Genetic Evidence for Differential Regulation of Corneal Epithelial and Stromal Thickness.

PURPOSE: Central corneal thickness (CCT) is a quantitative trait associated with keratoconus and primary open-angle glaucoma. Although CCT is highly heritable, known genetic variations explain only a fraction of the phenotypic variability. The purpose of this study was to identify additional CCT-influencing loci using inbred strains of mice. METHODS: Cohorts of 82 backcrossed (N2) and 99 intercrossed (F2) mice were generated from crosses between recombinant inbred BXD24/TyJ and wild-derived CAST/EiJ mice. Using anterior chamber optical coherence tomography, mice were phenotyped at 10 to 12 weeks of age, genotyped based on 96 genome-wide single nucleotide polymorphisms (SNPs), and subjected to quantitative trait locus (QTL) analysis. RESULTS: In an analysis of total CCT among all mice, two loci passed the significance threshold of P = 0.05. These were on Chr 3 and Chr 11 (Cctq4 and Cctq5, respectively). A third locus of interest was identified in a two-dimensional pairwise analysis; this locus on Chr 14 (Cctq6) exhibited a significant additive effect with Cctq5. Independent analyses of the dataset for epithelial and stromal thickness revealed that Cctq4 is specific to the epithelial layer and that Cctq5 and Cctq6 are specific to the stromal layer. CONCLUSIONS: Our findings demonstrate a quantitative multigenic pattern of CCT inheritance in mice and identify three previously unrecognized CCT-influencing loci: Cctq4, Cctq5, and Cctq6. This is the first demonstration that distinct layers of the cornea are under differential genetic control and highlights the need to refine the design of future genome-wide association studies of CCT.

Ketamine/Xylazine-Induced Corneal Damage in Mice.

PURPOSE: We have observed that the commonly used ketamine/xylazine anesthesia mix can induce a focally severe and permanent corneal opacity. The purpose of this study was to establish the clinical and histological features of this deleterious side effect, its sensitivity with respect to age and anesthesia protocol, and approaches for avoiding it. METHODS: Young C57BL/6J, C57BLKS/J, and SJL/J mice were treated with permutations of anesthesia protocols and compared using slit-lamp exams, optical coherence tomography, histologic analyses, and telemetric measurements of body temperature. RESULTS: Ketamine/xylazine induces corneal damage in mice with a variable frequency. Among 12 experimental cohorts, corneal damage associated with ketamine/xylazine was observed in 9 of them. Despite various treatments to avoid corneal dehydration during anesthesia, the frequency of corneas experiencing damage among responding cohorts was 42% (26% inclusive of all cohorts), which is significantly greater than the natural prevalence (5%). The damage was consistent with band keratopathy. It appeared as a white or gray horizontal band located proximal to the pupil and was positive for subepithelial calcium deposition with von Kossa stain. CONCLUSIONS: The sum of our clinical and histological observations is consistent with ketamine/xylazine-induced band keratopathy in mice. This finding is relevant for mouse studies involving the eye and/or vision-dependent behavioral assays, which would both be prone to artifact without appreciation of the damage caused by ketamine/xylazine anesthesia. Use of yohimbine is suggested as a practical means of avoiding this complication.

Support and challenges to the melanosomal casing model based on nanoscale distribution of metals within iris melanosomes detected by X-ray fluorescence analysis.

Melanin within melanosomes exists as eumelanin or pheomelanin. Distributions of these melanins have been studied extensively within tissues, but less often within individual melanosomes. Here, we apply X-ray fluorescence analysis with synchrotron radiation to survey the nanoscale distribution of metals within purified melanosomes of mice. The study allows a discovery-based characterization of melanosomal metals, and, because Cu is specifically associated with eumelanin, a hypothesis-based test of the 'casing model' predicting that melanosomes contain a pheomelanin core surrounded by a eumelanin shell. Analysis of Cu, Ca, and Zn shows variable concentrations and distributions, with Ca/Zn highly correlated, and at least three discrete patterns for the distribution of Cu vs. Ca/Zn in different melanosomes - including one with a Cu-rich shell surrounding a Ca/Zn-rich core. Thus, the results support predictions of the casing model, but also suggest that in at least some tissues and genetic contexts, other arrangements of melanin may co-exist.

Nano-scale morphology of melanosomes revealed by small-angle X-ray scattering.

Melanosomes are highly specialized organelles that produce and store the pigment melanin, thereby fulfilling essential functions within their host organism. Besides having obvious cosmetic consequences--determining the color of skin, hair and the iris--they contribute to photochemical protection from ultraviolet radiation, as well as to vision (by defining how much light enters the eye). Though melanosomes can be beneficial for health, abnormalities in their structure can lead to adverse effects. Knowledge of their ultrastructure will be crucial to gaining insight into the mechanisms that ultimately lead to melanosome-related diseases. However, due to their small size and electron-dense content, physiologically intact melanosomes are recalcitrant to study by common imaging techniques such as light and transmission electron microscopy. In contrast, X-ray-based methodologies offer both high spatial resolution and powerful penetrating capabilities, and thus are well suited to study the ultrastructure of electron-dense organelles in their natural, hydrated form. Here, we report on the application of small-angle X-ray scattering--a method effective in determining the three-dimensional structures of biomolecules--to whole, hydrated murine melanosomes. The use of complementary information from the scattering signal of a large ensemble of suspended organelles and from single, vitrified specimens revealed a melanosomal sub-structure whose surface and bulk properties differ in two commonly used inbred strains of laboratory mice. Whereas melanosomes in C57BL/6J mice have a well-defined surface and are densely packed with 40-nm units, their counterparts in DBA/2J mice feature a rough surface, are more granular and consist of 60-nm building blocks. The fact that these strains have different coat colors and distinct susceptibilities to pigment-related eye disease suggest that these differences in size and packing are of biological significance.

Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus.

Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10(-8)). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4-1.88, P = 2.7 × 10(-10), and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29-1.68, P = 4.9 × 10(-9)). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10(-4); tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.

Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma.

PURPOSE: Mutations in the myocilin gene (MYOC) are the most common known genetic cause of primary open-angle glaucoma (POAG). The purpose of this study was to determine whether topical ocular sodium 4-phenylbutyrate (PBA) treatment rescues glaucoma phenotypes in a mouse model of myocilin-associated glaucoma (Tg-MYOC(Y437H) mice). METHODS: Tg-MYOC(Y437H) mice were treated with PBA eye drops (n = 10) or sterile PBS (n = 8) twice daily for 5 months. Long-term safety and effectiveness of topical PBA (0.2%) on glaucoma phenotypes were examined by measuring intraocular pressure (IOP) and pattern ERG (PERG), performing slit lamp evaluation of the anterior chamber, analyzing histologic sections of the anterior segment, and comparing myocilin levels in the aqueous humor and trabecular meshwork of Tg-MYOC(Y437H) mice. RESULTS: Tg-MYOC(Y437H) mice developed elevated IOP at 3 months of age when compared with wild-type (WT) littermates (n = 24; P < 0.0001). Topical PBA did not alter IOP in WT mice. However, it significantly reduced elevated IOP in Tg-MYOC(Y437H) mice to the level of WT mice. Topical PBA-treated Tg-MYOC(Y437H) mice also preserved PERG amplitudes compared with vehicle-treated Tg-MYOC(Y437H) mice. No structural abnormalities were observed in the anterior chamber of PBA-treated WT and Tg-MYOC(Y437H) mice. Analysis of the myocilin in the aqueous humor and TM revealed that PBA significantly improved the secretion of myocilin and reduced myocilin accumulation as well as endoplasmic reticulum (ER) stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, topical PBA reduced IOP elevated by induction of ER stress via tunicamycin injections in WT mice. CONCLUSIONS: Topical ocular PBA reduces glaucomatous phenotypes in Tg-MYOC(Y437H) mice, most likely by reducing myocilin accumulation and ER stress in the TM. Topical ocular PBA could become a novel treatment for POAG patients with myocilin mutations.

Anterior segment dysgenesis and early-onset glaucoma in nee mice with mutation of Sh3pxd2b.

PURPOSE: Mutations in SH3PXD2B cause Frank-Ter Haar syndrome, a rare condition characterized by congenital glaucoma, as well as craniofacial, skeletal, and cardiac anomalies. The nee strain of mice carries a spontaneously arising mutation in Sh3pxd2b. The purpose of this study was to test whether nee mice develop glaucoma. METHODS: Eyes of nee mutants and strain-matched controls were comparatively analyzed at multiple ages by slit lamp examination, intraocular pressure recording, and histologic analysis. Cross sections of the optic nerve were analyzed to confirm glaucomatous progression. RESULTS: Slit lamp examination showed that, from an early age, nee mice uniformly exhibited severe iridocorneal adhesions around the entire circumference of the eye. Presumably as a consequence of aqueous humor outflow blockage, they rapidly developed multiple indices of glaucoma. By 3 to 4 months of age, they exhibited high intraocular pressure (30.8 ± 12.5 mm Hg; mean ± SD), corneal opacity, and enlarged anterior chambers. Although histologic analyses at P17 did not reveal any indices of damage, similar analysis at 3 to 4 months of age revealed a course of progressive retinal ganglion cell loss, optic nerve head excavation, and axon loss. CONCLUSIONS: Eyes of nee mice exhibit anterior segment dysgenesis and early-onset glaucoma. Because SH3PXD2B is predicted to be a podosome adaptor protein, these findings implicate podosomes in normal development of the iridocorneal angle and the genes influencing podosomes as candidates in glaucoma. Because of the early-onset, high-penetrance glaucoma, nee mice offer many potential advantages as a new mouse model of the disease.

Quantitative trait loci associated with murine central corneal thickness.

The cornea is a specialized transparent tissue responsible for refracting light, serving as a protective barrier, and lending structural support to eye shape. Given its importance, the cornea exhibits a surprising amount of phenotypic variability in some traits, including central corneal thickness (CCT). More than a mere anatomic curiosity, differences in CCT have recently been associated with risk for glaucoma. Although multiple lines of evidence support a strong role for heredity in regulating CCT, the responsible genes remain unknown. To better understand the genetic basis of CCT variability, we conducted a genomewide quantitative trait locus (QTL) analysis with (C57BLKS/J x SJL/J) F(2) mice. This experiment identified a locus, Cctq1 (central corneal thickness QTL 1) on chromosome 7 (Chr 7; peak, 105 Mb), that is significantly associated with CCT. To independently test the biological significance of these results, (C57BLKS/J x NZB/B1NJ) F(2) mice were generated and analyzed for associations with Chr 7. This experiment identified a significant association at 131 Mb. Furthermore, low-generation congenic mice in which the Chr 7 QTL interval from the SJL strain was transferred onto the KS background had CCT values significantly higher than inbred KS mice. These results demonstrate that the genetic dependence of CCT in mice is a multigenic trait, which in these contexts is significantly regulated by a region on Chr 7. Future identification of the genes for these QTL will provide improved understanding of the processes regulating CCT and the pathophysiology of glaucoma.

Tethering recombination initiation proteins in Saccharomyces cerevisiae promotes double strand break formation.

Meiotic recombination in Saccharomyces cerevisiae is initiated by the creation of DNA double strand breaks (DSBs), an event requiring 10 recombination initiation proteins. Published data indicate that these 10 proteins form three main interaction subgroups [(Spo11-Rec102-Rec104-Ski8), (Rec114-Rec107-Mei4), and (Mre11-Rad50-Xrs2)], but certain components from each subgroup may also interact. Although several of the protein-protein interactions have been defined, the mechanism for DSB formation has been challenging to define. Using a variation of the approach pioneered by others, we have tethered 8 of the 10 initiation proteins to a recombination coldspot and discovered that in addition to Spo11, 6 others (Rec102, Rec104, Ski8, Rec114, Rec107, and Mei4) promote DSB formation at the coldspot, albeit with different frequencies. Of the 8 proteins tested, only Mre11 was unable to cause DSBs even though it binds to UAS(GAL) at GAL2. Our results suggest there may be several ways that the recombination initiation proteins can associate to form a functional initiation complex that can create DSBs.