Cytosolic nucleic acid sensing and mitochondrial transcriptomic changes as early triggers of metabolic disease in db/db mice.

Animal models of diabetes, such as db/db mice, are a useful tool for deciphering the genetic background of molecular changes at the initial stages of disease development. Our goal was to find early transcriptomic changes in three tissues involved in metabolism regulation in db/db mice: adipose tissue, muscle tissue and liver tissue. Nine animals (three per time point) were studied. Tissues were collected at 8, 12 and 16 weeks of age. Transcriptome-wide analysis was performed using mRNA-seq. Libraries were sequenced on NextSeq (Illumina). Differential expression (DE) analysis was performed with edgeR. The analysis of the gene expression profile shared by all three tissues revealed eight upregulated genes (Irf7, Sp100, Neb, Stat2, Oas2, Rtp4, H2-T24 and Oasl2) as early as between 8 and 12 weeks of age. The most pronounced differences were found in liver tissue: nine DE genes between 8 and 12 weeks of age (Irf7, Ly6a, Ly6g6d, H2-Dma, Pld4, Ly86, Fcer1g, Ly6e and Idi1) and five between 12 and 16 weeks of age (Irf7, Plac8, Ifi44, Xaf1 and Ly6a) (adj. p-value < 0.05). The mitochondrial transcriptomic profile also changed with time: we found two downregulated genes in mice between 8 and 12 weeks old (Ckmt2 and Cox6a2) and five DE genes between 12 and 16 weeks of age (Mavs, Tomm40L, Mtfp1, Ckmt2 and Cox6a2). The KEGG pathway analysis showed significant enrichment in pathways related to the autoimmune response and cytosolic DNA sensing. Our results suggest an important involvement of the immunological response, mainly cytosolic nucleic acid sensing, and mitochondrial signalling in the early stages of diabetes and obesity.

Intratumor heterogeneity in colorectal cancer: Distribution of tumor suppressor gene variants with regard to patient lymph node status.

Intratumor heterogeneity (ITH) results from accumulation of somatic mutations in the fractions of successive cancer cell generations. We aimed to use deep sequencing to investigate ITH in colorectal tumors with particular emphasis on variants in oncogenes (ONC) and tumor suppressor genes (TSG). Samples were collected from 16 patients with colorectal cancer and negative or positive lymph node status (n = 8 each). We deep-sequenced a panel of 56 cancer-related genes in the central and peripheral locations of T3 size primary tumors and healthy mucosa. The central region of T3 tumors has a different frequency profile and composition of genetic variants. This mutation profile is capable of independently discriminating patients with different lymph node status (p = 0.028) in the central region. We noted an increasing number of mutations outside of the central region of the tumor and a higher number of mutations in tumors from node-positive patients. Unexpectedly, in the healthy mucosa, we identified somatic mutations with variant allele frequencies, characteristic not only of heterozygotes and homozygotes but also of other discrete peaks (e.g., around 10%, 20%), suggestive of clonal expansion of certain mutant alleles. We found differences in the distribution of variant allele frequencies in TSGs when comparing node-negative and node-positive tumors (p = 0.029), as well as central and peripheral regions (p = 0.00399). TSGs may play an important role in the escape of the tumor toward metastatic colonization.

Synthesis of Nonsymmetrically Substituted 2,3-Dialkoxyphenazine Derivatives and Preliminary Examination of Their Cytotoxicity.

Fourteen new 2,3-dialkoxyphenazine derivatives with two different alkoxy groups bearing R1 and R2 alkyl chains, defined as -CH2CH(CH3)2 and -(CH2)n-1CH3 for n = 1, 2, 4, 6, 8, and 10, were prepared via regioselective synthesis. The applied synthetic protocol is based on the following reactions: the Buchwald-Hartwig coupling of a nonsymmetrically substituted 4,5-dialkoxy-2-nitroaniline with a 1-bromo-2-nitrobenzene derivative featuring additional tert-butyl, trifluoromethyl or two methoxy groups; the reduction of bis(2-nitrophenyl)amine; and a final step of tandem-like oxidation that leads to the preparation of a heterocyclic phenazine system. The regioselectivity of these steps and the molecular structure of the compounds under investigation were confirmed by nuclear magnetic resonance and additionally by single-crystal X-ray diffraction performed for some examples of 5 and 6 phenazine series. For 7-(tert-butyl)-3-isobutoxy-2-(octyloxy)phenazine (5f), 3-(hexyloxy)-2-isobutoxy-7-(trifluoromethyl)phenazine (6e), and 2,3-bis(hexyloxy)-7,8-dimethoxyphenazine (7), viability and cytotoxicity assays were performed on the LoVo human colon adenocarcinoma cell line, with 5f confirmed to exhibit cytotoxicity.

No differences in miRNA expression in the stroma of ERG+ and ERG- prostate cancers.

Prostate cancer (PC) is one of the most common cancers in males. A significant proportion of PCs bear TMPRSS2-ETS translocation and overexpress ERG transcription factor, allowing classification into ERG+ and ERG- groups, which differ in several features including the tumor microenvironment. The aim of the study was to verify whether they differ in expression of the miRNA in the microenvironment. The material consisted of 150 radical prostatectomies. Immunohistochemistry (IHC) for ERG was done using a routine method. FISH for TMPRSS2-ETS translocation was done with a ZytoLight SPEC ERG/TMPRSS2 TriCheck Probe. From each case, a representative section was selected, and tumor and non-tumor were microdissected with the LMD7000 device. RNA was isolated using the RNeasy Mini Kit system (Qiagen) and miRNA libraries were prepared with the NEBNext Multiplex Small RNA Library Prep Set for Illumina and their sequencing was performed on the NexSeq 500. Statistical analysis was done with Statistica and R software. When analyzing the expression of miRNAs some differences could be seen, but after correction for multiple comparisons was applied, these were found to be non- significant.

MicroRNA composition of plasma extracellular vesicles: a harbinger of late cardiotoxicity of doxorubicin.

BACKGROUND: The use of doxorubicin is associated with an increased risk of acute and long-term cardiomyopathy. Despite the constantly growing number of cancer survivors, little is known about the transcriptional mechanisms which progress in the time leading to a severe cardiac outcome. It is also unclear whether long-term transcriptomic alterations related to doxorubicin use are similar to transcriptomic patterns present in patients suffering from other cardiomyopathies. METHODS: We have sequenced miRNA from total plasma and extracellular vesicles (EVs) from 66 acute lymphoblastic leukemia (ALL) survivors and 61 healthy controls (254 samples in total). We then analyzed processes regulated by differentially expressed circulating miRNAs and cross-validated results with the data of patients with clinically manifested cardiomyopathies. RESULTS: We found that especially miRNAs contained within EVs may be informative in terms of cardiomyopathy development and may regulate pathways related to neurotrophin signaling, transforming growth factor beta (TGFß) or epidermal growth factor receptors (ErbB). We identified vesicular miR-144-3p and miR-423-3p as the most variable between groups and significantly correlated with echocardiographic parameters and, respectively, for plasma: let-7g-5p and miR-16-2-3p. Moreover, vesicular miR-144-3p correlates with the highest number of echocardiographic parameters and is differentially expressed in the circulation of patients with dilated cardiomyopathy. We also found that distribution of particular miRNAs between of plasma and EVs (proportion between compartments) e.g., miR-184 in ALL, is altered, suggesting changes within secretory and miRNA sorting mechanisms. CONCLUSIONS: Our results show that transcriptomic changes resulting from doxorubicin induced myocardial injury are reflected in circulating miRNA levels and precede development of the late onset cardiomyopathy phenotype. Among miRNAs related to cardiac function, we found vesicular miR-144-3p and miR-423-3p, as well as let-7g-5p and miR-16-2-3p contained in the total plasma. Selection of source for such studies (plasma or EVs) is of critical importance, as distribution of some miRNA between plasma and EVs is altered in ALL survivors, in comparison to healthy people, which suggests that doxorubicin-induced changes include miRNA sorting and export to extracellular space.

Variants influencing age at diagnosis of HNF1A-MODY.

BACKGROUND: HNF1A-MODY is a monogenic form of diabetes caused by variants in the HNF1A gene. Different HNF1A variants are associated with differences in age of disease onset, but other factors are postulated to influence this trait. Here, we searched for genetic variants influencing age of HNF1A-MODY onset. METHODS: Blood samples from 843 HNF1A-MODY patients from Czech Republic, France, Poland, Slovakia, the UK and the US were collected. A validation set consisted of 121 patients from the US. We conducted a genome-wide association study in 843 HNF1A-MODY patients. Samples were genotyped using Illumina Human Core arrays. The core analysis was performed using the GENESIS package in R statistical software. Kinship coefficients were estimated with the KING and PC-Relate algorithms. In the linear mixed model, we accounted for year of birth, sex, and location of the HNF1A causative variant. RESULTS: A suggestive association with age of disease onset was observed for rs2305198 (p = 2.09E-07) and rs7079157 (p = 3.96E-06) in the HK1 gene, rs2637248 in the LRMDA gene (p = 2.44E-05), and intergenic variant rs2825115 (p = 2.04E-05). Variant rs2637248 reached nominal significance (p = 0.019), while rs7079157 (p = 0.058) and rs2825115 (p = 0.068) showed suggestive association with age at diabetes onset in the validation set. CONCLUSIONS: rs2637248 in the LRMDA gene is associated with age at diabetes onset in HNF1A-MODY patients.

The transcriptome-wide association search for genes and genetic variants which associate with BMI and gestational weight gain in women with type 1 diabetes.

BACKGROUND: Clinical data suggest that BMI and gestational weight gain (GWG) are strongly interconnected phenotypes; however, the genetic basis of the latter is rather unclear. Here we aim to find genes and genetic variants which influence BMI and/or GWG. METHODS: We have genotyped 316 type 1 diabetics using Illumina Infinium Omni Express Exome-8 v1.4 arrays. The GIANT, ARIC and T2D-GENES summary statistics were used for TWAS (performed with PrediXcan) in adipose tissue. Next, the analysis of association of imputed expression with BMI in the general and diabetic cohorts (Analysis 1 and 2) or GWG (Analysis 3 and 4) was performed, followed by variant association analysis (1 Mb around identified loci) with the mentioned phenotypes. RESULTS: In Analysis 1 we have found 175 BMI associated genes and 19 variants (p < 10-4) which influenced GWG, with the strongest association for rs11465293 in CCL24 (p = 3.18E-06). Analysis 2, with diabetes included in the model, led to discovery of 1812 BMI associated loci and 207 variants (p < 10-4) influencing GWG, with the strongest association for rs9690213 in PODXL (p = 9.86E-07). In Analysis 3, among 648 GWG associated loci, 2091 variants were associated with BMI (FDR < 0.05). In Analysis 4, 7 variants in GWG associated loci influenced BMI in the ARIC cohort. CONCLUSIONS: Here, we have shown that loci influencing BMI might have an impact on GWG and GWG associated loci might influence BMI, both in the general and T1DM cohorts. The results suggest that both phenotypes are related to insulin signaling, glucose homeostasis, mitochondrial metabolism, ubiquitinoylation and inflammatory responses.

Dysregulation of Transcription Factor Activity During Formation of Cancer-Associated Fibroblasts.

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with FER1L4 expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels.

Contribution of a Novel B3GLCT Variant to Peters Plus Syndrome Discovered by a Combination of Next-Generation Sequencing and Automated Text Mining.

Anterior segment dysgenesis (ASD) encompasses a spectrum of ocular disorders affecting the structures of the anterior eye chamber. Mutations in several genes, involved in eye development, are implicated in this disorder. ASD is often accompanied by diverse multisystemic symptoms and another genetic cause, such as variants in genes encoding collagen type IV. Thus, a wide spectrum of phenotypes and underlying genetic diversity make fast and proper diagnosis challenging. Here, we used AMELIE, an automatic text mining tool that enriches data with the most up-to-date information from literature, and wANNOVAR, which is based on well-documented databases and incorporates variant filtering strategy to identify genetic variants responsible for severely-manifested ASD in a newborn child. This strategy, applied to trio sequencing data in compliance with ACMG 2015 guidelines, helped us find two compound heterozygous variants of the B3GLCT gene, of which c.660+1G>A (rs80338851) was previously associated with the phenotype of Peters plus syndrome (PPS), while the second, NM_194318.3:c.755delC (p.T252fs), in exon 9 of the same gene was noted for the first time. PPS, a very rare subtype of ASD, is a glycosylation disorder, where the dysfunctional B3GLCT gene product, O-fucose-specific ß-1,3-glucosyltransferase, is ineffective in providing a noncanonical quality control system for proper protein folding in cells. Our study expands the mutation spectrum of the B3GLCT gene related to PPS. We suggest that the implementation of automatic text mining tools in combination with careful variant filtering could help translate sequencing results into diagnosis, thus, considerably accelerating the diagnostic process and, thereby, improving patient management.

The relationship between myocardial fibrosis and myocardial microRNAs in dilated cardiomyopathy: A link between mir-133a and cardiovascular events.

It is unknown whether fibrosis-associated microRNAs: miR-21, miR-26, miR-29, miR-30 and miR-133a are linked to cardiovascular (CV) outcome. The study evaluated the levels of extracellular matrix (ECM) fibrosis and the prevalence of particular microRNAs in patients with dilated cardiomyopathy (DCM) to investigate any correlation with CV events. METHODS: Seventy DCM patients (48 ± 12 years, EF 24.4 ± 7.4%) underwent right ventricular biopsy. The control group was comprised of 7 patients with CAD who underwent CABG and intraoperative biopsy. MicroRNAs were measured in blood and myocardial tissue via qPCR. The end-point was a combination of CV death and urgent HF hospitalization at the end of 12 months. There were differential levels of circulating and myocardial miR-26 and miR-29 as well as myocardial miR-133a when the DCM and CABG groups were compared. Corresponding circulating and myocardial microRNAs did not correlate with one another. There was no correlation between microRNA and ECM fibrosis. By the end of the 12-month period of the study, CV death had occurred in 6 patients, and a further 19 patients required urgent HF hospitalization. None of the circulating microRNAs was a predictor of the combined end-point; however, myocardial miR-133a was an independent predictor in unadjusted models (HR 1.53; 95% CI 1.14-2.05; P < .004) and adjusted models (HR 1.57; 95% CI 1.14-2.17; P < .005). The best cut-off value for the miR-133a level for the prediction of the combined end-point was 0.74 ΔCq, with an AUC of 0.67. The absence of a correlation between the corresponding circulating and myocardial microRNAs calls into question their cellular source. This study sheds new light on the role of microRNAs in ECM fibrosis in DCM, which warrants further exploration.

Comparative iTRAQ analysis of protein abundance in the human sinoatrial node and working cardiomyocytes.

Our objective was to assess the changes in protein abundance in the human sinoatrial node (SAN) compared with working cardiomyocytes to identify SAN-specific protein signatures. Four pairs of samples (the SAN and working cardiomyocytes) were obtained postmortem from four human donors with no evidence of cardiovascular disease. We performed protein identification and quantitation using two-dimensional chromatography-tandem mass spectrometry with isobaric peptide labeling (iTRAQ). We identified 451 different proteins expressed in both the SAN and working cardiomyocytes, 166 of which were differentially regulated (110 were upregulated in the SAN and 56 in the working cardiomyocytes). We identified sarcomere structural proteins in both tissues, although they were differently distributed among the tested samples. For example, myosin light chain 4, myosin regulatory light chain 2-atrial isoform, and tropomyosin alpha-3 chain levels were twofold higher in the SAN than in working cardiomyocytes, and myosin light chain 3 and myosin regulatory light chain 2-ventricular/cardiac muscle isoform levels were twofold higher in the ventricle tissue than in SAN. We identified many mitochondrial oxidative phosphorylation, ß-oxidation, and tricarboxylic acid cycle proteins that were predominantly associated with working cardiomyocytes tissue. We detected upregulation of the fatty acid omega activation pathway proteins in the SAN samples. Some proteins specific for smooth muscle tissue were highly upregulated in the SAN (e.g. transgelin), which indicates that the SAN tissue might act as the bridge between the working myocardium and the smooth muscle. Our results show possible implementation of proteomic strategies to identify in-depth functional differences between various heart sub-structures.

The presence of IL-8 +781 T/C polymorphism is associated with the parameters of severe Clostridium difficile infection.

PURPOSE: There is large variation in the clinical manifestations of Clostridium difficile infection (CDI). We also still can not predict which patients are more susceptible to reinfection with CDI. The aim of our study was to evaluate the effect of gene single nucleotide polymorphisms (SNP) of proinflammatory cytokines, specifically IL-1ß, IL-8 on the development, clinical course and recurrence of CDI. METHODS: We performed a prospective study of adults (130 people ≥ 18 years) including 65 patients with CDI treated in tertiary hospital and 65 healthy persons. The following 3 variants were analyzed for the occurrence of gene polymorphisms in patients with CDI versus the control group: IL-1ß +3953 A/G (rs1143634), IL-1ß -31 A/G (rs1143627), and IL-8 +781 T/C (rs2227306). Then, we assessed the correlation between these genetic polymorphisms and biochemical parameters important in CDI course, CDI severity as well as CDI recurrence. RESULTS AND CONCLUSIONS: The presence of genetic polymorphisms of IL-1ß +3953 A/G, -31 A/G and IL-8 +781 T/C did not have an effect on the development or recurrence of CDI. The presence of IL-8 +781 T/C polymorphism is associated with the severe CDI.

Fibrin Clot Permeability as a Predictor of Stroke and Bleeding in Anticoagulated Patients With Atrial Fibrillation.

BACKGROUND AND PURPOSE: Formation of denser fiber networks has been reported in atrial fibrillation and ischemic stroke. In this longitudinal cohort study, we evaluated whether fibrin clot density may predict thromboembolic and bleeding risk in patients with atrial fibrillation on vitamin K antagonists. METHODS: In 236 patients with atrial fibrillation receiving vitamin K antagonists treatment, we measured ex vivo plasma clot permeability (Ks), a measure of the pore size in fibrin networks. RESULTS: During a median follow-up of 4.3 (interquartile range, 3.7-4.8) years, annual rates of ischemic stroke or transient ischemic attack and major bleeds were 2.96% and 3.45%, respectively. In multivariate Cox regression analysis, patients with lower Ks (<6.8 cm2×10-9, median) had increased risk of ischemic stroke or transient ischemic attack (hazard ratio [HR], 6.55; 95% confidence interval [CI], 2.17-19.82) and major bleeds (HR, 10.65; 95% CI, 3.52-32.22). Patients with elevated Ks (≥6.8 cm2×10-9) had an increased rate of minor bleeding compared with the remainder (11.63% per year versus 3.55% per year; P<0.0001). The independent predictors of stroke or transient ischemic attack were low Ks (<6.8 cm2×10-9; HR, 7.24; 95% CI, 2.53-20.76), age (HR, 1.05; 95% CI, 1.01-1.10), and treatment with angiotensin-converting enzyme inhibitors (HR, 2.27; 95% CI, 1.08-4.77). Major bleeds were predicted by low Ks (<6.8 cm2×10-9; HR, 8.48; 95% CI, 2.99-24.1) and HAS-BLED score ≥3 (HR, 2.22; 95% CI, 1.12-4.38). CONCLUSIONS: This study is the first to show that unfavorable fibrin properties reflected by formation of denser fibrin networks determine, in part, the efficacy and safety of anticoagulation with vitamin K antagonists in patients with atrial fibrillation.

Association of plasma miR-223 and platelet reactivity in patients with coronary artery disease on dual antiplatelet therapy: A preliminary report.

Decreased plasma levels of microRNA-223 (miR-223), predominantly of platelet origin, were proposed as a surrogate marker of efficacy of antiplatelet therapy. However, higher on-treatment platelet reactivity was associated with lower plasma miR-223 in patients with coronary artery disease (CAD) on dual antiplatelet therapy (DAPT) including clopidogrel and aspirin. Our aim was to compare plasma miR-223 and platelet reactivity in CAD patients on DAPT with newer P2Y12 antagonists vs. clopidogrel. We studied 21 men with CAD admitted to our centre owing to a non-ST-elevation acute coronary syndrome, and with an uncomplicated hospital course. From the day of admission, the patients were receiving either clopidogrel (n = 11) or prasugrel/ticagrelor (n = 10) in addition to aspirin. Before discharge, miR-223 expression in plasma was estimated by quantitative polymerase chain reaction using the comparative Ct method relative to miR-16 as an endogenous control. Multiple electrode aggregometry was used to assess platelet aggregation in response to adenosine diphosphate (ADP). ADP-induced platelet reactivity was decreased in the patients treated with prasugrel or ticagrelor compared with those on clopidogrel (mean ± SD: 139 ± 71 vs. 313 ± 162 arbitrary units [AU]*min, p = 0.006), due to a more potent antiplatelet activity of the novel P2Y12 antagonists. Consequently, six out of seven patients in the lower tertile of the ADP-induced platelet aggregation were treated with the newer P2Y12 blockers, whereas six out of seven patients in the upper tertile were on clopidogrel. Plasma miR-223 was elevated with decreasing platelet reactivity (Spearman's rho = -0.52; p = 0.015 for trend), being significantly higher in the lower tertile of the ADP-induced platelet aggregation (median [range]: 1.06 [0.25-2.31]) vs. the upper tertile (0.20 [0.13-2.30]) (p = 0.04). In conclusion, our preliminary results argue against the notion of low plasma miR-223 as a marker of platelet responsiveness to DAPT. On the contrary, more potent platelet inhibition associated mainly with newer P2Y12 antagonists appears to coincide with higher miR-223 relative to the subjects with attenuated responsiveness to DAPT.

Whole-exome sequencing as a tool for searching for genetic background modifiers in MEN1 patients with neuroendocrine pancreatic tumours, including insulinomas.

INTRODUCTION: Multiple endocrine neoplasia type 1 (MEN1) is a monogenic disease caused by inactivating variants in the MEN1 gene. Although the reason for its development is well-known, disease phenotypes are unpredictable and differ even among carriers of the same pathogenic driver mutation. Genetic, epigenetic, and environmental factors may play a role in driving the individual phenotype. Those factors, however, still mostly remain unidentified. In our work, we focused on the inherited genetic background in pancreatic neuroendocrine neoplasms (pNENs) in MEN1 patients, and the pancreatic tumour subgroup with insulinoma. MATERIAL AND METHODS: Whole exome sequencing was performed in MEN1 patients. The symptoms of interest were pancreatic neuroendocrine tumours in one analysis and insulinoma in the second. The study included families as well as unrelated cases. Genes with variants that are not neutral to the encoded gene product were defined in symptom-positive patients as compared to symptom-negative controls. The interpretation of the results was based on functional annotations and pathways shared between all patients with the given symptom in the course of MEN1. RESULTS: Whole-exome screening of family members and unrelated patients with and without pNENs revealed a number of pathways that are common for all the analysed cases with pNENs. Those included pathways crucial for morphogenesis and development, proper insulin signalling, and structural cellular organization. An additional analysis of insulinoma pNEN patients revealed additional pathways engaged in glucose and lipid homeostasis, and several non-canonical insulin-regulating mechanisms. CONCLUSIONS: Our results show the existence of pathways that are identified in a non-literature-predefined manner, which might have a modifying function in MEN1, differentiating the specific clinical outcomes. Those results, although preliminary, provide evidence of the reasonableness of performing large-scale studies addressing the genetic background of MEN1 patients in determining their individual outcomes.

Expression of micro-ribonucleic acids in thyroid nodules and serum to discriminate between follicular adenoma and cancer in patients with a fine needle aspiration biopsy classified as suspicious for follicular neoplasm: A preliminary study.

BACKGROUND: Approximately 10% of thyroid nodules undergoing fine needle aspiration biopsy (FNAB) receive a suspicious for follicular neoplasm (SFN) classification. Currently, there is no diagnostic tool to preoperatively discriminate between follicular adenoma (FA) and thyroid cancer (TC), and most patients require surgery to exclude malignancy. OBJECTIVES: To characterize the micro-ribonucleic acid (miRNA) signature of tumors assessed as SFN and define circulating miRNA patterns to distinguish FA from follicular cancer in patients with thyroid nodules biopsied using FNAB. MATERIAL AND METHODS: The study included excised tumor and thyroid tissue samples from 80 consecutive patients collected by a pathologist in the operating theater. The miRNA was isolated from specimens at the Center for Medical Genomics OMICRON, and next-generation sequencing (NGS) was used to obtain target miRNAs. In addition, miRNA expression was detected in serum using polymerase chain reaction (PCR). RESULTS: Well-differentiated thyroid cancer (WDTC) samples had significantly higher expression levels of hsa-miR-146b-5p (p = 0.030) and hsa-miR-146b-3p (p = 0.032), while the expression levels of hsa-miR-195-3p were significantly lower (p = 0.032) in WDTC samples compared to FA specimens. The serum of TC patients showed markedly higher expression of the unique miRNA hsa-miR-195-3p (p = 0.039). CONCLUSIONS: The overexpression of hsa-miR-146b-5p and hsa-miR-146b-3p, and the downregulation of hsa-miR-195-3p expression could be used as biomarkers to distinguish FA from WDTC in patients with FNAB results classified as Bethesda tier IV. In addition, hsa-miR-195-3p could act as a serum biomarker for differentiating patients with FA from those with WDTC, and preoperative measurement of its expression would help avoid unnecessary surgeries. However, this concept needs further verification in a more substantial prospective study.

Targeted sequencing of a gene panel in patients with familial hypercholesterolemia from Southern Poland.

INTRODUCTION: Familial hypercholesterolemia (FH) is an autosomal dominant monogenic lipid metabolism disorder characterized by a significantly elevated level of low­density lipoprotein (LDL) cholesterol and leading to premature ischemic heart disease. FH is caused by mutations in the LDLR, APOB, and PCSK9 genes; however, these mutations account for only about 40% of FH cases. In order to obtain a genetic diagnosis of FH, sequencing of other genes involved in the lipid metabolism might be useful. OBJECTIVES: This study aimed to describe genetic variants in genes associated with FH in a group of patients from the Malopolska province in Southern Poland, using the targeted next generation sequencing (NGS) technology. PATIENTS AND METHODS: The study involved 90 unrelated adults (age range, 18-70 years) with FH diagnosed clinically according to the Simon Broome Register criteria. A custom­designed capture assay and the Illumina MiSeq platform were used. The panel included exons and exon / intron boundaries of known FH­causing genes: LDLR, APOB, and PCSK9, as well as genes previously associated with high cholesterol levels: APOE, ABCG5, ABCG8, LPL, NPC1, LDLRAP1, LIPC, STAP1, and CELSR2. Genetic variants were classified based on in silico predictions and ClinVar reports. RESULTS: We detected 4 patients with variants in the LDLR and APOB genes that had not been previously linked to FH in ClinVar. We also found APOB mutations outside the common LDL receptor-binding region, in exons 26 and 29. Interestingly, we observed a high frequency of pathogenic variants in exon 4 of the APOE gene: rs7412, probably damaging (4 patients) and rs429358, benign (16 patients). CONCLUSIONS: NGS is a useful and reliable method to detect new variants in genes related to FH. In addition, the results enable the detection of FH phenocopies and introduction of appropriate treatment.

Accelerated ageing and coronary microvascular dysfunction in chronic heart failure in Tgαq*44 mice.

Age represents a major risk factor in heart failure (HF). However, the mechanisms linking ageing and HF are not clear. We aimed to identify the functional, morphological and transcriptomic changes that could be attributed to cardiac ageing in a model of slowly progressing HF in Tgαq*44 mice in reference to the cardiac ageing process in FVB mice. In FVB mice, ageing resulted in the impairment of diastolic cardiac function and in basal coronary flow (CF), perivascular and interstitial fibrosis without changes in the cardiac activity of angiotensin-converting enzyme (ACE) or aldosterone plasma concentration. In Tgαq*44 mice, HF progression was featured by the impairment of systolic and diastolic cardiac function and in basal CF that was associated with a distinct rearrangement of the capillary architecture, pronounced perivascular and interstitial fibrosis, progressive activation of cardiac ACE and systemic angiotensin-aldosterone-dependent pathways. Interestingly, cardiac ageing genes and processes were represented in Tgαq*44 mice not only in late but also in early phases of HF, as evidenced by cardiac transcriptome analysis. Thirty-four genes and 8 biological processes, identified as being ageing related, occurred early and persisted along HF progression in Tgαq*44 mice and were mostly associated with extracellular matrix remodelling and fibrosis compatible with perivascular fibrosis resulting in coronary microvascular dysfunction (CMD) in Tgαq*44 mice. In conclusion, accelerated and persistent cardiac ageing contributes to the pathophysiology of chronic HF in Tgαq*44 mice. In particular, prominent perivascular fibrosis of microcirculation resulting in CMD represents an accelerated cardiac ageing phenotype that requires targeted treatment in chronic HF.

Elevated urinary excretion of immunoglobulins in nonproteinuric patients with type 1 diabetes.

Increased albuminuria is a hallmark of early diabetic nephropathy, whereas the role of immunoglobulins (Igs), such as IgG (its 1-4 subtypes), IgA, and IgM, different in charge and size, has not been examined in early nephropathy in the past due to lack of a sensitive and reliable method. Our study group consisted of subjects with type 1 diabetes (T1D) and normoalbuminuria (n = 78), microalbuminuria (n = 78), and of 75 healthy subjects (HS). A Luminex-based immunoassay (1,000 time more sensitive than nephelometry-based method) was validated for the urine matrix and used for the measurements of IgG1-4, IgA, and IgM in our study groups. The Luminex-based assay detected Igs in 87% of HS subjects and in 100% of T1D subjects. Recovery of known amounts of Igs added to urine was 92-118%. In the normoalbuminuria group, urinary concentrations of albumin, IgG2, IgA, and IgM were significantly higher than in HS, whereas in the microalbuminuria, further elevation of IgG2, IgG4, and IgA was the most pronounced. In all three groups, fractional excretion of Igs was at least 100 times lower than that of albumin. Fractional excretion of IgG2 was the highest among all Igs. We validated a sensitive method for measuring Igs in urine using Luminex. We found that elevated concentrations of Igs, particularly in IgG2 and IgA, is present in subjects with T1D and no proteinuria. Elevation of those particular Ig subtypes suggests a contribution of novel mechanisms in early diabetic nephropathy, different from charge and size barrier impairment.

Paraoxonase 2 C311S single nucleotide polymorphism is associated with type C lesions in coronary atherosclerosis.

BACKGROUND: Paraoxonases (PON) 1-3 are lactonases with antioxidant and atheroprotective properties. The best known single nucleotide polymorphisms (SNPs) within the PON family, include: Q192R (rs662), L55M (rs854560) in the PON1 gene and C311S (rs7493) in the PON2 gene. Their influence on the occurrence and course of coronary artery disease (CAD) is unclear. The aim of this study was to assess the association between the most common PON1 and PON2 genetic variants with the presence of CAD, as well as their relation to coronary lesion complexity in accordance with the ACC/AHA standard. METHODS: We included 1027 individuals: 367 CAD patients qualified for coronary angiography and 660 healthy volunteers as controls. We extracted DNA from circulating blood leukocytes, amplified the PON1 and PON2 genetic sequence and used restriction enzymes to identify the SNPs. Patients with CAD underwent coronary angiography and were assigned to two groups based on lesion severity: patients with at least one type C lesion and without a type C lesion. The former where categorized into those with a significant narrowing (≥50% diameter stenosis) and those without one. RESULTS: We found no association between the analyzed SNPs and symptomatic CAD. However, in patients with diagnosed CAD, the PON311S allele was independently associated with the risk of the most complex type C coronary lesion occurrence. CONCLUSIONS: Our study is the first report of an association between PON2 311S SNP and the type of coronary atherosclerotic lesions in humans.