Genomics

Saavedra JT, Schwartzman JA, Gilmore MS. Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR. Curr Protoc Mol Biol 2017;118:15.15.1-15.15.15.Abstract

Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to (1) amplify DNA spanning the transposon insertion junction, and (2) increase the specific yield of transposon insertion junction fragments for sequence analysis. The resulting sequence is mapped to a bacterial genome to identify the site of transposon insertion. In this protocol, AP-PCR as it is routinely used to map sites of transposon insertion within Staphylococcus aureus, is used to illustrate the principle. Guidelines are provided for adapting this protocol for mapping insertions in other bacterial genomes. Mapping transposon insertions using this method is typically achieved in 2 to 3 days if starting from a culture of the transposon insertion mutant. © 2017 by John Wiley & Sons, Inc.

Pacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer A, Mevel M, Turunen H, Barungi T, Penaud-Budloo M, Broucque F, Blouin V, Moullier P, Ayuso E, Vandenberghe LH. AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations. Mol Ther 2017;25(6):1375-1386.Abstract
Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.
Lindström S, Loomis S, Turman C, Huang H, Huang J, Aschard H, Chan AT, Choi H, Cornelis M, Curhan G, De Vivo I, Eliassen HA, Fuchs C, Gaziano M, Hankinson SE, Hu F, Jensen M, Kang JH, Kabrhel C, Liang L, Pasquale LR, Rimm E, Stampfer MJ, Tamimi RM, Tworoger SS, Wiggs JL, Hunter DJ, Kraft P. A comprehensive survey of genetic variation in 20,691 subjects from four large cohorts. PLoS One 2017;12(3):e0173997.Abstract

The Nurses' Health Study (NHS), Nurses' Health Study II (NHSII), Health Professionals Follow Up Study (HPFS) and the Physicians Health Study (PHS) have collected detailed longitudinal data on multiple exposures and traits for approximately 310,000 study participants over the last 35 years. Over 160,000 study participants across the cohorts have donated a DNA sample and to date, 20,691 subjects have been genotyped as part of genome-wide association studies (GWAS) of twelve primary outcomes. However, these studies utilized six different GWAS arrays making it difficult to conduct analyses of secondary phenotypes or share controls across studies. To allow for secondary analyses of these data, we have created three new datasets merged by platform family and performed imputation using a common reference panel, the 1,000 Genomes Phase I release. Here, we describe the methodology behind the data merging and imputation and present imputation quality statistics and association results from two GWAS of secondary phenotypes (body mass index (BMI) and venous thromboembolism (VTE)). We observed the strongest BMI association for the FTO SNP rs55872725 (β = 0.45, p = 3.48x10-22), and using a significance level of p = 0.05, we replicated 19 out of 32 known BMI SNPs. For VTE, we observed the strongest association for the rs2040445 SNP (OR = 2.17, 95% CI: 1.79-2.63, p = 2.70x10-15), located downstream of F5 and also observed significant associations for the known ABO and F11 regions. This pooled resource can be used to maximize power in GWAS of phenotypes collected across the cohorts and for studying gene-environment interactions as well as rare phenotypes and genotypes.

Bujakowska KM, Fernandez-Godino R, Place E, Consugar M, Navarro-Gomez D, White J, Bedoukian EC, Zhu X, Xie HM, Gai X, Leroy BP, Pierce EA. Copy-number variation is an important contributor to the genetic causality of inherited retinal degenerations. Genet Med 2017;19(6):643-651.Abstract
PURPOSE: Despite substantial progress in sequencing, current strategies can genetically solve only approximately 55-60% of inherited retinal degeneration (IRD) cases. This can be partially attributed to elusive mutations in the known IRD genes, which are not easily identified by the targeted next-generation sequencing (NGS) or Sanger sequencing approaches. We hypothesized that copy-number variations (CNVs) are a major contributor to the elusive genetic causality of IRDs. METHODS: Twenty-eight cases previously unsolved with a targeted NGS were investigated with whole-genome single-nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) arrays. RESULTS: Deletions in the IRD genes were detected in 5 of 28 families, including a de novo deletion. We suggest that the de novo deletion occurred through nonallelic homologous recombination (NAHR) and we constructed a genomic map of NAHR-prone regions with overlapping IRD genes. In this article, we also report an unusual case of recessive retinitis pigmentosa due to compound heterozygous mutations in SNRNP200, a gene that is typically associated with the dominant form of this disease. CONCLUSIONS: CNV mapping substantially increased the genetic diagnostic rate of IRDs, detecting genetic causality in 18% of previously unsolved cases. Extending the search to other structural variations will probably demonstrate an even higher contribution to genetic causality of IRDs.Genet Med advance online publication 13 October 2016.
Zeng Z, Zhang J, Jing S, Cheng Z, Bofill-Mas S, Maluquer de Motes C, Hundesa A, Girones R, Seto D, Zhang Q. Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom. Genome Announc 2016;4(6)Abstract

The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.

Lamont RE, Tan W-H, Innes MA, Parboosingh JS, Schneidman-Duhovny D, Rajkovic A, Pappas J, Altschwager P, DeWard S, Fulton A, Gray KJ, Krall M, Mehta L, Rodan LH, Saller DN, Steele D, Stein D, Yatsenko SA, Bernier FP, Slavotinek AM. Expansion of phenotype and genotypic data in CRB2-related syndrome. Eur J Hum Genet 2016;24(10):1436-44.Abstract

Sequence variants in CRB2 cause a syndrome with greatly elevated maternal serum alpha-fetoprotein and amniotic fluid alpha-fetoprotein levels, cerebral ventriculomegaly and renal findings similar to Finnish congenital nephrosis. All reported patients have been homozygotes or compound heterozygotes for sequence variants in the Crumbs, Drosophila, Homolog of, 2 (CRB2) genes. Variants affecting CRB2 function have also been identified in four families with steroid resistant nephrotic syndrome, but without any other known systemic findings. We ascertained five, previously unreported individuals with biallelic variants in CRB2 that were predicted to affect function. We compiled the clinical features of reported cases and reviewed available literature for cases with features suggestive of CRB2-related syndrome in order to better understand the phenotypic and genotypic manifestations. Phenotypic analyses showed that ventriculomegaly was a common clinical manifestation (9/11 confirmed cases), in contrast to the original reports, in which patients were ascertained due to renal disease. Two children had minor eye findings and one was diagnosed with a B-cell lymphoma. Further genetic analysis identified one family with two affected siblings who were both heterozygous for a variant in NPHS2 predicted to affect function and separate families with sequence variants in NPHS4 and BBS7 in addition to the CRB2 variants. Our report expands the clinical phenotype of CRB2-related syndrome and establishes ventriculomegaly and hydrocephalus as frequent manifestations. We found additional sequence variants in genes involved in kidney development and ciliopathies in patients with CRB2-related syndrome, suggesting that these variants may modify the phenotype.

Arno G, Hull S, Carss K, Dev-Borman A, Chakarova C, Bujakowska K, van den Born I, Robson AG, Holder GE, Michaelides M, Cremers FPM, Pierce E, Raymond LF, Moore AT, Webster AR. Reevaluation of the Retinal Dystrophy Due to Recessive Alleles of RGR With the Discovery of a Cis-Acting Mutation in CDHR1. Invest Ophthalmol Vis Sci 2016;57(11):4806-13.Abstract

PURPOSE: Mutation of RGR, encoding retinal G-protein coupled receptor was originally reported in association with retinal dystrophy in 1999. A single convincing recessive variant segregated perfectly in one family of five affected and two unaffected siblings. At least one further individual, homozygous for the same variant has since been reported. The aim of this report was to reevaluate the findings in consideration of data from a whole genome sequencing (WGS) study of a large cohort of retinal dystrophy families. METHODS: Whole genome sequencing was performed on 599 unrelated probands with inherited retinal disease. Detailed phenotyping was performed, including clinical evaluation, electroretinography, fundus photography, fundus autofluorescence imaging (FAF) and spectral-domain optical coherence tomography (OCT). RESULTS: Overall we confirmed that affected individuals from six unrelated families were homozygous for both the reported RGR p.Ser66Arg variant and a nearby frameshifting deletion in CDHR1 (p.Ile841Serfs119*). All had generalized rod and cone dysfunction with severe macular involvement. An additional proband was heterozygous for the same CDHR1/RGR haplotype but also carried a second null CDHR1 mutation on a different haplotype. A comparison of the clinical presentation of the probands reported here with other CDHR1-related retinopathy patients shows the phenotypes to be similar in presentation, severity, and rod/cone involvement. CONCLUSIONS: These data suggest that the recessive retinal disorder previously reported to be due to homozygous mutation in RGR is, at least in part, due to variants in CDHR1 and that the true consequences of RGR knock-out on human retinal structure and function are yet to be determined.

Gao X, Nannini DR, Corrao K, Torres M, Chen Y-DI, Fan BJ, Wiggs JL, Wiggs JL, Taylor KD, James Gauderman W, Rotter JI, Varma R. Genome-Wide Association Study Identifies WNT7B as a Novel Locus for Central Corneal Thickness in Latinos. Hum Mol Genet 2016;Abstract

The cornea is the outermost layer of the eye and is a vital component of focusing incoming light on the retina. Central corneal thickness (CCT) is now recognized to have a significant role in ocular health and is a risk factor for various ocular diseases, such as keratoconus and primary open angle glaucoma. Most previous genetic studies utilized European and Asian subjects to identify genetic loci associated with CCT. Minority populations, such as Latinos, may aid in identifying additional loci and improve our understanding of the genetic architecture of CCT. In this study, we conducted a genome-wide association study (GWAS) in Latinos, a traditionally understudied population in genetic research, to further identify loci contributing to CCT. Study participants were genotyped using either the Illumina OmniExpress BeadChip (∼730K markers) or the Illumina Hispanic/SOL BeadChip (∼2.5 million markers). All study participants were 40 years of age and older. We assessed the association between individual single nucleotide polymorphisms (SNPs) and CCT using linear regression, adjusting for age, gender, and principal components of genetic ancestry. To expand genomic coverage and to interrogate additional SNPs, we imputed SNPs from the 1000 Genomes Project reference panels. We identified a novel SNP, rs10453441 (P = 6.01E-09), in an intron of WNT7B that is associated with CCT. Furthermore, WNT7B is expressed in the human cornea. We also replicated 11 previously reported loci, including IBTK, RXRA-COL5A1, COL5A1, FOXO1, LRRK1, and ZNF469 (P < 1.25E-3). These findings provide further insight into the genetic architecture of CCT and illustrate that the use of minority groups in GWAS will help identify additional loci.

Igo RP, Cooke Bailey JN, Romm J, Haines JL, Wiggs JL. Quality Control for the Illumina HumanExome BeadChip. Curr Protoc Hum Genet 2016;90:2.14.1-2.14.16.Abstract

The Illumina HumanExome BeadChip and other exome-based genotyping arrays offer inexpensive genotyping of some 240,000 mostly nonsynonymous coding variants across the human genome. The HumanExome chip, with its highly non-uniform distribution of markers and emphasis on rare coding variants, presents some unique challenges for quality control (QC) and data cleaning. Here, we describe QC procedures for HumanExome data, with examples of challenges specific to exome arrays from our experience cleaning a data set of ∼7,500 samples from the NEIGHBORHOOD Consortium. We focus on standard procedures for QC of genome-wide array data including genotype calling, sex verification, sample identity verification, relationship checking, and population structure that are complicated by the HumanExome panel's enrichment in rare, exonic variation. © 2016 by John Wiley & Sons, Inc.

Greenwald SH, Charette JR, Staniszewska M, Shi LY, Brown SDM, Stone L, Liu Q, Hicks WL, Collin GB, Bowl MR, Krebs MP, Nishina PM, Pierce EA. Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease. Am J Pathol 2016;186(7):1925-38.Abstract

The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism.

Joshi AD, Andersson C, Buch S, Stender S, Noordam R, Weng L-C, Weeke PE, Auer PL, Boehm B, Chen C, Choi H, Curhan G, Denny JC, De Vivo I, Eicher JD, Ellinghaus D, Folsom AR, Fuchs C, Gala M, Haessler J, Hofman A, Hu F, Hunter DJ, Janssen HLA, Kang JH, Kooperberg C, Kraft P, Kratzer W, Lieb W, Lutsey PL, Darwish Murad S, Nordestgaard BG, Pasquale LR, Reiner AP, Ridker PM, Rimm E, Rose LM, Shaffer CM, Schafmayer C, Tamimi RM, Uitterlinden AG, Völker U, Völzke H, Wakabayashi Y, Wiggs JL, Zhu J, Roden DM, Stricker BH, Tang W, Teumer A, Hampe J, Tybjærg-Hansen A, Chasman DI, Chan AT, Johnson AD. Four Susceptibility Loci for Gallstone Disease Identified in a Meta-analysis of Genome-Wide Association Studies. Gastroenterology 2016;151(2):351-363.e28.Abstract

BACKGROUND & AIMS: A genome-wide association study (GWAS) of 280 cases identified the hepatic cholesterol transporter ABCG8 as a locus associated with risk for gallstone disease, but findings have not been reported from any other GWAS of this phenotype. We performed a large-scale, meta-analysis of GWASs of individuals of European ancestry with available prior genotype data, to identify additional genetic risk factors for gallstone disease. METHODS: We obtained per-allele odds ratio (OR) and standard error estimates using age- and sex-adjusted logistic regression models within each of the 10 discovery studies (8720 cases and 55,152 controls). We performed an inverse variance weighted, fixed-effects meta-analysis of study-specific estimates to identify single-nucleotide polymorphisms that were associated independently with gallstone disease. Associations were replicated in 6489 cases and 62,797 controls. RESULTS: We observed independent associations for 2 single-nucleotide polymorphisms at the ABCG8 locus: rs11887534 (OR, 1.69; 95% confidence interval [CI], 1.54-1.86; P = 2.44 × 10(-60)) and rs4245791 (OR, 1.27; P = 1.90 × 10(-34)). We also identified and/or replicated associations for rs9843304 in TM4SF4 (OR, 1.12; 95% CI, 1.08-1.16; P = 6.09 × 10(-11)), rs2547231 in SULT2A1 (encodes a sulfoconjugation enzyme that acts on hydroxysteroids and cholesterol-derived sterol bile acids) (OR, 1.17; 95% CI, 1.12-1.21; P = 2.24 × 10(-10)), rs1260326 in glucokinase regulatory protein (OR, 1.12; 95% CI, 1.07-1.17; P = 2.55 × 10(-10)), and rs6471717 near CYP7A1 (encodes an enzyme that catalyzes conversion of cholesterol to primary bile acids) (OR, 1.11; 95% CI, 1.08-1.15; P = 8.84 × 10(-9)). Among individuals of African American and Hispanic American ancestry, rs11887534 and rs4245791 were associated positively with gallstone disease risk, whereas the association for the rs1260326 variant was inverse. CONCLUSIONS: In this large-scale GWAS of gallstone disease, we identified 4 loci in genes that have putative functions in cholesterol metabolism and transport, and sulfonylation of bile acids or hydroxysteroids.

Bennett J, Wellman J, Marshall KA, McCague S, Ashtari M, DiStefano-Pappas J, Elci OU, Chung DC, Sun J, Wright FJ, Cross DR, Aravand P, Cyckowski LL, Bennicelli JL, Mingozzi F, Auricchio A, Pierce EA, Ruggiero J, Leroy BP, Simonelli F, High KA, Maguire AM. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial. Lancet 2016;Abstract

BACKGROUND: Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study. METHODS: In this follow-on phase 1 trial, one dose of AAV2-hRPE65v2 (1·5 × 10(11) vector genomes) in a total volume of 300 μL was subretinally injected into the contralateral, previously uninjected, eyes of 11 children and adults (aged 11-46 years at second administration) with inherited retinal dystrophy caused by RPE65 mutations, 1·71-4·58 years after the initial subretinal injection. We assessed safety, immune response, retinal and visual function, functional vision, and activation of the visual cortex from baseline until 3 year follow-up, with observations ongoing. This study is registered with ClinicalTrials.gov, number NCT01208389. FINDINGS: No adverse events related to the AAV were reported, and those related to the procedure were mostly mild (dellen formation in three patients and cataracts in two). One patient developed bacterial endophthalmitis and was excluded from analyses. We noted improvements in efficacy outcomes in most patients without significant immunogenicity. Compared with baseline, pooled analysis of ten participants showed improvements in mean mobility and full-field light sensitivity in the injected eye by day 30 that persisted to year 3 (mobility p=0·0003, white light full-field sensitivity p<0·0001), but no significant change was seen in the previously injected eyes over the same time period (mobility p=0·7398, white light full-field sensitivity p=0·6709). Changes in visual acuity from baseline to year 3 were not significant in pooled analysis in the second eyes or the previously injected eyes (p>0·49 for all time-points compared with baseline). INTERPRETATION: To our knowledge, AAV2-hRPE65v2 is the first successful gene therapy administered to the contralateral eye. The results highlight the use of several outcome measures and help to delineate the variables that contribute to maximal benefit from gene augmentation therapy in this disease. FUNDING: Center for Cellular and Molecular Therapeutics at The Children's Hospital of Philadelphia, Spark Therapeutics, US National Institutes of Health, Foundation Fighting Blindness, Institute for Translational Medicine and Therapeutics, Research to Prevent Blindness, Center for Advanced Retinal and Ocular Therapeutics, Mackall Foundation Trust, F M Kirby Foundation, and The Research Foundation-Flanders.

Wang YE, Kakigi C, Barbosa D, Porco T, Chen R, Wang S, Li Y, Singh K, Pasquale LR, Lin SC. Oral Contraceptive Use and Prevalence of Self-Reported Glaucoma or Ocular Hypertension in the United States. Ophthalmology 2016;123(4):729-36.Abstract

PURPOSE: To investigate the association between oral contraceptive (OC) use and glaucoma prevalence in the United States. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 3406 female participants, aged 40 years or older, from the 2005 to 2008 National Health and Nutrition Examination Survey, who reported a presence or absence of glaucoma or ocular hypertension completed both the vision and the reproductive health questionnaires and underwent eye examinations. METHODS: Multivariate regression analysis was used to assess the correlation between OC use and self-reported glaucoma or ocular hypertension (n = 231 cases), controlling for potential confounders, including age, ethnicity, systemic comorbidities such as hypertension and stroke, ocular diseases such as cataract and diabetic retinopathy, and reproductive health factors, including age at menopause, age at menarche, history of hormone replacement therapy, and gynecological surgical history. MAIN OUTCOME MEASURES: The outcome variable was self-reported glaucoma or ocular hypertension. RESULTS: After adjusting for confounders, those with ≥3 years of OC use had greater odds (odds ratio, 1.94; 95% confidence interval, 1.22-3.07) of self-reported glaucoma or ocular hypertension. Other factors associated with higher glaucoma or ocular hypertension prevalence included older age, African American race, and later age at menarche. CONCLUSIONS: Oral contraceptive use may be associated with increased risk of self-reported glaucoma or ocular hypertension.

Tang S, Hemberg M, Cansizoglu E, Belin S, Kosik K, Kreiman G, Steen H, Steen J. f-divergence cutoff index to simultaneously identify differential expression in the integrated transcriptome and proteome. Nucleic Acids Res 2016;44(10):e97.Abstract

The ability to integrate 'omics' (i.e. transcriptomics and proteomics) is becoming increasingly important to the understanding of regulatory mechanisms. There are currently no tools available to identify differentially expressed genes (DEGs) across different 'omics' data types or multi-dimensional data including time courses. We present fCI (f-divergence Cut-out Index), a model capable of simultaneously identifying DEGs from continuous and discrete transcriptomic, proteomic and integrated proteogenomic data. We show that fCI can be used across multiple diverse sets of data and can unambiguously find genes that show functional modulation, developmental changes or misregulation. Applying fCI to several proteogenomics datasets, we identified a number of important genes that showed distinctive regulation patterns. The package fCI is available at R Bioconductor and http://software.steenlab.org/fCI/.

Gao S, Jakobs TC. Mice Homozygous for a Deletion in the Glaucoma Susceptibility Locus INK4 Show Increased Vulnerability of Retinal Ganglion Cells to Elevated Intraocular Pressure. Am J Pathol 2016;186(4):985-1005.Abstract

A genomic region located on chromosome 9p21 is associated with primary open-angle glaucoma and normal tension glaucoma in genome-wide association studies. The genomic region contains the gene for a long noncoding RNA called CDKN2B-AS, two genes that code for cyclin-dependent kinase inhibitors 2A and 2B (CDKN2A/p16(INK4A) and CDKN2B/p15(INK4B)) and an additional protein (p14(ARF)). We used a transgenic mouse model in which 70 kb of murine chromosome 4, syntenic to human chromosome 9p21, are deleted to study whether this deletion leads to a discernible phenotype in ocular structures implicated in glaucoma. Homozygous mice of this strain were previously reported to show persistent hyperplastic primary vitreous. Fundus photography and optical coherence tomography confirmed that finding but showed no abnormalities for heterozygous mice. Optokinetic response, eletroretinogram, and histology indicated that the heterozygous and mutant retinas were normal functionally and morphologically, whereas glial cells were activated in the retina and optic nerve head of mutant eyes. In quantitative PCR, CDKN2B expression was reduced by approximately 50% in the heterozygous mice and by 90% in the homozygous mice, which suggested that the CDKN2B knock down had no deleterious consequences for the retina under normal conditions. However, compared with wild-type and heterozygous animals, the homozygous mice are more vulnerable to retinal ganglion cell loss in response to elevated intraocular pressure.

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