Association of BRCA1 germline mutations in young onset triple-negative breast cancer (TNBC).

BACKGROUND: BRCA1-associated breast cancers have been associated to a triple-negative phenotype. The prevalence of BRCA1 germline mutations in young onset TNBC based on informativeness of family history has not been reported. PATIENTS AND METHODS: From January 2008 to May 2009 were collected blood and tumor samples from patients with TNBC younger than 50 years and without a family history of breast and ovarian cancer in first- and second-degree relatives. Analysis of BRCA1 germline mutations was made. Age at diagnosis and informativeness of family history (presence of female in first- and second-degree relatives alive until age 45) was collected in all cases. Immunohistochemistry of basal-like features was performed centrally in all available tumors. RESULTS: Seven pathogenic mutations were detected in 92 patients (7.6 %), two of them in patients younger than 35 years (28.6 %) (Fisher's exact test, p = 0.631). Three non-classified variants were detected (3.2 %). Family history was informative in two patients with a pathogenic mutation (28.6 %) and not informative in five (71.4 %) (Fisher's exact test, p = 0.121). Of the seven patients with a pathogenic mutation, four had a basal-like phenotype. CONCLUSION: Patients with apparently sporadic TNBC younger than 50 years and a non-informative family history are candidates for germline genetic testing of BRCA1.

Genetic analysis of BRCA1 and BRCA2 in breast/ovarian cancer families from Aragon (Spain): two novel truncating mutations and a large genomic deletion in BRCA1.

We screened BRCA1 and BRCA2 germline mutations in 60 high-risk breast and/or ovarian cancer patients and 20 relatives from Aragon (Spain) by DHPLC (Denaturing High Performance Liquid Chromatography) and direct sequencing of the entire coding sequence and the splicing sites of both genes. We have identified 17 different pathogenic mutations: 8 in BRCA1 and 9 in BRCA2 in 60 unrelated patients and 50% of relatives were carriers. The prevalence of pathogenic mutations in this study was 33.33%. Two truncating mutations are novel: c.5024_5025delGA in exon 16 of BRCA1 and c.2929delC in exon 11 of BRCA2 (numbered after GenBank U14680 and U43746). Multiplex Ligation Dependent Probe Amplification (MLPA) was performed for large mutational scanning of both genes and a large genomic deletion in BRCA1 was found (DelEx8-13). Furthermore, five mutations are described for the first time in Spanish population: c.1191delC, c.3478_3479delTT and c.6633_6637delCTTAA (BRCA1) and c.3972_3975delTGAG and 3908_3909delTG (BRCA2). Three mutations have been reported previously once in Spain: c.3600_3610del11 (BRCA1), c.5804_5807delTTAA (BRCA2) and c.9246C>A (BRCA2). The mutation c.5374_5377delTATG has been found before only in two unrelated families from Castilla-Leon, Spain (BRCA2). Frequent mutations described in Spanish population have also been present: c.187_188delAG, c.5242C>A and c.5385insC in BRCA1 and c.3492_3493insT and c.9254_9258delATCAT in BRCA2. c.5242C>A, 3972_3975delTGAG and c.5804_5807delTTAA were the recurrent mutations found. Fifteen different unclassified variants were identified (25% families). Although specific BRCA1 and BRCA2 mutations are recurrently reported as a result of genetic founder effects we conclude that heterogeneous ethnicity increases the variety of mutations that can be found in Spanish populations.

Evaluación del test del azul de dimetilmetileno (DMB) en el screening de las mucopolisacaridosis y su comparación con la prueba del cetilpiridinio (CPC) / Dimethylmethylene (DMB) test in the screening of mucopolysaccharidosis compared with cetylpiridinium choloride (CPC) technique

En el presente trabajo se realiza un estudio comparativoentre las técnicas del cloruro de cetilpiridinio (CPC) y la del azulde dimetilmetileno (DMB), utilizadas en el diagnóstico o screeningde las mucopolisacaridosis, y se calculan para estas técnicasasí como para la del azul Alcian, a partir de los datos procedentesde Milani y col.10, la sensibilidad, especificidad, valorpronóstico de la prueba positiva y eficiencia del método conobjeto de comparar y recomendar una de ellas.De las 1160 muestras de orinas analizadas procedentes delHospital Infantil del complejo hospitalario del Miguel Servet, 14fueron mucopolisacaridosis verdaderas, diagnosticadas por eltest del azul de toluidina (++++) y cromatografía en capa finay, posteriormente, confirmadas por la realización de los correspondientestest enzimáticos. El método del CPC se llevó a caboen 1042 casos, de los cuales 108 resultaron ser falsos positivos(10,30 %), frente a los 3 casos de los 118 (2.54 %) queresultaron de entre los realizados con la técnica del DMB.El tratamiento estadístico de los datos no aporta resultadosconcluyentes a favor de ninguna de las dos técnicas. En cambio,fueron determinantes los análisis de la sensibilidad, especificidad,valor pronóstico de la prueba positiva y eficiencia dela misma, a favor de la técnica del DMB frente a las otras dostécnicas (CPC y azul Alcian). Estos datos permiten recomendardicha técnica y aportan la razón por la que la misma sea la deelección para el screening de las mucopolisacaridosis

This study compares the cetylpiridinium chloride (CPC) andthe dimethylmethylene blue (DMB) techniques, both of which areused in the diagnosis or screening of mucopolysaccharidosis,and in these techniques as well as in those of the Alcian blue,from the Milani et col.10 data, the sensibility, specificity, pronosticvalue of the positive test and efficiency of the technique areevaluated in order to compare and recommende one of them. Of the 1,160 urine samples analysed from the Children’sHospital of the Miguel Servet Hospital complex, 14 were truemucopolysaccharidosis, diagnosed by means of the toluidineblue test (++++) and thin layer chromatography and, posteriourly,confirmed by performing the corresponding enzymatictest. The CPC method was performed in 1,042 cases, of which108 (10.3 %) were false positives, against the 3 cases of the118 (2.54 %) resulting from the DMB technique.The statistical treatment of the data contributed no conclusiveresults in favour of either of the techniques but the analysesof sensitivity, specificity, prognostic value of the positivetest also its efficiency favoured the DMB in relation to the othertwo techniques (CPC and Alcian blue). These data support therecommendation of this technique and may be the reason bythe preference of the same one to perform the screening of themucopolysaccharidosis

Modificación de la técnica de determinación de mucopolisacáridos que utiliza azul de dimetilmetileno / Modification of the technique of determination of mucopolisacaccharides with dimethyl-metilene blue

Una de las técnicas con colorantes catiónicos (azul de dimetilmetileno) utilizadas en los laboratorios clínicos para el screening de mucopolisacaridosis recomienda la precipitación de los GAG o mucopolisacáridos con CPC. En este trabajo se introdujo una modificación que consistió en eliminar la fase de precipitación, con objeto de evitar las posibles pérdidas de muestra que pudieran tener lugar con las sucesivas decantaciones en las orinas con valores elevados y en las de valores más bajos, aparte de la simplificación que representa para la técnica. Los resultados confirmaron la hipótesis, encontrándose diferencias significativas (p<0.01) en los valores de GAG en orinas de dos mucopolisacaridosis (valores más altos) y un adulto normal (valores más bajos). Para el rango de valores intermedios, ambas técnicas, precipitando y sin precipitar, proporcionan idénticos resultados.En conclusión, con la modificación propuesta se simplifica la técnica y se gana fiabilidad en la determinación de los valores extremos de GAG en orina. (AU)

NADH oxidase activity of mitochondrial apoptosis-inducing factor.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFDelta1-120) exhibit NADH oxidase activity, whereas superoxide anion (O(2)(-)) is formed. AIFDelta1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFDelta1-120, which lacks FAD, has no NADH oxidase activity. However, native AIFDelta1-120, apoAIFDelta1-120, and the reconstituted (FAD-containing) holoAIFDelta1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFDelta1-120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFDelta1-120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.