SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system

Abstract

Background: Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds.

Hypothesis/objectives: To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds.

Animals: Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs.

Methods: Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA.

Results: In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown.

Conclusions and clinical importance: These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds.

Keywords: Hereditary disease; Metabolic disease; Nephropathy; Urolithiasis.

Fig. 1

Sequencing chromatograms displaying the mutations found and their effect on the protein sequence. A) A 1 bp deletion in SLC3A1 exon 1 in purebred and mixed breed Labrador Retriever and causing a frameshift and a premature stop codon to truncate the translated protein. B) A 6 bp in frame deletion in SLC3A1 exon 6 in Australian Cattle Dogs and a mixed breed dog deleting 2 threonines in a repeat of 3. C) A single nucleotide exchange in SLC7A9 exon 9 found heterozygous in cystinuric Miniature Pinschers, changing the amino acid from glycin to arginine.

Fig. 2

Pedigree of the Miniature Pinscher family displaying the genotype and the phenotype of the dogs studied, showing their correlation and the inheritance in an autosomal dominant trait

Fig. 3

Fragment length analysis on a 5% polyacrylamid gel, discriminating the normal DNA amplicon of 140 bp from the smaller fragment of 134 bp in presence of the 6 bp deletion in SLC3A1 exon 6 found in cystinuric Australian Cattle Dogs (see AUCD #) and a mixed breed dog (here Mx)

Fig. 4

Alignment of the amino acid sequences from different species, surrounding A) the deletion of two threonines, found in SLC3A1 exon 6 in cystinuric Australian Cattle Dogs and a mixed breed dog, and B) the exchange of a glycine to an arginine, found in cystinuric Miniature Pinschers, both showing a highly conserved sequence in the protein.