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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

AimTo determine the risk of myocardial infarction (MI) associated with pattern, frequency, and intensity of alcohol consumption, type of alcoholic beverage, and the combined effect of alcohol and smoking on risk of MI, inflammation, and lipid profile.MethodA total of 423 cases with a first MI and 465 controls from the Maltese Acute Myocardial Infarction (MAMI) Study were analysed. Data was collected through an extensive interviewer-led questionnaire, along with measurements of various blood parameters. Medians and the Mann–Whitney test were used to assess effect of different drinking patterns, frequency, intensity, and smoking and drinking combinations on hs-CRP and lipid profile. Odds ratios, adjusted for the conventional risk factors of MI (AdjORs), were calculated as an estimate of the relative risk of MI.ResultsRegular alcohol consumption protected against MI [AdjOR 0.6 (95% CI 0.4–0.9)] while daily binge drinking increased risk [AdjOR 5.0 (95% CI 1.6–15.0)] relative to regular drinkers who did not binge drink. Whereas moderate weekly consumption of wine protected against MI, high weekly consumption of beer conveyed a deleterious effect. Alcohol consumption decreased risk of MI independent of smoking status. Frequent alcohol consumption was associated with higher HDL-, non-HDL-, total cholesterol and triglycerides, and lower hs-CRP. Total and HDL-cholesterol increased and BMI decreased with increasing quantity of weekly alcohol consumption relative to the non-regular drinkers. The effect of smoking on lipid profile and hs-CRP was less pronounced in current drinkers than in those who were non-regular drinkers.ConclusionThe protective effect of alcohol consumption was dependent on the pattern, frequency, type, and intensity of alcohol consumed. Alcohol modified the effects of smoking on the lipid profile. Regular drinking attenuated the effect of smoking on hs-CRP and lipid profile.

The samples and data used in this study were collected as part of the 5th framework (FP5) EU funded Geoparkinson study, project number QLK4‐CT‐ 1999‐01133. The Maltese arm of this group included Prof Christian Scerri, Dr Joseph Borg, Dr Karen Cassar, Ms Wilma Cassar, Ms Ruth Galdies, Dr Norbert Vella, Dr Vicky Mifsud, Dr Josanne Aquilina and Dr Galea Debono., Background: Mutations in Leucine-rich repeat kinase 2 NM_198578 (LRRK2 c.6055G>A (p.G2019S), LRRK2 c.4321C>G (p.R1441G)) and alpha-synuclein NM_000345 (SNCA c.209G>A (p.A53T)) genes causing Parkinson's disease (PD) are common in Mediterranean populations. Variants in the Quinoid Dihydropteridine Reductase NM_000320 (QDPR c.68G>A (p.G23D)), Sepiapterin Reductase NM_003124 (SPR c.596-2A>G) and Methylenetetrahydrofolate Reductase NM_005957 (MTHFR c.677C>T and c.1298A>C) genes are frequent in Malta and potential candidates for PD. Methods: 178 cases and 402 control samples from Malta collected as part of the Geoparkinson project were genotyped for MTHFR polymorphisms, QDPR and SPR mutations. Only PD and parkinsonism cases were tested for SNCA and LRRK2 mutations. Results: LRRK2 c.4321C>G and SNCA c.209G>A were not detected. The LRRK2 c.6055G>A mutation was found in 3.1 % of Maltese PD cases. The QDPR mutation was found in both cases and controls and did not increase risk for PD. The SPR mutation was found in controls only. The odds ratios for MTHFR polymorphisms were not elevated. Conclusions: The LRRK2 c.6055G>A is a cause of PD in the Maltese, whilst QDPR c.68G>A, SPR c.596-2A>G and MTHFR c.677C>T and c.1298A>C are not important determinants of PD., The samples and data used in this study were collected as part of the 5th framework (FP5) EU funded Geoparkinson study, project number QLK4‐CT‐ 1999‐01133. This work was supported by research grants of SBW and RF from the University of Malta. The funders played no other part in the research or its interpretation., peer-reviewed

Background The overall effect of the major pro-inflammatory cytokine interleukin-1 (IL-1) on coagulation and fibrinolysis is prothrombotic. We recently found that haplotype 5 (H5) of the gene (IL1RN) coding for the interleukin-1 receptor antagonist (IL-1Ra), the natural inhibitor of IL-1, is associated with an increased risk of venous thrombosis. It is unclear whether variations in IL1RN affect the risk of myocardial infarction. Objectives The aim of this study was to investigate the effect of the five most common haplotype groups of IL1RN on the risk of myocardial infarction and on IL1RN mRNA levels. Patients/methods We genotyped 5 single nucleotide polymorphisms (SNPs) in IL1RN in 560 male patients and 646 male control subjects of a population-based case–control study on myocardial infarction, enabling us to tag the five common haplotype groups of IL1RN. For all haplotype groups the relationship with the risk of myocardial infarction and IL1RN mRNA levels was determined. Results An increased risk of myocardial infarction was found for haplotype 3 (H3) carriers (tagged by SNP 13760T/C, odds ratio = 1.3; 95% confidence interval: 1.1–1.7) compared to non-H3 carriers. No effect on myocardial infarction risk was found for the other haplotypes. H3 carriers had decreased IL1RN mRNA levels compared to non-H3 carriers (p < 0.01), whereas mRNA levels were higher in H2 carriers compared to non-H2 carriers (p < 0.01). Conclusions We found that H3 carriership increases the risk of myocardial infarction. This effect could be explained by the reduced IL1RN expression in H3 carriers, which is expected to result in reduced levels of IL-1Ra, the principal antagonist of IL-1.

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.

Atherosclerosis and the acute coronary syndromes (ACS) are very prevalent in the developed world, and they are on the increase. These complex diseases result from interplay between genes, lifestyle, and environment. Lifestyle and environmental factors are now largely known, and research efforts are shifting towards the discovery of predisposing genetic factors. Several approaches are being undertaken including family studies, genetic association studies, and studies in mice. Guided by our increasingly detailed understanding of the processes underlying cardiovascular disease many candidate genes have been evaluated including those encoding adhesion molecules, chemokines and cytokines, and matrix metalloproteinases. Genetic association studies using polymorphisms in these genes have often given conflicting results that are difficult to judge. The literature on this work will be reviewed here and some common themes emerge. These include the complexities surrounding polymorphisms found within a cluster of genes of related function, the possibility that an, underlying condition that predisposes to endothelial dysfunction is required for certain polymorphisms to exert their effect, the possibility that a gene, or another gene tightly linked to it, modifies risk for a traditional risk factor for atherosclerosis or its acute effects, and the notion that studying combinations of genotypes, or combinations of genotypes and predisposing traditional factors may be a better reflection of the situation in vivo. The practical difficulties of elucidating such complex influences are also discussed

Studies on the role of inflammation in cardiovascular disease focus on surrogate markers like plasma levels of C-reactive protein or interleukins that are affected by several factors. In this study we employ an approach in which the inflammatory mRNA profile of leucocytes is measured directly in a multigene system. We investigated the mRNA profile for 35 inflammatory markers in blood samples in a case-control study including 524 men with a history of myocardial infarction and 628 control subjects. Compared with controls, patients showed mRNA profiles with increased levels of most inflammatory mRNAs. The 2 most prominent mRNA risk indicators encoded the secreted protein macrophage migration inhibitory factor (crude odds ratio [OR], 3.4 for the highest quartile versus the lowest quartile (95% confidence interval [CI95], 2.3-4.9), and the intracellular regulator proteinase inhibitor 9 (OR, 2.5 for the highest versus the lowest quartile (CI95, 1.8-3.5), both showing an increase in odds ratio with increasing quartiles. Leucocytes in the blood of patients with myocardial infarction are more active in transcription of inflammatory genes, as evidenced by mRNA profiling. These data support the hypothesis that an inflammatory response involving leucocytes plays a role in the pathogenesis of myocardial infarction.