© 1992 Oxford University Press
Human Molecular Genetics, Vol. 1, No. 3
195-198
Molecular analysis of patients with Hunter syndrome: implication of a region prone to structural alterations within the IDS gene M.-LSte6n-Bondeson1, N.Dahl12, T.Tonnesen3, W.J.KIeijer4, G.Seidlitz5, K.-H.Gustavson2, P.J.Wilson6, C.P.Morris6, J.J.Hopwood6 and U.Pettersson1 1
Received March 3, 1992; Revised and Accepted May 13, 1992
ABSTRACT Hunter syndrome or mucopolysaccharidoses type II (MPSII), is a lysosomal storage disorder caused by a deficiency in the activity of the enzyme iduronate-2-sulphatase (IDS). We have investigated the occurrence of rearrangements and deletions of the IDS gene in a Southern analysis of 46 unrelated MPS-II patients of different ethnic origins using a cDNA clone containing the entire IDS gene as a probe. Structural alterations of the IDS gene were found in DNA from 9 patients two of whom showed large deletions including all coding sequences of the gene. The distal and proximal breakpoint of these deletions were determined by hybridization of markers flanking the IDS gene. Seven of the observed alterations constitute major rearrangements of the gene. Interestingly, six of these rearrangements showed similar or identical patterns by Southern analysis suggestive for a region prone to structural alterations within the IDS gene. We also demonstrate the potential use of the IDS probe for carrier detection in families with a rearranged IDS gene. A contiguous gene deletion syndrome characterized by Hunter syndrome and epilepsy is also discussed.
different mutations at the IDS locus affecting enzyme function and expression. The human IDS enzyme has been purified and sequenced (3, 4) and recently a cDNA clone containing the complete coding region of the enzyme has been isolated and characterized (4). The IDS gene has been mapped to Xq28 by cytogenetic analysis of a girl with Hunter syndrome widi a X:5 chromosomal translocation (5) and also by linkage analysis and physical mapping studies (6,7,8,9,10,11). The order of loci near the IDS locus defined on basis of physical mapping and linkage analysis is cen-DXS 297-DXS 2%-DXS 295-IDS-DXS 3 0 2 - DXS 304-qter (8, 9, 11). Recent studies have indicated that deletions or rearrangements of the IDS gene might occur in patients with Hunter syndrome (4,10,12,13). We have investigated DNA from 46 MPS-II index patients using a cDNA clone containing the entire coding region of IDS. Here we report 9 patients with structural alterations in the IDS gene. Two of these patients have large deletions spanning both the IDS gene and flanking markers. A remarkable finding was that six patients show indistinguishable rearrangements by Southern analysis suggestive of a region within the IDS gene that is prone to rearrangements.
INTRODUCTION
RESULTS Southern blot analysis of MPS-II patients Forty-six unrelated males of different ethnic origins of whom 45 had a severe form of Hunter syndrome were examined by Southern blot analysis using pB2Scl7 as a probe. Genomic DNA from the patients was analyzed after cleavage with EcoRI, Hindin and Pstl. Thirty-seven of the patients analyzed displayed patterns indistinguishable from those of the normal controls (data not shown). However, no hybridization was detected in two patients while 7 patients had an abnormal restriction pattern compared to normal individuals. Figs, la, lb and lc (patients no. 2 and 3), show that no hybridization was found even after long exposure times suggesting that the complete gene has been deleted in these two patients. Rehybridization of the filters with other probes demonstrated that sufficient amount of DNA was present on the filters (data not shown).
Hunter syndrome or MPS-II (McKusick 30990) is an X-Iinked recessive lysosomal storage disorder. A deficiency in the activity of the enzyme iduronate-2-sulphatase (IDS), responsible for degradation of dermatan sulphate and heparan sulphate, results in accumulation of these mucopolysaccharides in the lysosomes and their excretion in the urine. The incidence of MPS-II is about 1 in 132 000 male births (1). Clinically, the patients may present a wide spectrum of phenotypes from severe to a relatively milder course. The severe type appears in late infancy and is characterized by mental retardation, coarse facial features, short stature, skeletal deformities, joint stiffness and death before age 15 years. In its mild form the disease is manifested later, the intelligence is preserved and the patients survive to adulthood. The adult patients can develop somatic features similar to those in the severe form but with a slower progression (2). This observed clinical heterogeneity has been postulated to reflect
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Department of Medical Genetics, Uppsala University, Box 589, S-751 23 Uppsala, 2Department of Clinical Genetics, University Hospital, 751 85 Uppsala, Sweden, 3Department of Biochemistry and Molecular Genetics, The J.F.Kennedy Institute, 7 Gl. Landevej, DK-2600 Glostrup, Denmark, 4Department of Clinical Genetics, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands, institute for Medical Genetics, Ernst-Maritz-Arndt University, Fleischmannstrasse 42-44, DO-2200 Greifswald, Germany and 6Lysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide Children's Hospital, North Adelaide, SA 5006, Australia
196 Human Molecular Genetics, Vol. 1, No. 3
Mapping of IDS Deletion Breakpoints To map the extension of the deletions in the two individuals deleted for IDS, Southern blot analysis was carried out. Markers detecting loci located proximal (DXS 296 and DXS 295) and distal to IDS (DXS 466 and DXS 304) were used as probes. The results are shown in Table I. All loci but DXS 466 were present in DNA from one of the patients (patient no. 3; DDR/GPI)- DNA from the other patient, (no. 2; DK/TJK), failed to hybridize to both DXS 466 and the DXS 295 while the DXS 2 % and DXS 304 loci were present. From physical mapping data DXS 2 % is located approximately 800 kb proximal from IDS while DXS 304 is located within a distance of 900 kb distal of the IDS locus (8). These results thus implicate that the deletions cover a region of less than 1700 kb in the two patients. Family Study We also analyzed DNA from family members of one of the patients (F'g-1> patient no. 1) carrying a rearranged IDS gene.This study was performed to determine if an affected brother of a patient carried the same rearrangement and also to determine if his sisters were carriers The DNA samples were analyzed with EcoRI and Hindm. As shown in Fig.2, both the affected brothers displayed the same altered restriction pattern compared to normal controls. Analysis of the mother and one of the daughters revealed both the normal and the altered restriction pattern implying that they are carriers of the mutation (Fig.2).
DISCUSSION The wide spectrum of clinical phenotypes displayed in Hunter syndrome patients may be explained by observed genetic heterogeneity. The results reported here together with those of Wilson et al. (4,10) and Palmieri et al. (13) support the notion that the IDS gene may suffer several kinds of mutations. In the Southern analysis reported by Wilson et al. (4, 10) 7 out of 23 patients had restriction patterns which deviated from those of the normal controls, including two patients with a complete deletion of the IDS gene. In the Southern analysis performed by Palmieri et al. (13) four out of 25 Italian MPS-II patients showed an abnormal hybridization pattern. Here we
report another 9 patients with structural alterations of the IDS gene identified among 46 unrelated Hunter males analyzed. These 9 patients all had the severe form of the Hunter syndrome. Two of the patients carried a deletion spanning the entire coding sequence of the IDS gene. In previous studies of Wilson et al.
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Figure 1. Southern blot analysis with pB2Scl7 of samples digested with the restriction enzymes a) EcoRI b) Hindlll and c) PstI. C: Normal female. Lanes 2 - 1 0 : 9 MPS-II patients with structural alternations of the IDS gene. The 0.7 kb and 2.9 kb fragment in Fig. lb is present i all patients except no. 2 and 3. but not visible. The order of the patients is the same in both Figs, la, lb and lc. The positions of DNA size markers (in kb) are shown to the right.
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DNA samples from the remaining 7 patients with detectable alterations showed different patterns compared to normal controls when analyzed with EcoRI or Hindm (Figs, la and /3 respectively). Cleavage of DNA with EcoRI revealed five bands in normal controls with the sizes 9.4 kb, 8.1 kb, 7.2 kb, 6.6 kb and 3.7 kb. In one of the patients (patient no. 9, Fig. la) the 6.6 kb band was absent. In the remaining 6 patients with a deviant restriction pattern the 7.2 kb and 9.4 kb bands were missing and instead two bands of 11.5 kb and 5.8 kb were present (Fig. la). Hindm cleavage of normal controls revealed 8 different bands (9.4 kb, 6.6 kb, 4.8 kb, 4.7 kb, 2.9 kb, 2.7 kb, 1.6 kb and 0.7 kb) as depicted in Fig. lb. Two of these (9.4 kb and 6.6 kb) were absent in the 7 Hunter males with an altered restriction pattern. In one of the patients (patient no. 9) two new bands of 14.0 kb and 11.8 kb were revealed while new bands of 3.4 kb and 12.9 kb were seen in the remaining 6 patients. When these patients were analyzed with PstI (Fig. lc) structural alterations were detected in only one patient (patient no. 9) where a 8.0 kb band present in normal controls was absent.
Human Molecular Genetics, Vol. 1, No. 3 197 (4, 10) aberrant restriction patterns were observed in several patients after PstI cleavage of genomic DNA. However, in the analysis reported here all the structural alterations were observed when the genomic DNA was digested with HindlH or EcoRI while aberrant restriction patterns were detected in DNA from only one patient digested with PstI. The structural alterations observed after Hindlll and EcoRI cleavage are unlikely to TaWe 1. X chromosome deletion mapping of Hunter patients Locus
VK 21A VK 18A P B2Scl7 1110 U6.2
DXS DXS IDS DXS DXS
Patient DDR/GPI
DK/JTK 296 295 466 304
Xq-denved DNA markers used to define the breakpoints of the deletions in the two patients DDR/GPI and DK/TJK. DDR and DK indicate that the patients are from Germany and Denmark, respectively. GPI and TJK are the initials of the patients. The presence ( + ) and absence ( - ) of the different markers in the DNA from the patients are indicated.
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Figure 2. Southern hybridization of EcoRI (Fig.2a) and Hindlll (Fig. 2b) digested samples with the IDS cDNA probe. The pedigree of the affected and unaffected relatives are indicated above. The affected boy in lane 3 in Fig 2a and 2b is the same patient characterized as in Fig. 1 (patient no. 1). The sizes in kb of DNA markers are shown to the right.
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Probe
represent polymorphisms since no such polymorphisms have been detected previously with the probe (8). Furthermore, these mutations are more likely to represent rearrangements rather than single point mutations since aberrant restriction patterns were observed in these patients both when analyzed with EcoRI and HindlTJ. Interestingly, six of 7 patients showed similar or identical rearrangements which implicates a region prone to structural alterations within the IDS gene. These results are not likely to be caused by a founder effect since these patients represent different ethnic origins: two patients are from Poland and the remaining patients are from Czechoslovakia, Denmark, Finland and Norway. In addition, an Italian patient with a similar EcoRI rearrangement to those reported here has also been described recently (13). These structural alterations are most likely representing intragenic rearrangements since sequences representing the 5' end of the IDS gene (10) are present as well as 3' end sequences (Stee'n-Bondeson et al., unpublished results). Further characterization of this region is presently under investigation. Thus, in 20% of the Hunter patients reported here structural alterations of the IDS gene could be detected. Our results together with those of Wilson et al. (4,10) and Palmieri et al. (13), suggest
198 Human Molecular Genetics, Vol. 1, No. 3
MATERIALS AND METHODS DNA isolation and hybridization Forty-six unrelated Bulgarian, Czechoslovakian, Danish, Finnish, German. Hungarian, Italian, Jugoslavian, Norwegian, Polish, Portuguese and Spanish Hunter syndrome males with defective iduronate-2-sulphatase activity in cultured fibroblasts were studied. Genomic DNA was extracted from from normal human blood and cultured fibroblasts. Ten jig of genomic DNA was digested to completion with appropriate restriction enzymes (Pharmacia P-L Biochemicals) and fractionated by clectrophoresis on 0.8% agarose gels. Transfer onto nylon membranes (Hybond-N, Amersham) was performed according to the procedure of Southern (17). The probes were radiolabelled by random priming using an oligolabclling kit (27-9250-01, Pharmacia) and hybridized to the filters at 65° in a hybridization solution containing: 5 x SSPE (0 9M NaCI, 0.05M
NaPhosphate, pH 8.3, 5mM EDTA), 5 x Denhardt's buffer, 0.2% (w/v) SDS and 250 /tg/ml yeast RNA. The filters were washed 2h in 2xSSC, 0.5%SDS and30minin0.1xSSC, 0. l%SDSat65° and autoradiographed with intensifying screens. DNA probes For Southern blot analysis a 1760 bp fragment from the cDNA clone pB2Scl7 containing the entire coding region of the IDS (Wilson, unpublished) was used. In addition the following markers were used: DXS 295 (11), DXS 2% (11), DXS 466 (15) and DXS 304 (18).
ACKNOWLEDGEMENTS We thank Britt-Marie Carlberg for excellent technical assistance. We are grateful to Dr T.Hulsebos, University of Amsterdam for providing the marker II 10 and Dr G.R. Sutherland, Adelaide Children's Hospital, Australia, for sharing the markers VK18A and VK21 A. We would also like to thank colleagues who have referred their patients to us. Financial assistance for this project was provided by grants from the Beijer Foundation, the Swedish Medical Research Council and the Marcus Borgstrom Foundation.
ABBREVIATIONS Bp, basepair(s); IDS, iduronate-2-sulphatase; kb, kilobase(s) or 1000 bp; MPSII, mucopolysaccharidoses type II; no, number; RFLP(s), restriction fragment length polymorphism(s); SDS, sodium dodecylsulphate, SSC, 0.15M NaCI. 0.015M Na3. citrate, pH7.5.
REFERENCES 1. Young, I.D. and Harper, P.S. (1982) Hum. Genet., 60, 391-392. 2. Spranger, J. (1990) In Emery, A.E.H., Rimoin, D.L (eds). Principles and Practice of Medical Genetics. Vol. 2, pp 1800- 1802. Churchill Livingstone. 3. Bidicki, J , Freeman, C , Clements, P.R. and Hopwood, J J. (1990) Btochem. J., 271, 75-86. 4. Wilson, P.J., Morris, C.P., Anson, D.S., Occhiodoro, T., Bielicki, J., Clements, P.R. and Hopwood, J.J. (1990) Proc. Natl. Acad. Sci. USA, 87, 8531-8535. 5. Roberts, S.H., Upadhyaya, M., Sarfarazi, M and Harper, P.S. (1989) J. Med. Genet., 26, 309-313. 6. Upadhyaya, M., Sarfarazi, M., Bamforth, J.S., Thomas, N.S.T., Oberle\ I., Young, I. and Harper, P S. (1986) Hum. Genet., 74, 391 -398. 7. Thomas, N.S.T., Roberts, S.H., Upadhyaya, M., Knight, S. and Harper, P.S. (1989) Cytogenet. Cell Genet., 51, 1090. 8. Suthers, G.K., Oberle, I., Nancarrow, J., Mulley, J.C , Hyland. V.J., Wilson, P.J., McCure, J.. Morris, C.P., Hopwood, J.J., Manoel, J.L. and Sutherland, G.R. (1991) Genomks, 9, 37-43. 9. Suthers, G.K., Mulley, J . C , Voelckel, M.A., Dahl, N., Vaisanen, M.L., Steinbach, P., Glass, I A., Schwartz, C.E., van Oost, B.A., Thibodeau, S.N., Haites, N.E., Oostra, B.A., Gine, R., Carballo, M., Morris, C.P., HopwoodJ.J. and Sutherland, G.R. (1991) Am. J. Hum. Genet., 48, 460-467 10. Wilson, P.J., Suthers, G.K., Callen, 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. 11. Suthers, G.K., Hyland, V.J., Callen, D.F., Oberle\ I., Rocchi, M., Thomas, N.S., Morris, C.P., Schwartz, C.E., Schmidt, M.. Ropers. H.H., Baker, E., Oostra, B.A., Dahl, N.. Wilson, P.J., Hopwood, J.J. and Sutherland. G.R. (1990) Am. J. Hum. Genet., 47, 187-195. 12. Beck, M., Steglich, C , Zabel, B., Dahl, N., Schwinger, E., Hopwood. J.J. and Gal, A. (1992) Am. J. Med. Genet. In press. 13. Palmieri, G., Capra, V., Romano, G., D'Urso, M.. Johnson, S., Schlcssinger, D., Morris, P., Hopwood, J., Di Natale, P., Gatti, R. and Ballabio, A. (1992) Genomes, 12, 52-57. 14. Le Guern, E., Couillin. P.. Oberle. I., Ravise, N. and Boue, J. (1990) Genomics, 7, 358-362. 15. Hulscbos, T.J.M., Oostra, B.A., Broersen, S., Smits, A., van Oost, B.A. and Westerveld, A. (1991) Hum. Genet.. 87, 369-372. 16. Wraith, J.E., Cooper, A.. Thomley. M., Wilson, PJ., Nelson, P.V., Morris. C.P. and Hopwood, J.J. (1991) Hum. Genet., 87, 205-206. 17. Southern, E.M. (1975) J. Mol. Biol., 98, 503-517. 18. Dahl, N., Hammarstrom-Heeroma, K., Goonewardena, P , Wadelius. C . Gustavson, K.-H., Holmgren, G., van Ommen, G.J. B. and Pettersson, U. (1989) Hum. Genet., 82, 216-218.
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that most of the mutations (70-80%) in Hunter syndrome represent point mutations or small rearrangements of the IDS gene which escape detection by Southern analysis. The IDS cDNA probe pc2S15 has previously been reported to detect TaqI and StuI RFLPs in the IDS locus (8,9). Thus, this probe and other closely linked markers may provide useful information for carrier risk estimations in connection with Hunter syndrome (8, 14). In this study we have also demonstrated the potential use of the IDS cDNA in carrier detection analysis in families with a rearranged IDS gene. The altered hybridization pattern observed in Hunter patients is easily detectable in carriers, without use of flanking markers. In addition to point mutations and rearrangements, the IDS gene seems to be a target for different deletions. Three patients with complete deletion of the IDS coding sequences have previously been reported (4, 10, 12, 16). One of these (04-1; ref. 10) has a deletion extending proximally from the IDS gene including the locus DXS 296 (4, 10, 11). The deletion in the second patient is smaller and and does not include flanking loci (4, 10, 11). The third patient reported by Beck et al. (12) has a deletion extending distal from the IDS gene spanning the DXS 304 locus. As shown in Table I the two patients reported here both lack the locus DXS 466 which is located either within or distal to the IDS gene, since it is absent in the cell line CY34 derived from a girl with Hunter syndrome with a X:5 translocation (10, 15). Thus the distal breakpoints of the deletions are located between DXS 304 and DXS 466 in both DK/JTK and DDR/GPI. However, the proximal breakpoints differ. In patient DK/TJK it is located between DXS 295 and DXS 296 while it is positioned between IDS and DXS 295 in DDR/GPI. Clinically, both patient DK/TJK and DDR/GPI present the classical severe phenotype of Hunter syndrome. In addition, DK/JTK suffers from epileptic seizures. Epileptic seizures have also been described for the two patients reported by Wilson et al. (10) and Wraith et al. (16). A comparison of the breakpoints in these three patients suggests that a gene or genes located in the vicinity to the IDS gene may be involved in the development of epilepsy. Further investigation of location of the deletion breakpoints in DDR/GPI and DK/JTK is in progress. A more detailed phenotypic analysis of patients with different deletions together with a higher resolution map of the deletions may provide valuable information about the location of other genes in this region and their resulting phenotypes. A physical map of a 1.2Mb YAC contig spanning the IDS gene, recently reported by Palmieri et al. (13) reveals several putative CpG islands. This also indicates the presence of other genes in the vicinity of the IDS gene.
Human Molecular Genetics, Vol. 1, No. 3
195-198
Molecular analysis of patients with Hunter syndrome: implication of a region prone to structural alterations within the IDS gene M.-LSte6n-Bondeson1, N.Dahl12, T.Tonnesen3, W.J.KIeijer4, G.Seidlitz5, K.-H.Gustavson2, P.J.Wilson6, C.P.Morris6, J.J.Hopwood6 and U.Pettersson1 1
Received March 3, 1992; Revised and Accepted May 13, 1992
ABSTRACT Hunter syndrome or mucopolysaccharidoses type II (MPSII), is a lysosomal storage disorder caused by a deficiency in the activity of the enzyme iduronate-2-sulphatase (IDS). We have investigated the occurrence of rearrangements and deletions of the IDS gene in a Southern analysis of 46 unrelated MPS-II patients of different ethnic origins using a cDNA clone containing the entire IDS gene as a probe. Structural alterations of the IDS gene were found in DNA from 9 patients two of whom showed large deletions including all coding sequences of the gene. The distal and proximal breakpoint of these deletions were determined by hybridization of markers flanking the IDS gene. Seven of the observed alterations constitute major rearrangements of the gene. Interestingly, six of these rearrangements showed similar or identical patterns by Southern analysis suggestive for a region prone to structural alterations within the IDS gene. We also demonstrate the potential use of the IDS probe for carrier detection in families with a rearranged IDS gene. A contiguous gene deletion syndrome characterized by Hunter syndrome and epilepsy is also discussed.
different mutations at the IDS locus affecting enzyme function and expression. The human IDS enzyme has been purified and sequenced (3, 4) and recently a cDNA clone containing the complete coding region of the enzyme has been isolated and characterized (4). The IDS gene has been mapped to Xq28 by cytogenetic analysis of a girl with Hunter syndrome widi a X:5 chromosomal translocation (5) and also by linkage analysis and physical mapping studies (6,7,8,9,10,11). The order of loci near the IDS locus defined on basis of physical mapping and linkage analysis is cen-DXS 297-DXS 2%-DXS 295-IDS-DXS 3 0 2 - DXS 304-qter (8, 9, 11). Recent studies have indicated that deletions or rearrangements of the IDS gene might occur in patients with Hunter syndrome (4,10,12,13). We have investigated DNA from 46 MPS-II index patients using a cDNA clone containing the entire coding region of IDS. Here we report 9 patients with structural alterations in the IDS gene. Two of these patients have large deletions spanning both the IDS gene and flanking markers. A remarkable finding was that six patients show indistinguishable rearrangements by Southern analysis suggestive of a region within the IDS gene that is prone to rearrangements.
INTRODUCTION
RESULTS Southern blot analysis of MPS-II patients Forty-six unrelated males of different ethnic origins of whom 45 had a severe form of Hunter syndrome were examined by Southern blot analysis using pB2Scl7 as a probe. Genomic DNA from the patients was analyzed after cleavage with EcoRI, Hindin and Pstl. Thirty-seven of the patients analyzed displayed patterns indistinguishable from those of the normal controls (data not shown). However, no hybridization was detected in two patients while 7 patients had an abnormal restriction pattern compared to normal individuals. Figs, la, lb and lc (patients no. 2 and 3), show that no hybridization was found even after long exposure times suggesting that the complete gene has been deleted in these two patients. Rehybridization of the filters with other probes demonstrated that sufficient amount of DNA was present on the filters (data not shown).
Hunter syndrome or MPS-II (McKusick 30990) is an X-Iinked recessive lysosomal storage disorder. A deficiency in the activity of the enzyme iduronate-2-sulphatase (IDS), responsible for degradation of dermatan sulphate and heparan sulphate, results in accumulation of these mucopolysaccharides in the lysosomes and their excretion in the urine. The incidence of MPS-II is about 1 in 132 000 male births (1). Clinically, the patients may present a wide spectrum of phenotypes from severe to a relatively milder course. The severe type appears in late infancy and is characterized by mental retardation, coarse facial features, short stature, skeletal deformities, joint stiffness and death before age 15 years. In its mild form the disease is manifested later, the intelligence is preserved and the patients survive to adulthood. The adult patients can develop somatic features similar to those in the severe form but with a slower progression (2). This observed clinical heterogeneity has been postulated to reflect
Downloaded from http://hmg.oxfordjournals.org/ at University of California, San Francisco on March 11, 2015
Department of Medical Genetics, Uppsala University, Box 589, S-751 23 Uppsala, 2Department of Clinical Genetics, University Hospital, 751 85 Uppsala, Sweden, 3Department of Biochemistry and Molecular Genetics, The J.F.Kennedy Institute, 7 Gl. Landevej, DK-2600 Glostrup, Denmark, 4Department of Clinical Genetics, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands, institute for Medical Genetics, Ernst-Maritz-Arndt University, Fleischmannstrasse 42-44, DO-2200 Greifswald, Germany and 6Lysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide Children's Hospital, North Adelaide, SA 5006, Australia
196 Human Molecular Genetics, Vol. 1, No. 3
Mapping of IDS Deletion Breakpoints To map the extension of the deletions in the two individuals deleted for IDS, Southern blot analysis was carried out. Markers detecting loci located proximal (DXS 296 and DXS 295) and distal to IDS (DXS 466 and DXS 304) were used as probes. The results are shown in Table I. All loci but DXS 466 were present in DNA from one of the patients (patient no. 3; DDR/GPI)- DNA from the other patient, (no. 2; DK/TJK), failed to hybridize to both DXS 466 and the DXS 295 while the DXS 2 % and DXS 304 loci were present. From physical mapping data DXS 2 % is located approximately 800 kb proximal from IDS while DXS 304 is located within a distance of 900 kb distal of the IDS locus (8). These results thus implicate that the deletions cover a region of less than 1700 kb in the two patients. Family Study We also analyzed DNA from family members of one of the patients (F'g-1> patient no. 1) carrying a rearranged IDS gene.This study was performed to determine if an affected brother of a patient carried the same rearrangement and also to determine if his sisters were carriers The DNA samples were analyzed with EcoRI and Hindm. As shown in Fig.2, both the affected brothers displayed the same altered restriction pattern compared to normal controls. Analysis of the mother and one of the daughters revealed both the normal and the altered restriction pattern implying that they are carriers of the mutation (Fig.2).
DISCUSSION The wide spectrum of clinical phenotypes displayed in Hunter syndrome patients may be explained by observed genetic heterogeneity. The results reported here together with those of Wilson et al. (4,10) and Palmieri et al. (13) support the notion that the IDS gene may suffer several kinds of mutations. In the Southern analysis reported by Wilson et al. (4, 10) 7 out of 23 patients had restriction patterns which deviated from those of the normal controls, including two patients with a complete deletion of the IDS gene. In the Southern analysis performed by Palmieri et al. (13) four out of 25 Italian MPS-II patients showed an abnormal hybridization pattern. Here we
report another 9 patients with structural alterations of the IDS gene identified among 46 unrelated Hunter males analyzed. These 9 patients all had the severe form of the Hunter syndrome. Two of the patients carried a deletion spanning the entire coding sequence of the IDS gene. In previous studies of Wilson et al.
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Figure 1. Southern blot analysis with pB2Scl7 of samples digested with the restriction enzymes a) EcoRI b) Hindlll and c) PstI. C: Normal female. Lanes 2 - 1 0 : 9 MPS-II patients with structural alternations of the IDS gene. The 0.7 kb and 2.9 kb fragment in Fig. lb is present i all patients except no. 2 and 3. but not visible. The order of the patients is the same in both Figs, la, lb and lc. The positions of DNA size markers (in kb) are shown to the right.
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DNA samples from the remaining 7 patients with detectable alterations showed different patterns compared to normal controls when analyzed with EcoRI or Hindm (Figs, la and /3 respectively). Cleavage of DNA with EcoRI revealed five bands in normal controls with the sizes 9.4 kb, 8.1 kb, 7.2 kb, 6.6 kb and 3.7 kb. In one of the patients (patient no. 9, Fig. la) the 6.6 kb band was absent. In the remaining 6 patients with a deviant restriction pattern the 7.2 kb and 9.4 kb bands were missing and instead two bands of 11.5 kb and 5.8 kb were present (Fig. la). Hindm cleavage of normal controls revealed 8 different bands (9.4 kb, 6.6 kb, 4.8 kb, 4.7 kb, 2.9 kb, 2.7 kb, 1.6 kb and 0.7 kb) as depicted in Fig. lb. Two of these (9.4 kb and 6.6 kb) were absent in the 7 Hunter males with an altered restriction pattern. In one of the patients (patient no. 9) two new bands of 14.0 kb and 11.8 kb were revealed while new bands of 3.4 kb and 12.9 kb were seen in the remaining 6 patients. When these patients were analyzed with PstI (Fig. lc) structural alterations were detected in only one patient (patient no. 9) where a 8.0 kb band present in normal controls was absent.
Human Molecular Genetics, Vol. 1, No. 3 197 (4, 10) aberrant restriction patterns were observed in several patients after PstI cleavage of genomic DNA. However, in the analysis reported here all the structural alterations were observed when the genomic DNA was digested with HindlH or EcoRI while aberrant restriction patterns were detected in DNA from only one patient digested with PstI. The structural alterations observed after Hindlll and EcoRI cleavage are unlikely to TaWe 1. X chromosome deletion mapping of Hunter patients Locus
VK 21A VK 18A P B2Scl7 1110 U6.2
DXS DXS IDS DXS DXS
Patient DDR/GPI
DK/JTK 296 295 466 304
Xq-denved DNA markers used to define the breakpoints of the deletions in the two patients DDR/GPI and DK/TJK. DDR and DK indicate that the patients are from Germany and Denmark, respectively. GPI and TJK are the initials of the patients. The presence ( + ) and absence ( - ) of the different markers in the DNA from the patients are indicated.
B
9 1 2
9 1 2
ii(sT6 9 3
4
5
6
7
-23.1
ii®o 9 3
4
5
6
7 -23.1 -6.6 9.4 -4.4
-6.6
I - 4 .4 -2.3 -2.0
Figure 2. Southern hybridization of EcoRI (Fig.2a) and Hindlll (Fig. 2b) digested samples with the IDS cDNA probe. The pedigree of the affected and unaffected relatives are indicated above. The affected boy in lane 3 in Fig 2a and 2b is the same patient characterized as in Fig. 1 (patient no. 1). The sizes in kb of DNA markers are shown to the right.
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Probe
represent polymorphisms since no such polymorphisms have been detected previously with the probe (8). Furthermore, these mutations are more likely to represent rearrangements rather than single point mutations since aberrant restriction patterns were observed in these patients both when analyzed with EcoRI and HindlTJ. Interestingly, six of 7 patients showed similar or identical rearrangements which implicates a region prone to structural alterations within the IDS gene. These results are not likely to be caused by a founder effect since these patients represent different ethnic origins: two patients are from Poland and the remaining patients are from Czechoslovakia, Denmark, Finland and Norway. In addition, an Italian patient with a similar EcoRI rearrangement to those reported here has also been described recently (13). These structural alterations are most likely representing intragenic rearrangements since sequences representing the 5' end of the IDS gene (10) are present as well as 3' end sequences (Stee'n-Bondeson et al., unpublished results). Further characterization of this region is presently under investigation. Thus, in 20% of the Hunter patients reported here structural alterations of the IDS gene could be detected. Our results together with those of Wilson et al. (4,10) and Palmieri et al. (13), suggest
198 Human Molecular Genetics, Vol. 1, No. 3
MATERIALS AND METHODS DNA isolation and hybridization Forty-six unrelated Bulgarian, Czechoslovakian, Danish, Finnish, German. Hungarian, Italian, Jugoslavian, Norwegian, Polish, Portuguese and Spanish Hunter syndrome males with defective iduronate-2-sulphatase activity in cultured fibroblasts were studied. Genomic DNA was extracted from from normal human blood and cultured fibroblasts. Ten jig of genomic DNA was digested to completion with appropriate restriction enzymes (Pharmacia P-L Biochemicals) and fractionated by clectrophoresis on 0.8% agarose gels. Transfer onto nylon membranes (Hybond-N, Amersham) was performed according to the procedure of Southern (17). The probes were radiolabelled by random priming using an oligolabclling kit (27-9250-01, Pharmacia) and hybridized to the filters at 65° in a hybridization solution containing: 5 x SSPE (0 9M NaCI, 0.05M
NaPhosphate, pH 8.3, 5mM EDTA), 5 x Denhardt's buffer, 0.2% (w/v) SDS and 250 /tg/ml yeast RNA. The filters were washed 2h in 2xSSC, 0.5%SDS and30minin0.1xSSC, 0. l%SDSat65° and autoradiographed with intensifying screens. DNA probes For Southern blot analysis a 1760 bp fragment from the cDNA clone pB2Scl7 containing the entire coding region of the IDS (Wilson, unpublished) was used. In addition the following markers were used: DXS 295 (11), DXS 2% (11), DXS 466 (15) and DXS 304 (18).
ACKNOWLEDGEMENTS We thank Britt-Marie Carlberg for excellent technical assistance. We are grateful to Dr T.Hulsebos, University of Amsterdam for providing the marker II 10 and Dr G.R. Sutherland, Adelaide Children's Hospital, Australia, for sharing the markers VK18A and VK21 A. We would also like to thank colleagues who have referred their patients to us. Financial assistance for this project was provided by grants from the Beijer Foundation, the Swedish Medical Research Council and the Marcus Borgstrom Foundation.
ABBREVIATIONS Bp, basepair(s); IDS, iduronate-2-sulphatase; kb, kilobase(s) or 1000 bp; MPSII, mucopolysaccharidoses type II; no, number; RFLP(s), restriction fragment length polymorphism(s); SDS, sodium dodecylsulphate, SSC, 0.15M NaCI. 0.015M Na3. citrate, pH7.5.
REFERENCES 1. Young, I.D. and Harper, P.S. (1982) Hum. Genet., 60, 391-392. 2. Spranger, J. (1990) In Emery, A.E.H., Rimoin, D.L (eds). Principles and Practice of Medical Genetics. Vol. 2, pp 1800- 1802. Churchill Livingstone. 3. Bidicki, J , Freeman, C , Clements, P.R. and Hopwood, J J. (1990) Btochem. J., 271, 75-86. 4. Wilson, P.J., Morris, C.P., Anson, D.S., Occhiodoro, T., Bielicki, J., Clements, P.R. and Hopwood, J.J. (1990) Proc. Natl. Acad. Sci. USA, 87, 8531-8535. 5. Roberts, S.H., Upadhyaya, M., Sarfarazi, M and Harper, P.S. (1989) J. Med. Genet., 26, 309-313. 6. Upadhyaya, M., Sarfarazi, M., Bamforth, J.S., Thomas, N.S.T., Oberle\ I., Young, I. and Harper, P S. (1986) Hum. Genet., 74, 391 -398. 7. Thomas, N.S.T., Roberts, S.H., Upadhyaya, M., Knight, S. and Harper, P.S. (1989) Cytogenet. Cell Genet., 51, 1090. 8. Suthers, G.K., Oberle, I., Nancarrow, J., Mulley, J.C , Hyland. V.J., Wilson, P.J., McCure, J.. Morris, C.P., Hopwood, J.J., Manoel, J.L. and Sutherland, G.R. (1991) Genomks, 9, 37-43. 9. Suthers, G.K., Mulley, J . C , Voelckel, M.A., Dahl, N., Vaisanen, M.L., Steinbach, P., Glass, I A., Schwartz, C.E., van Oost, B.A., Thibodeau, S.N., Haites, N.E., Oostra, B.A., Gine, R., Carballo, M., Morris, C.P., HopwoodJ.J. and Sutherland, G.R. (1991) Am. J. Hum. Genet., 48, 460-467 10. Wilson, P.J., Suthers, G.K., Callen, 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. 11. Suthers, G.K., Hyland, V.J., Callen, D.F., Oberle\ I., Rocchi, M., Thomas, N.S., Morris, C.P., Schwartz, C.E., Schmidt, M.. Ropers. H.H., Baker, E., Oostra, B.A., Dahl, N.. Wilson, P.J., Hopwood, J.J. and Sutherland. G.R. (1990) Am. J. Hum. Genet., 47, 187-195. 12. Beck, M., Steglich, C , Zabel, B., Dahl, N., Schwinger, E., Hopwood. J.J. and Gal, A. (1992) Am. J. Med. Genet. In press. 13. Palmieri, G., Capra, V., Romano, G., D'Urso, M.. Johnson, S., Schlcssinger, D., Morris, P., Hopwood, J., Di Natale, P., Gatti, R. and Ballabio, A. (1992) Genomes, 12, 52-57. 14. Le Guern, E., Couillin. P.. Oberle. I., Ravise, N. and Boue, J. (1990) Genomics, 7, 358-362. 15. Hulscbos, T.J.M., Oostra, B.A., Broersen, S., Smits, A., van Oost, B.A. and Westerveld, A. (1991) Hum. Genet.. 87, 369-372. 16. Wraith, J.E., Cooper, A.. Thomley. M., Wilson, PJ., Nelson, P.V., Morris. C.P. and Hopwood, J.J. (1991) Hum. Genet., 87, 205-206. 17. Southern, E.M. (1975) J. Mol. Biol., 98, 503-517. 18. Dahl, N., Hammarstrom-Heeroma, K., Goonewardena, P , Wadelius. C . Gustavson, K.-H., Holmgren, G., van Ommen, G.J. B. and Pettersson, U. (1989) Hum. Genet., 82, 216-218.
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that most of the mutations (70-80%) in Hunter syndrome represent point mutations or small rearrangements of the IDS gene which escape detection by Southern analysis. The IDS cDNA probe pc2S15 has previously been reported to detect TaqI and StuI RFLPs in the IDS locus (8,9). Thus, this probe and other closely linked markers may provide useful information for carrier risk estimations in connection with Hunter syndrome (8, 14). In this study we have also demonstrated the potential use of the IDS cDNA in carrier detection analysis in families with a rearranged IDS gene. The altered hybridization pattern observed in Hunter patients is easily detectable in carriers, without use of flanking markers. In addition to point mutations and rearrangements, the IDS gene seems to be a target for different deletions. Three patients with complete deletion of the IDS coding sequences have previously been reported (4, 10, 12, 16). One of these (04-1; ref. 10) has a deletion extending proximally from the IDS gene including the locus DXS 296 (4, 10, 11). The deletion in the second patient is smaller and and does not include flanking loci (4, 10, 11). The third patient reported by Beck et al. (12) has a deletion extending distal from the IDS gene spanning the DXS 304 locus. As shown in Table I the two patients reported here both lack the locus DXS 466 which is located either within or distal to the IDS gene, since it is absent in the cell line CY34 derived from a girl with Hunter syndrome with a X:5 translocation (10, 15). Thus the distal breakpoints of the deletions are located between DXS 304 and DXS 466 in both DK/JTK and DDR/GPI. However, the proximal breakpoints differ. In patient DK/TJK it is located between DXS 295 and DXS 296 while it is positioned between IDS and DXS 295 in DDR/GPI. Clinically, both patient DK/TJK and DDR/GPI present the classical severe phenotype of Hunter syndrome. In addition, DK/JTK suffers from epileptic seizures. Epileptic seizures have also been described for the two patients reported by Wilson et al. (10) and Wraith et al. (16). A comparison of the breakpoints in these three patients suggests that a gene or genes located in the vicinity to the IDS gene may be involved in the development of epilepsy. Further investigation of location of the deletion breakpoints in DDR/GPI and DK/JTK is in progress. A more detailed phenotypic analysis of patients with different deletions together with a higher resolution map of the deletions may provide valuable information about the location of other genes in this region and their resulting phenotypes. A physical map of a 1.2Mb YAC contig spanning the IDS gene, recently reported by Palmieri et al. (13) reveals several putative CpG islands. This also indicates the presence of other genes in the vicinity of the IDS gene.
