XX Disorder of Sex Development is associated with an insertion on chromosome 9 and downregulation of RSPO1 in dogs (Canis lupus familiaris)

Abstract

Remarkable progress has been achieved in understanding the mechanisms controlling sex determination, yet the cause for many Disorders of Sex Development (DSD) remains unknown. Of particular interest is a rare XX DSD subtype in which individuals are negative for SRY, the testis determining factor on the Y chromosome, yet develop testes or ovotestes, and both of these phenotypes occur in the same family. This is a naturally occurring disorder in humans (Homo sapiens) and dogs (C. familiaris). Phenotypes in the canine XX DSD model are strikingly similar to those of the human XX DSD subtype. The purposes of this study were to identify 1) a variant associated with XX DSD in the canine model and 2) gene expression alterations in canine embryonic gonads that could be informative to causation. Using a genome wide association study (GWAS) and whole genome sequencing (WGS), we identified a variant on C. familiaris autosome 9 (CFA9) that is associated with XX DSD in the canine model and in affected purebred dogs. This is the first marker identified for inherited canine XX DSD. It lies upstream of SOX9 within the canine ortholog for the human disorder, which resides on 17q24. Inheritance of this variant indicates that XX DSD is a complex trait in which breed genetic background affects penetrance. Furthermore, the homozygous variant genotype is associated with embryonic lethality in at least one breed. Our analysis of gene expression studies (RNA-seq and PRO-seq) in embryonic gonads at risk of XX DSD from the canine model identified significant RSPO1 downregulation in comparison to XX controls, without significant upregulation of SOX9 or other known testis pathway genes. Based on these data, a novel mechanism is proposed in which molecular lesions acting upstream of RSPO1 induce epigenomic gonadal mosaicism.

Fig 1. GWAS Manhattan plot for logistic mixed model analysis showing association of XX DSD to CFA9 in the canine model pedigree.

The probability of association [-Log₁₀ (P)] is shown for single nucleotide polymorphisms (SNPs) on chromosomes 1–40. (Quantile-Quantile plot of the p-values is in S3 Fig).

Fig 2. Genomic regions on CFA9 associated with canine XX DSD, indicated by method of identification.

The top ideogram of CFA9 (UCSC browser, CanFam 3/3.1) shows the entire span in which regions associated with canine XX DSD were found in this study. Red lines indicate magnified portions of this span, which are screen shots from the browser. Horizontal bars approximate the location of each region identified. Text below the bars indicates the method by which a region was identified, and its specific coordinates. The bottom screen shot shows the location of the G+ insert (arrow) associated with canine XX DSD.

Fig 3. Embryonic lethality in association with the homozygous insertion at CFA9:6048201 was identified in breeding experiments.

At left are crosses between V4, a fertile GSHP XX DSD and GSHP males (PM6578, V31). At right are crosses between V4 and males from the XX DSD model pedigree (C752, C734), which vary in proportion of American cocker spaniel and beagle genetic background (ACS/BGL). Genotypes at the insertion locus (CFA9:6048201) are homozygous (G+G+), heterozygous (G+/-), or wild type (-/-). Females are indicated by open circles, XX DSD by filled circles, and XY males by squares. Each symbol represents one dog, except those containing a number, which indicates the number of dogs represented by that symbol. None of the offspring had the G+G+ genotype. Based on simple Mendelian inheritance, the expected number of -/-, G+/- and G+G+ offspring from the G+/- sires and dams is 4.25, 8.5, and 4.25, respectively (N = 17). This is significantly different from the numbers observed (x² = 8.1776, df = 2, Chi-squared test is significant at p<0.025 since 8.1776>7.38). Similarly, the expected number of G+/- and G+G+ offspring from a G+/- dam and G+G+ sire (V4 x C734) is 11 and 11, respectively (N = 22). This is a highly significant difference from the numbers observed (x² = 20.04, calculations include continuity correction for df = 1 [45]; Chi-squared test is significant at p<0.005 since 20.04>7.88). Since all offspring of C734 (G+G+) must have received the G+ allele from him, only those receiving the wild type allele from the GSHP V4 dam (G+/-) were born. Together, these results indicated that the G+G+ genotype, combined with ≥50% GSHP background, is associated with embryonic lethality.

Fig 4. RSPO1 and WNT4 expression in canine embryonic gonads was measured by RNA-seq.

(A) RSP01 and (B) WNT4 expression were lower in XX DSD gonads at risk (filled triangles) compared to those of XX age-matched controls (open triangles) at d37-39 and d42-44. Neither RSP01 nor WNT4 expression in XX DSD gonads at risk or those of XX age-matched controls was as low as those of XY littermate controls (filled inverted triangles) or XY age-matched controls (open inverted triangles). Each open circle represents RNA from one embryonic gonad pair, except for d34, which represents RNA from a gonad pool. Differential expression was statistically tested for XX DSD gonads compared to XX controls at d37-39 and d42-44 (* p-value<0.005; ** p-value ≤ 0.00005). FPKM is Fragments Per Kilobase of transcript per Million mapped reads.

Fig 5. SOX9 expression in canine embryonic gonads was measured by RNA-seq.

(A) At d37-39, SOX9 expression was significantly greater in XX DSD gonads at risk (filled triangles) compared to XX age-matched control gonads (open triangles), but <2 fold and primarily due to one sample (C3512). Each open circle represents RNA from one embryonic gonad pair, except for d34, which represents RNA from a gonad pool. Differential expression was statistically tested for XX DSD gonads compared to XX age-matched controls at d37-39 and d42-44 (** p-value ≤ 0.00005). (B) At all ages tested, SOX9 expression in XY littermate (filled inverted triangles) and XY age-matched control gonads (open inverted triangles) was much greater than in XX DSD gonads at risk (filled triangles) and XX age-matched controls (open triangles). Each open circle represents RNA from one embryonic gonad pair, except for d34, which represents RNA from a gonad pool. FPKM is Fragments Per Kilobase of transcript per Million mapped reads.

Fig 6. Genome browser view of the raw PRO-seq signal near the SOX9 gene in d39 gonads.

SOX9 expression was not significantly elevated in d39 XX DSD gonads at risk compared to those of XX age-matched controls. The SOX9 signal is highest in XY littermate control gonads (C3640, C3639, C3557) and much lower in the age-matched XX control (A1114) and XX DSD gonads at risk (C3641, C3682, C3556, C3681). For each sample, histograms show reads aligned to the plus strand (top, red) and minus strand (bottom, blue). Listed at left are embryo ID number, group (XX/XY) and genotype at the insertion locus: wild type (-/-), homozygous (G+G+), and heterozygous (G+/-) for the guanine insertion associated with XX DSD. RefSeq gene annotation is shown below the plot (CanFam3.1).