Preferential expression of mutant ABCD1 allele is common in adrenoleukodystrophy female carriers but unrelated to clinical symptoms

Abstract Background 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. Methods 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. Results 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. Conclusions 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.