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Cardiovascular Research ELSEVIER Cardlovascu[ar Rematch 35 0997) 2~0-255 Interactive effect of the p53 gene and cigarette smoking on coronary artery disease X.L. Wang, J. Wang, D.E.L. Wilcken " Department of Cardiovascular Medicine, University of New South Wales, Prince Henry/Prince of Wales Hospitals, Sydney, Australia Received 3 January 1997; accepted 15 April 1997 Abstract Objective: p53 is a tumour suppressor protein involved in the control of cell growth and has an established role in carcinogenesis, particularly in relfition to smoking. It may also be related to arteriosclerosis by affecting smooth muscle cell proliferation, a feature of atherogenesis. Methods: We explored a role for p53 in atherogenesis by assessing the a~ssociation between two DNA polymorphisms of the p53 gene (MspI at intron 6 and Haelll at intron 1) and angiographically documented coronary artery disease (CAD) in 654 Australian Caucasian patients. Results: There was a significant interactive effect of the two polymorphisms and cigarette smoking on CAD in a logistic regression analysis (P ~- 0.0039) but no association between CAD and either individual p53 polymorphic marker. CAD occurrence was more frequent in non-smoking patients with rare alleles at both sites (85.0%) compared to those homozygous for common alleles at both sites (70.4%). However, this was not seen in smokers (85.7 vs 82.8%). In all 654 patients cigarette smoking remained a significant predictor of CAD irrespective of p53 genotypes ( P = 0.0065). Conclusions: Our findings identify an interactive effect of both p53 polymorphisms and cigarette smoking on the occurrence of coronary artery disease in that non-smoking patients with rare alleles at both sites had increased incidence of CAD. They illustrate the relevance of genotype-specific and environment-dependent enhanced cardiovascular risk and foreshadow a need for further studies to establish functional changes. © 1997 Elsevier Science B.V. Keywords: p53 gene; DNA polymorphism; Cigarette smoking: Coronary artery disease; Gene-environment interaction I I I I I 1. Introduction A large body of evidence has established that both circulating and local arterial wall factors participate in the initiation and progression of atherosclerotie lesions [1,2]. Among the vascular wall changes are endothelial dysfunc- tion, uncontrollable proliferation of vascular smooth mus- cle cells (VSMC) and abnormal accumulation of extracel- lular matrix. Each of these may be influenced by both genetic and environmental factors [1-5] and so too may the relevant circulating variables [4,5]. Among the many, pathological processes occurring in the vascular wall during atherogenesis, endothelial dys- function has been thought a marker for the initial lesion and VSMC proliferation important for its progression [I-3]. • Corresponding author. Department of Cardiovascular Medicine, Clin- ical Sciences Building. Prince Henry Hospital, Linle Bay, NSW 2036, Australia. Tel.: +61 (2) 9382 5026: fax: +61 (2) 9382 5755; e-mail: x.l.wang@unsw.edu.au Many factors may be involved in enhancing abnormal proliferation, p53 is a mmour suppressor protein which is expressed in many types of human cells and is involved in the control of cell proliferation [6-9]. It also plays an important role in regulating the growth of VSMC [8-10]. Loss of p53 activity causes unrestrained growth while increased levels of p53 arrest cells in the G1 phase of the cell cycle. Mutant alleles of p53 are frequently found and also are often expressed at elevated levels in tumour cells [6-8,11]. They tend to bind to the wild-type subunit making it unable to function and in this way may alter the regulation of cell growth. Mutations have been reported in every codon of the five highly conserved domains from exon 5 through to 9 in various cancers [6]. Cigarette smoking appears to inactivate p53 by inducing mutations [12,13] and therefore we reasoned that p53 may influence the contribution of smoking to atherogenesis. Indeed, see- Thne for primal, review 31 days. 0008-6363/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0008-6363(97)00113-2 This article is tbr individual use only and may not be lbrther reproduced or stored electronically ~hthout written permission from the copyright holder. Unau~hor~2ec[ reproduction may re.suit fn tinancfa! and other pena~ftfes, fc) ELSEVIER ;SCIENCE: BV

i eral polymorphisms in the non-coding region of the p53 have also been shown to predispose an individual who ismokes, or who is a passive smoker, to the development of mmours [14,15], although this was not found in a recent Japanese study [16]. There are similarities between atherogenesis and benign Immour formation although in atherogenesis the process of proliferation is less aggressive and more chronic [17,18]. It is possible, therefore, that while major p53 mutations at iexons may lead to the development of malignant changes, some DNA variations at the p53 gene associated with quantitative changes in p53 production or minor alterations in p53 function may also be associated with the compara- tively mild enhancement found in atherogenesis. growth Data about such a relationship are sparse (only two stud- ies) and controversial [9,19]. Recently, SpoiLet al. showed la potential interactive effect of p53 and human cy- tomegalovirus on coronary re-stenosis after angioplasty [9]. A role for p53 in atberogenesis is also supported indirectly ibY the findings of Isner et al. [20]. They reported that apoptosis, a process to which p53 contributes [7,10], oc- curs in human atherosclerosis. However, D'Agostini et al. failed to show any relationship between a p53 DNA ipolymorphism and coronary artery disease (CAD) [19]. Atherogenesis is a muitifactodal process and many factors, particularly cigarette smoking [21,22], could con- Ifound a potential relationship between p53 and CAD. With this in mind, we explored a possible relation between p53 DNA polymorphisms and CAD documented angiographi- itally after controlling for other CAD risk factors including cigarette smoking. We specifically investigated the hypoth- esis that there are interactive effects between p53 and cigarette smoking which influence atherogenesis by assess- Iing this in Australian Caucasian CAD patients. We anal- ysed two polymorphic markers on the p53 gene. One was a HaelII polymorphism at intron 1 [23]; the other was the IMspI polymorphism at intron 6 [24] which is in the region (exons 5-9) of frequent mutations [6]. While many poly- morphic markers have been reported in various introns of p53 gene, we selected these two markers for the following I the HaeIII marker is located close the reasons. Firstly, to promoter region of the gene and could be in linkage with some functional changes in the regulatory region. Sec- Iondly, MspI polymorphisms at intron 6 may be in linkage disequilibrium with mutations responsible for functional changes since exons 5-9 are in a 'hot' region for mum- itions. i I I 2. Methods 2.1. The patients We studied Caucasian patients aged 65 years or less, both men and women, consecutively referred to the East- X.L. Wang et aL/ Cardiovascular Research 35 (1997) 250-255 251 em Heart Clinic at Prince Henry H~spital for coronary angiography over an 18-month period in 1994 and 1995. We excluded only patients shown to have significant left main disease (> 50% luminal obstruction) because it was difficult to categorise this small proportion of the total (5.0%) within the classification system we used (see be- low). A written consent was obtained from every patient after a full explanation of the study which was approved by the Ethics Committee of the University of New South Wales. A 4 ml venous blood sample was drawn into an EDTA sample tube from patients before the angiogram after a 6-14 h fast. The blood sample was centrifuged within 2 h and plasma and cellular components stored separately at -70°C in aliquot until analysis. DNA was extracted from the frozen cellular blood component by a salting-out method [25]. The extracted DNA was stored at 4°C until analysis. 2.2. Determination of the polymorphisms i~ the p53 gene The HaelII polymorphie marker is located at intron 1 of the p53 gene as described by Ito et al. [23]. The relevant DNA fragment was amplified by the 5'-'VI'CCGCTGTT- TCTTCCCATG-3' for the upstream and 5'- TGTGTGTAAATGCCACCTCG-3' for the downstream palmers in the PCR. The amplification was performed for 35 cycles with annealing temperature of 60°C in a Hybaid Thermal cycler in a total reaction of 50 p,l containing 180 ixM dN.TPs, 1.5 mM MgCI2, 50 mM KCI, 10 mM Tris/HCl (pH 8.3), 50 pmol of each palmer and 2 units Taq polymerase. 'The HaelII digest of the amplified frag- ment was subjected to electrophoresis through 8% acryl- anaide gel and identifies two alleles H1 58bp and 37bp, and H2 95bp in which H1 is a common allele. The MspI polymorphism is at intron 6, a G --* A substi- tution 61 bp downstream from exon 6 of the p53 gene (17p13) as described by McDaniel et al. [24]. This base change abolishes a Mspl restriction site and can be de- tected • after digestion of the relevant PCR fragment. The primers used for the PCR were 5'-AGGTCTGGTT- TGCAACTGGG-3' for the upstream and 5'-GAG- GTCAAATAAGCAGCAGG-3' for the downstream primers. The amplification was conducted for 35 cycles with the annealing tern .perature of 59°C in a total reaction volume of 50 ~,1 with contents the same a.~ the one above except for the specific pair of palmers. The amplified 107 bp fragment was digested by MspI (MI allele, 63 bp, 44 bp; M2 allele, 107 bp) in which M1 is a common allele. 2.3. Coronary angiographic documentation of CAD The presence and severity of coronary stenosis was determined by coronary angiograms which were assessed by two cardiologists who were unaware that the patients

252 X.I. Wang et al. / Cardiovascular Research 35 (1997) 250-255 were to be included in the study. Each angiogram was classified as revealing either no coronary lesion or lesions with less than or more than 50% luminal stenosis. For CAD incidence, patients were classified as having or not having angiographically demonstrable CAD; and for CAD severity we grouped the patients into those having one, two, or three major epicardial coronary arteries with more than 50% luminal obstruction. We also used the Green Lane coronary scoring system [25] which provides a nu- merical value for lesion severity and takes account of the amount of myocardium supplied by an affected vessel; the maximal score is 15. 2.4. Documentation of cigarette smoking We documented smoking status by grouping patients into those who were non-smokers and had never smoked and into those who were or had been smokers. Smokers were further grouped as current smokers who had regularly smoked at least 5 cigarettes per day for at least the previous 3 mOhths and ex-smokers who had stopped smok- ing for more than 1 year. The life-time smoking dose in pack-years (1 pack-year = smoking one pack of 20 cigarettes each day for 1 year) was recorded as described previously [25]. Table ! Characteristics of patients in the study (mean 5: s.e.m.) Without CAD With CAD N 128 526 Age (yr) 53.8 5:0.7 56.7 -I- 0.3 " " BMI (kg/m2) 27.7 + 0.4 28.3 5:0.2 Smokers (%) a65 (50.8%) 373 (70.9%) Life-time smoking dose (pack-years) 13.4 5:2.1 26.5 + 1.3 ° " Total cholesterol (retool/l) 5.3 5:0.08 5.5 d:0.04 HDL-cholesterol (retool/l) 1.215:0.03 1.05 5:0.01 " Triglyceride (retool/l) 1.684-0.08 2.075:0.05 ° TC/HDL-C 4.675:0.13 5.62+0.08 * Lp(a) (rag/l) 286 + 28 315 5:15 Male/Female 68/60 417/109 HaeHl polymorphism HI/I 104 (80.6%) 412 (78.5%) HI/2 24 (18.6%) 106 (20.2%) H2/2 I (0.8%) 7 0.3%) "H2" allele # 0.101 0.114 Mspl polymorphism MI/I 96(75.0%) 374(71.1%) MI/2 28 (21.9%) 144 (27.4%) M2/2 4 (3.1%) 8 (1.5%) 'M.2' allele0.141 0.152 a Smokers include both ex- and current smokers. ° * P < 0.01, ' * " P < 0.001 by Student's t-test or Xz comparison. Values are presented as means:s.e.m. # H2 is the rar~ allele of the p53 HaelII polymorphism and M2 is the rar~ allele of the p53 Mspl polymorphism. 2.5. Statistical analysis The frequencies of the alleles and genotypes among different subgroups were compared by X2-test. ANOVA was used for comparison.of quantitative variables among 3 groups or more and Student's t-test for two group compar- isons. A logistic regression analysis was used to assess the independent contributions to CAD of various risk factors while controlling for other variables using the SPSS-X statistical package. In the logistic analysis, predictive vari- ables are entered into the model either independently (described as main effect) or jointly (described as interac- tive effect). 3. Results 3.1. Genetic characteristics of the patients The demographic information for the 654 patients (male 441, female 161) with or without angiographically demon- strable CAD is shown in Table 1. Genotype distributions of the p53 MspI and HaellI potymorphisms between the two groups are not different and are in Hardy-Weinberg equilibrium as shown in Table 1. Although these two polymorphic markers are located more than 11 200 bp apart, they are in linkage disequilibrium (X2 = 9.89, dr-- 4, P --- 0.042). 3.2. p53 polymorphisms and occurrence of angiographi- cally documented CAD Among the 654 patients there were 68 men and 60 women who had angiographically normal coronary arteries (Table 1). Using ,a simple ×2-analysis, there were no relationships between the MspI or HaelII polymorphisms and the absence of CAD (X2 ~ 0.025, P ~ 0.98, and Xz ~ 1.64, P = 0.44, respectively). We assessed the relationship between the p53 polymor- phisms and CAD occurrence in a logistic regression analy- sis whilst controlling for the other potentially confounding variables age, sex, presence of hypertension or diabetes, body mass index, total cholesterol (TC), HDL cholesterol (HDL-C), lipoprotein(a), TC/HDL-C ratio and cigarette smoking. We entered both MspI, HaelII genotypes, smok- ing status [in terms of smokers (current and ex-smokers) and non-smokers], sex, presence of hypertension or dia- betes, BMI and other lipoprotein variables as individual terms, and MspI* smoking, HaellI*smoking, Mspl* HaelII*smoking, MspI* HaelIl*sex, Mspl * HaelII * age as interactive terms in a stepwise like- lihood ratio model. For current and ex-smokers in this patient population relationships with both CAD occurrence and severity were the same and depended upon the life-time smoking dose (in pack-years) as we have reported previ- ously [25]. Therefore we grouped current and ex-smokers |l 1 I 1 I

X.L. Wang et at. / Cardiovascular Research 35 (1997) 250-255 Table 2 Number of patients with CAD in relation to p53 HaelIl, Mspi polymor- phisms and cigarette smoking 253 p53 Polymorphisms * Non-smokers Smokers Males H1/I and Ml/! 56/70 (80.0%) 184/211 (87.2%) 'H2" or 'M2' 37/51 (72.5%) 102/115 (88.7%) "H2' and "M2' 12/12 (100%) 24/26 (92.3%) Females HI/I and MI/I 26/48 (54.1%) 34/53 (64.2%) 't-[2' or 'M2' 14/27 (51.9%) 26/29 (89.7%) 'I-I2' and 'M2' 5/8 (62.5%) 2/4 (50.0%) Total HI/I and MI/I 83/I 18 (70.3%) 218/264 (82.6%) "H2' or 'M2" 52/78 (66.6%) 127/144 (88.2%) 'H2' and "M2' 17/20 (85.0%) 26/30 (86.7%) '1 " H1/I and MI/I: common allele at both sites. 2" or 'M2': rare alleles at either site. 'H2' and 'M2': rare "alleles at both sites. Common alleles for the Haelll and MspI polymorphisms are 'HI" and 'Mi'; rare alleles for alleles for the HaellI and MspI polymorphisms are "H2" and i!"M2'. In the log-/inear regression analysis among all patients, smoking status is significantly correlated with the presence of CAD (×2 = 10.82, P ~ 0.001). WhiLst the p53 polymorphisms at both sites were not associ- ated with CAD as a main effect (×2. t.97, P-0.37), there is a I significant interactive effect of the polymorphisms and smoking on the presence of CAD (×z ~ 7.112, P - 0.028). I I ! I together in the model. While the analysis revealed that neither Mspl nor HaelII had any significant main effect ion CAD occurrence, the interactive term of MspI* HaelII* smoking did. There was a significant posi- tive effect on the presence of CAD (r ,~ 0.124, P ~" 0.0039) in the model which included all the main-effect terms and other interactive terms. The independent contributions of smoking (r-0.108, P-0.0065), age (r~0.200, P-- 0.00001), sex (r-- 0.214, P ~. 0.000001) and TC/HDL-C ratio (r== 0.13, P = 0.0016) were unaffected by this inter- active term and remained significant and independent pre- dictors. This interactive effect between the p53 polymor- phisms and smoking on CAD is shown for male and female patients in Table 2. Using log-linear analysis, there was a significant three-way interaction between smoking status, p53 polymorphisms and CAD (X2S 7.112, P ~ 0.028). To explain the interactive effect derived from the logis- tic regression, we have also presented the data in a tabu- lated form in Table 2. Although the association between smoking status and the presence of CAD was independent of the p53 polymorphisms, cigarette smoking appeared to weaken the association between p53 polymorphisms and CAD. Whilst the percentages of CAD in smokers with common alleles at both sites and rare alleles at either site (82.6 and 88.2%) was much higher than in those of non-smokers (70.3 and 66.6%), there was no difference in patients with rare alleles at both sites between smokers (86.7%) and non-smokers (85.0%). Furthermore, patients who had one or two rare alleles at both sites and were non-smokers were more likely to have CAD. However, this association was not observed in smokers (Table 2). We also explored the possibility that the p53 polymor- phisms could quantitatively modify the association be- tween cigarette smoking and CAD. As previously reported [25], the life-time smoking dose in patients who were smokers and who were assessed by coronary angiography was greater among those with than among those smokers without CAD (38.5 + 1.5 pack-years, n = 371, vs 29.5 + 2.8 pack-years, n = 67, P z 0,009). This difference was maintained among smoking patients who were homozy- gous' for the common alleles at both sites (30.5 ± 4.2 pack-years, n ~= 46 without CAD, vs 40.8 ± 2.1 p~ack-years, n ~ 218 with CAD, P- 0.035), but was not maintained among those with rare alleles at either of the two sites (28.6:1:3.4 pack-years, n ,= 21 without CAD, vs 35.9 q- 2.1 pack-years with CAD, tt= 153, P--0.08). This is consis- tent with the polymorphisms tending to minimise smoking-related vascular changes a/though there was only Table 3 Genotype distributions of the p53 MspI and HaeIII polymorphisms among all patients with different number of significantly diseased vessels I MspI genotypes a Number of significantly diseased vessels ( > 50% luminal obstruction) 0 I 2 3 MI / 1 133 (72.7%) 127 (69.4%) 96 (67.1%) 117 (78.0%) MI/2 or M2/2 50 (27.3%) 56 (30.6%) 47 (32.9%) 33 (22.0 .~,) HaeIll genotypes b H1/I 149 (81.4%) 139 (76.8%) 113 (79.6%) t~0 (78.9%). HI/2 or H2/2 34 (18.6%) 42 (23.2%) 29 (20.4%) 32 (21.1%) I p53 polymor~hisms ¢ H1/I and MI/I 111 (60.7%) 99 (55.3%) 82 (58.2%) 90 (60.4%) "H2' or "M2' 61 (33.3%) 64 (35.6%) 43 (30.3%) 54 (36.2%) I 'H2' and "M2' 11 (6.0%) 17 (9.5%) 17 (12.t%) 5 (3,4%) The observed number of cases and the colunm percentage (in brackets) are presented. The association between genotypes and the number of significantly diseased vessels was assessed by Xz-test a~ followings: (a) X2 w 5.08, P z 0.16; (b) ×2 ~ 0.85, P = 0.83; (c) X2 ~ 10.48, P = 0.10.

254 X.L. Wang et aL / Cardiovascular Research 35 (1997) 250-255 Table 4 Interactive effect of p53 Mspl and HaelIl polyraorphisms and smoking status on coronary scores (mean4-s.e.m.) p53 Non-smokers Smokers Total polymorphisms HI/I and MI/I 4.394-0.32 (118) 5.394-0.24 (264) 5.08:t:0.20 (382) "H2' or "M2" 4.01 +0.44 (78) 5.764-0.32 (144) 5.17±0.27 (222) 'H2" and "M2" 5.444-0.14 (20) 5.344-0.71 (230) 5.394-0.54 (50) F 0.71 0.34 0.11 p 0.491 0.713 0.90 F- and P-values wer~ obtained by ANOVA. a small number of patients with rare alleles in this sub- group. 3.3. p53 polymorphisras and CAD severity To explore a possible association between p53 and CAD severity, as opposed to occurrence, we first assessed severity from the number of significantly diseased vessels (Table 3). The, frequency distribution of the rare alleles was not different among those with or without signifi- cantly diseased vessels (> 50% luminal obstruction). In a ×2 comparison, neither of the p53 polymorphic markers was associated with the number of significantly diseased vessels (X2= 5.08, P ~ 0.16 for MspI polymorphism and X2-- 0.85, P = 0.83 for HaelII polymorphism and X2-= 10.48, P -- 0.105 for both polymorphisms). In a log-linear analysis, we also found no three-way interactions among p53 polymorphisms, cigarette smoking and the number of significantly diseased vessels (×z = 4.39, P ,- 0.623) whilst smoking remained a significant predictor for CAD severity (×2 = 19.5, P ~= 0.0002). Although non-smoking patients with the rare alleles at both sites tended to have higher coronary scores as shown in Table 4, the differences were not statistically significant. Using ANOVA the p53 polymorphisms had no main effect on the scores (P = 0.54). The p53 polymorphisms had no interactive effects with sex (P=0.194), smoking (P= 0.061), or age (P-~ 0.226) on coronary scores whilst sex (P = 0.0001), age (P ~,,0.00001) and cigarette smoking (P---0.007) all exerted major main effects on the scores. 4. Discussion The study identifies a significant interactive effect be- tween both p53 polymorphisms and cigarette smoking on CAD occurrence (P = 0.0039) whilst showing that the p53 HaeIII and MspI polymorphisms are not individually as- sociated with CAD occurrence and severity (Tables I and 3). The presence of the p53 rare alleles at both sites was associated with more frequent CAD in non-smokers, but not in smoking patients. Although the incidence of CAD among smokers was increased independent of genetic pro- files, the p53 polymorphism did appear to modify the association quantitatively. The frequency of CAD occur- rence in patients homozygous for the wild-type at both or either site was much higher in smokers than that in non- smokers (Table 2), an observation further supported by the differences in life-time smoking dose among smokers. However, this difference was not seen in patients with rare alleles at both sites. The findings of our study are consis- tent with the notion that certain genetically related CAD risk factors are environmentally modifiable, and that some environmental CAD risk factors could also be affected by specific genotypes. It is well established that cigarette smoking is a major risk factor for CAD. Smoking may enhance atherogenesis by many mechanisms as reviewed previously [21,22,26,27], but our present findings are consistent with the conclusion that p53 is one of the target molecules smoking affects in relation to atherogenesis. Denissenko et al. have also re- cently reported the involvement of p53 gene mutations in smokers who develop lung cancer 1"13]. Although the mechanism(s) remains unknown, we could speculate that smoking may quantitatively decrease the expression of functional p53, which in mm leads to abnormal prolifera- tion of many ceils including vascular smooth muscle cells and therefore promotes atherogenesis. Vascular smooth muscle cell proliferation contributes to atherogenesis. Fur- thermore, p53-related atherogenesis could also be mediated through p53-dependent apoptosis. Wild-type p53 induces apoptosis of vascular smooth muscle cells, particularly cells infected with viruses [10]. Thus expression of mutant p53 could block this wild-type p53 function and suppress apoptosis [10,20]. The occurrence of apoptosis in athero- matous lesions has been described although the mecha- nisms are not clear [20]. Specifically, our study shows that to determine the true association between p53 and CAD it is essential to control for smoking. Since both the MspI and HaelII polymorphie markers are on introns, we postu- late that they are in linkage with some functional effects which induce either quantitative or qualitative changes in p53 gene expression. Our results indicate that this relates to the presence of the rare alleles at both sites since altered CAD risk was only observed when both rare alleles were present. It has been shown that intron 4 of p53 influences gene expression [28,29], and Peller et al. have identified an association between the MspI polymorphism at intron 6 and cancer predisposition [30]. There is therefore a likeli- hood of a functional role for the polymorphisms we have studied. In vitro experiments are required to explore func- tional effects and to assess environment-dependent changes in p53 gene expression. Although the size of the patient population we studied is appropriate for evaluation of the main effect of the polymorphisms, for interactive effects the power is re- duced because of the degrees of freedom. This is particu- larly true for CAD severity and we found no association between the p53 polymorphisms and CAD severity. In conclusion, our current study is consistent with an associa-

X.I- Wang et al. / Cardiovascular Research 35 (1997) 250-255 255 tion between p53 polymorphisms and CAD occurrence which is influenced by cigarette smoking. Cigarette smok- ing remains a powerful CAD risk factor regardless of the p53 genotyp* and may mask the relationships with the p53 genotypes we assessed whereas in non-smoking patients with rare alleles at both sites the increased incidence of CAD is evident. Acknowledgements This work was supported by a grant from National Health and Medical Research Council of Australia. We wish to thank Ms Lily Fenech, Mr Steven Brouwer and all nurses in the Eastern Heart Clinic for their assistance in clinical data collection, and Ms Ah Siew Sirn for her laboratory assistance. We are also mos~ ~rateful to the cardiologists in the Department for allowing us to study their patients. References [1] Stary HC, Chandler AB, Dinsmore RE, et al. 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