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Despite significant advances in the diagnosis and treatment of cardiovascular diseases, recent calls have emphasized the unmet need to improve precision-based approaches in cardiovascular disease. Although some studies provide preliminary evidence of the diagnostic and prognostic potential of circulating coding and non-coding RNAs, the complex RNA biology and lack of standardization have hampered the translation of these markers into clinical practice. In this position paper of the CardioRNA COST action CA17129, we provide recommendations to standardize the RNA development process in order to catalyse efforts to investigate novel RNAs for clinical use. We list the unmet clinical needs in cardiovascular disease, such as the identification of high-risk patients with ischaemic heart disease or heart failure who require more intensive therapies. The advantages and pitfalls of the different sample types, including RNAs from plasma, extracellular vesicles, and whole blood, are discussed in the sample matrix, together with their respective analytical methods. The effect of patient demographics and highly prevalent comorbidities, such as metabolic disorders, on the expression of the candidate RNA is presented and should be reported in biomarker studies. We discuss the statistical and regulatory aspects to translate a candidate RNA from a research use only assay to an in-vitro diagnostic test for clinical use. Optimal planning of this development track is required, with input from the researcher, statistician, industry, and regulatory partners., peer-reviewed

In this study, DNA was extracted from whole blood which had been collected and stored at -20°C for 5–18 years, with the aim of determining the most suitable commercial DNA extraction kit for this purpose. DNA from nine cord blood samples collected in 1999, 2001 and 2012, with low blood volumes (

Objective To investigate the effect of passive smoking, active smoking and smoking cessation on inflammation, lipid profile and the risk of myocardial infarction (MI), Methods A total of 423 cases with a first MI and 465 population controls from the Maltese Acute Myocardial Infarction (MAMI) Study were analysed. Data were collected through an interviewer-led questionnaire, and morning fasting blood samples were obtained. ORs adjusted for the conventional risk factors of MI (aORs) were calculated as an estimate of the relative risk of MI. The influence of smoking on biochemical parameters was determined among controls., Results Current smokers had a 2.7-fold (95%CI 1.7 to 4.2) and ex-smokers a 1.6-fold (95%CI 1.0 to 2.4) increased risk of MI. Risk increased with increasing pack-years and was accompanied by an increase in highsensitivity C reactive protein levels and an abnormal lipid profile. Smoking cessation was associated with lower triglyceride levels. Exposure to passive smoking increased the risk of MI (aOR 3.2 (95% CI 1.7 to 6.3)), with the OR being higher for individuals exposed to passive smoking in a home rather than in a public setting (aOR 2.0 (95% CI 0.7 to 5.6) vs aOR 1.2 (95% CI 0.7 to 2.0)). Passive smoke exposure was associated with higher levels of total cholesterol, triglycerides and total cholesterol:high-density lipoprotein cholesterol ratio compared with individuals not exposed to passive smoking., Conclusions Both active and passive smoking are strong risk factors for MI. This risk increased with increasing pack-years and decreased with smoking cessation. Such effects may be partly mediated through the influence of smoking on inflammation and lipid metabolism., peer-reviewed

The biosynthesis of Hb F in place of the deficient Hb A could be a suitable treatment for beta hemoglobinopathies. Among newborn Hb F-Malta-I heterozygotes, it could be shown that the XmnI sequence alone had little, if any effect on gamma-globin gene expression, but interplay with the (AT)(X)T(Y) sites in cis and in trans may occur. In contrast, while the XmnI sequence is clearly correlated with gamma-globin levels in anemic adult beta-thalassemia (thal) homozygotes, the effect on F-erythrocyte numbers and Hb F/F-erythrocyte appears independent of the (AT)(X)T(Y) sites. Even at levels of hydroxyurea (HU) as low as 1.65 mg/kg/day (vs. 10 mg/kg/day on the high dose regime) it can be shown that although even a small increase of Hb F could be obtained, the effect was rarely translated into an increase in circulating hemoglobin (Hb). In most cases, the elevated Hb F level was dependent on the XmnI sequence and was due to increased numbers of F-erythrocytes or Hb F/F-erythrocyte or both. It seems that the bone marrow of thalassemia homozygotes may be more sensitive to myelosuppression by HU possibly due to medullary inflammation. While the data are consistent with loop models of globin switching mechanisms, there is urgent need for large, hypothesis driven, multicenter trials of molecules that could maintain or re-induce high Hb F levels in beta-thal and subject to genetic and epigenetic constraints including inflammation.

Hb F-Malta-I [Ggamma117(19)His--Arg, CAT--CGT] is a stable and benign variant of Hb F found in 1.8% of Maltese newborn. We studied 120 Hb F-Malta-I heterozygotes and four Hb F-Malta-I homozygotes. The mean proportion of Ggamma-F-Malta-I in Hb F was 0.26 +/- 0.03 for the Hb F-Malta-I heterozygotes and 0.58 +/- 0.06 for the Hb F-Malta-I homozygotes. The Hb F-Malta-I allele was shown to occur on a background of the common Mediterranean haplotype Va [+ + - - - - - + + -]. Furthermore, the common Mediterranean haplotypes Va, IIIb [- + + + - + + + + -], I [+ + - - - - - + + +] and II [- + - + + - + + + +] accounted for most (66.2%) of the wild-type alleles among the tested Hb F-Malta-I heterozygotes. Different genotypes at the 5' epsilon HincII, Ggamma and Agamma HindIII, and 3'psibeta HincII sites (but not at the 5' Ggamma XmnI site) were found to be linked to significant variations in the proportion of Ggamma-F-Malta-I and Ggamma-globins in the Hb F of newborn Hb F-Malta-I heterozygotes. Moreover, the 5' Ggamma XmnI site was found to be associated with variations in Hb F and Ggamma-globin levels in a population of adult Maltese beta-thalassemia (thal) homozygotes. This implies that a determinant linked to the XmnI site which effects Ggamma-globin gene expression is active in anemic adults but not in normal infants.