Tribal ethnicity and CYP2B6 genetics in Ugandan and Zimbabwean populations in the UK: implications for efavirenz dosing in HIV infection.

OBJECTIVES: To determine differences in CYP2B6 loss of function (LoF) single nucleotide polymorphisms (SNPs) and haplotypes between Zimbabweans and Ugandans, and within Ugandan populations (Bantu and Nilotic). METHODS: Genetic epidemiological study enrolling adult black African Ugandan and Zimbabwean patients attending a UK HIV-1 clinic, irrespective of antiretroviral therapy status. Genomic DNA was extracted from whole blood and the presence of CYP2B6 alleles was determined by direct sequencing of all nine exons of the CYP2B6 gene. Blood was also collected, where appropriate, for determination of efavirenz concentrations. Frequency of SNPs in all patients and LoF haplotype frequencies were calculated. The relationship between the number of LoF haplotype alleles possessed and efavirenz trough concentration (ETC) was determined. RESULTS: Thirty-six Zimbabweans and 74 Ugandans (58 Bantu and 16 Nilotic) were recruited. The definite haplotypes determined were *6, *18, *20 and *27 as LoF and *4 as gain of function. Among those with definite genotypes, the frequency of LoF alleles was 65% [95% confidence interval (95% CI): 51-80] of Zimbabweans versus 22% (95% CI: 12-31) of Ugandan Bantus (P = 10(-6)) and versus 39% (95% CI: 14-64) of Ugandan Nilotics (P = 0.09). Among the 19 patients with definite genotype and with available ETCs, log ETCs were associated with a greater number of LoF haplotype alleles [848 ng/mL (n = 12), 1069 ng/mL (n = 4) and 1813 ng/mL (n = 3) for 0, 1 or 2 LoF haplotypes, respectively (P = 0.016)]. CONCLUSIONS: Among Zimbabweans, LoF haplotypes constitute the majority of CYP2B6 alleles and are significantly higher in prevalence compared with Ugandans. Frequencies of LoF haplotypes and SNPs in Ugandan Nilotics appear to lie between those of Zimbabweans and Ugandan Bantus. These findings may have relevance to pharmacokinetics and dosing of efavirenz in African populations.

SHP-2 and PI3-kinase genes PTPN11 and PIK3R1 may influence serum apoB and LDL cholesterol levels in normal women.

Insulin regulates apoB metabolism via activation of PI3K or regulation of MTP via MAPK/ERK signalling. SHP-2 enhances both pathways through increased IRS-1 phosphorylation. We hypothesized that variants in the SHP-2 gene PTPN11 and PI3K p85alpha subunit gene PIK3R1 may influence fasting levels of plasma apoB and/or LDL cholesterol. We tested association of tagging SNPs (tSNPs) in each gene with serum lipids in a large sample of unselected population-based Caucasian female twins (n=2771, mean age 47.4+/-12.5 years) and then tested interaction between tSNPs in determining apoB and LDL levels. PTPN11 tSNP rs11066322 was associated with apoB (P=0.007) and rs11066320 was associated with LDL cholesterol (P=0.016). PIK3R1 tSNP rs251406 was associated with apoB (P=0.0003) and rs706713 was associated with LDL cholesterol (P=0.009). PTPN11 tSNP rs11066322 interacted with PIK3R1 tSNP rs251406 in determining serum apoB levels (P=0.012) and with PIK3R1 tSNP rs40318 in determining LDL cholesterol levels (P=0.009). Association of single tSNPs with both apoB and LDL cholesterol as well as interactions between the two genes suggest that variants influencing SHP-2 activity may modulate the acute pathway by which insulin regulates these lipids.

Phosphatidylinositol 3-kinase p85alpha regulatory subunit gene PIK3R1 haplotype is associated with body fat and serum leptin in a female twin population.

AIMS/HYPOTHESIS: Phosphatidylinositol 3-kinase (PI3K) couples the leptin and insulin signalling pathways via the insulin receptor substrates IRS1 and IRS2. Hence, defective activation of PI3K could be a novel mechanism of peripheral leptin or insulin resistance. We investigated associations of tagging single-nucleotide polymorphisms (tSNPs) in the PI3K p85alpha regulatory subunit gene PIK3R1 with anthropometry, leptin, body fat and insulin sensitivity in a female twin population of European extraction. MATERIALS AND METHODS: Eight tSNPs were genotyped in 2,778 women (mean age 47.4+/-12.5 years) from the St Thomas' UK Adult Twin Registry (Twins UK). RESULTS: SNP rs1550805 was associated with serum leptin (p=0.028), BMI (p=0.025), weight (p=0.019), total fat (p=0.004), total fat percentage (p=0.002), waist circumference (p=0.025), central fat (p=0.005) and central fat percentage (p=0.005). SNPs rs7713645 and rs7709243 were associated with BMI (p=0.020 and p=0.029, respectively), rs7709243 with weight, total and central fat (p=0.026, p=0.031 and p=0.023, respectively) and both SNPs with fasting glucose (p=0.003 and p=0.001, respectively) and glucose 2-h post OGTT (p=0.023 and p=0.007, respectively). Subjects with haplotype 222 (frequency 7.2%) showed higher serum leptin concentration (p=0.007) and body fat measures (p< or =0.001 for all), and those with haplotype 221 (frequency 38.7%) showed higher fasting and 2-h glucose (p=0.035 and p=0.021, respectively) compared with subjects with the most common haplotype, 111 (frequency 45.5%). CONCLUSIONS/INTERPRETATION: Association of the PIK3R1 SNP rs1550805 with serum leptin and body fat may reflect a diminished ability of PI3K to signal via IRS1 or IRS2 in response to leptin.

Common polymorphisms in SOCS3 are not associated with body weight, insulin sensitivity or lipid profile in normal female twins.

AIMS/HYPOTHESIS: Inhibition of signal transduction by suppressor of cytokine signalling-3 (SOCS-3) potentially influences resistance to insulin and leptin. The aim of this study was to test the association between three single-nucleotide polymorphisms (SNPs) representative of common linkage disequilibrium clusters in SOCS3 (rs4969169, rs12953258 and rs8064821) and obesity measures, insulin sensitivity measures and serum lipids in the general population. METHODS: The three SNPs, which had rare allele frequencies >0.06, were genotyped in 2,777 female twins of European extraction (mean age 47.4+/-12.5 years) from the St Thomas' UK Adult Twin Registry (Twins UK). RESULTS: Minor allele frequencies were as follows: rs4969169=0.067, rs12953258=0.097 and rs8064821=0.101. Individual SOCS3 SNPs were not associated with general or central obesity, or with two indices of insulin sensitivity (homeostasis model assessment and insulin sensitivity measure). CONCLUSIONS/INTERPRETATION: The results do not indicate that any of the three SNPs studied are associated with obesity, insulin measures or lipid measures.

Signal-transduction pathways involved in the hypertrophic effect of hsp56 in neonatal cardiomyocytes.

Heat shock protein (hsp) 56 (hsp56) is an immunophilin that acts as a cofactor with hsp90 and exhibits both peptidyl-prolyl isomerase activity and chaperone activity. Previous studies have shown that the hypertrophic effect of cardiotrophin-1 (CT-1) in cardiac cells is dependent on hsp56 induction. CT-1 activates a number of signal-transduction pathways. Therefore, we sought to determine whether these pathways were also important for hsp56-induced hypertrophy using overexpression with transiently transfected plasmid vectors in rat neonatal cardiomyocytes. Here we show that multiple signalling pathways are involved in hsp56-induced hypertrophy, namely the Janus kinase-signal-transducer and activator of transcription, extracellular signal-regulated protein kinase and PI3-kinase/Akt signalling pathways. Dominant-negative mutants and inhibitors of these pathways were able to block the hypertrophy observed as a result of hsp56 overexpression. However, an inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway was not able to block the hypertrophic effect of hsp56 overexpression. Furthermore, we show that domains I, II and IV of the hsp56 protein may be involved in its hypertrophic effect. These studies show for the first time that multiple signalling pathways are involved in the hypertrophic effect of hsp56 and that overexpression of hsp56 itself is able to activate the necessary signalling pathways, which induce hypertrophy.

Genetic determinants of the response to bezafibrate treatment in the lower extremity arterial disease event reduction (LEADER) trial.

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.

Endurance enhancement related to the human angiotensin I-converting enzyme I-D polymorphism is not due to differences in the cardiorespiratory response to training.

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.

Elite swimmers and the D allele of the ACE I/D polymorphism.

A polymorphism of the human angiotensin-1-converting enzyme (ACE) gene has been identified in which the presence (insertion, I allele) of a 287-bp fragment rather than the absence (deletion, D allele) is associated with lower ACE activity. Several recent studies have shown an association of the I allele with endurance performance, it being found with excess frequency in elite distance runners, rowers and mountaineers. Other workers using heterogeneous cohorts of athletes from mixed sporting disciplines have found no such association. An increasing linear trend of I allele frequency with the distance run amongst Olympic runners and an excess of the D allele amongst sprinters led us to examine whether the ratio of I and D alleles in swimmers competing over different distances would also vary. Swimmers (n=120) from the European and Commonwealth championships and an American college team had their ACE genotype determined and their gene and allele frequencies compared with several control groups, the most closely age-matched of which were 1,248 military recruits. Of the 103 Caucasians, there was a significant excess of the D allele compared with this control group only in the truly elite swimmers of the European and Commonwealth championships (P=0.004). This association remained in those competing over shorter distances (P=0.005 for 400 m and below) but not in the longer events. These findings were confirmed in three further large control groups. A population association study testing whether a genetic marker (the ACE I/D polymorphism) occurs more frequently in cases (elite athletes) than in controls therefore requires a homogeneous cohort of subjects from the same sporting discipline.

Variation in the PPARalpha gene is associated with altered function in vitro and plasma lipid concentrations in Type II diabetic subjects.

AIMS/HYPOTHESIS: Peroxisome proliferator activated receptor alpha (PPARalpha) regulates genes involved in lipid metabolism, haemostasis and inflammation, in response to fatty acids and fibrates, making it a candidate gene for risk of dyslipidaemia, atherosclerosis and coronary artery disease. Plasma non-esterified fatty acids are increased in subjects with Type II (non-insulin-dependent) diabetes mellitus, suggesting that PPARalpha could link Type II diabetes and dyslipidaemia, and affect response to fibrates. This has been investigated in association studies in healthy and diabetic subjects and in vitro studies. METHODS: The human PPARalpha gene was isolated and screened for variation by single strand conformation polymorphism analysis. Genotypes were determined for 129 Type II diabetic subjects and 2508 healthy men. The association with plasma lipid concentrations was examined. The function of the V162 variant was examined in co-transfection assays. RESULTS: We identified two polymorphisms, one in intron 3 and a missense mutation, leucine 162 to valine, in the DNA binding domain. In Type II diabetic patients, V162 allele carriers had higher total cholesterol, HDL cholesterol and apoAI whereas intron 3 rare allele carriers had higher apoAI concentrations. By contrast, no effect was observed in healthy rare allele carriers. In vitro, the V162 variant showed greater transactivation of a reporter gene construct. CONCLUSION/INTERPRETATION: Naturally occurring variation alters PPARalpha function, influencing plasma lipid concentrations in Type II diabetic patients but not healthy people. This demonstrates that PPARalpha is a link between diabetes and dyslipidaemia, and so could influence the risk of coronary artery disease, the greatest cause of morbidity and mortality in Type II diabetes.