Bone marrow transplantation for sickle cell anemia: summarizing comments.
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Anemia, Sickle Cell↗
Biomedical subjects
E Beutler
Publications and source records attributed to E Beutler.
High frequency of the Gaucher disease mutation at nucleotide 1226 among Ashkenazi Jews.
Reliable estimates of the frequency of Gaucher disease-producing mutations are not available. The high frequency of Gaucher disease in the Ashkenazi Jewish population is due to the occurrence of a mutation at nucleotide (nt) 1226. We have screened 593 DNA samples from normal Ashkenazi Jews, as well as 62 DNA samples from all our Ashkenazi Jewish patients with Gaucher disease, for the presence of the 1226 mutation. In the 593 presumed normal Ashkenazi Jewish individuals the 1226 mutation was identified in the heterozygous state in 37 and in the homozygous state in two, giving a gene frequency of .035 for the mutation. This 1226 mutation represented 73% of the 124 Gaucher disease alleles in Jewish Gaucher disease patients. Accordingly we estimate that the gene frequency for Gaucher disease among the Ashkenazi Jewish population is .047, which is equivalent to a carrier frequency of 8.9% and a birth incidence of 1:450.
Adolescent↗
The facile detection of the nt 1226 mutation of glucocerebrosidase by 'mismatched' PCR.
The most common Gaucher disease-producing mutation among Ashkenazi Jews is an A----G substitution at cDNA nt 1226 (genomic nt 5841). We describe a simple method for detecting this mutation both in genomic DNA and in cDNA by performing polymerase chain reaction (PCR) using a 5'-primer mismatched at one nucleotide so as to create an Xho I restriction site. When the mutation is present. the 105 bp fragment formed is cleaved to 89 and 16 nt fragments. The 89 bp fragment is easily visualized on a gel making it possible to distinguish individuals who do not have the mutation from heterozygotes and homozygotes.
Base Sequence↗
Misuse of marrow examination in the diagnosis of Gaucher disease.
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Bone Marrow Examination↗
Human red cell glucose-6-phosphate dehydrogenase: all active enzyme has sequence predicted by the X chromosome-encoded cDNA.
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Amino Acid Sequence↗
Lasting remissions in hairy-cell leukemia induced by a single infusion of 2-chlorodeoxyadenosine.
2-Chlorodeoxyadenosine is a simple purine nucleoside that has previously been shown to be effective in the treatment of low-grade malignant disorders of lymphoid tissue, including chronic lymphocytic leukemia and non-Hodgkin's lymphoma. Because of these encouraging results, we treated 12 patients with another low-grade B-cell neoplasm, hairy-cell leukemia. The patients received 2-chlorodeoxyadenosine (0.1 mg per kilogram of body weight per day) by continuous infusion for seven days. All the patients responded to treatment. Eleven had complete remissions characterized by the normalization of peripheral blood and bone marrow and disappearance of tumor masses. The longest remission has been 3.8 years. None of the patients have relapsed, and the median duration of remission has been 15.5 months. No serious toxic reactions occurred as a result of 2-chlorodeoxyadenosine therapy. These results suggest that 2-chlorodeoxyadenosine may be the most effective therapy available for hairy-cell leukemia. The administration of 2-chlorodeoxyadenosine resulted in a higher rate of complete remission than is observed with interferon alfa, and it required no maintenance therapy. Its toxicity may be lower than that of deoxycoformycin, and the responses were achieved with single courses of treatment.
2-Chloroadenosine↗
Gamma-glutamylcysteine synthetase deficiency and hemolytic anemia.
gamma-Glutamylcysteine synthetase is one of the enzymes of glutathione (GSH) synthesis. A deficiency of this enzyme has been found only once previously in humans: it was associated with spinocerebellar degeneration and hemolytic anemia. We report the case of a woman, daughter of fifth cousins, who was gamma-glutamylcysteine-synthetase-deficient. Modest decreases in the amount of GSH in cultured lymphoblasts and fibroblasts could be documented. The amount of residual enzyme was insufficient to permit detailed studies of the characteristics of the mutant enzymes, but no major abnormality in its Km for cysteine and glutamic acid or in its heat stability were found. In contrast to the earlier report, the only manifestation of the enzyme deficiency was hemolytic anemia. This leads us to conclude that either the occurrence of neurologic symptoms in the other reported family was a chance association or that the clinical expression of this rare defect is pleomorphic.
Adult↗
In vivo aging of red cell enzymes: study of biotinylated red blood cells in rabbits.
It is generally recognized that the activities of some of the red cell enzymes decline as the cell ages. However, there is still a controversy regarding the rate at which this aging occurs. In the present study we applied newly developed technology for the specific isolation of maturing reticulocytes/erythrocytes for a more comprehensive study of in vivo aging of red cell enzymes in rabbits. Anemia was induced by repeated phlebotomy, and reticulocyte-rich erythrocytes were labeled with N-hydroxy succinimido-biotin and then transfused into a normal rabbit. These biotinylated cells were isolated at various time points by their affinity for an avidin support, and the enzymatic activity of 19 red cell enzymes was measured. We observed a biphasic pattern of decay for the activity of six age-dependent enzymes--aldolase, glutamate-oxaloacetate transaminase, glucose 6-phosphate dehydrogenase, hexokinase, pyrimidine 5'-nucleotidase, and pyruvate kinase.
Animals↗
Linkage between a PvuII restriction fragment length polymorphism and G6PD A-202A/376G: evidence for a single origin of the common G6PD A- mutation.
DNA samples from 29 males with the G6PD A- phenotype and 14 males with a G6PD B phenotype were studied for the presence of each of four polymorphic restriction sites in the glucose-6-phosphate dehydrogenase locus. All G6PD A- subjects with the G6PD A-202A/376G genotype, regardless of population origin, shared identical haplotypes. In view of the fact that at least one of the restriction sites, the PvuII site in the intron between exon 5 and 6, has thus far been uncommon in the populations studied, it seems likely that the G6PD A- mutation at nucleotide 202 arose relatively recently and in a single individual.
Base Sequence↗
Detection of the 1226 (Jewish) mutation for Gaucher's disease by color PCR. A means for studying the gene frequency of the disorder.
The authors applied the polymerase chain reaction- (PCR) based color complementation assay for rapid detection of the 1226 ("Jewish") mutation of the glucocerebrosidase gene. Fifty-seven unrelated patients with Gaucher's disease and 50 unrelated normal Ashkenazi Jewish volunteers were studied. This mutation was identified in more than 75% of the Jewish Gaucher's disease alleles and in 4 (8%) of the 50 normal Jewish volunteers. The reliability of the technique was verified both by DNA sequencing and by leukocyte beta-glucosidase assay. This method is suggested as the simplest and most suitable one for a large-scale screening for the 1226 mutation for Gaucher's disease.
Adolescent↗
Gaucher disease associated with a unique KpnI restriction site: identification of the amino-acid substitution.
In an earlier survey of the glucocerebrosidase locus using 20 restriction enzymes and a 1039 bp probe we found that 1 of 9 Gaucher disease patients had a unique pattern with KpnI. a pattern that was not observed in any other Gaucher patient or in 31 controls. We have now localized the mutation in this patient to a T----A transversion in nucleotide 764 of the cDNA occurring on one of the two glucocerebrosidase alleles. The presumed abnormality in the other allele has not been identified. The deduced amino-acid change in the allele with the mutation at nucleotide 764 is a relatively drastic alteration of amino acid 255 from phenylalanine to tyrosine. The genomic DNAs of 31 other Gaucher disease patients (representing 62 Gaucher disease alleles) were examined for this mutation using the polymerase chain reaction. None were found to have this abnormality.
Base Sequence↗
A glucocerebrosidase fusion gene in Gaucher disease. Implications for the molecular anatomy, pathogenesis, and diagnosis of this disorder.
The molecular diagnosis of Gaucher disease has been difficult due to the existence of several different point mutations in the glucocerebrosidase gene and due to the presence of a tightly linked, highly homologous pseudogene. We now report the occurrence of a "Lepore-like" glucocerebrosidase fusion gene in which the 5' end is the functional gene and the 3' end is the pseudogene. This further complicates the molecular diagnosis of Gaucher disease but sheds light on the molecular anatomy of the glucocerebrosidase gene complex and on the pathogenesis of this important storage disease.
Adult↗
High level transcription of the glucocerebrosidase pseudogene in normal subjects and patients with Gaucher disease.
Gaucher disease is due to mutations involving the glucocerebrosidase gene. A closely homologous pseudogene is located approximately 16 kD downstream from the functional gene. Sequence analysis of clones from cDNA libraries made from skin fibroblast cultures showed several independent clones with the sequence of an aberrantly processed pseudogene message. Examination of cellular RNA from lymphoblasts or fibroblasts obtained from thirteen Gaucher disease patients, one Gaucher disease heterozygote, and four normal subjects showed that the pseudogene was consistently transcribed, and that in some cases the level of transcription seemed to be approximately equal to that of the functional gene. The transcription of the pseudogene must be taken into account when attempting to detect mutations of glucocerebrosidase by the study of cDNA libraries.
Base Sequence↗
Potent toxicity of 2-chlorodeoxyadenosine toward human monocytes in vitro and in vivo. A novel approach to immunosuppressive therapy.
Lymphoid cells were thought to be uniquely susceptible to excess 2'-deoxyadenosine (dAdo), when exposed to inhibitors of adenosine deaminase (ADA). However, we now find that human monocytes are as sensitive as lymphocytes to dAdo or to the ADA-resistant congener 2-chloro-2'-deoxyadenosine (CldAdo). Monocytes exposed in vitro to CldAdo, or to dAdo plus deoxycoformycin rapidly developed DNA strand breaks. Both the DNA damage and the toxicity of CldAdo or dAdo toward monocytes were blocked by deoxycytidine, but not by inhibitors of poly(ADP-ribose) polymerase. A partial decrease in RNA synthesis and a gradual decline of cellular NAD were early biochemical events associated with monocyte DNA damage. Low CldAdo concentrations (5-20 nM) inhibited monocyte phagocytosis and reduced the release of interleukin 6. Higher CldAdo concentrations led to a dose- and time-dependent loss of monocyte viability. Circulating monocytes disappeared within 1 wk in patients with cutaneous T cell lymphoma or with rheumatoid arthritis during continuous CldAdo infusion. The marked sensitivity of human monocyte function and survival to CldAdo in vitro, together with the monocyte depletion in patients receiving CldAdo chemotherapy, suggests that CldAdo or other dAdo analogues offer a novel therapeutic strategy for chronic inflammatory and autoimmune diseases characterized by inappropriate monocyte deployment or function.
2-Chloroadenosine↗
Linkage of the PvuII polymorphism with the common Jewish mutation for Gaucher disease.
We have localized the PvuII polymorphism of the glucocerebrosidase gene complex to intron 6 of the active gene. Using the polymerase chain reaction (PCR) to amplify intron 6 of DNA samples from Pv1.1-/Pv1.1+ individuals, we defined the mutation causing this polymorphism as a G----A single-base substitution at position 3931 of the active gene. By analyzing 54 unrelated Gaucher patients we show strong linkage disequilibrium between the Pv1.1- genotype and the common Jewish mutation 1226 causing the adult type of this disease. Gaucher disease patients heterozygous for the 1226 allele and one unidentified allele (1226/?), particularly those of Jewish ancestry, were predominantly of the Pv1.1-/PV1.1+ genotype. This suggests that one of the unknown alleles may be relatively common and linked to the Pv1.1+ genotype.
Cell Line↗
The NT 1311 polymorphism of G6PD: G6PD Mediterranean mutation may have originated independently in Europe and Asia.
A polymorphic site exists in exon 11 of G6PD: in the wild-type enzyme, nucleotide (NT) 1311 is a C, but is some individuals from diverse populations a T is present instead. Nine of 54 X chromosomes from Europeans of mixed origins, nine of 41 X chromosomes of Ashkenazi Jewish subjects, three of 18 X chromosomes of Sicilians, five of 20 African X chromosomes, and nine of 20 Asian Indian X chromosomes had the mutant genotype. In contrast, the mutation was found in only three of 59 Oriental X chromosomes and in three of 30 Central/South American X chromosomes. The mutation was absent from four samples of chimpanzee DNA. Twenty-one of 22 male subjects from Mediterranean countries who had the G6PD Mediterranean 563T genotype investigated in the present study or reported previously had a T at NT 1311. Only one had the normal C at NT 1311. In contrast, both G6PD Mediterranean563T males from the Indian subcontinent had the normal C at NT 1311. These findings suggest that the same mutation at nucleotide 563 giving rise to G6PD Mediterranean may have arisen independently in Europe and in Asia.
Animals↗
Gene transfer in the treatment of hematologic disease.
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Dependovirus↗
The genetics of glucose-6-phosphate dehydrogenase deficiency.
In the past 35 years, G-6-PD and its genetic variants have been a valuable resource for the geneticist and cell biologist. Over 380 putative variants have been described and have served as a paradigm, showing how many ways mutations can affect an enzyme. With the cloning and sequencing of G-6-PD, a new chapter has been opened in our understanding of G-6-PD. Variants that were thought to be different have proven to be identical, and those that were thought to be the same are now seen to be heterogeneous. Importantly, the ability to deduce quickly the amino acid substitution in a G-6-PD variant makes feasible, for the first time, the unraveling of relationships between the structure and function of this enzyme.
Animals↗