Germline and Somatic mutations in postmenopausal breast cancer patients.

OBJECTIVES: In breast cancer (BC) patients, the frequency of germline BRCA mutations (gBRCA) may vary according to the ethnic background, age, and family history of cancer. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is the second most common somatic mutated gene in BC; however, the association of mutations in both genes with cancer has not been thoroughly investigated. Thus, our aims were to investigate gBRCA mutation frequency in a cohort of postmenopausal Brazilian BC patients and the association of gBRCA1/BRCA2 and PIK3CA somatic mutations. METHODS: Forty-nine postmenopausal (>55 years) and forty-one young (≤35 years) BC patients were included in this study. The postmenopausal group included patients who reported a positive family history of cancer. For these patients, gBRCA1/BRCA2 were sequenced using next-generation sequencing (NGS) or Sanger sequencing. Data for gBRCA in young patients were already available from a previous study. DNA from formalin-fixed, paraffin-embedded (FFPE) tumors was obtained from 27 postmenopausal and 41 young patients for analyzing exons 9 and 20 of PIK3CA. The association between gBRCA1/BRCA2 and somatic mutations in PIK3CA was investigated. RESULTS: The overall frequency of gBRCA1/BRCA2 among the 49 postmenopausal patients was 10.2%. The frequencies of somatic mutations in PIK3CA in the postmenopausal and young patients were 37% and 17%, respectively (ns). The most common PIK3CA mutation was found to be E454A. Nonsense and frameshift mutations, which may counteract the oncogenic potential of PIK3CA were also detected. Regardless of age, 25% of BRCA1/BRCA2 mutation carriers and non-carriers , each, had PIK3CA somatic mutations. CONCLUSIONS: Data obtained indicate that BRCA1/BRCA2 gene testing may be considered for postmenopausal patients with BC who have a family history of cancer. Although some of them are not considered pathogenic, somatic variants of PIK3CA are frequently observed in BC patients, especially in postmenopausal patients.

Germline and Somatic mutations in postmenopausal breast cancer patients

OBJECTIVES: In breast cancer (BC) patients, the frequency of germline BRCA mutations (gBRCA) may vary according to the ethnic background, age, and family history of cancer. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is the second most common somatic mutated gene in BC; however, the association of mutations in both genes with cancer has not been thoroughly investigated. Thus, our aims were to investigate gBRCA mutation frequency in a cohort of postmenopausal Brazilian BC patients and the association of gBRCA1/BRCA2 and PIK3CA somatic mutations. METHODS: Forty-nine postmenopausal (>55 years) and forty-one young (≤35 years) BC patients were included in this study. The postmenopausal group included patients who reported a positive family history of cancer. For these patients, gBRCA1/BRCA2 were sequenced using next-generation sequencing (NGS) or Sanger sequencing. Data for gBRCA in young patients were already available from a previous study. DNA from formalin-fixed, paraffin-embedded (FFPE) tumors was obtained from 27 postmenopausal and 41 young patients for analyzing exons 9 and 20 of PIK3CA. The association between gBRCA1/BRCA2 and somatic mutations in PIK3CA was investigated. RESULTS: The overall frequency of gBRCA1/BRCA2 among the 49 postmenopausal patients was 10.2%. The frequencies of somatic mutations in PIK3CA in the postmenopausal and young patients were 37% and 17%, respectively (ns). The most common PIK3CA mutation was found to be E454A. Nonsense and frameshift mutations, which may counteract the oncogenic potential of PIK3CA were also detected. Regardless of age, 25% of BRCA1/BRCA2 mutation carriers and non-carriers , each, had PIK3CA somatic mutations. CONCLUSIONS: Data obtained indicate that BRCA1/BRCA2 gene testing may be considered for postmenopausal patients with BC who have a family history of cancer. Although some of them are not considered pathogenic, somatic variants of PIK3CA are frequently observed in BC patients, especially in postmenopausal patients.

Use of an automated pyrosequencing technique for confirmation of sickle cell disease.

BACKGROUND: The diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sß0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary. OBJECTIVES: To develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB gene. METHODS: We developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene. RESULTS: We identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant PyS = heterozygous S and HPLC, which would suggest SB-thalassemia or other heterozygous S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sß thalassemia genotype, 84 samples were classified as Sß0 thalassemia and 81 as Sß+ thalassemia. The most frequent beta thalassemia mutations of Sß0 and Sß+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively. DISCUSSION: The PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common ß+ and ß0 mutations in SCD patients with Sß-thalassemia in a large multi-institutional SCD cohort in Brazil.

Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil.

BACKGROUND: Approximately 8-15% epithelial ovarian cancer patients are BRCA1 or BRCA2 germline mutation carriers. Brazilian inhabitants may have peculiar genetic characteristics associated with ethnic diversity, and studies focusing on the entire BRCA1/BRCA2 gene sequencing in Brazilian ovarian cancer patients are still lacking. The aim of this study was to evaluate BRCA1/2 mutations, through entire gene sequencing, in a Brazilian population of women with epithelial ovarian cancer. METHODS: In a cross sectional study performed in one reference centre for cancer treatment in São Paulo, Brazil, 100 patients diagnosed with epithelial ovarian cancer unselected for family history of breast and/or ovarian cancer were included. The complete coding sequence of BRCA1/2 genes was evaluated through Next-Generation or capillary sequencing. Large deletions were investigated through Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: Nineteen pathogenic mutations (BRCA1: n = 17 and BRCA2: n = 2) featuring 14 different mutations, including two large deletions in BRCA1 (exon 1-2 deleted and exon 5-7 deleted) were identified. Three mutations were detected more than once (c.3331_3334delCAAG, c.5266dupC and c.4484G > T). Two novel frameshift mutations were identified, one in BRCA1 (c.961_962delTG) and one in BRCA2 (c.1963_1963delC). BRCA1/2 mutations were seen in 35.5% of the patients with first and/or second-degree relatives with breast and/or ovarian cancer. Nineteen variants of uncertain significance (VUS) were detected (BRCA1: n = 2 and BRCA2: n = 17), including five distinct missense variants (BRCA1: c.5348 T > C; BRCA2: c.2350A > G, c.3515C > T, c.7534C > T, and c.8351G > A). CONCLUSIONS: Among epithelial ovarian cancer patients unselected for family history of cancer, 19% were BRCA1/2 germline mutation carriers. Almost ¾ of the BRCA mutations, including two large deletions, were detected only once. Our work emphasizes the need of entire gene sequencing and MLPA screening in Brazil.

Anticorpos contra citoplasma de neutrófilos (ANCA) em portadores vasculites / Antibodies against neutrophil cytoplasm (ANCA) in bearing of vasculitis

A granulomatose de Wegener é uma vasculite sistêmica cujo diagnóstico se baseia em dados clínicos, radiológicos e histopatológicos. Recentemente foi descrito nesses pacientes a presença de anticorpos contra citoplasma de neutrófilos normais (ANCA) e sua detecçäo seria de valor no diagnóstico e na avaliaçäo de atividade de doença. Estudamos a freqüência desses anticorpos utilizando técnicas de ELISA e imunofluorescência indireta (IFI) em 103 pacientes com vasculites primárias ou secundárias a doenças difusas do tecido conjuntivo. Observamos positividade do ANCA por IFI em sete de 17 pacientes com granulomatose de Wegener (GW), quatro de 24 portadores de PAN, sendo que três desses apresentaram padräo atípico de IF, houve positividade microscópica e um paciente, vasculite näo caracterizada. Pela técnica de ELISA, houve positividade do ANCA em cinco de 24 casos de PAN, quatro de 17 casos de GW, um de 16 casos de polimialgia reumática, um caso de arterite microscópica e em uma, vasculite näo determinada. Esses dados confirmam o valor da detecçäo do ANCA em vasculites sistêmicas primárias, particularmente a GW

Anticorpos anticardiolipina em vasculites / Antiocardiolipin antibodies in vasculitis

Os autores estudaram os anticorpos anticardiolipina (aCL) em pacientes com vasculites. Esses anticorpos foram detectados em seis de 15 pacientes com vasculite reumatóide, três de 29 com poliarterite nodosa, dois de nove com síndrome de Behcet, dois de quatro com arterite de células gigantes, um de seis com vasculite de Churg-Strauss e nenhum dos pacientes com granulomatose de Wegener ou púrpura de Henoch-Schönlein. Os autores discutem os possíveis mecanismos patogênicos desses anticorpos