- Email: [email protected]
APOA5—a recent addition to genes determining plasma triglycerides
International Congress Series 1253 (2003) 79 – 84
APOA5—a recent addition to genes determining plasma triglycerides Philippa J. Talmud * Division of Cardiovascular Genetics, British Heart Foundation Laboratories, Rayne Building, Department of Medicine, Royal Free and University College Medical School, 5 University Street, London WC1E 6JJ, UK
Abstract Raised plasma triglyceride (TG) levels is an independent risk factor for coronary artery disease (CAD), thus, understanding the genetic and environmental determinants of TG levels is of major importance. TG metabolism is a process for delivering free fatty acids for energy storage or hoxidation, involving a number of different hydrolytic enzymes and apolipoproteins (apo). The genes encoding these proteins are, therefore, candidates for determining plasma TGs. There is evidence that common variations in LPL, apo CIII (APOC3) and apo E (APOE) have the strong effect on plasma TG levels at the population level. More recently, the latest recognized member of the apolipoprotein gene family, APOA5, identified by comparative sequencing between human and mouse DNA, was located approximately 27 kb distal to APOA4 in the APOA1 – C3 – A4 gene cluster on chromosome 11q23, and variation in APOA5 was shown to be associated with plasma TG levels. The involvement of variation in the APOA1 – C3 – A4 locus in determining differences in lipid levels is well documented. We have examined the relative influence of APOA5 variants on plasma lipids, compared to the impact of variation in other genes within the cluster to determine if these effects are independent or a consequence of the strong linkage disequilibrium across the locus. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Triglycerides; Animal models; APOA1 – C3 – A4 – A5 gene cluster
1. Introduction The strong inverse correlation between high-density lipoprotein cholesterol (HDL-C) (an independent risk factor for CAD) [1] and plasma TG levels raised doubts whether raised TG is an independent risk factor for CAD or merely reflects the HDL-C effect. The * Tel.: +44-20-7679-6968; fax: +44-20-7679-6212. E-mail address: [email protected] (P.J. Talmud). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(02)01275-X
80
P.J. Talmud / International Congress Series 1253 (2003) 79–84
meta-analysis by Hokanson and Austin [2] confirmed the positive association between raised plasma TG levels and CAD. The mechanism for this raised TG – CAD risk association results from the overproduction of TG-rich lipoproteins (TGRL) and partially hydrolyzed lipoproteins, which cannot be efficiently cleared from the circulation and, thus, accumulate in the plasma. TGs from these particles are inappropriately transferred to low-density lipoproteins (LDL), which become cholesterol-depleted and TG-enriched. Subsequent hydrolysis of this TG by hepatic lipase (HL) converts these LDL particles to smaller, denser particles (small dense LDL). Small, dense LDL particles are prone to oxidation because they are no longer cleared by the LDL-receptor (LDL-R), and remain in the circulation. Oxidized small, dense LDL particles cleared by the scavenger receptor on macrophages promote foam cell formation and, thus, atherogenesis [3]. These finding emphasizes the need to identify the genetic and environmental determinants of plasma TG concentrations.
2. Transgenic animal models Transgenic animal models have provided interesting insights into the in vivo impact of lipolytic enzymes and the apolipoproteins CII, CIII, AV and E on TG metabolism. However, it must be remembered that these are extreme situations where total absence of the protein under study (in the knockout animal) or its presence at very high levels (in transgenic animals) might have little or no physiological or pathophysiological relevance. An example of this comes from LPL-deficient mice (lpl / ). In the heterozygous form (lpl+/ ), these mice are mildly hypertriglyceridemic. However, homozygous pups (lpl / ) do not survive more than 18 h after suckling, and die of severe hypertriglyceridemia with capillaries engorged with chylomicrons, particularly prominent in the lungs, thus, causing severe cyanosis [4]. This phenotype is much more severe than homozygous LPL deficiency in humans and may reflect that the null mutation in the mice (i.e. no LPL protein) differs from the missense mutations most common in human LPL deficiency (i.e. small amounts of LPL of very low activity) which is compatible with life. It is also possible that the nonenzymatic bridging function of LPL in the missense protein might be sufficient for survival in humans. Conversely and as expected, mice overexpressing the human LPL gene show faster postprandial clearance, with a decrease in VLDL-TG and with cholesterol enrichment of LDL particles [5]. Interestingly, knockout mice expressing LPL in cardiac muscles display normal TG and HDL levels after weaning, demonstrating that cardiac muscle LPL alone is enough to maintain normal circulating TG levels [6]. Since apo CII is the activator of LPL, it was surprising that transgenic mice overexpressing APOC2 are severely hypertriglyceridemic due to the accumulation of VLDL particles [7]. This is thought to be primarily due to the inhibition of LPL hydrolysis. Thus, at low apo CII levels, lipolysis is stimulated, while at high levels, it is inhibited. In humans, plasma levels of apo CII are low and there is no evidence that they vary greatly in healthy subjects in the general population so the relevance of these transgenic mouse studies is unclear. As expected, consistent with its role as ligand, apoE knockout mice have an extreme phenotype. These mice develop very severe hypercholesterolemia and spontaneous
P.J. Talmud / International Congress Series 1253 (2003) 79–84
81
atherosclerosis and have been used extensively as a mouse model of atherosclerosis [8]. Transgenic mice expressing an apo E receptor-defective mutation Arg142Cys have remnants enriched for both cholesterol and TG [9]. This suggests that apo E might also have a direct effect on lipolysis and at high concentrations alter normal lipolytic activity as well as acting as ligand for remnant clearance, while in the knockout mice with no apo E, only remnant clearance, but not lipolysis, is affected. Confirmation of the role of apo CIII in the clearance of TGRLs comes from animal studies. Disruption of the apoC3 gene in mice results in protection from postprandial hypertriglyceridemia [10], whilst the overexpression of human APOC3 results in hypertriglyceridemia. This is due to an increased number of VLDL particles in the circulation, which contain more TG and apo CIII and less apo E, thus, reducing apo E-mediated lipoprotein uptake [11]. Animal studies indicate that an APOA5 transgene leads to a f 65% reduction in TG levels while apoav knockout mice have four times higher TG levels than control littermates [12]. Thus, in contrast to apo CIII, where high plasma levels are associated with high TG levels [13], apo AV levels appear to be inversely related to TG levels. The mechanism for this action remains unclear. A clue to the function of apo AV comes from the upregulation of expression after partial hepatectomy in the rat suggesting that apo AV may act by controlling the uptake of lipids by the liver [14]. Thus, these animal studies highlight those genes primarily determining TG levels, namely LPL, APOC2, APOC3 and APOE.
3. Rare versus common mutations affecting TG levels Severely raised TG levels most likely results from rare mutations (monogenic disorder) with drastic effects, in essence inborn errors of metabolism. However, it is likely that TG levels in subjects in the general population will be modulated by several gene variants each of small impact (polygenes), but which are more common in the population as a whole. Thus, subjects with high TG levels could have inherited several of these mutations by chance. As with most genetic effects, predisposition is often modulated by environmental factors and this is very relevant to plasma TG levels, which are highly sensitive to changes in diet, obesity, smoking and exercise. 3.1. Common mutations of LPL—AsAsn9Asp, Asn291Ser and Ser447Stop Within the coding region of the LPL gene, three common amino acid changes have been reported: Asp9Asn (D9N), Asn291Ser (N291S) and Ser447Stop (S447X) (reviewed in Ref. [15]). While Asn9 and Ser291 occur at carrier frequencies ranging from 1% to 6% in healthy Caucasian samples, Stop447 carrier frequency ranges from 11% to 23% [15]. To date, there have been three summary analyses examining the effects of Asp9Asn and Asn291Ser mutations on TG levels and CAD risk [16 –18]. All three agree on the modest but consistent TG-raising effect of these variants, which was estimated to be a 20% and 31% increase in TG levels for Asp9Asn and Asn291Ser, respectively. Unlike the other two common amino acid changes, the premature termination of LPL by two amino acids in the Stop447 variant
82
P.J. Talmud / International Congress Series 1253 (2003) 79–84
appears to result in a lowering of TG levels by 8% and it is protective of CAD with an odds ratio of 0.8 (95% CI 0.7 – 1.0) [18], although the exact mechanism is not known. 3.2. Common variants in APOC3 The first polymorphism in an apolipoprotein gene to be reported was the SstI polymorphism in the 3Vuntranslated region (3VUTR) of the APOC3 gene caused by a C –G change at position 3238 [19]. The rare C3238 allele has consistently been associated with raised plasma TG and CAD (see Ref. [20]). The SstI site itself is thought unlikely to be of functional significance, but acts as a marker for a functional mutation elsewhere at the gene locus, possibly in the promoter. The distal promoter of APOC3 is highly polymorphic, and some of these sites lie within an insulin responsive element (IRE) and affect the transcriptional activity of the gene [21]. Studies of the effect on APOC3 expression showed that 455T >C or 482C >T variants, both within the IRE, led to the abolition of insulin responsiveness of the APOC3 promoter [22]. These IRE variant sites are in strong allelic association with the SstI polymorphism and, furthermore, have been shown to explain the SstI effect, thus, clarifying that the SstI site is unlikely to be functional [23]. The 482C >T is associated with differences in the response of insulin and glucose levels after an oral glucose load in young healthy men. In the same study, a second APOC3 variant in the APOC3 –A4 intergenic region, 2854T >G, was associated with response to an oral fat tolerance test. The 2854 site is close to an HNF4 transcription factor binding site and the effects seen might be due to the allelic association between these two sites and the possibility that 2854T >G is acting as a marker of he HNF4 site. Thus, specific genetic variants at the APOC3 locus differentially affect postprandial TGs and response to an oral glucose load and suggest a novel mechanism of the effects of variation at this locus on risk of atherosclerosis [23]. 3.3. APOA5 variants affect plasma TG levels Pennacchio et al. [12] examined the effect of single nucleotide polymorphisms (SNPs) in APOA5. SNPs 1– 3 showed strong allelic association with each other and with plasma TG levels in two independent studies, SNP4, 11 kb upstream of APOA5in the APOA4 –A5 intergenic region had no effect on TG levels. Thus, variation within the APOA5 gene was associated with plasma TG levels in humans. The question we are currently investigating is what is the relative contribution of genes within the APOC3 – A4 – A5 cluster in determining TG levels. Is APOA5 an independent marker of TG levels or is it merely the linkage disequilibrium between APOC3 and APOA5, which might explain the association between APOA5 variants and TG or conversely between APOC3 and TG? Haplotype analysis of the cluster should help tease this out.
4. Conclusion A number of common mutations in LPL, APOE, APOC3 and APOA5 significantly impact on TG levels in the population as a whole. Since raised TG levels is a risk factor for
P.J. Talmud / International Congress Series 1253 (2003) 79–84
83
CAD [2], it is suggested that these variants would be associated with an increased risk of CAD or in some cases may even be protective. For example, the APOC3 SstI polymorphism has constantly shown a CAD case control difference in frequency, linking APOC3 gene variation with risk. By contrast, the LPL Ser447Stop polymorphism has a reduced frequency in cases than controls and in meta-analysis [18] is associated with an odds ratio of 0.8 (%CI 0.7– 1.0). Thus. it seems inevitable that the way forward, to tease out genetic factors affecting TG metabolism, and their effect on risk, must take into account environmental factors and other polygenic effects.
Acknowledgements This work was funded by the British Heart Foundation.
References [1] P.W. Wilson, R.D. Abbott, W.P. Castelli, W.B. Kannel, R.J. Garrison, S. Kalousdian, A.G. Bostom, L.A. Cupples, J.L. Jenner, J.M. Ordovas, L.J. Seman, E.J. Schaefer, High density lipoprotein cholesterol and mortality, Arteriosclerosis 276 (1996) 544 – 548. [2] J.E. Hokanson, M.A. Austin, Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies, J. Cardiovasc. Risk 3 (1996) 213 – 219. [3] M.J. Chapman, M. Guerin, E. Bruckert, Atherogenic, dense low-density lipoproteins. Pathophysiology and new therapeutic approaches, Eur. Heart J. 19 (Suppl. A) (1998) A24 – A30. [4] P.H. Weinstock, C.L. Bisgaier, K. Aalto-Seta¨la¨, H. Radner, R. Ramakrishnan, S. Levak-Frank, A.D. Essenburg, R. Zechner, J.L. Breslow, Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impared very low density lipoprotein clearance in heterozygotes, J. Clin. Invest. 96 (1995) 2555 – 2568. [5] M.-S. Liu, F.R. Jirik, R.C. LeBoeuf, H. Henderson, L.W. Castellani, A.J. Lusis, Y. Ma, I.J. Forysthe, H. Zhang, E. Kirk, J.D. Brunzell, M.R. Hayden, Alteration of lipid profiles in plasma of transgenic mice expressing human lipoprotein lipase, J. Biol. Chem. 269 (1994) 11417 – 11424. [6] S. Levak Frank, W. Hofmann, P.H. Weinstock, H. Radner, W. Sattler, J.L. Breslow, R. Zechner, Induced mutant mouse lines that express lipoprotein lipase in cardiac muscle, but not in skeletal muscle and adipose tissue, have normal plasma triglyceride and high-density lipoprotein-cholesterol levels, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 3165 – 3170. [7] N.S. Shachter, T. Hayek, T. Leff, J.D. Smith, D.W. Rosenberg, A. Walsh, R. Ramakrishnan, I.J. Goldberg, H.N. Ginsberg, J.L. Breslow, Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice, J. Clin. Invest. 93 (1994) 1683 – 1690. [8] A.S. Plump, J.D. Smith, T. Hayek, K. Aalto Setala, A. Walsh, J.G. Verstuyft, E.M. Rubin, J.L. Breslow, Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells, Cell 71 (1992) 343 – 353. [9] S. Fazio, Y.L. Lee, Z.S. Ji, S.C. Rall Jr., Type III hyperlipoproteinemic phenotype in transgenic mice expressing dysfunctional apolipoprotein E, J. Clin. Invest. 92 (1993) 1497 – 1503. [10] N. Maeda, H. Li, D. Lee, P. Oliver, S.H. Quarfordt, J. Osada, Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridaemia and protection from postprandial hypertriglyceridaenia, J. Biol. Chem. 269 (1994) 23610 – 23616. [11] K. Aalto Setala, E.A. Fisher, X. Chen, T. Chajek Shaul, T. Hayek, R. Zechner, A. Walsh, R. Ramakrishnan, H.N. Ginsberg, J.L. Breslow, Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles, J. Clin. Invest. 90 (1992) 1889 – 1900.
84
P.J. Talmud / International Congress Series 1253 (2003) 79–84
[12] L.A. Pennacchio, M. Olivier, J.A. Hubacek, J.C. Cohen, D.R. Cox, J.C. Fruchart, R.M. Krauss, E.M. Rubin, An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing, Science 294 (2001) 169 – 173. [13] C.C. Shoulders, P.J. Harry, L. Lagrost, S.E. White, N.F. Shah, J.D. North, M. Gilligan, P. Gambert, M.J. Ball, Variation at the apo AI/CIII/AIV gene complex is associated with elevated plasma levels of apo CIII, Atherosclerosis 87 (1991) 239 – 247. [14] H.N. Der Vliet, M.G. Sammels, A.C. Leegwater, J.H. Levels, P.H. Reitsma, W. Boers, R.A. Chamuleau, Apolipoprotein A-V. A novel apolipoprotein associated with an early phase of liver regeneration, J. Biol. Chem. 276 (2001) 44512 – 44520. [15] R.M. Fisher, S.E. Humphries, P.J. Talmud, Common variation in the lipoprotein lipase gene: effects on plasma lipids and risk of atherosclerosis, Atherosclerosis (1997) 145 – 159. [16] J.E. Hokanson, Lipoprotein lipase gene variants and risk of coronary disease: a quantitative analysis of population-based studies, Int. J. Clin. Lab. Res. 27 (1997) 24 – 34. [17] J.J. Kastelein, J.M. Ordovas, M.E. Wittekoek, S.N. Pimstone, W.F. Wilson, S.E. Gagne, M.G. Larson, E.J. Schaefer, J.M. Boer, C. Gerdes, M.R. Hayden, Two common mutations (D9N, N291S) in lipoprotein lipase: a cumulative analysis of their influence on plasma lipids and lipoproteins in men and women, Clin. Genet. 56 (1999) 297 – 305. [18] H.H. Wittrup, A. Tybjaerg Hansen, B.G. Nordestgaard, Lipoprotein lipase mutations, plasma lipids and lipoproteins, and risk of ischemic heart disease. A meta-analysis, Circulation 99 (1999) 2901 – 2907. [19] A. Rees, C.C. Shoulders, J. Stocks, D.J. Galton, F.E. Baralle, DNA polymorphism adjacent to human apoprotein A-1 gene: relation to hypertriglyceridaemia, Lancet 1 (1983) 444 – 446. [20] P.J. Talmud, S.E. Humphries, Apolipoprotein C-III gene variation and dyslipidaemia, Curr. Opin. Lipidol. 8 (1997) 154 – 158. [21] M. Dammerman, L.A. Sandkuijl, J.L. Halaas, W. Chung, J.L. Breslow, An apolipoprotein CIII haplotype protective against hypertriglyceridemia is specified by promoter and 3Vuntranslated region polymorphisms, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 4562 – 4566. [22] W.W. Li, M.M. Dammerman, J.D. Smith, S. Metzger, J.L. Breslow, T. Leff, Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia, J. Clin. Invest. 96 (1995) 2601 – 2605. [23] D.M. Waterworth, J. Ribalta, V. Nicaud, J. Dallongeville, S.E. Humphries, P. Talmud, ApoCIII gene variants modulate postprandial response to both glucose and fat tolerance tests, Circulation 99 (1999) 1872 – 1877.
APOA5—a recent addition to genes determining plasma triglycerides Philippa J. Talmud * Division of Cardiovascular Genetics, British Heart Foundation Laboratories, Rayne Building, Department of Medicine, Royal Free and University College Medical School, 5 University Street, London WC1E 6JJ, UK
Abstract Raised plasma triglyceride (TG) levels is an independent risk factor for coronary artery disease (CAD), thus, understanding the genetic and environmental determinants of TG levels is of major importance. TG metabolism is a process for delivering free fatty acids for energy storage or hoxidation, involving a number of different hydrolytic enzymes and apolipoproteins (apo). The genes encoding these proteins are, therefore, candidates for determining plasma TGs. There is evidence that common variations in LPL, apo CIII (APOC3) and apo E (APOE) have the strong effect on plasma TG levels at the population level. More recently, the latest recognized member of the apolipoprotein gene family, APOA5, identified by comparative sequencing between human and mouse DNA, was located approximately 27 kb distal to APOA4 in the APOA1 – C3 – A4 gene cluster on chromosome 11q23, and variation in APOA5 was shown to be associated with plasma TG levels. The involvement of variation in the APOA1 – C3 – A4 locus in determining differences in lipid levels is well documented. We have examined the relative influence of APOA5 variants on plasma lipids, compared to the impact of variation in other genes within the cluster to determine if these effects are independent or a consequence of the strong linkage disequilibrium across the locus. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Triglycerides; Animal models; APOA1 – C3 – A4 – A5 gene cluster
1. Introduction The strong inverse correlation between high-density lipoprotein cholesterol (HDL-C) (an independent risk factor for CAD) [1] and plasma TG levels raised doubts whether raised TG is an independent risk factor for CAD or merely reflects the HDL-C effect. The * Tel.: +44-20-7679-6968; fax: +44-20-7679-6212. E-mail address: [email protected] (P.J. Talmud). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(02)01275-X
80
P.J. Talmud / International Congress Series 1253 (2003) 79–84
meta-analysis by Hokanson and Austin [2] confirmed the positive association between raised plasma TG levels and CAD. The mechanism for this raised TG – CAD risk association results from the overproduction of TG-rich lipoproteins (TGRL) and partially hydrolyzed lipoproteins, which cannot be efficiently cleared from the circulation and, thus, accumulate in the plasma. TGs from these particles are inappropriately transferred to low-density lipoproteins (LDL), which become cholesterol-depleted and TG-enriched. Subsequent hydrolysis of this TG by hepatic lipase (HL) converts these LDL particles to smaller, denser particles (small dense LDL). Small, dense LDL particles are prone to oxidation because they are no longer cleared by the LDL-receptor (LDL-R), and remain in the circulation. Oxidized small, dense LDL particles cleared by the scavenger receptor on macrophages promote foam cell formation and, thus, atherogenesis [3]. These finding emphasizes the need to identify the genetic and environmental determinants of plasma TG concentrations.
2. Transgenic animal models Transgenic animal models have provided interesting insights into the in vivo impact of lipolytic enzymes and the apolipoproteins CII, CIII, AV and E on TG metabolism. However, it must be remembered that these are extreme situations where total absence of the protein under study (in the knockout animal) or its presence at very high levels (in transgenic animals) might have little or no physiological or pathophysiological relevance. An example of this comes from LPL-deficient mice (lpl / ). In the heterozygous form (lpl+/ ), these mice are mildly hypertriglyceridemic. However, homozygous pups (lpl / ) do not survive more than 18 h after suckling, and die of severe hypertriglyceridemia with capillaries engorged with chylomicrons, particularly prominent in the lungs, thus, causing severe cyanosis [4]. This phenotype is much more severe than homozygous LPL deficiency in humans and may reflect that the null mutation in the mice (i.e. no LPL protein) differs from the missense mutations most common in human LPL deficiency (i.e. small amounts of LPL of very low activity) which is compatible with life. It is also possible that the nonenzymatic bridging function of LPL in the missense protein might be sufficient for survival in humans. Conversely and as expected, mice overexpressing the human LPL gene show faster postprandial clearance, with a decrease in VLDL-TG and with cholesterol enrichment of LDL particles [5]. Interestingly, knockout mice expressing LPL in cardiac muscles display normal TG and HDL levels after weaning, demonstrating that cardiac muscle LPL alone is enough to maintain normal circulating TG levels [6]. Since apo CII is the activator of LPL, it was surprising that transgenic mice overexpressing APOC2 are severely hypertriglyceridemic due to the accumulation of VLDL particles [7]. This is thought to be primarily due to the inhibition of LPL hydrolysis. Thus, at low apo CII levels, lipolysis is stimulated, while at high levels, it is inhibited. In humans, plasma levels of apo CII are low and there is no evidence that they vary greatly in healthy subjects in the general population so the relevance of these transgenic mouse studies is unclear. As expected, consistent with its role as ligand, apoE knockout mice have an extreme phenotype. These mice develop very severe hypercholesterolemia and spontaneous
P.J. Talmud / International Congress Series 1253 (2003) 79–84
81
atherosclerosis and have been used extensively as a mouse model of atherosclerosis [8]. Transgenic mice expressing an apo E receptor-defective mutation Arg142Cys have remnants enriched for both cholesterol and TG [9]. This suggests that apo E might also have a direct effect on lipolysis and at high concentrations alter normal lipolytic activity as well as acting as ligand for remnant clearance, while in the knockout mice with no apo E, only remnant clearance, but not lipolysis, is affected. Confirmation of the role of apo CIII in the clearance of TGRLs comes from animal studies. Disruption of the apoC3 gene in mice results in protection from postprandial hypertriglyceridemia [10], whilst the overexpression of human APOC3 results in hypertriglyceridemia. This is due to an increased number of VLDL particles in the circulation, which contain more TG and apo CIII and less apo E, thus, reducing apo E-mediated lipoprotein uptake [11]. Animal studies indicate that an APOA5 transgene leads to a f 65% reduction in TG levels while apoav knockout mice have four times higher TG levels than control littermates [12]. Thus, in contrast to apo CIII, where high plasma levels are associated with high TG levels [13], apo AV levels appear to be inversely related to TG levels. The mechanism for this action remains unclear. A clue to the function of apo AV comes from the upregulation of expression after partial hepatectomy in the rat suggesting that apo AV may act by controlling the uptake of lipids by the liver [14]. Thus, these animal studies highlight those genes primarily determining TG levels, namely LPL, APOC2, APOC3 and APOE.
3. Rare versus common mutations affecting TG levels Severely raised TG levels most likely results from rare mutations (monogenic disorder) with drastic effects, in essence inborn errors of metabolism. However, it is likely that TG levels in subjects in the general population will be modulated by several gene variants each of small impact (polygenes), but which are more common in the population as a whole. Thus, subjects with high TG levels could have inherited several of these mutations by chance. As with most genetic effects, predisposition is often modulated by environmental factors and this is very relevant to plasma TG levels, which are highly sensitive to changes in diet, obesity, smoking and exercise. 3.1. Common mutations of LPL—AsAsn9Asp, Asn291Ser and Ser447Stop Within the coding region of the LPL gene, three common amino acid changes have been reported: Asp9Asn (D9N), Asn291Ser (N291S) and Ser447Stop (S447X) (reviewed in Ref. [15]). While Asn9 and Ser291 occur at carrier frequencies ranging from 1% to 6% in healthy Caucasian samples, Stop447 carrier frequency ranges from 11% to 23% [15]. To date, there have been three summary analyses examining the effects of Asp9Asn and Asn291Ser mutations on TG levels and CAD risk [16 –18]. All three agree on the modest but consistent TG-raising effect of these variants, which was estimated to be a 20% and 31% increase in TG levels for Asp9Asn and Asn291Ser, respectively. Unlike the other two common amino acid changes, the premature termination of LPL by two amino acids in the Stop447 variant
82
P.J. Talmud / International Congress Series 1253 (2003) 79–84
appears to result in a lowering of TG levels by 8% and it is protective of CAD with an odds ratio of 0.8 (95% CI 0.7 – 1.0) [18], although the exact mechanism is not known. 3.2. Common variants in APOC3 The first polymorphism in an apolipoprotein gene to be reported was the SstI polymorphism in the 3Vuntranslated region (3VUTR) of the APOC3 gene caused by a C –G change at position 3238 [19]. The rare C3238 allele has consistently been associated with raised plasma TG and CAD (see Ref. [20]). The SstI site itself is thought unlikely to be of functional significance, but acts as a marker for a functional mutation elsewhere at the gene locus, possibly in the promoter. The distal promoter of APOC3 is highly polymorphic, and some of these sites lie within an insulin responsive element (IRE) and affect the transcriptional activity of the gene [21]. Studies of the effect on APOC3 expression showed that 455T >C or 482C >T variants, both within the IRE, led to the abolition of insulin responsiveness of the APOC3 promoter [22]. These IRE variant sites are in strong allelic association with the SstI polymorphism and, furthermore, have been shown to explain the SstI effect, thus, clarifying that the SstI site is unlikely to be functional [23]. The 482C >T is associated with differences in the response of insulin and glucose levels after an oral glucose load in young healthy men. In the same study, a second APOC3 variant in the APOC3 –A4 intergenic region, 2854T >G, was associated with response to an oral fat tolerance test. The 2854 site is close to an HNF4 transcription factor binding site and the effects seen might be due to the allelic association between these two sites and the possibility that 2854T >G is acting as a marker of he HNF4 site. Thus, specific genetic variants at the APOC3 locus differentially affect postprandial TGs and response to an oral glucose load and suggest a novel mechanism of the effects of variation at this locus on risk of atherosclerosis [23]. 3.3. APOA5 variants affect plasma TG levels Pennacchio et al. [12] examined the effect of single nucleotide polymorphisms (SNPs) in APOA5. SNPs 1– 3 showed strong allelic association with each other and with plasma TG levels in two independent studies, SNP4, 11 kb upstream of APOA5in the APOA4 –A5 intergenic region had no effect on TG levels. Thus, variation within the APOA5 gene was associated with plasma TG levels in humans. The question we are currently investigating is what is the relative contribution of genes within the APOC3 – A4 – A5 cluster in determining TG levels. Is APOA5 an independent marker of TG levels or is it merely the linkage disequilibrium between APOC3 and APOA5, which might explain the association between APOA5 variants and TG or conversely between APOC3 and TG? Haplotype analysis of the cluster should help tease this out.
4. Conclusion A number of common mutations in LPL, APOE, APOC3 and APOA5 significantly impact on TG levels in the population as a whole. Since raised TG levels is a risk factor for
P.J. Talmud / International Congress Series 1253 (2003) 79–84
83
CAD [2], it is suggested that these variants would be associated with an increased risk of CAD or in some cases may even be protective. For example, the APOC3 SstI polymorphism has constantly shown a CAD case control difference in frequency, linking APOC3 gene variation with risk. By contrast, the LPL Ser447Stop polymorphism has a reduced frequency in cases than controls and in meta-analysis [18] is associated with an odds ratio of 0.8 (%CI 0.7– 1.0). Thus. it seems inevitable that the way forward, to tease out genetic factors affecting TG metabolism, and their effect on risk, must take into account environmental factors and other polygenic effects.
Acknowledgements This work was funded by the British Heart Foundation.
References [1] P.W. Wilson, R.D. Abbott, W.P. Castelli, W.B. Kannel, R.J. Garrison, S. Kalousdian, A.G. Bostom, L.A. Cupples, J.L. Jenner, J.M. Ordovas, L.J. Seman, E.J. Schaefer, High density lipoprotein cholesterol and mortality, Arteriosclerosis 276 (1996) 544 – 548. [2] J.E. Hokanson, M.A. Austin, Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies, J. Cardiovasc. Risk 3 (1996) 213 – 219. [3] M.J. Chapman, M. Guerin, E. Bruckert, Atherogenic, dense low-density lipoproteins. Pathophysiology and new therapeutic approaches, Eur. Heart J. 19 (Suppl. A) (1998) A24 – A30. [4] P.H. Weinstock, C.L. Bisgaier, K. Aalto-Seta¨la¨, H. Radner, R. Ramakrishnan, S. Levak-Frank, A.D. Essenburg, R. Zechner, J.L. Breslow, Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impared very low density lipoprotein clearance in heterozygotes, J. Clin. Invest. 96 (1995) 2555 – 2568. [5] M.-S. Liu, F.R. Jirik, R.C. LeBoeuf, H. Henderson, L.W. Castellani, A.J. Lusis, Y. Ma, I.J. Forysthe, H. Zhang, E. Kirk, J.D. Brunzell, M.R. Hayden, Alteration of lipid profiles in plasma of transgenic mice expressing human lipoprotein lipase, J. Biol. Chem. 269 (1994) 11417 – 11424. [6] S. Levak Frank, W. Hofmann, P.H. Weinstock, H. Radner, W. Sattler, J.L. Breslow, R. Zechner, Induced mutant mouse lines that express lipoprotein lipase in cardiac muscle, but not in skeletal muscle and adipose tissue, have normal plasma triglyceride and high-density lipoprotein-cholesterol levels, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 3165 – 3170. [7] N.S. Shachter, T. Hayek, T. Leff, J.D. Smith, D.W. Rosenberg, A. Walsh, R. Ramakrishnan, I.J. Goldberg, H.N. Ginsberg, J.L. Breslow, Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice, J. Clin. Invest. 93 (1994) 1683 – 1690. [8] A.S. Plump, J.D. Smith, T. Hayek, K. Aalto Setala, A. Walsh, J.G. Verstuyft, E.M. Rubin, J.L. Breslow, Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells, Cell 71 (1992) 343 – 353. [9] S. Fazio, Y.L. Lee, Z.S. Ji, S.C. Rall Jr., Type III hyperlipoproteinemic phenotype in transgenic mice expressing dysfunctional apolipoprotein E, J. Clin. Invest. 92 (1993) 1497 – 1503. [10] N. Maeda, H. Li, D. Lee, P. Oliver, S.H. Quarfordt, J. Osada, Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridaemia and protection from postprandial hypertriglyceridaenia, J. Biol. Chem. 269 (1994) 23610 – 23616. [11] K. Aalto Setala, E.A. Fisher, X. Chen, T. Chajek Shaul, T. Hayek, R. Zechner, A. Walsh, R. Ramakrishnan, H.N. Ginsberg, J.L. Breslow, Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles, J. Clin. Invest. 90 (1992) 1889 – 1900.
84
P.J. Talmud / International Congress Series 1253 (2003) 79–84
[12] L.A. Pennacchio, M. Olivier, J.A. Hubacek, J.C. Cohen, D.R. Cox, J.C. Fruchart, R.M. Krauss, E.M. Rubin, An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing, Science 294 (2001) 169 – 173. [13] C.C. Shoulders, P.J. Harry, L. Lagrost, S.E. White, N.F. Shah, J.D. North, M. Gilligan, P. Gambert, M.J. Ball, Variation at the apo AI/CIII/AIV gene complex is associated with elevated plasma levels of apo CIII, Atherosclerosis 87 (1991) 239 – 247. [14] H.N. Der Vliet, M.G. Sammels, A.C. Leegwater, J.H. Levels, P.H. Reitsma, W. Boers, R.A. Chamuleau, Apolipoprotein A-V. A novel apolipoprotein associated with an early phase of liver regeneration, J. Biol. Chem. 276 (2001) 44512 – 44520. [15] R.M. Fisher, S.E. Humphries, P.J. Talmud, Common variation in the lipoprotein lipase gene: effects on plasma lipids and risk of atherosclerosis, Atherosclerosis (1997) 145 – 159. [16] J.E. Hokanson, Lipoprotein lipase gene variants and risk of coronary disease: a quantitative analysis of population-based studies, Int. J. Clin. Lab. Res. 27 (1997) 24 – 34. [17] J.J. Kastelein, J.M. Ordovas, M.E. Wittekoek, S.N. Pimstone, W.F. Wilson, S.E. Gagne, M.G. Larson, E.J. Schaefer, J.M. Boer, C. Gerdes, M.R. Hayden, Two common mutations (D9N, N291S) in lipoprotein lipase: a cumulative analysis of their influence on plasma lipids and lipoproteins in men and women, Clin. Genet. 56 (1999) 297 – 305. [18] H.H. Wittrup, A. Tybjaerg Hansen, B.G. Nordestgaard, Lipoprotein lipase mutations, plasma lipids and lipoproteins, and risk of ischemic heart disease. A meta-analysis, Circulation 99 (1999) 2901 – 2907. [19] A. Rees, C.C. Shoulders, J. Stocks, D.J. Galton, F.E. Baralle, DNA polymorphism adjacent to human apoprotein A-1 gene: relation to hypertriglyceridaemia, Lancet 1 (1983) 444 – 446. [20] P.J. Talmud, S.E. Humphries, Apolipoprotein C-III gene variation and dyslipidaemia, Curr. Opin. Lipidol. 8 (1997) 154 – 158. [21] M. Dammerman, L.A. Sandkuijl, J.L. Halaas, W. Chung, J.L. Breslow, An apolipoprotein CIII haplotype protective against hypertriglyceridemia is specified by promoter and 3Vuntranslated region polymorphisms, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 4562 – 4566. [22] W.W. Li, M.M. Dammerman, J.D. Smith, S. Metzger, J.L. Breslow, T. Leff, Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia, J. Clin. Invest. 96 (1995) 2601 – 2605. [23] D.M. Waterworth, J. Ribalta, V. Nicaud, J. Dallongeville, S.E. Humphries, P. Talmud, ApoCIII gene variants modulate postprandial response to both glucose and fat tolerance tests, Circulation 99 (1999) 1872 – 1877.