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DNA RESEARCH 3, 269-271 (1996) Short Communication Structure and Sequence of the Human Sulphamidase Gene Litsa E. KARAGEORGOS,* Xiao-Hui Guo, Lianne BLANCH, Birgit WEBER, Donald S. ANSON, Hamish S. ScOTT,t and John J. HoPWOOD Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia (Received 30 July, 1996) Abstract Sanfilippo A syndrome (MPS-IIIA) is a mucopolysaccharide lysosomal storage disorder caused by a deficiency in the lysosomal enzyme, sulphamidase (EC 3.10.1.1), which is required for the degradation of heparan sulphate. A genomic clone containing the entire sulphamidase gene was isolated from a chromosome 17-specific gridded cosmid library. The structure of the gene and the sequence of the exon/intron boundaries and the 5' promoter region were determined. The sulphamidase gene is split into 8 exons spanning approximately 11 kb. Key words: sulphamidase; Sanfilippo A syndrome; lysosomal storage disorder Sulphamidase (heparan TV-sulphatase; EC 3.10.1.1) is a lysosomal exohydrolase required for the degradation of heparan sulphate in mammalian cells.1'2 In humans, a deficiency of sulphamidase function leads to the lysosomal storage of partially degraded glycosaminoglycans, causing the clinical disorder Sanfilippo A syndrome,3 otherwise known as mucopolysaccharidosis type IIIA (MPSIIIA). MPS-IIIA is inherited as an autosomal recessive disease with considerable variation in severity of clinical phenotypes.2 The disease pathology of MPS-IIIA is characterised by severe central nervous system (CNS) degeneration, unique among the MPS conditions.2 Clinical features can include delayed development (particularly speech), hyperactivity, aggressive behaviours, sleep disturbances, coarse hair, hirsutism and diarrhoea. These clinical features are progressive and usually appear between 2 and 6 years with patients appearing normal prior to onset. Mild skeletal pathology, hepatosplenomegaly and joint stiffness are found mostly in older patients. Diagnosis of less severe MPS-IIIA can be missed due to the relatively mild somatic and radiographic features, and to false negative screening for elevated heparan sulphaturia in urinary tests.2 The sulphamidase cDNA has recently been cloned and sequenced, and the chromosomal localisation of the sulphamidase gene has been reported as being 17q25.3.4 In * t t Communicated by Mituru Takanami To whom correspondence should be addressed. Tel. +61-8-2046682, Fax. +61-8-204-7100, E-mail: [email protected] Present address: Division of Medical Genetics, University of Geneva Medical School, Geneva, Switzerland. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. U60107, U60108, U60109, U60110 and U60111. this report, we describe the isolation of the sulphamidase gene and the determination of the sequence of the intron-exon boundaries. These partial sequence data are sufficient to allow definition of mutations in Sanfilippo syndrome patients from genomic DNA and to explore diagnostically useful polymorphisms in this region. The cDNA clone that contained the full coding sequence of sulphamidase, ANS6,4 was used to isolate 8 genomic clones from a gridded chromosome-17 specific cosmid ICRF library.5 Hybridization and restriction enzyme analysis revealed that the cosmid clone ICRFcl05HO237 contained the entire 11kb gene. The sequences surrounding the exon-intron boundaries were determined by direct polymerase chain reaction cycled sequencing using internal oligonucleotides to prime either on subclones of positive fragments or directly on the cosmid DNA. The size of each of the introns was determined by estimating their sizes by PCR between the exons. This analysis revealed that the sulphamidase gene contained a total of 8 exons that spanned approximately 11 kb (Fig. 1). It was found (Table 1) that the intron boundaries were flanked by highly conserved consensus splicing signals.6 Multiple Tissue Northern Blots have shown that the sulphamidase gene produces three major mRNA transcripts (3.1, 4.3, and 7.1 kb), with the 3.1-kb transcript being the predominant mRNA species in most tissues.4 No consensus polyadenylation site was found in the 3'untranslated region of the cDNA, although an alternative site has been proposed.4 Four copies of a 57-bp tandem repeat have been identified in the 3' UTR. Ten normal genomic DNA samples were analysed for the presence of these repeats, and these repeats were not found to be polymorphic. [Vol. 3, Human Sulphamidase Gene 270 8 88 bp 161 bp - ^ 151 bp 106 82 157 bp 233 bp 560 bp * -3.5 kb -2.2 kb 264 bp -500bp 284 bp -1.6 kb 1.06 kb -1.1kb 3'UTR -11 kb Figure 1. Structure of the human sulphamidase gene. Table 1. Exon-Intron sizes and nucleotide sequence of the exon-intron junctions in the human sulphamidase gene. Exon Size (bp) cDNA positionaa MOO 1 1-100 161 100-26 2 105 262-367 3 4 150 368-518 519-673 154 5 674-757 83 6 758-961 7 233 9628 >544 a Intron no. Size (kb) 5' splice donor 1 CTC CTC G gtgagtgccggc ~3.5 2 ~2.2 CTG CCC CAG gtgaggtgcaag 3 0.264 CGC ACA G gtgaggaccccg 4 ~0.5 GAT GAC CG gtatgagtcggg 0.284 5 GAC GTG CTG gtaggacggccc 6 GAC CAA G gtgggcttgcag 1.06 7 CTC CTA G gtatgcctttgt 3' splice acceptor cacctcacgcag CG GAT GAC ctttgctggcag CAT CAG AAT cttcccgcccag GC ATC ATC cctttccgttcag G CCT TTC tgcctgccccag GTG CCT TAC ccctcaccacag GA GTT GGA cgtcccttccag AC CTC ACG ' Numbering according to Scott et al.4 The genomic organization of sulphamidase does not appear to be similar to other sulphatases. The characterization of the genomic structures of the ARSD and ARSE genes were recently reported.7 These two new sulphatase genes showed perfect conservation of the intronexon junctions, with the splicing occurring at exactly the same position in the two genes. This conserved genomic organization was also shared by steroid sulphatase (STS), but was completely different from that of all the other reported sulphatase genes, including sulphamidase, as shown here. Furthermore, it is suggested that the ARSD, ARSE, and STS genes originated from a common ancestral gene through a series of duplication events which occurred recently during evolution,7 while it is likely that other sulphatases evolved due to mutation and selection for function over time. Having defined the structure and partial sequence of the sulphamidase gene, including the intron-exon boundaries, and defined a series of oligonucleotides for use in priming PCR reactions and for sequencing, we are now in a position to identify mutations in MPS-IIIA. The availability of the partial sequence of the sulphamidase gene should improve the ability to diagnose MPS-IIIA by direct mutation detection and the use of linked polymorphisms. Acknowledgments: This work was supported by grants from the National Health and Medical Research Council of Australia, the Women's and Children's Hos- pital Research Foundation and HSS was supported by the Raymond A Bryan IV Fellowship from the American MPS Society Inc. References 1. Hopwood, J. J. 1989, In: Lane, D. W. and Lindahl, U. (eds) Heparin: Chemical and Biological Properties, Clinical Applications, Arnold, London, pp. 190-229 2. Neufeld, E. F. and Muenzer, J. 1995, In: Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D. (eds) The Metabolic and Molecular Bases of Inherited Disease, 7th ed, McGraw-Hill, New York, pp. 2465-2494. 3. Sanfilippo, S. J., Podosin, R., Langer, L. O., Jr., and Good, R. A. 1963, Mental retardation associated with acid mucopolysacchariduria (heparitin sulfate type), J. Pediat, 631, 837-838. 4. Scott, H. S., Blanch, L., Guo, X. H., Freeman, C, Orsborn A., Baker, E., Sutherland, G. R., Morris, C. P., and Hopwood, J. J. 1995, Cloning of the sulphamidase gene and identification of mutations in Sanfilippo A syndrome, Nature Gen., 11, 1-6. 5. Lehrach, H., Drmanac, R., Hoheisel, J., Larin, Z., Lennon, G., Monaco, A. P., Nizetic, D., Zehetner, G., and Poustka, A. 1990, In: Davies, K. E. and Tilghman, S. M. (eds) Genome Analysis Volume 1: Genetic and Physical Mapping, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 39-81. 6. Penotti, F. E. 1991, Human pre-mRNA splicing signals, J. Theor. BioL, 150, 385-420. No. 4] L. E. Karageorgos et al. 7. Meroni, G., Franco, B., Archidiacono, N., Messali, S., Andolfi, G., Rocchi, M., and Ballabio, A. 1996, Characterization of a cluster of sulfatase genes on Xp22.3 sug- 271 gests gene duplications in an ancestral pseudoautosomal region, Hum. Mol. Genet, 5, 423-431.