METHODS AND PATIENTS
Patients
A 34-year-old healthy woman II2 had given birth to a boy with typical symptoms of MECP2 duplication syndrome. These symptoms included infantile hypotonia, delayed psychomotor development, poor speech development, intellectual disability, and recurrent respiratory infections. She wanted to give birth to a second child. To explore the genetic cause and phenotypic effects of skewed XCI, we performed a single-nucleotide polymorphism (SNP) array and whole-exome sequencing (WES) for her and her asymptomatic mother I2. Meanwhile, we detected the XCI states of the two females by HUMARA /RP2 assays and RNA-seq.
This study followed the Ethics Committee of Women’s Hospital’s recommendations, School of Medicine at Zhejiang University. All participants provided informed consent in accordance with the Declaration of Helsinki. The Review Board of the Women’s Hospital, School of Medicine, Zhejiang University in China approved the study protocol.
DNA/RNA extraction and qPCR
We extracted genomic DNA samples of peripheral blood and fetal amniotic fluid with the GentraPuregene Kit (Qiagen, Germany). We extracted total RNA with TRIzol reagent according to the manufacturer’s instructions (Invitrogen). Quantitative real-time (qRT-PCR) was carried out using SYBR Green PCR Master Mix (Takara, Japan) on the Applied Biosystems 7900HT system. Supp. Table S1 lists the primers. Melting curve analyses confirmed that all primers were specific for their respective transcript. We used the ΔΔCt method to determine relative DNA/cDNA levels and determined the fold change by the value of 2-ΔΔCt.
SNP array
We performed the SNP array using the CytoScan™ HD Array Kit (Affymetrix, USA) according to the manufacturer’s instruction, with around 2,600,000 markers, including 750,000 SNP probes and 1,900,000 non-polymorphism probes used for comprehensive whole-genome coverage. Data were analyzed by Chromosome Analysis Suite software (Affymetrix, Santa Clara, CA) based on GRCh38 assembly.
XCI analysis
XCI in the Xq28-duplication females was analyzed by PCR amplification of the HUMARA gene and retinitis pigmentosa (RP2 ) locus, aspreviously described (Allen, Zoghbi, Moseley, Rosenblatt, & Belmont, 1992; Machado et al., 2014).
RNA-Seq
We performed RNA-Seq by Biomarker Technologies (Beijing, China) and generated sequencing libraries using NEBNextR Ultra™ Directional RNA Library Prep Kit for IlluminaR (NEB, USA) following the manufacturer’s recommendations. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq Xten platform. Raw data (raw reads) in the FASTQ format were first processed through in-house Perl scripts, and clean data (clean reads) were obtained by removing reads containing an adapter, reads containing ploy-N, and low-quality reads from raw data. Then, the clean reads were mapped to the reference genome sequence. HISAT2 tools software was used for reference genome mapping. We used Picard-tools v1.41 and SAMtools v0.1.18 to sort through reads, remove duplicated reads, and merge each sample’s bam alignment results. GATK2 or SAMtools software was used to perform SNP calling. Raw VCF files were filtered using the GATK standard filter method, and only SNPs with a distance >5 were retained. Differential expression analysis of two groups was performed using the DESeq R package (1.10.1)
Whole-Exome Sequencing (WES)
WES was performed by Biomarker Technologies (Beijing, China). The sequencing libraries were generated using the NimbleGen SeqCap EZ Human Exome V3 (Roche, Basel, Swiss) following the manufacturer’s recommendations. Clustering of the index-coded samples was performed on a cBot Cluster Generation System (Illumina, USA) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq X Ten platform with a 150 bp paired-end module.
Burrows-Wheeler Aligner v0.7.13-r1126 was used to align each sample’s clean reads with the reference genome using default parameters. Alignment files were converted to BAM files using SAMtools software. Variant calling was performed for all samples by using the Haplotype Caller in GATK software.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) was performed in the female and her mother. Xq28 was tagged with BAC RP11-119A22 (Illumina) labeled in the red spectrum. The centromere probe was Vysis CEP X (DXZ1), which was labeled in the green spectrum (Abbott Laboratories).