Germline influences on processes that contribute to colorectal carcinogenesis

Abstract

Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world with incidence rising particularly in younger individuals in the West. Understanding of the genetic causes underlying CRC has increased greatly in recent years as a result of work on familial CRC syndromes, which has identified rarer but more impactful genetic mutations, and CRC genome-wide association studies (GWAS), which have identified more common forms of DNA variation. The common variants from CRC GWAS are less impactful individually but when combined have a non-trivial effect on CRC risk. However, there are still many gaps of knowledge in terms of CRC genetics, with one notable example being the mechanisms through which variants are affecting CRC risk. This project aimed to contribute to this area by first expanding the existing GWAS meta-analysis dataset through the addition of 2,356 CRC patients and 14,162 CRC-free control patients from the 100,000 Genomes Project (100kGP). Inclusion of 100kGP data led to the identification of eight novel CRC risk loci but also called into question six previously identified loci. This modest net gain of CRC risk loci indicates that substantial expansion of GWAS cohorts (perhaps the addition of > 100,000 patients) may be needed for meaningful improvements in understanding common causes of CRC. Several risk loci identified by CRC GWAS highlight genomic regions where the target gene may not be affecting CRC predisposition through effects on the colorectal epithelium, but instead via an intermediate phenotype. The project explored two of these potential intermediate phenotypes: blood cell traits and the microbiome. For blood cell traits, genetic fine-mapping of a locus at 12q24.12 was first conducted to identify whether pan-haematopoietic regulator SH2B3 was the CRC causal risk gene, but these results were not conclusive. Work was then expanded to examine the phenotype generally, initially by using Mendelian randomisation (MR) to establish whether causal links existed between blood cell trait variation and CRC risk. A modified PCA-based GWAS and MR approach was then used to try and mitigate the high levels of horizontal pleiotropy that are apparent in haematopoietic genetics. These results showed indications of red blood cell and platelet variation affecting CRC risk but were not definitive and require further investigation. For the microbiome, fine-mapping was conducted on four risk loci, where the possible CRC causal genes (GALNT12, B3GNT8, FUT2, FUT3, and FUT6) play roles in oligosaccharide modification processes such as glycosylation and fucosylation in the colon, thereby potentially affecting susceptibility to microbial infections and subsequent CRC risk. The previous study of rare variants in GALNT12 specifically has raised the question as to whether it is a moderate CRC risk gene; this hypothesis was tested using the larger 100kGP cohort, which suggested that it is not a moderate risk gene, and then expanded to include the other four potential microbiome CRC genes. Once again, work was then extended from individual loci to the overall mechanism. First, MR was conducted using reported variants from existing microbiome association studies performed in disease-free individuals to test whether there are causal links between levels of bacterial taxa and CRC risk. This analysis suggested that variation in the Escherichia/Shigella, Fusobacterium, Streptococcus, and Bifidobacterium genera may be causal for CRC risk. A new complementary approach running GWAS in CRC patients specifically using the 100kGP cohort was performed, with relative abundance of bacterial genera and species, counts from toxin-producing bacterial strains, as well as global bacterial measures taken from the tumour directly used as phenotypes. Variants identified from these GWAS were then used in MR analyses to see whether causality between bacterial measures derived from CRC patients and CRC risk could be established. This analysis instead suggested causal associations for Oscillibacter and Blautia genera, Bacteroides fragilis and Ruminococcus faecis species, and bacterial count per human cell. In summary, this project has contributed to the understanding of the genetics of CRC carcinogenesis using methods that are, in large part, based on the intrinsic robustness of genetic data. The challenges encountered in this project are common in the field, namely (i) the weakness of most genetic associations with both CRC and its risk factors and (ii) confounding and pleiotropic effects of genes and other unobserved factors. My work provides insight into common genetic variation in CRC, as well as interesting clues for the role of blood cell levels and gut bacteria in CRC risk