Mutations in the SLC2A9 Gene Cause Hyperuricosuria and Hyperuricemia in the Dog

Allantoin is the end product of purine catabolism in all mammals except humans, great apes, and one breed of dog, the Dalmatian. Humans and Dalmatian dogs produce uric acid during purine degradation, which leads to elevated levels of uric acid in blood and urine and can result in significant diseases in both species. The defect in Dalmatians results from inefficient transport of uric acid in both the liver and renal proximal tubules. Hyperuricosuria and hyperuricemia (huu) is a simple autosomal recessive trait for which all Dalmatian dogs are homozygous. Therefore, in order to map the locus, an interbreed backcross was used. Linkage mapping localized the huu trait to CFA03, which excluded the obvious urate transporter 1 gene, SLC22A12. Positional cloning placed the locus in a minimal interval of 2.5 Mb with a LOD score of 17.45. A critical interval of 333 kb containing only four genes was homozygous in all Dalmatians. Sequence and expression analyses of the SLC2A9 gene indicated three possible mutations, a missense mutation (G616T;C188F) and two promoter mutations that together appear to reduce the expression levels of one of the isoforms. The missense mutation is associated with hyperuricosuria in the Dalmatian, while the promoter SNPs occur in other unaffected breeds of dog. Verification of the causative nature of these changes was obtained when hyperuricosuric dogs from several other breeds were found to possess the same combination of mutations as found in the Dalmatian. The Dalmatian dog model of hyperuricosuria and hyperuricemia underscores the importance of SLC2A9 for uric acid transport in mammals

A LINE-1 Insertion in DLX6 Is Responsible for Cleft Palate and Mandibular Abnormalities in a Canine Model of Pierre Robin Sequence

<div><p>Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects in humans. In order to study cleft palate in a naturally occurring model system, we utilized the Nova Scotia Duck Tolling Retriever (NSDTR) dog breed. Micro-computed tomography analysis of CP NSDTR craniofacial structures revealed that these dogs exhibit defects similar to those observed in a recognizable subgroup of humans with CP: Pierre Robin Sequence (PRS). We refer to this phenotype in NSDTRs as CP1. Individuals with PRS have a triad of birth defects: shortened mandible, posteriorly placed tongue, and cleft palate. A genome-wide association study in 14 CP NSDTRs and 72 unaffected NSDTRs identified a significantly associated region on canine chromosome 14 (24.2 Mb–29.3 Mb; p_raw = 4.64×10⁻¹⁵). Sequencing of two regional candidate homeobox genes in NSDTRs, distal-less homeobox 5 (DLX5) and distal-less homeobox 6 (DLX6), identified a 2.1 kb LINE-1 insertion within DLX6 in CP1 NSDTRs. The LINE-1 insertion is predicted to insert a premature stop codon within the homeodomain of DLX6. This prompted the sequencing of DLX5 and DLX6 in a human cohort with CP, where a missense mutation within the highly conserved DLX5 homeobox of a patient with PRS was identified. This suggests the involvement of DLX5 in the development of PRS. These results demonstrate the power of the canine animal model as a genetically tractable approach to understanding naturally occurring craniofacial birth defects in humans.</p></div

Pedigree of 7 CP1 NSDTR families depicting segregation of the mutant allele with the LINE-1 element insertion.

<p>Filled symbols represent NSDTRs with the CP1 phenotype. Diagonal lines indicate that the NSDTR is deceased. “+” represents wildtype allele. “m” represents the mutant allele. [ ] genotypes were inferred if DNA was not available.</p

PCR amplification and expression analysis of LINE insertion in CP1 and WT NSDTRs.

<p>A. Gel image of long range PCR amplification of the DLX6 intronic LINE-1 insertion from genomic DNA of an unaffected NSDTR (WT; 2500 bp band), CP1 NSDTR (CP1; 4600 bp band), and negative water control (Neg). CP1 has a 2.1 kb LINE insertion. B. Gel image of RT-PCR amplification of DLX6 from WT and CP1 cDNA. CP1 expresses both a wildtype (1390 bp) and mutant (2600 bp) transcript. The mutant transcript has a 1.2 kb insertion. GAPDH was used to control for cDNA concentrations. C. Relative DLX6 gene expression ratios by transcript of cerebral cortex cDNA from 3 neonatal CP1 NSDTRs compared to 3 neonatal WT NSDTRs. Boxes represent the interquartile range, and the dotted lines within represent median gene expression. Whiskers of the boxplot represent minimum and maximum observations. Relative expression levels were normalized to the housekeeping gene, B2M. Statistical significance is reported as p<0.05(*). D. Summary of relative expression of wildtype and mutant DLX6 transcripts of CP1 NSDTRs compared to WT NSDTRs. Fold change and p-values were calculated using REST2009 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004257#pgen.1004257-Pfaffl1" target="_blank">[24]</a>.</p

Summary of allele frequencies of genotyping results.

a<p>Allele frequencies indicate the number of individuals with at least one copy of the mutant allele.</p

Minor allele frequency of DLX5 and DLX6 variants among human cleft cohort and control cohort.

<p>N Number of individuals sequenced; CP Cleft palate; NS Nonsyndromic; CLP Cleft lip and palate.</p><p>– Genotypes counts not available.</p>a,b<p>These SNPs have minor allele frequencies from 7–11% in non-European populations.</p><p>SNPs with MAF listed in bold are statistically significant when compared to MAF of 1000 genomes and ESP databases <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004257#pgen.1004257-Abecasis1" target="_blank">[25]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004257#pgen.1004257-Exome1" target="_blank">[26]</a>.</p

Schematic illustration of genomic and cDNA DLX6 gene structure in unaffected (WT) and CP1 NSDTRs (CP1).

<p>Nucleotides boxed and in bold are the 13 base pair target site duplication identified as part of the DLX6 LINE-1 insertion. Conservation represents the UCSC genome browser comparative genomics conservation track of human, dog, mouse, and rat sequence conservation. The region of conservation represented in red is the region disrupted by the LINE-1 insertion. Image is not drawn to scale.</p

Genome-wide association study results of NSDTRs with CP.

<p>A. Manhattan plot of −log₁₀ of raw p-values by chromosome. The p_genome value is the p-value after 100000 permutations. The lowest p_genome is found across 8 SNPs on cfa14: 25822897, 25832747, 25847915, 25854827, 25868609, 25995782, 26023199, and 26082330. B. Plot of the raw p-values by Mb on cfa14 depicting the associated region. C. Observed haplotypes in 12 CP NSDTRs. Horizontal bars represent haplotypes from the 12 CP NSDTRs with associated haplotype, with runs of homozygosity in grey. The critical interval is defined by the shared homozygous haplotype denoted by the black bars (cfa14. 24189817-29319290). The 2 CP NSDTRs without the associated haplotype are not included in this figure. D. Quantile-Quantile plot of genome-wide association results. Black dots represent the observed versus expected p-values of all SNPs (λ = 1.05). Grey dots represent the observed versus expected p-values after removal of all SNPs on cfa14. (λ = 1.02). The solid grey line represents the null hypothesis: observed p-values equal expected p-values.</p

Phenotype of neonatal CP1 NSDTRs.

<p>A. Neonatal CP1 NSDTR with an extensive cleft of the hard and soft palate. B. Neonatal NSDTR with a normal palate (WT). C. Lateral view of CP1 head exhibiting relative mandibular brachygnathia. D. Lateral view of WT head with a normal jaw relationship. E. Coronal CT image depicting the failure of the palatine processes and nasal septum to fuse in CP1 NSDTRs. F. Coronal CT image depicting midline fusion of palatal process and nasal septum in WT. P – Palatine process, NS – Nasal septum G. 3D reconstruction of microCT imaging of CP1 and WT skulls with mandibles removed. CP1 skull shows abnormally shaped palatine process and palatine bones. Bones colored blue are the palatine processes and palatine bones. WT skull shows anatomical location of normal palatine sutures and shape of palatine processes and palatine bones. H. 3D reconstruction of mandibles depicting abnormal angulation of the condylar process (*) in CP1 mandibles compared to WT mandibles.</p