Skewing of X-inactivation ratios in blood cells of aging women is confirmed by independent methodologies
ABSTRACT Nonrandom X-chromosome inactivation (XCI), also known as skewing, has been documented in the blood cells of a significant proportion of normal aging women by the use of methylation-based assays at the polymorphic human androgen receptor locus (HUMARA). Recent data obtained with a new transcription-based XCI determination method, termed suppressive polymerase chain reaction (PCR), has shed controversy over the validity of XCI ratio results obtained with HUMARA. To resolve this disparity, we analyzed XCI in polymorphonuclear leukocytes of a large cohort of women aged 43 to 100 years with the use of HUMARA (n=100), a TaqMan single nucleotide polymorphism (SNP) assay (n=90), and the suppressive polymerase chain reaction (PCR) assay (n=67). The 3 methods yielded similar skewing incidences (42%, 38%, and 40%, respectively), and highly concordant XCI ratios. This confirms that the skewing of XCI ratio seen in blood cells of aging women is a bona fide and robust biologic phenomenon.
- SourceAvailable from: Enrique Medina-Acosta
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- "The data were analyzed with GeneScan Analysis 3.7 and Genotyper 3.7 software (Applied Biosystems). Fluorescent peak areas representing true alleles were normalized for the occurrence of stutter products using the approach outlined in the literature . The degree of association between the percentages of the Xi/Xa referred by the methylation statuses at the RP2 GAAA onshore and AR CAG repeat loci across women with varying extents of random and non-random XCI was determined by calculating the Spearman correlation coefficient, CI95% and p value and visualized with a scatterplot using Graph Pad Prism 5.0. "
ABSTRACT: 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.PLoS ONE 07/2014; 9(7):e103714. DOI:10.1371/journal.pone.0103714 · 3.23 Impact Factor
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- "Fisher's exact test); moderately skewed XCI in 3/9 symptomatic and 11/21 asymptomatic carriers (p = 0.4397; Fisher's exact test); random XCI in 3/9 symptomatic and 4/21 asymptomatic carriers (p = 0.6402; Fisher's exact test). The increase of age has been associated, by other studies [16,17], with a more frequent occurrence of preferential XCI but, in our population, no correlation was found between the degree of XCI of the X-ALD carriers and the age at evaluation (p = 0.1550; Spearman ρ correlation). This suggests that ABCD1 mutations may influence the XCI process, thus making the possible correlation between older age and preferential XCI irrelevant. "
ABSTRACT: Approximately 20% of adrenoleukodystrophy (X-ALD) female carriers may develop clinical manifestations, typically consisting of progressive spastic gait, sensory deficits and bladder dysfunctions. A skewing in X Chromosome Inactivation (XCI), leading to the preferential expression of the X chromosome carrying the mutant ABCD1 allele, has been proposed as a mechanism influencing X-linked adrenoleukodystrophy (X-ALD) carrier phenotype, but reported data so far are conflicting. To shed light into this topic we assessed the XCI pattern in peripheral blood mononuclear cells (PBMCs) of 30 X-ALD carriers. Since a frequent problem with XCI studies is the underestimation of skewing due to an incomplete sample digestion by restriction enzymes, leading to variable results, we developed a pyrosequencing assay to identify samples completely digested, on which to perform the XCI assay. Pyrosequencing was also used to quantify ABCD1 allele-specific expression. Moreover, very long-chain fatty acid (VLCFA) levels were determined in the same patients. We found severely (≥90:10) or moderately (≥75:25) skewed XCI in 23 out of 30 (77%) X-ALD carriers and proved that preferential XCI is mainly associated with the preferential expression of the mutant ABCD1 allele, irrespective of the manifestation of symptoms. The expression of mutant ABCD1 allele also correlates with plasma VLCFA concentrations. Our results indicate that preferential XCI leads to the favored expression of the mutant ABCD1 allele. This emerges as a general phenomenon in X-ALD carriers not related to the presence of symptoms. Our data support the postulated growth advantage of cells with the preferential expression of the mutant ABCD1 allele, but argue against the use of XCI pattern, ABCD1 allele-specific expression pattern and VLCFA plasma concentration as biomarkers to predict the development of symptoms in X-ALD carriers.Orphanet Journal of Rare Diseases 01/2012; 7(1):10. DOI:10.1186/1750-1172-7-10 · 3.96 Impact Factor
Blood Cells Molecules and Diseases 03/2011; 15(46):288. DOI:10.1016/j.bcmd.2011.02.001. · 2.33 Impact Factor
- "j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d conclude that their results validate previous studies in which HUMARA assay document age-dependent skewing in females and that skewing of XCI ratio seen in blood cells of aging women is a genuine biologic phenomenon . Until recently it has been assumed that the XCI skewing intensity would be the same in all tissues. "