key: cord-0262239-z91lemqi authors: Lagou, V.; Jiang, L.; Ulrich, A.; Zudina, L.; Gutierrez Gonzalez, K. S.; Balkhiyarova, Z.; Faggian, A.; Chen, S.; Todorov, P.; Sharapov, S.; David, A.; Marullo, L.; Mägi, R.; Rujan, R.-M.; Ahlqvist, E.; Thorleifsson, G.; Gao, H.; Evangelou, E.; Benyamin, B.; Scott, R.; Isaacs, A.; Zhao, J. H.; Willems, S. M.; Johnson, T.; Gieger, C.; Grallert, H.; Meisinger, C.; Müller-Nurasyid, M.; Strawbridge, R. J.; Goel, A.; Rybin, D.; Albrecht, E.; Jackson, A. U.; Stringham, H. M.; Correa, I. R.; Farber-Eber, E.; Steinthorsdottir, V.; Uitterlinden, A. G.; Munroe, P. B.; Brown, M. J.; Schmidberger, J. title: Random glucose GWAS in 493,036 individuals provides insights into diabetes pathophysiology, complications and treatment stratification date: 2021-04-20 journal: nan DOI: 10.1101/2021.04.17.21255471 sha: ecc865573f67efa1f09b585519cdf0f536110061 doc_id: 262239 cord_uid: z91lemqi Homeostatic control of blood glucose requires different physiological responses in the fasting and post-prandial states. We reasoned that glucose measurements under non-standardised conditions (random glucose, RG) may capture diverse glucoregulatory processes more effectively than previous genome-wide association studies (GWAS) of fasting glycaemia or after standardised glucose loads. Through GWAS meta-analysis of RG in 493,036 individuals without diabetes of diverse ethnicities we identified 128 associated loci represented by 162 distinct signals, including 14 with sex-dimorphic effects, 9 discovered through trans-ethnic analysis, and 70 novel signals for glycaemic traits. Novel RG loci were particularly enriched in expression in the ileum and colon, indicating a prominent role for the gastrointestinal tract in the control of blood glucose. Functional studies and molecular dynamics simulations of coding variants of GLP1R, a well-established type 2 diabetes treatment target, provided a genetic framework for optimal selection of GLP-1R agonist therapy. We also provided new evidence from Mendelian randomisation that lung function is modulated by blood glucose and that pulmonary dysfunction is a diabetes complication. Thus, our approach based on RG GWAS provided wide-ranging insights into the biology of glucose regulation, diabetes complications and the potential for treatment stratification. significance of the RG effect estimates, although when all covariate models were individually 211 applied, nine additional signals at genome-wide significance were identified in UKBB (Table 212 1, Supplementary Table 4) . Table 3 ). Among the novel RG signals, USP47 was nominally significant in the 218 individuals of African, FAM46 and ACVR1C in the Indian and TRIM59/KPNA4 and ZC3H13 in 219 Chinese UKBB ancestry. Trans-ethnic meta-analyses combining Europeans and the other four 220 UKBB ancestral groups revealed seven novel RG signals, including those at FOXN3, EPS8 and 221 ISG20L2 (Table 1) . Overall, while being only 16,554 individuals larger in sample size than the 222 European meta-analysis, the trans-ethnic analysis expanded the novel locus discovery for RG 223 by one tenth (Supplementary Table 5) . 224 225 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. A316T has a single amino acid substitution in the core of the receptor transmembrane domain 291 (Figure 2c ) that leads to an alteration of the hydrogen bond network in close proximity (Video 292 S1). In A316T, residue T316 5.46 replaced Y242 3.45 in a persistent hydrogen bond with the 293 backbone of P312 5.42 one turn of the helix above T316 5.46 (Figures 2d-e, Video S1) . This 294 triggers a local structural rearrangement that could transmit to the intracellular G protein 295 binding site through transmembrane helix 3 (TM3) and TM5. A structural water molecule was 296 found close to position 5.46 in both A316T and WT (water cluster 5, Figure 2f ). The same 297 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint water bridged the backbone of Y241 3.44 and A316 5.46 in WT, or the backbone of Y241 3.44 and 298 the side chain of T316 5.46 in A316T. Given the importance of conserved water networks in the 299 process of activation of class A GPCRs 28,29 , the presence of a stable hydrated spot close to 300 position 5.46 30 corroborates this site as important for tuning the intracellular conformational 301 landscape of GLP-1R. Also, a stabilising role for the water molecules at the binding site of the 302 G protein (water cluster apha5, Figure 2f ) cannot be ruled out. Note that our results differ 303 from a previous analysis of A316T dynamics 22 , which used an early model that does not fully 304 capture the full structural features of the current active GLP-1R models. 305 In analogy with A316T, molecular simulations with the G168S variant indicate the formation 307 of a stable new hydrogen bond between the side chain of residue S168 1.63 and A164 1.59 , 308 located one turn above on the same helix (Video S2, Figure 2g ). This moved the C-terminal 309 end of TM1 closer to TM2 and reduced the overall flexibility of ICL1 (Figure 2h) , which could 310 potentially alter the role of ICL1 in G protein activation. In contrast to A316T and G168S, the 311 site of mutation R421W is consistent with persistent interactions with the G protein. 312 Simulations predicted a propensity of R421W to interact with a different region of the G 313 protein -subunit to that engaged by WT (Figure 2i) . 314 315 For a broader view of the impact of GLP1R coding variation, we screened an additional 178 316 missense variants identified from exome sequencing 31 for exendin-4-induced mini-Gs 317 coupling and endocytosis (Figures 2j-k, Supplementary Table 12) . 110 variants showed a 318 reduced response in either or both pathways ("LoF1"), and 67 displayed a specific response 319 deficit that was not fully explained by differences in GLP-1R surface expression ("LoF2"), with 320 many of these defects being larger than in the analysis in Figure 2a . 321 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint WARS; with combined results from ileum and colon also highly enriched, including the novel 346 NMT1 and the established FADS1/3 and MADD genes (Figure 1a, Supplementary Tables 15a-347 b). Moreover, epigenetic annotations using the GARFIELD tool highlighted significant 348 (P<2.5×10 -5 , Methods) enrichment of RG-associated variants in foetal large intestine, as well 349 as blood, liver and other tissues (Supplementary Figure 4, Supplementary Table 16 Figure 1a , Supplementary 367 Table 19 ). These traits represent highly branched galactosylated sialylated glycans (attached 368 to alpha1-acid protein -an acute-phase protein 41 ), known to lead to chronic low-grade 369 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. that patients with diabetes are at an increased risk of death from the viral infection COVID-391 19 52 , with pulmonary dysfunction contributing to mortality 53 . Our data therefore support the 392 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We previously highlighted diverse effects of FG and T2D loci on pathophysiological processes 410 related to T2D development by grouping associated loci in relation to their effects on multiple 411 phenotypes 6 . Cluster analysis of the RG signals with 45 related phenotypes identified three 412 separate clusters that give insights into the aetiology of glucose regulation and associated 413 disease states (Methods, Figure 1a , Supplementary Table 23, Supplementary Figures 5a-d) . 414 Cluster 1 ("metabolic syndrome" cluster) clearly separated 33 loci with effects on higher 415 waist-to-hip ratio, blood pressure, plasma triglycerides, insulin resistance (HOMA-IR) and 416 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint coronary artery disease risk, as well as lower testosterone and sex hormone binding globulin 417 levels in men. Cluster 3 was characterised in particular by insulin secretory defects 6 . Cluster 2 418 was less clearly defined by a primary effect on insulin release versus insulin action 3 , but 419 interestingly included a sub-cluster of 21 loci which exert protective effects on inflammatory 420 bowel disease. Moreover, cluster 2 was notable for generally reduced impact on T2D risk in 421 comparison to clusters 1 and 3, underscoring the partial overlap between genetic 422 determinants of glycaemia and T2D that is known to exist 55 . We used RG (mmol/l) measured in plasma or in whole blood (corrected to plasma level using 448 the correction factor of 1.13). Individuals were excluded from the analysis, if they had a 449 diagnosis of T2D or were on diabetes treatment (oral or insulin). Individual studies applied 450 further sample exclusions, including pregnancy, fasting plasma glucose equal to or greater 451 than 7 mmol/l in a separate visit, when available, and having type 1 diabetes. Detailed 452 descriptions of study-specific RG measurements are given in Supplementary Table 1 . All 453 studies were approved by local ethics committees and all participants gave informed consent. 454 We examined the distributions of untransformed and natural logarithmic transformed RG in 455 the first set of six available cohorts. We observed that RG was approximately normally 456 distributed after natural log transformation. We then determined the variables that could 457 have a significant effect on RG by fitting several regression models using naturally log-458 transformed RG as the outcome with age, sex, BMI and time since last meal as predictors. 459 Modelling of RG revealed significant effects (P<0.05) of age, sex, BMI and time since last meal 460 (accounted for as T, T 2 and T 3 ) in these cohorts (Supplementary Table 2 ). Compared to RG 461 models without T, inclusion of T, T 2 and T 3 increased the proportion of variance explained in 462 the range of 1-6%. Thus, inclusion of this covariate is potentially equivalent to 1-6% increase 463 in study sample size. For the GWAS, we included individuals based on two RG cut-offs: <20 464 mmol/l (20) to account for the effect of extreme RG values and <11.1 mmol/l (11), which is 465 an established threshold for T2D diagnosis. We then evaluated six different models in GWAS 466 according to covariates included and cut-offs used: 1) age (A) and sex (S), RG<20 mmol/L 467 (AS20), 2) age, sex and BMI (B), RG<20 mmol/L (ASB20), 3) age and sex, RG<11.1 mmol/L 468 (AS11), 4) age, sex and BMI, RG<11.1 mmol/L (ASB11), 5) age, sex, T, T 2 and T 3 , RG<20 mmol/L 469 (AST20) and 6) age, sex, T, T 2 and T 3 and BMI, RG<20 mmol/L (ASTB20 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. imputation method implemented in the SS-Imp v0.5.5 software 65 . SNPs with imputation 499 quality score <0.7 were excluded. We then conducted inverse variance meta-analyses to 500 combine the association summary statistics from all components using METAL (version from 501 2011-03-25) 66 . We focused our meta-analyses on models AS20 (17 cohorts, Nmax=481,150) 502 and AST20 (when time from last meal was available in the cohort) (12 cohorts, Nmax=438,678). 503 For FHS cohort, where no information was available for individuals with RG>11.1 (an 504 established threshold for 2hGlu concentration, which is a criterion for T2D diagnosis), AS11 505 model results were used. In order to maximise the association power while taking into 506 account T, we also performed meta-analysis using AST20 (when time from last meal was 507 available in the cohort) combined with AS20 (otherwise) and we termed this analysis as 508 AS20+AST20 in the following text (17 cohorts, Nmax=480,250). 509 A signal was considered to be associated with RG if it had reached genome-wide significance 510 (P<5x10 -8 ) in the meta-analysis of UKBB and other cohorts in either of our two models of 511 interest (AS20) or (AST20) or in their combination (AS20+AST20). We report the P-value from 512 the combined model, unless otherwise stated. Full results from all models are provided in the 513 Supplementary Table 3 . All the follow-up analyses were conducted using the combined 514 AS20+AST20 model. We checked for nominal significance (P<0.05) and directional consistency 515 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint of the effect sizes for the selected leads in the combined model in UKBB results vs other 516 cohort results. We further extended the check between UKBB results and meta-analysis of 517 other cohorts including FG GWAS meta-analysis 54 excluding overlapping cohorts. This meta-518 analysis conducted in METAL was sample size and P-value based due to the measures being 519 at different scale (natural logarithm transformed RG and untransformed FG). 520 521 We performed GWAS in those non-European populations within UKBB that had a sample size 523 of at least 1,500 individuals. These were Black (N=7,644), Indian (N=5,660), Pakistani 524 (N=1,747) and Chinese (N=1,503). We further meta-analysed our European cohorts with the 525 trans-ethnic UKBB cohorts. The analyses were performed with BOLT-LMM and METAL. 526 527 To evaluate sex-dimorphism in our results, we meta-analysed the UKBB and the Vanderbilt 529 cohort with the GMAMA software 67 , which provides a 2 degrees of freedom (df) test of 530 association assuming different effect sizes between the sexes. We considered a signal to show 531 evidence of sex-dimorphism if the 2 df test P-value was <5x10 -8 and if the sex heterogeneity 532 P-value (1 df) was <0.05. 533 534 We performed a standard clumping analysis [PLINK 1.9 (v1.90b6.4) 68 criteria: P≤510 -8 , 536 r 2 =0.01, window-size=1Mb, 1000 Genomes Phase 3 data as linkage disequilibrium (LD) 537 reference panel] to select a list of near-independent signals. We then performed a stepwise 538 model selection analysis (GCTA conditional analysis) to replicate the analysis using GCTA 539 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint SmBiT in a 9:1 ratio, followed by selection with 100 µg/ml hygromycin. The resulting cell lines 564 were maintained in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% 565 penicillin/streptomycin. 566 567 Assays were performed as previously described 71 . Where stable cell lines were used (i.e. 569 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The assay was performed as previously described 71 . Where stable cell lines were used (i.e. 587 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint assay performed 24 hours later; 2) the plate was imaged as above both prior to and after 611 ligand treatment (+subsequent Mesna cleavage); 3) surface labelling quantification was 612 obtained from the pre-treatment read, and total internalised receptor was obtained from the 613 post-treatment read. 614 615 Technical replicates within the same assay were averaged to give one biological replicate. For 617 concentration-response assays (Figures 2a and 2b) , ligand-induced responses were analysed 618 by 3-parameter fitting in Prism 8.0 (GraphPad Software). As a composite measure of 619 agonism 75 , log10-transformed Emax/EC50 values were obtained for each ligand/variant 620 response. The wild-type response was subtracted from the variant response to give 621 ∆log(max/EC50), a measure of gain-or loss-of-function for the variant relative to wild-type. For transient transfection assays (Figure 2j) , responses were normalised to wild-type 631 response and log10 transformed to give Log ∆ response. Additionally, the impact of differences 632 in surface expression on functional responses was determined by subtracting log-633 transformed normalised expression level from log-transformed normalised response. 634 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The active state structure of GLP-1R in complex with OXM 27 and Gs protein was modelled as 644 previously described 30 and used to simulate the WT GLP-1R and G168S, A316T and R421W. 645 The systems were prepared for molecular dynamics (MD) simulations and equilibrated as 646 reported in 30 . AceMD3 76 was employed for production runs (four MD replicas of 500 ns each). 647 AquaMMapS analysis 77 was performed as previously described 30 . 648 649 After selecting the signals with each region based on different M-A results from AS20, AST20 651 and AS20+AST20 models, we further performed a credible set analysis to obtain a list of 652 potential causal variants for each of the 143 selected signals. Based on the method adopted 653 from 78 under the assumption that there is one causal variant within each region, we created 654 99% credible sets. We also calculated credible sets for the trans-ethnic meta-analysis and 655 compared the results between the European only and trans-ethnic meta-analyses. 656 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We applied the GARFIELD tool v2 86 on the RG AS20+AST20 meta-analysis results to assess 703 enrichment of the RG-associated variants within functional and regulatory features. 704 GARFIELD integrates various types of data from a number of publicly available cell lines. Those 705 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. We further performed co-localization analysis using whole blood gene expression-QTL (eQTL) 724 data provided by eQTLGen 39 and AS20+AST20 meta-analysis results. Only cis-eQTL data from 725 eQTLGen was incorporated to reduce the computational burden. The COLOC2 Bayesian-726 based method 88 was used to interrogate the potential co-localization between RG GWAS 727 signals and the genetic control of gene expression. We first extracted the RG GWAS test 728 statistics of all the SNPs within +/-1Mb region around the 143 RG signals. Then, for each RG 729 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint signal, we matched the eQTLGen results with the RG results and performed COLOC2 analysis 730 evaluating the posterior probability (PP) of five hypotheses for each region: H0, no 731 association; H1, GWAS association only; H2, eQTL association only; H3, both GWAS and eQTL 732 association, but not co-localised; and H4, both GWAS and eQTL association and co-localised. 733 Only GWAS signals with at least one nearby gene/probe reaching PP (H4) ≥ 0.5 were reported. 734 735 We assessed the genetic association between 143 RG signals and 113 human blood plasma 737 N-glycome traits using previously published genome-wide summary association statistics 89 . 738 The description of the analysed traits and details of the association analysis can be found 739 elsewhere 40 . We considered associations to be significant when P-740 value<0.05/113/143=3.09e-6 (after Bonferroni correction). Association was considered as 741 suggestive when P-value<10 We considered P≤0.05 as the nominal significant level. 752 753 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We applied a bidirectional two-sample MR strategy to investigate causality between RG and 755 lung function, as well as T2D and lung function using independent genetic variants as 756 instruments. MR can provide estimates of the effect of modifiable exposures on an outcome 757 (e.g. disease) unaffected by classical confounding or reverse causation, whenever randomised 758 clinical trials are not feasible. We looked for evidence for the presence of a causal effect of 759 RG and T2D on two lung function phenotypes; FVC and FEV1 in a two-sample MR setting. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We tested the ability of the RG genetic effects to predict RG, T2D and HbA1c. We are clumped so that they are largely independent of each other and thus their effects can be 800 summed. To assess predictive power, PRS for RG, T2D and FG were regressed onto the 801 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint phenotypes of interest (i.e. RG, T2D and HbA1c) providing the coefficient of determination 802 (R 2 ) as an estimate for the correlation between the phenotype and the PRS in the VUMC 803 cohort. All models were adjusted for age, four principal components, sex and the cohort-804 specific batch effect. Since the optimal P-value threshold for including SNPs in the PRS is 805 unknown a priori, PRS are calculated over a range of thresholds and regressed onto the 806 phenotype of interest, optimising prediction accordingly. The R 2 estimates for each trait were 807 derived by subtracting the R 2 from the null model (Phenotype ~ sex + age + 4 principal 808 components + batch) from the R 2 from the full model (Phenotype ~ PRS + sex + age + 4 809 principal components + batch) which contains the PRS at the best predicting P-value (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Instrumentation Grant S10OD017985 and S10RR025141; and CTSA grants UL1TR002243, 1194 UL1TR000445, and UL1RR024975. Genomic data are also supported by investigator-led 1195 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 194,008 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Error bars represent standard errors for βRG and mini-Gs coupling in response to GLP-1 1329 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint represented as mean ± standard error after normalization to wild-type response and log10-1354 transformation. Variants are categorised as "LoF1" when the response 95% confidence 1355 interval falls below zero or "LoF2" where expression-normalised 95% confidence interval falls 1356 below zero. (k) GLP-1R snake plot created using gpcr.com summarizing the functional impact 1357 of missense variants; for residues with >1 variant, classification is applied as 1358 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.17.21255471 doi: medRxiv preprint Bi-directional drug-microbiome interactions of 960 anti-diabetics The metabochip, a custom genotyping array for genetic studies of 962 metabolic, cardiovascular, and anthropometric traits Genotype imputation A new multipoint 967 method for genome-wide association studies by imputation of genotypes A. minimac2: faster genotype imputation Mixed-model association 972 for biobank-scale datasets Efficient Bayesian mixed-model analysis increases association power 974 in large cohorts A global reference for human genetic variation Evaluation and application of summary statistic 978 imputation to discover new height-associated loci METAL: fast and efficient meta-analysis of 980 genomewide association scans GWAMA: software for genome-wide association meta-982 analysis Second-generation PLINK: rising to the challenge of larger and 984 richer datasets 988 70. Fang, Z. et al. The Influence of Peptide Context on Signaling and Trafficking of 989 Glucagon-like Peptide-1 Receptor Biased Agonists Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic 992 Trafficking and Degradation in Pancreatic Beta Cells Using the Flp-In T-Rex system to regulate 994 GPCR expression A BaSiC tool for background and shading correction of optical 996 microscopy images Automated method for the rapid and precise estimation of 998 adherent cell culture characteristics from phase contrast microscopy images A Scale of Agonism and Allosteric Modulation for Assessment of 1001 Bias, and Receptor Mutation ACEMD: Accelerating Biomolecular 1003 Dynamics in the Microsecond Time Scale An 1005 Alternative Tool to Monitor the Role of Water Molecules During Protein-Ligand 1006 Association A Bayesian measure of the probability of false discovery in genetic 1008 epidemiology studies 1011 80. Genomes Project, C. et al. A map of human genome variation from population-scale Fine-mapping type 2 diabetes loci to single-variant resolution 1052 using high-density imputation and islet-specific epigenome maps PRSice-2: Polygenic Risk Score software for biobank-scale Sex-dim: women SLC43A2 Chr: chromosome; Pos: Position GRCh37; nonsyn: non-synonymoys; sex-dim: sex-dimorphic EAF: allele frequency of the random glucose (RG) raising allele. A signal was annotated as 1277 "European" if it had reached genome-wide significance (P<5x10 -8 ) in the meta-analysis of European cohorts in either of our two models of interest with adjustment for age, sex with or 1279 without time since last meal (where available) along with exclusion of extreme 1280 hyperglycaemia (RG>20 mmol/L) or in their combination. A signal was annotated as 1281 "European, UKBB only" if it had reached genome-wide significance (P<5x10 -8 ) in UKBB in any 1282 of the six RG models (Methods). The EAF and P-values reported here are from the combined 1283 RG model. Heterogeneity among studies was assessed using the I 2 index. The Cochran's Q-1284 test (for sex heterogeneity representing the differences in allelic effects between sexes) P-1285 value is also shown No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Asterisks annotate novel for glycaemic traits RG 1304 signals. Track 1: RG Manhattan plot reporting -log10(P-value) for RG-GWAS meta-analysis, 1305 signals reaching genome-wide significance (P-value<510 -8 ) are coloured in red. Crosses 1306 annotate genome-wide significant loci that show evidence of sex heterogeneity (Methods): 1307 blue crosses indicate signals with larger effects in men, green crosses -signals with larger 1308 effects in women. Track 2: Effects of RG genome-wide significant on four GIP/GLP-1-related 1309 traits GWAS. The colours of the dotted lines indicate four GIP/GLP-1-related traits, grey dot -1310 signals reaching P-value<0.01 for a GIP/GLP-1-related trait of lead RG variants in 204 1314 gut-microbiome PheWAS. Light green dots -RG lead SNPs, red dots -variants with significant 1315 effects at P-value<10 -4 . Track 5: MetaXcan results for 10 selected tissues for RG GWAS meta-1316 analysis (Methods), signals colocalising with genes (P-value<510 -6 ) are plotted for each 1317 tissue. (b) Credible set analysis of RG associations in the European meta-analysis. Variants 1318 from each of the RG signal credible sets are grouped based on their posterior probability (the 1319 percentiles labelled on the sides of the bar). SNP variants with posterior probability >80%, 1320 along with their locus names are provided No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. stimulation. The grey shaded area corresponds to the 95% confidence interval of the slope of 1330 the regression analysis (β=-0.027, 95%CI[-0.036 -{-0.016}], P-value=0.0001), which explained 1331 65% of the variance in these associations 42 backbone computed during MD simulations of GLP-1R WT and A316T; the 1342 cut-off distance for hydrogen bond is shown. (e) Difference in the hydrogen bond network 1343 between GLP1-R WT and A316T. (f) Analysis of water molecules within the TMD of GLP1-R 1344 WT and A316T suggests minor changes in the local hydration of position 5.46 (unperturbed 1345 structural water molecule). (g) Distributions of the distance between position 168 1.63 and 1346 Y178 2.48 during molecular dynamics simulations of GLP-1R WT and G168S. (h) During MD 1347 simulations the GLP-1R isoform S168G showed increased flexibility of ICL1 and H8 compared 1348 to WT, suggesting a different influence on G protein intermediate states. (i) Contact 1349 differences between Gs and GLP-1R WT or W421R; the C terminal of W421R H8 made more 1350 interactions with N terminal segment of Gs  subunit. (j) Mini-Gs and GLP-1R We thank participants in deCODE genetic studies whose contribution made this work Alejandra Tomas has received grant funding from Sun Pharmaceuticals. 1243