Supplementary Material for: Methylation Patterns of SOX3, SOX9, and WNT4 Genes in Gonads of Dogs with XX (SRY-Negative) Disorder of Sexual Development

Ovotesticular or testicular disorder of sexual development in dogs with female karyotype and lack of <i>SRY</i> (XX DSD) is a common sexual anomaly diagnosed in numerous breeds. The molecular background, however, remains unclear, and epigenetic mechanisms, including DNA methylation, have not been studied. The aim of our study was comparative methylation analysis of CpG islands in promoters of candidate genes for XX DSD: <i>SOX9</i>, <i>SOX3</i>, and <i>WNT4</i>. Methylation studies were performed on DNA extracted from formalin-fixed/paraffin-embedded or frozen gonads from 2 dogs with ovotesticular and 2 dogs with testicular XX DSD as well as control females (<i>n</i> = 4) and males (<i>n</i> = 2). Bisulfite-converted DNA was used for CpG methylation analysis using quantitative pyrosequencing. Promoter regions of <i>SOX9</i> and <i>WNT4</i> showed similar CpG methylation in each group, ranging from 0 to 5.5% and from 39 to 74%, respectively. The <i>SOX3</i> promoter showed significantly higher methylation in the ovotesticular XX DSD cases and the testicular XX DSD and control males, suggesting that <i>SOX3</i> methylation may play a role in canine XX DSD pathogenesis.