Figure 1.
Chromosomal and physical map positions and sequence features of the locus encompassing the RP2 onshore tandem GAAA repeat.
The composite image above the DNA sequence is based on screenshots generated using the UCSC Genome Browser (http://genome.ucsc.edu) [49], with the RP2 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 RP2 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 RP2 5′coding region highlighted in light green. The locations of the forward and reverse primer sequences used for genotyping the RP2 onshore tandem GAAA repeat are highlighted in red and pink, respectively. The tandem GAAA repeat sequence is highlighted in black with white symbols. The 5meC-sensitive restriction endonuclease recognition sites analyzed in the XCI experiments are highlighted in blue and brown in white symbols.
Figure 2.
Reverse transcription-PCR across the GAAA repeat-containing region.
RP2 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 RP2 onshore tandem GAAA repeat-specific region (A) or the GAPDH-specific region (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 RP2 onshore tandem GAAA repeat-specific DNA amplimer is 350 to 391-bp. The GAPDH-specific DNA amplimers are as follows: 130-bp for GAPDHP63 (6∶80663360-80663489) and GAPDHP1 (X:39647022-39647151) and 220-bp for GAPDH (12∶6646089-6646308). The processed (mature) GAPDH-specific cDNA-derived product is 130- bp.
Figure 3.
Allelic distribution of the RP2 onshore tandem GAAA repeat.
(A) 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 AR 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 RP2 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) RP2 onshore tandem GAAA repeat-containing allele frequencies and heterozygosity (HE) rates observed in the population subsets consisting of Brazilian and Dutch women.
Figure 4.
Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat.
Random (A) and non-random (B) X-inactivation patterns generated for different CpG-containing 5meCpG-sensitive restriction endonuclease sites obtained using the 5meCpG-based PCR RP2/AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with HpaII, HhaI or BstUI 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).
Figure 5.
RP2 and AR repeat-based methylation results are highly concordant.
Scatterplot visual assessment of the strength of association between the percentages of the main inactive allele referred by the methylation statuses at the RP2 onshore tandem GAAA repeat (y-axis) and the AR CAG repeat (x-axis) loci. The methylation statuses are highly concordant (Spearman r = 0.9404, CI95% = 0.8950 to 0.9665; p<0.0001) across varying degrees of random (50–80%) and non-random (>80%) XCI. The regression line superimposed on the plot provides the best-fitting straight line for the scattered data.
Figure 6.
AR CAG and the RP2 GAAA polymorphisms refer to the same X-chromosomes.
Segregation analysis of either AR or RP2 alleles distinguishes the maternal origin of the preferentially skewed Xi present in the daughter. Xi is identified based on the 230-bp AR allele and the 368-bp RP2 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 RP2 onshore tandem GAAA repeat is minimal, in contrast with that at the AR CAG repeat.
Figure 7.
Hemophilia A occurs due to highly skewed XCI.
Electropherograms of alleles obtained using the 5meCpG-based RP2/AR repeat biplex PCR assay across the HpaII restriction site in a heterozygote female carrier of a one-base insertion, frameshift mutation in factor VIII domain B. The female is a hemophiliac due to highly skewed inactivation of the unaffected X-chromosome, represented by the AR 215-bp and RP2 368-bp alleles. The RP2 and AR repeat-based 5meCpG readouts refer to the skewed X-inactivation state. The F8 mutation was screened through conformational sensitive gel electrophoresis [19] and direct sequencing. Allele names (upper boxed numbers) are the lengths in base pairs of each fluorescence peak and the intensity of each peak is in relative fluorescence units (RFU). The lower boxed numbers correspond to the areas under the allele peaks.
Figure 8.
Molecular phylogenetic analysis using the maximum likelihood method.
The evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei model [50]. The bootstrap consensus tree inferred from 1,000 replicates [51] is taken to represent the evolutionary history of the analyzed taxa [51]. 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 [51]. 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 1st + 2nd + 3rd+ Noncoding. In total, there were 410 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [25]. The numbers in parentheses correspond to the lengths of the uninterrupted tandem arrays in GAAA repeat units.
Figure 9.
The RP2 onshore tandem GAAA repeat is polymorphic in marmosets.
Electropherograms of alleles observed in marmosets genotyped via quantitative fluorescent PCR. (A) 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) Representative random XCI pattern observed at the 5meCpG-sensitive BstUI recognition site within the RP2 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).