PAI-1 4G/5G repeat is a target in gastric carcinomas with microsatellite instability

Background: The plasminogen activator inhibitor-1 (PAL-1) plays an important role in the pathogenesis of cancer. The 4G/5G promoter polymorphism of PAI-1 is potentially involved in regulating gene transcription. Aims: To explore the role of the PAL-1 4G/5G repeat as target of microsatellite instability (MSI), 50 gastric carcinomas (GCs), characterized for MSI, were screened. Methods: PAI-1 4G/5G was analysed by direct sequencing. Results: Allelic imbalance was observed in 5 of the 50(10%) GCs. Specifically, 2 cases (40%) harboured a G deletion and 3(60%) a G insertion in tumour compared to normal DNA. These five cases were included in the subgroup of 20 GCs (25%) with high level of MSI (MSI-H). A statistically significant association emerged between PAL-1 mutations and MSI-H status (p = 0.0073). The frequency of PAL-1 mutations was also evaluated, together with other known target genes, by analysing MSI-H GCs for mutations at selected coding repeats within genes controlling cell growth, apoptosis and DNA repair. Overall, mutation frequency ranged from 56.3% to 5.3%. Conclusion: The frequency of PAL-1 mutations here reported in MSI-H GCs is, accordingly, comparable with values obtained for real targets. The relatively high incidence of PAL-1 mutations is suggestive of a positive pressure towards selection in MSI-H GCs. (C) 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine) at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine) at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala) of exon 1. (<i>Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733</i>

An AT-rich region in the APC gene may cause misinterpretation of familial adenomatous polyposis molecular screening

Familial adenomatous polyposis (FAP) is an autosomal-dominant condition mainly due to a mutation of the adenomatous polyposis coli (APC) gene. The present study reports evidence of a technical issue occurring during the mutational analysis of APC exon 4. Genetic conventional direct sequence analysis of a repetitive AT-rich region in the splice acceptor site of APC intron 3 could be misinterpreted as a pathogenetic frameshift result. However, this potential bias may be bypassed adopting a method for random mutagenesis of DNA based on the use of a triphosphate nucleoside analogues mixture. Using this method as a second-level analysis, we also demonstrated the nonpathogenic nature of the variant in the poly A trait in APC exon 4 region (c.423-4delA) that do not result in aberrant splicing of APC exons 3-4; conversely, we did not find a previously reported T deletion/insertion polymorphism

Prion Protein Gene M129V Polymorphism and Variability in Age at Migraine Onset

Prion protein, a sialoglycoprotein with neuroprotective properties on oxidative stress damage, has been related with the mechanisms leading to migraine. In the present case‐control study, we investigated the correlation between the common methionine/valine polymorphism at codon 129 within the prion protein gene (PRNP) and migraine. Genotyping of PRNP V129M variant was performed in 384 migraine patients and 185 age‐, sex‐, and race‐ethnicity‐matched healthy controls. The frequencies of the PRNP V129M genotype did not differ significantly between migraineurs and controls. The frequencies of 129VV genotype were significantly higher in patients with earlier age at migraine onset. No correlation was found between PRNP 129 genotype and demographics, and other clinical migraine features. Our data suggest that the PRNP 129VV polymorphism is not a direct migraine risk factor but is significantly associated with an earlier onset of the disease

VEGF-A gene promoter polymorphisms and microvascular complications in patients with essential hypertension

Objectives: We investigated the possible involvement of vascular endothelial growth factor (VEGF-A) gene promoter polymorphisms in essential hypertension (EH). Design and methods: 1225 bp of the VEGF-A gene promoter were screened for polymorphisms using PCR amplification and direct DNA sequence analysis in 62 EH and 62 normotensive (HS) individuals. Circulating VEGF-A levels were determined by immunoassay. Results: -152G/A (p = 0.009) and -116G/A (p = 0.016) polymorphisms were correlated to hypertension (p < 0.05). Median platelet VEGF-A load in EH was 2.10 fg/plt. Patients with microvascular complications (MC) had higher platelet VEGF-A load than those without (p = 0.005). Multivariate analyses showed that -116 A allele was an independent predictor of microalbuminuria (p = 0.014) and increased platelet VEGF-A load (p = 0.009) in EH. Platelet VEGF-A load independently predicted MC (p = 0.049) in addition to -116G/A polymorphism (p = 0.035). Conclusions: Abnormal regulation of VEGF-A due to polymorphism at position -116 might represent a genetic factor for increased VEGF-A production and MC in EH. (C) 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved

Preanalytical Procedures for DNA Studies: The Experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Preanalytical variables, including the anticoagulants and stabilizing agents, time, storage temperature, and methods of DNA extraction applied to blood samples, may affect quality and quantity of isolated nucleic acids for future genomic applications. Considering the large number of collected samples, standard operating procedure optimization for whole blood preservation before DNA extraction is a crucial step in a biological repository. Moreover, the future application of the biological material may not be known subsequent to its extraction. To define standard operating procedures for whole blood preservation before DNA extraction, we aimed to determine whether different combinations of anticoagulants, blood storage temperatures, and time intervals before storage at -80 degrees C might have an impact on quality and quantity of subsequent extracted DNA. After spectrophotometer quantification, the quality and integrity of DNA were assessed by agarose gel electrophoresis, polymerase chain reaction, and real-time polymerase chain reaction methods. We observed that decrease in DNA recovery during blood storage time was more pronounced at room temperature than at 4 degrees C. Based on our experience, we recommend as anticoagulants of choice sodium citrate and ethylenediaminetetraacetate, whereas sodium citrate theophylline adenosine dipyridamole could represent an alternative choice, excluding a priori lithium heparin and Fluoride-Oxalate. Based on the overall evaluation criteria, we conclude that the procedures necessary to preserve the whole blood before the DNA extraction may have a significant impact on downstream molecular biological applications

Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).
Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications

TNFA Gene Promoter Polymorphisms and Susceptibility to Recurrent Pregnancy Loss in Italian Women

The aim of this study was to investigate the relationship between serum tumor necrosis factor alpha (TNF-alpha) levels and single nucleotide polymorphisms (SNPs) of the TNFA gene promoter (-376G/A, -308G/A, and -238G/A) in 100 Italian Caucasian women with reproductive failure and 100 fertile controls. Molecular analysis of TNFA SNPs showed higher frequencies of -238G allele (P = .028) as well as the presence of a 3-loci haplotype (-376G/-308A/-238G; P = .020) in fertile controls compared to women with reproductive failure. Serum TNF-alpha levels were higher in study women compared to controls (P = .001). Of interest, the TNFA -376G/-308A/-238G haplotype was an independent predictor of low TNF-alpha levels (P = .021) and miscarriage (P = .023) in multivariate analyses. In conclusion, these findings support the concept of an association of TNFA polymorphisms and recurrent pregnancy loss (RPL). In particular, the TNFA -238GG variant and the TNFA -376G/-308A/-238G haplotype might represent protective factors, probably through reduced TNF-alpha production and/or mediated responses