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Master's Dissertation
DOI
https://doi.org/10.11606/D.17.2022.tde-03022023-104236
Document
Author
Full name
Raíssa Silva Tristão
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2022
Supervisor
Committee
Traina, Fabíola (President)
Lazarini, Mariana
Palma, Leonardo Carvalho
Title in Portuguese
Análise de polimorfismo em TET2 e TERT em neoplasias mieloproliferativas BCR-ABL1 negativas
Keywords in Portuguese
Citogenética convencional
Neoplasias mieloproliferativas
Polimorfismo TERT
Polimorfismo TET2
Abstract in Portuguese
As neoplasias mieloproliferativas (NMPs) são um grupo heterogêneo de distúrbios hematológicos sendo classificadas pela presença de alterações nas células da linhagem mieloide (granulócitos, eritrócitos e megacariócitos). As três principais entidades desse grupo conhecidas como NMP BCR::ABL1-negativas clássicas são a policitemia vera (PV), trombocitose essencial (TE) e mielofibrose primária (MFP). As mutações em JAK2, CALR e MLP estão relacionadas ao fenótipo neoplásico. Estudos de associação genômica ampla (GWAS) sugerem que genes condutores adicionais podem predispor para o risco de desenvolver NMP, e influenciam o desenvolvimento, fenótipo ou a severidade dessas doenças. Os estudos multicêntricos revelaram que o polimorfismo TET2 (metilcitosina deoxigenase 2) rs3733609 C>T e o polimorfismo TERT (transcriptase reversa da telomerase) rs2736100 A>C têm um papel na fisiopatologia das NMPs. Os escores prognósticos atuais utilizam os dados clínicos, laboratoriais, citogenéticas e moleculares para o diagnóstico, monitoramento do tratamento e prognóstico em NMP. O objetivo desta pesquisa foi investigar e descrever as características demográficas, clínicas, laboratoriais, citogenéticas e moleculares, a frequência e o impacto clínico dos polimorfismos TET2 rs3733609 e TERT rs2736100 nos pacientes com NMP BCR::ABL1-negativas clássicas: PV, TE e MFP do HC FMRP USP. A casuística incluiu uma coorte de 245 pacientes com diagnóstico de NMP BCR::ABL1-negativas clássicas: PV, TE e MFP, em acompanhamento regular no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HC FMRP-USP) no período de 13 anos, de dezembro de 2006 a dezembro de 2019. Os dados demográficos, clínicos e de desfechos foram obtidos a partir da revisão dos prontuários físicos e/ou eletrônicos. O cariótipo foi investigado por citogenética convencional. A investigação dos polimorfismos nos genes TET2 e TERT foi realizada pelo método de PCR em tempo real com sondas de hidrólise específicas. As analises estatísticas foram realizadas utilizando GraphPad Prism 9 (GraphPad Prism Software, Inc., CA, EUA). Duzentos e quarenta e cinco pacientes foram avaliados, 123 do sexo masculino e 122 do sexo feminino, mediana de idade de 73 anos. Cento e dezenove pacientes tiveram diagnóstico de MFP, nos quais a mutação JAK2V617F foi detectada em 71, mutação CALR em 25, mutação MPL em 5, e 18 foram considerados triplo-negativos. Setenta e dois pacientes tiveram diagnóstico de PV, nos quais a mutação JAK2V617F foi detectada em 64, mutação JAK2 éxon 12 em 1 paciente e JAK2 selvagem em 7 pacientes. Cinquenta e quatro pacientes tiveram diagnóstico de TE, nos quais a mutação JAK2V617F foi detectada em 25, mutação CALR em 12 pacientes e 17 foram triplo-negativos. Dentre os 124 pacientes submetidos à análise citogenética, 107 apresentaram cariótipo normal. Entre 17 pacientes com alterações citogenéticas, 10 tiveram diagnóstico de MFP sendo um com cariótipo complexo. A alteração citogenética mais frequentemente observada foi a trissomia do cromossomo 8. Cento e vinte e um pacientes não obtiveram metáfases ou não foram submetidos à análise citogenética. Dos 245 pacientes, 227 foram genotipados para o polimorfismo TET2 rs3733609, 36 portaram o alelo polimórfico (C), dos quais 4 eram homozigotos (CC). Para TERT rs2736100, 242 foram genotipados, 152 portavam o alelo polimórfico (A) dos quais 33 pacientes eram homozigotos (AA). O polimorfismo TET2 rs3733609 TC + CC esteve associado com maior frequência de hipertensão arterial sistêmica (p=0,046) em pacientes com NMP clássicas com mutação JAK2V617F, maior mediana de contagem de plaquetas (p=0,017) em pacientes com PV, e maior mediana de LDH (p=0,0087) e maior frequência de mutação em genes da via JAK/STAT (p=0,046) em pacientes com TE, comparado com pacientes com polimorfismo TET2 rs3733609 TT. O polimorfismo TERT rs2736100 CA + AA esteve associado com menor valor da mediana de hemoglobina (p=0,0036), hematócrito (p=0,0055), contagem de neutrófilos (p=0,032), menor frequência de mutações no gene da JAK2V617F (p=0,025) e maior frequência de diabetes mellitus tipo 2 (p<0,0001) na coorte de pacientes com NMP clássicas, menor valor da mediana de hemoglobina (p=0,0025) na coorte de pacientes com NMP clássicas com mutação JAK2V617F, menor mediana de contagem de leucócitos (p=0,02) e neutrófilos (p=0,009) na coorte de pacientes com PV, menor mediana de contagem de hemoglobina (p=0,0018) e hematócrito (p=0,005) em pacientes com MFP. A associação com uma importante entidade molecular de ordem etiológica para as NMP BCR::ABL1-negativas clássicas pode representar o envolvimento de fatores germinativos dignos de investigação não apenas para prática da genética médica, mas também pela dinâmica de aquisição da mutação condutora ao fenótipo neoplásico.
Title in English
TET2 and TERT polymorphisms analysis in BCR-ABL1 negative myeloproliferative neoplasms
Keywords in English
Conventional cytogenetics
Myeloproliferative neoplasms
TERT polymorphism
TET2 polymorphism
Abstract in English
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of hematological disorders classified by the presence of alterations in myeloid lineage cells (granulocytes, erythrocytes and megakaryocytes). The three main entities in this group known as classic BCR::ABL1-negative MPN are polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF). Mutations in JAK2, CALR and MPL often drive the neoplastic phenotype. Genomic-wide association studies (GWAS) suggest that additional driver genes may predispose to the risk of developing MPN, and influence the development, phenotype, or severity of the diseases. Multicenter studies revealed that the TET2 (methylcytosine deoxygenase 2) rs3733609 C>T polymorphism and the TERT (telomerase reverse transcriptase) rs2736100 A>C polymorphism play a role in the pathophysiology of MPNs. Current prognostic scores use clinical, laboratory, cytogenetic, and molecular data for diagnosis, treatment monitoring, and prognosis in MPN. The aim of this research was to investigate and describe the demographic, clinical, laboratory, cytogenetic and molecular features, frequency and clinical impact of TET2 rs3733609 and TERT rs2736100 polymorphisms in patients with classic BCR::ABL1-negative MPN: PV, ET and PMF from Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HC FMRP-USP). The casuistry was composed of 245 patients cohort diagnosed with classical BCR::ABL1- negative MPN: PV, ET and PMF, in regular follow-up at the HC FMRP USP for a period of 13 years, from December 2006 to December 2019. Demographic, clinical and outcome data were obtained from the review of physical and/or electronic medical records. The karyotype was investigated by conventional cytogenetics. The investigation of polymorphisms in the TET2 and TERT genes was performed by the real-time PCR method with specific hydrolysis probes. Statistical analyzes were performed using GraphPad Prism 9 (GraphPad Prism Software, Inc., CA, USA). Two hundred and forty-five patients were evaluated, 123 males and 122 females, median age 73 years. One hundred and nineteen patients were diagnosed with PMF, in which the JAK2V617F mutation was detected in 71, CALR mutation in 25, MPL mutation in 5, and 18 were considered triple-negative. Seventy-two patients were diagnosed with PV, in which JAK2V617F mutation was detected in 64, JAK2 exon 12 mutation in 1 patient and wild-type JAK2 mutation in 7 patients. Fifty-four patients were diagnosed with ET, in which the JAK2V617F mutation was detected in 25, CALR mutation in 12 patients and 17 were triple-negative. Among the 124 patients submitted to cytogenetic analysis, 107 had a normal karyotype. Among 17 patients with cytogenetic alterations, 10 were diagnosed with PMF, one with a complex karyotype. The most frequently observed cytogenetic alteration was chromosome 8 trisomy. One hundred and twenty-one patients did not obtain metaphases or were not submitted to cytogenetic analysis. Of the 245 patients, 227 were genotyped for the TET2 rs3733609 polymorphism, 36 carried the polymorphic allele (C), of which 4 were homozygous (CC). For TERT rs2736100, 242 were genotyped, 152 carried the polymorphic allele (A) of which 33 patients were homozygous (AA). The TET2 rs3733609 TC + CC polymorphism was associated with a higher frequency of systemic arterial hypertension (p=0.046) in patients with classic MPN with JAK2V617F mutation, higher median platelet counts (p=0.017) in patients with PV, and higher median of LDH (p=0.0087) and higher frequency of mutation in JAK/STAT pathway genes (p=0.046) in patients with ET compared with patients with TET2 rs3733609 TT polymorphism. The TERT rs2736100 CA + AA polymorphism was associated with lower median hemoglobin (p=0.0036), hematocrit (p=0.0055), neutrophil counts (p=0.032), lower frequency of mutations in the JAK2V617F gene (p=0.025) and higher frequency of type 2 diabetes mellitus (p<0.0001) in the cohort of patients with classic MPN, lower median hemoglobin (p=0.0025) in the cohort of patients with classic MPN with JAK2V617F mutation, lower median leukocyte (p=0.02) and neutrophil counts (p=0.009) in the cohort of patients with PV, lower median hemoglobin (p=0.0018) and hematocrit (p=0.005) in patients with PMF. The association with an important molecular entity of etiological order for classical BCR::ABL1-negative MPN may represent the involvement of germline factors worthy of investigation not only for the practice of medical genetics, but also for the dynamics of acquisition of the mutation leading to the neoplastic phenotype.
 
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Publishing Date
2023-02-08
 
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