March 2015

CONTEXT: A heterozygous de novo c.1228G>A mutation (E410K) in the TUBB3 gene encoding the neuronal-specific β-tubulin isotype 3 (TUBB3) causes the TUBB3 E410K syndrome characterized by congenital fibrosis of the extraocular muscles (CFEOM), facial weakness, intellectual and social disabilities, and Kallmann syndrome (anosmia with hypogonadotropic hypogonadism). All TUBB3 E410K subjects reported to date are sporadic cases. OBJECTIVE: This study aimed to report the clinical, genetic, and molecular features of a familial presentation of the TUBB3 E410K syndrome. DESIGN: Case report of a mother and three affected children with clinical features of the TUBB3 E410K syndrome. SETTING: Academic Medical Center. MAIN OUTCOME MEASURES: Genetic analysis of the TUBB3 gene and clinical evaluation of endocrine and nonendocrine phenotypes. RESULTS: A de novo TUBB3 c.1228G>A mutation arose in a female proband who displayed CFEOM, facial weakness, intellectual and social disabilities, and anosmia. However, she underwent normal sexual development at puberty and had three spontaneous pregnancies with subsequent autosomal-dominant inheritance of the mutation by her three boys. All sons displayed nonendocrine features of the TUBB3 E410K syndrome similar to their mother but, in addition, had variable features suggestive of additional endocrine abnormalities. CONCLUSIONS: This first report of an autosomal-dominant inheritance of the TUBB3 c.1228G>A mutation in a family provides new insights into the spectrum and variability of endocrine phenotypes associated with the TUBB3 E410K syndrome. These observations emphasize the need for appropriate clinical evaluation and complicate genetic counseling of patients and families with this syndrome.

The ability to form biofilms in a variety of environments is a common trait of bacteria, and may represent one of the earliest defenses against predation. Biofilms are multicellular communities usually held together by a polymeric matrix, ranging from capsular material to cell lysate. In a structure that imposes diffusion limits, environmental microgradients arise to which individual bacteria adapt their physiologies, resulting in the gamut of physiological diversity. Additionally, the proximity of cells within the biofilm creates the opportunity for coordinated behaviors through cell-cell communication using diffusible signals, the most well documented being quorum sensing. Biofilms form on abiotic or biotic surfaces, and because of that are associated with a large proportion of human infections. Biofilm formation imposes a limitation on the uses and design of ocular devices, such as intraocular lenses, posterior contact lenses, scleral buckles, conjunctival plugs, lacrimal intubation devices and orbital implants. In the absence of abiotic materials, biofilms have been observed on the capsule, and in the corneal stroma. As the evidence for the involvement of microbial biofilms in many ocular infections has become compelling, developing new strategies to prevent their formation or to eradicate them at the site of infection, has become a priority.

The paucity of animal models exhibiting full pathology of diabetic retinopathy (DR) has impeded understanding of the pathogenesis of DR and the development of therapeutic interventions. Here we investigated if hyperhexosemic marmosets (Callithrix jacchus) develop characteristic retinal vascular lesions including macular edema (ME), a leading cause of vision loss in DR. Marmosets maintained on 30% galactose (gal)-rich diet for two years were monitored for retinal vascular permeability, development of ME, and morphological characteristics including acellular capillaries (AC) and pericyte loss (PL), vessel tortuosity, and capillary basement membrane (BM) thickness. Excess vascular permeability, increased number of AC and PL, vascular BM thickening, and increased vessel tortuosity were observed in the retinas of gal-fed marmosets. Optical coherence tomography (OCT) images revealed significant thickening of the retinal foveal and the juxtafoveal area, and histological analysis showed incipient microaneurysms in retinas of gal-fed marmosets. Findings from this study indicate that hyperhexosemia can trigger retinal vascular changes similar to those seen in human DR including ME and microaneurysms. The striking similarities between the marmoset retina and the human retina, and the exceptionally small size of the monkey, offer significant advantages to this primate model of DR.

PURPOSE: To determine whether there is an association between response to intravitreal anti-vascular endothelial growth factor agents and genotype in patients with neovascular age-related macular degeneration. METHODS: Analysis of the current literature evaluating pharmacogenetics of treatment response in patients with neovascular age-related macular degeneration. RESULTS: Studies have demonstrated associations between various genotypes and response to intravitreal anti-vascular endothelial growth factor agents. Lower-risk genotypes of the CFH, ARMS2, HTRA1, and VEGF-A genes may be associated with improved visual outcomes. Additionally, frequency of injections may be associated with certain genotypes. CONCLUSION: Genetic background may influence an individual's response to treatment of neovascular age-related macular degeneration. Further studies to investigate biologic pathways of neovascular age-related macular degeneration and gene products that are directly involved might lead to better understanding of contribution of various genes to treatment response.

Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism. MECP2 encodes a methyl-DNA-binding protein that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.

Here we report that VEGF-A and IGF-1 differ in their ability to stabilize newly formed blood vessels and endothelial cell tubes. Although VEGF-A failed to support an enduring vascular response, IGF-1 stabilized neovessels generated from primary endothelial cells derived from various vascular beds and mouse retinal explants. In these experimental systems, destabilization/regression was driven by lysophosphatidic acid (LPA). Because previous studies have established that Erk antagonizes LPA-mediated regression, we considered whether Erk was an essential component of IGF-dependent stabilization. Indeed, IGF-1 lost its ability to stabilize neovessels when the Erk pathway was inhibited pharmacologically. Furthermore, stabilization was associated with prolonged Erk activity. In the presence of IGF-1, Erk activity persisted longer than in the presence of VEGF or LPA alone. These studies reveal that VEGF and IGF-1 can have distinct inputs in the angiogenic process. In contrast to VEGF, IGF-1 stabilizes neovessels, which is dependent on Erk activity and associated with prolonged activation.

PURPOSE: To compare immunohistochemical and genetic overlaps and differences between intraocular medulloepitheliomas and embryonal tumors with multilayered rosettes of the brain. DESIGN: Retrospective histopathologic, immunohistochemical and genetic analysis of 20 intraocular medulloepitheliomas. METHODS: 1) Review of clinical data and hematoxylin and eosin stained sections with 2) immunohistochemical staining of paraffin sections using a polyclonal antibody against the protein LIN28A, and 3) FISH testing for the amplification of the genetic locus 19q13.42 involving the C19MC cluster of miRNA. Ten retinoblastomas served as controls and to determine the specificity of these biomarkers for intraocular medulloepitheliomas. RESULTS: Nineteen of the 20 intraocular medulloepitheliomas were either diffusely or focally LIN28A positive (weak, moderate or strong). The most intense positivity correlated with aggressive behavior such as intraocular tissue invasion or extraocular extension. None of the cases studied by fluorescence in situ hybridization (FISH) harbored an amplicon for C19MC. The ten retinoblastomas were LIN28A and C19MC negative. CONCLUSION: LIN28A has a putative role in oncogenesis and is found only in embryonic cells and malignancies. Intraocular medulloepitheliomas and embryonal tumors with multilayered rosettes of the brain both display LIN28A positivity. Only the latter, however, display amplification of the 19q13.42 locus involving C19MC, implying that other causative factors are at play in intraocular medulloepitheliomas. More aggressive tumor behavior within the eye can be partially predicted by LIN28A staining intensity.

An unprecedented pigmented caruncular apocrine hidrocystoma with the additional feature of an oncocytic transformation of the cyst's lining cells is reported. Over a year, a 79-year-old woman developed a centrally pigmented lesion of her right caruncle with translucent borders. Because of concern about a melanoma, a carunculectomy with adjunctive cryotherapy and placement of an amniotic membrane graft were performed, and the excised specimen was evaluated microscopically. A large cyst dominated the caruncle and was lined by an inner layer of columnar eosinophilic and granular cells with an outer, interrupted layer of flattened myoepithelial cells. Phosphotungstic acid hematoxylin staining disclosed myriad cytoplasmic, dot-like mitochondria signifying an oncocytic change. Immunohistochemistry revealed gross cystic fluid disease protein-15 and cytokeratin 7-positivity indicative of apocrine differentiation. Oncocytic change is characteristically encountered in lacrimal ductal cysts and tumors.

A 47-year-old woman presented with a medial orbital tumor initially diagnosed as either a myxoid neurofibroma or myoepithelioma. Over 30 years the tumor recurred seven times and was serially debulked. Careful histopathologic analysis coupled with immunohistochemical studies performed on the last two biopsies established the rare diagnosis of a locally aggressive angiomyxoma (because of its local infiltrative growth) with myofibroblastic features (smooth muscle actin and calponin positivity and desmin negativity). The last recurrence manifested at a shorter interval than the earlier ones, suggesting an accelerating clinical course. By this late stage there was complete blindness, a frozen globe, and extreme, unmeasurable proptosis accompanied by massive chemosis and eyelid fullness. An exenteration was performed, and the orbital contents contained a persistent angiomyxoma, but additionally, another cellular population had emerged-mitotically active cells with a malignant rhabdoid phenotype (round shape, cytoplasmic hyaline/globoid inclusions composed of whorls of compact vimentin filaments as well as epithelial membrane antigen and focal cytokeratin positivity). This is the first orbital case of a rhabdoid transformation of a benign orbital mesenchymal tumor. Shortly after the exenteration, multifocal metastases, notably to the lungs, were found, leading to the introduction of chemotherapy, which was discontinued because of non-responsiveness of the tumor and patient intolerance. After 1 year of follow up, the patient is still alive, but has persistent active disease with widespread metastases and a guarded prognosis.

PURPOSE: We explored whether risk factor associations differed by primary open-angle glaucoma (POAG) subtypes defined by visual field (VF) loss pattern (i.e., paracentral or peripheral). METHODS: We included 77,157 women in the Nurses Health Study and 42,773 men in the Health Professionals Follow-up Study (1986-2010) and incident medical-record confirmed cases of paracentral (n=440) and peripheral (n=865) POAG subtypes. We evaluated African-heritage, glaucoma family history, body mass index (BMI), mean arterial blood pressure, diabetes mellitus, physical activity, smoking, caffeine and alcohol intakes. We used competing risk Cox regression analyses modeling age as the metameter and stratified by age, cohort and event type. We sequentially identified factors with the least significant differences in associations with POAG subtypes ("stepwise down" approach with P for heterogeneity [P-het]<0.10 as threshold). RESULTS: BMI was more inversely associated with the POAG paracentral VF loss subtype than the peripheral VF loss subtype (per 10 kg/m2; hazard ratio [HR]=0.67 [95% Confidence Interval [CI]: 0.52, 0.86] vs. HR=0.93 [95% CI: 0.78, 1.10]; P-het=0.03) as was smoking (per 10 pack-years; HR=0.92 [95% CI: 0.87, 0.98] vs. HR=0.98 [95% CI: 0.94, 1.01]; P-het=0.09). These findings were robust in sensitivity analyses using a "stepwise up" approach (identify factors that showed the most significant differences). Non-heterogeneous (p-het>0.10) adverse associations with both POAG subtypes were observed with glaucoma family history, diabetes, African-heritage, greater caffeine intake and higher mean arterial pressure. CONCLUSIONS: These data indicate that POAG with early paracentral VF loss has distinct as well as common determinants compared to POAG with peripheral VF loss.

While efforts have been made over the years, the exact cause of keratoconus (KC) remains unknown. The aim of this study was to identify alterations in endogenous metabolites in the tears of KC patients compared with age-matched healthy subjects. Three groups were tested: 1) Age-matched controls with no eye disease (N = 15), 2) KC - patients wearing Rigid Gas permeable lenses (N = 16), and 3) KC - No Correction (N = 14). All samples were processed for metabolomics analysis using LC-MS/MS. We identified a total of 296 different metabolites of which >40 were significantly regulated between groups. Glycolysis and gluconeogenesis had significant changes, such as 3-phosphoglycerate and 1,3 diphosphateglycerate. As a result the citric acid cycle (TCA) was also affected with notable changes in Isocitrate, aconitate, malate, and acetylphosphate, up regulated in Group 2 and/or 3. Urea cycle was also affected, especially in Group 3 where ornithine and aspartate were up-regulated by at least 3 fold. The oxidation state was also severely affected. Groups 2 and 3 were under severe oxidative stress causing multiple metabolites to be regulated when compared to Group 1. Group 2 and 3, both showed significant down regulation in GSH-to-GSSG ratio when compared to Group 1. Another indicator of oxidative stress, the ratio of lactate - pyruvate was also affected with Groups 2 and 3 showing at least a 2-fold up regulation. Overall, our data indicate that levels of metabolites related to urea cycle, TCA cycle and oxidative stress are highly altered in KC patients.

PURPOSE: In thyroid orbitopathy, surgical treatment of exophthalmos and compressive optic neuropathy is orbital decompression. Deep lateral wall decompression has been advocated alone or combined with the medial wall for a "balanced" decompression. The degree of lateral decompression is dependent on the volume of the sphenoid trigone comprising the deep lateral orbital wall. This study aims to compare the volume of the trigone in various races in men and women. METHODS: After Institutional Review Board approval, patients with normal sinus CT scans (Siemens Somatom 40-slice) were retrospectively reviewed. Inclusion criteria were men and women aged 30 to 60 years, no orbital disease or surgery, normal orbital CT scans, and self-reported race (Asian, black/African American, white). Scans were measured with imaging software (Synapse, Fujifilm USA). The superior and inferior extents of the measured trigone were the superior and inferior orbital fissures, respectively. In the axial CT plane, the areas of each slice of the right and left trigone were manually outlined with the software and volume subsequently calculated based on the slice thickness (2 mm). Comparisons between groups were made via repeated measures analysis of variance. RESULTS: One hundred twenty subjects were included, 20 from each subgroup, yielding 240 measured orbits. The overall volume of the sphenoid trigone for all groups combined was 1.53 cm (standard deviation 0.72 cm). Mean male volume was significantly larger than mean female volume (1.71 ± 0.83 cm vs. 1.35 ± 0.55 cm; p = 0.004). Average left side volume was larger than paired right side volume (1.58 ± 0.74 cm vs. 1.49 ± 0.71 cm; p = 0.02). There were no significant differences in average volumes between races (p = 0.17). CONCLUSIONS: The mean sphenoid trigone volume was larger in men than in women. There were no significant differences in volume between racial groups. The data showed significant interindividual and intraindividual variability. When analyzing these data for the purposes of orbital decompression, planning should be based on each side of each patient, as the expected degree of lateral decompression may vary greatly.

PURPOSE: To determine patient factors and eye conditions associated with artifacts in Spectralis optical coherence tomography (OCT) retinal nerve fiber layer (RNFL) scans. DESIGN: Retrospective cross-sectional study. METHODS: The prevalence of 12 artifact types were described in this review of 2313 eye scans from 1188 patients who underwent a complete eye examination with Spectralis OCT scanning during the period of September 2009 to July 2013. The generalized estimating equations model was used to analyze associations between increased artifact prevalence and 10 patient characteristics, which included age, sex, race, visual acuity, refractive error, astigmatism, cataract status, glaucoma staging, visual field reliability, and glaucoma diagnosis. RESULTS: A total of 1070 or 46.3% of the 2313 eye scans had at least 1 artifact. Decentration error was the most common artifact (27.8%), followed by posterior vitreous detachment artifacts (14.4%). Visual acuity of less than 20/40 (P < .0001), presence of moderate to severe cataracts (P < .0001), advanced stage of glaucoma (P < .0001), and a diagnosis of open-angle glaucoma (P = .0003) were associated with increased prevalence of artifacts. CONCLUSIONS: Clinicians should first assess scans for artifacts before making therapeutic decisions based on RNFL thickness measurements.

PURPOSE: Azithromycin and tetracyclines are commonly prescribed in the United States for the treatment of meibomian gland dysfunction (MGD). The efficacy of these antibiotics has been believed to be their antiinflammatory and antibacterial actions, which suppress MGD-associated posterior blepharitis and growth of lid bacteria. However, we recently discovered that azithromycin can act directly on human meibomian gland epithelial cells (HMGECs) to stimulate their function. In this study, we sought to determine whether tetracycline antibiotics can duplicate this azithromycin effect. METHODS: Immortalized HMGEC were cultured in the presence of a vehicle, azithromycin, doxycycline, minocycline, or tetracycline for 5 days. Cells were evaluated for cholesterol and neutral lipid staining, and the lipid composition of cellular lysates was analyzed by high-performance thin-layer chromatography. RESULTS: Our results demonstrate that azithromycin's ability to stimulate the differentiation of human meibomian gland cells is unique, and is not duplicated by doxycycline, minocycline, or tetracycline. Azithromycin, but not the other antibiotics, significantly increased the cellular accumulation of cholesterol, cholesterol esters, phospholipids, and lysosomes. These differentiative actions of azithromycin were paralleled by an increased expression of sterol regulatory element-binding protein 1. CONCLUSIONS: Our findings show that the stimulatory effects of azithromycin on HMGEC function are unique and are not duplicated by the antibiotics doxycycline, minocycline, or tetracycline. Our results further suggest that this stimulatory influence of azithromycin may contribute to its beneficial effect in treating MGD and its associated evaporative dry eye disease.

Analyses of genome-wide association study (GWAS) data have revealed that detectable genetic mosaicism involving large (>2 Mb) structural autosomal alterations occurs in a fraction of individuals. We present results for a set of 24,849 genotyped individuals (total GWAS set II [TGSII]) in whom 341 large autosomal abnormalities were observed in 168 (0.68%) individuals. Merging data from the new TGSII set with data from two prior reports (the Gene-Environment Association Studies and the total GWAS set I) generated a large dataset of 127,179 individuals; we then conducted a meta-analysis to investigate the patterns of detectable autosomal mosaicism (n = 1,315 events in 925 [0.73%] individuals). Restricting to events >2 Mb in size, we observed an increase in event frequency as event size decreased. The combined results underscore that the rate of detectable mosaicism increases with age (p value = 5.5 × 10(-31)) and is higher in men (p value = 0.002) but lower in participants of African ancestry (p value = 0.003). In a subset of 47 individuals from whom serial samples were collected up to 6 years apart, complex changes were noted over time and showed an overall increase in the proportion of mosaic cells as age increased. Our large combined sample allowed for a unique ability to characterize detectable genetic mosaicism involving large structural events and strengthens the emerging evidence of non-random erosion of the genome in the aging population.

PURPOSE: To categorize vitrectomy cytologic diagnoses and ancillary tests to address appropriate processing of low-volume vitreous samples. DESIGN: Retrospective case series. PARTICIPANTS: Five thousand seven hundred thirty-six vitreous samples. METHODS: Cytologic diagnoses of therapeutic and diagnostic vitrectomy samples and their processing protocols from 3 teaching institutions were reviewed. MAIN OUTCOME MEASURES: Diagnostic results were categorized as negative for malignancy, suspicious for malignancy, and positive for malignancy. All ancillary studies performed were documented, including special stains, immunohistochemistry analysis, cytokine levels, and polymerase chain reaction (PCR) analysis. RESULTS: Of the 5736 vitreous samples analyzed, 4683 (81.64%) were from Tufts Medical Center (TMC), 955 (16.65%) were from Boston Medical Center (BMC), and 98 (1.70%) were from Massachusetts Eye Research and Surgery Institution (MERSI). Cases from TMC and BMC were therapeutic and diagnostic vitrectomies, and MERSI cases were diagnostic vitrectomies. Most vitrectomies showed negative results for malignancy: 99.47% of TMC cases, 99.89% of BMC cases, and 79.6% of MERSI cases. These included vitreous hemorrhage and inflammatory or infectious findings. Ancillary studies performed in this category included Periodic Acid-Schiff staining for fungi, PCR analysis for toxoplasmosis, cytomegalovirus, Epstein-Barr virus (EBV), herpes simplex virus I and II, and vitreous cultures for infections (coagulase-negative Staphylococcus, Candida, Fusarium, and Propionibacterium species). Interleukin (IL) 10-to-IL-6 ratios were performed on 38.7% of cases from MERSI. Fourteen cases from TMC were suspicious for malignancy based on cytologic evaluation. Eleven cases from TMC, 1 case from BMC, and 20 cases from MERSI showed positive results for malignancy and included B-cell lymphoma, retinoblastoma, melanoma, and metastatic adenocarcinoma. The ancillary testing included PCR for heavy chain immunoglobulin gene rearrangements, immunohistochemistry for EBV, in situ hybridization for κ and λ light chains, and cytogenetics. CONCLUSIONS: This is the largest data pool of reported cytologic diagnoses of diagnostic and therapeutic vitrectomy samples. Cytologic evaluation of therapeutic vitrectomy samples provides a valuable baseline of nonpathologic findings that assist in differentiation between malignancy, infections, and inflammatory conditions. Allocation of small-volume vitreous samples to select ancillary testing from the plethora of available diagnostic tests requires preoperative communication between surgeons and pathologists to ensure appropriate and timely treatment methods.

Diagnosing Horner Syndrome can be difficult in the setting of an incomplete triad. A 27-year-old man presented with unilateral eyelid droop and intermittent ipsilateral headaches, having already seen 7 physicians. Physical examination revealed unilateral ptosis but no pupillary miosis or facial anhidrosis. Inspection of his clinical photographs revealed elevation of the ipsilateral lower eyelid, suggesting sympathetic dysfunction. On further questioning, he admitted to naphazoline dependence. Reexamination after ceasing the naphazoline unveiled the anisocoria. Vascular imaging subsequently revealed carotid dissection, and the patient was started on anticoagulant and antiplatelet therapy. The ptosis persisted after conjunctival Müllerectomy. External levator resection was recommended, but patient declined. This case underscores the importance of clinical photography, meticulous medical record review, and complete medication history including over-the-counter preparations. Clinicians should meticulously inspect the lower eyelid in cases of atypical blepharoptosis and consider the effects of eye drops when inspecting pupils for miosis.

PURPOSE: A pocket-sized collision warning device equipped with a video camera was developed to predict impending collisions based on time to collision rather than proximity. A study was conducted in a high density obstacle course to evaluate the effect of the device on collision avoidance in people with peripheral field loss (PFL). METHODS: The 41 meter long loop-shaped obstacle course consisted of 46 stationary obstacles from floor to head level, and oncoming pedestrians. Twenty five patients with tunnel vision (n = 13) or hemianopia (n = 12) completed 4 consecutive loops with and without the device, while not using any other habitual mobility aid. Walking direction and device usage order were counterbalanced. Number of collisions and preferred percentage of walking speed (PPWS) were compared within subjects. RESULTS: Collisions were reduced significantly by about 37% (p < 0.001) with the device (Floor-level obstacles were excluded because the device was not designed for them). No patient had more collisions when using the device. While the PPWS also reduced with the device from 52% to 49% (p = 0.053), this did not account for the lower number of collisions, as the changes in collisions and PPWS were not correlated (p = 0.516). CONCLUSIONS: The device may help patients with a wide range of PFL avoid collisions with high-level obstacles and barely affect their walking speed.