Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis. Identification and characterization of six novel mutations associated with familial PCT.

The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able to detect the underlying UROD mutation in 10 previously characterized DNA samples as well as a new mutation in each of six previously unexamined PCT patients. The six novel UROD mutations comprise three missense mutations (M01T, F229L, and M324T), two splice mutations (IVS3-2A-->T and IVS5-2A-->G) leading to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual catalytic activity, whereas the two missense mutants retained some residual activity. Furthermore, the missense mutants exhibited a considerable increase in thermolability. The six new mutations bring to a total of 29 the number of disease-related mutations in the UROD gene. The DGGE assay presented greatly improves the genetic diagnosis of fPCT and HEP, thereby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of human uroporphyrinogen decarboxylase.

A recombinant human uroporphyrinogen decarboxylase (E.C. 4.1.1.37, UROD) has been expressed in Escherichia coli and purified to homogeneity. Crystals grew by the hanging-drop vapor-diffusion technique from a starting solution containing 1.5 mg ml-1 protein. The crystals belong to the trigonal space group P3121 or its enantiomer P3221 and diffract to 3 A resolution. The unit-cell parameters are a = b = 103.4, c = 75.7 A and gamma = 120 degrees. The asymmetric unit contains one molecule. Preliminary structural predictions suggest for the protein a TIM-barrel type tertiary structure.

Hepatic uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients: the influence of virus C infection.

Porphyria cutanea tarda (PCT) is caused by a decreased activity of the hepatic enzyme uroporphyrinogen decarboxylase (URO-D). This deficiency causes overproduction, hepatic deposition, and increased excretion of uroporphyrin. Iron overload and hepatic viral infections are considered aggravating factors of the disease. Two forms of PCT have been described, as follows: a familial one with an inherited decrease of URO-D activity in all tissues and a sporadic one with a decreased activity of URO-D restricted to the liver. To assess whether the hepatic URO-D returns to normal during a remission of the disease, this activity was measured in liver biopsy samples in 24 sporadic PCT patients. The hepatic and urinary porphyrin concentrations were also measured. Viral status and histopathological findings were analyzed to assess their involvement in PCT. Six patients treated by phlebotomy to reduce hepatic iron and who were considered to be in clinical remission, characterized by a disappearance of cutaneous lesions, showed higher hepatic URO-D activities and lower hepatic porphyrin concentrations than did patients with overt PCT. The medians of these variables, however, did not achieve normal values. The hepatic URO-D activity showed a significant inverse relationship with both hepatic porphyrins and urinary uroporphyrin excretion. Hepatic URO-D activity was not reduced by hepatitis C virus (HCV) infection and liver damage. We conclude that the achievement of remission in PCT largely depends on the transient normalization of hepatic URO-D activity. A small increase in hepatic coproporphyrin in nonporphyric patients could reflect hepatic injury/iron/alcohol-induced oxidative stress oxidizing the accumulated heme precursors rather than a direct effect on hepatic URO-D enzyme.

Novel point mutation in the uroporphyrinogen III synthase gene causes congenital erythropoietic porphyria of a Japanese family.

The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in a member of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Günther's disease. The patient was homozygous for a novel missense mutation: a G to T transition of nucleotide 7 that predicted a valine to phenylalanine substitution at residue 3 (V3F). The parents were heterozygous for the same mutation. The loss of UROIIIS activity was verified by an in vitro assay system. The corresponding mutated protein was expressed in Escherichia coli and no residual activity was observed. Further studies are needed to determine whether the mutations of the UROIIIS gene (UROS) have a specific profile in Japan compared to European or American countries.

Prenatal diagnosis in congenital erythropoietic porphyria by metabolic measurement and DNA mutation analysis.

Identification of uroporphyrinogen III synthase (UROIIIS) gene mutations in patients with congenital erythropoietic porphyria (CEP) allows fast and reliable carrier detection and prenatal diagnosis. We describe here the first case of prenatal diagnosis by concomitant measurement of uroporphyrin I in amniotic fluid and direct detection of the gene mutation. A French couple, whose first child was diagnosed with CEP, requested prenatal diagnosis at 16 weeks of gestation. Uroporphyrin I was dramatically increased in amniotic fluid and the fetus was homozygous for the C73R mutation, the most common mutation in this disease. The pregnancy was then terminated.

[Congenital erythropoietic porphyria. Apropos of a fatal case in the neonatal period due to acute hemolysis with hepatic failure]. / Porphyrie érythropoïétique congénitale. A propos d'un cas fatal en période néonatale dû à une hémolyse aiguë avec insuffisance hépatique.

BACKGROUND: Congenital erythropoietic porphyria, an autosomal recessive disease, is characterized by deficiency of uroporphyrinogen III synthase. Clinical variability of the disease is related to the different mutations found in the patients. CASE REPORT: A newborn suffered one hour after birth from jaundice and polypnea with acute hemolysis. Severe cutaneous photosensitivity occurred after phototherapy. Congenital erythropoietic porphyria was suspected because of reddish-colored urine and confirmed by porphyrin analyses. The baby died one month later due to severe hemolytic anemia with hepatic failure. Uroporphyrinogen III synthase activity was decreased by 99% in bone marrow cells and established lymphoblastoid cells from the patient. Molecular biology studies demonstrated the presence of the Cys 73-->Arg substitution at the homozygous state in the patient. CONCLUSION: This mutation, the most frequently found in this disease, is responsible for a severe phenotype. Molecular characterization provides genotype/phenotype correlations in this porphyria and allows to clarify unusual cases of porphyrias.

Identification of two new mutations in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. Six missense mutations in the UROIIIS gene, a deletion and an insertion have already been described in CEP. This work brings further evidence for the heterogeneity in the genetic defect found in CEP. Two new mutations are described, a point mutation (V99A) and a frame-shift mutation (633insA) in the same patient who had a mild to moderate form of Günther's disease. The mutation (V99A) had a detectable residual activity when expressed in Escherichia coli while the insertion (633insA), which introduced a premature stop, had no activity. In the patients studied in our laboratory, the mutation C73R, associated with a severe phenotype, remains the most frequently seen.

The primary structure of the hemoglobins of a southern hemisphere lamprey (Mordacia mordax, Cyclostomata).

Mordacia mordax is a southern hemisphere lamprey belonging to Mordaciidae, a primitive family of Cyclostomata. Adult erythrocytes contain three monomeric hemoglobins which can be easily separated by cellulose acetate electrophoresis and isolated by ion-exchange chromatography. The N-terminal regions, and the tryptic peptides from each chain were submitted to automated Edman degradation; the alignment of the fragments was obtained by homology with the other Petromyzonoidea hemoglobins hitherto sequenced. Our results confirm the phylogenic distance between lampreys and hag-fish hemoglobins. As was observed for Petromyzon marinus species, two hemoglobins of Mordacia mordax are very close, as they differ only at 7 positions.

Carnivora: the primary structure of the alpha-chains of ferret (Mustela putorius furo, Mustelidae) hemoglobins.

Ferret erythrocytes contain two hemoglobins differing only by their alpha-chains. The primary structure of the common beta-chain has been previously described; the complete sequence of the two alpha-chains are reported in this paper. The globin chains were separated by ion-exchange chromatography; the alpha-chains (42 steps), their tryptic peptides as well as the prolyl-peptides were subjected to automatic liquid- and gas-phase Edman degradation. The two alpha-chains are very similar, differing at only one position (Asp15----Gly15). Comparison with human hemoglobin alpha-chain shows 16 and 17 exchanges, for alpha 1 and alpha II chains, respectively; two substitutions involve alpha 1/beta 1 contacts and one the heme contacts. A high degree of homology was noted when the alpha-chains were compared to the corresponding chains of other representatives of the Carnivora order.

[Qualitative and quantitative hemoglobin anomalies. Fundamental concepts, recent advances ]. / Anomalies qualitatives et quantitatives des hémoglobines. Notions fondamentales, acquisitions récentes.

The presence, in metropolitan France, of large foreign communities originating from Maghreb and from Black Africa, introduces hereditary haemoglobinopathies diagnosis in the routine work of the clinical laboratory. On the other hand, the sustained interest of the medical staff and of biologists arises principally from the dramatic progresses acquired since twenty years in the field of molecular anatomy and physiology of the oxygen carrier. Recent studies concerning human haemoglobin genes and their expression resulted in a new classification of haemoglobinopathies and in new possibilities in the prenatal diagnosis of these disease.

Primary structure of the minor haemoglobins from the sea lamprey (Petromyzon marinus, Cyclostomata).

Erythrocytes of the adult Sea Lamprey Petromyzon marinus contain several haemoglobin species, but only the main constituent has hitherto been sequenced. The present paper describes the determination of the primary structures of the two minor species, whose electrophoretic mobilities are higher and lower than that of the main component. Tryptic peptides from both chains were purified by high-performance liquid chromatography, then sequenced and aligned by homology with the main haemoglobin. The fast and the major components appeared to be very similar, differing in only four positions (pos. 5: Ser----Thr; pos. 33: Thr----Ser; pos. 86: Val----Ala; pos. 99: Gly----Arg). The slow haemoglobin component, however, differed from the main component with respect to 27 amino-acid residues. The position of the three globins of Petromyzon marinus in the phylogenetic tree of haemoglobins is discussed and a relationship with primitive alpha-chains is postulated.

Haemoglobins, LX. Primary structure of the major haemoglobin of the sea lamprey Petromyzon marinus (var. Garonne, Loire).

Lampreys belong to the class of Cyclostomata; practically no evolution of these Vertebrates can be noted since Paleozoïc times; lampreys thus appear as a choice material for studying several problems in the field of biochemical evolution. Several monomeric haemoglobins can be characterized in the erythrocytes of the sea lamprey (Petromyzon marinus). The major constituent was isolated by chromatography, and submitted to tryptic digestion; soluble tryptic peptides were separated by gel filtration into 5 fractions; the peptides of each fraction were isolated either by Dowex-50 chromatography or by HPLC; the insoluble core was oxidized and submitted to HPLC fractionation. The primary structure of the whole chain and of the purified tryptic peptides was determined using automatic sequencing; alignment of the peptides was achieved by homology with the previously established covalent structure of the globin of Lampetra fluviatilis. The sequence we established confirms the crystallographic data of Hendrickson and Love. Globin/haem contacts are discussed; a tentative explanation of the absence of tetramerization can be proposed after comparison with the aminoacid residues involved in alpha 1 beta 1 and alpha 1 beta 2 contacts. Petromyzon globin differs at three locations (Thr/Ser3, Leu/Met58, Thr/Ser60) from Lampetra fluviatilis globin. The monomeric chain of another Cyclostomata Myxine glutinosa, differs more considerably (88 residues). Our results corroborate recent paleontologic data which favour the separation of lampreys from hagfishes; Cyclostomata cannot be considered as a monophylic group. Finally, there is a closer relation between lamprey globin and alpha chains than between this monomeric globin and beta chains, and furthermore apomyoglobins of higher vertebrates.

The amino acid sequence of the alpha chain of badger (Meles meles) haemoglobin.

The complete amino acid sequence of the alpha chain from the badger (Meles meles) haemoglobin was elucidated using conventional methods chiefly performed on tryptic peptides separated by peptide "mapping" and comparison with human alpha chain. Sixteen differences were noted between the alpha chain of badger and man. Phylogenetic aspects and three-dimensional structure requirements are discussed.

Primary sequence of the beta-chain of Badger haemoglobin.

Badger (Meles meles) haemoglobin was purified by paper electrophoresis and converted into globin. Chain separation was carried out on a CM-cellulose column in the presence of 8 M urea. The beta-chain was aminoethylated, purified by gel filtration and submitted to tryptic digestion. A fingerprint obtained with the enzymic digests showed 17 distinct ninhydrin-positive spots from which 20 pure peptides were isolated by further electrochromatographic separations. These peptides were sequenced using Dansyl-Edman and Ptc-Edman degradation techniques. The presence of amide residues was confirmed after aminopeptidase M hydrolysis. Taking human haemoglobin beta-chain as a model, the covalent structure could be completely resolved without the help of any further overlapping technique. The following substitutions were noted (badger/human, position): Ala/Pro5, Ser/Ala13, Tyr/Phe41, Asp/Glu43, Ser/Ala70, Glu/Asp73, Lys/Ala76, Asn/His77, Lys/Thr87, Lys/Arg104 and Gln/Pro125. A comparison with other haemoglobin beta-chains already sequenced shows a greater similarity with dog haemoglobin, the only example of beta-chain of known structure in the order of Carnivores.