 So, we have a great second session for you guys. We'd like to bring up our first speaker, Sonia Sobrowski from the Mayo Clinic. We'll be talking about the role of genetic testing. I know this can be the bane of our existence in cardiac amyloid diagnosis, let alone just all of cardiomyopathy. So, really excited for her great talk. Thank you for your attention. Hopefully, you can all hear me. Okay. Thank you for that introduction, Amber. As she mentioned, I'm a genetic counselor here at Mayo Clinic, and I actually serve our adult general genetics population. So I see lots of patients with wide varieties of indications, but I'm particularly fond of cardiac genetics. So, today I'll be discussing the role of genetic testing in cardiac amyloidosis. So, no conflicts of interest to disclose, breeze through that. So, given that my focus area is in genetics, it's fitting that the majority of this discussion will focus on identifying hereditary or variant type, transthyritin amyloidosis. So, thinking about the types of amyloidosis most commonly associated with cardiac manifestations, we've already touched on AL and ATTR. In the ATTR cases, as we've also discussed, can be wild type or hereditary. And genetic testing can help differentiate between the two, which can then help us better understand implications for the patient and their family members. So, genetic testing for hereditary TTR amyloidosis involves analysis of the TTR gene, and this gene is located on chromosome 18Q12.1 and encodes the transthyritin protein, which again, as discussed previously, is one of the three pre-albumins. So I won't talk too much about this since we've already gone over most of it, but pathogenic variants, also known as mutations, in this gene are associated with amyloid deposition predominantly affecting the peripheral nerves or the heart, though there are a small percentage of gene mutations considered to be non-amyloidogenic. So TTR genetic variants are implicated in the etiology of several diseases, including amyloidotic polyneuropathy, urethroid hyperthyroxenemia, amyloidotic vitreous opacities, cardiomyopathy, and carpal tunnel syndrome, as we've discussed previously. And from a molecular standpoint, pathogenic variants in this gene are nearly always sequence variants rather than deletions or duplications. So thinking about the inheritance of hereditary ATTR, these variants are inherited in an autosomal dominant pattern. So that means that each child of an individual with hereditary TTR amyloidosis has a 50% chance to inherit this causative variant. And we expect that the affected individual inherited the variant from a parent. So we would also anticipate that other first-degree relatives, so siblings, parents, also have a 50% chance to share the same amyloidosis-causing variant. Theoretically, there's a chance that somebody's TTR variant could be de novo or new, arising for the first time in themselves. But the rate of de novo TTR mutations is currently unknown. And we'll continue to dive into the concepts of incomplete penetrance and variable expressivity as related to this condition. But just so we have kind of a framework for what those mean over the next couple of slides, incomplete penetrance really refers to the idea that not all individuals with a specific genotype will manifest a particular clinical symptoms or characteristics of that, while variable expressivity really refers to the variability of signs and symptoms that can occur in different people with the same genetic condition. And these things, incomplete penetrance, variable expressivity, are pretty common in the cardiogenetics world. And for hereditary TTR amyloidosis, there's been quite a bit of investigation done related to genotype-phenotype correlations for specific variants. So for example, the valine-50 methionine variant is considered the most widely studied TTR variant. And just to touch on nomenclature again, really what this means is that at amino acid position 50, there should be a valine, and now there's a methionine there. And autonomic neuropathy and peripheral neuropathy are most commonly associated with this variant, while cardiac involvement is much less common. And penetrance appears to vary by age, region, or ethnic group. So just for example, in Portugal, the cumulative disease risk in individuals with this variant is estimated to be at about 91%, whereas in individuals in France who are heterozygous for this variant, the risk is about 50% by age 70, and in Sweden, the penetrance is much lower, estimated to be at 36% by age 70. And in some cases, heterozygous do remain completely asymptomatic. And then looking at another example, we've also briefly touched on this one previously as well. Approximately 3% to 4% of African Americans are estimated to carry the valine 142 isoleucine variant, which may be present in more than 5% of the population in some areas of West Africa. And most individuals who are heterozygous for this variant are expected to develop late onset cardiac amylidosis. So over 140 disease-causing TTR variants have been identified, and this table shows a few additional genetic variants with a breakdown of how these variants have been characterized thus far in terms of prevalence, median age of onset, at symptom presentation, primary ethnicity, geographical region, cardiac phenotype, other manifestations. And genotyping doesn't provide us with a crystal ball to perfectly predict disease course or outcome, but it can help us anticipate general clinical presentation, age of onset, organ involvement, and potential prognosis based on what have been generally seen in individuals with these specific variants. And we do know that, essentially, all TTR variants exhibit age-dependent penetrance, so risk for symptoms increases with age. And research has also been done in individuals who are homozygous for specifically that valve 50-met variant, so people who have two of this variant want an either copy of their TTR gene. And this has suggested that homozygots present with a slightly more severe clinical course, so higher incidence rate, earlier onset than heterozygotes within the same family, or people with just one TTR mutation. And these people who are homozygous amyloid deposition was also found to be more widespread compared to heterozygotes in that study. And so another important piece of information to consider when reading a genetic test report or searching for specific TTR variants in the literature is that the nomenclature of these variants has changed over time. So when we look at that valve 50-met variant in one paper, it might also show up as the valve 30-met variant in another paper. And really this naming convention differentiation is just because one includes a 20-amino acid signal peptide in the count of residues, whereas the other doesn't. So that accounts for the difference between 30 and 50. But just important to keep in mind as we see these variants pop up in the research. And then as previously mentioned, you know, there are many more known TTR variants than just those five. So many have been studied to better understand the phenotypes associated with them, and that is available in the research to find. So knowing a patient's genotype and whether their TTR amyloidosis is wild type or hereditary may also have implications in treatment. And I am not an expert on the treatment side of things, but just to briefly review, options may include TTR tetramer stabilizers to address tetramer dissociation in fibril formation or gene silencing therapies which use antisense oligonucleotides or small interfering RNAs to reduce amyloid-fibril precursor protein. And there are other potential molecular therapies under investigation as well for hereditary TTR amyloidosis that may aim to also either inhibit synthesis or stabilize variant TTR or to disrupt insoluble amyloid fibrils. So gene editing therapies such as CRISPR-Cas9 technology is one of these methods that's currently under investigation. So we're probably all familiar with the clinicalchiles.gov website, so I won't talk about that too much. But some of the eligibility criteria for these research studies or clinical trials may require a known genetic variant to be present. So now that we've kind of touched on why genetic testing can be beneficial, it's important to know when it's indicated. So TTR genetic testing is certainly recommended for all patients with an established diagnosis of TTR cardiac amyloidosis regardless of age to differentiate between the wild type and hereditary ATTR given the disgust management implications. And at this time, genetic testing is not indicated for AL amyloidosis. Another situation that may arise or maybe you've interacted with patients who have done direct to consumer testing that can be purchased at the store, sometimes patients who do this type of testing and opt in for the medical assessment side of things may be identified to carry a common TTR variant. So for context, these tests are commercially available to purchase, healthcare provider involvement, insurance provider involvement is not necessary, and they might screen for the presence of really only three common TTR variants. So if a patient is found to carry one of these variants, there's a good chance that it's a true positive, but it's still recommended to follow up with confirmatory clinical genetic testing. Now it's also important to be especially wary of patients who might have done direct to consumer testing and they say I have a rare variant in the TTR gene. These may pop up when patients elect to use their data that they've gotten from direct to consumer testing and upload it to a third party interpretation service. These rare variants that pop up from that type of testing have been known to have a significant false positive rate. So certainly clinical confirmatory testing is really recommended in those cases. And on the flip side of that, it's also again important to recognize that this testing only really screens for those three common variants. So if somebody has a suspicious clinical or family history for hereditary amyloidosis, but they say, oh, I had negative direct to consumer testing, that really doesn't rule out the possibility that this is something hereditary and clinical TTR sequencing would be indicated in that case for a more comprehensive analysis. And then another indication for genetic testing would be if somebody has a known family history of a known TTR variant and they want to know what their risk is. And in that case, it's really helpful for the patient to obtain records of their affected family member's genetic test results. So we have reference of what variant we should know to be looking for. And it's important to keep in mind that a negative family history doesn't necessarily rule out the possibility of hereditary TTR amyloidosis. There are several reasons why a family history may appear non-contributory, such as the failure to recognize symptoms, early death of a parent or other relatives before onset of symptoms that really never had a chance to present, or given the variable nature of the condition, it's possible that it may not have yet presented itself in a parent or other family members. And then once we have identified, okay, this patient probably needs some genetic testing or is recommended to have genetic testing, how do we coordinate that? The testing itself is typically performed using a blood, buccal, or saliva sample. And there are many labs that offer TTR analysis as a genetic testing service. So some of these commercial labs who specialize in genetic testing may have convenient insurance billing assistance where they might be able to conduct a benefit investigation for the patient to provide them with an estimated cost of the testing before proceeding with testing, or they might provide them with a self-pay option for testing that's typically around $250. Some of these labs also have policies to not balance bill patients who are insured through Medicare or Medicaid, so oftentimes testing might be covered for them. Additionally, I have on here some sponsor testing programs as well. So these exist to offer no-charge genetic testing to patients who meet certain eligibility criteria, like a suspected diagnosis in themselves or a confirmed family history of hereditary amyloidosis. So there's often different eligibility or clinical features that you can go in and say, yes, this patient meets this criteria and they might qualify for free testing. So these sponsored programs have the goal of providing increased access to genetic testing and counseling for patients, and they then might receive de-identified patient data for research and commercial purposes. So just a couple of examples of those are the LNILM Act program through in VTAY laboratory, as well as the HATTR Compass Genetic Testing Program through AMBRE Genetics. So if you need assistance coordinating genetic testing, genetic counselors are here to help. In general, we can assist with performing risk assessments, providing genetics educations for patients as well as their family members, facilitating decision-making and coordinating that genetic testing as well, among other things that we can assist with. So if you have genetics questions, ask us. And where do we find genetic counselors? Your hospital or clinic may have genetic counselors on staff who can assist in answering genetics questions or point you in the right direction, but also many of these labs offer genetic counseling services. So you can call the lab and ask questions about variant interpretation or if you need help understanding a result, or sometimes genetic counseling for positive results is included for patients in the cost of testing, or they might partner with independent telehealth companies that can provide those genetic counseling services for patients. And a good resource I always provide too is www.findageneticcounselor.com that can help you locate local genetic counselors. So looking past the affected patient themselves and into their family and family dynamics, when a genetic variant is identified to be the cause of a patient's amyloidosis, their family members might be interested to know whether they're also at risk for hereditary amyloidosis as well. So testing of asymptomatic family members requires additional consideration and counseling. It's important that they have the opportunity to meet with a genetics provider to discuss their motivations for genetic testing as well as the benefits and limitations of doing genetic testing at that time. So they might have certain expectations of what testing can tell them and how it might impact their medical management or their family dynamics. So it's helpful to have a discussion of family history to understand what their experience has been with the condition and that affected family member or multiple family members and kind of to know what the predicted age of disease onset may be for that family, both in terms of what other family members have shown and what we might know from the research side of things about that particular familial variant. So it's been suggested to begin monitoring for symptoms around 10 years earlier than the predicted age of onset. And it's also important to consider that predictive testing in asymptomatic minors is considered inappropriate. So it's helpful to set expectations with a patient that if they're positive for the variant, we typically will not be testing their children if under 18. Something else to think about is psychosocial implications of testing. Really not everybody is going to react the same way to a positive or negative genetic test result, finding out about their risk for one of these, for this condition. So it can be helpful to prompt the patients to consider know how they would really feel either way. Maybe if a negative result, they might be really glad or relieved that they're not at risk or sometimes patients might feel guilty that everybody else in their family is positive or have undergone this medical experience and now they know that that's not going to be the case for them. Or on the flip side, if a positive test result, maybe they'll be really anxious or stressed about the potential health implications or they could feel empowered to know their risks and understand their management options. And then from an insurance standpoint, it's important to think about how the implications of learning about a genetic test result might impact different insurance eligibility. So something I talk about with patients is that there's a federal law called GINA or the Genetic Information Non-Discrimination Act that protects most people from their health insurance and employer asking about their genetic test results or using that risk information to discriminate against them. However, other insurance companies like Life Insurance, Long-Term Care, Disability Insurance Companies are legally allowed to ask about genetic test results and use them in their eligibility determination processes. So for some patients, this is something they really care about for others, something that's not too much on their mind. And another reason that individuals might want to know their TTR genetic status is for family planning purposes. So when a person is identified to have a TTR pathogenic variant, IVF and preimplantation genetic testing might be available to select for embryos that don't carry the variant. And there are differences in perspective about prenatal testing for familial TTR variants during pregnancy given the adult onset and variability of the condition. So really discussion of the considerations may be beneficial for reproductive decision making as well. So with that, I just have some references. And thank you so much for your time.