Long polymerase chain reaction-based fluorescence in situ hybridization analysis of female carriers of X-linked chronic granulomatous disease deletions

Abstract

Chronic granulomatous disease (CGD) is a rare inherited disorder in which antimicrobial activity of phagocytes is impaired due to the lack of reactive oxygen species, or oxidative burst, produced by NADPH oxidase. The X-linked form of CGD, representing approximately 70% of all cases, is caused by mutations in the cytochrome b beta subunit (CYBB) gene, which maps to chromosome Xp21.1. CYBB encodes the gp91-phox protein, a necessary component in the NADPH oxidase pathway. A wide variety of mutations have been identified in X-linked CGD patients, all of which lead to deletion of the functional protein and no oxidative burst activity. The mutations vary from single nucleotide substitutions to deletions of the entire gene. In this article, we report a mutation detection method for probands of female relatives at risk for carrier status of large deletions of the CYBB gene. Through fluorescent in situ hybridization of metaphase chromosomes, we were able to consistently distinguish carriers from noncarriers using polymerase chain reaction-derived, labeled DNA specific for exons 2 to 13 of the CYBB region at Xp21.1.

Figure 1

FISH using the CGD CYBB gene probe composed of exons 2 to 13 (red) and centromere control probe (green) on three separate study samples. A: Female relative of a CGD patient:noncarrier. B: Carrier of CYBB deletion showing no red signal on one X chromosome. C: CGD-affected male with CYBB deletion showing green control, but no red signal on X chromosome.