WAS is an X-linked disorder characterized by immune deficiency, thrombocytopenia and eczema. Previous studies of G-6-PD isozyme expression in rare females doubly heterozygous for WAS and G-6-PD found one of two X chromosomes to be preferentially active in T cells and platelets compared to their fibroblasts. Presumably this is due to selection against cells in which the X chromosome with the WAS allele is active. We have used a new strategy that distinguishes the active and inactive X chromosomes for the detection of WAS carriers. Genomic ONA is analyzed by Southern blot to detect heterozygosity for X chromosome restriction fragment length polymorphisms (RFLP) at the PGK or HGPRT loci. For both loci, the state of methylation is different and constant between the active and inactive X chromosomes. In females heterozygous for these RFLP's the active and inactive X's can thus be distinguished with with methylation-sensitive restriction endonucleases. We have screened 32 female relatives of WAS patients; 14 were heterozygous at one of the two loci (44% potentially informative). The methylation pattern of DNA from T cells of non-carriers showed random X-inactivation. In contrast, DNA from T cells of WAS carriers showed only one of the two X chromosomes to be active. Thus, X-inactivation analysis appears to directly identify WAS carriers and does not require the rare co-occurence of G-6-PD heterozygosity. We are currently using this method to determine the cell lineages affected by the WAS gene defect.
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USE OF X CHROMOSOME INACTIVATION ANALYSIS TO ESTABLISH CARRIER STATUS FOR X-LINKED SEVERE COMBINED IMMUNODEFICIENCY
The Lancet (1988)
Archives of Disease in Childhood (1988)