Abstract
Loss of heterozygosity of several specific genomic regions is frequently observed in neuroblastoma tumors and cell lines, but homozygous deletion (HD) is rare, and no neuroblastoma tumor suppressor gene(TSG) has yet been identified. We performed a systematic search for HD, indicative of a disrupted TSG, in a panel of 46 neuroblastoma cell lines. An initial search focused on a well-characterized consensus region of hemizygous deletion at 1p36.3, which occurs in 35% of primary neuroblastomas. Each cell line was screened with 162 1p36 markers, for a resolution of 13 kb within the consensus 1p36.3 deletion region and 350 kb throughout the remainder of 1p36. No HDs were detected. This approach was expanded to survey 21 known TSGs,specifically targeting intragenic regions frequently inactivated in other malignancies. HD was detected only at the CDKN2A(p16INK4a/p14ARF)gene at 9p21 and was observed in 4 of 46 cell lines. The observed region of HD included all exons of both CDKN2A and the closely linked CDKN2B(p15INK4b) gene for cell lines LA-N-6 and CHLA-174, all exons of CDKN2A but none of CDKN2B for CHLA-179, and only 104 bp within CDKN2A exon 2 for CHLA-101. All four deletions are predicted to inactivate the coding regions of both p16INK4a and p14ARF. HD was observed in corresponding primary tumor samples for CHLA-101 and CHLA-174 but was not present in constitutional samples. These results suggest that for neuroblastoma, large HDs do not occur within 1p36, most known TSGs are not homozygously deleted, and biallelic inactivation of CDKN2A may contribute to tumorigenicity in a subset of cases.
INTRODUCTION
The biallelic inactivation of TSGs3 is considered crucial for the genesis and evolution of neoplastic cells. Biallelic inactivation of a specific TSG can occur by several mechanisms, including chromosomal deletion of both alleles. Although uncommon, HD is occasionally observed in primary tumors and cell lines from a variety of malignancies, and the region of biallelic deletion is usually confined to a small genomic region surrounding a target TSG. As HDs usually span relatively short genomic regions, the detection and characterization of HD in various malignancies has been instrumental in the identification of several TSGs, including RB1, WT1, and CDKN2A(1, 2, 3).
Neuroblastoma is a common pediatric tumor of the peripheral nervous system and is often incurable when diagnosed after 1 year of age. Neuroblastoma displays remarkable clinical heterogeneity, ranging from spontaneous regression and/or differentiation to rapidly progressive and metastatic disease, and considerable genetic heterogeneity is also apparent. Cytogenetic, molecular genetic, and functional analyses of primary tumors and cell lines have identified a number of genomic regions frequently exhibiting hemizygous deletion (4). Deletion of 1p36 correlates strongly with advanced disease and is the most well-characterized region of deletion in neuroblastoma(5, 6, 7). LOH studies have narrowed the region of consistent overlapping deletion to 1p36.3, and this region is deleted in ∼35%of primary neuroblastomas (5, 8, 9). Several groups have also hypothesized the existence of one or more additional TSGs located elsewhere within 1p36 (10, 11, 12). Despite the characterization of numerous candidates, no 1p36 TSG has yet been identified, largely because of the paucity of tumors with localized 1p36 rearrangements (4).
Besides 1p36, several additional regions of the genome are frequently deleted in primary neuroblastomas, including 3p, 4p, 11q23, and 14q32(4). However, no TSG consistently mutated or rearranged in neuroblastoma tumors has yet been identified, and analyses of several known TSGs, including TP53, RET, CDKN2A, and MADH4, have detected few if any mutations in these genes (4). Furthermore, HD of known tumor suppressor loci has been reported only rarely in neuroblastoma(13, 14, 15, 16). The lack of evidence for genetic alterations in the genes encoding the DNA damage sensor p53 and the cyclin-dependent kinase inhibitory protein p16INK4a is notable, as these two genes are commonly disrupted in most malignancies (17, 18).
In the present study, we investigated whether HD occurs with significant frequency in neuroblastoma by systematically screening a large panel of cell lines with markers mapping to 1p36 and also with markers representing known TSGs. We report evidence for HD at CDKN2A but not within distal 1p or at other known TSGs in our cell line panel.
MATERIALS AND METHODS
Sample Collection and DNA Isolation.
A panel of 46 neuroblastoma cell lines were used for HD analysis. Twenty-four cell lines have been previously described (Table 1). Cell lines CHLA-10, CHLA-101, CHLA-103, CHLA-108, CHLA-124, CHLA-132,CHLA-136, CHLA-138, CHLA-140, CHLA-143, CHLA-152, CHLA-153, CHLA-171,CHLA-174, CHLA-178, CHLA-179, CHLA-185, CHLA-52, CHLA-54, CHLA-60,CHLA-95, and CHLA-98 were established as described from patients treated with myeloablative chemoradiotherapy (19). Cell lines CHP-901 and CHP-902R were established at the Children’s Hospital of Philadelphia from a bone marrow biopsy and a relapsed tumor mass,respectively, and cultured in RPMI 1640 (Life Technologies, Inc.,Gaithersburg, MD) with 10% fetal bovine serum, 25 μg/ml gentamicin,and 1% oxaloacetate/pyruvate/bovine insulin-media supplement(OPI; Life Technologies, Inc.). MYCN amplification and 1p allelic loss determinations were performed as described previously (7). Frozen tumor and corresponding normal marrow samples of patients from which cell lines CHLA-101, CHLA-174,and CHLA-179 were derived were obtained from the Children’s Cancer Group Neuroblastoma Biology Resource Laboratory at Children’s Hospital Los Angeles. To obtain primary tumor DNA from the patient who gave rise to the LA-N-6 cell line, cryopreserved marrow was thawed, washed to remove DMSO in Iscove’s DMEM with 10% fetal bovine serum, and treated with 10 units/ml of DNase for 1 h at 37°C in a 5%CO2 incubator. The mononuclear cells were separated using a Ficoll density centrifugation. Mononuclear cells were then treated with magnetic immunobeads (one bead/total cells) and tumor cells removed as described previously (20). The resulting mononuclear cells, which contained <0.1% tumor, were centrifuged to a pellet, medium-aspirated, and flash-frozen for future DNA extraction. The Jurkat T-cell line was kindly provided by S. Lessin (University of Pennsylvania). DNA was isolated from cell line pellets or frozen tissue using anion-exchange chromatography (Qiagen, Valencia, CA). DNA from Centre d’Etude du Polymorphisme Humain reference family member 1331-01 (Coriell Cell Repositories, Camden, NJ) was used as a normal control.
DNA Markers.
Details of the DNA markers used to screen for HD of 1p36 and other TSGs are listed in Tables 2 and 3, respectively. Primers used for detailed mapping of HD around CDKN2A are included in Table 3. Primer sequences were obtained from the Genome Database4and from literature reports of specific TSG characterizations or were generated from a representative sequence using Primer 3.5If possible, TSG primers targeted gene regions commonly disrupted in other tumors. Markers for 1p36 were mapped using a distal 1p-specific mapping panel6as described previously. Markers mapping to 9p were ordered using map information from the HUGO chromosome 9 integrated map (Genome Database accession no. 6276683) and genomic sequence tracts from GenBank.7
Genotyping, HD Detection, and DNA Sequencing.
A set of 46 neuroblastoma cell lines demonstrating unique genotypes at three highly polymorphic microsatellite markers (D3S1744, D7S796, and D12S391) were used in the HD study(Table 1). A pooled DNA template containing human RH cell lines 21-30 from the Stanford G3 panel (Research Genetics, Huntsville, AL),each of which had no human DNA fragment retention for markers within 1p36, was used as a control for detecting 1p36 HD. The 46 cell lines, a normal DNA control (1331-01), and a negative control (PCR reaction with no added template or the RH control) were amplified by PCR in 20 μl volumes containing 4 μl of 10× PCR buffer II (Perkin-Elmer, Norwalk,CT), 0.4 μm of each primer, 0.2 mm of each deoxynucleotide triphosphate, 0.2 units AmpliTaq Gold DNA polymerase (Perkin-Elmer), 1.5 mm MgCl2, 0.5%Ficoll, 0.00625% bromphenol blue, and 14.4 ng template DNA. Reactions were amplified for 1 cycle at 95°C (3 min); 15 cycles at 95°C (45 s) with the annealing/extension temperature starting at 70°C and decreasing by 0.7°C each cycle (1 min); 35 cycles at 95°C (45 s), 55°C (30 s), and 72°C (1 min); and 1 cycle at 72°C(10 min). Twenty μl of each reaction solution was analyzed by electrophoresis on an enhanced sensitivity gel system (Visigel;Stratagene, La Jolla, CA), with products detected by ethidium bromide staining. PCR reactions yielding no visible signal for a specific marker were repeated, followed by radiolabeled amplification, PAGE, and autoradiographic detection as described above. DNA sequences for CDKN2A exon 2 were obtained on an Applied Biosystems Model 377 DNA sequencer using the ABI Taq DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer).
Southern Analysis.
Ten μg of each genomic DNA sample was restriction digested for 16 h with PstI (Promega, Madison, WI) and electrophoresed on a 0.6% agarose gel in 1× TBE. Gels were sequentially immersed in 0.25 m HCl for 30 min,1.5 m NaCl/0.5 m NaOH for 30 min, and 0.5 m Tris (pH 7.4)/1.5 m NaCl for 30 min. Electrophoresed DNA was then transferred onto Hybond N+ membranes (Amersham Pharmacia, Uppsala, Sweden), washed in 6× SSC, UV cross-linked, and hybridized to either a 1.1-kb CDKN2A DNA probe amplified from genomic DNA using primers CDKN2ex2-1 and CDKN2ex2-3 (Table 3) or to a probe spanning exons 3–5 of the MLL gene at 11q23(kindly provided by Maureen Megonigal (Children’s Hospital of Philadelphia, PA). Probe radiolabeling was performed with the Rediprime Random Labeling Kit and Redivue[α-32P]dCTP (Amersham Pharmacia). Membranes were prehybridized for 3 h in 20 ml of prehybridization solution(10% dextran sulfate, 0.75 m NaCl, 0.04 NaPO4 (pH 7.2), 4 mm EDTA(pH 8), 0.5% N-lauryl sarcosine, 5× Denhardt’s solution, and 125μg/ml sheared herring sperm DNA), hybridized for 16 h at 65°C after the addition of labeled probe, and washed with 2× SSC and then in decreasing concentrations of SSC-0.2% N-lauryl sarcosine at 65°C,followed by autoradiography.
RESULTS
HD Screen of 1p36.
Neuroblastoma cell lines were used to simplify HD detection, because the absence of nonmalignant stromal cells allows implementation of a high-throughput true/false assay system. A set of 46 genetically distinct cell lines was selected for analysis (Table 1). Thirty-nine of the 46 cell lines (85%) had evidence of a 1p hemizygous deletion. Previously, we identified a smallest overlapping region of consistent deletion (SRO) spanning ∼1 Mb within 1p36.3 by LOH analyses of primary neuroblastomas (5). This region has been further characterized by the construction and analysis of a distal 1p-specific mapping panel, which subdivides 1p35–p36 into 50 distinct genomic intervals (21). The 1p36.3 SRO is entirely contained within mapping intervals 6–11 (Table 2; Ref. 5). Seventy-six markers within the SRO, providing an average HD detection resolution of approximately 13 kb, were used to survey the 46 neuroblastoma cell lines by PCR. Included in this set of markers were primers representing each gene or expressed sequence tag cluster known to map within or near the SRO, including the proposed candidate neuroblastoma TSGs TP73, TNFRSF12, and HKR3 (22, 23, 24). To our knowledge, HD for 1p36 has never been reported; therefore, we created a HD control by pooling DNA from 10 RH cell lines known to contain several human DNA fragments but no fragments within 1p36. A product of the predicted size was detected with all 76 markers for every neuroblastoma cell line but not for the HD control or a negative control (no template; Fig. 1 A).
Although all reported 1p36 deletions in neuroblastomas include allelic loss for the 1p36.3 SRO, additional 1p36 suppressor loci both distal and proximal to this region have been postulated and a number of candidate TSGs have been proposed (4). Therefore, a second set of 86 1p36 markers was used to assess whether HD might occur elsewhere within 1p36 (Table 3). Markers mapping within each of the 50 intervals defined by the distal 1p mapping panel were included to assure that each portion of 1p36 was well represented. We also designed markers for the proposed 1p36 TSGs (TNFRSF1B, RIZ, PAX7, NBL1, TCEB3, E2F2, C1ORF4, LAP18, and ID3; Ref. 4), as well as for twelve markers surrounding a reported 1p36.2 translocation breakpoint in the neuroblastoma cell line NGP (9, 25). The 86 markers, which provide an average 1p36 resolution of 350 kb, were used to survey the 46 neuroblastoma cell lines. A product of the predicted size was visible for each marker in every neuroblastoma cell line and in a normal control but not in a negative control.
HD Screen of Known TSGs.
Several known TSGs have been characterized for mutations in neuroblastomas, but no abnormalities have been detected with significant frequency. However, most of these analyses have included only small cohorts of tumors and/or cell lines, and many identified TSGs have not been investigated. Therefore, we extended our HD search to include 21 known TSGs throughout the genome (Table 3). Intragenic primers suitable for genomic PCR were designed for each TSG. Whenever possible, primers were selected to span intragenic regions previously demonstrated to be the most frequently deleted and/or mutated in malignant cells. The 21 TSG markers were each used to survey the neuroblastoma cell line panel and the Jurkat T-cell line, which is homozygously deleted for CDKN2A (26). Twenty of the 21 TSG markers yielded an amplification product in every cell line. However, the CDKN2A primers, which span exon 2 of the gene,did not amplify a product in three neuroblastoma cell lines (CHLA-174,CHLA-179, and LA-N-6), nor in the Jurkat control, in repeated trials using both ethidium staining and radioisotopic detection (Fig. 1,B). In addition, a CDKN2A product ∼100 bp shorter than the predicted length was consistently generated in cell line CHLA-101 (Fig. 1 B) and was the only product generated in this cell line.
Analysis of CDKN2A Deletions.
The CDKN2A HDs detected in the four neuroblastoma cell lines were investigated further with 28 additional markers located within or flanking CDKN2A to map the proximal and distal HD boundaries. Included were markers spanning exons 1α, 1β, and 3 and introns 1α, 1β, and 2 of CDKN2A; exons 1 and 2 of CDKN2B; and several proximal and distal loci (Fig. 2). LA-N-6, CHLA-174, and the control Jurkat line demonstrated HD for the entire CDKN2A gene including exon 1β, which encodes the 5′portion of p14ARF, and both exons of CDKN2B. CHLA-179 showed HD for all exons of CKDN2A but not for CDKN2B. No additional alterations were detected for CHLA-101. A survey of the remaining 42 neuroblastoma cell lines with the four CDKN2A and two CDKN2B exon-specific markers detected products of the expected size in all cases. The LA-N-6, CHLA-174, and CHLA-179 HDs were confined to a region of 9p21 flanked by IFNA and D9S171 (Fig. 2), and each deletion would be predicted to completely abolish production of both the p14ARFand p16INK4a protein products.
The CDKN2A HDs were then confirmed by Southern analysis(Fig. 3). Corresponding primary tumor and constitutional DNAs for LA-N-6,CHLA-174, and CHLA-179 were analyzed in parallel to determine whether HD occurred in vivo or only after cell-culture establishment. A CDKN2A exon 2 probe hybridized to the requisite 3.4-kb PstI fragment in constitutional DNA samples from the three cell lines showing HD by PCR, as well as in primary tumor DNA from CHLA-179. However, in agreement with the PCR results, no hybridization was observed for the three cell lines with CKDN2A HD. Furthermore, primary tumor DNA for CHLA-174 also failed to hybridize to the CDKN2A probe, suggesting that HD occurred in vivo in this tumor (no LA-N-6 tumor sample was available for analysis). Hybridization with a control probe from a different chromosome to the same filter yielded a band of identical size and approximately the same intensity for each sample (not shown).
Southern analysis of cell line CHLA-101 confirmed the PCR results of a truncated CDKN2A exon 2 (Fig. 3). Both techniques demonstrated an ∼100-bp deletion within exon 2. DNA sequencing of the truncated exon 2 PCR product showed a deletion of 104 bp (bp 246–349 and residues 69–103 of p16INK4a; and bp 390–493 and residues 83–118 of p14ARF) entirely contained within exon 2. PCR analysis of primary tumor DNA for the patient from which CHLA-101 was derived showed only the truncated exon 2 fragment, suggesting that the primary tumor contained an exon 2 HD identical to the cell line (not shown). Sequencing of CDKN2Aexon 2 from the primary tumor DNA confirmed these results. No corresponding constitutional DNA for CHLA-101 was available for analysis.
The 104-bp deletion of CDKN2A exon 2 in CHLA-101 is predicted to disrupt both p16INK4a and p14ARF, which use alternate reading frames within exon 2. For p16INK4a, the COOH-terminal 87 residues of the wild-type protein would be replaced with the 15 COOH-terminal residues of p14ARF because of a frameshift from the p16INK4a reading frame to the p14ARF reading frame. It is unlikely that such a fusion protein, if expressed in CHLA-101, would be functional, as the deleted region of p16INK4a contains the Cdk4/Cdk6 binding site where most loss-of-function point mutations localize(27). For p14ARF, the final 50 residues would be replaced by 42 frameshifted residues.
DISCUSSION
Hemizygous deletion of distal 1p, first identified in 1977, is frequently observed in advanced stage neuroblastomas (4). However, despite the identification of a 1 Mb SRO for LOH within 1p36.3(5), no distal 1p TSG has yet been identified. To date,>1000 primary tumors have been assessed for 1p allelic loss, but no small deletions or HD have been detected (4). Furthermore,germline, tumor, or cell line-specific 1p translocation breakpoints are rare and scattered throughout 1p (4). The lack of localized 1p rearrangements has led to alternate hypotheses for the mechanism of 1p-mediated tumor suppression. Several groups have pursued additional 1p36 TSG loci by: (a) mapping 1p36-specific primary tumor or cell line rearrangements identified in closely associated malignancies (28); (b)characterizing constitutional 1p36 rearrangements in patients subsequently developing neuroblastoma (9, 29); or(c) identifying correlations between 1p deletion size and the presence of MYCN amplification (11, 30). Moreover, although not directly supported by the present study,mechanisms other than genetically mediated biallelic inactivation of a TSG may be applicable, including haploinsufficiency of one or more 1p36 loci or imprinting as an epigenetic mechanism for inactivation of the remaining TSG allele.
Our findings of no HD within 1p36 are consistent with the possibility that disruption of only a single 1p36 homologue can facilitate neuroblastoma tumorigenesis. Although the marker density we used to survey HD within the 1p36.3 SRO was high (13 kb), very small regions of HD may have gone undetected. In most cases, biallelic inactivation of a TSG occurs by intragenic deletion or bp mutation of one allele, and then by a second, larger deletion event in the remaining allele, as is common with TP53 (31). Because we surveyed only cultured cell lines for HD at 1p36.3, it is formally possible that HD occurs more frequently in primary tumors. However, this seems unlikely,given that many of the cell lines in our study were derived from advanced neuroblastomas passaged repeatedly in vitro, and that most cell lines had hemizygous deletion of distal 1p.
With the exception of CDKN2A, HD was not detected for any of the 21 TSGs surveyed. Our inclusion of positive and negative controls for HD and the confirmation of CDKN2A HD by Southern analysis for all four identified HD cases suggests that our PCR-based HD assay is both sensitive and specific. It remains possible that HD of one or more TSGs went undetected with the exon primers used for TSG surveying. However, because we targeted the most commonly disrupted gene region of each TSG, it is likely that the majority of HD events would have been identified. HDs and/or inactivating mutations in neuroblastoma are known to be rare or absent in the TP53, DCC, MADH4, and RET tumor suppressor genes (4). Previously, four cases of HD have been reported for neuroblastoma: HD of several exons of the NF1 gene at 17q11.2 in a primary tumor from a patient with neurofibromatosis type 1(13) and in a neuroblastoma cell line (32);HD of the CASP8 gene at 2q33 in one cell line(16); and HD of CDKN2A in a single cell line(see below; Ref. 14). Whereas we did not detect HD of NF1, the NF1 locus has not yet been well characterized in neuroblastoma, although there appears to be no increased incidence of neuroblastoma in patients with neurofibromatosis or vice versa (32). The present study does not address whether HD occurs at other chromosomal loci than those included here. However, experiments using representational differential analysis did not detect HD in several neuroblastoma cell lines.8Furthermore, a lack of HD at a tumor suppressor locus does not preclude inactivation of the gene by other genetic or epigenetic events, such as gene mutation or methylation-mediated gene silencing.
The region of 9p21 is frequently deleted in a wide range of malignancies (33). Three loci in 9p21 have been implicated as TSGs: CDKN2A/p16INK4a and CDKN2A/p14ARF, which partially share a coding exon but are encoded by two distinct reading frames; and the highly homologous CDKN2B/p15INK4b(Fig. 2; Refs. 3, 34). Substantial genetic evidence suggests that disruption of both p16INK4a and p14ARF, but not p15INK4b,is crucial for tumor development (18). Almost all tumor-specific rearrangements of 9p21 alter exon 2, which is shared by p16INK4a and p14ARF, and only a few mutations affecting just one of the three implicated proteins have been reported (18, 35). Targeted deletion experiments of the three loci in mice also suggest a causative role for CDKN2A but not CDKN2B, as mice with germ-line disruptions of CDKN2A are cancer-prone (36). p16INK4a acts as an inhibitor of the cell cycle activators cdk4 and cdk6, which in turn inactivate the pRB tumor suppressor protein, whereas p14ARF is thought to derepress p53 by binding to and inactivating mdm2 (18). Disruptions of the CDKN2A locus that concomitantly eliminate functional p16INK4a and p14ARF are thus believed to inactivate both the p53 and Rb tumor suppression pathways.
Inactivation of both the Rb and p53 pathways are key events for tumorigenicity in almost all neoplasms (31, 37). Because mutations and gene rearrangements of TP53 and RB1are rare in neuroblastoma, these pathways might instead be disrupted by other mechanisms. One possible alternative is a CDKN2A gene rearrangement that simultaneously inactivates both p16INK4a and p14ARF. The four neuroblastoma HDs described in the present study all target CDKN2A exon 2 and disrupt both the p16INK4a and p14ARF coding regions, consistent with the type of CDKN2A rearrangements seen in other malignancies. The exon 2-specific 104-bp CDKN2A deletion observed in cell line CHLA-101 supports this hypothesis. Also, because the CHLA-101 and CHLA-179 HDs do not extend to CDKN2B, it is unlikely that biallelic disruption of CDKN2B plays a role in neuroblastoma tumorigenesis, although monoallelic deletion of this gene could conceivably have an ancillary effect. However, mutations or deletions limited to CDKN2Bhave not been detected in neuroblastomas (38).
Previous genetic analyses have reported evidence for infrequent disruption of CDKN2A in neuroblastoma. Several LOH studies cumulatively found 9p21 allelic loss in 29 of 131 (22%) primary neuroblastomas (14, 38, 39, 40, 41), with LOH most frequently observed in tumors identified by mass screening of urinary catecholamine metabolites. However, HD of CDKN2A has not been found in primary tumors (3, 14, 38, 39, 41, 42, 43),although Dicicianni et al. (14) reported HD for an adriamycin-resistant subclone of the Be2C cell line that was not observed in the parent cell line. Comparative genomic hybridization studies of primary neuroblastomas have also reported only a low frequency of 9p deletions (4). Furthermore, only a single CDKN2A mutation, a missense mutation at residue 52 of p16INK4a in exon 2 has been identified out of 178 primary tumors and 28 cell lines screened (14, 38, 39, 41, 42, 43). Notably, exon 1β (encoding p14ARF) has not been included in any of the mutation screens, but these results together suggest that a purely genetic disruption of the CDKN2A locus is uncommon. Our results agree with these findings in that only 4 of 46 (9%) cell lines demonstrate gross biallelic inactivation. Nevertheless, our identification of HD in two of three corresponding primary tumors suggests that CDKN2A inactivation is a contributing in vivo genetic event for a subset of neuroblastomas, rather than a strictly in vitro phenomenon.
It is also conceivable that p16INK4a and/or p14ARF are inactivated by alternative mechanisms. A correlation between promoter hypermethylation and decreased expression of the p16INK4a transcript, presumably providing an epigenetic mechanism for CDKN2A suppression,has been noted in other malignancies (44, 45, 46). Three studies of CDKN2A methylation in neuroblastoma cumulatively found exon 1α methylation in 35% of tumors and cell lines, but no correlations between methylation status and expression levels were apparent (14, 38, 41). The methylation status and mutational analysis of CDKN2A exon 1β in neuroblastoma has not been reported. The expression pattern of CDKN2A in primary tumors is varied and exhibits no obvious correlations with other parameters (38, 41, 47). Likewise, analyses of other genes in the CDKN2A pathway have found few abnormalities in neuroblastoma (14, 38, 42, 47, 48, 49).
Mutation of the TP53 gene is very rare in primary neuroblastomas, and deletion or LOH of 17p is uncommon(4). There is, however, evidence that inactivation of the p53 pathway is important in some neuroblastomas. Amplification of the p53-inhibitory MDM2 locus has been identified in several neuroblastoma cell lines and in a single tumor (50, 51, 52). Furthermore, immunohistochemistry studies suggest that p53 is sequestered in the cytoplasm in neuroblastomas, which has been postulated as a posttranslational mechanism for functional repression of p53 (53, 54). The CDKN2A deletions observed in the present study, which would be predicted to affect p53 function through p14ARF, are consistent with alterations in p53 signaling playing a role in the tumorigenesis of some neuroblastomas. More detailed analysis of p14ARFexpression and function in neuroblastoma will be needed to fully understand the contribution that this protein plays in neuroblastoma.
Survey of HD at D1S47 (1p36.3) and CDKN2A exon 2 (9p21). Cell lines were amplified with primers for D1S47 (A) and CDKN2A exon 2 (B) and resolved by horizontal gel electrophoresis. A, at D1S47, a product of the predicted size was detected in every neuroblastoma cell line and in a normal human control, but not in the HD control (RH) or a negative control (no template). B, the CDKN2A primers did not amplify a product in three neuroblastoma cell lines (CHLA-174, CHLA-179, and LA-N-6). In addition, a CDKN2A product ∼100 bp shorter is present only in cell line CHLA-101, which is consistent with DNA sequencing results showing a 104-bp deletion in this sample.
Survey of HD at D1S47 (1p36.3) and CDKN2A exon 2 (9p21). Cell lines were amplified with primers for D1S47 (A) and CDKN2A exon 2 (B) and resolved by horizontal gel electrophoresis. A, at D1S47, a product of the predicted size was detected in every neuroblastoma cell line and in a normal human control, but not in the HD control (RH) or a negative control (no template). B, the CDKN2A primers did not amplify a product in three neuroblastoma cell lines (CHLA-174, CHLA-179, and LA-N-6). In addition, a CDKN2A product ∼100 bp shorter is present only in cell line CHLA-101, which is consistent with DNA sequencing results showing a 104-bp deletion in this sample.
Extent of HD in four neuroblastoma cell lines. Top, map of 9p21 region surrounding CDKN2A. Shaded boxes indicate CDKN2B (p15) and CDKN2A (p16) exons. Markers used in the HD assay are depicted by solid vertical ticks. The map is to scale except for the most 5′ and 3′flanking markers and is oriented relative to the chromosome 9 centromere (cen) and 9p telomere (tel). Below, the extent of HD in the four neuroblastoma cell lines. Horizontal black bars indicate the presence of at least one copy of the region.
Extent of HD in four neuroblastoma cell lines. Top, map of 9p21 region surrounding CDKN2A. Shaded boxes indicate CDKN2B (p15) and CDKN2A (p16) exons. Markers used in the HD assay are depicted by solid vertical ticks. The map is to scale except for the most 5′ and 3′flanking markers and is oriented relative to the chromosome 9 centromere (cen) and 9p telomere (tel). Below, the extent of HD in the four neuroblastoma cell lines. Horizontal black bars indicate the presence of at least one copy of the region.
Southern analysis of CDKN2A. Genomic DNA samples were digested with PstI and hybridized with a CDKN2A exon 2 probe that recognizes a 3.5 kb PstI fragment. HD is apparent in the cell lines Jurkat (negative control), LA-N-6, CHLA-179, and CHLA-174 and in a corresponding primary tumor sample for CHLA-174. CHLA-101 exhibits a band shift that is the result of a 104-bp deletion entirely within exon 2. CL, cell line DNA; T, primary tumor DNA; N, normal(nonmalignant tissue) DNA. No tumor sample was available for LA-N-6.
Southern analysis of CDKN2A. Genomic DNA samples were digested with PstI and hybridized with a CDKN2A exon 2 probe that recognizes a 3.5 kb PstI fragment. HD is apparent in the cell lines Jurkat (negative control), LA-N-6, CHLA-179, and CHLA-174 and in a corresponding primary tumor sample for CHLA-174. CHLA-101 exhibits a band shift that is the result of a 104-bp deletion entirely within exon 2. CL, cell line DNA; T, primary tumor DNA; N, normal(nonmalignant tissue) DNA. No tumor sample was available for LA-N-6.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported in part by Grants CA39771 (to G. M. B.), CA78966 (to J. M. M.), CA60104 (to R. C. S.), and CA82830 (to C. P. R.) from the NIH, and the Audrey E. Evans Chair in Molecular Oncology (to G. M. B.).
The abbreviations used are: TSG, tumor suppressor gene; HD, homozygous deletion; LOH, loss of heterozygosity;SRO, smallest region of overlap; RH, radiation hybrid.
Internet address: www.gdb.org.
Internet address:www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi.
Internet address:compgen.rutgers.edu/chr1/data/1p36map/index.shtml.
Internet address: www.ncbi.nlm.nih.gov/.
J. M. Maris and N. Lisitsyn, unpublished results.
Neuroblastoma cell line panel
Cell line . | Reference . | MYCN statusa . | MYCN reference . | 1p statusb . | 1p reference . |
---|---|---|---|---|---|
CHLA-10 | CSc | Single copy | CS | LOH | CS |
CHLA-42 | (19) | Single copy | (19) | LOH | CS |
CHLA-51 | (19) | Single copy | (19) | LOH | CS |
CHLA-52 | CS | Amplified | CS | LOH | CS |
CHLA-54 | CS | Amplified | CS | LOH | CS |
CHLA-60 | CS | Single copy | CS | LOH | CS |
CHLA-79 | (19) | Single copy | (19) | LOH | CS |
CHLA-90 | (19) | Single copy | (19) | LOH | CS |
CHLA-95 | CS | Amplified | CS | LOH | CS |
CHLA-98 | CS | Amplified | CS | LOH | CS |
CHLA-101 | CS | Amplified | CS | LOH | CS |
CHLA-103 | CS | Amplified | CS | LOH | CS |
CHLA-108 | CS | Amplified | CS | LOH | CS |
CHLA-123 | CS | Amplified | CS | LOH | CS |
CHLA-124 | CS | Amplified | CS | LOH | CS |
CHLA-132 | CS | Single copy | CS | LOH | CS |
CHLA-136 | (55) | Amplified | (55) | LOH | CS |
CHLA-138 | CS | Amplified | CS | LOH | CS |
CHLA-140 | (55) | Single copy | (55) | LOH | CS |
CHLA-143 | CS | Amplified | CS | LOH | CS |
CHLA-150 | CS | Single copy | CS | LOH | CS |
CHLA-152 | CS | Amplified | CS | LOH | CS |
CHLA-153 | CS | Amplified | CS | LOH | CS |
CHLA-171 | (56) | Single copy | (56) | No LOH | CS |
CHLA-174 | CS | Single copy | CS | No LOH | CS |
CHLA-178 | CS | Amplified | CS | LOH | CS |
CHLA-179 | CS | Amplified | CS | LOH | CS |
CHLA-185 | CS | Amplified | CS | LOH | CS |
CHP-134 | (57) | Amplified | (58) | Deletion | (59) |
CHP-901 | CS | Amplified | CS | LOH | CS |
CHP-902R | CS | Amplified | CS | LOH | CS |
LA-N-5 | (60) | Amplified | (61) | Deletion | (62) |
LA-N-6 | (63) | Single copy | (61) | LOH | CS |
N206 | (64) | Amplified | (65) | Deletion | (65) |
NB-69 | (66) | Single copy | (66) | Deletion | (66) |
NBL-S | (67) | Single copy | (67) | No LOH | CS |
NGP | (68) | Amplified | (58) | No deletion | (68) |
NLF | (58) | Amplified | (58) | Deletion | (62) |
NMB | (68) | Amplified | (58) | Deletion | (69) |
SK-N-AS | (61) | Single copy | (61) | LOH | CS |
SK-N-BE | (70) | Amplified | (61) | Deletion | (71) |
SK-N-DZ | (72) | Amplified | (61) | No LOH | CS |
SK-N-FI | (72) | Single copy | (61) | No deletion | (65) |
SK-N-SH | (73) | Single copy | (58) | No deletion | (68) |
SMS-KAN | (71) | Amplified | (61) | Deletion | (71) |
SMS-MSN | (74) | Amplified | (61) | LOH | CS |
Cell line . | Reference . | MYCN statusa . | MYCN reference . | 1p statusb . | 1p reference . |
---|---|---|---|---|---|
CHLA-10 | CSc | Single copy | CS | LOH | CS |
CHLA-42 | (19) | Single copy | (19) | LOH | CS |
CHLA-51 | (19) | Single copy | (19) | LOH | CS |
CHLA-52 | CS | Amplified | CS | LOH | CS |
CHLA-54 | CS | Amplified | CS | LOH | CS |
CHLA-60 | CS | Single copy | CS | LOH | CS |
CHLA-79 | (19) | Single copy | (19) | LOH | CS |
CHLA-90 | (19) | Single copy | (19) | LOH | CS |
CHLA-95 | CS | Amplified | CS | LOH | CS |
CHLA-98 | CS | Amplified | CS | LOH | CS |
CHLA-101 | CS | Amplified | CS | LOH | CS |
CHLA-103 | CS | Amplified | CS | LOH | CS |
CHLA-108 | CS | Amplified | CS | LOH | CS |
CHLA-123 | CS | Amplified | CS | LOH | CS |
CHLA-124 | CS | Amplified | CS | LOH | CS |
CHLA-132 | CS | Single copy | CS | LOH | CS |
CHLA-136 | (55) | Amplified | (55) | LOH | CS |
CHLA-138 | CS | Amplified | CS | LOH | CS |
CHLA-140 | (55) | Single copy | (55) | LOH | CS |
CHLA-143 | CS | Amplified | CS | LOH | CS |
CHLA-150 | CS | Single copy | CS | LOH | CS |
CHLA-152 | CS | Amplified | CS | LOH | CS |
CHLA-153 | CS | Amplified | CS | LOH | CS |
CHLA-171 | (56) | Single copy | (56) | No LOH | CS |
CHLA-174 | CS | Single copy | CS | No LOH | CS |
CHLA-178 | CS | Amplified | CS | LOH | CS |
CHLA-179 | CS | Amplified | CS | LOH | CS |
CHLA-185 | CS | Amplified | CS | LOH | CS |
CHP-134 | (57) | Amplified | (58) | Deletion | (59) |
CHP-901 | CS | Amplified | CS | LOH | CS |
CHP-902R | CS | Amplified | CS | LOH | CS |
LA-N-5 | (60) | Amplified | (61) | Deletion | (62) |
LA-N-6 | (63) | Single copy | (61) | LOH | CS |
N206 | (64) | Amplified | (65) | Deletion | (65) |
NB-69 | (66) | Single copy | (66) | Deletion | (66) |
NBL-S | (67) | Single copy | (67) | No LOH | CS |
NGP | (68) | Amplified | (58) | No deletion | (68) |
NLF | (58) | Amplified | (58) | Deletion | (62) |
NMB | (68) | Amplified | (58) | Deletion | (69) |
SK-N-AS | (61) | Single copy | (61) | LOH | CS |
SK-N-BE | (70) | Amplified | (61) | Deletion | (71) |
SK-N-DZ | (72) | Amplified | (61) | No LOH | CS |
SK-N-FI | (72) | Single copy | (61) | No deletion | (65) |
SK-N-SH | (73) | Single copy | (58) | No deletion | (68) |
SMS-KAN | (71) | Amplified | (61) | Deletion | (71) |
SMS-MSN | (74) | Amplified | (61) | LOH | CS |
Amplified, presence of ≥3 diploid copies of the MYCN oncogene. Single copy, no evidence of MYCN amplification.
Chromosomal status of 1p36. LOH,loss of heterozygosity or regional homozygosity for 1p36 polymorphisms(see “Materials and Methods”); No LOH, loss of heterozygosity or homozygosity not observed; Deletion, cytogenetic evidence for deletion of one 1p36 homologue; No deletion, no cytogenetically visible deletion of 1p36.
Determined in the current study.
Markers within 1p36 used for HD analysis
Marker name . | Marker typea . | Map intervalb . | GDB IDc . |
---|---|---|---|
Within the 1p36.3 SROb | |||
SHGC-32565 | Genomic STS | 6 | GDB: 6454441 |
D1S1215 | Genomic STS | 6 | GDB: 9925924 |
TP73 | Gene | 6 | GDB: 9925922 |
NBR | Gene | 6 | GDB: 5218325 |
TIGR-A008H35 | EST | 6 | GDB: 6054525 |
KIAA0495-1 | Gene | 6 | GDB: 9878921 |
KIAA0495-2 | Gene | 6 | GDB: 9867124 |
SHGC-74093 | EST | 6 | GDB: 9897877 |
D1S468 | Polymorphism | 6 | GDB: 199719 |
D1S3578 | EST | 6 | GDB: 734773 |
KIAA0495-3 | Gene | 6 | GDB: 9897995 |
D1S1467 | Genomic STS | 6 | GDB: 330403 |
D1S1341 | Genomic STS | 6 | GDB: 330025 |
D1S1446 | Genomic STS | 6 | GDB: 330340 |
D1S3023 | EST | 6 | GDB: 626297 |
stSG4593 | EST | 6 | GDB: 4566301 |
SHGC-30623 | EST | 6.5 | GDB: 4567035 |
EIF3S2-1 | Gene | 6.5 | GDB: 6054578 |
stSG26492 | EST | 6.5 | GDB: 9870106 |
EIF3S2-2 | Gene | 6.5 | GDB: 9868174 |
stSG32237 | EST | 6.5 | GDB: 9883824 |
D1S2845 | Polymorphism | 7 | GDB: 613167 |
D1S2132 | Polymorphism | 7 | GDB: 685449 |
D1S2660 | Polymorphism | 7 | GDB: 606855 |
D1S1475 | Genomic STS | 7 | GDB: 330427 |
stSG10106 | EST | 7 | GDB: 4570133 |
D1S3378 | EST | 7 | GDB: 678361 |
WI-10480 | Genomic STS | 7 | GDB: 1220375 |
D1S2893 | Polymorphism | 7 | GDB: 614637 |
D1S1476 | Genomic STS | 7 | GDB: 330430 |
D1S1608 | Polymorphism | 7 | GDB: 685038 |
KIAA0673 | Gene | 7 | GDB: 441576 |
WI-21934 | EST | 7 | GDB: 7087737 |
D1S2306E | EST | 7 | GDB: 581241 |
D1S2145 | Polymorphism | 7 | GDB: 685923 |
D1S3405 | Genomic STS | 7 | GDB: 678641 |
D1S3676 | Genomic STS | 7 | GDB: 3750033 |
stSG28971 | Genomic STS | 7 | GDB: 6055170 |
D1S1371 | Genomic STS | 7 | GDB: 330115 |
D1S2795 | Polymorphism | 7 | GDB: 611883 |
D1S2633 | Polymorphism | 9 | GDB: 603389 |
TIGR-A009F43 | Genomic STS | 9 | GDB: 9867326 |
D1S47 | Genomic STS | 9 | GDB: 549224 |
WI-22579 | EST | 10 | GDB: 7087844 |
H96854 | EST | 10 | GDB: 9879415 |
KCNAB2 | Gene | 10 | GDB: 4565990 |
D1S1257 | Genomic STS | 10 | GDB: 329773 |
WI-15605 | EST | 10 | GDB: 4574060 |
D1S2731 | Polymorphism | 10 | GDB: 609633 |
D1S3701 | Genomic STS | 10 | GDB: 3949640 |
TNFRSF12-1 | Gene | 10 | GDB: 9838558 |
TNFRSF12-2 | Gene | 10 | GDB: 4565980 |
TIGR-A006V30 | EST | 10 | GDB: 4588048 |
D20409 | EST | 10 | GDB: 9876859 |
stSG28441 | EST | 10 | GDB: 9871600 |
D1S3041 | Polymorphism | 10 | GDB: 626374 |
D1S3702 | Genomic DNA | 10 | GDB: 3949645 |
SHGC-150 | EST | 10 | GDB: 4566749 |
D1S1873E | Genomic STS | 10 | GDB: 443043 |
N95458 | EST | 10 | GDB: 9886908 |
stSG4588 | EST | 10 | GDB: 4566297 |
stSG2139 | EST | 10 | GDB: 4564685 |
U91316 | EST | 10 | GDB: 9873033 |
WI-13857 | EST | 10 | GDB: 4576676 |
D1S2870 | Polymorphism | 10 | GDB: 613758 |
D1S253 | Polymorphism | 10 | GDB: 188555 |
D1S1250 | Genomic STS | 10 | GDB: 329752 |
D1S3100 | Genomic STS | 10 | GDB: 636122 |
HKR3 | Gene | 10 | GDB: 3945572 |
stSG22641 | EST | 10 | GDB: 9884431 |
D1S1365 | Genomic STS | 10 | GDB: 330097 |
D1S2642 | Polymorphism | 11 | GDB: 603794 |
D1S214 | Polymorphism | 11 | GDB: 188072 |
D1S1646 | Polymorphism | 11 | GDB: 684504 |
D1S1398 | Genomic STS | 11 | GDB: 330196 |
WI-11303 | EST | 11 | GDB: 1220330 |
Outside 1p36.3 SROb | |||
D1S3672 | Genomic STS | 0.5 | GDB: 3750025 |
D1S3674 | Genomic STS | 0.5 | GDB: 3750029 |
D1S1287 | Genomic STS | 0.5 | GDB: 329863 |
D1S80 | Polymorphism | 1 | GDB: 178639 |
TNFRSF14 | Gene | 1 | GDB: 440310 |
stSG9144 | EST | 1 | GDB: 4569901 |
D1S2515 | EST | 1 | GDB: 596514 |
RER1 | Gene | 1 | GDB: 4569901 |
D1S76 | Polymorphism | 1 | GDB: 168840 |
PEX10 | Gene | 1 | GDB: 4578612 |
Marker name . | Marker typea . | Map intervalb . | GDB IDc . |
---|---|---|---|
Within the 1p36.3 SROb | |||
SHGC-32565 | Genomic STS | 6 | GDB: 6454441 |
D1S1215 | Genomic STS | 6 | GDB: 9925924 |
TP73 | Gene | 6 | GDB: 9925922 |
NBR | Gene | 6 | GDB: 5218325 |
TIGR-A008H35 | EST | 6 | GDB: 6054525 |
KIAA0495-1 | Gene | 6 | GDB: 9878921 |
KIAA0495-2 | Gene | 6 | GDB: 9867124 |
SHGC-74093 | EST | 6 | GDB: 9897877 |
D1S468 | Polymorphism | 6 | GDB: 199719 |
D1S3578 | EST | 6 | GDB: 734773 |
KIAA0495-3 | Gene | 6 | GDB: 9897995 |
D1S1467 | Genomic STS | 6 | GDB: 330403 |
D1S1341 | Genomic STS | 6 | GDB: 330025 |
D1S1446 | Genomic STS | 6 | GDB: 330340 |
D1S3023 | EST | 6 | GDB: 626297 |
stSG4593 | EST | 6 | GDB: 4566301 |
SHGC-30623 | EST | 6.5 | GDB: 4567035 |
EIF3S2-1 | Gene | 6.5 | GDB: 6054578 |
stSG26492 | EST | 6.5 | GDB: 9870106 |
EIF3S2-2 | Gene | 6.5 | GDB: 9868174 |
stSG32237 | EST | 6.5 | GDB: 9883824 |
D1S2845 | Polymorphism | 7 | GDB: 613167 |
D1S2132 | Polymorphism | 7 | GDB: 685449 |
D1S2660 | Polymorphism | 7 | GDB: 606855 |
D1S1475 | Genomic STS | 7 | GDB: 330427 |
stSG10106 | EST | 7 | GDB: 4570133 |
D1S3378 | EST | 7 | GDB: 678361 |
WI-10480 | Genomic STS | 7 | GDB: 1220375 |
D1S2893 | Polymorphism | 7 | GDB: 614637 |
D1S1476 | Genomic STS | 7 | GDB: 330430 |
D1S1608 | Polymorphism | 7 | GDB: 685038 |
KIAA0673 | Gene | 7 | GDB: 441576 |
WI-21934 | EST | 7 | GDB: 7087737 |
D1S2306E | EST | 7 | GDB: 581241 |
D1S2145 | Polymorphism | 7 | GDB: 685923 |
D1S3405 | Genomic STS | 7 | GDB: 678641 |
D1S3676 | Genomic STS | 7 | GDB: 3750033 |
stSG28971 | Genomic STS | 7 | GDB: 6055170 |
D1S1371 | Genomic STS | 7 | GDB: 330115 |
D1S2795 | Polymorphism | 7 | GDB: 611883 |
D1S2633 | Polymorphism | 9 | GDB: 603389 |
TIGR-A009F43 | Genomic STS | 9 | GDB: 9867326 |
D1S47 | Genomic STS | 9 | GDB: 549224 |
WI-22579 | EST | 10 | GDB: 7087844 |
H96854 | EST | 10 | GDB: 9879415 |
KCNAB2 | Gene | 10 | GDB: 4565990 |
D1S1257 | Genomic STS | 10 | GDB: 329773 |
WI-15605 | EST | 10 | GDB: 4574060 |
D1S2731 | Polymorphism | 10 | GDB: 609633 |
D1S3701 | Genomic STS | 10 | GDB: 3949640 |
TNFRSF12-1 | Gene | 10 | GDB: 9838558 |
TNFRSF12-2 | Gene | 10 | GDB: 4565980 |
TIGR-A006V30 | EST | 10 | GDB: 4588048 |
D20409 | EST | 10 | GDB: 9876859 |
stSG28441 | EST | 10 | GDB: 9871600 |
D1S3041 | Polymorphism | 10 | GDB: 626374 |
D1S3702 | Genomic DNA | 10 | GDB: 3949645 |
SHGC-150 | EST | 10 | GDB: 4566749 |
D1S1873E | Genomic STS | 10 | GDB: 443043 |
N95458 | EST | 10 | GDB: 9886908 |
stSG4588 | EST | 10 | GDB: 4566297 |
stSG2139 | EST | 10 | GDB: 4564685 |
U91316 | EST | 10 | GDB: 9873033 |
WI-13857 | EST | 10 | GDB: 4576676 |
D1S2870 | Polymorphism | 10 | GDB: 613758 |
D1S253 | Polymorphism | 10 | GDB: 188555 |
D1S1250 | Genomic STS | 10 | GDB: 329752 |
D1S3100 | Genomic STS | 10 | GDB: 636122 |
HKR3 | Gene | 10 | GDB: 3945572 |
stSG22641 | EST | 10 | GDB: 9884431 |
D1S1365 | Genomic STS | 10 | GDB: 330097 |
D1S2642 | Polymorphism | 11 | GDB: 603794 |
D1S214 | Polymorphism | 11 | GDB: 188072 |
D1S1646 | Polymorphism | 11 | GDB: 684504 |
D1S1398 | Genomic STS | 11 | GDB: 330196 |
WI-11303 | EST | 11 | GDB: 1220330 |
Outside 1p36.3 SROb | |||
D1S3672 | Genomic STS | 0.5 | GDB: 3750025 |
D1S3674 | Genomic STS | 0.5 | GDB: 3750029 |
D1S1287 | Genomic STS | 0.5 | GDB: 329863 |
D1S80 | Polymorphism | 1 | GDB: 178639 |
TNFRSF14 | Gene | 1 | GDB: 440310 |
stSG9144 | EST | 1 | GDB: 4569901 |
D1S2515 | EST | 1 | GDB: 596514 |
RER1 | Gene | 1 | GDB: 4569901 |
D1S76 | Polymorphism | 1 | GDB: 168840 |
PEX10 | Gene | 1 | GDB: 4578612 |
Continued
Marker name . | Marker typea . | Map intervalb . | GDB IDc . |
---|---|---|---|
D1S243 | Polymorphism | 2 | GDB: 188393 |
WI-16874 | EST | 2 | GDB: 4582418 |
WI-15347 | EST | 2 | GDB: 4579637 |
A006A26 | STS | 2 | GDB: 4571021 |
SGC34147 | EST | 2 | GDB: 4582234 |
WI-14412 | EST | 2 | GDB: 4581970 |
SCNN1D | Gene | 2 | GDB: 5053412 |
SGC33169 | EST | 2 | GDB: 4580254 |
WI-11477 | EST | 3 | GDB: 1220275 |
TIGR-A007H27 | EST | 3 | GDB: 4588644 |
stSG9906 | EST | 3 | GDB: 4570099 |
WI-18288 | EST | 3 | GDB: 4582616 |
stSG4467 | EST | 3 | GDB: 4568853 |
KIAA0447 | Gene | 3 | GDB: 1220306 |
GNB1 | Gene | 4 | GDB: 636086 |
WI-13821 | EST | 5 | GDB: 4577655 |
D1S97 | Genomic STS | 5 | GDB: 636065 |
DVL1 | Gene | 5 | GDB: 4873202 |
D1S2694 | Polymorphism | 12 | GDB: 608406 |
D1S1306 | Genomic STS | 12 | GDB: 329920 |
D1S548 | Polymorphism | 13 | GDB: 686691 |
D1S508 | Polymorphism | 13 | GDB: 200120 |
DJ-1 | Gene | 15 | GDB: 1220388 |
TNFRSF9 | Gene | 15 | GDB: 3750021 |
SGC34994 | EST | 15 | GDB: 4575666 |
D1S1829E | EST | 15 | GDB: 441741 |
TNFRSF12-3 | Gene | 15 | GDB: 4579729 |
stSG4370 | EST | 15 | GDB: 4566135 |
D1S503 | Polymorphism | 15 | GDB: 200109 |
D1S1615 | Polymorphism | 15 | GDB: 686211 |
D1S3275 | EST | 15 | GDB: 675946 |
SLC2A5 | Gene | 15 | GDB: 188840 |
CA6 | Gene | 15 | GDB: 636125 |
D1S160 | Polymorphism | 15 | GDB: 182199 |
ENO1 | Gene | 16 | GDB: 549140 |
TIGR-A004Y04 | EST | 17 | GDB: 4583178 |
FRAP1 | Gene | 17 | GDB: 1220473 |
D1S450 | Polymorphism | 18 | GDB: 199528 |
D1S244 | Polymorphism | 18 | GDB: 188418 |
PIK3CD | Genomic STS | 18 | GDB: 1230531 |
D1S1768E | EST | 18 | GDB: 439878 |
SHGC-31453 | EST | 18 | GDB: 4567450 |
stSG1544 | EST | 19 | GDB: 4562131 |
D1S2028E | EST | 20 | GDB: 445905 |
TNFRSF8 | Gene | 21 | GDB: 549221 |
TNFRSF1B | Gene | 21 | GDB: 197005 |
RIZ | Gene | 21 | GDB: 4878792 |
SHGC-7147 | Genomic STS | 22 | GDB: 1236006 |
D1S1833E | EST | 23 | GDB: 441843 |
WI-13542 | EST | 24 | GDB: 4578031 |
D1S228 | Polymorphism | 24 | GDB: 188240 |
ZNF151 | Gene | 25 | GDB: 3750046 |
EPHA2 | Gene | 26 | GDB: 636074 |
PAX7 | Gene | 27 | GDB: 636107 |
D1S3017 | EST | 28 | GDB: 626241 |
HTR6 | EST | 29 | GDB: 678346 |
RNU1A | Gene | 30 | GDB: 5054970 |
WI-11927 | EST | 31 | GDB: 1220491 |
NBL1 | Gene | 31 | GDB: 636140 |
ECE1 | Gene | 32 | GDB: 4581562 |
ALPL | Gene | 33 | GDB: 549237 |
D1S2436 | Genomic STS | 33 | GDB: 588738 |
CDC42 | Gene | 33 | GDB: 1220325 |
RAP1GA1 | Gene | 33 | GDB: 3750043 |
TCEB3 | Gene | 34 | GDB: 5049874 |
E2F2 | Gene | 34 | GDB: 636071 |
HMGCL | Gene | 34 | GDB: 636092 |
LAP18 | Gene | 34 | GDB: 549193 |
ID3 | Gene | 35 | GDB: 636098 |
SHGC-2516 | EST | 36 | GDB: 3802031 |
C1ORF4 | Gene | 37 | GDB: 9925921 |
CHLC.GGAA2D04 | Polymorphism | 38 | GDB: 1220268 |
SLC9A1 | Gene | 39 | GDB: 549196 |
FGR | Gene | 40 | GDB: 188675 |
PTAFR | Gene | 40 | GDB: 636116 |
D1S247 | Polymorphism | 45 | GDB: 188453 |
Marker name . | Marker typea . | Map intervalb . | GDB IDc . |
---|---|---|---|
D1S243 | Polymorphism | 2 | GDB: 188393 |
WI-16874 | EST | 2 | GDB: 4582418 |
WI-15347 | EST | 2 | GDB: 4579637 |
A006A26 | STS | 2 | GDB: 4571021 |
SGC34147 | EST | 2 | GDB: 4582234 |
WI-14412 | EST | 2 | GDB: 4581970 |
SCNN1D | Gene | 2 | GDB: 5053412 |
SGC33169 | EST | 2 | GDB: 4580254 |
WI-11477 | EST | 3 | GDB: 1220275 |
TIGR-A007H27 | EST | 3 | GDB: 4588644 |
stSG9906 | EST | 3 | GDB: 4570099 |
WI-18288 | EST | 3 | GDB: 4582616 |
stSG4467 | EST | 3 | GDB: 4568853 |
KIAA0447 | Gene | 3 | GDB: 1220306 |
GNB1 | Gene | 4 | GDB: 636086 |
WI-13821 | EST | 5 | GDB: 4577655 |
D1S97 | Genomic STS | 5 | GDB: 636065 |
DVL1 | Gene | 5 | GDB: 4873202 |
D1S2694 | Polymorphism | 12 | GDB: 608406 |
D1S1306 | Genomic STS | 12 | GDB: 329920 |
D1S548 | Polymorphism | 13 | GDB: 686691 |
D1S508 | Polymorphism | 13 | GDB: 200120 |
DJ-1 | Gene | 15 | GDB: 1220388 |
TNFRSF9 | Gene | 15 | GDB: 3750021 |
SGC34994 | EST | 15 | GDB: 4575666 |
D1S1829E | EST | 15 | GDB: 441741 |
TNFRSF12-3 | Gene | 15 | GDB: 4579729 |
stSG4370 | EST | 15 | GDB: 4566135 |
D1S503 | Polymorphism | 15 | GDB: 200109 |
D1S1615 | Polymorphism | 15 | GDB: 686211 |
D1S3275 | EST | 15 | GDB: 675946 |
SLC2A5 | Gene | 15 | GDB: 188840 |
CA6 | Gene | 15 | GDB: 636125 |
D1S160 | Polymorphism | 15 | GDB: 182199 |
ENO1 | Gene | 16 | GDB: 549140 |
TIGR-A004Y04 | EST | 17 | GDB: 4583178 |
FRAP1 | Gene | 17 | GDB: 1220473 |
D1S450 | Polymorphism | 18 | GDB: 199528 |
D1S244 | Polymorphism | 18 | GDB: 188418 |
PIK3CD | Genomic STS | 18 | GDB: 1230531 |
D1S1768E | EST | 18 | GDB: 439878 |
SHGC-31453 | EST | 18 | GDB: 4567450 |
stSG1544 | EST | 19 | GDB: 4562131 |
D1S2028E | EST | 20 | GDB: 445905 |
TNFRSF8 | Gene | 21 | GDB: 549221 |
TNFRSF1B | Gene | 21 | GDB: 197005 |
RIZ | Gene | 21 | GDB: 4878792 |
SHGC-7147 | Genomic STS | 22 | GDB: 1236006 |
D1S1833E | EST | 23 | GDB: 441843 |
WI-13542 | EST | 24 | GDB: 4578031 |
D1S228 | Polymorphism | 24 | GDB: 188240 |
ZNF151 | Gene | 25 | GDB: 3750046 |
EPHA2 | Gene | 26 | GDB: 636074 |
PAX7 | Gene | 27 | GDB: 636107 |
D1S3017 | EST | 28 | GDB: 626241 |
HTR6 | EST | 29 | GDB: 678346 |
RNU1A | Gene | 30 | GDB: 5054970 |
WI-11927 | EST | 31 | GDB: 1220491 |
NBL1 | Gene | 31 | GDB: 636140 |
ECE1 | Gene | 32 | GDB: 4581562 |
ALPL | Gene | 33 | GDB: 549237 |
D1S2436 | Genomic STS | 33 | GDB: 588738 |
CDC42 | Gene | 33 | GDB: 1220325 |
RAP1GA1 | Gene | 33 | GDB: 3750043 |
TCEB3 | Gene | 34 | GDB: 5049874 |
E2F2 | Gene | 34 | GDB: 636071 |
HMGCL | Gene | 34 | GDB: 636092 |
LAP18 | Gene | 34 | GDB: 549193 |
ID3 | Gene | 35 | GDB: 636098 |
SHGC-2516 | EST | 36 | GDB: 3802031 |
C1ORF4 | Gene | 37 | GDB: 9925921 |
CHLC.GGAA2D04 | Polymorphism | 38 | GDB: 1220268 |
SLC9A1 | Gene | 39 | GDB: 549196 |
FGR | Gene | 40 | GDB: 188675 |
PTAFR | Gene | 40 | GDB: 636116 |
D1S247 | Polymorphism | 45 | GDB: 188453 |
EST, expressed sequence tag;STS, sequence-tagged site.
The 1p36.3 SRO is contained within map intervals 6–11 as defined in (21). Intervals 0–15,16–30, and 31–40 correspond approximately to cytogenetic bands 1p36.3, 1p36.2, and 1p36.1, respectively. The distal 1p36.2 translocation breakpoint in the NGP cell line maps within map interval 15. Markers are not ordered within specific intervals.
GDB ID, Genome Database unique identifier.4
Tumor suppressor and 9p loci analyzed for HD
Locus name . | Cytolocationa . | Positionb . | GDB IDc . | |||
---|---|---|---|---|---|---|
Tumor suppressor loci | ||||||
VHL | 3p25-p26 | Exon 2 | GDB: 361151 | |||
FHIT | 3p14.2 | Exon 6 | GDB: 4579455 | |||
APC | 5q21-q22 | Exon 8 | GDB: 439445 | |||
PTCH | 9q31 | Exon 11 | GDB: 9925913 | |||
TSC1 | 9q34 | Exon 15 | GDB: 9925919 | |||
RET | 10q11.2 | Exon 7 | GDB: 9925917 | |||
PTEN | 10q23.3 | Exon 7 | GDB: 9925915 | |||
WT1 | 11p13 | Exon 9 | GDB: 371667 | |||
MEN1 | 11q13 | Exon 3 | GDB: 9925907 | |||
ATM | 11q22-q23 | Exon 25 | GDB: 9925903 | |||
PPP2R1B | 11q23 | Exon 3 | GDB: 9925911 | |||
BRCA2 | 13q12.3 | Exon 9 | GDB: 6013909 | |||
RB1 | 13q14.2 | Exon 24 | GDB: 344171 | |||
TSC2 | 16p13.3 | Exon 23 | GDB: 6053988 | |||
TP53 | 17p13.1 | Exon 6 | GDB: 186921 | |||
NF1 | 17q11.2 | Exon 26 | GDB: 5887052 | |||
BRCA1 | 17q21 | Exon 8 | GDB: 389047 | |||
DCC | 18q21.1 | 3′ UTR | GDB: 190580 | |||
MADH4 | 18q21.1 | Exon 11 | GDB: 9925905 | |||
NF2 | 22q12.2 | Exon 7 | GDB: 9925909 | |||
9p markers | ||||||
D9S288 | 9p22-pter | 0.0227 | GDB: 200107 | |||
D9S286 | 9p22-pter | 0.0471 | GDB: 200054 | |||
D9S256 | 9p23-9p22 | 0.0663 | GDB: 199092 | |||
D9S162 | 9p22 | 0.1122 | GDB: 188003 | |||
IFNA 1&2 | 9p22 | 0.113–0.1156 | GDB: 181544 | |||
IFNA 3&4 | 9p22 | 0.113–0.1156 | GDB: 196683 | |||
D9S1749 | 9p21 | 0.1161 | GDB: 595876 | |||
1063.7 | 9p21 | 0.1161–0.1181 | GDB: 9925891 | |||
c18.b | 9p21 | 0.1161–0.1181; 8 kb 3′ of CDKN2A | GDB: 9925892 | |||
CDKN2ex3 | 9p21 | 0.1181–0.1182; CDKN2A exon 3 | GDB: 9925893 | |||
c5.1 | 9p21 | 0.1181–0.1182; CDKN2A intron 2 | GDB: 9925895 | |||
CDKN2ex2 | 9p21 | 0.1181–0.1182; CDKN2A exon 2 | GDB: 462451 | |||
CDKN2ex1 1+2 | 9p21 | 0.1181–0.1182; CDKN2A exon 1 | GDB: 462445 | |||
CDKN2ex1 5+6 | 9p21 | 0.1181–0.1182; CDKN2A exon 1 | GDB: 9925896 | |||
c5.3 | 9p21 | 0.1181–0.1182; CDKN2A intron 1 | GDB: 9925899 | |||
R2.3 | 9p21 | 0.1181–0.1182; CDKN2A intron 1 | GDB: 9925900 | |||
p14 | 9p21 | 0.1181–0.1182; CDKN2A exon 1 | GDB: 9925898 | |||
R2.7 | 9p21 | 0.1183–0.1184; 4 kb 5′ of CDKN2A | GDB: 9925901 | |||
D9S1752 | 9p21 | 0.1183–0.1184; 11 kb 5′ of CDKN2A | GDB: 595993 | |||
MTS2ex2 | 9p21 | 0.1183–0.1184; CDKN2B exon 2 | GDB: 679534 | |||
MTS2ex1 | 9p21 | 0.1183–0.1184; CDKN2B exon 1 | GDB: 5887034 | |||
D9S171 | 9p21 | 0.1745 | GDB: 188218 | |||
D9S1121 | 9pter-9qter | 0.1828–0.1831 | GDB: 685827 | |||
D9S169 | 9p21 | 0.1838 | GDB: 188142 | |||
D9S319 | 9p21 | 0.1895 | GDB: 228573 | |||
D9S1678 | 9p21 | 0.1906 | GDB: 582665 | |||
D9S165 | 9p21-9q21 | 0.192 | GDB: 188047 | |||
D9S301 | 9p21-9q21 | 0.4733 | GDB: 686844 |
Locus name . | Cytolocationa . | Positionb . | GDB IDc . | |||
---|---|---|---|---|---|---|
Tumor suppressor loci | ||||||
VHL | 3p25-p26 | Exon 2 | GDB: 361151 | |||
FHIT | 3p14.2 | Exon 6 | GDB: 4579455 | |||
APC | 5q21-q22 | Exon 8 | GDB: 439445 | |||
PTCH | 9q31 | Exon 11 | GDB: 9925913 | |||
TSC1 | 9q34 | Exon 15 | GDB: 9925919 | |||
RET | 10q11.2 | Exon 7 | GDB: 9925917 | |||
PTEN | 10q23.3 | Exon 7 | GDB: 9925915 | |||
WT1 | 11p13 | Exon 9 | GDB: 371667 | |||
MEN1 | 11q13 | Exon 3 | GDB: 9925907 | |||
ATM | 11q22-q23 | Exon 25 | GDB: 9925903 | |||
PPP2R1B | 11q23 | Exon 3 | GDB: 9925911 | |||
BRCA2 | 13q12.3 | Exon 9 | GDB: 6013909 | |||
RB1 | 13q14.2 | Exon 24 | GDB: 344171 | |||
TSC2 | 16p13.3 | Exon 23 | GDB: 6053988 | |||
TP53 | 17p13.1 | Exon 6 | GDB: 186921 | |||
NF1 | 17q11.2 | Exon 26 | GDB: 5887052 | |||
BRCA1 | 17q21 | Exon 8 | GDB: 389047 | |||
DCC | 18q21.1 | 3′ UTR | GDB: 190580 | |||
MADH4 | 18q21.1 | Exon 11 | GDB: 9925905 | |||
NF2 | 22q12.2 | Exon 7 | GDB: 9925909 | |||
9p markers | ||||||
D9S288 | 9p22-pter | 0.0227 | GDB: 200107 | |||
D9S286 | 9p22-pter | 0.0471 | GDB: 200054 | |||
D9S256 | 9p23-9p22 | 0.0663 | GDB: 199092 | |||
D9S162 | 9p22 | 0.1122 | GDB: 188003 | |||
IFNA 1&2 | 9p22 | 0.113–0.1156 | GDB: 181544 | |||
IFNA 3&4 | 9p22 | 0.113–0.1156 | GDB: 196683 | |||
D9S1749 | 9p21 | 0.1161 | GDB: 595876 | |||
1063.7 | 9p21 | 0.1161–0.1181 | GDB: 9925891 | |||
c18.b | 9p21 | 0.1161–0.1181; 8 kb 3′ of CDKN2A | GDB: 9925892 | |||
CDKN2ex3 | 9p21 | 0.1181–0.1182; CDKN2A exon 3 | GDB: 9925893 | |||
c5.1 | 9p21 | 0.1181–0.1182; CDKN2A intron 2 | GDB: 9925895 | |||
CDKN2ex2 | 9p21 | 0.1181–0.1182; CDKN2A exon 2 | GDB: 462451 | |||
CDKN2ex1 1+2 | 9p21 | 0.1181–0.1182; CDKN2A exon 1 | GDB: 462445 | |||
CDKN2ex1 5+6 | 9p21 | 0.1181–0.1182; CDKN2A exon 1 | GDB: 9925896 | |||
c5.3 | 9p21 | 0.1181–0.1182; CDKN2A intron 1 | GDB: 9925899 | |||
R2.3 | 9p21 | 0.1181–0.1182; CDKN2A intron 1 | GDB: 9925900 | |||
p14 | 9p21 | 0.1181–0.1182; CDKN2A exon 1 | GDB: 9925898 | |||
R2.7 | 9p21 | 0.1183–0.1184; 4 kb 5′ of CDKN2A | GDB: 9925901 | |||
D9S1752 | 9p21 | 0.1183–0.1184; 11 kb 5′ of CDKN2A | GDB: 595993 | |||
MTS2ex2 | 9p21 | 0.1183–0.1184; CDKN2B exon 2 | GDB: 679534 | |||
MTS2ex1 | 9p21 | 0.1183–0.1184; CDKN2B exon 1 | GDB: 5887034 | |||
D9S171 | 9p21 | 0.1745 | GDB: 188218 | |||
D9S1121 | 9pter-9qter | 0.1828–0.1831 | GDB: 685827 | |||
D9S169 | 9p21 | 0.1838 | GDB: 188142 | |||
D9S319 | 9p21 | 0.1895 | GDB: 228573 | |||
D9S1678 | 9p21 | 0.1906 | GDB: 582665 | |||
D9S165 | 9p21-9q21 | 0.192 | GDB: 188047 | |||
D9S301 | 9p21-9q21 | 0.4733 | GDB: 686844 |
Cytogenetic localizations are from the Genome Database.4
For tumor suppressor genes,exon/intron positions of markers are listed. For 9p markers, the fractional length position or range is shown for each marker according to the HUGO chromosome 9 integrated map (GDB: 6276683). Also listed for certain 9p markers are positions relative to CDKN2A/B;positions were deduced from available genomic sequence.
GDB ID, Genome Database unique identifier.
Acknowledgments
We gratefully acknowledge Naohiko Ikegaki, Frank Speleman, and Stuart Lessin for cell lines; Maureen Megonigal for the MLL probe; the Children’s Hospital of Philadelphia nucleic acid/protein research core facility for DNA sequencing; Nikolai Lisitsyn for representational differential analysis; Erik Sulman for helpful comments; and Cathy Lee for technical assistance.