Biomedical subjects
Publications and source records attributed to P Merel.
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To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an 'in house' protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and > or =97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was approximately 49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis.
Thirty renal transplant recipients, after transplantation, were tested weekly with the following assays: cytomegalovirus (CMV) antigenemia (pp65 Ag), plasma qualitative Amplicor CMV (P-AMP), plasma and peripheral blood leukocyte quantitative Amplicor CMV monitor (P- and PBL-CMM), peripheral blood leukocyte (PBL) quantitative Quantiplex bDNA CMV, version 2.0 (bDNA), and whole-blood Nuclisens pp67 CMV (pp67). Eleven patients developed symptomatic CMV disease, and 7 developed asymptomatic CMV infection. For prediction of CMV disease, the sensitivity, specificity, and positive and negative predictive values, respectively, were as follows: 100%, 63%, 61%, and 100% for pp65 Ag; 100%, 42%, 50%, and 100% for bDNA; 91%, 47%, 50%, and 90% for PBL-CMM; 55%, 74%, 55%, and 74% for P-AMP; 55%, 74%, 55%, and 74% for P-CMM; and 64%, 79%, 64%, and 79% for pp67. First positive results in PBL were obtained 9-10 days before symptoms of CMV disease, compared with 5-6 days in plasma and 0 days in whole blood. PBL assays appear to be more appropriate than plasma assays when pre-emptive therapy is required to prevent the rapid progression from the first detection of the virus to CMV disease.
OBJECTIVE: The pathogenic role of TT virus (TTV) is not well known, especially during chronic hepatitis C virus (HCV) infection. We retrospectively investigated the presence of TTV DNA in the plasma of patients with chronic HCV infection and compared the characteristics of TTV-DNA-positive and -negative groups. METHODS: Between November 1996 and November 1998, 234 patients were included. Inclusion criteria were persistently elevated serum alanine aminotransferase (ALT) levels, anti-HCV and HCV-RNA positivity, and seronegativity for hepatitis B virus and human immunodeficiency virus markers. TTV DNA was amplified in nested polymerase chain reaction with TTV-specific primers, and products were analyzed by agarose-gel electrophoresis. Data were analyzed using the chi2, Fisher's exact test, or Mann-Whitney test, as appropriate. RESULTS: TTV DNA was detected in 19 (8.1%; 95% confidence interval: 4.6-11.6%) patients. TTV-DNA-positive and TTV-DNA-negative patients did not differ statistically for age, gender ratio, source of HCV infection, HCV disease duration, biological parameters, histological grade, HCV-RNA load, or HCV genotype. Although nonsignificant (p = 0.21), there was a trend for a higher prevalence of TTV DNA in patients with an unknown cause of HCV infection (4/22, 18.2%) than in intravenous drug users (4/84; 4.8%), in those exposed to potential risk factors (4/49; 8.2%), or in those having received blood transfusion (7/79; 8.9%). CONCLUSIONS: Because the rates of HCV replication and the severity of liver lesions in TTV-DNA-negative and -positive patients were similar, the hepatic pathogenicity of TTV in chronic hepatitis C patients is questionable.
BACKGROUND AND OBJECTIVES: Posttransfusion hepatitis still occurs at an incidence of about 1 in 118,000 for HBV and 1 in 220,000 for HCV. This collaborative study aimed to determine the prevalence of a novel flavivirus, GBV-C/HGV, even though its role in transfusion-associated hepatitis is uncertain. MATERIALS AND METHODS: GBV-C/HGV RNA was detected by PCR using either the Boehringer detection kit or by primers previously described. HGV antibodies were detected by a serological assay from Boehringer. RESULTS: The observed GBV-C/HGV RNA frequency was 3.4%. HGV antibodies occurred in 9.5% of donors. CONCLUSION: In our study, 12. 9% of the donors had been in contact with the GBV-C/HGV virus.
Umbilical cord blood (UCB) cells from HLA-matched donors are used as an alternative to bone marrow for allogeneic transplantation and reports of successful UCB transplantation in patients with severe aplastic anemia (SAA) are scarce. SAA was discovered in a 4-year-old girl in February 1990. Transfusion support started in August 1990 and standard treatments were unsuccessful. The birth of an HLA-compatible brother in October 1993 permitted the cryopreservation of UCB. In December 1994 UCB transplantation was decided upon. No toxicity occurred. G-CSF was started at day 28. WBC and PMN reached 0.5 x 10(9)/l at days 33 and 37. RBC and platelet transfusion independence were reached at days 50 and 52. Mixed chimerism was demonstrated in blood cells at 1.5, 4 and 6 months after UCBT by molecular biology (VNTR). FISH studies yielded similar results at 15 and 18 months. Twenty months after UCBT, molecular biology showed full donor chimerism. Clinical follow-up (last follow-up: 32 months post transplant) is unremarkable. We suggest that CY and ATG may be a suitable regimen for related HLA-compatible UCBT in patients with SAA. Residual recipient cells can disappear even very late after UCBT, permitting the establishment of complete donor chimerism.
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Recurrent deletions of chromosome fragments observed in neoplasms are thought to participate in tumor development through the inactivation of tumor-suppressor genes. In gliomas, the most frequent deletions involve chromosome arms 9p, 10q, 17p, 19q and 22q. We have analysed deletions of chromosome 22 in gliomas by studying loss of heterozygosity (LOH) at 8 microsatellite loci. LOH for this chromosome fragment was observed in 17/70 (24%) cases, most of them encompassing the region which encodes the gene altered in neurofibromatosis 2 (NF2), an inherited disease which predisposes to tumors of the nervous system. To investigate the possible involvement of the NF2 tumor-suppressor gene in the tumorigenesis of gliomas, we searched for alterations in its genomic structure and in its mature transcript. Northern-blot and reverse transcriptase-PCR experiments showed that the NF2 transcript is expressed and does not demonstrate obvious structural alterations. Moreover, analysis, at the genomic level, of the 16 coding exons of the NF2 gene by denaturing gradient gel electrophoresis failed to detect any somatically acquired point mutations. Altogether, these data strongly suggest that, although gliomas demonstrate recurrent chromosome 22 deletions most frequently encompassing the NF2 region, the NF2 gene is not altered in these tumors.
A family is presented in which neurofibromatosis type 2 (NF2) and autosomal dominant hearing loss segregate in an apparently independent way. The presence of the latter condition caused anxiety in all family members at risk for NF2 in whom hearing loss became apparent. Previously, we identified a G-->A transition in the donor splice site of exon 5 of the NF2 gene in a family member with proven NF2. As expected, the mutation was present in two other family members who fulfilled the diagnostic criteria for NF2. Four out of five family members at risk for NF2 developed hearing loss. Two of these had the G-->A transition. The mutation was absent in the two other individuals with hearing loss and in the fifth family member without hearing loss or other clinical symptoms. In this family, the identification of the underlying NF2 gene mutation excluded NF2 as the cause of hearing loss in two potential carriers of the mutated gene. On the other hand, it enabled the identification of two carriers of the NF2 gene mutation who did not fulfill the diagnostic criteria for NF2. They will have to be monitored very carefully for the development of NF2-associated tumors. The consistent association within this family of a relatively mild clinical phenotype with the NF2 mutation, supports earlier suggestions that intrafamilial variability is small in NF2.
Schwannomas are benign tumors of cranial, spinal, and other nerve sheaths that develop sporadically or are inherited as part of neurofibromatosis type 2 (NF2). The NF2 gene (SCH) on chromosome 22 has recently been identified and shown to be inactivated by mutation and allele loss in some schwannomas. However, only limited regions in the SCH coding region were examined for mutations. We have extended these studies by screening virtually all coding sequences of the SCH gene (95% coverage) and adjacent splice site sequences for the presence of mutations in 48 schwannomas. All tumors (34 vestibular schwannomas and 14 schwannomas of other locations) were additionally characterized for allele loss on chromosome 22. By PCR-DGGE screening of the 16 known exons of the SCH gene, 22 mutations were found. Most of these give rise to a premature stop codon and are expected to result in the synthesis of a truncated gene product (schwannomin). Although there was no apparent hotspot for mutations, 16 of the 22 mutations occurred in the first eight exons or adjacent splice site sequences of the SCH gene. In several vestibular as well as other schwannomas loss of one SCH allele and mutational inactivation of the second allele were identified in the same tumor. Our data indicate that the SCH gene is implicated in the development of schwannomas of all locations in the nervous system.
Meningiomas are common central nervous system tumours which present usually in the 4th and 5th decades of life. Loss of constitutional heterozygosity on chromosome 22 in 60% of sporadic meningiomas has implied the involvement of a tumour suppressor gene. The neurofibromatosis type 2 gene (NF2), a prime candidate for involvement in meningioma, was screened for point mutations. After examining eight of the 16 known NF2 exons in 151 meningiomas, 24 inactivating mutations were characterized. Significantly, these aberrations were exclusively detected in tumours which lost the other chromosome 22 allele. These results provide strong evidence that the suppressor gene on chromosome 22, frequently inactivated in meningioma, is the NF2 gene, and suggest that another gene is involved in the development of 40% of meningiomas.
Schwannomas are tumors arising from schwann cells surrounding peripheral nerves. Although most schwannomas are sporadic, they are seen in approximately 90% of individuals with neurofibromatosis type 2 (NF2), an autosomal dominantly inherited disease with an incidence of 1:40000 live births. The NF2 gene has recently been isolated on chromosome 22 and encodes a putative membrane organizing protein named schwannomin. It is believed to act as a tumor suppressor gene based on the high frequency of loss of heterozygosity (LOH) on this autosome in both sporadic and NF2 associated schwannomas and meningiomas and the identification of inactivating mutation in NF2 patients. In this study we examined 61 schwannomas including 48 sporadic schwannomas (46 of which are vestibular schwannomas) and 12 schwannomas obtained from NF2 patients, for mutations in 10 of the 16 coding exons of the NF2 gene. Twelve inactivating mutations were identified, 8 in sporadic tumours and 4 in tumors from people with NF2. These results support the hypothesis that loss of function of schwannomin is a frequent and fundamental event in the genesis of schwannomas.
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Neurofibromatosis type 2 (NF2) is a monogenic dominantly inherited disease predisposing carriers to develop nervous system tumours. To identify the genetic defect, the region between two flanking polymorphic markers on chromosome 22 was cloned and several genes identified. One is the site of germ-line mutations in NF2 patients and of somatic mutations in NF2-related tumours. Its deduced product has homology with proteins at the plasma membrane and cytoskeleton interface, a previously unknown site of action of tumour suppressor genes in humans.
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We describe in this report a computer program for easy interpretation of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns generated during HLA Class II typing by the modified PCR-RFLP method. HLA-DQA1, HLA-DQB1, HLA-DRB1 and HLA-DPB1 typing result analysis are thus greatly simplified. The program also allows video capture and image storage, which are becoming a new standard in molecular biology laboratories.