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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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de Oliveira-Junior, E. B. [1] ; Prando, C. [2] ; Lopez, J. A. [3] ; Arango, J. C. [3] ; Buzolin, M. [4] ; Rehder, J. [4] ; Pedroza, L. A. [1] ; Frazao, J. B. [1] ; Dantas, V. M. [5] ; Roxo-Junior, P. [6] ; Grumach, A. S. [7] ; Costa-Carvalho, B. T. [8] ; Bustamante, J. [9, 10] ; Condino-Neto, A. [1]
Total Authors: 14
Affiliation:
[1] Univ Sao Paulo, Inst Biomed Sci, Dept Immunol, BR-05508000 Sao Paulo - Brazil
[2] Rockefeller Univ, Rockefeller Branch, St Giles Lab Human Genet Infect Dis, New York, NY 10021 - USA
[3] Sch Microbiol, Primary Immunodeficiency Grp, Medellin - Colombia
[4] Univ Estadual Campinas, Sch Med, Ctr Invest Pediat, Campinas, SP - Brazil
[5] Univ Fed Rio Grande do Norte, Univ Hosp, Pediat Allergy Immunol Div, BR-59072970 Natal, RN - Brazil
[6] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Pediat, BR-14049 Ribeirao Preto, SP - Brazil
[7] ABC Med Sch, Dept Med, Santo Andre, SP - Brazil
[8] Univ Fed Sao Paulo, Sch Med, Dept Pediat, Div Allergy Immunol & Rheumatol, Sao Paulo - Brazil
[9] Natl Inst Hlth & Med Res, Lab Human Genet Infect Dis, Necker Branch, U980, Paris - France
[10] Univ Paris 05, Necker Med Sch, Paris - France
Total Affiliations: 10
Document type: Journal article
Source: Scandinavian Journal of Immunology; v. 76, n. 2, p. 158-166, AUG 2012.
Web of Science Citations: 4
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. (AU)