PATH310_M02_onQ_CompanionGuide

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 3 Autopsy Report: Histology of Heart Muscle ................................................................................................. 26 Mr. Jones’ Cause of Death .............................................................................................................................. 26 Question – Location of Plaques and Occlusions ......................................................................................... 27 Culprits for Endothelial Dysfunction: Hemodynamic Forces ..................................................................... 28 Types of Blood Flow ........................................................................................................................................ 28 Turbulent Blood Flow and atherosclerosis .................................................................................................. 29 Question – Location of Atherosclerotic Lesions .......................................................................................... 30 Feedback: Localization of Atherosclerosis Lesions ..................................................................................... 30 Shear Stress, Endothelial Dysfunction, and Atherosclerosis ..................................................................... 31 Effect of Shear Stress on Endothelial Cell Phenotype ................................................................................. 31 Shear Stress and Key Regulator Genes of Inflammation ........................................................................... 32 Shear Stress and Master Regulator Genes .................................................................................................. 32 Question – The Effect of Shear Stress on Endothelial Phenotype ............................................................. 32 Feedback: The Effect of Shear Stress on Endothelial Phenotypes ............................................................ 34 Case Presentation: Mr Jones’ Son .................................................................................................................. 35 Question – Mr. Jones’ Risk Factors ................................................................................................................ 36 Culprits for Endothelial Dysfunction: Hyperlipidemia ................................................................................ 37 Review of Lipoproteins ............................................................................................................................... 38 Reverse Cholesterol Transport ...................................................................................................................... 39 The Importance of H D L in Reverse Cholesterol Transport ......................................................................... 39 Role of Fats in Atherosclerosis ....................................................................................................................... 40 Role of Fats in Atherosclerosis: Mr. Jones’ Son’s Blood ............................................................................... 40 Plasma Lipid Levels and Associated Health Risks ....................................................................................... 41 Question – T C/H D L Ratio ................................................................................................................................. 42 Question – “Good” vs “Bad” Cholesterol ....................................................................................................... 43 “Good” vs “bad” Cholesterol ........................................................................................................................... 43 Types of hyperlipidemia ................................................................................................................................. 44 Primary Hyperlipidemia .................................................................................................................................. 44 Using GW A S to Identify Genes Linked to Coronary Artery Disease ....................................................... 45 Question – Identifying the Type of Hyperlipidemia ..................................................................................... 46 Question – Familial Hypercholesterolemia .................................................................................................. 46 Feedback: Familial Hypercholesterolemia ................................................................................................... 46 Question – Dietary Sources of Cholesterol .................................................................................................. 48

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 6 INTRODUCTION Please see the online learning module for the full experience of interactions within this document. INTRODUCTION OT MODULE 02, CARDIOVASCULAR DISORDERS This content was retrieved from Introduction Slide 1 of 5 of the online learning module The cardiovascular system is made up of three components: the heart, the vasculature, and the blood. You focused on one of these components in Module 01, the blood, by exploring hemophilia and thrombophilia. In this module you will explore pathologies that originate in the heart and blood vessels. In Section 01, Dr. Marosh Manduch and Dr. Chris Nicol will introduce you to the topic of atherosclerosis, and in Section 02 you will learn about channelopathies from Dr. Kristopher Cunningham and Dr. Harriet Feilotter. Watch the video for an introduction to Module 02 from your course coordinator, Dr. Chris Nicol. (2:16) Page Link: https://player.vimeo.com/video/442038457 LEARNING OUTCOMES This content was retrieved from Introduction Slide 2 of 5 of the online learning module By the end of Module 02, you will be able to: 1. Describe the key pieces of information needed to understand cardiovascular diseases. 2. Compare and contrast the contributing disease paradigm factors for patients predisposed to atherosclerosis versus channelopathies. 3. Identify and explain the general approaches, considerations, and challenges with testing of patients for various types of cardiovascular diseases. 4. Apply your knowledge to a hypothetical cardiovascular disease scenario to describe a potential etiology, mechanism, testing approach, pathological features, and treatment. Reference the disease paradigm framework as you consider these module learning outcomes. MODULE ASSESSMENTS This content was retrieved from Introduction Slide 3 of 5 of the online learning module One assessment is associated with Module 02. View the assessment details. TEAM-BASED LEARNING ASSIGNMENT #1 - Refer to Page 7 Activities Throughout the Module

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 9 SECTION 01: ATHEROSCLEROSIS CASE PRESENTATION: MR. JONES This content was retrieved from Section 01 Slide 2 of 58 of the online learning module In this section, you will explore the case of Mr. Jones, a 59-year old male who came into the emergency department at Kingston General Hospital and passed away a few days later. Your goal is to determine how and why he died. Explore Mr. Jones’ patient profile. Mr. Jones’ Patient Profile • 59-year-old male who experienced 3 weeks of abdominal pain prior to admission. • Medical History: hypertensive, smoker, below normal high-density-lipoproteins (H D L) levels, and above normal low-density lipoprotein (L D L) and cholesterol levels. • Dec 31: admitted with severe chest pain; tests concluded a myocardial infarction (MI)* ; treatment started. • Jan 4: Mr. Jones became unresponsive with sudden pulseless electrical activity; ~50ml of blood was removed by a pericardiocentesis* but unfortunately Mr. Jones died. Navigate through the next few pages to view his autopsy report. Definitions*: Myocardial Infarction (M I): Also known as a heart attack, a myocardial infarction occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle. Pericardiocentesis: A medical procedure that removes excess fluid from the pericardial sac of the heart. It is performed with a needle and catheter. A procedure to alleviate pressure on the heart in cases when the heart is unable to pump blood. AUTOPSY REPORT: ABNORMALITIES IN THE HEART This content was retrieved from Section 01 Slide 3 of 58 of the online learning module During the autopsy the pathologist observed a bulge in the pericardial sac* , that was further identified to contain clotted blood. Wanting to take a closer look at the underlying tissue, the pathologist removed almost three cups of blood from the pericardial sac. Reveal what the pathologist found next.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 12 References: Diseased Heart: Image courtesy of Dr. Marosh Manduch. Normal Heart: Transverse slide of a healthy heart, depicting normal sized ventricles and no necrotic or adipose tissue. AUTOPSY REPORT: LOCATIONS OF BLOCKAGES This content was retrieved from Section 01 Slide 6 of 58 of the online learning module In examining Mr. Jones’ arteries, the pathologist found several blockages along his coronary arteries. This finding is significant, as a reduction in lumen diameter along the main vessels that supply blood to the heart can result in oxygen deprivation and tissue damage. For documentation, the extent of the other blockages was recorded. Listen to Dr. Manduch talk about the key postmortem findings. (01:54) View the audio transcript. Start of Audio Transcript: So this is an image that was drawn by the original pathologist that performed the post-mortem examination, and it depicts a representation of the main coronary arteries of the heart, which is pivotal to the examination of the heart; it's, for us, a pathologist, to be looking at the status of the coronary arteries. And so, in these situations, we look carefully at the coronary arteries while they're still attached to the heart itself, or in situ. We cut them coronally and longitudinally, so along the entire length of the artery, and we examine the lumen every two millimetres or so. And what we're looking for is abnormalities or potential blockages. So if we look at each section of the lumen and see whether it's patent or not, and in this particular case, it was found that the right coronary artery was approximately 75% occluded with a blood clot or an atheroma. And associated with the blockage, there was a possible hemorrhage which suggested that an acute change may have occurred in this patient that could have caused a sudden and complete occlusion of the vessel. This was

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 15 Explore the risk factors associated with atherosclerosis and consider which risk factors would align with Mr. Jones’ case. Age In general, the risk of atherosclerosis increases with age. The earliest lesions of atherosclerosis are present after the age of 10 years old and some believe that this is a disease present since infancy. Biological Sex An outdated notion is that females have less atherosclerosis than males; however, it is now recognized that females also develop atherosclerosis at significant rates. Estrogen is protective against atherosclerosis via signalling pathways that lower lipids, increase nitric oxide, upregulate antioxidative pathways and vascular tone. While atherosclerosis is more prevalent in males, females often "catch up" after menopause due to the subsequent reduction in estrogen levels. Family History Some families have increased susceptibility to atherosclerosis. This may be multifactorial in nature, based on many of the other factors listed. Genetics Inherited mutations can increase one’s predisposition to developing atherosclerosis. For example, a mutation in LD L receptors leads to an accumulation in cholesterol. Cigarette Smoking This risk factor is able to both initiate and accelerate atherosclerosis, as well as cause multiple malignancies. Some of its many effects on endothelial cells result in poor vascular tone, and increased vasoconstriction, oxidation and prothrombotic products. Diabetes Mellitus Associated with increased endothelial dysfunction and lipid levels. Hypertension Accelerates the development of atheromas. Hyperlipidemia Increases lipid levels in the blood, which increases the likelihood of plaque build up in blood vessels. QUESTION – MR. JONES’ RISK FACTORS This content was retrieved from Section 01 Slide 11 of 58 of the online learning module

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 18 RESPONSE-TO-INJURY HYPOTHESIS This content was retrieved from Section 01 Slide 15 of 58 of the online learning module The contemporary theory of the pathology of atherosclerosis is called the response-to-injury hypothesis . According to this theory, atherosclerosis develops as a healing response to endothelial injury . Hence, endothelial dysfunction is a critical triggering event. Learn about the sequence of events described by the response-to-injury hypothesis from Dr. Manduch. When prompted drag the slider. (03:17) Start of Audio Transcript: We will begin exploring the response to injury hypothesis by dragging the slider to the first step, which is endothelial injury. Recall that endothelial cells line the inner surface of all vessels and are in direct contact with flowing blood. Endothelial cell injury may be caused by a number of stimuli, including hemodynamic forces, hyperlipidemia, hypertension, smoking, toxins, and viruses. Once damaged, endothelial cells become dysfunctional, which may lead to increased vascular permeability, leukocyte adhesion, and thrombosis. Drag the slider to the second step so we can look at the accumulation of lipoproteins and macrophages. Once endothelial cell damage or dysfunction has occurred and the blood vessel permeability is increased, there will be many things coming into the intima of the blood vessel from the blood. For example, low-density lipoproteins, L D Ls, and very low-density lipoproteins, VL D Ls, will get oxidized. Monocytes also transform into macrophages in this step. This change is mediated by a receptor on the dysfunctional endothelial cells that allows monocytes to enter the tunica intima where they will transform. Now drag the slider to the third step so we can explore the role of smooth muscle cells and foam cells. Smooth muscle cells, or SMC, in the tunica media migrate into the tunica intima. The smooth muscle cells, along with the macrophages, begin to engulf the oxidized LD Ls to form foam cells. The disease progresses and creates what's called an atherosclerotic cap. Drag the slider now to the fourth step where fatty streaks have now formed. This is the first visual manifestation of atherosclerosis, and it is at this point where it is still reversible. You can get rid of these streaks with behaviour and diet modification since they are the result of fat accumulation and foam cells in the endothelial lining. Finally, drag the slider to the fifth step, where a complete plaque has formed. This final step is irreversible, and the structure formed now is called an atheromatous plaque or fibrofatty atheroma or just plaque. There is progressive lipid accumulation within the tunica intima, and the growing number of molecules and substances that accumulate form this atheromatous plaque, which, when fully formed, will have a necrotic centre and a fibrous cap. Note the additional information revealed in step 5. 1. Injury 2. Accumulation

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 21 The correct response is d) Proliferation of S M C s does not occur in the media. According to the response-to-injury hypothesis, this step occurs in the intima. STABLE AND VULNERABLE PLAQUES This content was retrieved from Section 01 Slide 17 of 58 of the online learning module The atheromatous plaque is clinically stable and not thrombosis-prone. However, it can progress into a structurally vulnerable (unstable) plaque that is prone to complications, leading to thrombosis. Examine the differences between the stable and vulnerable plaques in the illustration; then, view equivalent histological images. Notice the thicker fibrous cap in the stable plaque. Schematic Histology Reference: Schematic & Histology: Kumar, V., Abbas, A., Aster, J. (2015). Robbins & Cotran Pathologic Basis of Disease Ninth Edition (pp. 498-500). Saunders. THE NATURAL PROGRESSION OF ATHEROSCLEROSIS This content was retrieved from Section 18 of 58 of the online learning module In examining Mr. Jones’ arteries, the pathologist found several blockages along his coronary arteries. This finding is significant, as a reduction in lumen diameter along the main vessels that supply blood to the heart can result in oxygen deprivation and tissue damage. For documentation, the extent of the other blockages was recorded. Listen to Dr. Manduch briefly describe the natural progression of atherosclerosis. (04:46) View the audio transcript. Start of Audio Transcript: The preclinical phase of atherosclerosis usually occurs at a young age, and the patients aren't even aware that it is happening mostly because there are no symptoms, so they feel absolutely fine. And this creates an issue for the family doctor or medical professional to get the patient to modify or adjust their behaviour when they feel that they're perfectly healthy. From a normal artery, we have the progression, as we discussed, through a potential sort of fatty streak, the development of fibrofatty plaque, which can then become either advanced or vulnerable plaque. Then we cross -- or at this point, we cross into the clinical phase, which usually happens by middle age, such as mid- 40s or even later, and it is at this point that patients start getting symptoms.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 22 So what are these symptoms? The earliest symptom to understand is what happens when a patient develops critical stenosis. So nothing really happens other than, gradually over time, the plaque grows and starts to occlude the vessel. So for example, the plaque could occlude 50% of the artery, then there is 50% less blood flow able to go through that artery and supply the heart. But the patient is still able to manage and cope and do something that they need to do, and so there are no symptoms. However, once the occlusion goes to about 75% or more, that's called critical stenosis. And so 75% is the threshold where, if you need more oxygen, that the blood that can fit through that stenosed artery is able to deliver, you will then, or the patient will then start to get symptoms, and these symptoms are usually chest pain. So two other clinical presentations involve an acute or sudden change in the plaque itself. In this example, most commonly, you get a thrombus that is superimposed on top of the established plaque within the artery, or lumen of the artery. For example, with a plaque, it could be 70% occluded, and everything is fine. There are no symptoms. But then there's an acute change, and you could get another thrombus or a clot on top of that resulting in, let's say, an 85 to 90% occlusion. And now that's not enough, so now you do get symptoms. And this occlusion now, or change in the plaque, can be caused by plaque rupture where the thin fibrous cap can break. There can be an erosion of the plaque. You can get plaque hemorrhage where plaque gets inside the pre-existing plaque and expands it, or you can get thrombi formation within the walls of the vessel or even an embolization that lodges in another area downstream of the thrombus formation. Alternatively, in rare cases, you can get an aneurysm, which is a ballooning of the arterial wall. That's the aneurysm, and the aneurysm, when it gets big enough, it can actually rupture. If the blood escapes, the patient will be in life-threatening danger, and this can occur because the smooth muscle cells are, as you recall, they're sort of exiting the media and entering the intima where they're making that plaque. So the wall of the artery, which is really the thick portion of the artery, the muscular portion is the media, and that portion of the artery gets thinner and thinner until the pressure of the blood that's flowing through it sort of push on it and make this balloon or aneurysm that then may eventually rupture. End of Audio Transcript. Reference: Adapted from images from Servier Medical Art. Servier Medical Art. (n.d.). Arteries - Atherothrombosis. Retrie ved 1 May 2020, from https://smart.servier.com/image-set-download/ DEVELOPMENT OF AN ATHEROSCLEROTIC PLAQUE AND CLINICAL PRESENTATION This content was retrieved from Section 01 Slide 19 of 58 of the online learning module As you can now appreciate, after the initial development of an atherosclerotic plaque, if the general conditions that gave rise to the initial plaque do not change significantly, the plaque progresses and can lead to significant abnormal physiological effects and clinical symptoms. Review atherosclerotic plaque progression and how Mr. Jones could have conceivably developed his atherosclerotic condition.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 23 Early Lesion The first step involves injury to the endothelium. This step is asymptomatic, so Mr. Jones would not have realized he was developing atherosclerotic disease. Plaque Growth The gradual growth of a plaque damages the inner wall of an artery. Over time, fatty deposits composed of cholesterol and other cellular products also build up at the site of injury, narrowing the arteries. Mr. Jones’ risk factors, especially hyperlipidemia, increased the likelihood of this step occurring. Plaque Rupture Eventually, the thin fibrous cap of the plaque could have ruptured, spilling cholesterol and other cellular substances into the bloodstream. Blood Clot Formation The rupturing of the plaque may cause the formation of a blood clot. This can block blood flow to a specific part of the body. As seen in Mr. Jones’ autopsy report, it appears blood flow was significantly blocked from reaching his heart. For interest, learn how atherosclerosis progression can be visualized post-mortem - Refer to Pages 23-24 Reference: Mayo Clinic. (April 24, 2018) Arteriosclerosis / atherosclerosis. Retrieved June 19 2020, from https://www.mayoclinic.org/diseases-conditions/arteriosclerosis-atherosclerosis/symptoms- causes/syc-20350569 PROGRESSION OF AORTIC ATHEROSCLEROSIS Subpage of Section 01 Slide 19 of 58 – Learn how atherosclerosis progression can be visualized post-mortem 1/1 A common site to inspect for signs of atherosclerosis post-mortem is the thoracic or abdominal aorta. Listen to Dr. Manduch discuss features of aortic atherosclerosis progression (01:05) Start of Audio Transcript: Here are three examples of abdominal aortas from actual patients, and they show us examples from mild atherosclerosis on the left to moderate atherosclerosis in the middle and advanced atherosclerosis on the right side. In the mild atherosclerosis case, we have some raised irregularities, some yellow fat or yellow streaks, whereas in the advanced stage, you can see crusting and ulceration of the plaques.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 24 There's a lot of calcium in there that forms and makes the plaque brittle. And so these are the advanced or complicated plaques, and these are the ones that can cause serious complications for patients by sort of rupturing or even sort of causing mural thromboses. End of Audio Transcript. Reference: Images courtesy of Dr. Marosh Manduch. AUTOPSY REPORT: OCCLUSION BY THROMBUS This content was retrieved from Section 01 Slide 20 of 58 of the online learning module As part of Mr. Jones’ autopsy report, some histological cross sections of his coronary arteries were examined. As you can now appreciate, there is evidence of very thin fibrous caps which resulted in plaque rupture and subsequent complete occlusion of this portion of the coronary artery. Reference: Kumar, V., Abbas, A., Aster, J. (2015). Robbins & Cotran Pathologic Basis of Disease Ninth Edition (pp. 499). Saunders. AUTOPSY REPORT: RIGHT CORONARY ARTERY

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 25 This content was retrieved from Seciton 01 Slide 21 of 58 of the online learning module Recall, Mr. Jones’ right coronary artery was also extensively examined. A p articular histological cross section shows a substantial plaque. Learn how to interpret the diseased cross section; compare these findings to what can be seen in the healthy sample. Plaque The vessel is almost entirely occluded by the plaque, which is composed of fibronecrotic tissue. It does not stain pink like the muscular wall, which is still present but very thin. Lumen Normally, the lumen is very large in diameter, whereas in the diseased cross section from Mr. Jones, it is abnormally small. This further reduced the amount of blood available to Mr. Jones’ heart. Reference: Images courtesy of Dr. Marosh Manduch.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 26 MR. JONES’ CASE: OCCLUSIVE THROMBOSIS CAUSES ACUTE MYOCARDIAL INFRACTION This content was retrieved from Section 01 Slide 22 of 58 of the online learning module The observation of a significant coronary plaque caused by an atheroma associated with plaque rupture, and subsequent superimposed thrombus formation, is the significant event that restricted blood flow, and oxygen delivery, to M r. Jones’ heart. It is very likely that this caused further strain on Mr. Jones’ heart, leading to a first, acute myocardial infarction (M I or heart attack) which sent him to the hospital and was his initial diagnosis. Reference: NIH Image Gallery. (2017). Heart With Muscle Damage and a Blocked Artery . Flickr. Retrieved July 14, 2020, from https://www.flickr.com/photos/nihgov/33328945325 AUTOPSY REPORT: HISTOLOGY OF HEART MUSCLE This content was retrieved from Section 01 Slide 23 of 58 of the online learning module Under a microscope, the abnormalities of a diseased cardiac muscle can be seen on Mr. Jones’ samples. Typically, healthy cardiac muscle is striated with nuclei and intercalated discs present. In Mr. Jones’ case, the cardiomyocytes appear to be dying, as distinguished by coloured spots throughout the muscle. These spots are created as a result of inflammatory cells, like neutrophils, which are recruited to contain the necrotic tissue. Compare Mr. Jones’ diseased cardiac muscle to healthy cardiac muscle. MR. JONES’ SAMPLE HEALTHY SAMPLE Reference: Images courtesy of Dr. Marosh Manduch. MR. JONES’ CAUSE OF DEATH This content was retrieved form Section 01 Slide 24 of 58 of the online learning module As the M I worsened over the next few days in hospital, the muscle became necrotic and quite soft. The heart continued to pump blood; however, the soft diseased muscle tissue eventually ruptured, leading to blood exiting from inside the heart into the pericardial sac. This lead to a condition called cardiac tamponade* . Even though blood was removed from the area by the doctors, the damage done was irreversible and Mr. Jones never recovered. To review Mr. Jones’ autopsy photos, switch between a transverse and external view of Mr. Jones’ heart.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 27 TRANSVERSE SECTION OF THE HEART EXTERNAL VIEW Definition*: Cardiac tamponade: Accumulation of fluid in the pericardial sac, which compromises the heart muscle and blood flow. This is considered a medical emergency. Reference: Images courtesy of Dr. Marosh Manduch. QUESTION – LOCATION OF PLAQUES AND OCCLUSIONS This content was retrieved from Section 01 Slide 25 of 58 of the online learning module Based on what y ou have learned about atherosclerosis, answer the question. List potential reasons why during the autopsy the pathologist found plaques and occlusions

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 28 where they did. Feedback: Navigate to the next page to learn about the factors that influence where plaques and occlusions occur in blood vessels. View the autopsy report of the plaque locations. CULPRITS FOR ENDOTHELIAL DYSFUNCTION: HEMODYNAMIC FORCES This content was retrieved from Section 01 Slide 26 of 58 of the online learning module Endothelial dysfunction can be induced by several injurious stimuli. Hemodynamic forces are one of the key factors that are strongly correlated with endothelial dysfunction. Hemodynamic forces refer to the mechanical forces exerted due to the flow of blood. As blood flows through a vessel, it exerts two predominant forces on its walls: pressure (ρ) and shear stress (τ). See where each type of mechanical force affects the blood vessel. PRESSURE Pressure acts perpendicular to the vessel wall and affects the vascular smooth muscle (VSM) cells. SHEAR STRESS Shear stress acts parallel to the vessel wall, exerted longitudinally in the direction of blood flow. This type of mechanical force affects endothelial cell function. Reference: Hahn, C., & Schwartz, M. A. (2009). Mechanotransduction in vascular physiology and atherogenesis. Nature Reviews Molecular Cell Biology , 10 (1), 53-62. Retrieved July 22, 2020, from: https://proxy.queensu.ca/login?url=http://dx.doi.org/10.1038/nrm2596 TYPES OF BLOOD FLOW This content was retrieved from Section 01 Slide 27 of 58 of the online learning module Levels of shear stress vary throughout the vasculature and can be influenced by the type of blood flow. Compare the two types of blood flow, laminar and turbulent blood flow. Note the illustrated changes in the direction and magnitude of the flow. LAMINAR BLOOD FLOW Laminar flow is the normal condition of blood flow in the circulatory system. It is characterized by blood flow parallel to the blood vessel wall and usually occurs in straight regions of arteries. This can be compared to water flowing through an open ended garden hose with no resistance.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 29 TURBULENT BLOOD FLOW Laminar flow can be disrupted and become turbulent, in curved arterial regions, or where a blood vessel branches. In turbulent blood flow, conditions become disturbed and have low or oscillatory shear stress. This can be compared to putting your finger over the end of the garden hose that is running. You may also have noticed in a stream with rocks and curves that small swirls are created as the water diverts around them. References: Cunningham, K. S., & Gotlieb, A. I. (2005). The role of shear stress in the pathogenesis of atherosclerosis. Laboratory Investigation , 85 (1), 9-23. Retrieved July 22, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1038/labinvest.3700215 Steeley, R., Stephens, T., Tate, P. (1 January, 2013). Anatomy and Physiology 6th edition. McGraw-Hill. Pp 741. Retrieved June 23, 2020, from https://physiology.nuph.edu.ua/wp-content/uploads/2018/01/21- Cardiovascular-System-Peripheral-Circulation-and-Regulati1.pdf TURBULENT BLOOD FLOW AND ATHEROSCLEROSIS This content was retrieved from Section 01 Slide 28 of 58 of the online learning module It is in these areas of turbulent flow where the blood may swirl around and cause some dysfunction in the endothelial cells themselves or contribute to accumulation of fats that lead to plaque formation, thereby constricting the vessel. This influences the magnitude of the shear stress in the region, which can create a vicious cycle. Learn from Dr. Nicol how shear stress fluctuates between normal laminar flow and turbulent flow caused by atherosclerosis. (01:16) View the audio transcript. Start of Audio Transcript: In this graph the main take away is the difference in shear stress under laminar flow and turbulent flow. In a normal artery there's high shear stress because blood is being pumped out of the heart. Comparatively a normal vein has normal laminar flow. However, in a thrombotic or atherosclerotic region on the other hand, there's a significant decrease in shear stress in the veins, even going to the negative direction. But there's even more significant increase in the stress in your arteries. The drop in the venous stress may be related to the fact that blood is not flowing through the arteries as fast, so it's not getting to the veins. When considering the artery, it's much like a scenario where you have a hose and you place your finger over the opening and the water exists at a high pressure. The blood that gets through this constricted vessel region is shooting through at high pressure and that can cause some micro-tears and endothelial damage in the vessel wall as it goes through. Especially around curves and branch points beyond. But in addition to this, the blood that remains before the small opening, becomes turbulent and also very disruptive to the endothelial cells.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 30 End of Audio Transcript. Reference: Adapted from Malek, A. M., Alper, S. L., & Izumo, S. (1999). Hemodynamic Shear Stress and Its Role in Atherosclerosis. JAMA , 282 (21), 2035-2042. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1001/jama.282.21.2035 QUESTION – LOCATION OF ATHEROSCLEROTIC LESIONS This content was retrieved from Section 01 Slide 29 of 58 of the online learning module Hemodynamic forces are responsible for dictating which vascular sites are susceptible or resistant to developing atherosclerosis. Using what you have learned about blood flow, shear stress, and atherosclerosis, answer the question. Question: Which regions in the vasculature are prone to the development of an atherosclerotic lesion? Explain the role of hemodynamic forces in predisposing these regions to endothelial dysfunction and subsequently atherosclerosis. Feedback: Compare your response to Dr. Nicol’s response. FEEDBACK: LOCALIZATION OF ATHEROSCLEROSIS LESIONS This content was retrieved from Section 01 Slide 30 of 58 of the online learning module Question: Which regions in the vasculature are prone to the development of an atherosclerotic lesion? Explain the role of hemodynamic forces in predisposing these regions to endothelial dysfunction and subsequently atherosclerosis. Continue to compare your respon se to Dr. Nicol’s response. Your Response Dr. Nicol’s Response Atherosclerotic lesions develop predominantly in arterial curves, bifurcations, and branch sites. This includes the aorta, carotid arteries, coronary arteries, renal arteries, and arteries of the lower extremities. You can now appreciate how these regions would force changes in blood flow and shear stress, but there are also molecular underpinnings to these changes, which you will explore next. Reference: LadyofHats. (2010). Circulatory System no tags . Wikimedia Commons. Retrieved June 2020, from https://commons.wikimedia.org/wiki/File:Circulatory_System_no_tags.svg

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 31 SHEAR STRESS, ENDOTHELIAL DYSFUNCTION, AND ATHEROSCLEROSIS This content was retrieved from Section 01 Slide 31 of 58 of the online learning module Research has demonstrated a correlation between shear stress, endothelial dysfunction, and atherosclerosis. It is suggested that laminar blood flow induces endothelial cells to express genes that protect against atherosclerosis. These genes are implicated in normal endothelial function. Regions with turbulent blood flow can impair endothelial phenotype and function, and are prone to atherosclerosis. This is because disturbed blood flow exerts low/oscillatory shear stress on the vessel wall allowing deposits of fats to form or inducing other endothelial cell dysfunction. References: Cunningham, K. S., & Gotlieb, A. I. (2005). The role of shear stress in the pathogenesis of atherosclerosis. Laboratory Investigation , 85 (1), 9-23. Retrieved June 23, 2020 from: https://proxy.queensu.ca/login?url=https://doi.org/10.1038/labinvest.3700215 Mehta, V., & Tzima, E. (2016). A turbulent path to plaque formation. Nature , 540 (7634), 531-532. Retrieved June 23, 2020 from: https://proxy.queensu.ca/login?url=https://doi.org/10.1038/nature20489 EFFECT OF SHEAR STRESS ON ENDOTHELIAL CELL PHENOTYPE This content was retrieved from Section 01 Slide 32 of 58 of the online learning module Under experimental conditions, when normal endothelial cells are exposed to low shear stress, changes in the endothelial cell morphology are observed. Compare the effects on normal endothelial cell phenotypes when exposed to physiological compared to low shear stress. PHYSIOLOGICAL SHEAR STRESS Endothelial cells exposed to physiological arterial shear stress (> 15 dynes/c m 2 ); cells are elongated and aligned to the direction of blood flow to increase the aerodynamics of their shape and minimize interference with vessel components. LOW SHEAR STRESS Endothelial cells exposed to low arterial shear stress (0-1 dynes/c m 2 ); cells have a cobblestone appearance. Genetic changes also occur when these cells are impacted by changes in shear stress that will be explored on the next slide." Reference: Topper, J. N., & Gimbrone Jr, M. A. (1999). Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype. Molecular Medicine Today , 5(1), 40-46. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1016/S1357-4310(98)01372-0

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 32 SHEAR STRESS AND KEY REGULATOR GENES OF INFLAMMATION This content was retrieved from Section 01 Slide 33 of 58 of the online learning module Endothelial cell inflammatory phenotypes, in response to their shear stress environments, are controlled by a few key regulator genes. Learn about the changes induced by laminar and turbulent shear stress. LAMINAR SHEAR STRESS Under laminar shear stress, endothelial cells express genes like K L F2 or N rf2. These genes act in a protective manner and help inhibit the expression of NF ᴋ B , a pro-inflammatory transcription factor. TURBULENT SHEAR STRESS Under turbulent shear stress, the expression of protective genes is lost, therefore, N F ᴋ B is upregulated to create a pro-inflammatory environment in blood vessels. This allows for the recruitment of monocytes and macrophages to initiate plaque formation and enables smooth muscle cells to proliferate. SHEAR STRESS AND MASTER REGULATOR GENES This content was retrieved from Section 01 Slide 34 of 58 of the online learning module The presented diagram shows the complex interaction of many genes. It is not important to focus on the specific molecular pathways, but instead understand the impact that the master regulators, N rf2 and K L F2, have on the endothelial environment under laminar shear stress conditions. As you just learned, laminar shear stress will upregulate K L F2, which turns on Nr f2. Together, upregulation of these genes results in the blockade of oxidative stress and inflammatory pathways (including N FκB ), while upregulating factors that help block thrombosis. Upregulated K L F2 and N rf2 genes also increase the communication in vascular tone, which can help set the stage for improving conditions to minimize the development of atherosclerotic plaques. These two genes play an important role in minimizing the oxidation/oxidative stress of the lipids that get out from the lumen, under the tunica intima into the tunica media. Reference: Image courtesy of Dr. Christopher Nicol. QUESTION – THE EFFECT OF SHEAR STRESS ON ENDOTHELIAL PHENOTYPE This content was retrieved from Section 01 Slide 35 of 58 of the online learning module Consolidate what you know about hemostasis and atherosclerosis by choosing whether a certain component is increased/upregulated ( ↑ ) or decreased/downregulated ( ↓ ) in each region and phenotype.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 33 Options: ↑ , ↓ Activated Phenotype: Turbulent Shear Stress ↑ or ↓ ? Coagulation P GL2, NO, tP A Platelet aggregation Endothelium Proliferation Apoptosis Alignment Inflammatory State SM C proliferation Leukocyte adhesion Quiescent Phenotype: Laminar Shear Stress ↑ or ↓ ? Coagulation P GL2, NO, tP A Platelet aggregation Endothelium Proliferation Apoptosis Alignment Inflammatory State SM C proliferation Leukocyte adhesion Feedback: Activated Phenotype: Turbulent Shear Stress ↑ or ↓ ? Coagulation P GL2, NO, tP A ↑ Platelet aggregation ↓ Endothelium Proliferation ↓ Apoptosis ↓ Alignment ↑ Inflammatory State SM C proliferation ↓

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 34 Leukocyte adhesion ↓ Quiescent Phenotype: Laminar Shear Stress ↑ or ↓ ? Coagulation P GL2, NO, tP A ↓ Platelet aggregation ↑ Endothelium Proliferation ↑ Apoptosis ↑ Alignment ↓ Inflammatory State SM C proliferation ↑ Leukocyte adhesion ↑ Proceed to the next slide for further feedback on this consolidation activity. FEEDBACK: THE EFFECT OF SHEAR STRESS ON ENDOTHELIAL PHENOTYPES This content was retrieved from Section 01 Slide 36 of 58 of the online learning module The molecular knowledge of how endothelial cells are affected by shear stress helps explain the development of atherosclerotic plaques. The presented table summarizes these relationships. Review different representations of these processes. COMPARISON TABLE Quiescent Phenotype: Laminar Shear Stress Activated Phenotype: Turbulent Shear Stress Coagulation ↑ P GL2, NO, tP A ↓ Platelet aggregation ↓ PGL2, NO, t PA ↑ Platelet aggregation Endothelium ↓ Proliferation ↓ Apoptosis ↑ Alignment ↑ Proliferation ↑ Apoptosis ↓ Alignment Inflammatory State Anti-inflammatory ↓ S M C proliferation ↓ Leukocyte adhesion Anti-inflammatory ↑ S M C proliferation ↑ Leukocyte adhesion LAMINAR FLOW DIAGRAM

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 35 TURBULENT FLOW DIAGRAM Reference: Diagram: Bergan, J. J., Schmid-Sch o ̈ nbein, G. W., Coleridge Smith, P. D., Nicolaides, A. N., Boisseau, M. R., and Eklof, B. (2006). Chronic Venous Disease. New England Journal of Medicine, 355(5) : 494. Retrieved June 23, 2020, from: https://www-nejm-org.proxy.queensu.ca/doi/full/10.1056/NEJMra055289 CASE PRESENTATION: MR JONES’ SON This content was retrieved from Section 01 Slide 37 of 58 of the online learning module For the remainder of this section, you will focus on Mr. Jones’ son, who arrived at the emergency department a few months after his dad passed away, complaining of chest pains. View his patient history and test results. Patient Medical History • 35-year-old male of French-Canadian ancestry experiencing chest pain when active • Father recently passed away (at age 59) from acute M I due to atherosclerosis • History: smoker of 10 cigarettes per day for last 20 years • Xanthomas* were found on his eyelids and knees upon performing a physical exam Test Results

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 36 • Blood pressure: 136 mmHg/86 mmHg (high) • B MI* : 30 kg/m 2 (obese) • Blood test results: o Triglycerides (T G): 160 mg/dl o High Density Lipoproteins (H DL): 38 mg/dl o Low Density Lipoproteins (L D L): 207 mg/dl o Total Cholesterol: 245 mg/dl Definitions*: Xanthomas: Cholesterol deposits found under the skin. They can be found on joints (elbows, knees), eyelids, and ankle tendons. BM I: Body mass index; a measure of body fat based on height and weight that applies to adult men and women. QUESTION – MR. JONES’ RISK FACTORS This content was retrieved from Section 01 Slide 38 of 58 of the online learning module The underlying etiology of atherosclerosis is unknown, however, as you can recall, certain factors may increase the risk of developing atherosclerosis. These factors can be distinguished as modifiable* and non-modifiable*. Question: Based on what you know from Mr. Jones’ case, determine if his son’s risk factors are modifiable or non-modifiable. RISK FACTORS Modifiable Non-Modifiable Unknown Hyperlipidemia Family History Gender Hypertension Cigarette smoking Increasing age Feedback: RISK FACTORS Modifiable Non-Modifiable Unknown Hyperlipidemia Unknown Family History Non-Modifiable Gender Non-Modifiable Hypertension Modifiable Cigarette smoking Modifiable Increasing age Non-Modifiable Listen to Dr. Nicol’s feedback. (00:44) View the audio transcript. Start of Audio Transcript:

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 37 Based on the patient history and Mr. Jones' autopsy results, Mr. Jones' son is also suspected of having atherosclerosis. His family and medical history certainly predispose him to developing this disease. And you may also appreciate that Mr. Jones' son has a few modifiable risk factors related to his lifestyle. Such as cigarette smoking and hypertension. His hyperlipidemia may also be modifiable but we won't know for sure if this is true until more information is obtained. Using your knowledge from Mr. Jones' case you will assist the healthcare team in treating Mr. Jones' son through his modifiable risk factors. End of Audio Transcript. Definitions*: Modifiable: Health or medical measures can be taken to influence the outcome of a disease. Non-modifiable: No measures can be taken to change its influence on the disease. CULPRITS FOR ENDOTHELIAL DYSFUNCTION: HYPERLIPIDEMIA This content was retrieved from Section 01 Slide 39 of 58 of the online learning module In addition to changes in shear stress conditions, endothelial dysfunction can also be induced by hyperlipidemia, which refers to abnormally high levels of lipids in the blood. Given the medical history of his father and his high B M I observed on admission to the emergency department, you decide to investigate that the possibility that Mr. Jones' son may also have hyperlipidemia, which could predispose him to atherosclerosis. This may or may not be an modifiable risk factor. Recall from previous courses that lipids are a group of hydrophobic organic molecules that include triglycerides* and cholesterol*. Lipids are usually hydrophobic and hence remain insoluble in water, blood plasma, and other extracellular fluids. In order to transport these lipids in the blood plasma, specialized proteins such as lipoproteins are used. Review the types of lipoproteins. REVIEW OF LIPOPROTEINS - Refer to Pages 38-39 Definitions*:

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 38 Triglycerides: An ester derived from glycerol and three fatty acids. The principle component of body fat in humans. Cholesterol: A sterol, a type of lipid, involved in cell growth, cell division, membrane repair, steroid hormone production. Reference: Servier Medical Art. (n.d.). Atheroma. Retrieved May 11 2020, from https://smart.servier.com/smart_image/atherosclerosis-23/ REVIEW OF LIPOPROTEINS Subpage of Section 01 Slide 39 of 58 – Review of Lipoproteins 1/1 Lipoproteins organize triglycerides and cholesterol into a plasma-soluble complex and in doing so provide a lipid delivery system. Learn about the different protein and lipid compositions. CM Chylomicrons (C M) are the least dense lipoproteins assembled in the intestinal mucosal cells. They are primarily composed of triglycerides and a small amount of protein. Chylomicrons carry dietary triacylglycerol from the intestinal tract to the tissues. VL DL Very low density lipoproteins (VL D L) consist mainly of triglycerides, some cholesterol molecules, and very few protein molecules. Similar to the chylomicrons, VL D L delivers triglycerides to cells in the body for cellular processes. IDL VL D L is converted into intermediate density lipoprotein (ID L). After the triglycerides on V LD L have been broken down in cells, the lipid to protein ratio changes and the lipoprotein becomes denser. L DL Low density lipoprotein (L D L) is synthesized in the blood from VL D L/I D L and is very rich in cholesterol. L DL is the main transporter of cholesterol to peripheral tissues, a process known as cholesterol transport. H DL High density lipoprotein (H D L) consists of less cholesterol and more protein, making these lipoproteins the most dense. H D L is a key mediator of reverse cholesterol transport which involves carrying cholesterol from cells back to the liver. Reference:

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 39 Lumen Learning. (n.d.). Lipoproteins. Nutrition Flexbook. Retrieved 30 April 2020, from https://courses.lumenlearning.com/suny-nutrition/chapter/4-71-lipoproteins/ REVERSE CHOLESTEROL TRANSPORT This content was retrieved from Section 01 Slide 40 of 58 of the online learning module Reverse cholesterol transport is involved in the modulation of the total lipid profile. This is a mechanism by which the body removes excess cholesterol from peripheral tissues and delivers it to be broken down in the liver and excreted. The key mediator in this process is H D L. Listen to Dr. Nicol describe the process of lipid transport and the important role of reverse cholesterol transport. (02:41) View the audio transcript. Start of Audio Transcript: Normally the fats in your diet are absorbed and turned into triglycerides and cholesterol in the liver. These then associate with lipoproteins which transport to your fat cells, starting with the formation of very low density lipoproteins in the liver, a cycling process converts them to L D Ls which may be utilized by the normal body needs for lipids such as maintaining cell membranes, stored in fat cells, or broken down for excretion via the bile. L D L receptors located on cell surfaces bind circulating L D L and bring it inside the cell, helping to maintain normal blood cholesterol levels. In extrahepatic cells or scavenger cells such as macrophages, L D L receptors also soak up LD L to break it down and may either store it or prepare it for excretion. Normally the body will work to eliminate excess levels of cholesterol and does so with the help of high density lipoproteins or H D L. It's worth noting at this point that H D L is commonly thought of as, quote unquote, a good fat, while V L D L and L D L are considered the quote unquote, bad fats. Just remember, the body needs fats to function normally too. Therefore, it may be better to think about these in terms of having a normal balance and/or a good reserve capacity to break down and eliminate excess fats as being good and an imbalance that favours excess storage or circulation of fats being bad. Normally you have fats that you eat and fats that you excrete through the bile. However, some fats associate with chylomicrons and get broken down by lipoprotein lipase enzymes that form HD L, the good fat. That helps cycle the excess bad cholesterol back for elimination. When L D L is broken down, it releases cholesterol which associates with H D L and travels back to the liver so it can be used again or excreted in the bile as we talked about above. This process is known as reverse cholesterol transport. This is where the levels of fats in the body become important. When looking at indicators maintaining high levels of HD L are ideal to reduce any potentially high levels of V L D L or L D L to ensure an appropriate recycling through the reverse cholesterol transport system. End of Audio Transcript. Reference: Image courtesy of Dr. Christopher Nicol. THE IMPORTANCE OF HD L IN REVERSE CHOLESTEROL TRANSPORT This content was retrieved from Section 01 Slide 41 of 58 of the online learning module

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 40 HD L facilitates reverse cholesterol transport, an important process that can help reduce the presence of atherosclerotic plaques. Watch the video to solidify your understanding of the principles of this process. (03:23) Note: Knowledge of the names/details of the receptors and pathways are not expected, however your focus should be on the principles of this process. Page Link: https://www.youtube.com/embed/q0YiPqmsXRg Reference: Tagouz. (2013, October 18). H D L & Reverse Cholesterol Transport [HD] [ Video]. YouTube. Retrieved 8 July, 2020 from https://www.youtube.com/watch?v=q0YiPqmsXRg ROLE OF FATS IN ATHEROSCLEROSIS This content was retrieved from Section 01 Slide 42 of 58 of the online learning module Normally, endothelial cells do not adhere to leukocytes. However, recall from the pathophysiology of atherosclerosis that dysfunctional endothelial cells also are able to mediate the adhesion and migration of leukocytes (e.g. monocytes and T-lymphocytes), a process which is mediated by a receptor called VCAM-1. When an atherogenic diet high in saturated fats and cholesterol is introduced, scientists have observed an increase in V CAM-1 expression in endothelial cells. They have also observed an increase in leukocyte adhesion to the endothelium. This suggests that hyperlipidemia promotes atherosclerosis through leukocyte adhesion. A simple way to start to narrow down the possibility of Mr. Jones’ son having hyperlipidemia is to take a blood sample before a meal, which you dutifully conduct. ROLE OF FATS IN ATHEROSCLEROSIS: MR. JONES’ SON’S BLOOD This content was retrieved from Section 01 Slide 43 of 58 of the online learning module Review the presented vials of centrifuged blood; a healthy patient sample (A) is provided as a reference against a sample from Mr. Jones’ son (B). What do you observe? Once ready, review some reflections from Dr. Nicol. BLOOD SAMPLE ANALYSIS As you may appreciate, the bottom portion of each vial contains the erythrocyte component of the sample. In the case of the healthy patient, there is a semi-transparent serum present above the blood layer.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 41 In Mr. Jones’ son’s case, the serum is almost completely taken up by a layer of yellow -white, opaque lipid. For approximately the same amount of blood that was spun down, there is an equivalent amount of lipid flowing in his bloodstream. Imagine how difficult it must be for the blood to flow through the circulation with such a viscous substance. With this visual indication of such a significant presence of lipids in his circulation, it certainly suggests to you that he likely has an associated higher risk of developing atherosclerosis. Reference: Hyperlipemia and Hepatic Lipidosis in Large Animals - Digestive System. (n.d.). Retrieved 16 June 2020, from https://www.merckvetmanual.com/digestive-system/hepatic-disease-in-large- animals/hyperlipemia-and-hepatic-lipidosis-in-large-animals PLASMA LIPID LEVELS AND ASSOCIATED HEALTH RISKS This content was retrieved from Section 01 Slide 44 of 58 of the online learning module You decide to review Mr. Jones’ son’s blood test results again, specifically for the plasma lipid levels. These levels can provide additional clues as to the cause of the high presence of lipids in his blood and potential effect on his health. Mr. Jones’ Son Triglycerides (T G): 160 mg/dL High Density Lipoproteins (H DL): 38 mg/dL Low Density Lipoproteins (L D L): 207 mg/dL Total Cholesterol: 245 mg/dL Go through the different lipoprotein reference tables to learn their appropriate and abnormal levels and compare them to the patient’s blood test results. NORMAL PLASMA LIPID LEVELS Triglycerides ( mg / dL ) Total Cholesterol ( m g / dL ) H D L ( mg / dL ) TC/ H DL -C Adult Female 80 190 55 3.5 Adult Male 120 200 43 4.7 Neonate 35 70 35 2.0 Definition: TC/H D L-C: This is a ratio of Total Cholesterol to H D L. This number is obtained by dividing total cholesterol levels by H D L levels. A higher ratio means higher risk for disease, and a lower ratio means a lower risk for disease. It is optimal for a ratio to be below 5. TRIGLYCERIDE LEVELS

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 42 Triglyceride Level Category Less than 150 m g/dL Normal 150-199 m g/d L Borderline High 200-499 m g/d L High 500 m g/d L and above Very High H DL -CHOLESTEROL LEVELS H DL Cholesterol Level Category Less than 40 m g / dL A major risk factor for heart disease. 40-50 m g / d L The higher your H D L level, the better. 60 m g / d L and above Considered protective against heart disease. L DL -CHOLESTEROL LEVELS L DL Cholesterol Level Category Less than 100 m g / d L Optimal 100-129 m g / d L Near or above optimal 130-159 m g / d L Borderline high 160-189 m g / d L High 190 m g / d L and above Very high For interest, review a note about the terms H D L-C and L D L-C. Of minor technical interest, H D L-Cholesterol levels and L D L-Cholesterol levels represent the measure of cholesterol associated with each type of particle (e.g. tells how much HD L or L D L is present but is really an indirect measure) and often used interchangeably with H D L/L D L even though they are not the same. In this course, if referring to results of a blood test, H D L-C or L D L-C is likely best to use, but other places where we mean the particles themselves, we can just use the term H D L or L D L. QUESTION – TC/H D L RATIO This content was retrieved from Section 01 Slide 45 of 58 of the online learning module Based on what you just learned about total cholesterol and HD L ratios, answer the question. Question: If Mr. Jones’ son has an HDL level of 38 mg/dL, a total cholesterol of 245 mg/dL, and a triglyceride level of 160 mg/dL, what is the TC/H DL ratio and what is his risk of cardiovascular disease? a) Ratio is 4.2; risk is very high. b) Ratio is 6.4; risk is low. c) Ratio is 4.2; risk is very low. d) Ratio is 6.4; risk is high. Dr. Nicol’s Feedback: The correct response is d).

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 43 Mr. Jones’ son’s TC/H D L ratio is 6.4. You calculate this number by dividing 245 m g/d L by 38 m g/d L. Since the ratio is above 5, the optimal range, you can assume his ratio of developing cardiovascular disease is high. Mr. Jones’ son’s plasma lipid levels are all outside the optimal ranges of: • T G <150 m g/d L • LD L <100 m g/d L HD L >60 mg/dL QUESTION – “GOOD” VS “BAD” CHOLESTEROL This content was retrieved from Section 01 Slide 46 of 58 of the online learning module While lipids are not inherently “good” or “bad”, it is helpful to consider these qualifiers in terms of their relative impact on health. Based on your knowledge of lipid transport, answer the question. Question: You know that M r. Jones’ son has low HDL levels. What does this mean and why does it put him at risk? a) Low HD L means he is unable to efficiently eliminate ‘bad’ cholesterol from his bloodstream, which increases both the amount available to clog his arteries and his risk of developing atherosclerosis. b) Low HD L means he has lower total cholesterol in his bloodstream, which decreases both the amount available to clog his arteries and his risk of developing atherosclerosis. c) Low HD L means he is unable to breakdown circulating fats, which decreases both the amount of ‘bad’ cholesterol in his bloodstream and his risk of developing atherosclerosis. Feedback: The correct answer is a). Continue to the next slide for more feedback “GOOD” VS “BAD” CHOLESTEROL This content was retrieved from Section 01 Slide 47 of 58 of the online learning module Feedback: The correct answer is a). B is incorrect, firstly because cholesterol will likely get increased in total (even though H DL contributes to total amount), especially with time, and second because the bad cholesterol will be more available to clog arteries. C is incorrect because low H D L does not inhibit ability to break down L D L, it only reduces ability to carry cholesterol back for reuse or elimination in the liver.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 44 To help you contextualize this information, the figure outlines a normal and atherosclerotic vessel. Notice the difference in H D L vs L D L levels. TYPES OF HYPERLIPIDEMIA This content was retrieved from Section 01 Slide 48 of 58 of the online learning module Hyperlipidemia can be a primary or secondary condition. Primary hyperlipidemia is a result of a genetic disorder while secondary hyperlipidemia is acquired and usually a result of environmental influences or associated with a health condition. Reveal examples associated with the primary or secondary conditions of hyperlipidemia. Primary Hyperlipidemia • Single gene mutations • Receptor protein defects Secondary Hyperlipidemia • Diabetes • Hypothyroidism • Pancreatitis As Mr. Jones’ son is not currentl y diagnosed with additional health conditions, other than the acute chest pain that brought him to the emergency department, you will investigate if he may have primary hyperlipidemia. PRIMARY HYPERLIPIDEMIA This content was retrieved from Section 01 Slide 49 of 58 of the online learning module Scientists have identified numerous inherited single gene mutations that are associated with atherosclerosis. Most of this knowledge comes from epidemiological studies, Mendelian forms of the disease, and animal models, and accounts for approximately 40% of the genetic risk factors associated to atherosclerosis. A significant risk factor for atherosclerosis development is primary hyperlipidemia; there are five types, each differing in cause, occurrence , and the type of lipoprotein it affects. Type Disorder Cause Occurrence Elevated Plasma Lipoprotein I Familial lipoprotein lipase deficiency Genetic Very Rare Chylomicrons IIa Familial hypercholesterolemia Genetic Less Common LD L IIb Polygenic hypercholesterolemia Multifactorial Most Common LD L III Familial dysbetalipoproteinemia Genetic Rare ID L, Chylomicron remnants

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 45 IV Hypertriglyceridemia Multifactorial, Genetic Common VL D L V Familial combined hyperlipidemia Genetic Less Common VL D L, L D L For interest, learn about the impact of single gene mutations and the risk of developing atherosclerosis. - Refer to Pages 45-46 Reference: Verma, N. (2016). Introduction To Hyperlipidemia And Its Treatment: A Review. International Journal of Current Pharmaceutical Research, 9 (1), 6. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.22159/ijcpr.2017v9i1.16616 USING GW A S TO IDENTIFY GENES LINKED TO CORONARY ARTERY DISEASE Subpage of Section 01 Slide 49 of 58 – Learn about the impact of single gene mutations and the risk of developing atherosclerosis 1/1 The CARDIoGRAM study was a meta-analysis of 14 genome-wide association studies (G WA S) of coronary artery disease (C A D) to see if any particular genes are highly implicated in C A D development could be identified. Listen to Dr. Nicol discuss the implications of this study. (01:22) View the audio transcript. Start of Audio Transcript: In this study, 100,000 subjects were evaluated and approximately 30 loci of significance were identified. Loci have been organized across the x-axis according to chromosome number and significant loci can be identified as being above the baseline cutup point in the graph represented as a horizontal line. Significant loci identify genes where a variation or a single nucleotide polymorphism or SNP were identified. Of interest, some genes were anticipated to appear such as those linked to hypertension and LD L regulations are identified and highlighted in the figure. However, individually this loci only represent a modest increase in risk of developing coronary artery disease. Many other genes showed snips of unknown function with respect to atherosclerosis. In other words, even with the genetic predisposition, the risk of developing atherosclerosis is very low. This emphasizes that the single gene defects alone do not necessitate the development of atherosclerosis. And that there is likely an environmental or lifestyle component trigger to the disease that should always be factored in. End of Transcript. Reference: Schunkert, H., König, I. R., Kathiresan, S., Reilly, M. P., Assimes, T. L., Holm, H., Preuss, M., Stewart, A. F., Barbalic, M., Gieger, C., Absher, D., Aherrahrou, Z., Allayee, H., Altshuler, D., Anand, S. S., Andersen, K., Anderson, J. L., Ardissino, D., Ball, S. G., Balmforth, A. J., … Samani, N. J. (2011). Large -scale association

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 46 analysis identifies 13 new susceptibility loci for coronary artery disease. Nature genetics , 43 (4), 333-338. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1038/ng.784 QUESTION – IDENTIFYING THE TYPE OF HYPERLIPIDEMIA This content was retrieved from Section 01 Slide 50 of 58 of the online learning module Consolidate your current understanding by answering the question. Mr. Jones’ Son Triglycerides (T G): 160 mg/dL High Density Lipoproteins (H DL): 38 mg/dL Low Density Lipoproteins (L D L): 207 mg/dL Total Cholesterol: 245 mg/dL Question: Cross referencing Mr. Jones’ son’s plasma lipid levels with the types of primary hyperlipidemias you just learned about, which type do you think he has and why? Feedback: Dr. Nicol’s Feedback: Considering he has very high L D L levels, you can narrow down his primary hyperlipidemia to Type II. However, you cannot know for sure without genetic testing which subtype it may be. You refer Mr. Jones’ son to a genetic counsellor for further genetic testing. QUESTION – FAMILIAL HYPERCHOLESTEROLEMIA This content was retrieved from Section 01 Slide 51 of 58 of the online learning module The genetic tests came back, showing Mr. Jones’ son had familial hypercholesterolemia (FH), an inherited form of primary hyperlipidemia (Type II). This disease results in an L D L receptor gene mutation. Based on this information, answer the question in the space provided. Question: Based on your knowledge of reverse cholesterol transport, why would L DL receptor mutation cause increased L DL-C levels in plasma? Feedback: FEEDBACK: FAMILIAL HYPERCHOLESTEROLEMIA This content was retrieved from Section 01 Slide 52 of 58 of the online learning module Dr. Nicol’s Feedback

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 47 Disruption of the L D L receptor prevents binding/uptake and removal of L D L as it returns to the liver to be removed. This increases L DL half-life, leading to an accumulation of plasma L D L and cholesterol levels. Frequently patients with F H have xanthomas, which were noted in Mr. Jones’ son’s medical history. Recall from Mr. Jones’ son’s medical history that he is French -Canadian. FH happens to be a common autosomal dominant defect among French-Canadians with approximately 1 in 300-500 individuals affected. Consequently he may have inherited this mutation from his father. Reference: Adapted from: What is Familial Hypercholesterolemia. (n.d.) F H Foundation. Retrieved 12 June 2020, from: https://thefhfoundation.org/familial-hypercholesterolemia/what-is-familial-hypercholesterolemia

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 48 QUESTION – DIETARY SOURCES OF CHOLESTEROL This content was retrieved from Section 01 Slide 53 of 58 of the online learning module Clearly Mr. Jones’ son has multiple risk factors for atherosclerosis, which likely ultimately led him to the hospital for his chest pain. Luckily, we know that modifying dietary fat consumption is one way to help reduce the risk of developing atherosclerosis. Food awareness is an important aspect in making healthy dietary choices. A diet rich in fat and sugar increases an individual’s risk of developing hyperlipidemia and consequently, atherosclerosis. To the best of your ability, determine whether the dietary fat increases ( ↑ ), decreases ( ↓ ), or has no effect on L D L and H D L levels. L DL H DL ↑ ↓ Neutral ↑ ↓ Neutral Monosaturated Fats Polyunsaturated Fats Saturated Fats Trans Fats Feedback: L DL H DL ↑ ↓ Neutral ↑ ↓ Neutral Monosaturated Fats ↓ ↑ Polyunsaturated Fats ↓ ↑ Saturated Fats ↑ ↑ Trans Fats ↑ Neutral Navigate to the next page to learn how diet affects HD L and L D L levels. DIETARY SOURCES OF HD L AND LDL This content was retrieved from Section 01 Slide 54 of 58 of the online learning module Using this information you can advise Mr. Jones’ son regarding how he can adjust his diet to decrease his risk of further atherosclerotic plaque development. View examples of foods for different types of fat and how they influence cholesterol levels. Refer back to the previous activity to compare this information to your initial responses. TYPE OF FAT MAIN SOURCE EFFECT ON CHOLESTEROL LEVELS Monounsaturated Olives, olive oil, canola oil, peanut oil, cashews, almonds, peanuts and most other nuts; avocados Lowers L D L, Raises H D L Polyunsaturated Corn, soybean, safflower and cottonseed oil; fish Lowers L D L, Raises H D L

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 49 Saturated Whole milk, butter, cheese, ice cream; red meat; chocolate; coconut, coconut milk, coconut oil, egg yolks, chicken skin Raises both H D L and L D L Trans Most margarines; vegetable shortening; partially hydrogenated vegetable oil; deep fried chips; many fast foods; most commercial baked goods Raises L D L TREATMENT APPROACHES FOR HYPERLIPIDEMIA This content was retrieved from Section 01 Slide 55 of 58 of the online learning module In addition to treating hyperlipidemia with lifestyle changes such as increased exercise and a modified diet to reduce plasma cholesterol, a family of drugs known as statins can be prescribed (e.g. Atorvastatin (Lipitor), Lovastatin (Mecavor)). These drugs lower cholesterol levels; at the liver they inhibit cholesterol biosynthesis by increasing the expression of L D L receptors. Recall that K L F2 plays a role in maintaining endothelial function. You learned that laminar shear stress is important in maintaining normal endothelial phenotype through K L F2 expression. Recent research has revealed that statins can also increase K L F2 expression. In other words, statins can positively impact vascular function independent of their plasma lipid lowering action. Hence, statins can prevent or manage atherosclerosis from two fronts. Reference the master gene regulator network of shear stress References: Parmar, K. M., Nambudiri, V., Dai, G., Larman, H. B., Gimbrone, M. A., & García-Cardeña, G. (2005). Statins exert endothelial atheroprotective effects via the KLF2 transcription factor. The Journal of Biological Chemistry, 280(29), 26714-26719. Retrieved July 27, 2020, from: https://proxy.queensu.ca/login?url=http://dx.doi.org/10.1074/jbc.C500144200 (Statin): Pierson, R., & Berkrot, B. (2012, February 28). Statins Cause Diabetes, Memory Loss. Retrieved May 11, 2020, from https://homeopathicassociates.com/drug-harm/statins-cause-diabetes-memory- loss/ (Diagram): Image courtesy of Dr. Christopher Nicol. THE WAY FORWARD FOR MR. JONES’ SON This content was retrieved from Section 01 Slide 56 of 58 of the online learning module Although Mr. Jones unfortunately passed away, Mr. Jones’ son interacted with his healthcare team early such that many of the modifiable risk factors associated with developing atherosclerosis may be managed to reduce the potential of developing life-threatening complications. He is prescribed a statin drug to help manage his familial hypercholesterolemia but is also advised to adjust his lifestyle choices such as trying to quit smoking, adopt a well-balanced low-fat diet, and

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 50 conduct regular exercise to reduce the chances of developing life-threatening complications in the future. HEAR FROM THE EXPERTS – ATHEROSCLEROSIS This content was retrieved from Section 01 Slide 57 of 58 of the online learning module Now that you have learned about atherosclerosis and how it can be managed to limit the chance of severe clinical outcomes, click the photo to hear from the pathologists from this section. Dr. Manduch and Dr. Nicol will discuss their research and clinical practice as well as their thoughts on the field. Dr. Marosh Manduch (10:52) Professor Department of Pathology & Molecular Medicine Queen’s University Dr. Christopher Nicol (04:29) Associate Professor Department of Pathology & Molecular Medicine Queen’s University Page Links: https://player.vimeo.com/video/442044075 https://player.vimeo.com/video/444665700 References: Dr. Marosh Manduch. (n.d). School of Medicine Department of Pathology and Molecular Medicine. Retrieved May 24, 2020, from https://pathology.queensu.ca/faculty-staff/marosh-manduch Dr. Christopher Nicol. (n.d) Department of Pathology and Molecular Medicine. Retrieved July 31, 2020, from https://pathology.queensu.ca/faculty-staff/chris-nicol SECTION 01: SUMMARY – ATHEROSCLEROSIS This content was retrieved from Section 01 Slide 58 of 58 of the online learning module In this section, you learned about a cardiovascular disease called atherosclerosis by following the case study of Mr. Jones and his son. You learned how atheromas form in blood vessels and how they enable other clinical complications to manifest, for example an acute myocardial infarction in Mr. Jones. You also learned how cholesterol influences atherosclerosis, and what can be done for a patient who presents with high L D L levels. You will continue learning about cardiovascular diseases, but now shifting focus to channelopathies.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 51 SECTION 02: CHANNELOPATHIES CASE PRESENTATION: BENJAMIN This content was retrieved from Section 02 Slide 2 of 40 of the online learning module In this section, you will explore the case of Benjamin, a 17-year-old male, who unfortunately drowned while swimming with friends in a lake in August. He drowned despite being a strong swimmer with lifeguard certifications. Your goal is to determine how a young, certified lifeguard succumbed to this seemingly preventable event. Learn additional information about the incident. INCIDENT DETAILS • Water was not too cold; no major hazards were identified in the water • Suddenly, friends witnessed him fall below wa ter’s surface • After a few minutes, friends realized something was wrong and could not find Benjamin • Police dive team later found the young male at bottom of the lake, 30 feet below the surface Reference: Adnan, M. (2011, August 22). Drowning (Mechanical Asphyxia). Retrieved July 27, 2020, from https://www.medicinembbs.org/2011/08/drowning-mechanical- asphyxia.html?showComment=1572278473855 BENJAMIN’S AUTOPSY REPORT This content was retrieved from Section 02 Slide 3 of 40 of theo online learning module Revea l Benjamin’s general autopsy findings, toxicology report, and notes regarding his cardiac morphology. General Autopsy Findings • Frothy fluid from mouth and nares (pulmonary edema) • No injuries • Positive signs of drowning • No natural disease identified following full examination; no microscopic evidence of disease Toxicology Report • No alcohol or drugs detected in his system Cardiac Morphology • Heart morphology normal • 330 g (normal weight) • No heart muscle disease observed • No valve disease observed

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 52 • Coronary arteries widely patent* Definition*: Widely Patent: Open, unobstructed, and allowing the free passage of blood through the artery. BENAJMIN’S FAMILY HISTORY This content was retrieved from Section 02 Slide 4 of 40 of the online learning module After speaking with Benjamin’s family, it was discovered that two of his uncles and a cousin on his mother’s side of the family all died suddenly and unexpectedly under the age of 40, similarly to Benjamin. His family members all presumably died from heart attacks. Think about these questions as you continue through the rest of the section. • What are the implications of this familial history for the rest of his family members, including his younger sister and brother who are still alive today? • How should such a devastating event be managed? QUESTION – BENJAMIN’S CAUSE OF DEATH This content was retrieved from Section 02 Slide 5 of 40 of the online learning module After reading his autopsy report, you know that Benjamin died suddenly. It appears to be a sudden and unexpected death with no anatomical or toxicological cause to explain it. Using your current knowledge, answer the question. Question: What do you think might have happened to cause Benjamin’s death? Feedback: Dr. Cunningham’s Feedback: Based on your prior knowledge from this Module or Module 01, you may have predicted sudden death by thromboembolism/pulmonary embolism or perhaps complications due to atherosclerosis. Continue to the next page to learn about these forms of sudden death. SUDDEN CARDIAC DEATH This content was retrieved from Section 02 Slide 6 of 40 of the online learning module Sudden death is death that occurs within one hour of an acute clinical process. It is often abrupt and typically of cardiovascular nature. As you can appreciate from Section 01, one of the most common causes for sudden death in the North American population is as the result of cardiovascular and cerebrovascular disease. Sudden death usually involves the heart or blood vessels, or both. The sudden nature of the death typically means that death has occurred through a ventricular arrhythmia* or inhibition of the cardiac conduction system.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 53 Read examples of sudden death due to a cardiovascular nature. SUDDEN DEATH BY PULMONARY THROMBOEMBOLISM – Refer to Page 53 SUDDEN DEATH BY SEVERE STENOSIS – Refer to Pages 53 – 54 Definition*: Arrhythmia: Irregular heartbeat that does not allow proper filling or expulsion of blood from the heart. Leads to loss of consciousness and eventually death if not stopped. SUDDEN DEATH BY PULMONARY THROMBOEMBOLISM Subpage of Section 02 Slide 6 of 40 – Sudden Death by Pulmonary Thromboembolism 1/1 Sudden death can be the result of a pulmonary thromboembolism, originating from a deep venous thrombosis in the legs. Recall what you learned about thrombophilia and pulmonary embolisms in Module 01. The presence of a thrombus in the lungs hinders the ability of the cardiovascular system to adequately perfuse the heart. If the heart becomes underperfused, cardiac activity cannot be sustained, resulting in arrhythmias and ultimately death. Reference: Ege, V. (2019). Pulomoner Emboli . Varisege. Retrieved on June 10th, 2020 from http://varisege.com/upload/5/emboli.pdf SUDEEN DEATH BY SEVERE STENOSIS Subpage of Section 02 Slide 6 of 40 – Sudden Death by Severe Stenosis 1/1 In Section 01, you learned about atherosclerosis, which is the most common cause of sudden death associated with some form of normal physical activity, such as shovelling snow. When the heart is working harder, it needs extra blood to compensate. If the lumen of the coronary arteries are narrowed due to atherosclerot ic plaques, and the heart can’t get what it needs in terms of blood flow,

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 54 then the heart will start developing serious issues, ranging from electrical instability to arrythmias to sudden death. However, atherosclerosis isn’t the only cause of sudden death . The presented image of severe stenosis represents the case of a young teen who suddenly died as a result of a chronic inflammatory disorder that led to increasing scar build-up inside his coronary arteries. See the difference in diameter of a normal vessel compared to that with significant luminal stenosis. Normal Severe Stenosis References: Normal: Image courtesy of Dr. Kristopher Cunningham. Severe Stenosis: Nephron. (2009). RCA atherosclerosis. Wikimedia Commons. Retrieved June 23, 2020, from https://commons.wikimedia.org/w/index.php?curid=7437309 QUESTION – BENJAMIN’S CASE This content was retrieved from Section 02 Slide 7 of 40 of the online learning module Considering your new knowledge regarding sudden cardiac death and the results of Benjamin’s autopsy, you have a strong suspicion that his death was not the result of an acquired cardiovascular disease as he did not display any symptoms of VT E, occluded vessels, or morphological heart abnormalities. Answer the question. Question: How would you proceed in your attempt to discover the cause of Benjamin’s death? Suggest some approaches. Feedback: Dr. Cunningham’s Feedback : In order to determine the cause of B enjamin’s death you should perform a medical investigation. Navigate to the next page to learn what this entails . MOLECULAR AUTOPSIES AND MEDICAL INVESTIGATIONS This content was retrieved from Section 02 Slide 8 of 40 of the online learning module In cases of unexplained sudden cardiac death, where the autopsy doesn’t clearly indicate any underlying cause, post-mortem genetic testing is becoming an increasingly important tool. A post- mortem D NA analysis is called a molecular autopsy and can identify disease-causing mutations. This proportion of identifiable disease-causing mutations used to be quite small, but is growing every year.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 55 Knowing what mutations and epigenetic changes are present in the deceased patient can have prognostic, diagnostic, and surveillance implications for surviving family members with the same mutation. If Benjamin is found to have a mutation, it may guide testing and the nature of follow up for living relatives. Therefore to round-out the medical investigation, a thorough clinical investigation of living family members and collaboration with genetic counsellors is required to ensure affected family members are appropriately informed. Reference: Adaptation of Figure 2 from, Tester, D. J., Ackerman, M. J. (2009). Cardiomyopathic and Channelopathic Causes of Sudden Unexplained Death in Infants and Children. Annual Review of Medicine, 60 : 69-84. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1146/annurev.med.60.052907.103838 INHERITABLE CARDIOVASCULAR DISEASES This content was retrieved from Section 02 Slide 9 of 40 of the online leaning module Some ailments a medical investigation might uncover are inheritable cardiovascular diseases* . Benjamin’s family history of early cardiac death alread y suggests an inheritable cardiac disease, even before the completion of a molecular autopsy. Learn about two inheritable cardiovascular diseases. CHANNELOPATHIES In channelopathies, there are no visible changes to the heart but there are mutations in proteins which make up the cardiac ion channels (or associated proteins) in heart muscle. CARDIOMYOPATHIES In cardiomyopathies, a physical change in the structure of the heart muscle can be observed due to the inability for the heart to properly pump blood. Often in cardiomyopathies, there can be mutations in structural and/or functional cardiomyocyte (heart muscle cell) proteins.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 56 Recall that no morphological changes were observed in the heart during Benjamin’s autopsy. Therefore, it is suspected he had a channelopathy, but further investigation is required. First, you will learn more about channelopathies. Definition*: Inheritable cardiovascular diseases: Cardiac disorders which can be inherited. Can include arrhythmias, congenital heart disease, cardiomyopathy, and high blood cholesterol. Coronary artery disease leading to heart attack, stroke, and heart failure can also run in families, indicating inherited genetic risk factors. Reference: University of Ottawa Heart Institute. (2020). Inherited Cardiac Conditions. Retrieved June 9th, 2020 from: https://www.ottawaheart.ca/heart-condition/inherited-cardiac-conditions-genetic- disorders#:~:text=Many%20cardiac%20disorders%20can%20be,indicating%20inherited%20genetic%20 risk%20factors THE CARDIAC ACTION POTENTIAL AND CHANNELOPATHIES This content was retrieved from Section 02 Slide 10 of 40 of the online learning module The cardiac action potential provides the basis for conduction of electricity throughout the heart and electro-mechanical coupling, which is essential for normal heart function. The cardiac action potential represents the total electrical activity of an individual cardiomyocyte undergoing a contraction. The ion channel composition of each cardiac cell contributes to the formation of the cardiac action potential that is important for propagation of excitation. Many ion channels open and close throughout different points of each cycle of cardiac muscle contraction, even more than shown in the figure. The major ion channels, and ones you may be most familiar with are the C a 2+ , K + , N a + ion channels. It is when mutations occur to affect these ions channels, that channelopathy conditions can arise. References: Rahm, A. K., Lugenbiel, P., Schweizer, P. A., Katus, H. A., & Thomas, D. (2018). Role of ion channels in heart failure and channelopathies. Biophysical reviews , 10 (4), 1097-1106. Retrieved July 27 2020, from: https://proxy.queensu.ca/login?url=http://dx.doi.org/10.1007/s12551-018-0442-3 Roden, D. M., Darbar, D., & Kannankeril, P. J. (2007). Antiarrhythmic drugs. In Cardiovascular Medicine (pp. 2085-2102). Springer, London. Retrieved July 27 2020, from: https://proxy.queensu.ca/login?url=http://dx.doi.org/10.1007/978-1-84628-715-2_102 PREVALENCE OF CHANNELOPATHIES This content was retrieved from Section 02 Slide 11 of 40 of the online learning module Channelopathies are a heterogeneous group of disorders resulting from the dysfunction of ion channels located in the membranes of all cells and many cellular organelles. Each of the ion channels

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 57 presented on the previous slide can be associated with a different channelopathy of the heart if it becomes abnormal. The prevalence of these different channelopathies does vary, as some are more rare than others. Reveal the prevalence of each channelopathy of the cardiovascular system. LONG QT SYNDROME 1 : 3000 BRUGADA SYNDROME 1 : 4000-10 000 CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA 1 : 7000-10 000 SHORT QT SYNDROME Very Rare IDIOPATHIC VENTRICULAR DEFIBRILATION Incidence rate unknown; cause unknown Long Q T Syndromes are the most common type of channelopathy. Although it may not seem terribly common, if put into terms of a city population, thousands of people may be living with LQ T S. Given L Q T S is the most common of channelopathy syndromes, let’s learn more about its specific pathophysiology. PATHOPHYSIOLOGY OF CHANNELOPATHIES IN RELATION TO LQTS This content was retrieved from Section 02 Slide 12 of 40 of the online learning module Channelopathies are typically diagnosed through the use of an electrocardiogram (EC G) recording, which presents a map of electrical impulse throughout the heart ( click interest button for example of a normal labelled EC G recording ). Figure A depicts a typical EC G recording for a healthy patient. When analyzing the components of the waveform, one can visualize atrial depolarization (P wave), ventricular depolarization (Q R S complex), and ventricular repolarization (S-T wave). In the case of Long Q T Syndrome, the most common channelopathy, this typically presents on an EC G strip as an elongated Q-T interval (as shown in Figure B). The exact magnitude of the elongated Q T interval varies between patients, and approximately 40% of Long Q T patients actually present with Q-T intervals within a normal range. This variation can make diagnosing this condition rather difficult. If untreated, then these abnormal Q-T intervals lead to uncoordinated heart muscle function and fatal cardiac arrhythmias. Review a refresher on action potential wave nomenclature

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 58 Reference: Cleveland Clinic. (2019). Long Q-T Syndrome (LQ T S). Cleveland Clinic. Retrieved June 10th, 2020, from https://my.clevelandclinic.org/health/diseases/17183-long-q-t-syndrome-lqts DIFFERENT TYPES OF LQ T S This content was retrieved from Section 02 Slide 13 of 40 of the online learning module Currently available genetic information is providing insight into the underlying cause of channelopathies, and L Q T S in particular. There are many different types of L Q T S, each associated with a genetic mutation affecting a different ion channel found either on the membranes of cells or cellular organelles. After the first mutation in the K C NQ 1 gene was linked to L Q TS, it became easier to identify other genes involved in channelopathies. Researchers began to look at other similar genes related to cardiac channel proteins and reported mutations in potassium, sodium or calcium channel proteins may also be linked to L Q T S. Reference: Webster, G., & Berul, C. I. (2013). An update on channelopathies: from mechanisms to management. Circulation , 127 (1), 126-140. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1161/CIRCULATIONAHA.111.060343 GENES ASSOCIATED WITH LQ T S This content was retrieved from Section 02 Slide 14 of 40 of the online learning module With the wealth of genetic information now available to us, different genetic mutations are starting to be associated with L Q T S. The presented table provides a summary of the research that has been done to try and associate mutations in specific ion channel genes to 15 different types of L Q T S disorders. These diseases may be either autosomal dominant (AD) or autosomal recessive (A R) and affect the flow of ions in or out of the cell to affect the potentiation of these cells.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 59 Reference: Image courtesy of Dr. Harriet Feilotter. QUESTION – DISCOVERING GENES ASSOCIATED WITH LQT S This content was retrieved from Section 02 Slide 15 of 40 of the online learning module Considering what you have just learned, answer the question. Question: What would be the ramifications of gaining much of the data regarding genes associated with L QT S from post-symptomatic diagnosis? Feedback: Dr. Feilotter’s Feedback: It is very important to note that this information has been collected over time with bias. Typically, this genetic association was only identified once a patient presented with symptoms of a potential channelopathy and exploratory genetic testing was done to see if a mutation in an ion channel existed. So the question remains, how can we begin to understand the clinical relevance of this genetic data? And is all this genetic data relevant for the purposes of testing patients for the presence of disease and potential treatment? INFLUENCE OF GENETIC INFORMATION ON OUR UNDERSTANDING OF CHANNELOPATHIES This content was retrieved from Section 02 Slide 16 of 40 of the online learning module You can now appreciate there are many genes known to cause Long Q T Syndrome. There are dozens of more known genes that are involved in other channelopathies. In ~15 years scientists have gone from identifying 2 genes to more than 40 genes with recognized defects contributing to channelopathies. This has given rise to an understanding of two important characteristics of channelopathies: clinical heterogeneity and locus heterogeneity.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 60 LOCUS HETEROGENEITY Each of the channelopathy disorders may be caused by mutations in multiple genes. For example, mutations in more than 15 genes may lead to a Long Q T syndrome. CLINICAL HETEROGENEITY Mutations in many of these genes may have a role in more than one of these channelopathy subtypes. For example, mutations in K C NQ1 gene may present clinically as either Long Q T or Short QT syndromes. Reference: Prajapati, C., & Aalto-Setälä, K. (2019). Modelling of Genetic Cardiac Diseases. In Visions of Cardiomyocyte-Fundamental Concepts of Heart Life and Disease . IntechOpen. Retrieved July 27, 2020, from: https://proxy.queensu.ca/login?url=http://dx.doi.org/10.5772/intechopen.84965 UNDERSTANDING LOCUS AND CLINICAL HETEROGENEITY This content was retrieved from Section 02 Slide 17 of 40 of the online learning module The complexity of channelopathy disorders has actually been appreciated for quite some time, even before extensive genetic information was directly available for these disorders. In the past, the use of familial pedigrees allowed one to track who within a family may have had a disease. The concepts of clinical and locus heterogeneity were later supported by expansion of our understanding of genes and mutations associated with these diseases. Watch Dr. Feilotter discuss how the past practice of pedigree development and current mutational analyses help us understand the complexities of channelopathies. (04:20) Page Link: https://player.vimeo.com/video/442044599

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 61 QUESTION – HOW DO WE APPROACH GENETIC TESTING? This content was retrieved from Section 02 Slide 18 of 40 of the online learning module Considering your past knowledge, answer the following question. Given the known clinical and locus heterogeneity associated with channelopathies, testing for the presence of a channelopathy disorder after Benjamin’s passing is not stra ightforward. Question: How would you approach identifying the type of channelopathy he had? (i.e. would you look for all 15 mutations previously identified as associated with Long QT syndrome as part of a diagnostic testing panel? Feedback: Dr. Feilotter ’s Feedback: Keep in mind that the many mutations previously identified as associated with different Long Q T syndromes were often based on investigating the genome of individuals who presented to clinicians with channelopathy symptoms. Therefore, how do we determine if the presence of mutation is worth testing for? Navigate to the next page to learn about a common approach in clinical genetic labs to address this issue. HOW DO WE ASSESS THE ASSOCIATION BEWTWEEN CHANGES IN A GENE AND PARTICULAR DISEASE? This content was retrieved from Section 02 Slide 19 of 40 of the online learning module The rise of genetic testing has allowed us to identify many genetic mutations that may lead to a disease case. However, systematic efforts must be made in order to determine whether a gene, and/or changes to its normal expression or function, are associated with a disease state. Listen to Dr. Feilotter discuss the process to make this determination. (02:11)

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 62 View the audio transcript. Start of Audio Transcript: If the association between mutations in a gene and a disease is published just once in the literature, that is actually not good enough for us to start testing the gene and giving information to patients and clinicians. There needs to be a more comprehensive and standardized way of assessing the association between mutations in a gene and a particular disease. There are organizations, such as one known as ClinGen, a National Institutes of Health-funded resource that tried to quantify the extent to which a gene is truly associated with a given disease, including the genes that we've been talking about here. Really assessing gene disease associations represents a lot of work and requires scouring the literature for any and all examples of genetic or biochemical evidence of that association. The presented table shows the basic principles used in the process. If you believe that a gene is involved in a disease, you start by identifying the very first published reference to that association, and then identify as much genetic evidence in the literature as you can. You then need to read the papers and assess the information extremely rigorously, and you can assign points depending on the quality of the data. You then do the same thing, looking for evidence in the literature that is biochemical in nature, such as functional studies, that show that a particular mutation in the gene you're exploring results in a biochemical defect that would be in keeping with the disease in question. The point system allows the scoring of genetic evidence, such as the example from the family pedigree shown earlier, and experimental or biochemical evidence, as well as whether the evidence is replicated over time and whether there is any conflicting data. The point system determines the strength of the association of the gene with disease ranked from limited or disputed to strong. A strong rating becomes a definitive rating, which is the highest level, if the evidence is replicated over time and is not disproven. In the clinical world, we wouldn't want to be testing for mutations in a gene that has less than a strong rating in terms of its association with the disease in question. End of Audio Transcript. Reference: Strande, N. T., Riggs, E. R., Buchanan, A. H., Ceyhan-Birsoy, O., DiStefano, M., Dwight, S. S., ... & Wright, M. W. (2017). Evaluating the clinical validity of gene-disease associations: an evidence-based framework developed by the clinical genome resource. The A merican Journal of Human Genetics, 100 (6 ), 895-906. Retrieved June 2020, from https://proxy.queensu.ca/login?url=https://doi.org/10.1016/j.ajhg.2017.04.015 GENES ASSOCIATED WITH LQ T S This content was retrieved from Section 02 Slide 20 of 40 of the online learning module Let’s return to our exploration of the 15 genes experimentally associated with L Q T S. The quantitative criteria just described were used to evaluate genetic data to see if certain genes were truly associated with L Q T S disease. Currently, only genes K C NQ1, K C N H2, and S C N5A have definitive evidence for being associated with L Q T S 1 through 3, meaning the presence of mutations in these genes determines presence of LQ T S conditions. The remaining genes are classified as limited, moderate, or disputed. As a result, clinical lab testing today is only offered for the 3 definitively classified genes. Nevertheless, since this is a continuous

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 63 process, as additional evidence arises for any genes, listed or yet unknown, clinical lab testing for mutations associated with channelopathies may expand in the future. Reference: Image courtesy of Dr. Harriet Feilotter. QUESTION – ADDITIONAL CONSIDERATIONS OF GENETIC TESTING This content was retrieved from Section 02 Slide 21 of 40 of the online learning module You now know there are three definitive genes associated with the development of Long Q T syndromes. Before spending resources testing Benjamin’s samples for the presence of mutations in these three genes, reflect on the questions in the provided space. Question: What benefit would there be to doing this genetic testing on Benjamin (as he is deceased)? Question: What additional testing considerations would you weigh in order to decide to test for other genetic mutations associated with other categories of channelopathies such as Brugada Syndrome or Catecholaminergic Polymorphic Ventricular Tachycardia? Feedback: Navigate to the next page for information about these considerations. FEEDBACK: ADDITIONAL CONSIDERATIONS OF GENETIC TESTING This content was retrieved from Section 02 Slide 22 of 40 of the online learning module Questions:

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 64 • What benefit would there be to doing this genetic testing on Benjamin (as he is deceased)? • What additional testing considerations would you weigh in order to decide to test for other genetic mutations associated with other categories of channelopathies such as Brugada Syndrome or Catecholaminergic Polymorphic Ventricular Tachycardia? Listen to Dr. Feilotter describe additional considerations for genetic testing in this case. (01:55) View the audio transcript. Start of Audio Transcript: In the scenario where genetic data are available for a disease, one question becomes when we offer that testing to patients. So, for the genes that we've been talking about so far, and that you see on this slide, they all offer diagnostic information. So, the presence of a pathogenic mutation in any of these genes can actually confirm a specific disease in a patient. However, that doesn't always help a lot unless it can also provide additional information, such as the prognosis or the likely outcome or course of the disease in a particular patient. Even providing prognostic information is not always that helpful if there isn't an appropriate treatment for the disease. So, really, the best case scenario for genetic testing in the clinic is where you can provide a confident diagnosis of a disease, some information about the prognosis, and potentially lead to a potential therapy for that patient. Currently, genetic testing for CPVT or Brugada syndrome provides information about diagnosis and limited information about prognosis, in some cases for Brugada syndrome, but doesn't really provide a lot of information about options for therapy that might be effective. However, in the case of Long Q T syndrome, we are actually able to provide prognostic, diagnostic, and information about an effective treatment for these patients. Given that channelopathies are inherited, then testing Benjamin for mutations in the three genes that we've already definitively associated with Long Q T syndrome would probably be the best approach, as it could then begin to provide potential directions for further investigations in his other family members, and they could actually be eventually managed if it turns out that they were also carrying the gene mutation. End of Audio Transcript. Reference: Strande, N. T., Riggs, E. R., Buchanan, A. H., Ceyhan-Birsoy, O., DiStefano, M., Dwight, S. S., ... & Wright, M. W. (2017). Evaluating the clinical validity of gene-disease associations: an evidence-based framework developed by the clinical genome resource. The American Journal of Human Genetics , 100 (6), 895-906. Retrieved June 2020, from https://proxy.queensu.ca/login?url=https://doi.org/10.1016/j.ajhg.2017.04.015 CLINICAL PRESENTATIONS ASSOCIATED WITH LQT S GENES This content was retrieved from Section 02 Slide 23 of 40 of the online learning module Due to clinical genetic testing considerations, you are planning to test Benjamin’s cardiac samples* for mutations in three specific genes. It so happens that the Long Q T syndromes associated with these genes, L Q T S 1-3, have distinct clinical presentations, and are associated with different triggers.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 65 Read details about each gene associated with their specific L QT syndrome and clinical presentation. As you review these presentations, consider if Benjamin’s medical history aligns with any of these triggers. KC N Q1 : L QT 1 In L Q T 1, mutations occur in the gene KCNQ1 , accounting for approximately 35% of Long QT cases. This is the type of L Q T S that will cause cardiac arrhythmias while participating in physical exercise, such as swimming or playing basketball where there is some sort of exertion. This is usually the cause when hearing about athletes dying suddenly during practice. For interest, reveal the percent total of L Q T 1-associated deaths stratified by activity. Exercise: 68% Emotion: 14% Sleep: 9% Other: 9% KC NH2 : L QT 2 In L Q T 2, mutations occur in the gene K C NH2, accounting for approximately 30% of Long Q T cases. This is the type of L Q T S most commonly caused by auditory triggers as well as the postpartum period. For example, an individual with L Q T 2 might actually be killed by their alarm clock going off in the morning, as a startling sound is enough to evoke an arrhythmia. For interest, reveal the percent total of L Q T 2-associated deaths stratified by activity. Emotion: 29% Sleep: 42% Other: 22% S CN5A : L QT 3 In L Q T 3, mutations occur in the gene S C N5A , accounting for approximately 10% of Long Q T cases. Individuals with L Q T 3 often die during rest and is most often associated with sudden infant death syndrome (SIDS). For interest, reveal the percent total of L Q T 3- associated deaths stratified by activity. Exercise: 4% Emotion: 12%

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 66 Sleep: 64% Other: 20% Definition*: Cardiac samples: Although we could test Benjamin’s blood, D NA obtained from tissue at autopsy has more stability. Reference: Ackerman, M. Clinic. (2019). Cardiac Channelopathies. Mayo Clinic . Retrieved June 10th, 2020 from https://www.mayo.edu/research/labs/sudden-death-genomics/research/cardiac-channelopathies QUESTION – DETERMINING BENJAMIN’S DIAGNOSIS This content was retrieved from Section 02 Slide 24 of 40 of the online learning module Using your knowledge of L Q T S, answer the question. Question: After learning about the three types of clinically testable Long QT syndromes and their triggers, which type do you suspect Benjamin suffered from and why? Feedback: Dr. Cunningham’s Feedback : It is suspected that Benjamin has L Q T 1. As you learned in your medical investigation Benjamin was athletic and a strong swimmer. L Q T 1 is usually the cause of death in athletes who pass suddenly during physical activity. To finally confirm your suspicions, you order a panel for testing the three definitive L Q T S genes from a sample of Benjamin’s heart. BENJAMIN’S CASE: GENETIC TESTING RESULTS AND IMPLICATIONS FOR HIS FAMILY This content was retrieved from Section 02 Slide 25 of 40 of the online learning module Benjamin’s genetic test results came back. The results reveal that he had a mutation in his KCNQ1 gene. This confirms the suspected LQ T 1 diagnosis for Benjamin. As there is a noted history of sudden cardiac death at a young age on Benjamin’s mother’s side of the family, you should now direct your attention on Benjamin’s surviving family members family members, including his younger sister Tianna and younger brother David, to see if they are at risk of LQ T S. It is known that the younger the age at which someone passes away from L Q T S, the stronger the suggestion that it is an inherited condition.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 67 Reference: Crotti, L., Spazzolini, C., Schwartz, P. J., Shimizu, W., Denjoy, I., Schulze-Bahr, E., ... & Wilde, A. A. (2007). Clinical Perspective. Circulation , 116 (21), 2366-2375. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1161/CIRCULATIONAHA.107.726950 TESTING TIANNA FOR LQT S This content was retrieved from Section 02 Slide 26 of 40 of the online learning module Let's focus on Benjamin's younger sister Tianna for a moment and assume genetic testing is proceeding for her. In order to ensure you can properly interpret Tianna's results clinically, you order a panel for the three definitive LQ T S genes and test for variants. It was found that Tianna has the same K C N Q1 mutation as her brother Benjamin. Does this mean she has L Q T S? To answer this question, the concepts of penetrance and expressivity need to first be explored. PENETRANCE AND EXPRESSIVITY This content was retrieved from Section 02 Slide 27 of 40 of the online learning module Phenotypes may vary among individuals with the same mutations due to penetrance and expressivity, which determine how confidently a mutation may be associated with a given disease. Learn about each concept. PENETRANCE Penetrance refers to the extent to which an individual will develop some aspect of the disease, if possessing a mutation in the disease-causing gene. For example, if penetrance is 100%, then anyone with the mutation will show some symptom of the disease. EXPRESSIVITY

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 68 Expressivity refers to presentation of the disease or intensity of the phenotype. In the context of L Q T S, two family members may have the same mutation, but one family member may have arrhythmias while another family member may have some fainting. Their phenotypic expression of the disease is different. View pedigree examples for different combinations of penetrance and expressivity. 100% PENETRANCE AND CONSTANT EXPRESSIVITY – Refer to Page 68 <100% PENETRANCE AND CONSTANT EXPRESSIVITY - Refer to Pages 68 – 69 <100% PENETRANCE AND VARIABLE EXPRESSIVITY - Refer to Page 69 100% PENETRANCE AND CONSTANT EXPRESSIVITY Subpage of Section 02 Slide 27 of 40 – 100% Penetrance and Constant Expressivity 1/1 When penetrance is complete, every person with the mutation will display some symptom of the disease. When expressivity is constant, all individuals with the mutation show the same symptoms. The pedigree shows a family in which the channelopathy mutation has 100% penetrance and constant expressivity. All family members with the mutation present with the same symptoms (both in terms of EC G phenotype and cardiac events) for the channelopathy of interest. Note that none of the family members present only as genotype-positive, because the penetrance is complete. Reference: Giudicessi, J. R., Ackerman, M. J. (2012). Determinants of incomplete penetrance and variable expressivity in heritable cardiac arrhythmia syndromes. Translational research : the journal of laboratory and clinical medicine , 161 (1), 1-14. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1016/j.trsl.2012.08.005 <100% PENETRANCE AND CONSTANT EXPRESSIVITY Subpage of Section 02 Slide 27 of 40 – < 100% Penetrance and Constant Expressivity 1/1 When penetrance is incomplete, not every individual with the mutation will display symptoms associated with the disease. When expressivity is constant, all individuals with the mutation show the same symptoms. The pedigree shows a family in which the channelopathy mutation has incomplete penetrance and constant expressivity. Some family members with the mutation present only as genotype-positive, while others also present with the EC G phenotype and cardiac event. Note that none of the family members only present with an EC G phenotype without cardiac events, as expressivity is constant. Reference: Giudicessi, J. R., Ackerman, M. J. (2012). Determinants of incomplete penetrance and variable expressivity in heritable cardiac arrhythmia syndromes. Translational research : the journal of laboratory

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 69 and clinical medicine , 161 (1), 1-14. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1016/j.trsl.2012.08.005 < 100% PENETRANCE AND VARIABLE EXPRESSIVITY Subpage of Section 02 Slide 27 of 40 – < 100% Penetrance and Variable Expressivity 1/1 When penetrance is incomplete, not every individual with the mutation will display symptoms associated with the disease. When expressivity is variable, individuals with the mutation show a variety of symptoms. The pedigree shows a family in which the channelopathy mutation has incomplete penetrance and variable expressivity. Some family members are only genotype-positive for the mutation (no symptoms of the disease present), others present with the EC G phenotype and some family members have the EC G phenotype with a cardiac event. This is because neither penetrance nor expressivity are constant. Reference: Giudicessi, J. R., Ackerman, M. J. (2012). Determinants of incomplete penetrance and variable expressivity in heritable cardiac arrhythmia syndromes. Translational research : the journal of laboratory and clinical medicine , 161 (1), 1-14. Retrieved June 23, 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1016/j.trsl.2012.08.005 ACTIVITY – HYPOTHETICAL SCENARIO ON PENETRANCE AND EXPRESSIVITY This content was retrieved from Section 02 Slide 38 of 40 of the online learning module Read through the hypothetical scenario and then navigate to the next slide to complete an activity to confirm your understanding of the concept of penetrance and expressivity. Scenario: During fall orientation, the PATH 310 Coordinator starts a new tradition to increase course enrollment, and hands out orange backpacks to all third year students. At the end of the year, the Coordinator wants to know: • whether handing out the backpacks was effective in ensuring students would enroll in the course, • if those enrolled students were successful, and, • if enrolled students enjoyed the course. To complete the activity on the next slide, consider that the orange backpacks represent a gene mutation, taking PATH 310 represents the experience being examined, and both receiving A+ grades and enjoying the course are two features (symptoms) of the experience. Reference: Napolitano, C., Bloise, R., Monteforte, N., & Priori, S. G. (2012). Sudden cardiac death and genetic ion channelopathies: long Q T, Brugada, short Q T, catecholaminergic polymorphic ventricular tachycardia,

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 70 and idiopathic ventricular fibrillation. Circulation , 125 (16), 2027-2034. Retrieved July 27 2020, from https://proxy.queensu.ca/login?url=http://dx.doi.org/10.1161/CIRCULATIONAHA.111.055947 QUESTION – PENETRANCE AND EXPRESSIVITY This content was retrieved from Section 02 Slide 29 of 40 of the online learning module Use the drop-down menu to select the correct penetrance and expressivity conditions for each outcome. Options: <100% penetrance and variable expressivity, 100% penetrance and constant expressivity, <100% penetrance and constant expressivity Outcome 1: Half of the third year students who got orange backpacks take PATH 310, but only some of them receive a final grade of A+, while most enjoyed the course. Outcome 2: All third year students who got orange backpacks take PATH 310, and all of them receive A+ final grades and all enjoyed the course. Outcome 3: Half of the third year students who got orange backpacks take PATH 310, but all of them receive a final grade of A+ and all enjoyed the course. Feedback: Outcome 1: Half of the third year students who got orange backpacks take PATH 310, but only some of them receive a final grade of A+, while most enjoyed the course. <100% penetrance and variable expressivity Outcome 2: All third year students who got orange backpacks take PATH 310, and all of them receive A+ final grades and all enjoyed the course. 100% penetrance and constant expressivity Outcome 3: Half of the third year students who got orange backpacks take PATH 310, but all of them receive a final grade of A+ and all enjoyed the course. <100% penetrance and constant expressivity Listen to Dr. Nicol’s feedback. (01:32) View the audio transcript. Start of Audio Transcript: In the first outcome, not all students who got orange backpacks took the course. This is equivalent to scenario where not all people with a channelopathy mutation get the disease, which translates to less than 100% penetrance. In addition, not everyone with an orange backpack in the course got an A+ and most but not all enjoyed the course. This is equivalent to not everyone with the same channelopathy mutation showing the same symptoms.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 71 In the second outcome, everyone who got an orange backpack took the course, similar to everyone with a certain channelopathy mutation having the disease. In addition, everyone with the mutation (orange backpack) showed all the symptoms (got an A+ and enjoyed the course), which translates to 100% penetrance and 100% expressivity. As you can imagine, this is not a very common scenario. In the third outcome, penetrance is less than 100%, similar to the first outcome because not everyone with the backpack takes the course. However, here everyone who does have an orange backpack and takes the course does get an A+ and does enjoy it. As a result, those who have the mutation and get the disease show the same symptoms translating into a constant expressivity. End of Audio Transcript. INCORPORATING PENETRANCE AND EXPRESSIVITY INTO CLINICAL INTERPRETATION OF TIANNA’S TESTING RESULTS This content was retrieved from Section 02 Slide 30 of 40 of the online learning module Consider that it is a rare scenario that penetrance is 100% and expressivity is constant. Typically, penetrance is less than 100% and expressivity is variable. In other words, a person may have a mutation associated with a disease but may or may not show the same symptoms as another person diagnosed with the disease or having the same mutation. This could happen for reasons that are not clear: it could be due to some part of their genetic background, it could be influenced by their environment, or lifestyle. The less the penetrance is, the harder it is to make the association between the disease and the gene. You have determined that Tianna has an L Q T S mutation with a variant (genotype A341V), same as what Benjamin was found to have, but she has not necessarily shown any outward symptoms of L Q T S yet. How do we interpret this clinical outcome, keeping in mind our understanding of penetrance and expressivity? Should Tianna be concerned about the presence of this variant or not? GENETIC PURGATORY AND CHANNELOPATHIES This content was retrieved from Section 02 Slide 31 of 40 of the online learning module An MD who treats patients for channelopathies and arrhythmias, Dr. Michael Ackerman, wrote a paper several years ago called, “Genetic purgatory and the cardiac channelopathies: Exposing the variants of uncertain/unknown significance issue”. In this paper, he notes: “Genetic purgatory is a place where the genetic test-ordering physician and patients and their families are stuck when a variant of uncertain/unknown significance (V US ) has been elucidated.” (Ackerman, 2015). What this means, is that when we have genomic variants, there is a scale of clinical interpretations that can be provided back to the clinician and the patient, which can make clinical interpretation even more complicated due to our incomplete understanding of the disease. Learn about the possible interpretations of genomic variants. Benign

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 72 A benign genomic variant is one which does not increase a patient's susceptibility to a disease or disorder. Variance of unknown significance A variance of unknown significance is a genomic variant which has an undetermined or unknown effect on the patient susceptibility to disease. Pathogenic A pathogenic genomic variant is one which is known to increase the patient's susceptibility to a disease or disorder. Reference: Ackerman, M. J. (2015). Genetic purgatory and the cardiac channelopathies: exposing the variants of uncertain/unknown significance issue. Heart rhythm , 12 (11), 2325-2331. Retrieved June 2020, from: https://proxy.queensu.ca/login?url=https://doi.org/10.1016/j.hrthm.2015.07.002 HOW GENETICISTS INTERPRET VARIANT DATA FOR THE PATIENT This content was retrieved from Section 02 Slide 32 of 40 of the online learning module As you have learned, interpreting genomic variants in a clinically-relevant manner is not an easy task. While you do not need to memorize the specifics, this table can be used to give you a further appreciation of the types of evidence considered when interpreting variants. For interest, watch the video to listen to Dr. Feilotter explain the criteria to consider and additional data to investigate when clinically interpreting a genomic variant. (07:10) Note: You may want to consider this information in the context of completing your group assessments. Page Link: https://player.vimeo.com/video/442045659 Reference: Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., ... & Voelkerding, K. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in medicine , 17 (5), 405-423. Retrieved July 27, 2020, from: https://proxy.queensu.ca/login?url=http://dx.doi.org/10.1038/gim.2015.30 CHALLENGES OF CLINICAL INTERPRETATION OF GENOMIC VARIANTS IN CHANNELOPATHIES This content was retrieved from Section 02 Slide 33 of 40 of the online learning module

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 73 As with many diseases, there is a spectrum of L Q T phenotypes, making it more difficult to identify individuals with this channelopathy. This challenge can be highlighted by research that has shown the overlap in Q T length (QT c) between normal non-carriers and L Q T S mutation carriers. In other words, certain individuals with L Q T S genomic variants can have Q T c intervals which are identical to those of healthy non-carriers. So, in Tianna’s case, even if we measured her Q T interval to determine if she had L Q T S symptoms, we would not be completely sure that her Q T interval was due to the pathogenic variant or not. Reference: Schulte, B., Carlsson, J., and Neuzner, J. (2001). Electrical interference between two transvenous implantable defibrillator leads. Heart, 86 (1): 14. Retrieved June 23, 2020, from: http://dx.doi.org.proxy.queensu.ca/10.1136/heart.86.1.14 LQ T S RISK STRATIFICATION This content was retrieved from Section 02 Slide 34 of 40 of the online learning module At this point, you may appreciate that in the case of channelopathies, it is not just a matter of identifying a mutation to appropriately diagnose a patient. In fact, in L Q T S, patients are often stratified according to risk based on gender, their Q T interval, type of L Q T S, and even specific genotype, or presence of multiple mutations, among other characteristics. Review the table and appreciate how the associated hazard risk for Tianna’s L Q T 1 diagnosis, could simply increase from low to high depending on her long QT interval measurement.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 74 Reference: Napolitano, C., Bloise, R., Monteforte, N., & Priori, S. G. (2012). Sudden cardiac death and genetic ion channelopathies: long Q T, Brugada, short Q T, catecholaminergic polymorphic ventricular tachycardia, and idiopathic ventricular fibrillation. Circulation , 125 (16), 2027-2034. Retrieved July 27 2020, from https://proxy.queensu.ca/login?url=http://dx.doi.org/10.1161/CIRCULATIONAHA.111.055947 QUESTION – WHAT ARE THE NEXT STEPS FOR TIANNA? This content was retrieved from Section 02 Slide 35 of 40 of the online learning module Tianna and her family are not sure what to do next given the outcomes of Benjamin’s molecular autopsy and the subsequent medical investigation findings that Tianna has the same L QT S1 mutation as her brother. They visit a genetic counselor for guidance. Answer the question given your understanding of channelopathies and the patient case. Question: What do you think the genetic counsellor would discuss with Tianna and her parents? Feedback: Dr. Feilotter’s Feedback: Although Tianna does not currently present with symptoms of L Q T S, we know that the variant present may have variable expressivity. Given the extensive history of sudden cardiac arrest events in the family and given her young age, the genetic counsellor may suggest that management or treatment options be explored rather than ignoring these molecular findings. As part of this process, Tianna may chose to have her Q T interval measured to further aid in her risk stratification. QUESITON – MANAGING TIANNA’S LQ T S This content was retrieved from Section 02 Slide 36 of 40 of the online learning module Consider your knowledge of the pathophysiology of channelopathies, answer the question to the best of your ability.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 75 Question: Hypothesize an approach to treat Benjamin’s family membe rs who possess potential L QT S variants. Feedback: Navigate through the next pages to learn about potential LQ T S management options. MANAGING ARRHYTHMIAS IN CHANNELOPATHIES This content was retrieved from Section 02 Slide 37 of 40 of the online learning module Given Long Q T syndromes lead to abnormal cardiac potentiation, management, or prevention of arrhythmic events is the key approach in reducing the risk of sudden cardiac death. There are two primary strategies employed for the management of arrhythmias associated with channelopathies. Read about these management approaches. ARRHYTHMIA PREVENTION Βeta -blockers may be prescribed in order to prevent arrhythmia in those diagnosed with channelopathies. Beta-blockers work to limit adrenergic stimulation that can provoke an arrhythmia in the setting of L Q T S. Side effects in beta-blockers cause some individuals to feel fatigued, so they cannot participate in physical activity. These side effects lead to poor medication compliance, especially in young people. ARRHYTHMIA TERMINATION An implantable cardioverter-defibrillator (I C D) can be used to deliver electrical shock to the heart. Wires from the device attach to the surface of the heart and senses the beat and rhythms of the heart. If arrhythmias arise, it provides a shock to reset the heart to an appropriate beat. The device does not always work, but has saved many lives. Reference: BruceBlaus. (2017). Implantable Defibrillator. Wikimedia Commons. Retrieved June 2020, from: https://commons.wikimedia.org/wiki/File:Implantable_Defibrillator.png THE WAY FORWARD FOR BENJAMIN’S FAMILY This content was retrieved from Section 02 Slide 38 of 40 of the online learning module On follow-up testing, Tianna is identified to have a L Q T arrhythmia. Given this and detection of the same K C NQ1 mutation found in Benjamin, beta blockers may be a good non-invasive choice for Tianna to prevent future arrhythmias and keep her from experiencing serious cardiac events in the future. If Benjamin had survived his serious arrhythmia event, he may have received an I C D and been prescribed reduced physical activity moving forward.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 76 Moving forward, all of Benjamin's family will be informed of the outcomes of his molecular autopsy and offered genetic testing. They will also receive counselling for themselves and for family planning purposes to help prevent further tragedies in this family. HEAR FROM THE EXPERTS – CHANNELOPATHIES This content was retrieved from Section 02 Slide 39 of 40 of the online learning module Now that you have learned about channelopathies and the challenges associated to providing clinical testing for this types of disorder, click the photos to hear from Dr. Feilotter and Dr. Cunningham, the pathologists from this section, discuss their research and clinical practice as well as their thoughts on the field. Dr. Harriet Feilotter (05:07) Professor Department of Pathology & Molecular Medicine, Richardson Laboratory Queen’s University Director, Molecular Genetics, Kingston Health Sciences Centre Dr. Kristopher Cunningham, MD, PhD, FRCPC (09:13) Forensic and Cardiovascular Pathologist D irector, Kingston Regional Forensic Pathology Unit, Pathology and Molecular Medicine Queen’s University Page Links: https://player.vimeo.com/video/442046550 https://player.vimeo.com/video/442048215 SECTION 02: SUMMARY – CHANNELOPATHIES This content was retrieved from Section 02 Slide 40 of 40 of the online learning module In this section, you learned about channelopathies through the unfortunate sudden death of Benjamin, a young, athletic man. You learned about different causes of sudden cardiac death and specifically how channelopathies play a role in disrupting the normal cardiac action potential. The focus on this section was on Long Q T syndromes and how they can be tested for, given the prevalence of disease-causing mutations and variants. Finally, you gained an appreciation of how genetic testing and clinical interpretation of channelopathies in patients is particularly challenging due to the complex traits of the disease and involves understanding the penetrance and expressivity of many possible variants and the challenge of identifying clinical phenotypes. Ultimately, management of arrhythmias remains the key approach to limit sudden cardiac death due to a channelopathy.

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 77 CONCLUSION QUESTION – INTEGRATE YOUR KNOWLEDGE This content was retrieved from Conclusion Slide 2 of 5 of the online learning module Consider your new knowledge regarding hemophilia and thrombophilia, answer the question. Question: How do the disorders of atherosclerosis and channelopathies compare and contrast with respect to each of the considerations of the disease paradigm framework? Feedback: D r. Nicol’s Feedback: Carefully considering the relationship of these diseases to the disease paradigm framework will help prepare you for T B L assessments and exams where you will have to integrate your knowledge and apply it to novel situations. Reference the disease paradigm framework. LEARNING OUTCOMES This content was retrieved from Conclusion Slide 3 of 5 of the online learning module 1. Describe the key pieces of information needed to understand cardiovascular diseases. 2. Compare and contrast the contributing disease paradigm factors for patients predisposed to atherosclerosis versus channelopathies. 3. Identify and explain the general approaches, considerations, and challenges with testing of patients for various types of cardiovascular diseases. 4. Apply your knowledge to a hypothetical cardiovascular disease scenario to describe a potential etiology, mechanism, testing approach, pathological features, and treatment. Reference the disease paradigm framework as you consider these module learning outcomes. ACKNOWLEDGEMENTS This content was retrieved from Conclusion Slide 4 of 5 of the online learning module We would like to thank the following individuals for their expertise in preparing the content for this learning module. Dr. Marosh Manduch Professor Department of Pathology & Molecular Medicine Queen’s University Dr.Chris Nicol Associate Professor

MODULE 02 COMPANION GUIDE PATH 310 INTRODUCTION TO PATHOLOGY AND MOLECULAR MEDICINE | PATH 310 MODULE 02 PAGE 78 Department of Pathology & Molecular Medicine Queen’s University Dr. Harriet Feilotter Professor Department of Pathology & Molecular Medicine, Richardson Laboratory, Queen’s University Director , Molecular Genetics, Kingston Health Sciences Centre Dr. Kristopher Cunningham, M D, P h D, F RCP C Forensic and Cardiovascular Pathologist Director , Kingston Regional Forensic Pathology Unit, Pathology and Molecular Medicine Queen’s University This module was designed and produced in collaboration with the Queen’s University Faculty of Health Sciences Course Development Unit. MODULE 02 COMPLETE! This content was retrieved from Conclusion Slide 5 of 5 of the online learning module SECTION 01: ATHEROSCLEROSIS SECTION 02: CHANNELOPATHIES