The presence of A5935G, G5949A, G6081A, G6267A, T9540C mutations in MT-CO1 and MT-CO3 genes and other variants of MT-CO1 and MT-CO3 gene fragments in the study population diagnosed with endometrial cancer

Objectives: The specific purpose of this study was the assessment of A5935G, G5949A, G6081A, G6267A mutations in MT-CO1 and T9540C in MT-CO3, and alterations detected during the analysis of MT-CO gene fragments in subject and control groups. A secondary aim was to assess the relationship between MT-CO1 and MT-CO3 gene alterations and endometrial cancer incidence and evaluation of the prognostic value of MT-CO1 and MT-CO3 gene alterations. Material and methods: In this study, we investigated A5935G, G5949A, G6081A, G6267A mutations in MT-CO1 and T9540C in MT-CO3, and alterations detected during the analysis of MT-CO gene fragments in formalin-fixed, paraffin-embedded endometrial and benign endometrial hyperplasia of a cohort of 125 subjects. Results: The T9540C mutation in MT-CO3 was detected in one patient from the subject group. None of the remaining muta­tions were detected. The research showed that the presence of alterations in MT-CO1 and MT-CO3 typical of other types of cancer is not a risk factor for endometrial cancer. Analysis of MT-CO1 and MT-CO3 gene fragments revealed 10 alterations (6 and 4 respectively). The alterations detected were identified in 10% of the tested group and 8% of the control group. Conclusions: The research showed that the presence of alterations in MT-CO1 (A5935G, G5949A, G6081A, G6267A) typical of other types of cancer is not a risk factor for endometrial cancer. Three new alterations detected in this study (A6052G, A9545G, G9575A) were described for the first time

cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

<p>Abstract</p> <p>Background</p> <p>Recently published data showed discrepancies beteween <it>P53 </it>cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers.</p> <p>Methods</p> <p>To this end, we analyzed 23 colorectal cancers for <it>P53 </it>mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry.</p> <p>Results</p> <p>We found <it>P53 </it>gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of <it>P53 </it>mRNA was present in samples with and without <it>P53 </it>mutations.</p> <p>Conclusion</p> <p>In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated <it>P53 </it>mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without <it>P53 </it>mutation (normal cells and cells showing <it>K-RAS </it>and/or <it>APC </it>but not <it>P53 </it>mutation) in samples presenting <it>P53 </it>mutation; 3, heterozygous or hemizygous mutations of <it>P53 </it>gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in <it>P53 </it>cDNA and DNA sequencing analysis.</p

Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies

Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor—EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor—EGFRvIII

Childhood adversities are not a predictors of SSTR4met in alcoholics

Genome methylation may modulate synaptic plasticity, being a potential background for mental disorder. Adverse childhood experiences (ACEs), known to be frequently reported by patients with alcohol dependence (AD), have been proposed as one of environmental inequities influencing DNA methylation. The study is aiming 1.To assess a promoter region methylation in gene for somatostatin receptor subtype-4 (SSTR4), a receptor for somatostatin, a neurotransmitter engaged in neuroplasticity and memory formation, in patients with AD; 2. To verify if SSTR4 promoter methylation is associated with ACEs and other selected environmental factors. Methodology: 176 patients with AD and 127 healthy controls were interviewed regarding 13 categories of ACEs; a structured self-reported questionnaire - to measure the sociodemographic and clinical characteristics; a module of Catalogue of Healthy Behavior – to assess nutritional health habits; the Alcohol Use Disorders Identification Test – to assess drinking severity. The SSTR4 promoter region methylation status was performed via methylation-specific PCR, and the genotyping for the SSTR4 rs2567608 functional polymorphism - according to the manufacturer’s standard PCR protocol

SSTR4, childhood adversity, self-efficacy and suicide risk in alcoholics

Patients with alcohol dependence (AD) are known to develop poor social skills, to report a higher number of adverse childhood experiences (ACEs) and to attempt suicide more frequently than the general population. The background for the association between ACEs and a higher risk of suicide still remains understudied. SSTR4 rs2567608 is a functional polymorphism of the gene for somatostatin receptor subtype 4, predominantly found in the CA1 hippocampus area and involved in memory formation. We hypothesize that the functional polymorphism SSTR4 rs2567608, general self-efficacy, and adverse childhood experiences influence the risk of suicide attempt in patients with AD

SIRT1-SIRT7 Expression in Patients with Lymphoproliferative Disorders Undergoing Hematopoietic Stem Cell Mobilization

Sirtuins are involved in the fate of hematopoietic stem cells (HSCs), including their metabolism, stress response, differentiation, migration, and apoptosis. The aim of this study was to explore SIRT1-7 expression during HSC mobilization. The study included 50 patients with lymphoproliferative disorders (39 multiple myeloma, 11 lymphoma). Samples were taken before mobilization (day 0) and on the day of first apheresis (day A). The sirtuin expression was evaluated by the Droplet Digital PCR (ddPCR) method. A significant increase of the SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7 levels measured at day A as compared to baseline was observed. The study revealed a positive correlation between SIRT5, SIRT6, and SIRT7 expression and the CD34+ peak value in peripheral blood and the number of CD34+ cells collected on day A. Patients from the SIRT7 “high expressors” group collected more CD34+ cells on day A than “low expressors”. Upregulated expressions of SIRT3 and SIRT7 on the day of first apheresis were observed in patients in complete remission status (CR) as compared to the non-CR group. Our results suggest that the investigated sirtuins may influence the HSC migration and hematopoietic landscape during mobilization. SIRT5, SIRT6, and SIRT7 may be associated with the efficacy of HSC mobilization

Warianty genetyczne w polskiej populacji pacjentów z tętniczym nadciśnieniem płucnym – sekwencjonowanie genów BMPR2, ALK1 i ENG.

Background: Pulmonary arterial hypertension (PAH) is a rare disease with a very serious prognosis. It seems that mutations in genes related to transforming growth factor-b signalling pathway are often related to the development of the disease. No study covers this problem in a Polish population. Aim: To screen for genetic mutations in a Polish cohort of patients with pulmonary hypertension, especially with idiopathic PAH, treated in a single hospital in Poland. Methods: DNA sequencing method was used. Samples from 50 patients with pulmonary hypertension were screened for mutations in type 2 bone morphogenetic protein receptor of the transforming growth factor-b superfamily gene (BMPR2). Samples from 20 patients with idiopathic PAH (11 men, mean age 55 years) were also screened for mutations in activin A receptor-like type 1 gene (ALK1) and endoglin gene (ENG). Results: No genetic variations were found for the BMPR2 gene. In all 20 samples from idiopathic pulmonary hypertension patients we found heterozygosity of single nucleotide polymorphism (SNP) rs 372023206 in ALK1 gene. Three samples from these patients showed variations of ENG gene: we found one sample with heterozygosity of SNP rs 200525684, one with heterozygosity of SNP rs 3739817, and one with both. Conclusions: We detected benign polymorphisms or genetic variants of unknown importance. It is possible that the Polish population of PAH patients differs from the previously described populations of other countries in terms of the frequency and importance of mutations in BMPR2, ALK1 and ENG genes.Wstęp Tętnice nadciśnienie płucne (pulmonary arterial hypertension - PAH) to rzadka, ciężka choroba z bardzo poważnym rokowaniem. Jej patogeneza pozostaje niejasna.Na podstawie dotychczasowych badań wydaje się, że w rozwoju PAH szczególnie ważną rolę odgrywają mutacje genów kodujących białka związane ze szlakiem sygnałowym transformującego czynnika wzrostu beta (TGF-beta). Do tej pory nie przeprowadzano jednak skriningu genetycznego, który mógłby wykazać rozpowszechnienie takich mutacji w polskiej populacji chorych z PAH. CelCelem niniejszej pracy było zbadanie w grupie osób leczonych w pojedynczym referencyjnym ośrodku w Polsce, jaka jest częstość występowania mutacji w trzech genach najczęściej łączonych z patogenezą PAH. MetodyWykorzystano metodę sekwencjonowania DNA z zastosowaniem reakcji łańcuchowej polimerazy. Próbki materiału genetycznego pobrano od pięćdziesięciu chorych z nadciśnieniem płucnym o różnej etiologii. Do badania włączono dwadzieścia osób z idiopatycznym PAH (główna grupa badana), dziesięć osób z zespołem Eisenmengera oraz dwadzieścia z nadciśnieniem płucnym wtórnym do lewokomorowej niewydolności serca. Próbki od wszystkich 50 chorych zbadano pod kątem mutacji z genie BMPR2 kodującym receptor typu 2 dla białka morfogenetycznego kości, należący do nadrodziny TGF-beta. Ponadto, w 20 próbkach z głównej grupy badanej chorych z idiopatycznym PAH (11 mężczyzn, średni wiek 55 lat) przeprowadzono także skrining w kierunku mutacji w genach: aktywinoreceptoropodobnej kinazy typu 1 (ALK1) oraz endogliny (ENG).WynikiW żadnej próbce nie znaleziono mutacji ani wariantów genetycznych BMPR2. W grupie idiopatycznego PAH, w genie ALK1 we wszystkich 20 próbkach stwierdzono heterozygotyczny polimorfizm pojedynczego nukleotydu (Single Nucleotide Polymorphism) SNP rs 372023206. Ponadto w trzech próbkach wykazano warianty genetyczne w obrębie genu ENG: u jednej osoby wystąpił heterozygotyczny polimorfizm SNP rs 200525684; u jednej osoby znaleziono heterozygotyczny polimorfizm SNP rs 3739817; była też jedna osoba, u której stwierdzono występowanie obu polimorfizmów jednocześnie. WnioskiW grupie chorych z idiopatycznym tętniczym nadciśnieniem płucnym wykazaliśmy występowanie łagodnych polimorfizmów lub wariantów genetycznych o nieustalonym znaczeniu klinicznym. Nie wykryto przypadku mutacji chorobotwórczej. Być może w polskiej populacji częstość występowania i przez to znaczenie patogenetyczne mutacji genów BMPR2, ALK1 i ENG są odmienne niż w opisanych dotychczas w literaturze populacjach innych krajów