Uroporphyrinogen decarboxylase structural mutant (Gly281----Glu) in a case of porphyria.

Uroporphyrinogen decarboxylase deficiency in man is responsible for familial porphyria cutanea tarda and hepatoerythropoietic porphyria. A recent study of a family with hepatoerythropoietic porphyria showed that the enzyme defect resulted from rapid degradation of the protein in vivo. Cloning and sequencing of a complementary DNA for the mutated gene revealed that the mutation was due to the replacement of a glycine residue by a glutamic acid residue at position 281. This base change leads to a protein that is very rapidly degraded in the presence of cell lysate. Characterization of the mutation will allow comparison of this defect in a homozygous patient with defects in other patients with familial porphyria cutanea tarda.

Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.

Two different point G to A mutations in exon 10 of the porphobilinogen deaminase gene are responsible for acute intermittent porphyria.

Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector. Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein. Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme. One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.

A liver-type mutation in a case of pronounced erythrocyte phosphofructokinase deficiency without clinical expression.

A marked erythrocyte phosphofructokinase deficiency was detected in a healthy man. His enzymatic activity was only 25% that of normal controls. His father and his son had erythrocytic phosphofructokinase activities of 50-55% that of normal controls. The chromatographic separation of erythrocytic phosphofructokinase isozymes, as well as immunological studies revealed a decrease in L-type phosphofructokinase activity. The lowered erythrocytic L-type phosphofructokinase activity was not accompanied by a decreased level of L-type phosphofructokinase in proteins. The L/M subunit ratio was similar to that of normal subjects. The defect resulted from the synthesis of stable L-type mutant subunit with high electrophoretic mobility. White blood cells, which synthesize mostly the same isozyme as L-type phosphofructokinase also showed a decreased activity and a high electrophoretic mobility. In spite of this important deficiency, and of significant metabolic alterations (a slight decrease in ATP; 2,3-diphosphoglycerate; triose phosphate), hemolysis did not appear in the propositus.

Reaction mechanism of erythrocyte phosphofructokinase.

The reaction mechanism of erythrocyte phosphofructokinase (PFK) was investigated by the initial velocity and the product inhibition. Intersecting lines obtained with initial velocity studies are consistent with a sequential mechanism and the formation of ternary complex as an intermediate. The product inhibition studies support an ordered Bi Bi mechanism in which fructose 6 phosphate (F6P) is the first substrate binding and adenosine diphosphate (ADP) is dissociated from the enzyme before fructose-1,6-P2 (FDP).

Influence of free Mg2+ on the kinetics of human erythrocyte phosphofructokinase.

The influence of Mg2+ on the reaction catalyzed by human erythrocyte phosphofructokinase has been investigated using kinetic methods. The catalytic activity of PFK is dependent upon the presence of Mg2+ which constitutes with ATP the true Mg-ATP2- substrate. Free Mg2+ has no influence on the affinity of the enzyme for Mg-ATP2- substrate. Erythrocyte PFK is more inhibited by ATP4- and uncomplexed citrate than it is by Mg-ATP2- and Mg-citrate. Free Mg2+ relieves the MgATP2- and Mg-citrate inhibition under conditions where free ATP4-is negligible. We can assume that uncomplexed Mg2+ acts as positive effector by direct binding to the enzyme. These results emphasize the role of Mg2+ in the regulation of PFK activity in the erythrocyte.

A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer.

Quantification of specific RNA or DNA molecules that are present in minute amounts in biological samples has previously been performed using PCR in the presence of an internal standard. We have adapted this concept by introducing several modifications that facilitate the quantification of the products and obviate the need for radioisotopes. After amplification, individual products are separated on sequencing gels and directly quantified using a fluorescent automated DNA sequencer. We describe two applications of this approach: the quantitation of minute amounts of bcr-abl hybrid mRNA from malignant cells and the determination of gene copy number in cells stably transfected with a plasmid bearing a chloramphenicol acetyltransferase gene.

An efficient laboratory made apparatus for DNA amplification.

DNA amplification by the polymerase chain reaction (PCR) is a method capable of producing a selective and very high enrichment of a specific DNA sequence. Hence it seems to be useful in various fields from basic research to clinical applications. In order to automatize PCR we assembled for a very low cost a mechanical system designed to carry a test tube holder successively in three thermal baths set at the required temperatures for the reaction. Two examples of the use of this machine are given: (i) amplification of DNA of a particular subtype of acute intermittent porphyria; (ii) the detection of the chimeric c-abl/bcr message found in chronic myelogenous leukemia cells.

Immunological studies of PK and PFK deficiencies induced by chemotherapy.

Pyruvate kinase (PK) and phosphofructokinase (PFK) erythrocyte deficiencies induced by chemotherapy were studied in 6 patients. From immunological tests it may be assumed that the PK and PFK deficiencies were due to different direct mechanisms: a disturbance of the synthesis of one of the PFK subunit; mutation(s) in the structural gene of PK which result in the synthesis of mutant proteins. Several molecular mechanisms are probably at the origin of all the disturbances induced by chemotherapy in the red blood cells. The study of these alterations which mimic those detected in preleukaemic and leukaemic states, provides information on the molecular events in the possible production of chemo-induced cancers.

Studies on the mechanism of the erythrocyte enzyme abnormalities induced by chemotherapy.

With the aim of determining the possible mechanisms of the red cell enzyme deficiencies induced by chemotherapy, deficient red cell glucose-6-phosphate dehydrogenase (G-6-PD), pyruvate kinase (PK) and phosphofructokinase (PFK) from 17 patients were purified and characterized. In all cases G-6-PD showed normal kinetics, electrophoretic mobility and thermostability suggesting that a decreased enzyme synthesis was possible for the deficient enzyme activity. In each case studied, at least one of the PK properties was modified, either in affinity for phosphoenol pyruvate, thermal stability or electrophoretic mobility, indicating a primary or secondary molecular abnormality. In some patients PFK had significantly increased affinity for citrate inhibitor; however, neither the quantity nor quality of the M subunits seemed to be altered. Thus it appears that chemotherapy can induce qualitative as well as quantitative red cell enzyme abnormalities by different mechanisms. These are similar to those observed in spontaneous leukaemia and preleukaemic states. Such a similarity poses the question of whether or not the red cell enzyme abnormalities induced by chemotherapy could be considered as the first sign of secondary leukaemia due to treatment by oncostatic drugs.

A red cell pyruvate kinase mutant with normal L-type PK in the liver.

The erythrocytic and liver pyruvate kinases (PK) from a patient with congenital nonspherocytic hemolytic anemia have been studied. In red blood cells, the residual activity, 28% of the normal control, presented normal kinetic properties, instability to heat and urea, and slow electrophoretic mobility. The L-type PK from the patient's liver was characterized by normal activity, kinetic properties, stability to heat and urea, and electrophoretic mobility. The fact that erythrocyte mutant PK may, as in previous reports, or may not be associated, as in the present observation, with molecular abnormalities of the liver PK provides support for the hypothesis of a gene rearrangement compatible with two different tissue-specific mRNAs.

Phosphofructokinase isozymes from human organs and blood cells.

Phosphofructokinase (PFK) isozymes of blood cells and some human tissues were studied by starch gel electrophoresis and immunoprecipitation by anti-muscle and anti-erythrocyte PFK sera. PFK from muscle, heart, brain and placenta were totally precipitated by both antisera. PFK from blood cells (erythrocytes, lymphocytes, granulocytes, platelets) were precipitated more strongly by anti-erythrocyte PFK serum than by anti-muscle PFK serum. Liver, kidney and monoblast PFK were slightly precipitated by both antisera. From the electrophoretic patterns and the immunoprecipitation curves we may conclude that muscle contains the homotetrameric M4 forms; platelet, liver and kidney the homotetrameric E4 form, and blood cells the M-E hybrids. Monoblasts probably contain a E4 type PFK precursor, and heart, placenta and brain, a modified M4 type PFK. Other isozymes, unrelated with muscle and erythrocyte, were revealed in liver and kidney.

Functional analysis of DNase-I hypersensitive sites at the mouse porphobilinogen deaminase gene locus. Different requirements for position-independent expression from its two promoters.

Porphobilinogen deaminase (EC 4.3.1.8; PBG-D) is the third enzyme of the heme biosynthetic pathway. In both human and mouse, the gene encoding PBG-D possesses two promoters, lying in close proximity. We have previously reported the mapping of six nuclear DNase-I hypersensitive sites at the PBG-D locus which could contribute to the regulation of the gene. In the present study, and in order to define all the elements necessary for a high level of expression and an integration site independence, we studied the pattern and the level of expression of a cloned PBG-D gene following integration into a host genome. The longest construct that we tested (12.5 kilobases) contained sufficient regulatory elements to promote expression levels similar to that of the endogenous gene, both in transgenic mice and in transfected cells. The overall contribution of individual DNase-I hypersensitive sites to the expression of the gene was then studied using a series of mutants that were stably transfected into mouse erythroleukemia cells. Two regions seem to play a critical role in the erythroid-specific expression of the PBG-D gene: the proximal promoter and a region situated at -1000 relative to the initiation site. Study of individual clones of mouse erythroleukemia cells revealed that the erythroid-specific expression of the gene was submitted to position effects in the absence of the upstream region, although the housekeeping transcription is not sensitive to such effects. The tandem arrangement of the housekeeping and tissue-specific promoters of the PBG-D gene raises some questions about the functioning of these two overlapping transcriptional units in erythroid cells. Previous data have suggested that in erythroid cells most of the transcripts initiated at the upstream promoter stop downstream of the first ubiquitous exon, between the two promoters. Here, we show that the deletion of a constitutive DNase-I hypersensitive site that is located in the region of the elongation block results in opposite effects on the steady state levels of housekeeping and tissue-specific RNA. This finding is consistent with the hypothesis that this region promotes premature termination of the housekeeping transcripts therefore preventing promoter interference.

Inherited erythrocyte phosphofructokinase deficiency: molecular mechanism.

Erythrocyte PFK activity 50--60% that of normal controls was found in a mother and her son, without muscular or hematological symptoms. The PFK activity of the mother's muscle was normal in fresh preparations and partially unstable to storage at 4 degrees C. Electrophoresis of muscle PFK revealed two bands, on normal and one abnormal with an anodic mobility greater than normal. Both patients were characterized as heterozygotes for an unstable muscle PFK. Unstable M'subunits disappeared in erythrocytes which are old cells devoid fo protein synthesis. Consequently an increased E/M subunit ratio leads to a distribution of the five isozymes different from that of normal erythrocytes. In these patients, we observed a loss of the M4 enzyme together with an increase in the E4 isozyme. The kinetic and immunologic data were compatible with these modifications. Isoelectric focusing of hemolysates from the two patients revealed an acidification of the main activity band, suggesting that an increase in E4 isozyme resulted in a change of the total electric charge.

Red blood cell enzyme abnormalities in patients treated with chemotherapy.

Sxiteen red blood cell enzyme activities and fetal haemoglobin level have been assayed in 60 patients treated for haematologic or nonhaematologic malignant diseases with various combinations of cytostatic drugs. Acquired enzyme deficiency was found in 20 patients. The most frequently decreased activities were those of G6PD (12 cases), PK (seven cases), PFK (six cases) and AK (three cases). In many patients decreased activity of some enzymes contrasted with increased activity of others such as ALD, TPI, G3PD, PGK, ENOL and 6PGD. The number of abnormalities seems to be related to the duration of the treatment. Incidence of enzyme deficiencies was similar in patients previously treated or not with radiotherapy. Enzyme abnormalities were correlated neither with haemoglobin level nor with reticulocyte count. They were very similar to those observed in primary acquired dyserythropoietic and preleukaemic states. Their prognostic value and mechanism are discussed.

Erythrocytic pyruvate kinase deficiency and hemolytic anemia inherited as a dominant trait.

A nonspherocytic hemolytic anemia, associated with a pyruvate kinase (PK) deficiency apparently inherited as a dominant trait has been identified in a family. In the affected members, the residual PK activities were 20% that of normal controls, an unusually low level for heterozygous subjects. The anemia was mild except in the proband, a 2-year-old boy who suffered from a severe anemia. The PKs of the proband and of his both parents have been characterized as kinetically and electrophoretically normal enzymes. Immunoprecipitation tests indicated a large amount of L-type inactive protein in the proband and his father. Moreover, an M2 type PK was present in the father's hemolysate, suggesting the existence of a compensatory process that derepressed the corresponding structural gene. We suggest that the presence of one or more mutated subunits in the tetrameric forms of L-type PK leads to the inactivation of these tetramers. This hypothesis accounts for the low residual activity in affected heterozygous members of this family. The severity of the hematological symptoms in the proband, in comparison with the mild hemolysis observed in the other heterozygous members of the family points to the existence of a large spectrum of pathologic expression for an identical PK defect present in a family.

Gd(-) Rennes, a new deficient variant of glucose-6-phosphate dehydrogenase associated with congenital nonspherocytic hemolytic anemia found in France.

A new variant of G6PD with total enzyme deficiency associated with nonspherocytic hemolytic anemia in a 60 year old Frenchman is characterized. Partially purified enzyme revealed slow electrophoretic mobility, decreased G6P affinity, thermal instability, abnormal pH curve with a single peak at pH 5.0, abnormal utilization of 2-deoxy-G6P and deamino NADP. This variant differs from all previously reported variants associated with chronic nonspherocytic hemolytic anemia. Accordingly this variant is designated Gd(-) Rennes.

The mouse porphobilinogen deaminase gene. Structural organization, sequence, and transcriptional analysis.

The porphobilinogen deaminase gene encodes the third enzyme of the heme biosynthetic pathway. This gene is expressed in a tissue-specific manner and gives rise to two isoenzymatic forms encoded by mRNA species differing in their 5' extremity. Recent studies in human demonstrated that the tissue-specific expression of the porphobilinogen deaminase gene is determined in erythropoietic cells, by the utilization of a specific promoter situated 3' to the housekeeping promoter used in other cell types. This results, through differential splicing, in the mutually exclusive presence of either exon 1 or exon 2 in mature mRNAs. Here, we report the cloning and sequencing of the porphobilinogen deaminase gene from mouse. The overall organization of the mouse gene is similar to that of the human one. In the housekeeping promoter, only a short stretch of homology is found including two potential Sp1 binding sites; in contrast, more extensive similarity appears in the erythroid-specific promoter including two motifs also found in globin gene, a CACCC box, and a recently described Ery F1 consensus binding sequence. We derived a set of single-stranded probes corresponding to different parts of the mouse gene to carry out a detailed analysis of the transcriptional unit in various cell types, using a run-on transcription assay on isolated nuclei. In liver cells, the first (non-erythropoietic) exon is more actively transcribed than parts of the gene situated downstream, suggesting that the elongation of transcripts is blocked within the 5' part of the first intron. In erythropoietic cells, the downstream promoter becomes activated; surprisingly, the initiation of transcription is also enhanced from the upstream (housekeeping) promoter and most of the transcripts initiated at the housekeeping promoter stop downstream of the first exon, between the two promoters.

Prevalence of the 281 (Gly----Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda.

The prevalence of the 281 (Gly----Glu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.

Molecular heterogeneity of acute intermittent porphyria: identification of four additional mutations resulting in the CRIM-negative subtype of the disease.

Four mutations of the porphobilinogen (PBG) deaminase gene that result in cross-reacting immunological material (CRIM)-negative forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA from patients and by cloning of the amplified products in a bacterial expression vector. One mutation is a single base deletion which causes a frameshift and which is expected to result in the synthesis of a truncated protein. Two other mutations consist of single base substitutions and lead to amino acid changes. The fourth mutation is a single base substitution producing an aberrant splicing and resulting in an mRNA which would encode a protein missing three amino acids. DNAs from 16 unrelated CRIM-negative AIP patients were screened for the presence of these four mutations, by hybridization with oligonucleotides specific for each of the mutations, but none of the four mutations was identified in additional patients. The results indicate that mutations responsible for CRIM-negative AIP are highly heterogenous.