ESPE Abstracts

Background: The leptin signaling cascade is a crucial regulator of satiety and energy homeostasis in the hypothalamus, comprising extracellular leptin binding to its receptor, phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) and consecutively the generation of a central satiety signal via neuropeptide secretion. We identified three variants in the extracellular domains of the LEPR gene in two children with severe early-onset obesity and hyperphagia: a compound heterozygous deletion (p.Y411del) and missense mutation (p.W664R) in patient 1; and a heterozygous missense mutation (p.R612H) combined with two polymorphisms in patient 2. Whereas the p.W664R variant has been described in one obese patient with a homozygous mutation, the p.Y411del variant is completely unknown so far.

Objective: We investigated the functional impact of the detected variants as a potential cause for the severe obesity in our patients.

Material and methods: HEK293 cells were transfected with plasmids encoding either wildtype or mutant (p.Y411del, p.W664R or p.R612H, respectively) leptin receptor. LEPR expression and signaling following leptin stimulation was assessed via immunoblot and quantitative PCR analysis of cell lysates. Cell surface expression of the leptin receptor variants was evaluated with flow cytometry.

Results: Wildtype and mutant leptin receptors were expressed in HEK293 cells at comparable levels. Phosphorylation of STAT3 after leptin stimulation was absent in cells expressing the p.Y411del and the p.W664R variant, but only mildly reduced in cells with the p.R612H mutation. Similar results were obtained, when mimicking (compound) heterozygous leptin receptor expression with combined p.Y411del and p.W664R expression or a variant receptor in combination with wildtype receptor. Furthermore, we confirmed cell surface expression of leptin receptor variants by FACS analyses with only mild reduction in the expression of mutant leptin receptors.

Conclusion: For the first time, we detected a functional impact for the novel leptin receptor variant p.Y411del, as well as for (compound) heterozygous expression of the p.W664R variant, thus making it a likely cause for the early onset obesity in patient 1. The heterozygous p.R612H variant, however, appears unlikely to explain the phenotype of patient 2 from our experimental analyses.