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Human Molecular Genetics Advance Access originally published online on July 5, 2007
Human Molecular Genetics 2007 16(18):2215-2225; doi:10.1093/hmg/ddm173
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Tiling resolution array comparative genomic hybridization, expression and methylation analyses of dup(1q) in Burkitt lymphomas and pediatric high hyperdiploid acute lymphoblastic leukemias reveal clustered near-centromeric breakpoints and overexpression of genes in 1q22-32.3

Josef Davidsson1,*, Anna Andersson1, Kajsa Paulsson1, Markus Heidenblad1, Margareth Isaksson1, Åke Borg2,4, Jesper Heldrup3, Mikael Behrendtz5, Ioannis Panagopoulos1, Thoas Fioretos1 and Bertil Johansson1

1 Department of Clinical Genetics, 2 Department of Oncology and 3 Department of Pediatrics, Lund University Hospital, Lund University, SE-221 85 Lund, Sweden, 4 Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Sweden and 5 Department of Pediatrics, Linköping University Hospital, SE-581 85 Linköping, Sweden

* To whom correspondence should be addressed. Tel: +46 46173398; Fax: +46 46131061; Email: josef.davidsson{at}med.lu.se

Received May 25, 2007; Accepted July 2, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although gain of 1q occurs in 25% of Burkitt lymphomas (BLs) and 10% of pediatric high hyperdiploid acute lymphoblastic leukemias (ALLs), little is known about the origin, molecular genetic characteristics and functional outcome of dup(1q) in these disorders. Ten dup(1q)-positive BLs/ALLs were investigated by tiling resolution (32k) array CGH analysis, which revealed that the proximal breakpoints in all cases were near-centromeric, in eight of them clustering within a 1.4 Mb segment in 1q12-21.1. The 1q distal breakpoints were heterogeneous, being more distal in the ALLs than in the BLs. The minimally gained segments in the ALLs and BLs were 57.4 Mb [dup(1)(q22q32.3)] and 35 Mb [dup(1)(q12q25.2)], respectively. Satellite II DNA on 1q was not hypomethylated, as ascertained by Southern blot analyses of 15 BLs/ALLs with and without gain of 1q, indicating that aberrant methylation was not involved in the origin of dup(1q), as previously suggested for other neoplasms with 1q rearrangements. Global gene expression analyses revealed that five genes in the minimally 57.4 Mb gained region—B4GALT3, DAP3, RGS16, TMEM183A and UCK2—were significantly overexpressed in dup(1q)-positive ALLs compared with high hyperdiploid ALLs without dup(1q). The DAP3 and UCK2 genes were among the most overexpressed genes in the BL case with gain of 1q investigated. The DAP3 protein has been reported to be highly expressed in invasive glioblastoma multiforme cells, whereas expression of the UCK2 protein has been correlated with sensitivity to anticancer drugs. However, involvement of these genes in dup(1q)-positive ALLs and BLs has previously not been reported.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gain of 1q, through duplication, isochromosome formation or unbalanced translocations, is one of the most frequent acquired cytogenetic abnormalities in human neoplasia (1). In solid tumors, dup(1q) is particularly common in hepatoblastoma (2), melanoma (3), Wilms' tumor (4) and carcinomas of the breast (5), liver (6), lung (7) and pancreas (8). As regards hematological malignancies, dup(1q) has been reported in all major diagnostic subgroups (1), although mainly in B-lineage acute lymphoblastic leukemia (ALL) (9), multiple myeloma (MM) (10) and B-cell non-Hodgkin lymphoma (NHL) (11). In these disorders, the incidence of dup(1q) varies significantly among the different cytogenetic subgroups, being quite strongly associated with t(11;14)(q13;q32) in MM, t(3;14)(q27;q32), t(8;14)(q24;q32) and t(14;18)(q32;q21) in NHL and with high hyperdiploidy (>50 chromosomes) in pediatric ALL (1).

Despite the high prevalence of 1q gain in neoplasia, next to nothing is known about its origin, molecular genetic characteristics and functional outcome. However, methylation studies of Wilms' tumors and carcinomas of the breast, liver and ovary have revealed an association between hypomethylation of the heterochromatic 1q region and the presence of dup(1q), and it has been suggested that this undermethylation is the cause of the 1q rearrangements (12–15). Whether this holds true in hematological malignancies has not been investigated. In MM, a number of studies, using chromosome banding, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), single nucleotide polymorphism (SNP) and expression analyses, have revealed that the proximal breakpoints (PBs) often are near-centromeric and that several genes on 1q are overexpressed (16–20). The breakpoints in NHL, mainly follicular and diffuse large B-cell lymphomas, have also been shown to localize close to the centromere, often involving the satellite II (sat II) domain (17,21,22).

Surprisingly few studies have focused on dup(1q) in Burkitt lymphomas (BLs) and high hyperdiploid pediatric ALLs, which harbor 1q gains in approximately 25 and 10% of the cases, respectively (1,9,23–27). As in MM and NHL, the PBs in the relatively small number of dup(1q)-positive BLs analyzed have been shown to involve either the sat II domain or to be almost centromeric (17,21). To the best of our knowledge, no ALLs have been investigated in detail in this respect; however, a recent FISH mapping of two high hyperdiploid ALLs with 1q gain revealed near-centromeric PBs (28). In the present study, the PBs as well as the distal breakpoints (DBs) were analyzed in dup(1q)-positive high hyperdiploid ALLs and BLs, using tiling resolution (32k) array CGH. In addition, the methylation pattern of sat II was investigated in ALLs and BLs with and without dup(1q) in order to ascertain whether undermethylation of the heterochromatic region is associated with 1q rearrangements in these disorders. Finally, gene expression analyses, using 27k cDNA microarrays, were performed to elucidate the functional outcome of 1q gains in BLs and ALLs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cytogenetic characterization of 1q gains
The literature review revealed that the vast majority of the 1q PBs in dup(1q)-positive BLs and pediatric high hyperdiploid ALLs map cytogenetically close to the centromere, in 1q11-21, whereas the DBs are more heterogeneous. Although the duplications are quite large, often involving most of chromosome arm 1q, a minimally gained segment (1q23-25) could be delineated in both the disorders (Figs 1 and 2).


Figure 1
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  		Figure 1. Gain of 1q in high hyperdiploid pediatric ALLs. (A) Cytogenetic imbalance map based on chromosome banding analyses of 48 cases reported in the literature (1) and unpublished cases from our department. (B) Representative example of array CGH 1q findings in ALL (case 1). (C) Frequency plot based on array CGH analysis of the 1q gains in cases 1–6.

 


Figure 2
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  		Figure 2. Gain of 1q in BLs. (A) Cytogenetic imbalance map based on G-banding analyses of 116 cases reported in the literature (1) and unpublished cases from our department. (B) Representative example of array CGH 1q findings in BL (case 8). (C) Frequency plot based on array CGH analysis of the 1q gains in cases 7–10.

 
Array CGH findings
The array CGH analyses confirmed, refined and, in some cases, revised the aberrations identified by chromosome banding (Table 1). In addition, a total of 54 previously undetected abnormalities were revealed by the array CGH in the 10 cases investigated; many of the changes were submicroscopic, whereas a few larger ones most likely had escaped detection because of poor chromosome morphology. Of the 54 additional aberrations, 20 changes were potentially pathogenetic, whereas 25 involved regions known to harbor copy number polymorphisms (CNPs) and nine involved the IGK@ and IGH@ loci (Table 1). Excluding numerical aberrations, CNPs and the IGK@ and IGH@ rearrangements, 23 different genomic imbalances were found by array CGH (Table 2). All 54 imbalances in the 10 cases are summarized as a heat map in Figure 3.


Figure 3
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  		Figure 3. Heat map of genomic imbalances detected by array CGH analyses of six high hyperdiploid ALLs (cases 1–6) and four BLs (cases 7–10) with dup(1q).

 


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  		Table 1. Clinical and cytogenetic features of the six high hyperdiploid ALLs and the four BLs with gain of 1q

 


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  		Table 2. Imbalances detected by array CGH in the 10 analyzed cases

 
The mapped 1q breakpoints and hence the sizes of the duplicated segments differed quite extensively between the chromosome banding and array CGH results (Table 1). Because of the often poor chromosome morphology in BLs/ALLs and the difficulty in delineating breakpoints cytogenetically, the findings from the array CGH analyses are most likely more accurate. As seen in Table 2, the 1q PBs in all 10 cases analyzed were near-centromeric, clustering within a 1.4 Mb segment at 1q12-21.1 (between chr 1:141.4 Mb and chr 1:142.8 Mb) in eight of the cases. The 1q DBs were more heterogeneous, generally being more distal in the ALLs than in the BLs. Although no clear-cut clustering was observed, three of the ALLs harbored DBs close to the telomere, at chr 1:245 Mb, whereas all four BLs had breakpoints between chr 1:176 Mb and chr 1:205 Mb (Table 2). The duplicated regions were hence larger in the ALLs (68.1–103.7 Mb) than in the BLs (35.4–63.6 Mb); the minimally gained segment in the ALLs was 57.4 Mb (chr 1:153.5–210.9 Mb), corresponding to dup(1)(q22q32.3), and 35 Mb (chr 1:141.8–176.8 Mb) in BLs, corresponding to dup(1)(q12q25.2) (Table 2; Figs 1 and 2).

Excluding CNPs and immunoglobulin rearrangements, additional cryptic, partial genomic imbalances were detected in two of the six ALLs, namely dup(4)(p16.2p16.3) and del(7)(q34) in case 1 and del(9)(q22.3q22.3) and del(12)(p12.1p13.2) in case 2 (Tables 1 and 2). In addition, the unbalanced 1;5 translocation in case 5 could be revised to der(5)t(1;5)(q22;q21.3) since the array CGH analysis revealed that the terminal deletion of 5q started in sub-band 5q21.3. Further FISH mapping of the 5q breakpoint, using the bacterial artificial chromosome probes RP11-541H04, RP11-505D04 and RP11-345I03 (BACPAC Resources, Oakland, CA, USA) revealed the presence of the first probe, located upstream of the EFNA5 gene, weak signal of the second probe, covering EFNA5 and loss of signal of the third probe, located downstream of the gene, strongly suggesting rearrangement of the EFNA5 gene (data not shown). Despite several attempts to amplify a putative EFNA5 fusion transcript using 5'-rapid amplification of cDNA ends (Clontech, Mountain View, CA, USA) no such chimera was obtained. The 7p breakpoint in the idic (7)(p11.2) in case 6, previously mapped in detail by locus-specific FISH (29), was confirmed by array CGH. Among the four BLs, cryptic imbalances were identified in two of them—dup(13)(q31.1q32.3) in case 8 and dup(X)(q28q28), dup(8)(q24.2q24.3), dup(14)(q32.3q32.3) and dup(18)(p11.2p11.3) in case 10. The presence of dup(8) and dup(14), involving regions in which MYC and some of the IGH genes map, in the latter case revealed that the cytogenetically balanced t(8;14) was unbalanced on the molecular genetic level. The add(17)(p13) in case 10 most likely represented a der(17)t(17;18)(p13.2;p11.2), considering the observed imbalances of these two chromosome arms. Unfortunately, lack of material precluded FISH confirmation.

Methylation status of sat II DNA
The methylation pattern of sat II DNA was investigated in a total of eight ALLs, at diagnosis and during remission; four of these ALLs harbored gain of 1q. Similarly, seven BLs, four of which with dup(1q), were also analyzed at diagnosis and during remission. Compared with a positive control for sat II hypomethylation and to normal peripheral blood DNA, the diagnostic and remission samples from all 15 cases studied showed a sat II methylation status similar to that observed in normal blood, and there were no clear-cut differences in intensities between the diagnostic and remission samples (Fig. 4). Hence, there was no evidence for sat II undermethylation in ALLs and BLs with or without 1q gains.


Figure 4
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  		Figure 4. Representative Southern blot analyses of sat II DNA methylation with BstBI, using DNA from ICF B-cells as a positive control for hypomethylation and DNA from normal peripheral blood as a reference and the sat II oligonucleotide probe. (A) Examples of high hyperdiploid ALLs, one with and one without dup(1q), at diagnosis (D) and at remission (R). (B) Similar examples of BLs.

 
Expression patterns of genes in dup(1q)
The cDNA microarray analyses of 29 high hyperdiploid ALLs revealed that a large number of genes, mapping in the minimally gained 1q region identified herein (1q22-32.3), were highly expressed, irrespective of the genomic 1q status. In order to find genes in this chromosomal segment that were overexpressed in the dup(1q)-positive ALLs only, a t-test was performed, identifying six genes to be differentially expressed (B4GALT3, CRB1, DAP3, RGS16, TMEM183A and UCK2). All but one (CRB1) was highly expressed in the ALLs with 1q gain. Two of the genes were among the top 15 most highly expressed genes in the single dup(1q)-positive BL case analyzed, namely DAP3 (rank 12) and UCK2 (rank 3). The expression findings are summarized in Figure 5.


Figure 5
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  		Figure 5. Expression profile of cDNA clones in the minimally gained 1q region in dup(1q)-positive high hyperdiploid ALLs. (A) Heat map of expressed cDNA clones in cases 4–6, with the location of the DAP3 and UCK2 genes, overexpressed also in a dup(1q)-positive BL, indicated. (B) Plot of cDNA clones in relation to Log2 ratios and genomic position on 1q. The DAP3 and UCK2 harboring clones are indicated in blue and red, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
As expected, the array CGH analyses identified several imbalances in regions known to harbor CNPs (30) as well as deletions involving the IGK@ and IGH@ genes, the latter probably representing somatic immunoglobulin rearrangements clonotypic for the malignant lymphoid cells (31); these changes most likely did not contribute to the pathogenesis of the malignant disorders. On the other hand, submicroscopic, putative pathogenetic aberrations were found in two of four BLs and two of six pediatric ALLs. Similar high frequencies of additional changes, using array CGH, have previously been reported in high hyperdiploid ALLs (27,32) and in BLs (33,34). As regards BLs, none of the cryptic changes identified so far have been recurrent and their molecular genetic consequences remain to be elucidated, as do their clinical impact. Including the present series, array CGH findings in 15 high hyperdiploid ALLs, 12 of which analyzed with the 32k array set, have now been reported (27,32). Among these, a total of 14 additional genomic imbalances have been observed in seven cases. To date, only one change has been recurrent, namely a deletion of 12p including, among other genes, ETV6. Interestingly, very recently an SNP array analysis of 39 high hyperdiploid ALLs revealed ETV6 deletions in two of the cases (35). Thus, cryptic hemizygous loss of this gene occurs in 5–10% of pediatric high hyperdiploid ALLs, suggesting that haploinsufficiency or loss of tumor suppressor function of ETV6 may play a role in this common childhood leukemia.

The 1q PBs mapped close to the centromere in all BLs and ALLs investigated (Table 2 and Figs 1Go3), a finding similar to what has been reported for other tumor types with 1q duplications (1). In the recent SNP array analysis of pediatric ALLs (35), approximately 30% of the high hyperdiploid cases were reported to harbor 1q gains. By using the publicly available dataset (http://ftp.stjude.org/pub/data/ALL-SNP1/) and the dchip software (36), the 1q PBs were extracted and shown to localize in the same region as the PBs in the present ALLs (data not shown). However, it should be noted that due to the repetitive nature of DNA sequences in this segment, the SNP density is lower than elsewhere in the genome, making accurate mapping difficult. This notwithstanding, the available data strongly indicate that the near-centromeric region of 1q is breakprone. The reason(s) for this is presently unknown, although it has been hypothesized that demethylation of the pericentric heterochromatin is associated with decondensation and subsequent instability of satellite sequences, resulting in 1q rearrangements (16), with several studies having reported undermethylation of sat II DNA in different neoplastic disorders (12–15). However, we found no evidence for hypomethylation of sat II in the present series of dup(1q)-positive BLs and ALLs (Fig. 4). Thus, we deem it highly unlikely that aberrant methylation of this region is the underlying mechanism behind dup(1q), at least not in BLs and ALLs. Instead, the genomic architecture as such, in particular segmental duplications, could be responsible. In fact, the presence of low-copy repeats has been implicated in the formation of both constitutional and neoplasia-associated chromosomal abnormalities (37–39). Interestingly, such duplications are very common immediately distal to highly repetitive satellite DNA close to the centromere, such as sat II (40), and recently (41–43), segmental duplications have been mapped to the 1q region in which the PBs occurred in our cases (Table 2). It is hence tempting to speculate that the dup(1q) in BLs and ALLs arises as a consequence of these rearrangement hotspots.

The 1q DBs were clearly more variable than the proximal ones, strongly indicating that there is not a common breakprone segment on distal 1q; in fact, none of the DBs (Table 2) are located in regions known to harbor such hotspots (42). Furthermore, the DBs also differed between the BLs and the ALLs, generally being more distal in the latter group (Table 2 and Figs 1Go3). Thus, the minimally gained segment was larger in the ALLs (57.4 Mb) than in the BLs (35 Mb), corresponding to dup(1)(q22q32.3) and dup(1)(q12q25.2), respectively. Interestingly, in a previous array CGH analysis of high hyperdiploid ALLs (27) one case, without a cytogenetically identified dup(1q), harbored a submicroscopic 0.6 Mb gain of 1q22, close to the minimally gained segment identified herein. It has been reported that tumors arising in nude mice inoculated with a human leukemic B-cell clone carrying dup(1)(q11q32) were more tumorigenic, grew faster and resulted in more metastases than did those occurring in mice inoculated with clones harboring other chromosomal abnormalities, suggesting that gain of this 1q segment provides a proliferative advantage (44). It is noteworthy that the minimally gained regions in both the BLs and the ALLs are included in that segment. Hence, one or several genes mapping to the minimally gained region are potentially pathogenetically important.

The microarray analyses revealed that six genes, namely B4GALT3, CRB1, DAP3, RGS16, TMEM183A and UCK2, were significantly differentially expressed in the dup(1q)-positive ALLs compared with the other high hyperdiploid cases. Surprisingly, one of these, the CRB1 gene, was underexpressed. Thus, gain of chromosomal material does not necessarily lead to overexpression of all the duplicated genes, as previously reported also in other tumor types (45,46). As regards the five up-regulated genes, two of them, i.e. DAP3 and UCK2 (Fig. 5), were among the most overexpressed genes in the BL case with gain of 1q. Interestingly, the DAP3 (death associated protein 3) protein, which, if intact, is normally proapoptotic, has been reported to be highly expressed in invasive glioblastoma multiforme cells (47), whereas expression of the UCK2 protein has been correlated with sensitivity to a few anticancer drugs, i.e. certain inhibitors of RNA polymerases (48). However, the leukemogenic impact of these genes in dup(1q)-positive high hyperdiploid ALLs and BLs remains to be elucidated.

In conclusion, the salient findings in the present study of dup(1q)-positive BLs and pediatric high hyperdiploid ALLs, using tiling resolution array CGH, methylation and expression analyses, were that the PBs clustered close to the centromere in both these disorders, the DBs were more heterogeneous, the minimally gained region was much larger in the ALLs than in the BLs, the sat II domain was not hypomethylated, several additional changes were observed and that five genes on 1q were significantly overexpressed in the dup(1q)-positive cases compared with ALLs without gain of 1q.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Patients
The array CGH study comprised six cases of B-cell precursor childhood high hyperdiploid ALL (five males, one female; age 2–11 years) and four cases of BL (two males, two females; age 7–45 years) (Table 1), all of which harbored 1q gains in the form of dup(1q) or unbalanced translocations involving 1q. Because of lack of material, FISH characterization of the 1q gains could only be performed in case 5. In the subsequent methylation investigations, diagnostic and remission DNA samples from cases 1–4 and 7–10 as well as from four additional high hyperdiploid ALLs and three BLs, without 1q gains, were analyzed. The gene expression patterns of cases 4–6 and 7 were extracted from a previously reported dataset (49).

Cytogenetic mapping of 1q gains
The Mitelman Database of Chromosome Aberrations in Cancer (1) was used to retrieve all cytogenetically characterized BLs and pediatric high hyperdiploid ALLs with 1q gains reported in the literature. In addition, unpublished cases from our department were included. In total, 116 BLs and 48 ALLs were identified. The PBs and DBs and the corresponding genomic imbalances were ascertained in all these cases.

Array CGH and gene expression analyses
DNA was extracted from bone marrow or peripheral blood cells obtained at the time of diagnosis and stored at –80°C. Male genomic DNA (Promega, Madison, WI, USA) was used as reference in all hybridizations. The 32k slides, containing 32 433 tiling bacterial artificial chromosome clones covering at least 98% of the human genome, were produced at the SWEGENE DNA microarray resource center at Lund University, Sweden. Labeling of DNA, slide preparation and hybridization were performed as described with minor modifications (50). Analyses of the microarray images were performed with the GenePix Pro 4.0 software (Axon Instruments, Foster City, CA, USA). For each spot, the median pixel intensity minus the median local background for both dyes was used to obtain the ratio of test gene copy number to reference gene copy number. Data normalization was performed for each array subgrid using lowess curve fitting with a smoothing factor of 0.33 (51). For all cases, the sex chromosomes and, for the high hyperdiploid ALLs, also the tri- and tetrasomic chromosomes were excluded when calculating the correction factor in the normalization. All analyses were performed in the Bioarray software environment (BASE) database (52). To identify imbalances, the MATLAB toolbox CGH plotter (53), the SeeGH software (54) and the TM4 software suite (55) were applied using moving mean average over three clones and log2 limits of >0.2. Classification as gain or loss was based on identification as such by the CGH plotter and also by visual inspection of the log2 ratios. Ratios >0.5 in five adjacent clones were classified as aberrant, with ratios 0.5–1.0 interpreted as duplications/hemizygous deletions and >1.0 as amplifications/homozygous deletions.

The cDNA microarray analyses have previously been reported (49). In short, samples from 87 pediatric B-lineage ALLs, including 29 high hyperdiploid ALLs and two BLs, were hybridized to 27k microarray slides containing 25 648 cDNA clones (Swegene DNA Microarray Resource Center) representing 13 737 Unigene clusters and 11 592 Entrez gene entries, according to the Unigene build 195. RNA extraction, amplification, labeling, hybridization, scanning, post-hybridization washing and feature analysis were performed as described (49). The data analyses were all performed in BASE and in the CGH-Explorer software, and when investigating chromosome-specific expression patterns, genes were mean-centered and analyzed with a BASE implementation of the MATLAB toolbox CGH-plotter (53,56). Three of the six high hyperdiploid ALLs (cases 4–6) and one of the four BLs (case 7) with dup(1q) in the present study were included in the dataset. To identify differentially expressed genes in the dup(1q)-positive ALLs, a t-test comparing high hyperdiploid ALLs with and without gain of 1q was performed on the expression patterns of genes in the minimally gained 1q region. P-values <0.05 were considered significant.

Methylation analysis
Sat II methylation status was investigated by Southern blot analysis as described (12). Two micrograms of DNA were digested with 20 U of the CpG methyl-sensitive restriction enzyme BstBI (New England BioLabs, Ipswich, MA, USA) for 18 h in standard conditions. The digested and fractionated DNA was blotted on a GeneScreen Plus hybridization transfer membrane (Perkin-Elmer, Waltham, MA, USA). The sat II probes used were an 18-mer single-stranded oligonucleotide, with the consensus sequence 5'-TCGAGTCCATTCGATGAT-3' (57), and a cloned Chr1-specific insert excised from the recombinant plasmid pUC1.77 (58). Hybridization was performed in Rapid-Hyb buffer (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, USA) at 42°C for the oligonucleotide probe and at 65°C for the cloned DNA probe. The oligonucleotide probe was radiolabeled with 5'-[{gamma}32P]-end dATP and the cloned Chr1-specific insert with {alpha}32P-dCTP (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, USA). In each Southern blot analysis, normal peripheral blood and ICF B-lymphocyte (59) (Corriell Cell Repositories, Camden, NJ, USA) DNA was included as methylated and hypomethylated controls, respectively. Using a FLA 3000 phosphoimager (Fujifilm Corporation, Tokyo, Japan), the approximate amount of methylation was assessed by inspection, comparing the intensities of the hybridized fragments.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Cancer Society, the Swedish Children's Cancer Foundation, the Swedish Research Council and the Knut and Alice Wallenberg Foundation via the SWEGENE program.

Conflict of Interest statement. None declared.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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K. Paulsson, J.-B. Cazier, F. MacDougall, J. Stevens, I. Stasevich, N. Vrcelj, T. Chaplin, D. M. Lillington, T. A. Lister, and B. D. Young
Microdeletions are a general feature of adult and adolescent acute lymphoblastic leukemia: Unexpected similarities with pediatric disease
PNAS, May 6, 2008; 105(18): 6708 - 6713.
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