The leptin–melanocortin pathway is pivotal in appetite and energy homeostasis. Pathogenic variants in genes involved in this pathway lead to severe early-onset monogenic obesity (MO). The MC4R gene plays a central role in leptin–melanocortin signaling, and heterozygous variants in this gene are the most common cause of MO. A targeted gene panel consisting of 52 obesity-related genes was used to screen for variants associated with obesity. Variants were analyzed and filtered to identify potential disease-causing activity and validated using Sanger sequencing. We identified two novel heterozygous variants, c.253A>G p.Ser85Gly and c.802T>C p.Tyr268His, in the MC4R gene in two unrelated patients with morbid obesity and evaluated the functional impact of these variants. The impact of the variants on the MC4R gene was assessed using in silico prediction tools and molecular dynamics simulation. To further study the pathogenicity of the identified variants, GT1-7 cells were transfected with plasmid DNA encoding either wild-type or mutant MC4R variants. The effects of allelic variations in the MC4R gene on cAMP synthesis, MC4R protein level, and activation of PKA, ERB, and CREB signaling pathways in both stimulated and unstimulated ɑ-MSH paradigms were determined for their functional implications. In silico analysis suggested that the variants destabilized the MC4R structure and affected the overall dynamics of the MC4R protein, possibly leading to intracellular receptor retention. In vitro analysis of the functional impact of these variants showed a significant reduction in cell surface receptor expression and impaired extracellular ligand binding activity, leading to reduced cAMP production. Our analysis shows that the variants do not affect total protein expression; however, they are predicted to affect the post-translational localization of the MC4R protein to the cell surface and impair downstream signaling cascades such as PKA, ERK, and CREB signaling pathways. This finding might help our patients to benefit from the novel therapeutic advances for monogenic forms of obesity.

Nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by renal unresponsiveness to the hormone vasopressin, leading to excretion of large volumes of diluted urine. Mutations in the arginine vasopressin receptor-2 (AVPR2) gene cause congenital NDI and have an X-linked recessive inheritance. The disorder affects almost exclusively male family members, but female carriers occasionally present partial phenotypes due to skewed inactivation of the X-chromosome. Here, we report a rare case of a woman affected with X-linked recessive NDI, presenting an average urinary output of 12 L/day. Clinical and biochemical studies showed incomplete responses to water deprivation and vasopressin stimulation tests. Genetic analyses revealed a novel heterozygous missense mutation (c.493G > C, p.Ala165Pro) in the AVPR2 gene. Using a combination of in-silico protein modeling with human cellular models and molecular phenotyping, we provide functional evidence for phenotypic effects. The mutation destabilizes the helical structure of the AVPR2 transmembrane domains and disrupts its plasma membrane localization and downstream intracellular signaling pathways upon activation with its agonist vasopressin. These defects lead to deficient aquaporin 2 (AQP2) membrane translocation, explaining the inability to concentrate urine in this patient.

The leptin-melanocortin pathway is pivotal in appetite and energy homeostasis. Pathogenic variants in genes involved in this pathway lead to severe early-onset monogenic obesity (MO). The MC4R gene plays a central role in leptin-melanocortin signaling, and variants predominantly heterozygous in this gene are the most common cause of MO. We identified two novel heterozygous variants c,253A>G p.Ser85Gly and c.802T>C p.Tyr268His in the MC4R gene in two unrelated patients with morbid obesity and evaluated the functional impact of these variants. A targeted gene panel consisting of 52 obesity-related genes was undertaken. Variants were analyzed and filtered to identify potential disease-causing and validated using Sanger sequencing. The impact of the variants on the MC4R gene using in silico prediction tools and molecular dynamics simulation was assessed. To further study the pathogenicity of the identified variants, GT1-7 cells were transfected with plasmid DNA encoding either wild-type or mutant MC4R variants. The effects of allelic variations in MC4R gene on cAMP synthesis, MC4R protein level, and activation of ERB and CREB signaling pathways in both stimulated and unstimulated ɑ-MSH paradigms were determined for their functional implications. In-silico analysis suggested that the variants destabilized the MC4R structure and affect the overall dynamics of the MC4R protein, possibly leading to intracellular receptor retention. In-vitro analysis of the functional impact of these variants showed a significant reduction in cell surface receptor expression and impaired extracellular ligand binding activity, leading to reduced cAMP production. In-vitro analysis assays and in silico analysis revealed the pathogenicity of the two novel MC4R variants identified. Our analysis shows that the variants do not affect total protein expression; however, they are predicted to affect the post-translational localization of MC4R protein to the cell surface and impair downstream signaling cascades. This finding might help our patients to benefit from the novel therapeutic advances for monogenic forms of obesity.