Duchenne Muscular Dystrophy Promoters
Presented by:
Dystrophin gene and protein
The DMD gene is the largest known gene, consisting of approximately 0.1% of the human genome.
The main product of the gene in muscle is a 427 kDa rod-shaped protein called dystrophin.
Dystrophin consists of four domains: an N-terminal actin-binding domain, 24 spectrin-like triple-helix repeats, a cysteine-rich domain and a unique C-terminal domain.
Four 5' promoters
Four 5' promoters regulate, in a cell-type-specific manner, the expression of full-length gene product:
M promoter (Muscle dystrophin promoter)
B promoter (Brain dystrophin promoter)
P promoter (Purkinje cell dystrophin promoter)
L promoter (lymphoblastoid cell dystrophin promoter)
Four internal promoters
Four internal promoters regulate the expression of smaller proteins which lack important parts of dystrophin, and probably have different functions.
Dp71 promoter (ubiquitous protein Dp71)
Dp116 promoter
Dp140 promoter (cell-type-specific proteins)
Dp260 promoter
While the function of muscle dystrophin is partially understood, the function of nonmuscle dystrophin and that of the smaller products of the gene is yet to be uncovered.
Dystrophin promoters
Muscle dystrophin promoter (M promoter)
M promoter is active in skeletal muscle, heart muscle and smooth muscle fibers, and in glial cells.
Like many other muscle-specific promoters, the M promoter becomes active after differentiation of myogenic cells.
Sequence analysis and comparison of the muscle dystrophin promoter of human, mouse and chicken has revealed several conserved motifs which are characteristic of many muscle-specific genes.
Brain dystrophin promoter (B promoter, also called cortex or C promoter)
B promoter is located 75–300 kilobase pairs (kbp) upstream from the M promoter, and more than 400 kbp upstream from the common exon 2.
The promoter is active in subpopulations of neurons but not in glial cells.
Purkinje cell dystrophin promoter (P promoter)
P promoter is located between the M promoter and the common second exon.
The mRNA regulated by this promoter was found in Purkinje neurons of the brain.
Interestingly, the P promoter also regulates the expression of an alternatively spliced mRNA with a terminator codon positioned 27 nucleotides after the initiator AUG. The biological significance of this mRNA is not clear.
The lymphoblastoid cell dystrophin promoter (L promoter)
L promoter is positioned more than 500 kbp upstream from the B promoter.
While the unique first exon of the transcripts regulated by the M, B and P promoters is spliced to exon 2, the L first exon is spliced to exon 3.
Thus, the encoded protein lacks a large part of the actin binding domain of dystrophin.
Dp71 promoter
Dp71 consists of the C-terminal and the cysteine-rich domains of dystrophin. It is the most abundant DMD gene product in non muscle tissues.
Dp71 was found in most tissue tested, however, a detailed analysis of Dp71 promoter activity during development revealed a pattern of restricted activity with regard to cell type and developmental stage.
Dp116 promoter
Dp116 consists of the last two spectrin-like repeats, the cysteine-rich domain and the C-terminal domain of dystrophin. The promoter of Dp116 is localized in intron 55 of the DMD gene.
Since Dp116 and its mRNA were found only in Schwann cells of the peripheral nervous system, it is likely that the activity of Dp116 promoter is restricted to these cells.
Dp140 promoter
Dp140 consists of the last five spectrin-like repeats, the cysteine-rich domain and the C-terminal domain of dystrophin.
The first (unique) exon and probably the promoter of Dp140 are localized in intron 44 of the DMD gene.
Dp140 promoter is mainly active in various parts of the brain, and in the kidney.
Dp260 promoter
Dp260 consists of 15 spectrin-like repeats, the cysteine rich domain and the C-terminal domain of dystrophin.
Its first unique exon and most likely the promoter are situated in intron 29 of the DMD gene.
Dp260 is expressed in the retina, and at lower levels in the brain and in cardiac muscle.
Mutations in the dystrophin gene
The most common changes in dystrophin are intragenic deletions, which account for 65% of dystrophin mutations.
Deletions are found in about 60–65% of patients with DMD and BMD, and the frequency of duplications may range from 5% to 15%, possibly as a result of the different sensitivity of the techniques used.
Mutations in the dystrophin gene
The remaining cases are thought to be caused by a combination of small mutations (most commonly point mutations resulting in nonsense or frame-shift mutations), pure intronic deletions, or exonic insertion of repetitive sequences.
Disease severity: deletions, duplication and the frame-shift hypothesis
There is no simple relation between the size of the deletion and the resultant clinical disease.
For example, the deletion of small exons, such as exon 44, typically results in DMD.
However, large deletions, which may involve nearly 50% of the gene, have been described in patients with BMD.
The central and distal rod domains seem to be almost dispensable functionally with some deletions in this region being associated with myalgia and muscle cramps, but not weakness.
Disease severity: deletions, duplication and the frame-shift hypothesis
Some patients may even present with an isolated increase in the concentration of creatine kinase in the plasma.
This has been shown in patients with in frame deletions in exons 32–44, 48–51, or 48–53 all of whom had normal or near normal dystrophin concentrations.
The effects on the phenotype depend, therefore, not so much on the extent of a deletion (and the same applies for duplication) but on whether or not it disrupts the reading frame.
Disease severity: deletions, duplication and the frame-shift hypothesis
Effects of different genomic deletions on the reading frame of the
dystrophin gene (A). The removal of exon 4 (B) and of exon 7-11
(C) maintens the open-reading frame. The deletion of exon 7 leads
to the loss of the open-reading frame (D).
Disease severity: deletions, duplication and the frame-shift hypothesis
A further observation is that very different deletions (in term of size and position) may have a very similar severe phenotype. The reason for this effect might be the occurrence of nonsense-mediated RNA decay.
Mutations that maintain the reading frame (in-frame) generally result in abnormal but partly functional dystrophin and are associated with BMD.
Effects of different mutations on dystrophin protein. The complete protein (top). An in-frame deletion of part of the spectrin-like domain leads to a shortened but functional protein, that still contains the cysteine-rich and carboxy-terminal domains (middle). An out-of frame deletion (bottom) results in a truncated protein that is rapidly degraded in the muscle.
Disease severity: deletions, duplication and the frame-shift hypothesis
In patients with DMD, deletions and duplications disrupt the reading frame (frame-shift), resulting in unstable RNA that eventually leads to the production of nearly undetectable concentrations of truncated proteins.
This reading frame hypothesis holds for over 90% of cases and is commonly used both as a diagnostic confirmation of dystrophinopathies and for the differential diagnosis of DMD and BMD.
Disease severity: deletions, duplication and the frame-shift hypothesis
Exceptions to the reading-frame hypothesis
Exceptions to the reading-frame hypothesis do exist and these include patients with BMD who carry frame-shift deletions or duplications and patients with DMD with in frame deletions or duplications.
Patients with BMD with frame-shift mutations have several well-characterised deletions or duplications in the 5' end of the gene (exons 3–7; 5–7; 3–6) or further downstream (exons 51, 49–50, 47–52, 44 or 45).
The most common event that allows these patients to produce at least some dystrophin is exon skipping, which occurs via alternative splicing.
The reason for the discrepancy of the observed phenotype is not fully understood and might relate to the efficiency and type of exon skipping events, which create larger messenger RNA deletions, some of which are in-frame and therefore functional.
Exceptions to the reading-frame hypothesis
An alternative mechanism for the exception to the rule in patients with BMD and out of frame deletion in exons 3–7 is the presence of an additional translation start site in exon 8.
This hypothesis comes from RNA studies that did not show any exon skipping events in these patients and showed the presence of a correct junction of exon 2 to exon 8, which suggests there was an out-of-frame messenger.
Exceptions to the reading-frame hypothesis
Exon-skipping events
The mechanisms that lead to exon skipping in patients with out-of-frame mutations are poorly understood and are likely to be caused by several factors.
Several patients with out-of-frame mutations will be able to produce an appreciable amount of dystrophin.
Although exon-skipping events occur more commonly in particular regions of the dystrophin gene, the type of deletion does not necessarily predict if an exon-skipping event will occur.
Exon-skipping events
13 patients with an exon 44 deletion, eight had DMD, four had BMD, and one had an intermediate phenotype between DMD and BMD.
Of nine patients with an exon 45 deletion, seven developed DMD and two had BMD.
One likely contributory factor could be the differences between deletion breakpoints within introns.
Exon-skipping events
Patients with an apparently identical exonic deletion are therefore likely to have different genomic breakpoints.
For example, a deletion of exon 44 means that the 5' and the 3' breakpoints fall in intron 43 and intron 44, respectively.
In such cases it is likely that both introns will have lost different regions.
Exon-skipping events
Intronic sequences might contain motifs that affect gene splicing. Different deletion breakpoints might, therefore, affect exon-skipping events.
Recently described the occurrence of a complex exon-skipping event in skeletal muscle of patients with isolated exon 5 deletions, who had either DMD or BMD.
Patients with DMD produce a lot of molecules in which exons 6 and 9 are skipped.
Exon-skipping events
By contrast, patients with BMD had an abundant exon-5-deleted transcript, thought to be in frame.
Therefore it is suggested that different deletion breakpoints are implicated in this complex dystrophin splicing modulation.
Point mutations
20–35% of patients with DMD and BMD do not have deletions or duplications of the dystrophin gene.
The identification of mutations in these patients has been hampered by the size of the dystrophin gene.
One of the specialist techniques used is the protein truncation test, which can be applied to muscle RNA.
Point mutations
Most of the mutations so far identified in patients with DMD are nonsense point mutations or small frame-shift deletions or insertions; splice-site mutations are also quite common.