Skewing of X-chromosome inactivation in three generations of carriers with X-linked chronic granulomatous disease within one family

Background: Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by impairment of intracellular microbicidal activity of phagocytes. Mutations in one of the four known NADPH-oxidase components preclude generation of superoxide and related antimicrobial oxidants, leading to the phenotype of CGD. Defects in gp91-phox, encoded by CYBB, lead to X-linked CGD, responsible for approximately 70% of all CGD cases. The aim of the study was to evaluate the hypothesis that age-related skewing of X-chromosome inactivation, as described in several CGD families, is caused by preferential survival of bone marrow clones with an inactive NADPH oxidase. Materials and methods: We studied the neutrophils from three patients and four carriers in three generations of a Turkish family with X-linked CGD. Carrier detection was carried out by the dihydrorhodamine (DHR)-1,2,3 assay, which measures on a per-cell basis the NADPH oxidase-dependent oxidation of DHR by phagocytes. The X-chromosome inactivation pattern was determined with the HUMARA assay in DNA from leucocytes as well as in DNA from a buccal smear of the four carriers. Results: The three patients were identified by a negative DHR test, and the mutation in their CYBB gene was characterized by DNA sequencing. Moreover, we found an age-related degree of skewing of X-chromosome inactivation in the leucocytes of the four X-CGD carriers, both at the protein level (NADPH oxidase activity) and at the DNA level (HUMARA assay). However, similar skewing of X-chromosome inactivation was found in the buccal DNA from these women. Conclusions: These novel findings indicate that the age-related degree of skewing was probably a chance finding, not related to preferential survival of NADPH oxidase-deficient precursor cells, because this enzyme is not expressed in (buccal) epithelial cells. © 2006 Blackwell Publishing Ltd.