Genomic basis of mucopolysaccharidosis type IIID (MIM ) revealed by sequencing of GNS encoding N-acetylglucosamine-6-sulfatase

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1 Available online at R Genomics 81 (2003) Short Communication Genomic basis of mucopolysaccharidosis type IIID (MIM ) revealed by sequencing of GNS encoding N-acetylglucosamine-6-sulfatase Andrea Mok, Henian Cao, and Robert A. Hegele* Robarts Research Institute, London, Ontario, Canada N6A 5K8 Received 4 September 2002; accepted 5 November 2002 Abstract Mucopolysaccharidosis type IIID (MPS IIID; Sanfilippo syndrome type D; MIM ) is caused by deficiency of the activity of N-acetylglucosamine-6-sulfatase (GNS), which is normally required for degradation of heparan sulfate. The clinical features of MPS IIID include progressive neurodegeneration, with relatively mild somatic symptoms. Biochemical features include accumulation of heparan sulfate and N-acetylglucosamine-6-sulfate in the brain and viscera. To date, diagnosis required a specific lysosomal enzyme assay for GNS activity. From genomic DNA of a subject with MPS IIID, we amplified and sequenced the promoter and 14 exons of GNS. We found a homozygous nonsense mutation in exon 9 (1063C 3 T), which predicted premature termination of translation (R355X). We also identified two common synonymous coding single-nucleotide polymorphisms and genotyped these in samples from four ethnic groups. This first report of a mutation in GNS resulting in MPS IIID indicates the potential utility of molecular diagnosis for this rare condition Elsevier Science (USA). All rights reserved. Keywords: Inborn errors of metabolism; Lyases; Variation (genetics); Nonsense mutation; Metabolic disease The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases, which are caused by deficiencies of enzymes that are normally required for the catabolism of glycosaminoglycans (GAGs) [1]. One group of these diseases is known collectively as MPS III, or the Sanfilippo syndrome [1]. The clinical presentation of MPS III is heterogeneous, but key symptoms of all subtypes include progressive neurological degeneration and severe mental retardation. Affected subjects usually present between ages 2 and 6, and symptoms include hyperactivity, sleep disturbance, aggressive behavior, delayed speech onset, and regression of social and mental skills [1]. Unlike other MPS types, the somatic features of MPS III are relatively mild and can include growth retardation, facial dysmorphia, joint contractures, bone dysplasia, and hydrocephalus. There is no treatment for MPS III and life expectancy is less than 2 decades. * Corresponding author. Fax: address: hegele@robarts.ca (R.A. Hegele). MPS III results from deficiency in the activity of one of four enzymes that normally degrade heparan sulfate. These include heparan-n-sulfatase (SHSH; EC ), -Nacetylglucosaminidase (NAGLU; EC ), -glucosamine N-acetyltransferase, and N-acetylglucosamine-6- sulfatase (GNS or G6S; EC ), whose absent activity causes, respectively, MPS IIIA, IIIB, IIIC, and IIID (MIM , , , and , respectively). Like most lysosomal storage disorders, these subtypes follow an autosomal recessive pattern of inheritance. To date, 60 SHSH mutations have been reported for MPS IIIA, and 85 NAGLU mutations have been reported for MPS IIIB [2]. However, no mutations have yet been found for either MPS IIIC or MPS IIID, which each have a population frequency of 1 in 1,000,000 [3]. MPS IIID was first described more than 20 years ago: one of the first cell lines characterized came from a 7-yearold East Indian boy with typical neurological manifestations [4]. Additional cases have since been reported [5 11]. Biochemical and cellular investigations have helped to charac /03/$ see front matter 2003 Elsevier Science (USA). All rights reserved. doi: /s (02)

2 2 A. Mok et al. / Genomics 81 (2003) 1 5 Table 1 GNS genomic DNA amplification primers Exon Primer sequence Primer product size (bp) P exon 1 F: 5 -CTT CAA GGC ACG GCT TTT TA R: 5 -CAT AAA GGG CCT CAG GTG G-3 Exon 2 F: 5 -AAC AGG AAT TCT GGG CTG TG R: 5 -CCT TCC TTC AGA TCA GGC AT-3 Exon 3 F: 5 -CAA TGA TGA GAA GTC CAT CTT TCT T R: 5 -TTC ACA GAA AAC AAC GTA TAC CAA-3 Exon 4 F: 5 -TCC TGT TTC TTT CTG GTC TGG R: 5 -GAT TCT AGG CGT GAG CCA CT-3 Exon 5 F: 5 -TTC CAC TTT GTG CAC TGA CC R: 5 -TGA GCC AAC CCT GTA AGG AC-3 Exon 6 F: 5 -TGT CTG CTC ACC AGA AGA CAT T R: 5 -TGG TTG TAC TAG GGA CAG ATG C-3 Exon 7 F: 5 -TTT GAC TGT GGT GAC TTG GTG R: 5 -ATC TCT CTG CTT CTG GTG CC-3 Exon 8 F: 5 -GTG ATA CCT CAG CCA TTG CC R: 5 -GTC CTG AGG GCA AGA TCC TC-3 Exon 9 F: 5 -TTG GCT GTG GAT TAA ATG AGT G R: 5 -TTC TGC TGC TCA GCC CTA GT-3 Exon 10 F: 5 -GTG CTG TTG TCT CAT GGG C R: 5 -AAG ATT TTC TGC CCC CAA AT-3 Exon 11 F: 5 -TTC ACC ATC GTT TGT AAA GGC R: 5 -CAC CTT GCC ATG TTG TCA GT-3 Exon 12 F: 5 -TCC CCA TGG AAG GTT TTC TA R: 5 -GAA GCA CAG ATA AGA AAC ACA ACC-3 Exon 13 F: 5 -CCT CCC TTC CCC TTT AGA AAT R: 5 -AAA GCT TGA TTT TCC CAG CA-3 Exon 14 F: 5 -GTC GAG GGC TCA TGT TTT GT R: 5 -GCT GAC TGG GTT GCT TTT TC-3 F, forward; R, reverse; P, promoter. terize the activity of GNS [12,13]. Briefly, GNS desulfates the 6-sulfated N-acetylglucosamine in or linkage found in heparan sulfate or keratan sulfate, respectively [12,13]. Absence of enzyme activity leads to lysosomal accumulation and urinary excretion of heparan sulfate and N-acetylglucosamine 6-sulfate residues. While GNS cdna has been cloned and the gene localized to chromosome 12q14 [14,15], there has been no reported mutational analysis of patients with MPS IIID. We developed amplification primers for GNS and used these to find a GNS nonsense mutation in a subject with MPS IIID. The human fibroblast cell line of a MPS IIID patient (GM5093) was obtained from Coriell Cell Repositories (Camden, NJ). This cell line came from the 7-year index case of MPS IIID, who had developmental delay, mental retardation, and behavioral disturbance [4]. We cultured cells in Dulbecco s modified Eagle s medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, and 10% (v/v) heat-inactivated fetal calf serum. Genomic DNA was isolated from cells using a Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN), according to the manufacturer s instructions. To amplify coding regions and intron exon boundaries from genomic DNA, we developed primers using the genomic sequence for N-acetylglucosamine-6-sulfatase from GenBank (NT_ and NM_002076). Primer sequences for the GNS promoter and exons 1 to 14 are shown in Table 1. We designed the primer sequences to anneal at a single temperature, thus allowing use of a simple amplification reaction. However, difficulty with exon 1 required slight modification to the protocol. For exons 2 to 14, amplification conditions were 94 C for 5 min, followed by 30 cycles comprising 30 s each of denaturing at 94 C, annealing at 60 C, and extension at 72 C, and then a final extension step at 72 C for 10 min. Genomic DNA (100 ng) was used for a 60- l PCR containing 200 mm Tris HCl (ph 8.4), 500 mm KCl, 50 mm MgCl 2, 0.2 mm dntps, 0.4 M each primer, and 1.0 U Taq polymerase (Invitrogen, Burlington, Canada). For exon 1, this reaction mixture was used with the addition of 25% more Taq polymerase and 5% dimethyl sulfoxide. Amplification conditions for exon 1 were changed such that each amplification cycle lasted only 20 s and the annealing temperature was lowered to 58 C. Amplification products were run on 2% agarose gels and purified with the QIAEX II gel extraction kit (Qiagen, Inc., Valencia, CA). Purified PCR fragments were sequenced directly in both directions (ABI Prism 377; PE Applied Biosystems, Mississauga, Canada), and all sequence variations were verified by restriction enzyme analysis and/or repeat sequencing. ABI Sequence Navigator software was used to align DNA sequences. To screen for common nucleotide variants in GNS, we sequenced DNA from three unrelated, unaffected, normal

3 A. Mok et al. / Genomics 81 (2003) Fig. 1. Sequence analysis of GNS in an unaffected individual (A) and in a subject with Sanfilippo type D (B). There was a C 3 T change in exon 9, producing a premature stop codon (R355X), and the subject was homozygous for the mutation.

4 4 A. Mok et al. / Genomics 81 (2003) 1 5 Table 2 GNS SNP allele frequencies Exon SNP name Allele frequency 2 198A3G 198G in Caucasians in East Indians in Chinese in Africans T3C 1650C Absent from Caucasians, Chinese, East Indians in Africans control subjects and from one MPS IIID patient. For the 198A 3 G single-nucleotide polymorphism (SNP) in exon 2, we studied DNA samples of clinically normal subjects taken from four ethnic groups (29 European Caucasian, 28 East Indian, 30 Chinese, 30 African) to determine allele and genotype frequencies. For the 1650T 3 C SNP in exon 14, we analyzed DNA samples from 20 subjects each of the same four ethnic groups for allele and genotype frequencies. For the 1063C 3 T nonsense mutation in exon 9, we analyzed DNA samples from 200 European Caucasian and 300 East Indian subjects. Genomic DNA was amplified using the above primer set and conditions. The 198A 3 G SNP introduced a restriction endonuclease recognition site for AciI, allowing for rapid genotyping assay. Amplification of exon 2 yielded a 262-bp product. Digestion of the 198A allele produced a single fragment of 262 bp, whereas digestion of the 198G allele produced two fragments of 185 and 77 bp. The 1063C 3 T mutation in exon 9 disrupted a restriction endonuclease recognition site for TaqI. Amplification of exon 9 yielded a 307-bp product. Digestion of the 1063T allele produced a single fragment of 307 bp, whereas digestion of the 1063C allele produced two fragments, with lengths of 153 and 154 bp. All fragments were resolved by electrophoresis in 2% agarose gels. The 1650T 3 C SNP located in exon 14 did not affect a restriction endonuclease recognition site. DNA was amplified as above and was then purified using serum alkaline phosphatase (SAP) and exonuclease I (PE Biosystems). Treatment reaction mixture included 2.0 l of SAP (1.0 U/ l), 0.2 l ofexoi, 6.0 l of deionized water, and 4.0 l of PCR product. This was incubated at 37 C for 1 h and then at 72 C for 15 min to inactivate the enzyme. An additional primer was designed to terminate 1 nucleotide before the SNP: 5 -GGACTCGAAGATTTTCCAAACA- 3. The PCR template and primer were treated with an ABI Prism SNaPshot ddntp primer extension kit and analyzed on an ABI Prism 377 DNA sequencer (PE Biosystems). A novel nonsense mutation in GNS exon 9, 1063C 3 T, was found in subject GM5093, who was homozygous for the 1063T allele (Fig. 1). Codon 355 of the mutant allele specifies premature termination instead of the normal arginine (R355X). Homozygosity for 1063T was confirmed with repeated sequencing on another day and by the presence of the expected polymorphic fragment sizes after TaqI digestion of the amplified fragment containing exon 9. The 1063T allele was absent from 200 normal Caucasians and 300 normal East Indians (p , Fisher s exact test). GNS R355X was found in cell line GM5093 from a 7-year-old male patient of East Indian origin with MPS IIID who presented with mental deterioration and behavioral changes [4,16]. Assay of N-acetylglucosamine-6-sulfatase from cultured skin fibroblasts showed an intracellular accumulation of excess 35 S-containing macromolecules and 3% of mean normal enzyme activity at all ph values tested. All other enzymes assayed involved in heparan sulfate degradation were present at normal levels [16]. The mutation would predict a truncated product of 355 residues lacking the COOH-terminal 40% of the normal enzyme, which would explain the deficient residual enzyme activity seen in the biochemical assays of skin fibroblasts. Genomic DNA sequencing experiments also revealed one synonymous SNP each in exons 2 and 14, namely 198A 3 G and 1650T 3 C, respectively. Both of these SNPs had reference numbers in the National Center for Biotechnology Information SNP database ( and , respectively). Observed genotype frequencies did not deviate from the predictions of the Hardy Weinberg equation. Allele frequencies are shown in Table 2. The 1650T 3 C SNP was private to Africans in our samples. The variable and overlapping phenotypes among MPS subtypes present difficulties in early specific diagnosis. An increase in urinary concentrations of GAGs, while suggestive, is nonspecific. Also, in MPS IIIS urinary screening for GAG can have false negatives. Enzymatic assays permit a more specific diagnosis, but the deficiency of N-acetylglucosamine-6-sulfatase activity seen in MPS IIID can also result from multiple sulfatase deficiency, requiring a second assay to exclude this possibility [14 18]. As with other MPS III types, demonstrating deficiency in enzyme activity is not always conclusive. Because clinical severity does not always correlate with residual GNS activity, the ability to screen for human mutations will permit structure function studies of GNS and mutational correlation with phenotype. The only animal model of MPS IIID is the Nubian goat with caprine Sanfilippo type D. Similar to the human phenotype, the goat studied showed delayed motor development, growth retardation, and accumulation of gangliosides in the central nervous system [19]. The N-acetylglucosamine-6-sulfatase deficiency was due to a nonsense C 3 T mutation in codon 102 from GNS cdna of the affected animal [20]. This finding allowed for a convenient method of carrier detection and prenatal testing in this species [21], prompting a preliminary trial of enzyme therapy [22]. The ability to molecularly diagnose MPS IIID in humans may similarly guide the use of specific intervention strategies for this extremely debilitating phenotype. With the reagents described herein, it should also be possible to molecularly screen for carriers in affected families.

5 A. Mok et al. / Genomics 81 (2003) Acknowledgments Pearl Campbell, Ryan Dominski, and Lesley Mok provided outstanding technical assistance. Robert Hegele holds a Canada Research Chair (Tier I) in Human Genetics and a Career Investigator award from the Heart and Stroke Foundation of Ontario. This work was supported by grants from the Canadian Institutes for Health Research (MT13430), the Canadian Genetic Diseases Network, and the Blackburn Group. References [1] J.J. Hopwood, C.P. Morris, The mucopolysaccharidoses: diagnosis, molecular genetics and treatment, Mol. Biol. Med. 7 (1990) [2] G. Yongalingam, J.J. Hopwood, Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: diagnostic, clinical, and biological implications, Hum. Mutat. 18 (2001) [3] P.J. Meikle, J.J. Hopwood, A.E. Clague, W.F. Carey, Prevalence of lysosomal storage disorders, JAMA 281 (1999) [4] H. Kresse, E. Paschke, K. von Figura, W. Gilberg, W. Fuchs, Sanfilippo disease type D: deficiency of N-acetylglucosamine-6-sulfate sulfatase required for heparan sulfate degradation, Proc. Natl. Acad. Sci. USA 77 (1980) [5] R. Gatti, et al., Sanfilippo type D disease: clinical findings in two patients with a new variant of mucopolysaccharidosis III, Eur. J. Pediatr. 138 (1982) [6] P. Kaplan, L.S. Wolfe, Sanfilippo syndrome type D, J. Pediatr. 110 (1986) [7] L. Siciliano, et al., Sanfilippo syndrome type D in two adolescent sisters, J. Med. Genet. 28 (1991) [8] P.T. Ozand, et al., Sanfilippo type D presenting with acquired language disorder but without features of mucopolysaccharidosis, J. Child. Neurol. 9 (1994) [9] J. Alroy, et al., The ultrastructure of skin from a patient with mucopolysaccharidosis IIID, Acta Neuropathol. 93 (1997) [10] M.Z. Jones, et al., Human mucopolysaccharidosis IIID: clinical, biochemical, morphological and immunohistochemical characteristics, J. Neuropathol. Exp. Neurol. 56 (1997) [11] S.S. Liour, M.Z. Jones, M. Suzuki, E. Bierberich, R.K. Yu, Metabolic studies of glycosphingolipid accumulation in mucopolysaccharidosis IIID, Mol. Genet. Metab. 72 (2001) [12] C. Freeman, P.R. Clements, J.J. Hopwood, Human liver N-acetylglucosamine-6-sulphate sulphatase: purification and characterization, Biochem. J. 246 (1987) [13] C. Freeman, J.J. Hopwood, Human liver N-acetylglucosamine-6- sulphate sulphatase: catalytic properties, Biochem. J. 246 (1987) [14] D.A. Robertson, D.F. Callen, E.G. Baker, C. Phillip-Morris, J.J. Hopwood, Chromosomal localization of the gene for human glucosamine-6-sulphatase to 12q14, Hum. Genet. 79 (1988) [15] D.A. Robertson, C. Freeman, P.V. Nelson, C. Phillip-Morris, J.J. Hopwood, Human glucosamine-6-sulfatase cdna reveals homology with steroid sulfatase, Biochem. Biophys. Res. Commun. 30 (1988) [16] G.V. Coppa, et al., Clinical heterogeneity in Sanfilippo disease (mucopolysaccharidosis III) type D: presentation of two new cases, Eur. J. Pediatr. 140 (1983) [17] C. Freeman, J.J. Hopwood, Human glucosamine-6-sulphatase deficiency: diagnostic enzymology towards heparin-derived trisaccharide substrates, Biochem. J. 282 (1992) [18] W. He, et al., A fluorometric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID), J. Inherit. Metab. Dis. 16 (1993) [19] M.Z. Jones, et al., Caprine mucopolysaccharidosis-iiid: clinical, biochemical, morphological and immunohistochemical characteristics, J. Neuropathol. Exp. Neurol. 57 (1998) [20] K.T. Cavanagh, J.R. Leipprandt, M.Z. Jones, K. Friderici, Molecular defect of caprine N-acetylglucosamine-6-sulphatase deficiency. A single base substitution creates a stop codon in the 5 -region of the coding sequence, J. Inherit. Metab. Dis. 18 (1995) 96. [21] J.R. Leipprandt, K. Friderici, D.J. Sprecher, M.Z. Jones, Prenatal testing for caprine N-acetylglucosamine-6-sulphatase deficiency and sex identification, J. Inherit. Metab. Dis. 18 (1995) [22] E. Downs-Kelly, et al., Caprine mucopolysaccharidosis IIID: a preliminary trial of enzyme replacement therapy, J. Mol. Neurosci. 15 (2000)