Cytogenetic and molecular insight into the genetic background of disorders of sex development in seventeen cats

Abstract

The genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats (atypical in males). The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XX^SRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes (SRY, TAC3, CYP11B1 and LHCGR). Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 (modal), and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5'UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.

Figure 1

Characteristics of DSD case #7318: (a) tricolored coat pattern; (b) male external genitalia with bifid scrotum; (c) histology of gonads: testes with seminiferous tubules with normal Sertoli cells, normal Leydig cells, and lack of germinal cells; (d) FISH showing the SRY gene (green) on the X chromosome (red); (e) detection of genes translocated on the X chromosome. The Yp-linked genes were amplified for the DSD cat (SRY, HSFY, CYORF15, TETY2, ZFY) and the Yq-linked genes were absent (CUL4BY, TETY1 and FLJ36031Y). L: 100–1000 bp ladder, DSD case #7318, M: control male; FM: control female; 0: negative control; (f) random inactivation pattern of X chromosomes in case #7318 and two control females, FM1 and FM2.

Figure 2

Characteristics of DSD case #7355: (a, b) normal male external genitalia; (c) histology of gonads: testes with degenerated spermatogenesis and rare sperms; (d) FISH with X (red) and Y (green) chromosome painting probes showing two cell lines: normal (38,XY) and monosomic (37,X); detection of Y-linked genes in blood leukocytes: (e) SRY: 1022 bp band detected by PCR, and (f) ZFY: 448 bp band (391 bp band represents the X-linked ZFX gene) detected by PCR–RFLP, L: 100 – 1000 bp ladder, lanes 1, 2: case #7355 (duplicated), M1, M2: control males; FM1, FM2: control females; 0: negative control.

Figure 3

Characteristics of DSD case #7513: (a, b, c) ambiguous external genitalia: rudimentary penis and vulva; (d) histology of gonads: testes with sparse seminiferous tubules, hypertrophic Sertoli cells, numerous Leydig cells and lack of germinal epithelial cells; FISH with X (red) and Y (green) chromosome painting probes showing two cell lines: 38,XX (e) and 38,XY (f) in lymphocytes; (g) estimation of the Y/X ratio based on proportion of AMELY and AMELX genes in blood leukocytes (#7513B) and fibroblasts (#7513F). The upper chart shows amplification signals from chromosome X (green color) and Y (blue color). In fibroblasts of case #7513 (#7513F) the signal was detected from chromosome X only. Both signals were detected in blood leukocytes (#7513B). On the lower chart, an Y/X ratio of approximately 0.5 was detected in blood leukocytes (#7513B), indicating the presence of two cell lines. In fibroblasts of DSD case #7513 (#7513F) and in a control female (FM), the lack of Y chromosome resulted in a Y/X ratio of = 0, while a Y/X ratio of approximately 1 was detected in a control male (M); NC: negative control.

Figure 4

Variability of SRY copy number in XY DSD and control cats: (a) assay targeting SRY coding sequence; (b) assay targeting 5′-flanking region of SRY. FM: control female; NC: negative control.

Figure 5

Phenotypes of two DSD cats which were homozygous for the g.70455376delC variant in 5′UTR of the LHCGR gene. Case #7302: (a) external genitalia with atypical penis, hypospadias, abnormal mucosa in the perineal area, and bifid scrotum (b) histological examination of gonads: numerous seminiferous tubules and Sertoli cells, sperm, and decreased number of Leydig cells; Case #7455: (c) external genitalia with abnormal penis, hypospadias, atypical mucosa, and bifid scrotum, (d) examination of gonads revealed asymmetrical testes with numerous seminiferous tubules, in larger testis with sperm and in smaller testis without sperm.