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.

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.

Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles.

The catalytic behaviour of alpha-CT (alpha-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme-micelle interaction leads to an increase in both the V(max) and the affinity for the substrate p -nitrophenyl acetate, indicating higher catalytic efficiency for bound alpha-CT. The bell-shaped profile of alpha-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in alpha-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the alpha-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in alpha-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of alpha-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased alpha-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm(-1), corresponding to the alpha-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the beta-sheet content in micelle-bound alpha-CT. Our data suggest that the higher catalytic efficiency of micelle-bound alpha-CT results from significant conformational changes.