Isolation and analysis of candidate myeloid tumor suppressor genes from a commonly deleted segment of 7q22

Genomics 85 (2005) 600 – 607 www.elsevier.com/locate/ygeno

Isolation and analysis of candidate myeloid tumor suppressor genes from a commonly deleted segment of 7q22 Nicole P. Curtissa,1, Jeannette M. Bonifasa,1, Jennifer O. Lauchlea,1, Jason D. Balkmana, Christian P. Kratza, Brooke M. Emerlinga, Eric D. Greenb, Michelle M. Le Beauc, Kevin M. Shannona,T a

Department of Pediatrics, University of California at San Francisco, 513 Parnassus Avenue, HSE 302, San Francisco, CA 94143, USA b Genome Technology Branch, National Human Genome Research Institute, Bethesda, MD 20892, USA c Section of Hematology/Oncology, Department of Medicine, and The Cancer Research Center, University of Chicago, Chicago, IL 60637, USA Received 17 August 2004; accepted 25 January 2005

Abstract Monosomy 7 and deletions of 7q are recurring leukemia-associated cytogenetic abnormalities that correlate with adverse outcomes in children and adults. We describe a 2.52-Mb genomic DNA contig that spans a commonly deleted segment of chromosome band 7q22 identified in myeloid malignancies. This interval currently includes 14 genes, 19 predicted genes, and 5 predicted pseudogenes. We have extensively characterized the FBXL13, NAPE-PLD, and SVH genes as candidate myeloid tumor suppressors. FBXL13 encodes a novel F-box protein, SVHis a member of a gene family that contains Armadillo-like repeats, and NAPE-PLD encodes a phospholipase D-type phosphodiesterase. Analysis of a panel of leukemia specimens with monosomy 7 did not reveal mutations in these or in the candidate genes LRRC17, PRO1598,and SRPK2. This fully sequenced and annotated contig provides a resource for candidate myeloid tumor suppressor gene discovery. D 2005 Elsevier Inc. All rights reserved. Keywords: Chromosome 7; Acute myeloid leukemia; Myelodysplastic syndrome; Monosomy 7; F-box proteins

Monosomy 7 and del(7q) are among the most common cytogenetic alterations found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [1–3]. These abnormalities occur in ~10% of de novo MDS and AML, in ~50% of patients with therapy-related leukemias [1], and in myeloid malignancies that arise in the context of constitutional predispositions such as Fanconi anemia, neurofibromatosis type 1, and severe congenital neutropenia. Importantly, monosomy 7 and del(7q) are strongly associated with adverse clinical features and with resistance to existing therapies. A commonly deleted segment (CDS) of chromosome band 7q22 was defined by cytogenetic investigation of 81

T Corresponding author. Fax: +1 415 502 5127. E-mail address: [email protected] (K.M. Shannon). 1 These authors contributed equally to this work. 0888-7543/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2005.01.013

patients with a malignant myeloid disorder characterized by a del(7q) [3]. Primary bone marrow cells from 15 of these cases were analyzed by fluorescence in situ hybridization (FISH) to identify the smallest commonly deleted interval. These studies delineated a region within 7q22 that was estimated to span 2–3 Mb of genomic DNA. The proximal boundary of the CDS was defined by 2 patients, whereas the distal boundary was defined by 3 patients [3]. We previously assembled a partial contig of this interval and excluded five candidate tumor suppressor genes (TSGs) by analyzing leukemia samples with monosomy 7 or a del(7q) for mutations [4]. A homolog of Drosophila trithorax called MLL5 has also been cloned from the CDS; however, molecular analysis did not reveal bsecond hit Q mutations in primary leukemias [5]. Here we describe a fully annotated genomic contig spanning the entire 7q22 CDS and the identification and analysis of additional candidate myeloid TSGs from this interval.

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Results Fig. 1 presents a tiling path comprising 28 overlapping bacterial and P1 artificial chromosome (BAC and PAC) clones spanning the 7q22 CDS, which is flanked distally by D7S1841 and proximally by D7S1503. Selected polymorphic loci are shown within the contig, which includes 2.52 Mb of nonredundant DNA sequence. Additional information about the BAC and PAC clones that span the CDS, including names, insert sizes, and GenBank accession numbers, is available at http://itsa.ucsf.edu/~kmslab/data/ data.html. Genome databases were queried to identify known and predicted genes within the CDS. Candidate sequences were evaluated further by designing oligonucleotide primers to amplify and clone a segment of each predicted open reading frame from human bone marrow cDNA. These amplified fragments were used to probe Northern blots prepared from hematopoietic tissues and leukemia cell lines to characterize candidate genes and to ascertain the size(s) of the transcript(s). We have identified 14 genes from the 7q22 CDS that are expressed in normal bone marrow, including 11 known genes (LRRC17 (P37NB), PMPCB, ZRF1, PSMC2, RELN, ORC5L, SRPK2, PRO1598, PRES, LHFPL3,and MLL5) and 1 pseudogene (S100A14) [4,5]. Three novel candidate tumor suppressor genes that showed strong cross-species conservation and/or contained putative functional domains were characterized in detail. These studies uncovered the FBXL13, NAPE-PLD, and SVH genes, one of which (SVH) was entered into the National Center for Biotechnology Information (NCBI) database by others [6]. FBXL13 encodes a novel F-box protein that is most similar to the F-box with leucine-rich repeats protein 2 (FBL2). F-box proteins with leucine-rich repeats mediate substrate-specific binding to ubiquitin ligase complexes, which target proteins to the proteasome for degradation [7]. The VHL TSG encodes an adapter component of a mammalian ubiquitin ligase complex that degrades hypoxia-inducible factor 1a [8], and hCdc4/Fbw7 is an F-box protein that has been implicated as a TSG [9,10]. Northern blot analysis demonstrated FBXL13 RNA message sizes of ~1.6 and ~2.3 kb, respectively (Fig. 2A). The first transcript (GenBank Accession No. AY359238) is 1568 bp in length, has 13 exons, and contains an F-box protein domain and 5 leucine-rich repeats with the (cystine-containing) subfamily. The second FBXL13isoform (GenBank Accession No. AY359239) is 2274 bp, has 19 exons, and contains an Fbox protein domain with 12 leucine-rich repeats (Fig. 2A). The FBXL13 locus spans 261 kb. Most of the gene lies within BAC clones CTA-318M5 and RP11-645N11 (GenBank Accession Nos. AC005250 and AC073127) (Fig. 1), but it partially extends into the flanking BAC RP4-802G15 (GenBank Accession No. AC006477). We also identified a partial sequence that showed homology with the hypothetical Caenorhabditis elegans

Fig. 1. Genomic organization of the 7q22 CDS. Left: The interval is flanked by the centromeric microsatellite marker D7S1503 and by the telomeric marker D7S1841. Additional loci within the region are shown. Center: The CDS includes 28 overlapping BAC and PAC clones. GenBank accession numbers are shown. Right: The locations and approximate sizes of the 14 known genes are presented, with the 5V–3Vorientation of each gene indicated by the direction of the arrows. Leukemia samples have been screened for mutations in all of these genes except PRES and LHFPL3, which are in progress.

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Fig. 2. Characterization of FBXL13, NAPE-PLD, and SVH. (A) Northern blots of RNA extracted from normal bone marrow and from the leukemia cell lines RCV-ACV-A and MV-1 were probed with a partial FBXL13 cDNA (top). (Bottom) The protein domain structure of FBXL13 showing leucine repeats (L) and the F-box domain. (B) Northern blot analysis of NAPE-PLD expression in hematopoietic tissues (top). (Bottom) Predicted protein structure showing the putative hydrolase domain. (C) Northern blot analysis of SVH expression in hematopoietic tissues (top). (Bottom) The SVH protein contains tandem Armadillo-like repeats, which are labeled bAQ.

protein Y37E11AR.3a and the mouse expressed sequence tag (EST) AI448472. Northern blot analysis revealed a message size of 5.2 kb in hematopoietic tissues (Fig. 2B), which was verified by amplifying the entire coding region from human bone marrow cDNA and sequencing the PCR products. The predicted open reading frame (GenBank Accession No. AY357337) consists of 1179 nucleotides with a long 3V untranslated region. The gene spans 49 kb within BAC clone RP11-401L13 (GenBank Accession No. AC007683) and includes seven exons (Fig. 1). Bioinformatic analysis of the putative protein sequence through the PFAM and BLASTp databases identified a predicted zincdependent hydrolase of the metallo-h-lactamase fold (Fig. 2B). In the absence of functional data, the Human Genome Organization initially designated this gene C7orf18 (for chromosome 7 open reading frame 18). After our GenBank submission, the name was changed to NAPE-PLDbased on a recent report that this gene encodes a novel phospholipase D that generates N-acylethanolamines [11]. SVH is related to the ALEX1 (Armadillo-like proteins lost in epithelial cancers on chromosome X), ALEX2,and ALEX3 genes. Based on their homology to proteins in the APC-h catenin signaling pathway and lack of expression in some human tumors and tumor-derived cell lines, it has been speculated that ALEX genes might function as TSGs [12]. Northern blot analysis of SVH revealed an ~2.6-kb message and a second ~1.4-kb transcript, both of which are highly expressed in fetal liver and in a range of hematopoietic tissues (Fig. 2C). Full-length cloning revealed four splice variants ranging in size from 2.3 to 2.6 kb. These data are in agreement with Huang et al. [6],

who also found that SVH is up-regulated in hepatocellular carcinomas (GenBank Accession Nos. AY150854, AY150853, AY150852, and AY150851). All of the splice variants contain an Armadillo-like repeat located within the N-terminal half of the protein (Fig. 2C). The SVH locus spans ~25 kb within BACs RP11-645N11, RP11-792M22, and RP11-401L13 (GenBank Accession Nos. AC073127, AC108167, and AC007683). We performed single-strand conformation polymorphism analysis (SSCP), denaturing high-pressure liquid chromatography (DHPLC), and quantitative real-time PCR analyses of primary leukemia samples and cell lines to evaluate FBXL13, NAPE-PLD, and SVH as candidate TSGs. This panel included 35 cryopreserved myeloid malignancies from adults and children with monosomy 7 or (del)7q. Criteria used to select specimens for mutational analysis included (1) the availability of multiple frozen vials, (2) a range of different diagnoses and patient ages, and (3) a high proportion of monosomy 7 detected by cytogenetic analysis. Fifteen patients were diagnosed with de novo AML, 3 had de novo MDS, 8 had therapy-related AML or MDS, and 6 children had myeloproliferative disorders that were classified as juvenile myelomonocytic leukemia or monosomy 7 syndrome. The other 3 patients had Philadelphia chromosome-positive acute lymphoblastic leukemia, Fanconi anemia that evolved to AML, or familial MDS with monosomy 7 (1 case of each). Cytogenetic analyses were performed on 24 adult specimens and 1 pediatric case at the University of Chicago. Unstimulated bone marrow demonstrated monosomy 7 in 100% of the metaphase cells of 12 patients and in 91–97%

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in 11 other cases. The other 2 specimens showed monosomy 7 in 70 and 64% of unstimulated bone marrow cells, respectively. Because myeloid malignancies with monosomy 7 are relatively rare in children, the pediatric cases were obtained from many centers. Institutional cytogenetic studies revealed a high proportion of monosomy 7 in most cases. Two cell lines with monosomy 7 were also analyzed for mutations in candidate TSGs. UoCM1, which was established from a 68-year-old patient with AML, contains a deletion of 7q as well as multiple additional chromosomal aberrations, including four copies of the MLL gene [13]. The MONO-7 cell line was developed from a pediatric patient with AML; the karyotype is 45, XY, –7, and these cells also have an NRAS mutation [14]. Analysis of leukemia samples did not reveal mutations in FLXL13, NAPE-PLD, or SVH but uncovered two nonfunctional polymorphic changes and two amino acid substitutions in FBXL13 that were found at similar frequencies in leukemia and control samples (Table 1). We also found no pathogenic mutations in three of the other candidate TSGs shown in Fig. 1 (LRRC17, PRO1598,and SRPK2); however, one silent polymorphism and one amino acid substitution were detected in the LRRC17 coding region that were equally frequent in normal and patient specimens (Table 1). Each leukemia that contained a polymorphism within FBXL13 or LRRC17 demonstrated a single allele, which is consistent with the high proportion of cytogenetically abnormal cells in these specimens. We developed real-time quantitative PCR assays to evaluate the possibility that FBXL13, NAPE-PLD, and/or SVH might be silenced by epigenetic mechanisms in myeloid leukemia as reported for TSGs in some human cancers [15]. The primer and probe sequences were designed to encompass segments that were common to the known FBXL13 and SVH isoforms. The DC T for FBXL13 mRNA expression in normal bone marrow was 10.41 F 0.5 relative to GAPDH,and the DC T for SVH was 4.75 F 0.53. For FBXL13, 11 of 14 monosomy 7 leukemias

Table 1 Characteristics of nucleotide polymorphisms Gene

Nucleotide and substitution

Amino acid substitution

Frequency

FBXL13

519, G to C

A to P

FBXL13

1072, G to C

G to A

FBXL13

1521-10inAC

N/A

FBXL13 LRRC17

1625, T to C 572, G to A

None None

LRRC17

636, A to G

L to E

5/42 Leukemias; normals 1/42 Leukemias; normals 1/42 Leukemias; normals 2/42 Leukemias 2/42 Leukemias; normals 2/42 Leukemias; normals

2/50 6/50 3/50

1/28 1/28

603

had expression levels that were similar to those of normal bone marrow, while 2 samples showed a N7-fold increase and 1 a 6-fold decrease. One of 14 monosomy 7 leukemia samples analyzed showed a 10-fold decrease in SVH expression relative to normal bone marrow and the other 13 expressed levels of SVH similar to those of normal marrow. The DC T for NAPE-PLD expression in normal bone marrow was 7.39 F 0.35 relative to GAPDH. Eleven of the 14 patient samples had an average 4.1-fold decrease in mRNA levels (range 3.1- to 5.1-fold). However, 2 other samples showed a 2-fold increase in expression levels of NAPE-PLD relative to normal bone marrow and 1 sample showed no change. Therefore, the difference between normal and patient bone marrow (2.69 F 1.5-fold decrease) did not achieve statistical significance across the entire population. The monosomy 7 specimens that expressed normal or increased NAPE-PLD mRNA levels were from a child with MDS and from adult patients with AML-M4 and t-MDS. There were no obvious differences in these patients that distinguished them from the 11 cases that showed reduced levels of NAPE-PLD expression. We also performed real-time quantitative PCR to compare the UoC-M1 and MONO-7 cell lines with normal bone marrow. In UoCM1 cells, NAPE-PLD and FBXL13 mRNA levels were reduced 1.5- and 2.7-fold, respectively, relative to normal bone marrow. SVH expression was 1.3-fold higher than in normal marrow. The MONO-7 cell line showed reduced expression of NAPE-PLD (7-fold), FBXL13(4.8-fold), and SVH (2.5-fold). We are working to identify additional candidate myeloid tumor suppressor genes from the 7q22 CDS. Queries of the NCBI, Celera Discovery Systems, and Chromosome 7 Annotation Project (TCAG; http://www.chr7.org) [16] databases uncovered 19 other predicted genes and 5 pseudogenes within the 7q22 CDS (see supplemental table). The number of unique genes is likely to be lower, as 6 genes overlap with other predicted genes and are transcribed from the same strand. Partial cDNA clones corresponding to GenBank Accession Nos. AK093485 and BC011396 have been cloned from normal bone marrow. UniGene clusters Hs.93379 and Hs.512629 show extensive homology to the EIF4B gene; however, we detected nonsense mutations in all three potential reading frames and could not amplify this transcript from normal bone marrow. Based on these studies, we conclude that this likely represents a pseudogene. BLAST queries using partial mRNA sequences corresponding to the putative genes AK093485 and LOC442340 within BAC RP11-401L13 uncovered homology with BAC clones located on 7p14–p15. The syntenic DNA domain on 7p14–p15, which includes multiple repetitive elements, has been partially delineated in the TCAG database. Interestingly, SVH and NAPE-PLD coding sequences are also contained within BAC RP11-401L13. However, BLAST queries using the full-length SVH and NAPE-PLD mRNA sequences as inputs did not reveal significant homology with 7p14–p15. Of 2762 unique ESTs

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within the 7q22 CDS, 399 are not homologous to any known or predicted genes. A list of the single ESTs located within the 7q22 CDS is available at http://itsa.ucsf.edu/ ~kmslab/data/data.html.

Discussion We have annotated a 2.52-Mb physical map spanning a commonly deleted segment identified in myeloid leukemia samples with proximal or distal breakpoints within 7q22 [3] and have exploited this resource to identify candidate myeloid TSGs. To date, full-length transcripts corresponding to 14 genes that are expressed in normal bone marrow have been isolated from this interval. Mutational analysis is complete for 12 of these and has not revealed bsecond hitQ mutations in leukemia specimens with monosomy 7 or del(7q) (this study and [4,5]). Screening is in progress for the other 2 genes in the interval (PRESand LHFPL3). SSCP and DHPLC are widely used to screen candidate disease genes for mutations; however, neither technique has 100% sensitivity. Furthermore, detecting pathologic mutation in tissues in which cancer cells are admixed with substantial numbers of normal cells may be problematic. However, we have shown that SSCP and DHPLC are robust techniques for detecting leukemia-associated mutations in bone marrow specimens [17,18] and we selected samples with a high proportion of cytogenetically aberrant cells for our analyses of candidate 7q22 TSGs. Based on these considerations, we believe that it is unlikely that any of the candidate 7q22 genes screened to date is mutated in a significant percentage of leukemias with monosomy 7 or a del(7q). Real-time quantitative PCR was performed to compare MLL5, FBXL13, SVH, and NAPE-PLD mRNA levels in normal and leukemic bone marrow. Of these genes, only NAPE-PLD showed reduced expression in most of the specimens with monosomy 7 or del(7q). The significance of this finding is uncertain, particularly since there are no functional data regarding the effects of NAPE-PLD on cell growth. Whereas the quantitative PCR experiments that we performed do not support epigenetic silencing of a candidate 7q22 TSG, the different cellular populations in normal, MDS, and AML bone marrow preclude any comment about modest or moderate changes in expression levels that might be functionally significant. Studies of purified populations such as CD34+ cells would be required to address this question. The 7q22 CDS that we have defined by performing cytogenetic and FISH analysis of primary bone marrow specimens with a del(7q) is in agreement with most published data (Fig. 3). Kere and co-workers [19,20] localized proximal 7q breakpoints in 4 cases of MDS/ AML associated with a del(7q) to the interval between EPO and PLANH1. PLANH1 is 1.7 Mb proximal to the CDS that we have defined. Fischer et al. [21] studied 21

Fig. 3. Commonly deleted intervals on 7q22 defined by different investigators. The relative positions of bcritical regionsQ defined by analyzing bone marrow specimens from patients with malignant myeloid disorders and a del(7q) are shown. As described in the text, a variety of techniques were employed by different research groups to define each interval. We have focused on the 2.52-Mb interval shown with the dotted line.

patients who developed myeloid leukemia associated with abnormalities of 7q. Their data implicated two nonoverlapping segments within 7q22–q31, a proximal region defined by 2 patients with chronic myelogenous leukemia (CML) and a more distal 20-Mb segment defined by patients with either MDS or AML. The CDS defined by us is contained within this 20-Mb region. Liang and colleagues [22] used 24 polymorphic markers from 7q22–q31 to examine DNA from 22 leukemias for loss of heterozygosity (LOH), including 15 with complete monosomy 7 and 7 others with a del(7q). Their study confirmed a high frequency of allele loss in the interval that we defined by FISH analysis. Of particular interest was a leukemia with apparent monosomy 7 that retained heterozygosity at the distal 7q22 locus D7S2545 but showed LOH at the more proximal loci D7S2446, D7S2504, and D7S2509, each of which maps within our CDS [4]. More recently, Tosi et al. [23] used FISH to investigate marrow samples from 17 patients with myeloid disorders who had simple 7q deletions or complex rearrangements associated with loss of genetic material from 7q. Consistent with other reports, 14 deletions had breakpoints within or proximal to 7q22. One patient with a complex karyotype had a t(7;7) that was associated with a deletion of approximately 150 kb spanning the CUTL1 locus in 7q22. This deletion maps distal to PLANH1 and overlaps with the proximal region defined by Fischer et al. [21] in 2 CML patients, but is centromeric to our CDS. Mutation analysis of these genes in leukemia samples has not yet been reported. Finally, Johnson et al. [24] reported a three-generation pedigree in which 2 individuals with

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MDS have a constitutional inversion. In this family, the breakpoint is closer to the centromere than the 7q22 CDS that we have defined and is within a region that is retained in many leukemias with a del(7q). In summary, while the existing data paint a complex picture of 7q deletions in myeloid malignancies, 7q22 is involved in a majority of cases and the 2.52-Mb CDS that we have interrogated is supported by data from other studies. We are cloning additional candidate TSGs from this interval to pursue the hypothesis that one of the remaining uncharacterized genes within the CDS undergoes homozygous inactivation in leukemia cells. An alternative possibility to the btwo hitQ model of recessive cancer genes is that haploinsufficiency for one or more genes located on 7q contributes to deregulated myeloid growth. If this is true, standard positional cloning strategies are highly problematic, as they ultimately rely upon demonstrating point mutations or deletions in the retained allele. Recent reports implicate haploinsufficiency for TP53, p27 Kip1 , Dmp1, and RUNX1/AML1 in the development of murine and human cancers [25–28]. Our analysis of leukemia samples with monosomy 7 or del(7q) has neither revealed pathologic mutations in any of the 12 candidate TSGs screened to date nor uncovered homozygous deletions or rearrangements in 8 of these candidate genes that were investigated by Southern blotting. Monosomy 7 and del(7q) are frequently detected in leukemias from patients with inherited predispositions that do not map to chromosome 7, in myeloid malignancies with somatic NRAS/KRAS2 mutations, and in leukemias with other cytogenetic abnormalities such as monosomy 5/del(5q) [1,2,29–31]. While plausible, there is no experimental evidence that loss of one allele of a gene (or genes) located within 7q22 cooperates with other genetic lesions in leukemogenesis. The bchromosome engineeringQ technology pioneered by Bradley and co-workers [32] provides a rational in vivo strategy for analyzing DNA segments that perturb cell growth by haploinsufficiency in mice. This approach, which has been used to create a model of DiGeorge syndrome [33], is theoretically appealing for deleting DNA segments thought to harbor TSGs [34]. The 7q22 CDS is syntenic with a continuous DNA segment located on mouse chromosome 5 [4]. Together, the strong association of monosomy 7 and del(7q) with previous exposure to alkylating agents and with certain inherited predispositions, the consistently poor responses of children and adults with monosomy 7/del(7q) to conventional therapies, and the discrete expression profile found in therapy-related leukemias with monosomy 7 [35] imply that loss of a gene (or genes) located on 7q plays a fundamental role in the biology of a significant subset of human myeloid malignancies. The data reported here should facilitate the goal of identifying the relevant myeloid TSG, which is essential for understanding how its encoded protein regulates hematopoietic growth.

605

Materials and methods Patient materials DNA was extracted from bone marrow or peripheral blood mononuclear cells collected from pediatric and adult patients with hematologic malignancies using standard methods. The experimental protocols involving human subjects were independently reviewed and approved by the Committees for the Protection of Human Subjects at the University of California at San Francisco and at the University of Chicago. Gene prediction and cloning procedure Predicted gene sequences from the NCBI (http:// www.ncbi.nlm.nih.gov) and Celera Discovery System (http://www.celeradiscoverysystem.com) provided a starting point for gene discovery from the 7q22 CDS. These sequences were used as input data for BLAST searches of the GenBank database to detect matching EST clones and cDNAs that uncovered partial cDNA sequences corresponding to the FBXL13, NAPE-PLD, and SVH genes. Primers were designed based on predicted gene sequences, related ESTs, and cDNA sequences using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_ www.cgi). Segments of the respective coding regions were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from a normal bone marrow RNA template and cloned using the TA cloning kit (Invitrogen, Carlsbad, CA, USA). The clones were then used as probes for Northern blot analysis to determine the transcript size and the pattern of tissue expression. The GRAIL program (http://compbio. ornl.gov/) was used to predict additional FBXL13, NAPEPLD, and SVH exons that were subsequently confirmed by performing RT-PCR. These approaches allowed us to assemble the entire coding sequence for each gene, which was confirmed by amplifying the full-length cDNAs, followed by sequencing. Full-length transcripts for the open reading frames of FBXL13, NAPE-PLD,and SVHwere amplified from normal bone marrow cDNA using the eLONGase Enzyme Mix (Invitrogen). The PCR products were sequenced using the UCSF Biomolecular Resource Center and sequences were assembled using the Macvector program. Bioinformatics Genomic sequences generated from BAC and PAC clones were used to identify ESTs within dbEST, as well as genes, mRNAs, or cDNAs from the nonredundant (nr) nucleotide database using BLASTn (http://www.ncbi.nlm. nih.gov/blast). Subsequently, expressed sequences with significant matches were mapped within the CDS using Macvector and BLASTn. The protein family (PFAM) database was used to perform protein domain analyses.

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For high-throughput discovery of Hidden Markov Model protein domains in ESTs and cDNAs, we used the GeneWise program for UNIX in conjunction with the PFAM database (http://www.ebi.ac.uk/Wise2/index.shtml). Intron/exon boundaries were assigned by comparing composite cDNA sequences to BAC/PAC genomic sequences using the Macvector and BLASTn programs. The GRAIL program (http://compbio.ornl.gov/) was used for exon prediction and the Primer3 program (http://www-genome. wi.mit.edu) aided in the design of new custom primers for cloning and mutation detection. Primer sequences are available upon request. Mutation screening We screened 35 primary leukemia samples and the UoCM1 and MONO-7 cell lines, which were derived from patients with monosomy 7 or del(7q) [13,14], for mutations in candidate genes. Seven leukemia samples with monosomy 5 or del(5q) were also investigated. The complete coding regions of FBXL13, NAPE-PLD, SVH, and other candidate genes were examined using a combination of SSCP and DHPLC. A detailed description of the SSCP procedure employed in our laboratory has been published [17]. For DHPLC analysis, exons were PCR amplified from patient samples in a volume of 25 Al containing nucleasefree distilled water, GeneAmp PCR Buffer containing 1.5 mM MgCl2 (PE Applied Biosystems, Foster City, CA, USA), final concentrations of 0.2 mM each dNTP, 0.2 AM forward and reverse primers, 0.625 units of AmpliTaq Gold (PE Applied Biosystems), and ~100 ng of each patient DNA sample. PCR thermal cycler conditions were 958C for 2 min followed by 35 cycles of 958C for 45 s, 578C for 45 s, and 728C for 1 min. Samples were analyzed in pairs due to chromosome 7 monosomy and were subjected to the postPCR reannealing conditions, suggested by the manufacturer, of 958C for 3 min, followed by a decreasing temperature walk of 18C per minute, starting at 958C and ending at 658C. The Stanford Melt program (http://insertion.stanford. edu/melt.html) was used to obtain an accurate melt profile and the most optimal temperature for the DHPLC analysis. DHPLC was performed using the Varian Helix System (Varian, Walnut Creek, CA, USA). Northern analysis cDNA cloned fragments from FBXL13, NAPE-PLD, and SVH were labeled and hybridized to blots containing total RNA from normal bone marrow and cell lines using PerfectHyb (Sigma Chemical Co., St Louis, MO, USA). mRNA expression in normal and leukemic cells FBXL13, NAPE-PLD, and SVH expression in bone marrow samples was assayed by quantitative real-time PCR. RNA was extracted from normal bone marrow cells

and leukemia specimens using either a Qiagen RNAeasy kit (Qiagen, Valencia, CA, USA) or TRIzol (Invitrogen), and cDNA was synthesized using the SUPERSCRIPT FirstStrand Synthesis System for RT-PCR (Invitrogen). Primer Express software (PE Applied Biosystems) was used to design primers and probes for TaqMan hybridization of FBXL13, NAPE-PLD, and SVH. These sequences are available at http://itsa.ucsf.edu/~kmslab/data/data.html. GAPDH expression was measured in each sample to ascertain cDNA quality and to establish a reference standard for making comparisons between cases. This gene was selected based on its use in previous studies that measured minimal residual disease levels in human leukemias [36]. Fold increase or decrease was calculated using the formula 2 DDCT. We purchased all reagents from PE Applied Biosystems. The samples were amplified in an ABI Prism 7700 sequence detection system at the following thermal cycle parameters: 508C for 2 min, 958C for 10 min, followed by 40 cycles of 958C for 15 s and 608C for 1 min.

Acknowledgments We are indebted to Hiroyuki Fujisaki (Osaka University) for providing the MONO-7 cell line and to Kimberly Shannon for technical support. This work was supported by PHS Grants CA40046 (M.M.L. and K.M.S) and CA72614 (K.M.S.), by the Jeffrey and Karen Peterson Family Foundation and the Frank A. Campini Foundation (K.M.S.), by NIH Training Grant T32ES07106 (J.O.L), and by a fellowship grant from the Dr. Mildred Scheel Stiftung fqr Krebsforschung (C.K.).

Appendix A. Supplementary data Supplementary data for this article may be found on ScienceDirect.

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