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To investigate the causative genes of myocardial infarction (MI) in a pedigree with multiple members with premature myocardial infarction (PMI) by whole-exome sequencing and functional verification.
PMI was defined by MI diagnosed before the age of 50 for men and 60 for women. Peripheral blood of all the family members (n=17) was obtained under informed consent for DNA extraction. Whole-exome sequencing was performed on 8 individuals from a PMI pedigree with two siblings diagnosed with MI before the age of 45 as well as their uncle at the age of 53. Bioinformatics analysis served as a tool for causative gene screening. Another 100 PMI patients were also enrolled for population validation of the potential causative genes. Finally, the potential causative genes were introduced into corresponding model cell lines by CRISPR-Cas9 technology, after which the functional consequences of the mutations were evaluated by western-blot and enzyme-linked immune-sorbent assay (Elisa).
Upon exome-sequencing and subsequent Bioinformatics analysis, two variants in SCAP (c.3035C>T, p.Ala1012Val) and AGXT2 (c.1103C>T, p.Ala338Val) were identified as potential causative mutation for PMI. It was noteworthy that only patients that meet the diagnosis of PMI (n=2) harbored two variants all together, while other MI patients or members with no MI carried only one or no above variants.
We then screened the two genes in an independent population with PMI. Another variants was identified on SCAP (c.1403 T>C, p.Val468Ala), which was absent in 28, 000 east-Asian population. However, no potential causative mutation on AGXT2 was found in the present population.
Further, the two variants on SCAP and AGXT2 were introduced into H293T and EA. hy926 cell lines respectively utilizing CRISPR-Cas9 measure. Functional study revealed that the SCAP mutation impaired SCAP-SREBP feedback mechanism which may lead to a “consititutive activation” effect of cholesterol synthesis related genes, while the AGXT2 mutation caused a deficiency of its aminotransferase activity which would lead to a down-regulation of NO production by ADMA accumulation.
SCAP and AGXT2 are potential causative genes for MI. Digenic mutation carriers may manifest a more severe phenotype, namely premature MI. In addition, the present results also hints the role of digenic (or polygenic) mutations in the molecular genetic mechanism of PMI.