Locked nucleic acid-enhanced detection of 1100delC*CHEK2 germ-line mutation in Spanish patients with hematologic malignancies.
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Adolescent↗
Biomedical subjects
Olfert Landt
Publications and source records attributed to Olfert Landt.
Real-time PCR to detect minimal residual disease in childhood ALL.
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Child↗
Prevalence and clinical impact of Met Y1253D-activating point mutation in radiotherapy-treated squamous cell cancer of the oropharynx.
Aberrant signalling through the hepatocyte growth factor/scatter factor receptor Met has been implicated in various aspects of the development of human cancer including the promotion of tumour invasion, angiogenesis and metastasis. Moreover, experimental data indicate that activation of the Met receptor may be involved in cellular resistance towards antineoplastic treatments such as chemotherapy and ionizing radiation. We determined the prevalence and clinical impact of the Met-activating mutation Y1253D in patients with squamous cell cancer of the oropharynx treated by radical radiotherapy. To screen archival tissue for the presence of a low-abundance point mutation, we developed a sensitive screening method using real-time polymerase chain reaction along with peptide nucleic acid-based DNA clamping and melting curve analysis. By this approach, Met Y1253D was detected in tumours of 15 out of 138 patients (10.9%). Both univariate and multivariate survival analysis revealed Met Y1253D to be significantly associated with impaired local tumour control. Our results provide evidence that the Met-activating mutation Y1253D is present in a notable subset of patients with oropharyngeal cancer and indicate that it may interfere with radioresponsiveness of these tumours, supporting the notion of aberrant Met signalling as a potential target for radiosensitization.
Amino Acid Substitution↗
Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes.
The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.
Animals↗
Elevated telomerase activity, c-MYC-, and hTERT mRNA expression: association with tumour progression in malignant lipomatous tumours.
The role of telomerase activity in tumour progression of liposarcomas is not well understood. Therefore, we investigated 72 liposarcomas of different histological subtypes for an association between telomeric lengths and telomerase activity, and assessed the association between the catalytic subunit human telomerase reverse transcriptase (hTERT) and its activator c-MYC. Telomeric repeat fragment lengths were determined using radioactive DNA-fingerprint analysis with the telomere-specific probe (TTAGGG)(3), whereas telomerase activity was ascertained using the non-radioactive TRAP-assay. To evaluate the expression of hTERT and c-MYC, we applied real-time RT-PCR using a LightCycler. Eight tumours were investigated by microdissection. The MIB1-proliferation index and hTERT and c-MYC protein expression were determined immunohistochemically. Genetic alterations showed a high degree of tumour specificity. Highly malignant myxoid/round cell liposarcomas showed the longest telomeres, the strongest telomerase activity, and the highest hTERT and c-MYC expression levels compared with the pure myxoid variants (p < 0.001), which are of low malignancy. Pleomorphic liposarcoma was characterized by zero or low hTERT and c-MYC expression and telomerase activity, but long telomeres, underlining their different pathogenetic pathway. Elevated gene expression was accompanied by protein immunopositivity. MIB1-proliferation index did not correlate with other molecular markers. We conclude that hTERT and c-MYC expression are associated with telomerase activity in liposarcomas. Elevated hTERT and c-MYC expression as well as high telomerase activity play a role in the tumour progression of this sarcoma type. Nevertheless, each histological subtype of liposarcomas is defined by a specific molecular pattern. Telomerase activation is the most common pathway in liposarcomas maintaining telomeric length.
Biomarkers, Tumor↗
One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes.
The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.
Adolescent↗
Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run.
BACKGROUND: Human herpes viruses cause a spectrum of diseases that are usually self-limiting but can be reactive during immuno-suppression and may then lead to severe or even life-threatening diseases. The LightCycler technology allows rapid polymerase chain reaction (PCR) including product analysis within a closed system. This approach has been demonstrated to be suitable for routine diagnostic virus detection. Several LightCycler PCR assays have been established to the detection of human herpes viruses. The assays vary in their detection formats and PCR cycling protocols. So, they cannot be performed within a single LightCycler run. OBJECTIVES: Development of four LightCycler PCR assays for parallel detection of DNA derived from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) in a single run. STUDY DESIGN: Primers and hybridization probes were tailored to suit one LightCycler PCR program. LightCycler PCRs were established, detection limits were determined, and clinical samples were evaluated. RESULTS: With quantified herpes virus type specific DNA spiked into cerebrospinal fluid, serum or EDTA plasma the detection limits were found either at 500 or 250 viral DNA copies per ml depending on the virus DNA specific PCR and on the specimen type used. The applicability of the new LightCycler assays for routine molecular testing was evaluated by testing 96 clinical samples. CONCLUSION: The developed set of LightCycler PCRs permits parallel detection of CMV, EBV, HSV-1, HSV-2, and VZV in a single LightCycler run. The new molecular assays can easily be used to the rapid, simple, and convenient detection of herpes virus DNA in cerebrospinal fluid, serum and EDTA plasma in the routine diagnostic laboratory.
Anticoagulants↗
Rapid genotyping of melanocortin-1 receptor with use of fluorescence-labeled oligonucleotides.
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Fluorescent Dyes↗
Evidence that paternal expression of the epsilon-sarcoglycan gene accounts for reduced penetrance in myoclonus-dystonia.
Myoclonus-dystonia (M-D) is a movement disorder characterized by rapid muscle contractions and sustained twisting and repetitive movements and has recently been associated with mutations in the epsilon-sarcoglycan gene (SGCE). The mode of inheritance is autosomal dominant with reduced penetrance upon maternal transmission, suggesting a putative maternal imprinting mechanism. We present an apparently sporadic M-D case and two patients from an M-D family with seemingly autosomal recessive inheritance. In both families, we detected an SGCE mutation that was inherited from the patients' clinically unaffected fathers in an autosomal dominant fashion. Whereas, in the first family, RNA expression studies revealed expression of only the mutated allele in affected individuals and expression of the normal allele exclusively in unaffected mutation carriers, the affected individual of the second family expressed both alleles. In addition, we identified differentially methylated regions in the promoter region of the SGCE gene as a characteristic feature of imprinted genes. Using a rare polymorphism in the promoter region in a family unaffected with M-D as a marker, we demonstrated methylation of the maternal allele, in keeping with maternal imprinting of the SGCE gene. Loss of imprinting in the patient with M-D who had biallelic expression of the SGCE gene was associated with partial loss of methylation at several CpG dinucleotides.
Alleles↗
Association between head and neck cancer and microsomal epoxide hydrolase genotypes.
Tobacco-associated carcinogens are catalyzed by microsomal epoxide hydrolase (mEH). Combinations of the Y113H and H139R polymorphic EPHX1 variants have been assumed to alter the enzyme activity and thus the risk of squamous cell head and neck cancer (SCCHN). Based on in vitro data, a putative low, medium and high mEH activity has been associated with combinations of these genotypes, and the respective activity categories have been frequently used in the estimation of risks for smoking-related cancers. We investigated the SCCHN risk for EPHX1 genotypes among 280 cases and 289 controls. We could not detect main effects of the EPHX1 genotypes, but a smaller risk of the 139HR genotype in smokers (odds ratio, OR, 0.57; 95% confidence interval, CI, 0.34-0.95). We could not confirm an increase of the SCCHN risk for genotype combinations according to a putative medium and high enzyme activity (OR 1.28, 95% CI 0.84-1.96; OR 0.98, 95% CI 0.58-1.64, respectively), but a significant heterogeneity of the estimated risks for the singular genotypes within these categories among smokers ( P=0.02). Further, p53 mutations among smoking cases were less frequent in the group with a putative high enzyme activity, although insignificant due to small numbers (OR 0.54, 95% CI 0.13-2.17). This supports uncertainties in categorizing genotypes with respect to limited enzyme activity data, especially when taken from in vitro experiments.
Carcinoma, Squamous Cell↗
Tropical calcific pancreatitis: strong association with SPINK1 trypsin inhibitor mutations.
BACKGROUND & AIMS: Tropical calcific pancreatitis (TCP) is a chronic pancreatitis unique to developing countries in tropical regions. The cause of TCP is obscure. Whereas environmental factors, such as protein energy malnutrition and ingestion of cassava, have been implicated, a genetic predisposition to the disease also may be important. In the present study we report on mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene in north Indian patients with TCP. METHODS: We studied 66 unrelated TCP patients (44 men, 49 with diabetes, and 6 with family history of TCP), 25 relatives, and 92 healthy control subjects. Samples were analyzed for SPINK1 variants (-53C>T, L14P, N34S, P55S, and 272T>C) and cationic trypsinogen (PRSS1) variants (A16V, K23R, N29I, and R122H) by melting curve analysis. RESULTS: Twenty-nine patients (44%) carried the N34S missense mutation, of whom 9 (14%) were homozygotes. In contrast, only 2 (2.2%) control subjects were N34S heterozygotes (prevalence ratio 20.2; 95% confidence interval 5.0-81.8; P < 0.0001 vs. TCP). The severity of pancreatitis did not differ between TCP patients with or without N34S, or among those heterozygous or homozygous for N34S. Among TCP patients with or without diabetes, the frequency of N34S carriers (43% vs. 47%) and N34S homozygotes (14% vs. 12%) was similar. CONCLUSIONS: TCP is highly associated with the SPINK1 N34S mutation. The high prevalence of N34S in TCP patients with and without diabetes suggests that these 2 subtypes have a similar genetic predisposition. The genetic predisposition to TCP resembles, at least in part, the idiopathic chronic pancreatitis found in industrialized countries.
Adult↗
Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis.
A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.
Computer Systems↗
Differentiation between Mycobacterium farcinogenes and Mycobacterium senegalense strains based on 16S-23S ribosomal DNA internal transcribed spacer sequences.
16S ribosomal DNA (rDNA) and 16S-23S internal transcribed spacer rDNA sequence analyses were performed on Mycobacterium farcinogenes and M. senegalense strains and 26 strains of other rapidly growing mycobacteria to investigate the phylogenetic structure of bovine farcy mycobacteria within the M. fortuitum complex. M. farcinogenes and M. senegalense were indistinguishable in their 5"-end 16S rDNA but showed both considerable interspecies spacer sequence divergence and a high level of intraspecies sequence stability. A rapid detection assay using PCR and hybridization with species-specific probes was developed. The assay was specific among 46 species other than M. farcinogenes and M. senegalense and correctly identified all M. farcinogenes and M. senegalense strains. PCR- and 16S-23S rDNA sequence-based detection will be a valuable approach for diagnosis of the causal agents of African bovine farcy in cattle.
Animals↗
Genotyping of hepatitis C virus types 1, 2, 3, and 4 by a one-step LightCycler method using three different pairs of hybridization probes.
Determination of hepatitis C virus (HCV) genotypes has become increasingly important during the last years for prediction of the clinical course and the outcome of antiviral therapy. Therefore, numerous different methods have been developed to enable HCV genotyping. However, many of them are very laborious and expensive, leading to limited usage in daily routine diagnostics. We have established a method which combines the speed of the new LightCycler technology with the use of amplification products generated for diagnostic quantitative HCV RNA determination. Differentiation of HCV genotypes is performed with these amplicons in a single step by using fluorophore-labeled hybridization probes. Although currently only two different acceptor fluorophores are available for the LightCycler, types 1, 2, 3, and 4, which are by far the prevailing HCV genotypes in Europe and the United States, can be distinguished. Genotypes of specimens from 190 chronically HCV-infected patients were determined by the LightCycler method and compared with the results of nucleotide sequencing. Concordant results were obtained for all samples. This new method offers a fast and convenient possibility to determine the quantitative HCV RNA load and the genotype in large-scale settings within about 4 h.
Base Sequence↗
Homozygous proline at codon 72 of p53 as a potential risk factor favoring the development of undifferentiated thyroid carcinoma.
The p53 tumor suppressor gene is altered in human cancer. A common polymorphism occurs at codon 72 of exon 4, with two alleles encoding either arginine (CGC) or proline (CCC). No data exist about the association of a distinct codon 72 variant with the histological subtypes of thyroid carcinoma. We developed a new one-step real-time PCR assay on the LightCycler to detect codon 72 polymorphism in the p53 gene. We studied 21 papillary, 18 follicular and 22 anaplastic thyroid carcinomas and compared them with 15 adenomas and 36 normal thyroid tissues (controls); moreover, we compared the cases for histological, clinical and demographic variables and genotype prevalence. In controls, the frequency of the three genotypes Arg/Arg, Arg/Pro, and Pro/Pro was 41.7, 50.0 and 8.3%, respectively. The homozygous proline was not found in benign thyroid adenomas and differentiated thyroid carcinomas. In contrast, all undifferentiated thyroid carcinomas (100%) had the homozygous proline phenotype. The frequency of the two other genotypes Arg/Arg and Arg/Pro was 66.7% and 33.3% in adenomas, 81.0% and 19.0% in papillary thyroid carcinomas, and 83.3% and 16.7% in follicular thyroid carcinomas, respectively. Comparing the genotypes with tumor stage, no correlation was found. However, lymph node and distant metastases status showed a statistically significant prevalence for the homozygous phenotypes Arg/Arg and Pro/Pro. There was no association between a special genotype and age and sex. We conclude that homozygous proline is a potential risk factor favoring the development of an undifferentiated thyroid carcinoma, and that the homozygous phenotypes at codon 72 of p53 are associated with a poorer prognosis of thyroid carcinoma.
Adult↗
Selective loss of codon 72 proline p53 and frequent mutational inactivation of the retained arginine allele in colorectal cancer.
According to recent reports, some cancer types exhibit nonrandom allele loss at codon 72 in exon 4 of the p53 gene [coding for proline (72Pro) or arginine (72Arg)]. To clarify this phenomenon for colorectal cancer and to find out if this preferential loss might have any functional significance, p53 loss of heterozygosity (LOH) and p53 mutations were investigated in a group of 61 colorectal cancers and 28 liver metastases, and were correlated with clinicopathologic factors. A comparison of a patient's blood codon 72 status with a healthy control group did not reveal an enhanced risk of developing colorectal tumors for one of the two isoforms. p53-LOH and p53 mutations were found in 62.2% and 39.4% of primary tumors, respectively, and in 57.9% and 25% of hepatic metastases, respectively. In 14 heterozygous cases showing exon 4-LOH, only the 72Pro allele was lost and the retained 72Arg was preferentially mutated. In general, p53 mutations were significantly associated with the 72Arg tumor status (P < .001). Distal tumors showed allelic losses of the p53 gene more commonly than proximal tumors (P = .054). The prevalence of 72Arg increased in frequency with higher Dukes stage (P = .056). We suggest that either the preferential loss of 72Pro or the mutation of the 72Arg in colorectal cancer and hepatic metastases is associated with malignant potential and might reflect carcinogenic exposure, particularly in the distal part of the large intestines.
Adult↗