Methods

Study subjects
Between August 2001 and November 2010, blood samples were collected from 720 subjects, including 360 lung cancer patients and 360 normal controls without cancer. Lung cancer patients were recruited from the patient pool at the Genomic Research Center for Lung and Breast/Ovarian Cancer, and control subjects were randomly selected from a pool of healthy volunteers who had visited the Cardiovascular Genome Center and Genomic Research Center for Allergy and Respiratory Diseases. Detailed information on diet, smoking status, drinking status, lifestyle, and medical history were collected by trained interviewers using a structured questionnaire. Out of 360 cases, information was available for 352 on smoking status, 317 on stage, and 343 on cell type, while information was available for 272 on smoking status of 360 controls. All study subjects provided written consent and were ethnic Koreans, and all participating Institutional Review Boards approved the study protocol.

Preparation of genomic DNA and direct sequencing
Genomic DNA was prepared from peripheral blood samples using a Puregene blood DNA kit (Gentra, Minneapolis, MN, USA), following the manufacturer's protocol.
To identify polymorphisms in the promoter of the AKT2 and AKT3 genes, human genomic DNA was isolated from the whole blood of 24 lung cancer patients for direct sequencing, and the promoter region of the AKT2 and AKT3 genes was amplified. Polymerase chain reaction (PCR) amplifications were performed on a PTC-225 Peltier Thermal cycler (MJ Research Inc., Waltham, MA, USA) using Ampli-TagGold (Roche, Branchburg, NJ, USA). All amplifications were performed using 35 cycles of 30 s at 95℃, 1 min at 64℃, and 1 min at 72℃, followed by a single 10-min extension at 72℃. PCR products were purified using the Montage PCR96 Cleanup kit (Millipore, Bedford, MA, USA) and eluted in 20 µL of nuclease-free H2O. DNA cycle sequencing was carried out using the BigDye Terminator V 3.1 Cycle Sequencing kit (Perkin Elmer, Foster City, CA, USA). Multiscreen SEQ 384-well filter plates were used for dye removal, and sequences were then analyzed on an Applied Biosystems 3700 (ABI, Foster City, CA, USA). All polymorphisms and sequence alignments were analyzed using Polyphred.

Genotyping
After direct sequencing of the AKT2 and AKT3 genes, we performed genotyping for the 8 polymorphisms of the AKT2 and AKT3 genes. The genotypes of the AKT2 and AKT3 polymorphisms were screened by TaqMan fluorogenic 5' nuclease assay (ABI). The final volume of the PCR was 5 µL, containing 10 ng of genomic DNA, and 2.5 µL TaqMan Universal PCR Master Mix, with 0.13 µL of 40 × Assay Mix. Thermal cycle conditions were as follows: 50℃ for 2 min to activate uracil N-glycosylase and prevent carryover contamination, 95℃ for 10 min to activate the DNA polymerase, and 45 cycles of 95℃ for 15 s and 60℃ for 1 min. All PCRs were performed using 384-well plates on a Dual 384-Well GeneAmp PCR System 9700 (ABI), and the endpoint fluorescent readings were performed on an ABI PRISM 7900 HT Sequence Detection System (ABI). Duplicate samples and negative controls were included to ensure accuracy of genotyping.

Statistical analysis
Allele frequencies, genotype frequencies, and departures of genotype distributions from Hardy-Weinberg equilibrium (HWE) for each SNP were analyzed using chi-square test or Fisher's exact test. A p-value of <0.05 was considered statistically significant. Linkage disequilibrium (LD) was tested on pairwise combinations of polymorphisms using the absolute value of the standardized measure of LD, D', calculated by Haploview version 3.2. The haplotypes and their frequencies were estimated by Haploview version 3.2. Genotype-specific risks were estimated as odds ratios with associated 95% confidence intervals by unconditional logistic regression (SAS Institute Inc., Cary, NC, USA) and adjusted for age, gender, and smoking status.