Study Subjects. We analyzed 27 peripheral blood samples from patients who had surgical treatment ofprimary BC. All cases were invasive ductal carcinomas. None had any familial history ofBC. Histopathological characteristics (tumor stage and size, histological agressivity) were ascertained from medical records. The age ofthe patients ranged from 37-82 years old, with a mean age of 57.11 ± 12.98, and a median age of 54 years old. Control subjects (n = 30) were randomly selected from healthy female (free of diagnosed cancer) blood donors, matched by age and menopausal status. The age ranged from 19 to 81 years old, with a mean age of 51 -96 (35 ± 11) and a median age of 52 years old.
Genotyping the Polymorphism. Genomic DNA was extracted from buffy coat obtained from EDTA-blood, using Qiagen QIAamp blood kit. The (CA)n microsatellite was amplified using a fluoresce labeled primer [5'-GGT AAA CCA TGG TCT GTA CC-3' (forward)], and an un-labeled primer [5'-AAC AAA ATG TTG AAT GAG TGG G-3' (reverse)]. Aliquots (-100 ng ofDNA) were amplified in a 25 ul reaction volume, containing 1.5 mM MgCl2> 5 pM of each primer, 200 nM dNTP, and 1.25 units of Taq-polymerase. PCR amplification was performed by an initial denaturation at 94°C/2 min, followed by a 35 cycle amplification (94°C/45 sec, 53°C/45 sec, and 72°C/45 sec) followed by a final extension at 72°C/2 min. The PCR products were separated on a polyacrylamide gel by automated fluorescence detection in a sequencer . Allele sizes were interpolated using a size standard, 20 DNA fragments from 60 to 400 bp, and with a CEQ™ 8000 DNA Analysis System. The results were confirmed by re-running quality control samples.
Statistical Analysis. Mantel-Haenszel %2 and t-test statistics across matched case-control sets were used to compare the distribution of the different alleles. All p-values are from two-tailed tests. The influence ofthe genotype length on the risk of BC was tested with an unconditional linear logistic regression. Because the results of significance tests may depend on the choice of cut-points for the continuous variable, we tested the differences among the allele size distributions by use of the Mann Whitney U test. All analyses were performed with the use of STATISTICA 6.0 software (StatSoft Inc, USA). Statistical significance level was set at a p value = 0.05.
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