Characterization of a Homozygous Deletion of Steroid Hormone Biosynthesis Genes in Horse Chromosome 29 as a Risk Factor for Disorders of Sex Development and Reproduction

Abstract

Disorders of sex development (DSD) and reproduction are not uncommon among horses, though knowledge about their molecular causes is sparse. Here we characterized a ~200 kb homozygous deletion in chromosome 29 at 29.7–29.9 Mb. The region contains AKR1C genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens. Mutations in AKR1C genes are associated with human DSDs. Deletion boundaries, sequence properties and gene content were studied by PCR and whole genome sequencing of select deletion homozygotes and control animals. Deletion analysis by PCR in 940 horses, including 622 with DSDs and reproductive problems and 318 phenotypically normal controls, detected 67 deletion homozygotes of which 79% were developmentally or reproductively abnormal. Altogether, 8–9% of all abnormal horses were homozygous for the deletion, with the highest incidence (9.4%) among cryptorchids. The deletion was found in ~4% of our phenotypically normal cohort, ~1% of global warmblood horses and ponies, and ~7% of draught breeds of general horse population as retrieved from published data. Based on the abnormal phenotype of the carriers, the functionally relevant gene content, and the low incidence in general population, we consider the deletion in chromosome 29 as a risk factor for equine DSDs and reproductive disorders.

Horse Clinical Cytogenetics: Recurrent Themes and Novel Findings

“…In three families of different breeds, SRY -positive XY DSD was associated with androgen insensitivity syndrome and with different mutations in the androgen receptor gene [ 150 , 151 , 152 ]. In two related male pseudohermaphrodite Standardbreds with SRY -positive XY DSD [ 146 ], a large (~200 kb) homozygous deletion in chr29 was found and proposed as a likely cause because the deletion removed a cluster of genes ( AKR1C family) with known functions in steroid hormone biosynthesis, including androgens and estrogens [ 58 , 112 ]. However, AR mutations or the deletion in chr29 are not present in many other cases of SRY -positive XY DSDs [ 58 ], suggesting that the molecular causes of the condition are heterogeneous.…”

Two Novel Cases of Autosomal Translocations in the Horse: Warmblood Family Segregating t(4;30) and a Cloned Arabian with a de novo t(12;25)

“…Analysis by PCR showed that all XY individuals were positive and all XX individuals were negative for the SRY gene, which is normal. Finally, all horses were tested for a 200-kb homozygous deletion in ECA29 involving AKR1C genes -a risk factor for abnormal sex development and/or reproduction [Ghosh et al, 2020]. The results showed that 3 foals were homozygous for the deletion: 2 (ID: H713 and H714) carrying a balanced translocation and 1 (ID: H716, "shabby hair") with unbalanced translocation (Table 1).…”

“…Worth mentioning is also the fact that both cloned stallions carried a homozygous deletion (D/D) in ECA29. This approximately 200-kb deletion at 29.7-29.9 Mb involves AKR1C genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens [Ghosh et al, 2020]. Mutations in AKR1C genes are associated with a spectrum of human disorders of sex development such as cryptorchidism, undervirilized external genitalia, dihydrotestosterone deficiency, and sex reversal [Baetens et al, 2019].…”

“…[Khanshour et al, 2013] and 8 X-linked short tandem repeats (STRs) [Anaya et al, 2017] using an ABI PRISM 377 (Applied Biosystems, Foster City, CA, USA) following previously described methods [Juras et al, 2003]. All animals were tested by PCR for the Y-linked SRY gene and for a 200-kb homozygous deletion in ECA29, a risk factor for disorders of sex development and reproduction [Ghosh et al, 2020]. The X-linked androgen receptor (AR) gene served as a positive control for all PCR amplifications.…”

“…The X-linked androgen receptor (AR) gene served as a positive control for all PCR amplifications. The primers were as follows: SRY-forward 5′-TGCATTCATGGTGTGGTCTC-3′, SRY-reverse 5′-ATGGCAATTTTTCGGCTTC-3′, 131 bp [Paria et al, 2011]; AR-forward 5′-AGCAGCAACAGGAGACCAGT-3′, ARreverse 5′-GCTTAAGCCTGGGAAAGTG-3′, 294 bp; ECA29 deletion (3 primer pairs): E458-forward 5′-AAAGATGCCG-GTTTAACCAA-3′, E458-reverse 5′-TGCAAACAGGCTTG-TACTTGA-3′, 103 bp; E661-forward 5′-GCATTTTGTTCCAT-GTGTGC-3′, E661-reverse 5′-CACAGTCAAACCACCCA-CTG-3′, 365 bp; and E668-forward 5′-TGCTGT TGA TCA-TCCTTATTATCC-3′, E668-reverse 5′-AAATGAAAATGAGA-ATAAAGGAAAGTG-3′, 179 bp [Ghosh et al, 2020].…”