Our results indicate that HPV infection may play a limited role in ovarian carcinogenesis.
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the intracellular folate metabolism which plays an important role in carcinogenesis through DNA methylation and nucleotide synthesis. The common MTHFR single nucleotide polymorphism C677T has been reported to be associated with reduced enzymatic activity. In order to investigate the influence of this polymorphism on the risk of chronic myeloid leukemia (CML), we performed a case-control study in a Serbian population of 52 patients with CML and 53 healthy control subjects. MTHFR C677T polymorphism genotyping was assessed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The results demonstrated no statistical difference in MTHFR 677 frequency distribution between patient and control groups. Our findings suggest that MTHFR 677 gene variants have no significant influence on the susceptibility to CML in a Serbian population.
The widespread use of gene expression analyses has been limited by the lack of critical evaluations of the methods used to extract nucleic acids from human tissues. For evaluating gene expression patterns in whole blood or leukocytes, the method of RNA isolation needs to be considered as a critical variable in the design of the experiments. Quantitative real-time PCR (qPCR) is widely used for the quantification of gene expression in today's clinical practice. Blood samples as a preferred RNA source for qPCR should be carefully handled and prepared in order not to inhibit gene expression analyses. The present study was designed to compare the frequently employed guanidine thiocyanate-phenol-chloroform-based method (TRI Reagent ® ) with two alternative RNA isolation methods (6100 PrepStation and QIAamp ® ) from whole blood or leukocytes for the purpose of gene expression analysis in chronic myeloid leukemia (CML) patients. Based on the results of this study, for the best combination of yield and RNA extraction purity, taking into account the necessary amount of the clinical sample and performance time, the protocol using phenol-based TRI Reagent ® for RNA extraction from leukocytes is suggested as the most suitable protocol for this specific gene expression analysis.
Zahvaljujem se dr Radmili Janković, NS, šefu Laboratorije za molekularnu genetiku na konstantnom trudu i pomoći kako pri uspostavljanju korišćene naučne metodologije, tako i pri poteškoćama u njenoj realizaciji u kliničkoj praksi. Takođe, zahvaljujem se i na stručnoj pomoći pri izradi i pisanju ovog rada.Posebnu zahvalnost dugujem svojoj koleginici dr Emini Mališić, NS, na pomoći koju mi je pružila u prvim koracima mog eksperimentalnog i naučnog rada. Zahvaljujem joj se i na neizmernom strpljenju i podršci, korisnim savetima i sugestijama i uvek temeljnim, kritičkim analizama kako u svakodnevnom radu, tako i tokom izrade i pisanja doktorske disertacije. Rezultati: Tačkaste mutacije u kodonima 12 i 13 KRAS gena su bile prisutne u 35,1%analiziranih pacijenata sa CRC-om. Distribucija detektovanih mutacija je bila sledeća:p.G12D 42,4%, p.G12V 21,2%, p.G12A 10,6%, p.G12C 7,6%, p.G12S 6,1%, p.G12R 1,5% i p.G13D 10,6 %. U grupi pacijenata sa wild-type (wt) KRAS statusom gena analiziranih na prisustvo p.V600E BRAF mutacije (n=101), samo jedan pacijent je bio heterozigot za datu mutaciju, dok su ostali bili wt.Od testiranih kliničko-histolopatoloških osobina tumora i osobina bolesnika, KRAS mutacije su se statistički značajno češće javljale kod muškaraca nego kod žena, kao i kod pacijenata sa lošijim performans statusom u odnosu na one sa boljim. Analizirajući povezanost pojedinih KRAS mutacija sa ispitivanim parametrima, zapazili smo statistički višu zastupljenost p.G12V mutacije kod tumora koji ne prodiru u dublje slojeve zida kolona i rektuma, u odnosu na tumore koji ih zahvataju ili probijaju serozu.Iako nije pokazana statistička značajnost, učestalost KRAS mutacija raste sa porastom veličine tumora, prisustvom i brojem metastaza, i oko dva puta je veća kod bolesnika sa raširenom u odnosu na lokalizovanu bolest. Povezanost BRAF mutacije p.V600E sa pomenutim parametrima nismo mogli da ispitamo jer je samo jedan bolesnik bio nosilac ove mutacije.U podgrupi od 49 bolesnika koji nisu imali inicijalne metastaze i koji su primili adjuvantnu terapiju, nije postojala statistički značajna razlika u vremenu do pojave metastaza u odnosu na KRAS mutacioni status. Ipak, može se zapaziti trend boljeg preživljavanja bez metastaza kod pacijenata sa mutacijama KRAS-a (medijana 19,5 meseci) u odnosu na wt KRAS grupu (medijana 13,2 meseci).U grupi pacijenata sa wt KRAS genom koji su primali kombinovanu EGFR-ciljanu terapiju (n=72), 15,3% je postiglo terapijski odgovor.Zaključak: Na osnovu dobijenih rezultata značajna povezanost mutacionog statusa KRAS gena sa kliničko-histopatološkim karakteristikama CRC-a i karakteristikama obolelih nije utvrđena. Zastupljenost i distribucija mutacija u ovom genu su u skladu sa rezultatima drugih studija u svetu. Prediktivni značaj KRAS gena u odgovoru na adjuvantnu terapiju nije pokazan, ali je zapažen trend boljeg preživljavanja bez metastaza kod nosilaca KRAS mutacija. Zbog malog procenta BRAF p.V600E mutacije u našoj grupi nismo mogli da proverimo njen značaj kao biomarkera ispitivanog maligniteta...
e12020 Background: Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) are important enzymes of the folate metabolism and are suggested as new prognostic factors for lung cancer. Cytosine to thymine transition at nucleotide 677 (C677T) leads to reduced MTHFR activity. The TS promoter has a tandem repeat polymorphism (2R, 3R) and a guanine to cytosine transition in the 3R allele related to TS protein expression. The aim of this study was to analyze the association of MTHFR and TS polymorphisms with lung adenocarcinoma in Serbia. Methods: A case-control study including 55 late-stage lung adenocarcinoma patients and 53 healthy subjects was performed. Restriction fragment length polymorphism analysis was used for MTHFR and TS genotyping. Depending on the presence of high (3RG) or low (2R, 3RC) expression alleles, TS functional groups were subclassified into HH (3RG/3RG), HL (2R/3RG, 3RG/3RC) and LL (2R/2R, 2R/3RC, 3RC/3RC) groups. Descriptive analyses included genotype and allelic frequencies; the odds ratio (OR) and 95 % confidence interval (CI) were calculated as an estimate of relative risk. Significance was considered for p < 0.05. Results: The distribution of the MTHFR variants in patients vs. controls was 61.8 % vs. 24.5 % for CC, 32.7 % vs. 62.3 % for CT and 5.5 % vs. 13.2 % for TT. A significant difference in CC vs. TT+CT MTHFR genotype distribution was observed between patients and controls (χ2 = 13.79; OR = 4.98; 95 % CI, 2.14 – 11.61). There was no significant association between the TS polymorphisms and the risk of lung adenocarcinoma occurrence, but in the CT+TT MTHFR subgroup, a nonsignificant difference in LL vs. HL+HH TS genotype distribution was observed between patients and controls (χ2 = 0.04; OR = 1.07; 95% CI, 0.31 – 3.67). Conclusions: A significant corelation between the CC MTHFR genotype and lung adenocarcinoma occurence in Serbia was found. Also, there was no significant correlation with lung adenocarcinoma occurence in the CT+TT MTHFR subgroup for carriers of the LL TS genotype. As this study was performed on a relatively small sample size further large case-control studies including analysis of gene-gene interactions with genes coding for other folate metabolism enzymes is needed.
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