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A skewed view of X chromosome inactivation
Jakub Minks, Wendy P. Robinson, and Carolyn J. Brown
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
Address correspondence to: Carolyn J. Brown, Department of Medical Genetics, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Phone: (604) 822-0908; Fax: (604) 822-5348; E-mail: cbrown@interchange.ubc.ca.
Find articles by Minks, J. in: JCI | PubMed | Google Scholar
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
Address correspondence to: Carolyn J. Brown, Department of Medical Genetics, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Phone: (604) 822-0908; Fax: (604) 822-5348; E-mail: cbrown@interchange.ubc.ca.
Find articles by Robinson, W. in: JCI | PubMed | Google Scholar
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
Address correspondence to: Carolyn J. Brown, Department of Medical Genetics, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Phone: (604) 822-0908; Fax: (604) 822-5348; E-mail: cbrown@interchange.ubc.ca.
Find articles by Brown, C. in: JCI | PubMed | Google Scholar
Published December 20, 2007 - More info
J Clin Invest. 2008;118(1):20–23. https://doi.org/10.1172/JCI34470.
© 2007 The American Society for Clinical Investigation
Related article:
No evidence that skewing of X chromosome inactivation patterns is transmitted to offspring in humans
Abstract
Skewing of X chromosome inactivation (XCI) can occur in normal females and increases in tissues with age. The mechanisms underlying skewing in normal females, however, remain controversial. To better understand the phenomenon of XCI in nondisease states, we evaluated XCI patterns in epithelial and hematopoietic cells of over 500 healthy female mother-neonate pairs. The incidence of skewing observed in mothers was twice that observed in neonates, and in both cohorts, the incidence of XCI was lower in epithelial cells than hematopoietic cells. These results suggest that XCI incidence varies by tissue type and that age-dependent mechanisms can influence skewing in both epithelial and hematopoietic cells. In both cohorts, a correlation was identified in the direction of skewing in epithelial and hematopoietic cells, suggesting common underlying skewing mechanisms across tissues. However, there was no correlation between the XCI patterns of mothers and their respective neonates, and skewed mothers gave birth to skewed neonates at the same frequency as nonskewed mothers. Taken together, our data suggest that in humans, the XCI pattern observed at birth does not reflect a single heritable genetic locus, but rather corresponds to a complex trait determined, at least in part, by selection biases occurring after XCI.
Authors
Véronique Bolduc, Pierre Chagnon, Sylvie Provost, Marie-Pierre Dubé, Claude Belisle, Marianne Gingras, Luigina Mollica, Lambert Busque
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What is skewed X chromosome inactivation?
X chromosome inactivation (XCI) — a process originally hypothesized by Lyon in 1961 (1) and by which one of the two copies of the X chromosome present in females is inactivated — achieves dosage equivalency for X-linked genes between XY males and XX females. A critical tenet of this hypothesis was that the initial choice of which X (maternal or paternal) to inactivate was random but then this choice was stably inherited. An individual cell’s decision to inactivate either the paternal or maternal X is made early in development, at approximately the time of implantation (2). A deviation from equal (50%) inactivation of each parental allele is known as skewing, with common criteria for “skewed” inactivation being arbitrarily defined as the observation of inactivation of the same allele in 75% or 80% of cells (and very skewed inactivation resulting in 90% or 95% of cells with the same allele inactive).
A solid understanding of the causes of skewed XCI is needed because skewing is often used as a tool in the clinical setting (see The clinical relevance of skewed XCI). As shown in Figure 1A, some individuals will undergo skewed XCI by chance, as there is a limited pool of cells at the time of inactivation and only a limited precursor population that contributes to any tissue. Selection can also lead to a secondary (or acquired) skewed inactivation. In addition, there may be variants controlling the initial choice of which X to inactivate, resulting in primary nonrandom inactivation. For example, in mice there is a locus known as the X chromosome controlling element (Xce) that differs between strains and results in primary skewing of inactivation during animal crosses (3). The murine Xce is located downstream of X (inactive)–specific transcript (Xist) (4), a gene that codes for a non–protein coding RNA that localizes to the inactive X chromosome and has been shown to be sufficient for XCI (reviewed in ref. 5). Whether there is a similar locus involved in XCI skewing in humans remains an open question. In this issue of the JCI, the study by Bolduc et al. addresses the relative contribution to XCI skewing of primary and secondary influences by investigating skewing in a population of over 500 pairs of healthy female mothers and their neonates (6).
Figure 1Skewed XCI in females. (A) Skewed XCI can result from either a primary nonrandom inactivation or a secondary, acquired nonrandom inactivation. There are two active X chromosomes in the female embryo, one from each parent (maternal and paternal; Xm and Xp, respectively). Upon inactivation, one X forms the heterochromatic inactive X depicted here as a darkly staining oval. The cells are shaded to reflect the different origins of their remaining active X. Subsequent stochastic or selective growth of one cell population leads to skewing of inactivation. However, if the initial inactivation was influenced by an allele (analogous to the murine Xce allele) then inactivation could be directed primarily towards one allele. A reduction in the embryonic precursor pool size at the time of random choice would make skewed XCI more likely. (B) The common way to examine XCI skewing makes use of a highly polymorphic CAG repeat (shown here as having allele sizes a and b) in the androgen receptor gene and differential methylation (methylation shown as black lollipop while the unmethylated allele has open lollipops) of the active (Xa) and inactive (Xi) X. DNA is extracted from the cells to be tested and then digested with a methylation-sensitive restriction enzyme (shown as scissors). The methylated (Xi) DNA will remain intact for subsequent PCR amplification using primers (shown as arrows) flanking the cut site. The ratio of the alleles (a and b, distinguished by PCR product length) in the undigested compared with the digested DNA reflects the XCI skewing.
Assays to examine XCI skewing require the presence of a polymorphism to distinguish the two Xs and a means of determining which X is active. While examining RNA or protein is a more direct measure of X chromosome activity, DNA methylation of the inactive X is often used as a surrogate since DNA is more easily extracted, stored, and analyzed. Furthermore, coding region polymorphisms are generally less informative and can show allelic imbalances in expression levels. An assay monitoring DNA methylation of the highly polymorphic CAG trinucleotide polymorphism at the 5ι end of the gene encoding the androgen receptor, as used in the Bolduc study (6), has become the standard for measuring XCI skewing (Figure 1B).
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Consideration of the sources of skewed XCI
If the assessment of XCI skewing is to be used as a clinical tool, we need to understand the biological sources of skewing. There have now been several large-scale analyses of XCI in an effort to define the basal level of skewing, the largest of which examined skewing in over 1,000 women (14). This new paper from Bolduc and colleagues (6) provides another large dataset for the literature. There is variability among studies (extreme skewing in newborns ranging from 0.5% to 2.7% of the population examined), some of which might be attributable to differences in methodology, however, the most striking changes in frequency of skewing are seen with patient age and cell type studied.
Much of the skewing seen in adult hematopoietic cells is not observed in newborns. Bolduc et al. observed a doubling of the frequency of extreme skewing in mothers relative to their newborns (6). This increase in skewing seems to become even more pronounced as women age (15). The Bolduc study also analyzed both blood and buccal (epithelial) tissue for skewing (6). They observed a substantial correlation between the two tissues, with blood showing greater skewing in both the neonates and mothers in agreement with previous studies (16, 17). This is an important finding because it suggests that tissues of different germ-lineage origin show similar inactivation patterns. As the tissue(s) affected by an X-linked disorder may be inaccessible to study, it will be important to determine the extent to which accessible tissues (such as blood or buccal samples) reflect the XCI pattern in other tissues. Age-related, acquired XCI skewing can lead to late-onset disease in individuals, as has been reported for X-linked sideroblastic anemia (18). If age-related, acquired XCI skewing occurs in tissues other than blood then late-onset symptoms might be a concern for additional X-linked disorders. Within blood, substantial differences in skewing, particularly in older adults, have been reported for different subpopulations of hematopoietic cells (19), which means that any differences in an individual’s immune health or method of sample acquisition that might bias cell representation may contribute to variability between individuals and studies.
A critical question addressed by the Bolduc study was whether there is a genetic component to skewing of XCI (6). Several twin studies have shown a substantial genetic component to acquired or secondary XCI skewing (20, 21). However, there has been limited study of the intriguing question of whether there are primary influences in humans on the choice of which X chromosome to inactivate. To address this issue, Bolduc et al. examined concordance between mother-daughter pairs for XCI skewing. In both buccal and hematopoietic cells, the authors saw no evidence that neonates with skewed XCI are more likely to have had a mother with skewed XCI. There are considerable challenges to such studies as discussed by the authors. First, much of the skewing observed in the mothers could have been caused by selective pressures and acquired secondarily rather than reflecting an initial bias. Second, the low frequency of skewing in the newborn population means that sample sizes need to be very large to detect a significant heritable effect. Finally, the contribution of the paternal allele is difficult to address since males do not inactivate their only X chromosome. Therefore, drawing an analogy with the murine Xce, the authors examined polymorphisms in the XIST gene to determine whether there was a correlation between a particular allele or haplotype (combination of alleles) and the prevalence of XCI skewing. Again, no evidence for a genetic component to skewing was observed. Interestingly, however, the French-Canadian population examined in this study was relatively homogeneous for the XIST alleles studied, limiting the authors’ power to detect a significant effect. There are striking population differences between XIST haplotype frequencies (see Table 1; ref. 22), suggesting that studies in other populations (or heterogeneous populations) might reveal a different story.
There are many case reports in the literature of families that seem to have substantial heritable skewing of XCI (for example, refs. 23, 24). Unlike in mice, in which the Xce alleles play a critical role in this skewing, such cases may reflect acquired skewing due to selection or very rare mutations related to XIST (25). While the population differences for XIST suggest we cannot yet close the book on the existence in humans of an Xce-like gene, the results from the Bolduc study in this issue of the JCI (6) support the hypothesis that the majority of XCI skewing observed in the adult population is acquired secondarily.
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