Rare genetic variants, identified by in-detail resequencing of loci, may contribute to complex traits. We used the apolipoprotein A-I gene (APOA1), a major high-density lipoprotein (HDL) gene, and population-based resequencing to determine the spectrum of genetic variants, the phenotypic characteristics of these variants, and how these results compared with results based on resequencing only the extremes of the apolipoprotein A-I (apoA-I) distribution. First, we resequenced APOA1 in 10,330 population-based participants in the Copenhagen City Heart Study. The spectrum and distribution of genetic variants was determined as a function of the number of individuals resequenced. Second, apoA-I and HDL cholesterol phenotypes were determined for nonsynonymous (NS) and synonymous (S) variants and were validated in the Copenhagen General Population Study (n = 45,239). Third, observed phenotypes were compared with those predicted using an extreme phenotype approach based on the apoA-I distribution. Our results are as follows: First, population-based resequencing of APOA1 identified 40 variants of which only 7 (18%) had minor allele frequencies >1%, and most were exceedingly rare. Second, 0. 27% of individuals in the general population were heterozygous for NS variants which were associated with substantial reductions in apoA-I (up to 39 mg/dL) and/or HDL cholesterol (up to 0. 9 mmol/L) and, surprisingly, 0. 41% were heterozygous for variants predisposing to amyloidosis. NS variants associated with a hazard ratio of 1. 72 (1. 09–2. 70) for myocardial infarction (MI), largely driven by A164S, a variant not associated with apoA-I or HDL cholesterol levels. Third, using the extreme apoA-I phenotype approach, NS variants correctly predicted the apoA-I phenotype observed in the population-based resequencing. However, using the extreme approach, between 79% (screening 0–1st percentile) and 21% (screening 0–20th percentile) of all variants were not identified; among these were variants previously associated with amyloidosis. Population-based resequencing of APOA1 identified a majority of rare NS variants associated with reduced apoA-1 and HDL cholesterol levels and/or predisposing to amyloidosis. In addition, NS variants associated with increased risk of MI.
Genome-wide association studies have identified multiple loci associated with complex traits and diseases, but until now common genetic variants (minor allele frequency >5%) at these loci only explain small proportions of the heritability [1], [2]. For example, the estimated heritability of high density lipoprotein (HDL) cholesterol in twin-studies is 50% [3], but the common alleles together or in combination explain less than 5–10% of the variation in plasma levels of HDL cholesterol [4]. Rare genetic variants (minor allele frequency <1%), which are identified by in-detail screening or resequencing of loci, may contribute to unravel this unexplained heritability [1], [2]. Apolipoprotein A-I (apoA-I) is the major protein component of HDL in plasma, and is a cofactor for lecithin∶cholesterol acyltransferase (LCAT), playing a key role in the so-called reverse cholesterol transport, i. e. the transport of cholesterol from peripheral tissues to the liver for excretion [3]. APOA1 (MIM 107680) encodes a 267 amino acid prepropeptide, which is sequentially cleaved to yield the mature 243 amino acid protein. Mutations in apoA-I may associate with low levels of plasma HDL cholesterol and apoA-I due to defective LCAT activation or to amyloidosis, or to amyloidosis with only minor or no effects on apoA-I and HDL cholesterol levels [5]–[11]. However, at present we lack comprehensive information on the spectrum of genetic variants in this pleiotropic gene in the general population, on the phenotypic characteristics of such variants in individuals in the general population, and whether additional information is gained from resequencing a sample of the entire general population, rather than using an extreme phenotype approach, previously used by us and others [12]–[16]. In the first part of this study, the aim was to determine the spectrum and distribution of genetic variants in APOA1 using a population-based resequencing approach. In the second part of the study, the aim was to determine the association of nonsynonymous (NS) and synonymous (S) variants in APOA1 in the general population with plasma levels of apoA-I and HDL cholesterol, and with risk of myocardial infarction (MI). In the third part of the study, we compared results using an extreme apoA-I phenotype approach with results from the population-based resequencing. For these purposes, we resequenced APOA1 in 10,330 participants in the Copenhagen City Heart Study (CCHS), and used the Copenhagen General Population Study (CGPS; n = 45,239 participants) to validate phenotypic results.
Studies were approved by institutional review boards and Danish ethical committees (Nos. KF-100. 2039/91, KF-01-144/01, H-KF-01-144/01) and conducted according to the Declaration of Helsinki. Informed consent was obtained from all participants. All participants were white and of Danish descent. No participants appeared in more than one of the two studies, permitting independent confirmation of the findings in each group. In both studies of the general population, diagnoses of MI (WHO International Classification of Diseases; ICD8: 410, ICD10: I21-I22) were collected from 1977 through May 10th 2011, and verified by reviewing all hospital admissions and diagnoses entered in the National Danish Patient Registry, and all causes of death entered in the National Danish Causes of Death Registry. A diagnosis of MI followed the changing definitions over time [20], [21]. As shown in flowchart (Figure 1). We screened the translated region of APOA1 in all 10,330 participants in the CCHS using four PCR fragments covering 119 bp upstream of exon 2, exons 2–4, and exon-intron boundaries (APOA1 consensus sequence NC_000011. 9) (Table S1). Mutational analysis was performed using a LightScanner (Idaho Technology Inc. , Salt Lake City, UT, USA), followed by sequencing on an ABI 3730 DNA Analyzer (Applied Biosystems Inc. , Foster City, CA, USA). NS and S variants identified in more than two individuals in the CCHS (K12K, S25S, S36A, F71Y, K107del, L144R, A164S, A190A), were genotyped in the CGPS using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems Inc. , Foster City, CA, USA) and TaqMan-based assays. Colorimetric and turbidimetric assays were used to measure nonfasting plasma levels of apoA-I, HDL cholesterol, total cholesterol, triglycerides, and apoB (Boehringer Mannheim, Mannheim, Germany and Konelab, Helsinki, Finland). LDL cholesterol was calculated using the Friedewald equation [22] when plasma triglycerides were ≤4. 0 mmol/L, and otherwise measured directly (Thermo Fisher Scientific, Waltham, MA, USA). Body mass index was measured weight (kg) divided by measured height squared (m2). Lipid-lowering therapy was self-reported. Physical inactivity, drinking, smoking, hypertension and diabetes were dichotomized and defined as physical inactivity (less than 2–4 hours per week of light physical activity at leisure time), drinking (more than 1 drink per week), current smoking, hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, and/or use of antihypertensive therapy), and diabetes (self-reported disease, current use of anti-diabetic medication, and/or nonfasting plasma glucose >11. 0 mmol/L). We used Stata/S. E. 10. 1. Two-sided p<0. 05 was significant. χ2-tests evaluated Hardy-Weinberg equilibrium. To adjust for the effect of gender and age on absolute levels within studies, and for differences in absolute levels between studies, plasma levels of apoA-I and HDL cholesterol were converted to percentiles by gender and by age (in 10-year age groups) within each study, allowing for direct comparisons between percentiles for mutations both within and between studies (CCHS and CGPS). Mean apoA-I and HDL cholesterol percentiles in individuals with a specific mutation were compared with the mean percentile (= 50th percentile of the normalized distribution) within the CCHS or CGPS as a whole using a z-test [23]. Mann-Whitney U-test and Fisher' s exact test compared, respectively, continuous and categorical variables between heterozygotes for different mutations and noncarriers. Number of NS and S variants identified among individuals with extremely low or high plasma levels of apoA-I were compared using Fisher' s exact test. Risk of MI for heterozygotes for all NS (S36A, F71Y, K107del, L144R, A164S) and for all S (K12K, S25S, A190A) mutations genotyped in both studies, was determined prospectively in the CCHS and CGPS combined, using Cox proportional hazards regression models with age as time scale and delayed entry (left truncation) in 1977. Fify-one individuals with a previous MI were excluded from the risk analyses. Hazard ratios were adjusted for age and sex, or multifactorially for age, sex, diabetes, hypertension and smoking.
Resequencing APOA1 in the CCHS identified a total of 40 genetic variants (Figure 2 and Table S2). Only seven variants (18%) had MAFs >1% (all in Hardy-Weinberg equilibrium, P-values: 0. 12 to 0. 82). Of these, none were in the coding region of APOA1, that is in exons 1–4 coding for the 267 amino acid prepropeptide. The cumulative number of genetic variants in APOA1 as a function of the number of individuals resequenced showed that by resequencing fewer than 100 individuals all seven variants in APOA1 with a MAF >1% were identified (Figure 2). In contrast, the number of genetic variants identified with a 0. 005%<MAF<0. 1% increased almost logarithmically reaching a plateau around 5,000 individuals, while the number of singletons (minor allele frequency = 0. 005%) increased almost linearly. In agreement with this, the number of different genetic variants identified in APOA1 as a function of the number of individuals with each variant, corresponding to the MAF, showed that seventeen of 40 variants (43%) were exceedingly rare and identified in only one or two individuals in the CCHS (Figure 3).
Using population-based resequencing of APOA1 in 10,330 individuals allowed description of the spectrum and distribution of genetic variants in this gene. Our results showed that the vast majority of variants, including variants associated with apoA-I and HDL cholesterol phenotype, were individually rare, though collectively relatively common. These results are in complete agreement with results from two previous population-based resequencing studies of three other genes affecting, respectively, triglycerides and diabetes related traits [25], [26]. Novel findings, compared to previous population-based screenings of apoA-I using isoelectric focusing [27], [28], include: First, the number of NS variants identified and the number of heterozygotes for these variants were, respectively, 5–6 fold and 10–20 fold increased. Second, we showed that 0. 27% of the population were heterozygous for variants associated with substantial reductions in apoA-I and HDL cholesterol levels, and 0. 41% were heterozygous for variants previously associated with amyloidosis, although none had been diagnosed with this disease. In addition, S variants, not identified by isoelectric focusing, also associated with reductions in apoA-I levels in the CCHS and CGPS combined (n>55,000). Third, heterozygosity for NS variants in APOA1 associated with a 2-fold increased risk of MI, largely driven by A164S, a variant not associated with apoA-I and HDL cholesterol levels. Finally, while these rare variants might have some effects on the extremes of the population distribution of apoA-I and HDL cholesterol and on levels in the individual, the contribution of both rare and common variants in APOA1 to the total variation in plasma levels of apoA-1 and HDL cholesterol were very modest, respectively, 0. 03% and 0. 3%, in agreement with the very large number of genes found to associate with apoA-I and/or HDL cholesterol levels in genomewide association studies [29]. The effect of common variants on plasma levels were 10-fold higher than for rare variants, suggesting that rare variants in this gene do not contribute in any major way to the missing heritability on a population level. An advantage of population-based resequencing is that the genetic variants identified can be tested against multiple phenotypes. This becomes especially important, if the gene under study has pleiotropic effects, i. e. affects multiple phenotypic traits, as is the case for APOA1: mutations in APOA1 have been associated with an inability to activate LCAT and with hereditary amyloidosis [5]–[7]. While mutations that poorly activate LCAT associate with low apoA-I and HDL cholesterol levels due to the rapid removal of lipid-poor discoidal HDL from the circulation [30], mutations that cause amyloidosis may [5], [6] or may not [9]–[11] associate with low apoA-I and/or HDL cholesterol, most likely depending on the severity of the mutation [8]. Of the seventeen nonsynonymous variants identified in the present study, only seven have been reported by others (P4R, V11X, S36A, A37T, F71Y, K107del, L144R) [11], [13], [24], [27], [31]–[41]. Of these variants, five (P4R, V11X, S36A, K107del, L144R) associated with low apoA-I and/or HDL cholesterol levels in the CCHS and in other studies [13], [32]–[34], [40], [41]. L144R is unable to activate LCAT [13], while S36A, F71Y, K107del, have been associated with amyloidosis [24], [38]. A164S, a variant without any association with HDL cholesterol or apoA-I levels in the CCHS and CGPS, was associated with an increased risk of IHD, MI, and premature death, and with reduced survival after diagnosis of IHD in the CCHS, most likely due to an attenuated form of cardiac amyloidosis [13]. Thus, four variants in APOA1 identified in 42 individuals (0. 41% of the population) have either previously been associated with or suspected of causing amyloidosis. Only two of these variants, S36A and K107del, associated with low HDL cholesterol in the CCHS. This highlights two points: 1) The inherent weakness of the extreme approach when a gene has pleiotropic effects. Using this approach in the CCHS, both F71Y, a known amyloidosis mutation [24], and A164S, a suspected amyloidosis mutation associated with increased risk of ischemic heart disease and early mortality [13], would have been assumed to be nonfunctional; 2) That variants in APOA1 associated with amyloidosis are relatively common in the general population. Comparing the results from the population-based resequencing approach with the results obtained using only the extremes of the population distribution of apoA-I in the CCHS showed that NS variants were overrepresented in the lower percentiles of the apoA-I distribution, especially those predicted in silico to be more pathological, and correctly predicted association with low apoA-I and/or HDL cholesterol levels. Thus, 0. 27% of the total population were heterozygous for one of nine different variants associated with substantial reductions in apoA-I and/or HDL cholesterol levels of up to, respectively, 39 mg/dL and 0. 9 mmol/L. As previously shown in the CCHS [13] and validated in the present study including the CGPS, NS mutations in APOA1 may associate with increased risk of MI, without associating with reduced apoA-1 and HDL cholesterol levels. The most likley explanation for this is that these mutations represent less severe amyloidosis mutations manifesting clinically as MI, instead of the more severe restrictive cardiomyopathy [42]. Accordingly, we found that the combined NS mutations associated with an increased risk of MI, the main contributor to this increased risk was A164S, a presumed amyloidogenic mutation not associated with apoA-1 or HDL cholesterol levels in either the CCHS or the CGPS. Our study suggests that the extreme phenotype approach used in a number of previous studies [12]–[16] is a powerful analytical strategy to capture the effects of both common and rare genetic variants on a specific a priori specified complex trait, provided the gene in question does not have pleiotropic effects. However, the success of this strategy depends on using a gene-near phenotype, preferably the direct gene product, on the size of the underlying study, the cut-off point for the extreme percentiles screened, and the frequencies and effect sizes of the identified variants. In conclusion, population-based resequencing of APOA1 identified a majority of rare NS variants associated with reduced apoA-I and HDL cholesterol levels and/or predisposing to amyloidosis. In addition, NS variants associated with increased risk of MI.
Rare genetic variants, identified by in-detail resequencing of loci, may contribute to complex traits. We used the apolipoprotein A-I gene (APOA1), a major high-density lipoprotein (HDL) gene, and population-based resequencing to determine the spectrum of genetic variants, the phenotypic characteristics of these variants, and how these results compared with results based on resequencing only the extremes of the apolipoprotein A-I (apoA-I) distribution. By resequencing APOA1 in >10,000 Danes and genotyping an additional >45,000, we show that population-based resequencing of APOA1 identifies a majority of rare genetic variants that together are relatively frequent: 0. 27% of the population are heterozygous for nonsynonymous (NS) variants in APOA1 that associate with substantial reductions in apoA-I and HDL cholesterol, and 0. 41% are heterozygous for variants predisposing to amyloidosis. NS variants associated with a hazard ratio of 1. 72 (1. 09–2. 70) for myocardial infarction (MI), largely driven by A164S, a variant not associated with apoA-I or HDL cholesterol levels. Resequencing only the extremes of the apoA-I distribution, between 79% and 21% of all variants are not identified; among these are variants previously associated with amyloidosis. These results provide direct evidence that rare NS variants in APOA1 contribute to low apoA-I and HDL cholesterol levels, to susceptibility to amyloidosis, and to risk of MI in the general population.
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apolipoprotein genes
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Population-Based Resequencing of APOA1 in 10,330 Individuals: Spectrum of Genetic Variation, Phenotype, and Comparison with Extreme Phenotype Approach