Mutations

Introduction

A mutation in the gene containing the genetic code for the dystrophin protein is found in nearly all Duchenne patients. There are different types of deletions, but the result of each mutation is the same: no functional dystrophin can be produced (see explanation about exon skipping). Nevertheless the type of mutation matters for some therapeutic approaches (exon skipping and PTC124), as these only apply to some specific mutations. I will here describe the most common mutations found in the dystrophin gene. How mutation-specific therapies apply for different mutations, is explained here.

From gene to protein

Genes contain the blueprint (genetic code) for proteins. Genes consist of DNA and are dispersed over 23 pairs of chromosomes, that are located in the nucleus of every cell in our body. The protein factory (the system that translates the genetic code into protein) is located outside the nucleus in the so called cytoplasm. As the DNA cannot leave the nucleus, genes and the protein factory are physically separated. Therefore, when a protein needs to be generated, a temporary copy is made from a gene. You can compare this with a recipe (gene) you need from a book (DNA) that cannot leave the library (nucleus). In order to prepare the recipe at home (cytoplasm) you make a copy of the recipe that you take with you. Temporary copies of a gene consist of RNA and are transported from the nucleus to the protein factory where the copy is translated into protein.

The genetic code within a gene is not continuous, but is dispersed over so called exons. In between exons reside introns, that do not contain protein information. Before the RNA can be transported to the protein factory to be translated into protein, these introns have to be removed and the exons joined together (figure 1). This process, called splicing, takes place in the nucleus. The resulting product only contains the genetic code (exons). This "messenger" RNA (mRNA) is subsequently transported into the cytoplasm and translated into protein.

Figure 1. The genetic code is dispersed over exons, which are interrupted by pieces of DNA that do not contain protein information (introns). First, an RNA copy is generated of a gene, then the introns are removed and exons are joined during the splicing process. The resulting messenger RNA contains the genetic code for a protein and can be translated by the protein factory. The dystrophin gene contains 79 exons and 78 introns.

When RNA is translated into protein, 3 RNA units code for a single protein unit (amino acid). A simplified example is shown in figure 2.

Figure 2. Translation of messenger RNA (mRNA) to protein. Each three RNA subunits (squares) codes for a protein subunit (circle). The translation starts with a start code for the initiation protein subunit, which is the same for all proteins (the red circle that says "start"). After this first subunit, different subunits (amino acids) follow. In this simplified example only two types of subunits are used (blue and yellow), but in reality there are over 20 different protein subunits. Proteins consist of tens, hundreds and sometimes thousands of subunits. For instance, the dystrophin protein contains 3685 protein subunits. A stop code is present at the end of the mRNA to indicate that protein translation is finished. After this stop signal, the completed protein is transported to its site of action (e.g. close to the muscle membrane for dystrophin). Due to the different properties of the protein subunits (big or small, soluble in water or in lipids, having an electric charge or not etc), the properties of a protein are determined by the amount of subunits and their individual properties.

Mutations

Genes contain the blueprint for proteins. If a gene contains a mistake (mutation) this will affect the protein product. Duchenne patients can not produce functional dystrophin protein due to mutations in their dystrophin gene. There are three types of mutations commonly found in Duchenne patients: deletions (the absence) of one or several exons, duplications of one or more exons, and small mutations (also called "point mutations").

Deletions

The majority (65%) of Duchenne patients have a deletion of one or more exons (Figure 3).

Figure 3. A deletion in the dystrophin gene. A normal dystrophin gene contains 79 exons, interrupted by 78 introns. In some Duchenne patients, part of the gene is missing (deletion). In this example exon 48, 49 and 50 are gone; the deletion starts after exon 47, in intron 47 and ends after exon 50 in intron 50. The deletion thus encompasses part of intron 47, exon 48, intron 48, exon 49, intron 49, exon 50 and part of intron 50. However, as only the exons contain the genetic code for protein, usually only the exons that are deleted are mentioned for deletion mutations: in this case this is thus a deletion of exon 48-50.

Deletions in Duchenne patients disrupt the genetic code of the dystrophin gene (exon 47 and exon 51 do not fit in figure 3). The consequences of this disruption are disastrous (figure 4).

Figure 4. The consequences of a disruption of the genetic code. On top, the original RNA and protein from figure 3 are shown. Below the genetic code is disrupted (in this case by the deletion of two RNA subunits). As each 3 RNA subunits contain the code for one protein subunit, this deletion has huge consequences: instead of the blue-yellow-yellow RNA code, the code is shifted to yellow-blue-yellow, and each subsequent code is also shifted to something else (disruption of genetic code). As a result, aberrant protein subunits are encoded starting at the mutation (green and orange instead of blue and yellow). As the function of a protein is determined by the characteristics of the protein subunits, a protein that contains aberrant subunits is not functional (e.g. it is impossible to build a model car with parts of a model plane).

Duplications

In a small part of Duchenne patients (7%) one or more exons are duplicated (figure 5).

Figure 5. A duplication in the dystrophin gene. A normal dystrophin gene contains 79 exons interrupted by introns. For some patients part of the gene is duplicated (in this example exon 48, 49 and 50). As with deletions, the duplication also involves the introns, but generally only the exons are mentioned: in this case a duplication of exon 48-50.

The consequences of duplications are similar to those of deletions: the genetic code is broken (figure 6), since exon 48 and exon 50 do not fit together (figure 5).

Figure 6. Disruption of the genetic code by a duplication. At the top the original RNA and protein from figure 3 are shown. At the bottom the genetic code is disrupted (through the duplication of two yellow RNA subunits). The consequences are similar to those of the deletion (figure 4): new protein subunit codes appear instead of the original ones and starting with the mutation, aberrant protein subunits are used to generate the protein. The aberrant subunits differ from those used in the deletion, but they are not the blue and yellow subunits needed for protein function (you cannot build a model car with parts from a model train either).

Small mutations

In slightly over a quarter of all Duchenne patients (28%) a small mutation is found in the dystrophin gene. For these patients all exons are present, but a single DNA subunit (and thus of the RNA copy) is changed (figure 7). This change causes a premature stop signal and the protein translation stops too soon.

Figure 7. Point mutations change only a single subunit of the DNA. As the RNA copy is based on the DNA original, this mutation is also copied into the RNA copy. In the example one of the blue subunits is altered into a red subunit. The blue subunit started a blue-yellow-yellow code, that is translated into the blue protein subunit. However, the red-yellow-yellow subunit encodes a "stop signal" (normally only present at the end of the mRNA). Due to this stop signal protein translation stops prematurely. The consequence is a non functional protein, because half the subunits are not included (half a model car does not work properly).

In addition to point mutations (one DNA subunit is replaced by another) it can also occur that one or two DNA subunits disappear or are added in an exon. This disrupts the genetic code in a similar way as deletions and duplications (figure 4 and figure 6).

Finally, small mutations can disrupt the splicing process (where introns are removed and exons are joined to form the genetic code). The start and end of all introns contain the same, specific combination of subunits, which are recognized by the splicing machinery (figure 8). If one of these specific subunits is changed, an exon will no longer be recognized, which can disrupt the genetic code (figure 9 and 10).

Figure 8.During the splicing process introns are removed and exons are joined. The splicing machinery responsible for this process recognizes the first two and the final two subunits of an intron (splice sites) and in this way an exon is "defined". All introns end with a yellow and a green subunit, and start with a green and red subunit.

Figure 9. A change in a subunit of the splice site (the first two and last two subunits of an intron) abolishes the recognition of exons by the splicing machinery. In this example the yellow-green code is changed into a yellow-red code, that is not recognized by the splicing machinery. Thus the exon is not defined and not included into the mRNA (figure 10).

Figure 10. The consequences of a "splice site" mutation. Due to a change in the second exon of a gene (e.g see figure 9), this exon is no longer recognized by the splicing machinery (it resembles an intron). As a result this exon is not included in the mRNA. This disrupts the genetic code (the first and third exon do not fit).