Project Description

The Problem

Cardiovascular disease (CVD) is the leading cause of death globally, responsible for 17.9 million deaths each year¹. In the United States, CVD outcomes are affected by health inequality, as risk factors and mortality rates disproportionately affect minority communities². Venous thromboembolism, a common form of CVD, has incidence rates 30-60% higher in African Americans than persons of European descent^3,4. Recent studies have shown that the rate of American adults with unmet medical need has been on the rise since 1999, especially among racial and ethnic minorities⁵. Our team has identified CVD and medical inequity as serious and intersecting issues at both a global scale and in our local community⁶. With our project, we aim to use synthetic biology to advance accessible, personalized medicine towards addressing these challenges.

The rs773902 Single Nucleotide Polymorphism

While issues of inequality in the social determinants of health are significant contributors to CVD disparities in the US, CVD risk also has a well-established genetic component⁷. Recent research from Dr. Michael Holinstat's laboratory at the University of Michigan Medical School has suggested that genetic variant rs773902 in the F2RL3 gene has consequences for thromboembolic risk and treatment efficacy⁸. The rs773902 genetic variant is classified as a single nucleotide polymorphism (SNP), which is a genetic variation of a single nucleotide at a specific point within a gene. The rs773902 SNP changes the 120th residue in the PAR4 receptor of platelets from alanine to threonine. This threonine-containing variant of PAR4 may be associated with increased thrombin sensitivity and platelet aggregation, potentially contributing to increased risk of thrombosis⁸. Further, evidence suggests that this SNP may also be associated with resistance to standard-of-care antiplatelet medications aspirin and clopidogrel⁸. In contrast, alternative medications heparin and ticagrelor do not appear to be affected. While these alternate medications may not be the best choice for every patient, knowlege of the patient's genotype may help a provider administer the best possible medication for each unique person.

The rs773902 SNP is also hypothesized to have implications for racial CVD disparities. This genetic variant is more common in populations of African descent, with 63% of alleles in persons of African ancestry coding for PAR4-Thr120 as compared to 19% of alleles in persons of European ancestry⁹. Together, this suggests that rs773902 could be one of many contributing factors to racial disparities in CVD risk and treatment outcomes in our local community ⁶.

It is not standard practice for physicians to routinely test patients for SNPs such as rs773902. One reason for this is the complexity of current clinical SNP testing, which relies on methods that are not practical for point-of-care settings. Clinical SNP detection is currently done through next-generation sequencing methods ¹⁰, which can take 2-8 weeks ¹¹ and, if not covered by insurance, can cost the patient up to $2000 out of pocket ¹². There is currently no standard for clinical SNP detection, but a few commercial methods are feasibly adaptable. Taqman Analysis is the current standard of SNP detection in the research setting, and utilizes a vast SNP database with corresponding SNP-assays. These assays are run externally, and thus Taqman Analysis requires outsourcing ¹³. Another potential method for clinical SNP detection is Fragment Analysis, which can be run in house but requires a PCR clean-up kit, thermocycler, SNaP Shot SNP amplification kit, and capillary electrophoresis ¹⁴. These methods are suboptimal, as they require time-intensive outsourcing, aren’t economically feasible, or require expensive equipment. Ultimately, it is difficult for doctors to know which treatment plan would be most appropriate for a given patient without having resources for rapid, cost effective genetic variant testing. We aim to develop an alternative genetic testing platform to better inform antiplatelet pharmacotherapy in order to help reduce disparities in CVD.

Our Solution

In response, we are developing an inexpensive, generalizable SNP detection platform for use in point-of-care settings, with rs773902 as our initial target. Our system is based on recently published methodology, which combines Loop-Mediated Isothermal Amplification (LAMP) with machine learning-generated fluorescent probes¹⁵. We are working to adapt this technology to on-site, personalized, genomic SNP detection. In addition, we hope to support our test with a low-cost fluorescence reader and easy-to-use kit for ready implementation of the assay in clinic.

Development of our test is divided into three aims: DNA extraction, gene amplification, and differential fluorescent probe binding and detection. For genomic extraction, our project adapts a published protocol of chemical lysis and simple purification via cellulose dipstick that could be used to extract DNA out of a blood or saliva sample from a patient ¹⁶. We then designed and optimized a LAMP reaction for rapid, isothermal amplification of the SNP-containing segment of F2RL3. Finally, we adapt a published method to design a fluorescent probe and sink combination for SNP detection via differential fluorescence¹³. In a point-of-care setting, such as a doctor’s office, a fluorescent signal would indicate a patient's variant of rs773902.

Additionally, we recognized a need for cost-effective fluorescence detection machinery that could be used in a point-of-care setting, rather than a traditional qPCR machine or fluorometer. qPCR machines can cost between $15,000 and $90,000 ¹⁷, so we are attempting to make a basic fluorometer costing only $300. Therefore, we aimed to also develop our own lower-cost fluorometer for specific use in the context of our test. By making this fluorometer modular and open source, we envision it being slightly modified in the future to be sensitive to other fluorophores, for use to detect other SNPs and in other point-of-care fluorescence assays.

In contrast to cost-ineffective and time-consuming genetic testing, our SNP-LAMP-based platform will provide results in under 1.5 hours for less than $3 per patient sample, all while using simple, affordable equipment such as a heating block and basic fluorimeter. We anticipate that healthcare providers will be able to purchase our complete platform, including all reagents and labware, for less than $2,500. The speed and affordability of this system will ensure its accessibility to all patient populations and healthcare settings, especially those who are under-resourced or experience difficulty following up after an initial appointment.

A device able to perform quick and accurate detection of the rs773902 SNP will be a valuable technology for physicians to inform their care of individuals who are experiencing or at risk for cardiovascular disease. This quick, inexpensive test can assist physicians in determining prescriptions that meet the needs of individual patients in order to best improve their health and reduce adverse side effects, ultimately improving equity in patient treatment outcomes.

Generalizability

As advancements in personalized medicine bring more sophisticated scientific techniques to the clinic, it is important to consider equitable uses of these strategies and to leverage them to improve the health outcomes of historically underserved populations. Accessible detection of SNPs could help mitigate disparities in CVD and other common diseases and also contribute to better health outcomes for all. In the future, our system may be applied to other clinically relevant SNPs in order to empower physicians to acquire critical genetic information to inform patient care. The low-resource nature of our solution will ensure accessibility of patient-specific care. Ultimately, applying synthetic biology to enable accessible genetic testing could aid in reducing healthcare disparities and vastly improve medical decision-making and patient care.