An acquired acute hepatic porphyria: a novel type of delta-aminolevulinate dehydratase inhibition.
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Acute Disease↗
Biomedical subjects
J T Prchal
Publications and source records attributed to J T Prchal.
Band 3 Tuscaloosa: Pro327----Arg327 substitution in the cytoplasmic domain of erythrocyte band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4.2.
Protein 4.2 is a major red blood cell (RBC) protein that interacts with the band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A----G in nucleotide 166, changing codon 56 from AAG to GAG (Lys----Glu), and substitution C----G in nucleotide 980, changing codon 327 from CCC to CGC (Pro----Arg). Since the Lys56----Glu56 substitution is found in a common asymptomatic variant of the band 3 protein designated band 3 Memphis, we conclude that either the Pro327----Arg327 substitution itself, or in combination with the band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic phenotype.
Amino Acid Sequence↗
New glucose-6-phosphate dehydrogenase mutations from various ethnic groups.
Seven new mutations that produce glucose 6 phosphate dehydrogenase (G6PD) deficiency are described. Three are in variants that were biochemically characterized and described previously, while four were found in samples that had not been characterized biochemically. Several of the mutations affect the amino acids that are mutated in other G6PD variants. As had been noted previously, variants that are associated with nonspherocytic anemia are located either near the glucose 6 phosphate or the NADP binding sites. Variants more distant from these sites are not associated with chronic hemolysis.
Amino Acid Sequence↗
Familial and congenital polycythemia in three unrelated families.
Three families with polycythemia inherited through apparently different modes are described. Secondary causes of polycythemia were ruled out. Erythropoietin (EPO) levels were normal or low, even after phlebotomy. In vitro erythroid colony growth in standard assay cultures containing EPO was normal; however, in the absence of added EPO, a few progenitors from most of the affected individuals were able to generate recognizable colonies of mature erythroblasts, although these were smaller and proportionately less numerous than seen in polycythemia vera (PV). To search for EPO-receptor changes as a possible pathophysiologic mechanism, we examined, by Southern blot analysis, genomic DNA samples from affected and nonaffected family members, as well as three patients with PV. Two different probes, derived from the human EPO-receptor, were used. We found no evidence for chromosomal rearrangements or gene amplification in hereditary polycythemia or PV patients. Further, no nucleotide sequences were found that were homologous to the Friend spleen focus-forming virus glycoprotein gp55, which has been shown to bind to and activate the murine EPO-receptor. Functional studies examining number and binding affinity of the EPO-receptor on erythroid progenitors from three hereditary polycythemia patients demonstrated no abnormalities. We conclude that the mechanism(s) for the erythrocytosis in familial and congenital polycythemia and in PV may not involve the EPO-receptor and, therefore, may result from alterations of postreceptor responses.
2,3-Diphosphoglycerate↗
Molecular defect of truncated beta-spectrin associated with hereditary elliptocytosis. Beta-spectrin Gottingen.
A large German family with autosomal dominantly inherited elliptocytosis, associated with truncated beta-spectrin missing the phosphorylated C-terminal peptide, has been described (Eber, S.W., Morris, S. A., Schroeter, W., and Gratzer, W. B. (1988) J. Clin. Invest. 81, 523-530). We have attempted to delineate the molecular defect of this abnormality at the gene level. Southern blot analyses revealed no evidence of a partial gene deletion or rearrangement. We used polymerase chain reaction (PCR) and amplified several relevant portions of the beta-spectrin gene, using genomic DNA from two different heterozygous patients. No abnormality was found in the last four exons of the beta-spectrin gene produced by PCR. Examination of the introns connecting the last four exons revealed a T to A substitution in the 5' splice site following the exon X in four of eight clones prepared from two affected individuals, but not from a normal subject of this family. To examine the effect of the T to A substitution in these patients, we made cDNA from the reticulocyte mRNA of the patient and examined its composition by PCR. Two distinct PCR fragments produced from the patient's beta-spectrin cDNA were found. One matched the predicted size for normal spectrin, while the other was 197 base pairs shorter. The mutant cDNA sequence revealed that the entire exon preceding the T to A substitution was spliced out, while the two terminal exons were preserved. The deletion of this exon resulted in a frameshift giving a different terminal amino acid (serine instead of leucine) as well as a new termination codon causing a deletion of 129 amino acids including potentially phosphorylated residues.
Base Sequence↗
Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin.
While varying degrees of spectrin deficiency have been found in the majority of patients with hereditary spherocytosis (HS), a combined severe deficiency of both spectrin and the spectrin-binding protein, ankyrin, has been reported only in two patients with severe HS. To elucidate the molecular basis of these protein deficiencies, we have studied the synthesis, assembly, and the mRNA levels of spectrin and ankyrin in peripheral blood reticulocytes in one of the previously reported probands. Pulse-labeling studies showed that in HS reticulocytes, the synthesis of alpha-spectrin was comparable with control reticulocytes while that of beta-spectrin was increased about fourfold, presumably reflecting increased erythropoietic drive. On the HS reticulocyte membrane, the amount of newly assembled spectrin was reduced to about half of the control values, presumably reflecting a decrease in the synthesis of the spectrin binding protein, ankyrin: the ankyrin synthesis was nearly absent in the cytosol and the amounts of membrane-associated ankyrin were reduced to about half of the normal values. The changes in the amounts of spectrin and ankyrin mRNAs quantitated by slot blot and Northern blot analyses were comparable with changes in the synthesis of these proteins: The alpha spectrin mRNA was within a control range and the beta-spectrin mRNA was slightly increased, while the amounts of ankyrin mRNA were reduced to about 50% of control values. We conclude that the primary defect underlying the combined spectrin and ankyrin deficiency is a deficiency of ankyrin mRNA leading to a reduced synthesis of ankyrin which, in turn, underlies the decreased assembly of spectrin on the membrane.
Adult↗
Identification of an unusual deletion within homologous repeats of human reticulocyte beta-spectrin and probable peptide polymorphism.
We screened two different human reticulocyte cDNA libraries with beta-spectrin(beta Sp)-specific polyclonal antibodies and with our original radiolabeled human BSP cDNA probe (encoding beta Sp). Of the 20 independent clones, the largest had about a 2.5-kb insert corresponding to the deduced amino acid (aa) sequence of the beta-7 to beta-14 repetitive segments. Among these segments, segment 12 was 7 aa shorter than the other repetitive segments. We showed that this truncation was not a result of (i) cloning artifact, (ii) alternate splicing, or (iii) common genomic polymorphism by additional examination of 14 individual human chromosomes. Recently, another laboratory described the BSP nucleotide (nt) sequence overlapping partially with our sequence. These overlapping sequences were homologous with the exception of two nt differences at the positions 1342 and 1514. The discrepancy at nt 1342 changes the His to Arg. This newly derived probe has been used to find an additional example of BSP restriction fragment length polymorphism.
Amino Acid Sequence↗
Structural and functional heterogeneity of alpha spectrin mutations involving the spectrin heterodimer self-association site: relationships to hematologic expression of homozygous hereditary elliptocytosis and hereditary pyropoikilocytosis.
Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.
Adolescent↗
Hemoglobin Birmingham and hemoglobin Galicia: two unstable beta chain variants characterized by small deletions and insertions.
Two unstable hemoglobins (Hbs) causing rather severe hemolytic anemia have been characterized. The beta chain of Hb Birmingham, found in an adult black man, is characterized by the loss of -Leu-Ala-His-Lys- at positions 141, 142, 143, and 144 and their replacement by one Gln residue. These changes are the result of a deletion of nine nucleotides, namely two base pairs (bp) of codon 141, all of codons 142 and 143, and one bp of codon 144; the remaining CAG triplet (C from codon 141 and AG from codon 144) codes for the inserted glutamine. In the beta chain of Hb Galicia from a Spanish patient, His and Val at positions 97 and 98 are replaced by one Leu residue. This is due to an ACG deletion in codons 97 and 98, which causes the removal of one His and one Val residue, while the remaining CTG triplet (C from codon 97 and TG from codon 98) codes for the inserted leucine residue. Two mechanisms, namely slipped mispairing in the presence of short repeats, and misreading by DNA polymerase due to a local distortion of the DNA helix, are considered in explaining the origin of the small deletions.
Adult↗
Patterns of spectrin transcripts in erythroid and non-erythroid cells.
Spectrin is the major protein of the membrane erythrocyte skeleton. More recently, homologous but non-identical spectrins (fodrins) were also found in various non-erythroid tissues. Spectrin mRNA in erythroid and various non-erythroid cells was examined by direct hybridization with human alpha-spectrin, beta-spectrin (erythroid spectrins), and alpha-fodrin (non-erythroid spectrin) cDNA probes. Northern blot analysis of poly (A)+ RNA revealed a distinct pattern of expression in erythroid vs. non-erythroid cells. Erythroid cells from early erythroblasts to reticulocyte stage expressed two mRNA species of beta-spectrin, whereas they expressed only a single species of alpha-spectrin, and no alpha-fodrin mRNA. In contrast, non-erythroid cells (platelets, myeloid cells, liver, muscle, heart, cerebellum, and eye lens) expressed either no alpha-spectrin mRNA or a different molecular weight transcript(s) of this gene, and a single species of alpha-fodrin mRNA. Additionally, they also expressed from none to multiple species of beta-spectrin, and these were of different molecular size(s) from that found in erythroid cells (with the exception of platelets). Transcripts of non-erythroid spectrin, alpha-fodrin, were found as a single copy only in non-erythroid tissues. Human and murine erythroleukemia cells expressed both erythroid spectrin transcripts in addition to alpha-fodrin and raise the possibility that erythroid progenitors may have the potential to express both erythroid and non-erythroid species. These data indicated that several mRNA species of beta-spectrin could be detected in both erythroid and some non-erythroid cells. Whether multiple spectrin peptides could also be found with functional heterogeneity is unclear. However, in each case, the pattern combination observed appeared to be tissue-specific.
Blotting, Northern↗
Congenital methemoglobinemia due to methemoglobin reductase deficiency in two unrelated American black families.
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Adolescent↗
J. chain disease: a novel form of plasma cell dyscrasia.
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Adult↗
cDNA sequence for human erythrocyte ankyrin.
The cDNA for human erythrocyte ankyrin has been isolated from a series of overlapping clones obtained from a reticulocyte cDNA library. The composite cDNA sequence has a large open reading frame of 5636 base pairs (bp) with the complete coding sequence for a polypeptide of 1879 amino acids with a predicted molecular mass of 206 kDa. The derived amino acid sequence contained 194 residues that were identical to those obtained by direct amino acid sequencing of 11 ankyrin proteolytic peptides. The primary sequence contained 23 highly homologous repeat units of 33 amino acids within the 90-kDa band 3 binding domain. Two cDNA clones showed evidence of apparent mRNA processing, resulting in the deletions of 486 bp and 135 bp, respectively. The 486-bp deletion resulted in the removal of a 16-kDa highly acidic peptide, and the smaller deletion had the effect of altering the COOH terminus of the molecule. Radiolabeled ankyrin cDNAs recognized two erythroid message sizes by RNA blot analysis, one of which was predominantly associated with early erythroid cell types. An ankyrin message was also observed in RNA from the human cerebellum by the same method. The ankyrin gene is assigned to chromosome 8 using genomic DNA from a panel of sorted human chromosomes.
Amino Acid Sequence↗
Molecular heterogeneity of glucose-6-phosphate dehydrogenase A-.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is probably the most common disease-producing genetic polymorphism of humans. Virtually all G6PD-deficient Africans show the G6PD A- phenotype, an electrophoretically rapid, deficient enzyme. The recently acquired ability to identify the point mutations producing the different variants has given us new insights into the population genetics of G6PD variants. Twenty-nine males with the G6PD A- phenotype were studied. They were of African, Mexican, Spanish, and US white ethnic origin. All had the A---G transition at nucleotide 376 characteristic of G6PD A. In each case, one of three additional mutations was present, at nucleotides 202, 680, or 968. That in this population second mutations producing G6PD deficiency occurred only on the genetic background of G6PD A suggests that G6PD A was at one time the most common type of G6PD in Africa. However, the nucleotide sequence of the chimpanzee (Pan troglodytes) G6PD indicates that the primordial human type of G6PD was G6PD B.
Base Sequence↗
Systemic immunization for the induction of IgA responses.
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Adult↗
Mild sickle cell anemia associated with alpha globin mutant alpha Montgomery.
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Adolescent↗
Characterization of a new G6PD variant: G6PD Titusville.
We describe a new glucose-6-phosphate dehydrogenase mutant, G-6-PD Titusville. The propositus is a 7-month-old black male infant with a transient hemolytic episode. The mutant enzyme is characterized by abnormal electrophoretic mobility, thermolability, Km for NADP, abnormal deamino NADP use and a decreased sensitivity to inhibition by NADPH. G-6-PD activity of hemolysate, as measured under optimal in vitro conditions, was not initially decreased, whereas fibroblasts, granulocytes, and platelets showed a markedly decreased level of enzyme activity. These properties identify G6PD Titusville as a unique variant of this X-linked, housekeeping enzyme. We conclude that although the propositus with G6PD Titusville had a transient hemolytic episode, we cannot be certain whether this association was a causative one.
Enzyme Stability↗
Presence of erythroid and nonerythroid spectrin transcripts in human lens and cerebellum.
Spectrin is a major protein of the red cell membrane, and is composed of alpha- and beta-subunits. While spectrin was initially thought to be specific for erythrocytes, similar, but nonidentical peptides have recently been identified in other tissues, including lens, suggesting the existence of a spectrin gene family. To study the nature of spectrin-like peptides in the lens, we examined the transcription of erythroid and nonerythroid spectrin in human lens and cerebellum by direct hybridization with known human alpha- and beta-spectrin cDNA (erythroid probes), as well as with a human alpha-fodrin cDNA (nonerythroid probe). Northern blots of poly(A)+ RNA from erythroid, as well as several nonerythroid cells, and the total RNA from lens, showed that the beta-spectrin cDNA probe hybridized to two distinct bands of 8.6 and 7.4 kb mRNA in human lens, and was present in abundance. The erythroid beta-spectrin 11 kb mRNA transcript was also found in the human cerebellum. The beta-spectrin transcripts from these nonerythroid tissues were found to have different sizes compared to the major erythroid 7.8 kb beta-spectrin mRNA. Human lens contained a 7.5 kb transcript of the erythroid alpha-spectrin subunit. In addition, the human lens was found to have a 7.2 kb alpha-fodrin transcript; while the cerebellum had an 8.5 kb alpha-fodrin mRNA, the same size as that found in other nonerythroid tissues. The abundance of erythroid and nonerythroid spectrin transcripts in lens was significantly greater than in any other tissues examined. These data show that the human lens has abundant erythroid alpha- and beta-spectrin transcripts, as well as a unique alpha-fodrin transcript.(ABSTRACT TRUNCATED AT 250 WORDS)
Blotting, Northern↗