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The chromosome 9p21(Chr9p21) locus is the most replicated genetic marker associated with coronary artery disease (CAD). The onset of dense gene arrays allowed the investigation of genetic variation at a genome-wide level with a high resolution. Thus, many genome wide association studies GWAS were done to understand the genetic components of disease. Through thousands of single nucleotide polymorphisms (SNPs) studied across the genome the locus in chromosome 9p21 was strongly associated with CAD in 2007. Further GWAS studies have confirmed the association of the 9p21 locus to CAD in cohort studies and in multiple ethnic groups. In addition, it has been determined that the Chr9p21 locus has a haplotype block that contains many single nucleotide polymorphisms in linkage disequilibrium that span a region of approximately 53 kb (Figure 1). The CAD haplotype has also been described as a “gene desert,” devoid of annotated protein-coding genes. The closest protein-coding genes are the 2 cyclin-dependent kinase inhibitors, CDKN2B and CDKN2A (Figure 1). However, the region also contains a long antisense noncoding RNA in the INK4 locus (ANRIL) (Figure 1). Many studies hypothesize that ANRIL expression is regulated by Chr9p21 and could influence cardiovascular risk.



History
CAD is the number one killer in the world. Through many decades of research, many efforts have been attempted to try to change this fact. There are several associated risk factors with CAD that have been identified that increase the amount of deaths due to CAD. Through many clinical trails, factors such as abnormal lipids, smoking, hypertension, diabetes, lack of exercise, abdominal obesity, psychosocial factors, amount of fruits and vegetable consumption, and alcohol consumption increase the chance of death from CAD. But, many of these factors can be reduced by simple lifestyle choices. However, even though the amount of deaths from CAD has decreased, a plateau has been reached in reducing the amount of deaths from CAD. Since, a lot of diseases have genetic predisposition, scientists now have changed the direction of combating CAD deaths by focusing on genetics in order to reduce the amount of deaths from CAD.

The understanding of the genetics of CAD came from the power of family history. The Framingham Heart Disease study is a study that tried to understand the affect of family history on the susceptibility to CAD. This study was established in 1948 and terminated in 2001 and followed 5209 residents of Framingham Massachusetts from ages 28 to 62 years to determine if CAD is an independent predictor of offspring cardiovascular events. They found in this study that there is a 2.6-fold higher prevalence of CAD with men who have a family history of CAD and a 2.3-fold increased risk for women who have a family history of CAD as well. Indicating that family history plays a major role in the susceptibility to CAD. Furthermore, unlike some diseases that are controlled by a single gene the genetic susceptibility to CAD is controlled by multiple genes. Therefore, when these genes are summed together, an individual is susceptible to CAD.

Therefore, in order to detect and map where these genes are it would require a lot of DNA markers. A study like the Framingham Heart Disease, which relies on linkage based on genotyping pedigrees, lacks the ability to map the location of predisposing genes because of the analytical sensitivity required. Now, we have the technology to allow us to identify these genes. We can do this by mapping the chromosomal locations of genes that predispose CAD by using many DNA markers or single nucleotide polymorphisms (SNPS) at certain intervals. If an affected individual inherits one or more of the markers we say that the marker is genetically linked. This means that the gene that controls this phenotype is physically close to the DNA marker. The technology used to pursue predisposed genes is called (GWAS). The technology of GWAS has now allowed us to map the genetic predisposition to CAD so that we can not only treat CAD, but also prevent it.

=Genetic Mapping Approaches=

Mapping in Caucasian cohorts
The first attempt to map the locus that is genetically predisposed to CAD was done by a genome-wide association scan that studied six independent samples from four Caucasian populations. The first sample called the Ottawa Heart Study 1 (OHS1) used cases and controls from Caucasian men and women in Ottawa, Canada in 2007. Cases used had a documented onset of CAD before the age of 60 years and had no diabetes or plasma cholesterol levels consistent with monogenic hypercholesterolemia. Controls were healthy Caucasian men >65 years old and women >70 years old from Ottawa who had no symptoms or history of CAD. Custom oligonucleotide arrays were used to assay 100,00 SNPS throughout 322 cases and 312 controls. There were around 2586 SNPS associated with CAD at a significance threshold of .025. These 2586 SNPS were then genotyped in an independent sample from Ottawa called Ottawa Heart Study 2 (OHS2) using the same criteria as the (OHS1). There were 50 associated SNPs with CAD at a significance threshold of 0.025. There was another case-control comparison done called the Atherosclerosis Risk in communities (ARIC) which followed 11,478 Caucasians. However, only 2 of the 50 SNPS originally found in the Ottawa populations were significantly associated with CAD. These two SNPs, rs 10757274 and rs2383206 were in strong linkage disequilibrium (r2=0.89) with each other. To validate these two SNPs they were assayed in three additional cohorts: Copenhagen City Heart study (CCHS), a study of about 10,578 Danish men and women, the Dallas Heart Study (DHS) a sample of Dallas residents consisting of 154 cases and 527 controls, and finally a third sample called the Ottawa Heart Study 3 (OHS3) of about 647 cases and 847 controls from the Ottawa Heart Study population. In all three studies, both SNPS rs 1075724 and rs2383206 were significantly associated with CAD. The two SNPs portray an allele that was associated with a 15-20 % increase in risk in the 50 % of the individuals who were heterozygous for the allele and a 30 -40 % increase in risk in CAD in 25 % of individuals who were homozygous for the allele. To fine map the locus associated with CAD, they assayed SNPS spaced about 5kb across the region extending 175kb upstream and downstream of the two SNPS. They found eight additional SNPS spanning about a 53-58 kb region located on chromosome 9p21. All eight of the SNPs were in linkage disequilibrium with each other including the two SNPs rs1075724 and rs2383206 (Mcpherson et al., 2007). The data found confers that there is a risk allele that comprises a 53-58kb haplotype on chromosome 9p21. These six independent samples provided a starting point to map gene predisposition of CAD. In order to confirm this locus, additional GWAS studies took place. For example, one study used an Icelandic population, but used Myocardial infarction (MI) as a phenotype because CAD is a chronic multi-stage inflammatory disease with MI as an acute complication. After using a total of 4587 cases and 12, 767 controls, the identified variant on chromosome 9p21 portrayed high significance. About 21 percent of this population is homozygous for this allele and their risk for obtaining (MI) is 1.64 times as great as non-carriers. Other studies used populations such as British and German to confirm this locus. The Welcome Trust Case Control Consortium (WTCCC) was a GWAS study conducted on the British population looking for identification of genes involved in common human diseases. In particular, in relating to CAD they had 1988 case subjects that had a history of MI before the age of 66. The control group was 1500 healthy individuals with no complications of CAD that donated blood to the WTCC study. They found a powerful association of CAD on chromosome 9p21 .In the study relating to German population, they identified loci that were strongly associated with (CAD) from the (WTCC) study and looked for replication of this loci in this German population. The study involved 875 case subjects with MI and 1644 controls. It was found that once again the 9p21 loci had the strongest association with CAD .Both these studies confirmed the 9p21 locus. Across all these studies mentioned above over 60,000 Caucasians were genotyped and it was found that around 75% of this Caucasian population had the 9p21genetic risk variant. This locus is heterozygous in 50% of Caucasians and homozygous in 25% with an increased risk of 15-20% and 30-40% respectively.

Multi Ethnic Mapping
A lot of the early mapping studies of CAD were done on populations from European descent; however, to effectively prevent CAD we need to develop knowledge of the risk locus in different geographic regions especially in severely developed countries where rates of CAD have increased. The ADVANCE study was one of the first studies that tried to understand the association of this disease to other racial/ethnic groups. The study cohort included 1,201 cases who had white/European ancestry, 96 cases who were African Americans, 108 who were Hispanics, 117 who were East Asians, and 287 who were mixed ancestry. They sought to look at three SNPS associated in the 55-58 kb risk interval. These SNPs were rs10757274, rs2383206, and rs107557278. The SNPs, rs1075724 and rs2383206, were previously studied in the Ottawa Heart Studies mentioned earlier and it was found that in the ADVANCE study these SNPS were once again associated with CAD in whites. On the other hand, one or more of these SNPs were associated with CAD in US Hispanics, and US East Asians. However, none of the SNPS were associated with CAD in African Americans. Another study entitled, Multi-Ethnic study of Atherosclerosis (MESA) tried to confirm the findings in the ADVANCE study. The cohort they looked at was 2,329 Caucasians, 691 Chinese, 2482 African American and 2,012 Hispanics. They unlike ADVANCE study tried to look for association of around 66 previously published SNPS (from published GWAS studies) .They had a SNP significance defined by Bonferroni-corrected p< 7.6 x10-4. With a larger study, the MESA studied confirmed the findings that there were significant associations for CAD with SNPs in the 9p21 locus in Caucasian population, and Hispanic. In addition, they also found no significant associations for CAD in African American populations. The fact that there are no significant associations for CAD in African American populations in both the ADVANCE and MESA study raises a very interesting discussion about how GWAS is conducted and about the genetics of the African American population. In many of the early GWAS studies conducted on CAD, a lot of the populations looked at first were from European descent. A possible reason for this is the fact that this cohort was used before CAD was mapped in many other GWAS studies to map things such as cancer, diabetes, macular degeneration, and Crohn’s disease .Therefore, it was a lot easier and cheaper to collect data on CAD from this cohort. However, it interesting to note that in the African American CAD mapping studies a smaller cohort was used compared to a larger cohort of European descent. Also this trend holds true for many other GWAS studies. Of the current 2,267 GWAS studies in 2014, only 14, 3 and 1 percent accounted for groups such as African American, Hispanic, or Jewish populations respectively. There are many reasons to include more African American cohorts in studies. One reason is that it is critical to the understanding of human variation and complex diseases to study more diverse set of cohorts. Also in particular to the African cohort they harbor populations with rich history and include many variants that are associated with diseases that don’t always replicate in non-Europeans .GWAS studies offer a great technology so that we can more closely map the genetics of a disease. However, in relating specifically to CAD it has allowed us to more closely understand the risk locus of CAD. But, it is important as GWAS studies progress that we make sure we are cautious of the inclusion of ethnic diversity in these studies. Keeping this observation in mind, a metaanaylsis was conducted on many of these GWAS studies that found that the effect of the risk locus in CAD was dependent on the age of disease onset. Individuals that were aged 55 years or younger had an odds ratio (OR) of 1.35 compared with individuals aged 56 to 75 years who had an OR of 1.21 indicating that the 9p21 risk locus had a stronger effect at an earlier age.

=Functional characterization of 9p21 risk locus = After many GWAS studies conducted, it is important to understand the functional significance of the risk locus within the 9p21 chromosome so that new drugs or treatments can be developed to combat CAD. There have been a couple studies that have tried to investigate the functional significance of the risk locus within the 9p21 chromosome. However, it is important to understand that the risk locus which is around 53-58kb is adjacent to CDKN2A (p16, inhibits CDK4) and CDKN2B (p15, CDK4) both which are involved with cell cycle regulation and encode cyclin-dependent kinase inhibitors that inhibit cell growth and proliferation. Next to CDKN2A and CDKN2B is ANRIL that encodes an antisense noncoding RNA that is part of the 53-58 kb risk locus. Multiple studies have tried to understand the relationships between these different genes and the CAD risk allele. One study tried to understand the risk locus by deleting this interval in mice and looking at the effect. The study deleted an orthologous 70-kb noncoding interval on mouse chromosome 4 that is orthologous to chromosome 9p21 risk locus .This region is 20% larger than the human interval particularly because there is increased repeated sequence content. This deleted interval lead to affecting cardiac expression of neighboring genes. Particularly, cardiac expression of the two noncoding genes Cdkn2a and Cdkn2B were severely reduced in the Chr4 Δ70kb/ Δ70kb mice. This suggests that CAD risk interval controls regulation of cardiac CDKN2A/B expression.Furthermore, one study tried to determine the relationship of this risk locus to the expression of ANRIL and CDKN2A/B expression. ANRIL is expressed in arterial smooth muscles cells and vascular smooth muscle cell proliferation .They found in their findings that the risk allele contains a functional enhancer, which activity is altered to those who carry the risk allele. There is a 2.2 fold increase in whole blood RNA expression of the short variants of ANRIL and a 1.2-fold decrease in the long ANRIL variant in individuals homozygous for the risk allele. Furthermore, many studies have been able to correlate that increased expression of the long transcript of ANRIL is associated with increased expression of CDKN2A/B when no risk allele is present. But, when the risk allele is present there is an increase in the short ANRIL transcript and decreased expression of CDKN2A/B.

These studies offer a possible explanation that the risk locus might promote CAD by regulating expression of ANRIL, which is associated with altered expression of genes controlling cellular proliferation pathways. Thus, by controlling cellular proliferation and inducing something such as smooth muscle proliferation it could serve as a possible cause for CAD. These studies offer a possible role of the risk locus; however, it is not yet certain, as many more studies need to take place to fully understand the risk locus role. But, it should be highly noted the role of GWAS has had in allowing us to be closer than ever in learning how to prevent CAD. This thus raises the question of whether a routine of genotyping risk alleles should be a routine practiced in medicine. There are many answers to this controversial question. However, in regarding the 9p21 risk locus, we simply don’t know much about this locus’s mechanism to develop specific treatments for anyone that carries it. But, the discovery of new treatments will really dictate how important these genetic variants are, since we have yet to see how results such as the discovery of the 9p21 risk locus has altered the management of a patient with CAD. Yet, there is no longer a technological barrier to accomplish this and it is important to able reduce CAD deaths by not only treating it, but preventing it as well.

=Reference List=