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.