The amplification at 13q31-q32 has been reported in not only hematopoietic malignancies but also in other solid tumors. We identified previously frequent amplification of chromosomal band 13q31-q32 in 70 cases of diffuse large B-cell lymphoma patients by conventional comparative genomic hybridization analysis. In an attempt to identify a candidate gene within this region, we used array comparative genomic hybridization and fluorescent in situ hybridization to map the 13q31-q32 amplicon. We then screened the 65 expressed sequence tags and Glypican 5 (GPC5) by reverse transcription-PCR and Northern blotting. As a result, we identified a novel gene, designated Chromosome 13 open reading frame 25 (C13orf25), which was overexpressed in B-cell lymphoma cell lines and diffuse large B-cell lymphoma patients with 13q31-q32 amplifications. However, GPC5, which has been reported to be a target gene for 13q31-q32 amplification, was truncated in one cell line, Rec1, possessing the amplification, and its expression in various cell lines with amplification at 13q31-q32 was not significantly different from that in other cell lines without amplification, suggesting that GPC5 is not likely to be the candidate gene. Additional analysis identified two major transcripts in the C13orf25 gene. The two transcripts A and B predicted open reading frames of 32 and 70-amino acid polypeptides, respectively. The former has been reported as bA121J7.2, which is conserved among species. Transcript-B also contained seven mature microRNAs in its untranslated region. These results suggest that the C13orf25 gene is the most likely candidate gene for the 13q31-q32 amplicon found in hematopoietic malignancies.

Chromosomal amplification is a common mechanism by which genes achieve overexpression in tumors. Identification and characterization of oncogenes present in amplified regions can thus provide important insights into the pathogenesis of cancer (1). Activated oncogenes, such as MYCN in neuroblastomas or HER2 in breast cancer, also have prognostic relevance (2, 3, 4).

The high-level amplification seen at 13q21-qter has been observed in hematological and other solid neoplasms. Amplification at 13q21-qter has been reported in diffuse large B-cell lymphoma (DLBCL; Ref. 5), mantle cell lymphoma (6), follicular lymphoma (7), primary cutaneous B-cell lymphoma (8), and nasal-type natural killer/T-cell lymphoma (9). Additional cases of amplification at 13q21-qter have also been reported in solid tumors: glioma (10), non-small cell lung cancer (10), bladder cancer (10), squamous-cell carcinoma of the head and neck (10), peripheral nerve sheath tumor (11), malignant fibrous histiocytoma (12), and alveolar rhabdomyosarcoma (13). We also reported the results of cytogenetic analysis of 70 DLBCL patients (14) using conventional comparative genomic hybridization (CGH), which found amplification of 13q, including the 13q31-q32 region, in 18 of 70 DLBCL cases.

Glypican 5 (GPC5) has been reported recently as a possible target gene for amplification at 13q31-q32 in a study using fluorescent in situ hybridization (FISH) in lymphoma cell lines (15). However, the mRNA expressions of GPC5 and expression sequence tags (ESTs) located in the amplification region of 13q31-q32 were not dealt with in their study.

In the study presented here, we identified the common region of amplification at 13q31-q32 in DLBCL patients and lymphoma cell lines by CGH, FISH, and array CGH, which allows for rapid screening and more detailed analysis of genomic imbalance in tumor genomes (16, 17, 18). This report describes the fine mapping of the 13q31-q32 amplification and identification of a novel gene, designated C13orf25 (Chromosome 13 open reading frame 25) that showed parallel expression to that of genomic amplification of 13q31-q32 in lymphoma cell lines and DLBCL patients. We also provided evidence that GPC5 is not a likely target for 13q31-q32 amplification.

Cell Lines, Tumor Specimens, and CGH Method.

The cell lines used were Karpas 1718 (splenic lymphoma with villous lymphocytes; Ref. 18), OCI-Ly4, OCI-Ly7, and OCI-ly8 (DLBCL, kindly provided by Dr. Riccardo Dalla-Favera of Columbia University, New York, NY), Rec1 (mantle cell lymphoma, kindly provided by Dr. Martin J. S. Dyer of Leicester University, Leicester, United Kingdom; Ref. 19), Karpas 422 (B-cell lymphoma cell line; Ref. 20), and ATN-1 (adult T-cell lymphoma cell line, kindly provided by Dr. Tomoki Naoe of Nagoya University School of Medicine, Nagoya, Japan; Ref. 21). SUDHL6 (Southwestern University Diffuse Histiocytic Lymphoma cell line; B-cell lymphoma), SP49 (mantle cell lymphoma cell line), Jurkat (T-cell acute lymphocytic leukemia), and other cell lines were described elsewhere (22). Cell lines with variable copy numbers of the X chromosome were purchased from the National Institute of General Medical Sciences Human Genetics Cell Repository Coriell Institute for Medical Research (Camden, NJ). Patient samples were collected with informed consent, and this experiment was approved by the Institutional Review Board of Aichi Cancer Center. All of the cell lines were maintained in RPMI 1640 supplemented with 10% FCS. Genomic DNA was extracted according to standard procedures using proteinase K digestion and phenol chloroform extraction. Normal DNA for use with conventional CGH and array CGH was prepared by using peripheral-blood lymphocytes from a normal male. “Conventional” CGH was carried out according to the manufacturer’s protocol (Vysis, Downers Grove, IL).

Array CGH.

The array fabrication and hybridization was performed according to the method described by Hodgson et al.(23) and Pinkel et al.(17), respectively. The array consisted of 2,088 bacterial artificial chromosome (BAC) and P-1 derived artificial chromosome (PAC) clones, covering the human genome at roughly a 1.5-Mb resolution, from library RP11 and 13 for BAC clones and RP1, 3, 4, and 5 for PAC clones. Information on clone names and their location on chromosomes is available on request. These clones were obtained from the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute (Oakland, CA).4 Each clone was cultured in Terrific Broth medium with the chloramphenicol (25 μg/ml), and BAC and PAC DNA was extracted with a plasmid Mini-kit (Qiagen, Germantown, MD). The location of each clone was also confirmed by FISH analysis. Roughly 10% of these clones could not be assigned to their expected region and excluded from this study, whereas the confirmed clones were used for array CGH. We used 10 ng of BAC and PAC DNA as the template for degenerate oligonucleotide primed PCR with the primer 5′-CCGACTCGAGNNNNNNATGTGG-3′, where n = A, C, G, or T (24). Amplifications were performed on a TaKaRa PCR Thermal Cycler MP (TaKaRa, Tokyo, Japan) using ExTaq polymerase (TaKaRa). Degenerate oligonucleotide primed-PCR products were enriched by ethanol precipitation and dissolved in distilled water, and then equal volume of DNA spotting solution DSP0050 (MATSUNAMI, Osaka, Japan) was added (∼1 μg/μl). DNA was robotically spotted (NGK Insulators, Ltd., Nagoya, Japan) in duplicate onto CodeLink activated slides (Amersham Biosciences, Piscataway, NJ). Tested and reference DNA (1 μg each) was digested with DpnII and labeled with the Bio prime DNA labeling system (Invitrogen Life Technologies, Inc., Tokyo, Japan) using cyanine3-dUTP and cyanine5-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ), respectively. Unincorporated fluorescent nucleotides were removed with the aid of Sephadex G-50 spin columns (Amersham Biosciences). Labeled 1 μg of tested and reference DNA samples was mixed with 100 μg of Human Cot-1 DNA (Invitrogen Life Technologies, Inc.) and precipitated, after which the pellet was resuspended in the 45-μl hybridization mixture, which consisted of 50% formamide, 10% dextran sulfate, 2× SSC, 4% SDS, and 10 μg/μl yeast tRNA (Invitrogen Life Technologies, Inc.). The hybridization solution was heated to 73°C for 5 min to denature the DNA and then incubated for 45 min at 37°C to allow blocking of the repetitive sequences. The slides spotted with DNA were denatured in a solution consisting of 70% formamide/2× SSC at 73°C for 4 min, then dehydrated in cold 70%, 85%, and 100% ethanol for 5 min each and air-dried. Hybridization was performed for 48 h in a container placed on a slowly rocking table containing 200 μl of 50% formamide and 2× SSC to control moisture. It was followed by 15 min posthybridization washing in 50% formamide/2× SSC at 50°C, 30 min in 2× SSC/0.1% SDS at 50°C, 15 min in PN buffer consisting of 0.1 M NaH2PO4, 0.1 M Na2HPO4 at (pH 8.0), and 0.1% NP40 at room temperature, rinsing in 2× SSC at room temperature, and finally dehydration in 70%, 85%, 100% ethanol at room temperature for 2 min each and air-drying. Scanning analysis was basically carried out with the Agilent Micro Array Scanner (Agilent Technologies, Palo Alto, CA). Thus, acquired array images were analyzed using Genepix Pro 4.1 (Axon Instruments, Inc., Foster City, CA). DNA spots were automatically segmented, the local background was subtracted, and the total intensities and fluorescence intensity ratio of the two dyes for each spot were calculated. Fluorescence intensity ratio of the two dyes (Cy3 intensity/Cy5 intensity) were converted into log2 intensity ratios (log2 ratio).

For the array used in this study, six simultaneous hybridizations of normal male versus normal male were performed to define the normal variation for log2 ratio. In this experiment, 122 clones showed less than one-tenth the fluorescence intensity of the mean value for all of the clones, and were excluded from array CGH analysis. The remaining 1,966 clones were used for the array CGH analysis. More than 95% of the measured fluorescence log2 ratio values of each spot (2 × 1966 clones) ranged from +0.2 to −0.2. The thresholds for the log2 ratio of gains and losses were set at the log2 ratio of +0.2 and −0.2, respectively. We also normalized the log2 ratio of each sample according to the following method.

The medium log2 ratio value for all of the clones was computed, and the clones were selected with a log2 ratio more than “the median + SD × A ” or less than “ the median − SD × A.” “A” was visually defined as the normal region by referring to the log2 ratio plots of all clones for each experiment. “A” was also assigned an approximate ranged from 0.3 to 0.7. We then computed the mean log2 ratio value for the selected clones, and designated this mean log2 ratio value as “X.” Finally, we obtained the “Y” value by subtracting “X” from the log2 ratio for each clone. In this study, each log2 ratio was analyzed on the basis of the “Y” value. We visually selected the clones, computed the SD for each experiment, and confirmed that the SD did not exceed 0.15. If it did, the value was considered unreliable for CGH analyses.

Array-hybridization of normal male versus normal female was performed to check any change in one copy number of the X chromosome. To confirm linearity associated with a change in the copy number, we also performed array hybridization of normal versus each cell line with different number of the X chromosome (GM04626: 47XXX; GM01415D: 48XXXX; and GM05009C: 49XXXXX). These cell lines were obtained from the National Institute of General Medical Sciences Human Genetics Cell Repository Coriell Institute for Medical Research. Fifty seven BAC or PAC clones of the X chromosome were used for the analysis. We computed the mean log2 ratio value of those clones on each hybridization.

FISH Analysis.

We confirmed the location of BAC clones on 13q31-q32 from information archived by the Ensembl Genome Data Resources.5 FISH analysis using 19 BAC clones located around the region of high-level amplification of 13q31-q32 demonstrated by array CGH, covering ∼15 Mb, was used for three cell lines (Karpas 1718, OCI-Ly4, and Rec1). Each interphase chromosome slide of cell lines was prepared according to a standard method. FISH was carried out according to the method described elsewhere (25).

Location of ESTs and Genes.

ESTs and genes located on the region of chromosome 13q31.3 were referenced by the National Center for Biotechnology Information,6 the Ensembl genome data resource,5 and the University of California at Santa Cruz Biotechnology.7 Array CGH and FISH analysis demonstrated that the common region of amplification at 13q31-q32 extended from BAC, RP11–360A9 to BAC, RP11–93M14. This region contained 65 independent ESTs that do not overlap each other and GPC5 (Table 1).

Reverse Transcription-PCR (RT-PCR) Analysis.

Three cell lines, Rec1, Karpas 1718, and OCI-Ly4, which showed high amplification on 13q31.3 by FISH and array CGH, were used for RT-PCR analysis. cDNA derived from fetal brain was also included. To avoid amplification from contaminated genomic DNA in the RNA samples, RNA was treated with amplification grade DNaseI (Invitrogen Life Technologies, Inc.) before cDNA syntheses of the samples, which were performed using SuperScriptII (Life Technologies, Inc., Division of Life Technologies, Inc., Gaithersburg, MD). Briefly, each 5 μl of total RNA was reverse-transcribed into cDNA dissolved in 40 μl of distilled water. RT-PCR was performed for 65 ESTs and GPC5 using the specific primers (Table 1). Each primer was also designed so that the Tm value would be between 55°C and 60°C. Amplifications were performed on a Thermal Cycler (Perkin-Elmer Corporation, Norwalk, CT). RT-PCR was conducted with the touchdown PCR method described elsewhere (26). Briefly, the reactions were comprised of 10 cycles of denaturation (94°C, 0.5 min), annealing (63°C, 0.5 min, 1°C decrease per 2 cycles), and extension (72°C, 2.5 min), followed by 35 cycles of denaturation (94°C, 0.5 min), annealing (58°C, 0.5 min), and extension (72°C, 2.5 min), and a final extension of 5 min at 72°C. Basically, the annealing temperature of the reaction was from 63 to 58°C. Additionally, RT-PCR was also performed under different conditions by changing the annealing temperature from 65 to 60°C or 60°C to 55°C. If no PCR products were obtained, we designed new primer sets to confirm their true negativity. All of the PCR products were separated by electrophoresis and purified using the QIA Quick Gel Extraction kit (Qiagen). TA cloning to purified PCR products was performed by using pBluescriptII SK (−), and sequenced by using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Northern Blot Analysis.

Northern blotting was performed with 30 ESTs and GPC5 cDNA against five cell lines (Rec1, Karpas 1718, OCI-Ly4, Jurkat, and ATN-1) and human placenta. Additional analysis used the candidate genes BC040320 and GPC5, which was included because it has been reported to be a candidate gene for this region (15). We analyzed and compared the expression of each of the ESTs and GPC5 in the cell lines with high-level amplification at 13q31-q32 (Rec1, Karpas 1718, and OCI-Ly4) and in the cell lines without (Jurkat and ATN-1). To examine the expression of BC040320 and GPC5 in detail, Northern blot analysis was also performed for several cell lines and patients. Northern blot hybridization was performed with a standard method (21). Each RT-PCR product was used as a specific probe labeled by PCR. Briefly, 10 ng of the RT-PCR products were labeled with [α-32P]dCTP by PCR. The reactions were carried out with 25 cycles of denaturation (94°C, 0.5 min), annealing (55°C, 0.5 min), and extension (72°C, 2.5 min), and a final extension of 5 min at 72°C. Total cellular RNA (5 μg) was size-fractioned on 1% agarose/0.66 M formaldehyde gel, transferred onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Tokyo, Japan). The membranes were then hybridized overnight at 42°C with [α-32P]dCTP-labeled probes, washed, and then exposed to BIOMAX MS films (EKC, Rochester, NY).

Candidate Gene Analysis.

BC040320, a candidate gene in the 13q31-q32 amplification region, was additionally analyzed. To confirm the sequence of the candidate gene, RT-PCR was performed between exon 1 and exon 4 of BC040320 using the primers 5′-TCCGGTCGTAGTAAAGCGCAGGCG-3′, designed on the side of exon1 and 5′-CTGAAGTCTCAAGTGGGCAT-3′, designed on the side of exon4 of BC040320. The PCR reaction was the same as the one described in the RT-PCR section.

Array CGH Analysis.

Array CGH consisting of 1,966 BAC and PAC clones were examined with normal male versus female, and demonstrated that most of the signals from autosomal chromosomes are within log2 ratio of +0.2 to −0.2 (Fig. 1,A). The linearity of copy number changes was studied with cell lines having a different number of X chromosomes. As shown in Fig. 1,B, the result of the plot of each calculated mean fluorescence ratio demonstrated that the fluorescence ratio was proportional to the change of one copy number. The array CGH used for four cell lines (Karpas 1718, Rec1, OCI-Ly4, and OCI-Ly7) and one DLBCL patient (D778) demonstrated high-level gains in copy number changes at 13q31-q32. Fig. 2,A shows a representative result for Karpas 1718 of the array CGH. Detailed results of chromosome 13 from three cell lines (Karpas 1718, Rec1, and OCI-Ly7) and one DLBCL patient (D778) are shown in Fig. 2,B. Conventional CGH and FISH analyses clearly confirmed these array CGH data (Fig. 3).

The Common Region of Amplification at 13q31-q32.

In an attempt to narrow the amplicon at 13q31-q32, FISH analysis of three cell lines (Karpas 1718, Rec1, and OCI-Ly4), using 19 BAC/PAC probes located on 13q31-q32 (Fig. 4,A) were conducted, and it was found that the common amplified region at 13q31-q32 was located between RP11–29C8 and RP11–93M14. The high-resolution array CGH data are shown in Fig. 4 B. Karpas 1718 and D778 (DLBCL patient sample) showed a wide area of amplification extending over >50 Mb of chromosome 13q. A small genomic region showing high-level amplification (defined as log2 ratio >1) extended from 13q22.2 to 13q31.3, with the region of 13q31.3 in particular showing a higher log2 ratio of >2. In the same manner, OCI-Ly7 and Rec1 also showed high-level amplification, which confined to 13q31.3. These results identified the common region of high-level amplification to extend from RP11–360A9 to RP11–481A22. On the basis of the FISH and array CGH results, we defined the genomic region between RP11–360A9 and RP11–93M14 as the common and smallest region of amplification in four cell lines and one DLBCL patient.

RT-PCR Analysis for ESTs of Chromosome 13q31.3.

Expression of 65 ESTs and GPC5 located in the common region of amplification on 13q31.3 were examined by RT-PCR using cDNA derived from three cell lines (Karpas 1718, OCI-Ly4, and Rec1) and fetal brain. RT-PCR products were examined by gel electrophoresis. A positive signal was defined as detection of an expected size of band. The results are summarized in Table 1. Thirty ESTs and GPC5, which showed the expected size of band, were found to be positive in all of the three cell lines with amplification of 13q31.3, and they were confirmed by their nucleotide sequence. Fifteen ESTs also showed the expected size, but RT-PCR analysis demonstrated only one or two cell lines so that they were excluded as candidate ESTs associated with amplification at 13q31.3. The remaining 21 ESTs did not show any bands in either OCI-Ly4 or fetal brain. They were also examined with another set of primers, but again no bands were detected (data not shown) so that they were also excluded as the candidate ESTs. A total of 35 of 65 ESTs were thus excluded and not additionally analyzed.

Northern Blotting.

To identify the expression patterns of 30 ESTs and GPC5, Northern blot was used for six kinds of RNAs, which were human placenta, three B-cell lymphoma cell lines (Rec1, Karpas 1718, and OCI-Ly4) with 13q31.3 amplification, and two T-cell lymphoma cell lines (Jurkat and ATN-1) without 13q31.3 amplification. Twenty-two of the ESTs showed hardly any detectable bands in any of the cell lines (Table 2). Fig. 5 shows representative expression patterns of the ESTs. AF339828 and BC040320 showed the similar expression pattern of a transcript of ∼6 kb and a smeary band >6 kb. The signals were observed in only three B-cell lymphoma cell lines, but not in human placenta or the T-cell lines. GPC5, a gene incompletely included in the common region of amplification at 13q31-q32, showed weak expression of ∼5-kb transcript in all of the cell lines and the human placenta at similar intensity. The signals for the other ESTs did not reflect any difference in copy number at 13q31.3 between cell lines and human placenta. Therefore, we regarded AF339828 and BC040320 as the most possible target gene for amplification at 13q31.3. Additional study against various samples including patient samples and normal tissues was performed with these two ESTs, and GPC5, reportedly a target gene for 13q31.3 amplification, was also examined.

Northern blot results with the BC040320 probe are shown in Fig. 6. High-level expression of BC040320 was seen in five cell lines with amplification at 13q31-q32 (Rec1, Karpas 1718, OCI-Ly4, OCI-Ly7, and OCI-Ly8). Lower level of expression than that of the five cell lines was seen in three cell lines without amplification (Karpas 422, SP49, and SUDHL6). Furthermore, two patients with amplification showed higher expression than the other two patients without amplification. These results indicated that the expression of BC040320 paralleled the gain in copy number shown by both conventional and array CGH. On the same membrane, expression of GPC5 in five cell lines with amplification at 13q31-q32 was not significantly different from that of the other cell lines without amplification, suggesting that GPC5 is not a likely candidate gene. The expression pattern of BC040320 was examined against various hematopoietic cell lines (T-cell lymphoma, multiple myeloma, myeloid leukemia, and natural killer/T-cell lymphoma; Fig. 6,B). Some cell lines showed weak signals when compared with the two cell lines with high-level amplification. GPC5 cDNA again yielded very weak signals without significant differences but with some variation. When normal tissues were examined, the BC040320 signal was hardly observable except for lung, thymus, and lymph node (Fig. 6 C).

In conclusion, the result of Northern blot using each of the probes revealed that the expression of BC040320 paralleled the gain in copy number at 13q31-q32 and that BC040320 was, thus, most likely to be the candidate gene.

Full Length, Genomic Location, and Characterization of the Candidate Gene.

We focused on AF339828 and BC040320 as the most likely candidate gene on the basis of the results of FISH, array CGH, and Northern blot analysis. We named this candidate gene C13orf25 (Chromosome 13 open reading frame 25) according to the recommendation of HUGO Gene Nomenclature Committee.8

To additionally characterize this gene and cDNA structures, we performed RT-PCR on exon 1 to exon 4 of BC040320. Two transcripts were obtained and sequence analysis found the shorter one to be transcript-A and the longer one transcript-B (Fig. 7). Database search with the Vega Genome Browser showed that bA121J7.2 (Vega_gene ID) is also located in this region.9 The peptides of 32-amino acids (AA) were predicted as bA121J7.2. The peptides of 32-AA predicted in bA121J7.2 were also predicted transcript-A. Furthermore, the same initiation codon predicted the 70-AA polypeptide in the transcript-B. It should be noted that five kinds of precursor micro-RNAs (miRNAs; miR91-precursor-13 micro RNA, miR18- precursor-13 micro RNA, miR19a-precursor-13 micro RNA, miR19b-precursor-13 micro RNA, and miR92-precursor-13 micro RNA), including seven kinds of miRNA (microRNA miR-17, miR-91, miR-18, miR-19a, miR-20, miR-19b, and miR-92) were also recognized in the sequence of transcript-B (Fig. 7).

Genetic alteration in 13q has been reported in many human cancers, including hematopoietic malignancies. Recent molecular genetic studies using FISH and CGH have demonstrated that amplification at 13q31-q32 has been detected frequently in hematopoietic malignancies. Amplification at 13q21-qter was demonstrated frequently in B-cell malignancies (5, 6, 7, 8). GPC5 has been proposed recently as the candidate gene for 13q31-q32 amplification region in B-cell lymphoma cell lines (15). In the study reported here, we examined genomic alteration at the GPC5 loci using array CGH and the expression of GPC5 using Northern blotting. The GPC5 sequence in the 2-Mb genomic region at 13q31.3 approximately ranges from BAC, RP11–121J7 to BAC, RP11–268K13. Our array CGH data for Rec1 showed that the log2 ratio of BAC, RP11–481A22, which located the intron of GPC5 between exon 6 and exon 7, showed a loss in copy number (log2 ratio = −0.76). The array data indicated that other BACs located on the telomeric side of this BAC also showed a loss. Our FISH data with Rec1 using the new BAC clone, RP11–93M14, containing exon 3, exon 4, and exon 5 of GPC5, also showed a loss in copy number. These results demonstrated that the GPC5 locus was not fully included in the common region of amplification at 13q31-q32 in the cell lines, suggesting that GPC5 in this allele might not be functional.

Northern blotting also showed that expression of GPC5 in cell lines with amplification at 13q31-q32 was not significantly different from that of the other cell lines without amplification. On the other hand, both BC040320 and AF339828 were expressed only in B-cell lymphoma cell lines with 13q31-q32 amplification but not in T-cell lymphoma or human placenta without 13q31-q32 amplification. Their ESTs were fully included in the common region of amplification at 13q31-q32. Detailed analysis using Northern blot showed that the expression of BC040320 almost paralleled the gains in copy number shown by both conventional and array CGH. Northern blot analysis showed that BC040320 was especially overexpressed in B-cell lymphoma cell lines with amplification at 13q31-q32 and hardly expressed in normal tissues, including lymphoid tissues. Although we also found minor mRNA expression of BC040320 in SP49 (mantle cell lymphoma cell line) and SUDHL6 (B-cell lymphoma cell line) without amplification at 13q31-q32 (Fig. 6 B), this expression may well have been caused not by the gain in copy number but other reasons that are not yet fully understood. These results suggested that BC040320 was the most likely a candidate gene for the amplification at 13q31-q32. We named this candidate gene C13orf25 (Chromosome 13 open reading frame 25).

To confirm the validity of C13orf25 cDNA, RT-PCR was performed, and two transcripts (Transcript-A and -B) were obtained. The Vega genome browser9 predicted the presence of a gene, bA121J7.2, encoding 32-AA polypeptides in the Transcript-A cDNA. A possible open reading frame in Transcript-B was also predicted, encoding 70-AA polypeptides starting from the same ATG (Fig. 7). The genomic structure of C13orf25 might be incomplete on the 3′ side because AF339828, which showed the same pattern of hybridization as BC040320, was observed near C13orf25 and was located at 300-bp downstream of C13orf25. Because of the presence of multiple bands in Northern blot analysis with BC040320, various transcripts might also be produced, but RT-PCR demonstrated major transcripts. The result of computer analysis using National Center for Biotechnology Information BLAST10 showed that the predicted proteins of C13orf25 contained no putative domains in those transcripts. Further study is needed, however, to characterize the proteins.

Five precursor miRNAs (miR91-precursor-13 miRNA, miR18-precursor-13 miRNA, miR19a-precursor-13 miRNA, miR19b-precursor-13 miRNA, and miR92-precursor-13 miRNA), including seven mature miRNAs (miRNA miR-17, miR-91, miR-18, miR-19a, miR-20, miR-19b, and miR-92), were obtained from the transcript-B sequence. The function of miRNA reportedly is to regulate the expression of target genes in both humans (27) and Caenorhabditis elegans(28, 29, 30, 31). miRNAs also mediate a cleavage of mRNA in Arabidopsis thaliana(32, 33). Calin et al.(34) reported recently an association between chronic lymphocytic leukemia and deletion of a section of chromosome 13 that contains the genes for miR-15 and miR-16. The presence of these miRNAs on the C13orf25 gene may provide an insight into the processes of tumorigenesis.

In the study presented here, we were able to demonstrate that C13orf25 but not GPC5 is the most likely candidate gene for amplification. C13orf25 gene was expressed in association with genomic amplification, and may play an important role in tumorigenesis and resulting poor prognosis.

Additional investigation into the function of C13orf25, including the seven miRNAs, can be expected to provide an insight into the role of the C13orf25 in tumorigenesis.

Grant support: Grant-in-Aid for the 2nd-Term Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare, a Grant-in-Aid for Science on Primary Areas (Cancer Research) from the Ministry of Education, Science and Culture.

Note: A. Ota and H. Tagawa contributed equally to this work.

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.

Requests for reprints: Masao Seto, Division of Molecular Medicine, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. Phone: 81-52-762-6111, extension 7080/7082; Fax: 81-52-764-2982; E-mail: mseto@aichi-ml.jp

4

Internet address: http://bacpac.chori.org/.

5

Internet address: http://www.ensembl.org/.

6

Internet address: http://www.ncbi.nlm.nih.gov/.

7

Internet address: http://genome.ucsc.edu/.

8

Internet address: http://www.gene.ucl.ac.uk/nomenclature/.

9

Internet address: http://vega.sanger.ac.uk/.

10

Internet address: http://www.ncbi.nlm.nih.gov/BLAST/.

Fig. 1.

Array comparative genomic hybridization analysis of normal versus female. A, representative genomic profile of an array comparative genomic hybridization using normal male versus normal female DNAs. Six simultaneous hybridizations of normal male versus normal male were performed to define the normal variation in log2 ratio (log2 cy3/cy5). In the control experiment, >95% of the measured fluorescence log2 ratio values of each spot (2 × 1966 clones) ranged from +0.2 to −0.2 (data not shown). The thresholds for the log2 ratio of gains and loss were therefore set at log2 ratio of +0.2 and −0.2, respectively. Array data are plotted as the mean log2 ratio of duplicate spots for each clone. Vertical lines show the threshold for the log2 ratio of gains and loss. The log2 ratio for each of the bacterial artificial chromosome clone is plotted as a function of its genome location, with chromosome 1 to the left and X to the right; for each chromosome the order is short-arm telomeric to long-arm telomeric. B, normalized log2 ratio for the changes in copy number of X chromosome. The normal male DNA was used as reference for all hybridizations. Array hybridizations were performed with the test genomic DNA from a normal male (1 × chromosome), a normal female (2 × chromosomes), and three cell lines containing three, four, and five copies of the X chromosome. Each plot stands for the mean value of all normalized fluorescence ratio of 57 clones from the X chromosome. The ratio on each of the X chromosome clone was normalized by the mean fluorescence intensity ratio of autosomal chromosome clones. We defined the fluorescence intensity ratio of array-hybridization with normal male versus normal male as 0. Each plot was then computed on the basis of the normalized value. The line represents the linear regression through all of the data with a slope of 0.51 and an intercept of 0.72.

Fig. 1.

Array comparative genomic hybridization analysis of normal versus female. A, representative genomic profile of an array comparative genomic hybridization using normal male versus normal female DNAs. Six simultaneous hybridizations of normal male versus normal male were performed to define the normal variation in log2 ratio (log2 cy3/cy5). In the control experiment, >95% of the measured fluorescence log2 ratio values of each spot (2 × 1966 clones) ranged from +0.2 to −0.2 (data not shown). The thresholds for the log2 ratio of gains and loss were therefore set at log2 ratio of +0.2 and −0.2, respectively. Array data are plotted as the mean log2 ratio of duplicate spots for each clone. Vertical lines show the threshold for the log2 ratio of gains and loss. The log2 ratio for each of the bacterial artificial chromosome clone is plotted as a function of its genome location, with chromosome 1 to the left and X to the right; for each chromosome the order is short-arm telomeric to long-arm telomeric. B, normalized log2 ratio for the changes in copy number of X chromosome. The normal male DNA was used as reference for all hybridizations. Array hybridizations were performed with the test genomic DNA from a normal male (1 × chromosome), a normal female (2 × chromosomes), and three cell lines containing three, four, and five copies of the X chromosome. Each plot stands for the mean value of all normalized fluorescence ratio of 57 clones from the X chromosome. The ratio on each of the X chromosome clone was normalized by the mean fluorescence intensity ratio of autosomal chromosome clones. We defined the fluorescence intensity ratio of array-hybridization with normal male versus normal male as 0. Each plot was then computed on the basis of the normalized value. The line represents the linear regression through all of the data with a slope of 0.51 and an intercept of 0.72.

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Fig. 2.

Genomic profiles of array comparative genomic hybridization. A, the representative genomic profile of array comparative genomic hybridization with Karpas 1718. The bacterial artificial chromosomes are ordered by position in the genome beginning at the 1p telomere and ending at the Xq telomere. The black arrow above the graph indicates high-level amplification (defined as log2 ratio >1). B, detailed genomic profiles of chromosome 13 in the three cell lines (Karpas 1718, Rec1, and OCI-Ly7) and one diffuse large B cell lymphoma patient (D778). The log2 ratio for each of the 68 bacterial artificial chromosomes and P-1 derived artificial chromosome clones is plotted as a function of its genome location, with chromosome 13q-centromere to the left and 13q-telomere to the right. ····, show the threshold for gains and loss. Bold arrows indicate high-level amplification (defined as log2 ratio >1) and thin arrows moderate-level amplification (0.2 > log2 ratio >1). Karpas 1718 shows a wide region of amplification extending over >50-Mb of chromosome 13q. Furthermore, high-level amplification in Karpas 1718 is observed from 13q22.2 to 13q31.3, with 13q31.3 in particular showing the highest amplification (log2 ratio >2). In the same manner, high-level amplification of OCI-Ly7 and Rec1 are shown at 13q31.3. Rec1 also shows wide loss in the vicinity of 13q31.3. The patient sample (D778) shows a wide region of amplification at 13q21.2–13q31.3 and 13q33.3-qter, with 13q31.1-q31.3 in particular indicating high-level amplification.

Fig. 2.

Genomic profiles of array comparative genomic hybridization. A, the representative genomic profile of array comparative genomic hybridization with Karpas 1718. The bacterial artificial chromosomes are ordered by position in the genome beginning at the 1p telomere and ending at the Xq telomere. The black arrow above the graph indicates high-level amplification (defined as log2 ratio >1). B, detailed genomic profiles of chromosome 13 in the three cell lines (Karpas 1718, Rec1, and OCI-Ly7) and one diffuse large B cell lymphoma patient (D778). The log2 ratio for each of the 68 bacterial artificial chromosomes and P-1 derived artificial chromosome clones is plotted as a function of its genome location, with chromosome 13q-centromere to the left and 13q-telomere to the right. ····, show the threshold for gains and loss. Bold arrows indicate high-level amplification (defined as log2 ratio >1) and thin arrows moderate-level amplification (0.2 > log2 ratio >1). Karpas 1718 shows a wide region of amplification extending over >50-Mb of chromosome 13q. Furthermore, high-level amplification in Karpas 1718 is observed from 13q22.2 to 13q31.3, with 13q31.3 in particular showing the highest amplification (log2 ratio >2). In the same manner, high-level amplification of OCI-Ly7 and Rec1 are shown at 13q31.3. Rec1 also shows wide loss in the vicinity of 13q31.3. The patient sample (D778) shows a wide region of amplification at 13q21.2–13q31.3 and 13q33.3-qter, with 13q31.1-q31.3 in particular indicating high-level amplification.

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Fig. 3.

Fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) analysis of cell lines with or without amplification at 13q31-q32. CGH results for four cell lines (Karpas 1718, Rec1, OCI-Ly4, and Jurkat) are shown. The lines to the right (green) and to the left (red) of each chromosome indicate the region gained or lost, respectively. Representative results of metaphase FISH with bacterial artificial chromosome, RP11–487A2 are shown on the right side of each panel. Chromosome 13 examined by CGH is also shown beside each ideogram. Three B-cell lymphoma cell lines, Karpas 1718 (A), Rec1 (B), and OCI-Ly4 (C) show amplification at 13q31-q32, but Jurkat (D) does not show one. FISH analysis shows amplification in >15 copies in the three B-cell lines, but no amplification in Jurkat. Each metaphase chromosome was counterstained by 4′,6-diamidino-2-phenylindole.

Fig. 3.

Fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) analysis of cell lines with or without amplification at 13q31-q32. CGH results for four cell lines (Karpas 1718, Rec1, OCI-Ly4, and Jurkat) are shown. The lines to the right (green) and to the left (red) of each chromosome indicate the region gained or lost, respectively. Representative results of metaphase FISH with bacterial artificial chromosome, RP11–487A2 are shown on the right side of each panel. Chromosome 13 examined by CGH is also shown beside each ideogram. Three B-cell lymphoma cell lines, Karpas 1718 (A), Rec1 (B), and OCI-Ly4 (C) show amplification at 13q31-q32, but Jurkat (D) does not show one. FISH analysis shows amplification in >15 copies in the three B-cell lines, but no amplification in Jurkat. Each metaphase chromosome was counterstained by 4′,6-diamidino-2-phenylindole.

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Fig. 4.

Analysis of 13q31-q32 by a combination of array comparative genomic hybridization (CGH) and interphase fluorescent in situ hybridization (FISH). A, summarized data of DNA sequence copy numbers in three cell lines (Karpas 1718, OCI-Ly4, and Rec1) determined by interphase FISH using 19 bacterial artificial chromosome (BAC) clones of 13q31.3, including a new BAC, RP11–93M14, that was not used for array CGH. Ten interphase cells were analyzed and the average copy numbers of the BAC clone signals were counted for each cell line. The vertical line indicates the copy number and the horizontal dotted line indicates normal two copies. shows the common region of gain in copy number, which extended fromRP11–29C8 to RP11–93M14. The positions of STS markers and all BAC clones were confirmed from information archived by Ensembl Genome Data Resource.5 The underlined BAC clones were used for FISH and array CGH. The thin arrow indicates the GPC5 gene loci. B, summarized data of array CGH analysis of three cell lines (Rec1, Karpas 1718, and OCI-Ly7) and one DLBCL patient (D778). The vertical line shows log2 ratio. ···· show the threshold for gain and loss set at log2 ratios of +0.2 and −0.2, respectively. shows the common region of high-level amplification (log2 ratio >1) in the three cell lines, which is extended from RP11–360A9 to RP11–481A22.

Fig. 4.

Analysis of 13q31-q32 by a combination of array comparative genomic hybridization (CGH) and interphase fluorescent in situ hybridization (FISH). A, summarized data of DNA sequence copy numbers in three cell lines (Karpas 1718, OCI-Ly4, and Rec1) determined by interphase FISH using 19 bacterial artificial chromosome (BAC) clones of 13q31.3, including a new BAC, RP11–93M14, that was not used for array CGH. Ten interphase cells were analyzed and the average copy numbers of the BAC clone signals were counted for each cell line. The vertical line indicates the copy number and the horizontal dotted line indicates normal two copies. shows the common region of gain in copy number, which extended fromRP11–29C8 to RP11–93M14. The positions of STS markers and all BAC clones were confirmed from information archived by Ensembl Genome Data Resource.5 The underlined BAC clones were used for FISH and array CGH. The thin arrow indicates the GPC5 gene loci. B, summarized data of array CGH analysis of three cell lines (Rec1, Karpas 1718, and OCI-Ly7) and one DLBCL patient (D778). The vertical line shows log2 ratio. ···· show the threshold for gain and loss set at log2 ratios of +0.2 and −0.2, respectively. shows the common region of high-level amplification (log2 ratio >1) in the three cell lines, which is extended from RP11–360A9 to RP11–481A22.

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Fig. 5.

Northern blot analysis of the candidate gene for 13q31-q32 amplification. Northern hybridization was performed against 6 kinds of RNAs comprising human placenta (lane 1), 3 B-cell lymphoma cell lines (lane 2, Rec1; lane 3, Karpas 1718; and lane 4, OCI-Ly4) with amplification at 13q31-q32 and 2 T-cell lymphoma cell lines (lane 5, Jurkat and lane 6, ATN-1) without amplification. Representative and characteristic expression patterns of 8 of 30 ESTs and GPC5 are shown. Expression of GPC5 and BI481522 was not significantly different, whereas LOC160824, AF339828, BC040320, AF339802, LOC121734, AA705439, and N49442 showed clearly different patterns of expression. In particular, the expression of AF339828 and BC040320, which showed similar patterns of hybridization, demonstrates concordance with the gain in copy number at 13q31-q32.

Fig. 5.

Northern blot analysis of the candidate gene for 13q31-q32 amplification. Northern hybridization was performed against 6 kinds of RNAs comprising human placenta (lane 1), 3 B-cell lymphoma cell lines (lane 2, Rec1; lane 3, Karpas 1718; and lane 4, OCI-Ly4) with amplification at 13q31-q32 and 2 T-cell lymphoma cell lines (lane 5, Jurkat and lane 6, ATN-1) without amplification. Representative and characteristic expression patterns of 8 of 30 ESTs and GPC5 are shown. Expression of GPC5 and BI481522 was not significantly different, whereas LOC160824, AF339828, BC040320, AF339802, LOC121734, AA705439, and N49442 showed clearly different patterns of expression. In particular, the expression of AF339828 and BC040320, which showed similar patterns of hybridization, demonstrates concordance with the gain in copy number at 13q31-q32.

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Fig. 6.

Expression study of GPC5 and BC040320. Amplification status at 13q31-q32 in each of the cell lines and diffuse large B-cell lymphoma (DLBCL) patients examined by conventional comparative genomic hybridization is indicated above names of the samples. A, expression pattern of GPC5 and BC040320 in cell lines and DLBCL patients with or without amplification at 13q31-q32. Expression of GPC5 in five cell lines and two DLBCL patients with amplification at 13q31-q32 is not significantly different from that of the other cell lines and patients without amplification. BC040320 is expressed in cell lines with amplification at 13q31-q32 (lanes 1–5) and at much lower levels in cell lines without amplification (lanes 6–8). In the same manner, BC040320 is strongly expressed in DLBCL patients with amplification at 13q31-q32 (lanes 9 and 10), but very weakly in cell lines without amplification (lanes 11 and 12). B, expression pattern of GPC5 and BC040320 in multiple cell lines with hematopoietic malignancies. Some cell lines (lanes 9, 11, and 12) with amplification at 13q31-q32 show weak signals when compared with the two cell lines (lanes 1 and 2) with high-level amplification. Expression of GPC5 shows very weak signals with some variations but without significant differences. AML, acute myeloid leukemia cell line. MM, multiple myeloma cell line. NK/T, natural killer/T-cell lymphoma/leukemia cell line. C, expression pattern of BC040320 and GPC5 in multiple normal tissues. Expression of BC040320 is hardly visible in normal tissues except for lung, thymus, and lymph node when compared with that of the two cell lines (lanes 1 and 2) with high-level amplification at 13q31-q32.

Fig. 6.

Expression study of GPC5 and BC040320. Amplification status at 13q31-q32 in each of the cell lines and diffuse large B-cell lymphoma (DLBCL) patients examined by conventional comparative genomic hybridization is indicated above names of the samples. A, expression pattern of GPC5 and BC040320 in cell lines and DLBCL patients with or without amplification at 13q31-q32. Expression of GPC5 in five cell lines and two DLBCL patients with amplification at 13q31-q32 is not significantly different from that of the other cell lines and patients without amplification. BC040320 is expressed in cell lines with amplification at 13q31-q32 (lanes 1–5) and at much lower levels in cell lines without amplification (lanes 6–8). In the same manner, BC040320 is strongly expressed in DLBCL patients with amplification at 13q31-q32 (lanes 9 and 10), but very weakly in cell lines without amplification (lanes 11 and 12). B, expression pattern of GPC5 and BC040320 in multiple cell lines with hematopoietic malignancies. Some cell lines (lanes 9, 11, and 12) with amplification at 13q31-q32 show weak signals when compared with the two cell lines (lanes 1 and 2) with high-level amplification. Expression of GPC5 shows very weak signals with some variations but without significant differences. AML, acute myeloid leukemia cell line. MM, multiple myeloma cell line. NK/T, natural killer/T-cell lymphoma/leukemia cell line. C, expression pattern of BC040320 and GPC5 in multiple normal tissues. Expression of BC040320 is hardly visible in normal tissues except for lung, thymus, and lymph node when compared with that of the two cell lines (lanes 1 and 2) with high-level amplification at 13q31-q32.

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Fig. 7.

Exon-intron structure of the C13orf25 gene. A, two expressed sequence tags, BC040320 and AF339828, which are overexpressed in the cell lines with amplification at 13q31.3, are shown above the ····. BC040320 is split into four exons, encompassing two bacterial artificial chromosome clones, RP11–282D2 and RP11–121J7. AF339828 is located to the telomeric side of BC040320 and ∼300-bp apart from BC040320. The primer set used for reverse transcription-PCR is shown below the exons. B, two transcripts obtained by reverse transcription-PCR. One (Transcript-A) is the same as the BC040320 sequence consisting of four exons containing 965-bp nucleotides. The other (Transcript-B) consists of two exons containing 5058-bp nucleotides. Computer analysis showed that 32-AA polypeptides of bA121J7.2 (Vega gene ID) were encoded in the Transcript-A cDNA. Possible open reading frames are shown as . Five precursor microRNAs (miRNAs; miR91-precursor-13 micro RNA, miR18- precursor-13 micro RNA, miR19a- precursor-13 micro RNA, miR19b- precursor-13 micro RNA, and miR92- precursor-13 micro RNA), including seven mature microRNAs (microRNA miR-17, miR-91, miR-18, miR-19a, miR-20, miR-19b, and miR-92) were obtained from the Transcript-B sequence, and are shown by the ▪ in Transcript-B. C, polypeptides sequences are also shown below the structure. The polypeptides of 13 amino acids (AA) are shared by Transcript-A and Transcript-B, and are indicated by underlining.

Fig. 7.

Exon-intron structure of the C13orf25 gene. A, two expressed sequence tags, BC040320 and AF339828, which are overexpressed in the cell lines with amplification at 13q31.3, are shown above the ····. BC040320 is split into four exons, encompassing two bacterial artificial chromosome clones, RP11–282D2 and RP11–121J7. AF339828 is located to the telomeric side of BC040320 and ∼300-bp apart from BC040320. The primer set used for reverse transcription-PCR is shown below the exons. B, two transcripts obtained by reverse transcription-PCR. One (Transcript-A) is the same as the BC040320 sequence consisting of four exons containing 965-bp nucleotides. The other (Transcript-B) consists of two exons containing 5058-bp nucleotides. Computer analysis showed that 32-AA polypeptides of bA121J7.2 (Vega gene ID) were encoded in the Transcript-A cDNA. Possible open reading frames are shown as . Five precursor microRNAs (miRNAs; miR91-precursor-13 micro RNA, miR18- precursor-13 micro RNA, miR19a- precursor-13 micro RNA, miR19b- precursor-13 micro RNA, and miR92- precursor-13 micro RNA), including seven mature microRNAs (microRNA miR-17, miR-91, miR-18, miR-19a, miR-20, miR-19b, and miR-92) were obtained from the Transcript-B sequence, and are shown by the ▪ in Transcript-B. C, polypeptides sequences are also shown below the structure. The polypeptides of 13 amino acids (AA) are shared by Transcript-A and Transcript-B, and are indicated by underlining.

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Table 1

RT-PCRa analysis

BAC clones, EST and STS Marker refer to the information obtained from Ensembl genome data resource. All BAC clones are included in RPCI-11 human male BAC library.

BACEST/geneSTS MarkerForward primerReverse primerFBRec1Karpas 1718OCI-Ly4
360A9 AW664738 D13S1457 5′-gccacatggtcaggttaaac 5′-cttcactaccttgcgactct − ND ND − 
27D9 AA309162  5′-ggctctcatttagctaaatg 5′-aagaagtggttgagacaaca +b 
15N8 No EST        
321E13 AW236754 D13S1175 5′-gagatggcacagcagttgaa 5′-tagttcaaactctacctgca − ND ND − 
447M23 No EST        
370B1 LOC121723  5′-aactactggtgaggactgca 5′-caagttccccttctgcagaa − ND ND − 
 AW059867  5′-aaggcttagctattatgctg 5′-acaatgaggaaaatctccca ++c ++ ++ ++ 
275J18 BC042969 D13S1818 5′-ctggtcacacatccacaatg 5′-cagtggaatctgagtcctag ++ ++ 
 AA628299  5′-cagaaggagtgttaagtctg 5′-caagaagaagctgccagtat ++ ++ ++ ++ 
143O10 No EST        
309H8 BG183515 D13S1239 5′-ctcatgactgtaatcccagc 5′-gtgatgccgtgaaatgagtc 
 BG191981  5′-ttcagtgacctcactgactg 5′-gaggattttgcagtcatcgc − ND ND − 
114G1 LOC160824 D13S767 5′-aggttttgtcagccacacct 5′-gaactatccgtaccttgtcc − ++ 
75N6 AA398228  5′-ctgtaccattgtgcccagaa 5′-atgactcagtccttctggct − ND ND − 
86C3 No EST D13S265       
51A2 No EST        
18M3 No EST        
388D4 LOC144774  5′-cactgtggcagttatacggt 5′-caatggttttccgcaccagt − ND ND − 
 LOC121727  5′-cctgggaaggatggtttctt 5′-ttacaacacaaggggcacac ++ 
409J23 No EST        
392A19 LOC121729  5′-ggagccattacttcaagacc 5′-agaccagtacttgtccagct ++ ++ ++ 
 BG776186  5′-tggacacgtgagtgtgtttc 5′-atgagctcaggcagctctat ++ − 
 LOC121728  5′-tgataggagacagacctgac 5′-tccagtcatcctgaggtaga − ND ND − 
 BG186078  5′-tcacacagtgtgactcacag 5′-tgatctcctctctgtagtgc ND − − 
 AA487882  5′-acccaagggaatattgacac 5′-gaagctagggaagcagtatt ND − − 
 BM542991  5′-tgcagtaggtggcaatctca 5′-tcgacttggactccatgaga − ND ND − 
505P2 BM695971 D13S1234 5′-ccttggagtgcttaaggtag 5′-actagggctctttgtagacc ND − − ++ 
158A8 AI027278  5′-gctgtgattgcgaagaagtg 5′-tccatatgtgagtgtggcag ND − − 
 BG927281  5′-acgccaagctctaatacgac 5′-ctcgggctatggtatatgac − ND ND − 
 BI825411  5′-accctggacaggtatggaat 5′-accatgagcacagtgctcaa − ND ND − 
 AA888411  5′-ctcccattgcagttactatg 5′-gtcgacatgttgttgaggtt 
360H15 AW515966  5′-atgttcaccaggctggtctt 5′-actaactctgtgggcttgca − ND ND − 
 BF818219  5′-tctgcccactaacatctggt 5′-cttgagtagctgagaccaca − ND ND − 
114C3 No EST        
432D3 LOC144776 D13S1190 5′-cacttccttgaaggggtttc 5′-ctctgactcttgggcacaat ND 
 AI126313  5′-ttgagacacagtcctgctct 5′-gcagggcacaatgttttcag − ND ND − 
319L6 AV731092  5′-attggtgaagccacctcaaa 5′-caggctaacatggatctagt ND − − 
 BG941714  5′-agtgcctacttcctaagacc 5′-tcaggaatcagtgcccaaac ND − − 
 AI262947  5′-ttgtgttcctggtccaccta 5′-acattgggccggtcacttat ND − − 
 AI493127  5′-tcaaatcaactgcacctcag 5′-cagttcgagacttcttccat − ND ND − 
51B13 BX097335  5′-gaagtaggatggtgacacct 5′-ctcagcaagtcatcctttgc − ND ND − 
430K10 AL701000 D13S886 5′-tggcctggagtaattagctg 5′-atttagggggtaggagagca ND − − 
 LOC160827  5′-ctgtgcactatcacttggga 5′-taggctctaagccgttggtt − ND ND − 
 LOC160826  5′-atggaatcaggttccctcca 5′-ctgttcccttcatctgaatg − ND ND − 
 BQ477330  5′-caaagtgctgggattacagg 5′-gccagctttgctgcacatta − ND ND − 
 BQ477741  5′-caacagaagatcggcccttt 5′-actccctgaagcacagcatt ND 
 AV731847  5′-caggcacttgcttaagggat 5′-aactagcctgcttcagcttc − ND ND − 
 BI481522  5′-atgtgaagagaggtctcagc 5′-agcaaaccacctagaggctt ND 
 BU729287  5′-cttagcctaatctccctaggcgga 5′-tatcaggtaggtggtccagtctca ND ++ ++ ++ 
 BM703078  5′-caggatcttgccctgttagt 5′-tatcgggtggcgaacaagat ND 
 AA719672  5′-tgtccttaggtagacattgt 5′-ttcaacctctgagaaaccca ND ND ND − 
 LOC121734  5′-acttcactgtcaacagcgag 5′-agagaccacatgcttgccat ND ++ ++ ++ 
 BE466687  5′-agttctggaggctaaaagtccagt 5′-gccaaaatcacatggagagactac ND 
282D2 BF352993  5′-gagaacagtaatttctttcc 5′-tgcaattattggggtaaagc ND ND ND − 
 BC040320  5′-gtcatacacgtggacctaac 5′-ctgaagtctcaagtgggcat ND ++ ++ ++ 
121J7 AF339828  5′-ctgacaagttctcagatcac 5′-actctgcatgagcctagatt ND ++ ++ ++ 
 AA701926  5′-agaccctgatggtctcttta 5′-ggctcaatgttttcctacgg ND ++ ++ ++ 
 BF908089  5′-tggaagaaaggacatgaggt 5′-tctcatgaatccatgcccaa ND 
 AW868481  5′-aagtaaatgtgagaagtagc 5′-tgctcatcctcattgtata ND − − 
 BX107378  5′-taacctgagcagaatccagccttg 5′-atggacccaaatgctgagaggaac ND ++ ++ ++ 
 AA599001  5′-aagagggacttgctgtgttg 5′-tgcacagacggtacagaagt ND ++ ++ 
 GPC5  5′-cactggcgggtaaaggggac 5′-agtattcagggaactgtcagtcacacc ND 
 AL043638  5′-ccagtctatcattgatggac 5′-gaagtgcctctgtaattgga ND ++ ++ ++ 
 AL708734  5′-gtaatcccagcactttggga 5′-tcttgttcttgtcccccagt ND 
487A2 AF339802 D13S1490 5′-tacctgggtaaccaagactc 5′-ctctgttcactgcattgaag ND ++ ++ ++ 
 H56919  5′-tgttgaccgactgagtgaac 5′-ttatggtgaagtccttcccc ND ++ 
 AA705439  5′-cgtactctagagttaaccaa 5′-atgattgtaagttccctgag ND ++ ++ ++ 
 W86832  5′-atcctcatttctcaggggct 5′-cctgtctgctctatgaagct ND − − − 
 N49442  5′-tggctgggcagaaatctgaa 5′-tacaggtctgttcgccacat ND ++ ++ ++ 
 N33596  5′-tccctagcaatgtgatgtac 5′-ctaaggtattcctaggctca ND − − 
 T84913  5′-gtagtaggtagaactgtcct 5′-atctaccctcggcaattttc ND ++ ++ ++ 
 BU656134  5′-tgctagggctggagtacaat 5′-cattttctcttggctcaccc ND ++ ++ ++ 
93M14 AW105449  5′-ccagcaactgtaatacatgc 5′-tcttcaaatccttgcctctg ND − ++ 
 AV754681  5′-acagccttctttggagagtg 5′-tccaagggcacagtggaatt ND − ++ ++ 
BACEST/geneSTS MarkerForward primerReverse primerFBRec1Karpas 1718OCI-Ly4
360A9 AW664738 D13S1457 5′-gccacatggtcaggttaaac 5′-cttcactaccttgcgactct − ND ND − 
27D9 AA309162  5′-ggctctcatttagctaaatg 5′-aagaagtggttgagacaaca +b 
15N8 No EST        
321E13 AW236754 D13S1175 5′-gagatggcacagcagttgaa 5′-tagttcaaactctacctgca − ND ND − 
447M23 No EST        
370B1 LOC121723  5′-aactactggtgaggactgca 5′-caagttccccttctgcagaa − ND ND − 
 AW059867  5′-aaggcttagctattatgctg 5′-acaatgaggaaaatctccca ++c ++ ++ ++ 
275J18 BC042969 D13S1818 5′-ctggtcacacatccacaatg 5′-cagtggaatctgagtcctag ++ ++ 
 AA628299  5′-cagaaggagtgttaagtctg 5′-caagaagaagctgccagtat ++ ++ ++ ++ 
143O10 No EST        
309H8 BG183515 D13S1239 5′-ctcatgactgtaatcccagc 5′-gtgatgccgtgaaatgagtc 
 BG191981  5′-ttcagtgacctcactgactg 5′-gaggattttgcagtcatcgc − ND ND − 
114G1 LOC160824 D13S767 5′-aggttttgtcagccacacct 5′-gaactatccgtaccttgtcc − ++ 
75N6 AA398228  5′-ctgtaccattgtgcccagaa 5′-atgactcagtccttctggct − ND ND − 
86C3 No EST D13S265       
51A2 No EST        
18M3 No EST        
388D4 LOC144774  5′-cactgtggcagttatacggt 5′-caatggttttccgcaccagt − ND ND − 
 LOC121727  5′-cctgggaaggatggtttctt 5′-ttacaacacaaggggcacac ++ 
409J23 No EST        
392A19 LOC121729  5′-ggagccattacttcaagacc 5′-agaccagtacttgtccagct ++ ++ ++ 
 BG776186  5′-tggacacgtgagtgtgtttc 5′-atgagctcaggcagctctat ++ − 
 LOC121728  5′-tgataggagacagacctgac 5′-tccagtcatcctgaggtaga − ND ND − 
 BG186078  5′-tcacacagtgtgactcacag 5′-tgatctcctctctgtagtgc ND − − 
 AA487882  5′-acccaagggaatattgacac 5′-gaagctagggaagcagtatt ND − − 
 BM542991  5′-tgcagtaggtggcaatctca 5′-tcgacttggactccatgaga − ND ND − 
505P2 BM695971 D13S1234 5′-ccttggagtgcttaaggtag 5′-actagggctctttgtagacc ND − − ++ 
158A8 AI027278  5′-gctgtgattgcgaagaagtg 5′-tccatatgtgagtgtggcag ND − − 
 BG927281  5′-acgccaagctctaatacgac 5′-ctcgggctatggtatatgac − ND ND − 
 BI825411  5′-accctggacaggtatggaat 5′-accatgagcacagtgctcaa − ND ND − 
 AA888411  5′-ctcccattgcagttactatg 5′-gtcgacatgttgttgaggtt 
360H15 AW515966  5′-atgttcaccaggctggtctt 5′-actaactctgtgggcttgca − ND ND − 
 BF818219  5′-tctgcccactaacatctggt 5′-cttgagtagctgagaccaca − ND ND − 
114C3 No EST        
432D3 LOC144776 D13S1190 5′-cacttccttgaaggggtttc 5′-ctctgactcttgggcacaat ND 
 AI126313  5′-ttgagacacagtcctgctct 5′-gcagggcacaatgttttcag − ND ND − 
319L6 AV731092  5′-attggtgaagccacctcaaa 5′-caggctaacatggatctagt ND − − 
 BG941714  5′-agtgcctacttcctaagacc 5′-tcaggaatcagtgcccaaac ND − − 
 AI262947  5′-ttgtgttcctggtccaccta 5′-acattgggccggtcacttat ND − − 
 AI493127  5′-tcaaatcaactgcacctcag 5′-cagttcgagacttcttccat − ND ND − 
51B13 BX097335  5′-gaagtaggatggtgacacct 5′-ctcagcaagtcatcctttgc − ND ND − 
430K10 AL701000 D13S886 5′-tggcctggagtaattagctg 5′-atttagggggtaggagagca ND − − 
 LOC160827  5′-ctgtgcactatcacttggga 5′-taggctctaagccgttggtt − ND ND − 
 LOC160826  5′-atggaatcaggttccctcca 5′-ctgttcccttcatctgaatg − ND ND − 
 BQ477330  5′-caaagtgctgggattacagg 5′-gccagctttgctgcacatta − ND ND − 
 BQ477741  5′-caacagaagatcggcccttt 5′-actccctgaagcacagcatt ND 
 AV731847  5′-caggcacttgcttaagggat 5′-aactagcctgcttcagcttc − ND ND − 
 BI481522  5′-atgtgaagagaggtctcagc 5′-agcaaaccacctagaggctt ND 
 BU729287  5′-cttagcctaatctccctaggcgga 5′-tatcaggtaggtggtccagtctca ND ++ ++ ++ 
 BM703078  5′-caggatcttgccctgttagt 5′-tatcgggtggcgaacaagat ND 
 AA719672  5′-tgtccttaggtagacattgt 5′-ttcaacctctgagaaaccca ND ND ND − 
 LOC121734  5′-acttcactgtcaacagcgag 5′-agagaccacatgcttgccat ND ++ ++ ++ 
 BE466687  5′-agttctggaggctaaaagtccagt 5′-gccaaaatcacatggagagactac ND 
282D2 BF352993  5′-gagaacagtaatttctttcc 5′-tgcaattattggggtaaagc ND ND ND − 
 BC040320  5′-gtcatacacgtggacctaac 5′-ctgaagtctcaagtgggcat ND ++ ++ ++ 
121J7 AF339828  5′-ctgacaagttctcagatcac 5′-actctgcatgagcctagatt ND ++ ++ ++ 
 AA701926  5′-agaccctgatggtctcttta 5′-ggctcaatgttttcctacgg ND ++ ++ ++ 
 BF908089  5′-tggaagaaaggacatgaggt 5′-tctcatgaatccatgcccaa ND 
 AW868481  5′-aagtaaatgtgagaagtagc 5′-tgctcatcctcattgtata ND − − 
 BX107378  5′-taacctgagcagaatccagccttg 5′-atggacccaaatgctgagaggaac ND ++ ++ ++ 
 AA599001  5′-aagagggacttgctgtgttg 5′-tgcacagacggtacagaagt ND ++ ++ 
 GPC5  5′-cactggcgggtaaaggggac 5′-agtattcagggaactgtcagtcacacc ND 
 AL043638  5′-ccagtctatcattgatggac 5′-gaagtgcctctgtaattgga ND ++ ++ ++ 
 AL708734  5′-gtaatcccagcactttggga 5′-tcttgttcttgtcccccagt ND 
487A2 AF339802 D13S1490 5′-tacctgggtaaccaagactc 5′-ctctgttcactgcattgaag ND ++ ++ ++ 
 H56919  5′-tgttgaccgactgagtgaac 5′-ttatggtgaagtccttcccc ND ++ 
 AA705439  5′-cgtactctagagttaaccaa 5′-atgattgtaagttccctgag ND ++ ++ ++ 
 W86832  5′-atcctcatttctcaggggct 5′-cctgtctgctctatgaagct ND − − − 
 N49442  5′-tggctgggcagaaatctgaa 5′-tacaggtctgttcgccacat ND ++ ++ ++ 
 N33596  5′-tccctagcaatgtgatgtac 5′-ctaaggtattcctaggctca ND − − 
 T84913  5′-gtagtaggtagaactgtcct 5′-atctaccctcggcaattttc ND ++ ++ ++ 
 BU656134  5′-tgctagggctggagtacaat 5′-cattttctcttggctcaccc ND ++ ++ ++ 
93M14 AW105449  5′-ccagcaactgtaatacatgc 5′-tcttcaaatccttgcctctg ND − ++ 
 AV754681  5′-acagccttctttggagagtg 5′-tccaagggcacagtggaatt ND − ++ ++ 
a

RT-PCR, reverse transcription-PCR; BAC, bacterial artificial chromosome; EST, expressed sequence tag; FB, fetal brain; ND, not done; STS, sequenced tagged site.

b

Detection of a thin band from the result of electrophoresis.

c

Detection of a thick band from the result of electrophoresis.

Table 2

Northern blot analysis

Each signal of those ESTsa and GPC-5 was visually evaluated after 1-week expose.

BACEST/geneProbe size (bp)Size (kb)Northern Blot
PlacentaRec1Karpas 1718OCI-Ly4JurkatATN-1
RP11-27D9 AA309162 130 − − − − − − − 
RP11-370B1 AW059867 160 − − − − − − − 
RP11-275J18 BC042969 440 − − − − − − − 
 AA628299 220 − − − − − − − 
RP11-309H8 BG183515 440 − − − − − − − 
RP11-114G1 LOC160824 550 6.5 +++ +++++ ++++ +++ +++ +++ 
RP11-388D4 LOC121727 420 − − − − − − − 
RP11-392A19 LOC121729 350 1.5 − − − − − 
RP11-158A8 AA888411 300 − − − − − − − 
RP11-432D3 LOC144776 500 − − − − − − − 
RP11-430K10 BQ477741 240 − − − − − − − 
 BI481522 210 5.5 ++ ++ ++ 
 BU729287 460  − − − − − 
 BM703078 470 − − − − − − − 
 LOC121734 240 0.8 +++ ++ ++ ++ +++ 
 BE466687 390 − − − − − − − 
RP11-282D2 BC040320 400 − +++++ +++++ +++++ − − 
RP11-121J7 AF339828 410 − +++++ +++++ +++++ +/− − 
 AA701926 240 − − − − − − − 
 BF908089 100 − − − − − − − 
 BX107378 420 − − − − − − − 
 AA599001 200 − − − − − − − 
 GPC5 600 
 AL043638 200  − − − − − − 
 AL708734 250 − − − − − − − 
RP11-487A2 AF339802 320 +/− ++ ++ ++ 
 H56919 160 − − − − − − − 
 AA705439 290 15 ++ +++ +/− +/− 
 N49442 320 15 ++ +++ +/− +/− 
 T84913 200 − − − − − − − 
 BU656134 390 − − − − − − − 
BACEST/geneProbe size (bp)Size (kb)Northern Blot
PlacentaRec1Karpas 1718OCI-Ly4JurkatATN-1
RP11-27D9 AA309162 130 − − − − − − − 
RP11-370B1 AW059867 160 − − − − − − − 
RP11-275J18 BC042969 440 − − − − − − − 
 AA628299 220 − − − − − − − 
RP11-309H8 BG183515 440 − − − − − − − 
RP11-114G1 LOC160824 550 6.5 +++ +++++ ++++ +++ +++ +++ 
RP11-388D4 LOC121727 420 − − − − − − − 
RP11-392A19 LOC121729 350 1.5 − − − − − 
RP11-158A8 AA888411 300 − − − − − − − 
RP11-432D3 LOC144776 500 − − − − − − − 
RP11-430K10 BQ477741 240 − − − − − − − 
 BI481522 210 5.5 ++ ++ ++ 
 BU729287 460  − − − − − 
 BM703078 470 − − − − − − − 
 LOC121734 240 0.8 +++ ++ ++ ++ +++ 
 BE466687 390 − − − − − − − 
RP11-282D2 BC040320 400 − +++++ +++++ +++++ − − 
RP11-121J7 AF339828 410 − +++++ +++++ +++++ +/− − 
 AA701926 240 − − − − − − − 
 BF908089 100 − − − − − − − 
 BX107378 420 − − − − − − − 
 AA599001 200 − − − − − − − 
 GPC5 600 
 AL043638 200  − − − − − − 
 AL708734 250 − − − − − − − 
RP11-487A2 AF339802 320 +/− ++ ++ ++ 
 H56919 160 − − − − − − − 
 AA705439 290 15 ++ +++ +/− +/− 
 N49442 320 15 ++ +++ +/− +/− 
 T84913 200 − − − − − − − 
 BU656134 390 − − − − − − − 
a

EST, expression sequence tag; BAC, bacterial artificial chromosome.

We thank Drs. Wen-Lin Kuo and Joe Gray at University of California at San Francisco (San Francisco, CA) for kindly sending us a detailed protocol of array CGH. The outstanding technical assistance of Hiroko Suzuki and Yumiko Kasugai is also very much appreciated. Encouragement and advice of Mitsuo Kawase at Nihon Insulator, Inc. for array CGH technology are also acknowledged.

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