Background

Colorectal cancer (CRC) is the third most frequently diagnosed cancer worldwide and the fourth most common cause of death from cancer.^{1, 2} There is a genetic component to risk of the disease, which is thought to explain up to 35% of variability in CRC risk.^3–5 In addition, modifiable lifestyle factors, including obesity, consumption of processed meat, and alcohol are thought to increase CRC risk.^{2, 6–9} However, the underlying biological pathways remain unclear, which limits targeted prevention strategies. Whilst CRC has higher mortality rates when diagnosed at later stages, early-stage CRC or precancerous lesions are largely treatable, meaning colorectal cancer screening programmes have the potential to be highly effective.^{10, 11} Due to the lack of known predictive biomarkers for CRC, wide-scale screening (if implemented at all) is expensive and often targeted crudely by age range. Identifying biomarkers predictive of CRC, or with causal roles in disease development, is therefore vital.

One potential source of biomarkers for CRC risk is the circulating metabolome, which offers a dynamic insight into cellular processes and disease states. It is increasingly clear from mechanistic studies that both systemic and intracellular tumour metabolism play an important role in CRC development and progression.^{12, 13} Interestingly, several major risk factors for CRC are known to have profound effects on metabolism.¹⁴ For instance, obesity has been shown via conventional observational and Mendelian randomization (MR) analyses to strongly alter circulating metabolite levels.^{9, 15–17} This suggests that the circulating metabolome may play a mediating role in the relationship between at least some common risk factors, such as obesity, and CRC – or at least might be a useful biomarker for disease or intermediates thereof. In particular, previous work has highlighted polyunsaturated fatty acids (PUFA) as potentially having a role in colorectal cancer development. The term PUFA includes omega-3 and −6 fatty acids. Recent MR work has highlighted a possible link between PUFAs, in particular omega 6 PUFAs, and colorectal cancer risk.¹⁸ Further investigating the relationship between CRC and circulating metabolites may therefore provide powerful insights into the causal pathways underlying disease risk, or alternatively may be valuable in prediction and early diagnosis.

MR is a genetic epidemiological approach used to evaluate causal relationships between traits.^{19, 20} This method uses genetic variation as a proxy measure for traits in an instrumental variable framework to assess the causal relevance of the traits in disease development. As germline genetic variants are theoretically randomised between generations and fixed at conception, this approach should be less prone to bias and confounding than conventional analyses undertaken in an observational context. Conventionally, MR is used to investigate the effect of an exposure on a disease outcome. In reverse MR, genetic instruments proxy the association between liability to a disease and other traits.²¹ This approach can identify biomarkers which cause the disease, are predictive for the disease, or have diagnostic potential.²¹ Given the suspected importance of the circulating metabolome in CRC development, employing both reverse MR and conventional forward MR for metabolites in the same study may be an efficient approach for revealing causal and predictive biomarkers for CRC. Although previous observational studies have investigated associations between the circulating metabolome and CRC risk, these studies may have been influenced by confounding bias which should be less relevant to MR analyses.^22–31 Additionally, these studies focussed on adults, who commonly take medications which may confound metabolite associations, further complicating interpretations.

Here, we applied a reverse MR framework to identify circulating metabolites which are associated with CRC liability across different stages of the early life course (spanning childhood to young adulthood, when use of medications and CRC are both rare) using data from a birth cohort study. We then attempted to replicate these results using reverse two-sample MR in an independent cohort of middle-aged adults (UK Biobank). We then performed conventional ‘forward’ MR of metabolites onto CRC risk using large-scale cancer consortia data to identify metabolites which may have a causal role in CRC development.

Methods

Study populations

This study uses data from 2 cohort studies: the Avon Longitudinal Study of Parents and Children (ALSPAC) offspring (generation 1) cohort (individual-level data) and the UK Biobank cohort (summary-level data); plus summary-level data from a genome-wide association study (GWAS) meta-analysis of CRC comprising the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), Colorectal Transdisciplinary Study (CORECT), and Colon Cancer Family Registry (CCFR).

ALSPAC is a population-based birth cohort study in which 14,541 pregnant women with an expected delivery date between 1 April 1991 and 31 December 1992 were recruited from the former Avon County of southwest England.³² Since then, 13,988 offspring alive at one year have been followed repeatedly with questionnaire- and clinic-based assessments.^{33, 34} Sufficient information was available on 6,221 of these individuals to be included in our analysis, as metabolomics was not performed for all individuals in the ALSPAC study. Study data were collected and managed using REDCap electronic data capture tools hosted at the University of Bristol.³⁵ REDCap (Research Electronic Data Capture) is a secure, web-based software platform designed to support data capture for research studies. Offspring genotype was assessed using the Illumina HumanHap550 quad chip platform. Quality control measures included exclusion of participants with sex mismatch, minimal or excessive heterozygosity, disproportionately missing data, insufficient sample replication, cryptic relatedness, and non-European ancestry. Imputation was performed using the Haplotype Reference Consortium (HRC) panel. Offspring were considered for the current analyses if they had no older siblings in ALSPAC (203 excluded) and were of white ethnicity (based on reports by parents, 604 excluded) to reduce the potential for confounding by genotype. Written informed consent was provided and ethical approval was obtained from the ALSPAC Law and Ethics Committee and the local research ethics committee. Consent for biological samples has been collected in accordance with the Human Tissue Act (2004). Informed consent for the use of data collected via questionnaires and clinics was obtained from participants following the recommendations of the ALSPAC Ethics and Law Committee at the time. The study website contains details of all available data through a fully searchable data dictionary and variable search tool (http://www.bristol.ac.uk/alspac/researchers/our-data/).

UK Biobank is a population-based cohort study based in 22 centres across the UK.³⁶ The cohort is made up of around 500,000 adults aged 40-80 years old, who were enrolled between 2006 and 2010. Genotyping data is available for 488,377 participants.³⁷ Participants were genotyped using one of two arrays – either the Applied Biosystems UK BiLEVE Axiom Array by Affymetrix (now part of Thermo Fisher Scientific), or the closely related Applied Biosystems UK Biobank Axiom Array. Approaches based on Principal Component Analysis (PCA) were used to account for population structure. Individuals were excluded: if reported sex differed from inferred sex based on genotyping data; if they had sex chromosome karyotypes which were not XX or XY; if they were outliers in terms of heterozygosity and missing rates; or if they had high relatedness to another participant. Multiallelic SNPs or those with a minor allele frequency of below 1% were removed. Imputation was performed using the UK10K haplotype and HRC reference panels.

The GWAS meta-analysis for CRC included up to 52,775 cases and 45,940 controls.^{38, 39} This sample excluded cases and controls from UK Biobank to avoid potential bias due to sample overlap which may be problematic in MR analyses.⁴⁰ Cases were diagnosed by a physician and recorded overall and by site (colon, 28,736 cases; proximal colon, 14,416 cases; distal colon, 12,879 cases; and rectal, 14,150 cases). Colon cancer included proximal colon (any primary tumour arising in the cecum, ascending colon, hepatic flexure, or transverse colon), distal colon (any primary tumour arising in the pleenic flexure, descending colon or sigmoid colon), and colon cases with unspecified site. Rectal cancer included any primary tumour arising in the rectum or rectosigmoid junction.³⁸ Approximately 92% of participants in the overall CRC GWAS were white-European (∼8% were East Asian). All participants included in site-specific CRC analyses were of European ancestry. Imputation was performed using the Michigan imputation server and HRC r1.0 reference panel. Regression models were further adjusted for age, sex, genotyping platform, and genomic principal components as described previously.³⁸ Ethics were approved by respective institutional review boards.

Assessment of CRC genetic liability

Genetic liability to CRC was based on single nucleotide polymorphisms (SNPs) associated with CRC case status at genome-wide significance (P<5×10⁻⁸). 108 independent SNPs reported by two major GWAS meta-analyses were eligible for inclusion in a CRC genetic risk score (GRS).^{38, 41} The set of SNPs was filtered, excluding 36 SNPs that were in linkage disequilibrium based on R²>0.001 using the TwoSampleMR package (SNPs with the lowest P-values were retained).⁴² This left 72 SNPs independently associated with CRC (Supplementary Table (ST) 1), 65 of which were available in imputed ALSPAC genotype data post quality control. As GWAS of site-specific CRC have identified marked heterogeneity,⁴³ GRS describing site-specific CRCs were constructed for sensitivity analyses using the same process outlined above. The GRS for colon cancer, rectal cancer, proximal colon cancer and distal colon cancer were comprised of 38, 25, 20 and 24 variants, respectively (ST1). For overall CRC and site-specific CRC analyses, sensitivity analyses excluding any SNPs in the FADS cluster (i.e. within the gene regions of FADS1, FADS2, or FADS3) (ST1) were performed given a likely role for these SNPs in influencing circulating metabolite levels directly, in particular via lipid metabolism (i.e., not primarily due to _CRC).44–50

Assessment of circulating metabolites

Circulating metabolite measures were drawn from ALSPAC and UK Biobank using the same targeted metabolomics platform. In ALSPAC, participants provided non-fasting blood samples during a clinic visit while aged approximately 8y, and fasting blood samples from clinic visits while aged approximately 16y, 18y, and 25y. Proton nuclear magnetic resonance (¹H-NMR) spectroscopy was performed on Ethylenediaminetetraacetic acid (EDTA) plasma (stored at or below −70 degrees Celsius pre-processing) to quantify a maximum of 231 metabolites.⁵¹ Quantified metabolites included the cholesterol and triglyceride content of lipoprotein particles; the concentrations and diameter/size of these particles; apolipoprotein B and apolipoprotein A-1 concentrations; as well as fatty acids and their ratios to total fatty acid concentration, branched chain and aromatic amino acids, glucose and pre-glycaemic factors including lactate and citrate, fluid balance factors including albumin and creatinine, and the inflammatory marker glycoprotein acetyls (GlycA). This metabolomics platform has limited coverage of fatty acids. In UK Biobank, EDTA plasma samples from 117,121 participants, a random subset of the original ∼500,000 who provided samples at assessment centres between 2006 and 2013, were analysed between 2019 and 2020 for levels of 249 metabolic traits (168 concentrations plus 81 ratios) using the same high-throughput ¹H-NMR platform. Data pre-processing and QC steps are described previously.^51–53 To allow comparability between MR and GRS estimates all metabolite measures were standardised and normalised using rank-based inverse normal transformation. For descriptive purposes in ALSPAC, body mass index (BMI) was calculated at each time point as weight (kg) divided by squared height (m²) based on clinic measures of weight to the nearest 0.1 kg using a Tanita scale and height measured in light clothing without shoes to the nearest 0.1 cm using a Harpenden stadiometer.

CRC liability variants were combined into a GRS using PLINK 1.9, specifying the effect (risk raising) allele and coefficient (logOR) with estimates from the CRC GWAS used as external weights.^{38, 41} GRSs were calculated as the number of effect alleles (or dosages if imputed) at each SNP (0, 1, or 2) multiplied by its weighting, summing these, and dividing by the total number of SNPs used. Z-scores of GRS variables were calculated to standardize scoring.

Statistical approach

An overview of the study design is presented in Figure 1. To estimate the effect of increased genetic liability to CRC on circulating metabolites we conducted a GRS analysis in ALSPAC and reverse two-sample MR analyses in UK Biobank. Estimates were interpreted within a ‘reverse MR’ framework,⁵⁴ wherein results are taken to reflect ‘metabolic features’ of CRC liability which could capture causal or predictive metabolite-disease associations. To clarify the direction of metabolite-CRC associations, we additionally performed conventional ‘forward’ two-sample MR analyses to estimate the effect of circulating metabolites on CRC risk using large-scale GWAS data on metabolites and CRC.

Figure 1:

1. Associations of CRC liability with circulating metabolites in early life

Separate linear regression models with robust standard errors were used to estimate coefficients and 95% confidence intervals for associations of GRSs with each metabolite as a dependent variable measured on the same individuals at age 8y, 16y, 18y, and 25y, adjusted for sex and age at the time of metabolite assessment. To aid interpretations, estimates were multiplied by 0.693 (log_e2) to reflect SD-unit differences in metabolites per doubling of genetic liability to CRC.⁵⁵ The Benjamini-Hochberg method was used to adjust P-values for multiple testing and an adjusted P-value of <0.05 was used as a heuristic for evidence for association given current sample sizes.⁵⁶

2. Reverse MR of the effects of CRC liability on circulating metabolites in middle adulthood

“Reverse” MR analyses⁵⁴ were conducted using UK Biobank for outcome datasets in two sample MR to examine the effect of CRC liability on circulating metabolites. SNP-outcome (metabolite) estimates were obtained from a GWAS of metabolites in UK Biobank.^{57, 58} Prior to GWAS, all metabolite measures were standardised and normalised using rank-based inverse normal transformation. Genetic association data for metabolites were retrieved using the MRC IEU UK Biobank GWAS pipeline.⁵⁹ Full summary statistics are available via the IEU Open GWAS project.^{54, 60} Up to 3 statistical methods were used to generate reverse MR estimates of the effect of CRC liability on circulating metabolites using the TwoSampleMR package⁶¹: random-effects inverse variance weighted (IVW), weighted-median, and weighted-mode, which each make differing assumptions about directional pleiotropy and SNP heterogeneity.^62–64 The IVW MR model will produce biased effect estimates in the presence of horizontal pleiotropy, i.e. where one or more genetic variant(s) included in the instrument affect the outcome by a pathway other than through the exposure. In the weighted median model, each genetic variant is weighted according to its distance from the median effect of all genetic variants. Thus, the weighted median model will provide an unbiased estimate when at least 50% of the information in an instrument comes from genetic variants that are not horizontally pleiotropic. The weighted mode model uses a similar approach but weights genetic instruments according to the mean effect. In this model, over 50% of the weight of the genetic instrument can be contributed to by genetic variants which are horizontally pleiotropic, but the most common amount of pleiotropy must be zero (known as the Zero Modal Pleiotropy Assumption (ZEMPA))⁶⁵. As above, estimates were multiplied by 0.693 (log_e2) to reflect SD-unit differences in metabolites per doubling of genetic liability to CRC.⁶⁶

3. Forward MR of the effects of metabolites on CRC

Forward MR analyses were conducted using summary statistics from UK Biobank for the same NMR-measured metabolites (SNP-exposure) and from GECCO/CORECT/CCFR as outlined above (SNP-outcome). We identified SNPs that were independently associated (R²<0.001 and P<5×10⁻⁸) with metabolites from a GWAS of 249 metabolites in UK Biobank described above. As before, we used up to 3 statistical methods to generate MR estimates of the effect of circulating metabolites on CRC risk (overall and site-specific): random-effects IVW, weighted-median, and weighted-mode. The Benjamini-Hochberg method was used to adjust P-values for multiple testing and an adjusted P-value of <0.05 was used as a heuristic for nominal evidence for a causal effect.⁵⁶ MR outputs are beta coefficients representing the logOR for CRC per SD higher metabolite, exponentiated to reflect the OR for CRC per SD metabolite.

MR analyses were performed in R version 4.0.3.⁶⁷ and GRS analyses in Stata 16.1 (StataCorp, College Station, Texas, USA). The ggforestplot R package was used to generate results visualisations.⁶⁸

Results

Associations of CRC liability with circulating metabolites in early life

At the time the ALSPAC blood samples were taken, the mean age of participants was 7.5y (N=4,767), 15.5y (N=2,930), 17.8y (N=2,613), and 24.5y (N=2,559) for the childhood, early adolescence, late adolescence and young adulthood time points respectively. The proportion of participants which were male were 50.5%, 47.4%, 44.5%, and 39.1% and mean BMI was 16.2, 21.4, 22.7, and 24.8 kg/m² for each time point respectively. The socio-demographic profile of ALSPAC offspring participants has been reported previously.⁶⁹ Mean and standard deviation (SD) values for metabolites on each measurement occasion in ALSPAC are shown in ST2.

In the GRS analysis, there was no strong evidence of association of CRC liability with metabolites at age 8y (ST3). At age 16y, there was evidence for association with several lipid traits including higher cholesteryl esters to total lipids ratio in large low-density lipoprotein (LDL) (SD change per doubling CRC liability = 0.06, 95% CI = 0.02 to 0.10) and higher cholesterol in very small very low-density lipoprotein (VLDL) (SD change per doubling CRC liability = 0.06, 95% CI = 0.03 to 0.10). There was strong evidence for association with several traits at age 18y including higher non-high-density lipoprotein (non-HDL) lipids, e.g., a 1 doubling CRC liability was associated with higher levels of total cholesterol (SD change = 0.05 95% CI = 0.01 to 0.09), VLDL-cholesterol (SD change = 0.05, 95% CI = 0.01 to 0.09), LDL-cholesterol (SD change = 0.06, 95% CI = 0.02 to 0.09)), apolipoproteins (apolipoprotein B (SD change = 0.06, 95% CI = 0.02 to 0.09)), and fatty acids (omega-3 (SD change = 0.08, 95% CI = 0.04 to 0.11), docosahexaenoic acid (DHA) (SD change = 0.05, 95% CI = 0.02 to 0.09)) (ST3). Figure 2 shows results for all clinically validated metabolites. At age 25y, there was no strong evidence of association of CRC liability with metabolites. In anatomical site-specific analyses, there was strong evidence for association of liability to colon cancer with omega-3 (SD change = 0.07, 95% CI = 0.03 to 0.11) and DHA (SD change = 0.07, 95% CI = 0.03 to 0.10) at age 18y. There was little evidence for any associations at any other CRC site or age (ST3, Supplementary Figure (SF)1-4). When SNPs in the FADS cluster gene regions were excluded due to possible horizontal pleiotropy given the role of FADS in lipid metabolism, there was a reduction in strength of evidence for an association of liability to CRC with any metabolite measured, although estimates were in a largely consistent direction with the prior analysis (ST4, SF5-6).

Figure 2:

Reverse MR of the effects of CRC liability on circulating metabolites in middle adulthood

All instrument sets from the reverse MR analysis had an F-statistic greater than 10 (minimum F-statistic = 36, median = 40), suggesting our analyses did not suffer from weak instrument bias (ST5). There was little evidence of an association of CRC liability (overall or by anatomical site) on any of the circulating metabolites investigated, including when the SNP in the FADS gene region was excluded, based on our pre-determined cut-off of FDR-P < 0.05; however, the direction of effect estimates was largely consistent with those seen in ALSPAC GRS analyses, with higher CRC liability weakly associated with higher non-HDLs, lipoproteins and fatty acid levels (SF7-9, ST6-7). Figure 3 shows the results for clinically validated metabolites. In subsite stratified analyses, there was strong evidence for a causal effect of genetic liability to proximal colon cancer on several traits, including total fatty acids (SD change per doubling of liability = 0.02, 95% CI = 0.01 to 0.04) and omega-6 fatty acids (SD change per doubling of liability = 0.03, 95% CI = 0.01 to 0.05).

Figure 3:

Forward MR for the effects of metabolites on CRC risk

All instrument sets from the forward MR analysis had an F-statistic greater than 10 (minimum F-statistic = 54, median = 141), suggesting that our analyses were unlikely to suffer from weak instrument bias (ST8-9). There was strong evidence for an effect of several fatty acid traits on overall CRC risk, including of omega-3 fatty acids (CRC OR = 1.13, 95% CI = 1.06 to 1.21), DHA (OR CRC = 1.76, 95% CI = 1.08 to 1.28), ratio of omega-3 fatty acids to total fatty acids (OR CRC = 1.18, 95% CI = 1.11 to 1.25), ratio of DHA to total fatty acids (CRC OR = 1.20, 95% CI = 1.10 to 1.31), and ratio of omega-6 fatty acids to omega-3 fatty acids (CRC OR = 0.86, 95% CI = 0.80 to 9.13) (ST10, Figure 4). These estimates were overlapping with variable precision in MR sensitivity models. When SNPs in the FADS gene region were excluded, there was little evidence for a causal effect of any metabolite investigated on CRC risk based on the predetermined FDR-P cut of off < 0.05, although the directions of effect estimates were consistent with previous analyses (ST11, SF11-12).

Figure 4:

In anatomical subtype stratified analyses evidence was strongest for an effect of fatty acid traits on higher CRC risk, and this appeared specific to the distal colon, e.g., omega-3 (distal CRC OR = 1.20, 95% CI = 1.09 to 1.32), and ratio of DHA to total fatty acids (distal colon OR = 1.29, 95% CI = 1.16 to 1.43). There was also evidence of a negative effect of ratio of omega-6 to omega-3 fatty acids (distal CRC OR = 0.80, 95% CI = 0.74 to 0.88) and a positive effect of ratio of omega-3 fatty acids to total fatty acids (distal CRC = 1.24, 95% CI = 1.15 to 1.35; seen also for proximal CRC OR = 1.15, 95% CI = 1.07 to 1.23) (ST10, SF10). These estimates were also directionally consistent in MR sensitivity models.

Discussion

Here, we used a reverse MR framework to identify circulating metabolites which are associated with genetic CRC liability across different stages of the early life course and attempted to replicate results in an independent cohort of middle-aged adults. We then performed forward MR to characterise the causal direction of the relationship between metabolites and CRC. Our GRS analysis provided evidence for an association of genetic liability to CRC with higher circulating levels of lipoprotein lipids (including total cholesterol, VLDL-cholesterol, and LDL-cholesterol), apolipoproteins (including apolipoprotein B), and fatty acids (including omega-3 and DHA) in young adults. These results were largely consistent in direction (though smaller in magnitude and weaker in strength of evidence) in a two-sample MR analysis in an independent cohort of middle-aged adults. Results were attenuated, but consistent in direction, when potentially pleiotropic SNPs in the FADS gene regions were excluded. However, it should be noted that use of a narrow window for exclusion based on being within one of the three FADS genes may mean that some pleiotropic SNPs remain. Our subsequent forward MR analysis highlighted polyunsaturated fatty acids as potentially having a causal role in the development of CRC.

Our analyses highlight a potentially important role of polyunsaturated fatty acids in colorectal cancer liability. However, these analyses may be biased by substantial genetic pleiotropy among fatty acid traits. SNPs which are associated with levels of one fatty acid are generally associated with levels of many more fatty acid (and non-fatty acid) traits.^{70, 71} For instance, genetic instruments within the FADS cluster of genes will likely affect both omega-3 and omega-6 fatty acids, given FADS1 and FADS2 encode enzymes which catalyse the conversion of both from shorter chain into longer chain fatty acids.⁷¹ In addition, the NMR metabolomics platform utilised in the analyses outlined here has limited coverage of fatty acids, meaning many putative causal metabolites for CRC, for example arachidonic acid, could not be investigated. Therefore, although our results indicate that polyunsaturated fatty acids may be important in colorectal cancer risk, given the pleiotropic nature of the fatty acid genetic instruments and the limited coverage of the NMR platform, we are unable to determine with any certainty which specific classes of fatty acids may be driving these associations.

Our analyses featured evaluating the effect of genetic liability to CRC on circulating metabolites across repeated measures in the ALSPAC cohort. The mean ages at the time of the repeated measures were 8y, 16y, 18y, and 25y, representing childhood, early adolescence, late adolescence, and young adulthood respectively, and therefore individuals in this cohort are unlikely to be taking metabolite-altering medication such as statins, and unlikely to have CRC. The strongest evidence for an effect of liability to CRC on metabolite levels was seen in late adolescence. The reason for this remains unclear. It is possible that this represents a true biological phenomenon if late adolescence is a critical window in CRC development or metabolite variability, which may be likely given the limited variance in metabolite levels at the later age of 25y (ST2). The lack of an effect at the younger ages could be explained by the fact that the CRC GRS may capture many key life events or experiences which could impact the metabolome (e.g., initiation of smoking, higher category of BMI reached, educational attainment level set, etc) but may not have yet happened at younger ages, thus obscuring an effect of genetic liability to CRC on the metabolome. Our results suggest that puberty could be important, with an effect seen seemingly particularly at the end of puberty. Repeating our analysis with sex-stratified data may aid in determining whether this is likely to be the case; sex-stratified GWAS for metabolites are not currently available to replicate such analyses. An alternative explanation is selection bias due to loss of follow-up, leading to a change in sample characteristics over time.

Another key finding in the reverse MR analysis was that genetic liability to CRC was associated with increased levels of total cholesterol, VLDL-cholesterol, LDL-cholesterol, and apolipoprotein B, though we find little evidence for a causal effect of these traits on risk of CRC in the forward MR, replicating previous forward MR analyses for total and LDL-cholesterol.^{9, 72–74} This suggests that these traits may either be only predictive of (i.e., non-causal for) later CRC development, or may be influenced by the development of CRC and could have diagnostic or predictive potential. Given that the participants in the ALSPAC cohort are many decades younger than the average age of diagnosis for CRC (mean age 25 years in the latest repeated measure analysed in ALSPAC; whereas the median age at diagnosis of CRC is 64 years),⁷⁵ the former seems the most likely scenario. Previous conventional observational studies have presented conflicting results when investigating the association between measures of cholesterol and CRC risk with some finding an inverse association and others a positive association, possibly reflecting residual confounding in conventional observational analyses.^76–82 Previous MR studies have had similar findings to our forward MR analysis, in that there seems to be little evidence for a causal effect of cholesterol on CRC development.^72–74 One possible explanation for how circulating levels of total cholesterol, VLDL-cholesterol, LDL-cholesterol and apolipoprotein B could predict (without necessarily causing) future CRC development could be linked to diet. A previous MR analysis suggested an effect of increased BMI on several measures of circulating cholesterol.⁹ Consuming a diet which is high in fat may increase CRC risk both through and possibly independently of adiposity, alongside increasing levels of circulating cholesterol.^83–88 The potential for lipoprotein or apolipoprotein lipid measures in future CRC risk prediction should be further investigated.

Our analyses stratified by anatomical subsite highlighted fatty acids as being affected by genetic liability to colon and proximal colon cancer, with the forward MR confirming that fatty acid traits may be particularly important in the development of these subsites of CRC as well as distal colon cancer.

In our forward MR analyses we were unable to replicate the findings of three previous MR studies which found evidence for a causal effect of circulating linoleic acid levels on CRC development in terms of strength of evidence, though the direction of the effect estimate was similar to previous studies.^89–91 This is surprising as all three previous analyses had a much smaller sample size than that included in our analysis (the largest had sample size of 24,748 for exposure vs 118,466 presently; and 11,016 cases and 13,732 controls for outcome vs 52,775 cases and 45,940 controls presently). Our analysis using updated genetic instruments to proxy fatty acids may be more successful in accurately instrumenting heterogenous phenotypes such as metabolite levels compared with previous analyses. All other findings in our forward MR analysis are consistent with previous MR studies where they exist.^72–74

Limitations

The limitations of this study include firstly the relatively small sample size included in the ALSPAC analysis, which may have implications for power and precision. Secondly, mostly due to the longitudinal nature of the ASLAPC study, our sample at each time point is composed of slightly different individuals. This could be influencing our results, and should be taken into account when comparing across time points. Thirdly, our analyses involving genetic instruments for CRC liability may have suffered from horizontal pleiotropy, even after excluding genetic variants in or near the FADS gene. Fourthly, our analyses were mostly restricted to white Europeans, which limits the generalisability of our findings to other populations. Fifthly, our analysis would benefit from being repeated with sex-stratified data. Although such GWAS results for metabolites are not currently available, the data to perform such GWAS are available in UK Biobank for future analyses. Sixthly, for our forward MR analysis, we used the UK Biobank for our exposure data. The UK Biobank has a median age of 58 at the time these measurements were taken, meaning statin use may be widespread in this population, which could be attenuating our effect estimates. Future work could attempt to replicate our analysis in a population with lower prevalence of statins intake. Finally, we included only metabolites measured using NMR. Confirming whether our results replicate using metabolite data measured with an alternative method would strengthen our findings.

Conclusions

Our analysis provides evidence that genetic liability to CRC is associated with altered levels of metabolites at certain ages, some of which may have a causal role in CRC development. Further investigating the role of polyunsaturated fatty acids in CRC risk and circulating cholesterol in CRC prediction may be promising avenues for future research.

Acknowledgements

ALSPAC: We are extremely grateful to all the families who took part in this study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses.

EPICOLON: We are sincerely grateful to all patients participating in this study who were recruited as part of the EPICOLON project. We acknowledge the Spanish National DNA Bank, Biobank of Hospital Clínic–IDIBAPS and Biobanco Vasco for the availability of the samples. The work was carried out (in part) at the Esther Koplowitz Centre, Barcelona.

Data availability

Individual-level ALSPAC data are available following an application. This process of managed access is detailed at www.bristol.ac.uk/alspac/researchers/access. Cohort details and data descriptions for ALSPAC are publicly available at the same web address. Summary-level GWAS data used in this study from UK Biobank are publicly available without the need for application through the MR-Base platform, which is accessible at http://www.mrbase.org/. The summary-level GWAS data for CRC used in this study are available following an application to GECCO (managed access).

Funding

Author funding

JAB is supported by the Elizabeth Blackwell Institute for Health Research, University of Bristol and the Wellcome Trust Institutional Strategic Support Fund (204813/Z/16/Z) and works in a Unit funded by the Medical Research Council (MC_UU_00011/1) and the University of Bristol. EEV, DNL and CB are supported by Diabetes UK (17/0005587). NJT is a Wellcome Trust Investigator (202802/Z/16/Z), is the PI of the Avon Longitudinal Study of Parents and Children (MRC & WT 102215/2/13/2), is supported by the University of Bristol NIHR Biomedical Research Centre (BRC-1215-20011), the MRC Integrative Epidemiology Unit (MC_UU_00011/1) and works within the CRUK Integrative Cancer Epidemiology Programme (C18281/A19169). EH is supported by a Cancer Research UK Population Research Committee Studentship (C18281/A30905), is supported by the CRUK Integrative Cancer Epidemiology Programme (C18281/A29019) and is part of the Medical Research Council Integrative Epidemiology Unit at the University of Bristol which is supported by the Medical Research Council (MC_UU_00011/4) and the University of Bristol. AC acknowledges funding from grant MR/N0137941/1 for the GW4 BIOMED MRC DTP, awarded to the Universities of Bath, Bristol, Cardiff and Exeter from the Medical Research Council (MRC)/UKRI. MCB was supported by the UK Medical Research Council (MRC) Skills Development Fellowship (MR/P014054/1), University of Bristol Vice-Chancellor’s Fellowship and MRC Integrative Epidemiology Unit (MC_UU_00011/6). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work used the computational facilities of the Advanced Computing Research Centre, University of Bristol - http://www.bristol.ac.uk/acrc/.

Study funding

ALSPAC: The UK Medical Research Council and Wellcome (Grant ref: 217065/Z/19/Z) and the University of Bristol provide core support for ALSPAC. This publication is the work of the authors who will serve as guarantors for the contents of this paper. A comprehensive list of grants funding is available on the ALSPAC website (http://www.bristol.ac.uk/alspac/external/documents/grant-acknowledgements.pdf); This research was specifically funded by The UK Medical Research Council (Grant ref: MC_UU_12013/1). GWAS data was generated by Sample Logistics and Genotyping Facilities at Wellcome Sanger Institute and LabCorp (Laboratory Corporation of America) using support from 23andMe.

Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO): National Cancer Institute, National Institutes of Health, U.S. Department of Health and Human Services (U01 CA137088, R01 CA059045, R01 201407). Genotyping/Sequencing services were provided by the Center for Inherited Disease Research (CIDR) contract number HHSN268201700006I and HHSN268201200008I . This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA015704. Scientific Computing Infrastructure at Fred Hutch funded by ORIP grant S10OD028685.

ASTERISK: a Hospital Clinical Research Program (PHRC-BRD09/C) from the University Hospital Center of Nantes (CHU de Nantes) and supported by the Regional Council of Pays de la Loire, the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (GEFLUC), the Association Anne de Bretagne Génétique and the Ligue Régionale Contre le Cancer (LRCC).

The ATBC Study is supported by the Intramural Research Program of the U.S. National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

CLUE II funding was from the National Cancer Institute (U01 CA086308, Early Detection Research Network; P30 CA006973), National Institute on Aging (U01 AG018033), and the American Institute for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

Maryland Cancer Registry (MCR): Cancer data was provided by the Maryland Cancer Registry, Center for Cancer Prevention and Control, Maryland Department of Health, with funding from the State of Maryland and the Maryland Cigarette Restitution Fund. The collection and availability of cancer registry data is also supported by the Cooperative Agreement NU58DP006333, funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services.

ColoCare: This work was supported by the National Institutes of Health (grant numbers R01 CA189184 (Li/Ulrich), U01 CA206110 (Ulrich/Li/Siegel/Figueiredo/Colditz, 2P30CA015704-40 (Gilliland), R01 CA207371 (Ulrich/Li)), the Matthias Lackas-Foundation, the German Consortium for Translational Cancer Research, and the EU TRANSCAN initiative.

The Colon Cancer Family Registry (CCFR, www.coloncfr.org) is supported in part by funding from the National Cancer Institute (NCI), National Institutes of Health (NIH) (award U01 CA167551). Support for case ascertainment was provided in part from the Surveillance, Epidemiology, and End Results (SEER) Program and the following U.S. state cancer registries: AZ, CO, MN, NC, NH; and by the Victoria Cancer Registry (Australia) and Ontario Cancer Registry (Canada). The CCFR Set-1 (Illumina 1M/1M-Duo) and Set-2 (Illumina Omni1-Quad) scans were supported by NIH awards U01 CA122839 and R01 CA143237 (to GC). The CCFR Set-3 (Affymetrix Axiom CORECT Set array) was supported by NIH award U19 CA148107 and R01 CA81488 (to SBG). The CCFR Set-4 (Illumina OncoArray 600K SNP array) was supported by NIH award U19 CA148107 (to SBG) and by the Center for Inherited Disease Research (CIDR), which is funded by the NIH to the Johns Hopkins University, contract number HHSN268201200008I. Additional funding for the OFCCR/ARCTIC was through award GL201-043 from the Ontario Research Fund (to BWZ), award 112746 from the Canadian Institutes of Health Research (to TJH), through a Cancer Risk Evaluation (CaRE) Program grant from the Canadian Cancer Society (to SG), and through generous support from the Ontario Ministry of Research and Innovation. The SFCCR Illumina HumanCytoSNP array was supported in part through NCI/NIH awards U01/U24 CA074794 and R01 CA076366 (to PAN). The content of this manuscript does not necessarily reflect the views or policies of the NCI, NIH or any of the collaborating centers in the Colon Cancer Family Registry (CCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government, any cancer registry, or the CCFR.

COLON: The COLON study is sponsored by Wereld Kanker Onderzoek Fonds, including funds from grant 2014/1179 as part of the World Cancer Research Fund International Regular Grant Programme, by Alpe d’Huzes and the Dutch Cancer Society (UM 2012–5653, UW 2013-5927, UW2015-7946), and by TRANSCAN (JTC2012-MetaboCCC, JTC2013-FOCUS). The Nqplus study is sponsored by a ZonMW investment grant (98-10030); by PREVIEW, the project PREVention of diabetes through lifestyle intervention and population studies in Europe and around the World (PREVIEW) project which received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant no. 312057; by funds from TI Food and Nutrition (cardiovascular health theme), a public–private partnership on precompetitive research in food and nutrition; and by FOODBALL, the Food Biomarker Alliance, a project from JPI Healthy Diet for a Healthy Life.

COLO2&3: National Institutes of Health (R01 CA060987)

Colorectal Cancer Transdisciplinary (CORECT) Study: The CORECT Study was supported by the National Cancer Institute, National Institutes of Health (NCI/NIH), U.S. Department of Health and Human Services (grant numbers U19 CA148107, R01 CA081488, P30 CA014089, R01 CA197350; P01 CA196569; R01 CA201407; R01 CA242218), National Institutes of Environmental Health Sciences, National Institutes of Health (grant number T32 ES013678) and a generous gift from Daniel and Maryann Fong.

CORSA: The CORSA study was funded by Austrian Research Funding Agency (FFG) BRIDGE (grant 829675, to Andrea Gsur), the “Herzfelder’sche Familienstiftung” (grant to Andrea Gsur) and was supported by COST Action BM1206.

CPS-II: The American Cancer Society funds the creation, maintenance, and updating of the Cancer Prevention Study-II (CPS-II) cohort. The study protocol was approved by the institutional review boards of Emory University, and those of participating registries as required.

CRCGEN: Colorectal Cancer Genetics & Genomics, Spanish study was supported by Instituto de Salud Carlos III, co-funded by FEDER funds –a way to build Europe– (grants PI14-613 and PI09-1286), Agency for Management of University and Research Grants (AGAUR) of the Catalan Government (grant 2017SGR723), Junta de Castilla y León (grant LE22A10-2), the Spanish Association Against Cancer (AECC) Scientific Foundation grant GCTRA18022MORE and the Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), action Genrisk. Sample collection of this work was supported by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncología de Catalunya (XBTC), Plataforma Biobancos PT13/0010/0013 and ICOBIOBANC, sponsored by the Catalan Institute of Oncology. We thank CERCA Programme, Generalitat de Catalunya for institutional support.

Czech Republic CCS: This work was supported by the Grant Agency of the Czech Republic (21-04607X, 20-03997S), by the Grant Agency of the Ministry of Health of the Czech Republic (grants AZV NU21-07-00247 and AZV NU21-03-00506), and Charles University Research Fund (Cooperation 43-Surgical disciplines)

DACHS: This work was supported by the German Research Council (BR 1704/6-1, BR 1704/6-3, BR 1704/6-4, CH 117/1-1, HO 5117/2-1, HE 5998/2-1, KL 2354/3-1, RO 2270/8-1 and BR 1704/17-1), the Interdisciplinary Research Program of the National Center for Tumor Diseases (NCT), Germany, and the German Federal Ministry of Education and Research (01KH0404, 01ER0814, 01ER0815, 01ER1505A and 01ER1505B).

DALS: National Institutes of Health (R01 CA048998 to M. L. Slattery).

EDRN: This work is funded and supported by the NCI, EDRN Grant (U01-CA152753).

EPIC: The coordination of EPIC is financially supported by International Agency for Research on Cancer (IARC) and also by the Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London which has additional infrastructure support provided by the NIHR Imperial Biomedical Research Centre (BRC). The national cohorts are supported by: Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Générale de l’Education Nationale, Institut National de la Santé et de la Recherche Médicale (INSERM) (France); German Cancer Aid, German Cancer Research Center (DKFZ), German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Federal Ministry of Education and Research (BMBF) (Germany); Associazione Italiana per la Ricerca sul Cancro-AIRC-Italy, Compagnia di SanPaolo and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (The Netherlands); Health Research Fund (FIS) - Instituto de Salud Carlos III (ISCIII), Regional Governments of Andalucía, Asturias, Basque Country, Murcia and Navarra, and the Catalan Institute of Oncology - ICO (Spain); Swedish Cancer Society, Swedish Research Council and and Region Skåne and Region Västerbotten (Sweden); Cancer Research UK (14136 to EPIC-Norfolk; C8221/A29017 to EPIC-Oxford), Medical Research Council (1000143 to EPIC-Norfolk; MR/M012190/1 to EPIC-Oxford). (United Kingdom).

EPICOLON: This work was supported by grants from Fondo de Investigación Sanitaria/FEDER (PI08/0024, PI08/1276, PS09/02368, PI11/00219, PI11/00681, PI14/00173, PI14/00230, PI17/00509, 17/00878, PI20/00113, PI20/00226, Acción Transversal de Cáncer), Xunta de Galicia (PGIDIT07PXIB9101209PR), Ministerio de Economia y Competitividad (SAF07-64873, SAF 2010-19273, SAF2014-54453R), Fundación Científica de la Asociación Española contra el Cáncer (GCB13131592CAST, PRYGN211085CAST), Beca Grupo de Trabajo “Oncología” AEG (Asociación Española de Gastroenterología), Fundación Privada Olga Torres, FP7 CHIBCHA Consortium, Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, Generalitat de Catalunya, 2014SGR135, 2014SGR255, 2017SGR21, 2017SGR653, 2021SGR00716, 2021SGR01185), Catalan Tumour Bank Network (Pla Director d’Oncologia, Generalitat de Catalunya), PERIS (SLT002/16/00398, Generalitat de Catalunya), Marató TV3 (202008-10), CERCA Programme (Generalitat de Catalunya) and COST Actions BM1206 and CA17118. CIBERehd is funded by the Instituto de Salud Carlos III.ESTHER/VERDI. This work was supported by grants from the Baden-Württemberg Ministry of Science, Research and Arts and the German Cancer Aid.

Harvard cohorts: HPFS is supported by the National Institutes of Health (P01 CA055075, UM1 CA167552, U01 CA167552, R01 CA137178, R01 CA151993, and R35 CA197735), NHS by the National Institutes of Health (P01 CA087969, UM1 CA186107, R01 CA137178, R01 CA151993, and R35 CA197735), and PHS by the National Institutes of Health (R01 CA042182).

Hawaii Adenoma Study: NCI grants R01 CA072520.

HCES-CRC: the Hwasun Cancer Epidemiology Study–Colon and Rectum Cancer (HCES-CRC; grants from Chonnam National University Hwasun Hospital, HCRI15011-1).

Kentucky: This work was supported by the following grant support: Clinical Investigator Award from Damon Runyon Cancer Research Foundation (CI-8); NCI R01CA136726.

LCCS: The Leeds Colorectal Cancer Study was funded by the Food Standards Agency and Cancer Research UK Programme Award (C588/A19167).

MCCS cohort recruitment was funded by VicHealth and Cancer Council Victoria. The MCCS was further supported by Australian NHMRC grants 509348, 209057, 251553 and 504711 and by infrastructure provided by Cancer Council Victoria. Cases and their vital status were ascertained through the Victorian Cancer Registry (VCR) and the Australian Institute of Health and Welfare (AIHW), including the National Death Index and the Australian Cancer Database. BMLynch was supported by MCRF18005 from the Victorian Cancer Agency.

MEC: National Institutes of Health (R37 CA054281, P01 CA033619, and R01 CA063464).

MECC: This work was supported by the National Institutes of Health, U.S. Department of Health and Human Services (R01 CA081488, R01 CA197350, U19 CA148107, R01 CA242218, and a generous gift from Daniel and Maryann Fong.

MSKCC: The work at Sloan Kettering in New York was supported by the Robert and Kate Niehaus Center for Inherited Cancer Genomics and the Romeo Milio Foundation. Moffitt: This work was supported by funding from the National Institutes of Health (grant numbers R01 CA189184, P30 CA076292), Florida Department of Health Bankhead-Coley Grant 09BN-13, and the University of South Florida Oehler Foundation. Moffitt contributions were supported in part by the Total Cancer Care Initiative, Collaborative Data Services Core, and Tissue Core at the H. Lee Moffitt Cancer Center & Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (grant number P30 CA076292).

NCCCS I & II: We acknowledge funding support for this project from the National Institutes of Health, R01 CA066635 and P30 DK034987.

NFCCR: This work was supported by an Interdisciplinary Health Research Team award from the Canadian Institutes of Health Research (CRT 43821); the National Institutes of Health, U.S. Department of Health and Human Serivces (U01 CA074783); and National Cancer Institute of Canada grants (18223 and 18226). The authors wish to acknowledge the contribution of Alexandre Belisle and the genotyping team of the McGill University and Génome Québec Innovation Centre, Montréal, Canada, for genotyping the Sequenom panel in the NFCCR samples. Funding was provided to Michael O. Woods by the Canadian Cancer Society Research Institute.

NSHDS: The research was supported by Biobank Sweden through funding from the Swedish Research Council (VR 2017-00650, VR 2017-01737), the Swedish Cancer Society (CAN 2017/581), Region Västerbotten (VLL-841671, VLL-833291), Knut and Alice Wallenberg Foundation (VLL-765961), and the Lion’s Cancer Research Foundation (several grants) and Insamlingsstiftelsen, both at Umeå University.

OSUMC: OCCPI funding was provided by Pelotonia and HNPCC funding was provided by the NCI (CA016058 and CA067941).

PLCO: Intramural Research Program of the Division of Cancer Epidemiology and Genetics and supported by contracts from the Division of Cancer Prevention, National Cancer Institute, NIH, DHHS. Funding was provided by National Institutes of Health (NIH), Genes, Environment and Health Initiative (GEI) Z01 CP 010200, NIH U01 HG004446, and NIH GEI U01 HG 004438.

SEARCH: The University of Cambridge has received salary support in respect of PDPP from the NHS in the East of England through the Clinical Academic Reserve. Cancer Research UK (C490/A16561); the UK National Institute for Health Research Biomedical Research Centres at the University of Cambridge.

SELECT: Research reported in this publication was supported in part by the National Cancer Institute of the National Institutes of Health under Award Numbers U10 CA037429 (CD Blanke), and UM1 CA182883 (CM Tangen/IM Thompson). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

SMS and REACHS: This work was supported by the National Cancer Institute (grant P01 CA074184 to J.D.P. and P.A.N., grants R01 CA097325, R03 CA153323, and K05 CA152715 to P.A.N., and the National Center for Advancing Translational Sciences at the National Institutes of Health (grant KL2 TR000421 to A.N.B.-H.)

The Swedish Low-risk Colorectal Cancer Study: The study was supported by grants from the Swedish research council; K2015-55X-22674-01-4, K2008-55X-20157-03-3, K2006-72X-20157-01-2 and the Stockholm County Council (ALF project).

Swedish Mammography Cohort and Cohort of Swedish Men: This work is supported by the Swedish Research Council /Infrastructure grant, the Swedish Cancer Foundation, and the Karolinska Institutés Distinguished Professor Award to Alicja Wolk.

UK Biobank: This research has been conducted using the UK Biobank Resource under Application Number 8614

VITAL: National Institutes of Health (K05 CA154337).

The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, U.S. Department of Health and Human Services through contracts 75N92021D00001, 75N92021D00002, 75N92021D00003, 75N92021D00004, 75N92021D00005

Author contributions

All authors contributed to the planning of the study. CJB and EH had access to data and conducted analyses. CJB and EH wrote the first draft. All authors critically reviewed the intellectual content and edited manuscript drafts and approved the final version for submission.