeloid leukemia with variant t(9;22) reveals a different signature from cases with classic translocation.

BACKGROUND: The t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5-10% of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological differences between CML patients with classic and variant t(9;22) have never been clarified. FINDINGS: In this study, we performed gene expression microarray analysis to compare CML patients bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified 59 differentially expressed genes significantly associated with the two analyzed groups. The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (complement component 5a receptor 1) is discussed. CONCLUSIONS: Our results reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK pathway deregulation and show that kinases are a common target of molecular alterations in hematological disorders.

Clonal hematopoiesis onset in chronic myeloid leukemia patients developing an adverse cardiovascular event.

Life expectation of chronic myeloid leukemia patients in the tyrosine kinase inhibitors era is almost equal to that of healthy subjects. On the other hand, their long-term management must take into account a higher risk of adverse events, at least partly related to the treatment. Various studies reported a higher incidence of cardiovascular events in these patients. Clonal hematopoiesis is broadly considered a major independent risk factor for cardiovascular events. Of note, the underlying physiopathological mechanisms connect clonal hematopoiesis with a global proinflammatory status, triggering a vicious circle in which the somatic mutations and inflammation feed each other. All this considered, we investigated the occurrence of clonal hematopoiesis in chronic myeloid leukemia patients developing a cardiovascular event under tyrosine kinase inhibitor therapy.

Feasibility of Optical Genome Mapping in Cytogenetic Diagnostics of Hematological Neoplasms: A New Way to Look at DNA.

Optical genome mapping (OGM) is a new genome-wide technology that can reveal both structural genomic variations (SVs) and copy number variations (CNVs) in a single assay. OGM was initially employed to perform genome assembly and genome research, but it is now more widely used to study chromosome aberrations in genetic disorders and in human cancer. One of the most useful OGM applications is in hematological malignancies, where chromosomal rearrangements are frequent and conventional cytogenetic analysis alone is insufficient, necessitating further confirmation using ancillary techniques such as fluorescence in situ hybridization, chromosomal microarrays, or multiple ligation-dependent probe amplification. The first studies tested OGM efficiency and sensitivity for SV and CNV detection, comparing heterogeneous groups of lymphoid and myeloid hematological sample data with those obtained using standard cytogenetic diagnostic tests. Most of the work based on this innovative technology was focused on myelodysplastic syndromes (MDSs), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL), whereas little attention was paid to chronic lymphocytic leukemia (CLL) or multiple myeloma (MM), and none was paid to lymphomas. The studies showed that OGM can now be considered as a highly reliable method, concordant with standard cytogenetic techniques but able to detect novel clinically significant SVs, thus allowing better patient classification, prognostic stratification, and therapeutic choices in hematological malignancies.

Optical Genome Mapping as a Tool to Unveil New Molecular Findings in Hematological Patients with Complex Chromosomal Rearrangements.

Standard cytogenetic techniques (chromosomal banding analysis-CBA, and fluorescence in situ hybridization-FISH) show limits in characterizing complex chromosomal rearrangements and structural variants arising from two or more chromosomal breaks. In this study, we applied optical genome mapping (OGM) to fully characterize two cases of complex chromosomal rearrangements at high resolution. In case 1, an acute myeloid leukemia (AML) patient showing chromothripsis, OGM analysis was fully concordant with classic cytogenetic techniques and helped to better refine chromosomal breakpoints. The OGM results of case 2, a patient with non-Hodgkin lymphoma, were only partially in agreement with previous cytogenetic analyses and helped to better define clonal heterogeneity, overcoming the bias related to clonal selection due to cell culture of cytogenetic techniques. In both cases, OGM analysis led to the identification of molecular markers, helping to define the pathogenesis, classification, and prognosis of the analyzed patients. Despite extensive efforts to study hematologic diseases, standard cytogenetic methods display unsurmountable limits, while OGM is a tool that has the power to overcome these limitations and provide a cytogenetic analysis at higher resolution. As OGM also shows limits in defining regions of a repetitive nature, combining OGM with CBA to obtain a complete cytogenetic characterization would be desirable.

Case report: biallelic DNMT3A mutations in acute myeloid leukemia.

DNMT3A gene mutations, detected in 20-25% of de novo acute myeloid leukemia (AML) patients, are typically heterozygous. Biallelic variants are uncommon, affecting ~3% of cases and identifying a worse prognosis. Indeed, two concomitant DNMT3A mutations were recently associated with shorter event-free survival and overall survival in AML. We present an AML case bearing an unusual DNMT3A molecular status, strongly affecting its function and strangely impacting the global genomic methylation profile. A 56-year-old Caucasian male with a diagnosis of AML not otherwise specified (NOS) presented a complex DNMT3A molecular profile consisting of four different somatic variants mapping on different alleles (in trans). 3D modelling analysis predicted the effect of the DNMT3A mutational status, showing that all the investigated mutations decreased or abolished DNMT3A activity. Although unexpected, DNMT3A's severe loss of function resulted in a global genomic hypermethylation in genes generally involved in cell differentiation. The mechanisms through which DNMT3A contributes to AML remain elusive. We present a unique AML case bearing multiple biallelic DNMT3A variants abolishing its activity and resulting in an unexpected global hypermethylation. The unusual DNMT3A behavior described requires a reflection on its role in AML development and persistence, highlighting the heterogeneity of its deregulation.

Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NMDA/NR1 subunit.

High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD(67) and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1(tet/tet) mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1(tet/tet) ES cell lines, neurons derived from Dnmt1(tet/tet) cells showed abnormal dendritic arborization and branching. The Dnmt1(tet/tet) neuronal cells also showed elevated levels of functional N-methyl d-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD(67) in neuronal networking and excitatory/inhibitory balance, respectively, we studied methylation of these genes' promoters in Dnmt1(tet/tet) ES cells and neurons. Both reelin and GAD(67) promoters were not hypermethylated in the Dnmt1(tet/tet) ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.

The Ribosomal Protein RpL22 Interacts In Vitro with 5'-UTR Sequences Found in Some Drosophila melanogaster Transposons.

Mobility of eukaryotic transposable elements (TEs) are finely regulated to avoid an excessive mutational load caused by their movement. The transposition of retrotransposons is usually regulated through the interaction of host- and TE-encoded proteins, with non-coding regions (LTR and 5'-UTR) of the transposon. Examples of new potent cis-acting sequences, identified and characterized in the non-coding regions of retrotransposons, include the insulator of gypsy and Idefix, and the enhancer of ZAM of Drosophila melanogaster. Recently we have shown that in the 5'-UTR of the LTR-retrotransposon ZAM there is a sequence structured in tandem-repeat capable of operating as an insulator both in Drosophila (S2R+) and human cells (HEK293). Here, we test the hypothesis that tandem repeated 5'-UTR of a different LTR-retrotransposon could accommodate similar regulatory elements. The comparison of the 5'-UTR of some LTR-transposons allowed us to identify a shared motif of 13 bp, called Transposable Element Redundant Motif (TERM). Surprisingly, we demonstrated, by Yeast One-Hybrid assay, that TERM interacts with the D. melanogaster ribosomal protein RpL22. The Drosophila RpL22 has additional Ala-, Lys- and Pro-rich sequences at the amino terminus, which resembles the carboxy-terminal portion of histone H1 and histone H5. For this reason, it has been hypothesized that RpL22 might have two functions, namely the role in organizing the ribosome, and a potential regulatory role involving DNA-binding similar to histone H1, which represses transcription in Drosophila. In this paper, we show, by two independent sets of experiments, that DmRpL22 is able to directly and specifically bind DNA of Drosophila melanogaster.

Identification and Characterisation of pST1023 A Mosaic, Multidrug-Resistant and Mobilisable IncR Plasmid.

We report the identification and characterisation of a mosaic, multidrug-resistant and mobilisable IncR plasmid (pST1023) detected in Salmonella ST1023, a monophasic variant 4,[5],12:i: strain of widespread pandemic lineage, reported as a Southern European clone. pST1023 contains exogenous DNA regions, principally gained from pSLT-derivatives and IncI1 plasmids. Acquisition from IncI1 included oriT and nikAB and these conferred the ability to be mobilisable in the presence of a helper plasmid, as we demonstrated with the conjugative plasmids pST1007-1D (IncFII) or pVC1035 (IncC). A sul3-associated class 1 integron, conferring resistance to aminoglycosides, chloramphenicol and trimethoprim-sulphonamides, was also embedded in the acquired IncI1 DNA segment. pST1023 also harboured an additional site-specific recombination system (rfsF/rsdB) and IS elements of the IS1, IS5 (IS903 group) and IS6 families. Four of the six IS26 elements present constituted two pseudo-compound-transposons, named PCT-sil and PCT-Tn10 (identified here for the first time). The study further highlighted the mosaic genetic architecture and the clinical importance of IncR plasmids. Moreover, it provides the first experimental data on the ability of IncR plasmids to be mobilised and their potential role in the horizontal spread of antimicrobial-resistant genes.

The genomic analysis brings a new piece to the molecular jigsaw of idiopathic erythrocytosis.

Erythrocytosis is a clinical condition characterized by increased red cell mass, hemoglobin, and hematocrit values. A significant fraction of patients is described as having idiopathic erythrocytosis. We have previously demonstrated an association between erythrocytosis and the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. In the present study, we investigated genomic and clinical features of 80 erythrocytosis patients with the aim to provide useful information in clinical practice. Patients with idiopathic erythrocytosis could have a genomic germline background, eventually associated with somatic variants. Through association analysis, we show that male patients presenting with idiopathic erythrocytosis, and normal EPO levels could be the best candidates for the search for the JAK2 GGCC_46/1 haplotype and CALR rs1049481_G allele. Further studies are needed to confirm these findings and to depict detailed genomic and phenotypical characteristics of these patients.