GENETIC AND BIOCHEMICAL NORMALIZATION IN FEMALE CARRIERS OF DUCHENNE MUSCULAR-DYSTROPHY - EVIDENCE FOR FAILURE OF DYSTROPHIN PRODUCTION IN DYSTROPHIN-COMPETENT MYONUCLEI

We studied 19 symptomatic female carriers of the Duchenne muscular dys trophy (DMD) gene. Most of these dystrophinopathy patients had had an erroneous or ambiguous diagnosis prior to dystrophin immunofluorescenc e testing. We assessed clinical severity by a standardized protocol, m easured X-chromosome inactivation patterns in blood and muscle DNA, an d quantitated the dystrophin protein content of muscle. We found that patients could be separated into two groups: those showing equal numbe rs of normal and mutant dystrophin genes in peripheral blood DNA (''ra ndom'' X-inactivation), and those showing preferential use of the muta nt dystrophin gene (''skewed'' X-inactivation). In the random X-inacti vation carriers, the clinical phenotype ranged from asymptomatic to mi ld disability, the dystrophin content of muscle was >60% of normal, an d there were only minor histopathologic changes. In the skewed X-inact ivation patients, clinical manifestations ranged from mild to severe, but the patients with mild disease were young (5 to 10 years old). The low levels of dystrophin (<30% on average) and the severe symptoms of the older patients suggested a poor prognosis for those with skewed X -inactivation, and they all showed morphologic changes of dystrophy. T he random inactivation patients showed evidence of biochemical ''norma lization,'' with higher dystrophin content in muscle than predicted by the number of normal dystrophin genes. Seventy-nine percent of skewed X-inactivation patients (11/14) showed genetic ''normalization,'' wit h proportionally more dystrophin-positive nuclei in muscle than in blo od. In 65% of the skewed X-inactivation patients, dystrophin was not p roduced by dystrophin-positive nuclei; an average of 20% of myofiber n uclei were genetically dystrophin-positive but did not produce stable dystrophin. Biochemical normalization seems to be the main mechanism f or rescue of fibers from dystrophin deficiency in the random X-inactiv ation patients. In the skewed X-inactivation patients, genetic normali zation is active, but production failure of dystrophin by dystrophin-n ormal nuclei may counteract any effect of biochemical normalization. I n the skewed X-inactivation patients, the remodeling of the muscle thr ough cycles of degeneration and regeneration led to threefold increase in the number of dystrophin-competent nuclei in muscle myofibers (3.3 +/- 4.6), while dystrophin content was on the average 1.5-fold less t han expected (-1.54 +/- 3.38). Our results permit more accurate progno stic assessment of isolated female dystrophinopathy patients and provi de important data with which to estimate the potential effect of gene delivery (gene therapy) in DMD.