Sadia Saeed

Large, rare chromosomal deletions associated with severe early-onset obesity

Obesity is a highly heritable and genetically heterogeneous disorder. Here we investigated the contribution of copy number variation to obesity in 300 Caucasian patients with severe early-onset obesity, 143 of whom also had developmental delay. Large (>500 kilobases), rare (<1%) deletions were significantly enriched in patients compared to 7,366 controls (P < 0.001). We identified several rare copy number variants that were recurrent in patients but absent or at much lower prevalence in controls. We identified five patients with overlapping deletions on chromosome 16p11.2 that were found in 2 out of 7,366 controls (P < 5 × 10-5). In three patients the deletion co-segregated with severe obesity. Two patients harboured a larger de novo 16p11.2 deletion, extending through a 593-kilobase region previously associated with autism and mental retardation; both of these patients had mild developmental delay in addition to severe obesity. In an independent sample of 1,062 patients with severe obesity alone, the smaller 16p11.2 deletion was found in an additional two patients. All 16p11.2 deletions encompass several genes but include SH2B1, which is known to be involved in leptin and insulin signalling. Deletion carriers exhibited hyperphagia and severe insulin resistance disproportionate for the degree of obesity. We show that copy number variation contributes significantly to the genetic architecture of human obesity.

Rare variants in single-minded 1 (SIM1) are associated with severe obesity

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.

Highly Sensitive Diagnosis of 43 Monogenic Forms of Diabetes or Obesity Through One-Step PCR-Based Enrichment in Combination With Next-Generation Sequencing

OBJECTIVE Accurate etiological diagnosis of monogenic forms of diabetes and obesity is useful as it can lead to marked improvements in patient care and genetic counseling. Currently, molecular diagnosis based on Sanger sequencing is restricted to only a few genes, as this technology is expensive, time-consuming, and labor-intensive. High-throughput next-generation sequencing (NGS) provides an opportunity to develop innovative cost-efficient methods for sensitive diabetes and obesity multigene screening. RESEARCH DESIGN AND METHODS We assessed a new method based on PCR enrichment in microdroplets (RainDance Technologies) and NGS using the Illumina HiSeq2000 for the molecular diagnosis of 43 forms of monogenic diabetes or obesity. Forty patients carrying a known causal mutation for those subtypes according to diagnostic laboratories were blindly reanalyzed. RESULTS Except for one variant, we reidentified all causal mutations in each patient associated with an almost-perfect sequencing of the targets (mean of 98.6%). We failed to call one highly complex indel, although we identified a dramatic drop of coverage at this locus. In three patients, we detected other mutations with a putatively deleterious effect in addition to those reported by the genetic diagnostic laboratories. CONCLUSIONS Our NGS approach provides an efficient means of highly sensitive screening for mutations in genes associated with monogenic forms of diabetes and obesity. As cost and time to deliver results have been key barriers to uncovering a molecular cause in the many undiagnosed cases likely to exist, the present methodology should be considered in patients displaying features of monogenic diabetes or obesity.

High prevalence of leptin and melanocortin-4 receptor gene mutations in children with severe obesity from Pakistani consanguineous families

Recessive or co-dominant single-gene mutations disrupting leptin melanocortin pathway cause severe obesity and hyperphagia. Since Pakistan has a very high rate of consanguinity, therefore, a significantly higher incidence of monogenic obesity is expected in its population. We have assessed the incidence of LEP and MC4R mutations and associated hormonal profiles, in a cohort of randomly selected Pakistani children with early onset of severe obesity. Sixty two unrelated children of consanguineous parents, with a weight-for-age percentile >97 were recruited in the study. Screening for mutations in the coding regions of LEP and MC4R was performed by direct sequencing. Serum hormone concentrations were determined by immunoassay. LEP mutations were found in 16.1% of the probands. Of these, 9 probands carried the homozygous frameshift mutation, G133_VfsX14, whereas one patient had a homozygous mutation involving deletion of 3 base pairs, (I35del). In these probands, leptin levels were very low or undetectable and insulin levels were increased in 33%. Homozygous MC4R mutations, M161T and I316S, identified separately in 2 subjects (3.2%), were associated with severe obesity, hyperphagia, hyperleptinemia and hyperinsulinemia. The heterozygous M161T sibling had normal body weight and hormone levels and the parents were only mildly overweight. Based on genetic analysis of LEP and MC4R genes only, we elucidated genetic causality of severe obesity in 20% of our patients confirming high prevalence of monogenic form of obesity in this consanguineous population. Co-dominancy of MC4R is exacerbated in this group with non-penetrance of obesity in heterozygous loss-of-function MC4R mutation carriers. The sub-ethnic specificity of LEP mutation, G133_VfsX14, suggests a founder effect.

Novel LEPR mutations in obese Pakistani children identified by PCR-based enrichment and next generation sequencing

Mutations in leptin receptor gene (LEPR) result in early onset extreme adiposity. However, their prevalence in different populations is not known. Indeed, LEPR screening by gold standard Sanger sequencing has been limited by its large size and the cost. One‐step PCR‐based targeted enrichment could be an option for rapid and cost effective molecular diagnosis of monogenic forms of obesity.

Genetic variants in LEP, LEPR, and MC4R explain 30% of severe obesity in children from a consanguineous population

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.

Functional Characterization of Obesity-Associated Variants Involving the α and β Isoforms of Human SH2B1

We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1β enhancement of nerve growth factor-induced neurite outgrowth, and the individual with the T546A variant exhibits mild developmental delay. The other 3 variants (A663V, V695M, and A723V) lie in the C-terminal tail of SH2B1α. SH2B1α variant carriers were hyperinsulinemic but did not exhibit the behavioral phenotype observed in individuals with SH2B1 variants that disrupt all isoforms. In in vitro assays, SH2B1α, like SH2B1β, enhances insulin- and leptin-induced insulin receptor substrate 2 (IRS2) phosphorylation and GH-induced cell motility. None of the variants affect SH2B1α enhancement of insulin- and leptin-induced IRS2 phosphorylation. However, T546A, A663V, and A723V all impair the ability of SH2B1α to enhance GH-induced cell motility. In contrast to SH2B1β, SH2B1α does not enhance nerve growth factor-induced neurite outgrowth. These studies suggest that genetic variants that disrupt isoforms other than SH2B1β may be functionally significant. Further studies are needed to understand the mechanism by which the individual isoforms regulate energy homeostasis and behavior.

Loss-of-function mutations in ADCY3 cause monogenic severe obesity

Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin–melanocortin pathway in controlling energy balance, appetite and body weight1. 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 populations2. We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity3–5. 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.Genetic analysis of children with severe obesity identifies mutations in the ADCY3 gene (encoding adenylate cyclase 3). These variants are rare in public databases and affect the functional activity of the protein, indicating that ADCY3 is a potential pharmacological target for obesity treatment.

Evidence of male hypogonadism at an early age as a familial risk of type 2 diabetes.

OBJECTIVE Previous investigations provide evidence of an association of hypogonadism with type 2 diabetes in men, and low testosterone levels have been regarded a risk factor for the disease. Since a strong genetic predisposition to type 2 diabetes has been demonstrated, here we investigate a possible tendency towards hypogonadism in young male offspring of diabetic parents. MATERIAL AND METHODS The study compares 32 male offspring of diabetic parents with 31 male offspring of nondiabetic parents matched by age. The subjects comprised boys (9-17 years) and young adults (19-25 years). Anthropomorphic measurements were made in all subjects. Fasting blood samples were analyzed for glucose and serum concentrations of testosterone (T), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), insulin and leptin were measured by ELISA. Free testosterone (FT) was calculated using T and SHBG levels. RESULTS Serum T, FT and bioavailable T (BAT) levels in offspring of diabetic parents were significantly lower than those of offspring of nondiabetic parents across all age groups. Mean serum LH levels were also lower in offspring of diabetic parents compared to the controls. Although LH levels in young adults with diabetic parents, tended to be lower than those of age-matched controls but the difference was not statistically significant. Serum insulin and leptin, and insulin resistance measured by HOMA-IR were significantly raised in older offspring of diabetic parents but were within the normal range. CONCLUSION Whereas hypogonadism was the only indicator of a possible predisposition to metabolic dysfunction in peripubertal children of diabetic parents, a significant change in other metabolic markers becomes apparent at a more advanced age.

Genetics of Obesity in Consanguineous Populations: Toward Precision Medicine and the Discovery of Novel Obesity Genes: Obesity and Consanguinity

Consanguinity has been instrumental in the elucidation of many Mendelian genetic diseases. Here, the unique advantage of consanguineous populations was considered in the quest for genes causing obesity.