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Symptomatic female carriers of Duchenne Muscular Dystrophy (DMD): Genetic and clinical characterization

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the dystrophin gene and is characterized by muscle degeneration and death. DMD affects males; females being asymptomatic carriers of mutations. However, some of them manifest symptoms due to a translocation between X chromosome and an autosome or to a heterozygous mutation leading to inactivation of most of their normal X chromosome. Six symptomatic female carriers and two asymptomatic were analyzed by: I) Segregation of STRs-(CA)n and MLPA assays to detect a hemizygous alteration, and II) X chromosome inactivation pattern to uncover the reason for symptoms in these females. The symptomatic females shared mild but progressive muscular weakness and increased serum creatin kinase (CK) levels. Levels of dystrophin protein were below normal or absent in many fibers. Segregation of STRs-(CA)n revealed hemizygous patterns in three patients, which were confirmed by MLPA. In addition, this analysis showed a duplication in another patient. X chromosome inactivation assay revealed a skewed X inactivation pattern in the symptomatic females and a random inactivation pattern in the asymptomatic ones. Our results support the hypothesis that the DMD phenotype in female carriers of a dystrophin mutation has a direct correlation with a skewed X-chromosome inactivation pattern.


INTRODUCTION
Duchenne muscular dystrophy (DMD) and the milder allelic Becker muscular dystrophy (BMD) are X-linked recessive diseases caused by absent or abnormal dystrophin, respectively, in skeletal muscle, which results in early muscle degeneration. DMD is a fatal disease that affects 1:3500 newborn males, while BMD is less severe and less frequent [1]. Both dystrophies are caused by mutations, mostly deletions, in the dystrophin gene [2,3]. One third of the cases arise from new mutations, while the remaining two thirds are inherited [4,5]. DMD and BMD usually affect males, with the majority of females being asymptomatic carriers. However, some of these females can reveal symptoms that vary from mild muscle weakness to a more severe clinical course and are classified as manifesting or symptomatic carriers [6,7,8,9,10]. Studies on muscle biopsy suggest that female dystrophinopathy patients show a skewed X inactivation, where the X chromosome that carries the normal dystrophin gene is preferentially inactivated [11,12]. Many of these symptomatic carriers have gross chromosomal rearrangements due to translocation between the X chromosome and an autosome, with one breakpoint involving the DMD gene [13]. In these cases the normal X chromosomes appear to be preferentially inactivated since the dystrophin levels are below normal, leading to DMD or to a milder variant [14]. On the other hand, a small number of females heterozygous for dystrophin mutations, have most of their normal X chromosome randomly inactivated, and manifest mild DMD symptoms [15]. Furthermore, a different X inactivation pattern was seen in pairs of monozygotic twins heterozygous for mutation in the dystrophin gene, which resulted in clinical manifestation in only one of them [16].
It is more difficult to ascertain the DMD mutations in female carriers than those in males because the presence of the normal X chromosome masks the alteration in its mutant counterpart [17]. Thus, in order to detect mutations (mostly deletions) in females clinically diagnosed with muscle disease, more laborious and expensive methods need to be used than for analysis of male patients. These methods include segregation analysis of short tandem-repeat (STR) (CA)n polymorphisms and MLPA assay, which are able to detect hemizigous patterns and thereby reveal A C C E P T E D M A N U S C R I P T

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4 the mutated DMD gene in female-carriers. The use of (STR) (CA)n polymorphisms, located throughout the dystrophin gene including the deletion-prone regions [18], is not as informative as MLPA analysis, however, both methods are essential for validation of the identified mutations.
The determination of maternal and paternal X chromosome activation status is useful for the analysis of non-random X inactivation patterns. One of the methods to test the X inactivation is based on the methylation-sensitivity of the restriction enzymes HpaII and HhaI [19]. It was shown that the methylation of HpaII and HhaI sites in the first exon of the human androgen-receptor locus correlates with X inactivation, moreover, the presence of an STR close to this site allows the use of a PCR assay to identify the methylation pattern of the maternally and paternally derived X chromosomes.
This study aimed to analyze six symptomatic and two asymptomatic female carriers using a strategy that includes: i) identification of the hemizygous patterns in the dystrophin gene by segregation of STRs-(CA)n markers and MLPA assay; and ii) determination of the X chromosome inactivation pattern. Comparing these results with clinical and histopathologic data provides a correlation between genotype and phenotype and thus unable us to uncover the reason for the manifesting DMD status in these females.

Patients
Six females with muscular dystrophy and no DMD history and two females without these symptoms were referred for a differential diagnosis of DMD by several hospitals in Argentina. The a priori diagnosis was established by current clinical, biochemical and histological criteria. After approval of the study protocol by the Bioethical Committee of Hospital de Clinicas "Jose de San Martin", informed consent for genetic analyses was signed by the patients. The clinical features are depicted in Table 1. The six symptomatic females presented with muscle alterations compatible with dystrophinopaty and several of them, in addition, with learning and behavioural anomalies.
One of the females had a DMD associated with an autism syndrome. The two asymptomatic females showed minor myopathic signs such as muscle cramps, myalgia and one of them (#60), a mild cardiac dysfunction. Both females presented with high CK levels.

Mutation analysis
Mutation screening was performed by segregation analysis of Short Tandem Repeats of (CA)n (STRs-(CA)n) and Multiplex Ligation-dependent Probe Amplification (MLPA). The DNA from peripheral blood leukocytes of probands and their parents was isolated by the CTAB method [20].
recommendations (MRC Holland, Amsterdam, Netherlands) in order to screen all the dystrophin exons. PCR products were analyzed on an AB1 3130 automated sequencer using Genescan software (Applied Biosystem). The dosage quotient was calculated using Gene Marker software.
X-chromosome inactivation pattern analysis (HUMARA assay) Genomic DNA samples were isolated from peripheral blood leukocytes (PBL) by salting out procedure [21] to detect X-chromosome inactivation by the AR (human androgen receptor gene) assay as was originally described with minor modifications [19]. Briefly, for each patient's DNA sample, two reactions were prepared: two μg of DNA were either treated (T) with 20 U Hpa II highly skewed for values > 90%, according to Amos-Landgraf et al. [22].

RESULTS
Six female patients manifesting different grades of DMD plus two females not manifesting DMD but with some features suggesting a DMD carrier status, were studied ( Table 1). The females with DMD symptoms represent 6% of the total DMD carrier females studied in our laboratory, which is consistent with previously reported data [6]. The age of disease onset in five of the symptomatic females was between 7 and 10. One of these patients (#499) had an onset of autism at the age of 3 with symptoms that included hyperactivity, impulsiveness and loss of previously acquired functions plus an onset of DMD at the age of 10. All these females shared myopathic changes on electromyograms and a progressive muscular weakness, which was less severe than in DMD males. Most of them are still able to walk, but with some problems such as frequent falls, fatigue, lameness and an inability to run. One of these patients (#482) became wheel-chair bound at the age of 19. The sixth female (#514), who was born from consanguineous parents, had an earlier onset, before the age of 6, and presented with more severe symptoms. This female became wheelchair bound at the age of 6 and died at the age of 13 from heart failure. The pattern of X-chromosome inactivation (XIP) in the six symptomatic and the two asymptomatic females is depicted in Figure 2. All the females were found to be heterozygous at the AR locus. The symptomatic females showed an extremely skewed X-chromosome inactivation pattern: XIP>90%, whereas, the two females not expressing DMD (#60 and 645) showed a random pattern of inactivation: XIP 52% and 71% respectively ( Figure 2 and Table 1).

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DISCUSSION
In order to clarify the mechanism responsible for clinical symptoms in female DMD mutationcarriers we studied six females with a clinical diagnosis of dystrophinopathy and two females without dystrophinopathy, but with biochemical/histological data suggesting a DMD carrier status. Segregation of STRs and MLPA analyses allowed for the detection of deletions in three females and a duplication in one of the patients. Two of these mutations were clustered in the central hot spot region and two other mutations, one deletion and one duplication were located at the 5´ region of the gene, which is a common site for duplications [9]. All the mutations were out of frame. No gross deletions/duplications were found in two of the female patients (#482 and 514) nor were they found in the two females not expressing DMD (#60 and 645), suggesting an absence of gross rearrangements and the presence of point mutations as the cause of dystrophinopathy or carrier

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10 status. Interestingly, one of the patients (#514) who was homozygous for most STR loci due to her parents´ consanguinity (cousins), additionally showed a retraction in the paternal 49 STR-allele.
This patient was the one that had a severe muscular disease and the only one who died at an early age. The absence of gross rearrangements revealed by STRs and MLPA analyses suggests that there was no chromosomal translocation between X and an autosome. Thus, another type of mutation would be responsible for the dystrophinopathy, and the severe presentation may be due to additional alterations in a glycoprotein complex or in other factors resulting from parental consanguinity.
Skewed X chromosome inactivation has been proposed as a possible explanation for the presence of DMD-symptoms in female carriers of dystrophin gene mutations. Such females should have an active X chromosome with the mutation and an inactive normal X chromosome. This hypothesis has been proved in females with an X-autosome translocation, in whom the X-autosome can not be inactivated and thus, the normal X-chromosome became inactivated in all the muscle cells, leading to severe DMD-like symptoms [9]. However, in cases with a skewed X-inactivation in the majority of the fibers (for example 80%) some muscle cells should have dystrophin-positive fibers. In these cases the results concerning the correlation between the patient phenotype and an Xinactivation pattern are controversial [7,8,22]. All the symptomatic females studied in our laboratory presented a skewed X-inactivation pattern (XIP), showing a relationship between the clinical phenotype and the pattern of X-inactivation. No translocations have been revealed by the methods used for detection of gross rearrangements. These data suggest a different reason for a skewed X-inactivation than the presence of an autosomic fragment joined to X chromosome with a mutated dystrophin gene. The lack of translocation in the only patient with a severe form of dystrophinopathy (#514) correlates with the partially skewed pattern of X-inactivation, unlike to that observed in patients with an X-autosome translocation [9].
In summary, different findings concerning the correlation between DMD symptoms and a skewed X chromosome inactivation in females have been reported previously. Our results support

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11 the hypothesis that the DMD phenotype in female carriers of dystrophin mutations is connected to a skewed X chromosome inactivation pattern, leading to less expression of the wild type dystrophin.