CMT4 Next Steps

Next Steps

After learning that Stacy likely has a compound heterozygote mutation causing CMT4, I found out that SH3TC2 is a known CMT4 causal gene causing CMT4C. I decided to investigate what we know about SH3TC2 and CMT4 in general. My search led me to find out about the work being done by the CMTA (CMT Association). In 2010, the CMTA, working together with the NIH, finished testing ~350,000 compounds at 7 different dosage levels in the effort to find a treatment for CMT1A. CMT1A is somewhat related, but still different from CMT4C. CMT1A is caused by overexpression of the PMP22 gene whereas CMT4C is caused by having no functional copies of the SH3TC2 gene. Now that we have found the causal gene, I researched the state-of-the-art of CMT4 research. The following describes where CMT4 is in terms of developing a therapeutic intervention.

Final Set of Mutations

The previous post has identified what I believe to be the CMT4 causal mutations. For the sake of being thorough, I will wrap up the genetics analysis by checking all CMT causal mutations in this one single post. It will be constantly updated until I have covered every single disease associated mutation in every single CMT causal gene. This post is going to be long, so feel free to ignore it. I'm just consolidating all of the mutations on CMT causal genes and making them public in the spirit of this blog. Note: not all of the mutations I will check cause CMT. Some mutations on CMT causal genes cause different diseases (e.g. Macular Degeneration).

Likely Cause of CMT4 Identified: SH3TC2

Extremely Likely Cause for CMT4 Identified

I received an email from Brad Chapman who has an awesome blog that explains how to deal with next-generation sequencing data. He generously offered his assistance in tracking down the CMT4 causal variant(s) in Stacy. As opposed to manually inspecting each known causal variant, he proposed scanning all low frequency mutations within known causal genes. Thanks to his help, we now know that Stacy has two mutations in the known CMT4 gene SH3TC2  that are so rare, they never occur in the 1000 genomes dataset. These two mutations are...

Is MTMR2 Causing CMT4?

Commentary

A lot has happened over the weekend. I've received some very encouraging e-mails as more people find out about this project. I want to thank you all for your interest. I wasn't sure what to expect, but my hope is still to make this as accessible and easy as possible to remove some of the "aura of mystery" surrounding genetic analysis. We live in an era when we can go from receiving a digital file through the internet to literally searching for the genetic basis of a mendelian disease in a loved one. While manually inspecting variants should not replace expert analysis, it can only help. In fact, Stacy initially went to the hospital to determine the genetic cause of her disease. She went to the hospital and they told us that they would check only known genetic causes for CMT4 for ~$3,000 and that insurance wouldn't cover the cost. This is about the same time that I heard 23andMe was about to launch a pilot for sequencing 80x exomes for $1000. Stacy decided to wait for this and then have me personally analyze her exome. This blog is the result of that decision. We are on the brink of an era when full genome sequences for all of us will be common place. It is my hope that in the future, people will not have to wonder what genetic mutation is causing their mendelian disease. Even if the result is not currently actionable, they will at the very least know what technological advances need to be made before a symptom decreasing therapeutic intervention is developed.

Is GDAP1 Causing CMT4?

Background

We are moving on to the 3rd gene to test for CMT4 causing mutations. In the last two posts, we investigated genes FGD4 and FIG4. We found no known CMT4 causing mutations in Stacy's exome. We still have 4 out of 7 genes to check. As we move down the list, the chance that Stacy either was misdiagnosed or has a novel mutation causing CMT4 increases. However, we won't know for sure until we exhaust all known mutations. Here, we check GDAP1 for CMT4 causing mutations in Stacy's exome.

Is Gene FIG4 Responsible?

Background

In the last post, we investigated gene FGD4 and found no known CMT4 causing mutations in Stacy's exome. It was the first of 7 genes we plan to check. Although it would have been absolutely amazing to have found something in the very first gene we checked, there are plenty of mutations left to check. This time we will be investigating gene FIG4. Please review the last post in order to follow along in this analysis.

The FGD4 gene

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.

How to Visualize bam files & find mutations

1. Launch the IGV (integrated genomics viewer) maintained by the broad institute.
2. **Switch from the human hg18 reference genome to the human hg19 reference genome. The image below shows the drop down menu in the top left of the IGV
3. Go to File, choose Load from file and select the file Stacy's FGD4 gene (included are the FGD4.bam and FGD4.bam.bai files required for visiualization).
4. Type in FGD4 and press ENTER, as shown below...

This gene is large and you may have to zoom in to examine it up close.

In the same text box where the gene FGD4 was entered, type in the following region:

chr12:32,778,799-32,778,882 to zoom into a smaller and more visible region

You should now see something like this...

Database of CMT causing mutations

Stacy had her exome sequenced with 23andMe. I was fortunate enough to have been included in the 80x exome pilot study and they were nice enough to allow me to switch places with Stacy. We got her exome sent to us over this past weekend. The first order of business is finding a list of CMT4 mutations to check. Here is a database of CMT causing mutations I found maintained by the Molecular Genetics Department at the University of Antwerp. In addition, I'll be using the HGMD (Human Gene Mutation Database). These is great resources and will provide dozens of possible CMT4 causing mutations to analyze. I'll check all of the mutations known to cause CMT4 in these databases and proceed from there. There is always a possibility that there is a novel mutation causing CMT4 at work.

About this blog

The goal of this blog is to catalog the process of finding the genetic cause of an inherited disease. Stacy was diagnosed with CMT4 (Charcot-Marie-Tooth Type 4) in the 1980s following a nerve biopsy which showed the characteristic onion-bulb myelin sheath around peripheral nerves and after measuring nerve conduction velocities along the length of her leg. There are many different causes of CMT, many known and many unknown. Stacy recently had her exome sequenced. My goal is to find the genetic basis of this disease. My name is Erik Corona and I am PhD student in the Stanford Medical school studying the genetics of complex disease. I will be making all results public and I will also post all code and/or scripts I use to generate these results. I will be posting screenshots of candidate structural variants and making every step along the way transparent and clear. This blog will represent a detailed case study of how we now have the technology and ability to use sequence data to find the genetic cause of a rare inherited disease. I welcome all comments, advice, and suggestions.

Data

Stacy has decided to make all the genes relevant to CMT4 public. I will be posting bam files for the regions overlapping the genes causing CMT4 as we examine every one of these genes throughout the blog. This is done to allow others to join in and analyze Stacy's exome.

Motivation

Stacy and I have been together for almost the entire time I've been working towards my PhD here at Stanford. Ever since Stacy came into my life, I have been happier, more fulfilled, and have managed to trick people into thinking I have a sense of fashion. To say the least, I consider myself extremely fortunate to have Stacy in my life. Some of you already know how painful it can be to have someone you love affected by a serious disease. As a PhD student who studies the genetics of disease, I find myself in the most fortunate position to be able to do something about it. I intend to use all of my knowledge to first isolate the mutation causing this disease and then plan to focus on furthering research into this condition. I don't know what the end result of this work will be, but at the very least I hope that it will further our understanding of this disease and be informative for the curious or others wishing to do the same.

                                                            Stacy and Erik