Enigmatic in vivo iduronate-2-sulfatase (IDS) mutant transcript correction to wild-type in Hunter syndrome.
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 IDSmRNA-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. (c) 2010 Wiley-Liss, Inc.
Lualdi
Susanna
S
S.S.D. Lab. Diagnosi Pre-Postnatale Malattie Metaboliche, Dipartimento di Neuroscience, IRCCS G. Gaslini, Genoa, Italy.
Tappino
Barbara
B
Di Duca
Marco
M
Dardis
Andrea
A
Anderson
Christopher J
CJ
Biassoni
Roberto
R
Thompson
Peter W
PW
Corsolini
Fabio
F
Di Rocco
Maja
M
Bembi
Bruno
B
Regis
Stefano
S
Cooper
David N
DN
Filocamo
Mirella
M
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016532:Mucopolysaccharidosis II / Q000201:enzymology* / Q000235:genetics*; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010375:Pedigree; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012730:Sex Chromosomes / Q000235:genetics; D055815:Young Adult
D018076:DNA, Complementary; D006023:Glycoproteins; C018322:IDS protein, human; D050505:Mutant Proteins; D012333:RNA, Messenger