Prabakaran S, Hemberg M, Chauhan R, Winter D, Tweedie-Cullen RY, Dittrich C, Hong E, Gunawardena J, Steen H, Kreiman G, Steen JA. Quantitative profiling of peptides from RNAs classified as noncoding. Nat Commun 2014;5:5429.Abstract

Only a small fraction of the mammalian genome codes for messenger RNAs destined to be translated into proteins, and it is generally assumed that a large portion of transcribed sequences--including introns and several classes of noncoding RNAs (ncRNAs)--do not give rise to peptide products. A systematic examination of translation and physiological regulation of ncRNAs has not been conducted. Here we use computational methods to identify the products of non-canonical translation in mouse neurons by analysing unannotated transcripts in combination with proteomic data. This study supports the existence of non-canonical translation products from both intragenic and extragenic genomic regions, including peptides derived from antisense transcripts and introns. Moreover, the studied novel translation products exhibit temporal regulation similar to that of proteins known to be involved in neuronal activity processes. These observations highlight a potentially large and complex set of biologically regulated translational events from transcripts formerly thought to lack coding potential.

Cronin T, Vandenberghe LH, Hantz P, Juttner J, Reimann A, Kacsó A-E, Huckfeldt RM, Busskamp V, Kohler H, Lagali PS, Roska B, Bennett J. Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter. EMBO Mol Med 2014;6(9):1175-90.Abstract

In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (~100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.

Cepko C. Intrinsically different retinal progenitor cells produce specific types of progeny. Nat Rev Neurosci 2014;15(9):615-27.Abstract
Lineage studies conducted in the retina more than 25 years ago demonstrated the multipotency of retinal progenitor cells (RPCs). The number and types of cells produced by individual RPCs, even from a single time point in development, were found to be highly variable. This raised the question of whether this variability was due to intrinsic differences among RPCs or to extrinsic and/or stochastic effects on equivalent RPCs or their progeny. Newer lineage studies that have made use of molecular markers of RPCs, retrovirus-mediated lineage analyses of specific RPCs and live imaging have begun to provide answers to this question. RPCs that produce two postmitotic daughter cells - that is, terminally dividing RPCs - have been the most well characterized RPCs to date, and have been shown to produce specific types of daughter cells. In addition, recent studies have begun to shed light on the mechanisms that drive the temporal order in which retinal cells are born.
MacKinnon S, Oystreck DT, Andrews C, Chan W-M, Hunter DG, Engle EC. Diagnostic distinctions and genetic analysis of patients diagnosed with moebius syndrome. Ophthalmology 2014;121(7):1461-8.Abstract
OBJECTIVE: To improve diagnostic assessment in Moebius syndrome by (1) creating more selective diagnostic subgroups and (2) conducting genetic evaluation in a large patient cohort. DESIGN: Prospective, observational study. PARTICIPANTS: Attendees of 3 consecutive Moebius syndrome conferences held in the United States, with a prior diagnosis of Moebius syndrome, were invited to participate. METHODS: Participants underwent standardized ophthalmologic examination for Moebius syndrome minimum diagnostic criteria (MDC) (congenital, nonprogressive facial palsy, and abduction deficit) and genetic testing for HOXA1, HOXB1, and TUBB3 mutations. MAIN OUTCOME MEASURES: The number of patients meeting MDC and the number of patients with confirmed genetic mutation. RESULTS: A total of 112 participants from 107 families enrolled. Nineteen percent of participants (21/112) did not meet accepted MDC for Moebius syndrome because they had abduction deficits without facial palsy or facial palsy with full ocular motility. All 5 families with 2 affected individuals had at least 1 family member in this category, including 2 siblings with comitant strabismus who harbored a HOXB1 mutation. Four unrelated participants, also not meeting MDC, had large-angle exotropia, vertical gaze deficiency, and ptosis consistent with congenital fibrosis of the extraocular muscles type 3 (CFEOM3); 1 patient harbored a novel TUBB3 mutation, and 3 patients harbored previously reported de novo TUBB3 mutations. Three percent of participants (3/112) met MDC but also had restricted vertical gaze. The remaining 88 participants (79%) met MDC and had full vertical gaze. This group had relatively homogeneous findings, and none had a family history of Moebius syndrome. Two previously undescribed phenomena were observed in this category: (1) volitional Bell's phenomenon and (2) intorsion with fixation. CONCLUSIONS: Although the genetic contributors to classic Moebius syndrome remain elusive, accuracy in clinical evaluation will properly subdivide patients to facilitate genetic testing as new candidate genes are identified. Failure to test ocular motility may lead to misdiagnosis of Moebius syndrome, especially in patients who have facial palsy with full ductions. Patients with exotropia, vertical gaze limitation, and ptosis do not have classic Moebius syndrome and may have TUBB3 mutations associated with CFEOM3. To optimize genetic analysis, we propose adding "full vertical motility" to the MDC for Moebius syndrome.
Zinn E, Vandenberghe LH. Adeno-associated virus: fit to serve. Curr Opin Virol 2014;8C:90-97.Abstract

Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue tropism, and limited viral host response has made it an attractive vector system for gene therapy. The viral protein capsid, the primary interface with the host, is the main determinant for these phenotypes, is highly variable, and is most subject to pressures during replication. Here, we explore the evolutionary path of AAV and other parvoviruses in respect to these phenotypes, as well as directed evolution and engineering strategies that have exploited the lessons learned from natural selection in order to address remaining limitations of AAV as a therapeutic gene transfer platform.

Malik AN, Vierbuchen T, Hemberg M, Rubin AA, Ling E, Couch CH, Stroud H, Spiegel I, Farh KK-H, Harmin DA, Greenberg ME. Genome-wide identification and characterization of functional neuronal activity-dependent enhancers. Nat Neurosci 2014;17(10):1330-9.Abstract

Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in mouse cortical neurons. We find that the subset of enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1) and binding of the transcriptional coactivator CREBBP (also called CBP) that shows increased acetylation of histone H3 Lys27 (H3K27ac) after membrane depolarization of cortical neurons functions to regulate activity-dependent transcription. A subset of these enhancers appears to require binding of FOS, which was previously thought to bind primarily to promoters. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function and provide a resource of functional cis-regulatory elements that may give insight into the genetic variants that contribute to brain development and disease.

Ehrenberg M, Pierce EA, Cox GF, Fulton AB. CRB1: one gene, many phenotypes. Semin Ophthalmol 2013;28(5-6):397-405.Abstract
Mutations in the CRB1 gene cause severe retinal degenerations, which may present as Leber congenital amaurosis, early onset retinal dystrophy, retinitis pigmentosa, or cone-rod dystrophy. Some clinical features should alert the ophthalmologist to the possibility of CRB1 disease. These features are nummular pigmentation of the retina, atrophic macula, retinal degeneration associated with Coats disease, and a unique form of retinitis pigmentosa named para-arteriolar preservation of the retinal pigment epithelium (PPRPE). Retinal degenerations associated with nanophthalmos and hyperopia, or with keratoconus, can serve as further clinical cues to mutations in CRB1. Despite this, no clear genotype-phenotype relationship has been established in CRB1 disease. In CRB1-disease, as in other inherited retinal degenerations (IRDs), it is essential to diagnose the specific disease-causing gene for the disease as genetic therapy has progressed considerably in the last few years and might be applicable.
Singh G, Robinson CM, Dehghan S, Jones MS, Dyer DW, Seto D, Chodosh J. Homologous recombination in E3 genes of human adenovirus species D. J Virol 2013;87(22):12481-8.Abstract
Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1β, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber.
Shaaban S, Ramos-Platt L, Gilles FH, Chan W-M, Andrews C, De Girolami U, Demer J, Engle EC. RYR1 mutations as a cause of ophthalmoplegia, facial weakness, and malignant hyperthermia. JAMA Ophthalmol 2013;131(12):1532-40.Abstract
IMPORTANCE: Total ophthalmoplegia can result from ryanodine receptor 1 (RYR1) mutations without overt associated skeletal myopathy. Patients carrying RYR1 mutations are at high risk of developing malignant hyperthermia. Ophthalmologists should be familiar with these important clinical associations. OBJECTIVE: To determine the genetic cause of congenital ptosis, ophthalmoplegia, facial paralysis, and mild hypotonia segregating in 2 pedigrees diagnosed with atypical Moebius syndrome or congenital fibrosis of the extraocular muscles. DESIGN, SETTING, AND PARTICIPANTS: Clinical data including medical and family histories were collected at research laboratories at Boston Children's Hospital and Jules Stein Eye Institute (Engle and Demer labs) for affected and unaffected family members from 2 pedigrees in which patients presented with total ophthalmoplegia, facial weakness, and myopathy. INTERVENTION: Homozygosity mapping and whole-exome sequencing were conducted to identify causative mutations in affected family members. Histories, physical examinations, and clinical data were reviewed. MAIN OUTCOME AND MEASURE: Mutations in RYR1. RESULTS: Missense mutations resulting in 2 homozygous RYR1 amino acid substitutions (E989G and R3772W) and 2 compound heterozygous RYR1 substitutions (H283R and R3772W) were identified in a consanguineous and a nonconsanguineous pedigree, respectively. Orbital magnetic resonance imaging revealed marked hypoplasia of extraocular muscles and intraorbital cranial nerves. Skeletal muscle biopsy specimens revealed nonspecific myopathic changes. Clinically, the patients' ophthalmoplegia and facial weakness were far more significant than their hypotonia and limb weakness and were accompanied by an unrecognized susceptibility to malignant hyperthermia. CONCLUSIONS AND RELEVANCE: Affected children presenting with severe congenital ophthalmoplegia and facial weakness in the setting of only mild skeletal myopathy harbored recessive mutations in RYR1, encoding the ryanodine receptor 1, and were susceptible to malignant hyperthermia. While ophthalmoplegia occurs rarely in RYR1-related myopathies, these children were atypical because they lacked significant weakness, respiratory insufficiency, or scoliosis. RYR1-associated myopathies should be included in the differential diagnosis of congenital ophthalmoplegia and facial weakness, even without clinical skeletal myopathy. These patients should also be considered susceptible to malignant hyperthermia, a life-threatening anesthetic complication avoidable if anticipated presurgically.
Melo MB, Nguyen QP, Cordeiro C, Hassan MA, Yang N, McKell R, Rosowski EE, Julien L, Butty V, Dardé M-L, Ajzenberg D, Fitzgerald K, Young LH, Saeij JPJ. Transcriptional analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways. PLoS Pathog 2013;9(12):e1003779.Abstract
Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts.
Rose AM, Shah AZ, Alfano G, Bujakowska KM, Barker AF, Robertson LJ, Rahman S, Sánchez LV, Diaz-Corrales FJ, Chakarova CF, Krishna A, Bhattacharya SS. A Study into the Evolutionary Divergence of the Core Promoter Elements of PRPF31 and TFPT. J Mol Genet Med 2013;7(2)Abstract
Mutations in have been implicated in retinitis pigmentosa, a blinding disease caused by degeneration of rod photoreceptors. The disease mechanism in the majority of cases is haploinsufficiency. Crucially, attempts at generation of animal models of disease have proved unsuccessful, yielding animals with a visual phenotype that does not mirror human disease. This suggests that, in these animals, the transcriptional regulation of is different to humans and compared to other species. Study of the evolution of the core promoter has important implications for our understanding of human disease, as disease phenotype is modified by differentially expressed alleles in the population. lies in a head-to-head arrangement with , a gene involved in cellular apoptosis. The two genes were shown to share common regulatory elements in the human genome. In this study, the core promoters of and were characterised by dual-luciferase reporter assay using genomic DNA from the green monkey, domestic dog and house mouse. It was found that the core promoters were conserved between human and monkey. In dog, the core promoter was conserved, but different gene architecture meant the gene was controlled by a long-range promoter lying some 2000bp from the transcription start site. There was very low level of conservation (<20%) of the 5' region between mouse and human. It was shown that mouse populations did not show variable expression levels, revealing a potential explanation for the lack of phenotype observed in the knock-out mouse model.
Robinson CM, Singh G, Lee JY, Dehghan S, Rajaiya J, Liu EB, Yousuf MA, Betensky RA, Jones MS, Dyer DW, Seto D, Chodosh J. Molecular evolution of human adenoviruses. Sci Rep 2013;3:1812.Abstract
The recent emergence of highly virulent human adenoviruses (HAdVs) with new tissue tropisms underscores the need to determine their ontogeny. Here we report complete high quality genome sequences and analyses for all the previously unsequenced HAdV serotypes (n = 20) within HAdV species D. Analysis of nucleotide sequence variability for these in conjunction with another 40 HAdV prototypes, comprising all seven HAdV species, confirmed the uniquely hypervariable regions within species. The mutation rate among HAdV-Ds was low when compared to other HAdV species. Homologous recombination was identified in at least two of five examined hypervariable regions for every virus, suggesting the evolution of HAdV-Ds has been highly dependent on homologous recombination. Patterns of alternating GC and AT rich motifs correlated well with hypervariable region recombination sites across the HAdV-D genomes, suggesting foci of DNA instability lead to formulaic patterns of homologous recombination and confer agility to adenovirus evolution.
Lebreton F, Valentino MD, Duncan LB, Zeng Q, McGuire AM, Earl AM, Gilmore MS. High-Quality Draft Genome Sequence of Vagococcus lutrae Strain LBD1, Isolated from the Largemouth Bass Micropterus salmoides. Genome Announc 2013;1(6)Abstract
Vagococci are usually isolated from marine hosts and occasionally from endodontic infections. Using 16S rRNA gene comparison, the closest relatives are members of the genera Enterococcus and Carnobacterium. A draft sequence of Vagococcus lutrae was generated to clarify the relationship of Vagococcus to these and other related low-G+C Gram-positive bacteria.