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Osteogenesis imperfecta (OI) is a heritable and chronically debilitating skeletal dysplasia. Patients with OI typically present with reduced bone mass, tendency for recurrent fractures, short stature and bowing deformities of the long bones. Mutations causative of OI have been identified in over 20 genes involved in collagen folding, posttranslational modification and processing, and in bone mineralization and osteoblast development. In 2016, we described the first X-linked recessive form of OI caused by MBTPS2 missense variants in patients with moderate to severe phenotypes. MBTPS2 encodes site-2 protease, a Golgi transmembrane protein that activates membrane-tethered transcription factors. These transcription factors regulate genes involved in lipid metabolism, bone and cartilage development, and ER stress response. The interpretation of genetic variants in MBTPS2 is complicated by the gene's pleiotropic properties; MBTPS2 variants can also cause the dermatological conditions Ichthyosis Follicularis, Atrichia and Photophobia (IFAP), Keratosis Follicularis Spinulosa Decalvans (KFSD) and Olmsted syndrome (OS) without skeletal abnormalities typical of OI. Using control and patient-derived fibroblasts, we previously identified gene expression signatures that distinguish MBTPS2 -OI from MBTPS2 -IFAP/KFSD and observed stronger suppression of genes involved in fatty acid metabolism in MBTPS2 -OI than in MBTPS2 -IFAP/KFSD; this was coupled with alterations in the relative abundance of fatty acids in MBTPS2 -OI. Furthermore, we observed a reduction in collagen deposition in the extracellular matrix by MBTPS2 -OI fibroblasts. Here, we extrapolate our observations in the molecular signature unique to MBTPS2 -OI to infer the pathogenicity of a novel MBTPS2 c.516A>C (p.Glu172Asp) variant of unknown significance in a male proband. The pregnancy was terminated at gestational week 21 after ultrasound scans showed bowing of femurs and tibiae and shortening of long bones particularly of the lower extremity; these were further confirmed by autopsy. By performing transcriptional analyses, gas chromatography-tandem mass spectrometry-based quantification of fatty acids and immunocytochemistry on fibroblasts derived from the umbilical cord of the proband, we observed perturbations in fatty acid metabolism and collagen production similar to what we previously described in MBTPS2 -OI. These findings support pathogenicity of the MBTPS2 variant p.Glu172Asp as OI-causative and highlights the value of extrapolating molecular signatures identified in multiomics studies to characterize novel genetic variants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lim, Marcionelli, Srikanthan, Ndarugendamwo, Pinner, Rohrbach and Giunta.)

Osteogenesis imperfecta (OI) is an inherited skeletal dysplasia characterized by low bone density, bone fragility and recurrent fractures. The characterization of its heterogeneous genetic basis has allowed the identification of novel players in bone development. In 2016, we described the first X-linked recessive form of OI caused by hemizygous MBTPS2 missense variants resulting in moderate to severe phenotypes. MBTPS2 encodes site-2 protease (S2P), which activates transcription factors involved in bone (OASIS) and cartilage development (BBF2H7), ER stress response (ATF6) and lipid metabolism (SREBP) via regulated intramembrane proteolysis. In times of ER stress or sterol deficiency, the aforementioned transcription factors are sequentially cleaved by site-1 protease (S1P) and S2P. Their N-terminal fragments shuttle to the nucleus to activate gene transcription. Intriguingly, missense mutations at other positions of MBTPS2 cause the dermatological spectrum condition Ichthyosis Follicularis, Atrichia and Photophobia (IFAP) and Keratosis Follicularis Spinulosa Decalvans (KFSD) without clinical overlap with OI despite the proximity of some of the pathogenic variants. To understand how single amino acid substitutions in S2P can lead to non-overlapping phenotypes, we aimed to compare the molecular features of MBTPS2 -OI and MBTPS2 -IFAP/KFSD, with the ultimate goal to unravel the pathomechanisms underlying MBTPS2 -OI. RNA-sequencing-based transcriptome profiling of primary skin fibroblasts from healthy controls ( n = 4), MBTPS2 -OI ( n = 3), and MBTPS2- IFAP/KFSD ( n = 2) patients was performed to identify genes that are differentially expressed in MBTPS2 -OI and MBTPS2- IFAP/KFSD individuals compared to controls. We observed that SREBP-dependent genes are more downregulated in OI than in IFAP/KFSD. This is coupled to alterations in the relative abundance of fatty acids in MBTPS2 -OI fibroblasts in vitro , while no consistent alterations in the sterol profile were observed. Few OASIS-dependent genes are suppressed in MBTPS2 -OI, while BBF2H7- and ATF6-dependent genes are comparable between OI and IFAP/KFSD patients and control fibroblasts. Importantly, we identified genes involved in cartilage physiology that are differentially expressed in MBTPS2- OI but not in MBTPS2 -IFAP/KFSD fibroblasts. In conclusion, our data provide clues to how pathogenic MBTPS2 mutations cause skeletal deformities via altered fatty acid metabolism or cartilage development that may affect bone development, mineralization and endochondral ossification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lim, Marfurt, Lindert, Opitz, Ndarugendamwo, Srikanthan, Poms, Hersberger, Langhans, Haas, Rohrbach and Giunta.)