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Mutation Screen of the SIM1 Gene in Pediatric Patients with Early-Onset Obesity

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Mutation Screen of the SIM1 Gene in Pediatric Patients with Early-Onset Obesity International Journal of Obesity (2014) 38, 1000–1004 & 2014 Macmillan Publishers Limited All rights reserved 0307-0565/14 www.nature.com/ijo ORIGINAL ARTICLE Mutation screen of the SIM1 gene in pediatric patients with early-onset obesity D Zegers1, S Beckers1, R Hendrickx1, JK Van Camp1, V de Craemer1, A Verrijken2, K Van Hoorenbeeck3, SL Verhulst3, RP Rooman3, KN Desager3, G Massa4, LF Van Gaal2 and W Van Hul1 BACKGROUND: The transcription factor SIM1 (Single-minded 1) is involved in the control of food intake and in the pathogenesis of obesity. In mice, Sim1 is involved in the development of the paraventricular nucleus, and Sim1 deficiency leads to severe obesity and hyperphagia. In humans, chromosomal abnormalities in the SIM1 gene region have been reported in obese individuals. Furthermore, recent data also suggest that loss-of-function point mutations in SIM1 are responsible for SIM1 haplo-insufficiency that is involved in causing human obesity. In this study, we therefore wanted to expand the evidence regarding the involvement of SIM1 mutations in the pathogenesis of severe early-onset obesity. METHODS: We screened 561 severely overweight and obese children and adolescents and 453 lean adults for mutations in the coding region of the SIM1 gene. Mutation screening in all patients and lean individuals was performed by high-resolution melting curve analysis combined with direct sequencing. To evaluate the effect of the mutations on SIM1 transcriptional activity, luciferase reporter assays were performed. RESULTS: Mutation analysis identified four novel nonsynonymous coding variants in SIM1 in four unrelated obese individuals: p.L242V, p.T481K, p.A517V and p.D590E. Five synonymous variants, p.P57P, p.F93F, p.I183I, p.V208V and p.T653T, were also identified. Screening of the lean control population revealed the occurrence of four other rare SIM1 variants: p.G408R, p.R471P, p.S492P and p.S622F. For variants p.T481K and p.A517V, which were found in obese individuals, a decrease in SIM1 transcriptional activity was observed, whereas the transcriptional activity of all variants found in lean individuals resembled wild type. CONCLUSIONS: In this study, we have demonstrated the presence of rare SIM1 variants in both an obese pediatric population and a population of lean adult controls. Further, we have shown that functional in vitro analysis of SIM1 variants may help in distinguishing benign variants of no pathogenic significance from variants which contribute to the obesity phenotype. International Journal of Obesity (2014) 38, 1000–1004; doi:10.1038/ijo.2013.188 Keywords: mutation analysis; SIM1; melanocortin signaling INTRODUCTION hypothalamic oxytocin and PVN melanocortin 4 receptor (Mc4r) SIM1 (Single-minded 1) is a member of the bHLH-PAS (basic helix- mRNA, suggesting that the hyperphagic obesity in Sim1-deficient loop-helix Per-Arnt-Sim) family of transcription factors. In mice, mice may be attributable to changes in the leptin-melanocortin- 6 Sim1 is essential for the formation of the paraventricular nucleus oxytocin pathway. The hypothesis that SIM1 might be a factor (PVN) in the hypothalamus,1 a region that is known to be within the famous leptin-melanocortin signaling pathway is implicated in the regulation of body weight, as neurons in the PVN further supported by the investigation of transgenic mice express murine Mc4r and are a target of a-MSH.2,3 Homozygous overexpressing human SIM1. Overexpression of SIM1 completely Sim1-knockout mice (Sim1 À / À ) lack a PVN and die perinatally. rescued the hyperphagia and partially rescued the obesity In contrast, heterozygous Sim1 þ / À mice are viable, but they show exhibited by agouti yellow mice, in which melanocortin early-onset obesity, increased linear growth and are hyperphagic, signaling is interrupted, suggesting that Sim1 acts downstream making their phenotype resemble mc4r-mutant mice. Partial Sim1 of Mc4r to control food intake.7 deficiency results in hypodevelopment of the PVN that contains SIM1 deficiency in humans has also been reported: a girl with on average 24% fewer cells.4 As lesions in the PVN also induce early-onset severe obesity and a de novo translocation between increased appetite,5 it was suggested that this hypocellularity chromosomes 1p22.1 and 6q16.2 has been described by Holder causes the hyperphagia and obesity of the Sim þ / À mice.4 More et al.8 In this case, the translocation separates the 50 promoter recently, Tolson et al.6 demonstrated that the role of Sim1 in region and bHLH domain from the rest of the gene. It was feeding regulation is not limited to formation of the PVN. They hypothesized that haplo-insufficiency of SIM1 was responsible for conditionally deleted Sim1 in mice postnatally, thereby generating the severe obesity in the subject.8 Further, in multiple obese conditional Sim1 heterozygous mice and conditional Sim1 patients with a Prader-Willi-like phenotype, deletions of the homozygote mice and revealing a dosage-dependent effect of 6q16.2-6q16.3 region have been reported. These deletions on 6q Sim1 on obesity. These mice exhibited a remarkable decrease in disrupt or delete the SIM1 gene, possibly giving rise to obesity.9–11 1Department of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium; 2Department of Endocrinology, Diabetology and Metabolic Diseases, Antwerp University Hospital, Antwerp, Belgium; 3Department of Paediatrics, Antwerp University Hospital, Antwerp, Belgium and 4Department of Paediatrics, Jessa Hospital, Hasselt, Belgium. Correspondence: Professor Dr W Van Hul, Department of Medical Genetics, University of Antwerp, Universiteitsplein 1, Antwerp B-2610, Belgium. E-mail: [email protected] Received 11 April 2013; revised 12 September 2013; accepted 24 September 2013; accepted article preview online 7 October 2013; advance online publication, 29 October 2013 Involvement of SIM1 mutations in childhood obesity D Zegers et al 1001 More recent data also suggest that loss-of-function point standard PCR protocol. Real-time PCR was performed as previously mutations in SIM1 are responsible for SIM1 haplo-insufficiency described.19 After amplification, samples were first denaturated at 95 1C that is involved in causing human obesity12–14 and Prader- and subsequently renaturated at 40 1C and then heated during Willi-like syndrome.12,13 Because of the alleged role of SIM1 fluorescence acquisition between 70 and 95 1C. When melting curves downstream of the leptin-melanocortin pathway and the reports deviated from WT, direct sequencing using an ABI Prism 3130xl Genetic Analyzer with ABI BigDye Terminator v1.1 Cycle Sequencing kits (Applied on SIM1 haplo-insufficiency in obese humans, we hypothesized Biosystems, Foster City, CA, USA) was performed in order to characterize that heterozygous point mutations in the SIM1 gene might be the putative sequence variations. Primer sequences for high-resolution melting cause of severe early-onset obesity, and we set out to screen the curve analysis and direct sequencing are available on request. SIM1 gene region in an extensive cohort of obese children. Constructs MATERIALS AND METHODS Wild-type (WT) human SIM1 subcloned into a pDR2 vector was kindly provided by Professor Chrast (University of Lausanne, Lausanne, Switzer- Study population land). The entire coding region of the hSIM1 gene was amplified using Mutation screening of the SIM1 gene was performed in a cohort of 561 specific primers introducing a HindIII and an EcoRI restriction site 50 and 30 severely overweight and obese children and adolescents and 453 healthy, of the SIM1 sequence, respectively. The PCR product was subsequently lean adults. Children and adolescents (243 boys, 318 girls) were recruited cloned into a pcDNA3 expression vector (Invitrogen, Carlsbad, CA, USA). from the pediatrics departments of the Antwerp University Hospital and Mutations were introduced into WT SIM1, subcloned into the pcDNA3 the Jessa Hospital, Hasselt, both in Belgium. This group (age range 0–12) expression vector, using the QuikChange II Site-Directed Mutagenesis Kit included 257 children aged 8.7±2.5 years (mean±s.d.) and 304 (Stratagene, La Jolla, CA, USA). The WT sequence and the presence of the adolescents aged 15.2±2.3 years, (age range 12–21; summary of all mutations were checked and confirmed by direct sequencing using an ABI characteristics: Table 1). The cohort of healthy lean adults (18.5 kg m À 2 3130xl Genetic Analyzer (Applied Biosystems) according to the protocol Xbody mass index (BMI)X24.9 kg m À 2) was recruited among employees described above to ensure that no errors had been induced. Luciferase from the university and the university hospital and among couples seeking reporter plasmid pGL3-HRE-Luc was kindly provided by professor Whitelaw prenatal counseling at the Department of Medical Genetics. This control (University of Adelaide, Adelaide, Australia). Wild-type human ARNT2 cDNA group included 160 men and 293 women, aged 35.2±7.2 years, with a cloned in a pCMV6-XL4 expression vector was obtained from Origene mean BMI of 22.1±1.7 kg m À 2. The study was approved by the local ethics Technologies (Rockville, MD, USA). committee, and all participants gave their written informed consent once the aim and design of the study had been explained. Reporter assay SIM1 For monitoring SIM1 transcriptional activity in cells transiently expressing Anthropometry WT or mutant SIM1, a luciferase assay was performed. HEK293 cells were Height was measured to the nearest 0.5 cm, and body weight was maintained in DMEM supplemented with 10% fetal bovine serum, measured on a digital scale to the nearest 0.1 kg. BMI was calculated for all 100 units ml À 1 penicillin and 100 mgmlÀ 1 streptomycin (Invitrogen). Cells patients and BMI-for-age percentile (490th percentile for all patients were incubated at 37 1C in humidified air containing 5% CO2. Cells were included) and Z-scores were based on the ‘Flemish Growth Charts 2004’.15 plated in 24-well plates at a density of 50 000 cells per well.
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