Isabelle Hombrados

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.

The primary sequence of badger myoglobin.

Abstract The badger apomyoglobin was first submitted to tryptic digestion. The tryptic hydrolysate of whole protein was fractionated by resin chromatography and each tryptic peptide was sequenced. The apomyoglobin was also cleaved by cyanogen bromide and the resulting fragments were fractionated by gel filtration; three segments were obtained; the N-terminal segment (55 residues), the C-terminal segment (22 residues) and the median segment (76 residues). Each segment was submitted to a variety of enzymatic digestions and the complete amino acid sequence of resulting peptides was established. In addition, the whole protein and the median segment were analyzed by using a sequenator (Edman); the first 31 N-terminal residues of the intact protein and the first 28 N-terminal residues of the median segment of CNBr resulting peptide were confirmed. The complete sequence of the C-terminal segment (22 residues) was also established and confirmed by manual Edmans degradation. Between badger and harbor seal myoglobins, 19 differences out of 153 residues were found. Of these amino acid replacements, one corresponds to the exchange of two bases and 18 to the exchange of one base in the coding triplets.

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. Hum Mutat 14:222–232, 1999.

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.

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 Günthers 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.

Primary sequence of the β-chain of badger haemoglobin

Abstract 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 β-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 β-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 β-chains already sequenced shows a greater similarity with dog haemoglobin, the only example of β-chain of known structure in the order of Carnivores.

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

Abstract The complete amino acid sequence of the α 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 α chain. Sixteen differences were noted between the α chains of badger and man. Phylogenetic aspects and three-dimensional structure requirements are discussed.

The N-terminal and C-terminal amino acid sequence of badger myoglobin.

A comparative study of the covalent structure of several animal myoglobins has been undertaken by some of us (Lille group): the complete covalent structure of horse [ 11, ox [2] and sheep [3] myoglobin has been determined and the partial amino acid sequence of hog [4] and dog [5] myoglobins investigated. No information has been reported on the myoglobin of the badger (Meles me/es), a wild animal rather common in the Bordeaux region for which some biochemical data are already available [6, 71. Therefore, in order to extend the structural studies and for the purpose of establishing evolutionary and genetic informations, we are attempting to deepen the comparative studies of this peculiar protein, using different data reported in the literature [S-16].

The β-chain of badger haemoglbin: Amino acid composition of the tryptic peptides and the N-terminal sequence to position 42

We report here the partial structure of the P-chain of the haemoglobin of the Badger (Meles meles), an animal of the hitherto poorly studied group of carnivores. The tryptic hydrolysate was resolved by electrochromatography and the amino acid composition of 15 peptides is given. The amino acid sequence to position 40 has been determined by analysis of four tryptic peptides, which are compared to the homologous peptides isolated from human P-chain. The sequence is confirmed and extended to position 42 by stepwise degradation of the whole P-chain, using a sequencer.

Fingerprints of tryptic peptides of Carnivora myoglobins

Abstract The moderate evolution rate of apomyoglobins may be the support of a simplified strategy for determining unknown covalent structures within the order of Carnivora, taking the badger apomyoglobin as a model. The CNBr cleavage was followed by the isolation of three polypeptide fragments which were subsequently submitted to trypsin digestion. The fingerprints of the three hydrolysates as may be obtained from seven Carnivora species, show a fairly constant number of spots, often corresponding to identical or closely related peptides, espcially in the case of the N-terminal and C-terminal fragments.