Bujakowska KM, Zhang Q, Siemiatkowska AM, Liu Q, Place E, Falk MJ, Consugar M, Lancelot M-E, Antonio A, Lonjou C, Carpentier W, Mohand-Saïd S, den Hollander AI, Cremers FPM, Leroy BP, Gai X, Sahel J-A, van den Born IL, Collin RWJ, Zeitz C, Audo I, Pierce EA. Mutations in IFT172 cause isolated retinal degeneration and Bardet-Biedl syndrome. Hum Mol Genet 2015;24(1):230-42.Abstract
Primary cilia are sensory organelles present on most mammalian cells. The assembly and maintenance of primary cilia are facilitated by intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium. Mutations in genes coding for IFT components have been associated with a group of diseases called ciliopathies. These genetic disorders can affect a variety of organs including the retina. Using whole exome sequencing in three families, we identified mutations in Intraflagellar Transport 172 Homolog [IFT172 (Chlamydomonas)] that underlie an isolated retinal degeneration and Bardet-Biedl syndrome. Extensive functional analyses of the identified mutations in cell culture, rat retina and in zebrafish demonstrated their hypomorphic or null nature. It has recently been reported that mutations in IFT172 cause a severe ciliopathy syndrome involving skeletal, renal, hepatic and retinal abnormalities (Jeune and Mainzer-Saldino syndromes). Here, we report for the first time that mutations in this gene can also lead to an isolated form of retinal degeneration. The functional data for the mutations can partially explain milder phenotypes; however, the involvement of modifying alleles in the IFT172-associated phenotypes cannot be excluded. These findings expand the spectrum of disease associated with mutations in IFT172 and suggest that mutations in genes originally reported to be associated with syndromic ciliopathies should also be considered in subjects with non-syndromic retinal dystrophy.
Wiggs JL, Langgurth AM, Allen KF. Carrier frequency of CYP1B1 mutations in the United States (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc 2014;112:94-102.Abstract
PURPOSE: CYP1B1 mutations cause autosomal recessive congenital glaucoma. Disease risk assessment for families with CYP1B1 mutations requires knowledge of the population mutation carrier frequency. The purpose of this study is to determine the CYP1B1 mutation carrier frequency in clinically normal individuals residing in the United States. Because CYP1B1 mutations can exhibit variable expressivity, we hypothesize that the mutation carrier frequency is higher than expected. METHODS: Two hundred fifty individuals without glaucoma or a family history of glaucoma were enrolled. CYP1B1 mutations were identified by DNA sequencing, and pathogenicity was estimated by PolyPhen-2 or a previous report of disease causality. RESULTS: Based on the disease frequency (1 in 10,000) and prevalence of CYP1B1-related congenital glaucoma (15% to 20%), the frequency of CYP1B1-related congenital glaucoma in the United States is approximately 1 in 50,000. Assuming Hardy-Weinberg equilibrium, the expected CYP1B1 mutation carrier frequency would be 1 in 112, or 0.89%. Among the 250 study participants, 11 (4.4%) are carriers of a single pathogenic mutation, representing a carrier frequency of 1 in 22, which is 5.1 times the expected frequency. A higher-than-expected carrier frequency (1 in 33, 3.0%) was also observed in 4300 white individuals sequenced by the National Heart Lung and Blood Institute Exome Sequencing Project. CONCLUSIONS: Our results show that the CYP1B1 mutation carrier frequency in the US population is between 1 in 22 and 1 in 33, which is 5.1 to 3.4 times the expected frequency. These results suggest that more individuals than expected are carriers of a deleterious CYP1B1 mutation, and that the prevalence of CYP1B1-related disease may be higher than expected.
Yamada T, Yang Y, Hemberg M, Yoshida T, Cho HY, Murphy PJ, Fioravante D, Regehr WG, Gygi SP, Georgopoulos K, Bonni A. Promoter decommissioning by the NuRD chromatin remodeling complex triggers synaptic connectivity in the mammalian brain. Neuron 2014;83(1):122-34.Abstract
Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by in vivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex in vivo. By interfacing genome-wide sequencing of transcripts and ChIP-seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum in vivo. Finally, in a targeted in vivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally regulated programming mechanism that drives synaptic connectivity in the mammalian brain.
Thomas S, Thomas MG, Andrews C, Chan W-M, Proudlock FA, McLean RJ, Pradeep A, Engle EC, Gottlob I. Autosomal-dominant nystagmus, foveal hypoplasia and presenile cataract associated with a novel PAX6 mutation. Eur J Hum Genet 2014;22(3):344-9.Abstract
Autosomal-dominant idiopathic infantile nystagmus has been linked to 6p12 (OMIM 164100), 7p11.2 (OMIM 608345) and 13q31-q33 (OMIM 193003). PAX6 (11p13, OMIM 607108) mutations can also cause autosomal-dominant nystagmus, typically in association with aniridia or iris hypoplasia. We studied a large multigenerational white British family with autosomal-dominant nystagmus, normal irides and presenile cataracts. An SNP-based genome-wide analysis revealed a linkage to a 13.4-MB region on chromosome 11p13 with a maximum lod score of 2.93. A mutation analysis of the entire coding region and splice junctions of the PAX6 gene revealed a novel heterozygous missense mutation (c.227C>G) that segregated with the phenotype and is predicted to result in the amino-acid substitution of proline by arginine at codon 76 p.(P76R). The amino-acid variation p.(P76R) within the paired box domain is likely to destabilise the protein due to steric hindrance as a result of the introduction of a polar and larger amino acid. Eye movement recordings showed a significant intrafamilial variability of horizontal, vertical and torsional nystagmus. High-resolution in vivo imaging of the retina using optical coherence tomography (OCT) revealed features of foveal hypoplasia, including rudimentary foveal pit, incursion of inner retinal layers, short photoreceptor outer segments and optic nerve hypoplasia. Thus, this study presents a family that segregates a PAX6 mutation with nystagmus and foveal hypoplasia in the absence of iris abnormalities. Moreover, it is the first study showing detailed characteristics using eye movement recordings of autosomal-dominant nystagmus in a multigenerational family with a novel PAX6 mutation.
Cheng L, Desai J, Miranda CJ, Duncan JS, Qiu W, Nugent AA, Kolpak AL, Wu CC, Drokhlyansky E, DeLisle MM, Chan W-M, Wei Y, Propst F, Reck-Peterson SL, Fritzsch B, Engle EC. Human CFEOM1 mutations attenuate KIF21A autoinhibition and cause oculomotor axon stalling. Neuron 2014;82(2):334-49.Abstract
The ocular motility disorder "Congenital fibrosis of the extraocular muscles type 1" (CFEOM1) results from heterozygous mutations altering the motor and third coiled-coil stalk of the anterograde kinesin, KIF21A. We demonstrate that Kif21a knockin mice harboring the most common human mutation develop CFEOM. The developing axons of the oculomotor nerve's superior division stall in the proximal nerve; the growth cones enlarge, extend excessive filopodia, and assume random trajectories. Inferior division axons reach the orbit but branch ectopically. We establish a gain-of-function mechanism and find that human motor or stalk mutations attenuate Kif21a autoinhibition, providing in vivo evidence for mammalian kinesin autoregulation. We identify Map1b as a Kif21a-interacting protein and report that Map1b⁻/⁻ mice develop CFEOM. The interaction between Kif21a and Map1b is likely to play a critical role in the pathogenesis of CFEOM1 and highlights a selective vulnerability of the developing oculomotor nerve to perturbations of the axon cytoskeleton.
Benaglio P, San Jose PF, Avila-Fernandez A, Ascari G, Harper S, Manes G, Ayuso C, Hamel C, Berson EL, Rivolta C. Mutational screening of splicing factor genes in cases with autosomal dominant retinitis pigmentosa. Mol Vis 2014;20:843-51.Abstract

PURPOSE: Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. METHODS: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. RESULTS: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5' untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. CONCLUSIONS: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin.

Bujakowska KM, Consugar M, Place E, Harper S, Lena J, Taub DG, White J, Navarro-Gomez D, Weigel DiFranco C, Farkas MH, Gai X, Berson EL, Pierce EA. Targeted exon sequencing in Usher syndrome type I. Invest Ophthalmol Vis Sci 2014;55(12):8488-96.Abstract

PURPOSE: Patients with Usher syndrome type I (USH1) have retinitis pigmentosa, profound congenital hearing loss, and vestibular ataxia. This syndrome is currently thought to be associated with at least six genes, which are encoded by over 180 exons. Here, we present the use of state-of-the-art techniques in the molecular diagnosis of a cohort of 47 USH1 probands. METHODS: The cohort was studied with selective exon capture and next-generation sequencing of currently known inherited retinal degeneration genes, comparative genomic hybridization, and Sanger sequencing of new USH1 exons identified by human retinal transcriptome analysis. RESULTS: With this approach, we were able to genetically solve 14 of the 47 probands by confirming the biallelic inheritance of mutations. We detected two likely pathogenic variants in an additional 19 patients, for whom family members were not available for cosegregation analysis to confirm biallelic inheritance. Ten patients, in addition to primary disease-causing mutations, carried rare likely pathogenic USH1 alleles or variants in other genes associated with deaf-blindness, which may influence disease phenotype. Twenty-one of the identified mutations were novel among the 33 definite or likely solved patients. Here, we also present a clinical description of the studied cohort at their initial visits. CONCLUSIONS: We found a remarkable genetic heterogeneity in the studied USH1 cohort with multiplicity of mutations, of which many were novel. No obvious influence of genotype on phenotype was found, possibly due to small sample sizes of the genotypes under study.

Qi Q, Kilpeläinen TO, Downer MK, Tanaka T, Smith CE, Sluijs I, Sonestedt E, Chu AY, Renström F, Lin X, Angquist LH, Huang J, Liu Z, Li Y, Asif Ali M, Xu M, Ahluwalia TS, Boer JMA, Chen P, Daimon M, Eriksson J, Perola M, Friedlander Y, Gao Y-T, Heppe DHM, Holloway JW, Houston DK, Kanoni S, Kim Y-M, Laaksonen MA, Jääskeläinen T, Lee NR, Lehtimäki T, Lemaitre RN, Lu W, Luben RN, Manichaikul A, Männistö S, Marques-Vidal P, Monda KL, Ngwa JS, Perusse L, van Rooij FJA, Xiang Y-B, Wen W, Wojczynski MK, Zhu J, Borecki IB, Bouchard C, Cai Q, Cooper C, Dedoussis GV, Deloukas P, Ferrucci L, Forouhi NG, Hansen T, Christiansen L, Hofman A, Johansson I, Jørgensen T, Karasawa S, Khaw K-T, Kim M-K, Kristiansson K, Li H, Lin X, Liu Y, Lohman KK, Long J, Mikkilä V, Mozaffarian D, North K, Pedersen O, Raitakari O, Rissanen H, Tuomilehto J, van der Schouw YT, Uitterlinden AG, Zillikens CM, Franco OH, Shyong Tai E, Ou Shu X, Siscovick DS, Toft U, Verschuren MWM, Vollenweider P, Wareham NJ, Witteman JCM, Zheng W, Ridker PM, Kang JH, Liang L, Jensen MK, Curhan GC, Pasquale LR, Hunter DJ, Mohlke KL, Uusitupa M, Cupples AL, Rankinen T, Orho-Melander M, Wang T, Chasman DI, Franks PW, Sørensen TIA, Hu FB, Loos RJF, Nettleton JA, Qi L. FTO genetic variants, dietary intake and body mass index: insights from 177 330 individuals. Hum Mol Genet 2014;23(25):6961-72.Abstract

FTO is the strongest known genetic susceptibility locus for obesity. Experimental studies in animals suggest the potential roles of FTO in regulating food intake. The interactive relation among FTO variants, dietary intake and body mass index (BMI) is complex and results from previous often small-scale studies in humans are highly inconsistent. We performed large-scale analyses based on data from 177 330 adults (154 439 Whites, 5776 African Americans and 17 115 Asians) from 40 studies to examine: (i) the association between the FTO-rs9939609 variant (or a proxy single-nucleotide polymorphism) and total energy and macronutrient intake and (ii) the interaction between the FTO variant and dietary intake on BMI. The minor allele (A-allele) of the FTO-rs9939609 variant was associated with higher BMI in Whites (effect per allele = 0.34 [0.31, 0.37] kg/m(2), P = 1.9 × 10(-105)), and all participants (0.30 [0.30, 0.35] kg/m(2), P = 3.6 × 10(-107)). The BMI-increasing allele of the FTO variant showed a significant association with higher dietary protein intake (effect per allele = 0.08 [0.06, 0.10] %, P = 2.4 × 10(-16)), and relative weak associations with lower total energy intake (-6.4 [-10.1, -2.6] kcal/day, P = 0.001) and lower dietary carbohydrate intake (-0.07 [-0.11, -0.02] %, P = 0.004). The associations with protein (P = 7.5 × 10(-9)) and total energy (P = 0.002) were attenuated but remained significant after adjustment for BMI. We did not find significant interactions between the FTO variant and dietary intake of total energy, protein, carbohydrate or fat on BMI. Our findings suggest a positive association between the BMI-increasing allele of FTO variant and higher dietary protein intake and offer insight into potential link between FTO, dietary protein intake and adiposity.

Salcone EM, Hamdy S, Melki S, Hunter DG. Scleral perforations during routine traction test in a patient with osteogenesis imperfecta. J AAPOS 2014;18(6):610-2.Abstract

Osteogenesis imperfecta comprises a rare group of genetic disorders caused by abnormal collagen that results in increased bone fragility and other sequelae. We describe a 37-year-old woman with osteogenesis imperfecta in whom two full-thickness scleral perforations were created by adjacent teeth of 0.5 mm forceps during traction testing while undergoing routine strabismus surgery. This case reviews the ocular findings of osteogenesis imperfecta and highlights the potential risk of ocular surgical complications in these patients.

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.