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A Null Mutation in Human APOC3 Confers a Favorable Plasma Lipid Profile and Apparent Cardioprotection

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Science  12 Dec 2008:
Vol. 322, Issue 5908, pp. 1702-1705
DOI: 10.1126/science.1161524

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Abstract

Apolipoprotein C-III (apoC-III) inhibits triglyceride hydrolysis and has been implicated in coronary artery disease. Through a genome-wide association study, we have found that about 5% of the Lancaster Amish are heterozygous carriers of a null mutation (R19X) in the gene encoding apoC-III (APOC3) and, as a result, express half the amount of apoC-III present in noncarriers. Mutation carriers compared with noncarriers had lower fasting and postprandial serum triglycerides, higher levels of HDL-cholesterol and lower levels of LDL-cholesterol. Subclinical atherosclerosis, as measured by coronary artery calcification, was less common in carriers than noncarriers, which suggests that lifelong deficiency of apoC-III has a cardioprotective effect.

Elevated plasma levels of low density lipoprotein cholesterol (LDL-C) and triglycerides (TGs) are important contributors to premature coronary heart disease (CHD) (13), and genetic variants causing low LDL-C are associated with reduced risk of CHD (4). Recently, nonfasting TG was found to be an independent CHD risk factor (5, 6), showing higher predictive power in one study than fasting TG (FTG), the traditional measure, likely because of the atherogenic remnant lipoproteins generated during absorption and clearance of dietary fat (5).

To identify genetic factors contributing to FTG and the postprandial TG (ppTG) dietary response, we performed a single high-fat feeding intervention and genome-wide association study (GWAS) in 809 Old Order Amish individuals as part of the Heredity and Phenotype Intervention (HAPI) Heart Study (7). Characteristics of these participants are shown in table S1. These individuals were fed a milkshake containing 782 kcal/m2 body surface area, with 77.6% of these calories from fat, and had blood drawn for lipid levels 0, 1, 2, 3, 4, and 6 hours after the intervention. The Affymetrix GeneChip Human Mapping 500K Array Set was used for genotyping leukocyte DNA from these 809 participants. Traits were normalized, and analyses accounting for sex and sex-specific age and age2; body mass index (BMI) and relatedness among participants were performed as described in the Methods section (8).

Results of the GWAS of FTG and ppTG [as estimated by the incremental area under the curve, iAUCTG (8)], transformed by their natural logarithm (ln), are shown in table S2 and fig. S1. The strongest evidence for association with both ln-FTG (P = 3.8 × 10–14) and ln-iAUCTG(P = 2.8 × 10–10) occurred on chromosome 11q23 at single-nucleotide polymorphism (SNP) rs10892151, which had a minor allele frequency (MAF) of 0.028 (A allele) (table S2). SNP rs10892151 is located within an intron of the DSCAML1 (Down syndrome cell adhesion molecule like 1) gene and also lies 823 kb away from the apolipoprotein A and C APOA1/C3/A4/A5 region, a cluster of more likely candidate genes, given the established key roles of their products in lipid metabolism (9). SNP rs681524 (MAF = 0.062), 40 kb from the cluster, showed nominal association with ln-FTG (P = 1.1 × 10–5) and ln-iAUCTG (P = 0.004) and was moderately correlated with rs10892151 [standardized linkage disequilibrium coefficient (D′)= 0.85, squared correlation coefficient (r2) = 0.31] (fig. S2).

Rs10892151 A carriers evidenced markedly lower FTGs and ppTGs than noncarriers (table S3), consistent with effects of deleting the APOC3 gene in mice (10), which led to the hypothesis that SNP rs10892151 tagged a loss-of-function mutation in APOC3. Sequencing of the coding region of APOC3 revealed a C → T substitution at the terminal nucleotide of exon 3, the 55th nucleotide from the ATG start codon; this substitution resulted in a premature stop codon for an arginine residue at amino acid position 19 (R19X). This position is located in the signal peptide of the protein, normally cleaved before the secretion of the mature 79–amino acid apoC-III peptide (11). Thus, a complete lack of production of apoC-III from alleles containing this mutation would be predicted. Moreover, the location of the premature stop codon in the mRNA transcript of the mutated gene meets the criteria for nonsense-mediated mRNA, in which certain mRNA transcripts with premature stop codons are degraded, rather than translated into protein (12, 13). Indeed, in a sample of 20 study participants [10 carriers of the 19X allele, RX (CT), and 10 noncarriers, RR (CC)] who made up four two-generation families and one pair of siblings, apoC-III protein levels in R19X carriers were approximately half of that in their noncarrier relatives (39% versus 87% of pooled serum control level, P = 0.0002) (Fig. 1). ApoC-III levels were highly correlated with ln-FTG levels [partial correlation coefficient (r)= 0.71, P = 0.0002] (Fig. 1, nontransformed FTG shown).

Fig. 1.

Triglyceride levels as a function of apoC-III protein levels stratified by APOC3 R19X genotype in 20 individuals. Filled squares indicate individuals carrying the 19X allele and open squares, noncarriers.

The R19X mutation was in strong linkage disequilibrium with (i.e., highly correlated with) the most highly associated GWAS SNP rs10892151 (D′ = 1, r2 = 0.85) (fig. S2). Pedigree and haplotype analysis were consistent with a single copy of the mutated allele having entered the population before the year 1800 (supporting online text and figs. S3 and S4). Evaluation of the association of this novel R19X mutation with ln-FTG and ln-ppTG in 802 of 809 HAPI Heart GWAS subjects successfully genotyped revealed similar associations to those identified with rs10892151 (Fig. 2 and table S4). R19X heterozygotes had significantly reduced TG levels compared with their RR counterparts at all six time points, with a median FTG (interquartile range) of 31 (25 to 48) versus 57 (42 to 81) mg/dl (P = 4.1 × 10–13) and iAUCTG median of 214 (154 to 338) versus 410 (276 to 608) mg/dl over 6 hours (P = 2.0 × 10–10). Nominal association (P < 0.05) of the mutation with ln-TG at 2, 3, and 4 hours, as well as ln-tAUC, ln-iAUC, natural logarithm of maximum TG, and natural logarithm of incremental maximum TG persisted after controlling for ln-FTG. The segregation of the R19X mutation with hypo-TG and blunted ppTG response in a representative family is shown in fig. S5.

Fig. 2.

TG levels before and during the high-fat challenge by R19X APOC3 genotype. Shown are covariate-adjusted geometric means with 95% confidence intervals. Filled squares indicate individuals carrying the 19X allele and open squares, noncarriers.

R19X carriers had significantly higher HDL-cholesterol (HDL-C) levels (mean ± SD: 67 ± 17 versus 55 ± 14 mg/dl, P = 9.0 × 10–7) and lower total cholesterol (191 ± 35 versus 209 ± 46 mg/dl, P = 0.02) and LDL-C (116 ± 32 versus 140 ± 43 mg/dl, P = 0.001) levels (Table 1) than noncarriers. There were no differences in BMI (P = 0.98) or waist circumference (P = 0.50), and fasting glucose and insulin were similar between R19X carriers and noncarriers (P = 0.53 and 0.72, respectively). R19X carriers had significantly lower levels of non-HDL (P = 0.0005); very low density lipoprotein (VLDL, 1.5 × 10–10); the most-dense VLDL subfraction, VLDL3 (P = 2.8 × 10–10); intermediate density lipoprotein (IDL, P = 8.8 × 10–15); real LDL (P = 0.01); and remnant lipoprotein cholesterol (P = 3.0 × 10–20), as well as higher levels of both HDL2 (P = 7.0 × 10–6) and HDL3 (P = 3.0 × 10–5) cholesterol. On the basis of the mutation's effect on TGs, VLDL-C levels increased significantly less in carriers than in noncarriers at 4 hours after the high-fat challenge (P = 0.0009) (Table 1).

Table 1.

Morphometric and metabolic characteristics and lipid subfractions by APOC3 R19X genotype (all analyses except age adjusted for sex and sex-specific age and age2 and all analyses except age, BMI, and waist adjusted for BMI) in the HAPI Heart Study. Variables analyzed directly are presented as mean ± SD. Variables natural logarithm–transformed for normalization before analysis are presented as median (interquartile range). All values are fasting unless otherwise indicated. SBP, systolic blood pressure; DBP, diastolic blood pressure.

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The hypothesis that a mutation conferring such a favorable lipid profile would be cardioprotective was evaluated in the Amish Family Calcification Study (14), which includes 335 of the HAPI Heart fat-challenge participants along with 698 additional Amish individuals, all of whom underwent electron beam computed tomography to quantify coronary artery calcification (CAC), a subclinical measure of coronary atherosclerosis. A standard lipid panel was also obtained. The effect of the APOC3 R19X mutation on decreased FTGs and increased HDL-C levels replicated in the 698 nonoverlapping AFCS participants (P = 1.9 × 10–22 and P = 1.3 × 10–14, respectively; P = 2.9 × 10–29 and 1.3 × 10–17 in the full AFCS). LDL-C levels tended to be lower in R19X carriers than in noncarriers in the nonoverlapping subset, and the difference reached statistical significance in the full AFCS (P = 0.01) (table S5).

Among AFCS participants, according to evidence-based clinical guidelines set by the National Cholesterol Education Program Adult Treatment Panel III (ATP-III) (15), LDL-C levels, the primary CHD prevention target, were more likely to be optimal (<100 mg/dl) in R19X carriers [odds ratio with 95% confidence limits (OR) = 2.24 (95% CI 1.07 to 4.66)] than in noncarriers (Table 2), although the difference did not quite reach statistical significance (P = 0.07). In addition, high HDL-C (≥60 mg/dl), considered by the ATP-III to be cardioprotective enough to cancel out one additional CHD risk factor (15), was much more common in R19X carriers than in noncarriers [OR = 7.0 (95% CI: 3.3 to 14.8), P = 9.1 × 10–6]. Notably, ATP-III–defined low HDL-C (<40 mg/dl) was absent in the carriers but present in 13.5% of noncarriers (Fisher's exact test, P = 4.2 × 10–4). All R19X carriers had FTGs in the normal (<150 mg/dl) range (maximum of 77 mg/dl), whereas only 83.6% of noncarriers had normal FTGs (Fisher's exact test, P = 0.0004). Among AFCS participants in the baseline age range (30 to 74 years) of the Framingham Heart Study, mutation carriers had significantly lower natural logarithm–transformed 10-year Framingham CHD risk (16) than noncarriers [risk ratio = 0.68 (95% CI: 0.58 to 0.79), P = 3.9 × 10–7] (Table 2 and fig. S6).

Table 2.

National Cholesterol Education Program ATP-III protective lipid levels, Framingham 10-year CHD risk, and CAC as a function of APOC3 R19X genotype in the AFCS. Dichotomous traits analyzed by generalized estimating equations (GEE) adjusted for age, sex, and sibship, as described in the methods (8), except triglycerides, which contains a zero cell. Framingham 10-year CHD risk analyzed as a natural log-transformed quantitative trait with variance components (adjusted for sex and sex-specific age and age2) as described in the methods (8). Framingham Heart Study comparisons included only individuals age 30 to 74 years to match baseline ages in that study. MESA comparisons included only individuals age 45 to 84 years to match baseline ages in that study.

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Consistent with their protective lipid profile, R19X carriers were significantly less likely than noncarriers to have any detectable CAC [OR = 0.35 (95% CI: 0.21 to 0.60), P = 0.002] or CAC scores greater than 100 Agatston units [OR = 0.40 (95% CI: 0.18 to 0.85), P = 0.01] (fig. S7). CAC scores of 1 to 100 were previously associated with a fourfold and scores greater than 100 with a sevenfold increased risk of major coronary events in comparison with individuals with no detectable calcification in the population-based Multi-Ethnic Study of Atherosclerosis (MESA) after adjustment for standard risk factors (17). Among AFCS participants in the age range of the MESA study (45 to 84 years), R19X carriers were significantly less likely to have a score at or above their respective MESA-derived (18) ethnicity-, sex-, and age-specific 75th percentile [OR = 0.38 (95% CI: 0.19 to 0.77), P = 0.003] (Table 2), a level associated in MESA, after adjustment for traditional risk factors, with an eightfold risk of CHD events in comparison with the lowest two quartiles (19). Analysis of the quantitative trait ln(CAC score + 1), which approximated a normal distribution after adjustment for age and sex (fig. S8), yielded a significant negative association between CAC quantity and being a carrier [βR19X = –0.84 ± 0.30, e.g., mean ratio = 0.43 (95% CI: 0.24 to 0.78), P = 0.005] (table S5).

ApoC-III, secreted from the liver and to a lesser extent by the intestines, is a component of both HDL and apoB-containing lipoprotein particles (9, 20), impairs catabolism and hepatic uptake of apoB-containing lipoproteins (21, 22), appears to enhance the catabolism of HDL particles (23), enhances monocyte adhesion to vascular endothelial cells (24), and activates inflammatory signaling pathways (25). Along with the observed association of elevated apoC-III concentration with increased CHD risk (26), the roles of apoC-III in lipoprotein metabolism would predict that apoC-III deficiency would decrease atherothrombotic tendency.

Previous reports of apoC-III deficiency in humans were complicated by the prothrombotic effects of accompanying apoA-I and/or apoA-IV deficiency (2730), small sample sizes and/or structurally abnormal apoC-III (31, 32). The current report of a favorable lipid profile and reduced subclinical coronary artery atherosclerosis in R19X null mutation carriers provides strong evidence in a relatively large number of individuals that apoC-III deficiency (by ∼50% of normal levels) is indeed cardioprotective.

Indirectly lowered APOC3 expression is one mechanism of the lipid-lowering effect of fibrates (33), and the use of several other lipid-lowering agents, including statins, thiazolidinediones, ezetimibe, niacin, fish oil, and weight loss, has also been associated with decreases in apoC-III levels [reviewed in (20)]. That a naturally occurring null mutation in APOC3 confers a favorable lipid profile and apparent cardioprotection and does not result in any obvious detrimental effect raises the possibility that therapies aimed specifically at down-regulating apoC-III expression will be clinically efficacious and safe in reducing the morbidity and mortality associated with CHD.

Supporting Online Material

www.sciencemag.org/cgi/content/full/322/5908/1702/DC1

Materials and Methods

SOM Text

Figs. S1 to S8

Tables S1 to S5

References

References and Notes

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