Characterization of common SMPD1 mutations causing types A and B Niemann-Pick disease and generation of mutation-specific mouse models

Iwan Jones, Xingxuan He, Fourogh Katouzian, Peter I. Darroch, Edward H. Schuchman

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

Herein we describe detailed characterization of four common mutations (L302P, H421Y, R496L and ΔR608) within the acid sphingomyelinase (ASM) gene causing types A and B Niemann-Pick disease (NPD). In vitro and in situ enzyme assays revealed marked deficiencies of ASM activity in NPD cell lines homoallelic for each mutation, although Western blotting and fluorescent microscopy showed that the mutant ASM polypeptides were expressed at normal levels and trafficked to lysosomes. Co-immunoprecipitation of the polypeptides with the ER chaperone, BiP, confirmed these findings, as did in vitro expression of the mutant cDNAs in reticulocyte lysates. We further developed a computer assisted, three-dimensional model of human ASM based on homologies to known proteins, and used this model to map each NPD mutation in relation to putative substrate binding, hydrolysis and zinc-binding domains. Lastly, we generated transgenic mice expressing the R496L and ΔR608 mutations on the complete ASM knock-out background (ASMKO), and established breeding colonies for the future evaluation of enzyme enhancement therapies. Analysis of these mice demonstrated that the mutant ASM transgenes were expressed at high levels in the brain, and in the case of the ΔR608 mutation, produced residual ASM activity that was significantly above the ASMKO background.

Original languageEnglish
Pages (from-to)152-162
Number of pages11
JournalMolecular Genetics and Metabolism
Volume95
Issue number3
DOIs
StatePublished - Nov 2008

Keywords

  • Lysosomal storage disease
  • Mutations
  • Transgenic mouse

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