BACKGROUND: Higher lipoprotein-associated phospholipase A(2)(Lp-PLA2) activity is associated with increased risk of coronary heart disease (CHD), making Lp-PLA2 a potential therapeutic target. PLA2G7 variants associated with Lp-PLA2 activity could evaluate whether this relationship is causal. METHODS AND RESULTS: A meta-analysis including a total of 12 studies (5 prospective, 4 case-control, 1 case-only, and 2 cross-sectional studies; n=26 118) was undertaken to examine the association of the following: (1) Lp-PLA2 activity versus cardiovascular biomarkers and risk factors and CHD events (2 prospective studies; n=4884); (2) PLA2G7 single-nucleotide polymorphisms and Lp-PLA2 activity (3 prospective, 2 case-control, 2 cross-sectional studies; up to n=6094); and (3) PLA2G7 single-nucleotide polymorphisms and angiographic coronary artery disease (2 case-control, 1 case-only study; n=4971 cases) and CHD events (5 prospective, 2 case-control studies; n=5523). Lp-PLA2 activity correlated with several CHD risk markers. Hazard ratios for CHD events for the top versus bottom quartile of Lp-PLA2 activity were 1.61 (95% confidence interval, 1.31 to 1.99) and 1.17 (95% confidence interval, 0.91 to 1.51) after adjustment for baseline traits. Of 7 single-nucleotide polymorphisms, rs1051931 (A379V) showed the strongest association with Lp-PLA2 activity, with VV subjects having 7.2% higher activity than AAs. Genotype was not associated with risk markers, angiographic coronary disease (odds ratio, 1.03; 95% confidence interval, 0.80 to 1.32), or CHD events (odds ratio, 0.98; 95% confidence interval, 0.82 to 1.17). CONCLUSIONS: Unlike Lp-PLA2 activity, PLA2G7 variants associated with modest effects on Lp-PLA2 activity were not associated with cardiovascular risk markers, coronary atheroma, or CHD. Larger association studies, identification of single-nucleotide polymorphisms with larger effects, or randomized trials of specific Lp-PLA2 inhibitors are needed to confirm or refute a contributory role for Lp-PLA2 in CHD.
OBJECTIVE: The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. METHODS AND RESULTS: We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. CONCLUSIONS: The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression.
Title: Candidate gene genotypes, along with conventional risk factor assessment, improve estimation of coronary heart disease risk in healthy UK men Humphries SE, Cooper JA, Talmud PJ, Miller GJ Ref: Clinical Chemistry, 53:8, 2007 : PubMed
BACKGROUND: One of the aims of cardiovascular genetics is to test the efficacy of the use of genetic information to predict cardiovascular risk. We therefore investigated whether inclusion of a set of common variants in candidate genes along with conventional risk factor (CRF) assessment enhanced coronary heart disease (CHD)-risk algorithms. METHODS: We followed middle-aged men in the prospective Northwick Park Heart Study II (NPHSII) for 10.8 years and analyzed complete trait and genotype information available on 2057 men (183 CHD events). RESULTS: Of the 12 genes previously associated with CHD risk, in stepwise multivariate risk analysis, uncoupling protein 2 (UCP2; P = 0.0001), apolipoprotein E (APOE; P = 0.0003), lipoprotein lipase (LPL; P = 0.007), and apolipoprotein AIV (APOA4; P = 0.04) remained in the model. Their combined area under the ROC curve (A(ROC)) was 0.62 (0.58-0.66) [12.6% detection rate for a 5% false positive rate (DR(5))]. The A(ROC) for the CRFs age, triglyceride, cholesterol, systolic blood pressure, and smoking was 0.66 (0.61-0.70) (DR(5) = 14.2%). Combining CRFs and genotypes significantly improved discrimination (P = 0.001). Inclusion of previously demonstrated interactions of smoking with LPL, interleukin-6 (IL6), and platelet/endothelial cell adhesion molecule (PECAM1) genotypes increased the A(ROC) to 0.72 (0.68-0.76) for a DR(5) of 19.1% (P = 0.01 vs CRF combined with genotypes). CONCLUSIONS: For a modest panel of selected genotypes, CHD-risk estimates incorporating CRFs and genotype-risk factor interactions were more effective than risk estimates that used CRFs alone.
LVH [LV (left ventricular) hypertrophy] is an independent risk factor for CHD (coronary heart disease). During LVH, the preferred cardiac energy substrate switches from FAs (fatty acids) to glucose. LPL (lipoprotein lipase) is the key enzyme in triacylglycerol (triglyceride) hydrolysis and supplies FAs to the heart. To investigate whether substrate utilization influences cardiac growth and CHD risk, we examined the association between the functional LPL S447X (rs328) variant and hypertension-induced LV growth and CHD risk. LPL-X447 has been shown to be more hydrolytically efficient and would therefore release more free FAs than LPL-S477. In a cohort of 190 hypertensive subjects, LPL X447 was associated with a greater LV mass index [85.2 (1.7) in S/S compared with 91.1 (3.4) in S/X+X/X; P=0.01], but no such association was seen in normotensive controls (n=60). X447 allele frequency was higher in hypertensives with than those without LVH {0.14 [95% CI (confidence interval), 0.08-0.19] compared with 0.07 (95% CI, 0.05-0.10) respectively; odds ratio, 2.52 (95% CI, 1.17-5.40), P=0.02}. The association of LPL S447X with CHD risk was then examined in a prospective study of healthy middle-aged U.K. men (n=2716). In normotensive individuals, compared with S447 homozygotes, X447 carriers were protected from CHD risk [HR (hazard ratio), 0.48 (95% CI, 0.23-1.00); P=0.05], whereas, in the hypertensives, X447 carriers had increased risk [HR, 1.54 (95% CI, 1.13-2.09) for S/S (P=0.006) and 2.30 (95% CI, 1.53-3.45) for X447+ (P<0.0001)] and had a significant interaction with hypertension in CHD risk determination (P=0.007). In conclusion, hypertensive LPL X447 carriers have increased risk of LVH and CHD, suggesting that altered FA delivery constitutes a mechanism through which LVH and CHD are associated in hypertensive subjects.
Lipoprotein-associated PLA2 (Lp-PLA2) hydrolyses the sn-2 position of glycerophospholipids, in particular platelet activating factor (PAF), generating significant amounts of Lyso-PAF which in turn, via a remodelling pathway, can generate arachidonic acid (AA) from alkyl-acyl-glycerophosphorylcholine. AA is a precursor for prostaglandin synthesis, which regulates adipogenesis through the peroxisome proliferator-activated receptor subfamily. AA may also modulate skeletal muscle growth. We investigated the association of the PLA2G7 A379V variant with changes in body composition in a longitudinal study of 123 male Caucasian army recruits over 10 weeks of intensive physical training. There was no effect of genotype on baseline measures. However, after exercise training, homozygosity for the 379V allele was associated with a decrease in percentage adipose tissue mass (-3.61+/-1.14%), compared to AV (-1.67+/-0.38%) and AA (-1.09+/-0.24%) genotypes (p=0.01), and a significant mean increase (3.51+/-1.17%) in percentage lean mass, compared to AV (1.64+/-0.38%) and AA (1.10+/-0.24%) recruits (p=0.02). The association of this genotype with changes in body composition after training suggests a novel role for Lp-PLA2.
The adult heart relies predominantly on fatty acids (FA) for energy generation, and defects in FA catabolism cause dramatic left ventricular (LV) growth in early age. Since lipoprotein lipase (LPL) is the key enzyme in plasma triglyceride catabolism and is highly expressed in the myocardium, we investigated an association between the functional LPL gene serine 447 stop (S447X) variant and exercise-induced LV growth. The S447X variant was genotyped in 146 British Army recruits undergoing a 10-week exercise programme. Over the training period, X447 allele carriers showed less LV growth than S447 homozygotes (SS, 5.8+/-0.7%; SX, 2.2+/-1.5%; P=0.03) and a decrease in systolic blood pressure (DeltaSBP: SS, 1.9+/-1.3 mmHg; SX, -5.7+/-2.2 mmHg; P=0.015). Although LPL genotype did not significantly predict LV growth with DeltaSBP in statistical modelling (LPL, P=0.14; DeltaSBP, P=0.06), regression analysis indicated that LPL S447X genotype effect on DeltaSBP accounted for only 20% of the effect on LV growth. In multivariate analysis, LPL, peroxisome-proliferator-activated receptor alpha and angiotensin-converting enzyme genotypes were independent predictors of cardiac growth. Thus, LPL S447X genotype influenced exercise-induced changes in LV mass and SBP. Change in blood pressure accounted for a proportion of LV growth. These data suggest that increased myocardial FA availability may reduce exercise-induced LV growth.
Title: Interaction between insulin (VNTR) and hepatic lipase (LIPC-514C>T) variants on the response to an oral glucose tolerance test in the EARSII group of young healthy men Waterworth DM, Jansen H, Nicaud V, Humphries SE, Talmud PJ Ref: Biochimica & Biophysica Acta, 1740:375, 2005 : PubMed
Insulin resistance is polygenic in origin, and can be observed at an early age. We have shown that variations in APOC3-482T>C and hepatic lipase (LIPC)-514C>T), individually (APOC3 alone) and interactively, modulate insulin and glucose levels after an OGTT in young healthy men participating in the European Atherosclerosis Research Study II (EARSII). Variation in the insulin gene (INS) variable number tandem repeat (VNTR) has been found to predispose to type 1 and type 2 diabetes. We have evaluated the HphI site 23 bp upstream of the INS gene (a surrogate marker for the VNTR) in EARSII (n=822), to determine if variation in INS contributes to insulin resistance. Carriers of the INS VNTR class III (HphI-) allele (frequency=0.29 (95%CI 0.27, 0.31)) had significantly higher 60-min insulin concentrations after the OGTT (P=0.014) and a marginally higher AUC insulin (P=0.07), compared to class I (HphI+) homozygotes. However, this effect on AUC insulin was modified by the level of physical activity, displaying significant gene:environment interaction (P=0.03). We tested for gene:gene interaction between the INS VNTR and both the LIPC-514C>T and APOC3-482T>C. While there was a significant interaction between INS VNTR and LIPC-514C>T on AUC glucose (P=0.013) and on AUC insulin (P=0.015), there was no interaction with APOC3-482T>C. Thus, despite a modest effect of the INS VNTR alone, the influence of this variant on insulin sensitivity was modified by gene:environment and gene:gene interactions, illustrating the biological complexity of insulin resistance.
Title: Association between the Ala379Val variant of the lipoprotein associated phospholipase A2 and risk of myocardial infarction in the north and south of Europe Abuzeid AM, Hawe E, Humphries SE, Talmud PJ Ref: Atherosclerosis, 168:283, 2003 : PubMed
Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) has been identified as a coronary heart disease (CHD) risk predictor. Both its anti-inflammatory role by hydrolysing platelet activating factor, and pro-inflammatory generation of atherogenic mediators may influence CHD risk. We investigated the association of the activity-reducing A379V variant with risk of myocardial infarction (MI) in a large European case-control study, which compared 527 post-MI men with 566 age-matched controls from north and south Europe. Overall, the frequency of the V379 allele was 0.24 (95%CI 0.21-0.26), with no evidence for differences between centres. Homozygosity for the V379 allele was associated with lower risk of MI, (Odds Ratio (OR) 0.56, 95%CI 0.32-0.98), maintained after adjustment for lifestyle factors and levels of inflammatory risk factors (C-reactive protein, fibrinogen, IL-6) (OR 0.46, 0.22-0.93). There was no evidence of heterogeneity of effect between the centres in the north and south of Europe (P-value for interaction=0.80). Since homozygosity for V379 occurs in only 5-6% of subjects, this genotype is not a major determinant of population genetic risk of CHD, but the association of this genotype with low levels of Lp-PLA(2), strongly support the pro-inflammatory causative, and not consequential, role of Lp-PLA(2) in CHD.
Title: Genetic and environmental determinants of plasma high density lipoprotein cholesterol and apolipoprotein AI concentrations in healthy middle-aged men Talmud PJ, Hawe E, Robertson K, Miller GJ, Miller NE, Humphries SE Ref: Ann Hum Genet, 66:111, 2002 : PubMed
The effects of common variants of cholesteryl ester transfer protein (CETP) (TaqIB), hepatic lipase (HL) (-514C>T), lipoprotein lipase (LPL) (S447X) and lecithin cholesterol acyl transferase (LCAT) (S208T) on the determination of high density lipoprotein cholesterol (HDL-C) and apolipoprotein AI (apoAI) levels were examined in 2773 healthy middle-aged men participating in the second Northwick Park Heart Study. The extent of gene:gene, gene:smoking and gene:alcohol interactions were determined. For HDL-C levels, only CETP genotype was associated with significant effects (p&0.0001), with the B2 allele being associated with higher levels in both smokers and non-smokers. This interaction was significant at the lowest tertile of TG, suggesting that TG levels were rate limiting. As previously reported, CETP, LPL and HL genotypes were all associated with significant effects on apoAI levels (all p&0.01), with carriers of the rare alleles having higher levels and with no evidence of heterogeneity of effects in smokers and non-smokers. LCAT genotype was not associated with significant effects on either trait. There was no significant interaction between any of the genotypes and alcohol consumption on either HDL-C or apoAI levels. All genotypic effects were additive for HDL-C and apoAI. Environmental and TG levels explained more than 20% and 5.5% of the variance in HDL-C and apoAI, respectively. The novel aspect of this finding is that genetic variation at these loci explained in total only 2.5% of the variance in HDL-C and 1.89% of the variance in apoAI levels. Thus despite the key roles played by these enzymes in HDL metabolism, variation at these loci, at least as detected by these common genotypes, contributes minimally to the variance in HDL-C and apoAI levels in healthy men, highlighting the polygenic and multifactorial control of HDL-C.
We have determined the genotypes of two common polymorphisms in the lipoprotein lipase (S447X) and hepatic lipase (-480C/T) genes in a cohort of 285 representative selected Czech probands (131 male and 154 female), examined in 1988 and reinvestigated in 1996. The genotype distributions of both polymorphisms were in Hardy-Weinberg equilibrium and did not differ between male and female subjects. The rare allele frequency of the lipoprotein lipase polymorphism did not differ significantly from the other European populations. Compared to the German populations, the frequency of the hepatic lipase -480T allele was significantly higher in the Czech group (20% vs. 36%, p<0.0001). There were no significant associations between the lipoprotein lipase gene variants and lipid parameters measured either in 1988, or in 1996 or with changes of lipid parameters over the 8-year period. The carriers of the T-480 allele of the hepatic lipase polymorphism were found to have higher HDL cholesterol levels (p=0.02). However, this difference was confined to female subjects only. The male carriers of the -480T allele had higher concentrations of total cholesterol (p=0.03) as compared to CC-480 subjects. Both associations were observed in 1996 only. In the Slavic Czech population, a common polymorphism in the hepatic lipase gene (-480C/T), but not in the lipoprotein lipase gene (S447X), is a significant determinant of plasma HDL cholesterol in females and plasma total cholesterol in males and indicates the importance of gender-associated effects in the genetic determinations of plasma lipids.
Title: Lipoprotein lipase gene variation is associated with a paternal history of premature coronary artery disease and fasting and postprandial plasma triglycerides: the European Atherosclerosis Research Study (EARS) Humphries SE, Nicaud V, Margalef J, Tiret L, Talmud PJ Ref: Arterioscler Thromb Vasc Biol, 18:526, 1998 : PubMed
The H-allele of the intron 8 HindIII polymorphism in the lipoprotein lipase (LPL) gene has been associated with a lower risk of myocardial infarction (MI) and plasma levels of triglycerides (TG). To test whether the HindIII site was in linkage disequilibrium with the functional variant LPL Serine447Stop (S447X), subjects from the European Atherosclerosis Research Study (EARS I) were genotyped for both polymorphic sites. This study included 515 offspring of fathers with a premature (<55 years old) MI, who were designated cases, and 930 age- and sex-matched control subjects from five different regions of Europe. Linkage disequilibrium between the two sites was very strong (>.99), with only three of the four possible haplotypes identified: H+S447, H-S447, and H-X447. The frequency of the H-X447 but not of the H-S447 haplotype was significantly lower in cases than in control subjects (.090 versus .117, P<.01) suggesting a protective effect for MI, with this difference being consistent in all five regions of Europe. Compared with individuals homozygous for the H+S447 haplotype, the odds ratio of having a paternal history of premature MI for H-X447 heterozygotes (approximately 20% of the population) was 0.71 (95% confidence interval, 0.55 to 0.92). In addition, there was an increase of the H-X447 haplotype frequency from north to south in control subjects (0.119 in Finland to 0.143 in the Mediterranean region, P<.01). Compared with the H+S447 haplotype, the H-X447 haplotype was associated with significantly lower concentrations of plasma TGs (5.4% lower, P=.01), with this effect being consistent over the regions of Europe. There was no significant evidence for a heterogeneity of effect between males and females or between cases and control subjects, although the effect on TG levels appeared to be the greatest in male cases (11% lower, P=.05). In a second study (EARS II), of 332 cases and 342 control subjects, postprandial clearance of TGs after a standard fat meal was examined. The H-X447 haplotype was associated with significantly lower postprandial triglyceride levels than was the H+S447 haplotype (9.4% smaller area under the curve, P<.05). Thus, the effects on MI risk and plasma lipids associated with the H allele appeared to be mainly mediated by the X447 mutation, and although the lowering effects associated with the H-X447 haplotype on fasting and postprandial TGs are not large, they are consistent with the lowering effect observed on MI risk throughout Europe.
The lipoprotein lipase (LPL) promoter -93T/G transition has previously been reported as having a triglyceride (Tg)-lowering effect, whereas the D9N variant has been shown to have a Tg-raising effect. These two variants were studied in 66 healthy subjects of Hispanic and 42 subjects of African-American origin, who had participated in a study of postprandial lipemia. While the allele frequency of the -93G was significantly different in the Hispanics and African Americans (0.09: 95% CI 0.04-0.13 and 0.28: 95% CI 0.19-0.38; P=0.0001, respectively), the N9 allele frequency was not different (0.06: 95% CI 0.02-0.1 and 0.05: 95% CI 0.002-0.093, respectively). Linkage disequilibrium between the -93T/G and D9N was highly significant in Hispanics (delta=0.67. P=0.0001), compared to delta=0.09 (NS) in African-Americans. In the combined group, compared to individuals with the common genotype (TT/DD; n=71) with fasting plasma Tg of 1.34 (+/-4.5% SEM) mmol/l, carriers of the G/D haplotype (TG/DD + GG/DD; n=25) had significantly lower plasma Tg levels of 1.08 (+/-10% SEM) mmol/l (P < 0.02). After the fat meal, compared to individuals with neither mutation, TT/DD, the effect of the G/D haplotype was to reduce significantly postprandial Tg (P < 0.036). Retinyl palmitate concentration at 5 hrs was significantly lower in G/D carriers than TT/DD individuals (P < 0.05). The lipid-raising effect of the N9 allele in carriers of the -93G (TG/DN + GG/DN) and effect on postprandial Tg clearance was not significant in this group. Thus carriers of the G/D haplotype have lower fasting plasma Tg and reduced alimentary lipemia. This allele may be associated with reduced risk of coronary artery disease.
Title: Common variation in the lipoprotein lipase gene: effects on plasma lipids and risk of atherosclerosis Fisher RM, Humphries SE, Talmud PJ Ref: Atherosclerosis, 135:145, 1997 : PubMed
The importance of the enzyme lipoprotein lipase (LPL) in the development of dyslipidaemia and atherosclerosis is increasingly recognised. Variations in the LPL gene which are common in the general population have been shown to be associated with alterations in plasma lipids. D9N and N291S both occur at carrier frequencies of up to about 5% and have been associated with increased plasma triacylglycerol and decreased high density lipoprotein cholesterol concentrations, effects which seem to be magnified in more obese individuals. S447X carrier frequency is approximately 20%, but unlike carriers of N9 or S291, X447 carriers appear to have a more favourable lipid profile. A transition within the LPL promoter at position-93 may lead to increased LPL activity and have a beneficial effect on plasma lipids. Greater knowledge of the underlying mechanisms of these variations within the LPL gene may be of considerable importance in understanding genetic predisposition to atherosclerosis and heart disease.
Title: Lipoprotein lipase variants D9N and N291S are associated with increased plasma triglyceride and lower high-density lipoprotein cholesterol concentrations: studies in the fasting and postprandial states: the European Atherosclerosis Research Studies Gerdes C, Fisher RM, Nicaud V, Boer J, Humphries SE, Talmud PJ, Faergeman O Ref: Circulation, 96:733, 1997 : PubMed
BACKGROUND: Variations at the DNA level with moderate effects on biochemical variables may be important for the occurrence of disease at the population level, if they are common. Two mutations in the LPL gene, N9 and S291, are associated with variation in fasting plasma concentrations of HDL cholesterol (HDL-C) and triglycerides (TG). We investigated whether these mutants were more frequent in offspring of cases with premature coronary disease and analyzed the effects on fasting plasma lipids and postprandial TG. METHODS AND RESULTS: Students with and without paternal history of myocardial infarction (cases and control subjects [controls]) were studied in the European Atherosclerosis Research Studies I and II (EARS-I and -II). Allelic frequencies for the N9 and S291 mutations did not differ between cases and control subjects. The N9 mutation was identified in 4.2% of all subjects in EARS-I, and carriers had higher fasting TG levels (P<.001) than noncarriers. In an oral fat tolerance test, there were no differences in postprandial TG between carriers and noncarriers of the N9 allele. The S291 mutation was identified in 3.1% of all subjects in EARS-I, and carriers had lower fasting HDL-C levels (P<.005) than noncarriers. There was a significant interaction between S291 genotype and body mass index on fasting TG levels (P<.01). In the cases, carriers of the S291 allele had higher TG levels 6 hours postprandially (P<.04) than did noncarriers. CONCLUSIONS: The two LPL mutations are common and may predispose to elevated TG and decreased HDL-C concentrations, even in young subjects. In the case of the S291 mutation, this effect appears to be mediated via delayed postprandial TG clearance. Moreover, even moderate obesity potentiates the TG-raising and HDL-lowering effects associated with the S291 allele.
Title: A common mutation in the lipoprotein lipase gene promoter, -93T/G, is associated with lower plasma triglyceride levels and increased promoter activity in vitro Hall S, Chu G, Miller G, Cruickshank K, Cooper JA, Humphries SE, Talmud PJ Ref: Arterioscler Thromb Vasc Biol, 17:1969, 1997 : PubMed
Single-strand conformational polymorphism analysis of the lipoprotein lipase promoter identified a T-->G transition at position -93. The frequency in healthy white men was 3.4% (n = 1575). There was an 83% allelic association between -93T-->G and Asp9-->Asn (D9N); all N9 mutations occurred on a -93G allele, but not all -93G mutations occurred on an N9 allele. It was thus possible to assess the effect on plasma triglyceride (Tg) levels of the rare -93G mutation in the presence of the wild-type D9. Carriers of the -93G, with genotype TG/DD, had significantly lower Tg levels than TT/DD individuals (1.36 versus 1.78 mmol/L, P = .01); carriers of both mutations (TG/DN) had the highest Tg levels (1.93 mmol/L). When the group was stratified above and below the sample mean for body mass index (BMI), carriers of the -93G on a D9 allele (TG/DD) were "protected" against the Tg-raising effect of obesity, as assessed by BMI. In Afro-Caribbeans (n = 91), the carrier frequency of -93G was 18-fold higher (63%), with weaker (17%) allelic association between -93G and N9. In vitro, the -93G promoter had 24% higher activity than the -93T in a rat smooth muscle cell line and 18% higher activity in a human adrenal cell line. A protein identified by band-shift assays bound to the -93G but not to the -93T allele, which may explain the lower Tg levels in -93G carriers.
The aim of this study was to identify mutations in the lipoprotein lipase (LPL) gene in 20 unrelated patients with familial lipoprotein deficiency (FLLD) and to investigate the genotype/phenotype relationship. The previously reported G188E mutation (Monsalve et al., J Clin Invest 86:728-734, 1990) was screened for and found to be present in seven individuals (12/40 alleles). In addition, three patients were heterozygous for the 2.0 kb insertion (Langlois et al., Proc Nalt Acad Sci US 86:948-952, 1989). Two approaches were taken for new mutation detection; single-strand conformation polymorphism and sequencing to identify micro-mutations in the proximal promoter and exons 1-9 of the LPL gene and Southern blotting to identify gross mutations. Ten different point mutations were found (W86G, A158T, H183Q, G188E, S193R, P207L, L252X, N291S, M301T, L303P). Additionally, a two nucleotide deletion in exon 6 (delta1006-1007), a six nucleotide deletion in exon 8 (delta1441-1447), and a silent substitution in the wobble position of codon E118 were identified. In vitro mutagenesis and expression in COS-B cells suggested that the A158T and S193R substitutions virtually abolished enzyme activity. In analysing the genotype/phenotype relationship, there was no strong association between age at diagnosis, severity of symptoms, lipid levels, and the nature/position of the mutation. Triglyceride levels, however, were higher in compound heterozygotes compared to true homozygotes, possibly reflecting increased instability of heterodimers. Overall, 29 of 40 (72.5%) mutant alleles were identified. Failure to identify the mutation in 11 alleles might reflect the inadequacy of the method or the possibility that mutations lie within regions of the gene not screened in the study because of lack of availability of sequence.
Title: Variation at the lipoprotein lipase and apolipoprotein AI-CIII gene loci are associated with fasting lipid and lipoprotein traits in a population sample from Iceland: interaction between genotype, gender, and smoking status Peacock RE, Temple A, Gudnason V, Rosseneu M, Humphries SE Ref: Genet Epidemiol, 14:265, 1997 : PubMed
The effects of polymorphisms in the lipoprotein lipase (LPL) gene (HindIII and S447X) and in the apolipoprotein (apo) AI-CIII gene cluster (G75A and C1100T) on levels of fasting plasma triglycerides, apoCIII, high density lipoprotein cholesterol (HDL-C), and apoAI were examined in 315 healthy men and women from Iceland. Non-smoking and smoking men and women were examined separately because of the strong effects of smoking status and gender on lipoproteins. For the LPL gene, there were no significant associations between plasma traits and genotypes of the S447X polymorphism, but the LPL-HindIII polymorphism was associated with significant effects on levels of all traits, with the effect of genotype on triglycerides and apoAI being modulated by smoking status, (genotype x smoking interaction, P < .02). The H- allele was generally associated with slightly lower levels of apoCIII, with a lowering effect on triglycerides only in smokers and with a raising effect on ApoAI in non-smoking and smoking men and in non-smoking women. For the apoCIII C1100T polymorphism, smoking and non-smoking men with one or more T alleles had levels of triglycerides roughly 10% higher than those with only the C allele; in contrast, the women with the T allele had lower levels of triglycerides (15.7% lower in non-smokers, P = .04; gender x genotype interaction, P = .02). In males and females and in smokers and non-smokers, the T allele was associated with levels of apoCIII that were 9-20% higher than those with only the C allele (P = .004 overall). In the non-smoking men, nonlinear additive effects were observed with combinations of genotypes at the LPL and apoAI-CIII loci, with the HDL-C and apoAI raising effect associated with the A75 allele and H- allele seen only in those men with both alleles, and the apoCIII raising effect associated with the H+ and T alleles seen only in those with both alleles. Thus, variations at both of the LPL and apoAI-apoCIII loci influence levels of triglycerides, apoCIII, HDL-C, and apoAI, but these effects are strongly modulated by smoking and are different between men and women. The mechanisms for these interactions between smoking or gender and genes are unknown, but future studies should take such interactions into account.
Title: Genetic factors affecting the consistency and magnitude of changes in plasma cholesterol in response to dietary challenge Humphries SE, Talmud PJ, Cox C, Sutherland W, Mann J Ref: Qjm, 89:671, 1996 : PubMed
We examined the role of common genetic variation in determining the consistency and magnitude of change in plasma total cholesterol (TC) levels in response to two separate changes from a high-saturated (SFA) to a low-saturated/high-polyunsaturated-fat (PUFA) diet, in a group of free-living healthy men and women. Consistent responders were defined as those whose mean difference in the change in TC was within one SD of the mean for all participants, and the remainder were defined as variable responders. DNA was obtained from 55 individuals and genotype determined at the apolipoprotein (apo) B locus (signal peptide, SP), apoCIII (C1100-T) and lipoprotein lipase (LPL) gene loci (HindIII). In the 38 consistent responders, the apoBSP24 allele was significantly more common than in the 17 individuals with a variable response (0.29 vs. 0.12; p < 0.05). No other polymorphism showed a significant frequency difference between groups. In the group as a whole, the correlation between the change in TC level in response to the first and second dietary change was 0.28 (p = 0.05), but those with one or more apoB SP24 alleles and those with the apoCIII genotype CC had a significantly higher correlation than those with other genotypes (0.46 (p = 0.05) vs. 0.12 (NS) and 0.31 (p = 0.05) vs. 0.02 (NS), respectively). In the group as a whole, mean response left TC 10% higher on the SFA than on the PUFA diet, and neither apoB nor apoCIII genotypes affected the magnitude of this response. However, individuals with the LPL HindIII genotype H+ H+ had a significantly smaller change in mean TC in response to diet than those with one or more H- allele (9.3% vs. 14.4%; p = 0.03). Thus variation at the apoB and apoCIII loci affects the consistency of response to change in dietary fat content, while variation at the LPL gene locus affects magnitude of response.
Using polymerase chain reaction (PCR) based techniques, we have identified individuals in the ECTIM study of myocardial infarction survivors (cases) and healthy matched controls who are carriers for a mutation of the gene for lipoprotein lipase (LPL) which alters amino acid 9 from aspartic acid to asparagine (LPL-D9N). The frequency of carriers in the cases from Belfast and France (3 separate centres) was 2.5 and 3.7%, respectively (mean 3.3%, 95% CI 1.9-4.7) and in the controls 2.0 and 2.9%, respectively (mean 2.7%, 95% CI 1.6-3.8%), but this difference was not statistically significant. In the cases, carriers of the allele for LPL-N9 had higher levels of several plasma lipid traits including total triglycerides (TG) (30%), very low density lipoprotein (VLDL) cholesterol (19%), apo E (24%), apo C-III (17%), lipoprotein particles (Lp) containing both apo E and apo B (LpE:B) (32%), and particles containing both apo C-III and apo B (LpCIII:B) (39%), and this effect was consistent in cases both from Belfast and from the French centres combined. By contrast, in the controls there were no differences in any lipid trait between carriers and non-carriers of the mutation that was consistent between the French centres and Belfast. There were no significant differences in the levels of any measured factor between cases and controls that could explain the different effect on plasma lipid traits associated with the mutation. However, compared to the non-carriers, in both cases and controls who carried the mutation, plasma TG concentrations were higher in those whose body mass index (BMI) was above the mean of the sample (26.0 kg/m2), with statistically significant interaction seen between BMI and genotype and levels of apo C-III, and lipoprotein particles containing both apo C-III and apo B (P < 0.02). The data suggest that carriers for the LPL-N9 mutation have a mild genetic predisposition to developing hyperlipidaemia and an atherogenic lipid profile, but that this requires the presence of other genetic or environmental factors for full expression, one of which appears to be increasing obesity.
The role of the lipoprotein lipase (LPL) gene in familial combined hyperlipidaemia (FCH) is unclear at present. We screened a group of 28 probands with familial combined hyperlipidaemia and a group of 91 population controls for two LPL gene mutations, D9N and N291S. LPL-D9N was found in two probands and one normolipidaemic population control. LPL-N291S was found in four probands and four population controls. Subsequently, two pedigrees from probands with the D9N mutation and two pedigrees from probands with the N291S mutation were studied, representing a total of 24 subjects. Both LPL gene mutations were associated with a significant effect on plasma lipids and apolipoproteins. Presence of the D9N mutation (n = 7) was associated with hypertriglyceridaemia [2.69 +/- 1.43 (SD) mmol L-1] and reduced plasma high-density lipoprotein cholesterol (HDL-C) concentrations (0.92 +/- 0.21 mmol L-1) compared with 11 non-carriers (triglyceride 1.75 +/- 0.64 mmol L-1; HDL-C 1.23 +/- 0.30 mmol L-1, P = 0.03 and P = 0.025 respectively). LPL-D9N carriers had higher diastolic blood pressures than non-carriers. LPL-N291S carriers (n = 6) showed significantly higher (26%) apo B plasma concentrations (174 +/- 26 mg dL-1) than non-carriers (138 +/- 26 mg dL-1; P = 0.023), with normal post-heparin plasma LPL activities. Linkage analysis revealed no significant relationship between the D9N or N291S LPL gene mutations and the FCH phenotype (hypertriglyceridaemia, hypercholesterolaemia or increased apo B concentrations). It is concluded that the LPL gene did not represent the major single gene causing familial combined hyperlipidaemia in the four pedigrees studied, but that the LPL-D9N and LPL-N291S mutations had significant additional effects on lipid and apolipoprotein phenotype.
A mutation in the lipoprotein lipase (LPL) gene, resulting in the substitution of asparagine by serine at residue 291 (LPL-S291), was found to occur in young survivors of a myocardial infarction from Sweden, combined hyperlipidemic subjects from the United Kingdom, and type III hyperlipidemic subjects from Germany at allelic carrier frequencies no different from those found in companion healthy control subjects (3.63 vs. 3.37; 1.85 vs. 1.60; and 2.00 vs. 1.56%, respectively). In a group of 620 healthy middle-aged men from the United Kingdom with baseline and three subsequent annual lipid measurements, mean plasma triacylglycerol (TG), (but not plasma cholesterol) concentrations in carriers of the mutation were significantly elevated over non-carriers (1.95 vs. 1.61 mmol/l, P = 0.05, and 5.83 vs. 5.65 mmol/l, P = 0.29, respectively). When these healthy control subjects were divided according to tertiles of body mass index (BMI), as expected, non-carriers whose BMI was in the upper two tertiles (BMI > or = 25.0 kg/m2) had higher plasma TG concentrations than those in the lowest tertile (1.90 vs. 1.54 mmol/l), but this difference was much greater in LPL-S291 carriers (2.33 vs. 1.36 mmol/l, P = 0.01, BMI x genotype interaction, P = 0.02). To confirm this effect, a second group of 319 healthy subjects from the United Kingdom was screened for LPL-S291. The allelic frequency of the mutation was found to be 1.88% and the effect on plasma lipid concentrations was very similar to that observed in the first control group (plasma TG, 2.31 vs. 1.27 mmol/l, P < 0.001 for LPL-S291 carriers vs. non-carriers, respectively). As before, those carriers whose BMI was in the top two tertiles for this sample (BMI > or = 23.3 kg/m2) had higher plasma TG concentrations than non-carriers (2.31 vs. 1.42 mmol/l). Thus, the LPL-S291 variant may predispose individuals to elevated plasma TG concentrations under conditions such as increased BMI.
Title: Associations of genotypes at the apolipoprotein AI-CIII-AIV, apolipoprotein B and lipoprotein lipase gene loci with coronary atherosclerosis and high density lipoprotein subclasses Peacock RE, Hamsten A, Johansson J, Nilsson-Ehle P, Humphries SE Ref: Clin Genet, 46:273, 1994 : PubMed
Association studies were carried out in a sample of 86 patients from Sweden who had survived a myocardial infarction (MI) at a young age and 93 age-matched healthy individuals, to compare the impact of polymorphisms at the apolipoprotein (apo) AI-CIII-AIV gene cluster on among-individual differences in plasma lipid and lipoprotein traits, the five high density lipoprotein (HDL) subclasses (2b to 3c), lipoprotein lipase (LPL) activity and presence and progression of atherosclerosis. Individuals were genotyped for four polymorphisms; 5'apoAI (G/A-75), 3'apoAI (PstI; P +/-), apoCIII (C/T1100) and apoCIII (PvuII; V +/-), using PCR-based techniques. Allele frequencies were similar in healthy individuals and patients (frequencies of alleles in combined population: 5'apoAI-A-75 = 0.14, 3'apoAI-P- = 0.05, apoCIII-T1100 = 0.27 and apoCIII-V- = 0.18). In the healthy individuals, levels of low density lipoprotein (LDL) triglycerides were significantly associated with genotypes of the apoCIII-PvuII polymorphism (p = 0.02), but no other associations were found between lipids or HDL subclasses and single polymorphisms in the apoAI-CIII-AIV gene cluster. Levels of triglycerides and very low density lipoprotein (VLDL) triglycerides were significantly higher in the presence of the haplotype defined by the presence of apoCIII-T1100 and common alleles of the other three polymorphisms, explaining 5.8% and 7.8% (p = 0.03 and 0.01), respectively, of sample variance. In the patients, no associations were found between lipids or HDL subclasses and variation at the apoAI-CIII-AIV gene cluster. Associations were also examined between levels of HDL subclasses and variation at the apoE (common isoforms), apoB (signal peptide and XbaI polymorphisms) and lipoprotein lipase (PvuII, HindIII and Serine447/Stop polymorphisms) gene loci. In the patient group only, levels of protein in HDL2b, HDL2a and HDL3b subclasses were significantly associated with genotypes of the LPL-HindIII polymorphism (22.1, 19.3 and 11.4%, respectively, of sample variance; p < 0.05). Finally, associations were examined between genotypes at the apoAI-CIII-AIV gene cluster and the extent of coronary atherosclerosis. Global severity of atherosclerosis at the first angiography was weakly associated with genotypes of the apoCIII-C/T1100 polymorphism, presence of the T1100 allele being associated with 53% lower median score (1.6 vs 0.75; p = 0.09).(ABSTRACT TRUNCATED AT 400 WORDS)
Title: Associations between lipoprotein lipase gene polymorphisms and plasma correlations of lipids, lipoproteins and lipase activities in young myocardial infarction survivors and age-matched healthy individuals from Sweden Peacock RE, Hamsten A, Nilsson-Ehle P, Humphries SE Ref: Atherosclerosis, 97:171, 1992 : PubMed
Association studies were carried out on a sample of 87 patients from Sweden who had survived a myocardial infarction (MI) at a young age and 93 age-matched healthy individuals, to compare the impact of polymorphisms (PvuII, HindIII and Serine447-Stop) at the lipoprotein lipase (LPL) gene locus on among-individual differences in plasma lipid traits and progression of atherosclerosis. Significant linkage disequilibrium was detected between any two of these polymorphisms, with the Stop447 allele being only found on the same chromosome as the rare alleles (no cutting sites) of the PvuII and HindIII polymorphisms. In the healthy individuals, weak associations were found between genotypes of the HindIII polymorphism and triglycerides and the PvuII polymorphism and high density lipoprotein cholesterol explaining 7.4% and 5.6% of sample variance (P = 0.03 and 0.09), respectively. No associations were found between these traits and genotypes of the Serine447-Stop substitution, and thus it is unlikely to be the cause of the associations seen with the PvuII and HindIII polymorphisms even though it truncates the enzyme amino acid sequence. The presence of the rare allele, H-, of the HindIII polymorphism was associated with a smaller variance in triglycerides and both cholesterol and triglycerides in the very low density lipoprotein fraction, and with larger interdependent variation between these lipid traits, and also between LPL activity and these lipid traits. This implies that the H- allele, rather than the Stop447 allele, has the major impact on interdependence between traits which are directly or indirectly influenced by LPL activity. In the healthy individuals who were carriers of the apolipoprotein E2 allele, the inter-dependence between LPL activity and lipid traits was significantly smaller, and that between high density lipoprotein cholesterol and both cholesterol and triglycerides in the very low density lipoprotein fraction was much larger compared with non-carriers (P < 0.05). No significant associations were found between lipid traits or lipase activity and genotypes of the Serine447-Stop substitution. However, in the patients, global severity of coronary atherosclerosis at the first angiography was significantly associated with haplotype combinations of the HindIII and the Serine447-Stop polymorphisms, with the H-Stop haplotype being associated with the highest median score (P = 0.02). The data suggest that variation at the LPL gene locus is associated with a pleiotropic effect, that is not directly mediated by changes in lipids, on severity of coronary atherosclerosis.
1. We have used polyclonal antibodies and a complementary DNA clone for human lecithin:cholesterol acyltransferase (LCAT) to study LCAT protein and the structure of the LCAT gene, respectively, in patients with familial LCAT deficiency from Norway, Ireland, Germany and Italy. 2. The patients had low levels of non-functional LCAT protein in their serum as measured by rocket immunoelectrophoresis; its mol. wt. of approximately 68,000 was identical with that of LCAT in normal plasma, as judged by immunoblotting. 3. Enzymatic digestion of DNA samples from the patients produced LCAT gene fragments which were indistinguishable from those found in normal individuals. 4. We conclude that LCAT deficiency in these patients is not caused by a large deletion or rearrangement of the LCAT gene sequences.
We have used a cDNA clone for human lecithin:cholesterol acyl transferase (LCAT) and Southern blotting techniques to identify the human LCAT gene in DNA from a series of rodent X human somatic cell hybrids. Our results are compatible with the location of the gene on human chromosome 16, and this has been confirmed using in situ hybridization of the LCAT cDNA to human metaphase chromosomes. These results confirm the earlier studies on LCAT-deficient patients, indicating that the structural gene for LCAT is on human chromosome 16q22.
The protein sequencing of tryptic peptides from purified human lecithin: cholesterol acyltransferase (LCAT) identified sufficient amino-acid sequence to construct a corresponding mixed oligonucleotide probe. This was used to screen an adult human cDNA liver library, from which incomplete cDNA clones were isolated. The DNA sequence of these clones allows the prediction of the entire amino-acid sequence of the mature LCAT enzyme. The mature protein consists of 416 amino acids and contains several marked stretches of hydrophobic residues and four potential glycosylation sites. The cDNA probe detects LCAT mRNA sequences approx. 1500 bases long in human liver, but not intestine, RNA. The cDNA probe was used to isolate LCAT genomic recombinants from a human genomic library. Southern blotting data, and restriction site mapping, suggest that there is a single human LCAT structural gene between 4.3 and 5.5 kb in size.
