Supplementary Material for: Hypospadias in a Male (78,XY; <i>SRY</i>-Positive) Dog and Sex Reversal Female (78,XX; <i>SRY</i>-Negative) Dogs: Clinical, Histological and Genetic Studies

Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the <i>SRY</i> gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the <i>SRY</i> gene. This animal was classified as a compound sex reversal (78,XX, <i>SRY</i>-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the <i>SRY</i> gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, <i>SRY</i>-negative). Molecular studies were focused on coding sequences of the <i>SRY</i> gene (case 1) and 2 candidates for monogenic hypospadias, namely <i>MAMLD1</i> (mastermind-like domain containing 1) and <i>SRD5A2</i> (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire<i> SRY</i> gene, including 5′- and 3′-flanking regions, did not reveal any mutation. The entire coding sequence of <i>MAMLD1</i> and <i>SRD5A2</i> was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In <i>MAMLD1</i> 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in <i>SRD5A2</i> 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes.