Muscular Dystrophy, Duchenne Type

A number sign (#) is used with this entry because Duchenne muscular dystrophy is caused by mutation in the gene encoding dystrophin (DMD; 300377).

Dystrophin-associated muscular dystrophies range from the severe Duchenne muscular dystrophy (DMD) to the milder Becker muscular dystrophy (BMD; 300376). Mapping and molecular genetic studies indicate that both are the result of mutations in the huge gene that encodes dystrophin, also symbolized DMD. Approximately two-thirds of the mutations in both forms are deletions of one or many exons in the dystrophin gene. Although there is no clear correlation found between the extent of the deletion and the severity of the disorder, DMD deletions usually result in frameshift. Boland et al. (1996) studied a retrospective cohort of 33 male patients born between 1953 and 1983. The mean age at DMD diagnosis was 4.6 years; wheelchair dependency had a median age of 10 years; cardiac muscle failure developed in 15% of patients with a median age of 21.5 years; smooth muscle dysfunction in the digestive or urinary tract occurred in 21% and 6% of the patients, respectively, at a median age of 15 years. In this cohort, death occurred at a median age of 17 years. The authors commented that the diagnosis of DMD is being made at an earlier age but survival has not changed.

Skeletal Muscle

The most distinctive feature of Duchenne muscular dystrophy is a progressive proximal muscular dystrophy with characteristic pseudohypertrophy of the calves. The bulbar (extraocular) muscles are spared but the myocardium is affected. There is massive elevation of creatine kinase levels in the blood, myopathic changes by electromyography, and myofiber degeneration with fibrosis and fatty infiltration on muscle biopsy.The onset of Duchenne muscular dystrophy usually occurs before age 3 years, and the victim is chairridden by age 12 and dead by age 20. The onset of Becker muscular dystrophy is often in the 20s and 30s and survival to a relatively advanced age is frequent.

Moser and Emery (1974) found that some female heterozygotes had myopathy resembling autosomal recessive limb-girdle muscular dystrophy (253600). Serum creatine kinase was particularly elevated in these patients. In most populations, the frequency of manifesting heterozygotes is about the same as that of females with limb-girdle muscular dystrophy.

Soloway and Mudge (1979) remarked that patients with advanced muscular dystrophy may develop hypokalemia from insults (vomiting, diarrhea, diuretics) that would have little effect on normal persons. Reduced intracellular potassium stores are responsible for this perilous situation, which may be the mechanism of death.

In an Italian boy with congenital myopathy, born to nonconsanguineous parents, Prelle et al. (1992) found absence of dystrophin in the patient's muscle by immunohistochemical methods and a deletion of the 5-prime end of the dystrophin gene. Although the patient showed severe mental retardation, there was no cerebral atrophy. Cardiomyopathy was also present.

Frigeri et al. (1998) analyzed AQP4 expression in the skeletal muscle of mdx mice; immunofluorescence experiments showed a marked reduction of aquaporin-4 (AQP4; 600308) expression, suggesting a critical role in the membrane alteration of DMD.

Wakayama et al. (2002) analyzed skeletal muscle samples from 6 patients with DMD and found markedly reduced AQP4 expression by immunohistochemical staining and markedly decreased levels of AQP4 mRNA as measured by RT-PCR, compared to controls. Genomic analysis of the AQP4 gene revealed no abnormalities. The authors concluded that the reduced mRNA was due to either decreased transcription or increased degradation of the message.

Noguchi et al. (2003) performed cDNA microarray analysis on skeletal muscle biopsy specimens from 6 patients with DMD. There was increased expression of genes related to immune response, sarcomere, extracellular matrix proteins, and signaling or cell growth. Upregulation of these genes reflected dystrophic changes, myofiber necrosis, inflammation, and muscle regeneration. Genes related to muscle homeostasis and energy metabolism were downregulated.

Cardiac Muscle

Myocardial involvement appeared in a high percentage of DMD patients by about 6 years of age; it was present in 95% of cases by the last years of life. Severe cardiomyopathy did not develop before age 21 in BMD and few patients showed any cardiac signs before age 13 (Nigro et al., 1983).

Mirabella et al. (1993) noted that electrocardiographic abnormalities had been reported in 6.6 to 16.4% of DMD heterozygous females and that in one carrier female severe cardiomyopathy had been described in association with muscle weakness. They reported 2 carriers with dilated cardiomyopathy and increased serum CK but no symptoms of muscle weakness. Heart biopsies in both patients showed absence of dystrophin in many muscle fibers.

Smooth Muscle

Noting that in DMD functional impairment of smooth muscle in the gastrointestinal tract can cause acute gastric dilatation and intestinal pseudoobstruction that may be fatal, Barohn et al. (1988) studied gastric emptying in 11 patients with DMD. Strikingly delayed gastric emptying times were observed.

Enigmatically, the extraocular muscles (EOMs) remain clinically unaffected during the course of Duchenne muscular dystrophy (Kaminski et al., 1992). Khurana et al. (1995) showed that dystrophin deficiency does not result in myonecrosis or pathologically elevated levels of intracellular calcium in the EOMs. They reported in vitro experiments demonstrating that extraocular muscles are inherently more resistant to necrosis caused by pharmacologically elevated intracellular calcium levels when compared with pectoral musculature. They suggested that the EOMs are spared in DMD because of their intrinsic ability to maintain calcium homeostasis better than other striated muscle groups. This suggested further that modulating levels of intracellular calcium in muscle may be of potential therapeutic use in DMD.

Nervous System

Mental retardation of mild degree is a pleiotropic effect of the Duchenne gene (Zellweger and Niedermeyer, 1965). As indicated later, the finding of dystrophin mRNA in brain may bear a relationship to the mental retardation in DMD patients. Emery et al. (1979) sought heterogeneity in DMD as one explanation for the high birth incidence. Affected boys were categorized according to whether they had severe mental handicap or not. Those with severe mental defect had later age of onset and confinement to wheelchair, less marked fall in creatine kinase with age, and a greater urinary excretion of certain amino acids. In 50 DMD patients with a mean age of 11.1 years (range 3.5 to 20.3), Bresolin et al. (1994) found that 31% had a Wechsler full intelligence quotient (FIQ) lower than 75 and that only 24% had appropriate IQ levels by this index.

Bushby et al. (1995) examined the hypothesis that the nature of the dystrophin mutation may influence the development of mental retardation. Previously, it had been shown that deletions removing the brain-specific promoter were compatible with normal intelligence. Bushby et al. (1995) studied 74 boys with DMD, 18% of which had a full scale IQ of below 70. The authors found no significant IQ difference between the patients with promoter deletions and those without, nor did they find a relationship between the length of the deletion and full scale IQ. They found, however, that boys with distal deletions were more likely to be mentally retarded than were those with proximal deletions.

Retinal Function

Abnormal retinal neurotransmission as measured by electroretinography (ERG) was observed in boys with DMD by Cibis et al. (1993) and Pillers et al. (1993). Electroretinography is a recording of summed electrical signal produced by the retina when stimulated with a flash of light. The dark-adapted ERGs, recorded under scotopic testing conditions, have shown normal a-waves (a response of negative polarity generated by the photoreceptors) but reduced amplitude rod-isolated b-waves (a response of positive polarity originating primarily from the ON-bipolar cells) in DMD patients. This type of ERG abnormality with profound b-wave suppression is commonly associated with night blindness; however, there have been no reports of night blindness or any other visual abnormality in boys with DMD, and dark-adaptometry studies have been normal. Fitzgerald et al. (1994) used long-duration stimuli to separate ON (depolarizing bipolar cell) and OFF (hyperpolarizing bipolar cell) contributions to the cone-dominated ERG to understand better how the retina functions in boys with DMD. In the ERGs of 11 DMD boys, they found abnormal signal transmission at the level of the photoreceptor and ON-bipolar cell in both the rod and cone generated responses. Jensen et al. (1995) examined 16 boys with DMD/BMD of whom 10 had negative ERGs. Eight of the boys had DMD gene deletions downstream from exon 44. Normal dark adaptation thresholds were observed in all patients and there were no anomalous visual functions. Hence, negative ERG in DMD/BMD is not associated with eye disease. Normal ERGs were found in 6 boys with DMD/BMD. Jensen et al. (1995) speculated that a retinal or glial dystrophin may be truncated or absent in the boys with negative ERGs.

The ophthalmic features of DMD include normal ERG a-wave with reduced b-wave, normal visual acuity, and normal retinal morphology. Immunocytochemistry revealed strong AQP4 water channel expression in Muller cells in mouse retina and in fibrous astrocytes in optic nerve. Li et al. (2002) compared ERGs and retinal morphology in wildtype mice and transgenic knockout mice with no Aqp4. Significantly reduced ERG b-wave potentials were recorded in 10-month-old null mice with smaller changes in 1-month-old mice. Morphologic analysis of retina by light and electron microscopy showed no differences in retinal ultrastructure. That retinal function was mildly impaired in Aqp4-null mice suggested a role for Aqp4 in Muller cell fluid balance. The authors suggested that AQP4 expression in supportive cells in the nervous system facilitated neural signal transduction in nearby electrically excitable cells.

Costa et al. (2007) evaluated color vision in 44 patients with Duchenne muscular dystrophy using 4 different color tests. Patients were divided into 2 groups according to the region of deletion in the dystrophin gene: 12 patients had deletion upstream of exon 30, and 32 downstream of exon 30. Of the patients with DMD, 47% (21/44) had a red-green color vision defect. Of the patients with deletion downstream of exon 30, 66% had a red-green color defect. No color defect was found in the patients with a deletion upstream of exon 30. A negative correlation between the color thresholds and age was found for the controls and patients with DMD, suggesting a nonprogressive color defect. The percentage (66%) of patients with red-green defect was significantly higher than the expected value (less than 10%) for the normal male population (P less than 0.001). Costa et al. (2007) suggested that the findings might be partially explained by a retinal impairment related to dystrophin isoform Dp260.

Carrier Females

In a 9-year follow-up of study of 99 Dutch female carriers of DMD or BMD mutations, Schade van Westrum et al. (2011) found that 11 carriers (10%) (10 DMD and 1 BMD) fulfilled the criteria for dilated cardiomyopathy (DCM). Nine of the patients had developed DCM during the follow-up period. These carriers were on average older, were more symptomatic, and more often had hypertension, exertional dyspnea, and chest pain compared to mutation carriers without DCM. The findings suggested that female carriers of a mutation can develop progressive cardiac abnormalities and should undergo routine cardiac evaluation, preferably by echocardiology.

Mercier et al. (2013) reviewed the features of 26 female carriers of pathogenic mutations in the DMD gene who were referred for symptoms related to the disorder before 17 years of age. Five had a Duchenne-like phenotype with loss of ambulation before age 15 years, 13 had a Becker-like phenotype with muscle weakness but persistence of ambulation after age 15 years, and 8 had exercise intolerance. Initial symptoms included significant muscle weakness (88%), mostly affecting the lower limbs, or exercise intolerance (27%). Cardiac dysfunction was present in 19%, and cognitive impairment in 27%. Cognitive impairment was associated with mutations in the distal part of the gene. Muscle biopsy showed dystrophic changes in 83% and mosaic immunostaining for dystrophin in 81%. The X-chromosome inactivation pattern was biased in 62% of cases. Mercier et al. (2013) concluded that carrier females may have significant symptoms of the disorder.

Blau et al. (1983) suggested that the defect in DMD is intrinsic to the undifferentiated myoblast. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had a decreased proliferative capacity and aberrant morphology. The hypothesis was tested by determining whether the myoblast defect was X-linked. Webster et al. (1986) obtained muscle cells from 5 females heterozygous for both DMD and G6PD (305900). In a total of 1,355 muscle clones, although the proportion of defective clones was increased, the cellular defect did not consistently segregate with a single G6PD phenotype in the myoblast clones from any individual. The hypothesis that the DMD gene is expressed in skeletal myoblasts and limits proliferation, was further tested by Hurko et al. (1987) established primary muscle culture from a female who was heterozygous for both DMD and G6PD. Both cloned and mass cultures were grown until senescence and the G6PD phenotype was scored. Myoblasts expressing the 2 different alleles at the G6PD locus did not differ in proliferative capacity, suggesting that expression of the Duchenne gene does not result in a decrease in proliferative capacity of the myoblasts. Thus, the hypothesis of Blau et al. (1983) was disproved.

Baricordi et al. (1989) studied the capping phenomenon in lymphoblastoid cell lines and found that they retain an impairment of capping of the type seen in nontransformed lymphocytes (Verrill et al., 1977). This was taken to mean that the capping impairment is an intrinsic cellular defect in DMD and not a phenomenon secondary to progression or activity of the disease. Further, it may indicate that there is a generalized membrane disorder in this condition.

Haslett et al. (2002) used expression microarrays to compare individual gene expression profiles of skeletal muscle biopsies from 12 DMD patients with those of 12 unaffected control patients. They identified 105 genes that differed significantly in expression levels between unaffected and DMD muscle. Many of the differentially expressed genes reflected changes in histologic pathology; e.g., immune response signals and extracellular matrix genes were overexpressed in DMD muscle, indicating the infiltration of inflammatory cells and connective tissue. Significantly more genes were overexpressed than were underexpressed in dystrophic muscle, with dystrophin underexpressed, whereas other genes encoding muscle structure and regeneration processes were overexpressed, reflecting the regenerative nature of the disease.

Straub et al. (2002) found impaired expression of muscle membrane-associated neuronal nitric oxide synthase (NOS1; 163731) in Duchenne patients; mean exhaled nitric oxide was significantly reduced in 13 males with DMD compared to 11 age-matched and 17 adult controls.

In muscle biopsy samples from 13 of 16 DMD patients, Kleopa et al. (2006) observed an age-dependent increase in utrophin (UTRN; 128240) staining, resulting in a mean increase of 11-fold compared to that found in normal adult tissue. In disease tissue, utrophin was present along the whole circumference of the sarcolemma, whereas it was present only along vessels and nerve endings in controls. Expression of utrophin in disease tissue showed a positive correlation with age at wheelchair-dependency in DMD, suggesting that utrophin expression has an ameliorating effect on the severity of DMD.

Patients with DMD have increased blood loss during spinal surgery compared to non-DMD patients. In Duchenne patients, Labarque et al. (2008) found decreased expression of the dystrophin isoforms Dp71 and Dp116 in platelets and skin fibroblasts, respectively, compared to controls. Decreased expression of these isoforms was associated with increased Gs (see, e.g., GNAS; 139320) signaling and activity upon stimulation. Functional studies showed that DMD platelets had slower aggregation in response to collagen with extensive shape changes and reduced platelet adhesion under flow conditions. Platelet membrane receptors were normal. The decreased collagen activation was shown to result from both Gs activation and cytoskeletal disruption. Overall, the findings suggested that DMD platelets have a disorganized cytoskeleton due to dysfunctional dystrophin Dp71, and also manifest Gs hyperactivity with reduced platelet collagen reactivity, which may result in increased bleeding during surgery.

The Haldane rule (Haldane, 1935) predicts that one-third of cases of a genetic lethal X-linked recessive will be the consequence of new mutation. Haldane (1956) further suggested that the mutation rate for Duchenne muscular dystrophy might be higher in males. Such would result in a lower proportion of cases being new mutants. Caskey et al. (1980) concluded that in their series cases resulting from new mutation approached closely the theoretically expected one-third. Ionasescu et al. (1980) concluded that measures of ribosomal protein synthesis, analyzed by discriminant function, identify 95% of proved and presumptive DMD carriers. Bucher et al. (1980) used this measure to test the Haldane rule. They found that only 9 (16.4%) of 55 mothers were noncarriers. When only the mothers of isolated cases were studied, 23.1% (9 of 39) were classified as noncarriers. They felt that a higher male than female mutation rate was the cause of the discrepancy.

In a study of 514 probands who constituted two-thirds of the known cases in Japan, Yasuda and Kondo (1982) could not demonstrate an effect of maternal grandfather's age at birth of the proband's mother. They pointed out that the data relevant to a maternal grandfather age effect in hemophilia A are conflicting, just as the data for DMD are inconsistent with those of Bucher et al. (1980). Examining the frequency of affected boys among the next-born male sibs of 37 initially 'sporadic' cases of DMD, Lane et al. (1983) found that the frequency was significantly greater than predicted by the Haldane theory (p = 0.029). The estimated proportion of new mutant cases in the combined clinic population of 106 families was 0.127 (SE = 0.111). They proposed that the absence of affected males in earlier generations in families of isolated cases may be explained in part by a high ratio of male to female stillbirths and infant deaths which in this study was more than 3 times that in the general population. (Note that there is at least one other 'Haldane's rule' (Haldane, 1922): 'When in the F1 offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous, heterogametic or XY sex.' See discussion of Orr (1993).)

Danieli and Barbujani (1984) concluded that the proportion of sporadic cases was 0.227 +/- 0.048 in an Italian series of 135 families combined with other sets of data. In a segregation analysis of 1,885 DMD families, Barbujani et al. (1990) arrived at an estimate of sporadic cases of 0.229, a significant deviation from the expected 0.333 based on mutation-selection equilibrium. They mentioned the previously discussed possible explanations for the finding, such as sex differences in mutation rate, and added a new one, namely, the occurrence of multiple DMD cases in the same sibship as a consequence of mutational mosaicism of the maternal germ cells, a phenomenon documented in a number of instances.

As might perhaps have been anticipated, a report appeared concerning a man with DMD who had fathered 2 children, a normal son and a carrier daughter (Thompson, 1978).

By analysis of Xp21 DNA markers in a family with 2 affected brothers, Borresen et al. (1987) demonstrated that the mutation had most likely occurred in a grandpaternal sperm. Therefore, barring gonadal mosaicism, it is unlikely that the maternal aunts and their daughters are carriers of the DMD gene.

Miciak et al. (1992) studied 3 boys with DMD, 2 of whom were related as first cousins and the third as a second cousin, all being related through males. They demonstrated that the molecular defect was different in each and speculated about instability of the DMD gene and possible involvement of transposons. They referred to similar observations by Zatz et al. (1991) in 4 Brazilian families. Vitiello et al. (1992) found no instance of mutation in the muscle promoter region of the DMD gene in a series of 115 unrelated DMD and BMD patients from northeast Italy. In 3 cases in which dystrophin of normal size was expressed at low levels, the DNA sequence of the promoter region showed no abnormality.

Gonadal Mosaicism

A possible example of gonadal mosaicism for the DMD locus was discussed by Wood and McGillivray (1988), who described a family in which a female ancestor of an individual with Duchenne muscular dystrophy seemed to have transmitted 3 distinct types of X chromosome to her offspring, as indicated by RFLP analysis. The authors postulated that in this individual the mutation arose as a postzygotic deletion, resulting in germinal mosaicism.

Witkowski (1992) suggested another explanation for those cases in which gonadal mosaicism has been suspected: such a female may represent a chimera that has originated from 2 fertilized eggs, one carrying the mutation. This, of course, has quite different implications regarding the risk that a maternal aunt of the proband is a carrier. Melis et al. (1993) reported a 3-generation family in which 2 sibs were affected with DMD. Immunohistochemical analysis of muscle dystrophin and haplotype analysis of the DMD locus demonstrated that the X chromosome carrying the DMD gene was transmitted from the healthy maternal grandfather to his 3 daughters, including the proband's mother. The definition of carrier status in 2 possible carriers permitted accurate genetic counseling and the prevention of the birth of an affected boy.

Witkowski (1992) presented the pedigree of a family with a balanced autosomal translocation in 3 generations: a son of a carrier exhibited lymphocytes with a normal karyotype as well as lymphocytes with the balanced translocation. She also cited the 47,XXX karyotype as a possible alternative explanation to germline mosaicism; there are known sibships in which boys have received 3 different haplotypes on the X chromosome from the mother. Unexpectedly, Passos-Bueno et al. (1992) observed that among 24 proven germline mosaic cases, 19 (79%) had a proximal mutation, while only 5 (21%) had a distal mutation.

Somatic Mosaicism and Heterozygous Females

Yoshioka (1981) observed unusually severely affected heterozygous females and suggested that factor(s) other than lyonization may be involved. One of the women was the product of a consanguineous mating, suggesting modification of expression by homozygosity at an autosomal locus.

Burn et al. (1986) reported monozygotic twin girls, one of whom had typical clinical features of DMD despite a normal female karyotype and the second of whom was normal. Burn et al. (1986) proposed that differences in lyonization accounted for the findings. Hybridization of fibroblasts from each twin with RAG-mouse cell line deficient in HPRT showed that in the affected twin it was the mother's X chromosome that was predominantly the active one, whereas in the normal twin it was the father's. In female monozygotic twins discordant for muscular dystrophy, Richards et al. (1990) showed that there was a mutation in dystrophin in both twins. Uniparental disomy and chromosome abnormality were excluded, but on the basis of methylation differences of the paternal and maternal X chromosomes, Richards et al. (1990) concluded that uneven lyonization was the underlying mechanism for disease expression in the affected female.

Lupski et al. (1991) pointed out that discordance of the DMD phenotype had never been described in male monozygotic twins. Lupski et al. (1991) described female monozygotic twins who carried the same mutation involving duplication of exons 42 and 43 of the DMD gene. One was a manifesting heterozygote, whereas the other was normal. Unlike the study of Richards et al. (1990) in which the skewed inactivation pattern was symmetrical in opposite directions, one twin being affected with DMD and the other being normal, the skew in this case involved only the affected twin, while the normal twin showed a random X-inactivation pattern. They suggested that the result was consistent with the model of twinning and X-inactivation proposed by Nance (1990) in that these twins probably represented asymmetric splitting of the inner cell mass (ICM): the affected twin probably arose when a small proportion of the ICM split off after lyonization had occurred. In this situation, the original ICM could have given rise to the normal twin with random lyonization, while the newly split cells would experience catch-up growth and lead to the affected twin.

Many DMD patients have rare staining dystrophin-positive fibers. The possibility of somatic mosaicism can be raised, but somatic reversion/suppression is another possibility. Indeed, the dystrophin-positive fibers have been referred to as 'revertants.' The revertants are found in both familial and nonfamilial cases. Klein et al. (1992) found that in patients with deletions, revertants did not stain with antibodies raised to polypeptide sequences within the deletion. These results indicated that positively stained fibers were not the result of somatic mosaicism in deletion patients. Klein et al. (1992) concluded that the most likely mechanism giving rise to positively staining fibers is a second site in-frame deletion. Thanh et al. (1995) used exon-specific monoclonal antibodies to determine which exons are removed in order to correct the reading frame in individual revertant muscle fibers. They showed that 15 revertant fibers in a DMD patient with a frameshift deletion of exon 45 had correction of the frameshift by the additional deletion of exon 44 (or perhaps exon 46 in some fibers) from the dystrophin mRNA, but not by larger deletions. This result was consistent with RT-PCR and sequencing of a minor dystrophin mRNA with an exon 43/46 junction in the biopsy. The results were consistent with somatic mutations in revertant-fiber nuclei, which result in removal of additional exons from dystrophin mRNA. These data did not clearly distinguish between additional somatic deletions and somatic effects on dystrophin mRNA splicing, however, and both mechanisms may be operating.

Pena et al. (1987) reported an extraordinary case of DMD leading to death at age 28 years in a heterozygous monozygotic female twin. Her sister was clinically normal but had an affected son. Eleven affected males in 3 generations and 7 separate sibships of the kindred were known. An undetected monozygotic twinning event was proposed by Glass et al. (1992) to explain a manifesting female for Becker muscular dystrophy. They concluded that females heterozygous for BMD have less likelihood of showing manifestations of muscular dystrophy than do females heterozygous for DMD. Abbadi et al. (1994) reported a pair of female monozygotic twins heterozygous for a deletion in the DMD gene and discordant for the clinical manifestations of the disorder. Results in lymphocytes and skin fibroblast cell lines suggested a partial mirror inactivation with the normal X chromosome preferentially active in the unaffected twin, and the maternally deleted X chromosome preferentially active in the affected twin.

Pegoraro et al. (1994) studied 13 female dystrophinopathy patients--10 isolated cases and 3 with a positive family history for DMD in males. All 13 had skewed X-inactivation patterns in peripheral blood DNA. Of the 9 isolated cases informative in their assay, 8 showed inheritance of the dystrophin gene mutation from the paternal germline. Only a single case showed maternal inheritance. Pegoraro et al. (1994) estimated that the 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus they suggested that there is some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation.

Chelly et al. (1986) reported the first observation of a girl with typical DMD and typical 45,XO Turner syndrome. The one X chromosome in the girl was normal by high resolution banding, but DNA analysis by Southern blotting and hybridization with 7 cloned probes mapping in the Xp21 region showed a deletion of 3 of the probes. In this case, the paternal chromosome was lost and the maternal X chromosome suffered a deletion mutation in the Xp21.2 region. Suthers et al. (1989) described a man with Becker muscular dystrophy and the Klinefelter syndrome who was much more mildly affected than his 3 nephews. The mild expression may be due to the fact that he was heterozygous for the muscular dystrophy mutation. The nephews indeed may have had Duchenne muscular dystrophy.

Among 35 children produced by 34 deliveries in 13 women who were mothers of males attending a muscular dystrophy clinic, Geifman-Holtzman et al. (1997) found that 6 (17%) were delivered in the breech position, which is a 5-fold increase above the national standards for term pregnancies. Of the 6 infants with breech presentation, 2 were males affected with DMD, 1 was a female heterozygote, 1 was a male who died perinatally, and the carrier status of the other 2 females was unknown. Most DMD affected males (12/14) were delivered in the vertex position. Thus, the authors concluded that maternal rather than fetal muscle weakness was the significant factor in determining fetal position at term. They suggested that subtle changes in uterine or pelvic girdle muscle tone may contribute to a higher rate of fetal breech presentation in carriers of the DMD gene.

Yoshioka et al. (1998) analyzed X inactivation in 4 manifesting heterozygotes, 5 asymptomatic carriers, and 32 female controls. Ninety-two percent were heterozygous for the CAG repeat in the androgen receptor (AR; 313700) gene. All manifesting carriers showed 70 to 93% skewed inactivation, whereas the asymptomatic carriers showed random inactivation (50-60%). Of the control females, 6% showed greater than 70% skewed inactivation.

Reported genetic mechanisms for female DMD include (1) a skewed pattern of X-chromosome inactivation in female carriers of a DMD mutation (Azofeifa et al., 1995); (2) X;autosome translocations that disrupt the DMD gene (Cantagrel et al., 2004); (3) monosomy X, or Turner syndrome, associated with a DMD mutation in the remaining X chromosome (Chelly et al., 1986); and (4) maternal isodisomy for the X chromosome carrying a DMD mutation (Quan et al., 1997). Katayama et al. (2006) reported a fifth mechanism in a Vietnamese child with DMD confirmed by genetic analysis. Although the child was phenotypically female, the karyotype showed 46,XY, and she was found to have a mutation in the AR gene causing androgen insensitivity syndrome (AIS; 300068). The patient's sister also had the AR mutation and AIS, but did not have the DMD mutation. The unaffected mother was found to be heterozygous for the AR mutation, but did not have the DMD mutation, indicating it was de novo in the proband. Katayama et al. (2006) concluded that the cooccurrence of independent mutations in both the DMD and AR genes constituted a fifth mechanism underlying female DMD.

Rajakulendran et al. (2010) reported 2 unrelated female carriers of DMD mutations who presented in adulthood with marked right-sided hemiatrophy and weakness of the arm and leg muscles. MRI showed muscle atrophy and fatty replacement on the affected side, and histologic studies showed decreased dystrophin staining. Both had increased serum creatine kinase. The older woman had areflexia of the affected side, no family history of muscular dystrophy, and showed skewed ratio of X inactivation in lymphocytes. The younger woman had an affected son and showed normal X inactivation in lymphocytes. Rajakulendran et al. (2010) suggested that a combination of skewed X inactivation in muscle tissue and somatic mosaicism accounted for the marked asymmetric manifestations.

Greenstein et al. (1977) found DMD in a 16-year-old girl with a reciprocal X;11 translocation. The mother was thought not to be a carrier. Possibly the break at Xp21 caused a null mutation; the normal X chromosome was inactivated. Verellen et al. (1978) reported the same situation with X;21 translocation and break at Xp21. Canki et al. (1979) described similar findings in a girl with X;3 translocation with break at Xp21. The mother was thought to be heterozygous.

Zneimer et al. (1993) used a combination of conventional and molecular cytogenetic techniques to investigate the twins first reported by Richards et al. (1990). The twins carried a deletion of approximately 300 kb within the dystrophin gene on one X chromosome. A unique DNA fragment generated from an exon within the deletion was hybridized in situ to metaphase chromosomes of both twins, a probe that would presumably hybridize only to the normal X chromosome and not to the X chromosome carrying the deletion. The chromosomes were identified by reverse-banding (R-banding) and by the addition of 5-bromodeoxyuridine in culture to distinguish early and late replicating X chromosomes, corresponding to active and inactive X chromosomes, respectively. The experiment showed predominant inactivation of the normal X chromosome in the twin with DMD. With an improved method of high resolution R-banding, Werner and Spiegler (1988) showed deletion of Xp21.13 in an 8-year-old boy with normal intelligence and no disorder other than DMD. His healthy mother was heterozygous for the deletion, which was subject to random X inactivation in lymphocytes.

Saito-Ohara et al. (2002) studied a 16-year-old patient with Duchenne muscular dystrophy, profound mental retardation, athetosis, and nystagmus who was shown to have a pericentric inversion of the X chromosome, 46,Y,inv(X)(p21.2q22.2). His mother carried this inversion on one allele. The patient's condition was originally misdiagnosed as cerebral palsy. Because the DMD gene is located at Xp21.2, which is one breakpoint of the inv(X), and because its defects are rarely associated with severe mental retardation, the other clinical features of this patient were deemed likely to be associated with the opposite breakpoint at Xq22. The molecular-cytogenetic characterization of both breakpoints revealed 3 genetic events that probably had disastrous influence on neuromuscular and cognitive development: deletion of part of the DMD gene at Xp21.2, duplication of the proteolipid protein gene (PLP1; 300401) at Xq22.2, and disruption of the RAB40AL gene (300405). Saito-Ohara et al. (2002) speculated that disruption of RAB40AL was responsible for the patient's profound mental retardation.

Tran et al. (2013) reported a 3-year-old Japanese boy with Duchenne muscular dystrophy and moderate mental retardation associated with an intrachromosomal inversion, inv(X)(p21.2;q28), involving both the dystrophin and the KUCG1 (300892) genes. KUCG1 is a long noncoding RNA that shows brain expression. The first exon of KUCG1 was spliced to a dislocated part of the dystrophin gene, producing a chimeric dystrophin transcript. Brain MRI in the patient was normal. Tran et al. (2013) hypothesized that interruption of the KUCG1 gene may have contributed to mental retardation in this patient. However, sequencing of the KUCG1 gene in 10 additional Japanese families with X-linked mental retardation did not identify any mutations.

Duchenne muscular dystrophy is not linked to colorblindness or G6PD (Emery et al., 1969; Zatz et al., 1974). No linkage with Xg has been found; total lod scores were -14.6 and -2.4 for theta of 0.10 and 0.30, respectively (Race and Sanger, 1975).

Lindenbaum et al. (1979) found DMD with X-1 translocation and suggested that the DMD locus is at Xp1106 or Xp2107. A number of females with X-autosome translocations with the breakpoint in the Xp21 band have shown Duchenne muscular dystrophy. One interpretation is that the gene locus is in that region and that the locus on the normal X is inactivated. Murray et al. (1982) found linkage of DMD with a restriction enzyme polymorphism at a distance of about 10 cM. The cloned DNA sequence bearing the polymorphism (lambda RC8) was assigned to Xp22.3-p21 by study of somatic cell hybrids. Spowart et al. (1982) outlined reasons for doubting the location of the DMD gene at Xp21.

Wieacker et al. (1983) studied the linkage between the restriction fragment length polymorphism defined by the cloned DNA sequence RC8 and X-linked ichthyosis. At least 2 crossovers were found among 9 meioses in an informative family, suggesting that RC8 and STS may be about 25 cM apart. Since STS is 15 cM proximal to the Xg locus and since the RC8 and Duchenne muscular dystrophy are closely linked, DMD may be 50 cM or more from Xg. Worton et al. (1984) studied a female with DMD and an X;21 translocation which split the block of genes encoding ribosomal RNA on 21p. Thus, ribosomal RNA gene probes can be used to identify a junction fragment from the translocation site and to clone segments of the X at or near the DMD locus.

Kingston et al. (1983, 1984) found linkage of BMD with the cloned sequence L1.28 (designated DXS7 by the seventh Human Gene Mapping Workshop in Los Angeles; D = DNA, X = X chromosome, S = segment, 7 = sequence of delineation). The interval was estimated to be about 16 cM, which is also the approximate interval between DXS7 and DMD. DXS7 is located between Xp11.0 and Xp11.3. Thus, these 2 forms of X-linked muscular dystrophy appeared to be allelic, a possibility also supported by the finding of both severe and mild disease (Duchenne and Becker, if you will) in females with X-autosome translocations. Contrary to reports of others, Kingston et al. (1984) found no evidence of linkage of BMD to colorblindness; Xg also showed no linkage.

Francke et al. (1985) studied a male patient with 3 X-linked disorders: chronic granulomatous disease with cytochrome b(-245) deficiency and McLeod red cell phenotype, Duchenne muscular dystrophy, and retinitis pigmentosa (see RP3, 300029). A very subtle interstitial deletion of part of Xp21 was demonstrated as the presumed basis of this 'contiguous gene syndrome.' That this was a deletion and not a translocation was demonstrated by the absence of 1 DNA probe from the genome of the patient. Since this probe (called 754) was clearly very close to DMD and recognized a RFLP of high frequency, it proved highly useful for linkage studies of DMD. The close clustering of CGD, DMD, and RP suggested by these findings was inconsistent with separate linkage data, which indicated that McLeod and CGD were close to Xg and that DMD and RP are far away (perhaps at least 55 cM) and as much as 15 cM from each other. At least 4 possible explanations of the discrepancy were proposed by Francke et al. (1985). One suggestion was that the deletion contained a single defect affecting perhaps a cell membrane component with the several disorders following thereon.

Mulley et al. (1988) reported the recombination frequencies between DMD and intragenic markers from 8 informative families containing 30 informative meioses. No recombinants were observed. The authors commented that the average theta between intragenic markers and DMD may be 1 to 2%. Grimm et al.(1989) reported a recombination rate of 4% between 2 subclones of the DNA segment DXS164 within the dystrophin locus, indicating a hotspot for recombination.

Tuffery-Giraud et al. (2009) described a French database for mutations in the DMD gene that includes 2,411 entries consisting of 2,084 independent mutation events identified in 2,046 male patients and 38 expressing females. This corresponds to an estimated frequency of 39 per million with a genetic diagnosis of a 'dystrophinopathy' in France. Mutations in the database include 1,404 large deletions, 215 large duplications, and 465 small rearrangements, of which 39.8% are nonsense mutations. About 24% of the mutations are de novo events. The true frequency of BMD in France was found to be almost half (43%) that of DMD.

Among 624 index cases evaluated for DMD mutations, Oshima et al. (2009) reported that a genomic rearrangement was detected in 238 (38.1%) samples. Deletions were detected in 188 (79.0%), and included 31 cases with single-exon deletions and 157 cases with multi-exonic deletions. Most of the deletions fell between exons 45 and 52 and between exons 8 and 13 of the gene. Duplications were detected in 44 (18.5%) cases, of which 12 involved single exons and 32 multiple exons. Complex rearrangements were detected in 6 (2.5%) cases. The remaining 386 cases showed normal results. Oshima et al. (2009) selected 15 unique rearrangement, of which none shared a common breakpoint, and used array CGH and MLPA analyses to evaluate the mechanism rearrangements. Fourteen of the deletions had microhomology and small insertions at the breakpoints, consistent with a mechanism of nonhomologous end joining (NHEJ) after DNA damage and repair. Analysis of 3 complex intragenic DMD gene rearrangements identified several features that could result in genomic instability, including breakpoints that aligned with repetitive sequences, an inversion/deletion involving a stem-loop structure, replication-dependent fork stalling and template switching (FoSTeS), and duplications causing secondary deletions.

Modifier Genes

Pegoraro et al. (2011) examined 106 DMD patients for variations in 29 genes selected as candidate modifiers of disease severity. Skeletal muscle mRNA profiling identified the G allele of rs28357094 in the promoter of the SPP1 gene (166490), which encodes osteopontin, as having a significant effect on both disease progression and response to glucocorticoids. In an autosomal dominant model, carriers of the G allele (35% of subjects) had more rapid progression and 12 to 19% less grip strength. The association was validated in a second cohort of 156 patients.

Symptomatic Hemizygotes

Clinical diagnosis of males affected with DMD is straightforward. Gait difficulty beginning at age three, progressive myopathic weakness with pseudohypertrophy of calves and massive elevations of serum levels of creatine kinase permit diagnosis. Electromyography and muscle biopsy are confirmatory. Inflammatory changes seen in biopsies taken early in the course of the disorder can erroneously suggest a diagnosis of polymyositis if careful note is not made of the histologic hallmarks of dystrophy.

Heyck et al. (1966) documented a high level of CPK (and other enzymes) in a 9-day-old infant from a family at risk. According to Dubowitz (1976), elevation in cord blood in a proven case had not been documented. Furthermore, many perinatal factors seem to cause elevation of CPK. Mahoney et al. (1977) demonstrated elevated CPK in fetal blood obtained by placental puncture and validated this as a method of prenatal diagnosis by demonstrating histologic changes in the skeletal muscle of the aborted fetus.

Darras et al. (1987) reported experience suggesting that despite the large number of intragenic and flanking DNA polymorphisms then available, uncertainties often remain in the prenatal diagnosis of DMD.

Bartlett et al. (1988) pointed out that mapping of deletions is a more reliable and an easier way to do prenatal diagnosis and carrier detection than by use of RFLPs. They suggested that once the entire gene is available for screening, most DMD boys will show deletions. Katayama et al. (1988) demonstrated the usefulness of RFLPs in prenatal diagnosis and carrier detection of DMD. In some of the examples cited, the authors made use of creatine phosphokinase levels as well. Speer et al. (1989) reviewed the status of prenatal diagnosis and carrier detection using cDNA probes. Clemens et al. (1991) took advantage of the existence of approximately 50,000-100,000 (CA)n loci in the human genome (Tautz and Renz, 1984) for carrier detection and prenatal diagnosis in DMD and BMD. (CA)n loci are a subclass of all short tandem repeat (STR) sequences. Because they are frequently polymorphic, so-called pSTR, they are useful for linkage purposes and are readily studied by PCR.

Bieber et al. (1989) described the use of immunoblotting for dystrophin analysis in the diagnosis of DMD in cases in which a gene deletion cannot be identified and RFLPs are equivocal. Evans et al. (1991) used in utero fetal muscle biopsy to assess dystrophin in a male fetus with the same X chromosome as an affected sib. Evans et al. (1993) used the same procedure to evaluate a female fetus found on amniocentesis performed for advanced maternal age to be carrying a de novo X;1 translocation with a breakpoint at Xp21. In utero muscle biopsy at 20 weeks of gestation showed normal dystrophin, and serum creatine kinase was normal at the time of birth of the infant. Situations in which testing of dystrophin by fetal muscle biopsy may be indicated were reviewed. Sancho et al. (1993) demonstrated that when conventional DNA analysis is not informative for the prenatal and postnatal diagnosis of DMD, myogenesis can be induced in cultured skin fibroblasts, amniocytes, or chorionic-villus cells by infecting the cells with a retrovirus vector containing MYOD (159970), a gene regulating myogenesis. Immunocytochemical analysis of dystrophin in the MYOD-converted muscle cells is an effective way of demonstrating dystrophin deficiency.

Beggs and Kunkel (1990) presented a flow diagram illustrating procedures for the molecular diagnosis of DMD or BMD. For males with consistent clinical features, CPK levels, and muscle biopsy, they suggested that Western blot testing for dystrophin be done first. If this is normal, the patient should be studied for other neuromuscular diseases. If dystrophin is of reduced or increased size, with or without reduction in the amount of dystrophin, BMD should be suspected. If dystrophin is absent, DMD should be suspected. Thereafter, PCR testing and Southern blot analysis should be done, looking for deletion/duplication. These procedures detect about 65% of patients, and the Southern blot permits prognostication of severity by distinguishing in-frame versus frameshift mutations in over 90% of cases. If no deletion or duplication is found, it is then necessary to resort to RFLP-based linkage studies, which unfortunately are laborious and time consuming. Once the diagnosis has been made, the information can be used for carrier detection and prenatal diagnosis. In females who are having symptoms of muscular dystrophy, immunohistochemistry for dystrophin in muscle showing a patchy loss of dystrophin can be used, and when abnormality is found, the same procedures of PCR, Southern blot, and linkage studies can be pursued. If the immunohistochemistry is normal, the female can be studied for other neuromuscular diseases. (Abnormality is indicative of the manifesting carrier state.) Beggs and Kunkel (1990) provided useful illustrative case histories as well as a hypothetical case in which a newborn male was found to have elevated CPK on a screening program but normal physical examination and negative family history. If Western blotting revealed absence of detectable dystrophin in the muscle and the PCR analysis detected a deletion which was confirmed by Southern blotting, his mother might carry the deletion or be normal. Even if normal, prenatal diagnosis could be offered her because of the significant probability that she was a germline mosaic. The usefulness of such screening programs for diagnosing DMD at a stage when diagnosis can be useful to the parents in the planning of other pregnancies is worthy of consideration.

Kristjansson et al. (1994) used primer extension preamplification (PEP) to increase the scope and capacity of single cell genetic diagnosis by generating sufficient template to perform multiple subsequent DNA analyses using PCR. They reported the simultaneous analysis of single cells at 5 commonly deleted dystrophin exons. In 93% of PEP reactions with single amniocytes, chorionic villus cells and blastomeres, successful results were obtained, and a blinded analysis of single lymphoblasts from affected males resulted in 93% diagnostic accuracy. They suggested that transfer of unaffected male embryos and improved diagnostic reliability is achieved with the ability to perform replicate multilocus analyses from the same blastomere.

Parsons et al. (1996) discussed procedures used for disclosure of the diagnosis of Duchenne muscular dystrophy to parents after newborn screening. Newborn screening for DMD was introduced into Wales in 1990. While screening in the newborn period for DMD was still under evaluation, preliminary evidence indicated that the excessive trauma anticipated in making such a disclosure presymptomatically could be avoided by implementing a strict protocol of disclosure and support. Parental choice should be facilitated at every stage from screen to diagnosis, and parents should be provided with maximum unbiased information on which to base their decisions. The family should not experience delay in getting the results with the additional stress this may cause. Meetings with the primary health care team and with the pediatrician facilitated ongoing support for the family.

Heterozygotes

Roses et al. (1977) concluded that isoenzyme 5 of lactate dehydrogenase is as sensitive an indicator of carrier status as creatine phosphokinase. Indeed, some carrier females with normal CPK were identified with LDH-5. By combining the 2 enzyme determinations and screening pedigrees extensively, they found that 28 of 30 mothers were probably heterozygotes. This high proportion of carriers is consistent with a higher mutation rate in males than in females, a conclusion suggested also by data on Lesch-Nyhan syndrome (308000) and hemophilia (306700). Hemopexin (142290) is elevated in some DMD carriers. Percy et al. (1981) found that hemopexin, used in combination with creatine kinase, improved the identification of carriers. Sato et al. (1978) presented evidence that red cell membrane as well as muscle membrane is involved. Beckmann et al. (1978) pointed out that the diagnosis of carrier females with plasma CPK is best in the neonatal or infant period. They suggested screening of all infants.

Although analysis of DNA with probes complementary to the dystrophin gene clarifies the diagnosis in at least two-thirds of isolated adult male patients, this approach in female patients is frustrated by the obfuscation of molecular deletion by heterozygosity, when gene dosage alone is not sufficiently reliable. Pulsed field gel electrophoresis may allow detection of abnormal-sized fragments of the dystrophin gene in these patients, and analysis of the dystrophin protein itself may be helpful.

Tangorra et al. (1989) suggested that an increased tendency of erythrocytes to form echinocytes (spine cells) on exposure to L-alpha-lysophosphatidylcholine could be used as a means of detecting DMD carriers.

With increased utilization of dystrophin protein analysis of muscle biopsies for molecular diagnosis, many female myopathy patients with no previous family history of any neuromuscular disease have been found to have a mosaic dystrophin immunostaining pattern on muscle biopsy (Minetti et al., 1991). These patients generally were diagnosed as having limb-girdle muscular dystrophy (with presumed autosomal recessive inheritance) before reclassification, by dystrophin testing, as female dystrophinopathy patients (Arikawa et al., 1991). In a large follow-up study of 505 muscle biopsies from female myopathy patients, Hoffman et al. (1992) found that about 10% of women with hyperCKemia, myopathic pattern by muscle biopsy, and no family history of DMD could be identified as carriers of DMD when tested with the dystrophin immunofluorescence assay. It was assumed that such female dystrophinopathy patients were heterozygous carriers who showed preferential inactivation of the X chromosome harboring the normal dystrophin gene. Such was shown to be the case, for example, in 2 sets of discordant monozygotic twins (Bonilla et al., 1990; Richards et al., 1990).

However, mosaic staining patterns have only been detected in heterozygote females with elevated levels of creatine kinase in the blood. Diagnosis of asymptomatic women without deletions or elevated creatine kinase remains a problem. In a study of clonal myogenic cell cultures from a potential heterozygote for DMD who also was heterozygous for G6PD isozymes, Hurko et al. (1989) found that only those myogenic colonies expressing the G6PD-A isozyme also expressed dystrophin. He suggested that somatic cell testing of dystrophin expression may be useful in genetic carrier tests in ambiguous cases.

Hoogerwaard et al. (2005) examined skeletal muscle biopsies from 50 definite carriers of DMD and BMD, including 22 manifesting carriers, 5 carriers with exertion-dependent myalgia or cramps, and 23 nonmanifesting carriers. Although 42% of the biopsies showed nonspecific abnormalities, no association was found between histopathologic changes and muscle weakness, dilated cardiomyopathy, serum creatine kinase activities, dystrophin abnormalities, or age. For example, 5 carriers with cardiomyopathy had no dystrophin abnormalities, whereas 6 nonmanifesting carriers had abnormal immunohistochemical dystrophin patterns.

Intrafamilial Variability

Sifringer et al. (2004) investigated the differences between the expression profiles of skeletal muscle biopsies from a very rare instance of 2 brothers with a different clinical course of DMD. Comparison of important parameters in the development of the 2 brothers made clear that the older brother was far more affected by muscle weakness than the younger. The younger brother was able to sit 9 months earlier and to walk 22 months earlier than the older one. The older brother was wheelchair-bound at the age of 9 years, whereas the younger one was not expected to become wheelchair dependent at the same age. Furthermore, the older boy was mentally retarded. Though deletions or point mutations in the DMD gene were not detected, negative immunofluorescence in both brothers supported the diagnosis of dystrophinopathy and suggested compensating mechanisms for the younger less affected brother. Sifringer et al. (2004) compared the transcriptomes in skeletal muscle in the 2 brothers to identify overexpressed transcripts that might be responsible for the milder phenotype. Six genes were found to be overexpressed 3 to 20 times in the less affected patient compared with the more severely affected boy; casein kinase 1 (600505) showed a slightly higher expression. Upregulation of myosin light polypeptide 2 (MYL2; 160781), one of the most sensitive markers of muscle fiber regeneration, was found with the milder phenotype. The purpose of these studies was to identify modifiers that might be exploited therapeutically in Duchenne muscular dystrophy.

The management of DMD is largely symptomatic: providing assisting devices for walking, prevention of scoliosis, and respiratory toilet. Goertzen et al. (1995) reported on the efficacy of early release of the spina muscles, resection of the tensor fasciae latae muscle, and a lengthening of the tendo calcaneus in 32 patients with Duchenne muscular dystrophy at the mean age of 6.1 years in safely preventing severe contractures and delaying the progression of scoliosis.

In a kindred with 9 previous cases of DMD, Zatz et al. (1981) observed a boy who was unusually mildly affected, perhaps because of the coincidence of growth hormone deficiency. Following up on this observation (Zatz and Betti, 1986), Zatz et al. (1986) used a growth hormone inhibitor, mazindol, in one of monozygotic twins concordant for DMD. The other twin received a placebo. After 1 year, the 'code was broken' and the placebo-treated twin was found to be much worse than his mazindol-treated brother in whom the 'condition was practically arrested.'

From a 6-month trial study, Mendell et al. (1989) concluded that prednisone improves the strength and function of patients with DMD. The mechanism of the improvement was not known and it was not clear whether prolonged treatment with corticosteroids is warranted despite their side effects.

Studies had shown a correlative relationship between calpain (114220) activity in dystrophic muscle and muscle necrosis, but had not tested whether calpain activation precedes cell death or is a consequence of it. Spencer and Mellgren (2002) hypothesized that calpains may play an active role in necrotic processes in dystrophic muscle, and that inhibition of calpains might provide a therapeutic option for treatment of DMD.

Malik et al. (2010) found that 10 boys with DMD due to stop codon mutations in the dystrophin gene showed a 50% decrease in serum creatine kinase levels compared to baseline levels after a 14-day treatment with intravenous infusion of gentamicin. In contrast, this treatment had no effect on 8 boys with frameshift mutations. Among 12 patients treated for 6 months, 6 showed an increase of dystrophin levels in serial skeletal muscle biopsies, 3 of whom had increases into a potentially therapeutic range (300% or more increase in dystrophin levels). The average muscle scale in these patients did not decrease over the study period, and some patients even had a slight increase in forced vital capacity, suggesting a clinical benefit. Only 1 patient developed a T-cell immune response to a novel epitope. The results of the study indicated that long-term dosing of gentamicin over 6 months could be safely achieved, and supported the concept that gentamicin can induce a read-through of stop codons in DMD.

Gene Therapy

Donor myoblasts injected into muscles of patients with DMD could theoretically fuse with host muscle fibers, thus contributing their nuclei which could potentially replace deficient gene products. Mendell et al. (1995) injected muscle cells donated by unaffected fathers or brothers once a month for 6 months into the biceps brachii muscle of 1 arm of each of 12 boys with DMD. Although in 1 patient 10.3% of muscle fibers expressed donor-derived dystrophin after myoblast transfer and 3 other patients had a low level of donor dystrophin, there was no significant difference in muscle strength between arms injected with myoblasts and control arms.

Van Deutekom et al. (2001) explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, the authors devised an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from 2 unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. The authors hypothesized that this strategy may be applicable to not only more than 65% of DMD mutations, but also to many other genetic diseases.

Aartsma-Rus et al. (2003) expanded the application of the antisense rescue method (van Deutekom et al., 2001) to cultured muscle cells from 6 DMD patients carrying different deletions and a nonsense mutation. In each case, the specific skipping of the targeted exon was induced, restoring dystrophin synthesis in over 75% of cells. The protein was detectable 16 hours posttransfection, increased to significant levels at the membrane within 2 days, and was maintained for at least a week. Its proper function was further suggested by the restored membrane expression of 4 associated proteins from the dystrophin-glycoprotein complex.

Harper et al. (2002) performed a detailed functional analysis of dystrophin structural domains and showed that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and micro-dystrophins. Studies in transgenic mdx mice, a model for DMD, revealed that a wide variety of functional characteristics of dystrophy are prevented by some of these truncated dystrophins. Muscles expressing the smallest dystrophins were fully protected against damage caused by muscle activity and were not morphologically different from normal muscle. Moreover, injection of adeno-associated viruses carrying micro-dystrophins into dystrophic muscles of immunocompetent mdx mice resulted in a striking reversal of histopathologic features of the disease. Harper et al. (2002) concluded that the dystrophic pathology can be both prevented and reversed by gene therapy using micro-dystrophins.

To address the need for a drug capable of suppressing premature termination, Welch et al. (2007) identified PTC124, a chemical entity that selectively induces ribosomal read-through of premature but not normal termination codons. PTC124 is a 284.24-Da, achiral, 1,2,4-oxadiazole linked to fluorobenzene and benzoic acid rings. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2 to 8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well-characterized activity profile, oral bioavailability, and pharmacologic properties indicated that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options. Clinical trials had been initiated for the treatment of both cystic fibrosis (219700) and DMD at the time of the report.

Up to 50% of patients with Duchenne muscular dystrophy show evidence of rare, dystrophin-positive fibers (revertant fibers) caused by spontaneous, clonal, frame-restoring skipping of stretches of exons (Aartsma-Rus et al., 2004). This finding prompted the investigation of the potential for therapeutic conversion of DMD into its in-frame counterpart, Becker muscular dystrophy, with the use of antisense techniques. Because of their capacity to skip an exon specifically by blocking its inclusion during splicing, antisense oligonucleotides can correct the reading frame of DMD transcripts, yielding internally truncated dystrophins such as those associated with Becker muscular dystrophy. Aartsma-Rus et al. (2003) showed in cultured cells from DMD patients that an intraexonic antisense oligonucleotide, PRO051, efficiently induced specific exon-51 skipping. On the basis of the frequency of mutations in patients with DMD in the Leiden database (Aartsma-Rus et al., 2006), van Deutekom et al. (2007) concluded that PRO051 might correct the reading frame in 16% of all patients with the disease. The effectiveness of antisense compounds in correcting the open reading frame of the DMD gene and thus restoring dystrophin expression in vitro and in animal models in vivo prompted van Deutekom et al. (2007) to test the effect of intramuscular injection of PRO051 in patients with this disorder. Four patients were selected for treatment who had a positive exon-skipping response to PRO051 in vitro. A single injection was made in the tibialis anterior muscle and biopsy performed 28 days later. Each patient was found to show specific skipping of exon 51 and sarcolemmal dystrophin in 64 to 97% of myofibers. The amount of dystrophin in total protein extracts ranged from 3 to 12% of that found in the control specimen and from 17 to 35% of that of the control specimen in the quantitative ratio of dystrophin to laminin alpha-2 (156225). Hoffman (2007) reviewed the potential of this approach for a form of personalized molecular medicine.

Rodino-Klapac et al. (2007) provided a review of the state of research in gene therapy for DMD.

Mendell et al. (2010) reported on the delivery of a functional dystrophin transgene to skeletal muscle in 6 patients with Duchenne muscular dystrophy. Dystrophin-specific T cells were detected after treatment, providing evidence of transgene expression even when the functional protein was not visualized in skeletal muscle. Circulating dystrophin-specific T cells were unexpectedly detected in 2 patients before vector treatment. Revertant dystrophin fibers, which expressed functional, truncated dystrophin from the deleted endogenous gene after spontaneous in-frame splicing, contained epitopes targeted by the autoreactive T cells. Mendell et al. (2010) cautioned that the potential for T-cell immunity to self and nonself dystrophin epitopes should be considered in designing and monitoring experimental therapies for this disease.

In a 12-year prospective study in the Campania region of southern Italy, Nigro et al. (1983) found an incidence of DMD of 21.7 per 100,000 male live births and of BMD of 3.2 per 100,000. The latter might be underestimated because of lesser severity but surely not to an extent to explain an incidence one-seventh of that of DMD. Of the DMD patients, 38.5% were familial; of the BMD cases, 50%.

Williams et al. (1983) analyzed 244 Toronto pedigrees of DMD. The incidence of DMD in Ontario was estimated to be 292 per million male births. The proportion of sporadic cases was one-third, demonstrating equal mutation rates in males and females. A multifactorial component (H = 0.379) contributing to familial resemblance for CPK measurements was found. They illustrated use in genetic counseling of a computer program COUNSEL, which takes the multifactorial component in CPK into account.

Mostacciuolo et al. (1987) presented population data on the incidence and prevalence of the Becker and Duchenne forms of muscular dystrophy and estimated mutation rates for each. Muller and Grimm (1986) pointed out that by using X chromosomal RFLPs to establish DNA haplotypes in 3-generation DMD families, one can calculate the ratio of mutation rates in males and females from the proportion of DMD patients who have inherited their maternal grandfather's X-chromosome. In the Netherlands, van Essen et al. (1992) estimated the prevalence rate of DMD at birth to be 1:4,215 male live births. The prevalence rate in the male population on January 1, 1983 was estimated to be 1:18,496. An extensive tabulation of previous data was provided. Roddie and Bundey (1992) observed that in the West Midlands region of Britain, DMD is twice as common as expected in Asiatic Indians and less common than expected in Pakistanis. Although the numbers were small, they could not be explained by bias of ascertainment and were considered to be real. They suggested that a possible mechanism for the high frequency of DMD in Indians is the presence of repetitive elements in the wildtype gene that predispose to mutations.

Shomrat et al. (1994) suggested that in Israeli patients with either Duchenne or Becker muscular dystrophy, deletions in the DMD gene constitute a much smaller proportion of cases than is found in European and North American populations. The figures were 37% in Israelis as compared to 55 to 65% in the other populations. They pointed to reports suggesting that the proportion of deletions among mutant dystrophin alleles is lower also in some Asian populations such as Japanese and Chinese than it is in Western countries. They found no correlation between the size of the deletion and the severity of the disease. All of the deletions causing frameshift resulted in the DMD phenotype.

Onengut et al. (2000) compared patterns of DMD gene deletions in 4 populations: Turks, Europeans, North Indians, and Indians from all over India. Statistical tests revealed differences in the proportions of small deletions. In contrast, the distribution of deletion breakpoints and the frequencies of specific deletions commonly observed in the 4 populations were not significantly different. The variations strongly suggested that sequence differences exist in the introns, and that the differences are in agreement with genetic distances among populations. The similarities suggested that some intronic sequences have been conserved and that those will trigger recurrent deletions.

Krahn and Anderson (1994) studied the mdx mouse model of muscular dystrophy and showed that treatment with anabolic steroids increases myofiber damage.

Winand et al. (1994) found a deletion of the dystrophin (300377) promoter in a male domestic short-haired cat with a generalized muscle hypertrophy, stiffness, and mild histopathologic dystrophy. The mutation eliminated expression of the muscle and Purkinje neuronal dystrophin isoforms. The cortical neuronal isoform was expressed at a detectable level in skeletal muscle, but not in the heart.

Mann et al. (2001) reported a potential therapeutic approach to Duchenne muscular dystrophy as an alternative to the introduction of functional dystrophin into dystrophic tissue through either cell or gene replacement: the use of 2-prime-O-methyl antisense oligoribonucleotides to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting the antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, they induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide-liposome complexes. They suggested that this approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein with milder clinical expression.

Chamberlain (2002) reviewed the progress and pitfalls associated with gene therapy in the context of murine models of muscular dystrophy.

Because insulin-like growth factor I (IGF1; 147440) enhances muscle regeneration and protein synthetic pathways, Barton et al. (2002) hypothesized that muscle-specific expression of Igf1 could preserve muscle function in the mdx mouse model. Transgenic mdx mice overexpressing Igf1 in muscle showed increased muscle mass, increased force generation, reduced fibrosis, and decreased myonecrosis compared with mdx mice. In addition, signaling pathways associated with muscle regeneration and protection against apoptosis showed significantly elevated activities. Barton et al. (2002) concluded that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin.

Gilbert et al. (2003) injected the tibialis anterior (TA) muscle of neonatal and juvenile dystrophin-deficient (mdx) mice with HDCBDysM, a viral construct encoding 2 full-length murine dystrophin cDNAs regulated by the CMV enhancer/beta-actin promoter. After 10 days, 42% of the total number of TA fibers in neonatal muscles were dystrophin-positive (dys+), a value that did not decrease for 6 months (the study duration). In treated juveniles, maximal transduction occurred after 30 days (24% of TA fibers positive), but decreased by 51% after 6 months. In neonatally treated muscles, the percentage of dys+ fibers with centrally localized myonuclei remained low, localization of the dystrophin-associated protein complex was restored at the plasma membrane, muscle hypertrophy was reduced, and maximal force-generating capacity and resistance to contraction-induced injuries were increased. The same pathologic aspects were improved in the treated juveniles, except for reduction of muscle hypertrophy and maximal force-generating capacity. A strong humoral response against murine dystrophin was evident in both animal groups, but mild inflammatory response occurred only in the treated juveniles.

ADAM12 (602714) is a disintegrin and metalloprotease shown to prevent muscle cell necrosis in the mdx mouse (Kronqvist et al., 2002). Moghadaszadeh et al. (2003) found that transgenic mice overexpressing ADAM12 exhibited only mild myopathic changes and accelerated regeneration following acute injury. Only small changes in gene expression profiles were found between mdx/ADAM12 transgenic mice and mdx mice, suggesting that significant changes in mdx/ADAM12 muscle might occur posttranscriptionally. By immunostaining and immunoblotting, Moghadaszadeh et al. (2003) detected a 2-fold increase in expression and extrasynaptic localization of alpha-7B integrin (ITGA7; 600536) and utrophin (128240), the functional homolog of dystrophin. Expression of dystrophin-associated glycoproteins was also increased.

Bassett and Currie (2003) reviewed zebrafish models for muscular dystrophy and congenital myopathy.

Utrophin is a chromosome 6-encoded dystrophin-related protein that has functional motifs in common with dystrophin. The ability of utrophin to compensate for dystrophin during development and when transgenically overexpressed provided an important impetus for identifying activators of utrophin expression. The utrophin promoter A is transcriptionally regulated in part by heregulin (142445)-mediated, extracellular signal-related kinase-dependent activation of the GABP(alpha/beta) transcription factor complex (see 600610). Therefore, this pathway offers a potential mechanism to modulate utrophin expression in muscle. Krag et al. (2004) tested the ability of heregulin to improve the dystrophic phenotype in the mdx mouse model of DMD. Intraperitoneal injections of the small peptide encoding the epidermal growth factor-like region of heregulin ectodomain for 3 months in vivo resulted in upregulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction-mediated damage, and a reduction of muscle pathology. The amelioration of dystrophic phenotype by heregulin-mediated utrophin upregulation offered a pharmacologic therapeutic modality that obviates many of the toxicity and delivery issues associated with viral vector-based gene therapy for DMD.

Chakkalakal et al. (2004) showed that mice expressing enhanced muscle calcineurin (PPP3CA; 114105) activity (CnA*) displayed elevated levels of utrophin around their sarcolemma. The authors crossed CnA* mice with mdx mice to determine the suitability of elevating calcineurin activity in preventing dystrophic pathology. Muscles from mdx/CnA* displayed increased nuclear localization of Nfatc1 (600489) and a fiber type shift toward a slower phenotype. Measurements of utrophin levels in mdx/CnA* muscles revealed an 2-fold induction in utrophin expression. Members of the dystrophin-associated protein (DAP) complex were present at the sarcolemma of mdx/CnA* mouse muscle. Restoration of the utrophin/DAP complex was accompanied by significant reductions in the extent of central nucleation and fiber size variability. Assessment of myofiber sarcolemmal damage revealed a net amelioration of membrane integrity, and immunofluorescence studies showed a reduction in the number of infiltrating immune cells in muscles from mdx/CnA* mice. Chakkalakal et al. (2004) concluded that elevated calcineurin activity attenuates dystrophic pathology.

Goyenvalle et al. (2004) achieved persistent exon skipping that removed the mutated exon on the dystrophin (300377) mRNA of the mdx mouse by single administration of an adeno-associated virus (AAV) vector expressing antisense sequences linked to a modified U7 small nuclear RNA (RNU7-1; 617876). Goyenvalle et al. (2004) reported the sustained production of functional dystrophin at physiologic levels in entire groups of muscles and the correction of the muscular dystrophy.

Yue et al. (2004) generated female heterozygous mdx mice that persistently expressed the full-length dystrophin gene in 50% of cardiomyocytes. Heart function of mdx mice was normal in the absence of external stress. Using beta-isoproterenol challenge in 3-month-old mice, cardiomyocyte sarcolemma integrity was significantly impaired in mdx but not in heterozygous mdx and C57BL/10 mice. In vivo closed-chest hemodynamic assays revealed normal left ventricular function in beta-isoproterenol-stimulated heterozygous mdx mice. The nonuniform dystrophin expression pattern in heterozygous mdx mice resembled the pattern seen in viral gene transfer studies. Yue et al. (2004) concluded that gene therapy correction in 50% of the heart cells may be sufficient to treat cardiomyopathy in mdx mice.

In a review, Shelton and Engvall (2005) stated that canine models of DMD had been described in the golden retriever, beagle, Rottweiler, German shorthaired pointer, and Japanese spitz breeds. Feline DMD has also been reported in domestic shorthaired cats.

Sampaolesi et al. (2006) stated that the only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about one year of age as a result of failure of respiratory muscles. Sampaolesi et al. (2006) reported that intraarterial delivery of wildtype canine mesoangioblasts (vessel-associated stem cells) resulted in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibers). The authors concluded that the outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.

Wehling-Henricks et al. (2005) produced dystrophin-deficient mdx mice in which there was myocardial expression of a neuronal nitric oxide synthase (NOS1; 163731) transgene. Expression of the transgene prevented the progressive ventricular fibrosis of mdx mice and greatly reduced myocarditis. Electrocardiographs (ECG) of ambulatory mdx mice showed cardiac abnormalities that were characteristic of DMD patients. All of these ECG abnormalities in mdx mice were improved or corrected by NOS1 transgene expression. In addition, defects in mdx cardiac autonomic function, which were reflected by decreased heart rate variability, were significantly reduced by NOS1 transgene expression. Wehling-Henricks et al. (2005) concluded that their findings indicate that increasing NO production by dystrophic hearts may have therapeutic value.

In dystrophin-deficient mdx mice, Cohn et al. (2007) demonstrated that increased TGF-beta (TGFB1; 190180) activity caused failure of muscle regeneration. Systemic antagonism of TGF-beta through administration of TGF-beta-neutralizing antibody or AGTR1 (106165) blocker losartan improved muscle regeneration and diminished fibrosis. After 6 to 9 months of treatment with losartan, analysis of various muscle groups showed significant attenuation of disease progression in mdx mice, and in vivo grip-strength testing showed improvement after 6 months of losartan treatment. Physiologic analysis of explanted extensor digitorum longus muscles revealed a losartan-induced increase in muscle mass that correlated with a significant increase in the number of fibers per muscle, and the performance of the losartan-treated muscle in generating absolute force over a range of stimulation intensities was statistically indistinguishable from that of wildtype mice.

Peter et al. (2009) showed that myogenic Akt (164730) signaling in mouse models of DMD promoted increased expression of utrophin (UTRN; 128240), which replaced the function of dystrophin, thereby preventing sarcolemma damage and muscle wasting.

Bellinger et al. (2009) found that the calcium channel Ryr1 (180901) in skeletal muscle from mdx mice showed increased inducible nitric oxide (NOS2A; 163730)-mediated S-nitrosylation of cysteine residues, which depleted the channel complex of calstabin-1 (FKBP12; 186945). This resulted in leaky channels with increased calcium flux. These changes were age-dependent and coincided with dystrophic changes in muscle. Prevention of calstabin-1 depletion from Ryr1 with S107, a compound that binds the Ryr1 channel and enhances binding affinity, inhibited sarcoplasmic reticulum calcium leak, reduced biochemical and histologic evidence of muscle damage, improved muscle function, and increased exercise performance in mdx mice. Bellinger et al. (2009) proposed that the increased calcium flux via a defective Ryr1 channel contributes to muscle weakness and degeneration in DMD by increasing calcium-activated proteases.

Iwata et al. (2009) demonstrated that muscular dystrophy is ameliorated in mdx mice by dominant-negative inhibition of Trpv2 (606676), a principal candidate for Ca(2+)-entry pathways. When transgenic (Tg) mice expressing a Trpv2 mutant in muscle were crossed with mdx mice, the cytosolic Ca(2+) concentration increase in muscle fibers was reduced. Histologic, biochemical, and physiologic indices characterizing dystrophic pathology, such as an increased number of central nuclei and fiber size variability/fibrosis/apoptosis, elevated serum creatine kinase levels, and reduced muscle performance, were all ameliorated in the mdx/Tg mice. Iwata et al. (2009) proposed that TRPV2 is a principal Ca(2+)-entry route leading to a sustained Ca(2+) concentration increase and muscle degeneration.

Li et al. (2009) generated delta-sarcoglycan (SGCD; 601411)/dystrophin (300377) double-knockout mice (delta-Dko) in which residual sarcoglycans were completely eliminated from the sarcolemma. Utrophin levels were increased in these mice but did not mitigate disease. The clinical manifestation of delta-Dko mice was worse than that of mdx mice. They showed characteristic dystrophic signs, body emaciation, macrophage infiltration, decreased life span, less absolute muscle force, and greater susceptibility to contraction-induced injury. Li et al. (2009) suggested that subphysiologic sarcoglycan expression may play a role in ameliorating muscle disease in mdx mice.

Li et al. (2009) investigated the role and the mechanisms by which increased levels of matrix metalloproteinase-9 (MMP9; 120361) protein cause myopathy in dystrophin-deficient mdx mice. Levels of MMP9 but not tissue inhibitor of MMPs were drastically increased in skeletal muscle of mdx mice. Infiltrating macrophages also contributed to the elevated levels of MMP9 in dystrophic muscle. In vivo administration of a NFKB-inhibitory peptide NBD blocked the expression of MMP9 in dystrophic muscle of mdx mice. Deletion of the Mmp9 gene in mdx mice improved skeletal muscle structure and functions and reduced muscle injury, inflammation, and fiber necrosis. Inhibition of MMP9 increased the levels of cytoskeletal protein beta-dystroglycan (DAG1; 128239) and Nos1 and reduced the amounts of caveolin-3 (CAV3; 601253) and Tgfb in myofibers of mdx mice. Genetic ablation of MMP9 significantly augmented the skeletal muscle regeneration in mdx mice. Pharmacologic inhibition of MMP9 activity also ameliorated skeletal muscle pathogenesis and enhanced myofiber regeneration in mdx mice.

Willmann et al. (2009) provided a review of mammalian models of Duchenne muscular dystrophy, with emphasis on the models that are most effective for testing treatment options at the preclinical stage. The review included mouse, canine, and feline models. The mdx mouse was recommended as the model of choice for preclinical testing, and the canine model for use in well-controlled experimental settings.

Wehling-Henricks et al. (2009) tested whether the loss of neuronal nitric oxide synthase, nNOS (NOS1; 163731), contributes to the increased fatigability of mdx mice. The expression of a muscle-specific nNOS transgene increased the endurance of mdx mice and enhanced glycogen metabolism during treadmill running, but did not affect vascular perfusion of muscles. The specific activity of phosphofructokinase (PFK; 610681), the rate-limiting enzyme in glycolysis, was positively affected by nNOS in muscle; PFK-specific activity was significantly reduced in mdx muscles and the muscles of nNOS-null mutants, but significantly increased in nNOS transgenic muscles and muscles from mdx mice that expressed the nNOS transgene. PFK activity measured under allosteric conditions was significantly increased by nNOS, but unaffected by endothelial NOS or inducible NOS. The specific domain of nNOS that positively regulates PFK activity was assayed by cloning and expressing different domains of nNOS and assaying their effects on PFK activity. This approach yielded a polypeptide that included the flavin adenine dinucleotide (FAD)-binding domain of nNOS as the region of the molecule that promotes PFK activity. A 36-amino acid peptide in the FAD-binding domain was identified in which most of the positive allosteric activity of nNOS for PFK resides. Wehling-Henricks et al. (2009) proposed that defects in glycolytic metabolism and increased fatigability in dystrophic muscle may be caused in part by the loss of positive allosteric interactions between nNOS and PFK.

Miura et al. (2009) found that GW501516, a peroxisome proliferator-activated receptor PPAR-beta/delta (PPARD; 600409) agonist, stimulated utrophin A (UTRN; 128240) mRNA levels in mdx muscle cells, through an element in the utrophin A promoter. Expression of PPARD was greater in skeletal muscles of mdx versus wildtype mice. Over a 4-week trial, treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels, leading to its increased expression at the sarcolemma. Expression of alpha-1-syntrophin (SNTA1; 601017) and beta-dystroglycan (DAG1; 128239) was also restored to the sarcolemma. The mdx sarcolemmal integrity was improved, and treatment also conferred protection against eccentric contraction-induced damage of mdx skeletal muscles.

Dystrophin (300377) deficiency does not fully recapitulate the human disorder in mdx mice, which show milder skeletal muscle defects and potent regenerative capacity of the myofiber. Sacco et al. (2010) demonstrated that the milder mouse mdx phenotype resulted from a greater reserve of functional muscle stem cells caused by longer telomeres in inbred mice. Mdx mice also lacking the telomerase RNA component (TERC; 602322) (mTR) developed severe progressive muscular dystrophy more consistent with the human phenotype. Mdx/mTR double-mutant mice had shortened telomeres in muscle cells associated with a decline in muscle stem cell regenerative capacity. The defect in mdx/mTR double-mutant mice was ameliorated histologically by transplantation of wildtype muscle stem cells. Sacco et al. (2010) suggested that progression of the human disorder results, in part, from a cell-autonomous failure of muscle stem cells to maintain the damage-repair cycle initiated by dystrophin deficiency.

To obtain therapeutic levels of utrophin expression in dystrophic muscle, Di Certo et al. (2010) developed a strategy based on artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment, the authors generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. The artificial Jazz protein restored sarcolemmal integrity and prevented the development of the dystrophic disease in mdx mice.

In addition to its presence in muscle, dystrophin (300377) is also found in vasculature, and its absence results in vascular deficiency and abnormal blood flow. To create a mouse model of DMD with increased vasculature, Verma et al. (2010) crossed mdx mice with Flt1 knockout mice, which display increased endothelial cell proliferation and vascular density during embryogenesis. Flt1 +/- and mdx:Flt1 +/- adult mice displayed a developmentally increased vascular density in skeletal muscle compared with wildtype and mdx mice, respectively. The mdx:Flt1 +/- mice showed improved muscle histology compared with mdx mice, with decreased fibrosis, calcification, and membrane permeability. Functionally, the mdx:Flt1 +/- mice had an increase in muscle blood flow and force production compared with mdx mice. Because utrophin is upregulated in mdx mice and can compensate for the lacking function of dystrophin, Verma et al. (2010) created a triple-mutant mouse (mdx:utrophin -/-:Flt1 +/-). The mdx:utrophin -/-:Flt1 +/- mice also displayed improved muscle histology and significantly higher survival rates compared with mdx:utrophin -/- mice, which showed more severe muscle phenotypes than mdx mice. Verma et al. (2010) suggested that increasing the vasculature in DMD may ameliorate the histologic and functional phenotypes associated with this disease.

Menazza et al. (2010) investigated whether reactive oxygen species (ROS) produced in mitochondria by monoamine oxidase (MAO) contribute to muscular dystrophy pathogenesis. Pargyline, an MAO inhibitor, reduced ROS accumulation along with a beneficial effect on the dystrophic phenotype of Col6a1 (120220) -/- mice, a model of Bethlem myopathy (158810) and Ullrich congenital muscular dystrophy (UCMD; 254090), and mdx mice, a model of Duchenne muscular dystrophy. Oxidation of myofibrillar proteins, as probed by formation of disulfide crossbridges in tropomyosin (see 191010), was detected in both Col6a1 -/- and mdx muscles. Notably, pargyline significantly reduced myofiber apoptosis and ameliorated muscle strength in Col6a1 -/- mice. Menazza et al. (2010) concluded that there is a novel and determinant role of MAO in muscular dystrophies, adding evidence of the pivotal role of mitochondria and suggesting a therapeutic potential for MAO inhibition.

In the mdx mouse model, M1 macrophages play a major role in worsening muscle injury. However, mdx muscle contains M2c macrophages that promote tissue repair. Villalta et al. (2011) investigated factors regulating the balance between M1 and M2c macrophages in mdx mice. Ablation of Il10 (124092) expression in mdx mice increased muscle damage in vivo and reduced mouse strength. Treatment of mdx muscle macrophages with Il10 reduced activation of the M1 phenotype, as assessed by iNOS expression. Macrophages from mice lacking Il10 were more cytolytic than macrophages from wildtype mice. Real-time PCR and immunohistochemical analysis detected expression of Il10 receptor (IL10RA; 146933) in mdx muscle. Ablation of Il10 expression in mdx mice did not affect satellite cell numbers, but it increased myogenin (MYOG; 159980) expression in vivo during the acute and regenerative phases of mdx pathology. Villalta et al. (2011) concluded that IL10 plays a significant role in muscular dystrophy by reducing M1 macrophage activation and cytotoxicity, increasing M2c macrophage activation, and modulating muscle differentiation.

Gehrig et al. (2012) showed that increasing the expression of intramuscular heat-shock protein-72 (Hsp72; 140550) preserves muscle strength and ameliorates the dystrophic pathology in 2 mouse models of muscular dystrophy. Treatment with BGP-15, a pharmacologic inducer of Hsp72 that can protect against obesity-induced insulin resistance, improved muscular architecture, strength, and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe kyphosis, muscle weakness, and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles, and extended life span. Gehrig et al. (2012) found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA; 108730) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with Serca to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased Serca activity in dystrophic skeletal muscles. Gehrig et al. (2012) concluded that their results provided evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell, and pharmacologic therapies.

Cathepsin S (CTSS; 116845) is a cysteine protease that is actively secreted in areas of tissue injury and inflammation, where it participates in extracellular matrix remodeling and healing. Tjondrokoesoemo et al. (2016) observed significant induction of Ctss expression in injured wildtype mouse muscle or muscle from mdx mice. Deletion of Ctss in mdx mice resulted in protection from DMD pathogenesis, including reduced myofiber turnover and pathology, reduced fibrosis, and improved running capacity. Ctss deletion in mdx mice significantly increased myofiber sarcolemma membrane stability, with enhanced expression and membrane localization of utrophin, intregrins, and beta-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Transgenic mice overexpressing Ctss in skeletal muscle exhibited increased myofiber necrosis, muscle histopathology, and deficits similar to those of muscular dystrophy. Tjondrokoesoemo et al. (2016) concluded that CTSS induction during muscular dystrophy is a pathologic event that underlies disease pathogenesis.

Amoasii et al. (2018) used adeno-associated viruses to deliver CRISPR gene-editing components to 4 dogs with the deltaE50-MD dog model of DMD and examined dystrophin protein expression 6 weeks after intramuscular delivery in 2 dogs or 8 weeks after systemic delivery in 2 dogs. After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. Amoasii et al. (2018) concluded that these large-animal data supported the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.