In this study we assessed the pattern of both X Chromosome Inactivation (XCI) and ABCD1 allele-specific expression (ASE) in the peripheral blood mononuclear cells (PBMCs) of 30 female X-ALD carriers.
First, we observed, by a strictly controlled and very reproducible experimental procedure, that the skewing of XCI is significantly more common in X-ALD carriers than in healthy females, in agreement with previous findings .
Second, by ASE analyses, we provided the first evidence that, in X-ALD carriers, the active X chromosome is typically the chromosome carrying the mutant ABCD1 allele.
It is already known that the severity of clinical symptoms in X-linked disorders can be influenced by XCI skewing . Our finding of a skewed XCI favoring the inactivation of the wild-type allele is peculiar. Indeed, the skewing of XCI seems to commonly favor the inactivation of the mutant allele, thus explaining why, in many severe X-linked disorders, female carriers may be asymptomatic and have a severely skewed XCI [5, 6]. This is the case of Lesch-Nyhan syndrome, in which the cells expressing the wild-type allele of HPRT1 reproduce faster, thus gradually causing decrease of the cells in which the mutant allele is expressed . Similarly, in Wiskott-Aldrich syndrome, female carriers show preferential inactivation of the mutant allele in hematopoietic cells . In contrast, X-ALD had already been considered as the example of X-linked disease in which ABCD1 mutations confer a proliferative advantage rather than disadvantage, leading to XCI skewing in favor of the X chromosome with the mutation [5, 6, 8, 18], an evidence here demonstrated in vivo. This type of skewing could be due to a somatic selection in favor of cells carrying an active mutated X chromosome, after the X-inactivation process; in turn, this positive selection might be due to a growth advantage of cells expressing the defective ABCD1 allele . However, it remains enigmatic why cells expressing a mutant ABCD1 should have a growth advantage over normal cells. Reduced intracellular VLCFA β-oxidation, due to the mutant ABCD1, and the consequent change of lipid environment might be the basis for this phenomenon.
Moreover, we have investigated whether both the skewing of XCI or the preferential expression of the mutant ABCD1 allele were related to the neurological manifestations affecting a subgroup of female carriers. Our data do not support any significant correlation between neurological manifestations of X-ALD carriers and the XCI pattern or the degree of expression of the mutant ABCD1 allele. The lack of correlation between neurological manifestations and XCI pattern in PBMCs is in agreement with previous data of Watkiss et al and Jung et al [7, 21], and in contrast with those of Maier et al . Since XCI pattern evaluation could be biased by experimental procedures, it is conceivable that discrepancies between our results and previous results are due to the different methodologies used. Indeed, our data were obtained in a highly reproducible manner and in a cohort of X-ALD female carriers larger than those previously studied. The lack of correlation between neurological manifestations and XCI pattern or degree of expression of the mutant ABCD1 allele might be due to the tissue analyzed, as the degree of XCI or mutant ABCD1 expression might be different in the brain, which is the tissue mainly involved in X-ALD. Although XCI in brain from X-ALD carriers was not investigated, a similar XCI pattern in blood and brain has been observed . To explore the maintenance of XCI pattern in another tissue we performed XCI and ASE in urinary sediment of a small subset of our patients (n = 8) and we have found that XCI and ASE pattern were similar in the two cell types (data not shown).
Finally, our data reveal a significant and causative correlation between the VCLFA plasma concentrations and the degree of mutant ABCD1 allele-specific expression in vivo. This is in agreement with previous in vitro observations by Migeon et al .
In conclusion, we provide evidence that: i) in X-ALD carriers the high frequency of non-random XCI usually reflects the preferential expression of the mutant ABCD1 allele, thus supporting by in vivo data the hypothesis relating to the growth advantage of cells expressing the mutant ABCD1 ; ii) the quantitative allele-specific expression mirrors the plasma concentration of VCLFAs; iii) unfortunately, due to the lack of correlation between XCI/ASE pattern and clinical symptoms, our results prove that these molecular values cannot be considered in clinical practice as predictive markers for the development of symptoms in X-ALD carriers.