title: Rapid and accurate denaturating high performance liquid chromatography protocol for the detection of alpha-l-iduronidase mutations causing mucopolysaccharidosis type I.
abstract: BACKGROUND
  Mutations in the alpha-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.
authors: Department of Pediatrics and Adolescent Medicine, Laboratory for Inherited Metabolic Disorders, Medical University of Vienna, Vienna, Austria. david.kasper@meduniwien.ac.at
authors: David C
authors: DC
authors: Kasper
authors: Furhan
authors: F
authors: Iqbal
authors: Lenka
authors: L
authors: Dvorakova
authors: Jiri
authors: J
authors: Zeman
authors: Martin
authors: M
authors: Magner
authors: Olaf
authors: O
authors: Bodamer
authors: Arnold
authors: A
authors: Pollak
authors: Kurt R
authors: KR
authors: Herkner
authors: Chike B
authors: CB
authors: Item
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000191:economics; D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis / Q000191:economics; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D007068:Iduronidase / Q000032:analysis* / Q000378:metabolism; D008059:Mucopolysaccharidosis I / Q000235:genetics*; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000191:economics
chemical_list: D007068:Iduronidase