The melatonin hypothesis for the genetic causation of adolescent idiopathic scoliosis originated in 1959, when Thillard first reported that pinealectomy produced scoliosis in chickens, which was confirmed by Dubousset et al
Since those early studies, the association between melatonin and the development of scoliosis has remained a promising, but contentious field of research. A critique of the evidence for the melatonin hypothesis is that simply the surgery required to perform a pinealectomy induces scoliosis, while the low melatonin levels are merely a secondary effect of the surgery and not directly associated with the development of scoliosis. Machida et al
addressed this critique in a recent study where they induced experimental scoliosis in mice without pinealectomy and supplemented the scoliotic mice with melatonin.15
Twenty-nine of 30 bipedal mice with no melatonin treatment developed scoliosis, 5 of 20 control quadrupedal mice developed scoliosis, but none of the bipedal mice with melatonin treatment developed scoliosis, which supported the contention that restoration of melatonin levels can prevent the development of scoliosis. Additionally, Machida et al
designed a study that found pineal gland transplantation and melatonin supplementation following pinealectomy procedures prevented the development of spinal deformity.16
Several studies have proposed that melatonin-related receptors may be involved in scoliosis. Yong et al
found that melatonin receptor expression in bilateral paravertebral muscles in AIS is asymmetric, but recommended further research to determine whether the asymmetry was a primary cause or secondary effect of scoliosis.17
High-affinity melatonin receptors (hMel-1A and hMel-1B) have recently been cloned and appear to be the sites through which melatonin elicits its biologic effects.18
The melatonin receptors localize in the hypothalamic suprachiasmic nuclei, cerebellum and spinal cord, and nonneural sites including ovaries and blood vessels.19
Also, these receptors may be associated with human balance control and sensorimotor performance.20
Morcuende et al
studied hMel-1A and found no mutations in the coding region of hMel-1A.21
In this study we did not find any mutation in hMel-1B, which corroborates the study by Qiu et al
which failed to identify a single nucleotide polymorphism associated with the occurrence of AIS.22
A more recent study by Qiu et al
found that a SNP in the promoter region of hMel-1B was associated with AIS.23
We performed a screen of the SNP in 180 patients with AIS and 180 controls, and found no significant difference in genotype frequencies.
Recent research has also found evidence of a dysfunction in melatonin signaling pathway in patients with AIS. Moreau et al
investigated the ability of melatonin to block cAMP accumulation in osteoblast samples collected from patients with AIS, and found this ability to be significantly depressed when compared with controls.24
Later work by the same group showed evidence of a molecular interaction between hMel-1B and protein kinase C delta, and an improvement in the ability to suppress cAMP by melatonin in AIS osteoblasts when supplemented with estrogen.25, 26
Hardeland et al
recently provided an excellent review of melatonin, its receptors, and its signaling pathways.27
In particular, the review provided a comprehensive discussion of the highly pleiotropic effects of melatonin that depend on the distribution and density of its receptors, G protein directed signaling through membranous receptors, and tissue-dependent melatonin metabolites. The intimate regulation of melatonin-related transcription factors and protein kinases by G-alpha variants, beta-gamma dimers, beta-arrestin, or ion channels provides an expanding area that demands further research in relation to AIS.
GPR50 is an X-linked, orphan G protein-coupled receptor that has been associated with melatonin-receptor regulation. Levoye et al
found GPR50 heterodimerizes constitutively and specifically with hMel-1A and hMel-1B melatonin receptors, and abolished the high-affinity binding function of the hMel-1A melatonin receptor.12
RORα is a nuclear hormone receptor that can bind to the hormone response elements of several genes as a possible enhancer, though the specific functions of this protein are not known. Wiesenberg et al
proposed that RORα acts as a transcription factor that mediates nuclear melatonin signaling.11
In this study, we found two polymorphisms in the GPR50 receptor of scoliotic patients, but they were not present at a highly significant frequency when compared to the controls. The second exon of GPR50, in which the two cSNPs are found, encodes a transmembrane domain, which we would expect to be malfunctional in AIS patients. We initially hypothesized that the two polymorphisms in GPR50 could additively cause a severe conformational change in the protein structure of GPR50, thereby preventing GPR50 from properly binding to hMel-1A and hMel-1B and preventing GPR50 from abolishing the binding function of hMel-1A; however, this hypothesis was not supported by our data.
Several limitations of our study design should be addressed. First, we did not screen the non-coding (intronic) regions of GPR50, hMel-1B, and ROR-Alpha, and it is possible there may be a transcription binding site or enhancer region in the intronic sequence that could contain mutations. However, in genetic screens it is generally accepted not to evaluate the intronic regions given the excessive amount of DNA sequence information and the fact that the specific location of regulatory binding sites within the intronic regions are often unknown, as was the case in our genes. Second, we used relatively small patient and control populations in our initial screen, but our total population included over 400 patients and controls. Based on the initial sample size, it is possible that we missed rare mutations that cause a small proportion of scoliosis cases.
Overall, our results combined with the hMel-1A results of Morcuende et al
demonstrate that no mutations in any known melatonin-related receptor are associated with the phenotype of AIS.21
No significant differences were found in the GPR50, RORα, or hMel-1B coding, splice site, or promoter regions. The strong evidence of a melatonin-related cause for the development of idiopathic scoliosis still encourages research into undiscovered melatonin-related receptors, and hormones such as the melatonin-precursor serotonin, and the catalytic enzymes for the serotonin-melatonin pathway, HIOMT and NAT. The melatonin hypothesis remains a controversial, but promising field of research, and this study marks a significant finding in the understanding of the relationship between melatonin, melatonin receptors, and the development of adolescent idiopathic scoliosis.