Manejo de las muestras para test inmunohistoquímicos, moleculares y genéticos en el cáncer de mama / Management of samples for immunohistochemical, molecular and genetic tests in breast cancer

Las determinaciones inmunohistoquímicas y moleculares en las muestras tisulares son métodos utilizados para establecer factores pronósticos y predictivos en el cáncer de mama. Para que estas técnicas sean lo más eficaces posible se requiere de un correcto manejo de los especímenes, lo que incluye controles preanalíticos como el del tiempo de isquemia, una correcta fijación, tallado e inclusión en parafina, así como la adecuada selección de la representación del tumor que se someterá a estudio. La precisión en todos estos pasos repercutirá en un resultado óptimo de la técnica inmunohistoquímica o molecular aplicada (AU)

Molecular and immunohistochemical analysis of tissue samples are useful tools to establish prognostic and predictive factors in breast cancer. The efficiency of these techniques is based on a correct management of surgical specimens which includes pre-analytic controls such time of ischemia, appropriate fixation, gross manipulation and paraffin embedding. It is also important the right selection of tumor representation for study submission. The accuracy of all these steps will affect in the optimal result of the immunohistochemical and molecular test applied (AU)

Current molecular genetics strategies for the diagnosis of lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)].

Aplicación de la secuenciación masiva de nueva generación al diagnóstico molecular de la hipercolesterolemia familiar / Applicability of next generation sequencing to the molecular diagnosis of familial hypercholesterolemia

Introducción: La hipercolesterolemia familiar es una enfermedad autosómica dominante que cursa con niveles plasmáticos elevados de colesterol unido a lipoproteínas de baja densidad. La presencia de esta alteración en el metabolismo lipídico se asocia a un aumento del riesgo cardiovascular en los pacientes que la padecen, siendo de gran importancia la realización de un diagnóstico genético para ofrecer un tratamiento adecuado y disminuir la morbimortalidad. El objetivo de este trabajo es describir la aplicación de la secuenciación de nueva generación al diagnóstico genético de la hipercolesterolemia familiar, en comparación con la secuenciación Sanger, la técnica convencional usada hasta el momento. Material y métodos: Se analizaron 110 muestras de sangre venosa periférica procedente de pacientes que presentaban cuadro clínico de hipercolesterolemia familiar mediante secuenciación de nueva generación, utilizando un panel comercial que permite la identificación de mutaciones en los genes LDLR, APOB, PCSK9 yLDLRAP1 (SEQPRO Lipo, Progenika) con la tecnología GS JUNIOR 454 (Roche). Resultados: Aplicando esta tecnología fue posible secuenciar los genes asociados a la hipercolesterolemia familiar descritos hasta el momento en grupos de hasta 20 pacientes simultáneamente. Se detectaron un total de 35 mutaciones en las 110 muestras analizadas, localizándose el 94,29% en el gen LDLR. Todas las mutaciones identificadas fueron confirmadas mediante el método de secuenciación Sanger. Conclusiones: La utilización de la secuenciación masiva de nueva generación permite la realización de un diagnóstico genético más rápido y un análisis molecular más eficiente de los genes implicados en la hipercolesterolemia familiar con una fiabilidad similar a la técnica convencional Sanger (AU)

Introduction: Familial hypercholesterolemia is an autosomal dominant disorder that causes increased levels of cholesterol associated with low density lipoproteins in plasma. The presence of altered lipid metabolism in these patients increases their level of risk of suffering from a cardiovascular disease. The certainty of having this genetic disorder by making a timely and precise molecular diagnostic is crucial for the appropriate treatment and the reduction of the disease morbidity-mortality. The aim of this work is to describe the applicability of next generation sequencing technology to the genetic diagnosis of familial hypercholesterolemia and compare this novel method with the conventional Sanger sequencing method. Material and methods: A next generation sequencing commercial panel (SEQPRO Lipo, Progenika) was used to analyze 110 peripheral venous blood samples from patients with familial hypercholesterolemia. This enables the assessment of mutations in genes associated with the disease (e.g. LDLR, APOB, PCSK9 and LDLRAP1)with the GS JUNIOR 454 technology (Roche). Results: Application of next generation sequencing enables the sequencing of the genes involved in the familial hypercholesterolemias, described so far, in groups of 20 patients simultaneously. Using this novel technology, a total of 35 mutations were detected in the 110 analysed samples, with 94.29% being located in the LDLR gene. Mutations were confirmed by Sanger sequencing. Conclusion: Next generation sequencing enables a quick genetic diagnosis and a more efficient molecular analysis of all genes described so far to be involved in familial hypercholesterolemia, with similar reliability to that of conventional Sanger sequencing (AU)