Background
Obesity is an increasing global health problem accompanied by several significant health impairments, such as type-2 diabetes and cardiovascular diseases. Although excessive energy intake together with low levels of physical activity contribute to the ongoing obesity epidemic, genetic research has shown that heritage might account for as much as 70% of the population variation in BMI [
1]. Genome-wide association (GWA) studies have linked several genomic loci with a high BMI, and several new candidate genes have been identified [
2]. Most recently, the FTO gene has been given special attention because of its strong association with obesity in multiple cohorts differing in age [
3]. Basic research showed that FTO-deficient mice have less adipose tissue and a lower body mass compared with wild type mice, which supports the biological role proposed for the gene by the GWA analyses [
4].
Aside from FTO, GWA studies associated with obesity yet another gene, named TMEM18. TMEM18 was first proposed as an important obesity-related locus by the GIANT consortium [
5]. That study strongly associated the nearby SNP, rs6548238, to increased BMI and body weight. Subsequently, these results were replicated in the Icelandic GWA study for three other SNPs located in the close proximity of TMEM18 (rs2867125, rs4854344 and rs7561317), which all showed a strong association to both BMI and body weight [
6]. The FTO locus' association to BMI was confirmed therein. Furthermore, the TMEM18 locus' association to BMI has been replicated in two additional studies [
7,
8] and it has also been associated to the age of menarche [
9]. It should be noted that while the risk contribution of the TMEM18 locus to obesity in adults has been examined (6), no attempt to determine such association has been made in obese children. However, one previous study has associated the locus with BMI in children [
8].
The TMEM18 gene codes for a poorly characterized transmembrane protein. One study indicated that this protein is located in the nuclear envelope in neural stem cells [
10]. TMEM18 may be involved in cell migration as overexpression of the protein increases the migration of neural stem cells towards glioma in the rat brain. A preliminary expression profiling of TMEM18, that accompanied the first GWA study, suggested that it is ubiquitously expressed, but with certain differences between tissues [
5]. In line with the proposed role in the regulation of body weight, TMEM18 was shown to be expressed in the brain, including the hypothalamus, the region responsible for the control of energy homeostasis. Only few other brain areas were investigated in that project.
The current project provides the first detailed characterization of the human TMEM18 protein's sequence features, evolutionary history, gene expression profile in the rat and mouse, distribution in the mouse brain, and gene expression regulation in several murine feeding/body weight models. We also investigated the risk of obesity for variants upstream of the TMEM18 locus in the cohort of severely obese children and healthy controls, and performed the first examination of the locus association to several obesity-related traits.
Discussion
The TMEM18 gene has a remarkably long evolutionary history as it is found in most of the eukaryotic genomes we have looked at. It has therefore been present for at least 1500 MYA since the divergence of animals and plants/
T. pseudonana [
38]. Thus, it is one of the most ancient genes implicated in human obesity, such as FTO [
39] and MC4R [
40], that we are aware of. Most of the main genes involved in body weight regulation appeared after the radiation of vertebrates and very few are present beyond the animal kingdom. The amino acid sequences are highly conserved (Figures
2 and
3) with relatively small differences between vertebrates and other animals. Another very interesting feature (of curiosity, shared by the FTO gene), is that the gene is found in a single copy with no close relative in the genomes where it is present, despite that some genomes have undergone recent whole genome duplications such as
A. thaliana (38 MYA) [
41]. Surprisingly, we found two separate lineages where the TMEM18 gene is absent: fungi (yeasts, molds and mushrooms) and
pseudoocoelomata (
C. elegans). Both contain well analyzed genomes of high quality, suggesting that this gene has been lost at least two times on separate occasions in evolution and that the gene is thus not essential for life in all eukaryotes. The TMEM18 protein is predicted to have three transmembrane helices, a relatively uncommon property among human membrane proteins, as fewer than 5% of them share this feature [
42]. The membrane topology is mainly found among proteins with poorly characterized functions that do not have close relatives and are, therefore, sole representatives of their protein family in the human proteome. The TMEM18 protein has a nuclear localization signal that targets the protein for transportation to the nucleus, which also has been confirmed experimentally [
10]. Moreover, it contains a coiled-coil domain (Figure
1) that is common among DNA or RNA binding proteins, such a transcription factors, e.g. c-Fos [
43]. However, transcription factors are generally not membrane-bound proteins, but at least one exception exists in the gene named Myelin gene regulatory factor (MRF) that is predicted to have a transmembrane helix and plays a central regulatory role in myelination of the CNS [
44]. We found that there are only seven proteins, unrelated to each other, in the human proteome that contain three transmembrane helices and one coiled-coil domain, according to our queries in UniProt. Thus, the molecular structure of the TMEM18 protein is very uncommon in terms of other proteins in the human genome and surely very much different compared to other proteins involved in obesity, such as the enzyme FTO and the G protein-coupled melanocortin receptor, MC4R.
Other interesting features of the TMEM18 protein are the evolutionary conserved potential O-β-GlcNAc glycosylation and the phosphorylation sites, which are targets for the important posttranslational regulation of many proteins' functions and activities. Noteworthy, these sites are found in parts of the proteins that are supposed to face the inside of the nuclear envelope, i.e., the N-terminal and second loop (Figure
1) which, together with the beginning of the first and second transmembrane helices, are the most evolutionary conserved parts of the TMEM18 protein. This suggests that these parts of the protein constitute a potential active site, binding site or another important functional structure of the protein.
The TMEM18 gene is expressed in all the tissues we analyzed in the rat and mouse. Moreover, microarray data on TMEM18 from the fly (
D. melanogaster), found in the FlyAtlas database, (Figure
4) correspond well to the mammalian expression data and confirm the widespread expression with the abundant presence in the brain, although higher in several other tissues. Thus, the expression pattern is in agreement with the evolutionary conservation of the protein sequence. We performed a comprehensive immunohistochemical analysis as well as
in situ hybridization (Additional file
3). The immunohistochemical results showed that the TMEM18 protein is expressed throughout the mouse brain, which agrees with the real-time PCR expression profiles (Figure
4) and the
in situ hybridization (See Additional file
3). We performed co-localization with specific neuronal marker and assessed the extent of the expression in five major brain areas. We found that TMEM18 is expressed in most cells (71%), with a somewhat higher, but significant (p = 0.011; t-test), presence in neurons (73%) than in non-neurons (64%). The outcome is consistent for different brain areas studied (Figure
5), while the hypothalamus is the only region that has a relatively lower percentage of expression in neurons. The TMEM18 gene was presented as a hypothalamic gene by the earlier study reporting the GWA to obesity [
5]. We did not however see any evidence of significant enrichment in the TMEM18 expression level in the hypothalamus compared to other central regions, which is clear for, e.g., the aforementioned FTO gene and most neuroendocrine genes involved in body weight regulation. Thus, our results show that TMEM18 is likely to have a function in multiple tissues rather than being a solely hypothalamic gene. Nonetheless, the real-time PCR and immunohistochemical analyses confirmed that the TMEM18 is expressed abundantly in the hypothalamus and brainstem, regions that play a crucial role in the regulation of energy homeostasis: the hypothalamus, as the area that controls primarily feeding for calories and contains also components of the feeding reward system, and the brainstem that serves as the "relay station" for the gut-CNS signal cascade. Changes in energy needs of the organism, body weight as well as the rewarding value of food affect expression of genes known to participate in the regulation of consumption and body weight. Therefore, we studied the potential regulation of the TMEM18 gene expression in three different animal models that explore classical feeding paradigms. Feeding-related gene expression profile has previously been shown to be significantly changed in these tissues. For example, palatability affected hypothalamic expression of melanocortin receptor-4 (MC4R) and kappa (KOR) and mu (MOR) opioid receptors; an increased body weight influenced proopiomelanocortin (POMC), melanin concentrating hormone (MCH) and dynorphin (DYN) [
45] while restriction of energy intake through deprivation upregulated expression of the obesity-associated gene FTO. Somewhat surprisingly, the TMEM18's expression level in the hypothalamus and brainstem remained the same in all models utilized herein (Figure
6). In fact, it shows a more stable expression (GeNorm expression stability value; M = 0.493) than the commonly used housekeeping genes: Tubulin beta (M = 0.575) in the brainstem samples from the deprivation and increased body weight models (Experiment 1 and 3) and Cyclophilin (M = 0.505, TMEM18 M = 0.458) in the hypothalamus samples from the Sucrose vs Intralipid model (Experiment 2). The average expression stability value is 0.469, which is remarkable as it parallels housekeeping genes in these models. Still, TMEM18 would not be considered a true housekeeping gene as it is not found in all cells. It is however clear that TMEM18 is differently regulated compared to the classical neuropeptides and receptors, whose mRNA levels are altered in these models. It is interesting in this context that TMEM18 contains several potential phosphorylation and glycosylation sites (Figure
1) as both these processes are common regulatory pathways for nuclear proteins. It is also possible that this gene is so prevalent in central neurons (73% of them express it) that feeding-related changes in TMEM18 expression might have affected only a small subpopulation of TMEM18 cells and they were not detectable with the real-time PCR analysis of a large region, such as the hypothalamus or brainstem.
Our human genetic study showed a strong significance for the association of two SNPs (Table
1) located upstream of TMEM18 with childhood obesity in a Swedish cohort of obese children and a clear trend for an increase in BMI SDS for homozygote carriers of the major allele. Further, the estimations of the odds-ratios suggest that there are significantly higher risks for carriers of the major allele of both SNPs to develop obesity (Table
1). The odds-ratio for rs6548238 (OR = 1.450) is in the same magnitude as the calculations made by Renström and colleagues (OR = 1.100) for a cohort of 4923 Swedish adults [
7]. Moreover, the odds-ratios for the locus near TMEM18 are also as high as reported for the FTO locus (OR = 1.593) for the same cohort [
35]. This strengthens previous observations that this region is as strongly associated with obesity as FTO. We do not find however any significant association for the variations with any of the investigated obesity-related traits, many relevant for the detection of a diabetic phenotype, while there is a trend for a decrease in birth weight and length for homozygote carriers of the major alleles (Table
2). In conclusion, the results show that a region near TMEM18 is significantly contributing to obesity in severely obese children, a group that arguably has the highest genetic component and greatest need for healthcare among different population subgroups of obese individuals.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MSA designed, conceived and performed the bioinformatical and evolutionary analysis and drafted the manuscript. JAJ carried out the genetic studies and drafted the manuscript. JHAS did the immunohistochemical and in situ hybdridization experiments. PKO designed, performed and analyzed the feeding experiments and participated in the writing of the draft. JC and JA designed, performed and analyzed the expression studies and contributed to the discussion of the manuscript. ASL designed and conceived the feeding experiments. RF, MC and HBS conceived the study, and participated in its design and the writing of the draft. All authors read and approved the final manuscript.