Putting genome analysis to good use: Lessons from C-reactive protein and cardiovascular disease

The rise of genome-wide association studies

Over the past decade, much clinically useful information has been gathered in genome-wide association studies.

The rise of this type of study rested on our emerging understanding of the architecture of our genome. When the genomes of multiple humans were fully sequenced, we discovered that specific variants do not occur randomly in relation to each other. Rather, they tend to be inherited in particular blocks called haplotypes, and some SNPs or combinations of SNPS are very rare or essentially never seen.

In its first phase, the HapMap project organized these useful blocks of variants, genotyping 1 million SNPs for each of 270 individuals from mother-father-offspring trios from distinct geographic regions of the world.³ The second phase of the HapMap project extended the analysis to more than 3 million SNPs and to other populations.⁴

While the HapMap should be generally applicable to other populations not yet studied, limitations of the first two HapMap phases include rare SNPs or CNVs, or variants outside of haplotype regions.

The 1,000 Genomes Project, now under way, will develop an even more comprehensive catalog of human genetic variants in much broader populations.

The success of genome-wide association studies is also partly attributable to progress in DNA-sequencing technology. Using microarray chips, we can now look at millions of SNPs per patient or the entire coding sequence of the genome (termed the exome) in a single experiment that is both time-effecient and cost-effective.

What is a genome-wide association study?

A genome-wide association study generally compares genetic variants between patients with a particular clinical condition (cases) and people without the condition (controls), looking for statistically significant differences. As a tool for genetic discovery, these studies have revealed many avenues for further investigation in the pathogenesis of disease, as well as potential targets of therapy.

Using these studies, research groups around the world have found reproducible correlations between genetic variants and susceptibility to common adult-onset diseases.

Although many of the variants identified in these studies are associated with only a slightly higher risk of disease, the method is free of many of the inherent biases associated with clinical research. These studies permit a comprehensive, hypothesis-independent and unbiased scan of the genome to identify novel susceptibility factors, whereas earlier genetic epidemiology studies could take on only a handful of variables to evaluate at a time. Additionally, they are powered to detect very small increases (or decreases) in disease risk, previously outside the reach of linkage analysis. Polymorphisms (or, presumably, non-disease-causing DNA changes) discovered using these studies often correlate with clinical phenotypes or with levels of biomarkers, even if the genetic variants are not necessarily pathologic in themselves.

Thus, genome-wide association studies have led to important insights into the pathogenesis of multiple common diseases, such as inflammatory bowel disease and diabetes mellitus, and they are facilitating new treatment approaches. For instance, multiple studies have reproduced an association between Crohn disease and variation in the gene NOD2, whose protein product is implicated in bacterial product recognition, autophagy, and apoptosis.⁵ This discovery led to the investigation of new potential therapies for Crohn disease, ie, the tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva), known to inhibit NOD2 activity, and to the prognostic use of the NOD2 genotype in Crohn disease (a field of study known as genotype-phenotype correlation).

Future advances will likely come from looking at combinations of variants, which may carry a higher risk of disease than single variants.

CORONARY HEART DISEASE: FRESH INSIGHT INTO AN OLD PROBLEM

Cardiovascular disease accounts for 30% of deaths worldwide.⁶ Of all the cardiovascular disorders, coronary heart disease is rising most rapidly in incidence, as the rest of the world adopts Western practices such as a high-calorie, high-fat, high-glycemic diet.

Hundreds of risk factors for coronary heart disease have been described.⁷ Three of them are clearly modifiable participants in the pathogenesis of atherosclerosis: hypertension, smoking, and elevated LDL-C. These and others form the basis for risk-assessment tools such as the Framingham risk score and the Prospective Cardiovascular Münster (PROCAM) study score. Other possible markers require further evaluation as to whether they are clinically useful and are direct mediators of coronary heart disease.

Because up to 40% of coronary deaths occur in people who lack conventional risk factors for it (eg, they do not smoke and they have normal levels of LDL-C and blood pressure), researchers are searching hard for new, potentially treatable risk factors.⁸ Of particular interest are components of inflammatory pathways linked with atherosclerosis and coronary heart disease. The identity of the key inflammatory factors that cause arterial plaque formation and rupture continues to be studied.

CRP, an acute-phase reactant produced by the liver in response to inflammation, has received much attention, as serum CRP levels correlate strongly with coronary events. Researchers have used modifiers of CRP to try to alter the course of coronary heart disease, but traditional research has so far failed to establish a causal relationship between CRP and coronary heart disease.⁹

How we know that LDL-C is a mediator, not just a marker

As a risk factor, LDL-C resembles CRP in that its levels correlate with a number of other, confounding risk factors. Therefore, much basic research and clinical observation had to be done before we could say that LDL-C plays a role in the pathogenesis of coronary heart disease.

Initially an association between LDL-C and heart disease was noted.¹⁰ Then, studies of familial hypercholesterolemia uncovered genetic abnormalities that increase LDL-C levels and, thereby, the risk of coronary heart disease—eg, mutations in the LDL receptor gene,^11–14 the apolipoprotein B (APOB) gene at its LDL receptor-binding domain,¹⁵LDL-RAP1 (a gene encoding an accessory adaptor protein that interacts with the LDL receptor),¹⁶ and PCSK9 (a gene that codes for proprotein convertase subtilisin-kexin type 9 protease).¹⁷

Conversely, specific loss-of-function truncating mutations of PCSK9 that reduce LDL-C levels are associated with strong protection against coronary heart disease.¹⁸ Other gene mutations that reduce LDL-C also lower the risk.^19,20

Further, a genome-wide association study²¹ identified multiple genetic variations associated with different forms of dyslipidemia, uncovering additional links between LDL-C and coronary heart disease.

Finally, randomized controlled trials of niacin, fibrates, and statins showed that these potent LDL-C-lowering agents reduce the rate of development or progression of coronary heart disease.^22,23