Role of epigenetic m6 A RNA methylation in vascular development: mettl3 regulates vascular development through PHLPP2/mTOR-AKT signaling.

N6 -methyladenosine (m6A) methylation is the most prevalent RNA modification, and it emerges as an important regulatory mechanism of gene expression involved in many cellular and biological processes. However, the role of m6 A methylation in vascular development is not clear. The m6 A RNA methylation is regulated by dynamic interplay among methyltransferases, binding proteins, and demethylases. Mettl3 is a member of the mettl3-mettl14 methyltransferase complex, referred to as writers that catalyze m6A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl3+/- KO embryos show defective vascular development, which is directly visible in fli-EGFP and flk-EGFP zebrafish. Alkaline phosphatase staining and whole mount in situ hybridization with cdh5, and flk markers demonstrated defective development of intersegmental vessels (ISVs), subintestinal vessels (SIVs), interconnecting vessels (ICVs) and dorsal longitudinal anastomotic vessels (DLAV) in both heterozygous mettl3+/- and homozygous mettl3-/- KO zebrafish embryos. Similar phenotypes were observed in zebrafish embryos with morpholino knockdown (KD) of mettl3; however, the vascular defects were rescued fully by overexpression of constitutively active AKT1. KD of METTL3 in human endothelial cells inhibited cell proliferation, migration, and capillary tube formation. Mechanistically, mettl3 KO and KD significantly reduced the levels of m6 A RNA methylation, and AKT phosphorylation (S473) by an increase in the expression of phosphatase enzyme PHLPP2 and reduction in the phosphorylation of mTOR (S2481), a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. These data suggest that m6 A RNA methylation regulates vascular development via PHLPP2/mTOR-AKT signaling.

Analysis of causal effect of APOA5 variants on premature coronary artery disease.

Apolipoprotein A5 (APOA5) regulates the metabolisms of triglyceride and HDL. APOA5 variants have been linked to coronary artery disease (CAD), but their causal roles are not well studied yet. This study aims to identify the causal effects of APOA5 variants on premature CAD. Sequencing analysis of APOA5 in 128 premature, familiar CAD patients from GeneQuest identified 11 genomic variants, including p.S19W (rs3135506). SKAT analysis showed that all sequenced variants, in aggregate, significantly increased the risk of premature CAD (P-skat = 0.037). Individually, the p.S19W variant was significantly associated with risk of premature CAD (OR = 2.30, P = 0.008) in an independent set of 342 premature CAD patients and 537 controls after adjusting for covariates of sex, age, hypertension, body mass index, triglycerides (TGs), and total, LDL-, and HDL-cholesterol levels. Meanwhile, p.S19W significantly correlated with HDL-C levels (P = 0.048) and TG levels (P = 0.025). Mediation analysis yielded a mediation effect of p.S19W on risk of premature CAD through HDL-C (OR = 0.98, P = 0.040) and TG (OR = 0.98, P = 0.042), suggesting a causal relationship between p.S19W and premature CAD partially through its effects on HDL-C and TG levels. These results suggest that APOA5 variation regulates TG and HDL levels, thus displaying a causal role in the development of CAD.

Genome-Wide Linkage Analysis of Large Multiple Multigenerational Families Identifies Novel Genetic Loci for Coronary Artery Disease.

Coronary artery disease (CAD) is the leading cause of death, and genetic factors contribute significantly to risk of CAD. This study aims to identify new CAD genetic loci through a large-scale linkage analysis of 24 large and multigenerational families with 433 family members (GeneQuest II). All family members were genotyped with markers spaced by every 10 cM and a model-free nonparametric linkage (NPL-all) analysis was carried out. Two highly significant CAD loci were identified on chromosome 17q21.2 (NPL score of 6.20) and 7p22.2 (NPL score of 5.19). We also identified four loci with significant NPL scores between 4.09 and 4.99 on 2q33.3, 3q29, 5q13.2 and 9q22.33. Similar analyses in individual families confirmed the six significant CAD loci and identified seven new highly significant linkages on 9p24.2, 9q34.2, 12q13.13, 15q26.1, 17q22, 20p12.3, and 22q12.1, and two significant loci on 2q11.2 and 11q14.1. Two loci on 3q29 and 9q22.33 were also successfully replicated in our previous linkage analysis of 428 nuclear families. Moreover, two published risk variants, SNP rs46522 in UBE2Z and SNP rs6725887 in WDR12 by GWAS, were found within the 17q21.2 and 2q33.3 loci. These studies lay a foundation for future identification of causative variants and genes for CAD.