Endocrine Profile and Bone Turnover Markers in Severely Obese Children from a Pakistani Population

BACKGROUND AND OBJECTIVES: The rare single gene mutations resulting in early onset extreme obesity and hyperphagia have led to the discovery of the central leptin-dependent melanocortin signaling regulating energy homeostasis, food intake and body weight. Energy imbalance is known to influence other physiological mechanisms such as neuroendocrine, reproductive, metabolic and immune functions. Excessive obesity has also been shown to impact bone formation and mineralization as evidenced mainly through imaging techniques. However, the effects of obesity on bone metabolism have remained controversial and often conflicting in various reports presumably due to the heterogeneity of the disease and differences in age, sex and ethnicity of subjects under investigation. Monogenic obesity provides an exceptionally unique paradigm to study the physiological phenotype in relation to specific energy-impaired states in the human. In view of the foregoing, the present study aims to first identify cases of monogenic obesity by screening, a group of children with early onset severe obesity from consanguineous families and subsequently to assess bone metabolism in affected individuals using specific bone turnover biomarkers. In addition, associated changes in metabolic hormone levels are recorded. MATERIALS AND METHODS: Initially, 130 unrelated severely obese children from consanguineous families were recruited from the central Punjab province of Pakistan. The subjects, 0.3-13 years of age, had a body weight percentile >97 and a BMI SDS for age ≥3.0. Anthropometric data and information about family and medical history were recorded. In the first phase of investigation, DNA of all subjects was screened for leptin (LEP) and melanocortin-4 receptor (MC4R) genes mutations, in the coding regions. Subjects found negative for these mutations were subsequently screened by microdroplet PCR targeted against a panel of 27 known obesity associated genes and next generation sequencing. Serum from subjects identified with monogenic obesity and from a control group of 26 age-matched children with normal body weight, was analyzed for bone specific turnover biomarkers, osteocalcin (OC), osteopontin (OPN), osteoprotegerin (OPG) and sclerostin (SOST) using multiplex analyte profiling. In addition, serum levels of leptin, insulin and cortisol were assessed by enzyme linked immunosorbent assay (ELISA). Thyroid stimulating hormone (TSH) and thyroid hormones (T3 and T4) were determined by electro-chemiluminescence immunoassay (ECLIA). RESULTS: The two-step genetic analysis of 130 children with morbid obesity, identified 42 probands with lossof- function homozygous mutations in LEP, leptin receptor (LEPR), or MC4R genes. Amongst these, 23 probands were identified with mutations in LEP, 11 with mutations in LEPR and 8 children with mutations in the MC4R gene. Eleven of the 18 variants identified in the 3 genes associated with obesity, are reported here for the first time. Bone metabolism in affected subjects, was assessed by specific serum bone turnover markers. Serum levels of bone formation indicators, osteocalcin and osteopontin, were significantly lower in LEP and LEPR deficient subjects compared with controls. In contrast, in MC4R deficient children, levels of these two biomarkers were remarkably raised over values observed for all other groups. Serum concentration of bone resorption biomarkers, osteoprotegerin and sclerostin, for the three mutant groups were not remarkably different from the values of normal weight subjects. However, mean sclerostin levels in children with MC4R mutations tended to be lower than those with LEP and LEPR defects and of the control group. As expected, leptin levels were undetectable in subjects with LEP mutations. Hyperleptinemia was more pronounced in subjects with LEPR deficiency compared to those with MC4R deficiency. Insulin levels though raised in all affected subjects were significantly higher in children with MC4R deficiency whereas serum cortisol concentrations were significantly elevated in LEP deficient children compared to all other groups. Interestingly, TSH, T3 and T4 levels in all affected subjects were unremarkable and within the normal range. CONCLUSIONS: The present data in conformity with previous reports in this population, demonstrate a relatively high prevalence (32%) of monogenic obesity among severely obese children. Eighteen different known or novel loss-of-function mutations were identified in LEP, LEPR and MC4R genes. Assessment of bone metabolism in affected subjects revealed a consistent deficit in bone formation in subjects with leptin or leptin receptor deficiency. These results indicate an impaired osteogenic activity and further support a substantial role of leptin in bone homeostasis. Remarkably, opposite alterations in bone turnover presumably due to an up-regulation of bone formation, were associated with MC4R deficiency. The present data advocate investigation of bone health preferably using a combination of imaging and biochemical techniques in cases of severe obesity for individualized management or treatment.