Optic nerve hypoplasia in miniature poodle dogs: A preliminary genetic and candidate gene association study.

OBJECTIVE: To conduct a genetic and candidate gene association study with samples from phenotype-ascertained dogs to identify putative disease-associated gene/mutation for optic nerve hypoplasia (ONH) in the miniature poodle. ANIMALS STUDIED: A total of 43 miniature poodles from the United States and Europe, nine affected bilaterally with ONH, were included in the study. Pedigree information was recorded. PROCEDURES: A pedigree including all animals studied was assembled. Twenty-one genes typically expressed in ganglion cells or that are associated with ocular malformations and have a critical function in eye and neural retina development were selected. Exons and exon-intron boundaries of eight genes were sequenced in four ONH cases and four controls. Furthermore, cases and controls were genotyped with the Illumina CanineHD BeadChip to obtain genotypes for 13 additional candidate genes for haplotype association. RESULTS: The assembled pedigree connected all ONH-affected dogs to a possible common founder. Identified variants and haplotypes of the tested candidate genes did not segregate with the phenotype using Identity by Descent approach assuming autosomal recessive inheritance with variable but yet unknown penetrance. CONCLUSIONS: Pedigree analysis did not reveal the inheritance pattern. There is no evidence of association of the evaluated candidate genes with ONH; therefore, the screened candidate genes can provisionally be ruled out as causally associated with the disease.

Frameshift Variant in AMPD2 in Cirneco dell'Etna Dogs with Retinopathy and Tremors.

While the manifestations of many inherited retinal disorders are limited to loss of vision, others are part of a syndrome that affects multiple tissues, particularly the nervous system. Most syndromic retinal disorders are thought to be recessively inherited. Two dogs out of a litter of Cirneco dell' Etna dogs, both males, showed signs of retinal degeneration, along with tremors and signs described as either atypical seizures or paroxysmal dyskinesias, while the other two male littermates were normal. We named this oculo-neurological syndrome CONS (Cirneco oculo-neurological syndrome), and undertook homozygosity mapping and whole-genome sequencing to determine its potential genetic etiology. Notably, we detected a 1-bp deletion in chromosome 6 that was predicted to cause a frameshift and premature stop codon within the canine AMPD2 gene, which encodes adenosine monophosphate deaminase, an enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP). Genotyping of the available Cirneco population suggested perfect segregation between cases and controls for the variant. Moreover, this variant was absent in canine genomic databases comprised of thousands of unaffected dogs. The AMPD2 genetic variant we identified in dogs presents with retinal manifestations, adding to the spectrum of neurological manifestations associated with AMPD2 variants in humans.

A naturally occurring canine model of syndromic congenital microphthalmia.

In humans, the prevalence of congenital microphthalmia is estimated to be 0.2-3.0 for every 10,000 individuals, with nonocular involvement reported in ∼80% of cases. Inherited eye diseases have been widely and descriptively characterized in dogs, and canine models of ocular diseases have played an essential role in unraveling the pathophysiology and development of new therapies. A naturally occurring canine model of a syndromic disorder characterized by microphthalmia was discovered in the Portuguese water dog. As nonocular findings included tooth enamel malformations, stunted growth, anemia, and thrombocytopenia, we hence termed this disorder Canine Congenital Microphthalmos with Hematopoietic Defects. Genome-wide association study and homozygosity mapping detected a 2 Mb candidate region on canine chromosome 4. Whole-genome sequencing and mapping against the Canfam4 reference revealed a Short interspersed element insertion in exon 2 of the DNAJC1 gene (g.74,274,883ins[T70]TGCTGCTTGGATT). Subsequent real-time PCR-based mass genotyping of a larger Portuguese water dog population found that the homozygous mutant genotype was perfectly associated with the Canine Congenital Microphthalmos with Hematopoietic Defects phenotype. Biallelic variants in DNAJC21 are mostly found to be associated with bone marrow failure syndrome type 3, with a phenotype that has a certain degree of overlap with Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and reports of individuals showing thrombocytopenia, microdontia, and microphthalmia. We, therefore, propose Canine Congenital Microphthalmos with Hematopoietic Defects as a naturally occurring model for DNAJC21-associated syndromes.

CCDC66 frameshift variant associated with a new form of early-onset progressive retinal atrophy in Portuguese Water Dogs.

Aberrant photoreceptor function or morphogenesis leads to blinding retinal degenerative diseases, the majority of which have a genetic aetiology. A variant in PRCD previously identified in Portuguese Water Dogs (PWDs) underlies prcd (progressive rod-cone degeneration), an autosomal recessive progressive retinal atrophy (PRA) with a late onset at 3-6 years of age or older. Herein, we have identified a new form of early-onset PRA (EOPRA) in the same breed. Pedigree analysis suggested an autosomal recessive inheritance. Four PWD full-siblings affected with EOPRA diagnosed at 2-3 years of age were genotyped (173,661 SNPs) along with 2 unaffected siblings, 2 unaffected parents, and 15 unrelated control PWDs. GWAS, linkage analysis and homozygosity mapping defined a 26-Mb candidate region in canine chromosome 20. Whole-genome sequencing in one affected dog and its obligatory carrier parents identified a 1 bp insertion (CFA20:g.33,717,704_33,717,705insT (CanFam3.1); c.2262_c.2263insA) in CCDC66 predicted to cause a frameshift and truncation (p.Val747SerfsTer8). Screening of an extended PWD population confirmed perfect co-segregation of this genetic variant with the disease. Western blot analysis of COS-1 cells transfected with recombinant mutant CCDC66 expression constructs showed the mutant transcript translated into a truncated protein. Furthermore, in vitro studies suggest that the mutant CCDC66 is mislocalized to the nucleus relative to wild type CCDC66. CCDC66 variants have been associated with inherited retinal degenerations (RDs) including canine and murine ciliopathies. As genetic variants affecting the primary cilium can cause ciliopathies in which RD may be either the sole clinical manifestation or part of a syndrome, our findings further support a role for CCDC66 in retinal function and viability, potentially through its ciliary function.

Complex Structural PPT1 Variant Associated with Non-syndromic Canine Retinal Degeneration.

Rod and cone photoreceptors are specialized retinal neurons that have a fundamental role in visual perception, capturing light and transducing it into a neuronal signal. Aberrant functioning of rod and/or cone photoreceptors can ultimately lead to progressive degeneration and eventually blindness. In man, many rod and rod-cone degenerative diseases are classified as forms of retinitis pigmentosa (RP). Dogs also have a comparable disease grouping termed progressive retinal atrophy (PRA). These diseases are generally due to single gene defects and follow Mendelian inheritance.We collected 51 DNA samples from Miniature Schnauzers affected by PRA (average age of diagnosis ∼3.9 ±1 years), as well as from 56 clinically normal controls of the same breed (average age ∼6.6 ±2.8 years). Pedigree analysis suggested monogenic autosomal recessive inheritance of PRA. GWAS and homozygosity mapping defined a critical interval in the first 4,796,806 bp of CFA15. Whole genome sequencing of two affected cases, a carrier and a control identified two candidate variants within the critical interval. One was an intronic SNV in HIVEP3, and the other was a complex structural variant consisting of the duplication of exon 5 of the PPT1 gene along with a conversion and insertion (named PPT1dci ). PPT1dci was confirmed homozygous in a cohort of 22 cases, and 12 more cases were homozygous for the CFA15 haplotype. Additionally, the variant was found homozygous in 6 non-affected dogs of age higher than the average age of onset. The HIVEP3 variant was found heterozygous (n = 4) and homozygous wild-type (n = 1) in cases either homozygous for PPT1dci or for the mapped CFA15 haplotype. We detected the wildtype and three aberrant PPT1 transcripts in isolated white blood cell mRNA extracted from a PRA case homozygous for PPT1dci , and the aberrant transcripts involved inclusion of the duplicated exon 5 and novel exons following the activation of cryptic splice sites. No neurological signs were detected among the dogs homozygous for the PPT1dci variant. Therefore, we propose PPT1dci as causative for a non-syndromic form of PRA (PRA PPT1 ) that shows incomplete penetrance in Miniature Schnauzers, potentially related to the presence of the wild-type transcript. To our knowledge, this is the first case of isolated retinal degeneration associated with a PPT1 variant.