MTHFR (C677T) polymorphisms and stage III colon cancer: response to therapy.

BACKGROUND: Leucovorin and 5-fluorouracil (5-FU) chemotherapeutics are often used as coinhibitors of the thymidylate synthase pathway to thwart the growth of cancer cells in certain types of neoplasms. The metabolism of leucovorin is mediated through the enzyme methylenetetrahydrofolate reductase (MTHFR). A common polymorphism in the MTHFR gene has been reported to be responsible for as much as a 70% reduction in activity of this enzyme when present in the homozygous form. METHODS AND RESULTS: A total of 51 stage III colon cancer patients were identified through our tumor registry. Non-neoplastic, archived tissue was obtained for each patient and subjected to MTHFR C677T PCR-RFLP genotyping. The MTHFR C677T allele was present in 32 patients (28 heterozygotes and 4 homozygotes). The remaining 19 patients carried only the wild-type allele. Overall survival was 42.10% (8/19) for wild types and 43.757% (14/32) for those with at least one C677T allele. Of the four homozygotes identified, three have succumbed to their cancer and one is alive with cancer. CONCLUSIONS: We were unable to demonstrate a survival difference between those stage III colon cancer patients receiving leucovorin therapy that carried the MTHFR C677T allele and those that were wild type for this allele. The results of this study suggest that certain subgroups (ie, homozygotes) of patients may benefit from genotypic analysis of the MTHFR gene.

Simultaneous polymerase chain reaction restriction fragment length polymorphism identification of the factor V Leiden allele and the prothrombin 20210A mutation.

A novel mutation in the 3' untranslated region of the prothrombin gene, prothrombin 20210A, recently has been identified. This mutation is associated with increased serum prothrombin levels and an increased risk for venous thromboembolism. Patients who carry a mutation in the factor V gene (factor V Leiden) have also been demonstrated to be at increased risk for venous thromboembolism, and previous studies have identified a population prevalence of approximately 5% to 10% for the factor V Leiden allele. To simply and reliably identify patients who carry both genetic defects, a novel assay was developed that simultaneously determines the genotype of patients for the factor V Leiden allele and the prothrombin 20210A mutation. Representative samples (samples positive and negative for each mutation and a "double mutant") were then subjected to this single-tube genotyping assay. The results indicate that the simultaneous genotyping of these mutations will readily characterize the allelic status of patients for the two most frequent genetic mutations in the coagulation cascade.

The role of preoperative factor V Leiden screening in different geographic populations.

Patients harboring a specific mutation in the coagulation factor V gene have been identified as being at significantly increased risk for venous thrombosis. A simple genetic test that identifies carriers of this mutation (the factor V Leiden allele) is available and may have utility in various clinical settings, including preoperative risk assessment for thromboembolic complications. In this regard, it is generally agreed that prospective studies addressing the role of preoperative factor V Leiden mutational analysis are needed to clearly define the clinical prognostic/diagnostic significance of the presence of this mutation in surgical patients. This report questions the role that population dynamics (genetic and environmental backgrounds of individual populations) plays in the analysis of factor V genotypic data in relation to postsurgical thromboembolic complications. We have determined that the frequency of individuals carrying the factor V Leiden allele is 7.9 per cent for our South Central Pennsylvania population (395 wild type, 32 heterozygotes, 2 homozygotes) using a polymerase chain reaction-restriction fragment length polymorphism technique that specifically detects the factor V Leiden mutation. This baseline population information is useful from both a clinical and a basic science viewpoint. However, considering the various unknown genetic and environmental differences between geographically distinct populations, the significance of this result, in terms of clinical management of our surgical patients, is yet to be determined.

Cleavage of cottontail rabbit papillomavirus E7 RNA with an anti-E7 ribozyme.

We have designed and constructed a plasmid (pE7RZ-1) that contains a gene for a hammerhead ribozyme that specifically cleaves cottontail rabbit papillomavirus (CRPV) E7 RNA sequences in vitro. The in vitro produced transcripts of pE7RZ-1 cleave CRPV E7 target sequences in trans at 37 degrees C. Pretreatment of pE7RZ-1 RNA with poly-L-lysine (PLL) increases the stability of the RNA to greater than 2 hours in the presence of a crude papilloma cell extract and the pE7RZ-1 RNA/PLL mixture retains catalytic activity in this system. This is the first report of a papillomavirus specific ribozyme RNA cleavage and leads the way for the design of effective papillomavirus specific ribozyme based therapies.

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov.

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.

16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.

Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies.

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics.

The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.