Prevalence of the AMHR2 mutation in Miniature Schnauzers and genetic investigation of a Belgian Malinois with persistent Müllerian duct syndrome

Abstract

Persistent Müllerian duct syndrome (PMDS) is a sex-limited disorder in which males develop portions of the female reproductive tract. Important consequences of PMDS are cryptorchidism and its sequelae of infertility and increased risk of testicular cancer. Anti-Müllerian hormone (AMH) and its receptor (AMHR2) induce the regression of the Müllerian ducts in male embryos. In Miniature Schnauzer dogs, the genetic basis has been identified as an autosomal recessive nonsense mutation in AMHR2, but the allele frequency of the mutation is unknown. Thus, the primary objective of this study was to estimate the prevalence of the AMHR2 mutation in North American Miniature Schnauzers, in order to ascertain the value of genetic testing in this breed. An additional objective was to determine whether mutations in AMH or AMHR2 were responsible for PMDS in a Belgian Malinois; this would aid development of a genetic test for the Belgian Malinois breed. Genomic DNA from 216 Miniature Schnauzers (including one known PMDS case) was genotyped for the AMHR2 mutation, and DNA from a single PMDS-affected Belgian Malinois was sequenced for all coding exons of AMH and AMHR2. The Miniature Schnauzer cohort had an AMHR2 mutation allele frequency of 0.16 and a carrier genotypic frequency of 0.27. The genetic basis for PMDS in the Belgian Malinois was not determined, as no coding or splicing mutations were identified in either AMH or AMHR2. These findings support a benefit to AMHR2 mutation testing Miniature Schnauzers used for breeding or with cryptorchidism.

Keywords: Genetics < General reproduction; Miniature Schnauzer; Persistent Mullerian duct syndrome; dog.

Figure 1. Gel Image of Allele Specific Assay Testing for Miniature Schnauzer Mutation c.241C>T; p.R81*

This gel electrophoresis image demonstrates the allele specific assay results with three dogs of known genotype status. Each dog has two lanes, in which the normal and mutant AMHR2 alleles are separately tested for their presence. The first (left) column for each dog contains primers for the normal allele (indicated with “n”), and the second (right) column for each dog contains primers for the mutant allele (indicated with “a”). From left to right: a carrier dog (presence of both the normal and mutant allele, e.g., heterozygote), a normal/wild-type dog (homozygous for the normal allele), and an affected dog (homozygous for the mutant allele). Where no target allele was present, an additional primer for a 390 base pair product acted as an internal PCR control. The last two columns are template-free water controls for detecting any PCR-mixture contamination.

Figure 2. DNA Sequence Chromatographs Confirming Miniature Schnauzer Mutation c.241C>T; p.R81*

The three chromatographs demonstrate each genotype from a known/control dog, with the yellow arrows indicating the nucleotide of interest. Top = heterozygous (carrier dog), Middle = homozygous for the mutant nonsense allele (PMDS affected dog), Bottom = homozygous for the normal/reference allele (clear dog).

Figure 3. Genotype frequency of the c.241C>T AMHR2 mutation in Miniature Schnauzers

Dogs homozygous for the reference allele are indicated as NN, heterozygous for the mutant allele as DN, and homozygous for the mutant allele as DD.