Download Structure and Sequence of the Human Sulphamidase Gene

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
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