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).