ESPE Abstracts

Background: Monogenic obesity (MO) is a rare form of obesity caused by mutations in a single gene, and characterized by being severe and with early onset. The leptin-melanocortin pathway is the main pathway controlling satiety and food intake. Genetic variants in this pathway have been implicated in MO with many being identified in consanguineous populations. Another group of neuronal proteins important in the development of neuronal circuits linked to energy balance are the class 3 semaphorins (SEMA3A–G) and their receptors Plexin A (PLXNA1-4).

Aims: To investigate severe cases of early-onset obesity in the Qatari population and to functionally validate the role of PLXNA1 in obesity, using an in-vitro model by T-cell enrichment, and an in-vivo zebrafish model.

Methods: Children with negative MO gene panel findings (including 52 obesity-associated genes) were sequenced using WGS. Variant prioritization and filtration were conducted such that variants were considered when they met the following criteria: Snpeff moderate or high impact, CADD phred score => 20, and MAF < 1% in gnomAD, gnomAD middle east, GME, and QGP frequencies. Gene expression analysis was conducted on T-cells obtained from the patient and his family members for PLXNA1, and four lipid metabolism markers; PPARG, FASN, CPT1A, and SERBP-1c. Gene Knockdown on 1-4 cell stage zebrafish embryos was conducted using Morpholino Oligonucleotides (MOs) technology to induce transient PLXNA1 knockdown and confirmed using western blotting. Obesity was characterized using several assays such as yolk sac size assessment, lipid content quantification, metabolic rate assessment, and the measurement of leptin, cAMP, and triglycerides levels using ELISA.

Results: We identified three rare missense variants in the PLXNA1 gene (c.3510C>G, p.I1170M; c.4127A>C, p.E1376A; c.2476T>C, p.F826L) among two children with severe early-onset obesity inherited in a compound heterozygous pattern. The T-cell enrichment findings showed that the expression of PLXNA1 and adiposity markers in the patient were increased as compared to lean control. While zebrafish knockdown of PLXNA1 orthologs gave statistically significant increases in adiposity phenotypes such as increased yolk sac size, increased lipid content, increased leptin, triglycerides, and cAMP levels, and an impairment of cellular metabolism.

Conclusion: We revealed rare variants in PLXNA1 in severely obese children with early onset obesity, and we have functionally validated the role of the gene in the zebrafish model to show an adiposity phenotype with gene knockdown. Our research lays the groundwork for exploring the role of PLXNA1 in obesity development paving the way for targeted interventions and improved management strategies.