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.
Published in Clinica chimica acta; international journal of clinical chemistry. The full article is available on Pubmed.
Authors / corresponding author*
Kasper DC*,a,b; Iqbal F.*,a; Dvorakova L,c; Zeman J.,c; Magner M.,c; Bodamer O.,d; Pollak A.,a; Herkner KR,a,b; Item CB,a,b
a Department of Pediatrics and Adolescent Medicine, Laboratory for Inherited Metabolic Disorders, Medical University of Vienna, Vienna, Austria
b Research Core Unit (RCU) of Pediatric Biochemistry and Analytics, Medical University of Vienna, Vienna, Austria
c Institute of Inherited Metabolic Disorders and Department of Pediatrics, First Faculty of Medicine and General Teaching Hospital, Charles University in Prague, Prague, Czech Republic
d Paracelsus Medical University, University Children’s Hospital, Salzburg, Austria