Hereditary axonopathies are frequently caused by mutations in proteins that reside in the endoplasmic reticulum (ER). Which of the many ER functions are pathologically relevant, however, remains to be determined. REEP1 is an ER protein mutated in hereditary spastic paraplegia (HSP) and hereditary motor neuropathy (HMN). We found that HSP-associated missense variants at the N-terminus of REEP1 abolish ER targeting, whereas two more central variants are either rare benign SNPs or confer pathogenicity via a different mechanism. The mis-targeted variants accumulate at lipid droplets (LDs). N-terminal tagging, deletion of the N-terminus, and expression of a minor REEP1 isoform had the same effect. We also confirmed an increase in LD size upon cooverexpression of atlastins and REEP1. Neither wild-type REEP1, LD-targeted HSP variants, nor a non-LD-targeted HMN variant reproduced this effect when expressed alone. We conclude that the N-terminus of REEP1 is necessary for proper targeting to and/or retention in the ER. The protein's potential to also associate with LDs corroborates a synergistic effect with atlastins on LD size. Interestingly, LD size is also altered upon knockdown of seipin, mutations of which also cause HSP and HMN. Regulation of LDs may thus be an ER function critical for long-term axonal maintenance.

Osteoarthritis (OA) is a polygenic disease of older people resulting in the breakdown of cartilage within articular joints. Although a leading cause of disability, there are no disease-modifying therapies. Evidence is emerging to support the origins of OA in skeletogenesis. Whilst methylation QTLs (mQTLs) co-localizing with OA GWAS signals have been identified in aged human cartilage and used to identify effector genes and variants, such analyses have never been conducted during human development. Here, for the first time, we have investigated the developmental origins of OA genetic risk at seven well-characterized OA risk loci, comprising 39 OA-mQTL CpGs, in human foetal limb (FL) and cartilage (FC) tissues using a range of molecular genetic techniques. We compared our results to aged cartilage samples (AC) and identified significant OA-mQTLs at 14 CpGs and 29 CpGs in FL and FC tissues, respectively. Differential methylation was observed at 26 sites between foetal and aged cartilage, with the majority becoming actively hypermethylated in old age. Notably, 6/9 OA effector genes showed allelic expression imbalances during foetal development. Finally, we conducted ATAC-sequencing in cartilage from the developing and aged hip and knee to identify accessible chromatin regions, and found enrichment for transcription factor binding motifs including SOX9 and FOS/JUN. For the first time, we have demonstrated the activity of OA-mQTLs and expression imbalance of OA effector genes during skeletogenesis. We show striking differences in the spatiotemporal function of these loci, contributing to our understanding of OA aetiology, with implications for the timing and strategy of pharmacological interventions.

Objective Osteoarthritis (OA) is an age‐related disease characterized by articular cartilage degeneration. It is largely heritable, and genetic screening has identified single‐nucleotide polymorphisms (SNPs) marking genomic risk loci. One such locus is marked by the G>A SNP rs75621460, downstream of TGFB1. This gene encodes transforming growth factor β1, the correct expression of which is essential for cartilage maintenance. This study investigated the regulatory activity of rs75621460 to characterize its impact on TGFB1 expression in disease‐relevant patient samples (n = 319) and in Tc28a2 immortalized chondrocytes. Methods Articular cartilage samples from human patients were genotyped, and DNA methylation levels were quantified using pyrosequencing. Gene reporter and electrophoretic mobility shift assays were used to determine differential nuclear protein binding to the region. The functional impact of DNA methylation on TGFB1 expression was tested using targeted epigenome editing. Results The analyses showed that SNP rs75621460 was located within a TGFB1 enhancer region, and the OA risk allele A altered transcription factor binding, with decreased enhancer activity. Protein complexes binding to A (but not G) induced DNA methylation at flanking CG dinucleotides. Strong correlations between patient DNA methylation levels and TGFB1 expression were observed, with directly opposing effects in the cartilage and the synovium at this locus. This demonstrated biologic pleiotropy in the impact of the SNP within different tissues of the articulating joint. Conclusion The OA risk SNP rs75621460 impacts TGFB1 expression by modulating the function of a gene enhancer. We propose a mechanism by which the SNP impacts enhancer function, providing novel biologic insight into one mechanism of OA genetic risk, which may facilitate the development of future pharmacologic therapies.