Genetic Analysis of LPA Gene

Genetic analyses were performed at the Institute for Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana as previously described [20 (link),22 (link)]. Briefly, genomic DNA was extracted from venous blood samples by using FlexiGene DNA kit 250 (Qiagen, Hilden, Germany). The LPA genotyping was performed by using Predesigned TaqMan SNP genotyping assays (C_30016089_10, C_25930271_10; Applied Biosystems, Waltham, MA, USA) and TaqMan™ Genotyping Master Mix (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Analysis of LPA kringle repeats was performed by using custom TaqMan expression assay for exon 5 of the LPA gene. In brief, genomic DNA was diluted to 10 ng/µL with nuclease-free water. The PCR reaction mix contained 5 µL TaqMan Universal PCR Master Mix, 0.25 µL TaqMan Genotyping Assay Mix (40×), and 2.75 µL DNase-free water, to which we added 2 µL of the pre-diluted DNA. Genotyping PCR was performed with a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA).