title: The identification of novel mutations in the biotinidase gene using denaturing high pressure liquid chromatography (dHPLC).
abstract: Biotinidase deficiency (BD) is an autosomal recessive disorder of biotin metabolism that causes incomplete recycling of free biotin. The resulting depletion of intracellular biotin leads to impaired activities of biotin-dependent carboxylases. The ensuing clinical phenotype includes progressive neurologic deterioration with epileptic seizures, muscular hypotonia as well as skin eczema. BD may be readily diagnosed by analysing enzyme activity in dried blood spots during newborn screening but typically requires molecular confirmation. More than 100 different mutations in the biotinidase gene have been reported to date. To simplify molecular testing we have developed a rapid and accurate denaturing high pressure liquid chromatography (dHPLC) method of the promoter, 3'UTR, all exons including exon/intron boundaries as a first line screen followed by direct sequencing of the respective PCR products. To validate this method we used DNA from 23 different, newly diagnosed patients with biochemically proven BD from Austria, India, Morocco and Spain. A total of 11 mutations, missense 7, frameshift 3 and 1 nonsense, were screened. Six mutations were novel to this study. All mutations revealed distinct dHPLC pattern thus enabling their accurate detection. This study revealed that dHPLC method is robust, automated, economical and above all highly sensitive for the molecular analysis of biotinidase gene and should be used as a pre-analytical tool followed by sequencing of aberrant heteroduplex forming amplicons.
authors.affiliation: Department of Pediatrics and Adolescent Medicine, Laboratory for Inherited Metabolic Disorders, Medical University of Vienna, Austria. farhanzoologist@hotmail.com
authors.forename: Furhan
authors.initials: F
authors.lastname: Iqbal
authors.forename: Chike B
authors.initials: CB
authors.lastname: Item
authors.forename: Maria A
authors.initials: MA
authors.lastname: Vilaseca
authors.forename: Anil
authors.initials: A
authors.lastname: Jalan
authors.forename: Adolf
authors.initials: A
authors.lastname: Mühl
authors.forename: Maria L
authors.initials: ML
authors.lastname: Couce
authors.forename: Ana
authors.initials: A
authors.lastname: Duat
authors.forename: Maria P
authors.initials: MP
authors.lastname: Delgado
authors.forename: Joaquim
authors.initials: J
authors.lastname: Bosch
authors.forename: Alberto
authors.initials: A
authors.lastname: Puche
authors.forename: Jaume
authors.initials: J
authors.lastname: Campistol
authors.forename: Mercè
authors.initials: M
authors.lastname: Pineda
authors.forename: Olaf A
authors.initials: OA
authors.lastname: Bodamer
mesh_terms: D043523:Biotinidase / Q000235:genetics*; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D009154:Mutation; D011489:Protein Denaturation; D012680:Sensitivity and Specificity
chemical_list: D043523:Biotinidase