An ADAM9 mutation in canine cone-rod dystrophy 3 establishes homology with human cone-rod dystrophy 9

Abstract

Purpose: To identify the causative mutation in a canine cone-rod dystrophy (crd3) that segregates as an adult onset disorder in the Glen of Imaal Terrier breed of dog.

Methods: Glen of Imaal Terriers were ascertained for crd3 phenotype by clinical ophthalmoscopic examination, and in selected cases by electroretinography. Blood samples from affected cases and non-affected controls were collected and used, after DNA extraction, to undertake a genome-wide association study using Affymetrix Version 2 Canine single nucleotide polymorphism chips and 250K Sty Assay protocol. Positional candidate gene analysis was undertaken for genes identified within the peak-association signal region. Retinal morphology of selected crd3-affected dogs was evaluated by light and electron microscopy.

Results: A peak association signal exceeding genome-wide significance was identified on canine chromosome 16. Evaluation of genes in this region suggested A Disintegrin And Metalloprotease domain, family member 9 (ADAM9), identified concurrently elsewhere as the cause of human cone-rod dystrophy 9 (CORD9), as a strong positional candidate for canine crd3. Sequence analysis identified a large genomic deletion (over 20 kb) that removed exons 15 and 16 from the ADAM9 transcript, introduced a premature stop, and would remove critical domains from the encoded protein. Light and electron microscopy established that, as in ADAM9 knockout mice, the primary lesion in crd3 appears to be a failure of the apical microvilli of the retinal pigment epithelium to appropriately invest photoreceptor outer segments. By electroretinography, retinal function appears normal in very young crd3-affected dogs, but by 15 months of age, cone dysfunction is present. Subsequently, both rod and cone function degenerate.

Conclusions: Identification of this ADAM9 deletion in crd3-affected dogs establishes this canine disease as orthologous to CORD9 in humans, and offers opportunities for further characterization of the disease process, and potential for genetic therapeutic intervention.

Figure 1

Electroretinograms of normal and affected dogs. Electroretinograms (ERGs) from a 7 weeks old normal dog (A), a 12 weeks old dog affected with canine cone-rod dystrophy 3 (crd3; B), a crd3-affected dog aged 1.2 years (C), a 2 years old crd3-affected dog (D), and a 4.9 years old crd3-affected dog (E). Each vertical panel presents electroretinogram (ERG) responses to a red flash, a blue flash, a white flash, 5 Hz low-intensity white flashes (Rod), and 30 Hz high-intensity white light flicker (Cone). Short vertical arrows under the Rod and Cone flicker responses indicate the onset of the flickering light stimuli. Red and White traces represent mixed rod-cone responses, Blue and Rod traces are rod-specific, and Cone traces are cone-specific. Responses of the 12-weeks-old crd3-affected dog appear normal (B), but by 15 months of age, cone dysfunction is detected as reduced 30 Hz flicker responses (C), and is followed at later ages by continued deterioration of both cone and rod responses (D, E). At all ages, the loss of cone function is more prominent than that of rods. Vertical calibration bar=100 µV; horizontal=200 ms for rod flicker; and other responses are 100 ms.

Figure 2

Light- and electron-microscopic retinal morphology in normal and canine cone-rod dystrophy 3 (crd3) affected dogs. In the retina of a 27-weeks-old non-affected dog (A), the outer nuclear layer (ONL) comprises approximately 10 rows of rod nuclei and a single distal row of cone nuclei. The inner and outer segments of the photoreceptors (IS, OS) are of consistent proportions, tightly aligned, and parallel, and the distal OS tips are in close proximity to the apical membrane of the retinal pigment epithelium (RPE). In retinas of 4.7- and 13.4-weeks-old crd3-affected dogs (B, C), rod and cone IS and OS lack the tightly packed highly parallel organization of a normal photoreceptor layer, and the distal OS tips appear to be more distant from the RPE apical membrane than in normal dogs. In the retina of an 18-weeks-old crd3-affected dog (D), IS and OS are disarrayed and disorganized, and a distinct gap is observed between the RPE and the OS (arrows). The retinas of 26-weeks- and 5 years-old crd3-affected dogs (E, F) exhibit continued photoreceptor degeneration as evidenced by loss of cone and rod IS, OS, and nuclei. Electron micrograph of the retina of a 27-weeks-old nonaffected dog (G) shows that the microvilli from the RPE apical membrane extend to invest the photoreceptor OS. Electron micrographs of the retina of a 13.4-weeks-old crd3-affected dog (H, I) show that the RPE apical microvilli form a tangled flattened mat that does not extend to invest the photoreceptor OS (arrows).

Figure 3

Results of genome-wide association study in canine cone-rod dystrophy 3. The statistical signal (y-axis, negative Log10 [Fisher exact test 2-tailed probability]) for association between canine single-nucleotide polymorphism (SNP) genotype and canine cone-rod dystrophy 3 (crd3) phenotype, plotted against SNP chromosomal location (A), demonstrates a distinct peak on canine chromosome 16 (CFA16). Green dots are SNPs for which the association signal exceeded the Bonferroni threshold for genome-wide significance. Chromosome X is represented by the numbers 39 and 40. Homozygosity analysis of SNP genotypes (B), in the region of CFA16 yielding the peak association signal, reveals heterozygosity throughout the interval in 21 nonaffected control dogs, and demonstrates a 2.74 Mb homozygosity block in 20 crd3-affected dogs. Genotypes are color coded as follows: pink and green represent the major and minor genotypes observed in affecteds, respectively; yellow is heterozygous; and white is missing data. Black lines border the 2.74 Mb homozygosity block. Refseq genes screened as potential positional candidates for crd3 in the present study (arrowheads), and ADAM family genes identified within the crd3 minimal linkage disequilibrium interval (arrows) are indicated with annotation and order consistent with the CanFam2 canine genome assembly (C, not drawn to scale).

Figure 4

ADAM9 mutation in canine cone-rod dystrophy 3 (crd3) affected dogs. A schematic drawing, of part of the canine ADAM9 genomic sequence, aligning the normal and crd3 mutant alleles suggests a possible mutation mechanism. Bordered square boxes represent exons 14–17, lines between are introns; unbordered rectangular boxes represent long-terminal repeat sequences (LTR). Within the LTR are the nucleotides identified as single nucleotide polymorphisms (SNPs). A suggested mechanism of unbalanced recombination is illustrated, resulting in the deletion of part of intron 14, all of exon 15, intron 15, exon 16, and part of intron 16, as well as the formation of a single chimeric LTR. Arrows represent the location of primers used to identify the mutation. Primer pair F29/R31 amplifies the mutant allele and results in a 1,515 bp PCR product from genomic DNA. Primer pair F10/R10 amplifies the normal allele and results in a 602 bp PCR product from genomic DNA. B: Gel electrophoresis of multiplex PCR reaction identifies ADAM9 alleles from normal (N), crd3-carrier (C), and crd3-affected (A) dogs. The lower, 602 bp band is the normal allele, amplified by primer pair F10/R10 located within intron 15, which is deleted in the affected allele. The upper 1,515 bp band is the mutant allele, amplified by primer pair F29/R31, which flanks the >23 kb sequence deleted in the affecteds. Both bands are present in the heterozygous carrier dog. The normal and crd3 mutant canine ADAM9 transcripts, and their corresponding predicted translation products, are aligned (C) to illustrate their differences schematically. The protein domains represented are those predicted by Swiss-Prot for the human ADAM9 protein. Exons 15 and 16 are missing from the mutant transcript, and a premature stop codon is introduced (arrow). The mutant protein translated from this transcript is predicted to be truncated, lacking the last 287 amino acids of the C-terminus, part of the cysteine-rich domain, the complete epidermal growth factor (EGF)-like domain, the transmembrane domain, and the cytoplasmic tail.

Figure 5

ADAM9 RNA expression profile in normal and crd3-affected retinas. Northern blot, RNA expression of ADAM9 in frontal lobe, brain of 15.7 weeks-old normal dog (Lane 1); brain of 7.7 weeks-old normal dog (Lane 2); retina of 10.4-weeks old normal dog (Lane 3); retina of 8.6-weeks old normal dog (Lane 4); spleen of 22.1-weeks-old normal dog (Lane 5); retina of 12-weeks old normal dog (Lane 6); and retina of 13.4-weeks-old crd3-affected dog (Lane 7). A single band is observed at approximately 4.0 Kb. ADAM9 is highly expressed in retinas not affected with crd3 (lanes 3, 4, and 6), and at lower levels in brain and spleen (lanes 1, 2, and 5). In crd3-affected retina (lane 7), the mutant allele is observed as a slightly smaller band, and its level of expression appears slightly reduced compared to the normal.