Mutations & Reversions

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:

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