Blood and Buccal Swab Collection
The Genetic and Environmental Risk Factors for Hemorrhagic Stroke (GERFHS) study is a large case-control study of hemorrhagic stroke in the Cincinnati, Ohio region. This study protocol was approved by the Institutional Review Board of the University of Cincinnati, and all subjects provided written informed consent for genetic testing. To maximize participation and thereby maximize representativeness of the cohort, buccal cytobrush collection was performed on the majority of subjects. Buccal brushes for genetic analysis were collected beginning in 1997, and in mid-2000, the option of collection of blood samples was added to the protocol. Four individuals enrolled in 2000 or 2001 had both buccal brushes and blood samples collected, and were selected for this analysis. Four buccal brushes were collected on each participant (two right cheek, two left cheek) using CYTO-PAK Cytosoft Brushes (Medical Packaging Corp., Camarillo, CA), and two of the four collected buccal brushes were used for this analysis, except as noted below.
Study nurses were trained to collect buccal cytobrush samples by the following procedure: The subject would rinse their mouth with water gently prior to brush collection. Each brush was inserted into the subject's mouth and twirled firmly for 30 seconds against the subject's cheek in an up and down motion. The nurse returned the brush into the plastic tube and sealed the tube with the subject ID label. After all four brushes were collected, the sealed brushes were sealed again in an envelope labelled with the ID number and date and time of collection. These envelopes were stored in sealed freezer bags and stored at -80°C until they were transferred to the lab for DNA extraction. Blood samples were drawn by hospital nursing personnel using two 10 ml purple top tubes with EDTA solution from subjects during their hospital stay. The blood samples were spun down, DNA extracted and the DNA was stored at -80°C in the lab for future analysis.
DNA Extraction and Genotyping
For each buccal cytobrush, DNA was extracted using PureGene kits (Gentra Systems, Inc. Minneapolis, MN), resulting in eight separate buccal brush DNA samples for analysis (biological replicates). DNA was extracted from a single blood sample for each individual by Molecular Diagnostics Laboratories, Inc., and then separated into two aliquots for analysis (technical replicates). Throughout the paper, duplicate blood or buccal samples are designated either "replicate 1" or "replicate 2" to distinguish them; however, all samples for each individual were genotyped at the same time.
To extract blood DNA, a standard Proteinase K procedure was used. Briefly, this procedure included isolation and lysing white blood cells, treatment with Proteinase K (20 mg/μl), precipitation and washing DNA with isopropanol and resuspension of DNA in 500 μl of TE buffer. The Quant-it dsDNA HS assay kit and Qubit fluorometer (Invitrogen; Carlsbad, CA) were used for quantitating double-stranded DNA (dsDNA) in solution. This is a highly sensitive, dsDNA-specific assay employing a fluorescent nucleic acid stain.
Five buccal brush samples had initial concentrations of dsDNA <40 ng/μl (range: 3.4 – 39.8 ng/μl), and additional DNA was extracted from the tube that originally contained the buccal cytobrush. After this procedure, two buccal brushes (both from individual C) still had dsDNA concentrations <50 ng/μl (replicate 1: 40.4 ng/μl, replicate 2: 30.8 ng/μl). These samples were initially analyzed using 202 ng and 154 ng of dsDNA per GeneChip, respectively, but call rates were quite low (95% and 89%, respectively). The two remaining buccal brushes for individual C were then extracted and genotyped, and results from these latter two buccal brushes are reported.
Genotyping was performed on the Affymetrix GeneChip 3000 platform. We used the Nsp1 chip, which is an array of ~262 K SNPs. The recommended protocol as described in the Affymetrix manual was followed. All DNA samples were normalized to 50 ng/μl. Then, 5 μl (250 ng) of dsDNA was digested with NspI and ligated to adapters using T4 DNA ligase. Samples were then PCR amplified using TITANIUM Taq polymerase on an ABI9700 machine. PCR products were purified using the Clontech purification kit followed by fragmentation. Samples were then injected into cartridges, hybridized, washed and stained.
Mapping array images were obtained using the GeneChip Scanner 3000. Genotypes were called using the BRLMM software, analyzing all .cel files together. Any sample with a genotype call rate <95% was considered a QC failure, which would result in the sample being re-extracted and/or re-genotyped in a full-scale genetic study.
Statistical Methods
For each individual, two buccal samples (biological replicates) and two blood aliquots (technical replicates) were run, such that matched blood and buccal samples as well as matched blood samples from the same individual could be compared. Four possible comparisons of blood and buccal samples were analyzed: 1) blood replicate 1 with buccal replicate 1; 2) blood replicate 1 with buccal replicate 2; 3) blood replicate 2 with buccal replicate 1; and 4) blood replicate 2 with buccal replicate 2. Concordance of genotype calls between samples from the same individual was evaluated using % concordance and the Kappa statistic, which measures the agreement between methods exceeding that expected by chance. Percent concordance and Kappa statistics were calculated only among genotypes called in both samples being compared, excluding missing data. P ≤ 0.05 was considered significant.
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