This study involved unrelated HCC and LC patients of Korean ethnicity treated at the Asan Medical Center, Seoul, Korea. Disease diagnosis was confirmed by histopathology. Previous clinical history, enzyme-linked immunosorbent assay (ELISA)-based serum test results for hepatitis B virus (HBV) and hepatitis C virus (HCV), and clinical laboratory data were collected for these individuals; 89% of the HCC cases and 76% of the LC cases were chronically infected with either HBV or HCV. Two sources of controls were used. The first set of controls for our study was unrelated individuals from
the Asan Medical MK-8669 Center. The viral infection status of controls was not ascertained. A second set of controls was HBV+ individuals of Chinese origin (described previously).6 The local ethics committees and all subjects gave informed consent before inclusion in the study. A total of 386 Korean HCC cases, 86 Korean LC cases, 587 Korean controls (Supporting Table S1), and 100 Chinese controls passed the quality control evaluations (DNA integrity measurement, STRP genotyping for assessment of identity, and high SNP call rate from the Affymetrix 6.0 platform) described below. We confirmed through molecular assays that there is no population stratification among the Korean samples (see Supporting Methods). Individuals from the Korean population set were assigned to the discovery
(Stage 1) or validation Selleck XL765 (Stage 2) group based on their order of enrollment in
the study. Stage 1 included 271 Sitaxentan controls, 180 HCC cases, and 66 LC cases; Stage 2 had 316 controls, 206 HCC patients, and 20 individuals with LC. Key findings from the two-stage analysis were further validated using the Chinese control samples. Peripheral blood DNA was extracted using the Blood DNA Kit (Qiagen, Valencia, CA). DNA integrity and quantity were assessed using the Quantifiler Human DNA Quantification kit (Applied Biosystems, Foster City, CA). Polymerase chain reaction (PCR) products were analyzed with an ABI 3130XL Automated DNA Sequencer and the GeneMapper ID v3.2 software (Applied Biosystems, Foster City, CA). The Affymetrix SNP6.0 assay was performed according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA). Assay runs were performed in 96-well plates containing equal numbers of case and control samples, two Asian HapMap samples (chosen from NA18954, NA18971, NA18603, and NA18995) for external genotype validation, the Affymetrix Affy103 control DNA, and a water blank. Cases were randomly selected for each plate one-by-one using a random-number generator. Each case in the discovery phase was paired with its best match in sex and age among the control samples. Processing each Stage 1 case along with a matched control was aimed at minimizing technical variation in experimental results.