Deep sequencing of a candidate region, harboring the SOX9 gene, for the canine XX disorder of sex development. DSD dogs, SOX9 sequencing

Disorder of sex development (DSD) in dogs with female sex chromosomes (78, XX), a lack of the SRY gene and the presence of testes or ovotestes is commonly diagnosed in numerous breeds. The molecular background of DSD is not fully recognized but has been linked to the copy number variation (CNV) in the region harboring the SOX9 gene. We applied targeted next generation sequencing (NGS) techniques to compare DSD and normal female dogs. Targeted NGS of a 1.5 Mb region on canine chromosome 9 (CFA9) harboring the SOX9 gene revealed two putatively DSD associated CNVs in 355 kb upstream and 691 kb downstream of the SOX9, four blocks of low polymorphism and two blocks of an elevated heterozygosity. An initial NGS analysis showed the association with 2 SNPs, but validation in larger cohorts did not confirm this result. We identified a large homologous fragment (over 243.8 kb), named hfMAGI2, located upstream of the SOX9, that overlaps a known copy number variation region (CNVR). It shows a high sequence similarity with the 5’-flanking region of the MAGI2 gene located on CFA18, which encodes a protein involved in ovary formation during early embryonic development. Our study showed that the identified CNVR located upstream of the SOX9 gene contains potential regulatory sequences (long non-coding RNA and hfMAGI2) and led to assumption that a multiplication of this elements may alter expression of the SOX9 gene triggering the DSD phenotype.