Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin-melanocortin pathway in controlling energy balance, appetite and body weight . The majority of loss-of-function mutations (mostly recessive or co-dominant) have been identified in genes that are directly involved in leptin-melanocortin signaling. These genes, however, only explain obesity in<5% of cases, predominantly from outbred populations . We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity. These data suggested that new monogenic forms of obesity could also be identified in this population. Here we identify and functionally characterize homozygous mutations in the ADCY3 gene encoding adenylate cyclase 3 in children with severe obesity from consanguineous Pakistani families, as well as compound heterozygous mutations in a severely obese child of European-American descent. These findings highlight ADCY3 as an important mediator of energy homeostasis and an attractive pharmacological target in the treatment of obesity.

Objective: Single gene mutations leading to severe obesity have so far been identified in 3-5% cases in European populations. However, prevalence of these pathogenic mutations has not systematically been examined in specific consanguineous populations. Here we describe the incidence of obesity-associated mutations through a step-wise sequence analysis, in a cohort of 73 Pakistani children with severe obesity from consanguineous families. Methods: Initially, all subjects were screened for mutations in coding regions of leptin (LEP) and melanocortin 4 receptor (MC4R) genes by direct sequencing. Subjects negative for mutation in these genes were screened using microdroplet PCR enrichment and NGS. Genomic structural variation was assessed by genotyping. Serum leptin, insulin, and cortisol were determined by ELISA. Results: Among 73 children with severe obesity (BMI SDS > 3.0), we identified 22 probands and 5 relatives, carrying 10 different loss-of-function homozygous mutations in LEP, leptin receptor (LEPR), and MC4R genes, including 4 novel variants. Hypercortisolemia was significantly emphasized in LEP mutation carriers. Conclusions: The prevalence of pathogenic mutations in genes known to directly influence leptinmelanocortin signaling is 30% in our cohort. The results of this study emphasize the desirability of undertaking systematic and in-depth genetic analysis of cases with severe obesity in specific consanguineous populations.

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