A 6‐bp Deletion Variant in a Novel Canine Glutathione‐S‐Transferase Gene (GSTT5) Leads to Loss of Enzyme Function

Abstract

ObjectivesGlutathione‐S‐transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST‐theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs.MethodsBanked livers from 45 dogs of various breeds were screened for low GST‐theta activity by the substrate 1,2‐dichloro‐4‐nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole‐genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus.ResultsCanine livers ranged 190‐fold in GST‐theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in V max for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over‐represented in Pembroke Welsh corgis (P < 0.0001) based on available whole‐genome sequence data.ConclusionsAn Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST‐theta enzyme activity. This variant is significantly over‐represented in purpose‐bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens.

Glutathione‐S‐transferase‐theta genotypes and the risk of cyclophosphamide toxicity in dogs

“…We chose GST‐theta variants for this study because of marked variability in GST‐theta activities in dogs and functionally defective canine GST‐theta alleles . However, CP response in humans can be influenced by other GST gene variants, alone or in combination .…”

“…Genomic DNA was isolated from buccal swabs from each dog. GSTT1 and GSTT5 variants (Table ) were selected based on deleterious effects predicted by several modelling programs, and if the minor allele frequency was >0.01 in client‐owned dogs, banked canine liver samples or whole genome sequencing data from 281 domestic dogs . PCR products were subjected to dye‐terminator sequencing using BigDye reagents (Applied Biosystems, Carlsbad, California).…”

“…Listed variants were found in healthy client‐owned dogs, in a population of domestic dogs of various breeds, or in genotyped normal canine livers, with a minor allele frequency >0.01.…”

“…GST‐theta is highly polymorphic in dogs, with 2 GST‐theta genes expressed: GSTT1 , (CFA26, LOC477556) and GSTT5 (CFA26, LOC477558). We have characterized several canine GSTT1 polymorphisms that are predicted or shown to be functionally deleterious, and a GSTT5 polymorphism (del385_390) that leads to >90% loss of activity …”

Glutathione S‐transferase theta genotypes and environmental exposures in the risk of canine transitional cell carcinoma

“…Three polymorphic loci in canine GST‐theta genes were directly re‐sequenced: GSTT1 I2+28 G>A; the GSTT1 3′UTR haplotype containing 101_102 insT, and the 6 base pair coding deletion in exon 4 of GSTT5 (385_390delGACCAG; Asp129_Gln130del). Primers were designed from the canine genome assembly (CanFam3.1) as previously described . Sequence alignment and polymorphism screening were carried out using SerialCloner v2.6 (SerialBasics) and FinchTV chromatogram reader software (Geospiza Inc).…”