Classification performance of the BOAS Index.

<p>(A) Distribution of the BOAS Index for the French bulldog training dataset; (B) Box plots of the BOAS Index for the French bulldog training dataset according to functional grade. Boxes present lines at median, upper and lower quartiles; between whiskers = 95% confidence interval; circles = outliers within the inner fence; stars = outliers within the outer fence.</p

Signalment (median [minimum-maximum]) and details of French bulldogs and non-brachycephalic controls.

<p>Data are presented as median (minimum-maximum). BOAS refers to Brachycephalic Obstructive Airway Syndrome; BCS, body condition score.</p><p>^<b>a</b> Functional grades, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130741#pone.0130741.t001" target="_blank">Table 1</a>.</p><p>^<b>b</b> Breeds: English springer spaniel (n = 2), Border collie (n = 1), Jack Russell terrier (n = 1), Labrador retriever (n = 3), American bullterrier (n = 1), Beagles (n = 6), Dachshund (n = 1), Cairn terrier (n = 1), West Highland white terrier (n = 1), cross breeds (n = 3).</p><p>Signalment (median [minimum-maximum]) and details of French bulldogs and non-brachycephalic controls.</p

Representative WBBP flow waveforms for several study dogs.

<p>(A) non-brachycephalic control dog; (B) BOAS- French bulldog; (C) BOAS+ French bulldog, respiratory cycle Type 1; (D) BOAS+ French bulldog, Type 2; (E) BOAS+ French bulldog, Type 3.</p

Multiple Mechanisms Contribute to Leakiness of a Frameshift Mutation in Canine Cone-Rod Dystrophy

<div><h3></h3><p>Mutations in <em>RPGRIP1</em> are associated with early onset retinal degenerations in humans and dogs. Dogs homozygous for a 44 bp insertion including a polyA₂₉ tract potentially leading to premature truncation of the protein, show cone rod degeneration. This is rapid and blinding in a colony of dogs in which the mutation was characterised but in dogs with the same mutation in the pet population there is very variable disease severity and rate of progression.</p> <h3>Objective</h3><p>We hypothesized that this variability must be associated with leakiness of the <em>RPGRIP1</em> mutation, allowing continued RPGRIP1 production. The study was designed to discover mechanisms that might allow such leakiness.</p> <h3>Methods</h3><p>We analysed alternate start sites and splicing of <em>RPGRIP1</em> transcripts; variability of polyA_n length in the insertion and slippage at polyA_n during transcription/translation.</p> <h3>Results and Significance</h3><p>We observed a low rate of use of alternative start codons having potential to allow forms of transcript not including the insertion, with the possibility of encoding truncated functional RPGRIP1 protein isoforms. Complex alternative splicing was observed, but did not increase this potential. Variable polyA_n length was confirmed in DNA from different <em>RPGRIP1</em>^−/− dogs, yet polyA_n variability did not correspond with the clinical phenotypes and no individual was found that carried a polyA_n tract capable of encoding an in-frame variant. Remarkably though, in luciferase reporter gene assays, out-of-frame inserts still allowed downstream reporter gene expression at some 40% of the efficiency of in-frame controls. This indicates a major role of transcriptional or translational frameshifting in <em>RPGRIP1</em> expression. The known slippage of reverse transcriptases as well as RNA polymerases and thermostable DNA polymerases on oligoA homopolymers meant that we could not distinguish whether the majority of slippage was transcriptional or translational. This leakiness at the mutation site may allow escape from severe effects of the mutation for some dogs.</p> </div

Quantitation of cDNA fragments of different exonic regions of <i>RPGRIP1</i>.

<p>Retinal cDNA populations were analysed by quantitative RT-PCR in beagles of <i>RPGRIP1</i>^+/+ (blue) and <i>RPGRIP1</i>^−L/−L (red) genotypes. The absolute copy number of molecules in equal amounts of template cDNA is shown on a log scale. Each fragment was assayed in triplicate (technical replicates) and two replicate experiments and copy numbers of DNA molecules were calculated by comparison with control sequences cloned into plasmids.</p

Receiver operating characteristic (ROC) curve of the BOAS Index for diagnosis of functional BOAS+ French bulldogs.

<p>Bootstrapping was used to generate the associated 95% confidence intervals (area in blue) to delineate the expected range of screening performance. The black dot with whiskers (95% confidence interval) shows the position of the BOAS Index of 0.46 suggested as a cut off point for distinguishing functionally BOAS- and BOAS+ French bulldogs.</p

WBBP flow waveform illustration for a single respiratory cycle.

<p><b>The</b> flow cycle starts from inspiration (below the zero line of flow rate) then expiration (above the zero line of the flow rate).</p

Capillary electrophoresis of PCR products containing the polyA tract.

<p>PCR amplicon spanning the <i>RPGRIP1</i> polyA insertion was sized by capillary gel electrophoresis. The common electropherogram peak pattern from <i>RPGRIP1</i>^−/− MLHDs is represented by dogs MLD7 (b: late-onset affected, 9 y) and MLD4 (d: mid-onset affected, 5 y). In dogs MLD11 (a: clinically normal, 5 y) and MLD6 (c: clinically normal, 9 y), the highest peak in each electropherogram was shifted by 1 bp, to ‘113’, compared to the common PCR fragment peak pattern ‘114’. Note that the majority of the <i>RPGRIP1</i>^−/− dogs examined including both clinically affected and normal dogs showed the ‘114’ pattern. Direct cloning and haplotype analysis confirmed MLD6 as heterozygous for polyA_28/29 while the ‘114’ pattern corresponded to polyA_29/29.</p

p2 luc constructs used in dual-reporter luciferase assay.

<p>DNA sequences and the corresponding amino acids for plasmid constructs with polyA insertions (p2 luc/A_{28, 29 and 30}), and in-frame (p2 luc/F+) and <i>rluc</i>-only (p2 luc/F-, stop codon upstream of <i>fluc</i>). The polyA constructs shown indicate the three possible reading frames after the polyA sequence; only those with (3n-1) adenines, such as A₂₉, A₃₅, A₃₈ and A₄₁, lead to an in-frame <i>fluc</i>, unless the number of adenines is changed following transcription or the reading frame is altered during translation. (Note that this single base gain in the construct reading frame is specific to this reporter assay. In the cell, A₃₀, A₃₆, A₃₉ and A₄₂ would be in frame.) Blue and yellow highlights indicate <i>Renilla</i> and firefly gene sequences, respectively. The SalI and BamHI cloning sites are outlined. DNA sequence of the polyA tract is shown in red letters, while the flanking region (exon 3 of <i>RPGRIP1</i>) is in blue letters with the 15-bp duplication underlined.</p

Haplotypes spanning the 6.05 Mb flanking region of <i>RPGRIP1</i>.

<p> The ‘114’- major haplotype (yellow) corresponds to the haplotype predominant in <i>RPGRIP1</i>^−/− dogs, with the common PCR fragment pattern peaking at ‘114’ (A₂₉ insert) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051598#pone-0051598-g003" target="_blank">Figure 3</a>. Microsatellite marker alleles specific to the dogs with the ‘113’ pattern (A₂₈ insert) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051598#pone-0051598-g003" target="_blank">Figure 3</a> are indicated (blue). The polyA₂₈ allele was determined by cloning from genomic DNA (*) or PCR-fragment sizing (**).</p