Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements. 1. Introduction Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder resulting from deficiency of the enzyme α-L-iduronidase (IDUA, E.C. 3.2.1.76) and leads to the intralysosomal accumulation of undegraded glycosaminoglycans [1]. The gene encoding α-L-iduronidase (IDUA, OMIM？no. 252800) is located on chromosome 4p16.3 and contains 14 exons [2–4]. The most common IDUA mutation identified in MPS I patients is p.Trp402Ter (NG_008103.1:？g.20751G？>？A, NM_000203.3:？c.1205G？>？A, and NP_000194.2:？p.Trp402Ter) which represents at least 45% of the causal alleles in Northwestern Europe, North America, Australia, Portugal, and Spain, while in Russia, Italy, and Brazil its frequency has been estimated to be 4%, 11%, and 37%, respectively [5, 6]. Since p.Trp402Ter is the single most common causal mutation in MPS IH (OMIM no. 607014) patients of Western European origin and accounts for 60% of the alleles in unrelated Portuguese patients [7], we wanted to develop a rapid and reliable method for the convenient detection of this IDUA mutation (OMIM？no. 252800.0001). Denaturing high-performance liquid chromatography (dHPLC) was successfully applied to the screening of mutations involved in various diseases [8], including MPS I [9]. We optimized an efficient dHPLC method and validated its application to mutation screening using DBS as source of biological material. This semiautomated technique
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