Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in adults in western countries. A genome scan of CLL-prone families revealed a lod score of one in band 13q22.1. To investigate this finding, we selected 6 CLL families consisting of 63 individuals (CLL affected, n = 19; unaffected, n = 44) for fine mapping of a 23-megabase region in 13q14.2-q22.2. Interphase fluorescence in situ hybridization (FISH) revealed 13q14 deletion in 85% (11/13) of CLL patients. Four CLL families shared a 3.68-Mb minimal region in 13q21.33-q22.2. Two asymptomatic siblings who shared the 13q21.33-q22.2 at-risk haplotype exhibited CD5+ monoclonal B-cell lymphocytosis (MBL) on flow cytometry. One of these individuals also had a 13q14 deletion by FISH. These 2 individuals with MBL shared the at-risk haplotype with their CLL-affected relatives, providing further evidence of the relationship between CLL and MBL, as well as of the biologic significance of this novel region. Using direct DNA sequencing analysis, we screened 13 genes for mutations, but no frameshift or nonsense mutations were detected. Our studies revealed that 11 of the 13 genes in the candidate region were expressed in immune tissues, supporting their functional relevance in investigations of familial CLL. In conclusion, we identified a novel candidate region that may predispose to familial CLL.

B-cell chronic lymphocytic leukemia (CLL) is a monoclonal disorder characterized by a progressive accumulation of mature-appearing, but functionally incompetent, lymphocytes. It is the most common form of leukemia among adults in western countries. Data from the Surveillance, Epidemiology and End Results (SEER) Program estimate 9730 new cases of CLL in the United States for 2005 (http://seer.cancer.gov/csr/1975_2000/).1  Clinical diagnosis requires an absolute lymphocytosis higher than 5 × 109 mature-appearing lymphocytes/μL in the peripheral blood. Flow cytometry confirms the diagnosis by showing the presence of light chain–restricted B lymphocytes expressing CD5, CD19, dim CD20, CD23 antigens, and an absent or dim FMC-7 and CD79b staining.

Abnormalities of chromosome band 13q14 in CLL DNA were first reported by Fitchett et al in 1987.2  Frequent deletion of 13q14 has suggested that loss of this region may be involved in malignant transformation to CLL and has prompted efforts to define a minimal deleted region (MDR) predisposing to CLL. In 1993, several investigators3-5  reported an MDR in 13q14.3 in CLL tumor DNA (Figure 1), suggesting the presence of a putative tumor suppressor gene at this region. Molecular studies revealed a 1.6-megabase (Mb) deleted region bordered by the retinoblastoma gene (RB) and D13S25. Then, RB was excluded as a CLL candidate gene. Kitamura et al6  constructed a 650-kilobase (kb) contig derived from BAC clones surrounding the putative locus between D13S319 and D13S25 and identified potential CLL candidate genes in this region. Subsequently, molecular studies failed to identify pathologic changes related to CLL. Corcoran et al7  defined a 130-kb commonly deleted region overlapping the MDR defined by markers D13S272 and 6E3.2 (D13S319).8,9  Bullrich et al8  completely sequenced a 790-kb region spanning the CLL MDR, but failed to identify mutations in leukemia cases. Deletion 13q14 is the most frequent chromosomal aberration (∼ 55%) detected in sporadic CLL and is associated with the longest median treatment-free interval.10  Deletions of chromosome band 13q14 have also been found in non-Hodgkin lymphoma,11  acute lymphocytic leukemia,11  and mantle-cell lymphoma.12 

Figure 1

Map of previous genetic studies of CLL candidate region.

Figure 1

Map of previous genetic studies of CLL candidate region.

Close modal

Calin et al13  identified 2 microRNAs (miR15a and miR16-1) within a minimally deleted region of 13q14 and found reduced expression of miR15a and miR16-1 in 68% (41/60) of CLL samples analyzed in comparison with normal CD5+ B cells. The majority of these cases exhibits loss of heterozygosity (LOH) by microsatellite analysis.13  Mechanisms other than LOH (ie, promoter hypermethylation) were not identified as a causal factor for reduced miR expression in CLL samples. In 2005, Calin et al14  reported a nonsense polymorphism in ARLTS1, a gene with homology to the ADP-ribosylation factor family. The frequency of this polymorphism (Trp149Stop) was similar in controls and patients with sporadic CLL. Both of these studies were inconclusive in establishing a causal link between these genes and the pathogenesis of CLL.

There is accumulating evidence that a subset of CLL is due to genetic susceptibility.15-17  More than 80 families with aggregation of CLL have been reported.18  Population studies have shown a 7-fold increased risk of CLL and a 2-fold increased risk for lymphoproliferative diseases among first-degree relatives of patients with CLL.16  Goldin et al19  performed a whole genome scan of 94 individuals (38 affected individuals) from 18 families containing at least 2 living members with CLL. Lod scores of 1.0 or greater were found for loci on chromosomes 1, 3, 6, 12, 13, and 17. None of these loci achieved statistical significance19 ; however, 4 of these loci coincide with, or are adjacent to, regions of recurrent chromosomal abnormalities in CLL (6q, 13q, 12q, and 17p). Linkage results suggested a region of interest in band 13q22.1 (marker D13S156) among a subgroup of CLL families.19  To investigate this region for a CLL susceptibility gene, we selected 6 CLL-prone families for fine mapping of the 13q14.2-q22.2 region to determine if a shared minimal candidate region (MCR) may be defined.

This study was approved by the National Cancer Institute (NCI) institutional review board, and informed consent was obtained from all subjects in this report. Families with 2 or more cases of CLL have been enrolled in the NCI Familial B-CLL Registry since 1967. All available affected individuals and their first-degree relatives were evaluated, and biospecimens were collected at the NIH clinical center or on field trips. CLL diagnostic criteria were documented by available history, medical records, physical examination, clinical laboratory studies, and pathologic review of slides. Clinical data on families 1 to 4 were previously reported.20  Among these 6 families, there are 3 deceased individuals (1-2001, 5-1008, and 6-4004) with a history of CLL confirmed by pathology reports that did not have DNA available for genotyping and thus were not included in the analysis.

Genotyping

DNA was extracted from peripheral blood cells according to standard procedures. Genotyping was performed with fluorescent-labeled microsatellite markers from Applied Biosystems ([ABI], Foster City, CA). Seven markers were used in the initial assay to genotype the region from 13q14.2 to the telomeric recombinant marker D13S1306 at 13q22.2 identified by the whole genome scan.19  The order of the markers was as follows: centromere-D13S165-D13S273-D13S319-D13S1269-D13S1320-D13S1296-D13S156-D13S1306-telomere. Based on initial genotyping data, 6 additional microsatellite markers situated between D13S1296 and D13S156 (D13S279, D13S288, D13S1324, D13S1291, D13S1318, D13S166) and 3 between D13S156 and D13S1306 (D13S792, D13S162, D13S782) were typed in the 6 families to identify the centromeric and telomeric recombinant borders of the MCR. Positions of markers were confirmed with University of California, Santa Cruz (UCSC) BLAT21  and the latest genome assembly. Polymerase chain reaction (PCR) was performed per the manufacturer's direction using ABI Prism True Allele Premix. Alleles were separated on an ABI 3100 genetic analyzer and analyzed with ABI GeneMapper software 3.0.

Sequencing

DNA was extracted from peripheral blood leukocytes according to standard procedures. Bidirectional dye-terminator sequencing was used for mutation analysis as previously described.22  PCR conditions and primer sequences are available upon request. Exon-intron boundaries were determined by BLAT alignment with the assembled genomic sequence (NCBI Build 34; National Center for Biotechnology Information [NCBI], Bethesda, MD).

Tissue-expression profile

Tissue expression of 13 candidate genes from the minimal candidate region in 13q21.33-q22.2 was evaluated using the Human Immune cDNA panel (Clontech, Mountain View, CA). Primers were designed to amplify the 3′ UTR regions of the candidate genes. Titanium Taq (Clontech) was used for PCR amplification of cDNA from the Human Immune panel (Clontech). Primers that failed to amplify with the human control cDNA were tested for PCR efficiency using Centre d'Etude du Polymorphisme Humaine (CEPH) genomic DNA. PCR products were analyzed with a 2% agarose gel.

Functional studies

Total RNA was extracted from frozen lymphocytes using the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized using the cDNA Archive Kit (ABI). A sporadic CLL leukemic clone and normal B cells were isolated by flow cytometry for the purpose of studying differential expression of the candidate genes in 13q21.33-q22.2. Ten candidate genes showing expression in peripheral leukocytes and bone marrow were selected for gene expression analysis using real-time PCR and the TaqMan gene expression assay (ABI). PCR primers and Taqman probes for the 10 candidate genes (target genes) and β-actin (ACTB, calibrator gene) were designed by Applied Biosystems (ABI). The relative standard curve quantitation method was used to measure the expression level of the 10 target genes relative to ACTB gene-expression level.23  A standard curve using fibroblast cDNA was generated for the 10 target genes and the ACTB calibrator gene. cDNA archived from total RNA was diluted 1/10 for the CLL leukemic clone and 1/4 for the B-cell control and aliquoted in triplicate for real-time PCR quantitation of the 10 target genes and the ACTB calibrator gene. Mean mRNA expression levels of each target gene were normalized to the ACTB calibrator gene expression level measured by real-time PCR. The normalized target gene expression value(s) of the CLL leukemic clone was divided by the normalized value(s) of their respective target gene(s) from the B-cell control to derive a fold-difference change in target gene expression. Loc387937 and Chr13_441 were excluded from gene expression measurement as they were not expressed in the immune tissue assay. Loc387934 did not have unique coding sequence (as determined by NCBI BLAST24 ) necessary for design of Taqman probe/primers so it was not measured for gene expression.

Flow cytometry

Flow cytometry was performed on affected family members to confirm their CLL diagnosis. Whole-blood lysis and flow cytometric immunophenotyping were performed as previously described.25-31  Flow cytometric immunophenotyping was performed by 2- and 3-color immunofluorescent staining using a combination of directly conjugated reagents. Blood samples from patients with known sporadic CLL were used as positive controls for affected members of the families. Samples from blood-bank donors and unaffected spouses were used as controls for unaffected members. CellQuest (Becton Dickinson Biosciences [BDB], San Jose, CA) was used to acquire and analyze the flow cytometric immunophenotyping data. FACSComp (BDB) and QuickCal (Flow Standards, San Juan, PR) microbead standards were used to validate instrument performance and linearity. All monoclonal antibodies were obtained from BDB except anti-CD22 phycoerythrin (PE), which was obtained from Caltag (Burlingame, CA).

Cytogenetics

Buffy coats from fresh, heparinized peripheral blood were cultured in duplicate with each of the following mitogens: phytohemagglutinin (PHA), phorbol 12-myristate 13-acetate (PMA), pokeweed mitogen (PWM), and E coli lipopolysaccharide (LPS). After 72 (PHA) or 96 (PMA, PWM, LPS) hours in a humidified 5% CO2 incubator at 37°C, cells were harvested and fixed in 3:1 methanol–glacial acetic acid. Routine G-banded karyotype analysis and interphase fluorescence in situ hybridization (FISH) with a panel of 6 probes were performed according to standard protocols. The D13S319 and D13S25 probes (Vysis, Downers Grove, IL) were used to detect deletions in band 13q14. Minimums of 200 interphase nuclei were scored for hybridization signals for each probe. Based upon our results in healthy controls, losses and gains were interpreted as negative if they occurred in 4% or fewer of the nuclei; however, none of the positive results were equivocal as all were present in 12% or more of the nuclei.

Clinical description of CLL families

Six CLL-prone families were identified for fine mapping. Families were selected to maximize the number of available affected individuals and their unaffected first-degree relatives for molecular studies. Families 1, 2, 4, 5, and 6 had 3 or more affected members. Family 3 had 2 affected individuals and 5 unaffected first-degree relatives. Family 6 included 3 affected individuals (2 affected half-first cousins [6-2003 and 6-2012] and uncle-niece [6-2003 and 6-1001]). A total of 63 individuals (affected, n = 19; unaffected, n = 44) were available for genotyping (Figure 2). There were 19 family members affected with CLL with median age at diagnosis of 52 years (range, 38 to 76 years), and a slight preponderance of males (11 males, 8 females) (Table 1). The peripheral white blood cell (WBC) count of CLL cases ranged from 20.9 × 109/L to 186.8 × 109/L. Rai stage in CLL cases was as follows: stage 0, 47% (9/19); stage 1, 16% (3/19); stage 2, 16% (3/19); stage 3, 5% (1/19); and stage 4, 16% (3/19). Flow cytometry confirmed the diagnosis of CLL in all 14 cases tested (Table 1). Flow cytometry analysis of peripheral blood from members of family 2 revealed monoclonal B-cell lymphocytosis (MBL) in 2 asymptomatic siblings (2-1003 and 2-1006) (Figure 3) and no abnormalities in 2 other asymptomatic siblings (2-1004 and 2-1005). FISH analysis revealed 13q14 deletions in 85% (11/13) of CLL-affected individuals studied. An unaffected sibling (2-1006) in family 2 who had MBL was also found to have the 13q14 deletion by FISH, while a sibling (2-1005) who did not share the at-risk haplotype was found to be normal by FISH.

Figure 2

Pedigrees and haplotype analysis of 6 families in the study. A black square indicates individual affected with CLL; a black circle, individual with MBL (monoclonal B-cell lymphocytosis); a white-and-red box, individual with DNA available for analysis; *, reconstructed haplotype; and F, failed genotype.

Figure 2

Pedigrees and haplotype analysis of 6 families in the study. A black square indicates individual affected with CLL; a black circle, individual with MBL (monoclonal B-cell lymphocytosis); a white-and-red box, individual with DNA available for analysis; *, reconstructed haplotype; and F, failed genotype.

Close modal
Table 1

Clinical and cytogenetic features of CLL families

IndividualSexAge at diagnosis, yDiagnosisWBC count, × 109/LStageSurvival status and age, yFlow cytometryCytogenetic interphase FISH
1-1001 47 CLL 112 Rai 3 Dead, 76 CD5+ CLL del 13q14 
1-1003 52 CLL 59.6 Rai 1 Dead, 66 np np 
1-1005 47 CLL 144.6 Rai 1 Dead, 61 np np 
1-1008 58 CLL 32.2 Rai 0 Dead, 86 np no del 13q14 
2-2002 69 CLL 17.8 Rai 0 Living, 80 CD5+ CLL del 13q14 
2-1001 45 CLL 186.8 Rai 4 Dead, 49 CD5+ CLL del 13q14 
2-1002 50 CLL 251 Rai 2 Dead, 54 CD5+ CLL del 13q14 
2-1003 53 Asymptomatic 6.41 NA Living, 60 CD5+ MBL np 
2-1006 39 Asymptomatic 6.16 NA Living, 45 CD5+ MBL del 13q14 
3-2001 76 CLL 12.6 Rai 0 Dead, 82 CD5+ CLL np 
3-1001 49 CLL 68.0 Rai 0 Living, 59 CD5+ CLL del 13q14 
4-1001 47 CLL 98.4 Rai 0 Living, 82 CD5+ CLL np 
4-1002 70 CLL 24.6 Rai 0 Living, 81 CD5+ CLL np 
4-1003 68 CLL 52.1 Rai 2 Living, 76 CD5+ CLL np 
5-1002 62 CLL 63 Rai 4 Dead, 71 CD5+ CLL no del 13q14 
5-1003 47 CLL 64 Rai 0 Living, 61 CD5+ CLL del 13q14 
5-1005 58 CLL 56.7 Rai 0 Living, 69 CD5+ CLL del 13q14 
5-1007 73 CLL 27.1 Rai 4 Living, 81 CD5+ CLL del 13q14 
6-2003 59 CLL 20.9 Rai 0 Living, 64 np del 13q14 
6-2012 51 CLL 35.9 Rai 2 Living, 55 CD5+ CLL del 13q14 
6-1001 38 CLL 26.1 Rai 0 Living, 42 np del 13q14 
IndividualSexAge at diagnosis, yDiagnosisWBC count, × 109/LStageSurvival status and age, yFlow cytometryCytogenetic interphase FISH
1-1001 47 CLL 112 Rai 3 Dead, 76 CD5+ CLL del 13q14 
1-1003 52 CLL 59.6 Rai 1 Dead, 66 np np 
1-1005 47 CLL 144.6 Rai 1 Dead, 61 np np 
1-1008 58 CLL 32.2 Rai 0 Dead, 86 np no del 13q14 
2-2002 69 CLL 17.8 Rai 0 Living, 80 CD5+ CLL del 13q14 
2-1001 45 CLL 186.8 Rai 4 Dead, 49 CD5+ CLL del 13q14 
2-1002 50 CLL 251 Rai 2 Dead, 54 CD5+ CLL del 13q14 
2-1003 53 Asymptomatic 6.41 NA Living, 60 CD5+ MBL np 
2-1006 39 Asymptomatic 6.16 NA Living, 45 CD5+ MBL del 13q14 
3-2001 76 CLL 12.6 Rai 0 Dead, 82 CD5+ CLL np 
3-1001 49 CLL 68.0 Rai 0 Living, 59 CD5+ CLL del 13q14 
4-1001 47 CLL 98.4 Rai 0 Living, 82 CD5+ CLL np 
4-1002 70 CLL 24.6 Rai 0 Living, 81 CD5+ CLL np 
4-1003 68 CLL 52.1 Rai 2 Living, 76 CD5+ CLL np 
5-1002 62 CLL 63 Rai 4 Dead, 71 CD5+ CLL no del 13q14 
5-1003 47 CLL 64 Rai 0 Living, 61 CD5+ CLL del 13q14 
5-1005 58 CLL 56.7 Rai 0 Living, 69 CD5+ CLL del 13q14 
5-1007 73 CLL 27.1 Rai 4 Living, 81 CD5+ CLL del 13q14 
6-2003 59 CLL 20.9 Rai 0 Living, 64 np del 13q14 
6-2012 51 CLL 35.9 Rai 2 Living, 55 CD5+ CLL del 13q14 
6-1001 38 CLL 26.1 Rai 0 Living, 42 np del 13q14 

CLL indicates chronic lymphocytic leukemia; np, not performed; NA, not applicable; and MBL, monoclonal B-cell lymphocytosis.

Figure 3

Flow cytometry of 2 unaffected individuals with MBL. Panels A-C show data on 2-1003; panels D-F, on 2-1006. (A) Analysis of whole blood stained with CD19 PerCP. The plot shows forward scatter versus CD19-isolating B cells. (B) Overall kappa-positive clone. (C) A subgate showing that the CD5 population is monoclonal (kappa light chain restricted). (D) The analysis of StemSep-enriched B cells (StemSep, Vancouver, BC, Canada). The plot shows CD19PerCP Cy5.5 versus CD5 PE Cy7. The top box shows a bright CD5 population, and the bottom box shows a dim CD5 population. (E) A subgate showing that the brighter CD5 population shown in panel D is monoclonal (kappa light chain restricted). (F) A subgate showing that the lower dim CD5 population present in panel D is polyclonal.

Figure 3

Flow cytometry of 2 unaffected individuals with MBL. Panels A-C show data on 2-1003; panels D-F, on 2-1006. (A) Analysis of whole blood stained with CD19 PerCP. The plot shows forward scatter versus CD19-isolating B cells. (B) Overall kappa-positive clone. (C) A subgate showing that the CD5 population is monoclonal (kappa light chain restricted). (D) The analysis of StemSep-enriched B cells (StemSep, Vancouver, BC, Canada). The plot shows CD19PerCP Cy5.5 versus CD5 PE Cy7. The top box shows a bright CD5 population, and the bottom box shows a dim CD5 population. (E) A subgate showing that the brighter CD5 population shown in panel D is monoclonal (kappa light chain restricted). (F) A subgate showing that the lower dim CD5 population present in panel D is polyclonal.

Close modal

Mapping

Fine mapping of the 13q region allowed us to investigate whether the CLL susceptibility locus overlaps with the MDR and identify critical recombinants that define the MCR (MCR). Six polymorphic microsatellite markers between D13S165 and D13S1306 were selected for initial genotyping. Nine additional markers were typed to define the MCR. An integrated genetic and physical map of the region was constructed using information derived from public and private databases UCSC Genome Bioinformatics32 ; NCBI BLAST; Ensembl Genome Browser33 ; and Celera, Rockville, MD) (Figure 1). Haplotype analysis showed the individuals with CLL in families 1 to 4 shared a region from marker D13S165 to D13S1306 (Figure 2). Four individuals shared a smaller region due to recombination events at these markers: 1-1005 (D13S1324), 1-1008 (D13S162), 2-1002 (D13S1269), and 4-1002 (D13S782) (Figure 4). Two critical recombinants (1-1005, 1-1008) set the centromeric boundary at D13S1324 and telomeric boundary at D13S162, narrowing the CLL candidate region to a 3.68-Mb in 13q21.33-q22.2. Families 1 to 4 shared this critical region in all CLL-affected members. Family 5 did not share the minimal region at 13q21.33-q22.2 among all their CLL-affected members and was excluded from mutation analysis. Family 6 had a low probability of sharing this minimal candidate region as individuals 6-2003 and 6-1012 would both require double recombination events within a 3.68-Mb region (Figure 2F). The 6 families had the following backgrounds and ancestry: family 1 (Greek), family 2 (English/German/Scandinavian), family 3 (Irish/German/French), family 4 (German/Hungarian), family 5 (Eastern European/Jewish), and family 6 (Irish/German/Native American). The 4 families who shared the haplotype were of diverse ethnic backgrounds. We found no correlation between the families who shared the at-risk haplotype at 13q21.33-q22.2 and their ethnic origin.

Figure 4

Physical map of the CLL candidate region. (A) Locations of polymorphic markers, shown in red. A physical map of the region was constructed using information derived from public and private databases (UCSC Genome Bioinformatics32 ; NCBI; Ensembl Genome Browser33 ; and Celera). (B) Regions shared by the families (shown in green) with critical recombinants identifying the new minimal 3.68-Mb candidate region. (C) Location of the 13 genes within the new CLL candidate region.

Figure 4

Physical map of the CLL candidate region. (A) Locations of polymorphic markers, shown in red. A physical map of the region was constructed using information derived from public and private databases (UCSC Genome Bioinformatics32 ; NCBI; Ensembl Genome Browser33 ; and Celera). (B) Regions shared by the families (shown in green) with critical recombinants identifying the new minimal 3.68-Mb candidate region. (C) Location of the 13 genes within the new CLL candidate region.

Close modal

Sequence analysis

The 3.68-Mb critical region at 13q21.33-q22.2 contained 12 reference genes and one predicted gene in the UCSC Genome Bioinformatics database (Figure 4). There were no reference microRNAs in the Sanger miRBase34  for this candidate region. These 13 genes were screened for mutations using bidirectional sequencing and a panel of germ-line DNA from affected and unaffected individuals from families 1 to 4 and 6. Eighty-five polymorphisms were identified; 56 were located in exons and 29 were intronic (Table 2). Twenty-nine of 56 exonic polymorphisms were located in the 5′ and 3′ untranslated regions. Within the coding region, 15 nucleotide substitutions resulted in conservative amino acid changes, one codon triplication resulted in the insertion of 2 glycines in DACH1, and 11 substitutions resulted in synonymous changes (Table 2).

Table 2

Polymorphism summary

Gene exon/intronPosition, bpNucleotide changeAmino acid change
DIS3 (KIAA1008)NM_014953    
    Exon 5 c.794G>A G to A S265N 
    Intron 5 IVS + 81T>C T to C Noncoding 
    Exon 6 c.977C>G C to G T326R 
    Exon 14 c.1743A>G A to G T581T 
    Intron 16 IVS16 + 106C > T C to T Noncoding 
Chr13_441    
    Intron 2 IVS2−18T>C T to C Noncoding 
    Exon 3 c.198G>T G to T T66T 
FLJ22624NM_024808    
    Exon 1 c.115C>T C to T Noncoding 
    Intron 3 IVS3−69G>A G to A Noncoding 
    Intron 3 IVS3−31A>G A to G Noncoding 
    Exon 12 c.4G>A G to A Noncoding 
KLF5NM_001730    
    Intron 1 IVS1 + 168C>A C to A Noncoding 
    Intron 2 IVS2 + 56_ + 57dupGT Duplication GT Noncoding 
    Intron 2 IVS2 + 56_ + 57dupGTGT Duplication GTGT Noncoding 
Loc387934 XM_370729    
    Alt Exon 1 c.294G>A G to A A98A 
    Exon 1 c.438A>G A to G A146A 
Loc440145 XM_495961    
    Exon 3 c.300A>T A to T S100S 
Loc400145 XM_375031    
    Exon 1 c.85T>C T to C Noncoding 
    Exon 2 c.156C>T C to T H52H 
    Exon 2 c.245G>A G to A R82H 
    Exon 2 c.403G>A G to A G135S 
DACHINM_080759    
    Exon 1 c.374C>T C to T Noncoding 
    Exon 1 c.369T>C T to C Noncoding 
    Exon 1 c.308C>T C to T Noncoding 
    Exon 1 c.304C>T C to T Noncoding 
    Exon 1 c.299C>T C to T Noncoding 
    Exon 1 c.298C>A C to A Noncoding 
    Exon 1 c.298C>T C to T Noncoding 
    Exon 1 c.298delC Deletion C Noncoding 
    Exon 1 c.118C>T C to T Noncoding 
    Exon 1 c.293C>T C to T Noncoding 
    Exon 1 c.292C>T C to T Noncoding 
    Exon 1 c.270_−305del36 Deletion 36 Noncoding 
    Exon 1 c.201_−309del109 Deletion 109 Noncoding 
    Exon 1 c.190_−309del120 Deletion 120 Noncoding 
    Exon 1 c242_243dupCGGCGG Duplication CGGCGG G81_S82insGG 
    Intron 4 IVS4 + 14insT Insertion T Noncoding 
TBC1D4NM_014832    
    Exon 1 c.84C>G C to G P28R 
    Exon 1 c.302C>T C to T A101V 
    Exon 1 c.350T>C T to C F117S 
    Exon 1 c.363C>T C to T H121H 
    Intron 1 IVSI + 8G>A G to A Noncoding 
    Intron 1 IVSI + 18del61 Deletion 61 Noncoding 
    Intron 1 IVSI + 84A>G A to G Noncoding 
    Exon 2 c.606C>T C to T F202F 
    Alt intron 1 Alt IVS1 + 75insG Ins G Noncoding 
    Alt intron 4 Alt IVS4−92T>C T to C Noncoding 
    Alt intron 4 Alt IVS4−107A>G A to G Noncoding 
    Exon 7 c.1611T>G T to G S537S 
    Intron 7 IVS7 + 10insT Insertion T Noncoding 
    Intron 9 IVS9 + 139C>A C to A Noncoding 
    Intron 10 IVS10−59T>G T to G Noncoding 
    Intron 13 IVS13−3C>T C to T Noncoding 
    Exon 14 c.2455A>G A to G 1819V 
    Intron 14 IVS14−60C>T C to T Noncoding 
    Intron 15 IVS15 + 63_ + 64delTT Deletion TT Noncoding 
    Exon 16 c.2904C>T C to T L968L 
    Exon 19 c.3443C>T C to T T1148M 
    Exon 20 c.3620A>G A to G N1207S 
    Exon 20 c.3628C>G C to G L1210V 
    Intron 20 IVS20−19dupT Duplication T Noncoding 
    Exon 21 c.3827C>T C to T A1276V 
    Exon 21 c.980G>A G to A Noncoding 
    Exon 21 c.1599T>C T to C Noncoding 
PIBF1(C13orf24)NM_006346    
    Intron 3 IVS3 + 19A>T A to T Noncoding 
    Intron 3 IVS3 + 78T>G T to G Noncoding 
    Exon 4 c.499A>G A to G 1167V 
    Intron 4 IVS4 + 61T>C T to C Noncoding 
    Intron 8 IVS8 + 23C>T C to T Noncoding 
    Intron 10 IVS10-68_−71delGTAA Deletion GTAA Noncoding 
    Intron 12 IVS11 + 41delT Deletion T Noncoding 
    Exon 15 c.1888A>G A to G 1628V 
KLF12NM_007249    
    Exon 1 c.127_−128delTG Deletion TG Noncoding 
    Intron 2 IVS2−3T>C T to C Noncoding 
    Exon 4 c.272C>T C to T T91M 
    Exon 6 c.840C>A C to A G280G 
Loc387937 XM_373569    
    Exon 4 c.141C>T C to T Noncoding 
Loc400144 XM_378421    
    Exon 1 c.260C>T C to T Noncoding 
    Exon 2 c.412A>G A to G Noncoding 
    Exon 2 c.535A>G A to G Noncoding 
    Exon 2 c.597A>T A to T Noncoding 
    Exon 2 c.868(TG) 14-20 TG dinucleotide repeat 14-20 Noncoding 
    Exon 2 c.1712A>G A to G Noncoding 
    Exon 2 c.2097C>T C to T Noncoding 
    Exon 2 c.2442T>G T to G Noncoding 
Gene exon/intronPosition, bpNucleotide changeAmino acid change
DIS3 (KIAA1008)NM_014953    
    Exon 5 c.794G>A G to A S265N 
    Intron 5 IVS + 81T>C T to C Noncoding 
    Exon 6 c.977C>G C to G T326R 
    Exon 14 c.1743A>G A to G T581T 
    Intron 16 IVS16 + 106C > T C to T Noncoding 
Chr13_441    
    Intron 2 IVS2−18T>C T to C Noncoding 
    Exon 3 c.198G>T G to T T66T 
FLJ22624NM_024808    
    Exon 1 c.115C>T C to T Noncoding 
    Intron 3 IVS3−69G>A G to A Noncoding 
    Intron 3 IVS3−31A>G A to G Noncoding 
    Exon 12 c.4G>A G to A Noncoding 
KLF5NM_001730    
    Intron 1 IVS1 + 168C>A C to A Noncoding 
    Intron 2 IVS2 + 56_ + 57dupGT Duplication GT Noncoding 
    Intron 2 IVS2 + 56_ + 57dupGTGT Duplication GTGT Noncoding 
Loc387934 XM_370729    
    Alt Exon 1 c.294G>A G to A A98A 
    Exon 1 c.438A>G A to G A146A 
Loc440145 XM_495961    
    Exon 3 c.300A>T A to T S100S 
Loc400145 XM_375031    
    Exon 1 c.85T>C T to C Noncoding 
    Exon 2 c.156C>T C to T H52H 
    Exon 2 c.245G>A G to A R82H 
    Exon 2 c.403G>A G to A G135S 
DACHINM_080759    
    Exon 1 c.374C>T C to T Noncoding 
    Exon 1 c.369T>C T to C Noncoding 
    Exon 1 c.308C>T C to T Noncoding 
    Exon 1 c.304C>T C to T Noncoding 
    Exon 1 c.299C>T C to T Noncoding 
    Exon 1 c.298C>A C to A Noncoding 
    Exon 1 c.298C>T C to T Noncoding 
    Exon 1 c.298delC Deletion C Noncoding 
    Exon 1 c.118C>T C to T Noncoding 
    Exon 1 c.293C>T C to T Noncoding 
    Exon 1 c.292C>T C to T Noncoding 
    Exon 1 c.270_−305del36 Deletion 36 Noncoding 
    Exon 1 c.201_−309del109 Deletion 109 Noncoding 
    Exon 1 c.190_−309del120 Deletion 120 Noncoding 
    Exon 1 c242_243dupCGGCGG Duplication CGGCGG G81_S82insGG 
    Intron 4 IVS4 + 14insT Insertion T Noncoding 
TBC1D4NM_014832    
    Exon 1 c.84C>G C to G P28R 
    Exon 1 c.302C>T C to T A101V 
    Exon 1 c.350T>C T to C F117S 
    Exon 1 c.363C>T C to T H121H 
    Intron 1 IVSI + 8G>A G to A Noncoding 
    Intron 1 IVSI + 18del61 Deletion 61 Noncoding 
    Intron 1 IVSI + 84A>G A to G Noncoding 
    Exon 2 c.606C>T C to T F202F 
    Alt intron 1 Alt IVS1 + 75insG Ins G Noncoding 
    Alt intron 4 Alt IVS4−92T>C T to C Noncoding 
    Alt intron 4 Alt IVS4−107A>G A to G Noncoding 
    Exon 7 c.1611T>G T to G S537S 
    Intron 7 IVS7 + 10insT Insertion T Noncoding 
    Intron 9 IVS9 + 139C>A C to A Noncoding 
    Intron 10 IVS10−59T>G T to G Noncoding 
    Intron 13 IVS13−3C>T C to T Noncoding 
    Exon 14 c.2455A>G A to G 1819V 
    Intron 14 IVS14−60C>T C to T Noncoding 
    Intron 15 IVS15 + 63_ + 64delTT Deletion TT Noncoding 
    Exon 16 c.2904C>T C to T L968L 
    Exon 19 c.3443C>T C to T T1148M 
    Exon 20 c.3620A>G A to G N1207S 
    Exon 20 c.3628C>G C to G L1210V 
    Intron 20 IVS20−19dupT Duplication T Noncoding 
    Exon 21 c.3827C>T C to T A1276V 
    Exon 21 c.980G>A G to A Noncoding 
    Exon 21 c.1599T>C T to C Noncoding 
PIBF1(C13orf24)NM_006346    
    Intron 3 IVS3 + 19A>T A to T Noncoding 
    Intron 3 IVS3 + 78T>G T to G Noncoding 
    Exon 4 c.499A>G A to G 1167V 
    Intron 4 IVS4 + 61T>C T to C Noncoding 
    Intron 8 IVS8 + 23C>T C to T Noncoding 
    Intron 10 IVS10-68_−71delGTAA Deletion GTAA Noncoding 
    Intron 12 IVS11 + 41delT Deletion T Noncoding 
    Exon 15 c.1888A>G A to G 1628V 
KLF12NM_007249    
    Exon 1 c.127_−128delTG Deletion TG Noncoding 
    Intron 2 IVS2−3T>C T to C Noncoding 
    Exon 4 c.272C>T C to T T91M 
    Exon 6 c.840C>A C to A G280G 
Loc387937 XM_373569    
    Exon 4 c.141C>T C to T Noncoding 
Loc400144 XM_378421    
    Exon 1 c.260C>T C to T Noncoding 
    Exon 2 c.412A>G A to G Noncoding 
    Exon 2 c.535A>G A to G Noncoding 
    Exon 2 c.597A>T A to T Noncoding 
    Exon 2 c.868(TG) 14-20 TG dinucleotide repeat 14-20 Noncoding 
    Exon 2 c.1712A>G A to G Noncoding 
    Exon 2 c.2097C>T C to T Noncoding 
    Exon 2 c.2442T>G T to G Noncoding 

Small insertions/deletions were found within deep intronic regions of DACH1, KLF5, KLF12, TBC1D4, and PIBF1 (also known as C13orf24, chromosome 13 open-reading frame 24), (Table 2). One polymorphism resulted in the insertion of 2 glycines between codons 81 and 82 of DACH1. This amino acid change appears to be nondeleterious, did not cosegregate with disease, and was detected among all CEPH controls. A single nucleotide polymorphism (snp) located in the −3 position of the KLF12 intron 2 splice acceptor was detected in 4 affected individuals and 1 unaffected individual from 3 of 5 families. This polymorphism was previously reported by Rozenblum et al.35 

Five intronic polymorphisms and 2 conservative amino acid changes (I167V, I628V) detected in PIBF1 did not cosegregate with disease. A deep intronic polymorphism (IVS10−68_−71delGTAA) was detected in PIBF1 that cosegregated with 3 affected relatives (2-2002, 2-1001, 2-1002), and 2 unaffected relatives (2-1003, 2-1006) in family 2 who shared the affected haplotype in band 13q21.33-q22.2 (Figure 2B). This polymorphism was not detected in the other 4 families screened. PIBF1 IVS10−68_−71delGTAA polymorphism was detected at a frequency of 0.38 among 100 unrelated CEPH controls, indicating that this is a common polymorphism among Europeans. Three deep intronic polymorphisms were detected in KLF5 (Table 2). Two of these polymorphisms were located within intron 2 at a dinucleotide GT repeat and were present in both affected and unaffected individuals in 4 of 5 families. Both affected and unaffected individuals in families 3 and 4 were heterozygous for the KLF5 IVS2+56_+57dupGT or the KLF5 IVS2+56_+57dupGTGT polymorphisms. However, these polymorphisms in intron 2 of KLF5 did not cosegregate with disease. There were 13 intronic polymorphisms found in TBC1D4. These polymorphisms were detected in both affected and unaffected individuals among CLL families or solely in unaffected individuals who did not carry the affected haplotype at 13q21.33-q22.2. No additional polymorphisms were found that cosegregated with affected individuals among the remaining families analyzed. No frameshift or nonsense mutations were detected in the coding region of the 13 genes screened.

Functional studies

We examined the 13 genes in our candidate region for expression in a cDNA human immune panel that included peripheral leukocytes, spleen, bone marrow, liver, and thymus. Of the 13 genes identified in the candidate region, 11 were expressed in immune tissues, specifically leukocytes and bone marrow (Table 3). This finding supports that the genes analyzed in our study may have functional relevance in familial CLL.

Table 3

Tissue-expression profile of CLL candidate genes at 13q21.33-q22.2

Candidate geneSpleenLymph nodeThymusTonsilLeukocyteBone marrowLiverPositive control
loc440145 
TBC1D4 
PIBF1 
KLF5 
DIS3 
DACH1 
KLF12 
FLJ22624 
loc400144 − 
loc400145 − − 
loc387934 
loc387937 − − − − − − − 
CHR13_441 − − − − − − − 
Candidate geneSpleenLymph nodeThymusTonsilLeukocyteBone marrowLiverPositive control
loc440145 
TBC1D4 
PIBF1 
KLF5 
DIS3 
DACH1 
KLF12 
FLJ22624 
loc400144 − 
loc400145 − − 
loc387934 
loc387937 − − − − − − − 
CHR13_441 − − − − − − − 

+ indicates positive expression; −, negative expression.

The gene expression level of 10 candidate genes in the 13q21.33-q22.2 region was evaluated in a sporadic CLL leukemic clone compared with normal B cells. The CLL leukemic clone showed a decreased level of expression among 7 candidate genes compared with the normal B cells. The gene expression level of the CLL leukemic clone ranged from a decrease of −2.2-fold (FLJ22624) to −12.9-fold (TBC1D4) compared with normal B cells (Table 4). Two genes (DACH1 and loc400145) were not expressed in CLL cells or B cells. Loc400144 was not expressed in the CLL clone but showed low expression in the normal B cells.

Table 4

Candidate gene expression comparison between sporadic CLL and B cells

Candidate gene expressionCLL gene expression activity ratio to B cells, fold change
    loc440145/β-actin −2.8 
    TBC1D4/β-actin −12.9 
    PIBF1/β-actin −2.7 
    KLF5/β-actin −5.3 
    DIS3/β-actin −2.8 
    DACH1/β-actin Not expressed in lymphocytes 
    KLF12/β-actin −3.0 
    FLJ22624/β-actin −2.2 
    loc400144/β-actin Not expressed 
    Loc400145/β-actin Not expressed in lymphocytes 
Candidate gene expressionCLL gene expression activity ratio to B cells, fold change
    loc440145/β-actin −2.8 
    TBC1D4/β-actin −12.9 
    PIBF1/β-actin −2.7 
    KLF5/β-actin −5.3 
    DIS3/β-actin −2.8 
    DACH1/β-actin Not expressed in lymphocytes 
    KLF12/β-actin −3.0 
    FLJ22624/β-actin −2.2 
    loc400144/β-actin Not expressed 
    Loc400145/β-actin Not expressed in lymphocytes 

In this study, we present 6 families in which multiple members were diagnosed with CLL in successive generations, suggesting that inherited genes contribute to a subset of CLL.16  First-degree relatives of patients with CLL have an increased risk of developing CLL and other lymphoproliferative diseases, suggesting a shared genetic susceptibility in these families.16  Therefore, studies of familial CLL are particularly important in discovering CLL susceptibility genes.

Our study provides further evidence that familial CLL has unique clinical and molecular genetic features. In screening asymptomatic individuals using flow cytometry, we identified 2 individuals with CD5+ MBL who carried the at-risk haplotype at 13q21.33-q22.2 (Figure 3). These 2 individuals with MBL shared the at-risk haplotype with their CLL-affected relatives, providing further evidence for a relation between CLL and MBL. This finding is consistent with reports that MBL occurs in about 14% to 18% of asymptomatic relatives of familial CLL cases18,36  and supports the hypothesis that CD5+ MBL may be a precursor of CLL or a surrogate marker for carrier status. However, it remains to be determined through prospective studies if MBL is a predictor of eventual development of CLL among asymptomatic members of CLL families. The ability to detect MBL with flow cytometry should facilitate gene mapping studies in high-risk families.

Our study revealed that 85% of patients with familial CLL had deletions in 13q14. The frequency among familial cases is higher than reported in sporadic CLL (55%).10  In family 2, all affected individuals with CLL had deletions in 13q14 and shared a haplotype at 13q21.33-q22.2, suggesting that inherited factors may predispose to CLL. One asymptomatic individual who carried the at-risk haplotype at 13q21.33-q22.2 and exhibited CD5+ MBL on flow cytometry also had a 13q14 deletion by FISH. This is one of the first reported cases of an asymptomatic individual with 13q14 deletion. Deletions in 13q14 may represent a very early genomic change in a cascade of genetic events that predispose to developing CLL and/or MBL. Consistent with this is the finding that 13q14 deletion is the most frequent chromosomal aberration detected in sporadic CLL and that sporadic cases with 13q14 deletions as the sole abnormality have the longest estimated survival.10  However, the clinical significance of 13q14 deletion in an asymptomatic relative in the setting of familial CLL is unknown. It remains to be determined through prospective studies if the presence of a 13q14 deletion is a risk factor for development of CLL among asymptomatic members of CLL families.

This study identified a new candidate region 13q21.33-q22.2 that may predispose to familial CLL and MBL. Somatic deletions of 13q21-q22 have been observed in several tumors including sporadic37  and hereditary breast cancer,38  prostate cancer,39  and hepatoblastoma,40  but to our knowledge have not been reported in CLL. Using haplotype analysis, we identified a shared 3.68-Mb region at 13q21.33-q22.2 among 4 CLL families. Our studies revealed that 11 of the 13 genes identified in this 3.68-Mb candidate region were expressed in immune tissues, specifically leukocytes and bone marrow. This finding supports that the 11 genes in the candidate region have functional relevance in investigations of CLL. Using direct sequencing analysis, we screened 13 genes for germ-line mutations, and 85 polymorphisms were identified. In family 2, we found one deep intronic polymorphism in PIBF1 that cosegregated with the haplotype shared by 3 affected members. However, no conserved splice site or coding mutations were identified in this gene. PIBF1 is a lymphocyte-secreted immunomodulatory molecule41  that is expressed in spleen35  and overexpressed in breast tumor cell lines.42 PIBF1 is a microtubule-associated protein that localizes to centrosomes,42  where abnormalities are frequently observed in cancer cells, although their role in tumorigenesis is unknown.43 

Of the 12 reference genes at 13q21.33-q22.2, 5 candidate genes have putative function related to cell growth that makes them potentially important. DIS3 is the human homolog of mitotic control protein Dis3 of Saccharomyces pombe,44  and DACH1 inhibits transforming factor-β (TGF-β)–induced apoptosis.45 KLF5 is found to be deleted and down-regulated in prostate cancer46  and reintroduction of KLF5 in breast tumor cell lines with 13q21 deletion inhibited cell growth in vitro.47 KLF12 represses expression of activator protein-2 alpha (AP-2α).48  Decreased expression of AP-2α is associated with disease progression and metastases in breast cancer.49 DIS3 and KLF12 are expressed in peripheral leukocytes.35  Our gene expression investigations of a CLL clone showed a 2.2- to 12.9-fold decreased level of expression among 7 candidate genes including PIFB1 and KLF5. In addition, there was complete loss of expression of one gene (loc400144) compared with the normal B cells. It is unlikely that the decrease or loss of expression of genes in this region may be due to microRNAs since we found no reference microRNAs in this candidate region. Further gene expression studies of CLL clones will be conducted to confirm our findings. Future cytogenetic studies to determine whether large chromosomal deletions in 13q21.33-q22.2 are responsible for the decrease and/or loss of gene expression in this region are highly relevant since somatic deletions have been observed in tumors in this region.37-40 

We identified CpG islands in 7 of 13 genes near the promoter region of these respective genes. However, our literature search did not reveal evidence of maternal or paternal imprinting effects on chromosome 13 based on the reported normal phenotype of individuals who exhibit uniparental disomy of chromosome 13.50-52  Therefore, inherited methylation changes are unlikely to be relevant to the pathogenesis of CLL in these families. Our future studies will investigate the role of methylation in regulating gene expression in our new candidate region.

Two genome-wide linkage studies of CLL families have pointed to several potential candidate regions.19,53  Recently, Sellick et al53 conducted a genome-wide linkage analysis of 115 CLL families and found a nonparametric linkage score of 3.14 at locus 11p11 and HLOD scores higher than 1.15 at 4 loci (5q22-q23, 6p22, 10q25, and 14q32) consistent with genetic heterogeneity among these families.53  To date, a CLL susceptibility gene has not been identified using direct sequencing by our group and other investigators. Epigenetic mechanisms, microRNAs, and multiple genes may be involved in transcriptional silencing of gene(s) underlying CLL leukemogenesis. Several factors make the mapping and identification of CLL susceptibility genes quite challenging. First, the late onset of the disease makes it difficult to collect DNA from families with multigeneration-affected individuals. We will increase the power of linkage studies through consortial approaches that pool data and samples on CLL families to uncover CLL susceptibility genes. Second, familial CLL is a genetically heterogeneous disease with variable penetrance and a probable high phenocopy rate. In conclusion, we identified a novel candidate region that may predispose to familial CLL and MBL. Future cytogenetic and gene expression studies are planned to clarify the biological significance of this novel region.

Contribution: D.N., M.-H.W., and J.R.T. designed the research; L.F., J.F.F., L.R.G., and N.C. collected and analyzed the patient data; D.N., D.C.A., O.T., and F.A. performed research; D.N., D.C.A., G.E.M., and J.R.T. analyzed data; D.N. and J.R.T. wrote the paper; and all authors read and approved the final version of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Jorge R. Toro, Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Blvd, Executive Plaza South, Rm 7012,Rockville, MD 20892-7231; e-mail: [email protected].

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Contribution: D.N., M.-H.W., and J.R.T. designed the research; L.F., J.F.F., L.R.G., and N.C. collected and analyzed the patient data; D.N., D.C.A., O.T., and F.A. performed research; D.N., D.C.A., G.E.M., and J.R.T. analyzed data; D.N. and J.R.T. wrote the paper; and all authors read and approved the final version of the manuscript.

1
In Ries LAG, Eisner MP, Kosary CL (Eds.), et al.
SEER Cancer Statistics Review 1975-2000
2003
;Bethesda, MD National Cancer Institute.
2
Fitchett M, Griffiths MJ, Oscier DG, Johnson S, Seabright M. Chromosome abnormalities involving band 13q14 in hematologic malignancies.
Cancer Genet Cytogenet
1987
;
24
:
143
–150.
3
Liu Y, Szekely L, Grander D, et al. Chronic lymphocytic leukemia cells with allelic deletions at 13q14 commonly have one intact RB1 gene: evidence for a role of an adjacent locus.
Proc Natl Acad Sci U S A
1993
;
90
:
8697
–8701.
4
Hawthorn LA, Chapman R, Oscier D, Cowell JK. The consistent 13q14 translocation breakpoint seen in chronic B-cell leukaemia (BCLL) involves deletion of the D13S25 locus which lies distal to the retinoblastoma predisposition gene.
Oncogene
1993
;
8
:
1415
–1419.
5
Brown AG, Ross FM, Dunne EM, Steel CM, Weir-Thompson EM. Evidence for a new tumor suppressor (DBM) in human B-cell neoplasia telomeric to the retinoblastoma gene.
Nat Genet
1993
;
3
:
67
–72.
6
Kitamura E, Su G, Sossey-Alaoui K, et al. A transcription map of the minimally deleted region from 13q14 in B-cell chronic lymphocytic leukemia as defined by large scale sequencing of the 650 kb critical region.
Oncogene
2000
;
19
:
5772
–5780.
7
Corcoran MM, Rasool O, Liu Y, et al. Detailed molecular delineation of 13q14.3 loss in B-cell chronic lymphocytic leukemia.
Blood
1998
;
91
:
1382
–1390.
8
Bullrich F, Fujii H, Calin G, et al. Characterization of the 13q14 tumor suppressor locus in CLL: identification of ALT1, an alternative splice variant of the LEU2 gene.
Cancer Res
2001
;
61
:
6640
–6648.
9
Bouyge-Moreau I, Rondeau G, Avet-Loiseau H, et al. Construction of a 780-kb PAC, BAC, and cosmid contig encompassing the minimal critical deletion involved in B cell chronic lymphocytic leukemia at 13q14.3.
Genomics
1997
;
46
:
183
–190.
10
Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia.
N Engl J Med
2000
;
343
:
1910
–1916.
11
Liu Y, Hermanson M, Grander D, et al. 13q deletions in lymphoid malignancies.
Blood
1995
;
86
:
1911
–1915.
12
Stilgenbauer S, Nickolenko J, Wilhelm J, et al. Expressed sequences as candidates for a novel tumor suppressor gene at band 13q14 in B-cell chronic lymphocytic leukemia and mantle cell lymphoma.
Oncogene
1998
;
16
:
1891
–1897.
13
Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia.
Proc Natl Acad Sci U S A
2002
;
99
:
15524
–15529.
14
Calin GA, Trapasso F, Shimizu M, et al. Familial cancer associated with a polymorphism in ARLTS1.
N Engl J Med
2005
;
352
:
1667
–1676.
15
Capalbo S, Callea V, Musolino C, et al. Familial B-cell chronic lymphocytic leukemia in a population of patients from Southern Italy.
Int J Hematol
2004
;
79
:
354
–357.
16
Goldin LR, Pfeiffer RM, Li X, Hemminki K. Familial risk of lymphoproliferative tumors in families of patients with chronic lymphocytic leukemia: results from the Swedish Family-Cancer Database.
Blood
2004
;
104
:
1850
–1854.
17
Caporaso N, Marti GE, Goldin L. Perspectives on familial chronic lymphocytic leukemia: genes and the environment.
Semin Hematol
2004
;
42
:
201
–206.
18
Marti GE, Carter C, Abbasi F, et al. B-cell monoclonal lymphocytosis and B-cell abnormalities in the setting of familial B-cell chronic lymphocytic leukemia.
Cytometry B Clin Cytom
2003
;
52
:
1
–12.
19
Goldin LR, Ishibe N, Sgambati M, et al. A genome scan of 18 families with chronic lymphocytic leukaemia.
Br J Haematol
2003
;
121
:
866
–873.
20
Ishibe N, Sgambati MT, Fontaine L, et al. Clinical characteristics of familial B-CLL in the National Cancer Institute Familial Registry.
Leuk Lymph
2001
;
42
:
99
–108.
21
University of California, Santa Cruz. BLAT: Blast-Like Alignment Tool Available at: http://genome.ucsc.edu/cgi-bin/hgBlat. Accessed December 5, 2006.
22
Toro JR, Nickerson ML, Wei MH, et al. Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America.
Am J Hum Genet
2003
;
73
:
95
–106.
23
ABI: Guide to performing relative quantitation of gene expression using real-time quantitative PCR
2004
;Foster City, CA PE Applied Biosystems Tutorial part no. 4371095.
24
National Center for Biotechnology Information. Basic Local Alignment Search Tool (BLAST) http://www.ncbi.nlm.nih.gov/blast/. Accessed December 5, 2006.
25
Carter PH, Resto-Ruiz S, Washington GC, et al. Whole blood lysis: a flow cytometric analysis of three anticoagulants and five cell preparations.
Cytometry
1992
;
13
:
68
–74.
26
Duque RE, Andreef M, Braylan RC, Diamond LW, Peiper SC. Consensus review of the clinical utility of DNA flow cytometry in neoplastic hematopathology.
Cytometry
1993
;
14
:
492
–496.
27
Fleisher TA and Marti GE. Detection of unseparated human lymphocytes by flow cytometry. In Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W (Eds.).
Current Protocols in Immunology
1991
;New York, NY John Wiley and Sons Vol
1
: pp.
7.9.1
–7.9.7.
28
Kamihara S, Matutes E, Marco JG, et al. Flow cytometry detection of minimal DNA aneuploidy in mature lymphoid leukemias: comparison with metaphase and interphase cytogenetics.
Int J Oncol
1994
;
5
:
211
–214.
29
Zenger ZE, Vogt R, Mandy F, Schwartz A, Marti GE. Quantitative flow cytometry: inter-laboratory variation.
Cytometry
1998
;
33
:
138
–145.
30
Nicholson JKA, Rao PE, Calvelli T, et al. Artifactual staining of monoclonal antibodies in two-color combinations is due to an immunoglobulin in the serum and plasma.
Cytometry
1994
;
18
:
140
–146.
31
Shankey TV, Rabinovitch PS, Bagwell B, et al. Guidelines for implementation of clinical DNA cytometry: International Society for Analytical Cytology.
Cytometry
1993
;
14
:
472
–477.
32
University of California Santa Cruz. Genome Bioinformatics http://genome.ucsc.edu/. Accessed December 5, 2006.
33
European Bioinformatics Institute and Wellcome Trust Sanger Institute. Ensembl Genome Browser http://www.ensembl.org. Accessed December 5, 2006.
34
Wellcome Trust Sanger Institute. miRBase::Sequences http://microrna.sanger.ac.uk/sequences/. Accessed December 5, 2006.
35
Rozenblum E, Vahteristo P, Sandberg T, et al. A genomic map of a 6-Mb region at 13q21-q22 implicated in cancer development: identification and characterization of candidate genes.
Hum Genet
2002
;
110
:
111
–121.
36
Rawstron AC, Yuille MR, Fuller J, et al. Inherited predisposition to CLL is detectable as subclinical monoclonal B-lymphocyte expansion.
Blood
2002
;
100
:
2289
–2291.
37
Larramendy ML, Kushnikova T, Bjorkvist AM, et al. Comparative genomic hybridization reveals complex genetic changes in primary breast cancer tumors and their cell lines.
Cancer Genet Cytogenet
2000
;
119
:
132
–138.
38
Kainu T, Juo SH, Desper R, et al. Somatic deletions in hereditary breast cancers implicate 13q21 as a putative novel breast cancer susceptibility locus.
Proc Natl Acad Sci U S A
2000
;
97
:
9603
–9608.
39
Dong JT, Chen C, Stultz BG, Isaacs JT, Frierson HF Jr. Deletion at 13q21 is associated with aggressive prostate cancers.
Cancer Res
2000
;
60
:
3880
–3883.
40
Gray SG, Kytola S, Matsunaga T, Larsson C, Ekstrom TJ. Comparative genomic hybridization reveals population-based genetic alterations in hepatoblastomas.
Br J Cancer
2000
;
83
:
1020
–1025.
41
Szekeres-Bartho J, Kilar F, Falkay G, et al. The mechanism of the inhibitory effect of progesterone on lymphocyte cytotoxicity, I: progesterone-treated lymphocytes release a substance inhibiting cytotoxicity and prostaglandin synthesis.
Am J Reprod Immunol Microbiol
1985
;
9
:
19
–22.
42
Lachmann M, Gelbmann D, Kalman E, et al. PIBF (progesterone induced blocking factor) is overexpressed in highly proliferating cells and associated with the centrosome.
Int J Cancer
2004
;
112
:
51
–60.
43
Salisbury JL, Whitehead CM, Lingle WL, Barrett SL. Centrosomes and cancer.
Biol Cell
1999
;
91
:
451
–460.
44
Nagase T, Ishikawa K, Suyama M, et al. Prediction of the coding sequences of unidentified human genes, XIII: the complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.
DNA Res
1999
;
6
:
63
–70.
45
Wu K, Yang Y, Wang C, et al. DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.
J Biol Chem
2003
;
278
:
51673
–51684.
46
Chen C, Bhalala HV, Vessella RL, Dong JT. KLF5 is frequently deleted and down-regulated but rarely mutated in prostate cancer.
Prostate
2003
;
55
:
81
–88.
47
Chen C, Bhalala HV, Qiao H, Dong JT. A possible tumor suppressor role of the KLF5 transcription factor in human breast cancer.
Oncogene
2002
;
21
:
6567
–6572.
48
Imhof A, Schuierer M, Werner O, et al. Transcriptional regulation of the AP-2alpha promoter by BTEB-1 and AP-2rep, a novel wt-1/egr-related zinc finger repressor.
Mol Cell Biol
1999
;
19
:
194
–204.
49
Pellikainen J, Kataja V, Ropponen K, et al. Reduced nuclear expression of transcription factor AP-2 associates with aggressive breast cancer.
Clin Cancer Res
2002
;
8
:
3487
–3495.
50
Slater H, Shaw JH, Dawson G, et al. Maternal uniparental disomy 13 in a phenotypically normal child.
J Med Genet
1994
;
31
:
644
–646.
51
Berend SA, Feldman GL, McCaskill C, et al. Investigation of two cases of of paternal disomy 13 suggests timing of isochromosome formation and mechanisms leading to uniparental disomy.
Am J Med Genet
1999
;
82
:
275
–281.
52
Soler A, Margarit E, Queralt R, et al. Paternal isodisomy 13 in a normal newborn infant after trisomy rescue evidenced by prenatal diagnosis.
Am J Med Genet
2000
;
90
:
291
–293.
53
Sellick GS, Webb EL, Allinson R, et al. A high-density SNP genomewide linkage scan for chronic lymphocytic leukemia-susceptibility loci.
Am J Hum Genet
2005
;
77
:
420
–429.

Supplemental data

Sign in via your Institution