The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease

Abstract

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis -a simple, economical and rapid method -was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.

Libyan Boy with Autosomal Recessive Trait (p22-phox defect) of Chronic Granulomatous Disease

“…However, among the other subtypes of CGD, the autosomal recessive forms (40% of cases) may be associated with milder disease. The extent to which environmental factors and secondary genetic defects may influence the course of the disease is still not understood [10, 11]. …”

“…Currently, the yearly mortality rate is a little more than 1.5% per year for persons with autosomal recessive CGD and over 5% for those with X-linked CGD [10, 11]. The long-term survival rates of patients who present with symptoms after the end of their first year of age is significantly better than that of patients whose illness starts in infancy [13].…”

Pediatric allergy and immunology in Brazil

“…In the 1990's, other diseases started to be recognized (e.g., Chronic granulomatous disease [CGD], Wiskott–Aldrich syndrome, Chediak–Higashi syndrome, SCIDs) complement disorders, and cellular and phagocyte disorders. Functional and molecular diagnosis of PID improved with flow cytometry, and DNA sequencing technology revealing a novel genetic profile of PID . Several novel mutations were identified in a cohort of patients with CGD.…”

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

“…The cDNA was obtained by using SuperScript ® III Reverse Transcriptase (Invitrogen – Life Technologies), following the manufacturer’s instructions. For the PCR assay on cDNA, we used pairs of primers covering exons 1–5; 3–9; or 7–13 as requested [29].…”