Genome-wide association study and targeted metabolomics identifies sex-specific association of CPS1 with coronary artery disease

Abstract

Metabolites derived from dietary choline and L-carnitine, such as trimethylamine N-oxide and betaine, have recently been identified as novel risk factors for atherosclerosis in mice and humans. We sought to identify genetic factors associated with plasma betaine levels and determine their effect on risk of coronary artery disease (CAD). A two-stage genome-wide association study (GWAS) identified two significantly associated loci on chromosomes 2q34 and 5q14.1. The lead variant on 2q24 (rs715) localizes to carbamoyl-phosphate synthase 1 (CPS1), which encodes a mitochondrial enzyme that catalyses the first committed reaction and rate-limiting step in the urea cycle. Rs715 is also significantly associated with decreased levels of urea cycle metabolites and increased plasma glycine levels. Notably, rs715 yield a strikingly significant and protective association with decreased risk of CAD in only women. These results suggest that glycine metabolism and/or the urea cycle represent potentially novel sex-specific mechanisms for the development of atherosclerosis.

Figure 1. Results of a GWAS for plasma betaine levels in the GeneBank cohort.

The Manhattan plot for plasma betaine levels shows four significantly or suggestively associated loci on chromosomes 1q32.2, 2q34 5q14.1 and 16q24.1. The symbols for the genes closest to the lead SNPs are shown in italics and genome-wide thresholds for significant (P=5.0 × 10⁻⁸) and suggestive (P=5.0 × 10⁻⁶) association are indicated by the horizontal red and blue lines, respectively. P-values were obtained using linear regression with natural log transformed values and adjustment for age and sex.

Figure 2. Regional plots for the loci associated with plasma betaine levels.

The regions shown for chromosomes 5q14.1 (a) and 2q34 (b) are centred on the lead SNP (purple diamond) for each respective locus. The degree of LD (r²) between the lead SNP and other variants in the selected interval is given according the colour-coded legend in the box and genes are indicated in the bottom panel. Rs1047891 was formerly designated as rs7422339.

Figure 3. The genes and intermediates of the pathway linking choline metabolism to the urea cycle.

One route (green arrows) for the initial catabolism of choline is mediated by intestinal microbes and leads to the formation of trimethylamine (TMA). TMA is efficiently absorbed from the gastrointestinal tract and subsequently oxidized by the liver to form trimethylamine N-oxide (TMAO) through reactions catalysed by one or more of the flavin monooxygenase (FMO) family of enzymes. Alternatively (red arrows), choline can be oxidized to betaine through reactions catalysed by choline dehydrogenase (CHDH) and betaine aldehyde dehydrogenase (ALDH7A1). Betaine (also known as trimethylglycine) is demethylated to form dimethylglycine via the betaine-homocysteine S-methyltransferase enzymes (BHMT, BHMT2). This reaction simultaneously converts homocysteine to methionine. Dimethylglycine dehydrogenase (DMGDH) subsequently demethylates dimethylglycine to form sarcosine, which is then converted to glycine by sarcosine dehydrogenase (SDH) after removal of the remaining methyl group. Glycine is metabolized by a group of enzymes known as the glycine cleavage complex (GCC), which is the major route in animals for glycine degradation and the formation of ammonia (NH₃) and carbon dioxide (CO₂). NH₃ is converted to carbamoyl phosphate, which enters the urea cycle (blue arrows) through the rate-limiting reaction catalysed by carbamoyl-phosphate synthase 1 (CPS1), or can be converted back to glycine through the GCC. Carbamoyl phosphate is metabolized by ornithine transcarbamylase (OTC) to form citrulline and subsequently argininosuccinate through a reaction catalysed by argininosuccinate synthetase (ASS). This is followed by the formation of L-arginine by arginosuccinate lyase (ASL). L-Arginine is used as a substrate for the production of nitric oxide or metabolized by arginase (ARG1) to form urea for excretion and ornithine for re-entry back into the cycle. Metabolites that were available for analysis are shown in black, whereas unmeasured metabolites are shown in grey.