© 1992 Oxford University Press
Human Molecular Genetics, Vol. 1, No. 9
755-757
Mutation R468W of the iduronate-2-sulfatase gene in mild Hunter syndrome (mucopolysaccharidosis type II) confirmed by in vitro mutagenesis and expression Paul L.Crotty14, Stephen E.Braun24, Rose Ann Anderson34 and Chester B.Whitley23-4* 1
Department of Laboratory Medicine and Pathology, 2Department of Genetics and Cell Biology, department of Pediatrics and 4lnstitute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA
Received August 12, 1992; Revised and Accepted October 27, 1992
appears to have a relatively mild form of Hunter syndrome not associated with neurological impairment. For direct sequencing, RT-PCR products of this patient were purified on a silica matrix (Geneclean, Bio 101, La Jolla, CA) with subsequent centrifugal size exclusion chromatography. Using a series of primers (Fig. 1), direct DNA sequencing (15) of the products was performed with Sequenase Version 2.0 (USB, Cleveland, OH) according to the manufacturers recommendations with modifications: 1) double-stranded template was denatured by boiling and annealed with the primer by snap-cooling on dry ice; 2) 10% dimethylsulfoxide was included in the annealing reaction; 3) labelling reactions were performed on ice; 4) a primertemplate molar ratio of ^ 10:1 was used. For this patient, the entire coding region was found to be identical to the normal sequence (5) except for a single base substitution c 1402 —T replacing arginine468 with tryptophan, R468W. This change occurs in an area of predominantly hydrophilic amino acid residues thus reducing the hydrophobicity of the region. Prediction of secondary protein structure by the Gamier—Robson model (16) indicated that this amino acid, situated between two prolines, is interposed at a turn between two predominantly /3-sheet regions. To determine the effect of the R468W mutation on enzyme activity, GP+E86 cells (17) were transfected with retrovirus expression vectors containing no IDS sequence (LXSN), the normal IDS cDNA (L2SN) or the mutant cDNA (L2SN-R468W). For construction of L2SN, the 5'-end of the IDS coding region (extending to the EcoRI site at +1427) was obtained from the reference plasmid pc2S15 (5). The 3'-end of the coding region was derived by RT-PCR using oligonucleotide primers IDS3 and EDS2 (Fig. 1), the latter designed with a 2-base change to incorporate a BamlU site; a fragment of this product, extending from the EcoRI site at +1427 to the BamHl site in IDS2, was cloned into pBluescript II (pBS, Stratagene, La Jolla, CA). The two cloned fragments were combined by sequential ligation into the cloning site of pLXSN (18) to generate pL2SN. A plasmid containing the mutant sequence (pL2SN-R468W) was also constructed. Using the patient's RNA as template, RTPCR product was obtained with IDS2 and IDS3 (Fig. 1). A 299 bp fragment, extending from the HindUl site at +1128 to the £coRI site at +1427, was cloned into pBS yielding pBSHE299-R468W. The integrity of the insert and the C I 4 O 2 -T mutation were confirmed by sequencing the entire 299 bp and flanking regions. The 0.9 kb HindSl fragment of pL2SN was
* To whom correspondence should be addressed at: Box 446 UMHC, 420 Delaware Str SE, Minneapolis, MN 55455, USA
Downloaded from http://hmg.oxfordjournals.org/ at University of Manchester on April 8, 2015
Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is an X-linked disorder of glycosaminoglycan (GAG) metabolism resulting from deficiency of lysosomal iduronate-2-sulfatase (IDS) enzyme activity (1,2). In normal individuals IDS cleaves 2-sulfate from terminal iduronic acid residues of dermatan sulfate and heparan sulfate. Deficiency of IDS activity in patients with MPS n results in systemic accumulation of GAG. Severely affected individuals exhibit somatic abnormalities, mental retardation and die before age 15. In contrast, mildly affected patients may survive into adulthood incurring attenuated somatic manifestations and often without mental retardation (3,4). Cloning and sequencing of the normal IDS coding region (5) has made molecular analysis possible. Cumulated results of recent studies have determined that 14 of 62 MPS II patients have major gene abnormalities detectable either by Southern analysis (6 — 8) or by reverse transcription (RT) linked to polymerase chain reaction (PCR) (9,10). However, most affected individuals are likely to have single base substitutions or small deletions or insertions (11-13). We report a missense mutation in a patient with relatively mild Hunter syndrome. This mutation was identified by direct sequencing of RT-PCR products and confirmed by Mspl restriction digest. The functional significance of this mutation was established by correlation with serum IDS levels and by demonstrating defective enzyme activity in a retroviral expression system. Total RNA was isolated (14) from leukocytes or lymphoblastoid cell lines from several affected individuals, and RT-PCR reactions were carried out as previously described (10). The entire coding region of IDS was amplified as two overlapping segments. Both IDS segments were successfully amplified from 7 of the 8 patients studied; no products were detected for one patient despite successful amplification of/3-glucuronidase cDNA from the same RNA preparation. The RT-PCR products derived from these 7 patients were indistinguishable in size from those obtained from normal individuals indicating that most of these mutations were single base substitutions or small deletions or insertions. One of these 7 patients was selected for direct sequencing of the entire coding region. Regarding this patient, the diagnosis of Hunter syndrome was first suggested at 2.3 years of age and confirmed by measurement of elevated urinary glycosaminoglycan excretion and absence of serum IDS enzyme activity. At age 2.9 years, the child's IQ was 115 (Stanford-Binet Intelligence Scale, 4th edition). Currently, the patient is 5 years old and
756 Human Molecular Genetics, Vol. 1, No. 9
V
2^
i.
NORMAL
Msp\
I
AAA
16
8
14
4
12
2
10
6
iSSSSSSS^m^^ 478-
290-
277
Position
Sequence
IDS1 IDS2 IDS3 IDS4 IDS6 IDS7 IDS8 IDS10 IDS12 IDS14 IDS16
F-CGCGTCGAATTCGAAATGCC-y F-CATTTGGGATCCATGGTTGG-S' 5-ATGAAAACGTCAGCCAGTCC-3' S'-TTGGGGTATCTGAAGGGGAT-S1 5'-TCTAGATCCTCCrCTCACCA-31 51-ACCTCGCTGCCCCGTTCCTT-3* S-GTACTGGGGGATGGTGGAGA-y 5-GGATCCTCTTCCAAGTCACG-3' 5-CAATATCAGGGGAACATGGC-31 S'-GAGTrCTCCATCTGGCCCrC-S1 y-GGGACCTCACCAGCTTATCC-31
-14to+5 +1679 to +1660 +634 to +653 +707 to +668 +1719to+1700 +1257 to +1276 +366 to +347 +1352 to +1333 +1083 to +1064 +534to+515 +184to+165
MUTANT
S^SBSSS^^ 47R-
bp •567
bp 516/506
• 478
Figure 1. IDS mRNA with oligonucleotides used for amplification and for sequencing.
394 Table 1. Expression of normal and R468W mutant IDS in GP+E86 cells Vector (insert) LXSN (no insert) L2SN (normal IDS cDNA) L2SN-R468W (mutant cDNA)
IDS activity; mean ± S.D. (U/mg protein/hour) 3,492 ± 391 86,202 ± 10,340 4,070 ± 172
344
298
290/277
For each vector, a single transfection experiment was performed from which a heterogenous population of cells was selected (0.4 mg G418/ml medium), and then studied for IDS enzyme activity.
sizestd
also subcloned into pBS with selection of orientation allowing a 0.6 kb £coRI fragment to be isolated. This 0.6 kb EcoVl fragment was subcloned into pBS-HE299-R468W with selection of orientation allowing isolation of a 0.9 kb HindUl fragment. This 0.9 kb fragment containing the mutation was subcloned into pL2SN in place of the normal sequence, thus generating pL2SNR468W. The integrity of the construct was confirmed by a series of restriction digest experiments (BamHl, EcoRI, HinaTE, Nco\ and BsiEm). To evaluate expression of IDS activity, the plasmids pLXSN, pL2SN and pL2SN-R468W were linearized by cleavage with Ndel and then transfected into GP + E86 cells by calcium phosphate co-precipitation (19). After 48 hours of cultivation, transfectants were selected by growth in medium with G418 (0.4 mg/ml). After 7 days of selection, more than 100 drug-resistant clones of each construct were identified. Heterogeneous populations of stable transfectants were established by trypsinization and subculturing. Cells were harvested, washed twice in phosphate buffered saline, and then assayed for IDS activity (20). Cells transfected with L2SN containing the normal IDS sequence had very high IDS activity, levels approximately 20-fold greater than the endogenous levels of murine GP+E86 cells (Table 1). In contrast, cells transfected with vector L2SNR468W containing the mutant sequence had much lower enzyme activity comparable to cells transfected with LXSN.
proband
mother
normal
Figure 2. Mspl digestion of amplified IDS cDNA. The normal allele was digested to fragments of 478, 290 and 277 bp while the mutant R468W allele was digested to fragments of 567 and 478 bp. Mspl digestion of RT-PCR products from the patient showed the products predicted for the mutant allele. In contrast, the proband's mother showed the products anticipated for both the normal and mutant alleles.
The mutation R468W abolished an Mspl restriction site thus allowing confirmation of the mutant sequence and analysis in family members. IDS cDNA from the patient's mother was amplified with IDS2 and IDS3. Products of Mspl digestion were electrophoresed on a 4% agarose gel and visualized with ethidium bromide (Fig. 2). The patient showed bands predicted for the mutant allele. In contrast, his mother showed the products anticipated for both the normal and mutant alleles establishing her status as a heterozygote. This was consistent with a prior determination of her serum IDS enzyme activity (4.2 U/ml/h) which approximated the heterozygote range (4.7-11). In this fashion, characterization of specific molecular defects should remove the uncertainty which currently exists (21) when attempting carrier identification by means of enzyme analysis. Thus, several lines of evidence validate the significance of this mutation in a patient with mild Hunter syndrome, and exclude this change as a simple polymorphism: 1) full-length sequencing
Downloaded from http://hmg.oxfordjournals.org/ at University of Manchester on April 8, 2015
Primer
Human Molecular Genetics, Vol. 1, No. 9 757
Genotype-phenotype correlations may provide an alternate approach to estimating prognosis. Concurrent with our studies, sequencing of cloned IDS cDNA has been used to identify the mutation in a patient with Hunter syndrome of intermediate severity (13). Chemical cleavage of RT-PCR products has been applied to identify other mutations (9): of 6 patients studied, 1 had a deletion, 1 a premature termination codon, 1 a cryptic donor splice site, and 3 were found to have missense mutations. Most recently, single strand conformation polymorphism analysis has identified additional mutations including substitutions (2 missense, 2 nonsense) as well as deletions (1,2 and 60 bp) and a 22 bp insertion (11). As more patients are characterized at the molecular level, a greater understanding of the genotypic basis of phenotypic variability should evolve and facilitate prediction of severity in newly diagnosed patients. ACKNOWLEDGEMENTS We are indebted to Dr. John J.Hopwood for the partial-length IDS cDNA reference plasmid pc2S15 and Dr. William Sly for the full-length human 0-glucuronidase cDNA reference plasmid pHUGP13. We thank Dr. Bill Kaemmerer for molecular modelling. This work was supported by grants from the National Institutes of Health (RO1DK39891), the Ronald McDonald Children's Charities, and the Daniel Molinaro Foundation.
ABBREVIATIONS MPS - mucopolysaccharidosis IDS - kturonate-2-sulfatase GAG - glycoaminoglycan RT - reverse transcription PCR — polymerase chain reaction
REFERENCES 1. NeufeW, E.F. and Muenzer, J. (1989) In Scriver, C.R., Bcaudet, A.L., Sly, W.S. and Valle, D. (ed.), The metabolic basis of inherited diseases. McGrawHill, New York, Vol. II, pp. 1565-1587. 2. Whitley, C.B. (1992) In Beighton, P. (ed.), McKusick's Heritable Diseases of Connective Tissue. Mosby, St. Louis, Vol. pp. In press. 3. Young, I.D., Harper, P.S., Newcombe, R.G. and Archer, I.M. (1982) / Med Genet, 19, 408-411. 4. Young, I.D. and Harper, P.S. (1982) Arch Dis Child, 57, 828-836. 5. Wilson, P.J., Morris, C.P., Anson, D.S., Occhiodoro, T., Bielicki, J., Clements, P.R. and Hopwood, J.J. (1990) Proc Natl Acad Sd USA, 87, 8531-8535. 6. Wilson, PJ., Suthers, G.K., CaUen, D.F., Baker, E., Nelson, P.V., Cooper, A., Wraith, J.E., Sutherland, G.R., Moms, C.P. and Hopwood, J.J. (1991) Hum Genet, 86, 505-508. 7. Palrruen, G., Capra, V., Romano, G., D'Urso, M., Johnson, S., Schlessinger, D., Morris, P., Hopwood, J., Di Natale, P., Gam', R. and Ballabio, A. (1992) Genomes, 12, 52-57. 8. Wehncrt, M., Hopwood, J.J., Schroder, W. and Herrmann, F.H. (1992) Hum Genet, 89, 430-432. 9. Flomen, R.H., Green, P.M., Bentley, D.R., Giannelli, F. and Green, E.P. (1992) Genomics, 13, 543-550. 10. Crotty, P.L. and Whitley, C.B. (1992) Hum Genet, 90, In press. 11. Bunge, S., Steglich, C , Beck, M., Rosendranz, W., Schwinger, E., Hopwood, J.J. and Gal, A. (1992) Hum Mol Genet, 1, 335-339. 12. Gal, A., Beck, M., SeweU, A.C., Morris, C.P., Schwinger, E. and Hopwood, J.J. (1992) J Inherit Metab Dis, 15, 342-346. 13. Sukegawa, K., Tomatsu, S., Tamai, K., Dceda, M., Sasaki, T., Masue, M., Fukuda, S., Yamada, Y. and Orii, T. (1992) Biochem Biophys Res Commun, 183, 809-813. 14. Chomczynski, P. and Sacchi, N. (1987) Anal Biochem, 162, 156-159. 15. Sanger, F., Niklcn, S. and Coulson, A.R. (1977) Proc Natl Acad Sd USA, 74, 5463-5467. 16. Gamier, J., Osguthorpe, D.J. and Robson, B. (1978) J Mol Biol, 120, 97-120. 17. Markowitz, D.,Goff, S. and Bank, A. (1988)7 Virology, 62, 1120-1126. 18. Miller, A.D. and Rosman, G.J. (1989) Biotechniques, 7, 980-982. 19. Gorman, C. (1985) In Glover, D.M. (ed.), DNA cloning—a practical approach. IRL Press, Oxford, Vol. 2, pp. 143-190. 20. Wasteson, A. and Neufeld, E.F. (1982) Meth Enzymol, 83, 573-578. 21. Archer, I.M., Young, I.D., Rees, D.W., Oladimeji, A., Wusteman, F.S. and Harper, P.S. (1983) Am J Med Genet, 16, 61 - 6 9 . 22. Bemlohr, R.W. (1973) In Desnick, R.J., Bemlohr, R.W. and Krivit, W. (ed.), Birth Defects. March of Dimes, Baltimore, Vol. IX, pp. xi-xiii. 23. Conzelmann, E. and Sandhoff, K. (1983) Dev Neurosd, 6, 5 8 - 7 1 . 24. Leinekugel, P., Michel, S., Conzelmann, E. and Sandhoff, K. (1992) Hum Genet, 88, 513-23.
Downloaded from http://hmg.oxfordjournals.org/ at University of Manchester on April 8, 2015
determined that this is the only alteration in the IDS coding sequence; 2) the mutation causes a significant amino acid change replacing a basic hydrophilic amino acid with a hydrophobic residue; 3) study of the proband's mother showed the mutation to be co-inherited with enzyme levels approximating the carrier range; and 4) expression of the mutant cDNA in vitro confirms a lack of catalytic activity. At present, an early prediction of the severity of Hunter syndrome has not been possible either on the basis of clinical criteria or with currently available biochemical tests. However, early prognosis is becoming a key factor in making risk/benefit assessments as new therapeutic modalities are considered such as bone marrow transplantation, enzyme replacement therapy, and gene therapy. Bernlohr suggested that normal lysosomal enzyme levels are in excess of the metabolic demand, but that altered kinetics of an aberrant enzyme would lead to a higher steady-state concentration of substrate, or even progressive accumulation in affected individuals (22). More recently, Sandhoff and colleagues expanded upon this kinetic model proposing a relationship between the clinical phenotype (e.g., severity, age of onset) and small quantitative differences in residual enzyme activity (23); they subsequently provided experimental data correlating the residual activity of lysosomal enzymes and the turnover of substrate in cultured cells (24). By this model, severe Hunter syndrome might result from mutations which completely abolish in vivo enzyme activity (e.g., major gene deletions or rearrangements, mutations which prevent transcription of a stable mRNA, or mutations introducing early stop codons). In contrast, milder forms of Hunter syndrome might result from mutations with some residual activity. However, attempts to relate measured residual enzyme activity to severity of disease have proven unreliable for Hunter syndrome (21) presumably owing to differences between measured enzyme activity in vitro and the multitude of factors which determine the actual rate of substrate turnover in vivo. While expression of cDNA may serve to confirm specific mutations as demonstrated here, such experiments rely upon in vitro measurements of enzyme activity and would seem to provide no better handle on prognosis which thus remains a major challenge.
Human Molecular Genetics, Vol. 1, No. 9
755-757
Mutation R468W of the iduronate-2-sulfatase gene in mild Hunter syndrome (mucopolysaccharidosis type II) confirmed by in vitro mutagenesis and expression Paul L.Crotty14, Stephen E.Braun24, Rose Ann Anderson34 and Chester B.Whitley23-4* 1
Department of Laboratory Medicine and Pathology, 2Department of Genetics and Cell Biology, department of Pediatrics and 4lnstitute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA
Received August 12, 1992; Revised and Accepted October 27, 1992
appears to have a relatively mild form of Hunter syndrome not associated with neurological impairment. For direct sequencing, RT-PCR products of this patient were purified on a silica matrix (Geneclean, Bio 101, La Jolla, CA) with subsequent centrifugal size exclusion chromatography. Using a series of primers (Fig. 1), direct DNA sequencing (15) of the products was performed with Sequenase Version 2.0 (USB, Cleveland, OH) according to the manufacturers recommendations with modifications: 1) double-stranded template was denatured by boiling and annealed with the primer by snap-cooling on dry ice; 2) 10% dimethylsulfoxide was included in the annealing reaction; 3) labelling reactions were performed on ice; 4) a primertemplate molar ratio of ^ 10:1 was used. For this patient, the entire coding region was found to be identical to the normal sequence (5) except for a single base substitution c 1402 —T replacing arginine468 with tryptophan, R468W. This change occurs in an area of predominantly hydrophilic amino acid residues thus reducing the hydrophobicity of the region. Prediction of secondary protein structure by the Gamier—Robson model (16) indicated that this amino acid, situated between two prolines, is interposed at a turn between two predominantly /3-sheet regions. To determine the effect of the R468W mutation on enzyme activity, GP+E86 cells (17) were transfected with retrovirus expression vectors containing no IDS sequence (LXSN), the normal IDS cDNA (L2SN) or the mutant cDNA (L2SN-R468W). For construction of L2SN, the 5'-end of the IDS coding region (extending to the EcoRI site at +1427) was obtained from the reference plasmid pc2S15 (5). The 3'-end of the coding region was derived by RT-PCR using oligonucleotide primers IDS3 and EDS2 (Fig. 1), the latter designed with a 2-base change to incorporate a BamlU site; a fragment of this product, extending from the EcoRI site at +1427 to the BamHl site in IDS2, was cloned into pBluescript II (pBS, Stratagene, La Jolla, CA). The two cloned fragments were combined by sequential ligation into the cloning site of pLXSN (18) to generate pL2SN. A plasmid containing the mutant sequence (pL2SN-R468W) was also constructed. Using the patient's RNA as template, RTPCR product was obtained with IDS2 and IDS3 (Fig. 1). A 299 bp fragment, extending from the HindUl site at +1128 to the £coRI site at +1427, was cloned into pBS yielding pBSHE299-R468W. The integrity of the insert and the C I 4 O 2 -T mutation were confirmed by sequencing the entire 299 bp and flanking regions. The 0.9 kb HindSl fragment of pL2SN was
* To whom correspondence should be addressed at: Box 446 UMHC, 420 Delaware Str SE, Minneapolis, MN 55455, USA
Downloaded from http://hmg.oxfordjournals.org/ at University of Manchester on April 8, 2015
Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is an X-linked disorder of glycosaminoglycan (GAG) metabolism resulting from deficiency of lysosomal iduronate-2-sulfatase (IDS) enzyme activity (1,2). In normal individuals IDS cleaves 2-sulfate from terminal iduronic acid residues of dermatan sulfate and heparan sulfate. Deficiency of IDS activity in patients with MPS n results in systemic accumulation of GAG. Severely affected individuals exhibit somatic abnormalities, mental retardation and die before age 15. In contrast, mildly affected patients may survive into adulthood incurring attenuated somatic manifestations and often without mental retardation (3,4). Cloning and sequencing of the normal IDS coding region (5) has made molecular analysis possible. Cumulated results of recent studies have determined that 14 of 62 MPS II patients have major gene abnormalities detectable either by Southern analysis (6 — 8) or by reverse transcription (RT) linked to polymerase chain reaction (PCR) (9,10). However, most affected individuals are likely to have single base substitutions or small deletions or insertions (11-13). We report a missense mutation in a patient with relatively mild Hunter syndrome. This mutation was identified by direct sequencing of RT-PCR products and confirmed by Mspl restriction digest. The functional significance of this mutation was established by correlation with serum IDS levels and by demonstrating defective enzyme activity in a retroviral expression system. Total RNA was isolated (14) from leukocytes or lymphoblastoid cell lines from several affected individuals, and RT-PCR reactions were carried out as previously described (10). The entire coding region of IDS was amplified as two overlapping segments. Both IDS segments were successfully amplified from 7 of the 8 patients studied; no products were detected for one patient despite successful amplification of/3-glucuronidase cDNA from the same RNA preparation. The RT-PCR products derived from these 7 patients were indistinguishable in size from those obtained from normal individuals indicating that most of these mutations were single base substitutions or small deletions or insertions. One of these 7 patients was selected for direct sequencing of the entire coding region. Regarding this patient, the diagnosis of Hunter syndrome was first suggested at 2.3 years of age and confirmed by measurement of elevated urinary glycosaminoglycan excretion and absence of serum IDS enzyme activity. At age 2.9 years, the child's IQ was 115 (Stanford-Binet Intelligence Scale, 4th edition). Currently, the patient is 5 years old and
756 Human Molecular Genetics, Vol. 1, No. 9
V
2^
i.
NORMAL
Msp\
I
AAA
16
8
14
4
12
2
10
6
iSSSSSSS^m^^ 478-
290-
277
Position
Sequence
IDS1 IDS2 IDS3 IDS4 IDS6 IDS7 IDS8 IDS10 IDS12 IDS14 IDS16
F-CGCGTCGAATTCGAAATGCC-y F-CATTTGGGATCCATGGTTGG-S' 5-ATGAAAACGTCAGCCAGTCC-3' S'-TTGGGGTATCTGAAGGGGAT-S1 5'-TCTAGATCCTCCrCTCACCA-31 51-ACCTCGCTGCCCCGTTCCTT-3* S-GTACTGGGGGATGGTGGAGA-y 5-GGATCCTCTTCCAAGTCACG-3' 5-CAATATCAGGGGAACATGGC-31 S'-GAGTrCTCCATCTGGCCCrC-S1 y-GGGACCTCACCAGCTTATCC-31
-14to+5 +1679 to +1660 +634 to +653 +707 to +668 +1719to+1700 +1257 to +1276 +366 to +347 +1352 to +1333 +1083 to +1064 +534to+515 +184to+165
MUTANT
S^SBSSS^^ 47R-
bp •567
bp 516/506
• 478
Figure 1. IDS mRNA with oligonucleotides used for amplification and for sequencing.
394 Table 1. Expression of normal and R468W mutant IDS in GP+E86 cells Vector (insert) LXSN (no insert) L2SN (normal IDS cDNA) L2SN-R468W (mutant cDNA)
IDS activity; mean ± S.D. (U/mg protein/hour) 3,492 ± 391 86,202 ± 10,340 4,070 ± 172
344
298
290/277
For each vector, a single transfection experiment was performed from which a heterogenous population of cells was selected (0.4 mg G418/ml medium), and then studied for IDS enzyme activity.
sizestd
also subcloned into pBS with selection of orientation allowing a 0.6 kb £coRI fragment to be isolated. This 0.6 kb EcoVl fragment was subcloned into pBS-HE299-R468W with selection of orientation allowing isolation of a 0.9 kb HindUl fragment. This 0.9 kb fragment containing the mutation was subcloned into pL2SN in place of the normal sequence, thus generating pL2SNR468W. The integrity of the construct was confirmed by a series of restriction digest experiments (BamHl, EcoRI, HinaTE, Nco\ and BsiEm). To evaluate expression of IDS activity, the plasmids pLXSN, pL2SN and pL2SN-R468W were linearized by cleavage with Ndel and then transfected into GP + E86 cells by calcium phosphate co-precipitation (19). After 48 hours of cultivation, transfectants were selected by growth in medium with G418 (0.4 mg/ml). After 7 days of selection, more than 100 drug-resistant clones of each construct were identified. Heterogeneous populations of stable transfectants were established by trypsinization and subculturing. Cells were harvested, washed twice in phosphate buffered saline, and then assayed for IDS activity (20). Cells transfected with L2SN containing the normal IDS sequence had very high IDS activity, levels approximately 20-fold greater than the endogenous levels of murine GP+E86 cells (Table 1). In contrast, cells transfected with vector L2SNR468W containing the mutant sequence had much lower enzyme activity comparable to cells transfected with LXSN.
proband
mother
normal
Figure 2. Mspl digestion of amplified IDS cDNA. The normal allele was digested to fragments of 478, 290 and 277 bp while the mutant R468W allele was digested to fragments of 567 and 478 bp. Mspl digestion of RT-PCR products from the patient showed the products predicted for the mutant allele. In contrast, the proband's mother showed the products anticipated for both the normal and mutant alleles.
The mutation R468W abolished an Mspl restriction site thus allowing confirmation of the mutant sequence and analysis in family members. IDS cDNA from the patient's mother was amplified with IDS2 and IDS3. Products of Mspl digestion were electrophoresed on a 4% agarose gel and visualized with ethidium bromide (Fig. 2). The patient showed bands predicted for the mutant allele. In contrast, his mother showed the products anticipated for both the normal and mutant alleles establishing her status as a heterozygote. This was consistent with a prior determination of her serum IDS enzyme activity (4.2 U/ml/h) which approximated the heterozygote range (4.7-11). In this fashion, characterization of specific molecular defects should remove the uncertainty which currently exists (21) when attempting carrier identification by means of enzyme analysis. Thus, several lines of evidence validate the significance of this mutation in a patient with mild Hunter syndrome, and exclude this change as a simple polymorphism: 1) full-length sequencing
Downloaded from http://hmg.oxfordjournals.org/ at University of Manchester on April 8, 2015
Primer
Human Molecular Genetics, Vol. 1, No. 9 757
Genotype-phenotype correlations may provide an alternate approach to estimating prognosis. Concurrent with our studies, sequencing of cloned IDS cDNA has been used to identify the mutation in a patient with Hunter syndrome of intermediate severity (13). Chemical cleavage of RT-PCR products has been applied to identify other mutations (9): of 6 patients studied, 1 had a deletion, 1 a premature termination codon, 1 a cryptic donor splice site, and 3 were found to have missense mutations. Most recently, single strand conformation polymorphism analysis has identified additional mutations including substitutions (2 missense, 2 nonsense) as well as deletions (1,2 and 60 bp) and a 22 bp insertion (11). As more patients are characterized at the molecular level, a greater understanding of the genotypic basis of phenotypic variability should evolve and facilitate prediction of severity in newly diagnosed patients. ACKNOWLEDGEMENTS We are indebted to Dr. John J.Hopwood for the partial-length IDS cDNA reference plasmid pc2S15 and Dr. William Sly for the full-length human 0-glucuronidase cDNA reference plasmid pHUGP13. We thank Dr. Bill Kaemmerer for molecular modelling. This work was supported by grants from the National Institutes of Health (RO1DK39891), the Ronald McDonald Children's Charities, and the Daniel Molinaro Foundation.
ABBREVIATIONS MPS - mucopolysaccharidosis IDS - kturonate-2-sulfatase GAG - glycoaminoglycan RT - reverse transcription PCR — polymerase chain reaction
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determined that this is the only alteration in the IDS coding sequence; 2) the mutation causes a significant amino acid change replacing a basic hydrophilic amino acid with a hydrophobic residue; 3) study of the proband's mother showed the mutation to be co-inherited with enzyme levels approximating the carrier range; and 4) expression of the mutant cDNA in vitro confirms a lack of catalytic activity. At present, an early prediction of the severity of Hunter syndrome has not been possible either on the basis of clinical criteria or with currently available biochemical tests. However, early prognosis is becoming a key factor in making risk/benefit assessments as new therapeutic modalities are considered such as bone marrow transplantation, enzyme replacement therapy, and gene therapy. Bernlohr suggested that normal lysosomal enzyme levels are in excess of the metabolic demand, but that altered kinetics of an aberrant enzyme would lead to a higher steady-state concentration of substrate, or even progressive accumulation in affected individuals (22). More recently, Sandhoff and colleagues expanded upon this kinetic model proposing a relationship between the clinical phenotype (e.g., severity, age of onset) and small quantitative differences in residual enzyme activity (23); they subsequently provided experimental data correlating the residual activity of lysosomal enzymes and the turnover of substrate in cultured cells (24). By this model, severe Hunter syndrome might result from mutations which completely abolish in vivo enzyme activity (e.g., major gene deletions or rearrangements, mutations which prevent transcription of a stable mRNA, or mutations introducing early stop codons). In contrast, milder forms of Hunter syndrome might result from mutations with some residual activity. However, attempts to relate measured residual enzyme activity to severity of disease have proven unreliable for Hunter syndrome (21) presumably owing to differences between measured enzyme activity in vitro and the multitude of factors which determine the actual rate of substrate turnover in vivo. While expression of cDNA may serve to confirm specific mutations as demonstrated here, such experiments rely upon in vitro measurements of enzyme activity and would seem to provide no better handle on prognosis which thus remains a major challenge.
