Substantial genomic and functional evidence from primary tumors and cell lines indicates that a consistent region of distal chromosome 1p is deleted in a sizable proportion of human neuroblastomas, suggesting that this region contains one or more tumor suppressor genes. To determine systematically and precisely the location and extent of 1p deletion in neuroblastomas, we performed allelic loss studies of 737 primary neuroblastomas and genotype analysis of 46 neuroblastoma cell lines. Together, the results defined a single region within 1p36.3 that was consistently deleted in 25% of tumors and 87% of cell lines. Two neuroblastoma patients had constitutional deletions of distal 1p36 that overlapped the tumor-defined region. The tumor- and constitutionally-derived deletions together defined a smallest region of consistent deletion (SRD) between D1S2795 and D1S253. The 1p36.3 SRD was deleted in all but one of the 184 tumors with 1p deletion. Physical mapping and DNA sequencing determined that the SRD minimally spans an estimated 729 kb. Genomic content and sequence analysis of the SRD identified 15 characterized, nine uncharacterized, and six predicted genes in the region. The RNA expression profiles of 21 of the genes were investigated in a variety of normal tissues. The SHREW1 and KCNAB2 genes both had tissue-restricted expression patterns, including expression in the nervous system. In addition, a novel gene (CHD5) with strong homology to proteins involved in chromatin remodeling was expressed mainly in neural tissues. Together, these results suggest that one or more genes involved in neuroblastoma tumorigenesis or tumor progression are likely contained within this region.
Neuroblastoma is a common pediatric malignancy of the peripheral sympathetic nervous system. Despite recent advances in therapy, a large proportion of neuroblastoma patients succumb to the disease. Thus, identification and characterization of the genetic events underlying neuroblastoma tumorigenesis and progression are important priorities for management of this malignancy. Cytogenetic and molecular analyses of neuroblastoma tumors and cell lines have identified several frequently occurring genetic abnormalities, including deletion of chromosomes 1p, 11q, and 14q; allelic gain of 11p and 17q; and amplification of the MYCN proto-oncogene (Brodeur, 2003). Deletion of distal 1p is highly correlated with both MYCN amplification and an adverse patient outcome, suggesting the presence of one or more tumor suppressor genes (TSGs) within this region (Maris et al., 1995, 2000). Several lines of evidence support this hypothesis and further implicate 1p36 as the region most likely to contain a TSG. Numerous loss of heterozygosity (LOH) and molecular cytogenetic analyses of 1p in neuroblastoma have demonstrated allelic loss of 1p36 (Fong et al., 1989; Takayama et al., 1992; Schleiermacher et al., 1994; Takeda et al., 1994; White et al., 1995; Martinsson et al., 1997; Mora et al., 2000; Bauer et al., 2001; Maris et al., 2001; Godfried et al., 2002). Collectively, the incidence of LOH reported ranges from 25 to 35% of primary tumors. Furthermore, transfer of 1p chromosomal material into a neuroblastoma cell line has been shown to suppress tumorigenicity (Bader et al., 1991).
However, progress in narrowing the region and defining candidate TSGs within 1p36 has been slow, due in part to the fact that most 1p deletions are large. In addition, while several neuroblastoma cell line and constitutional chromosomal rearrangements involving 1p36 have been identified, the affected chromosomal regions do not tightly cluster (Barker et al., 1993; Savelyeva et al., 1994; Amler et al., 1995; van der Drift et al., 1995; Casciano et al., 1996; Ohira et al., 2000; Spieker et al., 2001; Satge et al., 2003). Moreover, linkage analysis of familial cases has excluded 1p36 as containing a familial predisposition locus (Maris et al., 1996).
We previously defined a region of allelic loss shared by virtually all primary neuroblastoma tumors with 1p deletions to 1p36.2–p36.3 by LOH analysis of 122 primary tumors (White et al., 1995), a finding which has subsequently been confirmed by other groups (Martinsson et al., 1997; Mora et al., 2000; Bauer et al., 2001; Spieker et al., 2001; Godfried et al., 2002). Our defined smallest region of consistent deletion (SRD) partially overlapped a constitutional 1p36 deletion in a patient subsequently diagnosed with neuroblastoma. In the present study, we undertook a comprehensive and large-scale molecular genetic approach to further characterize our initially defined SRD, with the objective of further narrowing the region and number of candidate genes to characterize. We expanded the LOH analysis to 737 primary tumors, along with genotype analyses of 46 neuroblastoma cell lines and of a second neuroblastoma patient with a constitutional 1p36 deletion. These data have allowed definition of a precisely bounded SRD within 1p36.3 that is consistently deleted in at least 25% of primary neuroblastomas. We also describe the physical mapping, sequencing, sequence analysis, and transcriptional profiles of genes within the defined SRD.
Paired primary tumor and normal DNA samples were collected from a cohort of 737 neuroblastoma patients. This cohort was generally representative of disease stage and age distributions, as well as for the frequency of MYCN amplification, with a slight bias towards tumors with a favorable outcome. Individual DNA pairs were genotyped for a subset of 61 polymorphic loci localizing to 1p (Table 1). The genotyping was performed in three phases. The first phase used a number of polymorphic loci throughout 1p in order to establish whether allelic loss was randomly distributed or concentrated within particular genomic regions of the chromosome arm. After establishment of 1p36 as the region of most consistent deletion, phase 2 primarily used polymorphic loci localized to distal 1p. As a distal 1p36 SRD was determined, phase 3 added 183 additional samples to the cohort (n=737), and additional markers within 1p36.3 were added in order to identify tumors with breakpoints within or near the defined SRD. After each phase, samples with interesting breakpoints were genotyped at further loci to confirm and localize the breakpoints. A tumor sample was considered to have LOH only if one allele signal was reduced >60% or more at two or more contiguous loci.
Within the entire primary tumor cohort, 184 of 737 tumors (25%) demonstrated allelic loss for two or more loci (Table 1). Of these, 117 cases showed LOH for all informative 1p loci surveyed, 65 cases showed partial 1p LOH that extended to the distal-most informative marker (partial terminal deletions), and two cases demonstrated interstitial deletion (Figure 1a). Of the cases with partial 1p deletions, the breakpoints were scattered throughout the chromosome arm, with no apparent clustering in specific regions. All but one of the 184 tumors with 1p LOH demonstrated LOH within a specific region of 1p36.3. This region was defined distally by tumor 670 with no LOH at D1S2660 but LOH at the adjacent locus D1S2795, and proximally by cases 216 and 428, both of which demonstrated no LOH at D1S214 but LOH distally (Figure 2). Case 216 showed LOH for the locus distally adjacent to D1S214 (D1S253), whereas case 428 was not informative for D1S253 but demonstrated LOH for the next adjacent locus distally (D1S2870). Together, these informative breakpoints defined a single SRD within 1p36.3.
Only one tumor demonstrated 1p LOH for a region other than the 1p36.3 SRD (Figure 1a). This tumor (case 222) demonstrated no allelic loss at 1p36.1 locus D1S3720 and also for five loci distal to D1S3720. However, this case showed LOH for three closely spaced loci within 1p32 (D1S1596, D1S1669, and D1S1643). No other tumor was found to contain an interstitial deletion of 1p exclusively proximal to the 1p36.3 SRD in our cohort.
We also determined the extent of 1p deletion within two individuals with neuroblastoma and known constitutional monosomy of 1p36. Clinical manifestations and preliminary genetic analysis of these cases have been described previously (Biegel et al., 1993; White et al., 1997). To determine the precise extent of the distal 1p regions deleted in these patients, genotyping of DNA from blood samples of the patients and corresponding parental samples was performed. Both 1p monosomy cases were found to have an interstitial deletion of the maternal copy of 1p36.2–1p36.3. Distally, case 1 showed heterozygosity for 1p36.3 locus D1S80 but hemizygosity for the next most proximal marker surveyed, D1S468. Proximally, case 1 was deleted for 1p36.2 locus D1S450, but six markers proximal to this locus were retained in both copies. For case 2, heterozygosity was noted for 1p36.3 marker D1S2660 and two informative loci distal to this locus (Figure 2). Proximally, case 2 was deleted for 1p36.2 locus D1S507 but not for informative loci proximal to this marker. The distal deletion boundary of case 2 thus coincided with and confirmed the distal boundary of the SRD defined by the primary tumor LOH results. The extent of deletion for each constitutional case entirely overlapped the tumor-defined 1p36.3 SRD (Figure 1b).
We also studied 46 genotype-unique neuroblastoma cell lines for 1p36 deletion using fluorescence in situ hybridization (FISH), cytogenetics, and/or inference of deletion from observing homozygosity at ⩾3 consecutive highly polymorphic 1p36 markers. In all, 40 of 46 (87%) cell lines demonstrated genomic abnormalities of 1p36 by these methods, either deletion (39 cases) or partial duplication and translocation (cell line NGP, t(1;15)(p36.1;q24),dup(1)(p36.2), see also Brodeur et al., 1977; Amler et al., 1995). All had relatively large deletions encompassing the entire SRD defined by the primary tumor results.
In combination, the primary tumor, constitutional case, and cell line deletion mapping results defined a single SRD within 1p36.3, extending minimally from D1S2795 to D1S253 and maximally from D1S2660 to D1S214. To create a physical map of the region, we first collected 381 PCR-formatted markers, including 227 representing unique transcripts that had been mapped previously to distal 1p. These were screened against a regional radiation-reduced hybrid cell line panel to determine the localization of each marker relative to the SRD. A total of 52 markers were localized within or near the SRD. These markers were then used to screen large-insert DNA clone libraries. Identified clones were assembled into contigs primarily by STS content mapping and endclone fingerprinting, yielding three contigs (Figure 3). A subset of clones representing minimal tiling paths for each contig was sequenced by the Sanger Institute, yielding 729 kb of finished sequence within the minimal SRD and 2.20 Mb within the maximal SRD. The two remaining gaps were cumulatively estimated to span 110 kb.
The consensus SRD was then analysed for the presence of transcriptional units by a variety of methods. First, our regional mapping work targeted all known genes and EST clusters previously localized to distal 1p. Besides the 52 markers used for large-insert clone identification, we assembled an additional 76 markers that localized to the SRD according to electronic-PCR or BLAST matches to the SRD DNA sequence, and also from localizations reported at eGenome and UCSC (White et al., 1999; Karolchik et al., 2003) (Figure 3). This set of 128 markers represented 11 characterized genes and five putative transcripts (Table 2). We also supplemented this transcriptional set by searching for additional genes and putative transcripts using a variety of approaches, including ab initio, transcript sequence homology, and comparative genomic homology-based gene prediction techniques, as well as incorporation and independent assessment of predictions made by other groups (e.g. Ensembl and UCSC). These efforts identified an additional 14 transcripts, for a total of 15 previously characterized genes and 15 uncharacterized and putative transcripts localized to the defined SRD (Table 2).
Finally, we determined the transcriptional expression profiles of 21 identified SRD genes in a panel of 17 normal human tissues, including four neural-derived tissues and adrenal gland, the latter of which is the site of origin for a large proportion of neuroblastomas. This analysis was initially performed both by our own experimentation, using a combination of semiquantitative (four genes) and real-time quantitative RT–PCR (17 genes) (Table 3). A total of 13 genes (CAMTA1, KIAA0469, TAS1R1, KIAA0720, ESPN, HES2, BACH, GPR153, LOC284509, MGC40168, CHD5, KCNAB2, and SHREW1) demonstrated some level of tissue specificity for the tissues surveyed. Of the genes, 17 were expressed in at least one neural-derived tissue; 10 of the genes (CAMTA1, FLJ10737, MGC33488, HKR3, GPR153, ICMT, FLJ32096, CHD5, NPHP4, and SHREW1) were expressed in all neural tissues surveyed; and 15 of the genes were expressed in the adrenal gland (Table 3). Of those genes with tissue specificity, six (CAMTA1, BACH, GPR152, LOC284509, CHD5, and SHREW1) were expressed at least partially in the nervous system, and only one (SHREW1) was found to be expressed preferentially in tissues of early development. Expression profiles for CHD5, KCNAB2, and SHREW1 are shown in Figure 4.
A sizable number of studies have independently identified and confirmed allelic loss within 1p for neuroblastoma cell lines and primary tumors (Fong et al., 1989; Takayama et al., 1992; Schleiermacher et al., 1994; Takeda et al., 1994; White et al., 1995; Martinsson et al., 1997; Mora et al., 2000; Bauer et al., 2001; Maris et al., 2001; Godfried et al., 2002). However, these studies have not identified a consistent region of deletion. Furthermore, these and other studies have proposed over 20 genes within these regions as candidate neuroblastoma tumor suppressors, none of which have yet been proven to play a significant causative role in neuroblastoma tumor development (Ejeskar et al., 2000; Judson et al., 2000; De Toledo et al., 2001; Huang et al., 2001; Abel et al., 2002; Cerignoli et al., 2002; Krona et al., 2003; Thompson et al., 2003; Mathysen et al., 2004). Our current work was designed to determine the location and extent of 1p deletion in a very large primary tumor cohort by extensive genotyping, sequencing, gene identification, and transcript characterization.
Our allelic loss studies initially targeted all of 1p. Subsequently, we increased the sample size and locus densities for distal 1p, and then for 1p36.3, as a single SRD emerged. Overall, allelic loss for 1p was identified in 184 of 737 (25%) primary neuroblastomas and 40 of 46 (87%) neuroblastoma cell lines, consistent with the frequencies of each that were identified in our earlier studies and those of other groups. Together, these results established a single SRD within 1p36.3, flanked by markers D1S2795 and D1S253, and spanning a minimum of 729 kb.
Our currently defined SRD confirms and more precisely defines the SRD found in our two previous studies, each of which used much smaller cohorts (Fong et al., 1989; White et al., 1995). All but one tumor with 1p LOH in our current study demonstrated allelic loss within this 1p36.3 SRD. The one exception was a tumor with a deletion extending proximally from the marker D1S56 in 1p36.1. However, 21 cases with 1p36.3 LOH did not have allelic loss extending to 1p36.1, so the exceptional case does not likely define a second SRD. The lack of additional SRDs is consistent with more recent LOH studies from other groups (Martinsson et al., 1997; Bauer et al., 2001; Caron et al., 2001), although see Schleiermacher et al. (1994). Of perhaps more significance, our defined 1p36.3 SRD is either completely encompassed within (Schleiermacher et al., 1994; Martinsson et al., 1997; Caron et al., 2001) or partially overlaps (Bauer et al., 2001) the defined SRDs of other groups analysing smaller tumor cohorts by LOH analysis. The overlap between our SRD and that of Bauer et al. is at most 260 kb, between D1S2731 and D1S214, and includes one characterized and three putative genes. Interestingly, although the Bauer Study surveyed only 49 primary neuroblastomas, three of 15 tumors with 1p36 LOH had interstitial deletions within 1p36.3, possibly indicating either a population variation or a difference in allelic loss assessment methodologies than with our current study, which identified only two interstitial deletions.
In contrast to recent LOH studies, there have been several investigations of individual neuroblastoma tumor- (Spieker et al., 2001; Van Roy et al., 2002) and cell-line-derived (Amler et al., 1995; Casciano et al., 1996; Ohira et al., 2000) chromosomal rearrangements, including a balanced constitutional translocation within 1p36.2 and a 500 kb homozygous deletion (HD) in a neuroblastoma cell line. Comparison of these rearrangements with our findings shows that each rearrangement maps proximal to our defined SRD. This suggests the possibility of additional neuroblastoma tumor suppressor loci located more proximal within 1p36, and that disruption of two or more TSGs within 1p36 may further contribute to the tumorigenicity or progression of neuroblastoma in those tumors with larger 1p36 deletions. However, we previously screened a large panel of neuroblastoma cell lines at high genomic density for homozygous deletions within 1p36 (Thompson et al., 2001), including several markers within the 500 kb HD identified by Ohira et al. (2000), but found no evidence of HD supporting these findings.
Our analysis of neuroblastoma cell lines and two neuroblastoma patients with constitutional deletions of 1p36 found that these samples also contained deletions that completely encompassed the primary tumor-defined SRD. Constitutional deletion patient 2's distal deletion breakpoint corresponded with the breakpoint of the primary tumor defining the distal SRD boundary, thus providing supporting evidence for this location. Monosomy 1p36 has been recognized recently as a relatively frequent constitutional chromosomal abnormality (Shapira et al., 1997; Heilstedt et al., 2003). Approximately 40% of all characterized cases have deletions that include a portion of our defined neuroblastoma SRD, and 30% are monosomic within the entire SRD. However, none of these other patients are reported to have neuroblastoma. Thus, if a causal relationship exists between 1p36 monosomy and neuroblastoma development, constitutional deletion of the SRD is not sufficient for the latter.
Using a variety of techniques, including LOH, cytogenetic, and STS content-based molecular analyses, our group and others have now cumulatively surveyed a large cohort of primary neuroblastomas (Bader et al., 1991; Schleiermacher et al., 1994; Martinsson et al., 1997; Caron et al., 2001). Nevertheless, only a few rare cases with informative breakpoints within 1p36.3 have been identified to date, and no gene-specific or small regional genomic abnormalities have yet been detected within the collective 1p36.3 SRD. Given the propensity for large, hemizygous 1p36 deletions, alternative hypotheses for tumor suppression have been suggested. These include the possibility of an additional, MYCN-associated TSG in proximal 1p36 due to the observation that MYCN-amplified tumors invariably delete at least the majority of 1p36 (Takeda et al., 1994; Caron et al., 2001); haploinsufficiency-based suppression accounting for the rarity of 1p36 homozygous deletions (Janoueix-Lerosey et al., 2004); the possibility of two or more nonoverlapping SRDs (White and Versteeg, 2000); and suppression of TSG expression from a hemizygous allele due to imprinting or other epigenetic modifications (Caron et al., 2001; Hogarty et al., 2002). Whether the lack of localized chromosomal abnormalities or HD is reflective of functional or structural mechanisms awaits additional molecular experimentation.
Genomic mapping, sequencing, and sequence analysis identified a total of 30 genes within the SRD, 15 of which were previously characterized to some extent. For 21 of these genes, we determined the expression patterns in 18 normal tissues. Of these, two genes are intriguing as candidate tumor suppressors. As we have reported previously (Thompson et al., 2003), CHD5 is a member of the chromodomain gene family, with high homology to CHD3 and CHD4, both of which are thought to be functional components of nucleosome remodeling and histone deacetylation complexes (Woodage et al., 1997). CHD5 is of special interest due to its preferential expression in the nervous system and the adrenal gland, as well as its lack of expression in 1p-deleted, high-risk neuroblastomas. The gene SHREW1 was expressed preferentially in fetal brain, total brain, spinal cord, cerebellum, and spleen. SHREW1 is predicted to encode a transmembrane-spanning protein and has recently been shown to interact specifically with E-cadherin/β-catenin complexes (Bharti et al., 2004), but little else is currently known regarding its function or that of its mouse homolog.
A number of additional SRD genes for which some function could be determined are plausible as having tumor suppressor roles, including the putative transcription factors MGC33488, PHF13, KIAA0469, HES2, FLJ32096, LOC284509, and CAMTA1. Of these, CAMTA1 is particularly intriguing due to its expression being mainly restricted to neural tissues, and also that it lies within the region of overlap shared by the 1p36.3 neuroblastoma SRDs defined by all recent studies. CAMTA1 is the first identified human homolog of a class of proteins whose members act as transcriptional activators, have a domain capable of binding calmodulin, and encode ankyrin repeats (Katoh, 2003). While CAMTA1 has not yet been well characterized functionally, Nakatani et al. (2004) report that CAMTA1 is highly expressed in N(euronal)-type neuroblastoma cell lines but absent in S(chwannian)-type cell lines.
HES2 is a member of a family of proteins with significant homology to the Drosophila hairy and enhancer of split transcription factors, both of which are required for sensory neurogenesis in late development (Katoh, 2004). Hairy also plays a crucial role as a pair-rule segmentation gene in early Drosophila development. Like other members of the human family, HES2 contains a bHLH domain, suggesting the ability to act as a transcription factor. The mouse ortholog of HES2 has been shown to bind to E-box and, with lower affinity, N-box regulatory sequences (Ishibashi et al., 1993); however, unlike other HES family members, mouse HES2 is not upregulated by the cellular differentiation factor Notch (Nishimura et al., 1998). Our studies found HES2 to be transcribed in most tissues surveyed, including brain and adrenal gland.
KIAA0720 encodes a gene with predicted pleckstrin and guanine nucleotide exchange factor domains, both of which indicate possible roles in intracellular signaling. GPR153 encodes a seven-transmembrane-spanning G-protein-coupled receptor with both neural and adrenal gland expression. In addition, NPHP4, which has recently been identified as the gene causative for the kidney disorders nephronophthisis-4 and Senior-Loken syndrome (Mollet et al., 2002), appears to be involved in early renal development but is also widely expressed. KCNAB2 is a member of the shaker subfamily of voltage-gated potassium channels and serves an auxiliary role to the functional α subunit of the channel (McCormack et al., 2002). Our analysis found this gene to be preferentially expressed in brain, which is consistent with previous findings indicating an association between deletion of KCNAB2 and an epileptic phenotype in patients with monosomy 1p36.3 (Heilstedt et al., 2001). Finally, the transcription factor HKR3 and the death domain receptor TNFRSF25 have previously been identified and extensively evaluated as candidate neuroblastoma tumor suppressor genes (Maris et al., 1997; Grenet et al., 1998), but no evidence to date implicates either in tumorigenesis.
In summary, our findings confirm, precisely refine, and characterize in detail a small region of allelic loss within 1p36.3 that is present in a substantial percentage of neuroblastoma primary tumors and cell lines. There is now a large body of evidence suggesting that one or more TSGs involved in neuroblastoma initiation and/or progression is localized to this region. A strong correlation between 1p36 deletion, MYCN amplification, and advanced stage disease indicates that this locus is an important contributor to neuroblastoma biology. Further characterization of the normal and neuroblastoma attributes of the genes within the SRD will assist in determining the gene(s) responsible for these biological effects.
Materials and methods
Paired tumor and constitutional samples were obtained from the Pediatric Oncology Group, the Children's Cancer Group, and the Children's Oncology Group. Cell lines were obtained from a variety of sources as previously described (Thompson et al., 2001). Both constitutional deletion patients have been described previously (Biegel et al., 1993; White et al., 1997). Tumor and constitutional DNAs were isolated as described (White et al., 1995). Tissue-specific RNAs were obtained from a commercial source (BD Biosciences; San Jose, CA, USA). The Children's Hospital of Philadelphia Institutional Review Board approved this research.
Genotyping and LOH analysis
Primers for PCR-formatted microsatellite and minisatellite polymorphisms D1S76, D1S243, D1S80, D1S50, D1S468, D1S2845, D1S2660, D1S2795, D1S2145, D1S2633, D1S2870, D1S253, D1S214, D1S1646, D1S2694, D1S548, D1S160, D1S503, D1S450, D1S244, D1S2667, D1S489, D1S434, D1S228, D1S507, D1S436, D1S2697, D1S170, D1S3669, D1S2644, D1S199, D1S3720, GGAA30B06, D1S1622, D1S247, D1S164, D1S201, D1S1596, D1S1669, D1S1643, and D1S481 were obtained from the Genome Data Base and eGenome (Letovsky et al., 1998; White et al., 1999). Assays for the remaining polymorphic markers have been previously described (Dracopoli et al., 1988; Fong et al., 1989; White et al., 1995). Samples were assayed using acrylamide gel electrophoresis and either radiolabeled PCR primers, autoradiography, and densitometric analysis (White et al., 1995), or using fluorescently labeled PCR primers analysed with ABI GeneScan and ABI Genotyper software (Applied Biosystems, Foster City, CA, USA) (Maris et al., 2000). Genotypes from all primary tumor pairs demonstrating LOH which defined tumor breakpoints were generated multiple times and confirmed by densitometric analysis with the threshold for LOH defined by an allelic intensity reduction of >60% for one allele. Genotype analyses of 46 unique neuroblastoma cell lines, the two constitutional deletion cases, and parental samples for each constitutional case, were also performed by PCR using radiolabeled primers and autoradiographic detection. Results from subsets of patients and loci used in this study have been reported elsewhere in detail (Fong et al., 1989; White et al., 1995) or preliminarily (White et al., 1997; Hogarty et al., 2000; White et al., 2001).
Genomic mapping, sequencing, and sequence annotations
PCR-formatted genomic markers representing polymorphisms, transcripts, and random STSs previously localized to 1p36 were derived from eGenome (White et al., 1999) and mapped by PCR against a distal 1p-specific radiation hybrid (RH) panel as described (Jensen et al., 1997), or by direct string matching of primer and genomic sequences using me-PCR (Murphy et al., 2004). Screening of large-insert clones and sequencing of PAC inserts, using a subset of the mapped genomic markers, has been described (Bentley et al., 2001). Sequence analysis was performed using a variety of approaches, including use of the in silico gene prediction algorithms Genscan (Burge and Karlin, 1997) and Metagene (http://www.goliath.ifrc.mcw.edu/MetaGene/), and the sequence homology algorithms BLAST, BLASTP, and BLASTX (Altschul et al., 1990), with the latest round of analyses performed using genome sequence build 34. In addition, gene predictions from the UCSC Genome Browser (Karolchik et al., 2003), the NCBI (Wheeler et al., 2004), and Ensembl (Birney et al., 2004) were assessed, and candidate transcripts were added if substantial evidence from transcript/partial cDNA/EST sequence representation, gene- and/or transcript-level comparative homology, and ab initio prediction was present. As transcript prediction is a subjective task, it is possible that we have over- and/or underinterpreted the validity of the 30 genes presented.
Transcript profiling assays
Real-time RT–PCR (TaqMan) assays were generated for the genes CAMTA1, FLJ10737, MGC33488, PHF13, KIAA0469, HKR3, TAS1R1, FLJ23323, KIAA0720, ESPN, HES2, GPR153, MGC40168, FLJ32096, CHD5, NPHP4, and SHREW1. A TaqMan assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a ubiquitously expressed housekeeping gene, was multiplexed along with each target gene to provide an internal control and for quantification. Gene-specific primers and fluorescent probes were obtained from Applied Biosystems. Each amplification reaction contained 1 × Master Mix (Applied Biosystems), 50 ng of cDNA, 5 μ M GAPDH detection primer, 5 μ M target gene detection primer, 10 μ M of each GAPDH amplification primer, and 10 μ M of each target gene amplification primer. Samples were amplified in duplicate in 20 μl reaction volumes in a 96-well format, using conditions of one cycle at 50°C for 2 min; one cycle at 95°C for 10 min; 55 cycles at 95°C for 15 s and then 60°C for 1 min. Standards with known DNA concentrations were included in every run for reaction controls. Assays were performed on an ABI Prism 7700 sequence detection system. Signals were normalized and quantified relative to GAPDH with the associated program Sequence Detector v1.7, using the comparative method. Transcript levels for the genes BACH, LOC284509, ICMT, CHD5, and KCNAB2 were assessed with a previously described semi-quantitative RT–PCR method (Eggert et al., 2000). cDNAs for all transcript profiles were generated using a Superscript II RT® Kit (Invitrogen, Carlsbad, CA, USA).
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This work was supported in part by NIH Grant CA039771 (GMB), CA087847 (JMM), the Abramson Family Cancer Research Institute, and the Audrey E Evans Endowed Chair (GMB). We gratefully acknowledge H Marshall, C Beltinger, E Sulman, M Fujimori, B Kaufman, S Jensen, and C Guo for previously reported LOH data used in the current study, and the Children's Oncology Group for access to patient material.
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