Initial Genome-Wide Case–Control Study for Genetic Background of Retinal Dysplasia in Czechoslovakian Wolfdog

Liu Y., Ye Z., Yu H., Zhang Y., Li Z.

Congenital cataract is one of the leading causes of vision loss in children, and a large proportion of cases are related to genetics. In a Chinese family, we reported a new missense mutation in CRYBA2 (c.223T>C: p.Tyr75His), which can cause autosomal dominant congenital bilateral cataract. We collected blood samples from family members (mother and two sons) and extracted DNA. Through whole-exome sequencing, we discovered a novel unreported mutation. According to relevant ACMG guidelines, this mutation was determined to be a variant of unknown clinical significance. This article further expands the site information on the CRYBA2 mutations.

Garcia M.J., Morales M.S., Yang T.S., Holden J., Bossardet O.L., Palmer S.A., Jhala M., Priest S., Namburu N., Beatty N., D’Empaire Salomon S.E., Vancel J., Wareham L.K., Padovani-Claudio D.A.

The family of pro-inflammatory and pro-angiogenic chemokines including Interleukin-8 (IL-8, aka CXCL8) and its homologues (CXCL1,2,3,5,6, and 7) exhibit promiscuous binding and activation of several G-protein-coupled receptors (i.e., CXCR2, CXCR1, and the atypical chemokine receptor (ACKR1)). A high proportion of their biological activity is attributed to CXCR2 activation, thus many CXCR2 inhibitors are in clinical trials for several chronic diseases. However, CXCR2 inhibition is often only investigated acutely in these trials or in Cxcr2−/− mice grown in gnotobiotic conditions. Since humans do not live in germ-free environments, our first goal is to highlight novel retinal and systemic observations in Cxcr2−/− mice grown in non-gnotobiotic conditions that suggest potential harmful consequences of long-term CXCR2 deficiency or blockade. Beyond confirmation of circulating blood/immune cell-related phenotypes, we report novel findings in Cxcr2−/− mice including: (1) delayed dye transit to the retinal vasculature, (2) alterations in the density and distribution of retinal vessels, astrocytes and microglia, (3) decreased electroretinogram a- and b-wave amplitudes, (4) reduced visual acuity, and (5) increased polymorphonuclear cell accumulation in vascular lumina abutting venular walls in the retina and in vital non-ocular tissues (lung and liver). Furthermore, PheWAS of CXCR2 CXCR1, and ACKR1 gene variants using data from UK Biobank participants suggest clinical associations with both retinal and vascular disease phenotypes. We conclude that chronic CXCR2 deficiency in mice contributes to functional damage to the retina and that the long-term safety of CXCR1/2 inhibitors designed for chronic use in humans should be explored before clinical adoption to safeguard sight and overall vascular health.

Gelatt K.N., Plummer C.E.

Shan S., Xu F., Brenig B.

Genome-wide association study (GWAS) using dog breed standard values as phenotypic measurements is an efficient way to identify genes associated with morphological and behavioral traits. As a result of strong human purposeful selections, several specialized behavioral traits such as herding and hunting have been formed in different modern dog breeds. However, genetic analyses on this topic are rather limited due to the accurate phenotyping difficulty for these complex behavioral traits. Here, 268 dog whole-genome sequences from 130 modern breeds were used to investigate candidate genes underlying dog herding, predation, temperament, and trainability by GWAS. Behavioral phenotypes were obtained from the American Kennel Club based on dog breed standard descriptions or groups (conventional categorization of dog historical roles). The GWAS results of herding behavior (without body size as a covariate) revealed 44 significantly associated sites within five chromosomes. Significantly associated sites on CFA7, 9, 10, and 20 were located either in or near neuropathological or neuronal genes including THOC1, ASIC2, MSRB3, LLPH, RFX8, and CHL1. MSRB3 and CHL1 genes were reported to be associated with dog fear. Since herding is a restricted hunting behavior by removing killing instinct, 36 hounds and 55 herding dogs were used to analyze predation behavior. Three neuronal-related genes (JAK2, MEIS1, and LRRTM4) were revealed as candidates for predation behavior. The significantly associated variant of temperament GWAS was located within ACSS3 gene. The highest associated variant in trainability GWAS is located on CFA22, with no variants detected above the Bonferroni threshold. Since dog behaviors are correlated with body size, we next incorporate body mass as covariates into GWAS; and significant signals around THOC1, MSRB3, LLPH, RFX8, CHL1, LRRTM4, and ACSS3 genes were still detected for dog herding, predation, and temperament behaviors. In humans, these candidate genes are either involved in nervous system development or associated with mental disorders. In conclusion, our results imply that these neuronal or psychiatric genes might be involved in biological processes underlying dog herding, predation, and temperament behavioral traits.

Momozawa Y., Merveille A., Battaille G., Wiberg M., Koch J., Willesen J.L., Proschowsky H.F., Gouni V., Chetboul V., Tiret L., Fredholm M., Seppälä E.H., Lohi H., Georges M., Lequarré A.

The domestic dog represents an ideal model for identifying susceptibility genes, many of which are shared with humans. In this study, we investigated the genetic contribution to individual differences in 40 clinically important measurements by a genome-wide association study (GWAS) in a multinational cohort of 472 healthy dogs from eight breeds. Meta-analysis using the binary effects model after breed-specific GWAS, identified 13 genome-wide significant associations, three of them showed experimental-wide significant associations. We detected a signal at chromosome 13 for the serum concentration of alanine aminotransferase (ALT) in which we detected four breed-specific signals. A large proportion of the variance of ALT (18.1–47.7%) was explained by this locus. Similarly, a single SNP was also responsible for a large proportion of the variance (6.8–78.4%) for other measurements such as fructosamine, stress during physical exam, glucose, and morphometric measurements. The genetic contribution of single variant was much larger than in humans. These findings illustrate the importance of performing meta-analysis after breed-specific GWAS to reveal the genetic contribution to individual differences in clinically important measurements, which would lead to improvement of veterinary medicine.

Iwabe S., Dufour V.L., Guzmán J.M., Holle D.M., Cohen J.A., Beltran W.A., Aguirre G.D.

PURPOSE: To examine the in vivo microanatomy of retinal folds and geographic lesions in dogs with acquired or inherited retinal dysplasia. MATERIAL AND METHODS: Thirteen dogs had retinal microanatomy evaluation under general anesthesia using cSLO/sdOCT; two eyes had noninherited multifocal retinal folds, five had inherited multifocal retinal folds (drd1 or drd2), and 10 geographic retinal dysplasia. Retinas from two drd2 carrier dogs were examined by histology and immunohistochemistry (IHC) after in vivo imaging. RESULTS: Retinal folds are the common feature of acquired focal/multifocal or geographic retinal dysplasia, are indistinguishable structurally from those associated with syndromic oculoskeletal dysplasia, and represent outer nuclear layer invaginations and rosettes visible by sdOCT. In dogs heterozygous for oculoskeletal dysplasia, the folds form clusters in a perivascular distribution along superior central vessels. IHC confirmed photoreceptor identity in the retinal folds. The geographic dysplasia plaques are not focally detached, but have inner retinal disorganization and intense autofluorescence in cSLO autofluorescence mode that is mainly limited to the geographic lesion, but is not uniform and in some extends beyond the plaques. CONCLUSION: We propose that the autofluorescent characteristic of the geographic lesions is associated with an inner retinal disruption associated with perivascular or infiltrating macrophages and phagocytosis of cellular debris. As well, we suggest restructuring the examination forms to distinguish the folds that are sporadically distributed from those that have a perivascular distribution as the latter likely represent carriers for drd. In this latter group, DNA testing would be a helpful tool to provide specific breeding advice.

Stavinohova R., Hartley C., Burmeister L.M., Ricketts S.L., Pettitt L., Tetas Pont R., Hitti R.J., Schofield E., Oliver J.A., Mellersh C.S.

Seven Northern Inuit Dogs (NID) were diagnosed by pedigree analysis with an autosomal recessive inherited oculoskeletal dysplasia (OSD). Short-limbed dwarfism, angular limb deformities and a variable combination of macroglobus, cataracts, lens coloboma, microphakia and vitreopathy were present in all seven dogs, while retinal detachment was diagnosed in five dogs. Autosomal recessive OSD caused by COL9A3 and COL9A2 mutations have previously been identified in the Labrador Retriever (dwarfism with retinal dysplasia 1—drd1) and Samoyed dog (dwarfism with retinal dysplasia 2—drd2) respectively; both of those mutations were excluded in all affected NID. Nine candidate genes were screened in whole genome sequence data; only one variant was identified that was homozygous in two affected NID but absent in controls. This variant was a nonsense single nucleotide polymorphism in COL9A3 predicted to result in a premature termination codon and a truncated protein product. This variant was genotyped in a total of 1,232 dogs. All seven affected NID were homozygous for the variant allele (T/T), while 31/116 OSD-unaffected NID were heterozygous for the variant (C/T) and 85/116 were homozygous for the wildtype allele (C/C); indicating a significant association with OSD (p = 1.41x10-11). A subset of 56 NID unrelated at the parent level were analysed to determine an allele frequency of 0.08, estimating carrier and affected rates to be 15% and 0.6% respectively in NID. All 1,109 non-NID were C/C, suggesting the variant is rare or absent in other breeds. Expression of retinal mRNA was similar between an OSD-affected NID and OSD-unaffected non-NID. In conclusion, a nonsense variant in COL9A3 is strongly associated with OSD in NID, and appears to be widespread in this breed.

Salen G., Steiner R.D.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of bile acid synthesis caused by mutations in the cytochrome P450 CYP27A1 gene that result in production of a defective sterol 27-hydroxylase enzyme. CTX is associated with abnormally high levels of cholestanol in the blood and accumulation of cholestanol and cholesterol in the brain, tendon xanthomas, and bile. Hallmark clinical manifestations of CTX include chronic diarrhea, bilateral cataracts, tendon xanthomas, and neurologic dysfunction. Although CTX is a rare disorder, it is thought to be underdiagnosed, as presenting signs and symptoms may be nonspecific with significant overlap with other more common conditions. There is marked variability in signs and symptoms, severity, and age of onset between patients. The disease course is progressive and potentially debilitating or fatal, particularly with respect to neurologic presentations that can include intellectual disability, autism, behavioral and psychiatric problems, and dementia, among others. Treatment with chenodeoxycholic acid (CDCA; chenodiol) is the current standard of care. CDCA can help restore normal sterol, bile acid, bile alcohol, and cholestanol levels. CDCA also appears to be generally effective in preventing adverse clinical manifestations of the disease from occurring or progressing if administered early enough. Improved screening and awareness of the condition may help facilitate early diagnosis and treatment.

Chang C.C., Chow C.C., Tellier L.C., Vattikuti S., Purcell S.M., Lee J.J.

Saadane A., Mast N., Charvet C.D., Omarova S., Zheng W., Huang S.S., Kern T.S., Peachey N.S., Pikuleva I.A.

Cholesterol elimination from nonhepatic cells involves metabolism to side-chain oxysterols, which serve as transport forms of cholesterol and bioactive molecules modulating a variety of cellular processes. Cholesterol metabolism is tissue specific, and its significance has not yet been established for the retina, where cytochromes P450 (CYP27A1 and CYP46A1) are the major cholesterol-metabolizing enzymes. We generated Cyp27a1(-/-)Cyp46a1(-/-) mice, which were lean and had normal serum cholesterol and glucose levels. These animals, however, had changes in the retinal vasculature, retina, and several nonocular organs (lungs, liver, and spleen). Changes in the retinal vasculature included structural abnormalities (retinal-choroidal anastomoses, arteriovenous shunts, increased permeability, dilation, nonperfusion, and capillary degeneration) and cholesterol deposition and oxidation in the vascular wall, which also exhibited increased adhesion of leukocytes and activation of the complement pathway. Changes in the retina included increased content of cholesterol and its metabolite, cholestanol, which were focally deposited at the apical and basal sides of the retinal pigment epithelium. Retinal macrophages of Cyp27a1(-/-)Cyp46a1(-/-) mice were activated, and oxidative stress was noted in their photoreceptor inner segments. Our findings demonstrate the importance of retinal cholesterol metabolism for maintenance of the normal retina, and suggest new targets for diseases affecting the retinal vasculature.

Mellersh C.S.

Inherited forms of eye disease are arguably the best described and best characterized of all inherited diseases in the dog, at both the clinical and molecular level and at the time of writing 29 different mutations have been documented in the scientific literature that are associated with an inherited ocular disorder in the dog. The dog has already played an important role in the identification of genes that are important for ocular development and function as well as emerging therapies for inherited blindness in humans. Similarities in disease phenotype and eye structure and function between dog and man, together with the increasingly sophisticated genetic tools that are available for the dog, mean that the dog is likely to play an ever increasing role in both our understanding of the normal functioning of the eye and in our ability to treat inherited eye disorders. This review summarises the mutations that have been associated with inherited eye disorders in the dog.

Machiela M.J., Chanock S.J.

Genome-wide association studies in canine models may help locate genomic susceptibility regions that are relevant to human disease. See related Research: http://genomebiology.com/2014/15/3/R25

Omarova S., Charvet C.D., Reem R.E., Mast N., Zheng W., Huang S., Peachey N.S., Pikuleva I.A.

Several lines of evidence suggest a link between age-related macular degeneration and retinal cholesterol maintenance. Cytochrome P450 27A1 (CYP27A1) is a ubiquitously expressed mitochondrial sterol 27-hydroxylase that plays an important role in the metabolism of cholesterol and cholesterol-related compounds. We conducted a comprehensive ophthalmic evaluation of mice lacking CYP27A1. We found that the loss of CYP27A1 led to dysregulation of retinal cholesterol homeostasis, including unexpected upregulation of retinal cholesterol biosynthesis. Cyp27a1-/- mice developed retinal lesions characterized by cholesterol deposition beneath the retinal pigment epithelium. Further, Cyp27a1-null mice showed pathological neovascularization, which likely arose from both the retina and the choroid, that led to the formation of retinal-choroidal anastomosis. Blood flow alterations and blood vessel leakage were noted in the areas of pathology. The Cyp27a1-/- retina was hypoxic and had activated Müller cells. We suggest a mechanism whereby abolished sterol 27-hydroxylase activity leads to vascular changes and identify Cyp27a1-/- mice as a model for one of the variants of type 3 retinal neovascularization occurring in some patients with age-related macular degeneration.

Aguilar I., Misztal I., Legarra A., Tsuruta S.

Genomic evaluations can be calculated using a unified procedure that combines phenotypic, pedigree and genomic information. Implementation of such a procedure requires the inverse of the relationship matrix based on pedigree and genomic relationships. The objective of this study was to investigate efficient computing options to create relationship matrices based on genomic markers and pedigree information as well as their inverses. SNP maker information was simulated for a panel of 40 K SNPs, with the number of genotyped animals up to 30 000. Matrix multiplication in the computation of the genomic relationship was by a simple 'do' loop, by two optimized versions of the loop, and by a specific matrix multiplication subroutine. Inversion was by a generalized inverse algorithm and by a LAPACK subroutine. With the most efficient choices and parallel processing, creation of matrices for 30 000 animals would take a few hours. Matrices required to implement a unified approach can be computed efficiently. Optimizations can be either by modifications of existing code or by the use of efficient automatic optimizations provided by open source or third-party libraries.

Puk O., Ahmad N., Wagner S., Hrabé de Angelis M., Graw J.

A new mouse mutant with small lenses was identified within a mutagenesis screen. The aim of the study was to determine its molecular and morphologic characterization.The offspring of paternally N-ethyl-N-nitrosourea (ENU)-treated C57BL/6J mice were analyzed for eye-size parameters by noninvasive in vivo laser interference biometry.A new mutant characterized by a clear, but significantly smaller lens without any changes for cornea thickness, anterior chamber depth, or aqueous humor size, was identified. The smaller size of the lens was more pronounced in the homozygous mutants, which were fully fertile and viable. The mutation was mapped to chromosome 1 between the markers D1Mit251 and D1Mit253. Using a positional candidate approach, the βA2-crystallin encoding gene Cryba2 was sequenced; a T→C exchange at cDNA position 139 led to a p.S47P amino-acid alteration. The eyes of newborn homozygous mutants showed no gross changes. At the age of three weeks, some clefts appeared at the cornea, but the lens and retina appeared without major changes. At the age of 25 weeks, the lenses of the heterozygous mutants develop a subcapsular cortical cataract, but the lenses of homozygous mutants were completely opaque.These findings demonstrate the first mutation in the Cryba2 gene. In contrast to the closely linked Cryg gene cluster, no congenital cataract mutation could be attributed to the Cryba2 gene. Therefore, the human CRYBA2 gene should be considered as a strong candidate gene for age-related cataracts, and the slightly smaller size of the lens might be recognized as an early biomarker for age-related cataracts.