In order to shed light on the mechanism responsible for the clinical manifestations in symptomatic female DMD carriers, we recruited a cohort of 18 female carriers of a mutation in the dystrophin gene, 7 symptomatic and 11 asymptomatic, and compared their genomic and transcription profiles.
In both symptomatic and asymptomatic females, immunostaining of muscle biopsy tissue with anti-dystrophin antibodies highlighted a mosaic pattern of dystrophin-positive and dystrophin-negative fibres, in addition to fibres with reduced dystrophin labelling. Results reported by other groups showed no correlation between the percentage of negative fibres and clinical phenotype [17, 27–29].
Skewed X-chromosome inactivation has been proposed as a possible explanation for the presence of symptoms in manifesting carriers; based on this hypothesis, the X chromosome carrying the mutated dystrophin gene should be active in the majority of nuclei in muscles from symptomatic carriers.
X chromosome inactivation can be tested by different methods. Methylation analysis at the androgen receptor locus represents the most used assay: in contrast to direct methods based on expression analysis of polymorphic X-linked genes, AR assay is an indirect method based on differential methylation on active and inactive X chromosome; however it is considered an accurate test as high correlation has been demonstrated with results obtained from direct expression analysis. Despite the fact that several studies assessing the role of X-inactivation have been performed, their findings have been inconclusive and even contradictory. This disparity in results is partly due to the non-homogeneity of the criteria adopted in different studies, such as the use of different cut-off levels to define skewed X-inactivation [15, 19, 21, 25]. Furthermore, these studies have frequently employed lymphocytes in evaluating X-inactivation, but these cells fail to mirror dystrophin expression, and considerable differences in X-inactivation pattern between different tissues from the same individual have been reported .
Nonetheless, the role of the X-inactivation pattern has been demonstrated in females with X-autosome translocation; cells featuring inactivation of the derivative X chromosome become partially monosomic for the translocated autosome, thereby conferring a selective advantage on cells having the derivative X chromosome active. This is the situation in our carrier 1, who features a balanced X;9 translocation; in this case the totally skewed pattern of 100:0 observed in her blood and muscle is not unexpected, and is in accordance with her severe phenotype. However, the absence of dystrophin labelling in the majority of fibres (80%), but not all as would be expected from a completely silenced normal X chromosome, raises the question of how and where dystrophin-positive fibres arise. Among the other symptomatic females informative for the polymorphism at the AR locus, the majority (4 out of 5) presented a random X-inactivation pattern, revealing a lack of relationship between X-inactivation pattern and clinical phenotype.
This is not surprising, as the absence of relationship between X chromosome inactivation pattern and phenotype has been highlighted for other X-linked disorders: in hemophilia A and B , Fabry disease  and myotubular myopathy  the occurrence of disease manifestations in females do not correlate with skewed X-inactivation.
Genomic analysis was integrated with RNA studies performed on muscle biopsy and revealed homogeneous transcriptional behaviour; in all carriers, both symptomatic and asymptomatic, the transcription of both mutated and wild-type mRNAs was observed. The coexistence of the two types of transcripts was demonstrated in almost all cases, either directly by RT-PCR or indirectly by the observation of the biallelic transcription of polymorphism-containing exons. Unfortunately, the huge size of the duplication and the absence of heterozygous exonic polymorphisms precluded identification of this evidence in carrier 10. Nonetheless, biallelic transcription was demonstrated in carriers 15 and 16, despite the presence of large deletions extending into the 3’-UTR of the gene, which might have reduced mRNA stability. In particular, we had the opportunity of further exploring carrier 16 by CGH-array; the 3’ breakpoint of the dystrophin rearrangement was thereby defined, and a 3.5 Mb deletion, extending outside the DMD genomic locus, was identified. In this case, the transcriptional behaviour seems to be very complex, presumably involving the formation of a fusion transcript comprising the DMD locus and another unidentified adjacent gene. However the evidence of monoallelic transcription of c.5234G>A polymorphism in dystrophin exon 37 suggests that the fusion breakpoint on the DMD gene transcript does not correspond to the breakpoint of the genomic deletion, which is located in intron 43.
The data gathered in the present study did highlight the absence of any relationship between X-inactivation pattern, transcriptional behaviour and dystrophic phenotype in females. Relative quantification of wild-type and mutated transcripts was performed in 9 carriers, revealing no relationship with the X-inactivation pattern. Among symptomatic females, this was particularly evident in carrier 4, characterized by skewed X-inactivation and similar levels of the two transcripts, and carrier 6, who, in contrast, was shown to possess a random X-inactivation pattern but a strong prevalence of the wild-type transcript. The absence of a relationship between X-inactivation pattern and transcription was also evident in asymptomatic carriers 11, 13, and 14.
The lack of relationship between phenotype and transcriptional behaviour is also evident from our results; among the 4 symptomatic carriers analysed with the High Sensitivity Chip, 3 showed the prevalence of the wild-type transcript over the mutated transcript, and 1 was characterized by similar levels of wild-type and mutated mRNAs. This discrepancy between transcript biallelic representation and X-inactivation strongly suggests that other, probably genetic, determinants may independently influence dystrophin transcription and X chromosome inactivation.
A genetic modifier of DMD severity in males has been recently described ; the G allele of the rs28357094 polymorphism in the osteopontin ( SPP1) promoter was demonstrated to be associated with increased muscle weakness and precocious loss of ambulation. Hence, we analysed this variant in our series of carriers. Despite the small size of the cohort precluding statistical significance, we did find similar proportions of T and G alleles in symptomatic ( T allele: 11/12; G allele; 1/12) and asymptomatic ( T allele: 17/22; G allele: 5/22) subjects.
Relative quantification of wild-type and mutated alleles was performed in females carrying out-of-frame mutations; these results could therefore be biased by nonsense-mediated decay of the mutated allele. In order to overcome this problem, we performed a relative quantification of wild-type transcript in respect to control females. Our data highlights the absence of relationship between phenotype and dystrophin transcriptional level. With the exception of carrier 4, characterized by very low dystrophin transcription rate (6-9% in respect to controls), in the other cases the levels quantified are very similar in symptomatic and asymptomatic carriers.
A reduction in dystrophin protein abundance appears to be a common feature in carriers: reduced protein levels have been described both in symptomatic [17, 28] and asymptomatic females . In our cohort of carriers, the manifesting female 1 for whom a precise protein quantification was performed showed a protein level of less than 15% of normal abundance; quantification in asymptomatic carrier 8 showed protein levels more than 70% of normal. These data raise the question of a possible relationship between dystrophin protein level and phenotype. This issue will need to be addressed in future studies of protein quantification with larger number of carriers.