An Objective Blood Test from Stimulated Gene Expression for Classification and Outcome Assessment in Clinical Trials of Gulf War Illness
- UT Southwestern Ambulatory Services
Robert Haley, M.D.
For aim 1, we will travel the approximately 150 GW-era veterans from the developmental and replication samples who participated in our prior studies to Dallas to obtain fresh whole blood. We will extract the peripheral blood monocytic cells (PBMCs) with Ficoll-Hypaque centrifugation, save an aliquot of PBMCs, and then use flow-cytometry to separate out the component cell types from the remaining PBMCs; divide each of these cell suspensions into aliquots; stimulate the aliquots of each cell type with saline, LPS alone, aCh alone, aCh followed by LPS (and an unstimulated aliquot); incubate the aliquots for 18 hours; extract the Rna from the cells of each aliquot perform next Generation Rna sequencing and quantification (Rna-seq) to measure the level of gene expression of the messenger Rnas and micro-Rnas by the pure cell types. in aim 2, we will perform bioinformatics statistical analyses to identify patterns of gene expression that accurately distinguish the 4 clinical groups. We will use standard bioinformatics software (e.g., Genespring) as well as a new multivariate classification method that maximizes statistical power to detect group differences in gene expression, developed by a member of our research team and used successfully in our initial pilot study.
We have previously collected peripheral blood samples from the same case and control subjects from the two study samples of Gulf War-era veterans that we have studied previously, who participated in our neuroimaging and biomarker studies,31,32 and who gave blood for our Pilot study gene expression study (funded by CDMRP). All of them have given written permission as part of the IRB-approved protocol to be contacted in the future for additional GWI studies.
The subjects include up to 51 Gulf War-era veterans from our long-term nested case-control study of the 24th Reserve Naval Construction Battalion known as the Developmental Sample, and up to 95 Gulf War-era veterans selected as a nested case-control study from the U.S. Military Health Survey (USMHS), a random sample survey (N=8,020) in a representative sample of the Gulf War-era military population,42 known as the Replication Sample. In the analysis of the gene expression data, we use the Developmental Sample as a
* sample, and the Replication Sample as a
* or validation sample to avoid type I errors due to multiple comparisons.
We expect approximately 140 subjects to agree to participate. In addition, we will draw blood from approximately 10 volunteers to test and validate the methods before proceeding with blood draw from the 140 study subjects.