Genotyping

Blood samples of the study population were collected by EDTA containing tubes with blinded unique identification number. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood draw. Genomic DNA was extracted from PBMCs or whole blood using a QIAcube (QIAGEN, Hilden, Germany). Then, most SNP genotyping was carried out using TaqMan SNP genotyping assays (Thermo Fisher Scientific, MA) according to the manufacturer’s protocol. In brief, two allele-specific TaqMan probes containing distinct fluorescent dyes and a PCR primer pair were used to detect specific SNP targets. Quantitative PCR, as a readout, was performed with Roter-GeneQ (QIAGEN). We describe the zygosity of A>G SNP as AA or GG, and AG in the case of homozygosity and heterozygosity, respectively.

Zygosity for the deletion (D)–insertion (I) polymorphism of the ACE gene was determined using an assay to distinguish D and I alleles based on PCR and visualization by electrophoresis (190bp and 490bp amplicon, respectively) (40). Samples classified as the deletion-homozygotic (DD) genotype were subjected to PCR again to eliminate DD mistyping by detecting a 319bp amplicon implied I allele (13).