Rapid and accurate denaturating high performance liquid chromatography protocol for the detection of α-l-iduronidase mutations causing mucopolysaccharidosis type I

David C. Kasper, Furhan Iqbal, Lenka Dvorakova, Jiri Zeman, Martin Magner, Olaf Bodamer, Arnold Pollak, Kurt R. Herkner, Chike B. Item

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Background: Mutations in the α-l-iduronidase A (IDUA) gene cause mucopolysaccharidosis type I (MPS I), a progressive multisystem disorder with features ranging over a continuum from mild to severe which is inherited in an autosomal recessive manner. To date over 100 mutations are known, nonetheless genotype-phenotype prediction is complicated and hampered due to attenuating polymorphisms, rare sequence variants, varied genetic backgrounds and environmental effects. Methods: In this study we report the first development of a denaturating high performance liquid chromatography (dHPLC) protocol for the rapid and accurate detection of recently described mutations in the IDUA gene. Optimal PCR running and dHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to 14 IDUA exons and their adjacent intronic/flanking sequences. Results: A total of 12 different mutations, 5 nonsense, 4 missense, 1 deletion, and 2 splice site (intron), in 10 MPS I patients were screened. All mutations revealed a distinct dHPLC pattern thus enabling their accurate detection. Conclusions: A dHPLC screening method was developed for the detection of mutations and sequence variants in the IDUA gene and the results presented in this study revealed that this promising method proved to be robust, automated, economical and above all, highly sensitive. Costs for the detection of mutations causing MPS I disease should be reduced by using this method as a pre-analytical tool followed by sequencing of aberrant heteroduplex-forming amplicons.

Original languageEnglish (US)
Pages (from-to)345-350
Number of pages6
JournalClinica Chimica Acta
Volume411
Issue number5-6
DOIs
StatePublished - Mar 2 2010

Keywords

  • α-l-Iduronidase
  • dHPLC
  • IDUA
  • MPS I
  • Mucopolysaccharidosis type I

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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    Kasper, D. C., Iqbal, F., Dvorakova, L., Zeman, J., Magner, M., Bodamer, O., Pollak, A., Herkner, K. R., & Item, C. B. (2010). Rapid and accurate denaturating high performance liquid chromatography protocol for the detection of α-l-iduronidase mutations causing mucopolysaccharidosis type I. Clinica Chimica Acta, 411(5-6), 345-350. https://doi.org/10.1016/j.cca.2009.11.027