* The application of cloned genes and their protein prod
ucts to molecular diagnostics has been an increasingly im
portant area of pathology. The first gene to be identified
by positional cloning was the Duchenne muscular dystro
phy gene, mutations of which cause one of the most com
mon and most devastating human inherited conditions. The
identification of the responsible gene and the encoded dys
trophin protein has resulted in a large series of studies con
cerning the other components of the membrane cytoskel
eton of myofibers and their involvement in different forms
of muscular dystrophy. Through the study of patients defi
cient in specific components of the muscle fiber, much is
being learned about normal myofiber structure and func
tion and dysfunction in disease states. A new frontier is the
application of the normal genes and proteins toward pa
tient therapeutics (gene therapy). Although highly experi
mental, delivery of therapeutic genes promises to become
an important medical practice.
(Arch Pathol Lab Med. 1999;123:1050-1052)
The application of molecular genetics to human disease has resulted in the identification of hundreds of genes and gene mutations responsible for a large variety of inherited and sporadic disorders. The prototype of the approach used for identification of disease genes in disorders of unknown pathogenesis was Duchenne muscular dystrophy; this was the first gene identified by positional cloning, where no knowledge of the underlying biochemical defect was known before the disease gene isolation.1,2
Duchenne muscular dystrophy is considered the most common lethal pediatric disorder worldwide. Whereas many of the more common inherited disorders show population specificity (cystic fibrosis in whites, sickle cell anemia in African Americans), Duchenne dystrophy is equally common in all world populations. The high incidence is a result of a high sporadic mutation rate: 1 in 10 000 sperm or eggs have the dystrophin gene destroyed as a new event. The dystrophin gene is approximately 3 million base pairs in length, making it nearly 10 times larger than the next largest gene.1 The large size makes it a large target for random mutational events.
The identification of the dystrophin gene immediately led to application of gene probes and protein antibodies toward patient diagnosis.3-5 Approximately 55% of patients with Duchenne dystrophy show a deletion mutation of one or more of the 79 exons of the dystrophin gene. Most deletion mutations are easily detected by multiplex polymerase chain reaction in standard use in many laboratories.7 DNA analysis of families through detection of deletion or duplication mutations or by linkage analysis using intragenic dinucleotide repeat polymorphisms8,9 has enabled carrier detection and prenatal diagnosis in most families. However, the high mutation rate complicates molecular diagnostics and genetic counseling of some families; it can be difficult to determine the carrier state of a mother of an isolated case of Duchenne dystrophy if a deletion cannot be found. At this point, the gene is too large to routinely search for private point mutations, and this, coupled with the high mutation rate, still leads to ambiguous risks assigned to some fetuses. Fetal muscle biopsy and dystrophin protein testing of the biopsy specimen have been the only options for unambiguous prenatal diagnosis in a number of cases.10
Loss of dystrophin in muscle leads to the clinically severe Duchenne muscular dystrophy; the protein tests are quite specific, with few if any clear cases of marked dystrophin deficiency as a secondary consequence of some other disorder. Present but abnormal dystrophin leads to a variety of clinical presentations and progressions, including localized weakness, asymptomatic high serum creative kinase levels, myalgia and cramps, and cardiomyopathy. These have been grouped under the moniker Becker muscular dystrophy, although some patients may not show clinical evidence of a skeletal muscle disease.11
Although Duchenne and Becker muscular dystrophy are X-linked recessive diseases, there is a high mutation rate that leads to many isolated cases where the mother is not a carrier. Moreover, a subset of female carriers, both isolated cases and familial cases, can show symptoms of muscular dystrophy. Indeed, about 10% of female patients with muscular dystrophy who show proximal weakness and high serum creative kinase levels can be shown to be manifesting carriers of Duchenne muscular dystrophy by muscle biopsy specimen immunostaining.12 These girls and women vary tremendously in clinical severity, although most or all show preferential use of the abnormal X chromosome (skewed X inactivation). Molecular diagnosis and genetic counseling of such isolated manifesting carriers are particularly challenging.13
With the advent of molecular diagnostics for the dystrophinopathies, it quickly became clear that some patients carrying the diagnosis of either Duchenne or Becker muscular dystrophy had normal dystrophin gene and protein findings and thus likely had some other disorder. Since nearly all such patients show family histories consistent with autosomal recessive inheritance (generally isolated cases), they were lumped into the diagnostic group of limb-girdle muscular dystrophy (generally milder disease) or severe childhood autosomal recessive muscular dystrophy (more severe or Duchennelike disease). Attention of researchers then turned to this presumed heterogeneous group of disorders to search for causative genes and proteins using two different experimental approaches. One approach was the candidate gene approach, where proteins functionally related to dystrophin (dystrophin-assodated proteins) were used as candidates for causing muscular dystrophy. The second approach was positional cloning, where extended muscular dystrophy families were used to localize and clone novel disease genes via molecular genetic technology. These approaches have led to the identification of 9 different genes that cause different types of muscular dystrophy but show normal dystrophin gene and protein findings. Those genes that cause proximal muscular dystrophies with elevated serum creatine phosphokinase (Duchenne and Beckerlike disorders) include the dystrophin-associated proteins a-sarcoglycan,14 beta-sarcoglycan,15 gamma-sarcoglycan,16 delta-sacroglycan,17 a muscle protease (calpain III),18 and caveolin.19 Other known dystrophy genes with some clinical distinctions include the late-onset, dominantly inherited oculopharyngeal muscular dystrophy (poly-A binding protein 2)20 and an X-linked recessive muscular dystrophy with early contractures and cardiac conduction disturbances (EmeryDreifus muscular dystrophy),21 which is caused by a nuclear membrane protein, emerin.22 About 50% of cases of congenital muscular dystrophy show abnormalities of the basal lamina of muscle fibers (laminin (x2), and most or all of these patients show dramatic white matter changes by magnetic resonance imaging, despite normal cognition.23,24 Most recently, the human homologue of a protein that, in worms, is involved in membrane fusion during spermatogenesis has also caused muscular dystrophy (dysferlin).25,26
Clinical cues can direct the molecular pathology studies (oculopharyngeal muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy). Given a patient with isolated proximal muscular dystrophy and high serum creatine kinase levels, dystrophin must usually be tested, and most males (~80%) and some females (~10%) will show a primary dystrophinopathy. In patients with normal dystrophin findings, the correct path toward molecular diagnosis is more convoluted, with no clear paradigms to date. Some muscle biopsy protein tests have been developed (sarcoglycans, calpain III), but there are problematic issues of specificity and sensitivity.27-29 Detection of gene mutations seems the most specific and sensitive route; however, it is technically and economically unfeasible to sequence each exon of all 9 genes.30 It is anticipated that methods to quickly, accurately, and inexpensively test patient DNA and/or RNA for mutations will continue to evolve and that large-scale molecular diagnosis can be done on many genes simultaneously at low cost sometime in the not too distant future.
Identification of the primary biochemical and genetic defect in a patient with muscular dystrophy is important for prognostic diagnosis and for genetic counseling of the patient and family. Currently, a precise molecular diagnosis does not substantially alter patient management, since there is little that can be done therapeutically for a patient with muscular dystrophy. Two emerging lines of research promise to make progress on the therapeutics of the muscular dystrophies. First, many homologous animal models of muscular dystrophy have been found: dogs, cats, and mice with naturally occurring dystrophin deficiency, hamsters with 8-sarcoglycan deficiency, and mice with merosin deficiency. These animals are being used to screen drugs that are able to mitigate the progression of the muscular dystrophy. For example, Drs Granchelli and Hudecki at the State University of New York in Buffalo are exercising dystrophin-deficient mdx mice so that weakness is observed and screening large numbers of compounds to test for mitigation of the weakness, with promising results.
The second route toward therapeutics is gene therapy: delivery of a normal copy of the faulty gene to the patient to complement the biochemical deficiency. Toward this end, substantial progress has been made during the last few years using two viral vectors: third-generation (gutted) adenovirus31 and adeno-associated virus.32 Adenovirus has been used as a gene vehicle for a number of inherited disorders; however, most have used variants of the virus that have elicited a major immune response from the host (first- or second-generation adenovirus). Thus, cells infected with the virus shuttling the therapeutic gene were relatively quickly rejected by the immune system of the host. One method to reduce the immune response has been to use immune suppressive agents. A more promising approach has been the development of an adenoviral vector that contains no viral genes and thus does not seem to provide much viral protein for antigenic presentation by the infected cell (third-generation or gutted adenovirus).31 The complete dystrophin gene has been placed into this virus and animal models of Duchenne dystrophy infected by intramuscular injection. This has resulted in therapeutic levels of dystrophin, although there are still issues of long-term persistence of expression, which may be due to low-level immune response. The latter is due, at least in part, to infection of dendritic cells with subsequent antigen presentation and contamination of the more immunogenic first-generation helper virus.33 In addition, this virus seems to have difficulty infecting mature myofibers, making efficient gene delivery in boys older than 1 year problematic.34
Adeno-associated virus has recently shown great romise for gene delivery to skeletal muscle. This small virus causes little or no immune response (partly due to lack of infection of dendritic cells), efficiently and stably infects adult muscle fibers, and shows excellent persistence of transgene expression (probably through integration into host cell chromosomes). Recent studies using this virus to deliver one of the sarcoglycan genes to a hamster model of severe childhood recessive muscular dystrophy have shown dramatic positive effects (J. Watchko, MD, D. Dressman, BS, E. Hoffman, PhD, and X. Xiao, PhD, unpublished data, 1999).32 Unfortunately, the limited gene-carrying capacity of this viral vector precludes use of the complete dystrophin gene. Studies are under way to use truncated, semifunctional forms of the dystrophin gene in animal models (X. Xiao, PhD, and T Ta-oka, MD, unpublished data).
The author notes the financial support of the National Institutes of Health, Muscular Dystrophy Association, and Duchenne Parent Project.
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Accepted for publication May 21, 1999.
From the Research Center for Genetic Medicine, Children's National
Medical Center, Washington, DC. The author is an Established Inves
tigator of the American Heart Association.
Presented at the College of American Pathologists Conference
XXXIV, Molecular Pathology: Role in Improving Patient Outcome, Be
thesda, Md, February 26-28, 1999.
Reprints: Eric P. Hoffman, PhD, Research Center for Genetic Medi
cine, Children's National Medical Center, 111 Michigan Ave NW,
Washington, DC 20010 (e-mail: email@example.com).
Copyright College of American Pathologists Nov 1999
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