Pterin Ring Research Articles

The molybdenum site in the xanthine oxidase-related aldehyde oxidoreductase from Desulfovibrio gigas and a catalytic mechanism for this class of enzymes

The crystal structure analysis of the aldehyde oxidoreductase from Desulfovibrio gigas was exceptionally revealing with regard to the ligands and structure of the molybdenum site and the mechanism of the hydroxylation reaction catalyzed. The metal is pentacoordinated by two sulfurs of the cis–dithiolene group of the molybdopterin cofactor and by facially arranged sulfido, oxo and water ligands. The latter is in hydrogen-bonding contact with the carboxylate group of Glu 869 and the hydroxyl group of an isopropanol molecule, a substrate analogue inhibitor. This steric arrangement strongly suggests a mechanism for the reductive half-cycle of the reaction with Glu 869 as the base, the metal-bound water as the source of the transferred hydroxyl group, and the sulfido group as the hydride acceptor. The geometry and the proposed mechanism are in agreement with density functional calculations on a model of the molybdenum site. In the oxidative half-reaction, electrons are withdrawn from Morv through the rigidly held pterin ring system, via the iron-sulfur clusters, to the protein surface.

D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH.

The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea. In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion. In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes. A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate. Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M. thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.

Electrostatic Characterization of Enzyme Complexes: Evaluation of the Mechanism of Catalysis of Dihydrofolate Reductase

The catalytic mechanism of dihydrofolate reductase is evaluated with Poisson−Boltzmann electrostatic and quantum chemical vibrational frequency calculations. The results indicate that an elevated pKa of 6.5 associated with the chemical step is due to the formation of the enol tautomer of the substrate's pterin ring. The tautomer is induced to form as a result of substrate binding, in which the substrate desolvates the active site and binds to the carboxylate of Asp 27. Although the binding reaction is favorable, burial of the negative charge on Asp 27 is not. Protonation of Asp 27 occurs, concerted with tautomerization of the substrate, resulting in active site electrical neutrality and activation of the substrate for catalysis. These results require a reinterpretation of previous data from Raman spectroscopy studies in which it was proposed that the reactive atom, the pterin N5, is directly protonated. Quantum chemical vibrational frequency calculations demonstrate that the enol tautomer undergoes a Raman active vibrational perturbation at a frequency similar to that observed experimentally. Furthermore, the calculations indicate that direct protonation of the pterin N5 due to classical electrostatic interactions is quite difficult, with the pKa for this residue being shifted from 2.6 in solution to below zero while bound to the protein. The conclusions of this work are independent of the protein dielectric constant in the range of 4−20.

Structure-based identification of a ricin inhibitor

Ricin is a potent cytotoxin which has been used widely in the construction of therapeutic agents such as immunotoxins. Recently it has been used by governments and underground groups as a poison. There is interest in identifying and designing effective inhibitors of the ricin A chain (RTA). In this study computer-assisted searches indicated that pterins might bind in the RTA active site which normally recognizes a specific adenine base on rRNA. Kinetic assays showed that pteroic acid could inhibit RTA activity with an apparent Ki of 0.6 mM. A 2.3 Å crystal structure of the complex revealed the mode of binding. The pterin ring displaces Tyr80 and binds in the adenine pocket making specific hydrogen bonds to active site residues. The benzoate moiety of pteroic acid binds on the opposite side of Tyr80 making van der Waals contact with the Tyr ring and forming a hydrogen bond with Asn78. Neopterin, a propane triol derivative of pterin, also binds to RTA as revealed by the X-ray structure of its complex with RTA. Neither pterin-6-carboxylic acid nor folic acid bind to the crystal or act as inhibitors. The models observed suggest alterations to the pterin moiety which may produce more potent and specific RTA inhibitors.

New Analogs of Methotrexate in Cancer and Arthritis

Abstract: Aminopterin and its N10-methyl analog; methotrexate, were synthesized in the late 1940s and methotrexate was subsequently incorporated into clinical use as an antitumor agent in the mid-1950s. In more recent years methotrexate has also become accepted as an effective agent for treatment of rheumatoid arthritis. Structure-activity studies have been ongoing over the past 40 years in an effort to develop a safer and more effective version of this powerful antifolate. Early work featured replacement of nitrogen atoms at various positions in the pterin ring, substitution about the phenyl ring in the side chain and alteration in the glutamic acid subunit. These studies shed considerable light on structural requirements for antitumor activity. However, new agents with serious potential for clinical introduction were not encountered until structural modification of the region bridging the pterin and benzoyl glutamate side chain took place. A series of 10-substituted-10-deazaaminopterin analogs was investigated and found to have activity superior to methotrexate in several tumor models in vivo. There was in vitro evidence for facilitated transport into tumor cells versus sensitive host tissue cells and increased polyglutamylation within the tumor cells such that a selective accumulation was achieved in tumor tissue. 10-Ethyl-10-deazaaminopterin (Edatrexate) was found to be clinically effective against lung and breast solid tumors. Our laboratories have continued to pursue structural modifications of 10-deaza, 5,10-dideaza and 8,10-dideazaaminopterins in an effort to improve upon the efficacy of edatrexate. Additional variation has been made in the "bridge region" and various heterocyclic rings have been introduced in place of the side chain phenyl ring. These modifications have resulted in additional new antitumor agents and compounds with activity exceeding that of methotrexate in rheumatoid arthritis.

3H-n.m.r. studies of multiple conformations and dynamic processes in complexes of folate and methotrexate with Lactobacillus casei dihydrofolate reductase

[7,3',5'-3H3]- and [7,9-3H3]-folic acid and [7,3',5'-3H3]methotrexate (MTX) have been prepared and 3H-n.m.r. spectra obtained for their complexes with Lactobacillus casei dihydrofolate reductase (DHFR). The 3H results confirm the presence of three pH-dependent different conformational forms in the complex DHFR.NADP+.folate. The folate benzoyl ring could be shown to be in essentially the same environment in the different forms, with the major differences being associated with the pterin ring. The appearance of a single resonance for the 3',5'-tritons showed that the benzoyl ring is flipping rapidly in all three forms. In contrast, the MTX complex was shown to exist as a single conformational state with the benzoyl ring flipping rate being too low to give a single averaged signal for the 3',5'-nuclei over the temperature range 283-313 K.

Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

Phosphorescence studies of folate

A room temperature phosphorescence analysis of folate was developed, resulting in the construction of a linear calibration curve within the range of 60–1000 ng. Relative standard deviations were approximately 2% for triplicate measurements. The phosphorescence properties of analytically pure folic acid were evaluated at 77 and 298 K on a sodium acetate-impregnated paper support and on other media. Evidence is presented to support the existence of an intricate intramolecular energy transfer scheme within the folate molecule. Our results indicate that only the pterin ring system within folate appears to yield phosphorescence when studied on a sodium acetate-impregnated paper support or in aqueous alcoholic rigid glasses. The specific phosphorescent properties of a series of folate constituents are also described. Some of the results presented within this paper are in contrast to earlier reports on the phosphorescence properties of folate, and explanations for these discrepancies are provided.

Theoretical Stereostructure of the Neutral Form of Natural Tetrahydrobiopterin

Summary The absolute conformation and the reactivity of atoms of the neutral form of natural tetrahydrobiopterin [(6R)-6-(L-erythro-1 ′,2′ -dihydroxypropyl)-5.6.7.8-tetrahydropterin] was determined by the Molecular-Orbital method within the MINDO/3 framework. Two different conformations (Form A and Form B) of the alkyl side chain relative to the pterin ring were obtained as the optimized form for the lowest energy conformation (ΔH= -174 kcal/lmol) of this molecular constitution with the same molecular width of 11.2 Å. Form A and B were distinguishable in the dihedral [01′ -C1′ -C6-H6J of - 88° and 30°. respectively. Net charges of atoms of Form A and B suggested that the predominant reactivity of the atoms of N1 C2. C4. O4, C4a. and C8a of the pterin ring and C1′. C2′. O1′ and O2′ of the side chain. It was also suggested that the position 2' is more active than the position 1′. in the alkyl side chain of Form A and B. toward NADP+ in the reverse reaction of sepiapterin reductase.

The mechanisms of inactivation of sulfite oxidase by periodate and arsenite.

The inactivation of sulfite oxidase, a molybdoenzyme containing the Mo cofactor, by arsenite and periodate was investigated. In contrast to ferricyanide (Gardlik, S., and Rajagopalan, K.V. (1991) J. Biol. Chem. 266, 4889-4895), neither of these reagents causes oxidation of the pterin ring of the Mo cofactor. Instead, inactivation by these reagents appears to involve attack on sulfhydryl groups at the active site of the enzyme. The inactivation of sulfite oxidase by arsenite was shown to be dependent on the presence of O2 and on the enzymatic oxidation of arsenite to arsenate. The inactivation was preventable by the presence of sulfite, or by the use of cytochrome c as the electron acceptor instead of O2. It is concluded that inactivation by arsenite is the result of arsenite displacement of Mo during enzymatic oxidation of arsenite to arsenate, when Mo transiently breaks its bond to protein or molybdopterin sulfhydryl(s) in order to provide a site for transfer of electrons to O2. Data indicate that arsenite is properly oriented to displace Mo only once every 20,800 turnovers, thus accounting for the slow rate of inactivation by this reagent. Inactivation of sulfite oxidase by periodate is believed to occur as the result of direct attack of periodate on the thiolate ligands of Mo, either those of the protein and/or molybdopterin, leading to Mo loss. Treatment of enzyme with even low levels of periodate resulted in loss of Mo and both sulfite:cytochrome c and sulfite:O2 activities. Molybdopterin of periodate-inactivated enzyme retained the ability to reconstitute nitrate reductase apoprotein in nit-1 extracts and the ability to reduce dichlorophenolindophenol, indicating that the pterin ring had not been oxidized.

Oxidation of molybdopterin in sulfite oxidase by ferricyanide. Effect on electron transfer activities.

The attenuation of the sulfite:cytochrome c activity of sulfite oxidase upon treatment with ferricyanide was demonstrated to be the result of oxidation of the pterin ring of the molybdenum cofactor in the enzyme. Oxidation of molybdopterin (MPT) was detected in several ways. Ferricyanide treatment not only abolished the ability of sulfite oxidase to serve as a source of MPT to reconstitute the aponitrate reductase in extracts of the Neurospora crassa mutant nit-1 but also eliminated the ability of sulfite oxidase to reduce dichlorobenzenoneindophenol after anaerobic denaturation. Additionally, the absorption spectrum of anaerobically denatured ferricyanide-treated molybdenum fragment of rat liver sulfite oxidase was typical of fully oxidized pterins. Ferricyanide treatment had no effect on the protein of sulfite oxidase or on the sulfhydryl-containing side chain of MPT. Quantitation of the ferricyanide reaction showed that 2 mol of ferricyanide were reduced per mol of MPT oxidized, yielding a fully oxidized pterin. These results corroborate the previously reported conclusion that the native state of reduction of MPT in sulfite oxidase is at the dihydro level (Gardlik, S., and Rajagopalan, K.V. (1990) J. Biol. Chem. 265, 13047-13054). As a result of oxidation of the pterin ring, the affinity of MPT for molybdenum is decreased, leading to eventual loss of molybdenum. Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT. These molecules retain sulfite:O2 activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased sulfite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observations suggest that the pterin ring of MPT participates in enzyme function, possibly in electron transfer, directly in catalysis, or by controlling the oxidation/reduction potential of molybdenum.

Partial proton NMR assignments of the Escherichia coli dihydrofolate reductase complex with folate: evidence for a unique conformation of bound folate

Sequence-specific 1H assignments have been made for over 25% of the amino acid side chains of Escherichia coli dihydrofolate reductase complexed with folate by using a variety of two-dimensional techniques. Proton resonances were assigned by using a combination of site-directed mutagenesis and a knowledge of the X-ray crystal structure. Unique sets of NOE connectivities present in hydrophobic pockets were matched with the X-ray structure and used to assign many of the residues. Other residues, particularly those near or in the active site, were assigned by site-directed mutagenesis. The ability to assign unambiguously the proton resonances of these catalytically important residues allowed for extensive networks of NOE connectivities to follow from these assignments. As a consequence of these assignments, the orientation of the pterin ring of folate could be determined, and its conformation is similar to that of the productive dihydrofolate complex. Under these experimental conditions, only one bound form of the pterin ring could be detected.

The state of reduction of molybdopterin in xanthine oxidase and sulfite oxidase.

Methods have been devised to examine the spectral properties and state of reduction of the pterin ring of molybdopterin (MPT) in milk xanthine oxidase and the Mo-containing domain of rat liver sulfite oxidase. The absorption spectrum of the native pterin was visualized by difference spectroscopy of each protein, denatured anaerobically in 6 M guanidine hydrochloride (GdnHCl), versus a sample containing the respective apoprotein and other necessary components. The state of reduction of MPT was also probed using 2,6-dichlorobenzenoneindophenol (DCIP) to measure reducing equivalents/MPT, after anaerobic denaturation of the protein in GdnHCl in the presence or absence of Hg2+. In the case of xanthine oxidase the data indicate that the terminal sulfide ligand of Mo causes the reduction of a native dihydro form of MPT to the tetrahydro level. This reduction does not occur if Hg2+ is added prior to denaturation of the protein. Based on its observed behavior, the native MPT in the Mo cofactor of xanthine oxidase is postulated to exist as a quinonoid dihydropterin. Quantitation of DCIP reduction by MPT of Mo fragment of sulfite oxidase showed a two-electron oxidation of MPT, even when the Mo fragment was denatured in the presence of Hg2+ to prevent internal reduction reactions due to sulfhydryls or sulfide. Difference spectra of DCIP-treated versus untreated Mo fragment showed that MPT had been fully oxidized. These data indicate that the native MPT in sulfite oxidase must be a dihydro isomer different from that in xanthine oxidase.

Anti-idiotypic antibodies elicited by pterin recognize active site epitopes in dihydrofolate reductases and dihydropteridine reductase

Monoclonal antibodies (mAbs) against antipterin immunoglobulin and dihydropteridine reductase (DHPR) and also polyclonal antibodies against human dihydrofolate reductase (DHFR) were obtained. The anti-idiotypic mAbs and anti-DHPR mAbs bind specifically to human DHFR, Escherichia coli DHFR, soybean seedling DHFR, and human DHPR in solidphase immunoassays. Further, the mAbs bind to the native but not to the denatured forms of DHFRs. The monoclonal antibodies also inhibit the enzymatic activity of human DHFR but not that of human DHPR. Competitive solid-phase immunoassays show stoichiometric inhibition by methotrexate and partial inhibition by NADPH of mAb binding to human DHFR. Cyanogen bromide fragments derived from human DHFR (residues 15–52 and 53–111), containing several active site residues, bind partially to some of the monoclonal anti-bodies. Accordingly, polyclonal antibodies to peptide 53–111 of human DHFR cross-react to some extent with human DHPR. Data from competitive immunoassays in which the binding of the various mAbs was tested singly and in combination with other mAbs suggest that these antibodies bind to a common region on human DHFR. The results also indicate that the mAbs display some heterogeneity with respect to specific epitopes. These data suggest that despite the absence of significant amino acid sequence homologies among the various DHFRs and DHPR, they have a fundamentally similar topography at the site of binding of the pterin moiety that is recognized by the anti-idiotypic mAbs generated by pterin. In the relatively simple structure of the pterin ring system there are different substituent groups at positions C 4 and C 6 in methotrexate, 7,8-dihydrofolate, and 7,8-dihydrobiopterin, suggesting that these antibodies are specific for regions on various proteins that interact with the remainder of the pterin moiety. These mAbs and similar mAbs specified by substituent groups on pterin may thus be used as specific probes or inhibitors of various folatedependent enzymes and transport proteins. They should also provide insights into some of the general features of antibody recognition of protein antigens.

Ab Initio Calculations on Keto/Enol Tautomers of Pterins. On the Significance of the Enol Tautomer of Pterin Ring in the Enzymatic Reduction of Dihydrofolate by DHFR

Abstract Ab initio and semi-empirical calculations were carried out on keto/enol tautomers of 6-methyl-7,8-dihydropterin, a model compound for the natural substrate of dihydrofolate reductase. The most likely conformer of pterin ring (inner enol 3) during the hydride transfer from NADPH was computationally chracterized.

Computational studies on pterins and speculations on the mechanism of action of dihydrofolate reductase

The significance of the enol form of the pterin ring in enzymatic reduction of dihydrofolate by DHFR is discussed on the basis of the results of ab initio calculations carried out on the keto/enol tautomers of 6-methyl-7,8-dihydropterin as the model compound for the natural substrate, dihydrofolate.

Structural elements of bactopterin from Pseudomonas carboxydoflava carbon monoxide dehydrogenase

Bactopterin is a novel pterin occurring in bacterial molybdoenzymes as the organic portion of the molybdenum cofactor. Its structure is investigated here. The compound contains a single pterin ring and carries a side chain at carbon atom 6 of the pterin nucleus as indicated by the formation of pterin-6-carboxylic acid upon alkaline permanganate oxidation. Studies with phosphate-cleaving enzymes revealed the presence of two monophosphoric acid monoesters. The affinity of reduced bactopterin for thiol-Sepharose points to the presence of thiol(s) in active bactopterin.

Studies on the oxidation state of molybdopterin

Molybdopterin, the organic component of the molybdenum cofactor common to all molybdenum-containing enzymes except nitrogenase, is proposed to contain two vicinal thiols capable of complexing Mo. The pterin ring is in an unknown state of reduction in the active cofactor. We have used subtractive difference spectroscopy for examining the reduction state of the pterin in sulfite oxidase and xanthine oxidase under rigorously anaerobic conditions in 6 M guanidinium chloride. It is concluded that the molybdopterin is in the tetrahydro form in both enzymes. Alkylation of molybdopterin with iodoacetamide in denatured samples of sulfite oxidase yields a derivative which contains two alkyl groups per phosphate and is in the tetrahydro form. These findings are in accord with the proposed presence of two thiols in the cofactor, and provide additional evidence for the state of reduction in the pterin.

Tetrahydrobiopterin analogues: solution conformations of 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyltetrahydropterins as determined by proton nuclear magnetic resonance.

The conformation of tetrahydrobiopterin analogues in aqueous solution at 23 degrees C has been determined by analyzing the 200-MHz 1H NMR spectral parameters of the enzymatically active 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyl-5,6,7,8-tetrahydropterins. Each of these cofactors, with the exception of the cis-6,7-dimethyl isomer, exhibited an unusually small trans 6H-7H spin-spin coupling (8.5-9.1 Hz). An empirical equation that accounts for the effects of substituent electronegativity and orientation on vicinal couplings [Haasnoot, C. A. G., deLeeuw, F. A. A. M., & Altona, C. (1980) Tetrahedron 36, 2783-2792] predicted this coupling to be 11.3-11.6 Hz. We attribute the discrepancy between the calculated and experimentally observed values of this coupling to hyperconjugation of the axially oriented C7-H bond with the pi orbital of the vinylogous amide protein of the pterin ring (N8-C8a = C4a-C4 = O) rather than conformational averaging. The trans 6H-7H interproton distance in the 6-methyl analogue is calculated to be 3.0 A from the measured decrease in the spin-lattice relaxation rate of the axially oriented C7 proton after specific deuteration at C6. This is consistent with the single-conformer interpretation. Chemical shift comparisons of the methyl resonances of these analogues, NOE measurements from selectively deuterated analogues, and the differential sensitivities of axially vs. equatorially disposed ring protons to protonation at N5 all indicate that (i) the methyl substituents at both the C6 and C7 positions markedly prefer equatorial-like orientations and (ii) the tetrahydropterin ring is, with the exception of a pronounced pucker at C6, nearly planar.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple molybdoenzyme deficiencies due to an inborn error of molybdenum cofactor metabolism: Two additional cases in a new family

Journal of Inherited Metabolic DiseaseVolume 6, Issue S2 p. 95-96 Short Communication Multiple molybdoenzyme deficiencies due to an inborn error of molybdenum cofactor metabolism: Two additional cases in a new family A. Munnich, A. Munnich Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorJ. M. Saudubray, J. M. Saudubray Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorC. Charpentier, C. Charpentier Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorH. Ogier, H. Ogier Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorF. X. Coudé, F. X. Coudé Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorJ. Frézal, J. Frézal Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorL. Yacoub, L. Yacoub Hôpital Universitaire, Sousse, TunisieSearch for more papers by this authorA. Harbi, A. Harbi Hôpital Universitaire, Sousse, TunisieSearch for more papers by this authorS. Snoussi, S. Snoussi Hôpital Universitaire, Sousse, TunisieSearch for more papers by this author A. Munnich, A. Munnich Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorJ. M. Saudubray, J. M. Saudubray Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorC. Charpentier, C. Charpentier Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorH. Ogier, H. Ogier Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorF. X. Coudé, F. X. Coudé Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorJ. Frézal, J. Frézal Clinique et Unité de Recherches de Génétique Médicale (INSERM U-12), Hôpital des Enfants-Malades, Paris, Cedex 15, 75743 FranceSearch for more papers by this authorL. Yacoub, L. Yacoub Hôpital Universitaire, Sousse, TunisieSearch for more papers by this authorA. Harbi, A. Harbi Hôpital Universitaire, Sousse, TunisieSearch for more papers by this authorS. Snoussi, S. Snoussi Hôpital Universitaire, Sousse, TunisieSearch for more papers by this author First published: 01 June 1983 https://doi.org/10.1007/BF01810344Citations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume6, IssueS2June 1983Pages 95-96 RelatedInformation