Muscular
dystrophies are a group of genetic
disorders that are characterized by progressive muscular weakness and atrophy.
The two related conditions inherited in an X-linked recessive pattern which
affect the skeletal muscles, are Duchenne and Becker muscular dystrophies (DMD
& BMD). These two conditions occur exclusively in males and together effect
1 in 3,500 to 5,000 newborn males worldwide. Though differing in severity, age
of onset and rate of progression the Duchenne and Becker muscular dystrophies
have similar signs and symptoms and are caused by different mutations in the
same gene located on the X chromosome. Becker muscular dystrophy is
distinguished from the Duchenne muscular dystrophy by its milder phenotype and
slower rate of progression. In Duchenne muscular dystrophy there is early onset
of progressive muscular weakness and by the age of 12 the patients are wheel
chair bound. In Becker muscular dystrophy has milder phenotype and remain
ambulatory till the age of 16 (Blake et al., 2002).
Dystrophin
Gene: The official name of the gene
is“dystrophin” and DMD is the official gene symbol. DMD provides instructions
for making a protein called dystrophin primarily located in the skeletal and
cardiac muscle. Dystrophin gene is the largest gene found in the nature, measuring
2.4 Mb. Location: Xp21.2. The number of exon count is 86.
The
Dystrophin Protein: Dystrophin is 427-kDa cytoskeletal protein can be
organized into four separate regions the actin-binding domain at the NH2
terminus, the central rod domain, the cysteine-rich domain, and the
COOH-terminal domain. Dystrophin functions in connecting the cytoskeleton of a
muscle fiber to the surrounding extracellular matrix through the cell membrane.
The dystrophin-associated protein
complex (DPC) was identified which spans the cell membrane and links the
actin-based cytoskeleton to the muscle basal lamina. Thus the DPC can be
thought of as a scaffold connecting the inside of a muscle fiber to the
outside. In striated and smooth muscles, the dystrophin complex normally spans
the plasma membrane and provides a strong connection between the cytoskeleton
and extracellular matrix (Blake et al., 2002).
The changes
that occur in dystrophin-deficient muscle are complex; absence of
dystrophin may cause pathology by more than a single distinct mechanism. The
dystrophin-deficient muscle cells have demonstrated the abnormalities in
calcium homeostasis, increased susceptibility to oxidative toxins, and stress
enhancable membrane permeability. The absence of dystrophin prevents assembly
of the DGC and resulting sarcolemma is fragile and highly susceptible to
injuries that can trigger muscle cell death. As muscle tissue is lost, it is
gradually replaced by connective tissue and adipose (fat) cells. Because
Duchenne and Becker muscular dystrophies result from faulty or missing
dystrophin, these conditions are classified as dystrophinopathies. The mutation
in DMD cause premature translation termination (nonsense and frame shift
mutations), while in BMD patients functional dystrophin is reduced either in
molecular weight (due to in-frame deletions) or in expression level.
Mutations
type in DMD and BMD.
Type of mutations
|
Duchenne Muscular Dystrophy
|
Becker Muscular Dystrophy
|
Deletions
|
65%
|
85%
|
Duplications
|
6-10%
|
6-10%
|
Point mutations
|
20-30%
|
20-30%
|
Small/deletions/insertions
|
||
Splicing mutations
|
Point
mutations:
Most of the point mutations lead to premature translational termination due to
nonsense (34%), frameshift (33%), splice site (29%) and missense (4%) mutations
in the dystrophin gene. (Shawky et al., 2014).
Tan et al. (2010) documented
the large deletions in the dystrophin gene in specific areas called
“hotspot”. Two main hotspot regions have
been detected, region I mid-distal region ranging from exon exons 42 - 45, 47,
48, 50 - 53, 60, and region I at the proximal portion of the gene extending
from exons 1, 3, 4, 5, 8, 13, 19.The documentation of Sironi et al. in
2006 and Walmsley et al. in 2010
revealed the deletion hotspots domains are in the exons 40–55 and exons 45–53 respectively. (Tan et al ., 2010).
Maher & Rath (2014) used the Digital
signal processing with 91% success rate to detect the mutation hot-spots and
exon-deletion in DMD gene(Maher &Rath , 2014).
Monaco et al. (1988) explained in the reading frame theory if a deletion does
not shift the normal open reading frame it will lead to the expression of an
internally truncated transcript consistent with BMD. If the deletion creates a
translational frameshift mutation the premature end in the translation will result
in the truncated protein or instability in the protein structure seen in DMD
phenotype (Monaco et al., 1988). The mutation leading to DMD results in the
absence or much reduced levels of dystrophin protein while in patients with BMD
some functional protein made. The exons do not necessarily contain the integral
number of triplet codons, the deletion of a DNA segment consisting of one or
more exons with a non-integral number of triplet codons would cause a shift in
the reading frame (Malhotra et al., 1988).
Malhotra et al. (1988) in the extensive study
done on the mutations of DMD gene in Duchenne and Becker muscular dystrophy did
not find a simple correlation between severity of phenotype and type of
deletion. They studied two out of six patients studied by Monaco et.al and
found that deletion occurring over the actin binding domain result in a mild
phenotype thus indicating that this region might not be essential to the
function of the protein. The patients having the deletion from 3 to 7 codon and
disruption of the translation in-frame
and resulting in BMD mild phenotype may be attributed to the differential
splicing strategy which will mask the frameshift mutations and will create the
in-frame mutation and thus a partially functional protein, though no concrete
evidence has been found. Another mechanism that can generate a partially
functional protein (with 3 to 7 exon deletion) can be the use of a new in-frame
translational start site immediately down-stream from the deletion. Three ATG
codon were found in exon 8 and the sequences flanking one of these ATGs conform
to Kozak’s consensus sequence. This reinitiation of internal ATG is never
observed in the eukaryotic mRNA and is only seen in synthetic constructs. The
third proposition for the exons 3 to 7 deletion cases is the presence of second
promoter possibly 110-kb intron downstream between exons 7 and 8 though the
evidence of existence of such promoter is not found. The consistent and
authentic proof of existence of phenotype and genotype of DMD is an open
research topic.
Goyenvalle et.al
(2004) have documented that most
mutations in the dystrophin gene create a frameshift mutation or a nonsense
mutation resulting in severe Duchenne muscular dystrophy. Alternative exons
splicing occurring naturally, as exon skipping at low frequency, sometime
eliminates mutation and produces a shorter but functional protein. This
phenomenon of Exon skipping and causing in-frame mutation that can naturally
occur during dystrophin mRNA processing can restore the reading frame and can
give rise to a rare “Reverent” that contain a shortened protein is being used
in the treatment of DMD. Adeno-Associated virus vector (AAV-2)
expressing antisense sequences which link with the small nuclear RNA and
facilitate their inclusion into mRNA processing spliceosome thus create a
possibility of a sustained and sequence specific modification of the targeted
mRNA structure (Goyenvalle et al., 2004).
Wilton et.al (2006) discussed the strategy
of exon skipping in the restoration of functional dystrophin protein. They described a panel of antisense
oligonucleotide (AO), appropriately designed and directed at splicing motifs
across the dystrophin gene transcript in vitro. These exon specific AO targeted
and bound with the exon and thus block exon recognition and/or spliceosome
assembly thus remove the targeted exon from the mature mRNA (Wilton et al.,
2009).
Kinali et.al(2009)
studied that oligonucleotides targeted
to splicing elements (Splice switching oligonucleotides) in DMD pre-mRNA can
lead to exon skipping and restoration of the open reading frame, and production
of functional dystrophin protein in vivo and vitro to benefit the patients
with this disorder. The team assessed and documented the dose efficacy and
safety of the drug (AVI-4658) that is a splice-switching oligonucleotide and
skips the exon 51 in dystrophin mRNA. The participants whose deletions could be
restored by skipping the exon 51 had the exon deletions as exon 45-50, 47-50,
48-50, 49-50, 50; 52 or 52-63. The team of researchers restored dystrophin
essentials domains for its functions with the use of potential disease
modifying drug for DMD in vivo. The researchers used a splice-switching
oligonucleotide called AVI-4658 that induces the skipping of dystrophin exon 51
and blocking its translation in patients with relevant deletion thus restoring
the open reading frame and induced the dystrophin protein expression after the
intramuscular injection. (Kinali et al., 2009).
Aartsma et.al (2002)
discussed the binding of the antisense
oligonucleotides to the exon-internal sequences in the pre-mRNA the splicing is
manipulated in the manner that the targeted exon is skipped and the slightly
shorter in-frame transcript is generated. The researches skipped the exon
46 efficiently via the oligonucleotide antisense in DMD gene with exon 45
deletion. They also identified other antisense oligonucleotides that could skip
11 other DMD exons. These antisense oligonucleotides could restore 50% of
deletions and 22% of duplications reported in the Leiden DMD-mutation database
(Aartsma-Rus et al., 2002).
The correlation between the phenotype and
type of deletion mutation is of diagnostic and prognostic significance. The
distribution and frequency of deletions spanning the entire locus of the gene
suggests that many “in-frame” deletions of the dystrophin gene are not detected
as individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD
clinical features.
Since the newly introduced molecular therapies have
bright chances to revolutionize outcome in patient’s genetic diagnosis is
essential to identify the molecular defect in each patient. I intend to study
the distribution of genotype and match it with the phenotype of the DMD gene to
re-enforce the “Reading frame theory” and find more clues of non-detection of
the DMD deletion not presenting as a phenotype.
References:
Aartsma-Rus, Annemieke, Mattie Bremmer-Bout, Anneke AM
Janson, Johan T. den Dunnen, Gert-Jan B. van Ommen, and Judith CT van Deutekom.
“Targeted Exon Skipping as a Potential Gene Correction Therapy for Duchenne
Muscular Dystrophy.” Neuromuscular Disorders 12 (2002): S71–77.
Aartsma-Rus, Annemieke, Anneke AM Janson, Wendy E.
Kaman, Mattie Bremmer-Bout, Johan T. den Dunnen, Frank Baas, Gert-Jan B. van
Ommen, and Judith CT van Deutekom. “Therapeutic Antisense-Induced Exon Skipping
in Cultured Muscle Cells from Six Different DMD Patients.” Human Molecular
Genetics 12, no. 8 (2003): 907–14.
Beggs, A. H., E. P. Hoffman, J. R. Snyder, Kiichi
Arahata, Linda Specht, Frederic Shapiro, Corrado Angelini, Hideo Sugita, and L.
M. Kunkel. “Exploring the Molecular Basis for Variability among Patients with
Becker Muscular Dystrophy: Dystrophin Gene and Protein Studies.” American
Journal of Human Genetics 49, no. 1 (1991): 54.
Blake, DerekJ. ,Andrew Weir, Sarah E.Newey, and Kay
E.Davies. “Function and Genetics of Dystrophin and Dystrophin-Related Proteins
in Muscle.” Physiological Reviews 82,
no.2 (2002):291-329
Cirak, Sebahattin, Virginia Arechavala-Gomeza, Michela
Guglieri, Lucy Feng, Silvia Torelli, Karen Anthony, Stephen Abbs, et al. “Exon
Skipping and Dystrophin Restoration in Patients with Duchenne Muscular
Dystrophy after Systemic Phosphorodiamidate Morpholino Oligomer Treatment: An
Open-Label, Phase 2, Dose-Escalation Study.” The Lancet 378, no. 9791
(2011): 595–605.
Cirak, Sebahattin, Lucy Feng, Karen Anthony, Virginia
Arechavala-Gomeza, Silvia Torelli, Caroline Sewry, Jennifer E. Morgan, and
Francesco Muntoni. “Restoration of the Dystrophin-Associated Glycoprotein
Complex after Exon Skipping Therapy in Duchenne Muscular Dystrophy.” Molecular
Therapy 20, no. 2 (2011): 462–67..
Goyenvalle, Aurélie, Adeline Vulin, Françoise
Fougerousse, France Leturcq, Jean-Claude Kaplan, Luis Garcia, and Olivier
Danos. “Rescue of Dystrophic Muscle through U7 snRNA-Mediated Exon Skipping.” Science
306, no. 5702 (2004): 1796–99.
Kinali, Maria, Virginia Arechavala-Gomeza, Lucy Feng,
Sebahattin Cirak, David Hunt, Carl Adkin, Michela Guglieri, et al. “Local
Restoration of Dystrophin Expression with the Morpholino Oligomer AVI-4658 in
Duchenne Muscular Dystrophy: A Single-Blind, Placebo-Controlled,
Dose-Escalation, Proof-of-Concept Study.” The Lancet Neurology 8, no. 10
(2009): 918–28..
Koenig, M., A. H. Beggs, M. Moyer, S. Scherpf, K.
Heindrich, T. Bettecken, G. Meng, et al. “The Molecular Basis for Duchenne
versus Becker Muscular Dystrophy: Correlation of Severity with Type of
Deletion.” American Journal of Human Genetics 45, no. 4 (1989): 498.
Koenig, Michel, and L. M. Kunkel. “Detailed Analysis
of the Repeat Domain of Dystrophin Reveals Four Potential Hinge Segments That
May Confer Flexibility.” Journal of Biological Chemistry 265, no. 8
(1990): 4560–66.
Koenig, M., A. P. Monaco, and L. M. Kunkel. “The
Complete Sequence of Dystrophin Predicts a Rod-Shaped Cytoskeletal Protein.” Cell
53, no. 2 (1988): 219–28.
Malhotra, S. B., K. A. Hart, H. J. Klamut, N. S.
Thomas, S. E. Bodrug, A. H. Burghes, M. Bobrow, et al. “Frame-Shift Deletions
in Patients with Duchenne and Becker Muscular Dystrophy.” Science 242,
no. 4879 (1988): 755–59.
Mann, Christopher J., Kaite Honeyman, Andy J. Cheng,
Tina Ly, Frances Lloyd, Sue Fletcher, Jennifer E. Morgan, Terry A. Partridge,
and Stephen D. Wilton. “Antisense-Induced Exon Skipping and Synthesis of
Dystrophin in the Mdx Mouse.” Proceedings of the National Academy of
Sciences 98, no. 1 (2001): 42–47.
Meher, J. K., and A. K. Rath. “Detection of
Mutation-Hotspots and Exon-Deletions Using Digital Signal Processing in
Duchenne Muscular Dystrophy (DMD) Gene.” Accessed October 27, 2014.
http://www.iosrjournals.org/iosr-jdms/papers/Vol13-issue1/Version-3/I013133439.pdf.
Monaco, Anthony P., Corlee J. Bertelson, Sabina
Liechti-Gallati, Hans Moser, and Louis M. Kunkel. “An Explanation for the
Phenotypic Differences between Patients Bearing Partial Deletions of the DMD
Locus.” Genomics 2, no. 1 (1988): 90–95.
Nakamura, Akinori, and Shin’ichi Takeda.
“Exon-Skipping Therapy for Duchenne Muscular Dystrophy.” The Lancet 378,
no. 9791 (2011): 546–47.
Petrof, Basil J. “The Molecular Basis of
Activity-Induced Muscle Injury in Duchenne Muscular Dystrophy.” Molecular
and Cellular Biochemistry 179, no. 1–2 (1998): 111–24.
Shawky, Rabah M., Solaf M. Elsayed, Theodor Todorov,
Andree Zibert, Salem Alawbathani, and Hartmut H.-J. Schmidt. “Non-Deletion
Mutations in Egyptian Patients with Duchenne Muscular Dystrophy.” Egyptian
Journal of Medical Human Genetics, 2014.
Tan, J., James Hsian-Meng Chan, Kim-Lian Tan, Azlina
Ahmad Annuar, Moon-Keen Lee, Khean-Jin Goh, and Kum-Thong Wong. “Dystrophin
Gene Analysis in Duchenne/Becker Dystrophy in a Malaysian Population Using
Multiplex Polymerase Chain Reaction.” Neurology Asia 15, no. 1 (2010):
19–25.
Tayeb, Mohammed T. “Deletion Mutations in Duchenne
Muscular Dystrophy (DMD) in Western Saudi Children.” Saudi Journal of
Biological Sciences 17, no. 3 (2010): 237–40.
Van Deutekom, Judith CT, Mattie Bremmer-Bout, Anneke
AM Janson, Ieke B. Ginjaar, Frank Baas, Johan T. den Dunnen, and Gert-Jan B.
van Ommen. “Antisense-Induced Exon Skipping Restores Dystrophin Expression in
DMD Patient Derived Muscle Cells.” Human Molecular Genetics 10, no. 15
(2001): 1547–54.
Wilton, Steve D., Abbie M. Fall, Penny L. Harding,
Graham McClorey, Catherine Coleman, and Susan Fletcher. “Antisense
Oligonucleotide-Induced Exon Skipping across the Human Dystrophin Gene
Transcript.” Molecular Therapy 15, no. 7 (2007): 1288–96
No comments:
Post a Comment