Introduction: Plasma proteins are critical mediators of cardiovascular processes and are the targets of many drugs. Previous efforts to characterize the genetic architecture of the plasma proteome have been limited by a focus on individuals of European descent and leveraged genotyping arrays and imputation. Hypothesis: Whole genome sequence analysis of the plasma proteome in individuals with greater African ancestry will increase power to identify novel genetic determinants. Methods: Proteomic profiling of 1,301 proteins was performed in 1852 Black adults from the Jackson Heart Study using aptamer-based proteomics (SomaScan®). Whole genome sequencing association analysis was ascertained for all variants with minor allele count ≥ 5. Results were validated using an alternative, antibody-based, proteomic platform (Olink®) as well as replicated in the Multi-Ethnic Study of Atherosclerosis and the HERITAGE Family Study. Results: We identify 569 genetic associations between 479 proteins and 438 unique genetic regions at a Bonferroni-adjusted significance level of 3.8 × 10-11. These associations include 134 novel locus-protein relationships and an additional 205 novel sentinel variant-protein relationships. Novel cardiovascular findings include new protein associations at the APOE gene locus including ZAP70 (sentinel single nucleotide polymorphism [SNP] rs7412-T, β = 0.61±0.05, p-value = 3.27 × 10-30) and MMP-3 (β = -0.60±0.05, p = 1.67 × 10-32), as well as a completely novel pleiotropic locus at the HPX gene, associated with nine proteins. Further, the associations suggest new mechanisms of genetically mediated cardiovascular disease linked to African ancestry; we identify a novel association between variants linked to APOL1 associated chronic kidney and heart disease and the protein CKAP2 (rs73885319-G, β = 0.34±0.04, p = 1.34 × 10-17) as well as an association between ATTR amyloidosis and RBP4 levels in community dwelling individuals without heart failure. Discussion: Taken together, these results provide evidence for the functional importance of variants in non-European populations, and suggest new biological mechanisms for ancestry-specific determinants of lipids, coagulation and myocardial function.

RATIONALE
Current guidelines do not sufficiently capture the heterogenous nature of asthma leading to suboptimal management and treatment strategies. A more comprehensive classification of asthma into biologically meaningful subgroups is needed. Given its position on the central biological dogma, as the ‘ome closest to phenotype, reflecting genetics, environment and their interactions, metabolomics represents a novel and compelling approach to accurately identifying asthma endotypes; i.e. subtypes defined by their functional or pathobiological mechanisms, with the potential for clinical translation.
METHODS
We performed plasma metabolomic profiling of 1155 asthmatic children from the Genetics of Asthma in Costa Rica Study (GACRS) across four profiling platforms covering a broad range of the metabolome. We generated patient similarity networks for each platform that connected asthmatics via edges representing patient-to-patient similarity in their metabolomic profiles (controlling for age, sex and body mass index). We then fused the four platform-specific networks using Similarity Network Fusion and performed spectral clustering on the resulting fused similarity network to identify metabo-endotypes. We explored phenotypic and clinical differences across the metabo-endotypes using ANOVA and chi-square tests. For validation we recapitulated the endotypes in an independent population of asthmatic children (CAMP, n=911) to determine whether the same between-endotype differences were observed. Finally, we identified metabolomic and genomic drivers of validated metabo-endotype membership, meta-analyzing findings from GACRS and CAMP.
RESULTS
We identified five metabo-endotypes in GACRS and observed significant differences across metabo-endotypes in asthma-relevant phenotypes pre-bronchodilator (p-ANOVA=8.3x10-5) and post-bronchodilator (p-ANOVA=1.8x10-5) FEV1/FVC ratio; use of inhaled (p-ANOVA=5.0x10-13) and oral (p-ANOVA=0.007) steroids, and airway hyperresponsiveness to methacholine (AHR PC20<16.8, p-ANOVA=8.4x10-7)p-ANOVA=8.4x10-7). Furthermore, there was a significant difference in eosinophil count (p-ANOVA=0.009). The recapitulated metabo-endotypes in CAMP displayed significant differences (p<0.05) in many of the same phenotypes. (Table 1). The “most-severe” asthma endotype was defined by the lowest FEV1/FVC ratio, and the highest prevalence of AHR and oral steroid usage. It was characterized by higher levels of glycerolipids and lysophospholipids, as well as lower levels of nucleotides relative to the other endotypes. These findings suggest dysregulation of pulmonary surfactant homeostasis in the “most-severe” endotype and were supported by the genetic analysis, which found that members of this endotype were more likely to carry variants in key pulmonary surfactant regulation genes such as BACH3 (meta-analyzed p=5.2x10-4) and BMP3 (meta-analyzed p=8.5x10-4).
CONCLUSIONS
These findings demonstrate that clinically meaningful endotypes can be derived and validated using metabolomic data, and that interrogating the drivers of these metabo-endotypes can help understand their pathophysiology and identify therapeutic targets.

Circulating metabolite levels are highly heritable, and may reflect the state of the human organism in health and disease. Numerous autosomal loci associated with metabolite levels have been reported, predominantly in individuals of European ancestry. The genetic architecture of metabolites is not fully understood, including the generalizability of previous findings in European ancestry to other ancestries, and the genetic contribution of sex chromosomes.

We performed a whole-genome sequencing association (WGS) analysis of ~64 million common SNPs (minor allele frequency ≥ 1%) in up to 11,840 participants (5,940 European-Americans, 1,843 African-Americans and 4,059 Hispanic/Latino participants) from the NHLBI Trans-Omics for Precision Medicine (TOPMed) Program and the NHGRI Centers for Common Disease Genomics (CCDG) with up to 1,666 circulating metabolites, quantified by liquid chromatography-mass spectrometry.

We discovered 2,741 novel variant-metabolite associations (spanning 257 genetic loci, including 93 novel), and validated 690 locus-metabolite associations reported previously (P < 3.0x10-11). The novel loci explained 1% - 28% of metabolite variation, and 27 associations were replicated in TwinsUK. Among the novel loci, ~27% contained genes involved in metabolism, and these loci were enriched in genes that cause inborn errors of metabolism (P = 2.3x10-10). We further demonstrated that the X chromosome was extensively involved in the metabolite regulation. Two missense variants located in ASMTL, encoding a protein implicated in cell division arrest, were associated with lower levels of three aromatic compounds (O-methylcatechol sulfate, tryptophan and 8-methoxykynurenate). Mendelian Randomization analysis revealed that several metabolites had levels increased based upon genotype that were associated with a 7%-14% risk change of coronary artery disease. These metabolites included succinoyltaurine, a derivative of taurine, which has long been suspected to be protective of atherosclerosis.

In summary, we conducted the first multi-ethnic WGS study of genetic determinants of circulating metabolites, and identified novel genetic loci, involved in miscellaneous biological processes, contributing to our understanding of human disease.

Introduction: Spirometric measures of pulmonary function form the basis for diagnosis and monitoring of chronic obstructive pulmonary disease (COPD) and predict morbidity and mortality. The molecular underpinnings of pulmonary function have not been fully characterized. Metabolomics holds promise to identify metabolic signatures involved in regulation of pulmonary function and develop biomarkers of early COPD.

Methods: Metabolomic profiling of blood samples was performed in seven TOPMed cohorts: the Atherosclerosis Risk in Communities (ARIC) Study, the Coronary Artery Risk Development in Young Adults (CARDIA) Study, the Cardiovascular Health Study (CHS), the Framingham Heart Study (FHS), the Jackson Heart Study (JHS) study, the Multi-Ethnic Study of Atherosclerosis (MESA) and the Hispanic Community Health Study/Study of Latinos (HCHS/SOL). We examined the cross-sectional associations by ethnic-study strata between each metabolite and three spirometic measures: forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and their ratio (FEV1/FVC), adjusting for age, age squared, sex, height, height squared, weight, smoking and kidney function. Fixed-effect inverse variance weighted meta-analysis was applied to produce trans-ethnic estimates.

Results: We meta-analyzed 13,622 participants with 808 metabolites (missing rate ≤ 25% in at least two studies), including 4,260 African Americans, 5,494 European Americans with 3,868 Hispanic Americans. There were 69, 75 and 3 metabolites significantly associated with FEV1, FVC and FEV1/FVC respectively (p<6.2×10-5, 0.05/808). Fifty-five metabolites were significantly associated with both FEV1 and FVC, where an SD increment of metabolite levels were associated with an average 29.5 ml and 36.8 ml change of FEV1 and FVC. Galactose and transcription/translation metabolism was enriched based on the Small Molecule Pathway Database. Among those 55 metabolites, we observed generally consistent effects across ethnic groups and across sex groups. We conducted sensitivity analyses by excluding COPD cases or smokers, which produced comparable results. We examined whether these 55 FEV1 and FVC related metabolites were also associated with COPD status (defined by FEV1/FVC and FEV1 being below the lower limit of normal, n = 742 cases). Eight metabolites met strict look-up level significance (p<9.0×10-4, 0.05/55) with an SD increment of metabolite levels associated with an average odds ratio for COPD of 1.21. (Figure).

Conclusion: We identified multiple metabolites that related to pulmonary function and COPD in multi-ethnic populations. These findings could provide novel insight into regulation of pulmonary function and COPD pathogenesis.