title: High-performance liquid chromatography under partially denaturing conditions (dHPLC) is a fast and cost-effective method for screening molecular defects: four novel mutations found in X-linked chronic granulomatous disease.
abstract: Implementing precise techniques in routine diagnosis of chronic granulomatous disease (CGD), which expedite the screening of molecular defects, may be critical for a quick assumption of patient prognosis. This study compared the efficacy of single-strand conformation polymorphism analysis (SSCP) and high-performance liquid chromatography under partially denaturing conditions (dHPLC) for screening mutations in CGD patients. We selected 10 male CGD patients with a clinical history of severe recurrent infections and abnormal respiratory burst function. gDNA, mRNA and cDNA samples were prepared by standard methods. CYBB exons were amplified by PCR and screened by SSCP or dHPLC. Abnormal DNA fragments were sequenced to reveal the nature of the mutations. The SSCP and dHPLC methods showed DNA abnormalities, respectively, in 55% and 100% of the cases. Sequencing of the abnormal DNA samples confirmed mutations in all cases. Four novel mutations in CYBB were identified which were picked up only by the dHPLC screening (c.904 insC, c.141+5 g>t, c.553 T>C, and c.665 A>T). This work highlights the relevance of dHPLC, a sensitive, fast, reliable and cost-effective method for screening mutations in CGD, which in combination with functional assays assessing the phagocyte respiratory burst will contribute to expedite the definitive diagnosis of X-linked CGD, direct treatment, genetic counselling and to have a clear assumption of the prognosis. This strategy is especially suitable for developing countries.
authors.affiliation: Department of Immunology - Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: E B
authors.initials: EB
authors.lastname: de Oliveira-Junior
authors.forename: C
authors.initials: C
authors.lastname: Prando
authors.forename: J A
authors.initials: JA
authors.lastname: Lopez
authors.forename: J C
authors.initials: JC
authors.lastname: Arango
authors.forename: M
authors.initials: M
authors.lastname: Buzolin
authors.forename: J
authors.initials: J
authors.lastname: Rehder
authors.forename: L A
authors.initials: LA
authors.lastname: Pedroza
authors.forename: J B
authors.initials: JB
authors.lastname: Frazão
authors.forename: V M
authors.initials: VM
authors.lastname: Dantas
authors.forename: P
authors.initials: P
authors.lastname: Roxo-Junior
authors.forename: A S
authors.initials: AS
authors.lastname: Grumach
authors.forename: B T
authors.initials: BT
authors.lastname: Costa-Carvalho
authors.forename: J
authors.initials: J
authors.lastname: Bustamante
authors.forename: A
authors.initials: A
authors.lastname: Condino-Neto
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000379:methods*; D003362:Cost-Benefit Analysis; D006105:Granulomatous Disease, Chronic / Q000235:genetics*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics*; D013997:Time Factors
chemical_list: D008562:Membrane Glycoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases