73 Preimplantation genetic diagnosis of glycogen branching enzyme deficiency and sex determination in equine in vitro-produced embryos

Glycogen branching enzyme deficiency (GBED) is an autosomal recessively inherited disease caused by a single base nonsense mutation (c.102C>A) in the GBE1 gene that results in a truncated enzyme, which generates a non-functional glycogen storage in mainly muscle and liver (Ward et al. 2004 Mamm. Genome 15, 570-577). The mutation is equally distributed between sexes and the crossing of heterozygous carriers (GBED ±) has the 25% chance to produce embryos affected by the disease (GBED −/−) whose pregnancies result in spontaneous abortions or newborn foals affected by severe cardiac or respiratory failure, seizures, muscle weakness and sudden death within 18 weeks of age (Valberg et al. 2001 J. Vet. Intern. Med. 15, 572-580). In adult quarter horses and related breeds, the mutant allele frequency has been estimated to be around 8% with at least 3% of spontaneous abortions caused by this disease (Wagner et al. 2006 J. Vet. Intern. Med. 20, 1207-1211). Preimplantation genetic analysis has been already reported by our laboratory for sex determination on trophoblastic biopsies (5–10 cells) of Day 7 to 9 preimplantation equine embryos obtained by intracytoplasmic sperm injection (ICSI) (Lazzari et al. 2020 J. Equine Vet. Sci. 89, 103097). Therefore, the aim of this study was to develop a screening test to determine both sex and genotype for GBED. The embryo biopsies were lysed by exposure to proteinase K solution, then part of the lysate was used for sex determination by simultaneous amplification by PCR of the constitutive glycoprotein α-galactosyltransferase 1 (GGTA1) gene and the equine sex-determining region of the Y chromosome gene (eSRY). With the remaining biopsy lysate, we amplified the GBE1 gene, assessing the presence of one (heterozygous) or rarely two (recessively homozygous) mutant alleles with Sanger sequencing analysis. Considering that GBED mutation eliminates a RsaI restriction site, we further confirmed the genotype by performing a restriction fragment length polymorphism analysis with RsaI on the remaining GBE1-specific PCR product. Next, electrophoresis analyses identified GBED carrier embryos due to the presence of two bands of 179 bp and 272 bp (36 + 236 bp) resulting from the elimination of the RsaI restriction site of the wild-type allele, identified instead by three bands (179, 36, and 236 bp). The sex and presence of the mutant allele was investigated on 11 biopsies collected from embryos generated by ICSI with semen of a GBED carrier quarter horse stallion and four biopsies collected from control embryos. Determination of sex was achieved with an efficiency of 93.3% (14/15: eight female and six male embryos), and GBED genotype was successfully determined for 13 biopsies (13/15: 86.6% efficiency), identifying three of them as embryo carriers of the mutation (one female and two males). We conclude that sex and GBED genotype can now be determined on equine embryo biopsies, which could prevent disease transmission by avoiding the transfer of affected embryos.