Quantification of Idua Enzymatic Activity Combined with Observation of Phenotypic Change in Zebrafish Embryos Provide a Preliminary Assessment of Mutated idua Correlated with Mucopolysaccharidosis Type I

Abstract

Mucopolysaccharidosis type I (MPS I) is an inherited autosomal recessive disease resulting from mutation of the α-l-Iduronidase (IDUA) gene. New unknown mutated nucleotides of idua have increasingly been discovered in newborn screening, and remain to be elucidated. In this study, we found that the z-Idua enzymatic activity of zebrafish idua-knockdown embryos was reduced, resulting in the accumulation of undegradable metabolite of heparin sulfate, as well as increased mortality and defective phenotypes similar to some symptoms of human MPS I. After microinjecting mutated z-idua-L346R, -T364M, -E398-deleted, and -E540-frameshifted mRNAs, corresponding to mutated human IDUA associated with MPS I, into zebrafish embryos, no increase in z-Idua enzymatic activity, except of z-idua-E540-frameshift-injected embryos, was noted compared with endogenous z-Idua of untreated embryos. Defective phenotypes were observed in the z-idua-L346R-injected embryos, suggesting that failed enzymatic activity of mutated z-Idua-L346R might have a dominant negative effect on endogenous z-Idua function. However, defective phenotypes were not observed in the z-idua-E540-frameshifted-mRNA-injected embryos, which provided partial enzymatic activity. Based on these results, we suggest that the z-Idua enzyme activity assay combined with phenotypic observation of mutated-idua-injected zebrafish embryos could serve as an alternative platform for a preliminary assessment of mutated idua not yet characterized for their role in MPS I.

Identification of an α‐l‐iduronidase (IDUA) M1T mutation in a Chinese family with autosomal recessive mucopolysaccharidosis I

“…The enzymatic activity of IDUA in zebrafish embryos was analyzed as previously described 11 . Control‐MO, IDUA‐MO, and IDUA‐MO + WT‐IDUA mRNA zebrafish embryos at 24 h post‐fertilization (hpf) were lysed and prepared as tissue homogenates.…”

“…The enzymatic activity of IDUA in zebrafish embryos was analyzed as previously described. 11 Control-MO, IDUA-MO, and IDUA-MO + WT-IDUA mRNA zebrafish embryos at 24 h post-fertilization (hpf) were lysed and prepared as tissue homogenates. The substrate of the IDUA, 4-methylumbelliferyl alpha-L-iduronide (4MU-I, Cayman chemical, #19543, USA) was diluted with sodium formate buffer (50 mM, pH 2.8) to the final volume of 20 μg/20 μL.…”

Anp32a Promotes Neuronal Regeneration after Spinal Cord Injury of Zebrafish Embryos

“…Although the use of mutants would be more convincing, no zebrafish Anp32a mutant is currently available. Despite some concerns over using the MO-base gene knockdown approach, Morpholino oligomers remain an essential tool to transiently inhibit gene expression in the zebrafish model, as attested by the numerous MO-related papers published by our lab [69][70][71][72]. As noted above, we employed MO-knockdown combined with rescue through Anp32a mRNA and Western blot analysis to demonstrate that the defective phenotype of spinal cord regeneration induced by Anp32a-MO was specific.…”