KaWah Li

Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study

BACKGROUNDnFamilial hypercholesterolaemia is a common autosomal-dominant disorder caused by mutations in three known genes. DNA-based cascade testing is recommended by UK guidelines to identify affected relatives; however, about 60% of patients are mutation-negative. We assessed the hypothesis that familial hypercholesterolaemia can also be caused by an accumulation of common small-effect LDL-C-raising alleles.nnnMETHODSnIn November, 2011, we assembled a sample of patients with familial hypercholesterolaemia from three UK-based sources and compared them with a healthy control sample from the UK Whitehall II (WHII) study. We also studied patients from a Belgian lipid clinic (Hôpital de Jolimont, Haine St-Paul, Belgium) for validation analyses. We genotyped participants for 12 common LDL-C-raising alleles identified by the Global Lipid Genetics Consortium and constructed a weighted LDL-C-raising gene score. We compared the gene score distribution among patients with familial hypercholesterolaemia with no confirmed mutation, those with an identified mutation, and controls from WHII.nnnFINDINGSnWe recruited 321 mutation-negative UK patients (451 Belgian), 319 mutation-positive UK patients (273 Belgian), and 3020 controls from WHII. The mean weighted LDL-C gene score of the WHII participants (0.90 [SD 0.23]) was strongly associated with LDL-C concentration (p=1.4u2008xu200810(-77); R(2)=0.11). Mutation-negative UK patients had a significantly higher mean weighted LDL-C score (1.0 [SD 0.21]) than did WHII controls (p=4.5u2008xu200810(-16)), as did the mutation-negative Belgian patients (0.99 [0.19]; p=5.2u2008xu200810(-20)). The score was also higher in UK (0.95 [0.20]; p=1.6u2008xu200810(-5)) and Belgian (0.92 [0.20]; p=0.04) mutation-positive patients than in WHII controls. 167 (52%) of 321 mutation-negative UK patients had a score within the top three deciles of the WHII weighted LDL-C gene score distribution, and only 35 (11%) fell within the lowest three deciles.nnnINTERPRETATIONnIn a substantial proportion of patients with familial hypercholesterolaemia without a known mutation, their raised LDL-C concentrations might have a polygenic cause, which could compromise the efficiency of cascade testing. In patients with a detected mutation, a substantial polygenic contribution might add to the variable penetrance of the disease.nnnFUNDINGnBritish Heart Foundation, Pfizer, AstraZeneca, Schering-Plough, National Institute for Health Research, Medical Research Council, Health and Safety Executive, Department of Health, National Heart Lung and Blood Institute, National Institute on Aging, Agency for Health Care Policy Research, John D and Catherine T MacArthur Foundation Research Networks on Successful Midlife Development and Socio-economic Status and Health, Unilever, and Departments of Health and Trade and Industry.

Gene-centric Association Signals for Lipids and Apolipoproteins Identified via the HumanCVD BeadChip

Blood lipids are important cardiovascular disease (CVD) risk factors with both genetic and environmental determinants. The Whitehall II study (n=5592) was genotyped with the gene-centric HumanCVD BeadChip (Illumina). We identified 195 SNPs in 16 genes/regions associated with 3 major lipid fractions and 2 apolipoprotein components at p<10(-5), with the associations being broadly concordant with prior genome-wide analysis. SNPs associated with LDL cholesterol and apolipoprotein B were located in LDLR, PCSK9, APOB, CELSR2, HMGCR, CETP, the TOMM40-APOE-C1-C2-C4 cluster, and the APOA5-A4-C3-A1 cluster; SNPs associated with HDL cholesterol and apolipoprotein AI were in CETP, LPL, LIPC, APOA5-A4-C3-A1, and ABCA1; and SNPs associated with triglycerides in GCKR, BAZ1B, MLXIPL, LPL, and APOA5-A4-C3-A1. For 48 SNPs in previously unreported loci that were significant at p<10(-4) in Whitehall II, in silico analysis including the British Womens Heart and Health Study, BRIGHT, ASCOT, and NORDIL studies (total n>12,500) revealed previously unreported associations of SH2B3 (p<2.2x10(-6)), BMPR2 (p<2.3x10(-7)), BCL3/PVRL2 (flanking APOE; p<4.4x10(-8)), and SMARCA4 (flanking LDLR; p<2.5x10(-7)) with LDL cholesterol. Common alleles in these genes explained 6.1%-14.7% of the variance in the five lipid-related traits, and individuals at opposite tails of the additive allele score exhibited substantial differences in trait levels (e.g., >1 mmol/L in LDL cholesterol [approximately 1 SD of the trait distribution]). These data suggest that multiple common alleles of small effect can make important contributions to individual differences in blood lipids potentially relevant to the assessment of CVD risk. These genes provide further insights into lipid metabolism and the likely effects of modifying the encoded targets therapeutically.

Refinement of Variant Selection for the LDL Cholesterol Genetic Risk Score in the Diagnosis of the Polygenic Form of Clinical Familial Hypercholesterolemia and Replication in Samples from 6 Countries

BACKGROUNDnFamilial hypercholesterolemia (FH) is an autosomal-dominant disorder caused by mutations in 1 of 3 genes. In the 60% of patients who are mutation negative, we have recently shown that the clinical phenotype can be associated with an accumulation of common small-effect LDL cholesterol (LDL-C)-raising alleles by use of a 12-single nucleotide polymorphism (12-SNP) score. The aims of the study were to improve the selection of SNPs and replicate the results in additional samples.nnnMETHODSnWe used ROC curves to determine the optimum number of LDL-C SNPs. For replication analysis, we genotyped patients with a clinical diagnosis of FH from 6 countries for 6 LDL-C-associated alleles. We compared the weighted SNP score among patients with no confirmed mutation (FH/M-), those with a mutation (FH/M+), and controls from a UK population sample (WHII).nnnRESULTSnIncreasing the number of SNPs to 33 did not improve the ability of the score to discriminate between FH/M- and controls, whereas sequential removal of SNPs with smaller effects/lower frequency showed that a weighted score of 6 SNPs performed as well as the 12-SNP score. Metaanalysis of the weighted 6-SNP score, on the basis of polymorphisms in CELSR2 (cadherin, EGF LAG 7-pass G-type receptor 2), APOB (apolipoprotein B), ABCG5/8 [ATP-binding cassette, sub-family G (WHITE), member 5/8], LDLR (low density lipoprotein receptor), and APOE (apolipoprotein E) loci, in the independent FH/M- cohorts showed a consistently higher score in comparison to the WHII population (P < 2.2 × 10(-16)). Modeling in individuals with a 6-SNP score in the top three-fourths of the score distribution indicated a >95% likelihood of a polygenic explanation of their increased LDL-C.nnnCONCLUSIONSnA 6-SNP LDL-C score consistently distinguishes FH/M- patients from healthy individuals. The hypercholesterolemia in 88% of mutation-negative patients is likely to have a polygenic basis.

Whole exome sequencing of familial hypercholesterolaemia patients negative for LDLR/APOB/PCSK9 mutations

Background Familial hypercholesterolaemia (FH) is an autosomal dominant disease of lipid metabolism, which leads to early coronary heart disease. Mutations in LDLR, APOB and PCSK9 can be detected in 80% of definite FH (DFH) patients. This study aimed to identify novel FH-causing genetic variants in patients with no detectable mutation. Methods and results Exomes of 125 unrelated DFH patients were sequenced, as part of the UK10K project. First, analysis of known FH genes identified 23 LDLR and two APOB mutations, and patients with explained causes of FH were excluded from further analysis. Second, common and rare variants in genes associated with low-density lipoprotein cholesterol (LDL-C) levels in genome-wide association study (GWAS) meta-analysis were examined. There was no clear rare variant association in LDL-C GWAS hits; however, there were 29 patients with a high LDL-C SNP score suggestive of polygenic hypercholesterolaemia. Finally, a gene-based burden test for an excess of rare (frequency <0.005) or novel variants in cases versus 1926 controls was performed, with variants with an unlikely functional effect (intronic, synonymous) filtered out. Conclusions No major novel locus for FH was detected, with no gene having a functional variant in more than three patients; however, an excess of novel variants was found in 18 genes, of which the strongest candidates included CH25H and INSIG2 (p<4.3×10−4 and p<3.7×10−3, respectively). This suggests that the genetic cause of FH in these unexplained cases is likely to be very heterogeneous, which complicates the diagnostic and novel gene discovery process.

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 (Cxa0>xa0T) 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], pxa0=xa00.01. This remained significant after adjustment for other risk factors. Ox-LDL:LDL ratio was significantly elevated in T allele carriers (CC vs. CT/TT: 16.3xa0±xa02.4 v 18.0xa0±xa02.9xa0U/mmol LDL, pxa0=xa00.02). (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], pxa0=xa00.02. 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.

Novel genetic approach to investigate the role of plasma secretory phospholipase A2 (sPLA2)-V isoenzyme in coronary heart disease: modified Mendelian randomization analysis using PLA2G5 expression levels.

Background—Secretory phospholipase A2 (sPLA2) enzymes are considered to play a role in atherosclerosis. sPLA2 activity encompasses several sPLA2 isoenzymes, including sPLA2-V. Although observational studies show a strong association between elevated sPLA2 activity and CHD, no assay to measure sPLA2-V levels exists, and the only evidence linking the sPLA2-V isoform to atherosclerosis progression comes from animal studies. In the absence of an assay that directly quantifies sPLA2-V levels, we used PLA2G5 mRNA levels in a novel, modified Mendelian randomization approach to investigate the hypothesized causal role of sPLA2-V in coronary heart disease (CHD) pathogenesis. Methods and Results—Using data from the Advanced Study of Aortic Pathology, we identified the single-nucleotide polymorphism in PLA2G5 showing the strongest association with PLA2G5 mRNA expression levels as a proxy for sPLA2-V levels. We tested the association of this SNP with sPLA2 activity and CHD events in 4 prospective and 14 case–control studies with 27 230 events and 70 500 controls. rs525380C>A showed the strongest association with PLA2G5 mRNA expression (P=5.1×10−6). There was no association of rs525380C>A with plasma sPLA2 activity (difference in geometric mean of sPLA2 activity per rs525380 A-allele 0.4% (95% confidence intervals [−0.9%, 1.6%]; P=0.56). In meta-analyses, the odds ratio for CHD per A-allele was 1.02 (95% confidence intervals [0.99, 1.04]; P=0.20). Conclusions—This novel approach for single-nucleotide polymorphism selection for this modified Mendelian randomization analysis showed no association between rs525380 (the lead single-nucleotide polymorphism for PLA2G5 expression, a surrogate for sPLA2-V levels) and CHD events. The evidence does not support a causal role for sPLA2-V in CHD.

Meta analysis of candidate gene variants outside the LPA locus with Lp(a) plasma levels in 14,500 participants of six White European cohorts.

BACKGROUNDnBoth genome-wide association studies and candidate gene studies have reported that the major determinant of plasma levels of the Lipoprotein (a) [Lp(a)] reside within the LPA locus on chromosome 6. We have used data from the HumanCVD BeadChip to explore the contribution of other candidate genes determining Lp(a) levels.nnnMETHODSn48,032 single nucleotide polymorphisms (SNPs) from the Illumina HumanCVD BeadChip were genotyped in 5059 participants of the Whitehall II study (WHII) of randomly ascertained healthy men and women. SNPs showing association with Lp(a) levels of p<10(-4) outside the LPA locus were selected for replication in a total of an additional 9463 participants of five European based studies (EAS, EPIC-Norfolk, NPHSII, PROCARDIS, and SAPHIR).nnnRESULTSnIn Whitehall II, apart from the LPA locus (where p values for several SNPs were <10(-30)) there was significant association at four loci GALNT2, FABP1, PPARGC1A and TNFRSFF11A. However, a meta-analysis of the six studies did not confirm any of these findings.nnnCONCLUSIONnResults from this meta analysis of 14,522 participants revealed no candidate genes from the HumanCVD BeadChip outside the LPA locus to have an effect on Lp(a) levels. Further studies with genome-wide and denser SNP coverage are required to confirm or refute this finding.

A systematic review and meta-analysis of 130,000 individuals shows smoking does not modify the association of APOE genotype on risk of coronary heart disease

Background Conflicting evidence exists on whether smoking acts as an effect modifier of the association between APOE genotype and risk of coronary heart disease (CHD). Methods and results We searched PubMed and EMBASE to June 11, 2013 for published studies reporting APOE genotype, smoking status and CHD events and added unpublished data from population cohorts. We tested for presence of effect modification by smoking status in the relationship between APOE genotype and risk of CHD using likelihood ratio test. In total 13 studies (including unpublished data from eight cohorts) with 10,134 CHD events in 130,004 individuals of European descent were identified. The odds ratio (OR) for CHD risk from APOE genotype (ε4 carriers versus non-carriers) was 1.06 (95% confidence interval (CI): 1.01, 1.12) and for smoking (present vs. past/never smokers) was OR 2.05 (95%CI: 1.95, 2.14). When the association between APOE genotype and CHD was stratified by smoking status, compared to non-ε4 carriers, ε4 carriers had an OR of 1.11 (95%CI: 1.02, 1.21) in 28,789 present smokers and an OR of 1.04 (95%CI 0.98, 1.10) in 101,215 previous/never smokers, with no evidence of effect modification (P-value for heterogeneity = 0.19). Analysis of pack years in individual participant data of >60,000 with adjustment for cardiovascular traits also failed to identify evidence of effect modification. Conclusions In the largest analysis to date, we identified no evidence for effect modification by smoking status in the association between APOE genotype and risk of CHD.

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.

Angiotensin-converting enzyme D allele does not influence susceptibility to acute hypoxic respiratory failure in children

ObjectiveThe D allele of the I/D polymorphism in the angiotensin-converting enzyme (ACE) gene has been associated with an increased risk of ARDS in critically ill adults and severity of bronchopulmonary dysplasia in pre-term infants. We hypothesised that the presence of the hypoxia-associated ACE D allele would increase susceptibility to acute hypoxic respiratory failure (AHRF) in a cohort of critically ill children.Design and settingSingle-centre prospective observational cohort study.PatientsChildren under 16xa0years of age requiring admission to a tertiary general PICU.Measurements and resultsA total of 216 Caucasian patients were enrolled. Thirty (13.9%) children developed AHRF and 13 were diagnosed with ARDS (6.0%). There was no significant difference in ACE D allele frequency between patient groups with or without AHRF (0.53 vs. 0.54).ConclusionsVariation in ACE activity does not influence the development of paediatric AHRF. This may reflect a different pathogenesis from adult ARDS.