The screening of Duchenne muscular dystrophy patients for submicroscopic deletions.

We have probed the DNA of 156 Duchenne muscular dystrophy (DMD) patients, representing 140 kindreds, with cloned DNA sequences derived from Xp21 and known to show deletions in some DMD patients. Sixteen cases showed a deletion, as defined by lack of hybridisation to one or more of the four probes used. However, two of these cases were brothers, so 15 independent deletions (10.7%) are represented. The deletion map is compatible with the suggested order for the sites of the probes used in the study, that is, telomere----pERT87.15----pERT87.8----pERT87.1----pX J1.1----754----centromere. Further mapping of these deletions and characterisation of the deletion breakpoints should facilitate more accurate molecular localisation of the gene or genes which, when mutated, are responsible for causing DMD.     

Comparisons between direct microscopic and cultural methods for recognition of Corynebacterium vaginale in women with vaginitis.

The frequency with which clue cells could be detected in Gram-stained vaginal smears and/or cervical Papanicolaou (Pap) smears was compared with the frequency of Corynebacterium vaginale (Haemophilus vaginalis) isolation in a group of 236 female patients, of whom 221 had vaginitis. Vaginal clue cells were found most often in women from whom C. vaginale was isolated (P = 0.00006) whereas, conversely, clue cells in cervical Pap smears were reported more frequently in women with negative cultures for this organism (P = 0.006). C. vaginale isolations were made more frequently from women with both vaginal and cervical clue cells reported (P = 0.000088). However, the combined false positive-false negative vaginal clue cell rate in the patients studied was 36.5%. Neither the detection of vaginal clue cells nor the isolation of C. vaginale was significantly affected by whether or not patients had trichomoniasis (P = 0.25). Trichomonas vaginalis detection in cervical Pap smears and vaginal isolation were related (P = 0.00005), whereas the same relationship was not significant for fungi (P = greater than 0.05).     

Characterization of translational frame exception patients in Duchenne/Becker muscular dystrophy

The clinical progression of Duchenne muscular dystrophy (DMD)patients with deletions can be predicted in 93% of cases bywhether the deletion maintains or disrupts the translationalreading frame (frameshift hypothesis). We have identified andstudied a number of patients who have deletions that do notconform to the translational frame hypothesis. The most commonexception to the frameshift hypothesis is the deletion of exons3 to 7 which disrupts the translational reading frame. We identifieda Becker muscular dystrophy (BMD) patient, an intermediate,and a DMD patient with this deletion. In all three cases, dystrophinwas detected and localized to the membrane. One DMD patientwith an inframe deletion of exons 4–18 produced no dystrophin.One patient with a mild intermediate phenotype and a deletionof exon 45, which shifts the reading frame, produced no dystrophin.Two patients with large inframe deletions had discordant phenotypes(exons 3–41, DMD; exons 13–48, BMD), but both produceddystrophin that localized to the sarcolemma. The DMD patient,113, indicates that dystrophin with an intact carboxy terminuscan be produced in Duchenne patients at levels equivalent tosome Beckers. The dystrophin analysis from these patients, togetherwith patients reported in the literature, indicate that morethan one domain can localize dystrophin to the sarcolemma. Lastely,the data shows that although most patients show correlationof clinical severity to molecular data, there are rare patientswhich do not conform.     

Effect of Shipment, Storage, Anticoagulant, and Cell Separation on Lymphocyte Proliferation Assays for Human Immunodeficiency Virus-Infected Patients

Lymphocyte proliferation assays (LPA), which can provide important information regarding the immune reconstitution of human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy, frequently involve shipment of specimens to central laboratories. In this study, we examine the effect of stimulant, anticoagulant, cell separation, storage, and transportation on LPA results. LPA responses of whole blood and separated peripheral blood mononuclear cells (PBMC) to different stimulants (cytomegalovirus, varicella-zoster virus, candida and tetanus toxoid antigens, and phytohemagglutinin) were measured using fresh specimens shipped overnight and frozen specimens collected in heparin, acid citrate dextrose (ACD), and citrate cell preparation tubes (CPT) from 12 HIV-infected patients and uninfected controls. Odds ratios for positive LPA responses were significantly higher in separated PBMC than in whole blood from ACD- and heparin-anticoagulated samples obtained from HIV-infected patients and from ACD-anticoagulated samples from uninfected controls. On separated PBMC, positive responses were significantly more frequent in fresh samples compared with overnight transportation for all antigens and compared with cryopreservation for the candida and tetanus antigens. In addition, viral antigen LPA responses were better preserved in frozen PBMC compared with specimens shipped overnight. CPT tubes yielded significantly more positive LPA results for all antigens, irrespective of the HIV patient status compared with ACD, but only for the candida and tetanus antigens and only in HIV-negative controls compared with heparin. Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples. We conclude that LPA should be performed on site, whenever feasible, by using separated PBMC from fresh blood samples collected in either heparin or ACD. However, if on-site testing is not available, optimal transportation conditions should be established for specific antigens.     

Production of genetically modified cells expressing specific transgenes by retroviral vectors for gene therapy

Summary Techniques and protocols are described for the generation of genetically modified cells that can be used for gene therapy. Primary fibroblast cultures are established from skin biopsies, maintained in culture, frozen for long-term storage, and retrieved when necessary. Retroviral packaging cell lines are generated by transfection of DNA into retroviral packaging cells by calcium-phosphate precipitation method or by lipofection method. To generate cell lines expressing high titer virus, individual colonies of cells are cloned and the virus titer is determined. Virus collected from packaging cells expressing high titer virus is then used to infect primary fibroblasts. To obtain fibroblast cell lines expressing high amounts of transgenes, individual cells can be cloned to generate clonal cell lines. Although the methods described here are for fibroblasts, the same methods or modification of the methods can be used for other cell types.     

Additive Effects of Older Brothers and Homosexual Brothers in the Prediction of Marriage and Cohabitation

Research has shown that male homosexuality tends to cluster in families and that homosexual males have, on average, a greater number of older brothers than do heterosexual males. This study investigated whether the former, between-families effect and the latter, within-families effect are additive. The subjects were 717 full siblings over age 40 reported by 343 heterosexual and homosexual male probands examined in Southern Ontario in 1994–1995. The sibling's history of legal marriage or cohabitation in a heterosexual relationship was taken as a proxy variable for sexual orientation. There were no significant findings for the female siblings. As expected, the never-married male siblings were more likely to come from the sibships of the homosexual probands, and they had a greater average number of older brothers. A bootstrapped logistic regression analysis showed that an additive model best explained the male siblings' data. The results suggest that the familial aggregation of male homosexuality cannot be explained by the birth order effect and that older brothers and family membership reflect separate influences on sexual orientation or sexual orientation-correlated behavior.     

Sequence of Echinochloa hoja blanca tenuivirus RNA-5

The sequence is presented of RNA-5 of Echinochloa hoja blanca tenuivirus, a second tenuivirus associated with rice cultivation in Latin America (after rice hoja blanca virus). The RNA is 1334 nucleotides long and contains in the complementary sense RNA a single long open reading frame. The deduced amino acid sequence of this open reading frame shows that it encodes a highly basic and hydrophilic 44 kD protein (pc5) with about 50% similarity to the pc5 protein of maize stripe virus (MStV). This and other features of the RNA are discussed.The GenBank accession number of the sequence reported in this paper is L47430.     

Norethisterone treatment to control timing of the IVF cycle

The use of norethisterone to control the timing of the precedingmenstrual cycle and in consequence the timing of the in-vitrofertilization (IVF) cycle has been evaluated in a therapeuticIVF programme in which oocyte recovery was limited to 2 dayseach week. A consecutive series of 181 cycles after norethisteroneand 29 untreated controls were compared. Menstruation occurred2– 3 days after norethisterone as planned in 82% of patientsoverall and in 87% of patients whose menstrual cycle lengthvaried by no more than 2 days about the median. Norethisteronetreatment did not significantly affect the outcome of IVF treatmentcompared with the controls in respect to cycles abandoned (12versus 0%, respectively), peak follicular diameter (mean 18.1mm versus 18.3 mm 48 h before laparoscopy), oocyte recoveryrate (4.6 versus 4.5 per patient), oocyte morphology (63% versus52% mature), or fertilization rate (72 versus 65% of matureoocytes). Clinical pregnancies were too few for comparison (rates27 versus 9% per laparoscopy) but the overall rate (23%) indicatedeffectiveness of the methods. Prior norethisterone treatmentappears to be an effective and useful means of controlling thetiming of the oocyte recovery in IVF treatment.