Aims. Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease. Methods. We conducted a transcriptome-wide association study (TWAS) of spinal canal stenosis by integrating genome-wide association study summary statistics (including 661 cases and 178,065 controls) derived from Biobank Japan, and pre-computed gene expression weights of skeletal muscle and whole blood implemented in FUSION software. To verify the TWAS results, the candidate genes were furthered compared with messenger RNA (mRNA) expression profiles of LSS to screen for common genes. Finally, Metascape software was used to perform enrichment analysis of the candidate genes and common genes. Results. TWAS identified 295 genes with permutation p-values < 0.05 for skeletal muscle and 79 genes associated for the whole blood, such as RCHY1 (PTWAS = 0.001). Those genes were enriched in 112 gene ontology (GO) terms and five Kyoto Encyclopedia of Genes and Genomes pathways, such as ‘chemical carcinogenesis - reactive oxygen species’ (LogP value = −2.139). Further comparing the TWAS significant genes with the differentially expressed genes identified by mRNA expression profiles of LSS found 18 overlapped genes, such as interleukin 15 receptor subunit alpha (IL15RA) (PTWAS = 0.040, PmRNA = 0.010). Moreover, 71 common GO terms were detected for the enrichment results of TWAS and mRNA expression profiles, such as negative regulation of cell differentiation (LogP value = −2.811). Conclusion. This study revealed the genetic mechanism behind the pathological changes in LSS, and may provide novel insights for the early diagnosis and intervention of LSS. Cite this article: Bone Joint Res 2023;12(6):387–396

Aims. Degenerative cervical spondylosis (DCS) is a common musculoskeletal disease that encompasses a wide range of progressive degenerative changes and affects all components of the cervical spine. DCS imposes very large social and economic burdens. However, its genetic basis remains elusive. Methods. Predicted whole-blood and skeletal muscle gene expression and genome-wide association study (GWAS) data from a DCS database were integrated, and functional summary-based imputation (FUSION) software was used on the integrated data. A transcriptome-wide association study (TWAS) was conducted using FUSION software to assess the association between predicted gene expression and DCS risk. The TWAS-identified genes were verified via comparison with differentially expressed genes (DEGs) in DCS RNA expression profiles in the Gene Expression Omnibus (GEO) (Accession Number: GSE153761). The Functional Mapping and Annotation (FUMA) tool for genome-wide association studies and Meta tools were used for gene functional enrichment and annotation analysis. Results. The TWAS detected 420 DCS genes with p < 0.05 in skeletal muscle, such as ribosomal protein S15A (RPS15A) (PTWAS = 0.001), and 110 genes in whole blood, such as selectin L (SELL) (PTWAS = 0.001). Comparison with the DCS RNA expression profile identified 12 common genes, including Apelin Receptor (APLNR) (PTWAS = 0.001, PDEG = 0.025). In total, 148 DCS-enriched Gene Ontology (GO) terms were identified, such as mast cell degranulation (GO:0043303); 15 DCS-enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified, such as the sphingolipid signalling pathway (ko04071). Nine terms, such as degradation of the extracellular matrix (R-HSA-1474228), were common to the TWAS enrichment results and the RNA expression profile. Conclusion. Our results identify putative susceptibility genes; these findings provide new ideas for exploration of the genetic mechanism of DCS development and new targets for preclinical intervention and clinical treatment. Cite this article: Bone Joint Res 2023;12(1):80–90

Introduction. Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity. It occurs mainly in girls and progresses during pre-pubertal and pubertal growth, which is a crucial period for bone mass acquisition. The cause and molecular mechanisms of AIS are not clear; at present the consensus is that AIS has a multifactor cause, with many genetic factors. During the past 5 years, considerable effort has been devoted to identify a gene or genes that cause a predisposition to AIS. Many loci for this disorder have been mapped to different chromosome regions, but no genes have been clearly identified as being responsible for AIS, and, most importantly, the resulting protein defects remain to be shown. We aimed to identify the gene(s) that could be involved in AIS and to validate their involvement by both genetic and functional analyses. Methods. A large multiplex AIS French family was chosen for this study on the basis of clinical and radiological data. Whole genome genotyping of the 20 members of this family led to the mapping of a dominant disease-causing gene to two critical genomic intervals (Edery and colleagues, Eur J Hum Genet, accepted [2011]), but the causative mutation remains to be identified. In parallel, gene expression profiling was investigated by microarray analysis in RNA samples isolated from osteoblasts derived from healthy individuals and those with AIS. RNA samples were extracted from osteoblasts, purified, fluorescently labelled, and then hybridised to gene expression microarrays with the Illumina expression BeadChips technology containing more than 46 000 probes for the human genome (HumanHT-12). Data analysis in R version 2.10.1 (Bioconductor packages oligo and limma) was done, and genes that had at least 1·5-fold change in expression were considered differentially regulated relative to controls. AIS candidate genes within the critical intervals were selected on the basis of their mRNA expression in AIS individuals and by their known functions. The coding regions of these candidate genes were then sequenced to identify potential mutations. The biological activity of mutant proteins is under evaluation by in-vivo functional studies in zebrafish. Results. In the AIS family, a maximum LOD score of 3·01 was reached on two specific chromosomal regions. The interval lengths of these regions were 7cM and 12cM. These two regions contain several genes that might be responsible for AIS. Microarray analysis showed many genes that are differentially regulated in AIS osteoblasts compared with control osteoblasts. We recorded that 2·6% of the 24000 genes examined were upregulated in AIS osteoblasts, whereas 2·16% of them were downregulated. We observed a roughly 3-fold increase or decrease in the transcripts of many genes in AIS osteoblasts. Some of the differentially regulated genes are located within the two chromosomal candidate regions. The sequencing of the candidate genes' coding sequences was done on the family members. Sequence analysis showed two rare SNPs located on the coding regions of a gene that we called CH5G1. These two SNPs are located on the C-terminal region of the CH5G1 protein and affect its structure and probably its cellular activity and biological process leading to the disease. The C-terminal region of this protein interacts with the mRNA of a gene whose defects cause scoliosis as a secondary phenotype. The pathogenic nature of these SNPs is being investigated in the zebrafish model. The results suggest that CH5G1 gene's defects could be associated with AIS in this family. Conclusions. Identification of susceptibility genes for AIS will facilitate the understanding of underlying biochemical pathways (functional studies) and ultimately the development of specific therapies (pharmacological studies). This is likely to have important implications, since the cause of AIS is unknown. Acknowledgments. This study is supported by the Fondation Yves Cotrel, Institut de France

Introduction. Within the intervertebral disc (IVD), nucleus pulposus (NP) cells reside within a unique microenvironment. Factors such as hypoxia, osmolality, pH and the presence of cytokines all dictate the function of NP cells and as such the cells must adapt to their environment to survive. Previously we have identified the expression of aquaporins (AQP) within human IVD tissue. AQPs allow the movement of water across the cell membrane and are important in cellular homeostasis. Here we investigated how AQP gene expression was regulated by the microenvironment of the IVD. Methods. Human NP cells were cultured in alginate beads prior to cytokine, osmolality, pH and hypoxia treatments and subsequent RT-qPCR to assess regulation of AQP gene expression. Results. Physiological conditions observed within the native IVD regulated AQP gene expression in human NP cells. Hyperosmotic treatment up-regulated the expression of AQP1 and 5 during hypoxic conditions, whereas AQP4 expression was down-regulated. During hypoxia and physiological pH treatments AQP5 expression was increased. Pro-inflammatory cytokines, increased during IVD degeneration, also altered AQP gene expression. Interleukin-1β (IL-1β) decreased expression of AQP1 and 3 yet up-regulated AQP9, interleukin-6 (IL-6) increased expression of AQP1, 3, and 9 and tumour necrosis factor α (TNFα) upregulated the gene expression of both AQP2 and 9. Conclusion. The microenvironment in which NP cells reside in vivo directly contributes to their correct function and survival. AQP gene expression was differentially regulated under healthy compared to degenerate conditions; this potentially highlights that during IVD degeneration NP cells differentially express AQPs. No conflicts of interest. Funded by BMRC, Sheffield Hallam University

Low bone mass and osteopenia have been described in the axial and peripheral skeleton of patients with adolescent idiopathic scoliosis (AIS). Recently, many studies have shown that gene polymorphism is related to osteoporosis. However, no studies have linked the association between IL6 gene polymorphism and bone mass in AIS. This study examined the association between bone mass and IL6 gene polymorphism in 198 girls with AIS. The polymorphisms of IL6-597 G→A, IL6-572 G→C and IL6-174 G→A and the bone mineral density in the lumbar spine and femoral neck were analysed and compared with their levels in healthy controls. The mean bone mineral density at both sites in patients with AIS was decreased compared with controls (p = 0.0022 and p = 0.0013, respectively). Comparison of genotype frequencies between AIS and healthy controls revealed a statistically significant difference in IL6-572 G→C polymorphism (p = 0.0305). There was a significant association between the IL6-572 G→C polymorphism and bone mineral density in the lumbar spine, with the CC genotype significantly higher with the GC (p = 0.0124) or GG (p = 0.0066) genotypes. These results suggest that the IL6-572 G→C polymorphism is associated with bone mineral density in the lumbar spine in Korean girls with AIS

Introduction. Idiopathic scoliosis is a spine disorder of unknown origin with a prevalence of 1·5-3% in the general population. Apart from the large multifactorial form sample of idiopathic scoliosis, there is a good evidence for the existence of a monogenic subgroup in which the disease is inherited in a dominant manner. However, results from published work suggest a strong heterogeneity in locations of the mutated genes. Methods. With a high resolution genome-wide scan, we undertook linkage analyses in three large multigenerational families with idiopathic scoliosis compatible with dominant inheritance, including 11–12 affected members or obligate carriers. Results. In two of these families, our results suggested intrafamilial genetic heterogeneity, whereas in the other we recorded a perfect marker disease co-segregation in two distinct chromosomal regions. We can state that one of these two locations is a novel idiopathic scoliosis disease gene locus, since the probability of having by chance this perfect co-segregation twice in the genome is very low (p=0·001). Lastly, in all three families studied, we excluded compatibility with linkage to the previously mapped dominant idiopathic scoliosis loci on chromosomes 19p13.3, 17p11.2, 9q34, 17q25, and 18q. Conclusions. Our findings confirm that there is a high genetic heterogeneity within the subgroup of dominant forms of idiopathic scoliosis

Introduction. Kyphoscoliosis is defined by a structural lateral curvature of the spine of 10° or more and an excessive thoracic kyphotic curve of 40° or more. Genetic analyses of families in which two or more members had kyphoscoliosis identified a 3·5 Mb area on chromosome 5p containing three genes of the Iroquois (IRX) homeobox family, IRX1, IRX2, and IRX4, which were then sequenced. Methods. Exons and highly conserved non-coding regions (HNCRs) 500 kb upstream and downstream fromIRX1, IRX2, and IRX4 were sequenced in 46 individuals from six families. Selection of these elements was based on PhastCons Placental Mammal Conserved Elements, Multiz Alignment. Single-nucleotide polymorphism (SNP) genotypes and sequence variants were obtained from all individuals. There were 431 SNPs, 61 in IRX4 regions, 80 in IRX2 regions, and 290 in IRX1 regions. 137 SNPs were novel. Mendelian inconsistencies were detected with PEDCHECK (inconsistency rate: 1·4%; missing data: 2·8%). SNPs and individuals with greater than 10% missing rate were excluded. Association analyses (ASSOC [SAGE version 6.0.1]) of the quantitative trait with patient's largest curve, were undertaken on 391 SNPs. Results. Association analyses resulted in 12 SNPs with p values less than 0·025, 11 of which were located upstream and downstream from IRX1. The most significant p value (p=0·000382) was obtained for rs35710183 (table). Multiple variants were found surrounding IRX1. The most prominent is a single base-pair deletion in all affected individuals genotyped in one family. All individuals with kyphoscoliosis and those with scoliotic curves greater than 35° had genotypes differing from the reference (unaffected) genotype for 23 SNPs. Several of these SNPs had significant p values for the association analyses done previously. Conclusions. The phenotype of kyphoscoliosis has been linked to sequence variants that lie within regulatory regions of the IRX homeobox gene family. Further analyses to establish the relevance of these findings will be done through in-vivo and in-vitro assays. The identification of spinal genetic determinants related to axial growth and maturation will help with the understanding of spinal pathology and potentially allow for development of directed therapeutic interventions

It has been suggested that matrix metalloproteinase-3 (MMP-3, stromelysin-1) has an important role in the degeneration of intervertebral discs (IVDs). A human MMP-3 promoter 5A/6A polymorphism was reported to be involved in the regulation of MMP-3 gene expression. We suggest that IVD degeneration is associated with 5A/6A polymorphism. We studied 54 young and 49 elderly Japanese subjects. Degeneration of the lumbar discs was graded using MRI in the younger group and by radiography in the elderly. 5A/6A polymorphism was determined by polymerase-chain reaction-based assays. We found that the 5A5A and 5A6A genotype in the elderly was associated with a significantly larger number of degenerative IVDs than the 6A6A (p < 0.05), but there was no significant difference in the young. In the elderly, the IVD degenerative scores were also distributed more highly in the 5A5A and 5A6A genotypes (p = 0.0029). Our findings indicate that the 5A allele is a possible risk factor for the acceleration of degenerative changes in the lumbar disc in the elderly

Aims. This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD). Methods. The gene expression profile of GSE23130 was downloaded from the Gene Expression Omnibus (GEO) database. Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases, and we used Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze the functions and pathways of EP-DEGs. STRING and Cytoscape were used to construct protein-protein interaction (PPI) networks and identify hub EP-DEGs. NetworkAnalyst was used to analyze transcription factors (TFs) and microRNAs (miRNAs) that regulate hub EP-DEGs. A search of the Drug Signatures Database (DSigDB) for hub EP-DEGs revealed multiple drug molecules and drug-target interactions. Results. A total of 56 EP-DEGs were identified in the differential expression analysis. EP-DEGs were enriched in the extracellular structure organization, ageing, collagen-activated signalling pathway, PI3K-Akt signalling pathway, and AGE-RAGE signalling pathway. PPI network analysis showed that the top ten hub EP-DEGs are closely related to IDD. Correlation analysis also demonstrated a significant correlation between the ten hub EP-DEGs (p＜0.05), which were selected to construct TF–gene interaction and TF–miRNA coregulatory networks. In addition, ten candidate drugs were screened for the treatment of IDD. Conclusion. The findings clarify the roles of extracellular proteins in IDD and highlight their potential as promising novel therapeutic targets. Cite this article: Bone Joint Res 2023;12(9):522–535

The aim of this study is to clarify the implication of ciliary pathway on the onset of the spinal curvature that occurs in Adolescent Idiopathic Scoliosis (AIS) patients through functional studies of two genes: POC5 and TTLL11. Since the genetic implication for AIS is accepted, many association and candidate gene analysis revealed the implication of ciliary genes. The characterisation of these two proteins was assessed by qPCR, WB and immunofluorescence in vitro using control cells and cells derived from AIS patients. The impact of genetic modification of these genes on the functionality of the proteins in vitro and in vivo was analysed in zebrafish model created by CRISPR/Cas9 using microCT and histologic analysis. Our study revealed that mutant cells, for both gene, were less ciliated and the primary cilia was significantly shorter compared to control cells. We also observed a default in cilia glutamylation by immunofluorescence and Western Blot. Moreover, we observed in both zebrafish model, a 3D spine curvature similar to the spinal deformation in AIS. Interestingly, our preliminary results of immunohistology showed a retinal defect, especially at the cone cell layer level. This study strongly supports the implication of the ciliary pathway in the onset of AIS and this is the first time that a mechanism is described for AIS. Indeed, we show that shorter cilia could be less sensitive to environmental factors due to lower glutamylation and result in altered signalling pathway. Identifying the biological mechanism involved is crucial for elucidating AIS pathogenesis

Background. Intervertebral disc degeneration (DD) is a complex age-related condition that constitutes the main risk factor for disabling back pain. DD is assessed using different traits extracted from MR imaging (MRI), normally combined to give summary measures (e.g. Pfirmann score). The aetiology of DD is poorly understood and despite its high heritability (75%), the precise genetic predisposition is yet to be defined. Genome wide association study (GWAS) is used to discover genetic variants associated with a disease or phenotype. It tests variants across the whole genome. It requires large samples to provide adequate but unfortunately there is poor availability of spine imaging data due to the high cost of MRI. We have adopted new methods to examine different MRI traits independently and use the information of those traits to boost GWAS power using specialized statistical software for jointly analyse correlated traits. Methods/Results. We examined DD MRI features disc narrowing, disc bulge, disc signal intensity and osteophyte formation in the TwinsUK cohort who had undergone T2-weighted sagittal spine MRI. GWAS were performed on the four traits. MTAG software was used to boost single trait GWAS power using the information in the other trait GWAS. 9 different loci were identified. Conclusions. Preliminary results suggest genes GDF6, SP1/SP7 are associated with individual trait signal intensity. In addition, novel associated genes with potential for shedding new light on pathogenic mechanisms are identified. Additional cohorts will be included in the design as a replication to test reproducibility of the results. Conflicts of interest: No conflicts of interest. Sources of funding: Funded by Disc4All, EU Horizon 2020, MSCA-2020-ITN-ETN GA: 955735

Backgrounds and aim. Low back pain resulting from Intervertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect, however, their behaviour in the harsh degenerate environment is unknown. Thus, we aimed to investigate and compare their physiological behaviour in in vitro niche that mimics the healthy and degenerated intervertebral disc environment. Methodology. Porcine NC cells were encapsulated in 3D alginate beads to maintain their phenotype then cultured in media to mimic the healthy and degenerate disc environment, together with control NC media for 1 week. Following which viability using PI and Calcein AM, RNA extraction and RT-PCR for NC cell markers, anabolic and catabolic genes analysed. Proteomic analysis was also performed using Digiwest technology. Results. A small increase in cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in NCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Discussion. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The initial work has been then translated to investigate the potential use of iPSCs differentiated into notochordal like cells as potential regenerative cell sources. Conflicts of interest: No conflicts of interest. Sources of funding: This project has received funding from the European Union Horizon 2020 research and innovation programme under grant agreement No 825925

Objectives. This study aims to investigate whether bacteria are present in intervertebral discs (IVDs) and their influence. Causality between chronic infection of the IVD and its degenerative process gained great interest recently. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in IVDs, from these 27 studies found, Cutibacterium acnes being the most abundant. However, whether bacteria identified were present in vivo or if they represent contamination remains unclear. Methods. Human IVD tissue was fixed in paraffin and Immunohistochemical stained for Gram-positive bacteria. NP cells in monolayer have been stimulated with LPS (0.1–50 µg/ml) and Peptidoglycan (0.1–50 µg/ml) for 24, 48 and 72 hrs to investigate their influence. The concentration of proinflammatory and catabolic cytokines in the media is being measured using ELISA. RNA extracted and RT-qPCR utilised for factors associated with disc degeneration matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors. Results. Bacteria were detected within IVD tissue. Bacteria was internalized by the NP cells and influenced the nuclei morphology. Preliminary results of the exposure of NP cells to bacterial components demonstrate that ADAMTS4 as well as IL-8 were showed an increase in gene expression after LPS and peptidoglycan treatment compared to the non-treated control. Underlining these results, IL-8 protein was increased in treated groups, whereas peptidoglycan treated groups showed a dose dependence. Conclusion. This study demonstrates that Gram positive bacteria are present within the IVD. The exposure of NP cells to peptidoglycans indicates that bacterial components trigger a stress response. Conflicts of Interest: No conflict of interest. Sources of Funding: This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

To determine whether spinal facet osteoblasts at the curve apex display a different phenotype to osteoblasts from outside the curve in patients with adolescent idiopathic scoliosis (AIS). Intrinsic differences in the phenotype of spinal facet bone tissue and in spinal osteoblasts have been implicated in the pathogenesis of AIS. However, no study has compared the phenotype of facet osteoblasts at the curve apex with the facet osteoblasts from outside the curve in patients with AIS. Facet bone tissue was collected from three sites, the concave and convex side at the curve apex and from outside the curve from three female patients with AIS (aged 13–16 years). Micro-CT analysis was used to determine the density and trabecular structure. Osteoblasts were then cultured from the sampled bone. Osteoblast phenotype was investigated by assessing cellular proliferation (MTS assay), cellular metabolism (alkaline phosphatase and Seahorse Analyser), bone nodule mineralisation (Alizarin red assay), and the mRNA expression of Wnt signalling genes (quantitative RT-PCR). Convex bone showed greater bone mineral density and trabecular thickness than did concave bone. The convex side of the curve apex exhibited a significantly higher proliferative and metabolic phenotype and a greater capacity to form mineralised bone nodules than did concave osteoblasts. mRNA expression of SKP2 was significantly greater in both concave and convex osteoblasts than in non-curve osteoblasts. The expression of SFRP1 was significantly downregulated in convex osteoblasts compared with either concave or non-curve. Intrinsic differences that affect osteoblast function are exhibited by spinal facet osteoblasts at the curve apex in patients with AIS

We previously reported that osteoblasts at the curve apex in adolescent idiopathic scoliosis (AIS) exhibit a differential phenotype, compared to non-curve osteoblasts(1). However, the Hueter-Volkmann principle on vertebral body growth in spinal deformities (2) suggests this could be secondary to altered biomechanics. This study examined whether non-curve osteoblasts subjected to mechanical strain resemble the transcriptomic phenotype of curve apex osteoblasts. Facet spinal tissue was collected perioperatively from three sites, (i) the concave and (ii) convex side at the curve apex and (iii) from outside the curve (non-curve) from six AIS female patients (age 13–18 years; NRES 19/WM/0083). Non-curve osteoblasts were subjected to strain using a 4-point bending device. Osteoblast phenotype was determined by RNA sequencing and bioinformatic pathway analysis. RNAseq revealed that curve apex osteoblasts exhibited a differential transcriptome, with 1014 and 1301 differentially expressed genes (DEGs; p<0.05, fold-change >1.5) between convex/non-curve and concave/non-curve sites respectively. Non-curve osteoblasts subjected to strain showed increased protein expression of the mechanoresponsive biomarkers COX2 and C-Fos. Comparing unstimulated vs strain-induced non-curve osteoblasts, 423 DEGs were identified (p<0.05, fold-change >1.5). Of these DEGs, only 5% and 6% were common to the DEGs found at either side of the curve apex, compared to non-curve cells. Bioinformatic analysis of these strain-induced DEGs revealed a different array of canonical signalling pathways and cellular processes, to those significantly affected in cells at the curve apex. Mechanical strain of AIS osteoblasts in vitro did not induce the differential transcriptomic phenotype of AIS osteoblasts at the curve apex

Study purpose and background. Novel regenerative therapies have the potential to restore function and relieve pain in patients with low back pain (LBP) caused by intervertebral disc (IVD) degeneration. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation into nucleus pulposus (NP) cells of the IVD, which have potential for IVD regeneration. We have also shown that GDF6 stimulation activates the Smad1/5/8 and ERK1/2 signalling cascades. The aim of this study was to progress our understanding of the immediate/early response mechanisms in ASCs (N=3) which may direct GDF6-induced differentiation. Methods and results. RNAseq was used to perform transcriptome-wide analysis across a 12-hour time course, post-stimulation. Gene ontology analysis revealed greater transcription factor and biological processes activity at 2hrs than at the 6hr and 12hr time points, where molecular and cellular activities appeared to stabilise. Interestingly, a number of lineage determining genes were identified as differentially expressed and work is ongoing to investigate whether the early response genes are maintained throughout differentiation, or whether they are responsible for early NP lineage commitment. Conclusion. This study is the first transcriptome-wide analysis on GDF6-mediated stimulation of ASCs, elucidating important early response mechanisms involved in directing appropriate differentiation. Identification of additional key markers and signalling pathways of differentiation will allow improved selection of ASCs for IVD regeneration. ‘No conflicts of interest’. Funding sources: NIHR Manchester Biomedical Research Centre and The RoseTrees Trust

Purpose of study and background. Degeneration of the intervertebral disc is a strong contributor of low back pain. Studies have shown that both, mechanical unloading and overloading, lead to disc degeneration. This is intuitively clear if one considers that an intervertebral disc essentially is a poro-elastic material embedded with cells, which depend on fluid flow for the transport of nutrients and waste products. As such, mechanical loading is also required for regeneration. It is unclear, however, how much loading is beneficial or detrimental for the healthy or degenerated disc. Methods and Results. We developed a loaded disc culture system for the long-term study of disc physiology. This way we could control both the mechanical and biochemical conditions. If no loading was applied, about half of the cells died within a week. Cells died under a low dynamic loading regime after three weeks. A diurnal loading regime rescued cell viability, gene expression profile and mechanical behavior of the discs. Both static and dynamic overloading induced damage to the discs and led to catabolic and inflammatory gene expressions. Conclusion. Intervertebral discs need a certain dosage of mechanical loading to remain viable. Under overloading, cells deform, change gene expression and become degenerative. The matrix is also remodeled, thereby further decreasing the hydrostatic pressure on the cells and increasing their deformation. This induces a vicious circle of disc degeneration, which needs to be reversed in order to repair the disc. The loaded disc culture system also allows evaluating new therapies for disc degeneration. There are no conflicts of interest. Funded by ZonMW program “Alternatives for live animal testing”, grant #11400090;. BioMedical Materials Program, grant # P2.01 IDiDas; Dutch Arthritis Funds, personal grant KSE

Objective. Modic changes (MC), a form of intervertebral disc degeneration visible as subchondral and vertebral bone marrow changes on spine magnetic resonance (MR), are known to be associated with low back pain. This study aimed to identify genes contributing to the development of MC using genome-wide association study. Methods. Presence of MC was evaluated in lumbar MR images in the Northern Finland Birth Cohort 1966 (NFBC1966, N=1182) and TwinsUK (N=647). Genome-wide association analyses were carried out in the cohorts separately using a linear regression model fitted to test for additive effects of SNPs and adjusting for age, sex, BMI, and either family relatedness via a kinship matrix (TwinsUK) or population stratification using principal components (NFBC1966). Meta-analysis of the two studies was carried out using the inverse-variance weighting approach. Results. A locus associated with MC reaching genome-wide significance (p<5e-8) was found on chromosome 9 with the lead SNP rs1934268 in intron 6 of the PTPRD gene. The SNP is located in the region of binding for a number of transcription factors which are involved in the development of the musculoskeletal system and spine cord. Conclusions. The first GWAS of MC has identified a likely functional intronic locus in PTPRD on chromosome 9 implicating musculoskeletal development. This work sheds light on the genesis of MC and paves the way for further studies on the shared genetic factors underlying the various features of spine degeneration. No conflicts of interest. Sources of Funding: The study was supported by EU FP7 project PainOMICs (grant agreement #602736), University of Oulu (grant #24000692), Oulu University Hospital (grant #24301140), and the European Regional Development Fund (grant # 539/2010 A31592). MBF, MK, and SS contributed equally to this study

Introduction. The intervertebral disc (IVD) is a highly hydrated tissue which is reduced during degeneration leading to loss of function. Aquaporins (AQP) are a family of 13 (AQP0-12) transmembrane channel proteins that selectively allow the passage of water and other small molecules in and out of cells and are responsible for maintaining water homeostasis. AQP1, 2, 3 and 5 have been identified in the IVD. Here gene and protein expression of all 13 AQPs was investigated in a large cohort of human IVDs to investigate expression during IVD degeneration. Methods. Gene expression of all 13 AQPs was investigated in non-degenerate and degenerate tissue from 102 human NP samples using RT-qPCR. AQPs which were expressed at gene level were further investigated in 30 IVD samples by Immunohistochemistry. Results. At gene level, AQP0, 1, 2, 3, 4, 5, 6, 7 and 9 were expressed in both non-degenerate and degenerate tissue. For the first time, protein expression of AQP6, 7 and 9 was identified in human IVD tissue, in which AQP2 and 3 were also identified. Conclusion. Hydration of the IVD is vital for its correct biomechanical function and a loss of water is associated with degeneration. The presence of many AQP isoforms within IVDs may suggest multiple roles for these water channels related to the survival and adaptation of NP cells, and physiology of the healthy IVD. The functional role of AQPs within the IVD is yet to be elucidated and thus warrants further investigation. No conflicts of interest. Funded by BMRC, Sheffield Hallam University

Objective. Low back pain (LBP) is a common debilitating condition with great socioeconomic impact. Identifying individuals at risk of LBP is challenging. We have shown IgG N-glycans are associated with LBP. Herewith, we used polygenic risk scores (PRS) from IgG-glycome to test predictability for LBP. Methods. Clusters of IgG-glycans were identified using weighted correlation network approach in TwinsUK (n = 4246). Genome-wide association studies were carried out for the clusters and top associated SNPs (p<5e-8) were extracted. Weighted PRS was calculated as the sum of the number of copies of effect allele from GWAS multiplied by their effect size using the UK Biobank data (n = 350000). The predictive capacity of the PRS for back pain in UK Biobank was estimated using logistic regression. Results. Multiple SNPs were found to be associated with the glycan clusters near genes known to be involved in glycosylation and the inflammatory response (e.g. ST6GAL1, B4GALT1, FUT8). A total of 175 SNPs was used to calculate weighted PRS. In UK Biobank the PRS was a statically significant, but poor, predictor of the risk of back pain (β = 0.126±0.050, p = 0.015, R. 2. = 2.6e-5). The SNPs on chromosome 14 in regulatory regions of FUT8 gene, one of the key governors of core fucosylation, were found to be significantly associated with back pain in UK Biobank (FDR-adjusted p-value < 0.05). Conclusions. These pilot data suggest that genetic component of glycosylation may be associated with the risk of LBP; however, its predictive ability is poor. Conflict of Interest: YSA is a co-owner of Maatschap PolyOmica. GL is a founder and CEO of Genos Glycoscience Research Laboratory. MBF and FMKW declare no conflict of interests. Sources of Funding: The research has been supported by the EC FP7 project PainOmics (grant agreement #602736) and conducted using the UK Biobank Resource (project # 18219)