Human Molecular Genetics, 2000, Vol. 9, No. 9 1321-1328
© 2000 Oxford University Press
Mutational analysis of the GPC3/GPC4 glypican gene cluster on Xq26 in patients with Simpson-Golabi-Behmel syndrome: identification of loss-of-function mutations in the GPC3 gene
Laboratory for Glycobiology and Developmental Genetics, 1Laboratory for Human Genome Analysis, 2University Hospital Leuven, Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, 3000 Leuven, Belgium, 3SKGZON, Department of Clinical Genetics, University Hospital of Maastricht, Maastricht, The Netherlands, 4Trondheim University Hospital, Department of Child and Adolescent Psychiatry, Trondheim, Norway and 5Departemento de Pediatria, Hospital de Clinicas, Universidade Federal do Parana, Parana, Curitiba, Brazil
Received 5 January 2000; Revised and Accepted 21 March 2000.
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ABSTRACT |
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Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked syndrome characterized by pre- and postnatal overgrowth (gigantism), which clinically resembles the autosomal Beckwith–Wiedemann syndrome (BWS). Deletions and translocations involving the glypican-3 gene (GPC3) have been shown to be associated with SGBS. Occasionally, these deletions also include the glypican-4 gene (GPC4). Glypicans are heparan sulfate proteoglycans which have a role in the control of cell growth and cell division. We have examined the mutational status of the GPC3 and GPC4 genes in one patient with Perlman syndrome, three patients with overgrowth without syndrome diagnosis, ten unrelated SGBS-patients and 11 BWS patients. We identified one SGBS patient with a deletion of a GPC3 exon. Six SGBS patients showed point mutations in GPC3. One frameshift, three nonsense, and one splice mutation predict a loss-of-function of the glypican-3 protein. One missense mutation, W296R, changes an amino acid that is conserved in all glypicans identified so far. A GPC3 protein that reproduces this mutation is poorly processed and fails to increase the cell surface expression of heparan sulfate, suggesting that this missense mutation is also a loss-of-function mutation. In three SGBS patients and in all non-SGBS patients, no mutations could be identified. We found three single nucleotide polymorphisms in the GPC4 gene but no evidence for loss-of-function mutations in GPC4 associated with SGBS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients with Simpson-Golabi-Behmel syndrome (SGBS, MIM: 312870) were first described in 1975 (1). This overgrowth syndrome is characterized by pre- and postnatal overgrowth, visceral and skeletal anomalies, ‘coarse’ face, supernummerary nipples, congenital heart defects and hypotonia (2). SGBS is also associated with an increased risk of developing embryonal tumours, mostly Wilms’ tumour and neuroblastoma (3,4). The spectrum of its clinical manifestations is broad, with phenotypes varying from very mild forms to infantile lethal forms (5). SGBS clinically resembles the Beckwith–Wiedemann syndrome (BWS) and Perlman syndrome (6,7). It is often the X-linked pattern of inheritance that indicates a diagnosis of SGBS (8). The gene for SGBS has been localised to Xq26 (9), and deletions and translocations affecting the glypican-3 gene, GPC3, which encodes a heparan sulfate proteoglycan of the glypican family, have been associated with SGBS (10).
Heparan sulfate proteoglycans are proteins that are substituted with heparan sulfate. Heparan sulfate is a complex polysaccharide that interacts with heparin-binding growth factors and influences the signalling activities of these factors (11). The glypicans compose a family of cell surface heparan sulfate proteoglycans that are linked to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. So far, six members of the glypican family are known in vertebrates (12,13). All glypicans share a characteristic cysteine motif, which is thought to result in a unique tertiary structure, and have consensus sequences for glycosaminoglycan attachment close to their C-termini. Glypicans play a role in the regulation of morphogen signalling (14). Mutations in dally, a glypican homologue in Drosophila, result in a disturbance of cell cycling and produce morphological defects in several adult tissues, including the eyes, antennae, wings and genitalia (15). Targeted deletion of Gpc3 in mice results in developmental overgrowth and some of the abnormalities typical of SGBS (16). Overexpression of glypican-3 induces apoptosis in a cell line-specific manner, and loss of this function might explain the overgrowth and anomalies like polydactyly and supernumerary nipples, which occur in SGBS (17).
Most mutations identified to date in SGBS are deletions that involve the GPC3 gene. In about 50% of patients with GPC3 deletions, the deletion involves exon-8 of GPC3 and extends to the centromere (18,19). We have recently identified one SGBS patient with a deletion that affects not only GPC3, but also GPC4, the gene for glypican-4, which flanks the centromeric end of GPC3 on Xq26 (20). A significant number of SGBS patients, however, have no detectable or gross GPC3 deletions. To clarify the relationship between these glypicans and SGBS we investigated whether mutations in GPC3 and/or GPC4 could be detected in SGBS patients. Since there is considerable overlap in clinical features between SGBS and other overgrowth syndromes, we also screened patients with BWS, Perlman syndrome and other unidentified forms of overgrowth. We screened the complete coding region of GPC3 and GPC4 by deletion polymerase chain reaction (PCR), single strand conformation analysis (SSCA) or direct sequencing of PCR-products and identified one SGBS-patient with a deletion of a GPC3 exon and six unrelated SGBS-patients with GPC3 point mutations. No point mutations in GPC4 could be detected, but we identified three polymorphisms in this gene.
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RESULTS |
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Deletion analysis
Deletion analysis by PCR, for all the GPC3 and GPC4 exons, identified one SGBS patient with a GPC3 deletion. Patient OG001 has a deletion of exon 7 of GPC3 (Fig. 1). This deletion leads to an aberrant glypican-3 transcript with a change in the reading frame in the terminal exon 8, introducing a stop codon. Moreover, the protein would lack the consensus sites for heparan sulfate attachment and GPI anchorage (Fig. 5). This deletion is therefore predicted to lead to a non-functional protein. None of the other patients showed deletions in GPC3 or GPC4.
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SSCA
DNA from patients, which had no demonstrable deletions of GPC3 or GPC4, was subjected to SSCA. The complete coding regions and all exon–intron boundaries of both genes were analysed. Seven patients showed aberrant migration patterns for GPC3 exons (Fig. 2A). Three exons from GPC4 also showed different migration patterns. However, these GPC4 patterns were also present in several controls, indicating the presence of polymorphisms (Fig. 2B).
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Sequencing of mutations/polymorphisms
All PCR products with aberrant migration patterns were directly sequenced and additionally TA-cloned and subsequently sequenced, leading to the identification of several point mutations (Table 1). Patient OG002 has a nonsense mutation in exon 2 (C65X).The patients AG0969 and OG003 have nonsense mutations in exon 3 (R199X in patient AG0969 and K340X in patient OG003). Patient AG0893 has a deletion of one nucleotide of exon 3 (957delT; base numbering is according to GenBank entry L47125) which predicts a frameshift in the transcript, leading to a premature stop codon and a truncated protein (Fig. 3). Patient GM13034 has a splice site mutation in exon 5 leading to the substitution of the consensus splice-donor site GT to TT. This mutation results in the substitution of the splice site with an AGT codon, coding for an arginine, followed by an in-frame stop codon (Fig. 3). Should a transcript be produced in this patient, the mutation would introduce a stop codon and result in a truncated protein. Unfortunately, the gene is not expressed in fibroblasts so we could not verify the effect of this mutation on the GPC3 mRNA. Patients AG0817 and AG0857 are cousins and both have the same mutation (T1076A) leading to the substitution of a conserved amino acid (W296R), which thus co-segregates with the disease. This amino acid is conserved in all known vertebrate glypicans and also in the Drosophila glypican dally, and a Caenorhabditis elegans glypican (Fig. 3). Three frequently occurring polymorphisms were found in the GPC4 gene, both in SGBS patients and control DNAs (T193C incidence: 7/17 chromosomes, T1385G incidence: 40/96 chromosomes, T1537C incidence: 40/96 chromosomes). Two of these polymorphisms give rise to conservative amino acid changes (D391E and V442A) in the GPC4 protein. The other polymorphism (T193C) is located in the 5'-untranslated region.
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Expression analysis of mutant GPC3
To test whether the T1076A missense mutation would impair the function of the glypican-3 protein, an expression vector was constructed with a GPC3 cDNA that reproduced the T1076A missense mutation. Namalwa cells were transfected with wild-type and mutant glypican-3 cDNAs, and after selection, analysed for the cell surface expression of heparan sulfate by fluorescence activated cell sorting (FACS) analysis. Namalwa cells express almost no endogenous heparan sulfate and we have previously shown that transfection of these cells with glypican cDNAs results in a large increase in the expression of heparan sulfate on the cell surface, as measured by quantitative cytofluorimetry (13). Namalwa cells transfected with wild-type GPC3 expressed >8-fold higher levels of heparan sulfate than control transfectants (empty pREP4). Namalwa cells transfected with cDNA for the mutant W296R GPC3, in contrast, showed almost no increase in heparan sulfate expression compared with the control (Fig. 4A).
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-heparan sulfate expression in Namalwa cells analysed by FACS. Non-digested and heparitinase-digested hygromycin-resistant cells were stained with the monoclonal antibodies 10E4 and 3G10. The antibody 10E4 recognizes native heparan sulfate chains while 3G10 reacts with the desaturated uronates that are generated by heparitinase and that remain in association with the core protein after the enzyme treatment. (Top: control) Namalwa cells transfected with empty pREP4 show little expression of heparan sulfate (recognized by 10E4) or To confirm this at the proteoglycan-level and also in other cell types, we also constructed epitope-tagged forms of wild-type glypican-3 and of the W296R mutant, and expressed these constructs in Madin–Darby canine kidney (MDCK) cells (Fig. 4B). Epitope-tagging confirmed that similar amounts of wild-type and mutant glypican-3 protein were expressed in these cells (not shown). Comparison of the detergent extracts of these cells, and assessment of the fraction of the tagged proteins in these extracts that accumulated as proteoglycan [as defined by diethylaminoethyl (DEAE-binding) and susceptibility to glycosidases] confirmed that the W296R mutant was much less substituted with glycosaminoglycan than the wild-type protein. Indeed, most of the wild-type protein bound to DEAE, yielding protein cores of ~65 kDa and 130 kDa after heparitinase and chondroitinase ABC digestion (migration as a series of staggered bands, due to a propensity for self-aggregation, is a common feature of cell surface proteoglycan core proteins). The mutant protein, in contrast, mostly failed to bind to DEAE. It migrated as two discrete bands of 65 kDa and 130 kDa (mimicking the two main bands observed after glycosidase digestion of the wild-type proteoglycan). Only a small fraction was substituted with glycosaminoglycan. Moreover, a significant fraction of the W296R mutant was not extracted by detergent.
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DISCUSSION |
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Molecular genetic analysis has greatly increased our knowledge of overgrowth syndromes. For example, it has been shown that multiple genes are causally related to BWS. This overgrowth syndrome can be caused by biallelic insulin-like growth factor (IGF-2) overexpression or point mutations in the p57Kip2 gene, while in a small number of patients, other genes and imprinting centres are involved (21). Although the first association of SGBS with GPC3 was made based on deletions and translocations in GPC3, which could also affect neighbouring genes (as has been shown for GPC4) and intron-embedded genes, our study strengthens this association by identifying point mutations in GPC3 in more than half of ten unrelated SGBS patient samples. Although we previously identified one SGBS patient with a GPC4 deletion (in addition to a partial GPC3 deletion), we did not find SGBS patients with a mutation in GPC4 only, suggesting that mutations in GPC4 only are not associated with SGBS.
To our knowledge this is the first study of a larger series of SGBS patients, where point mutations could be identified. Previously, one SGBS family with a micro-deletion of a GPC3 exon has been described. This deletion introduces a frameshift, resulting in premature termination of the protein (22). Recently, a sporadic case of SGBS was shown to be caused by the deletion of one nucleotide in exon 7 of GPC3 (23). Most GPC3 point mutations identified in the present study are found in exon 3, but this is also the largest exon (~40% of the coding region). All mutations are randomly distributed over the coding region with no evidence for a mutation hotspot (Fig. 5). Previous studies identified deletions associated with SGBS in all exons of GPC3 (18,19).
All GPC3 exonic deletions identified to date either remove the proper start codon, or introduce premature stop codons, should a stable message be produced. These premature terminations would lead to a truncated protein, which would lack in most cases one or more cysteines from the evolutionarily conserved cysteine motif, thought to be important for the unique tertiary structure of the glypicans. In addition, all these mutant proteins would lack the consensus sites for heparan sulfate attachment, which occur near the C-termini of the protein, resulting in a failure of heparan sulfate substitution. They would also lack the signal sequence for GPI attachment and cell surface anchorage. Likewise, the frameshift, nonsense and splice mutations identified in this study introduce premature stop codons in the sequence. Therefore all these mutations can be classified as complete loss-of-function mutations. From this perspective, the W296R mutation is of peculiar interest. It alters an amino acid that is conserved in all known vertebrate glypicans, including the Drosophila glypican dally, and a C.elegans glypican (Fig. 3). Such strict evolutionary conservation and the fact that it causes the SGBS phenotype upon mutation suggest that this mutation affects an amino acid that is critical for the function of the glypican-3 protein. From the analysis of the expression of recombinant glypican-3 proteins, we have evidence that the W296R mutation indeed results in a loss-of-function. Whereas wild-type glypican-3 is able to induce the expression of heparan sulfate on the cell surface of Namalwa cells, the mutant protein is not. This would indicate that the mutant protein does not accumulate in cells and/or at the cell surface, or that the mutant protein is produced, but that it can not be substituted with heparan sulfate. Further evidence in MDCK cells indicates that most of the mutant protein is indeed not properly substituted with heparan sulfate. Although the molecular basis for this defective processing remains to be clarified, the protein would appear to be severely reduced in its ability to regulate growth factor signalling, which is dependent on the presence of heparan sulfate on the core protein. It has been shown that glypican-1 can modulate fibroblast growth factor (FGF) signalling and that this is dependent on the presence of heparan sulfate chains (24). Glypican-3 is also able to bind FGF-2 through its heparan sulfate chains (25). How glypican-3 functions in development remains unclear, but all together, our results strongly suggest that the W296R mutation results in a less or non-functional protein.
Including the two previously mentioned patients (20), we identified exonic GPC3 deletions in only three out of ten molecularly confirmed unrelated cases of SGBS, confirming the observation that exonic deletions make up only a smaller part of SGBS cases (19). No mutations were found in three patients diagnosed as SGBS. These patients also did not show mutations in GPC4. It remains possible that these patients have a reduced expression of GPC3 (caused by promotor mutation or silencing of the gene). On the other hand, a new locus for a severe form of SGBS has recently been mapped to Xp22 (26). Although our SGBS patients did not show a severe form of SGBS, it is possible that these patients have mutations in the Xp22-linked SGBS gene. It has previously been concluded that no clear genotype–phenotype correlations can be made in SGBS (18). Although all mutations identified in this study are predicted to result in a loss-of-function, there is some phenotypic variability in SGBS. This may be explained by the presence of ‘modifier genes’ affecting the phenotype.
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MATERIALS AND METHODS |
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Patient samples
Genomic DNA was obtained from the lymphoblastic cell lines AG0817, AG0857, AG0893, FY0367 and AG0969 (European Collection of Animal Cell Cultures, Salisbury, UK); and from the fibroblastic cell line GM13034 (American Type Culture Collection, Manassas, VA), all from patients with diagnosis of SGBS (FY0367 with an uncertain SGBS diagnosis). With informed consent from the parents, we also screened DNA from patients with various overgrowth syndromes, examined at the Center for Human Genetics, University of Leuven, or elsewhere: one Perlman syndrome patient, two patients with overgrowth without syndrome diagnosis, six SGBS patients and 11 BWS patients. A summary of the phenotypes of the SGBS patients is described in Table1.
SSCA
Primers for the amplification of all GPC3 exons have been described (27). In addition, we also developed a set of intronic primers for optimal mutational analysis of GPC3 (Table 2A). Primer pairs designed for the amplification of all GPC4 coding exons were based on the characterisation of the exon–intron boundaries of the GPC4 gene (Table 2B). All patient DNAs were subjected to deletion analysis by PCR as previously described (20). Patient DNAs were also subjected to SSCA using fluorescent GPC3 and GPC4 primers. The reaction cycles were: 94°C for 30 s, annealing at 55°C for 30 s, 72°C for 30 s, for 35 cycles. Cycling was preceded by a 2.5 min incubation at 94°C. After 5 min denaturation at 95°C, PCR products were run overnight at 4°C, 400 V in 0.75% MDE–polacrylamide gels containing 5% glycerol as previously described (28). Gels were scanned using a Fluorimager (Molecular Dynamics, Sunnyvale, CA).
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Sequencing
PCR products with aberrant migration patterns were sequenced, either directly, after precipitation, or after T/A cloning in the vector pCR2.1 (Invitrogen). Both DNA strands were sequenced and, in the case of mutations, confirmed by repeating the PCR and sequencing. PCR-products for most GPC3 exons from the three SGBS patients, which did not show any deletions or aberrant migration in SSCA were also sequenced.
Expression plasmids, cell transfection, FACS and western blotting
The complete GPC3 cDNA was inserted into the KpnI site of the episomal vector pREP4, yielding the plasmid glyp3-pREP4. The missense mutation T1067A was introduced into the wild-type cDNA by Quickchange (Stratagene, La Jolla, CA) using the primers 5'-GGAGATTGACAAGTACAGGAGAGAATACATTC-3' and 5'-GAATGTATTCTCTCCTGTACTTGTCAATCTCC-3'. The complete mutant cDNA was resequenced to verify that only the SGBS mutation was present.
Namalwa cells (ATCC CRL 1432) were routinely grown in DMEF12 medium supplemented with 10% FCS and L-glutamine. For transfection, the cells were prewashed with Ca2+- and Mg2+-free phosphate buffered saline (PBS) and incubated for 10 min at 4°C (107 cells in 1 ml Ca2+/Mg2+-free PBS with 30 µg plasmid before electroporation at 240 V and 960 µF (Gene Pulser, Bio-rad, Nazareth, Belgium). Selection was started 48 h later with 250 µg/ml of hygromycin B. Stable transfectants were obtained after 12 days. Expression of heparan sulfate on the cell surface of the transfectant cells was analysed by FACS, using the monoclonal antibodies 10E4 and 3G10, as described before (13). The analyses were performed on a FACSort and data were analysed with the program Lysis II.
HA-epitope-tagged froms of wild-type and W296R mutant glypican-3 were constructed by subcloning the SmaI–BamHI restriction fragments from GPC3-pREP4 and GPC3W296R-pREP4, encoding the amino acid residues 22–580 of the proteins, into the blunted BglII site of the pDisplay vector (Invitrogen, Groningen, The Netherlands). Cell transfections and proteoglycan analyses (extractions, DEAE binding, enzyme digestions and western blotting) were as reported before (13,24). Briefly, stable transfectants were selected by subculture in medium supplemented with 500 µg/ml G418. GPC3-expressing drug-resistant clones were identified by immunocytochemistry and western blotting, using the monoclonal anti-HA antibodies 12CA5 and 3F10 (Boehringer Mannheim, Brussels, Belgium). Expressing cell clones were expanded and extracted with 1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl, pH 8.0, supplemented with a mixture of proteinase inhibitors (13), followed by an extraction with 60 mM octylglucoside in the same buffer. The detergent extracts were fractionated by ion-exchange chromatography over DEAE. Part of the fraction that was eluted from DEAE was digested with heparitinase and chondroitinase ABC. The detergent-insoluble fraction was scraped in SDS. All samples were fractionated by SDS–PAGE, blotted and stained with the anti-HA antibody 3F10.
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ACKNOWLEDGEMENTS |
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We thank all the patients and the families that participated in this study; Mrs E. Schollen for helpful discussions and suggestions about the SSCA; Dr G. Matthijs for a critical reading of the manuscript. We also thank Dr. M. Ireland (University of Newcastle upon Tyne, UK) for providing the clinical information relating to the patient DNA samples obtained from the ECACC. M.V. and B.D.C. are beneficiaries of a fellowship from the Vlaams Instituut voor de bevordering van het Wetenschappelijk – Technologisch onderzoek in de Industrie. G.D. is a research director of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. This work was supported by grants G.0234.95 and G.0219.99 from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, by the Geconcerteerde Onderzoeksacties 1996–2000, by the Interuniversitary Network for Fundamental Research sponsored by the Belgian Government, and by the Flanders Interuniversity Institute for Biotechnology.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Center for Human Genetics, University of Leuven, Campus Gasthuisberg, O&N6, Herestraat 49, B-3000 Leuven, Belgium. Tel: +32 16 345863; Fax: +32 16 345997; Email: guido.david@med.kuleuven.ac.be
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