By focusing on rare coding variants only (minor allele frequency [MAF] <1%), we identified 4 substitutions (p.I128T, p.Q152E, p.R581G, and p.T714A; Figure ) in 4 children presenting with clinical features of PWL syndrome, including severe obesity that started between 1 and 2 years of age (Table and Supplemental Table 1; supplemental material available online with this article; doi:
), and 4 other substitutions (p.T46R, p.E62K, p.H323Y, and p.D740H; Figure ) in 7 morbidly obese adults (Table and Supplemental Table 2).
Location of the 8 SIM1 rare substitutions.
Genetic and functional description of the 8 SIM1 rare substitutions
With regard to the obese child SIM1 mutant carriers presenting with PWL syndrome (Supplemental Table 1 and Supplemental Figure 1), the girl carrying the p.I128T variant received the substitution from her normal-weight father. The mother of the girl carrying the p.Q152E variant did not carry this substitution; the father’s DNA and phenotypes were not available. The girl carrying the p.R581G variant was an adopted child. In contrast, we were able to investigate the family of the boy carrying the p.T714A variant. His 3 siblings, his mother, and a maternal aunt also carried this substitution. The mother and her 4 children were severely obese and presented at least one other clinical feature associated with PWL syndrome, while the aunt was only overweight and without clinical abnormalities (Supplemental Table 3).
Among the morbidly obese adult SIM1 mutant carriers (Supplemental Table 2 and Supplemental Figure 2), 4 unrelated patients carried the same p.T46R variant. In the first mutated participant, the p.T46R variant had arisen de novo. In the second mutated participant, the p.T46R variant had been inherited from his obese mother. Furthermore, this participant had an obese sister who also carried the p.T46R variant. Although parental DNA samples were not available for the third p.T46R-carrying participant, her obese son carried the substitution, while her normal-weight sister and normal-weight daughter did not. The family of the fourth p.T46R-carrying participant was not available. The patient carrying the p.E62K variant had a normal-weight mother and 2 normal-weight daughters, none of whom carried the substitution. His severely obese father has died, and no DNA was available. The carrier of the p.H323Y variant had a morbidly obese sister who was also a p.H323Y carrier, while both parents had died. No DNA was available from relatives of the p.D740H carrier.
In summary, we identified 21 (adults and children) carriers of a rare heterozygous SIM1 variant (11 probands and 10 of their relatives) and 16 non-carrier relatives, and we found a strong contribution of SIM1 rare variants to intra-family risk for obesity (odds ratio[95% confidence interval]: OR = 20.9[3.5–126.5]; P = 9.3 × 10–4).
Of note, 3 of the 8 rare variants have been listed in the Single Nucleotide Polymorphism database (dbSNP 137) — p.E62K/rs201038781, p.I128T/rs138546433, and p.Q152E/rs140908824 — but are very rare in the general population, according to the NHLBI Exome Project and the 1000 Genomes Project (MAF <0.1%). When we sequenced 383 normal-weight controls, we found a single carrier of the p.I128T variant (in the heterozygous state). A previous study reported that p.I128T did not cosegregate with either obesity or overweight phenotype in a family of European descent (9
). Therefore, this variant is unlikely to have a significant effect on obesity or PWL-related clinical features. We then genotyped 2,896 normal-weight individuals but did not find any carriers of the substitutions p.T46R, p.E62K, p.Q152E, p.H323Y, p.R581G, p.T714A, or p.D740H.
Subsequently, we aimed to functionally assess the 8 rare substitutions. Using five types of in silico prediction software, we found that the mutation with the highest risk score of damaging effect was p.T46R, while the substitutions with the lowest risk score were p.Q152E and p.T714A (Table ). One of the aryl hydrocarbon receptor nuclear translocators, either ARNT or ARNT2, is required as a dimerization partner for SIM1 to function as a transcription factor, with mouse knockout studies suggesting that ARNT2 is the in vivo partner in the hypothalamus (10
). We constructed a homology model to estimate structural features of the SIM1:ARNT2 dimer at the bHLH and PASA domains, which was based upon the crystal structure for the N-terminal half of the CLOCK:BMAL1 dimer (12
). We found that the N-terminal amino acids (T46, E62, I128, and Q152) likely lie on the surface of the protein (Figure A). E62, I128, and Q152 amino acids were located in unstructured loops (Figure , B and C); thus, their possible contributions to perturbations of SIM1 structure could not be estimated. However, T46 lay in the middle of a helix that was in close proximity to a helix of ARNT2 (Figure B); thus, the larger arginine residue may critically perturb the dimerization interface.
Homology model of the SIM1:ARNT2 heterodimer.
We then assessed the functional effects of the 8 substitutions on the transcriptional properties of the SIM1:ARNT and SIM1:ARNT2 dimers in vitro using luciferase gene reporter assays in human 293 Flp-In T-Rex stable cell lines expressing the WT protein or one of the 8 SIM1 mutants (Figure , A and B). Importantly, with this system, only 1 copy of the mutated (or WT) SIM1 is integrated at a specific and predefined insertion site in the cell genome. This renders activity levels independent of the number of inserted copies and the insertion site, and allowed an accurate comparison of the mutants’ activities. We found a strong loss-of-function effect of p.T46R, p.H323Y, and p.T714A mutations on SIM1 transcriptional activity with both ARNT and ARNT2 (P < 0.001; Figure C). These mutations were located in the bHLH, PASB, and C-terminal domains, respectively (Figure ). The 3 mutations were identified in morbidly obese adults (n = 9 carriers of p.T46R or p.H323Y), in 1 overweight adult (carrier of p.T714A), and in severely obese subjects with PWL syndrome (n = 4 carriers of p.T714A). Together, these loss-of-function mutations were associated with a strong intra-family risk for obesity (OR = 28.0[2.7–295.7]; P = 5.6 × 10–3). The p.I128T and p.Q152E variants, which were identified either in normal-weight participants (n = 2 carriers of p.I128T) or in severely obese subjects with PWL syndrome (n = 2 carriers of p.I128T or p.Q152E), had a milder loss-of-function effect on SIM1 activity with ARNT or ARNT2 (Figure C). Two substitutions (p.E62K and p.D740H) which were identified in 2 morbidly obese participants, had a gain-of-function effect on SIM1 activity with ARNT only (Figure C). We did not detect any effect of p.R581G on SIM1 activity (Figure C), which was identified in a severely obese participant with PWL syndrome. Together, the rare variants with mild or no effects on SIM1 activity were not significantly associated with obesity within families (OR = 7.5[0.5–122.7]; P = 0.158). When we compared our functional data with the in silico predictions, only 62.5% concordance was found.
Functional assessment of each rare non-synonymous SIM1 variant.
Our genetic and functional studies, together with the findings of Ramachandrappa et al. (13
), convincingly demonstrate a link between SIM1 loss of function and severe to morbid obesity that may also be associated with PWL-related clinical features including developmental delay (or intellectual disability) and facial dysmorphism. The observed effects of SIM1
loss-of-function mutations on both development and body weight regulation are in line with the key role of SIM1
in development of the paraventricular nucleus of the hypothalamus (1
). Importantly, the necdin protein (NDN), which is believed to be involved in the Prader-Willi syndrome (14
), regulates the activity of the SIM1:ARNT2 dimer (15
), reinforcing the putative role of SIM1
in the PWL syndrome.
non-synonymous mutations were present in numerous patients with severe obesity associated with, or independent of, PWL-related clinical features. Some mutations, such as p.T46R, dramatically reduced SIM1 activity on a reporter gene and were only found in obese individuals, suggesting that loss-of-function SIM1 mutants may underpin a monogenic form of obesity. However, some substitutions with marked loss-of-function effect (e.g., p.T714A) did co-segregate with overweight/obesity but were not always associated with severe obesity or PWL syndrome. Moreover, the p.I128T variant, which caused mild loss of SIM1 function, was unlikely to contribute to obesity. Therefore, mutation of SIM1
is not always responsible for a fully penetrant form of obesity. Similar features have been repeatedly found in MC4R
loss-of function mutation carriers, where a permissive role of the environment on disease expression has been suggested (16
). The degree of penetrance is expected to be determined by the severity of loss of function of a particular mutant in combination with environment and genetic background. In obese patients presenting with the clinical features associated with Prader-Willi syndrome, if chromosomes 15q11 and 6q16 are found not to contain abnormalities, then SIM1 sequencing and subsequent molecular characterization should be performed, in order to demonstrate the presence of causative loss-of-function mutations.