Publications by authors named "Marco De Gobbi"

50 Publications

GITMO REGISTRY STUDY ON ALLOGENEIC TRANSPLANTATION IN PATIENTS AGED OVER 60 FROM 2000 TO 2017. IMPROVEMENTS AND CRITICISMS.

Transplant Cell Ther 2021 Nov 21. Epub 2021 Nov 21.

Unit of Haematology and Stem Cell Transplant Centre, "San Camillo" Hospital, Rome, Italy.

Background: Nowadays, allogeneic stem cell transplantation (Allo-SCT) can be offered to patients up to the age of 70-72 years and represents one of the most effective curative treatments for many hematological malignancies.

Objectives: The primary objective of the study is to collect data from the allo-SCTs performed in Italy from 2000 to 2017 in patients over 60 years of age to evaluate the changes in safety and efficacy outcomes as well as their distribution and characteristics over time.

Study Design: The GITMO AlloEld study (ClinicalTrials.gov: NCT04469985) is a retrospective, analysis of the allo-SCTs performed 30 Italian transplant Centers on older patients (≥ 60 years) from 2000 to 2017 (n=1,996).

Results: For the purpose of analysis, patients were grouped into three time periods: time A: 2000-2005, n=256 (12%); time B: 2006-2011, n=584 (29%); and time C: 2012-2017, n=1156 (59%). After a median follow-up of 5.6 years, the 5-year Non Relapse Mortality (NRM) remained stable (time A: 32.8%; time B: 36.2%; and time C: 35.0%, p = 0.5); the Overall Survival (OS) improved (time A: 28.4%; time B: 31.8%; and time C: 37.3%, p = 0.012); and the Cumulative Incidence of Relapse (CIR) reduced (time A: 45.3%; time B: 38.2%; time C: 30.0%, p < 0.0001). The 2-year incidence of extensive cGVHD reduced significantly (time A: 17.2%; time B: 15.8%; and time C: 12.2%, p = 0.004). Considering times A and B together (2000-2011), the 2-year NRM was positively correlated to the HCT-CI score; patients with HCT-CI of 0, 1 or 2, or ≥3 had rates of NRM of 25.2%, 33.9%, and 36.1%, respectively, (p < 0.001). Meanwhile, after 2012, the HCT-CI score was not significantlly predictive of NRM.

Conclusions: The study shows that the transplant procedure in elderly patients became more effective over time. Relapse incidence remains the major problem and strategies to prevent it are under investigation (e.g. post-transplant maintenance). Today, the selection of patients aged over 60 could be improved by combining HCT-CI and frailty assessments to better predict NRM.
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http://dx.doi.org/10.1016/j.jtct.2021.11.006DOI Listing
November 2021

Long-Term Clinical Outcomes of Hematopoietic Stem Cell Transplantation in Multiple Sclerosis.

Neurology 2021 01 20. Epub 2021 Jan 20.

Gianluigi Mancardi Department of Neurology, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa and Ospedale Policlinico San Martino, IRCCS, Genoa, Italy and IRCCS Scientific Clinical Institutes Maugeri, Pavia-Genoa Nervi/Italy.

Objective: To determine whether autologous hematopoietic stem cell transplantation (aHSCT) is able to induce durable disease remission in people with multiple sclerosis (MS), we analyzed the long-term outcomes after transplant in a large cohort of MS patients.

Methods: To be included, a minimum data set (consisting of age, MS phenotype, EDSS at baseline, information on transplant technology and at least 1 follow-up visit after transplant) was required.

Results: 210 patients were included [relapsing-remitting (RR)MS=122(58%)]. Median baseline EDSS was 6(1-9), mean follow-up was 6.2(±5.0) years. Among RRMS patients, disability worsening-free survival (95%CI) was 85.5%(76.9-94.1%) at 5 years and 71.3%(57.8-84.8%) at 10 years. In patients with progressive MS, disability worsening-free survival was 71.0%(59.4-82.6%) and 57.2%(41.8-72.7%) at 5 and 10 years, respectively. In RRMS patients, EDSS significantly reduced after aHSCT [p=0.001; mean EDSS change per year -0.09 (95%CI=-0.15 to -0.04%)]. In RRMS patients, the use of the BEAM+ATG conditioning protocol was independently associated with a reduced risk of NEDA-3 failure [HR=0.27(0.14-0.50), p<0.001]. Three patients died within 100-days from aHSCT (1.4%); no deaths occurred in patients transplanted after 2007.

Conclusions: aHSCT prevents disability worsening in the majority of patients and induces durable improvement in disability in patients with RRMS. The BEAM+ATG conditioning protocol is associated with a more pronounced suppression of clinical relapses and MRI inflammatory activity.

Classification Of Evidence: This study provides Class IV evidence that for people with MS, aHSCT induces durable disease remission in most patients.
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http://dx.doi.org/10.1212/WNL.0000000000011461DOI Listing
January 2021

Genetic Screening for Potential New Targets in Chronic Myeloid Leukemia Based on Drosophila Transgenic for Human BCR-ABL1.

Cancers (Basel) 2021 Jan 14;13(2). Epub 2021 Jan 14.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9;22)(q34;q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the () sequence and the () gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the transgenic as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.
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http://dx.doi.org/10.3390/cancers13020293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830713PMC
January 2021

Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells.

Int J Mol Sci 2020 Oct 16;21(20). Epub 2020 Oct 16.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

I is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.
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http://dx.doi.org/10.3390/ijms21207674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589297PMC
October 2020

Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms.

J Cell Mol Med 2020 09 13;24(18):10978-10986. Epub 2020 Aug 13.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.
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http://dx.doi.org/10.1111/jcmm.15730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521327PMC
September 2020

Autologous Hematopoietic Stem Cell Transplantation (AHSCT): Standard of Care for Relapsing-Remitting Multiple Sclerosis Patients.

Neurol Ther 2020 Dec 16;9(2):197-203. Epub 2020 Jun 16.

SSD Terapia oncoematologica intensiva e trapianto CSE, AOU San Luigi Gonzaga, Dipartimento di Scienze Cliniche e Biologiche, University of Turin, Turin, Italy.

Autologous hematopoietic stem cell transplantation (AHSCT) has been used in the treatment of highly active multiple sclerosis (MS) for over two decades. It has been demonstrated to be highly efficacious in relapsing-remitting (RR) MS patients failing to respond to disease-modifying drugs (DMDs). AHSCT guarantees higher rates of no evidence of disease activity (NEDA) than those achieved with any other DMDs, but it is also associated with greater short-term risks which have limited its use. In the 2019 updated EBMT and ASBMT guidelines, which review the clinical evidence of AHSCT in MS, AHSCT indication for highly active RRMS has changed from "clinical option" to "standard of care". On this basis, AHSCT must be proposed on equal footing with second-line DMDs to patients with highly active RRMS, instead of being considered as a last resort after failure of all available treatments. The decision-making process requires a close collaboration between transplant hematologists and neurologists and a full discussion of risk-benefit of AHSCT and alternative treatments. In this context, we propose a standardized protocol for decision-making and informed consent process.
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http://dx.doi.org/10.1007/s40120-020-00200-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606396PMC
December 2020

Highly Sensitive Detection of Mutations in Acute Myeloid Leukemia.

J Clin Med 2020 Jan 19;9(1). Epub 2020 Jan 19.

Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Regione Gonzole 10, 10043 Orbassano (Turin), Italy.

Background: Acute myeloid leukemia is a heterogeneous hematological disease, characterized by karyotypic and molecular alterations. Mutations in have a role in diagnosis and as a minimal residue disease marker. Often the variant allele frequency during follow up is less than 20%, which represents the limit of detection of Sanger sequencing. Therefore, the development of sensitive methodologies to identify mutations might help to monitor patients' response to therapy. We compared three different methods to identify and monitor mutations in patients' specimens.

Methods: Performances of PNA-PCR clamping, droplet digital PCR and Sanger for status identification were evaluated and compared in 96 DNA patients' specimens.

Results: In contrast with Sanger sequencing, our results highlighted the concordance between PNA clamping and digital PCR. Furthermore, PNA-PCR clamping was able to detect more mutated DNA with respect to Sanger sequencing that showed several false negatives independently from the allelic frequency.

Conclusions: We found that PNA-PCR clamping and digital PCR identified mutations in DNA samples with comparable results in a percentage significantly higher compared to Sanger sequencing. PNA-PCR clamping can be used even in laboratories not equipped for sophisticated analyses, decreasing cost and time for characterization.
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http://dx.doi.org/10.3390/jcm9010271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019902PMC
January 2020

Rhino-Orbital-Cerebral Mucormycosis after Allogeneic Hematopoietic Stem Cell Transplantation and Isavuconazole Therapeutic Drug Monitoring during Intestinal Graft versus Host Disease.

Mediterr J Hematol Infect Dis 2019 1;11(1):e2019061. Epub 2019 Nov 1.

Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano, Turin, Italy.

A diagnosis of rhino-orbital-cerebral mucormycosis was made in a 59-year-old man with a secondary acute myeloid leukemia a few days after hematopoietic stem cell transplantation. Prompt treatment with combined antifungal therapy (liposomal amphotericin B and isavuconazole) followed by a procedure of endoscopic sinus surgery resulted in the resolution of the infection. Therapeutic drug monitoring of isavuconazole was performed during the year of treatment showing an increment of plasma concentrations in correspondence with the improvement of intestinal GvHD, thus suggesting that in this or similar conditions TDM for isavuconazole can be of value. A literature review of cases of rhino-orbital-cerebral and rhino-cerebral mucormycosis in allogeneic hematopoietic stem cell transplant recipients was carried out.
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http://dx.doi.org/10.4084/MJHID.2019.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827600PMC
November 2019

Synergistic effect of eltrombopag and deferasirox in aplastic anemia: a clinical case and review of the literature.

Leuk Lymphoma 2020 01 10;61(1):234-236. Epub 2019 Sep 10.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

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http://dx.doi.org/10.1080/10428194.2019.1660969DOI Listing
January 2020

Transferrin Saturation Inversely Correlates with Platelet Function.

Thromb Haemost 2019 May 12;119(5):766-778. Epub 2019 Mar 12.

Department of Clinical and Biological Sciences, University of Turin, Orbassano, Turin, Italy.

Background:  The association between iron overload (IO) and risk of cardiovascular disease is controversial. Epidemiological studies have found a significant negative association of transferrin (Tf) saturation and cardiovascular events suggesting that higher body iron possibly confer a protective effect towards developing cardiovascular events. The biological mechanisms of this phenomenon are unknown.

Objective:  This article investigates the role of IO on platelet reactivity.

Materials And Methods:  This study was a prospective case-control study comparing 45 patients with IO, mostly characterized by the HFE gene mutations C282Y and/or H63D, with 32 healthy controls. We evaluated: (1) platelet aggregation in both platelet-rich plasma and whole blood, (2) platelet membrane expression of the activation marker CD62P, (3) activation of platelet signalling phosphoinositide 3-kinase /Akt and mitogen-activated protein kinase/extracellular signal-regulated kinases (Erk)-1/2 pathways, (4) a pattern of in vivo platelet activation markers, and (5) iron biomarker predictors of platelet reactivity.

Results:  IO patients had significantly lower platelet aggregability, expression of CD62P and phosphorylation amounts of pAkt and pErk-2 in response to agonists. Furthermore, patients with higher Tf saturation levels were characterized by lower circulating levels of sCD40L, PDGF-BB and thromboxane B. Platelet aggregation and activation parameters inversely correlated with Tf saturation and the stepwise multivariate regression analysis underlined the role of Tf saturation in predicting platelet reactivity. We also found that in vitro platelet exposure to diferric Tf, but not to iron-depleted TF, dose-dependently inhibited platelet function in all investigated subjects.

Conclusion:  Tf saturation is inversely associated with platelet reactivity and this could explain, at least in part, the association of high Tf and lower risk of cardiovascular diseases in IO.
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http://dx.doi.org/10.1055/s-0039-1681061DOI Listing
May 2019

Venetoclax plus decitabine induced complete remission with molecular response in acute myeloid leukemia relapsed after hematopoietic stem cell transplantation.

Am J Hematol 2019 02 29;94(2):E48-E50. Epub 2018 Nov 29.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

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http://dx.doi.org/10.1002/ajh.25352DOI Listing
February 2019

Analysis of Mesenchymal Stromal Cell Engraftment After Allogeneic HSCT in Pediatric Patients: A Large Multicenter Study.

J Pediatr Hematol Oncol 2018 11;40(8):e486-e489

Department of Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin.

The mesenchymal stem cell (MSC) role after allogeneic hematopoietic stem cell transplantation (HSCT) is still a matter of debate; in particular, MSC engraftment in recipient bone marrow (BM) is unclear. A total of 46 patients were analyzed for MSC and hemopoietic stem cell engraftment after HSCT. The majority of patients had the BM as the stem cell source, and acute leukemia was the main indication for HSCT. Mesenchymal and hematopoietic stem cell chimerism analysis was carried out through specific polymorphic tandemly repeated regions. All patients reached complete donor engraftment; no evidence of donor-derived MSC engraftment was noted. Our data indicate that MSCs after HSCT remain of recipient origin despite the following: (i) myeloablative conditioning; (ii) the stem cell source; (iii) the interval from HSCT to BM analysis; (iv) the underlying disease before HSCT; and (v) the patients' or the donors' age at HSCT.
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http://dx.doi.org/10.1097/MPH.0000000000001305DOI Listing
November 2018

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease.

Proc Natl Acad Sci U S A 2017 09 21;114(36):E7526-E7535. Epub 2017 Aug 21.

Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, United Kingdom;

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.
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http://dx.doi.org/10.1073/pnas.1703087114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594649PMC
September 2017

Identification of new BMP6 pro-peptide mutations in patients with iron overload.

Am J Hematol 2017 Jun 29;92(6):562-568. Epub 2017 Apr 29.

Department of Medicine, Section of Internal Medicine, University of Verona, Verona, Italy; Veneto Region Referral Center for Iron Disorders, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy.

Hereditary Hemochromatosis (HH) is a genetically heterogeneous disorder caused by mutations in at least five different genes (HFE, HJV, TFR2, SLC40A1, HAMP) involved in the production or activity of the liver hormone hepcidin, a key regulator of systemic iron homeostasis. Nevertheless, patients with an HH-like phenotype that remains completely/partially unexplained despite extensive sequencing of known genes are not infrequently seen at referral centers, suggesting a role of still unknown genetic factors. A compelling candidate is Bone Morphogenetic Protein 6 (BMP6), which acts as a major activator of the BMP-SMAD signaling pathway, ultimately leading to the upregulation of hepcidin gene transcription. A recent seminal study by French authors has described three heterozygous missense mutations in BMP6 associated with mild to moderate late-onset iron overload (IO). Using an updated next-generation sequencing (NGS)-based genetic test in IO patients negative for the classical HFE p.Cys282Tyr mutation, we found three BMP6 heterozygous missense mutations in four patients from three different families. One mutation (p.Leu96Pro) has already been described and proven to be functional. The other two (p.Glu112Gln, p.Arg257His) were novel, and both were located in the pro-peptide domain known to be crucial for appropriate BMP6 processing and secretion. In silico modeling also showed results consistent with their pathogenetic role. The patients' clinical phenotypes were similar to that of other patients with BMP6-related IO recently described. Our results independently add further evidence to the role of BMP6 mutations as likely contributing factors to late-onset moderate IO unrelated to mutations in the established five HH genes.
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http://dx.doi.org/10.1002/ajh.24730DOI Listing
June 2017

Enhancer deletion generates cellular phenotypic diversity due to bimodal gene expression.

Blood Cells Mol Dis 2017 05 1;64:10-12. Epub 2017 Mar 1.

The Roslin Institute, Developmental Biology Division, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK. Electronic address:

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http://dx.doi.org/10.1016/j.bcmd.2017.02.006DOI Listing
May 2017

A comparative study of myocardial molecular phenotypes of two tfr2β null mice: role in ischemia/reperfusion.

Biofactors 2015 Sep-Oct;41(5):360-71. Epub 2015 Oct 13.

Department of Clinical and Biological Sciences, University of Torino, AOU San Luigi Gonzaga, Orbassano, Torino, Italy.

Transferrin receptor 2 (Tfr2) is an iron-modulator transcribed in two isoforms, Tfr2α and Tfr2β. The latter is expressed in the heart. We obtained two mouse models with silencing of Tfr2β: one with a normal systemic iron amount (SIA), i.e., Tfr2-KI, and the other, i.e., LCKO-KI, with high SIA due to hepatic Tfr2α silencing. We aimed to assess whether Tfr2β might play a role in myocardial injury and whether Tfr2β silencing might modify proteins of iron metabolism, antioxidant, apoptotic, and survival enzyme activities in the heart undergoing ischemia/reperfusion (I/R). Isolated hearts of wild-type (WT) and Tfr2-null mice were studied before or after an I/R protocol, and proteins/RNA analyzed by Western blot and/or quantitative PCR. Tfr2β increased in WT hearts subject to I/R, and both Tfr2β null mice hearts were protected against I/R injury (about 40% smaller infarct-size compared to WT hearts). RISK kinases (ERK1/2-AKT-PKCε) were found up-regulated after I/R in Tfr2-KI, whereas SAFE enzyme (Stat3) and GSK3β resulted phosphorylated during I/R in LCKO-KI hearts. While HO-1 and HIF-2a were high in both Tfr2β-null mice, Catalase, and proapoptotic factors were upregulated only in LCKO-KI. Finally, Tfr2-KI hearts presented an increased Ferritin-H and a decreased Ferroportin1, whereas LCKO-KI hearts displayed an upregulation of Ferritin-L chain and DMT1/Hamp-RNA. In conclusion, Tfr2β isoform is involved in cardiac iron metabolism and its silencing leads to a protected phenotype (antioxidants, RISK, and/or SAFE upregulation) against I/R challenging. Iron-dependent signals involved in cardioprotection seem to be positively affected by Tfr2β downregulation and subsequent Ferritins upregulation.
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http://dx.doi.org/10.1002/biof.1237DOI Listing
September 2016

Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH.

Biomark Res 2015 3;3:15. Epub 2015 Jul 3.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Background: Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL (T315I) mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation.

Results: The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors.

Conclusions: We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.
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http://dx.doi.org/10.1186/s40364-015-0039-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490729PMC
July 2015

Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment.

Nat Genet 2014 Feb 12;46(2):205-12. Epub 2014 Jan 12.

Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, UK.

Gene expression during development and differentiation is regulated in a cell- and stage-specific manner by complex networks of intergenic and intragenic cis-regulatory elements whose numbers and representation in the genome far exceed those of structural genes. Using chromosome conformation capture, it is now possible to analyze in detail the interaction between enhancers, silencers, boundary elements and promoters at individual loci, but these techniques are not readily scalable. Here we present a high-throughput approach (Capture-C) to analyze cis interactions, interrogating hundreds of specific interactions at high resolution in a single experiment. We show how this approach will facilitate detailed, genome-wide analysis to elucidate the general principles by which cis-acting sequences control gene expression. In addition, we show how Capture-C will expedite identification of the target genes and functional effects of SNPs that are associated with complex diseases, which most frequently lie in intergenic cis-acting regulatory elements.
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http://dx.doi.org/10.1038/ng.2871DOI Listing
February 2014

Causes and consequences of chromatin variation between inbred mice.

PLoS Genet 2013 Jun 13;9(6):e1003570. Epub 2013 Jun 13.

Wellcome Trust Centre for Human Genetics, Oxford, UK.

Variation at regulatory elements, identified through hypersensitivity to digestion by DNase I, is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. Analysis of terminally differentiated erythroblasts in eight inbred strains of mice identified reproducible variation at approximately 6% of DNase I hypersensitive sites (DHS). Only 30% of such variable DHS contain a sequence variant predictive of site variation. Nevertheless, sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in variable DHS. Changes at a small proportion (less than 10%) of variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.
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http://dx.doi.org/10.1371/journal.pgen.1003570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681629PMC
June 2013

High-resolution analysis of cis-acting regulatory networks at the α-globin locus.

Philos Trans R Soc Lond B Biol Sci 2013 6;368(1620):20120361. Epub 2013 May 6.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, UK.

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.
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http://dx.doi.org/10.1098/rstb.2012.0361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682726PMC
December 2013

Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression.

Hum Mutat 2013 Aug 18;34(8):1140-8. Epub 2013 Jun 18.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.

Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.
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http://dx.doi.org/10.1002/humu.22343DOI Listing
August 2013

Intragenic enhancers act as alternative promoters.

Mol Cell 2012 Feb 19;45(4):447-58. Epub 2012 Jan 19.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK.

A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.
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http://dx.doi.org/10.1016/j.molcel.2011.12.021DOI Listing
February 2012

An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

EMBO J 2012 Jan 4;31(2):317-29. Epub 2011 Nov 4.

MRC Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.
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http://dx.doi.org/10.1038/emboj.2011.399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261560PMC
January 2012

Polycomb eviction as a new distant enhancer function.

Genes Dev 2011 Aug;25(15):1583-8

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. We reveal its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. We propose a new role of long-range regulatory elements in removing repressive PcG complexes.
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http://dx.doi.org/10.1101/gad.16985411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182025PMC
August 2011

Generation of bivalent chromatin domains during cell fate decisions.

Epigenetics Chromatin 2011 Jun 6;4(1). Epub 2011 Jun 6.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Background: In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined.

Results: Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3.

Conclusions: While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.
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http://dx.doi.org/10.1186/1756-8935-4-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131236PMC
June 2011
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