Type 2 diabetes mellitus T2D T2DM Single nucleotide polymorphism SNP Genetic risk score Pathogenesis CommentaryCoronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide [1]. A major risk factor for CAD that has long been recognised is type 2 diabetes mellitus (T2DM). In addition, obesity, sedentary lifestyle and stress are common shared risk factors that contribute to the close association between T2DM and CAD. It is not yet obvious whether and to what degree this close association between the two conditions can be attributed to shared genetic risk factors. In contrast to other risk factors shared between T2DM and CAD, genetic risk factors offer the advantage of being unaffected by the various confounders affecting the association between these two conditions. T2DM and CAD both have polygenic inheritance and genetic studies have identified numerous distinct loci for both diseases. For CAD, over 50 single nucleotide polymorphisms (SNPs) have been identified in genome-wide association studies (GWAS), but only a few of these also associated with traditional CAD risk factors (lipid and blood pressure traits) and none of these were associated with glyco-metabolic traits at a genome-wide significant level (p < 5 Â 10 À8 ) [2e4]. In the setting of T2DM, some components of the genetic predisposition to CAD appear to differ from those found in the general population. A variant related to the g-glutamyl cycle of amino acid transport that had not been previously identified in the general population was found to be associated with CAD at a genome wide-level of significance in T2DM, representing a novel genetic risk factor for CAD in T2DM [5].In this issue of Atherosclerosis, Jansen and colleagues used genetic markers as proxies for diabetes status to investigate whether T2DM e associated SNPs are also associated with increased risk of CAD in Caucasians, using the CARDIoGRAM Consortium dataset comprising~22,000 CAD cases and~65,000 controls [2,6,7]. They argued that if T2DM is a causal risk factor for CAD, then genetic variants associated with T2DM would consequently be expected to be associated with CAD. They investigated whether the risk alleles of T2DM -associated SNPs previously identified in GWAS also increase the risk of CAD. A systematic literature search identified 48 variants currently known to be robustly associated with T2DM, of which 4 were excluded (on technical grounds or because of statistically e defined pleiotropy) and the remaining 44 were included in the study. For 29 of these variants, the T2DM risk allele gave an effect in the expected direction, (i.e. increased risk of CAD) and for ten SNPs this was statistically significant (p < 0.05). Of these, only the T2DM SNP at the chromosome 2q36.3 locus, which contains the insulin receptor substrate 1 (IRS1) and LOC64673 genes, remained significantly associated with CAD after correcting for multiple testing (p ¼ 3.4 Â 10 À5 , Bonferroni corrected threshold p value 0.0011). Interestingly, for two of the remaining 15 variants (CILP2 and ...

MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells.

Aim: MicroRNAs (miRs) are involved in regulation of gene expression and may play a role in development of hypertension.Methods: Three months old C57BL/6J mice were treated angiotensin II (AngII, 490ng/kg/min) or control buffer using miniosmotic pumps for 2 weeks. Isolated RNA from aorta and perivascular adipose tissue (PVAT) was reverse transcribed using Megaplex primer pools and profiled with TaqMAn Rodent microRNA Array. Statistical tests were performed on normalized CT values in R (HTqPCR package) or DataAssist. Results: From 381 studied miRs 103 was differentially expressed in aortas and 134 in PVAT (Ct<32). Both principal components and hierarchical clustering analyses using all expressed miRs in aorta and PVAT separated hypertensive from control animals. In aortas, there were 29 and 24 differentially expressed miRs, at FDR adjusted p<0.05, between treatment groups, while using global mean or quantile normalization method respectively with 19 microRNAs overlapping. Out of these 7 miRs were overexpressed in aortas of hypertensive mice as compared to controls. Only mir-21 and mir-142-3p were significantly expressed after Bonferroni correction (3.5-fold induction) in hypertensive animals. Similar analysis identified 16 differently expressed miRs in PVAT between groups. Mir-214 was the only miR significantly overexpressed (8-fold induction) in hypertensive animals after Bonferroni correction. Effects of mir-21 and mir-214 on vascular function will be addressed in respective knockout models. Conclusions: AngII infusion changes miR profiles in murine aortas and PVAT. Mir-21 is the most upregulated in aortas whereas mir-214 is the most upregulated miR in PVAT, which can affect development of vascular dysfunction.