Welcome, everyone. Thank you all for joining today. We are excited to be presenting on our panel called Classification Investigation, Examining Pathogenicity of DMD Variants Detected on Carrier Screening. So this activity is intended for educational purposes only. These are the financial disclosures for the committee members. The ACMG is accredited by the ACCME to provide continuing medical education credits for this session. And this session is approved for one hour. A recording of this webinar will be available in two weeks in the ACMG Genetics Academy. All questions will be answered at the end of our talk today, and you can submit a question using the Q&A feature. Please only use the chat feature for indicating technical issues. To help prioritize a question, you can give it a thumbs up in the Q&A tab. These are today's learning objectives. Today's speakers include myself, Blake Vuccolo. I am a Certified Genetic Counselor at Baylor College of Medicine, and I work on several research projects. Kayla Banks is a Certified Genetic Counselor and the Community Research Project Director for Parent Project Muscular Dystrophy. And Sandra Darcilik is the Certified Genetic Counselor at Baylor College of Medicine and specializes in prenatal genetics and preconception counseling. So I will be starting our conversation today, and while I am not in the prenatal space professionally, this topic is very near and dear to my heart because I do actually have a friend who is found to be a carrier for a pathogenic DMD variant. And so I will be telling her story today from both a professional genetic counselor lens, but also from the lens of being her friend. So first I'd like to introduce you to my friend, we'll call her Cece. And at the time that this whole journey started, she was 35 years old. She's a social worker and a fitness coach. She's very strong-willed. She's a lot of fun to be around. And she has such a love for life. Cece is married to John, and she had been for about five years. And around this time, they had started having conversations about wanting to start a family. She mentioned this to her OB-GYN at a routine follow-up visit, and the OB-GYN recommended preconception carrier screening. And so Cece selected to have a 14-gene panel and was found to be heterozygous for a pathogenic duplication of exons 50 to 55 in the DMD gene, which I'm sure we're all aware is associated with dystrophinophathies, including Becker and Duchenne muscular dystrophies. So she didn't have a huge background in genetics, and at this point, she reached out to me to ask what this meant. And at the time, I was a genetic counseling student, and I knew that it would be important for her to talk to a prenatal genetic counselor. So I recommended her to go see one in a clinic. When she did that, the genetic counselor talked about the condition, and she also collected the family history for Cece. When the genetic counselor took the family history, she learned that there was no family history of Duchenne or Becker muscular dystrophy, and Cece did have two healthy brothers in their late 30s and early 40s. Both of Cece's parents did die from cancer, but the mother, importantly, did not have any symptoms associated with being a carrier for a DMD gene either. So at this point, knowing that Cece and John were interested in having children, the genetic counselor shared that there would be a 50% chance that Cece would pass down this variant, and she could either have an affected son or a daughter who was a carrier. There were several options on how to build a family that were discussed at this initial genetic counseling appointment, including natural conception with confirmatory prenatal or postnatal testing, or in vitro fertilization where preimplantation genetic testing for monogenic disorders could be studied. And so after this initial genetic counseling appointment, Cece met up with me and we were just talking about the different options that she had. There were several things that became apparent to me as I was talking to Cece. She mentioned and became very clear that she did not want a child with DMD or Becker muscular dystrophy. And so really her options were to try to conceive naturally, and if she did end up having a hemizygous male fetus, she would choose to terminate, or she would go the IVF route with PGTM. There were several things that factored into her decision. And on the natural side, there were several psychosocial implications. So for her, she wanted to be excited about pregnancy, and she was really worried that if she decided to conceive naturally, she would have fears and anxiety about going through the first several weeks not knowing if she was having a boy or girl, and then potentially subsequent weeks waiting for confirmatory testing to come back if she did find out she was having a male. And something that complicated this even further was that right around the time she was deciding to have a family, the abortion laws in Texas had just changed and had become very strict. And so if she needed to go the route of termination, she would have to go to another state to receive this care. In terms of IVF, one of the psychosocial factors that I saw come up in our conversations was that Cece and John had not anticipated this finding, especially given her negative family history. And so they had always imagined having children the natural way, and so they felt that there was a loss of intimacy in terms of having quote-unquote test-tube babies in her terms. And also, of course, with IVF, there are additional physical burdens of extracting the eggs and implanting embryos that you don't have with a natural pregnancy, as well as an additional cost. And this is something that they had to sit down as a couple to decide financially if it was feasible for them. It took several weeks for her to come to a decision with John, but they ultimately decided to pursue IVF, again, because they really wanted to minimize the chances that they would have an affected male. So just to give a little bit of background on PGTM technology, linkage analysis is the gold standard for PGTM testing. And linkage analysis looks for associations between the inheritance of short tandem repeats or STRs and the presence of a trait or a disease within families. STRs are repeating sequences of DNA base pairs that are highly polymorphic, and this makes them useful genetic markers for linkage analysis. Now the reason that direct variant testing isn't used commonly in PGT is that it can be very difficult to perform direct variant testing in PGTM, especially in genes like DMD, where there are often large duplications. And so therefore, most labs do not perform PGT through direct analysis alone. Now importantly, for linkage analysis to be set up, there must be another family member. And so typically, we would prefer parents or children who have this variant as well to be present and available to submit a sample to set up linkage analysis. Now the challenge in Cece's case is, if we look back at her family history, both of her parents are deceased, and she didn't have any children. She also did not have a sister and only had these two brothers, which we suspected would be negative, given that they didn't have any symptoms of DMD or BMD. And so at this point in time, she was counseled that linkage analysis could not be set up for PGTM since there was not another known carrier in the family. So what did she do? Well, the option for her and John was to create embryos and then test them using PGTA to transfer a female embryo, who at most would just be a carrier. They opted to do this, and so they underwent a cycle of IVF and had PGTA completed. And at the end, they had one female embryo and two male embryos that had normal PGTA testing. The couple had the female embryo implanted, and Cece successfully carried the pregnancy to term. And fortunately, Luna was born and she was healthy. And so on top of loving Luna and getting used to becoming parents, they were thinking in the back of their minds throughout that they did want to have a second child. And so they decided to test Luna for the DMD variant. Postnatal genetic testing did confirm that Luna was a carrier of the DMD duplication, and so linkage analysis was now possible. Cece and Luna's information was sent back to the PGTA testing company that could also perform the PGTM testing, and the lab let them know that there were no STRs within the duplicated exon range of the 50 to 55 duplications seen in Cece and Luna. That was available at the lab to use for a proxy for linkage analysis. However, the lab did identify a duplicated STR in the intron 49 of the DMD gene in both Cece and Luna. And so they felt that this intron STR could be tracked in the embryos. However, when testing of the two remaining male embryos, they found that this intron 49 duplication did not always track as expected with the haplotype for the duplication determined from the testing of Cece and Luna. And this was something that was very rare for the lab to see. People had talked about how they really had never seen this before. And at this point in time, the lab could not provide results for the remaining embryos. And so even though this tracked with Cece and Luna, it wasn't tracking correctly with the embryos. And they were left with a lot of questions of whether or not these embryos would be hemizygous for this pathogenic variant. At this point in time, Cece came to me. She was very confused. I was very confused because I had never heard of this. The genetic counselor was very confused and the lab was confused. One of the things that the lab thought might be an explanation is perhaps this duplication wasn't in fact in the DMD gene and was elsewhere in the genome. So Cece underwent FISH, which did confirm that the pathogenic duplication was located where we would expect. And so really at this point, we had more questions than answers. And the genetic counselor was really grasping at straws, trying to figure out where we could go from here to get more information. So what the counselor suggested was that we test Cece's brothers. Again, Cece has two brothers. One was 40 years old at the time. One was 37. Neither had any history of muscle weakness. And both were fairly athletic. So one was a marathon runner and the other one was a professional boxer. They both agreed to be tested for the pathogenic DMD duplication. And to everyone's surprise, the 40-year-old brother was hemizygous for the pathogenic duplication of exons 50 to 55 in the DMD gene. Now for a separate indication, he had had an echo before and it was normal. And really to just look at every avenue, we did also request that he get a CK level. And that did come back normal as well. So there was nothing subclinical going on that we could identify either. And so really just to recap, my friend found a pathogenic duplication in DMD on her carrier panel. She opted to proceed with IVF and selected for a female embryo, which was later confirmed to be a carrier. However, the duplication status of male embryos was unknown through the linkage analysis due to inconsistent haplotyping. And on top of all that, she had an unaffected brother that was found to be hemizygous for the pathogenic DMD duplication. So at this point in time, she did go back to the genetic counselor. And the genetic counselor shared that the status of the male embryos was unknown. However, given that the brother was unaffected and hemizygous for this variant, it was possible that even if the embryo did have the duplication, it might not have a dystrophinopathy. So again, the genetic counselor presented several avenues they could take to have another child. The first would be to try to conceive naturally, which at this point, there's a 75% chance that they could either have a girl or a boy that's unaffected or doesn't have this DMD variant. And even if they do have a boy with the DMD variant, it's potentially they wouldn't have a dystrophinopathy phenotype. However, if they go the natural conception route, there would be advanced maternal age risks related to aneuploidies, especially given she was a couple years older at this point. Or IVF was an option again. They could either do an entirely new round and select for another female embryo to reduce the risks, but that was cost prohibitive for my friend. So then the other option would be to implant one of the male embryos. Again, we don't know if they have this duplication, but they have been screened for aneuploidies. And so my friend talked about this decision for several months, honestly. And we talked through every single scenario on end. And at the end of the day, it became clear to me that she felt less concerned about this DMD variant at this point in time than she did about age-related risks. And so she and John together decided to pursue IVF and implant one of the male embryos. And so this was a really impactful case for me because not only is it interesting from a genetic standpoint, but I became really invested in this because it was happening to my friend and I got to see behind the scenes what this was like for her. And Cece, of course, talked to the genetic counselor a lot and shared some of her concerns, but I got to see in real time how these decisions and new information impacted her. Throughout our many talks about this journey with genetics and creating a family, Cece talked often about how she didn't regret going through IVF initially because she was able to have Luna and she's happy and healthy. But she does wonder if it was in fact necessary. So I mean, it's possible if her brothers had been tested before she had decided which route to take and they had learned that her brother was healthy and had this variant, she might have just tried to conceive naturally and it would have saved her from the physical and financial burden of IVF. We've also talked often about what would have happened if she had chosen to go the natural conception route to start with. Again, she would have terminated a pregnancy that was hemizygous for this variant in a male and so knowing what we know now, she could have potentially terminated an unaffected hemizygous male. Even though this isn't what she decided, there is still a level of guilt that she feels in knowing that this could have been an outcome for her and her family. We must also consider the psychosocial implications of carrier screening and subsequent genetic testing. A lot of times she had mentioned that she sometimes wished she didn't know any of this. She could have gone into pregnancy carefree, not having to worry about, you know, what decisions she's making and whether they were the right ones. And really throughout this very complex journey, she was sitting through uncertainty at every turn. This story shows us that as we are doing carrier screening more in healthy individuals and in broader populations, we may come across these variants more commonly that we initially believed to be pathogenic and fully penetrant, but perhaps that's not actually how they're acting in families. And so what we learned from this case in our clinic is that other family members should be tested for known familial DMD variants when one is detected on a carrier screen, especially when there's not a family history. The other thing that we need to keep in mind is that this may extend beyond DMD. You know, our clinic has anecdotal cases of other genes that came up on carrier screening that we found to be in healthy individuals in the family. And so this might be highlighting a bigger issue that we have with carrier screening and variant classification. And so this case, of course, as I mentioned before, was very interesting from a genetic standpoint, but because this was my friend, it really fueled my interest into digging into this further and learning more about what might be going on. And so this is when I actually connected with Sandra, who was her genetic counselor, and I connected with Kayla at one of the ACMG conferences a few years ago, asking if this is something that they've seen in other people. And when we started to hear that several individuals were having these experiences, we felt it was really important to have a panel like this. So this is all I have, and I'll pass it over to Sandra to talk about the current practices of carrier screening. And again, we're happy to answer any questions at the end of our panel. Thanks, Blake. So as Blake said, I am a genetic counselor that works in the preconception and the reproductive space. And so my part of the discussion today is going to talk a little bit about carrier screening, especially from the perspective of what we're doing with D&D from a carrier screening perspective. And for those of us that work in the reproductive space, I think the question that's becoming more and more relevant is, when did carrier screening become complicated? Because it certainly has become more nuanced over time. So what I'm going to do today is briefly review the evolution of carrier screening, compare and contrast the current society recommendations, particularly those from ACOG and ACMG, and then discuss some of the nuances and the complexities that we currently are living with, with carrier screening as it's currently being performed. As this audience is probably well aware, traditionally, historically, carrier screening was really based on ancestry, ethnicity-based recommendations and family history-based recommendations. So for a very long time, we would take an individual or a couple's reported ancestry, look at the conditions that were more common in those populations based on recommended guidelines, offer those conditions for carrier screening, and then also look at family history and see if there were any obvious risks for inherited genetic conditions for which carrier screening would be amenable. What's happened over time, particularly with new genetic testing technologies, with the reduction in cost of genetic testing, is that carrier screening has moved from kind of a one condition at a time type of approach to panel-based testing, to larger and larger panel-based approaches. And so now carrier screening is more of an approach from a pan-ethnic perspective in this kind of expanded carrier screening approach that we're taking. In the future, who knows? Carrier screening may become something that we're doing exome and genome sequencing and releasing carrier screening or carrier status. But right now we're kind of in the middle of the road where we're really looking at panel-based carrier screening and, quite frankly, larger and larger available panels. If we look at the guidance that's out there, the two major guidance that we have for carrier screening in the reproductive space come from ACMG and ACOG. As everyone on this webinar is probably aware, ACMG actually updated their carrier screening guidance back in 2021 after an evidence-based review. And in the guideline, they were working with this framework of a tiered type of approach to help decide what the best recommendation would be for general population carrier screening. They set out a tiered approach where Tier 1 would be kind of your basic cystic fibrosis, SMA, and family history risk-based screening, with Tier 2 being looking at autosomal recessive conditions that have a carrier frequency of greater than 1 percent in the general population, Tier 3 being looking at autosomal recessive conditions with a greater than 1 in 200 carrier frequency, and then including select X-linked conditions based on a slightly different calculation, and then Tier 4 being a panel or an approach that would be looking at conditions that have a less than 1 in 200 carrier frequency, understanding that in that scenario, genes and conditions could vary by lab. And so what the ACMG guidance settled on as far as the recommendation is that the recommendation would be that we would screen for disorders with a carrier frequency of greater than 1 in 200 in the general population for recessive conditions, and then also some select X-linked conditions. What this equated to is a panel of 97 autosomal recessive conditions and 16 X-linked conditions. For couples for which there is consanguinity at play, ACMG noted that doing a panel of disorders with a carrier frequency of less than 200 would be very appropriate for that population. ACMG's updated guideline is supported by ACOG, but it's not officially endorsed by ACOG. If you look at ACOG, on the other hand, their carrier screening guidance is still from 2017 and is different. And if you keep in mind that the majority of people who are ordering carrier screening are obstetricians and reproductive endocrinologists or fertility specialists, then they're typically looking to ACOG for their guidance. And so what ACOG currently recommends as far as carrier screening goes is for the general population CF, SMA, and then a CBC with screening for hemoglobinopathies. Beyond that, they recommend for individuals who are of Ashkenazi Jewish ancestry or who have a partner of Ashkenazi Jewish ancestry that a panel that includes conditions more common in the Ashkenazi Jewish population, Tay-Sachs disease carrier screening for individuals of French Canadian and Cajun ancestry. And then beyond that, they make note of Fragile X carrier screening for individuals who have a family history suggestive of Fragile X or a known family history of Fragile X syndrome or for individuals with a history of ovarian insufficiency. Again, they do make note that carrier screening for known inherited conditions for which there's a family history is appropriate. And in their guidance, they do note that ethnic specific pan-ethnic or expanded carrier screening approaches are acceptable strategies, but they don't particularly recommend those as an approach. So what are some of the challenges of carrier screening in the reproductive space? Well, first and foremost, like more and more genetic testing, most carrier screening is actually not ordered by genetics providers. It's ordered by non-genetics providers, your OBs, your fertility specialists, your PAs and NPs that work in those spaces. And so a lot of the testing that is being ordered and then ultimately interpreted is being ordered and interpreted by people that may not understand all of the nuances of genetic carrier screening or of the reporting of genetic carrier screening results. In addition to that, there are variations in what is actually covered by insurers. So while, yes, there are recommendations from ACMG and ACOG, a lot of what is driving carrier screening and what panels are being chosen really comes down to what's covered by patient insurance. And there's a lot of variety in this space. Some insurers will not cover carrier screening for more than six conditions. Some will not cover carrier screening panels that have greater than 15 conditions. And then for some insurers, there may be coverage and they may cover larger panels, but there may be specified minimums and maximums of what genes are and aren't included. And so there's a lot of gray and a lot of lack of transparency in what's covered by certain insurance policies. There's also very little consistency in panels between labs. So if you were to look at one individual lab, what you would typically find is they offer a variety of different carrier screening panels, everything from a very small panel that may include two or three conditions to larger panels that may be built on ACMG's guidance or may go beyond that. You may be looking at upwards of 600 conditions. What you'll also find if you look at different labs and you compare similar sized panels is that those similar sized panels are typically not identical. They don't all cover the same genes. They don't all have the same conditions. And so while you may look at a panel that's got 200 genes from lab A and a panel that has 200 genes from lab B, you may actually be getting different carrier screening depending on which lab that's being ordered from. And then also we have to keep in mind the nuances of variant classification in this space. So we certainly recognize that variant classification is much more challenging in the carrier screening space because there is no phenotype that's typically driving the variant classification. And very often there's not family history that's helping inform that variant classification. So we're much more relying on what's been reported before in the medical literature and what labs are doing for their variant curation. In addition to that, what we learn in some cases is that there are discrepancies between labs and their variant classification. And certainly this is not something that is specific to carrier screening. We see this in the diagnostic space as well. But in a space where you typically don't question a pathogenic or a likely pathogenic classification, very often in carrier screening we don't have a reason to question that variant curation and classification. There are times where we will need to question that because a patient may have had more than one carrier screening panel and we see discrepancies between those results. Sometimes that's because one lab classifies the variant as a variant of uncertain significance, which are typically not reported on carrier screening, while another lab reports it as likely pathogenic or pathogenic. Sometimes it's because of the evolution in time and the classification of the variant has changed over time. But in those cases where it's truly two labs that have differing classifications, that can become very confusing for those who are involved in the counseling of those patients as far as risk goes. It becomes even more so when you're dealing with an X-linked patient. If we think about carrier screening for DMD, and this very much probably applies to a lot of other X-linked conditions that we just haven't really been thinking about at this point. First and foremost, ACOG does not recommend DMD carrier screening for the general population. If there's a family history, yes, ACOG, that is part of their recommendation, but it's not a standard recommendation by ACOG. So for any provider who's using ACOG guidelines, typically DMD would not be something they would be thinking about doing carrier screening for. But for providers that are following ACMG guidance, then you are going to get DMD on the list of genes that's included in a carrier screening panel. What we also have to recognize is that DMD gene inclusion varies between labs. You would think probably that most of that is driven by the fact that DMD would only be included on larger gene panels and not smaller gene panels, but that's not quite the case. There are some labs that have smaller gene panels of, say, four, where DMD is actually one of the four genes on that small carrier screening panel. So it's not always about size of panel that dictates whether a gene like DMD will be included. For most carrier screening labs, when they are reporting on X-linked conditions like the DMD gene, only carrier status is reported or variant is reported if you're testing a female patient or an XX individual. However, there are some labs that run carrier screening where the panels are exactly the same in males and females, and they report out on both the recessive and the X-linked genes, regardless of the reported sex of the patient. So in some cases, you may be surprised by an X-linked variant in a male who's undergoing carrier screening. What we've been seeing in the reproductive space is that we are increasingly identifying patients who are reported to be carriers of DMD. This is true across the board. With increasing carrier screening and bigger panels, we are detecting more carriers in general, but we're also detecting more X-linked disorder carriers, and that includes carriers of dystrophinopathies. What we're also learning, though, and what Kayla will go into in a lot more detail in just a minute, is that we're also starting to recognize that we may need to do more when an individual is found to be a carrier of an X-linked condition like DMD in the absence of a known family history to really assess what the true reproductive risk is and what the personal health risk, if any, is. Cece's case is a very good example of how the absence of a family history really needs to drive helping us clarify the pathogenicity of a variant, and we can't necessarily rely on the data or the classification of the variant by the lab alone in all of the cases. I think that's particularly true of DMD when a lot of time we're dealing with large deletions, large multi-exon deletions or duplications and not point mutations where we may have much more clear defined information about that particular variant when we're dealing with a single nucleotide variant, but when we're dealing with multi-exon deletions and duplications, how certain are we that those deletions and duplications are exactly the same and having the same effect on the gene? This is particularly true because people who are found to be carriers have a lot of reproductive options that they have to choose between. Certainly they have the option of waiting until they deliver a child and then doing testing for that genetic condition, in this case a DMD variant at birth, and then figuring out things from there if the baby is affected and isn't affected. They also have the option of prenatal testing, and as Blake's narrative of the case described, a lot of times that's also deciding whether you're going to continue a pregnancy or not based off of those prenatal testing results. And of course, for those of us that work in the reproductive space, we want to make sure that people that are making those decisions are making those decisions when there is going to be a phenotype of concern at the end of the day. And then also there's the option of pre-implantation genetic testing, PGTM. And as Blake explained, the gold standard in PGTM is typically not direct variant analysis. And this is true not just of DMD, but most conditions that we're doing PGTM for. And most of that is driven by the fact that we've got concerns for allele dropout, and we want to make sure that this testing is as accurate as possible. But it's even more so true for conditions where we've got large multi-exon deletions and duplications, and that in those scenarios, linkage is really the only way the labs that do this testing are performing the testing. But that does require testing of other family members, other known carriers in the family to provide us informative marker patterns, and hopefully marker patterns that truly do end up being informative. And it's sometimes during these assessments when a couple is undergoing this kind of pre-setup for PGTM that we may learn that a variant isn't quite what we thought it was. So I think we've got a lot more questions about whether we can take things at face value in the carrier screening realm, especially for X-linked conditions. And to give you a deeper dive into that, I'm going to go ahead and pass it over to Kayla. Okay, so I'm going to dig a little bit deeper, more specifically into DMD variants and dystrophinopathies and what we know and what we're still trying to figure out. I know this audience is largely familiar with the DMD gene and dystrophinopathy, but just to start us off with a little bit of background information, when you think of the DMD gene and you're thinking about dystrophinopathy, most likely the first thing that comes to mind is Duchenne muscular dystrophy, which is an X-linked condition that is characterized by childhood onset, progressive muscular weakness, and cardiomyopathy. And the next thing to pop into your head might be Becker muscular dystrophy, which looks very similarly to Duchenne, but is generally associated with potentially a later onset or slower progression of symptoms and maybe a more variable progression. Historically, these may have been diagnosed initially on a muscle biopsy where they found that there was absent or significantly decreased dystrophin present in muscle tissue. As genetic testing has expanded, we've really seen documentation of this expansive spectrum that can be associated with dystrophinopathy variants. This might include someone who has something like a pseudometabolic phenotype where they have exertional myalgias or episodes of rhabdomyolysis. This might be someone who has isolated cardiac symptoms. This also includes the wide variety of symptomatic presentations that can be seen in heterozygotes or DMD variants. So we have manifesting carriers that maybe look very similarly to someone with Duchenne or Becker, or they may be clinically asymptomatic. When clinicians are encountering a DMD variant, there are a whole lot of factors that you may be looking to consider in your interpretation. If you're encountering a deletion of the DMD gene, which is something that we had kind of mentioned, a deletion of one or more exons, that is the most common type of variant that is seen in the DMD gene. The first thing that might come to your mind is the rating frame rule, which tells us that if a deletion is in frame, potentially we're still allowing for some production of dystrophin, though it might be shortened and it might be produced in less quantity, we still are seeing some dystrophin produced. And so generally that person may be expected to be on the Becker end of the spectrum. And that is compared to an out-of-frame deletion where it really stops dystrophin production, the body is not able to read past, and we get little to no production of dystrophin. And generally someone who is hemizygous for that type of variant would be expected to be on the Duchenne end of the spectrum. And we can look at the reading frame rule for about 90% accuracy. However, important notes about the rating frame rule, it seems to be less predictive in duplications and less predictive in variants that are more classically associated with Becker muscular dystrophy as opposed to Duchenne muscular dystrophy. Next, you may turn to the literature. So what has been seen before with this particular DMD variant? And can we use that to maybe get some prediction of what we would expect for this particular patient that you're working with? So the first two, Dove and Leiden are open access online databases that are specifically for DMD variants. We use them very frequently to help us with locating publications or case reports of DMD variants. I additionally will utilize the Duchenne registry, which if you are not familiar with the Duchenne registry, it is a self-report registry that is housed within PPMD to better understand the spectrum of dystrophinopathy. And so we ask for individuals to register. They can have a diagnosis of Duchenne, of Becker, of being a carrier, what have you. And they respond to surveys about their symptoms, about their genetic variant, about their family history to help us learn more. Taking a moment to highlight some of the phenotypes that we're seeing in the Duchenne registry. When someone registers with the Duchenne registry, they do self-report their diagnosis or their child's diagnosis if they are registering for their child. And you can, as expected, see that most of the participants that we have have a diagnosis of Duchenne. We do have representation from individuals who have a diagnosis of Becker or individuals whose diagnosis is not quite clear. They might be somewhere between Duchenne or Becker. They may be very young. And so they're not clinically sure if they will follow more of a Duchenne or Becker path. But I did want to draw attention specifically to a new category that has been added to the Duchenne registry and that we have had some uptake whenever individuals are registering. And that is there is an option to register yourself as an adult male with a pathogenic or likely pathogenic DMD variant and no symptoms. And we do currently have three individuals in the Duchenne registry who have registered themselves as an adult male with a pathogenic or likely pathogenic variant and no symptoms. In all of these cases, those individuals were either identified on their own expanded carrier screening or were identified through family follow-up testing after a family member was identified on expanded carrier screening. Earlier this year, we completed a data export that was specifically highlighting some of the carriers that are present within our registry. And I just wanted to take a moment to highlight some interesting data that was pulled from that. There is a survey which asks, how were you identified to be a carrier? And in the participants who responded to that survey, 77 of them indicated that they were identified on general carrier screening for Duchenne. 48 of which were solely on their carrier screening in the absence of symptoms or family history. I would like to now transition into a few case studies of families that I have had the opportunity to connect with. These were families who were originally identified on expanded carrier screening. They connected with us trying to discuss more what is known about their particular genetic variants, what next steps they might take, what other things they might need to consider. And each family did give their permission for me to share their story. And they did pursue additional testing through a sponsored genetic testing program that is housed within PPMD as well. So moving with case study number one, this was a woman in her 30s who was identified on expanded carrier screening to be a carrier of a deletion of exon 49 in the DMD gene. At that time, she decided to pursue diagnostic testing for her pregnancy. And the pregnancy was found to be an XY male who was positive for the DMD deletion. And they decided at that time, based on the information that they had, that they wanted to move forward with medical termination of the pregnancy. So whenever we look at a DMD variant, we want to think about, okay, what is known about this particular variant? If you're looking at a deletion of exon 49, it is an in-frame deletion. And so by reading frame rule, we would expect that a male with this deletion would be on the Becker end of the spectrum. And this particular variant has been reported before multiple times. So there was one publication out of India that had four patients identified with a deletion of 49. Three of them were given a diagnosis of Duchenne muscular dystrophy. And one of them was given a diagnosis of Becker muscular dystrophy. There was another publication out of France in 2022 that reported on five individuals who were incidentally identified with this particular deletion. And this group included a young boy who had a history of global developmental delay, but had normal creatine kinase levels and normal dystrophin staining on his muscle biopsy. It also included another boy who had a CK level at the upper limits of normal, but was otherwise unaffected. And a XX woman who was homozygous for this particular deletion, and also had a completely normal neurologic exam and normal CK levels. Even more recently in 2023, there was a publication in the American Journal of Medical Genetics from a group at Emory who identified a man in his fifties who presented to clinic with heart failure. He was a marathon runner and very healthy and no sign of muscular weakness, but they did pursue genetic testing on this individual and he was found to have a deletion of exon 49. So back to our family here and kind of what we did next. So this family decided to pursue some additional familial testing to help provide additional information about maybe what this particular deletion looks like within their family. The original patient's mother was identified to also be a heterozygous carrier of the DMD deletion. And they decided to additionally pursue testing for her apparently unaffected brother. And he was also found to have a deletion of exon 49. He is in his forties. He is a marathon runner, very healthy and doing very well, reportedly completely asymptomatic. I am happy to report that at the time that all of this additional familial testing was going on, the woman who I originally met with was pregnant again and did deliver a healthy boy who does not have the DMD deletion. We did additionally discuss that her brother does have a daughter. She has not yet been tested. She is an infant, but would need confirmatory testing though she is an obligate carrier and then monitoring for cardiac symptoms as she is getting pregnant. And then monitoring for cardiac symptoms as she gets older. So moving now to the second case study, this is another woman who I was connected with, again in her thirties, who was identified on expanded carrier screening to be a carrier of a duplication of exons 45 to 51 in the DMD gene. She connected with me because she wanted to have a conversation about what that might look like and what options there were for testing for her daughter in order to move forward with steps for building a probe for IVF testing for additional pregnancies. Initially kind of looking at that initial line of interpretation, this particular duplication, if it is inserted in tandem is expected to be an in-frame duplication. It has been reported previously. There are multiple entries in the Leiden database of individuals who have a diagnosis of MD or dystrophinopathy. Her daughter was confirmed to be a carrier of this same duplication. However, whenever testing was pursued for her daughter, my screen froze, apologies. There we go. The laboratory that did the testing for her daughter was initially completing next-gen sequencing-based deletion duplication analysis. The lab came back to us and said, something looks a little bit off. We're going to do some additional studies. So they pursued breakpoint analysis of the split reads and then confirmatory Sanger sequencing. And they identified that the duplicated region that was flagged on that original expanded carrier screening was actually extragenic and was not at all present on the X chromosome. It was actually located on 17q21.1. And they reclassified this variant in their interpretation as a variant of uncertain significance. Just anecdotally, I do know of another family who also has this same duplication, also extragenic. And as far as the families know, they are completely unrelated to each other. So going back to our case, what did they do next? They decided that they, again, wanted to pursue some additional familial testing just to get confirmation of this variant not interacting with or impacting how DMD is functioning. So they did pursue testing of her father and he was found to have this same VUS duplication. Her brother was also tested and he is negative for the duplication. So this may seem a bit obvious to say, but the message that I'm hoping to leave you all with is that we must be cautious in interpretation of variants that are identified on, incidentally identified on carrier screening or other testing in the absence of family history of dystrophinopathy. And a quick thank you to my team and our community members for always being willing to share their stories. All right, thank you again so much, everybody, for attending today. We do have some questions in about eight minutes to go through them. It looks like there are a couple of questions it looks like there was a question about whether in fact CC's duplication was pathogenic and someone later on said that there have been several cases of DMD with the duplication of exons 50 to 55, but there's still the questions of, how were the brothers clinically normal? Are there thoughts on whether there were different breakpoints, whether it wasn't in tandem or any thoughts? I know Sandra, you had dug into this case very deeply. So I'll pass that over to you to see if you could comment a little bit more on what else was performed for CC. Yeah, so we did additional fish studies to see if the duplication was where we would expect it to be most likely in tandem or somewhere else to answer that question, especially after the PGTM set up didn't go according to plan. And the fish results seem to indicate it's where we would expect it to be. It's in tandem. We don't have a reason to expect that it's definitely not on another chromosome like Kayla's cases. So, and so that didn't really give us a clear cut explanation. And so there are other reported cases like are mentioned in the medical literature with that duplication in individuals who have a diagnosis of a dystrophinopathy. So I think that's where a lot of the confusion originally stemmed from is, this is not, wasn't reported as likely pathogenic, was reported as pathogenic. There are cases in the medical literature with PubMed IDs that we reviewed that seem to indicate that this would be a risk for a clinically significant phenotype. But then, as we found, we've got a male in the family who is doing well, doesn't have any obvious feature. So we don't know why in this family it doesn't seem to be manifesting. And Kayla may have more insight into that. So I quickly pulled it up just to take a look at that particular duplication. It is an predicted in-frame duplication if it's in tandem. So theoretically, reading frame rule could indicate that maybe we're looking at something on the Becker end of the spectrum, which could be associated with a lot of variability. And also just thinking about potentially other modifiers that are contributing to the phenotype or absence of phenotype, just depending on each individual person. Thank you. Something that came up with both cases, what was the ethical conversation surrounding testing LUNA for carrier status with the purpose of setting up PGTM? And I know, Kayla, you also had an example of that as well. You know, it's interesting. And we do talk about, you do have to consider the implications for testing an asymptomatic minor and just kind of the general conversation surrounding that, potentially impacting life insurance coverage, those kinds of conversations. So I did ask for that person to, I don't perform full clinical counseling with my families, but I brought up some of those concerns and asked for them to kind of go back with their clinical team and have those discussions and make sure that they are comfortable with those risks. But it is their choice to utilize that medical information, right? And making those decisions for their family, so. I would say our conversations were right along those lines as, you know, it's kind of trying to weigh what you're gonna do with that information or the need for that information at this point versus what could that mean in the future for that child. Certainly a discussion of, you know, long-term care insurance, you know, potential discrimination based off of a genetic test result, those types of things. And then leaving it to the parents to make those decisions because quite frankly, if they wanted to do embryo testing and if this would have, you know, if this had been a variant that was clinically significant and, you know, more clearly associated with a phenotype, that would have made big impacts on what their reproductive decisions were. So I think because they knew how much impact that those decisions, what was driving those decisions for them knowing that information was really helpful. And I think they went into it fully understanding what they were taking on for that testing for their daughter at the same time. Thank you. It looks like we might have time for one more question. There were some questions about just the logistics of the linkage analysis. So someone asked if this was a de novo change and there wasn't someone else in the family to test for how that might be done. Or similarly, if there isn't a family history that allows for testing of, you know, a brother or another family member how can PGTM be set up? I guess I will take that one since that's more in my wheelhouse. There are times where PGTM will not be able to be set up and de novo cases are really good examples of that, particularly de novo variants in a child who's the first individual, but sometimes also when the patient is a carrier of a de novo variant. Some labs will do direct variant analysis like this and do direct variant analysis on PGTM, understanding that we may have a higher chance for a false negative due to an allele dropout event that we may not recognize is happening. But for them to feel comfortable in doing that they have to be able to clearly detect that variant on an embryo biopsy results. So some variants are easier to detect and for the labs to feel confident in their detection of large deletions and duplications, triplet repeats usually do not fall into those categories. And so for a lot of PGTM labs, if you're dealing with one of those multi-exon deletions or duplications or triplet repeat disorder, if there is not a way to set up linkage analysis either by testing other family members then there's not gonna be the ability to do PGTM. Thank you. And it looks like there is just one comment that fish will only tell you that the duplication is near the gene of interest and not that it will be in tandem. We are aware of its limitations, of course, and I believe in Cici's case, there was conversation about other testing, including genome, but that of course, clinically it's much more difficult to be doing those things to confirm. So with the information we had, there's of course, nothing to suggest that it's not in the region where it should be. So thank you so much for those questions. We do just also want to share that the ACMG annual clinical genetics meeting is in March from the 18th to 22nd in Los Angeles. So we hope to see you there. The program registration and hotel information is live and you are able to submit abstracts as well. And to have more information about the ACMG annual clinical genetics meeting, you can go to acmgeducation.net and you can submit the abstracts there as well. The abstract deadline is December 6th. We also are calling for abstract reviewers to review the abstracts that are submitted. Thank you so much for attending today. Thank you, everyone. We'll see you next month.

DMD variants

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