Subclinical atherosclerosis and its progression are modulated by PLIN2 through a feed-forward loop between LXR and autophagy.

BACKGROUND: Hyperlipidaemia is a major risk factor for cardiovascular disease, and atherosclerosis is the underlying cause of both myocardial infarction and stroke. We have previously shown that the Pro251 variant of perilipin-2 reduces plasma triglycerides and may therefore be beneficial to reduce atherosclerosis development. OBJECTIVE: We sought to delineate putative beneficial effects of the Pro251 variant of perlipin-2 on subclinical atherosclerosis and the mechanism by which it acts. METHODS: A pan-European cohort of high-risk individuals where carotid intima-media thickness has been assessed was adopted. Human primary monocyte-derived macrophages were prepared from whole blood from individuals recruited by perilipin-2 genotype or from buffy coats from the Karolinska University hospital blood central. RESULTS: The Pro251 variant of perilipin-2 is associated with decreased intima-media thickness at baseline and over 30 months of follow-up. Using human primary monocyte-derived macrophages from carriers of the beneficial Pro251 variant, we show that this variant increases autophagy activity, cholesterol efflux and a controlled inflammatory response. Through extensive mechanistic studies, we demonstrate that increase in autophagy activity is accompanied with an increase in liver-X-receptor (LXR) activity and that LXR and autophagy reciprocally activate each other in a feed-forward loop, regulated by CYP27A1 and 27OH-cholesterol. CONCLUSIONS: For the first time, we show that perilipin-2 affects susceptibility to human atherosclerosis through activation of autophagy and stimulation of cholesterol efflux. We demonstrate that perilipin-2 modulates levels of the LXR ligand 27OH-cholesterol and initiates a feed-forward loop where LXR and autophagy reciprocally activate each other; the mechanism by which perilipin-2 exerts its beneficial effects on subclinical atherosclerosis.

Integrative studies implicate matrix metalloproteinase-12 as a culprit gene for large-artery atherosclerotic stroke.

BACKGROUND: Ischaemic stroke and coronary heart disease are important contributors to the global disease burden and share atherosclerosis as the main underlying cause. Recent evidence from a genome-wide association study (GWAS) suggested that single nucleotide polymorphisms (SNP) near the MMP12 gene at chromosome 11q22.3 were associated with large-vessel ischaemic stroke. Here, we evaluated and extended these results by examining the relationship between MMP12 and atherosclerosis in clinical and experimental studies. METHODS AND RESULTS: Plasma concentrations of MMP12 were measured at baseline in 3394 subjects with high-risk for cardiovascular disease (CVD) using the Olink ProSeek CVD I array. The plasma MMP12 concentration showed association with incident cardiovascular and cerebrovascular events (130 and 67 events, respectively, over 36 months) and carotid intima-media thickness progression (P = 3.6 × 10-5 ). A GWAS of plasma MMP12 concentrations revealed that SNPs rs499459, rs613084 and rs1892971 at chr11q22.3 were independently associated with plasma MMP12 (P < 5 × 10-8 ). The lead SNPs showed associations with mRNA levels of MMP12 and adjacent MMPs in atherosclerotic plaques. MMP12 transcriptomic and proteomic levels were strongly significantly increased in carotid plaques compared with control arterial tissue and in plaques from symptomatic versus asymptomatic patients. By combining immunohistochemistry and proximity ligation assay, we demonstrated that MMP12 localizes to CD68 + macrophages and interacts with elastin in plaques. MMP12 silencing in human THP-1-derived macrophages resulted in reduced macrophage migration. CONCLUSIONS: Our study supports the notion that MMP12 is implicated in large-artery atherosclerotic stroke, functionally by enhancing elastin degradation and macrophage invasion in plaques.

Telomere shortening over 6 years is associated with increased subclinical carotid vascular damage and worse cardiovascular prognosis in the general population.

INTRODUCTION: Leucocyte telomere length (LTL) is an important determinant of telomere function and cellular replicative capacity. The aim of the present study was to examine prospectively the associations between telomere shortening (TS) and both the progression of atherosclerosis and the incidence of cardiovascular events (CVEs). MATERIALS AND METHODS: Leucocyte telomere length was measured by quantitative polymerase chain reaction to determine the ratio of telomere length to single-copy gene (T/S) in 768 subjects (462 female and 306 male) enrolled in a large general population survey [the Progressione della Lesione Intimale Carotidea (PLIC study)]. Common carotid artery intima-media thickness was determined at baseline and after 6 years of follow-up, and the associations between TS and the progression of atherosclerosis and incidence of CVEs were evaluated. RESULTS: Mean LTL was 1.25 ± 0.92 T/S (median 1.14) at baseline and 0.70 ± 0.37 T/S (median 0.70) after 6 years of follow-up. Median 6-year LTL change was -0.46 T/S [interquartile range (IQR) -0.57 to 1.06], equating to -0.078 T/S [IQR(-0.092 to 0.176)] per year. Of note, telomere lengthening occurred in 30.4% of subjects. After adjustment for classical cardiovascular disease (CVD) risk factors (age, gender, smoking, physical activity, alcohol consumption, systolic blood pressure, glucose levels, lipid profile and therapies), TS was associated with incident subclinical carotid vascular damage [hazard ratio (HR) 5.19, 95% confidence interval (CI) 1.20-22.4, P = 0.028]. Finally, subjects in whom LTL shortened over time showed an increased risk of incident CVE, compared to those in whom LTL lengthened (HR 1.69, CI 1.02-2.78, P = 0.041). CONCLUSION: These data indicate that TS is associated with increased risk of subclinical carotid vascular damage and increased incidence of CVEs beyond CVD risk factors in the general population, whereas LTL lengthening is protective.

Functional analysis of TCF7L2 genetic variants associated with type 2 diabetes.

BACKGROUND AND AIMS: Common non-coding variations within the TCF7L2 locus have a strong influence on type 2 diabetes (T2D) susceptibility through uncharacterised mechanisms. An islet-specific functional polymorphism has been identified, although this does not explain the association between genotype and gene expression in other cell types. This study sought to identify these other functional TCF7L2 variants. METHODS AND RESULTS: Alternative splicing and gene expression from TCF7L2 was examined from peripheral blood mononuclear cells from 100 healthy Caucasians using two T2D-associated SNPs, rs7903146 and rs12255372. Electrophoretic mobility shift assays and luciferase reporter assays were performed with these SNPs and those in strong LD to determine potential SNP functionality. Individuals homozygous for rs7903146 and rs12255372 T2D risk alleles (TT/TT) expressed 2.6-fold greater levels of TCF7L2 mRNA compared to individuals homozygous for the non-risk alleles (CC/GG, p = 0.006), although differentially spliced TCF7L2 transcripts did not differ by T2D risk-associated genotype. From SNPs identified to be in strong LD with the T2D-associated SNPs, rs7903146 and rs12255372, five (rs4132670, rs4506565, rs7903146, rs7901695, rs17747324) demonstrated allele-specific binding in electrophoretic mobility shift assays (EMSA). In luciferase reporter assays, rs4132670 exhibited 1.3-fold higher levels of enhancer activity in the Huh7 cell line (p = 3.8 × 10(-5)) and 2-fold higher levels in a WiDr colon carcinoma cell line (p = 0.008). CONCLUSIONS: These results suggest that rs4132670, located in a region of chromatin accessibility, is a non-tissue-specific candidate functional SNP that has the potential to play a role in TCF7L2 gene expression and T2D risk.

Effects of genetic variation on chromatin structure and the transcriptional machinery: analysis of the IL6 gene locus.

The presence of functional regulatory polymorphism at the interleukin 6 (IL6) locus is uncertain, with many conflicting in vitro findings. To examine the in vivo effect of the three putative functional IL6 promoter variants, -174G>C, -572G>C and -6331T>C, two complementary techniques, allele-specific chromatin immunoprecipitation and allele-specific formaldehyde-assisted isolation of regulatory elements, were carried out using unrelated lymphoblast cell lines of known genotype. There were no allele-specific effects for all three single-nucleotide polymorphisms (SNPs) under basal conditions. Upon IL-1ß stimulation, however, allele-specific effects were seen for the -6331 allele, which showed both increased RNA polymerase II loading (56%, P=0.001) and increased open chromatin (59%, P=0.004) for the T allele, which is in line with previous reports of this SNP and the effects from acute inflammation. These studies highlight the importance of examining chromatin under different environmental conditions when studying the functionality of regulatory polymorphisms.

IRS1 gene variants, dysglycaemic metabolic changes and type-2 diabetes risk.

BACKGROUND AND AIMS: A recent genome-wide association study identified rs2943641C > T, 500 kb from the insulin receptor substrate-1 gene (IRS1), as a type-2 diabetes (T2D) susceptibility locus. We aimed to replicate this association by meta-analysis and examine whether common variants within IRS1, present on the HumanCVD BeadChip, were associated with T2D risk. METHODS AND RESULTS: We genotyped rs2943641 in 2389 prevalent or incident T2D patients and 6494 controls from two prospective and three case studies based in UK and in the European Atherosclerosis Research Study-II (EARSII; n = 714). Thirty-three IRS1 variants had been genotyped in the prospective Whitehall-II study (n = 4752) using the HumanCVD BeadChip. In a fixed-effects meta-analysis of the UK study cohorts rs2943641T allele was associated with 6% lower risk of T2D (p = 0.18), with T-allele carriers having an odds ratio (OR) of 0.89 (95% confidence interval [CI]: 0.80-1.00, p = 0.056) compared to CC subjects. The T-allele was also associated with lower fasting insulin and homeostasis model assessment index of insulin resistance in Whitehall-II and with lower post-load insulin after an oral glucose tolerance test in EARSII (all p < 0.05). None of the IRS1 variants on the chip showed linkage disequilibrium with rs2943641. In silico analysis with follow-up genotyping (total n = 9313) identified that the rare allele of the IRS1 promoter variant rs6725556A > G showed association with reduced T2D risk (OR per G-allele: 0.82, 95%CI: 0.69-0.96, p = 0.015). CONCLUSIONS: We confirm the association of rs2943641T with T2D protection. There is a possible independent effect on risk of a putative IRS1 promoter variant.

Association between the rs1050450 glutathione peroxidase-1 (C > T) gene variant and peripheral neuropathy in two independent samples of subjects with diabetes mellitus.

Glutathione peroxidase-1 (GPx-1) is an endogenous anti-oxidant enzyme. The T allele of the GPx-1 rs1050450 (C > T) gene variant is associated with reduced enzyme activity. Our aim was to examine the association between this gene variant and peripheral neuropathy in two cross-sectional samples of subjects with diabetes: (i) 773 Caucasian subjects were genotyped from the UCL Diabetes and Cardiovascular disease Study (UDACS) and (ii) 382 Caucasian subjects from the Ealing Diabetes Study (EDS). Peripheral neuropathy status (and oxidised-LDL [Ox-LDL:LDL] and plasma Total Ant-ioxidant Status [TAOS] in UDACS), were analysed in relation to genotype. We observed that: (i) In UDACS, the odds ratio (OR) for peripheral neuropathy in the T allele carriers compared to the CC genotype was 1.61 [1.10-2.28], p = 0.01. This remained significant after adjustment for other risk factors. Ox-LDL:LDL ratio was significantly elevated in T allele carriers (CC vs. CT/TT: 16.3 ± 2.4 v 18.0 ± 2.9 U/mmol LDL, p = 0.02). (ii) In EDS, the OR for peripheral neuropathy in the T allele carriers compared to the CC genotype was 1.95 [1.11-3.42], p = 0.02. This remained significant after adjustment for other risk factors. In conclusion, we observed a significant association between the T allele and peripheral neuropathy and LDL oxidation. This is the first paper to examine the rs1050450 variant in two samples of Caucasian subjects with diabetes. Prospective analysis of the gene variant is required in diabetic and healthy cohorts with measured plasma markers of oxidative stress to investigate the described association further.

Variants of ADRA2A are associated with fasting glucose, blood pressure, body mass index and type 2 diabetes risk: meta-analysis of four prospective studies.

AIMS/HYPOTHESIS: We quantified the effect of ADRA2A (encoding α-2 adrenergic receptor) variants on metabolic traits and type 2 diabetes risk, as reported in four studies. METHODS: Genotype data for ADRA2A single nucleotide polymorphisms (SNPs) rs553668 and rs10885122 were analysed in >17,000 individuals (1,307 type 2 diabetes cases) with regard to metabolic traits and type 2 diabetes risk. Two studies (n = 9,437), genotyped using the Human Cardiovascular Disease BeadChip, provided 12 additional ADRA2A SNPs. RESULTS: Rs553668 was associated with per allele effects on fasting glucose (0.03 mmol/l, p = 0.016) and type 2 diabetes risk (OR 1.17, 95% CI 1.04-1.31; p = 0.01). No significant association was observed with rs10885122. Of the 12 SNPs, several showed associations with metabolic traits. Overall, after variable selection, rs553668 was associated with type 2 diabetes risk (OR 1.38, 95% CI 1.09-1.73; p = 0.007). rs553668 (per allele difference 0.036 mmol/l, 95% CI 0.008-0.065) and rs17186196 (per allele difference 0.066 mmol/l, 95% CI 0.017-0.115) were independently associated with fasting glucose, and rs17186196 with fasting insulin and HOMA of insulin resistance (4.3%, 95% CI 0.6-8.1 and 4.9%, 95% CI 1.0-9.0, respectively, per allele). Per-allele effects of rs491589 on systolic and diastolic blood pressure were 1.19 mmHg (95% CI 0.43-1.95) and 0.61 mmHg (95% CI 0.11-1.10), respectively, and those of rs36022820 on BMI 0.58 kg/m(2) (95% CI 0.15-1.02). CONCLUSIONS/INTERPRETATION: Multiple ADRA2A SNPs are associated with metabolic traits, blood pressure and type 2 diabetes risk. The α-2 adrenergic receptor should be revisited as a therapeutic target for reduction of the adverse consequences of metabolic trait disorders and type 2 diabetes.

Genetic variation within IL18 is associated with insulin levels, insulin resistance and postprandial measures.

BACKGROUND AND AIMS: IL-18 expression is up-regulated in atherosclerotic plaques, and higher levels are seen in obese and Type 2 Diabetic individuals. More recently, a possible role for IL-18 in glucose and energy homeostasis has been suggested. METHODS AND RESULTS: We investigated variation within the IL18 gene and its association with measures of obesity and the metabolic syndrome. Five IL18 tagging single nucleotide polymorphisms (rs1946519, rs2043055, rs549908, rs360729, rs3882891) were selected and genotyped in the Gene-Diet Attica Investigation on childhood obesity (GENDAI) (age range 10-14 yrs); in young European men in the second European Atherosclerosis Research offspring Study (EARSII), an offspring study (age range 18-28 yrs) and in a group of healthy women from the Greek Obese Women study (GrOW) (age range 18-74 yrs). Six common haplotypes were observed. In GrOW, Hap6 (Frequency-2.6%) was associated with higher insulin levels (p<0.0001), estimates of HOMA(-Insulin Resistance) (p<0.0001) and HOMA(-ß-cell) (p<0.0001) compared to the common haplotype Hap1 (Frequency-33.2%). In EARSII, rs2043055 was associated with peak and area under the curve triglycerides (p=0.001 and p=0.002, respectively) after an oral fat tolerance test in 'cases' but not 'controls'. None of the haplotypes were associated with measures of body fatness in any of the studies. CONCLUSION: Association of IL18 variation with insulin levels and estimates of insulin resistance were only observed in our adult study, suggesting that the effects of IL-18 are only associated with increasing age. Taken together with the association of IL18 variants with post-prandial measures, this provides support for IL-18 as a metabolic factor.

Mutation detection rate and spectrum in familial hypercholesterolaemia patients in the UK pilot cascade project.

Cascade testing using DNA-mutation information is now recommended in the UK for patients with familial hypercholesterolaemia (FH). We compared the detection rate and mutation spectrum in FH patients with a clinical diagnosis of definite (DFH) and possible (PFH) FH. Six hundred and thirty-five probands from six UK centres were tested for 18 low-density lipoprotein receptor gene (LDLR) mutations, APOB p.Arg3527Gln and PCSK9 p.Asp374Tyr using a commercial amplification refractory mutation system (ARMS) kit. Samples with no mutation detected were screened in all exons by single strand conformation polymorphism analysis (SSCP)/denaturing high performance liquid chromatography electrophoresis (dHPLC)/direct-sequencing, followed by multiplex ligation-dependent probe amplification (MLPA) to detect deletions and duplications in LDLR.The detection rate was significantly higher in the 190 DFH patients compared to the 394 PFH patients (56.3% and 28.4%, p > 0.00001). Fifty-one patients had inadequate information to determine PFH/DFH status, and in this group the detection rate was similar to the PFH group (25.5%, p = 0.63 vs PFH). Overall, 232 patients had detected mutations (107 different; 6.9% not previously reported). The ARMS kit detected 100 (44%) and the MLPA kit 11 (4.7%). Twenty-eight (12%) of the patients had the APOB p.Arg3527Gln and four (1.7%) had the PCSK9 p.Asp374Tyr mutation. Of the 296 relatives tested from 100 families, a mutation was identified in 56.1%. In 31 patients of Indian/Asian origin 10 mutations (two previously unreported) were identified. The utility of the ARMS kit was confirmed, but sequencing is still required in a comprehensive diagnostic service for FH. Even in subjects with a low clinical suspicion of FH, and in those of Indian origin, mutation testing has an acceptable detection rate.

APOE, CETP and LPL genes show strong association with lipid levels in Greek children.

BACKGROUND AND AIMS: Studies have consistently demonstrated that variants in a number of candidate genes are significant determinants of lipid levels in adults. However, few studies have investigated the impact of these variants in children. Therefore, in the present investigation we examined the influence of ten common variants in the genes for lipoprotein lipase (LPL-S447X), cholesterol ester transfer protein (CETP-Taq1B) apolipoprotein (APO) E (epsilon2, epsilon3, epsilon4), APOA5 (-1131C>T and S19W), APOA4 (S347T) and APOC3 (-482C>T; 1100C>T and 3238G>C) on lipoprotein levels children from the Gene-Diet Attica Investigation on childhood obesity (GENDAI). METHODS AND RESULTS: The ten variants selected were genotyped in 882 Greek children, mean age: 11.2+/-0.7 years (418 females and 464 males). Genotypes were assessed using TaqMan technology. Significantly higher total cholesterol (TC) (p=0.0001) and low-density lipoprotein cholesterol (LDL-C) (p<0.0001) were observed in APOE epsilon4 carriers compared to epsilon3/epsilon3 homozygotes and epsilon2 carriers. The association of APOE genotype with TC and high-density lipoprotein cholesterol (HDL-C) ratio (p=0.0008) was further modulated by body mass index. Carriers of the CETP TaqIB B2 allele had significantly higher HDL-C (p<0.0001) and significantly lower TC: HDL-C ratio (p<0.0001) compared to B1/B1 individuals. No significant associations were observed between APOA4, APOA5 and APOC3 variants and serum lipids. CONCLUSION: This study demonstrates that these common variants are associated with lipid levels in this healthy paediatric cohort, suggesting that even in these young children there may be potential in predicting their lifelong exposure to an adverse lipid profile.

Multiplex ligation-dependent probe amplification analysis to screen for deletions and duplications of the LDLR gene in patients with familial hypercholesterolaemia.

The most common genetic defect in patients with autosomal dominant hypercholesterolaemia is a mutation of the low-density lipoprotein receptor (LDLR) gene. An estimate of the frequency of major rearrangements has been limited by the availability of an effective analytical method and testing of large cohorts. We present data from a cohort of 611 patients referred with suspected heterozygous familial hypercholesterolaemia (FH) from five UK lipid clinics, who were initially screened for point mutations in LDLR and the common APOB and PCSK9 mutations. The 377 cases in whom no mutation was found were then screened for large rearrangements by multiplex ligation-dependent probe amplification (MLPA) analysis. A rearrangement was identified in 19 patients. This represents 7.5% of the total detected mutations of the cohort. Of these, the majority of mutations (12/19) were deletions of more than one exon, two were duplications of more than one exon and five were single exon deletions that need interpreting with care. Five rearrangements (26%) are previously unreported. We conclude that MLPA analysis is a simple and rapid method for detecting large rearrangements and should be included in diagnostic genetic testing for FH.

Association between periodontitis and common variants in the promoter of the interleukin-6 gene.

We recently reported an association between interleukin-6 (IL6) polymorphisms (SNPs) and haplotypes and aggressive periodontitis (AgP). The aim of this study was to investigate this association in a larger cohort of subjects, affected by either aggressive or chronic periodontitis. Five IL6 SNPs were analyzed in 765 subjects (167 generalized aggressive periodontitis, 57 localized aggressive, 310 chronic periodontitis and 231 periodontally healthy). Among Caucasians (n=454) there were moderate associations for -1363T allele (p=0.011) and for -174GG and -1363GG genotypes with diagnosis of periodontitis (respectively, p=0.044, OR=1.6, 95% CI=1.0-2.4, and p=0.017, OR=1.8, 95% CI=1.1-2.8, adjusted for age, gender and smoking). Haplotypes containing the -174G>C, -1363G>T and -1480C>G polymorphisms were associated with diagnosis of periodontitis (p=0.02). Subgroup analysis by disease phenotype showed associations for the localized AgP (LAgP) group and -1480C>G and -6106A>T SNPs (p=0.007 and 0.010, respectively). Among Caucasians the genotypes IL6 -1480 CC and -6106 TT increased the adjusted OR for LAgP (OR=3.09 and 2.27, respectively). This study supports the hypothesis that IL6 polymorphisms and haplotypes are moderately associated with periodontitis, possibly acting through influencing tissue levels of IL6. This association is stronger for LAgP than for other periodontal disease phenotypes.

Effects of six APOA5 variants, identified in patients with severe hypertriglyceridemia, on in vitro lipoprotein lipase activity and receptor binding.

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.

Family tracing to identify patients with familial hypercholesterolaemia: the second audit of the Department of Health Familial Hypercholesterolaemia Cascade Testing Project.

BACKGROUND: Family tracing is a method recognized to find new patients with familial hypercholesterolaemia (FH). We have implemented family tracing led by FH Nurses and have determined acceptability to patients, feasibility and costs. METHODS: Nurses were located at five National Health Service (NHS) Trusts; they identified FH patients and offered them family tracing. Responses and test results were recorded on a database and summarized on a family pedigree. RESULTS: The majority ( approximately 70%) of index cases participated; the proportion was lower when patients had been discharged from the clinics and in metropolitan areas. On average, 34% (range 13-50%) of relatives lived outside the catchment area of the clinics and could not attend the nurse-led FH clinics. Of the previously untested relatives, 76% who lived in the catchment area of the clinic came forward to be tested. One-third of the relatives who came forward for testing were children

Familial hypercholesterolaemia workshop for leveraging point-of-care testing and personalised medicine in association with the Lipid and Atherosclerosis Society of Southern Africa.

Familial hypercholesterolaemia (FH) is a common autosomal dominantly inherited disorder in which impaired clearance of plasma low-density lipoprotein cholesterol causes premature atherosclerotic vascular disease and tendon xanthomata. This workshop aimed to consolidate information on the diagnosis and management of FH in South Africa. The genetic causes include mutations in the LDL receptor, apolipoprotein B100 and proprotein convertase subtilisin/kexin type 9 (PCSK9). Additionally, the concatenation of multiple gene variants can result in polygenic FH. Therapeutic measures include a healthy lifestyle, statins and cholesterol-absorption inhibitors that will achieve control of the dyslipidaemia in the majority of cases. The recently introduced monoclonal antibodies to PCSK9 can improve achievement of target concentration in severe cases. FH is present in all sectors of the South African population but there is sparse documentation in the indigenous African populations. FH should be actively sought, diagnosed and treated with judicious pharmacotherapy and screening of relatives.

Multiplex MassARRAY spectrometry (iPLEX) produces a fast and economical test for 56 familial hypercholesterolaemia-causing mutations.

Familial hypercholesterolaemia (FH) is a common single gene disorder, pre-disposing to cardiovascular disease, which is most commonly caused by mutations in the LDL-receptor (LDLR) gene. About 5% of patients carry the p.R3527Q (previously R3500Q) mutation in the apolipoprotein B (APOB) gene and 2% carry the p.D374Y mutation in the PCSK9 gene, but the lack of high-throughput methods make routine genetic diagnosis difficult. In this study, we developed an iPLEX MassARRAY Spectrometry mutation test to identify 56 mutations (54 in the LDLR gene, 1 in the APOB gene and 1 in the PCSK9 gene). The iPLEX test was verified by analysing 150 DNA samples from FH patients with a previously characterized mutation and 96 no-mutation control samples. Mutations were identified in all 150 FH mutation-positive samples using the iPLEX assay, with 96% directly called by the software. The false-positive rate in no-mutation control samples was 0.015%. The overall specific mutation assay failure rate was 2.1%. In the UK, this gives an average detection rate of 75%.The FH iPLEX test is not only designed for large-scale targeted population screening for FH mutations, such as lipid clinic patients, but can also be used for population screening. The assay can easily be developed further to include additional FH-causing mutations, thus increasing the sensitivity of the diagnostic assay.

Identification of a novel regulatory region in the interleukin-6 gene promoter.

Interleukin-6 (IL6) is an important pleiotropic cytokine that is regulated at the transcriptional level. To date, most work on its regulation has focused on a 1.2kb region 5' from the start of transcription, similar to published reports on other cytokine genes. This report demonstrates for the first time that a cytokine gene can be regulated by cis-acting regions much further upstream than previously examined. Comparative genomic analysis showed that a 120 kb region contains blocks of sequence conservation between human and rodent genomes, and that a 15 kb region proximal to the start of transcription contains 10 highly homologous sequence blocks of between 100 and 250 bp. By means of a reporter gene assay, a novel transcriptionally active region located between -5307 and -5202 bp upstream from the start of transcription was identified. Electrophoretic mobility shift assays showed nuclear protein(s) binding to this region, thus raising the possibility that the regulatory activity shown by the reporter gene constructs may be mediated by these proteins. These results suggest that the regulation of IL6 expression involves a much larger upstream region than previously examined and the control of IL6 transcription is likely to be regulated by a complex mechanism of modular cis-regulatory elements.

Association of plasminogen activator inhibitor (PAI)-1 (SERPINE1) SNPs with myocardial infarction, plasma PAI-1, and metabolic parameters: the HIFMECH study.

OBJECTIVE: The purpose of this study was to investigate the effects of plasminogen activator inhibitor-1 (PAI-1) gene (SERPINE1) single nucleotide polymorphisms (SNPs) on the risk of myocardial infarction (MI), on PAI-1 levels, and factors related to the metabolic syndrome. METHODS AND RESULTS: Eleven SNPs capturing the common genetic variation of the SERPINE1 gene were genotyped in the HIFMECH study. In the 510 male cases and their 543 age-matched controls, a significant gene-smoking interaction was observed. In nonsmokers, the rs7242-G allele was more frequent in cases than in controls (0.486 versus 0.382, P=0.013) whereas the haplotype derived from the rs2227631 (-844A>G)-G and rs2227683-A alleles was approximately 3-fold lower in cases than in controls (0.042 versus 0.115, P=0.006). SERPINE1 haplotypes explained 3.5% (P=0.007) of the variability of PAI-1 levels, which was attributable to the combined effects of 3 SNPs, -844A>G, rs2227666, and rs2227694. The rs6092 (Ala15Thr) and rs7242 SNPs acted additively to explain 4.4% of the variability of plasma insulin levels and 1.6% of the variability of BMI (P<10(-3) and P=0.023, respectively). CONCLUSIONS: SERPINE1 haplotypes are mildly associated with plasma levels of PAI-1 and with the risk of MI in nonsmokers. They are also associated with insulin levels and BMI.

Candidate gene association studies in abdominal aortic aneurysm disease: a review and meta-analysis.

BACKGROUND: Candidate gene analysis has been frequently used in attempts to understand the pathological processes involved in many aspects of AAA disease. METHODS: This paper sets out a systems approach to reviewing AAA candidate gene analysis studies, whilst, explaining the key principles and design limitations of this universally applied technique. In addition we have performed a meta-analysis of six gene polymorphisms (ACE I/D, MTHFR+677C>T, MMP9-1562C>T, Il-1Beta/3953C>T, eNOS 4a/4b & TIMP1/+434C>T) reported in multiple case control studies. RESULTS AND CONCLUSIONS: Three of these polymorphisms were associated with a significant risk of AAA, ACE RR 1.33 [95% CI 1.20-1.48], MTHFR RR 1.14 [1.08-1.21] and MMP9 RR 1.09 [1.01-1.18]. These differences have been previously reported as equivocal, within a context of contradictory studies and as such this meta-analysis provides new evidence for their involvement in AAA disease. The plausibility of these findings is discussed within the context of a systems approach to the pathology of AAA disease.