versão impressa ISSN 1415-4757
Genet. Mol. Biol. vol.33 no.4 São Paulo 2010 Epub 12-Nov-2010
César Paz-y-MiñoI; Andrés López-CortésI; María José MuñozI; Bernardo CastroI,II; Alejandro CabreraI,II; María Eugenia SánchezI
IInstituto de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad de las Américas, Quito, Ecuador
IIEscuela de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Quito, Ecuador
The hRAD54 gene is a key member of the RAD52 epistasis group involved in repair of double-strand breaks (DSB) by homologous recombination (HR). Thus, alterations of the normal function of these genes could generate genetic instability, shifting the normal process of the cell cycle, leading the cells to develop into cancer. In this work we analyzed exon 18 of the hRAD54 gene, which has been previously reported by our group to carry a silent polymorphism, 2290 C/T (Ala730Ala), associated to meningiomas. We performed a PCR-SSCP method to detect the polymorphism in 239 samples including leukemia and normal control population. The results revealed that the 2290 C/T polymorphism has frequencies of 0.1 for the leukemia and 0.1 for the control group. These frequencies show no statistical differences. Additionally, we dissected the leukemia group in chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) to evaluate the polymorphism. The frequencies found in these subgroups were 0.14 for CML and 0.05 for ALL. We found statistically significant differences between CML patients and the control group (p < 0.05) but we did not find significant differences between ALL and the control group (p > 0.05). These results suggest a possible link between the 2290 C/T polymorphism of the hRAD54 gene and CML.
Key words: cancer, leukemia, CML, ALL, hRAD54, 2290 C/T polymorphism.
DNA repair is a major feature for maintenance of genome stability and integrity in living cells. DNA double-strand breaks (DSB) are lesions that threaten the integrity of the genome. If not properly processed, DSB can lead to cell cycle arrest or illegitimate DNA rearrangements such as translocations, inversions, or deletions, which contribute to cell dysfunction, cell death, or carcinogenesis (Wesoly et al., 2006).
It is known that the chromosomal aberrations that activate oncogenes and trigger the carcinogenic process always involve double-stranded DNA exchanges. This phenomenon may be produced by DSB (Lengauer et al., 1998). One major repair pathway in eukaryotic cells in order to eliminate deleterious effects is homologous recombination (HR) (Kooistra et al., 1997). HR is generally a precise way of resolving DSB and successful chromosome segregation during meiosis (Mazina and Mazin, 2008). Moreover, dysregulation of HR may lead to aberrant genetic rearrangements and genomic instability, resulting in various mutations (Park et al., 2008).
The hRAD54 gene plays an important role in homologous recombination and DNA double-strand break repair. Rad54 belongs to the Snf2/Swi2 protein family. It possesses a robust DNA-dependent ATPase activity, uses ATP hydrolysis to supercoil DNA and cooperates with the Rad51 recombinase in DNA joint formation (Smirnova et al., 2004; Rossi and Mazin, 2008). This gene has been mapped at chromosome 1p32 by fluorescent in situ hybridization (FISH) (Kanaar et al., 1996). The (1p32) region has been found to be a site of high rates of loss of heterozygosity, and concentrates six single nucleotide polymorphisms (SNP) that have been associated with genetic instability in meningiomas (Leone et al., 1999, 2003). hRAD54 also has been proposed as an oncosupressor in breast cancer and several point mutations in conserved regions of the hRAD54 gene have been found in primary tumors such as colonic adenocarcinoma, non-Hodgkin lymphoma and breast carcinoma (Smirnova et al., 2004). Moreover, the 1p32 region is a common partner for balanced translocation in hematological and lymphoid malignancies, e.g. t(1;14) (p32;q11) and t(1;3) (p32;q27) in leukemia and lymphoma, respectively (Cotter, 1998; Rubnitz and Pui, 1998).
In this work, we analyzed the status of the 2290 C/T silent polymorphism in exon 18 of the hRAD54 gene in the Ecuadorian population. It is reported that the polymorphism is associated with meningiomas (Leone et al., 2003), suggesting that this alteration could be involved in tumorigenesis. Therefore, the objective of this study was to relate the T allele distribution to leukemia development.
Leukemia is a malignant disease which is characterized by the presence of carcinogen cells in bone marrow and blood. According to the cancer cell type involved, this disease is classified as lymphocytic and myelogenous, which are divided in turn into acute and chronic. Therefore, leukemia is divided into four categories: acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (Gabert et al., 2003).
239 Ecuadorian individuals were analyzed, 137 individuals with clinical diagnosis of different types of leukemia (ALL = 61, CLL = 1, AML = 15, CML = 60) and 102 healthy individuals with no leukemia diagnosis as a normal control population. In Ecuador the most important ethnic group is known as "mestizo" (~60% of the total Ecuadorian population), the result of the mixture of Amerindians and Spaniards (Paz-y-Miño, 1998). The study was approved by the Ethical Committee of the Pontifical Catholic University of Ecuador. All study subjects received counseling and provided written consent for the study.
DNA extraction from bone marrow (leukemia patients and control population) was performed as described by (Sambrook et al., 1989). The bone marrow samples obtained from healthy individuals were taken prior to other medical diagnoses. The hRAD54 gene polymorphism 2290 C/T, in exon 18, was determined by the polymerase chain reaction - single-strand conformation polymorphism (PCR-SSCP) method. PCR was performed in a MJ Research PTC-200 thermal cycler (MJ Research, Watertown, Mass., USA) using primers designed by (Rasio et al., 1997). The amplification reaction was carried in a final volume of 20 µL, containing 1 X reaction buffer, 2.5 mM MgCl2 (Invitrogen, Carlsbad, CA), 200 µM of each dNTP, 2 µM of each sense and antisense primers, 1 U of Taq polymerase (Invitrogen, Carlsbad, CA) and 100 ng of genomic DNA as template. PCR conditions consisted of an initial denaturation at 94 ºC for 5 min; followed by 35 cycles of 94 ºC for 30 s, 54 ºC for 1 min, 1 min 30 s at 74 ºC, and a final extension at 72 ºC for 8 min. The amplified fragment of 242 bp was then denatured at 91 ºC for 5 min and snap-cooled for SSCP detection by electrophoresis on a 12% non-denaturing polyacrylamide (49:1 acrylamide:bisacrylamide) gel with 10% of glycerol.
Statistical analyses were done using PASW Statistics 17 for Windows (SPSS, Chicago). Allelic and genotypic frequencies were also calculated. The non-parametric Fisher's exact test was applied to determine significant differences between affected (CML = 60, ALL = 61) and healthy individuals.
The PCR-SSCP method allowed detection of the three genotypes of the hRAD54 gene polymorphism 2290 C/T (C/C, C/T, T/T) within the studied populations. From the total 239 individuals analyzed (478 alleles in total), 199 (83.26%) were homozygous for the normal allele (C/C), 38 (15.9%) had a heterozygous genotype (C/T) and two individuals (0.84%) were homozygous for the polymorphism (T/T). Of the 42 T alleles present in the studied population, 27 (64.29%) belonged to the study group and 15 (35.71%) to the control group. In the study group 17 (62.96%) individuals with CML and 6 (22.22%) individuals with ALL, presented the T allele (the percentages were calculated from the base of 27 alleles present in the total study group). The details of the genotypic distributions and allele frequencies of each group are presented in Table 1. The statistical analysis showed no significant differences between the control population and the study group (p > 0.05). When we applied the statistical test over the CML and ALL subgroups we found differences in the CML subgroup (p < 0.05), whereas the ALL showed no differences (p > 0.05).
HR is required to maintain genomic stability and its absence leads to potentially oncogenic translocations and other karyotypic changes. While this pathway exists to effect accurate and "safe" repair, it also has the potential to misrepair and generate deleterious products. Consequently, there is strong evolutionary pressure for mechanisms of apoptosis and cellular senescence which favor eliminating a cell with chromosomal damage from a dividing population over the risk of errant repair and oncogenic transformation (Ferguson and Frederick, 2001).
We found Hardy-Weinberg equilibrium in the allele frequency of the study population. Polymorphisms in genes that are responsible for maintenance of genome stability, such as hRAD54, could increase the risk to acquire cancer (Liu et al., 1999; Paz-y-Miño et al., 2003).
When the frequency of the polymorphism in the leukemia group (0.1) was compared with that in the normal control population (0.1), no statistical difference (p > 0.05) was observed. However, when we divided the study group into its two main subgroups, CML and ALL, the calculated frequencies for the CML subgroup (0.14) showed statistically significant differences (p < 0.05) from the control population, whereas those calculates for the ALL subgroup (0,05) did not. In spite of the results obtained from the statistical tests, it is important to note that size of the sample is not large; and that this may pose a limitation for the interpretation of the results.
It has been reported that t(9;22), responsible for some types of leukemia, especially CML, may be the result of an aberrant HR process, because the BCR and ABL genes involved in this translocation share some homology in their sequences (Bishop and Schiestl, 2002). These findings suggest that the recombinational repair of DSB by HR or spontaneous HR are both carried out by the RAD52 epistasis group (Mazina and Mazin, 2004); among which group the Rad54 protein plays an especially important role. The 2290 C/T polymorphism of the hRAD54 gene could be part of the mutator phenotype responsible for the appearance and progression of malignant transformation (Paz-y-Miño et al., 2003). However, this mutator phenotype seems to be linked to CML, but not to other types of leukemia (Sellick et al., 2008), suggesting that in fact the translocation involved in CML, t(9;22), is the result of mistakes of the DSB repair pathway by HR, in which the hRAD54 gene plays a key role (Bishop and Schiestl, 2002; Symington, 2002).
To SENACYT (Secretaría Nacional de Ciencia y Tecnología, Ecuador) for the financial support given to this investigation.
Bishop A and Schiestl R (2002) Homologous recombination and its role in carcinogenesis. J Biomed Biotechnol 2:75-85. [ Links ]
Cotter FE (1998) Lymphomas. In: Jameson JL (ed) Principles of Molecular Medicine. Humana Press, Aberdeen, pp 241-248. [ Links ]
Ferguson D and Frederick W (2001) DNA double strand break repair and chromosomal translocation: Lessons from animal models. Oncogene 20:5572-5579. [ Links ]
Gabert J, Beillard E, Van der Velden VHJ, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cavé H et al. (2003) Standarization and quality control studies of real-time quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - A Europe Against Cancer Program. Leukemia 17:2318-2357. [ Links ]
Kanaar R, Troelstra C, Swagemakers SMA, Essers J, Smit B, Franssen J, Pastink A, Bezzubova O, Buerstedde J, Clever B et al. (1996) Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: Evidence for functional conservation. Curr Biol 6:828-838. [ Links ]
Kooistra R, Vreeken K, Zonneveld JB, de Jong A, Eeken JC, Osgood CJ, Buerstedde JM, Lohman PH and Pastink A (1997) The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair radiation damage and recombination. Mol Cell Biol 17:6097-6104. [ Links ]
Lengauer C, Kinzler KW and Vogelstein B (1998) Genetic instabilities in human cancers. Nature 396:643-649. [ Links ]
Leone PE, Bello MJ, de Campos JM, Vaquero J, Sarasa JL, Pestaña A and Rey JA (1999) NF2 mutations and allelic status of 1p, 14q and 22qin sporadic meningiomas. Oncogene 18:2231-2239. [ Links ]
Leone PE, Mendiola M, Alonso J, Paz-y-Miño C and Pestaña A (2003) Implications of a RAD54L polymorphism (2290 C/T) in human meningiomas as a risk factor and/or genetic marker. BMC Cancer 3:e6. [ Links ]
Liu VF, Bhaumik D and Wang TS (1999) Mutator phenotype induced by aberrant replication. Mol Cell Biol 19:1126-1135. [ Links ]
Mazina O and Mazin A (2004) Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+. J Biol Chem 279:52042-52051. [ Links ]
Mazina O and Mazin A (2008) Human Rad54 protein stimulates human Mus81-Eme1 endonuclease. Proc Ntal Acad Sci USA 7:18249-18254. [ Links ]
Park JY, Yoo HW, Kim BR, Park R, Choi SY and Kim Y (2008) Identification of a novel human Rad51 variant that promotes DNA strand exchange. Nucleic Acids Res 36:3226-3234. [ Links ]
Paz-y-Miño C (1998) Ecuador. In: Penchaszadeh V (ed) Medical Genetic Services in Latin America. World Health Organization, New York, pp 14-16. [ Links ]
Paz-y-Miño C, Fiallo BF, Morillo SA, Acosta A, Giménez P, Ocampo L and Leone PE (2003) Analysis of the polymorphism [gIVS12-6T > C] in the hMSH2 gene in lymphoma and leukemia. Leuk Lymphoma 44:505-508. [ Links ]
Rasio D, Murakumo Y, Robbins D, Roth T, Silver A, Negrini M, Schmidt C, Burczak J, Fishel R and Croce CM (1997) Characterization of the human homologue of RAD54: A gene located on chromosome 1p32 at a region of high loss of heterozygosity in breast tumors. Cancer Res 57:2378-2383. [ Links ]
Rossi M and Mazin A (2008) Rad51 protein stimulates the branch migration activity of Rad54 protein. J Biol Chem 283:24698-24706. [ Links ]
Rubnitz JE and Pui CH (1998) Leukemias. In: Jameson JL (ed). Principles of Molecular Medicine. Humana Press, Aberdeen, pp 233-239. [ Links ]
Sambrook J, Fritsch E and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. [ Links ]
Sellick G, Foelding S, Qureshi M, Catocsky D, The International Familial CLL Consortium and Houlston R (2008) Germline mutations in RAD51, RAD51AP1, RAD51B, RAD51C, RAD51D, RAD52 and RAD54L do not contribute to familial chronic lymphocytic leukemia. Leuk Lymphoma 49:130-133. [ Links ]
Smirnova M, Van Komen S, Sung P and Klein HL (2004) Effects of tumor mutations on Rad54 functions. J Biol Chem 279:24081-24088. [ Links ]
Symington L (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 66:630-670. [ Links ]
Wesoly J, Agarwal S, Sigurdsson S, Bussen W, Van Komen S, Qin J, van Steeg H, van Benthem J, Wassenaar E et al. (2006) Differential contributions of mammalian Rad 54 paralogs to recombination, DNA repair and meiosis. Mol Cell Biol 976-989. [ Links ]
Send correspondence to:
Instituto de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad de las Américas
Av. de los Granados E12-41 y Colimes
Received: July 20, 2009; Accepted: July 13, 2010.
Associate Editor: Emmanuel Dias Neto
License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.