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(A) GOT2 western blot of HeLa cells stably silenced for GOT2 expression by shRNA transfection compared to non-silenced scrambled (Scr) control cells. β-actin was used as a loading control. (B) Number of GOT2 KD HeLa cells after transfection with empty vector (EV), GOT2- WT cDNA, and GOT2- c.357A>T. Cells were seeded into 12-well plates and incubated for various times, as indicated. The counts are reported as means (n=3). A t-test was applied to test for differences. n.s.: not significant.

(A) Aspartate/glutamate ratios assessed by LC-MS in GOT2 KD Hela cells transfected with empty vector (EV), GOT2 wild-type (WT) cDNA, GOT2- c.223T>G cDNA, and GOT2- c.357A>T cDNA. The ratios were reported as means (n=3). Error bars represent standard deviations. A t-test was applied to test for differences between GOT WT and GOT2- c.223T>G and - c.357A>T transfected cells. n.s.: not significant. (B) Succinate/fumarate ratios assessed by LC-MS in OGDHL KD Hela cells transfected with EV, OGDHL WT cDNA, and OGDHL- c.750G>T cDNA. The ratios were reported as means (n=3). Error bars represent standard deviations. A t-test was applied to test for differences. n.s.: not significant

DNA methylation (M-values) of the CpG island probes located within six PCC/PGL susceptibility genes encoding Krebs cycle enzymes (SDHA, SDHAF2, SDHB, SDHD, FH, and MDH2) in the 11 analyzed tumors, compared to in vitro methylated DNA (IVD).

Genes included in the targeted next-generation sequencing panel

Legends of the Supplementary Figures

(A) Quantitative PCR analysis of RBP1 expression of the 49 tumors included in the study compared to controls comprising five tumors carrying mutation in Krebs cycle genes and five cases carrying mutations in RET or NF1. RU: relative units. (B) SDHB immunohistochemistry of tumor_4 compared to a positive control tumor carrying a non-SDH mutation. The scale bars represent 50μm. (C) Representation of DNA methylation (M_values) of the CpG island probes located within the SDHC locus of tumor_4 compared to a blood sample from the same patient and a control DNA. (D) Immunohistochemical staining of 5-hmC in IDH3B-mutated tumor. Nuclear 5-hmC was observed only in sustentacular and some stromal cells. The scale bar represents 50μm.

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1. A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315–24. ©2017 AACR.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex disease whose polygenic component is mainly unidentified. We propose that levels of intermediate molecular phenotypes in the myocardium associated with histopathological damage could explain CDA susceptibility; so that variants of genes encoding these intermediate molecular phenotypes could identify patients susceptible to this complication. A genetically heterogeneous cohort of mice generated by backcrossing (N = 165) was treated with doxorubicin and docetaxel. Cardiac histopathological damage was measured by fibrosis and cardiomyocyte size by an Ariol slide scanner. We determine intramyocardial levels of intermediate molecular phenotypes of CDA associated with histopathological damage and quantitative trait loci (ipQTLs) linked to them. These ipQTLs seem to contribute to the missing heritability of CDA because they improve the heritability explained by QTL directly linked to CDA (cda-QTLs) through genetic models. Genes encoding these molecular subphenotypes were evaluated as genetic markers of CDA in three cancer patient cohorts (N = 517) whose cardiac damage was quantified by echocardiography or Cardiac Magnetic Resonance. Many SNPs associated with CDA were found using genetic models. LASSO multivariate regression identified two risk score models, one for pediatric cancer patients and the other for women with breast cancer. Molecular intermediate phenotypes associated with heart damage can identify genetic markers of CDA risk, thereby allowing a more personalized patient management. A similar strategy could be applied to identify genetic markers of other complex trait diseases.

Around 50% of the familial breast cancer (BC) cases are estimated to be caused by germline variants in known low-, moderate-, and high-risk susceptibility genes, while the other half is of unknown genetic origin. In the present study, we wanted to evaluate the role of the RECQ helicases, some of which have been studied in the past as candidates, with unclear results about their role in the disease. Using next-generation sequencing (NGS) technology, we analyzed the whole coding sequence of

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.

Background: tobacco is the main risk factor for developing lung cancer. Yet, some heavy smokers do not develop lung cancer at advanced ages while others develop it at young ages. Here, we assess for the first time the genetic background of these clinically relevant extreme phenotypes using whole exome sequencing (WES). Methods: we performed WES of germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age ( extreme cases, n=50) or did not present lung adenocarcinoma or other tumors at an advanced age (extreme controls, n=50). We selected non-synonymous variants located in exonic regions and consensus splice sites of the genes that showed significantly different allelic frequencies between both cohorts. We validated our results in all the additional extreme cases (i.e., heavy smokers who developed lung adenocarcinoma at an early age) available from The Cancer Genome Atlas (TCGA). Results: the mean age for the extreme cases and controls was respectively 49.7 and 77.5 years. Mean tobacco consumption was 43.6 and 56.8 pack-years. We identified 619 significantly different variants between both cohorts, and we validated 108 of these in extreme cases selected from TCGA. Nine validated variants, located in relevant cancer related genes, such as PARP4, HLA-A or NQO1, among others, achieved statistical significance in the False Discovery Rate test. The most significant validated variant (P=4.48x10(-5)) was located in the tumor-suppressor gene ALPK2. Conclusions: we describe genetic variants associated with extreme phenotypes of high and low risk for the development of tobacco-induced lung adenocarcinoma. Our results and our strategy may help to identify high-risk subjects and to develop new therapeutic strategies. This work was supported by the Spanish Society of Medical Oncology; Fundación SEOM and Fundación Salud 2000; and Government of Navarra. LMM research work is supported by Foundation for Applied Medical Research (FIMA), Fundación Científica de la Asociación Espanola Contra el Cáncer, Fundación Ramón Areces, and Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional 'Una manera de hacer Europa' (PI19/00098).

Resumen Objetivo Estratificacion de la poblacion general con base en las variantes genotipicas para seleccionar a aquellas mujeres de alto riesgo a desarrollar un cancer de mama que puedan ser candidatas a un seguimiento individualizado. Material y metodos Se ha realizado un estudio caso-control en 856 mujeres con cancer de mama y 839 mujeres controles de la poblacion general pareadas por edad, analizando la asociacion entre el riesgo a desarrollar cancer de mama y un grupo de variantes basado en 76 polimorfismos de un cambio de base (SNP) de susceptibilidad. Resultados Se han establecido 2 curvas de casos y controles con base en las odds ratio (OR) genotipicas que diferencian las 2 poblaciones con significacion estadistica (p = 2,293 × 10-15). Asimismo, se ha estratificado la poblacion de casos y controles e identificado un 14% de la poblacion que se encontraria en el grupo de alto riesgo con una OR > 2 (> 25% probabilidades de desarrollar un cancer de mama). Este grupo seria candidato a un seguimiento individualizado. Conclusiones El Polygenic Risk Score es un predictor del riesgo del cancer de mama independiente que puede ayudar a seleccionar mujeres con alto riesgo para establecer medidas de seguimiento y tratamiento individualizado en funcion del riesgo genetico.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and (13)C(5)-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

There is still around 50% of the familial breast cancer (BC) cases with an undefined genetic cause, here we have used next-generation sequencing (NGS) technology to identify new BC susceptibility genes. This approach has led to the identification of RECQL5, a member of RECQL-helicases family, as a new BC susceptibility candidate, which deserves further study. We have used a combination of whole exome sequencing in a family negative for mutations in BRCA1/2 throughout (BRCAX), in which we found a probably deleterious variant in RECQL5, and targeted NGS of the complete coding regions and exon-intron boundaries of the candidate gene in 699 BC Spanish BRCAX families and 665 controls. Functional characterization and in silico inference of pathogenicity were performed to evaluate the deleterious effect of detected variants. We found at least seven deleterious or likely deleterious variants among the cases and only one in controls. These results prompt us to propose RECQL5 as a gene that would be worth to analyze in larger studies to explore its possible implication in BC susceptibility.

OBJECTIVES Taxanes and anthracyclines are widely used in the treatment of breast cancer, although the benefit is limited to a proportion of patients and predictive biomarkers for clinical outcome remain elusive. PATIENTS AND METHODS We carried out a pharmacogenetic study in 181 patients with locally advanced breast cancer enrolled in a phase 2 randomized clinical trial (NCT00123929), where patients were randomly assigned to receive neoadjuvant single-agent docetaxel 100 mg/m(2) (n=84) or doxorubicin 75 mg/m(2) (n=97). We studied the association of 226 single nucleotide polymorphisms (SNPs) in 15 key drug biotransformation genes with neoadjuvant pathological tumor response residual cancer burden index to docetaxel and to doxorubicin. RESULTS We identified a significant association for rs162561, an intronic SNP located in the cytochrome P450 family 1 subfamily B member 1 (CYP1B1) gene, with tumor response in patients treated with single-agent docetaxel (dominant model: β=1.02, 95% confidence interval=0.49-1.55; P=1.77×10(-4)), and for rs717620, an SNP located in the promoter of the ATP-binding cassette subfamily C member 2 (ABCC2) gene, in patients treated with neoadjuvant doxorubicin (recessive model: β=1.67; 95% confidence interval=0.26-3.11; P=0.02). CONCLUSION We identified two polymorphisms in CYP1B1 and ABCC2 associated with tumor pathological response following docetaxel or doxorubicin neoadjuvant monotherapy, respectively. Although further validation is required, these variants could be potential predictive genetic markers for treatment outcome in breast cancer patients.

Motion planning for an autonomous vehicle is most challenging for scenarios such as large, multi-lane, and unsignalized intersections in the presence of dense traffic. In such situations, the motion planner has to deal with multiple crossing-points to reach an objective in a safe, comfortable, and efficient way. In addition, motion planning challenges include real-time computation and scalability to complex scenes with many objects and different road geometries. In this work, we propose a motion planning system addressing these challenges. We enable real-time applicability of a Monte Carlo Tree Search algorithm with a deep-learning heuristic. We learn a fast evaluation function from accurate, but non real-time models. While using Deep Reinforcement Learning techniques we maintain a clear separation between making predictions and making decisions. We reduce the complexity of the search model and benchmark the proposed agent against multiple methods: rules-based, MCTS, $A^{*}$ search, deep learning, and Model Predictive Control. We show that our agent outperforms these other agents in a variety of challenging scenarios, where we benchmark safety, comfort and efficiency metrics.

Capecitabine-induced hand-foot syndrome (CiHFS) is a common dermatological adverse reaction affecting around 30% of patients with capecitabine-treated cancer, and the main cause of dose reductions and chemotherapy delays. To identify novel genetic factors associated with CiHFS in patients with cancer, we carried out an extreme-phenotype genomewide association study in 166 patients with breast and colorectal capecitabine-treated cancer with replication in a second cohort of 85 patients. We discovered and replicated a cluster of four highly correlated single-nucleotide polymorphisms associated with susceptibility to CiHFS at 20q13.33 locus (top hit = rs6129058, hazard ratio = 2.40, 95% confidence interval = 1.78-3.20; P = 1.2 × 10-8 ). Using circular chromosome conformation capture sequencing, we identified a chromatin contact between the locus containing the risk alleles and the promoter of CDH4, located 90 kilobases away. The risk haplotype was associated with decreased levels of CDH4 mRNA and the protein it encodes, R-cadherin (RCAD), which mainly localizes in the granular layer of the epidermis. In human keratinocytes, CDH4 downregulation resulted in reduced expression of involucrin, a protein of the cornified envelope, an essential structure for skin barrier function. Immunohistochemical analyses revealed that skin from patients with severe CiHFS exhibited low levels of RCAD and involucrin before capecitabine treatment. Our results uncover a novel mechanism underlying individual genetic susceptibility to CiHFS with implications for clinically relevant risk prediction.

Testicular germ cell tumors (TGCTs) are a clinically and pathologically heterogeneous disease, and little is known of its genetic basis. Only low susceptibility risk loci have been identified for both sporadic and familial cases. Therefore, we tried to identify new susceptibility genes responsible for familial testicular cancer that may contribute to increasing our knowledge about the genetic basis of the disease. Nineteen Spanish families with at least two affected individuals with TGCT were selected. WES was performed on those individuals using an Illumina Hiseq2000 sequencing platform. Data were analyzed under a monogenic and polygenic model of inheritance, and candidate variants were evaluated in a case-control association study performed on 391 Spanish sporadic cases and 1,170 healthy Spanish controls. Results were replicated in a second series consisting of 101 TGCTs from the Cancer Genome Atlas (TGCA) and 27,000 controls from the Exome Aggregation Consortium (ExAC) database. Logistic regression was carried out to analyze the association strength (risk) of candidate variants obtained among cases and controls in different populations. Despite the sample size, we detected a significant earlier age of onset in familial TGCT (28y) than sporadic cases (33y), using a Mann-Whitney U test. We identified significant variants in the comparative study of TGCT cases (391) versus controls (almost 1,170), and three of them [PLEC (OR = 6.28, p = 6.42 × 10-23 ) (p.Arg2016Trp), EXO5 (OR = 3.37, p = 4.82 × 10-09 ) (p.Arg344AlafsTer10) and DNAH7 (OR = 1.64, p = 0.048)] were replicated as potential candidates that may contribute to explaining the genetic basis of TGCT.

The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. Whole-exome sequencing was applied to the germlines of a parent–proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

The estimation of the state of lateral dynamics of a vehicle is usually performed using information gathered by several sensors, such as cameras, radars, odometers, and accelerometers. However, some quantities and their variations cannot be measured directly in all driving situations whereas they are required for vehicle control. Among these unmeasured quantities, one can find road curvature. This paper proposes to apply nonlinear observers to provide such information, in addition to other quantities related to the vehicle state, in the context of Traffic Jump Pilot. The first contribution of this paper is to formulate the nonlinear dynamics model and then to design two nonlinear observers based on High-Gain and Sliding Mode techniques. Furthermore, in order to ensure their efficient applications in a real situation, an experimental methodology is developed in order to optimize their performances by finding an optimal set of gain values. In that respect, a large database of real driving conditions and different road curvature scenarios is recorded for the performances evaluation and then a recent multi-objective optimization algorithm is used to find the optimal tuning parameters of the observers. The effectiveness of the proposed methodology is illustrated through an application to a driving test, which is not in the database. The use of this optimization tool is a novelty in the context of nonlinear observation and control, which allows getting optimized observers that can be efficiently applied to experimental data.

PURPOSE: Anthracyclines are widely used chemotherapeutic drugs that can cause progressive and irreversible cardiac damage and fatal heart failure. Several genetic variants associated with anthracycline-induced cardiotoxicity (AIC) have been identified, but they explain only a small proportion of the interindividual differences in AIC susceptibility. METHODS: In this study, we evaluated the association of low-frequency variants with risk of chronic AIC using the Illumina HumanExome BeadChip array in a discovery cohort of 61 anthracycline-treated breast cancer patients with replication in a second independent cohort of 83 anthracycline-treated pediatric cancer patients, using gene-based tests (SKAT-O). RESULTS: The most significant associated gene in the discovery cohort was ETFB (electron transfer flavoprotein beta subunit) involved in mitochondrial β-oxidation and ATP production (P = 4.16 × 10-4) and this association was replicated in an independent set of anthracycline-treated cancer patients (P = 2.81 × 10-3). Within ETFB, we found that the missense variant rs79338777 (p.Pro52Leu; c.155C > T) made the greatest contribution to the observed gene association and it was associated with increased risk of chronic AIC in the two cohorts separately and when combined (OR 9.00, P = 1.95 × 10-4, 95% CI 2.83-28.6). CONCLUSIONS: We identified and replicated a novel gene, ETFB, strongly associated with chronic AIC independently of age at tumor onset and related to anthracycline-mediated mitochondrial dysfunction. Although experimental verification and further studies in larger patient cohorts are required to confirm our finding, we demonstrated that exome array data analysis represents a valuable strategy to identify novel genes contributing to the susceptibility to chronic AIC.

This paper presents a novel vehicle motion forecasting method based on multi-head attention. It produces joint forecasts for all vehicles on a road scene as sequences of multi-modal probability density functions of their positions. Its architecture uses multi-head attention to account for complete interactions between all vehicles, and long short-term memory layers for encoding and forecasting. It relies solely on vehicle position tracks, does not need maneuver definitions, and does not represent the scene with a spatial grid. This allows it to be more versatile than similar model while combining any forecasting capabilities, namely joint forecast with interactions, uncertainty estimation, and multi-modality. The resulting prediction likelihood outperforms state-of-the-art models on the same dataset., Comment: 7 pages, 4 figures, under review at ICRA and RA-L

In the recent vehicle trajectory prediction literature, the most common baselines are briefly introduced without the necessary information to reproduce it. In this article we produce reproducible vehicle prediction results from simple models. For that purpose, the process is explicit, and the code is available. Those baseline models are a constant velocity model and a single-vehicle prediction model. They are applied on the NGSIM US-101 and I-80 datasets using only relative positions. Thus, the process can be reproduced with any database containing tracking of vehicle positions. The evaluation reports Root Mean Squared Error (RMSE), Final Displacement Error (FDE), Negative Log-Likelihood (NLL), and Miss Rate (MR). The NLL estimation needs a careful definition because several formulations that differ from the mathematical definition are used in other works. This article is meant to be used along with the published code to establish baselines for further work. An extension is proposed to replace the constant velocity assumption with a learned model using a recurrent neural network. This brings good improvements in accuracy and uncertainty estimation and opens possibilities for both complex and interpretable models.

Throughout the past few years, a lively debate emerged about the timing and magnitude of the human migrations between the Iberian Peninsula and the Maghreb. Several pieces of evidence, including archaeological, anthropological, historical, and genetic data, have pointed to a complex and intermingled evolutionary history in the western Mediterranean area. To study to what extent connections across the Strait of Gibraltar and surrounding areas have shaped the present-day genomic diversity of its populations, we have performed a screening of 2.5 million single-nucleotide polymorphisms in 142 samples from southern Spain, southern Portugal, and Morocco. We built comprehensive data sets of the studied area and we implemented multistep bioinformatic approaches to assess population structure, demographic histories, and admixture dynamics. Both local and global ancestry inference showed an internal substructure in the Iberian Peninsula, mainly linked to a differential African ancestry. Western Iberia, from southern Portugal to Galicia, constituted an independent cluster within Iberia characterized by an enriched African genomic input. Migration time modeling showed recent historic dates for the admixture events occurring both in Iberia and in the North of Africa. However, an integrative vision of both paleogenomic and modern DNA data allowed us to detect chronological transitions and population turnovers that could be the result of transcontinental migrations dating back from Neolithic times. The present contribution aimed to fill the gaps in the modern human genomic record of a key geographic area, where the Mediterranean and the Atlantic come together.

Ego-motion estimation plays a crucial role in the achievement of autonomous driving and advanced driver assistance systems (ADAS). Classical approaches using wheel-based odometers suffers from errors due to the variation of wheels diameter, and are not robust to slippery conditions. This paper proposes an approach using static object detections from one or multiple low cost radars to improve the ego-motion estimation. First, the method determines a 2D motion of the ego vehicle for each radar available in the car. Three steps are applied: data association, linear regression, and outliers rejection. Then, the estimated 2D motions are fused with the proprioceptive sensors of the car in order to precisely estimate the speed and the yaw rate of the ego vehicle. In the tested scenarios, our approach drastically reduces the error speed with respect to classical wheel-based approach.

Importance Persistent chemotherapy-induced alopecia (pCIA) has been recently described in patients with breast cancer and in its most severe form occurs in up to 10% of these patients. Genetic risk factors associated with pCIA have not been adequately explored. Objective To identify genetic variants associated with pCIA. Design, Setting, and Participants In this genetic association study, 215 women with breast cancer treated with docetaxel-based chemotherapy with a follow-up of 1.5 to 10 years after the end of the treatment were recruited retrospectively through 3 hospital oncology units across Spain between 2005 and 2018. Severe pCIA was defined as lack of scalp hair recovery (Common Terminology Criteria for Adverse Events, version 3.0, grade 2) 18 months or more after the end of treatment. Patients with grade 2 pCIA were selected as cases, and those with no sign of residual alopecia 12 months after the end of docetaxel treatment were selected as controls. A genome-wide association study in a discovery phase was conducted, and logistic regression was used to identify variants associated with the risk to develop this adverse effect. The validity of the association was addressed through a replication phase. Exposures Docetaxel-based chemotherapy. Main Outcomes and Measures Genotypes of single-nucleotide variants associated with pCIA. Results In total, 215 women with breast cancer (median age, 51.6 years; interquartile range, 44-60 years) were recruited (173 patients for the discovery phase and 42 patients for the replication phase). In the discovery phase,ABCB1genetic variants were associated with risk to develop pCIA. In particular, single-nucleotide variation rs1202179, a regulatory variant located in an enhancer element that interacts with theABCB1promoter, was associated with the occurrence of pCIA. This finding was validated in the replication cohort (combined odds ratio, 4.05; 95% CI, 2.46-6.67;P = 3.946 × 10−8). This variant is associated withABCB1mRNA expression, and the risk allele was associated with decreasedABCB1expression levels (P = 1.64 × 10−20). Conclusions and Relevance This is the first study, to our knowledge, that identifies an association between a regulatory variant in theABCB1gene and the occurrence of pCIA in patients with breast cancer who were treated with docetaxel-based therapies. This finding suggests an important insight into the biological mechanisms underlying pCIA and opens the opportunity to explore personalized treatment of these patients.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the ‘iCOGS’ genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84−0.87; P=1.7 × 10−43) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology., Previous studies identified an association between the 2q35 locus and breast cancer. Here, the authors show that a SNP at 2q35, rs4442975, is associated with oestrogen receptor positive disease and suggest that this effect is mediated through the downregulation of a known breast cancer gene, IGFBP5.

Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome-wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced non-small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n = 3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (pcombined = 5.66 × 10-5 ; ORcombined = 2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (pcombined = 1.02 × 10-4 ; ORcombined = 2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early-stage NSCLC. PDE10A and ATP10DmRNA expressions correlated with survival in 821 stage I-II NSCLC patients (p = 0.01 and p

Mammographic density (MD) is an intermediate phenotype for breast cancer. Previous studies have identified genetic variants associated with MD; however, much of the genetic contribution to MD is unexplained. We conducted a two-stage genome-wide association analysis among the participants in the "Determinants of Density in Mammographies in Spain" study, together with a replication analysis in women from the Australian MD Twins and Sisters Study. Our discovery set covered a total of 3,351 Caucasian women aged 45 to 68 years, recruited from Spanish breast cancer screening centres. MD was blindly assessed by a single reader using Boyd's scale. A two-stage approach was employed, including a feature selection phase exploring 575,374 SNPs in 239 pairs of women with extreme phenotypes and a verification stage for the 183 selected SNPs in the remaining sample (2,873 women). Replication was conducted in 1,786 women aged 40 to 70 years old recruited via the Australian Twin Registry, where MD were measured using Cumulus-3.0, assessing 14 SNPs with a p value

1540 Background: Individual susceptibility to carcinogens may depend on the genetic background. We characterized the constitutional exome of individuals presenting extreme phenotypes of high sensitivity and resistance to develop tobacco induced LUAD, correlating the results to KRAS status. Methods: From an identification cohort (n=3,631) we selected 100 caucasian heavy smokers that either developed LUAD at early age (cancer cohort, n=50) or did not develop LUAD or other tumors at advanced age (cancer free cohort, n=50). We sequenced their germline DNA with the Agilent Human Exome Capture v5 (21,522 genes, 357,999 exons). Using logistic regression we selected the most significant variants between both cohorts and correlated them with KRAS mutation status of LUAD patients. Results: mean ages for the cancer and cancer free cohorts were 50 (range 34-55) and 78 years (72-90). Mean tobacco consumptions were 44 (range 6-72) and 55 pack-years (20-124). Median coverage was 96% at >10X; median depth was 97X. Table shows the most significant variants. rs7240666 ( ALPK2) achieved top significance (p=8.14x10-5, OR 0.18). rs78898229 ( ANKRD36C) and rs74866537 ( PTPN4) were predominantly represented in patients with KRAS+ tumors, OR: 16 (3.3-78) and 11.9 (3.3-43); and rs12426243 (CCDC41) in KRAS- tumors (OR: 13 (3-53). Conclusions: Our study characterizes for the first time the genotype of individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced LUAD according to KRAS status. Our results warrants further study to assess their value to screen these clinically relevant phenotypes; and to identify mechanisms of high susceptibility and resistance to carcinogens. [Table: see text]

Mammographic density (MD) is regarded as an intermediate phenotype in breast cancer development. This association study investigated the influence of 14 breast cancer susceptibility loci identified through previous genome-wide association studies on MD among the participants in the "Determinants of Density in Mammographies in Spain" (DDM-Spain) study.Our study covered a total of 3348 Caucasian women aged 45-68years, recruited from seven Spanish breast cancer screening centres having DNA available. Mammographic density was blindly assessed by a single reader using a semiquantitative scale. Ordinal logistic models, adjusted for age, body mass index and menopausal status, were used to estimate the association between each genotype and MD.Evidence of association with MD was found for variant rs3803662 (TOX3) (Odds Ratio (OR)=1.13, 95% Confidence Interval (CI)=1.03-1.25), and marginal evidence of association for susceptibility loci rs3817198 (LSP1) (OR=1.09, 95% CI=1.00-1.20) and rs2981582 (FGFR2) (OR=0.92, 95% CI=0.84-1.01). Two other loci were associated with MD solely among pre-menopausal women, namely, rs4973768 (SLC4A7) (OR=0.83, 95% CI=0.70-1.00) and rs4415084 (MEPS30) (OR=1.22, 95% CI=1.00-1.49).Our findings lend some support to the hypothesis which links these susceptibility loci to MD.

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes. Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases. Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1. A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio. Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315–24. ©2017 AACR.

Genome-wide association studies have found SNPs at 17q22 to be associated with breast cancer risk. To identify potential causal variants related to breast cancer risk, we performed a high resolution fine-mapping analysis that involved genotyping 517 SNPs using a custom Illumina iSelect array (iCOGS) followed by imputation of genotypes for 3,134 SNPs in more than 89,000 participants of European ancestry from the Breast Cancer Association Consortium (BCAC). We identified 28 highly correlated common variants, in a 53 Kb region spanning two introns of the STXBP4 gene, that are strong candidates for driving breast cancer risk (lead SNP rs2787486 (OR = 0.92; CI 0.90–0.94; P = 8.96 × 10−15)) and are correlated with two previously reported risk-associated variants at this locus, SNPs rs6504950 (OR = 0.94, P = 2.04 × 10−09, r2 = 0.73 with lead SNP) and rs1156287 (OR = 0.93, P = 3.41 × 10−11, r2 = 0.83 with lead SNP). Analyses indicate only one causal SNP in the region and several enhancer elements targeting STXBP4 are located within the 53 kb association signal. Expression studies in breast tumor tissues found SNP rs2787486 to be associated with increased STXBP4 expression, suggesting this may be a target gene of this locus., published version, peerReviewed

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limbgirdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O-glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces Oglucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle-specific adystroglycan hypoglycosylation not present in patients’ fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch-dependent loss of satellite cells. Junta de Andalucía PI-0017-2014

This work was supported by the Spanish Dirección General de Enseñanza Superior e Investigación Científica [grant numbers BFU2005-05931/BMC and BIO2005-05786], Ministerio de Ciencia e Innovación [grant number SAF2008-05436], Comunidad Autónoma de Madrid [grant numbers 11/BCB/010 and S-SAL-0253/2006], and an institutional grant from the Fundación Ramón Areces. The research group is a member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF) [grant number BFU2007-30688-E/BFI]. A.M. acknowledges the Comunidad Autónoma-de-Madrid for financial support through the AMAROUTO program to the Fundación Severo Ochoa., Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+–Cl−–glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.

J. Neurochem. (2011) 118, 195–204. Abstract Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na+/Cl−/glycine cotransport. GLYT1 displays a 2 : 1 : 1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3 : 1 : 1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na+-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.

Please cite this paper as: Genetic analysis of three important genes in pigmentation and melanoma susceptibility: CDKN2A, MC1R and HERC2/OCA2. Experimental Dermatology 2010; 19: 836–844. Abstract: The CDKN2A gene is regarded as the major familial malignant melanoma (MM) susceptibility gene. Human pigmentation is one of the main modulators of individual risk of developing MM. Therefore, the genes involved in the determination of skin colour and tanning response are potentially implicated in MM predisposition and may be useful predictors of MM risk in the general population. The human melanocortin-1 receptor gene (MC1R) plays a crucial role in pigmentation and also appears to be important in MM. The OCA2 gene has emerged as a new and significant determinant of human iris colour variation. We present a case–control study in Spanish population including 390 consecutive patients with melanoma and 254 control subjects. Sequence analysis of the entire coding region and genotyping of 5 tag-SNPs in the genomic region of MC1R was performed. We identified 27 variants, two reaching statistical significance [R160W (OR: 4.18, 95% CI: 1.24–14.04, P = 0.02) and D294H (OR: 3.10, 95% CI: 1.37–7.01, P = 0.01)] and we detected two novel non-synonymous changes: V92L and T308M. Odds ratio for carrying two functional variants was 4.25 (95% CI: 2.30–7.84, P = 3.63 × 10−6). Haplotypes of the entire MC1R region have been established, and we observed an enrichment of a rare European haplotype similar to African values carrying variants V92M and I155T. In addition, three potentially functional SNPs were selected in p16/CDKN2A and in the promoter region of OCA2/HERC2. Our data for CDKN2A gene did not reach statistically significant results for any of the two studied alleles. We found that the variant allele A > G of OCA2/HERC2 (rs12913832) was associated with pigmentation features: eye, hair and skin colour; P-values = 1.8 × 10−29, 9.2 × 10−16, 1.1 × 10−3, respectively, validating previous results.

The aim of this study is to assess if common genetic variants located in the CDKN1B locus, coding for the cell cycle inhibitor p27Kip1, are involved in thyroid cancer susceptibility. Based on the literature and functional predictions, we selected three polymorphisms within the CDKN1B gene (rs2066827 (T326G, V109G), rs34330 (−79C>T) and rs36228499 (−838C>A)) to perform the first case–control study in thyroid cancer involving this locus. We had 649 Spanish patients with sporadic thyroid cancer and 385 healthy representative controls available. Luciferase reporter gene assays, real-time quantitative reverse transcription-PCR and immunoblot experiments were carried out to demonstrate the putative effect of the associated variant. The polymorphism rs34330 (−79C>T) was identified as a risk factor for developing the follicular variant of papillary thyroid carcinoma (FVPTC), fitting a recessive model (odds ratio=2.12; 95% confidence interval=1.09–4.15; P value=0.023). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P value CDKN1B mRNA levels in lymphocytes, as well as at the protein level. This is the first study that identifies CDKN1B as a low-penetrance gene in thyroid cancer, and specifically in FVPTC subtype. We propose a reduced CDKN1B gene transcription depending on the genotype of the −79C>T (rs34330) variant as a novel mechanism underlying p27Kip1 downregulation.

The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168.

Background: Truncating mutations in ATM have been shown to increase the risk of breast cancer but the effect of missense variants remains contentious. Methods: We have genotyped five polymorphic (minor allele frequency, 0.9-2.6%) missense single nucleotide polymorphisms (SNP) in ATM (S49C, S707P, F858L, P1054R, and L1420F) in 26,101 breast cancer cases and 29,842 controls from 23 studies in the Breast Cancer Association Consortium. Results: Combining the data from all five SNPs, the odds ratio (OR) was 1.05 for being a heterozygote for any of the SNPs and 1.51 for being a rare homozygote for any of the SNPs with an overall trend OR of 1.06 (Ptrend = 0.04). The trend OR among bilateral and familial cases was 1.12 (95% confidence interval, 1.02-1.23; Ptrend = 0.02). Conclusions: In this large combined analysis, these five missense ATM SNPs were associated with a small increased risk of breast cancer, explaining an estimated 0.03% of the excess familial risk of breast cancer. Impact: Testing the combined effects of rare missense variants in known breast cancer genes in large collaborative studies should clarify their overall contribution to breast cancer susceptibility. Cancer Epidemiol Biomarkers Prev; 19(9); 2143–51. ©2010 AACR.

Despite all the research efforts made during the last few decades, most of the cases of families with breast cancer remain unexplained. Mutations in BRCA1 and BRCA2, and in other breast-cancer-susceptibility genes, account for about 25% of familial breast cancer. Linkage studies have failed to identify other breast-cancer-susceptibility genes. The selection criteria of the families, differences in the population background, or clinical and genetic heterogeneity, among other factors, might determine the power to detect the linkage signal. We have performed a SNP-based linkage scan with a total of 6000 SNP markers across the genome in 41 breast-cancer Spanish families, with an average of four breast-cancer cases per family not associated with BRCA1 or BRCA2 germline mutations. In addition, we have included three BRCA-positive families to test the power in linkage detection from a low-complexity family in which a high-penetrance mutation segregates. We have identified three regions of interest, located on 3q25, 6q24, and 21q22. The two former regions showed a suggestive linkage signal (HLOD scores 3.01 and 2.26, respectively), and the latter region showed a significant linkage signal (HLOD score 3.55). Moreover, we found that a subset of 13 families with bilateral breast cancer presented a HLOD of 3.13 on the 3q25 region. Our results suggest that several variables must be taken into account before performing a linkage study in familial breast cancer because of the high heterogeneity within non-BRCA1/2 families. Phenotypic and geographic homogeneity could be the most important factors.

Background: Epidemiological studies have linked adult height with breast cancer risk in women. However, the magnitude of the association, particularly by subtypes of breast cancer, has not been established. Furthermore, the mechanisms of the association remain unclear. Methods: We performed a meta-analysis to investigate associations between height and breast cancer risk using data from 159 prospective cohorts totaling 5 216 302 women, including 113 178 events. In a consortium with individual-level data from 46 325 case patients and 42 482 control subjects, we conducted a Mendelian randomization analysis using a genetic score that comprised 168 height-associated variants as an instrument. This association was further evaluated in a second consortium using summary statistics data from 16 003 case patients and 41 335 control subjects. Results: The pooled relative risk of breast cancer was 1.17 (95% confidence interval [CI] = 1.15 to 1.19) per 10 cm increase in height in the meta-analysis of prospective studies. In Mendelian randomization analysis, the odds ratio of breast cancer per 10 cm increase in genetically predicted height was 1.22 (95% CI = 1.13 to 1.32) in the first consortium and 1.21 (95% CI = 1.05 to 1.39) in the second consortium. The association was found in both premenopausal and postmenopausal women but restricted to hormone receptor-positive breast cancer. Analyses of height-associated variants identified eight new loci associated with breast cancer risk after adjusting for multiple comparisons, including three loci at 1q21.2, DNAJC27, and CCDC91 at genome-wide significance level P < 5 × 10-8. Conclusions: Our study provides strong evidence that adult height is a risk factor for breast cancer in women and certain genetic factors and biological pathways affecting adult height have an important role in the etiology of breast cancer.

Mammographic density (MD) is an intermediate phenotype for breast cancer. Previous studies have identified genetic variants associated with MD; however, much of the genetic contribution to MD is unexplained. We conducted a two-stage genome-wide association analysis among the participants in the "Determinants of Density in Mammographies in Spain" study, together with a replication analysis in women from the Australian MD Twins and Sisters Study. Our discovery set covered a total of 3,351 Caucasian women aged 45 to 68 years, recruited from Spanish breast cancer screening centres. MD was blindly assessed by a single reader using Boyd's scale. A two-stage approach was employed, including a feature selection phase exploring 575,374 SNPs in 239 pairs of women with extreme phenotypes and a verification stage for the 183 selected SNPs in the remaining sample (2,873 women). Replication was conducted in 1,786 women aged 40 to 70 years old recruited via the Australian Twin Registry, where MD were measured using Cumulus-3.0, assessing 14 SNPs with a p value0.10 in stage 2. Finally, two genetic variants in high linkage disequilibrium with our best hit were studied using the whole Spanish sample. Evidence of association with MD was found for variant rs11205277 (OR = 0.74; 95% CI = 0.67-0.81; p = 1.33 × 10(-10) ). In replication analysis, only a marginal association between this SNP and absolute dense area was found. There were also evidence of association between MD and SNPs in high linkage disequilibrium with rs11205277, rs11205303 in gene MTMR11 (OR = 0.73; 95% CI = 0.66-0.80; p = 2.64 × 10(-11) ) and rs67807996 in gene OTUD7B (OR = 0.72; 95% CI = 0.66-0.80; p = 2.03 × 10(-11)). Our findings provide additional evidence on common genetic variations that may contribute to MD.

OBJECTIVE A primary challenge in identifying replicable pharmacogenomic markers from clinical genomewide association study (GWAS) trials in oncology is the difficulty in performing a second large clinical trial with the same drugs and dosage regimen. We sought to overcome this challenge by incorporating GWAS results from cell-based studies using the same chemotherapy as a clinical cohort. METHODS In this study, we test whether the overlap between genetic variants identified in a preclinical study and a clinical study on capecitabine is more than expected by chance. A GWAS of capecitabine-induced cytotoxicity was performed in 164 lymphoblastoid cell lines derived from the CEU HapMap population and compared with a GWAS of hand-foot syndrome (HFS), the most frequent capecitabine-induced adverse drug reaction, in Spanish breast and colorectal cancer patients (n=160) treated with capecitabine. RESULTS We observed an overlap of 16 single nucleotide polymorphisms associated with capecitabine-induced cytotoxicity (P

Background.The approved capecitabine regimen as monotherapyinmetastaticbreastcancer(MBC)is1,250mg/m 2 twice daily for 2 weeks on and 1 weekoff (Cint). Dose modifications are often required because of severe hand-foot syndrome (HFS).Wetestedacontinuousregimenwithalowerdailydose but a similar cumulative dose in an attempt to reduce the severity of adverse events (AEs) while maintaining efficacy. Methods.We randomized 195 patients with HER-2/neunegativeMBCtocapecitabine800mg/m 2 twicedailythroughoutthe 21-daycycle (Ccont) or to Cintto assess noninferiority in the percentage of patients free of progression at 1 year. Secondary endpoints included efficacy and safety. Associations between polymorphisms in capecitabine metabolismrelated genes and drug response were assessed. Results.The percentage of patients free of progression at 1 year was 27.3% with Cint versus 25.3% with Ccont (difference of 22.0%; 95% confidence interval: 215.5% to 11.5%, exceeding the 15% deemed noninferior). Differences regarding other efficacy variables were also not found. Grade 3–4 HFS was the most frequent AE (41.1% in Cint vs. 42.3% in Ccont). Grade 3–4 neutropenia, thrombocytopenia, diarrhea, and stomatitis were more frequent with Cint. A 59 untranslated region polymorphism in the carboxylesterase 2 gene was associated with HFS. One polymorphism in cytidine deaminase and two in thymidine phosphorylase were associated with survival. Conclusion. Ourstudywasunabletoshow noninferioritywith the continuous capecitabine regimen (Ccont) compared with the approved intermittent regimen (Cint). Further investigation is required to improve HFS. Polymorphisms in several genes might contribute to interindividual differences in response to capecitabine.The Oncologist 2015;20:1–2 DISCUSSION In this patient population (Table 1), continuous, lower daily dosesofcapecitabinewerenotshowntobenoninferiorinefficacy tothestandardscheduledespitemaintainingthesamecumulative dose and dose intensity (Fig. 1).The percentage of patients free ofprogressionat1yearwere27.3%with1,250mg/m 2 twicedaily for 2 weeks on and 1 week off versus 25.3% with 800 mg/m 2 twice daily throughout the 21-day cycle (difference of 22.0%; 95% confidence interval: 215.5 to 11.5%), meaning that the margin deemed noninferior by the study design (15%) was exceeded. Median progression-free survival (PFS) and overall survival (OS) were numerically superior (although nonsignificant) with the approved intermittent administration schedule. Hand-foot syndrome (HFS) was not different between arms.

Background Peripheral neuropathy is the dose limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat solid tumours. This toxicity exhibits great inter-individual variability of unknown origin. The present study aimed to identify genetic variants associated with paclitaxel induced neuropathy via a whole genome approach. Methods A genome-wide association study (GWAS) was performed in 144 white European patients uniformly treated with paclitaxel/carboplatin and for whom detailed data on neuropathy was available. Per allele single nucleotide polymorphism (SNP) associations were assessed by Cox regression, modelling the cumulative dose of paclitaxel up to the development of grade 2 sensory neuropathy. Results The strongest evidence of association was observed for the ephrin type A receptor 4 ( EPHA4 ) locus (rs17348202, p=1.0×10–6), and EPHA6 and EPHA5 were among the top 25 and 50 hits (rs301927, p=3.4×10–5 and rs1159057, p=6.8×10–5), respectively. A meta-analysis of EPHA5 -rs7349683, the top marker for paclitaxel induced neuropathy in a previous GWAS (r2=0.79 with rs1159057), gave a hazard ratio (HR) estimate of 1.68 (p=1.4×10−9). Meta-analysis of the second hit of this GWAS, XKR4 -rs4737264, gave a HR of 1.71 (p=3.1×10−8). Imputed SNPs at LIMK2 locus were also strongly associated with this toxicity (HR=2.78, p=2.0×10−7). Conclusions This study provides independent support of EPHA5 -rs7349683 and XKR4 -rs4737264 as the first markers of risk of paclitaxel induced neuropathy. In addition, it suggests that other EPHA genes also involved in axonal guidance and repair following neural injury, as well as LIMK2 locus, may play an important role in the development of this toxicity. The identified SNPs could form the basis for individualised paclitaxel chemotherapy.

Common genetic variants contribute to the observed variation in breast cancer risk for BRCA2 mutation carriers; those known to date have all been found through population-based genome-wide association studies (GWAS). To comprehensively identify breast cancer risk modifying loci for BRCA2 mutation carriers, we conducted a deep replication of an ongoing GWAS discovery study. Using the ranked P-values of the breast cancer associations with the imputed genotype of 1.4 M SNPs, 19,029 SNPs were selected and designed for inclusion on a custom Illumina array that included a total of 211,155 SNPs as part of a multi-consortial project. DNA samples from 3,881 breast cancer affected and 4,330 unaffected BRCA2 mutation carriers from 47 studies belonging to the Consortium of Investigators of Modifiers of BRCA1/2 were genotyped and available for analysis. We replicated previously reported breast cancer susceptibility alleles in these BRCA2 mutation carriers and for several regions (including FGFR2, MAP3K1, CDKN2A/B, and PTHLH) identified SNPs that have stronger evidence of association than those previously published. We also identified a novel susceptibility allele at 6p24 that was inversely associated with risk in BRCA2 mutation carriers (rs9348512; per allele HR = 0.85, 95% CI 0.80–0.90, P = 3.9×10−8). This SNP was not associated with breast cancer risk either in the general population or in BRCA1 mutation carriers. The locus lies within a region containing TFAP2A, which encodes a transcriptional activation protein that interacts with several tumor suppressor genes. This report identifies the first breast cancer risk locus specific to a BRCA2 mutation background. This comprehensive update of novel and previously reported breast cancer susceptibility loci contributes to the establishment of a panel of SNPs that modify breast cancer risk in BRCA2 mutation carriers. This panel may have clinical utility for women with BRCA2 mutations weighing options for medical prevention of breast cancer., Author Summary Women who carry BRCA2 mutations have an increased risk of breast cancer that varies widely. To identify common genetic variants that modify the breast cancer risk associated with BRCA2 mutations, we have built upon our previous work in which we examined genetic variants across the genome in relation to breast cancer risk among BRCA2 mutation carriers. Using a custom genotyping platform with 211,155 genetic variants known as single nucleotide polymorphisms (SNPs), we genotyped 3,881 women who had breast cancer and 4,330 women without breast cancer, which represents the largest possible, international collection of BRCA2 mutation carriers. We identified that a SNP located at 6p24 in the genome was associated with lower risk of breast cancer. Importantly, this SNP was not associated with breast cancer in BRCA1 mutation carriers or in a general population of women, indicating that the breast cancer association with this SNP might be specific to BRCA2 mutation carriers. Combining this BRCA2-specific SNP with 13 other breast cancer risk SNPs also known to modify risk in BRCA2 mutation carriers, we were able to derive a risk prediction model that could be useful in helping women with BRCA2 mutations weigh their risk-reduction strategy options.

Lorente-Galdós, Belén et al., Association studies are the choice approach in the discovery of the genomic basis of complex traits. To carry out such analysis, researchers frequently need to (1) select optimally informative sets of Single Nucleotide Polymorphisms (SNPs) in candidate regions and (2) annotate the results of associations found by means of genome-wide SNP arrays. These are complex tasks, since many criteria have to be considered, including the SNPs' functional properties, technological information and haplotype frequencies in given populations. SYSNPs implements algorithms that allow for efficient and simultaneous consideration of all the relevant criteria to obtain sets of SNPs that properly cover arbitrarily large lists of genes or genomic regions. Complementarily, SYSNPs allows for comprehensive functional annotation of SNPs linked to any given marker SNP. SYSNPs dramatically reduces the effort needed for SNP selection from days of searching various databases to a few minutes using a simple browser. © 2012 Inderscience Enterprises Ltd.