Latin Americans are highly heterogeneous regarding the type of Native American ancestry. Consideration of specific associations with common diseases may lead to substantial advances in unraveling of disease etiology and disease prevention.Here we investigate possible associations between the type of Native American ancestry and leading causes of death. After an aggregate-data study based on genome-wide genotype data from 1805 admixed Chileans and 639,789 deaths, we validate an identified association with gallbladder cancer relying on individual data from 64 gallbladder cancer patients, with and without a family history, and 170 healthy controls. Native American proportions were markedly underestimated when the two main types of Native American ancestry in Chile, originated from the Mapuche and Aymara indigenous peoples, were combined together. Consideration of the type of Native American ancestry was crucial to identify disease associations. Native American ancestry showed no association with gallbladder cancer mortality (P = 0.26). By contrast, each 1% increase in the Mapuche proportion represented a 3.7% increased mortality risk by gallbladder cancer (95%CI 3.1–4.3%, P = 6×10−27). Individual-data results and extensive sensitivity analyses confirmed the association between Mapuche ancestry and gallbladder cancer. Increasing Mapuche proportions were also associated with an increased mortality due to asthma and, interestingly, with a decreased mortality by diabetes. The mortality due to skin, bladder, larynx, bronchus and lung cancers increased with increasing Aymara proportions. Described methods should be considered in future studies on human population genetics and human health. Complementary individual-based studies are needed to apportion the genetic and non-genetic components of associations identified relying on aggregate-data.

Background & aims: Gallbladder cancer (GBC) is a neglected disease with substantial geographical variability: Chile shows the highest incidence worldwide, while GBC is relatively rare in Europe. Here we investigate the causal effects of risk factors considered in current GBC prevention programmes as well as C-reactive protein (CRP) level as a marker of chronic inflammation. Approach & results: We applied two-sample Mendelian randomization (MR) using publicly available data and our own data from a retrospective Chilean and a prospective European study. Causality was assessed by inverse variance weighted (IVW), MR-Egger regression and weighted median estimates complemented with sensitivity analyses on potential heterogeneity and pleiotropy, two-step MR and mediation analysis. We found evidence for a causal effect of gallstone disease on GBC risk in Chileans (p = 9 × 10-5) and Europeans (p = 9 × 10-5). A genetically elevated body mass index (BMI) increased GBC risk in Chileans (p = 0.03), while higher CRP concentrations increased GBC risk in Europeans (p = 4.1 × 10-6). European results suggest causal effects of BMI on gallstone disease (p = 0.008); public Chilean data were not, however, available to enable assessment of the mediation effects among causal GBC risk factors. Conclusions: Two risk factors considered in the current Chilean programme for GBC prevention are causally linked to GBC risk: gallstones and BMI. For Europeans, BMI showed a causal effect on gallstone risk, which was itself causally linked to GBC risk.

Background & aims: Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary tract. Most cases of GBC are diagnosed in low-and middle-income countries and research into this disease has long been limited. In this study we therefore investigate the epigenetic changes along the model of GBC carcinogenesis represented by the sequence gallstone disease → dysplasia → GBC in Chile, the country with the highest incidence of GBC worldwide.Approach: To perform epigenome-wide methylation profiling, genomic DNA extracted from sections of FFPE gallbladder tissue was analyzed using Illumina Infinium MethylationEPIC BeadChips. Pre-processed, quality-controlled data from 82 samples (gallstones n=32, lowgrade dysplasia n=13, high-grade dysplasia n=9, GBC n=28) were available to identify differentially methylated markers, regions, and pathways as well as changes in copy number variations (CNVs). Main results:The number and magnitude of epigenetic changes increased with disease development and predominantly involved the hypermethylation of CpG islands and gene promoter regions. The methylation of genes implicated in Wnt signaling, Hedgehog signaling, and tumor suppression increased with tumor grade. CNVs also increased with GBC development and affected CDKN2A, MDM2, TP53, and CCND1. Gains in the targetable ERBB2 were detected in 14% of the GBC samples. Conclusions:Our results indicate that GBC carcinogenesis comprises three main methylation stages: early (gallstone disease and low-grade dysplasia), intermediate (highgrade dysplasia), and late (GBC). The identified gradual changes in methylation and CNVs may help to enhance our understanding of the mechanisms underlying this aggressive disease and eventually lead to improved treatment and early diagnosis of GBC.