Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0039730 (thalassemia)
 10,305 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.
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PMID:Detection of single base substitutions in total genomic DNA. 396 55

alpha-Globin is encoded by the two adjacent genes, alpha 1 and alpha 2. Although it is clearly established that both alpha-globin genes are expressed, their relative contributions to alpha-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in alpha-globin gene activity secondarily to the loss of function of one or more of these genes (alpha-thalassemia [Thal]) have not been directly investigated. This study further defines the expression of the two human alpha-globin genes by determining the relative levels of alpha 1 and alpha 2 mRNA in the reticulocytes of normal individuals and in individuals heterozygous for the common 3.7-kilobase deletion within the alpha-globin gene cluster that removes the alpha 2-globin gene (the rightward type alpha-Thal-2 deletion). To quantitate accurately the ratio of the two alpha-globin mRNAs, we have modified a previously reported S1 nuclease assay to include the use of 32P end-labeled probes isolated from alpha 1- and alpha 2-globin complementary DNA recombinant plasmids. In individuals with a normal alpha-globin genotype (as determined by Southern blot analysis [alpha alpha/alpha alpha]), alpha 2-globin mRNA is present at an average 2.8-fold excess to alpha 1. In individuals heterozygous for the rightward type alpha-Thal-2 deletion (-alpha/alpha alpha) the alpha 2/alpha 1 mRNA ratio is 1:1. These results suggest that the loss of the alpha 2-globin gene in the alpha-Thal-2 deletion is associated with a 1.8-fold compensatory increase alpha 1-globin gene expression.
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PMID:Compensatory increase in alpha 1-globin gene expression in individuals heterozygous for the alpha-thalassemia-2 deletion. 404 27

The molecular defect in four Kurdish Jews with homozygous, mRNA-deficient beta zero thalassemia was investigated. Electrophoretic profiles of pulse-labeled alpha- and beta-globin RNAs are similar to those of non-thalassemics; therefore, at least one of the thalassemic beta-globin alleles is transcribed. During a 30 min actinomycin D chase, most of the alpha- and beta-globin mRNA precursors and processing intermediates are converted to mRNA-sized RNA. Thalassemic and non-thalassemic beta-globin RNAs are indistinguishable, as determined by S1 nuclease mapping and RNA blotting. Non-thalassemic beta-globin mRNA is stable during a 30 min actinomycin chase, but 30%-75% of the thalassemic mRNA-sized molecules is degraded during that period. We conclude that the absence of beta-globin mRNA in this disease results from rapid turnover of beta-globin mRNA-sized molecules.
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PMID:Unstable beta-globin mRNA in mRNA-deficient beta o thalassemia. 610 Dec 6

Our studies have shown that globin-gene structure, as revealed by Southern blot analysis, is normal in most patients who are homozygous for beta-thalassemia. In many, but not all patients, evidence for mutations which alter the metabolism of beta-globin RNA molecules was obtained by several methods of analysis. The most specific and sensitive of these involves the use of single-stranded, highly radioactive probes generated using the M13 cloning system. Such probes allow detection of aberrantly processed RNA molecules by S1 nuclease analysis, thereby providing clues as to the position and nature of mutations within individual thalassemic globin genes. Of greatest potential interest are those genes which provide no evidence, in bone marrow RNA, of aberrantly processed intermediates. Analysis of these genes by molecular cloning and DNA sequencing may lead to identification of sequences which are important for initiation or termination of RNA transcription. Mutations which cause beta-thalassemia continue to provide a rich source of information about the nature of DNA sequences which are essential for efficient gene expression. By study of thalassemic patients, new insights are obtained into normal mRNA metabolism.
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PMID:Molecular defects in beta-thalassemia. 618 15

The reduced beta-globin synthesis characterizing the beta+ thalassemia phenotype has been shown to be caused by anomalous processing within the small intervening sequence (IVS1) of the beta-globin mRNA precursor. The beta-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3' junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3'-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with beta+ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned beta+-thalassemic gene preferentially generated the anomalously spliced RNA when expressed in monkey kidney cells. The anomalously spliced RNA constituted approximately 80%--90%, and normal beta RNA approximately 10%--20%, of the total beta mRNA. In contrast, the small amount of beta mRNA present in reticulocytes from such patients consisted predominantly of normal beta mRNA. These results suggest that the reduced amount of normally functioning beta mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.
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PMID:Abnormally spliced messenger RNA in erythroid cells from patients with beta+ thalassemia and monkey cells expressing a cloned beta+-thalassemic gene. 628 Aug 77

We have studied the expression of a cloned mutant human beta-globin gene in tissue culture cells. The gene, which was previously isolated from the chromosomal DNA of an individual with a low level of normal beta-globin expression (beta+-thalassemia), contains five mutations inside the large intervening sequence (IVS2), as well as a silent change in codon 2. This beta-thalassemia gene (thal) was inserted into a plasmid that is replicated and transcribed in a line of monkey kidney cells in culture. S1 nuclease mapping of the beta-globin RNA transcribed from this gene indicates that some of the beta-globin RNA is spliced abnormally by using a cryptic 3' splice sequence normally present in IVS2 but not used in processing the normal beta-globin transcript. The cryptic 3' splice site is not the site of a mutation in the thal gene. Because neither the 5' or 3' splice junction nor the cryptic site is mutated in this gene, it is most likely that the mutation at position 705 of IVS2, the only nonpolymorphic change in the gene, interferes indirectly with normal processing. These results suggest that certain sequences within IVS must be conserved to prevent abnormal splicing and loss of gene function.
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PMID:Abnormal splice in a mutant human beta-globin gene not at the site of a mutation. 629 82

We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O-thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O-thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5' flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta-mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.
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PMID:Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon. 673 65

We have analyzed the molecular basis of beta + thalassemia by studying the expression of a cloned beta-globin gene in HeLa cells. This beta-globin gene was isolated from a beta + thalassemic patient and differs from the normal beta-globin genes by only a single point mutation within the first intron. The beta + thalassemic and the normal beta-globin genes were cloned into an SV40-pBR328 vector and introduced into HeLa cells by calcium phosphate coprecipitation. We assayed the RNA from these transfected HeLa cells by S1 nuclease mapping and cDNA sequencing to detect the nature of the defect in beta-globin gene expression. While the transcripts of the normal beta-globin gene are processed correctly, the first intron of the beta + thalassemic beta-globin gene is incorrectly spliced in about 90% of the mRNA because of an additional 3; splice site that has been created by the point mutation. This incorrectly spliced mRNA is effectively exported to the cytoplasm, where it would conceivably be translated to give a truncated globin chain of 35 amino acids. The remaining 10% of the mRNA transcribed from the beta+ thalassemic globin gene is correctly spliced and can therefore be translated to give normal beta-globin. In addition to the incorrect splicing of the first intron, the splicing of both introns is retarded, which results in the accumulation of unspliced pre-mRNA. This suggests that removal of the first intron might facilitate splicing of the second intron.
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PMID:Beta + thalassemia: aberrant splicing results from a single point mutation in an intron. 689 66

We have developed a method that permits rapid identification of the consequences of mutations that alter beta-globin RNA processing in erythroid cells. S1 nuclease mapping techniques were used to analyze total bone marrow RNA obtained fron 15 patients who are clinically homozygous for beta-thalassemia and from 5 patients with erythroid hyperplasia from other causes. This analysis was facilitated by the use of single-stranded uniformly labeled DNA probes of high specific activity that were prepared by using recombinant phage M13-beta-globin DNA templates. Two abnormalities of RNA processing were found to occur with high frequency in these patients. Nine thalassemic patients were found to have increased levels of an RNA species that retains all sequences transcribed from intervening sequence 1, implying the presence of mutations that decrease the correct splicing of this intron. Seven of 15 thalassemic patients were found to have an abnormally processed RNA species that retains 19 nucleotides transcribed from the 3' end of intron 1; this abnormality is caused by the G leads to A substitution in intron 1 that is known to create an alternative splice acceptor site.
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PMID:RNA processing errors in patients with beta-thalassemia. 695 87

The human alpha-globin genes are duplicated and encode identical polypeptides. Recently we detected in cloned genomic DNAs characteristic sequence differences between the 3' untranslated regions of the 5' (alpha 2) and 3' (alpha 1) genes, not previously recognized by direct analysis of mRNA and cDNA transcripts. Based on these untranslated region differences, we have now used S1 nuclease mapping of RNA to detect and quantitate the two predicted alpha-mRNA species. With this assay we have examined the relative expression of the alpha-globin genes during normal development and in alpha-thalassemia syndromes. In normal adult reticulocytes, alpha 2 RNA is slightly more abundant than the alpha 1 species (ratio 60:40). This relative abundance of the alpha RNAs was consistently observed in fetal blood and liver RNA samples from 10 weeks of gestation to birth. In both deletion and nondeletion forms of alpha thalassemia, only the alpha 1 RNA and establish the normal pattern of relative alpha-gene expression during development independent of protein variants. RNA analysis also permits for the first time identification of the mutant genes in nondeletion forms of thalassemia.
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PMID:The duplicated human alpha-globin genes: their relative expression as measured by RNA analysis. 723 52


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