After computing G+C3% and CAI values, we selected the 10% of the genes having the lowest values for these indices.
The genes having the highest 50 scores (in module) of the discriminant components were extracted to give a biological meaning of the observed patient/control discrimination.
The initial complete list was first subdivided into tissue-specific gene sets that where then regrouped into sets of genes having the same expression phase within each tissue.
PCA defines single genes in the space of component scores, allowing for a biological association of components to groups of genes having the highest absolute scores and thus permitting a biological interpretation of the obtained discrimination.
Only 13 genes were significantly regulated by both groups, with 5 genes being concordant with respect to regulation pattern and the remaining 8 genes having the opposite pattern of regulation.
This was done by combining most informative canonical pathways identified using IPA, genes having the strongest association with the disease phenotype based on ANOVA analysis, and similarities to molecular patterns altered in other systemic inflammatory processes associated with endothelial dysfunction.
Biological responses are consequence of the concerted action of gene regulatory network, thus, limiting our attention to genes having the most significant variations is insufficient for a thorough understanding of emergent whole genome response.
The four genes having the same consensus promoter cannot, according to the annotation, be put into one functional class, but it is reasonable to believe that the consensus region might be important for the regulatory mechanism.
Next, using the DANASIS 2.0 system or sequencing of the promoter region, we screened for genes having the CCAAT sequence in the promoter region, because NF-YB is known to bind with high specificity to the CCAAT motif in the promoter region of a variety of genes (Table 1, gene names shown in red).
We identified that the two genes having the strongest association with the disease phenotype variable, encode two major immunomodulatory factors, the microsomal prostaglandin E synthase (PTGES/Agilent clone number A-24-P478940) and the complement regulatory protein CRIg/VSIG4, considered potent negative regulators of T and NK cell responses [43] [45].
The 60 genes having the largest counts constituted an ultimate expression profile of a dataset.
