Anormalidades genéticas e de metilação de DNA em uma coorte de leucemia linfoblástica aguda pediátrica com alterações no cromossomo 21 / Genetic and DNA methylation abnormalities in a cohort of acute lymphoblasticleukemia with chromosome 21 alterations

A leucemia linfoblástica aguda de células precursoras B (LLA-CPB) é uma neoplasia heterogênea. Aproximadamente 60% das LLA-CPB apresentam alterações envolvendo o cromossomo 21, incluindo hiperdiploidia, fusão gênica ETV6‐UNX1 e amplificação intracromossomal do cromossomo 21 (iAMP21). Mecanismos epigenéticos contribuem para a leucemogênese e a metilação do DNA, por sua vez, pode ser modulada por polimorfismos na via do folato. Portanto, este estudo tem como objetivo caracterizar o perfil genético e de metilação de DNA em LLA-CPB com alterações no cromossomo 21. Este estudo partiu de uma série de 1006 casos de LLA-CPB diagnosticados de 2002-2016 e foi desenhado em 3fases: 1) Identificação dos casos com alterações em número de cópias (CNA) no cromossomo 21 usando multiplex ligation probe amplification (MLPA) e FISH (RUNX1 e sondas para os cromossomos 4, 8, 10, 14, 17, 18, X e Y); 2) Caracterização do perfil de metilação e CNA por meio da técnica de microarranjo. Para tanto, o DNA foi modificado com EZ DNA Methylation™ Kit e o perfil de metilação analisado pelo Infinium Human Methylation 450 BeadChip Kit; e 3) Análises comparativas de metilação de DNA gene-específica, metilação em LINE-1(pirosequenciamento) e genotipagem para o polimorfismo MTHFR rs1801133 (PCR-RFLP) entre os diferentes subtipos moleculares de LLA-CPB, controles e amostras em remissão. Encontramos evidências de ganhos em CNA no cromossomo 21 em 83/374 (22%) das amostras (MLPA)...

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease. Approximately 60% of BCP-ALL present alterations involving chromosome 21 (chr 21), including high hyperdiploid, ETV6‐RUNX1 fusion and intrachromosomal amplification of thechromosome 21 (iAMP21). Contributing with this process, epigenetic mechanisms could regulate the transcription and induce leukemogenesis. Polymorphisms in genes involved in folate metabolism could influence this aberrant methylation. This study aimed to characterize the genetic and DNA methylation profile of BCP-ALL with chr 21 aberrations, as well as to identify the DNA methylation signatures of different BCP-ALL molecular subgroups. This study enrolled 1006 BCP-ALL diagnosed between 2002-2016 and was performed in tree steps: 1) Identification of copy number alterations (CNA) regarding the Chr 21 by multiplex ligation probe amplification (MLPA) and FISH (“LPH012 TEL/AML1 translocation, dual fusion probe and centromere probes to chr 4, 8, 10, 14, 17, 18, X and Y); 2) DNA methylation and CNAcharacterization by DNA methylation array. DNA from BCP-ALL cases, controls and remission samples was modified with EZ DNA Methylation™ Kit and analyzed by the InfiniumHumanMethylation450 BeadChip Kit; 3) Comparative gene methylation, LINE-1 methylation (pyrosequencing) and MTHFR rs1801133 genotype (PCR-RFLP) analysis between the different BCP-ALL subgroups. We found evidence of gains in chr 21 in 83/374 (22%) of the samples analyzed by MLPA. Among the BCP-ALL with chr 21 gains, 11/83 (13%) had ≥5 RUNX1 signals and 53/83 (64%) were classified as hyperdiploid...

Polymorphisms in CYP1B1, CYP3A5, GSTT1, and SULT1A1 Are Associated with Early Age Acute Leukemia.

Based on observational studies, early age leukemia (EAL) was associated with maternal hormone exposure during pregnancy. We studied the association between genetic polymorphisms of estrogen metabolism and EAL. Using data from the Brazilian Collaborative Study Group of Infant Acute Leukemia (2000-2012), 350 cases and 404 age-matched controls and 134 mothers of cases and controls were genotyped to explore polymorphisms in genes of the estrogen metabolism pathway: CYP1B1 (c.1294C>G, rs1056836), CYP3A4 (c.-392A>G, rs2740574), CYP3A5 (c.219-237G>A, rs776746), GSTM1/GSTT1 deletions, and SULT1A1 (c.638G>A, rs9282861; and c.667A>G, rs1801030). Logistic regression was used to calculate the odds ratios (OR) with 95% confidence intervals (CIs), and unconditional logistic regression was used to estimate adjusted odds ratios (aORs) by ethnicity. Because of multiple testing, p values < 0.01 were significant after Bonferroni correction. SULT1A1 (c.638G>A) was associated to infant acute lymphoblastic leukemia and acute myeloid leukemia (AML) risk in males (additive model: aOR = 0.52; 95% CI: 0.29-0.95, p = 0.03; dominant model: aOR = 2.18; 95% CI: 1.17-4.05, p = 0.01, respectively). CYP1B1 polymorphism was associated with a decreased risk of AML either for non-white or female children (additive model: OR = 0.24; 95% CI: 0.08-0.76, p < 0.01; additive model: aOR = 0.27; 95% CI: 0.08-0.89, p = 0.03, respectively). Since polymorphisms of Cytochrome P450 genes presented gender-specific risk associations, we also investigated their expression. CYP1B1 was not expressed in 57.1% of EAL cases, and its expression varied by genotype, gender, and leukemia subtype. Maternal-fetal GSTT1 null genotype was associated with risk of EAL. This study shows that polymorphisms in genes of estrogen metabolism confer genetic susceptibility to EAL, mainly in males, and maternal susceptibility genes modify the risk for developing EAL in newborns.

O papel dos polimorfismos nos genes RFC1, MTHFR, e SHMT1 na susceptibilidade e sobrevida em LLAs da primeira infância / The role of polymorphisms in genes RFC1, MTHFR, and SHMT1 susceptibilityand survival in early childhood acute lymphoblastic leukemia

Fatores de susceptibilidade genética modificam o risco para as leucemias linfoblástica aguda (LLA) pediátrica. Nesse estudo tivemos como objetivo esclarecer o papel dos polimorfismos MTHFR677C>T, MTHFR1298A>C, RFC180G>A e SHMT11420C>T na susceptibilidade e sobrevida de LLA da primeira infância. Foi realizada a extração de DNA de 219 casos (idade ≤ 24 meses) e 347 controles. A metodologia aplicada na detecção dos polimorfismos RFC1rs1051266 (80G>A), MTHFRrs1801133 (677C>T) e MTHFRrs1801131 (1298A>C) foi PCR-RFLP (restricted fragmento length polymorphism), e o polimorfismo SHMT1rs1979277 (1420C>T) foi analisado por pirosequenciamento. Diferenças na distribuição genotípica entre casos e controles foram testadas por regressão logística pelo cálculo de OR (IC 95%). A interação gene-gene foi avaliada através do método de fator de sinergia. As análises de sobrevida foram realizadas pelo método de Kaplan Meier e Log Hank, também foi feito o cálculo de OR (IC 95%) para analisar associação entre os polimorfismos e o status (vivo ou morto) dos pacientes. Todas as análises foram realizadas com o pacote estatístico SPSS 18, Chicago. Foi observada uma associação de risco para LLA conferida pela presença de pelo menos uma variante do gene RFC180GA+AA (OR, 1,74; IC 95% 1,05 –2,88), SHMT11420CT+TT (OR 1,95; IC 95%, 1,12–3,38) e MTHFR677 CT+TT (OR 1,78 ,IC 95%, 1,17–2,71) ajustado por gênero, idade e cor da pele. As combinações entre RFC180G>A e MTHFR677C>T (OR 1,26; IC 95%, 1,37 -5,48), RFC180G>A e SHMT11420C>T (OR 2,72 ; IC 95%, 1,25 –5,91), e SHMT11420C>T e MTHFR677C>T (OR 2,97; IC 95% 2,97, 1,50 –5,85) conferiram aumento no risco. O fator de sinergia para as variantes RFC180G>A e MTHFR1298A>C foi de 2,61 vezes, porém, não apresentou significância estatística (p=0.06). RFC180G>A, SHMT11420C>T não foram associados com LLA com MLL rearranjado, porém MTHFR677C>T conferiu um aumento no risco (OR 2,04; IC 95%, 1,19-3,49). Em conclusão, o efeito combinado das variantes RFC180G...

Genetic susceptibility modifies risk in childhood acute lymphoblastic leukemia (ALL). W e examined whether the combined effect of gene variants of folate cycle were associated with the susceptibility of ALL and whether age was an affecting factor. The over all survival (OS) was also analyzed to estimate the prognostic risks. Four single nucleotide polymorphisms (SNPs) in 3 genes (RFC1, SHMT1 and MTHFR) were genotyped in 219 B-cell precursor ALL (age ≤ 24 months-old) and 347 healthy controls. RFC1rs1051266 (80G>A), MTHFRrs1801133 (677C>T) and MTHFRrs1801131 (1298A>C) were performed by restricted fragment length polymorphism, whereas, SHMT1rs1979277 (1420C>T) by pyrosequencing. Comparison was performed through contingency tables to examine the associations between MTHFR, RFC1and SHMT1genotypes and ALL risk adjusted by skin color. Gene-gene interactions were evaluated by synergy factor test. OS analysis was performed by Kaplan–Meier and log-rank test. Increased risk for ALL was found associated with at least one variant allele RFC180GA+AA (OR, 1.74, 95% CI 1.05 –2.88), HMT11420CT+TT (OR 1.95, 95% CI, 1.12–3.38) and MTHFR677 CT+TT (OR 1.78 ,95% CI, 1.17–2.71) adjusted by gender, age and skin color. The combinations RFC180G>A/MTHFR677C>T (OR 1.26, 95% CI, 1.37 -5.48), RFC180G>A/SHMT11420C>T (OR 2.72 , 95% CI ,1.25 -5.91), and SHMT11420C>T /MTHFR677C>T (OR 2.97, 95% CI 2.97, 1.50 -5.85) confer enhanced risk.The synergy factor calculated with RFC180G>A and MTHFR1298A>C was 2.61-fold, although not statistically significant (p=0.06); RFC180G>A, SHMT11420C>T were not associated with ALL with MLLrearrangements, whereas the MTHFR677C> T confers an increased risk (OR 2.04, 95% CI, 1.19-3.49). In conclusion, the combined effect of RFC180G>A, SHMT11420C>T and MTHFR677C> T increases the risk for early Bcp-ALL, but has no influence in the OS