Genetic determinants of endogenous arginine derivatives and their role in cardiometabolic diseases : With special emphasis on ADMA, SDMA and L-homoarginine

Background: Cardiometabolic diseases are a significant burden to healthcare and one of the leading causes of morbidity and death across the world. A prominent feature of cardiometabolic diseases is endothelial dysfunction and subsequent diminished bioavailability of vasoactive molecule nitric oxide (NO). Asymmetric and symmetric dimethylarginines (ADMA and SDMA) reduce NO bioavailability and thus contribute to the loss of the protective effects of NO (e.g. antithrombotic and anti-inflammatory) in the vasculature, leading to cardiometabolic complications. High ADMA and SDMA and low Lhomoarginine (hArg), a naturally occurring non-proteinogenic amino acid, predict increased risk of cardiovascular and overall mortality although their genetic determinants and the underlying molecular mechanisms are not completely understood. Aims of the study: 1) identify novel genetic determinants of serum ADMA, SDMA and hArg by genome-wide association study (GWAS); 2) investigate associations of these newly identified genetic variants with cardiometabolic diseases and end points; and 3) characterise the biomarker and causal roles of serum hArg in the development of cardiometabolic diseases. Materials and methods: In studies I and II, GWASs were conducted by using the Cardiovascular Risk in Young Finns Study (YFS, N=2,083) and the Ludwigshafen Risk and Cardiovascular Health (LURIC, N=3,027) study materials. In study III, genetic associations with atrial fibrillation were studied in LURIC, in the Finnish Cardiovascular Study (FINCAVAS, N=3,862) and in the Corogene study. Associations with ischemic stroke were studied in the Wellcome Trust Case Control Concortium 2 (WTCCC2) GWAS cohorts (3,548 cases and 5,972 controls) and in FINCAVAS. In study IV, observational association analyses between hArg and cardiometabolic risk factors and diseases were investigated in YFS. The causal effect estimates between hArg and cardiometabolic risk factors and diseases were calculated using association summary statistics for SNP-outcome associations available in the public domain. This data involved tens to hundreds of thousands of individuals from GWA metaanalyses. In all studies, genotyping and imputation passed strict quality control measures. Serum levels of ADMA, SDMA and hArg were measured using the same reversed-phase high-performance liquid chromatography method from the YFS and LURIC studies. The GWASs were performed using the PLINK, ProbABEL, QUICKTEST and SNPTEST software programs while Mendelian randomisation and other statistical analyses were performed under the R statistical environment. Results: In study I, several intron variants at the DDAH1 locus (rs28489187 as the lead variant) were associated with circulating ADMA levels and two missense variants in AGXT2 (rs37369, Val140Ile; rs16899974, Val498Leu) were independently associated with SDMA levels. An additional association signal for SDMA was observed in SLC25A45 (rs34400381, Arg285Cys/Arg243Cys/etc. depending on the splice variant) encoding a mitochondrial solute carrier and thus sharing the same intracellular localisation as the mitochondrial aminotransferase enzyme AGXT2. Serum hArg levels were associated with variants at the GATM (rs1153858), CPS1 rs1047891 (Thr1406Asn, formerly rs7422339) and AGXT2 (rs37369, Val140Ile) loci (study II). The two functional AGXT2 variants (rs37369 and rs1873737) were associated in a consistent manner with the low-frequency to high-frequency (LF/HF) ratio of spectral heart rate variability (HRV) in YFS (study I). Moreover, the same AGXT2 variants showed strong associations with atrial fibrillation in LURIC and age at a first ischemic stroke diagnosis in FINCAVAS. However, these associations were not replicated in all the cohorts studied (study III). In longitudinal study (IV), serum hArg predicted incident hyperglycemia and abdominal obesity in young men in YFS. In young women of reproductive age, hArg levels were elevated in those who used oestrogen-containing oral contraceptives compared to non-users. In Mendelian randomisation studies, we found no evidence that life-long exposure to higher levels of serum hArg would causally increase the risk of developing cardiometabolic risk factors, type 2 diabetes mellitus or coronary artery disease. Conclusions: These results reveal novel genetic variants associated with circulating ADMA, SDMA and hArg levels and demonstrate that AGXT2 is involved in cardiometabolic diseases and their complications. Thus, AGXT2 might be a promising target for novel therapeutic interventions. Although not likely to be a causal mediator, serum hArg could potentially be used as a biomarker to detect individuals that are prone to developing abdominal obesity and associated comorbidities.