IRB review and consent in human tissue research.
Explore the source record for details and available documents.
Biomedical Research↗
Biomedical subjects
D G Leonard
Publications and source records attributed to D G Leonard.
Ataxin 1 and ataxin 3 in neuronal intranuclear inclusion disease.
Neuronal intranuclear inclusion disease (NIID) is a multisystem neurodegenerative disorder characterized by large intranuclear aggregates in neurons of the central and peripheral nervous system. These ubiquitinated intranuclear inclusions are morphologically similar to the intraneuronal aggregates that have been identified in the CAG/polyglutamine expansion diseases. As rare aggregates in NIID contain a polyglutamine epitope, we further investigated the relationship between this disease and the CAG/polyglutamine expansion diseases. Here, we show that ataxin 1 and ataxin 3 proteins are recruited into aggregates in NIID in the absence of a CAG expansion in the SCA1 and SCA3 genes. These data support an association of NIID with the polyglutamine disorders and provide evidence of in vivo recruitment of proteins with polyglutamine tracts into intraneuronal aggregates.
Adult↗
A novel, non-nested reverse-transcriptase polymerase chain reaction (RT-PCR) test for the detection of the t(15;17) translocation: a comparative study of RT-PCR cytogenetics, and fluorescence In situ hybridization.
BACKGROUND: The development of a rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) assay is described that identifies the promyelocytic leukemia- retinoic acid receptor alpha (PML-RARa) hybrid messenger RNA (mRNA), a characteristic feature of acute promyelocytic leukemia (APL). METHODS AND RESULTS: Randomly primed complementary (cDNA) is synthesized from leukocyte RNA and amplified in the presence of Taq Gold in 2 separate reaction tubes containing primer pairs specific for intron 3 (bcr 3, long [L] form mRNA transcript) and intron 6 (bcr 1, short [S] form)/exon 6 (bcr 2, variant [V] form) breakpoints in PML, respectively. The different sized products generated from each RNA transcript (S, L, or V forms) are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. The sensitivity of the assay is 1 in 10,000 to 1 in 100,000. The separate amplification of a b2-microglobulin transcript controls for adequate RNA and cDNA preparation. The newly developed assay was used clinically for the evaluation of 78 patients with APL. It was rapid and more sensitive than cytogenetic karyotyping, both for the diagnosis of APL and the assessment of minimal residual disease (MRD) after therapy. RT-PCR detected PML-RARa mRNA in all cases positive for the t(15;17) translocation by cytogenetics. However, as many as 50% and 80% of the diagnostic specimens and the specimens for MRD assessment, respectively, that were positive by RT-PCR were negative by cytogenetics. The ratio of cases with L-form to S-form PML-RARa fusion transcript was 2:1, whereas 3 cases (10%) had fusion sites in exon 6 of the PML gene (V forms). In addition, approximately 50% of the patients were diagnosed morphologically with microgranular M3V-type leukemia, but no significant correlation with PML breakpoints was found. CONCLUSION: The current assay is rapid, sensitive, and specific without using nested PCR or hybridization.
Biomarkers, Tumor↗
Molecular genetic testing from paraffin-embedded tissue distinguishes nonmolar hydropic abortion from hydatidiform mole.
BACKGROUND: Diagnosis of hydatidiform mole by histology and ploidy analysis is limited by overlap of criteria for nonmolar hydropic abortion, complete mole, and partial mole. With early presentation, diagnosis is difficult due to limited tissue and lack of clinical features. Accurate diagnosis of these entities is important for both prognosis and patient management. We assessed a polymerase chain reaction (PCR) assay for polymorphic short tandem repeats (STR) for discrimination between nonmolar hydropic abortion, complete mole, and partial mole based on the genetic composition of molar pregnancies. METHODS: Seventeen cases of products of conception (POC) diagnosed by histology and flow cytometry ploidy analysis were studied retrospectively. PCR was performed using maternal and chorionic villus DNA extracted from microdissected, formalin-fixed, paraffin-embedded tissue sections. Allelic patterns for up to eight well-characterized polymorphic STR loci were determined using the GenePrint Fluorescent STR System (Promega Corporation, Madison, WI). The presence of three villus alleles at a single locus was interpreted as partial mole. Detection of only one allele in the villi, different from all maternal allele(s) at the same locus, was interpreted as a complete mole. RESULTS: This technique identified eight complete moles previously diagnosed as complete mole (3), hydatidiform mole, otherwise unspecified (1), hydropic villi (2), hydropic villi versus partial mole (1), and partial mole (1). The diagnoses of five partial moles by the molecular assay were consistent with the diagnoses by histology and flow cytometry. One nonmolar gestation was identified, which had been diagnosed previously as hydropic villi. In three cases, maternal DNA amplification was insufficient for definitive diagnosis. CONCLUSION: Molecular genetic testing of POC from paraffin-embedded tissue accurately distinguishes complete mole, partial mole, and nonmolar hydropic abortion. Identification of triploidy by flow cytometry can confirm a histological impression of partial mole. Histological and ploidy analysis of POC results in underdiagnosis of complete moles.
Abortion, Spontaneous↗
The utility of clinical and radiographic features in the diagnosis of cytomegalovirus central nervous system disease in AIDS patients.
BACKGROUND: Polymerase chain reaction (PCR) assays of cerebrospinal fluid (CSF) for cytomegalovirus (CMV) DNA have facilitated the diagnosis of CMV-associated central nervous system disease in AIDS patients. We attempt to correlate clinical and radiographic features that are associated with CMV PCR- positivity in CSF from AIDS patients with neurologic disease. METHODS AND RESULTS: A retrospective case controlled comparison was made of CMV PCR-positive and PCR-negative patients. RESULTS: CMV PCR-positive patients were significantly more likely to have nystagmus, prior CMV retinitis, and CSF protein levels.90 mg/dL. Of patients with 0, 1, and $2 of these features, 5.6%, 55.2%, and 88.9%, respectively, were PCR-positive. Ependymal enhancement was present by magnetic resonance imaging in 9 of 12 PCR-positive, and in 8 of 30 PCR-negative patients. CONCLUSION: These clinical and radiographic features may serve as useful adjuncts toward the establishment of the diagnosis of CMV-associated neurologic disease in AIDS patients.
AIDS-Related Opportunistic Infections↗
Thrombosis in a patient with combined homozygosity for the factor V Leiden mutation and a mutation in the 3'-untranslated region of the prothrombin gene.
The factor V Leiden mutation and a guanine-to-adenine mutation at nucleotide 20210 in the 3'-untranslated region of the prothrombin gene are the most prevalent genetic defects in patients with deep venous thrombosis. Heterozygosity for the factor V Leiden and the prothrombin gene mutations is found in approximately 20 and 6% of unselected patients with deep venous thrombosis, respectively, whereas the prevalences of the two mutations in the general Caucasian population are approximately 6 and 2%, respectively. We evaluated an 18-year-old man presenting with a spontaneous episode of superficial venous thrombosis for the presence of an inherited thrombotic disorder. After excluding deficiencies of antithrombin, protein C, and protein S, genomic DNA from the patient was tested for the presence of the factor V Leiden and prothrombin gene mutations. Consanguinity was not present in the family. Genotyping demonstrated that the patient was homozygous for the factor V Leiden and prothrombin gene mutations. The likelihood of identifying an individual in the general population who is homozygous for both mutations similar to our patient is estimated to be less than 1 in 10 million.
3' Untranslated Regions↗
Primary peritoneal carcinoma: a review of the literature.
Explore the source record for details and available documents.
Carcinoma↗
Graft-versus-tumor induction with donor leukocyte infusions as primary therapy for patients with malignancies.
PURPOSE: Histocompatible allogeneic donor leukocyte infusions (DLIs) were administered as primary cancer therapy in a phase I trial to determine (1) whether mixed chimerism could be detected without a prior allogeneic transplantation, (2) the toxicity of primary DLI, and (3) whether a graft-versus-tumor (GVT) reaction could be observed. PATIENTS AND METHODS: Eighteen patients were studied. Patients received interferon alfa-2b for a minimum of 4 weeks, followed by DLI (level 1). Patients with no toxicity or engraftment were eligible to receive cytarabine or cyclophosphamide followed by another course of DLI (level 2). Engraftment was determined using polymerase chain reaction amplification of donor and host-specific DNA polymorphisms. RESULTS: Donor cells were detected in the blood in 14 of 16 assessable patients within 1 hour of DLI. Chimerism detectable 4 weeks after DLI was observed in four patients, and five patients were not assessable. Prior autologous transplantation was associated with late chimerism (P =.0014). Acute graft-versus-host disease (GVHD) occurred in four of 16 assessable patients (grade 1, two patients; grade 2, one patient; grade 4, one patient). One patient with grade 4 acute GVHD developed pancytopenia. Only the four patients treated after prior autologous transplantation developed acute GVHD (P =.0005). Three of four patients with acute GVHD and late chimerism responded to primary DLI, and one patient was not assessable for response. CONCLUSION: Allogeneic adoptive immunotherapy resulted in sustained chimerism, acute GVHD, and a GVT response in heavily pretreated patients. This indicates that it may be possible to generate a direct GVT response for patients with malignancies without the need for intensive conditioning therapy immediately before DLI. Immunosuppression may be required for sustained donor cell engraftment.
Adolescent↗
The future of molecular genetic testing.
The potential applications for genetic testing are immense, with most diseases having some aspect influenced by, if not directly caused by, changes in the genome of the patient. The translation of genetic information into medical applications will be influenced by our understanding of the human genome, technological advances, and social, ethical, and legal issues surrounding genetic testing. With time, new genetic information will be translated into clinical tests for the diagnosis of current illness and prediction of future disease risk, and will be used for the development of genetically directed therapies and preventive interventions. Most genetic testing will be highly automated, with only rare genetic disease tests performed manually. The challenge for the clinical genetic laboratory is to keep pace with this information explosion to provide state-of-the-art genetic testing and to ensure that the genetic test results are used in a morally, ethically, and socially responsible way.
DNA↗
Issues in genetic testing.
Explore the source record for details and available documents.
Chemistry, Clinical↗
Association of germline p53 mutation with MLL segmental jumping translocation in treatment-related leukemia.
Segmental jumping translocations are chromosomal abnormalities in treatment-related leukemias characterized by multiple copies of the ABL and/or MLL oncogenes dispersed throughout the genome and extrachromosomally. Because gene amplification potential accompanies loss of wild-type p53, we examined the p53 gene in a case of treatment-related acute myeloid leukemia (t-AML) with MLL segmental jumping translocation. The child was diagnosed with ganglioneuroma and embryonal rhabdomyosarcoma (ERMS) at 2 years of age. Therapy for ERMS included alkylating agents, DNA topoisomerase I and DNA topoisomerase II inhibitors, and local radiation. t-AML was diagnosed at 4 years of age. The complex karyotype of the t-AML showed structural and numerical abnormalities. Fluorescence in situ hybridization analysis showed multiple copies of the MLL gene, consistent with segmental jumping translocation. A genomic region including CD3, MLL, and a segment of band 11q24 was unrearranged and amplified by Southern blot analysis. There was no family history of a cancer predisposing syndrome, but single-strand conformation polymorphism (SSCP) analysis detected identical band shifts in the leukemia, ganglioneuroma, ERMS, and normal tissues, consistent with a germline p53 mutation, and there was loss of heterozygosity in the ERMS and the t-AML. Sequencing showed a CGA-->TGA nonsense mutation at codon 306 in exon 8. The results of this analysis indicate that loss of wild-type p53 may be associated with genomic instability after DNA-damaging chemotherapy and radiation, manifest as a complex karyotype and gene amplification in some cases of t-AML.
Acute Disease↗
Novel bcl-2 breakpoints in patients with follicular lymphoma.
Using genomic DNA from patients with follicular lymphoma, we performed polymerase chain reaction (PCR) amplifications to detect t(14;18) translocations. Unexpectedly large products of approximately 1 kilobase (kb) were detected by gel electrophoresis in 2 of 50 positive cases. In these 2 cases, sequence analyses showed novel breakpoints in the 3' untranslated region of bcl-2, approximately 800 bp downstream of the major breakpoint region (mbr). The breakpoints in IgH occurred in JH4 in one patient and JH5 in the other. Sequences just upstream of the new bcl-2 breakpoints suggest a mechanism of translocation that may include minisatellite core-mediated recombination. In one of our two patients with novel bcl-2 breakpoints, the approximately 1 kb product obtained using conventional mbr primers was detectable only when a nested PCR was performed. These findings have important implications for diagnosis and minimal residual disease detection in t(14;18)-positive lymphomas.
Adult↗
Proteolipid protein is necessary in peripheral as well as central myelin.
Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are major components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.
Adolescent↗
High incidence of Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus in tumor lesions and peripheral blood mononuclear cells from patients with Kaposi's sarcoma in Uganda.
With the advent of AIDS, Kaposi's sarcoma (KS) has become one of the leading malignancies in sub-Saharan Africa. Recently, DNA sequences from a new human herpesvirus called KS-associated herpesvirus (KSHV) or human herpesvirus type 8 have been found in KS tumor lesions in high frequency. Analyses of tumor lesions from 38 Ugandan KS patients indicated a uniform presence of KSHV in KS tumor lesions as revealed by polymerase chain reaction and Southern hybridization. In contrast, only 31% (11/36) of the normal skin biopsies from the same patient population were positive. The frequency of KSHV DNA detection in peripheral blood mononuclear cells (PBMC) of KS patients was also high (84%, 31/37). Similar analyses revealed the presence of cytomegalovirus (21% in KS lesions) to be discordant with KS development. A large number of KS lesions (87%, 33/38) and KS PBMC (70%, 26/37) were, however, positive for Epstein-Barr virus sequences. In addition, KSHV DNA was not found in the PBMC of Ugandans without KS.
Adult↗
Polymerase chain reaction detection and clinical significance of varicella-zoster virus in cerebrospinal fluid from human immunodeficiency virus-infected patients.
Varicella-zoster virus (VZV) causes ocular and other central nervous system (CNS) disease in human immunodeficiency virus (HIV)-infected persons. To study the prevalence of CNS disease due to VZV, cerebrospinal fluid (CSF) specimens from 84 consecutive HIV-infected patients with new neurologic symptoms were tested for VZV DNA by a polymerase chain reaction (PCR) assay. Six patients were PCR-positive for VZV in CSF; 3 additional patients were subsequently identified who were not part of the serial population sample. Among these 9 patients, all had clinical presentations consistent with ocular and other CNS disease due to VZV; 4 were without zoster on presentation. Sustained improvement in association with antiviral therapy was observed in 3. Therefore, VZV DNA was detected in the CSF of 7% of HIV-infected patients presenting with neurologic symptoms; the diagnosis of VZV-related CNS disease was facilitated by this assay; improvement in association with antiviral therapy was observed in some patients.
Adult↗
Kaposi sarcoma-associated herpesvirus/human herpesvirus type 8 and Epstein-Barr virus in iatrogenic Kaposi sarcoma.
Explore the source record for details and available documents.
Aged↗
Signal transduction pathway activating interferon-alpha-stimulated gene expression.
Interferon-alpha (IFN alpha) causes profound physiological changes following binding to susceptible target cells. These changes, which include induction of an antiviral state, inhibition of cellular proliferation, and modulation of differentiation, require the transcriptional activation of a set of genes. We have characterized the macromolecular components required for this stimulation of gene expression in order to define the biochemical mechanism of IFN alpha signal transduction. IFN alpha stimulated genes (ISGs) are immediate response genes which utilize a pre-existing set of proteins to mediate their induction. A 15 bp IFN alpha-inducible enhancer element present in the promoters of IFN alpha-stimulated genes, termed the IFN alpha stimulated response element (ISRE), is the genetic target for activation of ISGs. This DNA sequence is both necessary and sufficient for transcriptional activation and is the target for action of a positive transcription factor termed ISGF3. The active, DNA-binding form of ISGF3 is only found in cells which have been exposed to IFN alpha; however, it is activated from a silent form present in all responsive cells. ISGF3 is a multimeric complex assembled from cytoplasmic precursors which are translocated to the nucleus in response to IFN alpha. Assembly and translocation of ISGF3 is the earliest defined event in the IFN alpha response pathway.
Cytoplasm↗
A nerve growth factor-regulated messenger RNA encodes a new intermediate filament protein.
Differential screening of a cDNA library from the PC12 rat pheochromocytoma cell line previously revealed a clone, clone 73, whose corresponding mRNA is induced by nerve growth factor (NGF). Induction parallels NGF-stimulated PC12 differentiation from a chromaffinlike phenotype to a sympathetic neuronlike phenotype. We report that DNA sequence analysis reveals that clone 73 mRNA encodes an intermediate filament (IF) protein whose predicted amino acid sequence is distinct from the known sequences of other members of the IF protein family. The sequence has highest homology with desmin and vimentin and includes the highly conserved central alpha-helical rod domain with the characteristic heptad repeat of hydrophobic residues, but has lower homology in the amino-terminal head and carboxyl-terminal tail domains. The head domain contains a large number of serine residues which are potential phosphorylation sites. The expression of clone 73 in vivo in the nervous system of the adult rat was investigated by in situ hybridization of clone 73 probes to tissue sections. The mRNA is expressed at high levels in ganglia of the peripheral nervous system, including the superior cervical ganglion (sympathetic), ciliary ganglion (parasympathetic), and dorsal root ganglion (sensory). In the central nervous system, motor nuclei of cranial nerves III, IV, V, VI, VII, X, and XII as well as ventral horn motor neurons and a restricted set of other central nervous system nuclei express the clone 73 mRNA. Tissues apart from those of the nervous system did not in general express the mRNA, with only very low levels detected in adrenal gland. We discuss the implications of these results for the mechanism of NGF-induced PC12 cell differentiation, the pathways of neuronal development in vivo, and the possible function of the clone 73 IF protein and its relationship to other IF proteins.
Amino Acid Sequence↗