TAPT1-at the crossroads of extracellular matrix and signaling in Osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) is a hereditary skeletal disorder primarily affecting collagen type I structure and function, causing bone fragility and occasionally versatile extraskeletal symptoms. This study expands the spectrum of OI-causing TAPT1 mutations and links extracellular matrix changes to signaling regulation.

Figure 1. Consequences of p.Leu108Trp mutation in TAPT1 on disease phenotype and the underlying pathomechanism

(A) Pedigree of the family with the affected individual (black symbol) carrying a homozygous TAPT1 mutation. Heterozygous carriers (half‐shaded symbols) and consanguineous relationship (double line) are highlighted (upper panel). In the affected individual (II:3) with the clinical phenotype of OI (middle panel), dual‐energy X‐ray absorptiometry (DEXA) was used to assess areal bone mineral density (aBMD) and radiographs of tibia and hand/wrist show long bones with thin cortices (bone health index 3.04, −5.68 SDS) as well as disorganized calcifications around the growth plate (also called popcorn calcifications, lower panel). (B) Model for human TAPT1 protein and the c.323 T > G, p.Leu108Trp mutation were visualized employing ChimeraX and the AlphaFold 2 model of human TAPT1 (UniProt entry Q6NXT6). Leu108 is located within an N‐terminal alpha helix protruding into the membrane bilayer and projects into a cavity surrounded by several transmembrane helices (upper panel). Glu285, Asp306, Asp353, Lys356, His357 and Tyr371 are located on several helices and are engaged in salt bridges or connected by hydrogen bonds. In‐silico substitution of Leu108 by a tryptophane reveals severe clashes for all rotamers (lower panel). (C) Immunoblotting of TAPT1 protein in control and patient fibroblasts (upper panel) and quantification of band intensities by ImageJ analysis (lower panel) revealed slightly reduced TAPT1 levels in patient cells. Fold change difference relative to the mean of controls is plotted as individual values from n = 3 independent experiments. Statistical analysis: One way ANOVA; P‐value = 0.06; (D) qPCR analysis of relative gene expression of the collagen genes COL1A1 (upper panel) and COL1A2 (lower panel) in control and patient fibroblasts confirmed that expression was not downregulated in patient cells. Gene expression was normalized to GAPDH, calibrated to the mean of controls and fold changes are plotted on a logarithmic scale form at least n = 5 independent experiments. Statistical analysis: One‐way ANOVA with post hoc Bonferroni multiple comparison of patient to controls a, b or c; P‐value [COL1A1] = 0.004, **c; P‐value [COL1A2] = 0.007, **c. (E) Immunofluorescence analysis of collagen type I in control (upper panel) and patient fibroblasts (lower panel) detected reduced collagen network in patient cells. Representative pictures are shown from at least n = 3 independent experiments with three control cell populations each. Scale bar: 500 μm. (F) Negative staining and transmission electron microscopy of cell culture media from control and patient cells visualized impaired collagen fibril formation in patient cells. Higher magnifications of fibers are shown as inserts. Scale bars: 500 nm (overviews), 100 nm (inserts). (G, H) qPCR analysis of relative gene expression of SFRP1 in control and patient fibroblasts without stimulation (‐SAG, G) or upon stimulation with 1 μM smoothened agonist (+SAG, H) for 24 h hours uncovered increased SFRP1 expression in patient cells under both conditions. Fold changes plotted on a logarithmic scale from n = 6 independent experiments are shown. Gene expression was normalized to GAPDH and calibrated to the mean of nonstimulated control cells from (G). Statistical analysis: One‐way ANOVA with post hoc Bonferroni multiple comparison of patient to controls a, b or c; P‐value [‐SAG] = 0.0001, ***a, ***b, ***c; P‐value [+SAG] < 0.0001, ***a, ***b, ***c. (I) ELISA of SFRP1 protein levels in the serum of eight healthy controls or two independent serum samples of the patient determined increased SFRP1 levels in patient serum. No statistical analysis could be performed on this dataset due to only two available patient values. Source data are available online for this figure.