Limb-Girdle Muscular Dystrophies with Louise Rodino-Klapac and Livija Medne

How is it we find ourselves surrounded by such complexity, such elements? The genes of you and me, Jesus.

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Do you remember the DNA Today episode about epigenetic tests? It was with Greenwood Genetic Center's Dr. Ray Louis and genetic counselor Kelly Walden. We chatted about testing for epigenetic conditions and specifically their diagnostic lab's test, EpiSign. EpiSign included 40 conditions during this initial interview in 2021. So I'm so excited to share with you that version 4 is now available and includes over 70 conditions. They sure have made a lot of great strides in that time. For those that haven't listened to this episode yet, the diagnostic lab at the Greenwood Genetic Center is the only lab in the country to offer this epigenetic test called EpiSign. It's a novel, clinically validated test that analyzes methylation. If you're attending ACMG later this month in Salt Lake City, I recommend stopping by Booth 607 to chat with Greenwood to learn more. Brush up on your epigenetics before that by listening to episode 145 of DNA Today and visit ggc.org. Links in the show notes.

Surely you've heard of whole genome sequencing, but what about rapid and ultra-rapid whole genome sequencing? This is an emerging method of diagnosing genetic conditions for quick management. PerkinElmer Genomics offers this incredibly valuable test, which can be life-saving for ill babies and kids. Learn more in our full episode with PerkinElmer Genomics here on DNA Today. You can visit perkinelmergenomics.com for more information. The link is also available in the show notes and on our website dnatoday.com.

We're exploring limb girdle muscular dystrophy in this episode. Joining me for this conversation are two experts, not just one but two experts in the field of muscular dystrophy. We have Dr. Louise Rodino-Klapac, who is the Executive Vice President, Head of Research and Development, and Chief Scientific Officer at Sarepta Therapeutics, who has 15 years of experience researching and studying limb girdle muscular dystrophy. Livia Menda is a senior genetic counselor and systems director of genetic counseling at the Children's Hospital of Philadelphia, which many of you probably know is CHOP. She has 15 years also of experience in pediatric neuromuscular diagnoses, including limb girdle muscular dystrophy. So thank you both for taking the time to come on the show and help me explore limb girdle muscular dystrophy today.

Thank you. It's a pleasure to be with you today, and the second time we've recorded together, which is awesome. Not on this show — you guys gotta head over to the Final Tips podcast for that one. We'll put a link in the show notes for people that want to check that out.

But as we said, we're talking about limb girdle muscular dystrophy, which is a neuromuscular disorder that causes weakness and wasting of the muscles. Livia, I thought we could start with you. Just talking a little bit about, as we mentioned, you have an expertise in muscular dystrophies. So talking about limb girdle: what muscles are typically affected first? When do symptoms usually start for people? And I imagine it's a bit of a range.

Yes. So limb girdle muscular dystrophy is not one condition but rather a group of conditions that follow a similar pattern of muscle involvement. So number one, it's very clear: limb girdle muscular dystrophy is a genetic condition. It's not acquired, it's not autoimmune, it's not otherwise mediated — it is a genetic condition. Second, it is a condition that primarily affects the skeletal muscle, which is the predominantly affected muscle, and it affects it in a specific pattern, first targeting the muscles that are closer to the core of the body. Those would be the shoulder girdle and the hip girdle muscles, thus the limb girdle name. And the weakness has to be slowly progressive, not rapidly progressive. Eventually, more distal muscles can also become involved. What distinguishes it from other forms of muscular dystrophy, perhaps more severe muscular dystrophies, is that people do achieve independent walking with this group of conditions, and the onset of weakness follows that initial normal development. Generally, individuals also have elevated creatine kinase levels in their serum, which is a marker of muscle breakdown. All of us have some level of creatine kinase in our bloodstream because we use muscles, we break down muscles by exercising, but when that breakdown is ongoing, that marker is considerably elevated, and that's a true sign of muscular dystrophy. And the changes are progressive, and that's visible on two other aspects: that's visible on what we call histology — muscle biopsy when that is done — and that's also visible on something that we do more frequently now than 20 years ago, and that's muscle imaging, looking at muscle by ultrasound or MRI. Those degenerative changes are seen. So when we take that all together, we can recognize a pattern that fits the limb girdle muscular dystrophies. And you asked the first question: when is the age of onset? It's incredibly variable. There are forms of limb girdle muscular dystrophy that have age of onset during the first decade of life — five to seven years of age, perhaps in some cases even a little bit earlier — but there are forms of limb girdle muscular dystrophy where individuals notice muscle weakness only in their fifth or sixth decade.

Wow, that is a huge variability. And you might work mostly in the pediatric space, so a lot of patients that you're seeing are more the kid age.

Absolutely.

There's just so many different types within limb girdle. So when you're looking at this, someone has a muscular dystrophy, obviously you don't know which one it is as you're going through the diagnostic odyssey. What makes it stand out that you're like, 'Oh, this is a sign that I'm really thinking it could be limb girdle' as opposed to there's a lot of muscular dystrophies?

So that's why it's important that evaluation is done in experienced centers where there are pediatric or adult neuromuscular specialists — neurologists with additional training in neuromuscular conditions who work generally in teams, maybe with physical therapists or occupational therapists that are also skilled in evaluating various muscle groups. And more and more increasingly, the larger neuromuscular centers include genetic counselors to help guide genetic testing. Whereas 15, 20 years ago, the evaluation started with an EMG, electromyography and nerve conduction studies, and often a muscle biopsy. This day and age, we really want to see that elevated creatine kinase level to really show there's ongoing muscle breakdown, but then the next step, often with the same blood draw saving the specimen, is the genetic test. It's much more non-invasive, it's definitely specific. The changes on histology, muscle biopsy, it can be specific for certain types or subtypes of limb girdle muscular dystrophy, especially if those proteins are expressed at the muscle cell membrane. However, in many cases and in other subtypes of limb girdle muscular dystrophy, one sees dystrophic changes — changes in the muscle that are reflective of muscle undergoing breakdown, there's fatty tissue replacement, connective tissue replacement — but they're not specific to one category. So genetic testing has really moved as the tier one diagnostic test following really good and proper physical examination and that creatine kinase enzyme level.

Yeah, so it sounds like it's really those three aspects: it's the CK level, and then looking at the genetic testing, and then looking at the physical too of just evaluating that child, teenager, or adult depending on the type.

And I think what else has moved into the forefront of clinics is bedside muscle imaging. That was more done routinely in Europe first, but it's really luckily invaded the North American clinics as well. The bedside imaging that's done by neurologists, often by ultrasound, can really provide valuable clues because certain types of limb girdle muscular dystrophies will have a certain imaging pattern — sparing of one muscle, more involvement with another muscle subtype, either again in the shoulder girdle muscles or the hip girdle muscles — and that can also give an astute clinician some clues which subtype of limb girdle muscular dystrophy an individual may have.

Thank you for highlighting that again, because I hadn't heard of that before. That's surprising that that didn't come out for me during grad school or during my pediatric rotations.

It didn't come out because technology has also changed. Ultrasound machines have become the size of iPads pretty much, or with maybe some additional things. The other part is ultrasound is more utilized in children because it can be done without sedation. MRIs can be more utilized in adults as adults can be compliant and lie still in the MRI machine, whereas children often do need to be sedated for an MRI study, which in itself is a risk factor for assumption.

That makes sense. I'm sure parents have the option, they're going to say, 'Well, we'd rather do an ultrasound if we can instead of sedating for an MRI.' So then when it comes to the genetic testing, is it usually a panel that's ordered? Because now a lot of testing is moving more into whole genome, whole exome, but is it still like a limb girdle panel or muscular dystrophy panel that providers are ordering if they know, 'Okay, we have a muscle issue happening'?

No, you can't — you hit the nail on the head. Most providers these days will go with a muscular dystrophy panel, or sometimes other commercial labs will call it a limb girdle pattern weakness gene panel. And the advantage of a gene panel is that there's capture of the sequences of the known genes that cause the various subtypes of limb girdle muscular dystrophies, and it's done in one test instead of going through a stepwise approach. Exome and genome sequencing are wonderful tools but not necessarily needed for workup of limb girdle muscular dystrophy, as in many cases the diagnosis can be achieved by a comprehensive panel that does include the known genes for the subtypes of limb girdle muscular dystrophy. Exome sequencing has its role; it can identify cases that are maybe more complex where muscle involvement is part of broader systemic disease. And of course, exome and genome sequencing are also tools that are used to discover genetic causes of the yet unknown subtypes of limb girdle muscular dystrophy presentation.

So if providers are ordering a panel, then that's kind of — you're saying it's the standard approach. From that result, are we able to say, 'Oh, you have, let's say, limb girdle,' and then are we able to then identify and say, 'You have this type' and give more information at that point, or do we need a little bit more testing and variant curation to get to that point?

That's an excellent question. I think the answer is not unique to muscular dystrophies but to all genetic tests. So in many cases, the answer is very straightforward because the pathogenic gene variant is very clear, either because it's recurrent or it's pathogenic by nature because it's, for example, a loss-of-function allele due to splice sites or frameshift or nonsense variant. At other times, the variant is unique, and then we do need to put some work in to arrive at a diagnosis or maybe dismiss that variant. So I think most of us who work in this field really take a stepwise approach, and I think that's the right approach when there is no slam dunk definitive answer. And that's number one: the phenotype. Number two: the genotype. And if the variant is novel, how do you assess that? Well, then we go to the imaging and see: does the imaging fit any known pattern? And that can be by ultrasound or MRI. Then we also like to go to the histotype, so muscle biopsy. While no longer being the first step in diagnostic evaluation, it is an absolutely useful tool and a diagnostic workup step that is used. And in this day and age, we tend to use muscle biopsy to clarify variants of unknown significance, to see if the muscle itself looks similar to what's expected for that gene with pathogenic changes in one particular gene. So we go with phenotype, we go with genotype, we go with histotype, and imaging — I can't turn it into a type, but imaging also is very important to provide that. Once you have those four aspects aligning, then we can come up with a diagnosis, working together with the team. Of course, it involves an experienced neuromuscular neurologist, an experienced molecular geneticist lab director, an experienced pathologist, and an experienced genetic counselor for the cases that are not straightforward.

Yeah, so a lot of tools to be using in this. And it's interesting because a lot of times when I hear, 'Okay, we found a variant, we don't know what it means,' usually the next step is we do trio testing, we test the biological parents to see, 'Oh, do they have that?' But it sounds like that's not high on your list. You're more like, 'Well, let's look at what the muscle looks like. Is this fitting in terms of this patient?' So do you ever do trio testing, or do you have enough information with the muscle biopsy and everything?

That's an excellent question. Well, since most forms of limb girdle muscular dystrophy are recessive, parental testing perhaps is less contributory. If we find a parent carries it, it doesn't influence our interpretation. For those where a dominant inheritance is suspected, then testing the parent is absolutely critical, or testing unaffected relatives sometimes. For adult-onset forms, parents are not available, but siblings who are unaffected or similarly affected — testing them, cascade testing, is important for dominant conditions, absolutely.

And one aspect that I know if parents are listening or caregivers, it's like, 'Okay, well this sounds really expensive.' You know, as a genetic counselor, I'm sure you're involved with ordering this testing as well as being involved with patient care. Are you aware — are there only genetic testing available through insurance companies and cash options? Is there any programs that are running where there's not a cost to the patient themselves?

That's an excellent question. I think this day and age, there are multiple avenues how to achieve a molecular diagnosis. The traditional one is a test ordered through a clinic by a provider that's billed to insurance. In most cases, it requires an insurance authorization, but limb girdle muscular dystrophy, just like other neuromuscular diagnoses, has a relatively easy way to achieve insurance authorization when the letter of medical necessity is written correctly. Insurance companies are not looking to validate testing for academic purposes or to approve testing; they approve testing when arriving at a genetic diagnosis impacts management. So those of us in the field who've been in the field for a couple decades plus have kind of fine-tuned that skill. And for neuromuscular conditions, knowing the diagnosis absolutely impacts management. While we wait for more targeted, more curative treatments, not every individual with every type of limb girdle muscular dystrophy needs ongoing surveillance for cardiomyopathy or cardiac arrhythmias. Not every individual with LGMD needs monitoring for nocturnal hypoventilation — that's difficulties maintaining oxygenation during sleep when someone is laying down supine, relying just on the diaphragm as opposed to diaphragm and our intercostal muscles, our chest muscles, as well. For those who do, it's critically important to catch that, to diagnose it timely, and to treat it, because heart conditions and breathing conditions can be treated appropriately even in the settings where we cannot treat the underlying muscle pathology yet. So that's one approach: really having your providers write letters of medical necessity demonstrating that writing a diagnosis will impact management. Insurance companies are not interested in paying for yearly echocardiograms from age 23 throughout lifetime if that's not necessary. But there are also other programs. There are industry-supported programs at certain commercial laboratories where clinicians need to fill out a form attesting that an individual meets criteria for a limb girdle type muscular dystrophy. That generally involves the presentation of the features we discussed: muscle weakness, elevated creatine kinase level, perhaps a family history also of muscular dystrophy. And there are these sponsored programs that are available at no cost at all. And for some individuals, some commercial laboratories are also offering relatively price-friendly options where it's not covered by insurance but an individual pays out of pocket. Those prices or charges have dropped over the last 10 years from being well above a thousand, two thousand dollars to now the price being in a few hundred dollars. So of course everyone has a different financial situation, but I think this day and age there should be ability to achieve a genetic diagnosis if one can be identified, with the various testing options that are available to providers and to patients.

And we see how important that is, as you talked about, of knowing, 'Okay, you have this subtype, so this is important because we need to do this type of care or we don't have to do this type of care or surveillance.'

Genetic technology moves fast. The first genome took 13 years to finish a draft for reference; this was 20 years ago in 2003. And now a whole genome sequence can be performed in days, even hours. Since sequencing an entire genome can be done in such a short amount of time, this lowers the cost, which means more people can utilize whole genome sequencing now. This is particularly helpful in the NICU for ill infants. PerkinElmer Genomics explains more to me in our full episode here on DNA Today. It is remarkable how quick diagnosis from whole genome sequencing can save a baby's life with a change in treatment informed from the results of that test. I learned more about how genome data is mined to maximize useful information from one data set and other omic assays that help enhance diagnosis. Don't miss the full episode here on DNA Today. You can visit perkinelmergenomics.com for more information. The link is also available in the show notes and on our website dnatoday.com.

Which muscular dystrophy causes weakness of the muscles typically starting around the hips and shoulders? That would be limb girdle muscular dystrophy, or LGMD. LGMD is a group of neuromuscular diseases caused by mutations in genes responsible for proteins critical for muscle function, regulation, and repair. Sarepta is a global biotechnology company working on engineering precision genetic medicine with the goal of changing the lives of people living with rare muscular dystrophies. Their multi-platform precision genetic medicine engine includes gene therapy, RNA, and gene editing approaches. And that reminds me, ACMG is in March and Sarepta will be at Booth 504. You can also head over to limbgirdle.com to learn more. Again, that's limbgirdle.com.

I've enjoyed recording a few episodes about epigenetics. One of the interviews where I learned the most was with the diagnostic lab at the Greenwood Genetic Center. They taught me about EpiSign, which is a novel, clinically validated test that analyzes methylation. I just learned that since this episode aired in 2021, version 4 of EpiSign has been released, which has expanded to include over 70 conditions. If you're attending ACMG this month, stop by Booth 607 to chat with Greenwood Genetics. In the meantime, brush up on your epigenetics by listening to episode 145 of DNA Today and visit ggc.org. Link in the show notes.

So Louise, I'd love to pull you in and hear your expertise. Coming from Sarepta, you guys have a gene therapy program for what is it, the six most common subtypes of limb girdle?

So I was just gonna ask, so this covers the majority of limb girdle, those six subtypes. So can you explain a little bit more about what these subtypes are and a little more background on that?

So explain to me a little bit more about how the gene therapy works. So you're providing a gene that, quote-unquote, works as opposed to the broken gene that they have, and that's how they have this diagnosis. From my limited exposure to this, researching to prepare for this episode, I learned there's kind of three main components to gene therapy. Could you walk us through what those components are?

So with this gene therapy, then it's going to differ depending on those subtypes, I imagine. We talked about the most common six is what you guys are focused on at Sarepta. So how does that differ between subtypes? Because if you're delivering a quote-unquote normal wild-type, like just if you looked at my genome you said, 'Okay, this is probably just like a typical one,' right? So how does it differ between subtypes if you're just providing that healthy gene, I guess we can call it?

And important to know is that there's different genes for these different subtypes. It's not one limb girdle gene. So that's important, and that makes sense because I'm looking back in my head at carrier screening reports, like yes, there's a but it's limb girdle, so many times on carrier screening I'm like, that's because there's different genes. So okay, thank you for patiently explaining that to me. So for gene therapy, then that's how it works. What is the goal? Like, what do we want to see if a patient is being administered with this gene therapy? What do we want to see in terms of outcome for them?

Yeah, certainly to be able to kind of pause the symptoms where they are so that it's not developing any further. And we see that with other types of gene therapy treatments that if we can administer it even before symptoms are present, that's super ideal, which isn't always realistic though. So you mentioned clinical trials. Is that the status that gene therapy is in right now specifically for limb girdle, or are we looking at clinical trials? What's the current status?

Yeah, that's very good advice. Everybody should bookmark that because just about every guest that comes on mentions that link, so we'll throw it in the show notes for people. When do you expect, you know, of getting through these clinical trials? Obviously things have to happen and certain things have to be approved. Let's say you guys get great results. When would you expect FDA to approve that so that it's widely available so that a healthcare provider could prescribe that to a patient that's been identified? Or like, let's say in my case, I'm meeting with a couple, they're both a carrier and they're continuing the pregnancy, we're gonna say test the baby once the baby's born and we're identifying, okay, they have limb girdle. Like, how soon would I be able to tell patients, like, we're expecting maybe in this range of time? Which I imagine it's going to be a range; you don't have a crystal ball to say it's going to be exactly this year.

Yeah, very understandable. So I think what a lot of us face is that patients ask, 'Well, what can I do now while scientists are still working on fine-tuning the gene therapy treatments, on making sure that they're safe and that they are able to deliver what their promise is?'

So I think from the very clinical perspective, I always encourage all my patients to join a registry. Each LGMD subtype has its own registry based on the underlying genetic difference, based on the underlying gene mutation or pathogenic variant. Those registries are critical to establish really scientifically based, factually based natural history for that condition when it is untreated. In terms of looking at the primary muscle disease process, of course individuals who register with these natural history studies are treated with conventional methods — their hearts are treated, their breathing apparatus if that's affected is treated — but the skeletal muscle itself cannot be treated without the more advanced therapies that are in development. These natural history studies are absolutely critical, crucial, and even necessary for any clinical trial with phase two and phase three moving on from phase one, because one needs to show that the intervention — the gene therapy, RNA-based therapy, or another therapy — is making a difference. It is impacting the natural history of the disease process, and one can now demonstrate that if patients are not well characterized before that treatment arrives. And FDA absolutely looks for that, and FDA requires that there is a meaningful effect that's not just visible by measuring some compound in blood or in muscle; it has to provide impactful difference in function and also quality of life as well, and that's measured by various scales.

For patients that are interested in accessing the gene therapy programs, is there a way that their healthcare providers can help them enroll in this or see if they're even eligible?

Fantastic. And as we wrap up, my last question is: is there anything we didn't cover in terms of any myths about limb girdle that you guys wanted to clarify? Anything that patients will often ask you or you're like, 'Oh wait, let me explain, that's not actually how it works'? Anything come to mind for you guys?

I'd say for me, we talked about it: it's just that limb girdle is a general diagnosis. Now we know it's a collection of disorders with similar phenotypes but not the same. Each one is a distinct gene that is altered, and it's important to know your diagnosis so that we can best find treatments and pass forward for patients. But we're optimistic about what we can do for limb girdle, and I think the journey of genetic diagnosis and natural history studies will help us.

Yeah, definitely. Well, thank you both so much for coming on and sharing so much with our audience and myself, just educating me on how all of this works. I'm really intrigued to see the natural history studies once those are published and how the gene therapy is progressing over the years. So thank you so much for all of your work in muscular dystrophies over 15 plus years for each of you. So we've got 30 years plus of experience just in this one episode. So thank you so much, I really appreciate it.

Thank you for having us.

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