Abstract
Although the disorder of sex development in dogs with female karyotype (XX DSD) is quite common, its molecular basis is still unclear. Among mutations underlying XX DSD in mammals are duplication of a long sequence upstream of the SOX9 gene (RevSex) and duplication of the SOX9 gene (also observed in dogs). We performed a comparative analysis of 16 XX DSD and 30 control female dogs, using FISH and MLPA approaches. Our study was focused on a region harboring SOX9 and a region orthologous to the human RevSex (CanRevSex), which was located by in silico analysis downstream of SOX9. Two highly polymorphic copy number variable regions (CNVRs): CNVR1 upstream of SOX9 and CNVR2 encompassing CanRevSex were identified. Although none of the detected copy number variants were specific to either affected or control animals, we observed that the average number of copies in CNVR1 was higher in XX DSD. No copy variation of SOX9 was observed. Our extensive studies have excluded duplication of SOX9 as the common cause of XX DSD in analyzed samples. However, it remains possible that the causative mutation is hidden in highly polymorphic CNVR1.
Highlights
Enhancer regulatory sequence which duplicated may trigger SOX9 expression in the absence of the SRY gene product[9]
Results of the Fluorescence in Situ Hybridization (FISH) experiment indicated a potential copy number variable regions (CNVRs) in the region detected by BAC-3
Four groups of animals were included in the FISH study: XX disorder of sex development (DSD) dogs (A5, A6, A7, A8, B3, B4), healthy relatives of A6 and B4 XX DSD dogs (C2, C3, C4 and C5) and 4 reference, fertile female dogs (F1–F4)
Summary
Enhancer regulatory sequence which duplicated may trigger SOX9 expression in the absence of the SRY gene product[9]. Genome wide association study (GWAS) of related animals with XX DSD implicated the SOX9 region, but did not pinpoint the causative mutation[11]. It has recently been claimed that some canine XX DSD cases are caused by duplication of a 577 kb region containing the SOX9 gene, as detected by array comparative genome hybridization (aCGH) and confirmed by real time qPCR. It has to be noted that duplication of SOX9 in control samples was not detected in any of copy number variants (CNVs) discovery studies[19,20,21,22,23]. We describe the use of cytogenetic mapping (fluorescence in situ hybridization, FISH) and multiplex ligation-dependent probe amplification (MLPA) approaches to identify two highly variable CNVRs, and show that SOX9 is not duplicated in any of the samples analyzed
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