Validation of an NGS Panel Designed for Detection of Actionable Mutations in Tumors Common in Latin America.

Next-generation sequencing (NGS) is progressively being used in clinical practice. However, several barriers preclude using this technology for precision oncology in most Latin American countries. To overcome some of these barriers, we have designed a 25-gene panel that contains predictive biomarkers for most current and near-future available therapies in Chile and Latin America. Library preparation was optimized to account for low DNA integrity observed in formalin-fixed paraffin-embedded tissue. The workflow includes an automated bioinformatic pipeline that accounts for the underrepresentation of Latin Americans in genome databases. The panel detected small insertions, deletions, and single nucleotide variants down to allelic frequencies of 0.05 with high sensitivity, specificity, and reproducibility. The workflow was validated in 272 clinical samples from several solid tumor types, including gallbladder (GBC). More than 50 biomarkers were detected in these samples, mainly in BRCA1/2, KRAS, and PIK3CA genes. In GBC, biomarkers for PARP, EGFR, PIK3CA, mTOR, and Hedgehog signaling inhibitors were found. Thus, this small NGS panel is an accurate and sensitive method that may constitute a more cost-efficient alternative to multiple non-NGS assays and costly, large NGS panels. This kind of streamlined assay with automated bioinformatics analysis may facilitate the implementation of precision medicine in Latin America.

Inflammatory profiles in Chilean Mapuche and non-Mapuche women with gallstones at risk of developing gallbladder cancer.

Chile has high incidence rates of gallbladder cancer globally, particularly among Amerindian women, who also have a high prevalence of gallstones. We examined differences in inflammatory biomarkers between Mapuche and non-Mapuche women from the Chile Biliary Longitudinal Study, a cohort of women with ultrasound-detected gallstones. We randomly selected 200 Mapuche women frequency matched to non-Mapuche women on age and statin use Inflammatory biomarkers were analyzed using a multiplex assay and linear regression to assess associations of a priori markers (CCL20, CXCL10, IL-6, and IL-8) with ethnicity. Novel biomarkers were analyzed using exploratory factor analysis (EFA) and sufficient dimension reduction (SDR) to identify correlated marker groups, followed by linear regression to examine their association with ethnicity. The mean values of IL-8 were higher in Mapuche than non-Mapuche women (P = 0.04), while CCL20, CXCL10, and IL-6 did not differ significantly by ethnicity. EFA revealed two marker groups associated with ethnicity (P = 0.03 and P < 0.001). SDR analysis confirmed correlation between the biomarkers and ethnicity. We found higher IL-8 levels among Mapuche than non-Mapuche women. Novel inflammatory biomarkers were correlated with ethnicity and should be studied further for their role in gallbladder disease. These findings may elucidate underlying ethnic disparities in gallstones and carcinogenesis among Amerindians.

Polymorphisms PSCA rs2294008, IL-4 rs2243250 and MUC1 rs4072037 are associated with gastric cancer in a high risk population.

Genetic variants are considered risk factors for gastric cancer. To date, 61 polymorphisms have been identified as associated with this disease. The aim of the present study was to analyze the association of some of those polymorphisms with GC in Chile. We performed a case-control study including 310 gastric cancer cases and 311 controls to assess the association of 36 single-nucleotide polymorphisms genotyped by Global Screening Array (GSA). Three polymorphisms was significantly associated: PSCA rs2294008 (allele model, OR = 1.49, 95%CI 1.17-1.88, P = 1.08 × 10-3), IL-4 rs2243250 (allele model, OR = 1.28, 95%CI 1.01-1.62, P = 0.04), and MUC1 rs4072037 (allele model, OR = 0.78, 95%CI 0.61-0.99, P = 0.04).PSCA rs2294008, IL-4 rs2243250 and MUC1 rs4072037 are associated with gastric cancer in Chile. It suggests that those polymorphisms could be used as biomarkers to assess the genetic risk for this cancer outside of the previously studied populations, not only for East Asians and Caucasians populations.

Development of a small panel of SNPs to infer ancestry in Chileans that distinguishes Aymara and Mapuche components

BACKGROUND: Current South American populations trace their origins mainly to three continental ancestries, i.e. European, Amerindian and African. Individual variation in relative proportions of each of these ancestries may be confounded with socio-economic factors due to population stratification. Therefore, ancestry is a potential confounder variable that should be considered in epidemiologic studies and in public health plans. However, there are few studies that have assessed the ancestry of the current admixed Chilean population. This is partly due to the high cost of genome-scale technologies commonly used to estimate ancestry. In this study we have designed a small panel of SNPs to accurately assess ancestry in the largest sampling to date of the Chilean mestizo population (n = 3349) from eight cities. Our panel is also able to distinguish between the two main Amerindian components of Chileans: Aymara from the north and Mapuche from the south. RESULTS: A panel of 150 ancestry-informative markers (AIMs) of SNP type was selected to maximize ancestry informativeness and genome coverage. Of these, 147 were successfully genotyped by KASPar assays in 2843 samples, with an average missing rate of 0.012, and a 0.95 concordance with microarray data. The ancestries estimated with the panel of AIMs had relative high correlations (0.88 for European, 0.91 for Amerindian, 0.70 for Aymara, and 0.68 for Mapuche components) with those obtained with AXIOM LAT1 array. The country's average ancestry was 0.53 ± 0.14 European, 0.04 ± 0.04 African, and 0.42 ± 0.14 Amerindian, disaggregated into 0.18 ± 0.15 Aymara and 0.25 ± 0.13 Mapuche. However, Mapuche ancestry was highest in the south (40.03%) and Aymara in the north (35.61%) as expected from the historical location of these ethnic groups. We make our results available through an online app and demonstrate how it can be used to adjust for ancestry when testing association between incidence of a disease and nongenetic risk factors. CONCLUSIONS: We have conducted the most extensive sampling, across many different cities, of current Chilean population. Ancestry varied significantly by latitude and human development. The panel of AIMs is available to the community for estimating ancestry at low cost in Chileans and other populations with similar ancestry.

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

Graphical modeling of gene expression in monocytes suggests molecular mechanisms explaining increased atherosclerosis in smokers.

Smoking is a risk factor for atherosclerosis with reported widespread effects on gene expression in circulating blood cells. We hypothesized that a molecular signature mediating the relation between smoking and atherosclerosis may be found in the transcriptome of circulating monocytes. Genome-wide expression profiles and counts of atherosclerotic plaques in carotid arteries were collected in 248 smokers and 688 non-smokers from the general population. Patterns of co-expressed genes were identified by Independent Component Analysis (ICA) and network structure of the pattern-specific gene modules was inferred by the PC-algorithm. A likelihood-based causality test was implemented to select patterns that fit models containing a path "smoking→gene expression→plaques". Robustness of the causal inference was assessed by bootstrapping. At a FDR ≤0.10, 3,368 genes were associated to smoking or plaques, of which 93% were associated to smoking only. SASH1 showed the strongest association to smoking and PPARG the strongest association to plaques. Twenty-nine gene patterns were identified by ICA. Modules containing SASH1 and PPARG did not show evidence for the "smoking→gene expression→plaques" causality model. Conversely, three modules had good support for causal effects and exhibited a network topology consistent with gene expression mediating the relation between smoking and plaques. The network with the strongest support for causal effects was connected to plaques through SLC39A8, a gene with known association to HDL-cholesterol and cellular uptake of cadmium from tobacco, while smoking was directly connected to GAS6, a gene reported to have anti-inflammatory effects in atherosclerosis and to be up-regulated in the placenta of women smoking during pregnancy. Our analysis of the transcriptome of monocytes recovered genes relevant for association to smoking and atherosclerosis, and connected genes that before, were only studied in separate contexts. Inspection of correlation structure revealed candidates that would be missed by expression-phenotype association analysis alone.

Chromosome Y variants from different inbred mouse strains are linked to differences in the morphologic and molecular responses of cardiac cells to postpubertal testosterone.

BACKGROUND: We have reported previously that when chromosome Y (chrY) from the mouse strain C57BL/6J (ChrYC57) was substituted for that of A/J mice (ChrYA), cardiomyocytes from the resulting "chromosome substitution" C57BL/6J-chrYA strain were smaller than that of their C57BL/6J counterparts. In reverse, when chrYA from A/J mice was substituted for that of chrYC57, cardiomyocytes from the resulting A/J-chrYC57 strain were larger than in their A/J counterparts. We further used these strains to test whether: 1) the origin of chrY could also be linked to differences in the profile of gene expression in the hearts of adult male mice, and 2) post-pubertal testosterone could play a role in the differential morphologic and/or molecular effects of chrYC57 and chrYA. RESULTS: The increased size of cardiomyocytes from adult male C57BL/6J mice compared to C57BL/6J-chrYA resulted from the absence of hypertrophic effects of post-pubertal testosterone on cells from the latter strain. However, gene profiling revealed that the latter effect could not be explained on the basis of an insensitivity of cells from C57BL/6J-chrYA to androgens, since even more cardiac genes were affected by post-pubertal testosterone in C57BL/6J-chrYA hearts than in C57BL/6J. By testing for interaction between the effects of surgery and strain, we identified 249 "interaction genes" whose expression was affected by post-pubertal testosterone differentially according to the genetic origin of chrY. These interaction genes were found to be enriched within a limited number of signaling pathways, including: 1) p53 signaling, which comprises the interacting genes Ccnd1, Pten and Cdkn1a that are also potential co-regulators of the androgen receptors, and 2) circadian rhythm, which comprises Arntl/Bmal1, which may in turn regulate cell growth via the control of Cdkn1a. CONCLUSION: Although post-pubertal testosterone increased the size of cardiomyocytes from male C56BL/6J mice but not that from their C57BL/6J-chrYA counterparts, it affected gene expression in the hearts from both strains. However, several cardiac genes responded to post-pubertal testosterone in a strict strain-selective manner, which provides possible mechanisms explaining how chrY may, in part via interference with androgen regulatory events, be linked to morphologic differences of cardiac cells of adult male mice.