Genomic dna kit

WLS tagging SNPs (tagSNPs) were selected from the 1000 Genomes Chinese Han Beijing population (CHB) using Haploview^{23 (link)}. SNPs with minor allele frequencies (MAF) ≥0.05 were selected based on pairwise tagging with an r² threshold of 0.5. A final set of 40 tag SNPs was selected within the WLS gene (Supplemental Table S1). Genomic DNA was extracted from peripheral blood leukocytes according to the manufacturer’s protocol (Genomic DNA kit, Axygen Scientific Inc., California, USA). Genotyping was performed for all SNPs using the Sequenom Mass ARRAY RS1000 system (Sequenom, San Diego, California, USA). The results were processed using Typer Analyzer software (Sequenom), and genotype data were generated from the samples^{24 (link)}.

We searched for all SNPs with minor allele frequencies (MAFs) ≥ 0.02 within the region of the MDM4 gene in the 1000 Genomes Project Chinese Han Beijing population database. MAF ≥ 0.02 and tagging r² ≥ 0.8 were used as a screening standard in the selection of tag SNP, which generated 24 tag SNPs for our study. As a result, these 24 tag SNPs (rs3014610, rs2169137, rs117139931, rs137991330, rs4252707, rs190876924, rs12024619, rs72644182, rs117137314, rs76605997, rs76432362, rs116854458, rs12138846, rs61421373, rs191840558, rs116907825, rs115517182, rs12567161, rs150337092, rs80242302, rs3789044, rs3789043, rs884108 and rs61817485) were included in further analyses. All our selected SNPs had P values greater than 0.05 by the HWE test. Commercial kits were used to extract genomic DNA from peripheral blood leukocytes (Genomic DNA kit, Axygen Scientific Inc., CA, USA). Genotyping was conducted for 24 selected SNPs by using the platform of Sequenom Mass ARRAY RS1000 system (Sequenom, San Diego, CA, USA). Typer Analyzer software (Sequenom, San Diego, California, USA) was used to process signal results to ultimately generate genotype data^{20 (link)}. Case and control statuses were blinded during all genotyping processes for quality control. Five percent of the random samples were repeated, and the results were 100% concordant.

For SNP selection, 1000 Genomes Chinese Han Beijing (CHB) data were used as reference data. We extracted all SNPs with MAFs greater than 0.03 within the LTBP3 gene region in 1000 Genome CHB data, resulting in a SNP set of 19. Then, tag SNPs (r²≥0.8) were identified within this SNP set based on the method proposed by Gabriel et al. [14 (link)]. Finally, a total of 9 tag SNPs located within the LTBP3 gene region were selected for genotyping (Supplementary Table S1). We extracted genomic DNA from peripheral blood leukocytes according to the manufacturer’s protocol (Genomic DNA Kit, Axygen Scientific, Inc., CA, U.S.A.). A high-throughput Sequenom MassARRAY platform (Sequenom, San Diego, CA, U.S.A.) was utilized for SNP genotyping. Briefly, the signals from the platform were automatically analyzed using Sequenom Typer 4.0 software, and genotype data were generated from the processed results [15 (link)]. To estimate the genotyping quality, 5% of random samples were repeated for genotyping. With a concordance rate of 100%, the quality of genotyping data was confirmed. The case/control status of the samples was blinded to the technicians during the genotyping process [16 (link)].