Loci for insulin processing and secretion provide insight into type 2 diabetes risk.

Insulin secretion is critical for glucose homeostasis, and increased levels of the precursor proinsulin relative to insulin indicate pancreatic islet beta-cell stress and insufficient insulin secretory capacity in the setting of insulin resistance. We conducted meta-analyses of genome-wide association results for fasting proinsulin from 16 European-ancestry studies in 45,861 individuals. We found 36 independent signals at 30 loci (p value < 5 × 10-8), which validated 12 previously reported loci for proinsulin and ten additional loci previously identified for another glycemic trait. Half of the alleles associated with higher proinsulin showed higher rather than lower effects on glucose levels, corresponding to different mechanisms. Proinsulin loci included genes that affect prohormone convertases, beta-cell dysfunction, vesicle trafficking, beta-cell transcriptional regulation, and lysosomes/autophagy processes. We colocalized 11 proinsulin signals with islet expression quantitative trait locus (eQTL) data, suggesting candidate genes, including ARSG, WIPI1, SLC7A14, and SIX3. The NKX6-3/ANK1 proinsulin signal colocalized with a T2D signal and an adipose ANK1 eQTL signal but not the islet NKX6-3 eQTL. Signals were enriched for islet enhancers, and we showed a plausible islet regulatory mechanism for the lead signal in the MADD locus. These results show how detailed genetic studies of an intermediate phenotype can elucidate mechanisms that may predispose one to disease.

Integrating transcriptomics, metabolomics, and GWAS helps reveal molecular mechanisms for metabolite levels and disease risk.

Transcriptomics data have been integrated with genome-wide association studies (GWASs) to help understand disease/trait molecular mechanisms. The utility of metabolomics, integrated with transcriptomics and disease GWASs, to understand molecular mechanisms for metabolite levels or diseases has not been thoroughly evaluated. We performed probabilistic transcriptome-wide association and locus-level colocalization analyses to integrate transcriptomics results for 49 tissues in 706 individuals from the GTEx project, metabolomics results for 1,391 plasma metabolites in 6,136 Finnish men from the METSIM study, and GWAS results for 2,861 disease traits in 260,405 Finnish individuals from the FinnGen study. We found that genetic variants that regulate metabolite levels were more likely to influence gene expression and disease risk compared to the ones that do not. Integrating transcriptomics with metabolomics results prioritized 397 genes for 521 metabolites, including 496 previously identified gene-metabolite pairs with strong functional connections and suggested 33.3% of such gene-metabolite pairs shared the same causal variants with genetic associations of gene expression. Integrating transcriptomics and metabolomics individually with FinnGen GWAS results identified 1,597 genes for 790 disease traits. Integrating transcriptomics and metabolomics jointly with FinnGen GWAS results helped pinpoint metabolic pathways from genes to diseases. We identified putative causal effects of UGT1A1/UGT1A4 expression on gallbladder disorders through regulating plasma (E,E)-bilirubin levels, of SLC22A5 expression on nasal polyps and plasma carnitine levels through distinct pathways, and of LIPC expression on age-related macular degeneration through glycerophospholipid metabolic pathways. Our study highlights the power of integrating multiple sets of molecular traits and GWAS results to deepen understanding of disease pathophysiology.

Author Correction: Exome sequencing of Finnish isolates enhances rare-variant association power.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exome sequencing of Finnish isolates enhances rare-variant association power.

Exome-sequencing studies have generally been underpowered to identify deleterious alleles with a large effect on complex traits as such alleles are mostly rare. Because the population of northern and eastern Finland has expanded considerably and in isolation following a series of bottlenecks, individuals of these populations have numerous deleterious alleles at a relatively high frequency. Here, using exome sequencing of nearly 20,000 individuals from these regions, we investigate the role of rare coding variants in clinically relevant quantitative cardiometabolic traits. Exome-wide association studies for 64 quantitative traits identified 26 newly associated deleterious alleles. Of these 26 alleles, 19 are either unique to or more than 20 times more frequent in Finnish individuals than in other Europeans and show geographical clustering comparable to Mendelian disease mutations that are characteristic of the Finnish population. We estimate that sequencing studies of populations without this unique history would require hundreds of thousands to millions of participants to achieve comparable association power.

Exome sequencing of 20,791 cases of type 2 diabetes and 24,440 controls.

Protein-coding genetic variants that strongly affect disease risk can yield relevant clues to disease pathogenesis. Here we report exome-sequencing analyses of 20,791 individuals with type 2 diabetes (T2D) and 24,440 non-diabetic control participants from 5 ancestries. We identify gene-level associations of rare variants (with minor allele frequencies of less than 0.5%) in 4 genes at exome-wide significance, including a series of more than 30 SLC30A8 alleles that conveys protection against T2D, and in 12 gene sets, including those corresponding to T2D drug targets (P = 6.1 × 10-3) and candidate genes from knockout mice (P = 5.2 × 10-3). Within our study, the strongest T2D gene-level signals for rare variants explain at most 25% of the heritability of the strongest common single-variant signals, and the gene-level effect sizes of the rare variants that we observed in established T2D drug targets will require 75,000-185,000 sequenced cases to achieve exome-wide significance. We propose a method to interpret these modest rare-variant associations and to incorporate these associations into future target or gene prioritization efforts.

Adiponectin GWAS loci harboring extensive allelic heterogeneity exhibit distinct molecular consequences.

Loci identified in genome-wide association studies (GWAS) can include multiple distinct association signals. We sought to identify the molecular basis of multiple association signals for adiponectin, a hormone involved in glucose regulation secreted almost exclusively from adipose tissue, identified in the Metabolic Syndrome in Men (METSIM) study. With GWAS data for 9,262 men, four loci were significantly associated with adiponectin: ADIPOQ, CDH13, IRS1, and PBRM1. We performed stepwise conditional analyses to identify distinct association signals, a subset of which are also nearly independent (lead variant pairwise r2<0.01). Two loci exhibited allelic heterogeneity, ADIPOQ and CDH13. Of seven association signals at the ADIPOQ locus, two signals colocalized with adipose tissue expression quantitative trait loci (eQTLs) for three transcripts: trait-increasing alleles at one signal were associated with increased ADIPOQ and LINC02043, while trait-increasing alleles at the other signal were associated with decreased ADIPOQ-AS1. In reporter assays, adiponectin-increasing alleles at two signals showed corresponding directions of effect on transcriptional activity. Putative mechanisms for the seven ADIPOQ signals include a missense variant (ADIPOQ G90S), a splice variant, a promoter variant, and four enhancer variants. Of two association signals at the CDH13 locus, the first signal consisted of promoter variants, including the lead adipose tissue eQTL variant for CDH13, while a second signal included a distal intron 1 enhancer variant that showed ~2-fold allelic differences in transcriptional reporter activity. Fine-mapping and experimental validation demonstrated that multiple, distinct association signals at these loci can influence multiple transcripts through multiple molecular mechanisms.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Adipose Tissue Gene Expression Associations Reveal Hundreds of Candidate Genes for Cardiometabolic Traits.

Genome-wide association studies (GWASs) have identified thousands of genetic loci associated with cardiometabolic traits including type 2 diabetes (T2D), lipid levels, body fat distribution, and adiposity, although most causal genes remain unknown. We used subcutaneous adipose tissue RNA-seq data from 434 Finnish men from the METSIM study to identify 9,687 primary and 2,785 secondary cis-expression quantitative trait loci (eQTL; <1 Mb from TSS, FDR < 1%). Compared to primary eQTL signals, secondary eQTL signals were located further from transcription start sites, had smaller effect sizes, and were less enriched in adipose tissue regulatory elements compared to primary signals. Among 2,843 cardiometabolic GWAS signals, 262 colocalized by LD and conditional analysis with 318 transcripts as primary and conditionally distinct secondary cis-eQTLs, including some across ancestries. Of cardiometabolic traits examined for adipose tissue eQTL colocalizations, waist-hip ratio (WHR) and circulating lipid traits had the highest percentage of colocalized eQTLs (15% and 14%, respectively). Among alleles associated with increased cardiometabolic GWAS risk, approximately half (53%) were associated with decreased gene expression level. Mediation analyses of colocalized genes and cardiometabolic traits within the 434 individuals provided further evidence that gene expression influences variant-trait associations. These results identify hundreds of candidate genes that may act in adipose tissue to influence cardiometabolic traits.

ACE2 expression in adipose tissue is associated with cardio-metabolic risk factors and cell type composition-implications for COVID-19.

BACKGROUND: COVID-19 severity varies widely. Although some demographic and cardio-metabolic factors, including age and obesity, are associated with increasing risk of severe illness, the underlying mechanism(s) are uncertain. SUBJECTS/METHODS: In a meta-analysis of three independent studies of 1471 participants in total, we investigated phenotypic and genetic factors associated with subcutaneous adipose tissue expression of Angiotensin I Converting Enzyme 2 (ACE2), measured by RNA-Seq, which acts as a receptor for SARS-CoV-2 cellular entry. RESULTS: Lower adipose tissue ACE2 expression was associated with multiple adverse cardio-metabolic health indices, including type 2 diabetes (T2D) (P = 9.14 × 10-6), obesity status (P = 4.81 × 10-5), higher serum fasting insulin (P = 5.32 × 10-4), BMI (P = 3.94 × 10-4), and lower serum HDL levels (P = 1.92 × 10-7). ACE2 expression was also associated with estimated proportions of cell types in adipose tissue: lower expression was associated with a lower proportion of microvascular endothelial cells (P = 4.25 × 10-4) and higher proportion of macrophages (P = 2.74 × 10-5). Despite an estimated heritability of 32%, we did not identify any proximal or distal expression quantitative trait loci (eQTLs) associated with adipose tissue ACE2 expression. CONCLUSIONS: Our results demonstrate that individuals with cardio-metabolic features known to increase risk of severe COVID-19 have lower background ACE2 levels in this highly relevant tissue. Reduced adipose tissue ACE2 expression may contribute to the pathophysiology of cardio-metabolic diseases, as well as the associated increased risk of severe COVID-19.

Investigating rare pathogenic/likely pathogenic exonic variation in bipolar disorder.

Bipolar disorder (BD) is a serious mental illness with substantial common variant heritability. However, the role of rare coding variation in BD is not well established. We examined the protein-coding (exonic) sequences of 3,987 unrelated individuals with BD and 5,322 controls of predominantly European ancestry across four cohorts from the Bipolar Sequencing Consortium (BSC). We assessed the burden of rare, protein-altering, single nucleotide variants classified as pathogenic or likely pathogenic (P-LP) both exome-wide and within several groups of genes with phenotypic or biologic plausibility in BD. While we observed an increased burden of rare coding P-LP variants within 165 genes identified as BD GWAS regions in 3,987 BD cases (meta-analysis OR = 1.9, 95% CI = 1.3-2.8, one-sided p = 6.0 × 10-4), this enrichment did not replicate in an additional 9,929 BD cases and 14,018 controls (OR = 0.9, one-side p = 0.70). Although BD shares common variant heritability with schizophrenia, in the BSC sample we did not observe a significant enrichment of P-LP variants in SCZ GWAS genes, in two classes of neuronal synaptic genes (RBFOX2 and FMRP) associated with SCZ or in loss-of-function intolerant genes. In this study, the largest analysis of exonic variation in BD, individuals with BD do not carry a replicable enrichment of rare P-LP variants across the exome or in any of several groups of genes with biologic plausibility. Moreover, despite a strong shared susceptibility between BD and SCZ through common genetic variation, we do not observe an association between BD risk and rare P-LP coding variants in genes known to modulate risk for SCZ.

The genetic architecture of type 2 diabetes.

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of the heritability of this disease. Here, to test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole-genome sequencing in 2,657 European individuals with and without diabetes, and exome sequencing in 12,940 individuals from five ancestry groups. To increase statistical power, we expanded the sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support the idea that lower-frequency variants have a major role in predisposition to type 2 diabetes.

Integrative analysis of gene expression, DNA methylation, physiological traits, and genetic variation in human skeletal muscle.

We integrate comeasured gene expression and DNA methylation (DNAme) in 265 human skeletal muscle biopsies from the FUSION study with >7 million genetic variants and eight physiological traits: height, waist, weight, waist-hip ratio, body mass index, fasting serum insulin, fasting plasma glucose, and type 2 diabetes. We find hundreds of genes and DNAme sites associated with fasting insulin, waist, and body mass index, as well as thousands of DNAme sites associated with gene expression (eQTM). We find that controlling for heterogeneity in tissue/muscle fiber type reduces the number of physiological trait associations, and that long-range eQTMs (>1 Mb) are reduced when controlling for tissue/muscle fiber type or latent factors. We map genetic regulators (quantitative trait loci; QTLs) of expression (eQTLs) and DNAme (mQTLs). Using Mendelian randomization (MR) and mediation techniques, we leverage these genetic maps to predict 213 causal relationships between expression and DNAme, approximately two-thirds of which predict methylation to causally influence expression. We use MR to integrate FUSION mQTLs, FUSION eQTLs, and GTEx eQTLs for 48 tissues with genetic associations for 534 diseases and quantitative traits. We identify hundreds of genes and thousands of DNAme sites that may drive the reported disease/quantitative trait genetic associations. We identify 300 gene expression MR associations that are present in both FUSION and GTEx skeletal muscle and that show stronger evidence of MR association in skeletal muscle than other tissues, which may partially reflect differences in power across tissues. As one example, we find that increased RXRA muscle expression may decrease lean tissue mass.

Colocalization of GWAS and eQTL signals at loci with multiple signals identifies additional candidate genes for body fat distribution.

Integration of genome-wide association study (GWAS) signals with expression quantitative trait loci (eQTL) studies enables identification of candidate genes. However, evaluating whether nearby signals may share causal variants, termed colocalization, is affected by the presence of allelic heterogeneity, different variants at the same locus impacting the same phenotype. We previously identified eQTL in subcutaneous adipose tissue from 770 participants in the Metabolic Syndrome in Men (METSIM) study and detected 15 eQTL signals that colocalized with GWAS signals for waist-hip ratio adjusted for body mass index (WHRadjBMI) from the Genetic Investigation of Anthropometric Traits consortium. Here, we reevaluated evidence of colocalization using two approaches, conditional analysis and the Bayesian test COLOC, and show that providing COLOC with approximate conditional summary statistics at multi-signal GWAS loci can reconcile disagreements in colocalization classification between the two tests. Next, we performed conditional analysis on the METSIM subcutaneous adipose tissue data to identify conditionally distinct or secondary eQTL signals. We used the two approaches to test for colocalization with WHRadjBMI GWAS signals and evaluated the differences in colocalization classification between the two tests. Through these analyses, we identified four GWAS signals colocalized with secondary eQTL signals for FAM13A, SSR3, GRB14 and FMO1. Thus, at loci with multiple eQTL and/or GWAS signals, analyzing each signal independently enabled additional candidate genes to be identified.

ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn's mutant.

Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn's disease (CD), and phosphorylation of CD-associated ATG16L1 T300A (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Phosphorylated ATG16L1 localizes to the site of internalized bacteria and stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy, indicating a role for ATG16L1 phosphorylation in the promotion of anti-bacterial autophagy. In contrast to wild-type ATG16L1, ULK1-mediated phosphorylation of caATG16L1 drives its destabilization in response to stress. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1 signalling to ATG16L1 is a double-edged sword, enhancing the function of the wild-type ATG16L1, but promoting degradation of caATG16L1.

When Safety Event Reporting Is Seen as Punitive: "I've Been PSN-ed!"

STUDY OBJECTIVE: Reporting systems are designed to identify patient care issues so changes can be made to improve safety. However, a culture of blame discourages event reporting, and reporting seen as punitive can inhibit individual and system performance in patient safety. This study aimed to determine the frequency and factors related to punitive patient safety event report submissions, referred to as Patient Safety Net reports, or PSNs. METHODS: Three subject matter experts reviewed 513 PSNs submitted between January and June 2019. If the PSN was perceived as blaming an individual, it was coded as punitive. The experts had high agreement (κ=0.84 to 0.92), and identified relationships between PSN characteristics and punitive reporting were described. RESULTS: A total of 25% of PSNs were punitive, 7% were unclear, and 68% were designated nonpunitive. Punitive (vs nonpunitive) PSNs more likely focused on communication (41% vs 13%), employee behavior (38% vs 2%), and patient assessment issues (17% vs 4%). Nonpunitive (vs punitive) PSNs were more likely for equipment (19% vs 4%) and patient or family behavior issues (8% vs 2%). Punitive (vs nonpunitive) PSNs were more common with adverse reactions or complications (21% vs 10%), communication failures (25% vs 16%), and noncategorized events (19% vs 8%), and nonpunitive (vs punitive) PSNs were more frequent in falls (5% vs 0%) and radiology or laboratory events (17% vs 7%). CONCLUSION: Punitive reports have important implications for reporting systems because they may reflect a culture of blame and a failure to recognize system influences on behaviors. Nonpunitive wording better identifies factors contributing to safety concerns. Reporting systems should focus on patient outcomes and learning from systems issues, not blaming individuals.

Directional dominance on stature and cognition in diverse human populations.

Homozygosity has long been associated with rare, often devastating, Mendelian disorders, and Darwin was one of the first to recognize that inbreeding reduces evolutionary fitness. However, the effect of the more distant parental relatedness that is common in modern human populations is less well understood. Genomic data now allow us to investigate the effects of homozygosity on traits of public health importance by observing contiguous homozygous segments (runs of homozygosity), which are inferred to be homozygous along their complete length. Given the low levels of genome-wide homozygosity prevalent in most human populations, information is required on very large numbers of people to provide sufficient power. Here we use runs of homozygosity to study 16 health-related quantitative traits in 354,224 individuals from 102 cohorts, and find statistically significant associations between summed runs of homozygosity and four complex traits: height, forced expiratory lung volume in one second, general cognitive ability and educational attainment (P < 1 × 10(-300), 2.1 × 10(-6), 2.5 × 10(-10) and 1.8 × 10(-10), respectively). In each case, increased homozygosity was associated with decreased trait value, equivalent to the offspring of first cousins being 1.2 cm shorter and having 10 months' less education. Similar effect sizes were found across four continental groups and populations with different degrees of genome-wide homozygosity, providing evidence that homozygosity, rather than confounding, directly contributes to phenotypic variance. Contrary to earlier reports in substantially smaller samples, no evidence was seen of an influence of genome-wide homozygosity on blood pressure and low density lipoprotein cholesterol, or ten other cardio-metabolic traits. Since directional dominance is predicted for traits under directional evolutionary selection, this study provides evidence that increased stature and cognitive function have been positively selected in human evolution, whereas many important risk factors for late-onset complex diseases may not have been.

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms.

Genetic studies of body mass index yield new insights for obesity biology.

Obesity is heritable and predisposes to many diseases. To understand the genetic basis of obesity better, here we conduct a genome-wide association study and Metabochip meta-analysis of body mass index (BMI), a measure commonly used to define obesity and assess adiposity, in up to 339,224 individuals. This analysis identifies 97 BMI-associated loci (P < 5 × 10(-8)), 56 of which are novel. Five loci demonstrate clear evidence of several independent association signals, and many loci have significant effects on other metabolic phenotypes. The 97 loci account for ∼2.7% of BMI variation, and genome-wide estimates suggest that common variation accounts for >20% of BMI variation. Pathway analyses provide strong support for a role of the central nervous system in obesity susceptibility and implicate new genes and pathways, including those related to synaptic function, glutamate signalling, insulin secretion/action, energy metabolism, lipid biology and adipogenesis.

An innovative outpatient monitor service for gynecological patients in the United Kingdom: Case study evaluation of clinical effectiveness, economic outcomes, patient safety, and service improvement.

BACKGROUND: Failure to attend appointments places a huge strain on health care systems around the world, resulting in poorer care for the patients, waste of staff time and increased waiting times. This study looked at the impact of an Outpatient-Monitor-Service (OMS) on clinical, economic, patient safety and service improvement outcomes in gynaecology patients compared with care as usual (with no access to the OMS). METHODS: We conducted a retrospective match-pair controlled study at a London-based hospital. The cohort included adult women who received either (i) gynecological, (ii) hysteroscopy or (iii) vulval procedures. A cost-consequences analysis compared intervention (who received the OMS) with control (historical cases who did not receive the OMS. Main outcome measures were clinical-effectiveness, NHS-cost, patient safety, and service improvement. RESULTS: The intervention had positive impacts spanning clinical, patient safety and service improvement areas and showed cost saving results for the healthcare in terms of reduced follow-up consultations and did-not-attend occurrences. CONCLUSIONS: The OMS offered by Message Dynamics appears to be a successful digital health technology to monitor gynecological patients' conditions and inform clinical decision making via remote channels, which is particularly relevant in coronavirus disease pandemic.

Identification of seven novel loci associated with amino acid levels using single-variant and gene-based tests in 8545 Finnish men from the METSIM study.

Comprehensive metabolite profiling captures many highly heritable traits, including amino acid levels, which are potentially sensitive biomarkers for disease pathogenesis. To better understand the contribution of genetic variation to amino acid levels, we performed single variant and gene-based tests of association between nine serum amino acids (alanine, glutamine, glycine, histidine, isoleucine, leucine, phenylalanine, tyrosine, and valine) and 16.6 million genotyped and imputed variants in 8545 non-diabetic Finnish men from the METabolic Syndrome In Men (METSIM) study with replication in Northern Finland Birth Cohort (NFBC1966). We identified five novel loci associated with amino acid levels (P = < 5×10-8): LOC157273/PPP1R3B with glycine (rs9987289, P = 2.3×10-26); ZFHX3 (chr16:73326579, minor allele frequency (MAF) = 0.42%, P = 3.6×10-9), LIPC (rs10468017, P = 1.5×10-8), and WWOX (rs9937914, P = 3.8×10-8) with alanine; and TRIB1 with tyrosine (rs28601761, P = 8×10-9). Gene-based tests identified two novel genes harboring missense variants of MAF <1% that show aggregate association with amino acid levels: PYCR1 with glycine (Pgene = 1.5×10-6) and BCAT2 with valine (Pgene = 7.4×10-7); neither gene was implicated by single variant association tests. These findings are among the first applications of gene-based tests to identify new loci for amino acid levels. In addition to the seven novel gene associations, we identified five independent signals at established amino acid loci, including two rare variant signals at GLDC (rs138640017, MAF=0.95%, Pconditional = 5.8×10-40) with glycine levels and HAL (rs141635447, MAF = 0.46%, Pconditional = 9.4×10-11) with histidine levels. Examination of all single variant association results in our data revealed a strong inverse relationship between effect size and MAF (Ptrend<0.001). These novel signals provide further insight into the molecular mechanisms of amino acid metabolism and potentially, their perturbations in disease.