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ScienceDirect Journal of Genetics and Genomics 41 (2014) 197e203

ORIGINAL RESEARCH

Genetic Analysis of 17 Children with Hunter Syndrome: Identification and Functional Characterization of Four Novel Mutations in the Iduronate-2-Sulfatase Gene Dimitry A. Chistiakov a,b,*, Lyudmila M. Kuzenkova c, Kirill V. Savost’anov b, Anait K. Gevorkyan d, Alexander A. Pushkov b, Alexey G. Nikitin b, Nato D. Vashakmadze c, Natalia V. Zhurkova b, Tatiana V. Podkletnova c, Leila S. Namazova-Baranova d, Alexander A. Baranov e a

Department of Medical Nanobiotechnology, Pirogov Russian State Medical University, Moscow 117997, Russia b Department of Molecular and Genetic Diagnostics, Division of Laboratory Medicine, Institute of Pediatrics, Research Center for Children’s Health, Moscow 119991, Russia c Department of Psychoneurology and Psychosomatic Pathology, Institute of Pediatrics, Research Center for Children’s Health, Moscow 119991, Russia d Institute of Preventive Pediatrics and Rehabilitation, Research Center for Children’s Health, Moscow 119991, Russia e Research Center for Children’s Health, Moscow 119991, Russia Received 27 October 2013; revised 21 January 2014; accepted 26 January 2014 Available online 4 February 2014

ABSTRACT Mucopolysaccharidosis type II (MPS II) is a rare X-linked disorder caused by alterations in the iduronate-2-sulfatase (IDS ) gene. In this study, IDS activity in peripheral mononuclear blood monocytes (PMBCs) was measured with a fluorimetric enzyme assay. Urinary glycosaminoglycans (GAGs) were quantified using a colorimetric assay. All IDS exons and intronic flanks were bidirectionally sequenced. A total of 15 mutations (all exonic region) were found in 17 MPS II patients. In this cohort of MPS II patients, all alterations in the IDS gene were caused by point nucleotide substitutions or small deletions. Mutations p.Arg88His and p.Arg172* occurred twice. All mutations were inherited except for p.Gly489Alafs*7, a germline mutation. We found four new mutations (p.Ser142Phe, p.Arg233Gly, p.Glu430*, and p.Ile360Tyrfs*31). In Epstein-Barr virus (EBV)-immortalized PMBCs derived from the MPS II patients, no IDS protein was detected in case of the p.Ser142Phe and p.Ile360Tyrfs*31 mutants. For p.Arg233Gly and p.Glu430*, we observed a residual expression of IDS. The p.Arg233Gly and p.Glu430* mutants had a residuary enzymatic activity that was lowered by 14.3 and 76-fold, respectively, compared with healthy controls. This observation may help explain the mild disease phenotype in MPS II patients who had these two mutations whereas the p.Ser142Phe and p.Ile360Tyrfs*31 mutations caused the severe disease manifestation. KEYWORDS: Hunter syndrome; Mucopolysaccharidosis type II; Iduronate-2-sulfatase; Mutations; Glycosaminoglycans

INTRODUCTION

* Corresponding author. Tel: þ7 495 434 1301, fax: þ7 495 434 1422. E-mail addresses: [email protected], dmitry.chistyakov@ yahoo.com (D.A. Chistiakov).

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome (OMIM 309900), is a rare genetic disorder belonging to the group of lysosome storage diseases (Lampe et al., 2013). MPS II results from the lack or deficiency of

1673-8527/$ - see front matter Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. http://dx.doi.org/10.1016/j.jgg.2014.01.007

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iduronate-2-sulfatase (IDS, EC 3.1.6.13), a lysosomal enzyme that degrades glycosaminoglycans (GAGs) such as heparan sylfate and dermatan sulfate (Hopwood et al., 1993). GAGs are a molecular component of proteoglycans that constitute the extracellular matrix (ECM) (Sarrazin et al., 2011). The ECM serves many functions including mechanical support, separating cells from each other, involving in cellular uptake of macromolecules, and providing a network for intercellular communication and signaling (Iozzo, 1998). In turnover, ECM components are permanently replaced with newly synthesized molecules, with internalization and further degradation of previous constituents in prelysosomal compartments and lysosomes (Iozzo, 1998). IDS deficiency leads to the accumulation of partially digested GAGs in the cell and induces cytotoxicity. In MPS II, mucopolysaccharides build up in all tissues and organs throughout the body, causing in turn systemic alterations in their function. Hunter syndrome is an X-linked disease suggesting its transmission from a mother to her children (Berg et al., 1968). Indeed, this disorder occurs almost exclusively in males. MPS II is a sex-linked recessive disorder since the carriage of the abnormal IDS gene copy typically does not affect females due to the compensatory role of the second normal gene copy. However, several examples of this disorder in females mostly arisen from skewed inactivation of chromosome X are documented (Kloska et al., 2011). The prevalence of this disorder in Caucasians varies from 1:46,000 (Krabbi et al., 2012) to 1:320,000 live births (Nelson et al., 2003). An increased prevalence of Hunter syndrome (twice higher as compared to most other Caucasian populations) was reported in Ashkenazi and Sephardic Jews living in Israel (Zlotogora et al., 1985). At birth, infants with MPS II look visually normal. The disease is diagnosed usually at age of 1.5‒4.0 years. The affected children exhibit a delay in physical and mental development, dwarfism, coarse (gargoyle-like) faces, enlarged tongue, respiratory difficulties, bone and cardiovascular abnormalities, tendon and muscle contractures, increased liver and spleen size, deafness, progressive loss of vision, sleep disturbance, and aggression (Beck, 2011). Compared to severe (early-onset) MPS II, patients with mild (late) form of Hunter syndrome have mild or no mental retardation and delayed disease progression (Froissart et al., 2002). MPS II patients develop serious complications such as cardiac valve disease, heart failure, obstructive lung disease, and pulmonary hypertension that usually became fatal by the second decade of life (Braunlin et al., 2011). The IDS gene encoding iduronate-2-sulfatase is located on chromosome Xq28 (Wilson et al., 1991). The majority (over 80%) of genetic alterations causing Hunter syndrome result from the point mutations and small deletions/insertions of 1‒3 nucleotides in the IDS gene. Some MPS II mutations are not inherited from a mother and in fact are de novo mutations occurring preferentially during male meiosis (Froissart et al., 2007). The remaining 20% of genetic alterations are presented by large deletions and chromosome rearrangements at the Xq28 locus. The high frequency of large genetic rearrangements involving the IDS gene may be explained in part by the presence of the IDS pseudogene (IDSP1) located in a distance of 20 kb upstream of the 50

end of IDS (Timms et al., 1995). The pseudogene contains copies of two exons (3 and 7) and three introns (2, 3, and 7) of the functional gene whose sequence is over 95% similar to that of the IDS gene (Timms et al., 1995). The pseudogene is indeed responsible for a high incidence of homologous recombination events in Xq28 leading to various deletions and inversions in patients with MPS II (Bunge et al. 1998). According to the Human Genome Mutation Database (www.hgmd.org), over 360 MPS II-causing mutations with exonic point mutations comprising a half of the IDS mutations have been reported. Due to the high heterogeneity and increased frequency of IDS gene alterations in patients with Hunter syndrome, a growing list of MPS II mutations is regularly updated. In this study, we presented the mutation analysis in 17 patients affected with Hunter syndrome including the report of four new IDS mutations. RESULTS Clinical and biochemical analysis of MPS II patients Clinical and biochemical characteristics of patients studied are presented in Table 1. As expected, the patients with Hunter syndrome were characterized with abnormally large head, coarse facial features, physical disability, joint contractures, frequent mental and growth retardation, enlarged spleen and liver, impulsive behavior, chronic respiratory and nasal infections, hernias, and common cardiac, pulmonary, and neural (carpal tunnel syndrome) complications (Beck, 2011). All MPS II patients had a markedly reduced enzymatic activity of plasma IDS compared with that in 20 age-matched normal children (4.45  4.11 vs. 316  126 nmol/4 h/mL; P < 0.00001), thereby suggesting a serious defect in IDS activity due to the presence of Hunter syndrome. An average GAGs concentration normalized to creatinine was increased by 3-fold in urine of affected patients compared to normal subjects (33.7  11.9 vs. 11.0  6.5 mg/mmol/L creatinine; P ¼ 0.0099), reflecting a decreased degradation rate of mucopolysaccharides in MPS II. Genetic analysis of MPS II patients A total of 15 exonic mutations were found in the IDS gene (Table 2). Among those, there were nine missense mutations, three nonsense mutations, and three frameshift mutations. Mutations p.Arg88His and p.Arg172* were detected twice. It should be stressed that mutation p.Arg88His was found in each of two probands belonging to the same family. A single mutation was occurred in exons 6 and 7, two mutations were in exon 4, three mutations were detected in exon 3, and finally four mutations were observed in exons 5 and 9. None of the missense mutations were observed in 111 chromosomes X of healthy unrelated Russian subjects suggesting that they do not represent polymorphisms. Of 15 IDS mutations found, only one (p.Gly489Alafs*7) was de novo mutation because neither the patient’s mother nor the grandmother carried the mutation.

D.A. Chistiakov et al. / Journal of Genetics and Genomics 41 (2014) 197e203 Table 1 Clinical and biochemical characteristics of MPS II patients Clinical characteristics

Value

Gender (male/female)

17/0

Age at onset (years)

1.08  0.63 [0.33‒2.0]

Age at diagnosis (years)

3.03  1.35 [1.18‒5.0]

Disease severity (severe/mild)

13/2

IDS activity (nmol/4 h/mL)

4.45  4.11 [0.074‒22.6]

GAGs (mg/mmol/L creatinine)

33.7  11.9 [12.5‒88.2]

Tendon contractures (%)

17 (100)

Macrocephaly (%)

15 (88)

Mental retardation (%)

15 (88)

Hepatosplenomegalia (%)

13 (76)

Hyperactivity, impulsivity, aggression (%)

13 (76)

Frequent respiratory infections (%)

13 (76)

Cardiac valve disease (%)

11 (65)

Respiratory obstruction (%)

10 (59)

Umbilical and inguinal hernias (%)

10 (59)

Carpal tunnel syndrome (%)

9 (53)

Growth retardation (%)

8 (47)

Persistent rhinorrhea (%)

6 (35)

Pseudobulbar palsy (%)

6 (35)

Sleep apnea (%)

6 (35)

Retinopathy (%)

3 (18)

Dementia (%)

3 (18)

Corneal clouding (%)

2 (12)

Pulmonary hypertension (%)

2 (12)

Chronic diarrhea (%)

2 (12)

Dental abnormalities (%)

1 (6)

Hydrocephalus (%)

1 (6)

Spinal cord compression (%)

1 (6)

Values are mean  S.D. The range of values is shown in square brackets. The percentage is presented in parentheses. IDS, iduronate-2-sulfatase; GAGs, glycosaminoglycans.

We discovered four new mutations that have never been reported previously. There were a missense mutation p.Arg233Gly (exon 5) and a nonsense mutation p.Glu430* (exon 9), which are both associated with a mild phenotype of Hunter syndrome. Two other mutations including p.Ser142Phe (exon 4) and p.Ile360Tyrfs*31 (exon 7) caused a severe course of disease. All new mutations were inherited since p.Arg233Gly, p.Ser142Phe, and p.Ile360Tyrfs*31 were found in the patient’s mother while p.Glu430* was detected in both mother and grandmother. Western blot analysis of newly identified IDS mutants The Western blot analysis showed the absence of the IDS protein in Epstein-Barr virus (EBV) cell lines derived from MPS II patients with the p.Ser142Phe and p.Ile360Tyrfs*31 mutations. In contrast, EBV cell lines having the p.Arg233Gly and

199

p.Glu430* IDS mutants showed some residual expression of the enzyme that was markedly lowered compared to the normal subjects (Fig. 1). In the case of p.Glu430*, a translated polypeptide had a molecular weight (MW) of about 61 kDa that corresponds to that of the truncated p.Glu430* IDS mutant. In line with this, we failed to detect any IDS enzymatic activity in peripheral mononuclear blood monocytes (PMBCs) derived from the patients who had the p.Ser142Phe and p.Ile360Tyrfs*31 mutations. PMBCs from MPS II patients with the p.Arg233Gly and p.Glu430* IDS mutations exhibited very low IDS enzymatic activity that was reduced by 14.3- and 76-fold, respectively, in comparison with that of healthy individuals (Fig. 2). These findings correlate with the mild MPS II phenotype in the case of the p.Arg233Gly and p.Glu430* mutants and severe disease manifestation in subjects who had the p.Ser142Phe and p.Ile360Tyrfs*31 mutations. DISCUSSION As a result of genetic analysis of 17 patients affected with MPS II, we have identified four new mutations in the IDS gene. The novel mutation p.Ile360Tyrfs*31 leads to the protein translation frameshift at codon 360 with formation of a new translation frame that adds 31 new amino acid (aa) residues to the mutant protein and terminates at codon 391. This mutation results in the rapid degradation of translated polypeptide since neither the IDS protein itself nor IDS enzymatic activity has been detected in PMBCs from the MPS II patient who carries this mutation. Similarly, the p.Ser142Phe mutation also has deleterious effect on the enzyme due to the lack of both the IDS protein and its activity in PMBCs from the patient who has this mutation. The mutation affects serine at position 142 that is highly preserved across the vertebrates (Fig. 3A). Ser142 is located in the vicinity to Lys135, which is involved in sulfate binding and therefore is essential for catalytic activity of IDS and related sulfatases (von Bu¨low et al., 2001; Sa´enz et al., 2007). Indeed, substitution of the polar serine by the hydrophobic phenylalanine may seriously alter normal folding of the enzyme and abolish its activity. As a consequence, abnormally folded translation product may be subjected to rapid degradation through the mechanism of endoplasmic reticulum-associated degradation. The mutation p.Glu430* results in a truncated protein that lacks 120 C-terminal amino acid residues compared to the full-length IDS. The mutant IDS protein remains the highly conserved sulfatase catalytic domain spanning 33‒420 aa (Parenti et al., 1997). However, the p.Glu430* IDS mutant lacks two putative N-glycosylation sites at codons 513 and 537 and several secondary structural elements such as four bsheets and three a-helices (Sa´enz et al., 2007), leading to in turn the destabilization of the tertiary protein structure. Thus, the mutated enzyme has a very low, residual activity. The truncating mutation p.Glu430* seems not to affect synthesis of the IDS precursor because the truncated protein is produced in the presence of related mutations p.Arg443*, p.Glu521*, and p.Gln531* (Chang et al., 2005; SukegawaKawasaka et al., 2006). However, defects occur when further

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Table 2 Genetic analysis of MPS II patients Mutation

Consequence

Position No. of Phenotype Status occurrence

c.257C>T

p.Pro88Leu

Exon 3 1

Severe

Reported

c.263C>A

p.Arg88His

Exon 3 2

Severe

Reported

c.325T>C

p.Trp109Arg

Exon 3 1

Severe

Reported

c.395C>G

p.Ser132Trp

Exon 4 1

Severe

Reported

c.425C>T

p.Ser142Phe

Exon 4 1

Severe

Novel

c.514C>T

p.Arg172*

Exon 5 2

Severe

Reported

c.596_599del4 p.Lys199Argfs*13 Exon 5 1

Severe

Reported

c.598C>T

p.Gln200*

Exon 5 1

Severe

Reported

c.697C>G

p.Arg233Gly

Exon 5 1

Mild

Novel

c.795C>A

p.Asn265Lys

Exon 6 1

Severe

Reported

c.1077delG

p.Ile360Tyrfs*31

Exon 7 1

Severe

Novel

c.1288G>T

p.Glu430*

Exon 9 1

Mild

Novel

c.1295G>A

p.Cys432Tyr

Exon 9 1

Severe

Reported

c.1402C>G

p.Arg468Gly

Exon 9 1

Severe

Reported

c.1466delG

p.Gly489Alafs*7

Exon 9 1

Severe

Reported

IDS, iduronate-2-sulfatase; MPS II, mucopolysaccharidosis type 2.

Control 2

Control 1

p.Glu430*

p.Arg233Gly

p.IIe360Tyrfs*31

kDa 200 140 100 80 60 50

p.Ser142Phe

maturation of the precursor mutant. In the case of these mutations, the resulting mutant product remained residual enzymatic activity, had altered conformation and decreased stability, and was associated with a mild or intermediate but never with severe MPS II phenotype (Sukegawa et al., 1995; Chang et al., 2005; Sohn et al., 2010). The missense mutation p.Arg233Gly resides in the highly conserved IDS region across the vertebrates (Fig. 3B). The mutation is situated in the flexible hydrophilic loop, which is ˚ ) to relatively close (approximately in the distance of 10‒12 A the catalytic Cys84 and may contribute to the active site conformation (Fig. 4) (Sa´enz et al., 2007). Indeed, the substitution of the polar arginine by the neutral glycine should affect the loop conformation and flexibility. However, the mutation p.Arg233Gly does not fully inactivate the enzyme since the mutated protein remains significantly reduced catalytic activity and is associated with the mild MPS II phenotype.

Like other IDS mutations caused by an arginine substitution (p.Arg48Pro and p.Arg95Thr) and associated with the moderate MPS II severity (Sukegawa-Kawasaka, et al. 2006), the mutation p.Arg233Gly may not impair both precursor synthesis and processing but can alter the enzyme conformation and lysosomal trafficking. Interestingly, the loop containing the mutation site is recognized by the specific chicken egg yolk IgY antibodies developed to detect human IDS (Sosa and Barrera, 2005). In plasma of MPS II patients, mutated IDS has altered antigenicity suggesting the presence of conformational changes compared to the intact enzyme (Parkinson et al., 2004). Early studies revealed the presence of a total of 30 IDS mutations in 37 Russian MPS II patients (Karsten et al., 1998, 1999). Most of those mutations (26 of 30) were presented by point sequence substitutions whereas the remaining four mutations involved large deletions and complex rearrangements in the IDS locus. Similarly, in this study, we reported a total of 15 IDS mutations presented only by point nucleotide changes and small deletions, and found no large genetic alteration in the cohort of 17 Russian children with Hunter syndrome. These findings are in good agreement with data on other ethnic groups including those found in France (Froissart et al., 1998), Japan (Isogai et al., 1998), USA (Li et al., 1999), Italy (Filocamo et al., 2001), and Thailand (Keeratichamroen et al., 2008). A spectrum of IDS mutations detected in 36 Russian MPS II patients (Karsten et al., 1998) only slightly overlapped with that observed in our study. We found p.Arg88His in two cases (both are siblings) whereas this mutation was found only once by Karsten et al. (1998). All other mutations were unique for each dataset. The frequency of this mutation (once in each dataset of Russian MPS II subjects and a total of 2 of 53 affected patients) was unusually low compared to other populations such as Chinese (5/38) (Zhang et al., 2011), Japanese (5/43) (Isogai et al., 1998), Italians (5/40) (Filocamo et al., 2001), Taiwanese (6/14) (Lin et al., 2006), and UK (4/57) (Vafiadaki et al., 1998). In summary, our results provided further evidence of mutational heterogeneity of the IDS gene observed in patients with Hunter syndrome. Russian MPS II subjects share similarities to mutations of other populations while manifesting population-unique features. The novel mutations expand the IDS gene mutation spectrum. The functional analysis of these mutations presented in this study provided a new insight on the molecular mechanisms of Hunter syndrome. MATERIALS AND METHODS Patients

IDS precursor (74 kDa) Truncated IDS (61 kDa)

40 Fig. 1. Western blot analysis of protein extracts from EBV-transformed peripheral mononuclear blood cells, derived from patients with Hunter syndrome and healthy subjects, using an IDS specific monoclonal antibody.

This study was performed according to the ethical principles of the Declaration of Helsinki. The study protocol was approved by the local Institutional Review Ethics Committee. Informed consents were obtained from the parents of all patients. A total of 17 male patients with Hunter syndrome from 16 families were registered in the Research Center for Children’s Health. The patients had primary features of MPS II including delayed growth, dwarfism, coarse faces, mental

D.A. Chistiakov et al. / Journal of Genetics and Genomics 41 (2014) 197e203

IDS activity, nmol/4 h/mL

201

P < 0.00001 P = 0.00026

380 360 340 320 300 60 40 20

p.IIe360Tyrfs*31 p.Ser142Phe

p.Arg233Gly

p.Glu430*

Control

Fig. 2. IDS activity measured by the fluorimetric enzyme assay in peripheral mononuclear blood cells derived from patients with Hunter syndrome and healthy subjects.

retardation, bone and tendon abnormalities, hyperactivity, and frequent cardiovascular (heart valve disease, cardiomyopathy) and inflammatory (rhinitis, adenoiditis) complications. Due to the presence of mental regress, 15 patients had severe disorder while the remaining two had normal mental status and mild disease.

Zhang et al. (2011). PCR reactions were carried out on GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 mL. Reaction mixture

A

Biochemical measurements IDS activity was measured in PMBCs using a fluorimetric enzyme assay, and 4-methylumbelliferyl-a-iduronate 2-sulfate was used as a substrate (Voznyi et al., 2001). Urinary GAGs were quantified using a colorimetric assay by measuring absorbance of a complex between GAGs and 1,9dimethylmethylene blue (Alonso-Ferna´ndez et al., 2010). This approach has higher sensitivity and requires significantly less sample volume compared to the alcian blue-based method of measuring GAGs directly in urine (de Jong et al., 1994). The measurements were carried out on a microplate reader Infinite M200 (Tecan, Ma¨nnedorf, Switzerland). Urine values of GAGs were expressed as a GAG/creatinine ratio (mg/mmol/ L creatinine). For each sample, biochemical measurements were performed in triplicate.

B

DNA analysis Total human DNA from dried blood spots was extracted with help of the Quick Gene DNA Tissue Kit S (AutoGen, Holliston, USA) as described in the manufacturer’s protocol. All nine exons of the IDS gene with flanking intronic sequences were amplified by PCR using primers reported by

Fig. 3. The conservation analysis of mutations p.Ser142Phe (A) and pArg233Gly (B) of IDS in vertebrates. The mutation site is highlighted in gray. Three top amino acid sequences correspond to three human IDS isoforms.

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contained 2.5 mL of 10 PCR buffer (Promega, Madison, USA), 200 nmol/L of each dNTP, 200 mmol/L of each PCR primer, 0.5 U GoTaq DNA polymerase (Promega), and 10 ng of DNA. Exons 1, 3, 5, and 6‒9 were amplified by preheating at 95 C for 5 min followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 61 C for 30 s, and extension at 72 C for 1 min, and final extension for 7 min at 72 C. Exons 2 and 9 were amplified at the same PCR conditions but annealing temperatures were 60 C and 67 C respectively. PCR products were then purified using Wizard SV96 PCR CleanUp Kit (Promega) and sequenced on an ABI 3500 DNA Sequencer (Applied Biosystems) using an ABI PRISM Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Generation of Epstein-Barr virus (EBV) cell lines and detection of IDS by immunoblotting PMBCs (106 cells/mL) from MPS II patients and normal controls were cultured in RPMI 1640 supplemented with 2 mmol/L L-glutamine (GIBCO, Carlsbad, USA), 50 mg/mL streptomycin, and 50 U/mL penicillin (Cellgro, Manassas, USA) in the presence of 10 mg/mL of phytogemagglutinin M (Merck, Whitehouse Station, USA) and a 1:3 dilution of EBV supernatant obtained from the EBV-infected B95-8 cell line (ATCC CRL#1612). Cells were cultivated in EBV-containing medium for one month to be sure that they become immortalized. The immortalized cells were then used for immunoblot analysis. IDS protein was detected by Western blot according to standard protocols (Per et al. 1985). Briefly, a total of 30 mg of protein lysate of EBV cell lines were run on a gradient (8%‒ 16%) polyacrylamide gel, which was then blotted to a polyvinylidene difluoride membrane (Millipore, Bedford, USA) for 6 h at 115V in 6% isopropanol transfer buffer. Anti-IDS p.Glu430*

C-terminal Cys84 p.IIe360Tyrfs*31

p.Ser142Phe N-terminal p.Arg233Gly Fig. 4. 3D structure of IDS. Location of catalytically essential Cys84 in the enzyme active site is indicated by an arrow. Positions of four novel mutations p.Ser142Phe, Arg233Gly, p.Ile360Tyrfs*31, and Glu430* are shown.

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