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Query: UMLS:C0019045 (hemoglobinopathies)
 2,704 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Blood specimens dried on filter paper are now widely used for neonatal screening of hemoglobinopathies. These samples are perfectly suited for electrophoresis studies and HPLC analysis. They may also be used for DNA analysis. The structural characterization of a hemoglobin variant is also possible using protein chemistry methods. After elution of the hemoglobin from the paper, the different components are fractionated by a microscale preparative isoelectric focusing. The structural modification of the abnormal hemoglobin is then determined through a series of techniques including chain separation, aminoethylation, trypsin digestion, analysis of the peptides and determination of their aminoacid composition. The efficiency of this strategy is demonstrated by the study of an alpha-chain variant (Hb Hasharon) and three beta-chain variants (Hb S, Hb D Punjab, Hb E). Unambiguous identification of the structural abnormality was obtained with samples stored for up to 18 months and with abnormal fractions amounting to only approximately 10% of the total lysate.
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PMID:Structural characterization of abnormal hemoglobins from dried blood specimens in a neonatal screening program. 821 61

Gene transfer of human globin genes into human pluripotent stem cells via viral vectors may soon be realized. The high level of globin gene expression believed to be required for the treatment of severe hemoglobinopathies necessitated the inclusion of cis-acting sequences (LCR). Retroviral vectors containing the LCR elements are prone to rearrangement, low titer, and poor expression. Inclusion of a "minilocus" containing four HS sites linked to a globin gene resulted in higher expression in transplanted mice, but rearrangement of the provirus still occurs, and it is unclear what significance these experiments have with regard to human marrow stem cell transduction. Recombinant AAV is among the newest of genetic transfer vectors. This once obscure virus possesses unique properties that distinguish it from all other vectors. Its major advantage is the lack of pathogenicity in humans. Wild-type AAV has the unusual ability to selectively integrate into the mammalian genome at a specific region, thus reducing the concern for genomic disruption and insertional mutagenesis. The ability of AAV to carry regulatory elements without interference from the viral template may enable greater control of transferred gene expression. Disadvantages currently include the inferior packaging systems which yield low numbers of recombinant virions which are contaminated with wild-type adenovirus. The small AAV genome that can be packaged (approximately 5 kb) rules out its use for transfer of larger genes. Recombinant AAV viruses do not appear to demonstrate the same site-specific genomic integration as wild-type viruses. Elucidation of the mechanism of site-specific integration should prove useful in the development of safe vectors for gene transfer as well as provide insight into the nature of DNA recombination in humans.
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PMID:Gene therapy for human hemoglobinopathies. 823 72

The province of Ontario has a total population of approximately 10 million people, with approximately 20% being of African, Southeast Asian, East Indian, Mediterranean, or Middle Eastern ancestry in whom the gene frequency for hemoglobinopathies is relatively high. In 1989, the Ontario Ministry of Health funded the establishment of the Provincial Hemoglobinopathy DNA Diagnostic Laboratory located at the McMaster University Medical Centre in Hamilton, Ontario. The Laboratory provides DNA analysis to identify the globin gene mutations in carriers and affected individuals, and performs prenatal diagnosis for severe hemoglobinopathies. Annually, more than 400 patient samples are referred to the Laboratory for investigation, of which 25-35 are fetal samples from pregnancies at risk for either homozygous alpha-thalassemia, beta-thalassemia major, or sickling disorders. We have detected more than 70 different globin gene mutations, including several mutations not previously reported in the literature. Here we present examples of the approaches used to detect globin gene mutations in a heterogeneous "at risk" population such as in Ontario, and discuss the impact of this service on patient care, genetic counselling, and the incidence of severe hemoglobinopathies in Ontario.
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PMID:Carrier detection and prenatal diagnosis of hemoglobinopathies in Ontario. 826 89

At this time, the sole generally accepted use for DNA diagnosis in the hemoglobinopathies is for the prenatal detection of disease, which can be identified by these means early in the first trimester of pregnancy. By ascertaining genotype rather than phenotype, the confusion that results from diagnostic errors should be diminished. DNA diagnostics are the future of all genetic disease detection and this future will soon be upon us.
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PMID:DNA diagnosis for the detection of sickle hemoglobinopathies. 834 37

Five different enzymes, carbamyl phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC) argininosuccinate synthetase (AS), argininosuccinate lyase (AL) and arginase (AR) play a role in urea synthesis from ammonium. The structures of cDNA of all these enzymes and those of genome DNA of some enzymes (OTC, AL, AR) have been already clarified, and using of the information, the alleles of each enzyme deficiency have been identified. Alleles are extremely heterogeneous in all enzyme deficiencies, in sharp difference from other inborn errors of metabolism, such as cystic fibrosis and hemoglobinopathies.
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PMID:[Molecular basis of urea cycle disorders]. 846 64

Prenatal diagnosis is available for pregnancies at risk for virtually all inherited disorders of hemoglobin production. The field of reproductive genetics must confront many ethical, legal, and social concerns regarding its use, many of which derive from a woman's desire to bear children but legal right to abortion. The goal of more widespread utilization of prenatal diagnosis is sought in the context of questioning the ethical control to be exerted over the biological makeup of future generations. Its appropriate application would be facilitated greatly by the availability of reliable DNA markers of disease severity. Advances in fetal sampling and in detecting mutant globin genes have provided the safe, accurate methodology required for prenatal diagnosis. Chorionic villus sampling in the first trimester has become standard practice, but second trimester amniocentesis also is used for sampling fetal DNA. The use of preimplantation diagnosis and testing fetal cells from the maternal circulation will soon be practical. DNA-based detection of globin gene mutations has been facilitated greatly by the polymerase chain reaction revolution, and several reliable diagnostic methods are available. Polymerase chain reaction-based methods rely on restriction analysis, allele-specific hybridization or amplification, DNA sequence analysis, and new non-polymerase chain reaction methods for DNA amplification in vitro. These methods are available for detecting hemoglobinopathy, thalassemia, and thalassemic-hemoglobinopathy genes that affect alpha- or beta-globin loci.
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PMID:Advances in the prenatal and molecular diagnosis of the hemoglobinopathies and thalassemias. 853 29

Adeno-associated virus, serotype 2 (AAV2)-based chimeric plasmids that harbored a near-full-length human alpha- or beta-globin cDNA were constructed. The cDNAs were spliced into an AAV plasmid, pAAV delta K, downstream from the viral P40 promoter, substituting the capsid gene region. The correctness of the insertion with regard to the transcription polarity was ascertained by both restriction enzyme analysis and DNA sequencing. One of the constructs, pAAVcHBBLCR, contained the erythroid-specific enhancer elements, the locus control region, HS1 and HS2, to ensure an efficient and tissue-specific gene expression. Use of a defective complementing helper, pAVXB (Dixit, M.; et al. Gene 1991, 104, 253-257.) and adenovirus 2 made it possible to prepare recombinant AAVs (rAAVs). Infection of human 293 cells (embryonal kidney cell line) with the resultant rAAV (AAVcHBB) and cotransfection of mouse erythroleukemia (MEL) cells with the beta-globin construct (pAAVcHBBLCR) and an alpha-globin construct (pAAVcHAB) triggered efficient synthesis of human globin polypeptides in the cells, as analyzed by biochemical and immunohistochemical means. The LCR made the construct respond to an inducer, N,N-hexamethylenebisacetamide, the amount of expressed human beta-globin reaching a similar level as the endogenous mouse beta-globin in MEL cells. Electrotransfection of mouse bone marrow hematopoietic stem/progenitor cells with the constructs dramatically increased the number of benzidine-positive cells in liquid suspension culture, indicating expression and synthesis of a human hemoglobin in these cells. Thus, the rAAV constructs may be useful for gene therapy of hemoglobinopathies.
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PMID:Synthesis of human globin polypeptides mediated by recombinant adeno-associated virus vectors. 869 28

Prenatal diagnosis of hemoglobinopathies was performed in 250 fetuses at risk for hemoglobinopathies. The main diagnostic procedures were in vitro hemoglobin synthesis analysis in fetal blood and analysis of DNA obtained from chorionic villus samples. Sixty-six percent of the fetuses were at risk for beta thalassemia major and 28% for sickle cell anemia. Beta thalassemia mutations were heterogenous, and 51 fetuses examined by the DNA technique were found to be at risk for at least 20 different combinations.
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PMID:Prenatal diagnosis of hemoglobinopathies in Turkey: Hacettepe experience. 872 Oct 31

The thalassemia syndromes were the first of human diseases to become thoroughly examined for the underlying molecular lesions by the application of molecular genetic strategies and recombinant DNA methods. Students of thalassemia have now enjoyed over two decades of experience with this research paradigm. These experiences reveal both the awesome power and the limitations of the "reductionist, deterministic" approach of gene cloning and analysis. Incredibly precise and abundant information about the exact molecular lesions responsible for various forms of thalassemia were rapidly obtained by the use of molecular genetic approaches. The mechanisms by which these mutations deranged globin gene expression could be documented with extraordinary precision and efficiency. Precise, powerful methods for detecting disease early in fetal life were rapidly developed, made practical for field use, and disseminated widely. This resulted in a dramatic reduction in the incidence of new births of patients with homozygous beta thalassemia. These experiences demonstrate the extraordinary impact that recombinant DNA technology has upon our ability to understand disease processes, to detect disease long before its phenotypic expression is apparent, and to influence the prevalence of the abnormal alleles in the population. Experience with the antenatal diagnosis of the thalassemias also demonstrates, and should alert us to, the relative ease with which genetic information can be applied to societal and governmental initiatives to alter the reproductive behavior of individuals. While the benefits of reducing the incidence of beta thalassemia are clearcut, application of the strategies that were applied in this narrow situation to broader aspects of disease or genetic manipulation does raise concerns. The thalassemia syndromes demonstrate that genetic information does have more than a theoretical potential to have a major impact upon society. The struggles of many investigators to develop effective pharmacologic agents for the treatment of hemoglobinopathies have also revealed some of the limitations of an isolated molecular approach to the understanding of disease. The tortuous course by which a class of reagents has been identified for stimulation of HbF synthesis illustrates an important point. The application of recombinant DNA methods revealed an entirely new array of pathophysiologic facts that stimulated new hypotheses about the regulation of gene expression and opportunities to manipulate that regulation therapeutically. However, practical application and proper understanding of the molecular information were achieved only when those data were placed in the context of cell biology, tissue and organ-based clinical pathophysiology, and clinical pharmacology. Progress was possible only because of the productive interaction of talented individuals with expertise in these different fields. Our two decades of experience with the thalassemias illustrate very clearly the fact that biology and disease are extraordinarily complex, non-deterministic processes. They will be understood and treated properly only if thriving centers exist within which individuals with diverse interests, expertise, and perspectives about basic science and clinical medicine can exist, interact, and have sufficient time to employ their imaginations to the fullest benefit.
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PMID:The thalassemia syndromes: lessons from molecular medicines index case. 872 57

Anemia, mental status changes, and fatal respiratory failure complicated a febrile illness in a previously healthy 14-year-old black female. At autopsy, widespread fat emboli and bone marrow necrosis were found. Hemoglobin electrophoresis on an antemortem, pretransfusion specimen revealed hemoglobin S/beta+ thalassemia. Acute parvovirus B19 (PV B19) infection was suspected. Postmortem serum and a variety of paraffin-embedded tissues were assayed for PV B19 DNA using the polymerase chain reaction (PCR). The expected PCR product was identified in the serum specimen and in paraffin-embedded sections of bone marrow, kidney, spleen, parathyroid, thyroid, adrenal, and gastrointestinal tract: lung, liver, ovary, fallopian tube, uterus, brain, heart, and pancreas were negative. PV B19 infection is highly contagious and may be rapidly fatal in children with hemoglobinopathies by several mechanisms, including fat embolism. Therefore, there exists the risk of multiple deaths within a family. The acute infection may be easily and expeditiously diagnosed using serum or a variety of paraffin-embedded tissues.
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PMID:Fatal fat embolism syndrome in a child with undiagnosed hemoglobin S/beta+ thalassemia: a complication of acute parvovirus B19 infection. 896 32


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