Monoallelic pathogenic variants in LEPR do not cause obesity

Abstract

Individuals with obesity caused by biallelic pathogenic LEPR (leptin receptor) variants can benefit from setmelanotide, the novel MC4R agonist. An ongoing phase 3 clinical trial (NCT05093634) includes individuals with obesity who carry a heterozygous LEPR variant, although the obesogenic impact of these variants remains incompletely evaluated. The aim of this study was to functionally assess heterozygous variants in LEPR and to evaluate their effect on obesity. We sequenced LEPR in ∼10,000 participants from the French RaDiO study. We found 86 rare heterozygous variants. Each identified variant was then investigated in vitro using luciferase and western blot assays. Using the criteria of the American College of Medical Genetics and Genomics (ACMG), including the strong criterion related to functional assays, we found 12 pathogenic LEPR variants. Most heterozygotes did not present with obesity, and we found no association between these pathogenic variants and body mass index (BMI). This lack of association between pathogenic LEPR variants and obesity risk or BMI was confirmed using exome data from 200,000 individuals in the UK Biobank. In the literature, among 55 reported heterozygotes for of a rare pathogenic LEPR variant, only 27% had obesity. In conclusion, monoallelic pathogenic LEPR variants were functionally tested, and they do not elevate the risk of obesity or BMI levels. This raises questions about the use of setmelanotide, a costly drug with potential side effects, based solely on the presence of a heterozygous LEPR variant.

Keywords: LEPR; functional genetics; molecular diagnosis; monogenic obesity; precision medicine; rare variants.

Figure 1

Location of rare LEPR variants in the RaDiO study Variants highlighted in red represent P/LP variants. Purple bars represent fibronectin type-III 2 domains; the orange bar represents an immunoglobulin-like domain; the green bar represents the leptin-binding region; the light-blue bar represents a WSXWS motif; the dark-blue bar represents a box 1 motif; and the yellow bar represents the domain that is required for JAK2 activation and STAT3 phosphorylation. The source of protein domains was UniProt (#P48357).

Figure 2

Loss-of-function LEPR variants in the RaDiO study from in vitro assays (A) Loss-of-function LEPR variants from luciferase assays. HEK293 cells were transiently transfected with the plasmid encoding the β-galactosidase gene for normalization plus the LEPR expression plasmid (mutated or wild type) or the empty vector, the JAK2 construct, and the STAT3 reporter plasmid. Two days after transfection, cells were incubated with serum-free fresh medium supplemented with or without leptin for 6 h. Luciferase activity was calculated as relative luminescence units normalized against β-galactosidase activity. Data are fold changes to luciferase activity of wild-type condition without leptin (mean ± SEM). All experiments were performed in triplicate, and each experiment was repeated six times. p values were calculated related to “WT + L” by unpaired Mann-Whitney tests. ^∗p <0.05; ^∗∗p <0.01; ^∗∗∗p <0.001; ^∗∗∗∗p <0.0001. (B) Loss-of-function LEPR variants from western blots. HEK293 cells were transiently transfected with the empty vector, or the LEPR expression plasmid (mutated or wild type), along with the JAK2 construct. After 48 h, cells were switched to serum-free medium and stimulated (or not) with 200 ng/mL leptin. 30 μg of protein was electrophoresed on a 10% precast gel and transferred onto a membrane. Membranes were immunoblotted with antibodies against phosphorylated STAT3 and β-actin. These membranes are representative of three independent experiments. EV, empty vector; FC, fold change; L, leptin; NT, LEPR not transfected (negative control); WT, wild type.