| Human Molecular Genetics |
Pages 787-791 |
|
Molecular defects in Sanfilippo syndrome type A
Introduction
Results
Outline of strategy for mutation analysis
Identification of mutations
Discussion
Materials And Methods
Genomic DNA, RNA and cDNA preparation
Amplification of sulphamidase coding sequences
Preparation of single-stranded DNA for sequencing from PCR products
Direct sequencing of PCR products
Allele specific oligonucleotide (ASO) analysis
Acknowledgements
ABBREVIATION
References
Molecular defects in Sanfilippo syndrome type A
Molecular defects in Sanfilippo syndrome type A
Lianne Blanch, Birgit Weber, Xiao-Hui Guo, Hamish S. Scott+ and John J. Hopwood*
Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, Australia
Received December 26, 1996; Revised and Accepted February 25, 1997
Sanfilippo A syndrome (mucopolysaccharidosis type IIIA, MPS-IIIA) is an autosomal recessive neurodegenerative disorder due to an enzymatic defect of the lysosomal enzyme sulphamidase (EC 3.10.1.1) required for the degradation of heparan sulphate. In this study, molecular defects in the sulphamidase gene of MPS-IIIA patients were investigated in a group of 10 patients of Australian and American origin. The entire coding region of the sulphamidase gene was RT-PCR amplified and one polymorphism (R456H), four novel mutations (S66W, R245H, E447K, 1307 del 9) and one previously described mutation (1284 del 11) were identified by direct PCR sequencing. R245H was present in six patients including one severely affected homozygote. In three of the other patients with R245H, second mutant alleles were identified as S66W, 1284 del 11 and E447K, respectively. S66W was also detected in another patient where the other mutant allele remains undefined. In addition, 1307 del 9 was also detected in a patient with the other mutant allele remaining undefined. Allele specific oligonucleotide hybridisation was used to determine the incidence of these in a population of 26 MPS-IIIA patients (Australian and American) and 60 normal controls (Australian). R245H represented 27% (14/52 alleles) in this total patient population, while the other three changes ranged from 1.9 to 9.6% (1-5 of 52 alleles). The sequence variant, R456H, was shown to be polymorphic as it was present in 55% of normal and 38% of patient alleles. The total combined incidence of these five is 46% of alleles. This is the first study of the molecular defects in MPS-IIIA patients and will greatly assist the development of molecular analysis for MPS-IIIA patients and studies concerned with genotype to phenotype relationships.
Mucopolysaccharidosis type III (MPS-III, eponym: Sanfilippo syndrome) is inherited as an autosomal recessive disease in humans and is caused by the accumulation of undegraded heparan sulphate in lysosomes. There are four recognised subtypes (MPS-IIIA, B, C and D) that result from deficiencies of different enzymes involved in the lysosomal degradation of heparan sulphate. Sanfilippo patients develop severe central nervous system degeneration resulting in progressive dementia often combined with delayed speech, sleep disturbance, hirsutism, diarrhoea, hyperactivity and aggressive behaviour (1 ). Clinical onset in severely affected patients usually occurs following 2-3 years of apparently normal development. Mild skeletal pathology, joint stiffness and hepatosplenomegaly are often present in older patients. MPS-III patients may present and develop within a wide spectrum of clinical severity. Clinically mild patients can be missed because of the relatively less severe clinical presentation and a false negative test for elevated heparan sulphaturia obtained using some tests (1 ). The incidence of MPS-III in The Netherlands has been estimated at 1:24 000 (2 ) and in Australia at 1:55 000 (unpublished observations).
Genes coding for the enzymes involved in MPS-IIIA, IIIB and IIID have been cloned (3 -7 ) leaving the MPS-IIIC gene to be characterised. In this paper we describe the first study of the molecular defects in the sulphamidase gene leading to MPS-IIIA and the incidence of these five mutations in a group of 26 MPS-IIIA patients.
Ten MPS-IIIA patients chosen for investigation for molecular defects in their MPS-IIIA gene and confirmed to have deficient sulphamidase activities (10 ) in cultured fibroblasts are listed in Table 2 . Oligonucleotide primers were designed to amplify from cDNA and produce three overlapping fragments (1, 2 and 3) containing the entire coding region of sulphamidase. In all 10 patients the size of these fragments was indistinguishable from those obtained from normal controls. Each fragment was purified using Dynabeads (Dynal), with single-stranded products then subjected to direct PCR sequencing using internal primers (Table 1 ).
Table 1
Oligonucleotide primers for amplification of the sulphamidase gene
| Region amplifieda |
Primer no.b |
Primer sequence |
| 10-616 bp |
ns21 |
5'-GCGCGAATTCGCCATGAGCTGCCCCGTGCCCGCCT-3' |
| (fragment 1) |
ns79 |
5'-TCTCTCCGTTGCCAAACTTCTCACAGAAGG-3'c |
| 498-1030 bp |
ns11 |
5'-AAATTCCTGCAGACTCAGGATGACCGGCCT-3' |
| (fragment 2) |
ns80 |
5'-TCTTCGAGCCAAAGATGGCGTAGCTGGGGT-3'c |
| 875-1564 bp |
ns32 |
5'-CGGGCACTGCTGAACCCTTACTGGTGTCAT-3'c |
| (fragment 3) |
ns64 |
5'-TCTGGGACATGCCTGGGATGTGTG-3' |
acDNA sequence from reference 3.bPrimers ns1-50 are in the positive orientation while ns51-100 are in the negative orientation cBiotinylated primer.
Table 2
List of patient genotypes and phenotypes
| Patient |
Allele 1 |
Allele 2 |
Presence of
R456Ha |
Sourceb |
Age of
onset (years) |
| 2779 |
nd |
nd |
absent |
Australian |
5 |
| 2804 |
S66W |
R245H |
Heterozygous |
Australian |
5 |
| 3299 |
R245H |
nd |
Heterozygous |
Australian |
4 |
| 3348 |
R245H |
R245H |
Homozygous |
Australian |
3 |
| 3469 |
R245H |
1284 del 11 |
Heterozygous |
Australian |
4 |
| 4105 |
R245H |
nd |
Heterozygous |
GM 06110c |
6 |
| 4106 |
E447K |
R245H |
Heterozygous |
GM 00879c |
3 |
| 4107 |
1307 del 9 |
nd |
absent |
GM 01094c |
5 |
| 4112 |
S66W |
nd |
absent |
GM 00629c |
10 |
| 4113 |
nd |
nd |
absent |
GM 00643d |
3 |
aPolymorphism.
bCountry of origin of patient.
cUSA-patients from NIGMS Human Genetic Mutant Cell Repository.
nd, no changes detected.
A summary of the confirmed mutations and genotypes is presented in Table 2 .
Fragment 1, containing the first 500 bp of the sulphamidase coding sequence, yielded only one change, a C -> G substitution at nucleotide position 209 to change serine at residue 66 to a tryptophan (S66W). This change was found in two alleles of the 10 patients initially sequenced (Table 2 ). A second base pair substitution G -> A at nucleotide position 746 changed an arginine residue to a histidine (R245H) in fragment 2. This missense mutation was present in seven of 20 alleles from six of the 10 patients initially studied, including one patient homozygous for R245H (Table 2 ). Sequencing of fragment 3 (Table 1 ) revealed four changes. The deletion of 11 bp at nucleotide position 1284 results in the last base of amino acid 424 changing from C to G to alter the original tyrosine to a stop codon leading to a 78 amino acid shortened sulphamidase. The deletion of 9 bp at nucleotide position 1307 leads to the last two bases of the codon for amino acid 432 being changed from AC to GC leading to the substitution of a tyrosine by a tryptophan. In addition to this Y432W substitution, the 9 bp deletion results in the deletion of three amino acids (R433, A434, R435) before the normal reading frame is restored at W436. The 11 bp deletion was only found in one allele of the 10 patients. Two base pair substitutions, both G -> A (at nucleotide positions 1351 and 1379, respectively) lead to amino acid changes E447K and R456H, respectively. The 11 and 9 bp deletions, and E447K were each only found in one allele of the 10 patients by this sequence analysis. The R456H sequence variant was found in six of the 10 patients with it being homozygous in one patient.
All sequence variants observed, especially those that were not clearly pathogenic (i.e. S66W, R245H, E447K and R456H) were further analysed by ASO analysis of 120 alleles from a group of 60 unaffected Australians, and 52 MPS-IIIA alleles from 26 unrelated Australian and American MPS-IIIA patients (including the 10 originally used in this study). The sequence variant, R456H, was thus shown to be polymorphic as it was present in 55% of normal and 38% of patient alleles, including unaffected homozygotes. By this analysis, all other sequence variants described here are likely to be pathogenic as they were not detected in the 120 normal alleles. Indeed, R245H was present in 14 of 52 MPS-IIIA alleles (26.9%), S66W present in five of 52 alleles (9.6%), E447K in one allele (1.9%), 1307 del 9 in two alleles (3.9%) and a previously described deletion (3 ), 1284 del 11, in two alleles (3.9%). E447K is a non-conservative substitution causing a charge change and, therefore, is likely to be responsible for the dysfunction and/or instability of the resultant sulphamidase. However, as only one E447K substitution was identified in 52 MPS-IIIA mutant alleles and none in 120 alleles from unaffected individuals, it is still possible, although unlikely, that this substitution is not disease causing.
The recent isolation of a full-length cDNA encoding sulphamidase (3 ) permitted the characterisation of molecular defects causing the enzyme deficiency in patients with a Sanfilippo phenotype. In this study, using cultured skin fibroblasts from 10 patients, extraction of total RNA, subsequent reverse-transcription, amplification and sequencing of the sulphamidase cDNA revealed one deletion (1307 del 9), three missense mutations (S66W, R245H and E447K) and one polymorphism (R456H). Twelve alleles of the 20 mutant alleles were detected by direct sequencing of RT-PCR products. The nine alleles, not defined,may be within untranslated regions not amplified in this analysis, control regions orintronic sequence (for example splice site mutations or other mutations affecting the stability of the mRNA) and therefore would have been missed by this method which relies on the direct sequencing of RT-PCRproducts. Although each sequencing gel was thoroughly checked, it is possible that some changes within the coding region could have been missed. The genomic structure and sequence of the sulphamidase gene was not known when this study commenced. It has now been defined (11 ) and further analysis of these patient's alleles by sequencing and other techniques such as SSCP of PCR products from patient genomic DNA will enable us to determine why only 12/20 alleles were determined in this study.
The ASO analysis of 26 unrelated Australian and American MPS-IIIA patients,which includes 10 patients originally studied,revealed that R245H, S66W, E447K, 1307 del 9 and a previously described deletion (3 ), 1284 del 11, were present in 26.9, 9.6, 1.9, 3.9 and 3.9% of alleles, respectively. Seven of 26 patients had fully defined genotypes. None of these changes were present in 120 alleles screened from an unaffected Australian population. The total combined incidence of these fivemutationsin this American/Australian patient group is 46.4% of alleles accounting for the expected 21.5% of fully defined genotypes.
With 1284 del 11 (3 ) and 1307 del 9 described in this study, there are two described deletions in the sulphamidase gene. To understand the mechanisms causing the 9 bp deletion described in this paper and the 11 bp deletion previously described (3 ), the genomic structure was examined for possible tandem and inverse tandem repeats of the sequences deleted. None were identified. Also there is no exon or intron boundary in the region (11 ). Thus at this time we are unable to give a mechanism for these deletions. All mutations (S66W, R245H, E447K and the two deletions) can be considered to be pathological because they are found in a number of unrelated MPS-IIIA patients but not in 120 normal control alleles. S66, although not conserved among the 13 sulphatases on the database, is within three amino acids of a region (CTPSR), highly conserved in all mammalian sulphatases and thus, substitution of the bulkier tryptophan residue would be expected to disrupt a structure likely to be at the active site of all sulphatases (12 ). Again, R245 is not conserved amongst sulphatases, however the high (26.9%) incidence of R245H mutation in the analysed group of MPS-IIIA patients makes this change important in studies concerned with the relationship between genotype and clinical phenotype. Thus far, all 14 R245H alleles detected in this patient population also have the R456H substitution. Although this may suggest a possible founder effect for R245H, the high incidence of the R456H substitution in the normal population does not enable this conclusion to be firmly made. We are unable to predict the combined effect of R245H and R456H substitutions on sulphamidase activity. R456H without R245H does not appear to cause a Sanfilippo clinical phenotype and we have not observed low sulphamidase activities in our unaffected populations which would be expected to contain ~7-8% R456H homozygotes. It is unlikely, therefore, that the R456H substitution modifies the clinical severity of patients with R245H genotypes. However, to be sure, expression of R245H with and without R456H is required. Two unrelated patients homozygous for R245H were severely affected presenting at 1.5 and 2.5 years with developmental delay and coarse facial features, hirsutism and hepatosplenomegaly. Both patients had marked heparan sulphaturia (13 ). The patients with the R245H/S66W or R245H/1284 del 11 genotypes both presented early with developmental delay and behavioural disturbances and developed severe clinical phenotypes by 9-10 years of age.
This is the first study of the molecular defects in MPS-IIIA patients. As more complete genotypes are defined, and more complete clinical descriptions of patients ascertained, thiswill greatly assist the development of molecular analysis for MPS-IIIA patients and studies concerned with genotype to phenotype relationships. The work described herealso provides PCR based tests for the MPS-IIIA mutations described, which could be used in prenatal diagnosis or carrier detection.
Skin fibroblasts from American MPS-IIIA patients were obtained from NIGMS Human Genetic Mutant Cell Repository. Skin fibroblasts were obtained from Australian MPS-IIIA patients diagnosed by the Australasian Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders in the Department of Chemical Pathology at the Women's and Children's Hospital, Adelaide.
Normal and MPS-IIIA skin fibroblasts were cultured and genomic DNA was prepared as previously described (8 ). Total RNA was prepared as previously described (9 ). cDNA was prepared from skin fibroblasts by adding RNA (5 [mu]g) to a reaction mix containing 300 ng of random octamers and sterile water which was heated at 70oC for 10 min and then immediately placed on ice. To this mixture was added Moloney murine leukemia virus (Mo-MLV) reverse transcriptase buffer (BRL), deoxynucleotides to 0.5 mM (Boehringer Mannheim), DTT to 0.01 M and 200 U Mo-MLV reverse transcriptase (BRL) to a final reaction volume of 50 [mu]l. Incubation at 37oC for 1 h was followed by hydrolysis of the RNA by the addition of 5 [mu]l 3 M NaOH and further incubation at 37oC for 20 min. The NaOH was neutralised by the addition of 1.25 [mu]l 10.3 M HCl, and the cDNA was precipitated and resuspended in 50 [mu]l of water. Each PCR used 5 [mu]l of cDNA (14 ).
Table 3
Allele specific oligonucleotide primers, washing temperatures and primers for confirmation of sequence variants in the sulphamidase gene
Mutation/
polymorphism |
Region of
amplification |
PCR
producta |
ASO primer sequencesb |
ASO hybridisation
washing temp |
| R245H |
Exons 5c and 6 |
ns38/87 |
normal 5'-GTCGGCCGCATGGACC-3' |
|
| |
|
|
mutant 5'-GTCGGCCACATGGACCA-3' |
65oC |
| S66W |
105-258 bpd |
ns36/84 |
normal 5'-CTTCACCTCGGTCAGC-3' |
55oC |
| |
Exon 2 |
|
mutant 5'-CTTCACCTGGGTCAGC-3' |
55oC |
| E447K |
1082-1564 bp |
ns 16/64 |
normal 5'-ACCCCACGAGACCCAG-3' |
59oC |
| |
Exon 8 |
|
mutant 5'-ACCCCACAAGACCCAG-3' |
55oC |
| 1307 del 9 |
1082-1564 bp |
ns 16/64 |
normal 5'-TACTACCGGGCGCGCT-3' |
|
| |
Exon 8 |
|
mutant 5'-TTACT[and]ACTGCTGGGAG-3' |
50oC |
| |
|
|
del 9 |
|
| R456H |
1082-1564 bp |
ns 16/64 |
normal 5'-GACCCGCGCTTTGCTC-3' |
60oC |
| |
Exon 8 |
|
mutant 5'-GACCCGCACTTTGCTC-3' |
60oC |
aOligonucleotide combinations used to identify sequence variants are as follows:ns 16 5'-TCTGGGCCACCGTCTTTGGCAGCC-3';
ns 36 5'-TGACGGAGGCTTTGAGAGTGGCGC-3';
ns 37 5'-GAGGGCAGCTCCTGTGTGCTGAGG-3';
ns 38 5'-ACCTTGGCAATTAACCTCCTTCCG-3';
ns 64 5'-TCTGGGACATGCCTGGGATGTGTG-3';
ns 84 5'-GGGCAGGCCAGTGAGGAGGCTGGC-3';
ns 87 5'-CTCACCCACATTATGCCGTGACCT-3'.
bUnderlined bases signify those which are changed in the mutant sequence.
cGenomic sequences from reference 11.
dcDNA sequence from reference 3.
Table 4
Oligonucleotide primers for sequencing of PCR amplified product
| Primer no. |
Primer sequence |
Priming at
nucleotidesa |
| ns5 |
5'-AATGCCTTCACCTCGGTCAGCAGC-3' |
196-219 |
| ns10 |
5'-GCCAAGTCAGCGAGGCCTACGTGA-3' |
929-952 |
| ns12 |
5'-TACACCACCGTCGGCCGCATGGAC-3' |
730-753 |
| ns17 |
5'-TTCAAGATGCCCTTTCCCATCGAC-3' |
1186-1209 |
| ns70 |
5'-CTCCGCACCTTGTCGAAGGAGTTG-3' |
306-329 |
| ns72 |
5'-GAAGATCACCAGTGTGTCGTTCAGGACACC-3' |
793-822 |
acDNA sequence from reference 3.Primers ns1-50 are in the positive orientation and ns51-100 are in the negative orientation.
The coding region was amplified from cDNA in three overlapping fragments of ~500 bp using one biotinylated and one unbiotinylated oligonucleotide for each amplification(for sequence of primers see Table 1 ).After preparation of single stranded DNA for sequencing, as described below,each fragment was sequenced using two internal oligonucleotide primers(Table 3 ). Oligonucleotides used for amplificationfor either confirmation of the sequence variants observed, or to perform ASOare also listed in Table 3 .
PCR reactions were carried out in 1* Biotech PCR buffer (67 mM Tris-HCl pH 8.8, 16.6 mM (NH4)2SO4, 0.45% Triton X-100, 0.2 mg/ml gelatin) with 2.5 mM MgCl2, 400 [mu]M dNTPs, 1 U Taq polymerase (Boehringer Mannheim), 200 ng of each primer and 10% DMSO (all except ns21/79 and ns41/88), in a total volume of 50 [mu]l. All PCRs were 40 cycles, with an initial denaturation of 2 min at 94oC, then 15 s at 94oC, followed by 15 s at 94oC, 30 s at 55oC for genomic or 60oC for cDNA templates and 45 s at 72oC. PCRs were carried out on a Hybaid OmniGene thermal cycler using the tube control option.
The protocol was performed according to the manufacturer's instructions (Dynal, Solid-Phase DNA Sequencing, Hand Book, Second Edition). Briefly, as described above, in each of the three RT-PCRs one of the oligonucleotides was biotinylated (Table 1 ). The PCR products were purified onQiaquick PCR spin columns (Qiagen) to remove unincorporated primers and nucleotides and the DNA was subsequently immobilised onto streptavidin coated magnetic beads (Dynabeads Product No 112.05) by adding 40 [mu]l of washed bead suspension to 40 [mu]l purified PCR product and incubating for 15 min at 21oC while keeping the beads suspended by tipping the tube. The DNA/bead complex was pelleted and held to the tube wall by a magnet which allowed removal of the supernatant and washing of the pellet in 2* BW buffer (10 mM Tris-HCl pH 7.5, 1.0 mM EDTA, 2.0 M NaCl). Alkali denaturation (8 [mu]l 0.1 M NaOH for 2 min) of the DNA/bead pellet released the non-biotinylated strand into the supernatant which was collected with the magnet and neutralised with the addition of 4 [mu]l 0.2 M HCl and 1 [mu]l Tris-HCl pH 8.0. The biotinylated strand remained bound to the beads and was further washed before being taken up in 20 [mu]l H2O. The two strands were then sequenced separately with the appropriate sequencing oligonucleotides (Table 4 ).
Sequencing was performed in microtitre trays on a Hybaid OmniGene thermocycler (simulated tube calibration factor 400) using the fmol DNA Sequencing System (PromegaQ4100) via end-labelling of internal primers with [[gamma]-32P]dATP according to the manufacturer's instructions. All sequencing reactions were 30 cycles, with the time of each step being the same as for the PCR reaction but the temperature of annealing 2-5oC higher. Most of the RT-PCR products were sequenced in only one direction, with the opposite direction being sequenced only when necessary to confirm whether an aberrant band on a sequencing gel was actually a change in the DNA sequence.
ASOs were performed as outlined previously (8 ) to screen unrelated MPS-IIIA patients for the presence of the common and other mutations, as well as to screen normal individuals to determine whether these changes were polymorphisms rather than mutations. Genomic DNA from patients and normals was used to amplify the region of each mutation (see Table 3 ). The amplification products were then blotted on to nylon membrane (GeneScreen Plus) and probed with oligonucleotide primers specific for the normal or mutant sequence basically as described (8 ) but with the hybridisation and washing conditions as stated in Table 3 .
We thank Drs Don Anson and Phillip Morris for helpful discussions. This work was supported by grants from the National Health and Medical Research Council of Australia, the Women's and Children's Hospital Research Foundation and a Raymond A. Bryan IV Fellowship from the American MPS Society, Inc. BW was supported by a long term fellowship of the Human Frontier Science Program, Strasbourg.
MPS-IIIA, mucopolysaccharidosis type IIIA or Sanfilippo A syndrome.
1 Neufeld, E.F. and Muenzer, J. (1995) The mucopolysaccharidoses. In Scriber, C. R., Beaudet, A. L., Sly, W. S. and Valle, D. (eds) The Metabolic and Molecular Basis of Inherited Disease. 7th edn New York, McGraw-Hill, pp 2465-2494.
2 van de Kamp, J.J.P., Niemeijer, M.F., von Figura, K. and Giesberts, M.A.H. (1981) Genetic heterogeneity and clinical variability in the Sanfilippo syndrome (Types A, B and C). Clin. Genet., 20, 152-160.
3 Scott, H.S., Blanch, L., Guo, X-Hui, 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 Genet., 11, 465-467.
4 Weber, B., Blanch, L., Clements, P.R., Scott, H.S. and Hopwood, J.J. (1996) Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis IIIB). Hum. Mol. Genet., 5, 771-777. MEDLINE Abstract
5 Zhao, H.G., Li, H.H., Bach, G., Schmidtchen, A. and Neufeld, E.F. (1996) The molecular basis of Sanfilippo syndrome type B. Proc. Natl. Acad. Sci. USA, 93, 6101-6105.
6 Robertson, D.A., Freeman, C., Morris, C.P. and Hopwood, J.J. (1992) A cDNA clone for glucosamine-6-sulphatase reveals differences between aryl-and non-aryl sulphatases. Biochem. J., 288, 539-544. MEDLINE Abstract
7 Robertson, D.A., Freeman, C., Nelson, P.V., Morris, C.P. and Hopwood, J.J. (1988) Human glucosamine 6-sulphatase cDNA reveals homology with steroid sulphatase. Biochem. Biophys. Res. Comm., 157, 218-224. MEDLINE Abstract
8 Scott, H.S., Litjens, T., Nelson, P.V., Brooks, D.A., Hopwood, J.J. and Morris, C.P. (1992) [alpha]-L-Iduronidase mutations (Q70X and P533R) associated with a severe Hurler phenotype. Hum. Mutat., 1, 333-339. MEDLINE Abstract
9 Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156-159. MEDLINE Abstract
10 Hopwood, J.J. and Elliott, H. (1982) Diagnosis of Sanfilippo A syndrome by estimation of sulphamidase activity using a radiolabelled tetrasaccharide substrate. Clin. Chim. Acta, 123, 241-250. MEDLINE Abstract
11 Karageorgos, L. E., Guo, X-H., Blanch, L., Weber, B., Anson, D.S., Scott, H.S. and Hopwood, J.J. (1996) Structure and sequence of the human sulphamidase gene. DNA Res., 3, 269-271.
12 Schmidt, B., Selmer, T., Ingendoh, A. and von Figura, K. (1995) A novel amino acid modification in sulfatases that is defective in multiple sulfatase deficiency. Cell, 82, 271-278. MEDLINE Abstract
13 Hopwood, J.J. and Harrison, J.R. (1982) High-resolution electrophoresis of urinary glycosaminoglycans. Anal. Biochem., 119, 120-127 MEDLINE Abstract
14 Scott, H.S., Anson, D.A., Orsborn, A.M., Nelson P.V., Clements, P.R., Morris, C.P. and Hopwood, J.J. (1991) Human [alpha]-L-iduronidase: cDNA isolation and expression. Proc. Natl. Acad. Sci. USA, 88, 9695-9699. MEDLINE Abstract
*To whom correspondence should be addressed. Tel: +61 8 8204 7293; Fax: +61 8 8204 7100; Email: jhopwood@ medicine.adelaide.edu.au
+Present address: Department of Genetics and Microbiology, University of Geneva Medical School, Geneva, Switzerland
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