Dystrophinopathies

In 1987, the protein responsible for DMD, and its milder relative Beckers muscular dystrophy (BMD) was identified in humans and named dystrophin (Hoffman et al. 1987). DMD is an X-linked disease that typically presents itself early in childhood as muscle weakness and progresses to muscle loss and fibro-sis. Most boys with this disease die around the age of puberty or shortly thereafter from respiratory or cardiac failure. Dystrophin is a large cytoskeletal protein that is localized near the plasma membrane. The protein interacts with actin filaments, and members of the dystrophin-associated glycoprotein complex (DGC) that in turn spans the membrane, linking the cytoskeleton within the cell to the extracellular matrix. The majority of mutations in the human population are deletions, which are thought to be due to misalignment of dystrophin exons during meiosis, as these exons contain highly repetitive sequences. Mutations that maintain the dystrophin reading frame and allow the preservation of specific domains of the dystrophin protein often result in the milder form of the disease,

BMD, which may present as mild muscle weakness and may not be diagnosed until adulthood.

The classic murine model of DMD is the mdx mouse. This mutation was recognized in 1977 in the C57BL/10ScSn background. In 1989, the molecular basis of DMD in the mdx mouse was determined to be the substitution of T for C at position 3185, creating a nonsense codon (Sicinski et al. 1989). This premature stop occurs in exon 23 of the dystrophin protein. Mdx mice have a normal or nearly normal phenotype with a lifespan similar to that of the parental strain. Affected mice show central nucleation of many muscle fibers, indicating muscle degeneration and regeneration, but do not show weakness or fibrosis, with the exception of the diaphragm muscle, which is significantly affected (Stedman et al. 1991). If mdx mice are subject to eccentric contraction, such as walking on a downhill treadmill, they show significant muscle damage, leading the mice to become symptomatic (Sandri et al. 1997). Mdx mice show a significant number of the so called "revertant fibers", where fibers stain positive for dystrophin in affected animals. It is generally believed that few, if any of these fibers are true revertants of the point mutation, but rather, most are splicing variants that do not express the mutant exon 23. The elimination of exon 23 does not affect the reading frame, like a number of other exons of dystrophin, thus the remainder of the protein is produced in-frame. Because affected males achieve sexual maturity, it is possible to breed affected males to carrier and affected females thereby greatly increasing the relative number of affected mice per litter.

In an attempt to enhance the disease phenotype of the mdx mouse, it has been crossed with a model where the apparent paralog of dystrophin, utrophin, has been knocked out, generating the "double knockout" or dko model (Deconinck et al. 1997). Utrophin has been hypothesized to compensate for the lack of dystrophin in the mouse by a number of investigators, and utrophin knockout mice show no sign of the disease. However, dko mice show a phenotype that resembles that of human DMD, with a severe progressive muscular dystrophy and premature death.

Verne Chapman and colleagues created four additional models of DMD in mice in the late 1980s using using N-ethylynitrosourea (ENU) chemical mutagenesis (Chapman et al. 1989). These models were termed mdx 2-5CV. The mutation in the mdx 2CV strain has been determined to be a single base substitution in the splice acceptor sequence of intron 42. A series of different splicing variants therefore results, none of which maintain the normal open reading frame (Im et al. 1996). Similarly, mdx 3CV is due to a point mutation in intron 65 that creates a novel splice acceptor site, resulting in abnormally spliced RNA (Cox et al. 1993). The mdx 4CV model is due to a C to T transition in exon 53, resulting in a nonsense codon (CAA to TAA) (Im et al. 1996) and the mdx 5CV mutation is a 53 base pair deletion in the mRNA due to an A to T substitution in exon 10 that creates a novel splice donor site. This mutation results in the deletion of 53 bases and a frame-shift in the mRNA (Im et al. 1996). These models present unique opportunities to examine the contributions of dystrophin's multiple domains and promoters to contribute to disease presentation, progression, and pathogenesis. All four resemble mdx in the presentation of DMD. However, mdx 3CV mice show increased neonatal mortality (Cox et al. 1993).

In addition, this strain shows faint staining for dystrophin in muscles, similar to a subset of human patients. The mdx 4CV and 5CV strains show a tenfold reduction in revertant fibers when compared to the mdx model or mdx 2CV (Danko et al. 1992) (Table 1.1).

Spontaneously occurring DMD has been recognized in both dogs and cats. With the advent of molecular approaches and the identification of mutations in the dys-trophin gene as the cause of DMD in humans, it has become possible to confirm, that the cause of the disease in these intermediate models are mutations in the dys-trophin gene. Feline DMD is relatively rare, having been reported in three breeds of cat (Carpenter et al. 1989; Gaschen et al. 1992; Winand et al. 1994). The hallmark of this disease in cats is significant hypertrophy of muscle and the disease has been called hypertrophic feline muscular dystrophy (HFMD) due to this. Both the tongue and diaphragm can be severely affected resulting in the inability to drink due to swelling of the tongue and the inability to move food and water into the stomach when diaphragmatic hypertrophy constricts the esophagus. Some cats with DMD can lead nearly normal lives if they do not suffer these complications. Many do go on to develop sub-clinical cardiomyopathies and a few have been documented to develop acute, life-threatening rhabdomyolysis when given inhalational anesthesia. Interestingly, elevated serum creatine kinase (CK) levels cannot be used to identify affected or carrier animals at birth, but only begin to rise approximately two weeks postnatally.

Canine DMD has been diagnosed much more commonly than feline DMD, probably due to the severity of the disease in this species. Among the breeds in which DMD has been described are the Golden Retriever (Sharp et al. 1992), German Short-haired Pointer (Schatzberg et al. 1999), Rottweiler (Winand and Cooper 1994), Labrador Retriever (Bergman et al. 2002), Welsh Corgi (Woods et al. 1998), West Highland White (Smith, unpublished), English Springer Spaniel (Smith unpublished), Australian Labradoodle (Smith unpublished), Old English Sheepdog (Wieczorek et al. 2006) Grand Basset Griffon Vendeen (Klarenbeek et al. 2007) and Japanese Spitz (Jones et al. 2004). The Golden Retriever was the first

Table 1.1 Animal models of Duchenne muscular dystrophy

Species

Model

Comments

Mouse

Mdx

Mild disease

Mouse

Mdx 2CV

Mild disease

Mouse

Mdx 3CV

Mild disease, some faint staining

Mouse

Mdx 4CV

Mild disease, low number of revertant fibers

Mouse

Mdx 5CV

Mild disease, low number of revertant fibers

Mouse

Dko

Utrophin - dystrophin double knockout,

severe disease

Cat

Multiple breeds

Hypertrophic changes predominate

Dog

Golden retriever

Original model, some revertant fibers, intron 6

Dog

German shorthaired pointer

Spontaneous knockout model

Dog

Welsh corgi

Intron 13 insertion

Dog

Labrador retriever

Intron 19 insertion

dog model of DMD identified and the first in which the mutation was determined. The mutation in this breed was found to be a point mutation in the splice acceptor site of intron 6 (Sharp et al. 1992). This results in exon 7 being spliced out of the mRNA, causing a frame-shift and subsequent termination. Some revertant fibers are noted in the Golden Retriever and these have been shown to be due to alternative splicing out of exons 3-9 or 5-12 (Schatzberg et al. 1998). The Golden Retriever mutation has also been bred onto the Beagle background. Recently, a male Golden Retriever was described who has the published mutation, but is very mildly affected. This dog has had multiple litters and some of his affected male offspring also show the extremely mild phenotype, indicating that the source of the effect may be autosomal in origin (Ambrosio et al. 2008).

Several other breeds have reached a similar level of molecular analysis and can be considered as alternative models to the Golden Retriever. The German Short-haired Pointer was determined to be a spontaneous knockout mutation as these dogs are missing the entire dystrophin gene (Schatzberg et al. 1999). Consequently, there are no revertant fibers noted in the muscles of these dogs, making interpretation of results significantly easier. However, the lack of any dystrophin expression in these dogs also means that their immune systems are naive and that even canine dystrophin may elicit an immune response. The mutation in the Japanese Spitz has not yet been published, however analysis of the protein using a panel of antibodies indicates that a severely truncated protein of 70-80 kdal (Jones et al. 2004). The mutations in the Welsh Corgi and the Labrador Retriever have been determined to be intronic insertions, in introns 13 and 19 respectively (Smith unpublished). In both cases, the insertions occur downstream of AG dinucleotides in the insertion which function as splice acceptor sites. Downstream, within the insertion, are putative splice donor sites, allowing the inserted material to act as novel exons. In-frame termination codons are present in these novel exons resulting in truncated dystro-phin production. A small number of revertant fibers can be identified in both models with antibodies to epitopes beyond the mutation site, indicating that alternative splicing is most likely responsible for this low level expression.

Canine models exhibit many of the same clinical signs as boys with DMD. Affected puppies may be identified, often within hours after birth, by their elevated CK levels. These can be extremely high and while they often decrease during the first few weeks of life, CK levels remain elevated for the dog's life. Physical signs usually become apparent at 6-8 weeks of age when the puppies are noted to be smaller and to tire more readily than their normal siblings. Progression of the disease occurs over the ensuing 5-6 months, with loss of muscle mass, weakness and kyphosis as classical signs (Fig. 1.1). Microscopically, affected muscles show degeneration, regeneration, fatty infiltration, and fibrosis. Many dogs with DMD show similar cardiac disease to boys with this disease. Progression can vary between dogs with the same mutation, with some individuals requiring euthanasia within 6 months of birth, while other can survive into adulthood. However, with the exception of the Golden Retriever noted above, these "longer lived" affected dogs obviously show disease, need significant nursing care to be maintained and usually succumb around 2-3 years of age. In dogs with DMD, the typical reasons for euthanasia

Fig. 1.1 DMD affected male yellow Labrador Retriever at 5 months of age. This dog shows severe muscle loss and fibrosis, kyphosis, weakness and hyperextension of the carpus and tarsus. This represents the extreme of this phenotype

are inability to eat, recumbency, respiratory disease secondary to compromised respiration and heart failure.

Animal husbandry in colonies of DMD dogs can be challenging. Dystrophic puppies have a higher neonatal mortality rate than normal puppies. Enhanced survival of the affected dogs requires precise timing of pregnancies, systematic surveillance of pregnant female dogs for signs of impending birth, and intensive observation perinatally. Dedicated facilities for whelping will help facilitate this process. Newborn puppies must be weighed multiple times each day to monitor weight gain and puppies need to be checked for dehydration and chilling frequently. Affected puppies can require bottle-feeding, either as a supplement to maternal feeding (preferred) or as their sole source of nutrition. The affected puppies become robust within a week or two of birth, and require little specialized care for the next month or two. However, once clinical signs begin to appear, the affected puppies may require significant additional nursing care. This includes the feeding of softer diets, regular cleaning and grooming, continued attention to weight gain, limited exercise, regular monitoring for respiratory obstruction and infections and regular assessment of disease progression. In older affected dogs, the extreme fibrosis associated with the disease may present appearance issues with animal care workers and regulatory personnel who are not familiar with the model as they may mistake the appearance of the dog for starvation. Some affected male dogs may live past puberty, and as a consequence can be used to breed female carriers and produce affected female dogs.

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