SABRe CVD

Title/nature of data: Multiplexed Immunoassay Panel 11 (SABRe Project) – Lab Assay

Cohort: Offspring, Generation 3

Time Interval: Offspring Cohort Exam 7, Generation 3 Cohort Exam 1

Dataset name: l_mpimn11_2005_m_0855s

Study description

Cardiovascular Disease (CVD) has remained the leading cause of death in the United States for nearly all of the past century (Rosamond et al., 2007). Approximately one in three Americans live with a CVD at an estimated cost of $431 billion, nearly 3% of the total U.S. Gross Domestic Product (America Heart Association, 2007). If all major forms of CVD were eliminated, the average life expectancy would rise by almost seven years, more than twice the increase expected from curing cancer (Anderson RN, 1999). However, the future looks increasingly hopeful. Age-adjusted mortality from heart disease declined 16% between 2000 and 2004, continuing a long-term downward trend of over 60% since 1950 (Miniño et al., 2006).

This improvement is due in part to an extensive health survey of Framingham, Massachusetts. The Framingham Heart Study (FHS), including the original cohort of 5,209 individuals and eventually the next two generations, was the first epidemiological study to focus on a non-infectious disease and the first to try and connect lifestyle with disease (National Heart, 1998). The FHS was the first to identify cholesterol (Dawber et al., 1957), smoking (Doyle et al., 1962), obesity (Kannel et al., 1967), and diet (Gordon et al., 1981) as risk factors for CVD. In fact, the FHS was the first to introduce “risk factor” as a clinical term (Kannel et al., 1961). In the nearly 60 years since its inception, the FHS has led to over 1,300 peer-reviewed publications including an increasing number of genetic and biochemical linkage studies (Lindpaintner et al., 1996).

Despite work identifying the physiological and genetic signatures of CVD, the need remains for a comprehensive, proteomic approach to heart disease. Protein biomarkers may provide an up-to-the-minute view of a patient’s systemic biochemistry, especially when considered in a multiplex fashion (Rifai & Ridker, 2003). Unfortunately, protein biomarker assays with mainstream clinical acceptance, such as those for the cardiac troponins (McDonough & Van Eyk, 2004) or C-reactive protein (Ridker et al., 2001), have generally been considered in isolation. In addition, the FDA approval rate for new clinical biomarkers has decreased considerably since 1993 (Anderson & Anderson, 2002), reflecting the limitations of these single-analyte tests. Given the depth and complexity of the human plasma proteome, it is clear that any successful molecular assay for CVD must consider multiple biomarkers simultaneously.

This proposal is submitted in response to a call by the National Heart, Lung, and Blood Institute in November 2006. The following SABRE CVD Project-2 would utilize plasma samples from the FHS, along with their associated phenotypic data, to accomplish two aims:

1. Develop a workflow for high-throughput protein biomarker validation using immunoassays and statistical bioinformatics.
2. Identify a protein biomarker panel of minimal size with high diagnostic sensitivity and specificity for both atherosclerosis and metabolic syndrome components

The biomarker nominating committee was formed to handle the task and defining the process of identifying proteins of interest to the aims of Project 2, creating a transparent review process and then executing these tasks. The committee is comprised of the six full-time members. Since its inception, the committee has met bi-weekly and made excellent progress towards its goals.

Defining the biomarker nomination and review process

A formal biomarker nomination process was defined, reviewed, and put to use. A template was created with the required elements needed for a thorough review of each nominated protein target, including the following critical fields:

1. Source of existing data on proposed target
   1. Animal studies
   2. Gene expression data
   3. Proteomics data
   4. Genome-wide association studies (GWAS)
2. The diseases associated with the marker?
   1. Is this a priority condition based on the Project 2 aims?
3. Is the marker pathway dependent?
   1. Define the pathway and its importance
4. Is the marker pleiotropic?
   1. What other traits are associated
5. Have other large scale studies been conducted on this target?
   1. QC data available
   2. Assay type
   3. Results
6. Relevant literature linked to marker

Laboratory methods/technology—SABRE CVD Project 2

The technology being implemented to measure the 170 plasma biomarkers is the Luminex™ xMAP assay. This technique is an extension of the enzyme-linked immunosorbent assay (ELISA) performed with multiple analyte-specific capture antibodies bound to a set of fluorescent beads. An xMAP assay can simultaneously quantify up to 100 analytes at abundances as low as pg/ml in multiple samples (Johannisson, 2006). This multiplexed format allows higher throughput and reduced sample use as compared to other ELISA formats

Once a target has been identified, the assay workflow includes an initial phase, where the laboratory team will develop antibody pairs corresponding to distinct epitopes of each biomarker. Each pair receives extensive material validation to ensure compatibility with our immunoassay format.

Inclusion/Exclusion Criteria:

The Framingham Heart Study Offspring Exam 7 and Third Generation Exam 1 plasma samples are the samples used in this project.

The entire cohorts are being measured—the only exclusion being applied is based on available physical plasma samples in the Framingham archives.