Polyadenine insertion disrupting the G6PC1 gene in German Pinschers with glycogen storage disease type Ia (GSD1A)

Glycogen storage diseases (GSD) are a group of inherited disorders of carbohydrate metabolism that occur in humans and animals. Variants in 20 different genes are currently known to result in GSD in humans.1 One of the candidate genes is G6PC1 encoding glucose-6-phosphatase catalytic subunit 1. G6PC1 loss-of-function variants cause GSD1A, which is also termed von Gierke disease. The disease is characterized by severe hypoglycemia and hepatomegaly owing to hepatic glycogen accumulation early in life.2 A canine form of GSD1A has been identified in Maltese Terriers and is caused by a p.Met121Ile variant in the G6PC1 gene (OMIA 000418-9615).3 Dogs with GSD1A have become a popular, naturally occurring animal model for gene-therapy research in this disease.3-5 Two 4-week-old purebred German Pinscher puppies, one male and one female, showed an enlarged abdomen at clinical examination and poor weight gain since birth. Ultrasonography of the female puppy revealed an increased liver size. Furthermore, this puppy was reported to be bilaterally blind. The male puppy developed seizures. The affected puppies were euthanized owing to worsening of clinical signs and poor prognosis. At necropsy, both dogs showed severe hepatomegaly with light brown to yellow discoloration and fragile consistency of the liver. Histologically, diffuse swelling and vacuolization of hepatocytes with peripheral displacement of nuclei was found. Periodic acid Schiff-positive, diastase-sensitive material was present in hepatocytes, indicating glycogen accumulation. We isolated genomic DNA from both cases from liver tissue samples and sequenced the genome of one affected dog at approximately 18.6× coverage with Illumina 2 × 150 bp reads. The data were analyzed as described previously6 and submitted to the European Nucleotide Archive under sample accession no. SAMEA8157169. Filtering for variants present only in the sequenced dog and absent in 795 control genomes (Table␣S1) yielded no homozygous private protein-changing variant in a known GSD candidate gene1 (Table␣S2). We then visually inspected the short read alignments in the candidate genes for structural variants using the Integrative Genomics Viewer.7 This revealed a homozygous 76 bp insertion into exon 5 of the G6PC1 candidate gene, which probably causes a loss of function (chr9:g.20,134,857_20,134,858ins76; XM_038676372.1:c.634_635ins76). The insertion consisted of 60 consecutive adenines and an additional 16 bp duplication of the integration site (Figure␣1, File S1). The variant was confirmed by Sanger sequencing and an additional 208 unrelated German Pinscher dogs were genotyped. Only the two affected dogs carried the insertion in a homozygous state. However, 24 additional heterozygous carriers were identified corresponding to a carrier frequency of 12%. Based on the clinical and histological findings GSD was diagnosed in two German Pinscher puppies. A precision medicine approach identified a 76 bp SINE insertion in G6PC1 as the most likely candidate causative variant and allowed designation of the phenotype as GSD1A. The disease allele is present at non-negligible frequency in the German Pinscher dog population and the introduction of genetic testing for breeding animals is recommended. We thank the dog owners for providing samples and information about their dogs. Furthermore, we are grateful to the Next Generation Sequencing Platform and the Interfaculty Bioinformatics Unit of the University of Bern for performing whole-genome sequencing experiments and providing the computational infrastructure. We thank the Dog Biomedical Variant Database Consortium (Gus Aguirre, Catherine André, Danika Bannasch, Doreen Becker, Brian Davis, Cord Drögemüller, Kari Ekenstedt, Kiterie Faller, Oliver Forman, Steve Friedenberg, Eva Furrow, Urs Giger, Christophe Hitte, Marjo Hytönen, Vidhya Jagannathan, Tosso Leeb, Frode Lingaas, Hannes Lohi, Cathryn Mellersh, Jim Mickelson, Leonardo Murgiano, Anita Oberbauer, Sheila Schmutz, Jeffrey Schoenebeck, Kim Summers, Frank van Steenbeek and Claire Wade) for sharing whole genome sequencing data from control dogs. We also acknowledge all researchers who deposited dog or wolf whole genome sequencing data into public databases. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.