Publications by authors named "Fernanda Gutierrez-Rodrigues"

18 Publications

  • Page 1 of 1

Association between leukocyte telomere length and sex by quantile regression analysis.

Hematol Transfus Cell Ther 2021 Feb 6. Epub 2021 Feb 6.

Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP), Ribeirão Preto, SP, Brazil.

Introduction: Telomere length (TL) is a biomarker of cellular proliferative history. In healthy individuals, leukocyte TL shortens with age and associates with the lifespan of men and women. However, most of studies had used linear regression models to address the association of the TL attrition, aging and sex.

Methods: We evaluated the association between the TL, aging and sex in a cohort of 180 healthy subjects by quantile regression. The TL of nucleated blood cells was measured by fluorescent in situ hypridization (flow-FISH) in a cohort of 89 men, 81 women, and 10 umbilical cord samples. The results were validated by quantitative polymerase chain reaction (qPCR) and compared to a linear regression analysis.

Results: By quantile regression, telomere dynamics slightly differed between sexes with aging: women had longer telomeres at birth and slower attrition rate than men until the sixth decade of life; after that, TL eroded faster and became shorter than that in men. These differences were not observed by linear regression analysis, as the overall telomere attrition rates in women and men were similar (42 pb per year, p < 0.0001 vs. 45 pb kb per year, p < 0.0001). Also, qPCR did not recapitulate flow-FISH findings, as the telomere dynamics by qPCR followed a linear model.

Conclusion: The quantile regression analysis accurately reproduced a third-order polynomial TL attrition rate in both women and men, but it depended on the technique applied to measure TL. The Flow-FISH reproduced the expected telomere dynamics through life and, differently from the qPCR, was able to detect the subtle TL variations associated with sex and aging.
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http://dx.doi.org/10.1016/j.htct.2020.12.005DOI Listing
February 2021

Eltrombopag added to immunosuppression for children with treatment-naïve severe aplastic anaemia.

Br J Haematol 2021 Feb 7;192(3):605-614. Epub 2021 Jan 7.

Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD, USA.

Acquired severe aplastic anaemia (SAA) has an immune pathogenesis, and immunosuppressive therapy (IST) with anti-thymocyte globulin and cyclosporine is effective therapy. Eltrombopag (EPAG) added to standard IST was associated with higher overall and complete response rates in patients with treatment-naïve SAA compared to a historical IST cohort. We performed a paediatric subgroup analysis of this trial including all patients aged <18 years who received EPAG plus standard IST (n = 40 patients) compared to a historical cohort (n = 87) who received IST alone. Response, relapse, clonal evolution, event-free survival (EFS), and overall survival were assessed. There was no significant difference in either the overall response rate (ORR) or complete response rate at 6 months (ORR 70% in EPAG group, 72% in historical group, P = 0·78). Adults (≥18 years) had a significantly improved ORR of 82% with EPAG compared to 58% historically (P < 0·001). Younger children had lower response rates than did adolescents. The trend towards relapse was higher and EFS significantly lower in children who received EPAG compared to IST alone. Addition of EPAG added to standard IST did not improve outcomes in children with treatment-naïve SAA. EPAG in the paediatric population should not automatically be considered standard of care. Registration: clinicaltrials.gov (NCT01623167).
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http://dx.doi.org/10.1111/bjh.17232DOI Listing
February 2021

Eltrombopag monotherapy can improve hematopoiesis in patients with low to intermediate risk-1 myelodysplastic syndrome.

Haematologica 2020 12 1;105(12):2785-2794. Epub 2020 Dec 1.

National Heart, Lung, and Blood Institute.

Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by cytopenia and a propensity to develop acute myeloid leukemia (AML). The management of lower-risk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag (EPAG), a thrombopoietin receptor agonist, can improve platelet counts in LR-MDS and tri-lineage hematopoiesis in aplastic anemia (AA). We conducted a phase 2 dose modification study to investigate the safety and efficacy of EPAG in LR-MDS. EPAG dose was escalated from 50 mg/day, to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a PNH clone, marrow hypocellularity, thrombocytopenia with or without other cytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with previous EPAG studies in AA; no patients discontinued drug due to adverse events. Three patients developed reversible grade-3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued EPAG after achieving a robust response (median time 16 months); four of them reinitiated EPAG due to declining counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to AML on study. In conclusion, EPAG was well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.
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http://dx.doi.org/10.3324/haematol.2020.249995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716353PMC
December 2020

Somatic Mutations in and Severe Adult-Onset Autoinflammatory Disease.

N Engl J Med 2020 12 27;383(27):2628-2638. Epub 2020 Oct 27.

From the National Human Genome Research Institute (D.B.B., A.K.O., W.P., N.B., D.L.R., D.O.C., K.M., P.H., S.R., L.X., H.O., M.N., A.J., R.S.L., N.D., J.J.C., M.C.V.M., D.N., B.D.S., W.A.G., S.M.B., I.A., D.L.K.), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (M.A.F., K.A.S., S.N., W.L.T., S.G., C.C.-R., W.G., E.R., K.V.W., G.W., S.B., S.D., Z.D., R.A.C., M.J.K., M.G., P.C.G.), the National Institute of Dental and Craniofacial Research (J.C.C., A.J.A., A.W.), the Undiagnosed Diseases Program, Common Fund, Office of the Director (N.B., D.L.R., M.C.V.M., D.N., W.A.G.), the Hematology Branch, National Heart, Lung, and Blood Institute (Z.W., B.P., E.M.G., F.G.-R., L.W.D., C.S.H., N.S.Y.), the National Institute of Allergy and Infectious Diseases (K.S.B.), the Laboratory of Pathology, Center for Cancer Research, National Cancer Institute (C.-C.R.L.), the National Institutes of Health (NIH) Intramural Sequencing Center, National Human Genome Research Institute (J.M.), the Department of Laboratory Medicine (W.W., M.T., A.D.-F., K.R.C.), and the National Eye Institute (M.A.-A.), NIH, Bethesda, and GeneDx, Gaithersburg (K.R.) - both in Maryland; and the National Amyloidosis Centre, Royal Free Hospital London NHS Foundation Trust and University College London, London (D.R., H.J.L.), and the National Institute for Health Research-Leeds Biomedical Research Centre and Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds (S.S.) - both in the United Kingdom.

Background: Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders.

Methods: We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9-edited zebrafish were used as an in vivo model to assess gene function.

Results: We identified 25 men with somatic mutations affecting methionine-41 (p.Met41) in UBA1, the major E1 enzyme that initiates ubiquitylation. (The gene lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet's syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation.

Conclusions: Using a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.).
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http://dx.doi.org/10.1056/NEJMoa2026834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847551PMC
December 2020

Detectable mutations precede late myeloid neoplasia in aplastic anemia.

Haematologica 2021 02 1;106(2):647-650. Epub 2021 Feb 1.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI).

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http://dx.doi.org/10.3324/haematol.2020.263046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849750PMC
February 2021

Eltrombopag for patients with moderate aplastic anemia or uni-lineage cytopenias.

Blood Adv 2020 04;4(8):1700-1710

Translational Stem Cell Biology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

There is no standard or widely effective treatment of patients with moderate aplastic anemia (MAA) or hypo-productive uni-lineage cytopenias (UC). Eltrombopag (EPAG), a small molecule thrombopoietin mimetic, has previously been shown to result in durable multi-lineage hematologic responses with low toxicity in patients with refractory severe aplastic anemia (SAA). Its safety and efficacy in MAA are unknown. This prospective phase 2 study enrolled previously untreated and treated MAA and UC patients with clinically relevant cytopenias. EPAG was administered at doses escalating from 50 to 300 mg/d. Hematologic responses were assessed at 16 to 20 weeks. Responding patients were continued on EPAG until reaching defined robust or stable blood counts. EPAG was reinstituted for relapse. Thirty-four patients were enrolled between 2012 and 2017, including 31 with MAA and 3 with UC. Seventeen patients responded in at least 1 eligible lineage by the primary end point. A striking improvement in anemia was observed in a patient with Diamond-Blackfan anemia. EPAG was well tolerated, and it was discontinued for robust or stable blood counts in 12 of 17 patients after a median of 8 months. A majority required re-initiation of EPAG for declining counts, and all regained response. Two of 34 patients developed non-chromosome 7 bone marrow cytogenetic abnormalities while taking EPAG, without dysplasia or increased blasts. Somatic mutation allele frequencies in cancer genes did not increase overall on EPAG. EPAG is a well-tolerated oral treatment of cytopenias in patients with MAA/UC. This trial was registered at www.clinicaltrials.gov as #NCT01328587.
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http://dx.doi.org/10.1182/bloodadvances.2020001657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189303PMC
April 2020

A novel homozygous RTEL1 variant in a consanguineous Lebanese family: phenotypic heterogeneity and disease anticipation.

Hum Genet 2019 Dec 1;138(11-12):1323-1330. Epub 2019 Nov 1.

LAU Gilbert and Rose-Marie Chagoury School of Medicine, LAUMC/RH, Zahar Street, Achrafieh, Beirut, 1110, Lebanon.

Phenotypic heterogeneity is often observed in patients with telomeropathies caused by pathogenic variants in telomere biology genes. However, the roles of recessive variants in these different phenotypes are not fully characterized. Our goal is to describe the biological roles of a novel homozygous RTEL1 variant identified in a consanguineous Lebanese family with unusual presentation of telomeropathies. A proband was screened for germline variants in telomere biology genes by whole exome sequencing. Leukocytes' telomere length was measured in the proband and eight relatives. We identified a novel homozygous p.E665K RTEL1 variant in the proband, his mother, and seven siblings that associated with telomere shortening and a broad spectrum of clinical manifestations, ranging from mild unspecific findings to severe phenotypes. Consanguinity in at least three family generations led to increased frequency of the homozygous p.E665K variant in the youngest generation and progressive telomere shortening. The increased frequency of the homozygous RTEL1 variant due to consanguinity in this Lebanese family allowed us to infer novel behaviors of recessive RTEL1 variants, as the expressivity and penetrance of this gene are very heterogenous between inter- and intra-generations. Progressive telomere shortening was associated with disease anticipation, first reported in recessive autosomal telomeropathies. Both genetic testing and telomere length measurement were critical for the clinical diagnosis of this family with telomere diseases marked by phenotypic heterogeneity.
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http://dx.doi.org/10.1007/s00439-019-02076-8DOI Listing
December 2019

Telomere dynamics and hematopoietic differentiation of human DKC1-mutant induced pluripotent stem cells.

Stem Cell Res 2019 10 20;40:101540. Epub 2019 Aug 20.

Department of Medical Imaging, Hematology, and Clinical Oncology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil. Electronic address:

Telomeropathies are a group of phenotypically heterogeneous diseases molecularly unified by pathogenic mutations in telomere-maintenance genes causing critically short telomeres. X-linked dyskeratosis congenita (DC), the prototypical telomere disease, manifested with ectodermal dysplasia, cancer predisposition, and severe bone marrow failure, is caused by mutations in DKC1, encoding a protein responsible for telomerase holoenzyme complex stability. To investigate the effects of pathogenic DKC1 mutations on telomere repair and hematopoietic development, we derived induced pluripotent stem cells (iPSCs) from fibroblasts of a DC patient carrying the most frequent mutation: DKC1 p.A353V. Telomeres eroded immediately after reprogramming in DKC1-mutant iPSCs but stabilized in later passages. The telomerase activity of mutant iPSCs was comparable to that observed in human embryonic stem cells, and no evidence of alternative lengthening of telomere pathways was detected. Hematopoietic differentiation was carried out in DKC1-mutant iPSC clones that resulted in increased capacity to generate hematopoietic colony-forming units compared to controls. Our study indicates that telomerase-dependent telomere maintenance is defective in pluripotent stem cells harboring DKC1 mutation and unable to elongate telomeres, but sufficient to maintain cell proliferation and self-renewal, as well as to support the primitive hematopoiesis, the program that is recapitulated with our differentiation protocol.
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http://dx.doi.org/10.1016/j.scr.2019.101540DOI Listing
October 2019

Short telomere length in peripheral blood leukocytes in head and neck cancer: Findings in a Brazilian cohort.

Head Neck 2019 03 27;41(3):672-677. Epub 2018 Dec 27.

Department of Internal Medicine, University of São Paulo at Ribeirão Preto School of Medicine, São Paulo, Brazil.

Background: Telomeres are specialized DNA structures that are critical to maintain cell homeostasis and to avoid genomic instability. Epidemiological studies have examined the association between leukocyte telomere length (LTL) and risk of cancers, but the findings remain conflicting.

Methods: Mean LTL was measured by quantitative PCR in 97 patients with head and neck cancer (HNC) and 262 healthy controls. The association between LTL and patients' clinical status, such as smoke, alcoholism, and overall survival, were also evaluated.

Results: The age-adjusted LTL was significantly shorter in patients with HNC in comparison to healthy controls (P = .0003). Patients with shortest LTL had an increased risk to develop HNC (P < 0.0001). No significant correlation was observed between LTL and patients' clinical features and personal habits.

Conclusions: Our data support the hypothesis that LTL is a risk factor for HNC. The use of LTL as a biomarker can help physicians to identify high-risk individuals for HNC.
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http://dx.doi.org/10.1002/hed.25472DOI Listing
March 2019

Pathogenic TERT promoter variants in telomere diseases.

Genet Med 2019 07 7;21(7):1594-1602. Epub 2018 Dec 7.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA.

Purpose: The acquisition of pathogenic variants in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. In nonmalignant diseases, TERTp variants have been reported only in patients with idiopathic pulmonary fibrosis (IPF) due to germline variants in telomere biology genes.

Methods: We screened patients with a broad spectrum of telomeropathies (n = 136), their relatives (n = 52), and controls (n = 195) for TERTp variants using a customized massively parallel amplicon-based sequencing assay.

Results: Pathogenic -124 and -146 TERTp variants were identified in nine (7%) unrelated patients diagnosed with IPF (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere biology genes. TERTp clone selection did not associate with peripheral blood counts, telomere length, and response to danazol treatment. However, it was specific for patients with telomeropathies, more frequently co-occurring with TERT germline variants and associated with aging.

Conclusion: We extend the spectrum of nonmalignant diseases associated with pathogenic TERTp variants to marrow failure and liver disease due to inherited telomerase deficiency. Specificity of pathogenic TERTp variants for telomerase dysfunction may help to assess the pathogenicity of unclear constitutional variants in the telomere diseases.
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http://dx.doi.org/10.1038/s41436-018-0385-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555700PMC
July 2019

Epidemiological, clinical and genetic characterization of aplastic anemia patients in Pakistan.

Ann Hematol 2019 Feb 13;98(2):301-312. Epub 2018 Nov 13.

Department of Biochemistry, Quaid-i-Azam University, Islamabad, 44000, Pakistan.

Aplastic anemia (AA) is the most serious non-malignant blood disorder in Pakistan, ranked second in prevalence, after thalassemia. We investigated various epidemiological, clinical, and genetic factors of AA in a Pakistani cohort of 214 patients reporting at our hospital between June 2014 and December 2015. A control group of 214 healthy subjects was included for comparison of epidemiological and clinical features. Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between ages 10 and 29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence, and high rate of consanguineous marriages. Serum granulocyte colony-stimulating factor and thrombopoietin levels were significantly elevated in AA patients, compared to healthy controls (P < 0.0001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exons 3 and 7 of TERT gene. Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.
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http://dx.doi.org/10.1007/s00277-018-3542-zDOI Listing
February 2019

Circulating exosomal microRNAs in acquired aplastic anemia and myelodysplastic syndromes.

Haematologica 2018 07 19;103(7):1150-1159. Epub 2018 Apr 19.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD, USA.

Exosomal microRNAs modulate cancer cell metabolism and the immune response. Specific exosomal microRNAs have been reported to be reliable biomarkers of several solid and hematologic malignancies. We examined the possible diagnostic and prognostic values of exosomal microRNAs in two human bone marrow failure diseases: aplastic anemia and myelodysplastic syndromes. After screening 372 microRNAs in a discovery set (n=42) of plasma exosome samples, we constructed a customized PCR plate, including 42 microRNAs, for validation in a larger cohort (n=99). We identified 25 differentially expressed exosomal microRNAs uniquely or frequently present in aplastic anemia and/or myelodysplastic syndromes. These microRNAs could be related to intracellular functions, such as metabolism, cell survival, and proliferation. Clinical parameters and progression-free survival were correlated to microRNA expression levels in aplastic anemia and myelodysplastic syndrome patients before and after six months of immunosuppressive therapy. One microRNA, mir-126-5p, was negatively correlated with a response to therapy in aplastic anemia: patients with higher relative expression of miR-126-5p at diagnosis had the shortest progression-free survival compared to those with lower or normal levels. Our findings suggest utility of exosomal microRNAs in the differential diagnosis of bone marrow failure syndromes. (Registered at ).
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http://dx.doi.org/10.3324/haematol.2017.182824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029526PMC
July 2018

CRISPR/Cas9-mediated ASXL1 mutations in U937 cells disrupt myeloid differentiation.

Int J Oncol 2018 Apr 28;52(4):1209-1223. Epub 2018 Feb 28.

Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1202, USA.

Additional sex combs-like 1 (ASXL1) is a well‑known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells. Comparable cell growth and cell cycle progression were observed between wild-type (WT) and ASXL1-mutated U937 cells. Drug-induced cytotoxicity, as measured by growth inhibition and apoptosis in the presence of the cell-cycle active agent 5-fluorouracil, was variable among the mutated clones but was not significantly different from WT cells. In addition, ASXL1-mutated cells exhibited defects in monocyte/macrophage differentiation. Transcriptome analysis revealed that ASXL1 mutations altered differentiation of U937 cells by disturbing genes involved in myeloid differentiation, including cytochrome B-245 β chain and C-type lectin domain family 5, member A. Dysregulation of numerous gene sets associated with cell death and survival were also observed in ASXL1-mutated cells. These data provide evidence regarding the underlying molecular mechanisms induced by mutated ASXL1 in leukemogenesis.
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http://dx.doi.org/10.3892/ijo.2018.4290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843401PMC
April 2018

The interpretation of rare or novel variants: damaging vs. disease-causing.

Rev Bras Hematol Hemoter 2018 Jan - Mar;40(1):3-4. Epub 2017 Dec 6.

Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRPUSP), Ribeirão Preto, SP, Brazil; Fundação de Pesquisa São Paulo (FAPESP), Ribeirão Preto, SP, Brazil.

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http://dx.doi.org/10.1016/j.bjhh.2017.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003047PMC
December 2017

Heterozygous variants in bone marrow failure and myeloid neoplasms.

Blood Adv 2018 01 4;2(1):36-48. Epub 2018 Jan 4.

Department of Haematological Medicine, King's College Hospital, London, United Kingdom.

Biallelic germline mutations in (regulator of telomere elongation helicase 1) result in pathologic telomere erosion and cause dyskeratosis congenita. However, the role of mutations in other bone marrow failure (BMF) syndromes and myeloid neoplasms, and the contribution of monoallelic mutations to disease development are not well defined. We screened 516 patients for germline mutations in telomere-associated genes by next-generation sequencing in 2 independent cohorts; one constituting unselected patients with idiopathic BMF, unexplained cytopenia, or myeloid neoplasms (n = 457) and a second cohort comprising selected patients on the basis of the suspicion of constitutional/familial BMF (n = 59). Twenty-three variants were identified in 27 unrelated patients from both cohorts: 7 variants were likely pathogenic, 13 were of uncertain significance, and 3 were likely benign. Likely pathogenic variants were identified in 9 unrelated patients (7 heterozygous and 2 biallelic). Most patients were suspected to have constitutional BMF, which included aplastic anemia (AA), unexplained cytopenia, hypoplastic myelodysplastic syndrome, and macrocytosis with hypocellular bone marrow. In the other 18 patients, variants were likely benign or of uncertain significance. Telomeres were short in 21 patients (78%), and 3' telomeric overhangs were significantly eroded in 4. In summary, heterozygous variants were associated with marrow failure, and telomere length measurement alone may not identify patients with telomere dysfunction carrying variants. Pathogenicity assessment of heterozygous variants relied on a combination of clinical, computational, and functional data required to avoid misinterpretation of common variants.
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http://dx.doi.org/10.1182/bloodadvances.2017008110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761623PMC
January 2018

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.

Blood Cells Mol Dis 2018 03 4;69:10-22. Epub 2018 Jan 4.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892-1202, USA.

DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.
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http://dx.doi.org/10.1016/j.bcmd.2017.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728079PMC
March 2018

Consequences of acute oxidative stress in Leishmania amazonensis: From telomere shortening to the selection of the fittest parasites.

Biochim Biophys Acta Mol Cell Res 2017 Jan 9;1864(1):138-150. Epub 2016 Nov 9.

Genetics Department, Biosciences Institute, Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Botucatu, SP, Brazil. Electronic address:

Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects millions of people around the world. During infection, the parasites use different strategies to survive the host's defenses, including overcoming exposure to reactive oxidant species (ROS), responsible for causing damage to lipids, proteins and DNA. This damage especially affects telomeres, which frequently results in genome instability, senescence and cell death. Telomeres are the physical ends of the chromosomes composed of repetitive DNA coupled with proteins, whose function is to protect the chromosomes termini and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2mM hydrogen peroxide (HO) for 1h, which was able to increase intracellular ROS levels. In addition, oxidative stress induced DNA damage, as confirmed by 8-oxodGuo quantification and TUNEL assays and the dissociation of LaRPA-1 from the 3' G-overhang, leading to telomere shortening. Moreover, LaRPA-1 was observed to interact with newly formed C-rich single-stranded telomeric DNA, probably as a consequence of the DNA damage response. Nonetheless, acute oxidative stress caused the death of some of the L. amazonensis population and induced cell cycle arrest at the G2/M phase in survivor parasites, which were able to continue proliferating and replicating DNA and became more resistant to oxidative stress. Taken together, these results suggest that adaptation occurs through the selection of the fittest parasites in terms of repairing oxidative DNA damage at telomeres and maintaining genome stability in a stressful environment.
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http://dx.doi.org/10.1016/j.bbamcr.2016.11.001DOI Listing
January 2017

Direct comparison of flow-FISH and qPCR as diagnostic tests for telomere length measurement in humans.

PLoS One 2014 19;9(11):e113747. Epub 2014 Nov 19.

Department of Internal Medicine, University of São Paulo at Ribeirão Preto School of Medicine, Ribeirão Preto, São Paulo, Brazil; Center for Cell-based Therapy, São Paulo Research Foundation (FAPESP), Ribeirão Preto, São Paulo, Brazil.

Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2) = 0.60; p<0.0001) and patients (R(2) = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2) = 0.35; p<0.0001) and low correlation for patients (R(2) = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R(2) = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R(2) = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8 ± 7.1%) and qPCR (9.5 ± 7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6 ± 7.6% vs. 16 ± 19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113747PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237503PMC
December 2015