Biology subjects

Coucke, P. J.

Publications and source records attributed to Coucke, P. J..

3 recordsLinked to original sources

bioRxiv2025

Evaluating Variants of Uncertain Significance in Adult Knock-in Zebrafish: A Proof of Concept with a COL1A2 Variant

Genomic variants of uncertain significance (VUS) impede clinical decision-making. In this study, we use a knock-in strategy in zebrafish to evaluate the COL1A2 c.2123G>A VUS, identified in a 59-year-old female with recurrent fractures. Using prime editing, we obtained different zebrafish lines respectively harboring the VUS, a known pathogenic variant, or a known benign variant. Comprehensive skeletal phenotyping revealed no significant abnormalities in the zebrafish modeling the benign variant and the VUS, while zebrafish modeling the pathogenic variant showed scoliosis of the vertebral column, vertebral fusions, vertebral compressions, fractures, and increased mineralization of the notochord and intervertebral ligament. Our findings demonstrate for the first time, that COL1A2 variant modeling in zebrafish models informs functional validation and shows potential for elucidating associated pathogenic mechanisms. This approach can be extended to study VUS in other genes.

genetics↗

bioRxiv2020

b3galt6 knock-out zebrafish recapitulate β3GalT6-deficiency disorders in human and reveal a trisaccharide proteoglycan linkage region

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human {beta}3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

genetics↗

bioRxiv2018

Deep Phenotyping Enhances Somatic Mutant Detection and Mosaic Pattern Recognition in the Zebrafish Skeleton

Genetic mosaicism manifests as spatially variable phenotypes, whose detection and interpretation remains challenging. This study identifies biological factors influencing spatial phenotypic patterns in the skeletons of somatic mutant zebrafish, and tests methods for their analysis using deep phenotyping. We explore characteristics of loss-of-function clusters in the skeleton of CRISPR-edited G0 (\"crispant\") zebrafish, and identify a distinctive size distribution shown to arise from clonal fragmentation and merger events. Using microCT-based phenomics, we describe diverse phenotypic manifestations in somatic mutants for genes implicated in monogenic (plod2 and bmp1a) and polygenic (wnt16) bone diseases, each showing convergence with germline mutant phenomes. Finally, we describe statistical frameworks for phenomic analysis which confers heightened sensitivity in discriminating somatic mutant populations, and quantifies spatial phenotypic variation. Our studies provide strategies for decoding spatially variable phenotypes which, paired with CRISPR-based screens, can identify genes contributing to skeletal disease.

genetics↗