Positional cloning of a gene involved in hereditary multiple exostoses
Positional cloning of a gene involved in hereditary multiple exostosesWim Wuyts, Wim Van Hul*, Jan Wauters, Marina Nemtsova1, Edwin Reyniers, Els Van Hul, Kristel De Boulle, Bert B. A. de Vries2, Jan Hendrickx, Ilde Herrygers, Paul Bossuyt, Wendy Balemans, Erik Fransen, Lieve Vits, Paul Coucke, Norma J. Nowak3, Thomas B. Shows3, Laurence Mallet4, Ans M. W. van den Ouweland2, Julie McGaughran5, Dicky J. J. Halley2 and Patrick J. Willems
Department of Medical Genetics, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium, 1Research Center for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia, 2Department of Clinical Genetics, Erasmus University, Rotterdam, The Netherlands, 3Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA and 4Unité de Recherches sur les Handicaps Génétiques de l'Enfant, Inserm U393, Hôpital des Enfants-Malades, Paris, France
Received July 23, 1996;Revised and Accepted July 29, 1996
Hereditary multiple exostosis (EXT) is an autosomal dominant condition mainly characterized by the presence of multiple exostoses on the long bones. These exostoses are benign cartilaginous tumors (enchondromata). Three different EXT loci on chromosomes 8q (EXT1), 11p (EXT2) and 19p (EXT3) have been reported, and recently the EXT1 gene was identified by positional cloning. To isolate the EXT2 gene, we constructed a contig of yeast artificial chromosomes (YAC) and P1 clones covering the complete EXT2 candidate region on chromosome 11p11-p12. One of the transcribed sequences isolated from this region corresponds to a novel gene with homology to the EXT1 gene, and harbours inactivating mutations in different patients with hereditary multiple exostoses. This indicates that this gene is the EXT2 gene. EXT2 has an open reading frame encoding 718 amino acids with an overall homology of 30.9% with EXT1, suggesting that a family of related genes might be responsible for the development of EXT.
Multiple exostoses syndrome (EXT) is a heritable skeletal disorder of enchondral bone formation. The cardinal clinical symptom is the presence of multiple exostoses, most typically located in the metaphyseal bone cortex adjacent to the epiphyseal plate of long bones such as femur, tibia, humerus, ulna and radius. Exostoses are cartilage-capped osseous projections emanating from the bones which are rarely present at birth, but gradually develop and increase in size in the first decade of life (1 -9 ). Pathologic examination of these exostoses revealed that they consist of benign enchondromata (2 ,4 ). Apart from the typical exostoses, patients with EXT have a generalized defective (re)modeling of bone, especially of the metaphyses of long bones. This leads to abnormal skeletal growth resulting in short stature, and abnormal bone formation, which is responsible for club-shaped metaphyseal widening, cortical irregularities and in some cases gross deformities, especially of the forearm (2 -11 ). Sarcomatous degeneration of one of the enchondromata to chondrosarcoma or osteosarcoma occurs in ~2% of cases (7 -9 ).
Multiple exostoses has an estimated prevalence of 1/50 000 (7 ). It can occur as an isolated condition with autosomal dominant inheritance (EXT), or as part of microdeletion syndromes. EXT is an autosomal dominant disorder with incomplete penetrance and a skewed sex-ratio with more affected males than females. It is genetically heterogeneous with at least three loci. The EXT1 gene on chromosome 8q24.1 has recently been identified (12 ), whereas two other genes, EXT2 and EXT3, have been mapped to chromosomes 11p11-p12 (13 ,14 ) and 19p (15 ) respectively. As most EXT families show linkage to EXT1 or EXT2, the EXT3 locus is probably a minor locus (16 ). Although the EXT1 gene has been cloned, no homology to any other known gene was found, thereby hampering the elucidation of its function (12 ). However, as some chondrosarcoma and osteosarcoma show loss-of-heterozygosity (LOH) of the EXT regions on chromosomes 8 and 11, the EXT genes might be tumor-suppressor genes (17 -19 ). The EXT1 and EXT2 genes are also involved in two distinct microdeletion syndromes, both characterized by the presence of multiple exostoses. One copy of the EXT1 gene is constitutionally deleted in the Langer-Giedion syndrome, which is a contiguous gene syndrome caused by deletion of both the EXT1 and the TRPS I gene (12 ,20 -22 ). Also EXT2 has been implicated in a microdeletion syndrome, DEFECT 11 syndrome (23 -25 ). Patients with DEFECT 11 syndrome show interstitial Deletions of the proximal part of chromosome 11p encompassing the EXT2 candidate region, Enlarged parietal Foramina (FPP), multiple Exostoses, Craniofacial dysostosis and mental reTardation. It is possible that the DEFECT 11 syndrome is a contiguous gene syndrome resulting from the deletion of different genes, including EXT2 and FPP. This hypothesis is supported by the fact that also FPP, in analogy to EXT2, can occur as an isolated condition with an autosomal dominant mode of inheritance (26 ,27 ).
In this study we constructed a contig of YAC and P1 clones of the EXT2 candidate region. One of the transcribed sequences isolated from this region showed homology to the EXT1 gene and was found to harbour different inactivating mutations in patients with EXT2, thereby indicating that this gene is the EXT2 disease gene.
Linkage studies in large multiplex EXT2 families and analysis of key recombinational events have previously defined a minimal cosegregating region or genetic candidate region (14 ) between the anonymous microsatellite markers D11S1355 (telomeric side) and D11S1361/D11S554 (centromeric side). On the Généthon map (28 ) this region is ~3 cM. Markers D11S578, D11S1393 and D11S2095 are not integrated on this map, but were localized between D11S1355 and D11S1361 by PCR analysis on our YAC/P1 panel (see below), which is in agreement with the radiation hybrid map (29 ). Analysis of key recombinants in our EXT2 families revealed no crossover between EXT2 and these markers. One EXT2 family with a small molecular deletion only detected by D11S903 has been reported (16 ,18 ), but a detailed delineation of this deletion has not yet been reported. Deletions of the EXT2 region are also present in patients with Defect 11 syndrome (23 -25 ). In nearly all cases these deletions comprise the complete EXT2 candidate region as delineated by linkage analysis (23 -25 ). However, molecular analysis of a patient with characteristics of DEFECT 11 syndrome, including multiple exostoses, foramina parietale permagna (FPP), mental retardation and micropenis (30 ), showed a maternal deletion with a distal breakpoint between D11S914 and D11S915, and a proximal deletion breakpoint between D11S2095 and D11S935. As D11S2095 and markers centromeric of D11S2095 are not deleted in this patient with multiple exostoses, the candidate EXT2 region can be refined to the interval between D11S1355 and D11S2095 (Fig. 1 ).
To construct a yeast artificial chromosome (YAC) contig spanning the EXT2 candidate region, we first searched in the CEPH YAC database for YACs that were positive for microsatellite markers located in the EXT2 candidate region. For D11S1355, the distal boundary of the EXT2 candidate region, several YACs were present in the database (Table 1 ). Only two CEPH YACs (798A12 and 923B11) could be identified for D11S903, a marker located within the candidate interval. D11S2095, the flanking marker on proximal side was not present in the CEPH YAC database (Fig. 1 ). To extend this set of YACs we screened the CEPH, ICRF libraries and a chromosome 11-specific YAC library (31 ) with the markers mentioned above, and additionally also with D11S578 and D11S1393. The presence of these markers on the YACs was analyzed by PCR and hybridization. This resulted in the construction of a YAC contig bridging the complete EXT2 candidate region between D11S1355 and D11S2095 (Table 1 and Fig. 1 ). The EXT2 region is encompassed by two overlapping YACs with a size of 300 kb (YAC 650E6) and 400 kb (YAC 1B10) respectively. Therefore, the EXT2 candidate region is <700 kb assuming that no deletions in those two YACs are present. YAC 923B11 has a large size (1690 kb), but this might be due to its chimeric nature as it contains genomic fragments from chromosomes 2, 6 and 11. However, since all microsatellite markers within the EXT2 candidate region with the exception of D11S1355, were found to be present in YAC 923B11, this YAC was used for the screening of a P1 library.
Using DNA from the YAC 923B11 as a probe, 20 P1 clones were selected from gridded P1 membranes (ICRF). As it was not evident that these P1 clones are derived from chromosome 11p in view of the chimeric character of this YAC, we performed FISH analysis on metaphase chromosomes. P1 clones were mapped relative to cosmid ICRFcE07123, which is positive for D11S1355 and cosmid cCI11-388, which is positive for D11S1361. Out of the 20 clones 13 could be mapped back to the EXT2 region between D11S1355 and D11S1361. The relative ordering of these various P1 clones was obtained on interphase nuclei and by fiber FISH. PCR analysis of these 13 clones with D11S578, D11S1393, D11S903 and D11S2095 revealed six positive clones. Two clones were positive for D11S578 (M1637, H1623), two for D11S1393 (O1366, P14117), one for D11S903 (A1151) and one for D11S2095 (D0694).
cDNA selection experiments were performed to isolate cDNA clones from the EXT2 candidate region. Fragmented cDNA with a size of ~500 base pair (bp) obtained from human liver tissue was enriched for fragments from the EXT2 candidate region by hybridization in different rounds to P1 DNA. P1 clones used in this experiment were M1637, H1623, O1366, P14117, A1151 and D0694, all positive for one of the markers from the candidate region. After subcloning of the eluted fragments, clones were hybridized to these P1s to confirm their localization within the candidate region.
Hybridization of one of the cDNA selection clones, cDNAS, to the Soares' foetal brain cDNA library (34 ) resulted in a single positive cDNA clone, 288f10 (Fig. 1 ). The 1813 bp sequence of this clone showed an open reading frame (ORF) coding for 224 amino acids. Since clone 288f10 showed homology at the protein level with the EXT1 gene, it might represent part of the EXT2 cDNA, and was therefore pursued.
Family 1 is a large EXT family originating from Belgium. This family was used together with family 2 to map the EXT2 gene on chromosome 11 (13 ), and to refine the linkage interval afterwards (14 ). Lod scores for linkage with the EXT2 linkage interval exceed 3 in this family. SSCP analysis of a genomic PCR fragment of 463 bp amplified with primers A3 and A9 (Table 2 ) and digested with restriction enzyme SAU3AI, revealed an abnormal pattern which was present in all affected patients, but not in the unaffected family members nor in controls (Fig. 6 A). Direct sequencing of this PCR fragment showed heterozygosity for a C -> T mutation at nucleotide position 514 of the cDNA (Fig. 6 B). This mutation creates a stop codon TAA at amino acid position 172 (Q172X), leading to a severely truncated EXT2 protein. To confirm the C -> T mutation, a modified PCR reaction was developed. Using primers A4 and M1, a 117 bp fragment containing the mutation site was amplified. The mismatched T in primer M1 introduces an NdeI restriction site in the mutated sequence. Digestion with NdeI yields two fragments of 89 and 28 bp if the mutation is present, and a single 117 bp fragment if the mutation is absent. All patients of family 1 showed, in addition to the normal 117 bp amplification product, the fragments associated with the mutation, while unaffected family members and control individuals showed only the normal 117 bp fragment. Therefore, this nonsense mutation is the disease-causing mutation in this family.
SSCP analysis performed so far on patients from family 2 did not show any abberation yet, but further analysis is still in progress.
Family 3 is a multiplex EXT family of Belgian descent which shows linkage to the EXT2 region (maximal lod scores of 2.30 with D11S1393 at zero recombination). SSCP analysis of a 293 bp RT-PCR product from Epstein-Barr virus (EBV)-transformed lymphoblasts amplified by primers 96A and 96E (Table 2 ) showed an aberrant pattern in this family, not seen in any of the controls. Sequencing revealed the presence of two amplification products, one of expected length and one showing a 94 bp deletion, which interrupts the reading frame and results in a premature stop codon. As both a genomic deletion and aberrant splicing due to a splice site mutation could cause this cDNA deletion, we determined the genomic sequences surrounding these 94 bp. Therefore, P1 clones A1151 and D0494 were subcloned and the 96A-96E fragment was hybridized to the subclones. Sequence analysis of positive clones showed that one intron is located between nucleotides G1079 and A1080, whereas a second intron was found between nucleotides G1173 and G1174. This suggests that the 94 bp cDNA deletion corresponds to a single exon, and that exon skipping due to the presence of a splice site mutation occurs in this family. To prove this hypothesis, intron primers I2 and I4 were designed to amplify the full exon with its boundaries and direct sequencing of this amplification product was performed in two affected patients from family 3. This revealed a g -> a mutation at position +1 in the 5' splice site of the intron following the 94 bp exon (Fig. 7 ). Mutation of the completely conserved g to a at position +1 of the 5' splice site of the intron results in skipping of the 94 bp exon between cDNA nucleotides A1080 and G1173 and creates a frame shift after amino acid R360, which is followed by 43 novel amino acids and a premature stop codon at amino acid position 404 (Fig. 7 ). The mutation therefore yields a truncated EXT2 protein, indicating that this splice site mutation is disease-causing.
EXT2 mutation analysis was performed in three large multiplex families with multiple exostoses linked to the EXT2 region. Two of these families, family 1 of Belgian descent and family 2 of Dutch origin, have been reported before (13 ,14 ). From family 3, also originating from Belgium, DNA is available from 19 individuals, eight of them being affected with EXT. For several patients from these three families, RNA was also available.
The patient has WAGR syndrome with Wilms' tumor, aniridia, genital abnormalities and mental retardation. Clinical aspects of this patient have been described previously (30 ). The patient also shows multiple exostoses, and retrospectively was recognized by us to have the DEFECT 11 syndrome with multiple exostoses (EXT), enlarged foramina parietale (FPP) and mental retardation. Chromosomal studies revealed an interstitial deletion 11p11.2-p14.2. Molecular analysis was performed on DNA of the patient, his mother and his sister, but no DNA from the father was available. Chromosome 11 markers D11S1361, D11S2095, D11S903, D11S1355, D11S935, D11S914 and D11S915 were analyzed by PCR.
Southern blotting to Hybond N+ membranes (Amersham) was performed according to the recommendations of the manufacturers. Probes were labeled using the T7 QuickPrime Kit (Pharmacia). Hybridization and washing were performed using standard laboratory protocols. Hybridization of EXT2 cDNA yf69b06 to a commercially available Northern blot from Clontech was performed as recommended by the manufacturers.
The chromosome 11-specific YAC library was screened extensively by PCR with markers D11S1355, D11S578 and D11S903 using primers and annealing temperatures as described in GDB. The ICRF (43 ) and CEPH YAC libraries, a chromosome 11-specific cosmid library (ICRF) and a P1 library (ICRF) were screened by hybridization respectively, with an STS from D11S1355 and with YAC 923B11. The names of the ICRF P1 clones are abbreviated in this paper and should be preceded by the prefix ICRFP700 in order to obtain the full clone name. Cosmid ICRFc107E07123 is abbreviated to E07123.
Genomic DNA from EXT2 patients, the DEFECT11 patient, family members and controls was extracted from leukocytes by standard techniques. YAC DNA in solution was prepared according to standard laboratory protocols. To isolate the artificial chromosome from the yeast chromosomes, YAC 923B11 agarose plugs were prepared for loading on a preparative pulsed field gel. YAC 923B11 was grown in AHC medium, and 80 [mu]l 0.5% agarose plugs containing 1 * 108 cells were prepared. Plugs were incubated for 2 h at 37oC in CPE (40 mM citric acid, 120 mM Na2HPO4 and 20 mM EDTA, pH 8.0) containing 0.5 mg/ml zymolyase. Plugs were washed in TE-4, incubated for 48 h at 50oC in 0.5 M EDTA (pH 8.0) containing 1 mg/ml proteinase K and 1% N-lauroylsarcosine, washed again and stored in 0.5 M EDTA (pH 8.0) at 4oC. YAC 923B11 DNA was isolated by preparative pulsed field gel electrophoresis in 1% agarose gels in 0.5* TBE using a CHEF-DR III apparatus (BIO-RAD) at 6 V/cm for 38 h and an increasing switch time from 80 to 147 s. DNA from P1 clones, cosmids and plasmids were all prepared using the Qiaprep Spin Kits (Qiagen).
FISH and immunocytochemical detection of P1 and cosmid clones on mitotic chromosome spreads obtained from peripheral blood lymphocytes, were carried out as described previously (44 ), with R-banding according to Cherif et al. (45 ). Interphase FISH mapping was performed using the alkaline-borate treatment (46 ) at 1 mM borate concentration. For fiber FISH the procedure of Parra and Windle (47 ) was followed. An Olympus BX40 was used for fluorescence microscopy and the results were photographed on a PSI-probemaster 3 station.
Protocols used for cDNA selection were as described (48 ) with modifications. Pooled P1 DNA (1 [mu]g) was biotinylated by nick translation. This DNA was hybridized to cDNA obtained from human liver tissue that had previously been randomly primed and ligated to adaptors (B. Korn, unpublished information). Human cot-1 DNA and P1 vector DNA were added as competitors during this hybridization. Hybridization was performed at 65oC for 16 h. Heteroduplexes of genomic templates and cDNA were immobilized using Streptavidin-coated magnetic beads (Dynal). After washing of the magnetic beads with 0.1* SSC, the cDNA fragments are eluted by denaturation of the heteroduplexes at 95oC for 5 min. Eluted fragments were selected according to size using Chromaspin400 columns (Clontech), and amplified by PCR using the SK primer (5'-GCC GCT CTA GAA CTA GTG GAT C-3'). A second round of cDNA selection was performed using the SK-U primer (5'-CUA CUA CUA CUA GCC GCT CTA GAA CTA GTG GAT C-3'). Finally, amplified cDNAs were cloned using the CloneAmp System from BRL, and transformed into Escherichia coli DH5[alpha] cells.
5' RACE experiments were performed using the Human Leukocyte Marathon-Ready cDNA kit (Clontech) according to the recommendations of the manufacturer. Thirty PCR cycles of 30 s at 94oC, 30 s at 63oC and 4 min at 68oC were performed using adaptor primer AP1 and primer L2 (5'-CCATggACACTTCATTCgTCCACTCAgACTC-3') located in EXT2 cDNA clone yf69bo6 (Fig. 1 ). A second RACE experiment was performed with a nested PCR using adaptor primer AP2 and primer C2 (5'-ggtgattcgtacctcgatcccacc-3'). The amplification products were subcloned into pUC18 vector with the Sureclone Ligation Kit (Pharmacia), and transformed into competent E.coli DH5[alpha] cells. The size of the inserts of the clones was estimated by PCR and gel electrophoresis, and the 5' RACE products were sequenced.
Total RNA was isolated from EBV-transformed cell lines with trizol according to the manufacturer's instructions (Gibco BRL). For the synthesis of cDNA, 5 [mu]g of total RNA was reverse transcribed with an Oligo(dT)12-18 primer using the SuperScript Preamplification System (Gibco BRL). The cDNA products were directly used as templates for PCR amplification.
SSCP analysis of PCR (DNA) and RT-PCR (cDNA) fragments was performed according to standard protocols using HydroLink MDE Gel (J. T. Baker). Sequence analysis, based on the dideoxy method, was performed by dye terminator sequencing with Taq polymerase on an ABI-373 sequencer (Perkin-Elmer). For direct sequencing, PCR or RT-PCR fragments were purified from agarose gels with Spinbind columns (FMC). Sequencing reactions on PCR or RT-PCR products were performed with one of the amplification primers, whereas plasmid inserts were sequenced with vector primers.
Homology searches were performed with different BLAST programs, BLASTN, BLASTX, TBLASTN and TBLASTX (35 ). The PROSITE and Tmpred packages were used for protein analysis by direct access to the servers via the worldwide web (http://www.ebi.ac.uk/searches/prosite.html).
We are grateful to E. Bakker, M. Bühler, M. T. Dotti, P. De Jonghe, A. De Schepper, J. Dumon, B. Hamel, B. Horsthemke, C. Lagey, M. Le Merrer, H.-J. Ludecke, E. J. Meijers-Heijboer, L. Messiaen, F. Mollica, G. Mortier, A. Munnich, U. Pazzaglia, B. S. Sayli, F. Van Hoenacker, I. Van Riet and D. Zalatajev for collection of multiple exostoses families. We are indebted to B. Korn and A. Poustka for assistance with cDNA selection during the EMBO practical course, B. Oostra and H. Heus for cDNAs, S. Ramlakhan and E. de Laat for technical assistance, the Reference Library (Imperial Cancer Research Fund) for providing gridded membranes and clones, D. LePaslier (Centre d'Etudes du Polymorphism Humain) for providing CEPH YACs, the Japanese Cancer Research resources Bank for cosmid cCI11-330, F. Speleman and H. Van Roy for initial FISH experiments, B. Eussen for preparing the fiber-FISH illustrations, and R. Bernaerts and R. Kempenaers for secretarial assistance. This research is partly supported by an NFWO grant to W. Van Hul, a Concerted Action to P. J. Willems and an NIH grant CA63333 to N. Nowak and T. Shows. W. Wuyts is an aspirant of the IWT (Vlaams Instituut voor de bevordering van het Wetenschappelijk-Technologisch onderzoek in de industrie).
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