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Technical Briefs |
1 Laboratory of Molecular Biology, Department of Medical Biopathology, and3 Clinical Hematology, Service of Hematology, Hospital Universitario La Fe, Valencia, Spain;2 TIB MOLBIOL, Berlin, Germany;
aaddress correspondence to this author at: Laboratory of Molecular Biology, Escuela de Enfermería 7°, Hospital Universitario La Fe. Avda. Campanar 21, 46007 Valencia, Spain; fax 34-91-806-1206, e-mail bolufer_pas{at}gva.es
The prediction that there might be common DNA sequence variants that confer a small but appreciably enhanced risk of cancer has been validated with the discovery of the germ-line mutation 1100delC in the cell cycle checkpoint kinase gene (CHEK2; OMIM 604373) (1)(2). CHEK2 is located on chromosome 22q and encodes the human ortholog of yeast Cds1 and Rad53, which are G2-checkpoint kinases (3). CHEK2 is a protein kinase activated in response to DNA damage involved in cell-cycle arrest. It serves as a link in the ATM-CHEK2-CDC25A-CDK2 pathway that checks genomic integrity in response to DNA damage (4). The 1100delC mutation in exon 10 abolishes the kinase function of CHEK2 (5) and has been reported in patients with Li–Fraumeni syndrome in the United States and in Finnish families with a cancer phenotype suggestive of Li–Fraumeni syndrome, including breast cancer (5).
There have been recent reports of a higher incidence of the 1100delC mutated allele in patients with a family history of breast cancer who are not carriers of BRCA1 (OMIM 113705) or BRCA2 (OMIM 600185) mutations, compared with healthy controls (4.2% and 5.5% in breast cancer cases vs 1.4% and 1.1% in controls, respectively) (1)(6). The presence of the mutated allele approximately doubles the breast cancer risk in women and increases it 10-fold in men (1). It has also been reported that 4.8% of individuals with familial prostate cancer are carriers of distinct CHEK2 germ-line mutations (7). These mutations may contribute to prostate cancer risk, highlighting the importance of the integrity of DNA damage-signaling pathways in the development of prostate cancer.
The few reports for patients with hematologic malignancies have been concerned mainly with CHEK2 somatic mutations (8)(9)(10). Thus, in a series of 109 patients with leukemia and myelodysplastic syndrome (MDS), two somatic mutations were reported: one among 55 patients with acute myeloid leukemia and another among seven with non-Hodgkin lymphomas (8). Similarly, two somatic mutations were found in a study carried out on 10 patients with MDS and 3 with acute myeloid leukemia (9), and in a study carried out on 60 patients with non-Hodgkin lymphoma, there was a germ-line mutation in 1 with a mantle cell lymphoma (10). Thus, in general, there is a low incidence and low relevance of CHEK2 somatic mutations in the etiology of leukemia.
However, little is known about the relevance of the germ-line mutations of CHEK2 in the risk for developing leukemia and whether the germ line 1100delC CHEK2 sequence variant confers higher risk of leukemia, especially for treatment-related leukemia (TRL) and MDS. To address this point, we screened for the 1100delC CHEK2 germ-line mutations in patients with leukemia or TRL/MDS and in a control group.
We studied 107 patients with acute leukemia (AL). Two patients had AL (one type B, common; and one biphenotypic), and 105 had acute myeloid leukemia: 1 with French-American-British subtype Mo, 11 with subtype M1, 13 with subtype M2, 65 with subtype M3, 4 with subtype M4, 2 with subtype M4Eo, 5 with subtype M5, 2 with subtype M6, 1 who was not classified, and 1 with subtype M1 at relapse. There were 52 males and 55 females, and the median age was 46 years (range, 1–78 years). We also studied a group of 26 patients with TRL/MDS (15 males and 11 females) with a median age of 64 years (range, 7–87 years) at the time of diagnosis of the primary tumor (Table 1
). The control group consisted of 176 healthy volunteers (69 males and 107 females) who had a median age of 36.5 years (range, 16–75 years).
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DNA was extracted from 500 µL of whole blood anticoagulated with EDTA by use of MagNA Pure LC DNA Isolation Kits-Large Volume (Roche Diagnostics) with the MagNA Pure LC System (Roche).
One established method for detecting 1100delC CHEK2 starts with a long-range PCR amplifying an 
10-kb fragment, because there are at least six to eight genomic copies of similar genes containing identical exons 10–14 (Dr. Mieke Schutte, Medical Oncology, Rotterdam, The Netherlands, personal communication) (11). For the analysis of variants, we strongly prefer mutation-specific probes because the high melting temperature is valid proof of sequence identity and presence of the mutation. However, direct analysis of the gene using the primers CHEK2F (gi 6911603, positions 104284–104307) and CHEK2R (positions 104063–104084), as reported by Sodha et al. (11), in which a 245-bp fragment is amplified and melting curve analysis is performed with a 25mer probe specific for the mutation, failed, whereas the equivalent 26mer wild-type-specific probe gave a weak signal. The missing signal was probably caused by low probe binding because of high content of weak bases (80% AT). The substitution of 7 of 13 thymidine nucleotides by locked nucleic acid (LNA) derivatives (12) in the sensor probe enhanced the sensitivity and allowed the analysis to be performed without the initial long-range PCR step. We substituted only thymidine bases to study different substitution variants and also because LNA synthesis is less efficient and more expensive. The melting temperatures were calculated by use of the Tm calculator on the Exiqon Web page (http://www.exiqon.com).
Detection of 1100delC CHEK2 included two steps, starting with a conventional PCR using the primers CHEK2F and CHEK2R; the amplification product was then diluted 1:200 and subjected to a second round of amplification in the LightCycler (Roche) assay. The reaction mixture (final volume, 10 µL) contained 4 pmol of the same primers (0.4 µM), 2 pmol (0.2 µM) each of anchorLC probe (5'-LCRed640-CAC TCC AAG ATT TTG GGA GAG ACC TCT-phosphate-3'; gi 6911603, positions 104228–104254) and delCL sensor probe (5'-TTT TAG ATT ATGA TTT T-fluorescein-3'; positions 104258–104275; LNA-dT base positions are underlined, and the position of the deletion is shown as ), and 1 µL of Fast Start DNA Master Hybridization probes (3.0 mM MgCl2). The LC PCR program initiates with 10 min of denaturation at 95 °C to activate the DNA polymerase (Fast Start DNA Master Hybridization probes) followed by 50 cycles each of 95 °C for 2 s, 64 °C for 5 s, and 72 °C for 10 s. The final melting was performed by increasing the temperature from 40 °C to 90 °C at 0.2 °C/s and continuously reading the fluorescence (channels F2/F1). In every assay, an 1100delC-positive heterozygous control (kindly provided by Dr. Mieke Schutte) was run to ensure its detection. The control sample was verified by DNA sequencing.
We compared different DNA and LNA-modified hybridization probes specific for the wild-type and the mutated gene. Wild-type-specific DNA detected the mutation in PCR products without the preamplification step, whereas the mutation-specific DNA probes failed to produce a signal.
In contrast, the LNA-modified probes showed a substantially decreased melting temperature for the mismatched target. Both wild-type- and mutation-specific probes enabled detection of the deletion without the preamplification step. With the mutation-specific LNA-modified hybridization probe, we obtained melting temperatures of 54 °C for the deleted allele and 46 °C for the wild-type allele (Fig. 1
).
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None of the 309 samples analyzed among the three groups (AL, TRL/MDS, and controls) carried the 1100delC CHEK2 mutation, making the differences in the age ranges between the test and control populations irrelevant. Despite these negative results, in all samples tested, the reliability of the method was supported by the positive heterozygous control for 1100delC systematically used in every assay.
The results are in complete agreement with previous reports for Spanish patients with familial breast cancer (13) in which the authors were unable to detect the 1100delC CHEK2 mutant variant in any of 856 samples analyzed for both cases and controls. A very low incidence of 0.3% was described for this mutation in a population from New York (14). However, this mutation has been found at a frequency of 1.4% in a Finnish population (6) and 1.1% in control individuals from the United Kingdom, The Netherlands, and North America (1).
The lack of detection of this germ-line mutation among the 107 patients with AL is in agreement with most reports on hematologic malignancies, which show a scarcity of somatic CHEK2 mutations (8)(9)(10), but not the CHEK2*1100delC germ-line mutation.
The association of this mutation with cancer risk is dependent mainly on its incidence in the general population, which varies greatly among the groups studied. Thus, in the studies carried out in populations from The Netherlands or Finland, where the incidence of the 1100delC CHEK2 mutant in controls was highest, the presence of the 1100delC CHEK2 allele was associated with a high risk for breast cancer (1)(6). However, the absence of this mutation in the present study and in another study involving a Spanish population (13) and the very low incidence found in a study performed in New York (14) make the presence of this mutation clinically irrelevant.
Acknowledgments
We would like to express our gratitude to Dr. Mieke Schutte for providing a positive control sample. This study was supported by Grant PI020180 of the Spanish "Fondo de Investigacion Sanitaria" (FIS).
References
The following articles in journals at HighWire Press have cited this article:
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  M. Weischer, S. E. Bojesen, A. Tybjaerg-Hansen, C. K. Axelsson, and B. G. Nordestgaard Increased Risk of Breast Cancer Associated With CHEK2*1100delC J. Clin. Oncol., January 1, 2007; 25(1): 57 - 63. [Abstract] [Full Text] [PDF]
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