The pattern of XCI in PBMCs and CD4⁺ T-cell subsets derived from carriers of FOXP3 mutations is random. (A) Carriers 1, 2, 4, 6, and 9 have mutations in the forkhead/winged-helix (FKH) DNA-binding domain, whereas carrier 5 has a mutation in exon 6 between the C₂H₂ zinc finger and the leucine zipper domains. Carriers 3, 7, and 8 have a nucleotide substitution within the ATG translation start site, which completely abrogates the expression of the FOXP3 protein (FOXP3-null mutation; S.D.N., unpublished data, November 7, 2008). Carriers 2 and 4 and carriers 3, 7, and 8 are members of the same families. (B) Pherograms of XCI analyses of total PBMCs derived from carriers of FOXP3 mutations are shown (top panel). For carriers who are members of the same family (carriers 2 and 4, carriers 3, 7, and 8) only the pherogram of PBMCs derived from 1 representative subject is shown. CAG repeats of the HAR gene were amplified after digestion with the methylation-sensitive enzyme HpaII so that only methylated (inactive) sequences were amplified. For each HAR allele, the cosegregating WT or mut-FOXP3 allele is indicated according to CAG repeats that cosegregate with the mut-FOXP3 in the relevant IPEX patients (bottom panel). Genomic DNA derived from PBMCs of IPEX patients was not digested because in males the unique X-chromosome is unmethylated and active. (C) XCI was analyzed in CD4⁺ naive and memory T cells sorted according to their expression of CD45RA. The pherograms of CD4⁺ T-cell populations of the representative carrier 4 are shown. cr indicates carrier.