4. For both II2 and I2, the heterozygous SNP rs144237473 (chrX:73846325) in XIST only expressed in the X-chromosome with Xq28 duplication, which indicates that the Xq28-duplication chromosome was inactive
To identify variants that may cause primary skewing in I2, we perfomed RNA sequencing in the peripheral mononuclear blood cells (PMBCs), data quality summary of RNA sequencing was listed in Supp. Table S2. we identified eight SNPs near the XIST region in I2 and II2 by exome sequencing (Supp. Table S3). All SNPs were consistent in I2 and II2 and therefore not considered potential primary skewing causes in I2. We also quantified the degree of skewing of XCI using a metric based on theXIST ASE from paired RNA-seq and DNA-seq data. It showed that heterozygous alleles of rs144237473 (chrX:73846325) (G:0/A:288) were expressed in a homozygous condition in II2, which is maternal inheritance. Similarly, the allelic expressions of rs144237473 (G:0/A:291) in I2 were complete skewed (Supp. Table S3, Figure 4). We also verified the SNP in DNA and cDNA by Sanger sequencing (Figure 3A), which revealed that each female’s inactive X-chromosome was the same chromosome with Xq28 duplication.