Legends
FIGURE 1. Xq28 intrachromosomal duplications were identified in female I2 (daughter) and II2 (mother) by SNP array, qPCR, and FISH analyses. (A) Pedigree of the family. (B) Blue bars indicate a 437 kb duplication in Xq28 (chrX:153858452-154332213 in the genome build GRCh38) and a 284 kb duplication in Xq27.3 (chrX:144988272-145271978 in the genome build GRCh38) in I2 and II2. (C) The relative ratio of the Xq28 (MECP2 and IRAK1 ) regions in I2 and II2 by qPCR. (D) For I2 and II2, a FISH experiment showed that the MECP2-containing probe (detected by probe RP11-119A22, Spectrum red) occurred only at Xq28, the X-chromosome centromere was labeled by probe Vysis CEP X (DXZ1) in the green spectrum. It demonstrated that the Xq28 duplications are tandem repeats that had not integrated elsewhere in the genome.
FIGURE 2. Polymorphic repeats for HUMARA/RP2 detected X-chromosome inactivation (XCI) pattern analyses. (A, D) A peak of 267 bp for HUMARA (381 bp for RP2 ) was observed by assaying the undigested PCR product of I1, and no peaks were observed for the HpaII digested product. (B, E) The undigested PCR product of I2 gave two peaks of 281 and 287 bp for HUMARA (359 and 374 bp forRP2 ), each, while only one peak of 281 bp for HUMARA (374 bp for RP2 ) was observed with the HpaII digested product. I2 exhibited complete skewing of XCI, and the inactivated X-chromosome was linked with the 281 bp peak of HUMARA (374 bp peak of theRP2 ) PCR products. (C, F) The undigested PCR product of the proband II2 gave two peaks of 267 and 287 bp for HUMARA (359 and 381 bp for RP2 ). One X-chromosome linked with the 287 bp forHUMARA (359 bp for RP2 ) was inhibited from the mother I2, and the other was inhibited from the father I1. The product of HpaII digestion gave only one peak of 287 bp for HUMARA (359 bp forRP2 ) to II2, which also demonstrated complete skewing of XCI. Still, the inactivated X-chromosome was linked with the 287 bp forHUMARA (359 bp for RP2 ), which is different from that of the mother I2.
FIGURE 3. Sequence analysis of the genomic DNA and RT-PCR product of the XIST (chrX:73846325) and MECP2(chrX:154030040) cDNA from PMBCs. (A) The genotypes of XIST(chrX:73846325) were wild type, G>A Het, G>A Het, G>A Hom, G>A Hom in I:1 (DNA), I:2 (DNA), II:2 (DNA), I:2 (cDNA), II:2 (cDNA). (B) The genotypes of MECP2(chrX:154030040) were G>A Hom, wild type, G>A Het, wild type, G>A Hom, in I:1 (DNA), I:2 (DNA), II:2 (DNA), I:2 (cDNA), II:2 (cDNA). Yellow arrows indicate the SNPs.
FIGURE 4. XCI pattern and linkage analyses based on the polymorphic repeats for HUMARA/RP2 and RNA-seq. Schematic diagram of Xq28 duplication, HUMARA/RP2 PCR products, and RNA-seq in the pedigree. By HUMARA/RP2 assays, Xq28 duplication of I2 occurred in the X-chromosome, whose allele was linked with the 287 bp peak ofHUMARA PCR product and 359 bp peak of the RP2 PCR product. The X-chromosome was activated and delivered to the II2, but the X-chromosome of I2 was inactivated. Based on RNA-seq assays, for both I2 and II2, the heterozygous SNP rs144237473 (chrX:73846325) in XISTexpressed only in the X-chromosome with Xq28 duplication, and XCI genesMECP2 and IRAK1 expressions on the Xq28 duplicated alleles of the two females were not transcriptional expressions. Variable XCI genes HCFC1 , NAA10 and the escaping XCI gene RENBP , the duplicated alleles inherited from the mother I2 were in lower expression. RNA-seq indicated that the duplicated Xq28 chromosome was inactive.
* Reported XCI status refers to the XCI status in the list available in Tukiainenet al. (2017)
# It indicated that the X-chromosome was inactive based on the locus detected by DNA methylation assay or RNA-seq.
GT: genotype, the genotype of the locus.
() Allele depth; the number of reads supporting either the reference genotype or SNP genotype by RNA-seq.
FIGURE 5. Relative expression fold of MECP2 (A) andIRAK1 (B) from peripheral blood mononuclear cells (PMBCs) in control females, I2, and II2 by qPCR. Expression dosages of theMECP2 and IRAK1 genes showed no significant difference in I2, II2, and control females.