Population-Based Newborn Screening for Mucopolysaccharidosis Type II in Illinois: The First Year Experience.

OBJECTIVES: To assess the outcome of population-based newborn screening for mucopolysaccharidosis type II (MPS II) during the first year of screening in Illinois. STUDY DESIGN: Tandem mass spectrometry was used to measure iduronate-2-sulfatase (I2S) activity in dried blood spot specimens obtained from 162 000 infant samples sent to the Newborn Screening Laboratory of the Illinois Department of Public Health in Chicago. RESULTS: One case of MPS II and 14 infants with pseudodeficiency for I2S were identified. CONCLUSIONS: Newborn screening for MPS II by measurement of I2S enzyme activity was successfully integrated into the statewide newborn screening program in Illinois.

Noncovalent interactions in the gas-phase conformers of anionic iduronate (methyl 2-o-sulfo-α-L-iduronate): variation of subconformer versus ring conformer energetics for a prototypical anionic monosaccharide studied using computational methods.

The conformational preferences of the prototypical anionic monosaccharide (methyl 2-O-sulfo-α-L-iduronate) have been studied at various computational levels to investigate the energetic variation of 17 subconformers associated with the (4)C(1), (2)S(0), (5)S(1), and (1)C(4) ring conformers. These calculations include the first fully optimized MP2 calculations that have been performed for an anionic sugar system, and therefore allow an assessment of the performance of a group of DFT functionals (B3LYP, PW91PW91, and M05-2X) for treating the noncovalent dispersion and anomeric effects that are present in this system. We find that the recently developed M05-2X functional of Truhlar and co-workers [Y. Zhao, N. E. Schultz, D. G. Truhlar, J. Chem. Theory Comput., 2006, 2, 364] reproduces the MP2 results most closely, thus indicating that it may well be suitable for computational studies of larger ionic saccharides. Most importantly, the results presented indicate that it is crucial to consider the subconformers (which correspond to rearrangements of the sugar-ring side-chains) of the main ring-conformers in order to obtain a reliable overview of the potential energy surface of such systems. We find that the lowest isolated (gas-phase) conformer corresponds to a (4)C(1) chair conformer, which displays a pair of strong C(3)-OH···SO(3)(-) and OMe···SO(3)(-) electrostatic hydrogen-bonding interactions, coupled with a looser C(4)-OH···SO(3)(-) interaction. Overall, the relative energies of the subconformers appear to be straightforwardly related to the number of hydrogen-bonding interactions that each conformer displays among its pendant functional groups.

Use of estimated evolutionary strength at the codon level improves the prediction of disease-related protein mutations in humans.

Predicting the functional impact of protein variation is one of the most challenging problems in bioinformatics. A rapidly growing number of genome-scale studies provide large amounts of experimental data, allowing the application of rigorous statistical approaches for predicting whether a given single point mutation has an impact on human health. Up until now, existing methods have limited their source data to either protein or gene information. Novel in this work, we take advantage of both and focus on protein evolutionary information by using estimated selective pressures at the codon level. Here we introduce a new method (SeqProfCod) to predict the likelihood that a given protein variant is associated with human disease or not. Our method relies on a support vector machine (SVM) classifier trained using three sources of information: protein sequence, multiple protein sequence alignments, and the estimation of selective pressure at the codon level. SeqProfCod has been benchmarked with a large dataset of 8,987 single point mutations from 1,434 human proteins from SWISS-PROT. It achieves 82% overall accuracy and a correlation coefficient of 0.59, indicating that the estimation of the selective pressure helps in predicting the functional impact of single-point mutations. Moreover, this study demonstrates the synergic effect of combining two sources of information for predicting the functional effects of protein variants: protein sequence/profile-based information and the evolutionary estimation of the selective pressures at the codon level. The results of large-scale application of SeqProfCod over all annotated point mutations in SWISS-PROT (available for download at http://sgu.bioinfo.cipf.es/services/Omidios/; last accessed: 24 August 2007), could be used to support clinical studies.

Synthesis of uridine 5'-diphosphoiduronic acid: a potential substrate for the chemoenzymatic synthesis of heparin.

An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5'-diphosphoiduronic acid (UDP-IdoA) and uridine 5'-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5'-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity.

Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms.

The present study aimed to characterize mutant alleles in Mucopolysaccharidosis II and evaluate possible reduction of mRNA amount consequent to nonsense-mediated or nonstop mRNA decay pathways. A combination of different approaches, including real-time RT-PCR, were used to molecularly characterize seventeen patients. Fifteen alleles were identified and nine of them were new. The novel alleles consisted of three missense mutations (p.S71R, p.P197R, p.C432R), two nonsense (p.Q66X, p.L359X), two frameshifts (p.V136fs75X, p.C432fs8X), one allele carrying two in-cis mutations [p.D252N;p.S369X], and a large deletion (p.G394_X551). Analysing these results it emerged that most of the alterations resulted in mutants leading to mRNAs with premature termination codons, and therefore, potentially sensitive to mRNA surveillance pathway. By using real-time RT-PCR, the mRNAs resulting (i) from substitutions that changed one amino acid to a stop codon (L359X, and S369X), or caused the shifted reading frame with premature introduction of a stop codon (C432fs8X), (ii) from large deletion (p.G394_X551) that included the termination codon, seemed to be subject to degradation by nonsense-mediated (i) or nonstop decay (ii) mechanisms, as mRNA was strongly underexpressed. On the contrary, two mutations (Q66X and V136fs75X) produced transcripts evading mRNA surveillance pathway despite both of them fulfilled the known criteria. These results confirm the wide variability of the mRNA expression levels previously reported and represent a further exception to the rules governing susceptibility to nonsense-mediated decay. A close examination of the molecular basis of the disease is becoming increasingly important for optimising the choices of available or forthcoming therapies such as, enzyme replacement therapy or enzyme enhancement therapy.

Synthesis of a bicyclic analog of L-iduronic acid adopting the biologically relevant 2S0 conformation.

The synthesis of a bicyclic analogue of the naturally occurring alpha-L-iduronic acid locked in a biologically active (2)S0 skewboat conformation is disclosed. The desired (2)S0 conformation has been obtained by tethering the C-2 and C-5 carbon atoms of the sugar ring with a dimethyloxy bridge and confirmed by NMR and molecular modeling. The new mimic displays the exact hydroxyl pattern of alpha-L-iduronic acid, a major monosaccharide component of glycosaminoglycans and thus represents a closer mimic of the latter, compared to previously reported bicyclic analogs.

B3LYP/6-311++G** study of structure and spin-spin coupling constant in methyl 2-O-sulfo-alpha-L-iduronate.

Structures of three most stable conformers ((1)C4, (4)C1, (2)S0) of methyl 2-O-sulfo-alpha-L-iduronate monosodium salt have been analyzed by DFT using the B3LYP/6-311++G** method. The optimized geometries confirmed the influence of both 2-O-sulfate and carboxylate groups upon the pyranose ring geometry. The computed energies showed that the chair (1)C4 form is the most stable one. Time-averaged DFT-calculated proton-proton and proton-carbon spin-spin coupling constants agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium of methyl 2-O-sulfo-alpha-L-iduronate monosodium salt. The influence of the charged groups upon the magnitudes of spin-spin coupling constants is also discussed.

Direct assay of iduronate-2-sulfatase for Hunter disease using UPLC-tandem mass spectrometry and fluorogenic substrate.

OBJECTIVE: We devised iduronate-2-sulfatase (IDS) enzyme activity assays by combining fluorometric substrate and LC-MS/MS based detection. DESIGN AND METHODS: 4-Methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate (IDS-S) was used as a substrate for IDS. Its enzymatic product, 4-methylumbelliferyl α-L-idopyranosiduronic acid (IDS-P) and internal standard, 4-methylumbelliferyl α-L-idopyranoside (IDS-IS), were directly measured by UPLC-MS/MS. We determined the precision of our enzyme assay and the effects of sample amounts and incubation time based on the results. Dried blood spots (DBSs) of 110 normal newborns and three patients with Hunter disease were analyzed. RESULTS: IDS-IS, IDS-P and IDS-S were fully separated using UPLC without any ion suppressions. The intra- and inter-assay precisions were 8.5-10.5% and 11.9-15.3%, respectively. The amount of product obtained was proportional to the number of DBSs and increased linearly with the incubation period from 0 to 15 h. The enzyme activities in DBSs from three patients with MPS II were markedly lower than those in the DBSs of 110 normal newborns. CONCLUSION: To the best of our knowledge, this is the first report describing the use of LC-MS/MS for the diagnosis of Hunter disease with a commercially available substrate. Our method would be a rapid and effective screening tool for the diagnosis of Hunter disease with further study.

Synthesis and antimetastatic activity of L-iduronic acid-type 1-N-iminosugars.

L-Iduronic acid-type 1-N-iminosugars, (3R,4S,5R,6R)- and (3R,4S,5S,6R)-6-acetamido-4-amino-5-hydroxypiperidine-3-carboxylic acid (6 and 7, respectively), (3R,4S,5R,6R)-6-acetamido-4- guanidino-5-hydroxypiperidine-3-carboxylic acid (8), and (3R,4S,5R,6R)-4-amino- and -guanidino-5-hydroxy-6-(trifluoroacetamido) piperidine-3-carboxylic acid (9 and 10, respectively), were synthesized from siastatin B (1), isolated from Streptomyces culture, by the intramolecular Michael addition of O-imidate to its alpha,beta-unsaturated ester through cis oxiamination as a key step. Preincubation of B16 BL6 cells with these compounds inhibited invasion of the cells through reconstituted basement membranes. Pulmonary metastasis of B16 BL6 cells in mice was remarkably inhibited by pretreatment of the cells with these compounds in culture.

Fluorometric measurement of urinary alpha-L-iduronidase activity.

A fluorogenic substrate for alpha-L-iduronidase, 4-methylumbelliferyl alpha-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis. Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low alpha-L-iduronidase activity. The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.

A molecular dynamics description of the conformational flexibility of the L-iduronate ring in glycosaminoglycans.

For a synthetic hexasaccharide model it is shown that the conformational flexibility of the L-iduronate ring in glycosaminoglycans can be adequately described by using the PME methodology together with simulation protocols suitable for highly charged systems.

Substrates for the assay of alpha-L-iduronidase.

Procedures are described for the preparation of two disaccharides, 4-O-alpha-L-iduronosyl-2,5-anhydro[3H]mannitol and 3-O-alpha-L-iduronosyl-2,5-anhydro[3H]-talitol, from heparin and dermatan sulfate, respectively. These disaccharides lend themselves to an easy assay of alpha-L-iduronidase which is based on the fractionation of the liberated neutral anhydro[3H]mannitol or anhydro[3H]talitol from the unreacted substrate by adsorption of the latter to Dowex 1. Investigation of the reaction conditions showed that the alpha-L-iduronidase activity (enzyme from human fibroblasts and Helix pomatia) was optimal at pH 3.6 in acetate buffer containing 0.01 M NaCl with iduronosyl-2,5-anhydro[3H]mannitol as substrate. For iduronosyl-2,5-anhydro[3H]talitol the pH optimum was 4.0 with the H. pomatia enzyme. The KM for iduronosyl-2,5-anhydro[3H]mannitol was 0.23 mM with human fibroblasts and 0.04 mM with Helix enzyme; a KM value of 0.02 mM was determined for iduronosyl-2,5-anhydro[3H]talitol with the Helix alpha-L-iduronidase.

Metal binding to heparin monosaccharides: D-glucosamine-6-sulphate, D-glucuronic acid, and L-iduronic acid.

In order to ascertain which residues in heparin may be responsible for its metal binding capacities we have investigated metal binding to some of its component monosaccharides by 1H and 13C NMR. The diamagnetic Zn ion and the paramagnetic Ni ion were used as probes. 4-Methylumbelliferyl-2-deoxy-2-acetamido-6-O-sulpho-D-glucosamine was used as a model for O-sulphates. Only weak interactions with the sulphate group were found. The 4C1 ring conformation of sodium methyl-beta-D-glucopyranosiduronate was not perturbed by binding to its carboxylate and little evidence exists for chelation. By contrast, the ring conformation of the sodium methyl-alpha-L-idopyranosiduronate is affected by the addition of Zn greater than Pb greater than Cd greater than Ca much greater than K ions. The sodium salt is suggested to be an equilibrium mixture of the 2SO and 1C4 ring conformations. Cation binding to the carboxylate group shifts this equilibrium towards the 1C4 conformation and suggests additional binding to O5 or, less likely, O4. This effect appears to be electrostatic in nature, as excess Na and protonation produce similar shifts. Lead complexation is different from the other ions and suggests some covalent character. The control of the ring conformation of iduronic acid by metal ions may have biological implications for the action of heparin and heparin-like compounds.

L-iduronic acid derivatives as glycosyl donors.

O-[Methyl (2-O-acetyl-3-O-benzyl-4-O-levulinyl-alpha, and beta-L-idopyranosid)uronate] trichloroacetimidate and the corresponding n-pentenyl glycosides are efficient L-iduronic acid glycosyl donors. Both have been used for the high-yielding synthesis of basic disaccharide blocks which are useful for the subsequent synthesis of complex oligosaccharides related to heparin/heparan sulfate, and dermatan sulfate. In contrast, the corresponding thioethyl glycosides, thiophenyl glycosides, and fluoride, did not yield the expected disaccharides.

Efficient selective preparation of methyl-1,2,4-tri-O-acetyl-3-O-benzyl-beta-L-idopyranuronate from methyl 3-O-benzyl-L-iduronate.

Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-L-idopyranuronate 6beta/6alpha, prepared from methyl 3-O-benzyl-L-iduronate (4), is a key synthon in heparin/heparan sulfate synthesis. The 1H and 13C NMR spectra of the furanose-pyranose mixture of 4, after dissolution and equilibration in d(4)-methanol, were fully assigned allowing to expect that 4 could crystallise in the beta-pyranose form. New acetylation conditions able to trap this form were subsequently devised, allowing the isolation of 83% of pure 6beta by simple crystallisation, along with 9% of the 6beta/6alpha mixture. This represents a major advantage over the previously published procedure, especially on multigram scales.

Preparation of derivatives of L-idose and L-iduronic acid from 1,2-O-isopropylidene-alpha-D-glucofuranose by way of acetylenic intermediates.

The products (1) from the periodate oxidation of 1,2-O-isopropylidene-alpha-D-glucofuranose were converted by ethynylmagnesium bromide into a separable, 14:11 mixture of 6,7-dideoxy-1,2-O-isopropylidene-beta-L-ido-hept-6-ynofuranose (2) and its alpha-D-gluco analog 3. These crystalline products were further characterized as their respective 3,5-diacetates (5 and 7) and 3,5-dibenzoates (4 and 6). Ozonolysis of 2 and 3 led to 1,2-O-isopropylidene-beta-L-idofuranurono-6,3-lactone (8) and its alpha-D-gluco analog 9, respectively; similar ozonolysis of the dibenzoates 4 and 6, followed by treatment with diazomethane, gave methyl 3,5-di-O-benzoyl-1,2-O-isopropylidene-alpha-L-idofuranuronate (10) and its alpha-D-gluco analog 11, respectively. Diborane reduction of the ozonolysis products from 4 gave 1,2-O-isopropylidene-beta-tl-idofuranose (13) as its 3,5-dibenzoate (12), and a similar sequence was performed with 6. The propargylic alcohols 2 and 3 were reduced by lithium aluminum hydride, in high yield, to the allylic alcohol analogs 15 and 16, further characterized as their 3,5-dibenzoates 17 and 18; compounds 15 and 16 were also obtainable by vinylation of compounds 1. The two series of derivatives in this work, epimeric at C-5, were examined comparatively by polarimetry and p.m.r. spectroscopy.

Chemical change involved in the oxidative-reductive depolymerization of heparin.

A solution of hog intestinal heparin (average M(r) 12,000, anti-clotting activity 168 USP units/mg) in 0.2 M phosphate buffer (pH 7.2), was incubated in the presence of Fe2+ for 20 h at 50 degrees under an O2 atmosphere to yield oxidative-reductively depolymerized heparin (ORD heparin, average M(r) 3,000, anti-clotting activity 34 USP units/mg). Chemical analysis of the ORD heparin showed a 22, 26, and 14% loss of hexosamine, uronic acid, and N-acetyl group, respectively, but no remarkable loss of both total and N-sulfate groups. 1H and 13C NMR spectroscopic analysis indicated no decrease in the amount of L-iduronic acid 2-sulfate, but a marked loss of nonsulfated uronic acid (73 and 39% loss of D-glucuronic acid and L-iduronic acids, respectively, the sum of which corresponds to the chemically determined loss of total uronic acid). The results indicated that the ORD reaction of heparin proceeds essentially by destruction of monosaccharide units, except L-iduronic acid 2-sulfate residues, due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable residues.

4-O-methyl-beta-L-idopyranosyluronic acid linked to xylan from kraft pulp: isolation procedure and characterisation by NMR spectroscopy.

The tetrasaccharide 2"-O-(4-O-methyl-beta-L-idopyranosyluronic acid)xylotriose was isolated from enzymatically hydrolysed, unbleached, birch kraft pulp by anion-exchange chromatography in two steps. The primary structure of the tetrasaccharide was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. NOE data and 3JH,H coupling constants show that the 4-O-methyl-beta-L-idopyranosyluronic acid in the tetrasaccharide is predominantly in the 1C4 chair conformation. The pKa value (3.17) for 4-O-methyliduronic acid attached beta-(1-->2) to xylose was determined from the pH-dependent chemical shift of H-5. The amount of 4-O-methyliduronic acid (0.1-0.5 mol%) in surface xylan of unbleached birch and pine kraft pulps was determined by extensive xylanase treatment and further analysis by NMR spectroscopy and high-performance anion-exchange chromatography.

From D-glucuronic acid to L-iduronic acid derivatives via a radical tandem decarboxylation-cyclization.

A synthesis to L-iduronic derivatives, major components of heparin derived pentasaccharides was accomplished by formal inversion of configuration at C-5 of a D-glucuronic acid derivative through radical formation at C-5 using Barton decarboxylation followed by intramolecular radical addition on an acetylenic tether at O-4 giving exclusively a bicyclic sugar of L-ido configuration. Oxidation and ring opening of this bicyclic sugar led to a L-iduronate. This method opens the way to short syntheses of pentasaccharidic moiety of Idraparinux and congeners.

Importance of IdoA and IdoA(2S) ring conformations in computational studies of glycosaminoglycan-protein interactions.

Glycosaminoglycans (GAGs) interact with chemokines and growth factors in the extracellular matrix and, therefore, mediate cell communication processes. Heparin is one of the most studied GAGs, for which many experimental structures of its complexes with proteins are available. One of the monosaccharide components of heparin, sulfated iduronic acid (IdoA(2S)), is observed to adopt both (1)C4 and (2)S0 ring conformations. Despite the biological relevance of the sugar ring conformations for heparin-protein interactions, it is very challenging to take into account the conformational space of IdoA(2S) sugar ring for computational studies. Therefore, instead of systematically analyzing several ring conformations, which represents a combinatorial problem for a periodic heparin molecule, often only one ring conformation is taken into account. Here, we use docking and molecular dynamics (MD) to estimate how crucial this assumption could be for the conclusions being made in computational studies of heparin-protein interactions. We show that both docking solutions and free energy calculations from MD simulations are significantly affected by the conformations adopted by IdoA and IdoA(2S) rings. Therefore, in the application of computational approaches to heparin-protein systems the ring conformations should be treated properly to avoid misleading conclusions.