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Bone Abstracts (2019) 7 P37 | DOI: 10.1530/boneabs.7.P37

University of Pavia, Pavia, Italy.

Objectives: Osteogenesis Imperfecta (OI) type XIV is a recessive OI form characterized by bone fragility, multiple fractures and growth retardation. It is caused by mutation in TMEM38B gene encoding the endoplasmic reticulum (ER) channel TRIC-B. This channel allows the transport of K+ across the ER membrane modulating Ca2+ flux. Defective ER Ca2+ impaires collagen type I synthesis, likely affecting the activity of ER enzymes involved in its post translational modification. To investigate the role of TRIC-B in skeletal development an in vivo model of the disease will be of great relevance. Since OI type XIV murine model is lethal at birth, zebrafish could be an interesting alternative to study this heritable skeletal disease.

Methods: Temporal and spatial distribution of zebrafish tmem38b during development was evaluated by qPCR and in situ hybridization. To generate a zebrafish model for OI XIV we used CRISPR/Cas9 system. tmem38b gRNA and Cas9 mRNA were injected in fertilized embryos and the targeting was verified by T7 endonuclease assay. The mutations were determined by sequencing. Collagen type I extracted from skin and bone was analysed by SDS-PAGE.

Results: Tmem38b is present in a single copy in the zebrafish genome and we demonstrated by qPCR and whole mount in situ hybridization that its expression is detectable very early during development in cranial bones and in the swim bladder. Two mutant lines were generated: one carrying an in-frame deletion of 24 nucleotides, that eliminates in TRIC-B the highly conserved KEV domain and the second with a deletion causing an out-of-frame transcript with a 90% of non-sense mediated decay. The α(I) bands of collagen type I showed a faster migration suggesting a lower level of post-translational modification. A preliminary characterization of the knock-out fish showed no difference in standard length during larval stage, but a reduction at 4 months post fertilization.

Conclusion: We successfully generated two tmem38b knock-out zebrafish that will allow to investigate the effect of TRIC-B in vivo during skeletogenesis and that may represent a powerful tool to understand the OI type XIV pathophysiology. Telethon [grant n. GGP13098] and the European Community, FP7, ‘Sybil’ project [grant n. 602300].

Disclosure: The authors declared no competing interests.

Volume 7

9th International Conference on Children's Bone Health


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