br Genotype analysis br PARK promoter
2.3. Genotype analysis
PARK2 promoter variants rs2276201 and rs9347683 were selected for genotyping. These two SNPs were analyzed using Polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) assay. Primer sequences used for PCR, product size, restriction endonucleases (NEB, USA) and size of various digested fragments obtained are de-scribed in Supp. Table 1. PCR reactions were performed in a 25 μl re-action volume containing 100 ng genomic DNA, 10 pM of each primer, 0.2 mM dNTPs, 2.5 mM MgCl2, and 1 unit of Taq polymerase (Genei Bangalore India). The PCR α-CEHC conditions consisted of an initial de-naturation step at 95 °C for 5 min followed by 35 cycles of 30 s at 94 °C 30 s at 58 °C for rs2276201 and 59 °C for rs9347683; 30 s at 72 °C; and a final elongation at 72 °C for 10 min. Digested fragments were resolved on 2.5% agarose gel. 10% samples were randomly selected and were re-genotyped and 5% of randomly selected samples were directly se-quenced in order to validate the data generated by PCR-RFLP assay (Fig. 1). DNA sequencing was carried out at Chromos Bangalore, India.
2.4. Methylation specific-polymerase chain reaction (MS-PCR)
Characteristics Cases (%) Controls (%) P value
To determine the expression level of Parkin protein, Western blot analysis was performed. Briefly, proteins were extracted with a buﬀer containing 20 mM TRIS (pH 7.4), 2 mM EDTA, 0.5 mM EGTA, 50 mM mercaptoethanol, 0.32 mM sucrose and a protease inhibitor cocktail (Roche Diagnostics) by using a Teflon-glass homogenizer and were then sonicated twice for 20 s by using an ultrasonic probe, followed by centrifugation at 10,000g for 10 min at 4 °C. The concentration of the protein was determined by the BCA Protein Assay Kit (Thermo Scientific). Each sample containing about 30 μg protein homogenate was diluted in 2× Laemmli buﬀer (Biorad, Carlsbad, Calif., USA), he-ated at 90 °C for 5 min. Proteins were separated on Biorad Criterion XT BIS-TRIS 10% and then electrotransferred onto a nitrocellulose mem-brane (Biorad). Blots were blocked by using the Blocking Buﬀer (Hi-Media) and probed with appropriate antibodies: anti-Park2 polyclonal antibody (cat. no. STJ94956, St. Jones Laboratory, 1:500) and rabbit anti-β-actin (cat. no. sc-9104, Santa Cruz Biotechnology; 1:1500). The secondary antibodies, namely goat anti-rabbit IRDye 800CW (cat. no. 926-32211, Li-Cor Biosciences) or goat anti-mouse IRDye 680CW (cat. no. 926-68020D, Li-Cor Biosciences) were used at dilutions of 1:20,000 and 1:30,000, respectively. Blots were detected by chemiluminescence method.
2.6. Statistical analysis
Hardy–Weinberg equilibrium (HWE) testing of both the SNPs was consistent with the distribution. To compare the overall distribution of genotypes between patients and healthy controls 3 × 2 χ2 test was performed. Associations between individual genotypes and risk of col-orectal cancer were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs), using logistic regression model with correction for age. All statistical analysis were performed using SPSS
Fig. 1. Sequencing electropherogram of Park2 promoter polymorphisms. Representative sequencing pictures of rs2276201 polymorphism with genotypes T, C, T/C (Fig. 1a–c) and rs93476831 polymorphism with genotypes A, C, A/C (Fig. 1d–f).
Genotype and allele frequencies of PARK2 promoter polymorphisms in colorectal cancer cases and controls.
*P < 0.025 values significant after Bonferroni coeﬃcient. OR odds ratio, CI confidence interval. a P value for Hardy–Weinberg equilibrium testing. b P value for 3 × 2 χ2 test of comparision of overall genotype frequencies between cases and controls. c P value and corresponding age-adjusted OR (aOR) with 95% Cls[aOR(95% CI)] for comparision of genotype frequencies between cases and controls by logistic regression analysis (age is not adjusted in allele frequency comparisions).
statistical package, version 17 (SPSS Inc., Chicago, IL, USA) and P < 0.05 was considered statistically significant. All P-values were corrected for multiple comparisons according to Bonferroni method.
2.7. Transcription factor binding prediction
Transcription factor binding site (TFBS) in the polymorphic site was predicted using freely available online tools PROMO. PROMO is a freely available online tool for the identification of putative transcription
factor binding site from a species of interest. TRANSFAC database (version 8.3) is utilized by PROMO to predict specific binding site. Transcription factors binding is predicted within dissimilarity margin less or equal to fifteen percent.
The genotype frequencies in both colorectal cancer patients and controls were in agreement with Hardy-Weinberg equilibrium for both