Trisomy 21 alters DNA methylation in parent-of-origin-dependent and independent manners

The supernumerary chromosome 21 in Down syndrome differentially affects the methylation statuses at CpG dinucleotide sites and creates genome-wide transcriptional dysregulation of parental alleles, ultimately causing diverse pathologies. At present, it is unknown whether those effects are dependent or independent of the parental origin of the nondis-joined chromosome 21. Linkage analysis is a standard method for the determination of the parental origin of this aneuploidy, although it is inadequate in cases with deficiency of samples from the progenitors. Here, we assessed the reliability of the epigenetic 5(m)CpG imprints resulting in the maternally (oocyte)-derived allele methylation at a differentially methylated region (DMR) of the candidate imprinted WRB gene for asserting the parental origin of chromosome 21. We developed a methylation-sensitive restriction enzyme-specific PCR assay, based on the WRB DMR, across single nucleotide polymorphisms (SNPs) to examine the methylation statuses in the parental alleles. In genomic DNA from blood cells of either disomic or trisomic subjects, the maternal alleles were consistently methylated, while the paternal alleles were unmethylated. However, the supernumerary chromosome 21 did alter the methylation patterns at the RUNX1 (chromosome 21) and TMEM131 (chromosome 2) CpG sites in a parent-of-origin-independent manner. To evaluate the 5(m)CpG imprints, we conducted a computational comparative epigenomic analysis of transcriptome RNA sequencing (RNA-Seq) and histone modification expression patterns. We found allele fractions consistent with the transcriptional biallelic expression of WRB and ten neighboring genes, despite the similarities in the confluence of both a 17-histone modification activation backbone module and a 5-histone modification repressive module between the WRB DMR and the DMRs of six imprinted genes. We concluded that the maternally inherited 5(m)CpG imprints at the WRB DMR are uncoupled from the parental allele expression of WRB and ten neighboring genes in several tissues and that trisomy 21 alters DNA methylation in parent-of-origin-dependent and -independent manners

5meCpG epigenetic marks neighboring a primate-conserved core promoter short tandem repeat indicate X-chromosome inactivation.

X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates

Chromosomal and physical map positions and sequence features of the locus encompassing the <i>RP2</i> onshore tandem GAAA repeat.

<p>The composite image above the DNA sequence is based on screenshots generated using the UCSC Genome Browser (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Kent1" target="_blank">[49]</a>, with the <i>RP2</i> onshore tandem GAAA repeat-containing region viewing coordinates chrX:46,695,746-46,696,645 (GRCh37.p5/hg19 primary reference assembly of human X-chromosome; NC_000023.10), centered on the landmark CpG island of the <i>RP2</i> promoter. The GAAA repeat element maps within the Xp11.3. The presented features (from top to bottom) are annotated tracks for OMIM genes, UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics), reference mRNA, CpG and the tandem (GAAA)n repeat. The line drawing above the DNA sequence represents the physical map of the target locus, with the <i>RP2</i> 5′coding region highlighted in light green. The locations of the forward and reverse primer sequences used for genotyping the <i>RP2</i> onshore tandem GAAA repeat are highlighted in red and pink, respectively. The tandem GAAA repeat sequence is highlighted in black with white symbols. The 5^meC-sensitive restriction endonuclease recognition sites analyzed in the XCI experiments are highlighted in blue and brown in white symbols.</p

Methylation statuses at CpG sites near the <i>RP2</i> onshore tandem GAAA repeat.

<p>Random (<b>A</b>) and non-random (<b>B</b>) X-inactivation patterns generated for different CpG-containing 5^meCpG-sensitive restriction endonuclease sites obtained using the 5^meCpG-based PCR <i>RP2</i>/<i>AR</i> biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with <i>Hpa</i>II, <i>Hha</i>I or <i>Bst</i>UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).</p

<i>AR</i> CAG and the <i>RP2</i> GAAA polymorphisms refer to the same X-chromosomes.

<p>Segregation analysis of either <i>AR</i> or <i>RP2</i> alleles distinguishes the maternal origin of the preferentially skewed Xi present in the daughter. Xi is identified based on the 230-bp <i>AR</i> allele and the 368-bp <i>RP2</i> allele. The allele names are the lengths in base pairs of each fluorescence peak and the intensity of each peak is in relative fluorescence units (RFU). Note that the magnitude of stuttering at the <i>RP2</i> onshore tandem GAAA repeat is minimal, in contrast with that at the <i>AR</i> CAG repeat.</p

Molecular phylogenetic analysis using the maximum likelihood method.

<p>The evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Tamura2" target="_blank">[50]</a>. The bootstrap consensus tree inferred from 1,000 replicates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Felsenstein1" target="_blank">[51]</a> is taken to represent the evolutionary history of the analyzed taxa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Felsenstein1" target="_blank">[51]</a>. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Felsenstein1" target="_blank">[51]</a>. Initial tree(s) for the heuristic search were obtained automatically by applying the maximum parsimony method. The analysis involved 10 nucleotide sequences. The codon positions included were 1^st + 2^nd + 3^rd+ Noncoding. In total, there were 410 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Tamura1" target="_blank">[25]</a>. The numbers in parentheses correspond to the lengths of the uninterrupted tandem arrays in GAAA repeat units.</p

The <i>RP2</i> onshore tandem GAAA repeat is polymorphic in marmosets.

<p>Electropherograms of alleles observed in marmosets genotyped via quantitative fluorescent PCR. (<b>A</b>) Representative allele profiles from males, which exhibited only the major allele, and female animals with three distinct genotypes (homozygotes for either the minor or major allele or heterozygotes) are shown. (<b>B</b>) Representative random XCI pattern observed at the 5^meCpG-sensitive <i>Bst</i>UI recognition site within the <i>RP2</i> GAAA-containing amplimer, with an Xa/Xi ratio of approximately 65%. The allele names are the lengths in base pairs of each fluorescence peak and the intensity of each peak is in relative fluorescence units (RFU).</p

Reverse transcription-PCR across the GAAA repeat-containing region.

<p><i>RP2</i> onshore tandem GAAA repeat-specific steady-state RNA is not detected in mononucleated blood cells from two healthy female donors (21 years old) or a male donor (33 years old) or from the THP-1 male cell line. RNA samples were either reverse (+) or mock (−) transcribed (RT) prior to PCR amplification across the <i>RP2</i> onshore tandem GAAA repeat-specific region (<b>A</b>) or the <i>GAPDH</i>-specific region (<b>B</b>). Corresponding samples of genomic DNA were used as positive controls for the PCR assays. The amplification products were separated via electrophoresis in an 8% acrylamide: bis-acrylamide gel and silver-stained for detection. Lane L50 shows a standard 50-bp ladder (Invitrogen); lane H₂O is the negative PCR amplification control. The range of the <i>RP2</i> onshore tandem GAAA repeat-specific DNA amplimer is 350 to 391-bp. The <i>GAPDH</i>-specific DNA amplimers are as follows: 130-bp for <i>GAPDHP63</i> (6∶80663360-80663489) and <i>GAPDHP1</i> (X:39647022-39647151) and 220-bp for <i>GAPDH</i> (12∶6646089-6646308). The processed (mature) <i>GAPDH</i>-specific cDNA-derived product is 130- bp.</p

Allelic distribution of the <i>RP2</i> onshore tandem GAAA repeat.

<p>(<b>A</b>) Electropherogram of alleles observed in 60 unrelated Brazilian females genotyped via quantitative fluorescent PCR. The intensity of the red line tracing is related to the allele frequency. Smaller peaks preceding the designated allele peaks represent Taq polymerase stutter products corresponding to a mean of 2.6% of the amount of the true allele. In contrast, the mean stuttering for the <i>AR</i> disease-linked CAG repeat was 17.6% (not shown). Allele names are the lengths in base pairs of each fluorescence peak and the intensity of each peak is in relative fluorescence units (RFU). The <i>RP2</i> onshore tandem GAAA repeat locus exhibited an allelic span (the difference in length between the longest and the shortest allele per locus) of 41-bp in this population subset. (<b>B</b>) <i>RP2</i> onshore tandem GAAA repeat-containing allele frequencies and heterozygosity (H_E) rates observed in the population subsets consisting of Brazilian and Dutch women.</p

5^mCpG statuses at the <i>WRB</i> CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line.

<p>A consistent unmethylated pattern of CpG sites at the <i>WRB</i> CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole (<b>A</b>) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line (<b>B</b>). Electropherograms of the amplimers (see details of the assay in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.g002" target="_blank">Fig 2</a>) generated from either undigested DNA or <i>Hha</i>I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.</p