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.

Monogenic forms of obesity have been identified in ≤10% of severely obese European patients. However, the overall spectrum of deleterious variants (point mutations and structural variants) responsible for childhood severe obesity remains elusive. In this study, we genetically screened 225 severely obese children from consanguineous Pakistani families through a combination of techniques, including an in-house–developed augmented whole-exome sequencing method (CoDE-seq) that enables simultaneous detection of whole-exome copy number variations (CNVs) and point mutations in coding regions. We identified 110 (49%) probands carrying 55 different pathogenic point mutations and CNVs in 13 genes/loci responsible for nonsyndromic and syndromic monofactorial obesity. CoDE-seq also identified 28 rare or novel CNVs associated with intellectual disability in 22 additional obese subjects (10%). Additionally, we highlight variants in candidate genes for obesity warranting further investigation. Altogether, 59% of cases in the studied cohort are likely to have a discrete genetic cause, with 13% of these as a result of CNVs, demonstrating a remarkably higher prevalence of monofactorial obesity than hitherto reported and a plausible overlapping of obesity and intellectual disabilities in several cases. Finally, inbred populations with a high prevalence of obesity provide unique, genetically enriched material in the quest of new genes/variants influencing energy balance.

Recent advances in genetic analysis have significantly helped in progressively attenuating the heritability gap of obesity and have brought into focus monogenic variants that disrupt the melanocortin signalling. In a previous study, next generation sequencing has revealed a monogenic aetiology in ∼50% of the children with severe obesity from a consanguineous population in Pakistan. Here we assess rare variants in obesity causing genes in young adults with severe obesity from the same region. Genomic DNA from randomly selected 128 young adult obese subjects (BMI=37.2 ± 0.3; age=18.4 ± 0.3 years) was screened by conventional or augmented whole exome analysis for point mutations and copy number variants (CNVs). Leptin, insulin and cortisol levels were measured by ELISA. We identified thirteen subjects carrying 13 different pathogenic or likely pathogenic variants in LEPR, PCSK1, MC4R, NTRK2, POMC, SH2B1 and SIM1. We also identified for the first time in the human, two homozygous stop-gain mutations in ASNSD1 and IFI16 genes. Inactivation of these genes in mouse models has been shown to result in obesity. Additionally, we describe 9 homozygous mutations (7 missense, 1 stop-gain and 1 stop-loss) and 4 copy-loss CNVs in genes or genomic regions previously linked to obesity-associated traits by genome-wide association studies (GWAS). Unexpectedly, in contrast to obese children, pathogenic mutations in LEP and LEPR were either absent or rare, in this cohort of young adults. High morbidity and mortality risks and social disadvantage of children with LEP or LEPR deficiency may in part explain this difference between the two cohorts.

BACKGROUND: Biallelic pathogenic mutations in LEP, LEPR and MC4R genes controlling central leptin-melanocortin signalling cause early onset severe obesity. However, the long-term clinical outcomes of leptin signalling deficiency are unknown. DESIGN AND OBJECTIVES: We carried out a retrospective cross-sectional clinical investigation of a large cohort of children with LEP, LEPR or MC4R deficiency, to evaluate the progression of the disease and its impact on morbidity and mortality. PARTICIPANTS: Severely obese children from 454 consanguineous families of Pakistani origin were screened for mutations in the three genes using Sanger and exome sequencing. We identified 132 probands and 13 affected family members with homozygous pathogenic mutations in LEP, LEPR or MC4R. MAIN OUTCOME MEASURES: Weight, height, and head circumference were measured by trained technicians using standardized protocols. WHO-anthro and anthroplus were used to assess BMI-standard deviation score. All affected individuals underwent detailed physical and clinical investigations by expert paediatric endocrinologist. Metabolic and oxidative stress biomarkers were measured in serum. RESULTS: We report a very high mortality in children with LEP (26%) and LEPR-deficiency (9%), mainly due to recurrent pulmonary and gastro-intestinal infections. In addition, 40% of LEP- or LEPR-deficient surviving children experienced life-threatening episodes of pulmonary or gastro-intestinal infections. Oxidative stress as assessed by biomarkers, was significantly higher in LEP deficiency compared to the other two mutant groups. CONCLUSIONS: Children with congenital deficiency of leptin or its receptor suffer a high mortality rate, and severe morbidity. Although effective therapies are available for both but as yet (or to-date) are not accessible in Pakistan. An appreciation of the severe impact of leptin or leptin receptor deficiency on educational attainment, morbidity and early mortality risks should spur efforts to deliver available life-saving drugs to these children as a matter of urgency.

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