Genotyping for Detection of Polymorphisms Relevant to Inflammation (PCS3)Genetic samples (DNA) were recovered by buccal brushings for polymorphisms in 8 genes that regulate cytokine production, target cell responsiveness to cortisol, or both.
We performed whole genome amplification and then used real-time polymerase chain reaction (rtPCR) to amplify the allele variants for 5 genes associated with cytokines that either upregulate (interleukin [IL]-6 and tumor necrosis factor [TNF]-α) or downregulate (tissue growth factor [TGF]-β1, IL-10, and interferon [IFN]-γ) the inflammatory response to a viral upper respiratory infection. We also assayed for expression of polymorphisms in 3 (in addition to the TNF-α polymorphism) genes that affect cortisol production or target cell sensitivity: GABRA6, the gene encoding the γ-aminobutyric acid (GABA)Aα6 receptor subunit; ADRA2A, the gene encoding the α-2A adrenergic receptor; and CRHR1, the gene encoding the corticotropin-releasing hormone receptor 1.
Buccal brushings for recovery of participant DNA were performed at the screening physical exam (Visit 2), approximately 2 months prior to viral challenge. Specimens were collected by swabbing the inner cheek with a sterile cytobrush (Histobrush, Hardwood Products Company, USA). Swabs were then placed in a cryovial containing 500 µl of 0.9% physiological saline (one side/vial) and agitated to release buccal cells. Swabs were then pressed against the tube wall to release liquid from the fibers, and removed from the tube. This procedure was repeated three times per cheek for each subject. Vials containing specimens were capped tightly and stored at -80°C until assay.
Genomic DNA was extracted with the QIAamp DNA Blood Mini Kit (QIAGEN, Inc., Valencia, CA, USA), amplified using the QIAGEN REPLI-g whole genome amplification Kit (QIAGEN), and quantified with a Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). All assay procedures were performed following the methods provided by the manufacturer.
The primer and probe sequences used for genotype assay of the selected cytokines were constructed based on previous publications: TNF-α (-308 G/A), TGF-β1 (C/T codon 10, C/G codon 25), IL-10 (-1082 G/A, -819 T/C, -592 A/C), IL-6 (-174 G/C) and IFN-γ (+874 A/T). For the polymorphisms in the aforementioned genes associated with cortisol production or target cell sensitivity, we focused on the following 3 SNPs: GABRA6, rs3219151 (1519 T > C, 3'-UTR); ADRA2A, rs1800544 (-1291 G > C, 5'-UTR); and CRHR1, rs110402 (intron1). Genotyping was performed using TaqMan® Genotyping Master Mix (Applied Biosystems Inc., Foster City, CA, USA) following a published protocol. The 7300 System SDS software (version 1.4; Applied Biosystems) was used for instrument control, automated data collection, and genotype assignment1,2.
1 Gentile, D. A., Doyle, W. J., Zeevi, A., Howe-Adams, J., Kapadia, S., Trecki, J., & Skoner, D. P. (2003). Cytokine gene polymorphisms moderate illness severity in infants with respiratory syncytial virus infection. Human immunology, 64(3), 338-344.
2 Doyle, W. J., Casselbrant, M. L., Li-Korotky, H., Cullen Doyle, A. P., Lo, C., & Cohen, S. (2010). The IL-6 (-174, C/C) genotype predicts greater rhinovirus illness. The Journal of Infectious Diseases, 201, 199-206.