The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation

Mucopolysaccharidosis type I (MPS I; McKusick 25280; Hurler syndrome, Hurler-Scheie syndrome and Scheie syndrome) is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase (EC 3.2.1.76). MPS I patients present within a clinical spectrum bounded by the extremes of Hurler and Scheie syndromes. The alpha-L-iduronidase missense mutations R89Q and R89W were investigated and altered an important arginine residue proposed to be a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase. The R89Q alpha-L-iduronidase mutation was shown to result in a reduced level of alpha-L-iduronidase protein (< or =10% of normal control) compared to a normal control level of alpha-L-iduronidase protein that was detected for the R89W alpha-L-iduronidase mutation. When taking into account alpha-L-iduronidase specific activity, the R89W mutation had a greater effect on alpha-L-iduronidase activity than the R89Q mutation. However, overall the R89W mutation produced more residual alpha-L-iduronidase activity than the R89Q mutation. This was consistent with MPS I patients, with an R89W allele, having a less severe clinical presentation compared to MPS I patients with either a double or single allelic R89Q mutation. The effects of the R89Q and R89W mutations on enzyme activity supported the proposed role of R89 as a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase.     

Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations

The lysosomal storage disorder, mucopolysaccharidosis type I (MPS I), is caused by a deficiency of the enzyme alpha-L-iduronidase, which is involved in the breakdown of dermatan and heparan sulphates. There are three clinical phenotypes, ranging from the Hurler form characterised by skeletal abnormalities, hepatosplenomegaly and severe mental retardation, to the milder Scheie phenotype where there is aortic valve disease, corneal clouding, limited skeletal problems, but no mental retardation. In this study, 85 MPS I families (73 Hurler, 5 Hurler/Scheie, 7 Scheie) were screened for 9 known mutations (Q70X, A75T, 474-2a>g, L218P, A327P, W402X, P533R, R89Q, 678-7g>a). W402X was the most frequent mutation in our population (45.3%) and Q70X was the second most frequent (15.9%). In 30 families, either one or both of the mutations were not identified, which accounted for 25.9% of the total alleles. Therefore, all 14 exons of the alpha-L-iduronidase gene were screened in these patients and 23 different sequence changes were found, 17 of which were previously unknown. The novel sequence changes include 4 deletions (153delC, 628del5, 740delC, 747delG), 5 nonsense mutations (Q60X, Y167X, Q400X, R619X, R628X), 6 missense mutations (C205Y, G208V, H240R, A319V, P496R, S633L), a splice site mutation (IVS12+5g>a), and a rare polymorphism (A591T). The polymorphism and novel missense mutations were transiently expressed in COS-7 cells and all of them except the polymorphism showed complete loss of enzyme activity. In total, 165 of the 170 mutant alleles were identified in this study and despite the high frequency of W402X and Q70X, the identification of many novel mutations unique to individual families further highlights the genetic heterogeneity of MPS I.     

Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.

alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha-L-iduronidase premature stop codons and potential read-through in mucopolysaccharidosis type I patients

alpha-L-Iduronidase is a glycosyl hydrolase involved in the sequential degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. A deficiency in alpha-L-iduronidase results in the lysosomal accumulation and urinary secretion of partially degraded glycosaminoglycans and is the cause of the lysosomal storage disorder mucopolysaccharidosis type I (MPS I; Hurler and Scheie syndromes; McKusick 25280). The premature stop codons Q70X and W402X are two of the most common alpha-l-iduronidase gene (IDUA) mutations accounting for up to 70% of MPS I disease alleles in some populations. Here, we have reported a new mutation, making a total of 15 different mutations that can cause premature IDUA stop codons and have investigated the biochemistry of these mutations. Natural stop codon read-through was dependent on the fidelity of the codon when evaluated at Q70X and W402X in CHO-K1 cells, but the three possible stop codons TAA, TAG and TGA, had different effects on mRNA stability and this effect was context dependent. In CHO-K1 cells expressing the Q70X and W402X mutations, the level of gentamicin-enhanced stop codon read-through was slightly less than the increment in activity caused by a lower fidelity stop codon. In this system, gentamicin had more effect on read-through for the TAA and TGA stop codons when compared to the TAG stop codon. In an MPS I patient study, premature TGA stop codons were associated with a slightly attenuated clinical phenotype, when compared to classical Hurler syndrome (e.g. W402X/W402X and Q70X/Q70X genotypes with TAG stop codons). Natural read-through of premature stop codons is a potential explanation for variable clinical phenotype in MPS I patients. Enhanced stop codon read-through is a potential treatment strategy for a large sub-group of MPS I patients.     

Three novel α-L-iduronidase mutations in 10 unrelated Chinese mucopolysaccharidosis type I families

Mucopolysaccharidosis type I (MPS I) arises from a deficiency in the α-L-iduronidase (IDUA) enzyme. Although the clinical spectrum in MPS I patients is continuous, it was possible to recognize 3 phenotypes reflecting the severity of symptoms, viz., the Hurler, Scheie and Hurler/Scheie syndromes. In this study, 10 unrelated Chinese MPS I families (nine Hurler and one Hurler/Scheie) were investigated, and 16 mutant alleles were identified. Three novel mutations in IDUA genes, one missense p.R363H (c.1088G > A) and two splice-site mutations (c.1190-1G > A and c.792+1G > T), were found. Notably, 45% (nine out of 20) and 30% (six out of 20) of the mutant alleles in the 10 families studied were c.1190-1G > A and c.792+1G > T, respectively. The novel missense mutation p.R363H was transiently expressed in CHO cells, and showed retention of 2.3% IDUA activity. Neither p.W402X nor p.Q70X associated with the Hurler phenotype, or even p.R89Q associated with the Scheie phenotype, was found in this group. Finally, it was noted that the Chinese MPS I patients proved to be characterized with a unique set of IDUA gene mutations, not only entirely different from those encountered among Europeans and Americans, but also apparently not even the same as those found in other Asian countries.     

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.     

Cloning and characterization of cDNA encoding canine alpha-L-iduronidase. mRNA deficiency in mucopolysaccharidosis I dog.

alpha-L-Iduronidase is a lysosomal enzyme, the deficiency of which causes mucopolysaccharidosis I (MPS I); a canine MPS I colony has been bred to test therapeutic intervention. The enzyme was purified to apparent homogeneity from canine testis and found to consist of two electrophoretically separable proteins that had common internal peptides but differed at their amino termini. A 57-base oligonucleotide, corresponding to the most probable codons of the longest peptide, was used to screen a canine testis cDNA library. Three cDNAs were isolated, two of which lacked the 5'-end whereas the third was full-length except for a small internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The amino terminus of the larger protein, glutamic acid 26, is at the predicted signal peptide cleavage site, whereas the amino terminus of the smaller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of GenBank showed that the amino acid sequence of alpha-L-iduronidase has similarity to that of a bacterial beta-xylosidase. A full-length cDNA corresponding to the composite sequence was constructed (pcIdu) and inserted into the pSVL expression vector (pSVcIdu). Two days after Cos-1 cells were transfected with pSVcIdu, their intracellular and secreted level of alpha-L-iduronidase activity has increased 8- and 22-fold, respectively, over the endogenous activity. Fibroblasts of MPS I dogs, which have no alpha-L-iduronidase activity, lacked the normal alpha-L-iduronidase mRNA of 2.2 kilobases and contained instead a trace amount of a 2.8-kilobase species. Isolation and characterization of an expressible alpha-L-iduronidase cDNA represents the first step toward mutation analysis and replacement therapy.     

Identification of mutations in the alpha-L-iduronidase gene (IDUA) that cause Hurler and Scheie syndromes.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the lysosomal glycosidase alpha-L-iduronidase. Hurler (severe), Scheie (mild), and Hurler/Scheie (intermediate) syndromes are clinical subtypes of MPS-I, but it is difficult to distinguish between these subtypes by biochemical measurements. Mutation analysis was undertaken to provide a molecular explanation for the clinical variation seen in MPS-I. Using chemical cleavage and direct PCR sequencing, we have defined four previously undescribed mutations for MPS-I (delG1702, 1060 + 2t-->c, R89Q, and 678-7g-->a). R89Q and 678-7g-->a were found to be present in 40% of Scheie syndrome alleles. Expression of R89Q demonstrated reduced stability and activity of the mutant protein. The deleterious effect of R89Q may be potentiated by a polymorphism (A361T) to produce an intermediate phenotype. 678-7g-->a was found to be a mild mutation, since it was present in an index Scheie syndrome patient in combination with a severe allele (W402X). This mutation appears to allow a very small amount of normal mRNA to be produced from the allele which is likely to be responsible for the mild clinical phenotype observed. Both the 5' and 3' splice site mutations (1060 + 2t-->c and 678-7g-->a, respectively) result in high proportions of mature mRNAs containing introns, which has not been observed for other splicing mutations. The frameshift mutation (delG1702) and the 5' splice site mutation (1060 + 2t-->c) are both thought to be associated with severe MPS-I. The identification of these MPS-I mutations begins to document the expected genetic heterogeneity in MPS-I and provides the first molecular explanations for the broad range of clinical phenotypes observed.     

Hurler syndrome: a patient with abnormally high levels of alpha-L-iduronidase protein.

Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.     

An intron mutation in the human alpha 1(I) collagen gene alters the efficiency of pre-mRNA splicing and is associated with osteogenesis imperfecta type II

This study describes a homozygous, G----A transition at the moderately conserved +5 position within the splice donor site of intron 14 in the human alpha 1(I) collagen gene. The mutation reduced the efficiency of normal splice-site selection since the exon upstream of the mutation was spliced alternatively. Moreover, the extent of alternative splicing was sensitive to the temperature at which the mutant cells were grown, suggesting that the mutation directly affected spliceosome assembly. To achieve exon skipping, this effect must be propagated so as to disrupt the selection of a second splice site in the adjacent intron.     

Common antigenicity for two glycosidases

Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.     

Splicing Defects in the COL3A1 Gene: Marked Preference for 5′ (Donor) Splice-Site Mutations in Patients with Exon-Skipping Mutations and Ehlers-Danlos Syndrome Type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

Glycosidase active site mutations in human alpha-L-iduronidase

Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.     

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.