In the present study, we sequenced the coding region of the transcription factor SIM1 in 2,100 patients of mixed European descent with severe, early onset obesity from the GOOS cohort and 1680 patients from a UK population-based control cohort. Cosegregation studies in families of SIM1 variant carriers indicated a dominant form of inheritance with variable penetrance. Some rare variants found in patients were also found in controls (e.g., I128T, Q152E, D707H). These variants are unlikely to be sufficient to explain the severe obesity in patients in this study, although they may be contributors to overweight/obesity in individuals with a susceptible genetic background. The SIM1-containing locus has not emerged from genome-wide association studies of BMI and 2 large studies in European populations have failed to find a significant association between common variants in SIM1 and increased BMI (25
To explore the functional consequences of these variants, we generated stable cell lines expressing the mutant SIM1 proteins and tested their ability to activate the transcription of a SIM1-responsive reporter gene. Of the 13 variants examined, 9 had significantly reduced activity in this assay when paired with the proposed in vivo partner, ARNT2. A tenth variant, P497R, showed reduced activity but reached significance at P < 0.05 only with ARNT. No variants were found in the bHLH domain of the protein in this study, which mediates DNA binding and dimerization with ARNT and ARNT2. We identified 1 missense variant, S71R, in the sequence between the bHLH domain and the PAS region. This variant has weak activity, implying that this stretch of residues has a hitherto unrecognized functional role.
Three variants (I128T, Q152E, and R171H) were identified in the PAS-A domain and 1 (L238R) in the PAS-B domain. The PAS domains form conserved 3D folds and have been shown to perform multiple functions in bHLH/PAS proteins, including ligand binding (in the aryl hydrocarbon receptor), stabilization of heterodimerization (in CLOCK/BMAL; ref. 21
), and interaction with either transcription coactivators (HIF-1A/ARNT; ref. 26
) or repressive proteins (Cry interaction with Clock; ref. 27
). All 4 PAS region variants decreased SIM1 function, with R171H having the most profound effect on transcriptional activation. These findings suggest that the PAS-A and PAS-B domains in human SIM1 play a critical structural or functional role or roles that merit further investigation. Interestingly, I128T and Q152E were also found in controls, some, but not all, of whom were obese. The contribution of these variants to final body weight remains to be tested.
The remaining variants were found in the C terminus which is thought to be responsible for transcriptional regulation. R550H, D707H, and T712I were the most severely impaired, suggesting these are crucial residues in transcriptional regulation. As with other bHLH/PAS proteins (28
), there is evidence that the C terminus functions primarily as a transactivation or transrepression domain. By analogy, the closely related bHLH/PAS transcription factor SIM2 is known to regulate transcription differentially in a context-dependent manner (30
). SIM1 has variously been shown to activate and repress transcription in cell-based reporter assays. In addition to the reporter assays shown in Figure B, Ema et al. (32
) showed that Sim1 repressed the ability of a Gal4-ARNT fusion protein to activate a Gal4 reporter gene, while deletion of the C terminus of Sim1 abolished this repression. The altered activities of our C-terminal SIM1 variants confirm the need to embark on experiments aimed at discovering SIM1 interacting proteins and direct target genes.
The activity of 3 of the variant SIM1 proteins (R383G, S541L, R703Q), was indistinguishable from that of WT in assays with ARNT or ARNT2. It is possible that these variants are not pathogenic — there were no family data or detailed phenotypic data available for these patients, as they did not consent to further investigation (Supplemental Table 1). Alternatively, these variants may have a selective effect on aspects of SIM1 function that were not evaluated in our system. It is important to consider that, while this assay exclusively examined the transactivation properties of SIM1, the physiological relevance of this function remains to be established.
We have linked the development of severe early onset obesity with naturally occurring rare variants in SIM1 that cause reduced activity of this transcription factor. We report variants that reduce activity to varying degrees, from severely damaging variants that are likely to be major drivers of severe obesity (S71R, R171H, L238R, P497R, R550H, T712I) through moderately damaging variants that in some cases demonstrate variable penetrance (D707H) to variants that appear to result in a small reduction in activity whose precise contribution to obesity in carriers remains to be established (I128T, Q152E, R383G, S541L, P692L, R703Q). The reporter gene assay used in these studies can identify variants that are clearly deficient in activity, but may not be able to differentiate variants with mild loss of activity compared with WT SIM1. Given their location in the structure of SIM1, it is likely that some SIM1 variants may reduce function of the protein through a number of different mechanisms not tested here, including protein turnover, nuclear translocation, dimerization, DNA binding and binding to as-yet-unknown transcription cofactors.
Cumulatively, our genetic and functional data, together with the findings of Bonnefond and colleagues (33
), provide direct evidence for a role for SIM1 in the control of energy homeostasis in humans. SIM1 is expressed in several regions of the brain, including the PVN of the hypothalamus, which is involved in the regulation of food intake. Mouse models have shown Sim1 to play both a developmental and a postnatal physiological role in this nucleus (2
), with evidence divided as to whether 1 functional copy of Sim1
is sufficient for its developmental function (4
). Consistent with postnatal deletion of Sim1
causing hyperphagia (6
), stereotaxic injection of an adenovirus expressing shRNA directed against Sim1
into the PVN has shown that reduction of Sim1
expression in a subset of neurons is sufficient to affect food intake (36
Ad libitum energy intake was increased in patients with SIM1 variants with no evidence of a defect in basal energy expenditure. SIM1 variant carriers had lower blood pressure measurements, showed an attenuated increase in heart rate on waking, and had increased respiratory quotients when compared with obese controls. These later phenotypic characteristics were shared with MC4R
-deficient patients and are indicative of underlying autonomic dysfunction that is seen in animals and humans with reduced melanocortinergic tone (24
). These findings are consistent with a body of evidence that suggests a role for SIM1 in central melanocortin signaling.
Our results suggest that SIM1, or an as-yet-unidentified transcriptional target of SIM1, forms part of the melanocortin signaling pathway. The melanocortin agonist MTII and leptin administration have been shown to increase Sim1
gene expression in rodents, suggesting that Sim1
is upregulated as a consequence of Mc4r activation(4
). While the interaction between SIM1 and MC4R may operate at a number of levels, rescue of Mc4r
expression selectively in Sim1
-expressing neurons in Mc4r
-knockout mice normalizes food intake and improves the body weight phenotype (37
). This implies that Mc4r
are coexpressed in a subset of neurons and that Mc4r
in these neurons is critical to the central melanocortin pathway, which influences body weight by modulating food intake independently of energy expenditure (37
). However, targeted ablation of Sim1
-expressing neurons with diphtheria toxin resulted in obesity resultant from both hyperphagia and reduced energy expenditure, suggesting additional Sim1
independent roles exist in at least some Sim1
-expressing neurons to regulate energy balance (38
SIM1 patients share many features of melanocortin-deficient patients, but notably do not share the accelerated linear growth and increased final height seen in MC4R deficiency (23
). Further experiments are needed to elucidate the precise nature of the interaction between MC4R and SIM1 and the common pathways activated by MC4R and SIM1.
The transcriptional targets of SIM1 are unknown; however, the anorexigenic neuropeptide oxytocin is a potential candidate, as the hyperphagia of Sim1
haploinsufficient mice is partly ameliorated by the central administration of oxytocin and exacerbated by the administration of an oxytocin receptor antagonist (39
). PVN oxytocin neurons are activated by the central administration of a selective Mc4r agonist in mice; however, it is unclear whether oxytocin levels are modulated independently, or as a consequence, of defective melanocortin signaling. Impaired oxytocinergic signaling is one mechanism implicated in the hyperphagia and obesity seen in PWS, a clinical syndrome caused by lack of expression of a cluster of maternally imprinted snoRNAs on chromosome 15, which are thought to be involved in alternative mRNA splicing (40
). Patients with PWS have a distinctive appearance, with obesity, short stature, small hands, and characteristic facial features. They have developmental delay with prominent behavioral abnormalities, such as obsessive compulsive–type behaviors and emotional lability (40
). Some of these features are reminiscent of the cognitive and behavioral deficits seen in SIM1 variant carriers that were varied in manifestation and severity and could be influenced by impaired oxytocinergic signaling. The neurobehavioral features of SIM1 variant carriers are not recognized features of MC4R deficiency, implying that MC4R, SIM1, and OXT are unlikely to interact in an exclusively linear pathway. Further characterization of these features will have implications for the identification of patients in whom variants in SIM1 should be considered and for genetic counseling of SIM1 variant carriers.
Our data demonstrate that rare variants in SIM1 are associated with severe obesity and should be considered in patients with hyperphagic obesity of early onset. The study of patients with SIM1 variants that exhibit reduced activity in vitro provides strong evidence that this transcription factor plays a physiological role in central melanocortin signaling in a specific pathway that regulates food intake independently of energy expenditure, although the precise details of this circuitry remain to be established. Identifying the direct transcriptional targets of SIM1 will be critical to understanding the molecular mechanisms underlying its role in the regulation of food intake and body weight.