In the present study, we examined potential associations between 554 SNPs in 41 candidate gene regions proposed to be involved in lipid metabolism and MI incidence. We observed low nominal P-values (less than 0.01) for 10 genetic polymorphisms in our discovery sample. One of these associations, rs4149313 in the ABCA1 region, was consistent in two cohorts and one meta-analysis of studies from CARDIoGRAMplusC4D consortium including 177,719 European individuals in total.
, which is located on chromosome 9q31.1, codes for a cellular phospholipid and cholesterol transporter that mediates the formation of nascent HDL particles. This gene has an important role in HDL metabolism and certain mutations could lead to Tangier disease 
. rs4149313 is a non-synonymous SNP located in exon 18 of ABCA1
, which encodes an amino acid substitution, Ile>Met (Ile883Met). Ile883Met is located at N-terminal of the first nucleotide-binding motif, suggesting its potential function on formatting stable structure of ATP-binding cassette. In vitro
experiments showed that cholesterol efflux in transfected 293 cells with the I883M variant significantly differed from wild-type cells
, which supports the biological functionality of this variant. Furthermore, this variant is conserved in different species, including zebra fish, western clawed frog, chicken, common turkey, mouse, rat, guinea pig, dog, bovine, pig, and lowland gorilla
. The conservation of this variant also indicates its importance, as well as offers opportunities for functional studies of rs4149313 in different model animals. As rs4149313 is situated in a tight LD block in ABCA1
(), further fine-mapping using a re-sequencing approach target on rare causal variants in the region may also prove useful to further understand the role of ABCA1
in MI development.
Region plot of the ABCA1 locus (chromosome 9 co-ordinates 106157871–107157392) in the Swedish Twin Registry.
The analyses in STR showed that the minor allele of rs4149313 increased risk of MI in Swedish populations while not being associated with circulating HDL levels. This finding is in line with a prior study by Frikke-Schmidt and colleagues
in a Danish population. The recent study from the Global Lipids Genetics Consortium 
showed that the same allele that increased risk of MI/CAD in our study and in the prior Danish study also increase HDL and total cholesterol levels. This may seem conflicting, but recent Mendelian randomization studies
have indicated that plasma HDL cholesterol levels may not be a causal factor for CAD. Also, the majority of variants previously reported to be associated with both HDL and CAD have pleiotropic effects
. For example, CETP
variants are associated with decreasing plasma HDL and increasing LDL levels
. Considering that ABCA1
rs4149313 is also related with increasing total cholesterol level, it could affect CAD due to pleiotropic effects on other lipid fractions than HDL.
Several lipid-related loci have been identified in recent GWAS studies on MI/CAD, but cannot be replicated in our discovery study sample, for example, the LDLR
. The reason for this is that the effect of genetic loci is small, and that our discovery sample size is modest, leading to low statistical power. This illustrates the need for great caution when interpreting our negative results, as larger samples are needed to refuse associations.
We report evidence of a weak association between the ABCA1
locus and MI across three independent study samples including one meta-analysis of GWAS. The minor allele was associated with ~40% increased risk of incident MI during follow-up in two longitudinal cohorts while 3% increased odds in the CARDIoGRAMplusC4D consortium. This difference in effect sizes could reflect inflated effect estimates in the discovery sample (Winner's curse) or some degree of between-study heterogeneity for example due to differences in study design, age groups or ethnicity. In a sensitivity analysis, we rerun association analyses of rs4149313 and coronary heart disease (including also unstable angina, percutaneous transluminal coronary angioplasty and coronary artery bypass graft surgery in the endpoint definition) in our discovery sample, STR. These analyses were very similar to our main analyses in STR (HR, 1.41; 95% CI, 1.15–1.72). Since the association between ABCA1
rs4149313 and CAD showed such a weak effect (OR, 1.03) and just barely nominal significance (p
0.048), we cannot not entirely rule out the possibility for this finding to be a false positive. However, as rs4149313 is a missense variant predicted to be potentially functional and given our two-stage replication design with consistent findings (albeit decreasing effect sizes), we believe our finding to be of interest and likely a true positive.
In Sweden, all medical care is publically funded and available to all citizens, thus all in-patient care, medication and death information can be easily followed through national registries. In the present study, we decided to investigate the first incident MI through participants' entire life course via combining information from the Swedish national registries with information from questionnaires and blood sampling results in our studies. Therefore, we could include cases even before those individuals participated in STR or ULSAM which increased power of the present study, and avoided recall bias from self-reporting of disease. However, as the Swedish Patient Register was not completely introduced nationally until 1987, those who had their first incident MI before 1987 would be missed if MI was never recurrent again. Since MI is a disease developed across a long period, occurring during old age and repeatedly, we consider the use of age 18 as the entry age as a proper way to deal with our data maximizing the statistical power. In additional sensitivity analyses, the association of rs4149313 with incident MI was weaker when using blood drawing time as entering of follow up. This could be due to a potential survival bias caused by those whose first MI incidence occurred before 1987, or due to decreased power by losing 37% of the cases compared to our main analysis. Individuals experiencing fatal MI cases before baseline could for natural reasons not enter our study. The potential survival bias introduced by such early onset fatal cases could potentially decrease the effect size of our findings, and drive them towards the null.
The strengths of present study include the use of two cohorts and one meta-analysis of GWAS, which allowed independent replication; a large number of genetic loci studied in relevant gene regions and the stringent criteria for claiming association. There are also some limitations of our study. First, statistical power in our discovery sample and longitudinal replication sample was fairly low, which resulted in a non-significant association of ABCA1 rs4149313 with MI in STR and ULSAM after correction for multiple testing. Nevertheless, this association is replicated in two other independent study samples. The modest statistical power also means that several of our findings in the discovery sample that could not replicated could represent true associations with MI, and may merit further exploration in future studies. Second, candidate genes in this study were selected based on publications between 2003 to 2008 (largely before the GWAS era), and hence, many lipid-related loci that have been identified in later GWAS are not included in this study. Effects of such lipid loci on CHD would have to be explored in future studies. Third, since our study populations consisted of middle-aged to elderly individuals of European decent, the generalizability of our findings to younger individuals and other ethnicities is unknown.