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Methods and Applications: Genetic Insights into Glycine’s Protective Role Against CAD — European and East Asia, 2015 and 2020

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  • Abstract

    Introduction

    The purpose of this study is to examine the potential causal relationship between levels of circulating glycine and coronary artery disease (CAD) using a two-step Mendelian randomization (MR) analysis.

    Methods

    We analyzed data from genome-wide association studies (GWAS) conducted on European and East Asian populations. To assess the causal effects of circulating glycine levels on the risk of CAD. We used the inverse-variance weighting (IVW), weighted median (WM), MR-Egger, and Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) methods. Furthermore, we conducted mediation analysis to investigate the contribution of blood pressure and other cardiovascular disease-related traits.

    Results

    The two-step Mendelian randomization analysis revealed that higher levels of glycine in the blood were associated with a reduced risk of CAD in Europeans [odds ratio (OR)=0.84, 95% confidence interval (CI): 0.72, −0.98; P=0.029] and East Asians: (OR=0.76, 95% CI: 0.66, −0.89; P=3.57×10−4). Sensitivity analysis confirmed the robustness of these findings. Additionally, our results suggest that about 6.06% of the observed causal effect is mediated through genetically predicted systolic blood pressure (SBP) in the European population.

    Discussion

    Our results contribute to the current knowledge regarding the involvement of glycine in the progression of CAD, and provide valuable methodological insights for the prevention and treatment of this condition.

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  • Funding: Supported by the National Natural Science Foundation of China (82273612), and by Open Project of Key Laboratory of Science and Engineering for the Multi-Modal Prevention and Control of Major Chronic Diseases, Ministry of Industry and Information Technology (Grant No. MCD-2023-1-09)
  • [1] GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;385(9963):117 − 71. https://doi.org/10.1016/S0140-6736(14)61682-2CrossRef
    [2] Ballevre O, Cadenhead A, Calder AG, Rees WD, Lobley GE, Fuller MF, et al. Quantitative partition of threonine oxidation in pigs: effect of dietary threonine. Am J Physiol 1990;259(4 Pt 1):E483-91. http://dx.doi.org/10.1152/ajpendo.1990.259.4.E483.
    [3] Razak MA, Begum PS, Viswanath B, Rajagopal S. Multifarious beneficial effect of nonessential amino acid, glycine: a review. Oxid Med Cell Longev 2017;2017:1716701. https://doi.org/10.1155/2017/1716701CrossRef
    [4] Wittemans LBL, Lotta LA, Oliver-Williams C, Stewart ID, Surendran P, Karthikeyan S, et al. Assessing the causal association of glycine with risk of cardio-metabolic diseases. Nat Commun 2019;10(1):1060. https://doi.org/10.1038/s41467-019-08936-1CrossRef
    [5] Chang XL, Wang L, Guan SP, Kennedy BK, Liu JJ, Khor CC, et al. The association of genetically determined serum glycine with cardiovascular risk in East Asians. Nutr Metab Cardiovasc Dis 2021;31(6):1840 − 4. https://doi.org/10.1016/j.numecd.2021.03.010CrossRef
    [6] Jia Q, Han Y, Huang P, Woodward NC, Gukasyan J, Kettunen J, et al. Genetic determinants of circulating glycine levels and risk of coronary artery disease. J Am Heart Assoc 2019;8(10):e011922. https://doi.org/10.1161/JAHA.119.011922CrossRef
    [7] Burgess S, Davey Smith G, Davies NM, Dudbridge F, Gill D, Glymour MM, et al. Guidelines for performing Mendelian randomization investigations: update for summer 2023. Wellcome Open Res 2019;4:186. https://doi.org/10.12688/wellcomeopenres.15555.1CrossRef
    [8] Bowden J, Smith GD, Burgess S. Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. Int J Epidemiol 2015;44(2):512 − 25. https://doi.org/10.1093/ije/dyv080CrossRef
    [9] Bowden J, Davey Smith G, Haycock PC, Burgess S. Consistent estimation in mendelian randomization with some invalid instruments using a weighted median estimator. Genet Epidemiol 2016;40(4):304 − 14. https://doi.org/10.1002/gepi.21965CrossRef
    [10] Del Greco MF, Minelli C, Sheehan NA, Thompson JR. Detecting pleiotropy in Mendelian randomisation studies with summary data and a continuous outcome. Stat Med 2015;34(21):2926 − 40. https://doi.org/10.1002/sim.6522CrossRef
    [11] Stamler J, Brown IJ, Daviglus ML, Chan Q, Miura K, Okuda N, et al. Dietary glycine and blood pressure: the international study on macro/micronutrients and blood pressure. Am J Clin Nutr 2013;98(1):136 − 45. https://doi.org/10.3945/ajcn.112.043000CrossRef
    [12] Slomowitz LA, Gabbai FB, Khang SJ, Satriano J, Thareau S, Deng AH, et al. Protein intake regulates the vasodilatory function of the kidney and NMDA receptor expression. Am J Physiol Regul Integr Comp Physiol 2004;287(5):R1184 − 9. https://doi.org/10.1152/ajpregu.00169.2003CrossRef
    [13] Ding YP, Svingen GFT, Pedersen ER, Gregory JF, Ueland PM, Tell GS, et al. Plasma glycine and risk of acute myocardial infarction in patients with suspected stable angina pectoris. J Am Heart Assoc 2015;5(1):e002621. https://doi.org/10.1161/JAHA.115.002621CrossRef
  • FIGURE 1.  Schematic diagram of a two-step Mendelian randomization.

    FIGURE 2.  Forest plots showing effect sizes ± 95% confidence intervals for the association between genetically predicted circulating glycine levels and CAD. (A) European; (B) East Asian.

    Note: Four sets of IVs were used to estimate the association between circulating glycine levels and CAD risk: 1) significant glycine-related SNPs (19 SNPs) identified in the GWAS of circulating glycine; 2) loci near genes encoding enzymes-related to glycine metabolism (GLDC, PHGDH, PSPH, ALDH1L1, and CPS1, 4 SNPs); 3) loci near genes encoding enzymes related to glycine metabolism except for the pleiotropic CPS1 locus (which showed significant associations with multiple metabolites, 3 SNPs); and 4) the loci at GCSH and GLDC encoding enzymes of the glycine cleavage system (1 SNP).

    Abbreviation: SNP=single nucleotide polymorphism; MR-PRESSO=Mendelian Randomization Pleiotropy RESidual Sum and Outlier; GWAS=genome-wideassociation studies; ALDH1L1=aldehyde dehydrogenase 1 family member L1; CAD=coronary artery disease; CPS1=carbamoyl-phosphate synthase; GLDC=glycine decarboxylase; GCSH=glycine cleavage system protein H; PHGDH=phosphoglycerate dehydrogenase; PSPH=phosphoserine phosphatase; SBP=systolic blood pressure.

    TABLE 1.  Association of genetically predicted SBP with CAD risk in the Mendelian randomization analysis.

    Method OR 95% CI P
    WM 1.03 1.02 to 1.03 2.33×10−19
    IVW 1.03 1.03 to 1.04 1.76×10−56
    MR-Egger 1.04 1.03 to 1.05 1.73×10−11
    MR-PRESSO 1.03 1.03 to 1.04 1.96×10−44
    Note: OR>1 indicates that increased SBP was associated with an increased risk of CAD; The Cochran’s Q=589.23 (P=2.17×10−8), and I2=30.24%, indicating that there was heterogeneity. The MR-PRESSO global test (P<1×10−4) and MR-Egger intercept test (P=0.481) indicated that there was horizontal pleiotropy for the selected instruments.
    Abbreviation: CAD=coronary artery disease; CI=confidence interval; MR-PRESSO=Mendelian Randomization Pleiotropy RESidual Sum and Outlier; IVW=inverse variance weighted; OR=odds ratio; SBP=systolic blood pressure; WM=weighted median.
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Genetic Insights into Glycine’s Protective Role Against CAD — European and East Asia, 2015 and 2020

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Abstract

Introduction

The purpose of this study is to examine the potential causal relationship between levels of circulating glycine and coronary artery disease (CAD) using a two-step Mendelian randomization (MR) analysis.

Methods

We analyzed data from genome-wide association studies (GWAS) conducted on European and East Asian populations. To assess the causal effects of circulating glycine levels on the risk of CAD. We used the inverse-variance weighting (IVW), weighted median (WM), MR-Egger, and Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) methods. Furthermore, we conducted mediation analysis to investigate the contribution of blood pressure and other cardiovascular disease-related traits.

Results

The two-step Mendelian randomization analysis revealed that higher levels of glycine in the blood were associated with a reduced risk of CAD in Europeans [odds ratio (OR)=0.84, 95% confidence interval (CI): 0.72, −0.98; P=0.029] and East Asians: (OR=0.76, 95% CI: 0.66, −0.89; P=3.57×10−4). Sensitivity analysis confirmed the robustness of these findings. Additionally, our results suggest that about 6.06% of the observed causal effect is mediated through genetically predicted systolic blood pressure (SBP) in the European population.

Discussion

Our results contribute to the current knowledge regarding the involvement of glycine in the progression of CAD, and provide valuable methodological insights for the prevention and treatment of this condition.

  • 1. Department of Nutrition and Food Hygiene, School of Public Health, Harbin Medical University, Harbin City, Heilongjiang Province, China
  • 2. Key Laboratory of Precision Nutrition and Health of Ministry of Education, School of Public Health, Harbin Medical University, Harbin City, Heilongjiang Province, China
  • 3. Songyang County Center for Disease Prevention and Control, Songyang City, Zhejiang Province China
  • 4. Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China
  • 5. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York City, NY, USA
  • Corresponding authors:

    Rennan Feng, fengrennan@yeah.net

    Xiang Shu, shux@mskcc.org

  • Funding: Supported by the National Natural Science Foundation of China (82273612), and by Open Project of Key Laboratory of Science and Engineering for the Multi-Modal Prevention and Control of Major Chronic Diseases, Ministry of Industry and Information Technology (Grant No. MCD-2023-1-09)
  • Online Date: March 01 2024
    Issue Date: March 01 2024
    doi: 10.46234/ccdcw2024.034
  • Coronary artery disease (CAD), also known as atherosclerosis or coronary heart disease, is the leading cause of global mortality (1). Glycine, a non-essential amino acid, plays a critical role in cell growth, immune function, antioxidant response, and anti-inflammatory processes (2). Previous studies have shown positive effects of glycine on cardiovascular health (3). The therapeutic potential of glycine for metabolic disorders and cardiovascular diseases has been proposed. However, studies investigating the association between circulating glycine levels and CAD risk have yielded inconsistent results (46). Therefore, the causal relationship between glycine and CAD remains controversial, and if such a relationship exists, it may be influenced by metabolic factors such as blood pressure.

    Mendelian randomization (MR) is a statistical technique that uses genetic variants as instrumental variables to assess the causal impact of an exposure on an outcome (7). MR leverages the fact that genetic variants are randomly assigned at conception, making them immune to confounding factors typically found in observational studies. In order to explore the causal relationship between circulating glycine levels and the risk of CAD, as well as to uncover the underlying mechanisms, we conducted a comprehensive study using a two-step MR approach. This study aimed to investigate the potential causal effects of circulating glycine on CAD risk in individuals of European ancestry and East Asians.

    • In the study, we analyzed the relationship between specific genetic instruments and glycine levels in the UK Biobank (UKB), which included 114,972 individuals of European descent (Nightingale Health Plc; Biomarker Quantification Version 2020) and the study conducted by Wittemans et al. (4), which included 30,118 individuals of European ancestry.

      All details regarding the GWAS summary-level data are presented in Supplementary Table S1. In order to address potential weak instrumental bias, instrumental variables (IVs) should significantly associate with the exposure (P<5×10-8) and exhibit minimal linkage- disequilibrium (LD) with other single nucleotide polymorphisms (SNPs) (R2<0.001) within a clump distance of 1,000 kb (Supplementary Table S2). By utilizing the PhenoScanner database, we identified and subsequently excluded pleiotropic SNPs that are correlated with confounding factors (Supplementary Table S2).

      Our study utilized a two-step MR analysis design (Figure 1). The primary analysis was conducted using the inverse variance-weighted (IVW) method. In instances where heterogeneity was detected, we employed the IVW method with random effects. To further explore the robustness of our findings, we conducted sensitivity analyses using alternative approaches, including MR-Egger regression, the weighted median method, and Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) analysis, accounting for multiple genetic variants (89). To assess pleiotropy, we utilized the MR-Egger intercept test and the MR-PRESSO global test. Additionally, we evaluated heterogeneity of the MR findings using Cochran’s Q-statistic and the I2 index (10). The MR analyses were conducted using the ‘dplyr’ and ‘TwoSampleMR’ packages in R (version 4.0.5, R foundation for statistical computing, Vienna, Austria) The threshold of statistical significance was P<0.05.

      Figure 1. 

      Schematic diagram of a two-step Mendelian randomization.

    • The 19 identified SNPs collectively accounted for approximately 6.5% of the variation in circulating glycine levels (R2=6.5%). Furthermore, the F-statistic, which exceeds 18.66, indicates a low probability of weak instrument bias occurring in this study.

      We utilized a panel of 18 SNPs, excluding 1 palindromic SNP with intermediate allele frequencies, to assess the correlation between genetically predicted higher circulating glycine levels and decreased risk of CAD in Europeans. The analysis revealed a significant correlation [odds ratio (OR)=0.84, 95% confidence interval (CI): (0.72, 0.98), P=0.029, PCochran’s Q=0.018, PMR-PRESSO global test=0.03, PMR-Egger intercept test=0.069]. However, when using instruments consisting of 4, 3, and 1 SNPs, no significant associations were observed, despite consistently indicating the same direction of association (Figure 2A).

      Figure 2. 

      Forest plots showing effect sizes ± 95% confidence intervals for the association between genetically predicted circulating glycine levels and CAD. (A) European; (B) East Asian.

      Note: Four sets of IVs were used to estimate the association between circulating glycine levels and CAD risk: 1) significant glycine-related SNPs (19 SNPs) identified in the GWAS of circulating glycine; 2) loci near genes encoding enzymes-related to glycine metabolism (GLDC, PHGDH, PSPH, ALDH1L1, and CPS1, 4 SNPs); 3) loci near genes encoding enzymes related to glycine metabolism except for the pleiotropic CPS1 locus (which showed significant associations with multiple metabolites, 3 SNPs); and 4) the loci at GCSH and GLDC encoding enzymes of the glycine cleavage system (1 SNP).

      Abbreviation: SNP=single nucleotide polymorphism; MR-PRESSO=Mendelian Randomization Pleiotropy RESidual Sum and Outlier; GWAS=genome-wideassociation studies; ALDH1L1=aldehyde dehydrogenase 1 family member L1; CAD=coronary artery disease; CPS1=carbamoyl-phosphate synthase; GLDC=glycine decarboxylase; GCSH=glycine cleavage system protein H; PHGDH=phosphoglycerate dehydrogenase; PSPH=phosphoserine phosphatase; SBP=systolic blood pressure.

      Out of the initial 19 SNPs, 14 were included in our East Asian-focused MR analysis using data from Biobank Japan. Five SNPs were excluded due to missing data or palindromic status. Our analysis showed a consistent protective relationship between genetically predicted glycine levels and the risk of CAD (OR=0.76, 95% CI: 0.66, 0.89; P=3.57×10-4, PCochran’s Q=0.186, PMR-PRESSO global test=0.199, PMR-Egger intercept test=0.038), However, other IVs sets did not show a significant association, likely due to limited statistical power (Figure 2B).

      We found a significant relation between higher genetically predicted circulating glycine levels and lower genetically predicted SBP (β=−0.74, 95% CI: −1.28, −0.20; P=0.007, P Cochran’s Q =0.196, PMR-PRESSO global test=0.287], PMR-Egger intercept test=0.759). Using the CPS1 and GLDC instruments, we observed consistent effects of glycine on SBP (β=−0.62, 95% CI: −1.19, −0.06; P=0.03, PCochran’s Q=0.551, PMR-Egger intercept test=0.786] (Supplementary Figure S1).

      Furthermore, our analysis revealed that there was a negative relationship between genetically predicted circulating glycine levels and genetically predicted DBP (β=−0.30, 95% CI: −0.56, −0.05; P=0.02, PCochran’s Q=0.356, PMR-PRESSO global test=0.455, PMR-Egger intercept test=0.527). This association remained consistent when using alternative IV sets (Supplementary Figure S1). However, we did not find any association between predicted glycine levels and anthropometric, glycemic, inflammatory, or blood lipid traits (Supplementary Figures S2S5).

      Out of the 461 SNPs related to SBP, we included 412 in our MR analysis. Four SNPs were excluded due to insufficient data, and 45 SNPs were removed after an outlier test, using MR-PRESSO. We detected significant heterogeneity (PCochran’s Q=2.17×10-8), and the presence of horizontal pleiotropy was confirmed by using the MR-PRESSO global test (P<1×10-4). Our results demonstrated a positive association between SBP and the risk of CAD, with each unit increase in SBP associated with a 3% increase in CAD risk (OR=1.03, 95% CI: 1.02, 1.03; PWM=2.33×10-19) (Table 1).

      Method OR 95% CI P
      WM 1.03 1.02 to 1.03 2.33×10−19
      IVW 1.03 1.03 to 1.04 1.76×10−56
      MR-Egger 1.04 1.03 to 1.05 1.73×10−11
      MR-PRESSO 1.03 1.03 to 1.04 1.96×10−44
      Note: OR>1 indicates that increased SBP was associated with an increased risk of CAD; The Cochran’s Q=589.23 (P=2.17×10−8), and I2=30.24%, indicating that there was heterogeneity. The MR-PRESSO global test (P<1×10−4) and MR-Egger intercept test (P=0.481) indicated that there was horizontal pleiotropy for the selected instruments.
      Abbreviation: CAD=coronary artery disease; CI=confidence interval; MR-PRESSO=Mendelian Randomization Pleiotropy RESidual Sum and Outlier; IVW=inverse variance weighted; OR=odds ratio; SBP=systolic blood pressure; WM=weighted median.

      Table 1.  Association of genetically predicted SBP with CAD risk in the Mendelian randomization analysis.

      The potential mediation of systolic blood pressure (SBP) on the association between circulating glycine and CAD risk was investigated. The mediation effect involving SBP was found that 6.06% of the effect of circulating glycine, which was genetically predicted using 19 SNPs, was mediated through the genetically predicted SBP (Supplementary Table S3).

    • The two-step MR analysis demonstrated a significant causal association between decreased levels of genetically predicted circulating glycine and CAD. Our estimation revealed that approximately 6.06% of this potential causal effect is mediated through genetically predicted SBP, suggesting that the protective influence of circulating glycine may be attributed to its effect on reducing SBP. However, our findings suggest that other risk factors associated with CAD, such as glycemic characteristics, lipid profiles, and inflammatory markers, may not play a considerable role as mediators in this relationship.

      Previous studies conducted on European white populations have produced inconsistent results regarding the association between glycine levels and the risk of developing CAD. Specifically, one study on European whites did not provide strong evidence for a causal relationship between glycine and CAD risk (6). Additionally, the relationship between circulating glycine levels and the CAD risk in different racial groups remains uncertain. There is only one previous study that investigated this relationship in Singaporean Chinese individuals, and reported a similar protective effect (5). However, this study had limitations such as a relatively small sample size and the inclusion of only two SNPs, one of which was not validated. Consequently, the study design may have compromised the validity of their findings. In our research, we have cross-validated IVs using two independent GWAS datasets, which strengthens the reliability and robustness of our conclusions.

      Previous epidemiological studies have suggested that dietary glycine may have a protective effect on blood pressure regulation (11). Our MR analysis provides further support for this association. We found an inverse genetic correlation between circulating glycine levels and SBP, which accounted for approximately nearly 6.06% of the genetic association between glycine and CAD. In rat models of metabolic syndrome, diets rich in glycine have been shown to reduce hypertension by reducing free radical production and enhancing nitric oxide utilization (12). However, our study did not uncover any significant correlations between glycine and lipid traits or inflammatory markers (13). Further comprehensive investigations are needed to explore other potential mechanisms that may explain the genetic link between glycine and CAD.

      Our study had several strengths. First, we implemented a thorough process to select valid genetic instruments for MR analysis. This procedure reduced the potential bias caused by weak instruments and improved the statistical power of our study. We used different MR methodologies to ensure the reliability of our estimates regarding the causal relationship between circulating glycine and the risk of CAD. Additionally, we conducted MR analyses on two separate populations, obtaining consistent results.

      This study was subject to some limitations. First, the genetic instruments used in our analysis were derived from datasets consisting only of individuals of European descent. To date, there have been no large-scale GWAS studies investigating the relationship between circulating glycine and genetic instruments in East Asian populations. Additionally, our mediation analysis is subject to potential bias, as accurately establishing causal relationships can be challenging and distinguishing between mediation and confounding can be statistically complex.

      The present study utilized MR analysis to investigate the possible causal link between serum glycine levels and CAD. These findings contribute to our understanding of the role of glycine in the development of CAD and provide methodological insights for the prevention and treatment of the disease.

    • No conflicts of interest.

    • The study was based on the data provided by the Medical Research Council Integrative Epidemiology Unit. We thank the investigators of the previous studies.

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