MECHANISTIC INSIGHTS AND CLINICAL IMPLICATIONS FROM GENETIC STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 1

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1 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 1 Well I ll be talking to you primarily about cardiomyopathies today and I don t think anybody in this audience needs to be told what cardiomyopathies are, that they are primary disorders of the myocardium. Generally disorders that are associated with heart failure, arrhythmias and sudden cardiac death and most of these are mendelian single gene disorders inherited as autosomal dominant traits, less often autosomal recessive and even less often X linked but what s, what s interesting is that in the pediatric population some of the autosomal recessive and X linked forms can be a little bit more prevalent and we ll talk a little bit about that today. So what I ll talk about today primarily are going to be hypertrophic cardiomyopathy and I ll spend quite a bit of time on that because in fact this is the most common cardiomyopathy, and I ll also talk about some mimickers or phenocopies of hypertrophic cardiomyopathy or ACM including some of the storage cardiomyopathies and then other disorders that are associated with cardiac muscle thickening. Then we ll spend some time on dilated cardiomyopathy and then arrhythmogenic right ventricular cardiomyopathy as well. Now there is a lot of different areas that we can cover and obviously we can t do everything in an hour but I wanted to touch on some of the epidemiological and clinical features of these disorders and you know since this is a primarily clinical audience I do want to spend a little bit of time on just the clinical approach to symptoms and particularly with stratification for sudden cardiac death, which is one of the sort of the most worrisome aspects of these cardiomyopathies. And I ll touch a little bit on some of the differences between what we see on the adult side and what you see on the pediatric

2 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 2 side. And interspersed through all of this I will discuss what we know about the molecular genetics of these disorders, touch on some of the mechanistic aspects that we have discovered as a result of our genetic studies but it s kind of hard to put everything together comprehensively but at least you ll get a flavor as to some of what our recent insights are in this area. And I do want to spend a fair bit of time at the end just talking about the approach to genetic testing, which has really moved into the clinical realm especially in the past 5 or 6 years and some of that we are kind of doing routinely now in our, in our clinics. So let s talk about hypertrophic cardiomyopathy to begin with. And so it s the most common cardiomyopathy as I mentioned, prevalent at about 1 in 500 in the general population, affecting males and females just about equally. Overall there is about 600,000 patients that are affected in the U.S. and you know only about 1 out of 5 of these patients actually know of their diagnosis. There is a huge part of the iceberg that s under water and people just don t know they have this. We used to think that HCM was a particularly lethal disorder but it turns out that the animal mortality is only 1%, which is actually not very different for the general population. In the pediatric population it s actually 1 to 2% or so, but still relatively low. And unfortunately there are some subgroups that are at high risk of death where the mortality rate can go up to 3 to 6% and escalations that kind of behooves us to figure out who is at high risk and then offer appropriate therapy to them. So on the pediatric side about 10% of all cases of HCM are actually in patients under the age of 12 years, that s about 60,000 patients in the U.S. The symptoms and diagnosis will vary by age and

3 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 3 we ll talk a little bit about that. Overall the long term prognosis is actually pretty good except for a few sort of risk factors that put the mortality higher, especially if they actually manifest the clinical evidence of HCM early, within 1 year of age. So what does HCM look like? Well this is a normal heart right here, and in hypertrophic cardiomyopathy you get sort of typical remodeling which is abnormal thickening of the ventricular walls, and there is a variety of different pattens of thickening like in the curve, it can be primarily septo, it can be more concentric and if you look at the histology, this is a normal myocardium here with sort of parallel myocytes. So what happens with hypertrophic cardiomyopathy is that you get first what s called myofibrillar disarray which means that the myocytes are disorganized and not parallel and a considerable amount of fibrosis, interstitial fibrosis between the myocytes which is shown in blue here. And this may be one of the reasons for creating a substrate for arrhythmia as we ll discuss. So a number of things happen with HCM but one of the problems that occurs is that you can get if you get asymmetric septal hypertrophy you can get dynamic obstruction of left ventricle outflow, so that the blood has a tough time getting out into the aorta. And this is one of the reasons that patients can develop symptoms, including anginal symptoms, chest pain as well as dyspnea on exertion. The general cutoff for calling a patient obstructive is a gradient of 30 mm of mercury, which can occur in some patients at rest, in some patients it only occurs when you provoke the obstruction either by having them exercise or by other maneuvers that decrease the size of the left ventricle or increase the

4 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 4 conductivity of the left ventricle and exacerbate the dynamic obstruction to create a gradient of more than 30 mm of mercury. So how do we approach these patients clinically? As I mentioned the main symptoms that we see in these patients at least on the in adult patients is that they will have they can have chest pain or exertional chest pain, dyspnea on exertion and there is a variety of techniques that we can use to, to help these patients, a number of medications that I ll touch on briefly. If there is considerable obstruction that seems to be causing the symptoms we can offer surgical septal myectomy as well. A second area that we are always concerned about, as I mentioned before, is sudden cardiac death. These patients, at least some patients are at high risk for sudden cardiac death, in that setting some of them will need to be offered ICDs. Some patients, particularly as they get older, are at higher risk of developing atrial fibrillation, probably for a couple of reasons. The left atrium enlarges because it faces a diastolically dysfunctional left ventricle or a stiff left ventricle. Also some of the myocardium remodeling process that occurs in the ventricle probably also occurs in the atrium, so these patients can develop atrial fibrillation and are at risk of stroke. And one of the major issues that we face is that because this is a genetic disorder you are not just taking care of the patient, you are taking care of the family in many cases. And you have to screen the family because many relatives may in fact be at risk of HCM and not know it, they may be asymptomatic.

5 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 5 So what about kids then? So as I mentioned it s kind of dependent on the age at presentation. If they are infants then often there are manifest symptoms of congestive heart failure such as tachypnea and difficulty feeding, failure to thrive and obviously they may have heart murmurs on exam. when they are older their presentation becomes more and more similar to adults, so they may have some of the same sort of symptoms as young kids but also they may be presenting with syncope, palpitations, exercise intolerance, and as I said before if sometimes they may not have any symptoms but a relative may have had a diagnosis of HCM and therefore they may be at risk of having HCM and when they are evaluated clinically they are found in fact to have that diagnosis. So what do we do in our clinic in terms of just screening these patients? Generally when we have a new patient come in on the initial visit and actually every subsequent annual visit they ll obviously get a detailed history and physical exam, they will all get electrocardiograms, a trans-fluoroscopic echocardiogram and a Holter monitor. In addition at least on their initial visit we will get at least one cardiac MR imaging study done and exercise treadmill testing as well to determine their response to exercise in terms of blood pressure and arrhythmias. So why are we doing some of these things? So why is imaging so important? Some of it is pretty obvious, you know you want to do an echocardiogram as first line imaging to determine whether in fact they are hypertrophic and the pattern of hypertrophy and whether they have left ventricular outflow track obstruction. We generally will do cardiac MRI as well partly because it is free from some of the potential limitations of limited ultrasound windows, but also for other reasons, for risk

6 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 6 stratification. So what happens is that you know the cardiac MRI will give you anatomy, it will give you an idea of the extent of hypertrophy and the pattern of hypertrophy but you can also then characterize the amount of fibrosis that s present in the myocardium. And that is important because there is some evidence that the amount of fibrosis in the myocardium will correlate with the degree of risk for sudden death. We also combined the echocardiogram with a treadmill stress study, again to assess their response, their blood pressure response to exercise but also to determine what their peak left ventricular outflow track gradient is on exercise. Some of these patients will have only exertional symptoms so we want to figure out whether they are developing outflow track obstruction only with exercise, and this is one way to assess that. The general guidelines right now for clinical screening of family members who have a relative who has been diagnoses with HCM are as indicated in this slide. So when they are young under the age of 12 generally the recommendations are optional, and this is again this is kind of a consensus statement, nothing is cast in stone here but this is sort of general thought that some, some folks have put together which is that unless there is some high risk features, for example a high malignance or a family history of a lot of sudden cardiac death, or if the patient wants to engage in very intense comparative sports, or they obviously have some sort of symptoms generally you don t necessarily need to screen them as kids. Otherwise if you have some of these high risk histories they definitely should be screened at least once before the age of 12. Once they hit puberty then you probably want

7 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 7 to annual screening because that s the point at which the body is growing, the heart is growing and often HCM manifests during that period. After the age of 18 to 21 or so the recommendations are generally to screen about once every 5 years unless they develop symptoms or unless again there is a family history that is very concerning with a lot of sudden cardiac death. And the drugs that we use I think everybody is probably quite familiar with. We generally use beta blockers which will decrease the contractility of the heart and will decrease the heart rate too so that the left ventricle size increases and both of these will then decrease the amount of obstruction and hopefully relieve symptoms. Verapamil or other calcium channel blockers are used as second line therapy. And in adults we use Disopyramide as well, it s less commonly used in the pediatric population. Okay, so as I mentioned before, if drugs don t work one of the options is actually to do a surgical septal myectomy and this is actually a slide that s specific to children where we find in fact it is quite prevalent or common. If they present as a pediatric case of HCM you know by the time they are 10 years beyond diagnosis you know almost 30% of patients actually have a required surgical septal myectomy for refractory symptoms. And there seems to be survival benefits, so this is not just to help them with their symptoms, it turns out and this is true in adults, this is also true in children as in this slide, if patients undergo myectomy for refractory symptoms they have a much better survival than if they are just treated medically. So clearly there is not only a symptomatic benefit but also potentially a survival benefit.

8 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 8 What are the risk factors for sudden cardiac death? This is again the thing that we worry about the most. Well the traditional risk factors in the adult population are if they have a history of syncope or arrest or a documented ventricular tachycardia all that is bad, if they have a bad family history that s bad, in adults left ventricular wall thickness of more than 3 cm is a risk factor as is an abnormal or inability to raise blood pressure during exercise. What about adolescents and children? It s a little less clear. There has been a few studies out, much less data but this is one study which followed about 100 patients for about 6 years. There were 7 deaths. They could not actually identify any risk factors for sudden cardiac death. They did identify some risk factors for heart failure death, which included just extreme hypertrophy and abnormal blood pressure response to exercise. A second paper looked at again around 130 patients, followed up with them for around 6 years. They found some risk factors for sudden cardiac death including septal thickness of more than 2 cm, abnormal or inducible ventricular tachycardia on the electrophysiological study if the kids were diagnosed early but then are beyond the age of 13 years, if they have a history of syncope or presyncope. Interestingly some of the other risk factors in adults were not confirmed in this population. And finally a third Australian study looked at some of the determinant of survival and this is just overall survival. As you see it s actually pretty good, so even 10 years down the line there is about a 1 to 2% mortality rate per year. But other things that seemed to affect survival, if there was a

9 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 9 familial history the patients did better, if there is no family history of cardiomyopathy patients did worse. If the pattern of hypertrophy was asymmetric septal they did better, if it was concentric hypertrophy they did worse. And that s probably because the etiology of the cardiomyopathy is different depending on what the pattern is, the pattern of hypertrophy is or the family history is, and we ll talk a little bit about that in a few minutes. And finally if they present very early, within infancy, they do worse. And again that s maybe because the etiologies are somewhat different between the two age groups. All right, so let s talk a little bit about whether what s the genetic basis for HCM. So these are probably a reasonably comprehensive list of all the different genes that have now been associated with HCM. And the first was identified maybe 20 years ago now. So this was just a long list but actually most of these genes are essentially genes involved in cardiac contraction, so they are all part of proteins that are part of the sarcomere or some of the peripheral apparatus within the sarcomere. And this is a blowup of a sarcomere, here is the thick filament, the thin filament and these filaments passed one another during contraction, and so many of the proteins that are constituent of the sarcomere if they are mutated they will be associated with hypertrophic cardiomyopathy. And it turns out actually interestingly that there is only two genes that seem to account for most of the mutations we identify. If you just look at MYBPC3 and MYH7 both again sarcomere proteins, they account for about 80% of all the mutations we see. But then there is there are other genes that are involved at a lower frequency as well.

10 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 10 So what about this is experience basically from adult studies but what about pediatric HCM? Again most of these patients actually do have mutations in genes and contractile proteins but there are other etiologies that are a little bit more prevalent in the pediatric population as compared to the adult population. And we will discuss them briefly in a few minutes. What exactly are the genes? This is just a study of the Sequin Lab a few years ago where some 100 children with HCM were screened for mutations, and essentially the same sort of genes that were found in adults were found in these children as well. Around 55% of the patients were found to have a mutation which is actually similar to what we find in adults, and about 2/3 had a familial if they had a family history 2/3 of the patients were found to have a mutation. If there were sporadic cases only around 50% of the time was a mutation found. All right so what are the mechanisms of HCM? So now that we know many of the genetic mutations that cause HCM how can we dissect how these mutations actually lead to the phenotype? And so there is a lot of work that has gone on and we have partial elucidation of some of the mechanisms, it s not completely clear yet and there are obviously differences between different mutations and different genes. But in general, and this is I m summarizing years and years of work from multiple investigators in one slide here, but essentially the thought is that most of these mutations lead to basically a kind of gain of function, a hypercontractile state in the heart. So what happens is that you get increased ATPA activity, and as your consumption increased filament velocity of the sliding of

11 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 11 the thick and thin filaments in the sarcomere at least first generation. There is also evidence of associated with all of this of perturbation of calcium homeostasis within the cell. And there is different lines of evidence that suggest that if you have an abnormal calcium cycling or calcium handling in these myocytes this is associated with the HCM phenotype. And this is kind of a handwritten diagram which is mostly partly facts and a lot of conjecture, but some thought is that some of the you may have calcium sequestration within the sarcomere because the because of the mutations involved, and this will change the, the sort of the homeostasis of calcium within the cell lead to activation of abnormal cycling pathways that are calcium dependent within the nucleus of the cell leading ultimately to processes such as myocyte growth and hypertrophy or fibrosis as well. So this is partly hand-waving but there is actually some evidence for all of these conjectures. All right, so I mentioned the primary genetic mutations. I just want to touch on the fact that that doesn t give us the whole story. So if you look at patients in the same family with the same mutation they will still often have very different phenotypes and different disease outcomes, and the reasons for them, for that is multifactorial. One is definitely environmental differences, but another clearly documented contributor is that there is genetic heterogeneity in other genes in what are called modifiers that can impact on the severity of the phenotype. And this is an older slide from human data that suggests that variance in genes associated with the Angiotensin access and other signaling mechanisms for hypertrophy or more hormonal changes in the myocytes if you have variance in these, these variance can impact on the severity of HCM. And so we have to realize that it s

12 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 12 important to know what the genetic mutation is in a family but that is certainly not the whole story, that there is a lot of other genetic contributors to HCM as well as environmental contributors. So I ve gone through some of the data we have for hypertrophic cardiomyopathy, what about some of the mimickers? So in the pediatric population specifically we have to be more concerned about this because they do appear at greater frequency than they do in adults. And these are some of the genes that have been found to be mutated that lead to HCM phenotypes. And I m going to go through a couple of them pretty quickly. I ve included them in slides so that you can always access this later and go over them in more detail. But essentially most patients with left ventricular hypertrophy in the pediatric group will still have sarcomere gene mutations; however you can also there is a greater frequency of metabolic defects, other genetic disorders that are associated with HCM such as Noonan syndrome and neuromuscular disorders as well. And so if you look at the pediatric cardiomyopathy registry for patients with left ventricular hypertrophy about ¾ do have mutations in the sarcomere, but then roughly 10% of patients will have metabolic syndromes such as Pompe s, syndromic abnormalities, genetic abnormalities such as Noonan syndrome and then neuromuscular disorders. So it s important to realize that there are these sort of mimickers that are very different from sarcomere HCM and you know have different outcomes and in some cases have different treatments so it s important to keep that in mind. And also you know when you look at the infant population then in fact some of these other mimickers

13 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 13 become even a little bit more prevalent and probably because these other mimickers are more severe and does present earlier. And this is an example of one of the cardiomyopathies that you see that I don t see as often, left ventricular noncompaction which is associated with sort of deep trabeculations of the left ventricular wall and can be associated with what looks like hypertrophy on imaging can be associated with dilation and contractile dysfunction as well. The mutations that we found for this disorder many of them are similar to what we find in regular HCM but we also have other genes, some of which have clearly defined functions, some of which don t yet that can also be associated with left ventricular noncompaction. Then there are a number of different storage disorders that can cause cardiac hypertrophy that looks like HCM but is quite different. And let me just go through a couple of them. So one of the mutations that was identified pretty recently, it s been I guess in later on 2000 was in a protein called AMPK or AMP, Activated Protein Kinase. AMPK is a and a fuel gauge. It s present from yeast to man, it s highly conserved, present in every cell and it basically turns on when the cell is stressed and energy depleted. So when ATP levels are low and AMP levels are high this protein gets activated, it s a heterotrimer, catalytic alpha subunit and in regulatory and gamma subunits. And when it s turned on it basically activates a whole number of different processes that will increase ATV generation and decrease ATV consumption. So the gamma subunit is encoded by a gene called PLKG2, actually the gamma subunit that s cardiac specific is encoded by PLKG2 and

14 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 14 mutations in this particularly hetero this particular monomer within the heterotrimer can lead to a cardiac hypertrophy syndrome. So this is actually just a slide from one of my mouse models of this disorder, and what you see is that you don t get the other, the fibrosis and myofibrillar disarray that you see in regular HCM, instead you see these vacuoles sitting within the myocyte which are actually all glycogen, and if you actually do an assay for glycogen this is a wild type mouse, almost no glycogen in the heart, and in a huge astronomical amount of glycogen in the mutant now. So this is what actually happens in this disorder. It s not true hypertrophic cardiomyopathy, it s an entirely different disorder. And with a lot of work that s been done currently in my lab and in other labs we found that these mutations lead to basically again a function of the enzyme which leads to increased glucose entry. That glucose has nowhere to go because it s not really acquired within the cardiac myocyte and so it eventually gets shunted into glycogen which sits in the myocyte. And there s been a lot of work done on trying to figure out how that increased glucose entry is mediated and what we found is that actually there seems to be a novel glucose transfer in the heart called SGLT1 that is regulated partly by AMPK and is upregulated and actually causes this increased glucose uptake. And this is from one of our papers where we have data and we also have conjecture as to what all the different mechanisms are. But this transfer actually seems to be important in this disease and also in other potential other actually acquired heart disease as well, as some of you are actively investigating in our lab.

15 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 15 All right, let me skip through these slides because I don t want to run out of time. But I do want to say that there are other storage disorders that lead to increased deposition of glycogen or glycolipids or other proteins that are a little bit more prevalent in the pediatric population and can mimic hypertrophic cardiomyopathy. So Pompe s disease, Fabry disease which I think most people are familiar with, Noonan s syndrome is about a quarter of patients that have cardiac hypertrophy in addition to other more systemic abnormalities, and neuromuscular disorders such as Friedreich s Ataxia. All right, and finally some cardiomyopathies are actually not due to genomic, nuclear genomic mutations but rather due to either mitochondrial genomic genetic mutations or mutations in the nuclear genome that incorporate proteins that actually function in the mitochondria. So a few examples of these are Kearns-Sayre and a syndrome associated with multiple manifestations, myopathy, encephalopathy, lactic acidosis and stroke, so these are less common, they can either lead to hypertrophic or dilated cardiomyopathy and usually have a very poor outcome. They are manifested early in life and essentially the 50% survival is around 12 years of age, so quite a lethal group of disorders. All right let s talk a little bit about dilated cardiomyopathy. So dilated cardiomyopathy is less common than hypertrophic cardiomyopathy, at least the genetic form, instead of having just hypertrophy of the walls what you get is you can get some hypertrophy of the walls with dilated

16 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 16 cardiomyopathy but the major manifestation is as the name implies dilation of the ventricle and poor contractile activity. The genetic forms generally are incident at about 5 in 100,000, prevalent around 36 in 100,000. The mortality rate is quite high. Once the patient is symptomatic with symptoms of heart failure the one year mortality rate is around 25%. All right, this is a list that s reasonably complete of all the disease genes that have been associated with dilated cardiomyopathy. And I ll give you a general sort of thematic gestalt as to what these different genes are. What s kind of interesting is that very recently John Cricket-Sideman showed that Titin is actually a major cause of familiar and even sporadic dilated cardiomyopathy, account for almost 1/5 or 1/4 of patients. But one of the things that s kind of interesting is that when you look at these genes many of them are sarcomere protein genes, so the same genes that can cause hypertrophic cardiomyopathy are also associated with dilated cardiomyopathy; and so this was initially a little bit of unexpected surprise when the first genes were identified. But as we ve done more work we realized that some of this actually makes sense. So how is it that the same genes can lead to very distinct phenotypes? So this is a schematic representation of many of the proteins that are abnormal in dilated cardiomyopathy. The sarcomere again is affected just as it is in the HCM but you can also have other proteins such as those that connect the sarcomere to the sarcolemma and the extrasellar matrix. So this is these are proteins that are essentially transmit force out of the myocardium, or out of the myocyte into the adjacent cells and allow the heart to contract in a unified fashion. You can also have mutations in other

17 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 17 complexes sitting at the myocardium that are involved or sarcolemma that are involved in cell-cell adhesion or other cell-cell communication. So there is and finally I ll just briefly mention this, also mutations in genes that encode for calcium adding proteins. So a variety of different mechanisms seem to be at play here. And so to summarize this is sort of a summary that Jeff Tobin proposed a few years ago, different types of pathologies lead to this common phenotype of dilated cardiomyopathy such as impaired force transmission, impaired force generation just like in HCM except that in HCM the same genes lead to a sort of an increased force generation, the other mutations will lead to decreased force generation and ultimately dilated cardiomyopathy, changes in nuclear structure and function, changes in strengths, sensor machinery and changes in calcium regulation; so all of these ultimately lead to this common final pathway of dilated cardiomyopathy. This shows why the same genes with different mutations lead either to hypertrophic or dilated cardiomyopathy. And this is just one example of a few studies that have come out. The top two genes, the top two mutations here are mutations that are associated with hypertrophic cardiomyopathy. These mutations are also seen with dilated cardiomyopathy. And when you look at calcium binding activity and ATPase activity the HCM mutations lead to a gamma function, the dilated cardiomyopathy mutations generally lead to a decrease in function. So this may be one of the mechanisms by which gene mutations in the same genes lead to these distinct phenotypes, it really depends on how they affect the protein function.

18 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 18 And this is kind of a summary then of all the different functional changes for HCM mutations versus dilated cardiomyopathy mutations, divergent effects on calcium sensitivity, ATPase activity, filament velocity and force generation. And this is an example just of my own lab where we have a mouse model of a troponin mutation that leads to dilated cardiomyopathy. What we find is that these are calcium signals within the cardiac myocardium using optical mapping techniques. What we find is that there is actually an increase in diastolic calcium and a decrease in the rate of increase of calcium in systole. So this is associated with a decrease in calcium sensitivity that we ve documented and other people have documented. Perhaps as a compensatory mechanism what s happening is that the myocytes of these mice with dilated cardiomyopathy need more calcium onboard and more prolonged action potential to keep the calcium contributions high to compensate for the fact that these myocytes are calcium insensitive. All right, so as for HCM there are also genetic modifiers for DCM as well that can impact on the severity of the phenotype, so again we need to know what the primary mutation is but that s not the whole story. And so you can have mutations or rather variance in other genes again tending to be associated with neurohormonal function that can impact on the phenotype and severity of disease. All right let s talk a little bit about ARVC then. So arrhythmogenic right ventricle cardiomyopathy is a little bit of a distinct cardiomyopathy associated with replacement of the myocardium by fatty and fibrous tissue. And you can see it on gross pathology perhaps, it s kind of hard to see on this, but

19 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 19 basically you ve got the yellowing of the myocardium suggesting that there is fat there. And if you look at the histology definitely there is replacement of the myocytes by adipocytes here and there is also in blue here some fibrosis as well replacing the myocytes. And generally the patterns are actually quite, quite varied in different patients but generally what happens is that early on you will get some subepicardial replacement of myocardium by fat and fibrous tissue. Supposedly more in the right ventricle than the left ventricle, but that s why the name is ARVC, or right ventricular cardiomyopathy. In reality the left ventricle is in fact involved quite frequently as well and quite early. And with time the process then moves towards the endocardium and eventually you have basically end stage fibrosis of the it s more or less transmural. You know in the right ventricle but also in the left ventricle as well. And for reasons that we haven t fully elucidated but probably related to the fibrosis the cardiomyopathy is particularly prone to adipogenesis. So this is a relatively rare cardiomyopathy, it s present perhaps in 1 to 5 in 10,000 in the general population, it is again autosomal dominant and involves the replacement of myocardium by adipose and fibrous tissue with a high burden of ventricular arrhythmias. One of the problems is that often the clinical findings are a bit nonspecific especially if the pathology is mostly in the right ventricle initially and so sometimes this is a disease that s hard to diagnose. These are the genes that have been identified for ARVC. In fact to be honest some of these are probably not true associations with ARVC. But the ones that we are pretty confident about are all genes that are encoding proteins involved in the desmosome, so this is the center, the glue that connects adjacent cells together and allows the heart to contract in a coordinated fashion. If you

20 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 20 disrupt these structures you then lead to basically ARVC. So this is a disorder that is very much seems to be important in terms of cell-cell adhesion as a common mechanism. And the story is a little bit complex genetically, it turns out that in fact maybe 1 mutation is not enough, you may need multiple mutations to produce the true clinical phenotype and therefore you know we clinically sometimes perhaps rely a little bit too much on just identifying one mutation, particularly for ARVC. The story is actually more complex and we have to be cognizant of the fact that we may need to look at more than one variant running in the family that ultimately leads to the clinical phenotype. And how does this lead to this? We don t actually really know to be honest, so there are problems in the desmosome, we know that the desmosome that interacts with ion channels and gap junctions and constituents of the desmosome also are involved in nuclear signaling. So it is possible that through these mechanisms we get ventricular arrhythmias and through nuclear signal mechanisms you will get then changes, drop in myocytes and increased fibrosis and adipogenesis. But this is not clearly worked out at this point. So overall you know what we can probably say is that HCM is mainly a force generation and transmission disease. Storage cardiomyopathies are obviously metabolic disorders. ARVC is a disease of cell-cell adhesion and DCM is really a mixed bag involving many different processes that lead to the end stage of just you know a delegated phenotype. All right, let me just go through this. All right, let s just talk a little bit about moleculare genetic testing because we have about 10 minutes left. And this is really something that has moved into the clinical realm over the past 5, 6, 7 years. So why do we do genetic testing? Well the first is then to

21 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 21 identify family members who don t have symptoms. And so this is one of the problems we face, we identify a patient with a cardiomyopathy, great, we ll treat that patient and hopefully improve their outcome. But since these are genetic disorders obviously their relatives are at risk of having the same mutation, so one of the most important things is to see if we can identify them early on. This is a pedigree that was actually a research family from one of the earliest research families with HCM. And so the family was clinically assessed initially and in the older generation you can see that several patients or relatives were identified with to have HCM. But in the youngest generation ranging from age 2 to 20 only really one individual was identified and she was 19 years of age at the time. Once the genetic mutation was identified it was possible to have a genetic or molecular diagnosis and then you can see that you know roughly half the children in the third generation did have the mutation and therefore were at risk of having HCM. So clearly a genetic diagnosis in this setting, in the family setting can be very helpful for the family. Second is the issue of well prognosis. To see what the mutation is and see if you can correlate it with their outcomes. To be honest I don t think we are quite there yet, so genotype, phenotype correlations are something we are working on, but probably because the patient population isn t huge and many patients have kind of unique mutations we don t really have a good handle on saying well okay you have this gene involved, this mutation, this is what your outcome is going to be, or this is your risk of heart failure, this is your risk of arrhythmias, we are not really that good yet.

22 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 22 There is some early evidence that we can do some sort of gross genotype, phenotype correlation. This is these are survivor curves for patients with HCM who were found not to have mutations that we know of at this point versus those that were found to have mutations in routine clinical genetic screening. And the genotype positive patients had a worse outcome. Also if you look at heart failure again there is the same difference, genotype negative patients had a better heart failure free survival, the genotype positive patients had a worse survival. And what is of interest and we ve seen this clinically quite often actually is that patients can have more than one mutation for HCM as well as for other cardiomyopathies. Because it s a fairly prevalent disorder it s quite possible that two individuals will have children and each of the parents will have a distinct variant and both are then inherited by a child. So when that happens patients have two different mutations and distinct chains that will have a worse outcome. And this we ve found clinically. So it s important again to remember that genetic complexity is present in our patients and it behooves us to try to get the whole picture when we can. And some more evidence of potential genotype, phenotype correlations, this is a small study that just looked at pattern of hypertrophy in these cardiomyopathies depending on whether the patient was found to have a mutation or not. And so it turns out that for example if you have this type of risk curve septal hypertrophy these patients 80% of them actually do have a mutation in a sarcomere gene. Whereas if they have the apical parameter the hypertrophy is mostly down here then they are found to have a mutation about 30% of the time. So this is just suggestion suggestive that yes,

23 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 23 there are genotype correlates to the phenotype but we can only use this as a blunt instrument at this point, and not as a finely tuned scalpel. Where else will genetic testing help us? Well it helps us with reproductive choices, and this is as you move to a clinical realm we obviously work with a lot of young patients who are interested in starting families and in that setting it is now possible to do risk in vitro fertilization, identify implantation embryo tests which identify embryos that are free of the mutations and then to implant those in the mothers. So this has been done successfully now already and is certainly something that we can start offering our patients of reproductive age. Other possibilities with genetic testing, certainly we can offer them the opportunity to participate in research trials. We actually have research trials ongoing where patients who are gene type positive but don t have phenotype, can we offer them drugs that will prevent them from becoming phenotype positive over time? This is early work, there have been a couple, there is one trial that has just ended using Diltiazem as a possible agent based on animal work that had been done previously. That trial has not yet been reported. There is a second trial that will go on begin shortly using an ARB as again prophylaxis to help prevent the development of hypertrophic cardiomyopathy in patients who are genotype positive. So genetic testing may also offer us an opportunity to pursue further research.

24 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 24 And finally as I mentioned before some HCM is not HCM, it s actually a mimicker. Fabre disease is one example where enzyme replacement therapy is available, it s expensive but available and is basically almost curative. So it s important that we recognize that some patients don t have regular HCM, have something else that has a distinct treatment that will be helpful to our patients. So genetic testing is available clinically at a number of different centers and they all sort of a little bit different flavors of which diseases they test for, but essentially most of these cardiomyopathies are now testable from a genetic standpoint. For HCM overall we find a mutation about 50% of the time. If they have a family history the success rate goes up a bit to 2/3. And actually these numbers are probably even better now, these are numbers from a couple of years ago now. For dilated cardiomyopathy the numbers are not quite as good, we don t find mutation as often but again in certain settings especially for family reasons it s often helps to try to find a mutation if you can. Now when you get results back that s a challenge sometimes, so what are the possible outcomes of genetic testing? One is that you find a mutation, second is that you don t find a mutation or third, this happens around 7% of the time we find a variant that we just don t know what to do with. We don t know whether it causes disease or not. So if you find a positive result in somebody who has a clinical diagnosis, great, you know then this mutation is responsible for the clinical phenotype and then you can use that information to test other family members to see if they have the mutation or not. If you test a family member for a disease causing mutation and that relative doesn t have any

25 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 25 symptoms but has a positive result on genetic testing then you know that that patient has to be followed carefully over time. Now what about a negative result? So if somebody has a cardiomyopathy and we can t find a mutation then I m sorry, if a patient has a cardiomyopathy and we do find a mutation in that patient and we test the family member, that patient does not that relative does not have the genotype, then their risk of developing cardiomyopathy is not elevated and they don t need careful close followup. Another possibility is then that you have somebody who has a clinical diagnosis of cardiomyopathy but you were not able to find a mutation, what are the possible causes? One is that our clinical diagnosis is incorrect. That s not very common. We are reasonably good at making our clinical diagnoses now. More likely the diagnosis, the clinical diagnosis is correct but there is issues, we either our testing methodology is incomplete or there may be variants in his genes that are outside the coding sequence that we don t know are what the biological significance is and so we can t be sure they are disease causing. There are other genes that we either don t know about that can cause the phenotype or we don t test for because we don t know about them, or we know about them but they are just not part of our testing panels because they are present at a very low frequency in the patient population. So the worst thing for us unfortunately is when we get a variant of unknown significance. So we find a mutation or a variant, we don t know if it s disease causing. This happens fairly frequently because

26 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 26 even because we are still pretty early in the days of genetic testing and so a lot of patients in fact you find a mutation only in one family and it s not found in any other family. So over time as genetic testing continues hopefully we ll get more and more data and we ll see the same mutations in different families and be more confident they are disease causing. In some situations we are just not sure. So the first possibility, how can we help determine whether it s actually disease causing or not? One is you know if there are other affected members in the same family that are clinically affected you can test them to see if they have the variant or not. And by statistical analysis we use something called a log score, if it s more than 2 in the family that is sufficient evidence to suggest that that variant is actually disease causing if other people in the family have it as well. We do test panels of normal individuals to see if they have that variant. If they don t have the variant then it s more likely that this variant is actually disease causing. We can also use the fact that variants that cause the same disease they can sometimes to be in the same part of the same genes, so then the same functional domain. So if a variant is affecting the same domain, the protein that other known disease causing mutations are present in it s more likely to be disease causing. And finally we have a variety of computer modeling techniques that we use to predict what the effect of the mutation will be or the variant will be on protein function. And that can help us too. This is sort of I m not going to go through this now in detail, but it s in the slide. Essentially this is a protocol that we can use now to determine the utility of genetic testing in a family. And essentially

27 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 27 if the gene is mutated and known to be pathogenic you can test family members for its presence or absence. If it s present you have to counsel them and you can do surveil them clinically. If you don t find any mutation in that relative then they are not at risk of developing cardiomyopathy. We kind of discussed what happens with the variant of unknown significance, that s a situation where it gets a little tricky and we have to figure out if we can whether that variant is disease causing or not. All right, so generally what we do is we try to find the family member with the most severe phenotype and hopefully the best insurance to do this and we test them for the for mutations in the known genes. The sequencing studies, the tests are actually pretty expensive, they are in the thousands of dollars but some labs are now offering patient family caps of $100, which reduces the risk for the patient. And then testing for a known mutation in family relatives is relatively inexpensive, so even if insurance doesn t cover it or you don t have insurance it s not that expensive to confirm the presence of a known familial mutation in a new family member. The Genetic Nondiscrimination Act was passed in 2008, which is also somewhat reassuring to a lot of patients, it prohibits discrimination on the basis of genetic information by health insurance carriers and most, of course not all, but these protections do not extend to life insurance, disability insurance, long term care insurance. So there are still social issues that we need to address with our patients before genetic testing is conducted.

28 STUDIES OF CARDIOMYOPATHIES, FERHAAN AHMAD, MD, PhD 28 What I wanted to add a side, we do have a cardiovascular genetic center, we have a cardiomyopathy clinic here at Children s which are available to help you with evaluation of these patients and also the genetic counseling and genetic testing. And I think that s it, thank you very much.