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. 2022 Dec 31;6(1):4.
doi: 10.3390/mps6010004.

Analytical Validation of SOD2 Genotyping

Marija Debeljak  1 , Stacy Riel  1 , Ming-Tseh Lin  1 , James R Eshleman  1   2 , Channing J Paller  2   3
Affiliations

Analytical Validation of SOD2 Genotyping

Marija Debeljak et al. Methods Protoc. .

Abstract

Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells' protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.

Keywords: SOD2 gene; prostate cancer; prostate-specific antigen (PSA); pyrosequencing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SOD2 gene overview showing 2 exons out of total of 5 exons. Start codon, AUG (green), is shown in Exon 1. “X” (red) in Exon 2 denotes the polymorphism in codon 16. Blue arrows indicate primer position for PCR amplification. Amino acid sequence for Exon 1 and Exon 2 is listed for both wildtype and variant where start codon, M (green), amino acid polymorphism position (red), and mitochondrial targeting sequence (underlined) are indicated.
Figure 2
Figure 2
Pyrograms generated in silico using the software Pyromaker, which produces an expected pyrogram based on user input (A–C) and pyrograms experimentally generated by pyrosequencing (D–F). Panels (A) and (D) show pyrograms for homozygous WT, GCT/GCT (Ala/Ala) genotype. Panels (B) and (E) show pyrograms for heterozygous genotype, GCT/GTT (Ala/Val). Panels (C) and (F) show homozygous variant genotype, GTT/GTT (Val/Val). The three guanines, which contribute to the final 4G peak in the electropherograms, are not shown in the text.
See this image and copyright information in PMC

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