Professor of Medical Genetics and Ophthalmology
Description of research: research is directed to identifying the genetic causes of inherited blindness, identifying the mechanisms linking mutation to disease, and developing treatment approaches. Studies are done in the dog model which is affected a large variety of inherited photoreceptor diseases that are now being characterized at the molecular level. For "disease hunting" work, we have used two general approaches. The first, phenotype directed candidate gene analysis, directs selection of a small subset of candidate genes that, when mutated, would be expected to result in the observed phenotype. The second approach utilizes informative pedigree resources we have developed to carry out genome wide scans to identify the disease bearing chromosomal region. Subsequently, positional cloning of the gene and identification of the causative mutation is carried out. Once the genes and mutations are identified, we use molecular and proteomic approaches to examine the disease mechanism. In parallel, gene based therapies are developed with the goal of restoring function and preventing the degeneration of the mutant photoreceptor cells.
Molecular bases of inherited blindness
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PublicationExonic SINE Insertion in STK38L Causes Canine Early Retinal Degeneration (erd)(2010-12-01) Goldstein, Orly; Aguirre, Gustavo D; Aguirre, Gustavo D; Kukekova, Anna V; Acland, Gregory MFine mapping followed by candidate gene analysis of erd — a canine hereditary retinal degeneration characterized by aberrant photoreceptor development — established that the disease cosegregates with a SINE insertion in exon 4 of the canine STK38L/NDR2 gene. The mutation removes exon 4 from STK38L transcripts and is predicted to remove much of the N terminus from the translated protein, including binding sites for S100B and Mob proteins, part of the protein kinase domain, and a Thr-75 residue critical for autophosphorylation. Although known to have roles in neuronal cell function, the STK38L pathway has not previously been implicated in normal or abnormal photoreceptor development. Loss of STK38L function in erd provides novel potential insights into the role of the STK38L pathway in neuronal and photoreceptor cell function, and suggests that genes in this pathway need to be considered as candidate genes for hereditary retinal degenerations. PublicationBEST1 Gene Therapy Corrects a Diffuse Retina-Wide Microdetachment Modulated by Light Exposure(2018-03-20) Guziewicz, Karina E; Cideciyan, Artur V; Beltran, William A; Komáromy, András M; Guziewicz, Karina E; Cideciyan, Artur V; Aguirre, Gustavo D; Ruthel, Gordon; Komáromy, András M; Dufour, Valerie L; Swider, Malgorzata; Jacobson, Samuel G; Sumaroka, Alexander; Kendrick, Brian T; Ruthel, Gordon; Chiodo, Vince A; Heon, Elise; Hauswirth, William W; Jacobson, Samuel GMutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies. PublicationA Non-Stop S-Antigen Gene Mutation Is Associated With Late Onset Hereditary Retinal Degeneration in Dogs(2013-08-01) Aguirre, Gustavo D; Aguirre, Gustavo D; Jordan, Julie Ann; Acland, Gregory MPurpose: To identify the causative mutation of canine progressive retinal atrophy (PRA) segregating as an adult onset autosomal recessive disorder in the Basenji breed of dog. Methods: Basenji dogs were ascertained for the PRA phenotype by clinical ophthalmoscopic examination. Blood samples from six affected cases and three nonaffected controls were collected, and DNA extraction was used for a genome-wide association study using the canine HD Illumina single nucleotide polymorphism (SNP) array and PLINK. Positional candidate genes identified within the peak association signal region were evaluated. Results: The highest -Log10(P) value of 4.65 was obtained for 12 single nucleotide polymorphisms on three chromosomes. Homozygosity and linkage disequilibrium analyses favored one chromosome, CFA25, and screening of the S-antigen (SAG) gene identified a non-stop mutation (c.1216T>C), which would result in the addition of 25 amino acids (p.*405Rext*25). Conclusions: Identification of this non-stop SAG mutation in dogs affected with retinal degeneration establishes this canine disease as orthologous to Oguchi disease and SAG-associated retinitis pigmentosa in humans, and offers opportunities for genetic therapeutic intervention. PublicationIdentification of Genetic Variation and Haplotype Structure of the Canine ABCA4 Gene for Retinal Disease Association Studies(2010-10-01) Zangerl, Barbara; Aguirre, Gustavo D; Zangerl, Barbara; Aguirre, Gustavo D; Lindauer, Sarah J; Acland, Gregory MOver 200 mutations in the retina specific member of the ATP-binding cassette transporter superfamily (ABCA4) have been associated with a diverse group of human retinal diseases. The disease mechanisms, and genotype–phenotype associations, nonetheless, remain elusive in many cases. As orthologous genes are commonly mutated in canine models of human blinding disorders, canine ABCA4 appears to be an ideal candidate gene to identify and study sequence changes in dogs affected by various forms of inherited retinal degeneration. However, the size of the gene and lack of haplotype assignment significantly limit targeted association and/or linkage approaches. This study assessed the naturally observed sequence diversity of ABCA4 in the dog, identifying 80% of novel variations. While none of the observed polymorphisms have been associated with blinding disorders to date, breed and potentially disease specific haplotypes have been identified. Moreover, a tag SNP map of 17 (15) markers has been established that accurately predicts common ABCA4 haplotypes (frequency > 5%) explaining >85% (>80%) of the observed genetic diversity and will considerably advance future studies. Our sequence analysis of the complete canine ABCA4 coding region will clearly provide a baseline and tools for future association studies and comparative genomics to further delineate the role of ABCA4 in canine blinding disorders. PublicationCanine RD3 Mutation Establishes Rod-Cone Dysplasia Type 2 (rcd2) as Ortholog of Human and Murine rd3(2009-02-01) Kukekova, Anna V; Aguirre, Gustavo D; Aguirre, Gustavo D; Goldstein, Orly; Johnson, Jennifer L; Richardson, Malcolm A; Pearce-Kelling, Susan E; Swaroop, Anand; Friedman, James S; Acland, Gregory MRod-cone dysplasia type 2 (rcd2) is an autosomal recessive disorder that segregates in collie dogs. Linkage disequilibrium and meiotic linkage mapping were combined to take advantage of population structure within this breed and to fine map rcd2 to a 230-kb candidate region that included the gene C1orf36 responsible for human and murine rd3, and within which all affected dogs were homozygous for one haplotype. In one of three identified canine retinal RD3 splice variants, an insertion was found that cosegregates with rcd2 and is predicted to alter the last 61 codons of the normal open reading frame and further extend the open reading frame. Thus, combined meiotic linkage and LD mapping within a single canine breed can yield critical reduction of the disease interval when appropriate advantage is taken of within-breed population structure. This should permit a similar approach to tackle other hereditary traits that segregate in single closed populations. PublicationImprovement in Vision: A New Goal for Treatment of Hereditary Retinal Degenerations(2015-05-04) Jacobson, Samuel G; Cideciyan, Artur V; Jacobson, Samuel G; Cideciyan, Artur V; Aguirre, Gustavo D; Roman, Alejandro J; Sumaroka, Alexander; Hauswirth, William W; Palczewski, KrzysztofIntroduction: Inherited retinal degenerations (IRDs) have long been considered untreatable and incurable. Recently, one form of early-onset autosomal recessive IRD, Leber congenital amaurosis (LCA) caused by mutations in RPE65 (retinal pigment epithelium-specific protein 65 kDa) gene, has responded with some improvement of vision to gene augmentation therapy and oral retinoid administration. This early success now requires refinement of such therapeutics to fully realize the impact of these major scientific and clinical advances. Areas covered: Progress toward human therapy for RPE65-LCA is detailed from the understanding of molecular mechanisms to preclinical proof-of-concept research to clinical trials. Unexpected positive and complicating results in the patients receiving treatment are explained. Logical next steps to advance the clinical value of the therapeutics are suggested. Expert opinion: The first molecularly based early-phase therapies for an IRD are remarkably successful in that vision has improved and adverse events are mainly associated with surgical delivery to the subretinal space. Yet, there are features of the gene augmentation therapeutic response, such as slowed kinetics of night vision, lack of foveal cone function improvement and relentlessly progressive retinal degeneration despite therapy, that still require research attention. PublicationCloning of Canine Galactokinase (GALK1) and Evaluation as a Candidate Gene for Hereditary Cataracts in Labrador Retrievers(2005-06-01) Sidjanin, Duska J; Aguirre, Gustavo D; Aguirre, Gustavo D; McElwee, John L; Miller, BrianWe identified a pedigree of Labrador retrievers (LR) that develop hereditary cataracts between 6 and 18 months of age. In humans, galactokinase deficiency is an autosomal recessive disorder characterized by juvenile onset of cataracts.1 In order to evaluate GALK1 as a candidate gene, we cloned and sequenced the canine GALK1 gene and tested a single nucleotide polymorphism (SNP) in the gene for segregation with cataracts in the LR pedigree. PublicationWindow Into Retinal Studies(2010-10-01) Aguirre, Gustavo D; Aguirre, Gustavo DProfessor Gustavo Aguirre discusses the context of his research studies at the University of Pennsylvania, which are currently concentrating on the degenerative disease, retinitis pigmentosa. PublicationAge-Dependent Disease Expression Determines Remodeling of the Retinal Mosaic in Carriers of RPGR Exon ORFn15 Mutations(2009-08-01) Beltran, William; Aguirre, Gustavo D; Beltran, William; Aguirre, Gustavo D; Acland, Gregory MPURPOSE. To characterize the retinal histopathology in carriers of X-linked progressive retinal atrophy (XLPRA1 and XLPRA2), two canine models of X-linked retinitis pigmentosa caused, respectively, by a stop and a frameshift mutation in RPGRORF15. METHODS. Retinas of XLPRA2 and XLPRA1 carriers of different ages were processed for morphologic evaluation, TUNEL assay, and immunohistochemistry. Cell-specific markers were used to examine retinal remodeling events. RESULTS. A mosaic pattern composed of patches of diseased and normal retina was first detected in XLPRA2 carriers at 4.9 weeks of age. A peak of photoreceptor cell death led to focal rod loss; however, in these patches an increased density of cones was found to persist over time. Patches of disease gradually disappeared so that by 39 weeks of age the overall retinal morphology, albeit thinner, had improved lamination. In older XLPRA2 carriers (≥8.8 years), extended regions of severe degeneration occurred in the peripheral/mid-peripheral retina. In XLPRA1 carriers, opsin mislocalization and rare events of rod death were detected by TUNEL assay at 20 weeks of age; however, only patchy degeneration was seen by 1.4 years and was still apparent at 7.8 years. CONCLUSIONS. The time of onset and the progression of the disease differed between the two models. In the early-onset form (XLPRA2) the morphologic appearance of the retinal mosaic changed as a function of age, suggesting that structural plasticity persists in the early postnatal canine retina as mutant photoreceptors die. In the late-onset form (XLPRA1), patches of disease persisted until later ages. PublicationVariabilities in Retinal Function and Structure in a Canine Model of Cone-Rod Dystrophy Associated With RPGRIP1 Support Multigenic Etiology(2017-01-01) Aguirre, Gustavo D; Aguirre, Gustavo D; Marinho, Felipe P; Iwabe, Simone; Santana, Evelyn; McDaid, Kendra S; Miyadera, KeikoDefects in the cilia gene RPGRIP1 cause Leber congenital amaurosis and cone-rod dystrophy in humans. A form of canine cone-rod dystrophy (cord1) was originally associated with a homozygous insertion in RPGRIP1 (RPGRIP1ins/ins) as the primary disease locus while a homozygous deletion in MAP9(MAP9del/del) was later identified as a modifier associated with the early onset form. However, we find further variability in cone electroretinograms (ERGs) ranging from normal to absent in an extended RPGRIP1ins/ins canine colony, irrespective of the MAP9 genotype. Ophthalmoscopically, cone ERGabsentRPGRIP1ins/ins eyes show discolouration of the tapetal fundus with varying onset and disease progression, while sd-OCT reveals atrophic changes. Despite marked changes in cone ERG and retinal morphology, photopic vision-guided behaviour is comparable between normal and cone ERGabsentRPGRIP1ins/ins littermates. Cone morphology of the dogs lacking cone ERG are truncated with shortened outer and inner segments. Immunohistochemically, cone ERGabsentRPGRIP1ins/ins retinas have extensive L/M-opsin mislocalization, lack CNGB3 labelling in the L/M-cones, and lack GC1 in all cones. Our results indicate that cord1 is a multigenic disease in which mutations in neither RPGRIP1 nor MAP9 alone lead to visual deficits, and additional gene(s) contribute to cone-specific functional and morphologic defects.