Dr Francesca Bertola

Dr Mirella Filocamo
email: mirellafilocamo@ospedale-gaslini.ge.it

Giorgio Casati

Matthew Mort

Camillo Rosano

Anna Tylki-Szymańska

Beyhan Tuysuz

Orazio Gabrielli

Serena Grossi

Maurizio Scarpa

Anna Tylki-Szymanska

Beyhan Tüysüz

Giancarlo Parenti

Daniela Antuzzi

Jaime Dalmau

Maja Di

Carlo Dionisi Vici

Ilyas Okur

Jordi Rosell

Attilio Rovelli

Francesca Furlan

Miriam Rigoldi

Andrea Biondi

David N Cooper

Rossella Parini

Mark H Paalman

Patients with Mucopolysaccharidosis type I: Identification and Characterization of 35 Novel alpha-L-iduronidase (IDUA) Alleles

Keywords: IDUA mutations, mucoplysaccharidosis type I, Hurler disease, genotype-phenotype analysis, molecular modelling, MutPred analysis, in vitro splicing analysis IDUA, mucoplysaccharidosis type I, Hurler disease, genotype-phenotype analysis, molecular modelling, MutPred analysis, in vitro splicing analysis

published or not. The documents may come

INTRODUCTION

Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder resulting from deficiency of the lysosomal enzyme α-L-iduronidase (IDUA; E.C. 3.2.1.76) which is involved in the degradation of the glycosaminoglycans (GAGs), namely heparan sulfate and dermatan sulfate. IDUA deficiency leads to the intralysosomal accumulation of undegraded GAG substrates and this coincides with the onset of pathology [START_REF] Neufeld | The metabolic and molecular bases of inherited disease, 8th Ed[END_REF][START_REF] Clarke | Mucopolysaccharidosis Type I[END_REF]. Although the MPS-I clinical phenotype represents a continuous spectrum from the severe to the attenuated forms, three distinct phenotypes have been classically distinguished: 1) severe (Hurler syndrome, MPS IH; MIM# 607014) when the onset of symptoms is before 12 months of age, with survival no more than 10 years and mental retardation manifesting before the age of three; 2) intermediate (Hurler/Scheie syndrome, MPS IH/S; MIM# 607015) when onset of symptoms is between 1 and 6 years, survival is variable and mental retardation is absent or mild, but never before 3 years of age; and 3) 'attenuated' (Scheie syndrome, MPS IS; MIM# 607016) when symptoms first become apparent after the age of 5, survival is normal and mental retardation is never present [START_REF] Neufeld | The metabolic and molecular bases of inherited disease, 8th Ed[END_REF].

The gene encoding α-L-iduronidase (IDUA; MIM# 252800) maps to chromosome 4p16.3 and contains 14 exons; the cDNA open reading frame (ORF) is ~2 kb in length and encodes a polypeptide of 653 amino acids [START_REF] Scott | Chromosomal localization of the human alpha-L-iduronidase gene (IDUA) to 4p16.3[END_REF][START_REF] Scott | Human alpha-L-iduronidase: cDNA isolation and expression[END_REF](Scott et al., , 1992)). So far, more than 100 different disease-causing IDUA mutations have been reported (Human Gene Mutation Database; http://www.hgmd.org; [START_REF] Stenson | The Human Gene Mutation Database: 2008 update[END_REF]. Several different types of mutation have been documented: whereas missense mutations may allow for some residual enzyme activity and are associated with quite a variable clinical phenotype, those mutations that are likely to impact upon RNA processing (i.e. nonsense, frameshift and splice site mutations) almost invariably result in a more severe phenotype [START_REF] Terlato | Can mucopolysaccharidosis type I disease severity be predicted based on a patient's genotype? A comprehensive review of the literature[END_REF]. Non-pathogenic functional polymorphisms may also have a role in modifying the expression of IDUA mutant alleles, thereby contributing to the phenotypic and clinical heterogeneity characteristic of MPS I [START_REF] Scott | Molecular genetics of mucopolysaccharidosis type I: diagnostic, clinical, and biological implications[END_REF][START_REF] Beesley | Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations[END_REF][START_REF] Matte | Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients[END_REF].

Overall, most mutations are 'private', with only four mutations (p.W402X, p.Q70X, p.P533R, p.G51D) being common in specific populations. The most common IDUA mutation is p.W402X which has a frequency of around 50% in Northern Europe, the United Kingdom, North America [START_REF] Beesley | Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations[END_REF][START_REF] Clarke | Mutation analysis of 19 North American mucopolysaccharidosis type I patients: identification of two additional frequent mutations[END_REF][START_REF] Scott | Molecular genetics of mucopolysaccharidosis type I: diagnostic, clinical, and biological implications[END_REF] and Spain [START_REF] Gort | Analysis of five mutations in 20 mucopolysaccharidosis type 1 patients: high prevalence of the W402X mutation[END_REF], while in Russia, Italy and Brazil its frequency has been estimated to be 4% [START_REF] Voskoboeva | Molecular genetics of mucopolysaccharidosis type I: mutation analysis among the patients of the former Soviet Union[END_REF], 11% [START_REF] Gatti | Mutations among Italian mucopolysaccharidosis type I patients[END_REF][START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF] and 20% [Matte et al., 2000], respectively . By contrast, the p.Q70X mutation is much more frequent in Scandinavia and Russia (around 50% of alleles) than in other countries [START_REF] Bunge | Mucopolysaccharidosis type I: identification of 8 novel mutations and determination of the frequency of the two common alpha-L-iduronidase mutations (W402X and Q70X) among European patients[END_REF][START_REF] Voskoboeva | Molecular genetics of mucopolysaccharidosis type I: mutation analysis among the patients of the former Soviet Union[END_REF]]. The p.P533R mutation, which probably had a North African origin, has spread to Mediterranean countries, representing 13% and 10% of IDUA mutant alleles in Italy and Spain respectively [START_REF] Alif | Mucopolysaccharidosis type I in Morocco: clinical features and genetic profile[END_REF][START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF][START_REF] Voskoboeva | Molecular genetics of mucopolysaccharidosis type I: mutation analysis among the patients of the former Soviet Union[END_REF]]. Finally, the p.G51D mutation seems to be exclusively Italian with a relative frequency of 13% among IDUA mutant alleles [START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF].

We have previously reported IDUA gene mutations from a series of 30 Italian patients [START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF]]. Here we have characterized the underlying IDUA mutations in a group of 102 newly studied European patients, including 37 Italians, whose condition has been clinically and biochemically diagnosed as MPS I.

MATERIALS AND METHODS

Patients

The present series comprises a total of 102 European patients (pts) affected by MPS I. They are from diverse ethnic backgrounds: 37 were of Italian origin, 23 Polish, 21 Turkish, 18 Spanish, and 3 patients each from Hungary, Serbia and Greece, (Table 1). The diagnosis of MPS I was confirmed biochemically in all patients, demonstrating a defect of IDUA activity either in leukocytes or a fibroblast cell line. As reported in Table 1, the clinical phenotype, defined as previously reported [Neufeld and Muenzer, 2003], was available for all but three patients: 59% of patients presented with the severe form of the disease (MPS IH), 22% with the intermediate form (MPS IH/S) and 15% with the attenuated form (MPS IS); the remaining two patients (2%) manifested a clinical phenotype ranging between MPS IH-H/S (pt #43) and MPS IH/S-S (pt #92).

Thirty seven Italian cases and one Serbian case were recruited with the help of their attending clinicians. The remaining 64 patients from different countries were recruited and collected through a collaboration with Genzyme Corporation (Cambridge, MA, USA) as part of a pan-European project with the aim of performing the molecular characterization of all MPS I patients in Europe and collecting these data in an MPS I Registry (https://www.lsdregistry.net/mpsiregistry) [START_REF] Pastores | The MPS I registry: design, methodology, and early findings of a global disease registry for monitoring patients with mucopolysaccharidosis type I[END_REF]. Some of the samples from the Italian patients were supplied by the "Cell line and DNA Biobank from patients affected by Genetic Diseases" in Genova (http://dppm.gaslini.org/biobank/). Following ethical guidelines, all samples obtained for analysis and storage required prior written informed consent using a form approved by the Local Ethics Committee.

Cell culture

Fibroblast cells were cultured according to standard procedures. The cell lines were cultured and maintained in RPMI medium (EuroClone, Gibco, Paisley, UK) containing 15% FCS and penicillin/streptomycin, in a humidified atmosphere containing 5% CO 2 at 37°C.

Enzymatic assay

α-L-iduronidase activity was assayed in homogenates of leukocytes and/or fibroblast cell lines using 4methylumbelliferyl-α-L-iduronide (Glycosynth, Cheshire, UK) as a substrate [START_REF] Stirling | Fluorimetric assay for prenatal detection of Hurler and Scheie homozygotes or heterozygotes[END_REF]. Total protein was measured according to the Lowry method [START_REF] Lowry | Protein measurement with the Folin-Phenol reagents[END_REF].

Molecular analysis

Genomic DNA was extracted from circulating lymphocytes and/or cultured fibroblasts using either the Wizard Genomic DNA Purification kit (Promega, Madison, WI) or a standard phenol-chloroform-based method.

IDUA gene exons, exon-intron boundaries and part of the 5' untranslated region were PCR amplified using specific primers designed by reference to the genomic sequence (GenBank-EMBL Accession no. NG_008103.1). Intronic primers, designed for both PCR and sequence analyses, are listed in Supp. Table S1. PCR amplification reactions (except for exon 9) were carried out in 25µl vols containing 100ng genomic DNA, buffer 1X with 1.5mM MgCl 2 , 200µM dNTPs mix, 15pmol each primer, GC-RICH solution 1X and 0.5U proofreading PWO SuperYield DNA polymerase (Roche, Monza, Italy). Exon 9 PCR-amplification was carried out in 25µl vols containing 100ng genomic DNA, buffer 1X with 1.5mM MgCl 2 , 200µM dNTPs mix, 15pmol each primer and 0.5U GoTaq DNA polymerase (Promega, Madison, WI); owing to the use of a no-proofreading DNA polymerase, this reaction was performed in duplicate. Cycling conditions were: initial denaturation at 96°C for 5 min, 28 cycles denaturation at 96°C for 1 min, annealing at 63-70°C for 1 min, extension at 72°C for 1 min, followed by final extension at 72°C for 7 min. Control PCR reactions, in which no template was added, were included during each set of PCR reactions. PCR products were controlled for contamination by gel electrophoresis and were then purified using an enzymatic reaction containing 5U exonuclease I (Celbio, Italy) and 1U alkaline phosphatase (Promega, Madison, WI) under the following conditions: 15 min at 37°C followed by 15 min at 80°C.

Total RNA was prepared from peripheral blood lymphocytes isolated by the Fycoll method or from cultured fibroblasts using Trizol Reagent (Gibco, Paisley, UK) according to the manufacturer's instructions. First-strand cDNAs were synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and random hexamer primers. IDUA exonic fragments flanking splicing mutations were amplified and sequenced using primers and conditions listed in Supp. Table S1.

Sequence analysis of PCR and RT-PCR products was performed in the forward and reverse directions using BigDye v3.1 terminator technology (Applied Biosystems, Foster City, CA) and then purified with Wizard MagneSil Sequencing Reaction Clean-Up System (Promega, Madison, WI). Sequencing reactions were carried out, the products purified according to the manufacturer's instructions and analyzed on an ABI Prism 3130 Avant Automatic Sequencer (Applied Biosystems, Foster City, CA). Sequence alterations were confirmed by sequencing duplicate PCR products.

The question of the pathological authenticity of the novel IDUA sequence alterations detected was addressed by (i) searching dbSNP (http://www.ncbi.nlm.nih.gov/SNP) for their presence, (ii) screening 100 alleles from healthy control subjects for each alteration, (iii) modeling the amino acid changes into a homologous three-dimensional structure of the protein, (iv) employing the MutPred program [START_REF] Li | Automated inference of molecular mechanisms of disease from amino acid substitutions[END_REF][START_REF] Mort | In silico functional profiling of human disease-associated and polymorphic amino acid substitutions[END_REF] for the missense mutations, (v) using ESRPred and a neural network for splice site prediction [START_REF] Krawczak | Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing[END_REF] for the coding region variants upon splicing, and (vi) using PhyloP method [START_REF] Pollard | Detection of nonneutral substitution rates on mammalian phylogenies[END_REF] and ClustalW2 program [START_REF] Larkin | ClustalW and ClustalX version 2[END_REF] to measure evolutionary conservation of nucleotides and residues, respectively.

Molecular modeling of missense mutations

For modeling of the IDUA missense mutations, an homology model of the IDUA protein was used. This model was built by [START_REF] Rempel | A homology model for human α-l-iduronidase: Insights into human disease[END_REF] using the atomic coordinates of β-xylosidase (E.C.3.2.1.37) from Thermoanaerobacterium saccharolyticum as a template [PDB code 1Y24]. Visual inspection and graphical representations were then performed using the programs Coot and Chimera, respectively [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF][START_REF] Pettersen | UCSF Chimera -a visualization system for exploratory research and analysis[END_REF].

Bioinformatic analysis of IDUA variants

Missense variants in the IDUA gene were analysed with a computational model, MutPred [START_REF] Li | Automated inference of molecular mechanisms of disease from amino acid substitutions[END_REF][START_REF] Mort | In silico functional profiling of human disease-associated and polymorphic amino acid substitutions[END_REF]. MutPred is designed to assess the likely phenotypic consequences of missense substitutions occurring within sites of structural and/or functional importance, on the wild-type protein sequence. MutPred can be used to generate hypotheses as to the underlying molecular mechanism(s) responsible for disease pathogenesis.

The effect of the coding region variants upon splicing [splice site disruption, cryptic splice site activation and exon skipping via loss of exonic splicing enhancers (ESE) and/or gain of exonic splicing silencers (ESS)] was ascertained using ESRPred [M. Mort, unpuplished] and a neural network for splice site prediction [START_REF] Krawczak | Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing[END_REF].

Evolutionary sequence conservation across an alignment of IDUA orthologues from 44 vertebrate species was measured using the phyloP method [START_REF] Pollard | Detection of nonneutral substitution rates on mammalian phylogenies[END_REF]. PhyloP can measure accelerated evolution (more rapid evolution than expected under neutral drift) as well as evolutionary conservation (slower than expected evolution). A positive phyloP score represents an evolutionarily conserved nucleotide and a negative phyloP score indicates that the nucleotide has experienced more rapid evolution than would be expected under neutral drift.

Mutation nomenclature

Mutations

are described according to current mutation nomenclature guidelines (http://www.hgvs.org/mutnomen), ascribing the A of the first ATG translational initiation codon as nucleotide +1 [START_REF] Den Dunnen | Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion[END_REF][START_REF] Den Dunnen | Standardizing mutation nomenclature: why bother?[END_REF].

RESULTS AND DISCUSSION

A total of 102 European patients affected by MPS I were recruited by our centre. All patients underwent molecular characterization and the IDUA genotype was completely ascertained in 93 of the 102 patients.

Mutation detection

All 14 exons, splice junctions and proximal portions of the 5' and 3' untranslated regions of the IDUA genes of the 102 unrelated MPS I patients were investigated by DNA sequence analysis. Putatively pathological IDUA mutations were identified in 193 of 204 alleles. The genotypes, clinical phenotypes and geographic origins of the 102 patients studied here are listed in Table 1. The characteristics of the 55 distinct IDUA mutations identified are reported in Table 2. The mutational spectrum comprised 22 missense mutations, 14 splice site alterations, 9 microdeletions, 5 nonsense mutations, 3 micro-duplications, 1 translational initiation site mutation and 1 no-stop mutation. More than 60% (35/55) of these mutant alleles have not been previously reported, attesting to the extensive allelic heterogeneity of MPS I. The novel lesions resulted from (i) 11 missense mutations (p.Y76C, p.G84R, p.T103P, p.G219E, p.E276K, p.W306L, p.N384K, p.P385R, p.L396P, p.A436P, p.L535F) as well as two missense mutations located in cis to each other (p.[F188L;S423R]); (ii) 12 splicing mutations (c. 385+1G>A, c.493+1G>A, c.494-1G>A, c.956_972+9delinsTA, c.1189+4A>G, c.1189+5G>A, c.1190(iii) 5 micro-deletions (c.574delT, c.826_828del3, c.1045_1047del3, c.1398delC and c.1893delC), 1 micro-duplication (c.1166_1171dup), 2 nonsense mutations (p.201X and p.Y581X), 1 translational initiation site mutation (p.M1?) and 1 'no-stop' mutation (p.X654RextX62). In addition to these putative pathological mutations, a total of 37 putative intragenic IDUA polymorphisms were identified within either the exons or introns; eight novel SNPs were noted within the coding region (4) and in the introns (4) (Supp. Table S2). The allele frequencies of the novel variants, derived from 100 normal control alleles, are given in Supp. Table S2. Figure 1 depicts the location of the various mutations and polymorphisms detected in relation to the structure of the IDUA gene.

Missense mutations

A total of 14 novel IDUA missense mutations were detected during the course of this study; two being located in cis on the same IDUA allele (p.[F188L;S423R]). Consistent with their putative pathological authenticity, none of the underlying nucleotide changes were found in a screen of 100 control alleles. Nor were any of these substitutions listed as polymorphisms in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP).

Bioinformatic analysis of missense variants

MutPred predicted that most of the missense mutations listed in Table 2 are deleterious (91%; 21 out of 23; MutPred general score > 50). Further, confident in silico hypotheses for the underlying mechanism of pathogenesis were generated for 14 of the missense mutations (summarised in Table 2 and reported in full in Supp. Table S3). The two missense mutations (p.T103P and p.A436P) predicted not to be deleterious by MutPred, were analysed S4). PhyloP scores were used to measure evolutionary conservation at the nucleotide sequence level across IDUA orthologues derived from 44 vertebrate species. Both mutations (p.T103P and p.A436P) exhibited positive phyloP scores indicating that these mutations are located at conserved nucleotides. It should be noted that p.T103P displays only marginal conservation, with a phyloP score of 0.40. Analysis with SIFT [START_REF] Ng | Predicting deleterious amino acid substitutions[END_REF] predicted that both mutations would be tolerated in the protein. PolyPhen2 [START_REF] Adzhubei | A method and server for predicting damaging missense mutations[END_REF] however predicted that whilst p.T103P was benign, p.A436P mutation was likely to be potentially damaging. Neither mutation was predicted to disrupt splicing using a neural network [START_REF] Krawczak | Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing[END_REF]. MutPred analysis was also performed on the six polymorphic missense variants identified (listed in Supp. Table S2) but none of these were predicted to be deleterious (MutPred general scores invariably < 50).

Further evidence for the pathological/functional significance of the missense mutations was then sought from the analysis of the extent of evolutionary conservation of the mutated residues in 6 orthologous (vertebrate) IDUA proteins from human to Tetraodon nigroviridis. The computational analysis, carried out using ClustalW2 [http://www.ebi.ac.uk/clustalw; [START_REF] Larkin | ClustalW and ClustalX version 2[END_REF], revealed that among the 5 other vertebrate IDUA protein sequences examined, Y76, G84, F188, G219, R276, W306, N348, P385, L396, S423, L535 were invariant at these positions; such conservation over 500 Myrs of evolutionary time is supportive of the functional significance of these residues and hence the direct involvement of the respective mutations in disease causation. In agreement with the PolyPhen score results, residue A436 was found to be partially conserved (chimpanzee, dog and chicken) but T103 was not evolutionarily conserved. (Supp. Figure S1). It may be that the p.A436P mutation is mildly deleterious, whereas p.T103P may be tolerated and could therefore represent a rare polymorphism.

3D modelling of IDUA missense mutations

To shed further light on the deleterious consequences of the novel missense IDUA mutations at the protein level, we employed the homology model of IDUA constructed by [START_REF] Rempel | A homology model for human α-l-iduronidase: Insights into human disease[END_REF]. Since the IDUA model starts at residue 36 and terminates at residue 522, the p.L535F substitution could not be included in this analysis. As shown in Figure 2, and detailed in Supp. Table S5, several substitutions (p.G219E, p.P385R and p.S423R) appear to affect a hydrophobic buried region in the protein, one substitution occurs in the predicted active site residue (p.F188L), and one substitution in the active site cleft (p.W306L) [START_REF] Rempel | A homology model for human α-l-iduronidase: Insights into human disease[END_REF]. The remaining amino acid replacements (p.G84R, p.Y76C, p.T103P, p.E276K, p.N348K, p.L396P and p.A436P) affected solvent accessible residues. Consistent with previous findings [START_REF] Rempel | A homology model for human α-l-iduronidase: Insights into human disease[END_REF][START_REF] Vazna | Mucopolysaccharidosis type I in 21 Czech and Slovak patients: mutation analysis suggests a functional importance of C-terminus of the IDUA protein[END_REF][START_REF] Sugawara | Structural study on mutant alpha-L-iduronidases: insight into mucopolysaccharidosis type I[END_REF], those mutations that occur in the protein core (including the predicted active site), were associated with the severe (H) form of MPS I, whereas the missense mutations which affected surface-located residues were associated with the more attenuated clinical phenotypes (H/S, S) (Figure 2 and Supp. Table S5). Despite obtaining data from both structural and bioinformatic analyses, the pathological significance of the p.T103P, which occurs in a long loop of unknown function that protrudes into the solvent, remains unclear.

Splicing mutations

A substantial proportion of the novel IDUA mutations detected (34.3%) occurred in either the donor (c.385+1G>A, c.493+1G>A, c.1727+1G>A) or acceptor (c.494-1G>A, c.1190-1delG) splice sites or in the surrounding splicing motifs (c.1189+4A>G, c.1189+5G>A, c.1650+5G>C, c.1727+4C>T, c.1727+5G>C, c.1727+6T>A) (Table 2). All the single base-pair substitutions predicted to disrupt splicing were located at nucleotides which were evolutionarily conserved across an alignment of IDUA gene sequences from 44 vertebrate species. We also identified an indel mutation (c.956_972+9delinsTA) as the cause of aberrant splicing in one case. At the genomic level, the mutation comprised the deletion of 26 nucleotides and the insertion of the dinucleotide TA in a region overlapping the exon-intron 7 donor splice site.

Supp. Figure S2 graphically represents the intronic location of the 12 novel splicing mutations identified in MPS I patients in the present study. To assess the potential effect of these mutations on mRNA splicing, the relative strengths of the 5' and 3' splice site signals were evaluated using the NetGene2 server [http://www.cbs.dtu.dk/services/NetGene2]. According to this program, all 12 mutations would be predicted to impact upon RNA processing. In particular, donor splice sites would have been abrogated in the presence of c.385+1G>A, c.493+1G>A, c.1650+5G>C, c.1727+1G>A, c.1727+5G>C and c.1727+6T>A as would acceptor sites in the presence of c.494-1G>A and c.1190-1delG. The same analysis predicted a considerable decrease in the donor splice site confidence score in the cases of c.1189+4A>G, c.1189+5G>A and c.1727+4C>T. These predictions were then followed up by performing in vitro splicing analysis for those mutations identified in patients from whom appropriate samples were available. The functional relevance of six intronic mutations (c.494-1G>A, c.1189+4A>G, c.1189+5G>A, c.1727+4C>T, c.1727+5G>C and c.1727+6T>A) was assessed by carrying out reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on mRNA extracted from either lymphocytes (3 pts) or fibroblast cell lines (3 pts). In the case of c.1727+5G>C and c.1727+6T>A, the RT-PCR results clearly confirmed the abrogation of the expected donor splice site leading to the skipping of exon 12 or the activation of alternative splice sites (exonic or intronic) leading to frameshifts with consequent premature truncation of the protein (p.Y167TfsX, p.V371MfsX, p.L578VfsX). Full details are given in Table 3. 1. Some 45 (44%) of the patients (pts) were found to be either homozygous for the pan-ethnic IDUA mutations, p.W402X (12 pts), p.Q70X (9 pts) and p.P533R (3 pts), other known mutations (7 pts) or novel lesions (14 pts).

The remaining 54% of patients were compound heterozygotes (including 7 in whom the second mutant IDUA allele could not be identified). Finally, the mutant IDUA alleles in two patients (2%), in whom a defect in IDUA enzyme activity had been documented, remained unidentified.

Overall, we found 68 distinct genotypes including those (7) that were partially characterized. Our results confirm the high degree of mutational heterogeneity characteristic of MPS I. Extensive allelic heterogeneity often precludes the recognition of correlations between mutant genotypes and variant clinical phenotypes. This notwithstanding, in an attempt to obtain new insights into the genotype-phenotype relationship in MPS I, we have compared the genotypes present in our patient series with those of previously reported patients. Supp. Table S6, reports the comparable genotypes (13/68) and the respective number of patients per genotype reported in previous studies on this topic.

Among the analyzed group, 25 patients were either homozygous or compound heterozygous for the two common deleterious nonsense mutations (p.Q70X and p.W402X); their severe clinical phenotype concurred with those of 91 previously reported patients with comparable genotypes [START_REF] Bunge | Mucopolysaccharidosis type I: identification of 8 novel mutations and determination of the frequency of the two common alpha-L-iduronidase mutations (W402X and Q70X) among European patients[END_REF][START_REF] Gort | Analysis of five mutations in 20 mucopolysaccharidosis type 1 patients: high prevalence of the W402X mutation[END_REF][START_REF] Hein | [END_REF][START_REF] Li | Diversity of mutation and distribution of single nucleotide polymorphic alleles in the human alpha-L-iduronidase (IDUA) gene[END_REF][START_REF] Matte | Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients[END_REF]Scott et al., 1992;[START_REF] Vazna | Mucopolysaccharidosis type I in 21 Czech and Slovak patients: mutation analysis suggests a functional importance of C-terminus of the IDUA protein[END_REF][START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF][START_REF] Voskoboeva | Molecular genetics of mucopolysaccharidosis type I: mutation analysis among the patients of the former Soviet Union[END_REF]]. In agreement with previously reported genotype-phenotype correlations, a severe phenotype was not only associated with the p.W402X mutation in compound heterozygosity with both c.1650+5G>A and p.A327P [START_REF] Bunge | Mucopolysaccharidosis type I: identification of 8 novel mutations and determination of the frequency of the two common alpha-L-iduronidase mutations (W402X and Q70X) among European patients[END_REF][START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF][START_REF] Vazna | Mucopolysaccharidosis type I in 21 Czech and Slovak patients: mutation analysis suggests a functional importance of C-terminus of the IDUA protein[END_REF] but also with homozygosity for p.A327P and p.G51D [START_REF] Gatti | Mutations among Italian mucopolysaccharidosis type I patients[END_REF]. Also consistent with previous data [START_REF] Beesley | Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations[END_REF][START_REF] Tieu | Four novel mutations underlying mild or intermediate forms of alpha-L-iduronidase deficiency (MPS IS and MPS IH/S)[END_REF], homozygosity for p.L490P and compound heterozygosity for p.Q70X and c.1333_1335del3 yielded mild phenotypes in two other patients (Supp. Table S6). Although for these 38 patients (37%), the predicted genotype-phenotype relationship may be considered informative, for the remaining group with previously described genotypes, the correlations only partially concurred (Supp. Table S6). This latter group comprised 6 patients, 3 of whom were homozygous either for the p.P533R mutation or for the microdeletion c.46_57del12 (2 pts), and another patient who was compound heterozygous for p.G51D and p.P533R. Comparison between the genotype-phenotype correlation data from our 6 patients and 23 patients from other studies [Alif et al., 1999;[START_REF] Hein | The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation[END_REF][START_REF] Gatti | Mutations among Italian mucopolysaccharidosis type I patients[END_REF][START_REF] Hein | The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation[END_REF][START_REF] Laradi | Mucopolysaccharidosis I: alpha-L-Iduronidase mutations in three Tunisian families[END_REF][START_REF] Matte | Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients[END_REF]Scott et al., 1992;[START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF] confirmed the phenotypic variability of these mutations (p.P533R and c.46_57del12). Similarly, in our patient series (Table 1), these two latter mutations (p.P533R and c.46_57del12), although present in both the homozygous or compound heterozygous state, and in a significant number of individuals (viz. pts #5, #8, #37, #72, #80 and pts #24, #27, #45, #48, #84, #94, respectively), were found to be associated with a wide range of clinical severity.

Additional conclusions were drawn by individually comparing some mutations in our series (Table 1) with those from previous studies. According to [START_REF] Hein | The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation[END_REF], mildly deleterious consequences could be confirmed for the mutation p.R89W, contributing to the mild form of MPS I noted in pts #7 and #13, in whom this allele was in association with the already known severe mutations, p.G51D and p.P496R, respectively. In our own patients, this latter allele (p.P496R) was invariably found to be associated with the severe form of MPS I (pts #6, #17, #19, #22, #23, #30, #33); this finding confirms the assessment of its detrimental nature, made in vitro, by [START_REF] Beesley | Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations[END_REF]. The only patient harbouring p.P496R to exhibit an intermediate clinical form of MPS I (pt #1) was a compound heterozygote in whom the clinical effect of this detrimental allele was probably attenuated by the relative benign influence of p.G265R. In support of this latter supposition was the compound heterozygosity (p.G265R+p.Q70X) observed in the Scheie patient #44 (Table 1). Conversely, conclusions as to the genotypephenotype relationship manifested by mutation c.494-1G>A, found in either the homozygous (pts #81, #86, #90, #98) or compound heterozygous (pt #92) state, were equivocal since this lesion was associated with very different degrees of clinical severity, even in patients who appeared to share the same genetic background (Table 1). It may be that other genetic or non-genetic factors can modulate the clinical phenotype in MPS I. Among the possible genetic factors involved, as previously reported in the same MPS I [START_REF] Yogalingam | Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy[END_REF] or in other MPSs such as MPS II [START_REF] Lualdi | Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts[END_REF], are differentially spliced-RNA transcripts that might result from the splicing mutation c.494-1G>A, and could potentially be considered as modifiers of the clinical phenotype [START_REF] Nissim-Rafinia | Splicing regulation as a potential genetic modifier[END_REF]. Unfortunately, RNA samples were not available to permit experimental verification. Another factor limiting our ability to define effective genotype-phenotype correlations was probably our difficulty in attributing one or other clinical phenotype in a given case, bearing in mind that the clinical spectrum of disease in MPS I is in reality a continuum of phenotypes with gradually changing severity. This is particularly true for the so called 'intermediate form', where it is difficult to be truly objective, especially when the patients are young at the time of diagnosis and there is no standardized scoring index of severity.

Frequency and geographical distribution of common and rare mutations in the European polulation As reported in Table 2, in the whole series comprising patients with diverse genetic backgrounds, the frequency of the most common mutations was: p.Q70X (18.6%), p.W402X (18%), p.G51D (4.9%), p.P496R (4.4%) p.P533R (3.9%), p.A327P (2.4%) and c.46_57del12 (3.9%). Altogether, they represented 56% of IDUA alleles. Rare or private mutations comprised 49.5% of IDUA lesions, and 28.4.% of these were novel. However, when we analysed the geographical distribution and relative frequencies of the five most frequent mutations in different areas of Europe, we observed significant differences in the distribution of these mutations (Figure 3). In Western Europe, the most frequent mutation was p.W402X, representing 38.8% of alleles in Spain. Although its relative frequency in the other Mediterranean countries decreased sharply to 9.5% in both Italy and Turkey, p.W402X maintained a relatively high frequency in Northern Europe, accounting for 24% of IDUA alleles in our series of patients from Poland. Conversely, the p.Q70X mutation accounted for 39% of IDUA alleles in Poland but progressively decreased in terms of its frequency in a Southerly direction, constituting 14.9% in Italy, 8.3% in Spain and 7.1% in Turkey. On the basis of these results, and supported by previous data [START_REF] Terlato | Can mucopolysaccharidosis type I disease severity be predicted based on a patient's genotype? A comprehensive review of the literature[END_REF], it could be speculated that the European geographical diffusion of p.W402X occurred from the South-West in a Northerly direction, while that of p.Q70X occurred from the North-East in a Southerly direction. Mediterranean countries appear to have balanced the relatively low frequency of these two common mutations with other mutations specific to this geographical region viz. p.P533R, p.P496R and p.G51D, the latter two being specific to (and recurrent in) the Italian population, and c.494-1G>A, a Turkish-specific mutation (not shown in the figure). The relatively low frequency of the two usually common mutations (p.Q70X and p.W402X) in Italy and Turkey, results in an increase in IDUA allelic heterogeneity in these two countries as compared to Northern European countries. Thus, rare mutations accounted for 50% and 71.4% of alleles in Italy and Turkey, respectively, as compared to Poland and Spain where they were 37% and 41.6% respectively. The finding that European countries vary widely in terms of both the distribution and frequency of IDUA gene mutations has important implications for the selection of screening strategies in individual countries. The existence of dramatic differences in mutational heterogeneity and mutation prevalence highlights the importance of multi-national screening studies in helping to elucidate the genotype-phenotype relationship in disorders such as MPS I that are characterized by extensive allelic heterogeneity. Positions of mutations are marked by vertical boxes. Note that the translational initiation site substitution, predicted to produce either no protein or to move the initiation site up/downstream (p.M1?), was excluded from the analysis. 15,24,26 ,2 7,32,35,37,44,60 ,62,64, 68,69,70 ,7 2,73,75,76,79,80 ,81,82, 85,86,87 ,8 9,90, 92 ,93,98,99,102 Ex.1 rs10902762 c.60G>A (A20A) NA (0.44) . 15,24,26 ,2 7,32,35,37,44,60 ,62,64, 68,69,70 ,7 2,73,75,76,80,82 ,85,86, 87,89,90 ,9 2,93,98,99,102 Ex 17,20,21 ,2 3,25,30,33,39,40 ,41,43, 44,45,46 ,5 2,53,54,55,57,60 ,61,63, 64,65,76 ,7 9,91,96,100, 17,20,21 ,2 3,25,27,30,33,34 ,35,39,4 0,41,43,44 ,46,49,52,54,55,57,59,60 ,61,62,63,64,65,70,71,76,79,83,84, 91,96,10 18,19,22 ,2 4,26,29,31,32,36 ,37,39,4 7,48,50,51 ,55,56,58,65,66,68,69,70 ,74,75,76,77,81,82,85,86,88,90,92, 95,97,98

F

  of a Cohort of 102 European Patients with Mucopolysaccharidosis type I: Identification and Characterization of 35 Novel alpha-L-iduronidase (IDUA) Alleles

  4) . P resen t stud y Legen d: Loc = Location; Ex =ex on; Intr =intro n; del=deletion; d up=d uplication; Acc = Acceler ated; Co ns= C onserved; PTMs= Post-translatio nal m od ifications *I DUA gen e GenBank-EM BL accessio n no. NM _0 00203.3; * *I DUA gen e GenB an k-EMBL accessio n no. NP_00019 4; the no vel m utatio ns ar e given in bold; #Li et al. (2 009) and Mort et al. (2 010). Allele carrying 2 novel m utations in c is; note that th e sec ond change occu rs in exon 9 and it w as p reviously r eport ed as single allele as due to c.1269C>G by Yogalingam et al. (2004); § m utation pr eviously rep orted as d ue to a c.3G>A by Le e-Ch en and W ang (1997)

Figure 1 .

 1 Figure 1. Distribution of the 54 detected mutant alleles and 37 putatively neutral polymorphisms in relation to the structure of the IDUA gene. The schematic map of the IDUA gene depicts the positions of the 14 numbered exons (blue boxes) and associated unnumbered introns (blue lines). An indication of the clinical phenotype associated with each of the 54 different mutant alleles identified in the 102 patients is provided. Thus, on the right of the diagram are listed those mutations associated with severe and attenuated (intermediate+mild) phenotypes respectively. Mutations associated with variable or unknown clinical phenotypes are given in the gray dotted box. On the left of the diagram, the polymorphic variants detected during the course of this study are listed. Novel alleles (34) and novel polymorphic variants (8) are marked in red. SEVERE INTERMEDIATE ATTENUATED

  Summary of the results of the reverse transcriptase-PCR analysis on mRNA samples MUTATION site; ORF=open reading frame On the right: normal splicing is depicted as unbroken green lines and abnormal splicing as dotted fucsia lines; blue boxes indicate normally spliced exons and fucsia boxes genomic (exonic and intronic) regions involved in the altered splicing

Figure 3 .

 3 Figure 3. Geographical-distribution and relative proportion of the common mutations found in the present series.

Figure 3 :

 3 Figure 3: Geographical distribution and relative proportion of the common mutations found in the present series

  al.Supp. FigureS1. Evolutionary comparison of the protein sequences flanking the missense mutations identified in the human IDUA protein with their orthologous counterparts in 5 vertebrates (in silico data from http://www.ebi.ac.uk/clustalw/). From the top: From the top: Homo sapiens, Pan troglodytes, Canis familiaris, Mus musculus, Gallus gallus, Tetraodon nigrovisidis.

  accession no. NM_000203.3; * the primers are located 5' (upstream) and 3' (downstream) to the intronic sequences flanking each exon; **in this case, the size also includes intronic regions; ***internal primers used for sequencing

  Sup p. Ta ble S5. Pre dic tio n of c onsequences of n ovel missense m utations us in g the 3D h omology mode l o f the IDU A p rote in constructe d by Re mpe l et al. [2005] M U TATION PR OTEIN POSIT ION MODE LL ING ANALYSIS PR EDICTION p .Y76C Solvent acc e ssible Lar ge arom atic and polar side c hain subst ituted by a sm alle r, hig hly reac tive one. p.G84R Close to ac tive site Loss of m ain c hain flexibility and of solvent ac cessible surface . G ain of c harge. p.T103P Solvent acc e ssible Introduc tion of a new proline within a long loop (G 101-N 110) protruding int o the solvent. Ac quisition of loop rigidity. De cr ease in residue pola rity. p .F188L * Ac tive site F188 be longs to an am ino acid cluste r c om prising arom a tic residues (F143, W180, W 306, Y343) and re sidues bearing indole ring s (H56, H91, H185). The new ly acquired 188L re sidue w ould maintain the hydrophobic ity but would give r ise to a loss of a rom atic ity. F188 is consider ed to be an ac tive site residue p .S423R* Core Increase in size, altera tion in c harge. D isruption of loc al prote in fold due t o steric hindrance p.G 219E Core Increase in size, altera tion in c harg e. De sta bilisation of the local prote in fold due t o st eric hindrance p.E276K Solvent acc e ssible Altera tion in c harg e (negative to positive). Cha nge of the protein surface ele ctrostatic potential p.W306L Core W306 belongs to a n am ino acid cluster c om prising aroma tic re sidue s (F143, W180, W 306, Y343) and re sidues bearing indole ring s (H56, H91, H185). The new ly acquired 306L re sidue w ould maintain the hydrophobic ity but would give rise to a loss of arom aticity. p.N348K Solvent acc e ssible Altera tion in surfa ce loc al c harge. p.P385R Core Loss of loc al rigidity. Destabilisa tion of the loc al pr ot ein fold. p.L396P Solvent acc e ssible Loss of long and branc hed hydrophobic side chain (leucine). L oss of loc al flexibility in the edge of a turn. L oss of loc al protein fold. p .A 436P Solvent acc e ssibleGa in of m ain c hain rigidity (436P) w hic h would disr upt the supe r-seconda ry structure of the protein ow ing to its inability to form the m ain chain H-bonds that are required for β-sheet for ma tion. * mutations oc cur in c is on the same a llele

Table 1 .

 1 Clinical Phenotypes and Genotypes Encountered in 102 MPS Type 1 Patients with Distinct Ethnic Background

Table 1 :

 1 Clinical Phenotypes and Genotypes Encountered in 102 MPS type I patients with distinct ethnic background DNA considering the A of the ATG translation initiation start site as nucleotide +1) and GenBank-EMBL accession no. NP_000194 (protein); *Patient not analysed in our Lab; ^indicates parents' consanguinity; °Patient's parents belonging to the same village; Underlined patients belonging to the same geographical region

	Pt	Ethnic origin	Phenotype	Allele 1	§	Allele 2	§
	1	Italy	H/S	p.P496R	p.G265R
	2	Italy	H	p.A327P	p.A327P
	3	Italy	H	p.G51D	p.G51D
	4	Italy	H	p.Q70X	p.Q70X
	5	Italy	H/S	p.G51D	p.P533R
	6	Italy	H	p.G51D	p.P496R
	7	Italy	S	p.G51D	p.R89W
	8	Italy	H/S	p.P533R	p.P533R
	9	Italy	H/S	p.Q70X	?
	10	Italy	H/S	p.W402X	p.L535F
	11	Italy	H/S	p.W402X	p.E178K
	12	Italy	S	p.N348K	c.1727+5G>C
	13	Italy	H/S	p.R89W	p.P496R
	14	Italy	S	p.G51D	c.1333_1335del3
	15	Italy	S	p.Q70X	c.1333_1335del3
	16	Italy	H/S	p.[S423R;F188L]	p.[S423R;F188L]
	17	Italy	H	p.Q70X	p.P496R
	18	Italy	H	p.W402X	p.W402X
	19	Italy	H	c.1727+1G>A	p.P496R
	20	Italy	H	p.G51D	p.G51D
	21	Italy	S	p.Y581X	?
	22	Italy	H	p.G51D	p.P496R
	23	Italy	H	p.Q70X	p.P496R
	24	Italy	H	c.46_57del12	p.Y201X
	25	Italy	H	p.Q70X	p.Q70X
	26	Italy	H	?		?
	27	Italy	H/S	c.46_57del12	c.1189+5G>A
	28	Italy	H	c.380_382del3	c.380_382del3
	29	Italy	S	p.A327P	c.878_889dup
	30	Italy	H	p.Q70X	p.P496R
	31	Italy	H	c.1166_1171dup	c.1166_1171dup
	32	Italy	H/S	p.W402X	p.G265R
	33	Italy	H	p.Q70X	p.P496R
	34	Italy	H	p.T103P	?
	35	Italy	H	p.Q70X	c.398_403del6
	36	Italy	H	p.W402X	p.W402X
	37	Italy	H	p.P533R	?
	38*	Poland	H	p.A327P	p.W402X
	39	Poland	H	p.Q70X	p.W402X
	40	Poland	H	p.Q70X	p.Q70X
	41	Poland	H	p.Q70X	p.Q70X
	42	Poland	H	c.1045_1047del3	c.1045_1047del3
	43	Poland	H -H/S	p.Q70X	c.1045_1047del3
	44	Poland	S	p.Q70X	p.G265R
	45	Poland	S	p.Q70X	c.46_57del12
	46	Poland	S	p.Q70X	p.Y76C
	47	Poland	H	p.W402X	c.1650+5G>A
	48	Poland	S	c.46_57del12	p.X654R
	49	Poland	S	c.878_889dup	c.1181_1189dup
	50	Poland	H	p.W402X	p.W402X
	51	Poland	H	p.W402X	c.1398delC
	52	Poland	H	p.Q70X	c.972+2T>C

Table 2 .

 2 Characteristics of the IDUA gene mutations identified in the 102 MPS type I patients and MutPred analysis of the missense mutations

Table 2 :

 2 Characteristics of the IDUA gene mutations identified in the 102 MPS type I patients and MutPred analysis of the missense m utations

	Loc.	Site of nucleotide su bstitutio n*	P redicted effect on pro tein str uc ture **	Type of mutation	No. o f alleles (%)	Evolutio nary Conser vation (phyloP )	MutPre d analysis of missense mu tations # Deleterious Summary o f con fident in silico mutatio n pro bab ility hypotheses	Re ferences
	Ex. 1	c.1A>C c.152G>A	p .M1 ? p.G51 D	Translational initiation site Missense	2(0.01) 10(4.9)	Acc (-0.3) Con s (2.7)	0.99 0.97	Pro tein struct ure disrup tio n	Present st udy § Bunge et al. (1994 )
		c.46_57d el12	p .S16 _A19d el	In-fram e d el 8(3.9)	.	.		Bunge et al. (1994 )
		c.208 C>T	p.Q70X	Nonsense	3 8(18 )	Con s (2.1)	.		Sco tt e t al. (199 2)
		c.227A >G	p.Y 76C	Missense	1(0.05)	Con s (2.7)	0.73		Present st udy
	Ex. 2	c.2 50G>C	p.G84R	Missense	1(0.05)	Con s (5.5)	0.93	Gain of bi nding site and disrup tio n to pro tei n stru cture	Present st udy
		c.265 C>T	p.R89W	Missense	2(0.01)	Con s (0.3)	0.96		Bunge et al. (1995 )
	Ex. 3	c .307A>C c .3 80_382d el3	p.T10 3P p.L12 7del	Missense In-fram e d el 2(0.01) 1(0.05)	Con s (0.4) .	0.36 .		Present st udy Ventur i et al. (2002)
	Intr. 3	c.385+1G>A	r.spl?	Sp licing	1(0.05)	Con s (5.1)	.		Present st udy
	Ex. 4	c .3 98_403d el6	p .M 133_G134del In-fram e d el 1(0.05)	.	.		Ventur i et al. (2002)
	Intr. 4	c.493+1G>A c.494-1G>A	r.spl? p.R16 6TfsX27	Sp licing Sp licing	2(0.01) 9(4.4)	Con s (6.2) Con s (5.7)	. .		Present st udy P resen t stud y
		c.532G>A	p.E178K	Missense	1(0.05)	Con s (4.5)	0.97	Gain of fun ctional sites in cluding PTMs	Ventur i et al. (2002)
	Ex. 5	c.[5 62T>C;1269C>A]	p. [F188L ; S423R]	Missense	2(0.01) C ons(3.0;1.2) 0.95;0 .92	Gain of meth ylation at S423	P resen t stud y
		c.574delT	p.S192P fsX2	Frameshift 2(0.01)	.	.		P resen t stud y
	Ex. 6	c.6 03C>G c.656G>A	p .Y20 1X p .G2 19E	Non se nse Missense	1(0.05) 1(0.05)	Con s (0.7) Con s (5.9)	. 0.89	Pro tein struct ure disrup tio n	P resen t stud y P resen t stud y
		c.793G>C	p.G265R	Missense	3(1.4)	Con s (6.2)	0.95	Secon dary struct ure disr uption , gain of methylation at G265	Yogalingam et al.(20 04)
		c.826G>A	p.E27 6K	Missense	4(1.9)	Con s (6.2)	0.76		P resen t stud y
	Ex. 7	c.8 26_828del3	p.E276del	In-frame del 2(0.01)	.	.		P resen t stud y
		c.8 78_889du p	p .T29 3_Y296du p In-frame d up 2(0.01)	.	.		Bun ge et al. (1995)
		c .956_972+9delinsTA	r.spl?	Sp licing	2(0.01)	.	.		P resen t stud y
	Intr. 7	c.972+2T>C	r.spl?	Splicing	1(0.05)	Con s (1.6)	.		Sco tt e t al. (199 3)
		c .917G>T	p.W306L	Missense	1(0.05)	Con s (6.0)	0.82	Loss of catalytic r esidu e at P309	P resen t stud y
		9 79G>C	p .A32 7P	Missense	5(2.4)	Con s (1.2)	0.84	Pr otein stru cture disru ption and gain o f me thylation at K 324	Bun ge et al. (1995)
		c.1044C>G	p.N3 48K	Missense	1(0.05)	Acc (-0.4)	0.85	Decrease in p rotein stab ility and gain of PTMs	P resen t stud y
	Ex. 8	c.1045_1047 del3	p .D349del	In-frame del 3(1.4)	-	.		P resen t stud y
		c.1154C>G	p .P385R	Missense	1(0.05)	Con s (5.4)	0.82	Protein str uctur e d isr uption	P resen t stud y
		c. 1166_1171 dup	p .A38 9_M 390dup In-frame dup 2(0.01)		.		P resen t stud y
		c.11 87T>C	p.L396P	Missense	1(0.05)	Con s (3.1)	0.83	Decrease in pr otein stab ility and second ar y struct ure disr uption	P resen t stud y
		c.11 81_1189du p	p .A3 94_L396d up In-frame d up 1(0.05)	.	.		Bun ge et al. (1995)
		c.1189 +4 A>G	p.V 371MfsX43	Sp licing	2(0.01)	Con s (4.3)	.		P resen t stud y
	Intr. 8	c.1189 +5 G>A	p.V 371MfsX43	Sp licing	1(0.05)	Con s (2.9)	.		P resen t stud y
		c.1190 -1delG	r.spl?	Sp licing	1(0.05)	-	.		P resen t stud y
		c.1205G>A	p.W 402X	Nonsense	3 7(18 )	Con s (1.8)	.		Sco tt e t al. (199 2)
	Ex. 9	c.1306G>C c.13 33_1335del3	p .A436P p.D445 del	Missense In-fram e d el 2(0.01) 2(0.01)	Con s (2.8) .	0.45 .		P resen t stud y Bun ge et al. (1995)
		c.1 398d elC	p.P467RfsX58	Frameshift 1(0.05)	.	.		P resen t stud y
	Ex. 10	c.1469T>C c.147 5G>C	p.L490P p.R49 2P	Missense Missense	2(0.01) 1(0.05)	Con s (0.4) Con s (0.7)	0.86 0.83	Decr ease in prot ein stability an d pr otei n stru cture disru ption Loss of binding site	Tieu et al. (1995) Tieu et al. (1995)
		c.148 7C>G	p.P496R	Missense	9(4.4)	Con s (4.4)	0.95	Gain o f bin ding site	Beesley et al. (2001)
	Ex. 11	c.159 8C>G c.16 03C>T	p.P533R p.L53 5F	Missense Missense	8(3.9) 1(0.05)	Con s (4.1) Con s (1.7)	0.94 0.59	Gain o f bin ding site	Sco tt e t al. (199 2) P resen t stud y
	Intr. 11	c.1650 +5 G>A c.1 650+5G>C	r.spl? r.spl?	Splicing Sp licing	1(0.05) 1(0.05)	Con s (4.7) Con s (4.7)	. .		Ven turi et al. 2002 P resen t stud y
		c.1727 +1 G>A	r.spl?	Sp licing	1(0.05)	Con s (3.8)	.		P resen t stud y
	Intr. 12	c.172 7+4 C>T c.1 727+5G>C	p .L578V fsX14 Exon 12 skip pin g	Sp licing Sp licing	1(0.05) 1(0.05)	Con s (0.4) Con s (5.4)	. .		P resen t stud y P resen t stud y
		c.1727+6T>A	Exon 12 skip pin g	Sp licing	2(0.01)	Con s (1.1)	.		P resen t stud y
	Ex. 13	c.1743C>G	p .Y58 1X	Non se nse	1(0.05)	Acc (-1.1)	.		P resen t stud y
		c.1861C>T	p.R62 1X	Nonsense	1(0.05)	Con s (1.5)	.		Bun ge et al. (1994)
	Ex. 14	c.1 893d elC	p.F632SfsX105	Frameshift 1(0.05)		.		P resen t stud y
		c.19 60T>C	p.X6 54RextX62	No-stop	2(0.01)	Acc (-0 .

Table 3 .

 3 Summary of the results of the reverse transcriptase-PCR analysis on mRNA samplesGenotypes, clinical phenotypes and geographic origins of the 102 patients studied here are listed in Table

	F o
	r
	P
	e e r
	R e v i e w

Table S1 .

 S1 List of primers used for polymerase chain reaction (PCR) amplification of IDUA gene exons and exonintron boundaries Supp. TableS1: List of primers used for polymerase chain reaction (PCR) amplification of IDUA gene exons and exon-intron boundaries

						IDUA Gene Mutations 17
	Supp. Exons amplified	Forward (5'>3')*	Nucleotide position	Reverse (5'>3')*	Nucleotide position a	PCR product size (bp)	Annealing Temperature (°C)
			Genomic DNA Amplification a
	1	AGGCGGAACCGGCAGTGCAG	g.5035-5054	TCCGGTCTCTGAAGCTCTCA	g.5338-5319	304	67
	2	CGCTGCCAGCCATGCTGAGGCTCG	g.5760-5783	ACAAGGGGTCTTCCGAGC	g.6045-6028	286	67
	3-4**	ATCGGAGTCCTGTGTGGCACCT	g.18497-18518	TGCCCACCAATGTATCTCCTGA	g.19101-19080	605	65
	5	CATCACCTTGCACCCTCC	g.19391-19408	CGTCTACACCTGCCCTGG	g.19663-19646	273	65
	6	GAGGAAGGCAGGAGCAGAG	g.19611-19629	CTCAGCACCACCAGGGTC	g.19969-19952	359	65
	7-8**	TGCGGCTGGACTACATCTC	g.19854-19872	CTGGAGGAAGTGCGCTCC	g.20584-20567	731	63
	***	CCACCTTCCTCCCGAGAC (8F)	g.20199-20216	AGTAGCAGGTTCTGATGCTGC (7R)	g.20301-20281	-	-
	9-10** *** 11-12** 13-14**	TCCTTCACCAAGGGGAGG GGTGACCCTGCGGCTG (10F) o GTGTGGGTGGGAGGTGGA GGGCTTGAGGGAATGAGG r g.20631-20648 g.20911-20926 g.21244-21261 g.21911-21928	TCCTCAGGGTTCTCCAGG CTGACACTCAGGCCTCGG (9R) GAGCCCGAATGTCCATCTCATGTC GGTGATGGGAGGGCAGCA cDNA Amplification b	g.21331-21314 g.21030-21013 g.21853-21830 g.22483-22466	701 -610 573	63 -67 65
	3-4-5 8-9 12-13-14	CGGCATCTTGCTGGTCCGGACCC GGGCTGGTCCCTGCCACAGC TGCTGGTGCACGTGTGTGCGC	c.337-359 c.1000-1019 P c.1695-1715	CGCTGTCCCTCCGAGCAGGC CGTTGTCCAGGTAGCGCGTGAC ATGGGAGGGCAGCATGGGCAC	c.717-698 c.1523-1502 c.2132-2112	381 524 474	65 67 67
	IDUA gene:		r e e
				R e v i e w

  Supp. TableS2: Charac teristics of putative ly neutral SNPs identified in the IDUA gene

	Location	dbSNP no.	Site of nucleotide substitution* (amino acid change)**	Minor allele frequency § ( ) &	MutPred analysis Probability of Confident deleterious mutation in-silico hypotheses	Polymorphic background in the Patients # or control alleles
	Ex.1	rs11248061	c.24C>A (A8 A)	NA (0.42)	.

  Supp TableS6. Genotype-phenotype correlation: comparison with existing data from the literature

		NO. OF PATIENTS
		ANALYZED AND
	GENOTYPES*	(PHENOTYPE)
		THIS	OTHER
		STUDY	STUDIES

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGMENTS

European patient samples were obtained through a collaboration with Genzyme Corporation, Cambridge, MA, USA. Some samples were also obtained from the "Cell Line and DNA Biobank from Patients Affected by Genetic Diseases" (G. Gaslini Institute) -Telethon Genetic Biobank Network (Project No. GTB07001A). We also thank Prof. Generoso Andria (Napoli), Dr. Rita Barone (Catania), Prof. Cesare Danesino (Pavia), Prof. Paola Di Natale (Napoli), Dr. Graziella Uziel (Milano), for their contribution of clinical information and patient samples.

SUPPORTING INFORMATION

Page 18 of 24 John Wiley & Sons, Inc.

Human Mutation [START_REF] Bunge | Mucopolysaccharidosis type I: identification of 8 novel mutations and determination of the frequency of the two common alpha-L-iduronidase mutations (W402X and Q70X) among European patients[END_REF]; [START_REF] Gort | Analysis of five mutations in 20 mucopolysaccharidosis type 1 patients: high prevalence of the W402X mutation[END_REF]; [START_REF] Hein | The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation[END_REF]; [START_REF] Scott | Q70X[END_REF]; [START_REF] Vazna | Mucopolysaccharidosis type I in 21 Czech and Slovak patients: mutation analysis suggests a functional importance of C-terminus of the IDUA protein[END_REF]; [START_REF] Venturi | Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations[END_REF]