SNP Selection, Genotyping and Quality Control (QC) Filters

Several procedures were utilized to select SNPs. First, SNPs and genotypes of the TPCN2 gene were investigated in HapMap-HCB. Second, we calculated the linkage disequilibrium(LD) between SNPs in HapMap-HCB using the Genome Variation Server 138 (http://gvs.gs.washington.edu/GVS138/) and filtered all the monomorphic sites. The allele frequency cutoff was set to 20% and the r² threshold was set to 0.80. Finally, based on previous genetic studies [7,8] as well as public databases NCBI ClinVar database on the TPCN2 gene cluster, 6 candidate tag SNPs (rs35264875, rs267603153, rs267603154, rs3829241, rs1551305, and rs3750965) were selected.

Then, QC filters were applied to SNPs and samples before analysis to ensure robust association tests. We applied the same QC parameters to scans; the following SNPs were excluded: those a missing call rate of ≥2%, more than one discordance, significant deviations from HWE (P<1 × 10⁻⁴) or a minor allele frequency (MAF) of less than 1%. Three SNPs (rs3750965, rs3829241, and rs1551305) passed the QC criteria and were included ing the analysis.

Extracted DNA of the whole blood genome was analyzed by 1% agarose gel electrophoresis, and the DNA concentration and the degree of DNA degradation were subsequently estimated. Genotypes were determined using a Sequenom MassARRAY SNP genotyping system, which is based on detection through MALDI-TOF MS (Sequenom Inc., San Diego, CA, USA).