Open Access

Evidences showing wide presence of small genomic aberrations in chronic lymphocytic leukemia

  • Yeong C Kim1,
  • Yong-Chul Jung2, 6,
  • Jun Chen2, 7,
  • Ali H Alhasan3, 8,
  • Parawee Kaewsaard3, 9,
  • Yanming Zhang4,
  • Shuo Ma5,
  • Steve Rosen5 and
  • San Ming Wang1, 3Email author
Contributed equally
BMC Research Notes20103:341

DOI: 10.1186/1756-0500-3-341

Received: 23 August 2010

Accepted: 20 December 2010

Published: 20 December 2010

Abstract

Background

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western population. Although genetic factors are considered to contribute to CLL etiology, at present genomic aberrations identified in CLL are limited compared with those identified in other types of leukemia, which raises the question of the degree of genetic influence on CLL. We performed a high-resolution genome scanning study to address this issue.

Findings

Using the restriction paired-end-based Ditag Genome Scanning technique, we analyzed three primary CLL samples at a kilobase resolution, and further validated the results in eight primary CLL samples including the two used for ditag collection. From 51,632 paired-end tags commonly detected in the three CLL samples representing 5% of the HindIII restriction fragments in the genomes, we identified 230 paired-end tags that were present in all three CLL genomes but not in multiple normal human genome reference sequences. Mapping the full-length sequences of the fragments detected by these unmapped tags in seven additional CLL samples confirmed that these are the genomic aberrations caused by small insertions and deletions, and base changes spreading across coding and non-coding regions.

Conclusions

Our study identified hundreds of loci with insertion, deletion, base change, and restriction site polymorphism present in both coding and non-coding regions in CLL genomes, indicating the wide presence of small genomic aberrations in chronic lymphocytic leukemia. Our study supports the use of a whole genome sequencing approach for comprehensively decoding the CLL genome for better understanding of the genetic defects in CLL.

Findings

CLL (Chronic lymphocytic leukemia) is an incurable disease mainly affecting the B cell lineage in the western population, with a median age of diagnosis of 72 year old [1]. Determining the cause of CLL is crucial for understanding the acquisition and for clinical diagnosis, treatment and prognosis of CLL. Genetic factors have been linked to the etiology of CLL. Cytogenetic analyses identified chromosomal abnormalities including del11q23 affecting the ATM gene, tri12, del 13q14, and del17p13 affecting TP53 gene [2]. In addition, CGH studies found gains and losses in Xp11.2-p21 and Xq21-qter [3]. Molecular studies identified three genes: IgVH, CD38 and ZAP-70 that correlate with CLL prognosis [46]. A CLL-specific microRNA signature was also identified, suggesting that microRNA deletion could be involved in CLL [7]. SNP array studies identified 2q21.2, 6p22.1 and 18q21.1 abnormalities that follow a Mendelian inheritance pattern [8]. Whole genome association studies also identified multiple loci at 2q37.3, 8q24.21, 15q21.3 and 16q24.1 that appear to be associated with genetic susceptibility to CLL [9].

Although evidence supports the involvement of genetic factors in CLL, the frequency of genomic aberrations identified in CLL is relatively lower than those observed in the leukemias affecting other types of hematopoietic lineages [10]. This information suggests that the CLL genome is relatively intact with fewer aberrations than other types of leukemia. Alternatively, more genomic aberrations may exist in CLL but these could mainly be small lesions in the CLL genome that are difficult to detect using conventional technologies due to their limited resolution. With the rapid progress of genome sequencing technologies, enthusiasm is increasing for pursuing comprehensive detection of genomic aberrations in cancer by sequencing cancer genomes. In the case of CLL, a critical issue is to know the degree of genomic aberrations in order to justify the use of whole genome sequencing approach to analyze CLL genome. We reasoned if we can scan certain CLL genomes at sufficient high resolution and at reasonable genome coverage, we should gain first-hand information to estimate the degree of genomic aberrations in CLL.

We recently developed the DGS (Ditag Genome Scanning) technique that uses next-generation DNA sequencing technologies to collect paired-end sequences from restriction DNA fragments across a genome [11]. Using this technique, we analyzed CLL genomes. Nine samples of peripheral blood from untreated CLL patients diagnosed in Northwestern University Lurie Cancer Center and University of Chicago Medical Center were used in this study, of which three were used for paired-end tag collection, and eight including two used in paired-end tag collection were used for full-length sequencing analysis (Additional file 1: Supplemental Table S1). Informed consent was made by the patients, and the use of clinical CLL samples was approved by the institutional review board of University of Chicago and Northwestern University following institutional guidelines. The detailed experimental process followed the published protocol [11] and outlined in Figure 1. Briefly, mononuclear cells were isolated from each CLL peripheral blood or bone marrow sample by using NycoPrep™ A solution (Axis-Shield). Human genomic DNA was extracted from mononuclear cells by using QIAamp DNA Blood Kit (QIAGEN) following the manufacturer's protocol. To generate the DGS library, genomic DNA was fractionated by HindIII restriction digestion. The restriction fragments were dephosphorylated by CIP and cloned into pDGS-HindIII vector that contains two MmeI sites next to the HindIII cloning site. The genomic library was digested by MmeI to release two tags from the cloned DNA fragments. The tag-vector-tag fragments were then gel-purified, and re-ligated to form a ditag library. Ditags were released from the vectors by HindIII digestion, gel-purified, and concatemerized by using T4 DNA ligase (Promega). The concatemers at 200 to 500 bps were agarose-gel-purified and used for ditag sequencing by using a 454 GS20 sequencer (454 Life Sciences). Ditags were extracted from the resulting sequences based on the HindIII sites. Same ditags were combined to generate a unique ditag with the corresponding copy numbers.
https://static-content.springer.com/image/art%3A10.1186%2F1756-0500-3-341/MediaObjects/13104_2010_Article_745_Fig1_HTML.jpg
Figure 1

Outline of the experimental process. Genomic DNA samples were digested by restriction enzymes. Ditags (paired-end tags) were collected from both ends of restriction fragments and sequenced. The ditag sequences were compared to known human reference genome sequences. The unmapped ditags were used as sense and antisense PCR primers to amplify their original DNA fragments to generate full-length sequences. The sequences were mapped to reference genome sequences to determine the type of genomic aberrations.

To generate the reference ditag database, virtual HindIII restriction fragments were generated from known human genomic sequences. Two 16-bp virtual tags were extracted from the 5' and the 3' ends of each virtual fragment, and connected to form a reference ditag representing the virtual DNA fragment. The following sequences were used to extract the reference ditags:

1. Human genome reference sequences (hg18): http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/

2. Human dbSNP 126: ftp://ftp.ncbi.nih.gov/snp/organisms/human_9606

3. Chimpanzee genome reference sequences (PanTro2): http://hgdownload.cse.ucsc.edu/goldenPath/panTro2/bigZips/

4. Human GM15510 fosmid paired-end sequences:

http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?&cmd=retrieve&val=CENTER_PROJECT%20%3D%20%22G248%22&size=0&retrieve=Submit

5. Celera human genome sequences: http://www.ncbi.nlm.nih.gov/genomeprj/1431

6. Venter genome sequences: ftp://ftp.ncbi.nih.gov/pub/TraceDB/Personal_Genomics/Venter/

7. Watson genome sequences: ftp://ftp.ncbi.nih.gov/pub/TraceDB/Personal_Genomics/Watson/

8. Reference ditags were also extracted from HindIII fragments of E.coli K12 genome sequences to eliminate the ditags from E. coli DNA contaminated during library construction process.

Initial ditag mapping was performed with perfect match between experimental ditags and hg18 reference ditags. For the unmapped experimental ditags, a single-base mismatch in each single tag of the ditag was allowed to compensate for possible sequencing error or SNP. To identify the unmapped ditags related with homopolymer generated by 454 sequencing chemistry, the unmapped ditags with more than two homo-bases were stretched, e.g. AAA -> AAAA, or shortened, e.g. AAA -> AA, and mapped to reference ditags again. For the ditags remaining unmapped, they were mapped to the reference ditags of other sequence sources in the ditag reference database. The ditags remaining unmapped after these processes were defined as the unmapped ditags.

Unmapped ditag sequences were used to design sense primers and antisense (reverse/complementary) primers, with four extra bases CAGC added to the 5' end of sense primer and CGCC to the 5' end of antisense primer. Genomic DNA digested by HindIII was used as the templates for PCR amplification. PCR was performed with 35 cycles at 95°C 30 sec, 57°C 60 sec, and 72°C 3 min, followed by extension at 72°C for 10 min. The amplified products in each reaction were cloned into pGEM-T vector (Promega), transformed into E. coli TOP10 (Invitrogen), and plated in a single well of the 48-well Qtrays (Genetix). Four clones from each transformation were amplified by colony-PCR using M13F and M13R primers, and sequenced by Big-Dye Terminator v3.1 Cycle Sequencing Kit (ABI) using M13F primer. For the sequences that did not reach the full-length, second sequencing reactions were performed using M13R primer. To determine the genomic aberrations, each full-length sequence was mapped to hg18 using BLAT at a minimum of 90% identity as the cut-off.

The paired-end ditags were collected from three CLL samples. Genomic DNA from each sample was fractionated by HindIII digestion, which provides 3,561-bp resolution on average across the genome based on hg18 sequences [11]. Unique paired-end ditags of 272,193, 320,283, and 307,547 was collected from each CLL sample, covering 32%, 34% and 38% HindIII fragments in each CLL genome respectively. Comparing the three ditag sets shows that between 87,968 and 108,579 ditags are present between two CLL samples, and 51,632 ditags are commonly present in all three CLL samples (Table 1A). The ditags present only in individual CLL sample could be the ditags representing individual genomic differences, the ditags potentially originating from experimental artifacts, or ditags detected in one but not in others due to unsaturated ditag collection in each CLL under the sequencing scale. The 51,632 ditags detected in all three CLL samples cover 5% of genomic DNA fragments commonly detected in the three CLL genomes. In order to provide high confidence for further downstream studies, we focused on the 51,632 common ditags for further mapping analysis. We compared the 51,632 common ditags with multiple known human genome sequences, including the human genome reference sequence hg18, human SNP, human GM15510 genome sequences, chimpanzee genome sequences that are highly homologous to the humans, Watson genome sequences, and Venter genome sequences. Of the 51,632 ditags used for the mapping, 98.3% (50,799) map to hg18 that represent normal genomic fragments in the CLL genomes, 0.4% (230) are unmapped ditags that represent potential genomic aberrations commonly present in all three CLL genomes, and the remaining ditags map to other genomes that represent normal genome variations (Table 1B).
Table 1

Paired-end tags collected from three CLL samples

A. Ditag distribution in three CLL samples

 

CLL sample (%)

 

1

2

3

Total sequence reads

231,941

321,290

268,124

Total ditags

623,539

859,836

700,991

Unique ditag

272,188 (100)

320,278 (100)

307,542 (100)

Ditags common in two

99,815

99,815

 
 

87,968

 

87,968

  

108,579

108,579

Ditags common in three

51,632 (19)

51,632 (16)

51,632 (17)

B. Mapping ditags to reference human genome sequences

 

Mapped (%)

 

Unmapped (%)

Total common ditags

51,632 (100)

  

HG18

50,799 (98.3)

 

833 (1.7)

Homopolymer

22

 

816

Chimpanzee genome

195

 

616

Other human Genomes

386*

 

230

   GM15510 genome

28

  

   Celera genome

165

  

   Venter genome

352

  

   Watson genome

61

  

Total unmapped

  

230 (0.4)

*These mapped to multiple genomes was counted only once.

To determine the types of genomic aberrations for the unmapped ditags, we generated full-length sequence for the restriction DNA fragment detected by the unmapped ditags by using the "ditag-PCR" method, in which the ditag sequences were used as PCR sense and antisense primers to amplify the original DNA fragment that derived the unmapped ditag. We performed 192 reactions in eight CLL samples including two used in ditag collection and six additional CLL samples. Under the conditions that a full-length sequence must be longer than 50 bases and detected at least in the CLL used in ditag collection or at least in two additional CLL samples, 220 full-length sequences were generated from 100 unmapped ditags. Mapping the full-length sequences to hg18 identified different types of genomic aberrations caused by insertion, deletion and base change. Many of these aberrations created new HindIII restriction site that leads to the release of unmapped ditag, or the change of ditag sequence composition that prevents ditag mapping. These aberrations were observed in both coding and non-coding regions in CLL genome. For example, aberrations were detected in exons of NEK8, RUNX1 and MUC2 genes, and introns of 20 other genes (Table 2A, Additional file 2: Supplementary table S2). NEK8 encodes a member of the serine/threonine protein kinase family, which plays a role in cell cycle progression from G2 to M phase and is over-expressed in breast cancer [12]. A 353-base sequence converted from the unmapped ditag AAGCTTACCCTCTGGACGCCTGTATGAAGCTT maps to the last exon (Exon 15) coding for the 3' UTR of NEK8. Two HindIII restriction sites were inserted in the sequence that are not present in the wild-type NEK8 gene. RUNX1 is a gene involved in AML through its involvement in the t(8;21) [13]. A 434-base full-length sequence from a ditag AAGCTTCGGCCTATAG/ACAACCTAACAAGCTT was detected in all eight CLL samples, and maps to intron 3 and exon 4 of RUNX1. Analyzing the mapped region shows a T to C single-base change between the sequence and exon 4 of RUNX1 gene. Searching dbSNP reveals that this is a SNP (rs1235270). Due to the uncertainty of RUNX1 protein coding sequence itself, it is not certain if this germline SNP causes a coding amino acid change. Several bases are also changed in the mapped intron 3 of RUNX1 gene. These base changes raise an interesting question whether RUNX1 could be involved in CLL. MUC2 is a member of the MUCIN family, which codes for high molecular weight glycoproteins. The abnormalities of MUC2 is linked with colorectal and pancreas cancer [14]. A 410-base sequence derived from an unmapped ditag AAGCTTCCGGTCGGCTTCGCAGTAGAAAGCTT covers intron 29, exon 30 and intron 30 of MUC2 gene. This sequence also contains two HindIII restriction sites AAGCTT inserted at both its ends that do not exist in the wild-type MUC2 gene. Only three aberrations were detected in the exon of three known genes. This could be attributed to the limited genome coverage of the study and the low percentage of the exon-coding sequences in the genome. With increased genome coverage, it would be possible to identify the aberrations affecting more exons.
Table 2

Aberrations in exon and intron, and only present in CLL

Ditag

Full-length (bp)*

CLL Sample**

Chr.

Location

Aberration

Gene

  

1

2

3

4

5

6

7

8

   

Exon

Intron

A. Aberrations affecting exon and intron

             

AAGCTTACCCTCTGGACGCCTGTATGAAGCTT

353

+

-

-

-

-

-

-

-

17q11.2

24093147-24093483

Insertion

NEK8

 
 

268

+

+

-

-

-

-

-

-

19p13.2

10331021-10331266

Insertion

 

TYK2

AAGCTTCGGCCTATAGACAACCTAACAAGCTT

434

+

+

+

+

+

+

+

-

21q22.12

35949034-35949460

Base change

RUNX1

 

AAGCTTCCGGTCGGCTTCGCAGTAGAAAGCTT

410

-

+

-

+

-

-

-

-

11p15.5

1084558-1084956

Insertion

MUC2

MUC2

AAGCTTGAGGGTGGAGTTCCTTCTGTAAGCTT

181

-

+

+

-

-

-

-

-

2p11.2

87573183-87573358

Base change, insertion

 

BC070201

AAGCTTGGCCAGAGACAGGCATCATGAAGCTT

116

+

-

-

-

-

-

-

-

10q22.1

73213666-73213767

Base change

 

CDH23

AAGCTTGTGGACCACCGCTGTGAGTCAAGCTT

300

+

+

+

+

+

-

+

+

20p12.3

6033711-6034006

Base change

 

FERMT1

AAGCTTCATATGAGGATCAAAAACGAAAGCTT

283

+

+

-

+

+

+

-

-

3p14.2

61015245-61015527

Base change

 

FHIT

AAGCTTCTTTGTGATGCTCAGACATGAAGCTT

332

-

+

-

-

-

+

-

-

11q23.3

120263755-120264067

Base change

 

GRIK4

AAGCTTAGATCAGTGAGCCTACGGCGAAGCTT

605

+

+

+

+

+

+

-

+

16q22.2

69601759-69602345

Base change

 

HYDIN

AAGCTTCGCCGTGGGCTCACTGATCTAAGCTT

614

+

+

+

-

+

+

+

+

16q22.2

69601760-69602350

Base change, insertion

 

HYDIN

AAGCTTGCTGAACGCACCTGCGTGGAAAGCTT

448

+

-

+

-

-

-

+

+

5q22.2

112671099-112671541

Base change, insertion

 

MCC

AAGCTTGCTTCTTTGCTGATACTGGCAAGCTT

582

-

+

+

-

-

+

-

-

12q24.31

123563471-123564048

Base change, insertion

 

NCOR2

AAGCTTGGCGTCAATCCACACCAAAGAAGCTT

349

-

-

-

-

+

+

-

-

5q33.1

149870225-149870557

Insertion

 

NDST1

 

1306

-

-

-

+

-

+

-

-

8q22.2

99587880-99589168

Base change, insertion

 

STK3

AAGCTTGAAATAAGTGCTGCATCCTGAAGCTT

163

-

+

+

-

+

-

-

-

2q32.1

182858618-182858771

Base change, insertion

 

PDE1A

AAGCTTTCCTAGGGAGCTGGGTGGTGAAGCTT

638

-

-

-

+

+

-

-

-

17q25.1

68849242-68849864

Insertion

 

SDK2

 

211

+

-

-

-

-

-

-

-

9q34.11

129692280-129692481

Base change

 

ST6GALNAC6

AAGCTTGCAGAAGGGGAGCCAGGGTGAAGCTT

312

-

-

+

-

-

-

+

-

2p25.3

3382092-3382390

Insertion

 

TTC15

 

187

+

-

-

-

-

+

+

-

13q12.13

26098258-26098437

Base change, insertion

 

WASF3

AAGCTTCAGGAAAGTCCACTAGCAAAAAGCTT

197

+

+

+

+

+

+

+

-

9p13.3

33972115-33972294

Insertion

 

UBAP2

 

112

+

-

-

-

-

-

-

-

9p13.3

33972115-33972294

Insertion

 

UBAP2

 

170

+

-

-

-

-

-

-

-

9p13.3

33972115-33972294

Insertion

 

UBAP2

AAGCTTTAATGACTGAGGGGTTCTCAAAGCTT

1147

+

+

+

-

+

-

+

-

6q25.3

157931989-157933131

Base change

 

ZDHHC14

AAGCTTTGAGAACCCCTCAGTCATTAAAGCTT

1134

+

+

-

-

-

-

+

-

6q25.3

157931989-157933131

Base change

 

ZDHHC14

AAGCTTGCACAAGGGGCCCCTTGTGCAAGCTT

691

+

-

-

-

-

-

-

-

4p16.3

2372796-2373473

Insertion

 

ZFYVE28

B. Sequences only present in CLL genome

             

AAGCTTGATATCGTGATCACCTTAAGAAGCTT

332

-

+

+

-

-

-

-

-

-

-

-

-

-

AAGCTTAGATAGAGCGCAGTCAACTGAAGCTT

107

+

+

+

+

+

+

+

+

-

-

-

-

-

AAGCTTCCGGTCGGCTTCGCAGTAGAAAGCTT

159

+

+

+

+

-

+

-

-

-

-

-

-

-

AAGCTTCTCATCCTTCACCTTGGTCGAAGCTT

182***

+

-

-

-

-

-

-

-

-

-

-

-

-

 

540

-

+

-

-

-

-

+

+

-

-

-

-

-

 

362

-

-

-

-

-

-

+

+

-

-

-

-

-

AAGCTTGAAAAAGGTTCAGGCAAACTAAGCTT

84

+

-

-

-

-

-

-

-

-

-

-

-

-

AAGCTTGCTGAACGCACCTGCGTGGAAAGCTT

923

-

-

+

-

-

+

-

+

-

-

-

-

-

AAGCTTGGCGTCAATCCACACCAAAGAAGCTT

347

-

+

+

-

-

-

-

+

-

-

-

-

-

AAGCTTTCTTGATAAGGCTCCTACGCAAGCTT

250

-

+

-

-

-

-

-

+

-

-

-

-

-

*A full-length sequence must be detected in at least one of the two CLL sampes used in ditag collection, or at least in one other CLL samples.

** Sample #1 and 2 were used for ditag collection.

***tag1 part map to chr1:56672307-56672331

Aberrations also affect the introns of multiple genes. FHIT encodes diadenosine 5',5'''-P1,P3-triphosphate hydrolase involved in purine metabolism [15]. It is located in the common fragile site FRA3B on chromosome 3, where carcinogen-induced damage can lead to translocations in several cancers. A 283-base sequence maps to intron 8 of FHIT gene but its tag 1 contains GA to TG change. HYDIN encodes an axonemal protein; mutation of HYDIN is related to congenital hydrocephalus [16]. Two full-length sequences of 605-bp and 614-bp from two different unmapped ditags were obtained from seven CLL samples. Both sequences map to 21st intron of HYDIN. The 605-bp sequence contains CCTACGGCG in its tag 2 converted from wild-type gCcACaGCa (lowercase refers to the changed base), and the 614-bp sequence contains CGCC converted from wild-type tGCt in its tag 1 and an internal insertion. NCOR2 is a transcriptional regulator that recruits histone deacetylases to promoters [17]. A 582-base sequence maps to intron 1 of NCOR2, but its tag 1 contains an AAGC insertion, and tag 2 contains a C to T change, an AG deletion, and a T insertion. TYK2 is a member of the JAK family involving in IFN-g, IL-6, IL-10 and IL-12 signaling. Mutation in this gene is associated with hyperimmunoglobulin E syndrome [18]. A 268-base sequence maps to intron 14 of TYK2 but its tag 1 contains an AAGCTTA insertion and its tag 2 contains a TGAAGCTT insertion. Both insertions create HindIII restriction sites that lead to the generation of the unmapped ditag. A 197-base sequence was detected in seven CLL samples and two different sequences of 112-base and 170-base were generated from the CLL used in ditag collection. All three sequences map to UBAP2 located at 9p13.3, a gene involved in the ubiquitination pathway [19]. For the 197-base sequence, its 178 bases map to intron 6 of UBAP2 gene and the remaining 18 bases have no map, whereas the 112-base and 170-base sequences contain different insertions. Although the aberrations in many of these genes have been correlated with different types of cancer, most have not been linked with CLL.

Non-coding regions contribute to the majority of the genome, and contain important functional elements involving DNA replication, genome stability, regulation of gene expression, and coding for non-coding transcripts etc. Extensive characterization of non-coding region could provide rich candidate markers for clinical applications and identify the hotspots of genomic aberrations involving cancer development. A total of 37 sequences generated from 30 unmapped ditags mapped to the non-coding regions in the genome with various types of abnormalities (Table 3, Additional file 3: Supplemental Table S3). Although these loci are not directly located in the coding regions, many genes are located nearby the mapped locations. Of the 26 loci specifically mapped by the sequences, 15 have genes located either upstream, downstream or both within 100 kb distance. For example, a 614 base sequence maps to 5q35.1 between169443856-169444467, where DOCK2 is located 27,836 base upstream and FOXI1 is located 21028 downstream. A 398-base sequence maps to 15q26.1 between 88110782 and 88111168, where two homologous transcriptional factor genes, MESP1 and MESP2, are located 16,678-base upstream and 9,425-base downstream correspondingly. microRNA gene MIR663 are located 20,580 base upstream of 20p11.1 between 26157494-26158252 mapped by a 920-base sequence detected in seven CLL samples. Another microRNA gene MIR663B is located 10,964-base upstream of 2q21.2 between 132742087-132742356 mapped by a 290-base sequence, of which a non-coding RNA gene NCRNA00164 is located in between. The aberrations could affect the nearby genes through influencing the regulation of gene expression.
Table 3

Aberrations in the intergenic region

Ditag

FullL-length (bp)

CLL sample

chr.

Location

Aberration

Nearby genes

  

1

2

3

4

5

6

7

8

   

Upstream

Distance (bp)

Downstream

Distance (bp)

AAGCTTACTTTCTCGGTTCCATTACTAAGCTT

614

-

-

+

-

-

+

+

-

5q35.1

169443856-169444467

Base change

DOCK2

27,836

FOXI1

21,028

AAGCTTAGCCGGGCATCCTCTTTCCTAAGCTT

427

-

-

-

-

-

-

+

+

17q25.1

68688801-68689216

Base change

SSTR2

16,046

COG1

11,552

 

398

-

+

+

-

-

-

-

-

15q26.1

88110782-88111168

Insertion, base change

MESP1

16,678

MESP2

9,425

AAGCTTAGTTTGGCTGCATGAGACTGAAGCTT

737

-

-

+

-

-

-

-

+

16q23.1

74682385-74683114

Base change

    

AAGCTTATGATGATCCCCTGAGCTAAAAGCTT

358

+

-

-

-

-

-

-

-

1q23.3

162264795-162265148

Insertion, base change

    
 

264

+

-

-

-

-

-

-

-

5p15.33

2449552-2449774

Insertion

    

AAGCTTCAACGATAGTTCATCATCATAAGCTT

265

-

+

+

-

-

-

-

-

16p13.3

808762-808956

Base change

PRR25

4,900

LMF1

34,679

AAGCTTCAATAGCCGAAGCCAAACTAAAGCTT

556

+

+

-

-

+

+

+

-

12q15

66234005-66234554

Base change

  

DYRK2

94,467

AAGCTTCACTCAGTCATATGGCATGGAAGCTT

130

-

-

-

-

-

+

+

-

10q26.3

133154300-133154419

Insertion

    

AAGCTTCACTGCAGCTATAACACTGCAAGCTT

920

+

+

+

+

+

+

-

+

20p11.1

26157494-26158252

Insertion, base change

MIR663

20,580

  

AAGCTTCCTCTGTACTCACATTAACGAAGCTT

892

-

+

+

-

-

+

+

-

9q12

67914079-67914963

Base change

    

AAGCTTGAAATAAGTGCTGCATCCTGAAGCTT

606

+

-

-

-

-

-

-

-

1q41

220652161-220652760

Insertion, base change

    
 

252

-

-

+

-

-

-

-

+

20q13.13

46025615-46025861

Base change

    

AAGCTTGACTCATTGCGTCTTATTCTAAGCTT

1060

+

-

-

-

-

-

-

-

9q22.31

94477651-94478404

Insertion

IPPK

5,283

BICD2

35,062

AAGCTTGCACAAGGGGCCCCTTGTGCAAGCTT

606

+

-

-

-

-

-

-

-

2q35

220216964-220217558

Insertion

SLC4A3

16,038

  

AAGCTTGCAGAAGGGGAGCCAGGGTGAAGCTT

553

-

-

-

+

-

-

-

+

11q23.2

115071517-115072057

Insertion

    

AAGCTTGCTGAACGCACCTGCGTGGAAAGCTT

758

-

-

-

+

+

-

-

-

13q12.3

30403963-30404714

Insertion, base change

C13orf33

25,635

C13orf26

120

 

602

+

+

+

-

+

-

-

+

1q42.13

227318140-227318732

Insertion

    

AAGCTTGGAGCCCTAGCCACAATTGTAAGCTT

1453

-

+

-

-

+

+

+

-

13q21.33

70285628-70287080

Base change

    

AAGCTTGGCCAGAGACAGGCATCATGAAGCTT

900

+

-

+

+

-

+

-

+

6q23.2

135162469-135163363

Base change

    

AAGCTTTCACTTCATTGGAGTCAGTGAAGCTT

322

+

+

+

-

+

+

+

-

13q14.12

44367511-44367832

Base change

  

NUFIP1

43,552

AAGCTTTCCTAGGGAGCTGGGTGGTGAAGCTT

290

-

-

+

-

-

-

+

-

2q21.2

132742087-132742356

Insertion

NCRNA00164MIR663B

10,075; 10,964

  
 

120

+

-

-

-

-

-

-

-

4p15.33

14280120-14280230

Insertion, base change

    

AAGCTTTCCTTTTCCTTCTGCTCTTAAAGCTT

1071

+

-

+

-

-

+

-

-

6q27

164534304-164535365

Base change

    

AAGCTTTGCATTGGCAGAAGCCACCAAAGCTT

1039

-

+

-

-

-

-

+

-

9q12

69920049-69921093

Base change

    

AAGCTTTTAAGGGATCATGCCTCTCCAAGCTT

1534

+

-

+

-

+

+

+

-

1q21.2

148015439-148016961

Base change

  

FCGR1A

3,951

AAGCTTACCCTCTGGACGCCTGTATGAAGCTT

185

+

-

-

-

-

-

-

-

9q22.33

100893873-100893975

Insertion

  

TGFBR1

13,258

One hundred and forty seven full-length sequences converted from 57 unmapped tags map to the highly repetitive sequences in the non-coding regions. Of these sequences, 110 sequences map to the ALR/Alpha satellite sequences of the centromere, and chromosome 2, 10, and 17 are among the most frequent ones (Table 4, Additional file 4: Supplemental Table S4): 23 sequences converted from 13 unmapped tags map to the centromere of chromosome 2 at 2p11.1, 41 sequences converted from 16 ditags map to the centromere of chromosome 10 at 10q11.1, and 22 sequences converted from 6 unmapped ditags map to the centromere of chromosome 17 at 17p11.1. The presence of highly frequent aberrations in ALR/Alpha satellite sequences in these three chromosomes suggests that these could be the hot spot of genomic aberrations in CLL. Aberrations in repetitive sequences have been shown to contribute to cancer development [20]. However, it is difficult to analyze the aberrations in these highly repetitive regions using the hybridization-based approach due to the difficulty to designing specific probes. Our results show that restriction sequencing-based approach provides a useful tool to study the aberrations in these regions.
Table 4

Aberrations in the centromere region

Ditag

Full-length (bp)

CLL sample

Chr.

Location

Sequence type

  

1

2

3

4

5

6

7

8

   

AAGCTTTCATTGGGATAACAGTGTTGAAGCTT

562

-

+

+

-

-

+

+

+

2p11.1

132722630-132722850

ALR/Alpha

 

893

-

+

-

-

-

-

+

-

2p11.1

91677156-91682632

ALR/Alpha

 

217

+

+

-

+

-

-

+

-

2p11.1

91677835-91680254

ALR/Alpha

AAGCTTTCCAGTTAAGCTTTCTGGGGAAGCTT

1067

+

+

+

+

+

+

+

-

2p11.1

91257036-91258039

 
 

1002

+

-

-

-

-

-

+

-

2p11.1

91257036-91258039

 

AAGCTTCTTTATGAGGAACAGTGTTGAAGCTT

216

+

+

+

+

-

-

+

-

2p11.1

91670531-91670746

ALR/Alpha

 

896

+

+

-

+

-

-

-

-

2p11.1

91670531-91686712

ALR/Alpha

 

901

+

-

-

-

-

-

+

-

2p11.1

91670531-91686712

ALR/Alpha

 

560

+

-

-

-

+

-

-

-

2p11.1

91655191-91672448

ALR/Alpha

 

2231

-

-

-

-

+

+

-

-

2p11.1

91670550-91684334

ALR/Alpha

AAGCTTCTGAGAATGCCATCCCAATGAAGCTT

686

+

-

-

-

-

-

+

-

2p11.1

91677155-91689898

ALR/Alpha

AAGCTTATTTGAGATGAAAGGAGTAGAAGCTT

1234

+

+

-

-

-

-

-

-

2p11.1

91664565-91688410

ALR/Alpha

 

726

+

+

+

+

+

+

+

-

2p11.1

91676309-91680254

ALR/Alpha

AAGCTTCAACACTGTTGTTCCCAATGAAGCTT

612

-

+

+

-

-

+

-

-

2p11.1

91676431-91689428

ALR/Alpha

AAGCTTCAATGGGATGAAGAGTGTTGAAGCTT

556

+

+

+

-

+

+

+

-

2p11.1

91684461-91685014

ALR/Alpha

 

894

-

-

+

-

-

-

+

-

2p11.1

91677156-91680254

ALR/Alpha

AAGCTTCAATTGGGATAACAGTGTTGAAGCTT

555

-

-

-

+

-

+

+

+

2p11.1

91677836-91680592

ALR/Alpha

AAGCTTCATTAGGGATAACAGTGTTGAAGCTT

555

+

+

-

-

-

+

-

-

2p11.1

91677156-91677709

ALR/Alpha

AAGCTTCATTGGGAACAACAGTGTTGAAGCTT

269

-

+

-

-

-

-

+

-

2p11.1

91677155-91677709

ALR/Alpha

AAGCTTCATTGGGATGGCATTCTCAGAAGCTT

685

+

-

-

-

-

-

-

-

2p11.1

91674610-91684466

ALR/Alpha

AAGCTTCTATTGGGATAACAGTGTTGAAGCTT

556

+

+

+

-

-

+

+

+

2p11.1

91672232-91680592

ALR/Alpha

 

893

-

+

-

-

-

-

+

+

2p11.1

91653836-91682632

ALR/Alpha

AAGCTTGACTCATTGCGTCTTATTCTAAGCTT

1179

-

+

+

+

+

+

+

-

2p11.1

91031886-91033063

ERVL-B4-int

AAGCTTAAAACTCCTTTATGAAAAGAAAGCTT

637

+

-

-

-

-

-

-

-

10q11.1

41848823-41861037

ALR/Alpha

AAGCTTAAACTCCGTGCATCAAAAGAAAGCTT

689

+

+

-

-

-

-

+

-

10q11.1

41718813-41720661

ALR/Alpha

 

1407

+

-

-

-

-

-

-

-

10q11.1

41718474-41727608

ALR/Alpha

 

601

+

-

-

-

-

-

-

-

10q11.1

41718888-41720661

ALR/Alpha

AAGCTTAAACTTCTTGTATGAAAAGAAAGCTT

2067

-

+

+

-

-

-

+

-

10q11.1

41847790-41864775

ALR/Alpha

 

1023

+

+

-

-

-

-

-

-

10q11.1

41718797-41729299

ALR/Alpha

 

970

+

-

-

-

-

-

-

-

10q11.1

41850170-41864775

ALR/Alpha

 

346

+

-

-

-

-

-

+

-

10q11.1

41718460-41719477

ALR/Alpha

AAGCTTCAACGCTGCGCTATTGAAGGAAGCTT

345

-

+

+

-

-

-

-

-

10q11.1

41726415-41729786

ALR/Alpha

 

860

+

+

+

+

+

+

+

+

12p11.1

34724897-34729300

ALR/Alpha

AAGCTTCAACTCTGTCCGCCTAAAGGAAGCTT

175

-

-

-

-

+

-

+

-

10q11.1

41719301-41720316

ALR/Alpha

AAGCTTCAACTCTGTGCATTGGCCTCAAGCTT

279

+

+

+

+

+

+

+

+

10q11.1

41849321-41850275

ALR/Alpha

 

622

+

+

+

+

-

+

+

-

10q11.1

41849321-41858079

ALR/Alpha

 

619

-

+

-

-

-

+

+

-

10q11.1

41849321-41861137

ALR/Alpha

AAGCTTCAACTCTGTGCCGCTAAAGGAAGCTT

344

-

+

+

-

+

+

+

+

10q11.1

41718623-41720492

ALR/Alpha

 

2280

+

-

-

-

-

+

-

-

10q11.1

41717944-41720994

ALR/Alpha

 

1359

+

-

-

-

-

-

-

-

10q11.1

41717944-41729975

ALR/Alpha

 

1190

+

-

-

-

-

-

-

-

10q11.1

41718623-41720492

ALR/Alpha

 

681

+

-

-

-

-

-

-

-

10q11.1

41720147-41729975

ALR/Alpha

AAGCTTCCTTCAGAAACAAGGAGTTTAAGCTT

858

+

-

+

-

+

+

+

-

10q11.1

41718623-41720661

ALR/Alpha

AAGCTTCCTTTTAGGCCACAGAGTTGAAGCTT

348

-

+

+

-

-

-

-

-

10q11.1

41719301-41720492

ALR/Alpha

 

1029

-

+

+

-

-

-

-

-

10q11.1

41847960-41861361

ALR/Alpha

 

684

+

-

-

-

-

-

+

-

10q11.1

41718622-41721845

ALR/Alpha

AAGCTTCCTTTTCATACAAGGAGTTTAAGCTT

1498

-

+

-

-

-

+

-

-

10q11.1

41718461-41720661

ALR/Alpha

AAGCTTCTTTTTCATGCAAGGAGTTTAAGCTT

385

+

-

-

-

+

+

+

-

10q11.1

41718767-41719477

ALR/Alpha

 

724

+

-

-

-

-

-

-

-

10q11.1

41718767-41720661

ALR/Alpha

 

722

+

-

-

-

-

-

-

-

10q11.1

41847452-41858698

ALR/Alpha

AAGCTTTCCTTTAGGCCACAGAGTTGAAGCTT

1904

-

+

+

-

-

-

-

-

10q11.1

41847960-41863582

ALR/Alpha

 

345

-

-

+

+

-

-

+

-

10q11.1

41720491-41722352

ALR/Alpha

AAGCTTTCTTTTTCATCAAGGAGTTTAAGCTT

386

+

-

-

-

-

-

-

-

10q11.1

41720293-41720661

ALR/Alpha

AAGCTTTGAAATCTCCCACCTAAAGGAAGCTT

408

+

-

+

+

+

+

+

-

10q11.1

41718623-41722413

ALR/Alpha

 

750

-

-

+

+

-

-

-

-

10q11.1

41847563-41863586

ALR/Alpha

 

1262

+

-

-

-

-

-

-

-

10q11.1

41718623-41720552

ALR/Alpha

AAGCTTTTCTTTTCATCAAGGAGTTTAAGCTT

1379

-

-

+

-

-

-

+

-

10q11.1

41718797-41720661

ALR/Alpha

 

691

-

-

+

-

-

+

-

-

10q11.1

41718460-41720661

ALR/Alpha

 

2041

+

-

-

-

-

-

-

-

10q11.1

41847790-41866145

ALR/Alpha

 

832

+

-

-

-

-

-

-

-

10q11.1

41718460-41720661

ALR/Alpha

 

347

-

-

-

+

-

+

+

-

10q11.1

41718460-41720661

ALR/Alpha

 

1024

+

+

-

-

-

+

+

-

10q11.1

41718117-41720661

ALR/Alpha

AAGCTTTTGAGGCCAACACAGAGTTGAAGCTT

620

-

+

-

-

-

+

+

+

10q11.1

41849321-41855026

ALR/Alpha

 

281

+

+

-

-

-

-

-

-

10q11.1

41849321-41850276

ALR/Alpha

AAGCTTCCTGTGATGATTCGAGAGAGAAGCTT

1419

+

+

-

-

+

+

-

-

17p11.1

22175465-22179262

ALR/Alpha

 

576

-

-

-

-

-

+

+

+

17p11.1

22182601-22184019

ALR/Alpha

 

1966

+

-

+

-

-

-

-

-

17p11.1

22175465-22186396

ALR/Alpha

 

2314

-

-

+

-

-

+

-

-

17p11.1

22173089-22186396

ALR/Alpha

 

234

+

+

+

+

-

+

-

+

17p11.1

22170709-22172128

ALR/Alpha

 

578

+

+

+

-

-

+

-

+

17p11.1

22176307-22179262

ALR/Alpha

 

1090

-

+

-

-

-

-

+

+

17p11.1

22170709-22179262

ALR/Alpha

AAGCTTTCTCTCTCGACATCACAGAGAAGCTT

641

+

-

-

-

-

-

-

-

17p11.1

22178721-22179262

ALR/Alpha

 

1389

+

-

-

-

-

-

-

-

17p11.1

22175464-22184019

ALR/Alpha

 

1824

+

-

-

-

-

-

-

-

17p11.1

22175464-22181640

ALR/Alpha

 

579

-

+

-

-

+

+

+

-

17p11.1

22170709-22179262

ALR/Alpha

 

1420

-

+

+

-

+

+

-

-

17p11.1

22180222-22184019

ALR/Alpha

AAGCTTCTCTCTCGAACATCGCAGAGAAGCTT

1091

+

-

-

-

-

+

-

+

17p11.1

22173083-22184019

ALR/Alpha

 

749

-

-

+

-

-

-

-

+

17p11.1

22183291-22184019

ALR/Alpha

AAGCTTCTCTGAGATGTTCGAGAGAGAAGCTT

579

+

+

+

+

-

-

+

+

17p11.1

22181236-22184019

ALR/Alpha

 

918

+

+

+

-

-

-

-

-

17p11.1

22173087-22184019

ALR/Alpha

 

407

-

+

-

+

-

+

-

-

17p11.1

22174101-22184019

ALR/Alpha

 

406

-

-

+

-

-

-

-

+

17p11.1

22175464-22176883

ALR/Alpha

 

1087

+

-

-

-

-

-

-

-

17p11.1

22175459-22181640

ALR/Alpha

AAGCTTCTGAGAATGCTTTTCTGAAAAAGCTT

355

+

+

-

-

+

+

+

-

17p11.1

22184624-22184977

ALR/Alpha

 

1037

+

-

-

-

-

-

-

-

17p11.1

22184624-22186848

ALR/Alpha

AAGCTTTGAGACCTGTCTCAGAGTTGAAGCTT

799

+

+

+

-

+

+

+

-

17p11.1

21687309-21687527

ALR/Alpha

Ten full-length sequences generated from eight unmapped ditags did not map to known human genome sequences (Table 2B. Additional file 5: Supplementary table S5). For example, a 107-base full-length sequence converted from an unmapped ditag AAGCTTAGATAGAGCGCAGTCAACTGAAGCTT was detected in all eight CLL samples. However, it does not map to the reference genome sequences. These sequences represent the DNA contents present in CLL genomes but not in normal genomes.

Through high-resolution scanning of three CLL genomes and verifying the results using full-length sequences and additional CLL genomes, our study provides evidence showing the wide presence of genomic aberrations in CLL, of which most are small lesions. Studies with increased number of CLL samples and at high genome coverage will be required to better understand the genetic aberrations in CLL. Although the study used multiple genomics databases to eliminate the changes from normal genomic polymorphism, further studies with normal DNA from the same patient will be required to fully distinguish somatic mutations from germline variations in CLL.

Notes

Declarations

Acknowledgements

We wish to thank Dr. Janet Rowley for comments on the study. The study was supported by Mazza Foundation and Guglielmi Fidelity Charitable Fund (SW).

Authors’ Affiliations

(1)
Department of Genetics, Cell Biology & Anatomy, College of Medicine, University of Nebraska Medical Center
(2)
Northshore University Healthsystem Research Institute
(3)
McCormick School of Engineering and Applied Science, Northwestern University
(4)
Department of Medicine, University of Chicago Pritzker School of Medicine
(5)
Robert H. Lurie Comprehensive Cancer Center, Northwestern University
(6)
ACGT Inc.
(7)
School of Oceanography & Environment, Xiamen University
(8)
Interdepartmental Biological Science Graduate Program, Northwestern University
(9)
Food and Drug Administration

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© Wang et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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