Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

In Vivo Selection of Fluoroquinolone-Resistant Escherichia coli Isolates Expressing Plasmid-Mediated Quinolone Resistance and Expanded-Spectrum β-Lactamase

A ciprofloxacin-resistant Escherichia coli isolate, isolate 1B, was obtained from a urinary specimen of a Canadian patient treated with norfloxacin for infection due to a ciprofloxacin-susceptible isolate, isolate 1A. Both isolates harbored a plasmid-encoded sul1-type integron with qnrA1 and blaVEB-1 genes. Isolate 1B had amino acid substitutions in gyrase and topoisomerase.

Functional Variants in NOS1 and NOS2A Are Not Associated with Progressive Hearing Loss in Ménière's Disease in a European Caucasian Population

Hearing loss in Meniere's disease (MD) is associated with loss of spiral ganglion neurons and hair cells. In a guinea pig model of endolymphatic hydrops, nitric oxide synthases (NOS) and oxidative stress mediate loss of spiral ganglion neurons. To test the hypothesis that functional variants of NOS1 and NOS2A are associated with MD, wed genotyped three functional variants of NOS1 (rs41279104,rs2682826, and a cytosine-adenosine microsatellite repeat in exon 1f) and the CCTTT repeat in the promoter of NOS2A gene (rs3833912) in two independent MD sets(273 patients in total) and 550 controls. A third cohort of American patients was genotyped as replication cohort for the CCTTT repeat. Neither allele nor genotype frequencies of rs41279104 and rs2682826 were associated with MD, although longer alleles of the cytosine-adenosine microsatellite repeat were marginally significant (corrected p = 0.05) in the Mediterranean cohort but not in a second Galicia cohort. Shorter numbers of the CCTTT repeat in NOS2A were significantly more frequent in Galicia controls (OR = 0.37 [CI, 0.18-0.76], corrected p =0.04), but this finding could not be replicated in Mediterranean or American case-control populations. Meta-analysis did not support an association between CCTTT repeats and risk for MD. Severe hearing loss (>75 dB) was also not associated with any functional variants studied. Functional variants of NOS1 and and NOS2A do not confer susceptibility for MD.

Granada Virus: a Natural Phlebovirus Reassortant of the Sandfly Fever Naples Serocomplex with Low Seroprevalence in Humans

A new member of the phlebovirus genus, tentatively named Granada virus, was detected in sandflies collected in Spain. By showing the presence of specific neutralizing antibodies in human serum collected in Granada, we show that Granada virus infects humans. The analysis of the complete genome of Granada virus revealed that this agent is likely to be a natural reassortant of the recently described Massilia virus (donor of the long and short segments) with ayet unidentified phlebovirus (donor of the medium segment)

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

FABP4 Dynamics in Obesity: Discrepancies in Adipose Tissue and Liver Expression Regarding Circulating Plasma Levels.

BACKGROUND FABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile. OBJECTIVE In patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed. METHODS The expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot. RESULTS In obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group. CONCLUSION The inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity.

Inverse relation between FASN expression in human adipose tissue and the insulin resistance level.

BACKGROUND Adipose tissue is a key regulator of energy balance playing an active role in lipid storage and may be a dynamic buffer to control fatty acid flux. Just like PPARgamma, fatty acid synthesis enzymes such as FASN have been implicated in almost all aspects of human metabolic alterations such as obesity, insulin resistance or dyslipemia. The aim of this work is to investigate how FASN and PPARgamma expression in human adipose tissue is related to carbohydrate metabolism dysfunction and obesity. METHODS The study included eighty-seven patients which were classified according to their BMI and to their glycaemia levels in order to study FASN and PPARgamma gene expression levels, anthropometric and biochemical variables. RESULTS The main result of this work is the close relation between FASN expression level and the factors that lead to hyperglycemic state (increased values of glucose levels, HOMA-IR, HbA1c, BMI and triglycerides). The correlation of the enzyme with these parameters is inversely proportional. On the other hand, PPARgamma is not related to carbohydrate metabolism. CONCLUSIONS We can demonstrate that FASN expression is a good candidate to study the pathophysiology of type II diabetes and obesity in humans.

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

OBJECTIVE To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

Epigenetic mechanisms in non-alcoholic fatty liver disease: An emerging field.

Non-alcoholic fatty liver disease (NAFLD) is an emerging health concern in both developed and non-developed world, encompassing from simple steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis and liver cancer. Incidence and prevalence of this disease are increasing due to the socioeconomic transition and change to harmful diet. Currently, gold standard method in NAFLD diagnosis is liver biopsy, despite complications and lack of accuracy due to sampling error. Further, pathogenesis of NAFLD is not fully understood, but is well-known that obesity, diabetes and metabolic derangements played a major role in disease development and progression. Besides, gut microbioma and host genetic and epigenetic background could explain considerable interindividual variability. Knowledge that epigenetics, heritable events not caused by changes in DNA sequence, contribute to development of diseases has been a revolution in the last few years. Recently, evidences are accumulating revealing the important role of epigenetics in NAFLD pathogenesis and in NASH genesis. Histone modifications, changes in DNA methylation and aberrant profiles or microRNAs could boost development of NAFLD and transition into clinical relevant status. PNPLA3 genotype GG has been associated with a more progressive disease and epigenetics could modulate this effect. The impact of epigenetic on NAFLD progression could deserve further applications on therapeutic targets together with future non-invasive methods useful for the diagnosis and staging of NAFLD.

Epigenética: definición, bases moleculares e implicaciones en la salud y en la evolución humana

La Epigenética se refiere a los cambios heredables en el ADN e histonas que no implican alteraciones en la secuencia de nucleótidos y modifican la estructura y condensación de la cromatina, por lo que afectan la expresión génica y el fenotipo. Las modificaciones epigenéticas son metilación del ADN y modificaciones de histonas. Objetivo: hacer una revisión de la literatura sobre el concepto de epigenética y su impacto en la salud. Materiales y métodos: se realizó una revisión de la bibliografía sobre el concepto de epigenética, sus bases biológicas, el impacto sobre la salud y la enfermedad y su relación con la evolución. Resultados: los mecanismos epigenéticos han cobrado cada vez más importancia debido a la creciente asociación con enfermedades complejas y comunes, así como por su impacto en la salud de generaciones futuras y en la evolución humana. Conclusiones: la Epigenética tiene un claro impacto en la salud del individuo, en la de su descendencia y en la evolución de la especie humana.

Propuestas de secuencia. Educación plástica y visual.

Las dos secuencias del área de la educación plástica y visual que nos presenta este documento tratan de aportar al educador unas bases para desarrollar su programa de estudio. Los temas que tratará son los siguientes: lenguaje visual, elementos que lo configuran, espacio y volumen, representación de las formas planas, procedimientos y técnicas utilizados en los lenguajes visuales, apreciación del proceso de creación de las artes visuales y proporción y composición.

Bases para la elaboración de materiales curriculares de EPA en Castilla y León.

Se realiza un estudio que pretende ser un referente para la elaboración de materiales curriculares específicos de Educación de Personas Adultas para Castilla y León con la elaboración de un diseño curricular para la formación básica. Se abordan los contenidos curriculares referidos al segundo nivel o de consolidación de conocimientos y técnicas instrumentales con el fin de establecer un referente teórico para la elaboración de materiales curriculares de modo que sirva para unificar criterios de intervención y que, a la vez, integre como principio la flexibilidad para poder llevar a acabo las adaptaciones correspondientes al contexto del centro y del aula; se plantea una secuencia lógica de trabajo tras los contenidos del nivel 1. La oferta formativa para las personas adultas debe estar constituida por una formación básica como proceso que abarca desde la alfabetización a la obtención del título de Graduado en Educación Secundaria Obligatoria para las Personas Adultas (ESPA). Con la obtención del título, se han de cumplir los siguientes objetivos: adquirir y actualizar su formación básica y facilitar el acceso a los diferentes niveles del sistema educativo; mejorar su cualificación profesional o adquirir una preparación para el ejercicio de otras profesiones; desarrollar su capacidad de participación en la vida social, cultural, política y económica.

Del libro didáctico a la secuencia de formación: propuestas de intervención pedagógica

Esta nueva propuesta se constituye como una continuidad del proyecto El libro didáctico como material para la enseñanza de portugués LE - Perspectivas políticas: industria editorial y trabajo simbólico, culminado en 2011 y al mismo tiempo como una nueva etapa de investigación y producción de los materiales de estudio del PLE. El trabajo de análisis de los libros didácticos de portugués LE nos proporcionó bases teóricometodológicas para la reflexión sobre las configuraciones textuales, discursivas y semióticas en torno de la enseñanza del portugués como lengua extranjera en la Argentina y en nuestra región. Sin embargo, consideramos que nuestra tarea de investigación debe avanzar hacia una propuesta de intervención pedagógica que ayude a explorar nuevas estrategias y prácticas de comprensión y producción, a partir de materiales que favorezcan aprendizajes significativos. En esta instancia se propone profundizar las confrontaciones críticas entre las matrices discursivas de los LD y las experiencias educativas posibles de desplegarse en las aulas, tratando de eludir los modos estereotipados de abordar la enseñanza del PLE esto es, reelaborar y resignificar series de ejercitación gramaticales automatizadas y repetitivas; revisar y reinventar las modalidades de mediación narrativa y de sus lazos con otros universos discursivos que den cuenta de las identidades culturales de la brasilidad; cuestionar y poner en tensión la relación entre la lengua nacional, internacional y extranjera, las formas estandarizadas y locales con las producciones discursivas sociales disponibles; y los modos discursivos y genéricos en los cuales las autorías y las figuras de lectores se inscriben en los materiales de estudio. El punto de vista del AD, en relaciones transdisciplinarias con la glotopolítica, la semiótica y otras perspectivas críticas de la cultura y la educación, permitirá resituar, e interpretar alternativas didácticas para reorientar tanto los procesos de formación docente cuanto las experiencias áulicas en el contexto regional del Mercosur. En términos programáticos se expandirán los recorridos por diversos archivos discursivos y de tecnologías de la información y la comunicación vinculados a la LP; en este sentido, se deja abierta la posibilidad de incluir libros didácticos de reciente edición y archivos textuales en diferentes soportes tecnológicos que contrasten significativamente con el corpus ya analizado. Se trabajará en la elaboración de propuestas de intervención para la enseñanza de PLE que ponderen críticamente los posicionamientos ideológicos respecto de la lengua y las textualidades y propicien la condición dialógica del quehacer educativo. Dichas propuestas serán consideradas como secuencias de formación, entendidas como focos del proceso semiótico y polisémico de los saberes implicados en el aprendizaje de una lengua extranjera. Se discutirán criterios selectivos y operativos UNaM – FHyCS – SInvyP Acreditación de Proyectos de Investigación 5 de construcción de dichas secuencias en su dimensión curricular pedagógica y en escalas graduales e integradas de prácticas con el PLE.

Del libro didáctico a la secuencia de formación: propuestas de intervención pedagógica. 16H/342

Este proyecto se desprende de El LD como material para la enseñanza de PLE -Perspectivas políticas: industria editorial y trabajo simbólico culminado en 2011. El análisis de los LDs (libros didácticos) de PLE (Portugués Lengua Extranjera) nos proporcionó bases teórico-metodológicas para la reflexión sobre las configuraciones textuales, discursivas y semióticas acerca de la enseñanza del PLE en la Argentina y en nuestra región. Sin embargo, consideramos que nuestra tarea investigativa debe avanzar en una propuesta de intervención pedagógica que ayude a explorar nuevas estrategias y prácticas de comprensión y producción, a partir de materiales que favorezcan aprendizajes significativos. Para ello revisamos las conclusiones elaboradas en la etapa anterior en relación con las matrices discursivas predominantes de los LDs focalizadas en las dimensiones históricopolíticas y de gramaticalización de la lengua, en las matrices ético-culturales de la identidad brasileña y en las configuraciones editoriales del mundo de los negocios. Nuestro objetivo es elaborar propuestas de intervención para la enseñanza del PLE que ponderen críticamente los posicionamientos ideológicos respecto de la lengua y las textualidades que propicien la condición dialógica del quehacer educativo. Dichas propuestas serán consideradas como secuencias de formación, entendidas como focos del proceso polisémico de los saberes implicados en el aprendizaje de una lengua extranjera.