Update on the Corneal Dystrophies-Genetic Testing and Therapy.

ABSTRACT: One major purpose of the IC3D Corneal Dystrophy Nomenclature Revision was to include genetic information with a goal of facilitating investigation into the pathogenesis, treatment, and perhaps even prevention of the corneal dystrophies, an ambitious goal. Over a decade has passed since the first publication of the IC3D Corneal Dystrophy Nomenclature Revision. Gene therapy is available for an early-onset form of inherited retinal degeneration called Leber congenital amaurosis, but not yet for corneal degenerations. We review the current state of affairs regarding our original ambitious goal. We discuss genetic testing, gene therapy [RNA interference (RNAi) and genome editing], and ocular delivery of corneal gene therapy for the corneal dystrophies. Why have gene therapy techniques not yet been introduced for the corneal dystrophies?

New Insights on the Genetic Basis Underlying SHILCA Syndrome: Characterization of the NMNAT1 Pathological Alterations Due to Compound Heterozygous Mutations and Identification of a Novel Alternative Isoform.

This study aims to genetically characterize a two-year-old patient suffering from multiple systemic abnormalities, including skeletal, nervous and developmental involvements and Leber congenital amaurosis (LCA). Genetic screening by next-generation sequencing identified two heterozygous pathogenic variants in nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) as the molecular cause of the disease: c.439+5G>T and c.299+526_*968dup.This splice variant has never been reported to date, whereas pathogenic duplication has recently been associated with cases displaying an autosomal recessive disorder that includes a severe form of spondylo-epiphyseal dysplasia, sensorineural hearing loss, intellectual disability and LCA (SHILCA), as well as some brain anomalies. Our patient presented clinical manifestations which correlated strongly with this reported syndrome. To further study the possible transcriptional alterations resulting from these mutations, mRNA expression assays were performed in the patient and her father. The obtained results detected aberrant alternative transcripts and unbalanced levels of expression, consistent with severe systemic involvement. Moreover, these analyses also detected a novel NMNAT1 isoform, which is variably expressed in healthy human tissues. Altogether, these findings represent new evidence of the correlation of NMNAT1 and SHILCA syndrome, and provide additional insights into the healthy and pathogenic expression of this gene.

Coexistence of Meesmann Corneal Dystrophy and a Pseudo-Unilateral Lattice Corneal Dystrophy in a Patient With a Novel Pathogenic Variant in the Keratin K3 Gene: A Case Report.

PURPOSE: This study aims to clinically and genetically report a case of coexisting Meesmann corneal dystrophy (MECD) and pseudo-unilateral lattice corneal dystrophy (LCD). METHODS: Clinical characterization was supported by a complete ophthalmological evaluation, including visual acuity measurement and slit-lamp examination. Molecular diagnosis was performed by whole-exome sequencing analyzing the gelsolin, keratin K3 (KRT3), keratin K12, and transforming growth factor-beta-induced genes. RESULTS: A 57-year-old woman presented with recurrent corneal erosions over 17 years and visual impairment in both eyes. Ophthalmological evaluation revealed multiple central tiny cysts in the epithelium of both eyes and lattice linear lesions only in the right cornea. In both eyes, a corneal posterior crocodile shagreen degeneration could also be observed. These findings were compatible with a MECD and a unilateral LCD. Molecular analysis identified the novel heterozygous nucleotide substitution c.1492G>A (amino acid change p.Glu498Lys) in the KRT3 gene, in cosegregation with the MECD familial phenotype. However, no genetic evidence supported the unique LCD phenotype observed in the patient. CONCLUSIONS: To the best of our knowledge, this is the first report of a pseudo-unilateral LCD in a patient with coexistent MECD. Moreover, the genetic analysis showed a novel mutation in the previously MECD-associated gene KRT3.

Identification of a novel homozygous ARSG mutation as the second cause of Usher syndrome type 4.

PURPOSE: Usher syndrome is a genetic disease characterized by combined sensorineural hearing loss, retinitis pigmentosa, and vestibular areflexia, with 15 known causative genes. Depending on the severity and onset of the symptoms, 3 different subtypes of the pathology have been classically established, although an increasing number of rare cases are being accumulated as atypical forms. The present work aims to discover the genetic cause in a patient with atypical Usher syndrome, by performing whole exome sequencing in several family members. OBSERVATIONS: The obtained results identified a novel homozygous missense mutation (p.Asp44Asn) in the ARSG gene as the cause of the disease, which was characterized by late-onset progressive symptoms in the patient. A resembling phenotype, recently defined as the novel Usher syndrome type 4, was described in three families sharing another ARSG mutation. Both mutations affect two contiguous amino acid residues, which appear to be critical for the correct function of the protein. CONCLUSIONS AND IMPORTANCE: These findings support the identification of the second disease mutation in this gene and a new evidence of the implication of ARSG in the genetic basis of Usher syndrome type 4.

Expanding the retinal phenotype of RP1: from retinitis pigmentosa to a novel and singular macular dystrophy.

PURPOSE: This study aimed to identify the underlying genetic cause(s) of inherited retinal dystrophy (IRD) in 12 families of Kuwaiti origin affected by macular dystrophy and four Spanish patients affected by retinitis pigmentosa (RP). METHODS: Clinical diagnoses were based on standard ophthalmic evaluations (best-corrected visual acuity, retinography, fundus autofluorescence imaging, optical coherence tomography, electroretinography and visual field tests). Panel-based whole exome sequencing was used to simultaneously analyse 224 IRD genes in one affected member of each family. The putative causative variants were confirmed by Sanger sequencing and cosegregation analyses. Haplotype analysis was performed using single nucleotide polymorphisms. RESULTS: A homozygous missense mutation c.606C>A (p.Asp202Glu) in RP1 was found to be the molecular cause of IRD in all 12 families from Kuwait. These patients exhibited comparable symptoms, including progressive decline in visual acuity since adolescence. Fundus autofluorescence images revealed bilateral macular retinal pigment epithelium disturbances, with neither perimacular flecks nor peripheral alterations. A shared haplotype spanning at least 1.1 Mb was identified in all families, suggesting a founder effect. Furthermore, RP1 variants involving nonsense and/or frameshifting mutations (three of them novel) were identified in three Spanish autosomal-recessive RP families and one dominant RP pedigree. CONCLUSION: This study describes, for the first time, a macular dystrophy phenotype caused by an RP1 mutation; establishing a new genotype-phenotype correlation in this gene, expanding its mutation spectrum and further highlighting the clinical heterogeneity associated with IRD.

Generation of an induced pluripotent stem cell line (FRIMOi007-A) derived from an incomplete achromatopsia patient carrying a novel homozygous mutation in PDE6C gene.

Incomplete achromatopsia (ACHM) is a disorder in which there is function defect of cone photoreceptors in the retina and individuals with such disease retain residual color vision. Here, we have generated an induced pluripotent stem cell (iPSC) line carrying a homozygous mutation in the PDE6C gene, already related with this vision disorder. Skin fibroblasts from a patient with incomplete ACHM were reprogrammed to iPSCs by the non-integrative Sendai-virus method. Finally, the iPSC line has been characterized expressing the pluripotency markers and being capable to differentiate to endoderm, mesoderm and ectoderm in vitro.

Generation of Best disease-derived induced pluripotent stem cell line (FRIMOi006-A) carrying a novel dominant mutation in BEST1 gene.

Best disease, also known as Best vitelliform macular dystrophy, is an autosomal dominant form of macular degeneration. Here, we have generated an induced pluripotent stem cell (iPSC) line derived from a Best disease patient carrying a new dominant mutation in the BEST1 gene. Skin fibroblasts were reprogrammed to iPSCs by the non-integrative Sendai-virus method. The iPSC line has been characterized preserving the BEST1 mutation, expressing the pluripotency markers and being capable to differentiate to endoderm, mesoderm and ectoderm in vitro.

Characterization of the cone-rod dystrophy retinal phenotype caused by novel homozygous DRAM2 mutations.

Cone-rod dystrophies (CRD) are a group of Inherited Retinal Dystrophies (IRD) characterized by the primary involvement of cone photoreceptors, resulting in the degeneration of the central retina, or macula. Although there are more than 55 CRD genes, a considerable percentage of cases remain unsolved. In this context, the present study aimed to describe and characterize the phenoptype and the genetic cause of 3 CRD families from a cohort of IRD cases. Clinical evaluation in each patient was supported by a complete ophthalmological examination, including visual acuity measurement, fundus retinography, fundus autofluorescence imaging, optical coherence tomography and full-field electroretinography. Molecular diagnoses were performed by whole exome sequencing analyzing a group of 279 IRD genes, and cosegregation of the identified pathogenic variants was confirmed by Sanger sequencing. Three novel homozygous mutations in the autophagy gene DRAM2 were identified as the molecular cause of disease in the three families: c.518-1G>A, c.628_629insAG and c.693+2T>A. Clinical data revealed that the 3 patients presented a shared CRD phenotype with adult-onset macular involvement and later peripheral degeneration, although the age of onset, evolution and severity were variable. In order to characterize the transcription effects of these variants, mRNA expression studies were performed. The results showed alterations in the DRAM2 transcription, including alternative splicing forms and lower levels of mRNA, which correlated with the phenotypic variability observed between patients. For instance, frameshift mutations were related to a less severe phenotype, with circumscribed mid-peripheral involvement, and lower levels of mRNA, suggesting an activation of the nonsense-mediated decay (NMD) pathway; while a more severe and widespread retinal degeneration was associated to the inframe alternative splicing variant reported, possibly due to a malfunctioning or toxicity of the resulting protein. Following these findings, DRAM2 expression was assessed in several human tissues by semi-quantitative RT-PCR and two isoforms were detected ubiquitously, yet with a singular tissue-specific pattern in retina and brain. Altogether, although the unique retinal phenotype described did not correlate with the ubiquitous expression, the retinal-specific expression and the essential role of autophagy in the photoreceptor survival could be key arguments to explain this particular DRAM2 phenotype.

Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles.

Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.

mRNA expression analysis of the SUMO pathway genes in the adult mouse retina.

Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-PCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. The wide expression of the SUMO pathway genes, the transcriptional response under several light/dark conditions, and the diversity of expression patterns in different cell layers clearly support sumoylation as a relevant post-translational modification in the retina. This expression atlas intends to be a reference framework for retinal researchers and to depict a more comprehensive view of the SUMO-regulated processes in the retina.