Novel Genetic Variants for Cartilage Thickness and Hip Osteoarthritis.

Osteoarthritis is one of the most frequent and disabling diseases of the elderly. Only few genetic variants have been identified for osteoarthritis, which is partly due to large phenotype heterogeneity. To reduce heterogeneity, we here examined cartilage thickness, one of the structural components of joint health. We conducted a genome-wide association study of minimal joint space width (mJSW), a proxy for cartilage thickness, in a discovery set of 13,013 participants from five different cohorts and replication in 8,227 individuals from seven independent cohorts. We identified five genome-wide significant (GWS, P≤5·0×10-8) SNPs annotated to four distinct loci. In addition, we found two additional loci that were significantly replicated, but results of combined meta-analysis fell just below the genome wide significance threshold. The four novel associated genetic loci were located in/near TGFA (rs2862851), PIK3R1 (rs10471753), SLBP/FGFR3 (rs2236995), and TREH/DDX6 (rs496547), while the other two (DOT1L and SUPT3H/RUNX2) were previously identified. A systematic prioritization for underlying causal genes was performed using diverse lines of evidence. Exome sequencing data (n = 2,050 individuals) indicated that there were no rare exonic variants that could explain the identified associations. In addition, TGFA, FGFR3 and PIK3R1 were differentially expressed in OA cartilage lesions versus non-lesioned cartilage in the same individuals. In conclusion, we identified four novel loci (TGFA, PIK3R1, FGFR3 and TREH) and confirmed two loci known to be associated with cartilage thickness.The identified associations were not caused by rare exonic variants. This is the first report linking TGFA to human OA, which may serve as a new target for future therapies.

Intracellular trafficking of thyroid peroxidase to the cell surface.

For thyroid hormone synthesis, thyroid peroxidase (TPO) molecules must be transported from the endoplasmic reticulum via the Golgi complex to be delivered at the cell surface to catalyze iodination of secreted thyroglobulin. Like other glycoproteins, TPO molecules in transit to the cell surface have the potential to acquire endoglycosidase H resistance as a consequence of Golgi-based modification of their N-linked carbohydrates, and measurement of the intracellular distribution of TPO has often relied on this assumption. To examine TPO surface distribution in thyrocyte cell lines, we prepared new antibodies against rat TPO. Antibody reactivity was first established upon expression of recombinant rat (r) TPO in 293 cells, which were heterogeneous for surface expression as determined by flow cytometry. By cell fractionation, surface rTPO fractionated distinctly from internal pools of TPO (that co-fractionate with calnexin), yet surface TPO molecules remained endoglycosidase H (endo H)-sensitive. Although the FRTL5 (and PC Cl3) rat thyrocyte cell line also exhibits almost no endo H-resistant TPO, much of the endogenous rTPO is localized to the cell surface by immunofluorescence. Similar results were obtained by fractionation of FRTL5 cell membranes on sucrose gradients. We conclude that in FRTL5 cells, a large fraction of rTPO is delivered to the plasma membrane yet does not acquire Golgi-type processing of its N-glycans. Rat and mouse thyroid tissue TPO also shows little or no endo H resistance, although cell fractionation still needs to be optimized for these tissues.

Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus.

Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein. In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E. coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins. The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex. Furthermore, both the GST fusion protein and the cleaved HR2 show virus-cell fusion inhibition activity (IC(50) of 1.07-2.93 microM). The solubility of the GST-HR2 fusion protein is much higher than that of the corresponding peptide. Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors.