Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening

Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.     

Multiple forms of glycoproteins in the secretory product of the bovine subcommissural organ--an ancient glial structure

The glial subcommissural organ (SCO) is a conserved structure of the vertebrate brain that secretes a glycoprotein-rich product into both the extracellular matrix and the cerebrospinal fluid of the third ventricle that forms Reissner's fibre (RF). In order to identify specific secretory proteins of the subcommissural organ, a panel of antigen- and epitope-specific monoclonal antibodies was raised against bovine RF to study the distribution of epitopes in Western blots of bovine RF. Six groups of epitopes that were specific for SCO secretion were distinguished on the basis of their phylogenetic conservation and their different grades of resistance against chemical denaturation. The monoclonal antibody aRFME 4 recognised a carbohydrate-containing epitope that was strongly conserved in vertebrates and unique for SCO secretion. All epitopes showed essentially the same distribution pattern over 15 bovine RF glycoprotein fractions of different molecular masses in immunoblots indicating that the different RF fractions are closely related. They may represent multiple forms of SCO spondin.     

Association of oestrogen receptor alpha gene polymorphisms with postmenopausal bone loss, bone mass, and quantitative ultrasound properties of bone

Background: The gene encoding oestrogen receptor α (ESR1) appears to regulate bone mineral density (BMD) and other determinants of osteoporotic fracture risk.

Objective: To investigate the relation between common polymorphisms and haplotypes of the ESR1 gene and osteoporosis related phenotypes in a population based cohort of 3054 Scottish women.

Results: There was a significant association between a common haplotype "px", defined by the PvuII andXbaI restriction fragment length polymorphisms within intron 1 of the ESR1 gene, and femoral neck bone loss in postmenopausal women who had not received hormone replacement therapy (n = 945; p = 0.009). Annual rates of femoral neck bone loss were ~14% higher in subjects who carried one copy of px and 22% higher in those who carried two copies, compared with those who did not carry the px haplotype. The px haplotype was associated with lower femoral neck BMD in the postmenopausal women (p = 0.02), and with reduced calcaneal broadband ultrasound attenuation (BUA) values in the whole study population (p = 0.005). There was no association between a TA repeat polymorphism in the ESR1 promoter and any phenotype studied, though on long range haplotype analysis subjects with a smaller number of TA repeats who also carried the px haplotype had reduced BUA values.

Conclusions: The ESR1px haplotype is associated with reduced hip BMD values and increased rates of femoral neck bone loss in postmenopausal women. An association with BUA may explain the fact that ESR1 intron 1 alleles predict osteoporotic fractures by a mechanism partly independent of differences in BMD.

     

Studies on the C2-deficiency gene in man.

A one-step haemolytic assay using cellular intermediates was used to determine C2 levels in 50 HLA-A25 and B18 positive blood donors and four families suspected to have the C2-deficiency gene. The method clearly discriminated between homozygous normals and heterozygous deficient individuals, and it was found that approx. 50% of individuals with the haplotype HLA-A25, B18 had low levels of functional C2. In the four families studied, the close linkage of the C2-deficiency gene and the haplotype HLA-A25, B18 was confirmed. Furthermore, the C2-deficiency gene was shown to be a silent or null allele at the structural locus.     

Dystrophin disruption in enterovirus-induced myocarditis and dilated cardiomyopathy: from bench to bedside

Genetic defects of the dystrophin-glycoprotein complex (DGC) cause hereditary dilated cardiomyopathy. Enteroviruses can also cause cardiomyopathy and we have previously described a mechanism involved in enterovirus-induced dilated cardiomyopathy: The enteroviral protease 2A directly cleaves dystrophin in the hinge 3 region, leading to functional dystrophin impairment. During infection of mice with coxsackievirus B3, the DGC in the heart is disrupted and the sarcolemmal integrity is lost in virus-infected cardiomyocytes. Additionally, dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy in vivo, suggesting a pathogenetic role of the dystrophin cleavage in enterovirus-induced cardiomyopathy. Here, we extend these experimental findings to a patient with dilated cardiomyopathy due to a coxsackievirus B2 myocarditis. Endomyocardial biopsy specimens showed an inflammatory infiltrate and myocytolysis. Immunostaining for the enteroviral capsid antigen VP1 revealed virus-infected cardiomyocytes. Focal areas of cardiomyocytes displayed a loss of the sarcolemmal staining pattern for dystrophin and -sarcoglycan identical to previous findings in virus-infected mouse hearts. In vitro, coxsackievirus B2 protease 2A cleaved human dystrophin. These findings demonstrate that in human coxsackievirus B myocarditis a focal disruption of the DGC can principally occur and may contribute to the pathogenesis of human enterovirus-induced dilated cardiomyopathy.     

The effects of cryopreservation and thawing on the development in vitro and in vivo of biopsied 8-cell mouse embryos.

The possible impact of cryopreservation on biopsied 8-cell mouse embryos was investigated. Biopsied and control 8-cell embryos were cryopreserved using a slow freezing and quick thawing protocol with 1,2-propanediol as a cryoprotectant. The cryopreservation process did not affect either the recovery or the survival of biopsied embryos, when compared with intact controls; however, sham controls survived significantly better than biopsied 8-cell embryos (88.6 versus 74.2%, P less than 0.001). When fully and partially intact surviving embryos were cultured in vitro to the blastocyst stage, there was no difference in the proportions of embryos which formed blastocysts (biopsy 97.2%, intact control 98.4% and sham control 93.7%). The developmental potential and fetal development in vivo following embryo transfer were not impaired when assessed on day 17 of pregnancy. Cryopreservation of biopsied 8-cell mouse embryos is therefore a feasible approach to storing embryos while analysis of the biopsied material is carried out.     

Translocation (16;21)(p11;q22) in acute monoblastic leukemia with erythrophagocytosis

A patient with acute monoblastic leukemia with erythrophagocytosis and a t(16;21) (p11;q22), poor response to chemotherapy, early relapse, and a short survival of ten months is presented. Hematologically, this patient could be considered as a case of FAB M5b/t(8;16) but without the characteristic chromosomal translocation, i.e., there is no visible alteration on chromosome 8 and the breakpoint on chromosome 16 appears to be very proximal. These findings are briefly discussed in the light of other variants.     

Assessment of the viability and pregnancy potential of mouse embryos biopsied at different preimplantation stages of development

The developmental potential in vitro and in vivo of preimplantation mouse embryos biopsied at the 4-cell, 8-cell and morula stages were investigated. Biopsy had the least impact when performed at the 8-cell stage. There was no effect of biopsy on the development of 8-cells of blastocysts in vitro (95% compared with 99% of controls) or the implantation rate after transfers (82 versus 87%, P greater than 0.05); however, fewer embryos (52 versus 71%, P less than 0.05) resulted in viable fetuses. There was no effect of biopsy at the 8-cell stage on fetal weight on day 17. Blastocyst formation in vitro was significantly less for 4-cell biopsies compared with their controls (76 versus 90%, P less than 0.001) and biopsy also affected the implantation rate (44 versus 59%, P less than 0.01). Biopsy was most detrimental when performed on morulae, reducing the implantation rate from 65% for controls to 21% for biopsies (P less than 0.001). Fetal viability was also markedly affected with a reduction on day 17 from 42 to 26% accompanied by a significant reduction (24%, P = 0.02) of the mean fetal weight. Handling of embryos for biopsy at the morula stage, which involved removal of the zona pellucida, was a significant but not complete cause of the reduced implantation potential observed (sham-controls and intact-controls: 34 and 65%, P less than 0.001), while puncture of the zona during the biopsy of 4-cell and 8-cell embryos had no effect. Therefore, the 8-cell mouse embryo is the most suitable state for embryo biopsy.     

Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.     

Nested PCR for specific detection and rapid identification of human picornaviruses.

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.