Scientifically-Educational Course on Clinical Genetics Clinico-Genetic Aspects of a Cardiovascular Pathology

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Scientifically-Educational manual was developed in the frames of the Federal Targeted Programme Scientific and Scientific Pedagogical Personnel of the Innovation Russia in 2009-2013. Kursk State Medical University

PLAN
INTRODUCTION. URGENCY OF THE PROBLEM
PART I. THE GENETIC ASPECTS OF ISCHEMIC HEART DISEASE

AND ATHEROSCLEROSIS
PART II. THE HYPERTENSION GENETIC ASPECTS OF ESSENTIAL

I. INTRODUCTION. URGENCY OF THE PROBLEM

Until recently, a modest knowledge of genetics has been more than adequate for the day to day practice of clinical medicine and cardiology. However, this situation is rapidly changing. Advances in genetics and genomics over the past two decades, including the sequencing of the human genome, are shaping medicine to a greater extent than any other basic science endeavor. Indeed, the past 20 years of research has witnessed genetics becoming a foundational science. Information on genes, genetics, genetic testing, genomics, pharmacogenetics and related subjects has moved from highly specialized publications to the general medical literature.

Genomics and genetic science is changing the practice of medicine in fundamental ways. In cardiovascular medicine, the genetic basis of several forms of dyslipidemia, hypertension, diabetes, cardiomyopathies and vascular diseases have been identied.

The muscle that pumps blood received from veins into arteries throughout the body. It is positioned in the chest behind the sternum (breastbone; in front of the trachea, esophagus, and aorta; and above the diaphragm muscle that separates the chest and abdominal cavities. The normal heart is about the size of a closed fist, and weighs about 10.5 ounces. It is cone-shaped, with the point of the cone pointing down to the left. Two-thirds of the heart lies in the left side of the chest with the balance in the right chest.

Cardiovascular diseases are, more than in half of cases, the reason of death rate of the population of the country. This is the biggest group of diseases which come first on prevalence, death rates and incapacity in structure of all human somatopathology. Death rate from diseases of an atherosclerotic origin and essential hypertension makes about 85 % in the common death rate of the population from diseases of heart and vessels.
The heart is like any other muscle, requiring blood to supply oxygen and nutrients for it to function. It beats about 100,000 times a day, pumping blood through your circulatory system. The cycle of pumping blood throughout the body carries fresh oxygen to the lungs and nutrients to your body's tissues. Blood also takes waste, such as carbon dioxide, away from the tissues. Without this process, we could not live.

The present stage of perfection of diagnostics, treatment and prophylaxis of cardiovascular diseases is characterized by more and more wide application of methods of molecular diagnostics. In result essentially new opportunities in development of pathogenetic methods of diagnostics and treatment of diseases of heart and the vessels, based on a concrete pathology of genes are created.
In vivo gene transfer is the only option in tissues where the recipient cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells) or where cultured cells cannot be re-implanted efficiently in patients. Tissue targeting is an important consideration. The gene transfer construct may be emplaced directly into the target tissue, or it may be injected into the general circulation but designed in some way so as to be taken up only by the desired cell type. As there is no way of selecting and amplifying cells that have taken up and expressed the foreign gene, the success of this approach is crucially dependent on the general efficiency of gene transfer and expression.

The big variety of the pathological processes proceeding at a molecular level is combined with development of clinical manifestations of heart ischemic disease (HID). The knowledge of hereditary disorders of different stages of lipid metabolism will allow to expand knowledge of clinicians not only of molecular mechanisms of atherosclerosis development, but also about most genetically determined risk factors of disease development. Separation of these factors in each case of disease will permit to develop individual approaches to therapy of cardiovascular disorders and prophylaxis of complications in view of genetic bases of disease development, and also possible response of an organism to realizing medical actions.

PART I. THE GENETIC ASPECTS OF ISCHEMIC HEART DISEASE AND ATHEROSCLEROSIS

1. 2. 3. 4. INTRODUCTION LIPOPROTEIN METABOLISM PATHOGENESIS ETIOLOGY a) Contribution of genetic factors to atherosclerosis b) Contribution of environmental factors to atherosclerosis c) Family and twin studies d) The age of the individuals e) Animal models f) Population migration studies 5. MONOGENIC (MENDELIAN) FORMS a) Hyperlipidemias b) Classification c) Characteristics of hyperlipoproteinemias types 6. CAD AND MYOCARDIAL INFARCTION 7. CELLULAR FACTORS IN LESION INITITATION AND PROGRESSION 8. STROKE 9. METABOLIC SYNDROME AND DIABETES 10. HOMOCYSTEINE LEVELS 11. HEMOSTATIC FACTORS

12. CLINICAL IMPLICATIONS a) New Therapies b) Risk Stratification, Prevention, and Treatment c) Multilocus Testing d) Population Screening e) Pharmacogenetics 13. PREDICTION OF DISEASE PATHOLOGIES OF THE CARDIOVASCULAR SYSTEM IN KURSK. 14. PREDICTION OF CORONARY HEART DISEASE IN KURSK. 15. FORECAST OF THE MORBIDITY OF ACUTE MYOCARDIAL INFARCTION IN KURSK. 16. FORECASTING INCIDENCE OF ANGINA IN KURSK. 17. FORECAST OF CEREBROVASCULAR PATHOLOGY IN KURSK. 18. COMPARISON OF THE PROJECTED INCIDENCE OF DISEASES OF THE CIRCULATORY SYSTEM BETWEEN KURSK CITY AND KURSK REGION AS A WHOLE (PER 1000 PEOPLE) a) Angina b) Myocardial infarction c) Ischemic stroke d) Hemorrhagic stroke 19. RELATIONSHIP BETWEEN INCIDENCE OF CARDIOVASCULAR DISEASE WITH CHEMICAL POLLUTION OF KURSK REGION. 20. CONCLUSIONS

1. INTRODUCTION.
Atherosclerosis, the primary cause of coronary artery disease (CAD) and stroke, is a disorder with multiple genetic and environmental contributions. Genetic-epidemiologic studies have identified a surprisingly long list of genetic and non-genetic risk factors for CAD. However, such studies indicate that family history is the most significant independent risk factor. Many Mendelian disorders associated with atherosclerosis, such as familial hypercholesterolemia (FH), have been characterized, but they explain only a small percentage of disease susceptibility (although a substantial fraction of early CAD). Most cases of myocardial infarction (MI) and stroke result from the interactions of multiple genetic and environmental factors, none of which can cause disease by itself. Successful discovery of these genetic factors will require using complementary approaches with animal models, large-scale human genetic studies, and functional experiments. This review emphasizes the common, complex forms of CAD.

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries. The earliest lesions of atherosclerosis consist of sub-endothelial accumulations of cholesterol-engorged macrophages called foam cells. Such fatty streak lesions are not clinically significant, but they are the precursors of more advanced lesions characterized by the accumulation of lipid-rich necrotic debris and smooth muscle cells (SMCs). Such early atheromas typically have a fibrous cap consisting of SMCs and an extracellular matrix that encloses a lipid-rich necrotic core.
Symptoms of heart disease usually occur during exercise or activity. That's because the heart experiences increased demand for nutrients and oxygen that cannot be met because the coronary arteries are blocked. Other symptoms of heart disease include chest pain (angina), shortness of breath, jaw pain, and back pain, especially on the left side.

Athermanous plaques can become increasingly complex with calcification, ulceration at the luminal surface, and hemorrhage from small vessels that grow into the lesion from the media of the blood vessel wall. Although advanced lesions can grow sufficiently large to block blood flow, the most important clinical complication is an acute occlusion due to the formation of a thrombus or blood clot, resulting in a myocardial infarction (MI) or stroke. Usually, thrombosis is associated with rupture or erosion of a vulnerable plaque that has a thin fibrous cap.

Heart disease begins when cholesterol, fatty material, and calcium build up in the arteries. When this occurs in the arteries that supply the heart, this buildup, or plaque, causes the arteries to narrow, so that oxygen delivery to the heart is reduced. The reduction in oxygen delivery to the heart can create chest pain, also called angina.

2. Lipoprotein Metabolism
The most significant risk factors for CAD and stroke are related to lipoprotein metabolism. Figure 1 presents an overview of lipoprotein metabolism. Lipids are transported through the circulation as complexes with proteins, termed lipoproteins. In general, lipoproteins consist of a core of hydrophobic lipids (triglycerides and cholesterol esters) surrounded by a monolayer of amphipathic lipids (phospho-lipids and free cholesterol) and various apolipoproteins. Apolipoproteins function in the packaging and secretion of lipids from cells, in the processing of circulating lipoproteins, and as ligands for cellular receptors. The plasma apolipoproteins include apo(a), AI, All, AIV, AV, B48, B100, CI, CII, CIII, and E. Lipids can be transferred between lipoprotein particles by enzymes such as cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP).

Figure 1. Lipoprotein metabolism.

When plaque builds up to the point that it ruptures, it causes a blood clot to form in the coronary artery. The blood clot blocks blood from flowing to the heart muscle, leading to a heart attack. In a worst-case scenario, sudden cardiac arrest or fatal rhythm disturbance can occur.

Dietary lipids are packaged in intestinal mucosal cells and secreted into the lymph as large triglyceride-rich particles called chylomicrons. Cholesterol absorption is mediated in part by the ABCG5 and ABCG8 transporters. Upon entering the circulation, lipoprotein lipase (LPL) rapidly lipolyzes the chylomicrons, thereby delivering free fatty acids (FFAs) to tissues such as muscle and adipose. The LDL (apoB/E) receptor and the LDL receptorrelated protein (LRP) mediate the rapid uptake of the resulting remnant particles by liver (Figure 1).

The liver actively synthesizes triglycerides and cholesterol, which, along with intestinally derived lipids, are packaged and secreted as very low-density lipoproteins (VLDLs). Like chylomicrons, VLDL particles are large and triglyceride-rich, but they have a different metabolic fate. VLDLs undergo lipolysis in the circulation and give rise to intermediate-density lipoproteins (IDL). Hepatic receptors remove a significant fraction (about half) of IDLs from the circulation, and the remainder undergo further lipolysis by LPL and hepatic lipase (HL) to produce LDLs. LDLs have a long halflife in the circulation and, in most individuals, are the predominant cholesterol-carrying particles. The LDL (apoB/E) receptor, which is present in both liver and peripheral tissues, largely mediates the removal of LDL from the circulation (Figure 1).

HDLs are formed in the circulation from precursors secreted by liver and intestine, and from chylomicron and VLDL surface remnants. HDLs facilitate the removal of cholesterol from cells, a process mediated in part by ABCA1. The cholesterol is then esterified by lecithin cholesterol acyl transferase (LCAT) and transported to the liver, the only tissue capable of metabolizing and excreting excess cholesterol. The liver's uptake of HDL cholesterol is mediated partially by the scavenger receptor class BI (SR-BI). Hepatocytes can oxidize cholesterol to bile acids by a pathway involving cholesterol 7 -hydroxylase (Figure 1).

Heart attack is a sudden interruption in the heart's blood supply. This happens when there is a blockage in the coronary arteries, the vessels that carry blood to the heart muscle. When blood flow is blocked, heart muscle can be damaged very quickly and die. Prompt emergency treatments have reduced the number of deaths from heart attacks in recent years.

Most common forms of hyperlipidemia or low HDL levels are due to a combination of multiple genetic and environmental factors. Many of the genes for apolipoproteins, lipoprotein receptors, and enzymes in plasma lipoprotein metabolism have been examined as candidate genes to test for possible associations or linkages with levels of plasma lipoproteins and lipids. Most of the observed effects on lipoprotein metabolism are small and require confirmation, although some associations are convincing (Table 1). For example, three common variations of apoE exhibit marked effects on plasma cholesterol levels and contribute to type III hyperlipidemia and, in Western populations, explain 4% or more of total plasma cholesterol levels. Also, in several population studies, polymorphisms of the hepatic lipase gene were associated with HDL levels.

Table 1. Common genetic variations contributing to CAD and its risk factors. Only genes exhibiting evidence of linkage of association in multiple studies (see next page)
Trait Gene Variation Many mutations Three common missense alleles explain ~5% of variance of cholesterol Multiple polymorphisms LDL/VLDL LDL receptor ApoE

ApoAI-CIII-AIV-AV cluster

HDL levels

Hepatic lipase

Promoter polymorphism

Lp(a)

Apo(a)

Many alleles of apo(a) explain >90% variance

Homocysteine

Methylene tetrahydrofolate reductase

Missense polymorphism

Coagulation

Fibrinogen B Plasminogen activatorinhibitor type 1 Factor VIII

Promoter polymorphism Promoter polymorphism Common missense

Table 1 (cont.)
Trait Blood pressure Gene Angiotensinogen Variation Missense and promoter polymorphisms Missense polymorphism

B2-adrenergic receptor

Alpha-adducin

Missense polymorphism; support from studies in rats

CAD

Angiotensin converting enzyme

Insertion-deletion polymorphism

Serum paraoxonase

Missense polymorphism affecting enzymatic activity; animal studies support

Missense polymorphism Regulatory polymorphism

Toll-like receptor 4 Stroke Diabetes, obesity, and insulin resistance Phosphodiesterase 4D PPAR gamma calpain 10

2. Pathogenesis
The first observable event is the accumulation of lipoprotein particles and their aggregates in the intima at sites of lesion predilection. Subsequently, monocytes and lymphocytes adhere to the surface of the endothelium and then transmigrate across the endothelial monolayer into the intima. The monocytes proliferate, differentiate into macrophages, and take up the lipoproteins, forming cholesterol-engorged foam cells. With time, the foam cells die, contributing their lipid-filled contents to a growing necrotic core.

As lesions increase in size, smooth muscle cells migrate to the intima from the medial layer and secrete collagen to form a fibrous cap. Initially, such atheromatous lesions grow out toward the adventitia, but subsequently they encroach on the lumen. The lesions continue to grow by the migration of new mononuclear cells from the blood, which enter at the shoulder of the vessel, accompanied by the accumulation of extracellular lipid and fibrous elements. The factors involved in the development of advanced, complex lesions, frequently involving calcification and neovascularization, are less well understood. Of particular clinical relevance are the factors contributing to the development of vulnerable plaques that can rupture and trigger thrombosis.

Figure 2A,B summarizes some molecular and cellular interactions important in early and late stages of atherosclerosis. The endothelium, with its intercellular tight junctional complexes, acts as a selectively permeable barrier between blood and tissues, and it can regulate thrombosis, inflammation, vascular tone, and vascular remodeling. Among the important physical forces acting on endothelial cells (ECs) is fluid shear stress, which affects EC morphology. Cells in regions of arterial branching or curvature, where flow is disturbed, show increased permeability to macromolecules such as LDL, and are preferential sites for lesion formation.
A primary initiating event in atherosclerosis is LDL accumulation in the subendothelial matrix. Accumulation is greater when levels of circulating LDL are elevated, and both the transport and retention of LDL are increased in the preferred sites for lesion formation. LDL diffuses passively through EC junctions, and its retention in the vessel wall involves interactions between the LDL constituent apolipoprotein B (ApoB) and matrix proteoglycans. In addition to LDL, other ApoB-containing lipoproteins, namely lipoprotein(a) and remnants, can accumulate in the intima and promote atherosclerosis (Figure 2A).

Figure 2A

The trapped LDL undergoes modification, including oxidation, lipolysis, proteolysis, and aggregation. Enzymes involved in such modification include 12, 15-lipoxygenase (15-LO), inducible nitric oxide synthase (INOS), myeloperoxidase (MPO), and various lipases. One of the modifications most significant for early lesion formation is lipid oxidation, resulting from exposure to the oxidative waste of vascular cells. Such modifications initially give rise to minimally oxidized LDL (mmLDL) species that have pro-inflammatory activity but may not be sufficiently modified to be recognized by macrophage scavenger receptors. Mice lacking 15-LO have considerably diminished atherosclerosis, which suggests that this enzyme may be an important source of reactive oxygen species in LDL oxidation. High-density lipoprotein (HDL) protects against atherosclerosis. An important mechanism underlying this protective effect is HDL's role in removing excess cholesterol from peripheral tissues, but HDL also protects by inhibiting lipoprotein oxidation. The antioxidant properties of HDL are due in part to serum paraoxonase (PON1), an esterase carried on HDL that can degrade certain biologically active oxidized phospholipids. Platelet activating factor acetyl hydrolase (PAF-AH) and apolipoprotein A-I (Apo-Al) also have antioxidant properties (Figure 2A).

Atherosclerosis is characterized by its recruitment of monocytes and lymphocytes to the artery wall. A triggering event for this process is the accumulation of minimally oxidized LDL, which stimulates the overlying ECs to produce numerous proinflammatory molecules, including adhesion molecules and growth factors such as the macrophage colony-stimulating factor (M-CSF). The biological activity of minimally oxidized LDL occurs primarily in its phospholipid fraction, and scientists have identified active oxidation products resulting from the scission or rearrangement of unsaturated fatty acids. In addition to oxidized LDL, many other factors likely modulate inflammation, including homocysteine levels, sex hormones, and infection. Diabetes may promote inflammation in part by forming advanced glycation endproducts (AGEs) that interact with endothelial receptors. Adhesion molecules and chemotactic factors mediate the entry of particular types of leukocytes into the artery wall. The firm adhesion of monocytes and T cells to endothelium can be mediated by the integrin VLA-4 on these cells, which interacts with VCAM-1 on the endothelium and the CS-1 splice variant of fibronectin. Other molecules important in adhesion and migration include E-selectin and P-selectin (monocytes) and CXCR3, IP-10, I-TAC, and Mig (lymphocytes). Mice deficient in monocyte chemotactic protein (MCP-1) or its receptor CCR2 have significantly reduced atherosclerotic lesions, which suggests that MCP-1/CCR2 interaction has a role in monocyte recruitment in atherosclerosis. A variety of chemokines induced by interferon gamma (IFN ) may contribute to lymphocyte recruitment (Figure 2A).

A blood vessel (top) with ruptured atherosclerotic plaque, shown in yellow, is developing a blood clot.

The cytokine M-CSF stimulates the proliferation, differentiation, and survival of macrophages and influences various macrophage functions such as expression of scavenger receptors. Mice with a spontaneous null mutation of M-CSF have dramatically reduced lesions, which suggests an obligatory role for macrophages in lesion formation. LDL must be extensively modified before it can be taken up sufficiently rapidly by macrophages to form foam cells. A group of receptors that recognize a wide array of ligands mediates the rapid uptake of highly oxidized (and otherwise modified) LDL particles by macrophages, which leads to foam-cell formation. Two such scavenger receptors, SR-A and CD36, are of primary importance, and mice lacking either receptor show a modest reduction in atherosclerotic lesions. Secretion of cholesterol by macrophages, mediated by ABCA-1 and apolipoproteins such as apoE, may protect against atherosclerosis. Mice deficient in acyl-cholesterol acyl transferase (ACAT) cannot store cholesterol and have reduced lesions. The cholesterol-loaded macrophages undergo necrosis or apoptosis, donating their contents to a growing lipid-rich necrotic core. FAS and p53 partially mediate apoptosis (Figure 2A).

Fibrous plaques are characterized by a growing mass of extracellular lipid, mostly cholesterol and its ester, and by the accumulation of SMCs and SMC-derived extracellular matrix. Cytokines and growth factors secreted by macrophages and T cells are important for SMC migration and proliferation and extracellular matrix production. Recent studies show that the interaction of CD40 with its ligand CD40L (CD 154) makes an important contribution to the development of advanced lesions. The engagement of CD40 and CD40L results in the production of inflammatory cytokines, matrix-degrading proteases, and adhesion molecules. Although studies with immunodeficient mice originally indicated that lymphocytes were not required for atherogenesis, it is now clear that lymphocytes play a modulating role. For example, antibodies to oxidized LDL (oxLDL) epitopes and heat shock protein (HSP) influence disease progression. Also, studies of Tlymphocyte products, such as IFN-x, IL-4, IL-10, and IL-13, are consistent with an important influence in lesion development. Several risk factors contribute to the development of fibrous lesions, including elevated homocysteine, hypertension, and hormones. Elevated homocysteine injures ECs and stimulates proliferation of vascular SMC. Some effects of high blood pressure on atherosclerosis are mediated by components of the renin-angiotensin pathway. For example, angiotensin II directly stimulates SMC growth and extracellular matrix production. Studies with spontaneously hypertensive rats (SHRs) indicate that raised blood pressure stimulates expression of plateletderived growth factor (PDGF), a potent mitogen for SMCs. Estrogen has multiple anti-atherogenic properties, including effects on plasma lipoprotein levels and stimulation of prostacyclin and nitric oxide (NO) production (Figure 2B).

Figure 2B

Pathological studies suggest that the development of thrombus-mediated acute coronary events depends principally on the composition and vulnerability of a plaque rather than the severity of stenosis. Vulnerable plaques generally have thin fibrous caps and increased numbers of inflammatory cells. Maintenance of the fibrous cap reflects matrix production and degradation, and products of inflammatory cells likely influence both processes. For example, T cells produce INF- , which inhibits the production of matrix by SMCs, and macrophages produce various proteases, particularly matrix metalloproteinases (MMPs), that degrade extracellular matrix. Rupture frequently occurs at the lesion edges, which are rich in foam cells, which suggests that factors contributing to inflammation may also influence thrombosis. The stability of atherosclerotic lesions may also be influenced by calcification and neovascularization, common features of advanced lesions. Intimal calcification is an active process in which pericyte-like cells secrete a matrix scaffold, which becomes calcified, akin to bone formation. Oxysterols and cytokines regulate the process. The thrombogenicity of the lesion core likely depends on the presence of a tissue factor, a key protein in the initiation of the coagulation cascade. Production of the tissue factor by ECs and macrophages is enhanced by oxidized LDL, infection, or ligation of CD40 on ECs to CD40L on inflammatory cells (Figure 2B).

3. Etiology
In the past 50 years, epidemiological studies have revealed numerous risk factors for atherosclerosis (Table 2, Figure 3). These can be grouped into factors with an important genetic component and those that are largely environmental. The relative abundance of the different plasma lipoproteins is particularly important because raised levels of atherogenic lipoproteins are a prerequisite for most forms of the disease.

TABLE 2 Genetic and environmental risk factorsfor CVD

Risk factors with a significant genetic component (heritability)

Myocardial infarction (25% to 60%) Stroke Total cholesterol (40% to 60%) HDL-cholesterol (45% to 75%) Total triglycerides (40% to 80%) Body mass index (25% to 60%) Systolic blood pressure (50% to 70%) Diastolic blood pressure (50% to 65%) Lp(a) levels (90%) Homocysteine levels (45%) Type 2 diabetes (40% to 80%) Fibrinogen (20% to 50%) C-reactive protein Gender Age Environmental risk factors Smoking Diet Exercise Infection Fetal environment Air pollution (particulates)

Figure 3. Environmental risk factors for CVD

a) Contribution of genetic factors to atherosclerosis Atherosclerosis is probably one of the most complex diseases. Although we know only a small fraction of the genes involved in CAD, we can estimate a lower limit of the total number of genes by considering the genetics of the known genetic risk factors for CAD (Table 2, 3). With the exception of Lp(a) levels and gender, all involve multiple genetic factors and some, such as blood pressure, HDL levels, and total cholesterol levels, exhibit smooth population distributions characteristic of polygenic traits. Large genome scans for various risk factors, including diabetes, obesity, and hypertension, have failed to reveal loci accounting for a large fraction of the population variation, suggesting the involvement of many genes (Figure 4).

At least hundreds of genes are involved in susceptibility to CAD. Supporting this conclusion are genetic studies of traits such as diabetes, body fat, plasma lipoprotein levels, blood pressure, and atherosclerotic lesions in mice and rats, which together suggest that at least dozens of loci contribute to each of these traits when considering various crosses. Another level of complexity involves the interactions between risk factors. Frequently, these are not simply additive; for example, the effects of hypertension are considerably amplified if cholesterol levels are high.

Figure 4. Gene network example of predisposition to atherosclerosis

In attempting to understand the genetics of atherosclerosis, the investigator can include comparing the prevalence and incidence in various different population groups, the effects of migration, studying the incidence of the disease among relatives in family studies, comparing the incidence in identical and non-identical twins, determining the effect of environmental changes by adoption studies and studying the association of the disease with various other characteristics, such as the blood groups and biochemical or DNA polymorphisms.

Study can be made of the pathological components or biochemical factors of the disease in relatives, e.g. serum lipids among the relatives of patients with coronary artery disease. Study of diseases in animals which are homologous to diseases which occur in humans can also be helpful.

b) Contribution of environmental factors to atherosclerosis (see next

page)

Some of the common risk factors for heart disease include smoking, high blood pressure (hypertension), high cholesterol, diabetes, family history of heart disease, peripheral artery disease, and obesity.

Lifestyle risk factors that contribute to heart disease include lack of exercise, high-fat diet, emotional stress, and having a "type A" personality (aggressive, impatient, competitive).

c) Family and twin studies

Many family and twin studies have examined the importance of genetics and environment in human CAD. Within a population, the heritability of atherosclerosis (the fraction of disease explained by genetics) has been high in most studies, frequently exceeding 50%.

Table 2 lists heritability estimates and various risk factors for MI. Because the assumptions required for estimating heritability are not entirely correct, the calculated heritabilities must be considered approximate. In some cases, modeling the mode of inheritance has produced better estimates, but important assumptions remain.

Heritability also depends on the age of the individuals under study (heritability is much higher for early MI), the ethnic groups studied, and the state of the environment. Another measure of the importance of genetics is the increased risk to a relative of an affected individual compared to the general population. Because CAD is so common after 55 years of age, such calculations are usually presented for premature CAD. For example, one study observed that the relative risk of CAD for women under 65 years and for men under 55 years was five- to sevenfold increased if they had a first-degree relative with premature CAD.

Animal models of atherosclerosis also provide dramatic evidence for the importance of genetic contributions. For example, inbred strains of mice, each with a unique genetic background, differ widely in the development of atherosclerosis.

Population migration studies, on the other hand, show that the environment explains much of the variation in disease incidence between populations. For example, Japanese have a much lower incidence of CAD than Americans, but JapaneseAmericans who have adopted a Western lifestyle have about the same incidence as other Americans. Also, the Seven Countries Study, which examined plasma lipid levels and CAD in seven different populations, revealed striking differences that are not likely due to genetic differences.
A dramatic example of a stationary population that has undergone an environment change are the Pima Indians of Arizona. Prior to adapting a Western lifestyle, these Indians had a modest incidence of obesity and diabetes, but now obesity and diabetes are rampant. From these and other studies, it appears that the difference in CAD incidence between populations is due primarily to environmental factors. However, this is not to say that populations do not differ in genetic susceptibility.

Table 3. Common DNA sequence variations contributing to coronary heart disease (HD) and its risk factors. After Lusis et al.,2004. Only genes showing evidence of linkage or association in multiple studies are cited (see next page).

Table 3 (cont.)

4. Monogenic (Mendelian) Forms

Identification of the genes underlying monogenic disorders that cause atherosclerosis or affect risk factors for the disease has provided important insights into the fundamental biology (Tables 4, 5). In addition to enhancing our understanding of the biology of disease, the study of monogenic disorders can lead to successful drug therapies for common disease. For example, studies of FH have helped to unravel the molecular pathways that regulate cholesterol metabolism, knowledge which was important for developing cholesterol-lowering drugs. A recent study of a single large family with dominant inheritance of CAD and MI provides evidence for the involvement of MEF2A, a member of the myocyte enhancing factor-2 family. A rare mutation of the gene segregated with the disease in the family and MEF2A is strongly expressed in vascular endothelial cells and smooth muscle cells. The gene is not involved in several other families with familial CAD/MI.

In this image, the bulge seen on the right side is an enlarged left ventricle, the heart's main pumping chamber.

Table 4 Mendelian disorders relevant to atherosclerosis

Trait Disease Mutated gene Phenotype/mechanism

Elevated LDL

Familial hypercholesterolemia

LDLR

Decrease in LDL uptake by the liver due to a binding defect of LDLR

Familial ligand defective apoB-100

APOB

Reduced binding of apoB to LDLR slows the clearance of plasma LDL.

Sitosterolemia

ABCG5, ABCG8

Homozygous mutations in either gene results in increased cholesterol absorption by the intestine.

Autosomal recessive hypercholesterolemia

ARH

Decreased internal ization of LDLR in hepatocytes.

Table 4(cont.) Mendelian disorders relevant to

atherosclerosis
Mutated gene APOA1

Trait LowHDL

Disease

Phenotype/mechanism Homozygous null mutations result in the virtual absence of HDL and early CHD.

ApoAI deficiency

Tangier disease

ABCA1 transporter

This recessive disorder results in the inability of cells to export cholesterol and phospholipids, resulting in very low levels of HDL.

LCAT deficiency

Lecithin cholesterol acyltransferase

Patients with LCAT deficiency exhibit markedly low levels of HDL cholesterol, although there is no apparent increased risk of premature CVD.

Elevated homocysteine

Homocystinuria

Cystathionine -synthase

Inability to convert homocysteine to cystathionine results in very high levels of homocysteine and severe occlusive vascular disease.

Coronary artery disease

ad CAD1

MEF2A

A mutation in this transcription factor results in dominant familial vascular disease.

Table 5 Monogenic disorders of LDL-cholesterol (LDL-C metabolism).

a) Hyperlipidemias
Hyperlipidemia (hyperlipoproteinemia, or hyperlipidaemia) is the condition of abnormally elevated levels of any or all lipids and/or lipoproteins in the blood. It is the most common form of dyslipidemia (which also includes any decreased lipid levels). Lipids (fat-soluble molecules) are transported in a protein capsule, and the size of that capsule, or lipoprotein, determines its density. The lipoprotein density and type of apolipoproteins it contains determines the fate of the particle and its influence on metabolism. Hyperlipidemias are divided in primary and secondary subtypes. Primary hyperlipidemia is usually due to genetic causes (such as a mutation in a receptor protein), while secondary hyperlipidemia arises due to other underlying causes such as diabetes. Lipid and lipoprotein abnormalities are common in the general population, and are regarded as a modifiable risk factor for cardiovascular disease due to their influence on atherosclerosis. In addition, some forms may predispose to acute pancreatitis.

b) Classification
Hyperlipidemias may basically be classified as either familial (also called primary) caused by specific genetic abnormalities, or acquired (also called secondary) when resulting from another underlying disorder that leads to alterations in plasma lipid and lipoprotein metabolism. Also, hyperlipidemia may be idiopathic, that is, without known cause. Hyperlipidemias may also be classified directly into which types of lipids are elevated, that is hypercholesterolemia, hypertriglyceridemia or both in combined hyperlipidemia. Elevated levels of Lipoprotein may also be classified as a form of hyperlipidemia.
Familial hypercholesterolemia Xanthomatosis at area of ulnar joints

Familial (primary) Familial hyperlipidemias are classified according to the Fredrickson classification (Table 6, Figure 5) which is based on the pattern of lipoproteins on electrophoresis or ultracentrifugation. It was later adopted by the World Health Organization (WHO). It does not directly account for HDL, and it does not distinguish among the different genes that may be partially responsible for some of these conditions. It remains a popular system of classification, but is considered dated by many.
Familial hypercholesterolemia Tuberous xanthomas at extensor areas of knee joins

Table 6. Fredrickson classification of hyperlipidemias (see next page)

Hyperlipo- OMIM Synonyms Defect Increased Main symptoms proteinemia lipoprotein Decreased Chylomicrons Abdominal pain (from Type I a 238600 Buerger-Gruetz syndrome, or Familial lipoprotein pancreatitis), lipemia hyperchylomicronemia lipase retinalis, eruptive skin (LPL) xanthomas, hepatosplenomegaly b 207750 Familial apoprotein CII Altered deficiency ApoC2 LPL c 118830 inhibitor in blood LDL LDL Xanthelasma, arcus Type II a 143890 Familial hypercholesterolemia receptor senilis, tendon deficiency xanthomas b 144250 Familial combined hyperlipidemia Decreased LDL receptor and increased ApoB LDL and VLDL Treatment Diet control Serum Estimated appearance prevalence Creamy top 1 in layer 1,000,000

Bile acid Clear sequestrants, statins, niacin Statins, Clear niacin, fibrate

1 in 500 for heterozygotes

1 in 10

Table 6 (cont.)

Hyperlipo- OMIM Synonyms Defect Increased proteinemia lipoprotein Defect in Apo E IDL Type III 107741 Familial dysbetalipoproteinemia 2 synthesis

Type IV

144600 Familial hypertriglyceridemia

Type V

144650

Increased VLDL VLDL production and Decreased elimination Increased VLDL and VLDL Chylomicrons production and Decreased LPL

Main Treatment Serum Estimated symptoms appearance prevalence Tubo-Eruptive Fibrate, Turbid 1 in 10,000 Xanthomas & statins Palmar Xanthomas Fibrate, Turbid 1 in 100 niacin, statins

Niacin, fibrate

Creamy top layer & turbid bottom

Figure 5. Relative prevalence of familial forms of hyperlipoproteinemia

HYPERLIPOPROTEINEMIA, TYPE I (LIPOPROTEIN LIPASE DEFICIENCY).
HYPERCHOLESTEROLEMIA,

TYPES IIA AND IIB.

HYPERLIPOPROTEINEMIA, TYPE III. HYPERTRIGLYCERIDEMIA,

TYPE IV.
HYPERLIPOPROTEINEMIA, TYPE V.

c) Characteristics of hyperlipoproteinemias types

Hyperlipoproteinemia type I
Type I hyperlipoproteinemia exists in several forms: Lipoprotein lipase deficiency (Type Ia), due to a deficiency of lipoprotein lipase (LPL) or altered apolipoprotein C2, resulting in elevated chylomicrons, the particles that transfer fatty acids from the digestive tract to the liver. Familial apoprotein CII deficiency (Type Ib), a condition caused by a lack of lipoprotein lipase activator. Chylomicronemia due to circulating inhibitor of lipoprotein lipase (Type Ic) Type I hyperlipoproteinemia usually presents in childhood with eruptive xanthomata and abdominal colic. Complications include retinal vein occlusion, acute pancreatitis, steatosis and organomegaly, and lipaemia retinalis.

Hyperlipoproteinemia type II
Hyperlipoproteinemia type II, by far the most common form, is further classified into type IIa and type IIb, depending mainly on whether there is elevation in the triglyceride level in addition to LDL cholesterol. Type IIa: Familial hypercholesterolemia. This may be sporadic (due to dietary factors), polygenic, or truly familial as a result of a mutation either in the LDL receptor gene on chromosome 19 (0.2% of the population) or the ApoB gene (0.2%). The familial form is characterized by tendon xanthoma, xanthelasma and premature cardiovascular disease. The incidence of this disease is about 1 in 500 for heterozygotes, and 1 in 1,000,000 for homozygotes.

Type IIb The high VLDL levels are due to overproduction of substrates, including triglycerides, acetyl CoA, and an increase in B-100 synthesis. They may also be caused by the decreased clearance of LDL. Prevalence in the population is 10%. Familial combined hyperlipoproteinemia (FCH) Lysosomal acid lipase deficiency, often called (Cholesteryl ester storage disease) Secondary combined hyperlipoproteinemia (usually in the context of metabolic syndrome, for which it is a diagnostic criterion)

Familial hypercholesterolemia Xanthomas at extensor areas of hands and fingers

Hyperlipoproteinemia type III

This form is due to high chylomicrons and IDL (intermediate density lipoprotein). Also known as broad beta disease or dysbetalipoproteinemia, the most common cause for this form is the presence of ApoE E2/E2 genotype. It is due to cholesterol-rich VLDL (-VLDL). Its prevalence has been estimated to be approximately 1 in 10,000.

Familial hypertriglyceridemia is an autosomal dominant condition occurring in approximately 1% of the population.

Hyperlipoproteinemia type V
Hyperlipoproteinemia type V is very similar to type I, but with high VLDL in addition to chylomicrons. It is also associated with glucose intolerance and hyperuricemia

Non-classified forms are extremely rare: Hyperalphalipoproteinemia Polygenic hypercholesterolemia

Acquired (secondary)
Acquired hyperlipidemias (also called secondary dyslipoproteinemias) may mimic primary forms of hyperlipidemia and can have similar consequences. They may result in increased risk of premature atherosclerosis or, when associated with marked hypertriglyceridemia, may lead to pancreatitis and other complications of the chylomicronemia syndrome. The most common causes of acquired hyperlipidemia are:
diabetes mellitus Use of drugs such as diuretics, beta blockers, and estrogens Other conditions leading to acquired hyperlipidemia include: Hypothyroidism Renal failure Nephrotic syndrome Alcohol Some rare endocrine disorders and metabolic disorders Treatment of the underlying condition, when possible, or discontinuation of the offending drugs usually leads to an improvement in the hyperlipidemia. Specific lipidlowering therapy may be required in certain circumstances. Another acquired cause of hyperlipidemia, although not always included in this category, is postprandial hyperlipidemia, a normal increase following ingestion of food.

6. CAD and Myocardial Infarction

Whole-genome scans of families enriched for CAD have revealed several loci linked to CAD or MI (Table 7), but so far none of the underlying genes have been identified. Although the lod scores are significant, no replication of loci has been observed in the various studies. This could be due in part to the different populations tested and to the design of the studies. It is also possible, even likely, that measures of CAD, such as quantitative angiography, will give different results than MI because MI usually results from lesion rupture and is a function of the composition of the lesion as much as its size.

Table 7 Linkage studies of CAD or MI in human

populations
Trait Chromosome Lod score Population (number) Analytic method

MI

14qter

3.9

German (1406)

Variance component (SOLAR)

MI

lp34-36

11

United States (1163)

Allele sharing (SAGE)

CAD

2q21-22, xq23-26

3.2, 3.5

Finnish (364)

Allele sharing (MAPMAKER/SIBS)

CAD

16pl3-pter

North-Eastern Indian (535)

Allele sharing (MAPMAKER/SIBS)

Stroke

5q

4.4

Iceland (1772)

Allele sharing

7. Cellular Factors in Lesion Inititation and Progression

Because atherosclerosis involves an interaction of arterial cells with blood elements, genetic factors acting at the level of the artery wall or blood cells are likely to play an important role in genetic control of susceptibility to CAD and stroke. Evidence for the existence of such factors is based in part on the finding that the known risk factors do not fully account for the familial clustering of CAD. A study bearing on this point showed that intimal thickening of coronary arteries in infants who had died prior to 1 year of age was significantly associated with a family history of CAD.

The plaque deposited in the arteries is hard on the outside and soft and mushy on the inside. Sometimes the hard outer shell cracks. When this happens, a blood clot forms around the plaque. If the clot completely blocks the artery, it cuts off the blood supply to a portion of the heart. Without immediate treatment, that part of the heart muscle could be damaged or destroyed.

Recently, mutations of the MEF2A gene were shown to contribute to a rare, dominant form of familial CAD/MI. Many candidate genes for processes such as cellular growth, inflammation, and oxidative stress have been identified, but the genetic complexity of atherosclerosis makes it difficult to test the role of these genes directly. The difficulty of quantitating lesions in humans presents a problem for human genetic studies, and the endpoints most often used, such as MI, are relatively crude. For these reasons, studies in animal models are particularly informative. Recent animal studies provide conclusive evidence that genetic variations in vascular cell functions can dramatically influence susceptibility to atherosclerosis. Vascular permeability is increased at anatomical sites of atherosclerosis predilection, and the composition of the intimal matrix can influence LDL binding and retention. These processes suggest various candidate genes that could be examined in genetic studies, such as matrix proteins, but no definitive reports have been published.

There is abundant evidence that oxidation within the vessel wall is central to at least the early stages of atherogenesis, and molecular studies and studies with transgenic mice have suggested various processes and candidate proteins that could contribute to lipoprotein oxidation. These include the levels of metal ions, thiols, superoxide, lipoxygenase, glucose, nitric oxide, myeloperoxidase, antioxidants, and HDL-associated enzymes protecting against oxidation. Polymorphisms of some genes likely to influence oxidative stress or lipoprotein oxidation, including serum paraoxynase, nitric oxide synthase, and the hemachromatosis associated gene, are associated with CAD in population studies. The evidence that serum paraoxonase (PON1), a protein carried on HDL that inhibits the accumulation of oxidized lipids, plays an important role in genetic susceptibility to atherosclerosis is particularly convincing. Stimulated by observations that PON1 could inhibit the oxidation of LDL in vitro, many association studies of PON1 gene polymorphisms have been conducted.

Regular electrical impulses cause your heart to beat. But sometimes those impulses become erratic. The heart may race, slow down, or quiver. Arrhythmias are often harmless variations in rhythm that pass quickly. But some types make your heart less effective at pumping blood, and that can take a serious toll on the body. Let your doctor know if you've noticed your heart beating abnormally.

Two common missense polymorphisms, a leucine to methonine substitution at amino acid 55 and a glutamine to arginine substitution at amino acid 192, have been extensively studied. Currently, more than 50 PON1 associations studies have been conducted with many different populations, including many European groups, Japanese, Chinese, Asian Indians, and American Indians. As expected for a trait as complex as CAD, results differ between studies. However, taken together, the data are impressive because more than half of the studies yielded significant results, and studies involving the glutamine to arginine polymorphism, which affects the activity of the enzyme, consistently show that the arginine allele is associated with increased risk. Supporting the human data are studies in PON1 knockout and transgenic mice, which conclusively show that the enzyme can inhibit the development of atherosclerotic lesions. In response to lipoprotein oxidation or other influences, monocytes are recruited to the artery wall. This involves the expression of adhesion molecules, chemo-tactic proteins, and growth factors for monocyte-macrophages. Among the many potential candidate genes that could contribute to vascular inflammation, several have been examined in human association studies. Results with some, including TGF- 1, interleukin-1 receptor antagonist, and 5-lipoxygenase, are at least suggestive. A key process in foam-cell formation is the uptake of oxidized lipoproteins, mediated by cell surface receptors, which recognize oxidatively modified LDL. Null mutations of CD36, an important receptor for uptake of oxidized lipoproteins, commonly occur in the Japanese population, and these may influence CAD.

Factors influencing vascular smooth muscle cell growth and death are likely contributing factors in atherosclerosis. Polymorphisms of angiotensin converting enzyme (ACE), which converts angiotensin I to angiotensin II and also degrades the vasodilator bradykinin, have been associated with plasma levels of ACE and CAD in several populations. Angiotensin II promotes smooth muscle cell proliferation, whereas bradykinin is a growth inhibitor. One possible explanation for the proliferation of smooth muscle cells in atherogenesis is a nonmalignant transformation of the cells, resulting from mutations of genes involved in growth control. Benditt & Benditt provided evidence for monoclonal proliferation, but recent studies indicate that this results from normal development. Arterial calcification commonly occurs in advanced human atherosclerotic lesions, and such calcification may contribute to lesion rupture. Cell biology studies indicate that arterial calcification is an active process akin to bone formation, which suggests potential candidate genes. Studies with mice reveal the existence of distinct genetic factors contributing to arterial calcification and to myocardial cell calcification.
Coronary (balloon) angioplasty: A thin catheter is inserted into the blocked artery with a tiny balloon on the end. When the balloon is in the spot of the blockage, it is expanded to keep the artery open so blood can flow more freely, and the catheter is removed.

8. Stroke
Stroke has two main clinical phenotypes: ischemic stroke, responsible for 80% to 90% of events, and hemorrhagic stroke, responsible for the remainder. Ischemic stroke can be subdivided into forms due either to atherosclerosis of vessels in the brain or to a clot that travels from a diseased heart to occlude a cerebral artery. Atherosclerosis and hypertension are common denominators of all of these forms. Stroke, like MI, has a strong genetic component, as evidenced by family and twin studies and Mendelian forms.

Stroke is a medical emergency and the third leading cause of death in the U.S. It occurs when a blood vessel in the brain bursts or, more commonly, when a blockage develops. Without treatment, cells in the brain quickly begin to die. The result can be serious disability or death. If a loved one is having stroke symptoms, seek emergency medical attention without delay.

Recently, the gene encoding phosphodiesterase 4D (PDE4D) was implicated in ischemic stroke. Gretarsdottir et al. mapped a stroke susceptibility locus to chromosome 5q21 using linkage analysis of pedigrees in Iceland, a genetic isolate. They then saturated the region with microsatellite and SNP markers to test for association. The strongest association was with PDE4D, and the authors provided some evidence that the associated polymorphisms exhibit altered regulation of PDE4D splice isoforms. Although quite convincing, it will be important to carry out functional studies of the gene in rodent models of stroke.

Signs of a stroke may include: Sudden numbness or weakness of the body, especially on one side. Sudden vision changes in one or both eyes, or difficulty swallowing. Sudden, severe headache with unknown cause. Sudden problems with dizziness, walking, or balance. Sudden confusion, difficulty speaking or understanding others.

The most common type of stroke is known as an ischemic stroke. Nearly nine out of 10 strokes fall into this category. The culprit is a blood clot that obstructs a blood vessel inside the brain. The clot may develop on the spot or travel through the blood from elsewhere in the body.

Hemorrhagic strokes are less common but far more likely to be fatal. They occur when a weakened blood vessel in the brain bursts. The result is bleeding inside the brain that can be difficult to stop.

9. Metabolic Syndrome and Diabetes

Metabolic syndrome, also called the insulin resistance syndrome, is characterized by hyperinsulinemia, obesity, decreased HDL cholesterol levels, small dense LDL particles, hypertriglyceridemia, and hypertension, all of which contribute to an increased risk of heart disease. The strong clustering of insulin resistance, hyperlipidemia, and hypertension in the metabolic syndrome suggests that there are common genetic factors for these traits, but genes that are specific to each component most likely exist as well. A subset of insulinresistant individuals develop noninsulin-dependent diabetes mellitus (NIDDM), characterized by insufficient insulin secretory capacity to maintain normal glucose levels.

The five risk factors that define the metabolic syndrome are the products of some combination of aging, genetic predisposition, poor diet, and physical inactivity. The more of these risk factors an individual has, the greater are his or her risks for diabetes and CVD.

The metabolic symdrome is linked to heart disease as well as Diabetes.

In accordance with the definition of metabolic syndrome provided by World Health Organization (WHO) a person must have high level of insulin, fasting or post meal along with 2 other medical conditions from the following list: Obesity when the ration between waist and hip is more than 0.9, the BMI or Body Mass Index is 30kg/m2 or more or the waist circumference is more than 37in. Cholesterol Status when the Triglyceride level is 150mg/dl or more or the HDL level is less than 35mg/dl. Blood Pressure when the person is undergoing treatment for hypertension or he has been measured with a systolic blood pressure of 140mm Hg or more or a diastolic blood pressure of 90mm Hg or above.

Hyperglycemia causes increased nonenzymatic glycation of proteins, resulting in the production of advanced glycation end products (AGEs) and the formation of reactive oxygen species. Such AGEs may damage the artery wall, perhaps contributing to the accelerated atherosclerosis observed in diabetics. Among candidate genes that have shown significant associations to NIDDM or glucose and insulin levels are the insulin and insulin receptor genes, the glycogen synthase gene, the glucokinase gene, the b3-adrenergic receptor gene, and the PPARgamma gene. A genome scan in a MexicanAmerican population with a high rate of diabetes identified a major NIDDM susceptibility locus, designated NIDDM 1, on chromosome 2q. Using linkage disequilibrium mapping, scientists showed that an intronic polymorphism in the calpain-10 gene was strongly associated with the disease. A second locus for diabetes was identified on chromosome 1q in the Pima Indians of Arizona, in three independent sets of Caucasian pedigrees from Utah, France, and the United Kingdom, and in an Amish study sample.

It is interesting to note that the chromosome 1 locus was originally linked to the FCH phenotype in a set of Finnish families, and FCHL overlaps with diabetes. Obesity also exhibits considerable metabolic overlap with the abnormalities present in diabetes and is associated with hypertension. A variety of candidate genes have shown associations with various measures of obesity in human populations. These include the sodium-potassium-ATPase genes on chromosome 1, the alpha-2 adrenergic receptor on chromosome 10, the betahydroxysteroid dehydrogenase gene on chromosome 1, and the brown adipose tissue-uncoupling protein gene on chromosome 4. Genetic studies in mutant mouse strains have led to the identification of several genes contributing to obesity.

10. Homocysteine Levels

Total plasma homocysteine is an independent and significant risk factor for CAD. A common polymorphism in the methylene tetrahydrofolate reductase (MTHFR) gene is the underlying cause of moderate hyperhomocysteinemia in about half the cases. The most important known factor in determining whether the presence of the polymorphisms will result in elevated homocysteine levels is the intake and availability of folic acid. When plasma levels of folate are above 4 uM, homocysteine levels in individuals with the polymorphism are no different, on average, than those of unaffected individuals. At lower concentrations of folate, the likelihood of having high-basal homocysteine increases in all persons, but much more in individuals with the polymorphism.

11. Hemostatic Factors

Thrombosis triggered by atherosclerotic plaque rupture is the proximate cause of most morbidity and mortality associated with atherosclerotic vascular disease. Nonocclusive thrombosis with organization of the thrombus also contributes to plaque enlargement. Among studied blood hemostatic factors, plasminogen activator inhibitor type 1 (PAI-1) and Factor VII (FVII) show significant independent associations with arterial vascular disease, and there is strong evidence that common polymorphisms in each gene contribute moderately to the overall interindividual variability in their levels. A common theme, shared with the thermo-labile mutation of MTHFR affecting homocysteine levels, is that these polymorphisms influence the response to environmental factors (fibrinogen and smoking, PAI-1 and triglycerides, MTHFR and folate).

12. Clinical Implications

Knowledge of generic factors in the etiology of coronary heart disease has not so far been adequately utilized in attempts to combat premature CHD. The time has now come to utilize genetic information in a setting of family-oriented preventive medicine. This approach would greatly improve the efficiency of preventive efforts, utilizing predictive genetic testing and targeting counseling on those who need it most.

The optimistic predictions of the impact of genetics on the diagnosis and prevention of coronary heart disease (CHD) have not been realized, and whether DNA testing will be incorporated into clinical cardiology practice in the next decades is unclear. Most of the genetic variations identified thus far either are rare or influence traditional risk factors such as low HDL, elevated LDL, high blood pressure, and diabetes. Studies in humans and mice have begun to reveal highly penetrant genetic factors that contribute to CHD susceptibility independent of traditional risk factors. As technical advances bring down genotyping costs, we may pass a threshold for the cost-benefit ratio in the near future. One important application in diagnosis is likely to involve the identification of different forms of CHD to allow individualized treatment (pharmacogenetics). The application of multilocus genotyping to assess risk or to screen populations for individuals who are highly susceptible to CHD also is a possibility. Perhaps most important, genetic studies are leading to many insights into disease mechanisms, and these studies, along with biochemical approaches, are pointing to new therapeutic concepts and treatments.

a) New Therapies
Probably the most significant impact of genetic studies on medicine pertains to the development of therapies. The outstanding example is the impetus that genetic studies of familial hypercholesterolemia (FH) provided in the development of hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitors (statins). Several therapeutic approaches based on genetic studies are under development. The identification of a naturally occurring variant of apolipoprotein AI, known as apoAI Milano, in a village in northern Italy in 1980 led to investigations of the therapeutic potential of HDL and this protein variant as antiatherogenic therapy. Carriers of the apoAI Milano gene are characterized by extremely low levels of HDL cholesterol (10 to 30 mg/dL) and a decreased risk of atherosclerosis relative to the low level of HDL. The apoAI Milano protein differs from native apoAI in that a cysteine is substituted for arginine at position 173. This cysteine confers different properties to this protein as compared with normal apoAI, including the ability to form disulfide-bonded dimers with other apoAI Milano molecules and other HDL proteins such as apoAII.

Recombinant apoAI Milano has been formulated in a complex with phospholipids to mimic the properties of nascent HDL (ETC-216, Pfizer). Studies in mice and rabbits with experimental atherosclerosis have demonstrated that recombinant apoAI Milano/phospholipid complexes rapidly reduce the lipid and macrophage content of atherosclerotic plaques after a single infusion. The effect of short-term intravenous recombinant apoAI Milano/phospholipid complexes on atheroma burden in patients with acute coronary syndromes was studied in a recent clinical trial. The intravenous administration of ApoAI Milano for 5 doses at weekly intervals produced statistically significant regression in coronary atheroma volume in the target segment as compared with baseline measurements by intravascular ultrasound. No change was seen with saline placebo control. It remains to be determined whether apoAI Milano has unique properties that result in greater antiatherogenic potential than normal apoAI and whether the exciting results of this first study in humans can be confirmed in large-scale randomized clinical outcome trials.

Angioplasty is used to open a blocked heart artery and improve blood flow to the heart. The doctor inserts a thin catheter with a balloon on the end into the artery. When the balloon reaches the blockage, it is expanded, opening up the artery and improving blood flow. The doctor may also insert a small mesh tube, called a stent, to help keep the artery open after angioplasty.

HDL are involved in reverse cholesterol transport, the process by which cholesterol is transported from peripheral tissues to the liver, where it can be excreted in bile. Studies since the mid-1990s have provided strong evidence that HDL A< also may protect against atherosclerosis by virtue of its antiinflammatory properties. These studies, based in part on genetic variations that influence the antioxidant properties of HDL, indicate that HDL can selectively remove and destroy proinflammatory oxidized lipids from the vessel wall, thereby inhibiting the vicious cycle of LDL trapping, LDL oxidation, and inflammation. Navab et al have demonstrated that short, synthetic ampipathic helices, similar to those in apoAI, exert powerful protective effects against atherosclerosis when administered orally to mice or monkeys.

Another promising therapeutic target is the leukotriene pathway. Genetic studies implicated 5-lipoxygenase (5-LO) in atherosclerosis susceptibility in mice and then humans, and recent studies suggest that polymorphisms of other enzymes in leukotriene metabolism also are associated with CHD. 5-LO polymorphisms were implicated originally in asthma, and a variety of leukotriene synthesis inhibitors are widely used to treat asthma. Thus, these inhibitors also may protect against the development of CHD, possibly providing a useful complement to drugs that target risk factors, such as lipids and blood pressure.

b) Risk Stratification, Prevention, and Treatment

Current approaches to the management of asymptomatic individuals and those with documented CHD focus on assessing traditional risk factors, estimating risk, and modifying risk factors through therapeutic lifestyle modification or cardiovascular protective medications or both. National guidelines from the American Heart Association and the National Heart, Lung, and Blood Institute's National Cholesterol Education Program recommend an approach to initial global risk assessment of the asymptomatic patient to obtain an estimate of absolute cardiovascular risk. On the basis of standard risk factors and related risk correlates, the concept was set forth that asymptomatic patients can be placed into 1 of 3 risk categories: low, intermediate, and high.

The techniques used in office assessments include history, physical examination, laboratory testing, and ECG. The focus of the examination is on the detection of risk factors that either can be directly modified or will modify the overall intensity of riskreduction therapies. The major causal risk factors identified for routine assessment include age, smoking, elevated blood pressure, elevated serum LDL cholesterol, low HDL cholesterol, and diabetes. The approach to therapy is guided by the principle that the intensity of risk-factor management should be adjusted by the severity of risk. Lowrisk patients should be encouraged to adhere to healthy lifestyle habits. High-risk patients are advised to directly begin a regimen of aggressive risk reduction through a combination of nondrug and drug regimens. Patients at intermediate risk become candidates for further risk stratification through the measurement of the inflammatory marker high-sensitivity C-reactive protein or noninvasive procedures that test for the presence of myocardial ischemia or coronary atherosclerotic burden, or both techniques.

Although several of the conventional causal risk factors used in global risk assessment clearly have a genetic basis, specific genetic testing has not been recommended for routine clinical practice. Because conventional risk factors account for only 50% of the variability in risk, the identification of genetic differences influencing the pathways of measurable atherosclerotic risk factors or through novel pathways may allow for the determination of risk that is additive to the measurement of conventional risk factors. Moreover, some genetic differences, such as those in the genes for apoE, lipoprotein lipase (LPL). and interleukin-6, show evidence of specific environmental interactions (eg, with smoking), in which the overall risk is more than additive. Thus, the predictive models used in the Framingham risk score may be greatly enhanced by the addition of pivotal single-nucleotide polymorphisms and haplotypes that have been shown to affect CHD. It can be envisioned that genotypic information could transform the value of the risk score and be integrated in routine clinical practice to guide preventive therapy.

It also seems likely that genetic differences would be useful in predicting specific complications of atherosclerosis. It is interesting that the phosphodiesterase-4D (PDE4D) gene polymorphisms were specifically associated with stroke. The genetic linkage studies of Wang and colleagues suggest that genetic factors may be associated specifically with myocardial infarction rather than with atherosclerotic lesion size. Studies in mice have clearly demonstrated that genetic factors can influence the distribution of lesions in different vessel locations; for example, Teupser and colleagues observed striking differences in the development of lesions in the aortic sinus between strains C57BL/6 and FVB but no differences in the abdominal aorta. Studies in mice also have clearly shown that genetic factors distinct from those for lesion size influence vascular calcification and medial destruction leading to aneurysms.

c) Multilocus Testing
A number of genetic differences identified thus far could potentially serve as useful predictors of prevalent or incident CHD. Such markers, however, may not be useful clinically unless they possess additional characteristics. These characteristics include (1) ability to standardize the assay, (2) independence from established risk factors, (3) association with CHD clinical end points in observational studies and clinical trials, (4) presence of population norms to guide the interpretation of results, (5) ability to improve the overall prediction beyond that of traditional risk factors, (6) generalization of results to various population groups, and (7) acceptable cost of the assays. The use of genetic testing also would be affected by the type of relationship with atherosclerosis and other risk factors.
Many genes have been associated with the increased risk of atherosclerosis, acute myocardial infarction, or both, although a large fraction are likely to be false positives. Although most of these variations influence traditional risk factors, they may provide genetic information that is useful in assessing risk to relatives or guiding therapy (eg, LDL receptor, apoAV, apoB, apoE, cholesterol ester transfer protein, LPL, upstream transcription factor-1). Others may provide more reliable and cost-effective assays for risk factors. For example, apo(a) gene variations determine nearly all of the variance of Lp(a) levels, and although Lp(a) levels can be measured using immunoassays, the levels have proven difficult to standardize. DNA-based tests that distinguish the various apo(a) alleles according to haplotype could be developed to reliably predict Lp(a) levels.

Several genetic variations clearly influence CHD through mechanisms that are independent of traditional risk factors, although they may not be independent of markers of inflammation such as C-reactive protein or myeloperoxidase. The recently reported myocyte enhancer factor 2A variation is highly penetrant, although its frequency in the population is probably low. Common variations in the genes for PDE4D, lymphotoxin- , 5-LO, 5-LO-activating protein, and others are likely to improve the overall prediction of risk.

The plot viewer supports five major plot types: histograms, XY scatter plots, numeric value plots, heat maps, and interactive LD plots for detecting correlations between markers. Haplotypes can also be defined and visualized directly from an LD plot for specific regions or automatically across the genome.

The cover image shows Manhattan plot of P-values in a genome-wide association study (GWAS) for neutrophil count, the most abundant subtype of white blood cells (WBCs). This GWAS was performed using 5771 Japanese subjects under the support of BioBank Japan Project. Horizontal axis shows chromosomal positions. Vertical axis shows - log10 P-values of the single nucleotide polymorphisms (SNPs). The gray horizontal line in the plot represents the genome-wide significance threshold of P=5.0 108 Two genetic loci, PSMD3-CSF3 at 17q21 and PLCB4 at 20p12, demonstrated the significant associations (P <5.0=108).

The cost of such tests is, of course, an important issue. DNA is stable and simple to isolate, and with multiplexing, many tests (hundreds or thousands) can be performed on the amount of DNA in a few milliliters of blood. Until recently, the cost of typing DNA differences (genotyping) has been >$l/genotype, but new technologies are reducing this amount by more than a factor of 10. The number of tests that will be required per gene is another key issue. If, on the one hand, risk is determined largely by genetic differences that are common in human populations, such as the apoE alleles discussed above, then genotyping will be relatively inexpensive. If, on the other hand, susceptibility to CHD results largely from many different rare mutations, such as mutations of the LDL receptor in FH, then genotyping will be much more difficult, expensive, and less reliable. It has proven feasible, even for FH, to design rapid screens that detect a significant fraction of FH mutations. Most of the evidence suggests that common genetic variations will explain a significant fraction of the risk of common diseases, including CHD.

The value of genetic testing in cardiology will become clear only when it has been implemented on a large scale. Although the impact of the testing of individual polymorphisms could be estimated from published odds ratios for risk, these ratios are likely to vary between ethnic groups and to be influenced by genetic and Environmental interactions. Also most of the reported associations require confirmation. Stephens and Humphries have argued that genetic testing may not have much value because the presently known genetic differences may not predict risk over and above that of measured traits, such as plasma lipids. Quite reasonably, they posit that because an individual's personal characteristics and plasma risk-trait values reflect both genotype and exposure, they may be the superior predictors of clinical outcome. It is our opinion that several potential benefits to genetic testing exist. First, a number of recently identified genes are likely to be independent of measured traits. Second, they may guide therapy. Third, they may reveal highly penetrant disorders that could influence risk substantially for a patient as well as his or her family members. We suggest, therefore, that multilocus testing be evaluated in some large trials to determine its overall value and identify the most important risk factors.

d) Population Screening
Although the prevention of CHD is feasible and scientifically definable by preventing or controlling the major risk factors, the challenge of identifying and treating individuals with major CHD risk factors remains great. Nearly the entire US population can be considered at risk because of exposure to 1 unfavorable risk factor level or unhealthy lifestyle habit. Because atherosclerosis is clinically manifested later in life, knowledge of propensity for the disease could be available decades before the clinical disease develops, permitting early intervention. DNA tests could assess risk earlier in life, long before phenotype-based tests become useful in adults in assessing risk. A population-wide approach to identifying individuals at higher risk could be justified, allowing for targeted treatment for those found to be at high risk. As it becomes more Feasible and less expensive to perform genetic profiling, population screening to Identify individuals at risk should be considered.

e) Pharmacogenetics
Pharmacogenetics provides the experimental basis to understand the variability in response to drugs as a function of an individual's genetic makeup. Genetic polymorphisms may influence drug response through a number of mechanisms including pharmacokinetic interactions, pharmacodynamic gene-drug interactions that involve gene products expressed as receptors, and genes that are in the causal pathway of disease and are able to modify the effects of drugs.

Lowering serum lipid levels has been demonstrated to slow progression or even induce regression in atherosclerosis. However, as with any other drug treatment, the magnitude of plasma lipid responses to drug therapies varies considerably among individuals. Several genetic polymorphisms that may play a role in the different responses to lipidlowering therapy have been identified recently and thus may predict individual successes in hypolipemic drug treatment. Several studies have revealed significant interactions between an individual's apoE genotype and plasma lipoprotein lipid changes with statin therapy. In these studies, subjects with the E2 allele, and sometimes subjects with the E3 allele, were more likely to respond to statin therapy with a favorable reduction in total cholesterol and LDL cholesterol than were subjects with at least 1 E4 allele.

Variation in response to statins on disease progression also has been associated with genetic polymorphisms. A polymorphism for the cholesteryl ester transfer protein gene has been associated with the effectiveness of pravastatin on disease progression. About 16% of the Regression Growth Evaluation Statin Study (REGRESS) trial population (men with angiographically documented atherosclerosis) had the B2B2 genotype, and these subjects did not show any benefit from the use of statins on the progression of atherosclerosis, whereas individuals without this genotype did demonstrate benefit. In the pravastatin-randomized patients in the REGRESS trial, the risk of clinical events was associated with a common polymorphism in the promoter of the matrix metalloproteinase stromelysin-1 gene. These effects were independent of the effects of pravastatin on lipid levels. As previously discussed, the leukotriene pathway may make an important contribution to genetic susceptibility to atherosclerosis as well as to asthma. Genetic variations in 5-LO have been shown to contribute to the variable response to antileukotriene drugs in asthma. Genetic variants in the promoter region are believed to change the binding of these transcription factors and the rate of 5-LO transcriptional activation under inflammatory conditions. Common variations in the GC-rich region of the promoter were associated with the altered transcription of the 5-LO gene as compared with the common allele.

In people with asthma, carriers of these genetic variants demonstrated a diminished response to treatment with antileukotriene drugs, indicating a pharmacogenetic effect of the promoter sequences on responses to treatment. If antileukotriene medications are shown to be beneficial in atherosclerosis, then similar pharmacogenetic effects of the 5LO promoter sequences on responses to treatment would be expected. Genotyping may be used to identify individuals who would derive the greatest benefit of this antiinflammatory directed therapy. Genetic testing also may reveal useful dietary interactions. In the case of 5-LO, dietary arachidonic acid intake significantly enhanced the proatherogenic effect of 5-LO variants, whereas intake of n-3 polyunsaturated fatty acids attenuated this effect. Because n-3 polyunsaturated fatty acids decrease the formation of leukotrienes by competing with arachidonic acid as substrates for 5-LO, these findings suggest that the antiatherogenic effects of n-3 polyunsaturated fatty acids may be confined to or may be more prominent in individuals with genotypes that favor increased 5-LO activity.

Another interesting example is the interaction of a common thermolabile variant of methylene tetrahydrofolate reductase (MTHFR) and folate levels (determined largely by dietary consumption) in the control of homo-cysteine levels and susceptibility to CHD. The dependence of homocysteine levels on this interaction is evident when individuals in the study population were divided into those homozygous for the thermolabile allele (genotype TT), heterozygous for the thermolabile allele (genotype CT), or homozygous for the thermostable allele (genotype CC) and then subdivided according to folate levels. In individuals with high folate levels, the thermolabile allele had no effect on homocysteine levels, but in individuals with low folate (first tertile), homozygous individuals had a 2-fold increase in homocysteine levels as compared with the thermostable allele.

Usually, one of the defenses stops the drug interaction before it can produce an adverse consequence. In this case, the patients pharmacogenetic makeup protects against an adverse event. ADR=adverse drug reaction.

The relationship of MTHFR genotypes and folate levels to CHD is, of course, much more complex, and studies with large numbers of individuals are required to observe evidence of this relationship, given the considerable heterogeneity of CHD. Nevertheless, a meta-analysis of data from several studies showed that individuals with the thermostable MTHFR allele were protected against CHD as compared with individuals with the thermolabile allele and that this protection was dependent on the folate status of the individuals. Greater understanding of the genetic basis of atherosclerosis may provide mechanisms for creating personalized designer therapies for individual patients or groups of patients with similar diseases based on individual genotype. The current approach to patients with CHD or CHD risk equivalents involves therapeutic lifestyle modification and combinations of cardiovascular protective medications. Pharmacogenetics may allow for a more targeted approach that identifies the therapies to which individual patients will respond best. Ultimately, clinical outcomes in response to treatment could be predicted in advance.

13. Prediction of disease pathologies of the cardiovascular system in Kursk.

In the result of the analysis of cardiovascular system pathologies prognosis in Kursk city up to 2020 year it was concluded that it was extremely adverse and was characterized by a strongly pronounced tendency to growth. Expected incidence of the cardiovascular system pathologies in the regional center in 2020 would be about 21.8 people per 1000 population (in 2007, 14.6 per 1000 population). Growth happens almost in 1.5 times.
30

y = 0,4582x + 6,2552
25
incidence

20 15 10 5 0
19 87 19 89 19 91 19 93 19 95 19 97 19 99 20 01 20 03 20 05 20 07

years

incidence

prognosis

Prediction of disease pathologies of the cardiovascular system in Kursk up to 2020.

14. Prediction of coronary heart disease in Kursk.

Unfortunately, the negative trend will be observed in significant increase in incidence of coronary heart disease in Kursk up to 2020 year. According to expected level of coronary heart disease in Kursk in 2020 is 3.1 persons per 1000 population. In 2007 it was 2.3 people per 1000 population, consequently, the incidence of CHD would grow approximately 1.1 times.
4,0 3,5 3,0

y = 0,064x + 0,904

incidence

2,5 2,0 1,5 1,0 0,5 0,0

19 87 19 89 19 91 19 93 19 95 19 97 19 99 20 01 20 03 20 05 20 07

years
prognosis

incidence

Prediction of coronary heart disease in Kursk up to 2020.

15. Forecast of the morbidity of acute myocardial infarction in Kursk.

Forecast on incidence of acute myocardial infarction of the population of Kursk up to 2020, is characterized by a tendency to increase. Incidence rate will be1.2 persons per 1000 population. In 2007 it was 1.0 per 1000 population, that is, it would get older as compared with the level of 2007 and previous years.
1,40

y = 0,0145x + 0,6984
1,20

incidence

1,00 0,80 0,60 0,40 0,20 0,00

19 87 19 89 19 91 19 93 19 95 19 97 19 99 20 01 20 03 20 05 20 07

years
prognosis

incidence

Forecast of the morbidity of acute myocardial infarction in f Kursk up to 2020.

16. Forecasting incidence of angina in Kursk.

According to our calculations the incidence of angina per year will be around 0.8/1000 population. Thus, it will grow up compared with 2007 year (0.6/1000 people).
0,9 0,8 0,7

y = 0,0177x + 0,2183

incidence

0,6 0,5 0,4 0,3 0,2 0,1 0,0

19 87 19 89 19 91 19 93 19 95 19 97 19 99 20 01 20 03 20 05 20 07

prognosis incidence

years

Forecasting incidence of angina in Kursk up to 2020.

17. Forecast of cerebrovascular pathology in Kursk.

Forecast on incidence of cerebrovascular disease population of Kursk up to 2020, is characterized by significant trend downward. According to our calculations the incidence of cerebrovascular disease in a year will be about 2.5/1000 population. Presumably, it substantially would be reduced if compared with 2007 (3.1/1000 population), but would still be higher than in previous years in 1.25 times (2005 and 3,5 2006 -2/1000 people).
y = 0,0528x + 0,7016
3,0

incidence

2,5 2,0 1,5 1,0 0,5 0,0

19 87 19 89 19 91 19 93 19 95 19 97 19 99 20 01 20 03 20 05 20 07

years
incidence

prognosis

Forecast of cerebrovascular pathology in Kursk up to 2020.

Thus, studying the predictive results on cardiovascular morbidity among the population of the regional centre, it is worth noting some differences in the prediction of disease between the city of Kursk and Kursk region as a whole. In our forecast will be slight differences in changes in the dynamics on the morbidity of the above diseases among the population of the city and region: this is probably due, and with great anthropogenic pressure, higher levels of pollution in Kursk, including specific pollutants reducing morbidity of hypertensive disease is only in comparison with 2007 year but talk about completely favourable forecast cannot be, as in previous years still, those figures were below than expected up to 2020.
18. Comparison of the projected incidence of diseases of the circulatory system between Kursk city and Kursk region as a whole (per 1000 people)
The Incidence Of 1 2 Circulatory system pathology Hypertonic disease 3 4 Coronary heart disease Myocardial infarction 5 6 Angina Cerebrovascular Pathology growth decrease does not change decrease growth growth growth decrease Kursk City growth decrease Kursk Region growth growth

Genotypes correlations with predisposition to the cardiovascular diseases

Etiological role of genes was installed in the triggering of cardiovascular diseases in different populations of humans. Polymorphic variants were studied especially the relationship of genes with a predisposition to the cardiovascular diseases taking into account sexual dimorphism in their manifestation.

a) Angina
RISK OF ANGINA INCREASES

GENOTYPES CORRELATIONS

MEN
-50GT YP2J2 -50GT YP2J2 139HH EPHX1 114AA GSTP1 114AA GSTP1 (OR=6,55) 25RR TGF 1 (OR=4,30) 114AA GSTP1 (OR=2,11) 25RR TGF 1 (OR=2,56)

MEN
-50GG YP2J2 -50GG YP2J2 -50GG YP2J2 139HR EPHX1 114AV GSTP1 114AA GSTP1 139HR EPHX1 (OR=0,43) 114AV GSTP1 (OR=0,42) 1/2 PGC (OR=0,50) 1/2 PGC (OR=0,14) 1/2 PGC (OR=0,06) 25RP TGF 1 (OR=0,47) 25RR TGF 1 (OR=0,39)

WOMEN

114AV GSTP1

432VV CYP1B1 114AA GSTP1 114AA GSTP1 114AA GSTP1 10LP TGF 1 -509T TGF 1

10LP TGF 1 (OR=3,53 10LP TGF 1 (OR=2,46), ), -509T TGF 1 (OR=2,50), -511CT IL1 (OR=3,47), -511CT IL1 (OR=2,95), -511CT IL1 (OR=4,06).

WOMEN
432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 114AV GSTP1 (OR=0,06), 10LL TGF 1 (OR=0,21), -509 TGF 1 (OR=0,06), -511CC IL1 (OR=0,31), 192QQ PON1

RISK OF ANGINA DECREASES *The odds ratio (OR) is a measure of effect size, describing the strength of association or non-independence between two binary data values. It is used as a descriptive statistic, and plays an important role in logistic regression. Unlike other measures of association for paired binary data such as the relative risk, the odds ratio treats the two variables being compared symmetrically, and can be estimated using some types of non-random samples.

b) Myocardial infarction
GENOTYPES CORRELATIONS RISK OF MYOCARDIAL INFARCTION INCREASES

MEN
731AA CYP1A2 C1C1 CYP2E1 +61GA EGF 481FF NAT2 (OR=2,02), -50GT CYP2J2 (OR=3,80), 2/3(+) PGC (OR=7,97).

MEN
C1C1 CYP2E1 C1C1 CYP2E1 -50GG CYP2J2 -50GG CYP2J2 (OR=0,34), 2/3(-) PGC (OR=0,43), 2/3(-) PGC (OR=0,35).

WOMEN
m1m1 CYP1A1 m1m1 CYP1A1 m1m1 CYP1A1 m1m1 CYP1A1 m1m1 CYP1A1 432VL CYP1B1 114AA GSTP1 114AA GSTP1 114AA GSTP1 (OR=3,26), 3435TT MDR1 (OR=3,36), 10LP TGF 1 (OR=3,12), -509CT TGF 1 (OR=2,95), -511CT IL1 (OR=2,44), -511CT IL1 (OR=3,13), 10LP TGF 1 (OR=2,51), -509CT TGF 1 (OR=2,55),etc.

WOMEN
m1m1 CYP1A1 432LL CYP1B1 432LL CYP1B1 114AA GSTP1 114AA GSTP1 10LL TGF 1 25RR TGF 1 -509CC TGF 1 10LL TGF 1 (OR=0,32), 3435CC MDR1 (OR=0,13), 10LL TGF 1 (OR=0,22), 25RR TGF 1 (OR=0,24), -511CC IL1 (OR=0,41), -511CC IL1 (OR=0,11), -511CC IL1 (OR=0,32) -511CC IL1 (OR=0,25).

RISK OF MYOCARDIAL INFARCTION DECREASES

*The odds ratio (OR) is a measure of effect size, describing the strength of association or non-independence between two binary data values. It is used as a descriptive statistic, and plays an important role in logistic regression. Unlike other measures of association for paired binary data such as the relative risk, the odds ratio treats the two variables being compared symmetrically, and can be estimated using some types of non-random samples.

c) Ischemic stroke

GENOTYPES CORRELATIONS

RISK OF ISCHEMIC STROKE INCREASES

MEN
432VV CYP1B1 432VV CYP1B1 432VL CYP1B1 -50GT CYP2J2 -50GT CYP2J2 -511CT IL1 -511CT IL1 -50GT CYP2J2 (OR=15,69), -511CT IL1 (OR=5,40), -703CC IL5 (OR=2,10), 27SS IL3 (OR=7,74), -703CC IL5 (OR=23,44), 27PP IL3 (OR=5,40 -703CC IL5 (OR=2,36).

MEN

432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 -50GG CYP2J2

-50GG CYP2J2 (OR=0,38), 27SP IL3 (OR=0,09), -703CT IL5 (OR=0,19), -703CT IL5 (OR=0,48.)

WOMEN

WOMEN

-50GG CYP2J2 -50GT CYP2J2 -50GG CYP2J2 113HH EPHX1 113HH EPHX1 857FF NAT2 -511CT IL1 27SP IL3

113HH EPHX1 (OR=3,02), 857FF NAT2 (OR=4,38), -703CC IL5 (OR=2,10), 857FF NAT 2 (OR=3,96), -703CC IL5 (OR=4,33), -703CC IL5 (OR=2,67), -703CC IL5 (OR=2,29), -703CC IL5 (OR=3,03).

-50GG CYP2J2 -50GG CYP2J2 -50GG CYP2J2 113YH EPHX1 -511CC IL1 113YY EPHX1 113YH EPHX1 -511CC IL1 27SS IL3

113YH EPHX1 (OR=0,47), 857FS3 NAT2 (OR=0,18), -703CT IL5 (OR=0,38), -511CC IL1 (OR=0,40), -703CT IL5 (OR=0,45), -703CT IL5 (OR=0,40), -703CT IL5 (OR=0,40), -703CT IL5 (OR=0,45) -703CT IL5 (OR=0,50)

*The odds ratio (OR) is a measure of effect size, describing the strength of association or non-independence between two binary data values. It is used as a
descriptive statistic, and plays an important role in logistic regression. Unlike other measures of association for paired binary data such as the relative risk, the odds ratio treats the two variables being compared symmetrically, and can be estimated using some types of non-random samples.

RISK OF ISCHEMIC STROKE DECREASES

d) Hemorrhagic stroke

GENOTYPES CORRELATIONS

RISK OF HEMORRHAGIC STROKE INCREASES

MEN
731AA CYP1A2 731AA CYP1A2 -50GG CYP2J2 113HH EPHX1 590FS2 NAT2 (OR=2,93), 38AG CC16 (OR=3,40), 590FS2 NAT2 (OR=2,34), 590FS2 NAT2 (OR=2,60).

MEN
731CA CYP1A2 731CA CYP1A2 -50GG CYP2J2 -50GG CYP2J2 -50GG CYP2J2 113YH EPHX1 590FF NAT2 -50GG CYP2J2 (OR=0,30), 590FF NAT2 (OR=0,22), 113HH EPHX1 (OR=0,14), 113HR EPHX1 (OR=0,27), 590FF NAT2 (OR=0,27), 590FF NAT2 (OR=0,18), 38GG CC16 (OR=0,16).

WOMEN

WOMEN
-50GG CYP2J2 -50GG CYP2J2 -50GG CYP2J2 -50GG CYP2J2 -290AA CYP3A4 -290AA CYP3A4 -290AA CYP3A4 -290AA CYP3A4 -290AA CYP3A4 -290AA CYP3A4 (OR=0,26), 113YH EPHX1 (OR=0,34), -703CT IL5 (OR=0,31), -1111CC IL13 (OR=0,36), 113YH EPHX1 (OR=0,23), 181AAUGT1A6 (OR=0,07), 27SS IL3 (OR=0,37), -703CT IL5 (OR=0,20), -1111CC IL13 (OR=0,22), etc.

-50GG CYP2J2 -290AA CYP3A4 -290AG CYP3A4 -290AA CYP3A4 113HH EPHX1 EPHX1-113HH EPHX1 113HH EPHX1 113HH EPHX1

113HH EPHX1 (OR=3,40), 113HH EPHX1 (OR=3,73), 181TT UGT1A6 (OR=8,23), -703CC IL5 (OR=2,93), 181TT UGT1A6 (OR=5,00), 213HH SULT1A1 (OR=8,23), -511CC IL1 (OR=5,74), 27SP IL3 (OR=6,95), etc.

RISK OF HEMORRHAGIC STROKE DECREASES

*The odds ratio (OR) is a measure of effect size, describing the strength of association or non-independence between two binary data values. It is used as a descriptive statistic, and plays an important role in logistic regression. Unlike other measures of association for paired binary data such as the relative risk, the odds ratio treats the two variables being compared symmetrically, and can be estimated using some types of non-random samples.

19. Summing up we have received data of eco-epidemiological study on relationship between incidence of cardiovascular disease with chemical pollution of Kursk region. It should be noted that, despite some variations in the picture relationships of certain types of CVD and certain factors of environmental pollution, in general, we can select a range of toxic compounds, the products of anthropogenic origin, which appear to be implicated in the etiology of pathologically various cardiovascular diseases. Such factors include: emissions of various types of toxic substances from stationary sources and from road transport, such as carbohydrates, nitrogen oxides, sulphur dioxide, formaldehyde, phenol, pesticides Lontrel and TMTD (tetrametiltiuramdisulfid). With regard to the systems referred to endogenous detoxification of these compounds, it includes the whole ensemble of closely and functionally interacting classes of enzymes, decontaminating substances in an organism.

20.CONCLUSIONS
Investigations into the genetics of atherosclerosis have greatly advanced our knowledge of the mechanisms of this complex multifactorial disease. These investigations have led to some novel therapies that are undergoing testing in clinical trials. A number of genes that are associated with an increased risk for CHD have been identified recently. Several of these genes exhibit common variations that influence CHD risk independently of traditional risk factors (lymphotoxin- , PDE-4D, 5-LO, 5-LO-activating protein). Other genes are rare but are highly penetrant (myocyte enhancer factor 2A); still others are important markers of familial disease (upstream transcription factor-1). Prospective trials to evaluate the effectiveness and cost of genetic testing to risk-stratify individuals, guide therapy, and predict response are needed before these tests are integrated into routine clinical practice. Progress in this area will be driven by studying gene-gene, gene-phenotype, and gene-treatment interactions in large patient populations. Based on the rapid progress being made, an individual's genotype may well play an important clinical role in the assessment, prevention, and treatment of atherosclerosis.

PART II. THE GENETIC ASPECTS OF ESSENTIAL HYPERTENSION 1. INTRODUCTION 2. PATHOPHYSIOLOGY OF HYPERTENSION a) Oxidative stress b) Endothelial dysfunction c) Vascular remodeling 3. APPROACHES TO THE STUDY OF ESSENTIAL HYPERTENSION 4. IDENTIFICATION OF THE GENES INVOLVED IN HYPERTENSION a) The candidate gene approach b) Genome-wide linkage screens c) The study of monogenic hypertension d) Animal models of hypertension 5. METHODS USED TO DETERMINE THE INVOLVEMENT OF THE GENE OR QTL IN HUMAN HYPERTENSION a) Linkage analysis b) Association studies c) Epistatic interactions and haplotype analysis d) Combination of the above 6. STUDY OF MONOGENIC HYPERTENSION AND GENES RELATED TO ESSENTIAL HYPERTENSION a) Mutations of genes encoding epithelial sodium channel (Liddles syndrome) b) Mutations of aldosterone synthase gene c) 11- Hydroxylase deciency and 17-hydroxylase deciency

d) Apparent mineralocorticoid excess e) Mutation in the mineralocorticoid receptor f) Gordon syndrome g) Hypertension with brachydactyly h) Familial hyperaldosteronism type II i) Angiotensinogen j) 2-Adrenoceptor 7. PHARMACOGENOMICS 8. DRUG ISOLATION 9. INTERINDIVIDUAL RESPONSE TO TREATMENT 10. GENE THERAPY 11. DRUG SYNTHESIS 12. FUTURE PERSPECTIVES 13. PREDICTION OF INCIDENCE OF HYPERTENSION 14. THE ASSOCIATIONS OF COMBINATIONS OF GENOTYPES OF INTRAGENIC POLYMORPHISMS IN THE STUDIED GROUPS OF HEALTHY AND HYPERTENSIVE PATIENTS 15. HOPE FOR THE FUTURE

Numerous studies have revealed associations between elevated blood pressure, hyperlipidemia, insulin resistance, and CAD. There has been substantial progress in elucidating the genetic basis for certain Mendelian forms of hypertension, but the genetic factors contributing to common forms of hypertension are largely unknown. Numerous candidate genes, particularly members of the renin-angiotensin system, have been investigated in humans and animal models. Both linkage and association studies have provided strong evidence for the role of the angiotensinogen gene in hypertension in Caucasian and African-Caribbean populations as well as in pregnancy-induced hypertension. The angiotensin-converting enzyme gene is associated with coronary heart disease, but this appears to involve a mechanism independent of the effects on blood pressure. In addition to candidate gene studies, there have been several genome scans for essential hypertension and blood pressure in various populations.

1. Introduction

Hypertension, or high blood pressure, is a common condition that will catch up with most people who live into older age. Blood pressure is the force of blood pressing against the walls of the arteries. When it's too high, it raises the heart's workload and can cause serious damage to the arteries. Over time, uncontrolled high blood pressure increases the risk of heart disease, stroke, and kidney disease.

Hypertension affects approximately 25% of the adult population worldwide, and its prevalence is predicted to increase by 60% by 2025, when a total of 1.56 billion people may be affected. It is the major risk factor for cardiovascular disease and is responsible for most deaths worldwide.

Primary hypertension, also known as essential or idiopathic hypertension, accounts for as many as 95% of all cases of hypertension.

High blood pressure is sometimes called a silent killer because it may have no outward symptoms for years. In fact, one in five people with the condition don't know they have it. Internally, it can quietly damage the heart, lungs, blood vessels, brain, and kidneys if left untreated. It's a major risk factor for strokes and heart attacks.

Genetic factors account for 3060% of an individuals risk for developing hypertension. However, elucidation of the genes involved in hypertension has proven to be difcult. Blood pressure is inherited in a polygenic fashion, with multiple genes contributing to the nal phenotype.
The blood pressure of people with prehypertension is consistently just above the normal level -- falling anywhere between 120 and 139 for systolic pressure or 80 to 89 for the diastolic pressure. People in this range have twice the risk of developing heart disease than those with a lower reading. Your doctor may recommend lifestyle changes to help lower your blood pressure.

You have high blood pressure if readings average140/90 or higher -- for either number -- though you may still have no symptoms. At 180/110 and higher, you may be having a hypertensive crisis. Rest for a few minutes and take your blood pressure again. If it is still very high, call ambulance. A hypertensive crisis can lead to a stroke, heart attack, kidney damage, or loss of consciousness. Symptoms of a hypertensive crisis can include a severe headache, anxiety, nosebleeds, and feeling short of breath.

Environmental factors also make a major contribution to blood pressure, further complicating the understanding and investigation of the molecular and genetic aspects of hypertension. For example, increased salt intake has been linked to hypertension. Other environmental factors that may inuence the development of hypertension include smoking, excessive alcohol intake, psychologic stress, lack of aerobic exercise and central obesity.
Up to the age of 45, more men have high blood pressure than women. It becomes more common for both men and women as they age, and more women have hypertension by the time they reach 65. You have a greater risk if a close family member has high blood pressure or if you are diabetic. About 60% of people with diabetes have high blood pressure.

African-Americans are more likely to develop hypertension -- and to develop it at a younger age. Genetic research suggests that African-Americans seem to be more sensitive to salt. In people who have a gene that makes them salt-sensitive, just a half-teaspoon of salt can raise blood pressure by 5 mm Hg. Diet and excessive weight can play a role, as well.

In addition, there is some evidence that developmental factors may also predispose to high blood pressure. A low birth weight is associated with the later development of hypertension. It has been hypothesized that this is related to a congenital reduction of nephrons, which may subsequently impair salt excretion. Unfortunately, it is not always possible to dissect out the role of nature versus nature in the etiology of hypertension. For example, genetic causes may predispose towards smoking and excessive alcohol intake, which may then contribute to the development of hypertension.

Sodium, a major component of salt, can raise blood pressure by causing the body to retain fluid, which leads to a greater burden on the heart. The American Heart Association recommends eating less than 1,500 milligrams of sodium per day. You'll need to check food labels and menus carefully. Processed foods contribute up to 75% of our sodium intake. Canned soups and lunch meats are prime suspects.

However, the role of genetics in hypertension is not limited to the determination of its etiology; advances in the understanding of the genetics and molecular biology of hypertension may also improve the diagnosis and treatment of the disorder. A large proportion of hypertensive patients do not have adequate blood pressure control on their current medication. Although poor patient medication compliance is likely to account for a proportion of these, suboptimal drug efcacy may also contribute to this number. In addition, 1020% of treatment failures are a result of drug side effects.

Stress can make your blood pressure spike, but there's no evidence that it causes high blood pressure as an ongoing condition. However, stress may affect risk factors for heart disease, so it may have an indirect connection to hypertension. Stress may lead to other unhealthy habits, such as a poor diet, alcohol use, or smoking, which can contribute to high blood pressure and heart disease.

Thus, pharmacogenomic-based strategies for rationalizing treatment in hypertension would be a highly desirable goal. To some extent, clinicians are already matching patient proles with drug treatment. For example, a hypertensive patient with left ventricular hypertrophy is likely to benet from angiotensinconverting enzyme (ACE) inhibition, while patients with concurrent ischemic heart disease are better treated with a betablocker.

Being overweight places a strain on your heart and increases your risk of high blood pressure. That is why diets to lower blood pressure are often also designed to control calories. They typically call for cutting fatty foods and added sugars, while increasing fruits, vegetables, lean protein, and fiber. Even losing 10 pounds can make a difference.

However, much of the current treatment for hypertension is carried out on an empirical basis. A recent study suggests that there is great variation in patient response to antihypertensive drugs and selection of the best drug for a given patient is currently a matter of trial and error, a costly and timeconsuming process. In addition, the efcient control of hypertension can decrease the risk of blood pressure induced target organ damage, the prevalence of which increases with the length of time in which blood pressure remains uncontrolled. An improved understanding of the genes of hypertension may therefore enable the physician to better tailor medication to the patient with hypertension.

Drinking too much alcohol can increase your blood pressure. Guidelines from the American Heart Association state that if you drink alcohol, you should limit the amount to no more than two drinks a day for men, or one a day for women. They define a drink as one 12-ounce beer, four ounces of wine, 1.5 ounces of 80-proof spirits, or one ounce of 100-proof spirits.

If caffeine can make you jittery, can it also raise your blood pressure? It might have a temporary effect, but studies haven't shown any link between caffeine and the development of hypertension. You can safely drink one or two cups a day, according to the American Heart Association.

Gestational hypertension is a kind of high blood pressure that occurs in the second half of pregnancy. Without treatment, it may lead to a serious condition called preeclampsia that endangers both the mother and baby. The condition can limit blood and oxygen flow to the baby and can affect the mother's kidneys and brain. After the baby is born, the mother's blood pressure usually returns to its normal level.

Cold and flu medicines that contain decongestants are one of several classes of medicine that can cause your blood pressure to rise. Others include NSAID pain relievers, steroids, diet pills, birth control pills, and some antidepressants. If you have high blood pressure, talk to you doctor about what medicines and supplements you are taking that may affect blood pressure.

Some people only have a high reading in the doctor's office, perhaps because they're nervous. Some will only have blood pressure readings sporadically. Those people may have a higher chance of developing high blood pressure, a recent study shows. To get a more accurate reading, take your blood pressure at home, chart your readings, and share them with your doctor. It is also a good idea to bring in your home monitor in for a check of the device and your technique.

While hypertension is more often a problem for older people, even children can have high blood pressure. "Normal" blood pressure varies based on a child's age, height, and sex, so your doctor will need to tell you if there is a concern. Children are at greater risk if they are overweight, have a family history of the illness and if they're African-American.

2.Pathophysiology of hypertension
The regulation of extracellular uid volume by sodium excretion renal is responsible for the control of blood pressure. This mechanism is made possible by the ability of the kidneys to vary renal tubular sodium excretion in accordance with renal perfusion pressure. In the healthy individual, any increase in mean arterial blood pressure would lead to a proportional rise in renal perfusion pressure, which would increase renal sodium excretion.
Presentation of the hypertension mosaic showing the central placement of genes within the core of the mosaic. This molecular mosaic illustrates the key role of genetic factors in regulating the multiple pathways involved in the pathogenesis of essential hypertension. In contrast to conventional antihypertensive agents that treat surface facets of the Page mosaic, transcription-modulating drugs can be used to modify gene expression and attack essential hypertension at the core of the mosaic. Nonpharmacological therapies that manipulate environmental factors affecting gene transcription might provide another approach to attacking core molecular determinants of essential hypertension.

A diagram explaining factors affecting arterial pressure

The loss of sodium leads to shrinkage of extracellular uid volume, causing a drop in cardiac output, thus normalizing the blood pressure. A drop in renal perfusion pressure has the opposite effect and would result in sodium retention. Therefore, in order to maintain blood pressure homeostasis in the healthy individual, the kidney has to match sodium excretion to sodium intake. Hypertension results from the inability of the kidneys to excrete enough sodium to match intake or the inability of kidneys to excrete sufcient sodium in response to raised mean arterial pressure. Impairment of renal perfusion by renal artery stenosis, for example, results in a drop in renal perfusion pressure, leading to a rise in mean arterial pressure in order to maintain kidney perfusion. A similar mechanism accounts for the hypertensive change observed in coarctation of the aorta (Figure 6).

Other causes of hypertension include renal failure, where the remaining nephrons are unable to excrete enough sodium to match blood pressure, Conn and Cushing syndromes, and the various monogenetic forms of hypertension. However, up to 90% of patients with hypertension do not have a discernible cause for their high blood pressure. These patients are described as having essential hypertension. There is often no histologic abnormality in the kidneys of patients in the early stages of essential hypertension. However, a proportion of patients with essential hypertension, often referred to as salt-sensitive hypertensives, have a blunted natriuretic response to a rise in blood pressure (Figure 6).

Figure 6. Network of pathways and genes associated with blood pressure regulation

Numerous pathophysiologic factors have been associated with essential hypertension These include increased sympathetic nervous system activity, overproduction of sodium-retaining hormones and vasoconstrictors, increased renin secretion, lifestyle, insulin resistance, deficiencies of vasodilators such as NO and prostacyclin, altered cellular ion transport and abnormalities of resistance vessels (Figure 7,8,9,10,11,12) (Oparil et al. 2003). Recently, compelling evidence has shown that structural and functional abnormalities in the vasculature, including increased oxidative stress, endothelial dysfunction and vascular remodeling may contribute to the pathogenesis of hypertension (Koller 2002; Mulvany 2000; de Champlain et al. 2004). Many of the antihypertensive drugs interfere with one or more of the above factors.

Figure7. Pathophysiologic mechanisms of hypertension.

Figure 8. Molecular Mechanisms Implicated in the Retention of Sodium and Loss of Potassium by the Kidneys in Primary Hypertension.
Solid arrows indicate an increase or stimulation, and the broken arrow indicates inhibition. Numbers on the left denote the approximate percentage of reabsorption of filtered sodium in each nephronal segment during normal conditions. Several influences acting on the luminal sodium transporters and the basolateral sodium pump stimulate sodium retention and potassium loss. Promotion of sodium reabsorption by the activated epithelial sodium channel (ENaC) generates a more negative luminal membrane voltage (Vm) in the collecting duct that enhances potassium secretion through the luminal potassium channel and promotes kaliuresis. NHE-3 denotes sodiumhydrogen exchanger type 3, ACE angiotensin-converting enzyme, NKCC2 sodiumpotassium2 chloride cotransporter, and NCC sodiumchloride cotransporter. PST 2238 (rostafuroxin) antagonizes the effect of digitalis-like factor on the sodium pump.

Figure 9. Molecular Pathway Implicated in the Generation of Increased Arterial and Arteriolar Smooth-Muscle Tone by an Excess of Sodium and a Deficit of Potassium in Primary Hypertension.

Figure 10. Molecular Pathways Implicated in Potassium-Induced, Endothelium-Dependent Vasodilatation

Figure 11. Molecular Pathways Implicated in the Central Effects of Sodium and Potassium on Blood Pressure.

Figure 12.

a) Oxidative stress is a situation of serious imbalance between the production of reactive oxygen species (ROS) and antioxidant defense mechanisms. ROS include superoxide anions, hydroxyl radicals, and hydrogen peroxide. The primary sources for ROS generation include the mitochondrial electron transport chain and oxidase enzymes such as NAD(P)H oxidase, xanthine oxidase, cytochrome P450 enzymes and lipoxygenases (Mueller et al. 2005). Under normal conditions, only small quantities of ROS are generated and ROS are safely neutralized by the antioxidant system which consists of numerous enzymes and antioxidant molecules. Under pathological conditions, the rate of ROS generation exceeds the neutralizing capacity of the antioxidant defense system, leading to oxidative stress.
Cardiovascular continuum and the oxidative stress pathway. Pathwayomics depicted here as a cardiovascular continuum leading from the interrogation of multiple SNPs in genes of the oxidative stress pathway to risk haplotypes and future genetic testing. It is further stipulated that multiple subtle genetic polymorphisms interacting with environmental factors produce chronic imbalance between ROS and endogenous antioxidants. These functional abnormalities contribute to systemic endothelial dysfunction, hypertension, and other modifiable cardiovascular risk factors, which, in turn, lead to advanced atherosclerosis and target organ damage. CV indicates cardiovascular; BP, blood pressure; LVH, left ventricular hypertrophy; MI, myocardial infarction.

Nearly all animal studies performed in various models of hereditary and acquired hypertension have provided evidence to support the causative role of oxidative stress to the pathogenesis of hypertension. For example, a progressive increase in the production of superoxide anion in vascular and cardiac tissues during the development of hypertension has been demonstrated in various models of hypertension such as DOCAsalt, SHR, and the angiotensin or glucose-induced model of hypertension (de Champlain et al. 2004). There are three lines of evidence for the causal contribution of oxidative stress to hypertension. First, oxidative stress is present in various animal models of hypertension (Vaziri and Rodriguez-Iturbe 2006). Second, chronic treatment with potent antioxidant can prevent or attenuate the development of hypertension (de Champlain et al. 2004). Finally, induction of oxidative stress causes hypertension in genetically normal and healthy animals (Vaziri et al. 2000; Zhou et al. 2002).

b) Endothelial dysfunction. Vascular endothelial cells play an essential role in cardiovascular regulation by producing a number of potent local vasoactive agents, such as the vasodilator molecule nitric oxide (NO), and the vasoconstrictor peptide endothelin. Dysfunction of the endothelium has been implicated in human essential hypertension (MacGregor and Kaplan 2006; Quyyumi 1998). It is clear that antihypertensive drugs that interrupt the reninangiotensin system, including ACE inhibitors, angiotensin-receptor blockers and mineralocorticoid receptor antagonists, are effective in restoring endothelial dysfunction (MacGregor and Kaplan 2006). This action may at least partly account for their cardioprotective effects.

Endothelial dysfunction is a functional and reversible alteration of endothelial cells, resulting from impairment in NO availability. Endothelial cells release NO in response to several stimuli including increased shear stress during increased blood flow or muscarinic receptor stimulation (MacGregor and Kaplan 2006). Numerous studies have demonstrated a beneficial effect of acute and chronic L-arginine supplementation on NO production and endothelial function, and L-arginine has been shown to Reduce systemic blood pressure in some forms of experimental hypertension (Gokce 2004).
High density lipoprotein (HDL) exerts many cardiovascular protective effects beyond reverse cholesterol transport. Our most recent study explored a novel beneficial effect on vascular endothelial cells namely inhibition of tissue factor expression. This function may importantly contribute to the antiatherothrombotic effects of the lipid.

Endothelin-1 (ET-1), the main endothelin generated in the endothelium, acts in a paracrine or autocrine manner on ETA and ETB receptors on adjacent endothelial or smooth muscle cells (Pollock and Pollock 2005). An interaction of the NO and ET-1 systems may participate in the pathogenesis of endothelial dysfunction. It was found that the vasoconstrictor activity of ET-1 is increased along with diminished availability of NO (Haynes and Webb 1998). The imbalance between the two systems may enhance the vasoconstrictor and protrophic activity of ET-1, leading to the increase of blood pressure.

A destructive cross-talk between Rho/RhoKinase and Akt/eNOS causes vascular endothelial dysfunction.

Representative histological cross sections of coronary arteries 4 weeks after balloon injury A: Control; B: Probucol; C: LXS; D: HXS.
Disruption of the internal elastic lamina () was similar at the site of intimal reaction in all groups. Significant reduction of intimal hyperplasia was observed in groups treated with drugs, especially in HXS, compared with control.

c) Vascular remodeling . Peripheral resistance is consistently increased in hypertensive conditions (MacGregor and Kaplan 2006). Peripheral resistance is determined mainly by resistance vessels which consist of the small arteries (lumen diameters < 300 m) and arterioles. Examination of gluteal skin biopsy specimens obtained from patients with untreated essential hypertension revealed that essential hypertension is associated with reduced lumen areas and increased media-lumen ratios without an increase in medial area in resistance vessels (Mulvany 2000). This remodeling is known as inward eutrophic remodeling. Mulvany M (Mulvany 2002) suggests that essential hypertension is associated only with eutrophic, not hypertrophic, remodeling in small arteries. Although the mechanisms by which cause eutrophic remodeling is not clear, the following process is proposed: increased neurohumoral activity leads to vasoconstriction and increased blood pressure. On the basis of the Laplace relation, the wall stress remains normal by decreasing the diameter and increasing the wall thickness of the vessels (Mulvany 2002).

With time, the active vasoconstriction changes to a passive eutrophic remodeling as demonstrated by in vitro experiments (Bakker et al. 2000). Several classes of antihypertensive agents such as ACE inhibitors, angiotensin-receptor blockers and calcium-channel blockers normalize the vascular remodeling (Schiffrin 2001). But not all effective antihypertensive treatments are able to correct the abnormal vessel structure. For example, a -blocker does not reverse resistance artery remodeling even it effectively lowers blood pressure (Thybo et al. 1995).
An unfair tug-of-war causes atherosclerotic endothelial dysfunction
Competition between arginase and eNOS for the substrate L-arginine in endothelial cells decreases NO production, which represents a novel mechanism for atherosclerotic endothelial dysfunction and may explain the controversy of supplemental L-arginine therapy in patients with coronary heart disease.

3.Approaches to the study of essential hypertension

Currently there is controversy as to the best method for investigating the genes involved in the etiology of hypertension. Although there are numerous studies in this area of research, virtually all have provided conicting results. It is likely that in addition to the part played by the environment, epistatic interactions (i.e., interactions among various genes) may inuence the nal phenotype, large sample sizes are required if these interactions are to be studied in detail. In addition, the effect of age on the pathogenesis of hypertension should not be ignored. As hypertension is more common in older people, it is possible that the inuence of genes on blood pressure may also vary with age, or that gene changes related to aging may be among the risk factors for hypertension.

4.Identication of the genes involved in hypertension

The search for the genes involved in the causation of hypertension can be classied as methods that utilize: a) The candidate gene approach; b) Genome-wide linkage screens; c) The study of monogenic hypertension; and d) Animal models of hypertension.

a) Candidate genes
This approach investigates particular genes that are believed to be involved in blood pressure regulation. Most of the early studies on the genes involved in the etiology of hypertension were based in a traditional candidate gene approach and were often dependent on prior knowledge of the gene product in question. However, genes whose products are unknown or believed to be unimportant in the pathogenesis of hypertension are likely to be ignored (Table 8).

Table 8. Candidate genes that may have a role in essential hypertension.

Aldosterone synthase Alpha-adducin a2-Adrenoceptor Alpha subunit of Gs protein Angiotensin II receptor type I Angiotensin converting enzyme (ACE) Angiotensinogen Atrial natriuretic peptide Beta-adducin b2-Adrenoceptor b3-Adrenoceptor b2-Bradykinin receptor gene 11b-Hydroxysteroid dehydrogenase Chemokine receptor 2 gene Dopamine D2 receptor Endothelial nitric oxide synthase

Endothelin-1 Endothelin-2 Epithelial sodium channel beta-subunit Glucagon receptor Glucokinase Human natriuretic peptide receptor type A Human natriuretic peptide receptor type B Insulin receptor Lipoprotein lipase Pertussis toxin sensitive G protein bsubunit Renin Transforming growth factor b1 Tyrosine hydroxylase WNK1 WNK4

b) Genome-wide linkage screens

Genome-wide linkage screens utilize genetic markers spread throughout the genome. The use of densely spread markers in the genome allows the identication of quantitative trait loci (QTL) associated with hypertension. QTL refers to a position in the chromosome that may inuence the trait of interest. This approach eliminates the need for prior knowledge of gene function and allows the identication of loci that may not previously have been suspected to be involved in blood pressure regulation.

Ideogram of chromosome 3q, showing location of candidate genes in GSMA (genome-scan meta-analysis) bins 3.7 and 3.8.

Genome-wide scan revealed modifier loci on distal chromosomes 3 and 7. (A) Linkage obtained by genome-wide QTL mapping in 153 group 1 mice and empirical threshold calculated by permutation testing. Genetic distance calculated on the basis of recombination frequencies observed in the cohort. Yellow and black hatched lines represent empirical genome-wide thresholds for suggestive (P = 0.40) and significant (P = 0.05) linkage. (B) Heterozygosity at the D3Mit352 locus decreases proteinuria. Data are means SEM. (C) Linkage obtained by genome-wide QTL mapping in 243 group 2 mice and empirical threshold calculated by permutation testing. Yellow and black hatched lines represent empirical genome-wide thresholds for suggestive (P = 0.40) and significant (P = 0.05) linkage. (D and E) Heterozygosity at D7Mit44 locus leads to significant increases in plasma urea, creatinine, and K. Data are means SEM.

Although genome-wide scans are useful In detecting loci that may be responsible for hypertension, it should be noted that the possible pathophysiologic role(s) of these sites remain to be determined. A potential source of error in the use of genome-wide linkage screens is the absence of linkage disequilibrium between the gene of interest and the marker, because of recombination during meiosis. As the risk of recombination increases with the distance between the two loci, it follows that genome-wide scans require the use of closely spaced markers in order to produce a meaningful result. The simultaneous study of large numbers of loci increases the chance of obtaining a false positive result. Therefore, the conventional threshold of signicance of P <0.05 may be inappropriate in many genetic studies. Indeed, the independent replication of a positive result is of particular importance in the study of the genes of hypertension.

c) Study of monogenic hypertension

The identication of monogenic (Mendelian) forms of hypertension has contributed greatly to our understanding of the pathophysiology of hypertension. These forms of hypertension are rare but their Mendelian pattern of inheritance has greatly facilitated their study. However, studies aimed at determining whether the genes identied by this modality are involved in essential hypertension have often yielded conicting results.

d) Molecular tools in animal models of hypertension

Since the classic studies of Goldblatt et al. in 1934, animal models of hypertension have been crucial in advancing our understanding of the pathogenesis of this disorder. The use of animal studies overcomes many of the problems associated with human studies. For example, environmental conditions (e.g., salt intake) and genetic homogeneity can be strictly controlled. The identication of QTL or candidate genes in the rodent models of hypertension can lead to the study of homologous loci or genes in humans. Indeed, the public availability of genomic sequences in rats, mice and humans has greatly facilitated this process in recent times.

The selective breeding of animals displaying elevated blood pressure has resulted in the development of useful rodent models of hypertension, such as the SHR, Dahl salt-sensitive and Lyon strains. Indeed, Dahl was one of the rst researchers to show that the pressor effect of salt could be related to genetic factors through his work on the rat strain that bears his name. Crosses between inbred rodent strains can yield valuable information on the QTLs associated with blood pressure. A congenic strain can be dened as the strain in which the chromosomal region featuring the QTL of interest in one strain, the recipient, has been replaced by that of another, the donor. A difference in blood pressure between the recipient and congenic strains would indicate that the QTL in question is associated in the regulation of blood pressure.

The use of genetically engineered animals in the study of the genetics of hypertension was introduced in the late 1980s and has become increasingly widespread. This approach, often referred to as physiologic genomics, usually involves the use of animals in which the gene of interest is overexpressed, underexpressed or deleted. The RAS has been studied extensively in genetically engineered rodent strains. For example, the overexpression of the rat angiotensinogen gene in the mouse results in hypertension. In addition, angiotensinogen knockout mice are hypotensive. The application of this approach is of particular use in the study of complex diseases such as hypertension, as it allows the detailed dissection of the various pathophysiologic pathways. However, the results of experiments with knockout mice should be interpreted with care, as some of the functions of the deleted gene may be taken over by other genes.

Generation of transgenic animals by microinjection of fertilized oocytes, transfer to pseudopregnant females, and screening of progeny (e.g., by Southern blot analysis or PCR).

The introduction of techniques to silence a specic gene, without the manipulation of the host genome, are also useful in the study of hypertension. This can be achieved by one of two mechanisms: 1 The use of antisense molecules; or 2 By the introduction of RNA interference. Antisense technology refers to the use of oligonucleotide molecules that possess a complementary sequence to the mRNA sequence being targeted. This allows the antisense oligonucleotide to form a double-stranded complex with the target mRNA, which is then broken down by the host cell. Indeed, the admininistration of antisense oligonucleotides directed against the constituents of the RAS has resulted in hypotensive effects in rats.

RNA interference refers to an evolutionarily conserved mechanism by which gene expression is suppressed by the presence of homologous RNA. In RNA interference, the presence of short segments of double-stranded mRNA of 2123 nucleotide pairs has been shown to block the expression of homologous mRNA for a period of up to 3 weeks. RNA interference has an advantage over the use of knockout mice in that it allows the blockade of the expression of one or more genes without the need for time-consuming crosses. Interfering RNA has been used successfully to demonstrate the role of WNK-1 gene, which has a major effect on blood pressure, in inuencing the ERK-5 pathway (see section on Monogenic hypertension for a more detailed discussion on the WNK-1 gene). In addition, it is possible that RNA interference may be exploited as a therapeutic tool in the treatment of hypertension. For example, it has been shown that a single dose of interfering mRNA has benecial effects on blood pressure and renal function in a rat model of hypertension.

a) Linkage analysis Linkage analysis refers to the use of the link between the presence of the trait in question (e.g., hypertension) and the inheritance of a particular allele as studied either in sibling-pairs or in two to three generations of affected families. Linkage analysis has good specicity but poor sensitivity for the detection of genes involved in hypertension, and many of the linkage studies published over the past 10 years have been inconclusive because of the large sample size required.

5.Methods used to determine the involvement of the gene or QTL in human hypertension

b) Association studies Association studies are based on a casecontrol design, which allows the recruitment of cases that are unrelated to the healthy controls. In contrast to linkage studies, association studies have good sensitivity but are prone to false positive results. Another problem with many association studies is that such studies concentrate on each candidate gene individually, disregarding other candidate genes. Indeed, most of the association studies published to date have been used to test the involvement of candidate genes. This approach may be inappropriate, as it ignores the complex interactions between the genes involved in hypertension.

Williams et al. showed that interactions between the various alleles of different genes may be more important than allelic variation at a single locus in the determination of blood pressure. Potential confounding factors (e.g., linkage disequilibrium both within a gene and between neighboring genes) are also frequently ignored in most association studies. These shortcomings may explain the inconsistent and often contradictory results generated by the various studies on the genes involved in the etiology of hypertension. Another criticism of association studies in polygenic disorders is that many of these are of a poor quality. Finally, an association between a particular allele and high blood pressure does not imply cause.

c) Epistatic interactions and haplotype analysis Epistatic interactions refer to interactions between the allele of one particular gene and that of another. Studies on epistatic interactions may be of particular use in polygenic disorders such as essential hypertension and are often based on a modication of the association study. For example, a study by Staessen et al. has shown a strong association between hypertension and the presence of a DD homozygosity of the ACE gene, a CC homozygosity of the aldosterone synthase gene and a Gly460Trp heterozygosity of the -adducin gene.

Rearrangement of the physical map according to genetic mapping information. Data from each cohort is color coded (red, FXLE-LEXF; green, HXB-BXH; blue, GK BN). Outer circle, positions of informative SNPs for each cohort. Bars in the inner circle, conflicts in the genetic map. Arrows, relocation, according to minimal recombination fraction, of SNP markers that had extreme genetic distances compared to their physical distance from adjacent markers. For black lines, all crosses support the rearrangement; lime green, HXB-BXH and F2 cross support the rearrangement; orange, unresolved genomic conflicts.

Haplotype analysis refers to the simultaneous study of multiple allelic variations of a QTL or candidate gene. In a modied association study, BrandHerrmann et al. studied four different polymorphisms of the angiotensinogen gene C-532T, A-20C, C-18T and G-6A to determine which of these represented the best gene for functional analysis. They found that C-532T and G-6A had the strongest associations with blood pressure. The complexity of epistatic studies and haplotype analysis means that large numbers of patients are required for a meaningful result to be obtained.

d) Combination of the above The most convincing evidence for the involvement of a particular genes in the etiology of hypertension has come from combining the methods and models mentioned above. As the studies below illustrate, such combinations of methods using different models have proven to be of greatest effectiveness in developing an understanding of the genes underlying and inuencing essential hypertension, and in developing therapies for hypertension.

6.Study of monogenic hypertension and genes related to essential hypertension

Some rare forms of hypertension are caused by the mutation of a single gene. Many single gene forms of hypertension have been extensively studied, and this research has greatly contributed to our understanding of the pathophysiology and pharmacogenomics of hypertension. To date, all known forms of monogenic hypertension are caused by mutations of the genes involved in renal sodium handling.

a)Mutations of genes encoding epithelial sodium channel

Liddle syndrome is an autosomal dominant disorder characterized by low renin hypertension with hypokalemic alkalosis. The disease is caused by mutations affecting the epithelial sodium channel (ENac), which is made up of -, - and -subunits on chromosomes 12p13 (-subunit) and 16p12p13 (- and -subunits). Mutations that either delete or cause a frameshift mutation in the last 4576 amino acids of the Cterminal end of either the - or -subunits of the ENac can cause Liddle syndrome. These mutations reduce clearance of ENac from the cell surface, leading to an increased reabsorption of sodium [48]. In addition, an Asn530Ser mutation of the -subunit has been described in a Finnish patient with Liddle syndrome. The Asn530Ser mutation is believed to increase the probability of the sodium channel being open, with consequent increase in renal sodium reabsorption. As Liddle syndrome is brought about by a defect in the sodium channel, the condition responds to sodium channel antagonists such as triamterene and amiloride but not to spironolactone, an aldoster-one antagonist. Salt restriction is also important in the management of patients with Liddle syndrome. Thiazide and loop diuretics may be useful therapies but, as these agents can aggravate hypokalaemia, serum potassium needs to be monitored closely.
ENaC is regulated by aldosterone on the genomic level. The effect of aldosterone can be divided in 3 phases of: a latent phase, a phase of increasing Na+ transport, and a phase starting after 4 hours of stimulation of the basolateral Na+/K+-ATPase [Benos et al. 1995, J. Membr. Biol.]. The main component is the regulation of the epithelial Na+ channel: a blockade of ENaC by the diuretic amiloride stops the electrogenic Na+ transport (and the effect of aldosterone) completely [Benos 1982, Am. J. Physiol.].

b) Mutations of aldosterone synthase gene

Glucocorticoid suppressible hypertension (GSH) is a form of autosomal dominant hypertension characterized by decreased renin activity and normal or increased levels of aldosterone. The disorder is associated with hypokalemia and metabolic alkalosis. Aldosterone levels in GSH can be suppressed by the administration of exogenous glucocorticoid. GSH is caused by a chimeric gene on chromosome 8q2122 with the regulatory region of 11b-hydroxylase and the coding portion of aldosterone synthase, presumably the result of an unequal cross-over during meiosis. This gene is regulated by adrenocorticotrophic hormone (ACTH) but codes for aldosterone synthase. Therefore, adrenal production of aldosterone is stimulated by the presence of endogenous ACTH. Excess circulating aldosterone stimulates salt and water retention, which results in hypertension. The condition is treated with glucocorticoids, which suppresses ACTH production, with or without the addition of a conventional antihypertensive. Thiazide or loop diuretics should be used with care because of the risk of precipitating hypokalaemia.

The discovery of GSH has led to an interest in the role of the aldosterone synthase gene in the etiology of essential hypertension. A T-344C polymorphism in the promoter region of the gene may be associated with increased aldosterone levels. However, studies on the role of the T-344C polymorphism in the etiology of hypertension have been conicting [5962]. Some researchers have found an association between the T allele and hypertension, while others believe the C allele is more important in the pathophysiology of the disease. The fact that the T-344C single nucleotide polymorphism (SNP) is in complete linkage disequilibrium with another polymorphism, Lys173Arg, further complicates matters. It is uncertain which of these polymorphisms are of clinical importance. Recently, a small Japanese study has shown the additive benecial effect of spironolactone in the regression of left ventricular hypertrophy in hypertensive patients treated with ACE inhibition. This observation provides supplementary evidence of the benet of mineralocorticoid antagonism in cardiac hypertrophy/failure, a treatment that was recently shown to be benecial in severe heart failure patients by the Randomized Aldactone Evaluation Study (RALES)

c) 11- Hydroxylase deciency and 17-hydroxylase deciency

11- Hydroxylase deciency (11HD) is a rare cause of congenital adrenal hyperplasia. Classic 11HD patients present with masculinization of the female external genitalia, precocious pseudopuberty in both sexes and hypertension. Nonclassic forms may produce menstrual abnormalities, hirsutism and acne. The prevalence of nonclassic 11HD is unknown, but the condition may affect as many as 8.4% and 0.66.5% of women with polycystic ovary syndrome and hirsutism, respectively. 11- Hydroxylase is responsible for the production of cortisol. Multiple nonsense, missense and insertion mutations have been described in patients with 11HD. Defects in this enzyme result in an accumulation of steroid precursors which are shunted into the androgenic pathway, resulting in masculinization. Hypertension results from the elevated levels of steroid precursors with aldosterone-like actions (e.g., deoxycorticosterone). Milder forms of 11HD may well represent an intermediate hypertensive phenotype. Indeed, there is some evidence to suggest that 11- hydroxylase activity may be impaired in some essential hypertensives.

Cytochrome P450c17 has both 17-dehydroxyase and 17,20-lyase activity. Defects in cytochrome P450c17 are another cause of congenital adrenal hyperplasia. The pathophysiology of this condition s similar to that of 11HD in that there is impairment of secretion of cortisol, with compensatory hypersecretion of ACTH, which stimulates the production of large quantities of deoxycorticosterone by the adrenal glands. In addition, as 17,20-lyase is equired for the synthesis of gonadal sex hormones, affected males are usually born with female genialia. Females affected by the condition have primary amenorrhoea and hypogonadism. Multiple mutations of the P450c17 gene have been described o cause the disease.

d)Apparent mineralocorticoid excess

Patients with the autosomal recessive condition, apparent mineralocorticoid excess (AME), usually present with hypertension, hypokalaemic alkalosis, an increased cortisol : cortisone ratio, and reduced plasma renin activity. AME is caused by mutations affecting the gene coding for the enzyme 11-hydroxysteroid dehydrogenase type 2 isozyme (11HsD2). This enzyme is responsible for converting cortisol to cortisone in the kidney. This process protects the distal tubular mineralocorticoid receptor from activation by endogenous cortisol, which has the same afnity as aldosterone for the mineralocorticoid receptor. Mutations that deactivate or reduce the activity of 11HsD2 will result in excessive activation of the mineralocorticoid receptor, leading to salt retention. The clinical picture is virtually identical to hypertension that is caused by the chronic ingestion of liquorice, an inhibitor of 11HsD2. AME is treated with the aldosterone antagonist, spironolactone. Sodium channel blockade with amiloride and triamterene are effective alternatives. Mild forms of AME resemble essential hypertension. Li et al. described the heterozygote father of a child with AME with mineralocorticoid hypertension. This has given rise to the hypothesis that some cases of essential hypertension may be caused by defects of the 11HsD2.

Studies of essential hypertension have shown that the half-life of cortisol is signicantly prolonged in hypertensive patients. An impairment in the conversion of cortisol to inactive metabolites has also been reported in young men with hypertension. Lovati et al. have found that salt-sensitivity and 11HsD2 activity is associated with a polymorphic CA repeat in the 11HsD2 gene. A G534A mutation of the 11HsD2 may also be important in determining salt sensitivity, the G allele being associated with a greater increase in blood pressure with a salt load. These ndings may account for the observation that spironolactone may be a useful add-on therapy in the treatment of some hypertensives with low renin hypertension who are resistant to conventional antihypertensives. The relationship between the polymorphic CA repeat and hypertension, however, remains controversial and a recent study failed to nd an association between them.
Schematic diagram depicting the regulation of MR (mineralocorticoid receptors)activation by 11bHSD2 . Glucocorticoids, such as corticosterone, are normally prevented from activating the MR by pre-receptor enzyme inactivation (upper panel). However, when enzyme activity is absent, corticosterone can produce similar pathological cardiac responses to aldosterone (lower panel). Aldo, aldosterone; Cort, corticosterone; HRE, hormone responsive element.

e) Mutation in the mineralocorticoid receptor

A missense mutation that results in the substitution of leucine for serine at position 810 of the mineralocorticoid receptor has recently been described. Individuals heterozygous for the mutant receptor develop hypertension at a young age. The mutant receptor shows activity in the absence of added steroid, but normal activation by aldosterone. This receptor is also activated by the mineralocorticoid receptor antagonists, Progesterone and spironolactone. The former explains the pregnancy-induced exacerbation of hypertension in some of these patients. The mutation may also be related to the early onset of heart failure, providing more evidence in support of the role of mineralocorticoids in heart failure. As spironolactone activates the mutant receptor, unlike in the wild-type receptor where it acts as an antagonist, its use is likely to cause Deterioration in these patients. The exact prevalence of this mutation is unknown, although it is likely to be extremely rare.

f) Gordon syndrome
Gordon syndrome (pseudohypoaldosteronism type II) is characterized by low-renin hypertension, hyperkalaemia, hyperchloremic metabolic acidosis and normal or high plasma aldosterone levels, all of which respond well to thiazide diuretics. Some forms of this syndrome have been ascribed to mutations in either WNK1 or WNK4 genes, which belong to the WNK family of serine/threonine kinases [90]. Wild-type WNK4 is an inhibitor of the tubular thiazide-sensitive sodium-chloride co-transporter, an activity that is absent in the mutant variant associated with Gordon syndrome. WNK1 is believed to inhibit WNK4 activity. Therefore, gain of function mutations in the WNK1 gene would result in inhibition of WNK4 activity, leading to increased sodium and chloride reabsorption. An SNP near the promoter region of the WNK1 gene has been shown to be associated with blood pressure. It has therefore been suggested that increased expression of WNK1 may have a role in the pathogenesis of essential hypertension. Indeed, a recent study suggests that a common WNK1 polymorphism can predict response to treatment with thiazide diuretics. However, further studies are required to conrm this nding before any denite conclusions can be made.

g) Hypertension with brachydactyly

An autosomal dominant form of salt-resistant hypertension associated with brachydactyly has been described in Turkish, US and Canadian kindreds. The condition appears to be related to genetic changes in the short arm of chromosome 12. Indeed, a study has recently found a locus for essential hypertension on chromosome 12p in Chinese patients.

Absence of hypertensive retinopathy (left) and brachydactyly (right) in a Turkish kindred with autosomal dominant hypertension and brachydactyly

One additional piece of evidence against the "hypetensive nephrosclerosis" hypothesis is the existence of a rare, autosomal recessive disorder termed "Hypertension/Brachydactyly Syndrome", the gene for which has yet to be identified. These individuals suffer brachydactyly a relative shortening of the fingers and toes, see picture) and severe hypertension, with systolic blood pressures often well greater than 200. The mechanism of their hypertension is not entirely clear, but what is interesting in this small cohort of patients is that even after decades of exposure to blood pressures in the "malignant" range, very few develop renal damage. They do have stroke commonly, but not renal disease.

h) Familial hyperaldosteronism type II

This disorder has an autosomal dominant inheritance and is characterized by nonglucocorticoid remediable hyperaldosteronism with bilateral adrenal hyperplasia or aldosterone producing adenomas. The genetic cause of this condition is not known.

Although single-gene disorders have led to intriguing clues to hypertension, most of the research on essential hypertension has involved association and linkage studies focused on candidate genes. Although some gene variants appear to be associated with hypertension, results have not been consistently reproducible.

i) Angiotensinogen
Multiple SNPs have been described in the angiotensinogen gene. The M235T variant has been studied extensively. The T235 allele is associated with raised plasma angiotensinogen , which in turn may be correlated with blood pressure. T235 has been associated with hypertension in some studies, but not in others. M235T is in linkage disequilibrium with the G-6A and A-20C polymorphisms in the promoter region of the angiotensinogen gene. The G-6 allele is associated with a lower transcription rate for angiotensinogen, while the presence of A-20C allele can increase angiotensinogen transcription. M235T is also in linkage disequilibrium with at least three other polymorphisms: T+68C, C18T and T+31C.

Another polymorphism, M174T, may be involved in hypertension in some populations, but not others. Other angiotensinogen gene polymorphisms include C532T, which is in linkage disequilibrium with G-6A, and an Arg-30Pro substitution in the signal peptide of angiotensinogen. These polymorphisms may also inuence plasma angiotensinogen levels. Hunt et al. showed that individuals with the -6A allele are more salt sensitive and more likely to benet from salt restriction. Patients with the -6A allele may demonstrate a larger fall in blood pressure following weight loss when compared with -6G homozygotes. As Hunt et al. point out, there may be a particular genotype that would especially benet from increased vigilance in salt intake and/or weight monitoring. However, Giner et al. failed to nd an association between salt sensitivity and the M235T allele. Hypertensives with at least one T235 allele have been shown to require a higher dosage level of antihypertensive medication compared with M235 homozygotes. The angiotensinogen Gene locus may inuence an individuals response to ACE inhibition. However, more studies are required to conrm the pharmacogenomic role of the angiotensinogen locus.
Just like the components of a jigsaw puzzle, angiotensinogen and rennin have been created to be able to wrap around one another. Rennin changes the structure of the angiotensinogen molecule, and a brand-new molecule emergesangiotensin I.

j) 2-Adrenoceptor
The 2-adrenoceptor locus has been linked to hypertension. Two polymorphisms, Arg16Gly and Gln27Glu, have been studied in detail. The Gly16 variant of the receptor demonstrates increased downregulation after stimulation by isoproterenol compared to the Arg16 isoform. Such a response can lead to impaired vasodilatation to 2-adrenergic stimulation. Kotanko et al. found an association between the Gly16 allele and hypertension. However, Timmerman et al. discovered that the Gly16 allele is associated with lower blood pressure. Other studies have also shown conicting results. Atenolol and other beta-blockers used to treat hypertension can cause or aggravate bronchoconstriction.

A. Reverse transcription-polymerase chain reaction analysis of -adrenoceptor mRNA in porcine coronary arterioles and myocardial tissue was performed with the use of gene-specific primers for the 2-adrenoceptor (2-AR) and the 1-adrenoceptor (1-AR).

Therefore, it is of great clinical interest that the 2-adrenoceptor polymorphisms may have a role in determining airway reactivity. Patients homozygous for the Glu27 allele have decreased bronchial sensitivity to methacholine compared to those with at least one Gln27 allele. The presence of the Gly16 allele may be associated with increased downregulation, resulting in agonist desensitization when the patient is treated with beta-agonists. Indeed, asthmatic children with the Arg16 allele are more likely to respond to albuterol compared to Gly16 homozygotes. A better understanding of the interaction between beta-blockers, 2-adrenoceptor polymorphisms and bronchoconstriction could lead to the identication of a subset of patients who are likely to suffer from this adverse effect. However, it is premature to comment on the implications of the 2-adrenoceptor polymorphisms on the treatment of hypertension.

B. 2-AR transcripts from the subepicardial (EPI, n=3) and subendocardial (ENDO, n=3) arterioles were normalized with the corresponding GAPDH transcripts. *P<0.05 vs EPI arterioles. Marker=X174-DNA marker. n=number of independent experiments. B, Immunohistochemical analysis of 2-AR protein in EPI and ENDO arterioles, shown as a pseudocolor spectral display, was performed in vessels treated without (1 ) or with (+1 ) anti 2-AR primary antibody. Level of 2-AR protein expression was represented by the signal intensity of the color pallet. Similar results were obtained in 3 independent experiments.

7.Pharmacogenomics
Pharmacogenomics can be dened as the application of genomic technology, such as gene sequencing and microarray technology, to the development of a pharmacologic agent. The term thus encompasses the elds of pharmacogenetics and gene therapy, which elds refer, respectively, to the study of the effects of genotype on the response to a particular drug and the use of genetic material as a treatment for a disorder. Pharmacogenomic technology can therefore potentially be applied to any stage in drug development: from the initial development of the pharmacologic agent, through the preclinical tests, to the nal clinical trials. In addition, it is hoped that the application of pharmacogenomics can result in the development of personalized medicine, in which the use of a particular drug or drug combination can be tailored to the genotype of a patient. This is particularly true for a condition such as hypertension, for which a bewildering array of medications are available and which shows considerable interindividual variation to treatment by any given drug.

8.Drug isolation
Ferrari et al. have shown that a digitoxigenin derivative, PST 2238, can decrease the blood pressure and Na-K pump activity in the MHR but has no effect on normotensive controls. This agent also has more effect in the Na-K pump of cell lines expressing mutant -adducin compared with wild-type controls. These observations are of great interest, as they suggest PST2238 (or related compounds) may potentially be used in the future in the subgroup of hypertensives with the 460Trp allele.

9.Interindividual response to treatment

Early pharmacogenetic studies mostly focused on the effects of genetic variation on the metabolism (i.e., pharmacokinetics) of antihypertensive drugs. Cytochrome P450 CYP2D6 (CYP2D6) metabolizes many of the commonly used agents (including the betablocker, metoprolol), and polymorphisms in this enzyme can affect drug plasma levels and half-life. A clinically important polymorphism of the CYP2D6 enzyme was rst noticed during a trial involving the adrenergic ganglion-blocking antihypertensive drug debrisoquine. It was found that patients who are poor metabolizers of debrisoquine have an exaggerated hypotensive effect to treatment by the drug, which is one of the reasons why debrisoquine is not widely used in clinical practice. However, although individuals who are poor metabolizers of debrisoquine also have prolonged elimination half-life of metoprolol, they do not appear to have an increased incidence of adverse effects at therapeutic doses of the betablocker .

Catechol-O-methyltransferase (COMT) is reponsible for the methylation of catecholamines and drugs containing the catechol group (e.g., evodopa and methyldopa). Variation in COMT activity is most commonly because of two different alleles, one of which codes for an enzyme with high activity while the other has low activity. Individuals who have the high activity form of COMT exhibit enhanced breakdown of methyldopa. Therefore, these individuals may need a higher dose of the drug for the desired pharmacologic effect. Conversely, individuals with the low activity form of the enzyme may be more susceptible to the toxic effects of the drug. Another enzyme, acetyltransferase, is responsible for the acetylation of the drug hydralazine. Patients who are slow acetylators of the drug re at higher risk of developing hydralazinenduced systemic lupus erythematosus. More recently, efforts have been made to identify polymorphisms in the genes involved in blood pressure regulation that may be implicated in the interindividual variation to treatment by antihypertensive drugs. In other words, these studies aim to research the direct interaction (i.e., the pharmacodynamic relationship) between drugs and polymorphisms of these genes. Genes that have been evaluated for their pharmacodynamic effects include -adducin, angiotensinogen, pertussis toxin sensitive G protein -subunit, ACE I/D polymorphism, 1-adrenergic receptor, WNK1 and AGTR1. However, at present, there is no consensus as to the effect (if any) of polymorphisms of these genes on the pharmacodynamics of essential hypertension.

10.Gene therapy

The recent advances in our understanding of hypertension have stimulated interest into gene therapy for hypertension, although research in this eld is still restricted to animal studies. Research into gene therapy in hypertension can be classied into studies based on the overexpression of a gene known to have a hypotensive effect and those suppressing the effect of genes that have pressor effect on blood pressure. Both of these approaches require a thorough knowledge of the genetic sequence of the gene of interest. An example of the latter approach is the use of antisense oligonucleotides against the mRNA of the gene of interest. Antisense oligonucleotides are short segments of DNA which are designed to bind to the corresponding parts of the target mRNA. This interaction prevents the translation of the target mRNA by ribosomes and stimulates its breakdown by RNAse H. The use of antisense therapy has so far focused on the mRNA of genes of the RAS. Animal models have shown a marked reduction of blood pressure in rats after the injection of antisense DNA directed against AGTR1.

In addition, the hypotensive effect was prolonged, with a reduction in blood pressure for up to 9 weeks. However, it should be borne in mind that the study was carried out by the injection of the DNA-containing viral vector into the cerebral ventricles or the hypothalamus. More recently, it was shown that the use of intravenous antisense oligonucleotides directed against AGTR1 can reduce the blood pressure effectively for 9 days after a single injection. Some of the other genes that have been targeted successfully by the antisense approach: Angiotensinogen; AGTR1; 1-Adrenergic receptor; c-fos; CYP4A1; Thyrotropin releasing hormone; Thyrotropin releasing hormone receptor; Urinary kininase.
General scheme for the construction and production of viral gene therapy vectors. Although many different naturally occurring (wild-type) viruses have been used to make gene therapy vectors to carry therapeutic genes into cells and tissues, the general scheme for engineering these vectors is based on a common principle Essential viral genes (VG) are deleted from the wild-type virus genome, rendering the virus nonreplicating and incapable of causing human disease (1). This also creates space within the viral genome to insert foreign genetic material, such as a therapeutic gene (TG; 2). Once the therapeutic gene is inserted the viral vector is replicated in specialized cells that express the essential wild-type viral genes that are required by the virus (3). After purication steps the viral vector is introduced into targeted cells, organs or tissues into which it carries the TG (4). These normal human cells do not contain the essential viral Genes that are missing from the vector, thus the vector cannot replicate in these cells. The TG, however, is expressed in these cells.

The overexpression of genes known to have a hypotensive effect has also been shown to be successful in lowering blood pressure in animal models. The administration of the human kallikrein gene using an adenovirus vector resulted in a marked reduction in blood pressure in the deoxycorticosterone acetate salt-sensitive (DOCA) rat. In addition, the use of kallikrein gene therapy may prevent end organ damage in this rat model of hypertension. It has been hypothesized that the protective effect of kallikrein is mediated by the prevention of end organ brosisand the suppression of free radical production. Other genes whose overexpression has led to a decrease in blood pressure include the adrenomedullin gene, atrial natriuretic peptide and parathyroid hormone-related protein.

Viral vectors used in gene delivery.

11.Drug synthesis
For the past few hundred years, the medical treatment of disease has been by the use of chemical agents that are either extracted from natural sources or synthesized chemically. In the last decade, the use of recombinant biologic agents in the treatment of disease has become increasingly common. This is a development that would not have been possible without the use of genomic technology. An example of this class of agent is the use of the natriuretic peptides.

12.Future perspectives
The use of genomic technology has not led to the detection of a genetic polymorphism that may lead to essential hypertension in humans. However, there have been great advances in our understanding of the role of various genes in blood pressure regulation. Indeed, this has been marked by the increasing use of genetically engineered animals and inhibition of gene expression to investigate the effect of modulating the effect of the gene of interest. It is likely that a better knowledge of the molecular pathophysiology of hypertension will provide novel targets for pharmacologic intervention, such as PST 2238. In addition, better understanding may facilitate the development of gene therapy for the treatment of hypertension. Gene therapy for hypertension provides a potential route for effective prolonged blood pressure control with a single course of treatment, thus overcoming the problem of treatment failure resulting from poor compliance. Finally, a better understanding of the interaction between genes, the environment and drugs may facilitate the matching of a particular drug to the clinical requirements of the individual patient.

Clinical gene therapy trials in patients with cardiovascular diseases.

The modular structure of zinc nger proteins can be exploited to engineer highly specic transcriptio n factors or endonucleases. Transcription factors generally contain a DNA-binding domain that denes what gene(s) the factor will regulate, and a functional domain that denes what the nature of that regulation will be. Eachnger of a zinc nger protein (ZFP) transcription factor binds a specic 3 base pair (bp) sequence on the sense strand of the targeted gene (a). By combining multiple ngers together longer and longer DNA sequences can be specically targeted (e.g., a six nger ZFP will target an 18-bp sequence). By genetically fusing this DNA-binding domain to a specic functional domain (b), the ZFP ngers can be used to either turn on (activate) gene expression, or turn off (repress) gene expression. This same DNA-binding domain can be used to direct other functions such as DNA cutting (endonuclease function that can be used to repair defective genes) or site-specic insertion of genetic material (integrase function).

Correcting gene defects with engineered endonucleases. Many cardiovascular diseases are caused by or associated with gene mutations. The ability to engineer DNA-binding proteins that can bind with high specicity to targeted DNA sequences allows the creation of enzymes that will cut DNA at only the point specied. Two separate DNAbinding proteins are targeted to separate specic DNA sequences around a point mutation in a defective gene (a and b). These DNA-binding proteins are fused to the FOK1 endonuclease domain to create engineered sitespecic DNA-cutting enzymes. FOK1 requires dimerization with a second FOK1 domain to become functional. Thus, DNA cutting will not occur unless both engineered endonucleases bind to their closely separated and unique targeted sites (b). Creation of a site-specic double-strand break in the DNA facilitates recruitment of the cellular DNA repair machinery (b,c). A small double-strand DNA segment encoding the normal (corrected) gene sequence is provided during DNA repair, and the repair process results in incorporation of this corrected gene sequence (c,d).

13.Prediction of incidence of hypertension

Prediction of incidence of hypertension in Kursk city up to 2020 is relatively favorable and is characterized by some positive dynamics to reduce diseases. Allegedly in 2020, there will be a significant reduction in the incidence of hypertension compared with 2007 year (2007, 2 pers. per 1000 population). The incidence of hypertension in Kursk in 2020 would be about 1.3 persons per 1000 population and almost reaches the level of 2005 year (in 2007 1.2 pers. per 1000 population).
3,5 3

y = -0,0004x + 1,3076

incidence

2,5 2 1,5 1 0,5 0 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 .

years
prognosis

incidence

Prediction of incidence of hypertension in Kursk city up to 2020.

14.The associations of combinations of genotypes of intragenic polymorphisms in the studied groups of healthy and hypertensive patients taking into account sexual dimorphism were investigated.
GENOTYPES CORRELATIONS RISK OF ESSENTIAL HYPERTENSION INCREASES

MEN
731AA CYP1A2 731AA CYP1A2 731AA CYP1A2 731AA CYP1A2 731AA CYP1A2 731AA CYP1A2 731AA CYP1A2 731AA CYP1A2 287RR EPHX2 (OR=2,17), 590FS2 NAT2 (OR=2,93), 00 GSTT1 (OR=4,71), 114AA GSTP1 (OR=3,29), 3435CT MDR1 (OR=4,05), 25RR TGF 1 (OR=2,62), 460GW ADD1 (OR=3,25), 235TT AGT (OR=5,27), etc.

MEN
731CA CYP1A2 731CA CYP1A2 731CA CYP1A2 731CA CYP1A2 731CA CYP1A2 731CA CYP1A2 C1C1 CYP2E1 C1C1 CYP2E1 C1C1 CYP2E1 (OR=0,44), 287RQ EPHX2 (OR=0,15), 590FF NAT2 (OR=0,46), ++ GSTT1 (OR=0,45), 114AV GSTP1 (OR=0,07), 460GG ADD1 (OR=0,39), 287RQ EPHX2 (OR=0,38), ++ GSTT1 (OR=0,38), etc.

WOMEN WOMEN

432VL CYP1B1 432VL CYP1B1 432VL CYP1B1 432VL CYP1B1 -50GT CYP2J2 311SS PON2 311SS PON2

311SS PON2 (OR=2,35), 460GG ADD1 (OR=2,30), 1166AC AGTR1 (OR=2,06), 4582CC MLR (OR=2,77), 272GG GNB3 (OR=6,08), 114AA GSTP1 (OR=2,93), 3435TT MDR1 (OR=3,48),etc.

432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 432LL CYP1B1 -50GG CYP2J2

114AV GSTP1 (OR=0,14), 3435CC MDR1 (OR=0,38), 460GG ADD1 (OR=0,50), 272GS GNB3 (OR=0,05), 825CC GNB3 (OR=0,36), 1166AA AGTR1 (OR=0,40), 311SC PON2 (OR=0,44), etc.

RISK OF ESSENTIAL HYPERTENSION DECREASES

*The odds ratio (OR) is a measure of effect size, describing the strength of association or non-independence between two binary data values. It is used as a descriptive statistic, and plays an important role in logistic regression. Unlike other measures of association for paired binary data such as the relative risk, the odds ratio treats the two variables being compared symmetrically, and can be estimated using some types of non-random samples.

15.