Expresión diferencial de microRNAs en tumores de mujeres jóvenes con cáncer de mama: Cribado de dianas moleculares

El cáncer de mama constituye el cáncer invasivo más frecuente en mujeres, afectando a una de cada 8 mujeres a lo largo de su vida, siendo, además, la primera causa de muerte derivada de cáncer invasivo en la población española (15,5%). Las mujeres jóvenes representan únicamente el 3% del total de casos diagnosticados; sin embargo, estas pacientes presentan tumores de mayor tamaño e índice de proliferación, estando sobrerepresentados subtipos tumorales más agresivos y de peor pronóstico. Los microRNAs (miRNAs) son pequeñas moléculas de RNA no codificante, cuyo papel en la célula es regular la expresión de genes, bloqueando el paso de RNA mensajero a proteína. Su desregulación es típica en numerosas enfermedades, incluyendo cáncer, constituyen una herramienta muy útil como biomarcador diagnóstico y pronóstico debido a su estabilidad y reproducibilidad. El principal objetivo del trabajo de esta tesis ha sido establecer un perfil molecular característico del cáncer de mama en mujeres jóvenes. Para ello se seleccionaron 22 pacientes de cáncer de mama menores de 35 años y 12 pacientes mayores de 65 que no hubiesen recibido tratamiento neoadyuvante, no metastásicas y sin mutación en BRCA1/2, además de 3 mujeres sanas. Mediante un chip de hibridación de miRNAs se obtuvo la expresión en tejido tumoral de 1100 miRNAs humanos, de los cuales 91 mostraron una expresión diferente en mujeres menores de 35 años. Se obtuvo un perfil de miRNAs específico de mujeres jóvenes con cáncer de mama. Este perfil identificativo fue validado en líneas celulares procedentes de mujeres jóvenes (HBL100, HCC1500, HCC1937). Se estudió, además, en una serie de datos de 750 pacientes de cáncer de mama obtenidas a partir de datos depositados online de un estudio previo (METABRIC). Los miRNAs desregulados en mujeres jóvenes apuntaban a genes diana de rutas relacionadas con proliferación, supervivencia celular, migración y movilidad celular, y relacionadas directamente con procesos cancerosos. Se seleccionaron 18 miRNAs representativos, de los cuales, seis fueron validados mediante qRT-PCR en una nueva serie de 55 pacientes: hsa-miR-30c-5p, hsa-miR-92b, hsa-miR-139, hsa-miR-1228, hsa-miR-1275, hsa-miR-3196. Los miRNAs hsa-miR-92b y hsa-miR-139 mostraron una peor supervivencia asociada a su represión y sobre-expresión, respectivamente. Entre sus genes diana, se seleccionaron receptores de membrana relevantes desde un punto de vista traslacional: HER4, GRIN2B, GRIN3A y GRM3 y se evaluó su expresión mediante qRT-PCR, mostrando GRIN2B una expresión mayor en mujeres jóvenes. Se midió la cantidad de proteína HER4 en 31 cortes de tejido tumoral de pacientes jóvenes y 29 de mayores de 65 años, mediante tinción inmunohistoquímica in situ, lo que confirmó que existe una mayor expresión de HER4, localizada en membrana, en mujeres jóvenes. Los resultados respaldan la idea de que el cáncer de mama en mujeres jóvenes constituye una entidad clínica y biológica diferenciada dentro del cáncer de mama general, con una etiología particular que responde a una desregulación de procesos celulares relacionados con proliferación, supervivencia celular, migración y movilidad celular, que merece una mayor atención de cara a ofrecer un tratamiento personalizado más acorde a las necesidades particulares de este grupo de pacientes.Breast cancer is the most frequent cáncer in women, afecting one in eight women in their lifetime. It is also the first invasive cancer derived cause of death in Spanish population (15.5%). Young women only account for the 3% of total diagnosed cases, however, this patients usually present larger tumors with higher proliferation and overrepresentated in more aggressive tumor subtypes. MicroRNAs (miRNAs) are small non coding RNA molecules that regulates gene expression by silencing the protein translation. MiRNA deregulation tipycally affects in various diseases, including cancer, and constitute a useful tool as a diagnosis and prognosis biomarker, due to their stability and reproducibility. The aim of this study was to stablish a molecular profile unique of breast cancer in very young women. With that purpose, 22 breast cancer patients younger than 35 years and 12 patients older than 65 years were selected, besides of 3 healthy women. None of the patients recived neoadjuvant treatment, were metastasic nor/or have known BRCA1/2 mutation. Using a miRNA Affymetrix microarray, the expression of 1100 miRNAs was assessed in tumoral tissue. 91 of those miRNAs showed differential expression in patients younger than 35 years. The analyses performed revealed a molecular profile distinctive of young women with breast cancer. The profile was validated on cell lines derived from mammary tumor of young women (HBL100, HCC1500, HCC1937). The analyses were also carried out on a dataset from 750 breast cancer patients obtained from a published study (METABRIC repository). Deregulated miRNAs in young women pointed out to target genes related with proliferation, cell survival, migration and cell motility pathways, and with cancer-related events. Eighteen representative miRNAs were selected, six of those were validated by qRT-PCR in an independent serie of 55 patients: hsa-miR-30c-5p, hsa-miR-92b, hsa-miR-139, hsa-miR-1228, hsa-miR-1275, hsa-miR-3196. MiRNAs hsa-miR92b and hsa-miR-139 show differential survival prognosis when downregulated and upregulated, respectively. Among the target genes, relevant receptors were selected: HER4, GRIN2B, GRIN3A y GRM3 and their mRNA expression was measured using qRT-PCR assays. The results showed a higher expression of GRIN2B in tissue from young women. The HER4 protein was quantified by IHC in tumoral tissue from 31 young patients and 29 older patients, confirming a higher membrane expression of HER4 in young women. The results of this work support the idea that breast cancer in very young women constitutes a clinical and biological entity inside breast cancer, which particular etiology is due to the deregulation of cellular proceses related to proliferation, cell survival, migration and cell motility. Breast cancer in very young women, though, deserves utterly attention towards offering more focussed treatment that grants the needs of this particular group of patients

Gender is a major factor explaining discrepancies in eye colour prediction based on HERC2/OCA2 genotype and the IrisPlex model

In recent years, several studies have greatly increased our understanding of the genetic basis underlying human eye colour variation. A large percentage of the eye colour diversity present in humans can already be genetically explained, so much so that different DNA-based eye colour prediction models, such as IrisPlex, have been recently developed for forensic purposes. Though these models are already highly accurate, they are by no means perfect, with many genotype-phenotype discrepancies still remaining unresolved. In this work we have genotyped six SNPs associated with eye colour (IrisPlex) in 535 individuals from Spain, a Mediterranean population. Aside from different SNP frequencies in Spain compared to Northern Europe, the results for eye colour prediction are quite similar to other studies. However, we have found an association between gender and eye colour prediction. When comparing similar eye colour genetic profiles, females tend, as a whole, to have darker eyes than males (and, conversely, males lighter than females). These results are also corroborated by the revision and meta-analysis of data from previously published eye colour genetic studies in several Caucasian populations, which significantly support the fact that males are more likely to have blue eyes than females, while females tend to show higher frequencies of green and brown eyes than males. This significant gender difference would suggest that there is an as yet unidentified gender-related factor contributing to human eye colour variation

CSVS, a crowdsourcing database of the Spanish population genetic variability

The knowledge of the genetic variability of the local population is of utmost importance in personalized medicine and has been revealed as a critical factor for the discovery of new disease variants. Here, we present the Collaborative Spanish Variability Server (CSVS), which currently contains more than 2000 genomes and exomes of unrelated Spanish individuals. This database has been generated in a collaborative crowdsourcing effort collecting sequencing data produced by local genomic projects and for other purposes. Sequences have been grouped by ICD10 upper categories. A web interface allows querying the database removing one or more ICD10 categories. In this way, aggregated counts of allele frequencies of the pseudo-control Spanish population can be obtained for diseases belonging to the category removed. Interestingly, in addition to pseudo-control studies, some population studies can be made, as, for example, prevalence of pharmacogenomic variants, etc. In addition, this genomic data has been used to define the first Spanish Genome Reference Panel (SGRP1.0) for imputation. This is the first local repository of variability entirely produced by a crowdsourcing effort and constitutes an example for future initiatives to characterize local variability worldwide. CSVS is also part of the GA4GH Beacon network

Sex-specific genetic effects associated with pigmentation, sensitivity to sunlight, and melanoma in a population of Spanish origin

Background Human pigmentation is a polygenic quantitative trait with high heritability. In addition to genetic factors, it has been shown that pigmentation can be modulated by oestrogens and androgens via up- or down-regulation of melanin synthesis. Our aim was to identify possible sex differences in pigmentation phenotype as well as in melanoma association in a melanoma case-control population of Spanish origin. Methods Five hundred and ninety-nine females (316 melanoma cases and 283 controls) and 458 males (234 melanoma cases and 224 controls) were analysed. We genotyped 363 polymorphisms (single nucleotide polymorphisms (SNPs)) from 65 pigmentation gene regions. Results When samples were stratified by sex, we observed more SNPs associated with dark pigmentation and good sun tolerance in females than in males (107 versus 75; P = 2.32 × 10−6), who were instead associated with light pigmentation and poor sun tolerance. Furthermore, six SNPs in TYR, SILV/CDK2, GPR143, and F2RL1 showed strong differences in melanoma risk by sex (P < 0.01). Conclusions We demonstrate that these genetic variants are important for pigmentation as well as for melanoma risk, and also provide suggestive evidence for potential differences in genetic effects by sex.We thank the Madrid College of Lawyers and all patients from the different contributing Hospitals. We would like to thank Tais Moreno, M. Rosario Alonso, and Guillermo Pita for their expert technical assistance with Illumina genotyping, performed at the Spanish National Genotyping Centre (CeGen, Madrid). MI-V is funded by the “Ministry of Health Carlos III” under a Sara Borrell contract (CD15/00153). ML-C is funded by a Prometeo contract (2015/005). SSO is funded by the “ Ministry of Education, Culture and Sport” under a FPU fellowship (FPU13/04976). GR is funded by the “Ministry of Health Carlos III” under a Miquel Servet II contract (CPII14-00013). This work has also been partly funded by a research project from the Spanish Ministry of Economy and Competitiveness (CGL2014-58526-P), whose principal investigator is S

Genetic variants in PARP1 (rs3219090) and IRF4 (rs12203592) genes associated with melanoma susceptibility in a Spanish population

Background Few high penetrance genes are known in Malignant Melanoma (MM), however, the involvement of low-penetrance genes such as MC1R, OCA2, ASIP, SLC45A2 and TYR has been observed. Lately, genome-wide association studies (GWAS) have been the ideal strategy to identify new common, low-penetrance susceptibility loci. In this case–control study, we try to validate in our population nine melanoma associated markers selected from published GWAS in melanoma predisposition. Methods We genotyped the 9 markers corresponding to 8 genes (PARP1, MX2, ATM, CCND1, NADSYN1, CASP8, IRF4 and CYP2R1) in 566 cases and 347 controls from a Spanish population using KASPar probes. Genotypes were analyzed by logistic regression and adjusted by phenotypic characteristics. Results We confirm the protective role in MM of the rs3219090 located on the PARP1 gene (p-value 0.027). Additionally, this SNP was also associated with eye color (p-value 0.002). A second polymorphism, rs12203592, located on the IRF4 gene was associated with protection to develop MM for the dominant model (p-value 0.037). We have also observed an association of this SNP with both lentigines (p-value 0.014) and light eye color (p-value 3.76 × 10-4). Furthermore, we detected a novel association with rs1485993, located on the CCND1 gene, and dark eye color (p-value 4.96 × 10-4). Finally, rs1801516, located on the ATM gene, showed a trend towards a protective role in MM similar to the one firstly described in a GWAS study. Conclusions To our knowledge, this is the first time that these SNPs have been associated with MM in a Spanish population. We confirmed the proposed role of rs3219090, located on the PARP1 gene, and rs12203592, located on the IRF4 gene, as protective to MM along the same lines as have previous genome-wide associated works. Finally, we have seen associations between IRF4, PARP1, and CCND1 and phenotypic characteristics, confirming previous results for the IRF4 gene and presenting novel data for the last two, suggesting that pigmentation characteristics correlated with eye color are potential mediators between PARP1 and MM protection

Mutational Characterization of Cutaneous Melanoma Supports Divergent Pathways Model for Melanoma Development

This article belongs to the Special Issue Melanoma: Prevention and Molecular Epidemiology.[Simple Summary] The divergent pathway model established at least two approaches for melanoma development. One was related to a propensity to melanocytic proliferation (nevogenic), and the other was associated with an accumulation of solar damage (CSD). We conducted a retrospective study to examine whether this model had a molecular support using sequencing and bioinformatic tools on a set of cutaneous melanomas corresponding to these two groups. We found that the nevogenic melanomas were associated with mutations in BRAF, while the CSD melanomas were associated with mutations in NF1, ROS1, GNA11, and RAC1. We concluded that nevogenic and CSD melanomas constitute two different biological entities.[Abstract] According to the divergent pathway model, cutaneous melanoma comprises a nevogenic group with a propensity to melanocyte proliferation and another one associated with cumulative solar damage (CSD). While characterized clinically and epidemiologically, the differences in the molecular profiles between the groups have remained primarily uninvestigated. This study has used a custom gene panel and bioinformatics tools to investigate the potential molecular differences in a thoroughly characterized cohort of 119 melanoma patients belonging to nevogenic and CSD groups. We found that the nevogenic melanomas had a restricted set of mutations, with the prominently mutated gene being BRAF. The CSD melanomas, in contrast, showed mutations in a diverse group of genes that included NF1, ROS1, GNA11, and RAC1. We thus provide evidence that nevogenic and CSD melanomas constitute different biological entities and highlight the need to explore new targeted therapies.This study was supported by the Ministerio de Ciencia e Innovación-Instituto de Salud Carlos III (PI15/01860; PI19/00667), the Asociación Española Contra el Cáncer-Valencia through “Ayudas predoctorales en Oncología” grant, and the European Academy of Dermatology and Venereology (PPRC-2018-36)

A Comprehensive Analysis of 21 Actionable Pharmacogenes in the Spanish Population: From Genetic Characterisation to Clinical Impact

The implementation of pharmacogenetics (PGx) is a main milestones of precision medicine nowadays in order to achieve safer and more effective therapies. Nevertheless, the implementation of PGx diagnostics is extremely slow and unequal worldwide, in part due to a lack of ethnic PGx information. We analysed genetic data from 3006 Spanish individuals obtained by different high-throughput (HT) techniques. Allele frequencies were determined in our population for the main 21 actionable PGx genes associated with therapeutical changes. We found that 98% of the Spanish population harbours at least one allele associated with a therapeutical change and, thus, there would be a need for a therapeutical change in a mean of 3.31 of the 64 associated drugs. We also identified 326 putative deleterious variants that were not previously related with PGx in 18 out of the 21 main PGx genes evaluated and a total of 7122 putative deleterious variants for the 1045 PGx genes described. Additionally, we performed a comparison of the main HT diagnostic techniques, revealing that after whole genome sequencing, genotyping with the PGx HT array is the most suitable solution for PGx diagnostics. Finally, all this information was integrated in the Collaborative Spanish Variant Server to be available to and updated by the scientific community.CEGEN is part of the initiative IMPaCT-GENóMICA (IMP/00009) co-funded by ISCIII and the European Regional Development Fund (ERDF). Additional funding was obtained from the ISCIII Project PI18/01242, within the Health Strategic Action, which is co-funded by the European Regional Development Fund (ERDF).Peer reviewe

Involvement of ANXA5 and ILKAP in Susceptibility to Malignant Melanoma

Single nucleotide-polymorphisms (SNPs) are a source of diversity among human population, which may be responsible for the different individual susceptibility to diseases and/or response to drugs, among other phenotypic traits. Several low penetrance susceptibility genes associated with malignant melanoma (MM) have been described, including genes related to pigmentation, DNA damage repair and oxidative stress pathways. In the present work, we conducted a candidate gene association study based on proteins and genes whose expression we had detected altered in melanoma cell lines as compared to normal melanocytes. The result was the selection of 88 loci and 384 SNPs, of which 314 fulfilled our quality criteria for a case-control association study. The SNP rs6854854 in ANXA5 was statistically significant after conservative Bonferroni correction when 464 melanoma patients and 400 controls were analyzed in a discovery Phase I. However, this finding could not be replicated in the validation phase, perhaps because the minor allele frequency of SNP rs6854854 varies depending on the geographical region considered. Additionally, a second SNP (rs6431588) located on ILKAP was found to be associated with melanoma after considering a combined set of 1,883 MM cases and 1,358 disease-free controls. The OR was 1.29 (95% CI 1.12–1.48; p-value = 4×10−4). Both SNPs, rs6854854 in ANXA5 and rs6431588 in ILKAP, show population structure, which, assuming that the Spanish population is not significantly structured, suggests a role of these loci on a specific genetic adaptation to different environmental conditions. Furthermore, the biological relevance of these genes in MM is supported by in vitro experiments, which show a decrease in the transcription levels of ANXA5 and ILKAP in melanoma cells compared to normal melanocyte

A comprehensive database for integrated analysis of omics data in autoimmune diseases

This work is partially funded by FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento (Grant CV20-36723), Consejeria de Salud (Grant PI-0173-2017) and by EU/EFPIA Innovative Medicines Initiative Joint Undertaking PRECISESADS (115565). JMM is partially funded by Ministerio de Economia, Industria y Competitividad. None of the funding bodies played any role in the design of the study and collection, analysis, and interpretation of data nor in writing the manuscript.Background: Autoimmune diseases are heterogeneous pathologies with difficult diagnosis and few therapeutic options. In the last decade, several omics studies have provided significant insights into the molecular mechanisms of these diseases. Nevertheless, data from different cohorts and pathologies are stored independently in public repositories and a unified resource is imperative to assist researchers in this field. Results: Here, we present Autoimmune Diseases Explorer (https:// adex. genyo. es), a database that integrates 82 curated transcriptomics and methylation studies covering 5609 samples for some of the most common autoimmune diseases. The database provides, in an easy-to-use environment, advanced data analysis and statistical methods for exploring omics datasets, including meta-analysis, differential expression or pathway analysis. Conclusions: This is the first omics database focused on autoimmune diseases. This resource incorporates homogeneously processed data to facilitate integrative analyses among studies.FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento CV20-36723Consejeria de Salud PI-0173-2017EU/EFPIA Innovative Medicines Initiative Joint Undertaking PRECISESADS 115565Ministerio de Economia, Industria y Competitivida