Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.

SOX10 mutations in patients with Waardenburg-Hirschsprung disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung's disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon), piebald-lethal (sl) and lethal spotting (ls). The sl and ls phenotypes are caused by mutations in the genes encoding the Endothelin-B receptor (Ednrb) and Endothelin 3 (Edn3), respectively. The identification of Sox10 as the gene mutated in Dom mice (B.H. et al., manuscript submitted) prompted us to analyse the role of its human homologue SOX10 in neural crest defects. Here we show that patients from four families with WS4 have mutations in SOX10, whereas no mutation could be detected in patients with HSCR alone. These mutations are likely to result in haploinsufficiency of the SOX10 product. Our findings further define the locus heterogeneity of Waardenburg-Hirschsprung syndromes, and point to an essential role of SOX10 in the development of two neural crest-derived human cell lineages.

Human homology and candidate genes for the Dominant megacolon locus, a mouse model of Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital disorder of the enteric nervous system characterized by the absence of enteric ganglia. Three genes for HSCR have been identified: the RET proto-oncogene, the gene coding for the endothelin B receptor (EDNRB), and the endothelin 3 gene (EDN3). In mice, natural and in vitro-induced mutations affecting the Ret, Ednrb, and Edn3 genes generate a phenotype similar to human HSCR. Another model of HSCR disease is the Dominant megacolon (Dom), a spontaneous mouse mutation for which the target gene has not yet been identified. The Dom mutation has been mapped to the middle-terminal region of mouse chromosome 15, between D15Mit68 and D15Mit2. Using new or known polymorphisms for conserved human/mouse genes, we established the homology between the Dom locus and human chromosome 22q12-q13. Two genes, Smstr3 and Adsl, not previously mapped in the mouse genome, were located on mouse Chromosome 15. Three genes (Smstr3, Lgals1, and Pdgfb) are possible Dom candidates, as they do not recombine with the Dom mutation in a 252 Dom/+ animal backcross.

A high-resolution genetic map of mouse chromosome 15 encompassing the Dominant megacolon (Dom) locus.

Dominant megacolon (Dom) is one of four mutations in the mouse that can produce a phenotype similar to Hirschsprung disease in human. The Dom gene product is not known, and no candidate region has been defined for a possible human homolog. In this publication we report mapping the Dom locus with high definition, using several intra-and interspecific crosses and a set of 16 Chr 15-specific microsatellites flanking this locus.

[Prenatal diagnosis of homozygous pyruvate kinase deficiency]. / Déficit homozygote en pyruvate kinase à révélation anténatale.

Two consecutive cases of severe neonatal anaemia due to severe deficiency in pyruvate kinase were observed in the same sibhood. The first child died one hour after birth and the second required major transfusion support. Pyruvate kinase deficiency is a rare cause of congenital anaemia with recessive autosomic inheritance. Clinically, this deficiency has a very variable expression, and neonatal forms are not always very severe. Several variant molecules in pyruvate kinase deficiency have been described. Recent progress in our understanding of the gene would suggest the possibility of new diagnostic and prognostic approaches.

Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase. Functional consequences of substitutions of His10, His187 and Arg89.

Human bisphosphoglycerate mutase (GriP2 mutase) is a trifunctional enzyme which synthesizes and degrades GriP2 in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, His10 which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89-->Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of His10 by Gly (H10G) or Asp (H10D), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the His10 residue completely abolished the enzymatic activities. Concerning the His187 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at His187 could be distinguishable from those at His10 since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (GriP2). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55 degrees C but GriP2 was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55 degrees C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, Km values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas Km values for GriP2 were normal in mutase and phosphatase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

Natural and artificial mutants of the human 2,3-bisphosphoglycerate as a tool for the evaluation of structure-function relationships.

2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue. Using site directed mutagenesis, mutants were produced with shortened chains. Results indicated the importance of residues 252-256 for the function. A natural deficient mutant with the substitution 89 Arg----Cys was described. Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism.

Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism.

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.

Rabbit M type phosphoglyceromutase: comparative effects of two thiol reagents antibody reaction and hybridization studies.

1. Treatment of purified rabbit phosphoglyceromutase (M type) with N-ethylmaleimide or with iodoacetamide produces the concurrent loss of phosphoglyceromutase activity with its collateral glycerate-2,3-P2 phosphatase activity. 2. Differences are observed in the protective effect of glycerate-2,3-P2 and of glycolate-2-P against N-ethylmaleimide and iodoacetamide treatments. 3. Specific chicken antibodies obtained by injection of the purified rabbit M type phosphoglyceromutase do not cross-react with the B type but neutralize both rabbit and human M type phosphoglyceromutase. 4. Purified rabbit M type phosphoglyceromutase can hybridize in vitro with the purified human B type or with purified human glycerate-2,3-P2 synthase. 5. Its ability to hybridize with glycerate-2,3-P2 synthase is unchanged after iodoacetamide treatment.

Purification of the M-type phosphoglyceromutase from rabbit muscle.

A new method for the purification of M-type phosphoglyceromutase from rabbit muscle involving affinity chromatography on dye ligand media in the presence of 2,3-diphosphoglycerate is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate and by Cellogel electrophoresis. Immunological techniques showed that the anti-M antibodies are specific and do not cross-react with the B isozyme.

Possibility of prenatal diagnosis of hereditary triose phosphate isomerase deficiency.

Prenatal diagnosis has been performed on umbilical cord blood of an 18 weeks fetus of heterozygous triosephosphate isomerase (TPI) deficient parents. After excluding maternal blood contamination, TPI activity was measured and found to be 60 per cent of the normal mean whereas the value of glucose-6-phosphate dehydrogenase activity was in the normal range of fetal blood. In addition, the analysis of the characteristics of fetal TPI, i.e. Km measurements for glyceraldehyde-3-phosphate, heat stability tests and electrophoretic studies, did not show any evidence of a special form of TPI in fetal blood. These results were consistent with the heterozygous state and were confirmed at birth.

[Screening for hemoglobinopathies and G6PD deficiencies in Morocco]. / Dépistage des hémoglobinopathies et des déficits en G6PD au Maroc.

Screening for abnormal hemoglobins and G6PD deficiency was conducted in adult and new born Maroccans from Casablanca. The results presented deal with HbS and HbC traits, alpha, gamma, and delta mutations, the presence of detectable amount of Hb Bart's and the G6PD deficiency frequency.

Hereditary triose phosphate isomerase deficiency: seven new homozygous cases.

Seven new homozygous cases of hereditary triosephosphate isomerase (TPI) deficiency have been detected in five unrelated families. Two of the families originate in France, the others from Algeria, Yugoslavia, and Morocco. Only the parents coming from Algeria and Morocco were first cousins. In the other parents no evidence of consanguinity was found. All seven patients exhibited the same symptoms, i.e. hemolytic anemia appearing very early after birth associated with progressive neuromuscular symptoms. Expression of the deficiency is heterogeneous; this had previously been pointed out in the previously reported cases of TPI deficiency. Red cell TPI activity was 3 to 4% of the normal mean in the patients and 50 to 60% in the parents. The latter did not exhibit any clinical symptoms. The levels of red cell glycolytic intermediates and the characteristics of the mutated TPI could be studied in four of the patients only. Substantial increases of red cell dihydroxyacetone phosphate and of fructose 1,6-diphosphate, normal Km of TPI for glyceraldehyde phosphate, and thermoinstability of the enzyme were found. In addition the electrophoretic pattern showed no significant modification of the mobility of the TPI bands, but abnormal decreased staining of the two more anodal bands.

Biochemical and immunological arguments for homology between red cell and liver phosphoglyceromutase isozymes.

Phosphoglyceromutase (2,3-bisphospho-D-glycerate:2- phospho-D-glycerate phosphotransferase, EC 2.7.5.3) from the red cell is compared with the muscle and the liver forms of the enzyme obtained from rabbits. Partially purified samples were used for this study. The electrophoretic mobility of red cell phosphoglyceromutase is completely different from that of muscle and similar to that of liver. The muscle isozyme can hybridize with liver or red blood cell phosphoglyceromutase. N-Ethylmaleimide, an SH group reagent, completely inhibits muscle phosphoglyceromutase activity and slightly inhibits the activity of the enzyme from liver and from red cells. Both liver and red cell isozymes are completely inactivated by heating at 60 degrees C for 15 min, whereas a 25% decrease in inactivity is noted for the muscle enzyme. Finally, chicken antibody directed against human red cell phosphoglyceromutase reacts with the enzyme isolated from rabbit red cells and liver, but not with that obtained from muscle.

Evidence for the presence of a hybrid of phosphoglyceromutase/bisphosphoglyceromutase in the red cells: partial characterization of the hybrid.

Purified phosphoglyceromutase was hybridized in vitro with pure biphosphoglyceromutase . The hybrid showed an electrophoretic mobility identical to that of the intermediate band of red cell phosphoglyceromutase activity in the hemolysate patterns. Electrophoretic tests showed that the partially purified hybrid displayed both bisphosphoglyceromutase and phosphoglyceromutase activities and that the sample contained also a small portion of non-hybrid bisphosphoglyceromutase . By constrast to a non-hybrid mixture of the two purified mutases the hybrid exhibited heat instability of bisphosphoglyceromutase activity and neutralization of phosphoglyceromutase activity by anti- bisphosphoglyceromutase antibody.

[Diphosphoglyceromutase deficiency: new cases associated with erythrocytosis]. / Déficit en diphosphoglycérate mutase: nouveaux cas associés à une polyglobulie.

New cases of diphosphoglyceromutase (DPGM) have been detected, associated with erythrocytosis in two unrelated families. The deficiency appears to be inherited as an autosomal dominant trait. Diphosphoglycerate phosphatase activity paralleled DPGM activity in all the subjects. Three of the latter displayed complete DPGM deficiency with about 0.4% of the normal 2,3-diphosphoglycerate (2,3 DPG) level. The other four showed partial deficiency (about 50% normal mean) with a similar decrease in 2,3-DPG level. The P50 values are in agreement with the red cell 2,3-DPG concentrations. Il all the deficient subjects the ATP level was elevated and the pattern of glycolytic intermediates was disturbed, with an increase in fructose 1,6-diphosphate, triose-phosphates, 3-phosphoglycerate, glucose 1,6-diphosphate, and reduced or normal levels of glucose-6-phosphate and fructose-6-phosphate.

Partial characterization of the inactive mutant form of human red cell bisphosphoglyceromutase and comparison with an alkylated form.

The trifunctional enzyme bisphosphoglyceromutase (or diphosphoglycerate mutase) (EC 2.7.5.4) was purified from human red cells and injected into two chickens. Specific anti-bisphosphoglyceromutase antibodies were produced that displayed a single precipitation line on Ouchterlony plates and on immunoelectrophoresis. No cross-reaction of these antibodies was detected with phosphoglyceromutase, the common glycolytic enzyme. Immunoneutralization of bisphosphoglyceromutase and of its two other activities, i.e., bisphosphoglycerate phosphatase and phosphoglyceromutase, was observed for a purified preparation. The anti-bisphosphoglyceromutase antibody reacts with the inactive enzyme present in the hemolysate of a mutant human subject. It also binds bisphosphoglyceromutase inactivated by N-ethylmaleimide, a strong alkylating agent of SH groups. Active bisphosphoglyceromutase is stable at 55 degrees C, whereas the inactive forms of the mutant and of the alkylated hemolysates are thermolabile. These forms can be protected against thermal precipitation by 4 mM 2,3-diphosphoglycerate and 4 mM 3-phosphoglycerate. These findings afford evidence that the binding of the substrates on the bisphosphoglyceromutase molecule is not prevented by alkylation nor by the mutation of the hereditary inactive enzyme.