Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons.

BACKGROUND: Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses. METHODS: We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction. To evaluate this procedure, we examined the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with an increased risk for cardiovascular disease and neural tube defects. DNA was isolated from 10 microL of fresh EDTA-blood samples by use of a robotic workstation. The DNA samples were analyzed using molecular beacons as well as conventional methods. RESULTS: Both methods were compared, and no differences were found between outcomes of genotyping. CONCLUSIONS: The described assay enables robust and automated extraction of DNA and analysis of up to 96 samples (10 microL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high-throughput analyses.

Automated extraction and amplification of DNA from whole blood using a robotic workstation and an integrated thermocycler.

Growing knowledge of the genetic basis of inheritable diseases has resulted in a rapidly increasing demand for DNA mutation analysis. Current methods are reliable and suitable for low-throughput mutation analyses, but are unable to cope with the increasing demand for genetic analyses, necessitating the development of new, fully automated and reliable methods. We developed a semi-automated method for DNA mutation analysis by integrating a thermocycler into a robotic pipetting workstation. DNA was extracted from 84 samples of 10 microl of EDTA-treated whole blood using magnetic beads within 2 h. Directly after isolation, the DNA was automatically transferred to an integrated thermocycler for amplification. Our semi-automated method proved to be reliable and robust, showing unambiguously interpretable PCR signals without occurrence of contamination. It is also faster than conventional manual methods. Only a brief manual intervention is required to remove and refit the seal of the PCR plate. This semi-automated assay is a step forward in the development of fully automated assays for DNA mutation analysis.

Multiplex detection of four pathogenic retroviruses using molecular beacons.

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100, 000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.

Comparative analysis of p53 mutations in bladder washings and histologic specimens.

Mutations in the tumor suppressor gene p53, analyzed in bladder washings, have positive predictive value for the progression of superficial bladder cancer to invasive disease. Bladder washings reflect the general status of the urothelium, and because sampling of bladder washings can be performed as an outpatient procedure, patients can be monitored more carefully. To determine the actual value of bladder washing specimens in assessing the p53 status of histologic specimens, we used the technique of polymerase chain reaction-single-strand conformation polymorphism to analyze bladder washings and the synchronous tumors of 15 patients for the presence of p53 mutations. A significant correlation (2-tailed Fisher's exact test) between the p53 status of bladder washings and histologic specimens was observed if the 2 were compared among the specimens of a single patient. Overall, in 2 patients the mutation present in the tumors was not detected in the bladder washings, and in 1 patient the mutation in the bladder washing was not detected in the histologic specimens. These conflicting results obtained with bladder washings and histologic specimens could be explained mainly by the architecture of the tumors. The observed specificity of 86% and sensitivity of 75% emphasizes that although the correlation between the 2 methods is good, in a number of cases they are complementary to each other. The analysis of p53 mutations in at least 2 bladder washings gives insight into the p53 status of the synchronous tumors.

Molecular beacons: a new approach for semiautomated mutation analysis.

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We designed molecular beacons to detect a point mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, a mutation that has been related to an increased risk for cardiovascular disease and neural tube defects. The application of molecular beacons enables fast, semiautomated, accurate mutation detection. Moreover, the procedure is performed in a closed tube system, thereby avoiding carryover contamination. We believe these probes will find their way into nucleic acid research and diagnostics.

Differential expression of ferritin heavy chain in a rat transitional cell carcinoma progression model.

To identify molecular markers with predictive value for the progression of superficial bladder cancer we used the differential hybridization analysis approach. Since primary tumor material is heterogeneously composed of subpopulations that are poorly characterized, we used in this study a rat progression model system that phenotypically and cytogenetically resembles human superficial bladder cancer. In the differential hybridization analysis we compared the mRNA populations of low and high metastatic tumor lines. We observed an overexpression of ferritin Heavy chain (ferritin H) in the tumor line with the lower metastatic capacity and better differentiated phenotype. The exact clinical relevance for the differential expression of ferritin H in human bladder cancer remains to be determined.

Predictive value of p53 mutations analyzed in bladder washings for progression of high-risk superficial bladder cancer.

To assess the value of p53 mutations in predicting the progression of superficial bladder cancer [transitional cell carcinoma (TCC)] and to define exactly when p53 mutations occur in the process of tumor progression, 80 consecutive bladder washings from 26 high-risk (indicated by quantitative karyometric analysis) superficial TCC patients were examined by single-strand conformation polymorphism. Six of 13 patients who experienced clinical progression (progression to T2 or higher) were found to have a p53 mutation in one or more of their bladder washings. In the control group (no progression to invasive disease), only 1 of 13 patients had a p53 mutation. For these high-risk superficial TCC patients, the occurrence of a p53 mutation has a positive predictive value of 86% for the progression of disease. A negative predictive value of 63% was observed. Moreover, because p53 mutations were found in samples prior to progression (mean, 8 months), they could identify patients who need changes in their treatment strategies to prevent progression to invasive disease. Despite these promising results, it is obvious that to increase not only the positive predictive value but especially the negative predictive value of this procedure to predict progression, additional prognostic markers are still needed.

Comparison of P53 protein overexpression with P53 mutation in bladder cancer: clinical and biologic aspects.

BACKGROUND: Alterations of the tumor suppressor gene p53 are known to occur in bladder cancer. Although p53 overexpression is associated with mutation of the p53 gene, a substantial discrepancy between molecular genetic alteration in p53 and overexpression of the protein has been found. EXPERIMENTAL DESIGN: Tumor specimens of 39 bladder cancer patients were immunohistochemically analyzed for p53 overexpression, and the results were compared with the presence of a mutation as assessed by single strand conformation polymorphism (SSCP) and direct sequencing. Both clinical and biologic aspects were studied. RESULTS: A significant correlation between p53 overexpression and poor survival in the whole group studied was found (p < 0.01). No association between p53 overexpression and decreased survival was found for invasive tumors in contrast with other studies. Differences in treatment of the patients and different Ab and scoring systems used might explain these differences. In our study, the Kaplar-Meier curves showed the same result for p53 overexpression and p53 mutation when the whole group and the invasive tumors were studied. However, in the group of superficial tumors, which was unfortunately too small for statistical analysis, we found p53 overexpression in three tumors, and no p53 mutations were found. A good concordance between p53 mutation and p53 overexpression was found (p < 0.02). However, two out of eight tumors with an SSCP-proven p53 mutation showed no p53 immunoreactivity, probably as a result of loss of the nuclear localization signal. Twenty three percent (7/31) of the tumors showed p53 overexpression without any sign of a mutation. CONCLUSIONS: Our results indicate that, despite good concordance between p53 mutation and p53 overexpression, there is no direct casual relationship between mutation and protein accumulation. Apparently, other events than mutation can trigger p53 stability.

p53 mutations have no additional prognostic value over stage in bladder cancer.

Evidence is accumulating that the tumour-suppressor gene p53 is involved in the development of bladder cancer. Therefore we studied p53 mutations in 47 bladder cancers obtained from 45 patients using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. Eight out of 24 invasive tumours appeared to have a p53 mutation, while no p53 mutations were found in the superficial tumours. All the p53 mutations were found in grade 3 tumours. The tumours with altered p53 showed a higher frequency of allelic loss (FAL) than the tumours without a mutation (55.8% vs 21.1%, P < 0.05, chi 2 test). This increase in FAL suggests a correlation between p53 mutations and genetic instability. A significant correlation between mutated p53 and poor survival in the whole group studied was found (P < 0.001, log-rank test). However, within the group of muscle-invasive tumours the occurrence of p53 mutations had no additional prognostic value. Therefore, even though p53 mutations were found in aggressive tumours, the clinical usefulness of its detection seems limited. Nevertheless, these results imply that p53 is involved in the clinical behaviour of bladder cancer; its role in the progression of superficial cancer to invasive disease merits further attention.

Bacterial and clinical sequelae of the Twin bag system in continuous ambulatory peritoneal dialysis. A single centre study.

Since the introduction of disconnect systems, a marked reduction in continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis has been reported in the literature. At our centre too, a highly significant decline in the peritonitis rate was observed after the introduction of the Twin bag in 1990. In a multivariate analysis which we published recently, the Twin bag system, in conjunction with the more frequent use of the swan neck catheter, correlated significantly (p < 0.001) with an increase in the peritonitis-free interval. In the present study we retrospectively analyzed the bacteriological cultures of the peritonitis episodes, the antibiotic treatment prescribed, and the number of hospitalization days (HDs) before (non-Twin bag group; NTG) and after the introduction of the Twin bag system in our centre (Twin bag group; TG). In terms of absolute numbers, the decreased incidence of peritonitis in the TG was due by and large to a decline in all pathogenic micro-organisms, but mostly to a reduction of coagulase-negative staphylococci (CNS) compared with the NTG. The incidence of culture-negative episodes, however, showed no difference between the two groups. Proportionally, there was a significant increase in culture-negative peritonitis in the TG, whereas infections caused by CNS significantly decreased in comparison with the NTG (p < 0.01). The pattern of the antibiotics prescribed, i.e. mono- versus multi-drug regimens, did not differ between the two groups. Since, of all micro-organisms involved, CNS infections showed the largest decline in absolute numbers. Staphylococcus aureus increased relatively (43%) after the introduction of the Twin bag system.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular prognostic factors in bladder cancer.

In this paper the predictive value of molecular prognostic parameters for bladder cancer is discussed. DNA ploidy has additional prognostic value for grade 2 tumors, irrespective of stage, with aneuploid tumors having a poor prognosis. Overexpression of the epidermal growth factor receptor (EGFR) can be used as a prognostic factor for the group of superficial tumors. Both abnormal E-cadherin and retinoblastoma (RB) expression have additional prognostic value for invasive tumors. The exact predictive value for the superficial tumors needs further study. The results with respect to p53 are conflicting and its exact role especially in the progression of pT1g3 tumors has to be clarified. In view of the discordance concerning its prognostic value, c-erbB-2 overexpression also needs further study. It appears that at this moment only a few molecular markers seem to have potential prognostic value, but their precise clinical relevance has to be studied more extensively. In particular the value of progression markers in the superficial TCC needs more attention.

Long-term results of continuous ambulatory peritoneal dialysis: the first hundred patients. A single centre study.

In the present survey our experience on the first 100 patients on CAPD, treated from the start in 1982 till September 1991, is described. Sixteen were diabetics. Both the absolute numbers and the proportion of the total dialysis population have increased almost every year. Mean age did not change over the years due to an equilibrium between younger patients who received a transplant and elderly who stopped CAPD for other reasons. Patient survival at 3 years was 68%. Seventy patients stopped CAPD, of whom 25 died and 16 switched to haemodialysis. Twenty-four patients received a transplant, patient and transplant survival at 3 years being 89% and 77% respectively. Fifteen patients have had a follow-up period of 3 years or more, the longest being 123 months currently. Seventy-three CAPD-related complications occurred, the majority catheter-related. After the introduction of a 'break-in' period a significant reduction in leakage alongside the catheter was observed. In recent years there was a dramatic decrease in the incidence of CAPD-related peritonitis, from once every 8 to once every 30 months, which could be attributed mainly to the introduction of a new disconnect system in our centre, the so-called 'Twinbag'.

Major reduction of CAPD peritonitis after the introduction of the twin-bag system.

In recent years an impressive decrease in the incidence of CAPD-related peritonitis was observed in our centre, from 1.4 in the mid-eighties to 0.4 per patient year in 1991. In order to analyse which factors were most responsible for this decline, the present study was performed. From the start of our CAPD programme in 1982 until September 1991, 100 patients were enrolled. For each patient, time elapsed from catheter insertion until first peritonitis episode was recorded. Outcome was measured as the peritonitis-free interval in days. The following variables have been evaluated: age, gender, type of catheter, type of system, presence of diabetes mellitus, leakage, break-in period, presence of an exit-site infection, and performing surgeon. Data were analysed first by Kaplan-Meier product-limit estimate of survival (peritonitis-free interval). Thereafter Cox proportional hazard analysis was applied to the data, providing a conditional probability of peritonitis at each moment during follow-up, given a certain combination of risk factors. Our results show that the system, in conjunction with the type of catheter, was a decisive factor in the decline of the peritonitis rate in our centre. Patients on the twin-bag system (twin-bag group) showed a significant increase in the peritonitis-free interval in comparison with patients using other systems (non-twin bag group). Among the other variables analysed, only diabetes mellitus appeared to be relatively important. Episodes of culture negative peritonitis were more frequently observed in the twin-bag group, compared to the non-twin bag group. In absolute numbers Staph. non-aureus was the micro-organism most effectively reduced.