Our data demonstrate the utility of modern diagnostic methods in the clinical characterization of manifesting carriers; among 15 subjects, seven had point mutations that were detected by direct sequencing methods. We also demonstrate that in the absence of a family history of dystrophinopathy, muscle biopsy remains the common method of diagnosis, and suggest that in the presence of a family history it is appropriate for the clinician to consider genetic testing prior to biopsy. In addition, in the absence of a family history other findings associated with dystrophinopathy may be more likely to be misinterpreted; for example, after an incidental detection of elevated transaminases, subject #7 underwent liver biopsy at the age of 9 years before myopathic symptoms and the detection of an elevated creatine kinase (CK) level led to her muscle disease work-up. Carriers with a BMD relative are less frequently and less severely affected than carriers with a DMD relative2, 3, 8, 14
, which may explain why even though the UDP enrolls probands affected by either DMD or BMD, the proband had DMD for all eight of the MCs with a family history.
Consistent with previous reports7, 14
, the spectrum of clinical presentations in MCs was quite wide, ranging from a rapidly disabling DMD-like phenotype (subject #8) to a very mild late-onset presentation (subject #11). Later onset of symptoms suggests less severe disease; four patients (subjects #11, 12, 14, and 15) developed symptoms after age 25, and all were categorized as mild BMD-like severity at examination 5 to 34 years after symptom onset (). However, earlier age at symptom onset was not necessarily associated with a more severe phenotype; subjects #2 and #9, for example, had onset of symptoms by the age of 8, but were still classified as a mild BMD-like phenotype into their fourth or fifth decade. Most MCs presented initially with mild proximal lower extremity weakness and/or myalgia/muscle cramps. Muscle weakness was usually expressed as having difficulty climbing stairs or running. On manual muscle testing (MMT), weakness might be subtle and initially detectable only in one or two proximal lower extremity muscle groups, and none of our subjects showed weakness only in the arms, in contrast to a previous report8
. Asymmetry in muscle weakness has previously been described in between 15% and 81% of MCs5, 8
. The higher number8
may reflect the use of hand-held dynamometry to compare right-left muscle groups. Asymmetry can also be detected by skeletal muscle MRI imaging15
, and may be related to somatic mosaicism16
. Among our subjects, asymmetric muscle weakness was clinically notable in only 3/9 (33%) of patients with available detailed muscle strength data. Among the remaining 6 subjects, with limited or no detailed muscle examination data, asymmetric features were not reported.
As in DMD and BMD, cardiomyopathy should always be considered in manifesting carriers. Consistent with a previous report that up to 36% of MCs may have echocardiographic evidence of cardiac dysfunction17
, we found that 5 out of the 13 (38%) subjects who had available echo reports showed evidence of impaired systolic function (shortening fraction <28% or ejection fraction <55%). A decline of cardiac function in carriers may be relatively acute, or related to pregnancy: subject #4 showed a 15% drop in her ejection fraction in one year (28–29 years old), and subject #12 developed a postpartum cardiomyopathy that improved with medical treatment. Although not seen in isolation among the cohort we report, cardiomyopathy may be the only clinical manifestation in DMD
carriers; therefore, dystrophinopathy should be considered in the differential diagnosis of female patients with idiopathic cardiomyopathy18, 19
Immunolabeling of muscle biopsies with anti-dystrophin antibodies was reported to show scattered or patchy presence of fibers with reduced or absent dystrophin in all biopsied subjects. No association is expected between the degree of altered dystrophin expression and clinical variables including strength and serum CK7
or clinical course14, 20
, and dystrophin expression can vary in different muscle groups of DMD
. Nevertheless, we note that the two subjects reported to have either no appreciable dystrophin expression (subject #6) or a majority of fibers unstained (#8) were in the more severe clinical phenotype category (severe BMD-like and DMD-like).
We report what is to our knowledge the first example of a manifesting carrier with presumably compound heterozygous DMD
mutations: a deletion of exons 8–13 and an intron 69 splice site mutation (c.10086+2T>C). The out-of-frame exon 8–13 deletion has been reported in association with both DMD and BMD phenotypes3
, whereas the c.10086+2T>C has been included in the Leiden database (www.dmd.nl
) as DMD. Genetic analysis of the subject's mother did not reveal either mutation, and despite repeated efforts we could not obtain a blood or tissue sample from her father. No further archived muscle tissue was available for mRNA analysis, and the family declined repeated requests for a skin biopsy. Allele-specific genotyping was not feasible due to unavailability of an archived muscle biopsy sample, and the long genomic distance between the two identified mutations. Given her random XCI pattern, her severe BMD-like phenotype, and the absence of appreciable dystrophin staining on her biopsy, we assume that her two DMD
mutations lie in trans
rather than in cis
. In the absence of a family history, and given her father's apparently normal phenotype, they presumably occurred as de novo
germ line mutations. Our genetic results otherwise confirm that DMD
mutational mechanisms in MCs are heterogeneous. Our database is enriched for point mutations9
, so we presume that the actual relative frequency of MCs with point mutations is lower in the general population than in our data set, and equal to the distribution of DMD
mutations in non-referral cohorts22
Nonrandom X-chromosome inactivation (XCI) pattern has been proposed as an explanation for the development of symptoms in manifesting carriers (MCs) without chromosomal translocations14, 20, 23
. In peripheral blood, most women in the population have a random XCI pattern; only 8% have a skewed pattern of >80:2013
. The ratio of peripheral blood XCI is significantly correlated with other tissues24, 25
, including muscle24
, although cases exist in which muscle and peripheral blood XCI results diverge23
. Because skewed XCI can explain the development of symptoms in many instances20, 23, 26
, we expected to find skewed XCI in some portion of our subjects. Indeed, one subject (#8) had 100% skewed XCI, in accordance with her severe DMD-like phenotype and the absence of dystrophin in the majority of muscle fibers by immunohistochemistry (). However, excluding the single subject with an apparent compound heterozygous mutation, we observed nonrandom XCI in 5/13 informative subjects (38%). Our results are consistent with a previous study that showed both highly skewed and completely random XCI patterns in carriers23
. Unfortunately, insufficient archived muscle tissue was available with which to determine inactivation in muscle at the AR locus.
The lack of a clear correlation between phenotype and the XCI pattern as detected in peripheral blood might be attributed to several factors. First, the assumption that the methylation status of the androgen receptor (AR) locus reflects that of the DMD
locus on the X chromosomes in muscle tissue might not be correct; furthermore, the phase between the DMD
and androgen receptor loci has not been established in these patients, such that we cannot formally know which DMD
allele has been inactivated. Second, the pattern of XCI as ascertained from lymphocyte DNA may not reflect the XCI pattern in muscle in all patients23
. Third, the phenotype may be influenced by early clonal expansion of a skewed XCI precursor cell population in muscle. Finally, myotube formation requires interactions among multiple myoblasts, and muscle is made up of multinucleated cells. The functionality of a muscle fiber may depend in part upon the percentage of non-random XCI nuclei in a fiber, or the distribution pattern of these nuclei within it.
Our data raise the question of a correlation between mutation class and XCI pattern in symptomatic carriers. For example, all four subjects with nonsense DMD mutations (all of whom showed a relatively mild BMD-like phenotype) had random XCI patterns. In contrast, among seven informative subjects with deletions or duplications, only three had a random XCI pattern. We note that the definition of non-random XCI is arbitrary, and that some studies define XCI that equals 80:20 as non-random (rather than a value greater than 80:20)27, 28
. If our two subjects with a ratio of 80:20 are categorized as non-random, the correlation between mutation category (duplications/deletions versus point mutations) and XCI reaches statistical significance among the informative subjects (p value = 0.029 by Fisher's Exact Test). However, if we make the assumption that the uninformative subject #5 has random XCI, the significance of the correlation disappears. In summary, our data fail to demonstrate a significant association, and we are unaware of a model in which a given mutation class on a disease-causing X chromosome would have the effect of inducing inactivation at an X-chromosome locus in trans.
Whether an actual correlation exists will need to be addressed in future studies with larger numbers of subjects.