SOX9 gene variants in 27 French Bulldogs with disorder of sex development (XX, SRY-negative): identification of first case of skeletal abnormalities associated with SOX9 triplication

Introduction: The SOX9 gene encodes a transcription factor that acts downstream of the Y-linked SRY gene and plays a pivotal role in fetal testis development. Duplication of SOX9 or its regulatory sequences is a known cause of testicular or ovotesticular disorder of sex development (DSD) in chromosomal females (XX DSD). Numerous reports have described canine XX DSD, characterized by virilization (e.g., enlarged clitoris) and the presence of testes or ovotestes. This study aimed to identify SOX9 variants in a cohort of French bulldogs with XX (SRY-negative) DSD. Methods: In total, 27 DSD dogs were studied, including 20 with abdominal, spermatogenetically inactive testes, three with inactive testis and ovotestis, one with inactive testis and ovary, and one with ovotestes; in two dogs, analysis of gonads could not be performed. Moreover, 24 control females of the same breed, all with normal external female genitalia, were included. Results: Three known DNA variants were identified in SOX9: a 3 bp insertion/deletion (CCT/---, rs852828782), a T>C SNP (rs22704771) in the 5′ UTR, and an intronic T>G SNP (rs9183825). These variants were rare, and their distribution was similar in both cohorts. Additionally, the number of SOX9 gene copies was assessed using ddPCR. A single XX DSD case with additional skeletal malformations carried three copies of SOX9, while all other cases and control females had two copies. Conclusion: We conclude that SOX9 duplication is a rare cause of XX DSD in French bulldogs, and that the identified sequence variants in this gene are not associated with the disorder.