Periodic Reporting for period 2 - GUTSY (The gut microbiota and its systemic effects on metabolism and atherosclerotic disease) Reporting period: 2020-07-01 to 2021-12-31 Summary of the context and overall objectives of the project Cardiovascular disease (CVD) is the global leading cause of death. Atherosclerosis, or hardening of the arteries, is the main underlying pathologic process of CVD. Preventive efforts have reduced the incidence of CVD, but has not led all the way and new methods for prevention and treatment are needed. There is preliminary evidence that certain gut bacterial products increase the risk of atherosclerotic disease. The overall aim of this project is to identify gut bacterial species or products that accelerates or hampers the development of atherosclerotic disease and to provide easily accessible blood biomarkers for an atherosclerosis-enhancing gut microbiota. The field of microbiota research is of great attention, but the field lacks smart study designs that assess true causal effects of gut dysbiosis. Our research is anticipated to lead to identification of atherosclerosis-enhancing gut microbiota characteristics and their associated biomarkers that may open up new avenues for effective population-wide long-term prevention of atherosclerotic disease. The objectives are the following:1. Identification of fecal microflora characteristics associated with atherosclerosis 2. Identification of metabolites associated with an atherosclerosis-prone microbiota 3. Clarify the causal effects of gut microbiota characteristics on atherosclerosis, myocardial infarction and stroke. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Objective 1. We have analyzed 5,000 fecal samples from the Uppsala site of the Swedish CArdioPulmonary BioImage Study (SCAPIS). The analysis has been done through deep metagenomic sequencing. Moreover though collaboration with the SCAPIS Malmö site we have access to additional results from 5,000 additional samples. The quality control of the data shows very high quality, with high agreement in replicate samples. We have analyzed the association with atherosclerosis measured as coronary artery calcium score (CACS) measured with coronary CT angiography (CCTA). We included data from 8,271 participants aged 50-64 without previous cardiovascular events. We have identified >50 species with robust evidence for association with atherosclerosis in men and women. The results were presented at the International Human Microbiome Consortium Congress and the manuscript is planned to be submitted in September 2021. Objective 2.A total of 9,500 plasma samples has been analyzed for >1,300 metabolites (small compounds). We have assessed the association of all these compounds with the microbiota and found that some have a very strong relationship with certain gut bacteria. These data has been presented at the International Human Microbiome Consortium Congress and the manuscript is planned to be submitted in September 2021. We are currently investigating how well the atherosclerosis-linked bacteria can be predicted from the blood biomarkers. Objective 3. Distinguishing causal effects from mere correlation is necessary for prioritizing what bacteria that could be the target of novel treatments. To assess causal effects we will apply a special type of analysis relying on genetic variants in humans that alters the levels of gut bacteria and plasma metabolites. This method is called Mendelian Randomization and genetic information is needed. I am leading the work within SCAPIS to genotype the entire cohort of 30,000 individuals and the genotyping will be ready in March 2022, and imputation in March 2023. We have already recieved the results from the first 3,500 samples and are currently performing the quality control. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) These studies constitute the largest studies so far in the field and we have done detailed evaluation to identify the best statistical methods to analyze the material. We hope that our analysis pipeline can act as a blueprint for future studies in the field. Until the end of the project, we aim to be able to present a set of metabolites that is linked to an atherosclerosis-promoting gut flora and also to present evidence of causal effects of certain gut flora species on atherosclerosis. Moreover, we will analyze the association of these species with incident events of myocardial infarction and incident stroke.