Analysis of the polymorphisms in non-homologous DNA end joining (NHEJ) gene Ku70 and Ligase IV in sporadic breast cancer in women

Introduction
Breast cancer is an increasingly important cause of illness and death among women. Breast cancer affects one out of every 10 women in industrialized countries, and is a leading cause of cancer morbidity and mortality in women.
Genomic rearrangements such as translocations, deletions, and duplications are extremely frequent in cancer cells, particularly, in breast cancer cells [1–4]. Genomic rearrangements are believed to result from the aberrant repair of DNA double-strand breaks (DSBs).
A double-strand break (DSB) is the most lethal of all DNA lesions. If unrepaired, a DSB leads to loss of chromosome segments and threatens the survival of the cell. Equally detrimental to the organism are misrepaired DSBs that destabilize the genome and lead to genomic rearrangements. Genomic rearrangements become common in aging organisms, ultimately leading to deregulation of transcription and malignancies [5, 6]. DSBs in DNA are repaired by two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ) [7, 8].
During HR-mediated repair of DSB, the sister chromatid is used as a template to copy the missing information into the broken locus. In contrast, the NHEJ pathway simply fuses two broken ends with little or no regard for sequence homology. NHEJ starts with binding of Ku70/Ku80 heterodimer to the broken ends. Ku facilitates recruitment of Artemis-DNA-PKcs complex, which processes the ends to prepare them for ligation [9].
Next, the gaps are filled by DNA polymerase of the poIX family, and covalently joined by XRCC4-DNA ligase IV complex. NHEJ is rarely error-free, leading to deletions or insertions of filler DNA [10]. DSBs situated between two direct repeats can also be repaired by single-strand annealing (SSA), a highly mutagenic recombinational mechanism in which the sequence between the repeats is deleted.
Because single nucleotide polymorphism (SNP) is the most frequent and most subtle genetic variation in the human genome and has great potential for application to association studies of complex diseases [11], we used SNPs in the NHEJ genes Ku70 and Ligase IV to define their tumourigenic contribution to breast cancer development.
In the present work the association between Ku70, A46922G polymorphism and Ligase IV, A6008G (Ile591Val) polymorphism and breast cancer risk in Polish women was investigated.

Materials and methods

Patients
Blood was obtained from 135 postmenopausal women with node-negative (n = 55) and node-positive (n = 80) ductal breast carcinoma treated at the Department of Oncology, Institute of Polish Mother's Memorial Hospital. No distant metastases were found in patients at the time of treatment. The patients ranged in age from 43 to 82 years (median age 58 years). Median follow-up of patients at the time of analysis was 39 months (range: 2-71 months). The average tumour size was 20 mm (range: 17-32 mm). All tumours were graded by a method based on the criteria of Scarf-Bloom-Richardson. There were 31 (23.0%) tumours of grade I, 81 (60.7%) of grade II and 23 (16.3%) of grade III in total. Steroid receptor status was not determined in the investigated group. Blood samples from age-matched healthy women (n = 60) served as the controls.

Genotyping
We genotyped all patient and control samples for the listed polymorphisms (Table I) using a Big Day™ Terminator Cycle sequencing Ready Reaction Kit (Applied Biosystems) and the DNA ABI PRISM™ 377 sequence detection system (Applied Biosystems). TaqMan primers and probes were designed using Primer Express Oligo Design Software v1.0 (Applied Biosystems). Assays (15 µl) were carried out on 20 ng genomic DNA according to the manufacturer’s instructions. Amplifications were carried out on the GeneAmp 2400 PCR System (Perkin Elmer) at the annealing temperatures given in Table 1. Plates were read post-PCR on the ABI PRISM 377 sequence detector using the Big Day™ Terminator Cycle sequencing Ready Reaction Kit (Applied Biosystems).
The results were analysed by Sequencing Analysis Software™ ver. 3.4.1 (Applied Biosystems) and Factura™.

Statistical analysis
For each polymorphism, deviation of the genotype frequencies in the controls from those expected under the Hardy–Weinberg equilibrium was assessed using c2-test. Genotype frequencies in cases and controls were compared by c2-tests. P-values < 0.05 were considered to be significant.

Results
Table II shows the genotype distribution of Ku70 gene polymorphism between breast cancer patients and controls. It can be seen from the table that there were significant differences (p < 0.05) between the two investigated groups. The frequencies of the G and A alleles were 0.65/0.35 in patients and 0.54/0.46 in controls. In patients the observed frequencies of the G/G, G/A and A/A genotypes differed significantly (p < 0.05) from the distribution expected from the Hardy–Weinberg equilibrium.
Distributions of the A/A, A/G and G/G genotypes of the Ligase IV gene as well as the frequencies of the A and G alleles for breast cancer subjects and controls are displayed in Table III. It can be seen from the table that there were no significant differences between these two groups in both genotype distribution and allele frequencies (p > 0.05).
Dependencies of the distribution of genotypes and frequencies of alleles of both investigated polymorphisms on the tumour grade evaluated according to Scarf-Bloom-Richardson criteria in patients with breast cancer are displayed in Tables IV and V, respectively. There were no significant differences between distributions of genotypes in subgroups assigned to histological grades and the distribution predicted by the Hardy–Weinberg equilibrium (p > 0.05). There were no differences in frequencies of any of the alleles between subgroups either (p > 0.05).

Discussion
DNA is a precious molecule. It encodes vital information about cellular content and function. Damage to DNA can lead to cell cycle arrest, cell death or mutation.
In the NHEJ pathway, Ku70 and Ku80 then bind the DSB, followed by recruitment and activation of DNA-protein kinase (DNA-PK). XRCC4 and ligase IV (LIG4) are recruited by the DNA-PK holoenzyme, activated by DNA-PK-mediated phosphorylation, and then repair the break. Common variants in genes involved in DNA DSB repair are good candidates for low-penetrance breast cancer susceptibility. The aim of this study was to test the hypothesis that variants in genes Ku70 and LIG4 involved in DNA DSB repair confer increased susceptibility to breast cancer.
Kuschel et al. suggest that variants in XRCC2 and LIG4 alter breast cancer risk, together with stronger evidence that variants of XRCC3 are associated with risk. The variants in XRCC2 increase and those in LIG4 decrease breast cancer risk [12].
The variant alleles of 1310C>G (Ku70) and 2099-2408G>A (Ku80) are risk alleles for breast cancer as well as chromosomal radiosensitivity CRS. The homozygous variant genotype of 1781G>T (Ku70), on the other hand, seems to protect against breast cancer and ionizing radiation induced micronuclei [13].
The data show that two SNPs in Ku70 and XRCC4 may be associated with breast cancer risk. The increased risk of developing breast cancer was found in women harbouring a greater number of putative high-risk genotypes of NHEJ genes [14].
Fu et al. genotyped 30 SNPs in all five NHEJ genes (Ku70, Ku80, DNA-PKcs, Ligase IV, and XRCC4) in primary breast cancer patients. Two SNPs in Ku70 and XRCC4 were associated with breast cancer risk; this study provides new insights to suggest the role of the NHEJ pathway in breast cancer development and supports the possibility that breast cancer is initiated by oestrogen exposure, which causes DNA DSBs [14].
In premenopausal breast cancer in women four variants were associated with breast cancer risk: two in the XPF gene and two in the XRCC3 gene [15].
In the present work we did not find any correlation between occurrence of cancer and LIG4 gene polymorphism in Polish women. However, we detected a significant difference in distribution frequency of A and G alleles of Ku70 gene polymorphism between patients and controls (p < 0.05). The distribution of the genotypes G/G, G/A and A/A in patients differed from the one expected from the Hardy-Weinberg equilibrium, with an overrepresentation of G/G homozygotes. It is possible that the presence of the G allele is in linkage disequilibrium with another (so far unknown) mutation located outside the coding region in the Ku70 gene, which may be important for the Ku70 concentration in plasma.
On the other hand, we haven’t detected any significant difference between the genotypes in subgroups assigned to histological stages, which suggests a lack of association between the Ku70 polymorphism and breast cancer invasiveness.
Our study implies that polymorphism of the Ku70 gene may be associated with the occurrence of breast cancer in women from the Lodz region of Poland. Further studies, conducted on a larger group, are required to clarify this point.


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Address for correspondence
Beata Smolarz MD, PhD
Institute of Polish Mother’s Memorial Hospital
ul. Rzgowska 281/289
93-338 Łódź
e-mail: smolbea@wp.pl