Search

Abnormal patterns of fetal and infant growth have been associated with an increased risk of cardiovascular disease in adulthood. Catch-up growth during the first year of life has been associated with a higher prevalence of type 2 diabetes mellitus, whereas a lack of catch-up growth tracks with a risk of hypertension. The role of genetic factors influencing both growth and blood pressure have not been explored. We genotyped cord blood samples from 530 singleton, Caucasian, uncomplicated pregnancies, drawn from a larger cohort of 1650 pregnancies, and related polymorphism in the angiotensin converting enzyme (ACE) gene (alleles insertion (I) or deletion (D)) with measures of size at birth and at age of 1 year. ACE genotype did not significantly influence size at birth, although there was a greater proportion of individuals with the D/D genotype born with a birth weight less than the 10th centile (P=0.004). The ACE I/I genotype was significantly associated with higher weight (p=0.001), body mass index (p=0.001) and mid arm circumference (p=0.001) at 1 year of age compared to the ACE D/D and I/D genotypes. Individuals with the I/I genotype displayed catch-up (gain from birth size of >or=0.6 Standard Deviation Score) in weight (p=0.04), body mass index (p=0.03) and mid arm circumference (p=0.03) compared to the D/D group, the majority of which showed no change or catch-down. The I/D genotype was distributed equally across the catch up/catch down/no change categories. The effect was more marked in males, but ACE genotype and sex of the infant contributed independently to mid arm circumference measurements and there was no interaction between the two. There was no effect of maternal or paternal ACE genotype on birth size. In a multiple linear regression model ACE genotype, socioeconomic status and sex of the infant explained 10.9% of the variance in body mass index SDS at 1 year of age. We conclude that the ACE I/I genotype is associated with a higher weight and body mass index SDS at 1 year of age, along with catch-up in terms of these measures from birth to 1 year. The D/D genotype is associated with a greater proportion of babies, born at term, that at small for gestational age. These results suggest that due consideration should be given to the underlying genotype of an individual when evaluating the association of early human growth with the development of risk factors for cardiovascular disease. The observation of independent effects of genotype, sex of the individual and socioeconomic status on postnatal growth suggests the need to develop methodologies for the integration of genetic and environmental factors in causality modelling.

Background and Aims: An important part is played by inflammation in intracranial aneurysm formation. The hypothesis that there is an association of the proinflammatory cytokine interleukin-6 (IL-6) genotypes (-572G>C and -174G>C) with intracranial aneurysms was tested., Methods: IL-6 genotypes were determined in 91 Caucasian patients with aneurysms and compared with 2720 healthy UK controls., Results: For both polymorphisms, the distribution of the genotypes and estimated allele frequency were different between the control group and the aneurysm group. For -572G>C, a higher frequency of the C allele (p = 0.001) and more people homozygous for the C allele were found among those with aneurysms than among the controls (4.4% v 0.3%, p = 0.001). For -174G>C, more people homozygous for the C allele were found among the controls than among those with aneurysm (18% v 7%, p = 0.007). The 572C/174G haplotype was associated with an increased risk of aneurysms, with the relative risk compared with the common haplotype being 1.89 and that for the -572G/174C haplotype being 0.58 (p<0.0005)., Conclusion: This is the first study to show that IL-6 promoter polymorphisms are associated with intracranial aneurysmal disease. Whether this association is with the development, progression or rupture of such aneurysms, or represents survivor bias, is unclear.

Inflammation is thought to play an important role in intracranial aneurysm formation. Heme-oxygenase-1(HO-1) is a novel anti-inflammatory factor. A length polymorphic variant of the HO-1 gene promoter region, comprising (GT)n dinucleotide repeats, is associated with altered levels of gene transcription: long (= 36 GT) repeats are associated with decreased HO-1. We hypothesized that patients with aneurysmal subarachnoid haemorrhage were more likely to have long repeats than controls. Sixty-nine patients with aneurysms and 230 age-matched controls were genotyped, and allelic repeats were classed as <36 (short and medium repeats) and >36 (long repeats). Patients were more likely to have =36 repeats than controls (8 v. 4%, p = 0.037. Control patients without aneurysms were more likely to have short alleles. Thus, facilitated up-regulation of HO-1 may be a protective anti-inflammatory factor against the development of intracranial aneurysms, whilst a propensity to a more pro-inflammatory state may put individuals at risk. However, because of the relatively small sample size and modest statistical significance, the data must be interpreted with caution and the association needs to be confirmed in further samples.

Experimental and clinical observations suggest that innate immunity plays a major role in the pathogenesis and progression of atherosclerosis. A common C-260T polymorphism in the promoter of the CD14 gene, the trans-membrane receptor of lipopolysaccharides, has been inconsistently associated with coronary heart disease. Our objective was to evaluate the contribution of the CD14 polymorphism to the inflammatory response and to the risk of myocardial infarction (MI). We used an European case-control study, the HIFMECH study, comparing 533 men with MI and 575 sex- and age-matched controls. Associations between genotype and disease outcome, according to interleukin-6 (IL-6) and C-reactive protein (CRP) levels, were assessed using conditional logistic regression. The CD14/C-260T polymorphism was associated with plasma IL-6 levels, T/T subjects having higher plasma levels than C/C in cases but not in controls (mean+/-S.D.: 2.04+/-1.37 versus 1.70+/-1.15, p=0.01; 1.20+/-0.75 versus 1.35+/-0.88, p=0.31, respectively). Overall, the CD14/C-260T polymorphism was not associated with the risk of MI. However, in individuals with IL-6 plasma levels in the highest tertile, T allele carriers had a higher risk of MI than C/C (OR: 1.85; CI 95 1.05-3.25). IL-6 increased the risk of MI in carriers of the T allele (OR for first versus third IL-6 tertile: 4.02; CI 95 2.24-7.21), but not in C/C (OR: 0.75; CI 95 0.32-1.74, p=0.004 for interaction). The data indicate a role for CD14/C-260T in MI. The risk mediated by the polymorphism is highly dependent on IL-6 plasma levels.

Introduction: The tissue renin-angiotensin system is implicated in the pathogenesis of coronary artery disease (CAD). As locally synthesised aldosterone is a potential mediator of CAD, we have sought an association of the -344T>C variant of the aldosterone synthase (CYP11B2) gene with CAD events., Methods: Subjects comprised of the Second Northwick Park Heart Study (NPHSII), a prospective study of unrelated, healthy middle-aged Caucasian males. CAD events were recorded in 2490 subjects, and defined as a sudden cardiac death, myocardial infarction or coronary artery revascularisation procedure. Mean follow-up was 10.8 years. Aldosterone synthase genotype was determined in 2490 subjects. Power calculation suggests that we have 80% power (at a significance level of 0.05) to detect a difference in hazard ratio (HR) between homozygote groups of 0.45., Results: One hundred and eighty-seven CAD events were recorded in 2490 subjects. In the group overall, CAD events were independent of genotype with adjusted hazard ratios being 1.00 versus 1.25 versus 0.80 for TT versus TC versus CC genotypes, respectively, P = 0.07. Genotype interactions with smoking and blood pressure were sought. Whilst CAD events were independent of genotype amongst non-smokers, CC genotype in smokers was associated with a reduced risk HR 2.02 versus 2.28 versus 0.82 for TT versus TC versus CC genotypes, P = 0.05 (HR for TT + TC versus CC were 1.77 versus 0.67, P = 0.02). This apparent interaction remained after adjustment for conventional risk factors. No such interaction was found with blood pressure., Conclusions: Aldosterone synthase genotype is unrelated to overall CAD events risk. A possible interaction with smoking requires confirmation.

Unlabelled: Although the potential role of plasminogen activator inhibitor-1 (PAI-1) in the development of coronary artery disease is strongly supported by its biological characteristics, results of clinical studies remain controversial., Objectives: To investigate whether plasma PAI-1 concentrations and the -675 4G/5G polymorphism located in the PAI-1 gene could constitute risk markers for myocardial infarction (MI)., Patients and Methods: We used a European case-control study, the HIFMECH study, comparing 598 men with MI and 653 age-matched controls., Results: Insulin resistance explained a major part of the variation in PAI-1 (24%) whereas inflammation had only a minor contribution (0.01%). For both cases and controls plasma PAI-1 concentrations were significantly higher in the North than the South, and in both regions were higher in individuals with MI compared with control subjects [overall odds ratio (OR) for a 1 SD increase=1.54, 95% confidence interval (CI) 1.34, 1.77]. This difference was observed in all the centers studied. Overall, the difference between cases and control subjects remained significant after controlling for inflammation variables (OR=1.30, 95% CI 1.08, 1.57), but lost significance after controlling for insulin resistance variables (OR=1.17, 95% CI 0.98, 1.40). The 4G allele was associated with significantly higher PAI-1 levels in cases but not controls and, taken independently, did not modify the risk of MI (P=0.9). However, a significant interaction was observed with both insulin or proinsulin and the risk of MI (P=0.05 and 0.02, respectively), but not with triglycerides or body mass index (BMI). The insulin or proinsulin effect on risk was observed only in the carriers of the 4G/4G genotype. This interaction appeared not to be mediated by plasma PAI-1 antigen concentrations (P=0.01 and 0.02 after adjustment for PAI-1 plasma levels). The interaction with proinsulin but not insulin remained statistically significant after further adjustment for other factors associated with insulin resistance (triglycerides and BMI) and C-reactive protein (P=0.01)., Conclusion: This study suggests that PAI-1 has a role in risk of MI in the presence of underlying insulin resistance. A significant interaction between insulin or proinsulin and the -675 4G/5G polymorphism was observed in risk for MI. The mechanisms for these interactions remain to be determined.

Objective: C-reactive protein (CRP) concentrations are predictive of cardiovascular disease, and levels are heritable, in part. We identified novel polymorphisms in the CRP gene and assessed their influence on CRP level., Methods and Results: CRP was measured in 250 male army recruits before and after strenuous exercise and perioperatively in 193 coronary artery bypass graft (CABG) patients. Two novel polymorphisms were identified in the CRP gene, -717G>A in the promoter and +1444C>T in the 3'UTR. Among army recruits, CRP was higher in +1444TT homozygotes than +1444 C-allele carriers at baseline (1.04+/-0.38 versus 0.55+/-0.06, P=0.014) and at all time points after exercise (2.35+/-0.68 versus 1.07+/-0.12, 2.11+/-0.53 versus 0.88+/-0.09, and 1.77+/-0.44 versus 0.71+/-0.09, P=0.034, P=0.007, and P=0.013, at 2, 48, and 96 hours after exercise, respectively). In the CABG patients, mean CRP (mg/L) rose from 1.97+/-0.36 at baseline to 167.2+/-5.0 72 hours postoperatively. Genotype did not influence CRP at baseline; however, peak post-CABG CRP levels were higher in +1444TT homozygotes compared with +1444C-allele carriers (198+/-17 versus 164+/-5, P=0.03)., Conclusions: The CRP gene +1444C>T variant influences basal and stimulated CRP level. These findings have implications both for the prediction and pathogenesis of coronary heart disease.

IL-6 is a pleiotropic cytokine that plays a critical role in bone resorption. We describe two allelic variants in the IL-6 promoter, -572 and -174 G-->C, that alone and in combination influence IL-6 activity in vitro and in vivo. The association of IL-6 -572 genotypes and -572/-174 G-->C haplotypes with serum C-reactive protein (CRP), serum and urinary C-terminal cross-linking of type I collagen (a marker of bone resorption), and osteocalcin (a marker of bone formation) was investigated in a cohort of healthy postmenopausal women (n = 495; mean age +/- SD, 72 +/- 5.7 yr). Among IL-6 -572 genotypes, CRP was 37% higher (P = 0.02) and urinary C-terminal cross-linking of type I collagen was 20% higher (P = 0.01) in the presence of the C allele, whereas serum osteocalcin was not different. IL-6 -572/-174 haplotypes (G/C, G/G, and C/G) were significantly associated with all biochemical markers, and additive effects of the two polymorphic loci were found. Thus, there was a significant increase in the level of CRP (up to +79%; P = 0.007) and bone resorption markers (up to +32%; P = 0.017) with a decreasing number (from four to one) of IL-6 protective alleles -572G and -174C. In addition, there was a trend for lower age-adjusted bone mineral density at the lumbar spine in subjects with less IL-6 protective alleles (up to -9.6%; P = 0.037; P = 0.08 after further adjustment for weight). In conclusion, we describe two functional polymorphisms in the IL-6 gene regulatory region associated with IL-6 activity in postmenopausal women, both systemically (CRP) and locally in bone. As such, IL-6 polymorphisms are able to influence the risk of osteoporosis as well as other chronic disorders involving IL-6 activity.

Aims: Coronary stent deployment is a major advance in percutaneous treatment of ischaemic heart disease, but 10-40% of patients still develop angiographic restenosis by 6 months due to neointimal hyperplasia. Patient-specific factors, including genetic factors, can contribute to this process. We have conducted a prospective study to examine the involvement of genetic risk factors (eNOS, ACE, MMP-3, IL-6, and PECAM-1) in restenosis following coronary stent deployment., Methods and Results: A total of 226 patients who underwent elective and successful coronary artery stenting to de novo lesions in native coronary arteries were studied. Two hundred and five (90.7%) patients were restudied by coronary angiogram at 6 months and the stented lesions were assessed using an automated quantitative angiography system. Genotype was determined by polymerase chain reaction (PCR) and restriction enzyme digestion. Restenosis rate, defined as >or=50% diameter stenosis, was 29.3%. The overall genotype frequency distributions were in Hardy-Weinberg equilibrium for all variants. Carriers of the 298Asp allele of the eNOS Glu298Asp polymorphism showed a higher frequency of restenosis with an odds ratio of 1.88 (95%CI: 1.01-3.51, P=0.043) compared to 298Glu homozygotes. Carriers of the -786C allele of the eNOS -786T>C polymorphism also showed a higher frequency of restenosis with odds ratio of 2.06 (95%CI: 1.08-3.94, P=0.028). These effects were essentially additive and were independent of other classical risk factors. Other studied genes did not show significant association with coronary in-stent restenosis., Conclusion: In patients with coronary artery disease, the possession of the 298Asp and -786C variants of the eNOS gene are a risk factor for coronary in-stent restenosis, demonstrating the importance of the nitric oxide system in restenosis., (Copyright 2002 The European Society of Cardiology. Published by Elsevier Science Ltd.)

Genetic determinants of baseline levels and the fall in plasma triglyceride and fibrinogen levels in response to bezafibrate treatment were examined in 853 men taking part in the lower extremity arterial disease event reduction (LEADER) trial. Three polymorphisms in the peroxisome proliferator activated receptor alpha (PPARalpha) gene were investigated (L162V, G>A in intron 2 and G>C in intron 7), two in the apolipoprotein CIII (APOC3) gene (-482C>T and -455T>C) and one in the beta-fibrinogen (FIBB) gene (-455G>A). The presence of diabetes (n=158) was associated with 15% higher triglyceride levels at baseline compared to non-diabetics (n=654) (P<0.05). Among the diabetic group, carriers of the PPARalpha intron 7 C allele had 20% lower triglyceride levels compared to homozygotes for the common G allele (P<0.05), with a similar (non-significant) trend for the L162V polymorphism, which is in linkage disequilibrium with the intron 7 polymorphism. For the APOC3 gene, carriers of the -482T allele had 13% lower baseline triglyceride levels compared to -482C homozygotes (P<0.02), but no effect was observed with the -455T>C substitution. In the non-diabetic patients, the PPARalpha V162 allele was significantly associated with 9% higher baseline triglyceride levels (P<0.03) and a similar, but non-significant trend was seen for the intron 7 polymorphism. Overall, triglyceride levels fell by 26% with 3 months of bezafibrate treatment, and current smokers showed a poorer response compared to ex/non-smokers (23% fall compared to 28% P=0.03), but none of the genotypes examined had a significant influence on the magnitude of response. Carriers of the -455A polymorphism of the FIBB gene had, as expected, marginally higher baseline fibrinogen levels, 3.43 versus 3.36 g/l (P=0.055), but this polymorphism did not affect response to treatment. Overall, fibrinogen levels fell by 12%, with patients with the highest baseline fibrinogen levels showing the greatest decrease in response to bezafibrate. For both the intron 2 and the L162V polymorphisms of the PPARalpha gene there was a significant interaction (both P<0.01) between genotype and baseline levels of fibrinogen on the response of fibrinogen levels to bezafibrate, such that individuals carrying the rare alleles in the lowest tertile showed essentially no overall decrease compared to a 0.18 g/l fall in homozygotes for the common allele. Thus while these genotypes are a minor determinant of baseline triglyceride and fibrinogen levels, there is little evidence from this study that the magnitude of response to bezafibrate treatment in men with peripheral vascular disease is determined by variation at these loci.

Human physical performance is strongly influenced by genetic factors. We have previously reported that the I variant of the human angiotensin I-converting enzyme (ACE) gene is associated with greater endurance performance in mountaineers and Olympic runners and improved performance in army recruits. In this study we examined whether this effect is mediated by improvements in cardiovascular fitness with training in 58 army recruits homozygous for the insertion (I, ACE genotype II) or deletion (D, ACE genotype DD) allele. A submaximal and maximal exercise protocol was used to calculate both the heart rate/oxygen uptake (VO2) relationship and changes in maximal oxygen uptake (VO2max), respectively. There was no significant intergroup difference in VO2max at baseline (P=0.19) or after training (P=0.22). There was no difference between genotypes with training in the heart rate/VO2 elevation (P = 0.79 for the mean difference in mean adjusted heart rates). However, VO2 at all exercise intensities in the submaximal test was lower for all subjects after training and at 80 W the reduction in VO2 was greater for the II subjects compared to DD subjects [mean(SEM)] [1.6 (0.27) and 0.68 (0.27) ml kg(-1) min(-1), respectively; P = 0.02 for mean difference]. The I/D polymorphism may play a role in enhanced endurance performance but this is not mediated by differences in VO2max or the heart rate/VO2 relationship in response to training.

The aim of this study was to examine whether the well-established effect of the common TaqIB polymorphism in intron 1 of the gene for cholesterol ester transfer protein (CETP) on high density lipoprotein cholesterol (HDL-C) concentration and increased risk of myocardial infarction (MI), could be explained by the recently identified -629C>A functional polymorphism in the promoter. Non-fatal MI cases (388 male) and a control group of 794 healthy men were recruited from the 30 year long prospective Reykjavik Study. In the healthy men the frequency of the TaqIB B2 allele was 0.47 (95% CI: 0.44-0.50) and there was a strong allelic association with the -629A allele (D=-0.21, P<0.0001), which had a frequency of 0.52 (95% CI: 0.49-0.56). B2B2 homozygotes displayed 15% higher HDL-C levels than subjects homozygous for the B1 allele (P<0.0001). Homozygotes for the -629A allele displayed 14% higher HDL-C concentrations than subjects homozygous for the -629C allele (P<0.0001). The frequencies of the alleles associated with lower HDL-C were significantly higher in cases compared with controls, 0.59 versus 0.53 (TaqIB B1) and 0.52 versus 0.48 (-629 C) respectively (P<0.05 for both). There was a significantly higher risk for MI in B1B1 homozygotes (OR=1.44, 95% CI: 1.10-1.87, P<0.01), compared to the other genotypes combined. This was not observed for the CC homozygotes (OR=1.16, 95% CI: 0.87-1.54). In addition, homozygotes for the TaqI B2 allele experienced a first MI 2 years later than men with other genotypes, 59 versus 61 years (P<0.05). This effect was not seen for the promoter polymorphism. These results strongly confirm the role of the CETP gene and the TaqIB variant as a risk factor for MI and suggest that another functional polymorphism is yet to be discovered in the CETP gene, that will explain the effect on MI associated with TaqIB observed in this study.

Association studies between gene variants (polymorphisms) and measured intermediate phenotypes, such as lipid/lipoprotein levels, or disease endpoints such as coronary artery disease, are commonplace in the literature. But have we learnt anything from the shortcomings in study design and analytical strategies that have resulted in much controversy in this field over the last few years? This review highlights some of these problems. Using the lipoprotein lipase gene as an example, we evaluate new approaches to identifying polymorphisms that will stand up to linkage disequilibrium/association studies with complex disorders in this post Human Genome Project age, and emphasize the importance of gene-environment interaction in assessing the impact of gene variants.

Total blood levels of homocysteine (tHcy) have been shown to depend on both environmental and genetic factors, and to be associated with the risk of developing atherosclerosis with its complications of coronary heart disease (CHD) and stroke. In this study, 408 men and 346 women from two towns, Dewsbury and Maidstone were examined for tHcy levels and genotyped for the C677T and the A1298C polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene. Blood tHcy was significantly higher in men from the CHD high risk town of Dewsbury (12.7 micromol/l) than in the low CHD risk town of Maidstone (11.5 micromol/l) P<0.001, but not in women (10.7 vs. 10.5 micromol/l), with women in both towns, thus, showing significantly lower tHcy than men. There was no difference between towns in folate or vitamin B12 levels but the conventional inverse relationship with tHcy was seen. Smoking men and women from both towns had significantly higher tHcy and lower folate levels than non-smoking individuals (P<0.001). The frequency of the 677T allele in Dewsbury was 0.35 (95% CI; 0.32-0.39) compared with 0.29 (95% CI; 0.26-0.32) in Maidstone (P<0.01). Similar frequency difference of borderline statistical significance was seen both for men (P=0.054) and women (P=0.048) in both the towns, suggesting a true regional frequency difference. The effect of the 677T on tHcy was highly significant in the group as a whole with the most profound effect seen in men (12.0 micromol/l for CC vs. 14.1 micromol/l for TT, P<0.001). By contrast, there was no significant effect of the A1298C polymorphism on tHcy, folate or vitamin B12 levels, with no evidence for an interaction with the C677T genotype. The regional differences in tHcy levels were still present after the adjustment for folate and vitamin B12 levels, smoking and the effect of the C677T polymorphism. This suggests that there may be other unidentified factors, either environmental or genetic, affecting tHcy levels, and thus potentially having an impact on the risk of developing hyperhomocysteinaemia and CHD. These observations may have a bearing on regional differences in tHcy levels and the variation in CHD risk between regions in the UK.

Five polymorphisms in the thrombopoietin (TPO) gene were identified, one in the 5' untranslated region (UTR) (C1796T), two within intron 5 (C4830A and A4877C), and two in the 3' UTR (A5713G and A6160T). The allele frequencies were determined in a group of 450 healthy middle aged men from the UK and found to be 0.46 for 1796T, 0.38 for 4830A, 0.004 for 4877C, 0.47 for 5713G and 0.07 for 6160T. Genotypes for the three common polymorphisms were determined in a group of 176 young male Swedish survivors of a myocardial infarction (MI) and 186 age-matched controls and a group of 156 young Italian survivors of an MI and 147 age and sex matched controls. In both the Swedish and the Italian studies polymorphisms were found to be associated with increased risk of MI. In the Swedish sample the frequency of 4830A was significantly higher in controls (0.40) compared with patients (0.29) (P=0.003), with an odds ratio for AA homozygotes of 0.48 (0.25-0.92; P=0.03) compared with CC homozygotes. In the Italian sample the frequency of 5713G was significantly lower in controls (0.31) compared with cases (0.40) (P=0.03), with an odds ratio for GG homozygotes of 2.29 (1.08-4.89; P=0.03) compared with AA homozygotes. These risk associations are consistent since 4830A and 5713A show strong allelic association. After adjusting for other measured risk factors the effect on risk was still significant in the Italian sample 2.39 (1.02-5.58), but not in the Swedish sample 0.46 (0.16-1.32). The observation of frequency differences between cases and controls in two independent samples strongly suggests that the TPO gene is involved as a risk factor for developing MI at a young age, but the identified polymorphisms are probably acting as markers for an unidentified functional mutation elsewhere in the gene locus.

Background: Local cardiac renin-angiotensin systems may regulate left ventricular (LV) hypertrophic responses. The absence (deletion [D]) of a 287-bp marker in the ACE gene is associated with greater myocardial ACE levels and exercise-related LV growth than is its presence (insertion [I]), an effect potentially mediated through either increased activity of the cellular growth factor angiotensin II on the angiotensin type 1 (AT(1)) receptor or increased degradation of growth-inhibiting kinins. We sought to confirm ACE genotype-associated exertional LV growth and to clarify the role of the AT(1) receptor in this association., Methods and Results: One hundred forty-one British Army recruits homozygous for the ACE gene (79 DD and 62 II) were randomized to receive losartan (25 mg/d, a subhypotensive dose inhibiting tissue AT(1) receptors) or placebo throughout a 10-week physical training program. LV mass, determined by cardiac magnetic resonance, increased with training (8.4 g, P:<0.0001 overall; 12.1 versus 4.8 g for DD versus II genotype in the placebo limb, P:=0.022). LV growth was similar in the losartan arm: 11.0 versus 3.7 g for DD versus II genotypes (P:=0.034). When indexed to lean body mass, LV growth in the II subjects was abolished, whereas it remained in the DD subjects (-0.022 versus 0.131 g/kg, respectively; P:=0.0009)., Conclusions: ACE genotype dependence of exercise-induced LV hypertrophy is confirmed. Additionally, LV growth in DD (unlike II) subjects is in excess of the increase in lean body mass. These effects are not influenced by AT(1) receptor antagonism with the use of losartan (25 mg/d). The 2.4-fold greater LV growth in DD men may be due to the effects of angiotensin II on other receptors (eg, angiotensin type 4) or lower degradation of growth-inhibitory kinins.

Healthy middle-aged men (n = 1,470) from eight general practices across Britain were examined for plasma total homocysteine levels and genotyped for the A222V polymorphism in the methylene-tetrahydrofolate (MTHFR) gene, the 68 bp insertion polymorphism in exon 8 of the cystathionine b synthase (CBS) gene and the D919G polymorphism in the methionine synthase (MS) gene. The median value for plasma homocysteine was 11.90 micromol/l (25-75% Interquartile range 10.10-14.20) for the whole sample. Smokers had significantly higher homocysteine levels than non-smokers (12.90 vs 11.70 micromol/l and p < 0.00005) and levels significantly differed according to folate (p-value < 0.00005), with men in the lowest quartile of folate having the highest median homocysteine levels. Genotype at all three loci was associated with differences in plasma homocysteine level. Individuals homozygous for the MTHFR V222 allele had 1.6 micromol/l higher median homocysteine levels when compared to the other two genotypes (p < 0.00005), while for the CBS and MS genes, individuals carrying one or more of the rare alleles had lower median homocysteine than individuals homozygous for the common allele (0.80 micromol/l, p < 0.03, and 0.70 micromol/l, p < 0.04 respectively). The raising effect associated with homozygosity for the V222 allele was greater in men in the lowest quartile of folate (interaction between folate and genotype p = 0.02), but none of the genotype effects was significantly modulated by B12 levels. While the raising effects of V222 and MS D919 homozygosity on homocysteine level were essentially additive, the homocysteine lowering effect associated with the CBS 68bp allele was seen most strongly in men homozygous for the V222 allele (MTHFR-CBS genotype interaction p = 0.03) and the D919 allele (MS-CBS interaction p = 0.09). Age, folate, B12 and smoking explained 13.48% of the variance while the three genotypes combined and with interaction terms explained only an additional 2.63%. This interaction between CBS genotype and MTHFR and MS genotype points to a key role of the CBS transulphuration pathway in the metabolism of homocysteine that may be particularly important as a compensatory mechanism in subjects with low dietary folate.

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.

The D allele of the angiotensin-converting enzyme (ACE) I/D polymorphism is associated with elevated levels of serum and tissue ACE, increased production of the vasopressor angiotensin II and a reduction in the half-life of the vasodilator bradykinin. Several cardiac and renal conditions appear to have a worse prognosis in subjects homozygous for the D allele, whereas the I allele has been associated with enhanced endurance performance in elite distance runners, rowers and mountaineers. The nature of the gene-environment interaction between ACE I/D polymorphisms and physical training, an overview of recent findings and a discussion of possible underlying mechanisms is the subject of this review.

The aim of this controlled randomised clinical trial was to investigate the effects of regular low to moderate intensity physical activity on plasminogen activator inhibitor-1 (PAI-1) activity during three years while taking into account the 4G/5G polymorphism in the promoter of the PAI-1 gene. Male subjects (age 52-62 years, n = 132) were randomised into an exercise or a (non-intervention) reference group. Aerobic threshold increased by 8.8% (p = 0.025) in the exercise group, and decreased by 1.1% in the non-intervention group, while PAI-1 activity did not change significantly in either study group. However, homozygotes for the 4G allele in the exercise group showed a 36% reduction in PAI-1 (p = 0.025). In conclusion, although regular moderate physical activity did not decrease PAI-I activity in the whole group, regular exercise may be effective for controlling elevated PAI-1 level in subjects homozygous for the 4G allele.

There is a growing body of evidence supporting the roles of small, dense LDL and plasma triglyceride (TG), both features of the atherogenic lipoprotein phenotype, as risk factors for coronary heart disease. Although family studies and twin studies have demonstrated genetic influences on these risk factors, the specific genes involved remain to be determined definitively. The purpose of this study was to investigate genetic linkage between LDL size, TG, and related atherogenic lipoproteins and candidate genes known to be involved in lipid metabolism. The linkage analysis was based on a sample of 126 DZ women twin pairs, which avoids the potentially confounding effects of both age and gender, by use of a quantitative sib-pair linkage-analysis approach. Eight candidate genes were examined, including those for microsomal TG-transfer protein (MTP), hepatic lipase, hormone-sensitive lipase, apolipoprotein (apo) B, apo CIII, apo E, insulin receptor, and LDL receptor. The analysis suggested genetic linkage between markers for the apo B gene and LDL size, plasma levels of TG, of HDL cholesterol, and of apo B, all features of the atherogenic lipoprotein phenotype. Furthermore, evidence for linkage was maintained when the analysis was limited to women with a major LDL-subclass diameter >255 A, indicating that the apo B gene may influence LDL heterogeneity in the intermediate-to-large size range. In addition, linkage was found between the MTP gene and TG, among all the women. These findings add to the growing evidence for genetic influences on the atherogenic lipoprotein phenotype and its role in genetic susceptibility to atherosclerosis.

Abetalipoproteinaemia is a rare autosomal-recessive disorder caused by a defect in the large subunit of the microsomal triglyceride transfer protein (MTP) which is required for the assembly and secretion of apolipoprotein B-containing lipoproteins. We report here the use of a polymorphic CA dinucleotide repeat in intron 10, MTPIVS10, of the large subunit of the human MTP protein in the analysis of a pregnancy in a consanguineous family, in which abetalipoproteinaemia was suspected, although prenatal diagnosis was subsequently refused. The mutation in the family has been identified as a novel four-nucleotide insertion/duplication of exon 17 between nucleotides 2349 and 2350 of the cDNA sequence of the MTP gene. However, the marker, MTPIVS10, can be used as an alternative to the time-consuming mutation detection techniques.

In 1474 healthy Caucasoid men aged 45-65 y, insulin-like growth factor II (IGF2) Apal AA homozygotes showed a mean body weight 4 kg lower than Apal GG homozygotes (77.6 +/- 10.9 kg vs 81.6 +/- 11.5 kg, P = 0.003) with heterozygotes (GA) intermediate (80.1 +/- 11.9 kg). The mean serum IGF-II concentration in 44 Apal AA individuals was significantly higher than in 48 Apal GG individuals (683.3 +/- 146.9 ng/ml vs 614.0 +/- 124.0 ng/ml, P = 0.01). An INS Pstl polymorphism showed no association with weight and it was also found to be in minimal linkage disequilibrium with the IGF2 Apal site (coefficient 0.016). The IGF2 Apal AA genotype is therefore associated with lower mean body weight but higher serum IGF-II concentrations than the GG genotype. Apal GG homozygotes incur a 1.67-fold risk of pathological Body Mass index (BMI) (> 30 kg/m2) compared with AA homozygotes.

Three hundred and sixteen healthy Icelandic men and women were examined for the effect of the cholesteryl ester transfer protein (CETP) I405V polymorphism on plasma triglycerides, HDL cholesterol (HDLC) and apoAI concentration. Genotyping was performed using an allele specific oligomelting assay and the frequency of the V allele was 0.31 (95 CI for men 0.23-0.33 and for women 0.29-0.39). In women no significant difference was associated with the V405 genotype for any plasma lipid trait. However, men who were homozygous for the V405 allele had 9% higher apoAI and 14% higher HDLC levels (p < 0.05) than those homozygous for the common I405 allele. The genotype effect was seen only in the non-smokers (p = 0.07 and < 0.05, respectively), and in those consuming alcohol (p < 0.05 for both). Analysis of interaction between the environmental, life-style factors and genotype in men for the traits of HDLC and apoAI showed statistically significant interaction of the genotype only with alcohol consumption. The non-smoking men who reported alcohol consumption and who were homozygous for the CETP V405 allele had 16% higher plasma apoAI concentration than those who carried the I405 allele, and up to 20% higher apoAI level than smokers. On the basis of prospective studies carried out on the Icelandic population, non-smoking, alcohol-consuming men who are homozygous for the V405 allele could have from 32% to 40% lower risk of having a heart attack.

The beta-fibrinogen G/A-455 polymorphism genotype along with plasma fibrinogen levels was determined in 482 healthy middle-aged men, of whom 231 were smokers. Smokers had the highest plasma fibrinogen levels (2.92 g/l), ex-smokers the next (2.73 g/l), and never-smokers the lowest levels (2.66 g/l, P < 0.001). Those with one or two A-455 alleles had significantly higher plasma fibrinogen levels in never-smokers and ex-smokers (8.2% and 9.0%, respectively, P < 0.05), and the effect was larger in younger men (45 < 55 years, 11.6%, P = 0.002) than older men (> 65 years, 4.5%, NS), and was not significant in smokers (2.4%, P > 0.05). Allele frequencies were calculated and compared across age groups and between smokers and non-smokers. The difference in frequency of the A-455 allele between smokers and non-smokers varied significantly with age (P < 0.01), with the frequency of the A-455 allele being significantly lower in smokers than in non-smokers in subjects aged > 65 years (P < 0.05), but not in younger men. This demonstrates an interaction between age, smoking and allele frequency of the G/A-455 polymorphism in this population-based sample.

Association of the insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene with coronary artery disease with or without myocardial infarction (MI) was examined in a group of Chinese and Indian men in Singapore. The sample comprised an angiographically confirmed patient group of 276 Chinese and 102 Indians, of which 155 Chinese and 72 Indians had MI, and a matched healthy control group (147 Chinese and 166 Indians). The frequency of the D allele in the Chinese was 0.39 in those with CAD with MI, 0.43 in those with CAD but without MI, and 0.41 in the control group. The frequency of the D allele in Indians was 0.44 in CAD with or without MI, and 0.45 in the control group. There was no significant association of the ACE gene with CAD or MI in the Chinese or Indians, either in the entire sample or in different risk groups. The frequency of the D allele was significantly lower in the healthy Chinese and Indians than that reported in Caucasians. The association of the ACE gene with MI or CAD observed in other studies could not be confirmed in the present series of Chinese and Indians in Singapore.

The single strand conformational polymorphism (SSCP) method was used to screen patients with familial hypercholesterolemia (FH) for mutations in the 3' part of exon 4 of the low density lipoprotein receptor (LDLR) gene. In 311 patients, six previously described mutations were identified in 29 apparently unrelated individuals (9.3%); three of the mutations are null alleles producing no protein, while the other three lead to production of a defective protein. In the patients where no mutation was detected mean total plasma cholesterol levels were 9.4 mmol/l, compared to 11.3 mmol/l in those individuals with a mutation creating a null allele, and 11.2 mmol/l in those with a mutation that resulted in the production of a defective protein (p < 0.001). These data reinforce observations of others that specific mutations in the LDL receptor gene are associated with different effects on plasma lipids, and indicate that the phenotype is influenced by the genotype.

We have investigated the interrelationships of plasma PAI-1 activity, the PAI-1 4G/5G polymorphism and risk of myocardial infarction (MI) in the ECTIM study, a case-control study of MI based in Belfast, Lille, Strasbourg and Toulouse. Mean PAI-1 levels in cases were similar across all centres but in controls, levels in the French centres were significantly higher. Only in Belfast were PAIl1 levels higher in cases (11.7 AU/ml) than controls (10.5 AU/ml). The PAI-1 4G allele frequency was similar in cases and controls (0.55 and 0.54). In all groups, 4G homozygotes had the highest mean plasma PAI-1 level (4G4G vs 5G5G; cases overall: 14.2 vs 12.1AU/ml; controls overall: 15.0 vs 12.6AU/ml), with the heterozygotes generally intermediate. The data from Belfast are consistent with the literature implicating PAI-1 level as an MI risk factor. In ECTIM, the PAI-1 4G/5G polymorphism is not a genetic risk factor for MI but is associated with PAI-1 activity. Thus homozygosity for the 4G allele may predispose to elevated PAI-1 and impaired fibrinolysis, perhaps requiring interaction with other genetic or environmental factors to influence MI risk.

Objective: To determine in healthy men: (1) whether an extended genotype of the fibrinogen gene cluster using the G/A-455 and the BclI polymorphism of the beta fibrinogen gene and TaqI of the alpha fibrinogen gene explains a significantly larger proportion of variance in plasma fibrinogen levels in either smokers or non-smokers than a single polymorphism (G/A-455); (2) whether there is any evidence for genotype-smoking interaction in the determination of fibrinogen levels., Design: A cross sectional study of healthy, white men recruited at the screening for entry into the Thrombosis Prevention Trial., Setting: The subjects were drawn from four general practices in the United Kingdom., Results: The frequency of the rare alleles in the sample was 0.19 for the G/A-455 polymorphism (A-455), 0.15 for BclI (B+), and 0.27 for TaqI (T+) alleles. BMI and age made significant contributions to the variance in plasma fibrinogen levels only in non-smokers of 5.4% and 2.3% respectively and, in the group as a whole, smoking accounted for 6.6% of the variance. In the non-smokers, of the individual polymorphisms only the G/A-455 showed a significant association with plasma fibrinogen levels (p = 0.03). The mean fibrinogen in non-smokers homozygous for the G-455 allele was 2.54 g/l v 2.85 g/l in those homozygous for the A-455 allele, with the polymorphism explaining 3.6% of the variance in plasma fibrinogen levels in this group. On investigation of the association of fibrinogen levels with combined genotypes, the most significant effect was seen with the combination of the G/A-455 and TaqI polymorphisms, with those with no "fibrinogen raising alleles" having a mean fibrinogen of 2.57 g/l v 3.10 g/l for those with four "fibrinogen raising alleles" (p = 0.0036), and this combination explained 8.9% of the variance in plasma fibrinogen levels (p < 0.005). Although the contribution to variance was greater with the G/A-455/TaqI combination than the G/A-455 polymorphism alone (8.9% v 3.6%), this did not reach significance (p = 0.09). By contrast, in the smoking group, the only significant contribution to the difference in plasma fibrinogen levels was the G/A-455 genotype alone which, after adjustment for BMI and age, contributed 3.8% to the variance (p < 0.05). No interaction was shown between smoking and genotype., Conclusion: These data suggest that in non-smokers an extended genotype using the G/A-455 beta fibrinogen gene polymorphism and the TaqI alpha fibrinogen gene polymorphism explains a larger proportion of the variance in plasma fibrinogen levels than any one polymorphism alone, but that smoking has an overriding effect so that other variables such as age and BMI make little additional contribution.

Objective: To obtain rapid, high throughput genotyping of the angiotensin converting enzyme (ACE) gene intron 16 insertion/deletion polymorphism., Methods: DNA was obtained from whole blood samples by a simple liquid phase methanol extraction procedure. The ACE gene was amplified by the polymerase chain reaction (PCR) using two oligonucleotide primers (ACE1 and ACE3) outside the insertion sequence and one primer (ACE2) inside the sequence. Microtitre array diagonal gel electrophoresis (MADGE) was used to determine genotypes., Results: 84 and 65 bp PCR products indicating the presence of deletion (D) and insertion (I) alleles, respectively, were clearly resolved after electrophoresis on a 7.5% polyacrylamide gel. Up to 480 DNA samples on 5 gels could be genotyped in a single electrophoresis run, or up to 1000 samples in a working day., Conclusions: A simplified DNA extraction protocol coupled to the high throughput capability of the MADGE electrophoretic system for genotyping enables analysis of large populations for association studies of ACE genotype with cardiac disease events.

The effect associated with the substitution of adenine (A) for guanidine (G) in the promoter region of the apolipoprotein AI gene (-75 bp) with plasma apo AI and high-density lipoprotein (HDL) levels was investigated in the European Atherosclerosis Research Study (EARS). This is a study of healthy offspring (cases) of fathers who had suffered premature myocardial infarction (MI) before age 55 years (n = 565) and age- and sex-matched controls (n = 1,078) from 12 European countries, divided into 5 regions based on geography and language. The frequency of the polymorphism was not significantly different among the regions and the relative frequency of the rare A allele was similar in cases and controls (0.159 vs. 0.142) combining data from all regions. Individuals with one or more A allele had significantly higher plasma apo AI levels (P < 0.05) than individuals homozygous for the G allele. This effect was consistent in all regions. The data were analyzed separately in males and females. In females, those with one or more A allele had significantly higher apo AI levels (P = 0.05) than individuals homozygous for the G allele, and this raising effect of the A allele was greater in cases than controls for both apo AI (5.23% vs. 1.56%) and HDL (4.48% vs. 1.89%). In males, the A allele was associated with higher levels of apo AI and HDL, but the effect was much smaller and the differences did not reach statistical significance. In the females, where the effect of the A allele was strongest, the effect on apo AI associated with genotype was evident in non-smokers, and individuals with one or two A alleles had 3.6% higher apo AI and 3.14% higher HDL levels than individuals homozygous for the G allele. However, in the female smokers the raising effect of the A allele was greatly reduced (0.56%). Thus genetic variation in the promoter region of the apo AI gene is associated with differences in apo AI and HDL levels in healthy individuals throughout Europe, but the effect is modulated by gender, environmental factors such as smoking, and a family history of MI.

Three DNA polymorphisms (Ins/Del, XbaI and EcoRI) of the apolipoprotein B gene and their influence on body-mass index, serum lipids and apolipoprotein levels were studied in 181 healthy Indians of both sexes (121 males and 60 females), aged between 17 and 71 years. The frequencies of X+ (XbaI) and Del (Ins/Del) of the signal peptide region in Indians were found to be significantly lower (0.17 and 0.11, respectively) compared to the frequencies in Caucasians (0.50 and 0.32, respectively) (P < 0.025). The frequency of E- (EcoRI) was similar to that in Caucasians (0.10 vs 0.15). A highly significant linkage disequilibrium was observed between the XbaI site and Ins/Del polymorphism of the apo B gene in this sample (X2 = 31.9, P < 0.001). The simultaneous presence of Del and X+ allele was significantly associated with higher body mass index (X2 = 11.43, P < 0.005), serum total cholesterol (X2 = 5.11; P < 0.025) and triglyceride (X2 = 6.42; P < 0.025) levels. Mean values of adjusted BMI and serum triglyceride levels were found to be 29.0 +/- 1.92 vs 23.7 +/- 0.67 (P < 0.025) and 278.0 +/- 60.78 vs 140.4 +/- 15.43 mg/dl (P < 0.05), respectively, in subjects with Del and X+ compared to others. The multiple regression tests showed that 3.3 and 5.8% of the total variability of BMI is explained by Ins/Del and XbaI polymorphism, respectively, in this sample (P = 0.06 and 0.02), while 3.8% of serum triglyceride levels was explained by Ins/Del polymorphism of the apo B gene (P = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

Informative polymorphisms have been very difficult to detect in the elastin gene, and this has hampered the analysis of heritable connective tissue disorders, notably the Marfan syndrome. We have recently detected a dinucleotide repeat polymorphism in intron 17 of the human elastin gene consisting of 8 alleles with sizes between 161 and 175 bp. Analysis of 540 chromosomes from unrelated Caucasian individuals revealed a bimodal frequency distribution typical of (dC-dA)n.(dG-dT)n repeat polymorphisms, with allele frequencies ranging from 0.004 (161 bp) to 0.574 (163 bp). As the elastin gene was originally assigned to chromosome 2q31-ter and because more recent data have suggested an assignment to 7q11.1-21.1, we have genotyped a sub-set of the CEPH pedigrees and carried out pairwise linkage analysis with markers on chromosomes 7 and 2. Lod-scores of between +3.70 and +13.69 were obtained with markers spanning 7p13-q22.1, whilst negative lod-scores were observed with the chromosome 2 markers. Analysis of type II human ovarian teratomas placed the elastin gene within 11 cM of the centromere on chromosome 7. Additionally, we detected the dinucleotide repeat in human-rodent cell hybrids containing chromosome 7, but not those containing chromosome 2. These data confirm the assignment of elastin to chromosome 7 and provide a new, highly informative marker for the analysis of heritable disorders of connective tissue for which elastin is a candidate gene.

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.

It is well established that elevated plasma levels of low-density lipoprotein (LDL) particles are a risk factor for ischaemic heart disease with the distribution in LDL levels seen in the general population being the result of interaction between environmental factors, such as dietary fat intake, and genetic variation that is present in different individuals. One of the candidate genes where such variation is likely to occur, is the gene coding for apolipoprotein B (apo B). Many studies have reported an association between a common polymorphism of the apo B gene, detected using the restriction enzyme XbaI, and differences in plasma lipid levels, explaining 3-5% of the variance in LDL-cholesterol levels in samples representative of the healthy population. It has been proposed that the mechanism of this association is due to functional amino acid changes within the apo B protein, that affect LDL catabolism by altering binding affinity to the LDL-receptor. Several amino acid substitutions in the apo B gene have now been characterized, and these form the basis of the different epitopes that create the Ag marker system. Previous studies have reported that the Ag(x) epitope is associated with lower plasma lipid levels, and until recently the molecular basis for this association has been unclear. We have determined that the Ag(x) epitope is associated with both a Pro-Leu2712, and Asn-Ser4311 substitution, with the Leu-Ser allele being associated with significantly lower levels of plasma lipids in a sample of healthy individuals from Sweden.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiological studies have demonstrated that the risk of coronary artery disease (CAD) is increased in smokers but reduced in individuals with elevated plasma levels of high density lipoprotein cholesterol (HDL-C) and apolipoprotein (apo) A-I. In a sample of 315 men and women from Iceland, the mean levels of HDL-C and apo A-I in smokers were, respectively, 12% and 6% lower in men and 7% and 6% lower in women compared with nonsmokers. In the men who were nonsmokers, a guanine (G) to adenine (A) (G/A) substitution 75 bp upstream from the start of transcription of the apo A-I gene was associated with elevated levels of HDL-C and apo A-I, with those carrying the A allele having levels of HDL-C and apo A-I roughly 10% higher than those with only the G allele. This genotype effect was abolished in the men who smoked and absent in the sample of women. Based on the reported protective effect associated with elevated levels of apo A-I and HDL-C, men carrying the A allele would have roughly a 20% lower relative risk of CAD compared with those without the A allele, but only if they remained nonsmokers. This gene polymorphism would therefore be an important factor in determining those men who would most benefit from avoidance or cessation of smoking. Determining the mechanism of such interaction between genotype and environment will be important in understanding the etiology of CAD.

We have examined the associations between levels of plasma apolipoprotein (apo) AI, apo CIII and apo AIV and genetic variation in the apo AI-CIII-AIV gene cluster in 162 boys and young men from Belgium aged from 7 to 23 years. Genotypes were determined for six restriction enzymes XmnI, PstI, SstI, PvuIIA-CIII, PvuIIB-AIV and XbaI, and for the G to A substitution at -75 bp in the 5' region of the apo AI gene. The polymorphism most strongly associated with apo AI levels was the G to A substitution (P = 0.025, R2 x 100 = 3.6%) confirming previous observations. The polymorphism most strongly associated with apo CIII levels was that of PvuIIA-CIII (P = 0.023, R2 x 100 = 2.9%) in the apo CIII gene. This novel association must be interpreted with caution until it has been confirmed in an independent sample. The polymorphism associated with the largest effect on apo AIV levels was that detected with XbaI in the apo AIV gene, but this association was not statistically significant. Previously reported associations between the SstI polymorphism and triglyceride levels, and between the PstI polymorphism and apo AI levels, were weakly detected in the present sample. Our results show that variation associated with some of the polymorphisms in the apo AI-CIII-AIV cluster makes a small, but statistically significant, contribution to the determination of apo AI and apo CIII levels in this sample of young men and boys. These observations may, in part, explain reported associations between polymorphisms in this gene cluster, differences in plasma lipid and lipoprotein levels, and prevalence of coronary artery disease.

The genetic relationship amongst apolipoprotein B (apo B) gene polymorphisms (signal peptide insertion/deletion (Leu-Ala-Leu-16/-14), XbaI (Thr2488), EcoRI (Glu4154----Lys), Asn4311----Ser, 3'-VNTR) has been investigated in samples of South Asian (Indian) and Swedish individuals. The frequency distribution of alleles at all these sites was found to be significantly different between the South Asian and the Swedish samples (deletion allele: 0.20 v. 0.31, X+ (presence of XbaI cutting site): 0.29 v. 0.55, R- (absence of EcoRI cutting site): 0.11 v. 0.19, Ser4311: 0.45 v. 0.19). The distribution of allele frequencies at the VNTR site was bimodal in both populations. However, in South Asians, the most common allele was a 35 repeat unit allele, whilst in the Swedish sample, and in all other reports from Caucasian samples the 37 repeat unit allele was the most frequent (South Asian v. Swedish: 35 allele: 0.36 v. 0.19, 37 allele: 0.25 v. 0.48). Furthermore, four new alleles at the apo B gene 3'-VNTR site (15, 17, 32, 38 repeat unit alleles) were observed in South Asians, of which two (15 and 17 repeat unit alleles) were well outside the bimodal distribution. In both samples, strong linkage disequilibrium and allelic association were detected between alleles at the 3'-VNTR and each of the other sites, and also between the ins/del and XbaI sites and between the XbaI and Asn4311----Ser sites. The same five common haplotypes as defined by ins/del, XbaI, EcoRI and Asn4311----Ser were found to be present in both samples comprising 97 and 99% of the haplotypes observed in the South Asian and Swedish samples respectively. Detailed analysis revealed the predominant occurrence of certain 3'-VNTR alleles on specific haplotypes and demonstrates the usefulness of the VNTR site for haplotyping and representative association studies. A model for the evolutionary relationship of the major haplotypes including the 3'-VNTR site is presented. These findings support a mechanism of replication slippage as a major factor for the generation of new alleles at the apo B 3'-VNTR locus rather than unequal crossing over.

The frequency of restriction fragment length polymorphisms (RFLPs) of the apolipoprotein B (apo B) gene, detected by XbaI and EcoRI, and their influence on serum lipids and apolipoproteins were studied in healthy Chinese of both sexes in Singapore. A total of 221 subjects (150 males, 71 females) were investigated for the XbaI and 159 subjects for the EcoRI polymorphisms, while serum lipids and apolipoprotein levels were available for 196 subjects. The frequency of the X2 allele was found to be significantly lower in the Chinese than that reported in Caucasians from the United Kingdom (0.09 vs. 0.51, P less than 0.001). The haplotype frequencies were also significantly different between the Chinese and Caucasians with a higher frequency of X1R1 in the former compared to the latter (0.85 vs. 0.34, P less than 0.0001). The distribution of RFLP genotypes at both of the restriction sites was at Hardy-Weinberg equilibrium in all groups. The influence of the apo B RFLPs on serum lipids and apolipoprotein levels (apo AI, AII, and B) was studied by both residual and multiple regression analyses considering age, sex, body mass index (BMI), and genotypes as independent variables in all possible combinations. No association was observed between the apo B genotypes and serum lipids or apolipoprotein levels except for high density lipoprotein cholesterol (HDLC), apo AI and AII, with the X2 being associated with significantly lower levels of HDLC as well as apo AI and AII, the effect being stronger in males. These data raise the possibility that the mechanism of reported association between apo B polymorphism and coronary artery disease may be through effects on HDLC.

Three polymorphic sites of the apolipoprotein B gene - the insertion/deletion signal peptide, XbaI and EcoRI sites - were examined in a sample of 107 healthy men and in 46 men with evidence of coronary heart disease selected from a large population survey of South Asians aged 40-69 in London, U.K. There were no significant differences in allele frequencies between cases and controls. Frequencies of the ins (insertion) and X- (absence of XbaI cutting site) alleles were higher in South Asians than in Europeans studied previously (South Asians versus Europeans ins: 0.80 vs. 0.68, P less than 0.025; X-: 0.71 vs. 0.47-0.56, P less than 0.001). The del allele was associated with higher levels of total cholesterol (P less than 0.05) and the X+ allele with lower levels of HDL cholesterol (P less than 0.05), and thus both polymorphisms were associated with differences in the ratio of HDL cholesterol to total cholesterol (ins/del, P less than 0.01; XbaI, P less than 0.001). Mean waist-hip girth ratio was lower in the 10 men homozygous for the X+ allele than in the 42 men with X-/X+ and 55 men with X-/X- genotypes; the means (+/- SEM) were 0.92 +/- 0.02, 0.97 +/- 0.01 and 0.96 +/- 0.01 respectively (P = 0.03). These data suggest that genetic variation in linkage disequilibrium with the XbaI and ins/del polymorphisms of the apo B gene contributes to the determination of total cholesterol and HDL cholesterol levels and possibly to obesity in South Asians.

We investigated the association between fibrinogen levels and a HaeIII restriction fragment length polymorphism located at -453 bp from the start of transcription of the beta fibrinogen gene. 292 healthy men aged 45 to 69 years, recruited from general practices throughout Britain, were studied. None had a history of ischaemic heart disease. 41.1% (120) were smokers and fibrinogen levels were higher in this group. The frequency of the non-cutting allele (designated H2) was 0.19 and was the same in smokers and non-smokers. The H2 allele was associated with elevated levels of fibrinogen in both smokers and non-smokers and the effect of genotype was similar in both groups. After smoking, HaeIII genotype was the strongest predictor of fibrinogen levels and explained 3.1% of the variance in fibrinogen levels. These results confirm earlier studies that variation at the fibrinogen locus contributes to the between-individual differences in plasma fibrinogen level.

We have examined the association between variation at the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster and within-individual variation in serum cholesterol levels. Annual measurements were available over a period of 5-10 years in a group of 117 male patients with peripheral arterial disease. The overall within-individual coefficient of variation in cholesterol levels over time was 13.9%. For all patients, Restriction Fragment Length Polymorphism (RFLP) genotype at the apo B gene (XbaI and EcoRI) and apo AI-CIII-AIV gene cluster (XmnI, PstI and PvuII-CIII) had previously been determined. At the apo B locus, individuals heterozygous for either the XbaI or EcoRI RFLP showed significantly greater within-individual variability over time compared to individuals of other genotypes. At the apo AI-CIII-AIV gene cluster, individuals homozygous for the common allele of either the PstI or PvuIIA RFLPs showed the greatest within-individual variability over time but there was no difference in this estimate associated with XmnI genotype. Our observations suggest that variation at both the apo B and apo AI-CIII-AIV loci interacts with unidentified environmental factors to determine individual variability in serum cholesterol levels over time.

We have investigated the effect of apolipoprotein (apo) E polymorphism on serum lipid, lipoprotein, and apolipoprotein levels in a sample of 195 children, aged 8-11 years, from Sezze, Central Italy. The relative frequencies of e2, e3, and e4 alleles were 0.062, 0.867, and 0.072, respectively. Variation at the apo E gene locus explained 5.1% of the sample variance in serum total cholesterol levels, 7.6% in low-density lipoprotein (LDL) cholesterol levels, 7.3% in apo B levels, and 14.1% in high-density lipoprotein-apo E (HDL-E) levels. The effect of the e2 allele was to lower levels of total cholesterol, LDL-cholesterol, and apo B and to raise levels of HDL-E, while the effect of the e4 allele was the opposite. Variation at the apo E gene locus was not associated with differences in serum triglyceride, HDL-cholesterol, or apo AI levels. The effects of common apo E polymorphisms and genetic variation associated with the PvuII RFLP of the apo B gene on serum apo B levels were additive, explaining 11.3% of the phenotypic variance in this sample. When the effect of apo E polymorphism on serum lipid traits was estimated in boys and girls separately, variation at the apo E gene locus explained 10.4, 13.3, 13.3, and 13.5% of the phenotypic variance in serum total cholesterol, LDL-cholesterol, apo B, and HDL-E levels, respectively, in boys, while in girls only the effect on HDL-E levels (19.3%) reached statistical significance. This study has demonstrated that genetic variations at the apo E locus contribute to the determination of serum lipid, lipoprotein, and apolipoprotein levels in youths and that the effects are gender specific.

The detection of apolipoprotein E genotypes is of importance both for diagnostic and research purposes. We have previously used the polymerase chain reaction to amplify a specific region of the apolipoprotein E gene which, when used in conjunction with allele specific oligonucleotide probes, permits the detection of the six common apolipoprotein genotypes. In our present report we have modified the above procedure by immobilising the amplified DNA using Southern blotting. This enables the technique to be used in routine laboratories with minimal expenditure and little risk of cross-contamination between samples. Furthermore, it is inherently robust and may be rapidly performed.

An epitope of Apolipoprotein B (ApoB), recognised by a monoclonal antibody BIP-45, is associated with the development of ischaemic heart disease (Duriez et al. 1988). We have examined the genetic relationships between this epitope and three Restriction Fragment Length Polymorphisms (RFLPs) of the gene for ApoB detected with the enzymes EcoRI, PvuII and XbaI in a sample of 53 unrelated individuals from France. There is an association between binding affinity to BIP-45 and the XbaI RFLP; the 8.6 kb XbaI allele (absence of cutting site) being associated with low-affinity binding to BIP-45. In this sample of individuals there is no significant association between serum cholesterol levels and BIP-45 binding affinity, but there is a significant correlation between serum cholesterol levels and XbaI genotype, with individuals of the genotype X1X1 having the highest and those with the genotype X2X2 having the lowest levels of serum cholesterol. This suggests that variation at the ApoB locus may be involved independently in the determination of serum lipid levels and in the development of ischaemic heart disease.

We have determined the frequencies of the alleles at the EcoRI (E), XbaI (X) and PvuII (P) polymorphic restriction sites in the apo B gene in 124 white men with coronary artery disease (CAD) and in 146 white men free from CAD. The frequencies of the E- (restriction site absent) and X- alleles were both significantly higher in normocholesterolaemic men with CAD than in those without CAD, but the frequency of the P+ allele (restriction site present) was similar in the 2 groups. The frequency of the E- allele was significantly higher in CAD men with hypertriglyceridaemia than in normal men without hypertriglyceridaemia. In the normocholesterolaemic men without CAD, the mean serum cholesterol concentration was higher in those with genotype X++ than in those with genotype X--. Mean serum LDL-apo B and LDL-cholesterol concentrations did not differ significantly between men with different XbaI or EcoRI genotypes. Serum apo A-I levels differed significantly between normal men with different XbaI genotypes. Serum HDL-cholesterol levels differed significantly between CAD men with different XbaI genotypes. These results suggest that in white men the E- and X- alleles are in linkage disequilibrium with a nearby allele that is causally related to CAD. It is also possible that the amino acid substitution at position 4154 in apo B, brought about by the nucleotide change responsible for the EcoRI polymorphism, has a direct effect on the atherogenicity of LDL.