Abstract
The G-protein-coupled receptor accessory protein MRAP2 is implicated in energy control in rodents, notably via the melanocortin-4 receptor1. Although some MRAP2 mutations have been described in people with obesity1,2,3, their functional consequences on adiposity remain elusive. Using large-scale sequencing of MRAP2 in 9,418 people, we identified 23 rare heterozygous variants associated with increased obesity risk in both adults and children. Functional assessment of each variant shows that loss-of-function MRAP2 variants are pathogenic for monogenic hyperphagic obesity, hyperglycemia and hypertension. This contrasts with other monogenic forms of obesity characterized by excessive hunger, including melanocortin-4 receptor deficiency, that present with low blood pressure and normal glucose tolerance4. The pleiotropic metabolic effect of loss-of-function mutations in MRAP2 might be due to the failure of different MRAP2-regulated G-protein-coupled receptors in various tissues including pancreatic islets.
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Data availability
All relevant data have been included in the manuscript and/or in its supplementary tables and figures. Source data are available online for Extended Data Figs. 1, 2, 4 and 5. Targeted DNA-seq data of patients deficient in MRAP2 were deposited in the NCBI Sequence Read Archive under PRJNA564478.
Code availability
Code to perform analyses in this manuscript are available from the authors upon reasonable request (A.B., M.D. and M.C.).
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Acknowledgements
We are grateful to all individuals included in the different cohort studies. We thank A. Abderrahmani (University of Lille) for his technical advice. We thank L. Chan (Queen Mary University of London) for providing the plasmids including MRAP2 and MC4R. We thank F. Allegaert and N. Larcher for DNA collection and storage. We thank Endocells for providing the pancreatic beta cell line EndoC-βH1. We thank the Type 2 Diabetes Knowledge Portal (http://www.type2diabetesgenetics.org/gene/geneInfo/MRAP2) and the groups that provided data to this resource.
This work was supported by grants from the French-speaking Society of Diabetes (Société Française du Diabète) to A.B., from the European Foundation for the Study of Diabetes/Lilly (to A.B.), from the French National Research Agency (ANR-10-LABX-46 (European Genomics Institute for Diabetes) and ANR-10-EQPX-07-01 (LIGAN-PM) to P.F.), from the European Research Council (ERC GEPIDIAB-294785 to P.F. and ERC Reg-Seq-715575 to A.B.), from FEDER (to P.F.) and from the ‘Région Nord Pas-de-Calais’ (to P.F.). A.B. was supported by Inserm.
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P.F. and A.B. conceived the idea for the study and supervised the analyses. M.B., J.M., M.H., A.D., R.B., H.L., E.D., B.T., E.V., J.P., J.T., A.G., M.B., M.D., S.G., M.C. and A.B. performed the experiments and/or analyses. M.B. and A.B. wrote the first draft of the paper. P.F. revised the paper. S.F., G.C., J.-M.B., C.L.-M., M.T., R.S., J.W., C.A., J.K.-C., F.P., R.B., B.B., M.M. and P.F. contributed data (cohort studies or beta cell models). Furthermore, all authors critically reviewed the paper and approved the report for submission.
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Extended data
Extended Data Fig. 1 Design of the functional assessment of each MRAP2 mutation and its validation.
α-MSH, α-melanocyte-stimulating hormone; ACTH, adrenocorticotropic hormone; βGal, β-galactosidase; CHO, Chinese hamster ovary cells; pβGal, plasmid including the β-galactosidase gene; pCreLuc, plasmid including the firefly luciferase (Luc) gene under the control of cAMP response element (CRE); pMC4R, plasmid including MC4R; pMRAP2, plasmid including MRAP2; WT, wild-type. For the control of pMRAP2 transfection into CHO cells, we performed Western blot analyses in wild-type CHO cells and CHO cells transfected with pMRAP2. We confirmed that wild-type CHO cells did not endogenously express MRAP2 protein, whereas CHO cells transfected with pMRAP2 expressed MRAP2 protein (27 kDa, bottom asterisk), as well as its glycosylated form (29 kDa, top asterisk). For the negative control, data are cAMP reporter activity (CRE-Luc normalized with β-galactosidase), expressed as fold change after 0–30,000 pM α-MSH (left) or ACTH (right), relative to 0 pM. Data are the mean ± sem of three independent experiments in technical triplicates. Fold change was computed by dividing normalized luciferase (L*) by the mean of the baseline luciferase measures. This normalized luciferase fold change (FCL*) was analyzed using a linear regression model. The mutation (M), the agonist concentration (C), as an orthogonal polynomial function of degree 3 (PC) to enable possible nonlinear relations between FCL* and C and the interaction term (MPC) between M and PC were included in the model as covariates. The model was defined as follows: \(FC_{L^ \ast } = \beta _0 + \beta _1M + MP_C + P_C + {\it{\epsilon }}\) with, \(P_C = \alpha _1C^1 + \alpha _2C^2 + \alpha _3C^3\) \(MP_C = \theta _1C^1M + \theta _2C^2M + \theta _3C^3M\) ***P < 0.001, MC4R + p.Q4* MRAP2 (red) versus MC4R + wild-type MRAP2 (black).
Extended Data Fig. 2 Effect of MRAP2 variants on MC4R activity in response to α-MSH and ACTH in CHO cells.
Data are cAMP reporter activity (CRE-Luc normalized with β-galactosidase) in CHO cells cotransfected with MC4R plasmid and wild-type or mutated MRAP2 plasmid, expressed as fold change after 0–30,000 pM α-MSH or ACTH, relative to 0 pM. Data are the mean ± sem of three independent experiments in technical triplicates. Fold change was computed by dividing normalized luciferase (L*) by the mean of the baseline luciferase measures. This normalized luciferase fold change (FCL*) was analyzed using a linear regression model. The mutation (M), the agonist concentration (C), as an orthogonal polynomial function of degree 3 (PC) to enable possible nonlinear relations between FCL* and C, and the interaction term (MPC) between M and PC were included in the model as covariates. The model was defined as follows: \(FC_{L^ \ast } = \beta _0 + \beta _1M + MP_C + P_C + {\it{\epsilon }}\) with, \(P_C = \alpha _1C^1 + \alpha _2C^2 + \alpha _3C^3\) \(MP_C = \theta _1C^1M + \theta _2C^2M + \theta _3C^3M\) *P < 0.05, **P < 0.01, ***P < 0.001, MC4R + mutated MRAP2 (colors) versus MC4R + wild-type MRAP2 (black).
Extended Data Fig. 3 Co-segregation of p.N77S (carried by participants no. 4 and 9) and p.P195L (carried by participants no. 6 and 10) with obesity in two families.
HDL, high-density lipoprotein; HT, hypertension; MS, metabolic syndrome; strikethrough MS, no metabolic syndrome; NBP, normal blood pressure; NG, normal fasting glucose; NM, mutation carrier; NN, wild type; NW, normal weight; Ob, obese; PD, prediabetes; SOb, severely obese; TG, triglycerides.
Extended Data Fig. 4 Expression of MRAP2 in human pancreatic islets and beta cells.
a, PCR-free quantification of MRAP2 mRNA levels in a panel of human tissues. b, Western blot analyses of human islets and EndoC-βH1 cells using MRAP2 antibody. FACS-sorted beta cell, pancreatic beta cells sorted by flow cytometry; LCM beta cell, pancreatic beta cells obtained by laser capture microdissection. Three independent experiments showed similar results for Extended Data Fig. 4b.
Extended Data Fig. 5 Impaired insulin secretion from EndoC-βH1 cells treated with siRNA targeting MRAP2.
EndoC-βH1 cells were transfected with control nontargeting pool siRNA (siNTP) or MRAP2 siRNA (siMRAP2) and were analyzed 72 h thereafter. Insulin secretion (percentage of secretion of the total insulin content) was analyzed in response to 60 min of incubation with 0.5 mM glucose, followed by 60 min of incubation with 16.7 mM glucose. Data are box and whisker plots (with the minimum and the maximum) of four independent experiments (Left). Fold change data are mean values ± sem of four independent experiments (right). Fold change of insulin secretion for siMRAP2 was analyzed using a linear regression adjusted for experimental conditions (operator and date). Glc, glucose.
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Source Data Extended Data Fig. 2
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Baron, M., Maillet, J., Huyvaert, M. et al. Loss-of-function mutations in MRAP2 are pathogenic in hyperphagic obesity with hyperglycemia and hypertension. Nat Med 25, 1733–1738 (2019). https://doi.org/10.1038/s41591-019-0622-0
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DOI: https://doi.org/10.1038/s41591-019-0622-0
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