PROJECT 4: Somatic evolution of the hematopoietic system in cardiovascular disease

Cardiovascular disease (CVD) has recently been shown to associate with clonal expansions in the blood (clonal hematopoiesis, CH). These clonal expansions often carry mutations in characteristic driver genes and are thought to be causally linked to CVD progression. Therapeutic approaches targeting mutant blood cells are therefore in development. However, the causal link between CVD and CH remains incompletely understood. This application proposes to tackle the next set of crucial unsolved questions in this area. In Specific Aim 1, we will clarify the relationship between CVD and leukemogenic driver-free CH. All studies that so far observed an association with CVD have defined CH as clonal expansions carrying specific leukemogenic driver (LD) genes. This approach misses the bulk of positive selection in the blood, as most clonal expansions occur in the absence of LD mutations. It is currently unclear if the CVD-CH association is specific to CH that harbors (potentially pro- inflammatory) LD mutations. Currently, progress in this area is limited by a lack of methods for sensitive and scalable detection of LD-free CH. In Specific Aim 1A, we will make a decisive step toward facilitating the diagnosis of LD-free CH. We will adapt a novel microsatellite sequencing technology developed by our lab to enable ultra-sensitive detection of all (LD-free and LD-bearing) clonal expansions in the blood. We will then use this assay in combination with deep targeted sequencing to determine whether LD-free CH is associated with CVD in 100 patients and 100 age-matched controls. In Specific Aim 1B, we will determine whether LD-free CH associates with CVD to the same degree as LD-bearing CH in large-scale publicly available blood whole genome sequencing data. In Specific Aim 2, we will study the disease-specific selective pressures that CVD exerts on the hematopoietic system. Seminal genomics studies in illnesses such as chronic liver disease and inflammatory bowel disease have shown that pathological conditions exert disease-specific evolutionary pressures on tissues, resulting in the emergence of genetic variants that are not selected for during normal tissue evolution. In Specific Aim 2A, we will use the publicly available data sets that we also leverage in Aim 1B to search for genes that are under selective pressure specifically in CVD patients. To do so, we will calculate ratios of nonsynonymous to synonymous substitutions (dN/dS) across all protein-coding genes in the genomes of CVD patients and controls and search for genes that are under CVD-specific selective pressure. In Specific Aim 2B, we will use an in vivo screening platform to validate potential novel driver genes emerging from Aim 2A and study the effects of different CVD forms on known CH drivers. We will use custom CRISPR libraries to perform in vivo screens in the hematopoietic system of mice with atherosclerosis, hypertension, and myocardial infarction and ascertain whether distinct kinds of CVD select for different mutation patterns. Together, these experiments will provide a detailed portrait of the evolutionary pressures that shape the hematopoietic system in CVD.