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On-line version ISSN 1678-4685
Genet. Mol. Biol. vol.28 no.3 São Paulo July/Sept. 2005
HUMAN AND MEDICAL GENETICS
Márcia Cristina DuarteI; Jucimara ColomboI; Andréa Regina Baptista RossitII; Ana Elizabete SilvaI
IUniversidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, Departamento de Biologia, São José do Rio Preto, SP, Brazil
IIFaculdade de Medicina de São José do Rio Preto, Centro de Investigação de Microorganismos, São José do Rio Preto, SP, Brazil
In several DNA repair genes, polymorphisms may result in reduced repair capacity, which has been implicated as a risk factor for various types of cancer. The frequency of the polymorphic alleles varies among populations, suggesting an ethnic distribution of genotypes. We genotyped 300 healthy Southeastern Brazilian individuals (262 of European ancestry and 38 of African ancestry) for polymorphisms of codons 194 and 399 of the XRCC1 base excision repair pathway gene and of codon 241 of the XRCC3 homologous recombination repair pathway gene. The allele frequencies were 0.07 for the Arg194Trp and 0.33 for the Arg399Gln codons of the XRCC1 gene and 0.35 for the Thr241Met codon of the XRCC3 gene. The genotypic frequencies were within Hardy-Weinberg equilibrium. These frequencies showed ethnic variability when compared with those obtained for different populations from several countries.
Key words: DNA repair, XRCC1, XRCC3, polymorphism, ethnic variability.
Different DNA repair systems maintain the integrity of the human genome, so deficiency in the repair capacity due to mutations or polymorphisms in genes involved in DNA repair can lead to genomic instability that, in turn, is related to chromosomal instability syndromes and increased risk of developing various types of cancer (Mohrenweiser and Jones, 1998; Hansen and Kelley, 2000).
Several polymorphisms in DNA repair genes (XPD, XPF, ERCC1, XRCC1, XRCC3, XPA, XPB, XPC and hOGG1) representing different repair pathways have been reported (Shen et al., 1998; Ishida et al., 1999; Butkiewicz et al., 2000; Chavanne et al., 2000).
The human genes XRCC1 and XRCC3 belong to the X-Ray Repair Cross Complementing family and have been identified by their ability to restore DNA repair activity in Chinese hamster ovary (CHO) mutant cell lines EM9 (Thompson et al., 1990) and irs1SF (AA8) (Fuller and Painter, 1988), respectively. The XRCC1 protein plays a role in base excision repair (BER), interacts with DNA ligase III and complexes with DNA polymerase and PARP (poly ADP-ribose polymerase), facilitating the repair of DNA strand breaks and several types of DNA damage (Dianov et al., 2003). Shen et al. (1998) identified three polymorphisms in the XRCC1 gene at conserved sequences, resulting in amino acid substitutions at codons 194 (Arg194Trp; reported allele frequency, RAF = 0.25), 280 (Arg280His; RAF = 0.08) and 399 (Arg399Gln; RAF = 0.25) in only twelve healthy individuals from an unspecified population.
Many authors have analyzed these polymorphisms in human populations and found a significant association between the Arg194Trp and Arg399Gln variants and increased risk of early-onset colorectal carcinoma (Abdel-Rahman et al., 2000; Krupa and Blaviak, 2004) and gastric cardia cancer (Shen et al., 2000), besides head and neck cancer (Olshan et al., 2002) and skin cancer (Han et al., 2004) associated with the Arg194Trp variant and breast cancer (Duell et al., 2001), lung cancer (Zhou et al., 2003) and esophageal cancer (Yu et al., 2004), among others, associated with Arg399Gln polymorphism.
The product of the XRCC3 gene functions in the homologous recombination repair (HRR) for double-strand breaks (DSBs) and cross-link repair in mammalian cells (Dianov et al., 2003). During HRR the XRCC3 protein interacts with the Rad51C protein and possibly with the Rad51 protein itself, enabling Rad51 protein multimers to assemble at the site of damage (Brenneman et al., 2002). A common polymorphism in exon 7 of the XRCC3 gene results in an amino acid substitution at codon 241 (Thr241Met) that may affect the enzyme's function. The XRCC3 variant allele has been identified in healthy individuals at a frequency ranging from 0.23 to 0.38 (Shen et al., 1998; David-Beabs et al., 2001), and has been associated with increased risk of melanoma (Winsey et al., 2000), bladder cancer (Matullo et al., 2001), breast cancer (Smith et al., 2003) and lung cancer (Jacobsen et al., 2004).
The frequencies of polymorphisms of both metabolic and repair enzymes are distinct in different ethnic groups (Kato et al., 1992; Stephens et al., 1994; Lunn et al., 1999; Abdel-Rahman et al., 2000), based an which we conducted a study to estimate the frequency of the variants Arg194Trp and Arg399Gln in the XRCC1 BER gene and Thr241Met in the XRCC3 HRR gene in healthy individuals from a southeastern Brazilian population. As far as we know, this is the first study carried out to evaluate XRCC3 polymorphism in a Brazilian population.
Blood samples were collected from 300 healthy individuals (164 males and 136 females; mean age of 52 years, range 19 to 93 years) in the São José do Rio Preto region in the southeastern Brazilian state of São Paulo at a general hospital (Hospital de Base) and at the São José do Rio Preto campus of the Universidade Estadual Paulista - UNESP, São Paulo, Brazil. Based on their visual appearance and an interview the participants were ethnically classified as 262 individuals of European descent and 38 of African descent.
This study was approved by the National Research Ethics Committee, and written informed consent was obtained from all individuals.
The DNA from the blood samples was extracted as described by Abdel-Rahman et al. (1994). The XRCC1 genotypes for codons 194 (492 bp) and 399 (615 bp) were detected using multiplex PCR-restriction fragment length polymorphisms (RFLP) (Abdel-Rahman et al. 2000), to amplify the fragments and codon 241 of the XRCC3 gene was genotyped by the PCR-RFLP technique described by David-Beabs et al. (2001). The amplified fragments of codons 194 and 399 of the XRCC1 gene were digested with the MspI restriction enzyme and codon 241 of the XRCC3 gene with the NlaIII restriction enzyme and resolved on 2% 1000 agarose gel (Invitrogen, Brazil).
Statistical analyses were performed using the Statdisk v.9.5.5 computer software program and the chi-square test (c2) used to compare the genotype frequencies and ethnicity. c2 values with a probability (p) value greater than 0.05 being considered as coming from the same statistical population and hence not significant.
We found that the XRCC1 gene allele frequencies were 0.07 for the 194Trp polymorphism and 0.33 for the 399Gln polymorphisms, whereas the XRCC3 gene 241Met polymorphism allele frequency was 0.35. Genotypes distributions were within Hardy-Weinberg equilibrium (c2 = 0.24 for 194Trp, c2 = 1.69 for 399Gln and c2 = 0.72 for 241Met; P = 0.05). Figure 1 shows the banding patterns of the XRCC1 (A) and XRCC3 (B) polymorphisms.
No statistically significant differences in ethnicity were observed with respect to the 194Trp (p = 0.7337), 399Gln (p = 0.4048) and 241Met (p = 0.5306) allele frequencies. According to the literature, it seems that the genotype distribution of the XRCC1 and XRCC3 genes does not vary between sexes, but differences between ethnic groups have been suggested (Lunn et al., 1999; Abdel-Rahman et al., 2000; Shen et al., 2000). Table 1 shows the frequency distributions of the XRCC1 and XRCC3 genotypes in healthy individuals of our current study and compares these frequencies with those previously published for different ethnic groups.
In our study the allele frequencies of the 194Trp polymorphism in European descent (0.07) and African descent (0.09) were similar to those previously published by Rossit et al. (2002) for 96 healthy Brazilians from the same region. Our results also agree with those observed for American Caucasians (Lunn et al., 1999; David-Beabs and London, 2001; Smith et al., 2003), African Americans (Lunn et al., 1999; David-Beabs and London, 2001) and Egyptians (Abdel-Rahman et al., 2000) but not with the frequencies reported for Asians (Lunn et al., 1999; Shen et al., 2000).
The frequency of 0.34 for the XRCC1 399Gln allele in our European descent group was comparable with that described by Rossit et al. (2002) in a group of Brazilian Caucasians and in North American (Lunn et al., 1999; David-Beabs and London, 2001; Smith et al., 2003) and Italian Caucasians (Matullo et al., 2001), although it was statistically higher (p) than that reported for Asiatic populations (Lunn et al., 1999; Shen et al., 2000; Park et al., 2002; Yeh et al., 2005) and Egyptian (Abdel-Rahman et al., 2000). Although the difference observed in our African descent group was not significant (p > 0.05), the XRCC1 399Gln allele frequency (0.26) was lower than that of the European descent group but higher than that previously reported for African Americans (Lunn et al., 1999; David-Beabs and London, 2001).
We found that allele frequency of the XRCC3 241Met polymorphism was similar in Brazilians of European (0.36) and African (0.31) descent, these frequencies being comparable to those observed in North American (David-Beabs et al., 2001; Smith et al., 2003) and Italian (Matullo et al., 2001) studies but statistically higher (p < 0.05) than those observed in African American (David-Beabs et al., 2001) and Asiatic (Shen et al., 2004; Yeh et al., 2005) populations.
The Brazilian population is very heterogeneous, as a result of sexual congress between different ethnic groups, including native Indians and immigrants from Europe, Africa and Asia. Our results are in accordance with this fact, since no significant difference was found between the two Brazilians ethnic groups studied, although there were differences in the allele frequencies when these two groups were compared to Asiatic and Egyptian populations. Our data suggests that admixture plays an important role in the distribution of the genotypes studied and, because Brazil is large country, there may be differences in genotype distribution in the different States because of the diverse origins of the immigrants which settled in each state. The results reinforce the importance of further studies on polymorphisms of DNA repair genes that may play an important role in cancer susceptibility in different populations.
This study was supported by the Brazilian agency CAPES.
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Ana Elizabete Silva
Universidade Estadual Paulista
Instituto de Biociências, Letras e Ciências Exatas
Departamento de Biologia
Rua Cristóvão Colombo 2265
Jardim Nazareth, 15054-000
São José do Rio Preto, SP, Brazil
Received: December 23, 2004; Accepted: May 4, 2005.
Associate Editor: Francisco Mauro Salzano