Symptomatic female carriers of Duchenne muscular dystrophy (DMD): Genetic and clinical characterization

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Abstract

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 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 enable us to uncover the reason for the manifesting DMD status in these females.

Section snippets

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

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

Discussion

In order to clarify the mechanism responsible for clinical symptoms in female DMD mutation-carriers 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. Furthermore, their genotypic and phenotypic profiles were compared. The clinical presentation of 5 of the females, who are currently aged between 17 and 41, was similar to mild dystrophinopathy, characterized by a

Conflict of interests

The authors declare that they have no competing or other interests that might be perceived to influence the results and discussion reported in this paper.

Acknowledgments

This study was possible because of the invaluable assistance of the patients and their families. The authors want to thank Doctors: L. Francipane, (Hospital de Clinicas Jose de San Martin, Buenos Aires); R. Fauze, (Hospital del Niño Jesus, Servicio de Neurologia, San Miguel de Tucuman, Argentina) and L. Mesa (Buenos Aires), Argentina, for referral of the patients to our department and providing their clinical data and to Miss Laura O. Gaozza for language supervision. This work was supported by

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