Understanding the Role of Genetics in Peripheral Arterial Disease

Peripheral arterial disease (PAD) is a multifaceted condition, which affects more than ten percent of the general population older than 55. Although the prevalence of PAD is high and it is associated with high rates of morbidity and mortality, relatively little is known about its causes. In comparison to what is known about te traditional risk factors for PAD, very little is known about genetic risk factors. The aim of this paper is to define what is currently known about the role of genetics in PAD, along with discussing the genetic studies that have been performed, and the potential future implications of these studies. This information was obtained via a search of the English language medical literature between the years of 1966 and 2007. Retrieved articles pertaining to genetics and PAD were obtained through the PubMed search engine using the keywords genetics, PAD, and single nucleotide polymorphisms. Articles ranged from those which provided general information to those which postulated possible future treatments for PAD, entirely based on its genetic pathogenesis. This paper will provide a comprehensive overview of the genetic associations with PAD, along with offering some theories as to the potential future impact genetics may have in PAD research.

Genetic Factors Influencing Peripheral Arterial Disease

The most recent and ongoing genetic studies involve the use of large-scale genetic testing to detect single nucleotide polymorphisms (SNPs) that are significantly associated with PAD. Linking the SNP data with biological pathways leading to PAD may identify novel therapeutic targets for PAD, in addition to identifying those at high risk of developing the disease. Data on the subject is limited, but in the future, genetic testing may be recommended for those with a clear family history of PAD or those with other co-morbidities linked with a higher risk of PAD.

Diabetic patients with first-degree family members who underwent amputation were found to have a higher prevalence of PAD. This suggests that genetic factors may influence the severity of PAD in patients with diabetes. In a study of Hispanics over the age of 65 with type 2 diabetes, an association with the E-selectin gene was found in Mexican-Americans with a family history of premature cardiovascular disease. Gene polymorphisms on the E-selectin gene are a genetic variation of the gene which can be associated with an increased risk of the disease or the severity of the disease. The level of LPA has also been strongly associated with type 2 diabetes, which again could influence the severity of PAD in these patients.

Specific genes responsible for causing PAD are yet to be identified for a number of reasons. The genetic make-up of an individual is complex, and a combination of gene mutations, variants, and inherited risk factors contribute to the risk of developing PAD. However, in certain diseases where PAD is a common co-morbidity, for example in diabetes, genetic studies have been carried out in an attempt to establish a genetic basis for PAD.

Gene Mutations and Variants

It is also likely that certain SNPs contribute to the development of PAD risk factors such as diabetes and hypertension. By gaining a better understanding of how genetic variation influences PAD and its associated cardiovascular events, it may be possible to identify patients at an early stage who are at increased genetic risk and therefore tailor specific preventative measures.

Studies have shown that SNPs within specific genes may lead to increased risk of developing ischaemic heart disease and also result in abnormal clinical outcomes of the disease. In a study, patients with intermittent claudication who had previously undergone femoral-popliteal bypass surgery were evaluated for the composite outcome of cardiovascular death or myocardial infarction. It was found that patients with SNPs in the interleukin-6 gene had an increased risk of the composite outcome suggesting that genetic factors affecting inflammation may influence the systemic manifestations of atherosclerosis.

Gene mutations causing genetic diseases can occur at any one of the three billion sites in our DNA. The most common forms of genetic variation are known as single nucleotide polymorphisms (SNPs, or “snips”) in which a single base change occurs at a particular position in the human genome. Usually, SNPs have no harmful effects by themselves; they are normal variations that occur within the general population often with no known health problems occurring in carriers of an SNP.

Inherited Risk Factors

Inherited risk factors have been studied for more than 40 years as an important part of the epidemiology of PAD. Family history was considered the most prevalent inherited risk factor but is also a non-specific risk factor for many other diseases. Therefore, several large-scale studies have been undertaken to ascertain specific inherited risk factors of PAD, either by comparing the prevalence of risk factors in patients and controls or between siblings discordant for PAD. These studies have highlighted several inherited risk factors, which are also partly included in recent genome-wide association studies. Most prominently, these studies showed that type 2 diabetes is a heritable risk factor for PAD, not explained by its strong association with acquired risk factors. High lipoprotein (a) concentration was found to be a significant inherited risk factor for PAD that remained strongly associated with PAD after adjustment for acquired risk factors. Hyperhomocysteinemia was also demonstrated to be a heritable risk factor for PAD, which has possible implications for therapy of PAD with folic acid and B vitamins. As yet, genetic data on these risk factors is limited to candidate gene studies but holds potential for the discovery of new therapeutic targets for PAD. A family history of claudication has been shown to be more prevalent in patients than controls and to be associated with a younger age at onset of disease. A recent family-based survey has also discovered an association of paternal history of myocardial infarction with the onset of PAD in sons. This is additional to a well-established association of PAD with a history of myocardial infarction in the patient. These findings are consistent with the genetic theory of PAD and have provided further direction for research into specific inherited risk factors.

Gene-Environment Interactions

The interactions between genetic and environmental risk factors are complex and not well understood. However, in the multifactorial model that best describes the pathogenesis of PAD, gene-environment interactions are pivotal. It is clear that some individuals, particularly smokers, with a given genetic makeup are at a much higher risk for developing PAD. Smokers are up to ten times more likely to develop PAD than non-smokers. In addition, smokers with a family history of myocardial infarction had an earlier age of onset of the disease by up to 12 years when compared to non-smokers with no family history of CVD. In a study, heritability of PAD was shown to be 17%, but in current or former smokers heritability was 61%, suggesting that the presence of a deleterious genetic background significantly increases the risk of developing PAD among those exposed to environmental risk factors. Despite the strong evidence of gene-environment interactions, the specific genetic polymorphisms that modify the effect of environmental risk factors in PAD are largely unknown. An increased understanding of the nature of gene-environment interactions in PAD and the specific genetic polymorphisms involved may provide potential targets for the primary prevention of the disease in the future.

Genetic Testing and Diagnosis of Peripheral Arterial Disease

For genotyping methods, a variety of tests can be employed in detecting genetic markers. Restriction fragment length polymorphisms (RFLP) test is an older method where DNA is digested by restriction enzymes and gene alterations are then identified by changes in restriction fragment lengths. PCR and primer-extension based methods require designed probes to amplify and identify the specific gene sequence. High-throughput genotyping can be performed with microarray hybridization. With the decrease in costs, availability, and high throughput, SNP tests and whole genome sequencing can be applied to determine global gene differences between case control populations. The differing gene variants can also be functionally tested on animal models or in vitro studies to identify their effects on atherogenesis.

The genetic risk for developing PAD has only been tested by one research group in a small Israeli case control cohort. Since many of the genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) had only a modest effect on cardiovascular disease (CVD) risk, a more comprehensive tool would be required to estimate genetic risk in PAD. A discriminative test to identify individuals with or at increased risk of PAD could be used for recruiting high-risk patients into a prevention or clinical trial. The test could also be used for identifying individuals with a genetic predisposition to PAD. Currently, there are no known specific genetic markers for PAD. The field of genetic epidemiology analyzes the influence of genetic factors on the phenotypic expression of the disease in relation to environmental interactions. Any positive findings from current GWAS in CVD will be elaborated through further GWAS or candidate gene studies where the population is stratified according to CVD conditions.

Screening for genetic markers Genetic testing methods Diagnostic implications

Screening for Genetic Markers

As a first step in identifying genetic markers for PAD, patients and controls are compared to uncover genetic variations that are more prevalent in those with the disease. There are two types of studies that are used to compare genetic differences between these different groups. The first are candidate-gene association studies, in which genes with known function in relevant biological pathways are tested for their association with the disease. The second type of study is a genome-wide study, in which linkage analysis is used to detect which areas of the genome are likely to contain the gene of interest. With the recent advancement in bioinformatics, it is becoming increasingly more practical to conduct genome-wide studies using SNPs. These studies involve large-scale genotyping of thousands of SNPs in hopes of locating the genes responsible for PAD. SNPs that demonstrate a statistically significant difference in allele frequency between patients and controls are then sought after in replication studies to validate their association with the disease. If the replication studies are successful, these SNPs can be used as genetic markers to determine the risk of PAD. This information may be used in a clinical setting in the future to help identify patients at risk for PAD who lack symptomatic presentations of the disease.

Genetic Testing Methods

Genetic testing to identify the actual gene mutations that lead to certain types of PAD likely will not only help to identify persons at risk but will further aid elucidation of pathogenesis of the disease. Since identification of gene mutations is a specialized field that is costly and time consuming, this kind of research is likely most efficiently done through collaboration of investigators recruiting carefully characterized patient populations. These methodologies generally involve comparison of specific gene polymorphisms in patients with the disease to those without the disease or with different severity of disease. Usually it is best to start with studies of candidate gene polymorphisms based on what is known of the pathogenesis of the disease. If successful, the use of genome wide association studies using microarray technology can efficiently identify gene polymorphisms associated with the disease. Combining results of genetic studies with protein expression studies and functional studies of the gene polymorphisms can help to build a more complete understanding of the pathogenesis of PAD. At some point in the future, it may be possible to develop a clinical genetic test predicting risk of PAD or severity of PAD.

Diagnostic Implications

The potential future impact of genetic research on PAD is a more accurate identification of risk for the disease and development of more effective and personalized treatments. In the interim, family history remains the sole method of identifying a genetic predisposition to PAD. It is known that individuals with a history of familial hypercholesterolemia have a higher likelihood of developing PAD. Similarly, identification of PAD in a patient with a history of premature atherosclerosis will lead to a diagnosis of familial leg ischemia due to common risk factors shared by these diseases. Knowledge of a genetic predisposition to a specific cardiovascular risk factor will likely increase the frequency and intensity with which a patient is screened for PAD. This is especially important in diabetic patients as the high prevalence of PAD in this population has made leg artery auscultation a recommended part of routine physical exam of diabetic patients. Gene testing could potentially provide an explanation for cases of PAD due to genetic factors other than those tested for gene tests in this day and age; however, the effectiveness of these will likely parallel technological development in the field.

Implications for Treatment and Prevention

Analysis of genetic variations may also identify novel therapeutic targets for PAD. An improved understanding of the genetic determinants of processes such as atherosclerosis or aberrant angiogenesis, which underlie PAD, will provide valuable information about druggable targets to modify these disease processes. This may ultimately lead to development of new therapies that are more effective than those currently available.

Medical therapies which are currently used for PAD, such as antiplatelet agents or statins, have generally shown only modest efficacy in improving symptoms or altering disease course. An inability to identify patients who are most likely to respond to specific therapies may contribute to this lack of efficacy. In this context, genetic information can be used to match patients to therapies which are most likely to benefit them, thus improving the risk-benefit ratio of these therapies on a population basis.

A recent study by our group demonstrated the potential utility of a genetic risk score, based on 9p21 genotype, in predicting adverse cardiovascular outcomes in patients with symptomatic PAD. This is particularly relevant to prevention of disease complications, as it may be possible to use such genetic risk scores to identify PAD patients at very high risk of future events, who might derive the most benefit from aggressive lipid-lowering or antiplatelet therapies. In the future, it is foreseeable that gene array data considering multiple single nucleotide polymorphisms could be used to generate comprehensive genetic risk scores for individual PAD patients.

Personalized medicine, which focuses on tailoring medical treatments to individual patients, holds great promise in the management of patients with PAD. Genetic information can be used to predict an individual’s likelihood of developing PAD, as well as their likely progression to advanced disease states. Using a combination of clinical data and genetic information, it may be possible to stratify PAD patients according to their risk of disease progression, which would allow targeting of aggressive therapy to those patients most likely to benefit.

Personalized Medicine Approaches

More recently, the development of the direct factor Xa inhibitor Apixaban has shown a significant reduction of cardiovascular events compared to antiplatelet therapy in patients with and comparator trials with the GG genotype. Patients on Apixaban would offer a safe and effective therapy.

US approval for Ximelagatran, a thrombin inhibitor, was denied due to concerns of liver toxicity, despite it proving effective in preventing cardiovascular events in patients with recent myocardial infarction or stroke. This drug can potentially be used in PAD patients with non-obstructive disease, as they often have comorbidities that prevent the use of antiplatelet and anticlotting therapy.

This type of genetic information can also be used to reposition drugs that have failed in clinical trials. Information on the genetic pathway that the drug targets and the patients whom it is effective can enable the identification of a subset of patients who may benefit from the drug.

PAD is a complex disease, and drugs that have proven effective in other cardiovascular diseases may not work in PAD patients. It is becoming clear that statins are not effective in preventing cardiovascular events in PAD patients, and recent work has shown that patients with the GG genotype of the 9p21 locus have an increased risk of myocardial infarction and require more aggressive antiplatelet therapy. These patients, along with their response to different drugs, can be identified using genetic information.

Genetic research and discoveries in the future will enable the identification of high-risk patients well before the onset of disease. Personalized medicine, using genetic information, will enable the development of new drugs targeted to high-risk patients, potentially increasing the effectiveness of the drug for those who need it.

Targeted Therapies Based on Genetic Profiles

A potential alternative encompassed under the umbrella of targeted molecular therapies involves the use of specific biomarkers or genetic tests to identify patients with PAD who are most likely to benefit from therapies targeting a specific pathway. As more is learned about the genetic underpinnings of atherosclerosis and PAD, it is likely that specific genetic polymorphisms and/or transcriptional signatures will be identified which can predict with reasonable accuracy the likelihood of disease progression or the response to a specific therapy. If such genetic profiles can be identified, it may be feasible to conduct clinical trials of drugs aimed at specific pathways in patients who have been stratified by genetic profile and only include those who are most likely to benefit. An example of the type of study design in which this might work is a trial of cilostazol in patients with intermittent claudication. Though cilostazol has shown benefit compared to placebo in this population, it is likely that not all patients with claudication have the same underlying pathophysiology and some may benefit more than others. If a genetic profile could be identified which marks those more likely to benefit from PDE3 inhibition, it could be tested whether those patients do in fact respond better to cilostazol compared to placebo. This type of individualized approach to therapy has potential to maximize the benefit of a given treatment while minimizing risk and cost by avoiding treatment of patients unlikely to benefit. An even simpler scenario that could evolve in the near future is the use of a specific genetic test or panel of tests to identify the cause of a patient’s PAD such as a specific lipid abnormality or prothrombotic state. This could aid in selection of the most appropriate available therapy for that specific cause of PAD or in some instances provide an opportunity for prevention of severe PAD in patients identified as high risk before the onset of symptomatic disease.

New insights into genetic contributions to PAD pathogenesis suggest that myriad new therapeutic approaches may evolve over the coming years. Some of the most exciting possibilities involve targeted molecular therapies directed at gene products shown to have a role in the pathogenesis of atherosclerosis and PAD. However, translation of this knowledge from association studies and animal models into human studies and therapeutic recommendations will be a long and arduous process that may take decades in some instances. Early efforts at gene therapy for PAD have been quite modest and often focused on overexpression of single genes such as VEGF. Though modest success has been demonstrated in small clinical studies, these approaches are not yet ready for prime time and it may be some time before true gene-based therapies become routine for patients with PAD.

Lifestyle Modifications and Risk Reduction

A supervised exercise program is also the most effective non-invasive way to control high blood pressure. Weight loss and regular exercise can also be effective in treating dyslipidemia. If diet and lifestyle changes are insufficient, there are several medications available to treat high cholesterol. Patients with PAD who smoke cigarettes should enroll in a smoking cessation program. This is the single most effective and cost-effective intervention to improve health in persons with PAD. Aspirin and clopidogrel have both been shown to be more effective than placebo in preventing cardiovascular events in patients with symptomatic PAD.

Lifestyle modification is an important part of the treatment plan for PAD. Your healthcare provider may prescribe a supervised exercise training program to increase walking distance, improve symptoms, and functional status. This will usually be done at a supervised cardiac rehabilitation center, but could also be done at home. Several types of medications are available to treat underlying risk factors that may worsen the symptoms of PAD or modify symptoms. High blood pressure can be a significant factor in the progression of PAD. If you have high blood pressure, it is important that it be treated and controlled. The most effective means of doing this is often weight loss and limiting salt in the diet.

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