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Since the isolation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the characterization of the main mutation (delta F508) in 1989, a large number of rare mutations has been found. Full screening of the CFTR gene is difficult because it is split into 27 exons covering 250 kb of genomic DNA. This gene is essentially expressed in the lung and intestinal tract, neither of which are easily accessible for routine investigations. The recent description of a faint transcription of highly tissue-specific genes in any cell, a phenomenon known as illegitimate transcription, would facilitate the research of mutations and the characterization of truncated m-RNA caused by splicing mutations. Using the polymerase chain reaction on cDNA (cDNA-PCR), we detected transcripts of the CFTR gene in lymphocytes and lymphoblast cells at a very low level (about 300 times less than in lung or intestine). This strategy allowed us to obtain a sufficient amount of cDNA-PCR product compatible with further molecular analyses. We have, therefore, analyzed a cDNA fragment overlapping exons 10 and 11 by polyacrylamide gel electrophoresis and direct sequencing, and detected the delta F508 mutation at this level. Our protocol can be generalized to the investigation of the total 4.5-kb CFTR coding sequence.

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the alpha-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the alpha-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5'-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splicesite mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range.

The molecular basis of null alleles was investigated by cDNA polymerase chain reaction (PCR) in seven Tay-Sachs patients. Although mRNAs were undetectable by Northern blot, cDNA-PCR amplification allowed us to get a sufficient amount of cDNA to characterize abnormal transcripts. In two French patients (one homozygote and one compound heterozygote with a 4-bp insertion in exon 11 of the second allele) suffering an infantile form of the disease, we detected abnormal RNAs with a 17-bp insertion due to a GT to AT transition at the donor site of intron 9, resulting in the activation of a cryptic donor site in the intron. This mutation has been found in 9 out of 82 Tay-Sachs chromosomes (11%) in association with alleles responsible from different clinical courses. In the other five patients we found the 4-bp insertion in exon 11 and two nonsense mutations.

Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and greater than 30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, greater than 30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the greater than 30-kb fragment and is probably localized on chromosome 3 with the greater than 30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and greater than 30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes; the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.

Clinical, familial and biochemical studies from six unrelated Spanish patients with hereditary hemolytic anemia and erythrocyte pyruvate-kinase (PK) deficiency are described. A remarkable molecular heterogeneity of mutant PK variants involving kinetic properties, molecular stability or electrophoretic mobility is demonstrated. In two patients whose PK variants showed abnormal electrophoretic pattern and strongly aberrant kinetic properties, a chronic hemolytic anemia associated with several other clinical manifestations of chronic hemolysis occurred. In patients whose PK variants showed less abnormal kinetic and electrophoretic characteristics, there was only moderate or mild hemolytic anemia. One patient's PK variant with no obvious kinetic or electrophoretic alterations, showed a markedly decreased heat stability with severe diminution of antigenic concentration of the enzyme. This patient presented a spectacular clinical and hematological improvement after splenectomy. The purpose of the present study is to describe six new PK variants of Spanish origin. In addition, an attempt is made to find relationships between molecular abnormalities of mutant PK variants and the severity of hemolytic anemia, in these patients. The possible role of some kinetic alteration, such as fructose disphosphate (FDP) activation or ATP inhibition of PK variants, in the clinical manifestations of chronic hemolysis is also suggested.

A G-6PD deficiency was detected in a Negro patient from Senegal suffering from congenital nonspherocytic hemolytic anemia. The main characteristics of this variant were: profound defect of G-6PD activity in the red cells, decreased immunologic specific activity, fast electrophoretic mobility, decreased Km-G-6P and normal Km-NADP+, normal inhibition by ATP and NADPH, slightly increased utilization of the substrate analogues, slightly biphasic pH curve, high heat lability, subnormal activation energy. The characteristics of this variant being unique, it was called 'G-6PD Hôtel Dieu.'

A new G6PD variant, called G6PD Vientiane, has been discovered in a patient from Laos. The characteristics of this variant are: mild enzyme deficiency (about 50% of the normal activity) in the granulocytes and the red cells, with normal G6PD-related antigen concentration; increased stability; normal Km glucose 6-phosphate and NADP+; increased inhibition constant by NADPH; decreased inhibition by ATP; slightly increased utilization of the substrate analogue; abnormal pH curve, with maximum activity at pH 9.5; slightly reduced starch gel electrophoretic migration. The implications of the molecular stability of a deficient mutant variant are discussed.

By focusing in sucrose, gradient L-type pyruvate kinase from human liver could be separated into 2 major forms (pI 6.28 +/- 0.03 and 5.85 +/- 0.09) and a minor more acid form (pI = 5). These different forms could also be detected by focusing in acrylamide-ampholine slab gel. The major forms were interconvertible, the equilibrium being shifted toward the acid form by fructose 1,6-diphosphate and SH reagents, and toward the alkaline form by proteinic factors extracted by ammonium sulphate fractionation from liver extracts and from hemolysates. These factors seemed to be responsible for the stabilization of the liver crude extract enzyme in its alkaline conformation. By acrylamide slab gel electrofocusing, erythrocyte pyruvate kinase from whole hemolysates exhibited a complex pattern composed of at least 3 introconvertible forms. The in vitro aging of the red blood cells and the storage of the hemolysates resulted in a progressive disappearance of the acid forms and in a strengthening of the alkaline form. Partially purified erythrocyte enzyme focused in 2 major bands, interconvertible under the influence of the same factors as those described for L-type pyruvate kinase. Although closely related, the focusing patterns of L-type and erythrocyte-type were never exactly identical. Double immunodiffusion against antihuman erythrocyte-and L-type pyruvate kinases. Moreover, antihuman M2-type serum was unable to neutralize erythrocyte pyruvate kinase as well as to change its electrophoretic mobility. Consequently, we conclude that both human erythrocyte- and liver L-type pyruvate kinases existed under several conformers interconvertible under the influence of the same ligands or proteinic factors; erythrocyte-type enzyme seems to include L-type subunit and not M1- or M2-type subunits. The erythrocyte- and L-type enzymes, however, are not identical and the nature of the differences between them is discussed.

The defective PK variant of a patient with a severe form of hemolytic anemia was characterized by its inability to undergo a normal 'proteolytic maturation.' In obligatory heterozygotes it could be proved that red cells contained different PK species, some of them sensitive and the others partially resistant to the action of trypsin.

Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes. The M type isozymes have been studied by electrofocusing in thin layer acrylamide-ampholine gel. In adult tissues 4 types of isozymes were found, designated, from acid to alkaline pH, as M2 (predominant form in spleen, leukocytes, lung...), M3, M4 and M1 (predominant form in muscle and brain). In foetal tissues an extra band M2, called M2f, more anodic than M2, was added to the previously described isozymes. Except in brain (in which the isozymes M2, M3, M4 and M1 were found), the most anodic bands (M2f, M2 and M3) were predominant in all the foetal tissues. The isozymes M2f and M2 seem therefore to be the original M type pyruvate kinase forms from which the other isozymes issue. The rate of each isozyme seems to depend on tissue factors characterizing the state of differentiation of some tissues, as indicated by the ability of adult muscle extracts to change the isozymes M2 and M3 into more cathodic forms.

A case of hereditary nonspherocytic hemolytic anemia associated with partial erythrocyte PFK deficiency without muscular symptoms is reported: erythrocyte enzyme activity in the propositus was 60% of normal. Kinetic studies of erythrocyte PFK revealed increased sensitivity to ATP inhibition and decreased sensitivity to citrate inhibition. Muscle PFK from the patient had a normal enzymatic activity, but was highly unstable to heat, dilution without stabilizer and urea; furthermore its starch gel electrophoretic mobility was markedly faster than the one of a normal control. The results suggested that a muscle type's subunit was deficient in the erythrocyte PFK. The authors hypothesize that there was no PFK deficiency in the patient's muscle because of the active synthesis of proteins by this tissue. In contrast, the deficiency of PFK would be easily detected in erythrocytes, because of the absence of protein synthesis.

With an electro-immunodiffusion technique the authors immunologically titrated 10 deficient glucose-6-phosphate dehydrogenase variants in the leukocytes, platelets, and red blood cells.

A new G-6PD varient with enzyme deficiency is described in a 7-month-old Turkish boy without any hemolytic manifestation, except neonatal hyperbilirubinemia. The main characteristics of this variant were the following: Severe enzyme deficiency in erythrocytes (8% of normal), fast starch-gel-electrophoretic mobility (110% of normal), increased Ki NADPH with respect to NADP+, slightly biphasic pH curve, enzyme instability, in vivo and in vitro, decreased molecular specific activity (58% of normal).

In a 5-year-old Italian girl with severe congenital hemolytic anemia, red cell GPI deficiency was proven, and found to be due to a new variant, 'GPI Roma.' The parents are first cousins and have been proven to be heterozygous for this variant. GPI Roma was slightly unstable to heat and exhibited a slightly increased Michaelis constant for fructose-6-phosphate. A single predominant fast-migrating GPI form existed in the patient's white blood cells, while the electrophoretic pattern in the red cells was composed, in addition to this 'fast band,' of a major band migrating as normal GPI and of an additional slow band. It is shown that this phenomenon may be ascribed to postsynthetic events modifying the charge of the mutant enzyme.

Les auteurs ont dose l'activite et determine la migration electrophoretique de la glucose-6-phosphate deshydrogenase erythrocytaire chez 301 sujets mâles de race noire.

A low rate value of G6PD was found in red blood cells from a Cambodian boy. Enzyme mapping was performed according to the WHO standard methods. G6PD presented all the characteristics of the A(-) variant encountered in the Negroes and behaved distinct from fast migrating enzymes described in China. No negro was in the ancestry of the mother.

Erythrocyte pyruvate kinase (PK) deficiency was detected in a boy of dutch origin. Immunologic, electrophoretic, and kinetic studies of the enzymes of propositus and the members of his family demonstrated that the boy was heterozygote for two different mutant PK alleles. The mutant enzyme, for which his mother was heterozygote, was characterized by a lower immunologic specific activity, a decreased affinity for the substrate phospho-enol-pyruvate, and a loss of homotropic interactions toward this substrate, an increased affinity toward the allosteric inhibitor MgATP2-, a decreased affinity for the activator fructose-1,6-diphosphate, and a lowered pH optimum. Electrophoresis, Km app. for MgADP, and the reactivity toward the purine nucleotide substrate analogues were normal. The mutant enzyme for which his father was heterozygote was characterized by a decreased affinity for the substrate phospho-enol-pyruvate and a loss of homotropic interactions toward this substrate. All other parameters mentioned above were normal. The use of a kinetic study of mutant enzymes for the detection of heterozygotes is discussed.

Since the discovery by Carson et al. [3] of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients with primaquine-induced hemolytic crisis, more than 20 different red cell enzymopathies with red cell dysfunction have been described. In addition several metabolic diseases can be diagnosed through detection of a specific enzyme defect in red cells, but do not alter red cell function and viability or normal oxygen carriage and delivery to the tissues. The main progress made during recent years in knowledge of the earlier described enzyme disorders concerns molecular and genetic mechanisms of the defects and the relationship between molecular anomalies, red cell dysfunction and pathological expression. In some instances, such as for G6PD, pyruvate kinase (PK) and glucose phosphate isomerase (GPI), the association of secondary postsynthetic modifications with the genetic primitive alteration has been pointed out. Finally, some new enzyme disorders with congenital nonspherocytic hemolytic anemia (CNSHA) have been described, and the percentage of CNSHA of unknown cause is continuously decreasing.

Isozymic heterogeneity of human phosphofructokinase was investigated by means of ATP inhibition, immunoneutralization by antihuman muscle-type and antiliver-type phosphofructokinase antisera, solubility in (NH4)2SO4 solutions, and starch gel and polyacrylamide slab gel electrophoresis. The enzymes studied by these methods were purified from various normal and malignant human adult tissues by chromatography on blue Dextran Sepharose 4 B columns. From the results of these studied we suggest that three basic phosphofructokinase isozymes could exist: muscle-type, fibroblast-type, and liver-type isozymes. Muscle-type isozyme is the single form found in adult muscle, and is involved in the enzymes from heart, brain, red cell, and testis. Fibroblast-type isozyme is found mainly in the placenta, fibroblasts kidney, and some malignant tissues. Liver-type phosphofructokinase seems to be very definitely the predominant form in mature polymorphonuclear cells, platelets, and liver. Testis and red cell phosphofructokinase enzymes definitely include msucle-type aand liver-type subunits, associated in various hybrid forms.

Anomalies of the mutant pyruvate kinase variants and clinical symptoms have been compared in 22 unrelated patients with congenital red cell pyruvate kinase deficiency. This study suggests that some characteristics of the mutant enzymes could play a role in the intensity of haemolysis, namely residual activity, affinity for the substrate phosphoenolpyruvate and for the allosteric activator fructose 1,6 diphosphate and inhibition by ATP.

A deficient G-6PD variant was discovered in 4 males of one family from northwestern Germany. Five generations of this family could be studied. The deficient G-6PD was a new variant, called "Gd (--) Aachen". Its main characteristics are the following: severe enzyme deficiency in erythrocytes (3% of normal), contrasting with an almost normal activity in leukocytes; normal molecular specific activity (i.e., normal ratio enzyme activity/cross-reacting material); slow mobility in starch gel electrophoresis (92-94% of normal); increased Michaelis constant for glucoes-6-phosphate (60-70 muM) and NADP+ (20-25 muM); decreased inhibition constant by NADPH with respect to NADP+ (7 muM); increased inhibition by ATP; normal utilization of the substrate analogues; slightly biphasic pH curve; thermal instability, and normal activation energy of the enzymatic reaction. The relationships between the hematologic disorders (severe and frequent hemolytic crises) and the unfavorable kinetic modifications are discussed.

Two new deficient glucose-phosphate-isomerase (GPI) variants have been described in patients suffering from severe chronic hemolytic anemias. The patients' parents were consanguineous, such that the patients were true homozygotes for the mutated GPI genes. In both cases the main cause of the defect in enzyme activity was molecular instability of the mutated GPI molecules, their catalytic activity being nearly normal. GPI 'Paris' was characterized by a slow electrophoretic migration and, above all, a drastically altered affinity for the substrates glucose-6-phosphate (decreased) and fructose-6-phosphate (increased). GPI 'Enfants malades' exhibited a slightly reduced electrophoretic mobility, an abnormal curve of the activity in function of pH, and an abnormal ratio of maximal velocity in the backward direction (fructose-6-phosphate leads to glucose-6-phosphate) to that in the forward direction (glucose-6-phosphate leads to fructose-6-phosphate). No clear relation could be proved between the kinetic abnormalities of the mutant GPI variants on the one hand and the metabolic changes of the GPI-deficient red cells and the severity of hemolysis on the other. Finally we emphasized the possible role of the impairment of hexosemonophosphate pathway in the reduction of viability of the GPI-deficient red cells.