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.
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.
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.
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.
SIM1 (Single-minded 1) is a member of the bHLH-PAS (basic helix-loop-helix Per-Arnt-Sim) family of transcription factors. In mice, Sim1 is essential for the formation of the paraventricular nucleus (PVN) in the hypothalamus,1 a region that is known to be implicated in the regulation of body weight, as neurons in the PVN express murine Mc4r and are a target of α-MSH.2, 3 Homozygous Sim1-knockout mice (Sim1−/−) lack a PVN and die perinatally. In contrast, heterozygous Sim1+/− mice are viable, but they show early-onset obesity, increased linear growth and are hyperphagic, making their phenotype resemble mc4r-mutant mice. Partial Sim1 deficiency results in hypodevelopment of the PVN that contains on average 24% fewer cells.4 As lesions in the PVN also induce increased appetite,5 it was suggested that this hypocellularity causes the hyperphagia and obesity of the Sim+/− mice.4 More recently, Tolson et al.6 demonstrated that the role of Sim1 in feeding regulation is not limited to formation of the PVN. They conditionally deleted Sim1 in mice postnatally, thereby generating conditional Sim1 heterozygous mice and conditional Sim1 homozygote mice and revealing a dosage-dependent effect of Sim1 on obesity. These mice exhibited a remarkable decrease in hypothalamic oxytocin and PVN melanocortin 4 receptor (Mc4r) mRNA, suggesting that the hyperphagic obesity in Sim1-deficient mice may be attributable to changes in the leptin-melanocortin-oxytocin pathway.6 The hypothesis that SIM1 might be a factor within the famous leptin-melanocortin signaling pathway is further supported by the investigation of transgenic mice overexpressing human SIM1. Overexpression of SIM1 completely rescued the hyperphagia and partially rescued the obesity exhibited by agouti yellow mice, in which melanocortin signaling is interrupted, suggesting that Sim1 acts downstream of Mc4r to control food intake.7
SIM1 deficiency in humans has also been reported: a girl with early-onset severe obesity and a de novo translocation between chromosomes 1p22.1 and 6q16.2 has been described by Holder et al.8 In this case, the translocation separates the 5′ promoter region and bHLH domain from the rest of the gene. It was hypothesized that haplo-insufficiency of SIM1 was responsible for the severe obesity in the subject.8 Further, in multiple obese patients with a Prader-Willi-like phenotype, deletions of the 6q16.2-6q16.3 region have been reported. These deletions on 6q disrupt or delete the SIM1 gene, possibly giving rise to obesity.9, 10, 11 More recent data also suggest that loss-of-function point mutations in SIM1 are responsible for SIM1 haplo-insufficiency that is involved in causing human obesity12, 13, 14 and Prader-Willi-like syndrome.12, 13 Because of the alleged role of SIM1 downstream of the leptin-melanocortin pathway and the reports on SIM1 haplo-insufficiency in obese humans, we hypothesized that heterozygous point mutations in the SIM1 gene might be the cause of severe early-onset obesity, and we set out to screen the SIM1 gene region in an extensive cohort of obese children.
Materials and methods
Mutation screening of the SIM1 gene was performed in a cohort of 561 severely overweight and obese children and adolescents and 453 healthy, lean adults. Children and adolescents (243 boys, 318 girls) were recruited from the pediatrics departments of the Antwerp University Hospital and the Jessa Hospital, Hasselt, both in Belgium. This group (age range 0–12) included 257 children aged 8.7±2.5 years (mean±s.d.) and 304 adolescents aged 15.2±2.3 years, (age range 12–21; summary of all characteristics: Table 1). The cohort of healthy lean adults (18.5 kg m−2⩾body mass index (BMI)⩾24.9 kg m−2) was recruited among employees from the university and the university hospital and among couples seeking prenatal counseling at the Department of Medical Genetics. This control group included 160 men and 293 women, aged 35.2±7.2 years, with a mean BMI of 22.1±1.7 kg m−2. The study was approved by the local ethics committee, and all participants gave their written informed consent once the aim and design of the study had been explained.
Height was measured to the nearest 0.5 cm, and body weight was measured on a digital scale to the nearest 0.1 kg. BMI was calculated for all patients and BMI-for-age percentile (>90th percentile for all patients included) and Z-scores were based on the ‘Flemish Growth Charts 2004’.15 Using the ‘Flemish Growth Charts 2004’, the cutoff values for overweight and obesity are defined as the percentile line on the chart that crosses BMI 25 kg m−2 and BMI 30 kg m−2, respectively, at 18 years of age.
Blood samples from all patients and control subjects were obtained for extraction of genomic DNA by standard procedures.16 Mutation screening of the entire coding region of the SIM1 gene (GenBank accession number NM_005068.2), including intron-exon boundaries was performed by high-resolution melting curve analysis in combination with direct sequencing. High-resolution melting curve analysis (HRM), which is performed using the LightCycler 480 Real-Time PCR System (Roche Applied Science, Mannheim, Germany), is a highly sensitive technique to detect novel mutations in a large sample set.17, 18 For high-resolution melting curve analysis, the coding region of SIM1 (exon 1–exon 11, Figure 1) was divided into 14 amplicons, which were amplified using either a touch-down or a standard PCR protocol. Real-time PCR was performed as previously described.19 After amplification, samples were first denaturated at 95 °C and subsequently renaturated at 40 °C and then heated during fluorescence acquisition between 70 and 95 °C. When melting curves deviated from WT, direct sequencing using an ABI Prism 3130xl Genetic Analyzer with ABI BigDye Terminator v1.1 Cycle Sequencing kits (Applied Biosystems, Foster City, CA, USA) was performed in order to characterize putative sequence variations. Primer sequences for high-resolution melting curve analysis and direct sequencing are available on request.
Wild-type (WT) human SIM1 subcloned into a pDR2 vector was kindly provided by Professor Chrast (University of Lausanne, Lausanne, Switzerland). The entire coding region of the hSIM1 gene was amplified using specific primers introducing a HindIII and an EcoRI restriction site 5′ and 3′ of the SIM1 sequence, respectively. The PCR product was subsequently cloned into a pcDNA3 expression vector (Invitrogen, Carlsbad, CA, USA). Mutations were introduced into WT SIM1, subcloned into the pcDNA3 expression vector, using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The WT sequence and the presence of the mutations were checked and confirmed by direct sequencing using an ABI 3130xl Genetic Analyzer (Applied Biosystems) according to the protocol described above to ensure that no errors had been induced. Luciferase reporter plasmid pGL3-HRE-Luc was kindly provided by professor Whitelaw (University of Adelaide, Adelaide, Australia). Wild-type human ARNT2 cDNA cloned in a pCMV6-XL4 expression vector was obtained from Origene Technologies (Rockville, MD, USA).
Reporter assay SIM1
For monitoring SIM1 transcriptional activity in cells transiently expressing WT or mutant SIM1, a luciferase assay was performed. HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units ml−1 penicillin and 100 μg ml−1 streptomycin (Invitrogen). Cells were incubated at 37 °C in humidified air containing 5% CO2. Cells were plated in 24-well plates at a density of 50 000 cells per well. Eight hours later, cells were transiently transfected with 40 ng ARNT2 in pCMV6-XL4 expression vector, 120 ng pGL3-HRE-Luc, 10 ng pRL-TK and 40 ng WT or mutant SIM1 in pcDNA3 expression vector per well. Fourty-eight hours post-transfection, cells were then assayed for luciferase activity, using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) on a Glomax-multi Detection System (Promega). Firefly luciferase readings, measured as a reference for SIM1 transcriptional activity, were normalized to the readings of the pRL-SV40 Renilla luciferase control reporter. Relative ratios of Firefly luciferase readings over Renilla luciferase readings were normalized to the maximal relative ratio calculated for WT SIM1 (%). Each transfection was performed in duplicate, and the experiment was repeated three times with similar results. Data are expressed as mean values±s.d. P-values were obtained by performing Wilcoxon signed-rank test (IBM SPSS Statistics, version 20).
Screening of the coding region of the SIM1 gene in our population of obese children and adolescents led to the identification of multiple sequence variants. We detected five different synonymous variants, c.171A>T (P57P), c.279C>T (F93F; rs145361258), c.549C>T (I183I), c.624G>A (V208V) and c.1959C>T (T653T; rs41318041), that do not alter the protein sequence and two common nonsynonymous SNPs P352T (rs3734354) and A371V (rs3734355). Both variants, which are in complete linkage disequilibrium, were observed in the population of obese children and the population of lean controls with a frequency of 23% and 25.4% respectively (Pearson Chi-Square; P=0.376). Further, we also found four nonsynonymous coding variants in SIM1 (c.724C>G, c.1442C>A, c.1550C>T and c.1770C>G) leading to the amino-acid substitutions p.L242V, p.T481K, p.A517V and p.D590E, respectively. When we subsequently screened our lean control population, we found four of the synonymous variants also observed in the obese population. In addition, we found four nonsynonymous coding variants in the SIM1 gene (c.1222G>C, c.1412G>C, c.1474T>C and c.1865C>T) leading to the amino-acid substitutions p.G408R, p.R471P, p.S492P and p.S622F. An overview of the different coding variants found in our cohorts of obese children and lean control adults is given in Table 2.
We performed in silico analysis using a set of five different prediction programs (PolyPhen,20 PolyPhen-2,21 SIFT,22 SNPs&Go23 and MutPred24) for all SIM1 variants identified in the present study. For most of the eight reported nonsynonymous variants, there is large variation in prediction between the different programs used. However, the R471P variant, which was found in a lean control individual, was predicted to be benign by all programs. As this variant has also been identified in the 1000 genomes project25 and the NHLBI GO Exome Sequencing Project (ESP),26 it is expected to be a rare variant, which is not associated with obesity. Whereas none of the other variants have been reported in the 1000 genomes project, both D590E and S622F have been reported once in the current ESP6500 release of the NHLBI GO Exome Sequencing Project. However, as the ESP project cannot release phenotype information about any particular individual, no conclusions regarding causality of the variants can be drawn.
To evaluate the effect of the mutations on SIM1 trancriptional activity, we performed an in vitro luciferase reporter assay. For T481K and A517V-mutant SIM1, a decrease in transcriptional activity is observed, when co-transfected with the obligate dimerization partner ARNT2. Relative to wild-type SIM1 (100%), their transcriptional activities were reduced to 81.1±7.4% and 55.1±7.1%, respectively (P<0.05). However, transcriptional activity for all other variants (including the variants found in lean controls) resembled wild type (Figure 2).
Phenotype of T481K and A517V SIM1 variant carriers
The patient, in whom we discovered the T481K mutation, is a 20-year-old female presenting with obesity. Her body weight is 99 kg for 1.70 m, with a BMI of 34.3 kg m−2, the waist-to-hip ratio (WHR) is 0.98 and the fat mass percentage (measured by bioelectrical impedance analysis) is 48.1%. The patients father is also obese as well as the maternal grandparents. However, as no further family data were available, it was not possible to determine whether the mutation segregates with obesity in this family.
The patient harboring the A517V mutation is a 13-year-old boy, suffering from obesity with an early onset. Body weight reaches 87.5 kilograms with a BMI of 30.5 kg m−2. BMI Z-score is 2.23. The patient already suffered from several complications because of the extreme obesity and showed hypertriglyceridemia (248 mg dl−1), hyperinsulinism (36 μU ml−1) and hepatic steatosis. Both parents were overweight as well and the mother underwent gastric banding surgery. Unfortunately, both were not available for genetic analysis. As no family members were willing to cooperate for further research, it could not be determined whether the mutation was inherited from one of the overweight parents or had arisen de novo.
The involvement of SIM1 in the leptin-melanocortin signaling pathway that regulates energy homeostasis and food intake is firmly established. Mice heterozygous for Sim1 develop severe, early-onset obesity and are reported to be hyperphagic.4 In addition, SIM1 deficiency in humans is associated with a severe, early-onset obesity phenotype.8, 9, 10, 11, 27 Different research groups set out to determine the exact role of the SIM1 gene in human obesity by performing both mutation screens and association studies.28, 29, 30 Until recently, reports on SIM1 mutation screening in cohorts of obese individuals have been scarce, as only one missense mutation (I128T) had been reported in a 5-year-old Caucasian girl with severe early-onset obesity, hyperphagia and developmental delay for which disease causality could not be proven as the variant did not cosegregate with obesity in the family.29 Only recently, two novel studies on SIM1 rare mutations in obesity emerged.13, 14 In the paper by Bonnefond et al.,13 the authors report on four variants identified in children with Prader-Willi-like syndrome features and four other variants in morbidly obese adults. After performing luciferase gene reporter assays, three of the variants showed strong loss-of-function effects. Corroborated by functional experiments, they established a firm link between SIM1 loss-of-function mutations and severe obesity. The authors also reported that mutation of SIM1 is not always responsible for a fully penetrant form of obesity. Contradictory to our results, they did not identify any novel rare mutations in their population of 383 normal weight controls.13 In the paper by Ramachandrappa et al.,14 2100 individuals with early-onset obesity have been screened for mutations in the SIM1 gene. Nine out of thirteen different heterozygous SIM1 variants significantly reduced SIM1 transcriptional activity; however, they were reported to segregate with obesity with variable penetrance. Further, variant carriers presented with an increased ad libitum food intake at a test meal, normal basal metabolic rate and evidence of autonomic dysfunction.14
There are also several reports available in which the contribution of common variants in the SIM1 gene to human complex obesity is analyzed.28, 29, 30, 31 Results of these association studies are rather conflicting, as an initial association of the coding polymorphisms rs3734354 (P352T) and rs3734355 (A371V) with BMI in obese men29 was not replicated in a large scale association study performed in a French cohort comprising 1275 obese and 1395 lean control individuals.30 Further, the association of a linkage disequilibrium block spanning from 5′-UTR to intron 8 with obesity risk in a population of 6194 Pima Indians31 could also not be replicated in a cohort of French individuals.30 In the most recent study, homozygosity for the 352T/371V allele was associated with higher BMI in a white male population and three other SNPs (rs9390322, rs7746743 and rs3734353) were significantly associated with various adiposity measures,28 indicating that the role for common genetic variants in SIM1 is still not clearly defined.
In this study, we have demonstrated the presence of rare SIM1 variants in both an obese pediatric population as well as a population of lean adult controls. The majority of these variants is situated in the C-terminal domain of the transcription factor, whereas the L242V variant is located in the PAS2 domain (Figure 1). Observation from deletion studies showed that the HLH-PAS domain in SIM1 is a bipartite dimerization domain with the general dimerization partner ARNT2 and that the C-terminus contains the transcriptional regulatory domains.32 Further, there is a high level of homology between the human SIM1 and SIM2, mouse Sim1 and Sim2 and even drosophila sim genes in the amino-terminal part of the protein in which the conserved bHLH (basic helix-loop-helix), PAS (Per-Arnt-Sim) and PAC (PAS-associated C-terminal) domains are located. The carboxy-terminal part is only homologous between human SIM1 and mouse Sim1.33 When comparing peptide sequences, amino acids L242 and T481 show the strongest conservation compared with the other sim genes. However, as all variants identified in this study are situated in crucial protein domains, it is impossible to differentiate between benign and functionally relevant variants based on their location within the protein. Therefore, we performed in vitro functional experiments to evaluate the transcriptional activity of the (mutated) SIM1 protein. We were able to demonstrate that both the T481K and the A517V mutation affect SIM1 transcriptional activity, whereas transcriptional activity of all other identified variants resembled wild type.
While the ‘common disease, common variant’ hypothesis has led to inconsistent results regarding the influence of common SIM1 variants on obesity susceptibility, one might suggest that there could be a possibility for the ‘common disease, rare variant’ hypothesis to hold true for the SIM1 gene. This hypothesis suggests an important role of multiple rare gene variants of major effect, each of which is found in only a few people, to underlie susceptibility to common disease. However, our data emphasize that the firm identification of these rare, causative variants for obesity is not an easy task because of the background noise created by multiple rare variants also found in lean control individuals. The most decisive evidence to distinguish rare causal variants from benign, neutral variants can only be provided by extensive co-segregation analysis combined with comprehensive functional in vitro and even in vivo experiments.
As most of the variants identified in the present study were found scattered throughout the carboxy-terminal domain of the protein with no clear clustering pattern for the variants found in obese cases compared with the variants found in lean controls (Figure 1), there was no a priori indication for disease causality of any of the variants. Nevertheless, by performing in vitro experiments we demonstrated that two of the identified variants (T481K and A517V) decreased SIM1 transcriptional activity and that final conclusions regarding pathogenicity of the variants can only be based on functional studies.
Building on the recently generated and growing body of data regarding SIM1 mutations involved in the pathogenesis of obesity, we evaluated the prevalence of rare SIM1 mutations in human individuals with severe early-onset obesity as well as in lean controls in the current study. However, as rare variants were identified in both obese cases and lean controls and not all variants seem to affect transcriptional activity, this raises questions regarding the pathogenic impact of SIM1 variants on obesity. Therefore, future perspectives include more extensive and detailed studies to clarify the role of rare SIM1 mutations and to evaluate complementary mechanisms such as gene–gene and gene–environment interactions, as these may add to disease risk and contribute to the complex relationship between genotype and phenotype.
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Research was funded by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) to DZ. SB is a postdoctoral fellow of the Research Foundation—Flanders (FWO Vlaanderen). This work was supported by a grant (G0028.05) from the Research Foundation—Flanders (FWO Vlaanderen) to LVG and WVH and by a TOP-research grant from the University of Antwerp to WVH. This study was supported by an Interuniversity Attraction Pole Project (Phase VII project 43, BELSPO).
The authors declare no conflict of interest.
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Cite this article
Zegers, D., Beckers, S., Hendrickx, R. et al. Mutation screen of the SIM1 gene in pediatric patients with early-onset obesity. Int J Obes 38, 1000–1004 (2014). https://doi.org/10.1038/ijo.2013.188
- mutation analysis
- melanocortin signaling
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