2. HUMARA and RP2 assays showed that females I2 and II2 possessed complete skewed XCI, but in the opposite X-chromosome
As presented in Figure 2, we performed PCR-based HUMARA andRP2 assays to assess the XCI patterns in I2, II2. After digestion with the methylation-sensitive restriction enzyme HpaII, we found that only the inactive X-chromosome could synthesize the PCR products. We determined the origin of the inactivate X-chromosome by segregation analysis. The undigested PCR product of II2 gave two peaks (267/287 inHUMARA and 359/381 in RP2 ). A single peak represented a single peak (287 in HUMARA and 359 in RP2 ) for the HpaII-digested product (Figures 2C, F), which indicated that the inactivate allele was inherited from her mother I2. The assay of the undigested PCR product of the mother I2 gave two peaks (281/287 inHUMARA and 359/374 in RP2 ). A major peak (281 inHUMARA and 374 in RP2 ), detected by assaying the HpaII digested product (Figures 2B, E), was different from that in the inactivated product allele of II2. The above results indicate that complete skewing of XCI was found in the female II2 and her mother I2, and was different from that of the inactivate X-chromosome (Figures 2, 4). The 287 bp peak of HUMARA PCR products, 359 bp peak ofRP2 PCR products in II2, and the Xq28 duplication were derived from the mother I2, which were linked to the same X-chromosome. However, the allele, which gave a peak of 287 bp after HUMARA PCR, a peak of 359 bp after RP2 PCR, and the Xq28 duplication, was inactivated in II2 but activated in I2.