Conserved properties of hepatitis C genotype 1b proteinsas prognostic markers of response to pegylated interferon and ribavirin combination therapy

Uvod: Infekcija virusom hepatitisa C (HCV) predstavlja značajan globalni zdravstveni problem koji često vodi ka hroničnoj bolesti jetre i cirozi. Prema podacima Svetske zdravstvene organizacije registrovano je preko 170 miliona ljudi inficiranih virusom HCV. Sadašnja standardna terapija hroničnog HCV-a kod pacijenata inficiranih genotipom 1b, koji predstavlja najčešći genotip kako u SAD, zapadnoj Evropi i Srbiji, se sastoji od pegilovanog interferona u kombinaciji sa ribavirinom. Međutim, kombinovana terapija je praćena brojnim neželjenim efektima i dovodi do stabilnog virusološkog odgovora samo kod 50% pacijenata inficiranih genotipom 1. Stoga bi jednostavan i pouzdan test, koji bi pre početka terapije mogao da predvidi virusološki odgovor, bio od velike koristi u kliničkoj praksi. Metode: Identifikacija konzervirane informacije sadržane u proteinima HCV koja korelira sa odgovorom na standardnu kombinovanu terapiju rađena je bioinformatičkom analizom. Uzorci plazme 48 pacijenata sa hroničnom infekcijom HCV, genotipa 1b su klasifikovani u odnosu na odgovor na kombinovanu terapiju. Za određivanje primarne strukture proteina HCV primenjene su klasične metode molekularne biologije: reverzna transkripcija i lančana reakcija polimeraze (Rt-PCR), apsolutna kvantifikacija sa PCR-om u realnom vremenu i automatsko sekvenciranje. Rezultati: Na osnovu rezultata analize svih proteina virusa HCV utvrđeno je da je informacioni sadržaj proteina p7 u korelаciji sа odgovorom nа kombinovаnu terаpiju. Rezultati dobijeni analizom proteinskih sekvenci, 48 pacijenata sa teritorije Srbije su u saglasnosti sa predloženim bioinformatičkim kriterijumom. Posebna pažnja je posvećena optimizaciji eksperimentalnih protokola i formiranju homogenenih grupa u odnosu na osobenosti virusa (tip i podtip) i odgovora na antivirusnu terapiju pacijenata. Zaključak: Na osnovu rezultata ove studije predložen je bioinformatički kriterijum koji omogućаvа procenu odgovora hroničnih HCV bolesnikа inficirаnih genotipom 1b nа kombinovаnu terаpiju.Background: Hepatitis C virus (HCV) infection is a major and rising global health problem, affecting about 170 million people worldwide, according to WHO data, and often leading to chronic liver disease and cirrhosis. The current standard therapy for chronic HCV infection with pegylated interferon combined with ribavirin in patients with the genotype 1 infection, the most frequent genotype in the USA, Western Europe and Serbia, accompanied by numerous side effects, leads to a successful outcome in only about 50% of individuals. Therefore, simple and accurate prediction of hepatitis C treatment response is of great benefit to patients and clinicians. Methods: Identification of the conserved information of the HCV proteins that correlate with the combination therapy outcome was performed by bioinformatics analysis. Plasma samples of 48 chronic HCV patients from Serbia were classified according to the outcome of therapy. To determine primary structure of HCV proteins classical methods of molecular biology: reverse transcription and polymerase chain reaction (Rt-PCR), the absolute quantification-Real Time PCR and DNA sequencing were applied. Results: Among the HCV proteins that we have analyzed the informational property of the p7 of HCV genotype 1b was best related to the therapy outcome. Findings obtained from analyzing sequences 48 patients collected from Serbia were in perfect agreement with proposed bioinformatics criterion. Special attention was paid to optimization experimental protocols and forming homogeneous groups of patients regarding HCV genotype (type and subtype) and therapy response. Conclusions: On the basis of the results in the present study, a simple bioinformatics criterion that could be useful in assessment of the response of HCV-infected patients to the combination therapy has been proposed

Conserved properties of hepatitis C genotype 1b proteinsas prognostic markers of response to pegylated interferon and ribavirin combination therapy

Uvod: Infekcija virusom hepatitisa C (HCV) predstavlja značajan globalni zdravstveni problem koji često vodi ka hroničnoj bolesti jetre i cirozi. Prema podacima Svetske zdravstvene organizacije registrovano je preko 170 miliona ljudi inficiranih virusom HCV. Sadašnja standardna terapija hroničnog HCV-a kod pacijenata inficiranih genotipom 1b, koji predstavlja najčešći genotip kako u SAD, zapadnoj Evropi i Srbiji, se sastoji od pegilovanog interferona u kombinaciji sa ribavirinom. Međutim, kombinovana terapija je praćena brojnim neželjenim efektima i dovodi do stabilnog virusološkog odgovora samo kod 50% pacijenata inficiranih genotipom 1. Stoga bi jednostavan i pouzdan test, koji bi pre početka terapije mogao da predvidi virusološki odgovor, bio od velike koristi u kliničkoj praksi. Metode: Identifikacija konzervirane informacije sadržane u proteinima HCV koja korelira sa odgovorom na standardnu kombinovanu terapiju rađena je bioinformatičkom analizom. Uzorci plazme 48 pacijenata sa hroničnom infekcijom HCV, genotipa 1b su klasifikovani u odnosu na odgovor na kombinovanu terapiju. Za određivanje primarne strukture proteina HCV primenjene su klasične metode molekularne biologije: reverzna transkripcija i lančana reakcija polimeraze (Rt-PCR), apsolutna kvantifikacija sa PCR-om u realnom vremenu i automatsko sekvenciranje. Rezultati: Na osnovu rezultata analize svih proteina virusa HCV utvrđeno je da je informacioni sadržaj proteina p7 u korelаciji sа odgovorom nа kombinovаnu terаpiju. Rezultati dobijeni analizom proteinskih sekvenci, 48 pacijenata sa teritorije Srbije su u saglasnosti sa predloženim bioinformatičkim kriterijumom. Posebna pažnja je posvećena optimizaciji eksperimentalnih protokola i formiranju homogenenih grupa u odnosu na osobenosti virusa (tip i podtip) i odgovora na antivirusnu terapiju pacijenata. Zaključak: Na osnovu rezultata ove studije predložen je bioinformatički kriterijum koji omogućаvа procenu odgovora hroničnih HCV bolesnikа inficirаnih genotipom 1b nа kombinovаnu terаpiju.Background: Hepatitis C virus (HCV) infection is a major and rising global health problem, affecting about 170 million people worldwide, according to WHO data, and often leading to chronic liver disease and cirrhosis. The current standard therapy for chronic HCV infection with pegylated interferon combined with ribavirin in patients with the genotype 1 infection, the most frequent genotype in the USA, Western Europe and Serbia, accompanied by numerous side effects, leads to a successful outcome in only about 50% of individuals. Therefore, simple and accurate prediction of hepatitis C treatment response is of great benefit to patients and clinicians. Methods: Identification of the conserved information of the HCV proteins that correlate with the combination therapy outcome was performed by bioinformatics analysis. Plasma samples of 48 chronic HCV patients from Serbia were classified according to the outcome of therapy. To determine primary structure of HCV proteins classical methods of molecular biology: reverse transcription and polymerase chain reaction (Rt-PCR), the absolute quantification-Real Time PCR and DNA sequencing were applied. Results: Among the HCV proteins that we have analyzed the informational property of the p7 of HCV genotype 1b was best related to the therapy outcome. Findings obtained from analyzing sequences 48 patients collected from Serbia were in perfect agreement with proposed bioinformatics criterion. Special attention was paid to optimization experimental protocols and forming homogeneous groups of patients regarding HCV genotype (type and subtype) and therapy response. Conclusions: On the basis of the results in the present study, a simple bioinformatics criterion that could be useful in assessment of the response of HCV-infected patients to the combination therapy has been proposed

Genetic Polymorphisms of Neurocardiovascular Disorders

The autonomic nervous control of cardiovascular (CV) system plays a major role in the adaptation of the organism to the changes in external and internal environment. It’s dysfunction is the major pathophysiological factor in the development of neurocardiovascular diseases. The aim of this review is to present the state of the art on the role of candidate gene polymorphisms of the molecules in the signaling chain of neurocardiovascular transmission in neurocardiovascular diseases. Neurocardiovascular disorders can be classified as sympathetic vs. vagally mediated disorders, though in many disorders both systems are dysfunctional. A number of molecules along the signaling pathway can be functionally modified and be the background of the predisposition, faster progression or complicated form of the disease. When the disease is the consequence of the joined parasympathetic and sympathetic nervous system disequilibrium, the focus of neurogenetic research should be on molecules providing the cross-talk between the two systems: on intercellular and intracellular level and on the level of the signaling process integration. An aggregation of positive results for the association between certain genes and different neurocardiovascular phenotypes pointed on a specific "neurocardiovascular genetic hotspots". Identification of these genes could be of particular interest as a diagnostic tool in the clustered form of neurocardiovascular diseases. New data obtained from neurogenetic approach will improve the disease outcome by gene, pharmacologic and behavioral modulation of the autonomic nervous system

RASSF1A and p16 promoter methylation and treatment response in chronic hepatitis C genotype 1b patients treated with pegylated interferon/ribavirin

Prevention of chronic hepatitis C (CHC) and its complications is based on antiviral therapy and early detection of reliable molecular markers in persons under risk. We investigated whether the methylation status of RASSF1A and p16 genes, alone or in combination with host and viral factors, affects the response to therapy with pegylated interferon/ribavirin (PEG-IFN/RBV). Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of the target promoter sequences of RASSF1A and p16 in circulating-free DNA from the peripheral blood of 49 patients with CHC genotype 1b. The methylation status of the examined genes did not affect the response to therapy. However, the simultaneous presence of either RASSF1A or p16 methylation and the CC genotype of IL28B was significantly related to a sustained virologic response (P=0.009 and P=0.032, respectively). After Bonferroni correction, only the result concerning the RASSF1A gene remained significant (P<0.0125). Methylation of RASSF1A was associated with the CC genotype of the IL28B gene (P=0.024) and a higher viral load (≥400 000 IU/mL, P=0.009). Our results suggest that combined analysis of RASSF1A gene methylation and IL28B rs12979860 polymorphism could potentially help in the prediction of therapy response in CHC genotype 1b patients

Micro Rna-21 Expression Levels in Invasive Breast Carcinoma with a Non-Invasive Component

Invasive ductal carcinomas with a non-invasive component (IDC-DCIS) are classified as a group of invasive breast carcinomas, together with pure invasive ductal carcinomas of the breast (IDC). MicroRNA-21 (miR-21) has been characterized as a factor of breast cancer invasiveness, however the difference in miR-21 expression levels between IDC-DCIS and pure IDC tumors and the correlations with standard diagnostic and prognostic parameters inside the IDC-DCIS group are unknown. Our aim was to determine if miR-21 had the ability to distinguish these two invasive breast cancer groups. Levels of miR-21 expression were measured by a stem-loop quantitative Real-Time PCR (RT-qPCR) method. Expression levels of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her-2) and proliferative index Ki-67 were evaluated by immunohistochemistry. IDC-DCIS tumors had significantly lower levels of miR-21 expression in grade 2 (P=0.003, Mann-Whitney U test), ER positive (P=0.025, Mann-Whitney U test) and PR positive tumors (P=0.024, Mann-Whitney U test) than pure IDCs. miR-21 levels showed a different pattern of expression in IDC-DCIS compared to IDC tumors, which is based on the difference in miR-21 expression between Her-2 negative and Her-2 positive IDC-DCIS tumors (P=0.030, Mann-Whitney U test) and high negative correlation of miR-21 levels with PR levels (rho=-0.886, P=0.006, Spearman correlation). According to our results, IDC-DCIS breast carcinomas act in a different manner in pure IDC tumors with regard to the relations between miR-21 expression levels and the standard diagnostic and prognostic parameters, such as Her-2 status, ER and PR status and protein levels

Combined analysis of KRAS mutation and p16INK4a and p14ARF methylation status in locally advanced rectal carcinoma treated with preoperative chemoradiotherapy

Paper description:Patient responses to standard treatment of advanced stages of rectal carcinoma are variable, which emphasizes the need to define reliable predictive and prognostic molecular parameters.We propose a model of simultaneous analysis of KRAS gene mutation status and p16INK4a and p14ARF gene promoter methylation status in pre-treatment tumor biopsies.The simultaneous presence of p14ARF methylation and KRAS mutation was associated with more aggressive tumor behavior. The concurrent presence of alterations in all three examined genes was associated with shorter overall survival.Combined analysis of examined gene alterations revealed patient subgroups with a distinct pattern of tumor response and disease outcome.Abstract: Current management of locally advanced rectal carcinoma (LARC) involves preoperative chemoradiotherapy (preCRT) before surgery. Despite improved local control rate, the response to preCRT of individual patients is variable and may reflect heterogeneous biological properties among tumors of the same clinical stage. Identifying novel molecular parameters with predictive and/or prognostic value is of great clinical importance for a personalized therapeutic approach. In this study, KRAS mutation status was analyzed by direct sequencing, while methylation-specific polymerase chain reaction (MSP) was used to examine p16INK4a and p14ARF gene methylation status in pretreatment tumor biopsies of 60 patients with LARC. The examined molecular changes of KRAS, p16INK4a and p14ARF genes were mutually independent (p16INK4a/KRAS, P=0.272; p14ARF/KRAS, P=0.923; p16INK4a/p14ARF, P=0.715). However, the simultaneous presence of p14ARF methylation and KRAS mutation was associated with a more frequent appearance of local recurrences and distant metastasis (P=0.027). Moreover, patients with the simultaneous presence of p16INK4a and p14ARF methylation and KRAS mutation had significantly shorter overall survival (P=0.011). The obtained results strongly suggest that combined analyses of examined genetic and epigenetic molecular alterations could contribute to the identification of LARC patient subgroups with more aggressive tumor behavior and worse disease outcome

Response Factors to Pegylated Interferon-Alfa/Ribavirin Treatment in Chronic Hepatitis C Patients Genotype 1b

Hepatitis C virus infection is the most common chronic blood-borne infection and one of the most important causes of chronic liver disease. Knowing the predictors associated with pegylated interferon/ribavirin (PEG-IFN/RBV) combination therapy response is important for evidence-based treatment recommendations. The goal of this study was to identify host and viral factors of response to PEG-IFN/RBV treatment in chronic hepatitis C genotype 1b patients. We have examined the relationship between gender, age, level of alanine aminotransferase (ALT), viral load and liver fibrosis progression on therapy response. ALT level and viral load were evaluated before starting treatment with combination therapy. The elevated levels of ALT and route of HCV transmission were found to be significantly associated with the response to therapy in HCV-infected patients. Our findings may be useful for estimating a patients likelihood Of achieving sustained viral response

Methylation status of p16 and p14 genes in locally advanced rectal cancer: Potential clinical implication

Methylation of p16 and p14 genes is a common event in colorectal cancers; however, their exact role in the prediction of patients' outcome is unclear. We conducted this retrospective study to evaluate their potential predictive and/or prognostic roles. Methylation-specific PCR was used to examine the methylation status of p16 and p14 in pretherapeutic and preoperative biopsy specimens of 60 patients with locally advanced rectal cancer. The methylation status of the examined genes did not affect the response to preoperative chemoradiotherapy (CRT), recurrence rate and overall survival. However, patients with a simultaneous presence of either p16 or p14 methylation and high vascular endothelial growth factor (VEGF) expression showed a significantly worse response to CRT (p=0.005 and p=0.038, respectively). Moreover, patients with both p16 methylation and high VEGF expression had significantly shorter overall survival (p=0.010), while no such association was found in patients with p14 methylation and high VEGF expression. On the other hand, a subgroup of patients with p16 methylation and low VEGF and high epidermal growth factor receptor (EGFR) expression showed a significantly better response to CRT (p=0.024). The obtained results point to the importance of p16 and p14 methylation analyses in combination with VEGF and EGFR expression, aimed at better predicting treatment response and patient outcome. © 2018 by the Serbian Biological Society