Effect of Mutated ids Overexpression on IDS Enzyme Activity and Developmental Phenotypes in Zebrafish Embryos: A Valuable Index for Assessing Critical Point-Mutations Associated with Mucopolysaccharidosis Type II Occurrence in Humans

Abstract

Mucopolysaccharidosis type II (MPS II) is an X-linked disorder resulting from a deficiency in iduronate 2-sulfatase (IDS), which is reported to be caused by gene mutations in the iduronate 2-sulfatase (IDS) gene. Many IDS mutation sites have not yet had their causal relationship with MPS II characterized. We employed a gain-of-function strategy whereby we microinjected different mutated zebrafish ids (z-ids) mRNAs corresponded to human IDS gene into zebrafish embryos, and then measured their total IDS enzymatic activity and observed the occurrence of defective phenotypes during embryonic development. We examined three known mutation sites for human IDS genes (h-IDS) associated with MPS II symptoms, including h-IDS-P86L, -S333L and -R468W, which corresponded to z-ids-P80L, -S327L and -R454W. When these three mutated z-ids mRNAs were overexpressed in zebrafish embryos, the IDS enzymatic activity of the total proteins extracted from the injected embryos was not increased compared with the endogenous IDS of the untreated embryos, which suggests that the IDS enzymatic activity of these three mutated z-ids was totally lost, as expected. Additionally, we observed defective phenotypes in these injected embryos, resulting from the failed IDS enzyme breakdown, which, in turn, has a dominant negative effect on the endogenous wild-type IDS function. These phenotypes were similar to the clinical symptoms observed in MPS II pathogenesis. We further studied six uncharacterized IDS mutation sites as identified by the Taiwanese MPS newborn screening programs. We propose a novel IDS enzyme activity assay combined with phenotypic observation in zebrafish embryos, as an alternative platform for quickly providing a valuable index for preliminarily assessment of any identified IDS point mutation gene that has not yet been characterized, in the context of its role in MPS II development.

Global expression pattern of genes containing positively selected sites in European anchovy (Engraulis encrasicolus L.) may shed light on teleost reproduction

“…In another study, Bellesso et al utilized CRISPR/Cas9 technology to create a transgenic zebrafish line with a five-base-pair deletion in the z-ids gene, leading to a premature stop codon at amino acid 118 and a truncated form of z-IDS in cells. This loss of z-IDS function during early development decreased Fgf signaling and negatively impacted bone development at later stages [73]. Other notable PSGs exhibiting differentially expressed patterns in ovary tissue are methyltransferases (rnmt, dnmt1, trmt5) and it is well-known that oocyte growth is accompanied by dynamic epigenetic modifications [74].…”

Zebra-Sphinx: Modeling Sphingolipidoses in Zebrafish

“…In addition, KO of ids in zebrafish has provided novel information about the role of early deregulation of the fibroblast growth factor signaling pathway in the occurrence of irreversible skeletal defects before glycosaminoglycans' accumulation [163]. With a different approach, human-mutated IDS mRNAs have been injected into zebrafish embryos for a rapid preliminary study about novel IDS point mutations associated with MPSII [164].…”

Zebrafish, an In Vivo Platform to Screen Drugs and Proteins for Biomedical Use

“…To solve this puzzling issue, the zebrafish is also a simple but cost-effective animal model that can serve as an in vivo screening platform. In this particular case, since the preparation of mRNAs for a gain-of-function study is simpler, more effective and economical than that of MO-injection for a loss-of-function study, Lin et al [ 88 ] demonstrated that overexpression of some uncharacterized mutant mRNA, which obviously leads to developmental defects, might also contain a potentially malignant mutation because the enzymatic activity of IDS encoded by this mutant mRNA is inactive. Consequently, the exogenous mutated IDS interrupts the enzymatic activity of endogenous IDS through the dominant negative effect, which, in turn, causes to occur developmental defects.…”