Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.      

Falloposcopy in conjunction with laparoscopy: possibilities and limitations

Falloposcopy is a transvaginal microendoscopic technique to explore the human Fallopian tube from the uterotubal ostium to the fimbrial end. Falloposcopy provides a unique possibility to visualize endotubal disease and may be used therapeutically for removal of debris and for cutting down filmy intraluminal adhesions. To assess the clinical performance of falloposcopy as part of an infertility investigation, a total of 43 women scheduled for laparoscopy as part of an investigation of infertility had a falloposcopy performed in conjunction with the laparoscopy. All women were investigated at Danderyd Hospital, Stockholm and Akademiska Hospital, Uppsala, during 1995 and 1996. Images from the endosalpinx were obtained in 26 of 43 women (60.5%). In 10 women (23.3%), it was possible to obtain images from both tubes. No images were of sufficient quality to describe the entire tubal mucosa in detail. Falloposcopy represents a unique tool for visualization of endotubal disease and may provide a valuable instrument for in-vivo exploration of tubal physiology. However, certain technical problems limit the usefulness of this method in routine clinical practice. These technical problems have to be solved before falloposcopy can achieve a central position in investigation and treatment of tubal disease.      

Localization of a novel X-linked congenital stationary night blindness locus: close linkage to the RP3 type retinitis pigmentosa gene region

X-linked congenital stationary night blindness (CSNBX) is anon-progressive retinal disorder characterized by decreasedvisual acuity and loss of night vision. CSNBX Is clinicallyheterogeneous with respect to the involvement of retinal rodsand/or cones in the disease. in this study, we localize a newlocus for CSNBX to Xp21.1, thus providing evidence that CSNBXis also genetically heterogeneous. A clear correlation betweendifferent genotypes and phenotypes cannot be found yet. Thenew CSNBX gene described here is closely linked to the X-linkedretinitis pigmentosa type 3 gene region, which supports thehypothesis that there may be a functional relationship betweencongenital stationary night blindness and retinitis pigmentosa.     

Involving the patient: a prospective study on use, appreciation and effectiveness of an information system in head and neck cancer care

OBJECTIVE: To determine use, appreciation and effectiveness of an electronic health information support system in head and neck (H&N) cancer care. DESIGN: A prospective evaluation study. The evaluated system has four different functions: (1) communication amongst health care providers and between health care providers and patients, (2) information for health care providers and patients, (3) contact with fellow sufferers and (4) monitoring of discharged patients by means of electronic questionnaires. Evaluation of the system was done both objectively using automatically created log files and stored messages, and subjectively by using paper questionnaires from patients and general practitioners (GPs). SETTING: Department of Otorhinolaryngology and Head and Neck Surgery of a tertiary health care centre in the Netherlands. The system was put at patients' disposal for a period of 6 weeks following discharge from the hospital after surgery for H&N cancer, and was additional to standard care. PARTICIPANTS: Head and neck cancer patients, hospital physicians, members of a hospital-based support team, GPs, district nurses and speech therapists. MAIN OUTCOME MEASURES: Actual use of the system by patients and health care providers. Patients' appreciation for each of the system's four different functions. GPs' appreciation for the system. Capability to detect potential patient problems with the system. RESULTS: The system was used by 36 H&N cancer patients, 10 hospital physicians, 2 members of the support team, 8 GPs, 2 district nurses and 2 speech therapists. The total number of patient-sessions was 982: an average of 27.3 sessions per patient during the 6 weeks study period. In total, 456 monitoring questionnaires were completed. The support team in hospital responded with 231 actions. In 16 cases, an extra appointment was made for a patient with the hospital physician. Out of these cases, immediate action was considered necessary eight times. Patients appreciated the system highly, rating it with an average score of 8.0 on a 10-point scale. All patients used the monitoring function, and rated 'monitoring' with a mean score of 8.0 on a 10-point scale. Least used and appreciated was the 'contact with fellow sufferers' function. Only 8 out of possible 36 GPs used the system, rating it with an average of 5.6 on a 10-point scale. CONCLUSIONS: The electronic health information support system was used intensively and highly appreciated by H&N cancer patients. The system enabled the early detection of occurring health problems that required direct intervention. ICT can play an additional role in the management of patients, also in a relatively elderly and computer illiterate patient population.     

High level of unequal meiotic crossovers at the origin of the 22q11. 2 and 7q11.23 deletions

Interstitial chromosomal deletions at 22q11.2 and 7q11.23 are detected in the vast majority of patients affected by CATCH 22 syndromes and the Williams-Beuren syndrome, respectively. In a group of 15 Williams- Beuren patients, we have shown previously that a large number of 7q11.23 deletions occur in association with an interchromosomal rearrangement, indicative of an unequal crossing-over event between the two homologous chromosomes 7. In this study, we show that a similar mechanism also underlies the formation of the 22q11.2 deletions associated with CATCH 22. In eight out of 10 families with a proband affected by CATCH 22, we were able to show that a meiotic recombination had occurred at the critical deleted region based on segregation analysis of grandparental haplotypes. The incidences of crossovers observed between the closest informative markers, proximal and distal to the deletion, were compared with the expected recombination frequencies between the markers. A significant number of recombination events occur at the breakpoint of deletions in CATCH 22 patients (P = 2.99x10(-7)). The segregation analysis of haplotypes in three- generation families was also performed on an extended number of Williams-Beuren cases (22 cases in all). The statistically significant occurrence of meiotic crossovers (P = 4.45x10(-9)) further supports the previous findings. Thus, unequal meiotic crossover events appear to play a relevant role in the formation of the two interstitial deletions. The recurrence risk for healthy parents in cases where such meiotic recombinations can be demonstrated is probably negligible. Such a finding is in agreement with the predominantly sporadic occurrence of the 22q11.2 and 7q11. 23 deletions. No parent-of-origin bias was observed in the two groups of patients with regard to the origin of the deletion and to the occurrence of inter- versus intrachromosomal rearrangements.      

Fludarabine-based conditioning secures engraftment of second hematopoietic stem cell allografts (HSCT) in the treatment of initial graft failure.

Graft failure is associated with a high mortality rate. To date, regimens invoked for second transplants have resulted in inconsistent engraftment with high transplant-related mortality (TRM). We here report 16 consecutive patients, aged 4-59 years, who received second HSCT (HSCT-2) at a median of 45 days following primary or secondary failure of an initial unmodified (N = 3) or T cell-depleted (TCD) (N = 13) HSCT (HSCT-1). HSCT-1 was administered after myeloablative total body irradiation (TBI)- or alkylator-based conditioning for acute leukemias (N = 7), MDS (N = 6), CML (N = 2), and Fanconi anemia (N = 1). All patients experienced 1 or more infectious complications between HSCT-1 and HSCT-2, and 10 patients had active infections at the time of HSCT-2. Cytoreduction regimens used for HSCT-2 included fludarabine (Flu) in combination with cyclophosphamide (CTX) (N = 9), or thiotepa (Thio) (N = 5). In addition, 1 patient received Flu alone and 1 patient Thio combined with CTX. Antithymocyte globulin (ATG) (N = 11) or Alemtuzumab (N = 3) was added pretransplant to prevent rejection. For HSCT-2, donors included HLA-matched (N = 3) or mismatched (N = 8) related, or matched (N = 2) or mismatched (N = 3) unrelated donors. The primary graft donor was used in 6 of 16 cases. The grafts administered were unmodified peripheral blood stem cell transplantation (PBSCT) (N = 5) or bone marrow transplantation (BMT) (N = 3), TCD PBSCT (N = 8). All patients achieved engraftment at a median of 12 days and evaluable patients achieved complete donor chimerism. Six patients are alive with a median follow-up of 49 months, including 4/9 conditioned with Flu/CTX. In this series, outcome was statistically superior for younger patients (相似文献   

The specificity of the anti-dsDNA ELISA. A closer look

The anti-dsDNA ELISA is probably one of the most popular techniques for determining antibody reactivity towards dsDNA, since this assay system has proven high sensitivity and is easy to perform. An important difference from other ELISA systems is the use of an intermediate layer (e.g., protamine sulphate or poly-L-lysine) which has been found to be necessary in order to obtain sufficient coating of dsDNA to the plates. When a panel of monoclonal antibodies to DNA (n = 56), all reactive in this anti-dsDNA ELISA were tested on plates coated only with protamine sulphate (PS) a large number were positive, although with a lower reactivity than with DNA. Binding to protamine sulphate occurred via two mechanisms: (1) DNA/anti-DNA immune complexes, present in hybridoma culture supernatants and adherent to protamine sulphate and (2) some IgM antibodies appear to possess an intrinsic affinity for PS. The latter mechanism gives rise to a false positive reaction in the anti-dsDNA ELISA. It was found that 18% of the clones that were unreactive in any anti-dsDNA assay other than the anti-dsDNA ELISA were labelled 'anti-dsDNA' incorrectly. We therefore propose that antibody reactivity towards dsDNA in an ELISA system must be confirmed in other anti-dsDNA assays before such antibodies can be termed 'anti-dsDNA'.     

Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.