Mutations in the androgen receptor gene and other sex development key genes are associated with equine disorders of sex development

Abstract

Disorders of sex development (DSDs) occur when there is disagreement of chromosomal, gonadal, or anatomical sex. Although many cases of DSDs are reported in horses, the genomic basis of equine DSDs is not well understood, and most of the causative genes remain unidentified. The androgen receptor (AR) gene is composed of 8 exons and encodes for the AR protein, which plays a critical role in male sexual development by regulating gene expression in response to androgen signaling. Loss-of-function mutations in the AR gene are associated with several diseases, including androgen insensitivity syndrome (AIS) in which 64,XY individuals are female-presenting. More than 1,000 AR gene variants in humans contribute to AIS, whereas few AR gene variants are described in horses. In this work, we aim to advance the understanding of DSDs in horses by exploring the genomic basis of these conditions and identifying key sex development genes involved, focusing on AR and other critical genes involved in sexual differentiation. To achieve this, a comprehensive investigation was initiated into the genomics of 88 equine DSD cases, all determined to be chromosomally normal by karyotype analysis. These cases were categorized into 3 clinical phenotypes: 64,XX X-monosomy-like females, 64,XX intersex, and 64,XY SRY-pos female-like. The genomic analysis was conducted using short-read data-based variant VCF files of all DSD cases and ∼200 control horses. We developed and employed a pipeline in-house to investigate a set of 80 candidate genes, filtering for mutations of high to moderate effect and for homozygosity for alternate alleles with <1% frequency. Missense, nonsense, frameshift, or indel mutations were found in 15 sex development key genes in 25 DSD cases. Notably, 7 novel mutations were found in the AR gene in 7 of the 19 (37%) 64,XY SRY-pos female-like horses. These mutations, located in exons 1, 3, 4, and 7, represent 4 out of the 8 exons in the AR gene, and affect all major functional domains of the AR protein. The structural impacts of these mutations on protein folding are still under investigation. In addition, a hypothesis-free, genome-wide analysis is underway to identify additional potential variants that may contribute to these equine DSD cases, which were not captured in the initial candidate gene selection. This work provides insights that could lead to improved diagnostic methods for these conditions in equine medicine and help identify infertile animals, ultimately informing more effective breeding strategies.