Variation in Genes Related to Cochlear Biology Is Strongly Associated with Adult-Onset Deafness in Border Collies

WOS:000309817900003Peer reviewe

Partial Deletion of the Sulfate Transporter <em>SLC13A1</em> Is Associated with an Osteochondrodysplasia in the Miniature Poodle Breed

<div><p>A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8∶8) of genotype data from 173 k SNPs revealed a single associated locus on <em>CFA14</em> (P_raw <10^–8). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence <em>in situ</em> hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of <em>SLC13A1</em>, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an <em>Slc13a1</em> mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by <em>SLC26A2</em> than with the mouse <em>Slc13a1</em> model. <em>SLC26A2</em> is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of <em>SLC13A1</em> in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.</p> </div

Gross presentation of Miniature Poodle osteochondrodysplasia.

<p>(A) An affected dog (foreground) shows dwarfism relative to an age-matched normal (wildtype) dog (background). (B) The misshapen paw of an affected dog (Aff) relative to that of a wildtype (WT) dog. (C) Close-up of the affected foot, which is similar to talipes varus, or clubfoot, a distinguishing feature of human osteochondrodsyplasias.</p

Serum sulfate levels by <i>SLC13A1</i> genotypic class.

<p>A scatter plot for sulfate concentration measurements from serum obtained from several dogs of each genotypic class: <i>SLC13A1</i> homozygotes (n = 5); heterozygotes (n = 4); and Δ<i>slc13a1</i> homozygotes (affected dogs; n = 3). The minimum sensitivity threshold for the assay is 0.2 mM. Differences between genotypes were assessed with the Kruskal Wallis test. Although sample sizes were relatively small, the mean sulfate level of affected dogs was significantly lower than the means of the other genotypic classes (<i>p</i> = 0.02).</p

Cytogenetic confirmation of a deletion on chromosome 14 by <i>FISH</i>.

<p>Dual-color FISH probe results are shown for representative metaphase spreads obtained from (A) wildtype, (B) obligate carrier, and (C) affected Miniature Poodle dogs, respectively. The green LR-PCR 14–63.6 g FISH signal is within the deleted region and shows some centromeric background staining. The red BAC probe 14–59.1or signal distinguishes <i>CFA14</i> within the canine metaphase spread. Wildtype shows co-localization of red (control) and green (experimental) probes. This co-localization is absent in the affected dog (homozygous for deletion), and is heterozygous in the carrier dog.</p

Genetic mapping of the osteochondrodysplasia locus.

<p>(A) Manhattan plot of genome-wide results displaying a statistically significant peak of association on the terminal end of <i>CFA14</i>. Plotted <i>p</i>-values were calculated by Fisher’s Exact Test. The p-values for the six most strongly associated SNPs (overlapping on the graph), adjusted by permutation, were p = 0.0006. (B) Genotypic pattern of SNPs across the interval of interest on <i>CFA14</i> (62.5–64.0 Mb). Red, major allele in cases; orange, heterozygous SNP genotypes; yellow, homozygous minor allele in cases. Genotype data are shown horizontally for each of seven controls and eight cases. The arrow notes an historical crossover that bounds the ancestral haplotype shared among cases, upon which the founder mutation is predicted to have occurred. Nine SNPs systematically failed in the case samples (white block with orange), suggesting a large deletion. (C) Raw fluorescence intensity readings from the SNP array for loci across the region of interest (62.5–64.0 Mb). Although several SNPs were initially scored heterozygous (orange, in B), the raw intensity data shows systematic failure in cases for all 12 SNPs in this interval (red bracket), relative to controls. (D) The potential deletion was bounded by two SNPs that were scored in cases. The putative deletion includes a single gene, <i>SLC13A1</i>, drawn to scale, with exon structure in red. The location of physical breakpoints that were subsequently cloned are shown as opaque boxes.</p

DNA test of large scale deletion.

<p>(A) A three-primer, dual reporter design exploits known breakpoints to create differentially dye-labeled and differentially-sized PCR products for a co-dominant DNA test readout. (B) The assay converted to a high-throughput format by analysis with a semi-automated DNA sequencing instrument for fluorescence-based fragment analysis. The electropherogram shows standard fragments for sizing (red peaks), product corresponding to the deletion-specific allele (blue peak), and product corresponding to the wildtype reference allele (green peak). Test outcomes are shown for each of the three genotypes possible. There is a size difference in the products corresponding to the wildtype allele in the clear dog and the carrier dog. This is attributable to a small insertion/deletion in the interval that segregates in the Miniature Poodle population.</p

A Comprehensive Linkage Map of the Dog Genome

We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dog's unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with ∼3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with ∼1500 loci. An additional ∼1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from ∼22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patterns of linkage disequilibrium (LD), and thus benefit association, selective sweep, and phylogenetic mapping approaches. The computational and wet-bench strategies can be applied to the reference genome of any nonmodel organism to assemble a de novo linkage map