Methylation of AR locus does not always reflect X chromosome inactivation state

Department of Medicine, Division of Hematology, University of Utah School of Medicine Salt Lake City, UT 84132, USA.
Blood (Impact Factor: 10.45). 01/2012; 119(13):e100-9. DOI: 10.1182/blood-2011-11-390351
Source: PubMed


Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

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Available from: Andrew Wilson, Jan 06, 2014
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    • "Our data is in agreement with a previous study which did not find a statistical difference in XCI skewness among women with alcoholism in comparison to control group (Manzardo et al., 2012). Another possible explanation for the results obtained in our study and also alcoholic women's study may be based on a recent research reporting that the methylation of AR locus does not always correlate with XCI state (Swierczek et al., 2012). Although several studies have used this locus to evaluate XCI pattern, the statement of the mentioned research is a considerable note when discussing the data obtained from XCI studies. "
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    ABSTRACT: Introduction: X chromosome inactivation (XCI) is a process during which one of the two X chromosomes in female human is silenced leading to equal gene expression with males who have only one X chromosome. Here we have investigated XCI ratio in females with opioid addiction to see whether XCI skewness in women could be a risk factor for opioid addiction. Methods: 30 adult females meeting DSM IV criteria for opioid addiction and 30 control females with no known history of addiction were included in the study. Digested and undigested DNA samples which were extracted from blood were analyzed after amplification of the polymorphic androgen receptor (AR) gene located on the X chromosome. XCI skewness was studied in 3 ranges: 50:50-64:36 (random inactivation), 65:35-80:20 (moderately skewed) and >80:20 (highly skewed). Results: XCI from informative females in control group was 63% (N=19) random, 27% (N=8) moderately skewed and 10% (N=3) highly skewed. Addicted women showed 57%, 23% and 20%, respectively. The distribution and frequency of XCI status in women with opioid addiction was not significantly different from control group (P=0.55). Discussion: Our data did not approve our hypothesis of increased XCI skewness among women with opioid addiction or unbalanced (non-random) expression of genes associated with X chromosome in female opioid addicted subjects.
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    • "Skewing has traditionally been determined using the androgen receptor assay [22], which shows good correlation with expression-based determination of skewing [23]. A lack of agreement between some assays highlights the perils of using only a single gene to determine skewing [24]. To address this we instead averaged up to 177 genes (the subject training set) to determine the degree of skewing. "
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    ABSTRACT: X-chromosome inactivation (XCI) results in the silencing of most genes on one X chromosome, yielding mono-allelic expression in individual cells. However, random XCI results in expression of both alleles in most females. Allelic imbalance has been used genome-wide to detect mono-allelically expressed genes. Analysis of X-linked allelic imbalance in females with skewed XCI offers the opportunity to identify genes that escape XCI with bi-allelic expression in contrast to those with mono-allelic expression and which are therefore subject to XCI. We determine XCI status for 409 genes, all of which have at least five informative females in our dataset. The majority of genes are subject to XCI and genes that escape from XCI show a continuum of expression from the inactive X. Inactive X expression corresponds to differences in the level of histone modification detected by allelic imbalance after chromatin immunoprecipitation. Differences in XCI between populations and between cell lines derived from different tissues are observed. We demonstrate that allelic imbalance can be used to determine an inactivation status for X-linked genes, even without completely non-random XCI. There is a range of expression from the inactive X. Genes escaping XCI, including those that do so in only a subset of females, cluster together, demonstrating that XCI and location on the X chromosome are related. In addition to revealing mechanisms involved in cis-gene regulation, determining which genes escape XCI can expand our understanding of the contributions of X-linked genes to sexual dimorphism.
    Genome biology 11/2013; 14(11):R122. DOI:10.1186/gb-2013-14-11-r122 · 10.81 Impact Factor
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    • "Each of these techniques has some advantages and disadvantages or limitations [5]. However, DNA methylation does not always perfectly correlates with X-chromosome inactivation; in the most clonality assays, DNA methylation has been used to indicate X-inactivation [6] [7]. The problem appears particularly for some probes such as M27β, which would complicate studies of X-chromosome inactivation [8]. "
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    ABSTRACT: The aim of this study was to evaluate the patterns of X-chromosome inactivation during the remission in acute myeloid leukemia (AML) at the RNA level. Two hundred normal females and 45 female patients with AML entered the study. The frequency of heterozygosity was 48.5% (119/245) for P55, 40% (93/245) for IDS, and only 28.9% (71/245) for G6PD. Some individuals were heterozygous for more than one gene polymorphism. Overall, one hundred normal individuals proved showed to be heterozygous for at least one of the above polymorphisms. 92/100 (92%) normal females showed a polyclonal pattern. Clonal patterns were observed in 44/45 (98%) AML patients at presentation. Of 27 patients who were followed after remission, 23 (85.2%) patients showed a clonal pattern. Ten patients were available for a longer followup (up to 12 months) and the clonal pattern was observed in seven patients. It can be concluded that clonality at remission is a frequent event in AML and does not necessarily mean relapse of the disease. There is also a possibility of conversion of clonality to polyclonality over time.
    10/2012; 2012:971493. DOI:10.5402/2012/971493
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