© The Author 2007. Published by Oxford University Press.
ARTICLES |
The Role of EXT1 in Nonhereditary Osteochondroma: Identification of Homozygous Deletions
Affiliations of authors: Departments of Pathology (LH, AY, AMCJ, JVMGB, PCWH), Molecular Cell Biology (KS, JK), and Orthopedic Surgery (AHMT), Leiden University Medical Center, Leiden, The Netherlands; Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands (MVD, HVD)
Correspondence to: Pancras C. W. Hogendoorn, MD, PhD, Department of Pathology, Leiden University Medical Center, PO Box 9600 L1-Q, 2300 RC Leiden, The Netherlands (e-mail: p.c.w.hogendoorn{at}lumc.nl).
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ABSTRACT |
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Background: Multiple osteochondromas is a hereditary syndrome that is characterized by the formation of cartilage-capped bony neoplasms (osteochondromas), for which exostosis (multiple)-1 (EXT1) has been identified as a causative gene. However, 85% of all osteochondromas present as solitary (nonhereditary) lesions in which somatic mutations in EXT1 are extremely rare, but loss of heterozygosity and clonal rearrangement of 8q24 (the chromosomal locus of EXT1) are common. We examined whether EXT1 might act as a classical tumor suppressor gene for nonhereditary osteochondromas.
Methods: Eight nonhereditary osteochondromas were subjected to high-resolution array-based comparative genomic hybridization (array-CGH) analysis for chromosome 8q. The array-CGH results were validated by subjecting tumor DNA to multiple ligation-dependent probe amplification (MLPA) analysis for EXT1. EXT1 locus–specific fluorescent in situ hybridization (FISH) was performed on nuclei isolated from the three tissue components of osteochondroma (cartilage cap, perichondrium, bony stalk) to examine which parts of the tumor are of clonal origin.
Results: Array-CGH analysis of tumor DNA revealed that all eight osteochondromas had a large deletion of 8q; five tumors had an additional small deletion of the other allele of 8q that contained the EXT1 gene. MLPA analysis of tumor DNA confirmed these findings and identified two additional deletions that were smaller than the limit of resolution of array-CGH. FISH analysis of the cartilage cap, perichondrium, and bony stalk showed that these homozygous EXT1 deletions were present only in the cartilage cap of osteochondroma.
Conclusion: EXT1 functions as a classical tumor suppressor gene in the cartilage cap of nonhereditary osteochondromas.
Prior knowledge Mutations in the EXT1 gene cause multiple osteochondromas, a rare hereditary disorder characterized by multiple benign bone tumors. However, it is not known whether the EXT1 gene is also involved in the more common solitary (nonhereditary) osteochondromas. Study design Molecular cytogenetic study of eight nonhereditary osteochondromas and the three tissue components (cartilage cap, perichondrium, and bony stalk) of one osteochondroma. Contribution Inactivation of both copies of EXT1, which is one of the hallmarks of a classical tumor suppressor gene, occurs in nonhereditary osteochondromas, specifically in the cartilage cap. Implications All osteochondromas, both hereditary and nonhereditary, are neoplastic and develop as a result of the complete loss of one of the EXT genes. Limitations Due to the rarity of osteochondromas, few tumor samples were examined, and only one yielded sufficient material for analysis of tissue components. LOH due to mitotic recombination without copy number alterations could not be detected by the techniques used.
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Osteochondroma is the most common benign bone tumor and represents approximately 50% of all surgically treated benign bone tumors (1). Osteochondromas arise at the external surface of bones that are formed by endochondral ossification, and they consist of a cartilage cap that covers a bony stalk that is continuous with the underlying bone. Chondrocytes located in the cartilage cap have the same spatial organization as those in the epiphyseal growth plate and also undergo endochondral ossification (1). The cartilage cap is covered by a perichondrium that is continuous with the periosteum of the underlying bone. Approximately 15% of osteochondromas occur in the context of Multiple osteochondromas, a hereditary disorder that is characterized by multiple osteochondromas, the number of which can vary substantially between and within families, and is inherited in an autosomal dominant manner (2,3), but the vast majority of osteochondromas present as solitary (nonhereditary) lesions.
Multiple osteochondromas is caused by mutations in either of two genes: exostosis (multiple)-1 (EXT1; Online Mendelian Inheritance in Man [OMIM] No. 133700 [OMIM] ), which is located on chromosome 8q24.11–q24.13, and exostosis (multiple)-2 (EXT2; OMIM No. 133701 [OMIM] ), which is located on chromosome 11p11–12 (4–6). Both genes are ubiquitously expressed (4–6). Most of the germline mutations that have been identified in the EXT1 and EXT2 genes lead to premature truncation of the EXT proteins and the loss of protein function [reviewed in (7)]. Most hereditary osteochondromas have been reported to be heterozygous for one of the mutations in the EXT genes (8–10). However, the demonstration that some hereditary osteochondromas, in addition to carrying an EXT1 mutation, exhibit loss of the remaining wild-type allele of EXT1 (11), is consistent with Knudson's two-hit model of tumorigenesis (12), and indicates that EXT1 acts as a classical tumor suppressor gene in Multiple osteochondromas.
Somatic mutations in the EXT genes are extremely rare in nonhereditary osteochondromas and have been described in only three cases (13–15), one of which (15) was a nonhereditary secondary peripheral chondrosarcoma, which is a malignant cartilaginous tumor arising from a preexisting osteochondroma (16). However, the observation that loss of heterozygosity (LOH) and clonal rearrangement at 8q24 (the EXT1 locus) are as frequent in nonhereditary osteochondromas as are EXT1 gene mutations in patients with hereditary osteochondromas suggests that a gene on 8q24—most likely EXT1—is involved in the development of nonhereditary osteochondromas (11,17,18). By contrast, LOH at the EXT2 locus has been reported in only one nonhereditary osteochondroma (18) and, to our knowledge, no somatic mutations in EXT2 have been identified.
In a previous study (19), we examined EXT1 and EXT2 mRNA expression in hereditary and nonhereditary osteochondromas and found that patients with hereditary Multiple osteochondromas who had a germline mutation in either of the EXT genes had decreased mRNA expression of the corresponding EXT gene in their tumors compared with the expression found in normal epiphyseal growth plates. By contrast, in nonhereditary tumors, in which EXT1 or EXT2 gene mutations were absent, only EXT1 mRNA expression was decreased.
The gene products of EXT1 and EXT2, the EXT 1 and EXT2 proteins, form a heterooligomeric complex in the Golgi apparatus, where they function in heparan sulfate proteoglycan (HSPG) biosynthesis (20). HSPGs are large, multifunctional macroproteins that are involved in several growth signaling pathways in the epiphyseal growth plate (21,22). We previously found that decreased EXT1 or EXT2 mRNA expression in osteochondromas and chondrosarcomas was associated with intracellular accumulation of HSPGs in the Golgi apparatus. By contrast, in growth plates, where expression of HSPGs is extracellular, there is normal expression of the EXT genes (19). It has been shown that a lack of HSPGs at the cell surface affects growth signaling pathways in the growth plate (23) and, possibly, those in osteochondromas (24,25).
The decrease in EXT1 mRNA expression that we observed in nonhereditary osteochondromas (19) suggests that the loss of EXT1 mRNA expression is important for the development of these tumors. However, the fact that we and others (11,14,19,26,27) could find no evidence for somatic mutations or promoter methylation at the EXT1 gene in such tumors implies that other mechanisms may be used to inactivate EXT1 and decrease its mRNA expression. The LOH and clonal rearrangement found at 8q24 in nonhereditary osteochondromas might target the EXT1 gene itself or a regulatory element that affects EXT1 gene transcription. To evaluate this hypothesis, we subjected a series of nonhereditary osteochondromas to high-resolution array-based comparative genomic hybridization (array-CGH) and multiplex ligation-dependent probe amplification (MLPA) analyses. We also examined which part of the osteochondroma (the cartilage cap, bony stalk, or perichondrium) is of clonal origin using EXT1 locus–specific fluorescent in situ hybridization (FISH).
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Materials and Methods |
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Patient Material
Frozen and paraffin-embedded tumor tissues were retrieved from the tissue bank of the Leiden University Medical Center. Fresh-frozen material from the cartilage cap was available for eight patients with nonhereditary osteochondromas. Tumor-related data were obtained by review of pathology specimens and reports. We used bone scans and radiographs to confirm that the selected tumor samples were from patients who had only a single documented osteochondroma lesion. All samples were handled in a coded fashion, following the medical ethical guidelines described in the Code Proper Secondary Use of Human Tissue established by the Dutch Federation of Medical Sciences. Four tumors (those from patients L312, L657, L673, and L1247) were included in previous studies, in which they had been subjected to EXT mutation analysis (11,19). These results were included in this study.
Four patients (L312, L657, L1520, and L1649) provided peripheral blood leukocytes, which were used as the source of normal germline DNA. Normal connective tissue originating from the resection specimen was available for one patient (L1247) and was used as the source of normal germline DNA for that patient. Normal germline DNA was not available for three patients.
DNA and RNA Isolation
Sections (4-µm thick) of frozen tumor tissue were stained with hematoxylin–eosin to identify regions of the tumor sample in which at least 70% of the cells were tumor cells of the cartilage cap; tissue from those regions was used to isolate DNA and RNA. DNA from tumor samples and the connective tissue sample from patient L1247 was isolated with the use of a Wizard genomic DNA purification kit (Promega, Madison, WI). A salting-out procedure was used to extract normal germline DNA from freshly collected peripheral blood leukocytes (28). Total tumor RNA was isolated as described previously (29).
Mutation Analysis
We screened the entire coding sequences of the EXT1 and EXT2 genes in normal germline DNA (from five patients) and tumor-derived DNA from all eight osteochondromas by direct sequence analysis, as previously described (30).
Array-Based Comparative Genomic Hybridization Analysis of Chromosome 8q
A high-resolution 8q array containing a tiling bacterial artificial chromosome (BAC) clone set was constructed from a previously published clone set (31). This clone set spanned the entire long arm of chromosome 8 and consisted of 618 clones selected from the RPCI-11 library of BAC clones available from the BACPAC Resource Center (Children's Hospital Oakland Research Institute, Oakland, CA). Control BAC clones (n = 189) representing non–chromosome 8q locations were selected from the same library and used to normalize the signal intensities to the normal chromosome copy number (namely, two) (31). Genomic positions of the BAC clones were retrieved from the University of California Santa Cruz Genome Browser web page (http://genome.cse.ucsc.edu/), which used the Human May 2004 Assembly.
Polymerase chain reaction (PCR) amplification and spotting of the clones, hybridization, and image acquisition procedures were performed as previously described (32) with minor modifications. Briefly, each PCR-amplified clone set was spotted in triplicate per slide.
Genomic DNA (150 ng) was labeled with fluorescent dCTP in an overnight random primer–labeling reaction (BioPrime Random Prime Labeling Kit, Invitrogen, Carlsbad, CA). Test DNA (from tumor cells of the cartilage cap or normal leukocytes) and sex-mismatched reference DNA (Promega) were differentially labeled with Cy3-dCTP and Cy5-dCTP, respectively (GE Healthcare, Amersham,UK). Labeled DNA samples were pooled and precipitated in the presence of Cot1 DNA (Invitrogen). The precipitated DNA was resuspended in a formamide-based buffer and hybridized to the slides containing the 614 8q-specific and 189 control BAC clones for at least 48 hours, after which the slides were washed and dried.
The slides hybridized with labeled DNAs were scanned at 532 and 635 nm for Cy3 and Cy5, respectively, with the use of a GenePix Personal 4100A scanner (Axon Instruments, Union City, CA), and spot intensities were measured with the use of GenePix Pro 4.1 software. This software integrates pixel intensities for each spot and excludes spots for which the intensity of the reference DNA was less than five times that of the local background or spots for which more than 3% of the pixels were saturated. The fluorescent intensity ratios of the test (Cy3) and the reference (Cy5) samples of the entire clone set were normalized against the median of the fluorescent intensity ratios of the 189 control BAC clones by using a routine that we developed in Microsoft Excel 2000. For each clone, the average value from triplicate spots and its standard deviation were calculated. Only clones for which at least two of the three spots had fluorescent intensity ratios within the 20% confidence interval (CI) [empirically established and used in previous studies (31,32)] of the average intensity were used for further analysis. To display the data, the log2 value of the normalized average ratio was calculated for each clone. The threshold for gains and losses for each target was defined as ±0.33 on a log2 scale.
Multiplex Ligation-Dependent Probe Amplification
All osteochondromas were subjected to a two-color MLPA assay (using a kit supplied by Service XS, Leiden, The Netherlands) that was designed to identify deletions in the EXT1 and EXT2 genes that comprise several exons, as previously described (30). We used this assay to verify array-CGH analysis results for the EXT1 locus and to identify small deletions and amplifications of EXT1 that were beyond the limit of resolution of the tiling BAC array clone set (i.e., <150 kilobases [kb]). Each sample was analyzed in duplicate, and the assay was performed according to the supplier's instructions. Briefly, in a one-tube reaction, combinations of two adjacently annealing oligonucleotide probes were hybridized and ligated. Two probes for unlinked loci were included as a reference. After ligation, the common ends of the probes served as a template for PCR amplification with one primer pair of which the forward primer was labeled with a fluorescent dye. The resulting labeled PCR products were separated according to size using capillary electrophoresis on an ABI 3130 sequencer (Applied Biosystems, Foster City, CA). Data analysis of the different PCR products was performed with the use of GeneScan sequencing software (Applied Biosystems). The height of each exon-specific peak was divided by the sum of the heights of the two reference peaks to produce a ratio. The thresholds for genomic gains and hemizygous losses for each target were defined as ratios of higher than 1.2 and smaller than 0.8, respectively, on a linear scale. Homozygous deletions were defined as ratios smaller than 0.3 (i.e., 0.8 – 0.5 [the ratio of hemizygous loss).
Fluorescence In Situ Hybridization of Isolated Nuclei
To investigate which component(s) of the osteochondromas harbored a homozygous EXT1 deletion, we performed FISH on tumor cell nuclei that were isolated, as previously described (33), from the cartilage cap and perichondrium components of EDTA-decalcified formalin-fixed, paraffin-embedded tumor tissue from patient L1649. FISH was also performed on paraffin-embedded sections of tumor tissue from patient L1649 to investigate the different cell types in the bony stalk component of osteochondromas. After deparaffinization, the sections were pretreated with 0.01 M citrate (pH 6.0) at 80 °C for 80 minutes and predigested with 1 M sodium thiocyanate at 80 °C for 10 minutes, followed by incubation with 100 µg/mL RNase (Roche Applied Science, Penzburg, Germany) at 37 °C for 1 hour, followed by sequential enzymatic digestions with 0.02% collagenase (5 minutes at 37 °C) and 0.4% pepsin ([pH 2.0] at 37 °C for 15 minutes). After these treatments, the slides were rinsed in phosphate-buffered saline, dehydrated in an ethanol series, and dried in air.
We used DNA prepared from BAC clone RP11-357E22 and from fosmid clones, G248P86305D6 and G248P8030G11, (obtained from BACPAC Resource Center) as probes for FISH. DNA was isolated from the clones with the use of a High Pure PCR template preparation kit (Roche Applied Science) and labeled with either digoxigenin-11-dUTP or biotin-16-dUTP (both from Roche Applied Science) with the use of a BioPrime Random Prime Labeling Kit. The labeled clones were mixed with an alpha satellite DNA probe (34) specific for the centromere of chromosome 8 that had been directly labeled with Cy5-dUTP by nick translation, as previously described (35). Hybridization of the probe mixture to isolated nuclei and indirect detection of the hapten-conjugated probes were performed according to standard protocols, as previously described (36). Digital fluorescence imaging and image analysis were performed as previously described (37).
Quantitative Reverse Transcription–Polymerase Chain Reaction
For first-strand cDNA synthesis, 1 µg of total tumor RNA was reverse transcribed by avian myeloblastosis virus reverse transcriptase (Roche Applied Science) with the use of 100 ng oligo(dT)15 primer (Roche Applied Science) and 50 ng random primers (Invitrogen), according to the manufacturer's instructions. The sequences of the primers that were used for quantitative reverse transcription–polymerase chain reaction (qPCR) amplification of the RNAs for the EXT1 gene and for the genes to normalize the EXT1 mRNA expression (CPSF6, SRPR, GPR108, and HNRPH1) (19) as well as the expected sizes of the amplification products are provided in Table 1. qPCR was performed with the use of a qPCR Corekit for SybrGreen (Eurogentec, Seraing, Belgium) on an iCycler (BioRad, Hercules, CA). All samples were measured in duplicate. Fluorescent signals generated by PCR were collected in real time and translated to quantitative values using the iCycler software according to the manufacturer's instructions.
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Statistical Analysis
Normalization and data analysis were performed as previously described (38). Briefly, geometric averaging of expression of the four reference genes was performed to acquire reliable normalization of EXT1 gene expression using the geNorm programme (39). This method provides a normalization factor that is representative for the amount of mRNA in each sample. Because osteochondromas histologically resemble the epiphyseal growth plate (1), expression levels in the tumors were related to those of four normal growth plates, for which the EXT1 expression had been determined in a previous study (19). The average of the EXT1 expression level in the growth plates was set to 1. P values were computed by Student's t test and were considered statistically significant when less than or equal to .05.
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Results |
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Selection of Patient Material
Patients with a nonhereditary osteochondroma were selected based on review of pathologic, clinical, and radiologic data. Fresh-frozen material of nonhereditary osteochondromas was available for eight patients. Table 2 summarizes the clinical and tumor-related data for these patients.
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Mutation Analysis of the EXT1 and EXT2 Genes
To confirm that none of the patients included in this study was actually a multiple osteochondromas patient with very mild symptoms (i.e., only one osteochondroma documented) and that none of the tumors had somatic mutations in either the EXT1 or EXT2 gene, we analyzed the entire coding sequences of the EXT1 and EXT2 genes in normal germline DNA (from five patients) and tumor-derived DNA from all eight osteochondromas by direct sequencing. Apart from known polymorphisms (data not shown), no somatic or germline mutations were detected in either tumor or normal DNA.
High Resolution Array-CGH Analysis of Chromosome 8q in Nonhereditary Osteochondromas
We next examined chromosome 8q in the series of eight nonhereditary osteochondromas in more detail with a high-resolution array that contained a tiling BAC clone set. All eight nonhereditary osteochondromas had a large hemizygous deletion of 8q as detected by array-CGH analysis (Table 3). For seven tumors (from patients L657, L673, L1247, L1455, L1520, L1587, and L1649), this deletion extended to the telomere. The smallest region of overlap spanned approximately 2 megabases (Mb), from 8q24.11 (118.45 Mb position) to 8q24.12 (120.66 Mb position) and comprised six genes, including EXT1. In five tumors (from patients L657, L673, L1455, L1520, and L1649), we identified a homozygous deletion that covered the EXT1 gene, which indicated that a second independent deletion event had occurred in this region. The homozygous deletions were always smaller than the hemizygous deletions and ranged from 0.16 Mb (covered by clone RP11-1061J15) to 1.8 Mb (covered by 11 clones) (Fig. 1, A–D). None of the samples showed chromosomal gains.
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For DNA from tumor samples that have low levels of contamination with normal cells, the typical values of the log2-normalized average ratios for hemizygous and homozygous deletions are –1 and less than –2, respectively, on a log2 scale. However, the observed ratios were between –0.3 and –0.8 for hemizygous deletions and between –0.9 and 4.0 for homozygous deletions. The observed ratios are larger than expected and indicate that most tumor samples indeed contained amounts of normal cell contamination. Normal cells still had two copies of chromosome 8q.
All five of the patients for whom we had normal constitutional DNA (i.e., L312, L657, L1247, L1520, and L1649) had a normal genomic profile at 8q (Fig. 1, E). This finding indicated that in these nonhereditary osteochondromas, the 8q deletions, including the homozygous deletions, were of somatic origin.
Verification of Array-CGH Results by Multiplex Ligation-Dependent Probe Amplification Analysis of EXT1
We next used MLPA analysis to verify the array-CGH results and to facilitate identification of homozygous deletions involving individual exons, which are typically beyond the limit of resolution of the tiling array clone set. MLPA analysis of DNA from the leukocytes of a healthy donor was used to generate a normal profile for the EXT1 MLPA probe set (Fig. 2, A). MLPA analysis revealed the loss of at least one copy of the EXT1 gene in all eight osteochondromas (Fig. 2). MLPA analysis of tumor DNA also revealed homozygous deletions of the entire EXT1 gene in osteochondromas from patient L673 (data not shown) and L1649 (Fig. 2, B). These deletions were also identified by the array-CGH analysis. MLPA analysis also identified a homozygous deletion of exon 1 in osteochondroma from patient L1520 (Fig. 2, C), a result that was consistent with array-CGH results that showed homozygous deletion of the region covered by a single BAC clone, RP11-1061J15, which only included exon 1 of the EXT1 gene. In osteochondroma from patient L312, MLPA analysis revealed the complete loss of exons 2, 3, and 4 (Fig. 2, D), identifying a homozygous deletion of at least 7.3 kb. Osteochondroma from patient L1587 showed complete loss of exon 6 (Fig. 2, E), demonstrating a homozygous deletion of at least 119 bp (the size of this exon). Unlike the array-CGH analysis, MLPA analysis of osteochondroma from patient L657 did not show a homozygous deletion of the EXT1 gene but only a hemizygous deletion. This result probably reflects contamination of the tumor DNA sample with normal DNA, as was suggested by the log2 ratios found for hemizygous (–0.6) and homozygous deletions (–1.2) in the array-CGH experiments (Fig. 1, B). The PCR amplification for MLPA analysis of osteochondromas from patients L1247 and L1455 failed twice; because of the limited amount of material available, it was impossible to obtain MLPA data for these patients.
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EXT1 Locus–Specific Fluorescent In Situ Hybridization Analysis of Osteochondroma Tissue Components
Array-CGH analysis was performed with DNA isolated from cells of the cartilage cap, which is known to be of clonal origin (11,18). However, osteochondromas consist of three components: a cartilage cap, a bony stalk, and a perichondrium (1). Of the latter two components, it is still debated whether they are also of clonal or reactive/remodeled host bone origin. To examine whether the bony stalk and perichondrium of osteochondroma also harbored the homozygous deletion of EXT1, we performed FISH analysis on formalin-fixed paraffin-embedded material from patient L1649. It was not possible to perform EXT1 locus–specific FISH of the three tissue components in a single paraffin section because they behaved differently during pretreatment of the tissue section. Instead, we performed FISH on paraffin sections of the bony stalk only and on isolated nuclei of formalin-fixed, paraffin-embedded microdissected tissues containing either the cartilage cap or the perichondrium.
For FISH analysis, two fosmid clones covering the EXT1 gene were selected as EXT1 locus–specific probes. We used BAC clone RP11-357E22, which contains DNA located close to the EXT1 locus that was not altered in tumor DNA from patient L1649, and a centromeric probe for chromosome 8 as controls, allowing us to detect chromosome 8 and alterations in both EXT1 loci. FISH performed on normal metaphase spreads showed fluorescent signals for each of the three probes on two chromosomes, both of which were identified as chromosome 8 (Fig. 3, A). FISH signals for the fosmid clones were not observed in the nuclei from the cartilage cap, confirming the homozygous deletion of EXT1 (Fig. 3, B). By contrast, we observed two FISH signals of the EXT1 fosmid clones in nuclei isolated from the perichondrium of case L1649, indicating that both copies of the EXT1 gene were still present (Fig. 3, B). We also observed two FISH signals for the EXT1 fosmid clones in osteoblasts, osteocytes, bone marrow, and epithelial cells in the bony stalk (Fig. 3, B). The FISH results showed that the homozygous deletion of EXT1 was found only in nuclei of the cartilage cap and not in nuclei of the perichondrium and the bony stalk. Thus, in osteochondroma, only the cartilage cap appears to be of clonal origin.
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EXT1 mRNA Expression in Osteochondromas
Finally, to evaluate whether the homozygous deletion of EXT1 indeed abolished its mRNA expression, we examined EXT1 mRNA expression in the eight nonhereditary osteochondromas by qPCR and compared them with expression of four normal growth plates. For seven of the osteochondromas, sufficient amounts of RNA were available for determining EXT1 mRNA expression by qPCR. For each of these tumors, the EXT1 mRNA expression level is represented as the fraction of the average EXT1 mRNA expression in the growth plate (Table 4). The average EXT1 mRNA level was statistically significantly lower in osteochondromas than in four normal epiphyseal growth plates (0.12 versus 1.00; difference = 0.88, 95% CI = 0.51 to 1.15, P = .001 [Student's t test]). Thus, the decreased EXT1 mRNA expression in osteochondroma is consistent with the homozygous deletions of EXT1 that we identified with array-CGH and MLPA analyses.
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Discussion |
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TopAbstractContext and CaveatsMaterials and MethodsResultsDiscussionReferencesNotes |
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In this study, we identified homozygous deletions of the EXT1 gene in seven of eight nonhereditary osteochondromas by two types of analyses, array-CGH and MLPA. The smallest homozygous deletion identified by array-CGH analysis covered the EXT1 promoter region and exon 1, the largest exon of the gene (4); however, the higher resolution of the MLPA technique allowed us to identify homozygous deletions that were as small as single exons. All the homozygous deletions we identified resulted from a large deletion comprising a large part of the chromosome arm on one allele and a small deletion of only the EXT1 gene (or part of it) on the other allele. Frei et al. (40) have suggested that the small deletions were more likely to be the consequence of erroneous recombination events than to represent a somatic point mutation or replication errors that commonly occur in stretches of similar nucleotides. These results indicate that two distinct events (either simultaneous or consecutive) involving both alleles of chromosome 8 occurred in these tumors.
All the nonhereditary osteochondromas in our study demonstrated physical loss of 8q24 (and thus the EXT1 gene). By contrast, complete loss of the wild-type EXT1 gene by homologous recombination of the mutated allele has been described in hereditary osteochondromas (11) (Fig. 4). We speculate that, because small single–base pair mutations are rare in nonhereditary osteochondromas (13–15), physical loss of 8q24 is necessary to inactivate both copies of the EXT1 gene (Fig. 4).
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EXT1), inactivation of the second allele can be achieved either by physical loss of the remaining wild-type (WT) allele or by homologous recombination of the mutated allele. We show that in nonhereditary osteochondromas, both WT alleles are lost, usually by loss of 8q and a small EXT1 deletion, resulting in homozygous EXT1 deletion.To our knowledge, this is the first report to identify multiple cases of homozygous deletion of EXT1 in osteochondromas. Our results resolve questions about the role of EXT1 as potential tumor suppressor gene in the development of osteochondromas. An ongoing debate in the literature has been whether EXT1 fits the classical two-hit model for tumor suppressor genes (11,12) or whether haploinsufficiency for EXT1 via mutational inactivation (i.e., the loss of one allele) is sufficient for the formation of osteochondromas (8). This so-called haploinsufficiency model was based on studies in which 90% of the hereditary osteochondromas demonstrated heterozygous mutations in EXT1 or EXT2 (8–10). However, we have clearly demonstrated that biallelic inactivation of EXT1, which is one of the hallmarks of a classical tumor suppressor gene (41), also occurs in nonhereditary osteochondromas.
Previous studies (11,17,18) have demonstrated that the cartilage cap of osteochondroma is of clonal origin and thus is neoplastic. The clonal origin of the cartilage cap is confirmed by our identification of homozygous deletions in EXT1 in DNA isolated from the cartilage cap. However, it has remained unclear whether cells that form the bony stalk of an osteochondroma and the overlying perichondrium are also of clonal origin. By performing FISH analysis on the different components of an osteochondroma, we demonstrated that nuclei isolated from cells comprising the cartilage cap harbored a homozygous deletion of EXT1, whereas nuclei isolated from cells comprising the perichondrium and bony stalk had no such deletion. Thus, these results demonstrate that the perichondrium and the bony stalk of osteochondromas are not neoplastic.
Our finding that the cartilage cap is the only neoplastic component of osteochondroma revives a long-standing debate about the cell of origin of osteochondromas. Several theories have been suggested over the years (42,43), but so far, to our knowledge, no compelling evidence supporting any one of them has been reported in the literature. Recently, it was suggested that specific cells in the perichondrium may give rise to chondrocytes that are necessary for the development and continued growth of osteochondromas (44). However, this suggestion is inconsistent with our FISH results for the different components of osteochondroma, which suggest that the cell of origin most likely resides in the growth plate.
Conventional LOH analysis, which has been used to analyze osteochondromas in the past, usually does not detect homozygous deletions (11,17,18). To our knowledge, only one other study (14) has identified a homozygous deletion of EXT1 in a single nonhereditary osteochondroma. Even tumors that have little contamination by normal cells show retention of heterozygosity by conventional LOH analysis, which severely hampers the distinction between homozygous deletion and retention of heterozygosity. Homozygous deletions are typically detected only in cell lines that completely lack contamination (45). Our results show the benefit of using high-resolution array-CGH analysis with a tiling clone set to identify homozygous deletions.
Recently, we demonstrated that the loss of EXT expression in osteochondromas results in the disordered distribution of HSPGs (19). In these osteochondromas, HSPGs were no longer present at the cell surface but accumulated in the cytoplasm, where they were concentrated in the Golgi apparatus. HSPGs are known to be involved in several signaling pathways in the growth plate (21). It is possible that the lack of HSPGs at the cell surface in osteochondromas might have a functional effect on HSPG-dependent signaling pathways, e.g., Indian hedgehog (IHH) signaling. In the growth plate, IHH requires interaction with HSPGs to diffuse through the extracellular matrix to its receptor (23). However, two recent studies (24,25) showed that IHH signaling was still present in osteochondromas despite the absence of HSPGs at the cell surface. Benoist-Lasselin et al. (25) also demonstrated that IHH was expressed in all cells of the cartilage cap, whereas IHH expression in the growth plate is restricted to the transition zone (46). We speculate that osteochondroma cells circumvent the impaired diffusion capacities that result from diminished amounts of HSPGs at the cell surface by producing IHH in every cell of the cartilage cap, resulting in cell-autonomous (i.e., autocrine) IHH signaling. Other HSPG-dependent growth signaling pathways that are affected in osteochondroma are the parathyroid hormone–like hormone signaling pathway, which is a downstream target of IHH (46), and the fibroblast growth factor signaling pathway (47).
Our study has several limitations. One is the infrequent occurrence of osteochondromas, which inhibited the collection of a larger series of fresh-frozen tumors. Furthermore, the low cellularity and excess of extracellular matrix hampered DNA and RNA analyses. Therefore, we were unable to extend the series reported here. In general, array-CGH and MLPA are perfectly suitable for detecting physical loss of genomic regions (48). However, LOH due to mitotic recombination without copy number alterations cannot be detected by these techniques and will require complementary LOH analysis.
In conclusion, our identification of the homozygous deletion of EXT1 in the cartilaginous cells of nonhereditary (solitary) osteochondromas proves that EXT1 acts as a classical tumor suppressor gene in osteochondroma formation and supports the two-hit model for osteochondroma development. The absence of this deletion in the perichondrium and the bony stalk indicates that the cartilage cap constitutes the neoplastic lesion and that both hereditary and nonhereditary osteochondromas arise from cells of the growth plate rather than from the perichondrium, as previously suggested (44). Our results indicate that all osteochondromas develop as a result of loss of EXT1 and subsequent abrogation of HSPG-dependent signaling pathways.
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The Dutch Cancer Society (grant number: RUL 2002-2738) financially supported the experiments performed for the study. The departments of pathology of the Leiden University Medical Center and Erasmus Medical Center are partners of the EuroBoNeT consortium, a European Commission granted Network of Excellence for studying the pathology and genetics of bone tumors, which financed the collaboration between the different research groups who participated in this study. The funding agencies had no role in the design of the study; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.
We thank Marja van den Burg for expert assistance with the array experiments.
The chromosome 8 idiograms shown in Fig. 4 were obtained from http://www.pathology.washington.edu/research/cytopages. This study was presented at Human Genome Organisation's 11th Human Genome Meeting.
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Manuscript received September 11, 2006; revised December 15, 2006; accepted January 19, 2007.
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