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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 . S c , University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F M A S T E R OF S C I E N C E IN T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard  T H E 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.^3r^£^4W^^  The University of British Columbia Vancouver, Canada Date S e p V  DE-6 (2/88)  g>\  /  ^ V C ^ C A C O ?  {^OOCcJ^oc^  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 L P L 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 T G in addition to changes in plasma T C , 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, L P L 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 trimester of pregnancy were recruited. Fasting plasma T G , T C , HDL-C, LDL-C, rd  insulin, glucose and fractional esterification rate of HDL (FERHDL) were measured. Analysis of the L P L and apo E genes' polymorphisms were performed, in addition to  ii  sequencing of the L P L gene in 5 women with the highest T G levels. The frequencies of the L P L ( 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 % , E4, 11.0%)  polymorphisms were similar to previously published results in non-pregnant  women. Carriers of S 4 4 7 X had significantly lower T G levels ( p = 0 . 0 0 3 ) , and carriers of the N 2 9 1 S had significantly lower HDL-C levels ( p < 0 . 0 2 ) and higher F E R H D L ( p = 0 . 0 0 7 ) 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 T C , LDL-C and F E R H D L ( p < 0 . 0 5 ) compared to E 3 / E 3 , and carriers of the apo E4  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 L P L and apo E polymorphisms play an important role in T G metabolism. W e 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  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST O F AMINO ACIDS  vii  ABBREVIATIONS  viii  LIST O F T A B L E S  ix  LIST OF F I G U R E S  x  ACKNOWLEDGEMENTS  x'\  1. INTRODUCTION  1  1.1. LIPID A N D LIPOPROTEIN M E T A B O L I S M  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 A N D A T H E R O S C L E R O S I S  9  1.3. BIOCHEMICAL G E N E T I C S OF LIPOPROTEIN LIPASE  10  1.3.1. Overview  10  1.3.2. LPL Gene and Protein Structure  12  1.3.3. L P L and Hyperlipidemia  13  1.3.4. L P L Asp9Asn, Asn291 Ser, and Ser447X Polymorphisms  17  1.4. BIOCHEMICAL G E N E T I C S O F A P O L I P O P R O T E I N 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 F O R THIS S T U D Y 1.7. H Y P O T H E S E S 1.8. SPECIFIC AIMS MATERIALS AND METHODS 2.1. M A T E R I A L S 2.2. S T U D Y PARTICIPANTS 2.3. P L A S M A LIPID A N A L Y S I S 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 O F HDL ( F E R H D L )  2.5. INSULIN A N D G L U C O S E A S S A Y S 2.6. DNA A N A L Y S I S 2.6.1. P C R - B a s e d Detection of the Asp9Asn Allele 2.6.2. P C R - B a s e d Detection of the Asn291 Ser Allele 2.6.3. P C R - B a s e d Detection of the Ser447X Allele 2.6.4. P C R - B a s e d Detection of Apolipoprotein E Genotype 2.7. L P L 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 C H A R A C T E R I S T I C S  3.2. F R E Q U E N C Y OF LPL VARIANTS  45  3.3. E F F E C T O F 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 A P O L I P O P R O T E I N E V A R I A N T S  47  3.5. E F F E C T O F A P O L I P O P R O T E I N E G E N O T Y P E O N P L A S M A LIPIDS  48  3.6. E F F E C T O F A P O E A N D LPL P O L Y M O R P H I S M S  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 C H A R A C T E R I S T I C S  53  4.2. LIPOPROTEIN LIPASE P O L Y M O R P H I S M S A N D P L A S M A LIPIDS  56  4.3. A P O L I P O P R O T E I N E A N D P L A S M A LIPIDS  59  4.4. G E N E - G E N E INTERACTIONS O F LPL A N D A P O E P O L Y M O R P H I S M S O N P L A S M A LIPIDS  63  4.5. LPL S E Q U E N C I N G  65  4.6. G E N E T I C P O L Y M O R P H I S M S IN LPL A N D A P O E A N D RISK OF C A D  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 F O R C A D ?  70  4.8. F U T U R E DIRECTIONS  71  5. REFERENCES 6. APPENDICES  73 %  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 ANOVA  Analysis of variance  apo  Apolipoprotein  BMI  Body mass index  CE  Cholesterol ester  CETP  Cholesteryl ester transfer protein  CHD  Coronary heart disease  DNA  Deoxyribonucleic acid  EDTA  Ethylenediamine tetra-acetic acid  EDL  Endothelial lipase  FERHDL  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 R N A  PCR  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 L P L polymorphisms (D9N, N291S, S447X) 19 Table 1-•3: Apolipoprotein E alleles and association with lipid levels and C A D  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 W T 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 L P L D9N  37  Figure 2-2: Restriction digest and gel of L P L N291S  38  Figure 2-3: Restriction digest and gel of L P L 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  1.1.1  Lipid and Lipoprotein Metabolism  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 C H D [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 ( F E R H D L ) 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  Chylomicron VLDL  Chylomicron Remnant  10  20  40  Diameter (nM)  60  Figure 1-1: Classification by density of lipoprotein particles.  80  1000  T3 O  JZ Q. W  00  CO CM  O 00  CO  in  CO  o  CM  DO. O CD  < < i_ o o£  O  O >_  o (A 0) O  0  O  s  CD  c o a o a o a <  < O < LU ^ O  O  O  O  .c  a a a Q.J= < < < < O oo CM -^r o CO 00 T - T -  CQ O LU  o o o Q . CL  CD  Q.  CL_  _  < <<~ vO yfi 0  0  en in  vO  in m m CM m m s  s  i  i  Q. Q.  CO CM  t —  c '53  O  4-1  o Q. ©  ©  It E i , « flj «=» la- i=;  a> O) Q ^  O O CM  o  •  o  o  CO  CO 00  m  00 00 CM CD  m  CD O O  o  m  CD O O  V  c o o 1 o >» JZ o  CO  a >  O CM  O  m  CM  CM CO  ~  < < O mmo  m  in  00 CD O  CM  CD  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 V L D L . 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  Remnant?  J Chylomicrons  Reverse CETPj  Liver L  D  l  Cholesterol Transport  |_pj_ VLDL  Figure 1-2: Exogenous and endogenous pathways of lipid metabolism.  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 F F A and M G 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 T G which makes up roughly 90% of their total mass (Table 1-1). They are synthesized in the intestine and contain apolipoproteins A-l, A-ll, 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 T G . 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 T G , 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 V L D L by the liver. V L D L particles contain about 60% T G , 20% cholesterol ester, and 10% protein, which is almost exclusively apo B-100 (Table 1-1). Following secretion, V L D L takes up apo C (C-l, C-ll, C-lll) and apo E from HDL in exchange for T G [11]. These particles then undergo lipolysis by LPL. V L D L then takes up apo E from HDL forming V L D L 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 V L D L 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 R C T . 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 C H D 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 T G from LDL and V L D L via cholesterol ester transfer protein (CETP). C E T P enriches HDL with T G ; and it increases the cholesterol content of V L D L 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 T G ' s role in development of C H D has been controversial [32], it has been shown that high T G in combination with low HDL-C accounts for twice as many cases of C H D as low HDL-C alone [33]. In addition, T G rich particles of the apo B family represent significant predictors of C H D progression [34].  Approximately 80% patients with C H D 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 C H D 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 L C A T have proved to be important. In addition, the L P L and apo E gene are likely to be significantly involved in modulating the risk for dyslipidemia and atherosclerosis.  1.3  1.3.1  Biochemical Genetics of Lipoprotein Lipase  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 triglyceriderich 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 R N A (mRNA) is also detected in a wide variety of cell types, including macrophages. Unlike the adult liver, L P L is  synthesized in the fetal liver [49]. Following synthesis, L P L 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 L P L 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 L P L .  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 V L D L and chylomicrons. Following the hydrolysis of T G from the core of V L D L 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 L P L 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 L P L [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 L P L 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 T G , 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. L P L preferentially hydrolyzes the first and third ester bond of T G resulting in 2-monoglyceride. Monoglycerides are broken down more quickly if the first and third bonds are intact [59].  1.3.2  L P L 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. L P L 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 L P L [65, 66].  LPL and HL are bound to membrane proteoglycans and they are released into the plasma by intra venous heparin injection. L P L 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 T G . EDL has phospholipase activity and may play a role in modulating vessel wall lipid metabolism [63].  1.3.3  L P L 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 L P L deficiencies are autosomal codominant diseases meaning that both genes contribute equally to the phenotype. Complete deficiency in the L P L 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 L P L activity, that this unique syndrome was the result of a lipolytic defect [72].  13  Complete L P L 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 L P L deficiency, Breckenridge et. al. discovered that genetic absence of apo C-ll could result in a similar phenotype to L P L 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 L P L 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 L P L deficiency with two defective alleles, develop a phenotype of familial chylomicronemia previously called type I hyperlipidemia. Without LPL activity, chylomicrons and V L D L can not be catabolized properly resulting in the accumulation of these particles in the circulation and therefore, elevated plasma T G . L P L deficient individuals have very low levels of LDL-C due to their inability to properly process V L D L particles. Approximately 10-20% of V L D L is further metabolized into LDL [84]. There has been a great deal of research on the effects of L P L deficiency on the risk of atherosclerosis. Early studies suggested that L P L 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 L P L deficiency who had premature peripheral or coronary atherosclerosis before the age of 55. Impaired T G clearance may result in the increased exposure of lipoproteins to oxidation resulting in more atherogenic particles. In addition, inactive L P L 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 L P L knockout mouse pups had 3 times higher T G and 7 times higher V L D L - C than controls [88]. These animals became pale then cyanotic and finally died at around 18 hours of age. Total L P L 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 L P L in a single tissue type, such as muscle, was sufficient to rescue the L P L deficient pups [88], and maintain only a mild increase (1.5-2 times) in T G levels. Death from L P L knockout was also prevented by adenovirus mediated LPL 15  gene transfer to the liver [89]. This reduced V L D L levels and improved both oral and intravenous fat-load tolerance tests. Although heterozygous L P L 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 L P L 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 L P L 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 L P L deficiency. Peritz et. al. first characterized the deficiency in the LPL activity in hypertriglyceridemic cats [92]. These animals had high levels of L P L mass but no enzyme activity. The L P L 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 L P L 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 L P L deficiency is rare, heterozygosity for L P L 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 L P L 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 V L D L - C and V L D L - T G [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 C H D [99].  1.3.4  L P L 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 L P L 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 C H D 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 [105108]. A s 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 C H D risk [110-113]. However, a review paper reported a marginal negative association with C H D [114] and a meta-analysis suggested that carriers of the S447X mutation might possibly be at decreased risk of C H D [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  LO  oo o  o  X— I  o o  Q >>  o o o  CO  o o </)  <  a * * Q- to Q)  CO CO (/) ^ P CD o p O Q. —  ^  E CO ® .E "O CD to to — OO CDCDCC X3 to T3 CO _ c CD "C CD CO o CD E o  O i _i  12 re c E «2 re O  > ,o <  o  o o  Q X  c o  I  LO  Q  CD t/> CO CD  "D (D to CO  CD O CD Q i _  CD ^ to ^ CO > O O (1) CO Q  CD to CO CD  "O CD w CO  b 2 c o CD  —  Q  Q X  i—  O  c CO ]r o D) > CO CD rU) •=, O CO P o  o « g Z E co  O h-  T3 CD to CO CD o CD Q  T3  CD ^ CO > CD ~ O O CD °3 to  8.2  S 2  O  o c  CD  3  cr [it  i  CN  I  CO  CN  E  'I o E  X Q  CD O > _  CN  (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]. A s a ligand, apo E is responsible, in part, for uptake of dietary cholesterol in the form of chylomicron remnants, clearance of V L D L 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 T C (compared to E3) whereas E4 has been associated with higher T C [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  CN  CD CM  m  CM  CM  i  CM CM  CM 00 y  •- °.  •-  E  CD  o  0) O T J CO  sz  'Si  c o +5 (Q  to  "S | CD CO CO (0 CD =3 CD CO CO O CO O CD i _ CD £  CD CD "C h ~ co y CO CD  O O W  i=  CD  P  0)  "g CO  O  ™  12 C  £  —  •a  C  o—  CD  i »  CD  CLX:  O  T3 CD CO ! • • § CO C D o o i _ CD  co  CO ^  CD  ^  CO  roc"  <  ro « E g w <5  CD CD  CD CD CD CJ  a x o  a. O  C  o  Q Q -E  D)2  8 I g  CM V  o o  T3 C CO _co  CO (O CO  CD >  CD CD CD  •g  CD CD CD  CD  CO  >*-R E -o £ == 4 - CO *- o o  h J h ~o ~u ~u  co co to  — i i_  CO CD  Oi  X3 T3 L" (D (D T3 CO CO CD 1  o c  CD "CC O O v- Q CO CD — CO W, _ CL^Z — I c E  o a (J  12 a  O  00  CM CM CM 00  co co co  o o o c c c  Q.  o o  o  l _  4—  1—  C  CO  o o  CO CO CO  o  c  CO  &o G inO I  J? *= < < •o  CO  c o  <  o c 3 oo lit  CO  CD  JD  _Q)  CO  o  "CO  LU  c O  m  IS  m  o i _  Q. O Q. O  a.  CM LU  LU  TILL!  < 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 V L D L 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 V L D L (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  1.5.1  Pregnancy  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 T C 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 V L D L 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 V L D L 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 V L D L 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 L P L 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 L R P resembles the LDL receptor that is upregulated by estrogen [165, 166]. Yet in the liver, estrogen increases the secretion of T C and T G in the form of V L D L [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 V L D L - T G 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 L P L 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 L P L 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 T G absorption during pregnancy. The V L D L 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 T G 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 T G rich particles in the pregnant rat.  The contribution of L P L 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 L P L activity is reduced in humans by 85% during pregnancy [151]. Work by Knopp ef. al. has also demonstrated that L P L mediated T G 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 V L D L and LDL [187]. In addition, p migrating V L D L 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 L P L or apo E, or other causes such as diabetes, alcohol consumption or weight gain. In some cases extremely high T G 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 T G (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 T C , 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 T G levels, peaking during the third trimester. Because of the overproduction of TG-rich VLDL, normal pregnancy presents a challenge to the lipolytic capacity of L P L 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 L P L 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 T G level during the course of pregnancy. Thus, by using methods that are well established to detect three L P L polymorphisms and apo E genotypes in a group of normal pregnant women, it is possible to investigate whether selected polymorphisms in the L P L and apo E gene are associated with exaggerated hypertriglyceridemia in pregnancy, and to speculate as to the effect of these changes on C H D 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 L P L and apo E genes. Specific: 1. Carriers of the L P L N291S and D9N genotypes will have increased plasma T G levels and decreased HDL-C levels during pregnancy. 2. Carriers of the L P L S447X genotype will have decreased plasma T G and increased HDL-C levels during pregnancy. 3. Women who have the apo E2 allele will have lower levels of plasma T C & LDL-C, and higher levels of plasma T G , compared to wild type, during pregnancy. 4. Women who have the apo E4 allele will have higher levels of plasma T C , LDL-C and plasma T G , 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, T G , insulin, glucose, and fractional esterification rate of HDL (FERHDL).  3. To perform restriction fragment length polymorphism (RFLP) analysis to identify the three most common L P L polymorphisms. 4. To perform R F L P analysis to identify the apo E genotype. 5. Correlate the L P L gene polymorphisms and the apo E genotype with plasma lipid levels. 6. To sequence the L P L 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 W l , and the restriction enzymes (Hha I, Taq1, R s a 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 T G was determined as previously described [200], and plasma T C 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  FERHDL  was determined by an isotopic assay method that has been previously  described [205, 206]. Briefly, apo B-containing lipoproteins, V L D L and LDL, was precipitated from the plasma with the addition of phosphotungstic acid (PTA), and MgCI . A trace amount of tritiated cholesterol was applied to a paper disk, added to the 2  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 L P L gene and the apolipoprotein E variants by P C R and restriction endonuclease digestion of amplified product as described briefly bellow.  2.6.1  P C R - B a s e d Detection of the D9N Allele  The target sequence of the L P L gene (exon 2) was amplified by using 5'-AGG G C A A A T TTA C T T G C G A T G -3' as upstream primer and 5'-CTC C A G T T A A C C T C A 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  2l  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 L P L D9N polymorphism. Lane A heterozygous, lane B wildtype.  2.6.2  P C R - B a s e d Detection of the N291S Allele  The target sequence of the L P L gene (exon 6) was amplified by using 5 ' - G C C G A G A T A C A A T C T T G G T A -3' as upstream primer and 5'-ATA A T A T A A A A T A T A A A T A C T G C T T C T TTT G G C T C T G A C T G T 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 , 1% Triton X-100, 200 ng genomic DNA and final 2  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 L P L N291S polymorphism. Lane A heterozygous, lane B wildtype.  2.6.3  P C R - B a s e d 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 A A T G T C T A G G T G A-3* as upstream primer and 5'-TCA G C T TTA G C C C A G A A T 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  2.6.4  Figure 2-3: Restriction digest gels electrophoresis of the L P L S447X polymorphisms, lane A heterozygous lane B homozygous, lane C wildtype.  P C R - B a s e d Detection of Apolipoprotein E Genotype  The target sequence of the apo E (exon 4) gene was amplified by using 5'-ACA G A A T T C G C C C C G G C C T G G T A C A C - 3 ' as upstream primer and 5'-TAA G C T T G G C A C G G C T G T 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 D N A 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, D N A 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 L P L 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 A A T T G T A A A A C A C T C 3' and the upstream primer was 5' G T C A A A A T A T G C T G A G T G A A T 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 T G , all T G analyses were performed on logarithmically transformed values. Statistical analyses were performed using Microsoft Excel Data Analysis Package (Microsoft, Inc.).  40  CHAPTER III:  3.1  RESULTS  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%)  5 (2%)  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 T G , 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 T G , T C and LDL-C. Mean T G and T C were above the normal nonpregnant range whereas LDL-C remained within the non-pregnant value.  43  Normal Pregnant Range (non-pregnant range)  (n)  Range  Triglycerides (mmol/L]  2.74 + 1.01 (250)  0.95- 6.46  1.01 - 5 . 2 5 (<2.3)  HDL (mmol/L)  1.73 + 0.42 (249)  0 . 8 7 - -3.14  1.21 - 2 . 3 7 (>1.1)  LDL (mmol/L)  3.35 + 1.12 (249)  1.19- -7.14  2.32 - 5.56 (<3.4)  Cholesterol (mmol/L)  6.36 + 1.21 (250)  4 . 0 9 - 10.11  4.71 - 8 . 5 5 (<5.2)  FERHDL  19.52 + 5.37 (224)  8 . 5 4 - 38.43  14.57 + 8.22 (247)  3 . 7 3 - 59.58  4.18 + 1.60 (222)  0 . 3 - 17.8  Insulin (u.U/ml) Glucose (mmol/L)  —  (10.67 + 3.65) —  (6-24) —  (3.5-6)  Table 3-2: Cohort lipid levels.  The number of women with various T G 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 T G levels is skewed to the left.  a  40 35 30 25  r  H  L  rH  C  f  N  L  n CN  c  n  L  n  ^  i  n  i  n  L  f  CTi  t  ^  ir,  L  f  t  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].  D9N  N291S  S447X  +/+  0  0  1/240 (0.4%)  +/-  2/231 (0.9%)  10/236 (4.6%)  43/240(18.0%)  -/-  229/231 (99.1%)  226/236 (95.8%)  197/240 (82.1%)  Allele frequency  0.4%  2.3%  9.4%  Expected Carrier Frequency Y  2-4%  1-7%  17-22%  Genotype  Table 3-3: Frequency of L P L 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.  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  mmol/L  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  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 (43)  14.81 +8.67 (192)  0.51  Glucose  4.33 + 1.40 (40)  4.13 + 1.67 (170)  0.47  mmol/L  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 T C , LDL-C, F E R H D L , and insulin levels. Significantly lower levels of T C were found in the E 2 / E 4 individuals and apo E 2 carriers (E2/E2  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 E2/E3) E2/E3  (p<0.05). In addition, significantly lower F E R H D L values were found in the apo 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 T G , HDL-C, or glucose between any of the groups.  48  3  c  O  o CN  LO +1  CN  LO CN *  a: HI  CN O LO  LO  CN  CD  CN  d  +i  CD CN  oo CN  00 T  Q E I E  O o I- E E  00 CO  CD CN CN  d  +1  CO CN  CO LO  d  +1  CD CD LO  o  CO  CD  LO LO O CN  CD  ^ CO  2.  00  +1  d  •  CN CO  CD O LO  CO +l  CD CO O +l  00  d  d co  d  +i  +i  S  CO ~ — ' I--  + l  I-  CO LO  CD  CD  d —> +1 co o  ICO S  00 CN  +1  00  00  oo CD CN  d  +1 ° °  CD CN O  LO 00  +i  I00  CN OO  00 00  +1  «S  00  CD CO CN  d  00  *  •  o  CO 00  o  +1  oo  CN  o  d  CD LO 00 CD CD  S  o LO CN  +1  00  +1  00  +1 CD CM  ICM  00  d  00 CO  CD  oo  +1  +1  LO  CO  CO CN  CN  LO CN  CD  CD CN  +S i S$  I - ~'  +1  LO CD CO CN  *-  CO CD  +1  d  LO  +1  S  LO CN  oo  d  CN I--  CN 00 CO  00  LO CD  o o H E E  +1  CD  o  CN r--  —i o Q E E  +l  cn oo LO LO CO  +i  CO  CO  CN CO  CN 00  00 CD  .93 ±0. (24)  CO CN  LO 00  17 + 1. (131)  o  +1  CD CD CO  48 ±2.  00  .39 ±1  CD  CD O  S  h-  o  CD CN  CD CN  CO  CD  d  +i  CO CO CN  CD LO CN  N-  CN  * CD  +l  +l  LO CO CD  00  o  ICD 00 00 CD  S  .1  +l  00  LO CO +1  .03 ± 0.  o  01 CN  .38 ±1.  o  83 + 1.  O O  0)  o>  cb  CD CN  °CD +l CN CN CD LO  co LU O  CM LU  CO LU  LU  LU  LU  CO LU  CM LU  CN LU  CM LU  CO LU  LU  CO LU  CM CN LU + O CM  3.6  Effect of apo E and LPL Polymorphisms  The subjects with both known L P L 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 E2/E2 E2/E4 E2/E3 E3/E3 E3/E4 E4/E4  1 3 19 115 27 4  447+/-  291+/-  7 19 6  3 1  9+/-  1  447+/291+/-  447+/9+/-  447+/+  2 1  1  1  Table 3-8: Frequencies of the combined apo E and L P L 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) L P L 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 +1 CD c o  O  00 ° °  co  +1 ^ CN — CO CO  CN  + 1 T-:  +1 CO  o  or LU  co 2 ,  0 =d Jj o Q E 1 E  O o i- E E  o o H E E  •It  CN  CD  +i •sr I-  co d  +1  +1  CO 00  i— I-  CN  O  d  d  +1  +i CO CO  CN  CD CO  +1 CO CO CN  +1  o £ ^  cb  CD O  Q E E  *  s  CO CD  +i  00  00 CD  CD  s  d  +l I CN CO CD s  CN CO  °2  s  d  +l I CN s  00 CD  CN  CN  CO CO  CD  +1 CD  +l  d  Is  00  CD  CN  CD  LO  CD  I+l CN CN LO CO  CD CO  I-  LO  x—  o  CN  d  +l CD CN  s  LO  +i  CO  o  LO ^ +1 CD  CN  00  00  co  +i  oo 00  13.11 (26)  o  s  (23)  o  I-  LO CD  5.60  CD CO  LU  co lu  S  cm lu  C  S  co C LU  §  3.7  LPL Sequencing  The 10 exon and intron-exon boundaries of the L P L 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 L P L gene including the promoter and introns were found in three of these individuals. One individual was heterozygous for a new codon 117 ( G A G 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  Homozygous intron 5 mutation [-36]T/C  Heterozygous 117 mutation ( G A G to GAA)  None  None  None  TG (mmol/L) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L)  6.02  5.58  6.31  5.89  6.46  6.40  8.07  6.36  8.62  7.30  2.44  1.08  1.08  1.44  1.22  1.19  4.42  2.39  4.47  3.11  FERHDL  22.11  27.63  29.60  27.86  13.38  10.69  17.13  11.86  18.71  10.4  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  LPL Sequencing  (%/hr) Insulin (uU/ml) Glucose (mmol/L)  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 L P L 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. Selfreported environmental factors that play a role in lipid metabolism were also included in our investigation. W e 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 L P L 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 T C , LDL-C, apo (a), apo B and HDL-C returned to normal within six months [216, 217].  The mean T C , 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 32). 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 T G and T C 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 nonpregnancy 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 T G 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 T G levels during pregnancy [232]. In addition, pregnant rats fed cholesterol-rich diets have increased plasma T G and T C 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 V L D L [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 2 3 % 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 L P L 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 L P L 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 T G 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 F H 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 T G 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 L P L polymorphism on F E R H D L during the non-pregnant or the pregnant state.  When comparing S447X carriers to non-carriers, only T G 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 L P L 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 L P L protein and lipolytic activity [106, 108, 109]. The lower T G levels seen in the S447X carriers in our population are probably a direct result of the overall increase in L P L activity associated with the S447X polymorphism. W e also found a trend to increasing LDL-C in carriers (p<0.06), which is also likely due to the increased L P L activity leading to increased V L D L 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 [251253]. 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.  W e found that, in this population, there were significant differences in plasma T C , LDLC, F E R H D L , and insulin levels between the various genotypic apo E groups (apo E 2 / E 3 , E3/E3, E3/E4, E2/E4 E2/E4  and E 4 / E 4 ) ( T a b l e 3 - 7 ) . The T C was significantly lower in the  group than the E 3 / E 3 group. This has been previously reported in the non-  pregnant state, [ 1 2 3 , 2 6 1 ] . No significant differences in T C were found between E 2 / E 3 , E3/E4,  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 T C of these groups [ 2 5 8 ] . 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 [ 2 6 2 ] . W e did however, expect to find increased LDL-C in E 4 carriers, as has been previously reported [ 2 6 3 ] , 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 E2  allele and subjects with the apo E 3 / E 4 genotype than in individuals with the apo  E3/E3  genotype, this was only observed after the use of meta-analysis [ 2 5 8 ] . Plasma  T G levels vary widely among and within individuals [32], this was also evident in our cohort. This variability in T G levels could mask effects of the apo E phenotypes on T G levels [ 2 5 8 ] . 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 E3/E4  subjects [ 2 5 8 ] . 60  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 R C T 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 T C 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, V L D L 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. A s there is an overall decrease in LPL activity (approximately 85%) during pregnancy [151, 267], the conversion of chylomicrons and V L D L to chylomicron and V L D L 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. W e analyzed the participants' subgroups according to their apo E and L P L 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 E3/E4  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 L P L polymorphisms (Table 3 7). The only difference was in effects of the apo E polymorphism on plasma T C , which was no longer significant after the removal of the LPL polymorphism carriers. W e 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 L P L on lipid metabolism [ 2 7 3 - 2 7 9 ] . Salah et. al. assessed the effects of the L P L 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 L P L 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 63  higher levels of T C 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 T C but no differences were found between the ape E2 L P L 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 L P L 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 T C 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 L P L 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 L P L mutation results in decreased L P L activity and the apo E genotype is E2 heterozygote or homozygote, resulting in increased T G . 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. W e 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 T C (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 L P L 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 L P L 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 L P L 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 L P L 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 L P L activity and lipid metabolism is unknown. Several other intron 65  mutations have been identified in the L P L gene [280-284]. The majority of these mutations result in splice variants with a dramatically altered protein product and quite often low or no L P L 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 L P L 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 L P L 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 T G 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 L P L protein structure and activity would have to be investigated.  4.6  Genetic Polymorphisms in LPL and Apo E and Risk of CHD  As both L P L 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 C H D 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 C H D 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 T G 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 C H D .  A s 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 C H D [287] and recent studies have demonstrated the S447X polymorphism to be associated with significant protection against C H D in men [237]. It was further estimated that 9% of C H D 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. W e did not find a significant increase in HDL-C levels in the pregnant carriers of the S447X polymorphism. However, we did find significantly lower T G . Whether the changes in plasma T G play a role in protection against C H D 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 C H D [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 T G concentrations [171, 291] are associated with infant birthweight. With respect to T G , 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 C H D [133, 263, 292] and stroke [293]. A higher frequency of the apo E4 allele has been found in a C H D group compared with healthy controls. In contrast, the apo E2 allele was less frequent in the C H D group. The C H D risk association with the apo E4 allele is thought to result from higher T C or LDL-C levels associated with E4, with increased T G and decreased HDL-C as other contributing factors [258]. W e found no significant differences in T G , HDL-C, or T C 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 C H D in the apo E4 group or whether it increases the risk of C H D in the apo E3/E3 group. On the other hand, the apo E2 carriers had significantly lower T C and LDL-C but no significant differences in T G or HDL-C. This is consistent with the previous findings and suggests a lower risk of C H D 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 L P L N291S polymorphism, the increased T G 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 C H D 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 C H D risk.  4.7  Is Pregnancy a Risk Factor for CHD?  The impact of pregnancy on the development of C H D has been studied extensively [294-297], yet the role that lipid changes during pregnancy play a role in the outcome of C H D 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 C H D 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 C H D . 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 L P L 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. W e also do not fully understand how the lipoprotein changes in pregnancy affect the future risk of C A D 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 C A D .  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 C H D 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 C H D . 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A m J Epidemiol, 1992. 135(1): p. 68-78.  95  Appendix A: Mutations in the L P L gene  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46  Location Intronl Exon 2 - intron 2 lntron2 - Exon 3 Intron 3 Intron 3 Intron 3 Intron 4 Intron 6 Intron 6 Intron 6 Intron 8 Intron 9 Intron 9 -634 -95 -93 -79 -53 -39 Exon 1 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 4 Exon 4 Exon 4 Exon 4 Exon 4 Exon 4 Exon 5 Exon 5  Region non-coding non-coding non-coding non-coding non-coding non-coding non-coding non-coding non-coding non-coding non-coding non-coding non-coding promoter promoter promoter promoter promoter promoter 5'UTR coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding  Type 1 bp TV (SD site) 1 bp T R (SD site) 1 bp T R (SA site) 1 bp TV (20 bp from S D site) 1 bp T R (SA site) 1 bp T R (6 bp from S A site) 1 bp T R (SD site) 1 bp T V (SA site) 1 bp T R (1.57 kb from S A site) Repeate (3' of Alu sequence) 1 bp T V (485 bp from S D site)  1 bp TV 1 bp TV 1 bp TV 1 bp TV 1 bp TV 1 bp T R 13-19 (2bp Ins) Asp9Asn (M) Trp14Ter(T) Thr18Ter(11 bp Del) Asp21Val (M) Asn43Ser (M) His44Tyr (M) 1 bp Ins Tyr61Ter (T) Trp64Ter (T) Val69Leu (M) Tyr73Ter (T) Arg75Ser (M) Trp86Arg Trp86Gly Lys102 (1 bp Del/6bp Ins) Gln106Ter (T) Val108Val (S) 2 bp Ins Glu118Glu (S) N120 (4 bp Del) His136Arg (M) Gly139Ser (M) Gly142Glu (M) Val149Val (S) Gly154Ser (M) Asp156Asn (M)  47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95  Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon  5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 3-5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 A  6 - Intron 6 7 7 7 8 8 8  coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding  Asp156Gly (M) Asp156His(M) Pro157Arg (M) Ala158Thr(M) Glu163Asp (M) Glu163Glu (M) Ser172Cys (M) Ala176Thr(M) Asp180Glu (M) His183Gln (M) Gly188Glu (M) Gly188Arg (M) Ser193Arg (M) lle194Thr (M) Gly195Glu (M) His202His (S) Asp204Glu (M) lle205Ser (M) Pro207Leu (M) Gly209(1 bp Del) Cys216Ser Ala221(1 bp Del) lle225Thr (M) 6 Kb Deletion Cys239Ter (T) Arg243His (M) Arg243Cys (M) Arg243Leu (M) Ser244Thr (M) Asp250Asn (M) Ser251Cys (M) Leu252Arg (M) Leu252Ter (T) Ser259Arg (M) Ala261Thr (M) Tyr262His (M) Tyr262Ter (T) Ser266Pro (M) Leu286Pro (M) Asn291Ser (M) Met301Thr (M) Leu303Pro (M) 2 Kb Ins Ala334Thr (M) Thr352lle (M) Leu353 (2 bp Del) Thr361Thr (S) Leu365Val (M) 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 T R = transversion, T S = transition, S A = splice acceptor, S D = 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 U E C • What is your name?  First  • Date of Birth (d/m/y)  Last  ____ I I I  I  phone# (h): 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  Kg  • Present Weight  Kg  • Height  cm What did you eat?  • Time of Last Meal _  • 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 Q5-10 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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