A new gene responsible for at least two rare forms of Muscular Dystrophy has been found.On September 1, 1998 USA Today reported that there has been discovery of a new gene. This gene, LGMD2B, is responsible for at least two types of muscular dystrophy: limb girdle (LGMD) and a very rare form called Miyoshi myopathy (MM).
The LGMD2B gene, also called Dysferlin, was named after its gene product, the protein dysferlin. Research data show that mutation in this gene causes Miyoshi myopathy (MM) or limb girdle muscular dystrophy (LGMD2B). Large inbred families were used for the study because they share the same haplotype. The September 1998 issue of Nature Genetics simultaneously presented findings by two different groups of competing scientists, which link the "Dysferlin" gene to human chromosome 2p13 and to muscular dystrophy.
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II. What are Muscular Dystrophy Diseases?
Muscular dystrophy is an inherited genetic disease, but occasionally MD can result from a random mutation in one or more genes even if there is no known previous family history of muscular dystrophyt. "Dystrophy" is of Greek and Latin origin meaning "faulty nutrition". The name refers to the apparent muscle degeneration that is associated with the disease. The degeneration of the muscle is caused by defects in genes for muscle proteins or by deficiencies of the enzyme creatine kinase. For several major forms of MD, gene flaws have been linked to a single chromosome.
What muscular dystrophy does:It affects people regardless of age, race or gender. It causes a degeneration of skeletal muscles that are responsible for movement. A major symptom is weakness that affects gastrocnemius muscles first in young adults. It is usually transmitted as parental genes. The different forms may be x-linked, autosomal dominant or autosomal recessive.
Classification of MD There are at least nine major forms of MD of which a few are rare, such as Miyoshi myopathy. The different forms of MD are classified according to age of onset, the muscles they affect, severity, the types of muscles affected, symptoms, and the inheritance pattern of the disease. Histologically, abnormalities of the skeletal muscles can be used to differentiate between each form of MD. Some of the different forms of muscular dystrophy can be found in the following table.
Table 1. Classification of Muscular Dystrophy
| Name | Age of Onset | Muscles Affected |
| Becker | 2-16 years | pelvis, upper arms, upper legs |
| Distal | adulthood | hands or lower legs initially |
| Duchenne | 2-6 years | pelvis, upper arms, upper legs |
| Limb-girdle | teens to early adulthood | hips and shoulders initially |
| Mioshi myopathy | childhood or early adulthood | proximal hip and leg muscles first |
| Myotonic (aka Steinert's Disease) | Early childhood to early adulthood | face, feet, hands, front of neck |
| Oculopharyngeal (OPMD) | 40-60 years | eyelids and throat initially |
MD Diagnosis. There are many diagnostic tools that can identify the disease. A medical history and physical evaluation from a medical doctor is a very simple and effective method of diagnosing the disease. Other more technical diagnostic methods include a muscle biopsy in which a piece of muscle can be examined under a microscope for protein abnormalities, DNA testing, electromyograms which measures electrical impulses coming from the muscle, nerve conduction velocity (NCV) which tests for nerve function using electrodes, or a blood enzyme test. An enzyme called creatine kinase has elevated levels in patients with muscular dystrophy because degenerating muscles leak enzymes causing elevated blood serum levels. (cite MD home page)
MD Treatments include medications like corticosteroids or respiratory care. Deep breathing and coughing help to relieve some of the symptoms such as . Physical and occupational therapy are also helpful. (MDA home page) New genetic therapy is currently being tested by the Muscular Dystrophy Researchers.Figure 1. explains the process of proposed treatments. Basically, the therapeutic gene is put into a modified virus which can them deliver the gene to muscles cells. The muscle cells cna then "read" the encoded protein and express the correct, non mutated protein. A drawback to this kind of therapy is that the virus can illicit an immune response, and the body may reject the virus.
1. First the therapeutic gene is put into a modified virus. A virus is an efficient delivery vehicle because it can get the gene into many cells.
2. Billions of the modified virus are injected into a foot muscle of the participant.
3. The virus will then invade some of the individual muscle cells and the new gene can begin producing functional protein.
Illustration From: http://www.mdausa.org/news/genetherapy.html
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III. Methods for Locating the LGMD2B Gene
Locating the LGMD2B gene.Robert H. Brown Jr. and colleagues from Massachusetts General Hospital, mapped the Dysferlin or MM gene (cite) at almost the same time Kate Bushby’s team of scientists from the University of Newcastle in England mapped the LGMD2B gene to the same chromosome 2p13. (Brown, et al) Both genes for MM and LGMD were found in the same genetic interval between markers GGAA-P7430 and D2S2109.
Kate Bushby and colleagues identified the LGMD2B gene by positional cloning - a process by which genes are cloned based upon their location on a chromosome. Bushby's team identified approximately where the gene was by genotyping microsatellite markers. Exchanging of information with other scientists confirmed the approximate position. A YAC (yeast artificial chromosome was) was assembled. The LGMD2B gene is 36,133 bp long. After YAC assembly, a screening of the library is necessary. Chosen YACs were then analyzed by PFGE (pulse field gel electrophoresis) and the Southern-blot hybridization technique [link to 461 notes]. Long strands of DNA can be efficiently analyzed by chromosome walking. [Link to 461 chap 6] The ESTs predicted the genetic map because they correspond to different genes. The DNA corresponding to the ESTs was amplified using a technique called Polymerase Chain Reaction (PCR) [link to 461 notes]. Northern blot analysis was used to copy the cDNA sequence. A total of 6.2 kb of cDNA was sequenced and assembled by way of cDNA library screening.
Mutation Analysis. The goal of this experiment is to detect mutations [461 notes], which may be related to causes of muscular dystrophy. DNA from affected individuals or carriers was analyzed by SSCPand heteroduplexes. A total of nine different mutations were discovered. Three different frame-shift mutations were found at exons 3, 50, and 51. Two nonsense mutatations were found at exons 20 and 49. There are three missense mutations at exons 36, 54, and 50, and one altered protein at exon 52 (Brown, et al).
Table 2. Mutations and Consequences in the Dysferlin Gene
| Phenotype | Exon | Mutation | Base Pair Exchange | Consequence |
| MM | 3 | 537insA | insertion of A at 537 | Frameshift |
| MM | 20 | Q605X | CAG--> TAG at 2186 | Nonsense |
| DMAT | 50 | 596delG | deletion of G at 5966 | Frameshift |
| MM | 50 | 596delG | deletion of G at 5966 | Frameshift |
| MM | 49 | e1883x | GAG-->TAG at 5870 | Nonsesnse |
| MM | 52 | 6319+1G to A | Altered 5' splice site | Altered Protein |
| MM, LGMD | 36 | I1298V | ATC-->GTC at 4265 | Missense |
| MM | 50 | H1857R | CAT-->CGT at 5943 | Missense |
Methods for identifying MM gene.
Using basically the same methods as the Bushby group, Robert H. Brown and
colleagues first found PAC clones and identified repeat sequences. Repeat
markers were analyzed using PCR. ESTs within the appropriate interval on
the gene were analyzed using the northern-blot technique. Brown's group
of researchers concluded that the mutations in the dysferlin gene cause
muscular dystrophy and identical mutations can produce different phenotypes
of the disease.
Figure 3. Caenorhabditis elegans
Illustration from http://www.ed.ac.uk/~mbx/C_elegans/Ce_intro.html
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The dysferlin gene, also known as LGMD2B, is 6.9 kb long and was found between physical genetic markers GGAA-P7430 and D2S2109. Both groups of scientist found the gene between these markers. There are a total of 55 exons encoding the DNA. Sinle strand conformation polymorphism (SSCP) was used to screen each exon for mutations (Brown, et al).
Bushby and colleagues found the genetic interval by pooling DNA from ordered YACs and screening ESTs at random. PCR was used to localize ESTs (expressed sequence tags) to individual YACs. The ESTs that were found to have an expression pattern were linked to independent genes. The genes were all found in different muscles. The protein dysferlin was found in skeletal muscle, heart tissue and placenta.
The protein dysferlin is homologous to the C. elegans protein fer-1. The mutant protein fer-1 is found in C. elegans with abnormal spermatogenesis (sperm production). The sperm from a C. elegan with a mutant fer-1 protein have inadequate mobility, and present problems with fusion of membranous organelles in spermatozoa. Fer-1 is hydrophilic and is a very large protein, like dysferlin. It was suggested by Robert H. Brown and colleagues that the protein dysferlin may signal events such as membrane fusion, which is analogous to the fer-1 protein of C. elegans.
Mutations in the dysferlin gene are thought to cause muscular dystrophy. Nine mutations were detected (see figure 2 and table 2), and shown to be expressed when inherited homozygously (one allele from each parent). Six of the nine mutations are expected to block expression of the dysferlin protein. The mechanism of how deficiency of the protein dysferlin causes muscular dystrophy is not yet understood.
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Muscular dystrophy is a genetic disease that does not discriminate. It affects people of all ages and gender with variable degrees of severity, and remissions do not occur. Every year millions of dollars in research go into looking for a cause and a cure. The discovery of Dysferlin is a very important piece of the Muscular Dystrophy puzzle, since up to 10% of all muscular dystrophy cases are likely to be correlated with faulty dysferlin protein encoded by mutated genes.
Implications for future research and therapy. Future research will reveal the biological functions of the novel protein, dysferlin. Future research will also include studying of dysferlin mutant and normal, in tissues other than muscle as well as in muscle, the obvious target of MD. Dysferlin is thought to play a role in maintaining the integrity and strength of muscle cell membranes. Researchers can now expand genetic studies because dysferlin is the first MD gene found to affect only the distal muscles. A diagnostic tool for detection of LGMD2B mutations can be devised to provide genetic testing. The role of the LGMD2B in other forms of muscular dystrophy can now be examined. Other studies will be performed to determine if gene therapy with LGMD2B can reverse muscle deterioration.
To see frequently asked questions about muscular dystrophy go to the Muscular Dystrophy Association’s home page http://www.mdausa.org/.
