Genetic diagnosis of PLP gene duplications/deletions in patients with Pelizaeus-Merzbacher disease.

Genetic diagnosis of PLP gene duplications/deletions in patients with Pelizaeus-Merzbacher disease.PMD is an X-linked recessive disorder due to a proteolipid protein (PLP) deficiency. Duplications of PLP gene were shown to be the principle cause of the disorder, accounting for an estimated 50-70% of cases. To define a simple and reliable method for genetic diagnosis of PMD, a group of 42 patients with clinical manifestation of PMD was analyzed by means of real-time quantitative PCR. Parallel fluorescence in situ hybridization (FISH) analysis was performed on the same group of patients. Real-time PCR found seventeen samples had increased gene dosage, whereas FISH detected sixteen duplicated samples. Both methods identified a sample with PLP gene deletion. Our results indicate that real-time PCR is a sensitive and reliable method for the detection of gene duplications/deletions. We further discussed the advantages and limitations of each method in clinical diagnosis of PMD.

HER-2/neu expression in germ cell tumours.

AIMS: To determine the rate of HER-2/neu positivity of germ cell tumours by immunohistochemistry (IHC) and by fluorescence in situ hybridisation (FISH). PATIENTS/METHODS: Ninety six archival, paraffin wax embedded pathology specimens were chosen from four groups of germ cell tumours. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the INFORM assay and confirmed with a centromere 17 probe. RESULTS: Twenty two of 96 specimens overexpressed the HER-2/neu protein when measured by IHC. Only three specimens showed HER-2/neu gene amplification by FISH. There was no correlation between the results obtained by IHC and FISH. CONCLUSIONS: The lack of concordance between IHC and FISH makes it unlikely that overexpression of the HER-2/neu protein in germ cell tumours is of prognostic or therapeutic relevance. Because of the low rate of HER-2/neu gene amplification in germ cell tumours, a clinical trial of trastuzumab treatment in patients with germ cell tumours is not warranted.

Detection of monosomy 7 in bone marrow by fluorescence in situ hybridization. A study of Fanconi anemia patients and review of the literature.

Monosomy 7 is frequently found in the bone marrow of patients with Fanconi anemia (FA), marrow myelodysplasia, or acute myelogenous leukemia and is associated with poor prognosis. In our laboratory, cytogenetic analysis of bone marrow from an FA patient found 2 of 30 cells with monosomy 7, but the results of fluorescence in situ hybridization (FISH) indicated that 83 of 207 cells (40%) had monosomy 7. FISH was then used to analyze two earlier samples from the index case, neither of which had monosomy 7 as determined by standard cytogenetics. The FISH analysis determined that the first sample, taken 19 months earlier, had 8 of 200 cells (4%) with monosomy 7 and the second sample. taken 7 months later, contained 43 of 200 cells (21.5%) with monosomy 7. These results indicate a slow evolution toward monosomy 7 in the patient's bone marrow. Standard metaphase chromosome analysis represents only spontaneously dividing cells, leading us to hypothesize that FISH was detecting monosomy 7 in nondividing cells and that it might be useful in the early detection of abnormal clones. To test this hypothesis, FISH was performed on 13 bone marrow samples from nine patients with FA who did not exhibit monosomy 7 by cytogenetic analysis. Monosomy 7 was detected in 3.44% of nuclei in FA patients and in 3% of nuclei in normal controls. To date, none of these nine FA patients have developed monosomy 7 or leukemia. They are being monitored by standard cytogenetics and by FISH to determine whether monosomy 7 develops and whether it can be detected by FISH prior to its detection by standard cytogenetics. As standard practice, we have adopted FISH analysis for monosomy 7 in all patients with FA.

Nucleolar localization of the microtubule-associated protein tau in neuroblastomas using sense and anti-sense transfection strategies.

The Tau-1 monoclonal antibody was localized to the nucleolus of interphase cells and the nucleolar organizing regions (NORs) of acrocentric chromosomes in cultured human cells. Putative nucleolar and NOR tau was found in CG neuroblastoma cells which contain nucleolar tau and little cytoplasmic tau. To further establish the presence of tau in the nucleolus of these cells, sense and anti-sense transfection strategies were used. CG neuroblastoma cells were transfected with tau sense cDNA and immunostained with Tau-1. Cytoplasmic Tau-1 staining was greatly increased in CG cells which contain very little endogenous cytoplasmic tau. Nucleolar Tau-1 staining was also increased in certain CG cells indicating an increase in nucleolar tau in a subset of transfected cells. CG cells were also transfected with tau anti-sense cDNA which abolished Tau-1 staining in the nucleolus. These results contribute to a growing body of evidence defining tau as a multifunctional protein found in both the cytoplasm and nucleoli of primate cells.

Tau as a nucleolar protein in human nonneural cells in vitro and in vivo.

The Tau-1 monoclonal antibody was localized to the nucleolus of interphase cells and the nucleolar organizing regions (NORs) of acrocentric chromosomes in cultured human cells. Putative nucleolar and NOR tau was found in HeLa cells and lymphoblasts as well as in nontransformed fibroblasts and lymphocytes. To confirm the presence of tau in the nuclei of these nonneural cells, immunoblotting analysis was performed on isolated nuclei from lymphoblasts. Several tau bands were noted on the blot of the nuclear extract suggesting the presence of multiple tau isoforms. Tau-1 immunostaining demonstrated variable staining intensities between individual acrocentric chromosomes in all cells tested. In cultured peripheral lymphocytes, these staining patterns were the same from one chromosome spread to the next within an individual. This consistency of Tau-1 staining and its variability among NORs was reminiscent of staining patterns obtained using the silver-NOR procedure. Comparisons of Tau-1 immunostaining with silver staining of chromosome spreads from human lymphocytes demonstrated that Tau-1 did not immunostain all of the NORs that were silver stained. The intensity of Tau-1 fluorescence in nucleoli was further shown to be increased in phytohemagglutinin-stimulated lymphocytes, indicating an upregulation of nuclear tau when cells reentered the cell cycle. These results contribute to a growing body of evidence defining tau as a multifunctional protein that may be involved in ribosomal biogenesis and/or rRNA transcription in the nucleus of all cells as well as microtubule-stabilizing functions in the neuronal cytoplasm.