5. Most XCI genes in the X-chromosome with Xq28 duplication of the two females were not transcriptionally expressed
XCI statuses of X-linked genes were defined as escape, variable escape, and inactive. XCI genes, such as MECP2 and IRAK1 , were only expressed in the active X (Xa) chromosome. Escape XCI genes, located mostly in the pseudoautosomal region, were expressed both in Xa chromosomes and the inactive X (Xi) chromosome. Variable escape XCI genes were determined to be variable XCI statuses (Tukiainen et al., 2017).
We listed all SNPs information on the X-chromosome detected by both RNA sequencing and WES. For I2 and II2, in the non-pseudoautosomal region, where most SNPs of XCI genes in the X-chromosome with Xq28 duplication were no transcription, and there was a lower overall transcription for variable and escaping XCI genes (Supp. Table S4).
6. MECP2 and IRAK1 on the Xq28-duplicated alleles of the two females were not transcriptionally expressed by RNA sequencing
Then we focused on the Xq28-duplicated region (Supp. Table S5). All SNP expressions of XCI genes (MECP2, IRAK1, TMEM187, TKTL1 ) in II2 and I2 were in a homozygous condition, indicating complete skewed XCI in II2 and I2. In the Xq28-duplicated region, there are several informative SNPs, which can definite parental origin, such as MECP2(chrX:154030040), IRAK1 (chrX:154013378, chrX:154018741, chrX:154019032), TMEM187 (chrX:153982271, chrX:153982294, chrX:153982503, chrX:153982797) (Figure 4 and Supp. Table S5). DNA and cDNA genotypes of MECP2 (chrX:154030040) were also detected by PCR-sanger sequencing (Figure 3B). It showed that the alleles inheriting from the mother I2, which is with Xq28 duplication, were not transcriptionally expressed. Likewise, in the variable XCI genesHCFC1 , NAA10 and the escaping XCI gene RENBP , the duplicated alleles inherited from the mother I2 were in lower expression. As informative SNPs of XCI genes presented in Figure 4 and Supp. Table S5, we found that the XCI genes MECP2 andIRAK1 on the Xq28-duplicated alleles of II2 and I2 were in silencing transcription.