Genetic variants in CYP7A1 and risk of myocardial infarction and symptomatic gallstone disease

Genetic variants in CYP7A1 and risk of myocardial infarction and symptomatic gallstone disease Abstract Aims Myocardial infarction (MI) and gallstone disease (GSD) are intrinsically linked via cholesterol metabolism. We tested the hypothesis that genetic variants in the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver, are associated with risk of MI and GSD in the general population. Methods and results We performed tests of association between lipid levels and eight rare non-synonymous mutations and two common variants, rs2081687 and rs3808607, in CYP7A1 in 100 149 individuals from the general population. We further tested whether weighted allele scores for rs2081687 and rs3808607, which were associated with increased plasma levels of low-density lipoprotein (LDL) cholesterol, were associated with an increased risk of both MI and symptomatic GSD. During a mean follow-up of 7 years (0–23 years), MI developed in 2326 individuals and GSD in 2007. For rare mutations, CYP7A1 allele count was associated with an increase in LDL cholesterol of 12% (0.4 mmol/L) for individuals with the highest vs. the lowest allele count (P for trend = 3 × 10−4). For common variants, CYP7A1 weighted allele scores in individuals with a score >0.04 vs. ≤0 were associated with stepwise increases in LDL cholesterol of up to 2.4% (0.08 mmol/L), and with corresponding multifactorially adjusted hazard ratios of 1.25 [95% confidence interval (CI) 1.10–1.41] for MI and 1.39 (95% CI 1.22–1.59) for GSD (P for trend = 5 × 10−4 and 2 × 10−7, respectively). Results were similar in meta-analyses including publicly available data from large consortia. Conclusion Genetic variants in CYP7A1 which are associated with increased levels of LDL cholesterol, are associated with an increased risk of both MI and GSD. View largeDownload slide View largeDownload slide Genetics, Lipids, Cardiovascular disease, Gallstones Introduction Cholesterol plays a pivotal role in the pathogenesis of both myocardial infarction (MI) and gallstone disease (GSD), two exceedingly common and costly diseases.1,2 Elevated levels of plasma cholesterol are a well-known causal risk factor for atherosclerosis and MI, whereas elevated levels of biliary cholesterol promote the formation of cholesterol gallstones.3 Myocardial infarction and GSD, two seemingly unrelated diseases, are therefore intrinsically linked via cholesterol metabolism. Because cholesterol cannot be degraded, excess cholesterol is converted into bile salts (conjugated bile acids) or directly excreted into bile.4 Cholesterol 7 alpha-hydroxylase (CYP7A1), a liver-specific cytochrome P-450 monooxygenase, catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids in the classical pathway (Figure 1). Low CYP7A1 activity may, therefore, reduce the conversion of cholesterol to bile acids in the liver and increase hepatic cholesterol content, resulting in secondary hypercholesterolaemia due to down-regulation of hepatic low-density lipoprotein (LDL) receptors, and simultaneously reduce the ability to solubilize cholesterol in bile acid mixed micelles, predisposing to GSD. Taken together, this could lead to an increased risk of both MI and GSD (Take home figure). Figure 1 View largeDownload slide Biological function of cholesterol 7 alpha-hydroxylase. Cholesterol 7 alpha-hydroxylase catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids, the major pathway for catabolism of cholesterol in humans. Bile acids are transported as bile salts (conjugated bile acids) in to the bile via the bile salt export pump, ATP binding cassette subfamily B member 11. In the bile (right), increased conversion of cholesterol into bile acids increases the solubility of cholesterol in bile salt mixed micelles and hence prevents gallstone formation. Inhibition of the hepatic activity of cholesterol 7 alpha-hydroxylase by inactivating genetic variants in the CYP7A1 gene might therefore cause gallstone disease as a result of reduced bile acid secretion. Furthermore, the reduced conversion of cholesterol to bile acids might result in elevated liver cholesterol levels, down-regulated low-density lipoprotein receptors, secondary hypercholesterolaemia, and increased risk of atherosclerosis and ischaemic heart disease.6 ABCB11, ATP binding cassette subfamily B member 11; CYP7A1, Cytochrome P450, family 7, subfamily A, poplypeptide 1, the gene that encodes cholesterol 7 alpha-hydroxylase; LDL, low-density lipoprotein. Figure 1 View largeDownload slide Biological function of cholesterol 7 alpha-hydroxylase. Cholesterol 7 alpha-hydroxylase catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids, the major pathway for catabolism of cholesterol in humans. Bile acids are transported as bile salts (conjugated bile acids) in to the bile via the bile salt export pump, ATP binding cassette subfamily B member 11. In the bile (right), increased conversion of cholesterol into bile acids increases the solubility of cholesterol in bile salt mixed micelles and hence prevents gallstone formation. Inhibition of the hepatic activity of cholesterol 7 alpha-hydroxylase by inactivating genetic variants in the CYP7A1 gene might therefore cause gallstone disease as a result of reduced bile acid secretion. Furthermore, the reduced conversion of cholesterol to bile acids might result in elevated liver cholesterol levels, down-regulated low-density lipoprotein receptors, secondary hypercholesterolaemia, and increased risk of atherosclerosis and ischaemic heart disease.6 ABCB11, ATP binding cassette subfamily B member 11; CYP7A1, Cytochrome P450, family 7, subfamily A, poplypeptide 1, the gene that encodes cholesterol 7 alpha-hydroxylase; LDL, low-density lipoprotein. Take home figure View largeDownload slide We speculate that low activity of cholesterol 7 alpha-hydroxylase, which catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids in humans, results in a decrease in bile acid synthesis (1) and accumulation of cholesterol in the liver (2) leading to down-regulation of hepatic low-density lipoprotein receptors (3), hypercholesterolaemia (4), and secondary increased risk of myocardial infarction (5). Simultaneously, inability to solubilize cholesterol in bile salt mixed micelles predisposes to gallstone disease (6). Here, we show that common genetic variants in CYP7A1, which are associated with increased levels of low-density lipoprotein cholesterol mimicking the effect of a loss-of-function mutation in humans7 are associated with an increased risk of both myocardial infarction and gallstone disease. Therefore, increasing cholesterol 7 alpha-hydroxylase activity by bile acid sequestrants and other bile acid pool modulators may reduce risk of both myocardial infarction and gallstone disease. Take home figure View largeDownload slide We speculate that low activity of cholesterol 7 alpha-hydroxylase, which catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids in humans, results in a decrease in bile acid synthesis (1) and accumulation of cholesterol in the liver (2) leading to down-regulation of hepatic low-density lipoprotein receptors (3), hypercholesterolaemia (4), and secondary increased risk of myocardial infarction (5). Simultaneously, inability to solubilize cholesterol in bile salt mixed micelles predisposes to gallstone disease (6). Here, we show that common genetic variants in CYP7A1, which are associated with increased levels of low-density lipoprotein cholesterol mimicking the effect of a loss-of-function mutation in humans7 are associated with an increased risk of both myocardial infarction and gallstone disease. Therefore, increasing cholesterol 7 alpha-hydroxylase activity by bile acid sequestrants and other bile acid pool modulators may reduce risk of both myocardial infarction and gallstone disease. That plasma CYP7A1 activity is inversely correlated with plasma levels of LDL cholesterol in a monotonic dose–response fashion is supported by the following: (i) Treatment with bile acid sequestrants and more recently with elobixibat, an ileal bile acid transporter inhibitor, increase CYP7A1 activity and reduce LDL cholesterol in a dose-dependent manner,5,6 results which are in agreement with overexpression of CYP7A1 in animal models and (ii) Conversely, homozygosity for a loss-of-function mutation in CYP7A1 has previously been associated with loss of CYP7A1 enzyme activity, severe hypercholesterolaemia and increased hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and with premature GSD in a family study.7 Importantly, in that study there was a significant stepwise increase in plasma levels of LDL cholesterol as a function of genotype from wildtype to heterozygotes to homozygotes, implying that LDL cholesterol increased in a monotonic dose–response fashion with loss of enzyme activity. Taken together, these data suggest that there are an inverse and causal relationship between CYP7A1 activity and plasma LDL cholesterol levels. Therefore, we used the association between genetic variants in CYP7A1 and plasma levels of LDL cholesterol as proxies for CYP7A1 activity. This is clinically important because it suggests that bile acid sequestrants and other bile-acid pool modulators which lower LDL cholesterol by increasing CYP7A1 activity,6,8 may reduce risk of GSD in addition to reducing cardiovascular risk.9 We tested the hypothesis that lifelong high levels of LDL cholesterol associated with genetic variants in CYP7A1 are associated with both increased risk of MI and GSD in the general population. We genotyped eight non-synonymous mutations in CYP7A1 and two common variants previously associated with LDL cholesterol levels, in two prospective studies of the general population, the Copenhagen General Population Study (CGPS) and the Copenhagen City Heart Study (CCHS), totalling 100 149 individuals, of whom 2326 developed MI and 2007 developed symptomatic GSD. Methods The study was approved by institutional review boards and Danish ethics committees and was conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all individuals. Participants We included individuals in two similar prospective studies of the Danish general population, the CGPS and the CCHS.10 All individuals were white and of Danish descent. The Copenhagen General Population Study The CGPS was initiated in 2003, and enrolment is ongoing. Individuals were selected with the use of the National Danish Civil Registration System to reflect the adult Danish population aged 20 to 100 years or older. Data were obtained from a questionnaire, a physical examination, and from collection of blood samples. We included 89 944 consecutive individuals in the current analyses. During a mean follow-up of 6 years (0–11 years) (which ended in November 2014), 1448 had an incident MI and 1512 had incident symptomatic GSD. The Copenhagen City Heart Study In the CCHS, we included 10 205 individuals with DNA available from the 1991–94 and 2001–03 examinations in the current analyses. Individuals were recruited and examined as in the CGPS. During a mean follow-up of 16 years (0–23 years) (which ended in November 2014), 878 had an incident MI and 495 had incident symptomatic GSD. Combining the participants in the CGPS and the CCHS yielded a total of 100 149 participants at baseline (Table 1). During a mean follow-up of 7 years (0–23 years) (which ended in November 2014), MI developed in 2326 individuals and symptomatic GSD developed in 2007. Individuals with prevalent events at baseline were excluded from analyses of risk of MI and GSD (2179 individuals with MI and 3950 with GSD). In both studies, follow-up was 100% complete that is we did not lose track of even a single individual. DNA was available on all individuals, and lipid values were available on more than 98%. Table 1 Baseline characteristics of individuals by events No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a Values are median (interquartile range), or number of individuals (%). Events are prevalent and incident events. P-values by Mann-Whitney U-test or Pearson’s χ2 test. a P-value <0.001 vs. individuals with no event. b In women only. GSD, gallstone disease; MI, myocardial infarction. Table 1 Baseline characteristics of individuals by events No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a Values are median (interquartile range), or number of individuals (%). Events are prevalent and incident events. P-values by Mann-Whitney U-test or Pearson’s χ2 test. a P-value <0.001 vs. individuals with no event. b In women only. GSD, gallstone disease; MI, myocardial infarction. For additional information on clinical endpoints, laboratory analyses, and other covariates including measurement of liver fat content by computed tomography (CT) scans, see Supplementary material online, Appendix. Genotyping We genotyped all non-synonymous variants in CYP7A1 reported in Exome Variant Server (http://evs.gs.washington.edu/EVS/) with a minor allele frequency above 0.03% in European Americans (A13V, rs147162838; N36K, rs138113674; Y75C, rs377254635; T193I, rs72647413; R260L, rs139396617; G377S, rs117423932; P398A, rs142708991; G417R, rs201787113). In addition, we genotyped two common variants in CYP7A1, rs2081687 C>T and rs3808607 A>C (NM_000780.3: c.-267A>C; old nomenclature: -278A>C and -204A>C11,12). Both variants have previously been shown to associate with LDL cholesterol levels, either as the lead single nucleotide polymorphism (SNP) in published genome-wide association study (GWAS) studies (rs2081687)13 or with consistent stepwise associations as a function of genotype in association studies (rs3808607).11,12 These two common variants are in complete linkage disequilibrium (LD) with variants spanning the core promoter, coding region, and downstream region of CYP7A1 (Supplementary material online, Figure S1). We genotyped rs3808607 as a proxy for rs3824260 (Supplementary material online, Figure S1, right), because the latter variant proved technically challenging to genotype. There are no common non-synonymous variants in CYP7A1. Genotyping was by TaqMan-based assays (Applied Biosystems, Foster City, CA, USA) or by KASP genotyping technology (LGC Genomics Ltd, Hoddesdon, Herts, UK). Statistical analyses Data were analysed using Stata SE 13. The χ2 test evaluated the Hardy–Weinberg equilibrium. To compare characteristics in individuals by disease status or genotype, Mann–Whitney U-test, or Cuzick’s test for trend was used to compare continuous covariates, and Pearson’s χ2 test to compare categorical covariates. For trend tests, genotypes, CYP7A1 weighted allele scores (based on β-coefficients) or allele count were coded 0, 1, 2, and so forth. The genotype, CYP7A1 weighted allele score or allele count associated with the lowest LDL cholesterol level was used as the reference (coded 0). Cuzick’s test for trend was used to compare levels of continuous variables as a function of genotype, CYP7A1 allele count, and weighted allele scores. For both rare and common variants, we used an additive genetic model. Multifactorial adjustment in both logistic regression analyses (rare variants) and in Cox regression analyses (common variants) was for well-known risk factors for MI and GSD: age, gender, body mass index, hypertension, diabetes mellitus, physical activity, smoking, alcohol consumption, hormone replacement therapy (women only), and lipid-lowering therapy, i.e. the same characteristics as shown in Table 1. For the rare non-synonymous variants, we used a simple allele count based on the number of LDL-increasing alleles of the individual variants: Individuals heterozygous for T193I who had LDL cholesterol levels significantly lower than average were graded 0 (=reference group; N = 214); individuals with average LDL cholesterol levels were coded 1 (heterozygotes or homozygotes for the five variants without significant associations with LDL cholesterol, and all individuals who were wild type for any of the seven variants identified; N = 106 585); and individuals heterozygous for R260L who had LDL cholesterol levels significantly higher than average were graded 2 (N = 177) (Figure 2). The two common variants, rs2081687 C>T and rs3808607 A>C, were combined in weighted allele scores as follows: (i) the individual β-coefficients for LDL cholesterol for the minor allele of each variant was calculated by a regression analysis including both variants and adjusting for age, gender, and cohort (Supplementary material online, Table S1A; note that the effects of the minor alleles on LDL cholesterol are in opposite directions); (ii) the weighted allele scores were calculated by summation of these β-coefficients for each genotype combination (Supplementary material online, Table S1B, third column from left); and (iii) the weighted allele scores were divided into three groups by increasing score and LDL cholesterol levels: weighted allele score ≤0 (reference), >0 to 0.04, and >0.04 to achieve groups including a reasonable number of individuals in each group for analyses (Supplementary material online, Table S1B, third to fifth columns from left). For both variants, we show internal weights unadjusted and adjusted for the other variant, and unadjusted external weights from the Global Lipids Genetics Consortium.13 Figure 2 View largeDownload slide Lipid levels as a function of rare genetic variants in CYP7A1, individually and combined. The seven rare, non-synonymous variants were combined in a simple allele count based on the number of low-density lipoprotein cholesterol-increasing alleles for the individual genotypes. P-values by Mann–Whitney U-tests or for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Figure 2 View largeDownload slide Lipid levels as a function of rare genetic variants in CYP7A1, individually and combined. The seven rare, non-synonymous variants were combined in a simple allele count based on the number of low-density lipoprotein cholesterol-increasing alleles for the individual genotypes. P-values by Mann–Whitney U-tests or for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Fine-Grey curves and tests for trend evaluated the cumulative incidences of MI and symptomatic GSD as a function of age and CYP7A1 weighted allele scores with death as a competing event. Cox proportional hazards regression models using age as time scale and delayed entry (left truncation), which implies that age is automatically adjusted for, were used to estimate hazard ratios (HRs) for MI, symptomatic GSD, coronary intervention, cholecystectomy, non-alcoholic fatty liver disease (NAFLD), and cirrhosis as a function of CYP7A1 weighted allele scores. Multifactorial adjustments were for well-known risk factors for MI, and symptomatic GSD: age, gender, body mass index, hypertension, diabetes mellitus, physical activity, smoking, alcohol consumption, hormone replacement therapy (women only), and lipid-lowering therapy. Analyses were conducted from blood sampling at study entry (CCHS 1991–94 or 2001–03; CGPS 2003 and ongoing) through 10 November 2014. Only incident events were included, comprising a total of 2326 individuals with MI and 2007 with symptomatic GSD. In sensitivity analyses, we stratified the analyses by gender and study, grouped the weighted allele scores by other cut-points (4 and 5 groups), and excluded individuals on lipid-lowering therapy. Further, we determined the effect of a one standard deviation increase in CYP7A1 weighted allele score on risk of MI and symptomatic GSD. Finally, logistic regression analyses were used to assess whether risk of MI, risk of symptomatic GSD, or a one category increase in weighted allele score was associated with potential measured confounders (risk factors for MI and GSD: age ≥50 vs. <50 years; gender: male vs. female; body mass index ≥25 vs. <25 kg/m2; hypertension yes vs. no; diabetes mellitus yes vs. no; physical activity low vs. high; smoking yes vs. no; alcohol consumption high vs. low; hormone replacement therapy in women yes vs. no; and lipid-lowering therapy yes vs. no). Finally, we performed meta-analyses combining data from CCHS and CGPS with data from (i) The Global Lipids Genetics Consortium13 for LDL cholesterol; (ii) CARDIoGRAMplusC4D 1000 Genomes for risk of IHD14; and (iii) Recent GWAS data on GSD15 using the ‘metan’ command in Stata. Results Baseline characteristics of the 100 149 individuals by disease status, including both prevalent and incident events, are shown in Table 1. As expected, well-known risk factors for MI and GSD were over-represented in those with events. Baseline characteristics of individuals in the CGPS and CCHS are shown separately in Supplementary material online, Table S2; differences between studies reflect differences in lifestyle factors over time. In contrast to the observational data (Table 1), baseline characteristics as a function of weighted allele scores were similar, although use of lipid-lowering therapy tended to be slightly more frequent with increasing score, and hence increasing levels of LDL cholesterol (Table 2). Taken together, these data suggest that the genetic data based on stratification by LDL cholesterol, as opposed to the observational epidemiological data, are largely unconfounded by known, measured risk factors for MI and GSD. Genotype distributions did not differ from Hardy–Weinberg equilibrium (P > 0.25). There was moderate LD between the two common variants, CYP7A1 rs2081687 C>T and rs3808607 A>C (D´ = 0.93; R2 = 0.70), although in approximately 15% of individuals, or 14 778 individuals (8% of haplotypes) the minor alleles were not inherited on the same haplotype (Supplementary material online, Figure S1; minor allele frequencies 0.33 and 0.39, respectively). Table 2 Baseline characteristics as a function of CYP7A1 weighted allele score Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Values are median (interquartile range), or number of individuals (%). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test or Pearson’s χ2 test. a In women only. Table 2 Baseline characteristics as a function of CYP7A1 weighted allele score Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Values are median (interquartile range), or number of individuals (%). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test or Pearson’s χ2 test. a In women only. Cyp7A1 genotype, plasma lipids, and lipoproteins The effects of the individual rare, non-synonymous variants on LDL cholesterol levels and other lipids and lipoproteins are shown in Figure 2. Two of seven variants, T193I and R260L, were associated with LDL cholesterol levels which were, respectively, 3.7% lower and 7.9% higher than non-carriers (P-values 0.05 and 1 × 10−3), with similar trends in the CGPS and CCHS separately (Supplementary material online, Figures S2 and S3). Because our hypothesis was that lifelong high LDL cholesterol levels associated with genetic variants in CYP7A1 were associated with both increased risk of MI and GSD in the general population, we combined the seven rare variants (A13V, N36K, T193I, R260L, G377S, P398A, G417R; Y75C was not identified) by simple allele counts based on the number of LDL cholesterol-increasing alleles of the individual genotypes, using heterozygotes for the minor allele of T193I (GA genotype) as the reference group (=0; lower than average LDL cholesterol), heterozygotes and homozygotes for the five variants not associated with LDL cholesterol (A13V, N36K, G377S, P398A, and G417R) and wildtypes from all seven variants as the middle group (=1; average LDL cholesterol), and heterozygotes for R260L (CA genotype) as the top group (=2; higher than average LDL cholesterol). The corresponding stepwise increase in LDL cholesterol was up to 12% (0.4 mmol/L) for individuals with an allele count of two vs. 0 (Figure 2; P for trend = 3 × 10−4), compared to up to 2.4% (0.08 mmol/L) for common variants (Figure 3, see below). Rs2081687 and rs3808607 were individually associated with stepwise increases in LDL- and total cholesterol of up to 2.6% (0.09 mmol/L) and 1.7% (0.10 mmol/L) and up to 1.6% (0.05 mmol/L) and 1.1% (0.06 mmol/L), respectively, in homozygotes for the minor alleles vs. reference genotypes (Figure 3; P for trend from 2 × 10−7 to 4 × 10−17). However, as indicated by the individual adjusted β-coefficients (Supplementary material online, Table S1A), and as also evident when plotting LDL- and total cholesterol as a function of the individual genotypes on either the wildtype (CC or AA), heterozygote (CT or AC), or homozygote (TT or CC) background of the other variant (Supplementary material online, Figure S4), the minor alleles of the two variants were associated with LDL- and total cholesterol levels in opposite directions: higher levels for rs2081687 T-allele, and lower levels for rs3808607 C-allele. Therefore, weighted allele scores based on summation of the individual β-coefficients for LDL cholesterol for the two common variants (by linear regression analysis including both variants into the same analysis adjusting for age, gender, and cohort) were constructed and grouped into three groups: weighted allele score ≤0 (reference), 0–0.04, >0.04, each including a reasonable number of individuals for analyses (Supplementary material online, Table S1B). CYP7A1 weighted allele scores were associated with stepwise increases in plasma levels of LDL- and total cholesterol of up to 2.4% (0.08 mmol/L) and 1.6% (0.09 mmol/L), respectively, for individuals with a score of >0.04 vs. ≤0 (Figure 3; P for trend: 2 × 10−15 and 5 × 10−17). Results were similar in separate analyses of data from the CCHS and CGPS (Supplementary material online, Figure S5). Results for apolipoproteins B and -A-I were concordant with results for, respectively, LDL- and total cholesterol, and high-density lipoprotein cholesterol levels (Supplementary material online, Figure S6). Importantly, in a region comprising at least 15 megabases cantered on CYP7A1, no other variants associated with LDL cholesterol levels were in LD with variants in CYP7A1, suggesting that enrichment for pleiotropy due to LD with other variants seemed less likely.16 Cyp7A1 genotype, risk of myocardial infarction, and symptomatic gallstone disease In individuals with a weighted allele score ≤0 through >0–0.04 to >0.04, there was a stepwise increase in cumulative incidence of MI and symptomatic GSD as a function of age (Figure 4; P for trend = 2 × 10−4 and 8 × 10−6). For MI risk increased as a function of increasing weighted allele score from age 55, while risk of GSD increased from about age 30. As expected for very common variants, the increase in cumulative incidences as a function of allele score, were modest: at 60 years the cumulative incidence for MI was increased by 0.7%, and by age 80 by 2.5% in individuals with a score >0.04 vs. 0. For GSD, the corresponding increases were 1.0% at age 40, 2.4% at age 60, and 4.3% at age 80 (Figure 4). Results were similar in the CGPS and CCHS separately (Supplementary material online, Figures S7 and S8). Figure 3 View largeDownload slide Lipid levels as a function of common variants in CYP7A1, individually and combined. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score based on summation of the individual β-coefficients for low-density lipoprotein cholesterol (by logistic regression analysis including both variants into the same analysis adjusting for age, gender, and cohort) for the minor alleles (Supplementary material online, Table S1). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Figure 3 View largeDownload slide Lipid levels as a function of common variants in CYP7A1, individually and combined. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score based on summation of the individual β-coefficients for low-density lipoprotein cholesterol (by logistic regression analysis including both variants into the same analysis adjusting for age, gender, and cohort) for the minor alleles (Supplementary material online, Table S1). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Figure 4 View largeDownload slide Cumulative incidences of myocardial infarction and symptomatic gallstone disease as a function of age and CYP7A1 weighted allele score, after adjustment for death as competing event. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). P-values are for trend tests across sub-hazard ratios in Fine-Grey models. Figure 4 View largeDownload slide Cumulative incidences of myocardial infarction and symptomatic gallstone disease as a function of age and CYP7A1 weighted allele score, after adjustment for death as competing event. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). P-values are for trend tests across sub-hazard ratios in Fine-Grey models. The multifactorially adjusted [for well-known risk factors for MI and GSD: age, gender, body mass index, hypertension, diabetes mellitus, physical activity, smoking, alcohol consumption, hormone replacement therapy (women only), and lipid-lowering therapy, see Table 1] HRs for both MI and symptomatic GSD increased stepwise with increasing CYP7A1 weighted allele score up to 1.25 [95% confidence interval (CI) 1.10–1.41; P for trend= 5 × 10−4] for MI and 1.39 (95% CI 1.22–1.59; P for trend = 2 × 10−7) for symptomatic GSD, for individuals with a CYP7A1 weighted allele score ≥0.04 when compared with ≤0 (Figure 5, top). Results were similar in unadjusted analyses (Supplementary material online, Figure S9), or when stratified by gender or cohort (Figure 5, middle and bottom). In agreement, the HRs for coronary intervention (percutaneous coronary intervention (PCI) and/or coronary artery bypass grafting (CABG) and cholecystectomy increased stepwise with increasing allele score up to 1.20 (1.04–1.39) for coronary intervention and 1.47 (1.24–1.74) for cholecystectomy in individuals with an allele score >0.04 vs. 0 (Supplementary material online, Figure S10; P for trend: 0.02 and 8 × 10−6). Figure 5 View largeDownload slide Plasma low-density lipoprotein cholesterol levels, risk of myocardial infarction and symptomatic gallstone disease as a function of CYP7A1 weighted allele score: overall, stratified by gender, and by cohort. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). Hazard ratios were multifactorially adjusted for age, gender, body mass index, hypertension, diabetes, physical activity, smoking, alcohol consumption, hormone replacement therapy, and lipid-lowering therapy. P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test, or for trend tests of hazard ratios. CI, confidence interval; HR, hazard ratio; LDL, low-density lipoprotein; N, number. Figure 5 View largeDownload slide Plasma low-density lipoprotein cholesterol levels, risk of myocardial infarction and symptomatic gallstone disease as a function of CYP7A1 weighted allele score: overall, stratified by gender, and by cohort. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). Hazard ratios were multifactorially adjusted for age, gender, body mass index, hypertension, diabetes, physical activity, smoking, alcohol consumption, hormone replacement therapy, and lipid-lowering therapy. P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test, or for trend tests of hazard ratios. CI, confidence interval; HR, hazard ratio; LDL, low-density lipoprotein; N, number. Due to the relatively few individuals with rare variants, lack of statistical power did not allow reliable risk estimates for MI and symptomatic GSD (Supplementary material online, Figure S11). Meta-analyses on LDL cholesterol, risk of ischaemic heart disease, and gallstone disease In meta-analysis of CYP7A1 rs2081687 on LDL cholesterol including data from the Global Lipids Genetics Consortium,13 CCHS, and CGPS, the overall random effects beta-coefficient was 0.04 (95% CI 0.03–0.04) (P = 1 × 10−25) per risk increasing T-allele with an I2 = 0.0% (Supplementary material online, Figure S12A). In meta-analysis of CYP7A1 rs2081687 on risk of coronary artery disease including data from CARDIoGRAMplusC4D 1000 Genomes,14 CCHS, and CGPS, the overall random effects odds ratio was 1.01 (95% CI 1.00–1.03, P = 0.07) per risk increasing T-allele with an I2 = 0.0 (Supplementary material online, Figure S13A), while the overall random effects odds ratio for validated MI in the CCHS and CGPS was 1.07 (95% CI 1.02–1.12) (Supplementary material online, Figure S13C). For comparison, we also show data from the Global Lipids Genetics Consortium13 and CARDIoGRAMplusC4D 1000 Genomes14 on rs13277801 (Supplementary material online, Figures S12B and S13B), a variant in complete LD with the variant above (rs2081687). Rs13277801 was typed in 173 010 individuals as opposed to 89 888 for rs2081687 and the significance level for LDL cholesterol was therefore lower (Supplementary material online, Figure S1). Results for this variant were similar to those for rs2081687 (Supplementary material online, Figures S12 and S13A and B); the variant was not available in the GWAS of GSD. Finally, in meta-analysis of CYP7A1 rs6471717, a variant in complete LD with rs2081687 (R2 = 0.98), in the discovery cohorts of the largest GWAS study of GSD to date,15 the overall random effects odds ratio for GSD was 1.12 (95% CI 1.06–1.20) per risk increasing G-allele (Supplementary material online, Figure S14A) and 1.10 (95% CI 1.05–1.16) in the Copenhagen Studies (Supplementary material online, Figure S14B). Including data from the discovery cohorts on rs6471717 (Supplementary material online, Figure S14A) with data from the present study on rs2081687 (in complete LD with rs6471717) gave similar results (Supplementary material online, Figure S14C). Taken together, consortia data supported the results from the Copenhagen Studies on LDL cholesterol, risk of coronary artery disease, and risk of GSD as a function of common genetic variants in CYP7A1. Cyp7A1 genotype, liver parameters and inflammatory markers, liver fat content, and risk of non-alcoholic fatty liver disease and cirrhosis For the two common variants (rs2081687 and rs3808607), CYP7A1 weighted allele score was associated with a stepwise increase in gamma-glutamyl transferase—a sensitive marker of cholestasis—of up to 4.1% (1.58 U/L), for individuals with an allele score of >0.04 vs. ≤0 (P for trend = 3 × 10−4) (Supplementary material online, Figures S15 and S16, top middle column). In contrast, allele count was not associated with other plasma markers of liver function or inflammation. Furthermore, CYP7A1 weighted allele score was not associated with liver fat content based on liver attenuation on CT scans measured in Hounsfield Units, or with risk of NAFLD or liver cirrhosis (Supplementary material online, Figure S16, top). For comparison, PNPLA3 I148M genotype, a strong and specific cause of high liver fat content (=triglyceride content), was associated with stepwise increases in liver fat content (P for trend = 4 × 10−5) and alanine aminotransferase, a marker of liver cell damage (P for trend 2 × 10−42), and with HRs for NAFLD and liver cirrhosis of, respectively, 2.18 (95% CI 1.66–2.87) and 3.05 (95% CI 2.21–4.22) in homozygotes vs. non-carriers (Supplementary material online, Figure S16, bottom). Validation and sensitivity analyses To further test the robustness of our results, we conducted several sensitivity analyses. Using unadjusted data (Supplementary material online, Figure S9), stratifying analyses by gender or by study (CGPS and CCHS) (Figure 5 middle and bottom), dividing the weighted allele score at other cut points (4 or 5 groups) (Supplementary material online, Figure S17), or excluding individuals on lipid-lowering therapy (Supplementary material online, Figure S18), gave similar results. Furthermore, one unit (=1 standard deviation) increase in CYP7A1 weighted allele score was associated with HRs of 1.08 (1.04–1.12) for MI and 1.11 (1.06–1.16) for symptomatic GSD (P for trend: 3 × 10−4 and 2 × 10−6, respectively). For data on confounding and a discussion of this see Supplementary material online, Appendix and Supplementary material online, Figures S19 and S20. Discussion The main finding of this study is that lifelong, increased levels of plasma LDL cholesterol associated with genetic variants in CYP7A1 are associated with risk of both MI and GSD in the general population. These results suggest that MI and gallstones are intrinsically linked via the function of CYP7A1. The findings are of potential clinical importance because they suggest that CYP7A1 could be a relevant drug target for reducing risk of gallstones in addition to residual cardiovascular risk. It might be speculated, that bile acid sequestrants and other bile-acid pool modulators6,8 which lower plasma LDL cholesterol by increasing CYP7A1 activity and de novo synthesis of bile acids, may therefore simultaneously protect against ischaemic heart disease and GSD. To our knowledge, this is the first study to simultaneously assess plasma lipid levels, risk of MI, and risk of symptomatic GSD as a function of genetic variation in CYP7A1 in the general population, and the first study to use combined and weighted allele scores. Previous studies including between 379 and 2330 individuals have reported associations between rs3808607 (variously called -204/-278 A>C) and/or rs3824260 (variously called -496/-467/-554 T>C), two variants in complete LD,11 and lipid and lipoprotein levels,11,12,17–19 GSD,17,19 or progression of atherosclerosis.18 However, results have been conflicting, most likely because of the limited size of the studies, and because rs3808607 and rs3824260, as shown in the present study, are in LD with 3’variants (rs2081687 and others) which have opposite effects on plasma LDL cholesterol levels. Furthermore, in GWAS, rs2081687 has consistently been associated with plasma LDL cholesterol levels,13 and in a recent large GWAS, we have shown an association between rs6471717, a variant in complete LD with rs2081687 in our study (D’ = 0.99; R2 = 0.98), and risk of GSD.15 In agreement, in meta-analyses combining data from the Copenhagen Studies with data from the Global Lipids Genetics consortium,13 CARDIoGRAMplusC4D 1000 Genomes,14 or the studies included in the discovery cohort of the large gallstone GWAS mentioned above,15 the minor T-allele of rs2081687 (or G allele of rs6471717) was associated with high LDL cholesterol, a very modest increase in risk of coronary artery disease which became more significant for the hard endpoint MI, and an increase in risk of GSD. Because, we did not measure CYP7A1 activity, a major assumption of the present study is that as CYP7A1 enzyme function decreases, LDL cholesterol increases in a monotonic dose–response fashion, and that common genetic variants in CYP7A1 associated with a gene-dosage effect on plasma levels of LDL cholesterol can, therefore, be used as proxies for CYP7A1 activity. This is supported by several lines of evidence. First, treatment with bile-acid sequestrants which, by interrupting the enterohepatic circulation, deplete the bile acid pool, significantly increase CYP7A1 activity and de novo synthesis of bile acids, and lower plasma LDL cholesterol levels in a dose-dependent manner5 by increased receptor-mediated clearance of LDL.4,20,21 Treatment with bile-acid sequestrants decreases plasma LDL cholesterol by up to 25%, and reduces risk of coronary heart disease death and/or non-fatal MI when used as monotherapy.9,20–22 Due to the associated increase in CYP7A1 activity and bile acid synthesis, we speculate that bile acid sequestrants likely also reduce the risk of GSD, although this has not been tested directly. Second, Rudling et al.6 more recently showed that elobixibat, a minimally absorbed ileal bile acid transporter inhibitor, reduced LDL cholesterol and increased 7α-hydroxy-4-cholesten-3-one (C4) in a dose-dependent manner. C4 is a direct plasma marker of the enzymatic activity of CYP7A1 in the liver, the rate-limiting enzyme in bile acid synthesis for which cholesterol is the precursor.23 Both these studies therefore support that as CYP7A1 activity increases, LDL cholesterol decreases in a dose-dependent manner. Third, this interpretation is in agreement with results from animal models in which overexpression of the CYP7A1 gene resulted in a dose-dependent decrease in plasma cholesterol concentrations, mainly in the LDL sized lipoproteins, prevented diet-induced hypercholesterolaemia24,25 and protected against both atherosclerosis and GSD.24 Finally, homozygosity for a loss-of-function mutation in CYP7A1 has previously been associated with loss of CYP7A1 enzyme activity both in in vitro transfection studies and directly in human liver biopsies, with mean LDL cholesterol levels in three homozygotes of approximately 5 mmol/L corresponding to levels in heterozygous familial hypercholesterolaemia,7 with increased hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and with premature GSD in a family study.7 Importantly, in that study there was also a significant stepwise increase in plasma levels of LDL cholesterol as a function of genotype from wildtype to heterozygotes to homozygotes, implying that LDL cholesterol increased in a monotonic dose–response fashion with loss of enzyme activity. Taken together, these data suggest that there is an inverse and causal relationship between CYP7A1 activity and plasma levels of LDL cholesterol in humans, and suggest that differences in LDL cholesterol associated with genetic variants in CYP7A1 can be used as proxies for CYP7A1 activity. To date, however, there are no large-scale studies directly assessing CYP7A1 activity and plasma levels of LDL cholesterol, because the available methods (measurement of plasma C4) are not suitable for large-scale determination. It could be argued that some variants in CYP7A1 are pleiotropic due to LD with variants in other genes, and by selecting wholly on the LDL cholesterol association, there has been enrichment for such pleiotropy. This scenario can never be completely ruled out, however, because there are no other genes within at least 15 megabases of CYP7A1 which associate with LDL cholesterol and are in LD with variants in CYP7A1, this does not seem very likely. The mechanistic interpretation of the data may be straightforward. We speculate that as shown above,7 genetic variants inactivating CYP7A1 result in a decrease in bile acid synthesis and accumulation of cholesterol in the liver, leading to down-regulation of hepatic LDL receptors, hypercholesterolaemia, and increased risk of MI.7 Simultaneously, inability to solubilize cholesterol in bile salt mixed micelles predisposes to GSD.7 Conversely, gain-of-function variants which like bile acid sequestrants and elobixibat, up-regulate CYP7A1 activity, likely result in increased conversion of cholesterol to bile acids and depletion of cholesterol in the liver, leading to up-regulation of hepatic LDL receptors, hypocholesterolaemia and protection against both MI and GSD. Thus, the activity of the genetic variants in CYP7A1 might be reflected in their effect on LDL cholesterol levels. The cumulative incidences of MI as a function of weighted allele score increased slightly from age 55, while the corresponding risk of GSD increased from about age 30. As expected for very common genetic variants, the effects were modest but were internally consistent in the CGPS and CCHS. Because the diagnosis of MI was extensively validated by cardiologists by reviewing medical records, and because MI is a hard clinical endpoint with well-defined diagnostic criteria, major misclassification of the MI endpoint was very unlikely. The definition of symptomatic GSD was defined based on ICD codes for cholelithiasis or cholecystitis diagnosed in hospital. This definition most likely captured symptomatic gallstones verified by ultrasound. However, we cannot rule out that a small part of gallstone cases had asymptomatic gallstones diagnosed incidentally. Nevertheless, the prevalence of approximately 6% symptomatic gallstones in our study was comparable to results from previous studies that identified gallstones by similar registry-based methods.26 Because GSD is a hard endpoint with well-defined diagnostic criteria, the risk of misclassification is likely minor. In support of this, approximately 68% of individuals with symptomatic GSD in our cohort underwent cholecystectomy.26 Finally, the risk of MI and GSD as a function of weighted allele score was similar to the corresponding risk for coronary intervention and cholecystectomy, supporting reliability of the endpoints. Strengths and limitations A strength of our study is that we studied only white individuals from an ethnically homogeneous population. Although our results may therefore not necessarily apply to other ethnicities, we are not aware of data to suggest this. Additional strengths include the large sample size, the complete follow-up, the simultaneous assessment of plasma lipid levels, risk of MI, and risk of GSD as a function of genetic variation in CYP7A1 using weighted allele score, the association with LDL cholesterol for both common and rare genetic variants, and the association with increased risk of MI and GSD overall, in both prospective studies of the general population separately, and in both genders. Importantly, in addition to extensive independent replication internally between the CCHS and CGPS, adding data from available consortia in meta-analyses on LDL cholesterol, risk of coronary artery disease, and risk of GSD supported our findings. Because functional data were not available, and because we did not measure CYP7A1 activity, we can only speculate on the mechanisms underlying the association observed between genetic variants in CYP7A1 and plasma levels of LDL cholesterol, risk of MI and risk of symptomatic GSD. Nevertheless, our mechanistic interpretation of the data is in agreement with CYP7A1 as the initial and rate-limiting step in the conversion of cholesterol into bile acids in humans, with data on treatment with bile-acid-binding resins and elobixibat which up-regulate CYP7A1 activity and reduce LDL cholesterol in a dose-dependent manner, with functional data in humans on a truncating variant in CYP7A1 which reduced CYP7A1 activity in the liver and increased LDL cholesterol in a gene-dosage dependent manner, and with results from overexpression in animal models. Conclusion In conclusion, genetic variants in CYP7A1, which are associated with increased levels of LDL cholesterol are associated with an increased risk of MI and symptomatic GSD. It might be speculated that increasing CYP7A1 activity by bile acid sequestrants and other bile acid pool modulators may reduce risk of both MI and GSD. Supplementary material Supplementary material is available at European Heart Journal online. Acknowledgements We thank senior technician Mette Refstrup, Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, for her persistent attention to the details of the large-scale genotyping. Refstrup did not receive any compensation for her contribution. We are indebted to the staff and individuals of the Copenhagen General Population Study and the Copenhagen City Heart Study for their important contributions. Funding This work was supported by a grant from the European Union, Seventh Framework Programme Priority [FP7-HEALTH-2013-INNOVATION-1], contract #603091, The Danish Medical Research Council, The Research Fund at Rigshospitalet, Copenhagen University Hospital, Chief Physician Johan Boserup and Lise Boserup’s Fund, Ingeborg and Leo Dannin’s Grant, and Henry Hansen’s and Wife’s Grant, and a grant from the Odd Fellow Order. The study sponsors had no role in the conduct of the study, in the collection, management, analysis or interpretation of data or in the preparation, review, or approval of the manuscript. Conflict of interest: none declared. References 1 World Health Organization . Cardiovascular Diseases (CVDs). 2015. http://www.who.int/mediacentre/factsheets/fs317/en/ (29 December 2015). 2 Krawczyk M , Wang DQ-H , Portincasa P , Lammert F. Dissecting the genetic heterogeneity of gallbladder stone formation . Semin Liver Dis 2011 ; 31 : 157 – 172 . Google Scholar CrossRef Search ADS PubMed 3 Portincasa P , Moschetta A , Palasciano G. Cholesterol gallstone disease . Lancet 2006 ; 368 : 230 – 239 . Google Scholar CrossRef Search ADS PubMed 4 Cohen JC. Contribution of cholesterol 7alpha-hydroxylase to the regulation of lipoprotein metabolism . J Lipid Res 1999 ; 10 : 303 – 307 . 5 Davidson MH , Dillon MA , Gordon B , Jones P , Samuels J , Weiss S , Isaacsohn J , Toth P , Burke SK. Colesevelam hydrochloride (cholestagel)—a new, potent bile acid sequestrant associated with a low incidence of gastrointestinal side effects . Arch Intern Med 1999 ; 159 : 1893 – 1900 . Google Scholar CrossRef Search ADS PubMed 6 Rudling M , Camilleri M , Graffner H , Holst JJ , Rikner L. Specific inhibition of bile acid transport alters plasma lipids and GLP-1 . BMC Cardiovasc Disord 2015 ; 15 : 75. Google Scholar CrossRef Search ADS PubMed 7 Pullinger CR , Eng C , Salen G , Shefer S , Batta AK , Erickson SK , Verhagen A , Rivera CR , Mulvihill SJ , Malloy MJ , Kane JP. Human cholesterol 7α-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype . J Clin Invest 2002 ; 110 : 109 – 117 . Google Scholar CrossRef Search ADS PubMed 8 Thomas C , Pellicciari R , Pruzanski M , Auwerx J , Schoonjans K. Targeting bile-acid signalling for metabolic diseases . Nat Rev Drug Discov 2008 ; 7 : 678 – 693 . Google Scholar CrossRef Search ADS PubMed 9 Lipid Research Clinics Program . The Lipid Research Clinics Coronary Primary Prevention Trial Results: II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering . JAMA 1984 ; 251 : 365 – 374 . CrossRef Search ADS PubMed 10 Jørgensen AB , Frikke-Schmidt R , Nordestgaard BG , Tybjærg-Hansen A. Loss-of-function mutations in APOC3 and risk of ischemic vascular disease . N Engl J Med 2014 ; 371 : 32 – 41 . Google Scholar CrossRef Search ADS PubMed 11 Couture P , Otvos JD , Cupples LA , Wilson PW , Schaefer EJ , Ordovas JM. Association of the A-204C polymorphism in the cholesterol 7alpha-hydroxylase gene with variations in plasma low density lipoprotein cholesterol levels in the Framingham Offspring Study . J Lipid Res 1999 ; 40 : 1883 – 1889 . Google Scholar PubMed 12 Wang J , Freeman DJ , Grundy SM , Levine DM , Guerra R , Cohen JC. Linkage between cholesterol 7alpha-hydroxylase and high plasma low-density lipoprotein cholesterol concentrations . J Clin Invest 1998 ; 101 : 1283 – 1291 . Google Scholar CrossRef Search ADS PubMed 13 Global Lipids Genetics Consortium . Discovery and refinement of loci associated with lipid levels . Nat Genet 2013 ; 45 : 1274 – 1283 . CrossRef Search ADS PubMed 14 http://www.cardiogramplusc4d.org/data-downloads/ (26 October 2017). 15 Joshi AD , Andersson C , Buch S , Stender S , Noordam R , Weng LC , Weeke PE , Auer PL , Boehm B , Chen C , Choi H , Curhan G , Denny JC , De Vivo I , Eicher JD , Ellinghaus D , Folsom AR , Fuchs C , Gala M , Haessler J , Hofman A , Hu F , Hunter DJ , Janssen HL , Kang JH , Kooperberg C , Kraft P , Kratzer W , Lieb W , Lutsey PL , Darwish Murad S , Nordestgaard BG , Pasquale LR , Reiner AP , Ridker PM , Rimm E , Rose LM , Shaffer CM , Schafmayer C , Tamimi RM , Uitterlinden AG , Völker U , Völzke H , Wakabayashi Y , Wiggs JL , Zhu J , Roden DM , Stricker BH , Tang W , Teumer A , Hampe J , Tybjærg-Hansen A , Chasman DI , Chan AT , Johnson AD. Four susceptibility loci for gallstone disease identified in a meta-analysis of genome-wide association studies . Gastroenterology 2016 ; 151 : 351 – 363 . Google Scholar CrossRef Search ADS PubMed 16 http://locuszoom.sph.umich.edu/genform.php? type=ourdata (24 October 2017). 17 Jiang ZY , Han TQ , Suo GJ , Feng DX , Chen S , Cai XX , Jiang ZH , Shang J , Zhang Y , Jiang Y , Zhang SD. Polymorphisms at cholesterol 7alpha-hydroxylase, apolipoproteins B and E and low density lipoprotein receptor genes in patients with gallbladder stone disease . World J Gastroenterol 2004 ; 10 : 1508 – 1512 . Google Scholar CrossRef Search ADS PubMed 18 Hofman MK , Groenendijk M , Verkuijlen PJ , Jonkers IJ , Mohrschladt MF , Smelt AH , Princen HM. Modulating effect of the A-278C promoter polymorphism in the cholesterol 7alpha-hydroxylase gene on serum lipid levels in normolipidaemic and hypertriglyceridaemic individuals . Eur J Hum Genet 2004 ; 12 : 935 – 941 . 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Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alpha-hydroxy-4-cholesten-3-one in peripheral blood . J Lipid Res 2003 ; 44 : 859 – 866 . Google Scholar CrossRef Search ADS PubMed 24 Spady DK , Cuthbert JA , Willard MN , Meidell RS. Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol . J Clin Invest 1995 ; 96 : 700 – 709 . Google Scholar CrossRef Search ADS PubMed 25 Ratliff EP , Gutierrez A , Davis RA. Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia . J Lipid Res 2006 ; 47 : 1513 – 1520 . Google Scholar CrossRef Search ADS PubMed 26 Stender S , Nordestgaard BG , Tybjaerg-Hansen A. Elevated body mass index as a causal risk factor for symptomatic gallstone disease . Hepatology 2013 ; 58 : 2133 – 2141 . Google Scholar CrossRef Search ADS PubMed Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Heart Journal Oxford University Press

Genetic variants in CYP7A1 and risk of myocardial infarction and symptomatic gallstone disease

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Abstract

Abstract Aims Myocardial infarction (MI) and gallstone disease (GSD) are intrinsically linked via cholesterol metabolism. We tested the hypothesis that genetic variants in the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver, are associated with risk of MI and GSD in the general population. Methods and results We performed tests of association between lipid levels and eight rare non-synonymous mutations and two common variants, rs2081687 and rs3808607, in CYP7A1 in 100 149 individuals from the general population. We further tested whether weighted allele scores for rs2081687 and rs3808607, which were associated with increased plasma levels of low-density lipoprotein (LDL) cholesterol, were associated with an increased risk of both MI and symptomatic GSD. During a mean follow-up of 7 years (0–23 years), MI developed in 2326 individuals and GSD in 2007. For rare mutations, CYP7A1 allele count was associated with an increase in LDL cholesterol of 12% (0.4 mmol/L) for individuals with the highest vs. the lowest allele count (P for trend = 3 × 10−4). For common variants, CYP7A1 weighted allele scores in individuals with a score >0.04 vs. ≤0 were associated with stepwise increases in LDL cholesterol of up to 2.4% (0.08 mmol/L), and with corresponding multifactorially adjusted hazard ratios of 1.25 [95% confidence interval (CI) 1.10–1.41] for MI and 1.39 (95% CI 1.22–1.59) for GSD (P for trend = 5 × 10−4 and 2 × 10−7, respectively). Results were similar in meta-analyses including publicly available data from large consortia. Conclusion Genetic variants in CYP7A1 which are associated with increased levels of LDL cholesterol, are associated with an increased risk of both MI and GSD. View largeDownload slide View largeDownload slide Genetics, Lipids, Cardiovascular disease, Gallstones Introduction Cholesterol plays a pivotal role in the pathogenesis of both myocardial infarction (MI) and gallstone disease (GSD), two exceedingly common and costly diseases.1,2 Elevated levels of plasma cholesterol are a well-known causal risk factor for atherosclerosis and MI, whereas elevated levels of biliary cholesterol promote the formation of cholesterol gallstones.3 Myocardial infarction and GSD, two seemingly unrelated diseases, are therefore intrinsically linked via cholesterol metabolism. Because cholesterol cannot be degraded, excess cholesterol is converted into bile salts (conjugated bile acids) or directly excreted into bile.4 Cholesterol 7 alpha-hydroxylase (CYP7A1), a liver-specific cytochrome P-450 monooxygenase, catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids in the classical pathway (Figure 1). Low CYP7A1 activity may, therefore, reduce the conversion of cholesterol to bile acids in the liver and increase hepatic cholesterol content, resulting in secondary hypercholesterolaemia due to down-regulation of hepatic low-density lipoprotein (LDL) receptors, and simultaneously reduce the ability to solubilize cholesterol in bile acid mixed micelles, predisposing to GSD. Taken together, this could lead to an increased risk of both MI and GSD (Take home figure). Figure 1 View largeDownload slide Biological function of cholesterol 7 alpha-hydroxylase. Cholesterol 7 alpha-hydroxylase catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids, the major pathway for catabolism of cholesterol in humans. Bile acids are transported as bile salts (conjugated bile acids) in to the bile via the bile salt export pump, ATP binding cassette subfamily B member 11. In the bile (right), increased conversion of cholesterol into bile acids increases the solubility of cholesterol in bile salt mixed micelles and hence prevents gallstone formation. Inhibition of the hepatic activity of cholesterol 7 alpha-hydroxylase by inactivating genetic variants in the CYP7A1 gene might therefore cause gallstone disease as a result of reduced bile acid secretion. Furthermore, the reduced conversion of cholesterol to bile acids might result in elevated liver cholesterol levels, down-regulated low-density lipoprotein receptors, secondary hypercholesterolaemia, and increased risk of atherosclerosis and ischaemic heart disease.6 ABCB11, ATP binding cassette subfamily B member 11; CYP7A1, Cytochrome P450, family 7, subfamily A, poplypeptide 1, the gene that encodes cholesterol 7 alpha-hydroxylase; LDL, low-density lipoprotein. Figure 1 View largeDownload slide Biological function of cholesterol 7 alpha-hydroxylase. Cholesterol 7 alpha-hydroxylase catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids, the major pathway for catabolism of cholesterol in humans. Bile acids are transported as bile salts (conjugated bile acids) in to the bile via the bile salt export pump, ATP binding cassette subfamily B member 11. In the bile (right), increased conversion of cholesterol into bile acids increases the solubility of cholesterol in bile salt mixed micelles and hence prevents gallstone formation. Inhibition of the hepatic activity of cholesterol 7 alpha-hydroxylase by inactivating genetic variants in the CYP7A1 gene might therefore cause gallstone disease as a result of reduced bile acid secretion. Furthermore, the reduced conversion of cholesterol to bile acids might result in elevated liver cholesterol levels, down-regulated low-density lipoprotein receptors, secondary hypercholesterolaemia, and increased risk of atherosclerosis and ischaemic heart disease.6 ABCB11, ATP binding cassette subfamily B member 11; CYP7A1, Cytochrome P450, family 7, subfamily A, poplypeptide 1, the gene that encodes cholesterol 7 alpha-hydroxylase; LDL, low-density lipoprotein. Take home figure View largeDownload slide We speculate that low activity of cholesterol 7 alpha-hydroxylase, which catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids in humans, results in a decrease in bile acid synthesis (1) and accumulation of cholesterol in the liver (2) leading to down-regulation of hepatic low-density lipoprotein receptors (3), hypercholesterolaemia (4), and secondary increased risk of myocardial infarction (5). Simultaneously, inability to solubilize cholesterol in bile salt mixed micelles predisposes to gallstone disease (6). Here, we show that common genetic variants in CYP7A1, which are associated with increased levels of low-density lipoprotein cholesterol mimicking the effect of a loss-of-function mutation in humans7 are associated with an increased risk of both myocardial infarction and gallstone disease. Therefore, increasing cholesterol 7 alpha-hydroxylase activity by bile acid sequestrants and other bile acid pool modulators may reduce risk of both myocardial infarction and gallstone disease. Take home figure View largeDownload slide We speculate that low activity of cholesterol 7 alpha-hydroxylase, which catalyses the initial and rate-limiting step in the conversion of cholesterol into bile acids in humans, results in a decrease in bile acid synthesis (1) and accumulation of cholesterol in the liver (2) leading to down-regulation of hepatic low-density lipoprotein receptors (3), hypercholesterolaemia (4), and secondary increased risk of myocardial infarction (5). Simultaneously, inability to solubilize cholesterol in bile salt mixed micelles predisposes to gallstone disease (6). Here, we show that common genetic variants in CYP7A1, which are associated with increased levels of low-density lipoprotein cholesterol mimicking the effect of a loss-of-function mutation in humans7 are associated with an increased risk of both myocardial infarction and gallstone disease. Therefore, increasing cholesterol 7 alpha-hydroxylase activity by bile acid sequestrants and other bile acid pool modulators may reduce risk of both myocardial infarction and gallstone disease. That plasma CYP7A1 activity is inversely correlated with plasma levels of LDL cholesterol in a monotonic dose–response fashion is supported by the following: (i) Treatment with bile acid sequestrants and more recently with elobixibat, an ileal bile acid transporter inhibitor, increase CYP7A1 activity and reduce LDL cholesterol in a dose-dependent manner,5,6 results which are in agreement with overexpression of CYP7A1 in animal models and (ii) Conversely, homozygosity for a loss-of-function mutation in CYP7A1 has previously been associated with loss of CYP7A1 enzyme activity, severe hypercholesterolaemia and increased hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and with premature GSD in a family study.7 Importantly, in that study there was a significant stepwise increase in plasma levels of LDL cholesterol as a function of genotype from wildtype to heterozygotes to homozygotes, implying that LDL cholesterol increased in a monotonic dose–response fashion with loss of enzyme activity. Taken together, these data suggest that there are an inverse and causal relationship between CYP7A1 activity and plasma LDL cholesterol levels. Therefore, we used the association between genetic variants in CYP7A1 and plasma levels of LDL cholesterol as proxies for CYP7A1 activity. This is clinically important because it suggests that bile acid sequestrants and other bile-acid pool modulators which lower LDL cholesterol by increasing CYP7A1 activity,6,8 may reduce risk of GSD in addition to reducing cardiovascular risk.9 We tested the hypothesis that lifelong high levels of LDL cholesterol associated with genetic variants in CYP7A1 are associated with both increased risk of MI and GSD in the general population. We genotyped eight non-synonymous mutations in CYP7A1 and two common variants previously associated with LDL cholesterol levels, in two prospective studies of the general population, the Copenhagen General Population Study (CGPS) and the Copenhagen City Heart Study (CCHS), totalling 100 149 individuals, of whom 2326 developed MI and 2007 developed symptomatic GSD. Methods The study was approved by institutional review boards and Danish ethics committees and was conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all individuals. Participants We included individuals in two similar prospective studies of the Danish general population, the CGPS and the CCHS.10 All individuals were white and of Danish descent. The Copenhagen General Population Study The CGPS was initiated in 2003, and enrolment is ongoing. Individuals were selected with the use of the National Danish Civil Registration System to reflect the adult Danish population aged 20 to 100 years or older. Data were obtained from a questionnaire, a physical examination, and from collection of blood samples. We included 89 944 consecutive individuals in the current analyses. During a mean follow-up of 6 years (0–11 years) (which ended in November 2014), 1448 had an incident MI and 1512 had incident symptomatic GSD. The Copenhagen City Heart Study In the CCHS, we included 10 205 individuals with DNA available from the 1991–94 and 2001–03 examinations in the current analyses. Individuals were recruited and examined as in the CGPS. During a mean follow-up of 16 years (0–23 years) (which ended in November 2014), 878 had an incident MI and 495 had incident symptomatic GSD. Combining the participants in the CGPS and the CCHS yielded a total of 100 149 participants at baseline (Table 1). During a mean follow-up of 7 years (0–23 years) (which ended in November 2014), MI developed in 2326 individuals and symptomatic GSD developed in 2007. Individuals with prevalent events at baseline were excluded from analyses of risk of MI and GSD (2179 individuals with MI and 3950 with GSD). In both studies, follow-up was 100% complete that is we did not lose track of even a single individual. DNA was available on all individuals, and lipid values were available on more than 98%. Table 1 Baseline characteristics of individuals by events No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a Values are median (interquartile range), or number of individuals (%). Events are prevalent and incident events. P-values by Mann-Whitney U-test or Pearson’s χ2 test. a P-value <0.001 vs. individuals with no event. b In women only. GSD, gallstone disease; MI, myocardial infarction. Table 1 Baseline characteristics of individuals by events No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a No event MI only Symptomatic GSD only Both MI and GSD Number of individuals 90 065 4127 5579 378 Age (years) 57 (47–67) 68 (61–76)a 61 (51–70)a 70 (62–76)a Women (%) 49 978 (55) 1384 (34)a 3989 (72)a 186 (49)a Body mass index (kg/m2) 25 (23–28) 27 (24–30)a 27 (24–30)a 27 (25–30)a Hypertension (%) 10 364 (12) 1683 (41)a 1284 (23)a 186 (49)a Diabetes mellitus (%) 3310 (4) 581 (14)a 425 (8)a 69 (18)a Physical activity (%) 45 329 (50) 1629 (39)a 2180 (39)a 125 (33)a Smoking (%) 18 301 (20) 1245 (30)a 1317 (24)a 121 (32)a Alcohol consumption (%) 19 606 (22) 1132 (27)a 921 (17)a 86 (23) Hormone replacement therapyb (%) 5954 (11) 203 (10)a 652 (16)a 32 (13)a Lipid-lowering therapy (%) 8284 (9) 1594 (39)a 629 (11)a 137 (36)a Values are median (interquartile range), or number of individuals (%). Events are prevalent and incident events. P-values by Mann-Whitney U-test or Pearson’s χ2 test. a P-value <0.001 vs. individuals with no event. b In women only. GSD, gallstone disease; MI, myocardial infarction. For additional information on clinical endpoints, laboratory analyses, and other covariates including measurement of liver fat content by computed tomography (CT) scans, see Supplementary material online, Appendix. Genotyping We genotyped all non-synonymous variants in CYP7A1 reported in Exome Variant Server (http://evs.gs.washington.edu/EVS/) with a minor allele frequency above 0.03% in European Americans (A13V, rs147162838; N36K, rs138113674; Y75C, rs377254635; T193I, rs72647413; R260L, rs139396617; G377S, rs117423932; P398A, rs142708991; G417R, rs201787113). In addition, we genotyped two common variants in CYP7A1, rs2081687 C>T and rs3808607 A>C (NM_000780.3: c.-267A>C; old nomenclature: -278A>C and -204A>C11,12). Both variants have previously been shown to associate with LDL cholesterol levels, either as the lead single nucleotide polymorphism (SNP) in published genome-wide association study (GWAS) studies (rs2081687)13 or with consistent stepwise associations as a function of genotype in association studies (rs3808607).11,12 These two common variants are in complete linkage disequilibrium (LD) with variants spanning the core promoter, coding region, and downstream region of CYP7A1 (Supplementary material online, Figure S1). We genotyped rs3808607 as a proxy for rs3824260 (Supplementary material online, Figure S1, right), because the latter variant proved technically challenging to genotype. There are no common non-synonymous variants in CYP7A1. Genotyping was by TaqMan-based assays (Applied Biosystems, Foster City, CA, USA) or by KASP genotyping technology (LGC Genomics Ltd, Hoddesdon, Herts, UK). Statistical analyses Data were analysed using Stata SE 13. The χ2 test evaluated the Hardy–Weinberg equilibrium. To compare characteristics in individuals by disease status or genotype, Mann–Whitney U-test, or Cuzick’s test for trend was used to compare continuous covariates, and Pearson’s χ2 test to compare categorical covariates. For trend tests, genotypes, CYP7A1 weighted allele scores (based on β-coefficients) or allele count were coded 0, 1, 2, and so forth. The genotype, CYP7A1 weighted allele score or allele count associated with the lowest LDL cholesterol level was used as the reference (coded 0). Cuzick’s test for trend was used to compare levels of continuous variables as a function of genotype, CYP7A1 allele count, and weighted allele scores. For both rare and common variants, we used an additive genetic model. Multifactorial adjustment in both logistic regression analyses (rare variants) and in Cox regression analyses (common variants) was for well-known risk factors for MI and GSD: age, gender, body mass index, hypertension, diabetes mellitus, physical activity, smoking, alcohol consumption, hormone replacement therapy (women only), and lipid-lowering therapy, i.e. the same characteristics as shown in Table 1. For the rare non-synonymous variants, we used a simple allele count based on the number of LDL-increasing alleles of the individual variants: Individuals heterozygous for T193I who had LDL cholesterol levels significantly lower than average were graded 0 (=reference group; N = 214); individuals with average LDL cholesterol levels were coded 1 (heterozygotes or homozygotes for the five variants without significant associations with LDL cholesterol, and all individuals who were wild type for any of the seven variants identified; N = 106 585); and individuals heterozygous for R260L who had LDL cholesterol levels significantly higher than average were graded 2 (N = 177) (Figure 2). The two common variants, rs2081687 C>T and rs3808607 A>C, were combined in weighted allele scores as follows: (i) the individual β-coefficients for LDL cholesterol for the minor allele of each variant was calculated by a regression analysis including both variants and adjusting for age, gender, and cohort (Supplementary material online, Table S1A; note that the effects of the minor alleles on LDL cholesterol are in opposite directions); (ii) the weighted allele scores were calculated by summation of these β-coefficients for each genotype combination (Supplementary material online, Table S1B, third column from left); and (iii) the weighted allele scores were divided into three groups by increasing score and LDL cholesterol levels: weighted allele score ≤0 (reference), >0 to 0.04, and >0.04 to achieve groups including a reasonable number of individuals in each group for analyses (Supplementary material online, Table S1B, third to fifth columns from left). For both variants, we show internal weights unadjusted and adjusted for the other variant, and unadjusted external weights from the Global Lipids Genetics Consortium.13 Figure 2 View largeDownload slide Lipid levels as a function of rare genetic variants in CYP7A1, individually and combined. The seven rare, non-synonymous variants were combined in a simple allele count based on the number of low-density lipoprotein cholesterol-increasing alleles for the individual genotypes. P-values by Mann–Whitney U-tests or for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Figure 2 View largeDownload slide Lipid levels as a function of rare genetic variants in CYP7A1, individually and combined. The seven rare, non-synonymous variants were combined in a simple allele count based on the number of low-density lipoprotein cholesterol-increasing alleles for the individual genotypes. P-values by Mann–Whitney U-tests or for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Fine-Grey curves and tests for trend evaluated the cumulative incidences of MI and symptomatic GSD as a function of age and CYP7A1 weighted allele scores with death as a competing event. Cox proportional hazards regression models using age as time scale and delayed entry (left truncation), which implies that age is automatically adjusted for, were used to estimate hazard ratios (HRs) for MI, symptomatic GSD, coronary intervention, cholecystectomy, non-alcoholic fatty liver disease (NAFLD), and cirrhosis as a function of CYP7A1 weighted allele scores. Multifactorial adjustments were for well-known risk factors for MI, and symptomatic GSD: age, gender, body mass index, hypertension, diabetes mellitus, physical activity, smoking, alcohol consumption, hormone replacement therapy (women only), and lipid-lowering therapy. Analyses were conducted from blood sampling at study entry (CCHS 1991–94 or 2001–03; CGPS 2003 and ongoing) through 10 November 2014. Only incident events were included, comprising a total of 2326 individuals with MI and 2007 with symptomatic GSD. In sensitivity analyses, we stratified the analyses by gender and study, grouped the weighted allele scores by other cut-points (4 and 5 groups), and excluded individuals on lipid-lowering therapy. Further, we determined the effect of a one standard deviation increase in CYP7A1 weighted allele score on risk of MI and symptomatic GSD. Finally, logistic regression analyses were used to assess whether risk of MI, risk of symptomatic GSD, or a one category increase in weighted allele score was associated with potential measured confounders (risk factors for MI and GSD: age ≥50 vs. <50 years; gender: male vs. female; body mass index ≥25 vs. <25 kg/m2; hypertension yes vs. no; diabetes mellitus yes vs. no; physical activity low vs. high; smoking yes vs. no; alcohol consumption high vs. low; hormone replacement therapy in women yes vs. no; and lipid-lowering therapy yes vs. no). Finally, we performed meta-analyses combining data from CCHS and CGPS with data from (i) The Global Lipids Genetics Consortium13 for LDL cholesterol; (ii) CARDIoGRAMplusC4D 1000 Genomes for risk of IHD14; and (iii) Recent GWAS data on GSD15 using the ‘metan’ command in Stata. Results Baseline characteristics of the 100 149 individuals by disease status, including both prevalent and incident events, are shown in Table 1. As expected, well-known risk factors for MI and GSD were over-represented in those with events. Baseline characteristics of individuals in the CGPS and CCHS are shown separately in Supplementary material online, Table S2; differences between studies reflect differences in lifestyle factors over time. In contrast to the observational data (Table 1), baseline characteristics as a function of weighted allele scores were similar, although use of lipid-lowering therapy tended to be slightly more frequent with increasing score, and hence increasing levels of LDL cholesterol (Table 2). Taken together, these data suggest that the genetic data based on stratification by LDL cholesterol, as opposed to the observational epidemiological data, are largely unconfounded by known, measured risk factors for MI and GSD. Genotype distributions did not differ from Hardy–Weinberg equilibrium (P > 0.25). There was moderate LD between the two common variants, CYP7A1 rs2081687 C>T and rs3808607 A>C (D´ = 0.93; R2 = 0.70), although in approximately 15% of individuals, or 14 778 individuals (8% of haplotypes) the minor alleles were not inherited on the same haplotype (Supplementary material online, Figure S1; minor allele frequencies 0.33 and 0.39, respectively). Table 2 Baseline characteristics as a function of CYP7A1 weighted allele score Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Values are median (interquartile range), or number of individuals (%). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test or Pearson’s χ2 test. a In women only. Table 2 Baseline characteristics as a function of CYP7A1 weighted allele score Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Weighted allele score ≤0 >0–0.04 >0.04 P-value Number of participants 44 565 42 888 12 696 Age (years) 58 (48–67) 58 (48–67) 58 (48–67) 0.40 Women (%) 55 55 55 0.95 Body mass index (kg/m2) 26 (23–28) 26 (23–28) 25 (23–28) 0.18 Hypertension (%) 13 14 14 0.91 Diabetes mellitus (%) 4 4 4 0.64 Physical activity (%) 49 49 49 0.81 Smoking (%) 21 21 20 0.14 Alcohol consumption (%) 21 22 22 0.64 Hormone replacement therapya (%) 12 12 12 0.94 Lipid-lowering therapy (%) 10 11 12 <0.001 Values are median (interquartile range), or number of individuals (%). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test or Pearson’s χ2 test. a In women only. Cyp7A1 genotype, plasma lipids, and lipoproteins The effects of the individual rare, non-synonymous variants on LDL cholesterol levels and other lipids and lipoproteins are shown in Figure 2. Two of seven variants, T193I and R260L, were associated with LDL cholesterol levels which were, respectively, 3.7% lower and 7.9% higher than non-carriers (P-values 0.05 and 1 × 10−3), with similar trends in the CGPS and CCHS separately (Supplementary material online, Figures S2 and S3). Because our hypothesis was that lifelong high LDL cholesterol levels associated with genetic variants in CYP7A1 were associated with both increased risk of MI and GSD in the general population, we combined the seven rare variants (A13V, N36K, T193I, R260L, G377S, P398A, G417R; Y75C was not identified) by simple allele counts based on the number of LDL cholesterol-increasing alleles of the individual genotypes, using heterozygotes for the minor allele of T193I (GA genotype) as the reference group (=0; lower than average LDL cholesterol), heterozygotes and homozygotes for the five variants not associated with LDL cholesterol (A13V, N36K, G377S, P398A, and G417R) and wildtypes from all seven variants as the middle group (=1; average LDL cholesterol), and heterozygotes for R260L (CA genotype) as the top group (=2; higher than average LDL cholesterol). The corresponding stepwise increase in LDL cholesterol was up to 12% (0.4 mmol/L) for individuals with an allele count of two vs. 0 (Figure 2; P for trend = 3 × 10−4), compared to up to 2.4% (0.08 mmol/L) for common variants (Figure 3, see below). Rs2081687 and rs3808607 were individually associated with stepwise increases in LDL- and total cholesterol of up to 2.6% (0.09 mmol/L) and 1.7% (0.10 mmol/L) and up to 1.6% (0.05 mmol/L) and 1.1% (0.06 mmol/L), respectively, in homozygotes for the minor alleles vs. reference genotypes (Figure 3; P for trend from 2 × 10−7 to 4 × 10−17). However, as indicated by the individual adjusted β-coefficients (Supplementary material online, Table S1A), and as also evident when plotting LDL- and total cholesterol as a function of the individual genotypes on either the wildtype (CC or AA), heterozygote (CT or AC), or homozygote (TT or CC) background of the other variant (Supplementary material online, Figure S4), the minor alleles of the two variants were associated with LDL- and total cholesterol levels in opposite directions: higher levels for rs2081687 T-allele, and lower levels for rs3808607 C-allele. Therefore, weighted allele scores based on summation of the individual β-coefficients for LDL cholesterol for the two common variants (by linear regression analysis including both variants into the same analysis adjusting for age, gender, and cohort) were constructed and grouped into three groups: weighted allele score ≤0 (reference), 0–0.04, >0.04, each including a reasonable number of individuals for analyses (Supplementary material online, Table S1B). CYP7A1 weighted allele scores were associated with stepwise increases in plasma levels of LDL- and total cholesterol of up to 2.4% (0.08 mmol/L) and 1.6% (0.09 mmol/L), respectively, for individuals with a score of >0.04 vs. ≤0 (Figure 3; P for trend: 2 × 10−15 and 5 × 10−17). Results were similar in separate analyses of data from the CCHS and CGPS (Supplementary material online, Figure S5). Results for apolipoproteins B and -A-I were concordant with results for, respectively, LDL- and total cholesterol, and high-density lipoprotein cholesterol levels (Supplementary material online, Figure S6). Importantly, in a region comprising at least 15 megabases cantered on CYP7A1, no other variants associated with LDL cholesterol levels were in LD with variants in CYP7A1, suggesting that enrichment for pleiotropy due to LD with other variants seemed less likely.16 Cyp7A1 genotype, risk of myocardial infarction, and symptomatic gallstone disease In individuals with a weighted allele score ≤0 through >0–0.04 to >0.04, there was a stepwise increase in cumulative incidence of MI and symptomatic GSD as a function of age (Figure 4; P for trend = 2 × 10−4 and 8 × 10−6). For MI risk increased as a function of increasing weighted allele score from age 55, while risk of GSD increased from about age 30. As expected for very common variants, the increase in cumulative incidences as a function of allele score, were modest: at 60 years the cumulative incidence for MI was increased by 0.7%, and by age 80 by 2.5% in individuals with a score >0.04 vs. 0. For GSD, the corresponding increases were 1.0% at age 40, 2.4% at age 60, and 4.3% at age 80 (Figure 4). Results were similar in the CGPS and CCHS separately (Supplementary material online, Figures S7 and S8). Figure 3 View largeDownload slide Lipid levels as a function of common variants in CYP7A1, individually and combined. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score based on summation of the individual β-coefficients for low-density lipoprotein cholesterol (by logistic regression analysis including both variants into the same analysis adjusting for age, gender, and cohort) for the minor alleles (Supplementary material online, Table S1). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Figure 3 View largeDownload slide Lipid levels as a function of common variants in CYP7A1, individually and combined. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score based on summation of the individual β-coefficients for low-density lipoprotein cholesterol (by logistic regression analysis including both variants into the same analysis adjusting for age, gender, and cohort) for the minor alleles (Supplementary material online, Table S1). P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test. HDL, high-density lipoprotein; LDL, low-density lipoprotein; N, number. Figure 4 View largeDownload slide Cumulative incidences of myocardial infarction and symptomatic gallstone disease as a function of age and CYP7A1 weighted allele score, after adjustment for death as competing event. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). P-values are for trend tests across sub-hazard ratios in Fine-Grey models. Figure 4 View largeDownload slide Cumulative incidences of myocardial infarction and symptomatic gallstone disease as a function of age and CYP7A1 weighted allele score, after adjustment for death as competing event. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). P-values are for trend tests across sub-hazard ratios in Fine-Grey models. The multifactorially adjusted [for well-known risk factors for MI and GSD: age, gender, body mass index, hypertension, diabetes mellitus, physical activity, smoking, alcohol consumption, hormone replacement therapy (women only), and lipid-lowering therapy, see Table 1] HRs for both MI and symptomatic GSD increased stepwise with increasing CYP7A1 weighted allele score up to 1.25 [95% confidence interval (CI) 1.10–1.41; P for trend= 5 × 10−4] for MI and 1.39 (95% CI 1.22–1.59; P for trend = 2 × 10−7) for symptomatic GSD, for individuals with a CYP7A1 weighted allele score ≥0.04 when compared with ≤0 (Figure 5, top). Results were similar in unadjusted analyses (Supplementary material online, Figure S9), or when stratified by gender or cohort (Figure 5, middle and bottom). In agreement, the HRs for coronary intervention (percutaneous coronary intervention (PCI) and/or coronary artery bypass grafting (CABG) and cholecystectomy increased stepwise with increasing allele score up to 1.20 (1.04–1.39) for coronary intervention and 1.47 (1.24–1.74) for cholecystectomy in individuals with an allele score >0.04 vs. 0 (Supplementary material online, Figure S10; P for trend: 0.02 and 8 × 10−6). Figure 5 View largeDownload slide Plasma low-density lipoprotein cholesterol levels, risk of myocardial infarction and symptomatic gallstone disease as a function of CYP7A1 weighted allele score: overall, stratified by gender, and by cohort. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). Hazard ratios were multifactorially adjusted for age, gender, body mass index, hypertension, diabetes, physical activity, smoking, alcohol consumption, hormone replacement therapy, and lipid-lowering therapy. P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test, or for trend tests of hazard ratios. CI, confidence interval; HR, hazard ratio; LDL, low-density lipoprotein; N, number. Figure 5 View largeDownload slide Plasma low-density lipoprotein cholesterol levels, risk of myocardial infarction and symptomatic gallstone disease as a function of CYP7A1 weighted allele score: overall, stratified by gender, and by cohort. From the two common variants (rs2081687 and rs3808607), we generated a CYP7A1 weighted allele score (Supplementary material online, Table S1). Hazard ratios were multifactorially adjusted for age, gender, body mass index, hypertension, diabetes, physical activity, smoking, alcohol consumption, hormone replacement therapy, and lipid-lowering therapy. P-values are for trend tests by Cuzick’s extension of a Wilcoxon rank-sum test, or for trend tests of hazard ratios. CI, confidence interval; HR, hazard ratio; LDL, low-density lipoprotein; N, number. Due to the relatively few individuals with rare variants, lack of statistical power did not allow reliable risk estimates for MI and symptomatic GSD (Supplementary material online, Figure S11). Meta-analyses on LDL cholesterol, risk of ischaemic heart disease, and gallstone disease In meta-analysis of CYP7A1 rs2081687 on LDL cholesterol including data from the Global Lipids Genetics Consortium,13 CCHS, and CGPS, the overall random effects beta-coefficient was 0.04 (95% CI 0.03–0.04) (P = 1 × 10−25) per risk increasing T-allele with an I2 = 0.0% (Supplementary material online, Figure S12A). In meta-analysis of CYP7A1 rs2081687 on risk of coronary artery disease including data from CARDIoGRAMplusC4D 1000 Genomes,14 CCHS, and CGPS, the overall random effects odds ratio was 1.01 (95% CI 1.00–1.03, P = 0.07) per risk increasing T-allele with an I2 = 0.0 (Supplementary material online, Figure S13A), while the overall random effects odds ratio for validated MI in the CCHS and CGPS was 1.07 (95% CI 1.02–1.12) (Supplementary material online, Figure S13C). For comparison, we also show data from the Global Lipids Genetics Consortium13 and CARDIoGRAMplusC4D 1000 Genomes14 on rs13277801 (Supplementary material online, Figures S12B and S13B), a variant in complete LD with the variant above (rs2081687). Rs13277801 was typed in 173 010 individuals as opposed to 89 888 for rs2081687 and the significance level for LDL cholesterol was therefore lower (Supplementary material online, Figure S1). Results for this variant were similar to those for rs2081687 (Supplementary material online, Figures S12 and S13A and B); the variant was not available in the GWAS of GSD. Finally, in meta-analysis of CYP7A1 rs6471717, a variant in complete LD with rs2081687 (R2 = 0.98), in the discovery cohorts of the largest GWAS study of GSD to date,15 the overall random effects odds ratio for GSD was 1.12 (95% CI 1.06–1.20) per risk increasing G-allele (Supplementary material online, Figure S14A) and 1.10 (95% CI 1.05–1.16) in the Copenhagen Studies (Supplementary material online, Figure S14B). Including data from the discovery cohorts on rs6471717 (Supplementary material online, Figure S14A) with data from the present study on rs2081687 (in complete LD with rs6471717) gave similar results (Supplementary material online, Figure S14C). Taken together, consortia data supported the results from the Copenhagen Studies on LDL cholesterol, risk of coronary artery disease, and risk of GSD as a function of common genetic variants in CYP7A1. Cyp7A1 genotype, liver parameters and inflammatory markers, liver fat content, and risk of non-alcoholic fatty liver disease and cirrhosis For the two common variants (rs2081687 and rs3808607), CYP7A1 weighted allele score was associated with a stepwise increase in gamma-glutamyl transferase—a sensitive marker of cholestasis—of up to 4.1% (1.58 U/L), for individuals with an allele score of >0.04 vs. ≤0 (P for trend = 3 × 10−4) (Supplementary material online, Figures S15 and S16, top middle column). In contrast, allele count was not associated with other plasma markers of liver function or inflammation. Furthermore, CYP7A1 weighted allele score was not associated with liver fat content based on liver attenuation on CT scans measured in Hounsfield Units, or with risk of NAFLD or liver cirrhosis (Supplementary material online, Figure S16, top). For comparison, PNPLA3 I148M genotype, a strong and specific cause of high liver fat content (=triglyceride content), was associated with stepwise increases in liver fat content (P for trend = 4 × 10−5) and alanine aminotransferase, a marker of liver cell damage (P for trend 2 × 10−42), and with HRs for NAFLD and liver cirrhosis of, respectively, 2.18 (95% CI 1.66–2.87) and 3.05 (95% CI 2.21–4.22) in homozygotes vs. non-carriers (Supplementary material online, Figure S16, bottom). Validation and sensitivity analyses To further test the robustness of our results, we conducted several sensitivity analyses. Using unadjusted data (Supplementary material online, Figure S9), stratifying analyses by gender or by study (CGPS and CCHS) (Figure 5 middle and bottom), dividing the weighted allele score at other cut points (4 or 5 groups) (Supplementary material online, Figure S17), or excluding individuals on lipid-lowering therapy (Supplementary material online, Figure S18), gave similar results. Furthermore, one unit (=1 standard deviation) increase in CYP7A1 weighted allele score was associated with HRs of 1.08 (1.04–1.12) for MI and 1.11 (1.06–1.16) for symptomatic GSD (P for trend: 3 × 10−4 and 2 × 10−6, respectively). For data on confounding and a discussion of this see Supplementary material online, Appendix and Supplementary material online, Figures S19 and S20. Discussion The main finding of this study is that lifelong, increased levels of plasma LDL cholesterol associated with genetic variants in CYP7A1 are associated with risk of both MI and GSD in the general population. These results suggest that MI and gallstones are intrinsically linked via the function of CYP7A1. The findings are of potential clinical importance because they suggest that CYP7A1 could be a relevant drug target for reducing risk of gallstones in addition to residual cardiovascular risk. It might be speculated, that bile acid sequestrants and other bile-acid pool modulators6,8 which lower plasma LDL cholesterol by increasing CYP7A1 activity and de novo synthesis of bile acids, may therefore simultaneously protect against ischaemic heart disease and GSD. To our knowledge, this is the first study to simultaneously assess plasma lipid levels, risk of MI, and risk of symptomatic GSD as a function of genetic variation in CYP7A1 in the general population, and the first study to use combined and weighted allele scores. Previous studies including between 379 and 2330 individuals have reported associations between rs3808607 (variously called -204/-278 A>C) and/or rs3824260 (variously called -496/-467/-554 T>C), two variants in complete LD,11 and lipid and lipoprotein levels,11,12,17–19 GSD,17,19 or progression of atherosclerosis.18 However, results have been conflicting, most likely because of the limited size of the studies, and because rs3808607 and rs3824260, as shown in the present study, are in LD with 3’variants (rs2081687 and others) which have opposite effects on plasma LDL cholesterol levels. Furthermore, in GWAS, rs2081687 has consistently been associated with plasma LDL cholesterol levels,13 and in a recent large GWAS, we have shown an association between rs6471717, a variant in complete LD with rs2081687 in our study (D’ = 0.99; R2 = 0.98), and risk of GSD.15 In agreement, in meta-analyses combining data from the Copenhagen Studies with data from the Global Lipids Genetics consortium,13 CARDIoGRAMplusC4D 1000 Genomes,14 or the studies included in the discovery cohort of the large gallstone GWAS mentioned above,15 the minor T-allele of rs2081687 (or G allele of rs6471717) was associated with high LDL cholesterol, a very modest increase in risk of coronary artery disease which became more significant for the hard endpoint MI, and an increase in risk of GSD. Because, we did not measure CYP7A1 activity, a major assumption of the present study is that as CYP7A1 enzyme function decreases, LDL cholesterol increases in a monotonic dose–response fashion, and that common genetic variants in CYP7A1 associated with a gene-dosage effect on plasma levels of LDL cholesterol can, therefore, be used as proxies for CYP7A1 activity. This is supported by several lines of evidence. First, treatment with bile-acid sequestrants which, by interrupting the enterohepatic circulation, deplete the bile acid pool, significantly increase CYP7A1 activity and de novo synthesis of bile acids, and lower plasma LDL cholesterol levels in a dose-dependent manner5 by increased receptor-mediated clearance of LDL.4,20,21 Treatment with bile-acid sequestrants decreases plasma LDL cholesterol by up to 25%, and reduces risk of coronary heart disease death and/or non-fatal MI when used as monotherapy.9,20–22 Due to the associated increase in CYP7A1 activity and bile acid synthesis, we speculate that bile acid sequestrants likely also reduce the risk of GSD, although this has not been tested directly. Second, Rudling et al.6 more recently showed that elobixibat, a minimally absorbed ileal bile acid transporter inhibitor, reduced LDL cholesterol and increased 7α-hydroxy-4-cholesten-3-one (C4) in a dose-dependent manner. C4 is a direct plasma marker of the enzymatic activity of CYP7A1 in the liver, the rate-limiting enzyme in bile acid synthesis for which cholesterol is the precursor.23 Both these studies therefore support that as CYP7A1 activity increases, LDL cholesterol decreases in a dose-dependent manner. Third, this interpretation is in agreement with results from animal models in which overexpression of the CYP7A1 gene resulted in a dose-dependent decrease in plasma cholesterol concentrations, mainly in the LDL sized lipoproteins, prevented diet-induced hypercholesterolaemia24,25 and protected against both atherosclerosis and GSD.24 Finally, homozygosity for a loss-of-function mutation in CYP7A1 has previously been associated with loss of CYP7A1 enzyme activity both in in vitro transfection studies and directly in human liver biopsies, with mean LDL cholesterol levels in three homozygotes of approximately 5 mmol/L corresponding to levels in heterozygous familial hypercholesterolaemia,7 with increased hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and with premature GSD in a family study.7 Importantly, in that study there was also a significant stepwise increase in plasma levels of LDL cholesterol as a function of genotype from wildtype to heterozygotes to homozygotes, implying that LDL cholesterol increased in a monotonic dose–response fashion with loss of enzyme activity. Taken together, these data suggest that there is an inverse and causal relationship between CYP7A1 activity and plasma levels of LDL cholesterol in humans, and suggest that differences in LDL cholesterol associated with genetic variants in CYP7A1 can be used as proxies for CYP7A1 activity. To date, however, there are no large-scale studies directly assessing CYP7A1 activity and plasma levels of LDL cholesterol, because the available methods (measurement of plasma C4) are not suitable for large-scale determination. It could be argued that some variants in CYP7A1 are pleiotropic due to LD with variants in other genes, and by selecting wholly on the LDL cholesterol association, there has been enrichment for such pleiotropy. This scenario can never be completely ruled out, however, because there are no other genes within at least 15 megabases of CYP7A1 which associate with LDL cholesterol and are in LD with variants in CYP7A1, this does not seem very likely. The mechanistic interpretation of the data may be straightforward. We speculate that as shown above,7 genetic variants inactivating CYP7A1 result in a decrease in bile acid synthesis and accumulation of cholesterol in the liver, leading to down-regulation of hepatic LDL receptors, hypercholesterolaemia, and increased risk of MI.7 Simultaneously, inability to solubilize cholesterol in bile salt mixed micelles predisposes to GSD.7 Conversely, gain-of-function variants which like bile acid sequestrants and elobixibat, up-regulate CYP7A1 activity, likely result in increased conversion of cholesterol to bile acids and depletion of cholesterol in the liver, leading to up-regulation of hepatic LDL receptors, hypocholesterolaemia and protection against both MI and GSD. Thus, the activity of the genetic variants in CYP7A1 might be reflected in their effect on LDL cholesterol levels. The cumulative incidences of MI as a function of weighted allele score increased slightly from age 55, while the corresponding risk of GSD increased from about age 30. As expected for very common genetic variants, the effects were modest but were internally consistent in the CGPS and CCHS. Because the diagnosis of MI was extensively validated by cardiologists by reviewing medical records, and because MI is a hard clinical endpoint with well-defined diagnostic criteria, major misclassification of the MI endpoint was very unlikely. The definition of symptomatic GSD was defined based on ICD codes for cholelithiasis or cholecystitis diagnosed in hospital. This definition most likely captured symptomatic gallstones verified by ultrasound. However, we cannot rule out that a small part of gallstone cases had asymptomatic gallstones diagnosed incidentally. Nevertheless, the prevalence of approximately 6% symptomatic gallstones in our study was comparable to results from previous studies that identified gallstones by similar registry-based methods.26 Because GSD is a hard endpoint with well-defined diagnostic criteria, the risk of misclassification is likely minor. In support of this, approximately 68% of individuals with symptomatic GSD in our cohort underwent cholecystectomy.26 Finally, the risk of MI and GSD as a function of weighted allele score was similar to the corresponding risk for coronary intervention and cholecystectomy, supporting reliability of the endpoints. Strengths and limitations A strength of our study is that we studied only white individuals from an ethnically homogeneous population. Although our results may therefore not necessarily apply to other ethnicities, we are not aware of data to suggest this. Additional strengths include the large sample size, the complete follow-up, the simultaneous assessment of plasma lipid levels, risk of MI, and risk of GSD as a function of genetic variation in CYP7A1 using weighted allele score, the association with LDL cholesterol for both common and rare genetic variants, and the association with increased risk of MI and GSD overall, in both prospective studies of the general population separately, and in both genders. Importantly, in addition to extensive independent replication internally between the CCHS and CGPS, adding data from available consortia in meta-analyses on LDL cholesterol, risk of coronary artery disease, and risk of GSD supported our findings. Because functional data were not available, and because we did not measure CYP7A1 activity, we can only speculate on the mechanisms underlying the association observed between genetic variants in CYP7A1 and plasma levels of LDL cholesterol, risk of MI and risk of symptomatic GSD. Nevertheless, our mechanistic interpretation of the data is in agreement with CYP7A1 as the initial and rate-limiting step in the conversion of cholesterol into bile acids in humans, with data on treatment with bile-acid-binding resins and elobixibat which up-regulate CYP7A1 activity and reduce LDL cholesterol in a dose-dependent manner, with functional data in humans on a truncating variant in CYP7A1 which reduced CYP7A1 activity in the liver and increased LDL cholesterol in a gene-dosage dependent manner, and with results from overexpression in animal models. Conclusion In conclusion, genetic variants in CYP7A1, which are associated with increased levels of LDL cholesterol are associated with an increased risk of MI and symptomatic GSD. It might be speculated that increasing CYP7A1 activity by bile acid sequestrants and other bile acid pool modulators may reduce risk of both MI and GSD. Supplementary material Supplementary material is available at European Heart Journal online. Acknowledgements We thank senior technician Mette Refstrup, Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, for her persistent attention to the details of the large-scale genotyping. Refstrup did not receive any compensation for her contribution. We are indebted to the staff and individuals of the Copenhagen General Population Study and the Copenhagen City Heart Study for their important contributions. Funding This work was supported by a grant from the European Union, Seventh Framework Programme Priority [FP7-HEALTH-2013-INNOVATION-1], contract #603091, The Danish Medical Research Council, The Research Fund at Rigshospitalet, Copenhagen University Hospital, Chief Physician Johan Boserup and Lise Boserup’s Fund, Ingeborg and Leo Dannin’s Grant, and Henry Hansen’s and Wife’s Grant, and a grant from the Odd Fellow Order. The study sponsors had no role in the conduct of the study, in the collection, management, analysis or interpretation of data or in the preparation, review, or approval of the manuscript. Conflict of interest: none declared. References 1 World Health Organization . Cardiovascular Diseases (CVDs). 2015. http://www.who.int/mediacentre/factsheets/fs317/en/ (29 December 2015). 2 Krawczyk M , Wang DQ-H , Portincasa P , Lammert F. Dissecting the genetic heterogeneity of gallbladder stone formation . Semin Liver Dis 2011 ; 31 : 157 – 172 . Google Scholar CrossRef Search ADS PubMed 3 Portincasa P , Moschetta A , Palasciano G. Cholesterol gallstone disease . Lancet 2006 ; 368 : 230 – 239 . Google Scholar CrossRef Search ADS PubMed 4 Cohen JC. Contribution of cholesterol 7alpha-hydroxylase to the regulation of lipoprotein metabolism . J Lipid Res 1999 ; 10 : 303 – 307 . 5 Davidson MH , Dillon MA , Gordon B , Jones P , Samuels J , Weiss S , Isaacsohn J , Toth P , Burke SK. 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Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alpha-hydroxy-4-cholesten-3-one in peripheral blood . J Lipid Res 2003 ; 44 : 859 – 866 . Google Scholar CrossRef Search ADS PubMed 24 Spady DK , Cuthbert JA , Willard MN , Meidell RS. Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol . J Clin Invest 1995 ; 96 : 700 – 709 . Google Scholar CrossRef Search ADS PubMed 25 Ratliff EP , Gutierrez A , Davis RA. Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia . J Lipid Res 2006 ; 47 : 1513 – 1520 . Google Scholar CrossRef Search ADS PubMed 26 Stender S , Nordestgaard BG , Tybjaerg-Hansen A. Elevated body mass index as a causal risk factor for symptomatic gallstone disease . Hepatology 2013 ; 58 : 2133 – 2141 . Google Scholar CrossRef Search ADS PubMed Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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European Heart JournalOxford University Press

Published: Feb 23, 2018

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