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.

We previously demonstrated that 50% of children with obesity from consanguineous families from Pakistan carried pathogenic variants in known monogenic obesity genes. Here, we have discovered a novel monogenetic recessive form of severe childhood obesity, using an inhouse computational staged approach. This included analysis of whole-exome sequencing data of 366 children with severe obesity, 1,000 individuals of the Pakistani PROMIS study, and 200K participants of the UK Biobank, to prioritize genes harbouring rare homozygous variants with putative effect on human obesity. We identified five rare or novel homozygous missense mutations predicted deleterious in five consanguineous families in P4HTM encoding Prolyl 4-Hydroxylase Transmembrane (P4H-TM). We further found two additional homozygous missense mutations in children with severe obesity of Indian and Moroccan origin. Molecular dynamics simulation suggested that these mutations destabilized the active conformation of the substrate binding domain. Most carriers also presented with hypotonia, cognitive impairment and/or developmental delay. Three of the five probands died of pneumonia during the ∼2 years of the follow up. P4HTM deficiency is a novel form of syndromic obesity affecting 1.5% of our children with obesity associated with high mortality. P4H-TM is a hypoxia inducible factor that is necessary for survival and adaptation under oxygen deprivation but the role of this pathway in energy homeostasis and obesity pathophysiology remains to be elucidated.

<p> We previously demonstrated that 50% of children with obesity from consanguineous families from Pakistan carried pathogenic variants in known monogenic obesity genes. Here, we have discovered a novel monogenetic recessive form of severe childhood obesity, using an in-house computational staged approach. This included analysis of whole-exome sequencing data of 366 children with severe obesity, 1,000 individuals of the Pakistani PROMIS study, and 200K participants of the UK Biobank, to prioritize genes harbouring rare homozygous variants with putative effect on human obesity. We identified five rare or novel homozygous missense mutations predicted deleterious in five consanguineous families in <em>P4HTM</em> encoding Prolyl 4-Hydroxylase Transmembrane (P4H-TM). We further found two additional homozygous missense mutations in children with severe obesity of Indian and Moroccan origin. Molecular dynamics simulation suggested that these mutations destabilized the active conformation of the substrate binding domain. Most carriers also presented with hypotonia, cognitive impairment and/or developmental delay. Three of the five probands died of pneumonia during the ~2 years of the follow up. <em>P4HTM</em> deficiency is a novel form of syndromic obesity affecting 1.5% of our children with obesity associated with high mortality. P4H-TM is a hypoxia inducible factor that is necessary for survival and adaptation under oxygen deprivation but the role of this pathway in energy homeostasis and obesity pathophysiology remains to be elucidated. </p>

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 <i>LEPR</i>, <i>PCSK1</i>, <i>MC4R</i>, <i>NTRK2</i>, <i>POMC, SH2B1</i> and <i>SIM1</i>. We also identified for the first time in the human, two homozygous stop-gain mutations in <i>ASNSD1 </i>and <i>IFI16 </i>genes. Inactivation of these genes in mouse models has been shown to result in obesity. Additionally<i>, </i>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 <i>LEP</i> and <i>LEPR</i> were either absent or rare, in this cohort of young adults. High morbidity and mortality risks and social disadvantage of children with <i>LEP</i> or <i>LEPR</i> deficiency may in part explain this difference between the two cohorts.