GTG Mutation in the Start Codon of the Androgen Receptor Gene in a Family of Horses with 64,XY Disorder of Sex Development

Abstract

Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRY ⁺ disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.

Androgen Insensitivity Syndrome in a Family of Warmblood Horses Caused by a 25-bp Deletion of the DNA-Binding Domain of the Androgen Receptor Gene

“…The silent mutation in exon 1 (c.322C>T) identified by Révay et al [2012] in a family of American Quarter Horses was also observed in the current pedigree. In the previous report the 2 alleles (C>T) segregated among the animals, whereas all the individuals in the current family (Warmblood) invariably carried the T allele as a homozygous or hemizygous genotype, and therefore this SNP was excluded as a causative candidate.…”

“…Comparison of the sequences of the pedigree members to one another and to the horse AR reference sequence identified 2 variable positions: a substitution and a deletion. When compared to the reference C allele, the T allele of the previously described neutral exon 1 c.322C>T substitution [Révay et al, 2012;Bolzon et al, 2016] was observed in all 4 sequenced animals (DSD-M1, DSD-M2, N-M1, N-S1). Additionally, a 25-bp deletion in exon 2 (c.1630_1654del) was discovered, deleting 8 codons from the end of exon 2 ( Fig.…”

“…however, the molecular link between mutations of the AR gene and clinical characteristics of AIS were established in only 2 previous reports [Révay et al, 2012;Bolzon et al, 2016].…”

“…All 8 exons of the horse AR gene were sequenced in 4 pedigree members (DSD-M1, DSD-M2, N-M1, N-S1), using primer pairs designed to anneal to the surrounding introns [Révay et al, 2012]. Primer sequences are listed in online supplementary Table 1 (www.karger.com/doi/10.1159/000455114).…”

“…While male pseudohermaphroditism or AIS has been reported in several domestic species including dogs, cats, horses, and cattle [Howden, 2004;Lyle, 2007], detailed molecular investigations in horses are restricted to 2 studies. A missense mutation (c.2042G>C) was identified in members of a Thoroughbred family and presumed to result in the substitution of a very important tryptophan residue with serine in the AR LBD [Bolzon et al, 2016], and a rare start codon mutation (c.1A>G) in the American Quarter horse breed resulted in complete androgen insensitivity for 3 members of the horse family [Révay et al, 2012]. Both of these exonic mutations segregated in the pedigree according to the AIS phenotype.…”