Abstract
Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Highlights
Osteoporosis is a highly prevalent systemic skeletal disease characterized by loss of bone mass and low bone mineral density (BMD) that results in bone fragility and increased fracture risk.[1]
BMD and osteoporotic fractures are highly heritable with heritability estimated between 0.5 and 0.8, meaning that a substantial part of their variation is determined by genetic factors.[2]. Genomewide association studies (GWAS) in large cohorts from consortia such as GEFOS/GENOMOS and UK Biobank have identified hundreds of susceptibility loci for osteoporosis-related traits.[3,4,5] For example, BMD in lumbar spine and femoral neck was linked to 56 loci, of which 50 were replicated in an independent study that revealed 153 previously unknown loci.[4,6] Major risk loci, such as ESR1 and LRP5, have been verified in different GWAS studies.[6,7,8,9] for the vast majority of identified loci, both causal variants and perturbed genes and their molecular functional consequences remain largely unknown
Pronounced and well-described Wnt signaling in adult zebrafish occurs during fin regeneration, as this pathway is crucially important for different regeneration processes, including the differentiation and proliferation of osteoblasts, which are essential for the regeneration of the bony fin rays.[58,59] ) we primarily focused on caudal fin regeneration to monitor the consequences of loss of lrp5 in both lrp5À/À mutants and lrp5 crispants on canonical Wnt signaling
Summary
Osteoporosis is a highly prevalent systemic skeletal disease characterized by loss of bone mass and low bone mineral density (BMD) that results in bone fragility and increased fracture risk.[1] Osteoporosis is a complex disorder, influenced by both environmental and genetic factors. Small animal models—namely rodents—are well-established tools for osteoporosis studies.[10,11,12] since large-scale generation and phenotyping of KO mouse models is a slow and costly approach,(13,14) the role of most genes residing at GWAS loci still remains unknown. This indicates the need for alternative methods to accelerate validation of candidate osteoporosis genes
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