Insight into the Pathology of a COL1A1 Signal Peptide Heterozygous Mutation Leading to Severe Osteogenesis Imperfecta

Abstract

Osteogenesis imperfecta or “brittle bone disease” is a congenital disorder of connective tissue causing the bone to break easily. Around 85–90% of cases are due to autosomal dominant mutations in the genes encoding type I collagen, the major organic component of bone. Genotype–phenotype correlations have shown that quantitative defects of collagen type I lead to mild OI, whereas structural defects show a wide clinical range from mild to perinatal lethal. This may partially be explained by the type of amino acid substitution and the relative location in the domain structure. To fully understand the variability of the clinical manifestation and the underlying pathomechanisms, further investigations are required. Here we provide the first biochemical characterization of a mutation at the signal peptide cleavage site of COL1A1, a domain not yet characterized. By steady-state analysis, we observed reduced production of collagen type I. Furthermore, by pulse-chase analysis we detected delayed secretion and partial intracellular retention of collagen I. In the cellular fraction, the electrophoretic migration was abnormal; however, secreted type I collagen showed a normal migration pattern. The intracellular retention of collagen I was confirmed by immunofluorescent staining. Moreover, transmission electron microscopy of cultured fibroblasts revealed enlargement of ER cisternae. These results further support the hypothesis that mechanisms interfering with ER integrity play an important role in the pathology of severe OI.

Novel inactivating mutations in the FSH receptor cause premature ovarian insufficiency with resistant ovary syndrome

“…However, the SignalP 4.1 server revealed a marked reduction in the cleavage probability for the Asp15 substitution in FSHR. Efficient cleavage by the signal peptidase requires small and aliphatic residues (in particular Ala, Gly, Ser and Val) at the 1-and 3-positions of the cleavage site (Choo and Ranganathan, 2008;Lindert et al, 2017). Therefore, the Asp15 substitution in FSHR, at the 3-position of the cleavage site, might lead to subcellular mislocalization of FSHR (Bornemann et al, 2008;Chiereghin et al, 2017;Halic et al, 2006;Holtkamp et al, 2012;Rutz et al, 2015).…”

COL1‐related overlap disorder: A novel connective tissue disorder incorporating the osteogenesis imperfecta/Ehlers‐Danlos syndrome overlap

“…Fibroblasts of unaffected individuals of paediatric age were used as controls. Collagen steady‐state analysis in cultured fibroblasts was carried out as previously described . Radioactively labelled and pepsinized procollagens were separated on a 5% SDS‐PAGE and visualized by autoradiography.…”

“…Collagen steady-state analysis in cultured fibroblasts was carried out as previously described. 12 Radioactively labelled and pepsinized procollagens were separated on a 5% SDS-PAGE and visualized by autoradiography. Biochemical analysis was conducted prior to genetic analysis in P6 and P7, whereas in P18 it was performed in order to assess the outcome of the COL1A2 p.…”

XBP1s-Mediated ER Proteostasis Network Enhancement Can Selectively Improve the Folding and Secretion of an Osteogenesis Imperfecta-Causing Collagen-I Variant

“…We observed that, upon ascorbate (Asc) stimulation of collagen production, collagen-I secretion over a 24 h period was significantly reduced in the heterozygous G425S 1(I)-expressing fibroblasts compared to WT 1(I)-expressing control fibroblasts (Figure 2a; note that in this and other media immunoblots it is not possible to normalize to a 'housekeeping' protein like actin due to the absence of stable markers in media -instead, we carefully ensured equivalent cell counts, media volumes, and timecourses between samples as described in the Materials and Methods). Similar OI-associated collagen-I secretion defects have been observed across many OI genotypes 15,16,[45][46][47][48][49][50][51] . qPCR analysis of mRNA levels showed that classic UPR markers, including HSPA5, DNAJB9, and HYOU1, were not upregulated (Figure 2b) 23 .…”