Adolescent idiopathic scoliosis (AIS) still lacks a clear etiopathogenesis despite much clinical, epidemiological and basic science research, impeding the development of diagnostic and treatment strategies.¹ There is evidence to suggest that AIS has a strong dependence on genetic factors, though previous studies were not able to identify a specific cause.¹ Recently, a research team published the results of a genetic analysis of a large multicenter AIS cohort, which found that variants of the SLC6A9 gene may contribute to aberrant glycinergic neurotransmission.² The results indicate that dysfunction of synaptic neurotransmission and central pattern generators (CPGs)  is a potential common cause of AIS, thus providing a neuropathic origin for AIS.²

AIS is the most common type of scoliosis which affects 1-4% of the adolescent population and disproportionately affects young women.³ In severe cases, it can cause cardiopulmonary difficulties, leading to shortness of breath and potentially mortality.² Since AIS usually manifests in generally healthy patients and diagnosis can only be reached after eliminating all other common causes of scoliosis.⁴ As such, much of the etiology of AIS is unknown.⁴ This presents a hurdle to the development of early diagnostic, preventive and therapeutic strategies.² Earlier research has indicated a strong genetic influence on AIS.¹ However, despite extensive investigation, a specific locus could not be pinpointed and the precise causative factors remain unclear.¹

It was found that homozygous mutations in the SLC6A9 gene are linked to glycine encephalopathy, also known as nonketotic hyperglycinemia (NKH).⁵ This disease is characterized by significant elevation of glycine, an essential neurotransmitter and inhibitor in the central nervous system (CNS) in plasma and cerebrospinal fluid (CSF) and may lead to hypotonia and seizures.⁵ Patients who survive NKH tend to show progressive early-onset neuromuscular scoliosis, suggesting a link between SLC6A9 mutations and AIS.^2,6

To study this linkage, a genetic analysis of a large multicenter AIS cohort and identified disease-causing and predisposing SLC6A9 variants. The AIS cohort consisted of multigeneration families, trios, and around 1,700 sporadic patients.² 3219 ethnicity-matched controls were also included in the study.² Whole genome sequencing was performed on AIS individuals in the included families, which identified 2 rare variants of the SLC6A9 gene (c.1984C>T, p.R662W and c.617A>T, p.Y206F) and the amino acid substitutions were predicted to be deleterious or damaging, suggesting the gene variants are potential causes for AIS.² Subsequent SLC6A9-variant screening performed in sporadic patients yielded comparable results.² Several rare heterozygous missense variants of SLC6A9 were also identified and predicted to be deleterious or damaging.² All these variants altered conserved residues in various regions of glycine transporter 1 (GLYT1) and facilitated abnormally high levels of glycine.² Indeed, the study also reported relatively higher plasma glycine concentrations in AIS patients carrying SLC6A9 variants compared to the unaffected controls.² Cellular glycine uptake was significantly reduced in patients carrying the variants.² The results showed that most of the identified SLC6A9 variants from AIS patients caused loss of function and led to negative effects.²

To validate the association between SLC6A9 mutations and AIS, a zebrafish model was created by generating an SLC6A9 mutant zebrafish line.² It was found that at 7 days post fertilization (dpf), 65% of the SLC6A9 mutation-positive (slc6a9^m/^m) fish showed an apparent lateral axial curvature.² By 18 dpf, all slc6a9^m/m fish died, with spinal curvature observed in all of the dying subjects.² It was further suggested that excessive glycine caused by mutant GLYT1 interfered with the function of central pattern generators (CPGs), which are important in maintaining spinal alignment.² This further suggested that such mutations could lead to spinal developmental defects and cause scoliosis-like phenotypes.²

To investigate possible pharmacological prevention strategies, strychnine, an ionotropic glycine receptor (GlyR) antagonist, was given to slc6a9^m/^m zebrafish subjects, which reduced the percentage of fish with axial curvature from 70.2% to 30.3%.² When sodium benzoate, a glycine neutralizer used to treat glycine encephalopathy, was administered, the percentage of fish with the curvature phenotype reduced from 62.7% to 40.0%.² This suggests that the neutralization or blocking of glycine is a potential prevention strategy of AIS.²

In summary, this study has identified several variants in the GLYT1-encoding SLC6A9 gene which led to elevated extracellular glycine levels and abnormal glycinergic neurotransmission.² The zebrafish model showed that mutant GLYT1 led to the dysfunction of CPGs and dysregulations in spinal alignment.² Administration of glycine blockers or neutralizers lessened the effect of curvature.² These findings indicate a link between the SLC6A9 mutation, abnormal glycine levels and scoliosis, indicating an origin for AIS.²