The C677T mutation in the methylenetetrahydrofolate reductase gene: a genetic risk factor for methotrexate-related elevation of liver enzymes in rheumatoid arthritis patients.

OBJECTIVE: To study the possible relationship between the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene and the toxicity and efficacy of treatment with methotrexate (MTX) in patients with rheumatoid arthritis (RA). METHODS: Genotype analysis of the MTHFR gene was done in 236 patients who started MTX treatment with (n = 157) or without (n = 79) folic or folinic acid supplementation. Outcomes were parameters of efficacy of MTX treatment, patient withdrawal due to adverse events, discontinuation of MTX treatment because of elevated liver enzyme levels, and the total occurrence of elevated liver enzyme levels during the study. Multivariate logistic regression analysis was used to study the relationship between the presence of the MTHFR C677T mutation and toxicity outcomes of MTX treatment. RESULTS: Forty-eight percent of the patients showed the homozygous (T/T) or heterozygous (T/C) mutation. The presence of the C677CT or C677TT genotypes was associated with an increased risk of discontinuing MTX treatment because of adverse events (relative risk 2.01; 95% confidence interval 1.09, 3.70), mainly due to an increased risk of elevated liver enzyme levels (relative risk 2.38; 95% confidence interval 1.06, 5.34). Efficacy parameters were not significantly different between the patients with and those without the mutation. CONCLUSION: The C677T mutation is the first identified genetic risk factor for elevated alanine aminotransferase values during MTX treatment in patients with RA. We postulate that the incidence of clinically important elevation of liver enzyme levels during MTX treatment is mediated by homocysteine metabolism. Supplementation with folic or folinic acid reduced the risk of toxicity-related discontinuation of MTX treatment both in patients with and in patients without the mutation.

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.

Rapid identification of thermotolerant Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis from various geographic locations by a GTPase-based PCR-reverse hybridization assay.

Recently, a gene from Campylobacter jejuni encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from C. jejuni, C. coli, C. lari, and C. upsaliensis. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerant Campylobacter species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant Campylobacter species.

Influence of sulphasalazine, methotrexate, and the combination of both on plasma homocysteine concentrations in patients with rheumatoid arthritis.

OBJECTIVE: To study the influence of sulphasalazine (SSZ), methotrexate (MTX), and the combination (COMBI) of both on plasma homocysteine and to study the relation between plasma homocysteine and their clinical effects. METHODS: 105 patients with early rheumatoid arthritis (RA) were randomised between SSZ (2-3 g/day), MTX (7.5-15 mg/week), and the COMBI (same dose range) and evaluated double blindly during 52 weeks. Plasma homocysteine, serum folate concentrations, and vitamin B12 were measured. The influence of the C677T mutation of the enzyme methyl-enetetrahydrofolatereductase (MTHFR) gene was analysed. RESULTS: A slight trend towards increased efficacy and an increased occurrence of minor gastrointestinal toxicity was present in the COMBI group, no differences existed clinically between SSZ and MTX. Only a slight and temporary increase in plasma homocysteine was found in the SSZ group, in contrast with the persistent rise in the MTX group and the even greater increase in the COMBI patients. Patients homozygous for the mutation in the MTHFR gene had significantly higher baseline homocysteine, heterozygous MTHFR genotype induced a significantly higher plasma homoeysteine at week 52 compared with no mutation. No correlation was found between clinical efficacy variables and homocysteine. Patients with gastrointestinal toxicity had a significantly greater increase in homocysteine. CONCLUSION: A persistent increase in plasma homocysteine concentrations was observed in patients treated with MTX alone and more pronounced in combination with SSZ, in contrast with SSZ alone. An increase in plasma homocysteine is related to the C677T mutation in MTHFR. A relation in the change in homocysteine concentrations with (gastrointestinal) toxicity was found, no relation with clinical efficacy existed.

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.

Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene.

Polymerase chain reaction (PCR) mediated DNA fingerprinting has resulted in the identification of a novel Campylobacter jejuni gene, encoding a GTPase protein. The gene, consisting of 383 amino acids contained semi-conserved GTP-binding sites (designated G-1 to G-4), that are characteristic for members of the GTPase protein superfamily. Remarkably, this gene from C. Jejuni appears to encode a member of a novel family of GTP-binding proteins, containing two separate putative GTP-binding domains, each comprising a series of semi-conserved GTP-binding motifs. Spacing between these motifs is highly conserved. Based on this novel gene, a general PCR strategy for the identification of C. jejuni, C. coli, C. lari and C. upsaliensis was developed. PCR primers were deduced from GTP-binding motifs G-1 and G-3 of the first GTP-binding domain. These GTP-binding sites flank a variable region of precisely 117 bp in the four Campylobacter spp. that allowed the development of species-specific probes. This PCR-hybridization assay offers a novel tool for rapid molecular detection and specific identification of the thermophilic Campylobacter spp.

Generation of DNA probes for detection of microorganisms by polymerase chain reaction fingerprinting.

Identification of medically relevant microorganisms is important for diagnosis, treatment and prevention of infectious diseases. This has initiated the development of a large number of identification and typing techniques based on phenotypic and genetic characteristics. In general, these last mentioned nucleic acid-mediated techniques provide more detailed and consistent information on strain-specific characteristics. However, the development of clinically useful microbial DNA/RNA probes requires nucleotide sequence information and a set of well defined reference organisms for test validation in comparison with the current gold standard. This is a requirement for the development of accurate nucleic acid hybridisation and/or amplification tests. Recently, it has been demonstrated that polymerase chain reaction (PCR)-mediated genetic typing of microorganisms can lead to the immediate isolation of species-specific DNA probes by comparison of DNA fingerprints. This combines the sensitivity of PCR with the specificity of DNA probing without the need to generate nucleic acid sequence information prior to probe development. The implications of this procedure for clinical microbiology and epidemiological surveillance will be discussed. It is shown that specific probes can be developed for various taxonomic levels and that detection and identification can be combined into a single, fast procedure. The versatility and widely applicable principles of this procedure will be highlighted and exemplified by some newly developed tests and a review of the current literature.

Investigation of an outbreak of Campylobacter upsaliensis in day care centers in Brussels: analysis of relationships among isolates by phenotypic and genotypic typing methods.

An outbreak of Campylobacter upsaliensis in four Brussels day care centers (A, B-1, B-2, and C) affected 44 children. Diarrhea was the major symptom. From January 1991 to June 1992, the outbreak strain was isolated from 3, 1, and 21 (of 68) children in centers A, B-1, and B-2, respectively, and from 19 of 22 children in center C, IgG, IgM, and IgA antibodies were detected by Western blotting of serum specimens of 9 of 10 and 13 of 16 children in centers B-2 and C, respectively. Strains were typed by biotyping, DNA restriction-based and antibiotic susceptibility typing, whole cell protein and plasmid analysis, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR). On the basis of RFLP and PCR typing, the strains could be divided into two strongly related clonal variants: One was isolated only from the children of center A and the second only from children in the other day care centers.

Detection and identification of Campylobacter spp. using the polymerase chain reaction.

Since Campylobacters have fastidious growth requirements and conventional detection and identification requires at least 4-6 days, the development of fast but reliable detection procedures is needed. Although methods based on DNA probe technology have been developed, these are not sensitive enough for the detection of Campylobacter spp. in food products. Therefore a PCR procedure based on the amplification of the 16S rRNA gene was developed that specifically detects the thermophilic Campylobacter species. This assay provides an excellent tool for the rapid and sensitive isolation and identification of Campylobacter spp. from chicken samples. In order to further identify the different Campylobacter spp., which are difficult to distinguish by conventional methods, PCR mediated approximately DNA typing was used to select species-specific DNA probes. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. PCR mediated DNA typing was also used to study the epidemiology of diarrheal diseases caused by Campylobacter spp. Using primers complementary to dispersed repetitive DNA sequences and arbitrarily chosen DNA motifs PCR fingerprinting has proven to be a fast, highly discriminative and relatively simple method that can be applied in epidemiological investigations on Campylobacter infections. Besides this application of PCR fingerprinting for typing of Campylobacter spp. this method can also be used for the development of specific DNA probes.

Polymerase chain reaction-mediated DNA fingerprinting for epidemiological studies on Campylobacter spp.

The applicability of polymerase chain reaction (PCR)-mediated DNA typing, with primers complementary to dispersed repetitive DNA sequences and arbitrarily chosen DNA motifs, to study the epidemiology of campylobacter infection was evaluated. With a single PCR reaction and simple gel electrophoresis, strain-specific DNA banding patterns were observed for Campylobacter jejuni and C. upsaliensis. DNA from multiple strains isolated during an outbreak of C. jejuni meningitis generated identical banding patterns and could be distinguished from randomly isolated strains. Strains from a community outbreak of C. upsaliensis, that were all identical by conventional typing methods, could be divided into two genetically different groups. This report illustrates that PCR fingerprinting can be successfully applied in epidemiological investigations of campylobacter infections.

Discrimination of epidemic and sporadic isolates of Arcobacter butzleri by polymerase chain reaction-mediated DNA fingerprinting.

DNA polymorphisms of Arcobacter butzleri outbreak-related strains and Arcobacter reference strains were determined by use of the polymerase chain reaction with primers aimed at repetitive sequences. The epidemiological relationship among 14 outbreak-related strains was substantiated, as they showed virtually no genomic variations. Their DNA amplification patterns were, however, clearly different from those of all Arcobacter reference strains studied; each reference strain was characterized by a unique DNA fingerprint.

PCR-mediated DNA typing of Campylobacter jejuni isolated from patients with recurrent infections.

The polymerase chain reaction (PCR) and two primers aiming at the enterobacterial repetitive intergenic consensus (ERIC) and arbitrary DNA sequences, respectively, were used to fingerprint the genomic DNA of 24 Campylobacter jejuni strains isolated from five patients with recurrent C. jejuni infections. Results were compared with biotyping and serotyping. The latter two methods, when combined, distinguished 9 different types, whereas PCR-mediated DNA analysis discriminated 14 different patterns. For six strains, the results of PCR-mediated typing led to different interpretations. This method is proposed as an additional tool to further discriminate between C. jejuni strains that appear related by conventional typing methods. In view of its rapidity and simplicity, this method is a potential candidate to replace the relatively slow and laborious conventional methods. However, further study is needed to assess the sensitivity and specificity of PCR-mediated DNA analysis and to investigate the usefulness of this method as an epidemiological tool in outbreaks of Campylobacter infections.

Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting.

The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial repetitive intergenic consensus (ERIC) motifs generate isolate-specific DNA banding patterns. Analysis of these PCR fingerprints obtained for 33 isolates of Campylobacter jejuni, 30 isolates of Campylobacter coli, and 8 isolates of Campylobacter lari revealed that besides generation of isolate-specific fragments, species-specific DNA fragments of identical size were synthesized. It appeared that these DNA fragments could be used as species-specific probes, since they are unique for the pattern which they are deriving from. The probes do not cross-react with amplified DNA originating from a large panel of nonrelated microorganisms. Moreover, these probes displayed species specificity, as they reacted with a single restriction fragment on Southern blots containing DNA from C. jejuni, C. coli, and C. lari and other Campylobacter species. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. The principle of the procedure holds great promise for the rapid isolation of DNA probes which, in combination with a general PCR assay, may lead to efficient typing and detection procedures for a multitude of medically important nonviral microorganisms.

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.