Genetic and mechanical interactions in the pathogenesis of adolescent idiopathic scoliosis

Abstract

Abstract of the thesis entitled Genetic and Mechanical Interactions in the Pathogenesis of Adolescent Idiopathic Scoliosis Submitted by Vimalagopalan Divya For the degree of Master of Philosophy at The University of Hong Kong in March 2021

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity affecting adolescent-aged children. Affected children do not display outward spinal defects at birth but develop bodily curvature later. Despite much research, its pathogenesis has not been conclusively deemed to depend on a single factor. Although multiple other factors may cause AIS, interplay between genetics and the environment is likely to exist. For example, IVD wedging is a risk factor for AIS curvatures. A recent genome-wide association study listed several susceptible loci, including PAX1. A decrease in PAX1 levels due to single-nucleotide polymorphism in the enhancer region of PAX1 was found to be strongly associated with AIS in females. However, the functional mechanism has yet to be explored. This research attempts to understand the contribution of PAX1 to the pathogenesis of AIS. PAX1 codes for a transcription factor that regulates the development of the vertebral column and intervertebral discs (IVDs), but little is known about its postnatal functions. IVD is a fibrocartilage structure with a well-hydrated nucleus pulposus (NP) encapsulated by the concentric lamellae annulus fibrosus (AF) and separated from the adjacent vertebral body by the end plate. Together, they function as a unit for load bearing and distribution. The cellular composition enables the maintenance of IVD homeostasis. Loss of integrity in the IVD compartments can trigger a cellular stress response, and cascade of events disrupting spinal homeostasis and inducing curvatures. Asymmetrical mechanical loading will result in IVD wedging, inducing degenerative changes. Here we aim to study the effects of reduced Pax1 levels in IVDs on spinal homeostasis and the combined effects of Pax1 and IVD wedging on the progression of AIS. In this study, the dosage effects of Pax1 on IVD formation and maintenance were characterized in mice with Pax1-null allele(s). To study the environment interaction, a tail-looping model was adopted to initiate AIS wedging in IVDs, and the combined effect of asymmetrical loading and PAX1 deficiency was assessed to understand the contribution of Pax1 to curvature progression. Finally, bio-informatic analysis of RNA seq data was performed to determine the potential mechanism. Pax1 homozygous mice (HO) exhibited congenital scoliosis, including vertebral body and IVD defects at all spinal levels. Pax1 heterozygous mice (HE) appeared normal in gross appearance but had a mild distal kink. The histology of HE revealed early degenerative signs, such as chondrocyte-like cell clusters and honeycomb-like formations on the NP-AF boundaries, as well as chondrocyte-like cells with enlarged cell nuclei along AF fibers, which alters IVDs’ mechanical properties and triggers an altered cellular response upon mechanical loading. After wedging by tail-looping for two weeks and four weeks, Pax1 HE mice showed early deformations, such as decreased NP area, increased AF width on the compressed side, and increased AF height on the extended side, as well as increased apoptosis, which indicated a decreased resistance to loads. Unlooping followed by two weeks of recovery showed increased AF width on the compressed side and increased AF height on the extended side, indicating that AF fibers may have the potential for delayed/impaired self-repairing in Pax1 HE mice. Immunofluorescence and transcriptomic analysis demonstrated increased Pax1 expression upon wedging, clearly indicating that Pax1 is responsive to environmental forces. In summary, this study demonstrates that Pax1 levels play a significant role in the pathogenesis of AIS. Specifically, it was found that an environmental factor such as asymmetrical mechanical loading, together with a genetic factor Pax1, contributes to the progression of spinal curvature in AIS.

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