The genome sequence (1.9-fold coverage) of an inbred Abyssinian domestic cat was assembled, mapped, and annotated with a comparative approach that involved cross-reference to annotated genome assemblies of six mammals (human, chimpanzee, mouse, rat, dog, and cow). The results resolved chromosomal positions for 663,480 contigs, 20,285 putative feline gene orthologs, and 133,499 conserved sequence blocks (CSBs). Additional annotated features include repetitive elements, endogenous retroviral sequences, nuclear mitochondrial (numt) sequences, micro-RNAs, and evolutionary breakpoints that suggest historic balancing of translocation and inversion incidences in distinct mammalian lineages. Large numbers of single nucleotide polymorphisms (SNPs), deletion insertion polymorphisms (DIPs), and short tandem repeats (STRs), suitable for linkage or association studies were characterized in the context of long stretches of chromosome homozygosity. In spite of the light coverage capturing ∼65% of euchromatin sequence from the cat genome, these comparative insights shed new light on the tempo and mode of gene/genome evolution in mammals, promise several research applications for the cat, and also illustrate that a comparative approach using more deeply covered mammals provides an informative, preliminary annotation of a light (1.9-fold) coverage mammal genome sequence.

In 10 of 11 treated RPE65(-/-) eyes, gene transfer resulted in development of vision, both subjectively apparent by loss of nystagmus, and objectively recorded by ERG. Structurally, there was reversal of lipid droplet accumulation in the RPE. Uveitis developed in 75% of the transgene-treated eyes, a complication possibly due to an immunopathogenic response to the RPE65 molecule.

Scanning-laser ophthalmoscopy is a technique for confocal imaging of the eye in vivo. The use of lasers of different wavelengths allows to obtain information about specific tissues and layers due to their reflection and transmission characteristics. In addition, fluorescent dyes excitable in the blue and infrared range offer a unique access to the vascular structures associated with each layer. In animal models, a further enhancement in specificity can be obtained by GFP expression under control of tissue-specific promotors. Important fields of application are studies in retinal degenerations and the follow-up of therapeutic intervention.

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel −/−). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel −/− melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.

No abstract

A mutation in the CEP290 gene is reported in a cat pedigree segregating for autosomal recessive (AR) late-onset photoreceptor degeneration (rdAc). An initial screen of 39 candidate genes and genomic locations failed to detect linkage to cat rdAc. Linkage was ultimately established on cat B4 with 15 simple tandem repeat markers (logarithm of odds [LOD] range 4.83-15.53, Theta = 0.0), in a region demonstrating conserved synteny to human chromosome 12, 84.9-90.63 Mb. The sequence of 10 genes with feline retinal expression was examined in affected and unaffected individuals. A single-nucleotide polymorphism was characterized in intron 50 of CEP290 (IVS50 + 9T>G) that creates a strong canonical splice donor site, resulting in a 4-bp insertion and frameshift in the mRNA transcript, with subsequent introduction of a stop codon and premature truncation of the protein. A population genetic survey of 136 cats demonstrated that the rdAc mutation is in low frequency in Abyssinian populations (0.13, Sweden; 0.07, United States) and absent in breeds of non-Abyssinian heritage. Mutations in CEP290 have recently been shown to cause two human diseases, Joubert syndrome, a syndromic retinal degeneration, and Leber's congenital amaurosis, an AR early-onset retinal dystrophy. Human AR retinitis pigmentosa is among the most common causes of retinal degeneration and blindness, with no therapeutic intervention available. This identification of a large animal model for human retinal blindness offers considerable promise in developing gene-based therapies.

Progressive retinal atrophy (PRA), a hereditary eye disease leading to blindness, was found in the Abyssinian breed of cat. Sixty-eight cases of a bilateral generalized retinopathy, at different stages of the disease process, were seen in the breed during ophthalmoscopic examinations of cats throughout Sweden during a 2-year period. Forty-five percent of cats aged 2 years or older were affected in the examined group. The earliest case was diagnosed in a 16-month-old cat. At the age of 3-4 years a bilateral retinal atrophy was usually present in affected cats. Genetic analysis indicates that PRA in the Abyssinian cat is caused by an autosomal recessive gene.

Leber’s congenital amaurosis (LCA) is a group of severe inherited retinal degenerations that are symptomatic in infancy and lead to total blindness in adulthood. Recent clinical trials using recombinant adeno-associated virus serotype 2 (rAAV2) successfully reversed blindness in patients with LCA caused by RPE65 mutations after one subretinal injection. However, it was unclear whether treatment of the second eye in the same manner would be safe and efficacious, given the potential for a complicating immune response after the first injection. Here, we evaluated the immunological and functional consequences of readministration of rAAV2-hRPE65v2 to the contralateral eye using large animal models. Neither RPE65-mutant (affected; RPE65−/−) nor unaffected animals developed antibodies against the transgene product, but all developed neutralizing antibodies against the AAV2 capsid in sera and intraocular fluid after subretinal injection. Cell-mediated immune responses were benign, with only 1 of 10 animals in the study developing a persistent T cell immune response to AAV2, a response that was mediated by CD4+ T cells. Sequential bilateral injection caused minimal inflammation and improved visual function in affected animals. Thus, subretinal readministration of rAAV2 in animals is safe and effective, even in the setting of preexisting immunity to the vector, a parameter that has been used to exclude patients from gene therapy trials.