Bias in detection of instability of the (C)8 mononucleotide repeat of MSH6 in tumours from HNPCC patients.

Recently, we and others reported instability in the (C)8 repeat in exon 5 of MSH6 as a preferential target for somatic mutations in tumours from MSH6 germline mutation carriers. Here, we report that in 45% of tumours from MLH1, MSH2 and MSH6 germline mutation carriers no sequence change in the (C)8 repeat of MSH6 was found upon DNA sequencing analysis of PCR products with a shift in electrophoresis mobility. Using "standard" PCR primers a high frequency of instability (50-86%) of the (C)8 repeat was found, but using a modified PCR reverse primer, accomplishing modulation of non-templated addition of adenine during in vitro PCR amplification by the Taq polymerase, a markedly lower frequency of instability was found in tumours from MLH1, MSH2 and MSH6 mutation carriers (6, 13 and 40%, respectively). Furthermore, a significant difference of the frequency of instability of the (C)8 repeat in tumours from MSH6 mutation carriers was found compared to MLH1, MSH2 mutation carriers. These results might have important implications for the detection of instability of other short mononucleotide repeats, e.g. TGFbetaRII, BAX, IGFRII, PTEN, BRCA2.

E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain.

We have analysed a series of 49 human breast cancers for mutations in the entire coding region plus flanking intron sequences of the E-cadherin gene. The tumours included 41 infiltrating lobular carcinomas, two infiltrating ducto-lobular carcinomas and six infiltrative ductal carcinomas. In the lobular carcinomas 23 different somatic mutations were detected, of which seven were insertions, 11 deletions, two nonsense mutations and three splice site mutations. The other tumours showed no detectable E-cadherin mutations. All the frameshift and nonsense mutations are expected to generate a secreted E-cadherin fragment instead of a transmembrane protein with cell adhesion activity. The majority of the mutations (21 of 23) were found in combination with loss of heterozygosity of the wild type E-cadherin locus (16q22.1), a hallmark of classical tumour suppressor genes. The mutations were scattered over the whole coding region and no hot spots could be identified. All mutations described here were previously unreported. In conclusion, we have identified up to now E-cadherin mutations in 27 of 48 (56%) infiltrating lobular breast carcinomas and in 0 of 50 breast cancers of other histopathological subtypes. These data provide strong evidence that frequent E-cadherin mutations are involved in the particular etiology of sporadic lobular breast cancers.

Frequent allelic imbalance but infrequent microsatellite instability in gastric lymphoma.

Specific defects in DNA repair pathways are reflected by DNA microsatellite instability (MSI) and play an important role in carcinogenesis. Reported frequencies in gastric non-Hodgkin's lymphomas (NHL) vary from 14% to as high as 90%. Another form of genetic instability in tumours is allelic imbalance (AI) due to loss or gain of genetic material at a specific chromosomal region. This might point to the presence of a tumour suppressor gene or oncogene. We examined both MSI and AI in 26 gastric lymphomas (10 low-grade and 13 high-grade MALT lymphomas and three cases lacking MALT features and categorised as diffuse large B cell lymphoma (DLCL)). Tumour components and normal cells (epithelium, muscle) were microdissected from paraffin-embedded resection samples. Contrary to other studies we did not observe frequent MSI when investigating 18 different loci distributed over 12 chromosomes. Microsatellite instability of a single locus was found in 1/10 (10%) low-grade MALT lymphomas and 2/13 (15%) high-grade MALT lymphomas. These data indicate that DNA mismatch repair genes do not play a role in the pathogenesis of these lymphomas. Allelic imbalance was detected in 60% (6/10) of low-grade MALT lymphomas, in 62% (8/13) of high-grade MALT lymphoma and in 67% (2/3) of DLCL. In high-grade lymphomas more loci showed AI (one to seven loci, with a mean of 2.5 loci per case) than in the low-grade lymphomas (one to two loci, with a mean of 1.3 loci per case), possibly reflecting an increased genomic instability.

Atypical HNPCC owing to MSH6 germline mutations: analysis of a large Dutch pedigree.

Hereditary non-polyposis colorectal cancer (HNPCC) is the most common genetic susceptibility syndrome for colorectal cancer. HNPCC is most frequently caused by germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1. Recently, mutations in another MMR gene, MSH6 (also known as GTBP), have also been shown to result in HNPCC. Preliminary data indicate that the phenotype related to MSH6 mutations may differ from the classical HNPCC caused by defects in MSH2 and MLH1. Here, we describe an extended Dutch HNPCC family not fulfilling the Amsterdam criteria II and resulting from a MSH6 mutation. Overall, the penetrance of colorectal cancer appears to be significantly decreased (p<0.001) among the MSH6 mutation carriers in this family when compared with MSH2 and MLH1 carriers (32% by the age of 80 v >80%). Endometrial cancer is a frequent manifestation among female carriers (six out of 13 malignant tumours). Transitional cell carcinoma of the urinary tract is also relatively common in both male and female carriers (10% of the carriers). Moreover, the mean age of onset of both colorectal cancer (MSH6 v MSH2/MLH1 = 55 years v 44/41 years) and endometrial carcinomas (MSH6 v MSH2/MLH1 = 55 years v 49/48 years) is delayed. As previously reported, we confirm that the pattern of microsatellite instability, in combination with immunohistochemical analysis, can predict the presence of a MSH6 germline defect. The detailed characterisation of the clinical phenotype of this kindred contributes to the establishment of genotype-phenotype correlations in HNPCC owing to mutations in specific mismatch repair genes.

mRNA levels and methylation patterns of the tyrosine aminotransferase gene in aging inbred rats.

We have examined the mRNA levels and methylation patterns of the liver-specific tyrosine aminotransferase (TAT) gene in inbred female rats aged 6, 24 and 36 months. Northern hybridization analysis of total RNA showed a 65% decrease in the steady state transcript level of TAT in the liver of 24- and 36-month old rats as compared to 6-month old animals. The TAT gene as studied by Southern hybridization analysis using the isoschizomers Hpa II and Msp I was found to be hypomethylated in the liver as compared to spleen and brain at six CpG sites within the gene. Methylation at these sites remained unchanged during aging.

Messenger RNA levels and methylation patterns of GAPDH and beta-actin genes in rat liver, spleen and brain in relation to aging.

Messenger RNA levels and methylation patterns of the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and beta-actin genes were studied in spleen, liver and brain of 6-, 24- and 36-month old female inbred rats. In the spleen, the mRNA levels of both housekeeping genes significantly increased between 24 and 36 months. No age-related alterations in the expression of GAPDH or beta-actin mRNA were observed in brain or liver. A considerable intertissue and interindividual variation was observed in the mRNA levels of these genes in all age-groups as compared to the level of 28 S rRNA, which was used as an internal control. In this respect the interindividual variation in the level of GAPDH mRNA paralleled the variation observed in the beta-actin mRNA level in the three tissues studied. The methylation pattern of beta-actin was found to be tissue-specific in contrast to that of GAPDH, which was identical in all three tissues. No significant age-related alterations were observed in the GAPDH methylation pattern, whereas beta-actin appeared to become slightly demethylated with age in the spleen at the CpG site for which tissue-specificity was observed.

Plasmid rescue from transgenic mouse DNA using LacI repressor protein conjugated to magnetic beads.

A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described. The method is based on the high affinity of the LacI repressor protein for the lac operator sequence. Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA. After circularization, the plasmids were introduced into Escherichia coli by means of electroporation. Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging. Our results indicate that plasmid rescue is about 25 times more efficient. Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo.

Quality control in diagnostic molecular pathology in the Netherlands; proficiency testing for patient identification in tissue samples.

AIMS: To describe the evolution of proficiency testing for molecular diagnostic pathology with respect to determining unambiguously the patient identity of tissue samples by microsatellite analysis. METHOD: Four rounds of quality control exchanges of samples from different patients were sent with the purpose of identifying the correct origin of these samples. The samples were either paraffin wax embedded sections on glass, sections in tubes, or isolated DNA. Blinded samples were distributed to all participating laboratories. No restrictions to the method and short tandem repeat markers used for identification were imposed. RESULTS: In four subsequent rounds the number of participating laboratories increased from three to 10. The numbers of samples tested increased in time from five to 12. The microsatellite markers used by the different laboratories showed little overlap. In the first three rounds, in which isolated DNA was provided, all samples were accurately classified irrespective of the microsatellite markers used. In the last round, which also included paraffin wax embedded sections, a small number of laboratories experienced problems, either with amplification or incorrect classification of a few samples. CONCLUSION: Proficiency testing was useful, and showed country wide high quality and correct identification of (patient) samples with molecular techniques for diagnostic purposes.

LacZ transgenic mouse models: their application in genetic toxicology.

Gene mutations have been implicated in the etiology of cancer, developmental anomalies, genetic disease and aging. Many different methods for mutation detection have been developed and applied to obtain a more fundamental insight in the chain of molecular events that ultimately lead to mutations. Most of these methods, however, can only be applied to cultured cells and therefore do not allow comparative analysis of mutations in various organs and tissues in an intact organism. The main difficulty in studying mutagenesis in chromosomal DNA is to identify and isolate mutated genes with a high efficiency. Here we describe the development and application of LacZ transgenic mouse models for studying, in different organs and tissues, spontaneous or induced mutations. Such models allow study of the induction of DNA damage, repair, mutagenesis and carcinogenesis in one animal system. Accordingly, results obtained may ultimately provide greater insight into the chain of events from in vivo exposure to genotoxic agents to mutations and their ultimate physiological endpoints. In addition to their use in fundamental research, transgenic animal mutation models find a major application in the field of genetic toxicology testing, in particular with respect to organ specificity.

High somatic mutation frequencies in a LacZ transgene integrated on the mouse X-chromosome.

To study spontaneous and induced mutagenesis in vivo we recently constructed a series of transgenic mice harboring different numbers of bacteriophage lambda shuttle vectors, provided with a LacZ mutational target gene, integrated in their genome. The transgenic mice enabled analysis of spontaneous and induced mutation frequencies in postmitotic tissues like liver and brain. The obtained data indicated spontaneous mutation frequencies in the order of 10(-5)-10(-6). Here we report a 25-100 times higher spontaneous mutation frequency in liver and brain DNA of mice from strain 35.5, with the lambda-gt10LacZ concatemer integrated on the X-chromosome. These results indicate the presence of a mutational 'hot spot' in the mammalian somatic genome in vivo.

Identification and isolation of a specific antigen with diagnostic potential from Dictyocaulus viviparus.

The purpose of the investigation was to isolate and identify a specific antigen of Dictyocaulus viviparus that can be used to diagnose lungworm infections in cattle. Somatic, excretion and secretion antigens of adult D. viviparus and somatic antigens of L3 larvae were examined in an indirect enzyme-linked immunosorbent assay (ELISA) to determine whether they cross-reacted with sera collected from calves with mono-infections of Fasciola hepatica. Ostertagia ostertagi, Ascaris suum, or Cooperia oncophora. Serum samples containing antibodies directed against F. hepatica, A. suum, and O. ostertagi cross-reacted with somatic antigens of adult D. viviparus; these sera cross-reacted less with excretion and secretion antigens. When somatic antigens of adult D. viviparus were analysed in a Western blot, a 17-kDa protein that did not react with the heterologous sera was detected. This protein was isolated by ultrafiltration and anion chromatography. Sera collected from calves infected with D. viviparus was tested in indirect ELISAs with either somatic antigens of adult D. viviparus or with a low molecular antigen fraction of this preparation containing the 17-kDa protein. The extinction values that were measured in both assays correlated well. We conclude that the 17-kDa protein isolated from somatic antigens of adult D. viviparus may be useful in developing an improved immunoassay to diagnose lungworm infections in cattle.

Comparison of three enzyme immunoassays for diagnosis of Dictyocaulus viviparus infection.

Three enzyme-linked immunosorbent assays (ELISA) that detect antibodies against Dictyocaulus viviparus in cattle were compared for sensitivity, specificity and seroconversion after primary infection. These assays were (i) an indirect ELISA with crude somatic antigens from adult D. viviparus (ca-ELISA), (ii) an indirect ELISA with purified antigens (sa-ELISA) isolated from somatic antigens of adult D. viviparus and (iii) a competition ELISA with antigen purified with anion chromatography in combination with monoclonal antibodies against D. viviparus. Sera from helminth-naïve calves and sera from calves monospecifically infected with Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Ascaris suum or Fasciola hepatica were used to determine the specificity of the assays. Sera from calves and milk cows experimentally or naturally infected with D. viviparus, and from vaccinated calves, were used to test the sensitivity of the assays and to determine when the ELISAs detected seroconversion. The specificity of the competition and the sa-ELISA was 97%, whereas the specificity of the ca-ELISA was 67%. The sensitivities of the sa-ELISA, the competition ELISA and the ca-ELISA were 97, 73 and 99%, respectively. All three assays detected seroconversion between 4 and 6 weeks after primary infection. None of the assays detected seroconversion in calves receiving lungworm vaccination. We conclude that of these three tests, the sa-ELISA can be used most beneficially to diagnose lungworm disease.

Dictyocaulus viviparus in calves: effect of rotational grazing on the development of infections.

A study was made of the possibility of reducing lungworm infections in young grazing calves by rotational grazing for weekly periods on six paddocks. For this purpose three groups of four calves each were grazed on separate pastures in 1989, whereas a fourth group served as a permanently housed control group. Two groups of calves were infected experimentally with six doses of 10 larvae of Dictyocaulus viviparus during the first 3 weeks on pasture. In the third group, low natural infections with overwintered larvae occurred. One of the experimentally infected groups was rotationally grazed for weekly periods on six small plots while both other groups were set-stocked. Faecal larval counts and worm counts in tracer calves demonstrated lower lungworm infections in the rotationally grazed group than in both set-stocked groups. However, the numbers of worms found after challenge infection and subsequent necropsy were relatively high in the rotationally grazed group, indicating that development of immunity was less than in both other groups. Owing to the dry weather conditions in the summer of 1989, no serious clinical signs of husk developed in any of the three groups. These dry conditions, however, did not prevent the build-up of heavy pasture infectivity with gastrointestinal nematodes resulting in heavy worm burdens and serious clinical signs in tracer calves grazing for 4 days in August and September-October, respectively. This implies that rotational grazing did not have a clear effect on build-up of gastrointestinal nematode infections.

A comparison of the efficacy of four different long-acting boluses to prevent infections with Dictyocaulus viviparus in calves.

A field study of calves in their first grazing season tested the efficacy of four long-acting devices--a morantel sustained-release bolus, a levamisole sustained-release bolus, an oxfendazole interval bolus, and an albendazole interval bolus--against Dictyocaulus viviparus. The pasture had been previously contaminated by four calves orally inoculated with infective lungworm larvae. The calves were grazed together with four bolus-treated groups, each comprising four calves. Lungworm infection became patent in the experimentally inoculated calves between 22 and 26 days. Infection in the bolus-treated groups became patent after 54 days. The morantel bolus group excreted the most larvae, followed by the albendazole bolus group, and the levamisole bolus group. The oxfendazole bolus group excreted by far the least larvae. Eosinophil curves and ELISA titres showed that treated groups had essentially the same course of infection. The heavy infection to which the treated calves were exposed produced complete immunity in all groups. Challenge infection of 10,000 larvae at housing did not change any of the test parameters. Post-mortem examination showed only one positive calf with few worms. We concluded that when pastures are heavily infested with lungworm larvae, all boluses prevent severe clinical signs and allow build up of solid immunity, although none completely prevent excretion of larvae.

An epidemiological study of Fasciola hepatica in The Netherlands.

Transmission of F. hepatica under natural conditions was analysed in a three year programme. The variables used were the indirect haemagglutination (IHA) technique, worm establishment in tracer lambs and the population dynamics, infection rate and shedding pattern of Lymnaea truncatula. It is concluded that fluke eggs, infected snails and metacercariae on herbage can survive the winter in the Netherlands. Metacercarial availability was positively correlated to the amount of rainfall in the grazing period. The role developed eggs that survive the winter is important, because this results in earlier infections in the herd. The use of the serological diagnosis method IHA is important to detect F. hepatica infection in an early stage. Use of cellophane paper on floats is a useful method for determining the shedding pattern of cercariae from L. truncatula. It is concluded that collection of metacercariae on cellophane floats, inventarization of L. truncatula and its infection level are useful tools for the prediction of liverfluke infections.

Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep.

An enzyme-linked immunosorbent assay (ELISA) with somatic (S) or excretory-secretory antigens (ES) was compared with an indirect haemagglutination assay (IHA) for ability to detect antibodies against Fasciola hepatica in sheep. The specificity of both assays was determined by testing sera collected from sheep experimentally or naturally mono-infected with Fasciola hepatica, Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Taenia ovis, Eimeria spp., Trichostrongylus vitrinus, Trichostrongylus colubriformis or Nematodirus battus respectively. With S or ES antigens the specificity of the ELISA was 98% or 95% respectively, whereas the specificity of the IHA was 86%. Antibodies directed against Fasciola hepatica were detected by the ELISA with S or ES antigens from 2 weeks after infection until the end of the experiment, whereas the IHA detected antibodies from week 3. We conclude that the ELISA with S antigens compares favourably with the IHA and can be used for the serodiagnosis of ovine fasciolosis in the Netherlands.

An experimental field study on the build up of lungworm infections in cattle.

The build up of lungworm infections was studied in four groups of calves. Calves of Group 1 were infected experimentally with 6 x 10 larvae during the first 3 weeks after turnout. The pasture of Group 2 was contaminated with approximately 35,000 larvae in June and the pasture of Group 3 with approximately 1.3 million larvae in August. Group 4 served as the helminth free control group for challenge infections with 5,000 larvae in October. In Group 1 faecal larval counts increased 5 weeks after the beginning of patency and decreased after another 3 weeks, indicating the development of immunity after the second lungworm generation. In contrast, the development of immunity in Groups 2 and 3 occurred after the first lungworm generation as maximal faecal larval counts were seen within 3.5 weeks after the beginning of patency. Infection levels were highest in Group 3 which was the only group showing clinical signs. These signs became worse after oxfendazole treatment.

Comparison of a levamisole sustained-release bolus and ivermectin treatment to prevent bovine lungworm infection.

The efficacy of a levamisole sustained-release bolus to prevent parasitic bronchitis in calves in their first grazing season was compared to ivermectin treatment at three, eight and thirteen weeks after turn out. Contamination of the pasture was established by experimentally infected seeder calves. Twenty calves were split into two groups. Ten calves of one group received a bolus at the start of the experiment. In the other group the calves were treated with ivermectin at 21, 56 and 91 days. Two principal calves from each group were killed during the experiment to study histopathological changes. Pairs of tracer calves were introduced on both pastures at intervals of four weeks throughout the grazing period. The permanent calves were challenged with lungworm larvae at housing and slaughtered four weeks later. Both systems prevented parasitic bronchitis. Larval output was completely reduced in the ivermectin-treated calves while all bolus-treated calves excreted larvae at certain times. The highest group average was 4 larvae per gram faeces. Eosinophilia, ELISA-titres and histopathological changes confirmed the differences in larval uptake. Challenge infection was not successful in either group and no worms were found at slaughter. Weight gain was significantly different at housing in favour of the ivermectin-treated calves, but after challenge this was reduced due to a higher weight gain in the bolus-treated calves. The practical consequences of the results have been discussed.

The effect of rotational grazing for periods of one or two weeks on the build-up of lungworm and gastro-intestinal nematode infections in calves.

An experiment was carried out with three groups of grazing calves and one housed control group to study the effect of rotational grazing for periods of 1 and 2 weeks on the build up of lungworm and gastro-intestinal nematode infections respectively. The experiment demonstrated that rotational grazing for periods of 1 week on six plots prevented the build-up of heavy lungworm infections. A build up of heavier lungworm infections was observed in a group that was rotationally grazed for periods of 2 weeks on three plots and a group which remained on one plot throughout the grazing season; there was no difference between these two groups. In all three situations, there was an adequate development of immunity against D. viviparus, as measured by worm recovery after challenge infection at the end of the experiment in comparison with worm recovery of the similarly challenged control group. Neither rotational grazing scheme protected the calves against gastrointestinal helminthiasis, because tracer calves, which grazed for 4 days only in August or October, acquired infections which would have resulted in severe illness or even death if necropsy had been postponed for a week.