Computational protocol: Lack of association between the c.544G>A polymorphism of the heme oxygenase 2 gene and age related macular degeneration

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Protocol publication

[…] DNA fragment of HMOX2 gene containing the c.544G>A polymorphic site was amplified by polymerase chain reaction (PCR) in a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Total reaction volume for each sample was 25 μl and contained 10 ng of genomic DNA, 0.75 U Taq polymerase (Biotools, Madrid, Spain), 1 × reaction buffer, 0.5 mM dNTP, 1.5 mM MgCl2 and 0.25 μM of each primer (Sigma-Aldrich, St. Louis, MO, USA). Primers sequences were: forward 5′-AGGTGAGTGGCCTGTAAGTCC-3′, reverse 5′-TAGACCCAGAGCAGGAGGTG-3′. Thermal cycling conditions were: initial denaturation step at 95°C for 5 min, 34 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and amplification at 72°C for 1 min; final extension at 72°C for 5 min. Amplified fragments of 393 bp long, containing the polymorphic site were then digested with the restriction endonuclease HhaI (Fermentas, Burlington, Canada). The enzyme recognizes the G allele in the c.544G>A site and cleaves DNA generating 2 fragments: 204 and 189 bp long, whereas fragments carrying the A variant remained intact (). The digestion of 3 μl of PCR product in total volume of 15 μl was performed with 1 U of HhaI enzyme and 1 × digestion buffer for 6 min at 37°C. Then samples were separated on a 8% polyacrylamide gel in TBE buffer at 80 V. Gene Ruller (Fermentas, Burlington, Canada) was utilized as a molecular mass marker. [...] The allelic frequencies were estimated by gene counting, and genotypes were scored. The significance of the differences between distributions of alleles and genotypes was tested using the χ2 analysis. Unconditional logistic regression analysis was performed to assess the association between the genotypes of the polymorphism and AMD occurrence. The genotype-associated risk was expressed by crude odds ratio with 95% confidence intervals and the p value. Odds ratios were then adjusted for possible interfering factors. To verify a potential gene-environment interaction, the patients and controls were stratified depending on age, sex, living environment (rural or urban), smoking status and the occurrence of AMD among first-degree relatives. Multiple unconditioned logistic regression analyses were run to test the association of genotypes and environmental and social factors with AMD occurrence. Statistical analysis was performed using Statistica 9.0 package (Statsoft, Tulsa, OK, USA). […]

Pipeline specifications

Software tools Biotools, Statistica
Applications Miscellaneous, Population genetic analysis
Organisms Homo sapiens, Nicotiana tabacum
Diseases Macular Degeneration
Chemicals Heme, Iron, Oxygen