Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run.

Mitral valve prolapse (MVP) associated with severe mitral regurgitation is a debilitating disease with no pharmacological therapies available. MicroRNAs (miRNA) represent an emerging class of circulating biomarkers that have never been evaluated in MVP human plasma. Our aim was to identify a possible miRNA signature that is able to discriminate MVP patients from healthy subjects (CTRL) and to shed light on the putative altered molecular pathways in MVP. We evaluated a plasma miRNA profile using Human MicroRNA Card A followed by real-time PCR validations. In addition, to assess the discriminative power of selected miRNAs, we implemented a machine learning analysis. MiRNA profiling and validations revealed that miR-140-3p, 150-5p, 210-3p, 451a, and 487a-3p were significantly upregulated in MVP, while miR-223-3p, 323a-3p, 340-5p, and 361-5p were significantly downregulated in MVP compared to CTRL (p ≤ 0.01). Functional analysis identified several biological processes possible linked to MVP. In addition, machine learning analysis correctly classified MVP patients from CTRL with high accuracy (0.93) and an area under the receiving operator characteristic curve (AUC) of 0.97. To the best of our knowledge, this is the first study performed on human plasma, showing a strong association between miRNAs and MVP. Thus, a circulating molecular signature could be used as a first-line, fast, and cheap screening tool for MVP identification.

Circulating microRNAs (miRNA) have been proposed as specific biomarkers for several diseases. Quantitative Real-Time PCR (RT-qPCR) is the gold standard technique currently used to evaluate miRNAs expression from different sources. In the last few years, digital PCR (dPCR) emerged as a complementary and accurate detection method. When dealing with gene expression, the first and most delicate step is nucleic-acid isolation. However, all currently available protocols for RNA extraction suffer from the variable loss of RNA species due to the chemicals and number of steps involved, from sample lysis to nucleic acid elution. Here, we evaluated a new process for the detection of circulating miRNAs, consisting of sample lysis followed by direct evaluation by dPCR in plasma from healthy donors and in the cardiovascular setting. Our results showed that dPCR is able to detect, with high accuracy, low-copy-number as well as highly expressed miRNAs in human plasma samples without the need for RNA extraction. Moreover, we assessed a known myocardial infarction-related miR-133a in acute myocardial infarct patients vs. healthy subjects. In conclusion, our results show the suitability of the extraction-free quantification of circulating miRNAs as disease markers by direct dPCR.

Amyloid deposition within stenotic aortic valves (AVs) also appears frequent in the absence of cardiac amyloidosis, but its clinical and pathophysiological relevance has not been investigated. We will elucidate the rate of isolated AV amyloid deposition and its potential clinical and pathophysiological significance in aortic stenosis (AS). In 130 patients without systemic and/or cardiac amyloidosis, we collected the explanted AVs during cardiac surgery: 57 patients with calcific AS and 73 patients with AV insufficiency (41 with AV sclerosis and 32 without, who were used as controls). Amyloid deposition was found in 21 AS valves (37%), 4 sclerotic AVs (10%), and none of the controls. Patients with and without isolated AV amyloid deposition had similar clinical and echocardiographic characteristics and survival rates. Isolated AV amyloid deposition was associated with higher degrees of AV fibrosis (p = 0.0082) and calcification (p < 0.0001). Immunohistochemistry analysis suggested serum amyloid A1 (SAA1), in addition to transthyretin (TTR), as the protein possibly involved in AV amyloid deposition. Circulating SAA1 levels were within the normal range in all groups, and no difference was observed in AS patients with and without AV amyloid deposition. In vitro, AV interstitial cells (VICs) were stimulated with interleukin (IL)-1β which induced increased SAA1-mRNA both in the control VICs (+6.4 ± 0.5, p = 0.02) and the AS VICs (+7.6 ± 0.5, p = 0.008). In conclusion, isolated AV amyloid deposition is frequent in the context of AS, but it does not appear to have potential clinical relevance. Conversely, amyloid deposition within AV leaflets, probably promoted by local inflammation, could play a role in AS pathophysiology.

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition reduces plasma concentrations of low-density lipoprotein cholesterol (LDL-C), apolipoprotein B (apoB) and lipoprotein(a) [Lp(a)]. Atherogenic lipoprotein levels have been linked with calcific aortic valve stenosis (CAVS). Our objectives were to determine the association between variants inPCSK9and lipoprotein-lipid levels, coronary artery disease (CAD) and CAVS, and to evaluate if PCSK9 could be implicated in aortic valve interstitial cells (VICs) calcification.Methods: We built a genetic risk score weight for LDL-C levels (wGRS) using 10 independentPCSK9single nucleotide polymorphisms and determined its association with lipoprotein-lipid levels in 9692 participants of the EPIC-Norfolk study. We investigated the association between the wGRS and CAD and CAVS in the UK Biobank, as well as the association between thePCSK9R46L variant and CAVS in a meta-analysis of published prospective, population-based studies (Copenhagen studies, 1463 cases/101,620 controls) and unpublished studies (UK Biobank, 1350 cases/349,043 controls, Malmö Diet and Cancer study, 682 cases/5963 controls and EPIC-Norfolk study, 508 cases/20,421 controls). We evaluated PCSK9 expression and localization in explanted aortic valves by capillary Western blot and immunohistochemistry in patients with and without CAVS. Von Kossa staining was used to visualize aortic leaflet calcium deposits. PCSK9 expression under oxidative stress conditions in VICs was assessed.Results: The wGRS was significantly associated with lower LDL-C and apoB (p<0.001), but not with Lp(a). In the UK Biobank, the association ofPCSK9variants with CAD were positively correlated with their effects on apoB levels. CAVS was less prevalent in carriers of thePCSK9R46L variant [odds ratio=0.71 (95% confidence interval, 0.57-0.88), p<0.001]. PCSK9 expression was elevated in the aortic valves of patients with aortic sclerosis and CAVS compared to controls. In calcified leaflets, PCSK9 co-localized with calcium deposits. PCSK9 expression was induced by oxidative stress in VICs.Conclusion: Genetic inhibition ofPCSK9is associated with lifelong reductions in the levels of non-Lp(a) apoB-containing lipoproteins as well as lower odds of CAD and CAVS. PCSK9 is abundant in fibrotic and calcified aortic leaflets. Oxidative stress increases PCSK9 expression in VICs. These results provide a rationale for performing randomized clinical trials of PCSK9 inhibition in CAVS.