Enigmatic in vivo iduronate-2-sulfatase (IDS) mutant transcript correction to wild-type in hunter syndrome

Susanna Lualdi, Barbara Tappino, Marco Di Duca, Andrea Dardis, Christopher J. Anderson, Roberto Biassoni, Peter W. Thompson, Fabio Corsolini, Maja Di Rocco, Bruno Bembi, Stefano Regis, David N. Cooper, Mirella Filocamo

Research output: Contribution to journalArticle

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Abstract

Sequence analysis of the X-linked iduronate-2-sulfatase (IDS) gene in two Hunter syndrome patients revealed a lack of concordance between IDS genomic DNA and cDNA. These individuals were found to be hemizygous respectively for a nonsense mutation [c.22C>T;p.R8X] and a frameshift micro-insertion [c.10insT;p.P4Sfs] in their genomic DNA. However, both wild-type and mutant IDS sequences were evident upon cDNA analysis. Similar discrepant results were also obtained in a third unrelated patient carrying the same p.R8X mutation. Since both p.R8X mutations were inherited from carrier mothers, somatic mosaicism could be excluded. Although the presence of wild-type IDS mRNA-transcripts was confirmed in all three patients by restriction enzyme digestion, clone sequencing, pyrosequencing and single nucleotide primer extension (SNuPE), no wild-type IDS genomic sequence was detectable. The relative abundance of wild-type and mutation-bearing IDS-transcripts in different tissues was quantified by SNuPE. Although IDS transcript levels, as measured by real-time PCR, were reduced (51-71% normal) in these patients, some wild-type IDS protein was detectable by western blotting. Various possible explanations for these unprecedented findings (e.g. accidental contamination, artefactual in vitro nucleotide misincorporation, malsegregation of an extra maternal X-chromosome) were explored and experimentally excluded. PCR-based discriminant assay and segregation analysis of a linked IDS polymorphism (rs1141608) also served to exclude the presence of IDS cDNA derived from the maternal wild-type chromosome. Although it remains to be formally demonstrated by direct experimentation, the intriguing possibility arises that we have observed the in vivo correction of heritable gene lesions at the RNA level operating via a correction mechanism akin to RNA-editing.

Original languageEnglish
JournalHuman Mutation
Volume31
Issue number4
DOIs
Publication statusPublished - Apr 2010

Fingerprint

Iduronate Sulfatase
Mucopolysaccharidosis II
Nucleotides
Complementary DNA
Mothers
Mutation
RNA Editing
Mosaicism
Nonsense Codon
DNA
X Chromosome
Genes
Sequence Analysis
Real-Time Polymerase Chain Reaction
Digestion

Keywords

  • IDS gene mutation
  • Iduronate-2-sulfatase
  • In vivo mRNA transcript correction
  • Mucopolysaccharidosis type II

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Enigmatic in vivo iduronate-2-sulfatase (IDS) mutant transcript correction to wild-type in hunter syndrome. / Lualdi, Susanna; Tappino, Barbara; Di Duca, Marco; Dardis, Andrea; Anderson, Christopher J.; Biassoni, Roberto; Thompson, Peter W.; Corsolini, Fabio; Di Rocco, Maja; Bembi, Bruno; Regis, Stefano; Cooper, David N.; Filocamo, Mirella.

In: Human Mutation, Vol. 31, No. 4, 04.2010.

Research output: Contribution to journalArticle

Lualdi, Susanna ; Tappino, Barbara ; Di Duca, Marco ; Dardis, Andrea ; Anderson, Christopher J. ; Biassoni, Roberto ; Thompson, Peter W. ; Corsolini, Fabio ; Di Rocco, Maja ; Bembi, Bruno ; Regis, Stefano ; Cooper, David N. ; Filocamo, Mirella. / Enigmatic in vivo iduronate-2-sulfatase (IDS) mutant transcript correction to wild-type in hunter syndrome. In: Human Mutation. 2010 ; Vol. 31, No. 4.
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abstract = "Sequence analysis of the X-linked iduronate-2-sulfatase (IDS) gene in two Hunter syndrome patients revealed a lack of concordance between IDS genomic DNA and cDNA. These individuals were found to be hemizygous respectively for a nonsense mutation [c.22C>T;p.R8X] and a frameshift micro-insertion [c.10insT;p.P4Sfs] in their genomic DNA. However, both wild-type and mutant IDS sequences were evident upon cDNA analysis. Similar discrepant results were also obtained in a third unrelated patient carrying the same p.R8X mutation. Since both p.R8X mutations were inherited from carrier mothers, somatic mosaicism could be excluded. Although the presence of wild-type IDS mRNA-transcripts was confirmed in all three patients by restriction enzyme digestion, clone sequencing, pyrosequencing and single nucleotide primer extension (SNuPE), no wild-type IDS genomic sequence was detectable. The relative abundance of wild-type and mutation-bearing IDS-transcripts in different tissues was quantified by SNuPE. Although IDS transcript levels, as measured by real-time PCR, were reduced (51-71{\%} normal) in these patients, some wild-type IDS protein was detectable by western blotting. Various possible explanations for these unprecedented findings (e.g. accidental contamination, artefactual in vitro nucleotide misincorporation, malsegregation of an extra maternal X-chromosome) were explored and experimentally excluded. PCR-based discriminant assay and segregation analysis of a linked IDS polymorphism (rs1141608) also served to exclude the presence of IDS cDNA derived from the maternal wild-type chromosome. Although it remains to be formally demonstrated by direct experimentation, the intriguing possibility arises that we have observed the in vivo correction of heritable gene lesions at the RNA level operating via a correction mechanism akin to RNA-editing.",
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