BRCA1 and BRCA2 germline mutation analysis from a cohort of 1267 patients at high risk for breast cancer in Brazil.

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country.

Multidisciplinary diagnostics of chronic lymphocytic leukemia: European Research Initiative on CLL - ERIC recommendations.

Recent advances in chronic lymphocytic leukemia (CLL) includes description of disease genomic landscape, inclusion of prognostic relevant genetic tests in CLL workflow and evaluation of minimal residual disease (MRD)1 in parallel with the increase availability of novel therapy agents. In this review, the theoretical and practical aspects of response assessment have been discussed. These are based on updated recommendations of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) for genetic tests (TP53 mutation and IGHV status) and flow cytometry analysis for CLL. Methodological approaches and interpretation of results were also discussed.2,3.

Characterization of a human induced Pluripotent Stem (iPS) cell line (INCABRi002-A) derived from a primary myelofibrosis patient harboring the 5-bp insertion in CALR and the p.W146X mutation in TP53.

Primary myelofibrosis (PMF) is a hematological malignancy characterized by activation of the JAK/STAT pathway and risk of leukemic transformation. In this study, we generated an induced Pluripotent Stem (iPS) cell line derived from a 65-year old male PMF patient carrying the 5-pb insertion in the CALR gene (CALRins5) and the c.437 G > A mutation in the TP53 gene (p.W146X). The newly derived PMF3.17 iPS cell line harbors the original mutations and was characterized as bona fide iPS. Resource table.

Generation and characterization of a human induced pluripotent stem (iPS) cell line derived from an acute myeloid leukemia patient evolving from primary myelofibrosis carrying the CALR 52bp deletion and the ASXL1 p.R693X mutation.

Peripheral blood sample was donated by a 61years old female patient diagnosed with acute myeloid leukemia secondary to a primary myelofibrosis harboring the 52-bp deletion in the CALR gene (c.1092_1143del, p.L367fs*46) and the R693X mutation in the ASXL1 gene (c.2077C>T, p.R693X). CD34+ cells were isolated from the sample and subjected to the reprogramming procedure by using the Sendai virus carrying the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc. iPS colonies generated retained the original mutations and displayed all the features of bona fide iPS cells.

Caracterização Físico- Química da Asparaginase de Escherichia coli

A asparaginase tipo II de Escherichia coli é amplamente utilizada no tratamento da leucemia linfoblástica aguda. Ela é uma enzima homotetramérica, que catalisa a hidrólise de L-asparagina em ácido aspártico e amônia. Sua atividadeantineoplásica se baseia na depleção desse aminoácido no soro, pois as células leucêmicas são dependentes da L-asparagina plasmática. Por ser uma enzima de origem bacteriana, o tratamento pode causar reações alérgicas, além de efeitos colaterais que podem levar à sua suspensão. Portanto, para futuros avanços terapêuticos no tratamento, é importante o estudo das características cinéticas, termodinâmicas e estruturais da asparaginase. Neste trabalho, a caracterização proteica foi feita por fluorescência intrínseca, dicroísmo circular, aumento da pressão hidrostática, calorimetria diferencial de varredura, calorimetria de titulaçãoisotérmica, espectroscopia de absorção em UV-Visível e microscopia. A desnaturação térmica da asparaginase foi reversível de pH 7,5 a 9,5, com maiorreversibilidade entre pH 8,0 e 9,0. O pico de desnaturação, obtido por calorimetria diferencial de varredura, foi assimétrico e a desnaturação bastante cooperativa. O aumento da concentração proteica não causou aumento da temperatura de transição ou da entalpia calorimétrica, indicando a ausência de monômeros estáveisdurante a desnaturação. No entanto, a temperatura de transição foi dependente da velocidade de varredura, mostrando a presença de um intermediário dedesnaturação cineticamente controlado. A atividade e a estabilidade enzimáticaforam estudadas de pH 2,0 a 13,0. A enzima teve atividade máxima de pH 5,5, a 9,0 e apresentou pico de transição térmica de pH 2,0 a 12,0. A estrutura secundária não mudou de pH 3,0 a 12,0 e os resultados mostraram que a asparaginase desnaturouapenas em pH 13,0. Em pH 2,0, houve a formação de um intermediário do tipo“molten globule”. Em pH 8,0, a desnaturação da asparaginase por ureia ou por cloridrato...

Type II Escherichia coli asparginase is widely used in the treatment of acutelymphoblastic leukemia. It is a homotetrameric enzyme which catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. Its antineoplastic activity is based on the depletion of this amino acid in the serum, because leukemic cells aredependent on circulating L-asparagine. Because it is a bacterial enzyme, thetreatment can cause allergic reactions, as well as side effects that may lead to the suspension of the therapy. Thus, for future therapeutic advances in the treatment of acute lymphoblastic leukemia, it is important to study the kinetic, thermodynamic andstructural properties of asparaginase. In this work, protein characterization was done by intrinsic fluorescence, circular dichroism, high hydrostatic pressure, differential scanning calorimetry, isothermal titration calorimetry, UV-Visible absorption spectroscopy and microscopy. The thermal denaturation of asparaginase was reversible from pH 7.5 to 9.5, with higher reversibility values from pH 8.0 to 9.0. Thetransition peak, obtained by differential scanning calorimetry, was asymmetric and the thermal transition was highly cooperative. Increasing protein concentration did not increase the transition temperature or the calorimetric enthalpy, showing that thethermal transition occurred without stable monomers. However, the transition temperature was dependent on scan rate, indicating the presence of a transition intermediate kinetically controlled. The enzymatic activity and stability were studied from pH 2.0 to 13.0. The enzyme had maximum stability from pH 5.5 to 9.0, andpresented thermal transition from pH 2.0 to pH 12.0. The secondary structure did not change from pH 3.0 to 12.0, and our results showed that asparagine was denatured only at pH 13.0. At pH 2.0, we observed the formation of a “molten globule” intermediate...

Thermodynamic and structural characterization of zwitterionic micelles of the membrane protein solubilizing amidosulfobetaine surfactants ASB-14 and ASB-16.

Surface tension and isothermal titration calorimetry (ITC) were used to determine the critical micelle concentration (cmc) of the zwitterionic amidosulfobetaine surfactants ASB-14 and ASB-16 (linear-alkylamidopropyldimethylammoniopropanosulfonates) at 25 °C. The cmc and the heat of micellization were determined from 15 to 75 °C by ITC for both surfactants. The increase in temperature caused significant changes in the enthalpy and in the entropy of micellization, with small changes in the standard Gibbs energy (ΔG(mic)), which is consistent to an enthalpy−entropy compensation with a compensatory temperature of 311 K (ASB-14) and 314 K (ASB-16). In the studied temperature range, the heat capacity of micellization (ΔC(p)(mic)) was essentially constant. The experimental ΔC(p)(mic) was lower than that expected if only hydrophobic interactions were considered, suggesting that polar interactions at the head groups are of significant importance in the thermodynamics of micelle formation by these surfactants. Indeed, a NMR NOESY spectrum showed NOEs that are improbable to occur within the same monomer, resulting from interactions at the polar head groups involving more than one monomer. The ITC and NMR results indicate a tilt in the polar headgroup favoring the polar interactions. We have also observed COSY correlations typical of dipolar interactions that could be recovered with the partial alignment of the molecule in solution, which results in an anisotropic tumbling. The anisotropy suggested an ellipsoidal shape of the micelles, which results in a positive magnetic susceptibility, and ultimately in orientation induced by the magnetic field. Such an ellipsoidal shape was confirmed from results obtained by SAXS experiments that revealed aggregation numbers of 108 and 168 for ASB-14 and ASB-16 micelles, respectively. This study characterizes an interesting micelle system that can be used in the study of membrane proteins by solution NMR spectroscopy.