National Eye Institute

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies. Results for the final phase of the 1000 Genomes Project are presented including whole-genome sequencing, targeted exome sequencing, and genotyping on high-density SNP arrays for 2,504 individuals across 26 populations, providing a global reference data set to support biomedical genetics. The 1000 Genomes Project has sought to comprehensively catalogue human genetic variation across populations, providing a valuable public genomic resource. The data obtained so far have found applications ranging from association studies and fine mapping studies to the filtering of likely neutral variants in rare-disease cohorts. The authors now report on the final phase of the project, phase 3, which covers previously uncharacterized areas of human genetic diversity in terms of the populations sampled and categories of characterized variation. The sample now includes more than 2,500 individuals from 26 global populations, with low coverage whole-genome and deep exome sequencing, as well as dense microarray genotyping. They find that while most common variants are shared across populations, rarer variants are often restricted to closely related populations. The authors also demonstrate the use of the phase 3 dataset as a reference panel for imputation to improve the resolution in genetic association studies.

The 2005 National Institutes of Health (NIH) Consensus Conference proposed new criteria for diagnosing and scoring the severity of chronic graft-versus-host disease (GVHD). The 2014 NIH consensus maintains the framework of the prior consensus with further refinement based on new evidence. Revisions have been made to address areas of controversy or confusion, such as the overlap chronic GVHD subcategory and the distinction between active disease and past tissue damage. Diagnostic criteria for involvement of mouth, eyes, genitalia, and lungs have been revised. Categories of chronic GVHD should be defined in ways that indicate prognosis, guide treatment, and define eligibility for clinical trials. Revisions have been made to focus attention on the causes of organ-specific abnormalities. Attribution of organ-specific abnormalities to chronic GVHD has been addressed. This paradigm shift provides greater specificity and more accurately measures the global burden of disease attributed to GVHD, and it will facilitate biomarker association studies.

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly. We report a genome-wide screen of 96 cases and 50 controls for polymorphisms associated with AMD. Among 116,204 single-nucleotide polymorphisms genotyped, an intronic and common variant in the complement factor H gene (CFH) is strongly associated with AMD (nominal P value <10(-7)). In individuals homozygous for the risk allele, the likelihood of AMD is increased by a factor of 7.4 (95% confidence interval 2.9 to 19). Resequencing revealed a polymorphism in linkage disequilibrium with the risk allele representing a tyrosine-histidine change at amino acid 402. This polymorphism is in a region of CFH that binds heparin and C-reactive protein. The CFH gene is located on chromosome 1 in a region repeatedly linked to AMD in family-based studies.

We report here the adoptive transfer, to patients with metastatic melanoma, of highly selected tumor-reactive T cells directed against overexpressed self-derived differentiation antigens after a nonmyeloablative conditioning regimen. This approach resulted in the persistent clonal repopulation of T cells in those cancer patients, with the transferred cells proliferating in vivo, displaying functional activity, and trafficking to tumor sites. This led to regression of the patients' metastatic melanoma as well as to the onset of autoimmune melanocyte destruction. This approach presents new possibilities for the treatment of patients with cancer as well as patients with human immunodeficiency virus-related acquired immunodeficiency syndrome and other infectious diseases.

OBJECTIVE: To estimate the prevalence and distribution of age-related macular degeneration (AMD) in the United States by age, race/ethnicity, and gender. METHODS: Summary prevalence estimates of drusen 125 microm or larger, neovascular AMD, and geographic atrophy were prepared separately for black and white persons in 5-year age intervals starting at 40 years. The estimated rates were based on a meta-analysis of recent population-based studies in the United States, Australia, and Europe. These rates were applied to 2000 US Census data and to projected US population figures for 2020 to estimate the number of the US population with drusen and AMD. RESULTS: The overall prevalence of neovascular AMD and/or geographic atrophy in the US population 40 years and older is estimated to be 1.47% (95% confidence interval, 1.38%-1.55%), with 1.75 million citizens having AMD. The prevalence of AMD increased dramatically with age, with more than 15% of the white women older than 80 years having neovascular AMD and/or geographic atrophy. More than 7 million individuals had drusen measuring 125 microm or larger and were, therefore, at substantial risk of developing AMD. Owing to the rapidly aging population, the number of persons having AMD will increase by 50% to 2.95 million in 2020. Age-related macular degeneration was far more prevalent among white than among black persons. CONCLUSION: Age-related macular degeneration affects more than 1.75 million individuals in the United States. Owing to the rapid aging of the US population, this number will increase to almost 3 million by 2020.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Leber's congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.

Every eye movement produces a shift in the visual image on the retina. The receptive field, or retinal response area, of an individual visual neuron moves with the eyes so that after an eye movement it covers a new portion of visual space. For some parietal neurons, the location of the receptive field is shown to shift transiently before an eye movement. In addition, nearly all parietal neurons respond when an eye movement brings the site of a previously flashed stimulus into the receptive field. Parietal cortex both anticipates the retinal consequences of eye movements and updates the retinal coordinates of remembered stimuli to generate a continuously accurate representation of visual space.

The space around us is represented not once but many times in parietal cortex. These multiple representations encode locations and objects of interest in several egocentric reference frames. Stimulus representations are transformed from the coordinates of receptor surfaces, such as the retina or the cochlea, into the coordinates of effectors, such as the eye, head, or hand. The transformation is accomplished by dynamic updating of spatial representations in conjunction with voluntary movements. This direct sensory-to-motor coordinate transformation obviates the need for a single representation of space in environmental coordinates. In addition to representing object locations in motoric coordinates, parietal neurons exhibit strong modulation by attention. Both top-down and bottom-up mechanisms of attention contribute to the enhancement of visual responses. The saliance of a stimulus is the primary factor in determining the neural response to it. Although parietal neurons represent objects in motor coordinates, visual responses are independent of the intention to perform specific motor acts.

PURPOSE: We investigated the combination of lymphodepleting chemotherapy followed by the adoptive transfer of autologous tumor reactive lymphocytes for the treatment of patients with refractory metastatic melanoma. PATIENTS AND METHODS: Thirty-five patients with metastatic melanoma, all but one with disease refractory to treatment with high-dose interleukin (IL) -2 and many with progressive disease after chemotherapy, underwent lymphodepleting conditioning with two days of cyclophosphamide (60 mg/kg) followed by five days of fludarabine (25 mg/m(2)). On the day following the final dose of fludarabine, all patients received cell infusion with autologous tumor-reactive, rapidly expanded tumor infiltrating lymphocyte cultures and high-dose IL-2 therapy. RESULTS: Eighteen (51%) of 35 treated patients experienced objective clinical responses including three ongoing complete responses and 15 partial responses with a mean duration of 11.5 +/- 2.2 months. Sites of regression included metastases to lung, liver, lymph nodes, brain, and cutaneous and subcutaneous tissues. Toxicities of treatment included the expected hematologic toxicities of chemotherapy including neutropenia, thrombocytopenia, and lymphopenia, the transient toxicities of high-dose IL-2 therapy, two patients who developed Pneumocystis pneumonia and one patient who developed an Epstein-Barr virus-related lymphoproliferation. CONCLUSION: Lymphodepleting chemotherapy followed by the transfer of highly avid antitumor lymphocytes can mediate significant tumor regression in heavily pretreated patients with IL-2 refractory metastatic melanoma.

PURPOSE: To describe the Oxford Scheme for grading ocular surface staining in dry eye and to discuss optimization of stain detection using various dyes and filters. Also, to propose a sequence of testing for dry eye diagnosis. METHODS: The grading of corneal and conjunctival staining is described, using the Oxford Scheme, including biomicroscopy, optical filters, illumination conditions, and the characteristics of and instillation techniques used for, selected clinical dyes. RESULTS: A series of panels, labeled A-E, in order of increasing severity, reproducing the staining patterns encountered in dry eye, are used as a guide to grade the degree of staining seen in the patient. The amount of staining seen in each panel, represented by punctate dots, increases by 0.5 of the log of the number of dots between panels B to E. The use of the vital dyes fluorescein, lissamine green, and rose Bengal is described; fluorescein and lissamine green, used in conjunction with appropriate absorption filters, are recommended for use in clinical trials. The placement of staining in relation to the sequence of other diagnostic tests is discussed. CONCLUSIONS: The monitoring and assessment of corneal and conjunctival staining can be greatly enhanced by the use of a grading scale, controlled instillation of dyes, and standard evaluation techniques. This is of particular benefit in clinical trials, where ocular surface staining is commonly employed as an outcome measure

Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA(-) and CD45RO(+) after ex vivo expansion. After infusion, the persisting cells displayed a CD45RA(+) and CD45RO(-) phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss. Thus, T cells expressing highly reactive TCRs mediate cancer regression in humans and target rare cognate-antigen-containing cells throughout the body, a finding with important implications for the gene therapy of cancer. This trial was registered at www.ClinicalTrials.gov as NCI-07-C-0174 and NCI-07-C-0175.

Although the involvement in the striatum in the refinement and control of motor movement has long been recognized, recent description of discrete frontal corticobasal ganglia networks in a range of species has focused attention on the role particularly of the dorsal striatum in executive functions. Current evidence suggests that the dorsal striatum contributes directly to decision-making, especially to action selection and initiation, through the integration of sensorimotor, cognitive, and motivational/emotional information within specific corticostriatal circuits involving discrete regions of striatum. We review key evidence from recent studies in rodent, nonhuman primate, and human subjects.

Here we report the identification of a novel human opsin, melanopsin, that is expressed in cells of the mammalian inner retina. The human melanopsin gene consists of 10 exons and is mapped to chromosome 10q22. This chromosomal localization and gene structure differs significantly from that of other human opsins that typically have four to seven exons. A survey of 26 anatomical sites indicates that, in humans, melanopsin is expressed only in the eye. In situ hybridization histochemistry shows that melanopsin expression is restricted to cells within the ganglion and amacrine cell layers of the primate and murine retinas. Notably, expression is not observed in retinal photoreceptor cells, the opsin-containing cells of the outer retina that initiate vision. The unique inner retinal localization of melanopsin suggests that it is not involved in image formation but rather may mediate nonvisual photoreceptive tasks, such as the regulation of circadian rhythms and the acute suppression of pineal melatonin. The anatomical distribution of melanopsin-positive retinal cells is similar to the pattern of cells known to project from the retina to the suprachiasmatic nuclei of the hypothalamus, a primary circadian pacemaker.

One of the goals of the NIH Toolbox for Assessment of Neurological and Behavioral Function was to identify or develop brief measures of emotion for use in prospective epidemiologic and clinical research. Emotional health has significant links to physical health and exerts a powerful effect on perceptions of life quality. Based on an extensive literature review and expert input, the Emotion team identified 4 central subdomains: Negative Affect, Psychological Well-Being, Stress and Self-Efficacy, and Social Relationships. A subsequent psychometric review identified several existing self-report and proxy measures of these subdomains with measurement characteristics that met the NIH Toolbox criteria. In cases where adequate measures did not exist, robust item banks were developed to assess concepts of interest. A population-weighted sample was recruited by an online survey panel to provide initial item calibration and measure validation data. Participants aged 8 to 85 years completed self-report measures whereas parents/guardians responded for children aged 3 to 12 years. Data were analyzed using a combination of classic test theory and item response theory methods, yielding efficient measures of emotional health concepts. An overview of the development of the NIH Toolbox Emotion battery is presented along with preliminary results. Norming activities led to further refinement of the battery, thus enhancing the robustness of emotional health measurement for researchers using the NIH Toolbox.

BACKGROUND: Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders. METHODS: We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9-edited zebrafish were used as an in vivo model to assess gene function. RESULTS: lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet's syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation. CONCLUSIONS: Using a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.).

Purpose: We provide a standardized set of terminology, definitions, and thresholds of myopia and its main ocular complications. Methods: Critical review of current terminology and choice of myopia thresholds was done to ensure that the proposed standards are appropriate for clinical research purposes, relevant to the underlying biology of myopia, acceptable to researchers in the field, and useful for developing health policy. Results: We recommend that the many descriptive terms of myopia be consolidated into the following descriptive categories: myopia, secondary myopia, axial myopia, and refractive myopia. To provide a framework for research into myopia prevention, the condition of "pre-myopia" is defined. As a quantitative trait, we recommend that myopia be divided into myopia (i.e., all myopia), low myopia, and high myopia. The current consensus threshold value for myopia is a spherical equivalent refractive error ≤ -0.50 diopters (D), but this carries significant risks of classification bias. The current consensus threshold value for high myopia is a spherical equivalent refractive error ≤ -6.00 D. "Pathologic myopia" is proposed as the categorical term for the adverse, structural complications of myopia. A clinical classification is proposed to encompass the scope of such structural complications. Conclusions: Standardized definitions and consistent choice of thresholds are essential elements of evidence-based medicine. It is hoped that these proposals, or derivations from them, will facilitate rigorous, evidence-based approaches to the study and management of myopia.

Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.

Functional MRI (fMRI) has uncovered widespread hemodynamic fluctuations in the brain during rest. Recent electroencephalographic work in humans and microelectrode recordings in anesthetized monkeys have shown this activity to be correlated with slow changes in neural activity. Here we report that the spontaneous fluctuations in the local field potential (LFP) measured from a single cortical site in monkeys at rest exhibit widespread, positive correlations with fMRI signals over nearly the entire cerebral cortex. This correlation was especially consistent in a band of upper gamma-range frequencies (40-80 Hz), for which the hemodynamic signal lagged the neural signal by 6-8 s. A strong, positive correlation was also observed in a band of lower frequencies (2-15 Hz), albeit with a lag closer to zero. The global pattern of correlation with spontaneous fMRI fluctuations was similar whether the LFP signal was measured in occipital, parietal, or frontal electrodes. This coupling was, however, dependent on the monkey's behavioral state, being stronger and anticipatory when the animals' eyes were closed. These results indicate that the often discarded global component of fMRI fluctuations measured during the resting state is tightly coupled with underlying neural activity.

Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome. Recent reports claim that significantly larger vector genomes can be packaged intact. We examined the packaged vector genomes from plasmid-encoded AAV vectors that ranged from 4.7 to 8.7 kb in length, using AAV types 2, 5, and 8 capsids. Southern blot analysis indicated that packaged AAV vector genomes never exceeded 5.2 kb in length irrespective of the size of the plasmid-encoded vector or the capsid type. This result was confirmed by vector genome probing with strand-specific oligonucleotides. The packaged vector genomes derived from plasmid-encoded vectors exceeding 5 kb were heterogeneous in length and truncated on the 5′ end. Despite their truncated genomes, vector preparations produced from plasmid-encoded vectors exceeding 5.2 kb mediated reporter gene expression in vitro at high multiplicity of infection (MOI). The efficiency of expression was substantially lower than that of reporter vectors with genomes <5 kb in length. We propose that transcriptionally functional, intact vector genomes are generated in cells transduced at high MOI from the fragmentary genomes of these larger vectors, probably by recombination. Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome. Recent reports claim that significantly larger vector genomes can be packaged intact. We examined the packaged vector genomes from plasmid-encoded AAV vectors that ranged from 4.7 to 8.7 kb in length, using AAV types 2, 5, and 8 capsids. Southern blot analysis indicated that packaged AAV vector genomes never exceeded 5.2 kb in length irrespective of the size of the plasmid-encoded vector or the capsid type. This result was confirmed by vector genome probing with strand-specific oligonucleotides. The packaged vector genomes derived from plasmid-encoded vectors exceeding 5 kb were heterogeneous in length and truncated on the 5′ end. Despite their truncated genomes, vector preparations produced from plasmid-encoded vectors exceeding 5.2 kb mediated reporter gene expression in vitro at high multiplicity of infection (MOI). The efficiency of expression was substantially lower than that of reporter vectors with genomes <5 kb in length. We propose that transcriptionally functional, intact vector genomes are generated in cells transduced at high MOI from the fragmentary genomes of these larger vectors, probably by recombination.