Rapid and accurate denaturating high performance liquid chromatography protocol for the detection of α-L-iduronidase mutations causing mucopolysaccharidosis type I David C. Kasper a,b, ⁎ ,1 , Furhan Iqbal a,1 , Lenka Dvorakova c , Jiri Zeman c , Martin Magner c , Olaf Bodamer d , Arnold Pollak a , Kurt R. Herkner a,b , Chike B. Item 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 abstract article info Article history: Received 30 September 2009 Received in revised form 9 November 2009 Accepted 24 November 2009 Available online 29 November 2009 Keywords: Mucopolysaccharidosis type I MPS I α-L-Iduronidase IDUA dHPLC 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. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Mucopolysaccharidosis type I (MPS I; OMIM# 252800) is an autosomal recessive disorder caused by a deficiency of the lysosomal enzyme α-L-iduronidase (IDUA; EC 3.2.1.76). This enzyme is involved in the degradation of glycosaminoglycans dermatan and heparan sulfates in lysosomes, and an absence of IDUA activity leads to the lysosomal accumulation of partially degraded mucopolysaccharides [1]. The prevalence of MPS type I is reported to range from 1:45,000 to 1:111,000 births [2–4]. Three clinical classifications with a continuous spectrum of clinical symptoms from mild to severe have been described. First, the most severe form of IDUA deficiency, Hurler (MPS-IH; OMIM# 607014), followed by the second intermediate form, Hurler/Scheie (MPS-IH/S; OMIM# 607015) and third, the mild entity Scheie disease (MPS-IS; OMIM# 607016). The biochemical diagnosis is based on the detection of increased urinary excretion of dermatan and heparan sulfate and IDUA enzyme deficiency in leukocytes or skin fibroblasts [1]. These three clinical subtypes cannot definitely be distinguished alone by the quantification of the residual enzyme activity due to the hetero- genetic nature of mutant alleles at the IDUA locus. Consequently, the molecular biological analysis of MPS I is an important part of diagnosis. Molecular characterization revealed the identification of at least 100 distinct mutations including complex gene rearrangements, missense, nonsense and splice site mutations, small and large intragenic deletions and insertions in the IDUA gene (reported in the Human Gene Mutation Database (HGMD) database; www.hgmd.org). This reflects the high degree of molecular heterogeneity of the wide clinical variability observed in MPS I. However, several mutations demonstrated a higher prevalence in certain geographic locations, such as P533R and G51D suggesting a Mediterranean origin [5] or the high prevalence of p. W402X and p.Q70X among Caucasian patients [6–8]. The latter two mutations seemed to be associated with the most severe phenotype [8]. Nonetheless, prediction of a patient's clinical phenotype by genetic analysis of the IDUA gene is hampered by the high number of disease- Clinica Chimica Acta 411 (2010) 345–350 ⁎ Corresponding author. Medical University of Vienna, Vienna, Austria, Department of Pediatrics and Adolescent Medicine, Währinger Gürtel 18-20, A — 1090 Vienna, Austria. Tel.: +43 1 40400 2756; fax: +43 1 40400 3200. E-mail address: david.kasper@meduniwien.ac.at (D.C. Kasper). 1 These authors contributed equally to this work. 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.11.027 Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim