Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms

Susanna Lualdi, Maja Di Rocco, Fabio Corsolini, Marco Spada, Bruno Bembi, Giovanna Cotugno, Roberta Battini, Marina Stroppiano, Maria Gabriela Pittis, Mirella Filocamo

Research output: Contribution to journalArticlepeer-review

Abstract

The present study aimed to characterize mutant alleles in Mucopolysaccharidosis II and evaluate possible reduction of mRNA amount consequent to nonsense-mediated or nonstop mRNA decay pathways. A combination of different approaches, including real-time RT-PCR, were used to molecularly characterize seventeen patients. Fifteen alleles were identified and nine of them were new. The novel alleles consisted of three missense mutations (p.S71R, p.P197R, p.C432R), two nonsense (p.Q66X, p.L359X), two frameshifts (p.V136fs75X, p.C432fs8X), one allele carrying two in-cis mutations [p.D252N;p.S369X], and a large deletion (p.G394_X551). Analysing these results it emerged that most of the alterations resulted in mutants leading to mRNAs with premature termination codons, and therefore, potentially sensitive to mRNA surveillance pathway. By using real-time RT-PCR, the mRNAs resulting (i) from substitutions that changed one amino acid to a stop codon (L359X, and S369X), or caused the shifted reading frame with premature introduction of a stop codon (C432fs8X), (ii) from large deletion (p.G394_X551) that included the termination codon, seemed to be subject to degradation by nonsense-mediated (i) or nonstop decay (ii) mechanisms, as mRNA was strongly underexpressed. On the contrary, two mutations (Q66X and V136fs75X) produced transcripts evading mRNA surveillance pathway despite both of them fulfilled the known criteria. These results confirm the wide variability of the mRNA expression levels previously reported and represent a further exception to the rules governing susceptibility to nonsense-mediated decay. A close examination of the molecular basis of the disease is becoming increasingly important for optimising the choices of available or forthcoming therapies such as, enzyme replacement therapy or enzyme enhancement therapy.

Original languageEnglish
Pages (from-to)478-484
Number of pages7
JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
Volume1762
Issue number4
DOIs
Publication statusPublished - Apr 2006

Keywords

  • Genotype-phenotype correlation
  • Hunter
  • IDS
  • mRNA decay
  • Mucopolysaccharidosis
  • Real-time RT-PCR

ASJC Scopus subject areas

  • Biophysics
  • Molecular Biology
  • Molecular Medicine

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