Amino acids and risk of colon adenocarcinoma: a Mendelian randomization study

Initially, we employed a two sample Mendelian randomization approach to investigate the correlation between 20 amino acids and the risk of colon adenocarcinoma. Our findings revealed that alanine exhibited a significant association with the risk of colon adenocarcinoma. Subsequently, we conducted a reanalysis of the relationship between alanine and the risk of colon adenocarcinoma by utilizing three sets of alanine data while imposing the condition of linkage disequilibrium (LD). In order to examine the potential for reverse causality and mediating effects, we conducted a reverse analysis of the impact of colonic adenocarcinoma on alanine. Additionally, we investigated the possibility of ALT and gluconic serving as mediating factors in influencing the outcome. Finally, we utilized hospital data to observe the levels of alanine in the blood of patients diagnosed with colon adenocarcinoma and to explore the correlation between alanine levels and the stage and prognosis of the disease. A schematic of the study design is shown in Fig. 1.

The genome-wide association study (GWAS) datasets for 20 amino acids, colon adenocarcinoma, Alanine aminotransferase, and Fasting blood glucose were obtained from the IEU GWAS database (https://​gwas.​mrcieu.​ac.​uk/​), which were limited to the European population. Blood amino acid data, along with relevant clinical pathological and prognostic information, were collected from 83 healthy individuals and 236 patients with colon adenocarcinoma at the First Affiliated Hospital of Jinzhou Medical University. After a minimum of 12 h of fasting, blood samples were collected from all participants. Our previously published article provides a comprehensive method for detecting amino acids [7]. Blood amino acid data was collected from healthy individuals aged 50–80 years in addition to patients diagnosed with colon adenocarcinoma within the same age range. The gender composition of both groups was found to be similar. This study was approved by the institutional ethics committee of The First Affiliated Hospital of Jinzhou Medical University (No.202347).

The identification code for colon adenocarcinoma is finn-b-c3_COLON_ADENO, while the identification code for Alanine aminotransferase is bbj-a-6, and for Fasting blood glucose is ebi-a-GCST90002232. Table 1 presents comprehensive information on all amino acids.

In the present MR study, SNPs that exhibited a significant association with blood 20 amino acid at the genome-wide level of significance (P value < 5 × 10 – 8) and demonstrated no linkage disequilibrium (LD) with other SNPs (r2 < 0.001 within a clumping window of 10,000 kb) were employed as instruments for these blood amino acid. However, due to insufficient SNP availability for certain amino acids under the aforementioned conditions, the p value for arginine, proline, glutamic acid, and lysine was adjusted to 5e-7, while the p value for cysteine, threonine, and aspartic acid was set to 5e-6.

Upon conducting a screening, it was discovered that alanine exhibited an association with the risk of colon adenocarcinoma. To ensure accuracy, we utilized three sets of alanine exposure data from the IEU GWAS database and identified SNPs that demonstrated a significant association with blood alanine at the genome-wide level (P value < 5 × 10 – 8) and were not in linkage disequilibrium (LD) with other SNPs (r2 < 0.0001 within a clumping window of 10,000 kb) as instrumental variables for alanine. Due to a limited number of SNPs available for instrumental variable analysis in met-a-469, the p-value was adjusted to 5e-7.

Furthermore, a reverse Mendelian randomization analysis was conducted utilizing colon adenocarcinoma as the exposure and alanine as the outcome. SNPs as an instrumental variable associated with colon adenocarcinoma were established at the genome-wide significance level (P value < 5 × 10 – 6) and were required to exhibit no linkage disequilibrium (LD) with other SNPs (r2 < 0.01 within a clumping window of 10,000 kb).

The Inverse Variance Weighted (IVW) method was utilized as the primary approach for our MR analysis to investigate the causal impact of 20 blood amino acid levels on the risk of colon adenocarcinoma. Additionally, we employed other methods such as Weighted Median and Weighted Mode. Subsequently, a series of sensitivity analyses were conducted, including Heterogeneity (Cochran's Q statistics), Horizontal Pleiotropy (MR-Egger intercept analysis), and Leave-One-Out analysis. We also performed an MR-PRESSO analysis and utilized the MR-PRESSO outlier test method to remove outlier SNPs.

The present study conducted MR analysis, difference analysis of alanine level in blood among normal individuals and those diagnosed with colon adenocarcinoma, difference analysis of alanine level across various stages, Kaplan–Meier method was used to draw the survival curve, and the Log-rank test was used for comparison, with P < 0.05 as statistically significant. and univariate Cox analysis to examine the relationship between age, gender, stage, differentiation, and prognosis. All of the aforementioned analyses were deemed statistically significant at a p value of less than 0.05. Kaplan–Meier method was used to draw the survival curve, and the Log-rank test was used for comparison, with P < 0.05 as statistically significant.

Utilizing the IVW method, an MR analysis was conducted to investigate the correlation between 20 blood amino acids and the risk of colon adenocarcinoma. The results revealed a causal association between alanine and colonic adenocarcinoma, with a significance level of p < 0.05. Conversely, no significant relationship was observed between other amino acids and the risk of colon adenocarcinoma, with a p value exceeding 0.05. The detailed results are in Table 2.

To ascertain the precise association between alanine and the risk of colon adenocarcinoma, we employed three MR techniques to scrutinize the correlation between three distinct alanine exposure datasets and the risk of colon adenocarcinoma. Our analysis of alanine with an ID of met-d-Ala and the risk of colon adenocarcinoma revealed that the IVW method yielded a p value of less than 0.05, the Weighted median method yielded a p value close to 0.05, and the OR value was less than 1 (Fig. 2A). Further examination of the alanine exposure data from two additional groups, identified as met-c-840 and met- a-469, demonstrated that both the IVW and Weighted median methods produced p values below 0.05, and the OR value remained less than 1 (Fig. 2A). The examination of three sets of alanine exposure data revealed a negative correlation between alanine and the likelihood of colon adenocarcinoma (Fig. 2B-D). Additionally, the leave-one-out analysis indicated that a single SNP did not impact the results (Fig. 2E-G). Other sensitivity analyses also revealed that the MR-Egger Intercept p value and the Cochran Q test p value were both greater than 0.05. Detailed results can be found in supporting materials met-d-Ala, met-c-840, and met-a-469.

To investigate the potential reverse causal association between alanine and colon adenocarcinoma, a Mendelian randomization (MR) analysis was conducted with colon adenocarcinoma as the exposure variable and alanine as the outcome variable. The results of the analysis revealed a nonsignificant relationship between the two variables, with a p value greater than 0.05 (Fig. 3A). There is no obvious correlation between them (Fig. 3B). The Leave-one-out analysis also demonstrated that the relationship was not sensitive (Fig. 3C).

No significant causal relationship was observed between alanine and ALT, Fasting blood glucose (FBG), as well as between ALT, Fasting blood glucose, and colon adenocarcinoma, as indicated by P values greater than 0.05 (Table 3).

Through our data analysis, it was determined that the blood alanine levels of individuals diagnosed with colon adenocarcinoma were significantly lower in comparison to those of healthy individuals (Fig. 4A). This difference was found to be statistically significant (Fig. 4A). Furthermore, a significant decrease in alanine content was observed in patients with stage IV colon adenocarcinoma in comparison to those without metastasis (Fig. 4B). This difference was also found to be statistically significant (Fig. 4B). In addition, a comparison was conducted between the levels of alanine in patients diagnosed with left hemicolon and right hemicolon adenocarcinomas. The findings revealed that the levels of alanine in right hemicolon adenocarcinomas were comparatively lower than those observed in left hemicolon adenocarcinomas. However, it is worth noting that this disparity did not reach statistical significance (P = 0.059) (Fig. 4C). Prognostic analysis revealed that patients with elevated alanine levels exhibited a favorable prognosis (Fig. 4D). Additionally, univariate COX analysis indicated that alanine served as a positive prognostic factor for colon adenocarcinoma (Fig. 4E).