Figure 2.

X-inactivation patterns for a pair of identical twin females discordant for hemophilia A and their parents using HUMARA assay. Amplification was performed before (−) and after (+) HhaI digestion. Mother and twins are heterozygous for CAG repeats and both alleles are present before digestion represented by two major PCR products on the gel (maternal alleles, lane 3, M₁ and M₂; twin A and B alleles, lanes 5 and 7, M₂ and P). Mother and twin B show random X-inactivation with both alleles active and therefore equally amplified after digestion (lane 4, M₁ and M₂, and lane 8, M₂ and P), whereas twin A shows nonrandom inactivation with only the lower, paternally inherited allele represented (lane 6, P only). Father’s single X-chromosome (hemizygous) is always active (lane 1, P) and disappears after digestion (lane 2). Fainter stutter bands are products of slippage by DNA polymerase. The selective amplification of one allele in the affected twin shows that it is always methylated (i.e., inactive) and resistant to HhaI digestion.