The equine graying with age mutation of the STX17 gene: A copy number study using droplet digital PCR reveals a new pattern

Abstract

Summary The equine graying with age causative mutation in the syntaxin‐17 gene (STX17) has been known for over a decade, but proper genotyping of this variant remains challenging due to its molecular character (4.6‐kb tandem duplication). Precise information on gray mutation status is important for horse breeders and veterinarians, since gray homozygous horses are more prone to developing aggressive melanoma tumors than heterozygotes. Since recent studies have confirmed that droplet digital PCR is a valuable technique for copy number analysis, we decided to investigate whether this method can be used for accurate genotyping of the horse graying‐related variant and established the copy numbers of the 4.6‐kb fragment in the available cohort (n = 75) of gray and nongray horses of various breeds. Surprisingly, we found that our STX17 genotype results varied from what has been previously published, suggesting that gray phenotype is associated with the presence of six (GG) or four (Gg) copies of studied region. All the examined nongray horses (gg) have the two copies of these fragments. This new pattern and its inheritance were also confirmed by an analysis conducted for the Polish Warmblood horse family. We noted no further copy number variation in the entire tested samples set. Our study confirmed the usefulness and accuracy of droplet digital PCR for genotyping STX17 gene variant. Further studies on a broader range of materials are needed to fully understand the origin and molecular structure of the graying causative mutation in the horse STX17.

Equine STX17 intronic triplication confirmed by droplet digital PCR analysis of its breakpoints

“…Due to the presence of two functional MITF transcription factor binding sites, the region is considered a strong melanocyte‐specific enhancer 1 . Although previous studies recognized the causative variant as tandem duplication, 2 our latest investigation using droplet digital PCR (ddPCR) suggests a novel copy number pattern 3 . Since our results proved interesting to equine geneticists, horse breeders, and veterinarians, we decided to confirm whether the proposed model of STX17 intronic region triplication also holds true when we concentrate exclusively on unique sequences of multiplication breakpoints.…”

“…To estimate the exact copy number of DNA fragments, we used the equine F2 as a single‐copy reference gene 4 . The protocol of the ddPCR reaction and cycling conditions were identical to those in our previous work 3 . For each sample, approximately 20 000 droplets were generated using a QX200 Droplet Generator.…”

“…(a) Copy number analysis of three individuals carrying different gray genotypes using three different droplet digital PCR (ddPCR) assays: BP (located at multiplication breakpoints, designed for this study); B (located within the multiplicated region, Ref. 3) and C (located outside the multiplicated region, Ref. 3).…”

“…3) and C (located outside the multiplicated region, Ref. 3). (b) Schematic localization of the applied ddPCR assays dependently on gray genotype.…”

An intronic copy number variation inSyntaxin 17determines speed of greying and melanoma incidence in Grey horses

“…The causal mutation for Grey was originally identified as a 4.6 kb duplication in intron 6 of Syntaxin 17 ( STX17 ) 1 . However, a recent study 5 , based on digital PCR, indicated that the Grey haplotype carries three copies of the duplicated fragment. Horses homozygous for the Grey mutation ( G/G ) are reported to have significantly higher melanoma incidence than heterozygotes ( G/g ) 1 .…”