Common Genetic Variation in MC4R Does Not Affect Atherosclerotic Plaque Phenotypes and Cardiovascular Disease Outcomes

We analyzed the effects of the common BMI-increasing melanocortin 4 receptor (MC4R) rs17782313-C allele with a minor allele frequency of 0.22–0.25 on (1) cardiovascular disease outcomes in two large population-based cohorts (Copenhagen City Heart Study and Copenhagen General Population Study, n = 106,018; and UK Biobank, n = 357,426) and additionally in an elderly population at risk for cardiovascular disease (n = 5241), and on (2) atherosclerotic plaque phenotypes in samples of patients who underwent endarterectomy (n = 1439). Using regression models, we additionally analyzed whether potential associations were modified by sex or explained by changes in body mass index. We confirmed the BMI-increasing effects of +0.22 kg/m2 per additional copy of the C allele (p < 0.001). However, we found no evidence for an association of common MC4R genetic variation with coronary artery disease (HR 1.03; 95% CI 0.99, 1.07), ischemic vascular disease (HR 1.00; 95% CI 0.98, 1.03), myocardial infarction (HR 1.01; 95% CI 0.94, 1.08 and 1.02; 0.98, 1.07) or stroke (HR 0.93; 95% CI 0.85, 1.01), nor with any atherosclerotic plaque phenotype. Thus, common MC4R genetic variation, despite increasing BMI, does not affect cardiovascular disease risk in the general population or in populations at risk for cardiovascular disease.

The Copenhagen City Heart Study (CCHS) is a study of the general population that was initiated in 1976-1978 with follow-up examinations in 1981-1983, 1991-1994, and 2001-2003. Participants were selected with the use of the Danish Civil Registration System to reflect the adult Danish population 20 to 100 years of age or older. Data were obtained from a questionnaire, a physical examination, and from blood samples. During follow-up (which ended in May 2011), 2817 participants had incident ischemic vascular disease, of whom 2198 had ischemic heart disease. The Copenhagen General Population Study (CGPS) is a study of the general population that was initiated in 2003 with ongoing enrolment. Participants were recruited and examined exactly as in the CCHS. Information on diagnoses of ischemic heart disease (International Classification of Diseases, 8 th revision , codes 410 through 414; 10 th revision , codes I20 through I25) and ischemic cerebrovascular disease (ICD-8, codes 431 through 438; ICD-10, codes I60 through I69 and G45) was collected and verified through a review of all hospital admissions and diagnoses entered in the Danish National Patient Registry, all causes of death entered in the National Danish Causes of Death Registry, and medical records from hospitals and general practitioners. The Danish National Patient Registry has information on all patient contacts with all clinical hospital departments and outpatient clinics in Denmark, including emergency wards (from 1994). The National Danish Causes of Death Registry contains data on the causes of all deaths in Denmark, as reported by hospitals and general practitioners. Ischemic heart disease was fatal or non-fatal myocardial infarction or characteristic symptoms of angina pectoris, including revascularization procedures (ICD8: 410-414; ICD10: I20-I25). A diagnosis of myocardial infarction followed the changing definitions over time and required a typical rise and fall of biochemical markers (troponin or CK-MB), with later changes as indicated. Cases with ischemic cerebrovascular disease and ischemic stroke (ICD8: 431-438; ICD10: I60-I69, G45) were collected likewise.

General population: UK Biobank
The UK Biobank is a study of the general population of the United Kingdom. Baseline assessments took place between 2006 and 2010 in 22 different assessment centers across the country. [28] The project was completed under project number 56340. Information on diagnoses was collected through linkage with the NHS hospital admissions database based on ICD-10 coding (CAD, I21, I22, I24, I25; MI, I21; Stroke, I63 [ischemic stroke only]). More information on the genotyping processes can be found online. [29] Population at risk for cardiovascular disease: PROSPER Between December 1997 and May 1999, subjects were screened and enrolled in Scotland (Glasgow), Ireland (Cork), and the Netherlands (Leiden) for the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). Men and women aged 70-82 years were recruited if they had pre-existing vascular disease or increased risk of such disease because of smoking, hypertension, or diabetes. A total number of 5,804 subjects were randomly assigned to pravastatin or placebo. A large number of prospective tests were performed including Biobank tests and cognitive function measurements. The primary endpoint in the study was the combined endpoint of death from coronary heart disease (CHD), non-fatal myocardial infarct (MI), and occurrence of clinical stroke, either fatal or non-fatal. When death occurred following a non-fatal stroke within a period of 28 days, it was regarded as a fatal stroke. All endpoints were adjudicated by the study endpoint committee. A detailed description of the study has been published elsewhere. [19,30] A whole genome wide screening has been performed in the sequential PHASE project.
[21] Of 5,763 subjects DNA was available for genotyping. Genotyping was performed with the Illumina 660K beadchip, after quality control (call rate <95%) 5,244 subjects and 557,192 SNPs were left for analysis. These SNPs were imputed to 2.5 million SNPs based on the HAPMAP built 36 with MACH imputation software. Specific SNPs were extracted from the database with Plink software (http://zzz.bwh.harvard.edu/plink/).

Atherosclerotic plaque characteristics: Athero-Express study
Details on genotyping, quality control and imputation have been published previously.
[31] In brief, DNA was extracted from whole blood, or alternatively from plaque samples, following standardized in-house validated protocols. Genotyping was done using commercially available genotyping chips. The first batch (Athero-Express Genomics Study 1, AEGS1) was genotyped using Affymetrix Genome-Wide Human SNP Array 5.0, the second batch (Athero-Express Genomics Study 2, AEGS2) was genotyped using the Affymetrix Axiom® GW CEU 1 Array (Affymetrix, Santa Clara, CA, USA). We adhered to community standard quality control and assurance (QCA) procedures to clean the genotype data obtained in AEGS1 (N = 571) and AEGS2 (N = 868).
[32] The 998 phased haplotypes from the Genome of the Netherlands Project release 4 (GoNL4) encompassing 19,763,454 SNPs was used as the reference panel for imputation. [33] Histological sections were stained with haematoxylin and eosin for a general overview including calcifications and intraplaque hemorrhages, picrosirius red for collagen content, α-actin for smooth muscle cells, CD68 for macrophages, CD66b for neutrophils, mast cell tryptase for mast cells, and CD34 for microvessel density. Presence of collagen, and calcification were scored semi-quantitatively as no, minor, moderate or heavy staining in different locations in the plaque, and grouped for analysis in no/minor vs. moderate/heavy; intraplaque hemorrhage was scored as absent (no) vs. present (yes). Fat content was scored as no or less than 10%, and more than 10%. Macrophage and smooth muscle cell content were reported as percentage positive staining per plaque area. Microvessel density in the plaque was quantified in three hotspots and expressed as an average number of vessels per hotspot. In a subsample of the total population, neutrophil and mast cell content was quantified as total number of positive cells in the plaque.