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Lipoprotein lipase and apolipoprotein E polymorphisms : relationship to hypertriglyceridemia associated… McGladdery, Sandra Helen 2000

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Lipoprotein Lipase and Apolipoprotein E Polymorphisms: Relationship to Hypertriglyceridemia Associated with Pregnancy by Sandra Helen McGladdery B.M.L.Sc, University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 2000 ©Sandra McGladdery, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of r > ' ^ e . ^ 3 r ^ £ ^ 4 W ^ ^ {^OOCcJ^oc^ The University of British Columbia ^ V C ^ C A C O ? Vancouver, Canada Date S e p V g>\ / DE-6 (2/88) ABSTRACT Normal pregnancy is associated with a mild increase in plasma total cholesterol (TC) and a 3 to 4-fold increase in plasma triglycerides (TG). Plasma T G concentration is determined by the balance between the rate of production of TG-rich lipoproteins and the rate of removal of these lipoproteins from the circulation by lipolytic enzymes such as lipoprotein lipase (LPL) and hepatic lipase (HL) and its subsequent uptake by the liver through apo E receptor. Complete deficiency of LPL is manifested as chylomicronemia, a rare autosomal co-dominant disorder. While heterozygous individuals rarely present with chylomicronemia they may have a milder form of hypertriglyceridemia. Variations in the apo E gene have also been associated with increases in plasma TG in addition to changes in plasma TC, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Because of the overproduction of TG-rich VLDL, normal pregnancy challenges the lipolytic capacity of LPL and the ability to clear remnants via the apo E receptor. Cases of chylomicronemia and pancreatitis occurring during pregnancy have been reported in previously healthy women. During the course of pregnancy, LPL and apo E polymorphisms may cause T G levels to increase. It is hypothesized that pregnant women carrying some of these polymorphisms will develop more severe hypertriglyceridemia during the course of pregnancy. The objective of this thesis is to investigate the impact of three known LPL polymorphisms (Asp9Asn, Asn291 Ser, Ser447X) and the apo E genotype (4/4, 4/3, 3/3, 3/2, 2/4, 2/2) on lipid levels during pregnancy. Two hundred and fifty healthy women in the 3 r d trimester of pregnancy were recruited. Fasting plasma TG, TC, HDL-C, LDL-C, insulin, glucose and fractional esterification rate of HDL (FERHDL) were measured. Analysis of the LPL and apo E genes' polymorphisms were performed, in addition to ii sequencing of the LPL gene in 5 women with the highest T G levels. The frequencies of the LPL ( D 9 N , 0 . 9 % ; N 2 9 1 S , 4 . 6 % ; S 4 4 7 X , 1 8 % ) and the apo E ( E 2 , 7 . 6 % ; E 3 , 8 1 . 4 % , E 4 , 1 1 . 0 % ) polymorphisms were similar to previously published results in non-pregnant women. Carriers of S 4 4 7 X had significantly lower TG levels (p=0.003), and carriers of the N 2 9 1 S had significantly lower HDL-C levels (p<0.02) and higher F E R H D L (p=0.007) than the non-carriers. The small number of D 9 N carriers did not permit statistical analysis of the data. Possession of the E 2 allele was associated with significantly lower levels of TC, LDL-C and F E R H D L (p<0.05) compared to E 3 / E 3 , and carriers of the apo E 4 allele had increased plasma insulin levels compared to E 3 / E 3 . Sequencing of the LPL gene lead to the discovery of a new intron mutation in one of the women, which may be, at least in part, responsible for her increased T G levels. These findings support the notion that LPL and apo E polymorphisms play an important role in TG metabolism. We demonstrated that this observation extends to the pregnant state. The effect of these polymorphisms during pregnancy on lipid levels and its relationship to future risk of coronary artery disease in these women remains unclear and requires further study. in TABLE OF CONTENTS A B S T R A C T ii TABLE OF CONTENTS iv LIST OF AMINO ACIDS vii ABBREVIATIONS viii LIST OF TABLES ix LIST OF F IGURES x A C K N O W L E D G E M E N T S x'\ 1. INTRODUCTION 1 1.1. LIPID AND LIPOPROTEIN METABOLISM 1 1.1.1. Lipids 1 1.1.2. Lipoproteins 2 1.1.2.1. Exogenous Path way 6 1.1.2.2. Endogenous Pathway 7 1.1.2.2.1. Reverse Cholesterol Transport 7 1.2. DYSLIPIDEMIA AND A T H E R O S C L E R O S I S 9 1.3. BIOCHEMICAL GENETICS OF LIPOPROTEIN LIPASE 10 1.3.1. Overview 10 1.3.2. LPL Gene and Protein Structure 12 1.3.3. LPL and Hyperlipidemia 13 1.3.4. LPL Asp9Asn, Asn291 Ser, and Ser447X Polymorphisms 17 1.4. BIOCHEMICAL GENETICS OF APOLIPOPROTEIN E 20 1.4.1. Gene and Protein Structure 20 1.4.2. Allelic Effect in the Normal Population 21 iV 1.4.3. Apolipoprotein E and Hyperlipidemia 1.5. P R E G N A N C Y 1.5.1. Lipid Metabolism in Pregnancy 1.5.2. Role of Hormones in Lipid Metabolism in Pregnancy 1.5.3. Physiologic Mechanisms Affecting Lipoprotein Metabolism Pregnancy 1.5.4. Causes of Dyslipidemia in Pregnancy 1.6. RATIONALE FOR THIS STUDY 1.7. H Y P O T H E S E S 1.8. SPECIFIC AIMS MATERIALS AND METHODS 2.1. MATERIALS 2.2. STUDY PARTICIPANTS 2.3. P L A S M A LIPID ANALYSIS 2.4. F R A C T I O N A L E S T E R I F I C A T I O N R A T E OF HDL (FERHDL) 2.5. INSULIN AND G L U C O S E A S S A Y S 2.6. DNA ANALYSIS 2.6.1. PCR-Based Detection of the Asp9Asn Allele 2.6.2. PCR-Based Detection of the Asn291 Ser Allele 2.6.3. PCR-Based Detection of the Ser447X Allele 2.6.4. PCR-Based Detection of Apolipoprotein E Genotype 2.7. LPL G E N E S E Q U E N C I N G 2.8. STATISTICAL A N A L Y S E S RESULTS 3.1. C O H O R T CHARACTERISTICS 3.2. F R E Q U E N C Y OF LPL VARIANTS 45 3.3. E F F E C T OF LPL G E N O T Y P E ON P L A S M A LIPIDS 45 3.4. F R E Q U E N C Y OF APOLIPOPROTEIN E VARIANTS 47 3.5. E F F E C T OF APOLIPOPROTEIN E G E N O T Y P E ON P L A S M A LIPIDS 48 3.6. E F F E C T OF A P O E AND LPL POLYMORPHISMS 50 3.7. LPL S E Q U E N C I N G 52 4. DISCUSION 53 4.1. C O H O R T CHARACTERISTICS 53 4.2. LIPOPROTEIN LIPASE POLYMORPHISMS AND P L A S M A LIPIDS 56 4.3. APOLIPOPROTEIN E AND P L A S M A LIPIDS 59 4.4. G E N E - G E N E INTERACTIONS OF LPL AND A P O E POLYMORPHISMS ON P L A S M A LIPIDS 63 4.5. LPL S E Q U E N C I N G 65 4.6. GENETIC POLYMORPHISMS IN LPL AND A P O E AND RISK OF CAD 66 4.6.1. Lipoprotein Lipase 67 4.6.2. Apolipoprotein E 69 4.7. IS P R E G N A N C Y A RISK F A C T O R FOR CAD? 70 4.8. FUTURE DIRECTIONS 71 5. REFERENCES 73 6. APPENDICES % VI LIST OF AMINO ACIDS Amino Acid Single Letter Three Letter Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamic acid E Glu Glutamine Q Gin Glycine G Gly Histidine H His Isoleucine 1 He Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val LIST OF ABBRIVIATIONS A N O V A Analysis of variance apo Apolipoprotein BMI Body mass index C E Cholesterol ester C E T P Cholesteryl ester transfer protein CHD Coronary heart disease DNA Deoxyribonucleic acid EDTA Ethylenediamine tetra-acetic acid EDL Endothelial lipase F E R H D L Fractional esterification rate of HDL FFA Free fatty acids FH Familial hypercholesterolemia HDL High density lipoprotein HDL-C HDL cholesterol HL Hepatic lipase IDL Intermediate density lipoprotein LCAT Lecithin: cholesterol acyltransferase LDL Low density lipoprotein LDL-C Low density lipoprotein cholesterol LP Lipoprotein LPL Lipoprotein lipase MG Monoglycerides mRNA messenger RNA P C R Polymerase chain reaction PL Pancreatic lipase PTA Phosphotungstic acid RCT Reverse cholesterol transport TC Total cholesterol TG Triglyceride TLC Thin layer chromatography UTR Untranslated regions VLDL very low density lipoprotein WT wild type LIST OF TABLES Table 1-•1: Lipoprotein classes and properties 4 Table 1-•2: Characteristics of common LPL polymorphisms (D9N, N291S, S447X) 19 Table 1-•3: Apolipoprotein E alleles and association with lipid levels and CAD 22 Table 3--1: Study participants' personal and family history of various disorders 43 Table 3--2: Cohort lipid levels 44 Table 3--3: Frequency of LPL polymorphisms 45 Table 3--4: Lipid levels of N291S carriers and non-carriers 46 Table 3--5: Lipid levels of S447X carriers and non-carriers 47 Table 3-•6: Apo E allele frequencies 48 Table 3--7: Apo E polymorphisms and plasma lipid levels 49 Table 3--8: Frequency of the combined apo E and LPL genotypes 50 Table 3--9: Effect of apo E polymorphisms and WT LPL on lipid levels 51 Table 3--10: Sequencing results, lipid levels and genotypes of the five women with elevated T G levels 52 LIST OF FIGURES Figure 1-1: Classification by density of lipoprotein particles 3 Figure 1-2: Exogenous and endogenous pathways of lipid metabolism 5 Figure 2-1: Restriction digest and gel of LPL D9N 37 Figure 2-2: Restriction digest and gel of LPL N291S 38 Figure 2-3: Restriction digest and gel of LPL S447X 39 Figure 2-1: Restriction digest and gel of Apo E 40 Figure 3-1: Breakdown of the various ethnic groups within the cohort 42 Figure 3-2: Frequency of triglyceride levels during pregnancy 44 x Acknowledgments I would like to recognize my academic supervisor Dr. Jiri Frohlich for providing his valuable guidance, patience, and enthusiasm. I would also like to thank Drs. Haydn Pritchard and John Hill for giving me the opportunity to fully appreciate the technical aspects of my work. Lastly, I would like to thank all those from the Atherosclerosis Specialty Laboratory and individuals who have contributed to my work over the last three years. Dedication I dedicate this thesis to my family who has always provided their support, guidance, love and encouragement throughout the years. They are the reason was successful in completing this degree. CHAPTER I: INTRODUCTION 1.1 Lipid and Lipoprotein Metabolism 1.1.1 Lipids Lipids are a diverse group of molecules which are insoluble in water. They are divided into two large classes 1) fatty acid derivatives which include triglycerides (TG), phospholipids and fatty acids, and 2) isoprenoids which include cholesterol, and cholesteryl esters (CE). Most lipids are non-polar whereas others are amphipathic, containing both polar and non-polar regions. Another unique property of lipids is their ability to form micelles and lipoproteins spontaneously in water. It has been suggested that this trait is critical for the development of living organisms. Lipids perform a wide variety of functions. Phospholipids are the principal architectural components of plasma membranes, making up about 50% of their total mass. Cholesterol is also essential for the integrity of membranes by decreasing the permeability and increasing the stability and flexibility of the membrane. In addition, cholesterol is a precursor in the synthesis of many bioactive molecules such as steroids, vitamin D and bile acids. About 70% of the cholesterol in plasma is in the form of cholesteryl esters. Triglycerides are stored as droplets inside adipocytes and make up the majority of energy stores within the body. 1 1.1.2 Lipoproteins Cells are able to synthesize lipids but not very efficiently. The body has therefore developed an elaborate system for absorbing exogenous fats and for distributing lipids to the tissues in a soluble form. This is accomplished by specialized particles called lipoproteins. Lipoproteins are complex structures, which consist of a hydrophobic lipid core surrounded by a layer of phospholipids, free cholesterol, and amphipathic proteins called apolipoproteins. They are classified according to their density, size, and also by their apolipoprotein (apo) content such as apo B and other apolipoproteins (Lp A-l , Lp A-I:A-II, Lp A-IV, and Lp A-IV.A-I) [1] (Table 1-1). Classification of lipoprotein particles by size is clinically significant with respect to the likelihood of developing coronary heart disease (CHD). Higher levels of large, less dense high density lipoproteins (HDL) appear to protect individuals from CHD [2], whereas higher levels of small, dense low density lipoproteins (LDL) have a more atherogenic effect [3] (Figure 1.1). Particle size heterogeneity can be determined using the fractional esterification rate of apo B depleted plasma (FERHDL) as a probe for lipoprotein size distribution. F E R H D L is associated with HDL size distributions such that increased F E R H D L reflects higher levels of HDL3 and lower levels of HDL2 [4, 5]. F E R H D L is also governed by the T G content of the HDL particle and is correlated with LDL particle size [6]. The composition of apolipoproteins on the surface of lipoproteins determines these particles' affinity for lipids and also determines their metabolic fate by binding to specific receptors. Lipoproteins exchange apolipoproteins between themselves and also deliver lipids to the tissues. Plasma enzymes participate in these exchanges, resulting in a system that is constantly moving apolipoproteins and other components between different lipoprotein particles. A change in particle size also affects the conformation of apoproteins residing on the lipoprotein surface and thus their ability to bind various receptors. These interactions make up a complex and dynamic process in which the rate of metabolism of various particles' continuously changes as lipoprotein pools appear and disappear from the circulation. Density (g/mL) Lipoprotein Subclasses V L D L 10 20 40 60 Diameter (nM) Chylomicron Chylomicron Remnant 80 1000 Figure 1-1: Classification by density of lipoprotein particles. T3 O JZ Q . W O O >_ o (A 0) O c o a o a o a < c '53 4-1 o Q. © © I t E i , « flj «=» la- i=; a> O) Q ^ O < O < LU ^ O O O O . c a a a Q . J = < < < < O oo CM -^ r o CO 00 T - T -O O CM • o CO m o V c o o 1 o >» JZ o 00 CO 0 s CD CD CQ O LU o o o Q. CL C L _ < < < ~ vO yfi 0 s 0 s vO in m m CM m t— m O CD O O m CO a > CO CM in o CD DO. O Q. _ en in O CM o m m CO 00 CM o 00 CO 00 CM CD CD O O 00 CD O CD O 00 CO CM i i < < i_ o o £ Q. Q. ~ < < O m m CO CM O m o CM in CM oo CD o a X The metabolism of lipoproteins is influenced by environmental factors including diet, drugs, and disease, as well as genetic factors. High dietary intake of saturated fats will stimulate very low density lipoprotein (VLDL) synthesis and decrease high density lipoprotein (HDL) levels. Diets high in carbohydrates will also affect plasma VLDL. Sex hormones, insulin, and thyroid hormones also modulate lipoprotein synthesis and degradation through transcriptional or post-transcriptional mechanisms. There are two major pathways in lipid metabolism, the exogenous and the endogenous pathway, which includes reverse cholesterol metabolism (Figure 1-2). Exogenous Path Endogenous Path VLDL Liver Reverse C E T P j Cholesterol L D l Transport Remnant? J Chylomicrons |_pj_ Figure 1-2: Exogenous and endogenous pathways of lipid metabolism. VLDL 5 1.1.2.1 Exogenous Pathway The exogenous pathway is the initial step in the metabolism of dietary fat. Bile acids and phospholipids form micelles in the intestine. Subsequently, the dietary triglycerides are broken down to free fatty acids (FFA) and monoglycerides (MG) by pancreatic and intestinal lipases. The FFA and MG are taken up by the intestinal cells as is dietary cholesterol (mostly in the form of cholesteryl ester, which is hydrolyzed to free cholesterol and fatty acids by cholesterol esterase). The long chain fatty acids and cholesterol are coupled with apo B-48 to form chylomicrons [7]. Enterocytes then secrete the chylomicrons into the thoracic duct through which they enter into plasma. Chylomicrons are the largest lipoprotein with a diameter of about 1000 angstroms in size. They consist mostly of TG which makes up roughly 90% of their total mass (Table 1-1). They are synthesized in the intestine and contain apolipoproteins A- l , A- l l , A-IV and B-48 [8]. The total protein mass of a chylomicron is approximately 2%. While in the plasma they acquire apo C (apo C-l, apo C-ll, and apo C-lll) and apo E from HDL [9-11 ]. Apo C-ll is bound to the surface of the chylomicron and is crucial in the activation of lipoprotein lipase (LPL), which hydrolyses TG. Chylomicrons lose most of their T G via lipolysis by LPL. The released fatty acids bind serum albumin and are taken up by adipose and muscle tissues. The chylomicrons, now poor in TG, release phospholipids, apo A- l , apo A-IV, and apo C-l, II, and III to HDL in exchange for apo E and cholesteryl esters [12]. The new particles, called chylomicron remnants, are taken up by the liver via the apo E receptor. This pathway ensures that lipids are delivered to organs such as muscle, adipose, and liver. 6 1.1.2.2 Endogenous Pathway The endogenous pathway includes the synthesis of VLDL by the liver. VLDL particles contain about 60% TG, 20% cholesterol ester, and 10% protein, which is almost exclusively apo B-100 (Table 1-1). Following secretion, VLDL takes up apo C (C-l, C-l l , C-lll) and apo E from HDL in exchange for TG [11]. These particles then undergo lipolysis by LPL. VLDL then takes up apo E from HDL forming VLDL remnants or intermediate density lipoproteins (IDL). These particles may then either be removed by the liver (via apo E receptors) or be further catabolized by hepatic lipase (HL) to form low density lipoprotein (LDL). LDL, formed by the catabolism of VLDL, contains about 40% cholesteryl ester and a single molecule of apo B-100, which makes up 20% of its total mass. The apo B-100 targets LDL to the LDL receptor found on every cell type, but in higher levels on cells requiring cholesterol (adrenals and liver). Roughly 10% of LDL are found associated with apo (a) to form Lp (a). The role of this particle is unknown but it is associated with increased risk of atherosclerosis [13, 14]. 1.1.2.2.1 Reverse Cholesterol Transport Reverse cholesterol transport (RCT) is a hypothetical pathway describing the delivery of cholesterol from the peripheral tissues to the liver. The liver requires cholesterol for the synthesis of VLDL and primary bile acids, cholic and chenodeoxycholic acids, a step in which the rate limiting enzyme is 7-alpha hydroxylase. The bile acids are secreted into the intestine as bile salts where they are converted into secondary bile acids, deoxycholic and lithocholic. The bile acids emulsify fats in the gastrointestinal tract and are either reabsorbed by the liver or excreted in the feces [15, 16]. The process of cholesterol transport to the liver is mediated by HDL particles, which are secreted by the liver and the intestine as flattened disk-like particles. These nascent HDL's undergo maturation, which involves the gradual filling in of the core of the nascent HDL with cholesterol from peripheral tissues. Lecithin: cholesterol acyl transferase (LCAT) secreted by the intestine and liver converts free cholesterol of the tissue to cholesteryl esters at the surface of the HDL [17]. This reaction requires an activator, apo A-l that is also found on the surface of nascent HDL. As the nascent HDL mature, they get larger and can be classified by their density: HDL2 (1.063-1.125 g/ml) and HDL3 (1.1255-1.21 g/ml). HDL particles can also be classified by their apo A composition, either Lp A-l only or Lp A-l/A-ll containing particles. Lp A-l/A-ll is believed to originate mostly in the liver yet is less effective in RCT. Lp A-l, on the other hand, originates in the intestine and is associated with decreased risk of atherosclerosis. Multivariate analysis indicated that the apo A-l/A-ll ratio is independently associated with CHD but apo A-ll on its own was a less powerful predictor of disease [18]. HDL acts as a reservoir for apo E and apo C's, which shuttle between TG-rich particles and HDL. Cholesteryl ester from HDL is exchanged with TG from LDL and VLDL via cholesterol ester transfer protein (CETP). C E T P enriches HDL with TG; and it increases the cholesterol content of VLDL and LDL [19]. 8 1.2 Dyslipidemia and A therosclerosis The role of plasma lipids and lipoproteins in the development of coronary heart disease (CHD) has been studied for a number of years. There is now a large body of data based on epidemiological studies [20-24], experimental research [25-27], genetics [28, 29], and clinical trials [30, 31] that have shown that elevated total serum cholesterol and particularly LDL-cholesterol (LDL-C) as well as low serum HDL-cholesterol (HDL-C) results in increased risk of CHD. These findings are the basis for the current clinical guidelines. Although TG's role in development of CHD has been controversial [32], it has been shown that high TG in combination with low HDL-C accounts for twice as many cases of CHD as low HDL-C alone [33]. In addition, T G rich particles of the apo B family represent significant predictors of CHD progression [34]. Approximately 80% patients with CHD have abnormal lipids [35]. It is therefore of importance to understand the causes of dyslipidemia. Using twin and adoption studies, genetics has been shown to be a critical factor in the determination of lipid levels [36, 37]. In monozygotic twins, death of one twin from CHD at an early age was a strong predictor of risk of death in the other [38]. Although a very small percentage (10%) of survivors of myocardial infarction present with monogenic disorders of lipids and lipoproteins [39-41], studying large families suggest that major gene loci are modulated by other genes or environmental factors in the majority of patients with CHD. A good example of a polygenic form of dyslipidemia is type III hyperlipoproteinemia, which is characterized by the accumulation of IDL and can be caused by a variety of different mechanisms, such as apo E genotype and LPL polymorphism that will be discussed further below. The role of genetic factors in lipid and lipoprotein metabolism and in the development of dyslipidemias is being examined continuously and a number of candidate genes have been identified. Genes such as the LDL-receptor, apo A- l , apo B and LCAT have proved to be important. In addition, the LPL and apo E gene are likely to be significantly involved in modulating the risk for dyslipidemia and atherosclerosis. 1.3 Biochemical Genetics of Lipoprotein Lipase 1.3.1 Overview In 1943, Dr. Paul Hahn observed the elimination of alimentary lipemia in the plasma of dogs given intravenous injection of heparin [42]. In 1950 it was suggested that this phenomenon could result from the release of a "clearing factor" in plasma [43]. It was thought that heparin released an unknown surface-active agent promoting physical dispersion of lipids. It was not until 2 years later and 9 years after the initial observation that the phenomenon was demonstrated to be an enzyme [44], and was named lipoprotein lipase (LPL) [45]. Although its primary role is the hydrolysis of triglycerides from the core of triglyceride-rich lipoproteins in plasma, lipoprotein lipase is also critical in maintaining plasma lipid levels [46]. The free fatty acids from the reaction are released into the circulation and form water soluble complexes with albumin aiding in their delivery to other tissues for energy use or storage [47, 48]. LPL is primarily synthesized in the adipose tissue, skeletal muscles and the heart but its messenger RNA (mRNA) is also detected in a wide variety of cell types, including macrophages. Unlike the adult liver, LPL is synthesized in the fetal liver [49]. Following synthesis, LPL is secreted and transported in an unknown fashion to the luminal side of the capillary of the endothelium [50]. It then attaches itself through non-covalent bonds to heparan sulfate glycosaminoglycan [51, 52]. It has been suggested that LPL may be bound through an additional 116 kDa protein, which shares 100% identity with the amino terminal end of apo B [53]. LPL can be released from membrane bound heparan sulfate glycosaminoglycans into the circulation heparin which competes with the natural binding site of LPL. Lipoprotein lipase requires apo C-ll as a co-factor for its activation [45, 54-56]. Apo C-ll is a small protein of only 79 amino acids. It is found on the surface of HDL particles from which it is transferred to nascent VLDL and chylomicrons. Following the hydrolysis of T G from the core of VLDL and chylomicrons, excess surface material (and lipoproteins) are transferred back to HDL, thus allowing apo C-ll to be recycled [55]. The majority of active LPL is in the form of a homodimer [57]. The two sub-units are thought to lie in a head to tail orientation. The active dimer is in reverse equilibrium with the monomer. However the monomer is prone to reversible changes which result in loss of activity and eventual degradation by the liver. Each sub-unit has an apo C-ll binding site and two apo C-ll molecules are required for activation. Apo C-ll I, on the other hand, acts as an inhibitor of LPL [58] possibly by competing for the apo C-ll binding site on LPL. The ratio of apo C-ll to apo C-lll may therefore play a role in LPL activity and has been found to be different in the various types of dyslipidemia. LPL can hydrolyze a wide variety of substrates such as long and short chain TG, diglycerides, monoglycerides and phosphatidylcholine. The rate of the reaction 11 depends on the substrate. Phospholipids, for example, are hydrolyzed at only a fraction of the rate of triglycerides. LPL preferentially hydrolyzes the first and third ester bond of TG resulting in 2-monoglyceride. Monoglycerides are broken down more quickly if the first and third bonds are intact [59]. 1.3.2 LPL Gene and Protein Structure The gene for lipoprotein lipase (LPL) is located on chromosome 8 (8p22) [60]. It is comprised of 10 exons separated by 9 introns and has a total molecular weight of 30 Kb [61]. Exon 10 contains a 3' untranslated region (UTL) which contains 2 polyadenylation signals. These signals are thought to be used alternatively to produce 2 species of mRNA roughly 3350 and 3750 nucleotides long [62]. The translated gene forms a 475 amino acid protein from which a 27 amino acid signal peptide is cleaved, resulting in a 50 KD protein of 448 amino acids. LPL belongs to the lipase family together with hepatic lipase (HL), pancreatic lipase (PL), and recently described endothelial lipase (EDL) [63]. LPL, HL and PL are believed to have evolved from a single ancestral gene [64], but little is known thus far about the relationship to EDL. LPL has 53% homology with HL and 35% homology with PL. HL and PL are 36% homologous. It is thought that PL branched off earlier in evolution [64]. LPL, HL and PL belong to the family of serine esterases. However, they differ from the classic esterases in that they show specificity for substrates insoluble in water and are active when exposed to oil-water interfaces. Together with this group of enzymes, the lipases possess a catalytic triad consisting of serine, aspartate and histidine [62]. Only 12 PL has been crystallized and has been commonly used as a model for HL and LPL [65, 66]. LPL and HL are bound to membrane proteoglycans and they are released into the plasma by intra venous heparin injection. LPL is active as a dimer whereas PL is active as a monomer. HL appears to be active as a monomer in the liver [67] but a dimer in the ovaries [68]. HL is synthesized only in the liver and is bound only to the surface of the liver [69]. PL, on the other hand, is synthesized in the pancreas and is secreted into the duodenum where it is active and hydrolyses dietary TG. EDL has phospholipase activity and may play a role in modulating vessel wall lipid metabolism [63]. 1.3.3 LPL and Hyperlipidemia It was originally thought that LPL deficiencies were autosomal recessive disorders, meaning that two defective genes are required to cause biochemical and clinical sequelea. However, it has now been accepted that LPL deficiencies are autosomal co-dominant diseases meaning that both genes contribute equally to the phenotype. Complete deficiency in the LPL gene occurs in about one in a million people in the general population. There are exceptions, such as Northern Quebec, where the frequency can reach 1 in 400 due to the founder effect [46]. Burger and Gruts described the first case of chylomicronemia in 1932 [70], yet the familial pattern of inheritance was not reported until 1939 [71]. It was another 21 years before Havel and Gordon demonstrated, through the absence of LPL activity, that this unique syndrome was the result of a lipolytic defect [72]. 13 Complete LPL deficiency results in a massive elevation of plasma T G levels which can reach levels over 100 mmol/L (<2.3 mmol/L is the normal range). Most often the affected individuals present early in life with attacks of abdominal pain due to pancreatitis, failure to thrive, or later in life with hepatosplenomegaly and recurrent pancreatitis. Common physical signs include eruptive xanthomas and lipaemia retinalis. There is no cure for the disease, although following an extremely low fat diet [73] will reduce symptoms. Some time after the description of LPL deficiency, Breckenridge et. al. discovered that genetic absence of apo C-ll could result in a similar phenotype to LPL deficiency [74]. Since then, a number of studies have shown that apo C-ll deficiency is associated with increased serum T G and total cholesterol (TC) [75-78], as well as increased risk of premature vascular disease [79]. In addition to apo C-ll deficiency, other physiological conditions such as diabetes [80, 81] and renal disease [82, 83] can lower LPL activity, giving rise to a phenotype resembling LPL deficiency. Approximately 80 different mutations have been described in the coding region of the LPL gene and an additional 20 mutations have been reported in the non-coding region (Appendix A). The portions of the gene coding for the functional sites of the molecule have been found to contain the highest mutation density. Exon 5 contains around 30% of the known mutations and exon 6 contains roughly 22%. The majority of mutations discovered (86%) are point mutations with the remaining 14% being made up of insertions and deletions. 14 Homozygotes for LPL deficiency with two defective alleles, develop a phenotype of familial chylomicronemia previously called type I hyperlipidemia. Without LPL activity, chylomicrons and VLDL can not be catabolized properly resulting in the accumulation of these particles in the circulation and therefore, elevated plasma TG. LPL deficient individuals have very low levels of LDL-C due to their inability to properly process VLDL particles. Approximately 10-20% of VLDL is further metabolized into LDL [84]. There has been a great deal of research on the effects of LPL deficiency on the risk of atherosclerosis. Early studies suggested that LPL deficiency does not predispose individuals to atherosclerosis [72, 85] and preliminary studies revealed no serious atherosclerotic lesions in these individuals [86]. However, recent work by Benlian ef. al. suggests otherwise [87]. They reported 4 patients with LPL deficiency who had premature peripheral or coronary atherosclerosis before the age of 55. Impaired TG clearance may result in the increased exposure of lipoproteins to oxidation resulting in more atherogenic particles. In addition, inactive LPL protein may still be produced which can promote lipoprotein retention at the vessel wall thereby increasing the uptake of lipoproteins and resulting in foam cells and atherosclerotic plaque formation. Animal models have also been used to try to assess the consequences of LPL deficiency. Homozygous LPL knockout mouse pups had 3 times higher TG and 7 times higher VLDL-C than controls [88]. These animals became pale then cyanotic and finally died at around 18 hours of age. Total LPL deficiency in the mouse prevents T G removal from plasma causing death in the neonatal period. In further studies it was shown that expression of LPL in a single tissue type, such as muscle, was sufficient to rescue the LPL deficient pups [88], and maintain only a mild increase (1.5-2 times) in TG levels. Death from LPL knockout was also prevented by adenovirus mediated LPL 15 gene transfer to the liver [89]. This reduced VLDL levels and improved both oral and intravenous fat-load tolerance tests. Although heterozygous LPL deficiency is associated with increased T G levels, no differences in the extent of atherosclerosis were found in the aorta of heterozygous animals compared to the control animals [90]. It was suggested that LPL deficiency in the vascular wall could prevent the retention of atherogenic lipoproteins [90]. In addition to this, more recent studies have shown that expression of LPL in the macrophage promotes foam cell formation and atherosclerosis [91]. A naturally occurring LPL deficient feline model has also been used to study the effect of LPL deficiency. Peritz et. al. first characterized the deficiency in the LPL activity in hypertriglyceridemic cats [92]. These animals had high levels of LPL mass but no enzyme activity. The LPL protein fails to bind the endothelium and results in a phenotype similar to Class III type defect in humans [93]. The attempt to recover the LPL activity using liver-directed gene transfer and human enzyme replacement in these animals was limited by the immune response to both the human LPL protein and the adenovirus [94]. However, the use of this naturally occurring feline model may be useful in further development of LPL gene therapy as a viable option for clinical LPL deficiency. Although complete LPL deficiency is rare, heterozygosity for LPL deficiency occurs in 1 in 500 individuals. Heterozygotes do not present with any significant clinical manifestations and are usually identified as relatives of an affected proband. The impact of one dysfunctional allele is unknown. It has been shown that LPL activity can be decreased by 20-60% in these individuals. Postprandial studies revealed that the 16 impaired lipolysis leads to prolonged residence of circulating chylomicrons and VLDL [95]. Studies in fasting individuals revealed a decrease in HDL-C levels, particularly HDL2, reduced apo A-l and increased VLDL-C and VLDL-TG [96, 97]. Age, obesity, and diabetes also contribute to the expression of hypertriglyceridemia in heterozygotes [96, 98]. This potentially atherogenic lipid profile may suggest that heterozygotes for LPL deficiency may be at higher risk of CHD [99]. 1.3.4 LPL Asp9Asn, Asn291 Ser, and Ser447X Polymorphisms Many studies have reported that three common polymorphisms in the gene for LPL, Asp9Asn (D9N), Asn291Ser (N291S), and Ser447X (S447X) (premature truncation at codon 447) are associated with altered levels of both T G and HDL-C (Table 1-2). The D9N and the N291S have been identified in roughly 2-7% of the population depending on ethnic background [100]. They have both been found to be associated with a partial decrease in LPL activity resulting in increased plasma T G levels. In addition, both polymorphisms have been associated with decreased HDL-C levels [101-103]. Despite having only subtle effects on plasma lipids, the D9N variant has been found to lead to a more rapid progression of CHD and increased morbidity [103]. In addition, the N291S polymorphism has been associated with increased risk of ischemic heart disease in women [104]. By contrast, the S447X variant occurs at a much higher frequency in the population (18%) and has been associated with decreased T G and increased HDL-C [100]. However, studies on the catalytic function have lead to conflicting reports [105-108]. As a result, it has been suggested that decreased T G levels in S447X carriers are due to an increase in protein production rather than an increase in catalytic activity [106, 108, 109]. Several studies could not show a statistically significant association between LPL S447X and reduced CHD risk [110-113]. However, a review paper reported a marginal negative association with CHD [114] and a meta-analysis suggested that carriers of the S447X mutation might possibly be at decreased risk of CHD [100]. Our own studies involving a Cantonese Canadian population showed an increased frequency of S447X carriers in the highest tertile of HDL-C, suggesting a beneficial lipid profile in the S447X carriers [115]. 18 (/) CD o c 0) o Q X o c o CO o o </) < 12 re c E «2 re O > , o < o c CD 3 cr [it E ' I o E oo o X — I o o Q >> o o a * * Q- to Q) CO CO (/) ^ P CD o p O ^ Q . — CD "D t/> (D CO to CD CO CD i _ O CD Q CD ^ to ^ CO > O O (1) CO Q i CN Q o E CO ® .E "O CD to to — CO X3 CD CD CO T3 to CO _ CD "C CO o CD c CD E o O i _ i Q CD "O to CD CO w CD CO b 2 c o — CD Q T3 CD ^ to CO > CD ~ O O CD °3 O I CN CN LO I LO o o o Q X i — CD O O > _ c o CO CO ]r D) > CD r-U) •=, O CO P o o « g Z E co O h-T3 CD to CO CD o CD Q 8.2 S 2 CO X (0 1.4 Biochemical Genetics of Apolipoprotein E 1.4.1 Gene and Protein Structure Apolipoprotein E (apo E) is a structural component of plasma chylomicrons, VLDL, and HDL [116]. It plays a major role in regulating the metabolism of these particles via the apo E receptor and by the LDL (apo B, E) receptors on the surface of the liver and other peripheral tissues [117]. As a ligand, apo E is responsible, in part, for uptake of dietary cholesterol in the form of chylomicron remnants, clearance of VLDL remnants, and for removal of excess cholesterol from peripheral tissues through hepatic clearance of HDL containing apo E. Apo E is synthesized primarily in the liver, but also in a number of other tissues including brain [118]. Its preprotein undergoes intracellular proteolysis, glycosylation and desialylation resulting in a single polypeptide chain of 299 amino acids. It has a calculated molecular weight of 34, 135 Daltons [119, 120]. The gene for apo E is situated on chromosome 19 [121]. It is a polymorphic gene, which has three common alleles (s2, s3, e4) which encode for the three major isoforms of apo E. These isoforms differ by amino acid substitution at one or both of two sites (residues 112 and 158) on the 299 amino acid chain (Table 1-3) [122]. Apo E4 differs from apo E3 by the replacement of cysteine with arginine at position 112. Apo E2 differs form apo E3 by the replacement of arginine with cysteine at position 158. The amino acid change within the three isoforms also causes a charge shift with apo E3 being 0, E4 +1, and E2 - 1 . The combination of the three allele forms result in three homozygous and three heterozygous genotypes: E4/E4, E3/E3, E2/E2, E4/E3, E4/E2, and E3/E2. 20 1.4.2 Allelic Effect in the Normal Population Several studies from different research groups have shown that serum cholesterol levels are affected by apo E alleles (Table 1-3), and environmental factors and ethnic background seem to have little effect. Apo E2 has been found to be associated with lower TC (compared to E3) whereas E4 has been associated with higher TC [123, 124]. Functional differences have also been found between the isoforms. Apo E2 is associated with decreased ability to bind either the LDL receptor or the apo E receptor [122], which results in a reduced in vivo catabolism of apo E2 [125]. Apo E4 showed no change in receptor binding from E3 [122] but it has been associated with an increased in vivo catabolism [126]. 21 10 u c o sz c o +5 (Q O O W < 12 a ro « E g 1 w <5 a. O O D ) 2 8 I g o & J? < •*= < o o c 3 o-o l i t CN m CM i CM CM CM 00 • - y CD E 0) O TJ CO ' S i CD CO CO CD to £ CD CD "C h ~ co y CO CD i= C L X : "S | CD CO CO (0 =3 CD CO O O CD i _ C o — i » O X3 T3 L" (D (D T3 CO CO CD co co to CD CD i— i_ CD CD CD CJ Q Q -E CM V CO o I GO in < O CM LU CD C o o o CD c o m IS-LU CD CM CM CM CM 00 CM 00 •- °. CD P 0) " g 12 CO O ™ CD C £ co — CO •a ^ CD CD ^ CO roc" CD CD " C ! • • § o o CO CO CD >*-R E -o £ == 4- CO *- o o O v- Q T3 CD CO CO CD i _ o c CO CO CD — W, _ CL^Z I— c E CO O i o a (J h J h ~o ~u ~u CD CD CD CO (O CO co co co CD CD CD l _ 4— 1— o o o c c c o o CO o m TI-LL! a x o T3 C CO _co CD > •g Q. C o CO o o CO CO CO c CO CO J D _Q) "CO L U c o i _ Q. O Q. O a. < CO I a> Si CO 1.4.3 Apolipoprotein E and Hyperlipidemia Different apo E genotypes have been associated with specific lipid profiles. The majority of dysbetalipoproteinemia (characterized by p migrating VLDL) patients are E2 homozygotes [127]. However, it is estimated that less than 5% of E2/E2 individuals have type III disease [128], a disorder associated with palmar and tuboeruptive xanthomas, premature peripheral vascular and coronary artery disease and accumulation of abnormal cholesterol rich VLDL and IDL [129]. It is thought that the decreased ability of E2 to bind to lipoprotein receptors [130, 131] in addition to other genetic or environmental factor(s) leading to overproduction of VLDL (obesity, diabetes) [131, 138-142], may be the cause of this condition. Other genetic variants have been found in the apo E gene and have been identified with different forms of hyperlipoproteinemia [143-145]. The relationship of apo E genotype to atherosclerosis had, for a while, been controversial. However, an increased incidence of both E2 and E4 alleles has been found in patients with ischemic heart disease [132]. Conflicting reports of the effect on apo E4 allele on likelihood of myocardial infarction [133-136] have been now superseded by a new studie that reports a 2-fold increase of risk of dying following a myocardial infarction in carriers of the apo E4 allele compared with other patients [137]. 23 1.5 Pregnancy 1.5.1 Lipid Metabolism in Pregnancy Many research groups [146-153] have studied plasma lipoprotein changes during pregnancy in humans. It has been found that the plasma triglyceride concentrations increase 250% on average during the course of pregnancy and then rapidly decline postpartum. Plasma TC increases less dramatically by about 25% and declines more slowly after delivery. These changes occur primarily in the LDL fraction (60%) and less so in VLDL. HDL-C is also elevated throughout pregnancy but reaches a maximum at 20 weeks of gestation and then gradually declines to reach, by 39 weeks, levels slightly above the pre-pregnant level. The increase in HDL-C is mostly in HDL2 with relatively little increase in HDL3. Triglycerides increase in all lipoprotein fractions to a greater extent than cholesterol. The most pronounced increase is seen in VLDL and only a slight increase in LDL. There is a 25% elevation in HDL-TG, which is sustained until late gestation regardless of the concomitant decrease in HDL-C. Several investigations have also reported apolipoprotein changes during pregnancy [147, 150, 154-158]. Total apo B increases throughout gestation and is positively associated with the total cholesterol concentration [155]. A three-fold increase in apo B is seen in VLDL fraction at 36 weeks and to a smaller extent (40%) in the LDL fraction [155]. Apo A-l increases about 28% and this increase is sustained throughout gestation (unlike the HDL-C which rises and falls) [150]. Lp(a) increases about 100%, peaking at 20 weeks and declining towards the end of the pregnancy [157]. Various mechanisms may be responsible for the different changes in each lipoprotein fraction throughout 24 gestation. The increase in VLDL, LDL, HDL, and Lp(a) at the beginning of pregnancy may be due to sex-hormone dependent mechanisms while insulin and insulin-like hormones that increase late in gestation may drive VLDL and LDL higher while depressing HDL and preventing a further rise in apo A-l and mediate a decrease in Lp(a). These changes are discussed in more detail in the next section. 1.5.2 Role of Hormones in Lipid Metabolism of Pregnancy The role of estrogen in regulating lipoprotein metabolism has been extensively studied [159-161]. It has been suggested that LPL activity may be either slightly reduced or unchanged in the presence of estrogen alone [162, 163]. In individuals treated with oral-contraceptive, there is an enhanced uptake of chylomicron remnants by the LDL receptor related protein (LRP) [164]. This effect is thought to be a result of estrogen effect rather than progesterone because LRP resembles the LDL receptor that is upregulated by estrogen [165, 166]. Yet in the liver, estrogen increases the secretion of TC and T G in the form of VLDL [167], which is then metabolized much less rapidly than chylomicrons. However, due to the upregulation of the LDL receptor, the clearance of VLDL-remnants and LDL-C is enhanced. Progesterone, in combinations with estrogen, opposes most of the effects of estrogen either by acting as an anti-estrogen directly, or by exerting its own effect on lipid metabolism [159, 160, 168]. To understand the effect of hormones during pregnancy, correlative associations were studied between plasma lipoproteins and plasma hormones [147]. Plasma T G concentrations were positively associated with plasma estrogen concentrations, and VLDL-TG with insulin. LDL-C concentrations were inversely associated with 25 progesterone whereas HDL-C was positively associated with progesterone. Based on the major effects of estrogen and the minor effects of progesterone, the patterns seen in pregnancy primarily reflect the increase in estrogen with the exception of the marked increase in LDL-C. This may be a result of a combination of increased estrogen and progesterone, as also seen in subjects using oral contraceptives [169]. Since prolonged infusion of glucose in pregnant rats increases adipose LPL activity compared to that of virgin animals, it is proposed that resistance to insulin, occurring during late gestation, is primarily responsible for the reduction in LPL activity in adipose tissue [170]. Non-insulin dependent diabetes has been associated with hypertriglyceridemia [171, 172] and low serum HDL-C [172] during pregnancy. Type I diabetes results in reduced HDL3 which may alter the reverse cholesterol transport pathway and may lead to decreased cholesterol delivery to the fetus promoting infant macrosomia [173]. Experimental diabetes does not appear to alter placental LPL activity [147, 174] but may enhance oxidative stress on lipoproteins which may increase the uptake of oxidized or modified lipoproteins by placental macrophages via the scavenger receptor [175]. 1.5.3 Physiologic Mechanisms Affecting Lipoprotein Metabolism in Pregnancy Very few studies have investigated fat metabolism in pregnant humans. Consequently, much of the knowledge is based on work done in pregnant animal models. Following an oral dose of radioactive T G load, an increase in labeled lipids (d<1.006) was seen in the plasma of pregnant rats compared to non-pregnant controls [176]. This suggests that there is an increase in intestinal TG absorption during pregnancy. The VLDL protein 26 production is also increased in the pregnant rat. Another study, also done in rats, found that the clearance of radiolabeled chylomicrons was similar to that of non-pregnant animals indicating that the increase in T G was related less to differences in removal than to the production of TG particles [177]. T G secretion from the liver is also increased [178]. From these few studies it appears that the major contributor to the development of maternal hypertriglyceridemia may be the enhanced production of TG rich particles in the pregnant rat. The contribution of LPL activity in various tissues to hypertriglyceridemia is unclear. LPL activity is decreased in adipose tissue [151, 179-183] and increased in mammary gland [151, 180-183] and placenta [147, 151, 182, 184, 185] towards the end of term. Regardless of these changes, the total post-heparin LPL activity is reduced in humans by 85% during pregnancy [151]. Work by Knopp ef. al. has also demonstrated that LPL-mediated TG removal may be impaired, increasing plasma T G levels [186]. There have been few reports studying the clearance of remnant and LDL particles in pregnancy. Warth et. al. reported that the increase in intermediate density lipoproteins (IDL) parallels the increase in VLDL and LDL [187]. In addition, p migrating VLDL was seen in 1.1% of 36 week pregnant women and 1.6% of 6 week postpartum women but in none of the non-pregnant controls [158]. These findings suggest that pregnancy affects each lipoprotein fraction similarly and that the increase in remnants is proportional to that of other lipoproteins. There are no direct human data on the metabolism of LDL-C during pregnancy. However, Mabuchi ef. al. reported dramatic reductions of serum total and LDL 27 cholesterol levels in a woman with heterozygous-familial hypercholesterolemia during pregnancy [188]. This was interpreted to be the result of estrogens increasing LDL-C removal through the LDL-receptor. However, if LDL-C usually rises in pregnancy, and if LDL clearance is increased as a result of estrogen, the rate of entry of LDL into the circulation must be even greater than in the non-pregnant state. The cause of the elevation in serum HDL and apo A-l is also uncertain. Hepatic lipase (HL) activity has been found to be reduced in pregnancy [189]. This may contribute to the rise in HDL-C and HDL-TG. In addition, a strong negative association between HDL-TG and post-heparin HL has been found in all three trimesters as well as postpartum [186]. An increased HDL apoprotein secretion during pregnancy has also been reported [190]. It is possible that the reduced HL activity with the resulting decrease in removal of T G from IDL, LDL, and HDL, could help explain the increased T G content of LDL [147]. 1.5.4 Causes of Dyslipidemia in Pregnancy Severe hypertriglyceridemia can develop in the late gestation of pregnancy as a consequence of either genetic mutations in genes such as LPL or apo E, or other causes such as diabetes, alcohol consumption or weight gain. In some cases extremely high TG levels (chylomicronemia) may result in acute pancreatitis [191-195]. The only known treatment for the chylomicronemia is a dramatic reduction of dietary fat intake throughout the pregnancy. Triglyceride levels can also be dramatically reduced by the daily administration of 8-10 g of omega 3 fatty acid rich fish (salmon) oil [196]. Some of the hypertriglyceridemic women have normal T G levels postpartum, which 28 suggests that these individuals may be "prelipemic" (analogous to gestational diabetes) [154]. Some of the women have low serum HDL-C during pregnancy also have a low postpartum HDL-C [154]. Women with elevated LDL-C further increase LDL-C levels during pregnancy. A cholesterol lowering diet has been shown to lower LDL-C but the effect on the growth and development of the fetus is unknown [197, 198]. The majority of primary dyslipidemias have not yet been found to be associated with reproductive abnormality [199], with the exception of severe hypertriglyceridemia and pancreatitis, which can endanger the life of the mother as well as the fetus. 29 1.6 Rationale for this Study As discussed in previous sections, hypertriglyceridemia is due to a heterogeneous group of underlying disorders caused by genetic defects in the metabolism of triglyceride-rich lipoproteins or by a number of environmental or hormonal factors. Plasma triglyceride concentration is dependent on the balance between the rate of production of TG-rich lipoproteins and the rate of removal of these lipoproteins from the circulation by lipolytic enzymes such as lipoprotein lipase (LPL) and hepatic lipase (HL), and in subsequent uptake by the liver, through apo E receptor. Complete deficiency of LPL is manifested as chylomicronemia, a rare autosomal co-dominant disorder characterized by marked increases in fasting-state TG (usually >17 mmol/l), skin eruptions (eruptive xanthomas), lipemia retinalis, and recurrent episodes of abdominal pain and acute pancreatitis. The most common cause of this disorder is a mutation in the LPL gene that produces completely inactive LPL. Heterozygous individuals, however, rarely present with chylomicronemia but may have a milder form of hypertriglyceridemia. Variations in the apo E gene have also been associated with much less dramatic increases in T G in addition to changes in TC, LDL-C, and HDL-C. Alteration in sex hormones per se also triggers hypertriglyceridemia. One common example of hormonal changes leading to increase T G is pregnancy. Normal pregnancy is associated with a mild increase in plasma cholesterol levels and a 3-4 fold increase in TG levels, peaking during the third trimester. Because of the overproduction of TG-rich VLDL, normal pregnancy presents a challenge to the lipolytic capacity of LPL and the ability to clear remnants via the apo E receptor. Many cases of pregnancy-induced chylomicronemia causing pancreatitis have been reported. In some, chylomicronemia was noticed before pregnancy, in others it was not noticed until abdominal pain and 30 pancreatitis developed late in pregnancy. Numerous LPL gene mutations causing either complete or partial LPL deficiency as well as common polymorphisms, which have been shown to affect LPL activity and expression, have been described. It is hypothesized that pregnant women carrying some of these polymorphisms will have an associated increased TG level during the course of pregnancy. Thus, by using methods that are well established to detect three LPL polymorphisms and apo E genotypes in a group of normal pregnant women, it is possible to investigate whether selected polymorphisms in the LPL and apo E gene are associated with exaggerated hypertriglyceridemia in pregnancy, and to speculate as to the effect of these changes on CHD risk later in life. 1.7 Hypotheses General: 1. Differences in plasma lipids in the third trimester of pregnancy are due, at least in part, to genetic polymorphisms in the LPL and apo E genes. Specific: 1. Carriers of the LPL N291S and D9N genotypes will have increased plasma TG levels and decreased HDL-C levels during pregnancy. 2. Carriers of the LPL S447X genotype will have decreased plasma TG and increased HDL-C levels during pregnancy. 3. Women who have the apo E2 allele will have lower levels of plasma TC & LDL-C, and higher levels of plasma TG, compared to wild type, during pregnancy. 4. Women who have the apo E4 allele will have higher levels of plasma TC, LDL-C and plasma TG, compared to wild type, during pregnancy. 31 1.8 Specific Aims 1. To recruit 250 healthy women in their third trimester of pregnancy, according to specific selection criteria. 2. To collect a fasting blood sample from each individual and measure total cholesterol, plasma HDL-C, LDL-C, TG, insulin, glucose, and fractional esterification rate of HDL ( F E R H D L ) . 3. To perform restriction fragment length polymorphism (RFLP) analysis to identify the three most common LPL polymorphisms. 4. To perform R F L P analysis to identify the apo E genotype. 5. Correlate the LPL gene polymorphisms and the apo E genotype with plasma lipid levels. 6. To sequence the LPL gene of 5 women with the highest T G levels to identify possible mutations other than those analyzed in aim 3. 32 CHAPTER II: MATERIALS AND METHODS 2.1 Materials The QIAamp Blood Midi kits for DNA extraction and the HotstarTaq were obtained from QIAgen Co., Mississauga, Ontario. The agarose-1000 and primers were produced by Canadian Life Technologies, Mississauga, Ontario. The dNTP's were purchased from Promega Co., Madison Wl, and the restriction enzymes (Hha I, Taq1, Rsa I, and Mai I) were obtained from New England Biolabs Ltd., Mississauga, Ontario. The DNA ladder was purchased from Invitrogen, Carlsbad, California, and the glucose reagents were obtained from Bayer Corporation, New York City, USA. The radiolabeled cholesterol was from Amersham. All other reagent grade chemicals were purchased from Sigma Inc. Mississauga, Ontario. 2.2 Study Participants The cohort consisted of 250 unrelated pregnant women residing in the Greater Vancouver area. The subjects were recruited from prenatal classes, or by responding to an advertisement (Appendix 2). Informed consent, approved by University of British Columbia and St. Paul's Hospital ethics committees, was obtained from all study participants. All individuals completed a questionnaire (Appendix 3). It covered their personal and family medical history as well as other important parameters such as age, height, weight change, week gestation, current medication(s), diet, alcohol consumption, number of 33 previous pregnancies, and any past or present medical health information. There was no age restriction, but all individuals had to be in their third trimester and either pregnant for the first time or have had previous uncomplicated pregnancies. Individuals with disorders affecting lipoprotein metabolism including diabetes mellitus, thyroid, hepatic or renal disease were excluded from the study. In addition, subjects taking medications known to affect lipid metabolism (including diuretics, beta-blockers, and lipid lowering medications) and those with heavy alcohol intake were also excluded. 2.3 Plasma Lipid Analysis Fasting plasma samples were collected in 10ml EDTA-coated vacutainer tubes. Plasma was separated by centrifugation at 2000 rpm for 10 minutes. Plasma TG was determined as previously described [200], and plasma TC was determined using an enzymatic method [201]. HDL-C was determined following heparin manganese precipitation of apo B containing lipoproteins [202], and LDL-C was calculated using the Friedwald formula [203, 204]. The remainder of the plasma was stored at -70°C until needed. The red cells and buffy coat were stored at -20°C until DNA was extracted. 2.4 Fractional Esterification Rate of HDL ( F E R H D L J F E R H D L was determined by an isotopic assay method that has been previously described [205, 206]. Briefly, apo B-containing lipoproteins, VLDL and LDL, was precipitated from the plasma with the addition of phosphotungstic acid (PTA), and MgCI 2. A trace amount of tritiated cholesterol was applied to a paper disk, added to the plasma and incubated on ice for 18h to allow spontaneous transfer to occur. The 34 labeled samples were incubated in a shaking water bath at 37°C for 30 min during which time esterification by LCAT occurs. The reaction was stopped with the addition of 1ml of ethanol and the lipid extract was subjected to thin layer chromatography (TLC). The radioactivity of the free and esterifed cholesterol fractions was determined by liquid scintillography. The F E R H D L was then calculated as the percentage of radiolabel found in the esterifed cholesterol fraction, following incubation, over the total radioactivity in the sample. The normal value for healthy women is 10.6 + 3.6 %/hour [4]. 2.5 Insulin and Glucose Assays Insulin was measured by Dr. McNeill's lab at the University of British Columbia using the Human Insulin Specific RIA Kit (Linco Research Inc.) Briefly, a fixed concentration of labeled tracer antigen is incubated with a constant dilution of antiserum such that the concentration of antigen binding sites on the antibody is limited. When unlabeled antigen is added to this system, there is competition between labeled tracer and unlabeled antigen for the limited and constant number of binding sites on the antibody. Thus, the amount of tracer bound to antibody will decrease as the concentration of unlabeled antigen increases. This can be measured after separating antibody-bound from free tracer and counting one or the other, or both fractions. A standard curve is set up with increasing concentrations of standard unlabeled antigen and from this curve the amount of antigen in unknown samples can be calculated. Glucose was measured using the Glucose Reagent Kit (Technicon RA®1000/oreRA) provided by Bayer Corporation on a RA-1000 machine in our laboratory. The standard 35 protocol was followed which involves the phosphorylation of glucose by hexokinase and the action of glucose-6-phosphate dehydrogenase (G6PD) on the product [207, 208]. 2.6 DNA Analysis DNA was extracted from leukocytes using the standard protocol of the QIAamp Blood Midi Kit (QIAgen Co., Mississauga, Ontario). All the samples were then screened for the D9N, N291S and S447X mutations in the LPL gene and the apolipoprotein E variants by P C R and restriction endonuclease digestion of amplified product as described briefly bellow. 2.6.1 PCR-Based Detection of the D9N Allele The target sequence of the LPL gene (exon 2) was amplified by using 5 ' -AGG G C A AAT TTA CTT G C G A T G -3' as upstream primer and 5'-CTC C A G TTA A C C TCA TAT C C -3' as downstream primer [209, 210]. The amplification reactions were carried out in 10 mmol/L Tris-HCI (pH 9.0), 50 mmol/L KCI, 0.1% wt/vol gelatin, 1.5 mmol/L MgCI 2 l 1% Triton X-100, 200 ng genomic DNA and final concentrations of 50 umol/L dNTPs, 0.5 umol/L primers and 1.0 unit of HotStarTaq (QIAgen) in a total volume of 50 ul. Following an initial denaturation (15 minutes, 95°C), a touchdown P C R protocol was used. Simply, 10 cycles of touchdown P C R amplification 95°C (45 seconds), 60°C-50°C (30 seconds), 72°C (45 seconds) were followed by an additional 20 cycles of 95°C (45 seconds), 50°C (30 seconds), 72°C (45 seconds) with a final extension step of 7 minutes at 72°C. 30% of the P C R reaction product was used for digestion with 1.0 U 36 Taq1 according to the instructions of the manufacturer (New England Biolabs), in a total volume of 35 ul for 2 hours at 65°C. After electrophoresis of the digested P C R product in 4% agarose-1000 containing ethidium bromide, DNA restriction fragments were visualized and analyzed on a transilluminator (Figure 2-1). Figure 2-1: Restriction digest gel electrophoresis of the LPL D9N polymorphism. Lane A heterozygous, lane B wildtype. 2.6.2 PCR-Based Detection of the N291S Allele The target sequence of the LPL gene (exon 6) was amplified by using 5 ' -GCC G A G ATA C A A TCT T G G TA -3' as upstream primer and 5'-ATA ATA TAA AAT ATA AAT ACT G C T TCT TTT G G C TCT G A C TGT A -3' as downstream primer [211 ]. The amplification reactions were carried out in 10 mmol/L Tris-HCI (pH 9.0), 50 mmol/L KCI, 0.1% wt/vol gelatin, 1.5 mmol/L MgCI 2, 1% Triton X-100, 200 ng genomic DNA and final concentrations of 50 umol/L dNTPs, 0.5 umol/L primers and 1.0 unit of HotStarTaq (QIAgen) in a total volume of 50 ul. Following an initial denaturation (15 minutes, 95°C), a touchdown P C R protocol was used. Simply, 20 cycles of touchdown P C R amplification 95°C (1 minute), 60°C-50°C (45 seconds), 72°C (30 seconds) were followed by an additional 10 cycles of 95°C (1 minute), 50°C (45 seconds), 72°C (30 seconds) with a final extension step of 10 minutes at 72°C. 30% of the P C R reaction product was used for digestion with 1.0 U Rsa I according to the instructions of the manufacturer (New England Biolabs), in a total volume of 35 ul for 2 hours at 37°C. After electrophoresis of the digested P C R product in 3% agarose-1000 containing 37 ethidium bromide, DNA restriction fragments were visualized and analyzed on a transilluminator (Figure 2-2). Figure 2-2: Restriction digest gel electrophoresis of the LPL N291S polymorphism. Lane A heterozygous, lane B wildtype. 2.6.3 PCR-Based Detection of the S447X Allele The target sequence of the LPL (terminal part of exon 9) gene was amplified by using 5'-TAC A C T A G C AAT GTC T A G GTG A-3* as upstream primer and 5'-TCA G C T TTA G C C C A G AAT G C - 3 ' as downstream primer [212]. The amplification reactions were carried out in 10 mmol/L Tris-HCI (pH 9.0), 50 mmol/L KCI, 0.1% wt/vol gelatin, 1.5 mmol/L MgCl2,1% Triton X-100, 200 ng genomic DNA and final concentrations of 50 umol/L dNTPs, 0.5 umol/L primers and 1.0 unit of HotStarTaq (QIAgen) in a total volume of 50 ul. An initial denaturation (15 minutes, 95°C) was followed by 30 amplification cycles of 94°C (1 minute), 60°C (1 minute), 72°C (1 minute) with a final extension step of 10 minutes at 72°C. 30% of the P C R reaction product was used for digestion with 1.0 U Mn11 according to the instructions of the manufacturer (New England Biolabs), in a total volume of 35 ul for 2 hours at 37°C. After electrophoresis of the digested P C R product in 2% agarose-1000 containing ethidium bromide, DNA restriction fragments were visualized and analyzed on a transilluminator (Figure 2-3). 38 290bp 250bp 200bp Figure 2-3: Restriction digest gels electrophoresis of the LPL S447X polymorphisms, lane A heterozygous lane B homozygous, lane C wildtype. 2.6.4 PCR-Based Detection of Apolipoprotein E Genotype The target sequence of the apo E (exon 4) gene was amplified by using 5'-ACA GAA TTC G C C C C G G C C T G G TAC AC-3 ' as upstream primer and 5'-TAA G C T T G G C A C G G C TGT C C A A G G A-3' as downstream primer [213]. The amplification reactions were carried out in 10 mmol/L Tris-HCI (pH 9.0), 50 mmol/L KCI, 0.1% wt/vol gelatin, 1.5 mmol/L MgCl2,1% Triton X-100, 200 ng genomic DNA and final concentrations of 50 umol/L dNTPs, 0.5 umol/L primers and 1.0 unit of HotStarTaq (QIAgen) in a total volume of 50 ul. Following an initial denaturation (15 minutes, 95°C), a touchdown P C R protocol was used. Simply, 10 cycles of touchdown P C R amplification 95°C (45 seconds), 68°C-58°C (30 seconds), 72°C (45 seconds) were followed by an additional 25 cycles of 95°C (45 seconds), 58°C (30 seconds), 72°C (45 seconds) with a final extension step of 7 minutes at 72°C. 30% of the P C R reaction product was used for digestion with 1.0 U Hha I according to the instructions of the manufacturer (New England Biolabs), in a total volume of 35 ul for 1 hours at 37°C. After electrophoresis of the digested P C R product in 4% agarose-1000 containing ethidium bromide, DNA restriction fragments were visualized and analyzed on a transilluminator (Figure 2-4). 39 2/4 4/4 3/4 3/3 2/3 2/2 Figure 2-4: Hha I cleavage maps and electrophoretic separation of Hha I fragments after gene amplification of DNA from six subjects with the apo E isoforms. 2.4 LPL Gene Sequencing The sequencing of all ten exons and intron-exon boundaries of the LPL gene in the five study participants was performed by Dr. R. Hegele from University of Western Ontario. The method, previously described by Matsuru et. al., was used with the addition of exon 10 [214]. The downstream primer for exon 10 was 5' A C A G G C G G G AAT TGT AAA A C A C T C 3' and the upstream primer was 5' GTC AAA ATA T G C T G A G T G AAT C T G A C C 3'. 2.8 Statistical Analyses Between group comparison was performed using an A N O V A followed by the parametric t-test. Due to skewed distribution of TG, all TG analyses were performed on logarithmically transformed values. Statistical analyses were performed using Microsoft Excel Data Analysis Package (Microsoft, Inc.). 40 CHAPTER III: RESULTS 3.1 Cohort Characteristics The cohort consisted of 250 healthy pregnant women in their third trimester. They were of mixed ethnicity, recruited from the Lower Mainland and Greater Vancouver area of British Columbia, Canada between March 1996 and October 1998. The ethnic backgrounds are displayed in Figure 3-1. The majority (78.4%) of the women in this group reported being of European descent and nearly two thirds of those stated that they were of British, Irish, or Scottish ancestry. Roughly 9.6% of the cohort was Asian, predominantly Chinese, and 4.4% reported being of Indo Canadian, Punjabi, or Sri Lankan origin. The remainder of the group (7.6%) was of either other ethnic backgrounds or unknown. The average age of the group was 31.8 + 0.41 years with a range from 20 to 41 years. The average week of gestation was 35.4 + 0.14 with a range of 31-42 weeks. The number of previous pregnancies ranged from 0 to 5 with an average of 0.81 + 1.08. The majority of the women (53%) reported this being there first pregnancy, 25% had one previous pregnancy, 15% had 2 previous pregnancies, and the remaining 7% reported 3, 4, or 5 previous pregnancies. There was no significant difference in lipid levels between the women with various numbers of previous pregnancies. The average height of the subjects was 163.07 + 7.29 cm (132-197 cm) and their average weight gain during the current pregnancy was 12.77 + 4.04 kg (1.4-28.6 kg). 41 m European • Asian • Indo Canadian/Punjabi/Sri Lankan • Other/Unknown Figure 3-1: Breakdown of the various ethnic groups within the cohort. No subjects were included in the study if they had any diseases known to affect lipid metabolism or if they were on any medications known to effect lipid metabolism. Based on the questionnaires, none of the women were taking lipid altering medication and the majority reported consuming a regular diet, with the exception of 23 (9%) who reported following various vegetarian diets. None of the women reported frequent (>5 drinks per week) alcohol consumption and only 58 (23%) reported drinking occasionally (1-5 drinks per week) while the remainder stated they never drank. The self reported personal and family history is shown in Table 3-1. The women who reported having diabetes, with the exception of one whom reported gestational diabetes during previous pregnancy, were excluded from further analyses. The women with thyroid diseases who reported being well controlled and their lipid levels were not significantly different from the others (data not shown) were included in the study. 42 Personal History Diabetes Hypertension Thyroid Pancreatitis 6 (2.4%) (2%) 5 11 (4.4%) 2 (0.8%) Family History Diabetes Hypertension High Cholesterol 73 (29.2%) 75 (30%) 84 (33.6%) Table 3-1: Study participants' personal and family history. The lipid data, including F E R H D L , insulin and glucose, for the cohort are presented in Table 3-2. The mean values for TG, HDL-C, LDL-C and total cholesterol (TC) all fell within the normal range for pregnancy, yet some individual values were above the pregnant range for TG, TC and LDL-C. Mean TG and TC were above the normal non-pregnant range whereas LDL-C remained within the non-pregnant value. 43 Normal Pregnant Range (n) Range (non-pregnant range) Triglycerides (mmol/L] 2.74 + 1.01 0.95- 6.46 1.01 -5 .25 (250) (<2.3) HDL (mmol/L) 1.73 + 0.42 0.87- -3.14 1.21 - 2 . 3 7 (249) (>1.1) LDL (mmol/L) 3.35 + 1.12 1.19- -7.14 2.32 - 5.56 (249) (<3.4) Cholesterol (mmol/L) 6.36 + 1.21 4 . 0 9 - 10.11 4.71 - 8 . 5 5 (250) (<5.2) FERHDL 19.52 + 5.37 8 . 5 4 - 38.43 — (224) (10.67 + 3.65) Insulin (u.U/ml) 14.57 + 8.22 3 . 7 3 - 59.58 — (247) (6-24) Glucose (mmol/L) 4.18 + 1.60 0 . 3 - 17.8 — (222) (3.5-6) Table 3-2: Cohort lipid levels. The number of women with various TG levels is shown as a bar graph in Figure 3-2. Eight individuals had T G levels above the normal range associated with pregnancy and 2 individuals had T G levels below the normal range for pregnancy (dark bars). The frequency of TG levels is skewed to the left. 40 35 a 30 25 r H L C f N L n c n L n ^ i n i n L f t ^ L f t rH CN CTi ir, V£> T G (mmol/L) Figure 3-2: Frequency of triglyceride levels during pregnancy. 44 3.1 Frequency of LPL variants All 250 subjects were screened for the D9N, N291S, and S447X polymorphisms in the LPL gene. The gels for each restriction digest are displayed in the methods section (Figures 2-1 to 2-3) and the frequencies of homozygous and heterozygous carriers and non-carriers are presented in Table 3-3. The D9N and N291S mutations displayed low allele and carrier frequencies while the allele and carrier frequencies of the S447X were high, all of which were similar to previously published data [100]. Genotype +/+ +/--/-Allele frequency Expected Carrier Frequency Y D9N 0 2/231 (0.9%) 229/231 (99.1%) 0.4% 2-4% N291S 0 10/236 (4.6%) 226/236 (95.8%) 2.3% 1-7% S447X 1/240 (0.4%) 43/240(18.0%) 197/240 (82.1%) 9.4% 17-22% Table 3-3: Frequency of LPL Polymorphisms *F Wittrup ef. a/.,Circulation 1999, 99: 2901-2907 3.3 Effect of LPL Genotype on Plasma Lipids The lipid data for carriers of the N291S mutation verses non-carriers are summarized in Table 3-4. There was a significant decrease in HDL-C levels in the carrier group versus the non-carrier group (1.45 + 0.11 and 1.74 + 0.03, p<0.02). There was also a significant increase in F E R H D L in carriers when compared to non-carriers (23.83 + 7.02 45 and 19.34 + 5.47, p<0.01). Although there is a trend to increased T G in the carriers, this was not statistically significance. mmol/L Carriers (n) Non-carriers (n) p-value TC 6.31 + 0.49 (10) 6.34 + 0.08 (226) 0.9 TG 3.16 + 0.37 (10) 2.73 + 0.07 (226) 0.2 HDL-C 1.42 + 0.11 (10) 1.74 + 0.03 (225) <0.02 LDL-C 3.43 + 0.49 (10) 3.33 + 0.07 (225) 0.8 FERHDL 24.07 + 7.02 (10) 19.24 + 5.47 (197) 0.007 Insulin 12.33 + 3.45 (10) 14.70 + 8.39 (219) 0.38 Glucose 4.28 + 0.29 (8) 4.14 + 1.67 (196) 0.81 Table 3-4: Lipid levels of N291S carriers and non-carriers. A similar comparison was done for carriers of the S447X mutation verses non-carriers (Table 3-5). A significant decrease in T G in the carrier group was the only noticeable difference found between the two groups (2.34 + 0.12 and 2.84 + 0.07, p<0.003). Due to a small number, statistical analysis of D9N carriers verses non-carriers could not be performed. 46 mmol/L Carriers (n) Non-carriers (n) p-value TC 6.48 + 0.17 (43) 6.31 +0.09 (197) 0.4 TG 2.35 + 0.12 (43) 2.83 + 0.07 (197) 0.003 HDL-C 1.77 + 0.06 (43) 1.73 + 0.03 (198) 0.5 LDL-C 3.62 + 0.17 (43) 3.26 + 0.08 (198) 0.06 FERHDL 19.16 + 5.35 (40) 19.59 + 5.83 (173) 0.66 Insulin 13.90 + 6.53 14.81 +8.67 0.51 (43) (192) Glucose 4.33 + 1.40 4.13 + 1.67 0.47 (40) (170) Table 3-5: Lipid levels of S447X carriers and non-carriers. 3.4 Frequency of Apo E Variants The 250 subjects were also screened for apo E genotype. A gel displaying the restriction digest of the 6 possible genotypes is presented in the methods section (Figure 2-4), and the frequency of the alleles is shown in Table 3-6. The apo E3 allelic frequency is high (81 %), whereas the allelic frequency for apo E2 and apo E4 are low (7.9% and 10.7%) which is what was expected. 47 Apo E Allele Number Allelic Frequency E2/E2 1/223 0.4% E2/E3 28/223 12.6% E2/E4 4/223 1.8% E3/E4 37/223 16.6% E4/E4 4/223 1.8% E3/E3 149/223 66.8% apo E2 34/446 7.6% apo E3 363/446 81.4% apo E4 49/446 11.0% Table 3-6: Apo E allele frequencies. 3.5 Effect of apo E Genotype on Plasma Lipids The lipid data for the various apo E genotypes was analyzed and are displayed in Table 3 - 7 . Significant differences were found in TC, LDL-C, F E R H D L , and insulin levels. Significantly lower levels of TC were found in the E 2 / E 4 individuals and apo E 2 carriers ( E 2 / E 2 and E 2 / E 3 ) compared to the E 3 / E 3 individuals (p<0.05). There was also significantly higher plasma LDL-C in the E 3 / E 4 , E 4 / E 4 , E 3 / E 3 individuals as well as in the apo E 4 carriers ( E 3 / E 4 and E 4 / E 4 ) compared to the apo E 2 carriers ( E 2 / E 2 and E 2 / E 3 ) (p<0.05). In addition, significantly lower F E R H D L values were found in the apo E 2 / E 3 and apo E 2 carriers ( E 2 / E 2 and E 2 / E 3 ) when compared to the E 3 / E 3 carriers (p<0.05). Finally, a significantly higher insulin level was found in the apo E 3 / E 4 carriers compared the apo E 3 / E 3 carriers. No differences could be seen in TG, HDL-C, or glucose between any of the groups. 4 8 0) o o o 3 c CD 00 CO O O O +1 CD CD CO CN LO +1 LO CN CO CN CN 01 CN CD O LO 00 00 CD 83 + 1. .38 ±1. CN 00 48 ±2. 17 + 1. (131) .93 ±0. (24) CO CO o CN CN IS-- CO 00 CO 00 o *- CD LO +l cn oo LO + i $ IS- S ~' o oo +1 CN CD CD +1 CM CD +1 00 oo CN a: HI CN O LO * +i LO o CN LO CO +1 CD CN CO LO ^ +1 CO 2. LO O CN CD 00 +1 IS-CM d CN CD LO +1 00 CD CD CN OO * 00 +1 ° ° CD O CN —i o Q E E Q E I E O o I- E E o o H E E CD CN 00 CO CD CN CN CO LO CN r--d +i CD CN 00 T d +1 oo oo CN CO CN LO CD d +1 CD CN LO CD d +1 CD CD LO CO CN CO CN 00 CO d +1 LO CN o 00 d +1 LO LO CN +1 CO LO 00 CO 00 d —> +1 o IS-co CD CO d +1 o 00 CN « IS-00 d +1 CD O LO CD d +i oo CD CN LO CO +1 CD 00 CD IS-CO IS-00 • o CN • 00 .39 ±1 +l 00 CO 00 00 LO 00 00 d +i d +i co CO LO CO IS--~ — ' CD h-o .03 ± 0. +l CO CO 00 CN o N- CN +l +l o> LO CO 00 CD cb CD CN CD CO O +l CD CO CN 00 d .1 CD + l CN CO CD CN CD d +i CD LO CN * CD ° C D +l CN CN CD LO LU O CM CO CO LU LU LU LU LU LU CM CN CM CO CO LU LU LU LU LU LU co CM CN LU + O CM 3.6 Effect of apo E and LPL Polymorphisms The subjects with both known LPL and apo E genotype were further divided and the effect of these two gene alterations was evaluated. The number of subjects with the various genotype combinations is shown in Table 3-8. WT 447+/- 291+/- 9+/- 447+/-291+/-447+/-9+/-447+/+ E2/E2 1 E2/E4 3 E2/E3 19 7 E3/E3 115 19 3 2 1 1 E3/E4 27 6 1 1 1 E4/E4 4 Table 3-8: Frequencies of the combined apo E and LPL genotypes. Statistical analysis was performed on the sub-groups with the highest number of individuals (represented by the boxed area in Table 3-8). Comparison of lipids between the apo E2/E3, E3/E3, and E3/E4 carriers with wildtype (WT) LPL is presented in Table 3-9. The LDL-C in the apo E2/E3 subjects was significantly lower than the apo E3/E4 and the apo E3/E3 subjects (p<0.05). The F E R H D L was significantly higher in the apo E3/E4 than the apo E2/E3 (p<0.005) and the insulin was also higher in the apo E3/E4 when compared to the apo E3/E3 (p<0.05). The differences when comparing the S447X carriers and the non-carriers of their respective apo E groups did not reach statistical significance, nor did the analysis of differences in apo E genotype in S447X carriers (data not shown). 50 CD CO o o co or LU Q E E 0 =d Jj o Q E 1 E O o i - E E o o H E E LO CD +1 CD CD c o O 00 ° ° CN + 1 T - : co LO ^ +1 CD co 2 , CO CD CD O +1 CO 00 CN d +1 CN +1 CO CO CN LO +1 CD LU co LO +1 ^ CN — CO CO co d +1 i— Is-CN O d +i CO CO CD CD CD CO d +i °2 00 Is-CN CO CO d CD l u S +1 CD LO cm C l u S Is-+i oo 00 CN 5.60 13.11 (26) +1 CO +i (26) o 00 (26) 00 CO * o CN •It o CD +i £ +l (23) •sr ^ Is-CD x— (23) cb CN o 00 +l CN LO CO Is-CN CD CO d +l CO CD CN CO d +l 00 CD CN CD Is-CN Is-CN Is-CN +l 00 CD co C LU § 3.7 LPL Sequencing The 10 exon and intron-exon boundaries of the LPL gene from the five individuals with the highest T G levels were sequenced by Dr. Robert Hegele at the University of Western Ontario. No mutations within their LPL gene including the promoter and introns were found in three of these individuals. One individual was heterozygous for a new codon 117 (GAG to GAA), which resulted in a silent mutation (E117E). The fifth individual was homozygous for a novel intron 5 mutation [-36]T/C. The lipid levels and genotypes of the five individuals are displayed in Table 3-10. 1 2 3 4 5 LPL Sequencing Homozygous intron 5 mutation [-36]T/C Heterozygous 117 mutation (GAG to GAA) None None None TG (mmol/L) 6.02 5.58 6.31 5.89 6.46 TC (mmol/L) 6.40 8.07 6.36 8.62 7.30 HDL-C (mmol/L) 2.44 1.08 1.08 1.44 1.22 LDL-C (mmol/L) 1.19 4.42 2.39 4.47 3.11 FERHDL (%/hr) 22.11 27.63 29.60 27.86 13.38 Insulin (uU/ml) 10.69 17.13 11.86 18.71 10.4 Glucose (mmol/L) 17.8 4.4 3.9 4.8 4.5 D9N -/- -/- -/- -/- -/-N291N -/- -/- -/- -/- -/-S447X -/- -/- -/- -/- -/-apo E E3/E3 E3/E3 E3/E3 E3/E3 E3/E3 Table 3-10: Sequencing results, lipid levels and genotypes of the five women with elevated TG levels. 52 CHAPTER IV: DISCUSSION 4.1 Cohort Characteristics Pregnancy is a unique physiological condition that brings dramatic changes to the female body. These changes include alterations in the level of sex hormones, which in turn have profound effects on lipid metabolism. In addition, there are numerous genetic and environmental factors, which may play a role in altering lipid metabolism and could result in abnormal lipid levels. The goal of this project was to investigate the effect of three LPL polymorphisms (D9N, N291S, and S447X) and apo E genotypes (4/4, 4/3, 3/3, 3/2, 4/2, 2/2) on plasma lipid levels during the third trimester of pregnancy. Self-reported environmental factors that play a role in lipid metabolism were also included in our investigation. We hypothesized that differences in plasma lipid levels during pregnancy are due, in part, to the presence of genetic polymorphisms in LPL and apo E, and pregnancy is a "stress" that enhances the effect of these genetic factors. A cohort of 250 healthy pregnant women in their third trimester was studied. They were of mixed ethnicity with the majority being of European descent. There was a small fraction of Asian and Indo Canadians, which reflected the population diversity in British Columbia [215]. The ethnic background of the population was important to consider when analyzing the genetic frequencies of LPL and apo E polymorphisms, as the ethnic variation of these polymorphisms are well established. However, it should not play a significant role on the effect of the polymorphisms because it is relatively consistent between ethnic groups. 53 Although subjects were screened for diabetes at recruitment, six women were included who reported having a history of diabetes. One reported gestational diabetes in a previous pregnancy and she remained in the study. The other five were subsequently excluded from further analysis because the nature of their diabetic condition was unclear. None of the participants were specifically tested for diabetes; the diagnosis was based on self-reported data from the questionnaire. Two women had a history of pancreatitis or abdominal pain when non-pregnant. They were not excluded from the study because no clinical diagnosis was made and a variety of conditions could result in abdominal pain or pancreatitis. There were also 11 women who reported having some form of thyroid disease, but also reported being well controlled. Thyroid diseases have been shown to dramatically alter lipid levels. However, the effect of thyroid treatment on lipid levels has been studied and it was found that serum TC, LDL-C, apo (a), apo B and HDL-C returned to normal within six months [216, 217]. The mean TC, LDL-C, HDL-C, and T G levels for the cohort fell within the normal range for pregnancy, according to the reference ranges at 36 weeks gestation [158] (Table 3-2). The plasma lipid levels from our cohort agree with the large number of studies on lipid levels during pregnancy [218-221], and are representative of what is expected of a healthy pregnant population. The TG and TC levels in our study were elevated but it is difficult to determine to what extent since non-pregnant values were not available for comparison. Similarly, HDL-C was elevated but it was impossible to assess from cross sectional data whether the HDL-C values followed a rise and fall pattern peaking at 20 weeks gestation. A pregnancy range has not been established for F E R H D L but Ordovas et. al. showed that the esterification rate of HDL increases during the first half of gestation and remains steady or slightly decreased throughout the second half 54 regardless of the continuing increase of plasma lipid levels [222]. This corresponds to the rise and fall of HDL-C, which is seen primarily in the HDL2 fraction with little change in the HDL3 fraction [147]. The mean F E R H D L in this group was higher than the non-pregnancy value for women of this age [4], which supports the previous findings. Glucose levels were 4.18 + 1.60 mmol/L which is similar to previous studies that reported lower glucose levels during pregnancy [223] particularly during the third trimester of pregnancy [224]. Similarly, observed insulin levels were within the normal non-pregnant range, which was slightly below that the pregnant value [225, 226]. Several additional environmental and genetic factors, such as weight gain, dietary and alcohol habits, and family history of diabetes, hypertension, or high cholesterol may influence lipid metabolism during pregnancy. Weight gain during pregnancy has been associated with increased TG levels [227-229]. This, in turn, may increase the incidence of borderline insulin resistance at this time. The current guidelines for weight gain during pregnancy is 9-14 kg, but some studies have found the weight gain in healthy pregnancy to be much greater, with a mean of 15 kg and a range of 8-25 kg [230]. In our cohort the average weight gain was 12.77 + 4.04 kg, however the range (1.4-28.6 kg) was quite large and may be related to the inaccuracies of self reported data. It has been shown previously that self reported weights are less accurate in overweight individuals [231]. Therefore, it is difficult to assess the relative effect that this parameter may have on the lipid levels in this cohort. Diet and alcohol habits have also been associated with changes in lipid levels [227]. A diet rich in T G leads to increased plasma TG levels during pregnancy [232]. In addition, pregnant rats fed cholesterol-rich diets have increased plasma T G and TC compared to 55 virgin controls [233]. Chronic alcohol use also alters lipid levels during pregnancy, resulting in lower LDL-C and HDL2 and increased HDL3 and VLDL [234]. The majority of women in this cohort reported being on normal diets with only 9% following a vegetarian diet. Similarly, 77% of the women reported never drinking during their pregnancy, but 23% reported drinking occasionally. Data on family history of diabetes, high cholesterol and hypertension were also recorded in this cohort. Previous studies have shown that positive family history of elevated cholesterol levels is associated with increased cholesterol levels of a study participant [235, 236]. A high proportion of our cohort reported family history of high cholesterol (33.6%), however, there were no differences in lipid levels between those who reported a positive history compared to those that did not. Nor were there any differences in lipid parameters between those who reported a family history of diabetes (29.2%) or hypertension (30%) compared to those who did not. 4.2 Lipoprotein Lipase Polymorphisms and Plasma Lipids The LPL polymorphisms were analyzed in the 250 women and the carrier frequencies for the D9N, N291S, and S447X, were 0.9%, 4.2%, and 18% respectively. These levels were not different than what has been previously published in a meta-analysis by Wittrup et. al. [100] and the Framingham Offspring Study [237]. Various ethnic groups have been studied and it has been found that the frequency of the LPL polymorphism varies among these groups. For example, the D9N was significantly higher in blacks residing in London England when compared to whites and South Asians from the same area, but no differences were found in cases of N291S and the S447X [238]. This issue is yet to be clarified as some studies have reported lower frequencies of N291S and higher frequency S447X in some ethnic groups such as Chinese [115, 239] while other reports state the opposite in unique groups such as some French Canadian populations [240]. The lipid levels of carriers and non-carriers were compared only for the N291S and S447X polymorphism, but not for the D9N variant due to the small number of carriers. A significantly lower HDL-C level (p<0.015) and higher F E R H D L (p<0.007) were found in carriers compared to non-carriers of the N291S, but no significant differences were found in T G (Table 3-4). Lower HDL-C in carriers of the N291S mutation in this cohort has been found in some other studies of non-pregnant subjects [104, 211, 241, 242, 243], but not others [111, 244, 245]. Similar to our findings, in several studies there were no significant differences between N291S carriers and non-carriers in regards to plasma T G [111, 241, 245], while in others there were [104, 242-244, 246]. Two studies did not report an association between the N291S variant and either HDL-C or T G levels [111, 245]. In the non-pregnant state, the majority of the studies described so far find an association of the N291S polymorphism with lipid abnormalities, specifically with raised TG and low HDL-C. However, the segregation of T G and HDL-C in the N291S carriers that we find in the third trimester of pregnancy has been reported in one other study [241] involving non-pregnant heterozygotes for familial hypercholesterolemia (FH). It is possible that FH and pregnancy are two unique conditions that result in an altered lipid metabolism with similar phenotype. Both these conditions are associated with increased T G levels, which may mask the effects of the N291S variant on T G levels between carriers and non-carriers. In addition, they are both associated with 57 increased C E T P activity [247, 248], which may result in a decrease in HDL leading to low HDL-C levels in spite of similar TG levels. The difference in F E R H D L between carriers and non-carriers is interesting and is most likely the result of the differences in HDL-C observed between the two groups. F E R H D L is strongly correlated with HDL particle size and is also inversely related to HDL-C level [4, 5]. F E R H D L increases during pregnancy most likely as a result of the increasing HDL3 fraction [222, 249], but no documentation has reported the effect of LPL polymorphism on F E R H D L during the non-pregnant or the pregnant state. When comparing S447X carriers to non-carriers, only TG was found to be significantly lower (p<0.002). This has been previously reported in men in the Framingham Offspring Study, in addition to increased HDL-C [237]. The first report of S447X as a molecular basis for LPL deficiency was in a patient with type I hyperlipidemia [250]. Since then, in vitro analysis has lead to conflicting results concerning the variants' effect on the catalytic function [105-108]. It has been suggested that the S447X variant may result in increased production of LPL protein and lipolytic activity [106, 108, 109]. The lower TG levels seen in the S447X carriers in our population are probably a direct result of the overall increase in LPL activity associated with the S447X polymorphism. We also found a trend to increasing LDL-C in carriers (p<0.06), which is also likely due to the increased LPL activity leading to increased VLDL remnants being ultimately converted to LDL. This observation has not been previously reported and may be noticeable because of the increased T G levels during pregnancy. No differences were found in plasma HDL-C levels, which may be related to gender differences as increases 58 in HDL-C in carriers of the S447X variant have only been previously reported in men [212, 237]. 4.3 Apolipoprotein E and Plasma Lipids The apo E genotypes were determined in the 250 women. The frequencies of the E2, E3, and E4 alleles were 7.6%, 81.4%, and 11% respectively. These frequencies are not significantly different from what has been previously found in Caucasian cohorts [251-253]. However, the frequency of the apo E allele has also been shown to fluctuate greatly between ethnic populations. A large number of studies on African populations, both African American [254] as well as a number of African tribes [255, 256] have shown significantly higher frequency of the apo E4 allele. In addition, studies in Papua New Guineans have also shown significantly higher frequencies of apo E4 and lower apo E2 than in Caucasians [253]. On the other hand, Japanese populations have significantly lower frequency of apo E4 allele and the Malays have a higher frequency of the apo E2 allele [257]. Ethnic variations are an important consideration when discussing the frequency of the apo E alleles. However, it has been shown that the apo E alleles have a relatively uniform impact in different populations despite differences in genetic background and environmental factors such as age, obesity, presence of CHD, hyperlipidemia and diabetes [257, 258]. There is no significant difference in the apo E allele frequency between men and women, yet the impact of the apo E alleles on plasma lipids appears to be greater in women than in men [259]. Many groups have studied the effect of apo E genotype on lipid levels [124, 127, 128, 133, 257-260]. It has been well established that, regardless of ethnicity and under a 59 variety of conditions, there are distinct differences in plasma lipids among the various genotypes, yet this has not been studied during pregnancy. We found that, in this population, there were significant differences in plasma TC, LDL-C, F E R H D L , and insulin levels between the various genotypic apo E groups (apo E 2 / E 3 , E 3 / E 3 , E 3 / E 4 , E 2 / E 4 and E4/E4)(Table 3 -7) . The TC was significantly lower in the E 2 / E 4 group than the E 3 / E 3 group. This has been previously reported in the non-pregnant state, [ 123 , 2 6 1 ] . No significant differences in TC were found between E 2 / E 3 , E 3 / E 4 , or E 4 / E 4 when compared to the E 3 / E 3 group. This was unexpected because of the strong evidence that supports the effect of apo E genotype on plasma TC of these groups [258] . With respect to LDL-C, lower levels were found in the E 2 / E 3 group compared to the E 3 E 4 , E 4 / E 4 , and E 3 / E 3 groups. This agrees with previous reports published on the relationship of E 2 allele to plasma LDL-C [262] . We did however, expect to find increased LDL-C in E 4 carriers, as has been previously reported [263] , but this was not seen in our cohort. No significant differences in T G levels were observed between any of the groups. Although T G levels have been previously found to be higher in subjects carrying the apo E 2 allele and subjects with the apo E 3 / E 4 genotype than in individuals with the apo E 3 / E 3 genotype, this was only observed after the use of meta-analysis [258] . Plasma T G levels vary widely among and within individuals [32], this was also evident in our cohort. This variability in TG levels could mask effects of the apo E phenotypes on T G levels [258] . Similarly, no differences were found in HDL-C between any of the groups, while using a meta-analysis, HDL-C levels have been found to be lower in the apo E 3 / E 4 subjects [258] . 6 0 There were statistically significantly lower F E R H D L levels in the E2/E3 group compared to the E3/E3 group. This finding suggested that there might be a change in HDL composition associated with apo E2 allele. Apo E appears to play an important role in HDL-C metabolism and RCT by facilitating the expansion of the HDL core and enhancing its cholesterol-carrying capacity [264, 265]. It also directs the removal of cholesteryl-ester-enriched HDL by the liver [266]. The known lower binding capacity of the apo E2 allele may slow down the catabolism of HDL and prevent its removal by the liver resulting in an increase in the large HDL2 pool and thus lower F E R H D L in the carriers of the apo E2 allele. The effect of apo E genotype on plasma lipids may be unique to the physiological condition of pregnancy. In regards to both the TC and the LDL-C the altered lipid metabolism in pregnancy may change the phenotypic expression of the apo E alleles. Apo E is responsible for targeting remnant particles towards the uptake by the liver receptors. The apo B/E (LDL) receptor removes chylomicron remnants, VLDL remnants and IDL. In the non-pregnant state, apo E modifies the uptake of these particles, and is the rate limiting step in their catabolism. As there is an overall decrease in LPL activity (approximately 85%) during pregnancy [151, 267], the conversion of chylomicrons and VLDL to chylomicron and VLDL remnants may be impaired. This, in turn, lowers the apo E substrate pool and may decrease the need for uptake of apo E-containing particles by the liver. This may result in an elimination of the differences in lipid levels between the apo E4, E3 and E2 carriers. It is also possible that there were no differences in the levels of LDL-C in the apo E4 carriers because of the age of the 61 women studied. It has been shown that the differences in LDL-C levels are smaller in premenopausal women than in postmenopausal women [268]. Significantly higher plasma levels of insulin were found associated with the E3/E4 genotype when compared to the E3/E3 genotype. A number of studies have examined insulin levels and apo E genotype in various populations. Gonzalez et. al. reported significantly higher insulin levels in apo E4 carriers with Alzheimer's disease but not in the apo E4 control subjects [269]. In another study involving Alzheimer's disease patients, both apo E4 and insulin concentration were found to be associated with the disease. However, it was concluded that the insulin resistance syndrome was independent of apo E4 in Alzheimer's disease patients [270]. Similar to our findings, Uusitupa et. al. reported the highest insulin levels in apo E4 carriers with central obesity in a group of women classified by waist circumference and apo E genotype [271]. This finding suggests that apo E genotype, specifically apo E4, modifies the effect of central obesity on metabolic variables characteristic of insulin resistance. In another study, insulin levels were significantly higher in postmenopausal women under the age of 55 with apo E3/E4 genotype compared with apo E3/E3 and apo E2/E3 [272]. This may suggest that the effects of the apo E4 allele are modulated by hormonal changes, such as changing estrogen levels, which is common to both menopause and pregnancy. 62 4.4 Gene-Gene Interactions of LPL and Apo E Polymorphisms on Plasma Lipids Plasma apolipoprotein and lipid levels are likely to be determined by gene-gene or gene-environment interactions. The variation of an allele at a specific locus could alter the phenotypic expression of another polymorphic gene. Studying gene-gene interactions in a unique condition, such as pregnancy, may help to explain the variations in expression of a single trait. We analyzed the participants' subgroups according to their apo E and LPL gene polymorphisms. Although the numbers were small, we found significant differences in LDL-C, F E R H D L and insulin between the apo E 3 / E 3 , E 2 / E 3 , and E 3 / E 4 carriers with wildtype LPL (Table 3 -8) . This was similar to the analysis of the effects of apo E genotype without taking into account the LPL polymorphisms (Table 3 -7) . The only difference was in effects of the apo E polymorphism on plasma TC, which was no longer significant after the removal of the LPL polymorphism carriers. We were unable to show differences in lipids levels between the S 4 4 7 X carriers with the various apo E genotypes, or between the apo E groups with and without the S 4 4 7 X polymorphism. This is most likely due the small numbers in each group but may also be due to the changes in lipid levels during pregnancy. Several research groups have studied the interaction of apo E and LPL on lipid metabolism [ 2 7 3 - 2 7 9 ] . Salah et. al. assessed the effects of the LPL S 4 4 7 X polymorphism and apo E genotype on lipids in a normal (non-pregnant) population [273] . Their findings are similar to ours: plasma LDL-C was lower in the E 2 / E 3 wildtype LPL carriers compared to the E 3 / E 3 wildtype LPL carriers. However, they also reported that the carriers of the E 4 allele, both with and without the S 4 4 7 X polymorphism, had 6 3 higher levels of TC and LDL-C compared to the apo E3/E3 genotype but were not significantly different from each other. Similarly, the apo E2 genotype, with and without the S447X polymorphism, was linked to low plasma TC but no differences were found between the ape E2 LPL S447X carriers and the apo E2 S447X non-carriers. The only effect of the S447X polymorphism on the apo E alleles in a non-pregnant population was on T G levels, with S447X carriers having significantly lower T G values that their non-carrier counterparts. It was concluded that apo E and LPL act independently on lipid levels and that the apo E4 carriers without the S447X polymorphism had the highest T G levels in addition to high TC and LDL-C. Our study, was not statistically powered to prove this point. There are no cross sectional population studies of the effects of any of the LPL polymorphisms and apo E alleles during pregnancy. However, a small number of case studies have reported dramatically altered lipid levels in individuals with variations in both genes during pregnancy [194]. In the majority of these cases, the LPL mutation results in decreased LPL activity and the apo E genotype is E2 heterozygote or homozygote, resulting in increased TG. In our cohort there were no individuals fitting these criteria. Some of these individuals may have been diagnosed with hypertriglyceridemia, and would not have been eligible for our study. We did, however, have one apo E3/E4, N291S heterozygote and one apo E3/E4, D9N heterozygote. The lipid levels for the apo E3/E4, D9N heterozygote were not remarkable, yet the apo E3/E4, N291S heterozygote had above normal T G (6.12 mmol/L), high TC (5.83 mmol/L) and normal to low HDL-C (1.47 mmol/L) for this stage of pregnancy. Although no firm conclusions can be drawn from one case, the effect of variations in these two proteins during pregnancy merits further study. 64 4.5 LPL Sequencing The LPL gene of the five women with the highest plasma T G levels during the third trimester of pregnancy were sequenced to search for additional mutations (Table 3-10). In four of the five women the findings did not further explain their elevated T G levels. In one of these four women a heterozygous point mutation at positions 117 resulted in a silent mutation, thereby not altering LPL activity. When the questionnaire data of the four individuals was reassessed, no further explanations for increased T G became evident. Only one of the four women reported having family history of high cholesterol and diabetes, and they all had normal glucose and insulin levels. None of them drunk alcohol and all followed a normal diet. The weight gain for the four women (11.4-14.6 kg) was within the normal range for their state of pregnancy. None of the four women carried any of the other LPL polymorphisms and they all had apo E3/E3 genotype. There are a number of other environmental and genetic factors that play a role in lipid metabolism which may become significant during pregnancy. Although these four women did not have diabetes and they had normal insulin and glucose levels, glucose tolerance tests were not performed and they may have been glucose intolerant. In addition, small changes in apo C-ll may alter LPL binding to chylomicrons and may only become apparent during pregnancy. There is also the possibility that these women had not properly fasted for the blood test, which would have resulted in elevated T G levels. The sequencing results from the fifth woman revealed a homozygous intron 5 mutation at position [-36]. To our knowledge, this mutation has not been previously reported and its effect on LPL activity and lipid metabolism is unknown. Several other intron 65 mutations have been identified in the LPL gene [280-284]. The majority of these mutations result in splice variants with a dramatically altered protein product and quite often low or no LPL activity. Nakamara et. al. described a T to C transition in intron 3, 6 base pairs upstream form the splicing acceptor, in a group of hypertriglyceridemic individuals. These six individuals did not have any other LPL mutations. They were all male and four of them had impaired glucose tolerance or diabetes mellitus. This suggests that the intron 3 mutation combined with other factors may be related to hypertriglyceridemia [284]. Similarly, a homozygous intron mutation, as found in our participant, in addition to the stress of pregnancy, could result in increased T G levels. This individual did not have any other LPL mutations and her apo E genotype was apo E3/E3. She did not report any family history of disease and stated that she never drank and was on a normal diet. In addition, she reported a normal weight gain of 11 kg during pregnancy. However, she did have elevated plasma glucose levels (17.8 mmol/L), suggesting diabetes which could, in itself, lead to increased plasma TG levels. It is possible that the homozygous intron mutation detected in this woman is a contributing cause to her hypertriglyceridemia and further investigations into the effect of this mutation on LPL protein structure and activity would have to be investigated. 4.6 Genetic Polymorphisms in LPL and Apo E and Risk of CHD As both LPL and apo E play important roles in plasma lipid variations, either of these genes may contribute to the risk of developing coronary heart disease (CHD). In addition, we have to take into account the possible risk associated with the altered lipid phenotype temporarily brought on by these variants during pregnancy. 66 4.6.1 Lipoprotein Lipase Despite the association of the N291S polymorphism with lipid abnormalities, only one study has shown an increased prevalence of CHD in carriers compared to non-carriers [104]. In this study, which consisted of 9214 randomly recruited individuals and 948 patients with ischemic heart disease, female carriers of the N291S polymorphism were found at a higher frequency among patients with ischemic heart disease than among the controls. This association was not seen in men regardless of the association with an atherogenic lipid profile [285]. The increased frequency of CHD in the female N291S carriers may be a result of decreased HDL-C levels associated with the variant, leading to a lipid profile in these women which is similar to men. In contrast to our findings of no differences in plasma T G between N291S carriers compared to non-carriers, Wittrup et. al. reported a significant decrease in TG levels in the female (non-pregnant) N291S carriers compared to the non-carriers. Comparing our lipid data to the non-fasting plasma analysis done in a non-pregnant population containing a large number of ischemic heart disease individuals, may not be relevant. T G levels in the non-fasting state are usually higher than in the fasting state and may mask or enhance some of the impact of the N291S polymorphism. If increased risk is associated with decreased HDL-C and high T G levels in women carriers in the non-fasting state, women with the N291S polymorphism may be at an even higher risk during pregnancy, if the HDL-C levels remain low. These data, combined with a meta analysis by Hokanson et. al. [114] and a more recent study by Minnich et. al. [286], suggest that despite its influences on lipid levels, the presence of the N291S polymorphism alone may not be sufficient factor in the development of CHD. However, in combination with other 67 environmental factors, such as obesity [244, 246] and pregnancy [193], the N291S polymorphism may represent a predisposing genetic factor for CHD. As mentioned earlier, the S447X polymorphism is associated with increased HDL-C levels and decreased T G levels. Heterozygosity for the S447X polymorphism is associated with elevated HDL-C and has therefore been considered a potential benefit to carriers [212]. The S447X polymorphism has been found at lower frequencies in patients with CHD [287] and recent studies have demonstrated the S447X polymorphism to be associated with significant protection against CHD in men [237]. It was further estimated that 9% of CHD in the Framingham Offspring Study was prevented as a result of the S447X polymorphism [237]. How these findings relate to lipid changes during pregnancy is unclear. We did not find a significant increase in HDL-C levels in the pregnant carriers of the S447X polymorphism. However, we did find significantly lower TG. Whether the changes in plasma T G play a role in protection against CHD with respect to the S447X polymorphism has not been studied. However, there is now some consensus that increased T G level is an independent predictor of CHD [288, 289]. It is also unclear whether decreased T G levels during pregnancy will be detrimental to the development of the fetus as maternal free fatty acids [290] and TG concentrations [171, 291] are associated with infant birthweight. With respect to TG, the association with birthweight persists even when other maternal predictors are taken into account [171]. This does not appear to be clinically significant in our cohort but may play a role in pregnancies involving other environmental conditions leading to decreased T G levels such as malnutrition.[232] 68 4.6.2 Apolipoprotein E Apolipoprotein E plays an important role in the metabolism of lipoproteins as a ligand for both the LDL receptor and LDL receptor related protein (LRP). Various allele forms of apo E have a significant impact of plasma lipoprotein metabolism and thus on risk of CHD [133, 263, 292] and stroke [293]. A higher frequency of the apo E4 allele has been found in a CHD group compared with healthy controls. In contrast, the apo E2 allele was less frequent in the CHD group. The CHD risk association with the apo E4 allele is thought to result from higher TC or LDL-C levels associated with E4, with increased T G and decreased HDL-C as other contributing factors [258]. We found no significant differences in TG, HDL-C, or TC in the apo E4 carriers. In addition, there was no increase in plasma LDL-C when compared to the apo E3/E3 group. It is unclear whether the similarity in LDL-C levels between the apo E4 and the apo E3/E3 carriers reduces the risk of CHD in the apo E4 group or whether it increases the risk of CHD in the apo E3/E3 group. On the other hand, the apo E2 carriers had significantly lower TC and LDL-C but no significant differences in TG or HDL-C. This is consistent with the previous findings and suggests a lower risk of CHD compared to the apo E4 and the apo E3/E3 carriers. It was expected that in our cohort, the effects of the apo E alleles would be more pronounced because of pregnancy associated hypertriglyceridemia. However, as mentioned previously, with the LPL N291S polymorphism, the increased TG levels may mask the effect of the apo E alleles. Yet, on the other hand, these findings may reflect the true lipid profile during pregnancy. If so, the difference in plasma lipids and the risk of CHD between apo E4 and apo E3/E3 carriers may be less evident during pregnancy whereas apo E2 may still have a protective effect. It is clear that more work has to be done in this area before a complete understanding of how apo E alleles alter lipid levels during pregnancy and how these changes affect CHD risk. 4.7 Is Pregnancy a Risk Factor for CHD? The impact of pregnancy on the development of CHD has been studied extensively [294-297], yet the role that lipid changes during pregnancy play a role in the outcome of CHD is unclear. It has been shown in two prospective American studies, The Framingham Heart Study and the National Health and Nutrition Examination Survey National Epidemiological Follow-up Study, as well as smaller studies, that the risk of CHD is higher in women who have had six or more pregnancies [296, 298]. This finding remained significant even after adjustment for age and education level. One possible explanation for this observation was reported by Lewis et. al., who reported significantly lower HDL-C levels in women who have had a higher number of pregnancies [299]. D'Elio et. al. suggested that stress of child rearing may result in changes in stress levels and lifestyle but was unable to find an association between parity and stress [297]. Another study examined the effect of employment on HDL-C levels in nearly 2000 women living in Germany. It was found that the HDL-C levels of employed women were significantly higher than that of full-time homemakers. This association remained significant even after adjustment for age, body mass index (BMI), cigarette smoking, coffee and alcohol consumption, use of hormones, exercise, and reproductive history. [300]. The authors conclude that giving up employment may be related to lifestyle changes associated with decreased HDL-C levels leading to increased risk of CHD. 70 In addition to lifestyle changes, genetic variations, such as those examined in the current study, may explain why multiparous women are more likely to develop CHD. The N291S polymorphism was associated with decreased plasma HDL-C levels during pregnancy and occurred in 4.6% of our cohort. It is possible that in women with this polymorphism HDL-C levels stay low after the pregnancy and may further decrease with subsequent pregnancies. In addition, the beneficial effects of the S447X genotype (18%) seem to be abolished during pregnancy. More studies need to be done on the effect of genetic variations, such as LPL and apo E polymorphisms, that alter lipid metabolism during pregnancy. Our study showed, for the first time, that genetic factors affect lipid profile during pregnancy. The effect of gene-gene interaction between apolipoprotein E and lipoprotein lipase genotypes is not yet clearly understood. We also do not fully understand how the lipoprotein changes in pregnancy affect the future risk of CAD in childbearing women. Never the less, based on the reviewed literature and our own data it is reasonable to suggest that lipid abnormalities in pregnancy contribute to risk of CAD. 4.8 Future Directions This study offers valuable information on the role that polymorphisms play in lipid metabolism during pregnancy. It also allows to speculate as to the risk of CHD associated with pregnancy. Further research will be required to confirm our findings and delineate the basis of the gene-gene and gene-environment interactions responsible for abnormal lipid and lipoprotein phenotypes. An ideal future study will address the following areas: 71 1) Sample size: Although our sample size was appropriate to answer our research questions, it will be valualbe to repeat the analysis with a larger study group in a well designed prospective study. This will allow to analyze of additional variables and increase the statistical power of the study. 2) Data Collection: Collection of more detailed and accurate data on environmental factors such as weight gain, BMI, diet, exercise, smoking, and alcohol intake will provide a better assessment of the role of the environmental factors in lipid metabolism during pregnancy. This may be done by appropriately trained research coordinators, dietitians and nurses. 3) Other gene polymorphisms: There is also a number of other candidate genes, in addition to those examined in our study, that affect lipoprotein metabolism. Future studies should evaluate their roles individually as well as possible gene-gene interactions. 4) Study design Although a cross-sectional study is useful to evaluate a single time point, collection of plasma samples at various stages throughout the pregnancy and postpartum would be ideal to assess the changing dynamics during pregnancy. Longitudinal studies have been done during pregnancy but it would be useful in future studies to concentrate on the mechanisms involved and relate the specific genotypes and their interaction with others factors to the specific phenotypes. 72 In addition, pregnancy as a strong environmental factor may independently contribute to the risk of CHD. 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Gotoda, T., et al., Occurrence of multiple aberrantly spliced mRNAs upon a donor splice site mutation that causes familial lipoprotein lipase deficiency. J Biol Chem, 1991. 266(36): p. 24757-62. 284. Nakamura, T., et al., A novel nonsense mutation in exon 1 and a transition in intron 3 of the lipoprotein lipase gene. J Atheroscler Thromb, 1996. 3(1): p. 17-24. 285. Pimstone, S.N., et al., A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) results in altered postprandial chylomicron triglyceride and retinyl palmitate response in normolipidemic carriers. J Lipid Res, 1996. 37(8): p. 1675-84. 286. Minnich, A., et al., Lipoprotein lipase gene mutations in coronary artery disease. Can J Cardiol, 1998. 14(5): p. 711-6. 287. Galton, D. J . , et al., Identification of putative beneficial mutations for lipid transport. Z Gastroenterol, 1996. 34 Suppl 3: p. 56-8. 288. 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Am J Epidemiol, 1992. 135(1): p. 68-78. 95 Appendix A: Mutations in the LPL gene Location Region Type 1 Intronl non-coding 1 bp TV (SD site) 2 Exon 2 - intron 2 non-coding 1 bp TR (SD site) 3 lntron2 - Exon 3 non-coding 1 bp TR (SA site) 4 Intron 3 non-coding 1 bp TV (20 bp from SD site) 5 Intron 3 non-coding 1 bp TR (SA site) 6 Intron 3 non-coding 1 bp TR (6 bp from SA site) 7 Intron 4 non-coding 1 bp TR (SD site) 8 Intron 6 non-coding 1 bp TV (SA site) 9 Intron 6 non-coding 1 bp TR (1.57 kb from SA site) 10 Intron 6 non-coding Repeate (3' of Alu sequence) 11 Intron 8 non-coding 1 bp TV (485 bp from SD site) 12 Intron 9 non-coding 13 Intron 9 non-coding 14 -634 promoter 1 bp TV 15 -95 promoter 1 bp TV 16 -93 promoter 1 bp TV 17 -79 promoter 1 bp TV 18 -53 promoter 1 bp TV 19 -39 promoter 1 bp TR 20 Exon 1 5'UTR 13-19 (2bp Ins) 21 Exon 2 coding Asp9Asn (M) 22 Exon 2 coding Trp14Ter(T) 23 Exon 2 coding Thr18Ter(11 bp Del) 24 Exon 2 coding Asp21Val (M) 25 Exon 2 coding Asn43Ser (M) 26 Exon 2 coding His44Tyr (M) 27 Exon 2 coding 1 bp Ins 28 Exon 3 coding Tyr61Ter (T) 29 Exon 3 coding Trp64Ter (T) 30 Exon 3 coding Val69Leu (M) 31 Exon 3 coding Tyr73Ter (T) 32 Exon 3 coding Arg75Ser (M) 33 Exon 3 coding Trp86Arg 34 Exon 3 coding Trp86Gly 35 Exon 3 coding Lys102 (1 bp Del/6bp Ins) 36 Exon 3 coding Gln106Ter (T) 37 Exon 3 coding Val108Val (S) 38 Exon 3 coding 2 bp Ins 39 Exon 4 coding Glu118Glu (S) 40 Exon 4 coding N120 (4 bp Del) 41 Exon 4 coding His136Arg (M) 42 Exon 4 coding Gly139Ser (M) 43 Exon 4 coding Gly142Glu (M) 44 Exon 4 coding Val149Val (S) 45 Exon 5 coding Gly154Ser (M) 46 Exon 5 coding Asp156Asn (M) 47 Exon 5 coding Asp156Gly (M) 48 Exon 5 coding Asp156His(M) 49 Exon 5 coding Pro157Arg (M) 50 Exon 5 coding Ala158Thr(M) 51 Exon 5 coding Glu163Asp (M) 52 Exon 5 coding Glu163Glu (M) 53 Exon 5 coding Ser172Cys (M) 54 Exon 5 coding Ala176Thr(M) 55 Exon 5 coding Asp180Glu (M) 56 Exon 5 coding His183Gln (M) 57 Exon 5 coding Gly188Glu (M) 58 Exon 5 coding Gly188Arg (M) 59 Exon 5 coding Ser193Arg (M) 60 Exon 5 coding lle194Thr (M) 61 Exon 5 coding Gly195Glu (M) 62 Exon 5 coding His202His (S) 63 Exon 5 coding Asp204Glu (M) 64 Exon 5 coding lle205Ser (M) 65 Exon 5 coding Pro207Leu (M) 66 Exon 5 coding Gly209(1 bp Del) 67 Exon 5 coding Cys216Ser 68 Exon 5 coding Ala221(1 bp Del) 69 Exon 5 coding lle225Thr (M) 70 Exon 3-5 coding 6 Kb Deletion 71 Exon 6 coding Cys239Ter (T) 72 Exon 6 coding Arg243His (M) 73 Exon 6 coding Arg243Cys (M) 74 Exon 6 coding Arg243Leu (M) 75 Exon 6 coding Ser244Thr (M) 76 Exon 6 coding Asp250Asn (M) 77 Exon 6 coding Ser251Cys (M) 78 Exon 6 coding Leu252Arg (M) 79 Exon 6 coding Leu252Ter (T) 80 Exon 6 coding Ser259Arg (M) 81 Exon 6 coding Ala261Thr (M) 82 Exon 6 coding Tyr262His (M) 83 Exon 6 coding Tyr262Ter (T) 84 Exon 6 coding Ser266Pro (M) 85 Exon 6 coding Leu286Pro (M) 86 Exon 6 coding Asn291Ser (M) 87 Exon 6 coding Met301Thr (M) 88 Exon A coding Leu303Pro (M) 89 Exon 6 - Intron 6 coding 2 Kb Ins 90 Exon 7 coding Ala334Thr (M) 91 Exon 7 coding Thr352lle (M) 92 Exon 7 coding Leu353 (2 bp Del) 93 Exon 8 coding Thr361Thr (S) 94 Exon 8 coding Leu365Val (M) 95 Exon 8 coding Trp382Ter (T) 96 Exon 8 coding Trp382Ter (T) 97 Exon 8 coding Glu410Lys (M) 98 Exon 8 coding Glu410Val (M) 99 Exon 9 coding Cys418Tyr (M) 100 Exon 9 coding Ser447X (T) 101 Exon 9 coding 3 Kb Del TR = transversion, TS = transition, SA = splice acceptor, SD = splice donor, T = termination, M= missense. mutation, S = silent mutation, Ins = insertion, Del = deletion Appendix C Study of Lipoprotein Lipase Deficiency in Pregnancy Coordinated by: J Frchlich, MD Debbie DeAngeiis R.T., C C R C Heaithy Hean Program St. Paul's Hospital and UEC • What is your name? First Last _ _ _ _ phone# (h): • Date of Birth (d/m/y) I I I I phone# (w) • Famiiy Ethnic Background — (i.e. French, English, Chinese...) • Name of Physician • Number of previous pregnancies; Complications? Expected Date of Delivery • Are there any complication with this pregnancy? • Pre-Pregnant Weight • Present Weight • Height • Time of Last Meal _ Kg Kg cm What did you eat? • Are you on a regular or alternate type of diet (i.e. Vegetarian) • regular • other , Current Medications • none • yes •• What category best describes your alcohol drinking habits (pregnancy) • never • occasional (less than 1 drink a week) • 1 -5 Q 5 - 1 0 History of Disease Diabetes Hypertension Thyroid Bleeding Disorder Pancreatitis/ Severe Stomach Pain • Hepatitis Y N • • • • • • • • • • • • Famiiy History of Disease High Cholesterol Diabetes Mellitus Hypertension Bleeding Problems Angina / heart attack Strokes / amputation Pancreatitis/ Severe Stomach Pain Y N • • • • • • • • • • • • • For Lab Use Only!' Patient ID# • Date / time of blood collection Date questionnaire is answered PATIENT ADDRESS: 

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