A Low-Cost FSHD Diagnostic

Posted by Dr Premi Haynes on Jul 16, 2020

Recently Board member Premi Haynes interviewed Peter Jones regarding his low cost FSHD diagnostic that his lab is currently validating. Friends also recently funded this effort (see Improved, accessible, and affordable FSHD diagnostics).

Haynes: What made you decide to pursue research in FSHD?

Jones: My background training in the late 1990s was in epigenetics, chromatin and DNA methylation when these were just emerging as key regulators of gene expression. When I started my own lab in 2001 at the University of Illinois at Urbana-Champaign I was going to use my expertise to work on Rett Syndrome, which is a neurodegenerative disease associated aberrant regulation of methylated DNA. However, in 2002, Ryan Wuebbles, a new graduate student in the Cell Biology program at UIUC, asked if he could do his PhD in my lab since I was an epigenetics lab, but he wanted to work on a different disease called FSHD. He had heard it might have something to do with epigenetics.

I had never heard of FSHD so I asked why he chose that disease and he responded that he had recently been diagnosed with it. I looked it up and it seemed reasonable that it would have an epigenetic component so I agreed to give it a try and he became my first graduate student and we started an FSHD project despite not having any experience with studying muscle or muscle diseases.

Now we are a full-time FSHD lab with many projects on FSHD disease mechanism, therapeutic development, and diagnostics, all still focused around the epigenetic dysregulation in FSHD. We would not be doing any of this if an FSHD patient hadn’t taken this initiative to get involved.

Haynes: What is DNA methylation?

Jones: DNA consists of 4 different deoxynucleotides, CATG, which together make up the 3 billion bases of your genome sequence. However, deoxycytosine (C) can be modified with a methyl-group (CH3 - a carbon and 3 hydrogens) resulting in methyl-deoxycytosine (mC). Thus, the exact same DNA sequence can exist in an unmethylated form (e.g. ATCGATCT) and a methylated form (ATmCGATCT). However, this methylation of cytosine has profound effects as it provides a tag or a signal for the cellular machinery indicating that this sequence is different.

Often, parts of the human genome with a lot of DNA methylation are not expressed, or are OFF, while regions of the genome with low levels of DNA methylation are expressed, or are ON. The methylation of a particular gene may be different in different cell types or at different points in development. Thus, it provides a way for the genome to become compartmentalized and regulated without changing the sequence.

How does this relate to FSHD? The pathogenic gene in FSHD, DUX4, is supposed to be expressed (ON) only very early in human development and then turned off and not expressed in cells of healthy children or adults. As expected, the DUX4 gene is heavily methylated in healthy children and adults. However, in FSHD, the disease-causing mutations for FSHD1 and FSHD2 result in the failure to methylate the DUX4 gene so it is not epigenetically marked or tagged to be OFF later in development. Thus, in FSHD the DUX4 gene is not methylated. This is one of the factors that leads to the DUX4 gene being ON in FSHD, and resulting in the aberrant expression of DUX4 in FSHD skeletal muscles.

Haynes: How did you come up with the idea of testing saliva for a diagnostic test for FSHD and what does the test determine?

Jones: We did not set out to design a diagnostic for FSHD. Instead, we were trying to study the DNA methylation of the DUX4 gene, which for technical reasons of being in a DNA repeat, is harder than it sounds. The DUX4 gene resides in a piece of the genome called a D4Z4 repeat. There are many D4Z4 repeat units in healthy individuals as well as those with FSHD. These hundreds of repeats all have virtually the same DNA sequence, however, only one of the hundreds of D4Z4 repeats, the last one on chromosome 4, encodes the DUX4 gene that is expressed in FSHD.

My wife, Dr. Takako Jones, figured out how to very specifically determine the methylation status of this last chromosome 4 pathogenic D4Z4/DUX4 gene. She found she could use DNA from any source, as DUX4 is supposed to be OFF throughout the body, and also of (almost) any quality and clearly distinguish healthy levels of DNA methylation from FSHD levels. It was strictly a lab tool we used on well-characterized samples. Then, at an FSHD patient meeting in Boston, the researchers asked the patient audience what they wanted from the research community. A couple from Kenya spoke up, they had come all this way to learn about FSHD, but what they really wanted was a diagnosis. They had self-diagnosed the father with FSHD by watching YouTube videos and seeing who looked like them. There was no neurology expertise and definitely no diagnostics available in Kenya. I heard that and thought that if I had something with me to collect a sample they could spit for me and I could probably diagnose them before they went home. And it wouldn’t cost anything.

The lights went on in my head that 1) many people around the world do not have access to FSHD testing, 2) even though we don’t have a cure, people want to know their FSHD status, people see value in knowing what is wrong with them or the risk of their kids developing FSHD, and 3) our research test is fast, cheap, accurate, and can be done on saliva and does not need blood or specialized equipment. It seemed we (Takako) solved these problems all at once without even trying. We have since sent 25 saliva collection kits to Kenya and performed research testing on two large families afflicted with neuromuscular diseases, at no cost to them.

Haynes: Since this diagnostic test is in the research phase how can the FSHD community support this effort?

Jones: The key is to determine how accurate we can identify FSHD1, FSHD2, and not FSHD/healthy. It turns out that after testing hundreds of individuals it is clear that our approach is extremely accurate. However, as with anything, there are always some exceptions and there are a lot of unusual genetics once you start going around the US and the world. We are always looking for individuals with genetic confirmation of FSHD by deletion testing (FSHD1) or sequencing (FSHD2). In addition, the most difficult to find are individuals with a genetic confirmation of healthy, or not FSHD. This way we can more accurately determine our false positive and false negative rates. It is also very helpful to have healthy family members participate and those with a clinical diagnosis of FSHD but no genetic confirmation. Ours provides strictly a research result that is not clinically relevant, however, if you choose to see your results, they will provide information on one’s likely FSHD status. It could also suggest one might want to investigate other potential indications. So, in the end, participation in our study is the best way to support this effort. Fortunately, we have funding from Friends of FSH Research and individual donors to cover this project so there is no cost for participating.

Haynes: What and how long would it take to bring this test to the clinic?

Jones: This is an interesting question. In the US, all medical testing in which the results are used to make decisions about one’s healthcare must be performed in a government regulated lab environment, or a CLIA (Clinical Laboratory Improvement Amendments) lab. This ensures that a test does what it says it will do and is performed under the same strict controlled conditions every time to ensure accuracy. Getting CLIA approval and establishing a test in a CLIA lab is expensive. If one is going to bill Medicare or Medicaid for the testing, the test must also be FDA-approved, which is an extremely expensive process. Most rare disease testing is not FDA-approved and costs are covered at the whim of your insurance or by you. Thus, if you are on Medicare or Medicaid, the testing is not covered.

Any of this of course would add a large expense to our testing and also limits where the testing can be performed. Our initial goal was to make this a CLIA-approved test so that clinicians can use this for official diagnosis of FSHD and the results could be used to get into a clinical trial. However, we have heard from the FSHD community that there is a great desire for keeping this as a research test where the cost is very low, you can do it at home without a visit to a medical doctor, and the results are confidentially returned directly to the individual and do not become part of one’s official medical or insurance record.

Thus, we intend to keep performing this in our lab as a research test at no cost to the participants as a service to the worldwide FSHD community as long as we have funding to do so. And thank you again to Friends of FSH Research for providing funding. We have already helped people in 34 countries and across the US. In addition, we are negotiating with the Nevada State Health Lab on the UNR campus to establish a CLIA-approved version of this test in their CLIA laboratory. The only hold up is funding to modify space, buy dedicated equipment and pay for salaries, and then we can perform the CLIA certification and have the test available to the clinical community. Since the testing can be done on saliva DNA, it will be more accessible than any other testing and the technology for the testing means even the CLIA testing will be significantly less expensive than any testing currently being performed or developed (that we know of). With proper funding, a clinically relevant CLIA-approved test could be established in less than a year (once COVID is passed as that testing is taking up all resources at this time).