Serum chloride: epidemiology and genetic dissection of a novel marker for cardiovascular risk

Chloride (Cl-) is the principal anion in the human body and is essential for the maintenance of osmotic pressure, acid-base balance, the movement of water between fluid compartments and regulation of muscular activity. The ability of the kidney to reabsorb Cl- maintains serum Cl- within a narrow range. Hyper and hypochloraemia are associated with several disease states or can be the result of drugs. Serum Cl- is frequently measured in the clinical setting and although it is useful in a number of clinical conditions, it is often overlooked. Low serum Cl- has been shown in multiple phenotypically distinct populations to be associated with adverse outcomes, in particular cardiovascular outcomes, however the mechanism by which low serum Cl- increases mortality and/or cardiovascular events and its validity within the general population is unclear. This thesis aims to validate serum Cl- as a risk predictor in large scale epidemiological studies of two large patient cohorts; identify physiological pathways that affect serum Cl- and other electrolytes through metabolome-wide association study and identify genetic determinants of serum Cl- through a genome-wide association study. Chapter 1 provides an introduction to cardiovascular disease, cardiovascular risk and the use of serum biomarkers for risk stratification. The emphasis is on salt as a risk factor with focus on the epidemiological studies of serum Cl- and adverse outcomes. In Chapter 2 , the data sources (Glasgow Blood Pressure Clinic; NHS Greater Glasgow and Clyde Safe Haven; TwinsUK; Generation Scotland Family Health Study), data application process, methods for data cleaning and the preparation of data for analysis are detailed. Statistical methods for the epidemiological studies are described. Building upon previous work investigating the association of serum chloride and mortality outcomes in the Glasgow Blood Pressure Clinic (GBPC) hypertensive population, Chapter 3 explores the association of serum chloride and cause-specific morbidity and mortality outcomes in a new dataset linking the GBPC patient data with outcome data from the NHS Scotland Information Services Division. Multivariable Cox proportional hazard (Cox-PH) modelling and Cox-PH modelling with restricted cubic spline function were used investigate the associations. In 6,133 hypertensive patients attending the GBPC, using the multivariable Cox PH model, there was an inverse association of serum Cl- with all-cause and cardiovascular mortality, death from myocardial infarction, ischaemic heart disease and stroke at 10, 20 and 30 years follow up. The relationship between serum chloride with all-cause mortality, cardiovascular mortality and death from stroke was found to be U-shaped using Cox-PH modelling with restricted cubic spline function whilst for death from myocardial infarction and ischaemic heart disease the relationship was linear. The risk of hospital admission with heart failure was inversely related to serum Cl- in the multivariable Cox-PH model at 5 and 10 years follow up and this relationship was found to be U-shaped in the Cox-PH model with restricted cubic spline function. Chapter 4 sought to validate the findings from the GBPC population in a large general population study using data from the NHS Greater Glasgow and Clyde Safe Haven. A wider range of morbidity and mortality outcomes were studied, including non-cardiovascular outcomes which share common pathophysiological processes with cardiovascular disease, using multivariable Cox-PH models. In 519,957 patients from the NHSGGC Safe Haven, lower levels of serum Cl- were associated with higher risk of all-cause, cardiovascular mortality and heart failure admissions confirming the findings from the hypertension study described in Chapter 3. In addition, lower serum Cl- is also significantly associated with the following cause-specific cardiovascular outcomes – deaths due to MI, heart failure, arrhythmia, ischaemic stroke, intracerebral haemorrhage; admissions for PVD, ischaemic stroke and intracerebral haemorrhage. Furthermore, the following non-cardiovascular outcomes are also associated with lower serum Cl- - deaths due to COPD, cancer, chronic liver disease, alcohol related liver disease, diabetes mellitus; admissions for COPD, chronic liver disease, alcohol related liver disease, diabetes mellitus and CKD. Though serum chloride is inversely associated with adverse outcomes in Chapter 4 a causal mechanism is not established. In Chapter 5 a two-pronged approach is used to attempt to further understand the relationship between serum Cl- and risk. A metabolome wide association study (MWAS) was performed in the TwinsUK cohort to discover molecular markers and pathways associated with serum electrolyte levels through metabolomic profiling. Acid–base balance and electrolyte homeostasis are intricately connected to maintain the body’s internal environment within rigidly controlled limits therefore the study of metabolomic associations of electrolytes need to be considered in the context of these homeostatic processes. Common electrolyte and acid-base patterns were mapped, based on the HCO3--centric physicochemical model and electroneutrality, to metabolite associations. Na+ and Cl‐ showed the lowest number of independent metabolite associations (10 & 17 respectively) and K+ the highest number. Serum K+ showed the most significant associations with individual fatty acid metabolites with specific enrichment in fatty acid pathways. In contrast, serum Na+ and HCO3- showed associations predominantly with amino acids. Cl- and HCO3- association signals, however, helped map patterns based on the Stewart physicochemical model of acid-base chemistry enabling novel insights into electrolyte homeostasis. The heritability of serum electrolytes was estimated in the TwinsUK cohort with serum chloride showing low heritability (A 0.04 (0-0.19)). A genome wide association study meta-analysis was performed using the TwinsUK and Generation Scotland cohorts aiming to identify novel pathways to enhance understanding of electrolyte balance and inform Mendelian randomisation studies. The study was underpowered and there were no SNPs associated with serum Cl- at the conventional genome-wide significance level < 5x10-8. Chapter 6 bring the thesis to conclusion tying together the findings of the epidemiological, genetic and metabolomic studies to pave the path for future work. Serum Cl- is validated as an independent risk predictor for both cardiovascular and non-cardiovascular morbidity and mortality outcomes in the general population.