Volume 42, Issue 1-2

Next-generation sequencing (NGS) technologies in clinical diagnostics open vast opportunities through the ability to sequence all genes simultaneously at a cost and speed that is superior to traditional sequencing approaches. On the other hand, the practical implementation of NGS in routine diagnostics involves a variety of challenges, which need to be overcome. Among these are the generation, analysis and storage of large amounts of data, strict control of sequencing performance, validation of results, interpretation of detected variants and reporting. Here, we outline the Multiple Integration and Data Annotation Study, an approach for data integration in clinical diagnostics based on genotype-phenotype correlations. MIDAS aims to accelerate NGS data analysis and to enhance the validity of the results by computer-based variant prioritization using the clinical data of the patient. In this context, we present the MIDAS case reports of one patient with intellectual disability caused by a novel de novo loss-of-function variant in the GATAD2B gene [NM_020699.3: c.1426G>T (p.Glu476*)] identified by trio whole-exome sequencing, as well as two cardiac disease patients with severe phenotype and multiple variants in genes linked to cardiac arrhythmogenic disorders analyzed with multi-gene panel sequencing. Based on the data collected in the MIDAS cohort, the MIDAS software will be tested and optimized. Moreover, the MIDAS software concept can be extended modularly to include further data resources for improved data handling and interpretation in the broad field of diagnostics.

Background: Exosomes are small vesicles of sizes between 40 and 100 nm. They are actively segregated by numerous different cell types and they can be found in almost all body fluids. Thus, there is an emerging role of exosomes and exosomal deoxyribonucleic acid (exoDNA) in biomedical research, especially in molecular medicine. Exosomes are assembled and segregated actively and carry distinct surface markers for cellular communication. They are loaded with cargo such as DNA, ribonucleic acid (RNA) and proteins. As there are numerous different exosomal purification methods available, it is of essential need to select an appropriate technique to get reliable results. As neuropathology is faced with the challenge that brain tissue is not accessible in an easy fashion, exosomes represent an ideal tool for molecular neuropathology. Thus, disease-specific molecular alterations will be detectable in a minimally invasive way for early disease diagnosis and surveillance. Summary: The analysis of exoDNA as biomarkers in neuropathology will enable early diagnosis, monitoring and relapse detection of brain tumors and neuropsychiatric disorders. Outlook: It is assumed that the significance of exosomes will increase in the upcoming years. There are powerful approaches in development using exosomes in molecularly targeted therapy to ultimately cure devastating brain diseases.

Background: The metabolism of cholesteryl esters (CEs) is under the control of a gene network in macrophages. Several genes such as ATF3 and EGR2 are related to cholesterol metabolism. Methods: In this study, the ATF3 and EGR2 gene expression levels were evaluated in differentiated macrophages of subjects undergoing coronary artery angiography [controls (<5% stenosis), patients (>70% stenosis)] after treatment with small dense low density lipoprotein (sdLDL) particles. Monocytes were isolated using a RosetteSep Kit and were differentiated into macrophages using the M-CSF factor. A modified heparin-MgSO 4 -PEG method was used for the sdLDL preparation. The ATF3 and EGR2 gene expression levels were measured by the real-time quantitative polymerase chain reaction (RT-qPCR) technique. Results: In contrast with the control group (p=0.4), the ATF3 expression level reduced significantly in the differentiated macrophages from all patients [single vessel disease (SVD), fold change 17 times, p=0.02; two vessel disease (2VD), fold change 1.5 times, p=0.05; three vessel disease (3VD), fold change 3.5 times, p=0.04]. Also, the EGR2 expression level reduced significantly in all groups (p<0.02). The gene fold changes had no significant differences between the patients (p>0.8). Conclusions: We propose that the failure of ATF3 gene expression improves the CE synthesis after sdLDL influx. Furthermore, the reduced EGR2 gene expression level in the sdLDL-treated groups may be a negative factor in cholesterol homeostasis.

Background: Oxidative stress has been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Plasma thiols are major defense mechanisms against oxidative stress and undergo oxidation to form disulfides under oxidative conditions. This study was conducted to investigate thiol-disulfide homeostasis in NAFLD patients. Methods: Thirty patients with biopsy proven non-alcoholic steatohepatitis (NASH), 40 patients with simple steatosis and 50 healthy controls were included in the study. Serum total and native thiol concentrations and serum disulfide concentration were measured using the Erel and Neselioglu’s method. Results: The mean serum total thiol concentrations in the NASH, simple steatosis and control groups were 415±64 μmol/L, 447±38 μmol/L and 480±37 μmol/L, respectively (p<0.001). The mean serum native thiol concentrations in the NASH, simple steatosis and control groups were 378±62 μmol/L, 416±41 μmol/L and 451±36 μmol/L, respectively (p<0.001). The mean serum disulfide concentrations in the NASH, simple steatosis and control groups were 18.5±6.3 μmol/L, 15.5±4.8 μmol/L and 14.9±3.6 μmol/L, respectively (p=0.005). The native thiol/total thiol ratio was significantly lower and the disulfide/total thiol and disulfide/native thiol ratios were significantly higher in the NASH group than in the simple steatosis and control groups. Conclusions: Thiol-disulfide homeostasis is disturbed and shifted toward disulfide side in NAFLD and NASH patients.

Background: Ghrelin is a 28-amino acid peptide hormone which is produced in various tissues such as the kidney. It is proposed that this hormone exerts a broad spectrum of biological functions throughout the body. Ghrelin carries out endocrine and/or paracrine functions in the kidney, which seems to be one of the target tissues of this hormone. Results regarding circulating ghrelin levels in chronic kidney disease (CKD) and diabetic nephropathy (DN) patients are conflicting. We aimed to investigate the plasma ghrelin levels in type 2 diabetic patients with and without nephropathy. Methods: A total of 45 patients with DN and 45 patients with diabetes without diabetic nephropathy (NDN) were recruited for this study. Plasma ghrelin levels were determined using the enzyme-linked immunosorbent assay (ELISA) method. The association of plasma ghrelin with concentrations of fasting glucose (FBS), creatinine (Cr), blood urea nitrogen (BUN), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides (TG) and anthropometric parameters was analyzed. Results: Plasma ghrelin concentration in the DN group was 1.16 (1.01–1.38) ng/mL and in the NDN group was 1.16 (0.92–1.41) ng/mL, so there were no significant differences between the two groups (p=0.467). In the NDN group, ghrelin showed an inverse correlation with TG (r=−0.467, p=0.001) and a direct correlation with HDL (r=0.562, p=0.000) but in the DN group these correlations were not found. Conclusions: Our findings implicated no relationship between the plasma ghrelin level and renal dysfunction in type 2 diabetic patients. Therefore, plasma ghrelin level may not be a probable indicator of kidney insufficiency in patients with type 2 diabetes mellitus (T2DM). Furthermore, we also found a positive correlation between ghrelin and HDL and an inverse correlation with TG levels.

Background: Sepsis is characterized dysregulated inflammatory response and is the leading cause of organ failure or injury and death in critical care units. Ginkgo biloba extract 761 (EGb 761) is characterized by antioxidant activity. There is no clear data in the functional role of EGb 761 that inhibiting of proinflammatory cytokines exist in sepsis. The purpose of this study was to investigate the antiinflammatory effect of EGb 761 on the levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS) induced rat sepsis model. Methods: The animals were randomly divided into the following six groups: the control, LPS, flunixin meglumine (FM), EGb 761, LPS+FM and LPS+EGb 761 groups. Enzyme-linked immunosorbent assay (ELISA) analysis was applied to measure serum levels of VEGF, MMP-9, and iNOS. Results: Serum iNOs (p<0.05), VEGF (p<0.01) and MMP-9 (p<0.05) levels were significantly higher in the sepsis group. Treatment with EGb 761 decreased serum iNOs levels (p<0.01), VEGF and MMP-9 levels decreased significantly (p<0.05). Also, treatment with FM decreased iNOs (p<0.01), VEGF and MMP-9 levels significantly (p<0.05). Conclusions: It is suggested that sepsis may lead to increase serum iNOs, MMP-9, and VEGF levels and treatment of EGb 761 may have a potential antiinflammatory effect against LPS-induced sepsis.

Background: The role of oxidative stress in female reproduction is an area that needs more investigation. This study aims to assess the oxidative stress levels in follicular fluid (FF) samples of women undergoing in vitro fertilization (IVF) and to relate them to different diagnoses. Methods: A total of 199 woman were enrolled; 37 of them were diagnosed with polycystic ovary syndrome (PCOS), 71 with endometriosis and 41 with tubal factor infertility (TFI) and 50 of the patients were used as a control group. A sample of FF was collected from each patient at an oocyte retrieval day and analyzed for oxidative stress by measuring a class of reactive oxygen metabolites (ROMs) (dROMs test) and also analyzed for the plasma antioxidant test (PAT). Results: The data analyzed in FF were related to different diagnoses. Groups were not significantly different in age and body mass index (BMI), except for the PCOS group. There was a significant difference between dROMs and PAT levels in FF of patients vs. control group. The same finding was seen when the dROM/PAT ratio was used. Conclusions: We conclude that the evaluation of oxidative stress in FF needs more investigation with regard to markers in the follicular microenvironment.