A PCR based X-chromosome inactivation assay for carrier detection in X-linked immunodeficiencies using differential...

Carrier detection in X-linked immunodeficiencies (X-SCID, WAS, XLA) relies on the demonstration of non-random X inactivation patterns in blood cell lineages. Only a limited number of cells are available after cell separation methods. PCR-based techniques are therefore necessary to analyze active and inactive X chromosomes. Amplifying a polymorphic CAG repeat in the first exon of the androgen receptor gene after selective digestion of the active X chromosome with a methylation-sensitive restriction enzyme allows to distinguish between the paternal and maternal alleles and to identify their methylation status. DNA from B-, T-lymphocytes and total peripheral leukocytes of normal males, females and obligate carriers of X-linked immunodeficiencies were analyzed. The results of this PCR-based X inactivation assay are concordant with the standard methylation studies at the DXS255 locus using Southern blotting. This PCR assay provides a rapid and informative (heterozygosity > 90%) method in carrier detection of X-linked immunodeficiencies and other X-linked disorders, which show non-random X inactivation in cell lineages from the affected tissues.