Transcriptional dynamics of colorectal cancer risk associated variation at 11q23.1 correlate with tuft cell abundance and marker expression in silico

Colorectal cancer (CRC) is characterised by heritable risk that is not well understood. Heritable, genetic variation at 11q23.1 is associated with increased colorectal cancer (CRC) risk, demonstrating eQTL effects on 3 cis- and 23 trans-eQTL targets. We sought to determine the relationship between 11q23.1 cis- and trans-eQTL target expression and test for potential cell-specificity. scRNAseq from 32,361 healthy colonic epithelial cells was aggregated and subject to weighted gene co-expression network analysis (WGCNA). One module (blue) included 19 trans-eQTL targets and was correlated with POU2AF2 expression only. Following unsupervised clustering of single cells, the expression of 19 trans-eQTL targets was greatest and most variable in cluster number 11, which transcriptionally resembled tuft cells. 14 trans-eQTL targets were found to demarcate this cluster, 11 of which were corroborated in a second dataset. Intra-cluster WGCNA and module preservation analysis then identified twelve 11q23.1 trans-eQTL targets to comprise a network that was specific to cluster 11. Finally, linear modelling and differential abundance testing showed 11q23.1 trans-eQTL target expression was predictive of cluster 11 abundance. Our findings suggest 11q23.1 trans-eQTL targets comprise a POU2AF2-related network that is likely tuft cell-specific and reduced expression of these genes correlates with reduced tuft cell abundance in silico

Gene Co-Expression Network Analysis Identifies Vitamin D-Associated Gene Modules in Adult Normal Rectal Epithelium Following Supplementation

Colorectal cancer (CRC) is a common, multifactorial disease. While observational studies have identified an association between lower vitamin D and higher CRC risk, supplementation trials have been inconclusive and the mechanisms by which vitamin D may modulate CRC risk are not well understood. We sought to perform a weighted gene co-expression network analysis (WGCNA) to identify modules present after vitamin D supplementation (when plasma vitamin D level was sufficient) which were absent before supplementation, and then to identify influential genes in those modules. The transcriptome from normal rectal mucosa biopsies of 49 individuals free from CRC were assessed before and after 12 weeks of 3200IU/day vitamin D (Fultium-D3) supplementation using paired-end total RNAseq. While the effects on expression patterns following vitamin D supplementation were subtle, WGCNA identified highly correlated genes forming gene modules. Four of the 17 modules identified in the post-vitamin D network were not preserved in the pre-vitamin D network, shedding new light on the biochemical impact of supplementation. These modules were enriched for GO terms related to the immune system, hormone metabolism, cell growth and RNA metabolism. Across the four treatment-associated modules, 51 hub genes were identified, with enrichment of 40 different transcription factor motifs in promoter regions of those genes, including VDR:RXR. Six of the hub genes were nominally differentially expressed in studies of vitamin D effects on adult normal mucosa organoids: LCN2, HLA-C, AIF1L, PTPRU, PDE4B and IFI6. By taking a gene-correlation network approach, we have described vitamin D induced changes to gene modules in normal human rectal epithelium in vivo, the target tissue from which CRC develops

Deciphering colorectal cancer genetics through multi-omic analysis of 100,204 cases and 154,587 controls of European and east Asian ancestries

In the version of this article initially published, the author affiliations incorrectly listed “Candiolo Cancer Institute FPO-IRCCS, Candiolo (TO), Italy” as “Candiolo Cancer Institute, Candiolo, Italy.” The change has been made to the HTML and PDF versions of the article

Fine-mapping analysis including over 254,000 East Asian and European descendants identifies 136 putative colorectal cancer susceptibility genes

Genome-wide association studies (GWAS) have identified more than 200 common genetic variants independently associated with colorectal cancer (CRC) risk, but the causal variants and target genes are mostly unknown. We sought to fine-map all known CRC risk loci using GWAS data from 100,204 cases and 154,587 controls of East Asian and European ancestry. Our stepwise conditional analyses revealed 238 independent association signals of CRC risk, each with a set of credible causal variants (CCVs), of which 28 signals had a single CCV. Our cis-eQTL/mQTL and colocalization analyses using colorectal tissue-specific transcriptome and methylome data separately from 1299 and 321 individuals, along with functional genomic investigation, uncovered 136 putative CRC susceptibility genes, including 56 genes not previously reported. Analyses of single-cell RNA-seq data from colorectal tissues revealed 17 putative CRC susceptibility genes with distinct expression patterns in specific cell types. Analyses of whole exome sequencing data provided additional support for several target genes identified in this study as CRC susceptibility genes. Enrichment analyses of the 136 genes uncover pathways not previously linked to CRC risk. Our study substantially expanded association signals for CRC and provided additional insight into the biological mechanisms underlying CRC development

Functional genomics interrogation of gene-environment interactions between vitamin D and genetic variants associated with colorectal cancer susceptibility

Colorectal cancer (CRC) is the fourth most common cancer in the UK. It develops in a step-wise manner from normal tissue to cancerous tumour over many years. The heritability of CRC risk is estimated variously up to 35%. One possible explanation for thus far unexplained heritability is gene-environment interaction (GxE), where the effects of a particular environmental factor are moderated by genotype of an individual. Among numerous environmental factors implicated in CRC, low vitamin D intake and low plasma vitamin D concentration have been associated with increased risk of CRC in observational studies. While supplementation studies have failed to establish a beneficial relationship, these studies can be criticised variously and whether vitamin D can reduce CRC risk therefore remains an open question. This study utilises in vitro, ex vivo and in vivo approaches, combining wet-lab and in-silico analyses to investigate the differential effect of vitamin D on colorectal cell lines, organoids or tissue in relation to genotype at CRC risk loci. Vitamin D GxEs were first investigated in an in-house observational study of normal human colorectal mucosa. Genes were first identified which were correlated with expression of the vitamin D receptor gene (VDR). Using an agnostic approach to genomic variants associated both with CRC risk and with expression (cis-eQTLs) of nearby genes themselves correlated with VDR, three GxEs were identified for further study. These were then tested in a human supplementation study, with an interaction between rs7012462 and vitamin D on MYC expression being of particular interest. This GxE was then further explored in CRC cell lines. To demonstrate that this genomic region was definitively responsible for the GxE effect, efforts were then made to delete the region in CRC cell lines using a CRISPR-Cas9 approach. This study also utilised organoid models derived from adult normal colorectal mucosa (ANMO), adult colorectal cancer (CRCO) and foetal normal colorectal mucosa (FNMO) to investigate vitamin D effects in a model system isolated from many of the vagaries in heterogeneity found when studying a human population. Organoids were established in long-term culture and importantly validated as a model of human colorectal tissue. Vitamin D was shown to induce effects on ANMO phenotype, and to induce transcriptomic changes in ANMO derived from 9 individuals (447 upregulated and 423 downregulated genes, FDR p<0.05). KEGG analysis of differentially expressed genes implicated multiple pathways of relevance to CRC. Stemness-markers LRIG1 and MSI1 along with BMP pathway members BMP4 and SMAD7 were identified as genes of particular interest. Meta-analysis with another recently published ANMO vitamin D study (Fernandez-Barral et al 2019) provided a robust list of vitamin D responsive genes (795 differentially expressed genes). These differentially expressed genes were enriched for intolerance to loss of function mutations, and were enriched for functional mutations in the TCGA CRC dataset. CRCO appeared more resistant or to have a more idiosyncratic response to vitamin D: only 9 differentially expressed genes in CRCO derived from 4 individuals (8 upregulated and 1 downregulated, FDR p<0.05). Meta-analysis with the recently published CRCO vitamin D study did though identify 201 genes differentially expressed, of which 116 genes were common to ANMO. Gene-environment interactions were also investigated in ANMO, first involving vitamin D pathway gene variants, and then involving CRC risk variants. There was marked variation in CYP24A1 expression (the gene responsible for deactivating the active form of vitamin D) following calcitriol treatment between individuals, with a GxE noted involving rs209955. When CYP24A1 effects were taken into account in testing GxEs involving CRC risk variants, genotype dependent BMP4 expression changes in response to vitamin D were identified in ANMO involving rs35107139 at the 14q22.2 locus. This same GxE was also then identified in CRC cell lines, and in human supplementation and observational studies. Finally, the modifying effect of vitamin D on CRC risk associated with genetic variation at the BMP4 CRC locus suggested a predictive relationship to subsequent CRC onset in the UK Biobank. Taking a broader approach, this study has used ATACseq and VDR ChIPseq to identify vitamin D dependent open chromatin regions and VDR transcription factor binding regions in CRC cell lines and ANMO. While VDR-RXR motifs were identified in the majority of these regions, this was not among the most enriched motifs. The degree of overlap between different vitamin D dependent epigenetic regions, including from previously published ChIPseq, ChIPexo and FAIREseq studies, with each other and with genomic variants associated with CRC risk was reviewed (along with variants associated with risk of inflammatory bowel disease, rheumatoid arthritis, schizophrenia or intraocular pressure for comparison), showing that these relationships may be tissue, treatment and disease specific. Finally, this study reviewed effects of 12 weeks vitamin D supplementation on gene expression in adult normal rectal mucosa. While no single gene was differentially expressed at a genome-wide level following supplementation, by taking a gene-correlation network approach, vitamin D induced connectivity changes were identified involving groups (modules) of genes. By reviewing non-preserved modules before and after vitamin D supplementation, hub genes have been identified which may play a key role in modulating vitamin D actions in normal rectal epithelium. In summary, this project has identified vitamin D effects both at a genome-wide and single gene scale. Vitamin D epigenetic effects have been described, with enrichment of vitamin D dependent epigenetic peaks in proximity to genomic variants associated with CRC. It has also identified in a vitamin D supplementation study that while no single gene was differentially expressed in normal rectal mucosa, groups of genes changed their expression patterns. At the single-gene level, genotype-specific responses to vitamin D have also been identified. In particular GxEs involving vitamin D and rs7012462 on MYC expression and involving rs35107139 on BMP4 expression have been demonstrated. Importantly the modifying effect of vitamin D on CRC risk associated with genetic variation at the BMP4 CRC locus suggests a predictive relationship to subsequent CRC onset in the UK Biobank, and this provides compelling rationale for genotype-stratified RCTs to test protective effects of vitamin D on CRC risk