Background
In the last post, I covered how to visualize a bam file and find for mutations. In this case, we're calling anything that doesn't match the reference sequence a mutation.You can consider the "reference" sequence to roughly represent the DNA that the majority of people have. Not matching the reference sequence doesn't mean it's necessarily bad. All of us have millions of locations that will not match the reference sequence. However, not matching the reference sequence in a region known to cause a rare disease in someone who has said disease is a cause for further investigation.
Procedure
Start IGV, download Stacy's copy of FGD4, and type in FGD4 into the address bar at the top as shown in this previous post.
We are now ready to check the first gene in our list for CMT4 causing mutations. Each subtype of CMT4 is split up by the cuasal gene. If FGD4 is causing CMT4 in Stacy, then she will have CMT4H.
There are 5 mutations known to cause this disease in FGD4.
- FGD4 -- 5 mutations cause CMT4 Download Stacy's FGD4
- FIG4 -- 20 mutations cause CMT4
- GDAP1 -- 18 mutations cause CMT4
- MTMR2 -- 10 mutations cause CMT4
- PRX -- 2 mutations cause CMT4
- SBF2 -- 5 mutations cause CMT4
- SH3TC2 - 19 mutations cause CMT4
We are now going to look at the first gene FGD4 and check if Stacy has any of the mutations known to cause CMT4. See the previous post to learn how to open Stacy's copy of this gene. The five mutations in this gene that cause CMT4 are shown below.
| Mutation | Alias | Phenotype | Citations | |||
|---|---|---|---|---|---|---|
| 1 | c.[670C>T]+[670C>T] | [Arg224X]+[Arg224X] | CMT4H | Stendel et al., 2007 | ||
| 2 | c.[893T>C]+[893T>C] | [Met298Thr]+[Met298Thr] | CMT4H | Delague et al., 2007 | ||
| 3 | c.[893T>G]+[893T>G] | [Met298Arg]+[Met298Arg]/[Met298fs]+[Met298fs] | CMT4H | Delague et al., 2007 Stendel et al., 2007 | ||
| 4 | c.[1628_1629delAG]+[1628_1629delAG] | [Glu543fs]+[Glu543fs] | CMT4H | Stendel et al., 2007 | ||
| 5 | c.[1756G>T]+[1756G>T] | [Gly586X]+[Gly586X] | CMT4H | Stendel et al., 2007 | ||
Here are some details regarding the first mutation we will examine...
| Mutation Name | c.[670C>T]+[670C>T] |
|---|---|
| Mutation Alias | [Arg224X]+[Arg224X] |
| Description | Point mutation in coding region causing a premature stop mutation |
| Codon Change | From CGA to TGA (stop codon) |
| Phenotype | CMT4H |
- Open IGV and open FGD4.bam, as described in the previous post
- In the address bar (circled in yellow and labeled 1 in the image below) type in chr12:32,751,480-32,751,521 and press ENTER
Results
We can plainly see that the vertical region above the CGA circled in yellow is completely gray. This means there are no mutations in this region and she does not have TGA. This mutation is not the cause of CMT4 in Stacy. It would have been nice if we had been lucky and found the mutation causing CMT4 on our first try, but we'll have to keep looking.
| Mutation | Alias | Phenotype | Citations | |||
|---|---|---|---|---|---|---|
| 1 | ||||||
| 2 | c.[893T>C]+[893T>C] | [Met298Thr]+[Met298Thr] | CMT4H | Delague et al., 2007 | ||
| 3 | c.[893T>G]+[893T>G] | [Met298Arg]+[Met298Arg]/[Met298fs]+[Met298fs] | CMT4H | Delague et al., 2007 Stendel et al., 2007 | ||
| 4 | c.[1628_1629delAG]+[1628_1629delAG] | [Glu543fs]+[Glu543fs] | CMT4H | Stendel et al., 2007 | ||
| 5 | c.[1756G>T]+[1756G>T] | [Gly586X]+[Gly586X] | CMT4H | Stendel et al., 2007 | ||
Notice the blue blox in the image above. This blue box appears when we hover over an amino acid, which is in this case R (Arginine). This box contains the gene name FGD4 as well as the exon number where the amino acid we hovered over is located, which is Exon 5. Use this box to find the next CMT4 causing mutation along this gene, shown below.
| Mutation | Alias | Phenotype | Citations | |||
|---|---|---|---|---|---|---|
| 1 | ||||||
| 2 | ||||||
| 3 | Stendel et al., 2007 | |||||
| 4 | c.[1628_1629delAG]+[1628_1629delAG] | [Glu543fs]+[Glu543fs] | CMT4H | Stendel et al., 2007 | ||
| 5 | c.[1756G>T]+[1756G>T] | [Gly586X]+[Gly586X] | CMT4H | Stendel et al., 2007 | ||
This indicates that an amino acid change from Met (Methionine) to Thr (Threonine) at amino acid 298 may cause CMT4. Find amino acid 298 in Exon 7 by dragging the window from right to left. You may want to zoom out first since it's not too close by. You will find that there is another gray region, indicating that Stacy's 298th amino acid, located in in Exon 7 of the FGDP gene matches the reference sequence. This is not causing CMT4 either. Since the next mutation in the list is a Met to Arg amino acid change in the exact same location, we can exclude it as well.
Now, repeat these steps for mutations 4 to 5 at amino acids 543, and 586, respectively. These are located in Exons 13 and 14. These amino acids are completely gray, and therefore match the reference sequence.
| Mutation | Alias | Phenotype | Citations | |||
|---|---|---|---|---|---|---|
| 1 | ||||||
| 2 | ||||||
| 3 | Stendel et al., 2007 | |||||
| 4 | ||||||
| 5 | ||||||
Discussion
We have now examined all known CMT4 causing mutations in the gene FGD4. We have to keep in mind that there may be a CMT4 mutation in Stacy's FGD4 gene that has not previously been known to cause CMT4. We will finish checking all mutations already known to cause CMT4 before we start looking for novel mutations causing this disease.
**UPDATE
I also checked for the following CMT4 mutations:
**UPDATE
I also checked for the following CMT4 mutations:
- Arg275X (Arginine to stop codon mutation at the 275th codon in the gene)
- MUTATION WAS NOT FOUND
- A mutation in codon 542. Details were not available, but she matches the reference sequence in this codon, so whatever the mutation that causes CMT4 is, Stacy doesn't have it
- MUTATION WAS NOT FOUND
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