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Physical and transcript map of the minimally deleted region III on 17p implicated in the early development of Barrett's oesophageal adenocarcinoma

Abstract

Allelic imbalance (AI) studies on chromosome 17 (C17) in Barrett's oesophageal adenocarcinoma (BOA) tumours strongly suggest that a minimally deleted region on C17p harbours a BOA-associated gene with tumour suppressor function. This deleted region, designated minimal region III (MRIII), lies between the two microsatellite markers D17S1852 and D17S954. Computational sequence analysis techniques, BLAST and NIX, were used to assemble a physical map of MRIII, consisting of three overlapping bacterial artificial chromosome (BAC) clones, 297N7, 963H4 and 795F17, from the RPCI-11 library. The 270 kb genomic sequence of MRIII was analysed using the computational gene prediction methods NIX and TAP to identify putative BOA genes. A transcript map of MRIII has been generated and contains 25 candidate BOA genes, four of which are the named genes MYH3, SCO1, x006 and MAGOH-LIKE. The other candidates consist of seven genes predicted by TAP with associated ESTs identified by NIX, two genes predicted by TAP alone and 12 genes/ESTs (or pairs of ESTs) identified by NIX alone. No disease-specific mutations were identified in x006 or MAGOH-LIKE, although expression analysis of these genes suggests that they may show alternative splicing or be altered epigenetically or in regulatory regions in oesophageal cancer.

Introduction

Chromosome 17 is a cancer gene-rich chromosome, containing three known tumour suppressor genes (TSGs), p53, BRCA1, NF1, and seven putative TSGs, including HIC1, DEC1, OVCA1/OVCA2, ELAC2, TOC and DMC1 (Risk et al., 1994; Wales et al., 1995; Phillips et al., 1996; Schultz et al., 1996; Bruening et al., 1999; Harada et al., 2001; Tavtigian et al., 2001). We have previously shown that chromosome 17 (C17) is the most frequent site of allelic imbalance in Barrett's oesophageal adenocarcinoma (BOA) tumours (Dolan et al., 1998), and that C17 contains 13 common minimally deleted regions in BOA tumours (Dunn et al., 1999), many of which coincide with the location of known or putative TSGs.

Further evidence for the importance of these 13 minimally deleted regions (MRI-XIII) was provided by a more detailed analysis of allelic imbalance on C17 in premalignant tissues taken from numerous sites around five BOA tumours (Dunn et al., 2000). In that study, we demonstrated that the majority of the deletions (68%) observed in the BOA tumours also occurred in histologically defined premalignant tissue adjacent to the tumour, and it was apparent that each oesophagectomy specimen analysed demonstrated a unique pathway of clonally evolving loss of heterozygosity (LOH). From these data, we then identified minimally deleted region III (MRIII) as the most frequent clonally deleted region in the histologically defined earliest tissue, that is, Barrett's intestinal metaplasia (BIM) without dysplasia.

We suggest that MRIII is the site of an early molecular marker of malignant potential in BOA, which could be used to identify individuals at high risk of developing this disease. BOA has an extremely poor prognosis, with a 5-year survival rate of less than 5% (Rankin, 2000), which is often attributed to the frequent late presentation with disease. It has been shown that early intervention can significantly improve survival (Holscher et al., 1997), thus the early identification of high-risk patients is desirable. These individuals could be placed under increased surveillance and/or intervention, to improve the poor prognosis of this cancer.

The MRIII region is defined by four BOA tumours and is limited by the microsatellite markers D17S1852 and D17S954 in Dunn et al. (1999). The overall frequency of LOH at MRIII in BOA is 75% and the defining markers both map to 11.573 Mb on chromosome 17p. MRIII is distinct from p53 (Dunn et al., 2000), and multiplex PCR analysis of D17S1852 and D17S954 with chromosome 12 markers has shown that the allelic imbalance observed at these sites is because of loss of genetic information and not amplification (JR Dunn, unpublished). This paper describes the physical and transcript mapping of MRIII, which was undertaken in order to provide a scaffold from which to identify candidate BOA genes. The coding regions of two candidate genes were identified and analysed for mutations in BOA tumours.

Results

Physical map of MRIII

The Whitehead Institute Chromosome 17 Sequencing and Mapping web pages (http://www.genome.wi.mit.edu/seq/mapping.html) were searched by BLAST for clones containing D17S1852 and D17S954. Three overlapping sequenced clones from the RPCI-11 BAC library (Osoegawa et al., 2001) were identified, and these spanned MRIII. 297N7 (Accession No. AC002347) contained D17S1852, 795F17 (AC005284) contained D17S954 and an analysis of the sequence of these two BACs indicated that 963H4 (Accession No. AC015908) bridged the gap between the two clones (Figure 1).

Figure 1
figure1

Physical map of minimal region III (MRIII). Physical map of MRIII based on MS marker mapping and sequence analysis by NIX. ▪, MS marker mapped by PCR and electronic PCR (names in boxes); ——, sequenced BAC clone (names in bold)

Transcript map of MRIII

To identify candidate BOA genes within MRIII, we annotated the genomic sequence between D17S1852 and D17S954 using NIX (a suite of gene prediction and BLAST analysis programmes, http://www.hgmp.mrc.ac.uk/Registered/Webapp/nix/) (Williams, 1998) and TAP (a programme that predicts alternatively spliced products of the same gene, http://sapiens.wustl.edu/~zkan/TAP) (Kan et al., 2001).

A total of 25 candidate BOA genes were identified within MRIII, including two characterized genes (SCO1 and MYH3), one gene with a high degree of homology to a characterized Drosophila gene (MAGOH-LIKE) (Zhao et al., 1998) and one gene whose predicted protein product has been investigated with relation to myelodysplasia (x006) (Soenen et al., 1998) (Figure 2 and Table 1). The remaining 21 candidate genes consist of seven genes predicted by TAP with associated ESTs identified by NIX, two genes predicted by TAP alone and 12 genes/ESTs (or pairs of ESTs) identified by NIX alone (Table 1). A total of 19 genes were identified on the forward DNA strand and 10 on the reverse DNA strand. In five instances (genes 1, 6, 18, 20 and 21), a putative gene was identified in the same position on both DNA strands.

Figure 2
figure2

Transcript map of MRIII showing candidate BOA gene predictions by NIX and TAP (data from Table 1) (diagram is drawn a approximately to scale). Rectangle: named gene; rhombus: putative gene; diamond: EST; black vertical lines: scale in kb; heavy black horizontal line: genomic DNA. Candidate genes on the forward strand are above the genomic line, those on the reverse strand are below the genomic line. Predictions coincident on both strands span the genomic line. Gene symbols, where known, are written in italics. The prediction program used for each analysis is shown on the right

Table 1 List of candidate BOA genes within MRIII, identified by NIX and TAP

Of the 12 genes predicted by NIX alone, alignment of the EST sequences with other NIX predicted gene features (exons, promoters, CpG islands) was possible in 10/12 cases, thus lending more weight to their status as genuine genes (data not shown). Only genes 7, 17 and 25 did not align with any other features. Genes 1, 6, 20, 21, 22 and 24 were also aligned to Unigene clusters, and genes 3 and 21 to a poorly predicted protein. Genes 11 and 4 were predicted by TAP alone. Gene 11, predicted by TAP on the forward DNA strand, aligns with two ESTs identified by NIX, which were aligned to other transcripts on the reverse DNA strand using Sequencher™, thus these NIX ESTs were assigned by the authors to gene x006 (Table 1), and it is possible that the TAP-predicted Gene 11 is actually x006. Gene 4 (forward DNA strand) lies close to the start of the MYH3 gene predicted by NIX, and since the MYH3 gene was too large to input into the TAP programme in a single sequence fragment, it is thought that Gene 4 is actually at the start of MYH3. Therefore, we suggest that 22/25 of the predicted genes listed in Table 1 are genuine candidates, and it is possible that Genes 17, 7 and 25 are misassigned ESTs and should be treated with caution (although not disregarded) as prospective candidates.

In three out of the four named genes, there were discrepancies in both the size of gene and the number of exons predicted by the two methods (NIX and TAP), with TAP missing 5′ exons that were defined by NIX using cDNA sequence in two cases. Thus, MAGOH-LIKE was predicted by NIX to be 14.76 kb with nine exons, but to be only 10.874 kb with four exons by TAP (Table 1 and Figure 3). Similarly, MYH3 (a characterized gene) was predicted by NIX to be 26 kb with 36 exons, but TAP predicted a smaller (13.15 kb) gene with fewer (16) exons. In this case, TAP missed both 5′ and 3′ exons. The murine MYH3 has 40 exons so it is not clear whether the NIX prediction of this gene is also missing some exons. Gene x006, however, was predicted by TAP to be 8.9 kb with three exons, but NIX predicted a smaller gene (6.31 kb) with seven exons. In this case, TAP predicted an extra 5′ exon. SCO1 was predicted by both NIX and TAP to be 5.7 kb with four exons.

Figure 3
figure3

Figure showing the relative positions of candidate BOA gene predictions on BAC clone 297N7 as predicted by NIX and TAP (data from Table 1) (diagram is drawn approximately to scale). Gene names are given in italics. The number of predicted exons for each gene is shown, and alternative transcripts predicted by TAP are shown directly above the initial gene prediction. Rectangle: gene (shown above the line if on the forward strand and below the line if on the reverse strand); black vertical lines within genes: exon boundaries; black horizontal line: BAC 297N7. The prediction program used in each analysis is shown on the right. The positions of the EST primers used for expression analysis of MAGOH-LIKE and x006 are shown directly below their positions as predicted by NIX

Identification of MAGOH-LIKE and x006 as potential candidate BOA genes

Public domain websites contained some information about the function, expression pattern and conservation between the species of genes MYH3, SCO1, MAGOH-LIKE and x006. All four genes are both highly conserved and widely expressed in a variety of human tissues, and were thus all considered to be good candidates as a BOA TSG. This is because TSGs identified as being involved in the development of cancer by LOH analysis often play a role in the control of cell growth and death. They are, therefore, usually critical to the cell and are often expressed in many cell types. MYH3 and SCO1, however, do not have a cancer-related function. MYH3 is a fast skeletal muscle gene and SCO1 is involved in copper transport in the respiratory chain and these genes were thus listed for further investigation at a later date. Conversely, MAGOH-LIKE and x006 both have a putative cancer-related function. The human chromosome 1 homologue of MAGOH-LIKE has been associated with increased cell proliferation in mouse cell lines (Zhao et al., 1998), and the x006 protein is linked to myelodysplasia (Soenen et al., 1998). Therefore, the genes that were considered to have the highest priority in the search for the BOA genes were MAGOH-LIKE and x006, which were subjected to expression and mutational analyses.

Mutation and expression analysis of MAGOH-LIKE and x006

Mutation analysis

All of the predicted exons of MAGOH-LIKE (nine from NIX analysis, plus 1 additional from TAP analysis) and x006 (seven from NIX analysis) were examined for disease-specific mutations using SSCP and sequencing, respectively. Mutation analysis of the MAGOH-LIKE exons was performed by SSCP because all of the exons were small and it was the most rapid method. x006, however, had larger exons and was therefore analysed by sequencing.

The tumours used for SSCP analysis of MAGOH-LIKE were selected as described in Materials and Methods, and included a tumour that defined MRIII (i.e. showed LOH at MRIII bounded by retention of heterozygosity on either side). Tumours defining MRIII have probably ‘lost’ this second allele at MRIII following a hypothesized mutation in the first allele (Knudson's ‘two-hit’ hypothesis for tumour suppressor gene inactivation). By using 10 pairs of normal/tumour samples for the SSCP analysis, it was assumed that any causative mutations would be identified. This is because the overall frequency of LOH at MRIII in our earlier study was 75% and, in addition, we have found LOH at MRIII in Barrett's intestinal metaplasia in 4/5 samples tested (Dunn et al., 1999, 2000). The two tumours used in the sequence analysis of x006 were selected because they were the most likely tumours to show a mutation, since they only showed LOH at MRIII (i.e. defined MRIII). Thus the LOH must have originated in MRIII and the remaining copy of the cancer-causing gene located in this region would contain a mutation (the first ‘hit’ or Knudson's ‘two-hit’ hypothesis). OE33 BOA cell line DNA was also analysed for mutations in both genes.

No consistent disease-specific alterations were observed in either of the genes’ coding regions.

Expression analysis

In order to assess the expression patterns of the candidate BOA genes MAGOH-LIKE and x006 in normal and diseased oesophagus (BOA and oesophageal squamous cell carcinoma (OSCC)), and other human tissues, three ESTs from each gene were analysed by RT–PCR and SAGEmap virtual Northern analysis (http://www.ncbi.nlm.nih.gov/SAGE). The same ESTs were used for both analyses in order to obtain a direct comparison between the SAGEmap data and our RT–PCR results. Validation of DNase I treatment was by PCR using intronic primers. Unfortunately, there was no BOA tumour tissue RNA available for this analysis. In addition, there is no commercially available oesophageal adenocarcinoma RNA, thus the only available verified BOA cell line OE33 (ECACC Ref No. 96070808) was used as the source of BOA RNA.

The RT–PCR results indicated that the MAGOH-LIKE and x006 genes are expressed in normal and diseased (BOA and OSCC) oesophagus (Figure 4, Table 2). All three ESTs from each gene were expressed in normal oesophageal RNA, BOA cell line RNA and OSCC RNA. Expression data for other human tissue types from RT–PCR and SAGEmap analyses indicated that both genes are expressed in at least seven other human tissue types, thus both genes are confirmed as BOA candidate genes because of their wide tissue expression.

Figure 4
figure4

RT–PCR results for MAGOH-LIKE (a and b) and x006 (c and d) ESTs. (a) HS846185; (b) HS025225; (c) AW368449; (d) HS387329. N: normal oesophagus; BOA: Barrett's oesophageal adenocarcinoma; OSCC: oesophageal squamous cell carcinoma

Table 2 RT–PCR and SAGEmap virtual Northern expression results for three ESTs from each of the MAGOH-LIKE and x006 candidate genes

Furthermore, the RT–PCR data indicated that both MAGOH-LIKE and x006 appear to be differently expressed in BOA and OSCC compared to normal oesophagus, which suggests that they may be important genes in the development of oesophageal cancer (BOA and OSCC) (Figure 4). EST HS846185, which aligns with exon 1 of MAGOH-LIKE, demonstrated an additional 587 bp transcript in both BOA and OSCC compared to normal oesophagus, which only expressed the expected 251 bp transcript (Figure 4a data for BOA not shown). MAGOH-L1KE exon 7 EST, HS025225, appeared to be downregulated in BOA compared to normal oesophagus and OSCC (Figure 4b), although quantitative RT–PCR data would be necessary to confirm this. Expression of MAGOH-LIKE EST AW104858 (exon 8) was not altered in BOA and OSCC compared with normal oesophagus (data not shown). Similarly, x006 EST AW368449 (exon 1) demonstrated two additional transcription products in both BOA and OSCC compared to normal oesophagus (Figure 4c) and EST HS387329 (located between exons 6 and 7) appeared to be downregulated in BOA and OSCC compared to normal oesophagus (Figure 4d). Expression of x006 EST AI004998 (located between exons 6 and 7) was not altered in BOA and OSCC compared with normal oesophagus (data not shown). Only the MAGOH-LIKE exon 7 EST, HS025225, was identified in SAGEmap libraries, while two ESTs from the x006 gene were represented (AW368449 (exon 1) and HS387329 (between exons 6 and 7)).

In the light of the RT–PCR results, mutation analysis of MAGOH-LIKE and x006 was undertaken in Barrett's oesophageal adenocarcinoma cell line, OE33, in order to determine if genomic alterations could be responsible for the altered expression of these genes in this cell line. However, no mutations were identified in OE33, suggesting that the MAGOH-LIKE and x006 genes are altered epigenetically in oesophageal cancer.

Discussion

Our previous studies based on allelic imbalance data suggest the presence of a human Barrett's oesophageal adenocarcinoma (BOA) tumour suppressor gene (TSG) in a minimally deleted region of chromosome 17p (C17p) centromeric to the p53 TSG (Dunn et al., 1999, 2000), and multiplex PCR has confirmed that the allelic imbalance in this region is because of loss of heterozygosity (LOH) rather than amplification of the region (Dunn, unpublished). In this study, we have assembled physical and transcript maps of the deleted region (minimal region III–‘MRIII’) from which the candidate BOA TSG can be identified. The region is 270 kb in length.

The MRIII region has also been implicated in the development of oesophageal squamous cell carcinoma (OSCC) (Huang et al., 2000). The minimally deleted region in Huang et al.'s study however is limited by the two MS markers D17S804 (distal end at 10.641 Mb) and D17S799 (proximal end at 12.895 Mb), and is thus >2 Mb in size, more than four times the size of MRIII. LOH frequencies of 88% at D17S804 and 90% at D17S799 were observed. The LOH frequencies for the MS markers D17S954 and D17S1852 were not reported in this paper.

LOH analysis has been used successfully in the isolation of several candidate TSGs, most recently including an ovarian cancer TSG ‘Ov/Br septin’ on C17q25 (Russell et al., 2000), an oesophageal cancer TSG ‘DEC1’ on C9q32 (Nishiwaki et al., 2000), ‘LOH11CR2A’ (C11q23) implicated in breast and lung carcinomas (Monaco et al., 1997), and a renal cell carcinoma TSG ‘TU3A’ on C3p14.3–14.2 (Yamato et al., 1999). Each of these studies was conducted in a manner similar to that used to localize a BOA TSG in this project. Firstly, common region of allelic loss was identified in the tumours and the limiting MS markers determined; secondly, a physical contig of genomic segments was constructed to span the region of interest; and finally the candidate TSG was identified by sequence analysis of the region. This is currently considered the most appropriate approach for identifying novel TSGs in cancer research, when using genetic instability as the starting point.

Computational methods of gene identification have been used successfully in the identification of G-protein-coupled receptor genes in two separate studies (Marchese et al., 1999; Wittenberger and Chica Schaller, 2001), both of which utilized expressed sequence tag (EST) database searches. It is envisaged that many more human genes will be identified using such methods as the completion of the Human Genome Sequence progresses. Certainly, given the information we have accumulated leading up to a fully sequenced candidate BOA gene region, computational analyses have been the most rapid and effective method of identification of candidate genes for further investigation.

In all, 42% (11/26) of the candidate genes identified in MRIII were predicted by both NIX and TAP. Since the TAP program utilizes WU2BLASTN (Gish, 1996–2000) to search the EST database ‘dbEST’ (i.e. a different method to NIX), we believe that these gene predictions are more robust than those that were predicted by one method alone. We suggest that 22/25 of the predicted genes listed in Table 1 are genuine candidates, and it is possible that genes 17, 7 and 25 are mis-assigned ESTs and should be treated with caution (although not disregarded) as prospective candidates. ESTs are unedited single-pass sequencing reads and are therefore prone to error; at best there is a 97% accuracy rate (Hillier et al., 1996).

By using more than one method of gene finding (NIX suite of programmes and TAP) and the Sequencher™ sequence alignment programme, we believe that the candidate genes identified in this study are more likely to be genuine than if they had been identified using a single method. It is recognized however that before further investigation of candidate genes is undertaken (e.g. mutation analysis), it would be prudent to supplement the current information with laboratory-generated data such as RT–PCR, thus eliminating false exon predictions made by NIX or TAP.

The preliminary RT–PCR expression analysis results reported here provide evidence for alternative splicing in both MAGOH-LIKE and x006 in oesophageal cancer (BOA and OSCC) as alternative transcripts for MAGOH-LIKE exon 1 and x006 exon 1 are observed in both BOA and OSCC. In addition, the TAP predictions for MAGOH-LIKE and x006 lend further support to these results. TAP has predicted an alternatively spliced MAGOH-LIKE gene that had lost exon 1 and gained another exon between exons 2 and 3. It is noteworthy that since PCR primers for MAGOH-LIKE EST exon 1 gave a PCR product, it is most likely that exon 1 has been missed by the TAP programme. This is probably because TAP only uses EST data, whereas NIX uses cDNA data for their predictions. Thus, TAP may miss some 5′ ends of predicted genes.

Some apparent discrepancies were observed in the RT–PCR data for both MAGOH-LIKE and x006. Specifically, out of the three ESTs used for expression analysis of these genes, only one EST appeared to be downregulated in each case. Possible explanations for these discrepancies are as follows. Firstly, the ESTs that are apparently downregulated could represent alternatively spliced exons that are under-represented in tumour tissue. Secondly, the two ESTs that show no changes in expression in BOA or OSCC could actually be downregulated, but the change is too small to see in these experiments, and, conversely, it is possible that the ESTs that do show downregulation in BOA and/or OSCC are not actually downregulated. Quantitative RT–PCR data would determine if either of these alternatives were the case. Finally, it is possible that the sequence data at the site of the ESTs with unchanged expression levels (or indeed at the site of the apparently downregulated ESTs) may be poor and has led to inaccuracies in RT–PCR amplification.

The results in this study highlight the enormous potential of computational gene prediction methods coupled with laboratory-based methods. The bioinformatic approach provided an important sieve mechanism by which we are able to prioritize our analysis to two candidate genes. The RT–PCR analysis suggests that both the MAGOH-LIKE and x006 genes have an alternative form that is important in oesophageal cancer development (BOA and OSCC), but is not transcribed in the normal oesophagus. If these results are confirmed, and alternative gene products are made in oesophageal cancer, it will be important to ascertain whether these products are also found in other human cancers.

The major objective to this study was to identify the gene that could be considered to be an early marker of malignant potential specific to BOA. Even though there was compelling evidence that both MAGOH-LIKE and x006 were candidate genes based on the alternative splicing of exon 1, the results presented here indicate that both forms of each gene are found in both BOA and OSCC. In order to ascertain the frequency of occurrence of the alteration in BOA compared to OSCC, a larger data set will be required. The likelihood that either MAGOH-LIKE or x006 is involved in oesophageal cancer development is further strengthened by the apparent downregulation of 3′ ESTs in BOA and OSCC, respectively. Quantitative RT–PCR analysis will be required to expand on these results.

In summary, the expression analyses of MAGOH-LIKE and x006 by RT–PCR suggests that both genes remain good candidates for a tumour suppressor/cancer-related gene, albeit perhaps not specific for BOA, and both genes warrant further investigation in BOA and other cancers.

In conclusion, we have developed a transcript map of MRIII, a region of C17p that we believe contains an early molecular marker of malignant potential in BOA. This transcript map is the first of its kind in BOA, and will be the foundation for the identification of the BOA marker using a combination of computational- and laboratory-based methods.

Materials and methods

Identification of BAC clones spanning MRIII

BAC clones overlapping MRIII were identified by BLAST analysis of the chromosome 17 physical mapping and sequence data at the Whitehead Institute website (http://www.genome.wi.mit.edu) using the sequences of D17S1852 and D17S954. Clones containing these markers were purchased from Research Genetics and screened by PCR to verify the marker content. All PCR primers (including sequencing primers) used in this study were designed using the Primer 3 design programme (http://www.genome.wi.mit.edu) and purchased from MWG Biotech Ltd.

Computational sequence analysis: transcript mapping

20 kb segments (with 5 kb overlap) of the sequence of MRIII were analysed using the NIX and TAP programmes in order to predict genes and alternative splice products, respectively. Since NIX produces results for all predicted features, ranging from poor to excellent predictions, we employed set criteria for the selection of candidate genes predicted by NIX for further investigation as follows.

  1. 1

    Only ‘excellent’ predictions were noted. BLAST alignments for these predictions were also visually checked to verify if a significant proportion (95%) of the matched sequence was aligning to genomic sequence. In addition, all transcript alignments were verified using Sequencher™ (Gene Codes Corporation), using the following parameters: minimum 85% bp match and minimum 20 bp overlap.

  2. 2

    Exons predicted by fewer than two exon prediction program, which did not align with any other predicted features in NIX, and which were not predicted by TAP are not included in Table 1.

Expression analysis of candidate genes

Culture of OE33 and OE47 cell lines and RNA extraction

Cells were grown to 85% confluence in RPMI 1640 with 2 mM glutamine and 10% FBS in 550 ml tissue culture flasks, before detachment with 0.25% trypsin/EDTA and subsequent centrifugation at 100 g for 10 min to pellet the cells. Cell pellets were snap frozen on dry ice before storage at −80°C prior to RNA extraction. Each cell pellet contained 3 × 107 cells. Total RNA was extracted using the Qiagen Rneasy mini kit, and quantified using a GeneQuant II Spectrophotometer.

RT–PCR

RT–PCR reactions were carried out using the RetroScript™ first-strand synthesis RT–PCR kit (Ambion). Total RNA was purchased from Clontech (normal RNA from stomach, liver, prostate and brain) and Invitrogen (normal oesophagus, squamous cell tumour oesophagus and normal breast), and isolated from OE33 (BOA) and OE47 (colon adenocarcinoma). Cell line RNA samples were first treated with DNase enzyme (1 U) (Gibco BRL) to remove contaminating DNA. First strand cDNA was synthesized using 50 μ M. random decamers and 1–2 μg total RNA, according to kit instructions. Candidate gene transcripts (ESTs) were amplified by PCR (1 cycle of 94°C for 5 min; 30 cycles of 94°C for 30 s, 59°C for 30 s, 72°C for 30 s; 1 cycle of 72°C for 5 min) using Bioline Taq polymersase (1 U). EST primers were designed as described previously (Dunn et al., 2000).

SAGEmap virtual Northern analysis

In order to determine the breadth of expression of MAGOH-LIKE and x006 in a wider variety of other human tissues (cancer and normal), three EST sequences each from the MAGOH-LIKE and x006 candidate genes were analysed using the virtual Northern tool on the US National Centre for Biotechnology Information (NCBI) Serial Analysis of Gene Expression (SAGE) map website (http://www.ncbi.nlm.nih.gov/SAGE/SAGEtag.cgi). In brief, the EST sequence obtained from the NIX output was pasted into the SAGEmap virtual Northern query sequence box. The SAGEmap database was then automatically searched for matching SAGEmap library ‘TAGs’, and a list of the tissue libraries in which the EST was identified was given.

Mutation analysis of MAGOH-LIKE and x006

SSCP

All 10 of the predicted exons for the candidate gene MAGOH-LIKE were examined for mutations using the single-strand conformational polymorphism (SSCP) technique. Primers were designed in the flanking introns and all SSCP PCR products were <200 bp.

DNA (200 ng) was used for each template. DNA was derived from the BOA cell line, OE33, and the following BOA normal and tumour paired tissues:

  • DNA pair 1 defined MRIII at both proximal and distal limits,

  • DNA pair 2 showed LOH at MRIII, but did not define the region,

  • DNA pair 3 showed no LOH at MRIII.

DNA samples from the blood of six unaffected individuals were used as controls. Any exons demonstrating a polymorphism were subjected to a more rigorous SSCP analysis using a further 10 BOA DNA normal and tumour pairs. DNA was obtained as described in Dunn et al. (1999).

PCR primers were designed to fall immediately outside each exon such that the whole exon was amplified in each case. Verification of the amplification of a single, appropriately sized product was by agarose gel electrophoresis. SSCP DNA templates were prepared immediately prior to running SSCP gels as follows: 2 μl of PCR product and 10 μl of SSCP loading buffer (80% formamide, 100 mM NaOH, 10 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol) were denatured at 95°C for 3 min, then snap cooled on iced water. A volume of 1–4 μl of each denatured DNA sample was subjected to electrophoresis on polyacrylamide gels made with Protogel (30% acrylamide with 37.5 : acrylamide : bis acrylamide ratio, National Diagnostics) under the following eight running conditions.

  1. 1

    8 and 10% gel at 20°C,

  2. 2

    8 and 10% gel at 5°C,

  3. 3

    8 and 10% gel with 5% glycerol at 20°C,

  4. 4

    8 and 10% gel with 5% glycerol at 5°C.

Gels were then silver stained and analysed for polymorphisms.

Sequencing

All of the seven exons of candidate gene x006 were analysed for mutations by sequencing on an LiCor automated sequencer using an Epicentre SequiTherm EXCEL™II DNA Sequencing kit-LC. Sequencing DNA templates were derived using eLONGase® Enzyme mix (Gibco BRL) from 200 ng DNA from two BOA normal/tumour pairs that showed LOH at MRIII and the BOA cell line. DNA (150 ng) from BAC 297N7 was used as a positive control. Templates were amplified using primers (10 pmol/μl) designed to cover 800–1000 bp of sequence with the exon in the middle. Sequencing primers were designed immediately outside the exon sequence and were 5′labelled with IRD700. Templates were verified for sequencing by the presence of a single, appropriately sized product on agarose gel electrophoresis, then cleaned using the QIAquick™ PCR purification kit (Qiagen). Each exon was sequenced twice in each direction using a different PCR template. Sequence traces were analysed using the LiCor base-calling software, by eye, and using the Sequencher™ programme.

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Acknowledgements

We thank Dr Lakis Liloglou for advice on SSCP analysis. This study was supported by a grant from the North West Cancer Research Fund UK (Grant CR536).

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Correspondence to John K Field.

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Current address: Roy Castle International Centre for Lung Cancer Research

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Dunn, J., Risk, J., Langan, J. et al. Physical and transcript map of the minimally deleted region III on 17p implicated in the early development of Barrett's oesophageal adenocarcinoma. Oncogene 22, 4134–4142 (2003). https://doi.org/10.1038/sj.onc.1206466

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Keywords

  • chromosome 17
  • Barrett's oesophageal adenocarcinoma
  • transcript map
  • tumour suppressor gene
  • gene prediction programmes

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