Low miR-19b-1-5p Expression Is Related to Aspirin Resistance and Major Adverse Cardio- Cerebrovascular Events in Patients With Acute Coronary Syndrome.

Background Because of a nonresponse to aspirin (aspirin resistance), patients with acute coronary syndrome (ACS) are at increased risk of developing recurrent event. The in vitro platelet function tests have potential limitations, making them unsuitable for the detection of aspirin resistance. We investigated whether miR-19b-1-5p could be utilized as a biomarker for aspirin resistance and future major adverse cardio-cerebrovascular (MACCE) events in patients with ACS. Methods and Results In this cohort study, patients with ACS were enrolled from multiple tertiary hospitals in Christchurch, Hong Kong, Sarawak, and Singapore between 2011 and 2015. MiR-19b-1-5p expression was measured from buffy coat of patients with ACS (n=945) by reverse transcription quantitative polymerase chain reaction. Platelet function was determined by Multiplate aggregometry testing. MACCE was collected over a mean follow-up time of 1.01±0.43 years. Low miR-19b-1-5p expression was found to be related to aspirin resistance as could be observed from sustained platelet aggregation in the presence of aspirin (-Log-miR-19b-1-5p, [unstandardized beta, 44.50; 95% CI, 2.20-86.80; P<0.05]), even after adjusting for age, sex, ethnicity, and prior history of stroke. Lower miR-19b-1-5p expression was independently associated with a higher risk of MACCE (-Log-miR-19b-1-5p, [hazard ratio, 1.85; 95% CI, 1.23-2.80; P<0.05]). Furthermore, a significant interaction was noted between the inverse miR-19b-1-5p expression and family history of premature coronary artery disease (P=0.01) on the risk of MACCE. Conclusions Lower miR-19b-1-5p expression was found to be associated with sustained platelet aggregation on aspirin, and a higher risk of MACCE in patients with ACS. Therefore, miR-19b-1-5p could be a suitable marker for aspirin resistance and might predict recurrence of MACCE in patients with ACS.

MiR-223-3p and miR-122-5p as circulating biomarkers for plaque instability.

BACKGROUND: In this study, we discovered and validated candidate microRNA (miRNA) biomarkers for coronary artery disease (CAD). METHOD: Candidate tissue-derived miRNAs from atherosclerotic plaque material in patients with stable coronary artery disease (SCAD) (n=14) and unstable coronary artery disease (UCAD) (n=25) were discovered by qPCR-based arrays. We validated differentially expressed miRNAs, along with seven promising CAD-associated miRNAs from the literature, in the serum of two large cohorts (n=395 and n=1000) of patients with SCAD and UCAD and subclinical atherosclerosis (SubA) and controls, respectively. RESULT: From plaque materials (discovery phase), miR-125b-5p and miR-193b-3p were most upregulated in SCAD, whereas miR-223-3p and miR-142-3p were most upregulated in patients with UCAD. Subsequent validation in serum from patients with UCAD, SCAD, SubA and controls demonstrated significant upregulation of miR-223-3p, miR-133a-3p, miR-146-3p and miR-155-5p. The ischaemia-related miR-499-5p was also highly upregulated in patients with UCAD compared with the other groups (SCAD OR 20.63 (95% CI 11.16 to 38.15), SubA OR 96.10 (95% CI 40.13 to 230.14) and controls OR 15.73 (95% CI 7.80 to 31.72)). However, no significant difference in miR-499-5p expression was observed across SCAD, SubA and controls. MiR-122-5p was the only miRNA to be significantly upregulated in the serum of both patients with UCAD and SCAD. CONCLUSION: In conclusion, miR-122-5p and miR-223-3p might be markers of plaque instability.

A pooled-analysis of age and sex based coronary artery calcium scores percentiles.

BACKGROUND: Age and sex based coronary artery calcium score (CAC) percentiles have been used to improve coronary artery disease (CAD) risk prediction. However, the main limitation of the CACs percentiles currently in use is that they are often based on single studies. We performed a pooled analysis of all available studies that reported on CAC percentiles, in order to develop more generalizable age and sex nomograms. METHODS: PubMed/Medline and Embase were searched for studies that reported nomograms of age and sex-based CACs percentiles. Studies were included if they reported data collected among asymptomatic individuals without a history of cardiovascular disease. Absolute CACs for each specific percentile stratum were pooled and new percentiles were generated taking into account the sample size of the study. RESULTS: We found 831 studies, of which 12 met the inclusion criteria. Data on CACs percentiles of 134,336 Western and 33,488 Asians were pooled separately, rendering a weighted CACs percentile nomogram available at https://www.calciumscorecalculator.com. Our weighted percentiles differed by up to 24% from the nomograms in use today. CONCLUSIONS: Our pooled age and sex based CACs percentiles based on over 155,000 individuals should provide a measure of risk that is more applicable to a wider population than the ones currently in use and hopefully will lead to better risk assessment and treatment decisions.

Sudden cardiac death in families with premature cardiovascular disease.

OBJECTIVE: Sudden cardiac death (SCD) in families with premature atherosclerosis (PAS) is generally attributed to lethal arrhythmias during myocardial infarction. Yet, such arrhythmias may also arise from non-ischaemic inherited susceptibility. We aimed to test the hypothesis that Brugada syndrome is prevalent among families with PAS in which SCD occurred. METHODS: We investigated all patients who underwent Ajmaline testing to screen them for Brugada syndrome because of unexplained familial SCD in the Amsterdam University Medical Centers between 2004 and 2017. We divided the cohort into two groups based on a positive family history for PAS. All individuals with a positive Ajmaline test were screened for SCN5A-mutation. RESULTS: In families with SCD and PAS, the prevalence of positive Ajmaline test was similar to families with SCD alone (22% vs 19%). The number of SCD cases in families with SCD and PAS was higher (2.34 vs 1.63, p<0.001) and SCD occurred at older age in families with SCD and PAS (42 years vs 36 years, p<0.001), while the prevalence of SCN5A mutations was lower (3% vs 18%, p<0.05). CONCLUSIONS: Brugada syndrome has a similar prevalence in families with SCD and PAS as in families with SCD alone, although SCD in families with SCD and PAS occurs in more family members and at older age, while SCN5A mutations in these families are rare. This suggests that the SCD occurring in families with PAS could be related to an underlying genetic predisposition of arrhythmias, with a different genetic origin. It could be considered to screen families with SCD and PAS for Brugada syndrome.

Study Design and qPCR Data Analysis Guidelines for Reliable Circulating miRNA Biomarker Experiments: A Review.

BACKGROUND: In the past decade, the search for circulating microRNA (miRNA) biomarkers has yielded numerous associations between miRNAs and different types of disease. However, many of these relations could not be replicated in subsequent studies under similar experimental conditions. Although this lack of replicability may be explained by the variation in experimental design and analysis methods, guidelines on the most appropriate design and analysis methods to study circulating miRNAs are scarce. CONTENT: miRNA biomarker experiments generally consist of a discovery phase and a validation phase. In the discovery phase, typically hundreds of miRNAs are measured in parallel to identify candidate biomarkers. Because of the costs of such high-throughput experiments, the number of individuals included in those studies is often too small, which can easily lead to false positives and false negatives. In the validation phase, a small number of identified biomarker candidates are measured in a large cohort of cases and controls, generally by quantitative PCR (qPCR). Although qPCR is a sensitive method to measure miRNAs in the circulation, experimental design and qPCR data analysis remain challenging. Omitting some crucial steps in the design and analysis of the qPCR experiment or performing them incorrectly can cause serious biases, ultimately leading to false conclusions. SUMMARY: In this review, we aim to expose and discuss the most common sources of interstudy variation in miRNA research from a methodological point of view and to provide guidelines on how to perform these steps correctly to increase replicability of studies on circulating miRNAs.

Elevated lipoprotein(a) levels are associated with coronary artery calcium scores in asymptomatic individuals with a family history of premature atherosclerotic cardiovascular disease.

BACKGROUND: Elevated lipoprotein(a) (Lp(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease (ASCVD). Individuals with a family history of premature ASCVD are at increased cardiovascular risk with concomitantly a higher burden of (subclinical) atherosclerosis. However, whether Lp(a) contributes to the increased atherosclerotic burden in these individuals remains to be established. OBJECTIVE: In this study, we evaluated the association between Lp(a) levels and coronary atherosclerotic burden, assessed by coronary arterty calcium (CAC) scores, in asymptomatic individuals with a family history of premature ASCVD. METHODS: Lp(a) levels and other ASCVD risk factors were assessed in 432 individuals with premature ASCVD and in 937 healthy asymptomatic family members. CAC scores were only measured in asymptomatic family members. RESULTS: In this cohort, 16% had elevated Lp(a) levels, defined as ≥ 50 mg/dL. Among healthy family members, elevated Lp(a) levels were associated with both absolute CAC scores of ≥ 100 (odds ratio [OR] 1.79 [95% confidence interval {CI} 1.13-2.83]) as well as with age- and gender-corrected CAC scores ≥ 80th percentile (OR 1.69 [95% CI 1.14-2.50]). This coincides with a higher prevalence of cardiovascular events (OR 1.48 [95% CI 1.11-2.01]) in the whole cohort. CONCLUSION: Elevated Lp(a) levels were associated with higher CAC scores, both absolute as well as age- and gender-corrected percentiles, in individuals with a family history of premature ASCVD. These data imply that Lp(a) accelerates progression of atherosclerosis in these individuals, thereby contributing to their increased ASCVD risk.

Serially measured circulating microRNAs and adverse clinical outcomes in patients with acute heart failure.

AIMS: Previous studies have identified candidate circulating microRNAs (circmiRs) as biomarkers for heart failure (HF) using relatively insensitive arrays, validated in small cohorts. The present study used RNA sequencing to identify novel candidate circmiRs and compared these with previously identified circmiRs in a large, prospective cohort of patients with acute HF (AHF). METHODS AND RESULTS: RNA sequencing of plasma from instrumented pigs was used to identify circmiRs produced by myocardium. Production of known myomiRs and microRNA (miR)-1306-5p was identified. The prognostic values of this and 11 other circmiRs were tested in a prospective cohort of 496 AHF patients, from whom blood samples were collected at up to seven time-points during the study's 1-year follow-up. The primary endpoint was the composite of all-cause mortality and HF rehospitalization. In the prospective AHF cohort, 188 patients reached the primary endpoint, and higher values of repeatedly measured miR-1306-5p were positively associated with risk for reaching the primary endpoint at the same time-point [hazard ratio (HR) 4.69, 95% confidence interval (CI) 2.18-10.06], independent of clinical characteristics and NT-proBNP. Baseline miR-1306-5p did not improve model discrimination/reclassification significantly compared with NT-proBNP. For miR-320a, miR-378a-3p, miR-423-5p and miR-1254, associations with the primary endpoint were present after adjustment for age and sex (HR 1.38, 95% CI 1.12-1.70; HR 1.35, 95% CI 1.04-1.74; HR 1.45, 95% CI 1.10-1.92; HR 1.22, 95% CI 1.00-1.50, respectively). Rates of detection of myomiRs miR-208a-3p and miR-499a-5p were very low. CONCLUSIONS: Repeatedly measured miR-1306-5p was positively associated with adverse clinical outcome in AHF, even after multivariable adjustment including NT-proBNP. However, baseline miR-1306-5p did not add significant discriminatory value to NT-proBNP. Low-abundance, heart-enriched myomiRs are often undetectable, which mandates the development of more sensitive assays.

Prognostic value of circulating microRNAs on heart failure-related morbidity and mortality in two large diverse cohorts of general heart failure patients.

AIMS: Small studies suggested circulating microRNAs (miRNAs) as biomarkers for heart failure (HF). However, standardized approaches and quality assessment for measuring circulating miRNAs are not uniformly established, and most studies have been small, so that results are inconsistent. We used a standardized data handling protocol, optimized for circulating miRNA qPCRs to remove noise and used it to assess which circulating miRNAs robustly add prognostic information in patients with HF. METHODS AND RESULTS: We measured 12 miRNAs in two independent cohorts totalling 2203 subjects. Cohort I (Barcelona) comprised 834 chronic HF patients. Cohort II (Detroit) comprised 1369 chronic HF patients. Each sample was measured in duplicate, and normalized to a very abundant and stable miRNA (miR-486-5p). We used a multistep algorithm to distinguish false amplification signals and thus classify each miRNA measurement as 'valid', 'undetectable' or 'invalid'. Higher levels of miR-1254 and miR-1306-5p were significantly associated with risk of the combined endpoint of all-cause mortality and HF hospitalization in both cohorts, with hazard ratios ranging from 1.11 to 1.21 per log increase (P-values 0.004 to 0.009). However, adding these miRNAs to established predictors (age, sex, haemoglobin, renal function, and NT-proBNP) did not further augment the c-statistic beyond 0.69 (cohort I) or 0.70 (cohort II). CONCLUSION: We used a stringent quality assessment for miRNA testing, and were able to replicate the association of miR-1254 and miR-1306-5p with risk of death and HF hospitalization in HF patients of two independent cohorts. However, these two circulating miRNAs failed to improve prognostication over established predictors.

High miR-124-3p expression identifies smoking individuals susceptible to atherosclerosis.

BACKGROUND AND AIMS: The risk of developing cardiovascular disease (CVD) is twice as high among smoking individuals compared to non-smokers. Monocytes are involved in smoking-related atherosclerotic plaque formation. In this study, we investigated whether smokers with an increased risk of developing CVD can be identified on the basis of monocyte-derived miRNA expression levels. METHODS: We performed a miRNA microarray experiment on isolated monocytes from smoking, former smoking and non-smoking individuals in a cohort of patients with premature CVD and healthy controls (Cohort I, n = 76). RESULTS: We found miR-124-3p to be heterogeneously expressed among all smoking individuals, whereas expression was low in non-smokers. Subsequently, RT-qPCR measurements on whole blood showed that among smoking individuals an increase in miR-124-3p is associated with an increased risk for advanced atherosclerotic disease (cohort II, n = 24) (OR 11.72 95% CI 1.09-126.53) and subclinical atherosclerosis (coronary artery calcium score ≥ 80th percentile, cohort III n = 138) (OR 2.71, 95% CI 1.05-7.01). This was not observed among former smokers or non-smoking individuals. Flow cytometric analysis demonstrated that high miR-124-3p expression was associated with upregulation of the monocyte surface markers CD45RA, CD29 and CD206, indicating an altered monocyte phenotype. Finally, overexpression of miR-124-3p resulted in an upregulation of CD206 surface expression on monocytes. CONCLUSIONS: High miR-124-3p expression is associated with an increased risk of subclinical atherosclerosis in smoking individuals and with an altered monocyte phenotype. This may suggest that miR-124-3p identifies which smoking individuals are susceptible to the atherogenic effects of smoking.

Practical data handling pipeline improves performance of qPCR-based circulating miRNA measurements.

Since numerous miRNAs have been shown to be present in circulation, these so-called circulating miRNAs have emerged as potential biomarkers for disease. However, results of qPCR studies on circulating miRNA biomarkers vary greatly and many experiments cannot be reproduced. Missing data in qPCR experiments often occur due to off-target amplification, nonanalyzable qPCR curves and discordance between replicates. The low concentration of most miRNAs leads to most, but not all missing data. Therefore, failure to distinguish between missing data due to a low concentration and missing data due to randomly occurring technical errors partly explains the variation within and between otherwise similar studies. Based on qPCR kinetics, an analysis pipeline was developed to distinguish missing data due to technical errors from missing data due to a low concentration of the miRNA-equivalent cDNA in the PCR reaction. Furthermore, this pipeline incorporates a method to statistically decide whether concentrations from replicates are sufficiently concordant, which improves stability of results and avoids unnecessary data loss. By going through the pipeline's steps, the result of each measurement is categorized as "valid, invalid, or undetectable." Together with a set of imputation rules, the pipeline leads to more robust and reproducible data as was confirmed experimentally. Using two validation approaches, in two cohorts totaling 2214 heart failure patients, we showed that this pipeline increases both the accuracy and precision of qPCR measurements. In conclusion, this statistical data handling pipeline improves the performance of qPCR studies on low-expressed targets such as circulating miRNAs.

Reporting Weaknesses in Conference Abstracts of Diagnostic Accuracy Studies in Ophthalmology.

IMPORTANCE: Conference abstracts present information that helps clinicians and researchers to decide whether to attend a presentation. They also provide a source of unpublished research that could potentially be included in systematic reviews. We systematically assessed whether conference abstracts of studies that evaluated the accuracy of a diagnostic test were sufficiently informative. OBSERVATIONS: We identified all abstracts describing work presented at the 2010 Annual Meeting of the Association for Research in Vision and Ophthalmology. Abstracts were eligible if they included a measure of diagnostic accuracy, such as sensitivity, specificity, or likelihood ratios. Two independent reviewers evaluated each abstract using a list of 21 items, selected from published guidance for adequate reporting. A total of 126 of 6310 abstracts presented were eligible. Only a minority reported inclusion criteria (5%), clinical setting (24%), patient sampling (10%), reference standard (48%), whether test readers were masked (7%), 2 × 2 tables (16%), and confidence intervals around accuracy estimates (16%). The mean number of items reported was 8.9 of 21 (SD, 2.1; range, 4-17). CONCLUSIONS AND RELEVANCE: Crucial information about study methods and results is often missing in abstracts of diagnostic studies presented at the Association for Research in Vision and Ophthalmology Annual Meeting, making it difficult to assess risk for bias and applicability to specific clinical settings.

Circulating MicroRNAs Characterizing Patients with Insufficient Coronary Collateral Artery Function.

BACKGROUND: Coronary collateral arteries function as natural bypasses in the event of coronary obstruction. The degree of collateral network development significantly impacts the outcome of patients after an acute myocardial infarction (AMI). MicroRNAs (miRNAs, miRs) have arisen as biomarkers to identify heterogeneous patients, as well as new therapeutic targets in cardiovascular disease. We sought to identify miRNAs that are differentially expressed in chronic total occlusion (CTO) patients with well or poorly developed collateral arteries. METHODS AND RESULTS: Forty-one CTO patients undergoing coronary angiography and invasive assessment of their coronary collateralization were dichotomized based on their collateral flow index (CFI). After miRNA profiling was conducted on aortic plasma, four miRNAs were selected for validation by real-time quantitative reverse transcription polymerase chain reaction in patients with low (CFI<0.39) and high (CFI>0.39) collateral artery capacity. We confirmed significantly elevated levels of miR423-5p (p<0.05), miR10b (p<0.05), miR30d (p<0.05) and miR126 (p<0.001) in patients with insufficient collateral network development. We further demonstrated that each of these miRNAs could serve as circulating biomarkers to discriminate patients with low collateral capacity (p<0.01 for each miRNA). We also determined significantly greater expression of miR30d (p<0.05) and miR126 (p<0.001) in CTO patients relative to healthy controls. CONCLUSION: The present study identifies differentially expressed miRNAs in patients with high versus low coronary collateral capacity. We have shown that these miRNAs can function as circulating biomarkers to discriminate between patients with insufficient or sufficient collateralization. This is the first study to identify miRNAs linked to coronary collateral vessel function in humans.

[Severe parkinsonism due to metoclopramide: the importance of early recognition]. / Ernstig parkinsonisme door metoclopramide: het belang van tijdige herkenning.

In this article, we present 3 women aged 73, 85 and 88 years who developed metoclopramide-induced parkinsonism. Shortly after starting metoclopramide, bradykinesia and rigidity developed in all 3 patients; tremor and postural instability in 2 of them. We discontinued the metoclopramide after 3-6 months; 2 of the patients had fully recovered 4-6 months later. The 3rd patient died from pneumonia, however, 2 months after discontinuation. Metoclopramide, a dopamine D2-antagonist, is a frequently prescribed anti-emetic drug; however, evidence of its efficacy is limited. In many patients, domperidone, another dopamine D2-antagonist, seems to be a better alternative. Movement disorders due to domperidone are uncommon, presumably because it does not cross the blood-brain barrier. It is likely that metoclopramide-induced parkinsonism is not uncommon; however, it is under-recognized. Risk factors are female sex, advanced age, diabetes mellitus and polypharmacy. Follow-up on patients using metoclopramide is advised.

Platelets in patients with premature coronary artery disease exhibit upregulation of miRNA340* and miRNA624*.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. It remains unknown whether differences in miRNA expression levels in platelets can be found between patients with premature CAD and healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: In this case-control study we measured relative expression levels of platelet miRNAs using microarrays from 12 patients with premature CAD and 12 age- and sex-matched healthy controls. Six platelet microRNAs were significantly upregulated (miR340*, miR451, miR454*, miR545:9.1. miR615-5p and miR624*) and one miRNA (miR1280) was significantly downregulated in patients with CAD as compared to healthy controls. To validate these results, we measured the expression levels of these candidate miRNAs by qRT-PCR in platelets of individuals from two independent cohorts; validation cohort I consisted of 40 patients with premature CAD and 40 healthy controls and validation cohort II consisted of 27 patients with artery disease and 40 healthy relatives. MiR340* and miR624* were confirmed to be upregulated in patients with CAD as compared to healthy controls in both validation cohorts. CONCLUSION/SIGNIFICANCE: Two miRNAs in platelets are significantly upregulated in patients with CAD as compared to healthy controls. Whether the two identified miRNAs can be used as biomarkers and whether they are cause or consequence of the disease remains to be elucidated in a larger prospective study.