ANGPTL4 E40K and T266M
Effects on Plasma Triglyceride and HDL Levels, Postprandial Responses, and CHD Risk
Background— Angiopoietin-like 4 is a dual-function protein: an inhibitor of LPL, influencing plasma triglycerides (TGs), with angiogenic properties. We examined the association of common ANGPTL4 variants with CHD traits and risk in 5 studies (13 527 individuals).
Methods and Results— The effects on plasma lipids of 6 tagging SNPs and the recently identified E40K were examined in a study of 2772 men. Only T266M (rs1044250, MAF=30%) and E40K (MAF=2%) were significantly associated with TG-lowering (−10.4%, P<0.004 and −20.4%, P<0.0001), respectively. T266M no longer showed significant associations when K40 carriers (K40+) were excluded (P=0.2). Combining data from 5 studies confirmed the TG-lowering effect of K40+ (weighted mean difference: −0.12 [95% CI −0.18, −0.05] mmol/L TG P=0.0001). Surprisingly, in the 3 prospective studies, the combined OR for CHD was 1.48 (1.11 to 1.96, P=0.007), independent of TG. In individuals with a paternal history of MI (n=332) T266M, but not E40K, showed effects on postprandial AUC TG and glucose (P=0.009 and P=0.017, respectively) compared to controls (n=370).
Conclusion— Although associated with an atheroprotective lipid profile, E40K was associated with increased CHD risk, suggesting Angptl4 influences parameters beyond lipid levels. T266M showed effects only under conditions of postprandial stress. The functionality of these potential “loss-of-function” variants needs validation.
The angiopoietin-like proteins (Angptl) are a family of secreted proteins involved in energy metabolism which share tertiary structural domains with angiopoietins, with an N-terminal coiled-coil domain and a fibrinogen-like C-Ter.1 Angptl4 is also known as hepatic fibrinogen angiogenic-related protein, fasting induced adipose factor, and PPAR angiogenic related protein, and these names collectively give insight into the expression and function of the protein. Angptl4 expression under conditions of fasting increases in the liver,2 in adipose tissue in certain mouse strains,3 and is especially prominent in heart and skeletal muscle.4 Angptl4 is thus involved in the switch from fatty acids storage to β-oxidation and energy consumption.5 Angptl4 is cleaved proteolytically in plasma and circulates as N-Ter and C-Ter fragments as well as the full length protein, with the N-Ter and full length protein undergoing oligomerization.6 The involvement in lipid metabolism was demonstrated by intravenous injection of recombinant Angptl4 into mice, resulting in an increase in plasma TG levels attributable to lipoprotein lipase (LPL) inhibition.7 In vitro studies showed that the N-Ter domain is responsible for this, by acting as an unfolding molecular-chaperone, destabilizing the LPL active dimer to inactive monomer.8 The fibrinogen-like domain in angiopoietins bind Tie2 receptors, essential for angiogenic function, however Angptls do not bind Tie1 or Tie2 receptors and as such are orphan ligands.9 Angptl4 has been suggested to regulate angiogenesis,10 but depending on the experimental system both a proangiogenic role, for example during ischemia,11 and an antiangiogenic role, as in vascular permeability,12 have been reported. Yang et al recently showed that Angptl4 C-Ter inhibits angiogenesis by inhibiting the basic FGF signaling cascade, thus suppressing the MAP kinase pathway.13
To explore the effect of ANGPTL4 variation on lipid metabolism we identified 6 tagging SNPs (tSNPs) that capture >92% of the variation at that locus and examined their effects individually and as haplotypes on plasma lipids in the prospective Northwick Park Heart Study II (NPHSII).14 A single cSNP, T266M (rs1044250), showed association with TG levels. Concurrently Romeo et al identified a single cSNP, E40K, associated with lower TG levels, with effects replicated in 2 further studies.15 We have examined the association of T266M and E40K in a total of 13 527 individuals, participating in 3 prospective studies, an MI case-control study and an MI offspring study, to determine effects on plasma lipids, postprandial responses, and risk of CHD.
The recruitment protocols and baseline characteristics of the 5 studies have been extensively published before.16–20 Brief details are given in the supplemental Methods section (available online at http://atvb.ahajournal.org). Tagging SNP Identification and genotyping are presented in supplemental Methods. Details of tagging SNP Primers and probes for TaqMan assays are presented in supplemental Table I.
Hardy-Weinberg equilibrium was assessed using chi-squared tests. Linkage disequilibrium (LD) as measured by D′ was assessed using Haploview (http://www.broad.mit.edu/mpg/haploview/). All analyses were performed on normally-distributed data after appropriate transformation (log or square root). Results are presented as mean and standard deviation (SD) (or standard error of the mean [SEM] in Table 1). t tests and analysis of variance were used, where appropriate, to compare the changes of the continuous variables across the SNPs categories. Haplotypes were inferred using THESIAS21 excluding individuals with missing values, and differences in triglyceride by haplotype assessed assuming an additive model. The effect of genotype on risk in NPHSII was assessed by Cox proportional hazards models. Age and classical CHD risk factors were included as covariates and models were stratified by general practice. In HIFMECH conditional logistic regression models were used to take account of the matching for age and center. For EARSII because of the repeat measures the data were also analyzed by a repeated measures analysis of variance. Two-way analysis of variance for repeated measures (SAS PROC GLM/repeated time) were run to test for the overall significance of postprandial measurements over time and across genotypes. Results were combined over studies and forest plots constructed using the “metan” command in Stata. The level of statistical significance was taken as P<0.01. No adjustments were made for multiple testing as this has been suggested to lead to errors in interpretation.22
The baseline characteristics of the 5 studies are presented in Table 1. Detailed tSNP and haplotype analysis were initially carried out in NPHSII alone. The baseline characteristics for NPHSII men, stratified by CHD status, are published elsewhere.23 Men who developed CHD during follow-up (n=273) were older, had higher plasma total cholesterol levels, triglycerides, blood pressure, and lower HDL-cholesterol levels and the prevalence of smoking was higher than in those who remained CHD free (n=2499).
tSNP Frequencies and Association With Lipid Traits and BMI in NPHSII
Six tSNPs, which explained >92% of the genetic variation in ANGPTL4, were identified: rs4076317 (−207C>G), rs7255436 (3991A>C), rs1044250 (6959C>T, T266M), rs11672433 (9511A>G), rs7252574 (12574C>T), and rs1808536 (12651G>A) and genotyped in the study, together with E40K.15 The genotype distributions of all SNPs did not deviate from Hardy-Weinberg equilibrium (supplemental Table II). A map of ANGPTL4 is shown in supplemental Figure 1, demonstrating strong LD across the gene.
Of the 7 SNPs examined, only T266M (rs1044250) and E40K showed significant associations with lipid traits (Table 2). Results for the other tSNPs are presented in supplemental Tables IIIa through IIIe. M266 showed a recessive association with plasma TG levels with MM homozygotes having 10.4% lower TG levels (P<0.01) compared to men homozygous for the common allele. The effect of E40K was larger, showing a codominant effect with heterozygote men having 20.4% lower TG levels (P<0.0001) and 10% higher HDL-C levels (P<0.007), compared to EE homozygotes. Neither SNP was associated with effects on body mass index (data not shown). Seven haplotypes occurred at frequencies over 2%, but only haplotype H6, with rare alleles of both T266M and E40K (frequency 0.022), was associated with 34% lower TG-levels (P=0.0004) and borderline significantly higher HDL-C levels (by 16% P=0.02) compared to the common haplotype H1 (frequency=0.30; supplemental Table IV). TG levels associated with haplotype H2, which carries M266 independent of K40 (frequency 0.28), did not differ significantly from the common haplotype. To examine further whether T266M contributed to TG-lowering independent of E40K, whole genotypes defined by T266M and E40K were examined. Compared to the TG levels in EE/TT men (1.83±0.98 mmol/L), EK/TM and EK/MM men had 18.6% (1.49±0.83 mmol/L; P>0.0001) and 25% (1.38+0.66 mmol/L; P<0.0001) lower TGs. When E40K men were excluded from the reanalysis, there was no longer a significant association between T266M and TG levels (P=0.29; Table 2). Because ANGPTL4 expression is regulated by PPARs we examined whether these ANGPTL4 SNPs showed interaction with PPAR variants. Neither SNP showed significant evidence of interaction with PPARA SNPs L162V (rs1800206) and intron 7G>C (rs4253778) or PPARG P12A (rs1801282), nor LPL S447X (rs328) on lipid levels. Nor was there evidence of interaction with smoking on TG levels (data not shown). E40K explained 0.8% of the variance and T266M 0.3% of the variance in TG, and together explained 0.9% of the variance in plasma TG levels.
Frequency Distribution of E40K and T266M Across Europe (HIFMECH and EARSII)
Genotypes were in Hardy-Weinberg equilibrium by region, but with a significantly higher MAF frequency of both cSNPs in the Northern recruitment centers compared to the South in both studies (supplemental Figure IIa and IIb).
Effects of E40K and T266M on Lipid Traits in all Studies
The effect of both E40K and T266M on TG and HDL levels was assessed in all studies. The association of E40K, represented as a Forest plot examining the weighted mean difference in TG and HDL levels in K40carriers (K40+) versus E40 homozygotes, showed a consistent effect for TG levels with K40+ having a lower weighted mean of −0.12 (95% CI −0.18,−0.05) mmol/L, P=0.0001 (Figure 1a). The effect on HDL was also consistent, with K40+ having significantly higher weighted mean HDL levels 0.09 (95% CI 0.06 to 0.12) P=0.0001 (Figure 1b). To determine the effect of T266M independent of E40K, K40+ were excluded from the analysis. In confirmation of results from NPHSII, T266M (-K40+) did not show a significant effect on TG or HDL levels overall (data not shown).
Association Between of T266M and E40K and Risk of Future CHD
As shown in Figure 2, E40K was consistently associated with CHD risk with a pooled OR of 1.48 (1.11 to 1.96) P=0.007 for K40 carriers compared to E40 homozygotes, after adjustment for baseline TG levels, age, BMI, SBP, smoking, HDL, LDL and where appropriate gender. The effect of E40K on CHD risk is therefore independent of these classical risk factors. After exclusion of K40 carriers, the pooled OR for T266M showed no significant association with CHD risk with little consistency of effect (supplemental Figure III).
Association of T226M and E40K With Baseline Lipids and Postprandial Responses in EARSII
The association of the cSNPs with postprandial levels after an oral fat tolerance test (OFTT) and an oral glucose tolerance test (OGTT) was examined in EARSII, where young men were defined as “cases” on the basis of their father’s premature MI and “controls” when their father had no premature MI. In the repeated measures analysis of variance, the interaction between time and T266M was P=0.10 for triglyceride after the OFTT and P=0.0003 for glucose after the OGTT. However there was an interaction time*T266M*case-control status (P=0.023) with respect to triglyceride (Figure 3a). Further analyses were run on AUC and peak of triglyceride and glucose in cases and controls separately. As shown in Figure 3a, there was a significant genotype effect in cases, with M266 homozygous showing the lowest AUC and peak TG measures (P=0.009 and P=0.004, respectively). However, after further adjustment on fasting level, the significances were P=0.063 and P=0.033 respectively. After the glucose challenge, MM cases showed the lowest AUC glucose and peak glucose compared to other genotypes (P=0.017 and P=0.001, respectively; Figure 3b). The significances were unchanged after adjustment on fasting glucose value.
The principal findings of this study are a confirmation of a strong association of the E40K variant with lower TG levels in over 13 000 individuals. There were equally robust and consistent associations with higher HDL levels. However, despite these beneficial lipid associations we observed a borderline association of the K40 allele with increased CHD risk, independent of effects on TG and HDL levels and of other classical CHD risk factors. The association with measures of lipid and glucose levels of T266M, independent of the K40 allele, was only seen under conditions of postprandial stress.
Effect of ANPTL4 E40K on Baseline Lipid Levels
E40K is a rare variant and was reported to occur in only 1.3 to 3.0% of whites. Heterozygotes had 12% to 27% lower TG levels and homozygotes up to 56% lower TG levels compared to common allele homozygotes, and there was a significant but modest association with HDL levels.15 In the UK-based studies, NPHSII, WHII, and EPIC-Norfolk, E40K occurred at a similar frequency (1.6 to 2.0%) with up to 30% lower TG levels and 31% higher HDL levels in the K40 homozygotes. E40K, however, had no effect on mean fasting glucose in WHII where fasting glucose was measured at three recalls (P=0.89, data not shown).
This cSNP showed a significant frequency gradient across Europe (HIFMECH), occurring at a frequency of 4.2% in the North and 1.5% in the South, a difference confirmed in EARSII.
A number of other genetic variants show similar frequency gradients across Europe, for example the APOE ε4 allele, recently reviewed by Lao et al.24 The higher frequency of the K40 allele in the North compared to the South of Europe, and its association with increased CHD risk suggests that this variant might be contributing to the CHD gradient across Europe.
In EARSII, in the fasting state, E40K heterozygotes had significantly higher HDL levels (8.5%, P=0.01) but TGs were similar, although the size of the effect in the heterozygous state was of a similar magnitude as reported (12% lowering) but showed only borderline statistical significance (P=0.09).
T266M, tSNPs, and Effects on Baseline Lipids and Postprandial Responses
Of the initial 6 ANGPTL4 tSNPs identified, only T266M (rs1044250) showed a significant effect on TG levels, with a 10% TG-lowering effect in NPHSII. However, the impact of this variant on plasma TG levels was small (R2=0.8%). This is comparable to that of the APOA5 S19W and −1131T>C SNPs, which together explain only 0.9% of the TG variance in NPHSII, whereas LPL S447X explains only 0.3% (Talmud and Cooper, unpublished data). This highlights the polygenic determination of TG levels, whereas diet is likely to play a far greater role. Despite the fact that the tSNPs captured more than 92% of the genetic variation in ANGPTL4, none of them showed association with lipid levels and haplotype analysis provided no additional TG-lowering effects other than those seen with the haplotype defined by both K40 and M266. However, whole genotype analysis of the 2 cSNPs showed only a 5% difference in TG lowering in EK/TM men compared to EK/TT men, when K40 carriers were removed from the analysis T266M no longer showed a significant association with lower TG levels. This suggests that the effect of this SNP on baseline TG levels was minimal and reflected the LD with E40K (D′=1.0 and an R2=0.5). Thus E40K is acting as a marker or is itself the functional variant; this latter proposal is supported by the resequencing study,15 but functional studies are needed to verify this.
In EARSII, under the conditions of an oral fat load and an oral glucose load, those homozygous for the M266 showed better clearance of both TGs and glucose in the young men classified as “cases,” on the basis of a family history of premature paternal MI. No significant genotype effect was seen in the age and region matched “controls.” A similar case:control heterogeneity of effect in EARSII was reported for the USF1 variants on glucose clearance,25 and together these data highlight that even in these healthy young men, the genotypes effect are influenced and magnified in those with a family history of early MI. For E40K although a similar trend was seen, this did not approach statistical significance.
These results, except for the postprandial responses, confirm the findings of Staiger et al who examined 4 ANGPTL4 SNPs (3 of them studied here, including T266M) and reported that none of them were associated with anthropometric measures, fasting or postprandial lipids, or a family history of T2D.26
Although Angptl4 is induced in the fasting state it is possible that the 2 cSNPs behave differently under fasting and fed conditions. These potential “loss-of-function” variants are analogous to the angptl4 knock-out mice model which delineated the role of angplt4 to TG clearance rather than production, in both fed and fasted state.27 The effect on glucose metabolism is less clear. Adenoviral overexpression of Angptl4 improved glucose tolerance but led to lower glucose levels while inducing hepatosteatosis.28 However, Mandard et al reported no effect on fasting glucose in transgenic mice, but impaired glucose tolerance after chronic fat feeding,29 which supports the results seen in EARSII with M266 showing better clearance after the OGTT than wild-type T266. Romeo et al, reported borderline effects of E40K on fasting insulin levels but not glucose levels suggesting that to see the predicted modest effects on fasting levels, very large studies will be needed.15
Effects of E40K and T266M on CHD Risk
E40K, but not T266M, was associated with an unexpected increase in CHD risk, independent of lipid levels, with K40+ showing a pooled OR for CHD of 1.38 (1.05-.80) P=0.02 even after adjustment for TG levels, suggesting that these effects go beyond the association with TG, implicating the angiogenic properties of Angptl4.
Potential Impact of E40K and T266M on Angptl4 Function
Angptl4 undergoes oligomerization promoted by disulphide bonds in the coiled-coil N-Ter forming variable sized multimers,6 which undergo proteolytic cleavage releasing a C-terminal fibrinogen domain which circulates as a stable monomer.30 Both cleaved and full-length Angptl4 are present in the plasma.31 These data suggest that the N-Ter coiled-coil multimer and the monomeric C-Ter fibrinogen-like domain may have distinct functions. It is now clear that inhibition of LPL is mediated through the Angptl4 coiled-coil domain, which converts the active dimer to inactive monomer, described as an “unfolding molecular chaperone.”8 Both E40 and T266 are conserved across human, mouse, and rat,6 supporting a potential functional role. E40 lies in the N-terminal coil-coil domain and may therefore participate in LPL inactivation with “loss-of –functio” K40 showing less LPL-inhibition resulting in more TG hydrolysis and lower TG levels. T266 is positioned in the C-Ter fibrinogen domain. Under baseline conditions the association M266 with TG-lowering appears to reflect LD with E40K. However, under conditions of stress and in the fed state, we speculate that as part of the full-length Angptl4, it may influence LPL inhibition and might reflect differentially induced expression in the fed compared to the fasted state.
The function of the C-terminal fibrinogen domain remains unclear. It is stable in plasma as a monomer, and it has been suggested that it may have angiogenic properties. It has now been confirmed that Angptl4 inhibits angiogenesis by suppressing the Raf/MEK/extracellular signal regulated kinase (ERK) signaling cascade thus blocking bFGF-induced activation of MAP kinase.13 In its full length form it is possible that E40K influences the angiogenic properties of the C-Ter, and as a loss of function mutation would reduce the suppression of the Raf/MEK/ERK signaling pathway,13 thus promoting angiogenesis. Although angiogenesis may be protective, because vascular endothelial growth factor (VEGF) therapy in ischemic heart disease enhances the arterioprotective functions of the endothelium,32 its antiprotective effects are demonstrated by the fact that neovascularization promotes growth of the atherosclerotic lesion leading to plaque destabilization and rupture.33 Thus if we extrapolate this to a reduction in Angptl4 inhibition, this could promote neovascularization and thus plaque instability.
In conclusion, the replication of the association of ANGPTL4 variants with lower lipid levels, yet increased CHD risk in these studies, places Angptl4 in an interesting position, potentially influencing parameters beyond lipid levels. However, this study is not without its limitations. Although we analyzed 4 large studies, clarification of the associations with glucose levels is necessary. The tSNP approach captures the large majority of genetic variation, and we can rule out comprehensively the possibility that there are other common amino acid variants which might be functional, although there might be upstream variants in LD which affect the level of gene expression. Clearly, confirmation of these results is needed, together with in vitro studies of these 2 variants to validate their functionality.
The authors thank the participants, general practitioners, and staff in NPHSII and EPIC-NORFOLK.
Sources of Funding
NPHSII was supported by MRC UK, US NIH (grant NHLBI 33014), and Du Pont Pharma, Wilmington, USA. EPIC-Norfolk is supported by MRC UK and Cancer Research UK, with additional support from the EU, Stroke Association, British Heart Foundation (Grant PG2000/015), Department of Health, Food Standards Agency, and Wellcome Trust. The Whitehall Study II is supported by MRC UK, BHF, Department of Health, NHLBI (Hl36310); NIA (AG13196), and John D. and Catherine T McArthur Foundation. The HIFMECH study was supported by the European Commission (BMH4-CT96-0272), the full list of investigators is presented in supplemental information. EARSII was supported by the European Community (EU-Biomed 2 BMG4-98 to 3324), the full list of participants is presented in supplemental information. P.J.T., J.A.C., J.P., F.D., and S.E.H. are supported by BHF (PG2005/014). M.S. is supported by a Unilever/BBSRC Case studentship. M.G.M. is supported by an MRC professorship.
Original received July 8, 2008; final version accepted September 23, 2008.
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